TRP CHANNELS AS THERAPEUTIC TARGETS TRP CHANNELS AS THERAPEUTIC TARGETS from Basic Science to Clinical Use

TRP CHANNELS AS THERAPEUTIC TARGETS TRP CHANNELS AS THERAPEUTIC TARGETS From Basic Science to Clinical Use

Edited by

Arpad Szallasi MD, PhD Department of Pathology, Monmouth Medical Center, Long Branch, NJ, USA

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M. Allen McAlexander Neuronal Targets Team, Ana Charrua IBMC—Instituto de Biologia Respiratory Therapy Area, GlaxoSmithKline Phar- Molecular e Celular da Universidade do maceuticals, King of Prussia, Pennsylvania, USA Porto; Departamento de Urologia, Faculdade Ganesan Baranidharan Consultant in Pain de Medicina da Universidade do Porto and Medicine, Leeds Teaching Hospitals NHS trust: Departamento de Doenças Renais, Urológicas D ward, Seacroft Hospital, Leeds, LS14 6UH e Infecciosas, Faculdade de Medicina da Uni- versidade do Porto, Porto, Portugal Ralf Baron Division of Neurological Pain Research and Therapy, Department of Neurol- Francisco Cruz IBMC—Instituto de Biologia ogy, University Hospital Schleswig-Holstein, Molecular e Celular da Universidade do Kiel, Germany Porto; Departamento de Urologia, Faculdade de Medicina da Universidade do Porto; De- Arun K. Bhaskar Consultant in Pain Medicine, partamento de Doenças Renais, Urológicas e Leeds Teaching Hospitals NHS trust: D ward, Infecciosas, Faculdade de Medicina da Uni- Seacroft Hospital, Leeds, LS14 6UH versidade do Porto and Departamento de Mahendra Bishnoi Department of Nutritional Urologia, Hospital São João, Porto, Portugal Sciences, and Technology, National Agri-Food Matthew A.J. Duncton Renovis, Inc. (a wholly Biotechnology Institute (NABI), SAS Nagar, owned subsidiary of Evotec AG), South San India Francisco, California, USA Jill-Desiree Brederson Global Medical Commu- Madeleine Ennis Centre for Infection and Im- nications, Research and Development, AbbVie munity, School of Medicine, Dentistry and Inc., North Chicago, Illinois, USA Biomedical Sciences, Queen's University Bel- Dorothy Cimino Brown Veterinary Clinical In- fast, Belfast, Northern Ireland, UK vestigations Center and Department of Clinical Susan Fleetwood-Walker Centre for Integra- Studies, School of Veterinary Medicine, tive Physiology, School of Biomedical Sciences, University of Pennsylvania, Philadelphia, College of Medicine & Veterinary Medicine, Pennsylvania, USA University of Edinburgh, Scotland, UK Nigel W. Bunnett Monash Institute of Phar maceutical Sciences, and Department of Ehud Goldin SENS Research Foundation, Pharmacology, The University of Melbourne, Mountain View, CA, USA Parkville, Victoria, Australia Arthur Gomtsyan Department of Chemistry, Ingolf Cascorbi Institute of Experimental and Research and Development, AbbVie Inc., North Clinical Pharmacology, University Hospital Chicago, Illinois, USA Schleswig-Holstein, Kiel, Germany Huizhen Huang Department of Neurobiology, Michael J. Caterina Department of Neurosur- University of Pittsburgh; Pittsburgh Center for gery; Department of Biological Chemistry; Pain Research, Pittsburgh, PA, USA, and Tsing- Solomon H. Snyder Department of hua University School of Medicine, Beijing, China Neuroscience and Neurosurgery Pain Re- Gerald Hunsberger Neuronal Targets Team, Re- search Institute, Johns Hopkins School of spiratory Therapy Area, GlaxoSmithKline Phar- Medicine, Baltimore, Maryland, USA maceuticals, King of Prussia, Pennsylvania, USA

ix x CONTRIBUTORS

Michael J. Iadarola Anesthesia Section, Depart- Magdalene Moran Hydra Biosciences ment of Perioperative Medicine, Clinical Cen- Cambridge, MA, USA ter, NIH, Bethesda, Maryland, USA Christopher Neipp Flexible Discovery Unit, Neelima Khairatkar Joshi Glenmark Research GlaxoSmithKline Pharmaceuticals, King of Centre, Navi Mumbai, Glenmark Pharmaceuti- Prussia, Pennsylvania, USA cals Ltd, India Bernd Nilius Katholieke Universiteit of Leu- Pragyanshu Khare Department of Nutritional ven, Department of Cellular and Molecular Sciences and Technology, National Agri-Food Medicine, Laboratory of Ion Channel Research Biotechnology Institute (NABI), SAS Nagar, and TRP Research Platform Leuven (TRPLe), India Campus Gasthuisberg, Leuven, Belgium Kirill Kiselyov Department of Biological Sci- James C. Parker Department of Physiology and ences, University of Pittsburgh, Pittsburgh, Center for Lung Biology, University of South PA, USA Alabama, Mobile, Alabama, USA Ari Koivisto Research and Development, Ori- Antti Pertovaara Institute of Biomedicine/ onPharma, Orion Corporation, Turku, Finland Physiology, University of Helsinki, Helsinki, Kanthi K. Kondepudi Department of Food Sci- Finland ences and Technology, National Agri-Food Bio- Koenraad Philippaert Katholieke Universiteit technology Institute (NABI), SAS Nagar, India of Leuven, Department of Cellular and Molec- Artem Kondratskyi Inserm U-1003, Equipe la- ular Medicine, Laboratory of Ion Channel Re- bellisée par la Ligue Nationale contre le cancer, search and TRPLe (TRP Research Platform Laboratory of Excellence Ion Channels Science Leuven), Campus Gasthuisberg, Leuven, and Therapeutics, Université Lille 1, Villeneuve Belgium d’Ascq, France Daniel P. Poole Monash Institute of Pharma- Ina Kraus-Stojanowic Institute of Experimental ceutical Sciences; Department of Anatomy & and Clinical Pharmacology, University Hospi- Cell Biology, The University of Melbourne, tal Schleswig-Holstein, Kiel, Germany Parkville, Victoria, Australia TinaMarie Lieu Monash Institute of Pharma- Louis S. Premkumar Department of Pharma- ceutical Sciences, Parkville, Victoria, Australia cology, Southern Illinois University-School of Medicine, Springfield, Illinois, USA Daoyan Liu Department of Hypertension and Endocrinology, Center for Hypertension and Natalia Prevarskaya Inserm U-1003, Equipe la- Metabolic Diseases, Daping Hospital, Third bellisée par la Ligue Nationale contre le cancer, Military Medical University, Chongqing Insti- Laboratory of Excellence Ion Channels Science tute of Hypertension, Chongqing, China and Therapeutics, Université Lille 1, Villeneuve d’Ascq, France Nancy Luo Division of Cardiology, Department of Medicine, Duke University School of Medi- Pradeep Rajasekhar Monash Institute of Phar- cine, Durham, North Carolina, USA maceutical Sciences, Parkville, Victoria, Australia Lorcan McGarvey Centre for Infection and Im- munity, School of Medicine, Dentistry and Bio- Paul Rosenberg Division of Cardiology, De- medical Sciences, Queen's University Belfast, partment of Medicine, Duke University School Belfast, Northern Ireland, UK of Medicine, Durham, North Carolina, USA Rory Mitchell Centre for Integrative Physiol- Sarah E. Ross Department of Neurobiology; De- ogy, School of Biomedical Sciences, College of partment of Anesthesiology; Pittsburgh Center Medicine & Veterinary Medicine, University of for Pain Research, and Center for Neuroscience Edinburgh, Scotland, UK Research at the University of Pittsburgh, Pitts- burgh PA, USA CONTRIBUTORS xi

Kavisha Singh Division of Cardiology, Depart- Mary I. Townsley Department of Physiology ment of Medicine, Duke University School of and Center for Lung Biology, University of Medicine, Durham, North Carolina, USA South Alabama, Mobile, Alabama, USA Lindsey M. Snyder Department of Neurobiol- Nicholas A. Veldhuis Monash Institute of Phar- ogy, University of Pittsburgh; Pittsburgh Cen- maceutical Sciences; Department of Genetics, ter for Pain Research, and Center for The University of Melbourne, Parkville, Neuroscience Research at the University of Victoria, Australia Pittsburgh, Pittsburgh PA, USA Kartik Venkatachalam Department of Integra- Martin Steinhoff Department of Dermatology, tive Biology and Pharmacology, University of University of California San Francisco (UCSF), Texas School of Medicine, Houston, TX, USA San Francisco, California, USA; Charles Institute Rudi Vennekens Katholieke Universiteit of for Translational Dermatology, University Col- Leuven, Department of Cellular and Molecular lege Dublin (UCD), Dublin, Ireland Medicine, Laboratory of Ion Channel Research Mathias Sulk Department of Dermatology, Uni- and TRPLe (TRP Research Platform Leuven), versity of California San Francisco (UCSF), San Leuven, Belgium Francisco, California, USA; Department of Der- Donald G. Welsh Hotchkiss Brain Institute; matology, University Hospital Münster (UKM), Libin Cardiovascular Institute and Department Münster, Germany of Physiology and Pharmacology, University of Arpad Szallasi Department of Pathology, Mon- Calgary, Calgary, Alberta, Canada mouth Medical Center, Long Branch, NJ, USA Shiqiang Xiong Department of Hypertension Jessica Tan Department of Biology, College of and Endocrinology, Center for Hypertension Science and Technology, Temple University, and Metabolic Diseases, Daping Hospital, Philadelphia; Neuronal Targets Team, Respi Third Military Medical University, Chongqing ratory Therapy Area, GlaxoSmithKline Institute of Hypertension, Chongqing, China Pharmaceuticals, King of Prussia, Pennsylva- Anil Zechariah Hotchkiss Brain Institute; Libin nia, USA Cardiovascular Institute and Department of Physiology and Pharmacology, University of Balázs István Tóth Katholieke Universiteit of Calgary, Calgary, Alberta, Canada Leuven, Department of Cellular and Molecular Medicine, Laboratory of Ion Channel Research Alexander Zholos Department of Biophysics, and TRP Research Platform Leuven (TRPLe), Educational and Scientific Centre “Institute of Campus Gasthuisberg, Leuven, Belgium; DE- Biology”, Taras Shevchenko Kiev National Uni- MTA “Lendület” Cellular Physiology Research versity, Kiev, Ukraine Group, Department of Physiology, University Zhiming Zhu Department of Hypertension and of Debrecen, Medical and Health Science Cen- Endocrinology, Center for Hypertension and ter, Research Center for Molecular Medicine, Metabolic Diseases, Daping Hospital, Third Debrecen, Hungary Military Medical University, Chongqing Insti- tute of Hypertension, Chongqing, China Preface

Anyone venturing to edit a new book on a TRP channel activator or blocker prove transient receptor potential (TRP) channels clinically useful in the pharmacotherapy of has to be aware of the immense mountain any of these diseases? Although book edi- range of literature towering behind him: tors have many talents, precognition of the with over 500 reviews and a dozen or so future is not among them. Clinical trials books addressing them, TRP channels repre- themselves must provide the answers. As sent one of the best-reviewed topics in the this book will tell you, the obstacles facing current field of drug discovery and devel- the clinical development of drugs targeting opment. Despite the initial successes in this TRP channels are real but probably not in- field (only a decade elapsed between the surmountable, and the potential benefits cloning of TRPV1 in 1997 and clinical trials that pharmaceutical companies can reap of the first potent small molecule TRPV1 an- are huge. In China alone, for example, there tagonists for use as novel analgesic drugs), are over 100 million patients who are candi- the early euphoria has now been replaced by dates for a new antidiabetic medication. I am more measured expectations, because none giving this book to the reader in the sincere of the TRPV1 antagonists has yet progressed hope that it will facilitate the development of beyond phase II trials. At the same time, a clinical applications from the current excit- steady stream of exciting discoveries has ing findings in basic TRP channel research. extended the therapeutic potential of drugs targeting TRP channels into new disease Arpad Szallasi MD, PhD indications, including respiratory, cardio- Department of Pathology vascular, bladder, and metabolic diseases Monmouth Medical Center (e.g., obesity and diabetes), as well as neuro- Long Branch, NJ, USA logic disorders (e.g., stroke) and cancer. Will

xiii CHAPTER 1 An Introduction to Transient Receptor Potential Ion Channels and Their Roles in Disease Michael J. Caterina1,2,3,4,* 1Department of Neurosurgery, Johns Hopkins School of Medicine, Baltimore, Maryland, USA 2Department of Biological Chemistry, Johns Hopkins School of Medicine, Baltimore, Maryland, USA 3Solomon H. Snyder Department of Neuroscience, Johns Hopkins School of Medicine, Baltimore, Maryland, USA 4Neurosurgery Pain Research Institute, Johns Hopkins School of Medicine, Baltimore, Maryland, USA *Corresponding author: [email protected]

OUTLINE

Discovery and General Properties of TRP TRP Channels and the Brain 5 Channels 2 TRP Channels and Immune Function 5 TRP Channels and Development 6 TRP Channels in Normal Physiology 4 TRP Channels and Sensory Physiology 4 TRP Channels and Disease 6 TRP Channels and Cardiovascular Therapeutic Strategies Based on TRP Function 5 Channel Modulation 9 TRP Channels and Gastrointestinal Function 5 Acknowledgment 10 TRP Channels and Urological Function 5 References 10

TRP Channels as Therapeutic Targets 1 © 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/B978-0-12-420024-1.00001-1 2 1. INTRODUCTION TO TRP CHANNELS IN DISEASE DISCOVERY AND GENERAL PROPERTIES OF TRP CHANNELS

The diverse repertoire of ion channels expressed in mammalian and nonmammalian species is encoded by a multitude of gene families. Among these, the transient receptor potential (TRP) ion channel family exhibits an especially prevalent and complex link with disease. Fittingly for the theme of this book, the TRP channel name emerged as a consequence of a disease state, although the victims of this disease were not human beings, but rather members of a line of visually impaired fruit flies [1]. Electroretinograms recorded from photoreceptors of these flies revealed that the electrical response to a light pulse (receptor potential), instead of remaining robust throughout a pulse of several seconds, decayed prematurely. Subsequent molecular and physiological studies revealed that the gene mutated in these so-called transient receptor potential (trp) flies encoded an ion channel subunit that, together with a homologous channel subunit, TRPL, forms the functional photoreceptor channel. This channel is not gated directly by light, but rather is activated by a G protein-coupled phospholipase C signaling pathway following the photoisomerization of the light receptor protein, rhodopsin. Following the identification of the Drosophila TRP channel, numerous homologous proteins were discovered, both in invertebrate species, such as fruit flies and nematodes, and in vertebrate species from fish to mammals [2]. Based on their domain structure and details of their sequences, members of the TRP channel family can be divided into seven subfamilies: TRPA (ankyrin, 1 human member), TRPC (canonical, 6 human members, plus 1 human pseudogene), TRPM (melastatin, 8 human members), TRPML (mucolipin, 3 human members), TRPN (NompC, no human members), TRPP (polycystin, 3 human members), and TRPV (vanilloid, 6 human members). There is also a distantly related family, TRPY, found in yeast. Functional TRP channels consist of homomeric or heteromeric tetramers of subunits from these subfamilies. The domain structure of an example TRP channel subunit, TRPV1, is shown in Figure 1.1a. A common structural feature of all TRP channel subunits is a core of six transmembrane domains (S1-S6), flanked by intracellular amino- and carboxyl-termini. Between S5 and S6 there is a complex pore-loop structure, which breaches the extracellular plane of the plasma membrane and forms the ion selectivity filter. This overall architecture resembles that of the voltage-gated and cyclic nucleotide-gated channel families. The TRPC, TRPM, TRPV, TRPA, and TRPN subfamilies, referred to as Group I TRP channels, resemble one another more closely than they do the TRPP or TRPML subfamilies, which are classified as Group II. Two features found among most Group I TRP channels include a TRP box homology element (absent in the TRPA subfamily), just distal to the sixth transmembrane domain, that participates in channel multimerization and modulation of gating, and a string of 4-16 sequential ankyrin repeat domains in the amino terminus (absent in the TRPM subfamily) that serves as a site of channel regulation (Figure 1.1a). Several TRPM subfamily members also contain kinase or nucleotide binding domains within their carboxyl termini and are therefore referred to as “chanzymes.” Ion flux through TRP channels occurs via a central pore lined by the pore loop domains of the four channel subunits (Figure 1.1b). All known TRP channels are selective for cations, although their degree of discrimination among cations can vary. For example, although some channels such as TRPV5 and TRPV6 are highly selective for Ca2+, and TRPM4 and TRPM5 are relatively Ca2+ impermeant, most TRP channels are nonselective cation channels that can mediate flux of multiple monovalent and divalent cations [3]. Whereas most of these channels function at the plasma membrane, some are also found in organellar membranes. Discovery and General Properties of TRP Channels 3 Pore helix Selectivity filter S5 S6 S1 S2 S3 S4

Ankyrin repeat Linker Pre-S1 S4-S5 TRP domain C-terminal domain helix linker domain (a) DkTx protons

Extracellular Pore axis S1-S4 Capsaicin RTX

Intracellular

Pore module (S5-pore loop-S6) ATP (b) calmodulin Selectivity Extracellular filter

G643 Pore helix

S6 S5 I679

Intracellular (c) apo RTX/DkTx

FIGURE 1.1 Representative TRP channel structure. (a) Domain map of a TRPV1 subunit. Amino terminus is at left. (b) TRPV1 holochannel structure in the apo (closed) state, solved by cryo-electron microscopy. Each subunit is in a different color. At left the channel is viewed from the side, illustrating distinct sites at which several agonists and regulators bind to allosterically control gating. At right, the transmembrane portion of the channel is viewed from the bottom and illustrates the separation between the S1-S4 domain and the S5-pore loop-S6 domain that forms the pore module lining the central pore axis (RTX, resiniferatoxin; DkTx, tarantula double-knot toxin). (c) Comparison of the TRPV1 pore module in the apo form (left) vs. a strongly activated state (right) evoked by a combination of RTX and DkTx. Path available for ion permeation is marked by dotted volume. For clarity, only two opposing subunits are shown. Sites of maximal constriction (G643 in the upper pore and I679 in the lower pore) are indicated. Note widening of both constrictions on activation. Modified, with permission, from Liao et al. [3] and Cao et al. [4].

For example, TRPM2, TRPML, and TRPV2 channels can reside and function within the endolysosomal pathway [4]. A higher-resolution understanding of structural features of TRP channels has recently emerged with the solution of the atomic-level structure of one family member, TRPV1, by cryo-electron microscopy (Figure 1.1b and c) [5,6]. A few details and implications of this important advance will be described later in this chapter. 4 1. INTRODUCTION TO TRP CHANNELS IN DISEASE TRP CHANNELS IN NORMAL PHYSIOLOGY

TRP channels as a family are broadly expressed in mammalian tissues. In fact, every cell in the body likely expresses at least one family member, and often more. Moreover, these channels can be activated by a number of heterogeneous stimuli, including a plethora of endogenous and exogenous chemical ligands, physical stimuli such as temperature and mechanical force, free cytosolic Ca2+ ions, depletion of endoplasmic reticulum Ca2+ stores, and many others. It should therefore not be surprising that these channels have been linked to numerous physiological functions. The following examples provide a glimpse into the ubiquitous involvement of TRP channels in the fundamental processes of life. As will be emphasized later in this chapter, and throughout this book, the pervasiveness of TRP channels in normal mammalian biology sets the stage for them to serve as contributors to, modulators of, or even primary causes of numerous human diseases.

TRP Channels and Sensory Physiology Perhaps the best understood physiological functions of TRP channels are in the realm of sensory signal transduction. Just as the Drosophila TRP channel is a key effector in phototransduction, many other TRP channels serve either as primary transducers of environmental stimuli or as amplifiers or modulators of signals transduced by other receptors. The most extensively studied example from mammalian systems is TRPV1, a channel expressed at disproportionately high levels in a subpopulation of primary afferent nociceptors, sensory neurons that trigger the perception of pain [7]. TRPV1 was discovered on the basis of, and derives its name from, its ability to be gated by painful vanilloid compounds such as capsaicin (the main pungent ingredient in chili peppers) and resiniferatoxin (a highly potent irritant produced in the latex of Euphorbia plant species). Functional studies subsequently revealed that TRPV1 could alternatively be activated by other, nonvanilloid stimuli, most notably noxious heat (>42 °C), protons (calcium and sodium ion flux into nociceptor terminals, resulting in depolarization and consequent action potential firing. It also causes local neuronal release of bioactive peptides and consequent neurogenic inflammation. The necessity of TRPV1 for responsiveness to vanilloids and for normal heat-evoked pain has been demonstrated through the study of TRPV1 knockout mice, as well as the development and application of highly selective TRPV1 antagonists [7,8]. Subsequent to the discovery of TRPV1, a number of other TRP channels were identified as mediators of chemosensation, thermosensation, and perhaps most controversially, mechanosensation. Several examples include TRPA1, which is activated by pungent cysteine-reactive compounds such as mustard oil and acrolein, is a key mediator of itch sensation, and also appears to play roles in cold- and mechanically evoked pain [9]; TRPM8, which is activated by menthol and moderately cold temperatures and which is essential for mouse avoidance of such temperatures [10]; TRPC1, which has been implicated in transduction of low-intensity mechanical stimuli [11]; and TRPM3, another heat-gated channel that appears to contribute to heat-evoked pain [12]. In many cases, these channels appear to be functionally important not only at the peripheral terminals of sensory neurons, but also at their spinal and trigeminal central terminals, where they modulate the release of neurotransmitters onto second-order neurons [13,14]. Beyond somatosensory neurons, TRPM5 is a key participant in the detection TRP Channels in Normal Physiology 5 of taste stimuli [15], whereas TRPC6 and TRPC7 appear to be involved in phototransduction by intrinsically photosensitive retinal ganglion cells [16].

TRP Channels and Cardiovascular Function Many TRP channels, including TRPC1, TRPC3, TRPC6, TRPM4, TRPV2, and TRPV4, are expressed in endothelial or muscle cells within the heart and blood vessels, where they regulate vascular tone and permeability, as well as cardiac contractility [17]. These functions are accomplished predominantly by increasing free cytoplasmic calcium levels.

TRP Channels and Gastrointestinal Function Many TRP channels are expressed intrinsically within the gastrointestinal tract [18]. For example, TRPV6 is a key effector of vitamin D-stimulated calcium absorption in the gut, whereas TRPV2 is expressed in enteric neurons, where it appears to regulate gastrointestinal motility. There is also abundant innervation of the gastrointestinal tract by extrinsic sensory afferents that express TRPV1, TRPA1, and other TRP channels. These neuronal channels regulate such processes as mucosal bloodflow and sensitivity to luminal distension. Within the exocrine portion of the gastrointestinal tract, TRPC1 and TRPV4 regulate saliva production and/or pancreatic acinar cell secretion, through the modulation of intracellular calcium levels and the regulation of aquaporin water channels. In the endocrine pancreas, TRPM5 and TRPM2, among others, contribute to the regulation of beta cell insulin release.

TRP Channels and Urological Function TRP channels contribute to multiple processes within the kidneys and lower urinary tract [19–21]. These include regulation of renal glomerular filtration function by TRPC6, regulation of nephron osmoregulatory function by TRPV4, regulation of calcium and magnesium uptake by TRPV5 and TRPV6, and the contribution of TRPV1, TRPA1, and TRPV4 to the detection of stretch and irritation by bladder afferents and the urinary bladder epithelium.

TRP Channels and the Brain All the TRP channel subfamilies expressed in humans have been implicated in important functions within the central nervous system [22]. Examples include neurotransmitter release (TRPC3, TRPV1, TRPV2), neurogenesis (TRPC6), astrocyte calcium homeostasis (TRPA1), responses to oxidative stress (TRPM7, TRPM2), osmoregulation (TRPV1, TRPV4), respiratory control (TRPM4), and neuronal lysosomal function (TRPML).

TRP Channels and Immune Function Among the immune cell processes demonstrated to involve TRP channels are regulation of T-cell membrane excitability (TRPM4) and macrophage and microglial phagocytosis, lysosomal acidification, and cytokine release (TRPM2, TRPM7, TRPV2) [23,24]. Other cell types that contribute to innate immune function, including skin keratinocytes and primary sensory 6 1. INTRODUCTION TO TRP CHANNELS IN DISEASE afferents, also employ TRP channels to carry out these functions. For example, TRPA1 and TRPV4 were recently shown to mediate the release of immune modulatory molecules such as TSLP and endothelin-1 from keratinocytes [25,26], whereas TRPV1- and TRPA1-mediated neurogenic release of neuropeptides from sensory afferents can promote tissue swelling and regulate recruitment of immune cells [9,27].

TRP Channels and Development TRP channels also perform important functions related to reproduction and embryonic development. For example, elimination of TRPM7 is lethal at very early embryonic stages [28]. TRPV3 controls differentiation of the skin and hair, at least in part by regulating keratinocyte production of EGF receptor ligands [29], whereas TRPV2, TRPV4, and TRPM7 all may be involved in the differentiation of human adipocytes [30].

TRP CHANNELS AND DISEASE

Given the involvement of TRP channels in so many physiological processes, it should not be surprising that dysregulation of TRP channels has been linked to numerous pathophysiological conditions. In the ensuing chapters, the reader will encounter a host of situations in which an excess or shortage of TRP channel activity is a contributing factor to a human disease, or in which modulation of TRP channels provides a potential opportunity to circumvent a disease process. In some cases, the contribution of a particular TRP channel to a specific disease process is direct and paramount. As will be highlighted both in the chapter on hereditary TRP channelopathies (Chapter 2) and on TRP gene polymorphism (Chapter 4), as well as in chapters dedicated to diseases of specific organ systems, point mutations in a number of TRP channels are sufficient to produce clinical syndromes characterized by well-defined inheritance patterns [31,32]. Examples include mutations in TRPV4 leading to either sensorimotor axonopathies such as Type 2C Charcot-Marie-Tooth disease or heritable skeletal abnormalities such as brachyolmia, mutations in TRPA1 resulting in familial episodic pain syndrome, mutations in TRPC6 giving rise to focal segmental glomerulosclerosis, mutations in TRPV3 producing palmoplantar keratoderma and pruritus in Olmsted syndrome, mutations in TRPML3 causing the lysosomal storage disease type IV mucolipidosis, and mutations in TRPP1 and TRPP2 causing polycystic kidney disease. Although many of these are autosomal dominant conditions, this is not always so, and it is sometimes difficult to differentiate whether a given mutation produces disease through loss- or gain-of-function. There also exist situations in which the link between a given TRP channel and a given disease apparently arises from alterations in the expression of the channel or of factors that regulate the channel’s localization or activity. For example, following tissue inflammation or nerve injury, MAP kinase-dependent changes in the abundance of TRPV1 at the nociceptor terminal, protein kinase C and protein kinase A phosphorylation-mediated increase in TRPV1 sensitivity to its agonists or TRPV1 localization at the plasma membrane, and increases in the abundance of endogenous stimulators and potentiators of this channel, such as protons, endocannabinoids, and serotonin, all conspire to augment TRPV1 signaling [7,33]. A similarly TRP Channels and Disease 7 complex regulatory pattern appears to exist for TRPA1 [9]. As a consequence of these events, TRPV1, TRPA1, and other TRP channels have emerged as key contributors to chemical, thermal, and mechanical hypersensitivity in animal models of nerve injury or inflammation. In humans, immunostaining studies have revealed upregulation of TRPV1 in peripheral nerves in a number of pain states [34]. Furthermore, a recent study showed that epigenetic changes in TRPA1 promoter methylation in white blood cells and gene expression in skin biopsies are predictive of human thermal pain sensitivity [35]. Finally, as discussed in two later chapters, the development of selective TRPV1 (Chapter 8) and TRPA1 (Chapter 9) antagonists has set the stage for the direct pharmacological assessment of the involvement of these channels in human disease [36]. As will be detailed throughout this book, many nonsensory pathophysiological conditions also appear to involve hyper- or hypoactivity of TRP channels. For example, abnormalities in TRP channel function in immune or inflammatory processes contribute to conditions such as asthma, dermatitis, gastrointestinal inflammation, and autoimmunity. TRP channels are also involved in cardiovascular diseases such as heart failure and hypertension and in both vascular and nonvascular diseases of the central nervous system. There is a also a growing body of evidence to support the intimate involvement of TRP channels, including TRPM8, TRPC6, TRPV2, TRPV1, and TRPM1, in cancer [37]. Finally, links have emerged between channels such as TRPM2, TRPV1, and TRPV4 and diseases involving tissue and organismal homeostasis, such as diabetes and obesity. Factors that contribute to the many diverse consequences of TRP channel dysfunction and likely explain the pervasive association of TRP channels with disease: (1) Both widespread and specific expression patterns. As discussed earlier, every cell in the body is likely to express one or more TRP channels, providing numerous opportunities for their aberrant function to contribute to disease pathogenesis. At the same time, the exceptionally high levels of expression of TRP channels in specific cell types, such as particular sensory neuron populations, creates a scenario in which channel dysfunction can have a disproportionately large effect on that cell’s function or health. (2) Importance of calcium signaling. The ability of many TRP channels to mediate calcium influx into cells makes them gatekeepers for one of the most potent of biological signals. Cytoplasmic calcium levels are normally tightly controlled and regulate many cellular processes, such as secretion, motility, action potential firing and propagation, neurotransmitter release, and gene expression. At the same time, high levels of calcium can be cytotoxic. This creates a situation in which either gain or loss of TRP channel function can profoundly influence cellular behavior and survival. (3) Diversity of signaling outputs. Calcium is not the only ion that can flow through TRP channels. As discussed earlier, many TRP channels are nonselective cation channels that can pass sodium, potassium, and magnesium ions, which in turn can influence cell behavior through the control of membrane polarization, cellular osmolarity, or more specific functions of the particular ions involved. In addition, some TRP channels, such as TRPV1, can be induced to mediate the influx of unusually large cations, such as potentially toxic aminoglycosides [38]. The repertoire of TRP channel signaling is expanded even further by the existence of catalytic domains on some subtypes, such as TRPM7, and the ability of TRP channels to functionally interact with other signaling proteins, such as Homer and STIM family members [39,40]. 8 1. INTRODUCTION TO TRP CHANNELS IN DISEASE

(4) Polymodal regulation and polygating. A remarkable feature of many TRP channels is that a given subtype can be alternatively regulated by multiple diverse stimuli that might be chemical, thermal, or mechanical in nature. Furthermore, multiple stimuli can converge on a given TRP channel to evoke additive, supra-additive, or antagonistic activities. Mutagenesis studies on many TRP channels, coupled with the recent solution of the TRPV1 atomic structure, have provided some insights into TRP channel polymodality. Examination of TRPV1 structures, solved in the absence vs. presence of exogenous activators [5,6], has revealed that multiple domains previously implicated in responsiveness to various agonists (a vanilloid binding pocket between transmembrane domains 3 and 4, a pore turret domain adjacent to the selectivity filter, the ankyrin repeat region that can bind and be modulated by intracellular ATP and calmodulin) are connected via conserved motifs (a linker prior to the first transmembrane domain, a helix between transmembrane domains 4 and 5, the helical TRP domain distal to transmembrane domain 6, a short pore helix adjacent to the selectivity filter) to not one, but two gates within the channel pore. The first gate is located toward the cytoplasmic end of the pore, at a site where the sixth transmembrane helices of each of the four subunits come in close proximity to restrict ion access to the pore. A similar (though not identical) gate has long been recognized in the voltage-gated channel family. The second, and somewhat surprising, gate in TRPV1 is located within the selectivity filter itself, which widens measurably with strong stimulation. The existence of this second gate explains why stimuli like protons and the tarantula-derived double-knot vanillotoxin activate the channel on binding to an adjacent pore turret region and may provide a hint at the structural basis for the observation that large cation permeability through TRPV1 is augmented by strong, persistent channel stimulation. Although the extreme carboxyl terminal domain of TRPV1 was not included in the structures solved to date, previous work suggests that phosphorylation of this domain, or its interaction with calmodulin, phosphoinositides, and other regulators [41], offers further opportunities for coupling with the inner and outer gates, most likely via the allosteric linker domains described earlier. Mutagenesis studies further suggest that similarly multiple, parallel but interconnected mechanisms for channel activation exist in other TRP channels [42]. Moreover, TRPV1 subunits, and thus probably subunits in other TRP channels, are clearly not autonomous of one another within the tetrameric holochannel. Rather, their regulatory and allosteric coupling domains are intricately interlaced with one another [5,6]. This remarkable interconnectedness among channel domains and subunits renders plausible a view of TRP channels as being exceptionally well poised to respond to diverse environmental signals at the slightest provocation and to function as coincidence detectors of multiple stimuli. This view is consistent with the observation that point mutations leading to constitutively active TRP channel function have been observed in many of the regulatory domains described earlier [32]. It also provides a potential explanation for how disease- related changes in the multitude of TRP channel regulators might manifest as disease. Given the numerous structural interactions required to maintain such allosteric complexity and thereby avoid unfettered calcium influx into the cell, it is amazing that, outside of a few well-conserved regions, the primary sequences of the many TRP channel family are actually quite divergent. For the same reason, it is a wonder that there are not more TRP channelopathies. Perhaps there are, but their incompatibility with life masks their true frequency. Therapeutic Strategies Based on TRP Channel Modulation 9 THERAPEUTIC STRATEGIES BASED ON TRP CHANNEL MODULATION

The prevalence of TRP channel involvement in human disease creates numerous opportunities, in principle, to develop therapeutic strategies based on these channels. A number of these strategies are described in detail in later chapters. In general, they fall into several categories outlined here. (1) TRP channel antagonists. In the case of diseases arising from TRP channel hyperactivity, specific small molecule antagonists offer the prospect of directly suppressing this activity. This strategy has been greatly facilitated by the fact that many TRP channels are “druggable,” in that they possess binding sites for small molecules such as the natural products (e.g., capsaicin, resiniferatoxin, cannabinoids, menthol) that have been used to characterize their functions. High throughput screens, often based on TRP channel-mediated influx of calcium in response to chemical activation, have enabled the isolation of numerous small molecules with high affinity and, in some cases, exquisite selectivity for particular TRP channel subtypes. As will be seen in later chapters, one potential complication associated with the therapeutic application of TRP channel antagonists, even if they are highly target specific, is interference with desirable physiological functions such as regulation of body temperature [8]. One approach to circumventing this potential hazard has been to develop peptides that prevent TRP channels from interacting with sensitizing binding partners. For example, A-kinase anchoring protein (AKAP) is an adaptor protein that facilitates the phosphorylation of TRPV1 by protein kinase C or protein kinase A. In animal models, peptides that mimic the TRPV1-recognition motif of AKAP have been shown to interfere with this interaction and thereby suppress hyperalgesia without impairing normal acute nociceptive function [43]. As an alternative strategy to avoid side effects, the complexity of TRP channel regulation described earlier also creates the potential opportunity to finely tune antagonists to interfere only with selected modalities of channel activation [8]. (2) TRP channel agonists. In certain disease states, it may be therapeutically desirable to stimulate, rather than inhibit, one or more TRP channels. Sometimes, the goal might be to exaggerate the normal physiological function of the channel for positive cellular benefit. For example, as described in Chapter 14, activators of TRPM8 might be useful to treat chronic pain because primary afferent inputs from cool-sensitive neurons expressing TRPM8 can apparently antagonize, at the spinal circuit level, inputs related to noxious heat [44]. In other cases, the goal is to achieve cytotoxicity through TRP channel-mediated calcium influx and thereby remove or neutralize a particular cell type. In the case of TRPV1, humans have been practicing this latter strategy for millennia through the regular consumption of spicy foods [45]. Whereas, acutely, exposure to capsaicin causes pain, prolonged exposure to this compound, or a more potent TRPV1 agonist, resiniferatoxin, desensitizes nociceptive neurons and eventually results in degeneration of nociceptor terminals (Chapter 6). Indeed, it was this observation that first led to the recognition that nociceptors constitute a neurochemically distinct subset of sensory neurons. As described in Chapter 7, this strategy has been explored not only for the treatment of pain, but also for the treatment of hyperactive bladder. Of note, this 10 1. INTRODUCTION TO TRP CHANNELS IN DISEASE

strategy is not necessarily aimed at specifically reducing the activity of the target TRP channel, but rather at more generally eliminating the function of the cell in which it is expressed. Another proposed use of cytotoxic hyperstimulation of TRP channels is to bring about apoptosis in tumor cells (Chapter 22). (3) TRP channel cargos. An especially clever TRP channel based therapeutic strategy exploits the unusual ability of some TRP channels, mentioned earlier, to mediate the influx of relatively large cations (mol. wt. > 600 Da). The best example of this strategy again involves TRPV1 and is based on the coadministration of capsaicin with a relatively large cationic molecule (QX314) that inhibits voltage-gated sodium channels through action at the intracellular end of the sodium channel pore [46]. Because capsaicin facilitates the entry of this cation through TRPV1, nociceptive neurons that express TRPV1 can be selectively loaded with relatively high concentrations of the sodium channel blocker, achieving therapeutically useful local concentrations without subjecting the recipient to potentially toxic systemic doses. (4) TRP channel gene therapy. An admittedly more ambitious approach that might prove useful to treat diseases resulting from TRP channel gain-of-function is genetic manipulation of TRP channel expression or sequence. For example, one could introduce an exogenous copy of a TRP channel cDNA, by viral transduction or other methods, to either rescue TRP channel hypofunction or to selectively drive ectopic expression of cytotoxic TRP channels in target cells to enhance agonist-stimulated elimination of those cells. RNA interference, antisense cDNAs, might be used to selectively reduce the expression of gain-of-function mutant TRP channels, whereas dominant negative TRP channels could be used to suppress the function of hyperfunctional or overly abundant endogenous TRP channels. Alternatively, gene editing using Cas9/CRISPR [47] or related tools might be used to repair mutated TRP channel genes or manipulate their promoters to modify channel expression. The diversity of TRP channels, their extraordinary physiological and pathophysiological importance, and the plethora of review articles and book chapters on these topics have inspired the invention of many variations of the TRP acronym. As will be evident throughout this book, TRP channels can fairly be viewed as both the problem and the potential solution in many human disease states. In keeping with the acronym tradition, it is therefore suggested that the reader view this incredible family of ion channels through the lens of their promise as Targets for the Resourceful Physician.

Acknowledgment M.J.C. is an inventor on a patent on the use of products related to TRPV1 and TRPV2, which is licensed through UCSF and Merck. This conflict is being managed by the Johns Hopkins Office on Policy Coordination.

References [1] Montell C. The history of TRP channels, a commentary and reflection. Pflugers Arch 2011;461(5):499–506. [2] Venkatachalam K, Montell C. TRP channels. Annu Rev Biochem 2007;76:387–417. [3] Owsianik G, Talavera K, Voets T, Nilius B. Permeation and selectivity of TRP channels. Annu Rev Physiol 2006;68:685–717. [4] Dong XP, Wang X, Xu H. TRP channels of intracellular membranes. J Neurochem 2010;113(2):313–28. References 11

[5] Cao E, Liao M, Cheng Y, Julius D. TRPV1 structures in distinct conformations reveal activation mechanisms. Nature 2013;504(7478):113–18. [6] Liao M, Cao E, Julius D, Cheng Y. Structure of the TRPV1 ion channel determined by electron cryo-microscopy. Nature 2013;504(7478):107–12. [7] Caterina MJ, Julius D. The vanilloid receptor: a molecular gateway to the pain pathway. Annu Rev Neurosci 2001;24:487–517. [8] Kort ME, Kym PR. TRPV1 antagonists: clinical setbacks and prospects for future development. Prog Med Chem 2012;51:57–70. [9] Bautista DM, Pellegrino M, Tsunozaki M. TRPA1: a gatekeeper for inflammation. Annu Rev Physiol 2013;75:181–200. [10] McCoy DD, Knowlton WM, McKemy DD. Scraping through the ice: uncovering the role of TRPM8 in cold transduction. Am J Physiol Regul Integr Comp Physiol 2011;300(6):R1278–87. [11] Garrison SR, Dietrich A, Stucky CL. TRPC1 contributes to light-touch sensation and mechanical responses in low-threshold cutaneous sensory neurons. J Neurophysiol 2012;107(3):913–22. [12] Vriens J, Owsianik G, Hofmann T, Philipp SE, Stab J, Chen X, et al. TRPM3 is a nociceptor channel involved in the detection of noxious heat. Neuron 2011;70(3):482–94. [13] Matta JA, Ahern GP. TRPV1 and synaptic transmission. Curr Pharm Biotechnol 2011;12(1):95–101. [14] Tsuzuki K, Xing H, Ling J, Gu JG. Menthol-induced Ca2+ release from presynaptic Ca2+ stores potentiates sensory synaptic transmission. J Neurosci 2004;24(3):762–71. [15] Liman ER. TRPM5 and taste transduction. Handb Exp Pharmacol 2007;179:287–98. [16] Xue T, Do MT, Riccio A, Jiang Z, Hsieh J, Wang HC, et al. Melanopsin signalling in mammalian iris and retina. Nature 2011;479(7371):67–73. [17] Inoue R, Morita H, Ito Y. Newly emerging Ca2+ entry channel molecules that regulate the vascular tone. Expert Opin Ther Targets 2004;8(4):321–34. [18] Holzer P. TRP channels in the digestive system. Curr Pharm Biotechnol 2011;12(1):24–34. [19] Birder L, Andersson KE. Urothelial signaling. Physiol Rev 2013;93(2):653–80. [20] Everaerts W, Gevaert T, Nilius B, De Ridder D. On the origin of bladder sensing: Tr(i)ps in urology. Neurourol Urodyn 2008;27(4):264–73. [21] Mene P, Punzo G, Pirozzi N. TRP channels as therapeutic targets in kidney disease and hypertension. Curr Top Med Chem 2013;13(3):386–97. [22] Vennekens R, Menigoz A, Nilius B. TRPs in the brain. Rev Physiol Biochem Pharmacol 2012;163:27–64. [23] Massullo P, Sumoza-Toledo A, Bhagat H, Partida-Sanchez S. TRPM channels, calcium and redox sensors during innate immune responses. Semin Cell Dev Biol 2006;17(6):654–66. [24] Santoni G, Farfariello V, Liberati S, Morelli MB, Nabissi M, Santoni M, et al. The role of transient receptor potential vanilloid type-2 ion channels in innate and adaptive immune responses. Front Immunol 2013;4:34. [25] Moore C, Cevikbas F, Pasolli HA, Chen Y, Kong W, Kempkes C, et al. UVB radiation generates sunburn pain and affects skin by activating epidermal TRPV4 ion channels and triggering endothelin-1 signaling. Proc Natl Acad Sci USA 2013;110(34):E3225–34. [26] Wilson SR, The L, Batia LM, Beattie K, Katibah GE, McClain SP, et al. The epithelial cell-derived atopic dermatitis cytokine TSLP activates neurons to induce itch. Cell 2013;155(2):285–95. [27] Geppetti P, Nassini R, Materazzi S, Benemei S. The concept of neurogenic inflammation. BJU Int 2008;101(Suppl. 3):2–6. [28] Jin J, Desai BN, Navarro B, Donovan A, Andrews NC, Clapham DE. Deletion of Trpm7 disrupts embryonic development and thymopoiesis without altering Mg2+ homeostasis. Science 2008;322(5902):756–60. [29] Cheng X, Jin J, Hu L, Shen D, Dong XP, Samie MA, et al. TRP channel regulates EGFR signaling in hair morphogenesis and skin barrier formation. Cell 2010;141(2):331–43. [30] Che H, Yue J, Tse HF, Li GR. Functional TRPV and TRPM channels in human preadipocytes. Pflugers Arch 2014;466:947–59. [31] Lin Z, Chen Q, Lee M, Cao X, Zhang J, Ma D, et al. Exome sequencing reveals mutations in TRPV3 as a cause of Olmsted syndrome. Am J Hum Genet 2012;90(3):558–64. [32] Nilius B, Owsianik G. Transient receptor potential channelopathies. Pflugers Arch 2010;460(2):437–50. [33] Szolcsanyi J, Pinter E. Transient receptor potential vanilloid 1 as a therapeutic target in analgesia. Expert Opin Ther Targets 2013;17(6):641–57. 12 1. INTRODUCTION TO TRP CHANNELS IN DISEASE

[34] Gopinath P, Wan E, Holdcroft A, Facer P, Davis JB, Smith GD, et al. Increased capsaicin receptor TRPV1 in skin nerve fibres and related vanilloid receptors TRPV3 and TRPV4 in keratinocytes in human breast pain. BMC Womens Health 2005;5(1):2. [35] Bell JT, Loomis AK, Butcher LM, Gao F, Zhang B, Hyde CL, et al. Differential methylation of the TRPA1 promoter in pain sensitivity. Nat Commun 2014;5:1–11. [36] Moran MM, McAlexander MA, Biro T, Szallasi A. Transient receptor potential channels as therapeutic targets. Nat Rev Drug Discov 2011;10(8):601–20. [37] Liberati S, Morelli MB, Nabissi M, Santoni M, Santoni G. Oncogenic and anti-oncogenic effects of transient receptor potential channels. Curr Top Med Chem 2013;13(3):344–66. [38] Myrdal SE, Steyger PS. TRPV1 regulators mediate gentamicin penetration of cultured kidney cells. Hear Res 2005;204(1–2):170–82. [39] Worley PF, Zeng W, Huang GN, Yuan JP, Kim JY, Lee MG, et al. TRPC channels as STIM1-regulated store- operated channels. Cell Calcium 2007;42(2):205–11. [40] Yuan JP, Kiselyov K, Shin DM, Chen J, Shcheynikov N, Kang SH, et al. Homer binds TRPC family channels and is required for gating of TRPC1 by IP3 receptors. Cell 2003;114(6):777–89. [41] Winter Z, Buhala A, Otvos F, Josvay K, Vizler C, Dombi G, et al. Functionally important amino acid residues in the transient receptor potential vanilloid 1 (TRPV1) ion channel—an overview of the current mutational data. Mol Pain 2013;9:30. [42] Latorre R, Zaelzer C, Brauchi S. Structure-functional intimacies of transient receptor potential channels. Q Rev Biophys 2009;42(3):201–46. [43] Fischer MJ, Btesh J, McNaughton PA. Disrupting sensitization of transient receptor potential vanilloid subtype 1 inhibits inflammatory hyperalgesia. J Neurosci 2013;33(17):7407–14. [44] Proudfoot CJ, Garry EM, Cottrell DF, Rosie R, Anderson H, Robertson DC, et al. Analgesia mediated by the TRPM8 cold receptor in chronic neuropathic pain. Curr Biol 2006;16(16):1591–605. [45] Kissin I, Szallasi A. Therapeutic targeting of TRPV1 by resiniferatoxin, from preclinical studies to clinical trials. Curr Top Med Chem 2011;11(17):2159–70. [46] Roberson DP, Binshtok AM, Blasl F, Bean BP, Woolf CJ. Targeting of sodium channel blockers into nociceptors to produce long-duration analgesia: a systematic study and review. Br J Pharmacol 2011;164(1):48–58. [47] Terns RM, Terns MP. CRISPR-based technologies: prokaryotic defense weapons repurposed. Trends Genet 2014;30:111–18. CHAPTER 2 Transient Receptor Potential Dysfunctions in Hereditary Diseases: TRP Channelopathies and Beyond Balázs István Tóth,1,2,* Bernd Nilius1,* 1Katholieke Universiteit of Leuven, Department of Cellular and Molecular Medicine, Laboratory of Ion Channel Research and TRP Research Platform Leuven (TRPLe), Campus Gasthuisberg, Leuven, Belgium 2DE-MTA “Lendület” Cellular Physiology Research Group, Department of Physiology, University of Debrecen, Medical and Health Science Center, Research Center for Molecular Medicine, Debrecen, Hungary *Corresponding authors: [email protected]; [email protected]

OUTLINE

Introduction 13 TRPML Channelopathies 23 TRPC Channelopathies 14 TRPP Channelopathies 24 TRPV Channelopathies 17 Conclusions 25 TRPM Channelopathies 20 Acknowledgements 26 TRPA Channelopathies 23 References 26

INTRODUCTION

In the broadest sense, channelopathies can be defined as diseases associated with malfunction of ion channels or their regulatory proteins. Although this definition covers both congenital and acquired forms, generally only hereditary diseases are referred to as channelopathies

TRP Channels as Therapeutic Targets 13 © 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/B978-0-12-420024-1.00002-3 14 2. TRP DYSFUNCTIONS IN HEREDITARY DISEASES in which disease mutations in genes encoding ion channel subunits or regulatory proteins play an etiological role [1]. In hereditary transient receptor potential (TRP) channelopathies, a TRP channel is affected by the mutation. Recently, several hereditary TRP channelopathies have been described, and they have been discussed in many comprehensive reviews [2–5]. The increasing number of TRP channel-related diseases highlights these channels as novel pharmaceutical targets and also provide insight into its physiological function [6]. In this review, we describe hereditary channelopathies and also mention examples with available genetic evidence to explain several putative pathological conditions in which TRP dysfunction is suggested, although the primary mutations affect other genes. We refer also for in-depth information the OMIM link channelopathies.

TRPC CHANNELOPATHIES

TRP channels are linked to diseases since their first description. The discovery of the founding member of the TRP superfamily, the Drosophila TRP channel, was already due to a drosophila channelopathy disrupting the phototransduction and resulting in blindness of the fruit fly [7]. The closest mammalian relatives of the Drosophila TRP, members of the canonical (TRPC) subfamily, have been linked to many acquired diseases affecting, among else, cardiovascular and respiratory systems, skin, inflammatory processes, and probably neurodegenerative diseases [8], but there are only few examples for “real” hereditary TRPC channelopathies. In this review, we will not refer to hereditary diseases linked to store-operated (STIM/ORAI/TRPCs “?”) Ca2+ channels, although evidence has been reported for their involvement in several disease (e.g., severe combined immune deficiency [9], primary Sjogren’s syndrome [10], and tubular-aggregate myopathy [11]). TRPC1 can play a role in several skin diseases [12], including a few hereditary ones. Recently, it was discussed to be involved in the Gorlin (or Gorlin-Goltz) syndrome, a rare basal cell nevus syndrome with autosomal dominant hereditary (OMIM 109400). The syndrome has 100% penetrance and variable expressivity characterized by odontogenic keratocysts of the mandible, postnatal tumors, and multiple basal cell carcinomas (BCCs). Although it is mostly linked to mutations in the tumor suppressor gene PTCH1, a member of the patched gene family and receptor for sonic hedgehog, in some cases the TRPC1 gene was suggested to be involved in the development of many postnatal tumors [13]. Indeed, the lack of TRPC1 (and TRPC4) was also correlated with failure of differentiation in BCC cells [14]. The autosomal-dominant inherited skin malady, Darier(-White) disease (DD) or keratosis follicularis, characterized by hyperkeratotic papules, might also be connected to TRPC1 malfunction, although the primary causes are mutations in the atp2a2 gene encoding the SERCA2b endoplasmic reticulum Ca2+ pump [15]. In DD patients’ keratinocytes, increased protein expression and TRPC1-mediated Ca2+ influx were detected, which can contribute to the augmented proliferation and survival of DD keratinocytes [16]. Beyond the skin, TRPC1 can be associated with other hereditary diseases. For example, a novel spliced isoform of TRPC1 with exon 9 deletion (TRPC1E9del) was reported in a human ovarian adenocarcinoma cell line, and its role (together with other TRPC isoforms) in the proliferation and differentiation is also discussed [17]. In a genome-wide association study, SNPs in TRPC1 were discovered that were associated with type 2 diabetes [18]. The role of TRPC1 and ORAI1 might also be implicated in several angiogenesis syndromes leading to tumor neovascularization, which are frequently due to mutations in the Von Hippel Lindau tumor suppressor gene [19]. TRPC Channelopathies 15

TRPC3 is mostly linked to the central nervous system by hereditary diseases. In mice, a gain- of-function mutation in TRPC3 (T635A) caused degeneration of cerebellar Purkinje cells and a loss of type II unipolar brush cells, resulting in a cerebellar ataxia, the moonwalker mouse phenotype [20,21]. A single base pair polymorphism (rs13121031) located within the CpG island in the alternative promoter of the human TRPC3 gene was also connected to cerebellar ataxia and heart hypertrophy [22]. Although the link between TRPC3 and cerebellar ataxia is fairly strong in the aforementioned mouse models, there has not been any evidence presented in humans. However, a genetic screen for TRPC3 mutations in patients with late-onset cerebellar ataxia does not support a contribution of TRPC3 mutants to this disease [23]. TRPC3 might be indirectly targeted in various inherited diseases affecting the nervous system. One of them is the autosomal-dominant Spinocerebellar ataxia type 14 (SCA14) primary caused by mutations in PKCγ. Wild-type PKCγ negatively regulated TRPC3 channels, whose regulation was impaired in cerebellar Purkinje cells transfected with the S119P mutant isoform resulting in increased postsynaptic current amplitudes. This alteration could contribute to disruptive synapse pruning disturbing synaptic transmission and plasticity found in SCA14 patients [24]. TRPC3 might also be involved in another neurodevelopmental disorder, the Williams- Beuren syndrome, which is associated with hypercalcemia and heart or blood vessel problems. The main genetic defect generally lays in the transcription factor IIi gene that encodes TFII-I, which normally suppresses cell-surface accumulation of TRPC3, i.e., mutations in TFII-I can cause a TRPC3 gain-of-function due to increased protein expression in the plasma membrane [25]. The pervasive developmental disorder Rett syndrome (RTT), affecting mostly female patients and causing mental retardation, is a progressive neurodevelopmental disorder that can also be linked to TRPC3. RTT is caused by mutations in the gene MECP2 (methyl CpG binding protein 2) encoding a transcriptional regulator protein with mostly repressive functions [26]. TRPC3 has been identified recently as target of MeCP2 transcriptional regulation, and it was suggested to be involved in the impaired brain-derived neurotrophic factor signaling in RTT [27]. An SNP in TRPC3 (rs6820068) was also found to be associated with the risk to develop immunoglobulin A-induced nephropathy (IgA nephropathy, IgAN) in women; the prevalence of the SNP was 23% vs. 12% in female patients and healthy controls, respectively [28]. Some pharmacological evidence proposed that excessive Ca2+ influx via TRPC3 contributed to Ca2+ toxicity in pancreas and salivary gland, whose symptoms are characteristic for acute pancreatitis and Sjögren syndrome, a systemic autoimmune disease, in which immune cells destroy exocrine cells in tear glands, pancreas, and salivary glands [29]. TRPC4 has not been directly connected to any channelopathy yet. However, a genetic association study has shown some link between TRPC4 SNPs and generalized photosensitive epilepsies and related symptoms [30]. Furthermore, a missense SNP caused gain-of-function mutation in TRPC4 (I957V) that was suggested to be protective against myocardial infarction [31]. TRPC6, with other TRPC channels, was linked to infantile hypertrophic pyloric stenosis (IHPS) (OMIM 179010), the most common gastrointestinal obstruction disease in infancy with genetic background affecting the smooth muscle of the pylorus. A linkage analysis in IHPS identified SNPs in two genetic loci involving TRPC5 and TRPC6 [32] and later also SNPs affecting TRPC1. An SNP in the promoter region and a missense variant in exon 4 of TRPC6 are hypothesized as putative causal gene variants [33]. However, another study carried out on Chinese patients and healthy controls has not found association between IHPS and other SNPs in TRPC6 [34]. 16 2. TRP DYSFUNCTIONS IN HEREDITARY DISEASES

TRPC6 plays an important role in glomerular diseases in the kidney. Among them, several cases of focal and segmental glomerulosclerosis (FSGS type 2) (OMIM 603965) are considered as a real TRPC6 channelopathies; currently, at least 15 mutations in the N- and C-terminus of the TRPC6 gene have been described and linked to FSGS type 2 (for review, see Ref. [35], new mutations in Refs. [36,37]). FSGS is functionally characterized by proteinuria and progressive decline of renal function caused by malfunction or loss of podocytes. Podocytes are highly specialized epithelial cells lining the Bowman’s capsule and playing a key role in the function of the glomerular filtration barrier. Although it is not fully understood, yet, how mutations in TRPC6 lead to dysfunction or death of podocytes impairing glomerular permeability and filtration and finally resulting in FSGS, the investigation of the mutants’ phenotypes highlighted two most probably interdependent mechanisms: altered channel functions and impaired interactions with other proteins. The distorted protein-protein interaction can consequently alter regulation and/or trafficking of the channel, significantly influencing channel properties or expression. In podocytes, TRPC6 associates with the transmembrane protein nephrin, which is coupled to the nephrin-interacting adapter protein, CD2AP, and to podocin. This complex forms the slit diaphragm, the crucial component of the glomerular filter. Nephrin is known to negatively regulate the expression of TRPC6 in the plasma membrane ([38], for a review, see Ref. [39]). By the mechanism, nephrin was shown to inhibit TRPC6-PLC-γ1 interaction, which seems to be crucial in the membrane trafficking of the channel. Some of the described mutations (e.g., P112Q, N143S, S270T, R885C, E897K) may affect the nephrin binding site of the TRPC6, making it less sensitive for the nephrin-dependent negative regulation, which results in higher surface expression and enhanced TRPC6-mediated Ca2+ entry [40]. Although it is a fact that most of the TRPC6 mutations described in FSGS are associated with a gain-of-function phenotype and TRPC6-mediated calcium entry was found to mediate both angiotensine-II and albumin overload-induced loss of podocytes [41,42], downstream mechanisms, i.e., how overactivation of TRPC6 destroys the slit, are still under discussion. A very likely mechanism is the activation of nuclear factor of activated T-cells (NFAT) found in TRPC6 mutants. This effect was blocked by inhibitors of calcineurin, calmodulin-dependent kinase II, and phosphatidylinositol 3-kinase, but was found to be independent of Src, Yes, or Fyn ([43,44]; see for a review, Ref. [45]). Moreover, angiotensin II-induced Ca2+ entry via TRPC6 further increased the expression of the channel via calcineurin-NFAT signaling forming a positive feedback loop [41]. Recently, the Wnt/β-catenin and the MAP kinase ERK1/ 2-associated signaling pathways have also been suggested to be involved in the pathogenesis of TRPC6-mediated diabetic podocyte injury [46,47]. Interestingly, vitamin D downregulated the enhanced TRPC6 expression in podocytes through a direct effect on TRPC6 promoter activity, which might contribute to the antiproteinuric effect of vitamin D [48]. It has to be mentioned that the effect of TRPC6 overactivation can be context dependent: for example, acute activation of TRPC6, at least in mice, rescues podocytes from complement-mediated damage; however, chronic overactivation seems to play an etiological role in FSGS [49]. TRPC6 is also involved in the steroid-resistant nephrotic syndrome (SRNS) (OMIM 600995). Three mutations and an intronic nucleotide substitution were described in the sporadic form of this disease [50]. An additional SNP in the promoter region of TRPC6 was also described, which resulted in enhanced transcription in vitro and correlated with an increased protein expression in the kidney of SRNS patients [51]. Mutations in TRPC6 may also contribute to the idiopathic pulmonary arterial hypertension, where an SNP in the promoter region was found more TRPV Channelopathies 17

frequently in a cohort of patients. This mutation facilitated the binding of the inflammatory and carcinogenic transcription factor nuclear factor-κB and resulted in abnormally enhanced TRPC6 transcription [52]. TRPC6 is also mentioned as a candidate gene for Head and Neck Squamous Cell Carcinoma [53], and its overexpression in leukocytes was also demonstrated in primary open-angle glaucoma [54].

TRPV CHANNELOPATHIES

Transient receptor potential vanilloid 1 (TRPV1) the best characterized TRP channel, is not yet clearly linked to any hereditary disease. Although its central integrator role in nociception is widely accepted, only a few polymorphisms in TRPV1 are suggested associating with development and maintenance of chronic pain syndromes. Genetic variants of human TRPV1 with M315I mutation were found more frequently in Caucasian females suffering from neuropathic pain [55], and an intronic variant SNP (rs222741) in TRPV1 was found to be associated with migraine in a Spanish population [56]. Interestingly, the aforementioned M315I variation also showed higher frequency in type 1 diabetes-affected patients than in healthy controls in an Ashkenazi Jewish population [57]. In a patient with Miller-Dieker lissencephaly syndrome, an autosomal-dominant congenital disorder characterized by a developmental defect of the brain as a consequence of incomplete neuronal migration, a chromosome 17p13.3 deletion syndrome was identified, which includes, among else, deletion of TRPV1 [58]. A missense (I585V) variant of TRPV1 gene, showing decreased channel activity, was reported to be a potential genetic risk factor of painful knee osteoarthritis [59], and the same substitution also associated with lower risk of the symptoms of active asthma [60]. Another genetic TRPV1 variant (G315C) was linked to a functional dyspepsia in a Japanese population via influencing the upper gastrointestinal sensation [61]. Altered channel function and/or expression of TRPV2 has been widely connected to Duchenne muscular myopathy, diabetes, childhood asthma, and several forms of cancer [3,62–64]. Currently, elevated expression of TRPV2 has been described in induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) of patients affected by Hutchinson-Gillford progeria syndrome (HGPS; OMIM 176670), a rare genetic disorder in which the premature aging in multiple organs leads to early death. Elevated TRPV2 expression might be involved in the 2+ pathomechanism: it caused a sustained [Ca ]i elevation in HGPS iPSC-ECs induced by hypotonicity, which induced apoptosis. However, despite its potential role in several diseases, no “real” hereditary TRPV2 channelopathy has been detected so far. TRPV3 is one of the most abundantly expressed TRP channels in epidermal and follicular keratinocytes of both human and rodent skin (for recent reviews, see Refs. [12,65,66]). Olmsted syndrome (OS) (also known as mutilating palmoplantar keratoderma with periorificial keratotic plaques or Polykeratosis of Touraine), (OMIM 614594) is the first described hereditary cutaneous TRP channelopathy and also the first real TRPV3 channelopathy. It is a rare in- heritable skin disease characterized by the combination of periorificial, keratotic plaques, bilateral palmoplantar keratodermas, alopecia and associated with dermatosis and severe itching. In humans, three gain-of-function mutations (G573S, G573C, W692G) were identified causing OS (for recent reviews, see Refs. [65–68]). Recently, a new TRPV3 mutant, G573A, has been described in an OS patient, which also causes multiple immune dysfunctions, such 18 2. TRP DYSFUNCTIONS IN HEREDITARY DISEASES as hyper-IgE, elevated follicular T-cells, and persistent eosinophilia [69]. These findings turn the attention toward the immunological alterations of Olmsted syndrome and the potential role of TRPV3 mutants in the immunological dysregulation. The etiological role of TRPV3 mutants is also supported by rodent models DS-Nh mice and WBN/Kob-Ht rats, where two gain-of-function mutations, partly identical with the above ones found in OS, caused autosomal-dominant hairless phenotype associated with dermatitis [70]. Moreover, the hair growth regulatory role of TRPV3 was also evidenced in human [71]. Further supporting the role of TRPV3 as an important channel in skin pathophysiology, an upregulation of TRPV3 was reported in Rosacea, a frequent chronic inflammatory skin disease [72]. Recent studies have suggested that the pathophysiological role of TRPV3 can go beyond skin disorders. Genetic association studies have highlighted a potential role of the channel in primary headache disorders (like migraine, tension-type headache, and cluster headache) with a genetic preposition [73]. TRPV3 SNPs has also been identified in congenital hyperinsulinism of infancy [74]. The TRPV4 coding sequence is a real hot spot of mutations causing channelopathies. Currently, more than 50 mutations in the trpv4 gene have been discovered, causing at least nine different channelopathies. By their symptoms, TRPV4 channelopathies cover skeletal dysplasias and peripheral neuropathies, although mixed forms are also well known, and a clear distinction between these two groups of TRPV4 channelopathies is not always possible. The first recognized TRPV4-related channelopathy, the brachyolmia type 3 (OMIM 113500), affects the skeletal system. It is a relatively mild, autosomal-dominant skeletal dysplasia characterized by short stature, flattened vertebrae (platysspondyly) especially in the cervical region, reduced intervertebral spaces, and scoliosis or kyphosis [75]. This surprising finding triggered intensive research focusing on the newly recognized relationship between TRPV4 and skeletal disorders and resulted in the discovery of new TRPV4 channelopathies among skeletal dysplasias. These diseases share the main symptoms like short stature, platysspondyly, defects in bone ossification, and abnormalities in joints, but their severity shows a high variation not only among the different diseases but also among the different mutations underlying the same symptoms. Despite the variability in the symptoms’ severity, all diseases are probably due to dysfunction and differentiation abnormalities in chondrocytes of the bone growth plate. In the spondyloepimetaphyseal dysplasia Maroteaux pseudo-Morquio type 2 (SEDM-PM2) (OMIM 184095), the manifestations of the preceding symptoms are limited to the musculoskeletal system [76]. In the spondylometaphyseal dysplasia Kozlowski type (OMIM 184252), mainly the vertebrae and the metaphyses are affected. Although, like in the previous cases, the body length is normal at birth, it shows short stature by shortening of the trunk during the development, which reaches the clinical significance mostly between ages 1 and 4 years. Generally, the symptoms are more severe than in brachyolmia type 3 and SEDM-PM2: a prominent feature is platyspondyly again, but severe scoliosis and defects in the distal me- taphysis of the femur, the femoral neck, and trochanteric area are also observed. [77]. The most severe skeletal TRPV4 channelopathy is the metatropic dysplasia (OMIM 156530), which is sometimes combined with lethal fetal akinesia. The nonlethal forms are characterized by shortening of all long bones resulting in short limbs, serious enlargement of joints, heavy kyphoscoliosis, severe platyspondyly, and metaphyseal enlargement, as well as defects in ossification [77–79]. Parastremmic dysplasia (PD) (OMIM 168400) is characterized by severe dwarfism, thoracic kyphosis, and distortion and twisting of the limbs [parastremmic (Greek): TRPV Channelopathies 19 twisted], contractures of the large joints, malformations of the vertebrae and pelvis, and it can also associate with incontinence [76]. A recently described mild form of skeletal dysplasia is the familial digital arthropathy-brachydactyly (OMIM 606835) which appears in the first decade of life. Short fingers, deviations in finger joints, and irregularities in the articular surfaces characterize this arthropathy [80]. Following the description of an increasing number of TRPV4-caused skeletal dysplasias, the discovery of the causal role of TRPV4 in inherited neuropathies was a big surprise. As of today, three autosomal-dominant distal neuropathies are considered as hereditary TRPV4 channelopathies. Their main symptom is muscle atrophy caused by degeneration of the motoneurons in the spinal ventral horn, leading to muscle weakness and wasting in the distal limbs, but the respiratory system and the vocal cord can be also affected, and sometimes the motor symptoms are associated with sensory defects (for a review, see Ref. [81]). These diseases are congenital distal spinal muscle atrophy (CDSMA) (OMIM 600175), scapuloperoneal spinal muscle atrophy (SPSMA) (OMIM 181405), and hereditary motor sensory neuropathy type IIc (HMSN IIc or Charcot-Marie-Tooth neuropathy type 2C, CMT2C) (OMIM 606071). CDSMA is a nonprogressive lower motor neuron disorder restricted to the lower part of the body. It may associate with arthrogryposis (now also discovered in patients with mutations in the gene encoding the mechanosensory cation channel PIEZO2 [82]), bilateral talipes equino- varus, and flexion contractures of the knees and hips. Sometimes slight skeletal symptoms (e.g., lordosis, scoliosis, restricted joint movements) are also observed, but sensory defects are lacking [83]. SPSMA is a syndrome characterized by scapuloperoneal atrophy, scapular winging, muscle wasting in the lower limbs, absence of tendon reflexes, as well as laryngeal palsy and vocal-cord paralysis. Sometimes scoliosis and light sensory defects are reported [84–86]. In CMTC2C, a variable degree of muscle weakness of limbs, vocal cords, intercostal muscles, and sensoneurial hearing loss are the leading symptoms, but bladder urgency or incontinency are also common. It is often associated with slight skeletal or arthrial symptoms like club foot (talipes), congenital joint contractures (arthrogryposis), or scoliosis, but facial asymmetry, tongue fasciculations, and third and sixth cranial nerve palsies have also been reported. CMTC2 starts in infancy or childhood, and the life expectancy is shortened because of respiratory failure [85–88]. The exact pathomechanism by which mutations in TRPV4 are leading to the aforementioned diseases is vaguely understood, as are the reasons for the phenotype variability of TRPV4 channelopathies (i.e., why diverse mutations result in these different diseases) [89]. Although there are some exceptions and controversies, most of the disease-causing mutations show a gain-of-function phenotype, and there are speculations that the degree of channel overactivity might determine the severity of the disease ([90,91]; for reviews, see Refs. [89,92,93]). The increased, or at least altered, Ca2+ signaling via TRPV4 can result in altered neurogenesis, altered gene expression, or even cell death. On the other hand, mutations can affect the association of TRPV4 subunits with each other or other molecules influencing channel formation, interaction with cytoskeletal elements, cellular trafficking, or spatial distribution; the latter can have a significant effect on the differentiation of polarized cells like osteocytes or neurons. Indeed, if we have a look at the distribution of the mutations along the amino acid sequence of the channel, three hot spots can be identified for disease-causing mutations: (1) the ankyrin-repeat-domain (ARD) on the N-terminus, (2) the transmembrane region S3-S5, and (3) a C-terminal region were the channel associates with several members of the cytoskeleton, such as tubulin, actin, and MAP7 [94]. Regarding the 20 2. TRP DYSFUNCTIONS IN HEREDITARY DISEASES

ARD, the neuropathy-causing mutations are mainly localized in the convex surface of the ARD, but mutations causing skeletal dysplasia, although scattering through the whole length of the channel protein, seem to be more frequently located in the concave surface of the ARD. Because of our limited knowledge, the puzzle created by the large number of mutations often located in the same domain of the channel and the consequent (at least) nine different diseases is still challenging [89]. To make the picture of TRPV4 channelopathies even more complex, we have to mention that TRPV4 is highly expressed in the inner ear and the urothelium; therefore, it is not surprising that some patients also have hearing problems or bladder symptoms such as overactive bladder and incontinence (for a review, see Ref. [95]). Beyond the aforementioned real channelopathies, a recently recognized human TRPV4 variant (P19S) was linked to acquired chronic obstructive pulmonary disease (COPD). The presence of this mutant probably predisposes the carriers to COPD as a consequence of air pollution (e.g., diesel exhaust particles) because of a reduced airway clearance due to decreased cilia activity, which is supposed to be a TRPV4-dependent mechanism [96]. The same mutation/polymorphism can also cause hyponatremia [97]. TRPV5 and TRPV6, the two close relatives showing the highest Ca2+ selectivity in the TRP superfamily, function as Ca2+ (re)absorption channels in the kidney. Although none of the known human channelopathies has been affecting any of them yet, nonsynonymous SNPs in TRPV5 gene show high frequency among African Americans. Among the investigated mutations, A563T variant (and, with lower efficacy, also L712F) was found to increase Ca2+ permeability of TRPV5 resulting in increased Ca2+ reabsorption. This mechanism can contribute to increased Ca2+ retention found in the African-American population [98]. Both TRPV5 and TRPV6 can be involved in the pathomechanism of Pendred syndrome, a form of − − congenital deafness. The primary cause of the disease is a malfunction of the Cl / HCO3 exchanger, SLC26A4 (pendrin), which results in acidification of the endolymph of the inner ear. Because both TRPV5 and TRPV6 are sensitive to acidification, their inhibition by low pH leads to disturbances in the Ca2+ concentration of the endolymph [99]. Another carrier disease is Geitelman´s syndrome, which is characterized by salt-losing hypotension, hypomagnesemia, and hypokalemic metabolic alkalosis due to mutations in the thiazide-sensitive Na+/ Cl− cotransporter gene SLC12A3 and can also indirectly involve the malfunction of TRPV5 and TRPV6 [100]. Although no human equivalent exists so far, in HCALC1 mice model, an autosomal-dominant hypercalciuria can be considered as a real hereditary TRPV5 channelopathy caused by S682P mutation [101]. Their importance in the overall Ca2+ homeostasis is also supported by the fact that upregulation of both channels causes hypocalciuria [100]. However, rather controversially, an ancestral TRPV6 haplotype consisting three missense mutations by nonsynonymous polymorphisms showed a gain-of-function phenotype and seemed to increase the risk for calcium stone formation in certain forms of absorptive hypercalciuria [102,103].

TRPM CHANNELOPATHIES

Transient receptor potential melastatin 1 (TRPM1) was previously considered a tumor suppressor protein in melanoma cells, where the name of the whole melastatin subfamily stems from. Although the loss of TRPM1 channel protein is an excellent marker of melanoma aggressiveness, recently miRNA211 coded in an intron of TRPM1 was found to be responsible TRPM Channelopathies 21 for the tumor suppression [104]. Although its etiological role in melanomas was questioned, TRPM1 is a pathogenic factor to cause the autosomal recessive congenital stationary night blindness type 1C (CSNB1C) (OMIM 613216), a clinically and genetically heterogeneous group of retinal disorders. CSNB1C is characterized by nonprogressive impaired night vision and decreased visual acuity. On ON bipolar cells, TRPM1 channels are gated by the mGluR6 (GRM6) signaling cascade, and their opening is necessary for the depolarization evoked by light stimulation. The mutant channels show decreased light response, which causes the dysfunction of both rod and cone ON bipolar cells of the mammalian retina [105–110]. The same disease was discovered in the Appaloosa horse where CSNB was associated with coat spotting pattern. Although human patients also display myopia, reduced central vision, and nystagmus, unlike the Appaloosa horses and the anticipated TRPM1 function in melanocytes, none of the patients show abnormal skin pigmentation [111]. A DNA microdeletion involving, among else, the TRPM1 gene was found to cause a syndrome with severe central nervous system dysfunction, including mental retardation, extrapyramidal symptoms, refractory epilepsy, and encephalopathy. This deletion syndrome is also associated with congenital retinal dysfunction, suggested to be caused by the loss of TRPM1 [112]. TRPM2 and TRPM7, two chanzymes (i.e., ion channels that also possess enzymatic functions), have been suspected for a long time to cause the Guamanian amyotrophic lateral sclerosis (ALS-G) and Parkinsonism dementia-Guam (PD-G, or Parkinsonism dementia complex, PDC) (OMIM 105500), two related neurodegenerative disorders that are endemic on the Western Pacific (including Guam) [113]. Although ALS-G and PD-G have a multifactorial etiology involving special mineral composition of the soil and drinking water, as well as the presence of putative neurotoxin, l-beta-N-methylamino-l-alanine, derived from the traditionally consumed cycad plant, mutations in the TRPM2 and TRPM7 genes were identified in a subset of ALS-G and PD-G patients. These mutations resulted in a decreased channel activity in physiological circumstances, which, in the presence of the environmental triggers, may contribute to the pathomechanism of these diseases via decreased intracellular Mg2+ concentration ([114–116]; for a review, see Refs. [117,118]). However, a recent linkage analysis did not reveal any evidence in support of the linkage to the TRPM7 locus, indicating that at least TRPM7 is not associated with ALS-G/PDC [119]. TRPM2 has been implicated in various forms of bipolar disorder or psychosis maniaco- depressiva. One of the putative susceptibility locus of the bipolar disorder type I (BD-I), the “classical” form of the disease characterized by manic or mixed episodes usually alternating with major depressive episodes, is located in the TRPM2 encoding chromosomal region [120–122]. Furthermore, SNPs in the promoter region of TRPM2 are also linked to BD-II, in which form the maniac episodes are less dominant [123]. Most recently, a novel TRPM2 mutation (R755C) has been discovered in Crohn’s disease [124]. TRPM3 might possess the most transcript variants among TRP channels potentially resulting in functionally different channels [125], but the relations to genetic diseases have not been characterized, yet. TRPM3 has been recently discussed as a part of the genetic background for the comor- bidity between autism and muscular dystrophy Duchenne. Indeed, in some patients simultaneously suffering from both diseases, a deletion involving exons 1-9 of TRPM3 has been described [126]. TRPM3 is coded in a genetic locus that might be involved in the pathogenesis of the Kabuki syndrome (OMIM 147920), a multiple congenital mental retardation syndrome characterized by distinct facial appearance, heart defects, urinary tract anomalies, hearing loss, hypotonia, short stature, joint 22 2. TRP DYSFUNCTIONS IN HEREDITARY DISEASES laxity, and unusual dermatoglyphic patterns [127]. TRPM3 SNPs located in the splicing sites have been discovered in patients with metabolic syndrome and diabetes type 2 [128]. TRPM3 is also often mentioned as a gene involved in developmental failures of the vertebrate lens, which process is probably regulated by the miRNA204 coded in the intron 8 of the TRPM3 gene [129,130]. TRPM4 mutations are responsible for the development of the autosomal dominant progressive familial heart block type I (OMIN 113900), a progressive cardiac bundle branch disease in the His-Purkinje system. The disease-causing mutation E7K leads to a gain-of-function phenotype, probably due to an increased surface expression. This increased activity can lead to depolarization-induced defects in the conductive system and generate electrical gridlock [131,132]. Mutations were also identified in patients with atrioventricular block, but no mutations were found in other patients with sinus node dysfunction, Brugada syndrome, or long-QT syndrome [133]. It is still debated whether TRPM4 plays a genetic role in alteration of the arterial myogenic response (Bayliss effect) associated with stroke, cerebral autosomal-dominant ar- teriopathy with subcortical infarcts and leukoencephalopathy [134]. Recently, four TRPM4 mutants were described in patients with Brugada syndrome, which is characterized by a bifascicular block or a complete right bundle branch block. Two of the four mutants (P779R and K914X) resulted in a decreased expression, whereas the other two (T873I and L1075P) increased the expression of TRPM4 channels [135]. In the last year, a possible role of TRPM4 in multiple sclerosis has also been described [136]. TRPM5 has not been linked to any channelopathy yet. However, an SNP in the TRPM5 gene was associated with decreased risk of childhood leukemia [137]. TRPM6, like TRPV4, is also a hot spot of hereditary mutations leading to channelopathies. The channel has a key role in Mg2+ (re)absorption both in the intestines and kidney; therefore, its mutations result mostly in diseases associated with disturbances in Mg2+ homeostasis. More than 35 mutations in the TRPM6 gene have been described, causing the autosomal recessive disease hypomagnesemia with secondary hypocalcemia 1 (HSH1 or, HOMG1) (OMIM 602014) [138,139]. The leading symptoms of HSH1 are the very low serum levels of Mg2+ and Ca2+. It is diagnosed generally during the first 6 months of life, based on the characteristic secondary neurological symptoms. The primary defect is a decreased renal/intestinal Mg2+ reabsorption and the consecutively lowered parathyroid hormone (PTH) secretion by the parathyroid gland. The decrease in PTH and consequently also in serum Ca2+ levels (secondary hypocalcemia) result in generalized seizures, tetany, and muscle spasms, which are resistant to conventional anticonvulsive therapies. Without treatment, the disease leads to severe mental retardation or death. Successful causal therapies are based on the replenishment of the blood Mg2+ level, using intravenous Mg2+ application first, which has to be followed by lifelong high-dose oral Mg2+ supplementation. The overdosed Mg2+ increases the renal Mg2+ reabsorption via a paracellular pathway in the thick ascending limb of the loop of Henle, which has a lesser importance in healthy individuals, where the major part of the Mg2+ reabsorption occurs via TRPM6 expressed in the distal convoluted tubule. The disease-causing mutations in TRPM6 result in a loss-of-function phenotype. It mostly results from a truncated protein because introduction of stop codons, although single-point mutations, frameshift mutations, exon splicing, deletions and mutations affecting alternative splicing, as well as a pore mutation have been also described (for reviews and recent new mutations, see Refs. [4,140–142]). This form of hypomagnesaemia, which has to be separated from other forms (like HOMG2-6), caused mutations in other genes different from TRPM6. Beyond HSH1, TRPM6 deletion has TRPML Channelopathies 23 been described in a patient with epilepsy and intellectual disorder [143], and two genetic variants (V1393I and K1584E) have been identified in diabetes 2 patients [144]. TRPM8 is not linked to any distinct channelopathy yet. However, the pathomechanism of familial amyloid polyneuropathy (or familial amyloidotic neuropathy, neuropathic heredofamilial am- yloidosis, familial amyloid polyneuropathy), a rare group of autosomal-dominant neuropathies of autonomic and peripheral nerves, might involve TRPM8 [145]. Recently, SNPs in TRPM8 have been connected to hereditary forms of migraine [146,147]. The dry eye syndrome seems also to be related to TRPM8 dysfunction, but any hereditary evidence is still missing [148].

TRPA CHANNELOPATHIES

Transient receptor potential ankyrin 1 (TRPA1) mutation causes a painful channelopathy, the familial episodic pain syndrome 1 (FEPS1) (OMIM 615040). This is a rare autosomal-dominant disease. In affected patients, fasting and physical stress trigger episodes of debilitating upper body pain. The symptoms also involve enhanced cutaneous flare responses and secondary hyperalgesia to punctate stimuli. One point mutation was identified that causes an amino acid substitution (N855S) in the S4 voltage-sensing domain. Although its pharmacological profile is not altered, the mutant is characterized by a shift in gating properties; its activity is much higher at normal resting potential, resulting in a dramatic increase of inward currents. In vitro, specific TRPA1 antagonists inhibited the abnormal response of the mutant channel, which promises a potential cure for patients suffering from FEPS1 ([149,150]; see, for a recent review, Ref. [151]). Recently, a missense point mutation has been discovered in pain patients with paradoxical heat sensation, which causes the E179K substitution in the TRPA1 N-terminus [152]. Cold failed to activate the mutant channel, probably because of a disturbed interaction with associated proteins [153].

TRPML CHANNELOPATHIES

TRPML1, the funding member of the mucolipidosis subfamily of the TRP channels, was named after the channelopathy, mucolipidosis type IV (ML IV, ML4) (OMIM 252650), caused by mutations in the TRPML1 gene. ML4 is an autosomal recessive neurodegenerative disease with a lysosomal storage disorder background. The leading neurological and sensory symptoms are psychomotor retardation and ophthalmologic abnormalities. The latter include corneal opacity, retinal degeneration, and strabismus, but developmental defect of the corpus callosum was also found. Furthermore, blood iron deficiency and achlorhydria also characterize ML4 patients, whose majority belongs to the Ashkenazi Jews population [154–156]. Over 21 mainly loss-of-function mutations in TRPML1 have been identified in ML4 patients. Defects of channel function result in impaired Ca2+ release from the organelles, which is required for the correct order of cellular events involving membrane fusion/fission ([157]; for a recent review, see Ref. [158]). These malfunctions lead to lysosomal storage disease in which cells are unable to process the material captured during endocytosis, although in contrast to most storage diseases, the function of lysosomal hydrolases is normal in ML4. The pathomechanism involves a defect in transport along the endosomal/lysosomal pathway, affecting 24 2. TRP DYSFUNCTIONS IN HEREDITARY DISEASES membrane sorting, fusion of both endosomes and autophagosomes with lysosomes (for a review, see Ref. [159]). Defects in the late steps of endocytosis and autophagy cause intracellular accumulation of lysosomal substrates and formation of large vacuolar intracellular organelles containing amphiphilic lipids (phospholipids, sphingolipids, gangliosides, mucopolysaccha- rides, lipofucsins, etc.) and other materials from cell organelle debris [160,161]. Moreover, TRPML1 also functions as a Fe2+ and Zn2+ channel, which is important in the removal of these ions from the lysosomes. In the absence of this lysosomal Fe2+/Zn2+ leak in ML4, lysosomes can be overloaded with these heavy metals, leading to the further impairment of lysosomal functions [162]. Another lipid-storage disease, the Niemann-Pick type C disease (NPC) (OMIM 257220) is primarily caused by mutations in the lysosomal two-pore segment channel 1, but like ML4, it also shows dramatically reduced TRPML1-mediated lysosomal Ca2+ release. Sphingomyelins (SMs) undergo sphingomyelinase-mediated hydrolysis in normal lysosomes, but they are accumulated in lysosomes of cells of NPC patients. SMs were found to inhibit TRPML1 in vitro, and abnormal luminal accumulation of these lipids can also block TRPML1- and Ca2+-dependent lysosomal trafficking causing a secondary lysosome storage disorder [163,164]. TRPML3 mutation is responsible for the phenotype of the varitint-waddler mouse, characterized by deafness and altered fur pigmentation [165]. However, neither TRPML2 nor TRPML3 has been reported to cause human diseases yet.

TRPP CHANNELOPATHIES

The nomenclature of TRP channels in the polycystin (TRPP) family is still somewhat confusing due to the reclassification of some members. Although a recently suggested nomenclature numbers the “real” TRPP channels consecutively from TRPP1 to TRPP3 [166], in this review we still use the official HUGO nomenclature to prevent further confusions and remain consequent with most of the cited literature. In the TRPP family, we can find only three ion channels: (1) TRPP2 (in the new nomenclature TRPP1 but also known as PKD2), (2) TRPP3 (now TRPP2 or PKD2 like 1 (PKD2L1)), and (3) TRPP5 (now TRPP3 or PKD2L2). These real TRP channels, as well as the reclassified nonchannel members, have a strong relationship to polycystic kidney disease, which gave the name polycystin to the family. TRPP2 mutations are common causes of autosomal-dominant polycystic kidney disease (ADPKD) (OMIM 613095). The most characteristic symptom of this disease is the progressive development of large epithelial-lined cysts not only in the kidney but also in the liver, pancreas, seminal tract, and arachnoid membrane. In the kidneys, any segment of the nephron can be affected by formation of cysts. Developing cysts press the renal parenchyma, further increasing the circumference of the already dilated renal tubules. As they are growing, cysts occupy more and more space and thereby compress and destroy normal renal tissue, resulting in abnormally enlarged kidneys and impaired kidney function. Well-developed cysts are filled with fluid that is probably secreted by the epithelial cells of the cysts. Beside the kidney symptoms, ADPKD also causes cardiovascular abnormalities (e.g., coronary artery aneurysms or intracranial “berry” aneurysms), which often lead to vessel rupture, resulting in potentially fatal acute bleeding or chronic subdural hematomas [167]. Defects in the heart (e.g., defective septum formation) are also known consequences of TRPP2 mutations [168]. CONCLUSIONS 25

Mutations in PKD1 (formerly, TRPP1) and in TRPP2 (ca. 85% and 15% of the cases, respectively) are the major cases of ADPKD; more than 400 mutations in these genes have been described in ADPKD patients [169]. However, the exact pathomechanism and the contribution of PKD1 and TRPP2 to the pathophysiology of the polycystic kidney disease are largely unknown (for an excellent review, see Ref. [170]). TRPP2 was hypothesized to function as a putative flow-sensor in the primary cilia; to fulfill this role, it needs to associate with PKD1 forming the polycystin complex (for a review, see Ref. [171]). TRPP2 is involved in the regulation of cellular processes via several crucially important signaling pathways, like the JAK/ STAT, p53, mTOR, NFAT/AP-1, cAMP/PKA, cAMP-dependent ERK, cyclin-dependent kinases, or Wnt signaling pathways. All these pathways may be partially involved in the pathogenesis of ADPKD (for a review, see Ref. [172]). The disturbed connections of the mutant channel proteins with cytoskeletal interaction partners may also result in functional defects [173]. The complex of PKD1 with TRPP2 also prevents the nuclear translocation of a crucial regulator of cell proliferation and differentiation, the helix-loop-helix protein Id2. In patients with PKD1/TRPP2 mutations, Id2 accumulates in the nuclei of the renal epithelial cells to constitute a hyperproliferative phenotype, causing cyst formation [174,175]. Because ADPKD is a ciliopathy, an association of TRPP2 with other ciliopathies is also expected. In two sisters suffering from Joubert syndrome, a rare genetic disorder affecting the cerebellum and manifesting in balance and coordination disturbances, mutation of TCTN1 gene was identified. The translated protein Tectonic-1 is a regulator of the ciliogenesis, and it forms a complex with many ciliar proteins. Furthermore, it is needed for the correct ciliar localization of TRPP2 [176]. Another example is Meckel syndrome (also known as Meckel-Gruber syndrome, Gruber syndrome, or dysencephalia splanchnocystica), a rare, lethal, genetic ciliopathy. In a rat model of Meckel syndrome, significantly increased TRPP2 protein expression was found [177]. Interestingly, TRPP2 mutations can also lead to impaired morphogenesis; these mutations are reportedly involved in the deformation of the left-right lateralization due to a primary ciliar dyskinesia [178,179]. TRPP3 (earlier PKD2L1) is also needed for normal ciliar functions. It has been recently identified in primary cilia as a component (together with PKD1L1) of a Ca2+ permeable cation channel. This complex played a role in the regulation of GLI2 by smoothened protein, elements of the hedgehog pathway. TRPP3-PKD1L1 channels play an important role in the regulation of the Ca2+ concentration in a subciliar compartment [180,181]. Based on the literature, it is intriguing to hypothesize that TRPP3 (PKD2L1) may play an important role in neurodevelopmental disorders and ciliopathies (see for a review, Ref. [182]).

CONCLUSIONS

TRP channels are expressed practically all over the body, where they contribute to the regulation of several fundamental cellular functions. They act as cellular sensors that integrate external and endogenous stimuli, contribute to cell-to-cell communication, and take part in the maintenance of cellular homeostasis in many forms. Although connections of TRP channels to a plethora of diseases are well documented, the number of inherited channelopathies related to mutations in the TRP genes is relatively small. Furthermore, the role of the defective TRP channels in the pathomechanisms of these diseases is incompletely understood. 26 2. TRP DYSFUNCTIONS IN HEREDITARY DISEASES

This urgently requires more research. Without doubt, based on the well-documented crucial roles of TRPs in various cellular functions, discovery of additional TRP-related diseases is expected. We propose that TRP channels constitute promising targets for pharmacological development aimed at alleviation of the symptoms of TRP-related maladies.

Acknowledgments We thank all members of the Laboratory of Ion Channel Research, KU Leuven, Department Cellular and Molecular Medicine for constructive discussion. We thank especially Grzegorz Owsianik (Leuven) for his input in an early phase of this project. For the work on this review, B. I. T. was supported by the People Programme (Marie Curie Actions) of the European Union's Seventh Framework Programme (FP7/2007-2013) under REA Grant agreement no. 330489. B. N. was supported by the KU Leuven in his position as Emeritus met opdracht.

References [1] Kass RS. The channelopathies: novel insights into molecular and genetic mechanisms of human disease. J Clin Invest 2005;115:1986–9. [2] Abramowitz J, Birnbaumer L. Know thy neighbors: a survey of diseases and complex syndromes that map to chromosomal regions encoding TRP channels. In: Flockerzi V, Nilius B, editors. TRP channels. Berlin- Heidelberg: Springer Verlag; 2007. p. 377–406. [3] Nilius B, Owsianik G, Voets T, Peters JA. Transient receptor potential channels in disease. Physiol Rev 2007;87:165–217. [4] Nilius B, Owsianik G. Transient receptor potential channelopathies. Pflugers Arch 2010;460:437–50. [5] Everett KV. Transient receptor potential genes and human inherited disease. Adv Exp Med Biol 2011;704:1011–32. [6] Moran MM, McAlexander MA, Biro T, Szallasi A. Transient receptor potential channels as therapeutic targets. Nat Rev Drug Discov 2011;10:601–20. [7] Montell C. Visual transduction in Drosophila. Annu Rev Cell Dev Biol 1999;15:231–68. [8] Abramowitz J, Birnbaumer L. Physiology and pathophysiology of canonical transient receptor potential channels. FASEB J 2009;23:297–328. [9] Feske S. CRAC channelopathies. Pflugers Arch 2010;460:417–35. [10] Cheng KT, Alevizos I, Liu X, Swaim WD, Yin H, Feske S, et al. STIM1 and STIM2 protein deficiency in T lymphocytes underlies development of the exocrine gland autoimmune disease, Sjogren’s syndrome. Proc Natl Acad Sci USA 2012;109:14544–9. [11] Bohm J, Chevessier F, Maues De Paula A, Koch C, Attarian S, Feger C, et al. Constitutive activation of the calcium sensor STIM1 causes tubular-aggregate myopathy. Am J Hum Genet 2013;92:271–8. [12] Toth BI, Olah A, Szollosi AG, Biro T. TRP channels in the skin. Br J Pharmacol 2014;171:2568–81. [13] Sauer A, Blavin J, Lhermitte B, Speeg-Schatz C. Conjunctival ganglioglioma as a feature of basal cell nevus syndrome. J AAPOS 2011;15:387–8. [14] Beck B, Lehen’kyi V, Roudbaraki M, Flourakis M, Charveron M, Bordat P, et al. TRPC channels determine human keratinocyte differentiation: new insight into basal cell carcinoma. Cell Calcium 2008;43:492–505. [15] Pani B, Singh BB. Darier’s disease: a calcium-signaling perspective. Cell Mol Life Sci 2008;65:205–11. [16] Pani B, Cornatzer E, Cornatzer W, Shin DM, Pittelkow MR, Hovnanian A, et al. Up-regulation of transient receptor potential canonical 1 (TRPC1) following sarco(endo)plasmic reticulum Ca2+ ATPase 2 gene silencing promotes cell survival: a potential role for TRPC1 in Darier’s disease. Mol Biol Cell 2006;17:4446–58. [17] Zeng B, Yuan C, Yang X, Atkin SL, Xu SZ. TRPC channels and their splice variants are essential for promoting human ovarian cancer cell proliferation and tumorigenesis. Curr Cancer Drug Targets 2012;13:103–16. [18] Chen K, Jin X, Li Q, Wang W, Wang Y, Zhang J. Association of TRPC1 gene polymorphisms with Type 2 diabetes and diabetic nephropathy in Han Chinese population. Endocr Res 2013;38:59–68. [19] Moccia F, Dragoni S, Poletto V, Rosti V, Tanzi F, Ganini C, et al. Orai1 and transient receptor potential channels as novel molecular targets to impair tumor neovascularization in renal cell carcinoma and other malignancies. Anticancer Agents Med Chem 2014;14:296–312. References 27

[20] Becker EBE, Oliver PL, Glitsch MD, Banks GT, Achillic F, Hardy A, et al. A point mutation in TRPC3 causes abnormal Purkinje cell development and cerebellar ataxia in moonwalker mice. Proc Natl Acad Sci USA 2009;106:6706–11. [21] Sekerkova G, Kim JA, Nigro MJ, Becker EB, Hartmann J, Birnbaumer L, et al. Early onset of ataxia in moonwalker mice is accompanied by complete ablation of type II unipolar brush cells and Purkinje cell dysfunction. J Neurosci 2013;33:19689–94. [22] Martin-Trujillo A, Iglesias-Platas I, Coto E, Corral-Juan M, San Nicolas H, Corral J, et al. Genotype of an individual single nucleotide polymorphism regulates DNA methylation at the TRPC3 alternative promoter. Epigenetics 2011;6:1236–41. [23] Becker EB, Fogel BL, Rajakulendran S, Dulneva A, Hanna MG, Perlman SL, et al. Candidate screening of the TRPC3 gene in cerebellar ataxia. Cerebellum 2011;10:10. [24] Shuvaev AN, Horiuchi H, Seki T, Goenawan H, Irie T, Iizuka A, et al. Mutant PKC{gamma} in spinocerebellar ataxia type 14 disrupts synapse elimination and long-term depression in purkinje cells in vivo. J Neurosci 2011;31:14324–34. [25] Letavernier E, Rodenas A, Guerrot D, Haymann JP. Williams-Beuren syndrome hypercalcemia: is TRPC3 a novel mediator in calcium homeostasis? Pediatrics 2012;129:e1626–30. [26] Amir RE, Van den Veyver IB, Wan M, Tran CQ, Francke U, Zoghbi HY. Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2. Nat Genet 1999;23:185–8. [27] Li W, Calfa G, Larimore J, Pozzo-Miller L. Activity-dependent BDNF release and TRPC signaling is impaired in hippocampal neurons of Mecp2 mutant mice. Proc Natl Acad Sci USA 2012;109:17087–92. [28] Bertinetto FE, Calafell F, Roggero S, Chidichimo R, Garino E, Marcuccio C, et al. Search for genetic association between IgA nephropathy and candidate genes selected by function or by gene mapping at loci IGAN2 and IGAN3. Nephrol Dial Transplant 2012;27:2328–37. [29] Kim MS, Lee KP, Yang D, Shin DM, Abramowitz J, Kiyonaka S, et al. Genetic and pharmacological inhibition of the Ca2+ influx channel TRPC3 protects secretory epithelia from Ca2+-dependent toxicity. Gastroenterology 2011;140:2107–215. [30] von Spiczak S, Muhle H, Helbig I, de Kovel CG, Hampe J, Gaus V, et al. Association study of TRPC4 as a candidate gene for generalized epilepsy with photosensitivity. Neuromol Med 2010;12:292–9. [31] Jung C, Gene GG, Tomas M, Plata C, Selent J, Pastor M, et al. A gain-of-function SNP in TRPC4 cation channel protects against myocardial infarction. Cardiovasc Res 2011;91:465–71. [32] Everett KV, Chioza BA, Georgoula C, Reece A, Capon F, Parker KA, et al. Genome-wide high-density SNP-based linkage analysis of infantile hypertrophic pyloric stenosis identifies loci on chromosomes 11q14-q22 and Xq23. Am J Hum Genet 2008;16:1151–4. [33] Everett KV, Chioza BA, Georgoula C, Reece A, Gardiner RM, Chung EM. Infantile hypertrophic pyloric stenosis: evaluation of three positional candidate genes, TRPC1, TRPC5 and TRPC6, by association analysis and re-sequencing. Hum Genet 2009;26:819–31. [34] Ju JJ, Gao H, Li H, Lu Y, Wang LL, Yuan ZW. No association between the SNPs (rs56134796; rs3824934; rs41302375) in the TRPC6 gene promoter and infantile hypertrophic pyloric stenosis in Chinese people. Pediatr Surg Int 2011;27:1267–70. [35] Dryer SE, Reiser J. TRPC6 channels and their binding partners in podocytes: role in glomerular filtration and pathophysiology. Am J Physiol Renal Physiol 2010;299:F689–701. [36] Mottl AK, Lu M, Fine CA, Weck KE. A novel TRPC6 mutation in a family with podocytopathy and clinical variability. BMC Nephrol 2013;14:104. [37] Hofstra JM, Lainez S, van Kuijk WH, Schoots J, Baltissen MP, Hoefsloot LH, et al. New TRPC6 gain-of-function mutation in a non-consanguineous Dutch family with late-onset focal segmental glomerulosclerosis. Nephrol Dial Transplant 2013;28:1830–8. [38] Reiser J, Polu KR, Moller CC, Kenlan P, Altintas MM, Wei C, et al. TRPC6 is a glomerular slit diaphragm- associated channel required for normal renal function. Nat Genet 2005;37:739–44. [39] Moller CC, Flesche J, Reiser J. Sensitizing the slit diaphragm with TRPC6 ion channels. J Am Soc Nephrol 2009;20:950–3. [40] Kanda S, Harita Y, Shibagaki Y, Sekine T, Igarashi T, Inoue T, et al. Tyrosine phosphorylation-dependent activation of TRPC6 regulated by PLC-gamma1 and nephrin: effect of mutations associated with focal segmental glomerulosclerosis. Mol Biol Cell 2011;22:1824–35. [41] Nijenhuis T, Sloan AJ, Hoenderop JG, Flesche J, van Goor H, Kistler AD, et al. Angiotensin II contributes to podocyte injury by increasing TRPC6 expression via an NFAT-mediated positive feedback signaling pathway. Am J Pathol 2011;179:1719–32. 28 2. TRP DYSFUNCTIONS IN HEREDITARY DISEASES

[42] Chen S, He FF, Wang H, Fang Z, Shao N, Tian XJ, et al. Calcium entry via TRPC6 mediates albumin overload- induced endoplasmic reticulum stress and apoptosis in podocytes. Cell Calcium 2011;50:523–9. [43] Schlöndorff J, del Camino D, Carrasquillo R, Lacey V, Pollak MR. TRPC6 mutations associated with focal segmental glomerulosclerosis cause constitutive activation of NFAT-dependent transcription. Am J Physiol Cell Physiol 2009;296:C558–69. [44] Wang Y, Jarad G, Tripathi P, Pan M, Cunningham J, Martin DR, et al. Activation of NFAT signaling in podocytes causes glomerulosclerosis. J Am Soc Nephrol 2010;21:1657–66. [45] El Hindi S, Reiser J. TRPC channel modulation in podocytes-inching toward novel treatments for glomerular disease. Pediatr Nephrol 2010;26:1057–64. [46] Li Z, Xu J, Xu P, Liu S, Yang Z. Wnt/beta-catenin signalling pathway mediates high glucose induced cell injury through activation of TRPC6 in podocytes. Cell Prolif 2013;46:76–85. [47] Chiluiza D, Krishna S, Schumacher VA, Schlondorff J. Gain-of-function mutations in transient receptor potential C6 (TRPC6) activate extracellular signal-regulated kinases 1/2 (ERK1/2). J Biol Chem 2013;288:18407–20. [48] Sonneveld R, Ferre S, Hoenderop JG, Dijkman HB, Berden JH, Bindels RJ, et al. Vitamin D down-regulates TRPC6 expression in podocyte injury and proteinuric glomerular disease. Am J Pathol 2013;182:1196–204. [49] Kistler AD, Singh G, Pippin J, Altintas MM, Yu H, Fernandez IC, et al. TRPC6 protects podocytes during complement-mediated glomerular disease. J Biol Chem 2013;288:36598–609. [50] Mir S, Yavascan O, Berdeli A, Sozeri B. TRPC6 gene variants in Turkish children with steroid-resistant nephrotic syndrome. Nephrol Dial Transplant 2012;27:205–9. [51] Kuang XY, Huang WY, Xu H, Shi Y, Zhang XL, Niu XL, et al. 254C>G: a TRPC6 promoter variation associated with enhanced transcription and steroid-resistant nephrotic syndrome in Chinese children. Pediatr Res 2013;74:511–16. [52] Yu Y, Keller SH, Remillard CV, Safrina O, Nicholson A, Zhang SL, et al. A functional single-nucleotide polymorphism in the TRPC6 gene promoter associated with idiopathic pulmonary arterial hypertension. Circulation 2009;119:2313–22. [53] Bernaldo de Quiros S, Merlo A, Secades P, Zambrano I, Saenz de Santamaria I, Ugidos N, et al. Identification of TRPC6 as a possible candidate target gene within an amplicon at 11q21-q22.2 for migratory capacity in head and neck squamous cell carcinomas. BMC Cancer 2013;13:116. [54] Chen S, Fan Q, Gao X, Wang X, Huang R, Laties AM, et al. Increased expression of the transient receptor potential cation channel 6 gene in patients with primary open-angle glaucoma. Clin Exp Ophthalmol 2013;41:753–60. [55] Armero P, Muriel C, Lopez M, Santos J, Gonzalez-Sarmiento R. Analysis of TRPV1 gene polymorphisms in Spanish patients with neuropathic pain. Med Clin (Barc) 2012;139:1–4. [56] Carreno O, Corominas R, Fernandez-Morales J, Camina M, Sobrido MJ, Fernandez-Fernandez JM, et al. SNP variants within the vanilloid TRPV1 and TRPV3 receptor genes are associated with migraine in the Spanish population. Am J Med Genet B Neuropsychiatr Genet 2012;159B:94–103. [57] Sadeh M, Glazer B, Landau Z, Wainstein J, Bezaleli T, Dabby R, et al. Association of the M3151 variant in the transient receptor potential vanilloid receptor-1 (TRPV1) gene with type 1 diabetes in an Ashkenazi Jewish population. Isr Med Assoc J 2013;15:477–80. [58] Laurito S, Goldschmidt E, Marquez M, Roque M. Deletion of the LIS1, ASPA, TRPV1 and CAMTA2 genes in region 17p13.3 in a patient with Miller-Dieker syndrome. Rev Neurol 2011;52:189–91. [59] Valdes AM, De Wilde G, Doherty SA, Lories RJ, Vaughn FL, Laslett LL, et al. The Ile585Val TRPV1 variant is involved in risk of painful knee osteoarthritis. Ann Rheum Dis 2011;70:1556–61. [60] Cantero-Recasens G, Gonzalez JR, Fandos C, Duran E, Smit LA, Kauffmann F, et al. Loss-of-function of transient receptor potential vanilloid 1 (TRPV1) genetic variant is associated with lower risk of active childhood asthma. J Biol Chem 2010;285:27532–5. [61] Tahara T, Shibata T, Nakamura M, Yamashita H, Yoshioka D, Hirata I, et al. Homozygous TRPV1 315C influences the susceptibility to functional dyspepsia. J Clin Gastroenterol 2010;44:e1–7. [62] Harisseh R, Chatelier A, Magaud C, Deliot N, Constantin B. Involvement of TRPV2 and SOCE in Ca2+ influx disorder in DMD human myotubes with a specific contribution of alpha1-syntrophin and PLC/PKC in SOCE regulation. Am J Physiol Cell Physiol 2013;304:C881–94. [63] Cai X, Yang YC, Wang JF, Wang Q, Gao J, Fu WL, et al. Transient receptor potential vanilloid 2 (TRPV2), a potential novel biomarker in childhood asthma. J Asthma 2013;50:209–14. [64] Oulidi A, Bokhobza A, Gkika D, Vanden Abeele F, Lehen’kyi V, Ouafik L, et al. TRPV2 mediates adrenomedullin stimulation of prostate and urothelial cancer cell adhesion, migration and invasion. PLoS One 2013;8:e64885. References 29

[65] Nilius B, Biro T. TRPV3: a ‘more than skinny’ channel. Exp Dermatol 2013;22:447–52. [66] Nilius B, Biro T, Owsianik G. TRPV3: time to decipher a poorly understood family member! J Physiol 2014;15:295–304. [67] Lin Z, Chen Q, Lee M, Cao X, Zhang J, Ma D, et al. Exome sequencing reveals mutations in TRPV3 as a cause of Olmsted syndrome. Am J Hum Genet 2012;90:558–64. [68] Lai-Cheong JE, Sethuraman G, Ramam M, Stone K, Simpson MA, McGrath JA. Recurrent heterozygous missense mutation, p.Gly573Ser, in the TRPV3 gene in an Indian boy with sporadic Olmsted syndrome. Br J Dermatol 2012;167:440–2. [69] Danso-Abeam D, Zhang J, Dooley J, Staats KA, Van Eyck L, Van Brussel T, et al. Olmsted syndrome: exploration of the immunological phenotype. Orphanet J Rare Dis 2013;8:79. [70] Asakawa M, Yoshioka T, Matsutani T, Hikita I, Suzuki M, Oshima I, et al. Association of a mutation in TRPV3 with defective hair growth in rodents. J Invest Dermatol 2006;126:2664–72. [71] Borbiro I, Lisztes E, Toth BI, Czifra G, Olah A, Szollosi AG, et al. Activation of transient receptor potential vanilloid-3 inhibits human hair growth. J Invest Dermatol 2011;131:1605–14. [72] Sulk M, Seeliger S, Aubert J, Schwab VD, Cevikbas F, Rivier M, et al. Distribution and expression of non-neuronal transient receptor potential (TRPV) ion channels in rosacea. J Invest Dermatol 2012;132:1253–62. [73] Rainero I, Rubino E, Paemeleire K, Gai A, Vacca A, De Martino P, et al. Genes and primary headaches: discovering new potential therapeutic targets. J Headache Pain 2013;14:61. [74] Proverbio MC, Mangano E, Gessi A, Bordoni R, Spinelli R, Asselta R, et al. Whole genome SNP genotyping and exome sequencing reveal novel genetic variants and putative causative genes in congenital hyperinsulinism. PLoS One 2013;8:e68740. [75] Rock MJ, Prenen J, Funari VA, Funari TL, Merriman B, Nelson SF, et al. Gain-of-function mutations in TRPV4 cause autosomal dominant brachyolmia. Nat Genet 2008;40:999–1003. [76] Nishimura G, Dai J, Lausch E, Unger S, Megarbane A, Kitoh H, et al. Spondylo-epiphyseal dysplasia, Maroteaux type (pseudo-Morquio syndrome type 2), and parastremmatic dysplasia are caused by TRPV4 mutations. Am J Med Genet A 2010;152A:1443–9. [77] Krakow D, Vriens J, Camacho N, Luong P, Deixler H, Funari TL, et al. Mutations in the gene encoding the calcium-permeable ion channel TRPV4 produce spondylometaphyseal dysplasia, Kozlowski type and metatropic dysplasia. Am J Hum Genet 2009;84:307–15. [78] Camacho N, Krakow D, Johnykutty S, Katzman PJ, Pepkowitz S, Vriens J, et al. Dominant TRPV4 mutations in nonlethal and lethal metatropic dysplasia. Am J Med Genet A 2010;152A:1169–77. [79] Unger S, Lausch E, Stanzial F, Gillessen-Kaesbach G, Stefanova I, Di Stefano CM, et al. Fetal akinesia in metatropic dysplasia: the combined phenotype of chondrodysplasia and neuropathy? Am J Med Genet A 2011;155A:2860–4. [80] Lamande SR, Yuan Y, Gresshoff IL, Rowley L, Belluoccio D, Kaluarachchi K, et al. Mutations in TRPV4 cause an inherited arthropathy of hands and feet. Nat Genet 2011;43:1142–6. [81] McEntagart M. TRPV4 axonal neuropathy spectrum disorder. J Clin Neurosci 2012;19:927–33. [82] Coste B, Houge G, Murray MF, Stitziel N, Bandell M, Giovanni MA, et al. Gain-of-function mutations in the mechanically activated ion channel PIEZO2 cause a subtype of distal arthrogryposis. Proc Natl Acad Sci USA 2013;110:4667–72. [83] Zimon M, Baets J, Auer-Grumbach M, Berciano J, Garcia A, Lopez-Laso E, et al. Dominant mutations in the cation channel gene transient receptor potential vanilloid 4 cause an unusual spectrum of neuropathies. Brain 2010;33:1798–809. [84] DeLong R, Siddique T. A large New England kindred with autosomal dominant neurogenic scapuloperoneal amyotrophy with unique features. Arch Neurol 1992;49:905–8. [85] Deng H-X, Klein CJ, Yan J, Shi S, Wu Y, Fecto F, et al. Scapuloperoneal spinal muscular atrophy and hereditary motor and sensory neuropathy type IIC are allelic disorders caused by mutations in TRPV4. Nat Genet 2010;42:165–9. [86] Auer-Grumbach M, Olschewski A, Papic L, Kremer H, McEntagart ME, Uhrig S, et al. Mutations in the N-terminal ankyrin domain of TRPV4 cause congenital and scapuloperoneal spinal muscular atrophy, and hereditary motor and sensory neuropathy 2C. Nat Genet 2010;42:160–4. [87] Landouré G, Zdebik AA, Martinez TL, Burnett BG, Stanescu HC, Shi Y, et al. Mutations in TRPV4 cause Charcot-Marie-Tooth disease type 2C. Nat Genet 2010;42:170–4. [88] Rossor AM, Kalmar B, Greensmith L, Reilly MM. The distal hereditary motor neuropathies. J Neurol Neurosurg Psychiatry 2011;83:6–14. 30 2. TRP DYSFUNCTIONS IN HEREDITARY DISEASES

[89] Nilius B, Voets T. The puzzle of TRPV4 channelopathies. EMBO Rep 2013;14:152–63. [90] Loukin S, Su Z, Zhou X, Kung C. Forward genetic analysis reveals multiple gating mechanisms of TRPV4. J Biol Chem 2010;285:19884–90. [91] Loukin S, Zhou X, Su Z, Saimi Y, Kung C. Wild-type and brachyolmia-causing mutant TRPV4 channels respond directly to stretch force. J Biol Chem 2010;285:27176–81. [92] Nishimura G, Lausch E, Savarirayan R, Shiba M, Spranger J, Zabel B, et al. TRPV4-associated skeletal dysplasias. Am J Med Genet C Semin Med Genet 2012;160C:190–204. [93] Nilius B, Owsianik G. Channelopathies converge on TRPV4. Nat Genet 2010;42:98–100. [94] Inada H, Procko E, Sotomayor M, Gaudet R. Structural and biochemical consequences of disease-causing mutations in the ankyrin repeat domain of the human TRPV4 channel. Biochemistry 2012;51:6195–206. [95] Verma P, Kumar A, Goswami C. TRPV4-mediated channelopathies. Channels 2010;4:319–28. [96] Li J, Kanju P, Patterson M, Chew WL, Cho SH, Gilmour I, et al. TRPV4-mediated calcium influx into human bronchial epithelia upon exposure to diesel exhaust particles. Environ Health Perspect 2011;119:784–93. [97] Tian W, Fu Y, Garcia-Elias A, Fernandez-Fernandez JM, Vicente R, Kramer PL, et al. A loss-of-function nonsynonymous polymorphism in the osmoregulatory TRPV4 gene is associated with human hyponatremia. Proc Natl Acad Sci USA 2009;106:14034–9. [98] Na T, Zhang W, Jiang Y, Liang Y, Ma HP, Warnock DG, et al. The A563T variation of the renal epithelial calcium channel TRPV5 among African Americans enhances calcium influx. Am J Physiol Renal Physiol 2009;296:F1042–51. [99] Wangemann P, Nakaya K, Wu T, Maganti RJ, Itza EM, Sanneman JD, et al. Loss of cochlear HCO3-secretion causes deafness via endolymphatic acidification and inhibition of Ca2+ reabsorption in a Pendred syndrome mouse model. Am J Physiol Renal Physiol 2007;292:F1345–53. [100] Yang SS, Lo YF, Yu IS, Lin SW, Chang TH, Hsu YJ, et al. Generation and analysis of the thiazide-sensitive Na(+)-Cl(−) cotransporter (Ncc/Slc12a3) Ser707X knock-in mouse as a model of Gitelman syndrome. Hum Mutat 2010;31:1304–15. [101] Loh NY, Bentley L, Dimke H, Verkaart S, Tammaro P, Gorvin CM, et al. Autosomal dominant hypercalciuria in a mouse model due to a mutation of the epithelial calcium channel, TRPV5. PLoS One 2013;8:e55412. [102] Hughes DA, Tang K, Strotmann R, Schoneberg T, Prenen J, Nilius B, et al. Parallel selection on TRPV6 in human populations. PLoS One 2008;3:e1686. [103] Suzuki Y, Pasch A, Bonny O, Mohaupt MG, Hediger MA, Frey FJ. Gain of function haplotype in the epithelial calcium channel TRPV6 is a risk factor for renal calcium stone formation. Hum Mol Genet 2008;17:1613–18. [104] Guo H, Carlson JA, Slominski A. Role of TRPM in melanocytes and melanoma. Exp Dermatol 2012;21:650–4. [105] Audo I, Kohl S, Leroy BP, Munier FL, Guillonneau X, Mohand-Said S, et al. TRPM1 is mutated in patients with autosomal-recessive complete congenital stationary night blindness. Am J Hum Genet 2009;85:720–9. [106] Nakamura M, Sanuki R, Yasuma TR, Onishi A, Nishiguchi KM, Koike C, et al. TRPM1 mutations are associated with the complete form of congenital stationary night blindness. Mol Vis 2010;16:425–37. [107] Bijveld MM, Florijn RJ, Bergen AA, van den Born LI, Kamermans M, Prick L, et al. Genotype and phenotype of 101 dutch patients with congenital stationary night blindness. Ophthalmology 2013;120:2072–81. [108] Peachey NS, Pearring JN, Bojang Jr P, Hirschtritt ME, Sturgill-Short G, Ray TA, et al. Depolarizing bipolar cell dysfunction due to a TRPM1 point mutation. J Neurophysiol 2012;108:2442–51. [109] Audo I, Bujakowska KM, Leveillard T, Mohand-Said S, Lancelot ME, Germain A, et al. Development and application of a next-generation-sequencing (NGS) approach to detect known and novel gene defects underlying retinal diseases. Orphanet J Rare Dis 2012;7:8. [110] Bellone RR, Holl H, Setaluri V, Devi S, Maddodi N, Archer S, et al. Evidence for a retroviral insertion in TRPM1 as the cause of congenital stationary night blindness and leopard complex spotting in the horse. PLoS One 2013;8:e78280. [111] Li Z, Sergouniotis PI, Michaelides M, Mackay DS, Wright GA, Devery S, et al. Recessive mutations of the gene TRPM1 abrogate ON bipolar cell function and cause complete congenital stationary night blindness in humans. Am J Hum Genet 2009;85:711–19. [112] Spielmann M, Reichelt G, Hertzberg C, Trimborn M, Mundlos S, Horn D, et al. Homozygous deletion of chromosome 15q13.3 including CHRNA7 causes severe mental retardation, seizures, muscular hypotonia, and the loss of KLF13 and TRPM1 potentially cause macrocytosis and congenital retinal dysfunction in siblings. Eur J Med Genet 2011;54:E441–5. [113] Plato CC, Galasko D, Garruto RM, Plato M, Gamst A, Craig UK, et al. ALS and PDC of Guam: forty-year follow-up. Neurology 2002;58:765–73. References 31

[114] Hermosura M, Cui AM, Go G, Davenport B, Shetler C, Heizer J, et al. Altered functional properties of a TRPM2 variant in Guamanian ALS and PD. Proc Natl Acad Sci USA 2008;105:18029–34. [115] Hermosura MC, Garruto RM. TRPM7 and TRPM2-candidate susceptibility genes for Western Pacific ALS and PD? Biochim Biophys Acta 2007;1772:822–35. [116] Hermosura MC, Nayakanti H, Dorovkov MV, Calderon FR, Ryazanov AG, Haymer DS, et al. A TRPM7 variant shows altered sensitivity to magnesium that may contribute to the pathogenesis of two Guamanian neurodegenerative disorders. Proc Natl Acad Sci USA 2005;102:11510–15. [117] Benarroch EE. TRP channels: functions and involvement in neurologic disease. Neurology 2008;70:648–52. [118] Szydlowska K, Tymianski M. Calcium, ischemia and excitotoxicity. Cell Calcium 2010;47:122–9. [119] Hara K, Kokubo Y, Ishiura H, Fukuda Y, Miyashita A, Kuwano R, et al. TRPM7 is not associated with amyotrophic lateral sclerosis-parkinsonism dementia complex in the Kii peninsula of Japan. Am J Med Genet B Neuropsychiatr Genet 2010;153B:310–13. [120] Liu J, Juo SH, Terwilliger JD, Grunn A, Tong X, Brito M, et al. A follow-up linkage study supports evidence for a bipolar affective disorder locus on chromosome 21q22. Am J Med Genet 2001;105:189–94. [121] Xu C, Macciardi F, Li PP, Yoon IS, Cooke RG, Hughes B, et al. Association of the putative susceptibility gene, transient receptor potential protein melastatin type 2, with bipolar disorder. Am J Med Genet B Neuropsychiatr Genet 2006;141:36–43. [122] Xu C, Warsh JJ, Wang KS, Mao CX, Kennedy JL. Association of the iPLA2beta gene with bipolar disorder and assessment of its interaction with TRPM2 gene polymorphisms. Psychiatr Genet 2013;23:86–9. [123] Xu C, Li PP, Cooke RG, Parikh SV, Wang K, Kennedy JL, et al. TRPM2 variants and bipolar disorder risk: con- firmation in a family-based association study. Bipolar Disord 2009;11:1–10. [124] Zhang W, Hui KY, Gusev A, Warner N, Ng SM, Ferguson J, et al. Extended haplotype association study in Crohn’s disease identifies a novel, Ashkenazi Jewish-specific missense mutation in the NF-kappaB pathway gene, HEATR3. Genes Immun 2013;14:310–16. [125] Oberwinkler J, Phillipp SE. Trpm3. Handb Exp Pharmacol 2007;179:253–67. [126] Pagnamenta AT, Holt R, Yusuf M, Pinto D, Wing K, Betancur C, et al. A family with autism and rare copy number variants disrupting the Duchenne/Becker muscular dystrophy gene DMD and TRPM3. J Neurodev Disord 2011;3:124–31. [127] Kuniba H, Yoshiura KI, Kondoh T, Ohashi H, Kurosawa K, Tonoki H, et al. Molecular karyotyping in 17 patients and mutation screening in 41 patients with Kabuki syndrome. J Hum Genet 2009;54:304–9. [128] Park SH, Lee JY, Kim S. A methodology for multivariate phenotype-based genome-wide association studies to mine pleiotropic genes. BMC Syst Biol 2012;5(Suppl. 2):S13. [129] Shaham O, Gueta K, Mor E, Oren-Giladi P, Grinberg D, Xie Q, et al. Pax6 regulates gene expression in the vertebrate lens through miR-204. PLoS Genet 2013;9:e1003357. [130] Xie Q, Yang Y, Huang J, Ninkovic J, Walcher T, Wolf L, et al. Pax6 interactions with chromatin and identification of its novel direct target genes in lens and forebrain. PLoS One 2013;8:e54507. [131] Kruse M, Schulze-Bahr E, Corfield V, Beckmann A, Stallmeyer B, Kurtbay G, et al. Impaired endocytosis of the ion channel TRPM4 is associated with human progressive familial heart block type I. J Clin Invest 2009;119:2737–44. [132] Liu H, El Zein L, Kruse M, Guinamard R, Beckmann A, Bozio A, et al. Gain-of-function mutations in TRPM4 cause autosomal dominant isolated cardiac conduction disease. Circ Cardiovasc Genet 2010;3:374–85. [133] Stallmeyer B, Zumhagen S, Denjoy I, Duthoit G, Hebert JL, Ferrer X, et al. Mutational spectrum in the Ca(2+) activated cation channel gene TRPM4 in patients with cardiac conductance disturbances. Hum Mutat 2012;33:109–17. [134] Folgering JH, Sharif-Naeini R, Dedman A, Patel A, Delmas P, Honore E. Molecular basis of the mammalian pressure-sensitive ion channels: focus on vascular mechanotransduction. Prog Biophys Mol Biol 2008;97:180–95. [135] Liu H, Chatel S, Simard C, Syam N, Salle L, Probst V, et al. Molecular genetics and functional anomalies in a series of 248 Brugada cases with 11 mutations in the TRPM4 channel. PLoS One 2013;8:e54131. [136] Malhotra S, Castillo J, Negrotto L, Merino-Zamorano C, Montaner J, Vidal-Jordana A, et al. TRPM4 mRNA expression levels in peripheral blood mononuclear cells from multiple sclerosis patients. J Neuroimmunol 2013;261:146–8. [137] Han S, Koo HH, Lan Q, Lee KM, Park AK, Park SK, et al. Common variation in genes related to immune response and risk of childhood leukemia. Hum Immunol 2012;73:316–19. 32 2. TRP DYSFUNCTIONS IN HEREDITARY DISEASES

[138] Schlingmann KP, Waldegger S, Konrad M, Chubanov V, Gudermann T. TRPM6 and TRPM7—gatekeepers of human magnesium metabolism. Biochim Biophys Acta 2007;1772:813–21. [139] Schlingmann KP, Sassen MC, Weber S, Pechmann U, Kusch K, Pelken L, et al. Novel TRPM6 mutations in 21 families with primary hypomagnesemia and secondary hypocalcemia. J Am Soc Nephrol 2005;16:3061–9. [140] Woudenberg-Vrenken TE, Bindels RJ, Hoenderop JG. The role of transient receptor potential channels in kidney disease. Nat Rev Nephrol 2009;5:441–9. [141] Lainez S, Schlingmann KP, van der Wijst J, Dworniczak B, van Zeeland F, Konrad M, et al. New TRPM6 missense mutations linked to hypomagnesemia with secondary hypocalcemia. Eur J Hum Genet 2014;22:497–504. [142] Zhao Z, Pei Y, Huang X, Liu Y, Yang W, Sun J, et al. Novel TRPM6 mutations in familial hypomagnesemia with secondary hypocalcemia. Am J Nephrol 2013;37:541–8. [143] Baglietto MG, Caridi G, Gimelli G, Mancardi M, Prato G, Ronchetto P, et al. RORB gene and 9q21.13 microdeletion: report on a patient with epilepsy and mild intellectual disability. Eur J Med Genet 2014;57:44–6. [144] Nair AV, Hocher B, Verkaart S, van Zeeland F, Pfab T, Slowinski T, et al. Loss of insulin-induced activation of TRPM6 magnesium channels results in impaired glucose tolerance during pregnancy. Proc Natl Acad Sci USA 2012;109:11324–9. [145] Gasperini RJ, Small DH. Neurodegeneration in familal amyloidotic polyneuropathy. Clin Exp Pharmacol Physiol 2012;39:680–3. [146] Chasman DI, Schurks M, Anttila V, de Vries B, Schminke U, Launer LJ, et al. Genome-wide association study reveals three susceptibility loci for common migraine in the general population. Nat Genet 2011;43:695–8. [147] An XK, Ma QL, Lin Q, Zhang XR, Lu CX, Qu HL. PRDM16 rs2651899 variant is a risk factor for Chinese common migraine patients. Headache 2014;53:1595–601. [148] Kurose M, Meng ID. Dry eye modifies the thermal and menthol responses in rat corneal primary afferent cool cells. J Neurophysiol 2013;110:495–504. [149] Kremeyer B, Lopera F, Cox JJ, Momin A, Rugiero F, Marsh S, et al. A gain-of-function mutation in TRPA1 causes familial episodic pain syndrome. Neuron 2010;66:671–80. [150] Wood H. Pain: new familial pain syndrome caused by TRPA1 mutation. Nat Rev Neurol 2010;6:412. [151] Nilius B, Appendino G, Owsianik G. The transient receptor potential channel TRPA1: from gene to pathophysiology. Pflugers Arch 2012;464:425–58. [152] Binder A, May D, Baron R, Maier C, Tolle TR, Treede RD, et al. Transient receptor potential channel polymorphisms are associated with the somatosensory function in neuropathic pain patients. PLoS One 2011;6:e17387. [153] May D, Baastrup J, Nientit MR, Binder A, Schuenke M, Baron R, et al. Differential expression and functionality of TRPA1 genetic variants in conditions of thermal stimulation. J Biol Chem 2012;287:27087–94. [154] Slaugenhaupt SA. The molecular basis of mucolipidosis type IV. Curr Mol Med 2002;2:445–50. [155] Zeevi DA, Frumkin A, Offen-Glasner V, Kogot-Levin A, Bach G. A potentially dynamic lysosomal role for the endogenous TRPML proteins. J Pathol 2009;219:153–62. [156] Bach G, Zeevi DA, Frumkin A, Kogot-Levin A. Mucolipidosis type IV and the mucolipins. Biochem Soc Trans 2010;38:1432–5. [157] Lloyd-Evans E, Platt FM. Lysosomal Ca(2+) homeostasis: role in pathogenesis of lysosomal storage diseases. Cell Calcium 2011;50:200–5. [158] Wakabayashi K, Gustafson AM, Sidransky E, Goldin E. Mucolipidosis type IV: an update. Mol Genet Metab 2011;104:206–13. [159] Dong XP, Wang X, Xu H. TRP channels of intracellular membranes. J Neurochem 2010;113:313–28. [160] Micsenyi MC, Dobrenis K, Stephney G, Pickel J, Vanier MT, Slaugenhaupt SA, et al. Neuropathology of the Mcoln1(−/−) knockout mouse model of mucolipidosis type IV. J Neuropathol Exp Neurol 2009;68:125–35. [161] Vergarajauregui S, Puertollano R. Mucolipidosis type IV: the importance of functional lysosomes for efficient autophagy. Autophagy 2008;4:832–4. [162] Kiselyov K, Colletti GA, Terwilliger A, Ketchum K, Lyons CW, Quinn J, et al. TRPML: transporters of metals in lysosomes essential for cell survival? Cell Calcium 2011;50:288–94. [163] Shen D, Wang X, Li X, Zhang X, Yao Z, Dibble S, et al. Lipid storage disorders block lysosomal trafficking by inhibiting a TRP channel and lysosomal calcium release. Nat Commun 2012;13:731. [164] Tang Y, Li H, Liu JP. Niemann-Pick disease type C: from molecule to clinic. Clin Exp Pharmacol Physiol 2012;37:132–40. [165] Di Palma F, Belyantseva I, Kim H, Vogt T, Kachar B, Noben-Trauth K. Mutations in Mcoln3 associated with deafness and pigmentation defects in varitint-waddler (Va) mice. Proc Natl Acad Sci USA 2002;99:14994–9. References 33

[166] Wu LJ, Sweet TB, Clapham DE. International Union of Basic and Clinical Pharmacology. LXXVI. Current progress in the mammalian TRP ion channel family. Pharmacol Rev 2010;62:381–404. [167] Dietrich A, Chubanov V, Kalwa H, Rost BR, Gudermann T. Cation channels of the transient receptor potential superfamily: their role in physiological and pathophysiological processes of smooth muscle cells. Pharmacol Ther 2006;112:744–60. [168] Wu G, Markowitz GS, Li L, D’Agati VD, Factor SM, Geng L, et al. Cardiac defects and renal failure in mice with targeted mutations in Pkd2. Nat Genet 2000;24:75–8. [169] Audrezet MP, Cornec-Le Gall E, Chen JM, Redon S, Quere I, Creff J, et al. Autosomal dominant polycystic kidney disease: comprehensive mutation analysis of PKD1 and PKD2 in 700 unrelated patients. Human Mutat 2012;33:1239–50. [170] Chapin HC, Caplan MJ. The cell biology of polycystic kidney disease. J Cell Biol 2010;191:701–10. [171] Patel A, Honore E. Polycystins and renovascular mechanosensory transduction. Nat Rev Nephrol 2010;6:530–8. [172] Takiar V, Caplan MJ. Polycystic kidney disease: pathogenesis and potential therapies. Biochim Biophys Acta 2011;1812:1337–43. [173] Chen XZ, Li Q, Wu Y, Liang G, Lara CJ, Cantiello HF. Submembraneous microtubule cytoskeleton: interaction of TRPP2 with the cell cytoskeleton. FEBS J 2008;275:4675–83. [174] Li X, Luo Y, Starremans PG, McNamara CA, Pei Y, Zhou J. Polycystin-1 and polycystin-2 regulate the cell cycle through the helix-loop-helix inhibitor Id2. Nat Cell Biol 2005;7:1102–12. [175] Benezra R. Polycystins: inhibiting the inhibitors. Nat Cell Biol 2005;7:1064–5. [176] Garcia-Gonzalo FR, Corbit KC, Sirerol-Piquer MS, Ramaswami G, Otto EA, Noriega TR, et al. A transition zone complex regulates mammalian ciliogenesis and ciliary membrane composition. Nat Genet 2011;43:776–84. [177] Mason SB, Lai X, Ringham HN, Bacallao RL, Harris PC, Witzmann FA, et al. Differential expression of renal proteins in a rodent model of Meckel syndrome. Nephron Exp Nephrol 2010;117:e31–8. [178] Bataille S, Demoulin N, Devuyst O, Audrezet MP, Dahan K, Godin M, et al. Association of PKD2 (Polycystin 2) mutations with left-right laterality defects. Am J Kidney Dis 2011;58:456–60. [179] Yoshiba S, Shiratori H, Kuo IY, Kawasumi A, Shinohara K, Nonaka S, et al. Cilia at the node of mouse embryos sense fluid flow for left-right determination via Pkd2. Science 2012;338:226–31. [180] Delling M, DeCaen PG, Doerner JF, Febvay S, Clapham DE. Primary cilia are specialized calcium signalling organelles. Nature 2013;504:311–14. [181] DeCaen PG, Delling M, Vien TN, Clapham DE. Direct recording and molecular identification of the calcium channel of primary cilia. Nature 2013;504:315–18. [182] Valente EM, Rosti RO, Gibbs E, Gleeson JG. Primary cilia in neurodevelopmental disorders. Nat Rev Neurol 2014;10:27–36. CHAPTER 3 The Role of TRPV1 in Acquired Diseases: Therapeutic Potential of TRPV1 Modulators Mahendra Bishnoi,1 Pragyanshu Khare,1 Kanthi K. Kondepudi,2 Louis S. Premkumar3,* 1Department of Nutritional Sciences and Technology, National Agri-Food Biotechnology Institute (NABI), SAS Nagar, India 2Department of Food Sciences and Technology, National Agri-Food Biotechnology Institute (NABI), SAS Nagar, India 3Department of Pharmacology, Southern Illinois University-School of Medicine, Springfield, Illinois, USA *Corresponding author: [email protected]

OUTLINE

Introduction 36 TRPV1 and Cancer 42 TRPV1 and Pain 36 TRPV1 and Cardiovascular System- Related Diseases 42 TRPV1 and Gastrointestinal System- Related Disease 37 TRPV1 and Metabolic Diseases 44 TRPV1 and Urinary System-Related TRPV1 and Other Disease Conditions 45 Diseases 38 Conclusions 46 TRPV1 and Central Nervous System- Acknowledgment 46 Related Diseases 39 References 46 TRPV1 and Respiratory System-Related Diseases 41

TRPV1, the first member of the transient receptor potential vanilloid (TRPV) family of ion channels was cloned in 1997 by David Julius and colleagues [1]. TRPV1 is a 95-kDa, 838 amino acid protein consisting of six transmembrane (TM) segments with a pore-forming loop between the fifth and sixth TM segments and intracellular N- and C-terminals. The stoichi- ometry of TRPV1 is considered to be a homo- or heterotetramer. Recently, using electron cryomicroscopy, the detailed structure and possible allosteric modulation during activation of mammalian TRPV1 channel have been determined. TRPV1 exhibits fourfold symmetry around a central ion pore formed by TM segments 5-6 (S5-S6) with an open extracellular “mouth” with a short selectivity filter. Subunit organization is influenced by interactions among amino-terminal ankyrin repeats present in cytoplasmic domains. The opening on activation is associated with major structural changes in the outer pore and a pronounced dilation of a hydrophobic constriction at the lower gate, suggesting a dual-gating mechanism. This allosteric coupling between upper and lower gates may account for the rich physiological modulations exhibited by TRPV1 [2,3]. TRPV1 is expressed in sensory and nonsensory neurons and in nonneuronal cells. Neurons expressing TRPV1 are small to medium diameter and peptidergic, which gives rise to unmyelinated C-fibers and thinly myelinated A-δ fibers. These are mainly expressed in a subset of dorsal root ganglion (DRG), trigeminal ganglion (TG), and nodose ganglion neurons [1,4]. TRPV1 is expressed on both the peripheral and central terminals of the sensory neurons [1,5,6]. TRPV1 is expressed in various nonneuronal cells [7] including mast cells [8], epithelial cells of urinary bladder [9], stomach [10], palate [11], and airway [12–14], skin epidermal keratinocytes [15], hematopoietic cells [16] and preadipocytes [17]. On the basis of its widespread tissue distribution, loss- or gain-of-function can be associated with a variety of acquired diseases. Such effects can be brought about by changes in transcriptional, translational, and posttranslational modifications. The channels can be robustly sensitized by phosphorylation and undergo desensitization on overactivation by exogenous and endogenous ligands. This chapter focuses on role of TRPV1 ion channel in acquired disorders and development of TRPV1 modulators as therapeutic agents.

TRPV1 AND PAIN

Preclinical research has identified TRPV1 as an important target for developing novel analgesics. TRPV1 gene knockout mice are partially devoid of the thermal inflammatory hyperalgesia [18,19]. TRPV1 plays a central role in mediating peripheral [20] and central sensitization [21]. TRPV1 is involved in triggering central sensitization during repetitive C-fiber firing by releasing neuropeptides and neurotransmitters, which results in activation of glia [22–24]. One of the mechanisms involved in the peripheral sensitization is due to phosphorylation of the receptor, which robustly potentiates the channel [25–27]. Both TRPV1 antagonists and agonists are capable of inducing pain relief. As expected, TRPV1 antagonists prevent the generation of receptor potential in response to temperature or endogenous and exogenous ligands. However, while using potent exogenous agonists, the generation of receptor potential and subsequent generation of the action potential are TRPV1 and Gastrointestinal System-Related Disease 37 prevented either by desensitization of the receptor or by the depolarization block of the nerve terminals [28–30]. Desensitization of TRPV1 receptor is dependent on the concentration of the agonist and the extent of calcium influx through the receptor [28,31–33]. Persistent activation of TRPV1 by low concentrations of agonists can cause nerve terminal depolarization by maintaining the sodium channels in an inactivated state, thereby preventing nociceptive transmission in the short term and enhancing calcium influx into the nerve terminal causing nerve terminal ablation in the long term [28,32,34,35]. Therefore, both peripheral and central nerve terminals at the spinal cord can be targeted to induce pain relief by TRPV1 agonists/ antagonists [28,32,34]. Intrathecal administration of resiniferatoxin (RTX) reduces inflammatory thermal hypersensitivity without altering acute thermal sensitivity [28,34]. Rats treated with systemic RTX (200 μg/kg, i.p.) were devoid of both nocifensive behavior (manifested as the guarding behavior of the injured paw) and the evoked pain (quantified as thermal hyperalgesia in the hot plate test) [28,34]. Furthermore, it has been found in immunostaining studies that following intrathecal administration of RTX, TRPV1 staining in the central nerve terminals was completely abolished, whereas the staining of the cell bodies in the DRG and the peripheral terminals in the skin were intact [32,36]. Based on these findings, it is proposed that sensory efferent functions that are dependent on CGRP and SP release from the peripheral nerve terminals would not be affected following intrathecal administration of RTX [34–36]. To test the effectiveness of intrathecal administration of RTX in treating cancer pain, a clinical trial has been initiated by National Institute of Dental and Craniofacial Research (NCT008041). TRPV1 agonists and antagonists have been demonstrated to be beneficial in different preclinical models of pain. These include pain associated with multiple forms of arthritis, cancer, chemotherapy, diabetic peripheral neuropathy, Herpes zoster infection, inflammatory bowel disease (IBD), visceral pain, dental pain, and migraine [25,37–48]. Several newer TRPV1 antagonists have been synthesized, characterized, and validated, and some of them have entered clinical trials for these conditions [20,49–51]. Unfortunately, hyperthermia was a major side effect encountered following administration of TRPV1 antagonists in humans [52–54]. This unexpected side effect has halted the clinical trials and has steered away the efforts of the major pharmaceutical companies from pursuing TRPV1 antagonists as analgesics.

TRPV1 AND GASTROINTESTINAL SYSTEM-RELATED DISEASE

TRPV1-like immunoreactivity has been observed in myenteric ganglia, intraganglionic fiber tracts, blood vessels supplying GI tract, muscle layers, and mucosa. Eighty percent of sensory fibers that project into the visceral mucosa and 40-60% of vagal afferents arising from nodose ganglia are TRPV1 positive and play a major role in the sensation of bloating/discomfort, pain, nausea, and satiety [55]. TRPV1 labeling has also been found in the parietal cells. Higher density and number of TRPV1-immunoreactive axons have been found in the distal colon and rectum, whereas they are barely expressed in the transverse and proximal colon [56]. Using retrograde labeling techniques, it has been shown that a substantial proportion of spinal and vagal jejuna and colonic afferents express TRPV1 and/or in association with neuronal nitric oxide synthase (NOS), CGRP, and SP [57,58]. TRPV1 mRNA has been detected in cells of the rat gastric wall and rat gastric epithelial cell lines (RGM-1) [59,60]. 38 3. THE ROLE OF TRPV1 IN ACQUIRED DISEASES

Presence of TRPV1 on afferents innervating the mucosa of the human esophagus, its increased expression in disease conditions, and activation by acidic pH renders it as a potential target for the treatment of gastroesophageal reflux disease (GERD) [61,62]. TRPV1 knockout mice develop a lesser extent of esophagitis after acid exposure as compared to wild-type controls [63]. Surprisingly, in clinical trials TRPV1 antagonists showed a limited efficacy profile in GERD patients [64] (ClinicalTrials.Gov identifier: D9127C00002). TRPV1-positive neurons mediate visceral pain in response to inflammation and noxious rectal distension [65–67]. TRPV1 inhibition using antagonists and silencing by RNA interference has been reported to ameliorate visceral pain in rats [66,68,69]. Increased TRPV1-immunoreactivity has been observed in colonic sensory afferents in patients with IBD, both Crohn’s disease and ulcerative colitis [70], and in rectal sensory fibers with rectal hypersensitivity and fecal urgency [71]. TRPV1 also appears to be involved in the inflammation and hyperalgesia associated with dextran sodium sulfate-induced experimental colitis [72]. A correlation has been described between the number of TRPV1-immunoreactive fibers in the rectosigmoid colon and the abdominal pain score in patients with irritable bowel syndrome [73]. TRPV1 antagonists prevent the development of visceral hypersensitivity initiated by acetic acid treatment during the neonatal period in a rat model of irritable bowel syndrome [74,75]. TRPV1 modulation by both agonist (capsaicin) and antagonist (YL1421) has shown beneficial effects against trinitrobenzene sulfonic acid-induced rat model of IBD [76,77]. The pancreas is innervated by sensory neurons that play a central role in pancreatitis- associated inflammatory pain [78]. Recent studies suggest that efferent function of primary sensory neurons of the pancreas contributes to inflammation by releasing pro-inflammatory agents that can activate/sensitize TRPV1 [79]. Blockade of TRPV1 by capsazepine significantly attenuated pain associated with experimental pancreatitis, suggesting that the antagonists of TRPV1 could be useful to reduce the inflammatory response and ameliorate the excruciating pain associated with pancreatitis [80]. Activation of TRPV1 mediates neurogenic inflammation in cerulein-induced pancreatitis via activation of the neurokinin receptor 1 [81]. Enhanced TRPV1 immunoreactivity is also observed in the colons of patients with Hirschsprung’s disease, characterized by bowel obstruction [82].

TRPV1 AND URINARY SYSTEM-RELATED DISEASES

Multiple reports have suggested the TRPV1 expression in neuronal and nonneuronal cells of the urinary system, including bladder, renal pelvis, ureter, and urethra [34,83]. Activation of TRPV1 by agonists and physiological stimuli (heat and low pH) cause calcium influx and ATP release in rat and human urothelial cells [83], suggesting the presence of functional TRPV1. In TRPV1 knockout animals and following chemical/surgical denervation of rat or human bladder leads to loss of TRPV1 function [9,76,84–87]. Recently, Birder and colleagues have shown enhanced TRPV1 expression and TRPV1-dependent ATP release in overactive bladder patients [88]. Intravesical administration of capsaicin or RTX increases bladder capacity and partially restores continence [89,90] in the patients with neurogenic detrusor overactivity disorder. Intravesical administration of RTX in patients with detrusor overactivity has been found to be effective and safe [91–94]. TRPV1 and Central Nervous System-Related Diseases 39

The role of TRPV1 in micturition reflex dysfunction is well established [95–98]. Intravesical administration of TRPV1 agonist (capsaicin and RTX)-induced desensitization or denervation therapy results in controlled micturition in both neurogenic and nonneurogenic cases of overactive bladder [95,99]. TRPV1 antagonists have beneficial effects against pain and inflammation associated with interstitial cystitis [97,100]. GRC-6211, an orally active TRPV1 antagonist, counteracts the bladder hyperactivity and pain induced by cystitis [97]. Intravesicular RTX has been found to be safe, but less tolerable in a prospective, double-blind and randomized clinical trial for the treatment of interstitial cystitis [101]. In mice, genetic manipulation of the TRPV1 gene has been shown to be effective in bladder reflex hyperactivity and the spinal c-fos overexpression in response to cystitis [67]. Interestingly, recent reports have pointed out that severity of cystitis can be positively correlated with expression levels of TRPM2 and TRPV2 but not with TRPV1 [102].

TRPV1 AND CENTRAL NERVOUS SYSTEM-RELATED DISEASES

There are several studies using RT-PCR, radioligand binding, in situ hybridization, and autoradiography techniques, suggesting the presence of TRPV1 mRNA and protein in hippocampus (CA1 and CA3 regions); dentate gyrus; thalamic and hypothalamic nuclei; cortical structures such as prefrontal cortex (PFC), somatosensory cortex, anterior cingulate cortex, and insular cortex; limbic structures including central amygdala, caudate putamen, and substantia nigra; locus coeruleus; periaqueductal gray (PAG); ventral tagmental area; cerebellum; nucleus of solitary tract; ventral medulla; and olfactory bulb [103–106]. Interestingly, TRPV1 channels have also been reported to be present on glial cells of the CNS [104,107]. Although, studies have documented the presence of TRPV1 by various methods, there is limited functional evidence by the way of recording TRPV1-mediated membrane currents from CNS neurons. A recent work relying on a powerful combination of reporter mice, in situ hybridization, electrophysiological recordings, and calcium imaging suggests that TRPV1 expression is restricted to very few brain regions, most notably the caudate nucleus of the hypothalamus [108]. Furthermore, a recent study shows convincingly TRPV1 expression in the second-order inhibitory neurons in the spinal dorsal horn [109]. At present, it is unclear how one can reconcile with these strikingly different findings. Clearly, additional research has to be carried out to explain the differences that have been noted between wild- type and TRPV1 knockout mice. TRPV1-mediated modulation in miniature excitatory postsynaptic currents have been observed in the spinal cord, striatum, hippocampus, substantia gelatinosa, PAG, medial preoptic nucleus, substantia nigra, and locus coeruleus. In the CA1 region of the hippocampus, anandamide, an endogenous cannabinoid and a TRPV1 receptor agonist, has been shown to increase paired-pulse depression and inhibit evoked excitatory synaptic transmission that could be reversed by the TRPV1 antagonist, capsazepine but not by cannabinoid receptor 1 (C131) antagonist, AM281 [110,111]. TRPV1 activation facilitates long-term potentiation (LTP) and suppresses long-term depression (LTD) in the hippocampus. Acute stress-induced suppression of LTP and augmentation of LTD are reversed by capsaicin application [112]. Application of capsaicin or 12-(S)-HPETE induces a form of LTD that could be blocked by capsazepine, which is absent in TRPV1 knockout mice [112,113]. 40 3. THE ROLE OF TRPV1 IN ACQUIRED DISEASES

TRPV1-mediated calcium influx leads to mitochondrial damage, apoptosis, and cell death in neurons as well as in glial cells via different molecular mechanisms [114–118]. Using a gerbil model of global transient ischemia and ouabain-induced excitotoxicity, researchers have shown that TPRV1 has a neuroprotective effect [106,119–121]. An anandamide uptake inhibitor, AM404, was able to significantly reverse the hyperkinetic movements associated with Huntington’s disease in a 3-nitropropionic acid-induced model with Huntington’s disease. Hyperkinetic movements were reversed by TRPV1 antagonist, capsazepine, suggesting the involvement of TRPV1 [121]. TRPV1 has been implicated in the process of epileptogenesis due to a higher level of expression in the cortex and hippocampus [122,123]. Reduced inhibition within neuronal networks causes hyperexcitability and can enhance seizure susceptibility [124]. During epilepsy, anandamide levels increase due to enhanced neuronal activity [125], and both exogenous and endogenous anandamide display a proconvulsant activity [126]. In contrast, recent reports implicate an anticonvulsant action of anandamide in kainic-acid-induced seizures [123,127]. TRPV1 has been associated with neuropsychiatric disorders and addiction. Higher expression of TRPV1 in PFC, dorsolateral columns of PAG, and ventral hippocampal regions suggests a link between TRPVI and anxiety/aversive (fear) behavior. Ablation of TRPV1 expressing neurons or blockade of TRPV1 receptors can induce anxiolytic behavior. Systemic administration of capsazepine induces anxiolytic-like effects in rats [128]. In addition, TRPV1 knockout mice exhibit reduced anxiety-like behavior and impaired fear conditioning [129]. It has been recently shown that capsazepine induces anxiolytic-like effects when injected into the dorsolateral PAG [130,131], ventral hippocampus [132], and ventral medial PFC [133]. These studies suggest a role of TRPV1 in anxiety and fear conditioning. Studies in dopamine receptor 3 (D3R) knockout mice have suggested that altered endocannabinoid and endovanilloid systems in these animals are responsible for the excitotoxic and anxiogenic effects [134]. Intraperitoneal injections of a TRPV1 agonist, olvanil, decreased the time spent in the open arms of the elevated plus maze [135]. Further, TRPV1 has a role in cocaine-, nicotine-, and ethanol-induced addictive behavior and related psychiatric changes [136,137]. Methamphetamine and morphine administration is associated with increased TRPV1 expression [138,139]. SB366791, a TRPV1 antagonist, prevented morphine-induced tolerance/withdrawal symptoms as well as thermal hyperalgesia [139]. Systemic administration of capsaicin causes a significant reduction in movement in both the horizontal (locomotion) and vertical (rearing) planes, which is reversed by the TRPV1 antagonist, capsazepine [116]. l-Dihydroxyphenylalanine (l-DOPA) treatment in reserpine-treated rats elicits high levels of motor activity in both the horizontal and vertical planes, which is reversed by capsaicin [116]. Furthermore, intranigral administration of 12-(S)-HPETE in rats triggers dopaminergic neuronal death, whereas coadministration of capsazepine prevents this effect [140]. Anandamide and 12-(S)-HPETE have been implicated in l-DOPA-induced dyskinesias and cell death in cultured dopamine neurons by a TRPV1-mediated mechanism [116,140–142]. All these studies suggest a link between TRPV1 and dopaminergic circuits, with a potential role in Parkinson’s disease and dyskinesias. TRPV1 has a role in dopamine- mediated hyperactivity and schizophrenia [119,143]. TRPV1 is involved in the activation of glia (astroglia and microglia). In TRPV1 knockout animals, capsaicin and Complete Freund’s Adjuvant (CFA) administration or sciatic nerve ligation exhibit reduced glial activation, as suggested by reduced ionized calcium-binding adapter molecule 1 (Iba1) and astrocytes glial fibrillary acidic protein immunostaining [39]. TRPV1 and Respiratory System-Related Diseases 41

The TRPV1 antagonism is associated with a reduction in microglial activation, decrease in the production of proinflammatory cytokines (TNFα, IL-1β, and IL-6), and increase in the production of the anti-inflammatory cytokine IL1-R in different brain regions, including spinal cord and hippocampus [144–146]. Capsazepine and Iodo-RTX can significantly reduce the generation of Reactive Oxygen Species (ROS) in microglia [147].

TRPV1 AND RESPIRATORY SYSTEM-RELATED DISEASES

TRPV1 immunoreactivity is present in the lungs (in sensory nerve fibers), extra and intrapulmonary airways (within and beneath the epithelium, around blood vessels, and within airway smooth muscles and alveoli), tracheal epithelium, mast cells, pleura, and nasal mucosa [148–152]. Capsaicin induces calcium influx, causes membrane depolarization, and generates action potentials, mucus secretion and cough reflex through activation of TRPV1 [153,154]. Asthma, obstructive sleep apnea [155], chronic cough [156,157], cystic fibrosis [158], airway hyper-responsiveness [159], and acute lung injury [160] are all associated with acidic pH of the lower airway. Impaired CO2 clearance from the lungs in chronic obstructive pulmonary disease or excessive lactic acid production caused by tissue ischemia or hypoxia can lead to the acidification of pulmonary tissues. TRPV1 is activated by low pH, which suggests its involvement in airway-related diseases. Activation of TRPV1 induces the release of neuropeptides [161–163]. Stimulation of vagal pulmonary sensory neurons by bradykinin (BK) can initiate cough reflex [164]. TRPV1 is expressed in sensory airway nerve fibers and plays an important role in the cough reflex [153,165–167]. BK- and proton-induced activation or sensitization of TRPV1 underlies this response. One of the common side effects of angiotensin-converting enzyme inhibitors during the treatment of hypertension is cough, which is due to accumulation of BK because of the inhibition of its degradation. TRPV1 antagonists (capsazepine, iodo-RTX. BCTC, and JNJ17203212) have been shown to block capsaicin, citric acid-, and antigen-induced cough responses [168–171]. Capsaicin-induced desensitization of TRPV1 inhibits allergenic bronchoconstriction in sensitized guinea pigs [172]. Capsaicin-induced bronchoconstriction is not significant in humans, possibly due to lack of specific innervation [173–175]. Activation of TRPV1 may be associated with airway inflammation in chronic respiratory diseases [176,177]. Expression of TRPV1 in rat DRG neurons projecting to the lung and pleura has been investigated [178]. TRPV1 is coexpressed with CGRP and SP in the secretory cells of the airway and lungs. TRPV1-induced calcium influx can also trigger the release of SP and CGRP from both peripheral and central nerve terminals of sensory neurons resulting in the development of “neurogenic inflammatory reaction” in the airways [179,180]. Airway neurogenic inflammation is associated with the pathogenesis of rhinosinusitis [181,182]. Intranasal administration of capsaicin induces desensitization of TRPV1 and provides relief in patients with vasomotor rhinitis [183]. TRPV1 is also reported to be involved in endoplasmic reticulum-mediated stress and cell death in human bronchial epithelial and alveolar cells [184]. Endogenous 13-S-hydroxyoctadecadienoic acid (13-S-HODE) produced in high concentrations during mitochondrial degradation in reticulocytes plays a role in asthma. Inhibition of TRPV1 attenuates 13-S-HODE produced in both a mouse model and human bronchial epithelial cells [185]. Mucus hypersecretion occurs in chronic obstructive pulmonary disease, and TRPV1 plays a role in mucus secretion [177,186,187]. 42 3. THE ROLE OF TRPV1 IN ACQUIRED DISEASES TRPV1 AND CANCER

Tumorigenesis is associated with altered expression of TRP channels (TRPV1, TRPV6, TRPC1, TRPC6, TRPM1, TRPM4, TRPM5, and TRPM8) [188]. There are two strategies of targeting TRPV1 channels for the development of drugs to treat cancer. First, activation of TRPV1 can lead to sustained increase in cytoplasmic concentrations of calcium resulting in cancerous cell death either by apoptosis or necrosis [189,190]. Second, in TRPV1 expressing cancerous cells, TRPV1 ligands can act as carriers for the toxic payload (in radiotherapy or chemotherapy), thereby enhancing the efficacy and localization of drugs [191]. Several types of cancers such as prostate, bladder, brain, mammary gland, pancreas, tongue, skin, liver, and colon show altered expression of TRPV1 in the affected tissue. Overexpression of TRPV1 and TRPV1-mediated CGRP release have been demonstrated in colon adenocarcinoma by immunohistochemical studies. TRPV1 expression has also been reported in Glioblastoma multiforme, a deadly form of brain cancer [192]. TRPV1 receptor activation mediates cell death, thereby inhibiting the development of brain carcinoma [193]. Some studies have suggested that TRPV1 receptor is expressed and functional in human prostate cells as well as in prostate cancer cell lines [194]. Capsaicin, methanandamide, and RTX induce a dose-dependent increase in the intracellular calcium concentration in prostate cells, which is reversed by capsazepine [195]. There is a significant decrease in TRPV1 expression in human urothelial cancer cells [118,196]. Intrinsic and extrinsic apoptotic pathways are triggered in epithelial cancer cells by the application of capsaicin [118]. Expression of TRPV1 is significantly upregulated in human pancreatic cancer cells [189]. A recent study has indicated that TRPV1 receptor expression has a role in the prediction of hepatocellular carcinoma [197]. Capsaicin-stimulated calcium influx and cell migration are enhanced by hepatocyte growth factor/scatter factor (HGF/SF). Thus, TRPV1 may mediate, in part, the action of HGF/SF in increasing intracellular calcium and promoting tumor invasion in hepatoblastoma. TRPV1 gene expression is also identified in uveal melanoma cells [198] and oral squamous cell carcinoma [198–200]. Recently published studies demonstrate the presence of TRPV1 in human and canine mammary cancer cells [201]. TRPV1- immunoreactivity has been shown to be increased in all layers of the tongue epithelium in patients with squamous cell carcinoma of the tongue [199]. The incidence of skin cancer is increased significantly in mice lacking TRPV1 [202].

TRPV1 AND CARDIOVASCULAR SYSTEM-RELATED DISEASES

TRPV1 has been proven to be important in modulating cardiovascular functions. TRPV1 is expressed in sensory neurons innervating cardiac tissue, especially spinal sympathetic afferent fibers with nerve endings in the heart [203]. These afferent nerve endings may transduce tissue hypoxia- and inflammation-induced cardiac pain. Multiple studies have demonstrated the importance of the activation of TRPV1 and subsequent release of neuropeptides (CGRP, SP, and BK) and neurotransmitters (NE and ACh) in cardioprotection [204–208]. Blood vessels are densely innervated by TRPV1 expressing C-fiber terminals [209,210]. TRPV1-mediated CGRP (a potent vasodilator) release plays an important role in maintaining the microvascular circulation. CGRP is colocalized with SP in the sensory ganglia and perivascular nerve fibers of the heart [180,211]. CGRP is released by TRPV1 and Cardiovascular System-Related Diseases 43

TRPV1 activation and reduces vascular resistance and coronary perfusion pressure [212,213]. The endogenous cannabinoid, anandamide, induces profound vasodilation by direct activation of TRPV1 and subsequent release of CGRP [214]. Involvement of neurogenic release of CGRP in ischemia/reperfusion is altered in TRPV1 knockout and capsazepine-treated animals suggesting a role of TRPV1 [215]. Capsaicin-sensitive nerve terminals also express NOS and produce NO, which can affect vascular tone [49,216]. Increased spontaneous firing activity of sensory neurons innervating the heart following coronary artery occlusion is significantly decreased by the treatment with iodo-RTX [217]. TRPV1-mediated sympathetic responsiveness is enhanced in rats with the femoral artery occlusion as compared with sham-operated animals [218]. Ischemia-induced firing of nerve fibers carrying cardiac ischemic pain may be because of the activation of TRPV1 by acidosis. Loss of TRPV1 expressing nerve fibers in diabetic neuropathy may be responsible for silent myocardial ischemia in patients with long-standing diabetes. One of the areas where TRPV1 is expressed other than its predominant expression in sensory neurons is vascular smooth muscle cells [219]. Altered TRPV1 activity contributes to the development of hypertension and plays a counterbalancing role in preventing salt-induced increases in blood pressure. Data collected from genetic and experimental models of hypertension suggest that the destruction of capsaicin-sensitive sensory neurons renders a rat salt sensitive and hampers blood pressure regulation [220]. TRPV1 dysfunction is implicated in renal hypertension [221]. Ablation of the TRPV1 gene exacerbates renal damage induced by deoxycorticosterone (DOCA)-mediated salt hypertension, indicating that TRPV1 may play a protective role against end-organ damage induced by hypertension [222]. These renoprotective effects seem to be closely related to the inhibition of inflammatory response mediated via TRPV1. Involvement of protein kinase C pathway has been suggested [222]. Renal inflammation (monocyte/macrophage and lymphocyte recruitment, proinflammatory cytokine and chemokine production, nuclear factor-kappa B (NF-κB) activity, and adhesion molecule expression) is aggravated in DOCA- salt hypertensive TRPV1 knockout mice, suggesting that TRPV1 antagonists may lessen renal injury in DOCA-salt hypertensive animals [223]. TRPV1 expression and function are upregulated during high salt intake in Dahl salt-resistant (DR) rats, which prevent salt-induced increases in blood pressure, whereas the expression and function of TRPV1 are impaired in Dahl salt-sensitive (DS) rats [123]. TRPV1 function is impaired in the kidneys of DS rats, altering renal hemodynamics, and contributes to hypertension [123]. Activation of TRPV1 expressed in sensory nerves innervating the renal pelvis enhances afferent renal nerve activity and diuresis [224]. TRPV1 agonists capsaicin, RTX, and an orally active agonist, SA13353, prevent the ischemia/reperfusion-induced acute kidney injury, and the effect is proposed to be associated with the inhibition of inflammatory response mediated by TRPV1 [225,226]. Furthermore, TRPV1 knockout mice exhibit reduced serum arginine-vasopression response to increased serum osmolarity following high salt intake [227]. TRPV1 has a potential role to play in diabetes- and obesity-related cardiac complications. TRPV1 activation by capsaicin protects against diabetes-induced endothelial dysfunction through a mechanism involving the PKA/UCP2 pathway [228]. Obesity-related release of endothelium-derived contracting factors is also mediated by TRPV1 activation [229]. 17β-Estradiol potentiates mesenteric relaxation in response to anandamide through a TRPV1-dependent mechanism [230]. Reports confirm the expression of TRPV1 in duramater, which is associated with migraine headaches [231,232]. Coexpression of CGRP with TRPV1-expressing neurons and the requirement of 44 3. THE ROLE OF TRPV1 IN ACQUIRED DISEASES

TRPV1 activation for its release suggests that TRPV1 plays an important role in migraine [49,233–236]. Migraine-associated symptoms like phonophobia, photophobia, and facial allodynia may be associated with sensitization of TRPV1 expressed in TG neurons [237].

TRPV1 AND METABOLIC DISEASES

Multiple TRP channels including TRPV1 have shown to be expressed in pancreatic β-cells and play a significant role in insulin secretion and glucose homeostasis [238,239]. TRPV1 is reported to be expressed in nerve fibers supplying the pancreas, pancreatic islet endocrine cells, and pancreatic beta cell lines (RIN and INS1) [79,240,241]. Activation of TRPV1 expressed in islet beta cells increases intracellular calcium and facilitates insulin secretion [240], and the effect is inhibited by capsazepine or by removal of extracellular calcium [240]. TRPV1-mediated release of insulin can sensitize TRPV1 [242,243]. Activation of TRPV1 at nerve terminals can release neuropeptides such as CGRP and SP. SP promotes neurogenic inflammation, thereby creating an autocrine feedback loop involving TRPV1 [79]. Involvement of TRPV1 expressed at sensory neurons in diabetes, glucose homeostasis, and insulin sensitivity has been investigated by using different animal models [79,244–248]. Role of TRPV1 has been studied in obesity and other metabolic disorders. Consumption of chili peppers and related dietary ingredients generate heat sensation and increase the energy expenditure [249–251]. There are multiple clinical trials, suggesting the beneficial antiobesity effects of active ingredients in chili peppers, capsaicin and capsinoids (nonpungent capsaicin analogues), by virtue of their action on fat oxidation, energy expenditure, and induction thermogenesis [45,250–253]. In addition, experimental evidences through animal studies suggest that capsaicinoids (capsaicin, dihydrocapsaicin) possess antiobesity properties [45,250,254]. In a clinical trial, a Japanese group has found that capsinoid ingestion increases energy expenditure through brown adipose tissue activation [255]. Although it is controversial, multiple groups have shown the presence of TRPV1 channels in 3T3-L1 preadipocytes and adipose tissue of mice (wild type and ob/ob) and in fat tissue of obese humans [33,256]. Capsaicin dose-dependently induced calcium influx and prevented adipogenesis in 3T3-L1 preadipocytes [33]. Long-term capsaicin consumption along with the high-fat diet prevented diet- induced obesity in WT mice, but not in TRPV1-null mice [33]. Capsaicin (0.014% in the diet) did not influence the caloric intake but significantly decreased the weight of the visceral fat- pad in rodents [257]. Researchers have used genomic and proteomic tools to understand the molecular mechanisms of the antiobesity effect of capsaicin by analyzing the gene and protein expression levels in adipose tissue of rats in response to capsaicin treatment [33,36,258]. Using TRPV1 knockout animals, the role of TRPV1 in adipose tissue inflammation and sensory neuronal dysfunction has been studied [259]. TRPV1 activation by capsaicin or its nonpungent analogs capsiates has been reported to have modulatory effects on the cellular signaling pathways involved in energy metabolism, preadipocyte differentiation and obesity-induced inflammation. Capsaicin can cause activation of the serine/threonine kinase, AMP-activated kinase and peroxisome proliferator-activated receptor gamma inactivation of NF-κB, up- regulation of uncoupling protein (UCP) 1, and UCP2 expression in brown and white adipose tissues. Further, capsaicin also causes activation of caspase-3, pro-apoptotic genes Bax and Bak, cleavage of poly (ADP-ribose) polymerase (PARP), and down-regulation of antiapoptotic TRPV1 and Other Disease Conditions 45 factor Bcl-2, hence resultant promotion of apoptosis [33,242,243,258,260]. CGRP, which has a role in antagonizing insulin release, is primarily secreted by TRPV1-expressing nerve terminals. Capsaicin administration to young rats destroyed capsaicin-sensitive afferent autonomic nerves following prolonged sensitization and activation, which resulted in persistently high-circulating CGRP levels. High CGRP levels cause insulin resistance and result in obesity in the long term [213,261,262]. TRPV1 channels are functionally expressed in the coronary vessels of lean and obese male Ossabaw miniature swines and mediate endothelium-dependent vasodilatation [263]. Endogenous TRPV1 ligand, N-oleoylethanolamide reduces food intake in wild-type, but not in TRPV1 knockout mice, which suggests a novel role for TRPV1 in appetite regulation [264,265]. Although several reports suggest that capsaicin has antiobesity effects, because of its pungency, it has limited use. The nonpungent CH-19 sweet pepper (the major source of natural capsinoids), therefore, might be an attractive alternative. Capsiate, dihydrocapsiate, and nor- dihydrocapsiate present in capsinoids in CH-19 sweet pepper are relatively less pungent than capsaicin or dihydrocapsaicin [266]. Increased body temperature and oxygen consumption was noticed with a single dose of CH-19 sweet pepper, whereas regular consumption reduced body weight and promoted fat oxidation [267,268]. Capsiate, similar to capsaicin, enhances the energy expenditure and raises the core body temperature [269,270]. Capsinoids increased fat oxidation and thermogenesis in wild-type mice, but not in TRPV1 knockout mice, suggesting the involvement of TRPV1 in this action [271]. Capsinoids also increase the levels of UCP in adipose tissue [260]. Capsiate is available as a weight loss product in the market in Japan and the United States [266]. There are reports contradicting the role of TRPV1 in obesity. In several studies, TRPV1 knockout mice showed decreased body fat [262]; therefore, the exact role of TRPV1 in the regulation of body weight remains to be explored.

TRPV1 AND OTHER DISEASE CONDITIONS

Immune cells express TRPV1, and capsaicin modulates various immune responses, but the mechanism is still unclear. TRPV1 is shown to have beneficial effects on several immune diseases, including autoimmune encephalomyelitis, chronic fatigue syndrome, autoimmune diabetes, fibromyalgia syndrome, liver injury [272–275], and dermatitis [276]. TRPV1 has been shown to be involved in the progression of allergic contact dermatitis (ACD), as shown by its protective effect against oxazolone or 2,4-dinitrofluorobenzene-induced murine ACD [276,277]. Recent studies have shown that TRPV1 has a significant role in atopic dermatitis [278]. In an animal model of atopic dermatitis, increased coexpression of gastrin releasing peptide with TRPV1 was detected [279]. Capsaicin-induced desensitization of TRPV1 relieves scratching behavior in different animal models of atopic dermatitis [280]. TRPV1 antagonist, PAC-14028 ameliorates the atopic dermatitis-like symptoms in the NC/nga mice [281]. Interestingly, in another model of atopic dermatitis (NC/Tnd mice), stimulation of TRPV1 was found to be beneficial [282]. TRPV1 agonists have been suggested as novel ad- juvants in contact hypersensitivity, as suggested by the favorable outcomes in fluorescein isothiocyanate-induced mouse contact hypersensitivity model [283]. TRPV1 neurons respond to different itch-producing agents and can be classified as a central integrator of the itch mechanism, although itch sensitivity is carried by a specific population 46 3. THE ROLE OF TRPV1 IN ACQUIRED DISEASES of neurons at the level of the spinal cord [128,284]. TRPV1 is considered as a candidate to explain the amiloride-insensitive salty taste because TRPV1 knockout mice showed diminished gustatory nerve responses [285]. Immunohistochemical studies reveal the expression of TRPV1 in the hair cells, cells of the organ of Corti, and spiral ganglion cells of the cochlea [286]. TRPV1 may be involved in the regulation of human hair growth and hearing. It has been shown that TRPV1 agonists can inhibit hair growth. TRPV1 knockout mice exhibit a significant delay in hair follicle cycling as compared to wild-type counterparts [287]. Capsaicin markedly suppresses hair shaft elongation, induces apoptosis and catagen regression, and promotes the intrafollicular cytokine production in organ-cultured human hair follicles [288]. In addition, after noise exposure TRPV1 expression levels increase in all cochlear regions [289]. A recent report has suggested that a TRPV1-mediated inflammatory process is central to temporary hearing loss [290]. Also, cisplatin-induced hearing loss could be reduced by short interfering RNA against TRPV1 [291]. TRPV1 is also expressed in human sebaceous glands and in the immortalized SZ95 sebocyte cell line [292].

CONCLUSIONS

In summary, TRPV1 plays a significant role in many disease processes. The preclinical research from academia and industries has provided proof of concept that targeting TRPV1 by agonists or antagonist may be a useful strategy in treating some of the disease conditions. Contradictory data from clinical studies are discouraging, given the side effects associated with agonists (pungency) and antagonists (hyperthermia). However, localized effect by topical application or slow intrathecal administration can be useful to target peripheral and central terminals for pain relief. Further, oral administration of these agents could produce local effects in the GI tract by acting on the nerve terminals or nonneuronal cells (enteroendocrine cells) by releasing peptide hormones in a paracrine and endocrine fashion. The controversy over the extent of TRPV1 expression and distribution in different tissues stands in the way of making significant progress. Although the expression of TRPV1 is confirmed in certain tissues, the required concentrations of exogenous and endogenous ligands to activate the receptor leads to speculation about the role of TRPV1 in several physiological and pathophysiological conditions.

Acknowledgment This work is supported by the grant from NIDA (DA028017).

References [1] Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, Julius D. The capsaicin receptor: a heat- activated ion channel in the pain pathway. Nature 1997;389:816–24. [2] Cao E, Liao M, Cheng Y, Julius D. TRPV1 structures in distinct conformations reveal activation mechanisms. Nature 2013;504:113–18. [3] Liao M, Cao E, Julius D, Cheng Y. Structure of the TRPV1 ion channel determined by electron cryo-microscopy. Nature 2013;504:107–12. REFERENCES 47

[4] Holzer P. Local effector functions of capsaicin-sensitive sensory nerve endings: involvement of tachykinins, calcitonin gene-related peptide and other neuropeptides. Neuroscience 1988;24:739–68. [5] Dinh Q, Groneberg DA, Peiser C, Mingomataj E, Joachim RA, Witt C, et al. Substance P expression in TRPV1 and trkA-positive dorsal root ganglion neurons innervating the mouse lung. Respir Physiol Neurobiol 2004;144:15–24. [6] Lazzeri M, Vannucchi MG, Zardo C, Spinelli M, Beneforti P, Turini D, et al. Immunohistochemical evidence of vanilloid receptor 1 in normal human urinary bladder. Eur Urol 2004;46:792–8. [7] Szallasi A, Gunthorpe MJ. Peripheral TRPV1 receptors as targets for drug development: new molecules and mechanisms. Curr Pharm Des 2008;14:32–41. [8] Bíró T, Maurer M, Modarres S, Lewin NE, Brodie C, Acs G, et al. Characterization of functional vanilloid receptors expressed by mast cells. Blood 1998;91:1332–40. [9] Birder LA, Kanai AJ, de Groat WC, Kiss S, Nealen ML, Burke NE, et al. Vanilloid receptor expression suggests a sensory role for urinary bladder epithelial cells. Proc Natl Acad Sci USA 2001;98:13396–401. [10] Nozawa Y, Nishihara K, Yamamoto A, Nakano M, Ajioka H, Matsuura N. Distribution and characterization of vanilloid receptors in the rat stomach. Neurosci Lett 2001;309:33–6. [11] Kido MA, Muroya H, Yamaza T, Terada Y, Tanaka T. Vanilloid receptor expression in the rat tongue and palate. J Dent Res 2003;82:393–7. [12] Agopyan N, Bhatti T, Yu S, Simon SA. Vanilloid receptor activation by 2- and 10 micron particles induces responses leading to apoptosis in human airway epithelial cells. Toxicol Appl Pharmacol 2003;192:21–35. [13] Ahmed MK, Takumida M, Ishibashi T, Hamamoto T, Hirakawa K. Expression of transient receptor potential vanilloid (TRPV) families 1, 2, 3 and 4 in the mouse olfactory epithelium. Rhinology 2009;47:242–7. [14] Leonelli M, Martins DO, Kihara AH, Britto LRG. Ontogenetic expression of the vanilloid receptors TRPV1 and TRPV2 in the rat retina. Int J Dev Neurosci 2009;27:709–18. [15] Southall MD, Li T, Gharibova LS, Pei Y, Nicol GD, Travers JB. Activation of epidermal vanilloid receptor-1 induces release of proinflammatory mediators in human keratinocytes. J Pharmacol Exp Ther 2003;304:217–22. [16] Heiner I, Eisfeld J, Halaszovich C, Wehage E, Jungling E, Zitt C, et al. Expression profile of the transient receptor potential (TRP) family in neutrophil granulocytes: evidence for currents through long TRP channel 2 induced by ADP-ribose and NAD. Biochem J 2003;371:1045–53. [17] Bishnoi M, Kondepudi KK, Gupta A, Karmase A, Boparai RK. Expression of multiple transient receptor potential channel genes in murine 3T3-L1 cell lines and adipose tissue. Pharmacol Rep 2013;65:751–5. [18] Caterina MJ, Leffler A, Malmberg AB, Martin WJ, Trafton J, Petersen-Zeitz KR, et al. Impaired nociception and pain sensation in mice lacking the capsaicin receptor. Science 2000;288:306–13. [19] Davis JB, Gray J, Gunthorpe MJ, Hatcher JP, Davey PT, Overend P, et al. Vanilloid receptor-1 is essential for inflammatory thermal hyperalgesia. Nature 2000;405:183–7. [20] Immke DC, Gavva NR. The TRPV1 receptor and nociception. Semin Cell Dev Biol 2006;17:582–91. [21] Palazzo E, Rossi F, de Novellis V, Maione S. Endogenous modulators of TRP channels. Curr Top Med Chem 2013;13:398–407. [22] Malin SA, Molliver DC, Koerber HR, Cornuet P, Frye R, Albers KM, et al. Glial cell line-derived neurotrophic factor family members sensitize nociceptors in vitro and produce thermal hyperalgesia in vivo. J Neurosci 2006;26:8588–99. [23] Watkins LR, Milligan ED, Maier SF. Glial proinflammatory cytokines mediate exaggerated pain states: implications for clinical pain. Adv Exp Med Biol 2003;521:1–21. [24] Milligan ED, Langer SJ, Sloane EM, He L, Wieseler-Frank J, O’Connor K, et al. Controlling pathological pain by adenovirally driven spinal production of the anti-inflammatory cytokine, interleukin-10. Eur J Neurosci 2005;21:2136–48. [25] Xu H, Blair NT, Clapham DE. Camphor activates and strongly desensitizes the transient receptor potential vanilloid subtype 1 channel in a vanilloid-independent mechanism. J Neurosci 2005;25:8924–37. [26] Jeske NA, Patwardhan AM, Ruparel NB, Akopian AN, Shapiro MS, Henry MA. A-kinase anchoring protein 150 controls protein kinase C-mediated phosphorylation and sensitization of TRPV1. Pain 2009;146:301–7. [27] Fischer MJ, Btesh J, McNaughton PA. Disrupting sensitization of transient receptor potential vanilloid subtype 1 inhibits inflammatory hyperalgesia. J Neurosci 2013;33:7407–14. [28] Jeffry JA, Yu S-Q, Sikand P, Parihar A, Evans MS, Premkumar LS. Selective targeting of TRPV1 expressing sensory nerve terminals in the spinal cord for long lasting analgesia. PLoS One 2009;4:e7021. [29] Sikand P, Premkumar LS. Potentiation of glutamatergic synaptic transmission by protein kinase C-mediated sensitization of TRPV1 at the first sensory synapse. J Physiol 2007;581:631–47. 48 3. THE ROLE OF TRPV1 IN ACQUIRED DISEASES

[30] Raisinghani M, Pabbidi RM, Premkumar LS. Activation of transient receptor potential vanilloid 1 (TRPV1) by resiniferatoxin. J Physiol 2005;567:771–86. [31] Premkumar LS, Ahern GP. Induction of vanilloid receptor channel activity by protein kinase C. Nature 2000;408:985–90. [32] Rutter AR, Ma QP, Leveridge M, Bonnert TP. Heteromerization and colocalization of TrpV1 and TrpV2 in mammalian cell lines and rat dorsal root ganglia. Neuroreport 2005;16:1735–9. [33] Donnerer J, Liebmann I, Schicho R. Differential regulation of 3-beta-hydroxysteroid dehydrogenase and vanilloid receptor TRPV1 mRNA in sensory neurons by capsaicin and NGF. Pharmacology 2005;73:97–101. [34] Bishnoi M, Bosgraaf CA, Abooj M, Zhong L, Premkumar LS. Streptozotocin-induced early thermal hyperalgesia is independent of glycemic state of rats: role of transient receptor potential vanilloid 1 (TRPV1) and inflammatory mediators. Mol Pain 2011;7:52. [35] Brown DC, Iadarola MJ, Perkowski SZ, Erin H, Shofer F, Laszlo KJ, et al. Physiologic and antinociceptive effects of intrathecal resiniferatoxin in a canine bone cancer model. Anesthesiology 2005;103:1052–9. [36] Amaya F, Shimosato G, Nagano M, Ueda M, Hashimoto S, Tanaka Y, et al. NGF and GDNF differentially regulate TRPV1 expression that contributes to development of inflammatory thermal hyperalgesia. Eur J Neurosci 2004;20:2303–10. [37] Engler A, Aeschlimann A, Simmen BR, Michel BA, Gay RE, Gay S, et al. Expression of transient receptor potential vanilloid 1 (TRPV1) in synovial fibroblasts from patients with osteoarthritis and rheumatoid arthritis. Biochem Biophys Res Commun 2007;359:884–8. [38] Fernihough J, Gentry C, Bevan S, Winter J. Regulation of calcitonin gene-related peptide and TRPV1 in a rat model of osteoarthritis. Neurosci Lett 2005;388:75–80. [39] Chen Y, Willcockson HH, Valtschanoff JG. Influence of the vanilloid receptor TRPV1 on the activation of spinal cord glia in mouse models of pain. Exp Neurol 2009;220:383–90. [40] Park C-K, Kim MS, Fang Z, Li HY, Jung SJ, Choi S-Y, et al. Functional expression of thermo-transient receptor potential channels in dental primary afferent neurons: implications for tooth pain. J Biol Chem 2006;281:17304–11. [41] Chaudhari N, Roper SD. The cell biology of taste. J Cell Biol 2010;190:285–96. [42] Kim HY, Chung G, Jo HJ, Kim YS, Bae YC, Jung SJ, et al. Characterization of dental nociceptive neurons. J Dent Res 2011;90:771–6. [43] Breivik TR, Gundersen Y, Gjermo P, Fristad I, Opstad PK. Systemic chemical desensitization of peptidergic sensory neurons with resiniferatoxin inhibits experimental periodontitis. Open Dent J 2011;5:1–6. [44] McNamara FN, Randall A, Gunthorpe MJ. Effects of piperine, the pungent component of black pepper, at the human vanilloid receptor (TRPV1). Br J Pharmacol 2005;144:781–90. [45] Hu HZ, Gu Q, Wang C, Colton CK, Tang J, Kinoshita-Kawada M, et al. 2-Aminoethoxydiphenyl borate is a common activator of TRPV1, TRPV2, and TRPV3. J Biol Chem 2004;279:35741–8. [46] Ahern GP, Wang X, Miyares RL. Polyamines are potent ligands for the capsaicin receptor TRPV1. J Biol Chem 2006;281:8991–5. [47] Cuypers E, Yanagihara A, Rainier JD, Tytgat J. TRPV1 as a key determinant in ciguatera and neurotoxic shellfish poisoning. Biochem Biophys Res Commun 2007;361:214–17. [48] Siemens J, Zhou S, Piskorowski R, Nikai T, Lumpkin EA, Basbaum AI, et al. Spider toxins activate the capsaicin receptor to produce inflammatory pain. Nature 2006;444:208–12. [49] Szallasi A, Cortright DN, Blum CA, Eid SR. The vanilloid receptor TRPV1: 10 years from channel cloning to antagonist proof-of-concept. Nat Rev Drug Discov 2007;6:357–72. [50] Bishnoi M, Premkumar LS. Possible consequences of blocking transient receptor potential vanilloid 1. Curr Pharm Biotechnol 2011;12:102–14. [51] Khairatkar-Joshi N, Szallasi A. TRPV1 antagonists: the challenges for therapeutic targeting. Trends Mol Med 2009;15:14–22. [52] Jung J, Lee S-Y, Hwang SW, Cho H, Shin J, Kang Y-S, et al. Agonist recognition sites in the cytosolic tails of vanilloid receptor 1. J Biol Chem 2002;277:44448–54. [53] Chou MZ, Mtui T, Gao Y-D, Kohler M, Middleton RE. Resiniferatoxin binds to the capsaicin receptor (TRPV1) near the extracellular side of the S4 transmembrane domain. Biochemistry 2004;43:2501–11. [54] Giannantoni A, Mearini E, Di Stasi SM, Costantini E, Zucchi A, Mearini L, et al. New therapeutic options for refractory neurogenic detrusor overactivity. Minerva Urol Nefrol 2004;56:79–87. [55] Yamakuni H, Sawai-Nakayama H, Imazumi K, Maeda Y, Matsuo M, Manda T, et al. Resiniferatoxin antagonizes cisplatin-induced emesis in dogs and ferrets. Eur J Pharmacol 2002;442:273–8. REFERENCES 49

[56] Matsumoto K, Kurosawa E, Terui H, Hosoya T, Tashima K, Murayama T, et al. Localization of TRPV1 and contractile effect of capsaicin in mouse large intestine: high abundance and sensitivity in rectum and distal colon. Am J Physiol 2009;297:G348–60. [57] Tan LL, Bornstein JC, Anderson CR. Distinct chemical classes of medium-sized transient receptor potential channel vanilloid 1-immunoreactive dorsal root ganglion neurons innervate the adult mouse jejunum and colon. Neuroscience 2008;156:334–43. [58] Tan LL, Bornstein JC, Anderson CR. Neurochemical and morphological phenotypes of vagal afferent neurons innervating the adult mouse jejunum. Neurogastroenterol Motil 2009;21:994–1001. [59] Geppetti P, Trevisani M. Activation and sensitization of the vanilloid receptor: role in gastrointestinal inflammation and function. Br J Pharmacol 2004;141:1313–20. [60] Kato S, Aihara E, Nakamura A, Xin H, Matsui H, Kohama K, et al. Expression of vanilloid receptors in rat gastric epithelial cells: role in cellular protection. Biochem Pharmacol 2003;66:1115–21. [61] Matthews PJ, Aziz Q, Facer P, Davis JB, Thompson DG, Anand P. Increased capsaicin receptor TRPV1 nerve fibers in the inflamed human esophagus. Eur J Gastroenterol Hepatol 2004;16:897–902. [62] Bhat YM, Bielefeldt K. Capsaicin receptor (TRPV1) and non-erosive reflux disease. Eur J Gastroenterol Hepatol 2006;18:263–70. [63] Fujino K, de la Fuente SG, Takami Y, Takahashi T, Mantyh CR. Attenuation of acid induced oesophagitis in VR-1 deficient mice. Gut 2006;55:34–40. [64] Krarup AL, Ny L, Gunnarsson J, Hvid-Jensen F, Zetterstrand S, Simren M, et al. Randomized clinical trial: inhibition of the TRPV1 system in patients with nonerosive gastroesophageal reflux disease and a partial response to PPI treatment is not associated with analgesia to esophageal experimental pain. Scand J Gastroenterol 2013;48:274–84. [65] Spencer NJ, Kerrin A, Singer CA, Hennig GW, Gerthoffer WT, McDonnell O. Identification of capsaicin- sensitive rectal mechanoreceptors activated by rectal distension in mice. Neuroscience 2008;153:518–34. [66] Jones RCW, Xu L, Gebhart GF. The mechanosensitivity of mouse colon afferent fibers and their sensitization by inflammatory mediators require transient receptor potential vanilloid 1 and acid-sensing ion channel 3. J Neurosci 2005;25:10981–9. [67] Wang Z-Y, Wang P, Merriam FV, Bjorling DE. Lack of TRPV1 inhibits cystitis-induced increased mechanical sensitivity in mice. Pain 2008;139:158–67. [68] Christoph T, Grunweller A, Mika J, Schafer MKH, Wade EJ, Weihe E, et al. Silencing of vanilloid receptor TRPV1 by RNAi reduces neuropathic and visceral pain in vivo. Biochem Biophys Res Commun 2006;350:238–43. [69] Sugiura T, Bielefeldt K, Gebhart GF. Mouse colon sensory neurons detect extracellular acidosis via TRPV1. Am J Physiol Cell Physiol 2007;292:C1768–74. [70] Yiangou Y, Facer P, Dyer NHC, Chan CLH, Knowles C, Williams NS, et al. Vanilloid receptor 1 immunoreactivity in inflamed human bowel. Lancet 2001;357:1338–9. [71] Chan CLH, Facer P, Davis JB, Smith GD, Egerton J, Bountra C, et al. Sensory fibres expressing capsaicin receptor TRPV1 in patients with rectal hypersensitivity and faecal urgency. Lancet 2003;361:385–91. [72] Eijkelkamp N, Kavelaars A, Elsenbruch S, Schedlowski M, Holtmann G, Heijnen CJ. Increased visceral sensitivity to capsaicin after DSS-induced colitis in mice: spinal cord c-Fos expression and behavior. Am J Physiol Gastrointest Liver Physiol 2007;293:G749–57. [73] Akbar A, Yiangou Y, Facer P, Walters JRF, Anand P, Ghosh S. Increased capsaicin receptor TRPV1-expressing sensory fibres in irritable bowel syndrome and their correlation with abdominal pain. Gut 2008;57:923–9. [74] Winston J, Shenoy M, Medley D, Naniwadekar A, Pasricha PJ. The vanilloid receptor initiates and maintains colonic hypersensitivity induced by neonatal colon irritation in rats. Gastroenterology 2007;132:615–27. [75] Keszthelyi D, Troost FJ, Jonkers DM, Helyes Z, Hamer HM, Ludidi S, et al. Alterations in mucosal neuropeptides in patients with irritable bowel syndrome and ulcerative colitis in remission: a role in pain symptom generation? Eur J Pain 2013;17:1299–306. [76] Goso C, Evangelista S, Tramontana M, Manzini S, Blumberg PM, Szallasi A. Topical capsaicin administration protects against trinitrobenzene sulfonic acid-induced colitis in the rat. Eur J Pharmacol 1993;249:185–90. [77] Miranda A, Nordstrom E, Mannem A, Smith C, Banerjee B, Sengupta JN. The role of transient receptor potential vanilloid 1 in mechanical and chemical visceral hyperalgesia following experimental colitis. Eur J Neurosci 2007;25:1021–32. [78] Liddle RA. The role of transient receptor potential vanilloid 1 (TRPV1) channels in pancreatitis. Biochim Biophys Acta 2007;1772:869–78. [79] Razavi R, Chan Y, Afifiyan FN, Liu XJ, Wan X, Yantha J, et al. TRPV1 positive sensory neurons control β cell stress and islet inflammation in autoimmune diabetes. Cell 2006;127:1123–35. 50 3. THE ROLE OF TRPV1 IN ACQUIRED DISEASES

[80] Nilius B, Owsianik G, Voets T, Peters JA. Transient receptor potential cation channels in disease. Physiol Rev 2007;87:165–217. [81] Hutter MM, Wick EC, Day AL, Maa J, Zerega EC, Richmond AC, et al. Transient receptor potential vanilloid (TRPV-1) promotes neurogenic inflammation in the pancreas via activation of the neurokinin-1 receptor (NK-1R). Pancreas 2005;30:260–5. [82] Facer P, Knowles CH, Tam PKH, Ford AP, Dyer N, Baecker PA, et al. Novel capsaicin (VR1) and purinergic (P2X3) receptors in Hirschsprung’s intestine. J Pediatr Surg 2001;36:1679–84. [83] Avelino A, Charrua A, Frias B, Cruz C, Boudes M, de Ridder D, et al. Transient receptor potential channels in bladder function. Acta Physiol (Oxf) 2013;207:110–22. [84] Szallasi A, Conte B, Goso C, Blumberg PM, Manzini S. Vanilloid receptors in the urinary bladder: regional distribution, localization on sensory nerves, and species-related differences. Naunyn Schmiedebergs Arch Pharmacol 1993;347:624–9. [85] Apostolidis A, Brady CM, Yiangou Y, Davis J, Fowler CJ, Anand P. Capsaicin receptor TRPV1 in urothelium of neurogenic human bladders and effect of intravesical resiniferatoxin. Urology 2005;65:400–5. [86] Birder LA, Nakamura Y, Kiss S, Nealen ML, Barrick S, Kanai AJ, et al. Altered urinary bladder function in mice lacking the vanilloid receptor TRPV1. Nat Neurosci 2002;5:856–60. [87] Dinis P, Charrua A, Avelino A, Cruz F. Intravesical resiniferatoxin decreases spinal-fos expression and increases bladder volume to reflex micturition in rats with chronic inflamed urinary bladders. BJU Int 2004;94:153–7. [88] Birder LA, Wolf-Johnston AS, Sun Y, Chai TC. Alteration in TRPV1 and muscarinic (M3) receptor expression and function in idiopathic overactive bladder urothelial cells. Acta Physiol 2013;207:123–9. [89] Lazzeri M, Beneforti P, Turini D. Urodynamic effects of intravesical resiniferatoxin in humans: preliminary results in stable and unstable detrusor. J Urol 1997;158:2093–6. [90] Silva C, Silva Jo, Castro H, Reis F, Dinis P, Avelino AN, et al. Bladder sensory desensitization decreases urinary urgency. BMC Urol 2007;7:9. [91] Silva C, Avelino A, Souto-Moura C, Cruz F. A light and electron-microscopic histopathological study of human bladder mucosa after intravesical resiniferatoxin application. BJU Int 2001;88:355–60. [92] Silva C, Rio ME, Cruz F. Desensitization of bladder sensory fibers by intravesical resiniferatoxin, a capsaicin analog: long-term results for the treatment of detrusor hyperreflexia. Eur Urol 2000;38:444–52. [93] Silva C, Silva Jo, Ribeiro MJ, Avelino A, Cruz F. Urodynamic effect of intravesical resiniferatoxin in patients with neurogenic detrusor overactivity of spinal origin: results of a double-blind randomized placebo- controlled trial. Eur Urol 2005;48:650–5. [94] Watanabe T, Yokoyama T, Sasaki K, Nozaki K, Ozawa H, Kumon H. Intravesical resiniferatoxin for patients with neurogenic detrusor overactivity. Int J Urol 2004;11:200–5. [95] Avelino A, Cruz F. TRPV1 (vanilloid receptor) in the urinary tract: expression, function and clinical applications. Naunyn Schmiedebergs Arch Pharmacol 2006;373:287–99. [96] Juszczak K, Thor PJ. The basic neurophysiologic concept of lower urinary tract function—the role of vanilloid TRPV1 receptors of urinary bladder afferent nerve endings. Adv Clin Exp Med 2011;21:417. [97] Charrua A, Cruz CID, Narayanan S, Gharat L, Gullapalli S, Cruz F, et al. GRC-6211, a new oral specific TRPV1 antagonist, decreases bladder overactivity and noxious bladder input in cystitis animal models. J Urol 2009;181:379–86. [98] Kitagawa Y, Wada M, Kanehisa T, Miyai A, Usui K, Maekawa M, et al. JTS-653 blocks afferent nerve firing and attenuates bladder overactivity without affecting normal voiding function. J Urol 2013;189:1137–46. [99] Szallasi A, Fowler CJ. After a decade of intravesical vanilloid therapy: still more questions than answers. Lancet Neurol 2002;1:167–72. [100] Sculptoreanu A, de Groat WC, Tony Buffington CA, Birder LA. Abnormal excitability in capsaicin-responsive DRG neurons from cats with feline interstitial cystitis. Exp Neurol 2005;193:437–43. [101] Chen TYH, Corcos J, Camel M, Ponsot Y. Prospective, randomized, double-blind study of safety and tolerability of intravesical resiniferatoxin (RTX) in interstitial cystitis (IC). Int Urogynecol J 2005;16:293–7. [102] Homma Y, Nomiya A, Tagaya M, Oyama T, Takagaki K, Nishimatsu H, et al. Increased mRNA expression of genes involved in pronociceptive inflammatory reactions in bladder tissue of interstitial cystitis. J Urol 2013;190:1925–31. [103] Gibson HE, Edwards JG, Page RS, Van Hook MJ, Kauer JA. TRPV1 channels mediate long-term depression at synapses on hippocampal interneurons. Neuron 2008;57:746–59. [104] Szallasi A, Di Marzo V. New perspectives on enigmatic vanilloid receptors. Trends Neurosci 2000;23:491–7. [105] Steenland HW, Ko SW, Wu L-J, Zhuo M. Hot receptors in the brain. Mol Pain 2006;2:34. REFERENCES 51

[106] Lewinter RD, Skinner K, Julius D, Basbaum AI. Immunoreactive TRPV-2 (VRL-1), a capsaicin receptor homolog, in the spinal cord of the rat. J Comp Neurol 2004;470:400–8. [107] Al-Hayani A, Wease KN, Ross RA, Pertwee RG, Davies SN. The endogenous cannabinoid anandamide activates vanilloid receptors in the rat hippocampal slice. Neuropharmacology 2001;41:1000–5. [108] Cavanaugh DJ, Chesler AT, Jackson AC, Sigal YM, Yamanaka H, Grant R, et al. Trpv1 reporter mice reveal highly restricted brain distribution and functional expression in arteriolar smooth muscle cells. J Neurosci 2011;31:5067–77. [109] Kim YH, Back SK, Davies AJ, Jeong H, Jo HJ, Chung G, et al. TRPV1 in GABAergic interneurons mediates neuropathic mechanical allodynia and disinhibition of the nociceptive circuitry in the spinal cord. Neuron 2012;74:640–7. [110] Hajos N, Freund TF. Distinct cannabinoid sensitive receptors regulate hippocampal excitation and inhibition. Chem Phys Lipids 2002;121:73–82. [111] Li H-B, Mao R-R, Zhang J-C, Yang Y, Cao J, Xu L. Antistress effect of TRPV1 channel on synaptic plasticity and spatial memory. Biol Psychiatry 2008;64:286–92. [112] Marinelli S, Vaughan CW, Christie MJ, Connor M. Capsaicin activation of glutamatergic synaptic transmission in the rat locus coeruleus in vitro. J Physiol 2002;543:531–40. [113] Grueter BA, Brasnjo G, Malenka RC. Postsynaptic TRPV1 triggers cell type-specific long-term depression in the nucleus accumbens. Nat Neurosci 2010;13:1519–25. [114] Sappington RM, Sidorova T, Long DJ, Calkins DJ. TRPV1: contribution to retinal ganglion cell apoptosis and increased intracellular Ca2+ with exposure to hydrostatic pressure. Invest Ophthalmol Vis Sci 2009;50:717–28. [115] Kim SR, Kim SU, Oh U, Jin BK. Transient receptor potential vanilloid subtype 1 mediates microglial cell death in vivo and in vitro via Ca2+-mediated mitochondrial damage and cytochrome c release. J Immunol 2006;177:4322–9. [116] Kim SR, Lee DY, Chung ES, Oh UT, Kim SU, Jin BK. Transient receptor potential vanilloid subtype 1 mediates cell death of mesencephalic dopaminergic neurons in vivo and in vitro. J Neurosci 2005;25:662–71. [117] Shirakawa H, Yamaoka T, Sanpei K, Sasaoka H, Nakagawa T, Kaneko S. TRPV1 stimulation triggers apoptotic cell death of rat cortical neurons. Biochem Biophys Res Commun 2008;377:1211–15. [118] Amantini C, Ballarini P, Caprodossi S, Nabissi M, Morelli MB, Lucciarini R, et al. Triggering of transient receptor potential vanilloid type 1 (TRPV1) by capsaicin induces Fas/CD95-mediated apoptosis of urothelial cancer cells in an ATM-dependent manner. Carcinogenesis 2009;30:1320–9. [119] Pegorini S, Zani A, Braida D, Guerini-Rocco C, Sala M. Vanilloid VR1 receptor is involved in rimonabant- induced neuroprotection. Br J Pharmacol 2006;147:552–9. [120] Veldhuis WB, Van der Stelt M, Wadman MW, Van Zadelhoff G, Maccarrone M, Fezza F, et al. Neuroprotection by the endogenous cannabinoid anandamide and arvanil against in vivo excitotoxicity in the rat: role of vanilloid receptors and lipoxygenases. J Neurosci 2003;23:4127–33. [121] Lastres-Becker I, De Miguel R, De Petrocellis L, Makriyannis A, Di Marzo V, Fernandez Ruiz J. Compounds acting at the endocannabinoid and/or endovanilloid systems reduce hyperkinesia in a rat model of Huntington’s disease. J Neurochem 2003;84:1097–109. [122] Sun FJ, Guo W, Zheng DH, Zhang CQ, Li S, Liu SY, et al. Increased expression of TRPV1 in the cortex and hippocampus from patients with mesial temporal lobe epilepsy. J Mol Neurosci 2013;49:182–93. [123] Fu M, Xie Z, Zuo H. TRPV1: a potential target for antiepileptogenesis. Med Hypotheses 2009;73:100–2. [124] Steffens M, Schulze-Bonhage A, Surges R, Feuerstein TJ. Fatty acid amidohydrolase in human neocortex— activity in epileptic and non-epileptic brain tissue and inhibition by putative endocannabinoids. Neurosci Lett 2005;385:13–17. [125] Clement AB, Hawkins EG, Lichtman AH, Cravatt BF. Increased seizure susceptibility and proconvulsant activity of anandamide in mice lacking fatty acid amide hydrolase. J Neurosci 2003;23:3916–23. [126] Karanian DA, Karim SL, Wood JT, Williams JS, Lin S, Makriyannis A, et al. Endocannabinoid enhancement protects against kainic acid-induced seizures and associated brain damage. J Pharmacol Exp Ther 2007;322:1059–66. [127] Romigi A, Bari M, Placidi F, Marciani MG, Malaponti M, Torelli F, et al. Cerebrospinal fluid levels of the endocannabinoid anandamide are reduced in patients with untreated newly diagnosed temporal lobe epilepsy. Epilepsia 2010;51:768–72. [128] Patapoutian A, Tate S, Woolf CJ. Transient receptor potential channels: targeting pain at the source. Nat Rev Drug Discov 2009;8:55–68. 52 3. THE ROLE OF TRPV1 IN ACQUIRED DISEASES

[129] Rubino T, Realini N, Castiglioni C, Guidali C, Vigano D, Marras E, et al. Role in anxiety behavior of the endocannabinoid system in the prefrontal cortex. Cereb Cortex 2008;18:1292–301. [130] Campos AC, Guimarães FS. Evidence for a potential role for TRPV1 receptors in the dorsolateral periaqueductal gray in the attenuation of the anxiolytic effects of cannabinoids. Prog Neuropsychopharmacol Biol Psychiatry 2009;33:1517–21. [131] Santos CJPA, Stern CAJ, Bertoglio LJ. Attenuation of anxiety-related behaviour after the antagonism of transient receptor potential vanilloid type 1 channels in the rat ventral hippocampus. Behav Pharmacol 2008;19:357–60. [132] Aguiar DC, Terzian ALB, Guimaraes FS, Moreira FcA. Anxiolytic-like effects induced by blockade of transient receptor potential vanilloid type 1 (TRPV1) channels in the medial prefrontal cortex of rats. Psychopharmacology 2009;205:217–25. [133] Micale V, Cristino L, Tamburella A, Petrosino S, Leggio GM, Drago F, et al. Altered responses of dopamine D3 receptor null mice to excitotoxic or anxiogenic stimuli: possible involvement of the endocannabinoid and endovanilloid systems. Neurobiol Dis 2009;36:70–80. [134] Kim SR, Bok E, Chung YC, Chung ES, Jin BK. Interactions between CB1 receptors and TRPV1 channels mediated by 12-HPETE are cytotoxic to mesencephalic dopaminergic neurons. Br J Pharmacol 2008;155:253–64. [135] Kasckow JW, Mulchahey JJ, Geracioti Jr TD. Effects of the vanilloid agonist olvanil and antagonist capsazepine on rat behaviors. Prog Neuropsychopharmacol Biol Psychiatry 2004;28:291–5. [136] Blednov YA, Harris RA. Deletion of vanilloid receptor (TRPV1) in mice alters behavioral effects of ethanol. Neuropharmacology 2009;56:814–20. [137] Hayase T. Differential effects of TRPV1 receptor ligands against nicotine-induced depression-like behaviors. BMC Pharmacol 2011;11:6. [138] Tian Y-H, Lee S-Y, Kim H-C, Jang C-G. Repeated methamphetamine treatment increases expression of TRPV1 mRNA in the frontal cortex but not in the striatum or hippocampus of mice. Neurosci Lett 2010;472:61–4. [139] Chen Y, Geis C, Sommer C. Activation of TRPV1 contributes to morphine tolerance: involvement of the mitogen-activated protein kinase signaling pathway. J Neurosci 2008;28:5836–45. [140] Morgese MG, Cassano T, Cuomo V, Giuffrida A. Anti-dyskinetic effects of cannabinoids in a rat model of Parkinson’s disease: role of CB-1 and TRPV1 receptors. Exp Neurol 2007;208:110–19. [141] Lee J, Di Marzo V, Brotchie JM. A role for vanilloid receptor 1 (TRPV1) and endocannabinnoid signalling in the regulation of spontaneous and l-DOPA induced locomotion in normal and reserpine-treated rats. Neuropharmacology 2006;51:557–65. [142] Tzavara ET, Li DL, Moutsimilli L, Bisogno T, Di Marzo V, Phebus LA, et al. Endocannabinoids activate transient receptor potential vanilloid 1 receptors to reduce hyperdopaminergia-related hyperactivity: therapeutic implications. Biol Psychiatry 2006;59:508–15. [143] Newson P, Lynch-Frame A, Roach R, Bennett S, Carr V, Chahl LA. Intrinsic sensory deprivation induced by neonatal capsaicin treatment induces changes in rat brain and behaviour of possible relevance to schizophrenia. Br J Pharmacol 2005;146:408–18. [144] Sappington RM, Calkins DJ. Contribution of TRPV1 to microglia-derived IL-6 and NFκB translocation with elevated hydrostatic pressure. Invest Ophthalmol Vis Sci 2008;49:3004–17. [145] Marchalant Y, Brothers HM, Norman GJ, Karelina K, DeVries AC, Wenk GL. Cannabinoids attenuate the effects of aging upon neuroinflammation and neurogenesis. Neurobiol Dis 2009;34:300–7. [146] Talbot Sb, Dias JP, Lahjouji K, Bogo MR, Campos MM, Gaudreau P, et al. Activation of TRPV1 by capsaicin induces functional kinin B (1) receptor in rat spinal cord microglia. J Neuroinflammation 2012;9:16. [147] Schilling T, Eder C. Importance of the non-selective cation channel TRPV1 for microglial reactive oxygen species generation. J Neuroimmunol 2009;216:118–21. [148] Taylor-Clark TE, Kollarik M, MacGlashan Jr DW, Undem BJ. Nasal sensory nerve populations responding to histamine and capsaicin. J Allergy Clin Immunol 2005;116:1282–8. [149] Watanabe N, Horie S, Michael GJ, Keir S, Spina D, Page CP, et al. Immunohistochemical co-localization of transient receptor potential vanilloid (TRPV) 1 and sensory neuropeptides in the guinea-pig respiratory system. Neuroscience 2006;114:1533–43. [150] Watanabe N, Horie S, Michael GJ, Spina D, Page CP, Priestley JV. Immunohistochemical localization of vanilloid receptor subtype 1 (TRPV1) in the guinea pig respiratory system. Pulm Pharmacol Ther 2005;18:187–97. [151] Kollarik M, Undem BJ. Mechanisms of acid-induced activation of airway afferent nerve fibres in guinea-pig. J Physiol 2002;543:591–600. [152] Ricciardolo FLM. Mechanisms of citric acid-induced bronchoconstriction. Am J Med 2001;111(Suppl. 8A):18S–24S. REFERENCES 53

[153] Jia Y, Lee L-Y. Role of TRPV receptors in respiratory diseases. Biochim Biophys Acta 2007;1772:915–27. [154] Canning BJ, Mori N. Encoding of the cough reflex in anesthetized guinea pigs. Am J Physiol Regul Integr Comp Physiol 2011;300:R369–77. [155] Petrosyan M, Perraki E, Simoes D, Koutsourelakis I, Vagiakis E, Roussos C, et al. Exhaled breath markers in patients with obstructive sleep apnoea. Sleep Breath 2008;12:207–15. [156] Niimi A, Nguyen LT, Usmani O, Mann B, Chung KF. Reduced pH and chloride levels in exhaled breath condensate of patients with chronic cough. Thorax 2004;59:608–12. [157] Adcock JJ. TRPV1 receptors in sensitisation of cough and pain reflexes. Pulm Pharmacol Ther 2009;22:65–70. [158] Ojoo JC, Mulrennan SA, Kastelik JA, Morice AH, Redington AE. Exhaled breath condensate pH and exhaled nitric oxide in allergic asthma and in cystic fibrosis. Thorax 2005;60:22–6. [159] Delescluse I, Mace H, Adcock JJ. Inhibition of airway hyper-responsiveness by TRPV1 antagonists (SB-705498 and PF-04065463) in the unanaesthetized, ovalbumin-sensitized guinea pig. Br J Pharmacol 2012;166:1822–32. [160] Gessner C, Hammerschmidt S, Kuhn H, Seyfarth H-Jr, Sack U, Engelmann L, et al. Exhaled breath condensate acidification in acute lung injury. Respir Med 2003;97:1188–94.

[161] Berger KI, Ayappa I, Sorkin IB, Norman RG, Rapoport DM, Goldring RM. CO2 homeostasis during periodic breathing in obstructive sleep apnea. J Appl Physiol 2000;88:257–64. [162] Wasserman K, Whipp BJ, Casaburi R, Beaver WL. Carbon dioxide flow and exercise hyperpnea. Cause and effect. Am Rev Respir Dis 1977;115:225–37. [163] Rotto DM, Kaufman MP. Effect of metabolic products of muscular contraction on discharge of group III and IV afferents. J Appl Physiol 1988;64:2306–13. [164] Ni D, Gu Q, Hu HZ, Gao N, Zhu MX, Lee LY. Thermal sensitivity of isolated vagal pulmonary sensory neurons: role of transient receptor potential vanilloid receptors. Am J Physiol Regul Integr Comp Physiol 2006;29:R541–50. [165] Undem BJ, Chuaychoo B, Lee MG, Weinreich D, Myers AC, Kollarik M. Subtypes of vagal afferent C-fibres in guinea-pig lungs. J Physiol 2004;556:905–17. [166] De Swert KO, Joos GF. Extending the understanding of sensory neuropeptides. Eur J Pharmacol 2006;533:171–81. [167] Jia Y, McLeod RL, Wang X, Parra LE, Egan RW, Hey JA. Anandamide induces cough in conscious guinea-pigs through VR1 receptors. Br J Pharmacol 2002;137:831–6. [168] Lalloo UG, Fox AJ, Belvisi MG, Chung KF, Barnes PJ. Capsazepine inhibits cough induced by capsaicin and citric acid but not by hypertonic saline in guinea pigs. J Appl Physiol 1995;79:1082–7. [169] Trevisani M, Milan A, Gatti R, Zanasi A, Harrison S, Fontana G, et al. Antitussive activity of iodo-resiniferatoxin in guinea pigs. Thorax 2004;59:769–72. [170] Bhattacharya A, Scott BP, Nasser N, Ao H, Maher MP, Dubin AE, et al. Pharmacology and antitussive efficacy of 4-(3-trifluoromethyl-pyridin-2-yl)-piperazine-1-carboxylic acid (5-trifluoromethyl-pyridin-2-yl)-amide (JNJ17203212), a transient receptor potential vanilloid 1 antagonist in guinea pigs. J Pharmacol Exp Ther 2007;323:665–74. [171] McLeod RL, Fernandez X, Correll CC, Phelps TP, Jia Y, Wang X, et al. TRPV1 antagonists attenuate antigen-provoked cough in ovalbumin sensitized guinea pigs. Cough 2006;2:10. [172] Manzini S, Maggi CA, Geppetti P, Bacciarelli C. Capsaicin desensitization protects from antigen-induced bronchospasm in conscious guinea-pigs. Eur J Pharmacol 1987;138:307–8. [173] Molimard M, Martin CA, Naline E, Hirsch A, Advenier C. Contractile effects of bradykinin on the isolated human small bronchus. Am J Respir Crit Care Med 1994;149:123–7. [174] Ellis JL, Sham JS, Undem BJ. Tachykinin-independent effects of capsaicin on smooth muscle in human isolated bronchi. Am J Respir Crit Care Med 1997;155:751–5. [175] Luts A, Uddman R, Alm R, Basterra J, Sundler F. Peptide-containing nerve fibers in human airways: distribution and coexistence pattern. Int Arch Allergy Immunol 1993;101:52–60. [176] Reilly CA, Johansen ME, Lanza DL, Lee J, Lim JO, Yost GS. Calcium-dependent and independent mechanisms of capsaicin receptor (TRPV1)-mediated cytokine production and cell death in human bronchial epithelial cells. J Biochem Mol Toxicol 2005;19:266–75. [177] Karmouty Quintana H, Cannet C, Sugar R, Fozard JR, Page CP, Beckmann N. Capsaicin induced mucus secretion in rat airways assessed in vivo and non invasively by magnetic resonance imaging. Br J Pharmacol 2007;150:1022–30. [178] Groth M, Helbig T, Grau V, Kummer W, Haberberger RV. Spinal afferent neurons projecting to the rat lung and pleura express acid sensitive channels. Respir Res 2006;7:96. 54 3. THE ROLE OF TRPV1 IN ACQUIRED DISEASES

[179] Joos GF, Germonpre PR, Pauwels RA. Role of tachykinins in asthma. J Neurosci 2000;25:321–37. [180] Lundberg JM, Franco Cereceda A, Alving K, Delay Goyet P, Lou YP. Release of calcitonin gene related peptide from sensory neurons. Ann N Y Acad Sci 1992;657:187–93. [181] Baraniuk JN. Neurogenic mechanisms in rhinosinusitis. Curr Allergy Asthma Rep 2001;1:252–61. [182] Lacroix J-S, Landis BN. Neurogenic inflammation of the upper airway mucosa. Rhinology 2008;46:163–5. [183] Marabinil S, Ciabatti PG, Polli G, Fusco BM, Geppettil P. Beneficial effects of intranasal applications of capsaicin in patients with vasomotor rhinitis. Eur Arch Otorhinolaryngol 1991;248:191–4. [184] Thomas KC, Sabnis AS, Johansen ME, Lanza DL, Moos PJ, Yost GS, et al. Transient receptor potential vanilloid 1 agonists cause endoplasmic reticulum stress and cell death in human lung cells. J Pharmacol Exp Ther 2007;321:830–8. [185] Mabalirajan U, Rehman R, Ahmad T, Kumar S, Singh S, Leishangthem GD, et al. Linoleic acid metabolite drives severe asthma by causing airway epithelial injury. Sci Rep 2013;3:1349. [186] Rogers DF. Mucociliary dysfunction in COPD: effect of current pharmacotherapeutic options. Pulm Pharmacol Ther 2005;18:1–8. [187] Springer J, Groneberg DA, Pregla R, Fischer A. Inflammatory cells as source of tachykinin-induced mucus secretion in chronic bronchitis. Regul Pept 2005;124:195–201. [188] Prevarskaya N, Zhang L, Barritt G. TRP channels in cancer. Biochim Biophys Acta 2007;1772:937–46. [189] Hartel M, Di Mola FF, Selvaggi F, Mascetta G, Wente MN, Felix K, et al. Vanilloids in pancreatic cancer: potential for chemotherapy and pain management. Gut 2006;55:519–28. [190] Ziglioli F, Frattini A, Maestroni U, Dinale F, Ciufifeda M, Cortellini P. Vanilloid-mediated apoptosis in prostate cancer cells through a TRPV-1 dependent and a TRPV-1-independent mechanism. Acta Biomed 2009;80:13–20. [191] Gkika D, Prevarskaya N. Molecular mechanisms of TRP regulation in tumor growth and metastasis. Biochim Biophys Acta 2009;793:953–8. [192] Stock K, Kumar J, Synowitz M, Petrosino S, Imperatore R, Smith ESJ, et al. Neural precursor cells induce cell death of high-grade astrocytomas through stimulation of TRPV1. Nat Med 2010;18:1232–8. [193] Monaco III EA, Friedlander RM. Harnessing the brain’s tools for killing cancer cells could be a Key to treating high-grade gliomas. Neurosurgery 2012;71:N23–4. [194] Sanchez MG, Sanchez AM, Collado B, Malagarie-Cazenave S, Olea N, Carmena MJ, et al. Expression of the transient receptor potential vanilloid 1 (TRPV1) in LNCaP and PC-3 prostate cancer cells and in human prostate tissue. Eur J Pharmacol 2005;515:20–7. [195] Domotor A, Peidl Z, Vincze Ar, Hunyady B, Szolcsanyi J, Kereskay L, et al. Immunohistochemical distribution of vanilloid receptor, calcitonin-gene related peptide and substance P in gastrointestinal mucosa of patients with different gastrointestinal disorders. Inflammopharmacology 2005;13:161–77. [196] Lazzeri M, Vannucchi MG, Spinelli M, Bizzoco E, Beneforti P, Turini D, et al. Transient receptor potential vanilloid type 1 (TRPV1) expression changes from normal urothelium to transitional cell carcinoma of human bladder. Eur Urol 2005;48:691–8. [197] Miao X, Liu G, Xu X, Xie C, Sun F, Yang Y, et al. High expression of vanilloid receptor-1 is associated with better prognosis of patients with hepatocellular carcinoma. Cancer Genet Cytogenet 2008;186:25–32. [198] Mergler S, Derckx R, Reinach PS, Garreis F, Bohm A, Schmelzer L, et al. Calcium regulation by temperature-sensitive transient receptor potential channels in human uveal melanoma cells. Cell Signal 2014;26:56–69. [199] Marincsak R, Tath BI, Czifra G, Marton I, Radl P, Tar I, et al. Increased expression of TRPV1 in squamous cell carcinoma of the human tongue. Oral Dis 2009;15:328–35. [200] Gonzales CB, Kirma NB, De La Chapa JJ, Chen R, Henry MA, Luo S, et al. Vanilloids induce oral cancer apoptosis independent of TRPV1. Oral Oncol 2014;50:437–47, pii: S1368-8375(13)00811-7. [201] Vercelli C, Barbero R, Cuniberti B, Odore R, Re G. Expression and functionality of TRPV1 receptor in human MCF-7 and canine CF.41 cells. Vet Comp Oncol 2013. http://dx.doi.org/10.1111/vco.12028. [202] Bode AM, Cho Y-Y, Zheng D, Zhu F, Ericson ME, Ma W-Y, et al. Transient receptor potential type vanilloid 1 suppresses skin carcinogenesis. Cancer Res 2009;69:905–13. [203] Strecker T, Messlinger K, Weyand M, Reeh PW. Role of different proton-sensitive channels in releasing calcitonin gene-related peptide from isolated hearts of mutant mice. Cardiovasc Res 2005;65:405–10. [204] Vemula P, Gautam B, Abela GS, Wang DH. Myocardial ischemia/reperfusion injury: potential of TRPV1 agonists as cardioprotective agents. Cardiovasc Hematol Disord Drug Targets 2014;14:71–8 [Epub ahead of print]. [205] Tschope C, Koch M, Spillmann F, Heringer-Walther S, Mochmann H-C, Stauss H, et al. Upregulation of the cardiac bradykinin B2 receptors after myocardial infarction. Immunopharmacology 1999;44:111–17. REFERENCES 55

[206] Abbas SA, Sharma JN, Yusof APM. The effect of bradykinin and its antagonist on survival time after coronary artery occlusion in hypertensive rats. Immunopharmacology 1999;44:93–8. [207] Emanueli C, Maestri R, Corradi D, Marchione R, Minasi A, Tozzi MG, et al. Dilated and failing cardiomyopathy in bradykinin B2 receptor knockout mice. Circulation 1999;100:2359–65. [208] Fragasso G, Palloshi A, Piatti PM, Monti L, Rossetti E, Setola E, et al. Nitric-oxide mediated effects of trans- dermal capsaicin patches on the ischemic threshold in patients with stable coronary disease. J Cardiovasc Pharmacol 2004;44:340–7. [209] Premkumar LS, Raisinghani M. Nociceptors in cardiovascular functions: complex interplay as a result of cyclooxygenase inhibition. Mol Pain 2006;2:26. [210] Chiao H, Caldwell RW. The role of substance P in myocardial dysfunction during ischemia and reperfusion. Naunyn Schmiedebergs Arch Pharmacol 1996;353:400–7. [211] Bachelard H, St-Pierre S, Rioux F. Participation of capsaicin-sensitive neurons in the cardiovascular effects of neurotensin in guinea pigs. Peptides 1987;8:1079–87. [212] Mitchell JA, Williams FM, Williams TJ, Larkin SW. Role of nitric oxide in the dilator actions of capsaicin- sensitive nerves in the rabbit coronary circulation. Neuropeptides 1997;31:333–8. [213] Zygmunt PM, Petersson J, Andersson DA, Chuang H, Sorgard M, Di Marzo V, et al. Vanilloid receptors on sensory nerves mediate the vasodilator action of anandamide. Nature 1999;400:452–7. [214] Zhong B, Wang DH. TRPV1 gene knockout impairs preconditioning protection against myocardial injury in isolated perfused hearts in mice. Am J Physiol 2007;293:H1791–8. [215] Oroszi G, Szilvassy Z, Nemeth J, Tosaki A, Szolcsanyi J. Interplay between nitric oxide and CGRP by capsaicin in isolated guinea-pig heart. Pharmacol Res 1999;40:125–8. [216] Zahner MR, Li DP, Chen SR, Pan HL. Cardiac vanilloid receptor 1-expressing afferent nerves and their role in the cardiogenic sympathetic reflex in rats. J Physiol 2003;551:515–23. [217] Xing J, Gao Z, Lu J, Sinoway LI, Li J. Femoral artery occlusion augments TRPV1-mediated sympathetic responsiveness. Am J Physiol 2008;295:H1262. [218] Wang Y, Wang DH. Neural control of blood pressure: focusing on capsaicin-sensitive sensory nerves. Cardiovasc Hematol Disord Drug Targets 2007;7:37–46. [219] Cavanaugh DJ, Chesler AT, Braz JM, Shah NM, Julius D, Basbaum AI. Restriction of transient receptor potential vanilloid-1 to the peptidergic subset of primary afferent neurons follows its developmental downregulation in nonpeptidergic neurons. J Neurosci 2011;31:10119–27. [220] Woudenberg-Vrenken TE, Bindels RJM, Hoenderop JGJ. The role of transient receptor potential channels in kidney disease. Nat Rev Nephrol 2009;5:441–9. [221] Wang Y, Babankova D, Huang J, Swain GM, Wang DH. Deletion of transient receptor potential vanilloid type 1 receptors exaggerates renal damage in deoxycorticosterone acetate-salt hypertension. Hypertension 2008;52:264–70. [222] Wang Y, Wang DH. Aggravated renal inflammatory responses in TRPV1 gene knockout mice subjected to DOCA-salt hypertension. Am J Physiol 2009;297:F1550. [223] Li J, Wang DH. Role of TRPV1 channels in renal haemodynamics and function in Dahl salt-sensitive hypertensive rats. Exp Physiol 2008;93:945–53. [224] Mizutani A, Okajima K, Murakami K, Mizutani S, Kudo K, Uchino T, et al. Activation of sensory neurons reduces ischemia/reperfusion-induced acute renal injury in rats. Anesthesiology 2009;110:361–9. [225] Ueda K, Tsuji F, Hirata T, Takaoka M, Matsumura Y. Preventive effect of TRPV1 agonists capsaicin and resiniferatoxin on ischemia/reperfusion-induced renal injury in rats. J Cardiovasc Pharmacol 2008;51:513–20. [226] Sharif-Naeini R, Ciura S, Zhang Z, Bourque CW. Contribution of TRPV channels to osmosensory transduction, thirst, and vasopressin release. Kidney Int 2008;73:811–5. [227] Shimizu T. TRPV1 receptor as a therapeutical target for the treatment of migraine. Brain Nerve 2009;61:949–56. [228] Sun J, Pu Y, Wang P, Chen S, Zhao Y, Liu C, et al. TRPV1-mediated UCP2 upregulation ameliorates hyperglycemia-induced endothelial dysfunction. Cardiovasc Diabetol 2013;12:69. [229] Lobato NS, Filgueira FP, Prakash R, Giachini FR, Ergul A, Carvalho MHC, et al. Reduced endothelium-dependent relaxation to anandamide in mesenteric arteries from young obese Zucker rats. PLoS One 2013;8:e63449. [230] Ho WS. Modulation by 17β-estradiol of anandamide vasorelaxation in normotensive and hypertensive rats: a role for TRPV1 but not fatty acid amide hydrolase. Eur J Pharmacol 2013;701:49–56. 56 3. THE ROLE OF TRPV1 IN ACQUIRED DISEASES

[231] Lambert GA, Davis JB, Appleby JM, Chizh BA, Hoskin KL, Zagami AS. The effects of the TRPV1 receptor antagonist SB-705498 on trigeminovascular sensitisation and neurotransmission. Naunyn Schmiedebergs Arch Pharmacol 2009;380:311–25. [232] Goadsby PJ. Post-triptan era for the treatment of acute migraine. Curr Pain Headache Rep 2004;8:393–8. [233] Hoffmann J, Goadsby PJ. Emerging targets in migraine. CNS Drugs 2014;28:11–17. [234] Geppetti P, Capone JG, Trevisani M, Nicoletti P, Zagli G, Tola MR. CGRP and migraine: neurogenic inflammation revisited. J Headache Pain 2005;6:61–70. [235] Goadsby PJ. Calcitonin gene-related peptide antagonists as treatments of migraine and other primary headaches. Drugs 2005;65:2557–67. [236] Vass Z, Dai CF, Steyger PS, Jancso G, Trune DR, Nuttall AL. Co-localization of the vanilloid capsaicin receptor and substance P in sensory nerve fibers innervating cochlear and vertebro-basilar arteries. Neuroscience 2004;124:919–27. [237] Beltran J, Ghosh AK, Basu S. Immunotherapy of tumors with neuroimmune ligand capsaicin. J Immunol 2007;178:3260–4. [238] Uchida K, Tominaga M. The role of thermosensitive TRP (transient receptor potential) channels in insulin secretion. Endocr J 2011;58:1021–8. [239] Colsoul B, Nilius B, Vennekens R. Transient receptor potential (TRP) cation channels in diabetes. Curr Top Med Chem 2013;13:258–69. [240] Akiba Y, Kato S, Katsube K-i, Nakamura M, Takeuchi K, Ishii H, et al. Transient receptor potential vanilloid subfamily 1 expressed in pancreatic islet β cells modulates insulin secretion in rats. Biochem Biophys Res Commun 2004;321:219–25. [241] Gram DX, Ahren B, Nagy I, Olsen UB, Brand CL, Sundler F, et al. Capsaicin sensitive sensory fibers in the islets of Langerhans contribute to defective insulin secretion in Zucker diabetic rat, an animal model for some aspects of human type 2 diabetes. Eur J Neurosci 2007;25:213–23. [242] Van Buren JJ, Bhat S, Rotello R, Pauza ME, Premkumar LS. Sensitization and translocation of TRPV1 by insulin and IGF-I. Mol Pain 2005;1:17. [243] Sathianathan V, Avelino An, Charrua A, Santha P, Matesz K, Cruz F, et al. Insulin induces cobalt uptake in a subpopulation of rat cultured primary sensory neurons. Eur J Neurosci 2003;18:2477–86. [244] Guillot E, Coste A, Angel I. Involvement of capsaicin-sensitive nerves in the regulation of glucose tolerance in diabetic rats. Life Sci 1996;59:969–77. [245] Koopmans SJ, Leighton B, DeFronzo RA. Neonatal de-afferentation of capsaicin-sensitive sensory nerves increases in vivo insulin sensitivity in conscious adult rats. Diabetologia 1998;41:813–20. [246] Gram DX, Hansen AJ, Deacon CF, Brand CL, Ribel U, Wilken M, et al. Sensory nerve desensitization by resiniferatoxin improves glucose tolerance and increases insulin secretion in Zucker Diabetic Fatty rats and is associated with reduced plasma activity of dipeptidyl peptidase IV. Eur J Pharmacol 2005;509:211–17. [247] Moesgaard SG, Brand CL, Sturis J, Ahren B, Wilken M, Fleckner J, et al. Sensory nerve inactivation by resiniferatoxin improves insulin sensitivity in male obese Zucker rats. Am J Physiol 2005;288:E1137–45. [248] Melnyk A, Himms-Hagen J. Resistance to aging-associated obesity in capsaicin-desensitized rats one year after treatment. Obesity Res 1995;3:337–44. [249] Lopshire JC, Nicol GD. The cAMP transduction cascade mediates the prostaglandin E2 enhancement of the capsaicin-elicited current in rat sensory neurons: whole-cell and single-channel studies. J Neurosci 1998;18:6081–92. [250] Shin KO, Moritani T. Alterations of autonomic nervous activity and energy metabolism by capsaicin ingestion during aerobic exercise in healthy men. J Nutr Sci Vitaminol 2007;53:124–32. [251] Wahlqvist ML, Wattanapenpaiboon N. Hot foods-unexpected help with energy balance? Lancet 2001;358:348–9. [252] Ahuja KDK, Robertson IK, Geraghty DP, Ball MJ. Effects of chili consumption on postprandial glucose, insulin, and energy metabolism. Am J Clin Nutr 2006;84:63–9. [253] Inoue N, Matsunaga Y, Satoh H, Takahashi M. Enhanced energy expenditure and fat oxidation in humans with high BMI scores by the ingestion of novel and non-pungent capsaicin analogues (capsinoids). Biosci Biotechnol Biochem 2007;71:380. [254] Suri A, Szallasi A. The emerging role of TRPV1 in diabetes and obesity. Trends Pharmacol Sci 2008;29:29–36. [255] Yoneshiro T, Aita S, Kawai Y, Iwanaga T, Saito M. Nonpungent capsaicin analogs (capsinoids) increase energy expenditure through the activation of brown adipose tissue in humans. Am J Clin Nutr 2012;95:845–50. [256] Zhang LL, Liu DY, Ma LQ, Luo ZD, Cao TB, Zhong J, et al. Activation of transient receptor potential vanilloid type-1 channel prevents adipogenesis and obesity. Circ Res 2007;100:1063–70. REFERENCES 57

[257] Kawada T, Watanabe T, Takaishi T, Tanaka T, Iwai K. Capsaicin-induced beta-adrenergic action on energy metabolism in rats: influence of capsaicin on oxygen consumption, the respiratory quotient, and substrate utilization. Exp Biol Med 1986;183:250–6. [258] Joo JI, Kim DH, Choi J-W, Yun JW. Proteomic analysis for antiobesity potential of capsaicin on white adipose tissue in rats fed with a high fat diet. J Proteome Res 2010;9:2977–87. [259] Marshall NJ, Liang L, Bodkin J, Dessapt-Baradez C, Nandi M, Collot-Teixeira S, et al. A role for TRPV1 in influencing the onset of cardiovascular disease in obesity. Hypertension 2013;61:246–52. [260] Masuda Y, Haramizu S, Oki K, Ohnuki K, Watanabe T, Yazawa S, et al. Upregulation of uncoupling proteins by oral administration of capsiate, a nonpungent capsaicin analog. J Appl Physiol 2003;95:2408–15. [261] Cui J, Himms-Hagen J. Long-term decrease in body fat and in brown adipose tissue in capsaicin-desensitized rats. Am J Physiol 1992;262:R568–73. [262] Motter AL, Ahern GP. TRPV1-null mice are protected from diet-induced obesity. FEBS Lett 2008;582:2257–62. [263] Bratz IN, Dick GM, Tune JD, Edwards JM, Neeb ZP, Dincer UD, et al. Impaired capsaicin-induced relaxation of coronary arteries in a porcine model of the metabolic syndrome. Am J Physiol Heart Circ Physiol 2008;294:H2489–96. [264] Wang X, Miyares RL, Ahern GP. Oleoylethanolamide excites vagal sensory neurones, induces visceral pain and reduces short-term food intake in mice via capsaicin receptor TRPV1. J Physiol 2005;564:541–7. [265] Ahern GP. Activation of TRPV1 by the satiety factor oleoylethanolamide. J Biol Chem 2003;278:30429–34. [266] Luo X-J, Peng J, Li Y-J. Recent advances in the study on capsaicinoids and capsinoids. Eur J Pharmacol 2011;650:1–7. [267] Kwon DY, Kim YS, Ryu SY, Cha MR, Yon GH, Yang HJ, et al. Capsiate improves glucose metabolism by improving insulin sensitivity better than capsaicin in diabetic rats. J Nutr Biochem 2013;24:1078–85. [268] Ludy MJ, Moore GE, Mattes RD. The effects of capsaicin and capsiate on energy balance: critical review and meta-analyses of studies in humans. Chem Senses 2012;37:103–21. [269] Reinbach HC, Smeets A, Martinussen T, Muller P, Westerterp-Plantenga MS. Effects of capsaicin, green tea and CH-19 sweet pepper on appetite and energy mediated cytokine production and cell death in human bronchial epithelial cells. Clin Nutr 2009;28:260–5. [270] Faraut B, Giannesini B, Matarazzo V, Le Fur Y, Rougon G, Cozzone PJ, et al. Capsiate administration results in an uncoupling protein-3 downregulation, an enhanced muscle oxidative capacity and a decreased abdominal fat content in vivo. Int J Obes 2009;33:1348–55. [271] Kawabata F, Inoue N, Masamoto Y, Matsumura S, Kimura W, Kadowaki M, et al. Non-pungent capsaicin analogs (capsinoids) increase metabolic rate and enhance thermogenesis via gastrointestinal TRPV1 in mice. Biosci Biotechnol Biochem 2009;73:2690–7. [272] Cabranes A, Venderova K, de Lago E, Fezza F, Sanchez A, Mestre L, et al. Decreased endocannabinoid levels in the brain and beneficial effects of agents activating cannabinoid and/or vanilloid receptors in a rat model of multiple sclerosis. Neurobiol Dis 2005;20:207–17. [273] Light AR, White AT, Hughen RW, Light KC. Moderate exercise increases expression for sensory, adrenergic, and immune genes in chronic fatigue syndrome patients but not in normal subjects. J Pain 2009;10:1099–112. [274] Bang R, Biburger M, Neuhuber WL, Tiegs G. Neurokinin-1 receptor antagonists protect mice from CD95- and tumor necrosis factor-α-mediated apoptotic liver damage. J Pharmacol Exp Ther 2004;308:1174–80. [275] Banvolgyi ÃG, Palinkas L, Berki T, Clark N, Grant AD, Helyes Z, et al. Evidence for a novel protective role of the vanilloid TRPV1 receptor in a cutaneous contact allergic dermatitis model. J Neuropharmacol 2005;169:86–96. [276] Petrosino S, Cristino L, Karsak M, Gaffal E, Ueda N, Tuting T, et al. Protective role of palmitoylethanolamide in contact allergic dermatitis. Allergy 2010;65:698–711. [277] Ma J, Harnett KM, Behar J, Biancani P, Cao W. Signaling in TRPV1-induced platelet activating factor (PAF) in human esophageal epithelial cells. Am J Physiol 2010;298:G233–40. [278] Murota H, Izumi M, Abd El-Latif MIA, Nishioka M, Terao M, Tani M, et al. Artemin causes hypersensitivity to warm sensation, mimicking warmth-provoked pruritus in atopic dermatitis. J Allergy Clin Immunol 2012;130:671–82, e674. [279] Tominaga M, Ogawa H, Takamori K. Histological characterization of cutaneous nerve fibers containing gastrin-releasing peptide in NC/Nga mice: an atopic dermatitis model. J Invest Dermatol 2009;129:2901–5. [280] Mihara K, Kuratani K, Matsui T, Nakamura M, Yokota K. Vital role of the itch-scratch response in development of spontaneous dermatitis in NC/Nga mice. Br J Dermatol 2004;151:335–45. 58 3. THE ROLE OF TRPV1 IN ACQUIRED DISEASES

[281] Lim K-M, Park Y-H. Development of PAC-14028, a novel transient receptor potential vanilloid type 1 (TRPV1) channel antagonist as a new drug for refractory skin diseases. Arch Pharm Res 2012;35:393–6. [282] Amagai Y, Matsuda H, Tanaka A. Abnormalities in itch sensation and skin barrier function in atopic NC/Tnd mice. Biol Pharm Bull 2013;36:1248–52. [283] Sun YG, Zhao ZQ, Meng XL, Yin J, Liu XY, Chen ZF. Cellular basis of itch sensation. Science 2009;325:1531–4. [284] Imamachi N, Park GH, Lee H, Anderson DJ, Simon MI, Basbaum AI, et al. TRPV1-expressing primary afferents generate behavioral responses to pruritogens via multiple mechanisms. Proc Natl Acad Sci USA 2009;106:11330–5. [285] Zheng J, Dai C, Steyger PS, Kim Y, Vass Z, Ren T, et al. Vanilloid receptors in hearing: altered cochlear sensitivity by vanilloids and expression of TRPV1 in the organ of corti. J Neurophysiol 2003;90:444–55. [286] Bauer CA, Brozoski TJ, Myers KS. Acoustic injury and TRPV1 expression in the cochlear spiral ganglion. Int Tinnitus J 2003;13:21–8. [287] Bíró T, Bodó E, Telek A, Géczy T, Tychsen B, Kovács L, et al. Hair cycle control by vanilloid receptor-1 (TRPV1): evidence from TRPV1 knockout mice. J Invest Dermatol 2006;126:1909–12. [288] Bodó E, Bíró T, Telek A, Czifra G, Griger Z, Tóth BI, et al. A hot new twist to hair biology: involvement of vanilloid receptor-1 (VR1/TRPV1) signaling in human hair growth control. Am J Pathol 2005;166:985–98. [289] Gunthorpe MJ, Chizh BA. Clinical development of TRPV1 antagonists: targeting a pivotal point in the pain pathway. Drug Discov Today 2009;14:56–67. [290] Mukherjea D, Jajoo S, Sheehan K, Kaur T, Sheth S, Bunch J, et al. NOX3 NADPH oxidase couples transient receptor potential vanilloid 1 to signal transducer and activator of transcription 1-mediated inflammation and hearing loss. Antioxid Redox Signal 2011;14:999–1010. [291] Mukherjea D, Jajoo S, Whitworth C, Bunch JR, Turner JG, Rybak LP, et al. Short interfering RNA against transient receptor potential vanilloid 1 attenuates cisplatin-induced hearing loss in the rat. J Neurosci 2008;28:13056–65. [292] Toth BI, Geczy T, Griger Z, Dozsa A, Seltmann H, Kovacs L, et al. Transient receptor potential vanilloid-1 signaling as a regulator of human sebocyte biology. J Invest Dermatol 2009;129:329–39. CHAPTER 4 TRP Gene Polymorphism and Disease Risk Ina Kraus-Stojanowic,1 Ralf Baron,2 Ingolf Cascorbi1,* 1Institute of Experimental and Clinical Pharmacology, University Hospital Schleswig-Holstein, Kiel, Germany 2Division of Neurological Pain Research and Therapy, Department of Neurology, University Hospital Schleswig-Holstein, Kiel, Germany *Corresponding author: [email protected]

OUTLINE

TRP Gene Polymorphism 59 The TRPV Subfamily 79 TRPA1 74 Disease Risk, Clinical Diagnosis, and Personalized Medicine 81 The TRPC Subfamily 74 References 82 The TRPM Subfamily 76 The TRPML Subfamily 78

TRP GENE POLYMORPHISM

The TRP genes are listed in alphabetical and numerical order: TRPA1, single member of the TRPA subfamily, the TRPC subfamily with TRPC1, C3-C7, the TRPM subfamily with TRPM1-8, TRPML subfamily with TRMPL1, and the TRPV subfamily with V1, V3-V6 (Table 4.1). Rare mutations in transient receptor potential (TRP) channels that cause hereditary TRP channelopathies with dramatic phenotypes are described in Chapter 2 and are not discussed in detail here.

TRP Channels as Therapeutic Targets 59 © 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/B978-0-12-420024-1.00004-7 60 4. TRP GENE POLYMORPHISM AND DISEASE RISK Literature [ 1 ] [ 2 ] [ 3 ] [ 4 ] [ 5 ] [ 5 ] [ 5 ] [ 6 ] [ 5 ] [ 5 ] [ 5 ] [ 7 ] 371/253 controls, 371/253 controls, Caucasian Family study, Antioquian 735 normal subjects 844/2046, European 223/120, Chinese Han 223/120, Chinese Han 223/120, Chinese Han Family study, Caucasian 223/120, Chinese Han 223/120, Chinese Han 284/282, African American Population (cases/ controls) 223/120, Chinese Han Paradoxical heat sensation pain patients in neuropathic Familial episodic pain (FEPS) syndrome Pain tolerance to the cold stimuli Not with cough in subjects with or without asthma Not with T2D Not with T2D Not with T2D Not with infantile pyloric stenosis hypertrophic (IHPS) Not with T2D factor of Protective getting diabetic nephropathy in T2D Not with diabetic or end-stage nephropathy disease renal Association with Risk factor of T2D without diabetic nephropathy rs920829 rs11988795 rs7621642 rs3821647 rs1132030 rs12634067 rs2033912 rs953239 rs rs7638459 > A c.535G > A c.2564A > G c.2385 + 617G > A c.918G > A c.1929G > A c.2061G > A c.1480 − 111G c.1479 + 4558A > T c.172 + 2632A > C cDNA c.633 − 6323T > C p.Glu179Lys p.Asn855Ser – p.Ser306= p.Thr643= p.Arg687= – – – Protein – Exon Exon 22 Intron 29 SNPs Exon Exon Exon 8 Intron Intron Intron 7 SNPs Location Intron Genetic Variances of TRP Channels Reportedly Associated with Various Diseases or Behaviors of TRP Channels Reportedly Associated with Various Genetic Variances

4.1

TRPA1 TRPC1 TABLE Gene TRP GENE POLYMORPHISM 61 ] (Continued) [ 8 ] [ 8 ] [ 8 ] [ 8 ] [ 8 ] [ 9 ] [ 9 ] [ 10 ] [ 10 ] [ 10 ] [ 6 ] [ 10 ] [ 10 ] [ 10 ] [ 11 98/96 98/96 98/96 98/96 79/79 126/126 273/599 273/599 273/599 98/96 Family study, Family study, Caucasian 273/599 273/599 273/599 1172/1157; 1145/1142, European Not with episodic human ataxias cerebellar Not with episodic human ataxias cerebellar Not with episodic human ataxias cerebellar Not with episodic human ataxias cerebellar Idiopathic ataxia Not with cardiac hypertrophy response Photoparoxysmal (PPR), idiopathic generalized epilepsies (IGEs) PPR/IGE PPR/IGE Not with episodic human ataxias cerebellar Not with infantile pyloric hypertrophic stenosis (IHPS) PPR/IGE PPR/IGE PPR/IGE per day Cigarettes rs61742537 rs11732666 rs41278087 rs13121031 rs13121031 rs10507457 rs7329459 rs10507456 rs13121031 rs5942757 rs1535775 rs10161932 rs7338118 rs7050529 C c.585G > A c.2199G > A c.2271A > G c.2451A > G c.78C > G c.78C > G c.898 − 3204G > A c.1235 − 3135C > T c.1375 − 4324C > T c.2079 + 4443T > c.78C > G c.379 − 1950T > A c.*109G > A c.1884 + 769T > G c.900 + 286C > T p.Lys195= p.Arg733= p.Ser757= p.Glu817= – – – – – – p.Ala26= – – – – Exon 1 Exon 8 Exon 9 Exon 10 Promoter Promoter 3 Intron 3 Intron 5 Intron 5 Intron Exon 1 Intron 2 Intron Intron 5 Intron 5 Intron Intron TRPC4 TRPC3 TRPC5 62 4. TRP GENE POLYMORPHISM AND DISEASE RISK [ 12 ] [ 13 ] [ 14 ] [ 13 ] [ 15 ] [ 16 ] [ 17 ] [ 18 ] [ 16 ] [ 19 ] [ 20 ] [ 18 ] [ 6 ] [ 13 ] [ 21 ] [ 16 ] [ 18 ] Literature Population (cases/ controls) Family study, Family study, Czech Family study, Spanish Family study Family study, Spanish Family study Family study Family study, Dutch Family study, Italian Family study Family study Family study, Turkish Family study Family study, Caucasian Family study, Spanish Family study, Chinese Family study Family study, Italian Focal segmental (FSGS) glomerulosclerosis FSGS FSGS FSGS FSGS FSGS FSGS FSGS FSGS FSGS FSGS FSGS Infantile hypertrophic pyloric stenosis (IHPS) FSGS FSGS FSGS FSGS Association with rs3802829 rs121434390 rs146776939 rs121434391 rs121434392 rs36111323 rs36111323 rs121434394 rs cDNA c.43C > T c.325G > A c.335C > A c.374A > G c.495T > C c.428A > G c.524G > A c.653A > T c.808T > A c.1079G > A > T c.1211C > T c.1211C c.2339T > C c.2664C > A c.2683C > T c.2684G > T Protein p.Pro15Ser p.Gly109Ser p.Pro112Gln p.Asn125Ser p.Met132Thr p.Asn143Ser p.Arg175Gln p.His218Leu p.Ser270Thr p.Arg360His p.Leu395Ala p.Ala404Val p.Ala404Val p.Leu780Pro p.Glu889Lys p.Arg895Cys p.Arg895Leu Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon 4 Exon Exon Exon Exon Location Genetic Variances of TRP Channels Reportedly Associated with Various Diseases or Behaviors—Cont’d of TRP Channels Reportedly Associated with Various Genetic Variances

4.1

TABLE Gene TRPC6 TRP GENE POLYMORPHISM 63 (Continued) [ 16 ] [ 6 ] [ 6 ] [ 6 ] [ 22 ] [ 22 ] [ 23 ] [ 22 ] [ 24 ] [ 24 ] [ 24 ] [ 25 ] [ 26 ] [ 27 ] [ 26 , 28 ] [ 27 , 28 ] [ 28 ] [ 27 ] [ 28 ] [ 27 ] Family study, Family study, Caucasian Family study, Caucasian Family study, Caucasian 268/237, white 268/237, white 28/23 268/237, white 134/265 134/265 1050/879 Family study Family study Family study Family study Family study Family study Family study Family study Family study 134/265

Infantile hypertrophic Infantile hypertrophic pyloric stenosis (IHPS) IHPS IHPS Not with idiopathic pulmonary arterial hypertension (IPAH) IPAH nephritic Steroid-resistant (SNRS) syndrome Not IPAH Not with membranous (MGN) glomerulonephritis MGN MGN Nicotine dependence Complete congenital stationary night blindness (cCSNB) cCSNB cCSNB cCSNB cCSNB cCSNB cCSNB cCSNB FSGS rs11224883 rs7127346 rs3922961 rs3824934 rs3824934 rs3824935 rs17096918 rs4326755 rs2673931 rs191205969 rs121434395 c.170 + 430G > C c.1744 + 528G > A c.-914A > C c.-361A > T c.-254C > G c.-254C > G c.-218C > T c.-1768G > A c.170 + 70G > A c.170 + 4707C > T c.780 + 2860G > A c.167A > G c.215A > G c.220C > T c.296T > C c.1091T > G c.1418G > C c.1600G > A c.1622T > A c.2689G > A – – – – – – – – – – – p.Tyr56Cys p.Tyr72Cys p.Arg74Cys p.Leu99Pro p.Leu364Arg p.Arg473Pro p.Gly534Arg p.Met541Lys p.Glu897Lys Intron 1 Intron 6 Intron Promoter Promoter Promoter Promoter Promoter 5 ʹ Intron Intron Intron Exon Exon Exon Exon Exon Exon Exon Exon Exon TRPC7 TRPM1 64 4. TRP GENE POLYMORPHISM AND DISEASE RISK [ 29 ] [ 28 ] [ 30 ] [ 26 ] [ 30 ] [ 26 ] [ 26 ] [ 26 ] [ 30 ] [ 26 ] [ 31 ] [ 31 ] [ 32 ] [ 33 ] [ 34 ] [ 35 ] [ 33 ] Literature Family study Family study Family study Family study Family study Family study Family study Family study Family study Family study, Mexican Americans 600/450 family study, Caucasian Family study, Guamanian 67/20 Family study, Caucasian 783 cases Family study, Mexican Americans Population (cases/ controls) cCSNB cCSNB cCSNB cCSNB cCSNB cCSNB cCSNB cCSNB cCSNB levels Triglyceride Not with albuminuria (albumin-to-creatinine ratio, ACR) Bipolar disorder type I (BD-I) Bipolar disorder but not BD-II Guamanian amyotrophic (ALS-G) and lateral sclerosis parkinsonism-dementia (PD-G) Bipolar disorder Early age at onset in BD-I haplotype) families (C-T-A Effects of risperidone on Effects DSM-IV schizophrenia Association with rs11070811 rs1556314 rs1556314 rs145947009 rs1618355 rs1618355 rs17815774 rs cDNA c.1832C > A c.1870C > T c.2162G > A c.2645C > A c.2648A > G c.2885A > C c.3004A > T c.3224T > C c.4312A > G c.-306G > A c.1629T > G c.1629T > G c.3053C > T c.2791 − 15C > A c.2791 − 15C > A c.1813G > A Protein p.Pro611His p.Arg624Cys p.Arg721Gln p.Ser882Ter p.Glu883Gly p.Met962Thr p.Ile1002Phe p.Phe1075Ser p.Arg1438Gly – p.Asp543Glu p.Asp543Glu p.Pro1018Leu – – p.Val605Met Location Exon Exon Exon Exon Exon Exon Exon Exon Exon Promoter 18 SNPs Exon 11 Exon 11 Exon 18 Intron 18 Intron Exon Genetic Variances of TRP Channels Reportedly Associated with Various Diseases or Behaviors—Cont’d of TRP Channels Reportedly Associated with Various Genetic Variances

4.1

TRPM2 TABLE Gene TRP GENE POLYMORPHISM 65 (Continued) [ 35 ] [ 33 ] [ 33 ] [ 36 ] [ 36 ] [ 36 ] [ 37 ] [ 38 ] [ 38 ] [ 39 ] [ 40 ] [ 41 ] [ 40 ] [ 41 ] [ 40 ] [ 40 ] 67/20 Family study, Caucasian Family study, Caucasian 467/455, white 467/455, white 467/455, white Family study 1087 normal subjects 1087 normal subjects Family study 160 cardiac patients Family study, Libanese, and French 160 cardiac patients Family study, Libanese, and French patients 160 cardiac patients 160 cardiac in B 2 + lymphoblasts Early age at onset in BD-I haplotype) families (C-T-A Early age at onset in BD-I haplotype) families (C-T-A Beta-cell function (HOMA-%B) HOMA-%B HOMA-%B Not with bipolar affective (BPAD) disorder MeanHDL-C MeanHDL-C Intracellular Ca Progressive familial Progressive heart block type 1 (PFHB1B) PFHB1B PFHB1B PFHB1B PFHB1B PFHB1B PFHB1B rs933151 rs749909 rs2838553 rs2838554 rs4818917 rs688933 rs541326 rs1612472 rs267607142 rs172146854 rs387907216 rs172147855 rs201907325 rs172149856 rs172150857 A c.3147 − 1805C > T c.3975 − 207G > A c.254 + 3675T > A c.423 + 467G > C c.1215 + 200T > C c.177 + 62935A > G c.177 + 29143C > T c.19G > c.2963 − 789C > T c.393G > C c.490C > T c.878A > G c.1294G > A c.1744G > A c.2368T > C – – – – – – – p.Glu7Lys – p.Gln131His p.Arg164Trp p.Gln293Arg p.Ala432Thr p.Gly582Ser p.Tyr790His Intron 20 Intron 27 Intron Intron Intron Intron 14 SNPs Intron Intron Exon Intron 19 Intron Exon Exon Exon Exon Exon Exon TRPM3 TRPM4 66 4. TRP GENE POLYMORPHISM AND DISEASE RISK [ 41 ] [ 40 ] [ 40 ] [ 42 ] [ 43 ] [ 44 ] [ 45 ] [ 46 ] [ 46 ] [ 47 ] [ 46 ] [ 46 ] [ 48 ] [ 49 ] [ 47 ] Literature 160 cardiac 160 cardiac patients 160 cardiac patients 429/285, Chinese 179/182, Turkish Family study Family study Family study Family study 359/359 Family study Family study Family study 359/359 Family study, Family study, Libanese, and French Family study Population (cases/ controls) PFHB1B PFHB1B Risk of hepatitis B virus-related liver cirrhosis Primary open-angle glaucoma (POAG) Hypomagnesemia with secondary hypocalcemia (HSH or HOMG1) HSH HSH HSH T2D (women with 1393Ile- 1584Glu and low magnesium intake) HSH HSH HSH T2D (women with 1393Ile- 1584Glu and low magnesium intake) PFHB1B HSH Association with rs172151858 rs172152859 rs886277 rs34551253 rs121912625 rs3750425 rs2274924 rs200038418 rs cDNA c.2741A > G c.2908C > T c.704A > G c.1366G > A c.422C > T c.521T > G c.1060A > C > A c.1177G c.1437C > A c.3050C > G c.4577G > A c.4750A > G c.2531G > A c.2120G > A Protein p.Lys914Arg p.Pro970Ser p.Asn235Ser p.Ala456Thr p.Ser141Leu p.Glu157Stop p.Ile174Arg p.Thr354Pro p.Val1393Ile p.Tyr479Stop p.Pro1017Arg p.Lys1584Glu p.Gly844Asp p.Cys707Tyr Location Exon Exon Exon Exon 9 Exon Exon Exon 5 Exon 9 Exon 26 Exon 12 Exon Exon Exon 27 Exon Exon 17 Genetic Variances of TRP Channels Reportedly Associated with Various Diseases or Behaviors—Cont’d of TRP Channels Reportedly Associated with Various Genetic Variances

4.1

TRPM5 TRPM6 TABLE Gene TRP GENE POLYMORPHISM 67 (Continued) [ 58 ] [ 58 ] [ 59 ] [ 60 ] [ 3 ] [ 1 ] [ 50 ] [ 51 ] [ 52 ] [ 53 ] [ 54 ] [ 55 ] [ 56 ] [ 57 ] [ 53 ] [ 47 ] 133 normal subjects, Shorian 2731/10,747 5122/18,108, European 735 normal subjects 371/253, Caucasian 52,684 normal subjects, European 15,366 normal subjects, European 467/455, white Family study, Guamanian 24/27 688 + 210/1306 245/245, white 467/455, white 359/359 133 normal subjects, Shorian 471 normal subjects, Caucasian Total cholesterol, LDL LDL cholesterol, Total hip and waist cholesterol, circumference Migraine with Migraine compared nonmigraine headache Not with heat or cold pain Not with sensory parameters pain patients in neuropathic Glucose Hypomagnesia and bone mineral density Not with intermediate phenotypes or T2DM Guamanian amyotrophic (ALS-G) andlateral sclerosis parkinsonism-dementia (PD-G) ALS/PDC in Kii Not with peninsula Adenoma and hyperplastic polyps Not with risk of incident ischemic stroke Not with intermediate phenotypes or T2DM Not with diabetes risk cholesterol HDL Not with extracellular magnesium concentration rs11562975 rs17862920 rs10166942 rs2274924 rs11144134 rs8042919 rs8042919 rs8042919 rs28901637 rs2274924 c.750G > C c.-6 + 1918C > T c.-990T > C c.4750A > G c.33 + 2944A > G c.4445C > T c.4445C > T c.4445C > T c.747A > T c.4750A > G p.Leu250= – – p.Lys1584Glu – p.Thr1482Ile p.Thr1482Ile p.Thr1482Ile p.Pro249= p.Lys1584Glu Exon Intron 5 ʹ 12 SNPs 6 SNPs Exon 27 Intron 29 SNPs Exon Exon Exon 16 SNPs SNPs 11 5 SNPs Exon Exon 27 TRPM7 TRPM8 68 4. TRP GENE POLYMORPHISM AND DISEASE RISK [ 62 ] [ 63 ] [ 67 ] [ 64 ] [ 68 ] [ 69 ] [ 68 ] [ 70 ] [ 3 ] [ 69 ] [ 61 ] [ 62 ] [ 63 ] [ 62 ] [ 64 ] [ 64 ] [ 62 , 63 ] [ 61 , 65 ] [ 62–64 ] [ 66 ] [ 62 ] Literature Population (cases/ controls) Family study Family study Family study Family study 103/80, Korean 228/207, Dutch 103/80, Korean 195/205, Chinese 735 normal subjects 228/207, Dutch Family study Family study Family study Family study Family study Family study Family study Family study Family study Family study Family study MLIV MLIV MLIV MLIV Not with irritable bowel (IBS) syndrome pancreatitis Not with chronic Not with IBS Not with nonspecific chronic cough in children Not with heat or cold pain pancreatitis Not with chronic MLIV MLIV MLIV MLIV MLIV MLIV MLIV MLIV MLIV MLIV Mucolipidosis Type lV Mucolipidosis Type (MLIV) Association with rs9894618 rs222749 rs222749 rs222748 rs222747 rs222747 rs121908373 rs121908372

rs cDNA c.1465T > C c.1364C > T c.1395C > G c.6G > T c.271C > T c.271C > T c.501C > T c.945G > C c.945G > C c.304C > T c.442T > C c.639C > T c.694A > C c.964C > T c.1084G > T c.1207C > T c.346 − 1348delCTT c.1307A > G c.1461G > T c.1336G > A c.235C > T Protein p.Leu447Pro p.Ser456Leu p.Phe465Leu p.Lys2Asn p.Pro91Ser p.Pro91Ser p.His167= p.Met315Ile p.Met315Ile p.Arg102Ter p.Leu106Pro p.Arg172Stop p.Thr232Pro p.Arg322Stop p.Asp362Tyr p.Arg403Cys deltaPhe408 p.Tyr436Cys p.Val446Leu p.Val446Ile p.Gln79Stop Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Location Genetic Variances of TRP Channels Reportedly Associated with Various Diseases or Behaviors—Cont’d of TRP Channels Reportedly Associated with Various Genetic Variances

4.1

TRPV1 TABLE TRPML1 Gene TRP GENE POLYMORPHISM 69 (Continued) [ 71 ] [ 1 ] [ 68 ] [ 72 ] [ 69 ] [ 73 ] [ 3 ] [ 74 ] [ 69 ] [ 75 ] [ 1 ] [ 70 ] [ 76 ] [ 77 ] [ 78 ] [ 4 ] [ 4 ] [ 4 ] 371/253, German 103/80, Korean 146/205, Ashkenazi Jewish 228/207, Dutch 500 normal subjects 735 normal subjects 80 normal subjects 228/207, Dutch 301/470, Spanish 371/253, German 109/98, Japanese 195/205, Chinese 95 normal subjects 163 cases 1040/1037 844/2046, European 844/2046, European 844/2046, European Cold hypaesthesia in pain patients neuropathic Not with IBS 1 diabetes Type pancreatitis Not with chronic Longer cold withdrawal times Not with heat or cold pain Change in abdominal adiposity pancreatitis Not with chronic risk of current Lowered wheezing (active asthma) or cough Cold hypoalgesia, less hyperalgesia Functional dyspepsia Not with nonspecific chronic Not with nonspecific chronic cough in children Sensitivity of salt solutions disease Primary progressive (MS) in multiple sclerosis Migraine Cough symptoms in subjects without asthma Cough symptoms in subjects without asthma Cough symptoms in subjects without asthma rs222747 rs222747 rs222747 rs224534 rs8065080 rs8065080 rs8065080 rs8065080 rs8065080 rs8065080 rs222747 rs8065080 rs8065080 rs877610 rs222741 rs11655540 rs161365 rs17706630 c.945G > C c.945G > C c.945G > C c.1406C > T c.1753A > G c.1753A > G c.1753A > G c.1753A > G c.1753A > G c.1753A > G c.945G > C c.1753A > G c.1753A > G c.2157G > A c.-34 + 2841C > T c.2347 + 873A > C c.1547 + 274A > G c.1384 − 418C > T p.Met315Ile p.Met315Ile p.Met315Ile p.Thr469Ile p.Ile585Val p.Ile585Val p.Ile585Val p.Ile585Val p.Ile585Val p.Ile585Val p.Met315Ile p.Ile585Val p.Ile585Val p.Lys719= – – – – Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Intron Intron Intron Intron 70 4. TRP GENE POLYMORPHISM AND DISEASE RISK ] [ 79 ] [ 79 ] [ 80 ] [ 80 ] [ 80 ] [ 78 ] [ 4 ] [ 4 ] [ 4 ] [ 11 [ 81 ] [ 75 ] [ 82 ] [ 83 ] [ 84 ] [ 85 ] Literature 177/151, Chinese Family study Family study Family study 1040/1037 177/151, Chinese 844/2046, European 844/2046, European 1172/1157; 1145/1142, European 844/2046, European 301/470, Spanish 606/1285, Caucasian; 953/956, Norwegian Family study Family study Family study 1591 normal subjects Population (cases/ controls) Association with Childhood asthma (OLMS) Olmsted syndrome OLMS OLMS Migraine with aura Childhood asthma Cough symptoms in subjects without asthma Cough symptoms in subjects without asthma per day Cigarettes Cough symptoms in subjects without asthma Not with childhood asthma of wheezing or the presence pulmonary obstructive Chronic disease (COPD) Congenital distal spinal (CDSMA) muscular atrophy Spondyloepiphyseal type dysplasia Maroteaux dysplasia (MTD) Metatropic Lower sodium concentrations in (hyponatremia) serum rs rs4790522 rs199473704 rs199473704 rs199473705 rs7217270 rs4790521 rs150854 rs224498 rs4790520 rs2277675 rs3742030 rs3742030 rs387906324 rs3742030 > T cDNA c.*256T > G c.1717G > T c.1717G > A c.2074T > G c.2085 + 395T > C c.55C c.*343A > G T > G T > G c.-1900A > G c.-332A > G c.55C > T c.55C > T c.290C > G c.547G > A Protein – p.Gly573Cys p.Gly573Ser p.Trp692Gly – p.Pro19Ser – – – – – p.Pro19Ser p.Pro19Ser p.Pro97Arg p.Glu183Lys p.Leu199Phe Location 3 ʹ -UTR Exon Exon Exon Intron Exon 2 3 ʹ -UTR 5 ʹ 5 ʹ 5 ʹ 5 ʹ -UTR Exon 2 Exon 2 Exon Exon Exon Genetic Variances of TRP Channels Reportedly Associated with Various Diseases or Behaviors—Cont’d of TRP Channels Reportedly Associated with Various Genetic Variances

4.1

TRPV3 TRPV4 TABLE Gene TRP GENE POLYMORPHISM 71 (Continued) [ 83 , 86 ] [ 86 , 87 ] [ 86–89 ] [ 90 ] [ 90 ] [ 90 ] [ 85 ] [ 85 ] [ 89 , 91 ] [ 88 , 89 ] [ 86 ] [ 92 , 93 ] [ 85 ] [ 93 ] [ 85 ] [ 91 ] [ 85 ] [ 84 , 85 93 ] [ 85 ] [ 85 ] [ 84 ] Family study Family study Family study Family study Family study Family study Family study Family study Family study Family study Family study Family study Family study Family study Family study Family study Family study Family study Family study Family study Family study CMT2C and CMT2C CDSMA Familial digital arthropathy brachydactyly (FDAB) FDAB FDAB Spondylometaphyseal dysplasia, Kozlowski type (SMDK) MTD CMT2C CMT2C and scapuloperoneal spinal muscular atrophy (SPSMA) CMT2C MTD MTD SMDK MTD CMT2C MTD SMDK and parastremmatic dwarfism (PSTD) SMDK SMDK Spondyloepiphyseal type dysplasia Maroteaux and Charcot-Marie- CDSMA disease (CMT2C) Tooth rs267607145 rs387906905 rs121912636 rs121912634 rs387906902 rs267607150 rs387906904 c.805C > T c.806G > A c.809G > T c.812G > C c.819C > G c.832G > A c.883A > G c.943C > T c.946C > T c.947G > A c.991A > T c.992T > C c.998A > G c.1625C > A c.1781G > A c.1787T > C c.1798G > T c.1805A > G c.694C > T p.Arg269Cys p.Arg269His p.Gly270Val p.Arg271Pro p.Phe273Leu p.Glu278Lys p.Thr295Ala p.Arg315Trp p.Arg316Cys p.Arg316His p.Ile331Phe p.Ile331Thr p.Asp333Gly p.Val342Phe p.Ser542Tyr p.Phe592Leu p.Arg594His p.Leu596Pro p.Gly600Trp p.Tyr602Cys p.Arg232Cys Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon 72 4. TRP GENE POLYMORPHISM AND DISEASE RISK [ 84 , 85 92 93 ] [ 85 ] [ 85 ] [ 82 ] [ 82 ] [ 94 ] [ 92 ] [ 92 ] [ 94 ] [ 85 ] [ 85 ] [ 93 ] [ 85 ] [ 85 ] [ 84 , 85 92 ] [ 85 ] Literature Family study Family study Family study 606/1285, Caucasian; 953/956, Norwegian 606/1285, Caucasian; 953/956, Norwegian Family study Family study Family study Family study Family study Family study Family study Family study Family study Family study Family study Population (cases/ controls) loepiphyseal loepiphyseal Association with MTD and spondy type dysplasia Maroteaux MTD MTD pulmonary obstructive Chronic disease (COPD) COPD MTD MTD BRAC3 SMDK SMDK SMDK MTD SMDK MTD, SMDK, and spondyloepiphyseal type dysplasia Maroteaux MTD Brachyolmia type 3 (BRAC3) rs rs121912637 rs267607147 rs6606743 rs7971845 rs267607149 rs267607147 rs121912637

c.2396C > G c.2395C > T G > A c.-31 − 8070G > C c.1851C > A c.1853T > C c.1858G > A c.2125C > A c.2146G > T c.2324G > A c.2330G > A c.2389G > A c.2395C > G c.2396C > T c.1847G > A cDNA p.Pro799Arg p.Pro799Ser – p.Phe617Leu p.Leu618Pro p.Val620Ile p.Met625Ile p.Leu709Met p.Ala716Ser p.Arg775Lys p.Cys777Tyr p.Glu797Lys p.Pro799Ala p.Pro799Leu p.Arg616Gln Protein Exon Exon 1 Intron Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Location Genetic Variances of TRP Channels Reportedly Associated with Various Diseases or Behaviors—Cont’d of TRP Channels Reportedly Associated with Various Genetic Variances

4.1

TABLE Gene TRP GENE POLYMORPHISM 73 [ 96 ] [ 97 ] [ 82 ] [ 82 ] [ 82 ] [ 82 ] [ 4 ] [ 95 ] [ 96 ] [ 96 ] 141 cases 606/1285, Caucasian; 953/956, Norwegian 606/1285, Caucasian; 953/956, Norwegian 606/1285, Caucasian; 953/956, Norwegian 844/2046, European 20 cases 274/341, no African 274/341, no African 274/341, no African 606/1285, Caucasian; 953/956, Norwegian Not with prostate cancer, cancer, Not with prostate haplotype (157R + 378V 681T) COPD COPD COPD Not with cough in subjects or without asthma hypercalciuria Not with renal Calcium stone formation, haplotype (157R + 378V 681T) Calcium stone formation, haplotype (157R + 378V 681T) Calcium stone formation, haplotype (157R + 378V 681T) COPD

rs12579553 rs3825396 rs12578401 rs4987657 rs4987667 rs4987682 rs16940583

+ 787T > C c.1152 − 189G > A c.1153 c.1332 + 830G > A c.469T > C > G c.1132A c.2042T > C c.853 + 158T > C

– – – p.Cys157Arg p.Met378Val p.Met681Thr – 3 SNPs Intron 6 Intron 6 Intron Intron7 10 SNPs 8 SNPs Exon Exon Exon Intron 5 Intron TRPV5 TRPV6 74 4. TRP GENE POLYMORPHISM AND DISEASE RISK TRPA1

TRPA1 belongs to a group of nociceptors, mediating response to cold stimuli [98]. The channel is activated at temperatures below 17 °C, contributing to transduction of noxious cold in sensory neurons. Moreover, there is evidence that TRPA1 knockout mice have impaired noxious cold sensation [99]. To examine the genetic contribution to individual cold and heat pain sensitivity, genetic variations in TRPA1 were investigated in a few studies. Significant association was found between short-duration cold pain sensitivity and the intronic TRPA1 SNP rs11988795 (c.2385+617G>A). In American females of European ancestry, A/A homozygote variants showed less pain tolerance to cold stimuli compared to G/G homozygotes [3]. A genome-wide linkage scan in 13 affected and 10 unaffected family members with familial episodic pain syndrome and subsequent candidate gene sequencing in affected individuals identified an A to G transition in exon 22 of TRPA1 (c.2564A>G; p.Asn855Ser). This change was observed in all affected individuals but not in unaffected family members and not in ethnically matched unaffected controls [2]. In neuropathic pain patients, TRPA1 SNP rs920829 (c.535G>A; 710G>A, p.Glu179Lys) was associated with the presence of paradoxical heat sensation (PHS). Within the group of patients who suffered from PHS, heterozygous and homozygous carriers of the TRPA1 710G>A variant were significantly underrepresented as compared to neuropathic pain patients without PHS [1]. Associations between SNPs in TRPA1 and cough symptoms in subjects with or without asthma were not significant after adjusting for multiple testing [4].

THE TRPC SUBFAMILY

The TRPC (canonical) subfamily comprises the closest homology to Drosophila trp channels. They tend to form homotetramers and heterotetramers among TRPCs and other types of TRP protein. TRPC channels have substantial importance in vascular physiology and pathophysiology [100]. The TRPC1 gene resides within the linkage region for diabetic nephropathy in type 1 (T1D) and type 2 diabetes mellitus (T2D). A genetic association study was performed with two independent cohorts, including T1D patients with and without diabetic nephropathy and subjects with T2D-associated end-stage renal disease, or hypertensive (nondiabetic) end-stage renal disease, as well as nondiabetic controls. No significant association of examined TRPC1 DNA polymorphisms (rs953239, rs7638459, rs17624218, rs7621642, rs2033912, rs3821647, rs7610200) with diabetic nephropathy or end-stage renal disease was found [7]. In a Chinese Han population, two intronic TRPC1 SNPs were significantly associated with the development of T2D with and without diabetic nephropathy. In the SNP rs7638459, the CC genotype significantly increased the risk of getting T2D without DN, when compared with TT genotype. In SNP rs953239, the CC genotype significantly reduced the risk of getting T2D without DN, when compared with AA genotype. No significant association of TRPC1 SNPs rs7621642, rs2033912, rs3821647, and rs1132030 with diabetes mellitus among T2D without DN and with DN was found [5]. The TRPC Subfamily 75

In a cohort of ataxia patients and in individuals with cardiac hypertrophy, genotype frequency of the alternative promoter of TRPC3 (rs13121031, c.78C>G) was determined. This alternative promoter is regulated by allelic DNA methylation. The common G allele is associated with high levels of methylation, whereas the less prevalent C allele is unmethylated. These analyses revealed a statistical trend for the rare unmethylated homozygous C genotype to be present at a higher frequency in idiopathic ataxia patients, but not in those patients with known mutations or in individuals with cardiac hypertrophy, when compared to a control population [9]. In patients with genetically undefined late-onset cerebellar ataxia and patients with undefined episodic ataxia, all 11 coding exons and flanking exon/intron bound- aries of TRPC3 gene were sequenced. All found variations (c.78C>G, c.585G>A, c.2199G>A, c.2271A>G, c.2451A>G) were silent mutations in TRPC3 exons 1, 8, 9, and 10 and did not significantly contribute to the cause of late-onset and episodic human cerebellar ataxias [8]. Photoparoxysmal response (PPR, abnormal visual sensitivity of the brain to photic stimulation) is frequently associated with idiopathic generalized epilepsies (IGEs). An association of PPR with sequence variations of TRPC4 gene identified significant results with polymorphisms in intron 3 (rs10507457, rs7329459) and intron 5 (rs10507456, rs1535775, rs10161932, rs7338118). Corresponding haplotypes showed association with PPR/IGE, but correction for multiple comparisons were not significant [10]. A genome-wide and candidate gene association study of cigarette smoking behaviors revealed an association of TRPC5 intron polymorphism rs7050529 with cigarettes smoked per day [11]. Intron variant rs2673931 of TRPC7 was significantly associated with nicotine dependence in primary analysis, but not after correction for multiple tests [25]. A number of missense variants in TRPC6 have been reported to be associated with focal and segmental glomerulosclerosis type 2 (FSGS2) [12–21]. (See Chapter 2 for more details regarding this channelopathy.) Allele frequency of the TRPC6 SNP c.-254C>G (rs3824934) was significantly higher in patients with idiopathic pulmonary arterial hypertension (IPAH; 12%) than in normal subjects (6%). Percentage of c.-254G/G homozygotes in IPAH patients was 2.85 times that of normal subjects. Allele frequencies of c.-361A>T and c.-218C>T SNPs were not different between groups [22]. No statistically significant differences of TRPC6 gene polymorphisms rs3824935, rs17096918, and rs4326755 were found between patients with membranous glomerulonephritis and controls [24]. The allele frequency of the TRPC6 -254C>G SNP (rs3824934) in Chinese children with steroid-resistant nephritic syndrome (40.5%) was higher than that in steroid-sensitive nephritic syndrome subjects (27.1%) [23]. A genome scan in families with infantile hypertrophic pyloric stenosis (IHPS) identified chromosomal regions harboring genes for TRPC1, TRPC5, and TRPC6. Fine mapping and resequencing identified SNP rs3922961 (c.-914A>C) in the promoter region of TRPC6 that may act to prevent overexpression of TRPC6 thus protecting against the hypertrophy that is characteristic of IHPS [6]. TRPC6 has also been reported to play a role in pulmonary hypertension [101]. A missense variant in TRPC6 exon 4 (p.Ala404Val, rs36111323) was identified putatively affecting the splicing regulation of TRPC6. Moreover, homozygous carriers of intron 1 SNP rs11224883 were less frequent in cases than in controls, and homozygous carriers of the wild-type genotype of rs7127346 (intron 6) occurred at a higher frequency in cases than controls. Analyses did not provide compelling evidence for TRPC1 (rs12634067) and TRPC5 (rs5942757) being susceptibility factors for IHPS [6]. 76 4. TRP GENE POLYMORPHISM AND DISEASE RISK THE TRPM SUBFAMILY

The mammalian melastatin-related transient receptor potential (TRPM) subfamily consists of eight members. The highly divalent-permeable cation channels TRPM6 and TRPM7 are involved in the control of Mg2+ influx, whereas the Ca2+-impermeable channels TRPM4 and TRPM5 modulate cellular Ca2+ entry by determining the membrane potential. TRPM2, TRPM3, and TRPM8 mediate a direct influx of Ca2+ in response to specific stimuli [102]. A lot of variants in TRPM1 that cause amino acid substitutions are described to be associated with complete congenital stationary night blindness [26–28,30]. (See Chapter 2 for more details.) In a study among patients suffering from schizophrenia, a GWAS was performed to detect genetic variation underlying individual differences in response to treatment with antipsychotics. Applying the Positive and Negative Syndrome Scale, it was observed that a nonsynonymous SNP p.Val605Met (rs17815774) in TRPM1 modulated response to risperidone on negative symptoms [29]. In 39 large families with T2D background, 18 SNPs of the TRPM1 gene were screened for their association with albuminuria, a prognostic marker for cardiovascular and renal disease risk in diabetic and nondiabetic subjects. No association was found for the 18 SNPs tested with albumin-to-creatinine ratio; only SNP rs11070811 showed a modest association with triglyceride levels [31]. Allelic association in subjects with bipolar disorder (BD) showed significant results with TRPM2 SNP p.Asp543Glu (rs1556314) [32]. The association of this SNP was also found in both Caucasian case-control and family study designs; overtransmission of the c.1629G allele was observed essentially in BD type I families, whereas a trend toward undertransmission of this allele was seen in BD type II families [33]. SNP rs1618355 in intron 18 was significantly associated with BD as a whole and when stratified into BD-I and BD-II subgroups [35]. Haplotype analysis revealed statistically significant associations between BD-I probands with early age at onset and the C-T haplotype of rs1618355 and rs933151 in introns 18 and 20 and with the C-T-A haplotype of rs1618355, rs933151, and rs749909 in introns 18, 20, and 27 [33]. Basal calcium concentration in B-lymphoblast cell lines was significantly higher in BD-I patients, with the TRPM2 intron 19 SNP (rs1612472) T/T genotype compared to those with T/C and with C/C genotypes [35]. A genome-wide scan in bipolar affective disorder (BPAD) affected sib-pairs identified by linkage on chromosome 21 at 21q22, fine-mapping identified TRPM2 as a possible gene at 21q22. Fourteen SNPs spanning the TRPM2 gene (among them rs1556314 and rs933151) were tested for association but did not reveal any evidence for association of this gene with BPAD [37]. In the unique mineral environment in Guam, which is also found in Kii peninsula of Japan and southern West New Guinea, with severely low levels of Ca2+ and Mg2+ yet high in transition metals, a variant of TRPM2 (p.Pro1018Leu, rs145947009) was found in a subset of Guamanian amyotrophic lateral sclerosis (ALS-G) and parkinsonism-dementia (PD-G) [34]. In a case-control investigation of subjects with diabetes mellitus type 2 (T2D), the intronic TRPM2 variants rs2838553, rs2838554, and rs4818917 were inversely associated with beta-cell function (HOMA-%B) [36]. These variants also showed borderline significance for association with fasting plasma insulin levels with p < 0.05; however, none of these remained significant following correction for multiple testing. These results support the hypothesis that TRPM2 gene variation may modify β-cell function. Although HOMA-%B was negatively associated with the described SNPs, the present case-control investigation found no evidence for an The TRPM Subfamily 77

association of the TRPM2 variants tested with other diabetes-related intermediate phenotypes (HOMA-insulin resistance (HOMA-IR), fasting glucose levels, hemoglobin A1c (HbA1c) levels) nor with T2D [36]. Variants in TRP channels were also identified in participants of the Framingham Heart Study that seemed to contribute to blood lipid phenotypes. A genome-wide association study followed by multivariable linear regression using generalized estimating equations produced a significant association of TRPM3 SNPs rs688933 and rs541326 and mean HDL-cholesterol levels, but there was lack of family-based association. However, after three stages of replication, there was no convincing statistical evidence for an association between any of the tested SNPs and lipid phenotypes [38]. Different TRPM4 polymorphisms were identified in patients with progressive familial heart block type I. Variant c.19G>A (rs267607142, p.Glu7Lys) was exclusively detected in DNA samples of affected family members [39]. In each of the three families with autosomal dominant isolated cardiac conduction block, a heterozygous missense mutation of TRPM4 gene was found (rs387907216, p.Arg164Trp; rs201907325, p.Ala432Thr; and rs200038418, p.Gly844Asp, respectively). All three variants result in a gain of function by TRPM4 channel being highly expressed in cardiac Purkinje fibers [41]. In a cohort of unrelated patients with various types of inherited cardiac arrhythmic syndromes, additional TRPM4 variants were identified (p.Gln131His, p.Gln293Arg, p.Gly582Ser, p.Tyr790His, p.Lys914Arg, p.Pro970Ser) that play a major role in cardiac conduction disease [40]. In Chinese subjects with persistent hepatitis B virus infection, the TRPM5 rs886277 (p.Asn235Ser) polymorphism was associated with the risk of liver cirrhosis. The frequency of this SNP was also associated with the severity of decompensated cirrhosis based on the Child-Pugh classification [42]. To determine the effects of genetic polymorphisms in TRPM channel genes on the risk of primary open-angle glaucoma (POAG, progressive degeneration of the axons of the retinal ganglion cells), 26 SNPs from TRPM1-8 were studied in a Turkish population. The TRPM5 SNP rs34551253 (p.Ala456Thr) was significantly associated with POAG. The TT genotype frequency was significantly higher in patients with POAG than in the controls and could be a risk factor for developing POAG. There were no marked associations with the other 25 TRPM polymorphisms studied [43]. Numerous variants in TRPM6 gene have been associated with the rare autosomal recessive disorder hypomagnesemia with secondary hypocalcaemia, also called hypomagnesemia intestinal type 1 (HOMG1) [44–46,48,49,103] (for details regarding this channelopathy, see Chapter 2). In a genome-wide association study to identify loci influencing serum magnesium level, the T allele of intronic TRPM6 SNP rs11144134 was significantly associated with hypomagnesemia and femoral neck bone mineral density. The association was genome-wide significant when meta-analyzed with the replication dataset [52]. In another study, extracellular magnesium concentration was not significantly associated with TRPM6 p.Lys1584Glu (rs2274924) polymorphism [50]. In meta-analysis the interaction between magnesium intake and SNPs related to fasting glucose, insulin, and magnesium was quantified. No magnesium-related SNP or interaction between any SNP and magnesium reached significance after correction for multiple testing. However, rs2274924 in TRPM6 (p.Lys1584Glu) showed a nominal association with glucose [51]. To elucidate if common genetic variations in TRPM6 and TRPM7 contribute to risk of type 2 diabetes mellitus (T2D), SNPs were analyzed in incident diabetes cases and their controls. There was no robust and significant association 78 4. TRP GENE POLYMORPHISM AND DISEASE RISK

between any SNP nor common TRPM6 and TRPM7 haplotypes and diabetes risk. Only women with TRPM6 haplotype p.1393Ile-p.1584Glu (rs3750425 and rs2274924, respectively) had an increased risk of type 2 diabetes only under the prerequisite of low magnesium intake (<250 mg/day) [47]. A case-control study was not able to show any evidence of an association of the 29 tested TRPM6 and TRPM7 SNPs with diabetes-related intermediate phenotypes or presence of T2D after correction for multiple testing [53]. A TRPM7 variant (rs8042919, p.Thr1482Ile) was found in a subset of ALS-G and PD-G patients [54]. No association was found between this TRPM7 variant and ALS/PDC in the Kii peninsula of Japan [55]. Total magnesium consumption was linked to a significant lower risk of colorectal adenoma, particularly in those subjects with low Ca2+ and Mg2+ intake. The p.Thr1482Ile polymorphism in TRPM7 was associated with an elevated risk of both adeno- matous and hyperplastic polyps. Carriers of p.1482Ile who consumed diets with a high Ca2+ and Mg2+ content were at a higher risk of adenoma and hyperplastic polyps than subjects who did not carry the polymorphism [56]. To test the hypothesis that TRPM7 gene variations might play a role in the risk of ischemic stroke, a case-control study was performed. But no evidence for an association between the 16 SNPs tested and ischemic stroke was found [57]. Analysis of blood serum lipids in Shorians (a Turkish minority in Russia) and genotyping of TRPM8 SNPs revealed an association of the synonymous SNP rs11562975 (c.750G>C) with total cholesterol and LDL-cholesterol level and association of rs28901637 (c.747A>T) with blood serum HDL-cholesterol level. Mean level of total cholesterol and LDL cholesterol was higher in individuals with heterozygous genotype of c.750G>C as compared to the GG genotype. Individuals with heterozygous genotype in c.747A>T had lower HDL-cholesterol level than individuals with the AA genotype. Also an association of c.750G>C with anthropometric parameters (waist and hip circumference) characterizing lipid metabolism disturbances was found [58]. In a genome-wide association study, the intronic TRPM8 SNP rs17862920 showed significant association with migraine [59]. In a population-based genome-wide analysis including migraineurs and nonmigraineurs, TRPM8 SNP rs10166942 was associated with migraine compared to nonmigraine headache. This SNP was significant in meta-analysis among three replication cohorts and met genome-wide significance in meta-analysis combining discovery and replication cohorts [60]. To examine the genetic contribution to individual cold and heat pain sensitivity, variations in TRPM8 were investigated. The finding that there was no association between the investigated SNPs of TRPM8 and cold or heat pain sensitivity may be due to the used painful cold temperature (2-4 °C) in contrast to the higher range of threshold temperature of TRPM8 (8-28 °C) [3]. In neuropathic pain patients, none of the six TRPM8 SNPs tested showed any association with the parameter used for standardized quantitative sensory testing [1].

THE TRPML SUBFAMILY

The mucolipin family of transient receptor potential channels (TRPML) forms ion channels expressed in intracellular endosomes and lysosomes. Mutations can lead to endosomal/ lysosomal dysfunction and following neurodegeneration. TRPMLs fulfill multiple cellular functions including membrane trafficking, signal transduction, and organellar ion homeostasis [104]. The TRPV Subfamily 79

Variants in TRPML1 cause mucolipidosis type IV, an autosomal recessive neurodegenerative lysosomal storage disorder ([61–67,104]; for details, see Chapters 2 and 24).

THE TRPV SUBFAMILY

TRPV1 has various physiological functions. TRPV1 is predominantly expressed on small- diameter nociceptive neurons, likely to be C-fibers [105], and is involved in the transduction of noxious heat. Interestingly, it can be stimulated by the hot chili pepper constituent capsaicin [106]. Accordingly, the role of TRPV1 in human pain sensitivity was addressed in different studies. To examine the contribution of TRPV1 to individual cold and heat pain sensitivity, genetic variations in TRPV1 were investigated in certain association studies. Caucasian- American women with the TRPV1 585Val allele (p.Ile585Val, c.1753A>G, rs8065080) showed longer cold withdrawal times [73]. In a larger cohort, SNPs rs222747 (p.Met315Ile, c.945G>C) and rs8065080 (p.Ile585Val) showed no significant association with cold/heat pain sensitivity in Americans of European extraction [3]. To investigate genetic variations of TRPV1 in patients with chronic pancreatitis suffering from pancreatic pain, four SNPs (rs222749, rs222747, rs224534, rs8065050) were genotyped in patients and healthy controls. There was no significant difference in allele frequency between chronic pancreatitis patients and healthy controls. Furthermore, based on the SNP distribution, 17 diplotypes were generated. There was no significant difference in distribution of diplotypes between patients and controls [69]. In neuropathic pain patients with mainly preserved sensory function (a subgroup of the entire patient population), TRPV1 SNPs 1911A>G (rs8065080, c.1753A>G) and 1103C>G (rs222747, c.945G>C) had a significant relationship with the somatosensory function. The TRPV1 1911A>G (c.1753A>G) polymorphism was significantly associated with altered heat pain thresholds (HPT). Neuropathic pain patients with AA or AG genotype tended to show heat hyperalgesia, whereas GG homozygotes exhibited significantly higher, i.e., normal, HPT. TRPV1 1911A>G was also identified to be associated significantly to mechanical pain sensitivity (MPS). The presence of at least one G-variant allele was associated with lower, i.e., normalized, MPS to pinprick stimuli. Moreover, TRPV1 1911A>G wild-type carriers (AA) showed higher mechanical detection thresholds, i.e., mechanical hypoaesthesia, than variant subjects. The 1103C>G SNP (c.945G>C) was significantly associated with cold detection threshold. Homozygous variant carriers (GG) exhibited cold hypoaesthesia compared with heterozygous or wild-type carriers [1]. TRPV1 variants were also investigated for their association with a variety of other diseases. In a Japanese population, a significant inverse association between CC genotype of the TRPV1 SNP c.945G>C and functional dyspepsia was found. This genotype also had a lower risk of epigastric pain syndrome, postprandial syndrome, and Helicobacter pylori positive functional dyspepsia [71]. Because TRPV1 expression is known to be up-regulated in patients with irritable bowel syndrome (IBS), a Korean population was screened for their genotype frequencies of nonsynonymous TRPV1 SNPs rs9894618, rs222749, and rs222747. There was no significant difference in allele frequency of these three SNPs between controls and IBS group [68]. There was a significant increase in the rs222747 (p.Met315Ile) variant of the TRPV1 gene in the type 1 diabetes cohort compared to the control. Logistic regression analysis revealed that type 1 diabetes was significantly associated with p.Met315Ile. 80 4. TRP GENE POLYMORPHISM AND DISEASE RISK

No difference was found in the rs224534 (p.Thr469Ile) and rs8065080 (p.Ile585Val) allelic variants [72]. In a study where capsinoids were taken for weight loss, the TRPV1 SNP p.Ile585Val correlated significantly with change in abdominal adiposity. Subjects with the Val/Val and Val/Ile variants lost about twice as much abdominal fat as the study average, whereas Ile/ Ile subjects lost almost none [74]. The TRPV1 rs8065080 polymorphism was found to be associated with an individual’s perception of salt at suprathreshold levels [76]. TRPV1 expression and activity in the respiratory system appear to be altered under pathophysiological conditions such as chronic cough and airway hypersensitivity. In childhood asthma, carriers of the TRPV1 p.585Val variant showed a lower risk of current wheezing or cough [75]. No association with nonspecific chronic cough in children was found for TRPV1 SNPs rs222748 and rs8065080 [70]. Also the 3′-UTR region of TRPV1 was associated with childhood asthma. Allele frequency of SNP rs4790521 T>C was significantly increased in asthmatic children, but no significant difference was found in MAF of rs4790522 A>C. Genotype analysis showed that rs4790521 C/C and rs4790522 A/C were significantly associated with childhood asthma in Chinese of Han Nationality [79]. Statistically significant associations of six TRPV1 SNPs (rs11655540, rs161365, rs17706630, rs2277675, rs150854, rs224498) with cough symptoms were found in nonasthmatics after correction for multiple comparisons. Haplotype-based association analysis confirmed the SNP analyses for nocturnal cough and usual cough in subjects without asthma [4]. In multiple sclerosis (MS) patients, a selective risk-association of TRPV1 SNP rs877610 was found in primary progressive disease. Specific SNPs in the TRPV1 locus were either significantly overrepresented in DNA of patients with malignant MS or underrepresented in the ge- nomes of patients with benign MS [77]. A genome-wide and candidate gene association study of cigarette smoking behaviors revealed an association of TRPV1 polymorphism rs4790520 with cigarettes smoked per day [11]. To identify SNPs in TRP genes that may confer increased genetic susceptibility to migraine, a case-control genetic association study with replication was performed. After replication, nominal association was confirmed for two intronic SNPs, the T allele from TRPV1 rs222741 in the all-migraine group and the A allele from TRPV3 rs7217270 in the migraine with aura group. However, after applying Bonferroni correction for multiple comparisons, none of them remained significant [78]. Mutations in the TRPV3 gene are described to be associated with Olmsted syndrome, a rare congenital disorder characterized by palmoplantar and periorificial keratoderma. In six cases from China missense variants in TRPV3, which produced p.Gly573Ser, p.Gly573Cys, and p.Trp692Gly, were reported [80]. Numerous missense variations in TRPV4 lead to several skeletal diseases and neuropathies, e.g., metatropic dysplasia (MD) [85,92], brachyolmia type 3 [94], Charcot-Marie-Tooth disease [86–88,91], familial digital arthropathy brachydactyly [90], spondylometaphyseal dysplasia Kozlowski type [85,93], spondyloepiphyseal dysplasia Maroteaux type [84], scapuloperoneal spinal muscular atrophy [88,89], and congenital distal spinal muscle atrophy [83,89]. (For details, see Chapter 2.) To assess the impact of TRPV4 polymorphisms on chronic obstructive pulmonary disease (COPD)-related phenotypes, an association of 20 SNPs with COPD was performed in a family-based analysis. Seven SNPs (rs12578401 in intron 7, rs3825396 in intron 6, rs12579553 in intron 6, rs16940583 in intron 5, rs3742030 in exon 2 p.Pro19Ser, rs7971845 in intron 1, and rs6606743) showed significant association with COPD. Four of these SNPs (rs12578401, Disease Risk, Clinical Diagnosis, and Personalized Medicine 81 rs3825396, rs12579553, and rs16940583) were significantly associated with COPD even after a Bonferroni correction. The significant associations between the four SNPs and COPD in the case-control population replicated the results with the same effect directions (same risk allele in both populations) in the family data [82]. Hyponatremia (i.e., relative water excess, serum sodium concentration ≤138 mEq/L) was significantly associated with the TRPV4 p.Pro19Ser allele in two non-Hispanic Caucasian male populations. Mean serum sodium concentration was significantly lower in the TRPV4-p.Pro19Ser-positive subjects. Subjects with the minor allele were 2.43-6.45 times as likely to exhibit hyponatremia as subjects without the allele. Mean serum sodium concentration among subjects with one copy of the minor allele was significantly lower [81]. In childhood asthma, TRPV4 p.Pro19Ser showed no significant association with asthma or the presence of wheezing [75]. Associations between SNPs in TRPV4 and cough symptoms in subjects with or without asthma were also not significant after adjusting for multiple testing [4]. In patients with hypercalciuria and concomitant polyuria or decreased urinary pH synonymous polymorphisms of TRPV5 (p.Leu205=, p.Tyr222=, p.Tyr278=, p.Thr281=, p.Thr344=) and nonsynonymous SNPs (p.Ala8Val, p.Arg154His, p.Ala561Thr) were identified. In this specific research population, data do not support a primary role for TRPV5 in the pathogenesis of renal hypercalciuria [95]. Also, the relatively high frequency of TRPV5 p.Ala563Thr variant in African Americans, which exhibited an increased Ca2+ influx in in vitro assays [107], was not investigated for their function in Ca2+ hyperuria in African Americans. In renal calcium stone patients, three major nonsynonymous TRPV6 polymorphisms were identified (p.Cys157Arg, p.Met378Val, and p.Met681Thr). The frequency of the ancestral haplotype (157Arg+378Val+681Thr) was higher in Ca2+ stone formers when compared to a cohort of nonstone formers [96]. TRPV6 is overexpressed in prostatic adenocarcinoma tissue but is not detectable in healthy and benign prostate tissue [108]. The prostatic adenocarcinoma incidence within African Americans is two to three times increased compared to Caucasians. The ancestral haplotype of TRPV6 (157R+378V+681T), here named TRPV6a, is common among African populations. Within Caucasians ~87% exhibit the homozygous TRPV6b (157C, 378M, and 681M) genotype [109]. In the samples of prostatic adenocarcinoma tested, the TRPV6b allele was found in 87% without correlation with Gleason score and tumor stage. The occurrence of the TRPV6a allele did not correlated with a higher incidence of prostatic adenocarcinoma [97].

DISEASE RISK, CLINICAL DIAGNOSIS, AND PERSONALIZED MEDICINE

Sequencing of the human genome set the prerequisite to turn personalized medicine from an idea to a practice. In case of TRP channels, there is a lot of knowledge about genetic variants in patients suffering from the “classical” channelopathies (see Chapter 2). Moreover, there are a number of hints that TRP channels are involved in physiological disturbances. As described earlier, some “risky genes” have been identified leading to cardiovascular, renal, neurological, and inflammatory diseases. In particular, variants in the noxious pain receptors TRPA1 and TRPV1 contribute to the modulation of specific neuropathic pain characteristics. But most of those frequent genetic markers have not yet been proven as prognostic biomarker 82 4. TRP GENE POLYMORPHISM AND DISEASE RISK that could be applied for stratification of high or low risk patients or as a guide for patient information and monitoring. Aside from genetic information, there is a need to integrate molecular biological data not only with epigenetic modulation and expression profiles, but also with a patient’s physiological and anatomical characteristics to better understand the interplay of such factors in the development of defined human diseases or response to pharmacological interventions.

References [1] Binder A, May D, Baron R, Maier C, Tolle TR, Treede RD, et al. Transient receptor potential channel polymorphisms are associated with the somatosensory function in neuropathic pain patients. PLoS One 2011;6(3):e17387. [2] Kremeyer B, Lopera F, Cox JJ, Momin A, Rugiero F, Marsh S, et al. A gain-of-function mutation in TRPA1 causes familial episodic pain syndrome. Neuron 2010;66(5):671–80. [3] Kim H, Mittal DP, Iadarola MJ, Dionne RA. Genetic predictors for acute experimental cold and heat pain sensitivity in humans. J Med Genet 2006;43(8):e40. [4] Smit LA, Kogevinas M, Anto JM, Bouzigon E, Gonzalez JR, Le Moual N, et al. Transient receptor potential genes, smoking, occupational exposures and cough in adults. Respir Res 2012;13:26. [5] Chen K, Jin X, Li Q, Wang W, Wang Y, Zhang J. Association of TRPC1 gene polymorphisms with type 2 diabetes and diabetic nephropathy in Han Chinese population. Endocr Res 2013;38(2):59–68. [6] Everett KV, Chioza BA, Georgoula C, Reece A, Gardiner RM, Chung EM. Infantile hypertrophic pyloric stenosis: evaluation of three positional candidate genes, TRPC1, TRPC5 and TRPC6, by association analysis and re-sequencing. Hum Genet 2009;126(6):819–31. [7] Zhang D, Freedman BI, Flekac M, Santos E, Hicks PJ, Bowden DW, et al. Evaluation of genetic association and expression reduction of TRPC1 in the development of diabetic nephropathy. Am J Nephrol 2009;29(3):244–51. [8] Becker EB, Fogel BL, Rajakulendran S, Dulneva A, Hanna MG, Perlman SL, et al. Candidate screening of the TRPC3 gene in cerebellar ataxia. Cerebellum 2011;10(2):296–9. [9] Martin-Trujillo A, Iglesias-Platas I, Coto E, Corral-Juan M, San Nicolas H, Corral J, et al. Genotype of an individual single nucleotide polymorphism regulates DNA methylation at the TRPC3 alternative promoter. Epigenetics 2011;6(10):1236–41. [10] von Spiczak S, Muhle H, Helbig I, de Kovel CG, Hampe J, Gaus V, et al. Association study of TRPC4 as a candidate gene for generalized epilepsy with photosensitivity. Neuromol Med 2010;12(3):292–9. [11] Caporaso N, Gu F, Chatterjee N, Sheng-Chih J, Yu K, Yeager M, et al. Genome-wide and candidate gene association study of cigarette smoking behaviors. PLoS One 2009;4(2):e4653. [12] Obeidova L, Reiterova J, Lnenicka P, Stekrova J, Safrankova H, Kohoutova M, et al. TRPC6 gene variants in Czech adult patients with focal segmental glomerulosclerosis and minimal change disease. Folia Biol (Praha) 2012;58(4):173–6. [13] Santin S, Ars E, Rossetti S, Salido E, Silva I, Garcia-Maset R, et al. TRPC6 mutational analysis in a large cohort of patients with focal segmental glomerulosclerosis. Nephrol Dial Transplant 2009;24(10):3089–96. [14] Winn MP, Conlon PJ, Lynn KL, Farrington MK, Creazzo T, Hawkins AF, et al. A mutation in the TRPC6 cation channel causes familial focal segmental glomerulosclerosis. Science 2005;308(5729):1801–4. [15] Heeringa SF, Moller CC, Du J, Yue L, Hinkes B, Chernin G, et al. A novel TRPC6 mutation that causes childhood FSGS. PLoS One 2009;4(11):e7771. [16] Reiser J, Polu KR, Moller CC, Kenlan P, Altintas MM, Wei C, et al. TRPC6 is a glomerular slit diaphragm- associated channel required for normal renal function. Nat Genet 2005;37(7):739–44. [17] Hofstra JM, Lainez S, van Kuijk WH, Schoots J, Baltissen MP, Hoefsloot LH, et al. New TRPC6 gain-of-function mutation in a non-consanguineous Dutch family with late-onset focal segmental glomerulosclerosis. Nephrol Dial Transplant 2013;28(7):1830–8. [18] Gigante M, Caridi G, Montemurno E, Soccio M, d’Apolito M, Cerullo G, et al. TRPC6 mutations in children with steroid-resistant nephrotic syndrome and atypical phenotype. Clin J Am Soc Nephrol 2011;6(7):1626–34. [19] Buscher AK, Konrad M, Nagel M, Witzke O, Kribben A, Hoyer PF, et al. Mutations in podocyte genes are a rare cause of primary FSGS associated with ESRD in adult patients. Clin Nephrol 2012;78(1):47–53. REFERENCES 83

[20] Mir S, Yavascan O, Berdeli A, Sozeri B. TRPC6 gene variants in Turkish children with steroid-resistant nephrotic syndrome. Nephrol Dial Transplant 2012;27(1):205–9. [21] Zhu B, Chen N, Wang ZH, Pan XX, Ren H, Zhang W, et al. Identification and functional analysis of a novel TRPC6 mutation associated with late onset familial focal segmental glomerulosclerosis in Chinese patients. Mutat Res 2009;664(1–2):84–90. [22] Yu Y, Keller SH, Remillard CV, Safrina O, Nicholson A, Zhang SL, et al. A functional single-nucleotide polymorphism in the TRPC6 gene promoter associated with idiopathic pulmonary arterial hypertension. Circulation 2009;119(17):2313–22. [23] Kuang XY, Huang WY, Xu H, Shi Y, Zhang XL, Niu XL, et al. 254C>G: a TRPC6 promoter variation associated with enhanced transcription and steroid-resistant nephrotic syndrome in Chinese children. Pediatr Res 2013;74(5):511–16. [24] Chen WC, Chen SY, Chen CH, Chen HY, Lin YW, Ho TJ, et al. Lack of association between transient receptor potential cation channel 6 polymorphisms and primary membranous glomerulonephritis. Ren Fail 2010;32(6):666–72. [25] Bierut LJ, Madden PA, Breslau N, Johnson EO, Hatsukami D, Pomerleau OF, et al. Novel genes identified in a high-density genome wide association study for nicotine dependence. Hum Mol Genet 2007;16(1):24–35. [26] Li Z, Sergouniotis PI, Michaelides M, Mackay DS, Wright GA, Devery S, et al. Recessive mutations of the gene TRPM1 abrogate ON bipolar cell function and cause complete congenital stationary night blindness in humans. Am J Hum Genet 2009;85(5):711–19. [27] Audo I, Kohl S, Leroy BP, Munier FL, Guillonneau X, Mohand-Said S, et al. TRPM1 is mutated in patients with autosomal-recessive complete congenital stationary night blindness. Am J Hum Genet 2009;85(5):720–9. [28] van Genderen MM, Bijveld MM, Claassen YB, Florijn RJ, Pearring JN, Meire FM, et al. Mutations in TRPM1 are a common cause of complete congenital stationary night blindness. Am J Hum Genet 2009;85(5):730–6. [29] McClay JL, Adkins DE, Aberg K, Stroup S, Perkins DO, Vladimirov VI, et al. Genome-wide pharmacogenomic analysis of response to treatment with antipsychotics. Mol Psychiatry 2011;16(1):76–85. [30] Nakamura M, Sanuki R, Yasuma TR, Onishi A, Nishiguchi KM, Koike C, et al. TRPM1 mutations are associated with the complete form of congenital stationary night blindness. Mol Vis 2010;16:425–37. [31] Thameem F, Puppala S, Arar NH, Blangero J, Duggirala R, Abboud HE. Genetic variants in transient receptor potential cation channel, subfamily M 1 (TRPM1) and their risk of albuminuria-related traits in Mexican Americans. Clin Chim Acta 2011;412(23–24):2058–62. [32] McQuillin A, Bass NJ, Kalsi G, Lawrence J, Puri V, Choudhury K, et al. Fine mapping of a susceptibility locus for bipolar and genetically related unipolar affective disorders, to a region containing the C21ORF29 and TRPM2 genes on chromosome 21q22.3. Mol Psychiatry 2006;11(2):134–42. [33] Xu C, Li PP, Cooke RG, Parikh SV, Wang K, Kennedy JL, et al. TRPM2 variants and bipolar disorder risk: con- firmation in a family-based association study. Bipolar Disord 2009;11(1):1–10. [34] Hermosura MC, Cui AM, Go RC, Davenport B, Shetler CM, Heizer JW, et al. Altered functional properties of a TRPM2 variant in Guamanian ALS and PD. Proc Natl Acad Sci USA 2008;105(46):18029–34. [35] Xu C, Macciardi F, Li PP, Yoon IS, Cooke RG, Hughes B, et al. Association of the putative susceptibility gene, transient receptor potential protein melastatin type 2, with bipolar disorder. Am J Med Genet B Neuropsychiatr Genet 2006;141B(1):36–43. [36] Romero JR, Germer S, Castonguay AJ, Barton NS, Martin M, Zee RY. Gene variation of the transient receptor potential cation channel, subfamily M, member 2 (TRPM2) and type 2 diabetes mellitus: a case-control study. Clin Chim Acta 2010;411(19–20):1437–40. [37] Roche S, Cassidy F, Zhao C, Badger J, Claffey E, Mooney L, et al. Candidate gene analysis of 21q22: support for S100B as a susceptibility gene for bipolar affective disorder with psychosis. Am J Med Genet B Neuropsychiatr Genet 2007;144B(8):1094–6. [38] Kathiresan S, Manning AK, Demissie S, D’Agostino RB, Surti A, Guiducci C, et al. A genome-wide association study for blood lipid phenotypes in the Framingham Heart Study. BMC Med Genet 2007;8(Suppl. 1):S17. [39] Kruse M, Schulze-Bahr E, Corfield V, Beckmann A, Stallmeyer B, Kurtbay G, et al. Impaired endocytosis of the ion channel TRPM4 is associated with human progressive familial heart block type I. J Clin Invest 2009;119(9):2737–44. [40] Stallmeyer B, Zumhagen S, Denjoy I, Duthoit G, Hebert JL, Ferrer X, et al. Mutational spectrum in the Ca(2+)-activated cation channel gene TRPM4 in patients with cardiac conductance disturbances. Hum Mutat 2012;33(1):109–17. 84 4. TRP GENE POLYMORPHISM AND DISEASE RISK

[41] Liu H, El Zein L, Kruse M, Guinamard R, Beckmann A, Bozio A, et al. Gain-of-function mutations in TRPM4 cause autosomal dominant isolated cardiac conduction disease. Circ Cardiovasc Genet 2010;3(4):374–85. [42] Peng L, Guo J, Zhang Z, Liu L, Cao Y, Shi H, et al. A candidate gene study for the association of host single nucleotide polymorphisms with liver cirrhosis risk in chinese hepatitis B patients. Genet Test Mol Biomarkers 2013;17(9):681–6. [43] Okumus S, Demiryurek S, Gurler B, Coskun E, Bozgeyik I, Oztuzcu S, et al. Association transient receptor potential melastatin channel gene polymorphism with primary open angle glaucoma. Mol Vis 2013;19:1852–8. [44] Schlingmann KP, Weber S, Peters M, Niemann Nejsum L, Vitzthum H, Klingel K, et al. Hypomagnesemia with secondary hypocalcemia is caused by mutations in TRPM6, a new member of the TRPM gene family. Nat Genet 2002;31(2):166–70. [45] Apa H, Kayserili E, Agin H, Hizarcioglu M, Gulez P, Berdeli A. A case of hypomagnesemia with secondary hypocalcemia caused by Trpm6 gene mutation. Indian J Pediatr 2008;75(6):632–4. [46] Jalkanen R, Pronicka E, Tyynismaa H, Hanauer A, Walder R, Alitalo T. Genetic background of HSH in three Polish families and a patient with an X;9 translocation. Eur J Hum Genet 2006;14(1):55–62. [47] Song Y, Hsu YH, Niu T, Manson JE, Buring JE, Liu S. Common genetic variants of the ion channel transient receptor potential membrane melastatin 6 and 7 (TRPM6 and TRPM7), magnesium intake, and risk of type 2 diabetes in women. BMC Med Genet 2009;10:4. [48] Chubanov V, Schlingmann KP, Waring J, Heinzinger J, Kaske S, Waldegger S, et al. Hypomagnesemia with secondary hypocalcemia due to a missense mutation in the putative pore-forming region of TRPM6. J Biol Chem 2007;282(10):7656–67. [49] Zhao Z, Pei Y, Huang X, Liu Y, Yang W, Sun J, et al. Novel TRPM6 mutations in familial hypomagnesemia with secondary hypocalcemia. Am J Nephrol 2013;37(6):541–8. [50] Shuen AY, Wong BY, Wei C, Liu Z, Li M, Cole DE. Genetic determinants of extracellular magnesium concentration: analysis of multiple candidate genes, and evidence for association with the estrogen receptor alpha (ESR1) locus. Clin Chim Acta 2009;409(1–2):28–32. [51] Hruby A, Ngwa JS, Renstrom F, Wojczynski MK, Ganna A, Hallmans G, et al. Higher magnesium intake is associated with lower fasting glucose and insulin, with no evidence of interaction with select genetic loci, in a meta-analysis of 15 CHARGE Consortium Studies. J Nutr 2013;143(3):345–53. [52] Meyer TE, Verwoert GC, Hwang SJ, Glazer NL, Smith AV, van Rooij FJ, et al. Genome-wide association studies of serum magnesium, potassium, and sodium concentrations identify six Loci influencing serum magnesium levels. PLoS Genet 2010;6(8):e1001045. [53] Romero JR, Castonguay AJ, Barton NS, Germer S, Martin M, Zee RY. Gene variation of the transient receptor potential cation channel, subfamily M, members 6 (TRPM6) and 7 (TRPM7), and type 2 diabetes mellitus: a case-control study. Transl Res 2010;156(4):235–41. [54] Hermosura MC, Nayakanti H, Dorovkov MV, Calderon FR, Ryazanov AG, Haymer DS, et al. A TRPM7 variant shows altered sensitivity to magnesium that may contribute to the pathogenesis of two Guamanian neurodegenerative disorders. Proc Natl Acad Sci USA 2005;102(32):11510–15. [55] Hara K, Kokubo Y, Ishiura H, Fukuda Y, Miyashita A, Kuwano R, et al. TRPM7 is not associated with amyotrophic lateral sclerosis-parkinsonism dementia complex in the Kii peninsula of Japan. Am J Med Genet B Neuropsychiatr Genet 2010;153B(1):310–13. [56] Dai Q, Shrubsole MJ, Ness RM, Schlundt D, Cai Q, Smalley WE, et al. The relation of magnesium and calcium intakes and a genetic polymorphism in the magnesium transporter to colorectal neoplasia risk. Am J Clin Nutr 2007;86(3):743–51. [57] Romero JR, Ridker PM, Zee RY. Gene variation of the transient receptor potential cation channel, subfamily M, member 7 (TRPM7), and risk of incident ischemic stroke: prospective, nested, case-control study. Stroke 2009;40(9):2965–8. [58] Potapova TA, Yudin NS, Pilipenko IV, Kobsev VF, Romashchenko AG, Shakhtshneider EV, et al. Association of cold receptor TRPM8 gene polymorphism with blood lipid indices and anthropometric parameters in Shorians. Bull Exp Biol Med 2011;151(2):223–6. [59] Anttila V, Stefansson H, Kallela M, Todt U, Terwindt GM, Calafato MS, et al. Genome-wide association study of migraine implicates a common susceptibility variant on 8q22.1. Nat Genet 2010;42(10):869–73. [60] Chasman DI, Schurks M, Anttila V, de Vries B, Schminke U, Launer LJ, et al. Genome-wide association study reveals three susceptibility loci for common migraine in the general population. Nat Genet 2011;43(7):695–8. REFERENCES 85

[61] Bach G, Webb MB, Bargal R, Zeigler M, Ekstein J. The frequency of mucolipidosis type IV in the Ashkenazi Jewish population and the identification of 3 novel MCOLN1 mutations. Hum Mutat 2005;26(6):591. [62] Sun M, Goldin E, Stahl S, Falardeau JL, Kennedy JC, Acierno Jr JS, et al. Mucolipidosis type IV is caused by mutations in a gene encoding a novel transient receptor potential channel. Hum Mol Genet 2000;9(17):2471–8. [63] Altarescu G, Sun M, Moore DF, Smith JA, Wiggs EA, Solomon BI, et al. The neurogenetics of mucolipidosis type IV. Neurology 2002;59(3):306–13. [64] Bargal R, Avidan N, Olender T, Ben Asher E, Zeigler M, Raas-Rothschild A, et al. Mucolipidosis type IV: novel MCOLN1 mutations in Jewish and non-Jewish patients and the frequency of the disease in the Ashkenazi Jewish population. Hum Mutat 2001;17(5):397–402. [65] Goldin E, Stahl S, Cooney AM, Kaneski CR, Gupta S, Brady RO, et al. Transfer of a mitochondrial DNA fragment to MCOLN1 causes an inherited case of mucolipidosis IV. Hum Mutat 2004;24(6):460–5. [66] AlBakheet A, Qari A, Colak D, Rasheed A, Kaya N, Al-Sayed M. A novel mutation in a large family causes a unique phenotype of Mucolipidosis IV. Gene 2013;526(2):464–6. [67] Tuysuz B, Goldin E, Metin B, Korkmaz B, Yalcinkaya C. Mucolipidosis type IV in a Turkish boy associated with a novel MCOLN1 mutation. Brain Dev 2009;31(9):702–5. [68] Song YA, Park SY, Park YL, Chung CY, Lee GH, Cho DH, et al. Association between single nucleotide polymorphisms of the transient receptor potential vanilloid 1 (TRPV-1) gene and patients with irritable bowel syndrome in Korean populations. Acta Gastroenterol Belg 2012;75(2):222–7. [69] van Esch AA, Lamberts MP, te Morsche RH, van Oijen MG, Jansen JB, Drenth JP. Polymorphisms in gene encoding TRPV1-receptor involved in pain perception are unrelated to chronic pancreatitis. BMC Gastroenterol 2009;9:97. [70] Zhang XN, Yang J, Luo ZX, Luo J, Ren L, Li B, et al. Etiology of nonspecific chronic cough in children and relationship between TRPV1 gene polymorphisms and nonspecific chronic cough. Zhongguo Dang Dai Er Ke Za Zhi 2012;14(7):524–8. [71] Tahara T, Shibata T, Nakamura M, Yamashita H, Yoshioka D, Hirata I, et al. Homozygous TRPV1 315C influences the susceptibility to functional dyspepsia. J Clin Gastroenterol 2010;44(1):e1–7. [72] Sadeh M, Glazer B, Landau Z, Wainstein J, Bezaleli T, Dabby R, et al. Association of the M3151 variant in the transient receptor potential vanilloid receptor-1 (TRPV1) gene with type 1 diabetes in an Ashkenazi Jewish population. Isr Med Assoc J 2013;15(9):477–80. [73] Kim H, Neubert JK, San Miguel A, Xu K, Krishnaraju RK, Iadarola MJ, et al. Genetic influence on variability in human acute experimental pain sensitivity associated with gender, ethnicity and psychological temperament. Pain 2004;109(3):488–96. [74] Snitker S, Fujishima Y, Shen H, Ott S, Pi-Sunyer X, Furuhata Y, et al. Effects of novel capsinoid treatment on fatness and energy metabolism in humans: possible pharmacogenetic implications. Am J Clin Nutr 2009;89(1):45–50. [75] Cantero-Recasens G, Gonzalez JR, Fandos C, Duran-Tauleria E, Smit LA, Kauffmann F, et al. Loss of function of transient receptor potential vanilloid 1 (TRPV1) genetic variant is associated with lower risk of active childhood asthma. J Biol Chem 2010;285(36):27532–5. [76] Dias AG, Rousseau D, Duizer L, Cockburn M, Chiu W, Nielsen D, et al. Genetic variation in putative salt taste receptors and salt taste perception in humans. Chem Senses 2013;38(2):137–45. [77] Paltser G, Liu XJ, Yantha J, Winer S, Tsui H, Wu P, et al. TRPV1 gates tissue access and sustains pathogenicity in autoimmune encephalitis. Mol Med 2013;19:149–59. [78] Carreno O, Corominas R, Fernandez-Morales J, Camina M, Sobrido MJ, Fernandez-Fernandez JM, et al. SNP variants within the vanilloid TRPV1 and TRPV3 receptor genes are associated with migraine in the Spanish population. Am J Med Genet B Neuropsychiatr Genet 2012;159B(1):94–103. [79] Wang Q, Bai X, Xu D, Li H, Fang J, Zhu H, et al. TRPV1 UTR-3 polymorphism and susceptibility of childhood asthma of the Han Nationality in Beijing. Wei Sheng Yan Jiu 2009;38(5):516–21. [80] Lin Z, Chen Q, Lee M, Cao X, Zhang J, Ma D, et al. Exome sequencing reveals mutations in TRPV3 as a cause of Olmsted syndrome. Am J Hum Genet 2012;90(3):558–64. [81] Tian W, Fu Y, Garcia-Elias A, Fernandez-Fernandez JM, Vicente R, Kramer PL, et al. A loss-of-function nonsynonymous polymorphism in the osmoregulatory TRPV4 gene is associated with human hyponatremia. Proc Natl Acad Sci USA 2009;106(33):14034–9. [82] Zhu G, Gulsvik A, Bakke P, Ghatta S, Anderson W, Lomas DA, et al. Association of TRPV4 gene polymorphisms with chronic obstructive pulmonary disease. Hum Mol Genet 2009;18(11):2053–62. 86 4. TRP GENE POLYMORPHISM AND DISEASE RISK

[83] Fiorillo C, Moro F, Brisca G, Astrea G, Nesti C, Balint Z, et al. TRPV4 mutations in children with congenital distal spinal muscular atrophy. Neurogenetics 2012;13(3):195–203. [84] Nishimura G, Dai J, Lausch E, Unger S, Megarbane A, Kitoh H, et al. Spondylo-epiphyseal dysplasia, Maroteaux type (pseudo-Morquio syndrome type 2), and parastremmatic dysplasia are caused by TRPV4 mutations. Am J Med Genet A 2010;152A(6):1443–9. [85] Dai J, Kim OH, Cho TJ, Schmidt-Rimpler M, Tonoki H, Takikawa K, et al. Novel and recurrent TRPV4 mutations and their association with distinct phenotypes within the TRPV4 dysplasia family. J Med Genet 2010;47(10):704–9. [86] Klein CJ, Shi Y, Fecto F, Donaghy M, Nicholson G, McEntagart ME, et al. TRPV4 mutations and cytotoxic hypercalcemia in axonal Charcot-Marie-Tooth neuropathies. Neurology 2011;76(10):887–94. [87] Landoure G, Zdebik AA, Martinez TL, Burnett BG, Stanescu HC, Inada H, et al. Mutations in TRPV4 cause Charcot-Marie-Tooth disease type 2C. Nat Genet 2010;42(2):170–4. [88] Deng HX, Klein CJ, Yan J, Shi Y, Wu Y, Fecto F, et al. Scapuloperoneal spinal muscular atrophy and CMT2C are allelic disorders caused by alterations in TRPV4. Nat Genet 2010;42(2):165–9. [89] Auer-Grumbach M, Olschewski A, Papic L, Kremer H, McEntagart ME, Uhrig S, et al. Alterations in the ankyrin domain of TRPV4 cause congenital distal SMA, scapuloperoneal SMA and HMSN2C. Nat Genet 2010;42(2):160–4. [90] Lamande SR, Yuan Y, Gresshoff IL, Rowley L, Belluoccio D, Kaluarachchi K, et al. Mutations in TRPV4 cause an inherited arthropathy of hands and feet. Nat Genet 2011;43(11):1142–6. [91] Chen DH, Sul Y, Weiss M, Hillel A, Lipe H, Wolff J, et al. CMT2C with vocal cord paresis associated with short stature and mutations in the TRPV4 gene. Neurology 2010;75(22):1968–75. [92] Camacho N, Krakow D, Johnykutty S, Katzman PJ, Pepkowitz S, Vriens J, et al. Dominant TRPV4 mutations in nonlethal and lethal metatropic dysplasia. Am J Med Genet A 2010;152A(5):1169–77. [93] Krakow D, Vriens J, Camacho N, Luong P, Deixler H, Funari TL, et al. Mutations in the gene encoding the calcium-permeable ion channel TRPV4 produce spondylometaphyseal dysplasia, Kozlowski type and metatropic dysplasia. Am J Hum Genet 2009;84(3):307–15. [94] Rock MJ, Prenen J, Funari VA, Funari TL, Merriman B, Nelson SF, et al. Gain-of-function mutations in TRPV4 cause autosomal dominant brachyolmia. Nat Genet 2008;40(8):999–1003. [95] Renkema KY, Lee K, Topala CN, Goossens M, Houillier P, Bindels RJ, et al. TRPV5 gene polymorphisms in renal hypercalciuria. Nephrol Dial Transplant 2009;24(6):1919–24. [96] Suzuki Y, Pasch A, Bonny O, Mohaupt MG, Hediger MA, Frey FJ. Gain-of-function haplotype in the epithelial calcium channel TRPV6 is a risk factor for renal calcium stone formation. Hum Mol Genet 2008;17(11):1613–8. [97] Kessler T, Wissenbach U, Grobholz R, Flockerzi V. TRPV6 alleles do not influence prostate cancer progression. BMC Cancer 2009;9:380. [98] Story GM. The emerging role of TRP channels in mechanisms of temperature and pain sensation. Curr Neuropharmacol 2006;4(3):183–96. [99] Zurborg S, Yurgionas B, Jira JA, Caspani O, Heppenstall PA. Direct activation of the ion channel TRPA1 by Ca2+. Nat Neurosci 2007;10(3):277–9. [100] Beech DJ. Characteristics of transient receptor potential canonical calcium-permeable channels and their relevance to vascular physiology and disease. Circ J 2013;77(3):570–9. [101] Yu Y, Fantozzi I, Remillard CV, Landsberg JW, Kunichika N, Platoshyn O, et al. Enhanced expression of transient receptor potential channels in idiopathic pulmonary arterial hypertension. Proc Natl Acad Sci USA 2004;101(38):13861–6. [102] Kraft R, Harteneck C. The mammalian melastatin-related transient receptor potential cation channels: an overview. Pflugers Arch 2005;451(1):204–11. [103] Schlingmann KP, Sassen MC, Weber S, Pechmann U, Kusch K, Pelken L, et al. Novel TRPM6 mutations in 21 families with primary hypomagnesemia and secondary hypocalcemia. J Am Soc Nephrol 2005;16(10):3061–9. [104] Cheng X, Shen D, Samie M, Xu H. Mucolipins: intracellular TRPML1-3 channels. FEBS Lett 2010; 584(10):2013–21. [105] Tominaga M, Caterina MJ, Malmberg AB, Rosen TA, Gilbert H, Skinner K, et al. The cloned capsaicin receptor integrates multiple pain-producing stimuli. Neuron 1998;21(3):531–43. REFERENCES 87

[106] Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, Julius D. The capsaicin receptor: a heat- activated ion channel in the pain pathway. Nature 1997;389(6653):816–24. [107] Na T, Zhang W, Jiang Y, Liang Y, Ma HP, Warnock DG, et al. The A563T variation of the renal epithelial calcium channel TRPV5 among African Americans enhances calcium influx. Am J Physiol Renal Physiol 2009;296(5):F1042–51. [108] Wissenbach U, Niemeyer BA, Fixemer T, Schneidewind A, Trost C, Cavalie A, et al. Expression of CaT- like, a novel calcium-selective channel, correlates with the malignancy of prostate cancer. J Biol Chem 2001;276(22):19461–8. [109] Akey JM, Swanson WJ, Madeoy J, Eberle M, Shriver MD. TRPV6 exhibits unusual patterns of polymorphism and divergence in worldwide populations. Hum Mol Genet 2006;15(13):2106–13. CHAPTER 5 Use of Topical Capsaicin for Pain Relief Ganesan Baranidharan,* Arun K. Bhaskar Consultant in Pain Medicine, Leeds Teaching Hospitals NHS trust: D Ward, Seacroft Hospital, Leeds, LS14 6UH *Corresponding author: [email protected]

OUTLINE

Introduction 89 Safety and Tolerability 93 Pharmacodynamics 90 Delivery of Treatment with Capsaicin 8% Patch—Practical Aspects 94 Pharmacokinetics 91 Application Procedure for the Capsaicin Efficacy and Therapeutic Uses 91 8% Patch 96 Postherpetic Neuralgia 91 HIV-Associated Distal Sensory Postprocedural Instructions for Patients 97 Polyneuropathy 92 References 97 Other Neuropathic Pain States 93

INTRODUCTION

Capsaicin is an extract from chili peppers found in native America. They were used to spice the cuisines in Mexico and America and were then passed on to Asia by Africa by Europeans [1]. The hotness of chili peppers is measured in Scoville heat units, which are the number of times a chili extract must be diluted in water for it to lose its heat. Capsaicin scores about 16,000,000 in comparison to jalapeños, which measure about 4500 units. Turnbull described the use of chili as a hot alcoholic pepper extract to treat burning or itching extremities [2]. The pure crystalline form was isolated by Thresh in 1876 [3]. Capsaicin has

TRP Channels as Therapeutic Targets 89 © 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/B978-0-12-420024-1.00005-9 90 5. Use of Topical Capsaicin for Pain Relief been available for a long time as creams, lotions, or patches in low concentration. Capsaicin 0.025% and 0.075% could be purchased over the counter to treat neuropathic and nociceptive musculoskeletal pain, such as postherpetic neuralgia (PHN), diabetic neuropathy, postsurgical pain, osteoarthritis, and rheumatic arthritis. Chili extracts were also used to treat itching, psoriasis, cluster headaches, and migraines [4]. A Cochrane review on the use of low-concentration capsaicin concluded that there was insufficient evidence to recommend for routine clinical use. Capsaicin suffered two major disadvantages: (1) burning sensation and skin reactions not tolerated by many and (2) the need to apply multiple times in a day for 4-6 weeks. Studies also suggest that the effect is not more than placebo [5]. High-concentration capsaicin 8% patch, also known as NGX-4010 (Astellas Pharma/ NeurogesX, Inc.), provides rapid and longer lasting pain relief following a single application in different neuropathic pain states. In 2009, the Food and Drug Administration (FDA) and European Union (EU) approved the use of the 8% capsaicin patch after four-phase III clinical trials. The patch is currently approved for use in peripheral neuropathy in nondiabetic patients in the EU, whereas the FDA approved its use in the United States only to treat PHN. PHN [6] and HIV-associated distal sensory polyneuropathy (HIV-DSP) [7] were the two neuropathic pain models used for early clinical trials. There are also several case-series on the use of the capsaicin 8% patch in neuropathic pain states like scar pain, postsurgical pain, chemotherapy-induced neuropathy, and other localized peripheral states.

PHARMACODYNAMICS

Capsaicin comes from plants of the genus Capsicum, which belong to the Solanaceae family. Capsaicin is the main pungent chemical, the others being dihydrocapsaicin, nordihydrocap- saicin, homodihydrocapsaicin, and homocapsaicin [8]. The main site of action of capsaicin is the transient receptor potential vanilloid (TRPV1) channel. Although many endogenous and exogenous agonists nonselectively activate the channel, capsaicin is highly selective and highly potent at this channel. Capsaicin desensitizes nociceptive neurons. There are two distinct phenomena reported. The first is a classic pharmacological desensitization, where a prolonged and repeated application leads to progressive reduction in the size of subsequent response to capsaicin. The second phenomenon is a “functional desensitization,” where capsaicin leads to a reduction or loss of responsiveness of neurons to other stimuli. High-concentration capsaicin produces the functional desensitization [9]. Capsaicin increases the calcium:sodium permeability from a baseline of 8:1 to 25:1. There is a massive influx of calcium ions down the electrochemical gradient. In addition, there is also release of calcium through the TRPV1 channels activated on intracellular organelles like the endoplasmic reticulum. The excess intracellular calcium triggers calcium-dependent protease enzymes causing cytoskeletal breakdown. Microtubule depolymerization causes a halt in fast axonal transport [10,11]. Osmotic swelling is caused by the chloride influx. A TRPV1- independent mechanism also exists by causing direct inhibition of electron chain transport and subsequent mitochondrial dysfunction [12]. Thus, multiple mechanisms ultimately lead to loss of cellular integrity and defunctionalization of the nociceptor fibers. The nerve fibers retract to a Efficacy and Therapeutic Uses 91 depth at which mitochondrial function is preserved. Immunohistochemical studies have shown that capsaicin produces highly localized loss of nerve fibers in the epidermis and dermis [13]. This replaces the hypothesis in the mid-1980s about substance P depletion being responsible for capsaicin-induced pain relief. It is now known that substance P is one of the many neurotransmitters expressed by nociceptor fibers. Thus, nerve terminal defunctionalization and retraction, as caused by capsaicin, leads to decrease in all the neuropeptides released by the nerve terminals, and substance P is one among them [14].

PHARMACOKINETICS

Capsaicin 8% patch (Qutenza—Astellas Pharma Ltd.) contains 179 mg of capsaicin on a 280 cm2 patch (640 mg/cm2) and works locally in the skin and with little systemic absorption. Capsaicin is very lipophilic with little affinity for the aqueous blood phase and is readily absorbed into the epidermal and dermal layers. The amount of drug absorbed depends on the duration of exposure and also the surface area of application. Blood samples from 173 patients from the trials, at varying intervals after patch removal (0, 1, 3, 6, 24 h), had quantifiable levels of capsaicin in only 34 (20%) patients, and quantifiable plasma capsaicin above the lower limit (0.5 mg/ml) was identified in only 6% of them [15]. The mean maximum plasma concentration

(Cmax) after a 60-min application was 1.38 ng/ml and the Tmax to reach these levels was 1.46 h [15] as compared to 47 min after oral ingestion of 26.6 mg of capsaicin with a Cmax of 2.47 ng/ml [16]. Capsaicin is rapidly metabolized in the liver by the cytochrome enzymes. It has a high mean apparent clearance of 54,598 L/h and is therefore eliminated with a half-life of 1.64 h compared to an elimination t1/2 of 24.9 min for oral capsaicin [17]. Capsaicin is metabolized very slowly in the skin as shown by in vitro studies [6], and it has the clinical advantage of lasting longer on its site of application in the skin. So, there is no need for dose adjustments in hepatic or renal failure as there is rapidly clearance of any systemically absorbed drug.

EFFICACY AND THERAPEUTIC USES

Postherpetic Neuralgia A randomized double-blind pilot study (n = 44) comparing the high-concentration 8% patch with a low-concentration 0.04% patch as active control [18] had an initial 4-week study period followed by an open-label extension phase up to 48 weeks (n = 24), during which patients got three further applications of the study medication. The study population was comprised of adult patients with PHN of at least 6 months duration and having average numerical pain rating scores (NPRS) between 3 and 8/10. Patients using concomitant pain medications had to be on stable doses for at least 21 days prior to treatment, and patients using more than 60 mg/day of morphine did not meet the inclusion criteria. After pretreatment with 4% lidocaine cream for 1 h, the investigational capsaicin patch was applied for 60 min to a maximum surface area of 1000 cm2. Immediate treatment-related pain was managed by local cooling methods and oral oxycodone (1 mg/ml), and hydrocodone bi- tartarate/acetaminophen (5 mg/500 mg) were allowed as rescue medication up to day 5 only. 92 5. Use of Topical Capsaicin for Pain Relief

Reduction in NPRS scores, percentage of patients with 30% pain relief, and use of questionnaires such as BPI, Short form, SAT (self assessment to treatment) questionnaire, and patients 'global impression of change (PGIC) were measured, but the scores from the first week were discarded to avoid any confounding effect from the rescue medications. There was a 32.7% reduction in baseline NPRS scores in the NGX4010 study group compared to a mere 4.4% reduction in the control group (p = 0.003) [18]. This reduction was seen as early as the first week, and the effect seemed to be maintained through the study period (4-12 weeks), whereas in the control group pain scores returned to baseline in 2-4 weeks [18]. Fifty-three percent of patients had at least a 33% reduction in pain scores from weeks 2 to 4 with 8% capsaicin vs. none in the control group, and this was increased to 78% from weeks 9 to 12 with no patients again from the control group. Higher pain relief was seen in those patients not taking concomitant medications; this finding may suggest that patients on previous neuropathic medication may have more treatment-resistant pain. Overall effect of the 8% capsaicin patch was positive regardless of any concomitant medication used [19]. The two 12-week phase III trials had also significantly higher responders for 30% pain reduction in the capsaicin 8% patch group (44% vs. 33%; p = 0.05 and 47% vs. 35%; p = 0.021) [19,20] and were also significantly greater in the capsaicin group (30% vs. 21%; p = 0.035) for 50% pain reduction [20]. During weeks 2-12, 55% patients in the capsaicin group reported an improvement on the PGIC scale (−3 to +3 scale from very much worse to very much improved) compared to 43% in the control group [18] and similar results (41% vs. 26%; p = 0.001) were seen in other trials [19,20]. Integrated analysis of four trials in patients with PHN (n = 1079 total; n = 597 for capsaicin 8%; n = 482 for controls) showed significantly higher reductions in baseline pain scores in the capsaicin 8% group from weeks 2-12 (31.2% vs. 23.9%; p = 0.0002), and more patients achieved at least 30% pain reduction (45% vs. 36%; p = 0.0035) [21]. The capsaicin 8% patch was found to be significantly better than the 0.04% control patch in a Cochrane review of four studies (n = 1272) using capsaicin patches for managing PHN [22]. PGIC was regarded as first-tier evidence. The calculated NNT for “much” or “very much improvement” was 8.8 at 8 weeks (95% confidence interval 5.3-26) and NNT of 7.0 at 12 weeks (95% confidence interval of 4.6-15) [22]. Long-term efficacy for similar endpoints were studied in open-label extension studies, and patients received capsaicin 8% patch treatment at intervals of no less than 12 weeks and were followed up until 40 and 48 weeks in the two studies [18,23]. The mean percentage reduction in baseline NPRS scores after the first, second, and third treatments were −31.4%, −30.0%, and −34.1%, respectively [18], and the median duration of response was found to be 22 weeks; 14% maintained the response for 40 weeks [21,24].

HIV-Associated Distal Sensory Polyneuropathy HIV patients may develop distal sensory neuropathy due to the viral load or as a complication of the antiretroviral therapy; this can occur in 29-62% of HIV patients [25]. The efficacy of the capsaicin 8% patch has been studied in two-phase III trials [25,26] and an open-label study up to 48 weeks [27]. Patients who had moderate to severe pain for more than 2 months from HIV neuropathy were included in the study. If patients were on antiretroviral therapy, doses should have been stable for at least 8 weeks to fulfill the inclusion criteria. The capsaicin 8% patch was compared with a low-concentration capsaicin 0.04% patch as active control; Safety and Tolerability 93 as in the PHN studies, the durations of application were 30, 60, or 90 min. The mean reduction in NPRS scores at weeks 2-12 was much higher (22.7%) with the capsaicin 8% patch as compared to the control group (10.7%) (p = 0.0026) [25]. Mean reduction of pain of at least 30% was seen in 42%, 24%, and 36% patients in the 30-, 60-, and 90-min application groups, respectively, and improvement in PGIC scores was seen in a higher proportion of patients in the capsaicin 8% group (33%) compared to the control group (14%). The mean pain reduction scores in the capsaicin group were observed regardless of whether patients were on concomitant neuropathic drugs or on neurotoxic antiretroviral therapy [25]. An integrated pooled analysis of the two-phase II trials (n = 239 for capsaicin; n = 100 for controls) showed significantly greater reduction in NPRS scores from weeks 2 to 12 in the capsaicin group (27.0% vs. 15.7%; p = 0.0020) [28]. A larger proportion of patients also achieved greater than 30% reduction in pain (39% vs. 23%; p = 0.0051) in the active group [28]. The Cochrane review of two studies in HIV-AN patients (n = 801) treated with the high- concentration patch reported reduction in pain intensity, and the NNT for 30% pain intensity reduction from baseline was 11 [22]. PGIC was used as a reported outcome in only one study: based on this, the NNT for “much” or “very much improved” at 12 weeks was estimated to be 5.8 (95% confidence interval 3.8-12) [22]. A 48-week open-label study included 52 HIV-AN patients who had successful response to the capsaicin patch, and three to four further 60- or 90-min applications were allowed with an interval of at least 12 weeks between subsequent applications. The capsaicin 8% group showed 12.4% reduction in the baseline NPRS scores by week 48, and about 80% of the patients reported an improvement in the PGIC scale [27].

Other Neuropathic Pain States Capsaicin 8% patches have also been used in other peripheral neuropathic pain states. In a recently concluded open-label, randomized, multicenter study including more than 500 patients, capsaicin 8% patches showed noninferiority over pregabalin [29]. In early findings from a small group of carefully selected patients with cancer-associated neuropathic pain, 71% of patients had up to 90% pain relief [30]. Studies have also been done in patients with postamputation pain and stump-neuroma pain [31]. Another observational study, QUEPP, showed significant reduction in pain scores and functional improvement in patients diagnosed with localized neuropathic pain, including postsurgical pain, scar pain, and peripheral neuropathy in nondiabetic adults that had been unresponsive to other neuropathic agents [32]. There are also anecdotal reports of successfully treating localized neuropathic pain on the face and scalp after adequate precautions are taken to protect the eyes and nose as well as treating postherniorrhaphy neuropathic pain close to the genital mucosa.

SAFETY AND TOLERABILITY

The safety and tolerability of the high-concentration capsaicin patch has been evaluated both in phase III trials and subsequent open-label extension studies [18–20,23,25–27]. Adverse events were monitored. Vital signs, physical examination (including dermal and neurological assessments), treatment-related pain, and use of rescue medication were evaluated [19]. 94 5. Use of Topical Capsaicin for Pain Relief

In the open-label extension studies, which included both PHN and HIV-DSP patients, 98% of patients completed 90% of the treatment [27]. Similar results obtained in other trials suggest that treatment with capsaicin 8% patches was generally well tolerated. A total of 883 patients (67%) of the 1327 patients from various randomized controlled trials reported adverse reactions; most of these were transient minor application site-related problems. Only 0.8% patients discontinued treatment in the study group because of the adverse reactions, and this compared with 0.6% patients in the control group [6]. The pooled data suggested an overall dropout rate of 1.5% [7]. Nine deaths were reported, but none of these were related to the capsaicin treatment. Serious adverse events were uncommon, and only one, a case of accelerated hypertension possibly due to treatment-related pain, was attributed to capsaicin treatment [7,21]. The proportion of patients reporting a change in blood pressure (mild and transient, <8 mmg in average) over the course of the phase III studies was 1.7% in the capsaicin 8% group and 0.7% in the control group [1]. Though the incidence of cardiac events was low, the risk in patients with preexisting cardiovascular disease was higher (18%) compared to those without the cardiovascular risk (10.2%) when treated with capsaicin [7]. The most common adverse events were erythema, pain, edema, and pruritus at the patch application site; indeed, 96% of PHN patients and 75% of the HIV-DSP patients had application site reactions [27]. Dermal assessment scores above 3 were very rare and were mostly <2 (definite erythema, readily visible, minimal edema, or minimal popular response) for PHN patients and were <1 (minimal erythema, barely perceptible) for HIV-DSP patients [27]. These reactions were more common in the active group, peaking just after patch removal and resolved by day 3. Thirty-six percent of patients in the high-concentration patch group reported a 30% increase in baseline pain scores compared to 13% in controls during the duration of patch application, and the pain increased on days 0 and 1 and started resolving from day 2 [27]. In PHN patients (depending on the number of treatments), the proportion of patients reporting pain varied from 35% to 48%, and the mean dose of rescue analgesia (oxycodone) ranged from 12.2 to 17.1 mg. Thirty-two to 46% of patients among the HIV-AN group complained of pain during the four repeat treatments, and the mean dose of oxycodone used was 12.3-31.7 mg [2]. The use of rescue medication used from day 0 to day 5 was higher in HIV patients, and this equalized with PHN patients by day 5 [27]. Clinical neurological and sensory assessments, as well as QST, were carried out, and most subjects reported no significant changes. Those who did reported an improvement or return to normal sensation [27]. Other reported adverse events were coughing and sneezing, which occurred due to aerosolization of the capsaicin. Nausea was thought to be associated with use of oxycodone as rescue analgesia. Erythema was more common in PHN patients, whereas diarrhea, weight loss, and throat infections were more common in HIV-AN patients [27]. No cumulative toxicity was reported.

DELIVERY OF TREATMENT WITH CAPSAICIN 8% PATCH—PRACTICAL ASPECTS

Initially, the licensing agreement with the Medicines and Healthcare Regulatory Agency (United Kingdom) stipulated that clinical practitioners delivering the high-concentration Delivery of Treatment with Capsaicin 8% Patch—Practical Aspects 95 capsaicin patch treatment should undergo an initial training involving clinical information and a practical application workshop with placebo patches. This was followed by live training involving the use of the capsaicin 8% patches in the clinical setting under the supervision of an experienced trainer to gain competency in applying the patch and counseling patients appropriately before, during, and after administration of the patches [33,34]. This was replaced by an online training program, following which a live training or supervision of the patch application procedure may be arranged. The service was set up with one or more trained operators, mainly nurses, as a day case service in an appropriate clinical setting with adequate ventilation. The patients for the patch treatment are counseled in a clinic about what to expect, and any questions or concerns raised by the patient or their family members were addressed to their satisfaction. They are also given appropriate patient information leaflets outlining the treatment procedure and postprocedural care at home. Patients are advised not to shave or remove any body hair from the area to be treated within 48 h before treatment to prevent any breach of skin integrity at the site of application; any troublesome hair that prevents close contact of the patch to the skin are removed using scissors prior to the procedure. Patients are encouraged to bring their own rescue medication used for the flare-up of their neuropathic pain on the day of treatment and an audiovisual media to listen to music or watch an entertainment program during the treatment. The distraction provided may help to make the experience less uncomfortable. Pretreatment with EMLA (2.5% lidocaine and 2.5% prilocaine) and/or lidocaine cream is not routinely offered following the findings from the LIFT study, which showed that the use of even a low-dose analgesic like Tramadol 50 mg prior to the patch application can effectively reduce the treatment-related discomfort comparable with the application of the topical local anesthetic [35]. The capsaicin 8% patch measures 14 cm × 20 cm (280 cm2), and a maximum of four patches can be used at any given time to treat an area of 1120 cm2; it can be used to treat localized neuropathic pain in all areas of the body with the exception of the face, above the hairline on the scalp, and on or near mucous membranes of the face or perineum [34]. Most areas on the trunk or periphery require 1-2 patches, whereas treating both feet for peripheral neuropathy often requires 3-4 patches. The patch and cleansing gel are stored below 25 °C, and an unopened patch has a shelf life of 4 years. It is recommended that once opened from the sealed pack, the patch should be used within 2 h. Once the area to be treated is confirmed by the patient and the operator, the area is traced using a skin-marking pen, and a transpar- ency sheet is used to document the area as a record to compare the size of the treatment area for subsequent treatments. The use of well-fitting nitrile gloves ensures ease of application, as capsaicin can diffuse latex gloves and can contaminate the operator's hands [34]. The area is cleaned and dried prior to application of the patch to ensure close contact with the skin and is done in a manner to minimize aerosolization. After application, the patches are kept in place using suitable methods like wrapping the area with cling-film, bandages, or socks. Treatment of hands and feet are tedious due to the cutting the patches into little strips and wrapping around individual digits and covering the web spaces to get the best results. During the application period, the patient is fully monitored and rescue analgesia given as appropriate. Following the completion of treatment, the patch is removed by gently rolling inward to minimize aerosolization, and the area is removed of any residual capsaicin using 96 5. Use of Topical Capsaicin for Pain Relief a cleansing gel. The used patches and all the linen, gauze, and gloves are immediately disposed of safely using a sealed waste-disposal plastic bag. Patients are advised to avoid touching the treated area following the treatment as well as ensuring that there is no contact with contaminated clothes/linen. The vast majority of patients tolerate the procedure very well, and interactions/reassurance from the operator, as well as distraction strategies, help in managing the symptoms. The use of rescue analgesia is encouraged but is seldom needed. Dry cooling using a linen-wrapped cold pack applied over the treated area is possibly the best way to alleviate the symptoms. Wet compresses are not recommended prior to the cessation of treatment, as it interferes with patch adhesion, and postprocedure it can cause leaching of the capsaicin from the deeper layers of the skin. Patients who have had their feet treated for peripheral neuropathy found it comforting to wrap their feet in cling- film or plastic bags and immerse in water at room temperature after 24 h posttreatment. Most of the symptoms settle down within 48-72 h, and it is very rare that the symptoms persist after 5 days. Patients are routinely contacted by telephone 24 h and 72-96 h postprocedure to ensure that they feel well, as well as to address any queries. Patients are then reviewed in the pain clinic at 4 weeks and 8 weeks. During this time, systemic analgesics, including opioid and neuropathic drugs, are down-titrated or even tapered off if the pain control is good. The authors do not advocate repeating the patch routinely every 3 months, but wait for the symptoms to recur before offering the next treatment, as the duration of effect has been shown to vary between 3 and 15 months on average, depending on the underlying pathology [36].

APPLICATION PROCEDURE FOR THE CAPSAICIN 8% PATCH

• Identify the area to be treated; ensure that the skin is intact and unbroken. • Using the skin-marking pen, mark the area to be treated; if any hair is to be removed, it should be clipped closely to the skin using a pair of scissors. • The area to be treated is traced onto the tracing sheet clearly labeling the cephalo-caudal and right-left orientation. • Topical anesthetic cream (EMLA or 4% lidocaine) is applied for a period of time as per manufacturers' instruction—usually for an hour. • After the stipulated time, remove the topical anesthetic cream and dry the skin carefully; if patient has significant allodynia over the area to be treated, the use of a hair dryer is often helpful to dry and warm the area to facilitate adhesion. • Capsaicin 8% patches are cut to shape and orientation based on the tracing sheet template of the treatment area. • Apply the capsaicin patches to the treatment area, ensuring close adhesion to the skin; bandaging, cling-film, or socks may be used to facilitate the adhesion. The patch is left in place for 30 min if the feet are being treated or for 60 min for the rest of the body. • After the treatment duration has been completed, the capsaicin 8% patch is removed by carefully rolling inward to reduce the risk of aerosolization. • Clean the treated area of any residual capsaicin on the skin with the cleansing gel, and allow the area to dry spontaneously. REFERENCES 97 POSTPROCEDURAL INSTRUCTIONS FOR PATIENTS

• Counsel about avoiding contamination of clothes and towels with capsaicin at home. • Care should be taken to avoid contact of the treated area particularly with children and pets. • There may be delayed onset of burning sensation and pain a few hours after the treatment once the effect of the local anesthetic has worn off. • Discuss use of localized dry-cooling and analgesics for managing pain and discomfort. • Avoid hot baths/showers, exposure to direct sunlight to the treated area, and vigorous exercise for 24-48 h or until the acute symptoms have settled down. • Ensure not to abruptly discontinue preexisting prescribed analgesia and neuropathic pain medications without medical advice and supervision, even if there is a dramatic pain relief to avoid withdrawal effects. • Contact the treatment unit or the patient's general practitioner if there is any concerns of adverse side effects. • Provide contact details of the treatment unit.

References [1] Bode AM, Dong Z. The two faces of capsaicin. Cancer Res 2011;71:2809–14. [2] Turnbull A. Tincture of capsaicin as a remedy for chilblains and toothache. Dublin Free Press 1850;1:95–6. [3] Thresh JC. Isolation of capsaicin. Pharm J Trans 1876;6:941–7. [4] Anonymous. Martindale: the extra pharmacopoeia. 2nd ed. London: Royal Pharmaceutical Society; 1999. [5] Derry S, Moore RA. Topical capsaicin (low concentration) for chronic neuropathic pain in adults. Cochrane Database Syst Rev 2012;9:CD010111. [6] European Medicines Agency. Qutenza: summary of product characteristics. http://www.ema.europa.eu/docs/en_ GB/document_library/EPAR_-_Product_Information/human/000909/WC500040453.pdf [accessed 15 June 2013]. [7] Evaluation UFCFD. Medical review: Qutenza. http://www.accessdata.fda.gov/drugsatfda_docs/ nda/2009/022395s000Medr.pdf [accessed 15 June 2009]. [8] Luo X-J, Peng J, Li Y-J. Recent advances in the study on capsaicinoids and capsinoids. Eur J Pharmacol 2011;650:1–7. [9] Winter J, Bevan S, Campbell EA. Capsaicin and pain mechanisms. Br J Anaesth 1995;75:157–68. [10] Chard PS, Bleakman D, Savidge JR, Miller RJ. Capsaicin-induced neurotoxicity in cultured dorsal root ganglion neurons: involvement of calcium-activated proteases. Neuroscience 1995;65:1099–108. [11] Han P, McDonald HA, Bianchi BR, Kouhen RE, Vos MH, Jarvis MF, et al. Capsaicin causes protein synthesis inhibition and microtubule disassembly through TRPV1 activities both on the plasma membrane and intracellular membranes. Biochem Pharmacol 2007;73:1635–45. [12] Shimomura Y, Kawada T, Suzuki M. Capsaicin and its analogs inhibit the activity of NADH-coenzyme Q oxidoreductase of the mitochondrial respiratory chain. Arch Biochem Biophys 1989;270:573–7. [13] Polydefkis M, Hauer P, Sheth S, Sirdofsky M, Griffin JW, McArthur JC. The time course of epidermal nerve fibre regeneration: studies in normal controls and in people with diabetes, with and without neuropathy. Brain 2004;127:1606–15. [14] Anand P, Bley K. Topical capsaicin for pain management: therapeutic potential and mechanisms of action of the new high-concentration capsaicin 8% patch. Br J Anaesth 2011;107:490–502. [15] Babbar S, Marier J-F, Mouksassi M-S, Beliveau M, Vanhove GF, Chanda S, et al. Pharmacokinetic analysis of capsaicin after topical administration of a high-concentration capsaicin patch to patients with peripheral neuropathic pain. Ther Drug Monit 2009;31:502–10. 98 5. Use of Topical Capsaicin for Pain Relief

[16] Chaiyasit K, Khovidhunkit W, Wittayalertpanya S. Pharmacokinetic and the effect of capsaicin in Capsicum frutescens on decreasing plasma glucose level. J Med Assoc Thai 2009;92:108–13. [17] Jensen TS, Madsen CS, Finnerup NB. Pharmacology and treatment of neuropathic pains. Curr Opin Neurol 2009;22:467–74. [18] Backonja MM, Malan TP, Vanhove GF, Tobias JK, C102/106 Study Group. NGX-4010, a high-concentration capsaicin patch, for the treatment of postherpetic neuralgia: a randomized, double-blind, controlled study with an open-label extension. Pain Med 2010;11:600–8. [19] Backonja M, Wallace MS, Blonsky ER, Cutler BJ. NGX-4010, a high-concentration capsaicin patch, for the treatment of postherpetic neuralgia: a randomised, double-blind study. Lancet 2008;7:1106–12. [20] Irving GA, Backonja MM, Dunteman E, Blonsky ER, Vanhove GF, Lu SP, et al. A multicenter, randomized, double-blind, controlled study of NGX-4010, a high-concentration capsaicin patch, for the treatment of postherpetic neuralgia. Pain Med 2010;12:99–109. [21] McCormack PL. Capsaicin dermal patch: in non-diabetic peripheral neuropathic pain. Drugs 2010;70:1831–42. [22] Derry S, Sven-Rice A, Cole P, Tan T, Moore RA. Topical capsaicin (high concentration) for chronic neuropathic pain in adults. Cochrane Database Syst Rev 2013;2, CD007393. [23] Webster LR, Malan TP, Tuchman MM, Mollen MD, Tobias JK, Vanhove GF. A multicenter, randomized, double-blind, controlled dose finding study of NGX-4010, a high-concentration capsaicin patch, for the treatment of postherpetic neuralgia. J Pain 2010;11:972–82. [24] Webster RL, Malan PT, Tuchman M. Duration of treatment response to NGX-4010, a high-concentration capsaicin patch, in patients with post herpetic neuralgia. 20th Annual Scientific Meeting of the American Pain Society, Baltimore; 2010. [25] Simpson DM, Brown S, Tobias J, NGX-4010 C107 Study Group. Controlled trial of high-concentration capsaicin patch for treatment of painful HIV neuropathy. Neurology 2008;70:2305–13. [26] Clifford D, Simpson D, Brown S, Moyle G, Brew B, Conway B. A multicenter, randomized, double-blind, controlled study of NGX-4010, a high-concentration capsaicin patch, for the treatment of painful HIV-associated distal sensory polyneuropathy. In: 17th Conference on Retroviruses and Opportunistic Infections; 2010. [27] Simpson DM, Gazda S, Brown S, Webster LR, Lu SP, Tobias JK, et al. Long-term safety of NGX-4010, a high-concentration capsaicin patch, in patients with peripheral neuropathic pain. J Pain Symptom Manag 2010;39:1053–64. [28] Backonja MM, Irving AG, Webster RL. Efficacy of Qutenza (NGX-4010), a high concentration capsaicin cutaneous patch, in patients with peripheral neuropathic pain: results of integrated analyses. Eur J Neurol 2009;3:1046. [29] Haanpaa M, Ernault E, Siciliano T et al. Local or systemic treatment for neuropathic pain? ELEVATE: an open-label, randomised, multicentre, non-inferiority efficacy and tolerability study. Presented at 14th Asian Australasian Congress of Anaesthsiologists. 21–5 February, Auckland, New Zealand. [30] Bhaskar AK, England J, Lowe J. Management of neuropathic pain (NP) using the capsaicin 8% patch in patients with cancer. Palliative Medicine 2012;26(4):465. [31] Viel, Bredeau, Margaux, Fabre, Favier. Communications HOPIPHARM (Lille) and SFETD (Paris). Proceedings from 14th World Congress on Pain—IASP, Milan; 2012. [32] Maihöfner C, Heskamp ML. Prospective non-interventional study on the intolerability and analgesic effectiveness over 12 weeks after a single application of capsaicin 8% cutaneous patch in 1044 patients with peripheral neuropathic pain: first results of the Quepp study. Curr Med Res Opin 2013;29:673–83. [33] Baranidharan G, Das S, Bhaskar A. A review of the high-concentration capsaicin patch and experience in its use in the management of neuropathic pain. Ther Adv Neurol Disord 2013;6:287–97. [34] Summary of Product Characteristics: Qutenza 179 mg cutaneous patch. http://tinyurl.com/4y9hahb [accessed 15 June 2013]. [35] Astellas Pharma Europe Ltd. Tolerability of QUTENZA when applied after pre-treatment with lidocaine or tramadol in subjects with peripheral neuropathic pain—a randomized, multi-center, assessor-blinded study. [36] Bhaskar AK, S C, G B. Efficacy of using capsaicin 8% patch on-demand rather than the regular 3 monthly treatment for localised Neuropathic pain in Cancer Patients. EFIC. CHAPTER 6 TRPV1 Agonist Cytotoxicity for Chronic Pain Relief: From Mechanistic Understanding to Clinical Application Dorothy Cimino Brown,1,2,* Michael J. Iadarola3 1Veterinary Clinical Investigations Center, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA 2Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA 3Anesthesia Section, Department of Perioperative Medicine, Clinical Center, NIH, Bethesda, Maryland, USA *Corresponding author: [email protected]

OUTLINE

Introduction 100 RTX is Administered Locally 106 Mechanisms of TRPV1 Agonist Companion Dog and Human Studies 107 Therapeutic Action 101 Companion (Pet) Dog Models of Four Main Determinants of TRPV1 Chronic Pain 108 Agonist Actions 101 TRPV1 Agonists in the Companion Selectivity of RTX at the Cellular Level 101 Dog Model of Bone Cancer 108 RTX Produces a Prolonged Channel TRPV1 Agonists in the Companion Open Time 104 Dog Model of Osteoarthritis (OA) 111 TRPV1 is Selectively Expressed in Human Studies 114 Subpopulations of DRG Neurons 105 References 114

As fundamental knowledge of the molecules involved in nociception evolves, the utilization of this knowledge to formulate experimental molecular neurosurgical techniques also advances. The resulting dynamic combination is elucidating the role of specific receptors, neurons, and pathways in the generation and maintenance of pain states [1–3]. The numerous molecular targets that are identified provide an excellent mechanistic basis for clinical therapeutic intervention, either by molecular neurosurgical approaches or through subsequent adaptation to more conventional pharmacological approaches. The goal in using targeted cytotoxins in the clinical setting is to elicit profound and sustained pain relief through prolonged, even permanent, deactivation or deletion of subpopulations of primary afferent pain-sensing neurons via peripheral or central (spinal CSF) administration [4–7]. Translation of the mechanistic understanding of potential therapeutic interventions to clinically effective analgesic compounds has been a challenge in the field of chronic pain. Studies in laboratory animals, mainly rodents, with experimentally induced pain states have been only partially successful in predicting human clinical trial outcomes [8–15]. These experimentally induced conditions may not adequately model the natural disease process that leads to pain. Thus, supplementation of rodent models with additional models constitutes an infor- mative transitional step for drug development and application of novel treatments to human patients. In this regard, large animal pain conditions or disorders that closely mimic their human counterparts and human models for demonstrating target engagement are important new adjuncts [6,16,17]. There is growing interest in using the diseases that spontaneously develop in companion dogs to investigate efficacy of new pharmacological agents and interventional administration approaches. In the evaluation of TRPV1 agonist efficacy for chronic pain, the naturally occurring companion dog models of bone cancer and osteoarthritis have been instrumental in documenting analgesic efficacy and potential side effects, as well as informing future clinical trial design, justifying the starting dose, and selection of outcome measures and primary endpoints. The initial sections of this chapter examine several mechanistic neurobiological and pharmacological factors that determine optimal therapeutic actions of TRPV1 agonists. The mechanistic and practical insights gained from a succession of investigations involving in vitro ectopic expression systems, 3H-RTX binding, TRPV1 immunocytochemistry, in vivo preclinical studies in rodents, and the large animal veterinary models were key elements for understanding how to use TRPV1 agonists for the treatment of pain. TRPV1 agonists, unlike TRPV1 antagonists [16,17], are not administered systemically or orally; rather, they are used in an interventional fashion, being administered by injection into, or close to, the site that is generating the pain problem (reviewed in Refs. [18,19]). Although we understand much of the necessary underlying neurobiology and pharmacology, because resiniferatoxin (RTX) administration is an interventional technique, going forward into the multiple, potential human pain indications for which this approach may be appropriate will entail clinical experimentation to determine factors such as optimal administration protocols, optimal formulations, and development of expertise on the part of the specialist [20,21]. The second set of sections examines canine clinical pain conditions and the actions of RTX as a pain control agent in these disorders. Although such investigations establish RTX as a treatment in and of itself for veterinary patients, they also constitute a “transitional model” Selectivity of RTX at the Cellular Level 101 for human clinical trials. Control of pain from canine osteosarcoma by intrathecal RTX was one of the key results that provided incentive and determined progress to human clinical trials. We also present observations on intraarticular (IA) RTX for pain in canine osteoarthritis. Given the strong predictive information for human translation that emerged from the canine osteosarcoma pain control trials, we are optimistic about translating the osteoarthritis results into human and animal clinical practice.

MECHANISMS OF TRPV1 AGONIST THERAPEUTIC ACTION

First, it is important to acknowledge that RTX is an ultrapotent TRPV1 agonist. RTX causes extremely prolonged channel opening and calcium influx, which results in cytotoxicity to the TRPV1-positive pain fibers or cell bodies [22]. When applied peripherally, the nerve terminal is not just made unresponsive to further direct stimulation of the TRPV1 ion channel as in the case of antagonists, it is actually temporarily destroyed due to calcium cytotoxicity—making it incapable of being stimulated by any agonists of any algesic receptor targets normally expressed by these pain sensing nerve terminals [4,23,24]. Thus, the analgesia is far more complete than that attained by any single channel or receptor antagonist because the latter leave the nerve terminal intact and the nerve ending susceptible to stimulation via the other channels that are left unblocked [17,24].

FOUR MAIN DETERMINANTS OF TRPV1 AGONIST ACTIONS

Four aspects of the dorsal root ganglion (DRG) TRPV1 receptor system and TRPV1 agonist action are particularly relevant for optimal use of agonists as therapeutic agents. These involve (1) specificity of TRPV1 agonists, (2) TRPV1 agonist pharmacodynamics, and (3) selective cellular TRPV1 expression in DRG. All three of these attributes make important contributions to the profile of therapeutic action. A fourth element (4) is the requirement for localized administration. Placing the drug next to, or into, the peripheral site that is generating nociceptive activity adds a further level of specific action. Such peripheral generators can be from an identified source; these might include surgical incisions, trigger zones for abnormal activity in a chronic pain condition (e.g., a neuroma), or an arthritic joint. The four main criteria determining TRPV1 agonist actions are summarized in Table 6.1, and the basic research findings supporting the underlying mechanisms or properties are examined in the following sections.

SELECTIVITY OF RTX AT THE CELLULAR LEVEL

The presence of TRPV1 is the major determinant of RTX action. Without expression of TRPV1, RTX at concentrations used to lesion expressing cells, nerve terminals, or axons does not appear to produce any negative effects at the cellular level [5,6]. In fact, concentrations well above the effective dose (~1000×) do not appear to affect non-TRPV1-expressing cells. Even when nonexpressing neurons are adjacent to TRPV1 expressing neurons that are 102 6. TRPV1 AGONISTS AND PAIN CONTROL

TABLE 6.1 Determinants of TRPV1 Agonist Action That Influence Therapeutic Selectivity and Efficacy

1. RTX is highly selective for TRPV1: no TRPV1 expression, no RTX effect. 2. RTX is very potent and produces a prolonged channel open time: The ability to prop open the channel results in an excessive amount of calcium entry into the neuron or nerve ending producing a calcium cytotoxicity. 3. TRPV1 is selectively expressed: Only a subpopulation of DRG nociceptors express TRPV1. However, there is a variable level of TRPV1 expression among the expressors, and the level of expression may affect the susceptibility to RTX. The remainder of the DRG neurons do not express TRPV1 and are not susceptible to RTX. 4. RTX is administered locally: This produces a high local concentration that dissipates rapidly. It also confines the drug to the site of action and reduces, or completely circumvents, systemic effects.

undergoing damage from agonist activation, the nonexpressing cells appear to remain intact. These conclusions were reached using several techniques including live cell imaging of cultured DRG neurons, imaging of transiently, and stably transfected with TRPV1, and histology of ganglia injected with RTX [4–7,25,26]. An example of a live cell imaging study is shown in Figure 6.1 using cultured cells transiently transfected with a plasmid engineered to express TRPV1. This series of images, taken over a 45-min time period, show that only cells that take up the plasmid and express TRPV1

1 min 20 min

(a) (b) 25 min 45 min

FIGURE 6.1 Cellular specificity of RTX effects on TRPV1-expressing cells. (a) NIH3T3 cells ectopically expressing TRPV1 were stained with the vital dye MitoTracker Red and incubated in medium containing propidium iodide. (b) Upon exposure to 1.6 nM RTX, the cells expressing TRPV1 undergo rapid intracellular remodeling seen by fragmentation of the mitochondria and the endoplasmic reticulum (not visible here). (c,d) Transfected cells become permeable to propidium iodide, extrude fragments of cytoplasm, display blebs in the nuclear membrane, and eventually die. Surrounding cells remain intact and viable. Selectivity of RTX at the Cellular Level 103 are susceptible to vanilloid agonists. Surrounding nontransfected cells that have not taken up the plasmid and do not express TRPV1 are not susceptible to RTX. The process of cell destruction is rapid: the TRPV1 expressing cells undergo intracellular remodeling of the endoplasmic reticulum (ER) and the mitochondria and eventually the nuclear and plasma membranes that can be seen starting as soon as 1 min after exposure to RTX. Eventually the cell is irrevocably damaged and dies. The rapid and massive fragmentation of the ER and mitochondria is a direct result of the prolonged influx of calcium across the cell membrane. This process is illustrated in Figure 6.1. After transient transfection with a TRPV1-expressing plasmid, only a subpopulation of cells takes up the plasmid. At 24 h after transfection, the cells are incubated in MitoTracker Red, a vital dye that stains mitochondria in living cells and with the cell membrane impermeant dye, propidium iodide. Once the cell is damaged, it will become permeable to the propidium iodide. Upon addition of RTX, both the ER lattice (which can’t be seen in these images) and the elongate mitochondria fragment into vesicular structures [5,25]. Organelle fragmentation begins to occur within minutes and is coincident with the sharp, ++ agonist-induced rise in intracellular calcium ion concentration ([Ca ]i) [25]. In addition to the rapid actions on cell viability, Figure 6.1 also illustrates the specificity of the RTX-TRPV1 interaction. There is a notable lack of effect on non-TRPV1-expressing cells, even when in close proximity to RTX-compromised, TRPV1-expressing cells. At time zero, all the cell nuclei in the field of view are dark and exclude propidium iodide; also, all mitochondria in all the cells have a similar appearance. At 20 min after exposure to 1.6 nM RTX the mitochondria of several cells in the field of view have fragmented. The fragmentation is not so easy to appreciate at this magnification and can begin even earlier, within minutes of exposure [25]. By 25 min the TRPV1-expressing cells have become permeable to propidium iodide (the nuclei begin to turn red or brighter in the black-and-white image), and by 45 min the cells are irrevocably damaged (there are six cells with red/bright nuclei in the field of view, three in the upper half and three in the lower half of the frame; arrows indicate four of them). Small plasma-membrane-enclosed fragments of cytoplasm are also seen in the field at 45 min, which are visible as small red/bright droplets. The loss of plasma membrane can be detected using electrophysiological recording from DRG neurons and is measured as a decrease in membrane capacitance [26] reflecting the decreased ability of the membrane to store charge. Despite this level of cellular carnage, the nearby, nontransfected cells ostensibly appear un- damaged: Their mitochondria are intact, and they persist in excluding propidium iodide. Thus, the expression of TRPV1 is the major determinant of the cellular specificity of RTX and is the mechanism for TRPV1-dependent, RTX-triggered cytotoxicity. Experiments with TRPV1 ion channel blockers such as ruthenium red, orthosteric capsaicin antagonists, and RTX or capsaicin activation of TRPV1 in calcium free media demonstrate that the entire process of organelle fragmentation and cell death is attributable to calcium entering the cell through the pore of the TRPV1 ion channel using the complement of TRPV1 receptors located in the plasma membrane [25]. The exact same process occurs when cells are exposed to the calcium ionophore ionomycin [25,27]. Interestingly, DRG neurons and cells stably or transiently transfected with TRPV1-expressing plasmids also contain a population of TRPV1 protein located in the ER [22,25,28]. The ER localization was seen when we expressed a TRPV1eGFP fusion protein. Live cell imaging showed that the ER contained a prominent amount of fluorescently tagged TRPV1eGFP [25]. We were able to examine the activation kinetics of the ER TRPV1 population by incubating 104 6. TRPV1 AGONISTS AND PAIN CONTROL cells in calcium-free medium and performing RTX or capsaicin activation. Activation of the intracellular population of TRPV1 was possible because both agonist compounds are hydrophobic and readily cross the lipid bilayer of the plasma membrane. These studies showed that the ER contains enough TRPV1 and enough calcium that, when the ER TRPV1 is selectively activated (e.g., in zero extracellular calcium or when the plasma membrane TRPV1 channel is blocked with the membrane impermeant channel blocker ruthenium red), organelle fragmentation and cell death occur in a fashion identical to that achieved on activation of TRPV1 in the plasma membrane. However, the dose required to activate ER TRPV1 is about 10 times that needed to activate the plasma membrane TRPV1 population [22]. The reason for the difference in dose is not clear. However, based on the quantitative dose differential between TRPV1 in plasma membrane and ER, it appears that the ER TRPV1 does not directly influence the therapeutic pharmacodynamics because nearly all the calcium enters the cytoplasm via plasma membrane TRPV1 [22]. We have proposed that the ER TRPV1 may be used by cells to regulate intracellular calcium dynamics, and the dual localization has been explored in a variety of cell types and preparations, with cancer cells being the main cellular targets [29–32]. This same process of selective cell removal occurs in vivo in the sensory ganglia when RTX is microinjected directly into the sensory ganglia. With standard histological stains, some of the cells can be seen to be undergoing neuronophagia with the locations of the deleted cell bodies marked by hypercellular accumulation of lymphocytes called nodules of Nageotte. Nodules of Nageotte are frequently seen with infectious diseases such as leprosy that destroy sensory ganglion neurons [5,6]. The Nageotte nodules are eventually replaced by eosino- philic material surrounded by satellite cells [6]. During this process of cell death and reabsorption, adjacent cells remain intact, a situation similar to that seen in vitro in Figure 6.1. The very precise excision of pain-sensing neurons from the DRG reinforces the analogy of RTX to a chemical “molecular scalpel” for use in molecular neurosurgery [33]. The preceding paragraphs provide a fairly detailed account of the cellular mechanisms underlying the therapeutic actions of RTX. The behavioral concomitants are that (a) heat pain sensation is lost in the dermatomes innervated by axons or ganglia exposed to RTX as well as (b) thermal hyperalgesia as measured in experimental peripheral inflammation, and (c) sensations of clinical pain are obtunded as seen in canine osteosarcoma [5,6]. More recently, RTX has been evaluated in a wider variety of rodent models of clinical pain using various routes of administration [34–40]. Another set of studies has shown that a long-duration hyperalgesic state can be instated in mice following systemic exposure to RTX by intraperitoneal administration [41,42]. The effects of parenteral administration probably do not have a bearing on the local administration protocols discussed in this review. Recent use in humans includes evaluation of topical RTX for controlling premature ejaculation [43], and the long-investigated urological applications of RTX, among the earliest uses of RTX in humans, has been recently reviewed [44].

RTX PRODUCES A PROLONGED CHANNEL OPEN TIME

Two types of physiological experiments demonstrate the prolonged channel opening that is produced by RTX. First, using calcium imaging and DRG neurons in primary culture, it was possible to directly compare dynamic alterations in intracellular calcium induced by TRPV1 Is Selectively Expressed in Subpopulations of DRG Neurons 105 capsaicin to those induced by RTX [25]. When cells are exposed to a pulse of RTX, a large and prolonged increase in intracellular calcium occurs. This prolonged elevation occurs in both rat and human DGR neurons in primary culture [5]. A second line of cell-based evidence came from manipulating the extracellular calcium concentration with and without RTX and with and without ruthenium red, the membrane impermeant channel blocker [22]. Although we did not determine the full duration for which the channel can remain open, the duration was on the order of 20 min. This was determined by adding RTX to cultured DRG neurons in calcium-free medium, washing the RTX out for various times, and then switching back to calcium-containing buffer. The chamber was subjected to prolonged perfusion to remove any RTX. Nonetheless, on reinstating normal extracellular calcium concentration, a rapid and massive increase in intracellular calcium occurred. The calcium entry was attributed to the ability of RTX to keep the plasma membrane-localized TRPV1 ion channel in the open state because the calcium entry could be blocked by inclusion of ruthenium red, a TRPV1 channel blocker, in the perfusion media. This kind of dynamic assessment is not as readily apparent from an examination of in vitro binding kinetics; nonetheless, the binding studies show that [3H]-RTX is a high affinity ligand for TRPV1, which is consistent with the in vivo and in vitro potency of RTX. The affinity of [3H]-RTX for TRPV1-containing membrane preparations from various animals or HEK293 ectopically expressing rat TRPV1 appears to vary with the type of preparation, but the Ki is generally in the single-digit nanomolar range [45,46].

TRPV1 IS SELECTIVELY EXPRESSED IN SUBPOPULATIONS OF DRG NEURONS

Immunocytochemical staining of sections from TG or DRG shows that TRPV1 is expressed in a variety of small to medium-sized DRG neurons and exhibits a varying degree of expression (Figure 6.2). The small, very darkly stained neurons (midsize arrows) present a strong contrast to the medium and lightly stained neurons and especially to the large, unstained proprioceptive-somatosensory neurons illustrated in this section. Thus, although the degree of expression likely does not affect RTX specificity, it may be more of a determinant of RTX efficacy. A gradient of susceptibility to RTX can be hypothesized based on the amount of TRPV1 produced by any particular neuron such that the high expressors are the most susceptible to RTX, whereas lower expressing neurons are less susceptible. Evidence in support of this can be gleaned retrospectively from an earlier publication [5]. Panels C and D of Figure 6.2 in that report show that, after an intratrigeminal injection of RTX, most TRPV1+ neurons are eliminated, but the remaining TRPV1+ neurons all appear to meet the “lightly stained” criterion and would be classified as low expressors. This hypothesis needs more formal testing, but a gradient of expression among TRPV1+ DRG neurons seems to be a plausible explanation for the ability of some neurons to resist the actions of RTX. In these neurons, the expression of TRPV1 is low enough to allow them to sequester the incoming calcium and survive transient exposure to RTX. The therapeutic implications are several. A relatively high expression of TRPV1 confers a high degree of cellular vulnerability and specificity to the actions of RTX. Neurons or cells that express a low level of TRPV1 are less vulnerable to the RTX-induced calcium cytotoxicity. Cells that do not express TRPV1 are not vulnerable to RTX. Extrapolating these 106 6. TRPV1 AGONISTS AND PAIN CONTROL

FIGURE 6.2 Heterogeneity in level of expression of TRPV1 protein in rat DRG. TRPV1-immunoreactive neurons were detected by immunocytochemistry and visualized with diaminobenzidine. The counterstain was hematoxy- lin. Arrows: Note the variation in level of TRPV1 expression from very strong (small arrow), to intermediate (long arrow) to very light (medium arrow). The asterisks denote unstained neurons. These are generally large in size and correspond to proprioceptive or other large nonnociceptive myelinated afferents. These differences are addressed further in references [23,24].

considerations to therapeutic administration protocols suggests that the major effects of RTX will be manifested within the zone of highest RTX concentration. Clinically relevant effects may be observed at the edges of the concentration gradient, but these regions may eventually resume transmitting clinically relevant pain signals if some neurons or axons (a) remain intact, (b) are damaged but with time are repaired, (c) if nerve endings in an injected nerve terminal zone regenerate, and (d) if/when the disease process progresses. In such cases retreatment may be necessary.

RTX IS ADMINISTERED LOCALLY

The requirement for local administration is an advantage for the use of RTX; at the same time the elements of the administration protocol are probably the most critical variables for a clinically successful outcome. Variation in accessibility to the site of administration, ability to visualize the site of nerve damage/site of needle placement, the choice of sites (e.g., nerve terminal zone, peri- or intraneural, peri- or intraganglionic, intrathecal, intrajoint), the volume of injection solution, the concentration and/or absolute amount, rate of administration, iso- or hyperbaric formulation, and how many individual injections are all factors that need to be addressed and in part determined empirically. But once general guidelines are established, the range of parameters for optimization will become evident and manageable. These factors will allow the administration of RTX to be optimized and tailored to the individual Companion Dog and Human Studies 107 patient (reviewed in Ref. [19]). Nonetheless, the exact site of administration may still require experimentation to identify the most effective approach and parameters for administration. These types of considerations were recently demonstrated by the injections of lidocaine intrathecally and intraforaminally (peri-DRG) for phantom limb pain. The DRG was identified as a site of ectopic discharge that likely drives the cortical plasticity related to the phantom sensations. Such sensations were effectively blocked by local anesthetic administration to the DRG and not effectively suppressed by direct injection to the neuroma in the stump, even at low doses designed to inhibit spike electrogenesis rather than propagation [47]. Thus, if RTX were substituted for lidocaine, several routes of administration for a particular pain condition might need to be explored. One of the great benefits of local administration is that sensitivity to thermal pain is maintained throughout the portions of the body that are not exposed to RTX. This reduces the potential for inadvertent burns due to loss of thermal sensitivity to hot objects or liquids. The need for local administration is exemplified by results from clinical trials with TRPV1 antagonists. Antagonists block the ability to sense damaging heat throughout the body. As a result, patients treated with TRPV1 antagonists lack critical feedback on whether an object or liquid is hot and can cause tissue damage [16]. The TRPV1 antagonists have another side effect of causing hyperthermia to varying degrees, but that is not an issue with local agonist injections. It is noteworthy that the search for non-hyperthermia-inducing TRPV1 antagonists has continued [48,49]. It is also interesting to speculate about the potential uses of a non-hyperthermia-inducing antagonist. Given that antagonists seem to have temporally limited efficacy in acute traumatic tissue injury (e.g., third molar extraction) [16], it is possible that more chronic or diffuse pain problems such as fibromyalgia or myofacial pain [50] may respond, at least partially, to blocking TRPV1. Inhibition of the contribution of TRPV1 to peripheral sensitization may help to tip balance from a hypersensitive state to a more normo- or hyposensitive level yet allow the nociceptive nerve endings to respond to strong acute nociceptive stimuli. Anatomically targeted administration of TRPV1 agonists is a fairly straightforward approach to pain control and a relatively simple way to circumvent nonintended global effects. This is the essence of an interventional approach to personalized pain medicine. Although this seems straightforward, in some patients the exact site for needle placement may be difficult to define. For example, when the target is a damaged peripheral nerve ending that for whatever mechanism has begun to fire ectopically, certain imaging procedures might be used to enhance the precision of drug delivery. Procedures for imaging peripheral nerve endings or neuromas may be an appropriate preinjection step. Ultrasound has been used to visualize the neuroma that occurs in the foot with Morton’s neuroma. Other techniques may also be of value such as fluorescent imaging of nerves with labeled peptides [51] and use of PET ligands such as [18F]-saxitoxin for sodium channels [52].

COMPANION DOG AND HUMAN STUDIES

All the preceding considerations have been utilized to form the basis for companion dog studies in osteosarcoma and osteoarthritis pain and a human clinical trial for intractable pain in advanced cancer (a Phase I study of the intrathecal administration of RTX for treating 108 6. TRPV1 AGONISTS AND PAIN CONTROL

severe refractory pain associated with advanced cancer, http://clinicaltrials.gov/ct2/show/ NCT00804154). The initial dog osteosarcoma pain trials [5,6] are reviewed later; the results of a double-blind randomized clinical trial are being prepared for publication. In addition, we discuss here the results from a canine clinical study of IA RTX for treatment of pain from osteoarthritis. The human clinical trial in cancer pain is ongoing. Initial results have been presented in abstract form only [53] and are summarized later.

COMPANION (PET) DOG MODELS OF CHRONIC PAIN

Studies in companion dogs are a novel additional step in evaluating the safety and efficacy of interventional treatments for human translational therapy. Dogs spontaneously develop diseases that are pathologically, physiologically, and symptomatically analogous to those in people. The spontaneous pain caused by these naturally occurring diseases requires treatment for the animals’ sake, and carefully studying novel therapies in these dogs can provide greater insight into the potential efficacy in humans. Medical surveillance of dogs is second only to that of people, the dog’s state of health is observed in intimate detail on a day-today basis, and its ailments are attended by veterinary specialists using all the diagnostic approaches of modern medicine [54]. Dogs share the environment with people and thus the potential environmental risk factors for disease, and their large body size simplifies biologic sampling. The extended course of disease, compared to rodent models, allows for clinically relevant efficacy data collection, whereas the shorter overall life span of dogs, compared to humans, provides a time course of disease within a time frame reasonable for efficient data collection. In addition, because of the increasing interest in using these companion animal populations for translational research, outcome assessment instruments have been specifically developed to capture clinically and translationally relevant pain severity and pain impact data in these models [55–57].

TRPV1 AGONISTS IN THE COMPANION DOG MODEL OF BONE CANCER

Because bone cancer pain is a unique pain state that changes with the progression of the disease, the identification of effective novel interventions may be enhanced by utilizing an animal model that is specific to bone cancer and has a progression of disease that parallels the human condition. In addition to the similarities in presentation, pathology, physiology, and response to treatment, the issues associated with managing pain in human cancer patients are precisely mirrored in canine patients (Figure 6.3), where pain severity becomes refractory to conventional pain therapeutics as the disease progresses [5,6,55,58–60]. The frequency and intensity of the pain tends to increase over weeks or months. This is manifested in the need to give analgesics and increase or change the dose to allow continued weight bearing on the affected limb and improve the activity of the animal. As the disease progresses, weight bearing produces frequent episodes of breakthrough pain that are more difficult to control, even with large doses of opioids. These dogs often undergo euthanasia within several months of the diagnosis of bone cancer because of uncontrolled bone pain TRPV1 Agonists in the Companion Dog Model of Bone Cancer 109

FIGURE 6.3 Radiograph of canine osteosarcoma lesion. Anterior-posterior radiograph of the left antebrachium of a 12-year-old, male, mixed-breed dog that was diagnosed with osteosarcoma. Note the presence of an expansile moth-eaten lytic lesion in the distal radial metaphyseal region and sunburst periosteal reactions associated with it. and associated loss of function. This evolution of bone pain over weeks to months better approximates the human condition than the rapid progression in rodent models and allows enough time to evaluate the effectiveness of antinociceptive agents through the evolution of the pain process. This time is still short enough, however, to ensure rapid accrual of data through detailed prospective studies. Investigations of RTX in these dogs have highlighted both the promises and pitfalls of using a potent TRPV1 agonist centrally for the alleviation of bone cancer pain. The intrathecal administration of RTX requires a dedicated and knowledgeable team approach. Because RTX stimulates the TRPV1-sensitive neurons as it causes excessive 110 6. TRPV1 AGONISTS AND PAIN CONTROL

transmembrane calcium influx, there is a transient and intense activation of the nociceptive primary afferent neurons and axons eliciting a burning painful sensation, which must be managed during injection and in the peri-injection period. Although the dogs must be placed under general anesthesia to maintain immobility for the intrathecal injection, special attention is paid to premedicating the dogs with a strong opioid such as hydromorphone and then maintaining the dogs under general anesthesia with isoflurane and oxygen for 90 min following intrathecal RTX injection. The dogs need to be maintained at a surgical MAC for inhalant at injection and for the initial 45-60 min following injection. If the anesthetic depth is too light, the neuronal activation elicited by the RTX will cause the dogs to wake up during the TRPV1 activation phase. Dogs must be monitored with continuous electrocardiography and blood pressure monitoring while anesthetized, as RTX injection can cause a hemodynamic response with a peak effect within 5 min of injection, which resolves over 60 min in the average dog [6]. The increases in heart rate and blood pressure following RTX injection, although transient, can be quite profound with increases 100% above the steady-state level prior to injection. Although these effects tend to be self-limiting, a knowledgeable anesthetic team must be vigilant to rapidly manage the anesthetic depth and cardiovascular status of the animal. Anesthetic recovery can be prolonged, and core body temperature changes can be significant, but seemingly without long-term negative effects. Although the peri-injection period requires significant expertise and diligence, the efforts are rewarded in that these studies demonstrate a significant analgesic effect from a single intrathecal injection of RTX. In a blinded, controlled study, 72 companion dogs with bone cancer pain were randomized to standard of care analgesic therapy alone (controls, n = 36) or 1.2 mcg/kg intrathecal RTX in addition to standard of care analgesic therapy (n = 36). Significantly more dogs in the control group required unblinding and adjustment in their analgesic protocol or euthanasia within 6 weeks of randomization, than dogs that were treated with RTX (Figure 6.4). In addition to providing proof-of-concept data with regard to efficacy, these studies can also inform some of the most concerning potential adverse events. A major concern associated with neurolytic therapies is deafferentation pain syndromes. Indiscriminate destruction of ganglionic neurons produces extensive proliferation of glia, inflammatory, and connective tissue cells, leading to scar formation [61,62]. This can lead to denervation-associated pain syndromes by inadvertent compression of surviving fibers. This has been described as severe pain referred to the area treated, with or without the recovery of sensory function. This complication prevents the routine use of these therapies in patients with chronic, nonmalignant pain (e.g., chronic pancreatitis) [63]. It has been a concern that the selective loss of afferents through molecular neurosurgical techniques also could cause the formation of such abnormal anatomical structures and lead to a complex and difficult to treat neuropathic pain state. In animals, deafferentation often leads to self-mutilation, biting the region in which they might feel painful or paresthetic sensations [64–66]. Self-mutilation has not been documented in any dogs following intrathecal RTX injection, suggesting a lack of development of deafferentation pain syndromes. RTX treatment appears to excise the TRPV1-positive pain-sensing neurons in such a way that substantial scar formation is circumvented [5,6]. It is associated with proliferation of satellite cells and macrophages, and these cells are replacing the dead neurons, but without formation of significant scar tissue [6]. The lack of behaviors suggestive of subsequent neuropathic pain processes may also be due to the retention of afferents not expressing TRPV1, incomplete removal of C-fiber neurons, and the fact that C-fiber primary afferents TRPV1 Agonists in the Companion Dog Model of Osteoarthritis 111 10 15 20 25 of of euthanasia within 6 weeks intervention Number of dogs requiring additional analgesics 05 Standard-of-care alone (n=36) Standard-of-care + IT RTX (n=36)

FIGURE 6.4 Single-blind osteosarcoma trial. In a single-blind, controlled study, 72 companion dogs with bone cancer pain were randomized to standard of care analgesic therapy alone (control, n = 36) or 1.2 mcg/kg intrathecal RTX in addition to standard of care analgesic therapy (treated, n = 35). Significantly more dogs in the control group (74%) required unblinding and adjustment in analgesic protocol or euthanasia within 6 weeks of randomization, than dogs that were treated with RTX (44%).

ascend and descend five or more dermatomes [67]. All of these influences may prevent trophic disturbances and secondary neuronal alterations due to loss of synaptic contacts [6]. Caution is still warranted in this area, however, because long-term follow-up is still needed.

TRPV1 AGONISTS IN THE COMPANION DOG MODEL OF OSTEOARTHRITIS

IA injections provide a potential target step in the continuum of conservative management to surgical interventions for osteoarthritis (OA) pain [68]. However, few IA compounds are a recommended part of practice. Even the IA injection of hyaluronic acid, often advocated for treatment of symptomatic OA, has come into question regarding its efficacy [68,69]. Only IA injection of corticosteroids has demonstrated consistently positive clinical trial results, but its effects are short term (2-4 weeks) [68,70,71]. Due to concerns of serious side effects, including bone deterioration, and progressive cartilage damage, it is recommended that the same joint receives no more than three steroid injections per year [72,73]. This limits its use and highlights the tremendous unmet need for an IA agent that provides safer and longer-acting pain relief. Identifying novel IA interventions of OA pain has been limited by the significant dis- connect between the animal models used to study the structural versus symptomatic aspects 112 6. TRPV1 AGONISTS AND PAIN CONTROL of OA [74,75]. Although the spontaneous models best mimic the slow progression of human disease with pathology and pathogenesis most similar to those occurring in human OA, they are expensive and time consuming to use. As such, acute and severe chemically induced rodent models are most commonly used for the study of chronic OA pain [74,76–78]. However, the initiating event and many of the pathology changes in these chemically induced models are not typical of human OA [74,79]. Given these considerations, acute models appear subop- timal for studying chronic OA symptoms. Approximately 20% of the canine pet population over 1 year old in the United States spontaneously develops OA. That means 72 million dogs are afflicted with a disease that microscopically, macroscopically, physiologically, and symptomatically mimics the human condition [80–86]. Multiple factors (heredity, nutrition, trauma) interact to cause the disorder in both people and dogs, and it is a disease that progresses slowly over a period of years. Radiographs reveal osteophyte formation and increased density of subchondral bone (Figure 6.5). Pain, decreased activity, and joint stiffness are common [87]. In both humans and dogs, management of OA focuses on reducing pain and other symptoms, minimizing functional limitations, and avoiding side effects associated with pharmacologic therapy [88]. These similarities and parallels in disease and symptomatology make the companion dog an excellent model in which to study the effects of IA RTX. In a pilot study of 7 dogs with osteoarthritis, a single IA injection of 10 mcg RTX elicited clinically and statistically significant decreases in pain scores and improvement in limb use. Interestingly, the improvement in pain scores increased from day 7 to day 21 after

FIGURE 6.5 Canine osteoarthritis radiograph. Lateral radiograph of the right stifle (knee) of a 9-year-old boxer dog 2 years following surgical repair for cranial cruciate ligament rupture. There is marked periarticular osteophy- tosis affecting the trochlear ridges of the femur, patella, fabella, tibial, and femoral condyles. Metal crimps along the femur and bone tunnel in the proximal tibia are associated with the prior surgical repair. TRPV1 Agonists in the Companion Dog Model of Osteoarthritis 113

FIGURE 6.6 Osteoarthritis pain evaluated by Canine Brief Pain Inventory measurement. Canine Brief Pain Inventory pain severity and pain interference scores in 7 dogs with osteoarthritis treated with a single intraarticular injection of 10 mcg of resiniferatoxin on day 0.

injection (Figure 6.6). Based on the immediate mechanism of action, axonal terminal calcium cytotoxicity and immediate loss of TRPV1-containing primary afferent endings with peripheral injections, one might expect the maximum analgesic effect to be apparent immediately after injection with gradual loss of analgesic effect as terminals regenerate over 1-2 weeks. One explanation for this progressive improvement in pain scores over several weeks, as well as the owners’ perception that there was efficacy for many months, could be the fact that, in addition to the direct analgesic action elicited by eliminating the TRPV1-containing primary afferent terminals, RTX treatment blocks local neurogenic inflammation and that this has a sustained modifying effect on generation of nociceptive signals. Equally as important as the positive analgesic effect is the lack of adverse events documented in these dogs after injection. All dogs recovered uneventfully from the joint tap and injection. As is the case with IA corticosteroids and the high-profile antinerve growth factor compounds [89], a longer-term adverse event concern is a detrimental contribution to the natural progression of OA. Although accelerated joint destruction and osteonecrosis has not been documented in the dog, longer-term studies with systematic evaluation of the joint for progression of disease are needed. With these positive pilot results and lack of obvious adverse events, the next step in translating IA RTX to human application is to perform a randomized, controlled, owner-blinded study in a larger cohort of dogs with osteoarthritis in which a range of doses is tested and dogs are systematically followed with pain scores, radiographs, and gait analysis for at least 1 year after injection. 114 6. TRPV1 AGONISTS AND PAIN CONTROL HUMAN STUDIES

As noted earlier, previous studies in animal pain models demonstrated profound pain relief and improved mobility after intrathecal RTX. Presently RTX is undergoing a Phase I clinical trial to treat medically refractory severe pain in patients with advanced cancer using intrathecal administration into the lumbar cistern. The intrathecal route exposes the DRG neuronal perikarya and the dorsal roots to RTX, which can delete them by elevating intracellular calcium to induce apoptosis and/or necrosis, depending on the dose, or a selective chemoaxotomy. The procedure is conducted in the operating room using propofol sedation to prevent the acute pain that accompanies excitotoxic actions of RTX on TRPV1 neurons. To date 10 patients have been treated: a pilot group of 4, which received 3-13 μg of RTX and an additional 6, who have received 13 or 26 μg under a more comprehensive study with pre- and postinjection assessments of pain (numerical rating scale, NRS), quality of life, quantitative testing of regional thermal sensation, and safety measurements. It was reported that: “Patients in the initial 4 patient pilot study experienced variable amounts of pain relief. Patients in subsequent cohorts also reported less pain and improved mobility after RTX injection. NRS trended lower compared to pretreatment, although the NRS change was statistically insig- nificant at these doses. Thermal perception reduction was consistent with cell death of the TRPV1 neurons. There were no other sensory or motor changes post-treatment” [53]. These initial findings suggest that intrathecal RTX administration can selectively and irreversibly delete neurons that transmit pain. Additional accruals will further test the safety and efficacy of RTX with a focus on reducing refractory pain and improving quality of life in advanced cancer patients.

References [1] Piomelli D, Sasso O. Peripheral gating of pain signals by endogenous lipid mediators. Nat Neurosci 2014;17:164–74. [2] von Hehn CA, Baron R, Woolf CJ. Deconstructing the neuropathic pain phenotype to reveal neural mechanisms. Neuron 2012;73:638–52. [3] Binshtok AM. Mechanisms of nociceptive transduction and transmission: a machinery for pain sensation and tools for selective analgesia. Int Rev Neurobiol 2011;97:143–77. [4] Neubert JK, Karai L, Jun JH, Kim HS, Olah Z, Iadarola MJ. Peripherally induced resiniferatoxin analgesia. Pain 2003;104:219–28. [5] Karai L, Brown DC, Mannes AJ, Connelly ST, Brown J, Gandal M, et al. Deletion of TRPV1-expressing primary afferent neurons for pain control. J Clin Invest 2004;113:1344–52. [6] Brown DC, Iadarola MJ, Perkowski SZ, Hardem E, Shofer F, Karai L, et al. Physiologic and antinociceptive effects of intrathecal resiniferatoxin in a canine bone cancer model. Anesthesiology 2005;103:1052–9. [7] Tender GC, Walbridge S, Olah Z, Karai L, Iadarola MJ, Oldfield EH, et al. Selective ablation of nociceptive neurons for elimination of hyperalgesia and neurogenic inflammation. J Neurosurg 2005;102:522–5. [8] Blackburn-Munro G. Pain-like behaviours in animals—how human are they? Trends Pharmacol Sci 2004;25:299–305. [9] Dionne RA, Witter J. NIH-FDA Analgesic Drug Development Workshop: translating scientific advances into improved pain relief. Clin J Pain 2003;19:139–47. [10] Fisher K, Coderre TJ, Hagen NA. Targeting the N-methyl-D-aspartate receptor for chronic pain management. Preclinical animal studies, recent clinical experience and future research directions. J Pain Symptom Manage 2000;20:358–73. [11] Galer BS, Lee D, Ma T, Nagle B, Schlagheck TG. MorphiDex (morphine sulfate/dextromethorphan hydrobro- mide combination) in the treatment of chronic pain: three multicenter, randomized, double-blind, controlled clinical trials fail to demonstrate enhanced opioid analgesia or reduction in tolerance. Pain 2005;115:284–95. REFERENCES 115

[12] Hill R. NK1 (substance P) receptor antagonists—why are they not analgesic in humans? Trends Pharmacol Sci 2000;21:244–6. [13] Mao J. Translational pain research: bridging the gap between basic and clinical research. Pain 2002;97:183–7. [14] Mogil JS. Animal models of pain: progress and challenges. Nat Rev Neurosci 2009;10:283–94. [15] Mogil JS, Crager SE. What should we be measuring in behavioral studies of chronic pain in animals? Pain 2004;112:12–15. [16] Rowbotham MC, Nothaft W, Duan WR, Wang Y, Faltynek C, McGaraughty S, et al. Oral and cutaneous thermosensory profile of selective TRPV1 inhibition by ABT-102 in a randomized healthy volunteer trial. Pain 2011;152:1192–200. [17] Quiding H, Jonzon B, Svensson O, Webster L, Reimfelt A, Karin A, et al. TRPV1 antagonistic analgesic effect: a randomized study of AZD1386 in pain after third molar extraction. Pain 2013;154:808–12. [18] Iadarola MJ, Mannes AJ. The vanilloid agonist resiniferatoxin for interventional-based pain control. Curr Top Med Chem 2011;11(2171–2179):2011. [19] Iadarola MJ, Gonnella GL. Resiniferatoxin for pain management: an interventional approach to personalized pain medicine. Open Pain J 2013;6(Suppl. 1):95–107. [20] Raj PP, Lou L, Erdine S, Staats PS, Waldman SD, Racz G, et al. Interventional pain management: image-guided procedures. Philadelphia: Saunders-Elsevier; 2008. [21] Buvanendran A, Diwan S, Deer T. Intrathecal drug delivery for pain and spasticity. Philadelphia: Saunders- Elsevier; 2011. [22] Kárai LJ, Russell JT, Iadarola MJ, Oláh Z. Vanilloid receptor 1 regulates multiple calcium compartments and contributes to Ca2+-induced Ca2+ release in sensory neurons. J Biol Chem 2004;279:16377–87. [23] Mitchell K, Bates BD, Keller JM, Lopez M, Scholl L, Navarro J, et al. Ablation of rat TRPV1-expressing Aδ/ C-fibers with resiniferatoxin: analysis of withdrawal behaviors, recovery of function and molecular correlates. Mol Pain 2010;6:94. [24] Mitchell K, Lebovitz EE, Keller JM, Mannes AJ, Nemenov M, Iadarola MJ. Nociception and inflammatory hyperalgesia evaluated in rodents using infrared laser stimulation after Trpv1 gene knockout or resiniferatoxin lesion. Pain 2014;155:733–45. [25] Olah Z, Szabo T, Karai L, Hough C, Fields RD, Caudle RM, et al. Ligand-induced dynamic membrane changes and cell deletion conferred by vanilloid receptor 1. J Biol Chem 2001;276:11021–30. [26] Caudle RM, Kárai L, Mena N, Cooper BY, Mannes AJ, Perez FM, et al. Resiniferatoxin-induced loss of plasma membrane in vanilloid receptor expressing cells. Neurotoxicology 2003;24:895–908. [27] Subramanian K, Meyer T. Calcium-induced restructuring of nuclear envelope and endoplasmic reticulum calcium stores. Cell 1997;89:963–71. [28] Gallego-Sandín S, Rodríguez-García A, Alonso MT, García-Sancho J. The endoplasmic reticulum of dorsal root ganglion neurons contains functional TRPV1 channels. J Biol Chem 2009;284:32591–601. [29] Thomas KC, Ethirajan M, Shahrokh K, Sun H, Lee J, Cheatham 3rd TE, et al. Structure-activity relationship of capsaicin analogs and transient receptor potential vanilloid 1-mediated human lung epithelial cell toxicity. J Pharmacol Exp Ther 2011;337:400–10. [30] Ahn S, Park J, An I, Jung SJ, Hwang J. Transient receptor potential cation channel V1 (TRPV1) is degraded by starvation- and glucocorticoid-mediated autophagy. Mol Cells 2014;37:257–63. [31] Chien CS, Ma KH, Lee HS, Liu PS, Li YH, Huang YS, et al. Dual effect of capsaicin on cell death in human osteosarcoma G292 cells. Eur J Pharmacol 2013;718:350–60. [32] Stock K, Kumar J, Synowitz M, Petrosino S, Imperatore R, Smith ES, et al. Neural precursor cells induce cell death of high-grade astrocytomas through stimulation of TRPV1. Nat Med 2012;18:1232–8. [33] Martuza RL. Molecular neurosurgery for glial and neuronal disorders. Stereotact Funct Neurosurg 1992;59:92–9. [34] Lee MG, Huh BK, Choi SS, Lee DK, Lim BG, Lee M. The effect of epidural resiniferatoxin in the neuropathic pain rat model. Pain Physician 2012;15:287–96. [35] Bishnoi M, Bosgraaf CA, Premkumar LS. Preservation of acute pain and efferent functions following intrathecal resiniferatoxin-induced analgesia in rats. J Pain 2011;12:991–1003. [36] Abdelhamid RE, Kovács KJ, Honda CN, Nunez MG, Larson AA. Resiniferatoxin (RTX) causes a uniquely protracted musculoskeletal hyperalgesia in mice by activation of TRPV1 receptors. J Pain 2013;14:1629–41. [37] Aizawa N, Ogawa S, Sugiyama R, Homma Y, Igawa Y. Influence of urethane-anesthesia on the effect of resiniferatoxin treatment on bladder function in rats with spinal cord injury. Neurourol Urodyn 2013; http:// dx.doi.org/10.1002/nau.22549. 116 6. TRPV1 AGONISTS AND PAIN CONTROL

[38] Cruz LS, Kopruszinski CM, Chichorro JG. Intraganglionar resiniferatoxin prevents orofacial inflammatory and neuropathic hyperalgesia. Behav Pharmacol 2014;25:112–18. [39] Tender GC, Li YY, Cui JG. The role of nerve growth factor in neuropathic pain inhibition produced by resiniferatoxin treatment in the dorsal root ganglia. Neurosurgery 2013;73:158–65. [40] Wu CH, Lv ZT, Zhao Y, Gao Y, Li JQ, Gao F, et al. Electroacupuncture improves thermal and mechanical sensi- tivities in a rat model of postherpetic neuralgia. Mol Pain 2013;9:18. [41] Lin CL, Fu YS, Hsiao TH, Hsieh YL. Enhancement of purinergic signalling by excessive endogenous ATP in resiniferatoxin (RTX) neuropathy. Purinergic Signal 2013;9:249–57. [42] Danigo A, Magy L, Richard L, Sturtz F, Funalot B, Demiot C. A reversible functional sensory neuropathy model. Neurosci Lett 2014;571:39–44. [43] Shi B, Li X, Chen J, Su B, Li X, Yang S, et al. Resiniferatoxin for treatment of lifelong premature ejaculation: a preliminary study. Int J Urol 2014;21:923–6 [Epub ahead of print]. [44] Guo C, Yang B, Gu W, Peng B, Xia S, Yang F, et al. Intravesical resiniferatoxin for the treatment of storage lower urinary tract symptoms in patients with either interstitial cystitis or detrusor overactivity: a meta-analysis. PLoS One 2013;8:e82591. [45] Szallasi A, Blumberg PM. [3H]resiniferatoxin binding by the vanilloid receptor: species-related differences, effects of temperature and sulfhydryl reagents. Naunyn Schmiedebergs Arch Pharmacol 1993;347:84–91. [46] Kim MS, Ki Y, Ahn SY, Yoon S, Kim SE, Park HG, et al. Asymmetric synthesis and receptor activity of chiral simplified resiniferatoxin (sRTX) analogues as transient receptor potential vanilloid 1 (TRPV1) ligands. Bioorg Med Chem Lett 2014;24:382–5. [47] Vaso A, Adahan H, Gjika A, Zahaj S, Zhurda T, Vyshka G, et al. Peripheral nervous system origin of phantom limb pain. Pain 2014;155:1384–91. [48] Reilly RM, McDonald HA, Puttfarcken PS, Joshi SK, Lewis L, Pai M, et al. Pharmacology of modality-specific transient receptor potential vanilloid-1 antagonists that do not alter body temperature. J Pharmacol Exp Ther 2012;342:416–28. [49] Nash MS, McIntyre P, Groarke A, Lilley E, Culshaw A, Hallett A, et al. 7-tert-Butyl-6-(4-chloro-phenyl)-2-thioxo- 2,3-dihydro-1H-pyrido[2,3-d]pyrimidin-4-one, a classic polymodal inhibitor of transient receptor potential vanilloid type 1 with a reduced liability for hyperthermia, is analgesic and ameliorates visceral hypersensitivity. J Pharmacol Exp Ther 2012;342:389–98. [50] Fujii Y, Ozaki N, Taguchi T, Mizumura K, Furukawa K, Sugiura Y. TRP channels and ASICs mediate mechanical hyperalgesia in models of inflammatory muscle pain and delayed onset muscle soreness. Pain 2008;140:292–304. [51] Whitney MA, Crisp JL, Nguyen LT, Friedman B, Gross LA, Steinbach P, et al. Fluorescent peptides highlight peripheral nerves during surgery in mice. Nat Biotechnol 2011;29:352–6. [52] Hoehne A, Behera D, Parsons WH, James ML, Shen B, Borgohain P, et al. A 18F-labeled saxitoxin derivative for in vivo PET-MR imaging of voltage-gated sodium channel expression following nerve injury. J Am Chem Soc 2013;135:18012–15. [53] Heiss J, Iadarola M, Cantor F, Oughourli A, Smith R, Mannes A. A Phase I study of the intrathecal administration of resiniferatoxin for treating severe refractory pain associated with advanced cancer. J Pain 2014;15(4):S67. [54] Ostrander EA, Galibert F, Patterson DF. Canine genetics comes of age. Trends Genet 2000;16:117–24. [55] Brown D, Boston R, Coyne J, Farrar J. A novel approach to the use of animals in studies of pain: validation of the canine brief pain inventory in canine bone cancer. Pain Medicine 2009;10:133–42. [56] Brown DC, Boston RC, Coyne JC, Farrar JT. Development and psychometric testing of an instrument designed to measure chronic pain in dogs with osteoarthritis. Am J Vet Res 2007;68:631–7. [57] Brown DC, Boston RC, Coyne JC, Farrar JT. Ability of the canine brief pain inventory to detect response to treatment in dogs with osteoarthritis. J Am Vet Med Assoc 2008;233:1278–83. [58] Mueller F, Fuchs B, Kaser-Hotz B. Comparative biology of human and canine osteosarcoma. Anticancer Res 2007;27:155–64. [59] Withrow SJ, Powers BE, Straw RC, Wilkins RM. Comparative aspects of osteosarcoma. Dog versus man. Clin Orthop Relat Res 1991;270:159–68. [60] MacEwen EG. Spontaneous tumors in dogs and cats: models for the study of cancer biology and treatment. Cancer Metast Rev 1990;9:125–36. REFERENCES 117

[61] Sweet WH. Deafferentation pain after posterior rhizotomy, trauma to a limb, and herpes zoster. Neurosurgery 1984;15:928–32. [62] Hakanson S. Comparison of surgical treatments for trigeminal neuralgia: reevaluation of radiofrequency rhizotomy. Neurosurgery 1997;40:1106–7 [see comment]. [63] Whitworth LA, Feler CA. Application of spinal ablative techniques for the treatment of benign chronic painful conditions: history, methods, and outcomes. Spine 2002;27:2607–12. [64] Kriz N, Yamamotova A, Tobias J, Rokyta R. Tail-flick latency and self-mutilation following unilateral deafferentation in rats. Physiol Res 2006;55:213–20. [65] Kriz N, Rokyta R. Effect of unilateral deafferentation on extracellular potassium concentration levels in rat thalamic nuclei. Neuroscience 2000;96:101–8. [66] Albe-Fessard DG, Rampin O. Neurophysical studies in rats deafferented by dorsal root sections. In: Nashold B, Ovelmen-Levitt J, editors. Deafferentation pain syndromes: pathophysiology and treatments. New York: Raven Press; 1991. p. 125–39. [67] Traub RJ, Iadarola MJ, Ruda MA. Effect of multiple dorsal rhizotomies on calcitonin gene-related peptide-like immunoreactivity in the lumbosacral dorsal spinal cord of the cat: a radioimmunoassay analysis. Peptides 1989;10:979–83. [68] Cheng OT, Souzdalnitski D, Vrooman B, Cheng J. Evidence-based knee injections for the management of arthritis. Pain Med 2012;13:740–53. [69] Rutjes AWS, Juni P, da Costa BR, Trelle S, Nuesch E, Reichenbach S. Viscosupplementation for osteoarthritis of the knee: a systematic review and meta-analysis. Ann Intern Med 2013;157:180–91. [70] Godwin M, Dawes M. Intra-articular steroid injections for painful knees. Systematic review with meta-analysis. Can Fam Physician 2004;50:241–8. [71] Arroll B, Goodyear-Smith F. Corticosteroid injections for osteoarthritis of the knee: meta-analysis. BMJ 2004;328:869. [72] Habib GS, Saliba W, Nashashibi M. Local effects of intra-articular corticosteroids. Clin Rheumatol 2010;29:347–56. [73] Syed HM, Green L, Bianski B, Jobe CM, Wongworawat MD. Bupivacaine and triamcinolone may be toxic to human chondrocytes: a pilot study. Clin Orthop 2011;469:2941–7. [74] Little CB, Zaki S. What constitutes an "animal model of osteoarthritis"—the need for consensus? Osteoarthr Cartil 2012;20:261–7. [75] Baragi VM, Becher G, Bendele AM, Biesinger R, Bluhm H, Boer J, et al. A new class of potent matrix metalloproteinase 13 inhibitors for potential treatment of osteoarthritis: evidence of histologic and clinical efficacy without musculoskeletal toxicity in rat models. Arthritis Rheum 2009;60:2008–18. [76] Ameye LG, Young MF. Animal models of osteoarthritis: lessons learned while seeking the "Holy Grail". Curr Opin Rheumatol 2006;18:537–47. [77] Bendele A, McComb J, Gould T, McAbee T, Sennello G, Chlipala E, et al. Animal models of arthritis: relevance to human disease. Toxicol Pathol 1999;27:134–42. [78] Vincent TL, Williams RO, Maciewicz R, Silman A, Garside P, Arthritis Research UKamwg. Mapping pathogenesis of arthritis through small animal models. Rheumatology (Oxford) 2012;51:1931–41. [79] Barve RA, Minnerly JC, Weiss DJ, Meyer DM, Aguiar DJ, Sullivan PM, et al. Transcriptional profiling and pathway analysis of monosodium iodoacetate-induced experimental osteoarthritis in rats: relevance to human disease. Osteoarthr Cartil 2007;15:1190–8. [80] Henrotin Y, Sanchez C, Balligand M. Pharmaceutical and nutraceutical management of canine osteoarthritis: present and future perspectives. Vet J 2005;170:113–23. [81] Johnston SA. Osteoarthritis. Joint anatomy, physiology, and pathobiology. Vet Clin North Am Small Anim Pract 1997;27:699–723. [82] Vaino O. Translational animal models using veterinary patients—an example of canine osteoarthritis (OA). Scand J Pain 2012;3:84–9. [83] Burton-Wurster N, Hui-Chou CS, Greisen HA, Lust G. Reduced deposition of collagen in the degenerated articular cartilage of dogs with degenerative joint disease. Biochim Biophys Acta 1982;718:74–84. [84] Lust G, Pronsky W, Sherman DM. Biochemical and ultrastructural observations in normal and degenerative canine articular cartilage. Am J Vet Res 1972;33:2429–40. [85] Miller DR, Lust G. Accumulation of procollagen in the degenerative articular cartilage of dogs with osteoarthritis. Biochim Biophys Acta 1979;583:218–31. 118 6. TRPV1 AGONISTS AND PAIN CONTROL

[86] Liu W, Burton-Wurster N, Glant TT, Tashman S, Sumner DR, Kamath RV, et al. Spontaneous and experimental osteoarthritis in dog: similarities and differences in proteoglycan levels. J Orthop Res 2003;21:730–7. [87] Hinton R, Moody RL, Davis AW, Thomas SF. Osteoarthritis: diagnosis and therapeutic considerations. Am Family Physician 2002;65:841–8. [88] Shamoon M, Hochberg MC. Treatment of osteoarthritis with acetaminophen: efficacy, safety, and comparison with nonsteroidal anti-inflammatory drugs. Curr Rheumatol Rep 2000;2:454–8. [89] Brown MT, Murphy FT, Radin DM, Davignon I, Smith MD, West CR. Tanezumab reduces osteoarthritic knee pain: results of a randomized, double-blind, placebo-controlled phase III trial. J Pain 2012;13:790–8. CHAPTER 7 Intravesical Capsaicin and Resiniferatoxin for Bladder Disorders Ana Charrua,1,2,3,* Francisco Cruz1,2,3,4 1IBMC—Instituto de Biologia Molecular e Celular da Universidade do Porto, Porto, Portugal 2Departamento de Urologia, Faculdade de Medicina da Universidade do Porto, Porto, Portugal 3Departamento de Doenças Renais, Urológicas e Infecciosas, Faculdade de Medicina da Universidade do Porto, Porto, Portugal 4Departamento de Urologia, Hospital São João, Porto, Portugal *Corresponding author: [email protected]

OUTLINE

Capsaicin and Resiniferatoxin Overactivity and Idiopathic Detrusor Targets and Effects in the Overactivity 121 Urinary Bladder 120 Intravesical Application of Vanilloids in Patients with Bladder Pain Syndrome/ The Rationale for Using Vanilloids to Interstitial Cystitis 123 Overcome Lower Urinary Tract Symptoms 120 Is There a Future for Intravesical Instillation of Vanilloids in the Intravesical Application of Vanilloids 121 Urinary Bladder? 124 Intravesical Application of Vanilloids in Patients with Neurogenic Detrusor References 125

TRP Channels as Therapeutic Targets 119 © 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/B978-0-12-420024-1.00007-2 120 7. INTRAVESICAL CAPSAICIN AND RESINIFERATOXIN CAPSAICIN AND RESINIFERATOXIN TARGETS AND EFFECTS IN THE URINARY BLADDER

Capsaicin and resiniferatoxin (RTX) are clinically useful drugs because they specifically bind to, excite, and then desensitize the transient receptor potential vanilloid type 1 (TRPV1) [1,2]. TRPV1 is expressed in peptidergic primary afferents that course the mucosa and muscular layers of the urinary bladder [3–7] and in urothelial cells [6–8]. TRPV1 is known to be involved in the development of bladder pain associated with cystitis [9–11] and bladder hyperactivity of neurogenic and idiopathic origin [10–13].

THE RATIONALE FOR USING VANILLOIDS TO OVERCOME LOWER URINARY TRACT SYMPTOMS

Micturition is under the control of two distinct neuronal circuits [13]. In normal conditions, the circuit that prevails is the long, supraspinal pathway, initiated in Aδ bladder afferents and passing through the periaqueductal gray matter (PAG) and the pontine micturition center (PMC) [13]. Under certain abnormal conditions, another circuit, which is shorter and is entirely located in the lumbosacral spinal segments, may take over bladder control [13]. This circuit is initiated in C-type bladder afferents. The emergence of a micturition reflex commanded by the sacral micturition pathway was first observed in felines with chronic spinal cord transection between PMC and sacral spinal cord [13]. Posteriorly, this same reflex was shown to prevail in nonneurological conditions like chronic bladder obstruction [14] and inflammation [15]. The sacral micturition reflex, being entirely located in the lumbar-sacral spinal cord segments, escapes the control of supraspinal centers that regulate micturition, including PAG, PMC, and cortical areas that modulate those two centers [13]. The consequence is that the parasympathetic outflow generated by the sacral micturition reflex can generate detrusor contractions totally independent of vol- untary control [13]. This can generate sudden, imminent desire to void (usually known as urgency to void). If the impulse is strong enough to overcome sphincteric resistance, it may cause urinary leakage (urgency urinary incontinence) [16,17]. Being initiated by C fibers, desensitization of these primary afferents by capsaicin or RTX can decrease or suppress the abnormal sacral micturition reflex [13]. This possibility was first shown in chronic spinal cord transected cats. These animals, once treated with systemic capsaicin, lose the reflex micturition [13]. In contrast, capsaicin administration to the spinal cord of intact cats does not alter reflex micturition [13]. Likewise, capsaicin and RTX were shown to suppress bladder hyperactivity induced by cyclophosphamide-induced cystitis [10] and to decreased noxious input induced by bladder inflammation [10,18]. These experiments clearly suggested that the desensitization of bladder C fibers by capsaicin or RTX could have relevant clinical application. Two pertinent studies tested the application of capsaicin and RTX by intravesical route, taking advantage of the fact that capsaicin is part of the human diet, and RTX was used as a pain killer for centuries in the Roman Empire. The first clinical study used capsaicin [19] and the second study used RTX [15]; both studies involved patients with bladder overactivity of neurogenic origin. Intravesical Application of Vanilloids 121

Importantly, neither intravesical capsaicin nor RTX induces persistent morphological changes in the bladder. After 5 years of capsaicin instillation, there were no morphological changes in the bladder urothelium [20]. Bladder biopsies taken from patients with detrusor overactivity treated for 2 years with intravesical RTX showed no evidence of inflammatory reaction, hyperplastic response, or tumor formation in the bladder mucosa, or any damage in the urothelium and its basal membrane [21]. Furthermore, electron microscopic (ultrastructural) observations showed that RTX did not alter the structure of unmyelinated nerve fibers in the lamina propria of the aforementioned bladder samples [21]. As RTX was shown to desensitize bladder afferents without the intense initial excitation perceived by patients treated with capsaicin [22], most of the studies that are reviewed in this chapter have used RTX rather than capsaicin as the desensitizing agent.

INTRAVESICAL APPLICATION OF VANILLOIDS

Intravesical Application of Vanilloids in Patients with Neurogenic Detrusor Overactivity and Idiopathic Detrusor Overactivity Initial studies with capsaicin to ameliorate hyperreflexia were promising, with patients showing an increase in cystometric capacity and a decrease in maximum detrusor pressure up to 7 months posttreatment [19,23–31]. However, acutely capsaicin produces a strong abdominal burning pain after intravesical instillation. This adverse effect and the possibility of autonomic dysreflexia during capsaicin instillation discouraged the wider use of intravesical capsaicin [31–35]. The advantage of RTX over capsaicin to manage bladder hyperreflexia in neurogenic detrusor overactivity (NDO) patients was confirmed by de Sèze and coworkers in a double- blinded randomized clinical trial [36]. In this study, 21 patients were included in the RTX group, and 18 patients were included in the capsaicin group. One month after instillation, both groups (80% of patients for RTX and 78% of patients for capsaicin) had improvement in continence (5.5—0.5 leakage episodes/day in the capsaicin arm, and 4—0 leakage episodes/ day in the RTX arm), frequency (10—6 voids/day in the capsaicin arm and 8—6 voids/day in the RTX arm), and urgency, with increased maximum cystometric capacity (175–282 ml in the capsaicin arm and 221–263 ml in the RTX arm). The follow-up of these patients demonstrated that the duration of RTX and capsaicin desensitization was similar: 66.8 days in the capsaicin arm and 91.1 days in the RTX arm [36]. However, the comparison of the tolerability of these two compounds showed that suprapubic pain complaints were significantly more frequent in the capsaicin group [36]. Other side effects such as urethral pain, autonomic dysreflexia, or hematuria were rarely observed in either group. Also, no bladder changes were observed on cystoscopy [36]. In a placebo-controlled, randomized clinical trial involving 28 patients with NDO, Silva and coworkers [37] have demonstrated that intravesical instillation of 50 nM RTX (dissolved in 10% ethanol in saline) increased bladder volume to first involuntary detrusor contraction from 143 ± 95 to 184 ± 93 ml in NDO patients, whereas placebo instillation (10% ethanol) did not change that volume (from 115 ± 58 to 115 ± 61 ml). Similarly, maximal cystometric capacity increased from 189 ± 99 to 314 ± 135 ml in the RTX group, whereas the increase in the placebo 122 7. INTRAVESICAL CAPSAICIN AND RESINIFERATOXIN group was minimal (from 198 ± 111 to 204 ± 92 ml). Also, the mean frequency and daily incontinence improved 20% (from 9.5 ± 2.5 to 7.6 ± 2.1 voids/day, p = 0.01) and 60% (from 4.5 ± 4.5 to 1.6 ± 1.4 incontinence episodes/day, p = 0.03), respectively, in the RTX-treated group and were unaltered in the placebo group (from 10 ± 2 to 9.6 ± 2.6 voids/day, p = 0.6; 1.8 ± 2.5 to 1.0 ± 1.4 incontinence episodes/day). Because the etiology underlying NDO was diverse (each one represented by only a few cases), the authors did not analyze the effect of RTX and placebo per etiology [37]. The follow-up of these patients showed that RTX-induced improvement in urodynamic characteristic was maintained up to 3 months [37]. Furthermore, using a visual analog scale, the authors concluded that the intensity of the discomfort during the vehicle or the RTX solution instillation were similar [37]. Importantly, intravesical instillation of RTX did not cause autonomic dysreflexia, hematuria, or persistent infection [37]. These results were replicated by Kim and coworkers in 32 patients with NDO [38]. RTX (500-1000 nM) instilled into the bladder induced minimal pain, similar to that observed in the placebo arm [38]. Also, these authors observed a superior effect of RTX in mean cystometric capacity and in the mean number of incontinence episodes when compared to the placebo solution. RTX instillation was not associated with any significant complication. In another study, Kuo and coworkers treated 15 patients with NDO, 20 patients with bladder outlet obstruction, and 19 patients with idiopathic detrusor overactivity (IDO) with four intravesical RTX instillations (10 nM each), compared to placebo [39]. During the follow-up of these patients, the success rate of treatment was analyzed at 1, 3, 6, and 12 months follow-up [39]. Using the Kaplan-Meier analysis, the authors concluded that the success rate was higher in the RTX group than in placebo group (results were: 73% versus 29% (p < 0.001) at 1 month, 62% versus 21% (p < 0.001) at 3 months, 50% versus 11% (p < 0.001) at 6 months, and 12% versus 0.4% (p > 0.05) at 12 months after treatment, respectively). As in the previous studies, no adverse effects due to RTX intravesical installations were observed [39]. Silva and coworkers enrolled 23 overactive bladder (OAB) patients with refractory urgency into a clinical trial; these patients had their anticholinergic medication for bladder dysfunction interrupted for a month, after which they had filled out a 7-day voiding chart to score bladder sensations before each voiding [40]. On average, patients experienced 71 ± 12 episodes of severe urgency per week. Thereafter, patients received intravesical ethanol (10%), and 1 month later they were asked to fill out a second 7-day voiding chart. This time, the mean number of urgency episodes was 56 ± 11, with 17% of patients considering they had improved their urinary symptoms. In the last step of the study, the bladder of the OAB patients was treated with 50 nM RTX dissolved in ethanol (10%). One and 3 months later, patients filled in a third and fourth 7-day voiding chart. After RTX administration, the episodes of urgency decreased to 39 ± 9 and 37 ± 6, respectively, with 69% of patients reporting an improvement of urinary symptoms [40]. Of note, intravesical RTX is not the only therapeutic tool available to overcome bladder hyperreflexia in OAB patients. Botulinum toxin has become another option. Giannantoni and coworkers have performed a randomized clinical trial to compare the efficacy of these two treatments [41]. A total of 25 patients were randomly assigned to receive intravesically RTX (0.6 M) in saline or detrusor muscle injection with 300 units of botulinum toxin type-A (OnabotA). In both arms, there was a significant decrease in catheterization need and in- continent episodes and a significant increase in first detrusor contraction and maximum Intravesical Application of Vanilloids 123

bladder capacity at 6-, 12-, and 18-month follow-up. There were no local side effects with either treatment. However, OnabotA induced significantly greater improvements than RTX. At 6 months, compared to RTX, OnabotA decreased the number of daily incontinence episodes (from 5.4 ± 1.3 to 2.2 ± 1.2 in the RTX arm, and from 4.8 ± 1.1 to 1.4 ± 1.7 in the OnabotA arm, p < 0.001 in both arms), significantly increased the volume for involuntary detrusor contraction (from 200.5 ± 69.7 to 288.7 ± 83.7 ml in the RTX arm and from 190 ± 48.6 to 326.3 ± 80.9 ml in the OnabotA arm, p < 0.01 in the RTX arm versus OnabotA arm) and in maximum bladder capacity (from 223.3 ± 68.1 to 329.0 ± 72.3 ml in the RTX arm and from 211.9 ± 49.7 to 370.0 ± 79.6 ml in the OnabotA arm, p < 0.01 in the RTX arm versus OnabotA arm), and significantly decreased maximum detrusor pressure (from 70.2 ± 26.0 cm H2O to 63.1 ± 26.2 cm H2O in the RTX arm and from 78.5 ± 21.5 cm H2O to 47.0 ± 18.3 cm H2O in the OnabotA arm, p < 0.01 in the RTX arm versus OnabotA arm). The efficacy of RTX and botulinum toxin was also compared in patients with IDO [42]. Santos-Silva and coworkers enrolled 34 female patients into this study: 17 patients were treated with 100 nM RTX (in 10% ethanol), and another 17 patients were injected with 100 U OnabotA. As endpoints, the number of incontinence episodes in a 3-day bladder diary, the quality-of-life questionnaires, and urodynamic studies were used. These parameters were analyzed at baseline and at 3 months after treatment. OnabotA was more effective in abol- ishing urinary incontinence (from 3 to 0 incontinence episodes/day in the OnabotA arm and from 3.5 to 2.5 incontinence episodes/day in the RTX arm), in reducing frequency (from 11 to 6 episodes/day in the OnabotA arm and from 9.5 to 8.5 episodes/day in the RTX arm), in increasing quality of life (score increase from 29.2% to 45.9% in the OnabotA arm and from 12.4% to 15.7% in the RTX arm), and improving bladder capacity (from 363 to 490 ml in the OnabotA arm and from 450.5 to 558.5 ml in the RTX arm) [42].

Intravesical Application of Vanilloids in Patients with Bladder Pain Syndrome/Interstitial Cystitis Due to their ability to relieve pain by desensitizing nociceptive capsaicin-sensitive fibers, intravesical instillation of vanilloids was investigated as a tool to treat pain in patients with bladder pain syndrome/interstitial cystitis (BPS/IC) [43]. A pilot study was performed to assess the effect of multiple intravesical capsaicin instillations in increasing doses on the treatment of pain [43]. Most of the patients reported an improvement of symptoms (bladder capacity, frequency, nocturia) and pain, without any observed side effects. These results suggested intravesical capsaicin as a promising treatment for BPS/IC. In a randomized, placebo-controlled study, Lazzeri and coworkers evaluated the clinical benefit of intravesical application of 0.3 μM capsaicin (twice a week for a month) for severe bladder pain [44]. Before the study, all of the 36 patients had severe pelvic pain and all had undergone prior pain therapy such as nonsteroidal anti-inflammatory drugs, antihistamines, intravesical lidocaine hydrochloride, or transvaginal electrostimulation [44]. All patients complained about the warm and burning sensation in the pelvic region that accompanied the capsaicin intravesical instillation and was long-lasting but progressively less intense in the capsaicin group on successive instillations. In the placebo group, this discomfort had a shorter duration but was sustained on successive instillation of vehicle. An improvement in frequency (41.2-11.8% of patients that presented more than 10 voiding episodes after 6 months) 124 7. INTRAVESICAL CAPSAICIN AND RESINIFERATOXIN and in nocturia (29.4-5.9% of patients that presented more than 10 episodes after 6 months) was noted in the capsaicin-treated group only [44]. The authors have also observed a significant decrease in the pain score after capsaicin intravesical instillation (3.22 ± 0.42, p < 0.01 after treatment; 3.83 ± 0.47, p < 0.01 6 months after treatment) compared to before capsaicin treatment (5.61 ± 0.40). In fact, 71% of capsaicin-treated patients reported an improvement in quality of life, which lasted for 6 months without need for another medication for 22% of those patients. In the placebo group, pain score after treatment was 4.47 ± 0.36 after instillation and 4.48 ± 0.34 at 6 months after treatment. No histopathology changes or side effects were observed in either group after treatment [44]. Lazzeri and coworkers also evaluated the efficacy of prolonged intravesical infusion of 10 nM of RTX in the treatment of pain and lower urinary tract symptoms associated with BPS/ IC [45]. One month after RTX infusion, the five patients enrolled in this study had statistically significant reduction of pain (from 6.7 ± 0.83 to 2.4 ± 0.54, p < 0.01 1 month after treatment, and to 3.2 ± 0.44, p < 30.05 months after treatment), frequency (from 11.3 ± 1.39 to 7.4 ± 1.51, p < 0.01 1 month after treatment, and to 8.7 ± 1.76, 3 months after treatment), and nocturia (from 3.6 ± 0.54 to 1.2 ± 0.44, p < 0.01 1 month after treatment, and to 1.9 ± 0.74, 3 months after treatment). Again, no significant side effects were noticed during or after RTX infusion, which made RTX intravesical treatment preferred over the painful capsaicin intravesical instillation. These data prompted Payne and coworkers to perform a large randomized, double-blinded, multicenter, placebo-controlled study to evaluate the efficacy of intravesical RTX treatment (10, 50, and 100 nM) in 163 BPS/IC patients [46]. Three months after treatment neither placebo nor RTX-treated group presented significant changes at global response assessment score (p = 0.827), pain, nocturia, urgency, frequency, average voided volume, nor to responses to the O’Leary-Sant and Pelvic Pain and Urgency/Frequency Symptom Scale questionnaires [46]. These authors have observed that RTX instillation presented a dose-dependent increase in instillation pain (52% of patients from placebo arm and 72-80% of patients from RTX arm) and urgency. In fact, there was an increase in the percentage of patients who did not tolerate the 30 min required for RTX instillation. One of the patients treated with 10 nM RTX presented severe lower abdominal pain requiring hospitalization [46].

IS THERE A FUTURE FOR INTRAVESICAL INSTILLATION OF VANILLOIDS IN THE URINARY BLADDER?

Intravesical capsaicin instillation was initially promoted as a promising tool to overcome bladder hyperactivity. The pungency of capsaicin solutions was, however, not tolerated by many patients, and this compound was quickly replaced by RTX. Unfortunately, there are several problems with RTX treatment that need to be solved to render this compound clinically appealing. First and foremost, the optimal formulation of RTX is still unknown. RTX is unstable in aqueous solutions, which is thought to have contributed to some of the differences between the results of the studies where this molecule was used. Furthermore, RTX adheres to plastics, and if prepared in advance for instillation, a marked decay of active molecule rapidly occurs [47]. Therefore, studies to determine the adequate vehicle, the doses to be used, the number of applications, and the material used during administrations are still needed. REFERENCES 125

In fact, botulinum toxin type-A (which has a stable formulation) has largely replaced intravesical RTX. That said, RTX may present important advantages over botulinum toxin-A, such as that it does not exacerbate poor bladder emptying. In a recent large randomized clinical trial with OAB patients, botulinum toxin type-A induced almost 16% of urinary retention, which lasted for several weeks [48]. Furthermore, RTX instillation is easier and cheaper to administer than botulinum toxin type-A injection, which requires cystoscopy. In summary, if the adequate formulation and dosage are determined, the use of intravesical RTX solution may regain a therapeutic position in the treatment of some lower urinary tract symptoms, most probably placed between oral drugs and botulinum toxin injections.

References [1] Caterina MJ, Julius D. The vanilloid receptor: a molecular gateway to the pain pathway. Annu Rev Neurosci 2001;24:487–517. [2] Szallasi A, Blumberg PM. Vanilloid (capsaicin) receptors and mechanisms. Pharmacol Rev 1999;51(2):159–212. [3] Avelino A, Cruz C, Nagy I, Cruz F. Vanilloid receptor 1 expression in the rat urinary tract. Neuroscience 2002;109(4):787–98. [4] Apostolidis A, Popat R, Yiangou Y, et al. Decreased sensory receptors P2X3 and TRPV1 in suburothelial nerve fibers following intradetrusor injections of botulinum toxin for human detrusor overactivity. J Urol 2005;174(3):977–82, discussion 82–3. [5] Brady CM, Apostolidis AN, Harper M, et al. Parallel changes in bladder suburothelial vanilloid receptor TRPV1 and pan-neuronal marker PGP9.5 immunoreactivity in patients with neurogenic detrusor overactivity after intravesical resiniferatoxin treatment. BJU Int 2004;93(6):770–6. [6] Lazzeri M, Vannucchi MG, Zardo C, et al. Immunohistochemical evidence of vanilloid receptor 1 in normal human urinary bladder. Eur Urol 2004;46(6):792–8. [7] Yiangou Y, Facer P, Ford A, et al. Capsaicin receptor VR1 and ATP-gated ion channel P2X3 in human urinary bladder. BJU Int 2001;87(9):774–9. [8] Charrua A, Reguenga C, Cordeiro JM, et al. Functional transient receptor potential vanilloid 1 is expressed in human urothelial cells. J Urol 2009;182(6):2944–50. [9] Vizzard MA. Alterations in spinal cord Fos protein expression induced by bladder stimulation following cystitis. Am J Physiol Regul Integr Comp Physiol 2000;278(4):R1027–39. [10] Dinis P, Charrua A, Avelino A, Cruz F. Intravesical resiniferatoxin decreases spinal c-fos expression and increases bladder volume to reflex micturition in rats with chronic inflamed urinary bladders. BJU Int 2004;94(1):153–7. [11] Charrua A, Cruz CD, Cruz F, Avelino A. Transient receptor potential vanilloid subfamily 1 is essential for the generation of noxious bladder input and bladder overactivity in cystitis. J Urol 2007;177(4):1537–41. [12] Sculptoreanu A, de Groat WC, Buffington CA, Birder LA. Abnormal excitability in capsaicin-responsive DRG neurons from cats with feline interstitial cystitis. Exp Neurol 2005;193(2):437–43. [13] de Groat WC. A neurologic basis for the overactive bladder. Urology 1997;50(6A Suppl.):36–52, discussion 3–6. [14] Steers WD, De Groat WC. Effect of bladder outlet obstruction on micturition reflex pathways in the rat. J Urol 1988;140(4):864–71. [15] Cruz F, Guimaraes M, Silva C, Reis M. Suppression of bladder hyperreflexia by intravesical resiniferatoxin. Lancet 1997;350(9078):640–1. [16] Abrams P, Cardozo L, Fall M, et al. The standardisation of terminology of lower urinary tract function: report from the Standardisation Sub-committee of the International Continence Society. Neurourol Urodyn 2002;21(2):167–78. [17] Cruz F. Vanilloid receptor and detrusor instability. Urology 2002;59(5 Suppl. 1):51–60. [18] Cruz F, Avelino A, Coimbra A. Desensitization follows excitation of bladder primary afferents by intravesical capsaicin, as shown by c-fos activation in the rat spinal cord. Pain 1996;64(3):553–7. [19] Fowler CJ, Jewkes D, McDonald WI, Lynn B, de Groat WC. Intravesical capsaicin for neurogenic bladder dysfunction. Lancet 1992;339(8803):1239. 126 7. INTRAVESICAL CAPSAICIN AND RESINIFERATOXIN

[20] Dasgupta P, Chandiramani V, Parkinson MC, Beckett A, Fowler CJ. Treating the human bladder with capsaicin: is it safe? Eur Urol 1998;33(1):28–31. [21] Silva C, Avelino A, Souto-Moura C, Cruz F. A light- and electron-microscopic histopathological study of human bladder mucosa after intravesical resiniferatoxin application. BJU Int 2001;88(4):355–60. [22] Silva C, Rio ME, Cruz F. Desensitization of bladder sensory fibers by intravesical resiniferatoxin, a capsaicin analog: long-term results for the treatment of detrusor hyperreflexia. Eur Urol 2000;38(4):444–52. [23] Fowler CJ, Beck RO, Gerrard S, Betts CD, Fowler CG. Intravesical capsaicin for treatment of detrusor hyperreflexia. J Neurol Neurosurg Psychiatry 1994;57(2):169–73. [24] Geirsson G, Fall M, Sullivan L. Clinical and urodynamic effects of intravesical capsaicin treatment in patients with chronic traumatic spinal detrusor hyperreflexia. J Urol 1995;154(5):1825–9. [25] Chandiramani VA, Peterson T, Duthie GS, Fowler CJ. Urodynamic changes during therapeutic intravesical instillations of capsaicin. Br J Urol 1996;77(6):792–7. [26] Das A, Chancellor MB, Watanabe T, Sedor J, Rivas DA. Intravesical capsaicin in neurologic impaired patients with detrusor hyperreflexia. J Spinal Cord Med 1996;19(3):190–3. [27] Cruz F, Guimaraes M, Silva C, Rio ME, Coimbra A, Reis M. Desensitization of bladder sensory fibers by intravesical capsaicin has long lasting clinical and urodynamic effects in patients with hyperactive or hypersensitive bladder dysfunction. J Urol 1997;157(2):585–9. [28] De Ridder D, Chandiramani V, Dasgupta P, Van Poppel H, Baert L, Fowler CJ. Intravesical capsaicin as a treatment for refractory detrusor hyperreflexia: a dual center study with long-term followup. J Urol 1997;158(6):2087–92. [29] de Seze M, Wiart L, Joseph PA, Dosque JP, Mazaux JM, Barat M. Capsaicin and neurogenic detrusor hyperreflexia: a double-blind placebo-controlled study in 20 patients with spinal cord lesions. Neurourol Urodyn 1998;17(5):513–23. [30] Igawa Y, Satoh T, Mizusawa H, et al. The role of capsaicin-sensitive afferents in autonomic dysreflexia in patients with spinal cord injury. BJU Int 2003;91(7):637–41. [31] de Seze M, Wiart L, Ferriere J, de Seze MP, Joseph P, Barat M. Intravesical instillation of capsaicin in urology: a review of the literature. Eur Urol 1999;36(4):267–77. [32] de Seze M, Gallien P, Denys P, et al. Intravesical glucidic capsaicin versus glucidic solvent in neurogenic detrusor overactivity: a double blind controlled randomized study. Neurourol Urodyn 2006;25(7):752–7. [33] Wiart L, Joseph PA, Petit H, et al. The effects of capsaicin on the neurogenic hyperreflexic detrusor. A double blind placebo controlled study in patients with spinal cord disease. Preliminary results. Spinal Cord 1998;36(2):95–9. [34] Cruz F. Desensitization of bladder sensory fibers by intravesical capsaicin or capsaicin analogs. A new strategy for treatment of urge incontinence in patients with spinal detrusor hyperreflexia or bladder hypersensitivity disorders. Int Urogynecol J Pelvic Floor Dysfunct 1998;9(4):214–20. [35] Fowler CJ. Intravesical treatment of overactive bladder. Urology 2000;55(5A Suppl.):60–4, discussion 6. [36] de Sèze M, Wiart L, de Seze MP, et al. Intravesical capsaicin versus resiniferatoxin for the treatment of detrusor hyperreflexia in spinal cord injured patients: a double-blind, randomized, controlled study. J Urol 2004;171(1):251–5. [37] Silva C, Silva J, Ribeiro MJ, Avelino A, Cruz F. Urodynamic effect of intravesical resiniferatoxin in patients with neurogenic detrusor overactivity of spinal origin: results of a double-blind randomized placebo-controlled trial. Eur Urol 2005;48(4):650–5. [38] Kim JH, Rivas DA, Shenot PJ, et al. Intravesical resiniferatoxin for refractory detrusor hyperreflexia: a multicenter, blinded, randomized, placebo-controlled trial. J Spinal Cord Med 2003;26(4):358–63. [39] Kuo HC, Liu HT, Yang WC. Therapeutic effect of multiple resiniferatoxin intravesical instillations in patients with refractory detrusor overactivity: a randomized, double-blind, placebo controlled study. J Urol 2006;176(2):641–5. [40] Silva C, Silva J, Castro H, et al. Bladder sensory desensitization decreases urinary urgency. BMC Urol 2007;7:9. [41] Giannantoni A, Di Stasi SM, Stephen RL, Bini V, Costantini E, Porena M. Intravesical resiniferatoxin versus botulinum-A toxin injections for neurogenic detrusor overactivity: a prospective randomized study. J Urol 2004;172(1):240–3. [42] Santos-Silva A, Martins-Silva C, Lopes T, Silva J, Pinto R, Cruz F. Onabotulinum toxin a vs resiniferatoxin in the treatment of wet overactive bladder patients: a clinical and urodynamic prospective analysis. Neurourol Urodyn 2013;32(6):766. REFERENCES 127

[43] Fagerli J, Fraser MO, deGroat WC, et al. Intravesical capsaicin for the treatment of interstitial cystitis: a pilot study. Can J Urol 1999;6(2):737–44. [44] Lazzeri M, Beneforti P, Benaim G, Maggi CA, Lecci A, Turini D. Intravesical capsaicin for treatment of severe bladder pain: a randomized placebo controlled study. J Urol 1996;156(3):947–52. [45] Lazzeri M, Spinelli M, Beneforti P, Malaguti S, Giardiello G, Turini D. Intravesical infusion of resiniferatoxin by a temporary in situ drug delivery system to treat interstitial cystitis: a pilot study. Eur Urol 2004;45(1):98–102. [46] Payne CK, Mosbaugh PG, Forrest JB, et al. Intravesical resiniferatoxin for the treatment of interstitial cystitis: a randomized, double-blind, placebo controlled trial. J Urol 2005;173(5):1590–4. [47] Cruz F, Dinis P. Resiniferatoxin and botulinum toxin type A for treatment of lower urinary tract symptoms. Neurourol Urodyn 2007;26(6 Suppl.):920–7. [48] Chapple C, Sievert KD, MacDiarmid S, et al. OnabotulinumtoxinA 100 U significantly improves all idiopathic overactive bladder symptoms and quality of life in patients with overactive bladder and urinary incontinence: a randomised, double-blind, placebo-controlled trial. Eur Urol 2013;64(2):249–56. CHAPTER 8 Clinical and Preclinical Experience with TRPV1 Antagonists as Potential Analgesic Agents Arthur Gomtsyan,1,* Jill-Desiree Brederson2 1Department of Chemistry, Research and Development, AbbVie Inc., North Chicago, Illinois, USA 2Global Medical Communications, Research and Development, AbbVie Inc., North Chicago, Illinois, USA *Corresponding author: [email protected]

OUTLINE

Introduction 129 Perspective 139 Preclinical Overview of TRPV1 References 140 Antagonists 130 Clinical Overview of TRPV1 Antagonists 134

INTRODUCTION

It has been well established that capsaicin, the algesic component of chili peppers, produces tissue injury leading to neuronal activation and sensitization [1–4]. Psychophysical studies have demonstrated that mechanical and heat hyperalgesia and a cutaneous flare response follow intradermal injection of capsaicin into human skin and are correlated with pain magnitude and duration [1,4]. Neurophysiological studies in monkeys identified heat-induced sensitization of A and C fiber mechano-heat-sensitive (AMH, CMH) fibers and

capsaicin-induced sensitization of spinal dorsal horn neurons conducting along the spinothalamic tract [3,5,6]. Interestingly, heat-evoked pain in human subjects and heat-evoked neural activity in monkey CMH fibers overlapped, with an activation threshold of approximately 45 °C and a stimulus-response function that increased monotonically with increasing stimulus intensity [6]. Later, heat activation of the heterologously expressed cloned capsaicin receptor was defined to be in the noxious range with an activation threshold of 42 °C, thereby providing a molecular mechanism by which heat and capsaicin exert physiological effects [7,8]. Following this seminal discovery, pharmaceutical and academic laboratories have spent more than 15 years researching this protein as a therapeutic target for analgesia. The cloned capsaicin receptor, initially called the vanilloid receptor subtype 1 because of the unique gating by vanilloids such as capsaicin, was recognized as the first member of the transient receptor potential vanilloid family and was designated TRPV1. Structurally, TRPV1 is a tetrameric six-transmembrane ion channel protein with nonselective permeability to cations. Consistent with early neurophysiological studies demonstrating that capsaicin activated AMH and CMH fibers that travel along the spinothalamic tract, TRPV1 resides primarily on peptidergic small- and medium-diameter neurons in the peripheral nervous system [9–11]. Initially classified as the capsaicin receptor, TRPV1 functions as a molecular integrator of a variety of stimuli, including endogenous lipids, noxious heat, and acid. In addition to capsaicin, TRPV1 can be activated by plant products, including resiniferatoxin (RTX), piperine, gingerol, zingerone, as well as camphor and eugenol, ethanol, and spider and jellyfish venom ([12,13], and reviewed in Refs. [14,15]). Channel activation results in electrical and chemical activity in neurons. Channel opening results in signal transduction of nociceptive stimuli in neurons. Pronociceptive mediators including substance P, glutamate, and calcitonin gene-related peptide (CGRP) are released in response to TRPV1 activation, which in turn contributes to the development of neurogenic inflammation. The resulting proinflammatory milieu can sensitize TRPV1, leading to enhanced channel opening and thereby contributing to neuronal sensitization. Initially thought to exert effects primarily in the peripheral nervous system, TRPV1 expression and function in the central nervous system (CNS) is now widely reported, although the full extent of its physiological function in the CNS has not been elucidated [16–19]. TRPV1 functions as a molecular integrator of multiple physical and chemical stimuli, consistent with its localization on polymodal nociceptors in the peripheral nervous system. First- generation TRPV1 antagonists were designed to block all modes of TRPV1 activation. Clinical investigations of these compounds unveiled thermosensory deficits as a key limitation to further development. A breakthrough in the field was realized when TRPV1 antagonists could be designed to selectively block different modes of activation. The field quickly shifted toward the discovery of selective TRPV1 antagonists that could differentially block heat and capsaicin, but not acid activation of the channel to pharmacologically separate analgesic and thermoregulatory effects. Such modality-specific pharmacology has since been the focus for development of a novel analgesic-targeting TRPV1.

PRECLINICAL OVERVIEW OF TRPV1 ANTAGONISTS

In an effort to achieve analgesia through modulation of TRPV1 without the burning sensation associated with agonists such as capsaicin and RTX, the Sandoz group (now Novartis) reported on discovery of antagonists of TRPV1 and their assessment as pain relievers [20,21]. Preclinical Overview of TRPV1 Antagonists 131

S Cl HO HO H N N N O HO H O Capsaicin Capsazepine

FIGURE 8.1 Capsaicin and capsazepine.

Synthesis of small molecules with structural resemblance to capsaicin led to the development of capsazepine, the first TRPV1 antagonist pharmacological tool compound widely utilized in early TRPV1 research (Figure 8.1). The beginning of the 21st century witnessed a burst of TRPV1 drug discovery activities in major pharmaceutical and smaller biotechnology companies. The potential role of TRPV1 antagonists as analgesic agents was suggested based in part on attenuation of pain-like behaviors in TRPV1 knockout (KO) mice [22]. However, the first generation of TRPV1 antagonists was limited by chemotype-independent hyperthermia in preclinical species and humans ([23], and reviewed in Refs. [24,25]). These findings, along with hypothermia-inducing properties of TRPV1 agonists and absence of hyperthermia in KO mice treated with TRPV1 antagonists, unambiguously established a thermoregulatory role for the TRPV1 channel [26–28]. Therefore, pharmacological separation of analgesic and hyperthermic effects became the key challenge in developing TRPV1 antagonists as viable agents for pain management. One of the approaches to decouple undesired thermoregulatory effects from desired analgesic effects consisted of preventing TRPV1 antagonists from penetrating the brain where the hypothalamus is known to be involved in thermoregulation [29]. However, potent peripherally restricted TRPV1 antagonists still caused hyperthermia in rats, suggesting that peripheral restriction was not sufficient to attenuate the thermoregulatory effects and that the site of action for the hyperthermic effect was predominantly outside the CNS [30]. Direct administration of a selective but undifferentiated TRPV1 antagonist into the medial preoptic area of the hypothalamus did not affect core body temperature [18]. However, TRPV1-antagonist- induced increase in core body temperature was blocked by systemic RTX-mediated desensitization of TRPV1 [27]. Together, these studies suggest that visceral TRPV1 receptors are responsible for regulation of core body temperature. Selective pharmacological blockade of some but not all modes of TRPV1 activation emerged as a more promising direction toward discovery of TRPV1 antagonists that could provide pain relief without affecting body temperature. AMG-8562 (Figure 8.2) from Amgen was one of the early and most characterized modality-specific TRPV1 antagonists [31].

AMG-8562 blocked capsaicin activation of rat TRPV1 with an IC50 of 1.75 nM, did not affect heat activation, and potentiated pH 5 activation in a 45Ca2+ uptake assay using cells expressing recombinant rat TRPV1 [31]. Oral administration of AMG-8562 in rats either did not induce hyperthermia or had small hypothermic effect, but still showed efficacy in several preclinical pain models, albeit at rather high plasma concentrations. It should be noted that the pharmacological profile of AMG-8562 at human TRPV1 differs from the rat profile in that it partially blocked pH 5 activation in humans, whereas it potentiated acid activation at rat TRPV1. Significance of the blockade of proton activation for the thermoregulatory effects was confirmed in a systematic study of hyperthermic responses of rats, mice, and guinea pigs to TRPV1 antagonists displaying different pharmacological profiles [32]. It was concluded that 132 8. ANALGESIC POTENTIAL OF TRPV1 ANTAGONISTS

OH F3C H N H N N O O N N O

AMG-8562 (Amgen) AS-1928370 (Astellas)

O O H N N S O S O N O O F N F N N H H F F N F F F F

Cmpd 41 (Grünenthal) Cmpd 12 (Grünenthal)

Cl F F O O F N N H HN N H O H F N N S O H F O S O HN N N N F O H O Cmpd 15 (Grünenthal) Compound 10 (Abbvie) Compound 6 (Abbvie)

F F O O O O

HN N HN N H H HO Cl F N

A-1165442 (Abbvie) A-1106625* (Abbvie)

FIGURE 8.2 TRPV1 antagonists with differentiated pharmacology in preclinical species (full blockers of capsaicin and partial blockers of pH activation). *, structure of A-1106625 shown for comparison with A-1165442.

TRPV1 antagonists that do not block low pH activation would exhibit a hyperthermia-free profile, even if they are potent blockers of heat activation. AS-1928370 (Figure 8.2) from Astellas Pharma displays differential pharmacology in blocking the activation of TRPV1 [33]. This compound inhibited capsaicin-induced Ca2+ flux in rat

TRPV1 with an IC50 value of 880 nM and capsaicin-induced currents in electrophysiological assay with an IC50 value of 32 nM, but showed very small inhibitory activity against pH 6 Preclinical Overview of TRPV1 Antagonists 133 activation (<20% block at 10 μM). This profile was responsible for lack of effect on rectal body temperature in rats up to 10 mg/kg oral dose, although 30 mg/kg dose induced significant hypothermia. At an oral dose of 1 mg/kg, AS-1928370 fully attenuated pain-like behaviors evoked by intradermal capsaicin in a model of secondary hyperalgesia and exerted full efficacy in the rat spinal nerve ligation (SNL) model of mechanical allodynia (1-3 mg/kg dose range). These data provided evidence that analgesic effects in the SNL model of neuropathic pain were mediated by TRPV1. Such a conclusion was supported by high brain drug concentration, which sufficiently covered the IC50 value obtained from electrophysiological recordings (355 versus 32 nM). This was the first demonstration that a TRPV1 antagonist displaying no inhibitory effect on proton-induced activation can exhibit high efficacy in neuropathic pain model. Potent effects in rats were recapitulated in mice, where AS-1928370 significantly suppressed both capsaicin-induced acute nocifensive and withdrawal responses in the hot plate test at oral doses of 10-30 mg/kg [34]. Significant efficacy was obtained in the SNL model at lower oral doses of 0.3-1.0 mg/kg. Despite favorable preclinical pharmacological and safety profiles, there was no information reported on whether AS-1928370 or any of its analogs entered clinical trials. It should be noted that AS-1928370 did inhibit proton activation of human

TRPV1 with an IC50 value of 1.5 μM, while having no effect at rat TRPV1 (IC50 > 20 μM) [33]. Given the correlation established between inhibitory effects on proton-evoked activation of TRPV1 in vitro and changes in body temperature in vivo, there should be considerable caution in predicting a hyperthermia-free profile for AS-198370 in humans. Grünenthal described several chemotypes of TRPV1 antagonists, some representatives of which displayed differential pharmacology against capsaicin, low pH, and heat activation of TRPV1. Compound 41 (Figure 8.2), a diaryl acetamide, is a very potent TRPV1 blocker against capsaicin activation (Ki = 0.1 nM), but much weaker against pH 6 activation (IC50 = 87 nM) [35]. Several compounds from their propanamide series also exhibited large potency gaps inhibiting in vitro capsaicin and low pH. For example, compounds 12 (Figure 8.2) [36] and 15 (Figure 8.2) [37] were potent inhibitors of capsaicin activation with

IC50 values of 8 and 2 nM, but weak inhibitors of low pH activation with 0% and 15% effects correspondingly at 5 μM. Additional TRPV1 antagonists with differentiated pharmacology were discovered within carboxamide and urea containing series [38–40]. Grünenthal did not describe effects on core body temperature; therefore, it is unknown if modality specific in vitro pharmacology for some TRPV1 antagonists was predictive of lack of hyperthermia in preclinical species. In a series of patent applications AbbVie claimed TRPV1 antagonists of different chemical classes, several representatives of which did not produce thermosensation deficits in rats. Lack of thermosensory effects was demonstrated by increased average response latency for tail withdrawal from a 55 °C water bath. For example, Compound 10 (Figure 8.2) blocked capsaicin activation of TRPV1 with an IC50 value of 54 nM, but blocked only 28% of pH 5 activation at 37.5 μM [41]. As a result of differentiated pharmacology, Compound 10 had no effect on latency to tail withdrawal in the rat tail immersion assay, suggesting no in vivo effect on thermosensation. Similarly, no thermosensation deficits were observed for Compound 6 (Figure 8.2) in the same tail immersion assay [42]. Compound 6, representing a different chemical class of TRPV1 antagonists than Compound 10, was characterized as a full blocker of capsaicin activation with an

IC50 = 4 nM and partial blocker of acid activation with only 38% inhibition at 37.5 μM. Compound 6 also attenuated pain-like behaviors with 88% effect in the capsaicin-induced flinching model 134 8. ANALGESIC POTENTIAL OF TRPV1 ANTAGONISTS at an oral dose of 100 μmol/kg and with 59% effect in capsaicin-induced secondary mechanical hypersensitivity model at an oral dose of 10 μmol/kg. A more detailed account was reported from AbbVie on an additional series of differentiated TRPV1 antagonists [43]. A-1165442 (Figure 8.2) is a potent blocker of capsaicin activation of rat and human recombinant TRPV1 in FLIPR assays with IC50 values of 35 and 17 nM, respectively, as well as in whole cell patch clamp electrophysiology studies in rat dissociated dorsal root ganglia neurons (IC50 value 2.7 nM). In contrast, A-1165224 exhibited only a partial blockade of acid-evoked response at both rat and human TRPV1 measured in FLIPR (14% and 61% at 11 μM) and electrophysiology assays (66% block at 10 μM). Interestingly, biochemical analysis indicated that A-1165442 was less efficacious in blocking acid-evoked CGRP release compared with capsaicin-evoked CGRP release (22% vs. 100% block at 10 μM). A-1165442 and related analogues had a similar differentiated pharmacological profile highlighted by acid-sparring inhibition of TRPV1 and lack of or diminished body temperature elevations of <0.5 °C in telemetrized rats. The relationship between acid blocking of TRPV1 in vitro and hyperthermic effects of TRPV1 antagonists in vivo is consistent with results of earlier studies with other classes of TRPV1 antagonists [31–33] and was demonstrated with a structural analogue, A-1106625 (Figure 8.2), which is a potent TRPV1 blocker at all modes of activation [43]. Although acid partial blocker A-1165442 did not change rat core body temperature at plasma concentrations ~8.5-fold higher than its capsaicin blocking IC50, A-1106625 induced 1 °C temperature increase at lower multiples of plasma concentration. Both compounds exhibited comparable efficacy in a rat osteoarthritis (OA) pain model (ED50 ~ 30-35 μmol/kg). However, unlike A-1106625, acid partial blocker A-1165442 was ineffective in attenuating allodynia in a mouse bone cancer pain model. The latter result can be explained by the importance of osteoclast-induced acidosis for bone cancer pain generation [44,45].

CLINICAL OVERVIEW OF TRPV1 ANTAGONISTS

The first selective TRPV1 antagonist that was tested in humans was SB-705489 from GlaxoSmithKline. The compound effectively blocked TRPV1 activation in vitro by capsaicin, heat, and low pH and reduced inflammatory pain in rodents [46,47]. In target engagement studies during Phase 1 clinical trials, SB-705498 (Figure 8.3) at a single 400-mg oral dose significantly reduced the area of capsaicin-evoked flare versus placebo [48]. The same dose also reduced ultraviolet-B (UVB)-evoked flare area and heat hyperalgesia compared with placebo, albeit to a lesser degree. Reduction of flare area correlated with plasma exposure levels of SB-705495, suggesting effects were drug related and via a TRPV1-mediated mechanism. However, SB-705498 did not reduce the intensity of both capsaicin- and UVB-evoked flare as well as capsaicin-evoked thermal hyperalgesia. It is possible that the combination of capsaicin and heat resulted in more pronounced activation of TRPV1, blockade of which would require higher concentration of SB-705498. At the time point of pharmacodynamics assessments (~6 h postdose), total plasma concentration of SB-705498 was 0.5 ± 0.22 mg/mL, comparable to those predicted to be efficacious based on preclinical models. The maximum tolerated dose of

400 mg resulted in tmax of 2 h (0.75-4 h) and half-life of 54 h (35-93 h) with SB-705498 remaining quantifiable for the 168 h postdose sampling period. Effects of SB-705498 on heat and taste thresholds were also investigated. Heat threshold was elevated into the noxious range, in line Clinical Overview of TRPV1 Antagonists 135

Br O H H NN N N O HN N N CF3 H O N N CF AcHN 3 N N S H SB-705498 (GSK) AMG-517 (Amgen) ABT-102 (Abbvie) (Rami, 2006) (Doherty, 2007) (Gomtsyan, 2008)

CF3

O HN O F N N F3C HN N F N H H N F N N N MK-2295/NGD8243 (Merck) ABT-443* (Abbvie) AZD-1386 (AstraZeneca) (http://pubchem.ncbi.nlm.nih.gov/summary/ (US 2012/0245163) (Griffin, 2011) summary.cgi?cid=56603682)

Cl Cl O O N N O HN N N N N N H H CF3 N N DWP05195* (Daewoong ) H GRC-6211* (WO2006080821) (WO 2009128661) (WO2007042906) F3C

O O O O O O N N N N H H N O HO H H F C OH 3 F3C F MR-1817* (Mochida) (WO2010010935) (WO2008091021) (WO2010038803)

N I S I O O OH S S N N N H F3C O N H H N N OH OH H H MeO N O MeO H F3C JTS-653 (Japan Tobacco) PHE-377* (Kitagawa, 2012) (WO2005123666)(WO2006045498)

FIGURE 8.3 TRPV1 antagonists studied in humans. *, chemical structure not disclosed, but may represent chemical series published in patent applications. 136 8. ANALGESIC POTENTIAL OF TRPV1 ANTAGONISTS with the role of TRPV1 as thermosensor. Taste experiment results were not conclusive, but the threshold may have not changed after administering a series of diluted capsaicin solutions to volunteers [49]. Following Phase I, SB-705498 was investigated in clinical studies of acute migraine, dental pain, and rectal pain, but the results of these studies were not published. Clinical trials with Amgen’s AMG-517 (Figure 8.3) highlighted and raised wider aware- ness of thermoregulatory effects associated with the modulation of TRPV1 [50]. AMG-517 is an extremely potent TRPV1 antagonist blocking channel activation by a multiple agonists in human, rat, mouse, and monkey cells expressing TRPV1 (e.g., IC50 = 0.9 and 0.5 nM against capsaicin and pH5 activation of rat TRPV1) [51,52]. In addition to efficient attenuation of complete Freund’s adjuvant induced inflammatory pain in rats, AMG-517 also caused hyperthermia in rodents, dogs, and monkeys [52]. However, because repeating dosing of AMG-517 significantly attenuated hyperthermia in rats, dogs, and monkeys, the decision was made to test the compound in humans. In Phase 1 safety and pharmacokinetic studies, AMG-517 was administered to healthy volunteers at the oral dose range 1-25 mg. These doses induced plasma concentration-dependent hyperthermia, which was transient in nature, with body temperature returning to baseline values within 24 h. Maximum body temperature recorded was 39.9 °C. Repeated administration of AMG-517 (2, 5, and 10 mg) for 7 days elicited increased body temperature in the first day of administration to a mean maximum value of 38.3 °C, which was significantly attenuated at days 2-7, but not completely eliminated. To determine whether a drug concentration gap exists between analgesic and thermoregulatory effects, AMG-517 entered a Phase 1b dental pain study. Elevated body temperatures of 39-40.2 °C persisted for 1-4 days in 33% of patients receiving oral AMG-517 at doses 2, 8, and 15 mg administered immediately following extraction of 2 or 3 third molars. The highest body temperature of >40 °C was registered with the patient receiving the lowest dose of 2 mg. An unusually high magnitude of hyperthermia in some subjects with no clear correlation with drug concentration was explained by individual susceptibility, whereas long duration of hyperthermia was attributed to long half-life of AMG-517, which is reported to be approximately 13 days. As a consequence of pronounced hyperthermia in patients receiving AMG-517 after molar extraction, clinical trials were terminated without full assessment of the compound’s analgesic potential. Plasma concentrations of 20-30 ng/mL were found to be required for target coverage to elicit hyperthermia, but efficacious analgesic concentrations remained unknown [50,53]. Amgen reported a selection of the second clinical compound AMG-628 characterized by shorter half-life and improved drug-like properties compared with AMG-517 [54]. However, no data was reported on temperature effects of this compound, and it is unknown whether the compound entered clinical trials. There is no full consensus in the scientific community for the mechanism by which TRPV1 plays a role in body thermoregulation [28,55,56], as no clear thermoregulatory phenotype was observed in studies with TRPV1 KO mice [28,57,58]. However, hypothermic effects of TRPV1 agonists and hyperthermic effects of TRPV1 antagonists are suppressed in TRPV1 KO mice [22,27,50,56]. ABT-102 (Figure 8.3) from AbbVie is another clinically tested TRPV1 antagonist that potently blocked activation of the channel in vitro by multiple modes of stimulation [59,60]. One of the beneficial attributes of ABT-102 was its enhanced analgesic activity without accumulation in plasma or brain after repeated administration (for 5-12 days) in preclinical models of OA, postoperative, and bone cancer pain [61]. The analgesic potential of ABT-102 Clinical Overview of TRPV1 Antagonists 137 was assessed in an experimental pain study in healthy human volunteers [62]. Painful stimuli were induced by a CO2-laser on UVB-inflamed skin. Electroencephalography and visual analogue scale (VAS-pain) ratings were used to evaluate the effects of ABT-102 on human pain processing and perception. A single 6-mg dose of ABT-102 (Cmax, 15 ng/mL, coverage during experiment, 9 ng/mL) demonstrated superior analgesic efficacy to clinically effective etoricoxib (90 mg) and tramadol (100 mg). ABT-102 was safe and generally well tolerated by subjects in this study. However, ABT-102 produced a dose-dependent increase in body temperature up to 39 °C. Next, the effects of ABT-102 on body temperature were extensively studied in three separate Phase 1 single- and multiple-dosing studies in a combined 108 healthy volunteers [63]. Similar to hyperthermic effects observed in telemetrized rats [61], there was a significant correlation between ABT-102 plasma concentrations and initial transient increase in body temperature. Mean increase in body temperature at the highest dose and exposure of ABT-102 in humans (40 mg single dose, mean Cmax 73 ng/mL) [64] was 1.5 °C at tmax. However, transient increase in body temperature at the exposures found to be efficacious in experimental pain model [62] was only 0.6-0.8 °C. In addition, the temperature effects of ABT-102 were significantly attenuated after twice a day dosing for 3 days [63]. Authors of the study expressed little clinical concern based on relatively modest hyperthermia at predicted analgesic doses of ABT-102 and significant attenuation of hyperthermia after repeated administration of ABT- 102. However, due to the concerns that TRPV1 antagonists may also cause pronounced thermosensation deficits, another study in healthy volunteers was initiated to assess the potential of such impairment [65]. A variety of outcomes was measured after oral dosing of 1, 2, and 4 mg of ABT-102 twice a day for 7 days. ABT-102 dose-dependently increased oral and cutaneous pain thresholds measured by quantitative sensory testing (QST). Contrary to attenuation of hyperthermic effects in several human studies with ABT-102, thermosensation deficits were not attenuated after repeated administration of the compound. AbbVie subsequently advanced another TRPV1 antagonist, ABT-443 (Figure 8.3), into the clinic. This compound was characterized with modality-specific pharmacology in human, rat, and monkey recombinant cell lines [66]. In contrast to first-generation TRPV1 antagonists, ABT-443 fully blocked capsaicin and heat activation of the channel, but only partially blocked acid activation. Such differentiated pharmacology resulted in a benign thermoregulatory and thermosensory profile of ABT-443 in preclinical species. In Phase 1 safety and tolerability studies, ABT-443 at the dose of 60 mg (projected to provide analgesic efficacy corresponding to the EC50) had minimal effects on heat pain threshold and heat sensitivity in a water bath test [66]. However, the same dose caused significant reduction in the pain VAS score in a hot oral liquid test compared to placebo. Heat sensitivity impairments were more pronounced with administration of 450 mg of ABT-443. Despite a much improved window of projected efficacy versus thermosensation effects compared with ABT-102 and other first-generation TRPV1 antagonists, AbbVie has not pursued ABT-443 in Phase 2 trials with pain patients [66]. Significant heat sensing deficits were also reported in clinical trials with the multimodal TRPV1 antagonist MK-2295 (Figure 8.3) from Merck/Neurogen [67,68]. In QST and models imposing a suprathreshold heat stimulus, MK-2295 markedly increased the thresholds of sensing noxious heat. Risks of impaired heat sensitivity in real-life situations were also evaluated. In a study mimicking a bathing experience, subjects were given a single dose of 8 or 25 mg of MK-2295 and asked to withdraw their hand from a 49 °C water bath at the point 138 8. ANALGESIC POTENTIAL OF TRPV1 ANTAGONISTS of discomfort. It took 36 and 84 s longer, respectively, for drug-treated groups to feel heat pain and withdraw the hand compared with a placebo-controlled group. Similarly, only 77% and 60% of subjects dosed with 8 and 25 mg of MK-2295 perceived 70 °C water as too hot compared with 100% of the placebo group. These results were in agreement with reported preclinical effects of MK-2295 in hot plate and tail immersion assays. These effects were observed at doses and exposures comparable to those required for analgesic efficacy in inflammatory pain models [67]. TRPV1 KO mice administered with MK-2295 exhibited a phenotype with no thermosensation deficits, suggesting an on-target mechanism. AZD-1386 (Figure 8.3) from AstraZeneca exhibited concentration-dependent antihyperalgesic effects in preclinical models of inflammatory and neuropathic pain and, interestingly, at doses that did not cause hyperthermia following single or repeated administration [69]. However, AZD-1386 did produce hyperthermia in healthy volunteers in Phase 1 single- and multiple-dose tolerability studies, with the largest difference from placebo of 1.2 °C and the highest recorded body temperature of 38.0 °C [70]. The dose range in the single-dose study was 3-190 mg, and the dose range in the multiple-dose study was 20-150 mg. The magnitude of hyperthermia was deemed not clinically significant [70]. However, similar to TRPV1 antagonists previously tested in the clinic, AZD-1386 significantly increased the heat pain threshold in healthy volunteers (4.8 °C mean difference vs. placebo) [71]. In the same study, AZD-1386 decreased maximal pain after an intradermal capsaicin injection, thus producing an on-target pharmacodynamic effect. Because of an overall acceptable tolerability profile of AZD-1386 and its attenuation of heat- and capsaicin-induced pain in healthy volunteers, AstraZeneca sponsored two Phase 2 clinical trials to evaluate the analgesic potential of AZD-1386 in patients with pain from third molar extraction and in patients with OA of the knee. In the third molar extraction study, a 95-mg single dose of AZD-1386 produced analgesia from 15 min to 1 h; however, the effect rapidly faded away despite sustained plasma concentrations [72,73]. The rapid onset of analgesia and its short duration was explained by effective absorption of liquid AZD-1386 through injured mucosa and subsequent rapid elimination. The short duration of analgesia was in contrast to over 5 h maintenance of significant effects on heat pain and capsaicin pain thresholds in healthy volunteers [71]. Such discrepancy could be explained by a higher concentration of the drug needed to attenuate postoperative pain compared with heat pain and capsaicin-induced pain. It can also be explained by possible release/increase/synthesis of algogenic substances that target receptors other than TRPV1 and therefore do not respond to TRPV1 antagonists [73]. No major adverse events including hyperthermia were observed during that study. In the second Phase 2 study of patients with OA of the knee, 30 or 90 mg of AZD-1386 as a capsule was administered twice a day for 4 weeks [74]. Criteria for patient selection were insufficient pain relief with common anti-inflammatory drugs like NSAIDs and selective COX-2 inhibitors. Despite plasma exposures being in line with the study design expectations and the highest plasma concentration achieved was more than 7 μg/mL, AZD-1386 was not effective in reducing OA pain. The study was discontinued due to elevation of liver enzymes in patients receiving 90 mg of AZD-1386. Six patients had elevated S-ALT at three times above the upper limit of normal, whereas other hepatic enzymes were also increased in five of the six patients. AZD-1386 was also examined in an experimental esophageal pain model [75] in healthy volunteers followed by a study in patients with nonerosive gastroesophageal Perspective 139

reflux disease (NERD) [76]. The rationale for conducting such studies was based on NERD patients’ hypersensitivity to heat and acid and that TRPV1 is modulated by these stimuli. In addition, expression of TRPV1 in NERD patients is upregulated compared with healthy subjects. However, treatment of NERD patients with 95 mg of AZD-1386 (Figure 8.3) did not produce analgesia on esophageal heat pain or pain evoked by distension or electrical current. Consistent with previous studies, cutaneous heat tolerance in patients dosed with AZD-1386 was increased 4.9 °C versus the placebo group. A number of other companies advanced their TRPV1 antagonists into clinical trials, but the reports on the outcomes of these trials are scarce. After two successful Phase 1 studies in healthy volunteers, DWP05195 (structure not disclosed) from Daewoong Pharmaceutical entered Phase 2 efficacy trials in patients with postherpetic neuralgia [77]. This study, which evaluated oral doses of 100, 200, and 300 mg of DWP05195, was completed in December 2013 with no outcome information available. Also, no results were reported from the Phase 1 safety and tolerability clinical trials evaluating single ascending doses of MR-1817 (Figure 8.3) (structure not disclosed) from Mochida Pharmaceuticals, which were completed in 2011 [78]. GRC6211 (Figure 8.3) (structure not disclosed) discovered by Glenmark Pharmaceuticals and developed by Eli Lilly was advanced to Phase 2 studies in patients with OA pain. In 2008 Glenmark announced that Eli Lilly suspended further clinical developments of GRC-6211 [79]. JTS-653 (Figure 8.3) from Japan Tobacco is one of the most potent polymodal TRPV1 antagonists in preclinical in vitro assays, in vivo pain models [80,81], and a bladder overactivity model [82]. It has been shown that JTS-653 elicited transient hyperthermia in rats at the dose at which full reversal of carrageenan-induced mechanical hyperalgesia took place [80]. In 2010 JTS-653 advanced to Phase 2 clinical trials in Japan for pain and overactive bladder [83], but both trials were discontinued in 2011 for unknown reasons [84]. According to PharmEste’s website, the company’s TRPV1 antagonist PHE-377 (Figure 8.3) completed three Phase 1 clinical trials on safety, tolerability, and pharmacokinetics, as well as food interaction [85]. In addition, PHE377 has been evaluated in a multidose Phase 1b proof-of-principle study confirming its safety profile and demonstrating a positive pharmacodynamic profile as assessed on capsaicin-induced neurogenic flare. Encouraging results were also obtained using mechanical-evoked stimulation in naïve as well as UVB-irradiated skin following PHE377 oral daily dosing in healthy subjects. PHE-377, unlike previously discontinued TRPV1 antagonists, did not increase body temperature and did not affect heat pain thresholds in naïve skin. As of 2013 PharmEste was seeking a partnership to advance PHE-377 to proof-of-concept Phase 2 studies targeting chronic pain of different etiologies.

PERSPECTIVE

Decades of research on the hyperalgesic profile and analgesic potential of capsaicin, followed by the identification of TRPV1, has had profound impact on the field of pain research. Elucidation of activation of TRPV1 by polymodal stimuli represents a seminal discovery of physical and chemical stimuli acting through a single protein to transduce neuronal activity. The discovery of TRPV1 was followed by more than 15 years of pharmaceutical and academic research in an attempt to pharmacologically modulate the channel with exogenous ligands for robust analgesia in multiple etiologies of pain. Advancements in the field of pain because 140 8. ANALGESIC POTENTIAL OF TRPV1 ANTAGONISTS of the identification of TRPV1 have been unmatched. Despite this, a novel analgesic acting exclusively through antagonism of TRPV1 still lacks clinical proof of concept. Nevertheless, progress made in TRPV1 research has set the stage for specific targeting of other ion channels involved in the neurobiology of pain. Exploring the therapeutic potential of TRPV1 modulation for pain management led to identification of at least 11 TRPV1 antagonists suitable for human clinical trials. At least one compound, AZD-1386, was tested in Phase 2 clinical studies in patients with OA and pain associated with gastroesophageal reflux disease; however, no statistically significant pain relief was achieved in either study [74,76]. Clinical trials for most of the other compounds did not proceed to advanced stages of clinical development mainly because of drug-induced alterations in thermosensation. First-generation TRPV1 antagonists elevated core body temperature and increased heat pain thresholds in healthy volunteers during Phase 1 studies. Soon after the first setbacks, a new class of TRPV1 antagonists with a pharmacologically distinct profile was discovered. Unlike the first generation of TRPV1 antagonists, which blocked all modes of receptor activation, the new class of antagonists either maintained or partly inhibited acid activation of TRPV1, resulting in reduced temperature side effects in preclinical species. However, not much data is available on the outcomes of clinical studies in humans, and therefore, the analgesic potential of these TRPV1 antagonists is still not known. It is clear that in order to unequivocally determine the therapeutic practicality of TRPV1 inhibition for pain management in humans, drug molecules should be completely devoid of temperature effects, such as hyperthermia and thermosensation deficits. TRPV1 antagonists with differential pharmacology may provide a useful approach toward that goal. Recent breakthrough advances in obtaining high resolution structural information on TRPV1 may help to refine the design parameters for new TRPV1 antagonists with a thermal-neutral profile [86,87].

References [1] Simone DA, Baumann TK, LaMotte RH. Dose-dependent pain and mechanical hyperalgesia in humans after intradermal injection of capsaicin. Pain 1989;38(1):99–107. [2] Simone DA, Baumann TK, Collins JG, LaMotte RH. Sensitization of cat dorsal horn neurons to innocuous mechanical stimulation after intradermal injection of capsaicin. Brain Res 1989;486(1):185–9. [3] Simone DA, Sorkin LS, Oh U, Chung JM, Owens C, LaMotte RH, et al. Neurogenic hyperalgesia: central neural correlates in responses of spinothalamic tract neurons. J Neurophysiol 1991;66(1):228–46. [4] LaMotte RH, Shain CN, Simone DA, Tsai EF. Neurogenic hyperalgesia: psychophysical studies of underlying mechanisms. J Neurophysiol 1991;66(1):190–211. [5] Campbell JN, Meyer RA, LaMotte RH. Sensitization of myelinated nociceptive afferents that innervate monkey hand. J Neurophysiol 1979;42(6):1669–79. [6] Meyer RA, Campbell JN. Evidence for two distinct classes of unmyelinated nociceptive afferents in monkey. Brain Res 1981;224(1):149–52. [7] Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, Julius D. The capsaicin receptor: a heat- activated ion channel in the pain pathway. Nature 1997;389(6653):816–24. [8] Tominaga M, Caterina MJ, Malmberg AB, Rosen TA, Gilbert H, Skinner K, et al. The cloned capsaicin receptor integrates multiple pain-producing stimuli. Neuron 1998;21(3):531–43. [9] Guo A, Vulchanova L, Wang J, Li X, Elde R. Immunocytochemical localization of the vanilloid recep-

tor 1 (VR1): relationship to neuropeptides, the P2X3 purinoceptor and IB4 binding sites. Eur J Neurosci 1999;11(3):946–58. [10] Lauria G, Morbin M, Lombardi R, Capobianco R, Camozzi F, Pareyson D, et al. Expression of capsaicin receptor immunoreactivity in human peripheral nervous system and in painful neuropathies. J Peripher Nerv System 2006;11:262–71. References 141

[11] Brederson JD, Chu KL, Reilly RM, Brown BS, Kym PR, Jarvis MF, et al. TRPV1 antagonist, A-889425, inhibits mechanotransmission in a subclass of rat primary afferent neurons following peripheral inflammation. Synapse 2012;66(3):187–95. [12] Cuypers E, Yanagihara A, Karlsson E, Tytgat J. Jellyfish and other cnidarian envenomations cause pain by affecting TRPV1 channels. FEBS Lett 2006;580:5728–32. [13] Siemens J, Zhou S, Piskorowski R, Nikai T, Lumpkin EA, Basbaum AI, et al. Spider toxins activate the capsaicin receptor to produce inflammatory pain. Nature 2006;444:208–12. [14] Szallasi A, Cortright DN, Blum CA, Eid SR. The vanilloid receptor TRPV1: 10 years from channel cloning to antagonist proof-of-concept. Nat Rev Drug Discov 2007;6:357–72. [15] Brederson JD, Kym PR, Szallasi A. Targeting TRP channels for pain relief. Eur J Pharmacol 2013;716:61–76. [16] Cui M, Honore P, Zhong C, Gauvin D, Mikusa J, Hernandez G, et al. TRPV1 receptors in the CNS play a key role in broad-spectrum analgesia of TRPV1 antagonists. J Neurosci 2006;26:9385–93. [17] McGaraughty S, Bitner RS. TRPV1 and nicotinic receptors in descending modulation of nociception. In: Maione S, DiMarzo V, editors. Neurotransmission in the antinociceptive descending pathway. Research Signpost, Kerala, India; 2007. p. 97–117. [18] McGaraughty S, Segreti JA, Fryer RM, Brown BS, Faltynek CR, Kym PR. Antagonism of TRPV1 receptors indirectly modulates activity of thermoregulatory neurons in the medial preoptic area of rats. Brain Res 2009;1268:58–67. [19] Kim YS, Chu Y, Han L, Li M, Li Z, Lavinka PC, et al. Central terminal sensitization of TRPV1 by descending serotonergic facilitation modulates chronic pain. Neuron 2014;81(4):873–87. [20] Walpole CSJ, Wrigglesworth R. Structural requirements for capsaicin agonists and antagonists. In: Wood JN, editor. Capsaicin in the study of pain. San Diego: Academic Press; 1993. p. 63–82. [21] Walpole CS, Bevan S, Bowermann G, Boelsterle JJ, Breckenridge R, Davies JW, et al. The discovery of capsazepine, the first competitive antagonist of the sensory neuron excitants capsaicin and resiniferatoxin. J Med Chem 1994;37:1942–54. [22] Caterina MJ, Leffler A, Malmberg AB, Martin WJ, Trafton J, Petersen-Zeitz KR, et al. Impaired nociception and pain sensation in mice lacking the capsaicin receptor. Science 2000;288:306–13. [23] Gavva NR, Bannon AW, Surapaneni S, Hovland Jr DN, Lehto SG, Gore A, et al. The vanilloid receptor TRPV1 is tonically activated in vivo and involved in body temperature regulation. J Neurosci 2007;27:3366–74. [24] Kort ME, Kym PR. TRPV1 antagonists: clinical setbacks and prospects for future development. Prog Med Chem 2012;51:57–70. [25] Trevisani M, Gatti R. TRPV1 antagonists as analgesic agents. Open Pain J 2013;6:108–18. [26] Shimizu I, Iida T, Horiuchi N, Caterina MJ. 5-Iodoresiniferatoxin evokes hypothermia in mice and is a partial transient receptor potential vanilloid 1 agonist in vitro. J Pharmacol Exp Ther 2005;314:1378–85. [27] Steiner AA, Turek VF, Almeida MC, Burmeister JJ, Oliveira DL, Roberts JL, et al. Nonthermal activation of transient receptor potential vanilloid-1 channels in abdominal viscera tonically inhibits autonomic cold-defense effectors. J Neurosci 2007;27:7459–68. [28] Garami A, Pakai E, Oliveira DL, Steiner AA, Wanner SP, Camila Almeida MC, et al. Thermoregulatory phenotype of the Trpv1 knockout mouse: thermoeffector dysbalance with hyperkinesis. J Neurosci 2011;31(5):1721–33. [29] Romanovsky AA. Thermoregulation: some concepts have changed. Functional architecture of the thermoregulatory system. Am J Physiol Regul Integr Comp Physiol 2007;292:R37–46. [30] Tamayo N, Liao H, Stec MM, Wang X, Chakrabarti P, Retz D, et al. Design and synthesis of peripherally restricted transient receptor potential vanilloid 1 (TRPV1) antagonists. J Med Chem 2008;51:2744–57. [31] Lehto S, Tamir R, Deng H, Klionsky L, Kuang R, Le A, et al. Antihyperalgesic effects of AMG8562, a novel vanilloid receptor TRPV1 modulator that does not cause hyperthermia in rats. J Pharmacol Exp Ther 2008;326:218–29. [32] Garami A, Shimansky YP, Pakai E, Oliveira DL, Gavva NR, Romanovsky AA. Contributions of different modes of TRPV1 activation to TRPV1 antagonist induced hyperthermia. J Neurosci 2010;30:1435–40. [33] Watabiki T, Kiso T, Kuramochi T, Yonezawa K, Tsuji N, Kohara A, et al. Amelioration of neuropathic pain by novel transient receptor potential vanilloid 1 antagonist AS1928370 in rats without hyperthermic effect. J Pharmacol Exp Ther 2011;336:743–50. [34] Watabiki T, Kiso T, Tsukamoto M, Aoki T, Matsuoka N. Intrathecal administration of AS1928370, a transient receptor potential vanilloid 1 antagonist, attenuates mechanical allodynia in a mouse model of neuropathic pain. Biol Pharm Bull 2011;34(7):1105–8. [35] Frank R, Bahrenberg G, Christoph T, Schiene K, DeVry J, Saunders D, et al. PCT International Application WO 2008/125295; 2008a. 142 8. ANALGESIC POTENTIAL OF TRPV1 ANTAGONISTS

[36] Frank R, Bahrenberg G, Christoph T, Schiene K, DeVry J, Saunders D, et al. PCT International Application WO 2008/125296; 2008b. [37] Frank R, Bahrenberg G, Christoph T, Schiene K, DeVry J, Damann N, et al. US Patent Application US 2012/0258946; 2012b. [38] Frank R, Bahrenberg G, Christoph T, Schiene K, DeVry J, Damann N, et al. PCT International Application WO 2010/127855; 2010a. [39] Frank R, Bahrenberg G, Christoph T, Schiene K, DeVry J, Damann N, et al. PCT International Application WO 2010/127856; 2010b. [40] Frank R, Christoph T, Frormann S. US Patent Application US 2012/0115893; 2012a. [41] Gomtsyan A, Daanen J, Kort ME, Kym PR, Voight EA, Woller KR. US Patent Application US 2013/0345255; 2013. [42] Gomtsyan A, Daanen J, Gfesser GA, Lee C-H, McDonald HA, Puttfarcken PS, et al. US Patent Application US 2012/0245163; 2012. [43] Reilly RM, McDonald HA, Puttfarcken PS, Joshi SK, Lewis L, Pai M, et al. Pharmacology of modality-specific transient receptor potential vanilloid-1 antagonists that do not alter body temperature. J Pharmacol Exp Ther 2012;342:416–28. [44] Mantyh PW. Cancer pain and its impact on diagnosis, survival and quality of life. Nat Rev Neurosci 2006;7:797–809. [45] Jimenez-Andrade JM, Mantyh P. TRPV1 and bone cancer pain. In: Gomtsyan A, Faltynek CR, editors. Vanilloid Receptor TRPV1 in drug discovery. John Wiley & Sons, Inc., Hoboken, New Jersey, USA; 2010. p. 191–205. [46] Rami HK, Thompson M, Stemp G, Fell S, Jerman JC, Stevens AJ, et al. Discovery of SB-705498: a potent, selective and orally bioavailable TRPV1 antagonist suitable for clinical development. Bioorg Med Chem Lett 2006;16:3287–91. [47] Gunthorpe MJ, Hannan SL, Smart D, Jerman JC, Arpino S, Smith GD, et al. Characterization of SB-705498, a potent and selective vanilloid receptor-1 (VR1/TRPV1) antagonist that inhibits the capsaicin-, acid-, and heat-mediated activation of the receptor. J Pharmacol Exp Ther 2007;321(3):1183–92. [48] Chizh BA, O’Donnell MB, Napolitano A, Wang J, Brooke AC, Aylott MC, et al. The effects of the TRPV1 antagonist SB-705498 on TRPV1 receptor-mediated activity and inflammatory hyperalgesia in humans. Pain 2007;132(1–2):132–41. [49] Gunthorpe MJ, Chizh BA. Clinical development of TRPV1 antagonists: targeting a pivotal point in the pain pathway. Drug Discov Today 2009;14:56–67. [50] Gavva NR, Treanor JJ, Garami A, Fang L, Surapaneni S, Akrami A, et al. Pharmacological blockade of the vanilloid receptor TRPV1 elicits marked hyperthermia in humans. Pain 2008;136:202–10. [51] Doherty EM, Fotsch C, Bannon AW, Bo Y, Chen N, Dominguez C, et al. Novel vanilloid receptor-1 antagonists: 2. Structure-activity relationships of 4-oxopyrimidines leading to the selection of a clinical candidate. J Med Chem 2007;50:3515–27. [52] Gavva NR, Bannon AW, Hovland Jr DN, Lehto SG, Klionsky L, Surapaneni S, et al. Repeated administration of vanilloid receptor TRPV1 antagonists attenuates hyperthermia elicited by TRPV1 blockade. J Pharmacol Exp Ther 2007;323:128–37. [53] Wong GY, Gavva NR. Therapeutic potential of vanilloid receptor TRPV1 agonists and antagonists as analgesics: recent advances and setbacks. Brain Res Rev 2009;60:267–77. [54] Wang HL, Katon J, Balan C, Bannon AW, Bernard C, Doherty EM, et al. Novel vanilloid receptor-1 antagonists: 3. The identification of a second-generation clinical candidate with improved physicochemical and pharmacokinetic properties. J Med Chem 2007;50:3528–39. [55] Gavva NR. Body-temperature maintenance as the predominant function of the vanilloid receptor TRPV1. Trends Pharmacol Sci 2008;29:550–7. [56] Romanovsky AA, Almeida MC, Garami A, Steiner AA, Norman MH, Morrison SF, et al. The transient receptor potential vanilloid-1 channel in thermoregulation: a thermosensor it is not. Pharmacol Rev 2009;61:228–61. [57] Szelenyi Z, Hummel Z, Szolcsanyi J, Davis JB. Daily body temperature rhythm and heat tolerance in TRPV1 knockout and capsaicin pretreated mice. Eur J Neurosci 2004;19:1421–4. [58] Iida T, Shimizu I, Nealen ML, Campbell A, Caterina M. Attenuated fever response in mice lacking TRPV1. Neurosci Lett 2005;378:28–33. [59] Surowy CS, Neelands TR, Bianchi BR, McGaraughty S, El Kouhen R, Han P, et al. (R)-(5-tert-butyl-2,3-dihydro- 1H-inden-1-yl)-3-(1H-indazol-4-yl)-urea (ABT-102) blocks polymodal activation of transient receptor potential References 143

vanilloid 1 receptors in vitro and heat-evoked firing of spinal dorsal horn neurons in vivo. J Pharmacol Exp Ther 2008;326:879–88. [60] Gomtsyan A, Bayburt EK, Schmidt RG, Surowy CS, Honore P, Marsh KC, et al. Identification of (R)-1- (5-tertbutyl-2,3-dihydro-1H-inden-1-yl)-3-(1H-indazol-4-yl)urea (ABT-102) as a potent TRPV1 antagonist for pain management. J Med Chem 2008;51:392–5. [61] Honore P, Chandran P, Hernandez G, Gauvin DM, Mikusa JP, Zhong C, et al. Repeated dosing of ABT-102, a potent and selective TRPV1 antagonist, enhances TRPV1-mediated analgesic activity in rodents, but attenuates antagonist-induced hyperthermia. Pain 2009;142:27–35. [62] Schaffler K, Reeh P, Duan WR, Best AE, Othman AA, Faltynek CR, et al. An oral TRPV1 antagonist attenuates laser radiant-heat-evoked potentials and pain ratings from UVB-inflamed and normal skin. Br J Clin Pharmacol 2013;75:404–14. [63] Othman AA, Nothaft W, Awni WM, Dutta S. Effects of the TRPV1 antagonist ABT-102 on body temperature in healthy volunteers: pharmacokinetic/pharmacodynamic analysis of three phase 1 trials. Br J Clin Pharmacol 2013;75:1029–40. [64] Othman AA, Nothaft W, Awni WM, Dutta S. Pharmacokinetics of the TRPV1 antagonist ABT-102 in healthy human volunteers: population analysis of data from 3 phase 1 trials. J Clin Pharmacol 2013;52:1028–41. [65] Rowbotham MC, Nothaft W, Duan WR, Wang Y, Faltynek C, McGaraughty S, et al. Oral and cutaneous thermosensory profile of selective TRPV1 inhibition by ABT-102 in a randomized healthy volunteer trial. Pain 2011;152:1192–200. [66] Kym P. Highlights from Abbvie pain research: TRPV1, TrkA, Cav2.2 and Nav1.7. In: Presented at the Pain Therapeutics, London (UK), May 19; 2014. [67] Crutchlow M, Dong Y, Schulz V, Von Hoydonck P, Laethem T, Maes A, et al. Pharmacologic inhibition of TRPV1 impairs sensation of potentially injurious heat in healthy subjects (Abstract # LB-II-A-3). In: Presented at the 110th annual meeting of the American Society for Clinical Pharmacology and Therapeutics, National Harbor, MD, March 18-21; 2009. [68] Eid SR. Therapeutic targeting of TRP channels—the TR(i)P to pain relief. Curr Top Med Chem 2011;11:2118–30. [69] Laird GM, Martino G, Visser S, Oerther S, Yu XH, Perkins MN. Novel TRPV1 antagonist, AZD1386, produces plasma concentration-dependent reversal of heat hyperalgesia in inflammatory and neuropathic rat pain models in the absence of hyperthermic effects. In: Presented at the 13th World Congress on Pain, Montreal, Canada, August 29—September 2; 2010. [70] Ståhle L, Jonzon B, Björnsson M, Chetty R, Miller F. Single and multiple ascending dose studies of on oral TRPV-1 antagonist AZD1386 in healthy volunteers. In: Presented at the 13th World Congress on Pain, Montreal, Canada, August 29—September 2; 2010. [71] Karlsten R, Jonzon B, Quiding H, Carlsson M, Segerdahl M, Malamut R, et al. The TRPV1 antagonist AZD1386 inhibits capsaicin and heat evoked pain in healthy volunteers. In: Presented at the 13th World Congress on Pain, Montreal, Canada, August 29—September 2; 2010. [72] Quiding H, Webster L, Jonzon B, Karin A, Karlsten R, Reimfelt A, et al. Effects of TRPV1 antagonist AZD1386 in patients with pain after third molar extraction. In: Presented at the 13th World Congress on Pain, Montreal, Canada, August 29—September 2; 2010. [73] Quiding H, Jonzon B, Svensson O, Webster L, Reimfelt A, Karin A, et al. TRPV1 antagonistic analgesic effect: a randomized study of AZD1386 in pain after third molar extraction. Pain 2013;154:808–12. [74] Svensson O, Thorne C, Miller F, Björnsson M, Reimfelt A, Karlsten R. A Phase II randomized controlled trial evaluating efficacy and safety of the TRPV1 antagonists AZD1386 in osteoarthritis of the knee. In: Presented at the 13th World Congress on Pain, Montreal, Canada, August 29—September 2; 2010. [75] Krarup AL, Ny L, Åstrand M, Bajor A, Hvid-Jensen F, Hansen MB, et al. Randomised clinical trial: the efficacy of a transient receptor potential vanilloid 1 antagonist AZD1386 in human oesophageal pain. Aliment Pharmacol Ther 2011;33:1113–22. [76] Krarup AL, Ny L, Gunnarson J, Hvid-Jensen F, Zetterstrand S, Simrén M, et al. Randomised clinical trial: inhibition of TRPV1 system in patients with nonerosive oesophageal reflux disease and a partial response to PPI treatment is not associated to analgesia to oesophageal experimental pain. Scand J Gastroenterol 2013;48:274–84. [77] http://clinicaltrials.gov/ct2/show/NCT01557010?term=DWP05195&rank=3. [78] http://clinicaltrials.gov/ct2/show/NCT00960180?term=MR-1817&rank=1. [79] http://www.fiercebiotech.com/press-releases/further-clinical-trials-osteoarthritis-pain-suspended-grc-6211. 144 8. ANALGESIC POTENTIAL OF TRPV1 ANTAGONISTS

[80] Kitagawa Y, Miyai A, Usui K, Hamada Y, Deai K, Wada M, et al. Pharmacological characterization of (3S)-3- (hydroxymethyl)-4-(5-methylpyridin-2-yl)-N-[6-(2,2,2-trifluoroethoxy)pyrid in-3-yl]-3,4-dihydro-2H-benzo[b] [1,4]oxazine-8-carboxamide (JTS-653), a novel transient receptor potential vanilloid 1 antagonist. J Pharmacol Exp Ther 2012;342:520–8. [81] Kitagawa Y, Tamai I, Hamada Y, Usui K, Wada M, Sakata M, et al. The orally administered selective TRPV1 antagonist, JTS-653, attenuates chronic pain refractory to non-steroidal anti-inflammatory drugs in rats and mice including post-herpetic pain. J Pharmacol Sci 2013;122:128–37. [82] Kitagawa Y, Wada M, Kanehisa T, Atsuko A, Usui K, Maekawa M, et al. JTS-653 blocks afferent nerve firing and attenuates bladder overactivity without affecting normal voiding function. J Urol 2013;189:1137–46. [83] http://www.jt.com/investors/results/S_information/pharmaceuticals/pdf/P.L.20100729_E.pdf. [84] https://integrity.thomson-pharma.com/integrity/xmlxsl/pk_prod_list.productSummaryDetails?p_entry Number=642253. [85] http://www.pharmeste.com/repository/contenuti/paragrafi/file/PharmEste_Leaflet_Web.pdf. [86] Cao E, Liao M, Cheng Y, Julius D. TRPV1 structures indistinct conformations reveal activation mechanisms. Nature 2013;504(7478):113–18. [87] Liao M, Cao E, Julius D, Cheng Y. Structure of the TRPV1 ion channel determined by electron cryo-microscopy. Nature 2013;504(7478):107–12. CHAPTER 9 Transient Receptor Potential Ankyrin 1 Channel Antagonists for Pain Relief Ari Koivisto,1,* Antti Pertovaara2 1Research and Development, OrionPharma, Orion Corporation, Turku, Finland 2Institute of Biomedicine/Physiology, University of Helsinki, Helsinki, Finland *Corresponding author: [email protected]

OUTLINE

Introduction 146 Chemotherapy-Induced Neuropathy 149 Osteoarthritis 149 General Properties of the TRPA1 Postoperative Pain 150 Channel in Pain Transduction Bacterial Infection-Induced Pain and Amplification 146 and Inflammation 150 TRPA1 in Thermal and Chemical Migraine 151 Sensation 146 TRPA1 Agonists for Pain Relief? 153 TRPA1 in Mechanical Nociception TRPA1 Antagonists 153 and Primary Hyperalgesia 146 TRPA1 in Secondary (Central) Biomarkers 156 Mechanical Hyperalgesia 147 The Challenge of Who to Study in Proof-of-Concept 156 TRPA1 in Pathophysiological Pain Models 148 Conclusions 158 Sleep Deprivation-Induced Pain Acknowledgments 158 Hypersensitivity 148 Peripheral Diabetic Neuropathy 148 References 158 Peripheral Traumatic Neuropathy 149

Among the membrane receptors that contribute to the transduction of noxious signals on nociceptive nerve endings are those belonging to the transient receptor potential (TRP) family of ion channels. A member of the TRP family expressed on nociceptive nerve terminals is a calcium-permeable nonselective cation channel transient receptor potential ankyrin 1 (TRPA1) [1,2], the topic of this chapter. While on distal endings of nociceptive nerve fibers TRPA1 contributes to transduction of noxious signals, on their central terminals TRPA1 amplifies glutamatergic transmission to spinal interneurons (see for review Ref. [3]). Among nonneuronal cells expressing TRPA1 are epidermal keratinocytes, endothelium, vascular smooth muscle [4], and astrocytes [5]. The aim of this chapter is to introduce how TRPA1 contributes to the transduction and amplification of pain and how it thereby provides a target for pain relief.

GENERAL PROPERTIES OF THE TRPA1 CHANNEL IN PAIN TRANSDUCTION AND AMPLIFICATION

The biophysical, structural, and other basic properties of the TRPA1 channel are outside the focus of this chapter and extensively reviewed elsewhere (for recent reviews, see Ref. [6–9]).

TRPA1 in Thermal and Chemical Sensation TRPA1 is the only TRPA subfamily member of TRP channels in mammals, and it is expressed on a subpopulation of TRPV1-expressing nociceptive nerve fibers [1]. TRPA1 was originally considered to detect noxious cold (cold pain), whereas later studies indicate that it is involved in the detection of cold hypersensitivity rather than physiological cold pain [10]. TRPA1 of humans, unlike that of rattlesnakes or Drosophila, is not activated by heat [11]. The main function of TRPA1 in nerve terminals is to be a chemosensor of nociception. In line with this, TRPA1 is activated by various irritant chemicals and endogenous products of tissue injury. Among the large number of chemicals activating TRPA1 are pungent compounds in some spices (such as mustard oil, cinnamon, wasabi), acrolein (an irritant compound in tear gas), formalin, various reactive oxygen species (ROS), reactive nitrogen species, reactive carbonyl species, some general and local anesthetics (e.g., propofol and lidocaine), and cannabinoids (for an extensive list see Ref. [8]). In experimental studies, cinnamaldehyde has proved to be among the most selective chemical agonists of TRPA1 [12].

TRPA1 in Mechanical Nociception and Primary Hyperalgesia TRPA1 exerts a role in the mediation of mechanically evoked pain behavior as indicated by the finding that mechanically evoked responses of nociceptive primary afferent nerve fibers to noxious (high-intensity) stimulation were reduced following genetic ablation or pharmacological block of TRPA1 [13,14]. There is evidence to indicate that, as with heat General Properties of the TRPA1 Channel in Pain Transduction and Amplification 147 hypersensitivity, peripheral sensitization of primary afferent nociceptors may contribute to mechanical hyperalgesia, although the prevailing concept has until recently been that mechanical hyperalgesia is mainly due to central mechanisms [15]. TRPA1 may play a role in primary mechanical hyperalgesia (i.e., hyperalgesia at the site of injury) as shown by the electrophysiological findings that mechanically evoked responses were facilitated by inflammation only in a population of TRPA1-expressing nociceptive primary afferent nerve fibers [16], and that the mechanical sensitization of nociceptors was attenuated following pharmacological blockade of the TRPA1 [17]. These findings, however, leave open the question of whether the role of TRPA1 in mechanical hyperalgesia is direct or indirect. Among potential indirect TRPA1-related mechanisms is an interaction of nociceptors with keratinocytes, which play a role in nociception [18] and also express TRPA1 [19]. Another potential indirect mechanism is an interaction of the neuronal TRPA1 with other transducer molecules on the neuronal membrane.

TRPA1 in Secondary (Central) Mechanical Hyperalgesia After injury, the adjacent intact area will also be sensitized to mechanical stimulation [15]. This phenomenon is called secondary hyperalgesia, although it involves hypersensitivity to innocuous (tactile allodynia) as well as noxious mechanical stimulation. Earlier studies indicated that peripheral signals carried by mechanoreceptive (Aβ) fibers (humans) or low-threshold C fibers (rodents) elicit tactile allodynia due to central rather than peripheral mechanisms [20,21]. Interestingly, TRPA1 seems to have an important role in secondary mechanical hyperalgesia as indicated by the finding that a gain-of-function mutation in TRPA1 increases secondary pain hypersensitivity and neurogenic inflammation adjacent to the injured cutaneous area in humans [22]. In line with this, it has been shown in experimental animals that blocking the spinal TRPA1 channel reverses secondary pain hypersensitivity [23,24] and reduces the presumably dorsal root reflex-mediated cutaneous neurogenic inflammation adjacent to an injury site [25]. A potential mechanistic explanation for the involvement of spinal TRPA1 in secondary hyperalgesia is that injury discharge induces in the spinal dorsal horn ROS [26], which may at least partly be released from activated microglia [27]. ROS provides an endogenous agonist for TRPA1 [28] that, on central terminals of nociceptive nerve fibers, amplifies glutamatergic transmission to spinal interneurons [29]. In addition to ROS, injuries induce the generation of various endogenous TRPA1 agonists (e.g., hepoxilin A3 or 5,6-EET) in the spinal cord. Another cell type expressing TRPA1 is the astrocyte [5]. Activation of spinal astrocytes has been associated with their coupling to adjacent astrocytes or neurons that may promote spread of excitation; thereby activated astrocytes may contribute to pain hypersensitivity [30]. A recent study demonstrated that TRPA1 channels expressed on astrocytes contribute to basal Ca2+ levels and are required for constitutive d-serine release into the extracellular space, which contributes to N-methyl-D-aspartate (NMDA) receptor-dependent long-term potentiation (LTP) in the hippocampus [31]. Because NMDA receptor-dependent LTP in the spinal dorsal horn has been shown to increase excitability of pain-relay neurons [32], it may be speculated that TRPA1 on spinal astrocytes contributes to central pain hypersensitivity by enhancing LTP in pain-relay neurons of the spinal dorsal horn. 148 9. TRPA1 ANTAGONISTS FOR PAIN RELIEF TRPA1 IN PATHOPHYSIOLOGICAL PAIN MODELS

The contribution of the TRPA1 to pathophysiological pain has recently been studied in a large number of experimental animal models. Next, we briefly review results in some of the models.

Sleep Deprivation-Induced Pain Hypersensitivity Sleep deprivation may aggravate pain in clinical conditions and induce pain hypersensitivity in experimental conditions [33]. Conversely, pain may disturb sleep. Sleep deprivation-induced pain hypersensitivity is at least partly mediated by spinal TRPA1 as indicated by the finding that blocking spinal TRPA1 reversed sleep deprivation-induced pain hypersensitivity in the rat [24]. It may be hypothesized that the sleep-deprivation-induced pronociceptive loop involves a brainstem-spinal pathway that induces spinal generation of ROS, potentially through action on spinal astrocytes. In line with this, some of the brainstem-spinal pathways activate a spinal NMDA-nitric oxide cascade [34], which has been associated with activation of spinal astrocytes [35]. d-Amino acid oxidase (DAAO) in activated spinal astrocytes produces hydrogen peroxide, which, through action on TRPA1 on the central terminals of nociceptive nerve fibers, can promote transmission of nociceptive signals from peripheral to central pain-relay neurons. This hypothesis is supported by the findings that the facilitation of pain behavior induced by sleep deprivation was reduced not only by blocking the spinal TRPA1 but also by inhibiting spinal astrocytes with carbenoxolone [36] and by spinal administration of DAAO inhibitors [37]. Together these findings suggest that the spinal TRPA1 channel is a final common pathway for a pronociceptive cascade induced by sleep deprivation. This hypothesis, however, does not exclude the parallel involvement of other pronociceptive mechanisms.

Peripheral Diabetic Neuropathy Peripheral diabetic neuropathy (PDN) is a common disorder in patients with diabetes mellitus. PDN produces symptoms varying from paresthesia and spontaneous pain to numbness and sensory deficits. Small fibers and their functions (such as pain and warm sense) are affected early, and large fiber-mediated functions (such as vibrotactile sense) are affected later [38]. Although it is obvious that PDN has multiple underlying mechanisms, there is recent evidence to indicate that among the earliest mechanisms inducing PDN are those involving TRPA1. The role of TRPA1 in PDN is reviewed in more detail elsewhere [39], and therefore, this topic is only briefly introduced here. Diabetes mellitus generates endogenous compounds, such as 4-hydroxynonenal and methylglyoxal, which are TRPA1 agonists [40,41]. These endogenous TRPA1 agonists may produce a sustained influx of Ca2+ in nerve endings, due to prolonged activation of the TRPA1 channel [41]. This eventually results in axoplasmic calcium dysregulation and excitotoxicity [42]. Thereby, the prolonged TRPA1 channel-mediated calcium influx provides a potential mechanism for dysfunction and loss of small fiber endings, the hallmark of PDN. In line with this proposal, pharmacological block of TRPA1 reduced the maintenance [43] and development of pain hypersensitivity as well as loss of small fiber endings and their function in experimental diabetes [41]. The finding that elevated level of methylglyoxal induced by TRPA1 in Pathophysiological Pain Models 149 administration of a glyoxylase inhibitor resulted in a pain phenotype in wild-type, but not TRPA1 knockout, animals supports the hypothesis that a compound generated in diabetes (e.g., methylglyoxal) has an important role in the TRPA1-mediated development of PDN symptoms [44]. Although TRPA1 on distal nerve endings is likely to play a major role in the anatomical and physiological changes of small-diameter nerve fibers in PDN, amplification of pain signals by spinal TRPA1 is likely to have an important contribution to diabetic pain hypersensitivity. This was indicated by the finding that a TRPA1 antagonist suppressed diabetic pain hypersensitivity at a considerably lower dose following intrathecal than systemic or intraplantar application [23]. Interestingly, TRPA1 antagonists at a dose at which they suppress mechanical hypersensitivity failed to induce conditioned place-preference (an index of ongoing pain) in animals with streptozotocin-induced diabetes [45]. However, when interpreting this result, it should be noted that it is not clear whether the streptozotocin-induced diabetes model is associated with ongoing pain. Together, the experimental evidence predicts that blocking the TRPA1 channel would provide a mechanism-based treatment that suppresses or delays the development of PDN.

Peripheral Traumatic Neuropathy Various types of peripheral nerve injuries may induce pain that is associated with cutaneous hypersensitivity [38]. Blocking TRPA1 has proved to reduce pain hypersensitivity in peripheral neuropathy [46,47]. Amplification of the pain signal by spinal TRPA1 explains, at least in part, neuropathic hypersensitivity as indicated by the finding that a very low intrathecal dose of a TRPA1 antagonist markedly reduced hypersensitivity in animals with a spinal nerve ligation-induced neuropathy [24]. In spinal nerve-ligated animals, TRPA1 mRNA is down-regulated in the injured nerve and up-regulated in the adjacent intact nerves [48]. Concerning this finding, it is noteworthy that earlier evidence indicates that intact nerves adjacent to the nerve injury may significantly contribute to neuropathic pain [49].

Chemotherapy-Induced Neuropathy Among the most problematic side effects of some anticancer drugs (such as oxaliplatin) is neuropathy with severe mechanical and cold allodynia. In experimental animal studies, blocking TRPA1 has proved effective in suppressing the anticancer drug-induced mechanical and cold allodynia, indicating that TRPA1 has a significant role in chemotherapy-induced neuropathy [50].

Osteoarthritis Osteoarthritis is a common cause of joint pain. It typically leads to loss of articular cartilage, new bone formation in the subchondral region, and formation of new cartilage and bone at the joint margins. In the monosodium iodoacetate-induced model of experimental osteoarthritis, pharmacological blockade of TRPA1 had a mechanical antihypersensitivity effect on the responses of nociceptive spinal dorsal horn neurons [51], whereas TRPA1 block failed to influence the ongoing pain in osteoarthritic animals as revealed by the failure to 150 9. TRPA1 ANTAGONISTS FOR PAIN RELIEF induce conditioned place-preference [52]. Together, these findings suggest that TRPA1 may have a more important role in osteoarthritis-induced mechanical hypersensitivity than the ongoing pain.

Postoperative Pain Surgical operations induce ongoing pain that is accompanied by hypersensitivity of the affected region. In an experimental rat model of postoperative pain induced by incision of the skin and muscle tissue in the paw, pharmacological blockade of TRPA1 attenuated guarding behavior, an index of ongoing pain, and mechanical hyperalgesia [53]. The critical site of action for these effects proved to be the peripheral injury site because blocking TRPA1 in the operated plantar area attenuated guarding and mechanical hyperalgesia, whereas blocking TRPA1 in the intact contralateral site failed to influence pain behavior. In addition, spinal TRPA1 may have contributed to mechanical hypersensitivity at low-intensity stimuli because blocking the spinal TRPA1 reduced tactile allodynia-like behavior [53]. In contrast, a recent mouse study using a skin incision-induced model of postoperative pain reported that knockout of TRPA1 failed to influence the development of mechanical hypersensitivity [54]. It remains to be studied whether this difference in results is due to a species difference (rat vs. mouse), a difference in the injured tissue (skin + deep tissue incision vs. skin incision), or some other reason (e.g., pharmacological vs. genetic TRPA1 block).

Bacterial Infection-Induced Pain and Inflammation Several groups have reported that injection of the complete form of Freund’s adjuvant (CFA), a mixture that contains inactivated and dried mycobacteria, to the paw or knee of a rodent results in acute pain, mechanical allodynia, tissue swelling, and chronic inflammation that can be attenuated by selective TRPA1 antagonists or genetic abolition of TRPA1 [47,51,55–57]. This finding suggests that either bacteria can directly activate sensory neurons or that bacteria-induced inflammation may indirectly activate TRPA1 expressing sensory neurons and therefore result in pain and inflammation. Two recent elegant studies by Chiu et al. [58] and Meseguer et al. [59] shed light onto how microbes activate TRPA1 expressing sensory neurons. Live bacterial load rather than tissue swelling or immune activation was shown to correlate with pain or hyperalgesia in a Staphylococcus aureus infection mouse model [58]. S. aureus is a major cause of wound and surgical infection in humans. Bacteria were shown to activate sensory neurons by direct interaction. Further, sensory neuron activation was in part shown to be mediated by N-formyl peptides and α-hemolysin that are released from bacteria. In another study by Meseguer et al. [59], the bacterial cell wall constituent lipopolysaccharide (LPS), that is typical for Gram negative bacteria, was shown to directly activate TRPA1 and therefore cause pain and inflammation. This finding is intriguing because it was shown that toll-like receptor 4 of the innate immunity system, which was previously thought to be the major LPS receptor, was not needed for such an interaction. Further, the TRPA1-dependent neurogenic inflammation response was potentiated by simultaneous application of LPS and another inflammatory TRPA1 agonist such as 4-hydroxynonenal. Such an additive effect may be highly relevant in vivo, when a large number of inflammatory TRPA1 agonists are present and their levels TRPA1 in Pathophysiological Pain Models 151 are elevated. However, the LPS-induced hypothermic response is not mediated by TRPA1 because genetic abolition of TRPA1 did not modify the hypothermic response. Low-grade metabolic endotoxemia (i.e., elevated level of LPS in blood plasma) is prevalent in metabolic syndrome and diabetes [60]. Emerging evidence suggests that gut microflora acts as a source of elevated systemic LPS levels as a result of an excessive high-fat diet [61]. An elevated systemic LPS level may facilitate the appearance of pain in diabetes and promote progression of neuropathy because LPS acts as a TRPA1 agonist and potentiates the action of other TRPA1 agonists such as 4-hydroxynonenal, whose level is also elevated in diabetes. Lower back pain is a very common, hard to treat chronic pain condition. The pathophysiology of lower back pain is currently not well understood. Recently, a double-blind randomized clinical trial tested the hypothesis that chronic lower back pain is caused and maintained by a chronic low-grade bacterial inflammation. Treatment of lower back pain patients for 100 days with antibiotics improved statistically significantly the primary outcomes, disease-specific disability, and lumbar pain, as well as a number of secondary outcomes [62]. This finding strongly suggests that chronic, low-grade bacterial inflammation is involved in lower back pain pathophysiology. It is therefore possible that lower back pain patients may also benefit from treatment with selective TRPA1 antagonists without risks associated with long-term antibiotic treatment.

Migraine Migraine is an episodic headache that typically affects one side of the head. Pulsating migraine attack typically lasts from a couple of hours up to 3 days. Migraine is associated with sensitivity to light, sound, movement, and smell and is frequently accompanied by nausea and vomiting. About 30% of migraineurs experience transient aura before onset of migraine attack. The apparent absence of any exogenous cause of migraine headache has led to a search for putative migraine triggers. Several exogenous triggers have been identified such as tobacco smoke, occupational exposure to glyceryl trinitrate, certain odors, and drug medication over- use. However, the chemical diversity of migraine triggers presents a formidable challenge to understand at a molecular level how such triggers may bring about migraine headache in vulnerable individuals. Migraine is a neurovascular headache, which implies that experienced pain is tightly associated with changes in the cranial blood flow. Unmyelinated substance P and calcitonin gene-related peptide (CGRP) containing peptidergic pain-sensing C fibers originating from trigeminal ganglion surround large cerebral vessels, pial vessels, large venous sinuses, and dura mater in the cranium. Activation of afferent unmyelinated sensory neurons convey nociceptive information to the central nervous system (CNS) but, through the release of CGRP, contributes also to neurogenic inflammation in the innervated tissue [63]. Activation of capsaicin-sensitive TRPV1-expressing sensory neurons was shown to elicit meningeal vasodilation suggesting an important role in meningeal nociception [64]. This finding raises the possibility that meningeal TRPA1 activation may also play a role in migraine pain because TRPA1 and TRPV1 are colocalized to trigeminal peptidergic sensory neurons [65]. Also the fact that TRPA1 can be activated by an amazing diversity of chemical structures suggests that trigeminal TRPA1 activation may be a potentially unifying mechanism behind a wide range of migraine triggers. 152 9. TRPA1 ANTAGONISTS FOR PAIN RELIEF

Children who have experienced infantile colic in their early life are more likely to suffer from migraine attacks than those without colic. This suggests that colic may be a form of migraine attack and/or that colic may prime for future migraine attacks [66]. It is interesting here that TRPA1 in rodents is the last among the TRP channels in sensory neurons that is expressed during development [67]. Thus, it is possible that human TRPA1 expression coincides temporally with the emergence of colic symptoms suggesting a possible link between TRPA1 activation and colic symptoms. Putative food-borne or endogenously produced TRPA1 agonists can act as colic triggers because not all babies suffer from colic. Kunkler et al. [68] showed for the first time that intranasal application of the environmental and tobacco smoke irritant, acrolein, and a few other known TRPA1 agonists evoke neurogenic inflammation, increase meningeal blood flow, and stimulate CGRP release. Furthermore, all these effects were blocked by selective CGRP and TRPA1 antagonists. This study established that environmental exposure to TRPA1 agonists has the potential to cause migraine-like symptoms. Similarly, another environmental irritant and known migraine trigger, umbellulone, was shown to activate human and rodent TRPA1 ion channel in vitro and induce neurogenic inflammation and vasodilation, as well as migraine-related allodynia, effects that were blocked by the selective TRPA1 antagonist HC-030031 and the clinically effective migraine drug sumatriptan [69]. Cortical spreading depression is a self-propagating wave of cellular depolarization that travels across the cerebral cortex. It has been shown to correlate with migraine aura [70]. Recently, a technically challenging experimental study established that cortical spreading depression induces oxidative stress and results in subsequent TRPA1 activation in trigeminal ganglia. Local application of hydrogen peroxide, a known TRPA1 agonist, to the meninges was shown to induce electrical spiking of sensory neurons, which is a compelling sign of pronociceptive action. Hydrogen peroxide-induced excitation of trigeminal ganglia was mediated by TRPA1 because selective TRPA1 antagonist TCS-5861528 was able to abolish such an effect [71]. It is likely that several novel migraine-like headache mechanisms that depend on meningeal sensory neuron TRPA1 activation will be revealed in the near future. Among putative candidate mechanisms are nitroglycerin-induced migraine in which nitric oxide directly activates TRPA1 [72]. Bacterial meningitis headache may at least partly depend on bacterial and cellular leakage of LPS and formyl peptides both of which are known to activate TRPA1- expressing sensory neurons [58,59]. Mitochondrial encephalopathy with lactic acidosis and stroke-like episodes disorder may involve TRPA1-mediated lactic acidosis-induced migraine headache [73] because human TRPA1 is directly activated by acidosis [74]. Genetic and epigenetic factors that contribute to individual differences in migraine vulnerability and pathophysiology are slowly beginning to be understood. TRPM8 was recently found to be linked to migraine in a human genome-wide association genetic study [75], whereas no association with regard to TRPA1 has been found so far. Surprisingly, differential TRPA1 expression was recently shown to be strongly linked to individual heat pain thresholds in discordant twins and healthy volunteers. Increased methylation of the TRPA1 gene promoter was found to be associated with the elevated TRPA1 expression in tissues and the lowered heat pain thresholds [76]. This suggests that epigenetic changes that result in increased expression of TRPA1 in sensory neurons may amplify heat pain signaling in affected individuals, although TRPA1 is not directly activated by heat. In agreement with TRPA1 in Pathophysiological Pain Models 153 such a proposal, heat pain thresholds are lowered between migraine attacks in episodic and chronic migraineurs [77]. Future studies will hopefully shed light onto whether migraineurs and/or infants suffering from colic pain may have a hypermethylated TRPA1 promoter and increased TRPA1 expression. Triptans have revolutionized the acute treatment of migraine. However, a huge unmet need for more effective treatments exists. In a short period of time, compelling evidence has been collected to support the idea that TRPA1 antagonists have potential in the preventive and acute treatment of migraine pain and related symptoms.

TRPA1 Agonists for Pain Relief? A recent preclinical study suggests that TRPA1 agonism may be the long-sought mechanism behind the analgesic action of acetaminophen (paracetamol). It was suggested that the reactive metabolite of acetaminophen acts in vivo as a TRPA1 agonist and by such action attenuates pain through desensitizing TRPA1-expressing sensory neurons [78]. This finding raises an interesting question of whether TRPA1 agonism may be a general approach and useful for development of novel analgesics. From a drug discovery point of view, TRPA1 agonism appears exciting because the discovery of novel TRPA1 agonists is far easier than identifying a novel TRPA1 antagonist scaffold. However, several major challenges can be foreseen with regard to the TRPA1 agonism approach. First, because TRPA1 is a calcium-permeable nonselective cation channel, long- term sustained TRPA1 activation and possible pore dilation evoked by TRPA1 agonists can be predicted to lead to excitotoxicity [41]. Second, most known TRPA1 agonists are reactive compounds, which immediately raises serious concerns in drug discovery teams because of known high risks associated with reactive compounds and their metabolites regarding idio- syncratic adverse drug reactions (e.g., TRPA1 agonistic acetaminophen reactive metabolites are known liver toxicants). Third and last, production of a large number of chemically diverse endogenous TRPA1 agonists is elevated in several painful disease states. In such conditions, one needs to exclude the possibility that a synthetic TRPA1 agonist would add to the pronociceptive effect of the endogenous agonists rather than reducing it before using a synthetic TRPA1 agonist for pain treatment. For these reasons, the biased view of the authors is that developing a TRPA1 antagonist or negative allosteric modulator is going to be a more straightforward and successful path toward development of novel analgesics.

TRPA1 Antagonists In the drug discovery process potential therapeutics are designed and identified. The cloning and expression of human TRPA1 in a cell-based system in conjunction with automated intracellular fluorescence multiwell cellular screening assays as well as automated patch clamp screening assays have enabled the systematic search for novel small-molecule antagonist scaffolds [6]. Historically, most ion channel antagonist scaffolds have been originally discovered from natural sources such as animal and plant toxins. Unfortunately, the physiological role of TRPA1 as a broad irritancy and pain sensor has precluded the use of natural products as starting material for antagonist lead optimization because the vast majority of natural toxins are TRPA1 agonists rather than antagonists. Similarly, the high throughput screening of 154 9. TRPA1 ANTAGONISTS FOR PAIN RELIEF chemically diverse synthetic compound libraries has yielded an extremely low antagonist hit rate [79]. In contrast, a large number of chemically diverse TRPA1 agonists have been identified as false positives. Currently, the major drawback in TRPA1 antagonist development is the relatively small number of novel chemical leads that can be further optimized. Hope is that scaffold hopping in which novel antagonists are discovered through modification of a central core of known compounds will yield novel TRPA1 antagonist skeletons. Despite such cave- ats, progress has recently been made in identifying novel TRPA1 antagonist scaffolds that are suitable for further multiparameter optimization of drug-like properties such as potency, solubility, metabolic stability, toxicity, and patentability. Notable species differences in TRPA1 antagonist pharmacology became quickly clear after the discovery of the first generation of compounds [80]. These acted as antagonists in human TRPA1 but were inactive in rodent TRPA1, thereby effectively preventing preclinical efficacy and safety studies [81]. A major hurdle therefore has been to identify potent compounds that act as antagonists both in human and rodent TRPA1. Another major obstacle in the lead optimization of TRPA1 antagonists is the steep structure activity relationship of druggable chemical space in which an unusually small change in the compound structure results quickly in a loss of activity. This can be partly understood on the basis that small-molecule TRPA1 antagonists are negative allosteric modulators, which do not compete with the traditional agonist binding but bind instead to a site(s) that modulate or inhibit TRPA1 activation. Several pharmaceutical companies are currently actively pursuing novel small-molecule TRPA1 antagonists and optimizing existing scaffolds. Hydra Biosciences was the first company to disclose xanthine derivatives as TRPA1 antagonists, HC-030031 (Figure 9.1a) being a representative of such a series [82]. This was shown to be efficacious in vivo at 100 and 300 mg/kg. Another xanthine derivative, Chembridge-5861528, was explored by Orion Pharma as a tool compound, and it was shown to be about 10-fold more potent than HC- 030031 [43]. Abbott disclosed a highly potent oxime derivative A-967079 (WO/2009/089082) (Figure 9.1b), which did not show efficacy after oral administration in neuropathic pain animal models [83]. However, this result can be now understood as a consequence of limited access to the brain/spinal cord [84]. Amgen disclosed a series of trichloro(sulfanyl)ethyl benzamides as potent TRPA1 antagonists, which were active only in humans but not in rats (Figure 9.1c) [81]. Glenmark Pharmaceuticals has extensively characterized and expanded the chemical space identified by Hydra Biosciences. The most advanced compound developed by Glenmark Pharmaceuticals is GRC17536, but its structure is presently unknown. A typical representative of the Glenmark series is depicted in Figure 9.1d. Janssen disclosed a series of heterocyclic amides as modulators of TRPA1 in 2010 (Figure 9.1e) (2010/141805 A1). However, the promising compounds in this series suffer from poor solubility. Merck disclosed a decaline derivative as a potent TRPA1 antagonist (WO/2011/043954) (Figure 9.1f). Orion Pharma disclosed proline derivatives as promising TRPA1 antagonists in 2012 (WO/2012/152983 A1). A typical example is depicted in Figure 9.1g. Ajinomoto has explored heterocyclic amides as TRPA1 antagonists in 2013 (WO2013/108857 A1) (Figure 9.1h). AstraZeneca has disclosed recently N-1-alkyl-2-oxo-2-aryl amides, 4-aryloxy-1H-pyrrolo[3,2-c] pyridine, and a 1-aryloxyisoquinoline and aryl-N-(3-(alkylamino)-5(trifluoromethyl)phenyl) benzamides as TRPA1 antagonists [85–87]. A promising isoquinoline derivative compound 48 from Hu et al. [86] is depicted in Figure 9.1i. Orion Pharma disclosed recently novel arylamide derivatives as TRPA1 antagonists with improved potency, metabolic stability, and TRPA1 in Pathophysiological Pain Models 155

OR OH R O N N H S N N H N CI N O O N F CI CI CI

R = Me; HC-030031 R = H, Br, NO , OMe R = EI; Chembridge-5861528 2 (a) (b) (c)

F O Z S W O X Y Y F H HN N N HO N N H N Y Ar2 O R S 2 CF3 Ar O R1 O N S 1 O H (d) (e) (f)

O CF3 O X N Y R1 O NH R W 4 A O N O N Ar2 N S F O R3 Ar S O R2 1 F F O O (g) (h) (i)

F NH O O H N O N O N N CF3 H NH O OOCF3 F CI HN F S (j) (k) (l)

CF3

N N H (m)

FIGURE 9.1 Structures of TRPA1 antagonists. See text for further explanations. solubility (WO2014/053694). A representative arylamide is depicted in Figure 9.1j. Roche disclosed a series of substituted carbamate compounds as TRPA1 antagonists. A typical example of a Roche compound is shown in Figure 9.1k. Amgen is currently optimizing the solubility of azabenzofuran TRPA1 antagonists; promising compound 10 is depicted in Figure 9.1l [88]. 156 9. TRPA1 ANTAGONISTS FOR PAIN RELIEF

Novartis Research Institute published recently 5-(2-(trifluoromethyl)phenyl)-indazoles as a novel class of TRPA1 antagonists. A compound depicted in Figure 9.1m was shown to attenuate CFA-induced mechanical allodynia at 3-10 mg/kg after peroral administration [89]. Hydra Biosciences/Cubist Pharmaceuticals have successfully conducted a phase 1 study with their compound CB-625. Unfortunately, further development of CB-625 was discontinued by Cubist Pharmaceuticals due to poor solubility. Glenmark Pharmaceuticals is currently performing a phase 2 study for treatment of diabetic neuropathy and asthma with GRC17536. It is likely that additional phase 1 studies will start soon. Further, it is likely that improved understanding of the structure activity relationship of TRPA1 antagonism will speed up further effort to develop potent, selective, and drug-like TRPA1 antagonists.

BIOMARKERS

Despite intensive research activity and heavy investment from pharma industry, chronic pain remains a significant burden for many patients [90,91]. Clinically significant pain relief is achieved only in a subpopulation of patients with existing drugs. Those who benefit in terms of pain also benefit from significantly improved quality of life and ability to work [92]. Therefore a huge unmet need exists for improved pain relief with preferably mild or no side effects. Improving the survival of new drug candidates through preclinical research and clinical development is a key productivity driver for the pharmaceutical industry. Recent analysis of several phase 2 failures revealed the three pillars of survival: (1) exposure at the target site of action over a desired period of time, (2) binding to the pharmacological target as expected for its mode of action, and (3) expression of pharmacological activity commensurate with the demonstrated target exposure and target binding [93]. Thus, being able to prove pain target engagement in the first-in-man clinical trial is a highly valued asset that would increase confidence and willingness for continued investment toward a given target. A centrally acting TRPA1 antagonist is predicted to show robust efficacy in several human neuropathic pain conditions. Tactile hypersensitivity around the primary hyperalgesia site is defined as secondary hyperalgesia. As we have shown earlier that secondary hyperalgesia is maintained in several neuropathic pain animal models through spinal TRPA1 activation [23,24], a result that was independently confirmed by biochemical and electrophysiological measurements [94,95], and as mustard oil-induced secondary hyperalgesia is significantly enhanced in human TRPA1 gain-of-function mutation carriers [22], this strongly suggests that the widely used capsaicin and/or mustard oil-induced secondary hyperalgesia in human volunteers would be a rational target engagement biomarker for a centrally acting TRPA1 antagonist. The simple idea is to identify the plasma exposure that reaches sufficient CNS/ spinal exposure through attenuation of touch hypersensitivity in the secondary hyperalgesia area of the skin in a healthy volunteer. Such a dose is predicted to show efficacy in neuropathic pain patients, provided that the right patients are studied.

The Challenge of Who to Study in Proof-of-Concept Retrospective analysis of several human pain clinical trials have revealed that only a small subgroup of patients benefits significantly from a drug treatment [92]. Such a finding raises Biomarkers 157 the serious question whether the right patients, that is, responders for a putative treatment, have been correctly identified. Recent results suggest that the diabetes-specific toxic glycolysis by-product, methylglyoxal, acts as a TRPA1 agonist, as studied with the electrophysiological patch clamp technique, single-cell fluorescence imaging, as well as automated fluorescent imaging plate reader at the human and other species receptor [41]. This finding was elegantly confirmed in a study in which a selective glyoxalase inhibitor was used to elevate the methylglyoxal level in vivo [44]. As expected, the elevated methylglyoxal level resulted in a pain phenotype in wild-type mice but not in mice whose TRPA1 was genetically abolished. Together these findings strongly suggest that pain evoked by an elevated methylglyoxal level in vivo is solely mediated through TRPA1 activation. In line with this, an intradermal injection low dose of methylglyoxal induced intense pain when applied into the human skin [40]. Despite the best available management of diabetes, methylglyoxal plasma and intracellular levels are elevated in both type 1 and type 2 diabetic patients [96], and free plasma methylglyoxal levels of greater than 600 nM were recently shown to correlate with leg pain in diabetic neuropathy patients [97]. These preclinical and clinical findings support the hypothesis that diabetic neuropathy patients who have elevated plasma methylglyoxal levels and suffer from (leg) pain are the most likely responders for TRPA1 antagonist treatment. Therefore, it is expected that a reliable assessment of the plasma methylglyoxal levels from diabetic patients will help to identify likely responders to TRPA1 antagonist therapy. Assessing plasma-free methylglyoxal levels from diabetes patient plasma samples is currently rather resource craving and time consuming and uses sophisticated mass spectrometry technology that is not widely available. However, the recent discovery of a highly sensitive and selective methylglyoxal fluorescence sensor [98] can be predicted to be a game-changing technology in the clinical assessment of plasma-free methylglyoxal level. Diagnosis of diabetic peripheral sensory neuropathy in the clinic is aided by skin biopsy [99] because intraepidermal nerve fiber density is significantly reduced in diabetic neuropathy patients [100]. In an experimental animal model of diabetes, selective TRPA1 antagonist protected peptidergic intraepidermal sensory nerve fiber axon terminals during a 4-week treatment period [41]. It remains to be studied whether TRPA1 antagonism protects sensory neuron axon terminals from diabetes-induced damage and maintains sensory neuron functionality in clinical as well as experimental conditions. The neuropathy drugs presently on the market do not stop or reverse diabetic neuropathy progression. Studies in an experimental animal model of diabetes suggest that a TRPA1 antagonism might provide a unique disease-modifying treatment with the potential to even reverse progression of peripheral sensory neuropathy in diabetes patients. Although skin biopsy is quick and rather harmless to the patient, processing and analysis of skin biopsy samples is resource craving and time consuming. Therefore, a novel noninvasive technology in which corneal sensory nerves from diabetic patients can be studied with confocal microscopy provides a robust and highly sensitive method to assess alterations in sensory neuron densities in individual patients over time [101]. Intraepidermal nerve fiber density as studied from skin biopsies was recently shown to correlate with corneal sensory nerve fiber density [102]. Rapid automated diagnosis of PDN is now possible with corneal confocal microscopy [103]. It is clear that the use corneal confocal microscopy will be ad- vantageous in the study of the long-term disease-modifying efficacy of TRPA1 antagonist 158 9. TRPA1 ANTAGONISTS FOR PAIN RELIEF from diabetic peripheral neuropathic patients. Assessing corneal sensory nerve fiber density with confocal microscopy provides a highly relevant biomarker for disease progression assessment.

CONCLUSIONS

Blocking TRPA1 has proved effective in reducing mechanical and cold pain hypersensitivity in various pathophysiological pain models, with little or no side effects. When assessing TRPA1 antagonists in pain therapy, it should be noted that peripherally acting TRPA1 antagonists are likely to be optimal for suppression of primary hyperalgesia (such as inflammation-induced sensitization of peripheral nerve endings), whereas centrally (spinally) acting TRPA1 antagonists are needed for suppressing secondary hyperalgesia (in particular, tactile allodynia associated with various types of peripheral injuries). In an experimental model of PDN, prolonged blocking TRPA1 reduced the loss of nociceptive nerve endings and their function [41], thereby promising to provide a disease-modifying treatment for PDN. TRPA1 is a final common pathway for a large number of chemically diverse pronociceptive agonists generated in different disease conditions. Therefore TRPA1 antagonism can be expected to be superior over drugs targeting single nociceptive signaling pathways.

Acknowledgments The authors thank Hugh Chapman, Peteris Prusis, Patrik Holm, Ken Lindstedt, Niina Jalava, and Raymond Bratty for their comments and Riina Arvela for the illustration. The original work by the authors has been supported by OrionPharma (Espoo, Finland), the Sigrid Jusélius Foundation (Helsinki, Finland), and the Academy of Finland (Helsinki, Finland).

References [1] Story GM, Peier AM, Reeve AJ, Eid SR, Mosbacher J, Hricik TR, et al. ANKTM1, a TRP-like channel expressed in nociceptive neurons, is activated by cold temperatures. Cell 2003;112:819–29. [2] Jordt SE, Bautista DM, Chuang HH, McKemy DD, Zygmunt PM, Högestätt ED, et al. Mustard oils and cannabinoids excite sensory nerve fibres through the TRP channel ANKTM1. Nature 2004;427:260–5. [3] Pertovaara A, Koivisto A. TRPA1 ion channel in the spinal dorsal horn as a therapeutic target in central pain hypersensitivity and cutaneous neurogenic inflammation. Eur J Pharmacol 2011;666:1–4. [4] Fernandes ES, Fernandes MA, Keeble JE. The functions of TRPA1 and TRPV1: moving away from sensory nerves. Br J Pharmacol 2012;166:510–21. [5] Shigetomi E, Tong X, Kwan KY, Corey DP, Khakh BS. TRPA1 channels regulate astrocyte resting calcium and inhibitory synapse efficacy through GAT-3. Nat Neurosci 2011;15:70–80. [6] Moran MM, McAlexander MA, Bíró T, Szallasi A. Transient receptor potential channels as therapeutic targets. Nat Rev Drug Discov 2011;10:601–20. [7] Andrade EL, Meotti FC, Calixto JB. TRPA1 antagonists as potential analgesic drugs. Pharmacol Ther 2012;133:189–204. [8] Nilius B, Appendino G, Owsianik G. The transient receptor potential channel TRPA1: from gene to pathophysiology. Pflugers Arch 2012;464:425–58. [9] Bautista DM, Pellegrino M, Tsunozaki M. TRPA1: a gatekeeper for inflammation. Annu Rev Physiol 2013;75:181–200. [10] del Camino D, Murphy S, Heiry M, Barrett LB, Earley TJ, Cook CA, et al. TRPA1 contributes to cold hypersensitivity. J Neurosci 2010;30:15165–74. REFERENCES 159

[11] Cordero-Morales JF, Gracheva EO, Julius D. Cytoplasmic ankyrin repeats of transient receptor potential A1 (TRPA1) dictate sensitivity to thermal and chemical stimuli. Proc Natl Acad Sci USA 2011;108:E1184–91. [12] Bandell M, Story GM, Hwang SW, Viswanath V, Eid SR, Petrus MJ, et al. Noxious cold ion channel TRPA1 is activated by pungent compounds and bradykinin. Neuron 2004;41:849–57. [13] Kwan KY, Glazer JM, Corey DP, Rice FL, Stucky CL. TRPA1 modulates mechanotransduction in cutaneous sensory neurons. J Neurosci 2009;29:4808–19. [14] Kerstein PC, del Camino D, Moran MM, Stucky CL. Pharmacological blockade of TRPA1 inhibits mechanical firing in nociceptors. Mol Pain 2009;5:19. [15] Treede RD, Meyer RA, Raja SN, Campbell JN. Peripheral and central mechanisms of cutaneous hyperalgesia. Prog Neurobiol 1992;38:397–421. [16] Dunham JP, Kelly S, Donaldson LF. Inflammation reduces mechanical thresholds in a population of transient receptor potential channel A1-expressing nociceptors in the rat. Eur J Neurosci 2008;27:3151–60. [17] Lennertz RC, Kossyreva EA, Smith AK, Stucky CL. TRPA1 mediates mechanical sensitization in nociceptors during inflammation. PLoS ONE 2012;7:e43597. [18] Radtke C, Vogt PM, Devor M, Kocsis JD. Keratinocytes acting on injured afferents induce extreme neuronal hyperexcitability and chronic pain. Pain 2010;148:94–102. [19] Anand U, Otto WR, Facer P, Zebda N, Selmer I, Gunthorpe MJ, et al. TRPA1 receptor localisation in the human peripheral nervous system and functional studies in cultured human and rat sensory neurons. Neurosci Lett 2008;438:221–7. [20] Torebjörk HE, Lundberg LE, LaMotte RH. Central changes in processing of mechanoreceptive input in capsaicin-induced secondary hyperalgesia in humans. J Physiol 1992;448:765–80. [21] Seal RP, Wang X, Guan Y, Raja SN, Woodbury CJ, Basbaum AI, et al. Injury-induced mechanical hypersensitivity requires C-low threshold mechanoreceptors. Nature 2009;462:651–6. [22] Kremeyer B, Lopera F, Cox JJ, Momin A, Rugiero F, Marsh S, et al. A gain-of-function mutation in TRPA1 causes familial episodic pain syndrome. Neuron 2010;66:671–80. [23] Wei H, Chapman H, Saarnilehto M, Kuokkanen K, Koivisto A, Pertovaara A. Roles of cutaneous versus spinal TRPA1 channels in mechanical hypersensitivity in the diabetic or mustard oil-treated non-diabetic rat. Neuropharmacology 2010;58:578–84. [24] Wei H, Koivisto A, Saarnilehto M, Chapman H, Kuokkanen K, Hao B, et al. Spinal transient receptor potential ankyrin 1 channel contributes to central pain hypersensitivity in various pathophysiological conditions in the rat. Pain 2011;152:582–91. [25] Wei H, Koivisto A, Pertovaara A. Spinal TRPA1 ion channels contribute to cutaneous neurogenic inflammation in the rat. Neurosci Lett 2010;479:253–6. [26] Park ES, Gao X, Chung JM, Chung K. Levels of mitochondrial reactive oxygen species increase in rat neuropathic spinal dorsal horn neurons. Neurosci Lett 2006;391:108–11. [27] Kim D, You B, Jo EK, Han SK, Simon MI, Lee SJ. NADPH oxidase 2-derived reactive oxygen species in spinal cord microglia contribute to peripheral nerve injury-induced neuropathic pain. Proc Natl Acad Sci USA 2010;107:14851–6. [28] Andersson DA, Gentry C, Moss S, Bevan S. Transient receptor potential A1 is a sensory receptor for multiple products of oxidative stress. J Neurosci 2008;28:2485–94. [29] Kosugi M, Nakatsuka T, Fujita T, Kuroda Y, Kumamoto E. Activation of TRPA1 channel facilitates excitatory synaptic transmission in substantia gelatinosa neurons of the adult rat spinal cord. J Neurosci 2007;27:4443–51. [30] Spataro LE, Sloane EM, Milligan ED, Wieseler-Frank J, Schoeniger D, Jekich BM, et al. Spinal gap junctions: potential involvement in pain facilitation. J Pain 2004;5:392–405. [31] Shigetomi E, Jackson-Weaver O, Huckstepp RT, O’Dell TJ, Khakh BS. TRPA1 channels are regulators of astrocyte basal calcium levels and long-term potentiation via constitutive d-serine release. J Neurosci 2013;33:10143–53. [32] Liu XG, Sandkühler J. Long-term potentiation of C-fiber-evoked potentials in the rat spinal dorsal horn is prevented by spinal N-methyl-d-aspartic acid receptor blockage. Neurosci Lett 1995;191:43–6. [33] Lautenbacher S, Kundermann B, Krieg JC. Sleep deprivation and pain perception. Sleep Med Rev 2006;10:357–69. [34] Wiertelak EP, Furness LE, Horan R, Martinez J, Maier SF, Watkins LR. Subcutaneous formalin produces cen- trifugal hyperalgesia at a non-injected site via the NMDA-nitric oxide cascade. Brain Res 1994;649:19–26. [35] Zhang GH, Lv MM, Wang S, Chen L, Qian NS, Tang Y, et al. Spinal astrocytic activation is involved in a virally-induced rat model of neuropathic pain. PLoS ONE 2011;6:e23059. 160 9. TRPA1 ANTAGONISTS FOR PAIN RELIEF

[36] Wei H, Hao B, Huang JL, Ma AN, Li XY, Wang YX, et al. Intrathecal administration of a gap junction decoupler, + + − an inhibitor of Na -K -2Cl cotransporter 1, or a GABAA receptor agonist attenuates mechanical pain hypersensitivity induced by REM sleep deprivation in the rat. Pharmacol Biochem Behav 2010;97:377–83. [37] Wei H, Gong N, Huang JL, Fan H, Ma AN, Li XY, et al. Spinal d-amino acid oxidase contributes to mechanical pain hypersensitivity induced by sleep deprivation in the rat. Pharmacol Biochem Behav 2013;111:30–6. [38] Hoeijmakers JG, Faber CG, Lauria G, Merkies IS, Waxman SG. Small-fibre neuropathies—advances in diagnosis, pathophysiology and management. Nat Rev Neurol 2012;8:369–79. [39] Koivisto A, Pertovaara A. Transient receptor potential ankyrin 1 (TRPA1) ion channel in the pathophysiology of peripheral diabetic neuropathy. Scand J Pain 2013;4:129–36. [40] Eberhardt MJ, Filipovic MR, Leffler A, de la Roche J, Kistner K, Fischer MJ, et al. Methylglyoxal activates nociceptors through transient receptor potential channel A1 (TRPA1). A possible mechanism of metabolic neuropathies. J Biol Chem 2012;287:28291–306. [41] Koivisto A, Hukkanen M, Saarnilehto M, Chapman H, Kuokkanen K, Wei H, et al. Inhibiting TRPA1 ion channel reduces loss of cutaneous nerve fiber function in diabetic animals: sustained activation of the TRPA1 channel contributes to the pathogenesis of peripheral diabetic neuropathy. Pharmacol Res 2012;65:149–58. [42] Fernyhough P, Calcutt NA. Abnormal calcium homeostasis in peripheral neuropathies. Cell Calcium 2010;47:130–9. [43] Wei H, Hämäläinen MM, Saarnilehto M, Koivisto A, Pertovaara A. Attenuation of mechanical hypersensitivity by an antagonist of the TRPA1 ion channel in diabetic animals. Anesthesiology 2009;111:147–54. [44] Andersson DA, Gentry C, Light E, Vastani N, Vallortigara J, Bierhaus A, et al. Methylglyoxal evokes pain by stimulating TRPA1. PLoS ONE 2013;8:e77986. [45] Wei H, Viisanen H, Amorim D, Koivisto A, Pertovaara A. Dissociated modulation of conditioned place- preference and mechanical hypersensitivity by a TRPA1 channel antagonist in peripheral neuropathy. Pharmacol Biochem Behav 2013;104:90–6. [46] Obata K, Katsura H, Mizushima T, Yamanaka H, Kobayashi K, Dai Y, et al. TRPA1 induced in sensory neurons contributes to cold hyperalgesia after inflammation and nerve injury. J Clin Invest 2005;115:2393–401. [47] Eid SR, Crown ED, Moore EL, Liang HA, Choong KC, Dima S, et al. HC-030031, a TRPA1 selective antagonist, attenuates inflammatory- and neuropathy-induced mechanical hypersensitivity. Mol Pain 2008;4:48. [48] Katsura H, Obata K, Mizushima T, Yamanaka H, Kobayashi K, Dai Y, et al. Antisense knock down of TRPA1, but not TRPM8, alleviates cold hyperalgesia after spinal nerve ligation in rats. Exp Neurol 2006;200:112–23. [49] Ringkamp M, Meyer RA. Injured versus uninjured afferents: who is to blame for neuropathic pain? Anesthesiology 2005;103:221–3. [50] Nassini R, Gees M, Harrison S, De Siena G, Materazzi S, Moretto N, et al. Oxaliplatin elicits mechanical and cold allodynia in rodents via TRPA1 receptor stimulation. Pain 2011;152:1621–31. [51] McGaraughty S, Chu KL, Perner RJ, Didomenico S, Kort ME, Kym PR. TRPA1 modulation of spontaneous and mechanically evoked firing of spinal neurons in uninjured, osteoarthritic, and inflamed rats. Mol Pain 2010;6:14. [52] Okun A, Liu P, Davis P, Ren J, Remeniuk B, Brion T, et al. Afferent drive elicits ongoing pain in a model of advanced osteoarthritis. Pain 2012;153:924–33. [53] Wei H, Karimaa M, Korjamo T, Koivisto A, Pertovaara A. Transient receptor potential ankyrin 1 ion channel contributes to guarding pain and mechanical hypersensitivity in a rat model of postoperative pain. Anesthesiology 2012;117:137–48. [54] Barabas ME, Stucky CL. TRPV1, but not TRPA1, in primary sensory neurons contributes to cutaneous incision-mediated hypersensitivity. Mol Pain 2013;9:9. [55] Petrus M, Peier AM, Bandell M, Hwang SW, Huynh T, Olney N, et al. A role of TRPA1 in mechanical hyperalgesia is revealed by pharmacological inhibition. Mol Pain 2007;3:40. [56] da Costa DS, Meott FC, Andrade EL, Leal PC, Motta EM, Calixto JB. The involvement of the transient receptor potential A1 (TRPA1) in the maintenance of mechanical and cold hyperalgesia in persistent inflammation. Pain 2010;148:431–7. [57] Fernandes ES, Russell FA, Spina D, McDougall JJ, Graepel R, Gentry C, et al. A distinct role for transient receptor potential ankyrin 1, in addition to transient receptor potential vanilloid 1, in tumor necrosis factor α-induced inflammatory hyperalgesia and Freund’s complete adjuvant-induced monarthritis. Arthritis Rheum 2011;63:819–29. REFERENCES 161

[58] Chiu IM, Heesters BA, Ghasemlou N, Von Hehn CA, Zhao F, Tran J, et al. Bacteria activate sensory neurons that modulate pain and inflammation. Nature 2013;501:52–7. [59] Meseguer V, Alpizar YA, Luis E, Tajada S, Denlinger B, Fajardo O, et al. TRPA1 channels mediate acute neurogenic inflammation and pain produced by bacterial endotoxins. Nat Commun 2014;5:3125. [60] Lassenius MI, Pietiläinen KH, Kaartinen K, Pussinen PJ, Syrjänen J, Forsblom C, et al. Bacterial endotoxin activity in human serum is associated with dyslipidemia, insulin resistance, obesity, and chronic inflammation. Diabetes Care 2011;34:1809–15. [61] Musso G, Gambino R, Cassader M. Interactions between gut microbiota and host metabolism predisposing to obesity and diabetes. Annu Rev Med 2011;62:361–80. [62] Albert HB, Sorensen JS, Christensen BS, Manniche C. Antibiotic treatment in patients with chronic low back pain and vertebral bone edema (Modic type 1 changes): a double-blind randomized clinical controlled trial of efficacy. Eur Spine J 2013;22:697–707. [63] Benemei S, Nicoletti P, Capone JG, De Cesaris F, Geppetti P. Migraine. Handb Exp Pharmacol 2009;194:75–89. [64] Dux M, Sántha P, Jancsó G. Capsaicin-sensitive neurogenic sensory vasodilatation in the dura mater of the rat. J Physiol 2003;552:859–67. [65] Salas MM, Hargreaves KM, Akopian AN. TRPA1-mediated responses in trigeminal sensory neurons: interaction between TRPA1 and TRPV1. Eur J Neurosci 2009;29:1568–78. [66] Romanello S, Spiri D, Marcuzzi E, Zanin A, Boizeau P, Riviere S, et al. Association between childhood migraine and history of infantile colic. JAMA 2013;309:1607–12. [67] Hjerling-Leffler J, Alqatari M, Ernfors P, Koltzenburg M. Emergence of functional sensory subtypes as defined by transient receptor potential channel expression. J Neurosci 2007;27:2435–43. [68] Kunkler PE, Ballard CJ, Oxford GS, Hurley JH. TRPA1 receptors mediate environmental irritant-induced meningeal vasodilatation. Pain 2011;152:38–44. [69] Edelmayer RM, Le LN, Yan J, Wei X, Nassini R, Materazzi S, et al. Activation of TRPA1 on dural afferents: a potential mechanism of headache pain. Pain 2012;153:1949–58. [70] Hadjikhani N, Sanchez Del Rio M, Wu O, Schwartz D, Bakker D, Fischl B, et al. Mechanisms of migraine aura revealed by functional MRI in human visual cortex. Proc Natl Acad Sci USA 2001;98:4687–92. [71] Shatillo A, Koroleva K, Giniatullina R, Naumenko N, Slastnikova AA, Aliev RR, et al. Cortical spreading depression induces oxidative stress in the trigeminal nociceptive system. Neuroscience 2013;253:341–9. [72] Miyamoto T, Dubin AE, Petrus MJ, Patapoutian A. TRPV1 and TRPA1 mediate peripheral nitric oxide-induced nociception in mice. PLoS ONE 2009;4:e7596. [73] Koo B, Becker LE, Chuang S, Merante F, Robinson BH, MacGregor D, et al. Mitochondrial encephalomyop- athy, lactic acidosis, stroke-like episodes (MELAS): clinical, radiological, pathological, and genetic observations. Ann Neurol 1993;34:25–32. [74] de la Roche J, Eberhardt MJ, Klinger AB, Stanslowsky N, Wegner F, Koppert W, et al. The molecular basis for species-specific activation of human TRPA1 protein by protons involves poorly conserved residues within transmembrane domains 5 and 6. J Biol Chem 2013;288:20280–92. [75] Chasman DI, Schürks M, Anttila V, de Vries B, Schminke U, Launer LJ, et al. Genome-wide association study reveals three susceptibility loci for common migraine in the general population. Nat Genet 2011;43:695–8. [76] MuTHER Consortium. Differential methylation of the TRPA1 promoter in pain sensitivity. Nat Commun 2014;5:2978. [77] Schwedt TJ, Krauss MJ, Frey K, Gereau 4th. RW. Episodic and chronic migraineurs are hypersensitive to thermal stimuli between migraine attacks. Cephalalgia 2011;31:6–12. [78] Andersson DA, Gentry C, Alenmyr L, Killander D, Lewis SE, Andersson A, et al. TRPA1 mediates spinal antinociception induced by acetaminophen and the cannabinoid Δ(9)-tetrahydrocannabiorcol. Nat Commun 2011;2:551. [79] Chen J, Lake MR, Sabet RS, Niforatos W, Pratt SD, Cassar SC, et al. Utility of large-scale transiently transfected cells for cell-based high-throughput screens to identify transient receptor potential channel A1 (TRPA1) antagonists. J Biomol Screen 2007;12:61–9. [80] Chen J, Kym PR. TRPA1: the species difference. J Gen Physiol 2009;133:623–5. [81] Klionsky L, Tamir R, Gao B, Wang W, Immke DC, Nishimura N, et al. Species-specific pharmacology of trichloro(sulfanyl)ethyl benzamides as transient receptor potential ankyrin 1 (TRPA1) antagonists. Mol Pain 2007;3:39. 162 9. TRPA1 ANTAGONISTS FOR PAIN RELIEF

[82] McNamara CR, Mandel-Brehm J, Bautista DM, Siemens J, Deranian KL, Zhao M, et al. TRPA1 mediates formalin-induced pain. Proc Natl Acad Sci USA 2007;104:13525–30. [83] Chen J, Joshi SK, DiDomenico S, Perner RJ, Mikusa JP, Gauvin DM, et al. Selective blockade of TRPA1 channel attenuates pathological pain without altering noxious cold sensation or body temperature regulation. Pain 2011;152:1165–72. [84] Koivisto A, Chapman H, Jalava N, Korjamo T, Saarnilehto M, Lindstedt K, et al. TRPA1: a transducer and amplifier of pain and inflammation. Basic Clin Pharmacol Toxicol 2014;114:50–5. [85] Vallin KS, Sterky KJ, Nyman E, Bernström J, From R, Linde C, et al. N-1-Alkyl-2-oxo-2-aryl amides as novel antagonists of the TRPA1 receptor. Bioorg Med Chem Lett 2012;22:5485–92. [86] Hu YJ, St-Onge M, Laliberté S, Vallée F, Jin S, Bedard L, et al. Discovery of a 4-aryloxy-1H-pyrrolo[3,2-c]pyridine and a 1-aryloxyisoquinoline series of TRPA1 antagonists. Bioorg Med Chem Lett 2014;24:3199–203. [87] Laliberté S, Vallée F, Fournier PA, Bedard L, Labrecque J, Albert JS. Discovery of a series of aryl-N-(3-(alkylamino)-5(trifluoromethyl)phenyl)benzamides as TRPA1 antagonists. Bioorg Med Chem Lett 2014;24:3204–6. [88] Copeland KW, Boezio AA, Cheung E, Lee J, Olivieri P, Schenkel LB, et al. Development of novel azabenzofuran TRPA1 antagonists as in vivo tools. Bioorg Med Chem Lett 2014;24:3464–8. http://dx.doi.org/10.1016/j. bmcl.2014.05.069. [89] Rooney L, Vidal A, D’Souza AM, Devereux N, Masick B, Boissel V, et al. Discovery, optimization, and biological evaluation of 5-(2-(trifluoromethyl)phenyl)-indazoles as a novel class of transient receptor potential A1 (TRPA1) antagonists. J Med Chem 2014;57:5129–40. http://dx.doi.org/10.1021/jm401986p. [90] Finnerup NB, Sindrup SH, Jensen TS. The evidence for pharmacological treatment of neuropathic pain. Pain 2010;150:573–81. [91] Borsook D, Kalso E. Transforming pain medicine: adapting to science and society. Eur J Pain 2013;17:1109–25. [92] Moore RA, Straube S, Eccleston C, Derry S, Aldington D, Wiffen P, et al. Estimate at your peril: imputation methods for patient withdrawal can bias efficacy outcomes in chronic pain trials using responder analyses. Pain 2012;153:265–8. [93] Morgan P, Van Der Graaf PH, Arrowsmith J, Feltner DE, Drummond KS, Wegner CD, et al. Can the flow of medicines be improved? Fundamental pharmacokinetic and pharmacological principles toward improving Phase II survival. Drug Discov Today 2012;17:419–24. [94] Gregus AM, Doolen S, Dumlao DS, Buczynski MW, Takasusuki T, Fitzsimmons BL, et al. Spinal 12-lipoxygenase-derived hepoxilin A3 contributes to inflammatory hyperalgesia via activation of TRPV1 and TRPA1 receptors. Proc Natl Acad Sci USA 2012;109:6721–6. [95] Sisignano M, Park CK, Angioni C, Zhang DD, von Hehn C, Cobos EJ, et al. 5,6-EET is released upon neuronal activity and induces mechanical pain hypersensitivity via TRPA1 on central afferent terminals. J Neurosci 2012;32:6364–72. [96] Fleming T, Cuny J, Nawroth G, Djuric Z, Humpert PM, Zeier M, et al. Is diabetes an acquired disorder of reactive glucose metabolites and their intermediates? Diabetologia 2012;55:1151–5.

[97] Bierhaus A, Fleming T, Stoyanov S, Leffler A, Babes A, Neacsu C, et al. Methylglyoxal modification of Nav1.8 facilitates nociceptive neuron firing and causes hyperalgesia in diabetic neuropathy. Nat Med 2012;18:926–33. [98] Wang T, Douglass Jr EF, Fitzgerald KJ, Spiegel DA. A “turn-on” fluorescent sensor for methylglyoxal. J Am Chem Soc 2013;135:12429–33. [99] Dyck PJ, Herrmann DN, Staff NP, Dyck PJ. Assessing decreased sensation and increased sensory phenomena in diabetic polyneuropathies. Diabetes 2013;62:3677–86. [100] Polydefkis M, Griffin JW, McArthur J. New insights into diabetic polyneuropathy. JAMA 2003;290:1371–6. [101] Tavakoli M, Petropoulos IN, Malik RA. Corneal confocal microscopy to assess diabetic neuropathy: an eye on the foot. J Diabetes Sci Technol 2013;7:1179–89. [102] Sivaskandarajah GA, Halpern EM, Lovblom LE, Weisman A, Orlov S, Bril V, et al. Structure-function relationship between corneal nerves and conventional small-fiber tests in type 1 diabetes. Diabetes Care 2013;36:2748–55. [103] Petropoulos IN, Alam U, Fadavi H, Marshall A, Asghar O, Dabbah MA, et al. Rapid automated diagnosis of diabetic peripheral neuropathy with in vivo corneal confocal microscopy. Invest Ophthalmol Vis Sci 2014;55:2071–8. CHAPTER 10 TRPA1 Antagonists as Potential Therapeutics for Respiratory Diseases Jessica Tan,1,2 Gerald Hunsberger,2 Christopher Neipp,3 M. Allen McAlexander2,* 1Department of Biology, College of Science and Technology, Temple University, Philadelphia, Pennsylvania, USA 2Neuronal Targets Team, Respiratory Therapy Area, GlaxoSmithKline Pharmaceuticals, King of Prussia, Pennsylvania, USA 3Flexible Discovery Unit, GlaxoSmithKline Pharmaceuticals, King of Prussia, Pennsylvania, USA *Corresponding author: [email protected]

OUTLINE

TRPA1 as a Reactive, Noxious Irritant Ozone 170 Sensor 167 Isocyanates: Prototypical Occupational Hazards 171 Presence and Function of TRPA1 in TRPA1 Is the Primary Effector of the Mammalian Respiratory Tract 168 Respiratory Irritation Caused by Exogenous Agonists that Cause Multiple Classes of TRPA1 Activation in the Airways 168 Anesthetics 171 Non-Aldehyde TRPA1 Agonists Found Endogenous Molecules that Signal in Cigarette Smoke 169 Through TRPA1 in Mammals 172 Nicotine 169 Oxidative Tissue Damage and Its Cadmium 170 By-Products Activate TRPA1 172 Lipopolysaccharide 170 TRPA1 Is a Target of Cyclopentenone Prostaglandins Other Hazardous Air Pollutants that and Isoprostane 173 Activate TRPA1 170

TRPA1 Amplifies Neuronal Signals In Vivo Preclinical Pharmacology of Caused by Many Soluble TRPA1 Blockers 176 Inflammatory Mediators 174 TRPA1 Antagonists Claimed in Nonneuronal TRPA1 Expression the Patent Literature 177 and Function 175 Conclusions 187 TRPA1 and Small Cell Lung Carcinoma 176 References 188

The mammalian lung houses a heterogeneous assembly of cell types that works in concert to ensure proper oxygenation of blood and removal of the metabolic waste product CO2. Not surprisingly, inhaled foreign materials and the host’s response to them can impair this vital process. Threats to this exquisite system can be broadly classified into two categories: (1) gradual declines in the lung’s capacity to conduct gas exchange caused by inflammation, oxidative stress and aberrant tissue remodeling, and (2) acute chemical, physical, or other environmental threats to gas exchange that are detected by sensory nerves in the respiratory tract (Figure 10.1), triggering expulsion/dilution reflexes that may include sneeze, cough, bronchospasm, and airway secretions. These two categories are by no means mutually exclusive; by contrast, they are intimately linked, as inflamed and pathologically remodeled airways contain mediators that sensitize neuronal reflex arcs. This continued cycle can progress to the point where expulsion/dilution reflexes including airway fluid secretion and bronchospasm become exaggerated to the extent that they themselves now potentially become acute threats to gas exchange. One of the great challenges of treating respiratory ailments is finding points to intervene that will disrupt the vicious cycle of insult—pathological response—increased sensitivity to insult. The difficulty of this challenge is underscored by the observation that bronchospasm itself is sufficient to elevate indices of pathological airway remodeling in asthmatic airways [1]. The “pathological response” portion of this cycle is diverse and may feature aberrant remodeling including hyperplasia of resident cells such as airway smooth muscle and cells that produce mucus, high levels of oxidative stress, and nonresolving inflammation that may be driven by the innate and/or adaptive immune system [2]. Advances in fields including biochemistry and molecular biology have enabled rapid and pronounced advances in the identification and measurement of biomarkers associated with disease outcomes, and significant progress has been made in the understanding of this aspect of airway pathophysiology. Despite this impressive body of work, which has led to the discovery and development of multiple experimental therapeutics designed to ameliorate aspects of airway inflammation and/or pathological remodeling, we still do not have satisfactory explanations for what causes the sudden and often violent episodes of intermittent airflow obstruction in asthmatics or others with reactive airways. Atopy (allergy) is, without question, the single most important risk factor for asthma as there is a clear link and considerable overlap between the two phenomena. In fact, it could be argued that it is precisely the repeated validation and robustness of the link between these 10. TRPA1 ANTAGONISTS AS POTENTIAL THERAPEUTICS 165

Trigeminal nucleus (sensory)

Pons Nucleus tractussolitarius Nucleus ambiguus Trigeminal ganglion Spinal tract of trigeminal nerve Jugular ganglion

Nodose ganglion

Upper medulla

FIGURE 10.1 Sensory innervation of the respiratory tract. Sensory nerves that detect mechanical, thermal, and chemical stimuli in the nasal mucosa are carried by the ethmoidal branch of the trigeminal nerve (cranial nerve V) and synapse in the trigeminal nuclei (trigeminal nucleus principalis, oralis, or caudalis) within the brainstem. Sensory fibers that serve similar functions in the larynx, cartilaginous airways and lung parenchyma are carried within the vagus nerve (cranial nerve X), have cell bodies in either the inferior vagal (nodose) or superior vagal (jugular) ganglia, and synapse within the nucleus tractus solitarius. Inputs conveyed through this network regulate both sensations (e.g., urge to cough and dyspnea, or “air hunger”) and autonomic outflow, which profoundly influences airway secretions and caliber.

two phenomena that has limited investigations into other factors contributing to the asthmatic phenotype. This is not a trivial deficit, as many clinical observations should lead one to believe that allergic airway inflammation is not the sine qua non of asthma. Importantly, asthma and atopy frequently occur independent of one another in humans, and the population-attributable risk of asthma using either skin test sensitivity or total serum IgE as an index of atopy often does not exceed 50% [3]. Furthermore, hazardous air pollutants such as ozone [4] and sulfur dioxide [5] must be considered as agents capable of causing asthma-like symptoms, and the response to these irritants does not categorically involve mast 166 10. TRPA1 ANTAGONISTS AS POTENTIAL THERAPEUTICS cells [6], an observation consistent with the fact that many asthmatics cite their most frequent triggers as things that do not seem to readily involve allergic reactions [7]. As one might predict, asthmatics exhibit an exaggerated response to inhaled hazardous air pollutants; therefore, environmental and occupational irritants may both overtly trigger and increase the sensitivity of noxious respiratory sensations and respiratory expulsion/dilution reflexes. If in fact this increased irritant sensitivity is an aspect of airway pathophysiology that is separate from but partially overlapping with things such as responses to infectious pathogens and allergens, it stands to reason that airway irritability and the agents that increase airway irritability should be the subject of intense investigation. A large variety of chemicals cause respiratory irritation of some manner in both experimental animals and human subjects. Whereas the chemicals within this list are heterogeneous with regard to many chemical and physical properties, a considerable number of them share one distinctive property: chemical reactivity. This commonality can be readily observed when reading tables such as the one in Ref. [8], which provides a list of hazardous air pollutants thought to cause and/or exacerbate asthma in humans. Many hazardous irritants on this list are α,β- unsaturated carbonyls such as acrolein that can form adducts with electron-rich (nucleophilic) molecules including cysteines, histidines, and lysines within proteins. In many cases, these Michael addition reactions are irreversible under physiological conditions, creating adducted proteins that change conformation enough to exhibit altered activity or, eventually, cease to function. In addition, reactive molecules may also form adducts with and deplete cellular reduc- tants such as glutathione, causing an oxidant burden in cells. Although modern investigators have developed a sophisticated understanding of the chemical biology of oxidation-reduction reactions, the question of how irritant exposures relate to the fixed and gradual declines in lung function, as opposed to the sudden and potentially life-threatening episodes of airflow obstruction that can occur during a disease exacerbation, remains largely unanswered. Finally, and perhaps most important from a translational perspective, is the question of whether reactive irritants are broadly and nonspecifically toxic, or whether interrupting downstream biological effectors will ameliorate the damage caused by these hazardous reactive irritants. This question has been investigated in laboratory animals, where invasive experiments can yield mechanistic insight beyond what is readily achievable in humans. As one might expect, inhaled reactive irritants cause oxidative stress in airway cells [9], as well as inflammation and tissue damage [10]. One consistent theme that has emerged, however, is that in mammalian laboratory animal species, capsaicin-sensitive sensory neurons in the airways act as both the primary sensors of inhaled irritants and as the principal effector mechanism that serves to dilute and/or expel the irritant on acute exposure. These experiments are often performed by injecting rodents with high concentrations of capsaicin or resiniferatoxin (RTX), both of which selectively disable sensory neurons containing their receptor by triggering Ca2+ influx sufficient to cause persistent desensitization up to and including destruction of nerve terminals. This method has proven effective for years as a way to disable irritant-sensing nerves in the respiratory tract, as it largely abolishes neuronal responses to a diverse collection of respiratory irritants. Intranasal capsaicin desensitization protocols have also provided marked symptom relief in rhinitis patients, yielding strong evidence that similar mechanisms are also present in humans, at least within the nasal mucosa [11]. Although this technique pinpointed a subpopulation of irritant-sensing nerves as vital components of the response to noxious inhaled materials, it did not have the power to discriminate the molecular sensor(s) responsible for these effects. TRPA1 AS A REACTIVE, NOXIOUS IRRITANT SENSOR 167

In 1997, the seminal work of Caterina et al. [12] identified the ion channel then dubbed “VR1” (vanilloid receptor 1; later renamed transient receptor potential vanilloid 1, or TRPV1) as the receptor for capsaicin. This allowed for detailed mechanistic investigations of the heterologously expressed gene product and its pharmacology. Although these studies confirmed that TRPV1/VR1 was the sole sensor of both capsaicin and RTX, the TRPV1 channel failed to account for the broad irritant-sensing capacity of capsaicin-sensitive nerves, as many chemicals that triggered nocifensive reflexes through capsaicin-sensitive nerves did not appear to activate TRPV1 (see, for example, Ref. [13]). A reasonable conclusion that could be reached from this body of work was that although TRPV1 could serve as a critical gateway to introduce cytotoxic levels of calcium into these sensory nerves, it did not serve as the final common sensor of noxious respiratory irritants. This would mean that at least one other toxin sensor must exist within these nerves.

TRPA1 AS A REACTIVE, NOXIOUS IRRITANT SENSOR

TRPA1, originally named ANKTM1 due to its unusually large number (14+) of N-terminal ankyrin repeats, was cloned from lung fibroblasts [14], although its function was not examined until Story et al. [15] reported that it was an ion channel primarily expressed in capsaicin-sensitive sensory neurons that responded to cold temperatures in the noxious range. TRPA1 consists of a putative six-transmembrane (6TM) subunit that assembles as a te- tramer to form cation-permeable pores, with a putative pore-forming helix between TM5 and TM6. Further studies of the TRPA1 channel have consistently observed that its expression in naïve animals is predominantly limited to capsaicin-sensitive, TRPV1-expressing nociceptive neurons with cell bodies in the trigeminal, dorsal root, and vagal ganglia [16,17]. Following the characterization of TRPA1 as a nociceptor-enriched ion channel, multiple groups began observing its activation by a diverse array of naturally occurring pungent and algogenic compounds, including isothiocyanates such as allyl isothiocyanate (AITC) from mustard oil, cinnamaldehyde, and bradykinin (BK) (indirectly, via its B2 receptor) [16,18]. Numerous other chemicals that cause noxious sensations in humans were soon revealed to be TRPA1 agonists. This collection of chemicals was surprisingly large and structurally dissimi- lar. The puzzle of how TRPA1 could serve as such a promiscuous chemosensor was solved in two elegant studies that revealed that chemical reactivity was the property shared by many of these structurally diverse TRPA1 agonists [19,20]. These studies also identified specific cysteine (and, in the case of human TRPA1, a lysine) residues within the cytosolic N-terminus of TRPA1 that, when mutated, selectively inhibited channel gating by reactive stimuli. In aggregate, these early studies showed that TRPA1 was expressed in TRPV1-containing neurons that are the key sensors of chemical nociception and that its unusual ability to convert covalent modification of its intracellular residues into channel gating positioned it as a sensor of an unprecedented range of noxious stimuli. This set of properties made TRPA1 the leading candidate for the missing link between reactive chemicals and capsaicin-sensitive nerve activation. The generation of mice with genetic deletion of TRPA1 was the next major advance in TRPA1 biology. Two independent groups produced these mice using distinct targeting strategies, with results that were only subtly different. Both groups showed that Trpa1−/− mice demonstrated reduced avoidance or pain behaviors to AITC and reduced hyperalgesia 168 10. TRPA1 ANTAGONISTS AS POTENTIAL THERAPEUTICS

following intraplantar BK injections [21,22]. Intriguingly, Bautista et al. extended these findings by discovering that TRPA1 serves as the primary neuronal sensor of acrolein. This revealed the first mechanism via which a mediator of oxidative tissue damage could directly activate a transducer molecule within a nociceptor. This paper was also the first to generate interest in TRPA1 as a therapeutic target for respiratory disease because inhaled acrolein can cause violent respiratory irritation in humans [23], and it is also a hazardous air pollutant generated by multiple sources including auto emissions and cigarette smoke [24].

PRESENCE AND FUNCTION OF TRPA1 IN THE MAMMALIAN RESPIRATORY TRACT

Following the excitement generated by the results of Bautista and colleagues, other studies examining TRPA1’s role in respiratory irritant sensing soon followed. Nassenstein et al. [25] used single-cell RT-PCR to demonstrate that TRPA1 mRNA was present in TRPV1- containing vagal neurons that project to the airways of mice. Moreover, they demonstrated that the TRPA1 agonist cinnamaldehyde activated capsaicin-sensitive vagal neurons, caused pulmonary C-fiber discharge, and triggered respiratory irritation reflexes in mice. These experiments provided the first evidence of TRPA1 expression and function in the mammalian respiratory tract, but the authors did not use genetic or pharmacological interventions to confirm the role of TRPA1 in their mouse behavioral studies. Soon after, Bessac et al. employed Trpa1−/− mice to provide the first genetic evidence that TRPA1 was necessary for the sensory neuronal activation and respiratory irritation reflexes triggered by pro-oxidant mediators including HOCl [26]. These findings showed that TRPA1 activation was sufficient to trigger nocifensive respiratory reflexes in mammals and that multiple corrosive agents thought to activate nerves via nonspecific tissue damage act primarily through TRPA1. As alluded to earlier, multiple irritants trigger respiratory reflexes in laboratory animals via activation of capsaicin-sensitive nerves. Cigarette smoke, a causative factor in numerous cardiopulmonary diseases including COPD, is no exception. Lundberg and Saria [27] initially demonstrated that capsaicin desensitization prevents cigarette smoke-evoked plasma extravasation in rat airways. Andre et al. [28] were able to show that sensory nerve activation and neurogenic inflammatory responses caused by cigarette smoke in mouse and guinea pig are TRPA1-dependent and that α,β-unsaturated aldehydes such as acrolein and crotonaldehyde are the primary constituents of cigarette smoke that trigger these responses. These data proved that acute neuronal responses to cigarette smoke in mice and guinea pigs are mediated by TRPA1 and suggest that further investigations into the role of TRPA1 in chronic cigarette smoking-related illness are merited.

EXOGENOUS AGONISTS THAT CAUSE TRPA1 ACTIVATION IN THE AIRWAYS

Reactive xenobiotics beyond those found in cigarette smoke can act through TRPA1 to cause nerve activation. Acetaminophen/paracetamol is known to be hepatotoxic at high doses primarily due to the reactive metabolite N-acetyl-p-benzo-quinoneimine (NAPQI), although NON-ALDEHYDE TRPA1 AGONISTS FOUND IN CIGARETTE SMOKE 169 adverse effects of doses closer to therapeutic levels have not been widely explored. Nassini et al. [29] observed that dosing mice with acetaminophen (15-300 mg/kg) generates detectable levels of glutathione-conjugated NAPQI and that exogenously administered NAPQI causes neurogenic inflammatory responses in rodent lung and skin. Birrell et al. [30] first demonstrated that acrolein depolarizes vagus nerves of mouse, guinea pig, and human. The effect is abolished in tissue from TRPA1 knockout animals or by treatment with the TRPA1 antagonists HC-030031 or AP-18. Acute inhalational exposure to acrolein in rodents results in multiple respiratory reflexes, as well as delayed pulmonary neutrophilia and heightened reflex sensitivity [24,31]. Conscious guinea pigs cough following acrolein exposure, a reflex inhibited by the TRPA1 antagonist HC-030031 [30,32]. Importantly, although human studies such as those conducted by Sim and Pattle [23] would never earn approval from modern IRBs, the fact that their observations of the intensity of irritation caused by inhaled aldehydes in humans correlate strongly with the potency of those aldehydes at recombinant human TRPA1 is striking, as it ar- gues strongly, albeit indirectly, that TRPA1 is the primary human sensor of respiratory irritation caused by toxic aldehydes. Birrell et al. [30] were able to demonstrate translational value of their findings in a more ethically acceptable protocol in human subjects, where they showed that the TRPA1 agonist cinnamaldehyde (3-phenyl-acrolein) causes coughing in both humans and guinea pigs in a similar manner. Others have observed cough in guinea pigs caused by TRPA1 agonists [32,33], although TRPV1 may influence these responses under certain conditions [33].

NON-ALDEHYDE TRPA1 AGONISTS FOUND IN CIGARETTE SMOKE

Cigarette smoke contains many other toxins besides α,β-unsaturated aldehydes such as acrolein and crotonaldehyde, and it stands to reason that many of these toxins may activate TRPA1. Formaldehyde is one such chemical, although it is also an occupational and environmental respiratory hazard. Acute formaldehyde exposure causes ocular and respiratory irritation in humans [23], and sufficiently high exposures may cause chronic respiratory symptoms. In rodents, injections of formalin solutions that contain formaldehyde as the active ingredient have also been used in the study of pain for decades, and TRPA1 gene deletion or pharmacological inhibition yielded full efficacy in these models [34,35]. Based on these results, which also identified TRPA1 as the primary formaldehyde sensor in mouse sensory neurons, we speculated that TRPA1 would also serve as the principal effector of acute respiratory irritation caused by formalin, and this was verified when TRPA1-deficient mice did not respond measurably to formalin exposures that triggered robust respiratory irritation reflexes in wild-type (WT) mice [36]. This pathway may also be relevant in chronic exposure scenarios because the TRPA1 antagonist HC-030031 reduced airway inflammation and hyperresponsiveness in a model where BALB/c mice were exposed to both allergen and formaldehyde [37].

Nicotine In human subjects, nicotine, the primary addictive principle in tobacco, causes irritation and CNS reflex-mediated secretions when applied to the nasal mucosa [38] and retrosternal pain when inhaled [39]. Talavera et al. demonstrated that nicotine activates TRPA1 channels 170 10. TRPA1 ANTAGONISTS AS POTENTIAL THERAPEUTICS and that intranasal nicotine instillation causes only minor changes in the “enhanced pause” airflow resistance-related plethysmography value in freely behaving Trpa1−/− mice compared to WTs [40]. The ability of nicotine to cause respiratory irritation is likely multifactorial, as nicotinic receptors are also present on sensory neurons [40–42], and in humans, the retrosternal pain caused by inhaled nicotine is inhibited by the nicotinic receptor antagonist hexa- methonium [39]. Thus, TRPA1 may be one of several mechanisms through which nicotine evokes respiratory irritation.

Cadmium The heavy metal cadmium is in group 12 of the periodic table along with zinc and mercury. The identification of zinc as a TRPA1 agonist drew considerably more attention initially [43,44], although cadmium may arguably have comparable relevance in the respiratory tract due to its presence in tobacco. Indeed, as one might predict, because cadmium and zinc have similar chemistry as reflected by the fact that they belong to the same periodic table group, cadmium and zinc both cause comparable activation of TRPA1 [43], and cadmium causes pain behaviors [45] and airway sensory fiber activation [46] that are predominantly mediated by TRPA1 in acute experimental settings. Future results demonstrating how TRPA1 influences the overall toxicity profile of cadmium and whether mercury, one of the best-characterized toxins, exerts its toxicity in part through TRPA1 activation will be eagerly awaited.

Lipopolysaccharide Lipopolysaccharide (LPS) is a constituent of Gram negative bacteria that can trigger immune responses via the toll-like receptor 4 (TLR4) [47]. Recently, Meseguer et al. [48] demonstrated that LPS can activate a subset of mouse sensory neurons in vitro. Strikingly, these responses did not require TLR4, although they were abrogated by TRPA1 gene deletion. LPS activation of TRPA1 is also relevant in vivo, as TRPA1 was vital for the pain behaviors and acute tissue vasodilation/plasma extravasation reactions caused by LPS injections. These studies raise the intriguing possibility that TRPA1 may be a TLR4-independent contributor to the net circulatory dysfunction and organ failure caused by sepsis. Further investigations that will prove or disprove this hypothesis, and determine what role TRPA1 plays in the lung damage caused by this condition, are eagerly awaited.

OTHER HAZARDOUS AIR POLLUTANTS THAT ACTIVATE TRPA1

Ozone Ozone is a potent oxidant and an air pollutant that can impair lung function. Occupational exposures during the 1950s first illustrated the effects of ozone on pulmonary function, and studies have since shown that acute ozone inhalation results in noxious respiratory sensations and decrements in lung function, with these responses being at least partially sensitive to local anesthetic inhalation [4]. Ozone can cause rapid, shallow breathing that is blocked by Other Hazardous Air Pollutants that Activate TRPA1 171 inhibiting vagus nerve conduction [49], although the mechanism(s) by which ozone triggered vagal reflexes remained unclear. Recently, Taylor-Clark and Undem [50] demonstrated that the action of ozone on mouse pulmonary C-fibers is ruthenium red-sensitive and that ozone activation of mouse vagal neurons is mediated by TRPA1

Isocyanates: Prototypical Occupational Hazards Because AITC is a prototypical TRPA1 agonist, it was conceivable that isocyanates such as those used in the manufacture of polyurethane-containing products might also activate TRPA1. If so, this would be a significant discovery because isocyanate exposure can cause chronic airways disease in exposed workers [51], and this likely occurs via multiple mechanisms that are not readily clear [52]. Prior evidence from laboratory animal studies suggested that toluene diisocyanate (TDI), similar to other hazardous inhaled irritants, could cause respiratory responses such as sneezing and airway smooth muscle contraction that were inhibited by capsaicin desensitization [53,54]. These findings are consistent with the hypothesis that TRPA1 mediates the acute nerve activation and respiratory irritation reflexes caused by isocyanates, and indeed, TDI activates heterologously overexpressed human TRPA1 and causes calcium flux in sensory neurons from WT but not Trpa1−/− mice. Moreover, the respiratory irritation reflexes caused by acute intranasal TDI exposure were also dependent on the presence of TRPA1 [36]. Independently, Bessac et al. [55] investigated the effects of methyl isocyanate, the molecule released during the Bhopal disaster, and came to similar conclusions. Their studies showed that methyl isocyanate activates heterologously overexpressed TRPA1 in excised patch clamp recordings and causes TRPA1-dependent calcium flux in mouse sensory neurons. They also showed that either TRPA1 gene deletion or antagonism with HC-030031 markedly reduced irritation behaviors caused by methyl isocyanate. Taken together, these two studies employed multiple complementary approaches to prove that TRPA1 is the primary target and acute in vivo effector of multiple reactive isocyanates, molecules that have posed considerable occupational hazards in manufacturing workplaces.

TRPA1 Is the Primary Effector of Respiratory Irritation Caused by Multiple Classes of Anesthetics Volatile gas anesthetics such as desflurane and isoflurane can provoke effects in humans ranging from coughing to laryngospasm. The reasons for these adverse effects have been unclear. One clue to how volatile gas anesthetics cause airway irritation in humans comes from the observation that sevoflurane does not cause these responses to the same extent that isoflurane and desflurane do [56]. Based on this information, it follows that the mechanism by which gas anesthetics cause airway irritation in humans must be present in healthy individuals and engaged less robustly by sevoflurane than other gas anesthetics. Until the last several years, the only mechanistic evidence that animal studies added to this question was that gas anesthetics activate airway vagal capsaicin-sensitive C fibers and cause respiratory reflexes and that sevoflurane is the least potent anesthetic studied in this regard [57]. Thus, the transducer(s) of gas anesthetic reflex action must be located on airway nociceptive vagal sensory fibers. The search for specific 172 10. TRPA1 ANTAGONISTS AS POTENTIAL THERAPEUTICS molecular entities that could couple sensing of gas anesthetics to noxious respiratory sensations continued until the breakthrough study by Matta et al. [58], who discovered that TRPA1 is activated by multiple gas anesthetics, but not sevoflurane. Although other mechanisms may influence the respiratory effects of gas anesthetics (including potentiation of the capsaicin sensor TRPV1 [59]), the airway resistance increases caused by desflurane can be prevented by pretreating guinea pigs with the TRPA1 antagonist HC-030031 [60], and neuronal activity in the nucleus tractus solitarius (NTS, the brainstem site where many vagal afferent fibers synapse) triggered by laryngeal exposure to desflurane was inhibited by HC-030031 [61]. Volatile gas anesthetics are not the only class of anesthetic capable of activating TRPA1, as etomidate and propofol activate heterologously expressed hTRPA1 channels and native channels in mouse sensory neurons [58]. Voltage-gated sodium channel-blocking local anesthetics including lidocaine also activate TRPA1 [62], as well as TRPV1 [63,64]. Curiously, acute lidocaine inhalation causes a roughly threefold rightward shift in asthmatic airway reactivity to histamine, even though lidocaine itself reduces airflow in asthmatics [65]. One potential explanation for this apparent paradox that is only speculative at the moment is that lidocaine inhibits histamine reactivity by blocking voltage-gated sodium channels in sensory and/or parasympathetic nerves that may be involved in a CNS reflex-dependent component of the histamine response, whereas the asthmatic airway environment may lead to increased expression and/ or gating of TRPA1 and TRPV1 channels on airway sensory nerves such that lidocaine may activate them to an extent sufficient to initiate CNS-dependent parasympathetic reflex airflow obstruction.

ENDOGENOUS MOLECULES THAT SIGNAL THROUGH TRPA1 IN MAMMALS

Oxidative Tissue Damage and Its By-Products Activate TRPA1 Oxidative stress, a condition in which reactive oxygen species (ROS) overwhelm cellular reductant capacity to threaten tissue homeostasis, can be detected in most if not all pathological conditions, including diseases of the respiratory tract. When ROS react with unsaturated fatty acids, they generate lipid peroxidation products such as α,β-unsaturated carbonyls that can covalent modify proteins via Michael addition reactions with thiols. These products of lipid peroxidation include 4-hydroxy-2-hexenal, 4-hydroxy-2-nonenal (4-HNE), and 4-oxo- 2-nonenal (4-ONE) [66], alkenal lipid peroxidation products that possess chemical reactivity similar to acrolein. Indeed, 4-HNE activates heterologously overexpressed human TRPA1 and causes TRPA1-dependent Ca2+ mobilization in mouse sensory neurons, as well as TRPA1- dependent nocifensive behavior when administered acutely [67]. Unlike the majority of other tissues in the body, the respiratory tract encounters atmospheric oxygen and, as such, the oxidation-reduction balance/redox environment is different [68]. When oxygen tension varies markedly from atmospheric levels, by being either reduced (hypoxia) or elevated (hyperoxia), this redox environment becomes perturbed, triggering oxidative stress. Takahashi et al. [69] demonstrated that both hypoxic and hyperoxic conditions can activate TRPA1 through cysteine oxidation as well as through relief of prolyl hydroxylase-mediated inhibition. Endogenous Molecules that Signal Through TRPA1 in Mammals 173

Taylor-Clark et al. [70] also provided insight into how the unique redox environment of the respiratory tract could influence TRPA1 activity when they observed relatively modest mouse pulmonary C-fiber activation and neuropeptide-dependent guinea pig airway constriction in response to 4-HNE, although 4-HNE showed in vitro potency comparable to values obtained by others. Equal concentrations of the more-reactive 4-ONE elicited guinea pig airway constriction and vigorous mouse pulmonary C-fiber activation. The airway constriction caused by 4-ONE was abolished by capsaicin desensitization, blockade of tachykinin neurokinin 1 and 2 receptors, or by preincubating 4-ONE with glutathione to neutralize its reactive capacity. The pulmonary C-fiber activation was abolished by TRPA1 knockout, although TRPV1 also factored into the in vitro activation of mouse neurons caused by 4-ONE. In aggregate, these results demonstrate that lipid peroxidation products such as 4-ONE can activate capsaicin-sensitive sensory nerves in guinea pig airway tissue to elicit axon reflex, neuropeptide-dependent airway constriction. Moreover, these results highlight the possibility that the redox balance of the tissue microenvironment where alkenals such as 4-ONE are generated exerts a profound influence on nerve activation by this and related classes of chemically reactive mediators. Further emphasizing this concept, 9-nitro-oleic acid (9-OA-NO2), a product of the nitration reaction that occurs when reactive nitrogen species covalently modify nucleophiles, causes similar effects as 4-ONE, leading to robust TRPA1 activation and TRPA1- dependent mouse pulmonary C-fiber activation [71]. These findings underscore the role of TRPA1 as a potential integrator of multiple pathways linking oxidative stress and inflammation to noxious sensations and reflex hypersensitivity. Mitochondria provide the vast majority of energy in healthy cells via oxidative metabolism of acetyl CoA. When this process works efficiently, oxygen is reduced to water; however, when mitochondria are damaged and/or their rate of respiration is markedly altered, ROS including superoxide are generated at levels that can lead to cellular damage. Nesuashvili et al. [72] hypothesized that the mitochondrial complex III inhibitor antimycin A, which generates endogenously-produced ROS, could activate TRPA1, thereby creating the first direct link between dysfunctional mitochondria and TRPA1 activity. Their hypothesis proved correct, as antimycin A activated recombinant TRPA1 and caused mouse pulmonary nociceptor discharge predominantly through TRPA1. Although the specific products involved in this process were not identified, the effects of antimycin A were inhibited by multiple reagents that interfere with ROS production. These experiments provide very strong evidence that antimycin A generates mitochondrial ROS production that leads to TRPA1 activation.

TRPA1 Is a Target of Cyclopentenone Prostaglandins and Isoprostane Prostaglandins are lipid mediators generated by the metabolism of arachidonic acid by cyclo-oxygenase (COX) enzymes that are generally pro-inflammatory, as well as pro-algesic due to their ability to sensitize nerves including nociceptors [73]. Prostaglandin E2 (PGE2) is one of the most potent known pro-algesic lipid mediators, and it is produced in high levels by the COX-2 enzyme during inflammatory conditions. Prostaglandin D2 (PGD2) is a related lipid mediator produced by mast cells during conditions such as allergic inflammation [74]. The profound effects of both these mediators on sensory neurons have long been presumed to be entirely through their cognate G protein-coupled receptors (GPCRs), a notion supported by the fact that their acute effects are mediated by GPCRs [75]. Over time, however, 174 10. TRPA1 ANTAGONISTS AS POTENTIAL THERAPEUTICS

PGD2 and PGE2 are nonenzymatically degraded into prostaglandin A2 and 15 deoxyΔ12,14 prostaglandin J2, respectively. Neither of these mediators signals through any known prostaglandin GPCR; however, both molecules contain chemically reactive electron-deficient α,β-unsaturated carbonyls within their cyclopentenone rings. These molecules have numerous biological targets, but when Taylor-Clark et al. [76] revealed TRPA1 as a target of reactive cyclopentenone ring-containing prostaglandins, this provided the first mechanism through which COX products could activate nociceptive sensory neurons in a GPCR-independent manner. Nerve activity triggered by 15 deoxyΔ12,14 prostaglandin J2 is sufficient to generate nocifensive reflexes in vivo, as intraplantar injection causes pain behaviors that are absent in TRPA1 knockout mice [77], and intranasal instillation causes respiratory irritation reflexes (our unpublished observations). Together, these observations raise the intriguing possibility that chronic elevation of COX activity in vivo may raise concentrations of PGD2 and PGE2 to levels sufficient to cause a net accumulation of prostaglandin A2 and 15 deoxyΔ12,14 prostaglandin J2, and that activation of TRPA1 by these molecules may be a major effector mechanism of long-term, slowly reversible sensory neuronal hypersensitivity in disease states.

TRPA1 is also activated by 8-iso-PGA2, [76] an isoprostane produced by ROS oxidation of membrane phospholipids rather than COX activity. Materazzi et al. expanded on this observation by demonstrating that intraplantar injection of 8-iso-PGA2 evoked nocifensive reflexes in WT but not Trpa1−/− mice [78]. In total, these results add to the mounting body of evidence that multiple products of nonenzymatic metabolism of fatty acids directly activate TRPA1 and may contribute to COX inhibitor-refractory noxious sensations in humans.

TRPA1 Amplifies Neuronal Signals Caused by Many Soluble Inflammatory Mediators One of the earliest reports to characterize TRPA1 function [15] reported that the channel (at that point referred to as ANKTM1 due to its high number of ankyrin domains) could be activated when the muscarinic agonist carbachol was applied to cells co-expressing a muscarinic receptor and TRPA1. From this pioneering study, a model has emerged whereby TRPA1 may possibly be gated downstream of any Gq-coupled receptor expressed in the same cell membrane. How widely this occurs in physiological settings remains to be determined, although TRPA1 can be activated in experimental situations by activation of PAR2 [79] or BK’s B2 receptor [18]. The exact mechanism(s) through which this occurs have remained elusive, although it is reasonable to speculate that the intracellular Ca2+ elevation triggered by Gq signaling is one contributing factor because intracellular Ca2+ increases can potentiate channel function (and, at higher concentrations, inhibit it) [80].

BK is a nonapeptide pro-algesic and pro-inflammatory mediator that acts on its B2 (or, in some cases, its inducible B1) receptor to cause nerve activation and sensitization by multiple direct and indirect mechanisms [73]. Importantly, kinins including BK may be important mediators of viral symptoms in humans [81,82], and asthmatics demonstrate greater reactivity to BK than healthy subjects do [83]. Thus, whereas BK regulates the activity of many cellular targets (including ion channels) downstream of its Gq-coupled receptor [84], and it may be one of many mediators that activates or sensitizes airway nerve during inflammatory conditions, interrupting BK signaling in respiratory disease does have potential as a strategy to ameliorate respiratory symptoms. Bandell et al. [16] first demonstrated that TRPA1 is a downstream target of BK when they Nonneuronal TRPA1 Expression and Function 175 observed that BK produced outwardly rectifying cationic currents in Chinese Hamster Ovary

(CHO) cells dually transfected with both TRPA1 and the BK B2 receptor, but not either one sin- gly. Bautista et al. [22] demonstrated that TRPA1 is a downstream target of BK in native sensory neurons and that this is relevant in vivo when they showed that Ruthenium red, extracellular Ca2+ removal, or TRPA1 knockout markedly reduced BK-induced intracellular Ca2+ elevations in mouse trigeminal neurons and that TRPA knockout also abolished thermal hyperalgesia caused by intraplantar BK. In airway nerve terminals within mouse [85] and guinea pig [86,87] airways, BK causes robust action potential discharge in a manner dependent on the B2 receptor and downstream effectors that include TRPV1 and Ca2+-activated chloride channels [88]. Consistent with the concept that airway sensory fibers are BK-sensitive in a manner at least analogous to somatosensory fibers, BK also causes coughing, an expulsion/dilution reflex presumably triggered by noxious sensations, in guinea pigs. This coughing is markedly reduced by the TRPA1 antagonist HC-030031 [89]. This effect is most likely at the level of the sensory nerve because HC- 030031 inhibited BK-induced sensory neuron Ca2+ flux and vagus nerve depolarization in these studies. Taken together, these data indicate that TRPA1 is involved in BK-induced nociceptor sensitization, hyperalgesia, and cough, and that TRPA1 block may provide a target beyond the

B2 receptor that could ameliorate the noxious sensations caused by BK. Recently, TRPA1 has been identified as a downstream effector of multiple intracellular networks beyond direct signaling by reactive carbonyls and mediators acting via the canonical Gq-coupled pathway. Two cytokines that have been linked to the pathology of atopic dermatitis and asthma, IL-31 and thymic stromal lymphopoietin, activate a subset of mouse sensory neurons through their respective receptors to trigger signals that in turn activate TRPA1 [90,91]. In the case of both of these mediators, direct injection into the skin causes robust scratching in mice, and this behavior is markedly blunted by TRPA1 gene deletion. Although future studies will be necessary to determine what sensations and/or reflexes these mediators cause in the airways and what role TRPA1 could play in those phenomena, these results indicate that TRPA1 is capable of serving as a downstream effector that may amplify signals generated by multiple biochemical pathways initiated by diverse soluble ligands. How extensive—and context-dependent—this list is remains to be determined, however.

NONNEURONAL TRPA1 EXPRESSION AND FUNCTION

TRPA1 block or knockout is anti-inflammatory in models of both allergic [92] and cigarette smoke-induced inflammation [93]. Neurogenic inflammation triggered by TRPA1-dependent release of neuropeptides was presumed to be the mechanism that would explain this phenomenon, although, if true, this could be concerning because multiple molecules that block individual or combinations of the neurokinin receptors that are the predominant effector mechanisms of neurogenic inflammation in animal models have shown little benefit in human trials despite demonstrating pharmacodynamic effects against neuropeptide agonists [94]. However, Nassini et al. [93] demonstrated that TRPA1 immunoreactivity is present in nonneuronal cells within mouse and human airways, and that TRPA1 also plays a pro-inflammatory role in a mouse cigarette smoke exposure model that appears to be independent of neurogenic inflammation. These results expand both the potential pathological role of TRPA1 in human respiratory disease and the possible translational potential of this anti-inflammatory effect. 176 10. TRPA1 ANTAGONISTS AS POTENTIAL THERAPEUTICS TRPA1 and Small Cell Lung Carcinoma Although TRPA1 was initially identified due to an observed change in its expression in transformed cells, the role of TRPA1 in cancer is still largely unknown. A recent study [95] has implicated TRPA1 in small cell lung carcinoma (SCLC) cell resistance to serum starvation- induced apoptosis. The same study also demonstrated that siRNA-mediated knockdown of TRPA1 in H146 SCLC cells markedly inhibited their anchorage-independent growth, an in vitro measure of metastatic capability. Heightening the translational potential of these findings, the authors also found TRPA1 message in surgically resected SCLC specimens, but not in non-small cell carcinoma or lung tissue without visible tumor burden. These data suggest that TRPA1 activation may serve as a previously unappreciated mechanism linking cigarette smoke with SCLC metastasis and resistance to chemotherapeutics.

IN VIVO PRECLINICAL PHARMACOLOGY OF TRPA1 BLOCKERS

Mice with targeted deletion of the Trpa1 gene product have been used extensively to implicate TRPA1 in numerous pathological processes. These studies have provoked considerable interest in TRPA1 as a therapeutic target for respiratory diseases that may serve the unique function of limiting noxious respiratory sensations caused by acute inflammation and irritant exposures and perhaps even reversing long-term reflex hypersensitivity. What these studies do not provide, however, is insight into the pharmacology of TRPA1 and what properties a channel blocker may need to possess to achieve therapeutic efficacy. Current in vivo studies of TRPA1 blockers are limited by the number of structural scaffolds currently available. AP18 and HC-030031 (see Table 10.2 and Figure 10.2, respectively) were the first published small molecule blockers of the TRPA1 channel [16,96]. Both molecules display fully efficacious block against native and recombinant TRPA1 channels from multiple species within the low μM range, and studies have shown molecules of both structural tem- plates to be selective over many other molecular targets [97,98]. Obtaining consistent results with these two structurally distinct molecules, particularly when combined with genetic techniques such as antisense oligonucleotides or gene disruption, constitutes a data package that can boost confidence in the target as much as any rodent experiments could be expected to. A-967079, an α,β-unsaturated oxime, blocks TRPA1 channels with higher potency than the related AP18 and is also orally bioavailable in rodents, thereby rendering it a tool molecule sufficient for in vivo studies. Although A-967079 has not yet been studied in respiratory models, it has shown efficacy in mouse models of pain [98], pancreatitis [99], and contact dermatitis [100]. HC-030031 has been examined more extensively in preclinical models, where it has demonstrated consistent efficacy in respiratory models ranging from acute airway resistance caused by desflurane [60] to disease models such as the mouse ovalbumin assay, where it inhibited inflammation and increases in airway reactivity caused by allergen sensitization and challenge [92]. Moreover, Raemdonck et al. [101] reported that HC-030031 and the antimus- carinic tiotropium inhibited delayed increases in the whole-body plethysmography measurement Penh following allergen challenge in sensitized Brown Norway rats. The authors noted the presence of both rapid and delayed increases in this index that correlates with the early and late phase decrements in lung function asthmatics experience following allergen challenge TRPA1 Antagonists Claimed in the Patent Literature 177 and interpreted their results as evidence that this delayed phase reaction is dependent on the activation of TRPA1, which then triggers CNS-dependent parasympathetic reflex activity. TRPA1 is often considered a transducer of noxious stimuli, as it is expressed in sensory nerve endings, and its activation by multiple noxious stimuli can lead to action potential discharge in sensory fibers [70,102]. It bears nothing, however, that TRPA1 is also functionally expressed on the synaptic terminals of nociceptors. The TRPA1 agonist AITC increases the amplitude and frequency of glutamatergic excitatory postsynaptic potentials in substantia gelatinosa neurons of rat spinal cord slices [103] and intrathecal administration of AITC causes long-lasting sensitization of nociceptive behavior to mechanical and thermal stimuli [104]. Moreover, a series of elegant experiments where TRPA1 was blocked by the tool blocker Chembridge-5861528 via either intraplantar or intrathecal injections demonstrated that these two populations of channels appear to play distinct roles in pain models. In these experiments, the selective block of spinal TRPA1 channels yielded marked efficacy in a number of models in which no TRPA1 agonist was exogenously applied, ranging from secondary hyperalgesia caused by capsaicin application to REM sleep deprivation [105]. Based on this body of work, the authors suggested that TRPA1 channels in the spinal cord amplify the intensity of low-intensity mechanical inputs. According to this intriguing model, nerve terminal TRPA1 would be responsible for the direct initiation of noxious sensations caused by exposure of the terminals to noxious stimuli, whereas spinal TRPA1 channels would serve a separate role as amplifiers of a broad range of stimuli. Further evidence will prove how widely applicable this model is, which molecules produced within the dorsal horn serve as relevant endogenous openers of TRPA1 during pathological conditions (although this list may reasonably include multiple products of inflammation and oxidant stress), and whether an analogous concept applies within the NTS, where vagal sensory fibers synapse. Germane to this latter case, evidence suggests the presence of TRPA1 pharmacology within the NTS [61].

TRPA1 ANTAGONISTS CLAIMED IN THE PATENT LITERATURE

Despite the growing interest in TRPA1 as it pertains to numerous pathological conditions, published efforts describing TRPA1 antagonists have remained relatively limited. Of these, perhaps the most common targets contain a xanthine (caffeine)-related core. Figure 10.2 illustrates this core, as well as HC-030031 (Hydra Biosciences), the first reported xanthine-based TRPA1 blocker, which was discovered from a diverse small molecule screen [106].

O O O O H N HN N N N N N H

O N N O N N O N N H Xanthine Caffeine HC-030031 6.2 µM (AITC) 5.3 µM (Formalin) 7.5 µM (Mol. pain; AITC)

FIGURE 10.2 Structures of xanthine, caffeine and the xanthine-related TRPA1 antagonist HC-030031 (values represent literature-reported IC50’s) 178 10. TRPA1 ANTAGONISTS AS POTENTIAL THERAPEUTICS

O O O S N Aryl N N N H H N Aryl (5,6 or 6,6 fused O N N aromatic ring system)

1 2

FIGURE 10.3 General structures of xanthine-related TRPA1 antagonists

A number of other xanthine-related antagonists followed, with activities greatly improving on the micromolar potency of HC-030031. In general, they may be described as the xanthine core linked by a methylene amide to an aryl system, as demonstrated by the general structure 1 (Figure 10.3). However, considerable efforts have focused on replacing the xanthine portion with other 5,6- and 6,6-based fused ring systems, and these structures have more generally been attached to a thiazole-based biaryl group (structure 2). Table 10.1 illustrates some patented examples that may be characterized by the general structures 1 and 2, which have been prepared by both Hydra Biosciences/Cubist Pharmaceuticals and Glenmark Pharmaceuticals. These structures illustrate that there is flexibility in not only the heteroatom content of the fused 5,6 and 6,6 systems, but also the point of attachment to the amide linked biaryl group. Although these compounds usually exhibit nanomolar potency, they have been plagued by poor solubility. This issue was addressed by a patent filed by Glenmark, wherein a solubility-enhancing methylene phosphate group was incorporated on the thiazole ring (Ex. 13, 14). This change had a detrimental effect on potency relative to their parent compounds (Ex. 3, 4, respectively); however, both the solubility and rat Drug Metabolism and Pharmacokinetics (DMPK) profile of the phosphates was greatly improved. (Note: the solubility and DMPK data for Ex. 3 and 4 were reported in Ref. [110].) The last structure shown in Table 10.1 (Ex. 15), prepared by Cubist Pharmaceuticals, is the most recently reported compound. It was found to block inward currents in rat, dog, and human TRPA1 with a potency of ~100 nM and was at least 100-fold selective relative to seven other TRP channels, hERG, and hNaV1.2. Oral bioavailability in fed rats was >50%. Kinetic solubility of Ex. 15 was measured in several media. As an example, it was found that solubility in FaSSIF (fasted simulated small intestinal fluid) started at 3.5-4.5 mg/mL at 0 min and decreased to ~0.5-1 mg/ mL over 4 h. In the same year that HC-030031 was disclosed, Amgen reported a series of trichloro thiophenyl benzamides, as illustrated by AMG-9090 (Ex. 17) in Table 10.2. Although compounds such as AMG-9090 exhibited nanomolar potency at hTRPA1, some examples such as AMG- 9090 acted as agonists of the rat channel (rTRPA1). Also the same year, a patent described TRPA1 antagonism by the low MW α,β-unsaturated oxime, AP-18 (Ex. 18). Two years later, Abbott published a patent on related structures for treatment of nociceptive and neuropathic pain. These include A-967079 (Ex. 19), which has since been studied extensively in preclinical models [98]. Subsequently, Renovis elaborated on the SAR of this structural class. They showed that multiple modifications including the aryl ring substitution imparted agonist activity on the series, and only one new antagonist (Ex. 21) was identified from these efforts. Pfizer applied a pair-wise analysis method to the optimization of their TRPA1 targets. The strategy was to identify series for other biological targets (donor series) wherein the SAR for TRPA1 Antagonists Claimed in the Patent Literature 179 ] [ 107 ] [ 107 ] [ 110 Reference [ 108 ] [ 109 ] (Continued) h/ h/

ng ng

4000 7000

< < ng/mL ng/mL

250 250

< <

max max

DMPK (Rat) Data AUC0-24 mL AUC0-24 mL C C μ g/mL μ g/mL

0.2 0.1 < < Solubility in Water (nM) 50 hTRPA1 IC hTRPA1 < 50 50-100 < 50 < 50 < 50 3 3 3 N F F CF F Cl CF CF F F F N N N N S N S S NH S S N H N H N H O O N H O O O N N S H N N N N N O O O N O N O N N N N N Xanthine-related TRPA1 antagonists Xanthine-related TRPA1 Structure O O O O O

10.1

TABLE Example 2 3 1 4 5 180 10. TRPA1 ANTAGONISTS AS POTENTIAL THERAPEUTICS ] ] ] ] [ 110 [ 111 [ 111 [ 111 Reference

DMPK (Rat) Data

Solubility in Water (nM) 50 hTRPA1 IC hTRPA1 < 50 50-100 50-100 50-250 3 3 N F CF CF Cl F F Cl F F N N N N S NH S S S N H N H N H O N O O N O N N N N N N O O N O N O N N N N Xanthine-related TRPA1 antagonists—Cont’d Xanthine-related TRPA1 Structure O O O O

10.1

Example 7 8 9 6 TABLE TRPA1 Antagonists Claimed in the Patent Literature 181 ] ] ] ] [ 111 [ 111 Reference [ 112 [ 112 (Continued) 40 30

> >

% %

DMPK (Rat) Data F F μ M

μ M

21 5 > > Solubility in Water (nM) 50 hTRPA1 IC hTRPA1 50-100 < 50 250-499 < 250 3 CF 3 CF O F F 3 3 F 3 F CF CF CF F N N S S O O O N H HN HN HN N N O N N N S N N O N O N N O O N N N N Xanthine-related TRPA1 antagonists—Cont’d Xanthine-related TRPA1 Structure O O O O

10.1

TABLE Example 11 12 10 13 182 10. TRPA1 ANTAGONISTS AS POTENTIAL THERAPEUTICS ] ] ] Reference [ 113 [ 113 [ 114 h/

50,000

ng/mL ng/mL

99% 4000 7500

50 (fed; rat;

< > >

100,000

max max % F DMPK (Rat) Data C C AUC0-24 mL AUC0- 24 mL formula I) F % 25-50 (fasted; formula I) PPB μ g/mL

μ g/mL

1200 110 > > Solubility in Water (nM) 50 hTRPA1 IC hTRPA1 100-500 > 500 100 3 3 F F CF CF N N + + + + F F Na Na N - Na - Na - - O O O O O O P P OH N N N O S S O O S O O O O N N HN N N N S N N N O O N O N N N Xanthine-related TRPA1 antagonists—Cont’d Xanthine-related TRPA1 Structure O O O

10.1

TABLE Example 14 15 16 TABLE 10.2

Example Structure hTRPA1 IC50 rTRPA1 IC50 Reference 17 21 nM [115] H S N

O Cl Cl Cl Cl

OH 18 N 3.1 μM [93]

OH 19 N 74 nM 289 nM [95]

OH 20 N Agonist [116] (EC50 = 9.4 μM) Cl

21 OH 1.91 μM (pA vs. [116] N 2 cinnamaldehyde)

22 O 100 nM [117] NN O F3C O S N O

CN 23 O 60 nM [118]

N N H N O N

24 NO2 10 nM [119] O Cl N H O CF3

25 F 19 nM [120]

HN OH

H 184 10. TRPA1 ANTAGONISTS AS POTENTIAL THERAPEUTICS a single functional group matches the SAR seen for theirs. The optimal groups for the donor series would then be incorporated into their own. In this manner, it might be possible to find functional groups for potency/property enhancement that might not otherwise have been discovered through more traditional drug discovery processes. This strategy was applied to their TRPA1 thiazolopyrimidine dione series, which had up to that point failed to exceed a potency of 500 nM. A well-matched 5-HT6 donor series was identified that enabled further development of the amide SAR on their core. This resulted in the identification of the phenoxypyrrolidine amide (Ex. 22), which provided a fivefold increase in potency. In April of 2012, AstraZeneca published a patent on chiral piperazine amides such as the one shown in Ex. 23 for the treatment of disorders such as pain, asthma, pertussis, and nicotine addiction. This compound (R-enantiomer) was reported as having an IC50 of 60 nM against Zn(II) in a FLIPR assay. In June of the same year, Merck published a patent on aryl amides, wherein the central amide-racemic carbon-carbonyl sequence was determined to be crucial for the activity of the series. The potencies of the compounds tested against Zn(II) ranged from 0.79 μM to 200 nM, with the exception of Ex. 24, which was reported to be 10 nM. The nitro-containing analogs, which were the most potent, had low aqueous solubility. A series of decalin-based compounds, as exemplified by Ex. 25, was reported by Merck in 2011. Potencies against hTRPA1 activated by cinnamaldehyde ranged from 19 to 520 nM. No other biological data was given for this series. In December of 2010, a series of proline sulfonamides, many of which exhibited low nM potency, was reported by Jannsen Pharmaceuticals for the treatment of pain, arthritis, itch, cough, asthma, or inflammatory bowel disease (Table 10.3, Ex. 26-30). IC50s were given against

TABLE 10.3 Pyrrolidine sulfonamide TRPA1 antagonists

Example Structure hTRPA1 IC50 (nM) Reference

26 F3C 3 [121]

N N H N CF N 3 S O O O F

27 4 [121] O N N H N CF N 3 S O O O S Cl TRPA1 Antagonists Claimed in the Patent Literature 185

TABLE 10.3 Pyrrolidine sulfonamide TRPA1 antagonists—Cont’d

Example Structure hTRPA1 IC50 (nM) Reference 28 N 6 [121] H N O N CF S O O 3 O S Cl

29 9 [121] N N N H N CF N 3 S O O O S Cl

30 CF3 11 [121]

H N N S O O O S Cl

31 S CF3 30 [122] H N N N F S O O O F

32 S O 90 [122] H CF3 N N N S O O O S Cl 186 10. TRPA1 ANTAGONISTS AS POTENTIAL THERAPEUTICS

AITC challenge in CHO-TREX cells (applicable to the compounds shown in Table 10.3), or hTRPA1/TREx-HEK293 cells with 6,11-dihydro-5H-dibenzo[b,e]azepine-10-carboxylic acid methyl ester. Potencies ranging from 3 nM to 20 μM were reported. Subsequently, Ajinomoto published a patent claiming a series of proline sulfonamides (Ex. 31,32). In this claim, compounds were presented with potencies ranging from 60 nM to 5.84 μM against AITC. Janssen Pharmaceuticals (and later JNJ) developed a series of tricyclic ureas and thioureas (Ex. 33-35, Table 10.4). These compounds were tested in hTRPA1- and rTRPA1-inducible HEK293 cells with 6,11-dihydro-5H-dibenzo[b,e]azepine-10-carboxylic acid methyl ester as a challenge. It was found that potency was imparted only with the 4R enantiomers. These compounds suffered from low solubility, low metabolic stability, deep red color, and a potentially toxic thiourea. Four years later, Hydra Biosciences published on a series of tricyclic compounds (Ex. 36-40), that differed from the Janssen molecules in the incorporation of nitrogens within the aromatic portion of the tricyclic frame, as well as the addition of appendages on the urea/ thiourea moiety. No physicochemical properties were reported for these compounds.

TABLE 10.4 Tricyclic urea and thiourea TRPA1 antagonists

Example Structure hTRPA1 IC50 rTRPA1 IC50 Reference 33 S 130 nM 20 nM [123,124] HN NH

O O

34 O 3.9 μM 4.7 μM [123,124] HN NH

O O

35 S 13 nM 4 nM [123,124] HN NH

O O O

36 S <100 nM [125] HN NH

N O O O CONCLUSIONS 187

TABLE 10.4 Tricyclic urea and thiourea TRPA1 antagonists—Cont’d

Example Structure hTRPA1 IC50 rTRPA1 IC50 Reference 37 O <100 nM [125] N NH

N O O

38 NC O <100 nM [125] N NH

N O O

39 O <100 nM [125] N NH

N O O

40 O <100 nM [125] HN N NH

O O

CONCLUSIONS

TRPA1 is being actively pursued as a therapeutic target for respiratory disorders by multiple entities, as evidenced by an active patent literature. Blockade of the ion channel holds tremendous therapeutic potential to limit or reverse both acute noxious respiratory sensations and chronic elevations in irritant sensitivity, based on its specialized localization in irritant-sensing nerves and its ability to detect a range of hazardous pollutants, inflammatory mediators, and products of oxidative tissue damage. This potential must be tempered at present, however, by the facts that most of the functional data produced to-date are in rodent models and that the associations between the ability of hazardous pollutants to cause asthma-like symptoms in humans and to activate human TRPA1 ino vitr are no more than associations, albeit powerful ones. Further, despite its vital role in mediating acute effects of cigarette smoke in rodents, the question of how TRPA1 expression and function could change in chronically injured tissue where acute irritant reflexes may be dramatically altered (e.g., in the COPD lung) must go unanswered for now. 188 10. TRPA1 ANTAGONISTS AS POTENTIAL THERAPEUTICS

Fortunately, considerable advances have been made in the field, and at least two TRPA1 antagonists (GRC 17536 from Glenmark and CB-625 from Cubist in partnership with Hydra Biosciences) have entered clinical trials, although precious little data on these molecules (including their chemical structures) has been disclosed in the public domain. If these or other TRPA1 antagonists are sufficiently safe and efficacious, they will serve as tremendously powerful tools for clinical investigators to test hypotheses such as whether blocking TRPA1 reduces or eliminates adverse respiratory reactions caused by acute gas anesthetic inhalation, diminishes noxious sensations and/or inflammation caused by hazardous air pollutants, or perhaps even ameliorates the agonizing effects of tear gas that precipitate incapacitation. It is encouraging that multiple companies have identified molecules that selectively block TRPA1 in the low nM range, inhibit TRPA1 in preclinical disease models, and appear to be devoid of overt mechanism-based toxicity or tolerability limitations. Although these are significant achievements, they are only the first of many that must be overcome to one day deliver on the promise of TRPA1 antagonism in a meaningful manner. Now that in vitro potency and selectivity have been achieved through multiple chemical scaffolds, future medicinal chemistry efforts will be devoted to generating novel molecules that maintain these desirable traits while also improving the aqueous solubility as well as reducing lipophilicity and molecular weight.

References [1] Grainge CL, Lau LCK, Ward JA, Dulay V, Lahiff G, Wilson S, et al. Effect of bronchoconstriction on airway remodeling in asthma. N Engl J Med 2011;364(21):2006–15. [2] Shapiro SD, Ingenito EP. The pathogenesis of chronic obstructive pulmonary disease. Am J Respir Cell Mol Biol 2005;32(5):367–72. [3] Beasley R, Douwes J, Pekkanen J, Pearce NE. How much asthma can be attributed to atopic sensitization? In: Johnston SB, Holgate ST, editors. Asthma: critical debates. London: Blackwell Science Ltd.; 2002. p. 35–45. [4] Schelegle ES, Eldridge MW, Cross CE, Walby WF, Adams WC. Differential effects of airway anesthesia on ozone-induced pulmonary responses in human subjects. Am J Respir Crit Care Med 2001;163(5):1121–7. [5] Balmes JR, Fine JM, Sheppard D. Symptomatic bronchoconstriction after short-term inhalation of sulfur dioxide. Am Rev Respir Dis 1987;136(5):1117–21. [6] Shusterman D, Balmes J, Avila PC, Murphy MA, Matovinovic E. Chlorine inhalation produces nasal conges- tion in allergic rhinitics without mast cell degranulation. Eur Respir J 2003;21(4):652–7. [7] Ritz T, Steptoe A, Bobb C, Harris AHS, Edwards M. The asthma trigger inventory: validation of a questionnaire for perceived triggers of asthma. Psychosom Med 2006;68(6):956–65. [8] Leikauf GD. Hazardous air pollutants and asthma. Environ Health Perspect 2002;110(Suppl. 4):505–26. [9] Sunil VR, Vayas KN, Massa CB, Gow AJ, Laskin JD, Laskin DL. Ozone-induced injury and oxidative stress in bronchiolar epithelium are associated with altered pulmonary mechanics. Toxicol Sci 2013;133(2):309–19. [10] Triantaphyllopoulos K, Hussain F, Pinart M, Zhang M, Li F, Adcock I, et al. A model of chronic inflammation and pulmonary emphysema after multiple ozone exposures in mice. Am J Physiol Lung Cell Mol Physiol 2011;300(5):L691–700. [11] Bernstein JA, Davis BP, Picard JK, Cooper JP, Zheng S, Levin LS. A randomized, double-blind, parallel trial comparing capsaicin nasal spray with placebo in subjects with a significant component of nonallergic rhinitis. Ann Allergy Asthma Immunol 2011;107(2):171–8. [12] Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, Julius D. The capsaicin receptor: a heat- activated ion channel in the pain pathway. Nature 1997;389:816–24. [13] Symanowicz PT, Gianutsos G, Morris JB. Lack of role for the vanilloid receptor in response to several inspired irritant sir pollutants in the C57Bl/6J mouse. Neurosci Lett 2004;362(2):150–3. [14] Jaquemar D, Schenker T, Trueb B. An ankyrin-like protein with transmembrane domains is specifically lost after oncogenic transformation of human fibroblasts. J Biol Chem 1999;274(11):7325–33. REFERENCES 189

[15] Story GM, Peier AM, Reeve AJ, Eid SR, Mosbacher J, Hricik TR, et al. ANKTM1, a TRP-like channel expressed in nociceptive neurons, is activated by cold temperatures. Cell 2003;112(6):819–29. [16] Bandell M, Story GM, Hwang SW, Viswanath V, Eid SR, Petrus MJ, et al. Noxious cold ion channel TRPA1 is activated by pungent compounds and bradykinin. Neuron 2004;41(6):849–57. [17] Nagata K, Duggan A, Kumar G, Garcia-Anoveros J. Nociceptor and hair cell transducer properties of TRPA1, a channel for pain and hearing. J Neurosci 2005;25:4052–61. [18] Jordt SE, Bautista DM, Chuang HH, McKemy DD, Zygmunt PM, Hogestatt ED, et al. Mustard oils and cannabinoids excite sensory nerve fibres through the TRP channel ANKTM1. Nature 2004;427(6971):260–5. [19] Hinman A, Chuang HH, Bautista DM, Julius D. TRP channel activation by reversible covalent modification. Proc Natl Acad Sci USA 2006;103:19564–8. [20] Macpherson LJ, Dubin AE, Evans MJ, Marr F, Schultz PG, Cravalt BF, et al. Noxious compounds activate TRPA1 ion channels through covalent modification of cysteines. Nature 2007;445(7127):541–5. [21] Kwan KY, Allchorne AJ, Vollrath MA, Christensen AP, Zhang DS, Woolf CJ, et al. TRPA1 contributes to cold, mechanical, and chemical nociception but is not essential for hair-cell transduction. Neuron 2006;50:277–89. [22] Bautista DM, Jordt SE, Nikai T, Tsuruda PR, Read AJ, Poblete J, et al. TRPA1 mediates the inflammatory actions of environmental irritants and proalgesic agents. Cell 2006;124:1269–82. [23] Sim VM, Pattle RE. Effect of possible smog irritants on human subjects. J Am Med Assoc 1957;165(15):1908–13. [24] Bein K, Leikauf GD. Acrolein: a pulmonary hazard. Mol Nutr Food Res 2011;55(9):1342–60. [25] Nassenstein C, Kwong K, Taylor-Clark T, Kollarik M, MacGlashan DM, Braun A, et al. Expression and function of the ion channel TRPA1 in vagal afferent nerves innervating mouse lungs. J Physiol 2008;586(6):1595–604. [26] Bessac BF, Sivula M, von Hehn CA, Escalera J, Cohn L, Jordt SE. TRPA1 is a major oxidant sensor in murine airway sensory neurons. J Clin Invest 2008;118(5):1899–910. [27] Lundberg JM, Saria A. Capsaicin-induced desensitization of airway mucosa to cigarette smoke, mechanical and chemical irritants. Nature 1983;302(5905):251–3. [28] Andre E, Campi B, Materazzi S, Trevisani M, Amadesi S, Massi D, et al. Cigarette smoke-induced neurogenic inflammation is mediated by alpha, beta-unsaturated aldehydes and the TRPA1 receptor in rodents. J Clin Invest 2008;118(7):2574–82. [29] Nassini R, Materazzi S, Andre E, Sartiani L, Aldini G, Trevisani M, et al. Acetaminophen, via its reactive metabolite N-acetyl-p-benzo-quinoneimine and transient receptor potential ankyrin-1 stimulation, causes neurogenic inflammation in the airways and other tissues in rodents. FASEB J 2010;24:4904–16. [30] Birrell MA, Belvisi MG, Grace M, Sadofsky L, Faruqi S, Hele DJ, et al. TRPA1 agonists evoke coughing in guinea pig and human volunteers. Am J Respir Crit Care Med 2009;180(11):1042–7. [31] Hazari MS, Rowan WH, Winsett DW, Ledbetter AD, Haykal-Coates N, Watkinson WP, et al. Potentiation of pulmonary reflex response to capsaicin 24 h following whole-body acrolein exposure is mediated by TRPV1. Respir Physiol Neurobiol 2008;160(2):160–71. [32] Andre E, Gatti R, Trevisani M, Preti D, Baraldi PG, Patacchini R, et al. Transient receptor potential ankyrin receptor 1 is a novel target for pro-tussive agents. Br J Pharmacol 2009;158(6):1621–8. [33] Brozmanova M, Mazurova L, Ru F, Tatar M, Kollarik M. Comparison of TRPA1- versus TRPV1-mediated cough in guinea pigs. Eur J Pharmacol 2012;689:211–18. [34] Macpherson LJ, Xiao B, Kwan KY, Petrus MJ, Dubin AE, Hwang S, et al. An ion channel essential for sensing chemical damage. J Neurosci 2007;27(42):11412–5. [35] McNamara CR, Mandel-Brehm J, Bautista DM, Siemens J, Deranian KL, Zhao M, et al. TRPA1 mediates formalin-induced pain. PNAS 2007;104(33):13525–30. [36] Taylor-Clark TE, Kiros F, Carr MJ, McAlexander MA. Transient receptor potential ankyrin 1 mediates toluene diisocyanate-evoked respiratory irritation. Am J Respir Cell Mol Biol 2009;40(6):756–62. [37] Wu Y, You H, Ma P, Li L, Yuan Y, Li J, et al. Role of transient receptor potential ion channels and evoked levels of neuropeptides in a formaldehyde-induced model of asthma in Balb/c mice. PLoS ONE 2013;8(5):e62827. [38] Stjarne P, Lundblad L, Lundberg JM, Anggard A. Capsaicin and nicotine-sensitive afferent neurones and nasal secretion in healthy human volunteers and in patients with vasomotor rhinitis. Br J Pharmacol 1989;96(3):693–701. [39] Lee LY, Gerhardstein DC, Wang AL, Burki NK. Nicotine is responsible for airway irritation evoked by cigarette smoke inhalation in men. J Appl Physiol 1993;75(5):1955–61. [40] Talavera K, Gees M, Karashima Y, Meseguer VM, Vanoirbeek JAJ, Damann N, et al. Nicotine activates the chemosensory cation channel TRPA1. Nat Neurosci 2009;12(10):1293–9. 190 10. TRPA1 ANTAGONISTS AS POTENTIAL THERAPEUTICS

[41] Xu J, Yang W, Zhang G, Gu Q, Lee LY. Calcium transient evoked by nicotine in isolated rat vagal pulmonary sensory neurons. Am J Physiol Lung Cell Mol Physiol 2007;292(1):L54–61. [42] Kichko TI, Lennerz J, Eberhardt M, Babes RM, Neuhuber W, Kobal G, et al. Bimodal concentration-response of nicotine involves the nicotinic acetylcholine receptor, transient receptor potential vanilloid type 1, and transient receptor potential ankyrin 1 channels in mouse trachea and sensory neurons. J Pharmacol Exp Ther 2013;347(2):529–39. [43] Hu H, Bandell M, Petrus MJ, Zhu MX, Patapoutian A. Zinc activates damage-sensing TRPA1 ion channels. Nat Chem Biol 2009;5(3):183–90. [44] Andersson DA, Gentry C, Moss S, Bevan S. Clioquinol and pyrithione activate TRPA1 by increasing intracellular Zn2+. PNAS 2009;106(20):8374–9. [45] Miura S, Takahashi K, Imagawa T, Uchida K, Saito S, Tominaga M, et al. Involvement of TRPA1 activation in acute pain induced by cadmium in mice. Mol Pain 2013;9(1):7. [46] Gu Q, Lin RL. Heavy metals zinc, cadmium, and copper stimulate pulmonary sensory neurons via direct activation of TRPA1. J Appl Physiol 2010;108(4):891–7. [47] Hoshino K, Takeuchi O, Kawai T, Sanjo H, Ogawa T, Takeda Y, et al. Cutting edge: toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product. J Immunol 1999;162(7):3749–52. [48] Meseguer V, Alpizar YA, Luis E, Tajada S, Denlinger B, Fajardo O, et al. TRPA1 channels mediate acute neurogenic inflammation and pain produced by bacterial endotoxins. Nat Commun 2014;5:3125. [49] Lee LY, Dumont C, Djokic TD, Menzel TE, Nadel JA. Mechanism of rapid, shallow breathing after ozone exposure in conscious dogs. J Appl Physiol Respir Environ Exerc Physiol 1979;46(6):1108–14. [50] Taylor-Clark TE, Undem BJ. Ozone activates airway nerves via the selective stimulation of TRPA1 ion channels. J Physiol 2010;588:423–33. [51] Brugsch HG, Elkins HB. Toluene di-isocyanate (TDI) toxicity. N Engl J Med 1963;268(7):353–7. [52] Sangha GK, Alarie Y. Sensory irritation by toluene diisocyanate in single and repeated exposures. Toxicol Appl Pharmacol 1979;50:533–47. [53] Mapp CE, Graf PD, Boniotti A, Nadel JA. Toluene diisocyanate contracts guinea pig bronchial smooth muscle by activating capsaicin-sensitive sensory nerves. J Pharmacol Exp Ther 1991;256(3):1082–5. [54] Kalubi B, Takeda N, Irifune M, Ogino S, Abe Y, Su-Ling H, et al. Nasal mucosa sensitization with toluene diisocyanate (TDI) increases preprotachykinin A (PPTA) and preproCGRP mRNAs in guinea pig trigeminal ganglion neurons. Brain Res 1992;576(2):287–96. [55] Bessac BF, Sivula M, von Hehn CA, Caceres AI, Escalera J, Jordt SE. Transient receptor potential ankyrin 1 antagonists block the noxious effects of toxic industrial isocyanates and tear gases. FASEB J 2009;23(4):1102–14. [56] TerRiet MF, DeSouza GJA, Jacobs JS, Young D, Lewis MC, Herrington C, et al. Which is most pungent: isoflurane, sevoflurane or desflurane? Br J Anaesth 2000;85(2):305–7. [57] Mutoh T, Tsubone H, Nishimura R, Sasaki N. Effects of volatile anesthetics on vagal C-fiber activities and their reflexes in anesthetized dogs. Respir Physiol 1998;112(3):253–64. [58] Matta JA. General anesthetics activate a nociceptive ion channel to enhance pain and inflammation. Proc Natl Acad Sci USA 2008;105:8784–9. [59] Cornett PM, Matta JA, Ahern GP. General anesthetics sensitize the capsaicin receptor transient receptor potential V1. Mol Pharmacol 2008;74(5):1261–8. [60] Satoh J, Yamakage M. Desflurane induces airway contraction mainly by activating transient receptor potential A1 of sensory C-fibers. J Anesth 2009;23:620–3. [61] Mutoh T, Taki Y, Tsubone H. Desflurane but not sevoflurane augments laryngeal C-fiber inputs to nucleus tractus solitarii neurons by activating transient receptor potential-A1. Life Sci 2013;92(14–16):821–8. [62] Brenneis C, Kistner K, Puopolo M, Jo S, Roberson D, Sisignano M, et al. Bupivacaine-induced cellular entry of QX-314 and its contribution to differential nerve block. Br J Pharmacol 2014;171(2):438–51. [63] Leffler A, Fischer MJ, Rehner D, Kienel S, Kistner K, Sauer SK, et al. The vanilloid receptor TRPV1 is activated and sensitized by local anesthetics in rodent sensory neurons. J Clin Invest 2008;118:763–76. [64] Binshtok AM, Bean BP, Woolf CJ. Inhibition of nociceptors by TRPV1-mediated entry of impermeant sodium channel blockers. Nature 2007;449:607–10. [65] Groeben H, Grosswent T, Silvanus M, Beste M, Peters J. Lidocaine inhalation for local anaesthesia and attenuation of bronchial hyper-reactivity with least airway irritation. Eur J Anaesthesiol 2000;17(11):672–9. REFERENCES 191

[66] Catala A. Lipid peroxidation of membrane phospholipids generates hydroxy-alkenals and oxidized phospholipids active in physiological and/or pathological conditions. Chem Phys Lipids 2009;157(1):1–11. [67] Trevisani M, Siemens J, Materazzi S, Bautista DM, Nassini R, Campi B, et al. 4-Hydroxynonenal, an endogenous aldehyde, causes pain and neurogenic inflammation through activation of the irritant receptor TRPA1. PNAS 2007;104(33):13519–24. [68] Rahman I, Biswas SK, Kode A. Oxidant and antioxidant balance in the airways and airway diseases. Eur J Pharmacol 2006;533(1–3):222–39. [69] Takahashi N, Kuwaki T, Kiyonaka S, Numata T, Kozai D, Mizuno Y, et al. TRPA1 underlies a sensing mecha-

nism for O2. Nat Chem Biol 2011;7(10):701–11. [70] Taylor-Clark TE, McAlexander MA, Nassenstein C, Sheardown SA, Wilson S, Thornton J, et al. Relative contributions of TRPA1 and TRPV1 channels in the activation of vagal bronchopulmonary C-fibres by the endogenous autacoid 4-oxononenal. J Physiol 2008;586(14):3447–59. [71] Taylor-Clark TE, Ghatta S, Bettner W, Undem BJ. Nitrooleic acid, an endogenous product of nitrative stress, activates nociceptive sensory nerves via the direct activation of TRPA1. Mol Pharmacol 2009;75(4):820–9. [72] Nesuashvili L, Hadley SH, Bahia PK, Taylor-Clark TE. Sensory nerve terminal mitochondrial dysfunction activates airway sensory nerves via transient receptor potential (TRP) channels. Mol Pharmacol 2013;83(5):1007–19. [73] Petho G, Reeh PW. Sensory and signaling mechanisms of bradykinin, eicosanoids, platelet-activating factor, and nitric oxide in peripheral nociceptors. Physiol Rev 2012;92(4):1699–775. [74] Robinson C, Holgate ST. Mast cell-dependent inflammatory mediators and their putative role in bronchial asthma. Clin Sci (Lond) 1985;68(2):103–12. [75] Narumiya S, Sugimoto Y, Ushikubi F. Prostanoid receptors: structures, properties, and functions. Physiol Rev 1999;79:1193–226. [76] Taylor-Clark TE, Undem BJ, MacGlashan Jr DW, Ghatta S, Carr MJ, McAlexander MA. Prostaglandin-induced activation of nociceptive neurons via direct interaction with transient receptor potential A1 (TRPA1). Mol Pharmacol 2008;73(2):274–81. [77] Andersson DA, Gentry C, Moss S, Bevan S. Transient receptor potential A1 is a sensory receptor for multiple products of oxidative stress. J Neurosci 2008;28(10):2485–94. [78] Materazzi S, Nassini R, Andre E, Campi B, Amadesi S, Trevisani M, et al. Cox-dependent fatty acid metabolites cause pain through activation of the irritant receptor TRPA1. Proc Natl Acad Sci USA 2008;105(33):12045–50. [79] Dai Y, Wang S, Tominaga M, Yamamoto S, Fukuoka T, Higashi T, et al. Sensitization of TRPA1 by PAR2 contributes to the sensation of inflammatory pain. J Clin Investig 2007;117(7):1979–87. [80] Zurborg S, Yurgionas B, Jira JA, Caspani O, Heppenstall PA. Direct activation of the ion channel TRPA1 by Ca2+. Nat Neurosci 2007;10(3):277–9. [81] Proud D, Naclerio RM, Gwaltney JM, Hendley JO. Kinins are generated in nasal secretions during natural rhinovirus colds. J Infect Dis 1990;161(1):120–3. [82] Naclerio RM, Proud D, Lichtenstein LM, Kagey-Sobotka A, Hendley JO, Sorrentino J, et al. Kinins are generated during experimental rhinovirus colds. J Infect Dis 1988;157(1):133–42. [83] Reynolds CJ, Togias A, Proud D. Airways hyper-responsiveness to bradykinin and methacholine: effects of inhaled fluticasone. Clin Exp Allergy 2002;32(8):1174–9. [84] Liu B, Linley JE, Du X, Zhang X, Ooi L, Zhang H, et al. The acute nociceptive signals induced by bradykinin in rat sensory neurons are mediated by inhibition of M-type K+ channels and activation of Ca2+-activated Cl− channels. J Clin Invest 2010;120(4):1240–52. [85] Kollarik M, Undem BJ. Activation of bronchopulmonary vagal afferent nerves with bradykinin, acid and vanilloid receptor agonists in wild-type and TRPV1−/− mice. J Physiol Lond 2004;555(1):115–23. [86] Carr MJ, Kollarik M, Meeker SN, Undem BJ. A role for TRPV1 in bradykinin-induced excitation of vagal airway afferent nerve terminals. J Pharmacol Exp Ther 2003;304(3):1275–9. [87] Kajekar R, Proud D, Myers AC, Meeker SN, Undem BJ. Characterization of vagal afferent subtypes stimulated by bradykinin in guinea pig trachea. J Pharmacol Exp Ther 1999;289(2):682–7. [88] Lee MG, MacGlashan Jr DW, Undem BJ. Role of chloride channels in bradykinin-induced guinea pig airway vagal C-fibre activation. J Physiol Lond 2005;566(1):205–12. [89] Grace M, Birrell MA, Dubuis E, Maher SA, Belvisi MG. Transient receptor potential channels mediate the tussive response to prostaglandin E2 and bradykinin. Thorax 2012;67(10):891–900. 192 10. TRPA1 ANTAGONISTS AS POTENTIAL THERAPEUTICS

[90] Wilson SR, The L, Batia LM, Beattie K, Katibah GE, McClain SP, et al. The epithelial cell-derived atopic dermatitis cytokine TSLP activates neurons to induce itch. Cell 2013;155(2):285–95. [91] Cevikbas F, Wang X, Akiyama T, Kempkes C, Savinko T, Antal A, et al. A sensory neuron-expressed IL-31 receptor mediates T helper cell-dependent itch: involvement of TRPV1 and TRPA1. J Allergy Clin Immunol 2014;133(2):448–60. [92] Caceres AI, Brackmann M, Elia MD, Bessac BF, del Camino D, D’Amours M, et al. A sensory neuronal ion channel essential for airway inflammation and hyperreactivity in asthma. PNAS 2009;106(22):9099–104. [93] Nassini R, Pedretti P, Moretto N, Fusi C, Carnini C, Facchinetti F, et al. Transient receptor potential ankyrin 1 channel localized to non-neuronal airway cells promotes non-neurogenic inflammation. PLoS ONE 2012;7(8):e42454. [94] Ramalho R, Soares R, Couto N, Moreira A. Tachykinin receptors antagonism for asthma: a systematic review. BMC Pulm Med 2011;11(1):41. [95] Schaefer EAM, Stohr S, Meister M, Aigner A, Gudermann T, Buech TRH. Stimulation of the chemosensory TRPA1 cation channel by volatile toxic substances promotes cell survival of small cell lung cancer cells. Biochem Pharmacol 2013;85(3):426–38. [96] Petrus M, Peier AM, Bandell M, Hwang SW, Huynh T, Olney N, et al. A role of TRPA1 in mechanical hyperalgesia is revealed by pharmacological inhibition. Mol Pain 2007;3:40. [97] Eid SR, Crown ED, Moore EL, Liang HA, Choong KC, Dima S, et al. HC-030031, a TRPA1 selective antagonist, attenuates inflammatory- and neuropathy-induced mechanical hypersensitivity. Mol Pain 2008;4:48. [98] Chen J. Selective blockade of TRPA1 channel attenuates pathological pain without altering noxious cold sensation or body temperature regulation. Pain 2011;152:1165–72. [99] Schwartz ES, La JH, Scheff NN, Davis BM, Albers KM, Gebhart GF. TRPV1 and TRPA1 antagonists prevent the transition of acute to chronic inflammation and pain in chronic pancreatitis. J Neurosci 2013;33(13):5603–11. [100] Liu B, Escalera J, Balakrishna S, Fan L, Caceres AI, Robinson E, et al. TRPA1 controls inflammation and pruritogen responses in allergic contact dermatitis. FASEB J 2013;27(9):3549–63. [101] Raemdonck K, de Alba J, Birrell MA, Grace M, Maher SA, Irvin CG, et al. A role for sensory nerves in the late asthmatic response. Thorax 2012;67(1):19–25. [102] Kerstein P, del Camino D, Moran M, Stucky C. Pharmacological blockade of TRPA1 inhibits mechanical firing in nociceptors. Mol Pain 2009;5(1):19. [103] Kosugi M, Nakatsuka T, Fujita T, Kuroda Y, Kumamoto E. Activation of TRPA1 channel facilitates excitatory synaptic transmission in substantia gelatinosa neurons of the adult rat spinal cord. J Neurosci 2007;27(16):4443–51. [104] Raisinghani M, Zhong L, Jeffrey JA, Bishnoi M, Pabbidi RM, Pimentel F, et al. Activation characteristics of transient receptor potential ankyrin 1 and its role in nociception. Am J Physiol Cell Physiol 2011;301(3):C587–600. [105] Wei H, Koivisto A, Saarnilehto M, Chapman H, Kuokkanen K, Hao B, et al. Spinal transient receptor potential ankyrin 1 channel contributes to central pain hypersensitivity in various pathophysiological conditions in the rat. Pain 2011;152(3):582–91. [106] Moran MM, Fanger C, Chong JA, McNamara C, Zhen X, Mandel-Brehm J, Inventors. Preparation of purine and pyrimidine derivatives as TRPA1 modulators for treating pain. USA patent WO2007073505A2; 2007. [107] Muthuppalniappan M, Thomas A, Kumar S, Margal S, Neelima K-J, Mukhopadhyay I, et al., Inventors. Quinazolinedione derivatives as TRPA1 modulators. Patent US20090325987A1; 2009. [108] Kumar S, Thomas A, Waghmare NT, Margal S, Khaitratkar-Joshi N, Mukhopadhyay I, Inventors. Thienopyrimidinedione derivatives as TRPA1 modulators. Patent WO2010109334A2; 2010. [109] Chaudhari SS, Kumar S, Thomas A, Patil NP, Kadam AB, Deshmukh VG, et al., Inventors. Fused pyrimidine-dione derivatives as TRPA1 modulators. Patent WO2010109287A1; 2010. [110] Hang J, Inventor. Compounds useful for treating disorders related to TRPA1. Patent WO2010132838A1; 2010. [111] Kumar S, Thomas A, Gaikwad SS, Margal S, Phatangare SK, Irlapati NR, et al., Inventors. Pyrimidinedione- fused heterocyclic compounds as TRPA1 modulators. Patent WO2010125469A1; 2010. [112] Bakthavatchalam R, Inventor. Compounds useful for treating disorders related to TRPA1. Patent WO2010138879A1; 2010. [113] Kumar S, Thomas A, Chaudhari SS, Kansagra BP, Yemireddy VR, Khairatkar-Joshi N, et al., Inventors. 2-Amino-4-arylthiazole compounds as TRPA1 antagonists. Patent WO12085662A1; 2012. [114] Barker N, Gu J-Q, Naik G, Lai J-J, Machatha S, Hwang YS, et al., Inventors. Compositions with increased stability for inhibiting transient receptor potential ion channel TRPA1. Patent WO2014026073A1; 2014. REFERENCES 193

[115] Klionsky L, Tamir R, Gao B, Wang W, Immke DC, Nishimura N, et al. Species-specific pharmacology of trichloro(sulfanyl)ethyl benzamides as transient receptor potential ankyrin 1 (TRPA1) antagonists. Mol Pain 2007;3:39. [116] DeFalco J, Steiger D, Gustafson A, Emerling DE, Kelly M, Duncton MAJ. Oxime derivatives related to AP18: agonists and antagonists of the TRPA1 receptor. Bioorg Med Chem Lett 2010;20:276–9. [117] Mills JEJ, Brown AD, Ryckmans T, Miller DC, Skerratt SE, Barker CM, et al. SAR mining and its application to the design of TRPA1 antagonists. Med Chem Commun 2012;3(174):174–8. [118] Svensson M, Weigelt D, Inventors. TRPA1 receptor antagonist. Patent WO2012050512A1; 2012. [119] Vallin KS, Sterky KJ, Nyman E, Bernstrom J, From R, Linde C, et al. N-1-Alkyl-2-oxo-2-aryl amides as novel antagonists of the TRPA1 receptor. Bioorg Med Chem Lett 2012;22(17):5485–92. [120] Bilodeau MT, Egbertson M, Green A, Li Y, Hartnett JC, Inventors. Novel TRPA1 antagonists for treatment of pain and other TRPA1-associated diseases. Patent WO2011043954A1; 2011. [121] Berthelot DJC, Gijsen HJM, Zaja M, Rech J, Lebsack A, Xiao W, et al., Inventors. Heterocyclic amides as modulators of TRPA1. Patent WO2010141805A1; 2010. [122] Suzuki T, Kobayashi K, Asari S, Shiraishi S, Okuzumi T, Inventors. Heterocyclic amide derivative and pharmaceutical product containing same. Patent WO2013108857A1; 2013. [123] Gijsen HJM, Berthelot DJC, Decleyn MAJ, Inventors. 3,4-Dihydropyrimidine TRPA1 antagonists. Patent WO2009147079A1; 2009. [124] Gijsen HJ, Berthelot D, De Cleyn MA, Geuens I, Brone B, Mercken M. Tricyclic 3,4-dihydropyrimidine-2- thione derivatives as potent TRPA1 antagonists. Bioorg Med Chem Lett 2012;22(2):797–800. [125] Wu X, Spencer DK, Chen P, Zhou D, Peng S, Inventors. Compounds useful for treating disorders related to TRPA1. Patent US8530487B1; 2013. CHAPTER 11 Is TRPV3 a Drug Target?—A Decade of Learning Neelima Khairatkar Joshi* Glenmark Research Centre, Navi Mumbai, Glenmark Pharmaceuticals Ltd, India *Corresponding author: [email protected]

OUTLINE

Introduction 195 Neuropathic Pain 200 Osteoarthritic Pain 200 TRPV3 Tissue Expression 196 TRPV3 Activation 196 Safety of TRPV3 Antagonists 202 Functional Role of TRPV3 Channel: Pharma Efforts in Discovering TRPV3 Evidence from Genetic Studies 198 Antagonists in Treatment of Human Diseases 202 TRPV3 and Human Diseases 198 Skin Diseases 198 References 203

INTRODUCTION

Cloning of the TRPV3 channel gene was simultaneously reported by three independent laboratories. Human TRPV3 gene was cloned from the human testes library during database searches for TRP-related Expressed Sequence Tags (ESTs) by one group and during homology searches for the TRPV1-like gene of the Celera Human and public databases by another group [1,2]. Murine TRPV3 gene was cloned from the skin of a newborn mouse almost simultaneously [3]. TRPV3 channel protein has a prototype TRP structure with six transmembrane domains, a pore-loop region between the last two membrane-spanning domains, and three predicted ankyrin domains. The TRPV3 channel has low identity of ~43%, 42%,

41%, and 30% to TRPV1, TRPV4, TRPV2, and TRPV5 and TRPV6 channels, respectively [2]. Recent insights into structural and biophysical features are beyond the scope of this chapter, and interested readers can refer to Refs. [4,5].

TRPV3 TISSUE EXPRESSION

TRPV3 shows neuronal and nonneuronal expression in humans and mice. Although initial studies did not demonstrate TRPV3 expression in rodent dorsal root ganglia (DRG) neurons [3,6], subsequently its expression on human and rodent DRG, trigeminal, hippocampal, and superior cervical neurons and peripheral nerve endings and rat brain was convincingly shown [1,2,7–10]. In the brain, TRPV3 is expressed in ventral motor neurons, superior cervical ganglion neurons, deeper laminae of dorsal horn, and nigral dopaminergic neurons, although its physiological role in these areas remains to be investigated. There is ample evidence that TRPV3 channel is expressed in skin keratinocytes in man [1,2] and mice [3,6,11–13]. It is abundantly expressed in differentiated basal layer cells and undifferentiated suprabasal layers of mouse epidermis, suggesting a role in cutaneous thermosensation [14–16]. Heat-induced TRPV3 signals are believed to be transferred from keratinocytes to nearby free nerve endings in the dermis, thereby causing sensation of warmth or heat. Although no synapses are found between keratinocytes and sensory termini, it is possible that they transduce signals to surrounding nerves through chemical mediators. Its physiological role in skin barrier formation, hair growth, and cutaneous growth and survival is recently reviewed in depth elsewhere [4]. Apart from the skin, TRPV3 is also expressed in nasal and oral cavity, mainly in the tongue and palate [13]. In the mouth, TRPV3-like immunoreactivity is restricted to epithelial layers facing oral cavity. Its presence in the tongue and palate implies its function as a target for fla- vor actions of several plant-derived derivatives such as carvacrol and eugenol.

TRPV3 ACTIVATION

The mammalian nervous system detects temperature over a wide range, extending from noxious cold to noxious hot. Miscellaneous ThermoTRPs are implicated in this thermosensory function. TRPV1, TRPV2, TRPV3, and TRPV4 are responsible for warm to hot temperature sensing [16]. TRPV3 is activated by innocuous warmth (31-39 °C), which is further maintained at higher noxious temperatures. Specific structural domains involved in this are yet to be determined but could be associated with the TRP box region at C terminal as seen with some other thermoTRPs. TRPV3 channel can be alternatively activated by nonthermal stimuli as well. A variety of nonselective natural compounds such as camphor, menthol, carvacrol, and eugenol have been recognized as TRPV3 activators [6,13]. Camphor is a plant-derived monoterpene, a weak agonist of TRPV3 channel and known to sensitize human skin to warm temperatures [13]. A recent study has demonstrated that the cysteine residues at the pore region of the channel play a critical role in its camphor sensitivity [17]. Menthol is a monoterpene-based partial agonist of TRPV3 channel and shows much less (~65%) activation of the channel compared TRPV3 Activation 197 to that by camphor. Paradoxically, in humans, camphor and menthol exhibit the opposite thermal sensation of feeling warm and cool, respectively. The reason for this is not completely understood but could be due to nonselective and concentration dependent activation of other TRP channels by these agents. Carvacrol and eugenol are major ingredients of oregano and clove, respectively, and are nonselective activators of TRPV3 [13]. Overall, TRPV3 activation by these pungent, plant- derived chemicals implies a central role for TRPV3 in the protective, chemosensory mechanisms of skin. 2-Aminoethoxydiphenyl borate (2-APB) was first identified as a synthetic, nonterpenoid agonist of the TRPV3 channel. Although it is a widely used TRPV3 agonist in majority of investigations, caution should be exercised in interpreting the results because it is known to be a nonselective agent with simultaneous action on receptors other than TRPV3 as reviewed earlier [18]. The 2-APB activation of TRPV3 was very recently demonstrated to depend on presence of two residues H426 and R696 in the cytoplasmic region of channel and is separable from camphor or voltage response in human, dog, and frog [19]. Another recently described TRPV3 agonist is incensol acetate, a constituent of Boswellia cerrata resin [20]. However, its nociceptive properties remain to be described. Various agonists listed earlier are shown to reduce the heat activation threshold of TRPV3. Despite recognition of several exogenous activators as listed earlier, a specific and selective TRPV3 channel agonist with nociceptive properties is yet to be identified. Several endogenous biochemical mediators are also identified as TRPV3 sensitizers and activators. These are mainly produced during tissue injury or inflammation, and they include protein kinase C (PKC), nitric oxide (NO), and n-6 unsaturated fatty acids, in particular arachidonic acid (AA) as reviewed earlier [18]. Activation of PKC in skin cells and sensory neurons is an important event downstream of pro-inflammatory receptor activation. NO is one of the important and pleiotropic cell-signaling molecules that modulate diverse biological processes during inflammation and injury. NO-mediated signaling occurs via S-nitrosylation of TRP channel proteins as a posttranslational modification [21]. The TRPV3 channel belongs to a new category of cell surface receptors that can integrate NO and Ca2+ signals. Phospholipase C-coupled, G-protein coupled receptors (GPCRs) such as histamine and bradykinin may potentiate TRPV3 function under inflammatory conditions when these are produced and released in mass amounts. The TRPV3 channel could have an important role during inflammation as an integrator of signals from GPCRs at the level of neuronal input further to the PNS and CNS. Hence TRPV3 might be involved in the initiation and maintenance of sensory hypersensitivity during tissue inflammation. Arachidonic acid-mediated potentiation of the TRPV3 channel appears very interesting as it seems to be highly specific for TRPV3 because TRPV1, TRPV2, and TRPV4 are not activated by AA, and other saturated fatty acids are devoid of this effect [22]. Arachidonic acid is an endogenous mediator of inflammatory response in skin cells. It is released into extracellular milieu by infiltrating lymphocytes. In severe cases of “involved psoriasis,” AA concentration in the epidermis reaches very high levels, close to the concentration at which it shows robust TRPV3 potentiation under in vitro conditions [23,24]. Even under less severe conditions, the combined concentrations of free unsaturated fatty acids are likely to be sufficient to achieve TRPV3 activation. Moreover the in vitro studies demonstrate that TRPV3 activating effects of AA seem to increase with longer incubation. Hence AA seems to be a pathologically most relevant means for TRPV3 channel activation during skin inflammation. Overabundance 198 11. TRPV3 RECEPTORS AS DRUG TARGETS of AA is also found in patients with osteoarthritis. A recently published clinical trial shows association of AA with synovitis in the osteoarthritic (OA) knee [25]. High levels of (n-6) fatty acids in cancellous bone in osteoarthritis are also reported [26]. The TRPV3 channel has a unique mode of activation among thermoTRPs in that it is continuously sensitized on repeated agonist application [1,3,12,27]. This is in contrast to other structurally and functionally related TRP channels (such as TRPV1, TRPV4, and TRPA1), which are desensitized under such conditions. This feature has been observed both with recombinant TRPV3 and native TRPV3 on keratinocytes and is stimulus independent seen with all TRPV3 agonists [12]. The actual sensitization of TRPV3 channel is believed to involve relief from Ca2+-mediated inhibition of channel function [27].

FUNCTIONAL ROLE OF TRPV3 CHANNEL: EVIDENCE FROM GENETIC STUDIES

TRPV3 global knockout mice, as well as mice overexpressing TRPV3 in keratinocyte specific fashion, confirm role of TRPV3 in thermosensation and nociceptive signaling pathway [6,28]. This has been discussed in details earlier by us [18].

TRPV3 AND HUMAN DISEASES

The TRPV3 channel has a well-defined tissue distribution [6]. Its presence in peripheral nerves and its abundance in skin keratinocytes imply a pivotal role for TRPV3 in sensory pathways involved in pain and inflammatory skin diseases. Its confirmed role in thermal nociception and its implicated role in pain and cutaneous signaling following peripheral nerve activation and/or injury is amply discussed elsewhere [4,10,15,28–30].

Skin Diseases TRPV3 activation on keratinocytes may lead to the release of endogenous substances that, in turn, activate adjacent cutaneous sensory nerve terminals. A number of candidate media- 2+ tors of pathological nature such as prostaglandin E2 (PGE2), ATP, increased intracellular Ca , and IL-1α are released by activated TRPV3 on keratinocytes [13,15,28]. Keratinocytes cultured from TRPV3 overexpressing animals showed augmented TRPV3 ion channel activity and increased PGE2 mediator release in a gene-dosage dependent manner and in response to 2-APB, camphor, or heat [28]. TRPV3-dependant PGE2 release from keratinocytes may contribute to sensory functions, including acute nociception and hyperalgesia via the Prostaglandin E receptor (EP) receptor activation. TRPV3-dependant IL-1α release may have special importance because IL-1α is an important mediator of cutaneous inflammation. Release of IL-1 from keratinocytes in a TRPV3-dependant manner also supports the involvement of TRPV3 in the development of psoriasis [13]. Some of the antipsoriatic drugs such as hydrocortisone and

2-OH vitamin D3 work through IL-1 release inhibition [31]. Itch is an inflammatory and sensory response wherein cutaneous epidermal cells and neurites play a major role, and TRPV3 is expressed on both. TRPV3 and Human Diseases 199

There is ample evidence from TRPV3 gain-of-function (GOF) mutations in mice (as well as in humans) that support the hypothesized role of the TRPV3 channel in itchy skin diseases. The TRPV3 Gly573Ser GOF in TRPV3 is found to cause itchy dermatitis in rodents [32,33]. Another study on transgenic Nh mice overexpressing mutated TRPV3 channels showed enhanced release of nerve growth factor (NGF), a powerful sensitizer of sensory neurons, from epidermal sheets [34]. Further, the same GOF mutation is recently reported in human TRPV3 and was found to steer the skin to a hyperkeratotic itchy cutaneous condition, called Olmstead syndrome [35]. In atopic dermatitis patients, increased sprouting of epidermal C fibers is seen, inducing hypersensitivity to itching, which aggravates the disease, and this is accompanied by higher expressions of TRPV3 mRNA [29,36]. The postulated role of TRPV3 in skin-nerve cross talk and in modulating cutaneous hypersensitivity in inflammatory dermatoses such as itchy dermatitis and psoriasis is further strengthened by the fact (although histamine is the best-known pruritogen and a main target for antipruritic therapies) that histamine H1 receptor (H1R) antagonists are often ineffective in certain pruritic skin conditions, including dry skin pruritus [37]. There is increasing belief that miscellaneous inflammatory mediators other than histamine could have a key role in itching. Some of the newly recognized putative pruritogens, as depicted in Figure 11.1, are indeed activators of TRPV3 implicating its role in histamine-resistant itch. Taken together, the preceding observations strongly support the involvement of TRPV3 in skin inflammatory diseases accompanied by hyperkeratosis and itch, and few pharmaceutical industries are exploring the opportunity to make TRPV3 antagonist-based therapeutics [36,39]. Studies demonstrating in vitro attenuation of biochemical mediator release or reduction of psoriatic skin xenograft growth in vivo in severe combined immunodeficiency (SCID) mice by TRPV3 specific antagonist or siRNA are much awaited to endorse a functional role for TRPV3 in human skin diseases.

FIGURE 11.1 TRPV3-mediated cutaneous signaling between keratinocyte TRPV3 and nearby TRPV3 on free nerve endings via chemical mediators or enhanced TRPV3 activity due to gain-of-function mutation resulting in itchy skin disease condition [38]. 200 11. TRPV3 RECEPTORS AS DRUG TARGETS Neuropathic Pain Based on the unique property of TRPV3 channels of getting increasingly sensitized on repeated agonist stimulation, it is tempting to speculate that in chronic pain states continuous sensitization of TRPV3 could contribute to the ongoing pain signal transmission. In addition, there is good evidence that TRPV3 channel levels are increased in certain pain states. TRPV3 expression is up-regulated in the Chung model of neuropathic pain in rats [40] wherein the animals exhibited typical symptoms of neuropathic pain such as thermal hyperalgesia, mechanical hyperalgesia, and mechanical allodynia, along with a robust increase in the TRPV3 message at the L4 and L5 DRG neurons. As far as human pain is concerned, women experiencing breast pain (mastalgia) showed up-regulation of TRPV3 and TRPV4 in basal keratinocytes in the skin correlating with disease score [30]. Enhanced TRPV3-like immune-reactivity has also been reported in human neuropathic pain patients [2]. Skin and nerve preparations from patients with nerve injury, avulsed DRG, injured spinal roots, and diabetic neuropathy also showed significantly increased expression of the TRPV3 channel in injured brachial plexus nerves [10]. By contrast, decreased TRPV3-like immunoreactivity was seen in skin biopsies taken from diabetic neuropathy patients and in patients with injured ventral spinal cords. Reduced TRPV3 signals could be due to abnormally thin epidermis causing loss of TRPV3 channel in the former and impaired peripheral transport of channel in the latter case.

Osteoarthritic Pain There is good evidence to implicate TRPV3 activation in OA pain. NGF, NO, and prostanoids are prevalent inflammatory stimuli during OA and are implicated in synovial tissues damage and peripheral nerve activation (Figure 11.2). TRPV3 channel is expressed on peripheral nerves and the DRG neurons. There is excessive production of mediators such as NO and AA, which are known to cause direct activation of TRPV3 channels. Several preclinical and clinical studies have demonstrated increased AA levels in the cartilage, serum, and synovial fluid in OA patients as compared to nonOA controls. Very recently, clinical association was found between plasma AA levels and histological severity of damage to cartilage in OA patients [25]. In another study, cancellous bone from OA patients was found to contain almost double the amount of AA than that seen in a comparator osteoporotic bone and correlated with severity of the joint disease in OA patients [26]. In a canine study, dogs fed on low amounts of n-3 polyunsaturated fatty acids

(PUFA) had higher amounts of AA in the synovium and that correlated to [1] increased PGE2 in the synovial fluid [2], increased OA radiographic changes, and [3] decreased limb function. Conversely, dogs fed high levels of n-3 and low levels of n-6 PUFA had the opposite effects [41]. Taken together, these findings suggest that TRPV3 channels, which are activated during peripheral nerve injury and by inflammatory soup components (in particular by the overabundant AA in joint microenvironment during OA), represent a potential pain target. Another interesting association between OA and TRPV3 is the observation that postmenopausal women have reduced levels of estrogen and estrogen metabolites and have high incidence of OA. Estrogen metabolites are known to play an important role in the metabolism of AA, and increased AA levels in the postmenopausal women could be responsible for TRPV3 activation [42,43]. Given the abundance of AA during OA in rodents, dogs, and humans, the well-documented correlation TRPV3 and Human Diseases 201

FIGURE 11.2 TRPV3 activation on DRG neuron due to nerve injury, and by the inflammatory mediators such as AA and NO in the joint innervation can potentially cause pain signal transmission during OA. of AA levels with several cardinal features of OA (such as pain, synovitis, and bone loss), and the direct activation of TRPV3 by AA, TRPV3 blockade appears to be a clinically relevant approach for potential OA treatment. The contribution of TRPV3-mediated pain due to AA overproduction would largely depend on the proportion of AA directed toward TRPV3 activation versus cellular pro-inflammatory prostaglandin synthesis. It is important to point out that selective TRPV3 antagonists potently inhibit AA-induced TRPV3 channel activation in vitro and show robust efficacy in preventing inflammatory hyperalgesia in the Freund’s Complete Adjuvant (FCA) model, mechanical hyperalgesia in the Chronic Constriction Injury (CCI) model, and hyperalgesia in MIA- induced Monosodium Iodoacetate (OA) model in rats [44,45]. Of note, TRPV1 antagonists, too, had shown promising efficacy in animal models of OA but did not meet a primary efficacy end point in a clinical trial [46]. Currently, it is not known if TRPV1 and TRPV3 channels differentiate in terms of their contribution to human OA based on differential receptor regulation in chronic state or differential activation by receptor specific components in the inflammatory soup in the joint environment or both. Hence it remains to be seen if TRPV3 antagonists could be efficacious in clinical trials versus TRPV1 antagonists. 202 11. TRPV3 RECEPTORS AS DRUG TARGETS SAFETY OF TRPV3 ANTAGONISTS

Detailed investigation of TRPV3 knockout mice showed no gross anatomical skin defects. Epidermal and dermal layer thickness, keratinocyte specific markers, and skin epidermal barrier integrity were all found to be normal [6]. By contrast, it was shown that in rodents with a constitutively acting mutant TRPV3 channel, the channel overactivity results in a hair loss phenotype [32,47]. In summary, it is TRPV3 overactivity, and not blockade, that appears to be deleterious. Hyperthermia produced by the TRPV1 antagonists during preclinical studies and clinical trials has raised safety concerns around all “thermoTRP” channel targets. TRPV3 senses a physiological range of temperatures of 33-39 °C that overlaps with TRPV4 and also a nonphysiological range up to 50 °C. On the contrary, TRPV1 is activated only at nonphysiological temperatures. Hence, it seems likely that whereas TRPV1 functions as a temperature regulator, TRPV3 and TRPV4 may just be temperature sensors. Moreover, TRPV1 antagonist-associated hyperthermia could potentially be a consequence of suppression of agonist-induced hypothermia (i.e., blockade of a tonically active channel protein). On the contrary, there is no reported TRPV3 agonist that produces hypothermia. This is in accord with the observation that TRPV3 overexpressing transgenic mice do not show subnormal rectal temperature compared to the wild-type mice [28]. Evidence for lack of effect of TRPV3 antagonists on body temperature comes from preclinical studies with TRPV3 selective antagonists. TRPV3 selective antagonists synthesized at Glenmark did not produce any significant change in body temperature in rodents, ferrets, and humans (the author’s unpublished results). Scalding injury due to impaired noxious heat sensation is another safety concern associated with the TRPV1 antagonist [48]. It is speculated that there could be a significant functional redundancy between TRPV1, V3, and V4 for warmth sensation; therefore, TRPV3 channel blockade might not result in significant thermosensory deficit [4].

PHARMA EFFORTS IN DISCOVERING TRPV3 ANTAGONISTS IN TREATMENT OF HUMAN DISEASES

Due to its expression pattern, its unique property to undergo continuous sensitization, and its demonstrated role in hyperalgesia, pain transduction, and inflammatory signaling in preclinical studies, TRPV3 appears to be a valid target for treating chronic pain conditions such as osteoarthritis, neuropathic pain such as diabetic neuropathy, as well as skin disorders such as itch and dermatitis. This has attracted the attention of pharma companies to initiate TRPV3 antagonist discovery programs. Hydra Biosciences initiated drug discovery research around a TRPV3 target in a search of novel analgesics. Their program was partnered with Pfizer under a codevelopment agreement that eventually was terminated at the preclinical stage [49,50]. Glenmark’s TRPV3 antagonist entered into clinical trials after demonstrating promising preclinical efficacy and safety. The program was out-licensed to Sanofi-Aventis. After successful completion of a Phase I trial, SAR 292833/GRC 15300 is currently undergoing global Phase II, proof-of-concept clinical trials [51]. We predict that TRPV3 antagonists will remain in the focus for potential treatment of pain and itch-associated skin diseases. REFERENCES 203 References [1] Xu H, Ramsey IS, Kotecha SA, Moran MM, Chong JA, Lawson D, et al. TRPV3 is a calcium-permeable temperature-sensitive cation channel. Nature 2002;418:181–6. [2] Smith GD, Gunthrope MJ, Kelsell RE, Hayes PD, Rellly P, Facer P, et al. TRPV3 is a temperature-sensitive vanilloid receptor-like protein. Nature 2002;418:186–90. [3] Peier AM, Reeve AJ, Anderson DA, Moqrich A, Earley TJ, Hergarden AC, et al. A heat-sensitive TRP channel expressed in keratinocytes. Science 2002;296:2046–9. [4] Nilius B, Bíró T. TRPV3: a ‘more than skinny’ channel. Exp Dermatol 2013;22:447–52. [5] Nilius B, Bíró T, Owsianik G. TRPV3: time to decipher a poorly understood family member! J Physiol 2014;592(2):295–304. [6] Moqrich A, Hwang SW, Earley TJ, Petrus MJ, Murray AN, Spencer KSR, et al. Impaired thermosensation in mice lacking TRPV3, a heat and camphor sensor in the skin. Science 2005;307:1468–72. [7] Frederick J, Buck ME, Matson DJ, Cartwright DN. Increased TRPA1, TRPM8 and TRPV2 expression in dorsal root ganglia by nerve injury. Neurogenic Corporation, Branford, CT, USA. Biochem Biophys Res Commun 2007;358:1058–64. [8] Guatteo E, Chung KK, Bowel TK, Bernard G, Mercury NB, Lipski J. Temperature sensitivity of dopaminergic neurons of the substantia nigra pars compacta: involvement of transient receptor potential channels. J Neurophysiol 2005;94:3069–80. [9] Hu HZ, Gu Q, Wang C, Colton CK, Tang J, Kinoshita-Kwada M, et al. 2-Aminoethoxydiphenyl borate is a common activator of TRPV1, TRPV2, and TRPV3. J Biol Chem 2004;279:35741–8. [10] Facer P, Casula MA, Smith GD, Benham CD, Chessell IP, Bountra C, et al. Differential expression of the capsaicin receptor TRPV1 and related novel receptors TRPV3, TRPV4 and TRPM8 in normal human tissues and changes in traumatic and diabetic neuropathy. BMC Neurol 2007;7:1–12. [11] Chung M-K, Lee H, Caterina MJ. Warm temperatures activate TRPV4 in mouse 308 keratinocytes. J Biol Chem 2003;278(34):32037–46. [12] Chung M-K, Lee H, Mizuna A, Suzuki M, Caterina MJ. TRPV3 and TRPV4 mediate warmth-evoked currents in primary mouse keratinocytes. J Biol Chem 2004;279:21569–75. [13] Xu H, Delling M, Jun JC, Clapham DE. Oregano, thyme and clove-derived flavors and skin sensitizers activate specific TRP channels. Nat Neurosci 2006;9:628–35. [14] Lee H, Caterina MJ. TRPV channels as thermosensory receptors in epithelial cells. Pflugers Arch 2005;451:160–7. [15] Mandadi S, Sokabe T, Shibasaki K, Katanosaka K, Mizuno A, Moqrich A, et al. TRPV3 in keratinocytes trans- mits temperature information to sensory neurons via ATP. Pflugers Arch 2009;458:1093–102. [16] Dhaka A, Viswanath V, Patapoutian A. TRP ion channels and temperature sensation. Annu Rev Neurosci 2006;29:135–61. [17] Sherkheli MA, Vogt-Eisele AK, Weber K, Hatt H. Camphor modulates TRPV3 cation channels activity by interacting with critical pore-region cysteine residues. Pak J Pharm Sci 2013;26(3):431–8. [18] Khairatkar-Joshi N, Maharaj N, Thomas A. The TRPV3 receptor as a pain target: a therapeutic promise or just some more new biology? Open Drug Discov J 2010;2:89–97. [19] Hu H, Grandl J, Bandell M, Petrus M, Patapoutian A. Two amino acid residues determine 2-APB sensitivity of the ion channels TRPV3 and TRPV4. PNAS 2009;106:1626–31. [20] Moussaieff A, Rimmerman N, Bregman T, Straiker A, Felder CC, Shoham S, et al. Incensole acetate, an incense component, elicits psychoactivity by activating TRPV3 channels in the brain. FASEB J 2008;22:3024–34. [21] Yoshida T, Inoue R, Morii T, Takahashi N, Yamamoto S, Hara Y, et al. Nitric oxide activities TRP channels by cysteine S-nitrosylation. Nat Chem Biol 2006;2:596–607. [22] Hu H-Z, Xiao R, Wang C, Gao NA, Colton CK, Wood JD, et al. Potentiation of TRPV3 channel function by unsaturated fatty acids. J Cell Physiol 2006;208:201–12. [23] Hammarstrom S, Hamberg M, Samuelsson B, Duell EA, Stawiski M, Voorhees JJ. Increase concentrations of nonesterified arachidonic acid, 12 l-hydroxy-5,8,10,14-eicosatetraenoic acid, prostaglandin E2, and prostaglandin F2alpha in epidermis of psoriasis. Proc Natl Acad Sci USA 1975;72:5130–4. [24] Brash AR. Arachidonic acid as a bioactive molecule. J Clin Invest 2001;107:1339–45. [25] Baker KR, Matthan NR, Lichtenstein A, Niu J, Guermazi N, Roemer F, et al. Association of plasma n-6 and n-3 polyunsaturated fatty acids with synovitis in the knee: the MOST study. Osteoarthritis Cartilage 2012;20(5):382–7. http://dx.doi.org/10.1016/j.joca.2012.01.021. 204 11. TRPV3 RECEPTORS AS DRUG TARGETS

[26] Plumb MS, Aspden RM. High levels of fat and (n-6) fatty acids in cancellous bone in osteoarthritis. Lipids Health Dis 2004;3:12. http://dx.doi.org/10.1186/1476-511X-3-12. [27] Xiao R, Tang J, Wang C, Colton CK, Tian J, Zhu MX. Calcium plays a central role in the sensitization of TRPV3 channel to repetitive stimulations. J Biol Chem 2008;283:6162–74. [28] Huang SM, Lee H, Chung M-K, Park U, Yu YY, Bradshaw HB, et al. Overexpressed transient receptor potential vanilloid 3 ion channels in skin keratinocytes modulate pain sensitivity via prostaglandin E2. J Neurosci 2008;28:13727–37. [29] Paus R, Schmelz M, Bíró T, Steinhoff M. Frontiers in pruritus research: scratching the brain for more effective itch therapy. J Clin Invest 2006;116:1174–86. [30] Gopinath P, Wan E, Holdcroft A, Facer P, Davis J, Smith GD, et al. Increased capsaicin receptor TRPV1 in skin nerve fibres and related vanilloid receptors TRPV3 and TRPV4 in leratinocytes in human breast pain. BMC Womens Health 2005;5:1–9. [31] Maruyama K, Zhang J-Z, Nihei Y, Ono I, Kaneko F. Regulatory effects of antipsoriatic agents on interleukin-1 production by human keratinocytes stimulated with gamma interferon in vitro. J Pharmacol Biophys Res 1995;8(1-2):41–8. [32] Asakawa M, Yoshioka T, Matsutani T, Hikita I, Suzuki M, Oshima I, et al. Association of a mutation in TRPV3 with defective hair growth in rodents. J Invest Dermatol 2006;126:2664–72. [33] Imura K, Yoshioka T, Hirasawa T, Sakata T. Role of TRPV3 in immune response to development of dermatitis. J Inflamm 2009;6:1–10. [34] Yoshika T, Imura K, Asakawa M, Suzuki M, Oshima I, Hirasawa T, et al. Impact of the Gly573Ser substitution in TRPV3 on the development of allergic and pruritic dermatis in mice. J Invest Dermatol 2009;129:714–22. [35] Lin Z, Chen Q, Lee M, Cao A, Zhang J, Ma D, et al. Exome sequencing reveals mutations in TRPV3 as a cause of Olmsted syndrome. Am J Hum Genet 2012;90:558–64. [36] Yamamoto-Kasai E, Imura K, Yasui K, Shichijou M, Oshima I, Hirasawa T, et al. TRPV3 as a therapeutic target for itch. J Invest Dermatol 2012;132:2109–12. http://dx.doi.org/10.1038/jid.2012.97. [37] Wahlgren CF, Hagermark O, Bergstrom R. The antipruritic effect of a sedative and a non-sedative antihistamine in atopic dermatitis. Br J Dermatol 1990;122:545–51. [38] Damann N, Voets T, Nilius B. TRPs in our senses. Curr Biol 2008;18:R880–9. http://dx.doi.org/10.1016/j.cub.2008.07.063. [39] Hwang SW, Bang S-S. Compounds for inhibiting TRPV3 activity and uses thereof. US 2010/0137260A1; 2010. [40] Bevan S, Ganju P, McIntyre P, Patapoutian A, Peier A, Song C. Transient receptor potential channel TRPV3 and its tissue. US Patent 7,396,910 B2; 2008. [41] Bartges J, Budsberg SC, Pazak H. Effects of different N6:N3 fatty acid diets on canine stifle osteoarthritis. In: Orthopaedic research society 47th annual meeting. San Francisco, CA, February; 2001. [42] Sowers MR, McConnell D, Jannaisch M, Buyuktur AG, Hochberg M, Jamadar DA. Estradiol and its metabolites and their association with knee osteoarthritis. Arthritis Rheum 2006;54(8):2481–7. [43] Minerd J. Low estrogen linked to knee osteoarthritis in women. MedPage Today; 2006. medpagetoday.com. [44] Khairatkar-Joshi N. TRPV3 antagonist in pain management—generation next? In: SMi 8th annual conference— pain therapeutics. Copthorne, London; 2008. [45] Gullapalli S, Thomas A, Chaudhari SS, Lingam VSPR, Kattige V, Gudi GS, et al. GRC 15300—a novel, selective, orally active Trpv3 antagonist in clinical trial for potential treatment of chronic inflammatory and neuropathic pain. In: CHI’s—World Pharma Congress. Targeting pain with novel targets. Philadelphia, PA, USA; 2010. [46] Quiding H. Clinical experiences of TRPV1 antagonism, “The AZD1386 Story”. In: SMi, pain therapeutics; 2011. [47] Imura K, Yoshioka T, Hikita I, Tsukahara K, Arimura A, Sakata T. Influence of TRPV3 mutation on hair growth cycle in mice. Biochem Biophys Res Commun 2007;363:479–83. [48] Crutchlow M. Pharmacologic inhibition of TRPV1 impairs sensation of potentially injurious heat in healthy subjects. In: ASCPT 2009 in gaylord national resort & convention centre on the potomac, National Harbor, Maryland; 2009. [49] Hydra Biosciences and Pfizer Global Research & Development Sign Collaboration Agreement. Leverages Hydra’s Proprietary Ion Channel Technology in the area of pain. Cambridge, MA; 2007 www.hydrabiosciences. com/pdf/press_releases/2007_07_26.pdf. [50] Luke Timmerman 1/16/09. xconomy.com, [email protected]. [51] Glenmark completes Phase I trial for GRC 15300—a TRPV3 inhibitor for treatment of pain. Mumbai: Press Release; 2011. CHAPTER 12 Small Molecule Agonists and Antagonists of TRPV4 Matthew A.J. Duncton* Renovis, Inc. (a wholly owned subsidiary of Evotec AG), South San Francisco, California, USA *Corresponding author: [email protected]

OUTLINE

Introduction 205 TRPV4 Antagonists 210 Ruthenium red 210 TRPV4 Agonists 206 HC-067047 from Hydra Biosciences 211 4α-PDD and Related Phorbol Esters 206 RN-1734 and RN-9893 from Renovis 212 Proposed Endogenous Agonists Antagonists from GSK 212 of TRPV4 207 Antagonists from Pfizer 214 GSK1016790A and Relatives from Other Antagonists of TRPV4 215 GlaxoSmithKline 208 Conclusions 215 RN-1747 from Renovis 210 Natural Products from Herbal Extracts 210 References 216

INTRODUCTION

Transient receptor potential vanilloid 4 (TRPV4), originally identified in 2000 as an osmosensor [1–3], is a member of the transient receptor potential (TRP) superfamily of ion channels. This superfamily, which consists of approximately 28 members, is divided into 7 subfamilies (TRPA, TRPC, TRPM, TRPML, TRPN, TRPP, TRPV), based on sequence homology [4,5]. TRPV4 functions as a Ca2+-permeable, nonselective cationic ion channel and is activated by numerous stimuli such as endogenous and exogenous small molecule ligands,

TRP Channels as Therapeutic Targets 205 © 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/B978-0-12-420024-1.00012-6 206 12. Small Molecule Agonists and Antagonists of TRPV4 temperature (>27 °C), osmolarity, and phosphorylation by Src, protein kinase A (PKA), and protein kinase C (PKC) [1–3,6–11]. As documented by expression studies, TRPV4 seems to exhibit a broad tissue distribution [1–3,12–19]. Since its discovery in 2000, TRPV4 has been implicated in numerous biological processes and medical conditions. Examples include osmolarity sensing and regulation [1–3], thermosensation and regulation [6,12,20], mechanosensation [20,21], bone formation and remodeling [22–26], genetic disorders [24,25,27–30], pain and inflammatory conditions [31–38], bladder disorders [17,18], acute lung injury [39– 41], cardiovascular conditions [42], and metabolic disorders [43], among others. The purpose of this chapter is to highlight the most prominent small molecules that have been described as either agonists, or antagonists, of TRPV4. As such it should be regarded as an extension of two related articles published in 2010 and 2011 [44,45]. Because extensive coverage of the biology of TRPV4 is not included, readers are directed to some of the excellent reviews that already exist on this subject [46–50].

TRPV4 AGONISTS

4α-PDD and Related Phorbol Esters Phorbol esters, some of which are well-known tumor-promoters via activation of PKC, have also been described as agonists of TRP ion channels. For example, resiniferatoxin (RTX) 1 (Figure 12.1), a daphnane diterpene isolated from the latex of Euphorbia resinifera, is a potent activator of TRPV1, with a potency 103-105 times greater than pure capsaicin [51]. A search of related phorbol esters, by Nilius and coworkers, identified 4α-phorbol 12,13-didecanoate (4α-PDD) 2, as a potent activator of human and murine TRPV4 [52]. Significantly, 4α-PDD is not an activator of PKC (EC50 > 25 μM; [52]). A related phorbol ester, phorbol 12-myristate, 13-acetate (PMA) 3, was also found to be an activator of TRPV4, but was ca. 50 times less effective than 4α-PDD [52]. Unlike 4α-PDD, activation of PKC has been noted for PMA [52]. Structure-activity relationships (SARs) with 4α-PDD and relatives have also been investigated by the research groups of Nilius and Appendino [53]. This collaboration has revealed some fascinating SARs. For example, the 4-hydroxyl group was found to be essential for activity at TRPV4 because a deoxy congener of 4α-PDD, to give compound 4, was unable to

O O O O C9H19 O C13H27 O O O O C9H19 H O OH H H O H H OH HH OH HH O OMe OHO O HO O HO O OH OH Resinaferatoxin (RTX) 1 4α-PDD 2 PMA 3 TRPV4 EC50 = 0.37 µM

FIGURE 12.1 RTX, 4α-PDD, and PMA. TRPV4 Agonists 207

O O O O O O C9H19 O C5H11 O C7H15 O O O O C9H19 C5H11 C7H15 H H H

OH HH OH HH OH HH O OHO OHO OH OH OH Deoxygenated version of 4α-PDD 4 4α-PDH 5 Phorbol dioctanoate 6 TRPV4 EC50 = 0.07 µM TRPV4 EC50 > 50 µM TRPV4 EC50 > 50 µM O O O OH C9H19 O O O C9H19 C9H19 H OH

OH HH H HH O OHO HO OH OH Mono-decanoate 7 4α-LPDD 8

TRPV4 EC50 = 0.45 µM TRPV4 EC50 = 0.18 µM

FIGURE 12.2 Phorbol relatives of 4α-PDD. activate TRPV4 (Figure 12.2). Studies with a series of 12,13-diesters, in which the carbon chain length was varied, resulted in the identification of a particularly potent agonist, 4α-phorbol 12,13-dihexanoate (4α-PDH) 5, which was approximately fivefold more active at TRPV4 than 4α-PDD. Interestingly, 4α-PDH and 4α-PDD represent the most potent 12,13-diesters found in this study because other variations in chain length resulted in a substantial reduction in TRPV4 activity. For example, phorbol dioctanoate 6 was essentially inactive at TRPV4, with an EC50 greater than 50 μM. It was also possible to remove one of the decanoate chains from 4α-PDD and still retain activity at TRPV4. For instance, mono-decanoate analog 7, in which the 12-ester group of 4α-PDD was removed, showed diminished relative efficacy to 4α-PDD, but with a comparable EC50 value. Finally, observations with 4α-lumiphorbol didecanoate (4α-LPDD) 8, a unique (2π-2π) photocyclized adduct of 4α-PDD, revealed differences between the interactions of their respective diterpene cores with the TRPV4 receptor.

Proposed Endogenous Agonists of TRPV4 A variety of endocannabinoids, such as anandamide 9 and 2-arachidonoylglycerol (2-AG) 10 (Figure 12.3), have been shown to activate TRPV4 via an indirect mechanism [7,8,52]. Hydrolysis of anandamide, or 2-AG, by their respective degrading enzymes, fatty-acid amide hydrolase, or mono-acyl glycerol lipase, yields arachidonic acid (AA) 11, which also serves as an indirect agonist of TRPV4 [8]. Through a thorough examination of the downstream metabolic fate of AA, Nilius and coworkers were able to establish that TRPV4 activation was occurring via the downstream metabolites, 5,6-eopxyeicosatrienoic acid (5,6-EET) 12, and 208 12. Small Molecule Agonists and Antagonists of TRPV4

OH O O O OH N O OH H OH

Anandamide 9 2-Arachidonoylglycerol (2-AG) 10 Arachidonic acid (AA) 11 O O O O O O OH OH O P O P O O O

5,6-EET 12 8,9-EET 13 Dimethylallyl pyrophosphate (DMAPP) 14

FIGURE 12.3 Anandamide, 2-AG, AA, and endogenous agonists of TRPV4.

8,9-epoxyeicosatrienoic acid (8,9-EET) 13 [8]. These two epoxyeicosatrienoic acids are formed by the cellular oxidation of AA by a cytochrome P450 epoxygenase pathway, using the CYP 2C9 isoform. Thus, inhibitors of CYP 2C9 were able to abolish the activation of TRPV4 by AA [8]. The formation of 5,6-EET, or 8,9-EET, seems to be an important event for the activation of TRPV4 by osmotic cell swelling, as hypotonic cell swelling induces phospholipase-A2 activation and release of AA from the cell-membrane [7,8]. Inhibition of the P450 epoxygenase activity with miconazole (an inhibitor of CYP 2C9) strongly retarded the response of TRPV4 to hypotonic cell swelling. In contrast, the response of TRPV4 to other activating stimuli such as heat, or 4α-PDD, was unaffected by inhibition of P450 epoxygenase activity, indicating that these stimuli couple to TRPV4 by a pathway not involving AA. Dimethylallyl pyrophosphate (DMAPP) 14, an intermediate in the mevalonate metabolic pathway and found endogenously at nanomolar to micromolar concentrations, has also been identified as an agonist of TRPV4 (EC50 = 2.5 μM) [54]. At higher concentrations, DMAPP was also found to act as an antagonist at TRPV3 (IC50 = 10.4 μM). In vivo, DMAPP could promote inflammation in mice, the activity of which was blocked by codosing with the TRPV4 antagonists RN-1734 and HC-067047 [54]. Taken together with previous results on the action of mevalonate metabolites at TRP channels, a role for these metabolites as modulators of sensory TRP channels was proposed [54].

GSK1016790A and Relatives from GlaxoSmithKline GlaxoSmithKline (GSK) has described some aspects of the medicinal chemistry and detailed in vivo pharmacology with a potent TRPV4 agonist, GSK1016790A 19 (Figure 12.4), and a series of structurally related relatives [55–66]. The development of the medicinal chemistry was initiated with the identification of compound 15 as a submicromolar agonist of TRPV4

(EC50 = 0.7 μM). Previously, compound 15 was known to be a potent inhibitor of cathepsin

K (IC50 = 2 nM) [65,66]. Through a series of detailed structure-activity studies, the chemistry group at GSK was able to abolish the activity at cathepsin K, while dramatically improving the functional activity at TRPV4. This resulted in the identification of a number of azepine (e.g., 16), diaminobutane (e.g., 17), or diaminopropane (e.g., 18) analogs as potent agonists of TRPV4, with favorable rat pharmacokinetic profiles [65,66]. Further refinement furnished TRPV4 Agonists 209

O O O H H N N N N H N H S S O S N O N O O CN O O CN TRPV4 Hit (originally, a cathepsin K inhibitor) 15 Azepine selective TRPV4 agonist 16 TRPV4 EC50 = 707 nM TRPV4 EC50 (HAC assay) = 280 nM K = 2 nM Cat IC50 Cat K IC50 > 10,000 nM

F O Cl O OH H O O H H N N N N N S N S H H H S O S O O O Cl F Diaminobutane TRPV4 agonist 17 Diaminopropane TRPV4 agonist 18 TRPV4 EC50 FLIPR = 7 nM TRPV4 EC50 FLIPR = 20 nM TRPV4 EC50 (HAC assay) = 210 nM TRPV4 EC50 (HAC assay) = 20 nM Rat PK Rat PK CL = 8.8 mL/min/kg CL = 2.6 mL/min/kg Vd = 0.4 L/kg Vd = 0.3 L/kg %F = 88% %F = 41% HO O O O H N N N S S N H O O Cl Cl

GSK1016790A 19 TRPV4 potencies TRPV4 HEK FLIPR = 5.0 nM Human chondrocytes TRPV4 = 3.0 nM Bovine chondrocytes TRPV4 = 1.0 nM Mouse chondrocytes TRPV4 = 18.5 nM Rat chondrocytes TRPV4 = 10 nM Dog chondrocytes TRPV4 = 1.0 nM TRPV1 HEK FLIPR = 50 nM

FIGURE 12.4 GSK1016790A and related agonists from GlaxoSmithKline.

GSK1016790A 19, which is based around a piperazine linker [63–66]. Details of the in vitro and in vivo profile of GSK1016790A have been published [63,64]. For example, GSK1016790A is a potent agonist of TRPV4, with an EC50 of 3-5 nM at hTRPV4 and significant activity at bovine (EC50 = 1 nM), mouse (EC50 = 18.5 nM), rat (EC50 = 10 nM), and dog (EC50 = 1 nM) TRPV4 [63]. Significantly, the relative efficacy of GSK1016790A at TRPV4 is much greater than that of 4α-PDD. For example, the current density evoked by stimulation with GSK1016790A at 10 nM was over twice that recorded with 4α-PDD at a concentration of 10 μM [63]. In addition, GSK1016790A induces Ca2+ influx in HEK-293 cells, which do not express TRPV4

(EC50 = 50-100 nM). This calcium influx did not occur as a result of TRPV1 activation. In vivo, GSK1016790A induces bladder overactivity when infused directly into the bladders of mice. This effect was absent in TRPV4-knockout mice and led to the conclusion that TRPV4 plays a 210 12. Small Molecule Agonists and Antagonists of TRPV4 critical role in urinary bladder function [63]. Unfortunately, potent stimulation of the TRPV4 receptor has catastrophic side effects, as observed when GSK1016790A was administered in- travenously to mouse, rat, or dog [64]. Such administration resulted in endothelial failure and circulatory collapse, resulting in a dose-dependent drop in blood pressure and death [64]. In TRPV4-knockout mice, the effect of GSK1016790A on blood pressure, or heart rate, was absent at doses greater than 10-fold the lethal dose in wild-type counterparts, indicating the effect was mediated by TRPV4 [64]. Further evaluation in dogs indicated that cardiac output was severely reduced on exposure to GSK1016790A, which was associated with decreased stroke volume and delayed bradycardia [64]. Overall, the catastrophic side effects associated with systemic exposure to a potent TRPV4 agonist, such as GSK1016790A, have resulted in a natural avoidance of such compounds as potential therapeutic agents.

RN-1747 from Renovis Vincent et al. at Renovis have identified an arylsulfonamide TRPV4 agonist, RN-1747 20

(Figure 12.5) [67]. RN-1747 (EC50 = 0.77 μM) activates human TRPV4 with a similar potency to 4α-PDD, but is somewhat less active than 4α-PDD against rat TRPV4 (EC50 = 4.1 μM), or mouse TRPV4 (EC50 = 4.1 μM). At higher concentrations, RN-1747 serves as an antagonist of TRPM8 (IC50 = 4 μM), but is selective against other TRP channels, such as TRPV1 and TRPV3 [67].

Natural Products from Herbal Extracts Bisandrographolide A (BAA) 21 (Figure 12.6), a natural product isolated from an extract of

Andrographis paniculata, was shown to be an activator of mouse TRPV4 (EC50 = 0.79-0.95 μM) [68]. In addition, BAA was found to be relatively selective for TRPV4, with no activity noted at related TRP receptors, TRPV1, TRPV2, or TRPV3 [68].

TRPV4 ANTAGONISTS

Ruthenium red Ruthenium red 22 (Figure 12.7) was one of the first small molecules to be described as an antagonist of TRPV4 [52]. The compound, a well-known cationic dye dating from the nineteenth century, functions as a pore blocker of the TRPV4 channel. Although ruthenium

NO2 O Ph Cl S N N O

RN-1747 20

hTRPV4 EC50 = 0.77 µM

FIGURE 12.5 TRPV4 agonist, RN-1747. TRPV4 Antagonists 211

O O H O OH

H OH OH OH Bisandrographolide A (BAA) 21 TRPV4 EC50 = 0.79-0.95 µM

FIGURE 12.6 Bisandrographolide A (BAA), a natural product active as a TRPV4 agonist.

H2N NH2 H2N NH2 H2N NH2 Cl H2N Ru O Ru O Ru NH2 6 H2N NH2 H2N NH2 H2N NH2 Ruthenium red 22

FIGURE 12.7 TRPV4 antagonist, ruthenium red. red has been utilized extensively as a probe of TRPV4 function, it suffers from poor selectivity because it also interacts with numerous ion channels and biological targets [44,45]. Such polypharmacology may be highly undesirable in a proof-of-concept probe compound, and ruthenium red has now been superseded by more potent and/or selective TRPV4 antagonists such as HC-067047, RN-1734, and GSK2193847, among others (see following sections).

HC-067047 from Hydra Biosciences Hydra Biosciences have reported the structure of a potent TRPV4 antagonist, HC-067047 23 (Figure 12.8), and utilized the compound as a proof-of-concept probe in animal models of human disease. HC-067047 is a potent inhibitor of hTRPV4 (IC50 = 48 nM), rTRPV4 (IC50 = 133 nM), and mTRPV4 (IC50 = 17 nM) (Note: Various activating stimuli can be used.) [69]. When dosed to rodents, no effect on heart rate, core body temperature, thermal selection behavior, water

O CF3 HC-067047 23 NH hTRPV4 IC50 = 48 nM rTRPV4 IC50 = 133 nM N mTRPV4 IC50 = 17 nM Active at 10 mg/kg i.p. in model of cystitis N O

FIGURE 12.8 HC-067047 from Hydra Biosciences. 212 12. Small Molecule Agonists and Antagonists of TRPV4

intake, locomotion, or motor coordination were observed, a pleasing result given the severe side effects for some TRPV4 agonists discussed earlier. Significantly, HC-067047 improved bladder function in rodent models of cystitis induced by cyclophosphamide, being active at 10 mg/kg when dosed via the intraperitoneal route of administration [69]. Since its initial disclosure, HC-067047 has been used by many research groups to investigate the function of TRPV4, and the compound has been utilized extensively both in vitro and in vivo [54,70–78]. As a proof-of-concept probe of TRPV4 function, HC-067047 serves an excellent purpose.

However, activity at the hERG potassium channel (IC50 = 368 nM) [69], inhibition of which can lead to severe cardiovascular side effects, likely limits the usefulness of this compound to the preclinical setting.

RN-1734 and RN-9893 from Renovis As part of the same study that identified TRPV4 agonist RN-1747, Vincent et al. also reported the identification of TRPV4 antagonist RN-1734 24 (Figure 12.9) [67]. Interestingly, RN-1734 contains an arylsulfonamide motif, which is a substructural feature also present in antagonists from GSK and Pfizer (see following section). RN-1734 24 blocks the activity of human and rodent TRPV4 with IC50 values of 2.3 μM (human), 3.2 μM (rat), and 5.9 μM (mouse). Significantly, RN-1734 fully antagonized TRPV4 when stimulated with exogenous ligands, such as 4α-PDD, and mimics of endogenous activation, such as hypotonicity [67]. RN-1734 has been used as a proof-of-concept probe of TRPV4 function in both in vitro and in vivo settings [54,78–83]. For example, Hwang and colleagues have reported on the ability of RN-1734 to block the TRPV4-mediated pro-inflammatory effect of dimethylallyl pyrophosphate in rodents [54]. Separately, Renovis has also disclosed activity for RN-9893, a more potent antagonist of

TRPV4 than RN-1734, with Kb values of 100 nM, 57 nM, and 120 nM for hTRPV4, mTRPV4, and rTRPV4, respectively [84]. The compound also has acceptable pharmacokinetic behavior in rats, with bioavailability approaching 47% [84]. However, the chemical structure for RN- 9893 has not been revealed at this time.

Antagonists from GSK Scientists from GSK have been among the most prolific at identifying antagonists of TRPV4 (Figure 12.10). As of early 2014, their work has been detailed in approximately 13

Cl O Cl S N HN O RN-1734 24

hTRPV4 IC50 = 2.3 µM rTRPV4 IC50 = 3.2 µM mTRPV4 IC50 = 5.9 µM

FIGURE 12.9 TRPV4 antagonist RN-1734 from Renovis. TRPV4 Antagonists 213

O H H O N H H O O S Cl N O O N S N N N O N S H N N H H O O H Cl Cl Cl O Note: Presence of azetidine with a 2,4-dichlorophenylsulfonamide moiety in N TRPV4 antagonists from Pfizer (see Figure 12.11)

N NH S .HBr O NH N GSK205 25 N N

N SB-390570 26 N O O NH Cl N N N NH 28 Cl N

Br N hTRPV4 IC50 = 31.6 nM rTRPV4 = 32 nM Rat PK N CF3 CL = 13 mL/min/kg GSK2193874 27 Half-life = 2.1 h

hTRPV4 IC50 = 40 nM (GSK634775 as agonist) %F = 31% hTRPV4 IC50 = 50 nM (hypotonic stretch as agonist) Active at 10 mg/kg in a model rTRPV4 = 2 nM (GSK634775 as agonist) NC of pulmonary edema

FIGURE 12.10 Representative TRPV4 antagonists from GlaxoSmithKline. patent applications and numerous publications [85–100]. Early examples from the patent literature described chemotypes that shared many structural similarities to the TRPV4 agonists developed previously within GSK [85–90]. However, in recent years a more diverse set of chemotypes has started to emerge as antagonists of TRPV4 [91–100]. For example, GSK205 25, based around an aminothiazole core has been reported to act as a submicromolar antagonist at porcine TRPV4 (IC50 = 0.6 μM). Moreover, GSK205 was reported to be selective for TRPV4 [91]. Separately, GSK has disclosed a number of novel TRPV4 antagonists with potential to treat pulmonary edema-induced heart failure [99,100]. Within the first publication in this area, SB-390570 26 was identified as a nonselective inhibitor of TRPV4 with moderate activity at human (IC50 = 2 μM) and rat (IC50 = 0.3 μM) TRPV4 receptors [99]. SB-390570 was also a potent inhibitor of neurokinin-2/neurokinin-3 (IC50 = 100 nM and 8 nM, respectively) and suffered from high rat plasma clearance. Optimization of SB-390570 led to GSK2193874

27, a potent inhibitor of human (IC50 = 40-50 nM), rat (IC50 = 2 nM), mouse (IC50 = 5 nM), and dog (IC50 = 100 nM) TRPV4, with low clearance and good oral bioavailability in rat [99]. In addition, GSK2193874 was shown to be inactive against other TRP channels and selective 214 12. Small Molecule Agonists and Antagonists of TRPV4 for TRPV4 over important ion channels such as hERG and Cav1.2. Selectivity for TRPV4 was illustrated further by screening against ca. 200 receptors, ion channels, and enzymes. Significantly, GSK2193874 inhibited formation of pulmonary edema in a rodent model of heart failure due to high venous pressure in both acute and chronic settings [99]. An additional publication in this area detailed SAR and in vivo activity with an unrelated TRPV4 antagonist 28, based on a benzimidazole template [100]. Compound 28 exhibited good activity against hTRPV4 (IC50 = 31.6 nM) and rTRPV4 (IC50 = 32 nM) and showed acceptable pharmacokinetic properties in rat (clearance = 13 mL/min/kg and oral bioavailability (F) = 31%). A dose of compound 28 at 10 mg/kg was also active in vivo, blocking the decrease in mean arterial pressure in rodents challenged with an intravenous dose of the TRPV4 agonist, GSK1016790A [100]. Undoubtedly, the addition of selective TRPV4 antagonists GSK2193874 27 and compound 28 will be of great benefit to the research community and will complement existing TRPV4 antagonist tool compounds, ruthenium red, HC-067047, and RN-1734 (all of which may have inferior selectivity profiles than GSK2193874). The potential of TRPV4 antagonists in pulmonary indications may hold promise and is explored further in Chapter 13.

Antagonists from Pfizer Skerratt, Mills, and Mistry (Pfizer Neusentis) have described a method to triage hits from a screen of TRPV4 antagonists [101]. This led to a focus on piperidine alcohol 29 for hit-to- lead follow-up studies (Figure 12.11). Chemical modification of 29 to reduce cLogP (aimed at increasing metabolic stability), and then swapping the 4-pyridyl group to remove potential for drug-drug interactions, led to the identification of analog 30 as a potent inhibitor of hTRPV4 (IC50 = 49.7 nM), but with low activity at rTRPV4 (IC50 = 1.15 μM). Further optimization, guided in part by institutional experience with piperidine isosteres, led to azetidine alcohol 31 as a potent inhibitor of both human and rat TRPV4 (IC50 = 3.8 nM and 34.1 nM, respectively). Compound 31 showed good oral absorption in rat and was deemed a suitable

HO HO HO Cl Cl Cl N N N S S S O S F O O O O O Cl O O Cl Cl N NC NC Hit compound 29 30 Lead compound 31

hTRPV4 IC50 = 2200 nM hTRPV4 IC50 = 49.7 nM hTRPV4 IC50 = 3.8 nM rTRPV4 IC50 = 1150 nM rTRPV4 IC50 = 34.1 nM hTRPM8 IC = 9580 nM R 50 1 Cl Cl hERG IC50 = 9670 nM R1 R Rat PK 2 N N S R2 S CL = 23.5 mL/min/kg O O O O Cl Cl Vd = 4.3 L/kg Note: Presence of azetidine Note: Presence of 2,4-dichlorophenyl %F = 73% sulfonamide in antagonists sulfonamide in antagonists from from GSK (Figure 12.10) Renovis (Figure 12.9) & GSK (Figure 12.10)

FIGURE 12.11 Representative TRPV4 antagonists from Pfizer. Conclusions 215 tool compound with which to conduct further in vivo experiments [101]. Interestingly, lead compound 31 contains a 2,4-dichlorosulfonamide fragment that is also a prominent substruc- ture in previously published TRPV4 ligands such as RN-1734, an antagonist described by Renovis [67], and some agonists and antagonists described by GSK [55–66,85–100]. Because a 2,4-dichlorosulfonamide group seems to appear relatively frequently in TRPV4 ligands, it would seem that libraries built around this motif would be a good place to start if embarking on a de novo lead-searching campaign for TRPV4 antagonists or agonists. (Note: The lethal side effect seen with some potent TRPV4 agonists may limit the usefulness of such compounds—see earlier discussion.)

Other Antagonists of TRPV4 In addition to the antagonists detailed earlier, a plethora of other small molecules have been described as antagonists of TRPV4. Examples include capsazepine 32 (Figure 12.12), which although reported to inhibit hTRPV4 with an IC50 of 15 μM [67], is considerably more potent as an inhibitor of the related ion channel, TRPV1. Compound 33, a structurally related vanilloid from Kobayashi Pharmaceutical, has also been described as an antagonist of TRPV4 [102]. Citral 34, a terpene constituent of lemongrass oil, has also been documented as a weak inhibitor of TRPV4 (KD = 32 μM) [103]. Although the TRPV4 antagonist activity of capsazepine 32, compound 33, and citral 34 is potentially interesting, their use as selective probes of TRPV4 function is likely to be limited because more potent and/or selective tools are available.

CONCLUSIONS

Since its identification in 2000, TRPV4 has been implicated in a variety of physiological processes and human diseases. This has stimulated a search for proof-of-concept TRPV4 ligands with which to explore the TRPV4 mechanism in further detail. An early success was obtained in finding selective agonists of TRPV4, such as 4α-PDD, 4α-PDH, and GSK1016790A. However, TRPV4 agonists have sometimes come with severe cardiovascular side effects, which have limited the utility of these compounds. On the other hand, the last 3 years have seen much progress in identifying potent and selective antagonists of TRPV4, and a wide variety of small molecules, with diverse chemical structures, are now starting to emerge from the patent and primary literature. In the antagonist space, HC-067047, GSK2193874, and a benzimidazole antagonist from GSK stand out as particularly useful probes for investigating TRPV4 function.

Cl HO S O MeO C H HO N N N 9 19 H H O HO

Capsazepine 32 Vanilloid 33 Citral 34

FIGURE 12.12 Vanilloid and terpene TRPV4 antagonists. 216 12. Small Molecule Agonists and Antagonists of TRPV4

Given the pivotal role of TRPV4 in a variety of ailments with significant unmet clinical need, it is to be hoped that antagonists of TRPV4 may some day advance to the clinical setting, where the promising research may be translated into a benefit for human subjects.

References [1] Strotmann R, Harteneck C, Nunnenmacher K, Schultz G, Plant TD. OTRPC4, a nonselective cation channel that confers sensitivity to extracellular osmolarity. Nat Cell Biol 2000;2:695–702. [2] Liedtke W, Choe Y, Marti-Renom MA, Bell AM, Denis CS, Sali A, et al. Vanilloid receptor-related osmotically activated channel (VR-OAC), a candidate vertebrate osmoreceptor. Cell 2000;103:525–35. [3] Liedtke W, Friedman JM. Abnormal osmotic regulation in trpv4 −/− mice. Proc Natl Acad Sci USA 2003;100:13698–703. [4] Damann N, Voets T, Nilius B. TRPs in our senses. Curr Biol 2008;18:R880–9. [5] Venkatachalam K, Montell C. TRP channels. Annu Rev Biochem 2007;76:387–417. [6] Guler AD, Lee H, Iida T, Shimizu I, Tominaga M, Caterina M. Heat-evoked activation of the ion channel, TRPV4. J Neurosci 2002;22:6408–14. [7] Vriens J, Owsianik G, Fisslthaler B, Suzuki M, Janssens A, Voets T, et al. Modulation of the Ca2 permeable cation channel TRPV4 by cytochrome P450 epoxygenases in vascular endothelium. Circ Res 2005;97:908–15. [8] Watanabe H, Vriens J, Prenen J, Droogmans G, Voets T, Nilius B. Anandamide and arachidonic acid use epoxyeicosatrienoic acids to activate TRPV4 channels. Nature 2003;424:434–8. [9] Xu H, Zhao H, Tian W, Yoshida K, Roullet JB, Cohen DM. Regulation of a transient receptor potential (TRP) channel by tyrosine phosphorylation. SRC family kinase-dependent tyrosine phosphorylation of TRPV4 on TYR-253 mediates its response to hypotonic stress. J Biol Chem 2003;278:11520–7. [10] Fan HC, Zhang X, McNaughton PA. Activation of the TRPV4 ion channel is enhanced by phosphorylation. J Biol Chem 2009;284:27884–91. [11] Peng H, Lewandrowski U, Muller B, Sickmann A, Walz G, Wegierski T. Identification of a protein kinase C-dependent phosphorylation site involved in sensitization of TRPV4 channel. Biochem Biophys Res Commun 2010;391:1721–5. [12] Chung MK, Lee H, Caterina MJ. Warm temperatures activate TRPV4 in mouse 308 keratinocytes. J Biol Chem 2003;278:32037–46. [13] Fernandez-Fernandez JM, Nobles M, Currid A, Vazquez E, Valverde MA. Maxi K+ channel mediates regulatory volume decrease response in a human bronchial epithelial cell line. Am J Physiol Cell Physiol 2002;283:C1705–14. [14] Jia Y, Wang X, Varty L, Rizzo CA, Yang R, Correll CC, et al. Functional TRPV4 channels are expressed in human airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 2004;287:L272–8. [15] Wissenbach U, Bodding M, Freichel M, Flockerzi V. Trp12, a novel Trp related protein from kidney. FEBS Lett 2000;485:127–34. [16] Delany NS, Hurle M, Facer P, Alnadaf T, Plumpton C, Kinghorn I, et al. Identification and characterization of a novel human vanilloid receptor-like protein, VRL-2. Physiol Genomics 2001;4:165–74. [17] Birder L, Kullmann FA, Lee H, Barrick S, de Groat W, Kanai A, et al. Activation of urothelial transient receptor potential vanilloid 4 by 4alpha-phorbol 12,13-didecanoate contributes to altered bladder reflexes in the rat. J Pharmacol Exp Ther 2007;323:227–35. [18] Gevaert T, Vriens J, Segal A, Everaerts W, Roskams T, Talavera K, et al. Deletion of the transient receptor potential cation channel TRPV4 impairs murine bladder voiding. J Clin Invest 2007;117:3453–62. [19] Shen J, Harada N, Kubo N, Liu B, Mizuno A, Suzuki M, et al. Functional expression of transient receptor potential vanilloid 4 in the mouse cochlea. Neuroreport 2006;17:135–9. [20] Lowry CA, Lightman SL, Nutt DJ. That warm fuzzy feeling: brain serotonergic neurons and the regulation of emotion. J Psychopharmacol 2009;23:392–400. [21] Kohler R, Heyken WT, Heinau P, Schubert R, Si H, Kacik M, et al. Evidence for a functional role of endothelial transient receptor potential V4 in shear stress-induced vasodilatation. Arterioscler Thromb Vasc Biol 2006;26:1495–502. [22] Masuyama R, Vriens J, Voets T, Karashima Y, Owsianik G, Vennekens R, et al. TRPV4-mediated calcium influx regulates terminal differentiation of osteoclasts. Cell Metab 2008;8:257–65. REFERENCES 217

[23] Mizoguchi F, Mizuno A, Hayata T, Nakashima K, Heller S, Ushida T, et al. Transient receptor potential vanilloid 4 deficiency suppresses unloading-induced bone loss. J Cell Physiol 2008;216:47–53. [24] Rock MJ, Prenen J, Funari VA, Funari TL, Merriman B, Nelson SF, et al. Gain-of-function mutations in TRPV4 cause autosomal dominant brachyolmia. Nat Genet 2008;40:999–1003. [25] Krakow D, Vriens J, Camacho N, Luong P, Deixler H, Funari TL, et al. Mutations in the gene encoding the calcium-permeable ion channel TRPV4 produce spondylometaphyseal dysplasia, Kozlowski type and metatropic dysplasia. Am J Hum Genet 2009;84:307–15. [26] Muramatsu S, Wakabayashi M, Ohno T, Amano K, Ooishi R, Sugahara T, et al. Functional gene screening system identified TRPV4 as a regulator of chondrogenic differentiation. J Biol Chem 2007;282:32158–67. [27] Nilius B, Owsianik G. Channelopathies converge on TRPV4. Nat Genet 2010;42:98–100. [28] Landoure G, Zdebik AA, Martinez TL, Burnett BG, Stanescu HC, Inada H, et al. Mutations in TRPV4 cause Charcot-Marie-Tooth disease type 2C. Nat Genet 2010;42:170–4. [29] Deng HX, Klein CJ, Yan J, Shi Y, Wu Y, Fecto F, et al. Scapuloperoneal spinal muscular atrophy and CMT2C are allelic disorders caused by alterations in TRPV4. Nat Genet 2010;42:165–9. [30] Auer-Grumbach M, Olschewski A, Papic L, Kremer H, McEntagart ME, Uhrig S, et al. Alterations in the ankyrin domain of TRPV4 cause congenital distal SMA, scapuloperoneal SMA and HMSN2C. Nat Genet 2010;42:160–4. [31] Moore C, Cevikbas F, Pasolli HA, Chen Y, Kong W, Kempkes C, et al. UVB radiation generates sunburn pain and affects skin by activating epidermal TRPV4 ion channels and triggering endothelin-1 signaling. Proc Natl Acad Sci USA 2013;110:E3225–34. [32] Zhang Y, Wang YH, Ge HY, Arendt-Nielsen L, Wang R, Yue SW. A transient receptor potential vanilloid 4 contributes to mechanical allodynia following chronic compression of dorsal root ganglion in rats. Neurosci Lett 2008;432:222–7. [33] Alessandri-Haber N, Yeh JJ, Boyd AE, Parada CA, Chen X, Reichling DB, et al. Hypotonicity induces TRPV4- mediated nociception in rat. Neuron 2003;39:497–511. [34] Cenac N, Altier C, Chapman K, Liedtke W, Zamponi G, Vergnolle N. Transient receptor potential vanilloid-4 has a major role in visceral hypersensitivity symptoms. Gastroenterology 2008;135:937–46, 946 e931-932. [35] Alessandri-Haber N, Dina OA, Joseph EK, Reichling DB, Levine JD. Interaction of transient receptor potential vanilloid 4, integrin, and SRC tyrosine kinase in mechanical hyperalgesia. J Neurosci 2008;28:1046–57. [36] Levine JD, Alessandri-Haber N. TRP channels: targets for the relief of pain. Biochim Biophys Acta 2007;1772:989–1003. [37] Grant AD, Cottrell GS, Amadesi S, Trevisani M, Nicoletti P, Materazzi S, et al. Protease-activated receptor 2 sensitizes the transient receptor potential vanilloid 4 ion channel to cause mechanical hyperalgesia in mice. J Physiol 2007;578:715–33. [38] Suzuki M, Mizuno A, Kodaira K, Imai M. Impaired pressure sensation in mice lacking TRPV4. J Biol Chem 2003;278:22664–8. [39] Alvarez DF, King JA, Weber D, Addison E, Liedtke W, Townsley MI. Transient receptor potential vanilloid 4-mediated disruption of the alveolar septal barrier: a novel mechanism of acute lung injury. Circ Res 2006;99:988–95. [40] Hamanaka K, Jian MY, Townsley MI, King JA, Liedtke W, Weber DS, et al. TRPV4 channels augment macrophage activation and ventilator-induced lung injury. Am J Physiol Lung Cell Mol Physiol 2010;299:L353–62. [41] Hamanaka K, Jian MY, Weber DS, Alvarez DF, Townsley MI, Al-Mehdi AB, et al. TRPV4 initiates the acute calcium-dependent permeability increase during ventilator-induced lung injury in isolated mouse lungs. Am J Physiol Lung Cell Mol Physiol 2007;293:L923–32. [42] Inoue R, Jian Z, Kawarabayashi Y. Mechanosensitive TRP channels in cardiovascular pathophysiology. Pharmacol Ther 2009;123:371–85. [43] Ye L, Kleiner S, Wu J, Sah R, Gupta RK, Banks AS, et al. TRPV4 is a regulator of adipose oxidative metabolism, inflammation, and energy homeostasis. Cell 2012;151:96–110. [44] Vincent F, Duncton MAJ. TRPV4 agonists and antagonists. Curr Top Med Chem 2011;11:2216–26. [45] Vincent F., Duncton MAJ. TRPV4 and drug discovery. In: Szallasi A, Biro T, editors. TRP channels in drug discovery, vol. 1. Berlin: Springer; 2012;1:257–70. [46] Plant TD, Strotmann R. Trpv4. Handb Exp Pharmacol 2007;179:189–205. [47] Everaerts W, Nilius B, Owsianik G. The vallinoid transient receptor potential channel Trpv4: from structure to disease. Prog Biophys Mol Biol 2010;103:2–17. [48] Nilius B, Voets T. The puzzle of TRPV4 channelopathies. EMBO Rep 2013;14:152–63. 218 12. Small Molecule Agonists and Antagonists of TRPV4

[49] Sonkusare SK, Bonev AD, Ledoux J, Liedtke W, Kotlikoff MI, Heppner TJ, et al. Elementary Ca2+ signals through endothelial TRPV4 channels regulate vascular function. Science 2012;336:597–601. [50] Cortright DN, Szallazsi A. TRP channels and pain. Curr Pharm Des 2009;15:1736–9. [51] Szallasi A, Blumberg PM. Resiniferatoxin, a phorbol-related diterpene, acts as an ultrapotent analog of capsaicin, the irritant constituent in red pepper. Neuroscience 1989;30:515–20. [52] (a)Watanabe H, Davis JB, Smart D, Jerman JC, Smith GD, Hayes P, et al. Activation of TRPV4 channels (hVRL-2/ mTRP12) by phorbol derivatives. J Biol Chem 2002;277:13569–77. (b)Castagna M, Takai Y, Kaibuchi K, Sano K, Kikkawa U, Nishizuka Y. Direct activation of calcium-activated, phospholipid-dependent protein kinase by tumor-promoting phorbol esters. J Biol Chem 1982;257:7847–51. [53] Klausen TK, Pagani A, Minassi A, Ech-Chahad A, Prenen J, Owsianik G, et al. Modulation of the transient receptor potential vanilloid channel TRPV4 by 4alpha-phorbol esters: a structure-activity study. J Med Chem 2009;52:2933–9. [54] Bang S, Yoo S, Yang TJ, Cho S, Hwang SW. Nociceptive and pro-inflammatory effects of dimethylallyl pyrophosphate via TRPV4 activation. Br J Pharmacol 2012;166:1433–43. [55] Kumar S, Pratta MA, Votta BJ. WO2006029209. Chem Abstr 2006;144:286221. [56] Marquis RW. WO2007098393. Chem Abstr 2009;147:301493. [57] Casillas LN, Jeong JU, Marquis RW. WO2006029210. Chem Abstr 2006;144:311900. [58] Cichy-Knight M, Dunn AK, Marquis RW. WO2006105475. Chem Abstr 2006;145:397528. [59] Jeong JU, Marquis RW. WO2007030761. Chem Abstr 2007;146:338169. [60] Casillas LN, Marquis RW. WO2007070865. Chem Abstr 2007;147:95915. [61] Casillas LN, Marquis RW. WO2007082262. Chem Abstr 2007;147:166620. [62] Marquis RW. WO2007098393. Chem Abstr 2007;147:301493. [63] Thorneloe KS, Sulpizio AC, Lin Z, Figueroa DJ, Clouse AK, McCafferty GP, et al. N-((1S)-1-{[4-((2S)-2- {[(2,4-dichlorophenyl)sulfonyl]amino}-3-hydroxypropanoyl)-1-piperazinyl]carbonyl}-3-methylbutyl)- 1-benzothiophene-2-carboxamide (GSK1016790A), a novel and potent transient receptor potential vanilloid 4 channel agonist induces urinary bladder contraction and hyperactivity: part I. J Pharmacol Exp Ther 2008;326:432–42. [64] Willette RN, Bao W, Nerurkar S, Yue TL, Doe CP, Stankus G, et al. Systemic activation of the transient receptor potential vanilloid subtype 4 channel causes endothelial failure and circulatory collapse: part 2. J Pharmacol Exp Ther 2008;326:443–52. [65] Casillas LTR, Lin M, Rominger D, Donovan B, Brown BS, Kulkarni SG, et al. Identification and characterization of a potent and selective TRPV4 activator. In: 238th ACS national meeting, Washington, DC, United States, August 16–20, 2009; MEDI-451; 2009. [66] Jeong JU, Rahman A, Chen X, Casillas L, Lin M, Trout R, et al. Discovery and SAR studies of potent and orally bioavailable acyclic diaminoalkane derivatives as TRPV4 agonists. In: 238th ACS national meeting, Washington, DC, United States, August 16–20, 2009; MEDI-392; 2009. [67] Vincent F, Acevedo A, Nguyen MT, Dourado M, DeFalco J, Gustafson A, et al. Identification and characterization of novel TRPV4 modulators. Biochem Biophys Res Commun 2009;389:490–4. [68] Smith PL, Maloney KN, Pothen RG, Clardy J, Clapham DE. Bisandrographolide from Andrographis paniculata activates TRPV4 channels. J Biol Chem 2006;281:29897–904. [69] Everaerts W, Zhen X, Ghosh D, Vriens J, Gevaert T, Gilbert JP, et al. Inhibition of the cation channel TRPV4 improves bladder function in mice and rats with cyclophosphamide-induced cystitis. Proc Natl Acad Sci USA 2010;107:19084–9. [70] Qian X, Francis M, Koehler R, Solodushko V, Lin M, Taylor MS. Positive feedback regulation of agonist- stimulated endothelial Ca2+ dynamics by KCa3.1 channels in mouse mesenteric arteries. Arterioscler Thromb Vasc Biol 2014;34:127–35. [71] Xia Y, Fu Z, Hu J, Huang C, Paudel O, Cai S, et al. TRPV4 channel contributes to serotonin-induced pulmonary vasoconstriction and the enhanced vascular reactivity in chronic hypoxic pulmonary hypertension. Am J Physiol 2013;305:C704–15. [72] Li L, Yin J, Jie P, Lu Z, Zhou L, Chen L, et al. Transient receptor potential vanilloid 4 mediates hypotonicity- induced enhancement of synaptic transmission in hippocampal slices. CNS Neurosci Ther 2013;19:854–62. [73] Zhang L, Papadopoulos P, Hamel E. Endothelial TRPV4 channels mediate dilation of cerebral arteries: impairment and recovery in cerebrovascular pathologies related to Alzheimer’s disease. Br J Pharmacol 2013;170:661–70. [74] Mamenko M, Zaika OL, Boukelmoune N, Berrout J, O’Neil RG, Pochynyuk O. Discrete control of TRPV4 channel function in the distal nephron by protein kinases A and C. J Biol Chem 2013;288:20306–14. REFERENCES 219

[75] Dahan D, Ducret T, Quignard J, Marthan R, Savineau J, Esteve E. Implication of the ryanodine receptor in TRPV4-induced calcium response in pulmonary arterial smooth muscle cells from normoxic and chronically hypoxic rats. Am J Physiol Lung Cell Mol Physiol 2012;303:L824–33. [76] Materazzi S, Fusi C, Benemei S, Pedretti P, Patacchini R, Nilius B, et al. TRPA1 and TRPV4 mediate paclitaxel- induced peripheral neuropathy in mice via a glutathione-sensitive mechanism. Pfluegers Archiv 2012;463:561–9. [77] Jin M, Berrout J, Chen L, O’Neil RG. Hypotonicity-induced TRPV4 function in renal collecting duct cells: modulation by progressive cross-talk with Ca2+-activated K+ channels. Cell Calcium 2012;51:131–9. [78] Zheng X, Zinkevich NS, Gebremedhin D, Gauthier KM, Nishijima Y, Fang J, et al. Arachidonic acid-induced dilation in human coronary arterioles: convergence of signaling mechanisms on endothelial TRPV4-mediated Ca2+ entry. J Am Heart Assoc 2013;2:E000080. [79] Shahidullah M, Mandal A, Delamere NA. TRPV4 in porcine lens epithelium regulates hemichannel-mediated ATP release and Na-K-ATPase activity. Am J Physiol 2012;302:C1751–61. [80] Fichna I, Mokrowiecka A, Cycankiewicz AI, Zakrzewski PK, Malecka-Panas E, Janecka A, et al. Transient receptor potential vanilloid 4 blockade protects against experimental colitis in mice: a new strategy for inflammatory bowel diseases treatment. Neurogastroenterol Motil 2012;24:e557–60. [81] Aizawa N, Wyndaele J, Homma Y, Igawa Y. Effects of TRPV4 cation channel activation on the primary bladder afferent activities of the rat. Neurourol Urodynam 2012;31:148–55. [82] Butenko O, Dzamba D, Benesova J, Honsa P, Benfenati V, Rusnakova V, et al. The increased activity of TRPV4 channel in the astrocytes of the adult rat hippocampus after cerebral hypoxia/ischemia. PLoS One 2012;7:e39959. [83] Albert ES, Bec JM, Desmadryl G, Chekroud K, Travo C, Gaboyard S, et al. TRPV4 channels mediate the infrared laser-evoked response in sensory neurons. J Neurophysiol 2012;107:3227–34. [84] Wei Z-L, Nguyen MT, Acevedo A, Zipfel S, Defalco J, Dourado M, et al. Discovery of a proof-of-concept TRPV4 antagonist. In: Keystone symposia on the transient receptor potential ion channel superfamily, Breckenridge, Colorado, USA; 2007. [85] Cheung M, Eidam HS, Fox RM, Manas ES. WO2009146177. Chem Abstr 2009;152:27388. [86] Cheung M, Eidam HS, Fox RM, Manas ES. WO2009146182. Chem Abstr 2009;152:27389. [87] Cheung M, Eidam HS. WO2010011912. Chem Abstr 2010;152:215581. [88] Bury MJ, Cheung M, Eidam HS, Fox RM, Goodman K, Manas ES. WO2010011914. Chem Abstr 2010;152:192445. [89] Eidam HS, Fox RM. WO2011091407. Chem Abstr 2011;155:241261. [90] Eidam HS, Fox RM. WO2011091410. Chem Abstr 2011;155:261148. [91] Phan MN, Leddy HA, Votta BJ, Kumar S, Levy DS, Lipshutz DB, et al. Functional characterization of TRPV4 as an osmotically sensitive ion channel in porcine articular chondrocytes. Arthritis Rheum 2009;60:3028–37. [92] Brooks CA, Cheung M, Fox RM, Goodman KB, Hilfiker MA, Ye G. WO2011119693. Chem Abstr 2011;155:483971. [93] Brooks CA, Cheung M, Eidam HS, Fox RM, Hilfiker MA, Manas ES, et al. WO2011119701. Chem Abstr 2011;155:483972. [94] Brooks CA, Cheung M, Eidam HS, Fox RM, Hilfker MA, Manas ES, et al. WO2011119704. Chem Abstr 2011;155:483973. [95] Cheung M, Eidam HS, Goodman KB, Hilfiker MA. WO2011119694. Chem Abstr 2011;155:484122. [96] Brooks C, Cheung M, Eidam HS, Goodman KB, Hammond M, Hilfiker MA, et al. WO2012174342. Chem Abstr 2012;158:105110. [97] Brooks C, Cheung M, Goodman KB, Hammond M. WO2012174340. Chem Abstr 2012;158:105135. [98] Brooks C, Cheung M, Eidam HS, Goodman KB, Hammond M, Hilfiker MA, et al. WO2013012500. Chem Abstr 2013;158:215954. [99] Thorneloe KS, Cheung M, Bao W, Alsaid H, Lenhard S, Jian M, et al. An orally active TRPV4 channel blocker prevents and resolves pulmonary edema induced by heart failure. Sci Trans Med 2012;4:159ra148 1–11. [100] Hilfiker MA, Hoang TH, Cornil J, Eidam HS, Matasic DS, Roethke TJ, et al. Optimization of a novel series of TRPV4 antagonists with in vivo activity in a model of pulmonary edema. ACS Med Chem Lett 2013;4:293–6. [101] Skerratt SE, Mills JE, Mistry J. Identification of false positives in "HTS hits to lead": the application of Bayesian models in HTS triage to rapidly deliver a series of selective TRPV4 antagonists. Med Chem Commun 2013;4:244–51. [102] Tsukamoto S, Sawamura S. JP2009084290. Chem Abstr 2009;150:438732. [103] Stotz SC, Vriens J, Martyn D, Clardy J, Clapham DE. Citral sensing by transient receptor potential channels in dorsal root ganglion neurons. PLoS One 2008;3:e2082. CHAPTER 13 Potential Therapeutic Applications for TRPV4 Antagonists in Lung Disease Mary I. Townsley,* James C. Parker Department of Physiology and Center for Lung Biology, University of South Alabama, Mobile, Alabama, USA *Corresponding author: [email protected]

OUTLINE

Introduction 221 High Vascular Pressure-Induced Lung Injury 228 TRPV4 Localization in Lungs 222 Heart Failure and Pulmonary Edema 229 Acute Lung Injury via Direct Channel Activation 222 Pulmonary Arterial Hypertension 231 Airway Disease and Cough 232 Ventilator-Induced Lung Injury (VILI) 223 References 233

INTRODUCTION

TRPV4 is a polymodally gated nonselective cation channel [1]. Channel activation leads to permeation of both Ca2+ and Na+, with a permeability ratio of 6.3 [2]. Channel gating can be elicited by mechanical and shear stress, as well as cell swelling [3–6], all of which appear to be secondary to stress-induced activation of phospholipase A2 (PLA2) and subsequent synthesis of epoxyeicosatrienoic acids (EETs) [1,2,7,8]. Notably, deformation of cell-detached patches does not activate the channel [2]. Similarly, TRPV4 activation with endocannabinoids appears

TRP Channels as Therapeutic Targets 221 © 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/B978-0-12-420024-1.00013-8 222 13. TRPV4 IN LUNG DISEASE to be secondary to local hydrolysis of endocannabinoids to arachidonic acid and subsequent EET synthesis [9]. Synthetic phorbol esters such as 4α-phorbol-12,13-didecanoate (4αPDD) [10], small molecule activators GSK1016790A or GSK634775 [11,12], and heat [3] appear to act directly on channel proteins. Thus, there is significant potential for involvement of TRPV4 in lung disease through diverse activation pathways.

TRPV4 LOCALIZATION IN LUNGS

Using immunohistochemistry and Western blotting, we and others have documented expression patterns for TRPV4 protein in lung tissue and cells. TRPV4 is expressed in the alveolar septal wall of human, rat, and mouse lung, whereas expression in endothelium of extra-alveolar vessels is sporadic [11–13]. Immunostaining for TRPV4 in human, rat, and mouse lung is shown in Figure 13.1. In vitro, TRPV4 is also expressed in cultured rat lung endothelial cells [13,14]. Pulmonary vascular and airway smooth muscle also expresses TRPV4 in vivo and in vitro [13]. Vascular smooth muscle immunoreactivity for TRPV4 is particularly evident in human and rat lung (see Figure 13.1). TRPV4 is also expressed in airway epithelium and alveolar macrophages [13,15,16]. Though TRPV4 expression has been documented in sensory nerves from skin and colon [17,18], expression in airway sensory nerves has not been directly confirmed. Nonetheless, preliminary functional data supports a role for TRPV4 in Aδ nociceptive afferent fibers from mouse, guinea pig, and human airways [19].

ACUTE LUNG INJURY VIA DIRECT CHANNEL ACTIVATION

Direct activation of TRPV4 with the phorbol ester 4α-phorbol-12,13-didecanoate (4αPDD) or the small molecule agonist GSK1016790A increases the filtration coefficient (Kf) in isolated lungs, associated with disruption of the alveolar septal barrier, alveolar flooding, and lung edema [11–13,20]. The dose-dependent increase in Kf after treatment of isolated rat lung with

4αPDD is shown in Figure 13.1. Kf is a specific and sensitive measure of endothelial permeability, applicable to assessment of lung injury in any isolated mammalian lung [21,22]. The caveat to interpretation of Kf is that it measures the aggregate permeability of the entire perfused surface area in lungs. Interestingly, the TRPV4-mediated acute lung injury is localized to the alveolar septal compartment, with little evidence of injury to endothelium in extra-alveolar vessels. Disruption of the alveolar barrier appears to be due to cell detachment, blebbing, and/ or cell swelling rather than interendothelial cell gap formation [13,20]. Similarly, Willette and colleagues have reported that TRPV4 activation leads to detachment of endothelial cells in culture [12]. These perturbations in lungs elicited by TRPV4 activation are dependent on TRPV4- mediated calcium entry, as they are mitigated by perfusion with a low Ca2+ buffer, pretreatment of lungs with the TRPV4 antagonists, or use of lungs from Trpv4−/− mice [11,13]. Although the impact of TRPV4 activation on barrier function in lungs is clear, the downstream mechanisms linking TRPV4 activation and endothelial barrier disruption in the lungs needs to be further resolved. In airway epithelium, TRPV4 activation leads to release of the pro-inflammatory matrix metalloproteinase 1 (MMP1) [15]. Our own recent data suggests that TRPV4 activation initiates a signaling cascade leading to increased availability of active MMP2 and MMP9 in mouse lungs [23], which may contribute to matrix degradation and cell detachment. Ventilator-Induced Lung Injury (VILI) 223

FIGURE 13.1 TRPV4 expression and its impact on lung endothelial permeability. Immunohistochemistry doc- uments TRPV4 expression in alveolar septal walls, vascular endothelium, and vascular smooth muscle in human (A), rat (B), and mouse (C) lung. Similarly, TRPV4 is expressed in cultured rat microvascular (MV) and pulmonary artery (PA) endothelium (D). Activation of TRPV4 with the phorbol ester 4α-phorbol-12,13-didecanoate (4αPDD) increases the filtration coefficient (Kf) in isolated rat lung in a dose-dependent fashion (E). The related homovanillate compound 4αPDDHV, which targets TRPV1, has no impact on Kf. From Alvarez et al. [13], with permission.

VENTILATOR-INDUCED LUNG INJURY (VILI)

Although positive pressure mechanical ventilation is a lifesaving intervention in the setting of adult respiratory distress syndrome (ARDS) and acute lung injury (ALI), mechanical ventilation with excessive tidal volumes actually contributes to ventilator-induced lung injury (VILI) and increases mortality. Specifically, a large-scale clinical trial showed that decreasing the tidal volume from 12 mL/kg to 6 mL/kg reduced mortality by 22% [24]. The severity of VILI has been generally recognized to be both time and pressure (or volume) dependent in experimental studies as well as in clinical settings [25–28]. The syndrome includes a rapid 224 13. TRPV4 IN LUNG DISEASE

increase in vascular permeability followed by cytokine release and inflammatory cell infiltrates [25,26,28]. The rapid onset of the increase in vascular permeability, compared to the slower increase in cytokine levels and mobilization of inflammatory cell infiltrates some hours later, suggests a rapid intracellular signaling cascade of events followed by recruitment of inflammatory cells, which may then amplify the lung injury [29–31]. An increase in endothelial cell intracellular Ca2+ is a necessary component for increased vascular permeability induced by most mediators, and the permeability response to lung overdistention appears to be no exception [32–34]. Ca2+ entry occurs within seconds after mechanical perturbation and thus is one of the most rapid responses to mechanical strain in both isolated lungs and cultured cell preparations [35–37]. Early studies by Parker and colleagues on VILI in isolated rat lungs indicated that high peak inflation pressure (PIP) increases lung vascular permeability for the total lung, as well as individual arterial, venular, and capillary vascular segments, and these increases were attenuated by gadolinium, an inhibitor of stretch-activated cation channels [33,38]. The identity of these stretch-activated cation channels involved in initiating VILI was established by Hamanaka et al. [39], who measured Kf in isolated lungs of wild-type (WT) and −/− Trpv4 mice ventilated with high and low PIP. High PIP ventilation increased Kf in lungs of WT mice at 35 °C, an effect significantly augmented by increasing the perfusate temperature to 40 °C. The increase in Kf induced by high PIP in WT lungs was abolished by the TRPV antagonist ruthenium red and absent in lungs from Trpv4−/− mice at both 35 °C and 40 °C (Figure 13.2). In parallel studies, Hamanaka and colleagues showed that intracellular Ca2+ transients elicited in intact lungs during high PIP were abolished in the TRPV4 knockout (KO) lungs and ruthenium red treated lungs. Further, a morphometric evaluation of edema distribution indicated significant alveolar flooding in WT lungs compared to lungs from Trpv4−/− mice. The mechanism linking mechanical strain in VILI to activation of TRPV4 involves synthesis of arachidonic acid epoxygenase metabolites. In one of the first publications on TRPV4 in endothelial cells, Watanabe et al. reported that EETs derived from metabolism of arachidonic acid by P450 epoxygenases elicited TRPV4-mediated Ca2+ entry [9]. Alvarez et al. subsequently reported that EETs increased Kf in rat lungs [40,41]. As hypotonicity- induced cell swelling, a form of stretch, activates TRPV4 in a mechanism involving EET synthesis [7,8], we considered such a link in VILI (and in high vascular pressure-induced lung injury). Hamanaka et al. found that methanandamide (a competitive inhibitor of anandamide-derived arachidonic acid synthesis) and miconazole (a P450 inhibitor) abolished the increase in Kf induced by high PIP ventilation [39]. These findings were consistent with earlier work from the Parker laboratory that implicated PLA2 activation and subsequent synthesis of arachidonic acid metabolites in initiating the acute pulmonary vascular permeability increase in response to high PIP ventilation. Specifically, Yoshikawa et al. [42] showed that mice deficient in Clara cell secretory protein (CCSP), an inhibitor of cytosolic

PLA2 activity, had an increased susceptibility to acute VILI, whereas inhibition of PLA2 with arachidonyl trifluoromethyl ketone attenuated the lung vascular permeability increases and edema in both CCSP−/− and WT mice after 2 and 4 h of high PIP ventilation. Similarly,

Miyahara et al. [43] observed that either a cytosolic PLA2 inhibitor or a combination of cyclooxygenase, lipoxygenase, and P450 epoxygenase inhibitors prevented VILI-induced increase in Kf in isolated mouse lungs. Ventilator-Induced Lung Injury (VILI) 225

TRPV4-/-

TRPV4+/+

2.5 35 °C

2.0

O/100g) 1.5 2

1.0 **# *# Filtration coefficient (ml/min/cmH 0.5

0.0 Minutes 30 80 130 PIP (cmH2O) 9 25 35

40 °C 2.5 **#

2.0 O/100g) 2 1.5 # ion coefficient

at 1.0 Filtr (ml/min/cmH 0.5

0.0 Minutes 30 80 130 PIP (cmH2O) 9 25 35

FIGURE 13.2 Lung filtration coefficients (Kf) after 30-min periods of ventilation at increasing peak inflation pressures (PIP) in lungs from Trpv4+/+ and Trpv4−/− mice perfused at 35 °C (upper panel) or 40 °C (lower panel). *p < 0.05 versus 30 min in same group. **p < 0.05 versus 30 and 80 min in same group. #p < 0.05 versus Trpv4−/− group at same time period. From Hamanaka et al. [39], with permission.

Although these findings might be interpreted as a straightforward impact of stretch on lung endothelium, leading to EET synthesis and TRPV4 activation, other work suggests that the mechanism of VILI is more complex. Macrophages have been proposed as the major initiators of lung injury during mechanical ventilation because these cells produce copious amounts of pro-inflammatory cytokines during in vitro cyclical stretch compared to other lung cell types [44,45]. Further, TRPV4 agonists elicit Ca2+ entry and activate both human 226 13. TRPV4 IN LUNG DISEASE and murine macrophages [46,47]. More recently, macrophage depletion studies indicated protection against the lung permeability increase elicited by injurious ventilation [48,49]. The pattern of injury progression in VILI appears similar to that for other insults such as ischemia-reperfusion injury or sepsis, in that the early phase of injury is coordinated by lung macrophages, whereas the late phase of tissue injury is neutrophil dependent [50–52]. Both VILI and LPS-induced lung injury are attenuated by depletion of alveolar macrophages [39,48,49]. Alveolar macrophages activated by stretch secrete reactive oxygen species and platelet activating factor, which then activates endothelial NADPH oxidases [49,52,53]. Hamanaka et al. showed that VILI was blocked (Figure 13.3) by genetic deletion of TRPV4 and restored by instilling TRPV4-competent alveolar macrophages [46]. They also observed that 4αPDD-induced TRPV4 activation in mouse alveolar macrophages increased intracellular calcium, mitochondrial superoxide, and nitric oxide production and cell spreading on a glass surface (Figure 13.4), responses that were absent in macrophages lacking TRPV4. Further, the addition of WT macrophages restored lung nitrotyrosine staining in Trpv4−/− mice to the high level observed in WT lungs following high-pressure ventilation [46]. Collectively, these results are indicative of peroxynitrite formation with VILI due to production of excess superoxide and nitric oxide production [54]. The link between stress transmission and TRPV4 channel gating in macrophages may be direct. Alveolar macrophages are rapidly activated by high-volume and pressure ventilation and become firmly adherent within minutes after initiation of ventilation [48,49]. Although stretch-activated calcium transients in endothelial cells appear to be mediated by β1 integrins [35,55], macrophage adhesion and migration are

WT WT+KO macrophage KO KO+WT macrophage 1.0 *# *# 0.8 # *# #

O/100g) 0.6 2 #

0.4 Filtration coefficient (ml/min/cmH 0.2

0.0 Minutes 30 80 130

PIP (cmH2O) 9 25 35

FIGURE 13.3 Lung filtration coefficients (Kf) after 30-min periods of ventilation at increasing peak inflation pressures (PIP) in lungs from wild-type (WT) and TRPV4 knockout (KO) mice. These data are compared to the Kf responses to PIP in WT mice with KO alveolar macrophages instilled (WT + KO Macrophage) versus that in KO lungs with WT macrophages added (KO + WT Macrophage). *p < 0.05 versus the same group after 30 min. #p < 0.05 versus KO group within the same time period. From Hamanaka et al. [46], with permission. Ventilator-Induced Lung Injury (VILI) 227

FIGURE 13.4 Scanning electron micrographs of freshly harvested alveolar macrophages. Macrophages obtained by bronchoalveolar lavage from Trpv4−/− (A, B) and Trpv4+/+ (C, D) mice were imaged after 24-h incubation, under control conditions (A, C) or after 30-min treatment with 4αPDD (B, D). Note protrusions and spreading border (arrow) of TRPV4+/+ macrophage treated with 4αPDD. From Hamanaka et al. [47], with permission.

dependent on β2 and β3 integrins [56,57]. Thus, direct cell membrane distortion due to surface tension-dependent compression of macrophages likely leads to their activation and adher- ence to the alveolar surface at high lung distending pressures. Aside from clinical trials that definitively identified that low-volume ventilation reduced mortality, there has been little progress toward development of therapeutic interventions targeting TRPV4 in VILI. However, Jurek et al. recently proposed an approach with promise for clinical applicability, utilizing an intratracheal aerosol of ruthenium red-coated nanoparticles to target the critical macrophage TRPV4 in VILI [58]. Alveolar macrophages rapidly phagocytosed the nanoparticles, and a significant lung ruthenium red content was attained within minutes. Importantly, inhaled ruthenium red nanoparticles blocked the increase in lung microvascular permeability caused by high airway pressure mechanical ventilation. In the intact lung, treatment with inhaled ruthenium red nanoparticles was effective in blocking the Kf response to high-pressure mechanical ventilation for up to 3 days (Figure 13.5). Ruthenium red nanoparticles blocked calcium transients induced by 4αPDD in both alveolar macrophages and capillary endothelial cells, but they did not affect endothelial calcium transients due to ATP-dependent store depletion [58]. Development of such therapeutic strategies is important due not only to the impact of VILI itself, but also potentially due to exacerbation of underlying injury by mechanical ventilation and resultant multiple organ failure [59]. Huh and colleagues demonstrated that mechanical stretch dramatically amplifies interleukin-2 mediated injury to alveolar epithelium and endothelium in a lung-on-a-chip pulmonary edema model, effects completely abrogated by coadministration of a TRPV4 inhibitor [60]. 228 13. TRPV4 IN LUNG DISEASE

Blank NP + control ventilation at 4th RR NP + control ventilation at 4th Blank NP + HPMV at 4h RR NP + HPMV at 4h 2 Blank NP + HPMV at 1d RR NP + HPMV at 1d RR NP + HPMV at 3d * * 1.5 RR NP + HPMV at 7d * O/100g) 2

1 $# # ^

(mL/min/cmH 0.5 f K

0 Kf1 Kf2

FIGURE 13.5 Filtration coefficients measured at baseline (Kf1) and during experimental state (Kf2) in ex vivo mouse lungs exposed to control ventilation or high-pressure mechanical ventilation (HPMV) at 4 h or 1, 3, and 7 days after inhalation of nebulized blank or ruthenium red (RR) nanoparticles (NP). *p < 0.004 versus Kf1 in same group; p = 0.0027 versus Kf1 in same group; ^p = 0.007 versus Kf2 for Blank NP + HPMV at 4 h, #p < 0.05 versus Kf2 for Blank NP + HPMV at 1 day. From Jurek et al. [58], with permission.

HIGH VASCULAR PRESSURE-INDUCED LUNG INJURY

High vascular pressure in lung presents another form of mechanical stress that has long been recognized to elicit accumulation of edema fluid and to increase endothelial permeability [61,62]. Our early work provided support for the notion that mechanical force induced by acute high vascular pressure stress in the lung increased endothelial permeability and targeted the alveolar septal network with a threshold for injury of ~40 cmH2O in canine lungs [61,63]. Subsequently Kuebler et al. [64] identified that increased vascular pressure in rat lungs promotes Ca2+ entry into subpleural septal endothelium via a gadolinium-sensitive channel, but a channel candidate was not apparent at that time. It was only subsequent to the identification of TRPV4 as a channel responsive to mechanical stimulation that we specifically asked whether TRPV4 critically involved in high vascular pressure-induced lung injury. As in the work from Kuebler’s laboratory, we found that high vascular pressure elicited Ca2+ entry into subpleural endothelial cells in isolated mouse lung via activation of TRPV4 (Figure 13.6) 2+ [66]. Further, the high venous pressure-induced Ca entry through TRPV4 required PLA2- mediated arachidonic acid release and subsequent synthesis of EETs via cytochrome P450 expoxygenase. The Ca2+ response to high vascular pressure was similarly blunted in lungs from Trpv4−/− mice. The residual Ca2+ transient in these lungs was resistant to gadolinium, suggesting that all stretch-activated Ca2+ entry was indeed blocked. With acute high vascular pressure challenge, Kf increased once a threshold of ~30 cmH2O was reached and the sensitivity of lungs to pressure was increased with increasing perfusate temperature [66]. Either pretreatment of WT lungs with ruthenium red or use of lungs from Trpv4−/− mice abrogated 2+ the pressure-induced increase in Kf. As with the Ca responses, the permeability effect was Heart Failure and Pulmonary Edema 229

FIGURE 13.6 Ca2+ transients in subpleural microvessels after challenge with high vascular pressure (Pv). After fluo4-AM loading by perfusion, subpleural microvessels were imaged (A). Ca2+ transients were assessed at baseline and then during exposure to high vascular pressure (B, Pv = 5 or 30 cmH2O, respectively). Summary data show that Ca2+ transients elicited by high pressure were reproducible (C). The response to high pressure was blocked by pretreatment with an epoxygenase inhibitor (propargyloxyphenyl hexanoic acid, PPOH) or ruthenium red (RR) and −/− was similarly attenuated in lungs from Trpv4 mice (KO) with or without the addition of gadolium (GdCl3), an inhibitor of stretch activated channels. *p < 0.05 for Pv = 30 versus Pv = 5 cmH2O; #p < 0.05 for all treatment groups versus wild-type controls at this time point. From Jian et al. [66], with permission.

also abrogated by blocking PLA2 or P450 epoxygenases. Thus, mechanical stress, regardless of whether induced by high-volume/pressure ventilation or high vascular pressure, activates TRPV4 via an EET-dependent mechanism leading to acute lung injury.

HEART FAILURE AND PULMONARY EDEMA

Left heart failure is typically associated with chronic pulmonary venous hypertension and thus with increased pressures throughout the pulmonary circulation [65]. As a consequence, transvascular fluid filtration increases, regardless of whether pressures are sufficiently high to increase Kf or not [21,67]. If such filtration exceeds the capacity of lung lymphatics to clear fluid from the lung, pulmonary edema and potentially alveolar flooding result [62,67,68]. Alveolar flooding, in particular, detrimentally impacts gas exchange. The oxygen diffusing capacity decreases in proportion to the severity of heart failure, resulting in exercise intoler- ance and poor quality of life [69]. 230 13. TRPV4 IN LUNG DISEASE

Adaptations in lungs to chronic heart failure are multifaceted and complex, making interpretation of mechanisms involving TRPV4 challenging. After 2 months of rapid ventricular pacing and heart failure in dogs, Townsley et al. identified increased extravascular lung water, as expected, but also a ~30 cmH2O increase in the threshold for high vascular pressure- induced acute lung injury [63]. We had originally interpreted the increased resistance to high vascular pressure-induced injury as due to thickening of the alveolar septal barrier. Similar septal thickening can be found in guinea pigs after 5 months in an aortic banding model of chronic heart failure [70]. However, subsequent work in the pacing model as well as an arteriovenous fistula model in rats supported the notion of more complex adaptations in lung endothelial function. Heart failure and chronic pulmonary venous hypertension led to down-regulation of transient receptor potential (TRPC) channel proteins and thus loss of the permeability response to store depletion [41,71,72]. Nonetheless, the permeability response to the TRPV4 agonist 14,15-EET was retained in the arteriovenous fistula model of heart failure (i.e., evidence for retention of TRPV4 expression) [41]. Indeed, Thorneloe et al. found histologic evidence for pulmonary edema and increased endothelial expression of TRPV4 in lung sections from patients with heart failure [11]. Resolution of these data might lie in the circuitous mechanism linking mechanical stress to EET synthesis and thus to TRPV4 activation. Direct challenge with EETs may be effective in activating the channel, yet negative feedback mechanisms or remodeling might decrease the responsiveness of TRPV4 to mechanical stress. Several studies are relevant in that regard. First, Yin et al. reported that Ca2+ entry through TRPV4 acts to initiate a negative feedback mechanism blunting further Ca2+ entry through the channel, by recruiting nitric oxide synthesis and subsequent cyclic guanosine monophosphate (cGMP)-mediated desensitization of TRPV4 [73]. As a result, both the pressure-induced Ca2+ entry and the lung endothelial permeability response were attenuated. Further, they found that the phosphodiesterase 5 inhibitor sildenafil reduced lung edema in a myocardial infarct model of failure by reducing cGMP hydrolysis and thus exacerbation of this negative feedback pathway. Subsequently this group reported similar protection by sildenafil in an aortic banding model of heart failure [74]. Finally, Kerem et al. identified that lung endothelial Ca2+ responses to increased vascular pressure were markedly attenuated 9 weeks after aortic banding in rats [14]. They attributed this damped responsiveness to remodeling of the endothelial actin cytoskeleton, as cytochala- sin treatment reconstituted the Ca2+ response to increased pressure. The mechanism might be even more complex, however, as TRPV4 expression was decreased in lung endothelial cells from animals with heart failure. Extension of the observations implicating TRPV4 in high vascular pressure-induced lung injury to larger mammals and to chronic models of heart failure has been hampered by the lack of pharmacologic and/or genetic tools to specifically target TRPV4. However, recently Thorneloe and colleagues reported the development and characterization of small molecule antagonists that are selective for TRPV4 and have good oral bioavailability [11,75]. Using these pharmacologic tools, TRPV4 was identified as a requirement for the permeability response to acute high vascular pressure challenge in both mouse and canine lungs. As shown in Figure 13.7, pretreatment of rats with the specific TRPV4 antagonist GSK2193874 limited the development of pulmonary edema and hypoxemia in acute pulmonary venous hypertension induced by aortic banding (panels A and B). More important, GSK2193874 administered to mice during the second week post myocardial infarction was able to effectively promote resolution of pulmonary Pulmonary Arterial Hypertension 231

7 100 * 6 ** 80 5

4 60

3 /BW (mg/g) 40 LW 2 (mmHg) PaO2 20 1

0 0 (a) Sham (n = 7) Veh (n = 10) '874 (n = 11) (b) Sham (n = 7) Veh (n = 10) '874 (n = 11)

Sham (n = 6) Veh (n = 12) '874 (n = 13) 0

−1 −2

ter signal −3 −4

−5

∆ MRI lung wa −6 * −7 (c)

FIGURE 13.7 The TRPV4 antagonist GSK2193874 in rodent heart failure models. Pretreatment with the specific TRPV4 inhibitor GSK2193874 (’874) via oral gavage limited edema formation assessed by the lung weight/body

weight (LW/BW) ratio (A) and the reduction in arterial PO2 (PaO2, B) in rats following acute heart failure and pulmonary venous hypertension (left-ventricular end-diastolic pressure 30 cmH2O) induced by aortic banding. *p < 0.05; **p < 0.01 versus vehicle in A and C. In addition, ‘874 was effective in reversing the pulmonary edema resulting from heart failure secondary to myocardial infarction in mice (C). In this group, pulmonary edema was assessed 1 week following infarct by magnetic resonance imaging (MRI), then animals were randomly stratified to receive vehicle (Veh) or ‘874 daily for an additional 7 days. The change in MRI lung water intensity between days 7 and 14 was assessed. *p < 0.05 versus vehicle. From Thorneloe et al. [11], with permission. edema (panel C) [11]. Based on these promising outcomes, GlaxoSmithKline has recently registered a phase I clinical trial to assess the safety and pharmacokinetics of the TRPV4 inhibitor GSK2798745 in healthy humans and patients with stable heart failure [76].

PULMONARY ARTERIAL HYPERTENSION

Mechanisms underlying pulmonary arterial hypertension (PAH) have been much more extensively studied than those relevant to pulmonary venous hypertension in left heart disease. Dysfunction of pulmonary vascular endothelium, abnormal vasoconstriction, smooth muscle cell proliferation, and vascular remodeling all play key roles [77]. To date, serotonin and other ligands that activate store-operated channels (i.e., TRPC channels) have been implicated in 232 13. TRPV4 IN LUNG DISEASE the exaggerated vasoconstriction and smooth muscle proliferation in PAH. Nonetheless, several reports have recently emerged that potentially implicate TRPV4. As noted earlier, TRPV4 is expressed in pulmonary vascular endothelium and smooth muscle. The functional interplay between these two cellular components of pulmonary blood vessels leads to little impact of TRPV4 agonists on vascular tone in normal lungs, due to release of endothelial-derived nitric oxide [40,78]. With blockade of nitric oxide synthesis, TRPV4 activation leads to dose-dependent vasoconstriction [78]. In small intrapulmonary arteries and pulmonary arterial smooth muscle cells in culture, serotonin elicits Ca2+ responses, whole cell Ca2+ currents, and proliferation that are blunted by pretreatment with the nonspecific cyclooxygenase/lipoxygenase inhibitor eicosatetraynoic acid, the P450 ω-hydroxylase inhibitor 17-octadecynoic acid or ruthenium red, suggesting potential involvement of TRPV4 [79]. Further, Sham and colleagues reported that in the chronic hypoxia model of PAH, TRPV4 mRNA and protein expression was up-regulated in pulmonary arterial smooth muscle cells, and TRPV4 contributed to pulmonary vascular tone [80,81]. Although pulmonary blood vessels are not thought to exhibit myogenic responses (i.e., vasoconstriction evoked by increased intraluminal pressure), arteries from chronically hypoxic rats developed myogenic tone that could be inhibited by removal of Ca2+ from the bath solution or by treatment with the TRPV antagonist ruthenium red [82]. In intralobar pulmonary arteries from chronically hypoxic mice, either the TRPV4 antagonist HC-067047 or genetic deletion of TRPV4 blunted serotonin-induced vasoconstriction but not that induced by phenylephrine or endothelin-1 [80]. Similar impact of TRPV4 agonists on Ca2+ transients and proliferation of pulmonary artery smooth muscle cells in vitro has been reported [83,84]. Although the chronic hypoxia model does not appear to replicate the severity and complexity of human idiopathic PAH [85], these data do suggest that TRPV4 may potentially play a role. To date, there are no reports investigating a link between TRPV4 and idiopathic PAH.

AIRWAY DISEASE AND COUGH

Normal airways are designed to regulate airflow and to provide the fluid, mucus, and active beating of epithelial cilia that collectively subserve mucociliary clearance. The latter contributes to lung defense mechanisms, providing protection against inhaled particulates and pathogens. Sensory innervation of the airways provides mechanisms to detect noxious stimuli and reflexively evoke cough. These normal functions can be undermined in various diseases involving the airways, such as cystic fibrosis, airway injury and inflammation, asthma, and chronic obstructive pulmonary disease (COPD). Much of the work in this area has focused on other TRP channels, notably TRPV1, TRPM8, and TRPA1 [86]. Although the literature regarding TRPV4’s role in airways is not well developed, nonetheless, there are hints that targeting TRPV4 in airway disease might be beneficial. TRPV4 may play a role in functioning of airway epithelia, impacting mucociliary clearance. In murine tracheal epithelial cells, TRPV4 is expressed in apical cilia, colocalizing with ciliary tubulin [16]. In that model, Lorenzo and colleagues found that heat and 4αPDD elicited Ca2+ entry and increased ciliary beat frequency in a TRPV4-dependent manner; neither effect was evident in airway cells from Trpv4−/− mice. These responses to TRPV4 activation were implicated in a receptor-operated signaling cascade initiated by application of REFERENCES 233

extracellular ATP. Similar findings linking TRPV4 to regulation of ciliary beat frequency were obtained in human nasal epithelial cells [87]. The tight epithelial barrier and solute transport-driven fluid secretion to the airway surface are also required to maintain mucociliary clearance [88]. However, there is limited information about TRPV4 in this regard. Direct activation of TRPV4 with 4αPDD in cultured mammary gland epithelial cells increases mono- layer permeability by disrupting epithelial tight junctions [89]. In contrast, mechanical force such as shear actually enhanced barrier function (i.e., made the barrier tighter) in cultured primary human bronchial epithelial cells, a process that requires TRPV4-mediated Ca2+ entry and subsequent up-regulation of aquaporin-5 [90,91]. Epithelial cell swelling with hypotonicity (a stimulus for TRPV4 activation) initiates a Ca2+-dependent regulatory volume decrease, a compensatory response absent in epithelial from individuals with cystic fibrosis. Arniges and colleagues [92] reported that the cell-swelling induced TRPV4-dependent regulatory volume decrease was absent in a tracheal epithelial cell line from a cystic fibrosis patient, even though direct activation of TRPV4 with 4αPDD remained normal. Reconstitution of the cystic fibrosis transmembrane conductance regulator (CFTR) restored the TRPV4-dependent regulatory volume decrease in these cells. This group went on to show a requisite link between TRPV4- mediated Ca2+ entry and activation of large-conductance Ca2+-activated potassium channels in the regulatory volume decrease of human bronchial epithelial cells [93]. However, these findings might be context dependent, as hypotonicity-induced regulatory volume decreases remain normal in primary epithelial cells cultured from mainstem or lobar bronchi of cystic fibrosis patients [94]. Little information exists regarding expression and function of TRPV4 in airway smooth muscle and sensory nerves. A decade ago, Liedtke and Simon proposed a role for TRPV4 in airway hyperresponsiveness and asthma [95], based on the observation that human bronchial smooth muscle cells express TRPV4 and contract in response to TRPV4 activation by hypotonicity [96]. Since those initial reports, TRPV4 activation in airway smooth muscle has been shown to elicit tone development due to release of cysteinyl leukotrienes [47] and to initiate proliferation via a calcineurin/NFAT pathway [97]. With respect to TRPV4 in sensory neurons, only one preliminary report is available, which identifies GSK1016790A- and 4αPDD-dependent depolarization of Aδ sensory afferents from human, guinea pig, and mouse lungs [19], responses abolished in neurons from Trpv4−/− mice. At present, there are no reports translating these findings to animal models of asthma or clinical disease. Whereas Belvisi et al. found that GSK1016790A induced cough in conscious guinea pigs [19], an assessment of single nucleotide polymorphisms from normal individuals and those with asthma did not identify any association between the expression of TRPV4 variants and cough [98]. On a final note, Zhu and colleagues reported a strong association between TRPV4 single nucleotide polymorphisms and chronic obstructive pulmonary disease [99]. Several of the variants identified had previously been shown to impact assembly and trafficking of TRPV4 channels to the plasma membrane [100].

References [1] Nilius B, Vriens J, Prenen J, Droogmans G, Voets T. TRPV4 calcium entry channel: a paradigm for gating diversity. Am J Physiol Cell Physiol 2004;286(2):C195–205. [2] Strotmann R, Harteneck C, Nunnenmacher K, Schultz G, Plant TD. OTRPC4, a nonselective cation channel that confers sensitivity to extracellular osmolarity. Nat Cell Biol 2000;2(10):695–702. 234 13. TRPV4 IN LUNG DISEASE

[3] Gao X, Wu L, O’Neil RG. Temperature-modulated diversity of TRPV4 channel gating: activation by physical stresses and phorbol ester derivatives through protein kinase C-dependent and -independent pathways. J Biol Chem 2003;278(29):27129–37. [4] Liedtke W, Choe Y, Martí-Renom MA, Bell AM, Denis CS, Šali A, et al. Vanilloid receptor related osmotically activated channel (VR-OAC), a candidate vertebrate osmoreceptor. Cell 2000;103(3):525–35. [5] Liedtke W, Tobin DM, Bargmann CI, Friedman JM. Mammalian TRPV4 (VR-OAC) directs behavioral responses to osmotic and mechanical stimuli in Caenorhabditis elegans. Proc Natl Acad Sci USA 2003;100 (Suppl. 2):14531–6. [6] Mochizuki T, Sokabe T, Araki I, Fujishita K, Shibasaki K, Uchida K, et al. The TRPV4 cation channel mediates stretch-evoked Ca2+ influx and ATP release in primary urothelial cell cultures. J Biol Chem 2009;284(32):21257–64. [7] Vriens J, Owsianik G, Fisslthaler B, Suzuki M, Janssens A, Voets T, et al. Modulation of the Ca2+ permeable cation channel TRPV4 by cytochrome P450 epoxygenases in vascular endothelium. Circ Res 2005;97(9):908–15. [8] Vriens J, Watanabe H, Janssens A, Droogmans G, Voets T, Nilius B. Cell swelling, heat, and chemical agonists use distinct pathways for the activation of the cation channel TRPV4. Proc Natl Acad Sci USA 2004;101(1):396–401. [9] Watanabe H, Vriens J, Prenen J, Droogmans G, Voets T, Nilius B. Anandamide and arachidonic acid use epoxyeicosatrienoic acids to activate TRPV4 channels. Nature 2003;424(6947):434–8. [10] Watanabe H, Davis JB, Smart D, Jerman JC, Smith GD, Hayes P, et al. Activation of TRPV4 channels (hVRL-2/ mTRP12) by phorbol derivatives. J Biol Chem 2002;277(16):13569–77. [11] Thorneloe KS, Cheung M, Bao W, Alsaid H, Lenhard S, Jian MY, et al. An orally active TRPV4 channel blocker prevents and resolves pulmonary edema induced by heart failure. Sci Transl Med 2012;4(159):159ra48. [12] Willette RN, Bao W, Nerurkar S, Yue TL, Doe CP, Stankus G, et al. Systemic activation of the transient receptor potential vanilloid subtype 4 channel causes endothelial failure and circulatory collapse: Part 2. J Pharmacol Exp Ther 2008;326(2):443–52. [13] Alvarez DF, King JA, Weber D, Addison E, Liedtke W, Townsley MI. Transient receptor potential vanilloid 4-mediated disruption of the alveolar septal barrier: a novel mechanism of acute lung injury. Circ Res 2006;99(9):988–95. [14] Kerem A, Yin J, Kaestle SM, Hoffmann J, Schoene AM, Singh B, et al. Lung endothelial dysfunction in conges- tive heart failure. Circ Res 2010;106(6):1103–16. [15] Li J, Kanju P, Patterson M, Chew W-L, Cho S-H, Gilmour I, et al. TRPV4-mediated calcium influx into human bronchial epithelia upon exposure to diesel exhaust particles. Environ Health Perspect 2011;119(6):784–93. [16] Lorenzo IM, Liedtke W, Sanderson MJ, Valverde MA. TRPV4 channel participates in receptor-operated calcium entry and ciliary beat frequency regulation in mouse airway epithelial cells. Proc Natl Acad Sci USA 2008;105(34):12611–16. [17] Alessandri-Haber N, Dina OA, Joseph EK, Reichling DB, Levine JD. Interaction of transient receptor potential vanilloid 4, integrin, and SRC tyrosine kinase in mechanical hyperalgesia. J Neurosci 2008;28(5):1046–57. [18] Brierley SM, Page AJ, Hughes PA, Adam B, Liebregts T, Cooper NJ, et al. Selective role for TRPV4 ion channels in visceral sensory pathways. Gastroenterology 2008;134(7):2059–69. [19] Belvisi MG, Bonvini SJ, Grace MS, Ching YM, Dubuis E, Adcock JJ, et al. Activation of airway sensory nerves: A key role for the TRPV4 channel. Am J Resp Crit Care Med 2013;A5265. [20] Villalta PC, Townsley MI. Transient receptor potential channels and regulation of lung endothelial permeability. Pulm Circ 2013;3(4):802–15. [21] Parker JC, Townsley MI. Evaluation of lung injury in rats and mice. Am J Physiol Lung Cell Mol Physiol 2004;286(2):L231–46. [22] Parker JC, Townsley MI. Physiological determinants of the pulmonary filtration coefficient. Am J Physiol Lung Cell Mol Physiol 2008;295(2):L235–7. [23] Villalta PC, Rocic P, Townsley MI. Role of MMP2 and MMP9 in TRPV4-induced lung injury. Am J Physiol Lung Cell Mol Physiol 2014;307(8):L752–9. [24] Ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome. The Acute Respiratory Distress Syndrome Network. N Engl J Med 2000;342(18):1301–8. [25] Dreyfuss D, Saumon G. Ventilator-induced lung injury: lessons from experimental studies. Am J Respir Crit Care Med 1998;157(1):294–323. [26] Dos Santos CC, Slutsky AS. Invited review: mechanisms of ventilator-induced lung injury: a perspective. J Appl Physiol (1985) 2000;89(4):1645–55. REFERENCES 235

[27] Yoshikawa S, King JA, Reynolds SD, Stripp BR, Parker JC. Time and pressure dependence of transvascular Clara cell protein, albumin, and IgG transport during ventilator-induced lung injury in mice. Am J Physiol Lung Cell Mol Physiol 2004;286(3):L604–12. [28] Parker JC, Hernandez LA, Peevy KJ. Mechanisms of ventilator-induced lung injury. Crit Care Med 1993;21(1):131–43. [29] Quinn DA, Moufarrej RK, Volokhov A, Hales CA. Interactions of lung stretch, hyperoxia, and MIP-2 production in ventilator-induced lung injury. J Appl Physiol (1985) 2002;93(2):517–25. [30] Ricard JD, Dreyfuss D, Saumon G. Production of inflammatory cytokines in ventilator-induced lung injury: a reappraisal. Am J Respir Crit Care Med 2001;163(5):1176–80. [31] Yoshikawa S, King JA, Lausch RN, Penton AM, Eyal FG, Parker JC. Acute ventilator-induced vascular permeability and cytokine responses in isolated and in situ mouse lungs. J Appl Physiol 2004;97(6):2190–9. [32] Michel CC, Curry FE. Microvascular permeability. Physiol Rev 1999;79(3):703–61. [33] Parker JC, Ivey CL, Tucker JA. Gadolinium prevents high airway pressure-induced permeability increases in isolated rat lungs. J Appl Physiol 1998;84(4):1113–18. [34] Yin J, Kuebler WM. Mechanotransduction by TRP channels: general concepts and specific role in the vasculature. Cell Biochem Biophys 2010;56(1):1–18. [35] Matthews BD, Thodeti CK, Tytell JD, Mammoto A, Overby DR, Ingber DE. Ultra-rapid activation of TRPV4

ion channels by mechanical forces applied to cell surface β1 integrins. Integr Biol (Camb) 2010;2(9):435–42. [36] Kuebler WM, Ying X, Singh B, Issekutz AC, Bhattacharya J. Pressure is proinflammatory in lung venular capillaries. J Clin Invest 1999;104(4):495–502. [37] Davies PF. Flow-mediated endothelial mechanotransduction. Physiol Rev 1995;75(3):519–60. [38] Parker JC, Yoshikawa S. Vascular segmental permeabilities at high peak inflation pressure in isolated rat lungs. Am J Physiol Lung Cell Mol Physiol 2002;283(6):L1203–9. [39] Hamanaka K, Jian MY, Weber DS, Alvarez DF, Townsley MI, Al-Mehdi AB, et al. TRPV4 initiates the acute calcium-dependent permeability increase during ventilator-induced lung injury in isolated mouse lungs. Am J Physiol Lung Cell Mol Physiol 2007;293(4):L923–32. [40] Alvarez DF, Gjerde EA, Townsley MI. Role of EETs in regulation of endothelial permeability in rat lung. Am J Physiol Lung Cell Mol Physiol 2004;286(2):L445–51. [41] Alvarez DF, King JA, Townsley MI. Resistance to store depletion-induced endothelial injury in rat lung after chronic heart failure. Am J Resp Crit Care Med 2005;172:1153–60. [42] Yoshikawa S, Miyahara T, Reynolds SD, Stripp BR, Anghelescu M, Eyal FG, et al. Clara cell secretory pro-

tein and phospholipase A2 activity modulate acute ventilator-induced lung injury in mice. J Appl Physiol 2005;98(4):1264–71.

[43] Miyahara T, Hamanaka K, Weber DS, Anghelescu M, Frost JR, King JA, et al. Cytosolic phospholipase A2 and arachidonic acid metabolites modulate ventilator-induced permeability increases in isolated mouse lungs. J Appl Physiol 2008;104(2):354–62. [44] Edwards YS, Sutherland LM, Murray AW. NO protects alveolar type II cells from stretch-induced apoptosis. A novel role for macrophages in the lung. Am J Physiol Lung Cell Mol Physiol 2000;279(6):L1236–42. [45] Pugin J, Verghese G, Widmer MC, Matthay MA. The alveolar space is the site of intense inflammatory and pro- fibrotic reactions in the early phase of acute respiratory distress syndrome. Crit Care Med 1999;27(2):304–12. [46] Hamanaka K, Jian MY, Townsley MI, King JA, Liedtke W, Weber DS, et al. TRPV4 channels augment macrophage activation and ventilator-induced lung injury. Am J Physiol Lung Cell Mol Physiol 2010;299(3):L353–62. [47] McAlexander MA, Luttmann MA, Hunsberger GE, Undem BJ. Transient receptor potential vanilloid 4 activation constricts the human bronchus via the release of cysteinyl leukotrienes. J Pharmacol Exp Ther 2014;349(1):118–25. [48] Eyal FG, Hamm CR, Parker JC. Reduction in alveolar macrophages attenuates acute ventilator induced lung injury in rats. Intensive Care Med 2007;33(7):1212–18. [49] Frank JA, Wray CM, McAuley DF, Schwendener R, Matthay MA. Alveolar macrophages contribute to alveolar barrier dysfunction in ventilator-induced lung injury. Am J Physiol Lung Cell Mol Physiol 2006;291(6):L1191–8. [50] Prakash A, Mesa KR, Wilhelmsen K, Xu F, Dodd-o JM, Hellman J. Alveolar macrophages and Toll-like receptor 4 mediate ventilated lung ischemia reperfusion injury in mice. Anesthesiology 2012;117(4):822–35. [51] Zhao M, Fernandez LG, Doctor A, Sharma AK, Zarbock A, Tribble CG, et al. Alveolar macrophage activation is a key initiation signal for acute lung ischemia-reperfusion injury. Am J Physiol Lung Cell Mol Physiol 2006;291(5):L1018–26. 236 13. TRPV4 IN LUNG DISEASE

[52] Wang Z, Rui T, Yang M, Valiyeva F, Kvietys PR. Alveolar macrophages from septic mice promote polymorpho- nuclear leukocyte transendothelial migration via an endothelial cell Src kinase/NADPH oxidase pathway. J Immunol 2008;181(12):8735–44. [53] Broeke RT, Leusink-Muis T, Hilberdink R, Van Ark I, van den Worm E, Villain M, et al. Specific modulation of calmodulin activity induces a dramatic production of superoxide by alveolar macrophages. Lab Invest 2004;84(1):29–40. [54] Pacher P, Beckman JS, Liaudet L. Nitric oxide and peroxynitrite in health and disease. Physiol Rev 2007;87(1):315–424. [55] Thodeti CK, Matthews B, Ravi A, Mammoto A, Ghosh K, Bracha AL, et al. TRPV4 channels mediate cyclic strain-induced endothelial cell reorientation through integrin-to-integrin signaling. Circ Res 2009;104(9):1123–30. [56] Paine 3rd R, Morris SB, Jin H, Baleeiro CE, Wilcoxen SE. ICAM-1 facilitates alveolar macrophage phagocytic activity through effects on migration over the AEC surface. Am J Physiol Lung Cell Mol Physiol 2002;283(1):L180–7. [57] Furundzija V, Fritzsche J, Kaufmann J, Meyborg H, Fleck E, Kappert K, et al. IGF-1 increases macrophage motility via PKC/p38-dependent αvβ3-integrin inside-out signaling. Biochem Biophys Res Commun 2010;394(3):786–91. [58] Jurek SC, Hirano-Kobayashi M, Chiang H, Kohane DS, Matthews BD. Prevention of ventilator-induced lung edema by inhalation of nanoparticles releasing ruthenium red. Am J Respir Cell Mol Biol 2014;50(6):1107–17. [59] Muller-Redetzky HC, Will D, Hellwig K, Kummer W, Tschernig T, Pfeil U, et al. Mechanical ventilation drives pneumococcal pneumonia into lung injury and sepsis in mice: protection by adrenomedullin. Crit Care 2014;18(2):R73. [60] Huh D, Leslie DC, Matthews BD, Fraser JP, Jurek S, Hamilton GA, et al. A human disease model of drug toxicity-induced pulmonary edema in a lung-on-a-chip microdevice. Sci Transl Med 2012;4(159):159ra47. [61] Rippe B, Townsley M, Thigpen J, Parker JC, Korthuis RJ, Taylor AE. Effects of vascular pressure on the pulmonary microvasculature in isolated dog lungs. J Appl Physiol 1984;57(1):233–9. [62] Guyton AC, Lindsey AW, Howell JO, Williams JW, Franklin MA. Effect of elevated left atrial pressure and decreased plasma protein concentration on the development of pulmonary edema. Circ Res 1959;7(4):649–57. [63] Townsley MI, Fu Z, Mathieu-Costello O, West JB. Pulmonary microvascular permeability: responses to high vascular pressure after induction of pacing-induced heart failure in dogs. Circ Res 1995;77(2):317–25. [64] Kuebler WM, Ying X, Bhattacharya J. Pressure-induced endothelial Ca2+ oscillations in lung capillaries. Am J Physiol Lung Cell Mol Physiol 2002;282(5):L917–23. [65] Guazzi M, Galie N. Pulmonary hypertension in left heart disease. Eur Respir Rev 2012;21(126):338–46. [66] Jian M-Y, King JA, Al-Mehdi A-B, Liedtke W, Townsley MI. High vascular pressure-induced lung injury requires P450 epoxygenase-dependent activation of TRPV4. Am J Resp Cell Mol Biol 2008;38(4):386–92. [67] Staub NC. Pulmonary edema. Physiol Rev 1974;54(3):678–811. [68] Staub NC, Nagano H, Pearce ML. Pulmonary edema in dogs, especially the sequence of fluid accumulation in lungs. J Appl Physiol 1967;22(2):227–40. [69] Guazzi M. Alveolar-capillary membrane dysfunction in heart failure: evidence of a pathophysiologic role. Chest 2003;124(3):1090–102. [70] Huang W, Kingsbury MP, Turner MA, Donnelly JL, Flores NA, Sheridan DJ. Capillary filtration is reduced in lungs adapted to chronic heart failure: morphological and haemodynamic correlates. Cardiovas Res 2001;49(1):207–17. [71] Ivey CL, Roy BJ, Townsley MI. Ablation of lung endothelial injury after pacing-induced heart failure is related to alterations in Ca2+ signaling. Am J Physiol 1998;275(3 Pt 2):H844–51. [72] Roy BJ, Pitts VH, Townsley MI. Pulmonary vascular response to angiotensin II in canine pacing-induced heart failure. Am J Physiol 1996;271(1 Pt 2):H222–7. [73] Yin J, Hoffmann J, Kaestle SM, Neye N, Wang L, Baeurle J, et al. Negative-feedback loop attenuates hydrostatic lung edema via a cGMP-dependent regulation of transient receptor potential vanilloid 4. Circ Res 2008;102(8):966–74. [74] Yin J, Kukucka M, Hoffmann J, Sterner-Kock A, Burhenne J, Haefeli WE, et al. Sildenafil preserves lung endothelial function and prevents pulmonary vascular remodeling in a rat model of diastolic heart failure. Circ Heart Fail 2011;4(2):198–206. REFERENCES 237

[75] Hilfiker MA, Hoang TH, Cornil J, Eidam HS, Matasic DS, Roethke TJ, et al. Optimization of a novel series of TRPV4 antagonists with in vivo activity in a model of pulmonary edema. ACS Med Chem Lett 2013;4:293–6. [76] GlaxoSmithKline. A first time in human study to evaluate the safety, tolerability, pharmacokinetics, and pharmacodynamics of GSK2798745 in healthy subjects and stable heart failure patients. ClinicalTrials.gov [Internet] 2014 ed. Bethesda (MD): National Library of Medicine (US); 2014. [77] Morrell NW, Adnot S, Archer SL, Dupuis J, Jones PL, MacLean MR, et al. Cellular and molecular basis of pulmonary arterial hypertension. J Am Coll Cardiol 2009;54(1 Suppl.):S20–31. [78] Pankey EA, Zsombok A, Lasker GF, Kadowitz PJ. Analysis of responses to the TRPV4 agonist GSK1016790A in the pulmonary vascular bed of the intact-chest rat. Am J Physiol Heart Circ Physiol 2014;306(1):H33–40. [79] Ducret T, Guibert C, Marthan R, Savineau JP. Serotonin-induced activation of TRPV4-like current in rat intrapulmonary arterial smooth muscle cells. Cell Calcium 2008;43(4):315–23. [80] Xia Y, Fu Z, Hu J, Huang C, Paudel O, Cai S, et al. TRPV4 channel contributes to serotonin-induced pulmonary vasoconstriction and the enhanced vascular reactivity in chronic hypoxic pulmonary hypertension. Am J Physiol Cell Physiol 2013;305(7):C704–15. [81] Yang X-R, Lin M-J, McIntosh LS, Sham JSK. Functional expression of transient receptor potential melastatin- and vanilloid-related channels in pulmonary arterial and aortic smooth muscle. Am J Physiol Lung Cell Mol Physiol 2006;290(6):L1267–76. [82] Yang XR, Lin AH, Hughes JM, Flavahan NA, Cao YN, Liedtke W, et al. Upregulation of osmo-mechanosensitive TRPV4 channel facilitates chronic hypoxia-induced myogenic tone and pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol 2012;302(6):L555–68. [83] Martin E, Dahan D, Cardouat G, Gillibert-Duplantier J, Marthan R, Savineau JP, et al. Involvement of TRPV1 and TRPV4 channels in migration of rat pulmonary arterial smooth muscle cells. Pflugers Arch 2012;464(3):261–72. [84] Dahan D, Ducret T, Quignard JF, Marthan R, Savineau JP, Esteve E. Implication of the ryanodine receptor in TRPV4-induced calcium response in pulmonary arterial smooth muscle cells from normoxic and chronically hypoxic rats. Am J Physiol Lung Cell Mol Physiol 2012;303(9):L824–33. [85] Stenmark KR, Meyrick B, Galie N, Mooi WJ, McMurtry IF. Animal models of pulmonary arterial hypertension: the hope for etiological discovery and pharmacological cure. Am J Physiol Lung Cell Mol Physiol 2009;297(6):L1013–32. [86] Banner KH, Igney F, Poll C. TRP channels: emerging targets for respiratory disease. Pharmacol Ther 2011;130(3):371–84. [87] Alenmyr L, Uller L, Greiff L, Hogestatt ED, Zygmunt PM. TRPV4-mediated calcium influx and ciliary activity in human native airway epithelial cells. Basic Clin Pharmacol Toxicol 2014;114(2):210–16. [88] Trout L, Townsley MI, Bowden AL, Ballard ST. Disruptive effects of anion secretion inhibitors on airway mucus morphology in isolated perfused pig lung. J Physiol 2003;549(Pt 3):845–53. [89] Reiter B, Kraft R, Gunzel D, Zeissig S, Schulzke JD, Fromm M, et al. TRPV4-mediated regulation of epithelial permeability. FASEB J 2006;20(11):1802–12. [90] Sidhaye VK, Guler AD, Schweitzer KS, D’Alessio F, Caterina MJ, King LS. Transient receptor potential vanilloid 4 regulates aquaporin-5 abundance under hypotonic conditions. Proc Natl Acad Sci USA 2006;103(12):4747–52. [91] Sidhaye VK, Schweitzer KS, Caterina MJ, Shimoda L, King LS. Shear stress regulates aquaporin-5 and airway epithelial barrier function. Proc Natl Acad Sci USA 2008;105(9):3345–50. [92] Arniges M, Vazquez E, Fernandez-Fernandez JM, Valverde MA. Swelling-activated Ca2+ entry via TRPV4 channel is defective in cystic fibrosis airway epithelia. J Biol Chem 2004;279(52):54062–8. [93] Fernandez-Fernandez JM, Andrade YN, Arniges M, Fernandes J, Plata C, Rubio-Moscardo F, et al. Functional coupling of TRPV4 cationic channel and large conductance, calcium-dependent potassium channel in human bronchial epithelial cell lines. Pflugers Arch 2008;457(1):149–59. [94] Okada SF, Nicholas RA, Kreda SM, Lazarowski ER, Boucher RC. Physiological regulation of ATP release at the apical surface of human airway epithelia. J Biol Chem 2006;281(32):22992–3002. [95] Liedtke W, Simon SA. A possible role for TRPV4 receptors in asthma. Am J Physiol Lung Cell Mol Physiol 2004;287(2):L269–71. [96] Jia Y, Wang X, Varty L, Rizzo CA, Yang R, Correll CC, et al. Functional TRPV4 channels are expressed in human airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 2004;287(2):L272–8. [97] Zhao L, Sullivan MN, Chase M, Gonzales AL, Earley S. Calcineurin/NFAT-coupled TRPV4 Ca2+ sparklets stimulate airway smooth muscle cell proliferation. Am J Respir Cell Mol Biol 2014;50(6):1064–75. 238 13. TRPV4 IN LUNG DISEASE

[98] Smit LA, Kogevinas M, Anto JM, Bouzigon E, Gonzalez JR, Le Moual N, et al. Transient receptor potential genes, smoking, occupational exposures and cough in adults. Respir Res 2012;13:26. [99] Zhu G, Investigators I, Gulsvik A, Bakke P, Ghatta S, Anderson W, et al. Association of TRPV4 gene polymorphisms with chronic obstructive pulmonary disease. Hum Mol Genet 2009;18(11):2053–62. [100] Arniges M, Fernández-Fernández JM, Albrecht N, Schaefer M, Valverde MA. Human TRPV4 channel splice variants revealed a key role of ankyrin domains in multimerization and trafficking. J Biol Chem 2006;281(3):1580–6. CHAPTER 14 TRPM8 as a Target for Analgesia Rory Mitchell,1,* Susan Fleetwood-Walker1,* 1Centre for Integrative Physiology, School of Biomedical Sciences, College of Medicine & Veterinary Medicine, University of Edinburgh, Scotland, UK *Corresponding authors: [email protected]; [email protected]

OUTLINE

TRPM8 Structure 240 TRPM8-Mediated Analgesia 244

Localization of TRPM8 in the TRPM8 In Thermoregulation 246 Nervous System 240 Pharmacology of TRPM8 Antagonists and Agonists 246 In Vitro Studies on the Physiological TRPM8 Antagonists 246 Role of TRPM8 in Sensing TRPM8 Agonists 248 Cool/Cold Temperatures 241 Modulation of TRPM8 Function In Vivo Studies on the Physiological by Cellular Signaling 249 Role of TRPM8 in Sensing Lipids 249 Cool/Cold Temperatures 242 Kinases 250 G Protein-Coupled Receptor Signaling 250 Setting of Thermal Sensitivity in TRPM8-Expressing Sensory CNS Processes Activated by Neurons 243 TRPM8-Positive Afferents 252

The Impact of Pain States on Conclusions 254 TRPM8 Function 244 References 254

Only just over 10 years ago, the TRPM8 channel was cloned by several independent groups as the mediator of cool and menthol responsiveness in sensory ganglia and as an unknown transcript from prostate that was up-regulated in cancer [1–3]. Since that time, evidence has steadily and consistently accumulated to confirm a key physiological role of TRPM8 in the

TRP Channels as Therapeutic Targets 239 © 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/B978-0-12-420024-1.00014-X 240 14. TRPM8 AND ANALGESIA detection of innocuous cool. In addition, there is increasing interest in the channel as a potential target for new analgesics, especially in the context of chronic pain states that respond poorly to currently available therapeutics.

TRPM8 STRUCTURE

The TRPM8 channel is a nonselective cation channel assembled as a homotetramer of the 1104 amino acid protein, which individually has a molecular weight of around 128 kDa. The monomer has extended intracellular N- and C-termini and six transmembrane domains with a loop between domains 5 and 6 that is thought to contribute to the channel pore. Various reports implicate the C- or N-terminal segments in subunit assembly [4–6]. The C-terminal segment contains a short TRP box motif that is highly conserved throughout the family, including residues 1005 and 1009, involved in binding the allosteric potentiator PIP2 (phosphatidylinositol 4,5-bisphosphate), and is also necessary for agonist responsiveness and setting the cool temperature response threshold [2,3,7–9]. The initial part of the N-terminal segment has also been implicated in moderating responsiveness to cool and menthol [10]. Chemical agonists that activate TRPM8 include menthol and the synthetic compound icilin, both of which evoke a sensation of coolness. However, differences in channel residues involved in their recognition indicate that the two agonists bind to distinct sites, consistent with evidence that activation by icilin or cooling but not by menthol shows clear pH dependence and that icilin but not menthol action appears to require an increase in intracellular Ca2+ levels [11,12]. Mutation of residues 745, 842 and 856 in transmembrane domain 2 and the loop between domains 4 and 5 disrupts menthol activation whereas residues 799, 802 and 805 in transmembrane domain 3 are required for icilin effects [12–15]. TRPM8 also shows voltage sensitivity, with residues 842 and 856 probably contributing to voltage sensing [14,16,17]. Interactions between the different modes of activation are observed in that agonists not only increase open probability and conductance of the channel but also shift both its thermal threshold and voltage sensitivity toward more normal physiological levels [14,16–19]. The overall responses of a cell expressing TRPM8 to its activating stimuli will be further influenced by the effects of other ion channels on membrane potential and by signals from other receptors/intracellular signaling cascades.

LOCALIZATION OF TRPM8 IN THE NERVOUS SYSTEM

TRPM8 has a highly selective tissue distribution. Apart from primary somatosensory neurons and prostate tissue (from where it was cloned), it is expressed at only low levels in other tissues. These include visceral afferents, bladder, vascular smooth muscle, stomach, liver, colon, lung and airways, sperm, and some primary tumors [20–28]. In the nervous system, TRPM8 is strongly expressed in trigeminal (TG) and dorsal root ganglia (DRG) and has subsequently been observed in autonomic ganglia [29–31]. However, there appears to be extremely limited TRPM8 expression within the central nervous system (CNS) other than that associated with afferent terminals [32]. This idea was fully corroborated by observations on mutant mice expressing green fluorescent protein (GFP) under the control of the TRPM8 promoter [33,34]. IN VITRO STUDIES ON THE PHYSIOLOGICAL ROLE OF TRPM8 241

TABLE 14.1 Immunofluorescence assessment of TRPV1/TRPM8 colocalization in small DRG neurons: Sixteen μm unfixed cryostat sections (12-19 from 4 to 5 rats in each case) were incubated with guinea pig anti-TRPV1 and rabbit anti-TRPM8 primary antibodies, then appropriate secondaries labeled with distinct ALEXA-fluorophores. Cells were counted at ×40 magnification from confocal images using a Zeiss LS510 Axiovert microscope

Colocalized as Colocalized as Condition TRPV1 alone TRPM8 alone TRPV1 + TRPM8 % TRPV1 % TRPM8 Naïve 211 61 8 3.7 11.6 CFA 119 42 5 4.0 10.6 CCI 155 55 8 4.9 12.7

Furthermore, only 5-10% of DRG and TG neurons display the responsiveness to mild cooling (threshold around 25 °C), menthol and icilin that is characteristic of TRPM8, emphasizing the highly selective outcome to be expected from any intervention that might usefully target the channel. Early observations indicated that these neurons were small in diameter and distinct from those expressing classical nociceptive markers such as calcitonin gene-related peptide (CGRP), TRPV1, or binding sites for the lectin IB4 [3]. Most TRPM8-positive sensory neurons appear to be C fibers staining for peripherin, with a minor group of Aδ fibers, whose small cell bodies stain for neurofilament antigens [2,33–36]. Extensive in situ hybridization experiments in DRG indicated a pattern of cellular expression almost entirely distinct from that of the nociceptive channels TRPV1 and TRPA1 [37]. Other studies combining immunohistochemical and alternative localization techniques indicate a modest degree of overlap [33, 234,38], and our own dual immunofluorescence data from TRPM8 and TRPV1 (Table 14.1) concur in finding TRPV1 expression in a very small proportion of TRPM8-positive cells. A recent study utilizing genetically targeted ablation of TRPV1- and TRPM8-expressing cells reinforced the idea of minimal overlap [39]. Technical issues such as differences in sensitivity and selectivity of the reagents and methods used may contribute to the range of findings reported. A further factor to consider is that the extent of overlap may differ between somatic and autonomic sensory neurons where phenotypic subdivisions may differ from those of somatic afferents [20,24,40]. Studies exploring functional responses of TRPM8 have also tried to explore the extent to which any overlap is significant.

IN VITRO STUDIES ON THE PHYSIOLOGICAL ROLE OF TRPM8 IN SENSING COOL/COLD TEMPERATURES

When cloned TRPM8 is expressed in oocytes or fibroblasts, the cells are reported to show a temperature activation range from around 22-25 °C (threshold) to 8-10 °C (maximal) [2,3]. Correspondingly, native TG neurons that show Ca2+ elevation responses selectively to menthol have thermal activation thresholds around 25 °C [41]. DRG or TG neurons from TRPM8−/− mice show significantly reduced responses to cooling stimuli, which ranged in different studies from 22 °C through to 9 °C, and in C fiber firing induced by cooling from 32 °C to 2 °C in a skin- nerve preparation [42–45]. With such readouts of responses to imposed progressive cooling, it 242 14. TRPM8 AND ANALGESIA is not clear whether components of responses in the noxious temperature range are lacking in TRPM8−/− mice as well as those from innocuous temperatures. These data remain equivocal in deciding whether TRPM8 is involved in physiological cold pain under normal conditions. A number of studies on cultured TG or DRG cells describe subpopulations responding to both mild cooling (generally through the range 25-17 °C) and to menthol that are considered to reflect TRPM8-expressing cells [46–49]. Some caution is needed, though, as menthol has only modest selectivity for TRPM8 over TRPA1 [50], which also displays sensitivity to reduced temperatures and is expressed in a subset of TRPV1-positive nociceptors [41,51]. A careful comparison of the temperature sensitivity of individual menthol-responsive/allyl isothiocyanate (TRPA1 agonist)-unresponsive TG neurons (likely to express TRPM8) and those activated by both menthol and allyl isothiocyanate (likely to express TRPA1) points to higher (innocuous range) temperature thresholds in the TRPM8 group but with substantial overlap [41]. Although menthol responsiveness in itself does not decisively implicate TRPM8, some studies have explored responsiveness of sensory neuron populations to menthol and capsaicin [48]. Neurons with dual responsiveness could be taken as evidence for functionally significant TRPM8 channels in nociceptors, but the debate remains equivocal because of menthol’s limited selectivity for TRPM8 over TRPA1. In addition, the electrophysiological responses of menthol-activated (putative TRPM8-positive) DRG neurons to mild cooling to 24 °C, involving tetrodotoxin-sensitive Na+ channels, appeared to be attenuated at the more intense cold level of 10 °C, whereas the intense cold-induced firing in neurons with tetrodotoxin-insensitive Na+ channels (putative nociceptors) remained robust [52]. The development of highly selective agonist and antagonist tools for TRPM8 should help to further elucidate the situation. An additional variable in experiments with cultured sensory neurons is the possibility of a change in phenotype relating to duration in vitro [34], a parameter that can differ considerably between studies and may also undergo some transition during chronic pain states [53].

IN VIVO STUDIES ON THE PHYSIOLOGICAL ROLE OF TRPM8 IN SENSING COOL/COLD TEMPERATURES

C fiber recordings in rodents identify two distinct populations of cold-sensitive neurons [54]. The first of these comprises low temperature threshold, mechanoinsensitive, heat-insensitive afferents, sensitive to small reductions in skin temperature of as little as 2 °C, that are activated by menthol (10% topical) and by evaporative cooling due to acetone (i.e., nonnociceptive thermoreceptors that may express TRPM8). The second comprises high threshold cold-, mechano- and heat-sensitive nociceptive afferents, firing at 12 °C or below that are indifferent to menthol. These findings are consistent with a significant role for TRPM8 in innocuous cool detection but not cold pain in normal animals. Similar profiles are reported from microneurography experiments in normal human volunteers [55]. Very high concentrations of menthol (up to 40%) applied topically to the skin are perceived as noxious [56–58] [a situation mirrored by behavioral experiments in rodents [36]], but interpretative caution is needed as TRPM8 selectivity is uncertain at such concentrations. In the special circumstance of the cornea, highly sensitive cool thermoreceptors predominate, which respond vigorously to small reductions in temperature (as little as 0.5 °C reduction) and contain abundant TRPM8 [59]. Setting of Thermal Sensitivity in TRPM8-Expressing Sensory Neurons 243

Thermal place preference tests with TRPM8−/− mice indicate lack of sensation across the innocuous cooling temperature range [42–44,60]. Whereas colder temperatures (10 °C and below) showed return of temperature preference and cold plate paw withdrawal responses (10 °C and below) were normal in TRPM8−/− mice [42,44,45], some attenuation of thermal preference was seen, to a greater or lesser extent, at temperatures as low as 5 °C. Correspondingly, the flicking responses due to evaporative cooling from acetone, which can cool the skin to temperatures around 14-18 °C [43,61], were substantially reduced in TRPM8−/− mice [42,44] or by systemic administration of the high-affinity TRPM8 antagonist, PBMC [61]. As this temperature range corresponds to the loosely defined border between innocuous and noxious cooling in man [62] and the precise thermal consequences of differing laboratory protocols are uncertain, it is not clear that the test explicitly reflects cold pain as opposed to a response to innocuous cooling. Intraplantar injection of the selective TRPM8 agonist icilin at high local concentrations (8 mM solution) can evoke flinching behavior and spinal cord c-Fos expression, which are reduced in TRPM8−/− mice [43–45,63]. Intraplantar injections of icilin or menthol at high concentrations, however, cause activation of a wide variety of sensory neurons of different types through apparently TRPM8-independent processes [64]. Furthermore, many agents when injected directly into the skin, presumably adjacent to sensory nerve terminals, can elicit nociceptive responses that they would not normally cause through other routes such as topical administration [65]. Although intraplantar icilin-evoked nocifensive behavior appears to involve TRPM8, it is not clear that this relates to physiological cold sensing. As adaptive compensatory responses are possible in constitutive knockout animals, a targeted ablation strategy has also been investigated. In TRPM8 neuron-ablated mice (which express a diphtheria toxin (DTx) receptor transgene under the control of the TRPM8 promoter and were treated with DTx) results corroborated those in TRPM8−/− mice [39,60]. TRPM8 ablation abrogated behavioral responses to acetone-induced evaporative cooling and thermal preference through the innocuous cooling range of 30-10 °C [39,60]. However, avoidance of 0-10 °C cold surfaces and paw withdrawal/flinching to severe noxious cold were also attenuated in TRPM8 neuron-ablated mice, and this was to a greater extent than in TRPM8−/− mice [39,60]. Some of these data were obtained with a new sensitive forepaw-flinching assay, but the precise extent of temperature reduction reached in the forepaws may be affected by guarding behavior. Furthermore because the strategy ablates neuronal populations rather than individual candidate molecular mediators, additional proteins in subsets of TRPM8- expressing cells could be key to noxious cold sensing.

SETTING OF THERMAL SENSITIVITY IN TRPM8-EXPRESSING SENSORY NEURONS

Preexposure to chemical agonists such as menthol markedly increases cold-induced Ca2+ entry in TRPM8-expressing oocytes or fibroblasts and menthol-sensitive TG cells, as well as cold-induced firing in a skin-nerve preparation [2,3,66], a phenomenon also reported with TRPA1 [67]. The effective temperature threshold for cellular responses to cooling can also be influenced by coexpressed K+ channels acting to hyperpolarize the membrane and op- pose TRPM8-mediated depolarization. Both Kv1 and Kv7 family channels are coexpressed with TRPM8, notably in nociceptors where high K+ channel: modest TRPM8 expression ratios 244 14. TRPM8 AND ANALGESIA may drive the cold threshold into the noxious temperature range [66,68]. In contrast, low- threshold nonnociceptive cool-sensing afferents appear to have high ratios of TRPM8: K+ channel expression [59,66,68]. In addition, K2P TREK family channels, some of which are directly closed by temperature reductions, are present in subsets of TRPM8-positive cells [69] and may impact on their thermal sensitivity. Effects of cooling on A-type K+ channels and tetrodotoxin-sensitive or resistant Na+ channels may also modulate firing in cool thermoreceptors and cold nociceptors [52,70], whereas the notable resistance of Nav1.8 to cooling-induced desensitization is crucial for transmission in cold-sensitive afferents [71]. TRPM8 function (and indeed that of any other threshold-setting channels) is, of course, also subject to a variety of modulatory influences from intracellular signaling events (see later).

THE IMPACT OF PAIN STATES ON TRPM8 FUNCTION

Chronic pain states of either inflammatory or neuropathic origin crucially involve central hypersensitivity that is brought about by neurochemical changes ensuing from maintained nociceptor firing [72]. This will manifest as exaggerated responses to noxious stimuli (hyperalgesia) and perception of normally innocuous stimuli as noxious (allodynia). This central resetting of excitability will most likely apply to thermal, mechanical, and cool sensory inputs, so in the case of TRPM8-mediated inputs a degree of cool allodynia would be entirely expected. Whether there are any specific adaptive responses in TRPM8-expressing neurons themselves has been investigated by a number of groups, with varying results. There is little evidence that inflammation alters TRPM8 expression but there is clearly amplified responsiveness to acetone-induced cooling in the intraplantar Complete Freund’s Adjuvant (CFA) model of inflammatory pain [43]. This could potentially reflect increased expression of TRPA1 [73,74] or simply the expected central hypersensitivity to inputs from the TRPM8-mediated innocuous cool afferents [60,61]. In some neuropathic pain models, TRPM8 expression is reported to increase [36,75–77], although little change, or reduced expression, has been reported in others [73,78,79]. Cool allodynia is apparent after nerve injury, and both pharmacological and genetic interventions indicate that this is likely to involve TRPM8 [43,60,61,76–78]. This may, however, reflect simply the TRPM8-mediated reportage of innocuous cooling that, like any other somatosensory input, becomes amplified due to injury-induced central sensitization. Indeed, a recent electrophysiological study reported that acetone-evoked evaporative cooling responses, but not other sensory responses of spinal cord neurons, were inhibited by a selective TRPM8 antagonist in nerve injured but not naive rats [80]. Interestingly, however, in patients with established cool allodynia due to nerve injury, the topical administration of menthol does not aggravate hypersensitivity [81].

TRPM8-MEDIATED ANALGESIA

Although cooling and mint extracts containing menthol have been widely used for many years due to their soothing, antinociceptive effects, the molecular basis was long unknown [82–85]. The cloning of TRPM8 and its identification in a subset of DRG/TG sensory neurons provided a likely framework. In 2006, TRPM8 was specifically demonstrated for the first time to mediate analgesia due to cooling or the chemical agonists menthol and icilin, applied topically TRPM8-Mediated Analgesia 245 or intrathecally in animal models of both chronic neuropathic pain (CCI, chronic constriction injury) and inflammatory pain [36]. The pharmacological identification of TRPM8 mediation was corroborated by antisense knockdown experiments indicating that active functional TRPM8 was required as opposed to any potential for an effect due to agonist-induced channel desensitization. Both thermal (heat) hyperalgesia and mechanical allodynia were reversed, but cool allodynia was not addressed because of the likelihood of a complex, mixed influence. Interestingly, there was no effect on unsensitized responses in naive animals or in unaffected limbs until much higher concentrations, which produced pronociceptive effects. Experiments investigating the effects of relatively high concentrations of topically applied menthol in naive animals report attenuation of noxious thermal responses, mixed effects on cool/cold responses, and sensitization of innocuous mechanical responses [86], although mediation by TRPM8 was not ascertained, and off-target effects may contribute. The original observations of TRPM8 analgesia were subsequently confirmed in the CCI model of neuropathic pain, in which intrathecal menthol was similarly found to strongly reduce thermal hyperalgesia and mechanical allodynia but increase withdrawal responses from a 4 °C cold plate [77]. Mediator specificity was established by antisense knockdown in this study, too. Interestingly the analgesic effects of TRPM8 agonists were not observed in an alternative neuropathic pain model (SNL, spinal nerve ligation; [87]) in which TRPA1 has been implicated [88]. Evidence for TRPM8-mediated analgesia was also provided in the formalin-induced flinching model, in which cool-induced analgesia was attenuated in TRPM8−/− mice compared to controls [44]. Recent work provides robust support for the idea of TRPM8-mediated analgesia, showing that systemic or topical administration of menthol diminishes pain behavior due to noxious heat, TRPV1 or TRPA1 activators or intraperitoneal acidification as well as attenuating inflammation-induced mechanical hypersensitivity [89]. The critical role of TRPM8 was clearly demonstrated through abrogation of effects in the presence of a highly selective TRPM8 antagonist or in TRPM8−/− mice. Powerful analgesic effects of systemic menthol against formalin-induced flinching and inflammatory hypersensitivity have also been recently described at rather higher dosage levels, although in this case TRPM8-independent mechanisms may also contribute significantly [90]. Key supportive data have also been provided through experiments on targeted ablation of TRPM8-expressing neurons where the cooling-evoked attenuation of mechanical allodynia seen in the CCI neuropathic pain model in control animals was abrogated by toxin-evoked TRPM8 ablation [60]. Corresponding results were seen in TRPM8−/− mice. Further evidence shows that pain state-induced synaptic hypersensitivity not only at the spinal cord but also forebrain levels can be reversed by topical administration of TRPM8 agonist; with the involvement of TRPM8 established through blockade by a highly selective antagonist [91]. Taken together, these observations firmly establish that TRPM8 activation is able to gate-out hypersensitive nociceptive inputs and activation of the CNS in chronic pain states, most likely through the spinal influence of the TRPM8-expressing subset of sensory afferents. So a case can be made for the use of either antagonists or agonists at TRPM8 in the treatment of pain. Antagonists may be useful to treat the cool allodynia associated with chronic pain states. Effects are likely to be limited to this modality, however, as they would influence only the sensory detection of cool that involves TRPM8 and not the central sensitization that leads to parallel problems of mechanical allodynia and thermal hyperalgesia. Antagonists could potentially be considered for treating acute cold pain in naive subjects, but any evidence to validate this is much less strong than that illustrating the role of TRPM8 in innocuous cool thermosensation. It may well be that other factors play a key part in noxious cold sensing, so any 246 14. TRPM8 AND ANALGESIA effect of TRPM8 blockade may be less robust; this will become clear in future work. Agonists show great promise in that they are now well documented to produce efficacious analgesia in hypersensitive pain states where they appear to inhibit central sensitization and therefore reverse chronic pain of a number of different modalities. Both neuropathic and inflammatory pain hypersensitivity can be effectively targeted. One caveat with this approach would be that cool allodynia may be exacerbated, although it would be predicted that the analgesic effects of TRPM8 agonists suppressing central hypersensitivity act in opposition to any enhancement of peripheral cool sensing and thereby ameliorate any cool allodynia. Clinical evidence in chronic pain patients supports this idea, as cool allodynia does not seem to be a problematic issue [81,92,93]. Care is also needed in evaluation of the therapeutic window because of the possibility of noxious sensations if supratherapeutic concentrations of agonists are reached. Either strategy (as with any analgesic intervention) could potentially encounter on-target side effects in other tissues or off-target effects due to insufficient pharmacological specificity, a medicinal chemistry issue around the particular pharmacophore utilized. As TRPM8 is expressed in relatively few tissues, the on-target side-effect issue may be relatively unproblematic, especially if agents are applied topically to dermatomes around the site of chronic pain to access selectively the relevant TRPM8 afferents and limit the systemic drug load. For both antagonists and agonists, a further possible issue that would need to be evaluated might be disruption of central thermoregulation, as identified in the case of TRPV1 antagonists.

TRPM8 IN THERMOREGULATION

When antagonists of the noxious-heat sensing channel TRPV1 were tested in vivo as potential analgesics, significant effects on regulation of core body temperature became apparent. TRPV1 antagonists caused hyperthermia, thermogenesis, and vasoconstriction in wild-type but not TRPV1−/− mice [94,95]. Although in contrast to TRPV1, TRPM8 is absent from central thermoregulatory centers in the hypothalamus, potential effects of TRPM8 agonists or antagonists on core temperature have been investigated. Intraperitoneal injection of icilin at high concentrations produces a characteristic acute shivering behavior known as “wet dog shakes,” presumably by stimulating visceral afferents, and this response is attenuated in TRPM8−/− mice [45,96,97]. Systemic or topical administration of menthol or icilin (at relatively high doses) leads to a transient increase in core temperature, presumably an attempt at compensatory thermoregulation [98–103]. Accordingly, systemic TRPM8 antagonists produce a transient reduction in core temperature [61,80,103,104]. In both cases, some studies confirmed lack of effects in TRPM8−/− mice. Whether any significant thermoregulatory changes are observed at the doses therapeutically relevant for the treatment of chronic pain remains to be established.

PHARMACOLOGY OF TRPM8 ANTAGONISTS AND AGONISTS

TRPM8 Antagonists For a number of years no highly selective antagonists of TRPM8 were available to help validate the inferred role of the channel in thermosensation and modulation of pain processing. Early studies identified that some TRPV1 antagonists also had affinity for TRPM8, Pharmacology of TRPM8 Antagonists and Agonists 247

providing the first useful but fairly nonselective tools. Capsazepine, BCTC, and SB-452533 were shown to be effective TRPM8 antagonists but with clearly lower potency than at TRPV1, posing difficulties for data interpretation in complex in vivo situations [105,106]. The antifungal agent clotrimazole was identified as a relatively potent TRPM8 antagonist [107], but it additionally blocks K+ channels and activates both TRPV1 and TRPA1. Tryptamine derivatives such as 5-benzoyloxytryptamine were also unexpectedly shown to antagonize TRPM8 but may well impact on 5-HT receptor function, and any potential effects on other pain-relevant channels are unknown [108]. The first clearly selective TRPM8 agonist widely disclosed was AMTB, developed by Bayer [109], although its affinity was still only moderate. Other pharma (including Glenmark, Amgen, Janssen, and Johnson and Johnson) have now developed a number of highly potent and selective TRPM8 antagonists with diverse chemical structures. These include benzothiophene sulphonamides and phosphonates, fused ox- azoles and thiazoles, benzimidazoles, fused piperidines, aryl glycines, menthylamines, and ylidenephthalides (although the last two may have some TRPA1/TRPV1 activity) [110–118]. Additional potent and effective TRPM8 antagonists from further structural series have been produced by Pfizer and Takeda [61,80], and even endogenous and plant cannabinoids inhibit at submicromolar concentrations [119], yielding a truly diverse array of pharmacoph- ores as TRPM8 blockers (Figure 14.1).

FIGURE 14.1 Examples of recently produced TRPM8 antagonists reported to be active at submicromolar concentrations in vitro. Compounds represent several distinct structural series. Although certainly potent TRPM8 antagonists, the pharmacological selectivity profiles are generally not described in detail. 248 14. TRPM8 AND ANALGESIA TRPM8 Agonists The prototypical TRPM8 agonist menthol (more precisely (−) menthol or IR, 2S, 5R-2- isopropyl-5-methylcyclohexanol) is an effective but not particularly selective TRPM8 agonist. In our studies measuring Ca2+ fluorescence responses of HEK-293 cells stably expressing human TRPM8, it shows a mean EC50 of 11.2 μM with efficacy 66% that of the benchmark agonist icilin, but a mean EC50 of 29.6 μM and 95% efficacy compared to allyl isothiocyanate (mustard oil) at TRPA1. The stereoisomers show an unremarkable structure-activity relationship [13,36], but an extensive range of analogs has been produced, notably WS-12 and D-3263, which show greatly increased potency at TRPM8 [120–125]. The tetrahydropyrimidine-2-one, icilin, was reported to produce marked sensations of cold [126] and is established as a potent

TRPM8 agonist with an EC50 of 0.3 μM and considerable selectivity against TRPA1 (about 300-fold in our assays) and even more against TRPV1 [2,105]. However, icilin has substantial affinity as an antagonist at TRPV3 [127]. Thienopyrimidine compounds [124] display moderate potency and selectivity, with a hydroxyethyl-substituted example showing an EC50 of 10 μM at TRPM8 and at least 20-fold selectivity over TRPA1 and TRPV1 in our hands. Perhaps the most enigmatic of TRPM8 agonists is an aliphatic phosphonate, WS-148, (1-(di- sec-butyl- phosphinoyl)-heptane), which is reported to show an EC50 of 4.1 μM at TRPM8, although its off-target profile has not been described [121]. A recent report describes a series of isoxazole compounds as potent partial agonists in causing Ca2+ elevation in a DRG/neuroblastoma cell line that responds to menthol and expresses TRPM8 mRNA [128]. Further characterization of these compounds will be interesting, especially as modeling of interactions with a quaternary structural model of the TRPM8 channel [129] indicates a potential for both menthol-like and icilin-like interactions in the series [128] (Figure 14.2). The development of new highly

FIGURE 14.2 TRPM8 agonists of different structural series. WS-12 and D-3263 are analogs of menthol with considerably increased potency, showing EC50 at submicromolar concentrations. D-3263 has undergone a Phase 1, Open Label Trial in relation to potential antitumor activity. Icilin shows submicromolar potency and moderate selectivity. Thienopyrimidines, phosphonates, and isoxazoles need to be further assessed to determine target selectivity. Modulation of TRPM8 Function by Cellular Signaling 249

selective TRPM8 agonists as tools will be an important step forward, as the majority of physiological studies carried out to date with TRPM8 agonists have utilized either menthol or icilin, and unless additional strategies are incorporated to validate TRPM8 mediation, some caution is needed in interpretation. Menthol can exert a wide range of TRPM8-independent effects, generally at somewhat higher concentrations than those that adequately activate TRPM8. These include potentiation of GABA and glycine currents, inhibition of α4β2- and α7-nicotinic cholinergic receptors, 2+ + inhibition of 5-HT3 receptors, inhibition of Ca and Na channels and local anesthetic-like suppression of action potential firing, and even at very high concentrations, desensitization of TRPV3 and cell cycle arrest in proliferating cells [90,130–143]. Systemic menthol at low doses produces analgesia that principally involves TRPM8 [89], whereas effects at considerably higher doses appear to involve disruption of Ca2+ and Na+ channels [90]. Apart from its significant affinity for TRPV3, icilin has a number of additional effects. Inhibition of L-type Ca2+ channels is reported at similar submicromolar concentrations similar to those causing activation of TRPM8, but the basis is rather unclear as the effect increased slowly, without saturating, over five orders of magnitude of concentration [87]. Icilin at somewhat higher concentrations is also reported to activate epithelial Na+ channels, evoke a distinct inhibitory effect on TRPM8 gating that is separate from Ca2+-dependent channel desensitization, and even lead to cell cycle arrest in proliferating cells [15,144–146].

MODULATION OF TRPM8 FUNCTION BY CELLULAR SIGNALING

Lipids

Like other TRP channels, TRPM8 is prominently modulated by PIP2, which slows channel rundown in isolated membrane patches and acts to facilitate channel activation by cooling or chemical agents [147–149]. Mutation of TRP box PIP2-binding residues greatly reduces TRPM8 responses to stimuli including chemical agonists [91,148]. Enzymatic activity of phospholipase C (PLC), including that evoked by agonist stimulation of PLC-coupled receptors, can lead to dynamic local depletion of PIP2 concentrations and suppression of TRPM8 function [147,148,150,151]. A PIP2 binding protein, PIRT, is widely expressed in DRG cells and enhances menthol responses in HEK-293 cells cotransfected with TRPM8, whereas PIRT−/− mice show partially attenuated avoidance of cool and cold surfaces [152].

Lysophospholipids and polyunsaturated fatty acids, products of phospholipase A2 (PLA2) action on membrane phospholipids, reciprocally enhance or inhibit TRPM8 function, respectively [153–155]. Inhibition of PLA2 enzyme activity attenuated the hypersensitivity to 10 °C cold induced by the intraplantar injection of icilin at a high concentration, but not due to menthol [63]. Corresponding experiments in mutant mice indicated the involvement of TRPM8 in hypersensitivity due to intraplantar icilin but TRPA1 in that induced by menthol.

PLA2 activation downstream of receptor activation and kinase cascades could therefore lead to complex profiles of TRPM8 modulation. Changes in membrane lipid composition may also influence the localization of TRPM8 within cholesterol-rich lipid rafts, which appears to normally down-regulate its responsiveness to stimuli [156]. Volatile general anesthetics 250 14. TRPM8 AND ANALGESIA have been reported to affect TRPM8 with a transient enhancement followed by a sustained inhibition of function, effects that may reflect alterations in the membrane protein:lipid interface [157].

Kinases The intracellular domains of TRPM8 contain a number of potential regulatory phosphorylation sites that match more or less closely to the consensus target sequences of several common kinases [158]. Phorbol ester or receptor-mediated activation of protein kinase C (PKC) inhibits TRPM8 function [159–162], but there is little evidence that direct phosphorylation is responsible, rather than a downstream calcineurin-dependent dephosphorylation mechanism [159]. Protein kinase A (PKA) activation brought about by forskolin or 8-Br-cAMP is reported to reduce chemical agonist-evoked TRPM8 responses or DRG neuronal responses to cooling [119,161], although in our experiments with TRPM8-expressing HEK-293 cells, we observe significant inhibitory effects of phorbol 12,13-dibutyrate but not forskolin. A similar lack of effect of forskolin or 8-Br-cAMP was reported in cultured DRG cells [162], and another study even indicated that basal function of TRPM8 was dependent on patent phosphorylation by PKA [163]. TRPM8-positive sensory neurons are thought to be dependent on the tyrosine kinase receptor for nerve growth factor, TrkA during development and during cell culture [164,165] so tyrosine phosphorylation-dependent signaling cascades might modulate TRPM8. Although TrkA appears not to be generally coexpressed with TRPM8 in adult sensory neurons [165], around half of the TRPM8-positive TG and DRG cells express the GFRα3 receptor for another neuronal growth factor, artemin, and a subset of these also contain TrkA [166]. Intraplantar injection of artemin enhanced behavioral responses to evaporative cooling with acetone, a response that was absent in TRPM8−/− mice, but also reduced withdrawal latencies to noxious heat [166]. Intraplantar NGF had a similar but less marked effect on cooling responses. The intracellular signaling cascades underlying these modulatory effects remain to be elucidated.

G Protein-Coupled Receptor Signaling

Intracellular signaling from the mainly Gi/Go-coupled α2A-adrenoreceptor (most likely the inhibition of adenylyl cyclases) has been reported to inhibit TRPM8 function both in a heterologous cell expression system and in a small subpopulation of menthol-sensitive sensory neurons [163], with reducing phosphorylation status of PKA target sites thought to be responsible. The mainly Gq/G11-coupled M3 muscarinic receptor also inhibits TRPM8 in cotransfected HEK-293 cells [155]. Rather than through anticipated candidate mechanisms such as PLC-dependent PIP2 depletion, this appears to involve PLA2-dependent arachidonic acid production through a process unaffected by a selective PLC inhibitor. In addition the 2+ mainly Gq/G11-coupled B2 bradykinin receptor can reduce TRPM8 channel currents and Ca elevation in cotransfected cells and in a subpopulation of DRG cells, through a process not significantly affected by inhibitors of PLC or PKC [167]. The direct activator of PKC phorbol 12-myristate, 13-acetate had no apparent effect on TRPM8 activity in these experiments, in contrast to earlier reports [159–162]. Evidence was provided in support of a PLC-independent Modulation of TRPM8 Function by Cellular Signaling 251

mechanism of Gq binding to the channel to cause its inhibition, centering on effects of a chi- meric Gαq/Gαi construct to avoid PLC activation as wild-type, constitutively active and a previously described PLC-disabled mutant Gαq were able to trigger the earlier proposed inhibitory mechanism of PIP2 depletion. Wild-type and constitutively active Gαq were reported to be associated with TRPM8 in chelation-affinity and immunoprecipitation experiments; controls were provided using cells transfected with a single construct alone. In addition Gαq from cell lysates was captured by glutathione S-transferase (GST) fusion constructs of both N- and C-terminal domains of TRPM8. An additional protein that may be of relevance to these findings is G protein-coupled receptor kinase 2 (GRK2), which is recruited to the vicinity of activated receptors and is known to sequester Gαq [168]. Interestingly the region of

Gαq replaced in the Gαq/Gαi chimera is important for GRK2- as well as PLC-β-interaction [169]. GRK2 could potentially be associated with the B2 receptor in transfected cells or in vivo and affect Gαq localization and availability. An activated construct of the Gαq congener Gα11, is reported to have a lesser effect on TRPM8 compared to Gαq [170] and the same pattern is seen in the impairment of inositol phosphate signaling caused by GRK2 binding to the two

G proteins [171]. Whether or not GRK2 plays any part in Gαq: TRPM8 interactions is yet to be explored. Surprisingly though, the intraplantar injection of bradykinin (at doses effective in causing heat hypersensitivity [172]) had no effect at all on behavioral responses to evaporative cooling in mice [166]. Additional data consistent with Gαq inhibition of TRPM8 was provided in experiments investigating the pruritogenic receptor MrgprA3 in a small minority of DRG neurons that respond to capsaicin, allyl isothiocyanate, and menthol [173]. MrgprA3 activation partially reduced subsequent menthol responses in around half of these cells, whereas capsaicin responses were enhanced through a distinct mechanism [173]. The difficulties in distilling a simple unified model of TRPM8 channel regulation are emphasized by data indicating a completely contrasting process, the activation of Gαq and PLC by TRPM8 itself [174]. This report describes menthol-evoked Ca2+ mobilization from intracellular stores through a TRPM8-dependent process involving PLC activation, although menthol at high concentrations is also known to exert TRPM8-independent effects on endoplasmic reticulum [175]. Correspondingly, fluorescence resonance energy transfer (FRET) measurements indicated close proximity between fluorophore-tagged TRPM8 and Gαq that was considered to underlie G protein and PLC activation. The overall picture remains to be clarified, although potentially multiple processes could operate concurrently. A recent report describes a novel mechanism for the up-regulation of TRPM8 function that centers on a newly discovered molecular signaling complex between the 5-HT1B receptor and TRPM8 [91] (Figure 14.3). The channel associates directly with the 5-HT1B receptor but not with other G protein-coupled receptors, in HEK-293 cells and in small DRG neurons. 5-HT1B receptor activation amplifies TRPM8 responses to icilin or menthol through a mechanism dependent on phospholipase D1 (PLD1), which is also incorporated into the signaling complex. Evidence was provided that the process by which PLD can enhance channel function involves a stimulatory effect on phosphatidylinositol 4-phosphate (PIP)

5-kinase, leading to elevated levels of PIP2, the allosteric enhancer of TRPM8. In chronic pain models the combined treatment with 5-HT1B agonist and TRPM8 agonist was shown to amplify icilin (or menthol)-induced reversal of synaptic hypersensitivity in the CNS and enhance TRPM8 analgesia. 252 14. TRPM8 AND ANALGESIA

FIGURE 14.3 Schematic diagram illustrating the analgesic gating-out of hypersensitive spinal nociceptive transmission in chronic pain by TRPM8-expressing nonnociceptive afferents and the molecular mechanism through which 5-HT1B receptors act to enhance this process.

CNS PROCESSES ACTIVATED BY TRPM8-POSITIVE AFFERENTS

In the superficial dorsal horn of the spinal cord there are various categories of supra- spinally projecting neurons, including polymodal nociceptors and nonnociceptive cool receptors, indicating at least partially distinct spinal processing and onward transmission of innocuous cool sensation [176–179]. TRPM8-expressing afferents, visualized in mice with TRPM8 promoter-targeted GFP constructs, terminate in outer laminae II and laminae I of spinal dorsal horn [33,34]. These terminals overlap with the zone of peptidergic nociceptor termination as exemplified by CGRP and substance P immunofluorescence (but show minimal costaining at the single fiber level), while essentially avoiding the region of IB4-reactive nonpeptidergic nociceptor termination in inner laminae II. Cadherin-8 is expressed in laminae I, IIouter and especially IIinner of the dorsal horn and is also present in a subset of small DRG cells, many of which coexpress TRPM8 [180]. Ultrastructural analysis indicated both pre- and postsynaptic cadherin-8 expression within complex glomerular synapses. Recording from dorsal horn neurons in slices of wild-type mice, menthol-evoked increased mEPSP frequency and this effect was abolished in cadherin-8−/− animals, indicating that cadherin-8 may be required for functional connectivity between TRPM8 afferents and target neurons in superficial dorsal horn [180]. A further ultrastructural study showed that TRPM8-positive C- and Aδ- fiber terminals entered into synaptic connections that were generally simple dendritic, less commonly complex glomerular and occasionally axo-axonic in nature [181]. In the trigeminal sensory nuclei, there was evidence for a segregated localization of TRPM8 afferents that were positive for CGRP and those that expressed TRPM8 alone. In a transgenic mouse line that expresses GFP in a subset of GABAergic laminae II interneurons, the tonic central cells [182], menthol or icilin increase mEPSP frequency in these neurons, consistent with a direct input from TRPM8 positive afferents and a role in cool sensing [183]. Interneuron cross-connections [184,185] could then lead to inhibitory effects CNS Processes Activated by TRPM8-Positive Afferents 253 on vertical and transient central excitatory interneurons involved in processing Aδ- and C fiber nociceptive inputs [183]. Such pathways could contribute to the gating-out of hypersensitive responses to nociceptive inputs and reversal of pain behavior seen when icilin or menthol are administered at low doses in chronic pain models [36,91]. In those studies any major contribution of opioid receptors at the spinal level was excluded but a key mediating role of Group II and Group III metabotropic glutamate receptors was identified, each known to be expressed at both pre- and postsynaptic locations. There is evidence, however, to support an involvement of κ-opioid receptors in the acute analgesic effects of systemic menthol, but the site of action is likely to be an unknown supraspinal location as effects were attenuated when κ-opioid antagonist was delivered intracerebroventricularly [85]. A further study reported that acute analgesic effects of the menthol congener WS-12 (at a rather high intraperitoneal dose) in the hot plate test were diminished by systemic naloxone, a general opioid receptor antagonist [89]. These reports could both be consistent with a supraspinal role of κ-opioid receptors in TRPM8 ligand-induced analgesia, but in neither case was it shown explicitly that effects were TRPM8-mediated. Mice in which the developmental transcription factor Bhlhb5 is constitutively deleted lack a further subpopulation of inhibitory GABAergic interneurons in superficial dorsal horn and display pathological itch [186]. When these neurons were genetically tagged, they were shown to coexpress either galanin/dynorphin or neuronal nitric oxide synthase and be activated by capsaicin, allyl isothiocyanate, and menthol, each of which is reported to exert antipruritic influences [187]. Thus these cells may receive input from TRPM8 afferents and may influence itch through dynorphin/κ-opioid receptor mechanisms, although preprodynorphin−/− mice do not replicate the phenotype. Importantly though, Bhlhb5−/− mice do not exhibit any pain phenotype [186], so perhaps are dissociated from pathways underpinning TRPM8 analgesia. A further recent study reported that in isolated DRG neurons, the μ-opioid receptor agonist morphine, at a relatively high concentration, causes a small (15%) attenuation of menthol-induced currents and disruption of menthol-induced Ca2+ entry [188]. Data were provided to suggest that μ-opioid receptor immunoreactivity was found in TRPM8 immunoprecipitates from HEK-293 cells transfected with TRPM8 plus μ-opioid receptor but not cells transfected with TRPM8 alone and also that morphine may promote TRPM8 internalization. Evidence that μ-opioid receptors are expressed in native TRPM8-positive afferents is yet to be provided by immunofluorescence or equivalent approaches. Morphine-evoked analgesia was tested on cold responses of TRPM8−/− mice and found to be less marked than in the wild type, but the marked resetting of pain thresholds in the mutant mouse line means that it is hard to interpret the comparison with certainty. The robust antinociceptive effects of μ-opioid receptors that are known to be exerted at spinal dorsal horn and brainstem levels would need to be taken into account for a holistic analysis. A further report relating to the idea of functional interaction between different groups of afferent inputs describes the effects of toxin ablation of CGRP-expressing nociceptive afferents [189]. Interestingly, their removal amplifies behavioral responses to an evaporative cooling stimulus provided by acetone and reduces withdrawal latency from a cold plate at noxious temperatures. This is consistent with the general concept of crossover gating between different classes of afferent pathways and indicates that concurrent activation of analgesic inputs associated with innocuous cool sensation and blockade of nociceptive inputs may bring about a synergistic, greater than additive, therapeutic outcome. 254 14. TRPM8 AND ANALGESIA CONCLUSIONS

Over the small number of years since its discovery, the TRPM8 ion channel has become established as a promising target for analgesic intervention. Abundant evidence indicates that TRPM8 is key to the sensing of innocuous cool temperatures and is expressed in a subpopulation of small DRG/TG neurons, which course to the superficial dorsal horn in parallel to nociceptive afferents. In chronic pain states, the central sensitization that underpins hypersensitive pain behavior is attenuated by this TRPM8-positive input, acting to gate-out nociceptive processing and produce an analgesic effect. TRPM8 agonists can usefully target this mechanism to provide efficacious analgesia in chronic neuropathic or inflammatory pain. TRPM8 agonists are thought to exert mixed effects on one particular sign of chronic pain, that is cool allodynia, where they may both amplify innocuous cool sensing and attenuate central sensitization; the overall outcome therefore is to ameliorate any allodynia. TRPM8 antagonists are being investigated for the treatment of cool allodynia; whereas they are likely to be effective in reducing the cool sensing input itself, there is no basis to expect any effect on central sensitization or any impact on other modalities of chronic hypersensitive pain. Any participation of TRPM8 in noxious cold sensing in naive subjects is less clearly documented at present, with only a minority of TRPM8-positive DRG/TG cells expressing nociceptive markers, so any potential for intervention there remains to be validated. In the future the development of new highly selective chemical ligands for TRPM8 will be crucial, not only in providing tools to help unravel the complex neurobiology underlying its impact on pain processing, but also in the search for new drugs that are efficacious in the treatment of chronic pain.

References [1] Tsavaler L, Shapero MH, Morkowski S, Laus R. Trp-p8, a novel prostate-specific gene, is up-regulated in prostate cancer and other malignancies and shares high homology with transient receptor potential calcium channel proteins. Cancer Res 2001;61(9):3760–9. [2] McKemy DD, Neuhausser WM, Julius D. Identification of a cold receptor reveals a general role for TRP channels in thermosensation. Nature 2002;416(6876):52–8. [3] Peier AM, Moqrich A, Hergarden AC, Reeve AJ, Andersson DA, Story GM, et al. A TRP channel that senses cold stimuli and menthol. Cell 2002;108(5):705–15. [4] Erler I, Al-Ansary DM, Wissenbach U, Wagner TF, Flockerzi V, Niemeyer BA. Trafficking and assembly of the cold-sensitive TRPM8 channel. J Biol Chem 2006;281(50):38396–404. [5] Tsuruda PR, Julius D, Minor Jr DL. Coiled coils direct assembly of a cold-activated TRP channel. Neuron 2006;51(2):201–12. [6] Phelps CB, Gaudet R. The role of the N terminus and transmembrane domain of TRPM8 in channel localization and tetramerization. J Biol Chem 2007;282(50):36474–80. [7] Bandell M, Story GM, Hwang SW, Viswanath V, Eid SR, Earley MJ, et al. Noxious cold ion channel TRPA1 is activated by pungent compounds and bradykinin. Neuron 2004;41(6):849–57. [8] Brauchi S, Orta G, Salazar M, Rosenmann E, Latorre R. A hot-sensing cold receptor: C-terminal domain determines thermosensation in transient receptor potential channels. J Neurosci 2006;26(18):4835–40. [9] Brauchi S, Orta G, Mascayano C, Salazar M, Raddatz N, Urbina H, et al. Dissection of the components for PIP2 activation and thermosensation in TRP channels. Proc Natl Acad Sci USA 2007;104(24):10246–51. [10] Pertusa MI, González A, Hardy P, Madrid R, Viana FE. Bidirectional modulation of thermal and chemical sensitivity of TRPM8 channels by the initial region of the N-terminal domain. J Biol Chem 2014;289:21828–43. [11] Andersson DA, Chase HW, Bevan S. TRPM8 activation by menthol, icilin, and cold is differentially modulated by intracellular pH. J Neurosci 2004;24(23):5364–9. REFERENCES 255

[12] Chuang HH, Neuhausser WM, Julius D. The super-cooling agent icilin reveals a mechanism of coincidence detection by a temperature-sensitive TRP channel. Neuron 2004;43(6):859–69. [13] Bandell M, Dubin AE, Petrus MJ, Orth A, Mathur J, Hwang SW, et al. High-throughput random mutagenesis screen reveals TRPM8 residues specifically required for activation by menthol. Nat Neurosci 2006;9(4):493–500. [14] Voets T, Owsianik G, Janssens A, Talavera K, Nilius B. TRPM8 voltage sensor mutants reveal a mechanism for integrating thermal and chemical stimuli. Nat Chem Biol 2007;3(3):174–82. [15] Kuhn FJ, Kuhn C, Luckhoff A. Inhibition of TRPM8 by icilin distinct from desensitization induced by menthol and menthol derivatives. J Biol Chem 2009;284(7):4102–11. [16] Brauchi S, Orio P, Latorre R. Clues to understanding cold sensation: thermodynamics and electrophysiological analysis of the cold receptor TRPM8. Proc Natl Acad Sci USA 2004;101(43):15494–9. [17] Voets T, Droogmans G, Wissenbach U, Janssens A, Flockerzi V, Nilius B. The principle of temperature- dependent gating in cold- and heat-sensitive TRP channels. Nature 2004;430(7001):748–54. [18] Hui K, Guo Y, Feng ZP. Biophysical properties of menthol-activated cold receptor TRPM8 channels. Biochem Biophys Res Commun 2005;333(2):374–82. [19] Matta JA, Ahern GP. Voltage is a partial activator of rat thermosensitive TRP channels. J Physiol 2007;585 (Pt 2):469–82. [20] Hayashi T, Kondo T, Ishimatsu M, Yamada S, Nakamura K, Matsuoka K, et al. Expression of the TRPM8- immunoreactivity in dorsal root ganglion neurons innervating the rat urinary bladder. Neurosci Res 2009;65(3):245–51. [21] Johnson CD, Melanaphy D, Purse A, Stokesberry SA, Dickson P, Zholos AV. Transient receptor potential melastatin 8 channel involvement in the regulation of vascular tone. Am J Physiol Heart Circ Physiol 2009;296(6):H1868–77. [22] Materazzi S, Nassini R, Gatti R, Trevisani M, Geppetti P. Cough sensors. II. Transient receptor potential membrane receptors on cough sensors. Handb Exp Pharmacol 2009;187:49–61. [23] Babes A, Ciobanu AC, Neacsu C, Babes RM. TRPM8, a sensor for mild cooling in mammalian sensory nerve endings. Curr Pharm Biotechnol 2011;12(1):78–88. [24] Harrington AM, Hughes PA, Martin CM, Yang J, Castro J, Isaacs NJ, et al. A novel role for TRPM8 in visceral afferent function. Pain 2011;152(7):1459–68. [25] Jun JH, Kang HJ, Jin MH, Lee HY, Im YJ, Jung HJ, et al. Function of the cold receptor (TRPM8) associated with voiding dysfunction in bladder outlet obstruction in rats. Int Neurourol J 2012;16(2):69–76. [26] Plevkova J, Kollarik M, Poliacek I, Brozmanova M, Surdenikova L, Tatar M, et al. The role of trigeminal nasal TRPM8-expressing afferent neurons in the antitussive effects of menthol. J Appl Physiol 2013;115(2):268–74. [27] Ramachandran R, Hyun E, Zhao L, Lapointe TK, Chapman K, Hirota CL, et al. TRPM8 activation attenuates inflammatory responses in mouse models of colitis. Proc Natl Acad Sci USA 2013;110(18):7476–81. [28] Blackshaw LA. Transient receptor potential cation channels in visceral sensory pathways. Br J Pharmacol 2014;171(10):2528–36. [29] Zhang L, Jones S, Brody K, Costa M, Brookes SJ. Thermosensitive transient receptor potential channels in vagal afferent neurons of the mouse. Am J Physiol Gastrointest Liver Physiol 2004;286(6):G983–91. [30] Hondoh A, Ishida Y, Ugawa S, Ueda T, Shibata Y, Yamada T, et al. Distinct expression of cold receptors (TRPM8 and TRPA1) in the rat nodose-petrosal ganglion complex. Brain Res 2010;1319:60–9. [31] Zhao H, Sprunger LK, Simasko SM. Expression of transient receptor potential channels and two-pore potassium channels in subtypes of vagal afferent neurons in rat. Am J Physiol Gastrointest Liver Physiol 2010;298(2):G212–21. [32] Du J, Yang X, Zhang L, Zeng YM. Expression of TRPM8 in the distal cerebrospinal fluid-contacting neurons in the brain mesencephalon of rats. Cerebrospinal Fluid Res 2009;6:3. [33] Takashima Y, Daniels RL, Knowlton W, Teng J, Liman ER, McKemy DD. Diversity in the neural circuitry of cold sensing revealed by genetic axonal labeling of transient receptor potential melastatin 8 neurons. J Neurosci 2007;27(51):14147–57. [34] Dhaka A, Earley TJ, Watson J, Patapoutian A. Visualizing cold spots: TRPM8-expressing sensory neurons and their projections. J Neurosci 2008;28(3):566–75. [35] Nealen ML, Gold MS, Thut PD, Caterina MJ. TRPM8 mRNA is expressed in a subset of cold-responsive trigeminal neurons from rat. J Neurophysiol 2003;90(1):515–20. [36] Proudfoot CJ, Garry EM, Cottrell DF, Rosie R, Anderson H, Robertson DC, et al. Analgesia mediated by the TRPM8 cold receptor in chronic neuropathic pain. Curr Biol 2006;16(16):1591–605. 256 14. TRPM8 AND ANALGESIA

[37] Kobayashi K, Fukuoka T, Obata K, Yamanaka H, Dai Y, Tokunaga A, et al. Distinct expression of TRPM8, TRPA1, and TRPV1 mRNAs in rat primary afferent neurons with adelta/c-fibers and colocalization with trk receptors. J Comp Neurol 2005;493(4):596–606. [38] Okazawa M, Inoue W, Hori A, Hosokawa H, Matsumura K, Kobayashi S. Noxious heat receptors present in cold-sensory cells in rats. Neurosci Lett 2004;359(1-2):33–6. [39] Pogorzala LA, Mishra SK, Hoon MA. The cellular code for mammalian thermosensation. J Neurosci 2013;33(13):5533–41. [40] Hwang SJ, Oh JM, Valtschanoff JG. The majority of bladder sensory afferents to the rat lumbosacral spinal cord are both IB4- and CGRP-positive. Brain Res 2005;1062(1-2):86–91. [41] Karashima Y, Talavera K, Everaerts W, Janssens A, Kwan KY, Vennekens R, et al. TRPA1 acts as a cold sensor in vitro and in vivo. Proc Natl Acad Sci USA 2009;106(4):1273–8. [42] Bautista DM, Siemens J, Glazer JM, Tsuruda PR, Basbaum AI, Stucky CL, et al. The menthol receptor TRPM8 is the principal detector of environmental cold. Nature 2007;448(7150):204–8. [43] Colburn RW, Lubin ML, Stone Jr DJ, Wang Y, Lawrence D, D’Andrea MR, et al. Attenuated cold sensitivity in TRPM8 null mice. Neuron 2007;54(3):379–86. [44] Dhaka A, Murray AN, Mathur J, Earley TJ, Petrus MJ, Patapoutian A. TRPM8 is required for cold sensation in mice. Neuron 2007;54(3):371–8. [45] Knowlton WM, Bifolck-Fisher A, Bautista DM, McKemy DD. TRPM8, but not TRPA1, is required for neural and behavioral responses to acute noxious cold temperatures and cold-mimetics in vivo. Pain 2010;150(2):340–50. [46] Reid G, Flonta ML. Ion channels activated by cold and menthol in cultured rat dorsal root ganglion neurones. Neurosci Lett 2002;324(2):164–8. [47] de la Pena E, Malkia A, Cabedo H, Belmonte C, Viana F. The contribution of TRPM8 channels to cold sensing in mammalian neurones. J Physiol 2005;567(Pt 2):415–26. [48] Xing H, Ling J, Chen M, Gu JG. Chemical and cold sensitivity of two distinct populations of TRPM8-expressing somatosensory neurons. J Neurophysiol 2006;95(2):1221–30. [49] Teichert RW, Raghuraman S, Memon T, Cox JL, Foulkes T, Rivier JE, et al. Characterization of two neuronal subclasses through constellation pharmacology. Proc Natl Acad Sci USA 2012;109(31):12758–63. [50] Karashima Y, Damann N, Prenen J, Talavera K, Seqal A, Voets T, et al. Bimodal action of menthol on the transient receptor potential channel TRPA1. J Neurosci 2007;27(37):9874–84. [51] Story GM, Peier AM, Reeve AJ, Eid SR, Mosbacher J, Hricik TR, et al. ANKTM1, a TRP-like channel expressed in nociceptive neurons, is activated by cold temperatures. Cell 2003;112(6):819–29. [52] Sarria I, Ling J, Xu GY, Gu JG. Sensory discrimination between innocuous and noxious cold by TRPM8- expressing DRG neurons of rats. Mol Pain 2012;8:79. [53] Amaya F, Oh-hashi K, Naruse Y, Iijima N, Ueda M, Shimosato G, et al. Local inflammation increases vanilloid receptor 1 expression within distinct subgroups of DRG neurons. Brain Res 2003;963(1-2):190–6. [54] Teliban A, Bartsch F, Struck M, Baron R, Janig W. Responses of intact and injured sural nerve fibers to cooling and menthol. J Neurophysiol 2014;111(10):2071–83. [55] Campero M, Baumann TK, Bostock H, Ochoa JL. Human cutaneous C fibres activated by cooling, heating and menthol. J Physiol 2009;587(Pt 23):5633–52. [56] Wasner G, Schattschneider J, Binder A, Baron R. Topical menthol—a human model for cold pain by activation and sensitization of C nociceptors. Brain 2004;127(Pt 5):1159–71. [57] Namer B, Seifert F, Handwerker HO, Maihofner C. TRPA1 and TRPM8 activation in humans: effects of cinnamaldehyde and menthol. Neuroreport 2005;16(9):955–9. [58] Hatem S, Attal N, Willer JC, Bouhassira D. Psychophysical study of the effects of topical application of menthol in healthy volunteers. Pain 2006;122(1-2):190–6. [59] Parra A, Madrid R, Echevarria D, del Olmo S, Morenilla-Palao C, Acosta MC, et al. Ocular surface wetness is regulated by TRPM8-dependent cold thermoreceptors of the cornea. Nat Med 2010;16(12):1396–9. [60] Knowlton WM, Palkar R, Lippoldt EK, McCoy DD, Baluch F, Chen J, et al. A sensory-labeled line for cold: TRPM8-expressing sensory neurons define the cellular basis for cold, cold pain, and cooling-mediated analgesia. J Neurosci 2013;33(7):2837–48. [61] Knowlton WM, Daniels RL, Palkar R, McCoy DD, McKemy DD. Pharmacological blockade of TRPM8 ion channels alters cold and cold pain responses in mice. PLoS One 2011;6(9):e25894. [62] Morin C, Bushnell MC. Temporal and qualitative properties of cold pain and heat pain: a psychophysical study. Pain 1998;74(1):67–73. REFERENCES 257

[63] Gentry C, Stoakley N, Andersson DA, Bevan S. The roles of iPLA2, TRPM8 and TRPA1 in chemically induced cold hypersensitivity. Mol Pain 2010;6:4. [64] Braz JM, Basbaum AI. Differential ATF3 expression in dorsal root ganglion neurons reveals the profile of primary afferents engaged by diverse noxious chemical stimuli. Pain 2010;150(2):290–301. [65] Ross SE. Pain and itch: insights into the neural circuits of aversive somatosensation in health and disease. Curr Opin Neurobiol 2011;21(6):880–7. [66] Vetter I, Hein A, Sattler S, Hessler S, Touska F, Bressan E, et al. Amplified cold transduction in native nociceptors by M-channel inhibition. J Neurosci 2013;33(42):16627–41. [67] del Camino D, Murphy S, Heiry M, Barrett LB, Earley TJ, Cook CA, et al. TRPA1 contributes to cold hypersensitivity. J Neurosci 2010;30(45):15165–74. [68] Madrid R, de la Pena E, Donovan-Rodriguez T, Belmonte C, Viana F. Variable threshold of trigeminal cold- thermosensitive neurons is determined by a balance between TRPM8 and Kv1 potassium channels. J Neurosci 2009;29(10):3120–31. [69] Yamamoto Y, Hatakeyama T, Taniguchi K. Immunohistochemical colocalization of TREK-1, TREK-2 and TRAAK with TRP channels in the trigeminal ganglion cells. Neurosci Lett 2009;454(2):129–33. [70] Sarria I, Ling J, Gu JG. Thermal sensitivity of voltage-gated Na+ channels and A-type K+ channels contributes to somatosensory neuron excitability at cooling temperatures. J Neurochem 2012;122(6):1145–54. [71] Zimmermann K, Leffler A, Babes A, Cendan CM, Carr RW, Kobayashi J, et al. Sensory neuron sodium channel Nav1.8 is essential for pain at low temperatures. Nature 2007;447(7146):855–8. [72] Latremoliere A, Woolf CJ. Central sensitization: a generator of pain hypersensitivity by central neural plasticity. J Pain 2009;10(9):895–926. [73] Obata K, Katsura H, Mizushima T, Yamanaka H, Kobayashi K, Dai Y, et al. TRPA1 induced in sensory neurons contributes to cold hyperalgesia after inflammation and nerve injury. J Clin Invest 2005;115(9):2393–401. [74] Ji G, Zhou S, Carlton SM. Intact adelta-fibers up-regulate transient receptor potential A1 and contribute to cold hypersensitivity in neuropathic rats. Neuroscience 2008;154(3):1054–66. [75] Frederick J, Buck ME, Matson DJ, Cortright DN. Increased TRPA1, TRPM8, and TRPV2 expression in dorsal root ganglia by nerve injury. Biochem Biophys Res Commun 2007;358(4):1058–64. [76] Xing H, Chen M, Ling J, Tan W, Gu JG. TRPM8 mechanism of cold allodynia after chronic nerve injury. J Neurosci 2007;27(50):13680–90. [77] Su L, Wang C, Yu YH, Ren YY, Xie KL, Wang GL. Role of TRPM8 in dorsal root ganglion in nerve injury- induced chronic pain. BMC Neurosci 2011;12:120. [78] Caspani O, Zurborg S, Labuz D, Heppenstall PA. The contribution of TRPM8 and TRPA1 channels to cold allodynia and neuropathic pain. PLoS One 2009;4(10):e7383. [79] Staaf S, Oerther S, Lucas G, Mattsson JP, Ernfors P. Differential regulation of TRP channels in a rat model of neuropathic pain. Pain 2009;144(1-2):187–99. [80] Patel R, Goncalves L, Newman R, Jiang FL, Goldby A, Reeve J, et al. Novel TRPM8 antagonist attenuates cold hypersensitivity after peripheral nerve injury in rats. J Pharmacol Exp Ther 2014;349(1):47–55. [81] Wasner G, Naleschinski D, Binder A, Schattschneider J, McLachlan EM, Baron R. The effect of menthol on cold allodynia in patients with neuropathic pain. Pain Med 2008;9(3):354–8. [82] Wright A. Oil of peppermint as a local anaesthetic. Lancet 1870;2464:726. [83] Green BG, McAuliffe BL. Menthol desensitization of capsaicin irritation. Evidence of a short-term antinociceptive effect. Physiol Behav 2000;68(5):631–9. [84] Davies SJ, Harding LM, Baranowski AP. A novel treatment of postherpetic neuralgia using peppermint oil. Clin J Pain 2002;18(3):200–2. [85] Galeotti N, Di Cesare ML, Mazzanti G, Bartolini A, Ghelardini C. Menthol: a natural analgesic compound. Neurosci Lett 2002;322(3):145–8. [86] Klein AH, Sawyer CM, Carstens MI, Tsagareli MG, Tsiklauri N, Carstens E. Topical application of l-menthol induces heat analgesia, mechanical allodynia, and a biphasic effect on cold sensitivity in rats. Behav Brain Res 2010;212(2):179–86. [87] Hagenacker T, Lampe M, Schafers M. Icilin reduces voltage-gated calcium channel currents in naive and injured DRG neurons in the rat spinal nerve ligation model. Brain Res 2014;1557:171–9. [88] Katsura H, Obata K, Mizushima T, Yamanaka H, Kobayashi K, Dai Y, et al. Antisense knock down of TRPA1, but not TRPM8, alleviates cold hyperalgesia after spinal nerve ligation in rats. Exp Neurol 2006;200(1):112–23. 258 14. TRPM8 AND ANALGESIA

[89] Liu B, Fan L, Balakrishna S, Sui A, Morris JB, Jordt SE. TRPM8 is the principal mediator of menthol-induced analgesia of acute and inflammatory pain. Pain 2013;154(10):2169–77. [90] Pan R, Tian Y, Gao R, Li H, Zhao X, Barrett JE, et al. Central mechanisms of menthol-induced analgesia. J Pharmacol Exp Ther 2012;343(3):661–72. [91] Vinuela-Fernandez I, Sun L, Jerina H, Curtis J, Allchorne A, Gooding H, et al. The TRPM8 channel forms a complex with the 5-HT(1B) receptor and phospholipase D that amplifies its reversal of pain hypersensitivity. Neuropharmacology 2014;79:136–51. [92] Colvin LA, Johnson PR, Mitchell R, Fleetwood-Walker SM, Fallon M. From bench to bedside: a case of rapid reversal of bortezomib-induced neuropathic pain by the TRPM8 activator, menthol. J Clin Oncol 2008;26(27):4519–20. [93] Storey DJ, Colvin LA, Mackean MJ, Mitchell R, Fleetwood-Walker SM, Fallon MT. Reversal of dose-limiting carboplatin-induced peripheral neuropathy with TRPM8 activator, menthol, enables further effective chemotherapy delivery. J Pain Symptom Manage 2010;39(6):e2–4. [94] Gavva NR, Bannon AW, Surapaneni S, Hovland Jr DN, Lehto SG, Gore A, et al. The vanilloid receptor TRPV1 is tonically activated in vivo and involved in body temperature regulation. J Neurosci 2007;27(13):3366–74. [95] Steiner AA, Turek VF, Almeida MC, Burmeister JJ, Oliveira DL, Roberts JL, et al. Nonthermal activation of transient receptor potential vanilloid-1 channels in abdominal viscera tonically inhibits autonomic cold- defense effectors. J Neurosci 2007;27(28):7459–68. [96] Wei ET. Pharmacological aspects of shaking behavior produced by TRH, AG-3-5, and morphine withdrawal. Fed Proc 1981;40(5):1491–6. [97] Werkheiser J, Cowan A, Gomez T, Henry C, Parekh S, Chau S, et al. Icilin-induced wet-dog shakes in rats are dependent on NMDA receptor activation and nitric oxide production. Pharmacol Biochem Behav 2009;92(3):543–8. [98] Almeida MC, Steiner AA, Branco LG, Romanovsky AA. Cold-seeking behavior as a thermoregulatory strategy in systemic inflammation. Eur J Neurosci 2006;23(12):3359–67. [99] Ruskin DN, Anand R, LaHoste GJ. Menthol and nicotine oppositely modulate body temperature in the rat. Eur J Pharmacol 2007;559(2-3):161–4. [100] Tajino K, Matsumura K, Kosada K, Shibakusa T, Inoue K, Fushiki T, et al. Application of menthol to the skin of whole trunk in mice induces autonomic and behavioral heat-gain responses. Am J Physiol 2007;293(5):R2128–35. [101] Ding Z, Gomez T, Werkheiser JL, Cowan A, Rawls SM. Icilin induces a hyperthermia in rats that is dependent on nitric oxide production and NMDA receptor activation. Eur J Pharmacol 2008;578(2-3):201–8. [102] Tajino K, Hosokawa H, Maegawa S, Matsumura K, Dhaka A, Kobayashi S. Cooling-sensitive TRPM8 is ther- mostat of skin temperature against cooling. PLoS One 2011;6(3):e17504. [103] Almeida MC, Hew-Butler T, Soriano RN, Rao S, Wang W, Wang J, et al. Pharmacological blockade of the cold receptor TRPM8 attenuates autonomic and behavioral cold defenses and decreases deep body temperature. J Neurosci 2012;32(6):2086–99. [104] Gavva NR, Davis C, Lehto SG, Rao S, Wang W, Zhu DX. Transient receptor potential melastatin 8 (TRPM8) channels are involved in body temperature regulation. Mol Pain 2012;8:36. [105] Behrendt HJ, Germann T, Gillen C, Hatt H, Jostock R. Characterization of the mouse cold-menthol receptor TRPM8 and vanilloid receptor type-1 VR1 using a fluorometric imaging plate reader (FLIPR) assay. Br J Pharmacol 2004;141(4):737–45. [106] Weil A, Moore SE, Waite NJ, Randall A, Gunthorpe MJ. Conservation of functional and pharmacological properties in the distantly related temperature sensors TRVP1 and TRPM8. Mol Pharmacol 2005;68(2):518–27. [107] Meseguer V, Karashima Y, Talavera K, D’Hoedt D, Donovan-Rodríguez T, Viana F, et al. Transient receptor potential channels in sensory neurons are targets of the antimycotic agent clotrimazole. J Neurosci 2008;28(3):576–86. [108] DeFalco J, Steiger D, Dourado M, Emerling D, Duncton MA. 5-Benzyloxytryptamine as an antagonist of TRPM8. Bioorg Med Chem Lett 2010;20(23):7076–9. [109] Lashinger ES, Steiginga MS, Hieble JP, Leon LA, Gardner SD, Nagilla R, et al. AMTB, a TRPM8 channel blocker: evidence in rats for activity in overactive bladder and painful bladder syndrome. Am J Physiol Renal Physiol 2008;295(3):F803–10. [110] Branum ST, Colburn RW, Dax SL, Flores CM, Jetter MC, Liu Y, et al. N-Benzothienyl sulfonamides as TRPM8 modulators and their preparation, pharmaceutical compositions and use in the treatment of diseases. Chem Abstr 2009;150:168153. REFERENCES 259

[111] Irlapati NR, Thomas AL, Kurhe DK, Shelke SY, Khairatkar JN, Viswanadha S, et al. Preparation of fused oxaz- ole and thiazole derivatives as TRPM8 modulators. Chem Abstr 2010;152:192106. [112] Ortar G, De Petrocellis L, Morera L, Moriello AS, Orlando P, Morera E, et al. (−)-Menthylamine derivatives as potent and selective antagonists of transient receptor potential melastatin type-8 (TRPM8) channels. Bioorg Med Chem Lett 2010;20(9):2729–32. [113] Parks DJ, Parsons WH, Colburn RW, Meegalla SK, Ballentine SK, Illiq CR, et al. Design and optimization of benzimidazole-containing transient receptor potential melastatin 8 (TRPM8) antagonists. J Med Chem 2011;54(1):233–47. [114] Calvo RR, Meegalla SK, Parks DJ, Parsons WH, Ballentine SK, Lubin ML, et al. Discovery of vinylcycloalkyl- substituted benzimidazole TRPM8 antagonists effective in the treatment of cold allodynia. Bioorg Med Chem Lett 2012;22(5):1903–7. [115] Matthews JM, Qin N, Colburn RW, Dax SL, Hawkins M, McNally JJ, et al. The design and synthesis of novel, phosphonate-containing transient receptor potential melastatin 8 (TRPM8) antagonists. Bioorg Med Chem Lett 2012;22(8):2922–6. [116] Tamayo NA, Bo Y, Gore V, Ma V, Nishimura N, Tanq P, et al. Fused piperidines as a novel class of potent and orally available transient receptor potential melastatin type 8 (TRPM8) antagonists. J Med Chem 2012;55(4):1593–611. [117] Ortar G, Schiano Moriello A, Morera E, Nalli M, Di Marzo V, De Petrocellis L. 3-Ylidenephthalides as a new class of transient receptor potential channel TRPA1 and TRPM8 modulators. Bioorg Med Chem Lett 2013;23(20):5614–18. [118] Zhu B, Xia M, Xu X, Ludovici DW, Tennakoon M, Youngman MA, et al. Arylglycine derivatives as potent transient receptor potential melastatin 8 (TRPM8) antagonists. Bioorg Med Chem Lett 2013;23(7):2234–7. [119] De Petrocellis L, Starowicz K, Moriello AS, Vivese M, Orlando P, Di Marzo V. Regulation of transient receptor potential channels of melastatin type 8 (TRPM8): effect of cAMP, cannabinoid CB(1) receptors and endovanilloids. Exp Cell Res 2007;313(9):1911–20. [120] Beck B, Bidaux G, Bavencoffe A, Lemonnier L, Thebault S, Shuba Y, et al. Prospects for prostate cancer imaging and therapy using high-affinity TRPM8 activators. Cell Calcium 2007;41(3):285–94. [121] Bodding M, Wissenbach U, Flockerzi V. Characterisation of TRPM8 as a pharmacophore receptor. Cell Calcium 2007;42(6):618–28. [122] Duncan DF, Stewart F, Frolich MW, Urdeal D. Preclinical evaluation of the TRPM8 ion channel agonist D-3263 for benign prostatic hyperplasia. J Urol 2009;181(4):503. [123] Sherkheli MA, Vogt-Eisele AK, Bura D, Beltran Marques LR, Gisselmann G, Hatt H. Characterization of selective TRPM8 ligands and their structure activity response (S.A.R) relationship. J Pharm Pharm Sci 2010;13(2):242–53. [124] Bharate SS, Bharate SB. Modulation of thermoreceptor TRPM8 by cooling compounds. ACS Chem Neurosci 2012;3(4):248–67. [125] Journigan VB, Zaveri NT. TRPM8 ion channel ligands for new therapeutic applications and as probes to study menthol pharmacology. Life Sci 2013;92(8-9):425–37. [126] Wei ET, Seid DA. AG-3-5: a chemical producing sensations of cold. J Pharm Pharmacol 1983;35(2):110–12. [127] Sherkheli MA, Gisselmann G, Hatt H. Supercooling agent icilin blocks a warmth-sensing ion channel TRPV3. Scientific World Journal 2012;2012:982725. [128] Ostacolo C, Ambrosino P, Russo R, Lo Monte M, Soldovieri MV, Laneri S, et al. Isoxazole derivatives as potent transient receptor potential melastatin type 8 (TRPM8) agonists. Eur J Med Chem 2013;69:659–69. [129] Pedretti A, Marconi C, Bettinelli I, Vistoli G. Comparative modeling of the quaternary structure for the human TRPM8 channel and analysis of its binding features. Biochim Biophys Acta 2009;1788(5):973–82. [130] Swandulla D, Carbone E, Schafer K, Lux HD. Effect of menthol on two types of Ca currents in cultured sensory neurons of vertebrates. Pflugers Arch 1987;409(1-2):52–9. [131] Haeseler G, Maue D, Grosskreutz J, Bufler J, Nentwig B, Piepenbrock S, et al. Voltage-dependent block of neuronal and skeletal muscle sodium channels by thymol and menthol. Eur J Anaesthesiol 2002;19(8):571–9. [132] Hall AC, Turcotte CM, Betts BA, Yeung WY, Agyeman AS, Burk LA. Modulation of human GABAA and glycine receptor currents by menthol and related monoterpenoids. Eur J Pharmacol 2004;506(1):9–16. [133] Watt EE, Betts BA, Kotey FO, Humbert DJ, Griffith TN, Kelly EW, et al. Menthol shares general anesthetic activity and sites of action on the GABA(A) receptor with the intravenous agent, propofol. Eur J Pharmacol 2008;590(1-3):120–6. 260 14. TRPM8 AND ANALGESIA

[134] Zhang XB, Jiang P, Gong N, Hu XL, Fei D, Xiong ZQ, et al. A-type GABA receptor as a central target of TRPM8 agonist menthol. PLoS One 2008;3(10):e3386. [135] Sherkheli MA, Benecke H, Doerner JF, Kletke O, Voqt-Eisele AK, Gisselmann G, et al. Monoterpenoids induce agonist-specific desensitization of transient receptor potential vanilloid-3 (TRPV3) ion channels. J Pharm Pharm Sci 2009;12(1):116–28. [136] Baylie RL, Cheng H, Langton PD, James AF. Inhibition of the cardiac L-type calcium channel current by the TRPM8 agonist, (−)-menthol. J Physiol Pharmacol 2010;61(5):543–50. [137] Gaudioso C, Hao J, Martin-Eauclaire MF, Gabriac M, Delmas P. Menthol pain relief through cumulative inactivation of voltage-gated sodium channels. Pain 2012;153(2):473–84. [138] Hans M, Wilhelm M, Swandulla D. Menthol suppresses nicotinic acetylcholine receptor functioning in sensory neurons via allosteric modulation. Chem Senses 2012;37(5):463–9. [139] Kim SH, Lee S, Piccolo SR, Allen-Brady K, Park EJ, Chun JN, et al. Menthol induces cell-cycle arrest in PC-3 cells by down-regulating G2/M genes, including polo-like kinase 1. Biochem Biophys Res Commun 2012;422(3):436–41. [140] Ashoor A, Nordman JC, Veltri D, Yang KH, Al Kury L, Shuba Y, et al. Menthol binding and inhibition of alpha7-nicotinic acetylcholine receptors. PLoS One 2013;8(7):e67674. [141] Kawasaki H, Mizuta K, Fujita T, Kumamoto E. Inhibition by menthol and its related chemicals of compound action potentials in frog sciatic nerves. Life Sci 2013;92(6-7):359–67. [142] Lau BK, Karim S, Goodchild AK, Vaughan CW, Drew GM. Menthol enhances phasic and tonic GABAA receptor-mediated currents in midbrain periaqueductal grey neurons. Br J Pharmacol 2014;171(11):2803–13. [143] Walstab J, Wohlfarth C, Hovius R, Schmitteckert S, Röth R, Lasitschka F, et al. Natural compounds boldine and menthol are antagonists of human 5-HT receptors: implications for treating gastrointestinal disorders. Neurogastroenterol Motil 2014;26(6):810–20. [144] Yamamura H, Ugawa S, Ueda T, Nagao M, Shimada S. Icilin activates the delta-subunit of the human epithelial Na+ channel. Mol Pharmacol 2005;68(4):1142–7. [145] Kim SH, Kim SY, Park EJ, Kim J, Park HH, So I, et al. Icilin induces G1 arrest through activating JNK and p38 kinase in a TRPM8-independent manner. Biochem Biophys Res Commun 2011;406(1):30–5. [146] Lee S, Chun JN, Kim SH, So I, Jeon JH. Icilin inhibits E2F1-mediated cell cycle regulatory programs in prostate cancer. Biochem Biophys Res Commun 2013;441(4):1005–10. [147] Liu B, Qin F. Functional control of cold- and menthol-sensitive TRPM8 ion channels by phosphatidylinositol 4,5-bisphosphate. J Neurosci 2005;25(7):1674–81. [148] Rohacs T, Lopes CM, Michailidis I, Logothetis DE. PI(4,5)P2 regulates the activation and desensitization of TRPM8 channels through the TRP domain. Nat Neurosci 2005;8(5):626–34. [149] Zakharian E, Cao C, Rohacs T. Gating of transient receptor potential melastatin 8 (TRPM8) channels activated by cold and chemical agonists in planar lipid bilayers. J Neurosci 2010;30(37):12526–34. [150] Daniels RL, Takashima Y, McKemy DD. Activity of the neuronal cold sensor TRPM8 is regulated by phospholipase C via the phospholipid phosphoinositol 4,5-bisphosphate. J Biol Chem 2009;284(3):1570–82. [151] Yudin Y, Lukacs V, Cao C, Rohacs T. Decrease in phosphatidylinositol 4,5-bisphosphate levels mediates desensitization of the cold sensor TRPM8 channels. J Physiol 2011;589(Pt 24):6007–27. [152] Tang Z, Kim A, Masuch T, Park K, Weng H, Wetzel C, et al. Pirt functions as an endogenous regulator of TRPM8. Nat Commun 2013;4:2179. [153] Vanden Abeele F, Zholos A, Bidaux G, Shuba Y, Thebault S, Beck B, et al. Ca2+-independent phospholipase A2-dependent gating of TRPM8 by lysophospholipids. J Biol Chem 2006;281(52):40174–82. [154] Andersson DA, Nash M, Bevan S. Modulation of the cold-activated channel TRPM8 by lysophospholipids and polyunsaturated fatty acids. J Neurosci 2007;27(12):3347–55. [155] Bavencoffe A, Kondratskyi A, Gkika D, Mauroy B, Shuba Y, Prevarskaya N, et al. Complex regulation of the TRPM8 cold receptor channel: role of arachidonic acid release following M3 muscarinic receptor stimulation. J Biol Chem 2011;286(11):9849–55. [156] Morenilla-Palao C, Pertusa M, Meseguer V, Cabedo H, Viana F. Lipid raft segregation modulates TRPM8 channel activity. J Biol Chem 2009;284(14):9215–24. [157] Vanden Abeele F, Kondratskyi A, Dubois C, Shapovalov G, Gkika D, Busserolles J, et al. Complex modulation of the cold receptor TRPM8 by volatile anaesthetics and its role in complications of general anaesthesia. J Cell Sci 2013;126(Pt 19):4479–89. REFERENCES 261

[158] Latorre R, Brauchi S, Orta G, Zaelzer C, Vargas G. ThermoTRP channels as modular proteins with allosteric gating. Cell Calcium 2007;42(4-5):427–38. [159] Premkumar LS, Raisinghani M, Pingle SC, Long C, Pimentel F. Downregulation of transient receptor potential melastatin 8 by protein kinase C-mediated dephosphorylation. J Neurosci 2005;25(49):11322–9. [160] Abe J, Hosokawa H, Sawada Y, Matsumura K, Kobayashi S. Ca2+-dependent PKC activation mediates menthol-induced desensitization of transient receptor potential M8. Neurosci Lett 2006;397(1-2):140–4. [161] Linte RM, Ciobanu C, Reid G, Babes A. Desensitization of cold- and menthol-sensitive rat dorsal root ganglion neurones by inflammatory mediators. Exp Brain Res 2007;178(1):89–98. [162] Sarria I, Gu J. Menthol response and adaptation in nociceptive-like and nonnociceptive-like neurons: role of protein kinases. Mol Pain 2010;6:47. [163] Bavencoffe A, Gkika D, Kondratskyi A, Beck B, Borowiec AS, Bidaux G, et al. The transient receptor potential channel TRPM8 is inhibited via the alpha 2A adrenoreceptor signaling pathway. J Biol Chem 2010;285(13):9410–9. [164] Babes A, Zorzon D, Reid G. Two populations of cold-sensitive neurons in rat dorsal root ganglia and their modulation by nerve growth factor. Eur J Neurosci 2004;20(9):2276–82. [165] Takashima Y, Ma L, McKemy DD. The development of peripheral cold neural circuits based on TRPM8 expression. Neuroscience 2010;169(2):828–42. [166] Lippoldt EK, Elmes RR, McCoy DD, Knowlton WM, McKemy DD. Artemin, a glial cell line-derived neurotrophic factor family member, induces TRPM8-dependent cold pain. J Neurosci 2013;33(30):12543–52. [167] Zhang X, Mak S, Li L, Parra A, Denlinger B, Belmonte C, et al. Direct inhibition of the cold-activated TRPM8 ion channel by Galpha(q). Nat Cell Biol 2012;14(8):851–8. [168] Carman CV, Parent JL, Day PW, Pronin AN, Sternweis PM, Wedeqaertner PB, et al. Selective regulation of Galpha(q/11) by an RGS domain in the G protein-coupled receptor kinase, GRK2. J Biol Chem 1999;274(48):34483–92. [169] Tesmer VM, Kawano T, Shankaranarayanan A, Kozasa T, Tesmer JJ. Snapshot of activated G proteins at the membrane: the Galphaq-GRK2-Gbetagamma complex. Science 2005;310(5754):1686–90. [170] Li L, Zhang X. Differential inhibition of the TRPM8 ion channel by Galphaq and Galpha 11. Channels 2013;7(2):115–18. [171] Day PW, Carman CV, Sterne-Marr R, Benovic JL, Wedegaertner PB. Differential interaction of GRK2 with members of the G alpha q family. Biochemistry 2003;42(30):9176–84. [172] Chuang HH, Prescott ED, Kong H, Shields S, Jordt SE, Basbaum AL, et al. Bradykinin and nerve growth factor release the capsaicin receptor from PtdIns(4,5)P2-mediated inhibition. Nature 2001;411(6840):957–62. [173] Than JY, Li L, Hasan R, Zhang X. Excitation and modulation of TRPA1, TRPV1, and TRPM8 channel- expressing sensory neurons by the pruritogen chloroquine. J Biol Chem 2013;288(18):12818–27. [174] Klasen K, Hollatz D, Zielke S, Gisselmann G, Hatt H, Wetzel CH. The TRPM8 ion channel comprises direct Gq protein-activating capacity. Pflugers Arch 2012;463(6):779–97. [175] Mahieu F, Owsianik G, Verbert L, Janssens A, De Smedt H, Nilius B, et al. TRPM8-independent menthol- induced Ca2+ release from endoplasmic reticulum and Golgi. J Biol Chem 2007;282(5):3325–36. [176] Christensen BN, Perl ER. Spinal neurons specifically excited by noxious or thermal stimuli: marginal zone of the dorsal horn. J Neurophysiol 1970;33(2):293–307. [177] Dostrovsky JO, Craig AD. Cooling-specific spinothalamic neurons in the monkey. J Neurophysiol 1996;76(6):3656–65. [178] Han ZS, Zhang ET, Craig AD. Nociceptive and thermoreceptive lamina I neurons are anatomically distinct. Nat Neurosci 1998;1(3):218–25. [179] Craig AD, Krout K, Andrew D. Quantitative response characteristics of thermoreceptive and nociceptive lamina I spinothalamic neurons in the cat. J Neurophysiol 2001;86(3):1459–80. [180] Suzuki SC, Furue H, Koga K, Jiang N, Nohmi M, Shimazaki Y, et al. Cadherin-8 is required for the first relay synapses to receive functional inputs from primary sensory afferents for cold sensation. J Neurosci 2007;27(13):3466–76. [181] Kim YS, Park JH, Choi SJ, Bae JY, Ahn DK, McKemy DD, et al. Central connectivity of transient receptor potential melastatin 8-expressing axons in the brain stem and spinal dorsal horn. PLoS One 2014;9(4):e94080. [182] Hantman AW, Perl ER. Molecular and genetic features of a labeled class of spinal substantia gelatinosa neurons in a transgenic mouse. J Comp Neurol 2005;492(1):90–100. 262 14. TRPM8 AND ANALGESIA

[183] Zheng J, Lu Y, Perl ER. Inhibitory neurones of the spinal substantia gelatinosa mediate interaction of signals from primary afferents. J Physiol 2010;588(Pt 12):2065–75. [184] Yasaka T, Kato G, Furue H, Rashid MH, Sonohata M, Tamae A, et al. Cell-type-specific excitatory and inhibitory circuits involving primary afferents in the substantia gelatinosa of the rat spinal dorsal horn in vitro. J Physiol 2007;581(Pt 2):603–18. [185] Todd AJ. Neuronal circuitry for pain processing in the dorsal horn. Nat Rev Neurosci 2010;11(12):823–36. [186] Ross SE, Mardinly AR, McCord AE, Zurawski J, Cohen S, Jung C, et al. Loss of inhibitory interneurons in the dorsal spinal cord and elevated itch in Bhlhb5 mutant mice. Neuron 2010;65(6):886–98. [187] Kardon AP, Polgar E, Hachisuka J, Snyder LM, Cameron D, Savage S, et al. Dynorphin acts as a neuromodu- lator to inhibit itch in the dorsal horn of the spinal cord. Neuron 2014;82(3):573–86. [188] Shapovalov G, Gkika D, Devilliers M, Kondratskyi A, Gordienko D, Busserolles J, et al. Opiates modulate thermosensation by internalizing cold receptor TRPM8. Cell Rep 2013;4(3):504–15. [189] McCoy ES, Taylor-Blake B, Street SE, Pribisko AL, Zheng J, Zylka MJ. Peptidergic CGRPalpha primary sensory neurons encode heat and itch and tonically suppress sensitivity to cold. Neuron 2013;78(1):138–51. CHAPTER 15 Connecting TRP Channels and Cerebrovascular Diseases Anil Zechariah,1,2,3 Donald G. Welsh1,2,3,* 1Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta, Canada 2Libin Cardiovascular Institute, University of Calgary, Calgary, Alberta, Canada 3Department of Physiology and Pharmacology, University of Calgary, Calgary, Alberta, Canada *Corresponding author: [email protected]

OUTLINE

Overview: TRP Channels: Types, TRP Channels and Vasodilatation 268 Distribution, and Function 264 TRP Channels: Endothelial Proliferation, Angiogenesis, TRP Channels in Diseases 265 and Vascular Remodeling 268 TRP Channels in Smooth Muscle Cells 265 TRP Channels and Hypertension 269 Role in Smooth Muscle Proliferation 266 Role in Smooth Muscle Control TRP Channels and Stroke 272 of Blood Flow 266 Future Directions 272 Role in Myogenic Tone 266 References 273 TRP Channels and Endothelial Function 267 TRP Channel and Vascular Barrier Function 267

TRP Channels as Therapeutic Targets 263 © 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/B978-0-12-420024-1.00015-1 264 15. TRPs IN CEREBROVASCULAR FUNCTION AND DYSFUNCTION OVERVIEW: TRP CHANNELS: TYPES, DISTRIBUTION, AND FUNCTION

Transient receptor potential (TRP) channels are a large family of cation-specific ion channels that have acquired immense attention, due to their involvement with a variety of cellular functions. TRP multigene family sequence is conserved, both in vertebrates and invertebrates [1,2] and member proteins are classified in subfamilies named TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPA (ankyrin), TRPML (mucolipin), TRPP (polycystin), and TRPN (NOMPC-like; Figure 15.1). Among the subfamilies, 28 members are expressed in vertebrates (with the exception of TRPN) of which 27 are found in humans (excluding TRPC2, which is encoded by a pseudogene) [1,2] (Figure 15.1). TRP channels are expressed in a range of different human cell types and are tied to a variety of cellular processes ranging from stimuli sensation to ion homeostasis [3]. In this chapter, we plan to provide an overview of TRP channels in humans, emphasizing on the function and dysfunction of the cerebral circulatory system. The TRPC subfamily consists of six members (TRPC1, TRPC3, TRPC4, TRPC5, TRPC6, and TRPC7), which are robustly expressed in the human brain. In regard to the vasculature, TRPC1 displays a ubiquitous distribution pattern, whereas C3, C4, and C6 are expressed in vascular smooth muscle cells and C4 in the endothelium (Figure 15.1). These channels are engaged in brain development, cell differentiation, mechanosensation, arterial tone control, smooth muscle cell proliferation, and angiogenesis (for detailed review, see Ref. [2]). The human TRPV subfamily also contains six members (TRPV1, TRPV2, TRPV3, TRPV4, TRPV5, and TRPV6), all of them being represented in the brain tissue (Figure 15.1). V2 subunit was found in smooth muscle cells and V3, V4 in the vascular endothelium, playing key roles in thermosensation, mechano/osmolarity sensation, calcium reabsorption, and synaptic adaptation [2,3]. In humans, there are eight members of the TRPM subfamily (TRPM1, TRPM2, TRPM3, TRPM4, TRPM5, TRPM6, TRPM7, and TRPM8) among which M7 is distributed ubiquitously, whereas M2, M3, and M5 variants are expressed in the brain and M4 and M8 are also found in the smooth muscle cell layer [2] (Figure 15.1). These subunits are thought to play an integral part in myogenic tone development, apoptosis, cell cycle control, thermosensation, and Mg2+ homeostasis [2,3]. The TRPA subfamily is comprised of a sole member (TRPA1) (Figure 15.1), typically associated with thermosensation, olfactory responses, and endothelium-dependent dilation [2,3]. The TRPML subunit consists of TRPML1, TRPML2, and TRPML3 (Figure 15.1); however, there is

TRP canonical TRP vanilloid TRP melastatin TRP ankyrin TRP mucolipin TRP poly-cystin

TRPC1 TRPC7 TRPV1 TRPV6 TRPM1 TRPM8

TRPM7 TRPC3 TRPC6 TRPV2 TRPV5 TRPM2 TRPM6 TRPML1 TRPML3 TRPP2 TRPP5

TRPC4 TRPV3 TRPM3 TRPM5

TRPC5 TRPV4 TRPM4 TRPA1 TRPML2 TRPP3

FIGURE 15.1 Representation of the human TRP super family subunits. The subunits expressed in the cerebral circulatory system are indicated in red. TRP Channels in Smooth Muscle Cells 265 no evidence of TRPML expression in the resistance vasculature of the brain [2,3]. On the other hand all three members of the TRPP subfamily (TRPP2, TRPP3, and TRPP5) are present in arteries. These subunits have been linked to pressure and flow sensing along with the maintenance of vessel wall integrity [2,3].

TRP CHANNELS IN DISEASES

TRP channels are associated with a variety of physiological processes in humans including signal transmission, nociception, thermosensation, osmolarity regulation, and arterial tone development [4]. TRP channel activity is triggered by changes in cellular temperature, pressure, chemical agents, osmolarity, and pH [4,5] and by naturally occurring spices, herbs, toxins, and venoms [4,6]. Changes in intercellular calcium (Ca2+), induced by the activation of G-protein coupled receptors, phosphatidylinositol 4,5-bisphosphate, diacylglycerol (DAG), adenosine triphosphate (ATP), and calmodulin are also known to influence TRP channel activities [4,7]. In addition, interactions of TRP channels with intercellular proteins forming “channelosomes” are now believed to effect its trafficking, positioning, and activity [4,8,9]. Consequently, mutations in TRP channel encoding genes may be responsible for several diseases of the cerebrovascular and cardiovascular systems [4]. TRPC3, TRPC5, TRPC6, TRPM2, TRPM4, TRPM7, and TRPML1 have been implicated in CNS disorders, whereas TRPC1, TRPC3, TRPC6, and TRPM4 have been linked to cardiovascular abnormalities [4]. Although human genetic studies have demonstrated the role of TRP gene mutations in at least 12 hereditary diseases (e.g., polycystic kidney disease, hereditary motor and sensory neuropathy, spinal muscular atrophy, and progressive familial heart block), transgenic and knockout animal experiments, on the other hand, have revealed the role of TRP channels in the development of disease pathologies [4,10]. By targeting TRP channels, novel treatment strategies could be deducted for these so-called TRP channelopathies, a rapidly expanding category of diseases. TRP channel targeting molecules have been developed and tested clinically in recent years with minimum success [11]. For example, TRPV1 antagonist underwent clinical trials for the treatment of inflammatory, visceral, and neuropathic pain; however, this resulted in adverse side effects such as hyperthermia and impaired heat sensation in patients [11].

TRP CHANNELS IN SMOOTH MUSCLE CELLS

Vascular smooth muscle cells (VSMCs) account for the majority of blood vessel wall composition and hence are widely distributed, playing major roles in circulatory, respiratory, gastrointestinal, and urogenital systems [12,13]. Although blood flow regulation by contracting and relaxing the blood vessel remains the primary function of VSMCs, they have been implicated in many other physiological functions such as proliferation and migration, production of cytokines/chemokines/matrix proteins/growth factors/cell surface adhesion molecules, and in antigen presentation [12–17]. Smooth muscle activity, in large part, is determined by the changes in Ca2+ concentration, a process dependent on Ca2+ entry from the extracellular space through ion channels and on the Ca2+ release from the intracellular calcium stores [12]. Extracellular Ca2+ entry is facilitated by voltage-operated calcium 266 15. TRPs IN CEREBROVASCULAR FUNCTION AND DYSFUNCTION

channels (L-type and T-type) and voltage-independent calcium channels (receptor-operated channels, store-operated channels (SOC), and stretch-activated channels). TRP channels have been reported to be a key component of the voltage-independent calcium channels in the SMCs; their dysfunction is responsible for vascular pathologies ranging from arterial hypertension and arthrosclerosis, to stroke and cardiovascular diseases [12,18].

Role in Smooth Muscle Proliferation TRP channels have been implicated in the proliferation and migration of SMCs following vascular injury in humans. Smooth muscle proliferation requires a shift in the smooth muscle profile from a contractile phenotype to a synthetic phenotype, which is facilitated by transcriptional changes in regulatory proteins and is associated with the changes in the expression of ion channels [19]. Of note is the observation that the shift in smooth muscle phenotype also contributes to atherosclerosis and the formation of hypertensive microvessels [19]. Among TRP subtypes, TRPC (especially TRPC1, TRPC4, and TRPC5) are all known to be key players in smooth muscle proliferation [19]. Moreover, TRPC up-regulation was observed in patients with idiopathic pulmonary arterial hypertension, characterized by the abnormal increase of SMCs in the pulmonary artery [20].

Role in Smooth Muscle Control of Blood Flow Many TRP channel subtypes are linked with the regulation of contractile function in smooth muscle cells. TRPC1 is shown to be an essential component of SOC in inducing vessel contractions, whereas, on the other hand, vessel contraction resulting from TRPC6-activated DAG appears to work independent of store depletion [21,22]. TRPC3 is known to be involved with the regulation of blood flow in cerebral vessels, the suppression of which deceased constriction response to uridine triphosphate [23]. Conversely, TRPV4 has been shown to be associated with smooth muscle induced arterial dilation, as Ca2+ influx through TRPV4 activates ryanodine receptor, which in turn influences large conductance Ca2+ and voltage-activated potassium channels (BKCa) channels leading to vasodilation [24]. Members of the TRPM subunits are also represented in smooth muscle function, as TRPM3 agonist was shown to modulate contractile responses in the aorta [25]. In addition, TRPM4 is known to mediate pressure-induced vasoconstriction of the cerebral arterial myocytes, a process regulated by Ca2+ release from the IP3 receptor on the sarcoplasmic reticulum [26].

Role in Myogenic Tone Myogenic tone is an intrinsic vasomotor mechanism through which resistance arteries respond to changes in intravascular pressure. Stretch-activated channels respond to changes in intravascular pressure by facilitating Ca2+ entry and/or by membrane depolarization, activating voltage-operated Ca2+ channels. Elevation of intracellular Ca2+ leads eventually to the constriction of the vessel, by activation of myosin light chain kinase and/or by protein kinase C (PKC)/Rho-kinase dependent mechanisms [12,27]. Among the 27 human TRP subunits, at least 11 (TRPC1, TRPC5, TRPC6, TRPV1, TRPV2, TRPV4, TRPM3, TRPM4, TRPM7, TRPP2, and TRPA1) are known to be mechanosensitive stretch-activated channels [12]. Recent studies TRP Channels and Endothelial Function 267 in aged hypertensive mice suggest that TRPC channel suppression mitigates the myogenic constriction of the middle cerebral artery and the loss of the TRPC channel-dependent component of myogenic tone resulting in a diminished response to intraluminal pressure [28]. Furthermore, in rat cerebral arteries, smooth muscle depolarization and constriction response to increased intraluminal pressure was attenuated by down-regulation of TRPC6 [29]. The expression of TRPM4-like channels in cerebral artery myocytes can contribute to the depolarization and vasoconstriction of cerebral arteries, after activation by membrane-stretch [30]. Suppression of TRPM4 attenuated pressure-induced smooth muscle depolarization and myogenic vasoconstriction in intact rat cerebral arteries [31]. TRPM4 inhibition also resulted in decreased myogenic constriction and impaired autoregulation of cerebral blood flow in rats [32]. The role of PKC-dependent regulation of TRPM4 in the development of myogenic tone was demonstrated when suppression of TRPM4 resulted in the weakening of PKC-induced depolarization and vasoconstriction in cerebral arteries [33]. TRPV2 expression has been demonstrated in the aortic, mesenteric, and basilar arterial smooth muscle cells, where their down-regulation attenuated swelling-induced inward current and Ca2+ release [34]; however, there is no evidence of their role in the cerebral circulation.

TRP CHANNELS AND ENDOTHELIAL FUNCTION

TRP Channel and Vascular Barrier Function Endothelial cells form a semipermeable barrier allowing selective passage of molecules between the lumen and the interstitium. Increased endothelial permeability during inflammatory processes has been shown to be induced by actin polymerization-dependent endothelial cell rounding and by the formation of interendothelial gaps [25,35,36]. In addition, protein kinase C isoform (PKC) alpha is shown to be activated by Ca2+ influx into the cell, inducing endothelial contraction and disassembly of VE-cadherin junctions, leading to endothelial permeability changes [25,35,36]. TRPC1 and TRPC4 were among the first TRP subunits connected to abnormal permeability of endothelial cells, as TRPC4 knockout mice exhibited attenuated lung microvascular permeability in response to thrombin receptor activation, and TRPC1/C4 induced Ca2+ entry were proposed to disrupt the endothelial cell barrier and increase permeability in response to an inflammatory agonist [25,35,37]. Furthermore, TRPC6 was shown to activate Rho kinase through a PKC alpha mediated pathway, consequently leading to endothelial permeability [38]. Interestingly, vascular endothelial growth factor (VEGF) induced hyperpermeability of the endothelial barrier, which is a common denominator for many neurovascular diseases; was 2+ demonstrated to be mediated through the PLC-IP3 pathway, activating Ca entry via TRPC1 [39]. Furthermore, it has been observed that Ca2+ entry through TRPM4 leads to the formation of interendothelial gaps and to hyperpermeability in the capillary segment of the pulmonary artery [40]. Similarly, pulmonary microvessel permeability was also connected to TRPM2 (a nonselective cation channel mediating oxidant-induced Ca2+ entry) activation by neutrophilic oxidants, a process regulated by PKC alpha [41]. However, the role of these channels in cerebral microvascular permeability is yet to be demonstrated. Investigations on the role of endothelial permeability in the functionality of the blood-brain barrier (BBB) have demonstrated some interesting findings. Changes in Ca2+ dynamics are now believed to influence BBB permeability as 268 15. TRPs IN CEREBROVASCULAR FUNCTION AND DYSFUNCTION bradykinin, an inflammatory peptide increased Ca2+ oscillations resulting in hyperpermeability of the BBB [42]. Interestingly, this effect was abolished by interfering with connexin channels (i.e., through a peptide blocker) and also by the knockdown of connexin 37/40 [42]. The role of TRP channels in BBB permeability is indeed intriguing and observations indicate that controlling Ca2+ dynamics through these channels may attenuate the BBB permeability alterations associated with many neurological disorders.

TRP Channels and Vasodilatation Endothelial expression of 21 TRP channels has been documented, and at least five are associated with the endothelial-dependent vasodilation and blood flow delivery [43]. TRPC4 was the first TRP subunit thought to be involved in endothelium-dependent vasodilatation, as its knockout mice showed reduced agonist-induced Ca2+ entry and vasorelaxation [44]. TRPA1, a Ca2+ permeable nonselective cation channel, activated by electrophilic compounds such as allicin and allyl isothiocynate (AITC), was also associated with endothelium-mediated tone regulation [45]. AITC induced dilation of cerebral vessels in a concentration dependent manner, an effect that was selectively attenuated by the TRPA1 channel blocker HC-030031 [45]. Interestingly, AITC-induced vasodilation is insensitive to nitric oxide synthase inhibition but was blocked by the small and intermediate conductance Ca2+ activated potassium (K+) channel blockers, apamin and TRAM34 [45]. In agreement with these results, a recent study demonstrated the correlation between the AITC-induced increase in Ca2+ signals and consequent dilation of cerebral arteries in a concentration-dependent manner [46]. In addition, carvacrol (a compound present in oregano) was shown to trigger a concentration-dependent increase of intracellular Ca2+ in cerebral arterial endothelial cells and induced dilation of cerebral arteries, an effect inhibited by the presence of Ca2+-activated K+ channel blockers in the lumen and + by the presence of the inwardly rectifying K channel blocker BaCl2 [47]. Although TRPA1 and TRPV3 were both expressed in endothelial cells, carvacrol induced dilation was inhibited only by TRPV blocker ruthenium red, whereas it was unaffected by TRPA1 antagonist [47].

TRP Channels: Endothelial Proliferation, Angiogenesis, and Vascular Remodeling Angiogenesis is the formation of new vessels from preexisting vessels, a process normally dormant during adulthood with the exception of wound healing and physiological formation of vessels. Angiogenesis as a therapeutic paradigm is a “double edged sword.” Proangiogenic therapy aims to increase new vessel formation to increase blood flow to the damaged tissue, enhancing or encouraging recovery; whereas, antiangiogenic therapies target tumor vascularization, a process critical for tumor metastasis and thereby a fascinating target for cancer treatment. Tumor vascularization has been linked to many different Ca2+ channels, as Ca2+ signaling is quintessential for cellular processes such as gene transcription, cell proliferation, migration, and angiogenesis [48]. As TRP channels influence intracellular Ca2+ concentrations impacting crucial cytosolic and nuclear events, a growing body of evidence points toward their role in cancer incitation and progression [48]. Tumor tissue expansion induces drastic changes in the cellular environment and TRP channels with their ubiquitous expression, and multidimensional activation patterns may be involved in the regulation of different stages TRP Channels and Hypertension 269 of cancer cell development [48–50]. Out of the TRP channel subfamilies, endothelial TRPC1, TRPC4, TRPC6, TRPV4, and TRPM7 have been implicated in angiogenesis [48,51]. The first evidence for the role of TRPs in vessel formation came from a study demonstrating disrupted formation of intersegmental vessels during zebra fish development after TRPC1 knockdown [52]. TRPC1 was also shown to be a downstream component of the VEGF-induced angiogenesis pathway where it participated in the phosphorylation of extracellular signal regulated kinase (ERK) [52]. In addition, TRPC1 (along with TRPC4) was shown to be involved in tube formation by primary human umbilical vein endothelial cells (HUVECs) [53] and was also shown to regulate the growth of endothelial progenitor cells by participating in store- operated Ca2+ entry [54]. In the kidney, it has been demonstrated that TRPC4 loss in renal cell carcinoma (RCC) leads to impaired Ca2+ intake and diminished secretion of antiangiogenic protein thrombopsondin-1, enabling RCC progression [55]. Another study showed the role of TRPC4 in the transition of endothelial cells from a proliferating to quiescent phenotype by functioning as a stimulated Ca2+ entry channel [56]. The overexpression of TRPC6 increased proliferation and migration of human microvascular endothelial cells [57], whereas its inhibition arrested VEGF-induced proliferation and tube formation in HUVECs [58], delineating its role in VEGF-mediated angiogenesis. In addition, TRPC6 was found to cause a sustained elevation of intercellular Ca2+ in glioblastoma multiforme (GBM), which is coupled with the activation of calcineurin-nuclear factor of activated T-cell pathway [59]. Also, TRPC6 was elevated in clinical specimens of GBM, and knockdown of TRPC6 was related to reduced glioma growth, invasion, and angiogenesis [59]. Phosphatase and tensin homologue was shown to serve as a scaffold for TRPC6, enabling its cell surface expression and facilitating Ca2+ entry, inducing an increase in endothelial permeability and angiogenesis [60]. Stimulation by cyclic mechanical strain of pulsatile blood flow has been shown to activate the mechanosensitive TRPV4, promoting cytoskeletal remodeling and cell reorientation, processes critical for angiogenesis [61]. Activation of TRPV4 by fluid shear stress also lead to proliferation of vascular cells, and treatment with a specific TRPV4 activator, 4alpha-Phorbol 12, 13-didecanoate (4alphaPDD), triggered collateral growth and facilitated recovery in rats after femoral artery ligature [62]. Endothelial proliferation and angiogenesis are also influenced by magnesium dynamics, hence the magnesium transporter TRPM7 has also been linked to these processes. An inhibitory role of TRPM7 in the angiogenic process was revealed when its knockdown resulted in the proliferation of human microvascular endothelial cells, an effect modulated at least in part by the ERK pathway [63]. Taken together, these investigations have delineated several roles of TRP channels in endothelial cell proliferation and angiogenesis that could be targeted for developing effective proangiogenic and antiangiogenic therapies.

TRP CHANNELS AND HYPERTENSION

Hypertension is considered a primary risk factor of vascular diseases and has been implicated in atherosclerosis, stroke, heart failure, and renal complications [64,65]. Among the several factors that contribute to the development of hypertension, dysregulation of Ca2+ homeostasis and the consequent vascular dysfunction are most common. Advances in the TRP channel field have enabled us to understand that these cation channels, and variations 270 15. TRPs IN CEREBROVASCULAR FUNCTION AND DYSFUNCTION in their expression and/or regulation, are responsible for the development of many disorders, including hypertension. The TRP channel subunits TRPC1, TRPC3, TRPC5, TRPC6, TRPV1, TRPV4, TRPV5, TRPM4, TRPM6, TRPM7, and TRPA1 have all been connected to the development and/or progression of hypertension. TRPC3, TRPC5, and TRPM6 were found to be reduced significantly in patients after hypertensive intracerebral hemorrhage in comparison with controls [66]. In addition, the authors also found a correlation between the expression of TRPC3 and hypoxia inducible factor 1a, indicating its association with hypoxic conditions in the human cerebral tissue [66]. In spontaneously hypertensive rats, the expression of TRPC1, C3, and C5 were significantly increased in the mesenteric arteries and was associated with an increase in norepinephrine-induced vasomotion in comparison with normotensive rats [67]. Chronic treatment with candesartan or telmisartan significantly reduced the expression of these TRPs and norepinephrine-induced vasomotion in these animals [67]. Activation of the TRPC3 channel leads to myocyte depolarization and Ca2+ entry through VDCC resulting in vasoconstriction [68]. VDCC Ca2+ entry is suggested to be crucial for the autoregulation of cerebral blood flow and hence could be an interesting target to address dysfunctional vascular contractility, as during hypertension [68]. In addition, Xi et al. have demonstrated a mechanism of IP3-induced vasoconstriction of cerebral arteries, which occurs independent of SR Ca2+ release and mediated by TRPC3 channels and IP3 receptor-dependent I(Cat) activation [69]. Monocytes of erythropoietin-treated, hemodialysis patients revealed an increased expression of TRPC5 mRNA accompanied by increased systolic blood pressure, indicating a role of TRPC5 in erythropoietin induced hypertension [70]. In HUVECs, TRPC6 and TRPV1 mRNA expression levels were significantly increased after exposure to a pulsatile atheroprone shear stress flow in comparison to an atheroprotective flow profile [71]. Furthermore, the authors also found an association between TRPC6 mRNA expression and tumor necrosis factor-α mRNA in the human vascular tissue, delineating its connection to the inflammatory processes in the vasculature [71]. Recently, the involvement of TRPC6 and 20-hydroxyeicosatetraenoic acid (HETE) in the autoregulation of cerebral blood flow was elucidated under conditions of hypertension, where the authors described the loss of autoregulation in aged, Ang II-induced hypertensive mice along with exacerbated disruption of the BBB, increased neuroinflammation, and impaired hippocampal-dependent cognitive function [72]. In addition, it has been shown that 20-HETE activation of TRPV1, in part, mediates pressure-induced myogenic constriction and underlines 20-HETE-induced elevations in blood pressure and coronary resistance [73]. The connection between TRPV1 and intraluminal pressure changes were first described by Scotland et al. as an elevation in intraluminal pressure generated 20-HETE, activating TRPV1 on C fiber nerve endings and resulting in a myogenic response [74]. TRPV1 activation by dietary capsaicin enhanced endothelium dependent relaxation in mice, an effect absent in TRPV1 knockout mice [75]. Furthermore, chronic stimulation of TRPV1-activated PKA, resulted in increased eNOS phosphorylation, improved vasorelaxation, and the lowering of blood pressure in genetically hypertensive rats [75]. Remarkably, long-term administration of capsaicin significantly delayed the onset of stroke and increased survival time in spontaneously hypertensive rats, an effect mediated through eNOS-dependent vasorelaxation and induced by TRPV1 activation [76]. Moreover, long-term activation of capsaicin increased ATP binding cassette transporter A1 and reduced low-density lipoprotein-related protein1 expression in the aorta of atherosclerotic mice (ApoE−/−), reducing lipid storage and atherosclerotic lesions in the aorta, an effect absent in ApoE(−/−)/TRPV1 (−/−) double knockout mice [77]. TRP Channels and Hypertension 271

Rodent experiments have suggested activation of the sympathetic nervous system through the osmosensitive channel TRPV4 to be responsible for the robust increase in blood pressure after water ingestion, observed in human subjects with impaired baroreflex function [78]. Even though TRPV4 has been shown to be involved in pulmonary vasoreactivity [79] and hypoxia-induced pulmonary hypertension [80], and that nitric oxide synthase-inhibition induced hypertension is exacerbated in TRPV4 knockout mice [81], its role in the cerebral vasculature and its dysfunction, remains to be clearly elucidated. The inhibition of WKN4, a protein serine/threonine kinase that positively regulates the TRPV5-mediated Ca2+ transport process, is believed to be involved in the pathogenesis of familial hyperkalemic hypertension [82]. Moreover, TRPV5 knockout mice present phenotypic defects of renal disease such as hypercalciuria and impaired bone mineral density, exemplifying the role of this channel in renal physiology, hypertension, and genetic disorders of the kidney [82,83]. The role of TRPM subunits in hypertension is mainly represented by TRPM4, 6, and 7, where TRPM4 was first shown to be increasingly expressed in ventricular samples of spontaneously hypertensive rats in comparison with normotensive controls [84]. Interestingly, TRPM4 has been also connected with the electrophysiological perturbations associated with cardiac hypertrophy, of which hypertension is an important risk factor [85]. TRPM4 knockout mice present dysregulation of blood pressure toward hypertensive levels as TRPM4 regulates catecholamine release from chromaffin cells, the dysfunction of which leads to increased sympathetic tone and hypertension [86]. Mg2+ influences many cellular functions and variations in Mg2+ in the serum and tissue has been known to inversely affect blood pressure [87]. As TRPM6 and TRPM7 are associated with Mg2+ transport in the tissue, they are also involved in the development of vascular dysfunctions and hypertension. Whereas TRPM6 is expressed in epithelial cells, TRPM7 is globally expressed and is known as an important regulator of Mg2+. Membrane-associated Ang II- and aldosterone-regulated TRPM7 are involved in the DNA and protein synthesis in both rodent and human VSMCs [88]. Importantly, the expression of TRPM7 was blunted in spontaneously hypertensive rats, which resulted in decreased Mg2+ in the VSMC [89]. Similarly, in low intracellular Mg2+-exhibiting mice, the expression of TRPM7 was significantly elevated in the vasculature, whereas its downstream target, anti-inflammatory annexin-1, was reduced [90]. These animals also exhibited endothelial dysfunction, changes in vascular structure, and inflammation, revealing the role of TRPM7 signaling in the maintenance of vascular integrity and function [90]. A recent investigation has also revealed the role of TRPM7 in vascular remodeling, where its expression increased with the pressure overload-induced vascular wall thickening [91]. It was also associated with simultaneous accumulation of macrophages in the ad- ventitia and decreased expression of annexin-1, an effect reversed by TRPM7 inhibitor 2-aminoethoxydiphenyl borate [91]. TRPA1 are a class of mechanosensitive Ca2+ permeable channels that are activated by irritant chemicals and pungent natural compounds [92,93]. L-type calcium channel antagonist 1,4-dihydropyridines, including nifedipine, nimodipine, nicardipine, and nitrendipin, commonly used in the treatment of hypertension, have been shown to exert powerful excitatory effects on TRPA1 channels [92]. In addition, nifedipine is known to induce large elevations of Ca2+ in mouse nociceptors expressing TRPA1, an effect abrogated in the TRPA1 knockout mice [92]. 272 15. TRPs IN CEREBROVASCULAR FUNCTION AND DYSFUNCTION TRP CHANNELS AND STROKE

Ischemia refers to the inadequate supply of oxygen and nutrients to an organ, resulting in cell death and degeneration. Stroke is the fourth major cause of death and is the leading cause of adult disability in the Western population [94]. As stroke results from the blockage or disruption of a supplying blood vessel, vascular dysfunction is a vital risk factor in its development and during recovery. Hypoxia resulting from ischemia, activates a myriad of cellular responses including the rise in intercellular Ca2+ [95]. As the rise in intercellular Ca2+ depends on the activation of Ca2+ permeable channels and/or by the activation of intercellular Ca2+ stores, TRP channels, with their known influence on these mechanisms, are likely to play a key role. The first demonstration of the involvement of a TRP subunit in hypoxia was the observation that TRPM7 suppression blocked Ca2+ uptake, reactive oxygen species production and anoxic cell death in cortical neurons subjected to prolonged oxygen glucose deprivation [96]. Electrophysiological investigation on acute hippocampal slices showed that TRP channels are activated by cellular stress, contributing to ischemia-induced membrane depolarization, intracellular calcium accumulation, and cell swelling [97]. Likewise, TRPM2 mRNA expression was increased at 1 and 4 weeks following ischemic injury in a rat middle cerebral artery occlusion model of stroke [98]. In mouse hippocampal neurons, it was demonstrated that TRPM7 channels were involved in detecting the reduction in extracellular divalent ions, known to contribute to neuronal death after brain ischemia [99]. TRPM7 suppression by shRNA made neurons resistant to neuronal damage after brain ischemia, preserving neuronal morphology and function, leading to long-term potentiation and animal survival [100]. However, an assessment of 16 tag-single-nucleotide polymorphisms of the TRPM7 gene in humans showed no evidence for its role as a risk factor for ischemic stroke incidence [101]. An increase in TRPV4 expression in the cortical and hippocampal astrocytes of adult rat was shown to coincide with the development of astrogliosis after ischemia [102]. In addition, cultured hippocampal astrocytes have been found to respond to the TRPV4 activator 4-alpha-phorbol-12,-13-didecanoate as detected by an increase in intracellular Ca2+; this effect was augmented after hypoxic/ischemic injury, demonstrating the possible role of TRPM4 in astroglial reactivity after ischemia [102]. An investigation on a primate model of stroke has revealed promising results that Tat-NR2B9c, a PSD-95 interfering peptide known to disrupt TRPM7/PSD-95/neuronal NOS pathway, reduced stroke volume, maintained gene transcription, and preserved neurological function after ischemia [103,104]. The emerging role of TRP channels in neuronal injury development after stroke opens up a potential paradigm for clinical interventions in patients, extending even beyond the acute phase of stroke. However, the expressional, regulatory and functional characteristics of the many different TRP subunits need to be thoroughly elucidated so that a feasible therapeutic strategy could be derived for interventions in stroke pathology.

FUTURE DIRECTIONS

Our understanding of TRP channels has exploded in the past decade (>1600 manuscripts annually [105]), fueled by its potential as a drug target. However, the conundrum surrounding TRP channel functionality and its connection to human disease is currently underdeveloped. REFERENCES 273

The role of TRP channels in the cerebral circulation and in cerebrovascular diseases is still in its infancy. As TRP channels have been implicated in myogenic tone blood flow control along with the permeability of the BBB, collateral formation, angiogenesis and vascular remodeling, its significance in the cerebral circulation and cerebrovascular disorders have gained appreci- ation. The varied and global expression of TRP channels demonstrate its multifaceted roles in cellular physiology and at the same time presents itself as a challenging barrier for drug design and implementation. This task is further complicated by the ability of TRPs to heteromultim- erize and to interact with other intercellular proteins (channelosomes). The fact that many of TRP members are activated by natural compounds (e.g., carvacrol, capsaicin) is enthralling, as plant extracts have been used in traditional medicine for many centuries. Hence, it is conceivable that the regulation of TRP channels and subsequent intervention of their associated pathologies may be possible through dietary supplements via naturally occurring compounds, thereby minimizing the risk of side effects in patients. Given the ubiquitous nature and the presence of multidimensional activation pathways, a comprehensive understanding of TRP channel biology remains the “sine qua non” for extracting its therapeutic potential.

References [1] Vennekens R, Menigoz A, Nilius B. TRPs in the Brain. Rev Physiol Biochem Pharmacol 2012;163:27–64. [2] Nilius B, Owsianik G. The transient receptor potential family of ion channels. Genome Biol 2011;12:218–29. [3] Billeter AT, Hellmann JL, Bhatnagar A, Polk Jr HC. Transient receptor potential ion channels: powerful regulators of cell function. Ann Surg 2014;259(2):229–35. [4] Kaneko Y, Szallasi A. Transient receptor potential (TRP) channels: a clinical perspective. Br J Pharmacol 2014;171:2474–504. [5] Moran MM, McAlexander MA, Bíró T, Szallasi A. Transient receptor potential channels as therapeutic targets. Nat Rev Drug Discov 2011;10:601–20. [6] Vriens J, Nilius B, Vennekens R. Herbal compounds and toxins modulating TRP channels. Curr Neuropharmacol 2008;6:79–96. [7] Wu LJ, Sweet TB, Clapham DE. International Union of Basic and Clinical Pharmacology. LXXVI. Current progress in the mammalian TRP ion channel family. Pharmacol Rev 2010;62:381–404. [8] Planells-Cases R, Ferrer-Montiel A. TRP channel trafficking. In: Liedtke WB, Heller S, editors. TRP ion channel function in sensory transduction and cellular signaling cascades. Boca Raton, FL: CRC Press, Taylor & Francis; 2007. p. 319–30. [9] Goswami C. TRP-mediated cytoskeletal reorganization: implications for disease and drug development. In: Szallasi A, Bíró T, editors. TRP channels in drug discovery. Methods in pharmacology and toxicology, Springer protocols. New York: Humana Press; 2012. p. 13–39. [10] Everett KV. Transient receptor potential genes and human inherited disease. Adv Exp Med Biol 2011; 704:1011–32. [11] Brederson JD, Kym PR, Szallasi A. Targeting TRP channels for pain relief. Eur J Pharmacol 2013; 716(1–3):61–76. [12] Guibert C, Ducret T, Savineau JP. Expression and physiological roles of TRP channels in smooth muscle cells. Adv Exp Med Biol 2011;704:687–706. [13] Berridge MJ. Smooth muscle cell calcium activation mechanisms. J Physiol 2008;586:5047–61. [14] Gerthoffer WT. Mechanisms of vascular smooth muscle cell migration. Circ Res 2007;100:607–21. [15] House SJ, Potier M, Bisaillon J, Singer HA, Trebak M. The non-excitable smooth muscle: calcium signaling and phenotypic switching during vascular disease. Pflugers Arch 2008;456:769–85. [16] Tliba O, Panettieri Jr RA. Noncontractile functions of airway smooth muscle cells in asthma. Annu Rev Physiol 2009;71:509–35. [17] Wamhoff BR, Bowles DK, Owens GK. Excitation-transcription coupling in arterial smooth muscle. Circ Res 2006;98:868–78. 274 15. TRPs IN CEREBROVASCULAR FUNCTION AND DYSFUNCTION

[18] Gonzalez-Cobos JC, Trebak M. TRPC channels in smooth muscle cells. Front Biosci (Landmark Ed) 2010;15:1023–39. [19] Dietrich A, Kalwa H, Gudermann T. TRPC channels in vascular cell function. Thromb Haemost 2010;103(2):262–70. [20] Yu Y, Fantozzi I, Remillard CV, Landsberg JW, Kunichika N, Platoshyn O, et al. Enhanced expression of transient receptor potential channels in idiopathic pulmonary arterial hypertension. Proc Natl Acad Sci USA 2004;101:13861–6. [21] Kunichika N, Yu Y, Remillard CV, Platoshyn O, Zhang S, Yuan JX. Overexpression of TRPC1 enhances pulmonary vasoconstriction induced by capacitative Ca2+ entry. Am J Physiol Lung Cell Mol Physiol 2004;287:L962–9. [22] Dietrich A, Mederos y Schnitzler M, Kalwa H, Storch U, Gudermann T. Functional characterization and physiological relevance of the TRPC3/6/7 subfamily of cation channels. Naunyn Schmiedebergs Arch Pharmacol 2005;371:257–65. [23] Reading SA, Earley S, Waldron BJ, Welsh DG, Brayden JE. TRPC3 mediates pyrimidine receptor-induced depolarization of cerebral arteries. Am J Physiol Heart Circ Physiol 2005;288:H2055–61. [24] Earley S, Heppner TJ, Nelson MT, Brayden JE. TRPV4 forms a novel Ca2+ signaling complex with ryanodine receptors and BKCa channels. Circ Res 2005;97:1270–9. [25] Kumar B, Dreja K, Shah SS, Cheong A, Xu SZ, Sukumar P, et al. Upregulated TRPC1 channel in vascular injury in vivo and its role in human neointimal hyperplasia. Circ Res 2006;98:557–63. [26] Gonzales AL, Amberg GC, Earley S. Ca2+ release from the sarcoplasmic reticulum is required for sustained TRPM4 activity in cerebral artery smooth muscle cells. Am J Physiol Cell Physiol 2010;299(2):C279–88. [27] Hill MA, Zou H, Potocnik SJ, Meininger GA, Davis MJ. Invited review: arteriolar smooth muscle mechanotransduction: Ca(2+) signaling pathways underlying myogenic reactivity. J Appl Physiol 2001;91:973–83. [28] Toth P, Csiszar A, Tucsek Z, Sosnowska D, Gautam T, Koller A, et al. Role of 20-HETE, TRPC channels, and BKCa in dysregulation of pressure-induced Ca2+ signaling and myogenic constriction of cerebral arteries in aged hypertensive mice. Am J Physiol Heart Circ Physiol 2013;305(12):H1698–708. [29] Welsh DG, Morielli AD, Nelson MT, Brayden JE. Transient receptor potential channels regulate myogenic tone of resistance arteries. Circ Res 2002;90:248–50. [30] Morita H, Honda A, Inoue R, Ito Y, Abe K, Nelson MT, et al. Membrane stretch-induced activation of a TRPM4- like nonselective cation channel in cerebral artery myocytes. J Pharmacol Sci 2007;103(4):417–26. [31] Earley S, Waldron BJ, Brayden JE. Critical role for transient receptor potential channel TRPM4 in myogenic constriction of cerebral arteries. Circ Res 2004;95(9):922–9. [32] Reading SA, Brayden JE. Central role of TRPM4 channels in cerebral blood flow regulation. Stroke 2007;38(8):2322–8. [33] Earley S, Straub SV, Brayden JE. Protein kinase C regulates vascular myogenic tone through activation of TRPM4. Am J Physiol Heart Circ Physiol 2007;292(6):H2613–22. [34] Muraki K, Iwata Y, Katanosaka Y, Ito T, Ohya S, Shigekawa M, et al. TRPV2 is a component of osmotically sensitive cation channels in murine aortic myocytes. Circ Res 2003;93:829–38. [35] Tiruppathi C, Minshall RD, Paria BC, Vogel SM, Malik AB. Role of Ca2+ signaling in the regulation of endothelial permeability. Vascul Pharmacol 2002;39(4–5):173–85. [36] Ahmmed GU, Malik AB. Functional role of TRPC channels in the regulation of endothelial permeability. Pflugers Arch 2005;451(1):131–42. [37] Cioffi DL, Stevens T. Regulation of endothelial cell barrier function by store-operated calcium entry. Microcirculation 2006;13(8):709–23. [38] Singh I, Knezevic N, Ahmmed GU, Kini V, Malik AB, Mehta D. Galphaq-TRPC6-mediated Ca2+ entry induces RhoA activation and resultant endothelial cell shape change in response to thrombin. J Biol Chem 2007;282:7833–43. [39] Jho D, Mehta D, Ahmmed G, Gao XP, Tiruppathi C, Broman M, et al. Angiopoietin-1 opposes VEGF-induced increase in endothelial permeability by inhibiting TRPC1-dependent Ca2 influx. Circ Res 2005;96:1282–90. [40] Cioffi DL, Lowe K, Alvarez DF, Barry C, Stevens T. TRPing on the lung endothelium: calcium channels that regulate barrier function. Antioxid Redox Signal 2009;11(4):765–76. [41] Hecquet CM, Ahmmed GU, Malik AB. TRPM2 channel regulates endothelial barrier function. Adv Exp Med Biol 2010;661:155–67. REFERENCES 275

[42] De Bock M, Culot M, Wang N, Bol M, Decrock E, De Vuyst E, et al. Connexin channels provide a target to manipulate brain endothelial calcium dynamics and blood-brain barrier permeability. J Cereb Blood Flow Metab 2011;31(9):1942–57. [43] Sullivan MN, Earley S. TRP channel Ca(2+) sparklets: fundamental signals underlying endothelium-dependent hyperpolarization. Am J Physiol Cell Physiol 2013;305(10):C999–1008. [44] Freichel M, Suh SH, Pfeifer A, Schweig U, Trost C, Weissgerber P, et al. Lack of an endothelial store-operated Ca2+ current impairs agonist-dependent vasorelaxation in TRP4−/− mice. Nat Cell Biol 2001;3(2):121–7. [45] Earley S, Gonzales AL, Crnich R. Endothelium-dependent cerebral artery dilation mediated by TRPA1 and Ca2+-activated K+channels. Circ Res 2009;104(8):987–94. [46] Qian X, Francis M, Solodushko V, Earley S, Taylor MS. Recruitment of dynamic endothelial Ca2+ signals by the TRPA1 channel activator AITC in rat cerebral arteries. Microcirculation 2013;20(2):138–48. [47] Earley S, Gonzales AL, Garcia ZI. A dietary agonist of transient receptor potential cation channel V3 elicits endothelium-dependent vasodilation. Mol Pharmacol 2010;77(4):612–20. [48] Fiorio Pla A, Gkika D. Emerging role of TRP channels in cell migration: from tumor vascularization to metastasis. Front Physiol 2013;4:311. [49] Nilius B, Owsianik G, Voets T, Peters JA. Transient receptor potential cation channels in disease. Physiol Rev 2007;87:165–217. [50] Ouadid-Ahidouch H, Dhennin-Duthille I, Gautier M, Sevestre H, Ahidouch A. TRP channels: diagnostic markers and therapeutic targets for breast cancer. Trends Mol Med 2013;19:117–24. [51] Wong CO, Yao X. TRP channels in vascular endothelial cells. Adv Exp Med Biol 2011;704:759–80. [52] Yu PC, Gu SY, Bu JW, Du JL. TRPC1 is essential for in vivo angiogenesis in zebrafish. Circ Res 2010;106(7):1221–32. [53] Antigny F, Girardin N, Frieden M. Transient receptor potential canonical channels are required for in vitro endothelial tube formation. J Biol Chem 2012;287(8):5917–27. [54] Lodola F, Laforenza U, Bonetti E, Lim D, Dragoni S, Bottino C, et al. Store-operated Ca2+ entry is remodelled and controls in vitro angiogenesis in endothelial progenitor cells isolated from tumoral patients. PLoS One 2012;7(9):e42541. [55] Veliceasa D, Ivanovic M, Hoepfner FT, Thumbikat P, Volpert OV, Smith ND. Transient potential receptor channel 4 controls thrombospondin-1 secretion and angiogenesis in renal cell carcinoma. FEBS J 2007;274(24):6365–77. [56] Graziani A, Poteser M, Heupel WM, Schleifer H, Krenn M, Drenckhahn D, et al. Cell-cell contact formation governs Ca2+ signaling by TRPC4 in the vascular endothelium: evidence for a regulatory TRPC4-beta-catenin interaction. J Biol Chem 2010;285(6):4213–23. [57] Hamdollah Zadeh MA, Glass CA, Magnussen A, Hancox JC, Bates DO. VEGF-mediated elevated intracellular calcium and angiogenesis in human microvascular endothelial cells in vitro are inhibited by dominant negative TRPC6. Microcirculation 2008;15(7):605–14. [58] Ge R, Tai Y, Sun Y, Zhou K, Yang S, Cheng T, et al. Critical role of TRPC6 channels in VEGF-mediated angiogenesis. Cancer Lett 2009;283(1):43–51. [59] Chigurupati S, Venkataraman R, Barrera D, Naganathan A, Madan M, Paul L, et al. Receptor channel TRPC6 is a key mediator of Notch-driven glioblastoma growth and invasiveness. Cancer Res 2010;70(1):418–27. [60] Kini V, Chavez A, Mehta D. A new role for PTEN in regulating transient receptor potential canonical channel 6-mediated Ca2+ entry, endothelial permeability, and angiogenesis. J Biol Chem 2010;285(43):33082–91. [61] Thodeti CK, Matthews B, Ravi A, Mammoto A, Ghosh K, Bracha AL, et al. TRPV4 channels mediate cyclic strain-induced endothelial cell reorientation through integrin-to-integrin signaling. Circ Res 2009;104(9):1123–30. [62] Troidl C, Troidl K, Schierling W, Cai WJ, Nef H, Möllmann H, et al. Trpv4 induces collateral vessel growth during regeneration of the arterial circulation. J Cell Mol Med 2009;13(8B):2613–21. [63] Baldoli E, Maier JA. Silencing TRPM7 mimics the effects of magnesium deficiency in human microvascular endothelial cells. Angiogenesis 2012;15(1):47–57. [64] Liu D, Scholze A, Zhu Z, Krueger K, Thilo F, Burkert A, et al. Transient receptor potential channels in essential hypertension. J Hypertens 2006;24:1105–14. [65] Wang P, Liu D, Tepel M, Zhu Z. Transient receptor potential canonical type 3 channels—their evolving role in hypertension and its related complications. J Cardiovasc Pharmacol 2013;61(6):455–60. [66] Thilo F, Suess O, Liu Y, Tepel M. Decreased expression of transient receptor potential channels in cerebral vascular tissue from patients after hypertensive intracerebral hemorrhage. Clin Exp Hypertens 2011;33(8):533–7. 276 15. TRPs IN CEREBROVASCULAR FUNCTION AND DYSFUNCTION

[67] Chen X, Yang D, Ma S, He H, Luo Z, Feng X, et al. Increased rhythmicity in hypertensive arterial smooth muscle is linked to transient receptor potential canonical channels. J Cell Mol Med 2010;14(10):2483–94. [68] Brayden JE, Earley S, Nelson MT, Reading S. Transient receptor potential (TRP) channels, vascular tone and autoregulation of cerebral blood flow. Clin Exp Pharmacol Physiol 2008;35(9):1116–20. [69] Xi Q, Adebiyi A, Zhao G, Chapman KE, Waters CM, Hassid A, et al. IP3 constricts cerebral arteries via IP3 receptor-mediated TRPC3 channel activation and independently of sarcoplasmic reticulum Ca2+ release. Circ Res 2008;102(9):1118–26. [70] Liu Y, Xu Y, Thilo F, Friis UG, Jensen BL, Scholze A, et al. Erythropoietin increases expression and function of transient receptor potential canonical 5 channels. Hypertension 2011;58(2):317–24. [71] Thilo F, Vorderwülbecke BJ, Marki A, Krueger K, Liu Y, Baumunk D, et al. Pulsatile atheroprone shear stress affects the expression of transient receptor potential channels in human endothelial cells. Hypertension 2012;59(6):1232–40. [72] Toth P, Tucsek Z, Sosnowska D, Gautam T, Mitschelen M, Tarantini S, et al. Age-related autoregulatory dysfunction and cerebromicrovascular injury in mice with angiotensin II-induced hypertension. J Cereb Blood Flow Metab 2013;33(11):1732–42. [73] Bubb KJ, Wen H, Panayiotou CM, Finsterbusch M, Khan FJ, Chan MV, et al. Activation of neuronal transient receptor potential vanilloid 1 channel underlies 20-hydroxyeicosatetraenoic acid-induced vasoactivity: role for protein kinase A. Hypertension 2013;62(2):426–33. [74] Scotland RS, Chauhan S, Davis C, De Felipe C, Hunt S, Kabir J, et al. Vanilloid receptor TRPV1, sensory C-fibers, and vascular autoregulation: a novel mechanism involved in myogenic constriction. Circ Res 2004;95(10):1027–34. [75] Yang D, Luo Z, Ma S, Wong WT, Ma L, Zhong J, et al. Activation of TRPV1 by dietary capsaicin improves endothelium-dependent vasorelaxation and prevents hypertension. Cell Metab 2010;12(2):130–41. [76] Xu X, Wang P, Zhao Z, Cao T, He H, Luo Z, et al. Activation of transient receptor potential vanilloid 1 by dietary capsaicin delays the onset of stroke in stroke-prone spontaneously hypertensive rats. Stroke 2011;42(11):3245–51. [77] Ma L, Zhong J, Zhao Z, Luo Z, Ma S, Sun J, et al. Activation of TRPV1 reduces vascular lipid accumulation and attenuates atherosclerosis. Cardiovasc Res 2011;92(3):504–13. [78] McHugh J, Keller NR, Appalsamy M, Thomas SA, Raj SR, Diedrich A, et al. Portal osmopressor mechanism linked to transient receptor potential vanilloid 4 and blood pressure control. Hypertension 2010;55(6):1438–43. [79] Xia Y, Fu Z, Hu J, Huang C, Paudel O, Cai S, et al. TRPV4 channel contributes to serotonin-induced pulmonary vasoconstriction and the enhanced vascular reactivity in chronic hypoxic pulmonary hypertension. Am J Physiol Cell Physiol 2013;305(7):C704–15. [80] Yang XR, Lin AH, Hughes JM, Flavahan NA, Cao YN, Liedtke W, et al. Upregulation of osmo-mechanosensitive TRPV4 channel facilitates chronic hypoxia-induced myogenic tone and pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol 2012;302(6):L555–68. [81] Earley S, Brayden JE. Transient receptor potential channels and vascular function. Clin Sci (Lond) 2010;119(1):19–36. [82] Jiang Y, Ferguson WB, Peng JB. WNK4 enhances TRPV5-mediated calcium transport: potential role in hypercalciuria of familial hyperkalemic hypertension caused by gene mutation of WNK4. Am J Physiol Renal Physiol 2007;292(2):F545–54. [83] Menè P, Punzo G, Pirozzi N. TRP channels as therapeutic targets in kidney disease and hypertension. Curr Top Med Chem 2013;13(3):386–97. [84] Guinamard R, Demion M, Magaud C, Potreau D, Bois P. Functional expression of the TRPM4 cationic current in ventricular cardiomyocytes from spontaneously hypertensive rats. Hypertension 2006;48(4):587–94. [85] Guinamard R, Bois P. Involvement of transient receptor potential proteins in cardiac hypertrophy. Biochim Biophys Acta 2007;1772(8):885–94. [86] Mathar I, Vennekens R, Meissner M, Kees F, Van der Mieren G, Camacho Londoño JE, et al. Increased catecholamine secretion contributes to hypertension in TRPM4-deficient mice. J Clin Invest 2010;120(9):3267–79. [87] Sontia B, Touyz RM. Magnesium transport in hypertension. Pathophysiology 2007;14(3–4):205–11. [88] He Y, Yao G, Savoia C, Touyz RM. Transient receptor potential melastatin 7 ion channels regulate magnesium homeostasis in vascular smooth muscle cells: role of angiotensin II. Circ Res 2005;96(2):207–15. [89] Touyz RM, He Y, Montezano AC, Yao G, Chubanov V, Gudermann T, et al. Differential regulation of transient receptor potential melastatin 6 and 7 cation channels by ANG II in vascular smooth muscle cells from spontaneously hypertensive rats. Am J Physiol Regul Integr Comp Physiol 2006;290(1):R73–8. REFERENCES 277

[90] Paravicini TM, Yogi A, Mazur A, Touyz RM. Dysregulation of vascular TRPM7 and annexin-1 is associated with endothelial dysfunction in inherited hypomagnesemia. Hypertension 2009;53(2):423–9. [91] Li Y, Jiang H, Ruan C, Zhong J, Gao P, Zhu D, et al. The interaction of transient receptor potential melastatin 7 with macrophages promotes vascular adventitial remodeling in transverse aortic constriction rats. Hypertens Res 2014;37(1):35–42. [92] Inoue R, Jian Z, Kawarabayashi Y. Mechanosensitive TRP channels in cardiovascular pathophysiology. Pharmacol Ther 2009;123(3):371–85. [93] Fajardo O, Meseguer V, Belmonte C, Viana F. TRPA1 channels: novel targets of 1, 4-dihydropyridines. Channels (Austin) 2008;2(6):429–38. [94] Go AS, Mozaffarian D, Roger VL, Benjamin EJ, Berry JD, Blaha MJ, et al. Heart disease and stroke statistics–2014 update: a report from the American Heart Association. Circulation 2014;129(3):e28–292. [95] Toescu EC. Hypoxia sensing and pathways of cytosolic Ca2+ increases. Cell Calcium 2004;36(3–4):187–99. [96] Aarts M, Iihara K, Wei WL, Xiong ZG, Arundine M, Cerwinski W, et al. A key role for TRPM7 channels in anoxic neuronal death. Cell 2003;115(7):863–77. [97] Lipski J, Park TI, Li D, Lee SC, Trevarton AJ, Chung KK, et al. Involvement of TRP-like channels in the acute ischemic response of hippocampal CA1 neurons in brain slices. Brain Res 2006;1077(1):187–99. [98] Fonfria E, Mattei C, Hill K, Brown JT, Randall A, Benham CD, et al. TRPM2 is elevated in the tMCAO stroke model, transcriptionally regulated, and functionally expressed in C13 microglia. J Recept Signal Transduct Res 2006;26(3):179–98. [99] Wei WL, Sun HS, Olah ME, Sun X, Czerwinska E, Czerwinski W, et al. TRPM7 channels in hippocampal neurons detect levels of extracellular divalent cations. Proc Natl Acad Sci USA 2007;104(41):16323–8. [100] Sun HS, Jackson MF, Martin LJ, Jansen K, Teves L, Cui H, et al. Suppression of hippocampal TRPM7 protein prevents delayed neuronal death in brain ischemia. Nat Neurosci 2009;12(10):1300–7. [101] Romero JR, Ridker PM, Zee RY. Gene variation of the transient receptor potential cation channel, subfamily M, member 7 (TRPM7), and risk of incident ischemic stroke: prospective, nested, case-control study. Stroke 2009;40(9):2965–8. [102] Butenko O, Dzamba D, Benesova J, Honsa P, Benfenati V, Rusnakova V, et al. The increased activity of TRPV4 channel in the astrocytes of the adult rat hippocampus after cerebral hypoxia/ischemia. PLoS One 2012;7(6):e39959. [103] Cook DJ, Teves L, Tymianski M. Treatment of stroke with a PSD-95 inhibitor in the gyrencephalic primate brain. Nature 2012;483(7388):213–17. [104] Weilinger NL, Maslieieva V, Bialecki J, Sridharan SS, Tang PL, Thompson RJ. Ionotropic receptors and ion channels in ischemic neuronal death and dysfunction. Acta Pharmacol Sin 2013;34(1):39–48. [105] Nilius B. Transient receptor potential TRP channels as therapeutic drug targets: next round! Curr Top Med Chem 2013;13(3):244–6. CHAPTER 16 Activating, Inhibiting, and Highjacking TRP Channels for Relief from Itch Lindsey M. Snyder,1,3,4 Huizhen Huang,1,3,5 Sarah E. Ross1,2,3,4,* 1Department of Neurobiology, University of Pittsburgh, Pittsburgh, PA, USA 2Department of Anesthesiology, University of Pittsburgh, Pittsburgh, PA, USA 3Pittsburgh Center for Pain Research, Pittsburgh, PA, USA 4Center for Neuroscience Research at the University of Pittsburgh, Pittsburgh PA, USA 5Tsinghua University School of Medicine, Beijing, China *Corresponding author: [email protected]

OUTLINE

Introduction 279 An Unexpected Role for TprV3: Mutations in TRPV3 Result in Itchy Lesions Which Sensory Neurons Signal Itch? 280 Found in Olmsted Syndrome 287 Activating TRP Channels to Block Itch 282 Conclusions 289 Inhibiting TRP Channels to Block Itch 284 References 289 Highjacking TRP Channels to Deliver Relief from Itch 286

INTRODUCTION

Itch is an unpleasant sensation that produces the desire to scratch [1]. Moreover, scratching relieves the itch, at least temporarily. This behavior—scratching in response to an aversive stimulus—is highly conserved across species. All mammals and birds show itch behavior. Fish will rub themselves against a rough surface to remove an irritant. Even fruit flies display

TRP Channels as Therapeutic Targets 279 © 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/B978-0-12-420024-1.00016-3 280 16. TRP CHANNELS AND ITCH site-directed grooming behavior in response to an irritating substance [2]. The conservation of this behavioral response across the animal kingdom underscores the idea that itch has an important protective function: itch triggers a behavior that removes harmful agents from the body’s surface in the short term, and because itch feels unpleasant, an organism learns to avoid itch-inducing situations in the long term. However, there are many pathological conditions for which itch is no longer protective. In such cases, itch becomes a chronic condition that significantly decreases the quality of life. Numerous diseases are associated with severe itch, including atopic dermatitis, liver disease, postherpetic itch, small fiber neuropathies, and even some cancers [3]. Pathological itch from these and other diseases can be just as debilitating as pain, and there is a great unmet need for new treatment options [4]. In many ways, itch is like pain—both are aversive sensations that evolved to protect us, and both are initially detected by similar (though likely distinct) subsets of primary afferents [5]. Indeed, the transformation from an aversive somatosensory stimulus in the periphery to the sensory percept of either pain or itch in the brain appears to involve parallel neural pathways comprised of peripheral neurons that detect pruritogens, the superficial dorsal horn of the spinal cord, the parabrachial nucleus, the dorsal raphe, the thalamus, and several regions of the cortex. Thus, functional magnetic resonance imaging performed on the brain of a person experiencing either pain or itch might be almost indistinguishable [6]. Indeed, we do not yet understand how itch and pain are differentially encoded in the nervous system. Nevertheless, we know that they must be because itch and pain feel qualitatively different and elicit distinct behavioral responses.

WHICH SENSORY NEURONS SIGNAL ITCH?

There is great interest in determining which sensory neurons mediate itch and how these afferents are different than those that are involved in signaling pain. Identifying such fibers has important therapeutic implications because this knowledge could potentially allow the development of selective therapies for pruritus. Several years ago, microneurography experiments in humans led to the long-awaited discovery of itch-specific sensory neurons—these fibers responded to histamine, and their activity corresponded to the sensation of itch [7]. However, there was no molecular marker that allowed us to identify these cells. (Histamine responsiveness on its own is insufficient to identify an itch fiber because histamine activates numerous sensory neurons, likely including those that are involved in signaling pain). Thus, although there was good evidence for the existence of itch-selective sensory afferents, their identity remained elusive. In the last few years, several groups have reported the discovery of markers that define itch-mediating sensory afferents, including gastrin releasing peptide (GRP), Mas-related G-protein receptor A3 (MrgprA3), and Naturietic Polypeptide b (Nppb) [8–11]. But these reports are somewhat contradictory, and so the identity of itch fibers remains controversial. Both GRP and Nppb are attractive candidates as potential markers of itch afferents because both of these peptides cause itch when injected intrathecally [9,10]. Thus, it stands to reason that primary afferents that release these peptides might be involved in itch. However, the idea that GRP is a marker of itch neurons has come under scrutiny of late. The analysis of GRP expression in dorsal root ganglia (DRG) using antibodies originally suggested that Which Sensory Neurons Signal Itch? 281

GRP is expressed in subsets of primary sensory afferents [9]. Yet, it has been difficult to corroborate these findings with methods that detect GRP mRNA, and the inability to detect GRP message in DRG sensory neurons has now called the specificity of the GRP antibody into question [12], though this subject remains controversial [13]. Rather, GRP mRNA is found in abundance in spinal interneurons, and so it seems likely that these spinal interneurons (rather than DRG neurons) are the main source of endogenous GRP in the spinal cord. Nppb, on the other hand, remains a potential marker for itch neurons. Nppb mRNA is clearly expressed in subsets of primary afferents, and Nppb is required for itch sensation [10]. What remains to be determined is the degree to which Nppb is a bona fide marker of itch neurons—in other words, one that marks all itch afferents and only itch afferents. The strongest evidence for a marker that defines a specific population of sensory afferents that are tuned for itch comes from the analysis of a small population of DRG neurons that coexpress the MrgprA3 and MrgprC11. These related GPCRs were originally discovered in a screen for genes that are selectively expressed in peptidergic afferents [14]. Subsequently it was found that these two proteins are receptors for pruritogens. Specifically, MrgprA3 is a receptor for chloroquine, whereas MrgprC11 is activated by Bam8-22 and SLIGRL [15,16]. Because MrgprA3 and MrgprC11 are activated by pruritogens, it was logical to infer that the neurons that express these receptors are itch afferents. However, direct evidence that MrgprA3/C11-expressing neurons mediate itch was only possible on the genetic targeting of this population. The selective expression of cre recombinase in these cells made it possible to visualize this population clearly and enabled loss- and gain-of-function (LOF and GOF) studies to rigorously test the role of these afferents in itch. Importantly, the ablation of the MrgprA3 population with diphtheria toxin significantly reduced (although did not completely eliminate) itch, and selective activation of the MrgprA3 population resulted in scratching behavior [11]. The human counterpart to these receptors is thought to be MrgprX1, which also responds to a broad array of pruritogens when ectopically expressed, but whether this gene is a marker for itch-mediating neurons in humans is unknown [15,17]. The genetic labeling of the MrgprA3-expressing population of sensory neurons in a mouse was an important advance because it allowed us to see itch fibers for the first time [11]. Intriguingly, this population of neurons coexpresses CGRP and IB4, two mostly nonoverlap- ping markers that are thought to define distinct subsets of C fibers, the so-called peptidergic and nonpeptidergic classes, respectively. Furthermore, the central terminals of the MrgprA3 population ramify within a very narrow band of lamina II within the superficial spinal cord (newly termed II middle) that is between the peptidergic and nonpeptidergic layers. Hence the identification of MrgprA3-expressing itch afferents has challenged the conventional classification scheme for sensory neurons by revealing the existence of yet a third subtype that mediates itch and shows intermediate properties with respect to neurochemical expression as well as the laminar distribution of its central terminals. Importantly, the peripheral targeting of these neurons is entirely consistent with a role for these cells in itch: these neurons exclusively target structures in which we feel itch, such as the skin, but not any other regions of the body such as muscle or internal organs. Furthermore, within the skin, these neurons show an extremely superficial pattern of innervation, with terminations in the stratum granulosum. The loss- and gain-of function studies along with the specialized distribution of MrgprA3- expressing cells provide very compelling evidence that MrgprA3-expressing neurons mediate itch. Nonetheless, it should be noted that these fibers likely represent just one of several subpopulations of itch afferents [13,18]. 282 16. TRP CHANNELS AND ITCH ACTIVATING TRP CHANNELS TO BLOCK ITCH

Although we are still sorting out identity of all of the subtypes of primary sensory afferents that mediate itch, there is good evidence that these fibers belong to a larger population of sensory neurons that express TRPV1. This idea implies that, although TRPV1-targeted therapies may not be specific, they should nevertheless be effective at blocking itch. In mice, chemical ablation of TRPV1-expressing sensory neurons caused an almost complete loss of itch behavior [19]. Similarly in humans, repeated topical application of capsaicin is commonly used as a treatment for itch [20]. For instance, in a double-blind trial, low-concentration capsaicin treatment (0.025%, four times daily) caused a significant decrease in pruritic psoriasis [21]. This treatment seems paradoxical, and in some ways it is, for the initial response to capsaicin is intense burning, stinging, and itch. However, when TRPV1-expressing sensory nerve fibers are exposed to repeated applications of TRPV1 agonists, TRPV1-expressing sensory neurons show retraction of peripheral processes and impaired signaling (Figure 16.1), an effect that has been termed defunctionalization [22]. More recently, the use of a high-concentration capsaicin treatment (8% capsaicin patch) has been developed [23]. The advantage to this approach is that a single application can cause the defunctionalization of TRPV1-expressing sensory neurons. In anecdotal reports, an 8% capsaicin patch was successfully used to treat notalgia paraesthetica [24]. In addition, the fact that capsaicin patches significantly reduce postherpetic neuralgia strongly suggests that such treatment will also be effective for pos therpetic itch [25]. However, the treatment of intractable itch with capsaicin, though it can be effective, is a long way from a perfect solution. The treatment itself (not surprisingly) can be quite painful—indeed, concentrated capsaicin is applied in the presence of a local anesthetic. Furthermore, on the retraction of TRPV1-expressing fibers, there is a multimodal loss in sensitivity, including pain, itch, and warm/hot temperatures, and so the treatment is not particularly specific for itch. For these reasons, capsaicin therapy for pruritus only makes sense if the itch is localized to a specific region of the skin. Finally, the effects of capsai cin treatment are temporary, lasting up to 12 weeks, whereas the peripheral terminals from TRPV1-expressing sensory neurons regenerate and reinnervate the skin. Despite these shortcomings, low-dose capsaicin treatment is commonly used for the treatment of itch [20], and the newer high-concentration formulas have the potential to be broadly effective for numerous types of localized, neuropathic itch. Activating TRPV1 is not the only strategy to combat itch—the other is via TRPM8. Many over-the-counter itch remedies contain menthol as an active ingredient. Now research from our own lab may have uncovered the underlying mechanism for how menthol inhibits itch [26]. Menthol is known to activate TRPM8, which is expressed on sensory afferents that convey cool [27]. Our work suggests that menthol acts as a counterstimulus that inhibits itch via the activation of a specific population of inhibitory neurons in the dorsal spinal cord, which we have termed B5-I neurons. In particular, we show that B5-I neurons, which function to inhibit itch, get direct input from TRPM8 afferents. In addition, we find that, whereas menthol inhibits itch in wild-type mice, this counterstimulus has no effect in mice that are lacking B5-I neurons. These data suggest that B5-I neurons are the cellular basis for the inhibition of itch by menthol. In this regard, menthol and scratching may be two types of counterstimulation that work via analogous spinal circuits. But activation of TRPM8 as a strategy to inhibit itch Activating TRP Channels to Block Itch 283

FIGURE 16.1 Defunctionalizing TRPV1-expressing neurons is likely to block itch. Under conditions of pathological itch, sensory afferents that respond to pruritogens are abnormally active. Topical application of capsaicin at high concentration (or multiple applications at a low concentration) causes excessive calcium influx into TRPV1- expressing sensory afferents in the skin resulting in mitochondrial dysfunction and subsequent retraction of peripheral processes and impaired signaling (defunctionalization). As a consequence, cutaneous TRPV1-expressing sensory neurons (which include but are not limited to itch-responsive afferents) are silenced for a period of up to 12 weeks while the nerve terminals repair and reinnervate the skin.

has a notable advantage over scratching because TRPM8 activation—unlike scratching—does not cause tissue damage that further exacerbates itch. These findings suggest that TRPM8 agonists are a promising therapy for pruritus and that they reduce itch by harnessing endogenous anti-itch circuits. 284 16. TRP CHANNELS AND ITCH INHIBITING TRP CHANNELS TO BLOCK ITCH

Numerous chemical agents cause itch, and recently we have begun to get a clearer picture of how they do so. Activated dermal mast cells release histamine and serotonin, which bind to receptors on sensory neurons and cause itch. Foreign materials from plants and insects often have protease activity that can cause itch [28]. For instance, spicules from the tropical plant cowhage contain a protease that activates PAR2, and proteolytic cleavage products including BAM8-22 and SLIGRL cause itch by activating MrgprC11 [15,16,28]. Often itch is part of an immune response that involves the release of numerous cytokines, such as interleukin 31 and TSLP, which act on cytokine receptors [29,30]. What these itch receptors have in common is that they are all metabotropic rather than ionotropic receptors—in other words, they are not sufficient to trigger an action potential by themselves. Instead, these metabotropic receptors need to be coupled through signaling pathways to a channel that allows current influx to support the generation of an action potential. Notably, in every case that has been examined in detail, it has been found that the channels that are activated downstream of itch receptors are TRP channels. In particular, H1 receptor for histamine is coupled to TRPV1 via phospholipase A2 [19,31]. Analogously, MrgprA3, MrgprC11, and TSPLR appear to be coupled to TRPA1, whereas IL-31R may be able to couple to either TRP channel [29,30,32]. These studies emphasize the important idea that TRPV1 and TRPA1 are more than just pain sensors. Rather, it is now emerging that these channels are integrators of diverse noxious stimuli, including those that induce sensations of itch (Figure 16.2). Whether antagonists for TRPV1 and TRPA1 can block itch in humans is currently unknown. Several TRPV1 antagonists have failed in clinical trials for the treatment of pain because, at least when delivered systemically, these antagonists caused dangerous increases in body temperature in some patients [35]. This hyperthermia is likely due an underappreciated role for TRPV1 in sensing and regulating temperature. But the disappointing outcome of this clinical trial does not rule out the possibility that TRPV1 (and possibly TRPA1) antagonists could be used safely for the treatment of localized itch. In particular, the topical application of a TRPV1 antagonist to a delimited area of affected skin would be unlikely to trigger hyperthermia that

FIGURE 16.2 Pruritogens activate metabotropic receptors that require TRPV1 and/or TRPA1 for signaling. Many pruritogens signal through G-protein coupled receptors (e.g., histamine, SLIGRL, Bam8-22) or cytokine receptors (e.g., IL-31, TSLP), which cannot, by themselves, trigger an action potential. In many instances, these receptors appear to couple to either TRPV1 or TRPA1, which allow current influx and provide the depolarization potential. (A notable exception is hydrogen peroxide-mediated itch, which may be due to its ability to activate TRPA1 directly [33,34]). Thus, TRP antagonists may block itch that is caused by numerous, diverse pruritogens. Inhibiting TRP Channels to Block Itch 285 is seen on systemic treatment with the drug. Thus, TRPV1 and TRPA1 antagonists remain an attractive candidate for the treatment of localized itch. Indeed, there is now good evidence for the idea that blocking TRPs—particularly TRPA1— may be an effective way to treat some types of pruritus based on the work from two groups who have used several different animal models of chronic itch. In particular, allergic contact dermatitis was modeled by repeated exposure of the mice to urishiol (allergen found in poison ivy, poison oak, and sumac) or the chemical allergen oxazolone, which caused itchy skin accompanied by edema formation and increases in skin thickness [36]. Dry skin-induced itch was modeled through the repeated application of acetone/ether followed by water (AEW model) to disrupt barrier integrity of the skin, resulting in spontaneous itch [37]. Importantly, irrespective of which model was used, in all these experiments, TRPA1 knockout mice consistently showed a significant decrease in scratching behavior as well as reduced severity of dermatitis relative to wild-type controls. Moreover, acute inhibition of TRPA1 with TRPA1 antagonists also reduced scratching behavior in these animal models of chronic itch. These findings raise the possibility that TRPA1 antagonists may reduce chronic pruritus in humans, just as they do in rodent models. Intriguingly, not only did TRPA1-deficient mice display lower dermatitis pathology relative to controls, but these mice also lacked changes in gene and protein expression observed in wild-type mice as a result of these models of dermatitis. Specifically, although mice lacking TRPA1 showed a normal systemic immune response, they did not show an induction of pro-inflammatory cytokines and peptides that is seen in a normal cutaneous inflammatory response [36]. These findings suggest that TRPA1 activation in primary afferent neurons not only regulates the transmission of itch sensation but also may play a role in regulating the expression of genes involved in the neurogenic inflammatory response in chronic itch conditions. Normally scratching relieves itch sensation, but in chronic conditions the itch sensation is unrelenting and only temporarily relieved by scratching. Excessive scratching causes damage of the skin and release of inflammatory mediators that further sensitizes cutaneous afferents in the skin involved in transmitting itch and hence amplify the itch sensation. Interestingly, the sensitization to allergens caused an increase in substance P gene expression and protein levels in the skin that was reduced in TRPA1−/− mice [36]. Substance P is a peptide released by local immune cells and cutaneous afferent nerve terminals in the skin and has been found to be up-regulated in the skin of atopic dermatitis patients and correlated with disease severity [38]. Substance P binds to its receptor, the neurokinin-1 receptor (NK1R), which is expressed on mast cells in the skin. This leads to release of inflammatory mediators that further sensitize afferent nerve terminals [5]. There is also some evidence the NK1R is expressed on cutaneous afferent nerve terminals [39,40]. An increase in substance P can intensify the itch signal by activating and sensitizing afferent terminals through either mechanism. This, in turn, increases the desire to scratch, thus perpetuating the cycle (Figure 16.3). Interestingly, an NK1R antagonist during treatment was found to significantly reduce dermatitis scores and scratching behavior [36]. This discovery suggests that TRPA1 channel activation may be a critical component of SP-NK1R-induced enhancement of primary afferent responses in chronic pruritus. Although these studies highlight the key role of TRPA1 in chronic itch, there is also some evidence for the involvement of TRPV1. In particular, in two models of allergic contact dermatitis (oxazolone and exposure to dust mite extract), it was found that antagonism of TRPV1 286 16. TRP CHANNELS AND ITCH

FIGURE 16.3 TRP-mediated neurogenic inflammation and itch. Local release of peptides such as Substance P may contribute to a pathological cycle of itch by triggering the release of pruritogens from immune cells, thereby activating nerve terminals causing yet more peptide release. Blocking TRP channels may halt this pathological cycle. reduced atopic dermatitis-like symptoms by accelerating skin barrier recovery [41]. For instance, mice treated with the TRPV1 antagonist PAC-14028, during sensitization to a dust mite allergen showed significantly reduced dermatitis scores and scratching behavior. These findings underscore the notion that TRPV1 activation may be involved in perpetuating the cycle of inflammation that is seen in dermatitis. In keeping with this idea, the chemical ablation of TRPV1 cells prevented psoriasis-like inflammation that is seen in response to imiquimod [42]. Thus, there is good evidence that inhibition of either TRPA1 or TRPV1 can reduce itch and dermatitis-like symptoms in animal models, but whether this finding will translate to humans remains to be addressed.

HIGHJACKING TRP CHANNELS TO DELIVER RELIEF FROM ITCH

The local anesthetic lidocaine is very effective at blocking itch—but it is not specific. Instead, this drug diffuses into all cells, blocks voltage-gated sodium channels, and prevents action potential propagation resulting in numbness. However, a minor modification of lidocaine results in a derivative, QX-314, that is positively charged and cannot pass through a lipid bilayer. When it stays outside the cell, QX-314 is without effect. However, on activation, TRP channels (particularly TRPV1, but also TRPA1) create large, cation-permeable pores that allow QX-314 access into the cell [43]. Thus, QX-314 selectively targets sensory neurons that have open TRP channels. AN UNEXPECTED ROLE FOR TPRV3 287

FIGURE 16.4 Highjacking TRP channels to deliver relief from itch. QX-314 is a cell impermeant sodium channel blocker that can only gain access to the cell upon entry through a large diameter channel such as a TRP channel. Thus, QX-314 has the potential to selectively inactivate neurons that are pathologically activated in chronic itch.

Importantly, active itch-sensing neurons likely belong to this class. Evidence for the idea that QX-314 can inhibit itch came from a recent study that used QX-314 as a tool to reveal the existence of two distinct populations of itch fibers [44]. In this clever experiment, Robertson et al. took advantage of the fact that metabotropic itch receptors couple to TRPV1 and/or TRPA1 for function. Thus, coadministration of histamine and QX-314 resulted in the opening and then silencing of TRPV1 channels on histamine-responsive sensory neurons. Because this population had been silenced, histamine could no longer cause itch when it was reapplied 30 minutes later. However, chloroquine—a pruritogen that is thought to act on a distinct population—was still able to cause itch in these mice. Conversely, the coadministration of chloroquine and QX-314 rendered chloroquine-responsive cells insensitive to a second chloroquine challenge, but these mice retained sensitivity to histamine. These experiments were used as evidence of the existence of two separate populations of itch-sensing neurons. However, the implications of this study are far greater, for this study showed that both histamine- dependent and histamine-independent itch can be silenced with QX-314. Moreover, by specifically silencing sensory neurons that are overactive under conditions of pathological pruritus, it may be possible to selectively target itch (Figure 16.4).

AN UNEXPECTED ROLE FOR TPRV3: MUTATIONS IN TRPV3 RESULT IN ITCHY LESIONS FOUND IN OLMSTED SYNDROME

Several years ago, a spontaneous dominant mutation occurred in the DS mouse strain. These mutant mice never grew hair and hence were termed DS-Nh for no hair. In addition, Nh mice developed dermatitis and showed abnormal scratching behavior indicative of itch [45–49]. A few years later, a dominant mutation in an inbred rat strain likewise resulted in the absence of fur and signs of dermatitis (named Ht for Hypotrochotic, having abnormal hair) [50,51]. Nh mice and Ht rats showed many features that were similar to atopic dermatitis in humans, including elevated levels of IgE and IL-4, increased numbers of mast and other immune cells, and the presence of Staphylococcus aureus in the skin. Through positional cloning, it was subsequently discovered that the hairless and dermatitis phenotypes in both the Nh mice and Ht rats were caused by an amino acid substitution in TRPV3 at Gly573 [52]. The amino acid substitutions at this site turned out to be a gain-of-function mutation, which sensitizes TRPV3 such that it is abnormally active [53]. TRPV3 is highly expressed in keratinocytes [54], 288 16. TRP CHANNELS AND ITCH where it plays an important role in keratinocyte function that supports skin barrier formation and hair growth [54–58]. These findings suggest that abnormally elevated TRPV3 activity in keratinocytes results in disruptions of the epidermal barrier, immune infiltration, and pathological itch, as well as the loss of fur (Figure 16.5). Now humans expressing variants in the TRPV3 gene have been discovered, and such variants cause a rare condition called Olmsted syndrome involving itchy skin lesions, particularly on the palms, soles, and face, as well as hair loss. The first described cases were also dominant, gain-of-function variants at Gly573, just as seen in rodents. However, subsequently other variants in TRPV3 have also been found to cause Olmsted syndrome [59–62]. Intriguingly, some involve variants in TRPV3 that are recessive in nature and are thought to result in loss of TRPV3 function [63,64]. Patients with recessive variants in TRPV3 also have thickened, lesioned skin on the soles of their feet that is associated with severe itch and some loss of eyelashes. These findings raise the possibility that gain- and loss-of-function variants in the TRPV3 channel result in overlapping phenotypes, suggesting that either too much or too little activity of this channel can result in skin barrier dysfunction that is accompanied by immune infiltration and itch at the site of lesions, as well as alopecia. What is not currently known is whether abnormal TRPV3 activity underlies other, more common types of dermatological itch. There is some precedent for this idea because TRPV3 mRNA is significantly up-regulated in the area of lesional skin of patients with atopic dermatitis [65]. Furthermore, the activation of TRPV3 in keratinocytes results in the release of numerous inflammatory mediators that could potentially trigger itch [66–69]. Recently, several small-molecule TRPV3 antagonists have been identified, but whether they inhibit itch remains to be seen.

FIGURE 16.5 Mutations in TRPV3 causing itch due to Olmsted syndrome (OS). Two dominant mutations (G573C and G573S) in TRPV3 were first discovered in rodents, where they caused atopic-dermatitis like symptoms, skin barrier dysfunction, abnormal hair growth, and itch. Since then, dominant gain-of-function (GOF) mutations at this site have been described in humans where they cause Olmsted syndrome, a rare skin disease that involves the thickening of the skin, barrier dysfunction, and itchy lesions as well as alopecia. Most recently, recessive mutations that likely result in loss of function (LOF) have also been found to cause this syndrome, suggesting that appropriate TRPV3 function—neither too much, nor too little—is essential for normal keratinocyte function to support skin and hair growth. Whether abnormal TRPV3 activity underlies more common forms of itch is unknown. REFERENCES 289 CONCLUSIONS

The knowledge that TRPV1 and TRPA1 are key molecular integrators of itch has raised the possibility that these channels may be a good therapeutic target for the treatment of pruritus. There are many possible ways that TRPs could be targeted—from activating, to inhibiting, and even highjacking these channels to silence itch-mediating primary afferents. Moreover, there are numerous new TRP agonists and antagonists that, though originally developed as analgesics, should be considered for the treatment of itch. Even TRP compounds that have failed when given systemically have the potential to be effective and safe for the treatment of localized pruritus.

References [1] Ross SE. Pain and itch: insights into the neural circuits of aversive somatosensation in health and disease. Curr Opin Neurobiol 2011;21(6):880–7. [2] Phillis RW, Bramlage AT, Wotus C, Whittaker A, Gramates LS, Seppala D, et al. Isolation of mutations affecting neural circuitry required for grooming behavior in Drosophila melanogaster. Genetics 1993;133(3):581–92. [3] Weisshaar E, Dalgard F. Epidemiology of itch: adding to the burden of skin morbidity. Acta Derm Venereol 2009;89(4):339–50. [4] Bathe A, Weisshaar E, Matterne U. Chronic pruritus–more than a symptom: a qualitative investigation into patients’ subjective illness perceptions. J Adv Nurs 2013;69(2):316–26. [5] Ikoma A, Steinhoff M, Stander S, Yosipovitch G, Schmelz M. The neurobiology of itch. Nat Rev Neurosci 2006;7(7):535–47. [6] Pfab F, Valet M, Napadow V, Tolle TR, Behrendt H, Ring J, et al. Itch and the brain. Chem Immunol Allergy 2012;98:253–65. [7] Schmelz M, Schmidt R, Bickel A, Handwerker HO, Torebjork HE. Specific C-receptors for itch in human skin. J Neurosci 1997;17(20):8003–8. [8] Sun YG, Zhao ZQ, Meng XL, Yin J, Liu XY, Chen ZF. Cellular basis of itch sensation. Science 2009;325(5947):1531–4. [9] Sun YG, Chen ZF. A gastrin-releasing peptide receptor mediates the itch sensation in the spinal cord. Nature 2007;448(7154):700–3. [10] Mishra SK, Hoon MA. The cells and circuitry for itch responses in mice. Science 2013;340(6135):968–71. [11] Han L, Ma C, Liu Q, Weng HJ, Cui Y, Tang Z, et al. A subpopulation of nociceptors specifically linked to itch. Nat Neurosci 2013;16(2):174–82. [12] Fleming MS, Ramos D, Han SB, Zhao J, Son YJ, Luo W. The majority of dorsal spinal cord gastrin releasing peptide is synthesized locally whereas neuromedin B is highly expressed in pain- and itch-sensing somatosensory neurons. Mol Pain 2012;8:52. [13] Liu XY, Wan L, Huo FQ, Barry DM, Li H, Zhao ZQ, et al. B-type natriuretic peptide is neither itch-specific nor functions upstream of the GRP-GRPR signaling pathway. Mol Pain 2014;10(1):4. [14] Dong X, Han S, Zylka MJ, Simon MI, Anderson DJ. A diverse family of GPCRs expressed in specific subsets of nociceptive sensory neurons. Cell 2001;106(5):619–32. [15] Liu Q, Tang Z, Surdenikova L, Kim S, Patel KN, Kim A, et al. Sensory neuron-specific GPCR Mrgprs are itch receptors mediating chloroquine-induced pruritus. Cell 2009;139(7):1353–65. [16] Liu Q, Weng HJ, Patel KN, Tang Z, Bai H, Steinhoff M, et al. The distinct roles of two GPCRs, MrgprC11 and PAR2, in itch and hyperalgesia. Sci Signal 2011;4(181):ra45. [17] Sikand P, Dong X, LaMotte RH. BAM8-22 peptide produces itch and nociceptive sensations in humans independent of histamine release. J Neurosci 2011;31(20):7563–7. [18] Liu Q, Sikand P, Ma C, Tang Z, Han L, Li Z, et al. Mechanisms of itch evoked by beta-alanine. J Neurosci 2012;32(42):14532–7. [19] Imamachi N, Park GH, Lee H, Anderson DJ, Simon MI, Basbaum AI, et al. TRPV1-expressing primary afferents generate behavioral responses to pruritogens via multiple mechanisms. Proc Natl Acad Sci USA 2009;106(27):11330–5. 290 16. TRP CHANNELS AND ITCH

[20] Papoiu AD, Yosipovitch G. Topical capsaicin. The fire of a ‘hot’ medicine is reignited. Expert Opin Pharmacother 2010;11(8):1359–71. [21] Ellis CN, Berberian B, Sulica VI, Dodd WA, Jarratt MT, Katz HI, et al. A double-blind evaluation of topical capsaicin in pruritic psoriasis. J Am Acad Dermatol 1993;29(3):438–42. [22] Anand P, Bley K. Topical capsaicin for pain management: therapeutic potential and mechanisms of action of the new high-concentration capsaicin 8% patch. Br J Anaesth 2011;107(4):490–502. [23] Capsaicin patch (Qutenza) for postherpetic neuralgia. Med Lett Drugs Ther 2011;53(1365):42–3. [24] Metz M, Krause K, Maurer M, Magerl M. Treatment of notalgia paraesthetica with an 8% capsaicin patch. Br J Dermatol 2011;165(6):1359–61. [25] Wallace M, Pappagallo M. Qutenza(R): a capsaicin 8% patch for the management of postherpetic neuralgia. Expert Rev Neurother 2011;11(1):15–27. [26] Kardon AP, Polgar E, Hachisuka J, Snyder LM, Cameron D, Savage S, et al. Dynorphin acts as a neuromodula- tor to inhibit itch in the dorsal horn of the spinal cord. Neuron 2014;82(3):573–86. [27] Knowlton WM, McKemy DD. TRPM8: from cold to cancer, peppermint to pain. Curr Pharm Biotechnol 2011;12(1):68–77. [28] Reddy VB, Iuga AO, Shimada SG, LaMotte RH, Lerner EA. Cowhage-evoked itch is mediated by a novel cysteine protease: a ligand of protease-activated receptors. J Neurosci 2008;28(17):4331–5. [29] Cevikbas F, Wang X, Akiyama T, Kempkes C, Savinko T, Antal A, et al. A sensory neuron-expressed IL-31 receptor mediates T helper cell-dependent itch: involvement of TRPV1 and TRPA1. J Allergy Clin Immunol 2014;133(2):448–60, e7. [30] Wilson SR, The L, Batia LM, Beattie K, Katibah GE, McClain SP, et al. The epithelial cell-derived atopic dermatitis cytokine TSLP activates neurons to induce itch. Cell 2013;155(2):285–95. [31] Shim WS, Tak MH, Lee MH, Kim M, Koo JY, Lee CH, et al. TRPV1 mediates histamine-induced itching via the activation of phospholipase A2 and 12-lipoxygenase. J Neurosci 2007;27(9):2331–7. [32] Wilson SR, Gerhold KA, Bifolck-Fisher A, Liu Q, Patel KN, Dong X, et al. TRPA1 is required for histamine- independent, Mas-related G protein-coupled receptor-mediated itch. Nat Neurosci 2011;14(5):595–602. [33] Liu T, Ji RR. Oxidative stress induces itch via activation of transient receptor potential subtype ankyrin 1 in mice. Neurosci Bull 2012;28(2):145–54. [34] Sawada Y, Hosokawa H, Matsumura K, Kobayashi S. Activation of transient receptor potential ankyrin 1 by hydrogen peroxide. Eur J Neurosci 2008;27(5):1131–42. [35] Brederson JD, Kym PR, Szallasi A. Targeting TRP channels for pain relief. Eur J Pharmacol 2013; 716(1–3):61–76. [36] Liu B, Escalera J, Balakrishna S, Fan L, Caceres AI, Robinson E, et al. TRPA1 controls inflammation and pruritogen responses in allergic contact dermatitis. Faseb J 2013;27(9):3549–63. [37] Wilson SR, Nelson AM, Batia L, Morita T, Estandian D, Owens DM, et al. The ion channel TRPA1 is required for chronic itch. J Neurosci 2013;33(22):9283–94. [38] Toyoda M, Nakamura M, Makino T, Hino T, Kagoura M, Morohashi M. Nerve growth factor and substance P are useful plasma markers of disease activity in atopic dermatitis. Br J Dermatol 2002;147(1):71–9. [39] Carlton SM, Zhou S, Coggeshall RE. Localization and activation of substance P receptors in unmyelinated axons of rat glabrous skin. Brain Res 1996;734(1–2):103–8. [40] von Banchet GS, Schaible HG. Localization of the neurokinin 1 receptor on a subset of substance P-positive and isolectin B4-negative dorsal root ganglion neurons of the rat. Neurosci Lett 1999;274(3):175–8. [41] Yun JW, Seo JA, Jang WH, Koh HJ, Bae IH, Park YH, et al. Antipruritic effects of TRPV1 antagonist in murine atopic dermatitis and itching models. J Invest Dermatol 2011;131(7):1576–9. [42] Riol-Blanco L, Ordovas-Montanes J, Perro M, Naval E, Thiriot A, Alvarez D, et al. Nociceptive sensory neurons drive interleukin-23-mediated psoriasiform skin inflammation. Nature 2014;510:157–61. [43] Binshtok AM, Bean BP, Woolf CJ. Inhibition of nociceptors by TRPV1-mediated entry of impermeant sodium channel blockers. Nature 2007;449(7162):607–10. [44] Roberson DP, Gudes S, Sprague JM, Patoski HA, Robson VK, Blasl F, et al. Activity-dependent silencing reveals functionally distinct itch-generating sensory neurons. Nat Neurosci 2013;16(7):910–18. [45] Haraguchi M, Hino M, Tanaka H, Maru M. Naturally occurring dermatitis associated with Staphylococcus aureus in DS-Nh mice. Exp Anim 1997;46(3):225–9. REFERENCES 291

[46] Hikita I, Yoshioka T, Mizoguchi T, Tsukahara K, Tsuru K, Nagai H, et al. Characterization of dermatitis arising spontaneously in DS-Nh mice maintained under conventional conditions: another possible model for atopic dermatitis. J Dermatol Sci 2002;30(2):142–53. [47] Yoshioka T, Hikita I, Asakawa M, Hirasawa T, Deguchi M, Matsutani T, et al. Spontaneous scratching behaviour in DS-Nh mice as a possible model for pruritus in atopic dermatitis. Immunology 2006;118(3):293–301. [48] Watanabe A, Takeuchi M, Nagata M, Nakamura K, Hirasawa T, Nakao H, et al. Spontaneous development of dermatitis in DS-Nh mice under specific pathogen-free conditions. Exp Anim 2003;52(1):77–80. [49] Watanabe A, Takeuchi M, Nagata M, Nakamura K, Nakao H, Yamashita H, et al. Role of the Nh (Non-hair) mutation in the development of dermatitis and hyperproduction of IgE in DS-Nh mice. Exp Anim 2003;52(5):419–23. [50] Asakawa M, Yoshioka T, Hikita I, Matsutani T, Hirasawa T, Arimura A, et al. WBN/Kob-Ht rats spontaneously develop dermatitis under conventional conditions: another possible model for atopic dermatitis. Exp Anim 2005;54(5):461–5. [51] Tani S, Noguchi M, Hosoda Y, Sugibayasi K, Morimoto Y. Characteristics of spontaneous erythema appeared in hairless rats. Exp Anim 1998;47(4):253–6. [52] Asakawa M, Yoshioka T, Matsutani T, Hikita I, Suzuki M, Oshima I, et al. Association of a mutation in TRPV3 with defective hair growth in rodents. J Invest Dermatol 2006;126(12):2664–72. [53] Xiao R, Tian J, Tang J, Zhu MX. The TRPV3 mutation associated with the hairless phenotype in rodents is constitutively active. Cell Calcium 2008;43(4):334–43. [54] Peier AM, Reeve AJ, Andersson DA, Moqrich A, Earley TJ, Hergarden AC, et al. A heat-sensitive TRP channel expressed in keratinocytes. Science 2002;296(5575):2046–9. [55] Imura K, Yoshioka T, Hikita I, Tsukahara K, Hirasawa T, Higashino K, et al. Influence of TRPV3 mutation on hair growth cycle in mice. Biochem Biophys Res Commun 2007;363(3):479–83. [56] Cheng X, Jin J, Hu L, Shen D, Dong XP, Samie MA, et al. TRP channel regulates EGFR signaling in hair morphogenesis and skin barrier formation. Cell 2010;141(2):331–43. [57] Yoshioka T, Imura K, Asakawa M, Suzuki M, Oshima I, Hirasawa T, et al. Impact of the Gly573Ser substitution in TRPV3 on the development of allergic and pruritic dermatitis in mice. J Invest Dermatol 2009;129(3):714–22. [58] Borbiro I, Lisztes E, Toth BI, Czifra G, Olah A, Szollosi AG, et al. Activation of transient receptor potential vanilloid-3 inhibits human hair growth. J Invest Dermatol 2011;131(8):1605–14. [59] Duchatelet S, Pruvost S, de Veer S, Fraitag S, Nitschke P, Bole-Feysot C, et al. A new TRPV3 missense mutation in a patient with Olmsted syndrome and erythromelalgia. JAMA Dermatol 2014;150(3):303–6. [60] Danso-Abeam D, Zhang J, Dooley J, Staats KA, Van Eyck L, Van Brussel T, et al. Olmsted syndrome: exploration of the immunological phenotype. Orphanet J Rare Dis 2013;8:79. [61] Lai-Cheong JE, Sethuraman G, Ramam M, Stone K, Simpson MA, McGrath JA. Recurrent heterozygous missense mutation, p.Gly573Ser, in the TRPV3 gene in an Indian boy with sporadic Olmsted syndrome. Br J Dermatol 2012;167(2):440–2. [62] Lin Z, Chen Q, Lee M, Cao X, Zhang J, Ma D, et al. Exome sequencing reveals mutations in TRPV3 as a cause of Olmsted syndrome. Am J Hum Genet 2012;90(3):558–64. [63] Duchatelet S, Guibbal L, de Veer S, Fraitag S, Nitschke P, Zarhrate M, et al. Olmsted syndrome with erythromelalgia caused by recessive TRPV3 mutations. Br J Dermatol 2014;171:675–8. [64] Eytan O, Fuchs-Telem D, Mevorach B, Indelman M, Bergman R, Sarig O, et al. Olmsted syndrome caused by a homozygous recessive mutation in TRPV3. J Invest Dermatol 2014;134:1752–4. [65] Bianchi P, Ribet V, Casas C, Lejeune O, Schmitt AM, Redoules D. Analysis of gene expression in atopic dermatitis using a microabrasive method. J Invest Dermatol 2012;132(2):469–72. [66] Mandadi S, Sokabe T, Shibasaki K, Katanosaka K, Mizuno A, Moqrich A, et al. TRPV3 in keratinocytes trans- mits temperature information to sensory neurons via ATP. Pflugers Arch 2009;458(6):1093–102. [67] Xu H, Delling M, Jun JC, Clapham DE. Oregano, thyme and clove-derived flavors and skin sensitizers activate specific TRP channels. Nat Neurosci 2006;9(5):628–35. [68] Huang SM, Lee H, Chung MK, Park U, Yu YY, Bradshaw HB, et al. Overexpressed transient receptor potential vanilloid 3 ion channels in skin keratinocytes modulate pain sensitivity via prostaglandin E2. J Neurosci 2008;28(51):13727–37. [69] Phelps CB, Wang RR, Choo SS, Gaudet R. Differential regulation of TRPV1, TRPV3, and TRPV4 sensitivity through a conserved binding site on the ankyrin repeat domain. J Biol Chem 2010;285(1):731–40. CHAPTER 17 Role of TRP Channels in Skin Diseases Mathias Sulk,1,2 Martin Steinhoff1,3,* 1Department of Dermatology, University of California San Francisco (UCSF), San Francisco, California, USA 2Department of Dermatology, University Hospital Münster (UKM), Münster, Germany 3Charles Institute for Translational Dermatology, University College Dublin (UCD), Dublin, Ireland *Corresponding author: [email protected]

OUTLINE

Introduction 294 TRPV1 is Important for Epidermal Barrier Formation 299 TRPV Subfamily 294 TRPV2 299 TRPV1 295 TRPV3 300 TRPV1 Mediates Neuronal The Keratinocyte as a “Forefront” Sensations in Response to of Neural Signaling: TRPV3’s Skin Stimuli 295 Role in Thermosensation, Nonneuronal Expression of Pain and Itch 300 TRPV1 in the Skin 295 TRPV3 and Skin Barrier Activation of TRPV1 Under Function, Keratinocyte Inflammatory Conditions Proliferation, Skin Homeostasis, and Its Role in Inflammatory and Wound Healing 301 Skin Diseases 296 TRPV3 and Hair Growth 301 Antiproliferative Effects of TRPV1 TRPV3 and Inflammation 302 in Hair Follicles and TRPV4 303 Keratinocytes 298

TRPV4 in the Skin—A Sensor The TRPC (Canonical) Family 308 and Nociceptor for Outside TRPC Channels are Expressed in Skin (and inside) Stimuli 303 Cells and in Skin-Innervating Nerves 308 TRPV4 and Inflammation 304 TRPC Channels are Involved in TRPV4 and Skin Barrier 304 Keratinocyte Differentiation and TRPV4 and Skin Cancer 305 Proliferation 309 TRPV5 and TRPV6 305 TRPMs 310 Neuronal TRPM Expression and Cold TRPA1 305 Sensation 310 Role of TRPA1 in Thermosensation, Nonneuronal TRPM-Expression: Mechanosensation, Skin Keratinocytes and Immune Cells 311 Sensitization, Pain, and Itch 306 TRPM Channels are Implicated in Nonneuronal Expression of TRPA1 Melanocyte Function and Malignant in the Skin 306 Melanoma 311 TRPA1 in Keratinocytes: Differentiation and Barrier Function 307 TRPML3 313 TRPA1 Plays a Major Role in Skin Summary 313 Inflammation 307 TRPA1 is Functional in Melanocytes 308 References 313

INTRODUCTION

Transient receptor potential (TRP) ion channels are a heterogeneous group of nonselective cation channels. They are expressed on many cells or structures in the skin such as sensory nerves, keratinocytes, melanocytes, immune cells, endothelial cells, and fibroblasts. Therefore, TRP channels play a significant role for normal skin function (e.g. skin barrier, temperature sensation), but moreover, are also crucial under pathologic conditions. An involvement of many TRP channels is suggested for a large variety of skin diseases, like inflammation, skin cancer, and hair disorders.

TRPV SUBFAMILY

The vanilloid (TRPV) subfamily of TRP channels consists of four nonselective cation channels (TRPV1, TRPV2, TRPV3, and TRPV4), which are modestly Ca2+-permeable and two highly Ca2+-selective channels (TRPV5 and TRPV6). The nonselective cation channels are expressed by sensory nerves (TRPV1, -2, -3) and skin cells (TRPV1, -2, -3, -4), and are mainly known for their activation by different temperatures, therefore defined as “thermo TRPs.” In addition, the two highly Ca2+-selective channels (TRPV5 and TRPV6) exist, which are major calcium transporters in epithelial cells, in particular TRPV5 in the kidney and TRPV6 in the intestine. As TRPV1-4 and TRPV6 are expressed in skin cells, they have been extensively studied for their role in skin homeostasis as well as diseases. TRPV Subfamily 295 TRPV1 Capsaicin, the pungent ingredient in hot chili peppers, activates the founding member among the TRPV-channels, TRPV1. After activation through capsaicin or elevated temperatures (∼42 °C), TRPV1 mediates pain [1] and leads to neurogenic inflammation [2]. Moreover, recently, a role in histamine-induced itch was described [3], and therefore, TRPV1 is considered to be a good target to treat pain or itch (see Chapters 5, 6, 8, and 16 within this book for further details). Classically, TRPV1 is expressed by sensory neurons of the dorsal root and trigeminal ganglia. However, because TRPV1 is also expressed in many nonneuronal tissues, including human skin and immune cells, a role of TRPV1 in skin diseases (in addition to painful and pruritic conditions) can be anticipated. TRPV1 Mediates Neuronal Sensations in Response to Skin Stimuli Because TRPV1 plays a pivotal role in pain perception, it was also described to mediate nociceptive side effects associated with different topical skin treatments. A common side effect of tacrolimus, clotrimazole, and retinoids is a burning sensation. Recently, tacrolimus was shown to have direct effects on cultured sensory neurons by regulating the phosphorylation of TRPV1, which might be an explanation for its transient burning sensation [4]. Moreover, using TRPV1-knockout (KO) mice, TRPV1 was shown to induce nocifensive behavior after intraplantar injections of clotrimazole [5] and retinoids [6]. Notably, TRPV1 is also assumed to play a role in sensitive skin, and therefore TRPV1- antagonism might help the stinging, burning, and tightness that patients report in various skin diseases such as eczema, lupus erythematodes, or rosacea, for example [7]. Because patients with rosacea often report neuronal sensations (in particular, burning pain), a role of TRPV1 in rosacea is assumed [8,9], where activation of TRPV1 by rosacea trigger factors (e.g., UV radiation, temperature changes, exercising, alcohol) might lead to neurogenic inflammation associated with flushing, prolonged, erythema, burning pain, and leukocyte recruitment. Thermosensation and thermal hyperalgesia are major roles of TRPV1 in human and murine skin. Initially, it was shown in vitro that temperatures around 42 °C activate TRPV1 [10]. Using TRPV1-KO mice, a role of TRPV1 in thermosensation and inflammation-induced thermal hyperalgesia was also demonstrated in vivo [1]. Notably, also other thermosensors, other than TRPV1, are considered to be involved in the response to noxious heat [11], such as TRPV2, for example. In line with these data, a study with human healthy volunteers using a TRPV1-antagonist also confirmed the role of TRPV1 in thermosensation and thermal hyperalgesia in human skin [12]. TRPV1 is also considered to play an important role in mediating pruritic stimuli and histamine-induced itch. Furthermore, it was shown to be differentially expressed in human pruritic skin diseases and that TRPV1-modulation might be helpful to improve itch. Indeed, TRPV1-antagonists improve itch in murine atopic dermatitis [13,14]. Accordingly, capsaicin treatment improved itch in patients with prurigo nodularis [15] and notalgia paresthetica [16]. In addition, an increased staining intensity of TRPV1 in keratinocytes and nerve fibers was found in prurigo nodularis [17]. Nonneuronal Expression of TRPV1 in the Skin As a classical neuronal receptor, TRPV1 is expressed in the peripheral (e.g., trigeminal and dorsal root ganglia; skin C and Aδ fiber afferents; Ref. [18]) as well as the central nervous system (e.g., spinal cord dorsal horn, area postrema; Ref. [10]). 296 17. ROLE OF TRP CHANNELS IN SKIN DISEASES

In the skin, nonneuronal cells are also considered to express TRPV1. The most abundant cell type in the epidermis is keratinocytes. An expression of TRPV1 was demonstrated in many studies in murine and human skin on the mRNA and protein level, but a functional role is still discussed because not all groups were able to demonstrate TRPV1 functionality using calcium assays [19]. This is underlined by a recent study demonstrating that not all antibodies against TRPV1 are specific, and that certain anti-TRPV1 antibodies produce unspecific staining in TRPV1-KO mice of mouse urothelium [20]. Within the epidermis, basal keratinocytes show a stronger immunostaining as compared to suprabasal keratinocytes [17,21]. A modulation of TRPV1 expression levels (on the protein and mRNA-level) was observed after UV-radiation of human skin. After using a twofold minimal erythema dose (MED) of ultraviolet A/ultraviolet B (UVA/UVB) radiation, epidermal TRPV1 mRNA and protein-level up-regulation was found [22], whereas down-regulation of TRPV1 mRNA was observed using a fivefold MED after UVC-radiation in whole skin [23]. This indicates a different modulation of TRPV1 mRNA in the epidermal and dermal compartment, dependent on the type and dosage of UV-irritation. In addition to epidermal keratinocytes, the inner and outer root sheath and infundibulum of hair follicles, differentiated sebocytes, sweat gland ducts, and the secretory portion of eccrine sweat glands express TRPV1 [17,21,24]. However, because of the unclear situation of the specificity of antihuman TRPV1 antibodies and the contradictory calcium assays, the exact role of a functional TRPV1 in those structures awaits further clarification. Notably, capsaicin was described to improve apocrine chromhidrosis [25], which may be at least in part explained via TRPV1 activation in human sweat gland epithelial cells, or the surrounding peripheral nerves, which regulate sweat gland function. Here, further studies are demanded to clarify the role of TRPV1 in the regulation of human skin appendices. Among other skin cells, melanocytes [26] and fibroblasts [27,28] have also been discussed to express TRPV1, albeit conflicting data exist [17,21]. Thus, further studies are also needed to clarify the role of TRPV1 on human melanocytes and fibroblasts. A potential role of extraneural TRPV1 on blood vessels is clearer, at least in mice [29]. Within the dermal compartment of the skin, expression of TRPV1 on vascular tissue (endothelium, smooth muscle cells) is assumed [21,30], and also a TRPV1-dependent release of vasoactive mediators by perivascular nerves indicates a role of TRPV1 in vasodilation. Supporting this hypothesis, it has been reported in vitro that TRPV1-activation on endothelial cells could mediate nitric oxide (NO) release [31], and moreover, in humans, capsaicin increased the blood flow and produced erythema [32]. These findings correlate well with the historical studies of Jancso and Szolcsanyi, who had already demonstrated the pivotal role of capsaicin of vasoregulation and neurogenic inflammation 45 years ago [33]. Finally, immune cells (mast cells [17,21], neutrophils [34], primary human CD4-T-cells [35], dendritic cells [36–38], and epidermal Langerhans cells [21]) have also been demonstrated to express TRPV1. However, based on the aforementioned problems with specific antibodies and assays, the functional relevance of TRPV1 in immune cells needs further exploration.

Activation of TRPV1 Under Inflammatory Conditions and Its Role in Inflammatory Skin Diseases The first described activators of TRPV1 were capsaicin (and its ultrapotent analogue resin- feratoxin) and temperature changes [33,39]. Later, also other substances or mediators have TRPV Subfamily 297 been demonstrated to activate TRPV1 that are ultimately involved in inflammation. In particular, TRPV1 is activated by low PH [40] and lipoxygenase products such as leukotriene B4 [41] and is sensitized by ATP [42], bradykinin [43], prostaglandins (PGE2, PGI2) [44], histamine [40], reactive oxygen species [45], proteases, and PAR2 [46]. Thus, various components of the “inflammatory soup” can potentially activate or sensitize TRPV1 signaling. This fact not only explains the role of TRPV1 in inflammatory hyperalgesia, but also suggests a critical role in modulating immune responses and therefore the development of inflammatory skin diseases in general. Neuronal TRPV1 also induces acute inflammatory conditions via a neurogenic mechanism by releasing calcitonin gene related peptide (CGRP) and substance P (SP) from sensory nerve endings, defined as neurogenic inflammation [2]. Less understood is the role of TRPV1 in chronic inflammatory conditions, and this awaits further clarification. Especially for chronic inflammatory diseases, the role of TRPV1 on nonneuronal skin cells (e.g., endothelial cells, keratinocytes, fibroblasts, immune cells) has to be considered, but awaits further clarification because of conflicting findings regarding a rather proinflammatory or anti-inflammatory role of TRPV1 in allergic contact dermatitis [13,47]. However, most studies suggest a rather pro-inflammatory effect of TRPV1 on nonneuronal skin cells. In vitro, it was demonstrated that TRPV1-activation of cultured keratinocytes with capsaicin induced the up-regulation of cyclooxygenase-2 (COX-2) and increased the release of proinflammatory mediators like interleukin 8 (IL-8) and prostaglandin E2 [48,49]. In outer root sheath keratinocytes, interleukin 1 beta was inducible by TRPV1 [24]. Moreover, TRPV1 mediates the UV- and heat-shock-induced up-regulation of matrix metalloproteinase-1 (MMP-1) on the protein and mRNA-level in cultured keratinocytes via a calcium-dependent mechanism [50–52]. In line with these data, capsaicin evokes the release of leukotriene B4 (but reduced PGE2 levels) in human keratinocytes and dermal fibroblasts [28]. In mast cells, capsaicin treatment induced the release of IL-4, but failed to mediate mast cell degranulation [53]. So far, conflicting results exist about TRPV1’s role in dendritic cell function where capsaicin was suggested to effect cell maturation (increased antigen-presenting and costimulatory molecules) or cell migration to the lymph nodes [38,54]. Of note, it was elucidated that nickel, which is associated with nickel-induced contact dermatitis, directly activates TRPV1 in vitro using HEK293 and CHO cells [55], which might also suggest a role for TRPV1 in dermatitis. Indeed, several in vivo studies demonstrated the proinflammatory involvement of TRPV1 in inflammatory skin diseases. For example, capsaicin enhanced allergic contact dermatitis in the guinea pig [56]. In hairless mice, TRPV1-antagonism reduced the UV-induced mRNA and protein expression levels of MMP-2, MMP-3, MMP-9, or MMP-13, and of the proinflammatory cytokines IL-1β, IL-2, IL-4, TNF-α, and COX-2 [57]. Formaldehyde (an inducer of inflammation and pain) provoked skin inflammation (edema and neurotrophin-expression) that was improved in TRPV1-KO mice [58]. Because formalin is an activator of TRPA1, and the receptors for formaldehyde in the setting of human skin are not exactly clear, this observation has to await further clarification in the human system. Of further notice, TRPV1-antagonism in NC/Nga-mice with atopic-like dermatitis diminished the degranulation of mast cells, attenuated the TH2 type-immune response (decreased serum IgG1-levels), and led to decreased scratching behaviors in mice [13]. Based on these findings, it was concluded that repeated capsaicin applications could improve inflammatory responses by depleting the release of pro-inflammatory neuropeptides 298 17. ROLE OF TRP CHANNELS IN SKIN DISEASES

(e.g., SP, CGRP) from TRPV1-positive sensory nerve endings. Indeed, topical capsaicin (4-6 times daily, for 2 weeks) decreased symptoms and inflammation in patients with prurigo nodularis [15], indicating an anti-inflammatory and antipruritic effect of continued TRPV1 stimulation in humans. In a mouse model of psoriasis, topical capsaicin application over 5 days reduced the expression levels of TNFα, NF-κB, IL-17, IL-23 and thus improved skin inflammation [59]. Therefore, an anti-inflammatory role of continued TRPV1 application can be assumed in addition to its proinflammatory role in acute (neurogenic) inflammation, which has its maximum 1 h after stimulation and decreases within 6 h. In vitro, TRPV1-activation appears to regulate cytokine function. In monocyte- derived dendritic cells, TRPV1 inhibited cytokine-induced dendritic cell differentiation and activation and thus suppressed phagocytotic activity, as well as pro-inflammatory cytokine secretion [36]. In sebocytes, capsaicin suppressed lipid synthesis and expression levels of the proinflammatory cytokine IL-1β, suggesting a beneficial effect of TRPV1-agonism in patients with acne vulgaris [60]. In addition, two further in vivo studies support the idea of an anti-inflammatory component of TRPV1 activation in certain skin diseases. Using TRPV1-KO mice, a protective role of TRPV1 in acute oxazolone-induced contact dermatitis was demonstrated where TRPV1-KO mice showed enhanced edema and increased levels of TNF-α, whereas the accumulation of immune cells was not changed. After comparison with NK-1-receptor- and CGRP-receptor KO mice, which showed a pro-inflammatory role for both neuropeptide receptors in acute contact dermatitis, it was concluded that TRPV1 might down-regulate the sensitivity to inflammatory stimuli in the skin under certain conditions [47]. Moreover, TRPV1-KO mice exhibited more severe ulceration after trichloroacetic acid-peeling, which may be due to decreased release of protective cytokines [61]. In summary, the aforementioned results indicate that neural TRPV1 modulates acute neurogenic inflammation (1-6 h). Under other acute inflammatory condition such as acute allergic contact dermatitis (2-3 days), TRPV1 may be pro- as well as anti-inflammatory. One possible explanation is species differences, the timing when the inflammation was measured (first hours vs. 2-3 days), and the models used (different stimuli and triggers). For example, TRPV1-KO mice showed normal edema formation and scratching behavior in the oxazolone-model of acute contact dermatitis [62]. Thus, TRPV1 is able to modulate acute inflammatory conditions, but conflicting data regarding a rather pro-inflammatory or protective role still exist. Less information exists about a potential role of TRPV1 in chronic inflammatory skin diseases. Notably, very recently a pivotal role of TRPV1 (and the sodium channel Nav1.8) has been demonstrated in a mouse model of psoriasis (through topical imiquimod-application). TRPV1 and Nav1.8 were able to modulate immune reactions by regulating dermal dendritic cell function [63]. Together, these preliminary results underline the importance of TRPV1- nociceptors in chronic inflammation [8]. However, further studies, especially in human disease states and inflammatory models of different acuity, are demanded to finally clarify the involvement of TRPV1 on neuronal and nonneuronal cells in inflammatory skin diseases.

Antiproliferative Effects of TRPV1 in Hair Follicles and Keratinocytes Because keratinocytes in the root sheath and infundibulum of human hair follicles express TRPV1 [17,21], a role for TRPV1 in hair growth was suggested. Indeed, in human cultured outer root sheath keratinocytes, TRPV1-activation enhanced the expression levels of TRPV Subfamily 299 hair-growth-inhibitory mediators/cytokines IL-1β or TGF-β2 and reduced the expression levels of certain hair growth promoters (hepatocyte growth factor, insulin-like growth factor-I, stem cell factor) [24]. Moreover, in organ-cultured human scalp hair follicles, capsaicin- treatment induced diminished hair shaft elongation and proliferation and induced apoptosis and thus hair follicle regression [24]. These in vitro findings were in line with an in vivo study using TRPV1-KO mice, which demonstrated a delay in the hair follicle cycling [64]. Thus, TRPV1-antagonism may be beneficial for the treatment of alopecia, whereas TRPV1-agonism might improve hirsutism. In accord with TRPV1’s antiproliferative role in hair follicle function, several studies also provide evidence for antiproliferative effects of TRPV1 activation in keratinocytes. Capsaicin induced apoptosis and suppressed proliferation in cultured human keratinocytes [24,65] and sebocytes [60]. Moreover, TRPV1-KO mice developed higher numbers of skin tumors, which was explained by the ability of TRPV1 to degrade the EGF (epidermal growth factor) receptor, a receptor that is markedly up-regulated in cancer cells [66]. Therefore, an antiproliferative effect of TRPV1 can be concluded. Consequently, TRPV1 agonism might help skin conditions with increased levels of proliferation (e.g., skin cancer, psoriasis), whereas chronic TRPV1 antagonism (e.g., as a treatment for chronic pain or itch) could potentially increase the risk for skin cancer. This, however, has to be thoroughly investigated in humans and mice. TRPV1 is Important for Epidermal Barrier Formation The skin serves as a water-impermeable barrier to prevent excessive water loss. As temperature or other physical or chemical factors could alter barrier function, a role of TRPV1 in epidermal barrier homeostasis was considered. Accordingly, it was found in vivo that TRPV1-agonism with capsaicin hampered barrier recovery after tape stripping of hairless mouse skin, whereas TRPV1-antagonism improved barrier recovery [67]. Moreover, TRPV1- antagonism led to improved transepidermal water loss, a reformation of the neutral lipid layer and normalization of loricrin and filaggrin expression levels after tape stripping in mice [14]. Therefore, TRPV1 may play a protective role for epidermal barrier formation.

TRPV2 TRPV2 was the second described member of the TRPV-Ion channel family. It is also modestly Ca2+ permeable and probably involved in certain skin diseases. Recently it was described that TRPV2 plays a role in innate immunity by regulating immune cell function (e.g., macrophages and mast cells), although TRPV2 was initially shown to be expressed on neuronal tissue (e.g., brain, dorsal root ganglia [DRGs]) [68–70]. TRPV2 is activated by extreme heat (>52 °C), and because immunostainings revealed colocalization with TRPV1 in DRGs [71], a role of TRPV2 as a thermosensor for noxious heat was postulated. On the other hand, a recent study denied a relevant functional role of TRPV2 for thermosensation in vivo by demonstrating that TRPV2-KO mice did not show any behavioral differences to noxious heat under normal or inflammatory conditions [11]. Of note, hypo- osmolarity [72] and cell stretching [73] also activated murine TRPV2, indicating a potential role of this channel in mechanotransduction. Because TRPV2 is colocalized with CGRP and SP on DRGs [18], an indirect role of TRPV2 in promoting neurogenic inflammation was assumed, albeit direct evidence is still lacking both in rodents and humans. 300 17. ROLE OF TRP CHANNELS IN SKIN DISEASES

However, TRPV2 has been implicated in inflammatory processes. Many immune cells express TRPV2, among them macrophages (including dendritic cells and epidermal Langerhans cells), mast cells, neutrophil granulocytes [34], and lymphocytes [74], although functional evidence is still lacking in several of those cells. Interestingly, recent studies concluded that TRPV2 is essential for mast cell and macrophage function. Indeed, TRPV2 was essential for phagocytosis [75], migration [76], and cytokine (TNFα, IL-6) production in murine macrophages [77]. In contrast, TRPV2 mediated degranulation and activation in murine [75] and human [76] cultured mast cell lines. Therefore, targeting TRPV2 may play a beneficial role in diseases in which macrophages and mast cells play a pivotal role. In line with these data, increased expression levels of TRPV2 were observed in dermal immune cells of patients with rosacea, which exhibits increased levels of macrophages and mast cells, beside Th1 lymphocytes [9]. Of note, keratinocytes of human skin were also shown to express TRPV2 [9,18,78], but the exact role of keratinocyte-derived TRPV2 remains unclear, and further studies are needed.

TRPV3 Another important TRPV channel implicated in many skin diseases is TRPV3 [79,80]. It is only modestly Ca2+-permeable, and high expression levels were found in human [81–83] and murine [84–86] keratinocytes. In addition to skin, TRPV3 was also found in other organs like tongue [87], mouse testis [88], cornea [89,90], colon [91], larynx [92], and inner ear [93] and neuronal tissue (brain, peripheral nerves), but only low—if any—expression in mouse DRGs [70,82,84,94]. In addition, B- and T-lymphocytes [95], as well as dermal dendritic cells [96], were described to express TRPV3.

The Keratinocyte as a “Forefront” of Neural Signaling: TRPV3’s Role in Thermosensation, Pain and Itch Keratinocytes build the outer layer of the skin and are able to integrate different stimuli. As TRPV3 is a member of the “thermo-TRPs,” keratinocyte TRPV3 was originally shown to be activated by innocuous temperatures (range from 31-39 °C) [84]. Recent studies [97], however, have questioned its originally believed role [98,99] as an important temperature sensor under physiological temperatures in murine skin. Furthermore, several chemical skin “sensitizers” activate TRPV3 such as camphor [98], carvacrol, thymol, or eugenol [87]. Keratinocyte TRPV3 is assumed to mediate their neuronal sensations via ATP [99] and PGE2 release that stimulate their corresponding receptors on primary afferent neurons [100]. Hence, TRPV3 was shown to play a role in pain under inflammatory conditions (mechanical hyperalgesia) [100–102]. Thus, a TRPV3 antagonist (in particular 17(R)-resolvin D1) may be used as an analgesic [103]. (See Chapter 11 for further details.) In addition, TRPV3 was reported to be involved in itch, another neuronal skin sensation. Animal studies showed that mice with a TRPV3 gain-of-function mutation (Gly573Ser) developed a pruritic dermatitis [88]. Thus, TRPV3-antagonism may be a future therapy for chronic histamine-independent itch [104,105]. (See Chapter 16 for further details.) TRPV Subfamily 301

TRPV3 and Skin Barrier Function, Keratinocyte Proliferation, Skin Homeostasis, and Wound Healing TRPV3 may also play a role in skin barrier function, keratinocyte proliferation, skin homeostasis, and wound healing. Recently, TRPV3 was shown to build a signaling complex with TGF-α/EGFR [106], two growth factors that regulate keratinocyte proliferation and differentiation [107], to modulate the activity of transglutaminases to induce keratinocyte terminal differentiation [108]. Therefore, a reduced activity of the TGF-α/EGFR-complex in TRPV3-KO mice resulted in a reduced transglutaminase-activity, reduced terminal differentiation, and thus impaired barrier integrity [106]. In HaCaT-keratinocytes, TRPV3 was sensitized by cholesterol, which has been suggested to control keratinocyte differentiation and to induce epidermal cornification [109,110]. Thus, TRPV3 may be critically involved in skin barrier function, although more conclusive human data are still lacking. A recent in vitro study reported that α-hydroxyl acids, which are extensively used as exfoliative substances in skin cosmetic products, mediate their high epidermal cell turnover via TRPV3. Thus, α-hydroxyl acids, in particular the mild glycolic acid, activate TRPV3 by intracellular acidification to induce apoptosis, which helps skin renewal [111]. In line with this data, it was demonstrated in human organ-cultured hair follicles and cultures of human outer root sheath keratinocytes that TRPV3 stimulation suppresses cell proliferation and induces keratinocyte apoptosis [83]. Furthermore, TRPV3 antagonism prolonged cell survival of thermally stressed cells in a primary human keratinocyte culture [78]. Of note, TRPV3 also plays a role in skin homeostasis, NO-metabolism, and wound healing. Activation of TRPV3 induced NO production in cultured keratinocytes, which facilitated keratinocyte migration and—in vivo—improved wound healing [112]. Based on the fact that NO regulates vascular tone and vasodilation, it can be hypothesized that NO released by keratinocytes via TRPV3-activation affects the vascular tone and mediates vasodilation and therefore erythema [113]. In line with this, functional TRPV3 was detected in endothelial cells, and carvacrol (a TRPV3 agonist) was able to induce vasodilation in cerebral arteries [114]. Together, these studies in humans and rodents imply a role for TRPV3 in skin vasodilation and erythema.

TRPV3 and Hair Growth As TRPV3 is highly expressed in human and rodent skin (hair follicle keratinocytes included), and TRPV3-KO mice and mice with a TRPV3-gain-of-function mutation exhibit an abnormal or defective hair morphology, a role of TRPV3 in hair growth has been implicated. Indeed, it was demonstrated that TRPV3 activation inhibits hair growth in human organ- cultured hair follicles and cultures of human outer root sheath keratinocytes. The stimulation of TRPV3 stopped hair shaft elongation, inhibited proliferation, and induced apoptosis and hair follicle regression [83]. These in vitro findings of an antiproliferative, inhibitory role of TRPV3 on hair growth were confirmed in two different in vivo models: (1) TRPV3 gain-of-function mutations are associated with spontaneous hairlessness [88,115]. Two rodent strains, in particular DS-Nh mice (bearing a TRPV3 Gly573Ser mutation) and WBN/Kob-Ht rats (bearing a TRPV3 Gly573Cys mutation; Ref. [115]) were described to express a gain-of-function mutation, where the TRPV3-channel is constitutively active 302 17. ROLE OF TRP CHANNELS IN SKIN DISEASES

[116] and, therefore, have a hairless phenotype. Notably, DS-Nh mice persisted in the anagen phase, whereas no regeneration phase (telogen) was observed [117]. (2) TRPV3-KO mice exhibit a wavy hair coat and curly whiskers, similar to the abnormal hair morphogenesis in mice with loss-of-function mutations in the genes for TGF-α/ EGFR [106,107]. Thus, hair-follicle derived TRPV3 might be a future target to treat hair disorders, and TRPV3 antagonism might improve certain forms of alopecia, whereas TRPV3 agonists might help patients with hirsutism.

TRPV3 and Inflammation Because keratinocytes link the body surface with the environment and, thus, are the first target cells for harmful stimuli from the “outside,” keratinocyte-derived TRPV3 may also be involved in skin barrier function and inflammatory processes. Because keratinocytes have been shown to express functional TRPV3, which can be activated by several external stimuli, a role of keratinocyte-derived TRPV3 in inflammation can be anticipated. However, the precise mechanism of how TRPV3 may regulate epidermal function in human disease is still unexplored. However, in vitro observations reported that murine keratinocytes release proinflammatory mediators after TRPV3-activation and, moreover, that TRPV3 is sensitized under inflammatory conditions. Indeed, after stimulation with a TRPV3-agonist (eugenol), mouse keratinocytes were shown to release the proinflammatory cytokine IL1-α, and that bradykinin, as well as histamine, sensitize TRPV3 [87]. Interestingly, it was demonstrated that primary mouse keratinocytes from transgenic mice that overexpress TRPV3 release PGE2 in response to TRPV3 stimulation [100]. In addition, in mouse keratinocyte and DRG coculture systems, keratinocyte-derived TRPV3 was involved in the release of ATP after heating [99], and ATP was demonstrated to reduce the sensitivity of TRPV3 to its agonists [118]. Furthermore, arachidonic acids, which regulate important inflammatory processes, were shown to potentiate TRPV3 channel function in mouse keratinocytes [119]. In vivo studies confirmed the proinflammatory role of TRPV3 in mice. As mentioned earlier, mice with a TRPV3-Gly573 mutation (gain-of-function; Ref. [116]) have a hairless phenotype, but also develop spontaneous dermatitis and show elevated mast-cell-numbers [115]. DS-Nh mice (TRPV3-Gly573Ser mutation) and WBN/Kob-Ht rats (TRPV3-Gly573Cys mutation) both show elevated serum IGE-levels [120,121]. In addition, DS-Nh mice show increased serum levels for IL-4 and IL-13, increased nerve growth factor (NGF) serum and tissue levels, and a severe scratching behavior and, therefore, were described as a potential model for atopic dermatitis [122,123]. In line with these data, Gly573Ser-transgenic mice had also significantly elevated levels of IgE, CCL11, CCL17, IL-13, IL-17, MCP-1, thymic stromal lymphopoietin, increased mast cell numbers, and higher NGF-release from keratinocytes [88,124]. Further studies showed that the Gly573Ser mutation might contribute to the development of hapten-induced dermatitis and increased dendritic cell responses [124,125]. Thus, TRPV3 may be also involved in adaptive immunity. Importantly, TRPV3 also appears to play an important role in human skin. In accord with the earlier described in vitro and in vivo findings, the first TRPV3-related skin “TRP channelopathy” was recently identified in humans. It is caused by mutations in the trpv3-gene in TRPV Subfamily 303 humans, defined as Olmsted syndrome. It is a rare congenital keratinizing disorder characterized by bilateral mutilating excessive epidermal thickening of the palms and soles (palmoplantar keratoderma) and periorificial keratotic plaques. Clinically, it is often heterogenic, and different mutations (not only in the trpv3-gene) underlie this skin disease. In addition to hyperkeratosis, alopecia and severe itching occur in most cases, sometimes infections, and, rarely, squamous cell carcinomas in the keratotic areas. It was recently found that Olmsted syndrome is caused by missense gain-of-function mutations in the trpv3 gene (Gly573Ser, Gly573Cys, Trp692Gly) [126,127], leading to elevated apoptosis of keratinocytes and, thus, skin hyperkeratosis. In addition, other point mutations, particularly Trp521Ser [128], G573Ala [129], and Leu673Phe [130] were detected, of which the G573Ala mutation was associated with dermal infections, eosinophilia, and elevated IgE-levels [129]. Moreover, the Leu673Phe mutation in the trpv3-gene caused acute flares of inflammation and erythromelalgia [130]. Thus, modulation and possibly antagonism of TRPV3 might be a future therapy for Olmsted syndrome or erythromelalgia. Importantly, TRPV3 is thought to play a role in other inflammatory skin diseases. In line with the assumption that DS-Nh-TRPV3 gain-of-function mice could be considered as a model for human atopic dermatitis, higher expression levels of TRPV3 were found in the skin of patients with atopic dermatitis [124]. In addition, TRPV3 was implicated in rosacea, a common chronic inflammatory skin disease in which increased gene expression levels for TRPV3 mRNA and enhanced immunoreactivity for TRPV3 were found [9]. In summary, TRPV3 plays an important role for many processes in the skin. As it is expressed by keratinocytes, which subsequently release inflammatory mediators on TRPV3 stimulation, TRPV3 may be an important epidermal receptor to “sense” “outside danger” during inflammation and to regulate epidermal barrier function. TRPV3 may also regulate neuronal stimuli such as pain or itch, although this is not fully understood in humans. Thus, TRPV3 may regulate skin homeostasis, barrier integrity, and hair growth, and TRPV3- stimulation results in the release of different factors from keratinocytes, which are able to initiate and maintain inflammation.

TRPV4 Like TRPV3, TRPV4 is also a nonselective cation channel, which is modestly permeable to Ca2+, activated by moderate heat (25-34 °C), endogenous inflammatory metabolites (e.g., arachidonic acid metabolites), and exogenous compounds (phorbol esters, like 4α-PDD) and is robustly expressed in murine and human skin. Mouse [86,131] and human [132,133] keratinocytes, as well as mast cells, macrophages, fibroblasts, vascular tissue [134,135], and Merkel cells [136] were shown to express TRPV4, albeit a functional role for most cells is unclear. In addition, TRPV4 was found to be expressed in the trachea, kidney, salivary gland [137], liver, heart [138], inner ear hair cells, and neuronal tissue (brain, sympathetic, and parasympathetic nerve fibers and only at low levels in DRGs) [70,137,139,140].

TRPV4 in the Skin—A Sensor and Nociceptor for Outside (and Inside) Stimuli As a member of the “thermo-TRPs” family, TRPV4 was described to be activated by moderate heat (25-34 °C) [134,140]. Indeed, mice lacking TRPV4 showed an altered temperature behavior under normal [141] and under inflammatory [132,142] conditions. Conflicting with these 304 17. ROLE OF TRP CHANNELS IN SKIN DISEASES findings, other studies did not observe a role of TRPV4 as a temperature sensor and regarded the differences in KO mice compared to wild-type mice rather as strain-dependent [97]. Interestingly, as TRPV4 is expressed in vascular endothelium and skin sensory nerve endings, and hypotonicity or osmotic changes activate TRPV4 [136,138,143], this channel may be involved in hypotonic stimulus-induced nociception [139]. In addition, a role for TRPV4 in mechanosensation and mechanical hyperalgesia is assumed. Recent observations reported that TRPV4 is capable of sensing mechanical stimuli [144,145] and that it is involved in mechanical hyperalgesia under inflammatory conditions [132,146] or after DRG-compression [147].

TRPV4 and Inflammation TRPV4 plays also a role in inflammation, as inflammatory mediators activate TRPV4. It is also found on CGRP-coexpressing sensory nerves [148] and on several immune cells. Indeed, inflammatory mediators (e.g., metabolides of arachidonic acid [135], PGE2 [139], and histamine [149]) and receptors involved in inflammation (e.g., PAR2 [146]) were shown to activate and/or sensitize TRPV4. Moreover, it was shown that mast cells [150,151] and macrophages [152] express functional TRPV4, whereas the expression of TRPV4 in human leukocytes [153] and dendritic cells [37] was demonstrated, but its functionality in these cells remains unclear. TRPV4 on keratinocytes was also described to be involved in inflammatory conditions. Stimulation of TRPV4 in a keratinocyte cell line caused the release of IL-8 indicating a role of TRPV4 in neutrophil recruitment [154]. An in vivo study performed in keratinocyte-specific TRPV4-KO mice demonstrated that epidermal TRPV4-KO leads to a reduced release of the proinflammatory cytokine IL-6 and consequently, diminished macrophage and neutrophil numbers after generating UVB- induced photodermatitis. Of importance, epidermal TRPV4 increased the concentration of the proalgesic and inflammatory mediator endothelin 1 (ET-1). Thus, TRPV4 and ET-1 may play a role in human acute photodermatitis (“sunburn”), indicating a role of keratinocyte-derived TRPV4 as a potential target for UVB-induced sunburn [132]. In addition to the aforementioned in vitro and in vivo studies, the role of TRPV4 in inflammation was also investigated in human skin. TRPV4-expression levels were increased in dermal cells of rosacea, a chronic inflammatory skin disease [9].

TRPV4 and Skin Barrier Recent studies report that TRPV4 plays an important role in mediating skin barrier integrity and accelerating barrier recovery. Initially, Denda et al. described a temperature- dependent change in barrier recovery after tape stripping of murine and human skin. Using TRPV4 agonists and antagonists, it was elucidated that TRPV4 elevates Ca2+ levels in mouse primary keratinocytes, and thus activation of TRPV4 may accelerate barrier recovery [67]. In line with this, using TRPV4-deficient mice, it was found that epidermal TRPV4 promotes the development and maturation of intercellular junctions by binding to β-catenin, an adaptor protein linking intercellular adhesion molecules (E-cadherin) and the cytoskeleton (actin fibers) [131,155]. In human cultured keratinocytes it was shown that TRPV4-activation was important for barrier formation by mediating Ca2+-influx and thus promoting cell-cell junction development to augment intercellular barrier integrity. Supporting this hypothesis in a translational setting, the same authors demonstrated ex vivo in human skin tissue that TRPA1 305

TRPV4-activation leads to accelerated barrier recovery after the disruption of the stratum corneum [133]. In human epidermal keratinocytes, TRPV4-activation strengthened the tight junction- associated barrier, measured by an increased transepithelial electric resistance, and up- regulated tight junction-structural proteins (occludin and claudin-4) [156]. In summary, keratinocyte TRPV4 appears to be important for skin barrier integrity and contributes to cell-cell junction development, which prevents excess skin dehydration.

TRPV4 and Skin Cancer Due to its expression on keratinocytes, it was recently found that TRPV4 might also play a role in nonmelanoma skin (NMS) cancer [154]. Immunohistochemical studies with human skin samples of NMS cancer showed that TRPV4 expression was down-regulated in malignant lesions. Therefore, TRPV4 might serve as a “biomarker” for skin certain NMS cancers [154] and, similar to protease-activated receptor 2 (PAR-2), may be a negative regulator during carcinogenesis [157].

TRPV5 and TRPV6 Within the TRPV channel subfamily, TRPV5 and TRPV6 are exemptions regarding their Ca2+ permeability. Whereas TRPV1-V4 are only modestly permeable to calcium, TRPV5 and TRPV6 are highly Ca2+ selective and, therefore, are major calcium transporters in epithelial cells. In particular, TRPV5 appears to play an important role in the kidneys and TRPV6 in the intestine [158]. Moreover, TRPV6 is also expressed by keratinocytes [159,160], whereas a role for TRPV5 in human skin is unknown. Calcium homeostasis is important for keratinocyte proliferation, differentiation, and barrier function [158]. As TRPV6 is a selective calcium channel, it was shown in vitro that silencing of TRPV6 in human primary keratinocytes decreased the calcium-induced expression of keratinocyte differentiation markers (involucrin, transglutaminase-1, cytokeratin-10). Moreover, TRPV6 was demonstrated to mediate, at least in part, the pro-differentiating effects of vitamin D3. Thus, it is concluded that TRPV6 is essential for calcium- and/or vitamin D3-mediated keratinocyte differentiation and, therefore, also plays a role in promoting skin barrier function [160]. Moreover, TRPV6 may be involved in skin repair and in wound healing [161]. In line with these in vitro data, it was shown in vivo that deletion of the trpv6 gene in mice leads to decreased skin calcium levels, thinner layers of stratum corneum and in 20% of animals to alopecia and dermatitis [159]. In summary, TRPV6 might be important for skin barrier function, keratinocyte proliferation, and differentiation and, therefore, could be a promising target in inflammatory responses, hair growth disorders, and wound healing.

TRPA1

A number of endogenous and exogenous molecules, which act in and on the skin, activate TRPA1 (TRP-ankyrin) to mediate inflammation, pain, and itch. 306 17. ROLE OF TRP CHANNELS IN SKIN DISEASES Role of TRPA1 in Thermosensation, Mechanosensation, Skin Sensitization, Pain, and Itch Similar to TRPV1 to V4, TRPA1 was believed to be activated by temperature changes and thus to mediate thermosensation. Initially, it was described that TRPA1 is activated by temperatures <17 °C, which resembles the threshold for noxious cold in humans [162]. This is supported by findings that TRPA1 is colocalized with TRPV1 on DRGs [163]. Thus, TRPA1 was linked to the detection and sensation of noxious cold [164]. On the other hand, TRPA1 was not required for the initial detection of noxious cold in TRPA1-KO mice [165]. Therefore, it was speculated that TRPA1 mediates cold hypersensitivity (and is activated by cold temperatures) only under pathological conditions, for example, when endogenous activators [sensitizing inflammatory mediators] are present [166]. However, highly conflicting results exist with regard to the heat activation of TRPA1 [164,167], which could in part be explained by species differences [168]. For example, it was shown that cold activates rat and mouse TRPA1 but not human or rhesus monkey TRPA1 [168]. Consequently, the exact role of TRPA1 in the detection of noxious cold in various species remains controversial. In addition to temperature, mechanical stimuli might also be able to activate TRPA1 in the skin [169]. This is underlined by the finding that TRPA1 was expressed in structures of the skin that mediate mechanosensation: for example, it is present in the mechanopercep- tive nerve endings in epidermis and around Meissner corpuscles [170]. Therefore, a role for TRPA1 in mechanosensation [171–174] and mechanical hyperalgesia was postulated [175]. Conflicting results obtained with TRPA1-KO mice, however, did not confirm the activation of TRPA1 by mechanical stimuli and questioned the postulated role for TRPA1 in mechanosensation and mechanical hyperalgesia [165]. Thus, TRPA1’s role in mechanosensation and mechanical hyperalgesia needs further clarification in various species. Many skin sensitizers, pungent natural products, and irritant substances activate TRPA1 and are able to produce inflammation and pain. In particular, cinnamaldehyde (active ingredient in cinnamon), isothiocyanates (pungent substances in mustard oil, horseradish, and wasabi), allicin (garlic), and formalin (skin irritant) have been shown to activate TRPA1 [165,176]. Moreover, TRPA1 is activated by environmental chemicals such as formalin [177] and cigarette smoke [178–180], which contain abundant aldehydes. Thus, TRPA1 might also be implicated in the skin ageing effects of cigarette smoke. Skin sensitizers are capable of producing pain, which may also be mediated by TRPA1. Indeed, TRPA1 plays an important role in nociception [162]: Activation of TRPA1 produces pain behavior in mice [165], and recently a gain-of-function mutation in the human TRPA1 gene has been linked familial episodic pain syndrome [181]. (See Chapter 9 for further details.) In addition to nociception, TRPA1 is also considered to be important in the sensation and perception of itch. In particular, TRPA1 mediates histamine independent itch and is necessary for chronic itch, which might suggest TRPA1 as a good target for novel therapies against pruritic skin diseases [182–185]. (See Chapter 16 for further details.)

Nonneuronal Expression of TRPA1 in the Skin In mice, TRPA1 is predominantly found in TRPV1-expressing (CGRP- and SP-positive) nociceptive (DRG and TRG) neurons [163] that innervate the skin and terminate around TRPA1 307

Meissner corpuscles [170]. Human peripheral nerves also express TRPA1 [186,187]. Moreover, nonneuronal cells in the skin also express TRPA1, including both murine [170,188] and human [28,154,186,189] epidermal and hair follicle keratinocytes. In addition, up-regulated TRPA1 was found in keratinocytes of lesional skin from patients with atopic dermatitis [187] and solar keratosis [154]. However, the expression of functional TRPA1 in keratinocytes is currently debated because conflicting results exist [62,163]. Immune cells also express TRPA1. In a murine atopic dermatitis model, a colocalization of TRPA1 was found with eosinophils, Langerhans cells, macrophages, T cells, and mast cells [187]. Furthermore, endothelial cells [190], melanocytes [189,191], and fibroblasts [28,189] were found to express TRPA1. Of note, the specificity of the antimurine and antihuman TRPA1 antibodies is still a matter of debate. In addition, clear functional data for TRPA1 in human cells is still lacking. That said, the activation of TRPA1 on murine endothelial cells results in vasodilation [190], which might indicate a role of TRPA1 in erythema [28].

TRPA1 in Keratinocytes: Differentiation and Barrier Function A role for TRPA1 in keratinocyte proliferation, differentiation, and barrier function has been proposed. TRPA1 activation was demonstrated to evoke increased calcium levels in undifferentiated keratinocytes [192]. After activation of TRPA1 in primary human keratinocytes, genes involved in keratinocyte differentiation and proliferation (e.g., heat shock proteins, cyclins, and cyclin-dependent kinases) showed changes [189]. In line with these data, a recent study found that TRPA1 staining was increased in solar keratosis [154]. Following tape stripping of mice skin, TRPA1 activation was demonstrated to accelerate skin barrier recovery and increase lamellar body secretion [188], implicating a protective role for TRPA1 in skin barrier function.

TRPA1 Plays a Major Role in Skin Inflammation In addition to its role in “sensing” environmental skin sensitizers and irritants, TRPA1 can be also activated under inflammatory conditions by endogenous trigger factors. For example, endogenous pro-inflammatory and nociceptive mediators (prostaglandins, bradykinin, proteases, oxidative stress; Refs. [162], [179], [193], [194]) were shown to activate and sensitize TRPA1. Moreover, TRPA1 appears to be up-regulated under inflammatory conditions [187,195]. Activation of TRPA1 results in inflammation via the release of SP and CGRP by sensory nerve endings (neurogenic inflammation) or by the activation of TRPA1 on nonneuronal cells. It was shown in vitro that the activation of TRPA1 on keratinocytes results in the release of IL1-alpha, IL1-beta [189], and PGE2 [28]. In vivo, activation of TRPA1 with cinnamaldehyde evokes ear edema (SP-dependent) and leukocyte infiltration, which are prevented by specific TRPA1 antagonists [196]. Thus, chemicals that activate TRPA1 are able to enhance acute contact dermatitis induced by fluorescein isothiocyanate (FITC) and increase ear swelling and the migration of dendritic cells [197]. Also, desensitizing TRPA1 by application of topical allyl isothiocyanate leads to a reduced ear edema and inhibited dendritic cell trafficking and maturation [198]. Moreover, substances, which induce acute contact dermatitis, are also able to directly activate TRPA1. For example, it was shown that oxazolone directly activates TRPA1 and that TRPA1 is a major regulator of 308 17. ROLE OF TRP CHANNELS IN SKIN DISEASES inflammatory responses in contact dermatitis [62]. In acute (oxazolone-induced) contact dermatitis, TRPA1-KO mice showed decreased ear edema and leukocyte infiltration, diminished CXCL2, IL-4 and IL-6 cytokine levels, and reduced scratching behavior [62]. In chronic, atopic-like dermatitis induced by repeated oxazolone challenges, TRPA1-KO mice displayed less severe skin dermatitis and reduced levels of NGF, SP, and 5-HT [62]. In line with these findings, a role of TRPA1 in atopic dermatitis (caused by transgenic IL-13- production in mouse skin keratinocytes) was indicated. In these mice, an increased number of TRPA1-positive nerve fibers and mast cells was observed. Moreover, TRPA1 was shown to mediate calcium signals in a cultured mast cell line [187]. Whether the in vitro obtained calcium concentrations are sufficient to play a biological role in humans in vivo is currently under investigation. In addition, in human atopic dermatitis patients, an enhanced expression of TRPA1 was found in keratinocytes and dermal cells (including mast cells) as compared to healthy patients [187], indicating an essential role of TRPA1 in inflammatory and pruritic skin diseases (including atopic dermatitis).

TRPA1 is Functional in Melanocytes As melanocytes were shown to express TRPA1, a functional role of this ion channel in photo-transduction was assumed. This hypothesis was strengthened by findings that UVA evoked typical TRPA1 currents in electrophysiological recordings in a HEK293-cell line. Consequently, indirect activation of TRPA1 via the generation of oxidative stress was hypothesized [199]. Moreover, TRPA1 activation is able to increase intracellular calcium levels in melanoma cell lines, and TRPA1 was shown to be functional in melanocytes [200]. In accord, it was demonstrated that physiological doses of UVA radiation can induce retinal-dependent G protein-coupled calcium responses, which activate TRPA1 in human epidermal melanocytes. This provides evidence that TRPA1—together with calcium release from intracellular stores [201]—mediates extraocular phototransduction in melanocytes, which results in melanin synthesis [191].

THE TRPC (CANONICAL) FAMILY

The seven members (TRPC1-C7) of the modestly calcium-permeable TRPC family by sequence homology could be subdivided into two groups: (1) TRPC1/C4/C5 and (2) TRPC3/ C6/C7. (Of note, TRPC2 is only a pseudogene in humans [202,203]). Moreover, heteromultimeric TRPC-channel formations for TRPC1/TRPC4/TRPC5 and TRPC3/TRPC6/TRPC7 have been described [204]. Because TRPC channels are modest calcium permeable, they are assumed to contribute to receptor-operated Ca2+ entry (activation of TRPC3, TRPC6, TRPC7 in a diacylglycerol (DAG)-dependent mechanism), and also a role in store-operated Ca2+- entry has been discussed for TRPC1, TRPC4, TRPC5 (activation via PLC) [203,205].

TRPC Channels are Expressed in Skin Cells and in Skin-Innervating Nerves In the skin, TRPC channels (TRPC1, TRPC3, TRPC4, TRPC5, and TRPC6) are expressed by keratinocytes [206], where they are considered to be involved in keratinocyte differentiation The TRPC (Canonical) Family 309 and proliferation. In addition, fibroblasts [207] and immune cells were described to express TRPC channels. In particular, B-cells express TRPC7 [208], whereas primary human CD4+ T-cells express TRPC1 and TRPC3 [35]. Vascular tissue (endothelium and vascular smooth muscle cells) also expresses TRPC: these channels are thought to modulate vascular function, implying a role for TRPC channels in erythema and edema formation (reviewed in [205]). Some TRPC channels (TRPC1, TRPC3, TRPC4, and TRPC5) are expressed on DRG and trigeminal ganglia (TG) neurons (70). TRPC5 was shown to be expressed on peripheral intraepidermal nerve endings involved in the sensation of cold temperatures [209]. Another TRPC family member, TRPC1, was demonstrated to mediate (directly or indirectly) mechanical stimuli in cultured DRG neurons [210], which was strengthened by similar findings using TRPC1-deficient mice [211].

TRPC Channels are Involved in Keratinocyte Differentiation and Proliferation All TRPC channels (TRPC1 to TRPC7) are expressed on keratinocytes [206,212], where they are involved in calcium homeostasis. As calcium is a major regulator of the epidermal keratinocyte turnover, several studies indicate a role of TRPC channels in keratinocyte differentiation and proliferation. TRPC1 expression was described in human keratinocytes [213], and an involvement in store-operated calcium signaling in human cultured primary keratinocytes has been suggested [206]. Moreover, it is expressed in human gingival keratinocytes, and it was shown that TRPC1 mediates calcium-induced keratinocyte differentiation, as indicated by decreased involucrin levels after TRPC1 knockdown using siRNA [214]. Increased TRPC1 signaling was shown in Darier’s disease [215]. In this human skin disease, loss-of-function mutations in the gene (ATP2A2) that encodes SERCA2 lead to impaired keratinocyte differentiation, increased proliferation, and lower rates of apoptosis [216]. By a combination of immunohistochemistry and Western blots, it was demonstrated that TRPC1 protein was overexpressed in keratinocytes of patients with Darier’s disease and also in SERCA2+/− mice. Therefore, Darier’s disease keratinocytes show an increased calcium signaling and, thus, an increased proliferation rate (as compared to normal keratinocytes). Moreover, TRPC1 overexpression in a keratinocyte cell line promotes cell survival by inhibiting apoptosis (higher levels of antiapoptotic proteins) indicating that TRPC1 signaling might be a key factor for increased proliferation in Darier’s disease [215]. TRPC4 shows a high basal expression in differentiated keratinocytes [213,217] and was described to be involved in store-operated calcium signaling in human cultured primary keratinocytes [206,217]. Based on these findings, TRPC1 and TRPC4 have been implicated in the pathomechanism of basal cell carcinoma. Indeed, basal cell carcinoma cells did not express TRPC1/TRPC4, which might explain the lack of differentiation in these tumor cells [218]. In vitro and ex vivo, TRPC6 mediates calcium signals and promotes keratinocyte differentiation (keratin 10 expression). In addition, in human skin, triterpenes induced the up- regulation of TRPC6 in keratinocytes. Moreover, triterpenes improve actinic keratosis as indicated by up-regulation of the differentiation marker keratin 10. Thus, TRPC6 promotes keratinocyte differentiation and modulates keratinocyte proliferation, and therefore, TRPC6- activation might be helpful for actinic keratosis [213]. These findings were confirmed by 310 17. ROLE OF TRP CHANNELS IN SKIN DISEASES

another study [219], where keratinocyte-TRPC6 was studied in vitro and ex vivo. It was shown that a specific TRPC6-activator (hyperforin) evoked TRPC6-mediated calcium influx, and TRPC6 activation subsequently induced keratinocyte differentiation and inhibited proliferation. Therefore, a role of TRPC6 in chronic skin diseases with impaired keratinocyte differentiation and proliferation like psoriasis and atopic dermatitis can be assumed [219,220]. Furthermore, TRPC6 was demonstrated to be essential for the transdifferentiation from fibroblasts into myofibroblasts, and therefore, it might play a role in wound healing and fibrotic diseases [207]. In vitro, cultured fibroblasts from TRPC6 KO mice did not transform into myofibroblasts after TGF-β treatment and in vivo, TRPC6 KO mice showed impaired wound healing [207]. TRPC7 was shown to be involved in DAG-evoked calcium signaling in HaCaT human keratinocytes [212]. However, further in vivo and studies in primary cells are demanded to clarify its role in human disease. In line with the aforementioned studies that strongly suggest involvement of TRPCs in cell differentiation, proliferation, and apoptosis, it was demonstrated that TRPC channel expression is decreased in patients with psoriasis [221]. In accord, psoriatic keratinocytes show impaired calcium homeostasis (reduced calcium influx). Conversely, TRPC6 activation nor- malizes differentiation and proliferation levels of psoriatic keratinocytes [221]. Clearly, further studies are needed to clarify the specific role of each TRPC channel in keratinocyte proliferation and differentiation. Apparently (and somewhat confusingly), TRPC1 plays different role in Darier’s disease (TRPC1-overexpression might be pro-proliferative; Ref. [215]) and psoriasis (TRPC1-down-regulation might be pro-proliferative; Ref. [221]).

TRPMs

The group of TRPM channels consists of eight members. Based on their amino acid sequence, they could be subdivided into three groups, namely TRPM1/M3, TRPM4/M5, and TRPM6/7. The remaining two family members, TRPM2 and TRPM8, exhibit only low sequence homology and are therefore ungrouped [202]. TRPM channels are nonselective cation channels with specificity for monovalent (e.g., TRPM4, TRPM5) or divalent (TRPM6, TRPM7) cations [203]. Interestingly, TRPM4 is activated by calcium and can modulate cacium entry into the cells by regulating membrane potential [222]. TRPM channels have been implicated in taste sensation, magnesium-homeostasis, and detection of cold.

Neuronal TRPM Expression and Cold Sensation TRPM channels are expressed on neuronal tissue like DRGs (TRPM2, TRPM3, TRPM4, TRPM5, TRPM6, TRPM7, TRPM8) [70], as well as in epidermal and dermal nerve fibers where TRPM colocalizes with CGRP and SP [18]. Two members of the TRPM family have been implicated in temperature sensation: TRPM8 is activated at temperatures below 25 °C and therefore serves as a “cold sensor” [176] whereas TRPM3 is considered as a sensor for nociceptive heat [223]. Skin-sensitizing and cooling agents like menthol and icilin also activate TRPM8. In line with its role as a “cold sensor,” TRPM8-KO mice show behavioral deficits in response to cold TRPMs 311 stimuli [176]. Cold is able to alleviate pain and itch, and TRPM8-activation was shown to modulate nociception and to mediate analgesia in mice [224]. The burning sensation caused by clotrimazole [a treatment of yeast infections of the skin] was explained by its antagonism at TRPM8 [5].

Nonneuronal TRPM-Expression: Keratinocytes and Immune Cells In the skin, TRPM ion channels are expressed on melanocytes and in malignant melanoma (TRPM1, TRPM2, TRPM7, TRPM8) [225]. Keratinocytes and different immune cells (T cells, mast cells, granulocytes, and monocytes) express these ion channels, making them a potential target for therapies in skin diseases [226]. However, their functional role in disease state is still uncertain. In particular, TRPM8-activation on mouse-keratinocytes was demonstrated to improve skin barrier recovery after tape stripping and to mediate keratinocyte proliferation, suggesting a role of TRPM8 in wound healing [226]. Inflammatory skin diseases may be improved by modulating immune cell function. A functional role for TRPM2, TRPM4, and TRPM7 in regulating immune cells has been described. For example, TRPM2 is expressed in primary human CD4+-T cells and up-regulated after T cell-stimulation [35]. TRPM2 is functional in T cells [203] although its role in T cell regulation is still uncertain. In addition, TRPM2 is relevant for mast cell degranulation after antigen stimulation [227], as well as granulocyte and monocyte chemokine production [228] regulated by ADP-ribose [229]. TRPM4 was described to mediate membrane depolarization in immune cells. Therefore, TRPM4 activation is considered to modulate calcium signals in T cells affecting cytokine production [230] and mast cell function (alleviated mast cell degranulation) [222]. On the other hand, TRPM4 was implicated in dendritic cell migration [231]. Thus, the pro- or anti- inflammatory role of TRPM4 depends on the specific immune cell, and further studies are needed to determine its role in inflammatory skin diseases. Of further note, TRPM7 is also expressed in immune cells (lymphocytes, mast cells) and was suggested to be in involved in lymphocyte and mast cell proliferation [203].

TRPM Channels are Implicated in Melanocyte Function and Malignant Melanoma TRPM1, TRPM2, TRPM7 and TRPM8 are expressed by melanocytes and in malignant melanoma cells, and are thus suggested to play an important role in melanocyte function and malignant melanoma pathophysiology. However, direct evidence for a critical role in human melanoma is still poor. TRPM1 (Melastatin-1/MLSN-1), the founding member of the TRPM-ion channels, is considered the most important TRP channel in melanocyte function and malignant melanoma pathophysiology. Initially, it was found that highly metastatic, undifferentiated malignant melanomas exhibit a decreased expression of TRPM1 as compared to benign, highly differentiated nevi [232]. Also, TRPM1-expression correlates positively with the differentiation status of melanocytes, and, inversely, with the aggressiveness and tumor thickness of 312 17. ROLE OF TRP CHANNELS IN SKIN DISEASES

malignant melanoma [232]. Therefore, TRPM1 could serve as a prognostic marker in malignant melanoma [225]. Further studies confirmed this hypothesis [233,234] and even suggested that the TRPM1 mRNA expression pattern could be helpful for the differentiation of Spitz nevi compared to nodular malignant melanomas [235]. Of note, the expression levels of TRPM1 can also be altered during normal melanocyte maturation, which correlated also with its coexpression of its transcription factor MITF (microphthalmia transcription factor) [236]. To provide explanations for the postulated role of TRPM1 as a factor for melanocyte differentiation, further in vitro studies were performed [237,238]. These data suggest a role of TRPM1 as a key factor for melanocyte calcium homeostasis, controlling melanocyte proliferation, differentiation, and melanogenesis. Accordingly, involvement of TRPM1 in calcium homeostasis and melanogenesis was demonstrated using TRPM1-knockdown in cultured primary human melanocytes. Interestingly, TRPM1 knockdown resulted in a decrease of intracellular melanin pigment, and TRPM1 expression levels were reduced by UVB that subsequently decreased calcium influx [237]. This was also confirmed in another study in vitro with primary human neonatal epidermal melanocytes and mouse melanoma cells, where TRPM1 expression was found to correlate with melanin content. Thus, TRPM1 could be important for melanocyte calcium homeostasis and melanogenesis and might serve as a new target for pigmentation disorders [238]. This is also underlined by findings of a decreased TRPM1 expression in unpigmented skin of the Appaloosa horse [239]. Recent studies have found a microRNA (miR-211), which is located within the sixth intron of the TRPM1 gene, to be a key factor in malignant melanoma. MiR-211 is also regulated by the TRPM1 promotor with its transcription factor MITF and may thus play an important role in tumor suppression. In line with this, miR-211 expression was down-regulated in melanoma cells. Therefore, miR-211 in conjunction with TRPM1 may be a key factor for tumor invasiveness and aggressiveness in malignant melanoma. In particular, an increased expression of miR-211, but not TRPM1 decreased the aggressiveness (migration and invasion) of highly malignant human melanomas [240]. MicroRNA miR-211 was also shown to regulate many genes involved in melanoma pathophysiology [241] and therefore could serve as a new target to treat metastatic melanoma [242]. Other than TRPM1, two transcripts of TRPM2 (TRPM2-antisense and TRPM2-tumor enriched) were demonstrated to be up-regulated in melanoma cells. Moreover, overexpression of wild-type TRPM2 was shown to mediate melanoma apoptosis and necrosis [243]. Therefore, TRPM2 could serve as a potential new target for melanoma therapy, although this field of research is still at an infant stage. TRPM7 was shown to be functional in melanocytes and, different from TRPM1, increased expression levels were found in metastatic melanoma cells [244]. Using the zebrafish with a TRPM7-homozygous null-allele, it was delineated that TRPM7 protects melanocytes from cell death. Hereby, TRMP7 seems to be necessary to detoxify melanin intermediates, and thus, TRPM7 might serve as an important factor for melanocyte homeostasis [244]. Therefore, it could be hypothesized that a decreased expression of TRPM7 could lead to vitiligo, but further studies are necessary to confirm this hypothesis. TRPM8 could serve as a target in melanoma therapy. Although it was initially shown (albeit only in a small number of patients) that TRPM8 expression directly correlates with melanoma aggressiveness [245], recently a more protective role of TRPM8 was suggested. It was demonstrated in a human melanoma G-361 cell line that TRPM8-activation with menthol decreased REFERENCES 313 the viability of a cultured melanoma cell line in vitro and therefore is able to inhibit melanoma proliferation [246]. Because conflicting data exist, the exact role of TRPM8 in melanoma proliferation, and its expression in different types of melanoma need further investigation.

TRPML3

Last, a member of the transient receptor potential mucolipin subfamily, TRPML3, has recently been shown to be necessary for melanocyte function. These findings arise from mice with a varitint-waddler phenotype, which are deaf, have vestibular defects, and also exhibit pigmentation abnormalities. Here, TRPML3 was shown to be abundantly expressed in melanocytes and that the pigmentation defects are a result of a TRPML3 gain-of-function mutation that causes high intracellular calcium-levels resulting in melanocyte cell death [247].

SUMMARY

In conclusion, TRP ion channels play an important role in the regulation of various the skin cells and structures during health and disease. In addition to their contribution to the normal skin homeostasis, they could serve as targets for new therapies for many different skin disorders including inflammatory skin diseases, genetic disorders (e.g., Darier’s disease), autoimmunity, fibrotic diseases, and delayed wound healing, as well as pigmentation disorders, malignant melanoma, or nonmelanoma skin cancer. Further translational research in human tissue and cells are denuded to finally clarify their importance in disease state.

References [1] Caterina MJ, Leffler A, Malmberg AB, Martin WJ, Trafton J, Petersen-Zeitz KR, et al. Impaired nociception and pain sensation in mice lacking the capsaicin receptor. Science 2000;288(5464):306–13. [2] Roosterman D, Goerge T, Schneider SW, Bunnett NW, Steinhoff M. Neuronal control of skin function: the skin as a neuroimmunoendocrine organ. Physiol Rev 2006;86(4):1309–79. [3] Shim WS, Tak MH, Lee MH, Kim M, Kim M, Koo JY, et al. TRPV1 mediates histamine-induced itching via the activation of phospholipase A2 and 12-lipoxygenase. J Neurosci 2007;27(9):2331–7. [4] Pereira U, Boulais N, Lebonvallet N, Pennec JP, Dorange G, Misery L. Mechanisms of the sensory effects of tacrolimus on the skin. Br J Dermatol 2010;163(1):70–7. [5] Meseguer V, Karashima Y, Talavera K, D’Hoedt D, Donovan-Rodriguez T, Viana F, et al. Transient receptor potential channels in sensory neurons are targets of the antimycotic agent clotrimazole. J Neurosci 2008;28(3):576–86. [6] Yin S, Luo J, Qian A, Du J, Yang Q, Zhou S, et al. Retinoids activate the irritant receptor TRPV1 and produce sensory hypersensitivity. J Clin Invest 2013;123(9):3941–51. [7] Kueper T, Krohn M, Haustedt LO, Hatt H, Schmaus G, Vielhaber G. Inhibition of TRPV1 for the treatment of sensitive skin. Exp Dermatol 2010;19(11):980–6. [8] Steinhoff M, Buddenkotte J, Aubert J, Sulk M, Novak P, Schwab VD, et al. Clinical, cellular, and molecular aspects in the pathophysiology of rosacea. J Investig Dermatol Symp Proc 2011;15(1):2–11. [9] Sulk M, Seeliger S, Aubert J, Schwab VD, Cevikbas F, Rivier M, et al. Distribution and expression of nonneuronal transient receptor potential (TRPV) ion channels in rosacea. J Invest Dermatol 2012;132(4):1253–62. [10] Tominaga M, Caterina MJ, Malmberg AB, Rosen TA, Gilbert H, Skinner K, et al. The cloned capsaicin receptor integrates multiple pain-producing stimuli. Neuron 1998;21(3):531–43. 314 17. ROLE OF TRP CHANNELS IN SKIN DISEASES

[11] Park U, Vastani N, Guan Y, Raja SN, Koltzenburg M, Caterina MJ. TRP vanilloid 2 knock-out mice are susceptible to perinatal lethality but display normal thermal and mechanical nociception. J Neurosci 2011;31(32):11425–36. [12] Chizh BA, O’Donnell MB, Napolitano A, Wang J, Brooke AC, Aylott MC, et al. The effects of the TRPV1 antagonist SB-705498 on TRPV1 receptor-mediated activity and inflammatory hyperalgesia in humans. Pain 2007;132(1–2):132–41. [13] Yun JW, Seo JA, Jang WH, Koh HJ, Bae IH, Park YH, et al. Antipruritic effects of TRPV1 antagonist in murine atopic dermatitis and itching models. J Invest Dermatol 2011;131(7):1576–9. [14] Yun JW, Seo JA, Jeong YS, Bae IH, Jang WH, Lee J, et al. TRPV1 antagonist can suppress the atopic dermatitis-like symptoms by accelerating skin barrier recovery. J Dermatol Sci 2011;62(1):8–15. [15] Stander S, Luger T, Metze D. Treatment of prurigo nodularis with topical capsaicin. J Am Acad Dermatol 2001;44(3):471–8. [16] Wallengren J, Klinker M. Successful treatment of notalgia paresthetica with topical capsaicin: vehicle- controlled, double-blind, crossover study. J Am Acad Dermatol 1995;32(2 Pt 1):287–9. [17] Stander S, Moormann C, Schumacher M, Buddenkotte J, Artuc M, Shpacovitch V, et al. Expression of vanilloid receptor subtype 1 in cutaneous sensory nerve fibers, mast cells, and epithelial cells of appendage structures. Exp Dermatol 2004;13(3):129–39. [18] Axelsson HE, Minde JK, Sonesson A, Toolanen G, Hogestatt ED, Zygmunt PM. Transient receptor potential vanilloid 1, vanilloid 2 and melastatin 8 immunoreactive nerve fibers in human skin from individuals with and without Norrbottnian congenital insensitivity to pain. Neuroscience 2009;162(4):1322–32. [19] Pecze L, Szabo K, Szell M, Josvay K, Kaszas K, Kusz E, et al. Human keratinocytes are vanilloid resistant. PLoS One 2008;3(10):e3419. [20] Everaerts W, Sepulveda MR, Gevaert T, Roskams T, Nilius B, De Ridder D. Where is TRPV1 expressed in the bladder, do we see the real channel? Naunyn Schmiedebergs Arch Pharmacol 2009;379(4):421–5. [21] Bodo E, Kovacs I, Telek A, Varga A, Paus R, Kovacs L, et al. Vanilloid receptor-1 (VR1) is widely expressed on various epithelial and mesenchymal cell types of human skin. J Invest Dermatol 2004;123(2):410–13. [22] Lee YM, Kim YK, Chung JH. Increased expression of TRPV1 channel in intrinsically aged and photoaged human skin in vivo. Exp Dermatol 2009;18(5):431–6. [23] Weinkauf B, Rukwied R, Quiding H, Dahllund L, Johansson P, Schmelz M. Local gene expression changes after UV-irradiation of human skin. PLoS One 2012;7(6):e39411. [24] Bodo E, Biro T, Telek A, Czifra G, Griger Z, Toth BI, et al. A hot new twist to hair biology: involvement of vanilloid receptor-1 (VR1/TRPV1) signaling in human hair growth control. Am J Pathol 2005;166(4):985–98. [25] Gandhi V, Vij A, Bhattacharya SN. Apocrine chromhidrosis localized to the areola in an Indian female treated with topical capsaicin. Indian J Dermatol Venereol Leprol 2006;72(5):382–3. [26] Choi TY, Park SY, Jo JY, Kang G, Park JB, Kim JG, et al. Endogenous expression of TRPV1 channel in cultured human melanocytes. J Dermatol Sci 2009;56(2):128–30. [27] Kim SJ, Lee SA, Yun SJ, Kim JK, Park JS, Jeong HS, et al. Expression of vanilloid receptor 1 in cultured fibroblast. Exp Dermatol 2006;15(5):362–7. [28] Jain A, Bronneke S, Kolbe L, Stab F, Wenck H, Neufang G. TRP-channel-specific cutaneous eicosanoid release patterns. Pain 2011;152(12):2765–72. [29] Cavanaugh DJ, Chesler AT, Jackson AC, Sigal YM, Yamanaka H, Grant R, et al. Trpv1 reporter mice reveal highly restricted brain distribution and functional expression in arteriolar smooth muscle cells. J Neurosci 2011;31(13):5067–77. [30] Baylie RL, Brayden JE. TRPV channels and vascular function. Acta Physiol (Oxf) 2011;203(1):99–116. [31] Poblete IM, Orliac ML, Briones R, Adler-Graschinsky E, Huidobro-Toro JP. Anandamide elicits an acute release of nitric oxide through endothelial TRPV1 receptor activation in the rat arterial mesenteric bed. J Physiol 2005;568(Pt 2):539–51. [32] Nielsen TA, da Silva LB, Arendt-Nielsen L, Gazerani P. The effect of topical capsaicin-induced sensitization on heat-evoked cutaneous vasomotor responses. Int J Physiol Pathophysiol Pharmacol 2013;5(3):148–60. [33] Jancso N, Jancso-Gabor A, Szolcsanyi J. Direct evidence for neurogenic inflammation and its prevention by denervation and by pretreatment with capsaicin. Br J Pharmacol Chemother 1967;31(1):138–51. [34] Heiner I, Eisfeld J, Luckhoff A. Role and regulation of TRP channels in neutrophil granulocytes. Cell Calcium 2003;33(5–6):533–40. [35] Wenning AS, Neblung K, Strauss B, Wolfs MJ, Sappok A, Hoth M, et al. TRP expression pattern and the functional importance of TRPC3 in primary human T-cells. Biochim Biophys Acta 2011;1813(3):412–23. REFERENCES 315

[36] Toth BI, Benko S, Szollosi AG, Kovacs L, Rajnavolgyi E, Biro T. Transient receptor potential vanilloid-1 signaling inhibits differentiation and activation of human dendritic cells. FEBS Lett 2009;583(10):1619–24. [37] Szollosi AG, Olah A, Toth IB, Papp F, Czifra G, Panyi G, et al. Transient receptor potential vanilloid-2 mediates the effects of transient heat shock on endocytosis of human monocyte-derived dendritic cells. FEBS Lett 2013;587(9):1440–5. [38] Basu S, Srivastava P. Immunological role of neuronal receptor vanilloid receptor 1 expressed on dendritic cells. Proc Natl Acad Sci USA 2005;102(14):5120–5. [39] Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, Julius D. The capsaicin receptor: a heat- activated ion channel in the pain pathway. Nature 1997;389(6653):816–24. [40] Kajihara Y, Murakami M, Imagawa T, Otsuguro K, Ito S, Ohta T. Histamine potentiates acid-induced responses mediating transient receptor potential V1 in mouse primary sensory neurons. Neuroscience 2010;166(1):292–304. [41] Hwang SW, Cho H, Kwak J, Lee SY, Kang CJ, Jung J, et al. Direct activation of capsaicin receptors by products of lipoxygenases: endogenous capsaicin-like substances. Proc Natl Acad Sci USA 2000;97(11):6155–60. [42] Tominaga M, Wada M, Masu M. Potentiation of capsaicin receptor activity by metabotropic ATP receptors as a possible mechanism for ATP-evoked pain and hyperalgesia. Proc Natl Acad Sci USA 2001;98(12):6951–6. [43] Sugiura T, Tominaga M, Katsuya H, Mizumura K. Bradykinin lowers the threshold temperature for heat activation of vanilloid receptor 1. J Neurophysiol 2002;88(1):544–8. [44] Moriyama T, Higashi T, Togashi K, Iida T, Segi E, Sugimoto Y, et al. Sensitization of TRPV1 by EP1 and IP reveals peripheral nociceptive mechanism of prostaglandins. Mol Pain 2005;1:3. [45] Starr A, Graepel R, Keeble J, Schmidhuber S, Clark N, Grant A, et al. A reactive oxygen species-mediated component in neurogenic vasodilatation. Cardiovasc Res 2008;78(1):139–47. [46] Amadesi S, Nie J, Vergnolle N, Cottrell GS, Grady EF, Trevisani M, et al. Protease-activated receptor 2 sensitizes the capsaicin receptor transient receptor potential vanilloid receptor 1 to induce hyperalgesia. J Neurosci 2004;24(18):4300–12. [47] Banvolgyi A, Palinkas L, Berki T, Clark N, Grant AD, Helyes Z, et al. Evidence for a novel protective role of the vanilloid TRPV1 receptor in a cutaneous contact allergic dermatitis model. J Neuroimmunol 2005;169(1–2):86–96. [48] Southall MD, Li T, Gharibova LS, Pei Y, Nicol GD, Travers JB. Activation of epidermal vanilloid receptor-1 induces release of proinflammatory mediators in human keratinocytes. J Pharmacol Exp Ther 2003;304(1):217–22. [49] Huang J, Qiu L, Ding L, Wang S, Wang J, Zhu Q, et al. Ginsenoside Rb1 and paeoniflorin inhibit transient receptor potential vanilloid-1-activated IL-8 and PGE(2) production in a human keratinocyte cell line HaCaT. Int Immunopharmacol 2010;10(10):1279–83. [50] Li WH, Lee YM, Kim JY, Kang S, Kim S, Kim KH, et al. Transient receptor potential vanilloid-1 mediates heat- shock-induced matrix metalloproteinase-1 expression in human epidermal keratinocytes. J Invest Dermatol 2007;127(10):2328–35. [51] Lee YM, Li WH, Kim YK, Kim KH, Chung JH. Heat-induced MMP-1 expression is mediated by TRPV1 through PKCalpha signaling in HaCaT cells. Exp Dermatol 2008;17(10):864–70. [52] Lee YM, Kim YK, Kim KH, Park SJ, Kim SJ, Chung JH. A novel role for the TRPV1 channel in UV-induced matrix metalloproteinase (MMP)-1 expression in HaCaT cells. J Cell Physiol 2009;219(3):766–75. [53] Biro T, Maurer M, Modarres S, Lewin NE, Brodie C, Acs G, et al. Characterization of functional vanilloid receptors expressed by mast cells. Blood 1998;91(4):1332–40. [54] O’Connell PJ, Pingle SC, Ahern GP. Dendritic cells do not transduce inflammatory stimuli via the capsaicin receptor TRPV1. FEBS Lett 2005;579(23):5135–9. [55] Luebbert M, Radtke D, Wodarski R, Damann N, Hatt H, Wetzel CH. Direct activation of transient receptor potential V1 by nickel ions. Pflugers Arch 2010;459(5):737–50. [56] Wallengren J, Ekman R, Moller H. Capsaicin enhances allergic contact dermatitis in the guinea pig. Contact Dermatitis 1991;24(1):30–4. [57] Lee YM, Kang SM, Lee SR, Kong KH, Lee JY, Kim EJ, et al. Inhibitory effects of TRPV1 blocker on UV-induced responses in the hairless mice. Arch Dermatol Res 2011;303(10):727–36. [58] Usuda H, Endo T, Shimouchi A, Saito A, Tominaga M, Yamashita H, et al. Transient receptor potential vanilloid 1 - a polymodal nociceptive receptor - plays a crucial role in formaldehyde-induced skin inflammation in mice. J Pharmacol Sci 2012;118(2):266–74. 316 17. ROLE OF TRP CHANNELS IN SKIN DISEASES

[59] Desai PR, Marepally S, Patel AR, Voshavar C, Chaudhuri A, Singh M. Topical delivery of anti-TNFalpha siRNA and capsaicin via novel lipid-polymer hybrid nanoparticles efficiently inhibits skin inflammation in vivo. J Control Release 2013;170(1):51–63. [60] Toth BI, Geczy T, Griger Z, Dozsa A, Seltmann H, Kovacs L, et al. Transient receptor potential vanilloid-1 signaling as a regulator of human sebocyte biology. J Invest Dermatol 2009;129(2):329–39. [61] Li HJ, Kanazawa N, Kimura A, Kaminaka C, Yonei N, Yamamoto Y, et al. Severe ulceration with impaired induction of growth factors and cytokines in keratinocytes after trichloroacetic acid application on TRPV1- deficient mice. Eur J Dermatol 2012;22(5):614–21. [62] Liu B, Escalera J, Balakrishna S, Fan L, Caceres AI, Robinson E, et al. TRPA1 controls inflammation and pruritogen responses in allergic contact dermatitis. FASEB J 2013;27(9):3549–63. [63] Riol-Blanco L, Ordovas-Montanes J, Perro M, Naval E, Thiriot A, Alvarez D, et al. Nociceptive sensory neurons drive interleukin-23-mediated psoriasiform skin inflammation. Nature 2014;510:157–61. [64] Biro T, Bodo E, Telek A, Geczy T, Tychsen B, Kovacs L, et al. Hair cycle control by vanilloid receptor-1 (TRPV1): evidence from TRPV1 knockout mice. J Invest Dermatol 2006;126(8):1909–12. [65] Toth BI, Dobrosi N, Dajnoki A, Czifra G, Olah A, Szollosi AG, et al. Endocannabinoids modulate human epidermal keratinocyte proliferation and survival via the sequential engagement of cannabinoid receptor-1 and transient receptor potential vanilloid-1. J Invest Dermatol 2011;131(5):1095–104. [66] Bode AM, Cho YY, Zheng D, Zhu F, Ericson ME, Ma WY, et al. Transient receptor potential type vanilloid 1 suppresses skin carcinogenesis. Cancer Res 2009;69(3):905–13. [67] Denda M, Sokabe T, Fukumi-Tominaga T, Tominaga M. Effects of skin surface temperature on epidermal permeability barrier homeostasis. J Invest Dermatol 2007;127(3):654–9. [68] Peralvarez-Marin A, Donate-Macian P, Gaudet R. What do we know about the transient receptor potential vanilloid 2 (TRPV2) ion channel? FEBS J 2013;280(21):5471–87. [69] Santoni G, Farfariello V, Liberati S, Morelli MB, Nabissi M, Santoni M, et al. The role of transient receptor potential vanilloid type-2 ion channels in innate and adaptive immune responses. Front Immunol 2013;4:34. [70] Vandewauw I, Owsianik G, Voets T. Systematic and quantitative mRNA expression analysis of TRP channel genes at the single trigeminal and dorsal root ganglion level in mouse. BMC Neurosci 2013;14:21. [71] Liapi A, Wood JN. Extensive co-localization and heteromultimer formation of the vanilloid receptor-like protein TRPV2 and the capsaicin receptor TRPV1 in the adult rat cerebral cortex. Eur J Neurosci 2005;22(4):825–34. [72] Muraki K, Iwata Y, Katanosaka Y, Ito T, Ohya S, Shigekawa M, et al. TRPV2 is a component of osmotically sensitive cation channels in murine aortic myocytes. Circ Res 2003;93(9):829–38. [73] Shibasaki K, Murayama N, Ono K, Ishizaki Y, Tominaga M. TRPV2 enhances axon outgrowth through its activation by membrane stretch in developing sensory and motor neurons. J Neurosci 2010;30(13):4601–12. [74] Saunders CI, Kunde DA, Crawford A, Geraghty DP. Expression of transient receptor potential vanilloid 1 (TRPV1) and 2 (TRPV2) in human peripheral blood. Mol Immunol 2007;44(6):1429–35. [75] Link TM, Park U, Vonakis BM, Raben DM, Soloski MJ, Caterina MJ. TRPV2 has a pivotal role in macrophage particle binding and phagocytosis. Nat Immunol 2010;11(3):232–9. [76] Nagasawa M, Nakagawa Y, Tanaka S, Kojima I. Chemotactic peptide fMetLeuPhe induces translocation of the TRPV2 channel in macrophages. J Cell Physiol 2007;210(3):692–702. [77] Yamashiro K, Sasano T, Tojo K, Namekata I, Kurokawa J, Sawada N, et al. Role of transient receptor potential vanilloid 2 in LPS-induced cytokine production in macrophages. Biochem Biophys Res Commun 2010;398(2):284–9. [78] Radtke C, Sinis N, Sauter M, Jahn S, Kraushaar U, Guenther E, et al. TRPV channel expression in human skin and possible role in thermally induced cell death. J Burn Care Res 2011;32(1):150–9. [79] Nilius B, Biro T, Owsianik G. TRPV3: time to decipher a poorly understood family member!. J Physiol 2014;592(Pt 2):295–304. [80] Nilius B, Biro T. TRPV3: a ‘more than skinny’ channel. Exp Dermatol 2013;22(7):447–52. [81] Gopinath P, Wan E, Holdcroft A, Facer P, Davis JB, Smith GD, et al. Increased capsaicin receptor TRPV1 in skin nerve fibres and related vanilloid receptors TRPV3 and TRPV4 in keratinocytes in human breast pain. BMC Womens Health 2005;5(1):2. [82] Facer P, Casula MA, Smith GD, Benham CD, Chessell IP, Bountra C, et al. Differential expression of the capsaicin receptor TRPV1 and related novel receptors TRPV3, TRPV4 and TRPM8 in normal human tissues and changes in traumatic and diabetic neuropathy. BMC Neurol 2007;7:11. REFERENCES 317

[83] Borbiro I, Lisztes E, Toth BI, Czifra G, Olah A, Szollosi AG, et al. Activation of transient receptor potential vanilloid-3 inhibits human hair growth. J Invest Dermatol 2011;131(8):1605–14. [84] Peier AM, Reeve AJ, Andersson DA, Moqrich A, Earley TJ, Hergarden AC, et al. A heat-sensitive TRP channel expressed in keratinocytes. Science 2002;296(5575):2046–9. [85] Chung MK, Lee H, Caterina MJ. Warm temperatures activate TRPV4 in mouse 308 keratinocytes. J Biol Chem 2003;278(34):32037–46. [86] Chung MK, Lee H, Mizuno A, Suzuki M, Caterina MJ. TRPV3 and TRPV4 mediate warmth-evoked currents in primary mouse keratinocytes. J Biol Chem 2004;279(20):21569–75. [87] Xu H, Delling M, Jun JC, Clapham DE. Oregano, thyme and clove-derived flavors and skin sensitizers activate specific TRP channels. Nat Neurosci 2006;9(5):628–35. [88] Yoshioka T, Imura K, Asakawa M, Suzuki M, Oshima I, Hirasawa T, et al. Impact of the Gly573Ser substitution in TRPV3 on the development of allergic and pruritic dermatitis in mice. J Invest Dermatol 2009;129(3):714–22. [89] Yamada T, Ueda T, Ugawa S, Ishida Y, Imayasu M, Koyama S, et al. Functional expression of transient receptor potential vanilloid 3 (TRPV3) in corneal epithelial cells: involvement in thermosensation and wound healing. Exp Eye Res 2010;90(1):121–9. [90] Mergler S, Garreis F, Sahlmuller M, Reinach PS, Paulsen F, Pleyer U. Thermosensitive transient receptor potential channels in human corneal epithelial cells. J Cell Physiol 2011;226(7):1828–42. [91] Ueda T, Yamada T, Ugawa S, Ishida Y, Shimada S. TRPV3, a thermosensitive channel is expressed in mouse distal colon epithelium. Biochem Biophys Res Commun 2009;383(1):130–4. [92] Hamamoto T, Takumida M, Hirakawa K, Takeno S, Tatsukawa T. Localization of transient receptor potential channel vanilloid subfamilies in the mouse larynx. Acta Otolaryngol 2008;128(6):685–93. [93] Ishibashi T, Takumida M, Akagi N, Hirakawa K, Anniko M. Expression of transient receptor potential vanilloid (TRPV) 1, 2, 3, and 4 in mouse inner ear. Acta Otolaryngol 2008;128(12):1286–93. [94] Xu H, Ramsey IS, Kotecha SA, Moran MM, Chong JA, Lawson D, et al. TRPV3 is a calcium-permeable temperature-sensitive cation channel. Nature 2002;418(6894):181–6. [95] Inada H, Iida T, Tominaga M. Different expression patterns of TRP genes in murine B and T lymphocytes. Biochem Biophys Res Commun 2006;350(3):762–7. [96] Heng TS, Painter MWImmunological Genome Project C. The Immunological Genome Project: networks of gene expression in immune cells. Nat Immunol 2008;9(10):1091–4. [97] Huang SM, Li X, Yu Y, Wang J, Caterina MJ. TRPV3 and TRPV4 ion channels are not major contributors to mouse heat sensation. Mol Pain 2011;7:37. [98] Moqrich A, Hwang SW, Earley TJ, Petrus MJ, Murray AN, Spencer KS, et al. Impaired thermosensation in mice lacking TRPV3, a heat and camphor sensor in the skin. Science 2005;307(5714):1468–72. [99] Mandadi S, Sokabe T, Shibasaki K, Katanosaka K, Mizuno A, Moqrich A, et al. TRPV3 in keratinocytes trans- mits temperature information to sensory neurons via ATP. Pflugers Arch 2009;458(6):1093–102. [100] Huang SM, Lee H, Chung MK, Park U, Yu YY, Bradshaw HB, et al. Overexpressed transient receptor potential vanilloid 3 ion channels in skin keratinocytes modulate pain sensitivity via prostaglandin E2. J Neurosci 2008;28(51):13727–37. [101] Bang S, Yoo S, Yang TJ, Cho H, Hwang SW. Farnesyl pyrophosphate is a novel pain-producing molecule via specific activation of TRPV3. J Biol Chem 2010;285(25):19362–71. [102] Bang S, Yoo S, Yang TJ, Cho H, Hwang SW. Isopentenyl pyrophosphate is a novel antinociceptive substance that inhibits TRPV3 and TRPA1 ion channels. Pain 2011;152(5):1156–64. [103] Bang S, Yoo S, Yang TJ, Cho H, Hwang SW. 17(R)-resolvin D1 specifically inhibits transient receptor potential ion channel vanilloid 3 leading to peripheral antinociception. Br J Pharmacol 2012;165(3):683–92. [104] Steinhoff M, Biro T. A TR(I)P to pruritus research: role of TRPV3 in inflammation and itch. J Invest Dermatol 2009;129(3):531–5. [105] Yamamoto-Kasai E, Imura K, Yasui K, Shichijou M, Oshima I, Hirasawa T, et al. TRPV3 as a therapeutic target for itch. J Invest Dermatol 2012;132(8):2109–12. [106] Cheng X, Jin J, Hu L, Shen D, Dong XP, Samie MA, et al. TRP channel regulates EGFR signaling in hair morphogenesis and skin barrier formation. Cell 2010;141(2):331–43. [107] Schneider MR, Werner S, Paus R, Wolf E. Beyond wavy hairs: the epidermal growth factor receptor and its ligands in skin biology and pathology. Am J Pathol 2008;173(1):14–24. [108] Lorand L, Graham RM. Transglutaminases: crosslinking enzymes with pleiotropic functions. Nat Rev Mol Cell Biol 2003;4(2):140–56. 318 17. ROLE OF TRP CHANNELS IN SKIN DISEASES

[109] Klein AS, Tannert A, Schaefer M. Cholesterol sensitises the transient receptor potential channel TRPV3 to lower temperatures and activator concentrations. Cell Calcium 2014;55:59–68. [110] Schmidt R, Parish EJ, Dionisius V, Cathelineau C, Michel S, Shroot B, et al. Modulation of cellular cholesterol and its effect on cornified envelope formation in cultured human epidermal keratinocytes. J Invest Dermatol 1991;97(5):771–5. [111] Cao X, Yang F, Zheng J, Wang K. Intracellular proton-mediated activation of TRPV3 channels accounts for the exfoliation effect of alpha-hydroxyl acids on keratinocytes. J Biol Chem 2012;287(31):25905–16. [112] Miyamoto T, Petrus MJ, Dubin AE, Patapoutian A. TRPV3 regulates nitric oxide synthase-independent nitric oxide synthesis in the skin. Nat Commun 2011;2:369. [113] Cals-Grierson MM, Ormerod AD. Nitric oxide function in the skin. Nitric Oxide 2004;10(4):179–93. [114] Earley S, Gonzales AL, Garcia ZI. A dietary agonist of transient receptor potential cation channel V3 elicits endothelium-dependent vasodilation. Mol Pharmacol 2010;77(4):612–20. [115] Asakawa M, Yoshioka T, Matsutani T, Hikita I, Suzuki M, Oshima I, et al. Association of a mutation in TRPV3 with defective hair growth in rodents. J Invest Dermatol 2006;126(12):2664–72. [116] Xiao R, Tian J, Tang J, Zhu MX. The TRPV3 mutation associated with the hairless phenotype in rodents is constitutively active. Cell Calcium 2008;43(4):334–43. [117] Imura K, Yoshioka T, Hikita I, Tsukahara K, Hirasawa T, Higashino K, et al. Influence of TRPV3 mutation on hair growth cycle in mice. Biochem Biophys Res Commun 2007;363(3):479–83. [118] Phelps CB, Wang RR, Choo SS, Gaudet R. Differential regulation of TRPV1, TRPV3, and TRPV4 sensitivity through a conserved binding site on the ankyrin repeat domain. J Biol Chem 2010;285(1):731–40. [119] Hu HZ, Xiao R, Wang C, Gao N, Colton CK, Wood JD, et al. Potentiation of TRPV3 channel function by unsaturated fatty acids. J Cell Physiol 2006;208(1):201–12. [120] Watanabe A, Takeuchi M, Nagata M, Nakamura K, Nakao H, Yamashita H, et al. Role of the Nh (Non- hair) mutation in the development of dermatitis and hyperproduction of IgE in DS-Nh mice. Exp Anim 2003;52(5):419–23. [121] Asakawa M, Yoshioka T, Hikita I, Matsutani T, Hirasawa T, Arimura A, et al. WBN/Kob-Ht rats spontaneously develop dermatitis under conventional conditions: another possible model for atopic dermatitis. Exp Anim 2005;54(5):461–5. [122] Hikita I, Yoshioka T, Mizoguchi T, Tsukahara K, Tsuru K, Nagai H, et al. Characterization of dermatitis arising spontaneously in DS-Nh mice maintained under conventional conditions: another possible model for atopic dermatitis. J Dermatol Sci 2002;30(2):142–53. [123] Yoshioka T, Hikita I, Asakawa M, Hirasawa T, Deguchi M, Matsutani T, et al. Spontaneous scratching behaviour in DS-Nh mice as a possible model for pruritus in atopic dermatitis. Immunology 2006;118(3):293–301. [124] Yamamoto-Kasai E, Yasui K, Shichijo M, Sakata T, Yoshioka T. Impact of TRPV3 on the development of allergic dermatitis as a dendritic cell modulator. Exp Dermatol 2013;22(12):820–4. [125] Imura K, Yoshioka T, Hirasawa T, Sakata T. Role of TRPV3 in immune response to development of dermatitis. J Inflamm (Lond) 2009;6:17. [126] Lin Z, Chen Q, Lee M, Cao X, Zhang J, Ma D, et al. Exome sequencing reveals mutations in TRPV3 as a cause of Olmsted syndrome. Am J Hum Genet 2012;90(3):558–64. [127] Lai-Cheong JE, Sethuraman G, Ramam M, Stone K, Simpson MA, McGrath JA. Recurrent heterozygous missense mutation, p.Gly573Ser, in the TRPV3 gene in an Indian boy with sporadic Olmsted syndrome. Br J Dermatol 2012;167(2):440–2. [128] Eytan O, Fuchs-Telem D, Mevorach B, Indelman M, Bergman R, Sarig O, et al. Olmsted syndrome caused by a homozygous recessive mutation in TRPV3. J Invest Dermatol 2014;134:1752–4. [129] Danso-Abeam D, Zhang J, Dooley J, Staats KA, Van Eyck L, Van Brussel T, et al. Olmsted syndrome: exploration of the immunological phenotype. Orphanet J Rare Dis 2013;8:79. [130] Duchatelet S, Pruvost S, de Veer S, Fraitag S, Nitschke P, Bole-Feysot C, et al. A new TRPV3 missense mutation in a patient with Olmsted syndrome and erythromelalgia. JAMA Dermatol 2014;150:303–6. [131] Sokabe T, Fukumi-Tominaga T, Yonemura S, Mizuno A, Tominaga M. The TRPV4 channel contributes to intercellular junction formation in keratinocytes. J Biol Chem 2010;285(24):18749–58. [132] Moore C, Cevikbas F, Pasolli HA, Chen Y, Kong W, Kempkes C, et al. UVB radiation generates sunburn pain and affects skin by activating epidermal TRPV4 ion channels and triggering endothelin-1 signaling. Proc Natl Acad Sci USA 2013;110(34):E3225–34. REFERENCES 319

[133] Kida N, Sokabe T, Kashio M, Haruna K, Mizuno Y, Suga Y, et al. Importance of transient receptor potential vanilloid 4 (TRPV4) in epidermal barrier function in human skin keratinocytes. Pflugers Arch 2012;463(5):715–25. [134] Watanabe H, Vriens J, Suh SH, Benham CD, Droogmans G, Nilius B. Heat-evoked activation of TRPV4 channels in a HEK293 cell expression system and in native mouse aorta endothelial cells. J Biol Chem 2002;277(49):47044–51. [135] Watanabe H, Vriens J, Prenen J, Droogmans G, Voets T, Nilius B. Anandamide and arachidonic acid use epoxyeicosatrienoic acids to activate TRPV4 channels. Nature 2003;424(6947):434–8. [136] Liedtke W, Choe Y, Marti-Renom MA, Bell AM, Denis CS, Sali A, et al. Vanilloid receptor-related osmotically activated channel (VR-OAC), a candidate vertebrate osmoreceptor. Cell 2000;103(3):525–35. [137] Delany NS, Hurle M, Facer P, Alnadaf T, Plumpton C, Kinghorn I, et al. Identification and characterization of a novel human vanilloid receptor-like protein, VRL-2. Physiol Genomics 2001;4(3):165–74. [138] Strotmann R, Harteneck C, Nunnenmacher K, Schultz G, Plant TD. OTRPC4, a nonselective cation channel that confers sensitivity to extracellular osmolarity. Nat Cell Biol 2000;2(10):695–702. [139] Alessandri-Haber N, Yeh JJ, Boyd AE, Parada CA, Chen X, Reichling DB, et al. Hypotonicity induces TRPV4- mediated nociception in rat. Neuron 2003;39(3):497–511. [140] Guler AD, Lee H, Iida T, Shimizu I, Tominaga M, Caterina M. Heat-evoked activation of the ion channel, TRPV4. J Neurosci 2002;22(15):6408–14. [141] Lee H, Iida T, Mizuno A, Suzuki M, Caterina MJ. Altered thermal selection behavior in mice lacking transient receptor potential vanilloid 4. J Neurosci 2005;25(5):1304–10. [142] Todaka H, Taniguchi J, Satoh J, Mizuno A, Suzuki M. Warm temperature-sensitive transient receptor potential vanilloid 4 (TRPV4) plays an essential role in thermal hyperalgesia. J Biol Chem 2004;279(34):35133–8. [143] Liedtke W, Friedman JM. Abnormal osmotic regulation in trpv4−/− mice. Proc Natl Acad Sci USA 2003;100(23):13698–703. [144] Wu L, Gao X, Brown RC, Heller S, O’Neil RG. Dual role of the TRPV4 channel as a sensor of flow and osmolality in renal epithelial cells. Am J Physiol Renal Physiol 2007;293(5):F1699–713. [145] Mochizuki T, Sokabe T, Araki I, Fujishita K, Shibasaki K, Uchida K, et al. The TRPV4 cation channel mediates stretch-evoked Ca2+ influx and ATP release in primary urothelial cell cultures. J Biol Chem 2009;284(32):21257–64. [146] Grant AD, Cottrell GS, Amadesi S, Trevisani M, Nicoletti P, Materazzi S, et al. Protease-activated receptor 2 sensitizes the transient receptor potential vanilloid 4 ion channel to cause mechanical hyperalgesia in mice. J Physiol 2007;578(Pt 3):715–33. [147] Zhang Y, Wang YH, Ge HY, Arendt-Nielsen L, Wang R, Yue SW. A transient receptor potential vanilloid 4 contributes to mechanical allodynia following chronic compression of dorsal root ganglion in rats. Neurosci Lett 2008;432(3):222–7. [148] Gao F, Wang DH. Hypotension induced by activation of the transient receptor potential vanilloid 4 channels: role of Ca2+-activated K+ channels and sensory nerves. J Hypertens 2010;28(1):102–10. [149] Cenac N, Altier C, Motta JP, d’Aldebert E, Galeano S, Zamponi GW, et al. Potentiation of TRPV4 signalling by histamine and serotonin: an important mechanism for visceral hypersensitivity. Gut 2010;59(4):481–8. [150] Kim KS, Shin DH, Nam JH, Park KS, Zhang YH, Kim WK, et al. Functional expression of TRPV4 cation channels in human mast cell line (HMC-1). Korean J Physiol Pharmacol 2010;14(6):419–25. [151] Yang WZ, Chen JY, Yu JT, Zhou LW. Effects of low power laser irradiation on intracellular calcium and histamine release in RBL-2H3 mast cells. Photochem Photobiol 2007;83(4):979–84. [152] Hamanaka K, Jian MY, Townsley MI, King JA, Liedtke W, Weber DS, et al. TRPV4 channels augment macrophage activation and ventilator-induced lung injury. Am J Physiol Lung Cell Mol Physiol 2010;299(3):L353–62. [153] Spinsanti G, Zannolli R, Panti C, Ceccarelli I, Marsili L, Bachiocco V, et al. Quantitative Real-Time PCR detection of TRPV1-4 gene expression in human leukocytes from healthy and hyposensitive subjects. Mol Pain 2008;4:51. [154] Fusi C, Materazzi S, Minocci D, Maio V, Oranges T, Massi D, et al. Transient receptor potential vanilloid 4 (TRPV4) is down-regulated in keratinocytes in human non-melanoma skin cancer. J Invest Dermatol 2014;134:2408–17. [155] Sokabe T, Tominaga M. The TRPV4 cation channel: a molecule linking skin temperature and barrier function. Commun Integr Biol 2010;3(6):619–21. [156] Akazawa Y, Yuki T, Yoshida H, Sugiyama Y, Inoue S. Activation of TRPV4 strengthens the tight-junction barrier in human epidermal keratinocytes. Skin Pharmacol Physiol 2013;26(1):15–21. 320 17. ROLE OF TRP CHANNELS IN SKIN DISEASES

[157] Rattenholl A, Seeliger S, Buddenkotte J, Schon M, Schon MP, Stander S, et al. Proteinase-activated receptor-2 (PAR2): a tumor suppressor in skin carcinogenesis. J Invest Dermatol 2007;127(9):2245–52. [158] Bouillon R, Verstuyf A, Mathieu C, Van Cromphaut S, Masuyama R, Dehaes P, et al. Vitamin D resistance. Best Pract Res Clin Endocrinol Metab 2006;20(4):627–45. [159] Bianco SD, Peng JB, Takanaga H, Suzuki Y, Crescenzi A, Kos CH, et al. Marked disturbance of calcium homeostasis in mice with targeted disruption of the Trpv6 calcium channel gene. J Bone Miner Res 2007;22(2):274–85. [160] Lehen’kyi V, Beck B, Polakowska R, Charveron M, Bordat P, Skryma R, et al. TRPV6 is a Ca2+ entry channel essential for Ca2+-induced differentiation of human keratinocytes. J Biol Chem 2007;282(31):22582–91. [161] Lehen’kyi V, Vandenberghe M, Belaubre F, Julie S, Castex-Rizzi N, Skryma R, et al. Acceleration of keratinocyte differentiation by transient receptor potential vanilloid (TRPV6) channel activation. J Eur Acad Dermatol Venereol 2011;25(Suppl 1):12–18. [162] Bandell M, Story GM, Hwang SW, Viswanath V, Eid SR, Petrus MJ, et al. Noxious cold ion channel TRPA1 is activated by pungent compounds and bradykinin. Neuron 2004;41(6):849–57. [163] Story GM, Peier AM, Reeve AJ, Eid SR, Mosbacher J, Hricik TR, et al. ANKTM1, a TRP-like channel expressed in nociceptive neurons, is activated by cold temperatures. Cell 2003;112(6):819–29. [164] Caspani O, Heppenstall PA. TRPA1 and cold transduction: an unresolved issue? J Gen Physiol 2009;133(3):245–9. [165] Bautista DM, Jordt SE, Nikai T, Tsuruda PR, Read AJ, Poblete J, et al. TRPA1 mediates the inflammatory actions of environmental irritants and proalgesic agents. Cell 2006;124(6):1269–82. [166] del Camino D, Murphy S, Heiry M, Barrett LB, Earley TJ, Cook CA, et al. TRPA1 contributes to cold hypersensitivity. J Neurosci 2010;30(45):15165–74. [167] Kwan KY, Corey DP. Burning cold: involvement of TRPA1 in noxious cold sensation. J Gen Physiol 2009;133(3):251–6. [168] Chen J, Kang D, Xu J, Lake M, Hogan JO, Sun C, et al. Species differences and molecular determinant of TRPA1 cold sensitivity. Nat Commun 2013;4:2501. [169] Baraldi PG, Preti D, Materazzi S, Geppetti P. Transient receptor potential ankyrin 1 (TRPA1) channel as emerging target for novel analgesics and anti-inflammatory agents. J Med Chem 2010;53(14):5085–107. [170] Kwan KY, Glazer JM, Corey DP, Rice FL, Stucky CL. TRPA1 modulates mechanotransduction in cutaneous sensory neurons. J Neurosci 2009;29(15):4808–19. [171] Kindt KS, Viswanath V, Macpherson L, Quast K, Hu H, Patapoutian A, et al. Caenorhabditis elegans TRPA-1 functions in mechanosensation. Nat Neurosci 2007;10(5):568–77. [172] Hill K, Schaefer M. TRPA1 is differentially modulated by the amphipathic molecules trinitrophenol and chlor- promazine. J Biol Chem 2007;282(10):7145–53. [173] Kwan KY, Allchorne AJ, Vollrath MA, Christensen AP, Zhang DS, Woolf CJ, et al. TRPA1 contributes to cold, mechanical, and chemical nociception but is not essential for hair-cell transduction. Neuron 2006;50(2):277–89. [174] McGaraughty S, Chu KL, Perner RJ, Didomenico S, Kort ME, Kym PR. TRPA1 modulation of spontaneous and mechanically evoked firing of spinal neurons in uninjured, osteoarthritic, and inflamed rats. Mol Pain 2010;6:14. [175] Petrus M, Peier AM, Bandell M, Hwang SW, Huynh T, Olney N, et al. A role of TRPA1 in mechanical hyperalgesia is revealed by pharmacological inhibition. Mol Pain 2007;3:40. [176] Moran MM, McAlexander MA, Biro T, Szallasi A. Transient receptor potential channels as therapeutic targets. Nat Rev Drug Discov 2011;10(8):601–20. [177] Yonemitsu T, Kuroki C, Takahashi N, Mori Y, Kanmura Y, Kashiwadani H, et al. TRPA1 detects environmental chemicals and induces avoidance behavior and arousal from sleep. Sci Rep 2013;3:3100. [178] Andre E, Campi B, Materazzi S, Trevisani M, Amadesi S, Massi D, et al. Cigarette smoke-induced neurogenic inflammation is mediated by alpha, beta-unsaturated aldehydes and the TRPA1 receptor in rodents. J Clin Invest 2008;118(7):2574–82. [179] Trevisani M, Siemens J, Materazzi S, Bautista DM, Nassini R, Campi B, et al. 4-Hydroxynonenal, an endogenous aldehyde, causes pain and neurogenic inflammation through activation of the irritant receptor TRPA1. Proc Natl Acad Sci USA 2007;104(33):13519–24. [180] Shapiro D, Deering-Rice CE, Romero EG, Hughen RW, Light AR, Veranth JM, et al. Activation of transient receptor potential ankyrin-1 (TRPA1) in lung cells by wood smoke particulate material. Chem Res Toxicol 2013;26(5):750–8. [181] Kremeyer B, Lopera F, Cox JJ, Momin A, Rugiero F, Marsh S, et al. A gain-of-function mutation in TRPA1 causes familial episodic pain syndrome. Neuron 2010;66(5):671–80. REFERENCES 321

[182] Bautista DM, Wilson SR, Hoon MA. Why we scratch an itch: the molecules, cells and circuits of itch. Nat Neurosci 2014;17(2):175–82. [183] Wilson SR, The L, Batia LM, Beattie K, Katibah GE, McClain SP, et al. The epithelial cell-derived atopic dermatitis cytokine TSLP activates neurons to induce itch. Cell 2013;155(2):285–95. [184] Cevikbas F, Wang X, Akiyama T, Kempkes C, Savinko T, Antal A, et al. A sensory neuron-expressed IL-31 receptor mediates T helper cell-dependent itch: Involvement of TRPV1 and TRPA1. J Allergy Clin Immunol 2014;133(2):448–60. [185] Akiyama T, Carstens E. Neural processing of itch. Neuroscience 2013;250:697–714. [186] Anand U, Otto WR, Facer P, Zebda N, Selmer I, Gunthorpe MJ, et al. TRPA1 receptor localisation in the human peripheral nervous system and functional studies in cultured human and rat sensory neurons. Neurosci Lett 2008;438(2):221–7. [187] Oh MH, Oh SY, Lu J, Lou H, Myers AC, Zhu Z, et al. TRPA1-dependent pruritus in IL-13-induced chronic atopic dermatitis. J Immunol 2013;191(11):5371–82. [188] Tsutsumi M, Denda S, Ikeyama K, Goto M, Denda M. Exposure to low temperature induces elevation of intracellular calcium in cultured human keratinocytes. J Invest Dermatol 2010;130(7):1945–8. [189] Atoyan R, Shander D, Botchkareva NV. Non-neuronal expression of transient receptor potential type A1 (TRPA1) in human skin. J Invest Dermatol 2009;129(9):2312–15. [190] Earley S, Gonzales AL, Crnich R. Endothelium-dependent cerebral artery dilation mediated by TRPA1 and Ca2+-activated K+ channels. Circ Res 2009;104(8):987–94. [191] Bellono NW, Kammel LG, Zimmerman AL, Oancea E. UV light phototransduction activates transient receptor potential A1 ion channels in human melanocytes. Proc Natl Acad Sci USA 2013;110(6):2383–8. [192] Denda M, Tsutsumi M, Goto M, Ikeyama K, Denda S. Topical application of TRPA1 agonists and brief cold exposure accelerate skin permeability barrier recovery. J Invest Dermatol 2010;130(7):1942–5. [193] Materazzi S, Nassini R, Andre E, Campi B, Amadesi S, Trevisani M, et al. Cox-dependent fatty acid metabolites cause pain through activation of the irritant receptor TRPA1. Proc Natl Acad Sci USA 2008;105(33):12045–50. [194] Andersson DA, Gentry C, Moss S, Bevan S. Transient receptor potential A1 is a sensory receptor for multiple products of oxidative stress. J Neurosci 2008;28(10):2485–94. [195] Obata K, Katsura H, Mizushima T, Yamanaka H, Kobayashi K, Dai Y, et al. TRPA1 induced in sensory neurons contributes to cold hyperalgesia after inflammation and nerve injury. J Clin Invest 2005;115(9):2393–401. [196] Silva CR, Oliveira SM, Rossato MF, Dalmolin GD, Guerra GP, da Silveira Prudente A, et al. The involvement of TRPA1 channel activation in the inflammatory response evoked by topical application of cinnamaldehyde to mice. Life Sci 2011;88(25–26):1077–87. [197] Shiba T, Tamai T, Sahara Y, Kurohane K, Watanabe T, Imai Y. Transient receptor potential ankyrin 1 activation enhances hapten sensitization in a T-helper type 2-driven fluorescein isothiocyanate-induced contact hypersensitivity mouse model. Toxicol Appl Pharmacol 2012;264(3):370–6. [198] Maruyama T, Iizuka H, Tobisawa Y, Shiba T, Matsuda T, Kurohane K, et al. Influence of local treatments with capsaicin or allyl isothiocyanate in the sensitization phase of a fluorescein-isothiocyanate-induced contact sensitivity model. Int Arch Allergy Immunol 2007;143(2):144–54. [199] Hill K, Schaefer M. Ultraviolet light and photosensitising agents activate TRPA1 via generation of oxidative stress. Cell Calcium 2009;45(2):155–64. [200] Oehler B, Scholze A, Schaefer M, Hill K. TRPA1 is functionally expressed in melanoma cells but is not critical for impaired proliferation caused by allyl isothiocyanate or cinnamaldehyde. Naunyn Schmiedebergs Arch Pharmacol 2012;385(6):555–63. [201] Bellono NW, Najera JA, Oancea E. UV light activates a Galphaq/11-coupled phototransduction pathway in human melanocytes. J Gen Physiol 2014;143(2):203–14. [202] Wu LJ, Sweet TB, Clapham DE. International Union of Basic and Clinical Pharmacology. LXXVI. Current progress in the mammalian TRP ion channel family. Pharmacol Rev 2010;62(3):381–404. [203] Saul S, Stanisz H, Backes CS, Schwarz EC, Hoth M. How ORAI and TRP channels interfere with each other: Interaction models and examples from the immune system and the skin. Eur J Pharmacol 2014;739:49–59. [204] Goel M, Sinkins WG, Schilling WP. Selective association of TRPC channel subunits in rat brain synaptosomes. J Biol Chem 2002;277(50):48303–10. [205] Dietrich A, Kalwa H, Gudermann T. TRPC channels in vascular cell function. Thromb Haemost 2010;103(2):262–70. 322 17. ROLE OF TRP CHANNELS IN SKIN DISEASES

[206] Tu CL, Chang W, Bikle DD. Phospholipase cgamma1 is required for activation of store-operated channels in human keratinocytes. J Invest Dermatol 2005;124(1):187–97. [207] Davis J, Burr AR, Davis GF, Birnbaumer L, Molkentin JD. A TRPC6-dependent pathway for myofibroblast transdifferentiation and wound healing in vivo. Dev Cell 2012;23(4):705–15. [208] Lievremont JP, Numaga T, Vazquez G, Lemonnier L, Hara Y, Mori E, et al. The role of canonical transient receptor potential 7 in B-cell receptor-activated channels. J Biol Chem 2005;280(42):35346–51. [209] Zimmermann K, Lennerz JK, Hein A, Link AS, Kaczmarek JS, Delling M, et al. Transient receptor potential cation channel, subfamily C, member 5 (TRPC5) is a cold-transducer in the peripheral nervous system. Proc Natl Acad Sci USA 2011;108(44):18114–19. [210] Staaf S, Maxvall I, Lind U, Husmark J, Mattsson JP, Ernfors P, et al. Down regulation of TRPC1 by shRNA reduces mechanosensitivity in mouse dorsal root ganglion neurons in vitro. Neurosci Lett 2009;457(1):3–7. [211] Garrison SR, Dietrich A, Stucky CL. TRPC1 contributes to light-touch sensation and mechanical responses in low-threshold cutaneous sensory neurons. J Neurophysiol 2012;107(3):913–22. [212] Beck B, Zholos A, Sydorenko V, Roudbaraki M, Lehen’kyi V, Bordat P, et al. TRPC7 is a receptor-operated DAG-activated channel in human keratinocytes. J Invest Dermatol 2006;126(9):1982–93. [213] Woelfle U, Laszczyk MN, Kraus M, Leuner K, Kersten A, Simon-Haarhaus B, et al. Triterpenes promote keratinocyte differentiation in vitro, ex vivo and in vivo: a role for the transient receptor potential canonical (subtype) 6. J Invest Dermatol 2010;130(1):113–23. [214] Cai S, Fatherazi S, Presland RB, Belton CM, Roberts FA, Goodwin PC, et al. Evidence that TRPC1 contributes to calcium-induced differentiation of human keratinocytes. Pflugers Arch 2006;452(1):43–52. [215] Pani B, Cornatzer E, Cornatzer W, Shin DM, Pittelkow MR, Hovnanian A, et al. Up-regulation of transient receptor potential canonical 1 (TRPC1) following sarco(endo)plasmic reticulum Ca2+ ATPase 2 gene silencing promotes cell survival: a potential role for TRPC1 in Darier’s disease. Mol Biol Cell 2006;17(10):4446–58. [216] Sakuntabhai A, Ruiz-Perez V, Carter S, Jacobsen N, Burge S, Monk S, et al. Mutations in ATP2A2, encoding a Ca2+ pump, cause Darier disease. Nat Genet 1999;21(3):271–7. [217] Fatherazi S, Presland RB, Belton CM, Goodwin P, Al-Qutub M, Trbic Z, et al. Evidence that TRPC4 supports the calcium selective I(CRAC)-like current in human gingival keratinocytes. Pflugers Arch 2007;453(6):879–89. [218] Beck B, Lehen’kyi V, Roudbaraki M, Flourakis M, Charveron M, Bordat P, et al. TRPC channels determine human keratinocyte differentiation: new insight into basal cell carcinoma. Cell Calcium 2008;43(5):492–505. [219] Muller M, Essin K, Hill K, Beschmann H, Rubant S, Schempp CM, et al. Specific TRPC6 channel activation, a novel approach to stimulate keratinocyte differentiation. J Biol Chem 2008;283(49):33942–54. [220] Sun XD, You Y, Zhang L, Zheng S, Hong Y, Li J, et al. The possible role of TRPC6 in atopic dermatitis. Med Hypotheses 2012;78(1):42–4. [221] Leuner K, Kraus M, Woelfle U, Beschmann H, Harteneck C, Boehncke WH, et al. Reduced TRPC channel expression in psoriatic keratinocytes is associated with impaired differentiation and enhanced proliferation. PLoS One 2011;6(2):e14716. [222] Vennekens R, Olausson J, Meissner M, Bloch W, Mathar I, Philipp SE, et al. Increased IgE-dependent mast cell activation and anaphylactic responses in mice lacking the calcium-activated nonselective cation channel TRPM4. Nat Immunol 2007;8(3):312–20. [223] Vriens J, Owsianik G, Hofmann T, Philipp SE, Stab J, Chen X, et al. TRPM3 is a nociceptor channel involved in the detection of noxious heat. Neuron 2011;70(3):482–94. [224] Proudfoot CJ, Garry EM, Cottrell DF, Rosie R, Anderson H, Robertson DC, et al. Analgesia mediated by the TRPM8 cold receptor in chronic neuropathic pain. Curr Biol 2006;16(16):1591–605. [225] Guo H, Carlson JA, Slominski A. Role of TRPM in melanocytes and melanoma. Exp Dermatol 2012;21(9):650–4. [226] Denda M, Tsutsumi M, Denda S. Topical application of TRPM8 agonists accelerates skin permeability barrier recovery and reduces epidermal proliferation induced by barrier insult: role of cold-sensitive TRP receptors in epidermal permeability barrier homoeostasis. Exp Dermatol 2010;19(9):791–5. [227] Oda S, Uchida K, Wang X, Lee J, Shimada Y, Tominaga M, et al. TRPM2 contributes to antigen-stimulated Ca(2)(+) influx in mucosal mast cells. Pflugers Arch 2013;465(7):1023–30. [228] Yamamoto S, Shimizu S, Kiyonaka S, Takahashi N, Wajima T, Hara Y, et al. TRPM2-mediated Ca2+influx induces chemokine production in monocytes that aggravates inflammatory neutrophil infiltration. Nat Med 2008;14(7):738–47. [229] Massullo P, Sumoza-Toledo A, Bhagat H, Partida-Sanchez S. TRPM channels, calcium and redox sensors during innate immune responses. Semin Cell Dev Biol 2006;17(6):654–66. REFERENCES 323

[230] Launay P, Cheng H, Srivatsan S, Penner R, Fleig A, Kinet JP. TRPM4 regulates calcium oscillations after T cell activation. Science 2004;306(5700):1374–7. [231] Barbet G, Demion M, Moura IC, Serafini N, Leger T, Vrtovsnik F, et al. The calcium-activated nonselective cation channel TRPM4 is essential for the migration but not the maturation of dendritic cells. Nat Immunol 2008;9(10):1148–56. [232] Duncan LM, Deeds J, Hunter J, Shao J, Holmgren LM, Woolf EA, et al. Down-regulation of the novel gene melastatin correlates with potential for melanoma metastasis. Cancer Res 1998;58(7):1515–20. [233] Deeds J, Cronin F, Duncan LM. Patterns of melastatin mRNA expression in melanocytic tumors. Hum Pathol 2000;31(11):1346–56. [234] Duncan LM, Deeds J, Cronin FE, Donovan M, Sober AJ, Kauffman M, et al. Melastatin expression and prognosis in cutaneous malignant melanoma. J Clin Oncol 2001;19(2):568–76. [235] Erickson LA, Letts GA, Shah SM, Shackelton JB, Duncan LM. TRPM1 (Melastatin-1/MLSN1) mRNA expression in Spitz nevi and nodular melanomas. Mod Pathol 2009;22(7):969–76. [236] Lu S, Slominski A, Yang SE, Sheehan C, Ross J, Carlson JA. The correlation of TRPM1 (Melastatin) mRNA expression with microphthalmia-associated transcription factor (MITF) and other melanogenesis-related proteins in normal and pathological skin, hair follicles and melanocytic nevi. J Cutan Pathol 2010;37 (Suppl. 1):26–40. [237] Devi S, Kedlaya R, Maddodi N, Bhat KM, Weber CS, Valdivia H, et al. Calcium homeostasis in human melanocytes: role of transient receptor potential melastatin 1 (TRPM1) and its regulation by ultraviolet light. Am J Physiol Cell Physiol 2009;297(3):C679–87. [238] Oancea E, Vriens J, Brauchi S, Jun J, Splawski I, Clapham DE. TRPM1 forms ion channels associated with melanin content in melanocytes. Sci Signal 2009;2(70):ra21. [239] Bellone RR, Brooks SA, Sandmeyer L, Murphy BA, Forsyth G, Archer S, et al. Differential gene expression of TRPM1, the potential cause of congenital stationary night blindness and coat spotting patterns (LP) in the Appaloosa horse (Equus caballus). Genetics 2008;179(4):1861–70. [240] Levy C, Khaled M, Iliopoulos D, Janas MM, Schubert S, Pinner S, et al. Intronic miR-211 assumes the tumor suppressive function of its host gene in melanoma. Mol Cell 2010;40(5):841–9. [241] Margue C, Philippidou D, Reinsbach SE, Schmitt M, Behrmann I, Kreis S. New target genes of MITF-induced microRNA-211 contribute to melanoma cell invasion. PLoS One 2013;8(9):e73473. [242] Segura MF, Greenwald HS, Hanniford D, Osman I, Hernando E. MicroRNA and cutaneous melanoma: from discovery to prognosis and therapy. Carcinogenesis 2012;33(10):1823–32. [243] Orfanelli U, Wenke AK, Doglioni C, Russo V, Bosserhoff AK, Lavorgna G. Identification of novel sense and antisense transcription at the TRPM2 locus in cancer. Cell Res 2008;18(11):1128–40. [244] McNeill MS, Paulsen J, Bonde G, Burnight E, Hsu MY, Cornell RA. Cell death of melanophores in zebrafish trpm7 mutant embryos depends on melanin synthesis. J Invest Dermatol 2007;127(8):2020–30. [245] Tsavaler L, Shapero MH, Morkowski S, Laus R. Trp-p8, a novel prostate-specific gene, is up-regulated in prostate cancer and other malignancies and shares high homology with transient receptor potential calcium channel proteins. Cancer Res 2001;61(9):3760–9. [246] Yamamura H, Ugawa S, Ueda T, Morita A, Shimada S. TRPM8 activation suppresses cellular viability in human melanoma. Am J Physiol Cell Physiol 2008;295(2):C296–301. [247] Xu H, Delling M, Li L, Dong X, Clapham DE. Activating mutation in a mucolipin transient receptor potential channel leads to melanocyte loss in varitint-waddler mice. Proc Natl Acad Sci USA 2007;104(46):18321–6. CHAPTER 18 Transient Receptor Potential Channels in Hypertension and Metabolic Syndrome Zhiming Zhu,* Daoyan Liu, Shiqiang Xiong Department of Hypertension and Endocrinology, Center for Hypertension and Metabolic Diseases, Daping Hospital, Third Military Medical University, Chongqing Institute of Hypertension, Chongqing, China *Corresponding author: [email protected]

OUTLINE

TRP Channels in Hypertension 326 Summary 331 Introduction 326 TRP Channels in Metabolic Syndrome 332 Distribution of TRP Channels in the Introduction 332 Vasculature 326 TRP Channels in Obesity 333 TRP Channels Participate in the TRP Channels in Diabetes 334 Regulation of Vascular Function 326 TRP Channels Link with Lipid TRP Channels in the Pathogenesis of Metabolism and Atherosclerosis 335 Hypertension 327 TRP Channels Implicated in Human Perspective 337 Hypertension 327 References 337 TRP Channels in Hypertensive Animal Models 327 Dysfunction of TRP Channels is Associated with Hypertension- Related Target Organ Damage 329

TRP Channels as Therapeutic Targets 325 © 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/B978-0-12-420024-1.00018-7 326 18. TRP CHANNELS REGULATE VASCULAR AND METABOLIC FUNCTION TRP CHANNELS IN HYPERTENSION

Introduction Hypertension, traditionally defined as a condition associated with systolic blood pressure ≥ 140 mmHg or diastolic blood pressure ≥ 90 mmHg, affects 1 billion people worldwide [1,2]. As one of the leading risk factors for deadly cardiovascular disease, hypertension is emerging as an important global public-health challenge. The pathogenesis of hypertension is complex, involving both genetic and environmental factors. Hypertension has been traditionally thought to be due to neural and humoral stimulation of vascular constriction and to endocrine and renal stimuli that control blood volume. Of these stimuli, intracellular Ca2+ homeostasis is essential for vascular function and blood pressure regulation [3]. Dysregulation and disturbance of Ca2+ homeostasis has been noted in experimental and human hypertension [4,5]. Although increased transmembrane calcium influx and enhanced calcium release via store- operated calcium (SOC) entry or receptor-operated calcium (ROC) entry have been observed in the vasculature of hypertensive rats, the mechanism underlying the abnormal cellular calcium handling remains to be elucidated. During the past two decades of research in vascular function regulation, a family of nonselective cation channels, called transient receptor potential (TRP) channels, has received attention because these channels play a unique role in regulating intracellular Ca2+ concentration and vascular function [6]. Mounting evidence indicates that TRP channels participate in vascular dysfunction and the development of hypertension.

Distribution of TRP Channels in the Vasculature It has been reported that all transient receptor potential canonical (TRPC) channel isoforms are expressed in endothelial cells [7]. Transient receptor potential melastatin 4 (TRPM4) and transient receptor potential vanilloid 4 (TRPV4) are also present in vascular endothelial cells, although they appear less abundant. All the TRP channels (except TRPV5, TRPV6, and TRPM1) are reportedly present in arterial smooth muscle from various segments of the vasculature [8].

TRP Channels Participate in the Regulation of Vascular Function Several TRP channels contribute to the contraction of blood vessels. Overexpression of the human TRPC1 gene in rat pulmonary artery enhances vasoconstriction via store depletion-mediated Ca2+ influx [9]. Thapsigargin-stimulated store-operated current was reduced in both TRPC1 siRNA- and TRPC1 antisense-expressing cells [10]. These findings suggest that TRPC1 is a component of the store-operated Ca2+ entry pathway. TRPC3 is involved in Uridine Triphosphate (UTP)-mediated depolarization and vasoconstriction of both coronary and cerebrovascular smooth muscle cells (SMCs) [11]. TRPC6 is implicated in pressure-induced depolarization and vasoconstriction and contributes to myogenic constriction of cerebral arteries [12]. Dietrich et al. showed that constitutively active TRPC3 channels were up-regulated in TRPC6-deficient SMCs and that vascular SMC contractility was increased [13]. TRPC6 channels are thought to play a fundamental role in the regulation of smooth muscle tone in blood vessels [14]. Kark et al. demonstrated that functional expression of TRPV1 in vascular SMCs mediated vasoconstriction of the resistance arteries [15]. TRPM4 was discovered to contribute to pressure-induced SMC TRP Channels in Hypertension 327 depolarization and vasoconstriction [16] and cerebral blood flow regulation [17]. In contrast, some TRP channels are involved in vasodilation. TRPC1/TRPC3 is inhibited by the NO/cyclic guanosine monophosphate/Protein Kinase G (cGMP/PKG) pathway in SMCs and contributes to NO-induced vasorelaxation [18]. An essential role for TRPC4 channels in endothelium- dependent regulation of vascular tone has been recently proposed [19]. Agonist-induced Ca2+ entry is dramatically reduced in aortic endothelial cells of mice that lack TRPC4; the endothelium- dependent vascular relaxation in response to ATP and acetylcholine is also impaired [19]. TRPV1 is expressed abundantly in the endothelium. In isolated rat and mouse mesenteric arteries, activation of TPRV1 by its agonist capsaicin elicits an acute release of NO from endothelial cells and leads to vasodilation [20]. In contrast, this effect is reduced by TRPV1 receptor antagonists and is absent in arteries of the TRPV1−/− mouse [20]. TRPV4 is also involved in vasodilation induced by epoxyeicosatrienoic acid compounds [21], which cause endothelium-dependent vasodilation in rat coronary and cerebral arteries [22,23]. Both endothelial and smooth muscle TRPV4 channels are critical for vasodilation of mesenteric arteries in response to endothelial-derived factors [24]. Earley et al. reported that TRPA1 channels were present in the endothelium of rat cerebral and cerebellar pial arteries [25]. Activation of TRPA1 channels causes endothelium-dependent SMC hyperpolarization and vasodilation in this vascular bed [25].

TRP Channels in the Pathogenesis of Hypertension TRP Channels Implicated in Human Hypertension Abnormal expression and function of TRPC channels have been reported in both essential hypertension patients and rat models. Thilo et al. observed a significant correlation between TRPC3 transcripts and systolic blood pressure, expression of IL-1β, and TNF-α in monocytes from patients with essential hypertension [26]. They observed an approximately eightfold increase in TRPC3 transcripts in monocytes from patients with essential hypertension compared with normotensive control subjects [26]. Liu et al. compared the expression level and function of TRPCs between essential hypertensive patients and normotensive control subjects, and for the first time, they noted increased TRPC3 and TRPC5 protein expression and an increase in the gadolinium/calcium-influx ratio in essential hypertensive patients via TRPCs [27]. In addition, increased TRPC3 and TRPC5 expression and a subsequent SOC influx and increased 1-oleoyl-2-acetyl-sn-glycerol-induced cation influx in monocytes of patients with essential hypertension were observed ([28]; Table 18.1). Mutations in the gene encoding WNK lysine deficient protein kinase 4 (WNK4), which is a WNK family kinase that regulates the expression of TRPV4, have been linked to monogenic hypertension. Fu et al. reported that coexpression of WNK4 down-regulated TRPV4 function by decreasing its cell surface expression in HEK-293 cells [39]. They demonstrated functional regulation of TRPV4 by WNK4 and speculated that this pathway may influence systemic Ca2+ balance [39].

TRP Channels in Hypertensive Animal Models Disturbances in the regulation of the cytosolic calcium concentration play a key role in the pathogenesis of primary hypertension. Liu et al. evaluated the expression and function of calcium-permeable TRPCs in normotensive WKY and spontaneously hypertensive rats (SHRs) and demonstrated an increase in TRPC3 channel expression, increased TRPC3- related calcium influx, which was associated with increased contraction, and an increase in 328 18. TRP CHANNELS REGULATE VASCULAR AND METABOLIC FUNCTION

TABLE 18.1 Roles of TRP Channels in the Regulation of Vascular Function and the Pathogenesis of Hypertension

TRP channels Distribution Proposed roles Main References TRPC1 VSMC A component of store-operated Ca2+ entry pathway. [9,10] TRPC1, TRPC3 VSMC 1. Inhibited by the NO/cGMP/PKG pathway in [18] VSMC, contributes to NO-induced vasodilation [29] 2. Increased expression and contribution to increased vasomotion in hypertension TRPC3 VSMC Involved in UTP-mediated depolarization and [11] vasoconstriction TRPC3, TRPC5 VSMC Increased expression and calcium influx in VSMC [27,28] Monocyte and monocytes from hypertensive animals and patients TRPC6 VSMC TRPC6 knockout mice show elevated blood pressure [13] and enhanced vasoconstriction TRPV1 VSMC 1. Expressed in VSMC and mediating [15,20,30–34] Endothelial cell vasoconstriction of the resistance arteries 2. Involved in endothelial-dependent vasodilation 3. TRPV1 activation prevents hypertension 4. TRPV1 reduction is related to salt-induced increase in blood pressure 5. Regulation of salt-intake behaviors that are associated with the development of salt-sensitive hypertension TRPV4 VSMC 1. TRPV4 activation in the endothelium and VSMC [24,35] Endothelial cell promotes the vasodilation of mesenteric arteries 2. Enhanced expression of TRPV4 may counterbalance salt-induced increases in blood pressure TRPM4 VSMC Contribution to pressure-induced vasoconstriction [16,17] and cerebral blood flow regulation TRPM7 VSMC Reduced TRPM7 in SHRs contributes to [36] vasoconstriction in VSMC TRPM8 VSMC 1. Regulation of vascular tone [37,38] 2. Reduced TRPM8 may contribute to the enhanced vasoreactivity in PH TRPA1 VSMC TRPA1 activation elicits cerebral artery vasodilation [25] Endothelial cell

For details and references, see text.

angiotensin II-induced TRPC3 expression in vasculature from SHRs [5,39]. Furthermore, they noted that increased rhythmicity in hypertensive arterial smooth muscle is linked to TRPCs [29]. Norepinephrine-induced vasomotion and calcium influx were increased in mesenteric arterioles from SHRs, and TRPC1, TRPC3, and TRPC5 expression was also up-regulated [29]. Administration of candesartan or telmisartan, but not amlodipine, significantly reduced TRP Channels in Hypertension 329 the expression of TRPC1, TRPC3, and TRPC5 and norepinephrine-induced vasomotion in mesenteric arterioles from SHRs [29]. In addition, TRPC3 channel expression was greatly enhanced in TRPC6 knockout mice; however, up-regulation of TRPC3 did not functionally replace TRPC6 [13]. TRPC6 knockout mice show increased blood pressure and enhanced agonist-induced arterial vasoconstriction [13]. Increased TRPC3 expression relative to that of TRPC6 may predispose mice to hypertension [40]. Bae et al. reported that expression of TRPC6 and ROC currents were increased in mesenteric arteries from deoxycorticosterone acetate-salt hypertensive rats [41]. The presence of TRPC1 and TRPC6 is essential for the full development of hypoxic pulmonary hypertension (PH) in the mouse model [42]. Chronic hypoxia increased serotonin-induced vasoconstriction significantly; the augmented vasoreactivity was attenuated in TRPC1−/− and eliminated in TRPC6−/− pulmonary arteries [42]. Intracellular Mg2+ depletion has been implicated in vascular dysfunction in hypertension. Reduced TRPM7 expression is associated with reduced cytosolic Mg2+ concentration in mesenteric arterial SMCs from SHRs, which may facilitate vasoconstriction [36]. TRPM8 is involved in the regulation of vascular tone [37]. Liu et al. reported that down-regulation of TRPM8 may contribute to the enhanced vasoreactivity in PH [38]. Emerging evidence has shown that the TRPV1 channel is implicated in hypertension. TRPV1 activation exerts antihypertension effects by stimulating the release of calcitonin gene-related peptide (CGRP) from capsaicin-sensitive nerves and NO from endothelial cells [30]. Plasma concentrations of CGRP rise transiently after acute administration of capsaicin in adult rats and is accompanied by a decrease in blood pressure [31]. Our study demonstrated that activation of TRPV1 increased the phosphorylation of Protein Kinase A (PKA) and endothelial nitric oxide synthase (eNOS) and, thus, the production of NO in endothelial cells. Long-term stimulation of TRPV1 by dietary capsaicin lowered blood pressure in SHRs, but there was no change in plasma concentration of CGRP and substance P in SHR after long- term consumption of capsaicin [20]. TRPV1 has also been proposed to be involved in the pathogenesis of salt-induced hypertension. Wang demonstrated that TRPV1 was activated and its expression was up-regulated during high salt intake in Dahl salt-resistant rats, which prevented the salt-induced increase in blood pressure [32]. In contrast, TRPV1 expression and function was impaired in Dahl salt-sensitive rats, which rendered Dahl salt-sensitive rats to salt load in terms of blood pressure regulation [32]. Our study revealed that chronic administration of capsaicin reduced the high-salt-intake-induced endothelial dysfunction and nocturnal hypertension in part by preventing the generation of superoxide anions and via NO reduction in mesenteric arteries by activating vascular TRPV1 [33]. TRPV1 receptors may also mediate a general aversive response to salt [34], indicating a role for TRPV1 in regulating salt intake behaviors that are linked to the development of salt-sensitive hypertension. TRPV4 channels are also critically associated with salt-sensitive hypertension. Salt intake may enhance the expression of TRPV4 to counterbalance salt-induced increases in blood pressure in a salt-resistant strain of rats [35]. These findings highlight a promising role for TRPV4 in the treatment of salt-sensitive hypertension [35].

Dysfunction of TRP Channels is Associated with Hypertension-Related Target Organ Damage High blood pressure frequently causes target organ damage including atherosclerosis, cardiac hypertrophy, stroke, myocardial infarction, and end-stage renal failure [43]. Recent 330 18. TRP CHANNELS REGULATE VASCULAR AND METABOLIC FUNCTION studies have shown that some TRP channels participate in the pathogenesis of cerebrovascular dysfunction, cardiac hypertrophy, renal damage, and atherosclerosis.

TRP CHANNELS IN CEREBROVASCULAR DYSFUNCTION TRPC3 channels play a fundamental role in the regulation of vascular smooth muscle tone and in autoregulation of cerebral blood flow due to their role in the regulation of cerebral vascular contractility [44]. Suppression of TRPC3 expression in arterial vascular smooth muscle significantly decreased the depolarization and constriction of intact cerebral arteries in 2+ 2+ response to UTP [11]. In isolated cerebral artery myocytes, SR Ca release, IP3-induced [Ca ]i elevation, and vasoconstriction were reduced after TRPC3 knockdown and treatment with voltage-dependent Ca2+ channel blockers [45]. Thilo et al. proposed that TRPC3 expression was associated with hypertension and hypoxic conditions in human cerebral vascular tissue [46]. Reading and Brayden suggested that TRPM4 channels in cerebrovascular myocytes contributed to the autoregulation of cerebral blood flow in vivo [17]. In vivo suppression of TRPM4 decreased cerebral artery myogenic constriction and impaired autoregulation [17]. Gerzanich et al. showed a role for TRPM4 in secondary hemorrhage following central nervous system injury [47]. The up-regulation of TRPM4 led to cellular swelling and oncotic cell death [47]. TRPM6 was significantly reduced in cerebral vascular tissue taken from patients after hypertensive intracerebral hemorrhage when compared with control tissue [46]. Xu et al. demonstrated that activation of TRPV1 channels by dietary capsaicin caused increased phosphorylation of eNOS, delayed the onset of stroke, and further increased survival time in stroke-prone SHRs [48].

TRP CHANNELS AND CARDIAC HYPERTROPHY Hypertension plays an important role in the development of cardiac hypertrophy and heart failure. Emerging evidence indicates that TRP channels are critical regulators of microdomain signaling in the heart that controls pathological hypertrophy together with signaling via effectors including calcineurin and nuclear factor of activated T cells (NFAT) [49]. Ohba et al. first reported a potential role for TRPC1 channels in pressure overload-induced hypertrophy [50]. Expression of TRPC1 was significantly increased in the hearts of abdominal aortic-banded rats compared with sham-operated rats. ET-1 treatment resulted in increased expression of brain natriuretic protein, atrial natriuretic factor, and TRPC1 and increased cell surface area in neonatal myocytes. Silencing of TRPC1 with siRNA attenuated store-operated calcium entry (SOCE) and cardiac hypertrophy. TRPC1 gene-deleted mice were profoundly protected from cardiac hypertrophy following pressure overload [51]. NFAT is a Ca2+-dependent transcription factor that is activated in pathological hypertrophy [52]. Several subtypes of TRPC mediate Ca2+ influx, which is essential for NFAT-mediated hypertrophy. Nakayama et al. demonstrated that TRPC3-overexpressing transgenic mice showed significant increases in SOCE and developed cardiomyopathy with a loss of ventricular functional performance [53]. In addition, cardiac hypertrophy was synergistically increased in TRPC3 transgenic mice when they were subjected to pressure overload or Ang II phenylephrine infusion [53]. However, the augmented hypertrophic phenotype in TRPC3 transgenic mice was abolished when calcineurin Aβ was deleted [53]. Selective inhibition of TRPC3 via Pyr3 was reported to attenuate activation of NFAT and block cardiac hypertrophy in mice subjected to pressure overload [54]. Similarly, studies from other groups showed that cardiac-specific TRPC6 transgenic mice TRP Channels in Hypertension 331 showed heightened sensitivity to pressure overload and agonist-induced cardiac hypertrophy [55]. TRPC6 acts as a positive regulator of calcineurin-NFAT signaling that drives pathological hypertrophy [55]. Recently, Wu et al. generated cardiac-specific transgenic mice that express dominant-negative (dn) TRPC3, dnTRPC6, or dnTRPC4 that block the activity of the TRPC3/6/7 or TRPC1/4/5 subfamily of channels in the heart [56]. Remarkably, all three dn transgenic strategies attenuated the cardiac hypertrophic response following either neuroendocrine agonist infusion or press-overload treatment. In addition, dnTRPC4 cross-inhibited the activity of the TRPC3/6/7 subfamily in the heart, suggesting that TRPC subfamilies function as a coordinated complex [56]. The prohypertrophic effects of TRPC channels have also been shown in vitro in cultured cardiomyocytes [49]. The expression of TRPM4 protein is increased in cardiomyocytes from SHRs and is associated with left ventricular hypertrophy relative to normotensive WKY rats [57].

TRP CHANNELS IN RENAL DYSFUNCTION The kidney is one of the major target organs for hypertension, which regulates blood pressure via sodium excretion [37]. TRP channels expressed along different parts of the nephron suggest their involvement in renal function and/or pathogenesis of renal diseases [58]. TRPC6 channels have been proposed to influence the filtration barrier function of podocytes in the glomerulus [59]. Transient overexpression of TRPC6 in the mice slit diaphragm resulted in proteinuria [59]. This abnormally high expression led to disturbed Ca2+ regulation and disruption of the podocyte actin cytoskeleton, which in turn led to impaired podocyte function and proteinuria [59]. Thilo et al. demonstrated that VEGF regulated TRPC6 expression in podocytes and proteinuria [60]. Activation of TRPV1 in vivo or in isolated perfused kidneys increased the glomerular filtration rate and enhanced renal sodium and water excretion [61,62]. TRPV1 dysfunction led to impaired renal excretory function and disturbed hemodynamic homoeostasis [63]. A protective role of TRPV1 was observed in uninephrec- tomized mice administered with Deoxycortone Acetate (DOCA)-salt [64]..Renal inflammation was aggravated in TRPV1 knockout mice subjected to DOCA-salt hypertension [65]. These findings imply that TRPV1 mediates a protective signal pathway in salt-induced renal damage. Disturbance of the intracellular Mg2+ concentration leads to vascular dysfunction in hypertension [36]. TRPM6 is crucial for transcellular Mg2+ transport in the kidney. Loss-of-function mutations in TRPM6 lead to hypomagnesemia with secondary hypocalcemia [66]. TRPV4 may function as an osmoreceptor in the kidney and participate in the regulation of sodium and water balance [67]. These TRP channels may play a role in hypertension by regulating the total body divalent cation homeostasis and renal perfusion/hemodynamics.

Summary Mounting evidence reveals a critical role of TRP channels in the physiological regulation of vascular function and blood pressure. Functional equilibrium between TRP channels plays a critical role in maintaining vascular physiological function and blood pressure. Enhanced TRPC3/5-mediated vasoconstriction and impaired TRPV1-induced vasodilation was detected in hypertension [5,20,29,68,69]. Based on these findings, we propose that the imbalance in TRP channel function may be one etiology of hypertension. The impaired balance in TRP 332 18. TRP CHANNELS REGULATE VASCULAR AND METABOLIC FUNCTION

FIGURE 18.1 The functional balance in TRP channels in the regulation of vascular function and blood pressure. Multiple TRP channels are present in VSMC and the endothelium. Activation of TRPC3/5 results in an increase in VSMC [Ca2+]i via SOC- and diacylgylcerol (DAG)-mediated Ca2+ influx. Ang II and norepinephrine upregulate TRPC3/5 expression and lead to consequent Ca2+ influx and vasoconstriction. Activation of TRPV1 increases the phosphorylation of PKA and eNOS, thereby leading to production of NO in endothelial cells and vasodilation. A disturbance to this balance leads eventually to hypertension. channel function results in elevated [Ca2+]i and enhanced vasoconstriction and/or reduced vasodilation, thus contributing to the development of hypertension (Figure 18.1).

TRP CHANNELS IN METABOLIC SYNDROME

Introduction Metabolic syndrome is a cluster of closely related abnormalities that are associated with insulin resistance and increased cardiovascular risks, which include visceral obesity, hypertension, hyperglycemia, and dyslipidemia [70]. Based on the modified NCEP-ATP III report [71], metabolic syndrome is defined as being the composite of three or more of the following conditions: abdominal obesity with a waist circumference ≥ 90 cm in men and ≥ 80 cm in women; high blood pressure including systolic blood pressure ≥ 130 mmHg or diastolic blood pressure ≥ 85 mmHg; high fasting blood glucose ≥ 110 mg/dL (6.1 mmol/L); hypertriglyceridemia ≥ 150 mg/dL (1.7 mmol/L); or high-density lipoprotein cholesterol < 40 mg/dL (1.0 mmol/L) in men or < 50 mg/dL (1.29 mmol/L) in women. Evidence from clinical trials indicates that metabolic syndrome is associated with increased cardiovascular risk, including a higher risk of 18-year total mortality, cardiovascular disease mortality, coronary heart disease mortality, myocardial infarction, and stroke compared with those without metabolic syndrome [72–74]. TRP channels are broadly distributed in the vascular SMCs and endothelial cells and in the liver, pancreas, adipose tissue, and skeletal muscle, which explains their widespread function [75]. Moreover, TRP channels participate in hypertension, and dysfunction of TRP channels also contributes to the pathogenesis of obesity, diabetes, dyslipidemia, and TRP Channels in Metabolic Syndrome 333 atherosclerosis. Thus, changes in expression or dysfunction of TRP channels likely results in the development of metabolic syndrome.

TRP Channels in Obesity Obesity is not only a cardiometabolic risk factor but also a hallmark of metabolic syndrome [75]. Several TRP channels are associated with adipocytes and obesity. Studies from both humans and animals indicate that TRPV1 and its agonist capsaicin play a role in energy expenditure and the development of obesity. Capsaicin metabolic stability was evaluated in rat, dog, and mouse microsomes. These results suggest that capsaicin can be extensively and rapidly metabolized in the liver, consequently reducing systemic exposure. Metabolites of capsaicin, including hydroxy-capsaicin, 16,17-dehydrocapsaicin, and vanillylamine, were detected [76]. Furthermore, vanillylamine-like capsaicin is a structural analog of eugenol, a TRPV1 agonist [77,78]. Capsaicin can affect cardiovascular function through stimulating the release of CGRP and substance P from perivascular sensory nerve endings [79]. Capsaicin may exert direct effects on the vasculature, such as relaxing coronary, mesenteric, hepatic, basilar, and meningeal arteries of pigs and rats [80,81]. We assessed the time-dependent response to intragastric administration of capsaicin (15 mg/kg body weight) in rats. The absolute bioavailability of oral capsaicin was approximately 0.106% [20]. This small bioavailability can be caused by rapid hepatic metabolism of capsaicin in the liver [76,82]. Epidemiological data revealed that consumption of foods containing capsaicin was associated with a lower prevalence of obesity [83]. Rodents fed a diet containing 0.014% capsaicin showed a significant reduction in visceral fat-pad weight but no change in caloric intake [84]. Red pepper intake was shown to increase diet-induced thermogenesis and lipid oxidation [85]. Consumption of nonpungent capsaicin analogs (capsinoids) for 4-12 weeks both enhanced fat oxidation and significantly reduced abdominal adiposity in subjects with high Body Mass Index (BMIs) [86]. However, the mechanisms underlying these effects are not fully understood. One study has shown that intragastric administration of capsiate promotes energy consumption by triggering thermogenic sympathetic responses in a time- and dose-dependent manner in rats [87]. Another study in humans proposed that capsaicin stimulated sensory neurons in the mouth and gastrointestinal tract, resulting in increased noradrenaline levels. We showed that the activation of TRPV1 channels by dietary capsaicin increased cytosolic calcium and prevented adipogenesis of 3T3-L1- preadipocytes in vitro [88]. This effect was attenuated following TRPV1 knockdown. TRPV1 expression and capsaicin-mediated calcium influx were reduced in visceral adipose tissue from obese db/db and ob/ob mice and from obese human male subjects. Chronic dietary capsaicin consumption prevented obesity in WT mice fed a high-fat diet but not in TRPV1 knockout mice [88]. This study indicates that capsaicin may prevent adipogenesis and obesity via a direct action on TRPV1 channels in adipocytes. Furthermore, endogenous TRPV1 ligands can reduce food intake in WT mice but not in TRPV1-null mice [89]. Taken together, these findings further support a link between TRPV1 channels and energy metabolism related to obesity. TRPV4, originally characterized as a Ca2+-preferred cation channel, is a regulator of adipose oxidative metabolism, inflammation, and energy homeostasis [90]. TRPV4 deficiency protects mice from diet-induced obesity, adipose inflammation, and insulin resistance [90]. However, TRPV4 deficient mice have also been reported to show an increased susceptibility to obesity when fed a high-fat diet [91]. These conflicting phenotypes may result from different animal models used for each study. 334 18. TRP CHANNELS REGULATE VASCULAR AND METABOLIC FUNCTION

TRPM channels have also been reported to play a role in energy expenditure regulation. TRPM2 deletion protects mice from diet-induced obesity, which is associated with increased energy expenditure and expression of Peroxisome proliferator-activated receptor coactivator-1 α (PGC-1α), PGC-1β, Peroxisome proliferator-activated receptor α, Estrogen related receptors α, Mitochondrial transcription factor A, and Medium-chain acyl-CoA dehydrogenase in white adipose tissue [92]. TRPM5 plays a central role in sweet, bitter, and umami taste signaling and is also reportedly involved in fatty acid transduction in mouse taste cells [93]. Drugs designed to modulate TRPM5 may be useful for weight loss by impacting taste sensory signals and reducing energy consumption [94]. Chen et al. demonstrated the expression of TRPM7 in 3T3-LI preadipocytes [95]. Knocking down TRPM7 inhibited both proliferation and adipogenesis in 3T3-LI cells [95]. Our study showed that the cold-sensing TRPM8 channel was functionally present in mouse brown adipose tissue and that dietary obesity was prevented by activating TRPM8 with menthol [96]. Expression of UCP1 was up-regulated in adipocytes treated with menthol. Chronic dietary menthol significantly increased the core temperatures and locomotor activity in wild-type mice; these effects were absent in both TRPM8−/− and UCP1−/− mice [96]. These findings reveal a novel role for TRPM8, which could constitute a promising method for treating obesity.

TRP Channels in Diabetes Diabetes mellitus is characterized by high blood glucose levels, which results from defective insulin secretion and/or insulin sensitivity. Several TRP channels are implicated in the diabetic process. The role of TRPV1 in glucose homeostasis is controversial. Evidence indicates that TRPV1 may promote insulin secretion via its role as a calcium channel in β-cells [97], which is different from its role in TRPV1+ fibers [98]. The relationship between TRPV1 in β-cells and TRPV1 in neuronal cells remains unclear. Therefore, how TRPV1 affects insulin synthesis, degradation, and secretion requires further investigation. Several studies show that the removal of TRPV1+ fibers in Zucker diabetic fatty (ZDF) rats by TRPV1 agonists prevents the deterioration in glucose homeostasis by increasing insulin secretion and insulin responses [99,100]. Karlsson et al. reported that sensory denervation by capsaicin results in increased glucose tolerance in mice, which was due in part to a potentiated early insulin response to glucose [101]. Wang et al. noted that TRPV1 was localized to secretin tumor cell-1 (STC-1) cells and the ileum [102]. Capsaicin stimulated glucagon-like peptide-1 (GLP-1) secretion from STC-1 cells in a calcium-dependent manner via activation of TRPV1. Acute administration of capsaicin by gastric gavage increased GLP-1 and insulin secretion in WT but not TRPV1−/− mice [102]. Chronic dietary capsaicin not only improved glucose tolerance and increased insulin levels but also lowered daily blood profiles and increased plasma GLP-1 levels in WT mice but not TRPV1−/− mice [102]. Furthermore, TRPV1 activation by dietary capsaicin ameliorated abnormal glucose homeostasis and increased GLP-1 levels in the plasma and ileum of diabetic mice. There is also a report showing that glucose-stimulated insulin secretion is attenuated in capsaicin-treated rats, although their glucose levels did not change [103]. Administration of a TRPV1 antagonist suppresses CGRP secretion in pancreatic nerve fiber cells, thereby stimulating insulin secretion [104]. Several TRP channels are expressed in the pancreas and may be involved in the regulation of insulin secretion. TRPV5 locates to the secretory granules of pancreatic β-cells and may play a role in the regulation of endocrine Ca2+ homeostasis [105]. The expression level of TRP Channels in Metabolic Syndrome 335

TRPV5 is reduced in the pancreas of ZDF rats compared with nondiabetic rats [105]. TRPV6 has also been determined to be present in mouse and human exocrine pancreas cells but not β-cells [106,107]. It was recently observed that glucose tolerance and insulin secretion are impaired in TRPM2−/− mice [108]. Insulin secretion from islets of TRPM2−/− mice in response to glucose treatment was reduced [108]. TRPM5 acts as a regulator of insulin secretion in β-cells [109]. Glucose-induced insulin secretion is reduced in TRPM5−/− mice [109]. TRPM1, TRPM6, and TRPM7 are confirmed as susceptibility genes for diabetes in humans. However, further research is required to elucidate how these TRP channels regulate insulin secretion and the precise interactions between these TRP channels in the pathogenesis of diabetes [110,111]. TRP channels are also implicated in diabetic complications. Niehof and Borlak showed that dysfunction of TRPC1 is linked to the pathogenesis of diabetic nephropathy [112]. High glucose reduced TRPC6 expression significantly in cultured mesangial cells [113]. An increase in expression of TRPC6 protein was observed in glomeruli from patients with diabetic nephropathy compared with controls [60]. Vessels from diabetic patients are significantly more contracted than those of nondiabetic patients [114]. This contraction is inhibited by a TRPC channel inhibitor [114]. The expression level of TRPV1 in diabetes mellitus hearts is significantly lower. Diabetes- induced TRPV1 alterations are associated with poor recovery of cardiac function after myocardial ischemia [115]. The remodeling of voltage- and ligand-gated ion channels, such as TRP channels, can increase excitability of the sensory neurons and may play a role in diabetic peripheral neuropathy [116]. Facer et al. reported that the expression level of TRPV1 was reduced in nerve fibers in diabetic neuropathy skin samples, and a significant decrease in TRPV3 in diabetic skin was also observed [117]. Wuensch et al. showed that high glucose-induced oxidative stress increases TRPC6 expression and calcium influx in human monocytes [118]. Liu et al. reported that TRPC6 expression is increased in platelets from patients with type 2 diabetes mellitus [119]. These findings point to a novel pathway causing increased activation of monocytes and platelets and hence increased atherosclerosis in patient with diabetes. These studies also indicate the important role of TPR channels in hyperglycemia-induced target organ damage.

TRP Channels Link with Lipid Metabolism and Atherosclerosis The activity of TRP channels can be modulated by different lipid products or mediators [75]. Both membrane potential and level of phosphatidylinositol phosphates are efficient regulators of TRP channel gating [120]. Meanwhile, the physiological relevance of TRP channels in lipid metabolism has also been reported. Capsaicin-mediated activation of TRPV1 channels reduces diet-induced hypertriglyceridemia in rats [121], and rats fed a high-fat diet display higher lipoprotein lipase activity in adipose tissues following capsaicin administration [122]. Li et al. reported that chronic stimulation of TRPV1 by dietary capsaicin reduced circulating lipid levels significantly and prevented high-fat-induced fatty liver in WT mice by up-regulating UCP2; however, these effects were absent in TRPV1−/− mice [123]. Atherosclerosis is a major complication of metabolic syndrome. Several types of cells including VSMCs, endothelial cells, monocytes/macrophages, and platelets are involved in the pathogenesis of atherosclerosis. The level of TRPC1 expression in VSMCs correlates with the severity of atherosclerosis in Ossabaw swine fed a high-fat diet [124]. Ang II and subsequent NF-κB activation induces VSMC hypertrophy by increasing TRPC1 expression [125]. Recent studies suggest that dysregulation of TRPC1, TRPC3, TRPC4, and TRPC6 is 336 18. TRP CHANNELS REGULATE VASCULAR AND METABOLIC FUNCTION associated with vascular endothelial barrier dysfunction [126]. Increased activation of monocytes is associated with accelerated atherosclerosis in diabetes and hypertension [75]. High glucose-induced oxidative stress may increase TRPC6 expression and calcium influx in human monocytes [118]. Our studies indicate that high glucose increases phosphatidylinositol 3-kinase activity, which enhances the translocation of TRPC6 channels to the platelet surface and then enhances platelet activation [119] and increases TRPC3 and TRPC5 expression with a subsequent increase in SOC influx in the monocytes of patients with essential hypertension [28]. Ma et al. showed that TRPV1 activation significantly inhibited the accumulation of lipids in VSMCs by increasing ATP-binding cassette transporter A1 expression and reducing low-density lipoprotein-related protein 1 expression via calcineurin- and protein kinase A-dependent mechanisms [127]. Long-term activation of TRPV1 by capsaicin reduced atherosclerotic lesions in aorta from ApoE−/− mice but not from ApoE−/−/TRPV1−/− mice. These data indicate that TRPV1 activation ameliorates high-fat diet-induced atherosclerosis [127]. Moreover, these findings underscore the pathophysiological relevance of TRP channels in the development of atherosclerosis. Evidence indicates that some dietary factors or compounds including apigenin, cinnamaldehyde, menthol, and capsaicin are specific agonists for TRPV4, TRPA1, TRPM8, and TRPV1, respectively [128–130]. Capsaicin-mediated activation of TRPV1 causes multiple effects in cardiometabolic diseases (Figure 18.2) [20,48,88,102,127,131].

FIGURE 18.2 Activation of TRPV1 by capsaicin prevents cardiometabolic diseases. Activation of TRPV1 by dietary capsaicin leads to increased cerebrovascular vasodilation, cholesterol efflux, artery vasodilation, insulin secretion, thermogenesis, reduced lipidosis, insulin resistance, and adipogenesis and thereby prevents cardiometabolic diseases. REFERENCES 337

Cavanaugh et al. genetically modified the TRPV1 locus to reveal, with excellent sensitivity and specificity, the distribution of TRPV1 in all neuronal and non-neuronal tissues [132]. Restricted expression of TRPV1 in the Central Nerve System is conserved across species. Although TRPV1 expresses in other tissues and its non-neuronal function is reported, its nature is worth further investigation [132].

PERSPECTIVE

TRP channels exert multiple functions in the physiological regulation of vascular function and metabolism. Disturbance of the functional balance between TRP channels in these processes causes many deleterious effects that may be an important cause of metabolic syndrome. It is of great interest to further elucidate how TRP channels accomplish their roles in the physiology and pathology of vascular function and metabolism. In-depth studies of not only the role of specific TRP channels themselves but also the functional network of these channels must be completed. To accomplish this goal, new strategies including systems biology, engineering of novel animal models, optogenetics, and channelomics may be helpful. Although TRP channels play a crucial role in the pathogenesis of cardiometabolic diseases, the lack of specific channel modulators prevents our ability to use them to treat cardiometabolic diseases. Thus, the development of novel modulators of TRP channels is urgent. In addition, the translational research of TRP should be accelerated, and in particular, novel research findings should be promoted from the bench to the bedside.

References [1] Kearney PM, Whelton M, Reynolds K, Muntner P, Whelton PK, He J. Global burden of hypertension: analysis of worldwide data. Lancet 2005;365(9455):217–23. [2] Cowley Jr AW, Nadeau JH, Baccarelli A, Berecek K, Fornage M, Gibbons GH, et al. Report of the National Heart, Lung, and Blood Institute Working Group on epigenetics and hypertension. Hypertension 2012;59(5):899–905. [3] Firth AL, Remillard CV, Yuan JX. TRP channels in hypertension. Biochim Biophys Acta 2007;1772(8):895–906. [4] Thilo F, Loddenkemper C, Berg E, Zidek W, Tepel M. Increased TRPC3 expression in vascular endothelium of patients with malignant hypertension. Mod Pathol 2009;22(3):426–30. [5] Liu D, Yang D, He H, Chen X, Cao T, Feng X, et al. Increased transient receptor potential canonical type 3 channels in vasculature from hypertensive rats. Hypertension 2009;53(1):70–6. [6] Earley S, Reading S, Brayden JE. Functional Significance of Transient Receptor Potential Channels in Vascular Function. In: Liedtke WB, Heller S, editors. TRP Ion Channel Function in Sensory Transduction and Cellular Signaling Cascades. Boca Raton (FL);2007. [7] Yao X, Garland CJ. Recent developments in vascular endothelial cell transient receptor potential channels. Circ Res 2005;97(9):853–63. [8] Earley S. Vanilloid and melastatin transient receptor potential channels in vascular smooth muscle. Microcirculation 2010;17(4):237–49. [9] Kunichika N, Yu Y, Remillard CV, Platoshyn O, Zhang S, Yuan JXJ. Overexpression of TRPC1 enhances pulmonary vasoconstriction induced by capacitative Ca2+ entry. Am J Physiol Lung Cell Mol Physiol 2004;287(5):L962–9. [10] Brueggemann LI, Markun DR, Henderson KK, Cribbs LL, Byron KL. Pharmacological and electrophysiological characterization of store-operated currents and capacitative Ca2+ entry in vascular smooth muscle cells. J Pharmacol Exp Ther 2006;317(2):488–99. [11] Reading SA, Earley S, Waldron BJ, Welsh DG, Brayden JE. TRPC3 mediates pyrimidine receptor-induced depolarization of cerebral arteries. Am J Physiol Heart Circ Physiol 2005;288(5):H2055–61. 338 18. TRP CHANNELS REGULATE VASCULAR AND METABOLIC FUNCTION

[12] Welsh DG, Morielli AD, Nelson MT, Brayden JE. Transient receptor potential channels regulate myogenic tone of resistance arteries. Circ Res 2002;90(3):248–50. [13] Dietrich A, Mederos YSM, Gollasch M, Gross V, Storch U, Dubrovska G, et al. Increased vascular smooth muscle contractility in TRPC6−/− mice. Mol Cell Biol 2005;25(16):6980–9. [14] Freichel M, Vennekens R, Olausson J, Hoffmann M, Muller C, Stolz S, et al. Functional role of TRPC proteins in vivo: lessons from TRPC-deficient mouse models. Biochem Biophys Res Commun 2004;322(4):1352–8. [15] Kark T, Bagi Z, Lizanecz E, Pasztor ET, Erdei N, Czikora A, et al. Tissue-specific regulation of microvascular diameter: opposite functional roles of neuronal and smooth muscle located vanilloid receptor-1. Mol Pharmacol 2008;73(5):1405–12. [16] Earley S, Waldron BJ, Brayden JE. Critical role for transient receptor potential channel TRPM4 in myogenic constriction of cerebral arteries. Circ Res 2004;95(9):922–9. [17] Reading SA, Brayden JE. Central role of TRPM4 channels in cerebral blood flow regulation. Stroke 2007;38(8):2322–8. [18] Chen J, Crossland RF, Noorani MMZ, Marrelli SP. Inhibition of TRPC1/TRPC3 by PKG contributes to NO- mediated vasorelaxation. Am J Physiol Heart Circ Physiol 2009;297(1):H417–24. [19] Freichel M, Suh SH, Pfeifer A, Schweig U, Trost C, Weissgerber P, et al. Lack of an endothelial store-operated Ca2+ current impairs agonist-dependent vasorelaxation in TRP4−/− mice. Nat Cell Biol 2001;3(2):121–7. [20] Yang D, Luo Z, Ma S, Wong WT, Ma L, Zhong J, et al. Activation of TRPV1 by dietary capsaicin improves endothelium-dependent vasorelaxation and prevents hypertension. Cell Metab 2010;12(2):130–41. [21] Earley S, Heppner TJ, Nelson MT, Brayden JE. TRPV4 forms a novel Ca2+ signaling complex with ryanodine receptors and BKCa channels. Circ Res 2005;97(12):1270–9. [22] Kohler R, Heyken WT, Heinau P, Schubert R, Si H, Kacik M, et al. Evidence for a functional role of endothelial transient receptor potential V4 in shear stress-induced vasodilatation. Arterioscler Thromb Vasc Biol 2006;26(7):1495–502. [23] Marrelli SP, O’Neil RG, Brown RC, Bryan Jr RM. PLA2 and TRPV4 channels regulate endothelial calcium in cerebral arteries. Am J Physiol Heart Circ Physiol 2007;292(3):H1390–7. [24] Earley S, Pauyo T, Drapp R, Tavares MJ, Liedtke W, Brayden JE. TRPV4-dependent dilation of peripheral resistance arteries influences arterial pressure. Am J Physiol Heart Circ Physiol 2009;297(3):H1096–102. [25] Earley S, Gonzales AL, Crnich R. Endothelium-dependent cerebral artery dilation mediated by TRPA1 and Ca2+ -Activated K + channels. Circ Res 2009;104(8):987–94. [26] Thilo F, Scholze A, Liu DY, Zidek W, Tepel M. Association of transient receptor potential canonical type 3 (TRPC3) channel transcripts with proinflammatory cytokines. Arch Biochem Biophys 2008;471(1):57–62. [27] Liu D, Scholze A, Zhu Z, Krueger K, Thilo F, Burkert A, et al. Transient receptor potential channels in essential hypertension. J Hypertens 2006;24(6):1105–14. [28] Liu DY, Thilo F, Scholze A, Wittstock A, Zhao ZG, Harteneck C, et al. Increased store-operated and 1-oleoyl-2- acetyl-sn-glycerol-induced calcium influx in monocytes is mediated by transient receptor potential canonical channels in human essential hypertension. J Hypertens 2007;25(4):799–808. [29] Chen X, Yang D, Ma S, He H, Luo Z, Feng X, et al. Increased rhythmicity in hypertensive arterial smooth muscle is linked to transient receptor potential canonical channels. J Cell Mol Med 2010;14(10):2483–94. [30] Wang DH. The vanilloid receptor and hypertension. Acta Pharmacol Sin 2005;26(3):286–94. [31] Deng PY, Li YJ. Calcitonin gene-related peptide and hypertension. Peptides 2005;26(9):1676–85. [32] Wang Y, Wang DH. A novel mechanism contributing to development of Dahl salt-sensitive hypertension: role of the transient receptor potential vanilloid type 1. Hypertension 2006;47(3):609–14. [33] Hao X, Chen J, Luo Z, He H, Yu H, Ma L, et al. TRPV1 activation prevents high-salt diet-induced nocturnal hypertension in mice. Pflugers Arch 2011;461(3):345–53. [34] Ruiz C, Gutknecht S, Delay E, Kinnamon S. Detection of NaCl and KCl in TRPV1 knockout mice. Chem Senses 2006;31(9):813–20. [35] Gao F, Wang DH. Impairment in function and expression of transient receptor potential vanilloid type 4 in Dahl salt-sensitive rats: significance and mechanism. Hypertension 2010;55(4):1018–25. [36] Touyz RM, He Y, Montezano AC, Yao G, Chubanov V, Gudermann T, et al. Differential regulation of transient receptor potential melastatin 6 and 7 cation channels by ANG II in vascular smooth muscle cells from spontaneously hypertensive rats. Am J Physiol Regul Integr Comp Physiol 2006;290(1):R73–8. [37] Collins AJ, Foley RN, Herzog C, Chavers B, Gilbertson D, Ishani A, et al. Excerpts from the United States Renal Data System, 2008 Annual Data Report: atlas of chronic kidney disease and end-stage renal disease in the United States, National Institutes of Health NIDDK/DKUHD. Am J Kidney Dis 2009;53(1):S1–374. REFERENCES 339

[38] Liu XR, Liu Q, Chen GY, Hu Y, Sham JS, Lin MJ. Down-regulation of TRPM8 in pulmonary arteries of pulmonary hypertensive rats. Cell Physiol Biochem 2013;31(6):892–904. [39] Fu Y, Subramanya A, Rozansky D, Cohen DM. WNK kinases influence TRPV4 channel function and localization. Am J Physiol Renal Physiol 2006;290(6):F1305–14. [40] Liu D, Zhu Z, Tepel M. The role of transient receptor potential channels in metabolic syndrome. Hypertens Res 2008;31(11):1989–95. [41] Bae YM, Kim A, Lee YJ, Lim W, Noh YH, Kim EJ, et al. Enhancement of receptor-operated cation current and TRPC6 expression in arterial smooth muscle cells of deoxycorticosterone acetate-salt hypertensive rats. J Hypertens 2007;25(4):809–17. [42] Xia Y, Yang XR, Fu ZZ, Paudel O, Abramowitz J, Birnbaumer L, et al. Classical transient receptor potential 1 and 6 contribute to hypoxic pulmonary hypertension through differential regulation of pulmonary vascular functions. Hypertension 2014;63(1):173–80. [43] Chobanian AV, Bakris GL, Black HR, Cushman WC, Green LA, Izzo Jr JL, et al. The seventh report of the Joint National Committee on prevention, detection, evaluation, and treatment of high blood pressure: the JNC 7 report. JAMA 2003;289(19):2560–72. [44] Brayden JE, Earley S, Nelson MT, Reading S. Transient receptor potential (TRP) channels, vascular tone and autoregulation of cerebral blood flow. Clin Exp Pharmacol Physiol 2008;35(9):1116–20. [45] Xi Q, Adebiyi A, Zhao G, Chapman KE, Waters CM, Hassid A, et al. IP3 constricts cerebral arteries via IP3 receptor-mediated TRPC3 channel activation and independently of sarcoplasmic reticulum Ca2+ release. Circ Res 2008;102(9):1118–26. [46] Thilo F, Suess O, Liu Y, Tepel M. Decreased expression of transient receptor potential channels in cerebral vascular tissue from patients after hypertensive intracerebral hemorrhage. Clin Exp Hypertens 2011;33(8):533–7. [47] Gerzanich V, Woo SK, Vennekens R, Tsymbalyuk O, Ivanova S, Ivanov A, et al. De novo expression of Trpm4 initiates secondary hemorrhage in spinal cord injury. Nat Med 2009;15(2):185–91. [48] Xu X, Wang P, Zhao Z, Cao T, He H, Luo Z, et al. Activation of transient receptor potential vanilloid 1 by dietary capsaicin delays the onset of stroke in stroke-prone spontaneously hypertensive rats. Stroke 2011;42(11):3245–51. [49] Eder P, Molkentin JD. TRPC channels as effectors of cardiac hypertrophy. Circ Res 2011;108(2):265–72. [50] Ohba T, Watanabe H, Murakami M, Takahashi Y, Iino K, Kuromitsu S, et al. Upregulation of TRPC1 in the development of cardiac hypertrophy. J Mol Cell Cardiol 2007;42(3):498–507. [51] Seth M, Zhang ZS, Mao L, Graham V, Burch J, Stiber J, et al. TRPC1 channels are critical for hypertrophic signaling in the heart. Circ Res 2009;105(10):1023–30. [52] Wilkins BJ, Dai YS, Bueno OF, Parsons SA, Xu J, Plank DM, et al. Calcineurin/NFAT coupling participates in pathological, but not physiological, cardiac hypertrophy. Circ Res 2004;94(1):110–18. [53] Nakayama H, Wilkin BJ, Bodi I, Molkentin JD. Calcineurin-dependent cardiomyopathy is activated by TRPC in the adult mouse heart. FASEB J 2006;20(10):1660–70. [54] Kiyonaka S, Kato K, Nishida M, Mio K, Numaga T, Sawaguchi Y, et al. Selective and direct inhibition of TRPC3 channels underlies biological activities of a pyrazole compound. Proc Natl Acad Sci USA 2009;106(13):5400–5. [55] Kuwahara K, Wang Y, McAnally J, Richardson JA, Bassel-Duby R, Hill JA, et al. TRPC6 fulfills a calcineurin signaling circuit during pathologic cardiac remodeling. J Clin Invest 2006;116(12):3114–26. [56] Wu X, Eder P, Chang B, Molkentin JD. TRPC channels are necessary mediators of pathologic cardiac hypertrophy. Proc Natl Acad Sci USA 2010;107(15):7000–5. [57] Guinamard R, Demion M, Magaud C, Potreau D, Bois P. Functional expression of the TRPM4 cationic current in ventricular cardiomyocytes from spontaneously hypertensive rats. Hypertension 2006;48(4):587–94. [58] Woudenberg-Vrenken TE, Bindels RJ, Hoenderop JG. The role of transient receptor potential channels in kidney disease. Nat Rev Nephrol 2009;5(8):441–9. [59] Moller CC, Wei C, Altintas MM, Li J, Greka A, Ohse T, et al. Induction of TRPC6 channel in acquired forms of proteinuric kidney disease. J Am Soc Nephrol 2007;18(1):29–36. [60] Thilo F, Liu Y, Loddenkemper C, Schuelein R, Schmidt A, Yan Z, et al. VEGF regulates TRPC6 channels in podocytes. Nephrol Dial Transplant 2012;27(3):921–9. [61] Li J, Wang DH. Increased GFR and renal excretory function by activation of TRPV1 in the isolated perfused kidney. Pharmacol Res 2008;57(3):239–46. [62] Zhu Y, Wang DH. Segmental regulation of sodium and water excretion by TRPV1 activation in the kidney. J Cardiovasc Pharmacol 2008;51(5):437–42. 340 18. TRP CHANNELS REGULATE VASCULAR AND METABOLIC FUNCTION

[63] Xie C, Wang DH. Effects of a high-salt diet on TRPV-1-dependent renal nerve activity in Dahl salt-sensitive rats. Am J Nephrol 2010;32(3):194–200. [64] Wang Y, Wang DH. Protective effect of TRPV1 against renal fibrosis via inhibition of TGF-beta/Smad signaling in DOCA-salt hypertension. Mol Med 2011;17(11-12):1204–12. [65] Wang Y, Wang DH. Aggravated renal inflammatory responses in TRPV1 gene knockout mice subjected to DOCA-salt hypertension. Am J Physiol Renal Physiol 2009;297(6):F1550–9. [66] Schlingmann KP, Weber S, Peters M, Niemann Nejsum L, Vitzthum H, Klingel K, et al. Hypomagnesemia with secondary hypocalcemia is caused by mutations in TRPM6, a new member of the TRPM gene family. Nat Genet 2002;31(2):166–70. [67] Hsu YJ, Hoenderop JGJ, Bindels RJM. TRP channels in kidney disease. Biochim Biophys Acta 2007;1772(8):928–36. [68] Liu D, Scholze A, Zhu Z, Kreutz R, Wehland-von-Trebra M, Zidek W, et al. Increased transient receptor potential channel TRPC3 expression in spontaneously hypertensive rats. Am J Hypertens 2005;18(11):1503–7. [69] Liu DY, Siong SQ, Zu ZM. Imbalance and disfunction of transient receptor potential channels contribute to the pathogenesis of hypertension. Sci China-Life Sci 2014;57(8):818–25. [70] Eckel RH, Grundy SM, Zimmet PZ. The metabolic syndrome. Lancet 2005;365(9468):1415–28. [71] Grundy SM, Cleeman JI, Daniels SR, Donato KA, Eckel RH, Franklin BA, et al. Diagnosis and management of the metabolic syndrome: an American Heart Association/National Heart, Lung, and Blood Institute Scientific Statement. Circulation 2005;112(17):2735–52. [72] Eberly LE, Prineas R, Cohen JD, Vazquez G, Zhi X, Neaton JD, et al. Metabolic syndrome: risk factor distribution and 18-year mortality in the multiple risk factor intervention trial. Diabetes Care 2006;29(1):123–30. [73] Ninomiya JK, L’Italien G, Criqui MH, Whyte JL, Gamst A, Chen RS. Association of the metabolic syndrome with history of myocardial infarction and stroke in the Third National Health and Nutrition Examination Survey. Circulation 2004;109(1):42–6. [74] Malik S, Wong ND, Franklin SS, Kamath TV, L’Italien GJ, Pio JR, et al. Impact of the metabolic syndrome on mortality from coronary heart disease, cardiovascular disease, and all causes in United States adults. Circulation 2004;110(10):1245–50. [75] Zhu Z, Luo Z, Ma S, Liu D. TRP channels and their implications in metabolic diseases. Pflugers Arch 2011;461(2):211–23. [76] Chanda S, Bashir M, Babbar S, Koganti A, Bley K. In vitro hepatic and skin metabolism of capsaicin. Drug Metab Dispos 2008;36(4):670–5. [77] Garces-Claver A, Arnedo-Andres MS, Abadia J, Gil-Ortega R, Alvarez-Fernandez A. Determination of capsaicin and dihydrocapsaicin in capsicum fruits by liquid chromatography-electrospray/time-of-flight mass spectrometry. J Agric Food Chem 2006;54(25):9303–11. [78] Guenette SA, Ross A, Marier JF, Beaudry F, Vachon P. Pharmacokinetics of eugenol and its effects on thermal hypersensitivity in rats. Eur J Pharmacol 2007;562(1-2):60–7. [79] Li J, Wang DH. High-salt-induced increase in blood pressure: role of capsaicin-sensitive sensory nerves. J Hypertens 2003;21(3):577–82. [80] Bratz IN, Dick GM, Tune JD, Edwards JM, Neeb ZP, Dincer UD, et al. Impaired capsaicin-induced relaxation of coronary arteries in a porcine model of the metabolic syndrome. Am J Physiol Heart Circ Physiol 2008;294(6):H2489–96. [81] Lo YC, Hsiao HC, Wu DC, Lin RJ, Liang JC, Yeh JL, et al. A novel capsaicin derivative VOA induced relaxation in rat mesenteric and aortic arteries: involvement of CGRP, NO, cGMP, and endothelium-dependent activities. J Cardiovasc Pharmacol 2003;42(4):511–20. [82] Beaudry F, Vachon P. Quantitative determination of capsaicin, a transient receptor potential channel vanilloid 1 agonist, by liquid chromatography quadrupole ion trap mass spectrometry: evaluation of in vitro metabolic stability. Biomed Chromatogr 2009;23(2):204–11. [83] Wahlqvist ML, Wattanapenpaiboon N. Hot foods—unexpected help with energy balance? Lancet 2001;358(9279):348–9. [84] Kawada T, Hagihara K, Iwai K. Effects of capsaicin on lipid metabolism in rats fed a high fat diet. J Nutr 1986;116(7):1272–8. [85] Yoshioka M, St-Pierre S, Suzuki M, Tremblay A. Effects of red pepper added to high-fat and high-carbohydrate meals on energy metabolism and substrate utilization in Japanese women. Br J Nutr 1998;80(6):503–10. [86] Inoue N, Matsunaga Y, Satoh H, Takahashi M. Enhanced energy expenditure and fat oxidation in humans with high BMI scores by the ingestion of novel and non-pungent capsaicin analogues (capsinoids). Biosci Biotechnol Biochem 2007;71(2):380–9. REFERENCES 341

[87] Ono K, Tsukamoto-Yasui M, Hara-Kimura Y, Inoue N, Nogusa Y, Okabe Y, et al. Intragastric administration of capsiate, a transient receptor potential channel agonist, triggers thermogenic sympathetic responses. J Appl Physiol (1985) 2011;110(3):789–98. [88] Zhang LL, Yan Liu D, Ma LQ, Luo ZD, Cao TB, Zhong J, et al. Activation of transient receptor potential vanilloid type-1 channel prevents adipogenesis and obesity. Circ Res 2007;100(7):1063–70. [89] Wang XB, Miyares RL, Ahern GP. Oleoylethanolamide excites vagal sensory neurones, induces visceral pain and reduces short-term food intake in mice via capsaicin receptor TRPV1. J Physiol 2005;564(2):541–7. [90] Ye L, Kleiner S, Wu J, Sah R, Gupta RK, Banks AS, et al. TRPV4 is a regulator of adipose oxidative metabolism, inflammation, and energy homeostasis. Cell 2012;151(1):96–110. [91] O’Conor CJ, Griffin TM, Liedtke W, Guilak F. Increased susceptibility of Trpv4-deficient mice to obesity and obesity-induced osteoarthritis with very high-fat diet. Ann Rheum Dis 2013;72(2):300–4. [92] Zhang ZY, Zhang WY, Jung DY, Ko HJ, Lee Y, Friedline RH, et al. TRPM2 Ca2+ channel regulates energy balance and glucose metabolism. Am J Physiol Endocrinol Metab 2012;302(7):E807–16. [93] Liu P, Shah BP, Croasdell S, Gilbertson TA. Transient receptor potential channel type M5 is essential for fat taste. J Neurosci 2011;31(23):8634–42. [94] Palmer RK, Lunn CA. TRP channels as targets for therapeutic intervention in obesity: focus on TRPV1 and TRPM5. Curr Top Med Chem 2013;13(3):247–57. [95] Chen KH, Xu XH, Liu Y, Hu Y, Jin MW, Li GR. TRPM7 channels regulate proliferation and adipogenesis in 3T3-L1 preadipocytes. J Cell Physiol 2014;229(1):60–7. [96] Ma ST, Yu H, Zhao ZG, Luo ZD, Chen J, Ni YX, et al. Activation of the cold-sensing TRPM8 channel triggers UCP1-dependent thermogenesis and prevents obesity. J Mol Cell Biol 2012;4(2):88–96. [97] Akiba Y, Kato S, Katsube K, Nakamura M, Takeuchi K, Ishii H, et al. Transient receptor potential vanilloid subfamily 1 expressed in pancreatic islet beta cells modulates insulin secretion in rats. Biochem Biophys Res Commun 2004;321(1):219–25. [98] Pettersson M, Ahren B, Bottcher G, Sundler F. Calcitonin gene-related peptide: occurrence in pancreatic islets in the mouse and the rat and inhibition of insulin secretion in the mouse. Endocrinology 1986;119(2):865–9. [99] Gram DX, Ahren B, Nagy I, Olsen UB, Brand CL, Sundler F, et al. Capsaicin-sensitive sensory fibers in the islets of Langerhans contribute to defective insulin secretion in Zucker diabetic rat, an animal model for some aspects of human type 2 diabetes. Eur J Neurosci 2007;25(1):213–23. [100] Gram DX, Hansen AJ, Deacon CF, Brand CL, Ribel U, Wilken M, et al. Sensory nerve desensitization by resiniferatoxin improves glucose tolerance and increases insulin secretion in Zucker diabetic fatty rats and is associated with reduced plasma activity of dipeptidyl peptidase IV. Eur J Pharmacol 2005;509(2-3):211–17. [101] Karlsson S, Scheurink AJ, Steffens AB, Ahren B. Involvement of capsaicin-sensitive nerves in regulation of insulin secretion and glucose tolerance in conscious mice. Am J Physiol 1994;267(4 Pt 2):R1071–7. [102] Wang PJ, Yan ZC, Zhong J, Chen J, Ni YX, Li L, et al. Transient receptor potential vanilloid 1 activation enhances gut glucagon-like peptide-1 secretion and improves glucose homeostasis. Diabetes 2012;61(8):2155–65. [103] van de Wall EH, Gram DX, Strubbe JH, Scheurink AJ, Koolhaas JM. Ablation of capsaicin-sensitive afferent nerves affects insulin response during an intravenous glucose tolerance test. Life Sci 2005;77(11):1283–92. [104] Tanaka H, Shimaya A, Kiso T, Kuramochi T, Shimokawa T, Shibasaki M. Enhanced insulin secretion and sensitization in diabetic mice on chronic treatment with a transient receptor potential vanilloid 1 antagonist. Life Sci 2011;88(11-12):559–63. [105] Janssen SW, Hoenderop JG, Hermus AR, Sweep FC, Martens GJ, Bindels RJ. Expression of the novel epithelial Ca2+ channel ECaC1 in rat pancreatic islets. J Histochem Cytochem 2002;50(6):789–98. [106] Wissenbach U, Niemeyer BA, Fixemer T, Schneidewind A, Trost C, Cavalie A, et al. Expression of CaT- like, a novel calcium-selective channel, correlates with the malignancy of prostate cancer. J Biol Chem 2001;276(22):19461–8. [107] Zhuang L, Peng JB, Tou L, Takanaga H, Adam RM, Hediger MA, et al. Calcium-selective ion channel, CaT1, is apically localized in gastrointestinal tract epithelia and is aberrantly expressed in human malignancies. Lab Invest 2002;82(12):1755–64. [108] Uchida K, Dezaki K, Damdindorj B, Inada H, Shiuchi T, Mori Y, et al. Lack of TRPM2 impaired insulin secretion and glucose metabolisms in mice. Diabetes 2011;60(1):119–26. [109] Brixel LR, Monteilh-Zoller MK, Ingenbrandt CS, Fleig A, Penner R, Enklaar T, et al. TRPM5 regulates glucose-stimulated insulin secretion. Pflugers Arch 2010;460(1):69–76. 342 18. TRP CHANNELS REGULATE VASCULAR AND METABOLIC FUNCTION

[110] Mori Y, Otabe S, Dina C, Yasuda K, Populaire C, Lecoeur C, et al. Genome-wide search for type 2 diabetes in Japanese affected sib-pairs confirms susceptibility genes on 3q, 15q, and 20q and identifies two new candidate Loci on 7p and 11p. Diabetes 2002;51(4):1247–55. [111] Song Y, Hsu YH, Niu T, Manson JE, Buring JE, Liu S. Common genetic variants of the ion channel transient receptor potential membrane melastatin 6 and 7 (TRPM6 and TRPM7), magnesium intake, and risk of type 2 diabetes in women. BMC Med Genet 2009;10:4. [112] Niehof M, Borlak J. HNF4 alpha and the Ca-channel TRPC1 are novel disease candidate genes in diabetic nephropathy. Diabetes 2008;57(4):1069–77. [113] Graham S, Ding M, Sours-Brothers S, Yorio T, Ma JX, Ma R. Downregulation of TRPC6 protein expression by high glucose, a possible mechanism for the impaired Ca2+ signaling in glomerular mesangial cells in diabetes. Am J Physiol Renal Physiol 2007;293(4):F1381–90. [114] Chung AW, Au Yeung K, Chum E, Okon EB, van Breemen C. Diabetes modulates capacitative calcium entry and expression of transient receptor potential canonical channels in human saphenous vein. Eur J Pharmacol 2009;613(1-3):114–18. [115] Wei Z, Wang L, Han J, Song J, Yao L, Shao L, et al. Decreased expression of transient receptor potential vanilloid 1 impaires the postischemic recovery of diabetic mouse hearts. Circ J 2009;73(6):1127–32. [116] Naziroglu M, Dikici DM, Dursun S. Role of oxidative stress and Ca2+ signaling on molecular pathways of neuropathic pain in diabetes: focus on TRP channels. Neurochem Res 2012;37(10):2065–75. [117] Facer P, Casula MA, Smith GD, Benham CD, Chessell IP, Bountra C, et al. Differential expression of the capsaicin receptor TRPV1 and related novel receptors TRPV3, TRPV4 and TRPM8 in normal human tissues and changes in traumatic and diabetic neuropathy. BMC Neurol 2007;7:11. [118] Wuensch T, Thilo F, Krueger K, Scholze A, Ristow M, Tepel M. High glucose-induced oxidative stress increases transient receptor potential channel expression in human monocytes. Diabetes 2010;59(4):844–9. [119] Liu D, Maier A, Scholze A, Rauch U, Boltzen U, Zhao Z, et al. High glucose enhances transient receptor potential channel canonical type 6-dependent calcium influx in human platelets via phosphatidylinositol 3-kinase-dependent pathway. Arterioscler Thromb Vasc Biol 2008;28(4):746–51. [120] Nilius B, Mahieu F, Karashima Y, Voets T. Regulation of TRP channels: a voltage-lipid connection. Biochem Soc Trans 2007;35(Pt 1):105–8. [121] Manjunatha H, Srinivasan K. Hypolipidemic and antioxidant effects of curcumin and capsaicin in high-fat-fed rats. Can J Physiol Pharmacol 2007;85(6):588–96. [122] Tani Y, Fujioka T, Sumioka M, Furuichi Y, Hamada H, Watanabe T. Effects of capsinoid on serum and liver lipids in hyperlipidemic rats. J Nutr Sci Vitaminol (Tokyo) 2004;50(5):351–5. [123] Li L, Chen J, Ni Y, Feng X, Zhao Z, Wang P, et al. TRPV1 activation prevents nonalcoholic fatty liver through UCP2 upregulation in mice. Pflugers Arch 2012;463(5):727–32. [124] Edwards JM, Neeb ZP, Alloosh MA, Long X, Bratz IN, Peller CR, et al. Exercise training decreases store- operated Ca2 + entry associated with metabolic syndrome and coronary atherosclerosis. Cardiovasc Res 2010;85(3):631–40. [125] Takahashi Y, Watanabe H, Murakami M, Ohba T, Radovanovic M, Ono K, et al. Involvement of transient receptor potential canonical 1 (TRPC1) in angiotensin II-induced vascular smooth muscle cell hypertrophy. Atherosclerosis 2007;195(2):287–96. [126] Ahmmed GU, Malik AB. Functional role of TRPC channels in the regulation of endothelial permeability. Pflugers Arch 2005;451(1):131–42. [127] Ma L, Zhong J, Zhao Z, Luo Z, Ma S, Sun J, et al. Activation of TRPV1 reduces vascular lipid accumulation and attenuates atherosclerosis. Cardiovasc Res 2011;92(3):504–13. [128] Ma X, He D, Ru X, Chen Y, Cai Y, Bruce IC, et al. Apigenin, a plant-derived flavone, activates transient receptor potential vanilloid 4 cation channel. Br J Pharmacol 2012;166(1):349–58. [129] Namer B, Seifert F, Handwerker HO, Maihofner C. TRPA1 and TRPM8 activation in humans: effects of cinnamaldehyde and menthol. Neuroreport 2005;16(9):955–9. [130] Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, Julius D. The capsaicin receptor: a heat- activated ion channel in the pain pathway. Nature 1997;389(6653):816–24. [131] Cavanaugh DJ, Chesler AT, Jackson AC, Sigal YM, Yamanaka H, Grant R, et al. Trpv1 reporter mice reveal highly restricted brain distribution and functional expression in arteriolar smooth muscle cells. J Neurosci 2011;31(13):5067–77. CHAPTER 19 Transient Receptor Potential (TRP) Cation Channels in Diabetes Koenraad Philippaert,* Rudi Vennekens Katholieke Universiteit of Leuven, Department of Cellular and Molecular Medicine, Laboratory of Ion Channel Research and TRPLe (TRP Research Platform Leuven), Campus Gasthuisberg, Leuven, Belgium *Corresponding author: [email protected]

OUTLINE

TRP’s In Pancreatic ß Cells 344 TRP Channels in Diabetes-Associated TRPCs 345 Complications 353 TRPM2 345 TRPCs in Diabetic Vasculopathy 353 TRPM3 347 TRPCs in Diabetic Nephropathy 354 TRPM4 348 TRPV1 And TRPA1 in Diabetic TRPM5 348 Neuropathy 354 TRPM8 349 TRPV1 354 TRPV1 349 TRPA1 355 TRPV2 350 TRP Channels as Drug Target TRPV4 350 in T1DM and T2DM 356 TRPA1 351 Conclusions 357 TRPV1-Expressing Sensory Neurons in β Cell Function and Diabetes Acknowledgments 357 Mellitus 351 References 357 TRPV1 In Type 1 Diabetes Mellitus 352 TRPV1 In Type 2 Diabetes Mellitus 352

The transient receptor potential (TRP) family of cation channels consists of 28 mammalian members, which can be subdivided into six main subfamilies based on amino acid homology [1]. Most TRP channels are nonselective Ca2+-permeable channels, although the permeability ratios PCa/PNa vary considerably [2]. Gating of TRP channels is very diverse [3–7]. TRP channels can be activated by a plethora of stimuli, including ligand binding, voltage, cell swelling, and temperature. As a consequence, they are involved in diverse physiological processes such as heat sensing in sensory nerve endings, taste perception, and the modulation of allergic reactions. Moreover, these channels have been implicated in severe human diseases, such as polycystic kidney disease and mucolipidosis [8,9]. An increasing amount of data points to a role for TRP channels in pancreatic β cells, where they regulate insulin secretion. These data suggest that TRP channels might be interesting drug targets for the treatment of diabetes, and that they could be involved in the development of this important disease. Apart from the pancreatic pathology, diabetes is also associated with neuropathy, vasculopathy, and nephropathy. Considering the wide expression pattern of TRP channels, it is not surprising that TRP channels have also been associated with these aspects of diabetes, and increasing data indicates that their potential as drug targets is even more important in this area as compared to the pancreatic β cell.

TRP’s IN PANCREATIC ß CELLS

TRP channels have been described both in primary β cells and insulin-secreting cell lines [10,11]. TRPV2, TRPV4, TRPC1, TRPC4, TRPC6, TRPM2, TRPM3, TRPM4, and TRPM5 are identified in insulinoma cell lines such as MIN6 (mouse) or INS-1 (rat). Expression of TRPV2, TRPV4, TRPC1, TRPM2, TRPM3, TRPM4, and TRPM5 was reported in mouse islets and TRPM2, TRPV5, TRPC1, and TRPC4 in rat islets or β cells. Interestingly, no expression could be found in mouse islets for TRPV5, TRPM1, TRPM8, or TRPP3 (Colsoul, Nilius, & Vennekens, unpublished). Unfortunately, sparse data is available from human tissue: TRPV5 and TRPV6 are detected in human pancreas, and TRPM2, TRPM4, and TRPM5 transcripts are detected in human islets [12–14]. The pancreatic islet is a complex structure consisting of five different cell types: insulin- secreting β cells (which constitute 65-90% of the islet cell population), glucagon-releasing α cells, somatostatin-producing δ cells, polypeptide-containing PP cells, and ghrelin-secreting ε cells. Insulin is synthesized and secreted into the blood by the β cells, mainly in response to glucose but also in response to other nutrients (such as amino acids and fatty acids), hormones (e.g., the incretin hormones GLP-1 and GIP), and neurotransmitters (e.g., ACh) [15,16]. The secretion of insulin by the pancreatic β cell is a complex process driven by electrical activity 2+ 2+ and oscillations of the intracellular Ca concentration ([Ca ]cyt) [17]. Briefly, glucose enters the β cell via the high-affinity glucose transporter (GLUT-2), and glucose metabolism increases intracellular ATP levels, which closes ATP-sensitive K+ channels. This increases the input resistance of the β cell, allowing a small inward current, whose molecular identity is still elusive, to generate a significant depolarization [18–20]. Notably, TRP channels were often hypothesized to be interesting candidates for this background depolarizing current [11,21]. What follows is a typical pattern of bursts of action potentials from a depolarized plateau and parallel oscilla- 2+ tory increases of [Ca ]cyt. This pattern results from a complex interplay between ATP-sensitive TRP’s IN PANCREATIC ß CELLS 345

K+ channels, voltage-dependent Ca2+ and K+ channels, and the cellular metabolism of the β cell 2+ [21–23]. Increased [Ca ]cyt triggers exocytosis of insulin-containing vesicles [24]. Insulin lowers the blood glucose by promoting glucose uptake and nutrient storage in muscle, fat, and liver [25]. Disturbances in insulin secretion and glucose homeostasis lead to diabetes mellitus, a metabolic disorder characterized by hyperglycemia. In type 1 diabetes (T1DM; “Insulin-Dependent Diabetes Mellitus”), immune-mediated reduction of functional β cell mass results in a diminished or abolished insulin secretion. This results in the inability of the body to maintain normoglycemia. In type 2 diabetes (T2DM) the cause of hyperglycemia is more complex. It ranges from predominantly insulin resistance with relative insulin deficiency to a predominantly insulin secretory defect with insulin resistance. There are several risk factors toward the development of T2DM such as genetic and functional alterations in TRP channels. Furthermore, there is a connection between T2DM and a high-calorie diet or old age [26]. A subset of T2DM patients develops hyperglycemia secondary to hypoinsulinemia instead of peripheral insulin resistance; this is induced by a loss of functional β cell mass [27]. In the next sections, we describe the current knowledge of the function of TRP channels in the regulation of insulin release.

TRPCs TRPC channels are nonselective Ca2+-permeable cation channels, with the selectivity ratio

PCa/PNa varying significantly between the different family members [28]. TRPC channels are widely expressed, and their characterization is complicated by the possible occurrence of heterotetramers. TRPC1 mRNA could be detected in INS-1 cells and rat β cells and at high levels in mouse islets and MIN6 cells, whereas it could not be detected in another mouse insulinoma cell line βTC3 [29,30]. Expression of TRPC1 was found in whole human pancreas [31]. Four transcripts of mouse TRPC1 RNA, representing different splice variants, have been found in mouse islets and the β cell line MIN6 [29]. Polymorphisms in the TRPC1 gene have recently been associated with development of T2DM [32]. However, the influence of TRPC1 on insulin release still needs to be determined. Equally little is known about other TRPC channels in insulin release. Although TRPC4 could be detected in βTC3 and INS-1 cells, rat β cells, mouse islets, and human pancreas [30,31,33,34], analysis of blood glucose homeostasis by glucose tolerance tests did not reveal differences between wild-type (WT) and Trpc4-deficient mice both regarding basal glucose levels under fasting conditions, as well as following intraperitoneal glucose challenge [35]. Because TRPC4 is activated in the phospolipase C pathway, it is possible that the channel is involved in ACh- or glucagon- induced amplification of insulin release. TRPC6 transcripts have been detected, although to a low level, in βTC-3 insulin-secreting cells. Thus, it is clear that more research is needed to clarify the possible function of TRPC channels in the insulin release of the pancreatic β cell. It has been suggested that TRPC channels mediate the unknown depolarizing current that accounts for the Ca2+-release-activated cation current characterized earlier in βTC3 cells [34].

TRPM2 TRPM2 is a nonselective Ca2+-permeable cation channel fused C-terminally to an enzymatic ADP-ribose pyrophosphatase domain [36]. The channel is expressed in insulin-secreting cell 346 19. TRP CATION CHANNELS IN DIABETES lines, such as the rat cell lines CRI-G1 and RIN-5 F, and in human and mouse pancreatic islets [14,37–39]. Moreover, the channel coexpresses with insulin, but not with glucagon, indicating expression in β cells [39]. TRPM2 is activated by various stimuli, including adenine dinucleotides (ADPR, cADPR, − 2+ NAADP, β-NAD), reactive oxygen species (ROS) such as H2O2 and OH , and intracellular Ca [36,37,40,41]. A current with TRPM2-like properties could be detected in the rat insulinoma cell line CRI-G1, INS-1 cells, and primary mouse β cells. In Trpm2−/− primary β cells, no ADPR- elicited current could be detected, suggesting that TRPM2 is natively expressed and forms a functional channel in β cells [38,42]. TRPM2 could contribute to insulin release induced by heat, glucose, and incretin hormones [39,43]. Indeed, forskolin- (an activator of adenylyl cyclase) and exendin-4- (a GLP-1 receptor agonist) induced insulin release from rat pancreatic islets was significantly reduced in islets after shRNA-mediated knockdown of TRPM2 expression [39]. Furthermore, 2-aminoethoxydiphenyl borate (2-APB), a rapid and reversible inhibitor of TRPM2, inhibits both heat- and exendin-4-evoked insulin release from rat pancreatic islets [44]. These indications are further substantiated in the Trpm2−/− mouse: insulin secretion is induced by glucose, and GLP-1 is impaired in Trpm2-deficient islets, whereas the response to tolbutamide, a KATP channel inhibitor, is unchanged [43]. This results in higher basal glucose levels and an impaired glucose tolerance in Trpm2-deficient mice [43]. The 2+ impairment of insulin secretion is caused by reduced increases in [Ca ]cyt, indicating that TRPM2 mediates Ca2+ influx on glucose and/or GLP-1 stimulation. However, the situation might be even more complex because glucose-stimulated insulin secretion evoked by diazoxide and high K+ (conditions designed to “clamp” intracellular Ca2+ and inactivate the KATP channel-mediated pathways) was lost in Trpm2-deficient islets. Because the intracellular Ca2+ under these conditions was not altered between WT and Trpm2−/− islets, these data suggest that TRPM2 mediates insulin secretion independent of its role as a Ca2+ entry channel [43]. TRPM2 is important in ß cell apoptosis, a feature linked to the activation of the channel − by H2O2 and OH . These reactive oxygen species that are produced by oxidative stress are thought to play a central role in β cell death and the development of type 1 and type 2 diabe- 2+ tes [45,46]. Indeed, activation of TRPM2 by H2O2 has been shown to mediate Ca influx and β cell death in a rat β cell line RIN-5 F that natively expresses TRPM2 [37,47]. Moreover, INS-1 cells with suppressed TRPM2 expression are 72% less affected by H2O2-induced cell death 2+ + [42]. The H2O2-induced Ca influx is thought to be mediated by increasing levels of NAD or ADP-ribose that bind directly to the Nudix motif in the cytosolic C-terminal of TRPM2 [36,37]. Finally, TRPM2 has been reported to have an additional role as an intracellular Ca2+ release channel in pancreatic β cells [42]. Indeed, internally applied ADPR gives rise to a single Ca2+ transient both in INS-1 and in primary mouse β cells, and this effect was completely abolished in Trpm2−/− primary mouse β cells. Furthermore, TRPM2 colocalizes with lysosome- associated membrane protein-1 (LAMP1), a specific marker for lysosomes. In agreement with this, ADPR-induced intracellular Ca2+ release was abolished in INS-1 cells treated with bafi- lomycin A, a macrolide antibiotic that empties lysosomal calcium stores without affecting ER stores [48]. These data indicate that ADPR-dependent TRPM2-mediated Ca2+ release occurs predominantly from a lysosomal store. In addition, it was suggested that TRPM2-mediated TRP’s IN PANCREATIC ß CELLS 347

2+ Ca release contributes to H2O2-induced apoptosis [42]. Indeed, H2O2 induces significant cell death in INS-1 cells in the absence of extracellular Ca2+, albeit with a reduced severity. This effect was reduced in cells with reduced TRPM2 expression, indicating that not only Ca2+ influx through plasma membrane TRPM2 but also TRPM2-dependent lysosomal Ca2+ release plays a critical role in H2O2-mediated β cell death [42].

TRPM3 The TRPM3 gene encodes for different TRPM3 isoforms due to alternative splicing and exon usage, leading to channels with divergent pore and gating properties [49]. We refer here only to TRPM3α2 (1709 amino acids: the pore lacks 12 aa in comparison to the longest form TRPM3α1 of 1721 aa). Interestingly, the TRPM3 contains a recognition site for miR-204, which may regulate a variety of target genes at the transcriptional level [50,51]. TRPM3 is expressed in a variety of neuronal and nonneuronal tissue [51], including whole pancreas [52,53], INS-1 cells, and mouse pancreatic islets [54,55]. TRPM3 channels are directly activated by the neurosteroid hormone pregnenolone sulphate (PS). Pancreatic β cells and INS-1 cells express PS-sensitive channels that share several pharmacological and biophysical properties of recombinant TRPM3 channels (such as sensitivity to nifedipine and block by monovalent cations [54]). Moreover, PS elicits a large Ca2+ increase in INS-1 cells and pancreatic islets, an action dependent on TRPM3 expression. This PS-induced Ca2+ increase could be blocked by the selective and potent TRPM3 blocker mefenamic acid in INS-1E cells and mouse pancreatic islets [55]. Remarkably, mefenamic acid did not block glucose- or tolbutamide-induced Ca2+ increase, indicating that TRPM3 is not involved in the 2+ KATP-dependent Ca signaling of the β cell. PS, however, did increase glucose-induced insulin secretion from pancreatic islets, an effect abolished by mefenamic acid [54,55]. Thus, TRPM3 is an interesting target for the development of insulin secretagogues. Interestingly, PS activation of β cells (via TRPM3 and voltage-gated Ca2+ channels) induces the biosynthesis of a gene regulatory protein, the zinc finger transcription factor Egr-1, and in this way leads to increased biosynthesis of insulin [56]. However, the pharmacological concentrations of PS (50 μM) used to demonstrate enhancement of insulin secretion do not occur in vivo. It is possible that TRPM3 plays a more profound role in conditions where elevated plasma PS levels and changes in glucose homeostasis co-occur, such as pregnancy or 21-hydroxylase-deficiency. TRPM3 channels are also proposed to constitute a regulated Zn2+ entry pathway in pancreatic ß cells [57]. Zinc is important for insulin release as it is packed into cocrystals with insulin in the exocytotic vesicles. The formation of insulin crystals in β cells depends, among others, on the ZnT8 transporter, which contributes to the packaging efficiency of stored insulin [58]. Because Zn2+ ions are coreleased with insulin, pancreatic β cells need to continuously replenish their Zn2+ stores by taking up Zn2+ ions from the extracellular space. Insufficient Zn2+ uptake leads to impaired insulin synthesis and aggravated diabetic symptoms [59]. TRPM3 channels in ß cells have been shown to be highly permeable for Zn2+ and capable of mediating Zn2+ uptake under physiological conditions. The depolarization caused by the activation of TRPM3 channels could lead to the activation of voltage-dependent L-type Ca2+ channels and would in this way also lead to Zn2+ influx through these channels [54,60]. Whether Zn2+ influx regulation via TRPM3 channels is functionally relevant in β cells remains to be elucidated 348 19. TRP CATION CHANNELS IN DIABETES TRPM4 TRPM4 is a Ca2+-activated nonselective monovalent cation channel that is impermeable to divalent cations. The channel has been proposed to control insulin secretion in a rat insulinoma cell line INS-1, where TRPM4 protein is abundantly expressed [61]. TRPM4 expression and TRPM4-like channel activity could be detected in several β cell lines—INS-1, HIT-T15, RINm5F, β-TC3, and MIN-6—and also in the alpha cell line INR1G9 [12,61]. Inhibition of TRPM4 decreases the magnitude of the Ca2+ signal and insulin release in response to glucose, AVP (arginine-vasopressin, a Gq-coupled receptor agonist in β-cells), and glibenclamide in INS-1 cells [12,61]. These data suggest that depolarizing currents generated by TRPM4 are an important component in the control of intracellular Ca2+ signals necessary for insulin secretion. Furthermore, it is suggested that TRPM4-containing vesicles are translocated to the plasma membrane via Ca2+-dependent exocytosis, which may represent a regulatory mechanism by which β cells regulate electrical activity [12,61]. However, all these studies have been performed on cell lines. Although TRPM4 protein expression could be found within insulin-producing human β cells and mouse pancreatic islets [12], studies on Trpm4−/− mice revealed no difference in glucose-induced insulin secretion from freshly isolated pancreatic islets [62]. Moreover, these mice did not suffer from an impaired glucose tolerance after an intraperitoneal injection of glucose. These data suggest that TRPM4 is probably not involved in the signal mechanism following glucose stimulation. On the other hand, this does not exclude a possible role for TRPM4 in Gq- or Gs-receptor-coupled signaling pathways, for example during stimulation with glucagon or GLP-1. In addition, TRPM4 is proposed to be involved in glucagon secretion by the pancreatic alpha cell line αTC1-6 [63]: TRPM4 inhibition decreased the magnitude of intracellular Ca2+ signals and glucagon secretion in response to + several agonists such as the Gq-protein-coupled receptor agonist AVP and high K [63].

TRPM5 TRPM5, like its closest homologue TRPM4, is a Ca2+-activated nonselective monovalent cation channel that is impermeable to divalent cations [7]. The channel is expressed in β cells from pancreatic islets. A Ca2+-activated nonselective monovalent cation channel could be measured in β cells and was largely reduced in Trpm5−/− mice, indicating that TRPM5 is an important constituent of the Ca2+-activated cation current in β cells [64]. The TRPM5- 2+ dependent current small, ~2pA/pF at –80 mV and 1.5 μM [Ca ]cyt [64], suggesting that the channel can only influence the electrical activity of β cells under conditions of high input 2+ membrane resistance. Whereas no difference could be detected in Vm or intracellular Ca in nonstimulatory (low glucose, high KATP activity) conditions, TRPM5 seems to influence electrical activity during glucose stimulation (a condition with low KATP activity and consequently a high electrical resistance). Whereas normal WT islets respond to glucose stimulation with three types of oscillations (slow, mixed, or fast), Trpm5−/− islets displayed specifically 2+ a lack of fast glucose-induced oscillations in Vm and Ca [64]. TRPM5 contributes to the slow depolarization in the slow interburst interval of the glucose-induced electrical activity, in this way shortening the interburst interval and leading to faster glucose-induced oscillations in 2+ Vm and Ca . Why TRPM5 is only functionally relevant in a (fast-oscillating) subpopulation of the islets remains unclear, but it might be that the relative weight of the TRPM5-mediated TRP’s IN PANCREATIC ß CELLS 349 depolarizing conductance is coupled to the glycolytic rate in the cell. According to the dual oscillator model [23], fast oscillations are characterized by a high glycolytic rate (and a resulting high ATP production). This situation would make TRPM5 activity able to depolarize Vm in the interburst interval, as the hyperpolarizing KATP current is largely inactive at that point. This is in contrast with the situation in slow oscillating islets, where the oscillating glycolytic rate and the resulting high activity of KATP in the interburst interval would make TRPM5 2+ insufficient to depolarize Vm. Fast Ca oscillations are shown to be more efficient than slow oscillations in triggering exocytosis of secretory vesicles and insulin release [65]. In line with this, glucose-induced insulin release was reduced in isolated pancreatic islets from Trpm5−/− mice. Moreover, Trpm5-deficient mice display an impaired glucose tolerance during oral and intraperitoneal glucose tolerance tests [64,66]. This is caused by reduced glucose-induced insulin secretion from the β cells resulting in lower plasma insulin levels. These data suggest that Trpm5- deficient mice display a prediabetic phenotype caused by β cell dysfunction. The relevance of this prediabetic phenotype during conditions of higher insulin demand (such as pregnancy, obesity, aging) remains to be shown. Stimulation of TRPM5 increases the glucose tolerance in mice, by increasing calcium oscillation frequency in islets (Philippaert & Vennekens, unpublished). In leptin-deficient mice, a model for type II diabetes, a vast decrease of TRPM5 expression in the pancreatic islets was observed. Moreover, the glucose- induced calcium activity in islets from leptin-deficient mice resembles those of Trpm5−/− mice [67]. In this regard it is also interesting to note that Trpm5 expression is negatively correlated with blood glucose concentrations in the small intestine from diabetic patients [68]. Moreover, a recent study reports an association of TRPM5 variants with prediabetic phenotypes in subjects at risk for type 2 diabetes [69]. The functional impact of these mutations on the TRPM5 channel activity has not been clarified. However, these data indicate a possible link between TRPM5 and the development of type 2 diabetes mellitus.

TRPM8 TRPM8 is a cold sensing TRP channel that is activated by chemical ligands such as menthol and icilin [70,71]. It is highly expressed in prostate tissue; however, its functional role there remains to be elucidated [53]. TRPM8 is functionally expressed in dorsal root ganglia and trigeminal ganglia. In the sensory nervous system, TRPM8 has a prominent role in the sensing of cold temperatures and maintaining core body temperature [72]. There is no expression of TRPM8 in the endocrine pancreas [53]. However, mice lacking TRPM8 have an increased insulin sensitivity, which results from a compensatory mechanism following enhanced insulin clearance in TRPM8−/− mice. This compensatory mechanism includes up-regulation of the insulin-degrading enzyme (IDE) in the liver, which might result from the loss of TRPM8 mediated neuronal signals in hepatic neural innervations. TRPM8 positive sensory afferents innervate the hepatic vein [73]. TRPM8 has recently been indicated in association with pancreatic adenocarcinoma, where it might serve as a potential biomarker [74,75].

TRPV1 TRPV1 is best known for its function in nociceptive neurons, detecting noxious heat and pain. It is activated by heat and capsaicin, the pungent compound in chili peppers [76]. 350 19. TRP CATION CHANNELS IN DIABETES

There is functional expression of TRPV1 in the brain and sensory neurons [77]. TRPV1 transcripts have been detected in the pancreas, but it is currently unclear whether there is expression of TRPV1 in the β cells or merely limited to the innervation of the pancreas [78–80]. Treatment of mice with the TRPV1 activator capsaicin increased the postprandial insulin secretion, an effect that was absent in the Trpv1−/− mouse. No direct secretagogue effect of TRPV1 activation on the β cell could be observed. Instead, the insulin-promoting effect of activating TRPV1 can be contributed to increased incretin release from GLP-1 secreting intestinal cells [81]. Antibody reactivity indicates expression of TRPV1 in the afferent neurons innervating the pancreas, but not in the β cells. In nonobese diabetic (NOD) mice, a mutation leading to a hypofunctional TRPV1 channel was identified, which is linked to the onset of T1DM. Congenic NODxB6Idd4 mice carrying a fully functional WT TRPV1 gene do not have a diabetic phenotype. The onset of islet inflammation and diabetes in these mice might be mediated by the TRPV1+ insulin responsive sensory neurons [82]. A similar mutation was found in association with diabetes in a human population [83]. Clearly, TRPV1 could be important to evoke neuronal and hormonal signals to promote β cell activity (see following). However, current knowledge doesn’t support a direct role of TRPV1 in the β cell.

TRPV2 Expression of TRPV2 could be detected in MIN6 cells and in mouse primary β cells [84,85]. When MIN6 cells are cultured in a serum-free condition, immunoreactivity of TRPV2 is localized in an intracellular compartment, whereas addition of serum induces translocation of TRPV2 to the plasma membrane [86]. Both insulin and IGF-1 have been shown to be responsible for the translocation and insertion of TRPV2 from an intracellular compartment into the plasma membrane [85,86]. Translocation of TRPV2 was also induced by insulin secretagogues (including glucose) and knockdown of the insulin receptor attenuated insulin-induced translocation of TRPV2 [85]. Furthermore, elevation of Ca2+ entry caused by pretreatment of either MIN-6 cells or cultured mouse β cells with insulin is reduced by inhibition of TRPV2 [85]. Inhibition of TRPV2 also reduces glucose- or KCl-induced insulin secretion [85]. These data indicate that insulin released from the β cells further augments Ca2+ entry by recruiting TRPV2 to the plasma membrane and via this feed-forward mechanism accelerates insulin secretion. Indeed, it has been shown that insulin treatment induces the acceleration of the exocytotic response during the glucose-induced first-phase response by the insertion of TRPV2 into the plasma membrane in a PI3K-dependent manner [84]. Although the autocrine effect of insulin on β cell function has been a matter of debate [87], the β cell specific knockout of the insulin receptor gene shows impaired glucose-induced insulin secretion and reduced β cell mass, showing an important functional role for the insulin receptor in glucose sensing by the pancreatic β cell [88]. In this regard, more knowledge concerning the in vivo role of TRPV2 in β cells would expand the knowledge of insulin signaling in the healthy and the diabetic β cell.

TRPV4 TRPV4 is a Ca2+ permeable cation channel [89]. TRPV4 is expressed in MIN6 cells, INS-1E cells, and mouse pancreas [90]. Activation of TRPV4 enhances glucose-stimulated insulin secretion from INS-1E cells [91]. Reduction of Trpv4 expression significantly protected MIN6 TRPV1-EXPRESSING SENSORY NEURONS IN β CELL FUNCTION AND DIABETES MELLITUS 351 cells against hIAPP-induced calcium elevation, ER stress, and apoptosis [90]. hIAPP or amylin, the main component of amyloid, is strongly associated with the progressive loss of pancreatic β cell mass in type 2 diabetes. This may result from disruption of Ca2+ homeostasis. hIAPP forms insoluble aggregates and triggers Ca2+ changes that are associated with the induction of apoptosis via a pathway involving activation of the ER stress response [92]. The cytotoxicity of hIAPP is initiated on the cell surface and requires close contact between the aggregation pores and the β cell plasma membrane [93]. Because TRPV4 might be mechanosensitive, it has been proposed that TRPV4 senses the physical changes in the plasma membrane induced by hIAPP aggregation and in this way enables calcium entry, membrane depolarization, and activation of L-type Ca2+ channels, leading to cell death [90].

TRPA1 TRPA1 is a Ca2+-permeable nonselective cation channel, which is expressed in trigeminal and dorsal root ganglion neurons. TRPA1 is activated by several reactive electrophilic substances like allyl isothiocyanate (AITC), cinnamaldehyde, allicin, and acrolein, but also by nonreactive compounds like methylsalicylate, menthol, and icilin. TRPA1 is involved in various sensory processes, such as the detection of noxious cold and inflammatory hyperalgesia. TRPA1 is abundantly expressed in a rat pancreatic ß cell line and freshly isolated rat pancreatic β cells, but apparently not in pancreatic alpha cells [94,95]. Application of compounds such as mustard oil and 4-hydroxy-2-nonenal, which activate TRPA1, induces an inward membrane current, depolarization, and a rise of the intracellular Ca2+ level, leading to the release of insulin. This indicates that TRPA1 agonists could function as insulin secretagogues [94,95]. Strikingly, it was also reported that glucose-induced insulin release could be inhibited by the TRPA1 antagonist HC030031 [94]. This might suggest a role for TRPA1 during glucose-induced signaling in the ß cell, though it should be mentioned that data from Trpa1 knockout mice, which are indispensable controls for the selectivity of the antagonist, are lacking at this point.

TRPV1-EXPRESSING SENSORY NEURONS IN β CELL FUNCTION AND DIABETES MELLITUS

A local feedback loop exists between TRPV1-expressing sensory neurons and insulin- secreting β cells. Insulin released by the β cell will activate insulin receptors on the nerve terminals [96]. This increases TRPV1-mediated membrane currents by enhancing receptor sensitivity and translocation of TRPV1 from cytosol to plasma membrane [96] and by lowering the thermal activation threshold to room temperature [97]. The increase in TRPV1 currents leads to local release of neuropeptides such as substance P and CGRP that will sustain β cell physiology in an optimal range [82]: CGRP reduces insulin release from β cells [98], whereas substance P promotes neurogenic inflammation in the pancreas [99]. This local feedback loop has been proposed to be disturbed in type 1 diabetes mellitus. TRPV1 is highly expressed in primary sensory neurons, where it functions as a polymodal nociceptor [100]. Some controversy exists about expression of TRPV1 in endocrine islet cells. Whereas one study reports the expression of TRPV1 in Sprague-Dawley rat islets and rat 352 19. TRP CATION CHANNELS IN DIABETES

β cells lines (RIN and INS-1) and shows capsaicin-induced insulin release from insulinoma cells [78], others failed to show expression in mouse pancreatic islets [82] or in ß cells from Zucker diabetic fatty (ZDF) rat [80]. However, both the exocrine and the endocrine pancreas are innervated by TRPV1-positive neurons [80,101,102]. More and more evidence points to an important role for these neurons in type 1 and type 2 diabetes mellitus [100,103].

TRPV1 in Type 1 Diabetes Mellitus Ablation of TRPV1-positive neurons by agonist (capsaicin or RTX) administration in rat neonates improves glucose tolerance in those made diabetic by streptozotocin (STZ) treatment [104]. Nonobese diabetic (NOD) mice that are genetically prone to develop type 1 diabetes carry a hypofunctional TRPV1 mutant that is localized to the Idd4.1 diabetes-risk locus [82]. The hypofunctional NOD mutant causes a decreased secretion of substance P by the pancreatic sensory neurons. Interestingly, direct administration of substance P into the pancreas transiently reverses hyperglycemia in NOD mice and clears islet cell inflammation. The congenic NOD.Idd4.1 mouse strain, which expresses the WT TRPV1, is protected from diabetes [82,105]. However, injecting NOD mice with high doses of capsaicin (and thus eliminating TRPV1-positive neurons) also protects mice from diabetes [82]. Islet infiltration and the proportions and absolute numbers of effector T lymphocytes in pancreatic lymph nodes were reduced in capsaicin treated NOD mice [82]. The different effects of the neuropeptides on β cell function, depending on their concentration, might explain these apparently contradicting results. High concentrations generate a strong trophic signal, promoting β cells survival and function, whereas low concentrations have the opposite result, with deleterious effects on β cell function and viability [106,107]. It has been proposed that a fine balance in the local microenvironment between products such as substance P and islet β cell stress might determine the ensuing inflammatory reactions [82].

TRPV1 in Type 2 Diabetes Mellitus Systemic application of capsaicin (or resiniferatoxin [RTX], another TRPV1 agonist) ablates TRPV1-expressing nerve fibers and prevents aging-associated obesity and insulin resistance in rats and mice [108,109]. Furthermore, this treatment prevents the deterioration of glucose homeostasis in Zucker diabetic rats via increased insulin secretion [80]. The channel might also be linked with appetite regulation and obesity (a well-known risk factor for T2DM), as obese monkeys show markedly reduced capsaicin-evoked flare responses in their skin, which may point to down-regulated TRPV1 expression and/or loss of TRPV1-expressing nerve fibers [110]. The anorexic lipid mediator N-oleoylethanolamide is an endogenous TRPV1 ligand that reduces food intake in WT but not in Trpv1−/− mice [111]. As both obesity and T2DM might in part be an inflammatory disorder (as evidenced by increased levels of inflammatory markers [112–114]) and TRPV1 can be activated by inflammatory components [100,115], it has been suggested that TRPV1-expressing nerves are activated by proinflammatory substances in T2DM patients and that the resulting sustained CGRP release and high circulating CGRP levels promote insulin resistance [103]. Interestingly, treatment of ob/ob mice (a mouse model of obesity, insulin resistance, and T2DM) with a TRPV1 antagonist not only enhances insulin secretion but also decreases insulin resistance [116]. TRP Channels in Diabetes-Associated Complications 353 TRP CHANNELS IN DIABETES-ASSOCIATED COMPLICATIONS

TRPCs in Diabetic Vasculopathy Type 2 diabetes mellitus contributes to the development of both macrovascular and microvascular disorders. The major microvascular complications include nephropathy, reti- nopathy, and neuropathy, whereas the macrovascular complications manifest themselves as accelerated atherosclerosis, clinically resulting in premature ischemic heart disease, increased risk of cerebrovascular disease, and severe peripheral vascular disease [117]. These complications are a frequent cause of morbidity and mortality in patients with diabetes mellitus. Platelet dysfunction, leading to a prothrombotic state, contributes to diabetic microan- giopathy and macroangiopathy. Platelets from type 2 diabetic donors show enhanced ad- hesiveness and ability to aggregate more readily than those from healthy subjects [118]. This platelet hyperaggregability and hyperactivity is associated with abnormal intracellular Ca2+ homeostasis [118,119]. Several studies show that Ca2+ entry induced by the SERCA inhibitor thapsigargin is enhanced in platelets from diabetic donors, suggesting increased store-operated Ca2+ entry (SOCE) [120,121]. TRPC channels are well-known candidates of store-operated channels [122] and are identified in human platelets [123]. Interestingly, expression of TRPC3, Orai1, and STIM1 is enhanced in type 2 diabetes subjects as compared to controls [124]. Furthermore, hyperglycemia has been shown to cause enhanced TRPC6-mediated OAG-induced Ca2+ entry [125]. High glucose increases TRPC6 channel protein expression on the platelet surface, which is mediated by a phosphatidylinositol 3-kinase-dependent pathway [125]. Platelets from patients with type 2 diabetes mellitus showed increased TRPC6 expression as compared to nondiabetic individuals [125]. These data provide an explanation to the enhanced Ca2+ entry induced by physiological agonists in platelets from type 2 diabetes patients. However, reduced expression of TRPC6 in platelets from these patients has also been reported [124]. Moreover, a recent study shows that store-operated divalent cation entry might be attenuated in platelets from type 2 diabetic donors, although the overall Ca2+ entry in these cells stimulated by agonists or thapsigargin is still enhanced [126]. The reduced SOCE might be explained by impairment of the association between the Ca2+ sensor STIM1 and the proteins Orai1, hTRPC1, and hTRPC6 [126]. These data suggest that other noncapacitative Ca2+ entry pathways might be responsible for the enhanced Ca2+ entry observed after platelet stimulation with agonist or the SERCA inhibitor TG. Taken together, the pathogenesis of platelet disorders during type 2 diabetes mellitus remains largely unclear. SOCE is also active in smooth muscle cells and contributes to vasoconstriction [127]. Diabetes is associated with a perturbation of signaling pathways in vascular tissue. This causes vasomotor dysfunction characterized by impaired responsiveness to vasodilators or an exacerbated response to vasoconstrictors, leading to hypertension [128]. Vessels from diabetic patients are more contractile than those from nondiabetic patients [129]. This higher contractility could be partly due to the involvement of Ca2+ entry through the store-operated channels, as well as the regulation of the expression of TRPC1, 4, and 6. In caudal artery strips of type 2 diabetic Goto-Kakizaki (GK) rats, TRPC1 and TRPC6 expression was increased, and TRPC4 expression was found in diabetic, but not in nondiabetic, animals [130]. The situation in humans might be a bit different, as vessels from diabetic patients show up-regulation of 354 19. TRP CATION CHANNELS IN DIABETES

TRPC4 but down-regulation of TRPC1 and TRPC6 [129]. Nevertheless, these data suggest that diabetes modulates capacitative Ca2+ entry through the store-operated calcium channel specifically via the regulation of TRPC channels.

TRPCs in Diabetic Nephropathy Diabetic nephropathy is a progressive disorder that causes a decrease in the glomerular filtration rate and is the leading cause of end-stage renal disease. TRPC1 is localized on human chromosome 3q22-24, that is, a region considered to be a hot spot for diabetic nephropathy [131]. TRPC1 gene expression is down-regulated in the kidneys of several animal models of type 1 (STZ-administered rats) and type 2 diabetes (diabetic Zucker diabetic fatty fa/fa (ZDF) and db/db mice) [131,132]. Moreover, TRPC1 protein expression was repressed in kidneys of diabetic patients diagnosed with nodular glomerulosclerosis [131], although no association of TRPC1 polymorphisms with diabetic nephropathy or T2D-associated end-stage renal disease could be found [132]. Thus, TRPC1 dysfunction might be important in the development of diabetic nephropathy. The early stage of diabetic mellitus is characterized by renal hyperfiltration, which promotes the eventual development of diabetic nephropathy. Diabetic hyperfiltration results from a combination of decreased responsiveness of both the renal afferent arterioles and glomerular mesangial cells (MCs) to vasoconstrictors. MCs physiologically regulate glomerular hemodynamics. In diabetes, mesangial contractile function is impaired, and reduced Ca2+ influx is believed to be a major contributing factor to the hypocontractility. Agonist- stimulated Ca2+ entry is significantly inhibited by high glucose in cultured MCs, and this involves a decrease in TRPC6 protein expression. High glucose-induced down-regulation is specific for TRPC6 as TRPC1 and TRPC3 protein expression levels were not affected by high glucose [133].

TRPV1 and TRPA1 in Diabetic Neuropathy TRPV1 Diabetic peripheral neuropathy (DPN) is a common chronic complication in early to intermediate stages of diabetes mellitus [134]. It manifests as one or more kinds of stimulus-evoked pain including increased responsiveness to noxious stimuli (hyperalgesia), decreased sensitivity to painful stimuli (hypoalgesia), and hyperresponsiveness to normally innocuous stimuli (allodynia). The mechanisms underlying diabetic neuropathy remain poorly understood. TRPV1 is predominantly expressed in primary sensory nerves [76]. Its activation can result in the release of neuropeptides such as calcitonin gene-related peptide (CGRP) and substance P (SP). Studies with TRPV1-deficient mice demonstrate that TRPV1 is essential for thermal hyperalgesia induced by tissue injury and inflammation [135]. Of interest, treatment with capsaicin, a TRPV1 agonist, has been shown to improve sensory perception in humans with DPN [136], likely because overactivation of TRPV1 with capsaicin leads to Ca2+ overload in and degeneration of TRPV1-positive nerve endings. A similar strategy has been applied recently to the treatment of idiopathic rhinitis in humans [137]. TRP Channels in Diabetes-Associated Complications 355

DRG neurons isolated from STZ-induced diabetic rats showed significant increases in capsaicin- and proton-activated currents, implying that painful diabetic neuropathy is associated with enhanced function of TRPV1. This enhanced function involves increased phosphorylation, oligomerization, reallocation of channels to cell surface plasma membrane, and impaired desensitization of TRPV1 [138]. These features contribute to the excitability of sensory nerves that mediate neuropathic pain. In STZ-injected mice, TRPV1 expression varies with the neuropathic phenotype: diabetic neuropathy manifests as an initial phase of thermal hyperalgesia and a late phase of thermal hypoalgesia, and these phenotypes are accompanied by up- and down-regulation of TRPV1, respectively [139]. Moreover, STZ treatment failed to influence thermal nociception in TRPV1-deficient mice [139]. Finally, both TRPV1 expressing sub- and intraepidermal fibers and expression in surviving fibers were shown to be decreased in human diabetic neuropathy skin [140]. Thus, it might be suggested that TRPV1 plays an essential role in DPN. The cardiac muscle is also innervated by TRPV1-positive sensory nerves [141]. Interestingly, protein and mRNA expression of TRPV1 and its main neuropeptides CGRP and SP in the cardiac muscle of STZ-injected type 1 diabetic mice were markedly decreased [142]. It might be suggested that decreased expression of TRPV1 leads to depletion of CGRP and SP in diabetic hearts, which renders diabetic patients prone to myocardial infection, which is a common complication of diabetes mellitus. Central sensitization is also involved in neuropathic pain [143]. The periaqueductal gray (PAG) is a midbrain structure whose role in descending control of pain is quite well established [144] and is an important site for localization and actions of TRPV1 receptors in pain modulation [145]. Activation of glutamatergic projecting neurons in midbrain ventrolateral PAG induces antinociception, and agonists of TRPV1 increase firing of these neurons. The antinociceptive effect of capsaicin injected in the PAG was attenuated in STZ-treated rats, probably caused by a reduction in TRPV1 receptor expression [146]. Thus, down-regulation of TRPV1 expression in this brain area might contribute to diabetic hyperalgesia. Many of the aforementioned results are derived from STZ-induced diabetic animals. It should be noted that precaution must be taken when interpreting these results, as STZ has been shown to have a direct effect on TRPV1 expression and function. Indeed, STZ treatment, irrespective of the glycemic state of the animal, caused increased expression and function of TRPV1 in spinal dorsal horn and dorsal root ganglion, probably via the ROS-mediated pathway [147,148].

TRPA1 The TRPA1 ion channel is expressed mainly in nociceptive primary afferent neurons and is activated by reactive chemicals such as cinnamaldehyde and mustard oil. STZ-induced diabetes increases TRPA1 expression in DRG neurons [149]. Interestingly, the channel is activated by 4-hydroxynonenal (4-HNE) and methylglyoxal, reactive compounds that are generated under hyperglycemic conditions and believed to contribute to the development of diabetic neuropathy [150,151]. This activation is sustained [152], leading to the working hypothesis that sustained activation of the TRPA1 channel by compounds generated in diabetes mellitus contributes to diabetic neuropathy. Indeed, prolonged treatment with the TRPA1 channel antagonist Chembridge-5861528 prevents development of diabetic hypersensitivity and reduces the DM-induced attenuation of the cutaneous axon reflex in STZ-treated rats [152,153]. 356 19. TRP CATION CHANNELS IN DIABETES

Interestingly, spinal administration of Chembridge-5861528 produced a marked suppression of diabetic hypersensitivity, whereas cutaneous administration, even at a considerably higher dose, had only a weak antihypersensitivity effect [154]. This finding indicates that the attenuation of diabetic hypersensitivity by a systemically administered TRPA1 channel antagonist is rather due to blockade of spinal than cutaneous TRPA1 channels. It might be that activation of cutaneous TRPA1 channels is important in the induction of diabetic-induced hypersensitivity, whereas spinally located TRPA1 channels play an important role in maintenance of the hypersensitivity [154]. These findings suggest that prolonged treatment with a TRPA1 channel antagonist might prevent development of diabetic hypersensitivity. In this regard, it is interesting to note that paclitaxel, an anticancer drug that is known to potentiate cold hyperalgesia in diabetic patients, has been shown to activate TRPA1 via the production of ROS [149].

TRP CHANNELS AS DRUG TARGET IN T1DM AND T2DM

Whereas in T1DM insulin administration is the most important treatment, the most obvious treatment for T2DM is a change in lifestyle, accomplished by adaptation of exercise and diet. However, in many cases these measures fail to control the elevated blood glucose, and additional treatments are needed [155]. Historically, sulfonylureas were often used, but these drugs come with many unwanted effects, including the risk for hypoglycemia, weight gain, and exhaustion of the pancreas. Currently, the first-line drug of choice is metformin, an oral antidiabetic drug affecting peripheral insulin resistance. However, as beta-cell dysfunction is also associated with type 2 diabetes, antidiabetic drugs should also improve beta-cell function. TRP channels might be interesting drug targets in this regard. TRPM3 activators have been shown to enhance glucose- and GLP-1-dependent insulin release. TRPV1 might also be an interesting drug target as compounds modulating channel activity influence insulin resistance but also insulin release, GLP-1 secretion, and diabetic nephropathy in mice. Indeed, considering that multiple TRP channels are also involved in diabetes-associated complications (as outlined earlier), pharmacological targeting of these channels could affect both insulin secretion of the ß cell and diabetes-associated complications in peripheral tissue. Considering the side effects of sulfonylureas, novel insulin secretagogues are obviously wanted. An essential requirement for a new type of these drugs would be the glucose dependency of its action to avoid the risk of hypoglycemia. Incretin mimetics (GLP-1 receptor agonists that bind specific receptors and mimic the action of natural GLP-1) and incretin enhancers (inhibitors of the enzyme that degrades the incretin hormones and thus prolong their activity) are potential candidates. However, GLP-1R agonists require subcutaneous administration, and both incretin mimetics and enhancers are associated with unwanted effects, such as nausea and diarrhea for incretin mimetics and higher occurrence of infections for incretin enhancers [156], Furthermore, pharmacological activation of TRPM5 would enhance insulin release only under conditions of elevated glucose levels with no associated risk to hypoglycemia. As TRPM5 is also implicated in transduction of sweet taste, activators of TRPM5 might be expected to increase sweet sensation and in this way reduce intake of sugar. REFERENCES 357 CONCLUSIONS

Type 2 diabetes has a tremendous impact on public health worldwide. It is generally ac- knowledged that the disease results from a combination of peripheral insulin resistance and genetically determined susceptibility to β cell dysfunction. There is good evidence that TRP channels can regulate insulin release and β cell function. Specifically, through their ability to regulate intracellular Ca2+ levels and membrane depolarization, TRP channels might be interesting targets for the development of insulin secretagogues. TRP channels might be even more important drug targets in pathology secondary to diabetes, such as diabetic neuropathy, vasculopathy, and nephropathy. Although much of the available evidence is generated in cell lines, data are increasingly being confirmed in knockout mice. However, a functional role for any TRP channel in human islets has not been shown to date. This requires selective and potent pharmacology, which is only available for a few TRP channels. Clearly, this is one of the main hurdles that need to be taken to fully explore the potential of TRP channels in different aspects of diabetes mellitus.

Acknowledgments The authors wish to thank all the members of the Laboratory of Ion Channel Research (KU Leuven) and Professor Frans Schuit for stimulating discussions. The FWO Vlaanderen, the Interuniversitary Attraction Poles (IUAP) program of the Belgian federal government and the Bijzonder Onderzoeksfonds Leuven have granted support for the authors’ work. The authors are members of the TRP Research Platform Leuven (TRPLe) consortium Leuven.

References [1] Pedersen SF, Owsianik G, Nilius B. TRP channels: an overview. Cell Calcium 2005;38(3–4):233–52. [2] Owsianik G, D’Hoedt D, Voets T, Nilius B. Structure-function relationship of the TRP channel superfamily. Rev Physiol Biochem Pharmacol 2006;156:61–90. [3] Nilius B, Mahieu F, Karashima Y, Voets T. Regulation of TRP channels: a voltage-lipid connection. Biochem Soc Trans 2007;35(Pt 1):105–8. [4] Nilius B, Talavera K, Owsianik G, Prenen J, Droogmans G, Voets T. Gating of TRP channels: a voltage connection? J Physiol 2005;567(Pt 1):35–44. [5] Voets T, Nilius B. Modulation of TRPs by PIPs. J Physiol 2007;582(Pt 3):939–44. [6] Rohacs T, Nilius B. Regulation of transient receptor potential (TRP) channels by phosphoinositides. Pflugers Arch 2007;455(1):157–68. [7] Hofmann T, Chubanov V, Gudermann T, Montell C. TRPM5 is a voltage-modulated and Ca(2+)-activated monovalent selective cation channel. Curr Biol 2003;13(13):1153–8. [8] Nilius B, Owsianik G, Voets T, Peters JA. Transient receptor potential cation channels in disease. Physiol Rev 2007;87(1):165–217. [9] Nilius B, Voets T. TRP channels: a TR(I)P through a world of multifunctional cation channels. Pflugers Arch 2005;451(1):1–10. [10] Colsoul B, Vennekens R, Nilius B. Transient Receptor Potential Cation Channels in Pancreatic beta Cells. Rev Physiol Biochem Pharmacol 2011;161:87–110. [11] Jacobson DA, Philipson LH. TRP channels of the pancreatic beta cell. Handb Exp Pharmacol 2007;179:409–24. [12] Marigo V, Courville K, Hsu WH, Feng JM, Cheng H. TRPM4 impacts Ca(2+) signals during agonist-induced insulin secretion in pancreatic beta-cells. Mol Cell Endocrinol 2009;299:194–203. [13] Prawitt D, Monteilh-Zoller MK, Brixel L, Spangenberg C, Zabel B, Fleig A, et al. TRPM5 is a transient Ca2+−activated cation channel responding to rapid changes in [Ca2+]i. Proc Natl Acad Sci USA 2003;100(25):15166–71. 358 19. TRP CATION CHANNELS IN DIABETES

[14] Qian F, Huang P, Ma L, Kuznetsov A, Tamarina N, Philipson LH. TRP genes: candidates for nonselective cation channels and store-operated channels in insulin-secreting cells. Diabetes 2002;51(Suppl 1):S183–9. [15] Winzell MS, Ahren B. G-protein-coupled receptors and islet function-implications for treatment of type 2 diabetes. Pharmacol Ther 2007;116(3):437–48. [16] Newsholme P, Gaudel C, McClenaghan NH. Nutrient regulation of insulin secretion and beta-cell functional integrity. Adv Exp Med Biol 2010;654:91–114. [17] MacDonald PE, Rorsman P. Oscillations, intercellular coupling, and insulin secretion in pancreatic beta cells. PLoS Biol 2006;4(2):e49. [18] Rorsman P, Trube G. Glucose dependent K+−channels in pancreatic beta-cells are regulated by intracellular ATP. Arch Eur J Physiol 1985;405(4):305–9. [19] Henquin JC. Regulation of insulin secretion: a matter of phase control and amplitude modulation. Diabetologia 2009;52(5):739–51. [20] Henquin JC, Nenquin M, Ravier MA, Szollosi A. Shortcomings of current models of glucose-induced insulin secretion. Diabetes Obes Metab 2009;11(Suppl 4):168–79. [21] Drews G, Krippeit-Drews P, Dufer M. Electrophysiology of islet cells. Adv Exp Med Biol 2010;654:115–63. [22] Ashcroft FM, Rorsman P. Electrophysiology of the pancreatic beta-cell. Prog Biophys Mol Biol 1989;54(2):87–143. [23] Bertram R, Sherman A, Satin LS. Metabolic and electrical oscillations: partners in controlling pulsatile insulin secretion. Am J Physiol Endocrinol Metab 2007;293(4):E890–900. [24] Lemmens R, Larsson O, Berggren PO, Islam MS. Ca2+−induced Ca2+ release from the endoplasmic reticulum amplifies the Ca2+ signal mediated by activation of voltage-gated L-type Ca2+ channels in pancreatic beta-cells. J Biol Chem 2001;276(13):9971–7. [25] LeRoith D. Beta-cell dysfunction and insulin resistance in type 2 diabetes: role of metabolic and genetic abnormalities. Am J Med 2002;113(Suppl 6A):3S–11S. [26] Herder C, Roden M. Genetics of type 2 diabetes: pathophysiologic and clinical relevance. Eur J Clin Invest 2011;41(6):679–92. [27] Peiris H, Raghupathi R, Jessup CF, Zanin MP, Mohanasundaram D, Mackenzie KD, et al. Increased expression of the glucose-responsive gene, RCAN1, causes hypoinsulinemia, beta-cell dysfunction, and diabetes. Endocrinology 2012;153(11):5212–21. [28] Gees M, Colsoul B, Nilius B. The role of transient receptor potential cation channels in Ca2+ signaling. Cold Spring Harb Perspect Biol 2010;2:a003962. [29] Sakura H, Ashcroft FM. Identification of four trp1 gene variants murine pancreatic beta-cells. Diabetologia 1997;40(5):528–32. [30] Li F, Zhang ZM. Comparative identification of Ca2+ channel expression in INS-1 and rat pancreatic beta cells. World J Gastroenterol 2009;15(24):3046–50. [31] Riccio A, Medhurst AD, Mattei C, Kelsell RE, Calver AR, Randall AD, et al. mRNA distribution analysis of human TRPC family in CNS and peripheral tissues. Brain Res Mol Brain Res 2002;109(1–2):95–104. [32] Chen K, Jin X, Li Q, Wang W, Wang Y, Zhang J. Association of TRPC1 gene polymorphisms with type 2 diabetes and diabetic nephropathy in Han Chinese population. Endocr Res 2013;38(2):59–68. [33] Freichel M, Vennekens R, Olausson J, Hoffmann M, Muller C, Stolz S, et al. Functional role of TRPC proteins in vivo: lessons from TRPC-deficient mouse models. Biochem Biophys Res Commun 2004;322(4):1352–8. [34] Roe MW, Worley 3rd JF, Qian F, Tamarina N, Mittal AA, Dralyuk F, et al. Characterization of a Ca2+ release- activated nonselective cation current regulating membrane potential and [Ca2+]i oscillations in transgenically derived beta-cells. J Biol Chem 1998;273(17):10402–10. [35] Freichel M, Philipp S, Cavalie A, Flockerzi V. TRPC4 and TRPC4-deficient mice. Novartis Found Symp 2004;258:189–99, discussion 99–203, 63–6. [36] Perraud AL, Fleig A, Dunn CA, Bagley LA, Launay P, Schmitz C, et al. ADP-ribose gating of the calcium- permeable LTRPC2 channel revealed by Nudix motif homology. Nature 2001;411(6837):595–9. [37] Hara Y, Wakamori M, Ishii M, Maeno E, Nishida M, Yoshida T, et al. LTRPC2 Ca2+−permeable channel activated by changes in redox status confers susceptibility to cell death. Mol Cell 2002;9(1):163–73. [38] Inamura K, Sano Y, Mochizuki S, Yokoi H, Miyake A, Nozawa K, et al. Response to ADP-ribose by activation of TRPM2 in the CRI-G1 insulinoma cell line. J Membr Biol 2003;191(3):201–7. [39] Togashi K, Hara Y, Tominaga T, Higashi T, Konishi Y, Mori Y, et al. TRPM2 activation by cyclic ADP-ribose at body temperature is involved in insulin secretion. EMBO J 2006;25(9):1804–15. REFERENCES 359

[40] Du J, Xie J, Yue L. Intracellular calcium activates TRPM2 and its alternative spliced isoforms. Proc Natl Acad Sci USA 2009;106(17):7239–44. [41] Sano Y, Inamura K, Miyake A, Mochizuki S, Yokoi H, Matsushime H, et al. Immunocyte Ca2+ influx system mediated by LTRPC2. Science 2001;293(5533):1327–30. [42] Lange I, Yamamoto S, Partida-Sanchez S, Mori Y, Fleig A, Penner R. TRPM2 functions as a lysosomal Ca2+−release channel in beta cells. Sci Signal 2009;2(71):ra23. [43] Uchida K, Dezaki K, Damdindorj B, Inada H, Shiuchi T, Mori Y, et al. Lack of TRPM2 impaired insulin secretion and glucose metabolisms in mice. Diabetes 2011;60:119–26. [44] Togashi K, Inada H, Tominaga M. Inhibition of the transient receptor potential cation channel TRPM2 by 2-aminoethoxydiphenyl borate (2-APB). Br J Pharmacol 2008;153(6):1324–30. [45] Mandrup-Poulsen T. Apoptotic signal transduction pathways in diabetes. Biochem Pharmacol 2003;66(8):1433–40. [46] Rhodes CJ. Type 2 diabetes-a matter of beta-cell life and death? Science 2005;307(5708):380–4. [47] Ishii M, Shimizu S, Hara Y, Hagiwara T, Miyazaki A, Mori Y, et al. Intracellular-produced hydroxyl radical mediates H2O2-induced Ca2+ influx and cell death in rat beta-cell line RIN-5 F. Cell Calcium 2006;39(6):487–94. [48] Bowman EJ, Siebers A, Altendorf K. Bafilomycins: a class of inhibitors of membrane ATPases from microorgan- isms, animal cells, and plant cells. Proc Natl Acad Sci USA 1988;85(21):7972–6. [49] Oberwinkler J, Lis A, Giehl KM, Flockerzi V, Philipp SE. Alternative splicing switches the divalent cation selectivity of TRPM3 channels. J Biol Chem 2005;280(23):22540–8. [50] Weber MJ. New human and mouse microRNA genes found by homology search. FEBS J 2005;272:59–73. [51] Oberwinkler J, Phillipp SE. Trpm3. Handb Exp Pharmacol 2007;179:253–67. [52] Grimm C, Kraft R, Sauerbruch S, Schultz G, Harteneck C. Molecular and functional characterization of the melastatin-related cation channel TRPM3. J Biol Chem 2003;278(24):21493–501. [53] Fonfria E, Murdock PR, Cusdin FS, Benham CD, Kelsell RE, McNulty S. Tissue distribution profiles of the human TRPM cation channel family. J Recept Signal Transduct Res 2006;26(3):159–78. [54] Wagner TF, Loch S, Lambert S, Straub I, Mannebach S, Mathar I, et al. Transient receptor potential M3 channels are ionotropic steroid receptors in pancreatic beta cells. Nat Cell Biol 2008;10(12):1421–30. [55] Klose C, Straub I, Riehle M, Ranta F, Krautwurst D, Ullrich S, et al. Fenamates as TRP channel blockers: mefenamic acid selectively blocks TRPM3. Br J Pharmacol 2011;162:1757–69. [56] Mayer SI, Muller I, Mannebach S, Endo T, Thiel G. Signal Transduction of Pregnenolone Sulfate in Insulinoma Cells: activation of egr-1 expression involving trpm3, voltage-gated calcium channels, erk, and ternary complex factors. J Biol Chem 2011;286(12):10084–96. [57] Wagner TF, Drews A, Loch S, Mohr F, Philipp SE, Lambert S, et al. TRPM3 channels provide a regulated influx pathway for zinc in pancreatic beta cells. Pflugers Arch 2010;460(4):755–65. [58] Lemaire K, Ravier MA, Schraenen A, Creemers JW, Van de Plas R, Granvik M, et al. Insulin crystallization depends on zinc transporter ZnT8 expression, but is not required for normal glucose homeostasis in mice. Proc Natl Acad Sci USA 2009;106(35):14872–7. [59] Chausmer AB. Zinc, insulin and diabetes. J Am Coll Nutr 1998;17(2):109–15. [60] Gyulkhandanyan AV, Lee SC, Bikopoulos G, Dai F, Wheeler MB. The Zn2+−transporting pathways in pancreatic beta-cells: a role for the L-type voltage-gated Ca2+ channel. J Biol Chem 2006;281(14):9361–72. [61] Cheng H, Beck A, Launay P, Gross SA, Stokes AJ, Kinet JP, et al. TRPM4 controls insulin secretion in pancreatic beta-cells. Cell Calcium 2007;41(1):51–61. [62] Vennekens R, Olausson J, Meissner M, Bloch W, Mathar I, Philipp SE, et al. Increased IgE-dependent mast cell activation and anaphylactic responses in mice lacking the calcium-activated nonselective cation channel TRPM4. Nat Immunol 2007;8(3):312–20. [63] Nelson PL, Zolochevska O, Figueiredo ML, Soliman A, Hsu WH, Feng JM, et al. Regulation of Ca(2+)-entry in pancreatic alpha-cell line by transient receptor potential melastatin 4 plays a vital role in glucagon release. Mol Cell Endocrinol 2011;335(2):126–34. [64] Colsoul B, Schraenen A, Lemaire K, Quintens R, Van Lommel L, Segal A, et al. Loss of high-frequency glucose-induced Ca2+ oscillations in pancreatic islets correlates with impaired glucose tolerance in Trpm5−/− mice. Proc Natl Acad Sci USA 2010;107(11):5208–13. [65] Berggren PO, Yang SN, Murakami M, Efanov AM, Uhles S, Kohler M, et al. Removal of Ca2+ channel beta3 subunit enhances Ca2+ oscillation frequency and insulin exocytosis. Cell 2004;119(2):273–84. 360 19. TRP CATION CHANNELS IN DIABETES

[66] Brixel LR, Monteilh-Zoller MK, Ingenbrandt CS, Fleig A, Penner R, Enklaar T, et al. TRPM5 regulates glucose-stimulated insulin secretion. Pflugers Arch 2010;460(1):69–76. [67] Colsoul B, Jacobs G, Philippaert K, Owsianik G, Segal A, Nilius B, et al. Insulin downregulates the expression of the Ca2+−activated nonselective cation channel TRPM5 in pancreatic islets from leptin-deficient mouse models. Pflugers Arch 2013;466(3):611–21. [68] Young RL, Sutherland K, Pezos N, Brierley SM, Horowitz M, Rayner CK, et al. Expression of taste molecules in the upper gastrointestinal tract in humans with and without type 2 diabetes. Gut 2009;58(3):337–46. [69] Ketterer C, Mussig K, Heni M, Dudziak K, Randrianarisoa E, Wagner R, et al. Genetic variation within the TRPM5 locus associates with prediabetic phenotypes in subjects at increased risk for type 2 diabetes. Metabolism 2011;60(9):1325–33. [70] McKemy DD, Neuhausser WM, Julius D. Identification of a cold receptor reveals a general role for TRP channels in thermosensation. Nature 2002;416(6876):52–8. [71] Peier AM, Moqrich A, Hergarden AC, Reeve AJ, Andersson DA, Story GM, et al. A TRP channel that senses cold stimuli and menthol. Cell 2002;108(5):705–15. [72] Gavva NR, Davis C, Lehto SG, Rao S, Wang W, Zhu DX. Transient receptor potential melastatin 8 (TRPM8) channels are involved in body temperature regulation. Mol Pain 2012;8:36. [73] McCoy DD, Zhou L, Nguyen AK, Watts AG, Donovan CM, McKemy DD. Enhanced insulin clearance in mice lacking TRPM8 channels. Am J Physiol Endocrinol Metab 2013;305(1):E78–88. [74] Yee NS, Chan AS, Yee JD, Yee RK. TRPM7 and TRPM8 Ion Channels in Pancreatic Adenocarcinoma: Potential Roles as Cancer Biomarkers and Targets. Scientifica (Cairo) 2012;2012:415158. [75] Yee NS, Brown RD, Lee MS, Zhou W, Jensen C, Gerke H, et al. TRPM8 ion channel is aberrantly expressed and required for preventing replicative senescence in pancreatic adenocarcinoma: potential role of TRPM8 as a biomarker and target. Cancer Biol Ther 2012;13(8):592–9. [76] Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, Julius D. The capsaicin receptor: a heat- activated ion channel in the pain pathway. Nature 1997;389(6653):816–24. [77] Cavanaugh DJ, Chesler AT, Jackson AC, Sigal YM, Yamanaka H, Grant R, et al. Trpv1 reporter mice reveal highly restricted brain distribution and functional expression in arteriolar smooth muscle cells. J Neurosci 2011;31(13):5067–77. [78] Akiba Y, Kato S, Katsube K, Nakamura M, Takeuchi K, Ishii H, et al. Transient receptor potential vanilloid subfamily 1 expressed in pancreatic islet beta cells modulates insulin secretion in rats. Biochem Biophys Res Commun 2004;321(1):219–25. [79] Fagelskiold AJ, Kannisto K, Bostrom A, Hadrovic B, Farre C, Eweida M, et al. Insulin-secreting INS-1E cells express functional TRPV1 channels. Islets 2012;4(1):56–63. [80] Gram DX, Ahren B, Nagy I, Olsen UB, Brand CL, Sundler F, et al. Capsaicin-sensitive sensory fibers in the islets of Langerhans contribute to defective insulin secretion in Zucker diabetic rat, an animal model for some aspects of human type 2 diabetes. Eur J Neurosci 2007;25(1):213–23. [81] Wang P, Yan Z, Zhong J, Chen J, Ni Y, Li L, et al. Transient receptor potential vanilloid 1 activation enhances gut glucagon-like peptide-1 secretion and improves glucose homeostasis. Diabetes 2012;61(8):2155–65. [82] Razavi R, Chan Y, Afifiyan FN, Liu XJ, Wan X, Yantha J, et al. TRPV1+ sensory neurons control beta cell stress and islet inflammation in autoimmune diabetes. Cell 2006;127(6):1123–35. [83] Sadeh M, Glazer B, Landau Z, Wainstein J, Bezaleli T, Dabby R, et al. Association of the M3151 variant in the transient receptor potential vanilloid receptor-1 (TRPV1) gene with type 1 diabetes in an Ashkenazi Jewish population. Isr Med Assoc J 2013;15(9):477–80. [84] Aoyagi K, Ohara-Imaizumi M, Nishiwaki C, Nakamichi Y, Nagamatsu S. Insulin/phosphoinositide 3-kinase pathway accelerates the glucose-induced first-phase insulin secretion through TrpV2 recruitment in pancreatic beta-cells. Biochem J 2010;432(2):375–86. [85] Hisanaga E, Nagasawa M, Ueki K, Kulkarni RN, Mori M, Kojima I. Regulation of Calcium-permeable TRPV2 Channel by Insulin in Pancreatic {beta} Cells. Diabetes 2008;58:174–84. [86] Kanzaki M, Zhang YQ, Mashima H, Li L, Shibata H, Kojima I. Translocation of a calcium-permeable cation channel induced by insulin-like growth factor-I. Nat Cell Biol 1999;1(3):165–70. [87] Leibiger IB, Leibiger B, Berggren PO. Insulin feedback action on pancreatic beta-cell function. FEBS Lett 2002;532(1–2):1–6. [88] Kulkarni RN, Bruning JC, Winnay JN, Postic C, Magnuson MA, Kahn CR. Tissue-specific knockout of the insulin receptor in pancreatic beta cells creates an insulin secretory defect similar to that in type 2 diabetes. Cell 1999;96(3):329–39. REFERENCES 361

[89] Everaerts W, Nilius B, Owsianik G. The vanilloid transient receptor potential channel TRPV4: From structure to disease. Prog Biophys Mol Biol 2009;103:2–17. [90] Casas S, Novials A, Reimann F, Gomis R, Gribble FM. Calcium elevation in mouse pancreatic beta cells evoked by extracellular human islet amyloid polypeptide involves activation of the mechanosensitive ion channel TRPV4. Diabetologia 2008;51(12):2252–62. [91] Skrzypski M, Kakkassery M, Mergler S, Grotzinger C, Khajavi N, Sassek M, et al. Activation of TRPV4 channel in pancreatic INS-1E beta cells enhances glucose-stimulated insulin secretion via calcium-dependent mechanisms. FEBS Lett 2013;587(19):3281–7. [92] Casas S, Gomis R, Gribble FM, Altirriba J, Knuutila S, Novials A. Impairment of the ubiquitin-proteasome pathway is a downstream endoplasmic reticulum stress response induced by extracellular human islet amyloid polypeptide and contributes to pancreatic beta-cell apoptosis. Diabetes 2007;56(9):2284–94. [93] Lorenzo A, Razzaboni B, Weir GC, Yankner BA. Pancreatic islet cell toxicity of amylin associated with type-2 diabetes mellitus. Nature 1994;368(6473):756–60. [94] Cao DS, Zhong L, Hsieh TH, Abooj M, Bishnoi M, Hughes L, et al. Expression of transient receptor potential ankyrin 1 (TRPA1) and its role in insulin release from rat pancreatic beta cells. PLoS One 2012;7(5):e38005. [95] Numazawa S, Takase M, Ahiko T, Ishii M, Shimizu S, Yoshida T. Possible involvement of transient receptor potential channels in electrophile-induced insulin secretion from RINm5F cells. Biol Pharm Bull 2012;35(3):346–54. [96] Van Buren JJ, Bhat S, Rotello R, Pauza ME, Premkumar LS. Sensitization and translocation of TRPV1 by insulin and IGF-I. Mol Pain 2005;1:17. [97] Sathianathan V, Avelino A, Charrua A, Santha P, Matesz K, Cruz F, et al. Insulin induces cobalt uptake in a subpopulation of rat cultured primary sensory neurons. Eur J Neurosci 2003;18(9):2477–86. [98] Pettersson M, Ahren B, Bottcher G, Sundler F. Calcitonin gene-related peptide: occurrence in pancreatic islets in the mouse and the rat and inhibition of insulin secretion in the mouse. Endocrinology 1986;119(2):865–9. [99] Noble MD, Romac J, Wang Y, Hsu J, Humphrey JE, Liddle RA. Local disruption of the celiac ganglion inhibits substance P release and ameliorates caerulein-induced pancreatitis in rats. Am J Physiol Gastrointest Liver Physiol 2006;291(1):G128–34. [100] Szallasi A, Cortright DN, Blum CA, Eid SR. The vanilloid receptor TRPV1: 10 years from channel cloning to antagonist proof-of-concept. Nat Rev Drug Discov 2007;6(5):357–72. [101] Winston JH, He ZJ, Shenoy M, Xiao SY, Pasricha PJ. Molecular and behavioral changes in nociception in a novel rat model of chronic pancreatitis for the study of pain. Pain 2005;117(1–2):214–22. [102] Wick EC, Hoge SG, Grahn SW, Kim E, Divino LA, Grady EF, et al. Transient receptor potential vanilloid 1, calcitonin gene-related peptide, and substance P mediate nociception in acute pancreatitis. Am J Physiol Gastrointest Liver Physiol 2006;290(5):G959–69. [103] Suri A, Szallasi A. The emerging role of TRPV1 in diabetes and obesity. Trends Pharmacol Sci 2008;29(1):29–36. [104] Guillot E, Coste A, Angel I. Involvement of capsaicin-sensitive nerves in the regulation of glucose tolerance in diabetic rats. Life Sci 1996;59(12):969–77. [105] Grattan M, Mi QS, Meagher C, Delovitch TL. Congenic mapping of the diabetogenic locus Idd4 to a 5.2- cM region of chromosome 11 in NOD mice: identification of two potential candidate subloci. Diabetes 2002;51(1):215–23. [106] Hermansen K, Ahren B. Dual effects of calcitonin gene-related peptide on insulin secretion in the perfused dog pancreas. Regul Pept 1990;27(1):149–57. [107] Tsui H, Razavi R, Chan Y, Yantha J, Dosch HM. ’Sensing’ autoimmunity in type 1 diabetes. Trends Mol Med 2007;13(10):405–13. [108] Cui J, Himms-Hagen J. Long-term decrease in body fat and in brown adipose tissue in capsaicin-desensitized rats. Am J Physiol 1992;262(4 Pt 2):R568–73. [109] Melnyk A, Himms-Hagen J. Resistance to aging-associated obesity in capsaicin-desensitized rats one year after treatment. Obes Res 1995;3(4):337–44. [110] Pare M, Albrecht PJ, Noto CJ, Bodkin NL, Pittenger GL, Schreyer DJ, et al. Differential hypertrophy and atrophy among all types of cutaneous innervation in the glabrous skin of the monkey hand during aging and naturally occurring type 2 diabetes. J Comp Neurol 2007;501(4):543–67. [111] Wang X, Miyares RL, Ahern GP. Oleoylethanolamide excites vagal sensory neurones, induces visceral pain and reduces short-term food intake in mice via capsaicin receptor TRPV1. J Physiol 2005;564 (Pt 2):541–7. 362 19. TRP CATION CHANNELS IN DIABETES

[112] Festa A, D’Agostino Jr R, Howard G, Mykkanen L, Tracy RP, Haffner SM. Chronic subclinical inflammation as part of the insulin resistance syndrome: the Insulin Resistance Atherosclerosis Study (IRAS). Circulation 2000;102(1):42–7. [113] Herder C, Schneitler S, Rathmann W, Haastert B, Schneitler H, Winkler H, et al. Low-grade inflammation, obesity, and insulin resistance in adolescents. J Clin Endocrinol Metab 2007;92(12):4569–74. [114] Wu T, Dorn JP, Donahue RP, Sempos CT, Trevisan M. Associations of serum C-reactive protein with fasting insulin, glucose, and glycosylated hemoglobin: the Third National Health and Nutrition Examination Survey, 1988–1994. Am J Epidemiol 2002;155(1):65–71. [115] Pingle SC, Matta JA, Ahern GP. Capsaicin receptor: TRPV1 a promiscuous TRP channel. Handb Exp Pharmacol 2007;179:155–71. [116] Tanaka H, Shimaya A, Kiso T, Kuramochi T, Shimokawa T, Shibasaki M. Enhanced insulin secretion and sensitization in diabetic mice on chronic treatment with a transient receptor potential vanilloid 1 antagonist. Life Sci 2011;88(11–12):559–63. [117] Cooper ME, Bonnet F, Oldfield M, Jandeleit-Dahm K. Mechanisms of diabetic vasculopathy: an overview. Am J Hypertens 2001;14(5 Pt 1):475–86. [118] Carr ME. Diabetes mellitus: a hypercoagulable state. J Diabetes Complicat 2001;15(1):44–54. [119] Rosado JA, Saavedra FR, Redondo PC, Hernandez-Cruz JM, Salido GM, Pariente JA. Reduced plasma membrane Ca2+−ATPase function in platelets from patients with non-insulin-dependent diabetes mellitus. Haematologica 2004;89(9):1142–4. [120] Redondo PC, Jardin I, Hernandez-Cruz JM, Pariente JA, Salido GM, Rosado JA. Hydrogen peroxide and peroxynitrite enhance Ca2+ mobilization and aggregation in platelets from type 2 diabetic patients. Biochem Biophys Res Commun 2005;333(3):794–802. [121] Schaeffer G, Wascher TC, Kostner GM, Graier WF. Alterations in platelet Ca2+ signalling in diabetic patients is due to increased formation of superoxide anions and reduced nitric oxide production. Diabetologia 1999;42(2):167–76. [122] Berna-Erro A, Redondo PC, Rosado JA. Store-operated ca(2+) entry. Adv Exp Med Biol 2012;740:349–82. [123] Hassock SR, Zhu MX, Trost C, Flockerzi V, Authi KS. Expression and role of TRPC proteins in human platelets: evidence that TRPC6 forms the store-independent calcium entry channel. Blood 2002;100(8):2801–11. [124] Zbidi H, Lopez JJ, Amor NB, Bartegi A, Salido GM, Rosado JA. Enhanced expression of STIM1/Orai1 and TRPC3 in platelets from patients with type 2 diabetes mellitus. Blood Cells Mol Dis 2009;43(2):211–13. [125] Liu D, Maier A, Scholze A, Rauch U, Boltzen U, Zhao Z, et al. High glucose enhances transient receptor potential channel canonical type 6-dependent calcium influx in human platelets via phosphatidylinositol 3-kinase-dependent pathway. Arterioscler Thromb Vasc Biol 2008;28(4):746–51. [126] Jardin I, Lopez JJ, Zbidi H, Bartegi A, Salido GM, Rosado JA. Attenuated store-operated divalent cation entry and association between STIM1, Orai1, hTRPC1 and hTRPC6 in platelets from type 2 diabetic patients. Blood Cells Mol Dis 2011;46(3):252–60. [127] Lee CH, Poburko D, Kuo KH, Seow C, van Breemen C. Relationship between the sarcoplasmic reticulum and the plasma membrane. Novartis Found Symp 2002;246:26–41 discussion 41−47, 48–51. [128] Chaudhuri A. Vascular reactivity in diabetes mellitus. Curr Diab Rep 2002;2(4):305–10. [129] Chung AW, Au Yeung K, Chum E, Okon EB, van Breemen C. Diabetes modulates capacitative calcium entry and expression of transient receptor potential canonical channels in human saphenous vein. Eur J Pharmacol 2009;613(1–3):114–18. [130] Mita M, Ito K, Taira K, Nakagawa J, Walsh MP, Shoji M. Attenuation of store-operated Ca2+ entry and enhanced expression of TRPC channels in caudal artery smooth muscle from Type 2 diabetic Goto-Kakizaki rats. Clin Exp Pharmacol Physiol 2010;37(7):670–8. [131] Niehof M, Borlak J. HNF4 alpha and the Ca-channel TRPC1 are novel disease candidate genes in diabetic nephropathy. Diabetes 2008;57(4):1069–77. [132] Zhang D, Freedman BI, Flekac M, Santos E, Hicks PJ, Bowden DW, et al. Evaluation of genetic association and expression reduction of TRPC1 in the development of diabetic nephropathy. Am J Nephrol 2009;29(3):244–51. [133] Graham S, Ding M, Sours-Brothers S, Yorio T, Ma JX, Ma R. Downregulation of TRPC6 protein expression by high glucose, a possible mechanism for the impaired Ca2+ signaling in glomerular mesangial cells in diabetes. Am J Physiol Renal Physiol 2007;293(4):F1381–90. [134] Vinik AI, Park TS, Stansberry KB, Pittenger GL. Diabetic neuropathies. Diabetologia 2000;43(8):957–73. [135] Caterina MJ, Leffler A, Malmberg AB, Martin WJ, Trafton J, Petersen-Zeitz KR, et al. Impaired nociception and pain sensation in mice lacking the capsaicin receptor. Science 2000;288(5464):306–13. REFERENCES 363

[136] Forst T, Pohlmann T, Kunt T, Goitom K, Schulz G, Lobig M, et al. The influence of local capsaicin treatment on small nerve fibre function and neurovascular control in symptomatic diabetic neuropathy. Acta Diabetol 2002;39(1):1–6. [137] Van Gerven L, Alpizar YA, Wouters MM, Hox V, Hauben E, Jorissen M, et al. Capsaicin treatment reduces nasal hyperreactivity and transient receptor potential cation channel subfamily V, receptor 1 (TRPV1) overexpression in patients with idiopathic rhinitis. J Allergy Clin Immunol 2013;133:1332–9. [138] Hong S, Wiley JW. Early painful diabetic neuropathy is associated with differential changes in the expression and function of vanilloid receptor 1. J Biol Chem 2005;280(1):618–27. [139] Pabbidi RM, Yu SQ, Peng S, Khardori R, Pauza ME, Premkumar LS. Influence of TRPV1 on diabetes-induced alterations in thermal pain sensitivity. Mol Pain 2008;4:9. [140] Facer P, Casula MA, Smith GD, Benham CD, Chessell IP, Bountra C, et al. Differential expression of the capsaicin receptor TRPV1 and related novel receptors TRPV3, TRPV4 and TRPM8 in normal human tissues and changes in traumatic and diabetic neuropathy. BMC Neurol 2007;7:11. [141] Zahner MR, Li DP, Chen SR, Pan HL. Cardiac vanilloid receptor 1-expressing afferent nerves and their role in the cardiogenic sympathetic reflex in rats. J Physiol 2003;551(Pt 2):515–23. [142] Song JX, Wang LH, Yao L, Xu C, Wei ZH, Zheng LR. Impaired transient receptor potential vanilloid 1 in streptozotocin-induced diabetic hearts. Int J Cardiol 2009;134(2):290–2. [143] Zimmermann M. Pathobiology of neuropathic pain. Eur J Pharmacol 2001;429(1–3):23–37. [144] Millan MJ. Descending control of pain. Prog Neurobiol 2002;66(6):355–474. [145] Palazzo E, Luongo L, de Novellis V, Rossi F, Marabese I, Maione S. Transient receptor potential vanilloid type 1 and pain development. Curr Opin Pharmacol 2012;12(1):9–17. [146] Mohammadi-Farani A, Sahebgharani M, Sepehrizadeh Z, Jaberi E, Ghazi-Khansari M. Diabetic thermal hyperalgesia: role of TRPV1 and CB1 receptors of periaqueductal gray. Brain Res 2010;1328:49–56. [147] Bishnoi M, Bosgraaf CA, Abooj M, Zhong L, Premkumar LS. Streptozotocin-induced early thermal hyperalgesia is independent of glycemic state of rats: role of transient receptor potential vanilloid 1(TRPV1) and inflammatory mediators. Mol Pain 2011;7:52. [148] Pabbidi RM, Cao DS, Parihar A, Pauza ME, Premkumar LS. Direct role of streptozotocin in inducing thermal hyperalgesia by enhanced expression of transient receptor potential vanilloid 1 in sensory neurons. Mol Pharmacol 2008;73(3):995–1004. [149] Barriere DA, Rieusset J, Chanteranne D, Busserolles J, Chauvin MA, Chapuis L, et al. Paclitaxel therapy potentiates cold hyperalgesia in streptozotocin-induced diabetic rats through enhanced mitochondrial reactive oxygen species production and TRPA1 sensitization. Pain 2012;153(3):553–61. [150] Ohkawara S, Tanaka-Kagawa T, Furukawa Y, Jinno H. Methylglyoxal activates the human transient receptor potential ankyrin 1 channel. J Toxicol Sci 2012;37(4):831–5. [151] Trevisani M, Siemens J, Materazzi S, Bautista DM, Nassini R, Campi B, et al. 4-Hydroxynonenal, an endogenous aldehyde, causes pain and neurogenic inflammation through activation of the irritant receptor TRPA1. Proc Natl Acad Sci USA 2007;104(33):13519–24. [152] Koivisto A, Hukkanen M, Saarnilehto M, Chapman H, Kuokkanen K, Wei H, et al. Inhibiting TRPA1 ion channel reduces loss of cutaneous nerve fiber function in diabetic animals: sustained activation of the TRPA1 channel contributes to the pathogenesis of peripheral diabetic neuropathy. Pharmacol Res 2012;65(1):149–58. [153] Wei H, Hamalainen MM, Saarnilehto M, Koivisto A, Pertovaara A. Attenuation of mechanical hypersensitivity by an antagonist of the TRPA1 ion channel in diabetic animals. Anesthesiology 2009;111(1):147–54. [154] Wei H, Chapman H, Saarnilehto M, Kuokkanen K, Koivisto A, Pertovaara A. Roles of cutaneous versus spinal TRPA1 channels in mechanical hypersensitivity in the diabetic or mustard oil-treated non-diabetic rat. Neuropharmacology 2010;58(3):578–84. [155] Wajchenberg BL. Clinical approaches to preserve beta-cell function in diabetes. Adv Exp Med Biol 2010;654:515–35. [156] Cernea S, Raz I. Therapy in the early stage: incretins. Diabetes Care 2011;34(Suppl 2):S264–71. CHAPTER 20 TRP Channels in Cardiovascular Disease Kavisha Singh, Nancy Luo, Paul Rosenberg* Division of Cardiology, Department of Medicine, Duke University School of Medicine, Durham, North Carolina, USA *Corresponding author: [email protected]

OUTLINE

Calcium Signaling in the TRP Channels and Cardiac Cardiovascular System 365 Arrhythmias 372 TRP Channels and Cardiovascular TRPM4 and Cardiac Conduction Disease 366 Disease 373 TRP Channels in Cardiac TRPM7 Channels and Atrial Hypertrophy 367 Fibrillation 374 Role of TRP Channels in Cardiac TRPC Channels in Pulmonary Hypertrophy Signaling Pathways 369 Hypertension 375 Pharmacological Developments Acknowledgment 378 Targeting TRP Channels in References 378 Cardiac Hypertrophy 370

CALCIUM SIGNALING IN THE CARDIOVASCULAR SYSTEM

In virtually all cell types in the heart and vasculature, changes in the frequency and amplitude of calcium transients have been recognized as an important response to the changing workload [1,2]. Encoded in these calcium transients are signals that alter not only the immediate contractile response, as occurs with excitation contraction coupling, but also signals

TRP Channels as Therapeutic Targets 365 © 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/B978-0-12-420024-1.00020-5 366 20. TRP CHANNELS IN CARDIAC PHYSIOLOGY that initiate and maintain a remodeling response that adjusts cellular mass, ionic currents, kinetic properties of contractile proteins, and metabolic capacity [3]. Indeed, increased Ca2+ loading inside the cell, resulting in prolonged Ca2+ transients and elevated end diastolic Ca2+ concentrations, may be one of the pathophysiological changes that occurs in myocytes of the heart (cardiomyocytes) and vasculature [4,5]. Whether the handling of Ca2+ and the downstream signaling are the same in these different cell types would have important therapeutic implications. In fact, recent attention has begun to focus on transient receptor potential (TRP) channels because they may link mechanical activity to cell signaling in all muscle cell types [6]. Once considered the purview of only nonexcitable cells, TRP channels are now believed to be a key downstream target of activated G-protein coupled receptors (GPRCs) in excitable muscle cells. Hypertrophic agonists use GPRC signaling to change the activation of TRPC channels and promote calcium entry, thereby triggering growth signaling. One of the key pathways thought to be a cornerstone in the development of pathological hypertrophy of cardiac and smooth muscle myocytes is the calcineurin-NFAT (nuclear factor of activated T-cells) pathway. Calcineurin is a Ca2+-sensitive serine/threonine phosphatase that dephosphorylates NFAT and causes its translocation to the nucleus. In the nucleus, NFAT works with the cardiac-restricted zinc finger transcription factor GATA4 to activate the transcription of hypertrophic response genes encoding transcription factors such as NFAT and myocyte enhancing factor 2 (MEF2) [7]. Activation of the Ca2+-calcineurin-NFAT pathway has been strongly associated with the development of ventricular hypertrophy and found to be both sufficient and necessary for this pathological process [8,9]. MEF2, which is regulated independently by the class II histone deacytelases, is also turned on by calcineurin-induced des- phosphorylation. The downstream effects of these Ca2+-calcineurin-activated pathways result in a hypertrophic response that is mediated by growth factors such as transforming growth factor-β (TGF-β), connective tissue growth factor, and a switch to cardiac fetal gene expression patterns [10]. Many of the same signaling events contribute to the pathologic changes that occur in response to injury of the pulmonary artery. Recently, there has been increased interest in understanding these intracellular Ca2+ transients, which could potentially have a bigger role in controlling these transcription pathways. How the cell discriminates between different Ca2+ signals remains a poorly understood area of research, but nevertheless important in truly understanding how a ubiquitous second messenger has the ability to modulate a wide variety of intracellular mechanisms, both in physiological and pathological states.

TRP CHANNELS AND CARDIOVASCULAR DISEASE

TRP channels were the last superfamily of ion channels to be identified and represent a large family of nonselective cation channels that operate at the plasma membrane [11]. The individual channels in the family are unique in their biophysical and electrical properties and are activated by different mechanisms [12]. TRP channels are expressed in various cell types including cardiomyocytes, smooth muscle cells, and Purkinje fibers, but their role in cardiovascular disease is currently being defined [13–16]. There is a growing body of evidence that certain TRP subfamilies, such as TRPC and TRP melastatin (TRPM), are more critically important in various cardiovascular pathologies, from cardiac hypertrophy and arrhythmogenesis to pulmonary arterial hypertension (PAH) [17]. TRP Channels in Cardiac Hypertrophy 367

TRP channels contribute to Ca2+ entry via their action at the plasma membrane as nonselective cation channels. Regulation of intracellular Ca2+ by extracellular mechanisms occurs through several pathways, including Ca2+ release from intracellular organelles, activation of voltage-gated channels, Ca2+ entry through ligand-gated channels, and receptor-operated Ca2+ entry (ROCE). One of the postulated mechanisms by which TRP channels control Ca2+ is through store-operated Ca2+ entry (SOCE), where a drop in sarcoplasmic reticulum (SR) Ca2+ stores triggers the influx of Ca2+ through channels like Orai1 and TRPs at the plasma membrane [18]. SOCE has been implicated as a distinct mechanism of regulating intracellular calcium apart from L-type calcium channel and sodium-calcium exchanger and has been demonstrated in cardiomyocytes at the embryonic and neonatal stages [19]. With the discovery of Stromal Interaction Molecule 1 (STIM1) and Orai1 and their role in executing SOCE, it is not completely clear how critical a role TRP channels play or if they represent a distinct form of SOC channels [18]. Because many TRP channels are activated by a rise in intracellular Ca2+ levels, it has been difficult to distinguish channel gating between Ca2+ versus store dependence. However, there are several studies postulating that TRPCs, STIM1, and Orai1 interact with each other at a subcellular level to control SOCE, suggesting that at least some of the members of the TRP family are important in this mechanism [20]. Another mechanism by which TRP channels are activated to regulate intracellular Ca2+ is receptor-mediated Ca2+ entry. Agonist binding to a membrane receptor that is distinct from the TRP channel stimulates the production of intracellular second messengers that then lead to the activation of TRP channels to cause Ca2+ influx [21]. Most literature suggests that TRP channels function downstream of GPCRs, which then activate phospholipase C and a downstream cascade of second messengers. Diacylglycerol (DAG) has been postulated as one of the downstream signals responsible for direct activation of TRP channels in receptor- mediated Ca2+ entry. Some TRP channels have been proposed to be more involved in SOCE, and some are thought to be more receptive to receptor-mediated activation [22]. Within the TRPC subfamily, TRPC1/4/5 are thought to be activated through SOCE whereas TRPC3/6/7 are thought to be activated mainly by second messenger DAG [11] (Figure 20.1).

TRP CHANNELS IN CARDIAC HYPERTROPHY

Pathological cardiac hypertrophy is a typically maladaptive state of cardiac muscle that is associated with a variety of disease states, including hypertension, ischemic injury, heart failure, and valvular disease. Once cardiac hypertrophy has developed, the risks of adverse cardiac events increase along with associated morbidity and mortality [23]. Understanding the pathophysiology behind cardiac hypertrophy is critical to any pharmacological approach that attempts to alter disease mechanisms in the heart [24]. There is a growing amount of evidence that the canonical TRP subfamily (TRPC) is involved in mediating the development of cardiac hypertrophy, especially in disease models of pressure overload and neurohormonal excess [17,25,26]. Overexpression of TRPC channels (especially TRPC1/C3/C6) has been associated with an exaggerated response of cardiac myocytes to stressors such as pressure overload and stimulation via neuroendocrine agonists. TRPC3 is overexpressed in neonatal rat ventricular myocytes subjected to phenylephrine (PE) infusions, suggesting that TRPC channels are activated in pathological states [27]. TRPC3 transgenic mice also show an age-dependent 368 20. TRP CHANNELS IN CARDIAC PHYSIOLOGY

FIGURE 20.1 Mechanism of activation of TRP channels.

cardiomyopathy resulting from cardiac hypertrophy [28]. This cardiomyopathy also proved itself to be dose dependent, with high expressing TRPC3 transgenic mice developing pathology much earlier than the corresponding low-dose TRPC3 mouse line. Especially when subjected to agonists such as PE and angiotensin II (ATII) infusions for prolonged periods of time and transverse aortic constriction (TAC) to simulate pressure overload situations, mice overexpressing TRPC3 developed greater cardiac hypertrophy compared to wild-type controls. In certain studies, merely overexpressing TRPC3 was insufficient to stimulate the expression of genes associated with hypertrophy such as BNP (brain natriuretic peptide), but costimulation with hypertrophic agonists like PE did produce this response [27]. This finding suggests that even if altering TRPC channel expression at baseline does not induce pathological hypertrophy, the heart may become more susceptible to stresses such as pressure overload and neurohormonal excesses. ATII, which has been shown to induce the nuclear translocation of NFAT, also activates TRPC3 and TRPC6 channels through DAG, which results in the Ca2+ influx that is necessary in activating the calcineurin-NFAT pathway leading to cardiac hypertrophy [29]. In addition, combined deletion of TRPC3 and TRPC6 was protective against cardiac hypertrophy, suggesting a role for pharmacological inhibitors of these channels [30]. Similar studies were conducted in mouse hearts with transgenic expression of TRPC6. The transgene dosage of TRPC6 in mouse hearts directly correlated with the heart weight/ birth weight ratios in these mice, with the highest dosage of TRPC6 inducing the earliest and greatest signs of cardiac hypertrophy. Even though the transgenic mice with the lowest expression of TRPC6 showed no evidence of hypertrophy or heart failure at an adult stage, subjecting them to pressure overload by TAC immediately induced an increase in heart weight/birth weight ratios and a decrease in systolic function [31]. There is also evidence for the role of TRPC1 in the development of cardiac hypertrophy. Unlike TRPC3/6, which are DAG activated, TRPC1 may operate as a SOCE channel. Looking at TRPC1−/− mice after subjecting them to pressure overload via TAC, we found that mice lacking TRPC1 only showed Role of TRP Channels in Cardiac Hypertrophy Signaling Pathways 369 a modest increase in cardiac mass and had relatively preserved cardiac function when compared to wild type (WT) mice subjected to the same stresses. TRPC1−/− mice also showed decreased NFAT activation post-TAC compared to their WT counterparts, suggesting that the absence of TRPC1−/− was cardioprotective in the pathological situation of pressure overload [32]. Another study showed that TRPC1 channels stimulated the calcineurin/NFAT pathway through serotonin receptors in cardiomyoblasts, lending more support to the importance of TRPC1 in the development of cardiac hypertrophy [33].

ROLE OF TRP CHANNELS IN CARDIAC HYPERTROPHY SIGNALING PATHWAYS

The pathological changes that occur in cardiac hypertrophy along with the alteration of the functional properties of the myocardium are likely mediated by altered gene expression patterns. Pathological cardiac hypertrophy is a result of cardiac remodeling and a change in the functional properties of the myocardium that can only be a result of altered gene expressions. Altered Ca2+ signaling directly affects signaling pathways involved in the development of cardiac hypertrophy [24]. Because many of these pathways engage in cross talk with each other, understanding the choreographic details of these pathways is important if effective pharmacological approaches are to be developed in the future. It is beyond the scope of this chapter to discuss all the signaling mechanisms that are involved in the development of cardiac hypertrophy. We will focus the discussion on the pathways involved in the development of cardiac hypertrophy with which TRP channels have been associated. Several studies have implicated the importance of the calcineurin-NFAT pathway in the development of cardiac hypertrophy [10]. Calcineurin (Ca2+/calmodulin-dependent protein phosphatase 2B) links changes in intracellular Ca2+ to altered gene expression through NFAT. It is a serine/threonine phosphatase that is activated by increases in intracellular Ca2+. Once activated, it causes NFAT to become dephosphorylated and translocated to the nucleus. NFAT in the nucleus activates the transcription of several genes mediating cardiac hypertrophy through the cardiac-restricted zinc finger transcription factor GATA4 [34]. Fetal cardiac genes such as beta-myosin heavy chain (β-MHC), atrial natriuretic peptide (ANP), and BNP are all transcribed by the activation of the calcineurin-NFAT signaling pathway, with which TRP channels have been associated [35]. TRPC3 transgenic mice showed greater increases in the expression of a NFAT-luciferase transgene, especially when subjected to pressure overload and agonist stimulation, indicating that NFAT activation occurs downstream of TRPC channel activity. TRPC3 overexpression also stimulated translocation of NFAT to the nucleus, and TRPC6 was found to be a positive regulator of the calcineurin-NFAT pathway resulting in β-MHC expression [31,36]. TRPC3 and TRPC6 are directly activated by DAG, which links receptor activation to Ca2+ influx through the TRPC channels and subsequently to the activation of the calcineurin-NFAT pathway [37]. TRP channels have also been linked to regulation by antihypertrophic pathways. ANP and BNP are antihypertrophic cardiac fetal proteins that are up-regulated by calcineurin-NFAT activation, possibly to modulate the complex process of pathological cardiac hypertrophy. ANP and BNP have a common receptor, guanylyl cyclase-A (GC-A), which leads to the activation of protein kinase G (PKG) by synthesis of cyclic guanosine monophosphate (cGMP). 370 20. TRP CHANNELS IN CARDIAC PHYSIOLOGY

Initially, only the antiadrenergic effects of ANP, nitric oxide (NO), and cGMP through the inhibition of β and α adrenergic effects in cardiofibroblasts and myocytes were described [38]. In addition, the negative inotropic and growth-inhibiting effects of ANP were partly attributed to its ability to inhibit Ca2+ influx by blocking L-type Ca2+ channels [38]. Recently, the ANP-GC-A pathway has been better described in cardiac remodeling, opening up potential therapeutic avenues [39]. In mice with cardiomyocyte-specific deletion of (GC)-A, the heart weight/body weight ratios are increased, suggesting cardiac hypertrophy, and biomarkers of cardiac hypertrophy such as ANP and β-MHC are up-regulated [40]. These effects are even more exaggerated once the mice are subjected to pressure overload, indicating that preserving the ANP-GC-A pathway is important for prevention of cardiac hypertrophy. Studies are now showing that PKG, which is downstream of this pathway, catalyzes the phosphorylation of highly conserved threonine and serine residues on TRPC3/6, which attenuates the activities of these TRPC channels [41]. GC-A may even form a complex with TRPC3/6 in mice, suggesting that the pathway may be activated by TRPC channels independent of ANP [42]. Given that cardiac hypertrophy involves up-regulation of TRPC3/6, which contributes to NFAT activation, ANP/BNP may be acting as in vivo inhibitors of TRPC3/6 to produce an antihypertrophic effect [43,44]. In cultured neonatal rat ventricular myocytes, it was found that endothelin-1-activated NFAT expression was suppressed by ANP both at baseline and in models of TRPC6 overexpression [45]. Furthermore, when the PKG phosphorylation site, Thr69, on TRPC6 was mutated, the inhibitory effects of ANP were completely abolished, suggesting that ANP acts on TRPC channels through PKG-mediated phosphorylation of these conserved residues [45]. Thus, the calcineurin-NFAT pathway, which is prohypertrophic, and the ANP-GC-A-cGMP-PKG pathway, which is antihypertrophic, may cross talk with each other through TRPC channels to regulate cardiac remodeling [44].

PHARMACOLOGICAL DEVELOPMENTS TARGETING TRP CHANNELS IN CARDIAC HYPERTROPHY

Using TRP channels as a therapeutic target is potentially useful given its role in the pathophysiology of cardiac hypertrophy, but it presents several pharmacological challenges. Defining a single-channel TRPC current has proven difficult, as TRPC channels work in a highly heterogeneous system and are present in vivo as homo and heterotetramers. Within the TRPC subfamily, the individual properties of each channel have not been well defined. In addition, TRPC channels most likely act in concert with STIM1 and Orai1 to regulate SOCE [20,46]. Understanding how these channels function collectively presents a significant challenge in finding antagonists to specific TRPC channels. Gene deletion could result in compensatory overexpression of other channels involved in SOCE and ROCE in ways that are not yet well understood. Aside from these challenges, there has been a relative paucity of selective small-molecule inhibitors of TRP channels. Kaneko and Szallasi provide a comprehensive review of the various TRP agonists and antagonists that have been implicated as potential therapeutic options [47]. BTP2 (N-[4-(3,5-bis(trifluoromethyl)-1H- pyrazol-1-yl)phenyl]-4-methyl-1,2,3-thiadiazole-5-carboxamide) has been known to inhibit both TRPC and calcium release activated channels (CRAC), and SKF96365, which has been used in many of the studies investigating the physiology of TRPC channels, is an inositol triphosphate 3 (IP3) receptor that blocks both TRPC and T-type Ca2+ channels [48–50]. Pharmacological Developments Targeting TRP Channels in Cardiac Hypertrophy 371

For many years, only nonselective inhibitors of TRPC channels were available. One of the earliest TRPC inhibitors, known as BTP2, was initially identified as an inhibitor of SOCE current in T lymphocytes [50] but was eventually shown to be an inhibitor of TRPC3,5,6 and possibly even TRPM channels in human embryonic kidney (HEK293) cells [51]. Given the relative potency of BTP2 for TRPC channels and its ability to modify SOCE, it was a definite advance in the search for selective blockers of TRPC channels [52]. This discovery led to the development of other TRPC channel blockers by looking at the structural moieties that made up the family of BTPs. In 2008, a pyrazole compound, ethyl-1-(4-(2,3,3-trichloroacrylamide)phenyl)-5-(trifluoromethyl)- 1H-pyrazole-4-carboxylate was identified and named Pyr3 [53]. By stimulating HEK293 cells with carbachol and DAG to activate TRPC channels, the study showed that application of Pyr3 inhibited the activity of the TRPC3 channel with some selectivity when compared to Pyr1, Pyr2, and Pyr4. Looking at rat neonatal cardiomyocytes subjected to ATII infusions, it was found that treatment with Pyr3 decreased the translocation of NFAT and the expression of BNP, both of which are implicated in the process of pathological cardiac remodeling. When subjected to TAC, chronic treatment with Pyr3 resulted in attenuation of both concentric and dilated cardiomyopathy, as well as a decrease in ANP expression [53]. Pyr3 represents one of the first compounds showing relatively increased selectivity for TRPC3 channels compared to others in its class. However, because it blocks TRPC3 selectively and leaves other channels like TRPC6 and TRPC1 relatively untouched, it remains to be seen whether it can accomplish sufficient SOCE current blockade to prevent the development of cardiac hypertrophy. Other studies have suggested that individual TRPC channel blockade may not be enough to overcome the compensation by other members of the TRPC subfamily mediating the same pathological processes [30]. On the other hand, because TRPC channels function in heteromers, individual channel blockade may be sufficient to disrupt the normal functioning of the other channels making up the heteromer. Pyr3 has been shown to block Orai1 as well at concentrations that are approachable to the ones used for TRPC3 blockade, suggesting that it may not be as selective as predicted [54]. As part of the search for selective TRP channel inhibitors, a high throughput screen of piperidine and isoquinolone analogs based on an aniline-thiazide core helped identify high-potency analogs that blocked the increased TRPC3 and TRPC6 current in HEK cells when they were challenged with carbachol and a DAG analog [55]. Two of the novel compounds identified through this screen were used to model dual inhibition of TRPC3/6 in mouse and rat myocytes [30]. This study was unable to look at in vivo effects of the novel compounds GSK503A and GSK255B because of pharmacodynamic challenges in both animal models. However, when looking at cultured myocytes, it showed that the calcineurin-NFAT activation seen on stimulation by endothelin-1 was blunted by GSK503A in a dose-dependent manner. On subjecting mice to TAC to simulate pressure overload and then stimulating the cultured myocytes with ET-1, it showed increases in expression of TRPC3, TRPC6, and fetal gene markers of cardiac hypertrophy as well as increased NFAT activation. All these changes were attenuated when the myocytes were coincubated with GSK503A. Interestingly, when GSK503A was present, the overexpression of TRPC1 was also prevented, suggesting that blocking TRPC3/6 might prevent expression of other TRPC channels in the same subfamily. The results of this study go back to the original question of single versus dual inhibition of TRPC channels in future pharmacological approaches. Their single knockout models of TRPC3 and TRPC6, respectively, did not show any attenuation in cardiac hypertrophy when 372 20. TRP CHANNELS IN CARDIAC PHYSIOLOGY stimulated, suggesting that the methodologies of future studies modeling single TRPC channel inhibition will have to be scrutinized for their use of dominant negative proteins versus gene deletion models. The method of inhibition may be significant in the extrapolation of the data to the pharmacological potential of the compounds under study. The ability of native proteins to regulate TRPC channels is also being discovered. A type I membrane protein called Klotho produced in the kidney, parathyroid glands, and the choroid plexus has been shown to inhibit TRPC6 and thus act as a potentially cardioprotective agent at baseline [56]. Murine hearts that were stressed by isoproterenol infusions showed exaggerated cardiac hypertrophy and cardiac fibrosis in Klotho deficient mouse lines. Even though underexpression or overexpression of Klotho produced no changes at baseline, under isoproterenol stress, overexpressing Klotho was cardioprotective. Targeting the ANP-GC-A-cGMP-PKG-TRPC pathway is another strategy currently under study. Several mouse lines that were deficient in endothelial nitric oxide synthase (eNOS) demonstrated the expected phenotype of arterial hypertension but no cardiac hypertrophy [43]. In another study, the eNOS−/− mouse line had increased expression of ANP in left ventricular myocytes, suggesting that the increase in ANP might be protecting the heart against hypertrophic processes [43]. When GC-A was knocked out in these eNOS−/− mice, the cardio protective effect against hypertrophy was lost. Given that the production of NO increases the synthesis of cGMP, thus activating the pathway that eventually results in TRPC6 inhibition, this is a therapeutic target that could potentially be exploited to mitigate the prohypertrophic signaling mechanisms governed by the TRPC channels [57,58]. Sildenafil is a cGMP specific phosphodiesterase 5 (PDE5) inhibitor clinically used for the treatment of pulmonary hypertension with new pharmacological roles in the attenuation of cardiac remodeling [39]. In experiments that looked at how sildenafil could affect TRPC activity, it was found that PDE5 inhibition led to PKG activation and subsequent phosphorylation of TRPC6 at Thr69. This phosphorylation by PKG was found to be necessary to suppress the activity of TRPC channels because altering the Thr69 residue abolished this effect [45,59]. When cultured cardiomyocytes stimulated by endothelin were treated with sildenafil, NFAT activation was suppressed, and the overexpression of TRPC1/3/6 was attenuated as well [60]. Using sildenafil to prevent cardiac hypertrophy is a tempting pharmacological approach, given that PDE5 inhibitors are already in clinical use. There is suggestion in the literature that phosphorylation mediated by PDE3 inhibitors like cilostazol may also impact the activity of TRPC channels [57].

TRP CHANNELS AND CARDIAC ARRHYTHMIAS

The role of TRP channels in various cardiac conduction abnormalities is now being described. Several studies showed the presence of TRP channels in excitable tissue across the cardiac conduction system, from sinoatrial node cells to Purkinje cells to atrial myocytes [61,62]. In 2006, a Ca2+-activated, nonselective cation channel current was described in human atrial myocytes, which was similar to the current described in rat ventricular myocytes 2 years earlier [63,64]. The fact that this channel was activated by micromolar concentrations of intracellular Ca2+ led to the hypothesis that it might be involved in the development of cardiac arrhythmias such as atrial flutter and fibrillation in conditions like heart failure, where TRPM4 and Cardiac Conduction Disease 373

Ca2+ homeostasis is disrupted. Furthermore, the biophysical properties of this channel were found to be similar to that of TRPM4 and TRPM5, which suggested that these channels might be one and the same [65]. Since then, there has been increased evidence from genetic studies of families with conduction disease and arrhythmias that the TRPM channels are indeed involved in abnormal cardiac arrhythmias [66].

TRPM4 AND CARDIAC CONDUCTION DISEASE

TRPM4 is a member of the melastatin TRP subfamily. Unlike other TRP channels that are permeable to Ca2+ in varying degrees, the TRPM4 channel is Ca2+ impermeable and is only activated by an increase in intracellular Ca2+. It is only permeable to monovalent cations, being the most permeable to Na+ and K+. It is closely related to TRPM5 in these properties [65]. The presence of TRPM4 has been detected in various cardiac tissues, whether directly or indirectly. Ca2+-activated nonselective cation channel currents have been observed in the sinoatrial node whose properties are very similar to the TRPM4 current [67,68]. Ventricular myocytes have also shown evidence of a TRPM4-like current, even though detecting TRPM4 mRNA levels in the ventricle has proved to be challenging [64]. Staining for TRPM4 has shown in both atrial myocytes and the Purkinje fibers, suggesting its role in the normal physiology of the cardiac conduction system [69]. In fact, there is speculation that TRPM4 currents could be responsible for the phenomenon of supernormal conduction in the excitable tissue of the atria and His bundles of the heart [35,70]. Interestingly, many mutations in TRPM4 are being identified in several types of familial conduction disease. Progressive familial heart block type I (PFHBI) is a type of autosomal dominant cardiac conduction disease that has been genetically mapped to the locus on chromosome 19 that encodes TRPM4 [71]. The families with PFHBI show initial right bundle branch blocks (RBBB) that progress to bifascicular block and eventually complete heart block (CHB). Liu et al. recently described three families from Lebanon and France with mutations in the region on chromosome 19 that spans TRPM4 presenting with RBBB, left axis deviation, right axis deviation, and atrioventricular (AV) blocks [69]. The inheritance pattern of this isolated cardiac conduction disease (ICCD) was autosomal dominant with incomplete penetrance. Both studies found that TRPM4 was expressed at higher levels at the cell surface in the mutants compared to the wild types. They also proposed that defects in post-translational processes such as endocytosis and Small Ubiquitin MOdifier Conjugation (SUMOylation) caused the increased trafficking of TRPM4 to the plasma membrane. Endocytotic packaging of proteins and the balance between SUMOylation/deSUMOylation affect the degree of protein expression and degradation, ultimately impacting the functional intracellular protein levels. Both ICCD and PFHBI suggest that increased TRPM4 levels lead to an increase in the supernormal conduction, causing delays in normal conduction pathway impulse propagation, expressed clinically as RBBB, CHB, or AV blocks on the electrocardiogram. Another six novel mutations in TRPM4, including both amino acid substitutions and in-frame deletions, were discovered in both familial and sporadic cases of cardiac conduction disease by Stallmeyer et al., bringing the total number of known TRPM4 mutations linked with cardiac conduction abnormalities to 10 [72]. These mutations also resulted in conduction abnormalities such as RBBB and varying degrees of AV block. 374 20. TRP CHANNELS IN CARDIAC PHYSIOLOGY

Given that TRPM4 channelopathies are being discovered in families with cardiac conduction disease, TRPM4 may become a potentially useful therapeutic target for prevention of cardiac arrhythmias. In fact, various compounds with TRPM4 inhibitory and activation properties have already been described. 9-Phenanthrol, a benzo(c)quinolizinium derivative, has been described as an inhibitor of TRPM4 with high selectivity for this channel. 9-Phenanthrol was found to decrease the frequency of early after depolarizations induced by hypoxia and reoxygenation, suggesting that its antiarrhythmic activity may be linked to its direct inhibition of TRPM4 [73,74]. MPB-104 is another compound in the same pharmacological family as 9-phenanthrol, which is also known to have effects on the cystic fibrosis transmembrane conductance regulator [75]. Flufenamic acid is the TRPM4 inhibitor that has been most widely used in experimental models, but is also fairly nonselective, with actions on Ca2+ activated Cl− channels as well [76]. BTP2, which is an inhibitor of TRPC3 and TRPC5 as well as other CRAC channels, is thought to be a TRPM4 inhibitor. All these compounds are potential pharmacological targets that need to be further investigated for their applicability in the cardiac conduction diseases associated with TRPM4. In addition, the discovery that endocytic dysregulation is associated with the increased TRPM4 expression in both PFHBI and ICCD is raising interest about the possibility of regulation of ion channel density by controlling the mechanisms of endocytosis as a novel pharmaceutical approach. It remains to be seen whether any of these drugs can make it to the market as potential antiarrhythmics.

TRPM7 CHANNELS AND ATRIAL FIBRILLATION

Abnormal Ca2+ handling is thought to be one of the mechanisms underlying the development of atrial fibrillation (AF). Increased delay after depolarizations due to ryanodine receptor dysregulation causing increased SR Ca2+ load and subsequent Ca2+ leak is thought to be one of the arrhythmogenic factors resulting in paroxysmal AF [74,77]. These Ca2+ handling abnormalities can result in electrical and structural remodeling that leads to chronic AF [78]. The role of Ca2+-permeable channels in maintaining Ca2+ homeostasis in cardiomyocytes is obviously important for prevention of arrhythmogenesis. However, another important pathological process underlying the development of AF is cardiac fibrosis. The degree of fibrotic changes in the atrium of the heart correlates directly with the persistence of AF [79]. Atrial fibrosis is one of the key structural arrhythmogenic changes that underlie the development of AF [77]. Fibrosis in the myocardium is usually triggered by insults like ischemia, oxidative stress, hypertension, and so on. In response to these stimuli, fibroblasts undergo proliferation and differentiation and adversely remodel the extracellular matrix incident to fibrosis. Evidence from multiple sources suggests that Ca2+ has an important role to play in fibroblast growth and proliferation [80]. Because fibroblasts are nonexcitable and lack voltage-gated Ca2+ channels, TRP channels have been suggested as one of the candidates regulating Ca2+ entry into fibroblasts [81]. Given that TRP channels might play a part in mediating Ca2+ homeostasis in conduction tissue and in the pathophysiology of cardiac fibrosis, their role in AF is being explored. Both TRPM4 and TRPM7 are expressed in human atrial myocytes. However, TRPM4 is not Ca2+ permeable, and there is currently no evidence linking its function to the development of AF. TRPM7 belongs to the same TRP melastatin subfamily as TRPM4, but it is permeable to TRPC Channels in Pulmonary Hypertension 375 divalent cations like Ca2+ and is constitutively active [82]. In fact, immunoblotting has shown that not only is TRPM7 present in atrial myocytes along with TRPM4, but also its protein levels are highly up-regulated in patients with AF, whereas the TRPM4 levels remain unchanged [83]. TRPC1 is also expressed in both atrial myocytes and atrial fibroblasts, but although a functional TRPC1 current was discovered in atrial myocytes, the same could not be found in atrial fibroblasts, suggesting that the TRPC1 in fibroblasts does not contribute to the Ca2+ entry and subsequent signaling mechanisms controlling the proliferation of fibroblasts [16,35]. Fibroblasts from AF patients tended to undergo increased differentiation into myofibroblasts compared to patients with normal sinus rhythm. Knocking down TRPM7 decreased this differentiation process [83]. TRPM7 expression was also found to be responsive to TGF-β1 treatment suggesting that TRPM7 and TGF-β1 work in concert to induce the differentiation process. There is also supportive evidence that TRPM7 is important in the early stages of cardiogenesis, and deletions of TRPM7 can result in abnormal ventricular structure and conduction properties [84]. TRPM7 is inhibited by Mg2+, both intracellularly and extracellularly, but there are currently not many pharmacological strategies targeting TRPM7 to prevent the arrhythmias associated with this channel. La3+ and 2-aminoethoxydiphenylborate (2-APB) have been used as inhibitors of the TRPM7 channel in biological systems such as hepatic stellate cells and gastric adenocarcinoma cell lines, but none have been used to evaluate their effect on prevention of AF [85–87]. Because atrial fibrosis is also a process mediated by the renin-angiotensin system and the oxidative stress pathway apart from the TGF-β1 signaling pathway, it is not certain whether therapeutic approaches targeting TRPM7 will be sufficient for the abolishment of arrhythmias and the prevention of pathological atrial fibrosis. Research efforts are being directed toward identifying molecular players through high-throughput assays to search for effective TRPM7 inhibitors [88]. However, like other channels in the TRP family, TRPM7 is expressed in a variety of tissue types, and there is much more work to be done before targeting TRPM7 specifically for the purpose of AF therapy.

TRPC CHANNELS IN PULMONARY HYPERTENSION

PAH is a panvasculopathy, with pathology found in every layer of the blood vessel. Histological findings include intimal hyperplasia, medial hypertrophy, distal muscularization of peripheral pulmonary arteries, adventitial thickening, and thrombosis in situ. Increased proliferation, decreased apoptosis, and hypertrophy of pulmonary artery smooth muscle cells (PASMC), sustained pulmonary vasoconstriction, and increased apoptosis of endothelial cells converge to create the obstruction and obliteration of the vascular lumen (reviewed in Ref. [89]). A major trigger for pulmonary vasoconstriction is an elevation in cytoplasmic free calcium concentration [Ca2+]i. Intracellular calcium is required for phosphorylation of myosin, a necessary step in the actin-myosin binding that results in cell contraction and ultimately pulmonary vasoconstriction. In addition, calcium serves as a second messenger in cell signaling, gene expression, and cell proliferation through nuclear transcription factors such as NFAT [90]. NFAT is a Ca2+-calcineurin-dependent transcription factor that promotes PASMC proliferation [91]. As such, alterations in intracellular calcium homeostasis regulate pulmonary vascular tone and PASMC growth [92–96]. Through their 376 20. TRP CHANNELS IN CARDIAC PHYSIOLOGY role in non-voltage-dependent calcium entry channels, TRPC channels have recently been implicated to support PASMC function and the development of PAH [97]. TRPC1 and TRPC6 are the two major TRPC channels expressed in human pulmonary arteries and PASMCs [94,96,98,99], with major contributing roles from TRPC3 and TRPC4 as well. In addition, mRNA and protein levels of TRPC1 and TRPC6 were found to be higher in distal than proximal pulmonary arteries (PAs) of rats, suggesting some heterogeneity of distribution (and its role in hypoxic vasoconstriction; Ref. [100]). In PASMCs derived from idiopathic PAH patients, TRPC6 and TRPC3 show excessive levels of expression compared to normal controls as well as patients with secondary (World Health Organization (WHO) group 2–5) pulmonary hypertension (PH) [98]. This finding suggests a unique role for the TRPC family in the pathogenesis of primary PAH as differentiated from the greater PH group. TRPC1 also plays a critical role in proliferation of human PASMCs [92,94,101]. Down-regulation of TRPC1 using antisense oligo- nucleotide inhibited the enhancement in cell proliferation by 50% and lowered the amplitude of capacitative calcium entry induced by serum and growth factors [94]. Experimental animal models of PAH show increased expression of TRPC1 and TRPC4 [102]. Increased expression/ activity of platelet-derived growth factor receptor, an important mitogen involved in the vascular pathology of PAH [103], mediates cell proliferation at least partially by up-regulating TRPC6 expression [95]. Bosentan and sildenafil, oral antagonist of endothelin receptors and PDE5 inhibitor, respectively, both suppress PASMC proliferation as well as TRPC expression [101,104]. Capacitative calcium entry through TRPC family encoded receptors appears to represent a common downstream signaling pathway for many molecular mechanisms implicated in PAH. At least 6% of PAH cases occur within a familial context [105], and greater than 70% of them display loss-of-function mutations in bone morphogenetic protein receptor type 2 that promote cell proliferation [106]. These mutations have also been detected in idiopathic cases without an obvious family history. Epigenetic mechanisms of inheriting PAH or influencing disease susceptibility also exist. Single nucleotide polymorphism (SNP) variants in genes invoked in PAH have been found in the serotonin transporter, Kv1.5, as well as TRPC6 [99,107,108]. A gain-of-function −254(C → G) SNP within the regulatory regions of the TRPC6 gene is statistically associated with idiopathic PAH. Specifically, this SNP creates a binding sequence in TRPC6 that activates the ubiquitously expressed, inflammatory transcription factor, nuclear factor-kB (NF-kB). NF-kB then translocates into the nucleus, where it up-regulates TRPC6 expression and enhances calcium entry in idiopathic PAH PASMCs with the −254G allele. Inhibition of nuclear translocation of NF-kB attenuates TRPC6 expression [99]. NF-kB regulates cellular responses activated by inflammation, oxidative stress, and response to pathogens by controlling other transcription factors [109]. In this manner, the −254G allele primes the PASMCs in idiopathic PAH to an exaggerated response to NF-kB, creating a mechanistic link between PAH and the heightened inflammation described in its pathophysiology [89]. Allele carriers who are exposed to inflammatory triggers in the lung (e.g., collagen vascular autoimmune conditions, schistosomiasis, HIV) may have increased risk in developing vascular remodeling and PAH. Chronic hypoxia is a common cause of secondary pulmonary hypertension. Physiologically, pulmonary arteries constrict under conditions of alveolar hypoxia to divert blood flow away from poor- to well-ventilated areas of the lung. This phenomenon of hypoxic pulmonary vasoconstriction (HPV) occurs in an acute phase within seconds and a sustained phase over hours. If hypoxia becomes chronic, it induces pulmonary vascular remodeling that will elevate TRPC Channels in Pulmonary Hypertension 377 pulmonary vascular resistance; it is a common experimental model of WHO group 3 PH. Animal pulmonary arteries develop medial hypertrophy, but there is no intimal fibrosis or plexiform lesions, unlike in PAH. PASMCs show proliferation and extend to distal small pulmonary arteries [110]. Hypoxia inhibits potassium current through the voltage-dependent Kv1.5 channel, which depolarizes the cell and increases intracellular calcium through L-type voltage-operated calcium channels as well as TRPC channels [111–113]. Indeed, following exposure to chronic hypoxia, PASMCs showed twofold expression of TRPC1 and TRPC6 [114–116]. In TRPC6−/− mice, the acute phase of HPV was completely absent. However, the second sustained phase of vasoconstriction was not affected at all. In addition, when exposed to chronic hypoxia for 3 weeks, TRPC6 deficient mice developed the same vascular remodeling and PH as wild-type mice [115,117]. TRPC6 appears only indispensible for acute HPV and not essential for non-hypoxia- induced vasoconstrictor response and remodeling. In contrast, TRPC1−/− mice had suppression of PH after chronic hypoxia [115]. There was less PASMC migration and muscularization of distal vessels, but a similar degree of right ventricular hypertrophy (RVH) compared to wild- type mice [115,116]. In combined TRPC1 and TRPC6 knockout mice, however, markers such as PA pressure, RVH, and muscularization of distal vessels were all further suppressed compared to single TRPC1 knockout mice [115]. These studies show how specific TRPC isoforms can act in a temporal manner to influence vascular remodeling. Future development of therapeutic strategies will need to take into account how these channels intersect (Table 20.1).

TABLE 20.1 Selectivity of the inhibitors of various TRP channels

TRP channel Inhibiting compound Selectivity References TRPC1 PDE5 inhibitors Nonselective [59,60] TRPC3 Pyr3 Selective [53] BTP2 Nonselective [50,52,118] PDE5 inhibitors Nonselective [59,60] GSK503A Selective [30,55] TRPC5 BTP2 Nonselective [50–52] TRPC6 BTP2 Nonselective [50–52] GSK503A Selective [30,55] PDE5 inhibitors Nonselective [59,60] Klotho Selective [56] TRPM4 9-Phenanthrol Selective [73,74] MPB-104 Nonselective [75] Flufenamic acid Non selective [76] BTP2 Nonselective [50–52] TRPM7 La3+ Nonselective [86,87] 2-APB Nonselective [87,119–121] 378 20. TRP CHANNELS IN CARDIAC PHYSIOLOGY Acknowledgment We thank the members of the Rosenberg Lab and the Ion Channel Research Group (ICRU) at Duke University School of Medicine for helpful discussions of this topic. This work was supported in part by the NHLBI, NIH: R01HL093470.

References [1] Hill JA, Olson EN. Cardiac plasticity. N Engl J Med 2008;358(13):1370–80. [2] Williams RS, Rosenberg P. Calcium-dependent gene regulation in myocyte hypertrophy and remodeling. Cold Spring Harb Symp Quant Biol 2002;67:339–44. [3] Berridge MJ. Microdomains and elemental events in calcium signalling. Cell Calcium 1996;20(2):95–6. [4] Dibb KM, Graham HK, Venetucci LA, Eisner DA, Trafford AW. Analysis of cellular calcium fluxes in cardiac muscle to understand calcium homeostasis in the heart. Cell Calcium 2007;42(4-5):503–12. [5] O’Rourke B. The ins and outs of calcium in heart failure. Circ Res 2008;102(11):1301–3. [6] McKinsey TA, Kass DA. Small-molecule therapies for cardiac hypertrophy: moving beneath the cell surface. Nat Rev Drug Discov 2007;6(8):617–35. [7] Bers DM. Calcium cycling and signaling in cardiac myocytes. Annu Rev Physiol 2008;70:23–49. [8] Molkentin JD, Lu JR, Antos CL, Markham B, Richardson J, Robbins J, et al. A calcineurin-dependent transcriptional pathway for cardiac hypertrophy. Cell 1998;93(2):215–28. [9] Sussman MA, Lim HW, Gude N, Taigen T, Olson EN, Robbins J, et al. Prevention of cardiac hypertrophy in mice by calcineurin inhibition. Science 1998;281(5383):1690–3. [10] Wilkins BJ, Dai YS, Bueno OF, Parsons SA, Xu J, Plank DM, et al. Calcineurin/NFAT coupling participates in pathological, but not physiological, cardiac hypertrophy. Circ Res 2004;94(1):110–18. [11] Montell C. The latest waves in calcium signaling. Cell 2005;122(2):157–63. [12] Zheng J. Molecular mechanism of TRP channels. Compr Physiol 2013;3(1):221–42. [13] Huang F, Rock JR, Harfe BD, Cheng T, Huang X, Jan YN, et al. Studies on expression and function of the TMEM16A calcium-activated chloride channel. Proc Natl Acad Sci USA 2009;106(50):21413–18. [14] Rubinstein J, Lasko VM, Koch SE, Singh VP, Carreira V, Robbins N, et al. Novel role of transient receptor potential vanilloid 2 in the regulation of cardiac performance. Am J Physiol Heart Circ Physiol 2014;306(4):H574–84. [15] Zhang Z, Wang M, Fan XH, Chen JH, Guan YY, Tang YB. Upregulation of TRPM7 channels by angiotensin II triggers phenotypic switching of vascular smooth muscle cells of ascending aorta. Circ Res 2012;111(9):1137–46. [16] Zhang SS, Shaw RM. Multilayered regulation of cardiac ion channels. Biochim Biophys Acta 2013;1833(4):876–85. [17] Rowell J, Koitabashi N, Kass DA. TRP-ing up heart and vessels: canonical transient receptor potential channels and cardiovascular disease. J Cardiovasc Transl Res 2010;3(5):516–24. [18] Putney Jr JW. Recent breakthroughs in the molecular mechanism of capacitative calcium entry (with thoughts on how we got here). Cell Calcium 2007;42:103–10. [19] Uehara A, Yasukochi M, Imanaga I, Nishi M, Takeshima H. Store-operated Ca2+ entry uncoupled with ryanodine receptor and junctional membrane complex in heart muscle cells. Cell Calcium 2002;31(2):89–96. [20] Pani B, Bollimuntha S, Singh BB. The TR (i)P to Ca(2)(+) signaling just got STIMy: an update on STIM1 activated TRPC channels. Front Biosci 2012;17:805–23. [21] Hofmann T, Schaefer M, Schultz G, Gudermann T. Transient receptor potential channels as molecular substrates of receptor-mediated cation entry. J Mol Med (Berl) 2000;78(1):14–25. [22] Schaefer M, Plant TD, Obukhov AG, Hofmann T, Gudermann T, Schultz G. Receptor-mediated regulation of the nonselective cation channels TRPC4 and TRPC5. J Biol Chem 2000;275(23):17517–26. [23] Berenji K, Drazner MH, Rothermel BA, Hill JA. Does load-induced ventricular hypertrophy progress to systolic heart failure? Am J Physiol Heart Circ Physiol 2005;289(1):H8–16. [24] Heineke J, Molkentin JD. Regulation of cardiac hypertrophy by intracellular signalling pathways. Nat Rev Mol Cell Biol 2006;7(8):589–600. [25] Kurdi M, Booz GW. New take on the role of angiotensin II in cardiac hypertrophy and fibrosis. Hypertension 2011;57(6):1034–8. [26] Vennekens R. Emerging concepts for the role of TRP channels in the cardiovascular system. J Physiol 2011;589 (Pt 7):1527–34. REFERENCES 379

[27] Brenner JS, Dolmetsch RE. TrpC3 regulates hypertrophy-associated gene expression without affecting myocyte beating or cell size. PLoS One 2007;2(8):e802. [28] Nakayama H, Wilkin BJ, Bodi I, Molkentin JD. Calcineurin-dependent cardiomyopathy is activated by TRPC in the adult mouse heart. FASEB J 2006;20(10):1660–70. [29] Onohara N, Nishida M, Inoue R, Kobayashi H, Sumimoto H, Sato Y, et al. TRPC3 and TRPC6 are essential for angiotensin II-induced cardiac hypertrophy. EMBO J 2006;25(22):5305–16. [30] Seo K, Rainer PP, Shalkey Hahn V, Lee DI, Jo SH, Andersen A, et al. Combined TRPC3 and TRPC6 blockade by selective small-molecule or genetic deletion inhibits pathological cardiac hypertrophy. Proc Natl Acad Sci USA 2014;111(4):1551–6. [31] Kuwahara K, Wang Y, McAnally J, Richardson JA, Bassel-Duby R, Hill JA, et al. TRPC6 fulfills a calcineurin signaling circuit during pathologic cardiac remodeling. J Clin Invest 2006;116(12):3114–26. [32] Seth M, Zhang ZS, Mao L, Graham V, Burch J, Stiber J, et al. TRPC1 channels are critical for hypertrophic signaling in the heart. Circ Res 2009;105(10):1023–30. [33] Vindis C, D’Angelo R, Mucher E, Negre-Salvayre A, Parini A, Mialet-Perez J. Essential role of TRPC1 channels in cardiomyoblasts hypertrophy mediated by 5-HT2A serotonin receptors. Biochem Biophys Res Commun 2010;391(1):979–83. [34] Hunton DL, Lucchesi PA, Pang Y, Cheng X, Dell’Italia LJ, Marchase RB. Capacitative calcium entry contributes to nuclear factor of activated T-cells nuclear translocation and hypertrophy in cardiomyocytes. J Biol Chem 2002;277(16):14266–73. [35] Watanabe H, Iino K, Ohba T, Ito H. Possible involvement of TRP channels in cardiac hypertrophy and arrhythmia. Curr Top Med Chem 2013;13(3):283–94. [36] Bush EW, Hood DB, Papst PJ, Chapo JA, Minobe W, Bristow MR, et al. Canonical transient receptor potential channels promote cardiomyocyte hypertrophy through activation of calcineurin signaling. J Biol Chem 2006;281(44):33487–96. [37] Nishida M. Roles of heterotrimeric GTP-binding proteins in the progression of heart failure. J Pharmacol Sci 2011;117(1):1–5. [38] Calderone A, Thaik CM, Takahashi N, Chang DL, Colucci WS. Nitric oxide, atrial natriuretic peptide, and cyclic GMP inhibit the growth-promoting effects of norepinephrine in cardiac myocytes and fibroblasts. J Clin Invest 1998;101(4):812–18. [39] Blanton RM, Takimoto E, Lane AM, Aronovitz M, Piotrowski R, Karas RH, et al. Protein kinase g ialpha inhibits pressure overload-induced cardiac remodeling and is required for the cardioprotective effect of sildenafil in vivo. J Am Heart Assoc 2012;1(5):e003731. [40] Holtwick R, van Eickels M, Skryabin BV, Baba HA, Bubikat A, Begrow F, et al. Pressure-independent cardiac hypertrophy in mice with cardiomyocyte-restricted inactivation of the atrial natriuretic peptide receptor guanylyl cyclase-A. J Clin Invest 2003;111(9):1399–407. [41] Kwan HY, Huang Y, Yao X. Regulation of canonical transient receptor potential isoform 3 (TRPC3) channel by protein kinase G. Proc Natl Acad Sci USA 2004;101(8):2625–30. [42] Klaiber M, Dankworth B, Kruse M, Hartmann M, Nikolaev VO, Yang RB, et al. A cardiac pathway of cyclic GMP-independent signaling of guanylyl cyclase A, the receptor for atrial natriuretic peptide. Proc Natl Acad Sci USA 2011;108(45):18500–5. [43] Bubikat A, De Windt LJ, Zetsche B, Fabritz L, Sickler H, Eckardt D, et al. Local atrial natriuretic peptide signaling prevents hypertensive cardiac hypertrophy in endothelial nitric-oxide synthase-deficient mice. J Biol Chem 2005;280(22):21594–9. [44] Kilic A, Bubikat A, Gassner B, Baba HA, Kuhn M. Local actions of atrial natriuretic peptide counteract angiotensin II stimulated cardiac remodeling. Endocrinology 2007;148(9):4162–9. [45] Kinoshita H, Kuwahara K, Nishida M, Jian Z, Rong X, Kiyonaka S, et al. Inhibition of TRPC6 channel activity contributes to the antihypertrophic effects of natriuretic peptides-guanylyl cyclase-A signaling in the heart. Circ Res 2010;106(12):1849–60. [46] Yuan JP, Lee KP, Hong JH, Muallem S. The closing and opening of TRPC channels by Homer1 and STIM1. Acta Physiol (Oxf) 2012;204(2):238–47. [47] Kaneko Y, Szallasi A. TRP channels as therapeutic targets. Curr Top Med Chem 2013;13(3):241–3. [48] Chung SC, McDonald TV, Gardner P. Inhibition by SK&F 96365 of Ca2+ current, IL-2 production and activation in T lymphocytes. Br J Pharmacol 1994;113(3):861–8. 380 20. TRP CHANNELS IN CARDIAC PHYSIOLOGY

[49] Franzius D, Hoth M, Penner R. Non-specific effects of calcium entry antagonists in mast cells. Pflugers Arch 1994;428(5-6):433–8. [50] Zitt C, Strauss B, Schwarz EC, Spaeth N, Rast G, Hatzelmann A, et al. Potent inhibition of Ca2+ release-activated Ca2+ channels and T-lymphocyte activation by the pyrazole derivative BTP2. J Biol Chem 2004;279(13):12427–37. [51] He Y, Yao G, Savoia C, Touyz RM. Transient receptor potential melastatin 7 ion channels regulate magnesium homeostasis in vascular smooth muscle cells: role of angiotensin II. Circ Res 2005;96(2):207–15. [52] Kuwahara K, Nakao K. New molecular mechanisms for cardiovascular disease: transcriptional pathways and novel therapeutic targets in heart failure. J Pharmacol Sci 2011;116(4):337–42. [53] Kiyonaka S, Kato K, Nishida M, Mio K, Numaga T, Sawaguchi Y, et al. Selective and direct inhibition of TRPC3 channels underlies biological activities of a pyrazole compound. Proc Natl Acad Sci USA 2009;106(13):5400–5. [54] Schleifer H, Doleschal B, Lichtenegger M, Oppenrieder R, Derler I, Frischauf I, et al. Novel pyrazole compounds for pharmacological discrimination between receptor-operated and store-operated Ca(2+) entry pathways. Br J Pharmacol 2012;167(8):1712–22. [55] Washburn DG, Holt DA, Dodson J, McAtee JJ, Terrell LR, Barton L, et al. The discovery of potent blockers of the canonical transient receptor channels, TRPC3 and TRPC6, based on an anilino-thiazole pharmacophore. Bioorg Med Chem Lett 2013;23(17):4979–84. [56] Xie J, Cha SK, An SW, Kuro OM, Birnbaumer L, Huang CL. Cardioprotection by Klotho through downregulation of TRPC6 channels in the mouse heart. Nat Commun 2012;3:1238. [57] Nishioka K, Nishida M, Ariyoshi M, Jian Z, Saiki S, Hirano M, et al. Cilostazol suppresses angiotensin II- induced vasoconstriction via protein kinase A-mediated phosphorylation of the transient receptor potential canonical 6 channel. Arterioscler Thromb Vasc Biol 2011;31(10):2278–86. [58] Takahashi S, Lin H, Geshi N, Mori Y, Kawarabayashi Y, Takami N, et al. Nitric oxide-cGMP-protein kinase G pathway negatively regulates vascular transient receptor potential channel TRPC6. J Physiol 2008;586(Pt 17):4209–23. [59] Koitabashi N, Aiba T, Hesketh GG, Rowell J, Zhang M, Takimoto E, et al. Cyclic GMP/PKG-dependent inhibition of TRPC6 channel activity and expression negatively regulates cardiomyocyte NFAT activation Novel mechanism of cardiac stress modulation by PDE5 inhibition. J Mol Cell Cardiol 2010;48(4):713–24. [60] Kiso H, Ohba T, Iino K, Sato K, Terata Y, Murakami M, et al. Sildenafil prevents the up-regulation of transient receptor potential canonical channels in the development of cardiomyocyte hypertrophy. Biochem Biophys Res Commun 2013;436(3):514–18. [61] Zhang BT, Whitehead NP, Gervasio OL, Reardon TF, Vale M, Fatkin D, et al. Pathways of Ca(2)(+) entry and cytoskeletal damage following eccentric contractions in mouse skeletal muscle. J Appl Physiol (1985) 2012;112(12):2077–86. [62] Zhang BT, Yeung SS, Cheung KK, Chai ZY, Yeung EW. Adaptive responses of TRPC1 and TRPC3 during skeletal muscle atrophy and regrowth. Muscle Nerve 2014;49(5):691–9. [63] Guinamard R, Chatelier A, Demion M, Potreau D, Patri S, Rahmati M, et al. Functional characterization of a Ca(2+)-activated non-selective cation channel in human atrial cardiomyocytes. J Physiol 2004;558(Pt 1):75–83. [64] Guinamard R, Demion M, Magaud C, Potreau D, Bois P. Functional expression of the TRPM4 cationic current in ventricular cardiomyocytes from spontaneously hypertensive rats. Hypertension 2006;48(4):587–94. [65] Launay P, Fleig A, Perraud AL, Scharenberg AM, Penner R, Kinet JP. TRPM4 is a Ca2+-activated nonselective cation channel mediating cell membrane depolarization. Cell 2002;109(3):397–407. [66] Vassort G, Alvarez J. Transient receptor potential: a large family of new channels of which several are involved in cardiac arrhythmia. Can J Physiol Pharmacol 2009;87(2):100–7. [67] Guinamard R, Demion M, Launay P. Physiological roles of the TRPM4 channel extracted from background currents. Physiology (Bethesda) 2010;25(3):155–64. [68] Simard C, Hof T, Keddache Z, Launay P, Guinamard R. The TRPM4 non-selective cation channel contributes to the mammalian atrial action potential. J Mol Cell Cardiol 2013;59:11–19. [69] Liu H, El Zein L, Kruse M, Guinamard R, Beckmann A, Bozio A, et al. Gain-of-function mutations in TRPM4 cause autosomal dominant isolated cardiac conduction disease. Circ Cardiovasc Genet 2010;3(4):374–85. REFERENCES 381

[70] Nilius B, Mahieu F, Prenen J, Janssens A, Owsianik G, Vennekens R, et al. The Ca2+-activated cation channel TRPM4 is regulated by phosphatidylinositol 4,5-biphosphate. EMBO J 2006;25(3):467–78. [71] Kruse M, Schulze-Bahr E, Corfield V, Beckmann A, Stallmeyer B, Kurtbay G, et al. Impaired endocytosis of the ion channel TRPM4 is associated with human progressive familial heart block type I. J Clin Invest 2009;119(9):2737–44. [72] Stallmeyer B, Koopmann M, Schulze-Bahr E. Identification of novel mutations in LMNA associated with familial forms of dilated cardiomyopathy. Genet Test Mol Biomarkers 2012;16(6):543–9. [73] Simard C, Salle L, Rouet R, Guinamard R. Transient receptor potential melastatin 4 inhibitor 9-phenanthr ol abolishes arrhythmias induced by hypoxia and re-oxygenation in mouse ventricle. Br J Pharmacol 2012;165(7):2354–64. [74] Voigt N, Heijman J, Wang Q, Chiang DY, Li N, Karck M, et al. Cellular and molecular mechanisms of atrial arrhythmogenesis in patients with paroxysmal atrial fibrillation. Circulation 2014;129(2):145–56. [75] Macianskiene R, Gwanyanya A, Sipido KR, Vereecke J, Mubagwa K. Induction of a novel cation current in cardiac ventricular myocytes by flufenamic acid and related drugs. Br J Pharmacol 2010;161(2):416–29. [76] Abriel H, Syam N, Sottas V, Amarouch MY, Rougier JS. TRPM4 channels in the cardiovascular system: physiology, pathophysiology, and pharmacology. Biochem Pharmacol 2012;84(7):873–81. [77] Yue L, Xie J, Nattel S. Molecular determinants of cardiac fibroblast electrical function and therapeutic implications for atrial fibrillation. Cardiovasc Res 2011;89(4):744–53. [78] Harada M, Luo X, Qi XY, Tadevosyan A, Maguy A, Ordog B, et al. Transient receptor potential canonical-3 channel-dependent fibroblast regulation in atrial fibrillation. Circulation 2012;126(17):2051–64. [79] Xu J, Cui G, Esmailian F, Plunkett M, Marelli D, Ardehali A, et al. Atrial extracellular matrix remodeling and the maintenance of atrial fibrillation. Circulation 2004;109(3):363–8. [80] Rose RA, Belke DD, Maleckar MM, Giles WR. Ca(2+) entry through TRP-C channels regulates fibroblast biology in chronic atrial fibrillation. Circulation 2012;126(17):2039–41. [81] Yue Z, Zhang Y, Xie J, Jiang J, Yue L. Transient receptor potential (TRP) channels and cardiac fibrosis. Curr Top Med Chem 2013;13(3):270–82. [82] Zhang YH, Sun HY, Chen KH, Du XL, Liu B, Cheng LC, et al. Evidence for functional expression of TRPM7 channels in human atrial myocytes. Basic Res Cardiol 2012;107(5):282. [83] Du J, Xie J, Zhang Z, Tsujikawa H, Fusco D, Silverman D, et al. TRPM7-mediated Ca2+ signals confer fibrogen- esis in human atrial fibrillation. Circ Res 2010;106(5):992–1003. [84] Sah R, Mesirca P, Van den Boogert M, Rosen J, Mably J, Mangoni ME, et al. Ion channel-kinase TRPM7 is required for maintaining cardiac automaticity. Proc Natl Acad Sci USA 2013;110(32):E3037–46. [85] Chokshi R, Matsushita M, Kozak JA. Detailed examination of Mg2+ and pH sensitivity of human TRPM7 channels. Am J Physiol Cell Physiol 2012;302(7):C1004–11. [86] Kim BJ, Kim SY, Lee S, Jeon JH, Matsui H, Kwon YK, et al. The role of transient receptor potential channel blockers in human gastric cancer cell viability. Can J Physiol Pharmacol 2012;90(2):175–86. [87] Kim BJ, Park EJ, Lee JH, Jeon J-H, Kim SJ, So I. Suppression of transient receptor potential melastatin 7 channel induces cell death in gastric cancer. Cancer Sci 2008;99(12):2502–9. [88] Castillo B, Porzgen P, Penner R, Horgen FD, Fleig A. Development and optimization of a high-throughput bioassay for TRPM7 ion channel inhibitors. J Biomol Screen 2010;15(5):498–507. [89] Archer SL, Weir EK, Wilkins MR. Basic science of pulmonary arterial hypertension for clinicians: new concepts and experimental therapies. Circulation 2010;121(18):2045–66. [90] Remillard CV, Yuan JX. TRP channels, CCE, and the pulmonary vascular smooth muscle. Microcirculation 2006;13(8):671–92. [91] Bonnet S, Rochefort G, Sutendra G, Archer SL, Haromy A, Webster L, et al. The nuclear factor of activated T cells in pulmonary arterial hypertension can be therapeutically targeted. Proc Natl Acad Sci USA 2007;104(27):11418–23. [92] Golovina VA, Platoshyn O, Bailey CL, Wang J, Limsuwan A, Sweeney M, et al. Upregulated TRP and enhanced capacitative Ca(2+) entry in human pulmonary artery myocytes during proliferation. Am J Physiol Heart Circ Physiol 2001;280(2):H746–55. [93] McDaniel SS, Platoshyn O, Wang J, Yu Y, Sweeney M, Krick S, et al. Capacitative Ca(2+) entry in agonist- induced pulmonary vasoconstriction. Am J Physiol Lung Cell Mol Physiol 2001;280(5):L870–80. [94] Sweeney M, Yu Y, Platoshyn O, Zhang S, McDaniel SS, Yuan JX. Inhibition of endogenous TRP1 decreases capacitative Ca2+ entry and attenuates pulmonary artery smooth muscle cell proliferation. Am J Physiol Lung Cell Mol Physiol 2002;283(1):L144–55. 382 20. TRP CHANNELS IN CARDIAC PHYSIOLOGY

[95] Yu Y, Sweeney M, Zhang S, Platoshyn O, Landsberg J, Rothman A, et al. PDGF stimulates pulmonary vascular smooth muscle cell proliferation by upregulating TRPC6 expression. Am J Physiol Cell Physiol 2003;284(2):C316–30. [96] Kunichika N, Yu Y, Remillard CV, Platoshyn O, Zhang S, Yuan JX. Overexpression of TRPC1 enhances pulmonary vasoconstriction induced by capacitative Ca2+ entry. Am J Physiol Lung Cell Mol Physiol 2004;287(5):L962–9. [97] Landsberg JW, Yuan JX. Calcium and TRP channels in pulmonary vascular smooth muscle cell proliferation. News Physiol Sci 2004;19:44–50. [98] Yu Y, Fantozzi I, Remillard CV, Landsberg JW, Kunichika N, Platoshyn O, et al. Enhanced expression of transient receptor potential channels in idiopathic pulmonary arterial hypertension. Proc Natl Acad Sci USA 2004;101(38):13861–6. [99] Yu Y, Keller SH, Remillard CV, Safrina O, Nicholson A, Zhang SL, et al. A functional single-nucleotide polymorphism in the TRPC6 gene promoter associated with idiopathic pulmonary arterial hypertension. Circulation 2009;119(17):2313–22. [100] Lu W, Wang J, Shimoda LA, Sylvester JT. Differences in STIM1 and TRPC expression in proximal and distal pulmonary arterial smooth muscle are associated with differences in Ca2+ responses to hypoxia. Am J Physiol Lung Cell Mol Physiol 2008;295(1):L104–13. [101] Wang C, Li JF, Zhao L, Liu J, Wan J, Wang YX, et al. Inhibition of SOC/Ca2+/NFAT pathway is involved in the anti-proliferative effect of sildenafil on pulmonary artery smooth muscle cells. Respir Res 2009;10:123. [102] Liu XR, Zhang MF, Yang N, Liu Q, Wang RX, Cao YN, et al. Enhanced store-operated Ca(2)+ entry and TRPC channel expression in pulmonary arteries of monocrotaline-induced pulmonary hypertensive rats. Am J Physiol Cell Physiol 2012;302(1):C77–87. [103] Schermuly RT, Dony E, Ghofrani HA, Pullamsetti S, Savai R, Roth M, et al. Reversal of experimental pulmonary hypertension by PDGF inhibition. J Clin Invest 2005;115(10):2811–21. [104] Kunichika N, Landsberg JW, Yu Y, Kunichika H, Thistlethwaite PA, Rubin LJ, et al. Bosentan inhibits transient receptor potential channel expression in pulmonary vascular myocytes. Am J Respir Crit Care Med 2004;170(10):1101–7. [105] Rich S, Dantzker DR, Ayres SM, Bergofsky EH, Brundage BH, Detre KM, et al. Primary pulmonary hypertension. A national prospective study. Ann Intern Med 1987;107(2):216–23. [106] Simonneau G, Robbins IM, Beghetti M, Channick RN, Delcroix M, Denton CP, et al. Updated clinical classification of pulmonary hypertension. J Am Coll Cardiol 2009;54(1 Suppl.):S43–54. [107] Eddahibi S, Chaouat A, Morrell N, Fadel E, Fuhrman C, Bugnet AS, et al. Polymorphism of the serotonin transporter gene and pulmonary hypertension in chronic obstructive pulmonary disease. Circulation 2003;108(15):1839–44. [108] Remillard CV, Tigno DD, Platoshyn O, Burg ED, Brevnova EE, Conger D, et al. Function of Kv1.5 channels and genetic variations of KCNA5 in patients with idiopathic pulmonary arterial hypertension. Am J Physiol Cell Physiol 2007;292(5):C1837–53. [109] Ghosh S, Hayden MS. New regulators of NF-kappaB in inflammation. Nat Rev Immunol 2008;8(11):837–48. [110] Ryan J, Bloch K, Archer SL. Rodent models of pulmonary hypertension: harmonisation with the world health organisation’s categorisation of human PH. Int J Clin Pract Suppl 2011;172:15–34. [111] Weir EK, Lopez-Barneo J, Buckler KJ, Archer SL. Acute oxygen-sensing mechanisms. N Engl J Med 2005;353(19):2042–55. [112] Wang J, Shimoda LA, Weigand L, Wang W, Sun D, Sylvester JT. Acute hypoxia increases intracellular [Ca2+] in pulmonary arterial smooth muscle by enhancing capacitative Ca2+ entry. Am J Physiol Lung Cell Mol Physiol 2005;288(6):L1059–69. [113] Lin MJ, Leung GP, Zhang WM, Yang XR, Yip KP, Tse CM, et al. Chronic hypoxia-induced upregulation of store-operated and receptor-operated Ca2+ channels in pulmonary arterial smooth muscle cells: a novel mechanism of hypoxic pulmonary hypertension. Circ Res 2004;95(5):496–505. [114] Wang J, Weigand L, Lu W, Sylvester JT, Semenza GL, Shimoda LA. Hypoxia inducible factor 1 mediates hypoxia-induced TRPC expression and elevated intracellular Ca2+ in pulmonary arterial smooth muscle cells. Circ Res 2006;98(12):1528–37. [115] Xia Y, Yang XR, Fu Z, Paudel O, Abramowitz J, Birnbaumer L, et al. Classical transient receptor potential 1 and 6 contribute to hypoxic pulmonary hypertension through differential regulation of pulmonary vascular functions. Hypertension 2014;63(1):173–80. REFERENCES 383

[116] Malczyk M, Veith C, Fuchs B, Hofmann K, Storch U, Schermuly RT, et al. Classical transient receptor potential channel 1 in hypoxia-induced pulmonary hypertension. Am J Respir Crit Care Med 2013;188(12):1451–9. [117] Weissmann N, Dietrich A, Fuchs B, Kalwa H, Ay M, Dumitrascu R, et al. Classical transient receptor potential channel 6 (TRPC6) is essential for hypoxic pulmonary vasoconstriction and alveolar gas exchange. Proc Natl Acad Sci USA 2006;103(50):19093–8. [118] He F, Tan YH, Zhang G. [Isolation and identification of human dental pulp stem cells]. West China Journal of Stomatology 2005;23(1):75–8. [119] Chokshi R, Fruasaha P, Kozak JA. 2-Aminoethyl diphenyl borinate (2-APB) inhibits TRPM7 channels through an intracellular acidification mechanism. Channels (Austin) 2012;6(5):362–9. [120] Li M, Jiang J, Yue L. Functional characterization of homo- and heteromeric channel kinases TRPM6 and TRPM7. J Gen Physiol 2006;127(5):525–37. [121] Chokshi R, Matsushita M, Kozak JA. Sensitivity of TRPM7 channels to Mg2+ characterized in cell-free patches of Jurkat T lymphocytes. Am J Physiol Cell Physiol 2012;302(11):C1642–51.

CHAPTER 21 Targeting of Transient Receptor Potential Channels in Digestive Disease Daniel P. Poole,1,2,* TinaMarie Lieu,1 Nicholas A. Veldhuis,1,3 Pradeep Rajasekhar,1 Nigel W. Bunnett1,4 1Monash Institute of Pharmaceutical Sciences, Parkville, Victoria, Australia 2Department of Anatomy & Neuroscience, The University of Melbourne, Parkville, Victoria, Australia 3Department of Genetics, The University of Melbourne, Parkville, Victoria, Australia 4Department of Pharmacology, The University of Melbourne, Parkville, Victoria, Australia *Corresponding author: [email protected]

OUTLINE

Overview 386 TRP Channels as Targets for Prokinetic Drugs 391 Expression of TRP Channels in the TRPA1 Agonists 391 Gut and Their Role in GI Motility Dai Kenchu To 391 and Secretion 386 Peppermint Oil 392 TRPA1 389 TRPV1 389 Roles of TRP Channels in the TRPV2 390 Development of IBD 392 TRPV3 390 TRP Channels, Neurogenic TRPM8 390 Inflammation, and Visceral TRP Channel Expression in Smooth Hypersensitivity 392 Muscle and ICC 390 TRPV1 392 TRPV4 392

TRP Channels as Therapeutic Targets 385 © 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/B978-0-12-420024-1.00021-7 386 21. TARGETING OF TRANSIENT RECEPTOR POTENTIAL CHANNELS IN DIGESTIVE DISEASE

TRPA1 392 Therapeutic Targeting of TRP TRPM8 393 Channels for Sensory Disorders 394 Other TRP Channels 393 Summary 395 TRP Channels and Clinical IBD 393 Complementary Medicines and IBD: Acknowledgments 396 Role for TRP Channels 393 References 396 TRP Channel Sensitization in IBS- and IBD-Related Pain 394

OVERVIEW

Transient receptor potential (TRP) channels are integral components of a network of receptors and ion channels that detect chemical and mechanical stimuli. They are widely expressed throughout the digestive tract, with important roles in taste, visceral sensation, gastrointestinal (GI) motility, as well as absorptive and secretory functions [1]. Dysregulation of these functions through increased expression or aberrant sensitivity also plays a major role in the etiology of digestive diseases. These changes may underlie both the initiation and maintenance of visceral hyperalgesia and inflammation associated with inflammatory bowel disease (IBD), gastroesophageal reflux disease (GERD), pancreatitis, and functional disorders, including irritable bowel syndrome (IBS; see Figure 21.1). The GI tract is a major source and target of TRP channel activators, including protons, prostaglandin and arachidonic acid derivatives, G protein-coupled receptor (GPCR) agonists, and mechanical stimuli. A wide variety of pungent substances commonly associated with spices are potent TRP channel agonists and are often key active ingredients in traditional treatments for gut-related disorders [3]. This chapter provides an overview of the expression and function of TRP channels in the GI tract in health and disease and discusses the contributions of TRP channels to disease development and progression. Emphasis is placed on disorders related to GI motility, neurogenic inflammation, and sensation. The potential for TRP-targeted therapies in GI disease is discussed in detail where appropriate.

EXPRESSION OF TRP CHANNELS IN THE GUT AND THEIR ROLE IN GI MOTILITY AND SECRETION

The GI tract is intrinsically innervated by the enteric nervous system (ENS), comprising two major ganglionated plexuses: the myenteric plexus, which controls motility, and the submucosal plexus, which regulates fluid exchange and blood flow [2]. The gut also receives extrinsic innervation by vagal and spinal nerves that also regulate GI functions, notably sensory signaling [4]. Studies using plant extracts or selective pharmacological tools suggest that TRP channels influence GI function and provide a possible explanation for the anecdotal effects of commonly ingested spices and foods on the gut. The presence of established TRP channel activators in traditional herbal remedies establishes a mechanism through which these may exert their proposed therapeutic actions. Certain plant-derived TRP channel activators TRP CHANNELS AND GI MOTILITY AND SECRETION 387

FIGURE 21.1 Potential use of TRP channel targeted therapeutics for the treatment of GI diseases and disorders. TRP channels play integral roles in the development and maintenance of pain and neurogenic inflammation, conditions in which TRP channel inhibition is likely to be beneficial. TRP agonists have spasmolytic, prokinetic, and prosecretory effects that may alleviate constipation and symptoms of IBS. See text for details. alter GI motility and secretion in isolated preparations, including capsaicin [5], piperine [6], carvacrol/thymol [7], allyl isothiocyanate (AITC)/cinnamaldehyde [8], zingerone [9], and menthol [10]. However, the involvement of TRP channels and the molecular targets of many of these compounds in the gut are unresolved or under debate. To determine the cellular sites of TRP channel-mediated effects, a thorough understanding of the distribution of TRP channels within the gut is required. Key cell types that influence motility include myenteric neurons, extrinsic nerves, smooth muscle of the muscularis externa, 388 21. TARGETING OF TRANSIENT RECEPTOR POTENTIAL CHANNELS IN DIGESTIVE DISEASE interstitial cells of Cajal (ICC), and enteroendocrine cells (see Figure 21.2). The expression of TRP channels by enteric neurons is an area of much debate. At present, there is evidence that TRPV2, TRPA1, and TRPV1 are expressed in the ENS of a number of species, including humans. However, in the case of TRPV1 in particular, there is relatively limited direct functional data to support these findings (see the section “TRPV1”). Moreover, the presence of TRP channel immunoreactivity is not always supported by pharmacological studies using selective agonists and antagonists, and key controls to confirm antibody specificity are often omitted. There is also no definitive evidence for expression of mRNA for TRP channels by enteric neurons.

FIGURE 21.2 Potential therapeutic targeting of TRP channels for treatment of GI motility and secretion disorders. TRP channels are expressed by major cell types that regulate motility and secretion, as outlined. TRP agonists acting on these cells have spasmolytic, prokinetic, and prosecretory effects that may alleviate constipation and symptoms of IBS. (1) Enteric neurons: TRPA1, TRPV2, TRPC. (2) Enterochromaffin cells: TRPA1. (3) Smooth muscle: TRPC1, 3, 4, 6; TRPM4. (4) Interstitial cells of Cajal: TRPC1, TRPC2, TRPC4, TRPM7, TRPC4. (5) Epithelial cells: TRPC1; TRPV1, TRPV3, TRPV4, TRPV5, TRPV6; TRPM6, TRPM7. (6) Extrinsic primary afferent neurons: TRPV1, TRPV2, TRPV4; TRPA1, TRPM8; TRPC4. Details are provided in the text. Figure is modified from Ref. [11]. TRP CHANNELS AND GI MOTILITY AND SECRETION 389

Although the expression of TRP channels in the GI tract and the effects of TRP channel activators and inhibitors on gut function have been widely examined, there is a lack of con- sistency across these studies. Many of these differences may be attributable to species- or region-specific effects and to differences in methods and selectivity of reagents. The involvement of TRP channels is often implied, but not specifically examined. Moreover, there are a number of examples in which promising results obtained using laboratory species have not yielded equivalent results in clinical trials. Examples include the use of TRPV1 antagonists for alleviation of symptoms of esophageal reflux disease [12,13] and the use of traditional medicines in constipation [14]. An examination of the current literature also highlights the lack of a thorough understanding of the distribution of TRP channels in the human GI tract. Regulating GI function using TRP activators holds some therapeutic promise, given the fact that many of these compounds are regularly ingested as part of the diet. Most of the current preclinical and clinical studies have focused on the therapeutic application of TRP channel modulators for treatment of constipation-related disorders, functional dyspepsia, and GERD. Notably, much of the current work relates to the validation of traditional remedies and natural products, which may target TRPA1, TRPV1, and TRPM8. These compounds have functional effects in animal and human tissues, in addition to experimental models of digestive disease. However, these studies have not necessarily translated to effectiveness in clinical trials. One remedy that has shown promise is peppermint oil for the treatment of IBS, with a number of clinical studies suggesting efficacy [15]. It should be noted that there is no clear identification of molecular targets underlying these effects of menthol or peppermint oil, although it is likely to be through TRPM8 or TRPA1 and involve a neurogenic mechanism (see the section “TRPM8”). The use of peppermint oil in clinical trials without definitive knowledge of where it acts demonstrates a key difference between studies involving novel synthetic drugs and those of established, commonly used traditional remedies.

TRPA1 TRPA1 is expressed by cell types involved in controlling GI motility including enterochromaffin (EC) cells [8,16] and enteric neurons [17,18]. Importantly, these studies also identify equivalent expression across species, including humans. Studies in isolated intestine have identified two different mechanisms through which AITC may exert its effects, namely, release of 5-hydroxytryptamine (5-HT) from EC cells [8,19] and activation of enteric reflexes [18,20]. Non-TRPA1-dependent effects of AITC have been identified, although the precise molecular targets involved remain undefined [21,22]. The relative role of 5-HT release in stimulating propulsive motility is debatable given that AITC contracts the mucosa-free ileum [22] and recent reexamination of the role of mucosally derived 5-HT in propulsive motility [23].

TRPV1 Most studies conclude that functional TRPV1 expression in the gut is restricted to extrinsic primary afferent neurons of spinal or vagal origin, and that TRPV1 is not expressed by enteric neurons [24–26]. The pharmacological effects of capsaicin on intestinal contractility are generally tetrodotoxin resistant and are blocked by neurokinin receptor antagonists, suggesting that they are mediated through substance P (SP) release from extrinsic nerve terminals [5]. 390 21. TARGETING OF TRANSIENT RECEPTOR POTENTIAL CHANNELS IN DIGESTIVE DISEASE

Capsaicin simulates secretion in the human colon through the same mechanism [27]. Capsaicin can also inhibit GI contractions through release of nitric oxide (NO), 5-HT, opioids, or calcitonin gene-related peptide (CGRP) from these nerves [28]. Some effects of established TRPV1 activators on intestinal motility may be TRPV1-independent [29], highlighting potential issues with translation from basic science to the clinic, and the importance of studies using human tissues.

TRPV2 TRPV2 is expressed by gastric and intestinal enteric neurons [30–32]. TRPV2 activation relaxes the stomach and intestine and promotes GI transit [31]. These studies are limited by agonist specificity, although selective antagonists have been used to confirm TRPV2- dependence. The mechanosensitivity of TRPV2 is probably of most relevance to the GI tract, given that viscera are unlikely to be exposed to noxious heat. TRPV2 has been proposed as a therapeutic target for the treatment of functional dyspepsia associated with impaired gastric adaptive relaxation [32]. However, there are no equivalent preliminary studies to determine the expression and role of TRPV2 in human tissues.

TRPV3 TRPV3 is expressed by colonic epithelial cells, but expression by smooth muscle and the ENS has not been examined [33]. The TRPV3 activators thymol and eugenol stimulate 5-HT release from EC cells [34], with implications for colonic motility and secretion [8]. Active compounds from oregano, thyme, and cloves (carvacrol, thymol, and eugenol) have spasmolytic effects on the GI tract through both direct actions on smooth muscle and via the ENS [35]. Although activation of TRPA1 and TRPV3 by these compounds has since been determined [36], subsequent studies have not been performed to determine the respective role of these channels.

TRPM8 Both a TRPM8GFP reporter mouse and immunofluorescence have been used to define TRPM8 expression in the mouse colon, where it was localized to the epithelium and extrinsic nerve fibers, with little convincing evidence for expression by the ENS [37,38]. Although there is a consistent inhibitory effect of menthol on contractile activity, the molecular target of menthol in the GI tract remains undetermined [10].

TRP Channel Expression in Smooth Muscle and ICC ICCs, smooth muscle cells, and EC cells also regulate GI motility, and consideration of possible effects on these cells must be given with regard to therapeutic targeting of TRP channels. These cells do not express the major neuronal TRP channels outlined earlier. ICCs are the major interface between the ENS and smooth muscle contraction, and their activity is required for slow wave generation. TRPs act as nonselective cation channels in both ICCs and smooth muscle, where their activity is coupled to GPCRs. TRP expression by different subsets TRP Channels as Targets for Prokinetic Drugs 391 of ICC has been characterized, with TRPC1, 2, and 4, and TRPM4 and TRPM7 identified at the mRNA level [39,40]. Functional expression of TRPC4 [41] and TRPM7 [42] have been confirmed and may play a role in slow wave generation. Smooth muscle cells mainly express TRPC channels [43], which are involved in receptor- and store-operated Ca2+ entry and release. TRPC4 and TRPC6 couple to muscarinic receptor activation; their deletion attenuates muscarinic receptor signaling and delays GI transit [44].

TRP CHANNELS AS TARGETS FOR PROKINETIC DRUGS

Constipation may be due to a combination of two factors: reduced or nonpropulsive motility and increased relative absorptive activity. Delayed transit is correlated with an increased time for water absorption, and the two factors are therefore interrelated. Chronic constipation, including constipation-predominant IBS and chronic idiopathic constipation, is poorly managed by current bulk-forming, osmotic, and secretory laxa- tives [45]. Severe constipation is also a significant limiting side effect of opiate use and affects the majority of patients taking this class of drugs. Regulating GI function using TRP activators certainly holds some promise. However, a more rigorous experimental examination of the proposed actions of these agents and the specific involvement of TRP channels is required before the effectiveness of TRP channel activators in modifying GI motility can be proven.

TRPA1 Agonists Anecdotal and experimental evidence suggest that TRPA1 agonists are prokinetic and reverse constipation, although specific TRPA1 involvement is unproven [46,47]. Oral administration of AITC restores transit in mouse models of atonic and spastic constipation [47], and activation of TRPA1 on colonic epithelial cells stimulates anion secretion [7,48]. Thus, TRPA1 is an ideal therapeutic target for the treatment of nonobstructive constipation associated with delayed transit or dry stool. Whether TRPA1 activation in the colon is desirable given the proinflammatory and proalgesic effects of AITC remains to be determined.

Dai Kenchu To There has been renewed interest in the prokinetic actions of the traditional Japanese medicine Dai Kenchu To and its defined commercial formulation TU-100. TU-100 contains established TRPA1 and TRPV1 channel activators, including (6)-shogaol and hydroxyl-β-sanshool [49]. TU-100 has been proposed as a treatment for postoperative ileus, severe constipation, and symptoms of IBS and Crohn’s disease. TU-100 reverses opioid-induced constipation [50,51] and enhances colonic contractions [52]. However, there is no evidence for TRP channel involvement in these effects. Clinical trials have examined the therapeutic potential of TU- 100 with limited success, with no significant effect on gastric emptying or on colonic motility [14,53]. The apparent inability to translate data from rodents to humans highlights potential limitations to this experimental approach. 392 21. TARGETING OF TRANSIENT RECEPTOR POTENTIAL CHANNELS IN DIGESTIVE DISEASE Peppermint Oil Peppermint oil has been traditionally used to aid digestion, and one of its biologically active constituents is the TRPM8 activator menthol. Despite the prevalent use of peppermint oil to alleviate IBS symptoms and use as a spasmolytic, there have been very few studies of the effects of peppermint oil, menthol, or icillin on GI motility. Several clinical studies have reported effectiveness of peppermint oil as a spasmolytic [15]. Although menthol consistently inhibits GI contractions, the specific involvement of TRP channels has not been examined [10].

ROLES OF TRP CHANNELS IN THE DEVELOPMENT OF IBD

TRP Channels, Neurogenic Inflammation, and Visceral Hypersensitivity Activation of TRP channels expressed by primary spinal afferents promotes neurogenic inflammation and visceral pain through neuropeptide release. Both TRP function and expression are upregulated in IBD, leading to the enhancement and prolongation of tissue damage and pain. Moreover, there is an increase in the density of TRPV1-positive innervation of the colon in both IBD and IBS. Thus, pharmacological targeting of key TRP channels may attenuate disease-associated proinflammatory and nociceptive signaling. For the sake of brevity, this chapter will focus on TRP channel expression by colonic afferents and expression and functional changes associated with IBD and IBS.

TRPV1 TRPV1 plays a role in both the initiation and maintenance of colitis. Conversely, there is also evidence of a protective role for TRPV1 in experimental colitis. Genetic deletion of TRPV1, TRPV1 inhibition, and afferent desensitization by capsaicin all exacerbate the early, acute phases of colitis [54]. In particular, the TRP channel-dependent release of SP from these terminals is of primary significance [55]. Capsazepine is protective against dextran sulfate sodium (DSS) colitis [56], as is treatment with capsaicin, the latter presumably acting through desensitization of TRPV1 signaling [57]. Long-term inhibition of TRPV1 or TRPA1 attenuates development of chronic colitis [58,59]. Whether these observations translate to human disease remains to be determined.

TRPV4 TRPV4 is expressed by visceral afferents and the colonic epithelium [60–63]. TRPV4 is upregulated in colonic epithelial cells in experimental and clinical IBD [63–65]. Altered expression of TRPV4 in nerve fibers in disease and increased functional TRPV4 activity in IBD were not specifically examined.

TRPA1 TRPA1 is an attractive target for potential anti-inflammatory therapeutics. Intracolonic administration of mustard oil induces experimental colitis [66], TRPA1 mRNA is upregulated in Roles of TRP Channels in the Development of IBD 393 colitis [66], and endogenous TRPA1 activators are generated in inflamed tissues [67]. TRPA1 promotes, inhibits, or has no effect on experimental colitis [68–70].

TRPM8 TRPM8 is expressed by colonic afferents and exposure to the TRPM8 agonist icillin inhibits chemo- and mechanosensory signaling by these neurons [37]. Icillin inhibits capsaicin-stimulated release of CGRP and reduces colitis severity [38]. Moreover, menthol and eucalyptol reduce acute visceral pain primarily through actions at TRPM8, possibly through opioid release [71].

Other TRP Channels TRPM2 is expressed by monocytes, where it is involved in the release of CXCL8 (Interleukin-8). TRPM2 deletion reduces neutrophil infiltration and associated ulceration in DSS colitis [72]. TRPM2 also plays a role in inflammatory pain signaling, including mechanical and thermal pain [73]. The involvement of TRPM2 in visceral pain signaling is unexplored but is potentially very interesting given the role of TRPM2 in the development of colitis and the importance of mechanosensory input in visceral pain. Another possible therapeutic target is TRPC4. TRPC4 deletion reduces visceral pain associated with mustard oil-induced colitis [74], although expression of TRPC4 by colonic afferents and a role in neurogenic inflammation has yet to be demonstrated.

TRP Channels and Clinical IBD The expression and association of TRP channels with clinical IBD and IBS is best characterized for TRPV1, with almost no information on the other TRP channels. TRPV1- immunoreactive nerve fibers and mRNA are increased in IBS, active or quiescent IBD, and rectal hypersensitivity [75–79]. The density of TRPV1-specific innervation has been positively correlated with abdominal pain scores [77,78] and with thermal and mechanical sensory thresholds [76]. Increased TRPV1-positive innervation of intestinal blood vessels occurs in Crohn’s disease [80], suggesting a role in IBD-related vascular leak and altered blood flow. IBS is often associated with hypersensitivity to colorectal distension [81]. Increased innervation density and expression of TRPV1-positive nerve fibers within colon biopsies has been correlated with pain scores [77,78,82]. However, the roles and expression of other TRP channels in the etiology of IBS and IBD in humans remains largely unknown. This will no doubt change, given the emerging and established literature from animal models that have defined a clear role for TRP channels in intestinal inflammation. Whether hyperalgesia and increased sensitivity associated with clinical IBD is simply due to an increase in the extent to which the colon is innervated or also involves TRP sensitization has yet to be established.

Complementary Medicines and IBD: Role for TRP Channels Complementary and alternative medicines, including herbal medications, are commonly used by IBD patients, primarily stemming from a desire to discontinue steroid treatment [83]. Herbal formulations used to treat IBD commonly contain TRP agonists. Examples include 394 21. TARGETING OF TRANSIENT RECEPTOR POTENTIAL CHANNELS IN DIGESTIVE DISEASE peppermint oil, thyme and oregano oil, and Japanese Kampo. At present only limited studies into their effectiveness and the specific involvement of TRP channels in their effects have been conducted. Many of these compounds can activate multiple TRP channels and other targets, thus caution must be taken when ascribing either a TRP-dependent or TRP-specific mechanism of action. Oregano and thyme oils reduce the severity of 2,4,6-trinitrobenzenesulfonic acid (TNBS) colitis in rats and mice. Carvacrol attenuates TNBS colitis in mice through TRPA1- dependent suppression of NO production by macrophages and TRPA1 desensitization [69]. DKT/TU-100 suppresses TNBS colitis through a TRPA1 and adrenomedullin-dependent vasodilatatory action [84,85]. Eugenol protects against gastric ulceration through increased mucus production [86]. An equivalent role in experimental colitis has not been examined in detail. Curcumin, present in turmeric, has protective effects in TNBS colitis that are blocked by capsazepine [87]. However, curcumin had no effect on TRPV1 currents in Xenopus oocytes, and critical experiments to examine curcumin-dependent regulation of TRPV1 activity were not conducted. Thus, at present there is little evidence that the protective effects of curcumin are indeed TRPV1-mediated.

TRP CHANNEL SENSITIZATION IN IBS- AND IBD-RELATED PAIN

The etiology of IBS is unclear and is confounded by the diverse array of symptoms with which patients may present. Current theories suggest that IBS represents a postinfectious state [88,89] or a result of other stressors [90]. Abdominal pain associated with IBS and IBD is likely to involve hyperexcitability of visceral afferents [81,91]. The contribution of TRPs has recently been reviewed [1,92] and includes TRP sensitization, increased expression by afferents, and enhanced neuropeptide release. TRP sensitization is characterized by a reduction in activation threshold or augmented responsiveness to established activators or to mechanical distension [1]. TRPs expressed by colonic afferents are sensitized in disease models and following exposure to receptor agonists, including bradykinin [93], 5-HT [94–96], histamine [94,95], prostaglandins [95], proteases [62,97–99], growth factors [100], and cytokines [101]. In contrast, activation of protease-activated

receptor-4 (PAR4) by cathepsin G is analgesic [102,103], suggesting that the balance of inflammatory proalgesic and analgesic agonists determines the extent to which TRP-dependent visceral hypersensitivity develops. Mechanisms underlying TRP channel sensitization include TRP phosphorylation [104], Phosphatidylinositol 4,5-bisphosphate (PIP2) depletion [105], and TRP recruitment to the cell surface [106], leading to rapid, transit upregulation of channel activity. Hyperexcitability is maintained long after the resolution of inflammation and presumably involves other mechanisms such as TRP channel upregulation or increased nerve fiber sprouting. Elevated expression of TRPV1 and TRPA1 by colonic afferents occurs in experimental colitis [68,100,107–109] and IBS/IBD [75–78].

Therapeutic Targeting of TRP Channels for Sensory Disorders Inhibition of TRP channels expressed by afferent terminals may represent a viable therapeutic target and may prove efficacious [12,110]. Alternatively, activation of TRPM8 suppresses Summary 395 colitis and visceral hyperalgesia [38,71]. However, peripheral expression of TRP channels, particularly TRPA1 and TRPV4, is not restricted to afferent terminals (see Figure 21.2), and global inhibition of these channels may have undesirable on-target effects on gut function. Desensitization or functional denervation of visceral afferents (e.g., by capsaicin) is also possible [57] but will effectively eliminate normal sensory signaling from the bowel. Delivery of RNAi to visceral afferents theoretically enables long-term block of TRP channel expression, eliminating the requirement for regular drug administration. Viral vectors are the most effective means of delivery and could enable selective targeting of afferent neurons. However, selective delivery to these neurons remains a significant challenge [111]. Although intracellular signaling molecules, including protein kinase C (PKC), are key mediators of TRP sensitization [104], therapeutic targeting of kinases is limited by their ubiquitous expression, functional redundancy, and the lack of specificity of inhibitors [112]. Isoform-selective inhibition of PKC is possible and may prove to be more effective [113–115] but is limited by drug delivery and duration of action [111]. As our basic understanding of GPCR-TRP channel interactions develops, it is likely that novel kinase targets with more discrete cellular distributions will be identified. A novel target is the TRP domain, and TRPducin-like peptides derived from this sequence block TRPV1 activation and neuropeptide release, but not responses to mechanical stimuli [116]. The utility of these inhibitors in suppressing visceral pain is untested. Augmented TRP channel activity and pore dilation may be harnessed to specifically target visceral afferents. The coadministration of both capsaicin and the membrane impermeant voltage-dependent sodium channel inhibitor QX-314 blocks action potential firing exclusively in TRPV1 expressing neurons [117]. However, other TRPs that mediate visceral pain, including TRPA1, are ineffective at delivering QX-314 [118]. To our knowledge the potential for treating chronic visceral pain has yet to be examined. GPCR agonists that suppress TRP channel activity may be another therapeutic option.

PAR 4 agonists have protective roles in neurogenic inflammation and pain [103]. Similarly, there is potential for somatostatin receptor-dependent modulation of these channels, particularly TRPV1 [119,120].

SUMMARY

Considerable evidence points to a major role of TRP channels in the regulation of GI functions in health and disease states. TRP channels are expressed by important regulatory cell types, including intrinsic and extrinsic neurons, enteroendocrine cells, myocytes, and ICCs, and activation and inhibition of these channels affects motility, secretion, inflammation, and visceral sensitivity (Table 21.1). TRP channel activity modifiers are potential therapeutics in the GI tract and may account for the beneficial actions of certain traditional and complementary medications. However, although TRP channels are major homeostatic regulators of GI function, contribute to the etiology of digestive disease, and are potential therapeutic targets, many unresolved questions remain. There is a distinct need to translate observations from laboratory species to human subjects. Many reports have failed to use sufficiently selective agonists and antagonists, which has severely limited the utility of these findings. This is particularly the case in those involving herbal remedies, which may contain a number of different TRP channel activators. 396 21. TARGETING OF TRANSIENT RECEPTOR POTENTIAL CHANNELS IN DIGESTIVE DISEASE

TABLE 21.1 Summary of TRP Channels Involved in Regulating GI Function

Function/disease TRPA1 TRPV1 TRPV2 TRPV4 TRPM8 Other GERD/NERD, [121,122] [123–129] esophagitis Gastric ulcer/ [130,131] gastritis Gastric pain [132–135] [136–138] Gastric motility [46,139,140] [32] Pancreatitis [141–144] [142,143,145–148] [141] Pancreatic pain [141–143] [149,150] [141] IBS/IBD pain [70,93,100,101, [94–96,108,153–156] [60–62,94,103] TRPC4: [74] 107,151,152] IBD [68] [54,58,68,76–78,157] [63,64] [38,71] TRPM2: [72] Intestinal motility [8,18,20,46,47] [5,27] [31] TRPC4: [41,44] TRPC6: [44] TRPM7: [42] Intestinal secretion [7,48,85] [27,158,159]

Acknowledgments We thank Professor John Furness and Dr. Hyun Jung Cho (The Department of Anatomy & Neuroscience, The University of Melbourne, Australia) for assistance with figure preparation. Research in the authors’ laboratories has been supported by the National Health & Medical Research Council (Australia), The Australian Research Council, and Monash University.

References [1] Blackshaw LA, Brierley SM, Hughes PA. TRP channels: new targets for visceral pain. Gut 2010;59(1):126–35. [2] Vriens J, Nilius B, Vennekens R. Herbal compounds and toxins modulating TRP channels. Curr Neuropharmacol 2008;6(1):79–96. [3] Furness JB. The enteric nervous system and neurogastroenterology. Nat Rev Gastroenterol Hepatol 2012;9:286–94. [4] Brookes SJ, Spencer NJ, Costa M, Zagorodnyuk VP. Extrinsic primary afferent signalling in the gut. Nat Rev Gastroenterol Hepatol 2013;10(5):286–96. [5] de Man JG, Boeckx S, Anguille S, de Winter BY, de Schepper HU, Herman AG, et al. Functional study on TRPV1- mediated signalling in the mouse small intestine: involvement of tachykinin receptors. Neurogastroenterol Motil 2008;20(5):546–56. [6] Takaki M, Jin JG, Lu YF, Nakayama S. Effects of piperine on the motility of the isolated guinea-pig ileum: comparison with capsaicin. Eur J Pharmacol 1990;186(1):71–7. [7] Kaji I, Karaki S, Kuwahara A. Effects of luminal thymol on epithelial transport in human and rat colon. Am J Physiol Gastrointest Liver Physiol 2011;300(6):G1132–43. [8] Nozawa K, Kawabata-Shoda E, Doihara H, Kojima R, Okada H, Mochizuki S, et al. TRPA1 regulates gastrointestinal motility through serotonin release from enterochromaffin cells. Proc Natl Acad Sci USA 2009;106(9):3408–13. [9] Borrelli F, Capasso R, Pinto A, Izzo AA. Inhibitory effect of ginger (Zingiber officinale) on rat ileal motility in vitro. Life Sci 2004;74(23):2889–96. REFERENCES 397

[10] Amato A, Baldassano S, Serio R, Mule F. Tetrodotoxin-dependent effects of menthol on mouse gastric motor function. Eur J Pharmacol 2013;718(1–3):131–7. [11] Furness JB, Rivera LR, Cho HJ, Bravo DM, Callaghan B. The gut as a sensory organ. Nat Rev Gastroenterol Hepatol 2013;10(12):729–40. [12] Kindt S, Vos R, Blondeau K, Tack J. Influence of intra-oesophageal capsaicin instillation on heartburn induction and oesophageal sensitivity in man. Neurogastroenterol Motil 2009;21(10), 1032-e82. [13] Krarup AL, Ny L, Gunnarsson J, Hvid-Jensen F, Zetterstrand S, Simren M, et al. Randomized clinical trial: inhibition of the TRPV1 system in patients with nonerosive gastroesophageal reflux disease and a partial response to PPI treatment is not associated with analgesia to esophageal experimental pain. Scand J Gastroenterol 2013;48(3):274–84. [14] Iturrino J, Camilleri M, Wong BS, Linker Nord SJ, Burton D, Zinsmeister AR. Randomised clinical trial: the effects of daikenchuto, TU-100, on gastrointestinal and colonic transit, anorectal and bowel function in female patients with functional constipation. Aliment Pharmacol Ther 2013;37(8):776–85. [15] Grigoleit HG, Grigoleit P. Gastrointestinal clinical pharmacology of peppermint oil. Phytomedicine 2005;12(8):607–11. [16] Cho HJ, Callaghan B, Bron R, Bravo DM, Furness JB. Identification of enteroendocrine cells that express TRPA1 channels in the mouse intestine. Cell Tissue Res 2014;356(1):77–82. [17] Anand U, Otto WR, Facer P, Zebda N, Selmer I, Gunthorpe MJ, et al. TRPA1 receptor localisation in the human peripheral nervous system and functional studies in cultured human and rat sensory neurons. Neurosci Lett 2008;438(2):221–7. [18] Poole DP, Pelayo JC, Cattaruzza F, Kuo YM, Gai G, Chiu JV, et al. Transient receptor potential ankyrin 1 is expressed by inhibitory motoneurons of the mouse intestine. Gastroenterology 2011;141(2):565–75, 575.e1-4. [19] Doihara H, Nozawa K, Kojima R, Kawabata-Shoda E, Yokoyama T, Ito H. QGP-1 cells release 5-HT via TRPA1 activation; a model of human enterochromaffin cells. Mol Cell Biochem 2009;331:239–45. [20] Penuelas A, Tashima K, Tsuchiya S, Matsumoto K, Nakamura T, Horie S, et al. Contractile effect of TRPA1 receptor agonists in the isolated mouse intestine. Eur J Pharmacol 2007;576(1–3):143–50. [21] Capasso R, Aviello G, Romano B, Borrelli F, De Petrocellis L, Di Marzo V, et al. Modulation of mouse gastrointestinal motility by allyl isothiocyanate, a constituent of cruciferous vegetables (Brassicaceae): evidence for TRPA1-independent effects. Br J Pharmacol 2012;165(6):1966–77. [22] Bartho L, Nordtveit E, Szombati V, Benko R. Purinoceptor-mediated, capsaicin-resistant excitatory effect of allyl isothiocyanate on neurons of the guinea-pig small intestine. Basic Clin Pharmacol Toxicol 2013;113(2):141–3. [23] Keating DJ, Spencer NJ. Release of 5-hydroxytryptamine from the mucosa is not required for the generation or propagation of colonic migrating motor complexes. Gastroenterology 2010;138(2):659–70, 70.e1-2. [24] Patterson LM, Zheng H, Ward SM, Berthoud HR. Vanilloid receptor (VR1) expression in vagal afferent neurons innervating the gastrointestinal tract. Cell Tissue Res 2003;311(3):277–87. [25] Ward SM, Bayguinov J, Won KJ, Grundy D, Berthoud HR. Distribution of the vanilloid receptor (VR1) in the gastrointestinal tract. J Comp Neurol 2003;465(1):121–35. [26] Matsumoto K, Hosoya T, Tashima K, Namiki T, Murayama T, Horie S. Distribution of transient receptor potential vanilloid 1 channel-expressing nerve fibers in mouse rectal and colonic enteric nervous system: relationship to peptidergic and nitrergic neurons. Neuroscience 2011;172:518–34. [27] Krueger D, Foerster M, Mueller K, Zeller F, Slotta-Huspenina J, Donovan J, et al. Signaling mechanisms involved in the intestinal pro-secretory actions of hydrogen sulfide. Neurogastroenterol Motil 2010;22(11):1224– 31, e319–20. [28] Shibata C, Jin XL, Naito H, Matsuno S, Sasaki I. Intraileal capsaicin inhibits gastrointestinal contractions via a neural reflex in conscious dogs. Gastroenterology 2002;123(6):1904–11. [29] Fujimoto S, Mori M, Tsushima H, Kunimatsu M. Capsaicin-induced, capsazepine-insensitive relaxation of the guinea-pig ileum. Eur J Pharmacol 2006;530(1–2):144–51. [30] Kashiba H, Uchida Y, Takeda D, Nishigori A, Ueda Y, Kuribayashi K, et al. TRPV2-immunoreactive intrinsic neurons in the rat intestine. Neurosci Lett 2004;366(2):193–6. [31] Mihara H, Boudaka A, Shibasaki K, Yamanaka A, Sugiyama T, Tominaga M. Involvement of TRPV2 activation in intestinal movement through nitric oxide production in mice. J Neurosci 2010;30:16536–44. [32] Mihara H, Suzuki N, Yamawaki H, Tominaga M, Sugiyama T. TRPV2 ion channels expressed in inhibitory motor neurons of gastric myenteric plexus contribute to gastric adaptive relaxation and gastric emptying in mice. Am J Physiol Gastrointest Liver Physiol 2013;304(3):G235–40. 398 21. TARGETING OF TRANSIENT RECEPTOR POTENTIAL CHANNELS IN DIGESTIVE DISEASE

[33] Ueda T, Yamada T, Ugawa S, Ishida Y, Shimada S. TRPV3, a thermosensitive channel is expressed in mouse distal colon epithelium. Biochem Biophys Res Commun 2009;383(1):130–4. [34] Kidd M, Modlin IM, Gustafsson BI, Drozdov I, Hauso O, Pfragner R. Luminal regulation of normal and neo- plastic human EC cell serotonin release is mediated by bile salts, amines, tastants, and olfactants. Am J Physiol Gastrointest Liver Physiol 2008;295(2):G260–72. [35] Leal-Cardoso JH, Lahlou S, Coelho-de-Souza AN, Criddle DN, Pinto Duarte GI, Santos MA, et al. Inhibitory actions of eugenol on rat isolated ileum. Can J Physiol Pharmacol 2002;80(9):901–6. [36] Xu H, Delling M, Jun JC, Clapham DE. Oregano, thyme and clove-derived flavors and skin sensitizers activate specific TRP channels. Nat Neurosci 2006;9:628–35. [37] Harrington AM, Hughes PA, Martin CM, Yang J, Castro J, Isaacs NJ, et al. A novel role for TRPM8 in visceral afferent function. Pain 2011;152:1459–68. [38] Ramachandran R, Hyun E, Zhao L, Lapointe TK, Chapman K, Hirota CL, et al. TRPM8 activation attenuates inflammatory responses in mouse models of colitis. Proc Natl Acad Sci USA 2013;110:7476–81. [39] Epperson A, Hatton WJ, Callaghan B, Doherty P, Walker RL, Sanders KM, et al. Molecular markers expressed in cultured and freshly isolated interstitial cells of Cajal. Am J Physiol Cell Physiol 2000;279(2):C529–39. [40] Chen H, Ordög T, Chen J, Young DL, Bardsley MR, Redelman D, et al. Differential gene expression in functional classes of interstitial cells of Cajal in murine small intestine. Physiol Genomics 2007;31:492–509. [41] Torihashi S, Fujimoto T, Trost C, Nakayama S. Calcium oscillation linked to pacemaking of interstitial cells of Cajal: requirement of calcium influx and localization of TRP4 in caveolae. J Biol Chem 2002;277(21):19191–7. [42] Kim BJ, Lim HH, Yang DK, Jun JY, Chang IY, Park CS, et al. Melastatin-type transient receptor potential channel 7 is required for intestinal pacemaking activity. Gastroenterology 2005;129(5):1504–17. [43] Dwyer L, Rhee PL, Lowe V, Zheng H, Peri L, Ro S, et al. Basally activated nonselective cation currents regulate the resting membrane potential in human and monkey colonic smooth muscle. Am J Physiol Gastrointest Liver Physiol 2011;301(2):G287–96. [44] Tsvilovskyy VV, Zholos AV, Aberle T, Philipp SE, Dietrich A, Zhu MX, et al. Deletion of TRPC4 and TRPC6 in mice impairs smooth muscle contraction and intestinal motility in vivo. Gastroenterology 2009;137(4):1415–24. [45] Pohl D, Tutuian R, Fried M. Pharmacologic treatment of constipation: what is new? Curr Opin Pharmacol 2008;8(6):724–8. [46] Doihara H, Nozawa K, Kawabata-Shoda E, Kojima R, Yokoyama T, Ito H. Molecular cloning and characterization of dog TRPA1 and AITC stimulate the gastrointestinal motility through TRPA1 in conscious dogs. Eur J Pharmacol 2009;617:124–9. [47] Kojima R, Doihara H, Nozawa K, Kawabata-Shoda E, Yokoyama T, Ito H. Characterization of two models of drug-induced constipation in mice and evaluation of mustard oil in these models. Pharmacology 2009;84:227–33. [48] Kaji I, Yasuoka Y, Karaki S, Kuwahara A. Activation of TRPA1 by luminal stimuli induces EP4-mediated anion secretion in human and rat colon. Am J Physiol Gastrointest Liver Physiol 2012;302(7):G690–701. [49] Riera CE, Menozzi-Smarrito C, Affolter M, Michlig S, Munari C, Robert F, et al. Compounds from Sichuan and Melegueta peppers activate, covalently and non-covalently, TRPA1 and TRPV1 channels. Br J Pharmacol 2009;157(8):1398–409. [50] Nakamura T, Sakai A, Isogami I, Noda K, Ueno K, Yano S. Abatement of morphine-induced slowing in gastrointestinal transit by Dai-kenchu-to, a traditional Japanese herbal medicine. Jpn J Pharmacol 2002;88:217–21. [51] Wood MJ, Hyman NH, Mawe GM. The effects of daikenchuto (DKT) on propulsive motility in the colon. J Surg Res 2010;164(1):84–90. [52] Kikuchi D, Shibata C, Imoto H, Naitoh T, Miura K, Unno M. Intragastric Dai-Kenchu-To, a japanese herbal medicine stimulates colonic motility via transient receptor potential cation channel subfamily V Member 1 in dogs. Tohoku J Exp Med 2013;230:197–204. [53] Manabe N, Camilleri M, Rao A, Wong BS, Burton D, Busciglio I, et al. Effect of daikenchuto (TU-100) on gastrointestinal and colonic transit in humans. Am J Physiol Gastrointest Liver Physiol 2010;298(6):G970–5. [54] Massa F, Sibaev A, Marsicano G, Blaudzun H, Storr M, Lutz B. Vanilloid receptor (TRPV1)-deficient mice show increased susceptibility to dinitrobenzene sulfonic acid induced colitis. J Mol Med 2006;84:142–6. [55] Gad M, Pedersen AE, Kristensen NN, Fernandez Cde F, Claesson MH. Blockage of the neurokinin 1 receptor and capsaicin-induced ablation of the enteric afferent nerves protect SCID mice against T-cell-induced chronic colitis. Inflamm Bowel Dis 2009;15(8):1174–82. [56] Kimball ES, Wallace NH, Schneider CR, D’Andrea MR, Hornby PJ. Vanilloid receptor 1 antagonists attenuate disease severity in dextran sulphate sodium-induced colitis in mice. Neurogastroenterol Motil 2004;16(6):811–18. REFERENCES 399

[57] Storr M. TRPV1 in colitis: is it a good or a bad receptor?—a viewpoint. Neurogastroenterol Motil 2007;19(8):625–9. [58] Miranda A, Nordstrom E, Mannem A, Smith C, Banerjee B, Sengupta JN. The role of transient receptor potential vanilloid 1 in mechanical and chemical visceral hyperalgesia following experimental colitis. Neuroscience 2007;148(4):1021–32. [59] Engel MA, Leffler A, Niedermirtl F, Babes A, Zimmermann K, Filipovic MR, et al. TRPA1 and substance P mediate colitis in mice. Gastroenterology 2011;141(4):1346–58. [60] Brierley SM, Page AJ, Hughes PA, Adam B, Liebregts T, Cooper NJ, et al. Selective role for TRPV4 ion channels in visceral sensory pathways. Gastroenterology 2008;134(7):2059–69. [61] Cenac N, Altier C, Chapman K, Liedtke W, Zamponi G, Vergnolle N. Transient receptor potential vanilloid-4 has a major role in visceral hypersensitivity symptoms. Gastroenterology 2008;135:937–46, 46.e1-2. [62] Sipe WE, Brierley SM, Martin CM, Phillis BD, Cruz FB, Grady EF, et al. Transient receptor potential vanilloid 4 mediates protease activated receptor 2-induced sensitization of colonic afferent nerves and visceral hyperalgesia. Am J Physiol Gastrointest Liver Physiol 2008;294(5):G1288–98. [63] D’Aldebert E, Cenac N, Rousset P, Martin L, Rolland C, Chapman K, et al. Transient receptor potential vanilloid 4 activated inflammatory signals by intestinal epithelial cells and colitis in mice. Gastroenterology 2011;140(1):275–85. [64] Fichna J, Mokrowiecka A, Cygankiewicz AI, Zakrzewski PK, Malecka-Panas E, Janecka A, et al. Transient receptor potential vanilloid 4 blockade protects against experimental colitis in mice: a new strategy for inflammatory bowel diseases treatment? Neurogastroenterol Motil 2012;24(11):e557–60. [65] Distrutti E, Cipriani S, Mencarelli A, Renga B, Fiorucci S. Probiotics VSL#3 protect against development of visceral pain in murine model of irritable bowel syndrome. PLoS One 2013;8(5):e63893. [66] Kimball ES, Prouty SP, Pavlick KP, Wallace NH, Schneider CR, Hornby PJ. Stimulation of neuronal receptors, neuropeptides and cytokines during experimental oil of mustard colitis. Neurogastroenterol Motil 2007;19(5):390–400. [67] Trevisani M, Siemens J, Materazzi S, Bautista DM, Nassini R, Campi B, et al. 4-Hydroxynonenal, an endogenous aldehyde, causes pain and neurogenic inflammation through activation of the irritant receptor TRPA1. Proc Natl Acad Sci USA 2007;104(33):13519–24. [68] Engel MA, Khalil M, Mueller-Tribbensee SM, Becker C, Neuhuber WL, Neurath MF, et al. The proximodistal aggravation of colitis depends on substance P released from TRPV1-expressing sensory neurons. J Gastroenterol 2012;47(3):256–65. [69] Romano B, Borrelli F, Fasolino I, Capasso R, Piscitelli F, Cascio M, et al. The cannabinoid TRPA1 agonist can- nabichromene inhibits nitric oxide production in macrophages and ameliorates murine colitis. Br J Pharmacol 2013;169:213–29. [70] Cattaruzza F, Spreadbury I, Miranda-Morales M, Grady EF, Vanner S, Bunnett NW. Transient receptor potential ankyrin-1 has a major role in mediating visceral pain in mice. Am J Physiol Gastrointest Liver Physiol 2010;298(1):G81–91. [71] Liu B, Fan L, Balakrishna S, Sui A, Morris JB, Jordt SE. TRPM8 is the principal mediator of menthol-induced analgesia of acute and inflammatory pain. Pain 2013;154(10):2169–77. [72] Yamamoto S, Shimizu S, Kiyonaka S, Takahashi N, Wajima T, Hara Y, et al. TRPM2-mediated Ca2 + influx induces chemokine production in monocytes that aggravates inflammatory neutrophil infiltration. Nat Med 2008;14(7):738–47. [73] Haraguchi K, Kawamoto A, Isami K, Maeda S, Kusano A, Asakura K, et al. TRPM2 contributes to inflammatory and neuropathic pain through the aggravation of pronociceptive inflammatory responses in mice. J Neurosci 2012;32:3931–41. [74] Westlund KN, Zhang LP, Ma F, Nesemeier R, Ruiz JC, Ostertag EM, et al. A rat knockout model implicates TRPC4 in visceral pain sensation. Neuroscience 2014;262:165–75. [75] Yiangou Y, Facer P, Dyer NH, Chan CL, Knowles C, Williams NS, et al. Vanilloid receptor 1 immunoreactivity in inflamed human bowel. Lancet 2001;357:1338–9. [76] Chan CL, Facer P, Davis JB, Smith GD, Egerton J, Bountra C, et al. Sensory fibres expressing capsaicin receptor TRPV1 in patients with rectal hypersensitivity and faecal urgency. Lancet 2003;361(9355):385–91. [77] Akbar A, Yiangou Y, Facer P, Walters JR, Anand P, Ghosh S. Increased capsaicin receptor TRPV1-expressing sensory fibres in irritable bowel syndrome and their correlation with abdominal pain. Gut 2008;57(7):923–9. [78] Akbar A, Yiangou Y, Facer P, Brydon WG, Walters JR, Anand P, et al. Expression of the TRPV1 receptor differs in quiescent inflammatory bowel disease with or without abdominal pain. Gut 2010;59(6):767–74. 400 21. TARGETING OF TRANSIENT RECEPTOR POTENTIAL CHANNELS IN DIGESTIVE DISEASE

[79] Keszthelyi D, Troost FJ, Jonkers DM, Helyes Z, Hamer HM, Ludidi S, et al. Alterations in mucosal neuropeptides in patients with irritable bowel syndrome and ulcerative colitis in remission: a role in pain symptom generation? Eur J Pain 2013;17(9):1299–306. [80] de Fontgalland D, Brookes SJ, Gibbins I, Sia TC, Wattchow DA. The neurochemical changes in the innervation of human colonic mesenteric and submucosal blood vessels in Ulcerative colitis and Crohn’s disease. Neurogastroenterol Motil 2014;26(5):731–44. [81] Ritchie J. Pain from distension of the pelvic colon by inflating a balloon in the irritable colon syndrome. Gut 1973;14(2):125–32. [82] Akbar A, Walters JR, Ghosh S. Review article: visceral hypersensitivity in irritable bowel syndrome: molecular mechanisms and therapeutic agents. Aliment Pharmacol Ther 2009;30(5):423–35. [83] Langhorst J, Anthonisen IB, Steder-Neukamm U, Ludtke R, Spahn G, Michalsen A, et al. Amount of systemic steroid medication is a strong predictor for the use of complementary and alternative medicine in patients with inflammatory bowel disease: results from a German national survey. Inflamm Bowel Dis 2005;11(3):287–95. [84] Kaneko A, Kono T, Miura N, Tsuchiya N, Yamamoto M. Preventive effect of TU-100 on a type-2 model of Colitis in mice: possible involvement of enhancing adrenomedullin in intestinal epithelial cells. Gastroenterol Res Pract 2013;2013:384057. [85] Kono T, Kaneko A, Omiya Y, Ohbuchi K, Ohno N, Yamamoto M. Epithelial transient receptor potential ankyrin 1 (TRPA1)-dependent adrenomedullin upregulates blood flow in rat small intestine. Am J Physiol Gastrointest Liver Physiol 2013;304(4):G428–36. [86] Santin JR, Lemos M, Klein-Junior LC, Machado ID, Costa P, de Oliveira AP, et al. Gastroprotective activity of essential oil of the Syzygium aromaticum and its major component eugenol in different animal models. Naunyn Schmiedeberg’s Arch Pharmacol 2011;383(2):149–58. [87] Martelli L, Ragazzi E, di Mario F, Martelli M, Castagliuolo I, Dal Maschio M, et al. A potential role for the vanilloid receptor TRPV1 in the therapeutic effect of curcumin in dinitrobenzene sulphonic acid-induced colitis in mice. Neurogastroenterol Motil 2007;19(8):668–74. [88] Marshall JK, Thabane M, Garg AX, Clark WF, Moayyedi P, Collins SM. Eight year prognosis of postinfectious irritable bowel syndrome following waterborne bacterial dysentery. Gut 2010;59(5):605–11. [89] Simren M, Barbara G, Flint HJ, Spiegel BM, Spiller RC, Vanner S, et al. Intestinal microbiota in functional bowel disorders: a Rome foundation report. Gut 2013;62(1):159–76. [90] Al-Chaer ED, Kawasaki M, Pasricha PJ. A new model of chronic visceral hypersensitivity in adult rats induced by colon irritation during postnatal development. Gastroenterology 2000;119(5):1276–85. [91] Beyak MJ, Ramji N, Krol KM, Kawaja MD, Vanner SJ. Two TTX-resistant Na+ currents in mouse colonic dorsal root ganglia neurons and their role in colitis-induced hyperexcitability. Am J Physiol Gastrointest Liver Physiol 2004;287(4):G845–55. [92] Boesmans W, Owsianik G, Tack J, Voets T, Vanden Berghe P. TRP channels in neurogastroenterology: opportunities for therapeutic intervention. Br J Pharmacol 2011;162(1):18–37. [93] Brierley SM, Hughes PA, Page AJ, Kwan KY, Martin CM, O’Donnell TA, et al. The ion channel TRPA1 is required for normal mechanosensation and is modulated by algesic stimuli. Gastroenterology 2009;137(6) 2084–95e3. [94] Cenac N, Altier C, Motta JP, d’Aldebert E, Galeano S, Zamponi GW, et al. Potentiation of TRPV4 signalling by histamine and serotonin: an important mechanism for visceral hypersensitivity. Gut 2010;59(4):481–8. [95] Jones 3rd RC, Xu L, Gebhart GF. The mechanosensitivity of mouse colon afferent fibers and their sensitization by inflammatory mediators require transient receptor potential vanilloid 1 and acid-sensing ion channel 3. J Neurosci 2005;25(47):10981–9. [96] Sugiuar T, Bielefeldt K, Gebhart GF. TRPV1 function in mouse colon sensory neurons is enhanced by metabotropic 5-hydroxytryptamine receptor activation. J Neurosci 2004;24(43):9521–30. [97] Cenac N, Andrews CN, Holzhausen M, Chapman K, Cottrell G, Andrade-Gordon P, et al. Role for protease activity in visceral pain in irritable bowel syndrome. J Clin Invest 2007;117(3):636–47. [98] Kawabata A, Kawao N, Kitano T, Matsunami M, Satoh R, Ishiki T, et al. Colonic hyperalgesia triggered by proteinase-activated receptor-2 in mice: involvement of endogenous bradykinin. Neurosci Lett 2006;402:167–72. [99] Kawao N, Ikeda H, Kitano T, Kuroda R, Sekiguchi F, Kataoka K, et al. Modulation of capsaicin-evoked visceral pain and referred hyperalgesia by protease-activated receptors 1 and 2. J Pharmacol Sci 2004;94(3):277–85. [100] Malin S, Molliver D, Christianson JA, Schwartz ES, Cornuet P, Albers KM, et al. TRPV1 and TRPA1 function and modulation are target tissue dependent. J Neurosci 2011;31:10516–28. REFERENCES 401

[101] Hughes PA, Harrington AM, Castro J, Liebregts T, Adam B, Grasby DJ, et al. Sensory neuro-immune interactions differ between irritable bowel syndrome subtypes. Gut 2013;62:1456–65. [102] Annahazi A, Gecse K, Dabek M, Ait-Belgnaoui A, Rosztoczy A, Roka R, et al. Fecal proteases from diarrheic-IBS and ulcerative colitis patients exert opposite effect on visceral sensitivity in mice. Pain 2009;144(1–2):209–17. [103] Auge C, Balz-Hara D, Steinhoff M, Vergnolle N, Cenac N. Protease-activated receptor-4 (PAR 4): a role as inhibitor of visceral pain and hypersensitivity. Neurogastroenterol Motil 2009;21(11), 1189-e107. [104] Amadesi S, Nie J, Vergnolle N, Cottrell GS, Grady EF, Trevisani M, et al. Protease-activated receptor 2 sensitizes the capsaicin receptor transient receptor potential vanilloid receptor 1 to induce hyperalgesia. J Neurosci 2004;24(18):4300–12. [105] Dai Y, Wang S, Tominaga M, Yamamoto S, Fukuoka T, Higashi T, et al. Sensitization of TRPA1 by PAR2 contributes to the sensation of inflammatory pain. J Clin Invest 2007;117(7):1979–87. [106] Zhang X, Huang J, McNaughton PA. NGF rapidly increases membrane expression of TRPV1 heat-gated ion channels. EMBO J 2005;24:4211–23. [107] Christianson JA, Bielefeldt K, Malin SA, Davis BM. Neonatal colon insult alters growth factor expression and TRPA1 responses in adult mice. Pain 2010;151(2):540–9. [108] De Schepper HU, De Man JG, Ruyssers NE, Deiteren A, Van Nassauw L, Timmermans J-P, et al. TRPV1 receptor signaling mediates afferent nerve sensitization during colitis-induced motility disorders in rats. Am J Physiol Gastrointest Liver Physiol 2008;294:G245–53. [109] Winston J, Shenoy M, Medley D, Naniwadekar A, Pasricha PJ. The vanilloid receptor initiates and maintains colonic hypersensitivity induced by neonatal colon irritation in rats. Gastroenterology 2007;132:615–27. [110] Krarup AL, Ny L, Astrand M, Bajor A, Hvid-Jensen F, Hansen MB, et al. Randomised clinical trial: the efficacy of a transient receptor potential vanilloid 1 antagonist AZD1386 in human oesophageal pain. Aliment Pharmacol Ther 2011;33(10):1113–22. [111] Terashima T, Oka K, Kritz AB, Kojima H, Baker AH, Chan L. DRG-targeted helper-dependent adenovi- ruses mediate selective gene delivery for therapeutic rescue of sensory neuronopathies in mice. J Clin Invest 2009;119(7):2100–12. [112] Bain J, Plater L, Elliott M, Shpiro N, Hastie CJ, McLauchlan H, et al. The selectivity of protein kinase inhibitors: a further update. Biochem J 2007;408(3):297–315. [113] Aley KO, Messing RO, Mochly-Rosen D, Levine JD. Chronic hypersensitivity for inflammatory nociceptor sensitization mediated by the epsilon isozyme of protein kinase C. J Neurosci 2000;20(12):4680–5. [114] Shumilla JA, Liron T, Mochly-Rosen D, Kendig JJ, Sweitzer SM. Ethanol withdrawal-associated allodynia and hyperalgesia: age-dependent regulation by protein kinase C epsilon and gamma isoenzymes. J Pain 2005;6(8):535–49. [115] Hucho T, Levine JD. Signaling pathways in sensitization: toward a nociceptor cell biology. Neuron 2007;55(3):365–76. [116] Valente P, Fernandez-Carvajal A, Camprubi-Robles M, Gomis A, Quirce S, Viana F, et al. Membrane-tethered peptides patterned after the TRP domain (TRPducins) selectively inhibit TRPV1 channel activity. FASEB J 2011;25(5):1628–40. [117] Binshtok AM, Bean BP, Woolf CJ. Inhibition of nociceptors by TRPV1-mediated entry of impermeant sodium channel blockers. Nature 2007;449(7162):607–10. [118] Nakagawa H, Hiura A. Comparison of the transport of QX-314 through TRPA1, TRPM8, and TRPV1 channels. J Pain Res 2013;6:223–30. [119] Carlton SM, Du J, Davidson E, Zhou S, Coggeshall RE. Somatostatin receptors on peripheral primary afferent terminals: inhibition of sensitized nociceptors. Pain 2001;90(3):233–44. [120] Su X, Burton MB, Gebhart GF. Effects of octreotide on responses to colorectal distension in the rat. Gut 2001;48(5):676–82. [121] Yu S, Ouyang A. TRPA1 in bradykinin-induced mechanical hypersensitivity of vagal C fibers in guinea pig esophagus. Am J Physiol Gastrointest Liver Physiol 2009;296(2):G255–65. [122] Yu S, Gao G, Peterson BZ, Ouyang A. TRPA1 in mast cell activation-induced long-lasting mechanical hypersensitivity of vagal afferent C-fibers in guinea pig esophagus. Am J Physiol Gastrointest Liver Physiol 2009;297(1):G34–42. [123] Matthews PJ, Aziz Q, Facer P, Davis JB, Thompson DG, Anand P. Increased capsaicin receptor TRPV1 nerve fibres in the inflamed human oesophagus. Eur J Gastroenterol Hepatol 2004;16(9):897–902. 402 21. TARGETING OF TRANSIENT RECEPTOR POTENTIAL CHANNELS IN DIGESTIVE DISEASE

[124] Bhat YM, Bielefeldt K. Capsaicin receptor (TRPV1) and non-erosive reflux disease. Eur J Gastroenterol Hepatol 2006;18(3):263–70. [125] Banerjee B, Medda BK, Lazarova Z, Bansal N, Shaker R, Sengupta JN. Effect of reflux-induced inflammation on transient receptor potential vanilloid one (TRPV1) expression in primary sensory neurons innervating the oesophagus of rats. Neurogastroenterol Motil 2007;19(8):681–91. [126] Peles S, Medda BK, Zhang Z, Banerjee B, Lehmann A, Shaker R, et al. Differential effects of transient receptor vanilloid one (TRPV1) antagonists in acid-induced excitation of esophageal vagal afferent fibers of rats. Neuroscience 2009;161(2):515–25. [127] Shieh KR, Yi CH, Liu TT, Tseng HL, Ho HC, Hsieh HT, et al. Evidence for neurotrophic factors associating with TRPV1 gene expression in the inflamed human esophagus. Neurogastroenterol Motil 2010;22(9):971–7, e252. [128] Yoshida N, Kuroda M, Suzuki T, Kamada K, Uchiyama K, Handa O, et al. Role of nociceptors/neuropeptides in the pathogenesis of visceral hypersensitivity of nonerosive reflux disease. Dig Dis Sci 2013;58(8):2237–43. [129] Harrington AM, Brierley SM, Isaacs NJ, Young RL, Ashley Blackshaw L. Identifying spinal sensory pathways activated by noxious esophageal acid. Neurogastroenterol Motil 2013;25(10):e660–8. [130] Gazzieri D, Trevisani M, Springer J, Harrison S, Cottrell GS, Andre E, et al. Substance P released by TRPV1- expressing neurons produces reactive oxygen species that mediate ethanol-induced gastric injury. Free Radic Biol Med 2007;43(4):581–9. [131] Domotor A, Kereskay L, Szekeres G, Hunyady B, Szolcsanyi J, Mozsik G. Participation of capsaicin-sensitive afferent nerves in the gastric mucosa of patients with Helicobacter pylori-positive or-negative chronic gastritis. Dig Dis Sci 2007;52(2):411–7. [132] Sakurai J, Obata K, Ozaki N, Tokunaga A, Kobayashi K, Yamanaka H, et al. Activation of extracellular signal-regulated protein kinase in sensory neurons after noxious gastric distention and its involvement in acute visceral pain in rats. Gastroenterology 2008;134(4):1094–103. [133] Kondo T, Sakurai J, Miwa H, Noguchi K. Activation of p38 MAPK through transient receptor potential A1 in a rat model of gastric distension-induced visceral pain. Neuroreport 2013;24(2):68–72. [134] Kondo T, Oshima T, Obata K, Sakurai J, Knowles CH, Matsumoto T, et al. Role of transient receptor potential A1 in gastric nociception. Digestion 2010;82(3):150–5. [135] Kondo T, Obata K, Miyoshi K, Sakurai J, Tanaka J, Miwa H, et al. Transient receptor potential A1 mediates gastric distention-induced visceral pain in rats. Gut 2009;58(10):1342–52. [136] Sugiura T, Dang K, Lamb K, Bielefeldt K, Gebhart GF. Acid-sensing properties in rat gastric sensory neurons from normal and ulcerated stomach. J Neurosci 2005;25(10):2617–27. [137] Schicho R, Donnerer J, Liebmann I, Lippe IT. Nociceptive transmitter release in the dorsal spinal cord by capsaicin-sensitive fibers after noxious gastric stimulation. Brain Res 2005;1039(1–2):108–15. [138] Bielefeldt K, Davis BM. Differential effects of ASIC3 and TRPV1 deletion on gastroesophageal sensation in mice. Am J Physiol Gastrointest Liver Physiol 2008;294(1):G130–8. [139] Koseki J, Oshima T, Kondo T, Tomita T, Fukui H, Watari J, et al. Role of transient receptor potential ankyrin 1 in gastric accommodation in conscious guinea pigs. J Pharmacol Exp Ther 2012;341(1):205–12. [140] Kim MJ, Son HJ, Song SH, Jung M, Kim Y, Rhyu MR. The TRPA1 agonist, methyl syringate suppresses food intake and gastric emptying. PLoS One 2013;8(8):e71603. [141] Ceppa E, Cattaruzza F, Lyo V, Amadesi S, Pelayo JC, Poole DP, et al. Transient receptor potential ion channels V4 and A1 contribute to pancreatitis pain in mice. Am J Physiol Gastrointest Liver Physiol 2010;299(3):G556–71. [142] Schwartz ES, Christianson JA, Chen X, La JH, Davis BM, Albers KM, et al. Synergistic role of TRPV1 and TRPA1 in pancreatic pain and inflammation. Gastroenterology 2011;140(4), 1283–91e1-2. [143] Schwartz ES, La JH, Scheff NN, Davis BM, Albers KM, Gebhart GF. TRPV1 and TRPA1 antagonists prevent the transition of acute to chronic inflammation and pain in chronic pancreatitis. J Neurosci 2013;33(13):5603–11. [144] Li C, Zhu Y, Shenoy M, Pai R, Liu L, Pasricha PJ. Anatomical and functional characterization of a duodeno-pancreatic neural reflex that can induce acute pancreatitis. Am J Physiol Gastrointest Liver Physiol 2013;304(5):G490–500. [145] Xu GY, Winston JH, Shenoy M, Yin H, Pendyala S, Pasricha PJ. Transient receptor potential vanilloid 1 mediates hyperalgesia and is up-regulated in rats with chronic pancreatitis. Gastroenterology 2007;133(4):1282–92. [146] Zhu Y, Colak T, Shenoy M, Liu L, Pai R, Li C, et al. Nerve growth factor modulates TRPV1 expression and function and mediates pain in chronic pancreatitis. Gastroenterology 2011;141(1):370–7. [147] Nathan JD, Patel AA, McVey DC, Thomas JE, Prpic V, Vigna SR, et al. Capsaicin vanilloid receptor-1 mediates substance P release in experimental pancreatitis. Am J Physiol Gastrointest Liver Physiol 2001;281(5):G1322–8. REFERENCES 403

[148] Noble MD, Romac J, Wang Y, Hsu J, Humphrey JE, Liddle RA. Local disruption of the celiac ganglion inhibits substance P release and ameliorates caerulein-induced pancreatitis in rats. Am J Physiol Gastrointest Liver Physiol 2006;291(1):G128–34. [149] Wick EC, Hoge SG, Grahn SW, Kim E, Divino LA, Grady EF, et al. Transient receptor potential vanilloid 1, calcitonin gene-related peptide, and substance P mediate nociception in acute pancreatitis. Am J Physiol Gastrointest Liver Physiol 2006;290(5):G959–69. [150] Hartel M, di Mola FF, Selvaggi F, Mascetta G, Wente MN, Felix K, et al. Vanilloids in pancreatic cancer: potential for chemotherapy and pain management. Gut 2006;55(4):519–28. [151] Wang S, Dai Y, Fukuoka T, Yamanaka H, Kobayashi K, Obata K, et al. Phospholipase C and protein kinase A mediate bradykinin sensitization of TRPA1: a molecular mechanism of inflammatory pain. Brain 2008;131:1241–51. [152] Mitrovic M, Shahbazian A, Bock E, Pabst MA, Holzer P. Chemo-nociceptive signalling from the colon is enhanced by mild colitis and blocked by inhibition of transient receptor potential ankyrin 1 channels. Br J Pharmacol 2010;160:1430–42. [153] Okayama M, Tsubouchi R, Kato S, Takeuchi K. Protective effect of lafutidine, a novel histamine H2-receptor antagonist, on dextran sulfate sodium-induced colonic inflammation through capsaicin-sensitive afferent neurons in rats. Dig Dis Sci 2004;49:1696–704. [154] Brierley SM, Jones 3rd. RC, Xu L, Gebhart GF, Blackshaw LA. Activation of splanchnic and pelvic colonic afferents by bradykinin in mice. Neurogastroenterol Motil 2005;17(6):854–62. [155] Yang C-Q, Wei Y-Y, Zhong C-J, Duan L-P. Essential role of mast cells in the visceral hyperalgesia induced by T. spiralis infection and stress in rats. Mediat Inflamm 2012;2012:294070. [156] Yu Y-B, Yang J, Zuo X-L, Gao L-J, Wang P, Li Y-Q. Transient receptor potential vanilloid-1 (TRPV1) and ankyrin-1 (TRPA1) participate in visceral hyperalgesia in chronic water avoidance stress rat model. Neurochem Res 2010;35:797–803. [157] Engel MA, Becker C, Reeh PW, Neurath MF. Role of sensory neurons in colitis: increasing evidence for a neuroimmune link in the gut. Inflamm Bowel Dis 2011;17:1030–3. [158] Bouyer PG, Tang X, Weber CR, Shen L, Turner JR, Matthews JB. Capsaicin induces NKCC1 internalization and inhibits chloride secretion in colonic epithelial cells independently of TRPV1. Am J Physiol Gastrointest Liver Physiol 2013;304(2):G142–56. [159] Baird AW, Skelly MM, O’Donoghue DP, Barrett KE, Keely SJ. Bradykinin regulates human colonic ion transport in vitro. Br J Pharmacol 2008;155(4):558–66. CHAPTER 22 TRP Channels in Cancer Artem Kondratskyi,1 Natalia Prevarskaya,1,* 1Inserm U-1003, Equipe labellisée par la Ligue Nationale contre le cancer, Laboratory of Excellence Ion Channels Science and Therapeutics, Université Lille 1, Villeneuve d’Ascq, France *Corresponding author: [email protected]

OUTLINE

Introduction 405 TRPC Channels 411 The Role of TRP Channels in Cancer 406 Clinical Implications and Concluding TRPM Channels 407 Remarks 412 TRPV Channels 410 References 413

INTRODUCTION

Cancer is a group of diseases characterized by out-of-control growth and dissemina- tion of abnormal cells. Cancer represents one of the leading causes of death worldwide, accounting for 8.2 million deaths in 2012 [1]. Both external (radiation, tobacco, car exhaust fumes, and other carcinogens) and internal factors (inherited mutations, hormones, immune conditions) could be the causes of cancer. Moreover, these factors may act together or in sequence to initiate or promote the development of cancer [2]. There are more than 100 different types of cancer, including breast cancer, skin cancer, lung cancer, colon cancer, prostate cancer, lymphoma, and others. Different cancers are characterized by the different set of mutated genes (either oncogenes or tumor suppressor genes) and thus behave differently. However, Hanahan and Weinberg have proposed several general hallmarks, characteristic to most forms of cancer [3]. These hallmarks include sustaining proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative

immortality, inducing angiogenesis, activating invasion and metastasis, avoiding immune destruction, deregulating cellular energetics, tumor-promoting inflammation as well as genome instability and mutation [3]. Interestingly, calcium signaling has been previously reported to regulate most if not all of these phenomena [4,5]. Changes in the cytosolic-free Ca2+ concentration play a central role in many fundamental cellular processes, including muscle contraction, transmitter release, cell proliferation, differentiation, gene transcription, and cell death [6]. Given that Ca2+ controls so many vital processes, disturbance of the Ca2+ homeostasis regulatory mechanisms leads to a vast variety of severe pathologies, including cancer. Indeed, the role of Ca2+ is well established in many cell signaling pathways involved in carcinogenesis [7–9]. The increase in cytosolic calcium can occur as a result of Ca2+ influx from the extracellular space and/or Ca2+ release from intracellular sources. Both Ca2+ influx and Ca2+ release are tightly controlled by numerous regulatory systems that provide the specific spatial and temporal characteristics of an intracellular calcium signal that are required for sustaining certain cellular functions [6]. Mitochondrial, endoplasmic reticulum (ER), lysosomal, and cytosolic calcium levels are regulated by calcium-permeable ion channels localized either on the membranes of the intracellular organelles or on the plasma membrane [10,11]. The calcium-permeable channels, including families of transient receptor potential (TRP) channels, store-operated channels, voltage-gated calcium channels, two-pore channels, mitochondrial permeability transition pore, mitochondrial calcium uniporter, inositol 1,4,5-trisphosphate receptor 2+ (IP3) and ryanodine receptors, and others contribute to changes in [Ca ]i by providing Ca2+ entry pathways, by modulating the driving force for the Ca2+ entry, and also by providing intracellular pathways for Ca2+ uptake/release into/from cellular organelles [10– 13]. Thus, modulation of a calcium-permeable ion channel’s expression/function affects intracellular Ca2+ concentrations and consequently calcium-dependent processes, such as proliferation, apoptosis, and autophagy [14–16]. The growing number of studies suggests that malignant transformation is often accompanied by the changes in ion channel expression/function [17]. Among these channels, the members of the TRP channels superfamily have been shown to regulate a variety of calcium-dependent cellular processes altered in cancer [18]. In this chapter, we will summarize the information available on the role of TRP channels in different cancers and discuss the possible use of these channels as biomarkers and therapeutic targets to treat cancer.

THE ROLE OF TRP CHANNELS IN CANCER

Among 28 members of the mammalian TRP superfamily grouped into six subfamilies (TRPC (canonical), TRPM (melastatin), TRPV (valinoid), TRPA (ankyrin-like), TRPP (polycystin), and TRPML (mucolipin), over 10 members were reported to have a role in cancer [18]. Further, we discuss specific roles of various TRP channels in different cancers, categorized by TRP subfamily. The Role of TRP Channels in Cancer 407 TRPM Channels TRPM1 (melastatin), the founding member of TRPM subfamily, was originally identified as a melanoma metastasis suppressor gene based on its decreased expression in metastatic mouse melanoma cell lines [19]. TRPM1 is involved in the regulation of calcium homeostasis in melanocytes and mediate an endogenous current that can be suppressed by TRPM1 knockdown [20,21]. Moreover, it was shown that ultraviolet B radiation could regulate the expression of TRPM1 [20]. Also, TRPM1 expression has been linked to melanocyte proliferation and differentiation in vitro. TRPM1 expression is significantly lower in rapidly proliferating melanocytes compared to the differentiated melanocytes [20]. TRPM1 is alternatively spliced, resulting in the production of long and short isoforms [21–23]. Full-length TRPM1 (1533 amino acids) localizes to the plasma membrane and mediates Ca2+ entry when expressed in human embryonic kidney cells, whereas a cytosolic N-terminal truncation variant (500 amino acids) interacts directly with and suppresses the activity of full-length TRPM1. This suppression seems to result from the inhibition of translocation of TRPM1 to the plasma membrane [24]. Thus, alternative splicing of TRPM1 can potentially affect melanoma progression. It was reported that TRPM1 is expressed at high levels in poorly metastatic variants of B16-F1 melanoma cell line, and its expression was much less in highly metastatic B16-F10 melanoma cell line [19]. Moreover, Duncan and colleagues demonstrated high TRPM1 levels in normal human epidermal melanocytes and benign melanocytic nevi, whereas in primary melanomas TRPM1 expression was decreased with virtually no expression detected in metastatic melanoma [19]. Further, TRPM1 mRNA expression appears to inversely correlate with melanocytic tumor progression, melanoma tumor thickness, and the potential for melanoma metastasis [22,25,26]. These patterns of TRPM1 expression have led to the suggestion that TRPM1 could serve as a valuable diagnostic and prognostic marker for the development and progression of melanomas [27]. TRPM2 is a chanzyme, that is, an ion channel with enzyme activity. Its structure contains an enzymatic region with ADP-ribose (ADPR)-hydrolase activity [28]. TRPM2 is widely expressed in mammalian cell types, including neurons [29], immune cells [30], insulin-secreting cells [31], and neutrophil granulocytes [32]. It is thought that the principal physiological role of TRPM2 is to control cytokine release in human monocytes, including tumor necrosis factor-alpha (TNFα) [33], insulin secretion [31], and others. TRPM2 is activated by a number of signals, such as intracellular ADPR, hydrogen peroxide, and TNFα [34–36]. One of the most remarkable features of TRPM2 is its modulation by oxidative stress [34]. Recently it has been shown that TRPM2 could play a role in different cancers. It has been reported that insertion of TRPM2 into human glioblastoma cells facilitates hydrogen peroxide-induced cell death with no effect on proliferation, migration, and invasion [37]. In melanoma cell lines, TRPM2 locus has been found to transcribe TRPM2-AS (antisense) and TRPM2-TE (tumor-enriched dominant negative) transcripts, in addition to full-length TRPM2 [38]. Both TRPM2-AS and TRPM2-TE transcripts are up-regulated in melanoma and several other cancers, and thus, they down-regulate full-length TRPM2. Functional knockout of TRPM2-TE or overexpression of wild-type TRPM2 increases melanoma susceptibility to apoptosis and necrosis. Thus, restoration of full-length TRPM2 activity in cancer could be an attractive therapeutic opportunity. Interestingly, a somewhat different role of TRPM2 was 408 22. TRP Channels in Cancer demonstrated in prostate cancer [39]. siRNA-mediated knockdown of TRPM2 inhibits the growth of prostate cancer cells but not of noncancerous cells. This is due to the difference in its subcellular localization. In noncancerous cells TRPM2 is homogenously located near the plasma membrane and in the cytoplasm, whereas in the cancerous cells, a significant amount of the TRPM2 protein is located in the nuclei. Thus, it was suggested that TRPM2 is essential for prostate cancer cell proliferation and may be a potential target for the selective treatment of prostate cancer [39]. TRPM4 and TRPM5 are closely related channels, which are impermeable for calcium, in contrast to all other TRPs [40,41]. TRPM4 is mostly expressed in the pancreas, heart, and placenta, whereas TRPM5 is detected in the lungs, testis, tongue, digestive system, and brain [42]. It was reported that TRPM4 levels are elevated in prostate cancer, diffuse large B-cell non-Hodgkin lymphoma, and cervical cancer [43–45]. Further, it was demonstrated that TRPM4 enhances cell proliferation of HeLa cells, a cervical cancer-derived cell line, through up-regulation of the β-catenin signaling pathway [46]. In a recent study on the role of genetic polymorphisms in immune response genes in the development of childhood leukemia, Han and coauthors suggested that some genetic variants of TRPM5 are associated with the risk of childhood leukemia [47]. It is worthwhile to note that the Trpm5 gene is located on chromosome 11, aberrations of which have been linked to a variety of malignances including leukemia, ovarian cancer, and rhabdoid tumors [48–50]. Moreover, TRPM5 mRNA was found to be expressed in a large proportion of Wilms’ tumors and rhabdomyosarcomas [49]. TRPM7 represents a Ca2+-permeable nonselective cation channel with enzyme activity. It contains an atypical serine/threonine protein kinase within the C-terminal domain [51]. It is ubiquitously expressed and involved in the regulation of cellular magnesium homeostasis [52,53]. TRPM7 has been shown to regulate lymphocyte survival [54], anoxic neuronal cell death [55], cell volume in human epithelial cells [56], cell adhesion in neuroblastoma [57], as well as osteoblast proliferation and migration [58]. In cancer, TRPM7 has been reported to be overexpressed in high-grade breast cancer samples. Moreover, its expression positively correlates with tumor size [59], which makes it the putative diagnostic/prognostic marker for the development and progression of breast cancer. Silencing of TRPM7 was shown to inhibit growth and proliferation of human head and neck squamous carcinoma cell lines [60], human retinoblastoma cells [61], as well as breast cancer cells MCF-7 [59]. Recently it was reported that high levels of TRPM7 expression independently predict poor outcome in breast cancer patients and that it is functionally required for metastasis formation in a mouse xenograft model of human breast cancer [62]. Mechanistic studies revealed that TRPM7 regulates myosin II-based cellular tension, thereby modifying focal adhesion number, cell-cell adhesion, and polarized cell movement [62]. Another group of scientists suggested that TRPM7 mediates breast cancer cell migration and invasion through the mitogen-activated protein kinase (MAPK) pathway [63]. The importance of TRPM7 for human nasopharyngeal carcinoma cells migration [64], as well as for proliferation, migration, and invasion of pancreatic adenocarcinoma cells has also been reported [65]. TRPM8, another member of TRPM subfamily, was first cloned from the human prostate as a prostate-specific gene up-regulated in cancer [66]. Later, it was shown that TRPM8 has a major role in the cold sensation in sensory neurons [67,68]. In normal tissues, the expression of the channel could be detected in a subpopulation of primary sensory neurons [67,68], as The Role of TRP Channels in Cancer 409 well as in the male reproductive system [66,69–71]. In situ hybridization analysis showed that TRPM8 mRNA expression was at moderate levels in normal prostate tissue and appears to be elevated in prostate cancer [66]. Other than prostate, TRPM8 mRNA was expressed in a number of primary tumors of breast, colon, lung, and skin origin, whereas transcripts encoding TRPM8 were hardly detected or not detected in the corresponding normal human tissues [66]. Moreover, a significant difference in the expression level of TRPM8 mRNA between malignant and nonmalignant tissue specimens has been detected in prostate cancer tumors [70]. It was proposed that in prostate cancer cells TRPM8 could form a calcium-permeable channel at both plasma membrane and ER [72–75]. TRPM8 is subjected to alternative splicing, which generates several splice variants of the full-length and truncated forms. The full-length isoform is expressed at the plasma membrane, whereas the short isoform is localized to ER, where it forms a Ca2+-release channel [72,74,75]. TRPM8 localization and activity was reported to be regulated depending on the differentiation and oncogenic status of prostate cancer cells [72,75]. Thus, only highly differentiated prostate cancer cells expressed functional plasmalemmal TRPM8 channels. This was further confirmed by the finding that the TRPM8-mediated current density was significantly higher in prostate cancer cells obtained from in situ prostate cancer biopsies than in normal prostate or benign prostate hyperplasia cells. However, following dedifferentiation and loss of the luminal secretory phenotype, activity of plasmalemmal TRPM8 was abolished. In contrast, the ER-TRPM8 remained functional regardless of the differentiation status of prostate cells [72]. This differential regulation of TRPM8 activity has been explained by the complex regulation of the TRPM8 isoforms by the androgen receptor [73,75]. Indeed, it has been suggested that TRPM8 is directly regulated by androgen receptor in both normal and cancer prostate cells [73,75]. It appears that Trpm8 gene expression requires a functional androgen receptor and is down-regulated in cells losing androgen sensitivity [73,76]. Moreover, overexpression of androgen receptor in PNT1A cells (human postpubertal prostate normal, immortalized with SV40), that normally lack it, induced TRPM8 expression [73]. TRPM8 overexpression and overactivity in androgen-dependent prostate cancer was reported to correlate with the increased cell growth [77]. On the other hand, antiandrogen therapy as well as transition to androgen independence is associated with the loss of TRPM8 in prostate cancer cells, suggesting that the loss of TRPM8 could be a potential marker of the transition to a more advanced form of the prostate cancer [76]. Furthermore, it has been shown that pharmacological activation of TRPM8 as well as silencing of the channel with siRNA in human prostate carcinoma LNCaP cells negatively influences cell survival, likely by perturbing Ca2+ homeostasis [75]. However, plasma membrane TRPM8 might have a protective effect because stimulation of TRPM8 activity by PSA (prostate-specific antigen) reduced prostate cancer cell motility [78]. Therefore, the gradual loss of the plasmalemmal TRPM8 during tumor progression to late, invasive stages may be an adaptive mechanism of prostate cancer epithelial cells to reduce constant stimulation of the PSA/TRPM8 pathway [78]. Along with prostate cancer, the role of TRPM8 has been also demonstrated in other cancers. Activation of TRPM8 with menthol was reported to decrease the viability of melanoma cells [79]. In breast cancer, TRPM8 is functional at the plasma membrane and expressed in early primitive breast cancers presenting a well-differentiated status [80]. Further, TRPM8 was shown to be overexpressed and required for proliferation of pancreatic adenocarcinoma cells [65]. In human bladder cancer cells, menthol induces cell death via the TRPM8 channel [81]. 410 22. TRP Channels in Cancer

Thus, TRPM8 seems to have a great potential to be used as a diagnostic marker and/or therapeutic target in the treatment of different cancers.

TRPV Channels The TRPV1 channel, also known as capsaicin receptor, was the first discovered TRPV channel. TRPV1 was originally identified in sensory neurons as a heat-activated ion channel, which functions as a transducer of painful thermal stimuli in vivo [82]. Increased expression of TRPV1 has been found in different cancers, including cancers of prostate, colon, pancreas, and bladder [83–86]. High expression of TRPV1 was reported to be associated with a better prognosis of patients with hepatocellular carcinoma [87]. A progressive loss of TRPV1 expression has been found in the urothelium, as transitional cell carcinoma stage increased and cell differentiation was lower [85]. In glioma, TRPV1 expression was reported to inversely correlate with tumor grade with a complete loss observed in 93% of high grade (IV) glioblastomas [88]. Also, it was reported that TRPV1 suppresses skin carcinogenesis [89]. These results suggest that TRPV1 might function as a tumor suppressor, and down-regulation of TRPV1 mRNA expression may represent an independent negative prognostic factor for cancer patients. Moreover, TRPV1 has been proposed to be involved in cancer pain transduction [84,90]. TRPV2 exhibits 50% sequence identity to TRPV1 and is activated by noxious heat (> 53 °C) [91]. TRPV2 is also expressed in sensory neurons, as well as in other tissues, including smooth muscle cells and GI tract [12]. Overexpression of TRPV2 has been reported in several cancers and cancer cell lines [92]. In prostate cancer, TRPV2 expression levels are higher in metastatic cancers (stage M1) compared with primary solid tumors (stages T2a and T2b). This property makes TRPV2 a potential marker for aggressive advanced prostate cancer [93]. TRPV2 was also demonstrated to promote prostate cancer cell migration by regulating the expression of invasive enzymes MMP2, MMP9, and cathepsin B [93]. In liver cancers, TRPV2 demonstrates high expression levels in human hepatocarcinoma cells (HepG2) [94]. Expression of TRPV2 at both the mRNA and protein levels was also shown to be increased in cirrhotic livers compared with chronic hepatitis, suggesting that it might be a prognostic marker of patients with hepatocellular carcinoma [95]. Moderately and well-differentiated liver tumors were also characterized by the increased TRPV2 expression compared with that of poorly differentiated tumors [95]. In bladder cancer, enhanced TRPV2 mRNA and protein expression was found in high-grade urothelial carcinoma specimens and cell lines. Both the full-length TRPV2 and a short splice-variant were detected in normal human urothelial cells and normal bladder specimens, whereas a progressive loss of short-TRPV2 accompanied by a marked increase of full-length TRPV2 expression was found in high-grade urothelial carcinoma tissues [96]. TRPV2 mRNA was reported to be abundantly expressed in a poorly differentiated urothelial carcinoma T24 cells. Moreover, the TRPV2 agonist cannabidiol induced urothelial carcinoma cell death via apoptosis, suggesting that TRPV2 could serve as a potential therapeutic target for the treatment of bladder cancer [97]. In contrast, TRPV2 mRNA and protein expression progressively decline in high-grade glioma tissues as histological grade increases compared to benign astrocyte tissues [98]. Furthermore, TRPV2 channel negatively controls glioma cell survival and proliferation [98]. TRPV6 channel was first identified as a channel-like transporter mediating intestinal calcium absorption in the duodenum [99]. TRPV6 is expressed in a variety of tissues with The Role of TRP Channels in Cancer 411 predominant expression in the duodenum, jejunum, colon, and kidneys. The expression of TRPV6 is substantially increased in prostate cancer tissue as well as human carcinomas of the colon, breast, thyroid, and ovary (see Ref. [100] for review). In prostate cancer, TRPV6 expression correlates with tumor grade. TRPV6 mRNA levels are elevated in prostate cancer specimens in comparison to benign prostatic hyperplasia (BPH) specimens and positively correlate with the Gleason grade [101,102]. Thus, TRPV6 has been suggested as a prognostic marker and a promising target for new therapeutic strategies to treat advanced prostate cancer [101,102]. TRPV6 was also shown to be directly involved in the control of LNCaP cell proliferation, as siRNA-mediated TRPV6 silencing slowed down the proliferation rate, decreased the accumulation of LNCaP cells in the S phase of the cell cycle, and lowered the expression of the proliferating cell nuclear antigen [103]. An elevated expression of TRPV6 was also detected in colon carcinoma cells, where siRNA-mediated TRPV6 knockdown inhibited proliferation and induced apoptosis [104]. TRPV6 is mainly overexpressed in the invasive breast cancer cells and the selective silencing of TRPV6-inhibited human prostate carcinoma MDA-MB-231 cell migration and invasion, as well as MCF-7 migration [105]. Breast cancer cell proliferation was also reported to be regulated by TRPV6 [106]. Thus, it seems that in many cancers TRPV6 represents an oncogene; however, this is not true for all cancers. Indeed, no TRPV6 up-regulation was observed in pancreatic cancer. Furthermore, TRPV6 has been demonstrated to mediate capsaicin-induced apoptosis in gastric cancer cells [107].

TRPC Channels TRPC1 is a nonselective ubiquitously expressed cation channel [108]. TRPC1 has been proposed to be involved in store-operated calcium entry in cooperation with Orai1 and STIM1 [109,110]. TRPC1 contributes to multiple important physiological processes and provides an important route for agonist-, growth factor-, and protein kinase C (PKC)-induced Ca2+ entry in a variety of cell types (see Ref. [12] for review). TRPC1 has been also shown to have a role in cancer cell migration and proliferation. Thus, the TRPC1 channel was reported to be essential for glioma cell chemotaxis in response to stimuli, such as Epidermal growth factor (EGF) [111]. Another report has shown that TRPC1 depletion induced cell growth arrest by causing incomplete cytokinesis in gliomas [112]. Further, it was suggested that extracellular signal-regulated kinases 1 and 2 and TRPC1 channels are required for calcium-sensing receptor-stimulated MCF-7 breast cancer cell proliferation [113]. The inhibitory effect of TRPC1 depletion on proliferation was also demonstrated in non-small-cell lung carcinoma cells [114]. TRPC1 has been also associated with proliferative phenotype in breast cancer and was demonstrated to be overexpressed in human breast cancer epithelial primary culture [105]. Recently, the role of TRPC1, C3, C4, and C6 channels in lung and ovarian cancers was demonstrated [115,116]. The expression of TRPC1, C3, C4, and C6 was correlated to the differentiation grade of both non- small-cell lung cancer and ovarian cancer. Moreover, siRNA-mediated knockdown of TRPC1, C3, C4, or C6 channels inhibited cell proliferation in both cancers [115,116]. Recently, a functional expression of TRPC3 has been demonstrated in MCF-7 breast cancer cells. Here, inhibition of TRPC3 by exogenous polyunsaturated fatty acids consistently attenuated breast cancer cell proliferation and migration [117]. Also, high levels of TRPC3 expression in tumor cells have been proposed as an independent predictor of a better prognosis in patients with adenocarcinoma of the lung [118]. 412 22. TRP Channels in Cancer

The role of another member of TRPC subfamily, TRPC6, has been described in multiple cancers, including prostate [119,120], liver [121], renal [122], and breast cancer [123,124]. Thus, alpha1-adrenergic receptor signaling was reported to require the coupled activation of TRPC6 channels and nuclear factor of activated T-cells (NFAT) to promote proliferation of primary human prostate cancer epithelial cells, thereby suggesting TRPC6 as a novel potential therapeutic target [119]. In liver, TRPC6 expression levels were higher in tumors compared to normal he- patocytes. Moreover, TRPC6 overexpression stimulated proliferation of the human hepatoma cell line Huh-7 [121]. Expression of TRPC6 was demonstrated to be markedly increased in renal cell carcinoma specimens and was associated with renal cell carcinoma histological grade. The positive regulatory role of TRPC6 in proliferation of renal cell carcinoma ACHN cells has been also emphasized [122]. In breast carcinoma specimens, TRPC6 mRNA and protein were found to be elevated in comparison to normal breast tissue. However, no correlation was found between TRPC6 protein levels and tumor grades or metastases [124]. Moreover, activation of TRPC6 significantly reduced the growth and viability of the breast cancer cell lines [123]. Recently, it was reported that TRPC6 promotes cancer cell migration in head and neck squamous cell carcinomas (HNSCC) [125] and glioblastoma [126]. The increase in TRPC6 levels was demonstrated in HNSCC tumor samples and cancer cell lines. siRNA-mediated knockdown of TRPC6 expression in HNSCC-derived cells dramatically inhibited HNSCC- cell invasion but did not significantly alter cell proliferation [125]. In glioblastoma, TRPC6 has been shown to be required for the development of the aggressive phenotype because knockdown of TRPC6 expression inhibits glioma growth, invasion, and angiogenesis [126].

CLINICAL IMPLICATIONS AND CONCLUDING REMARKS

It’s now clear that TRP channels play an important role in cancer-related processes, including proliferation, differentiation, apoptosis, migration, and angiogenesis. Thus, it’s not surprising that a number of TRP channels have been proposed as potential anticancer therapeutic targets and/or diagnostic tools. Recently, synthetic vanilloid arvanil was proposed as therapeutics for high-grade astro- cytoma [127]. By activating the ER-localized TRPV1 channel, which is significantly overexpressed in this type of cancer cells, it induces ER-stress thereby promoting tumor cell death [127]. In 2009, the small molecule TRPM8 agonist D-3263 was presented as a potential treatment for BPH alone or in combination with finasteride, a current treatment for BPH. Further, high-affinity TRPM8 agonists WS-12 and WS-12F (WS-12 compound with incorporated fluorine) were proposed as potential drugs for prostate cancer imaging and therapy [128]. Moreover, it has been suggested that incorporation of a radioisotope into WS-12 could allow radiodiagnostics as well as radiotherapy of prostate cancer with all the advantages of selective targeting of TRPM8-expressing cells [128]. At present, there is a huge need in selective pharmacological modulators for most of the TRP channels, and there are no doubts that design of such specific molecules will greatly advance the field toward practical clinical implications of the TRP channel-based anticancer therapies. Given that calcium overload is detrimental to all cells, prolonged stimulation or overexpression of calcium-permeable channels in cancer cells will lead to substantial increase in cytoplasmic calcium followed by apoptotic/necrotic cell death [129]. In this respect, all the REFERENCES 413 calcium-permeable TRP channels could potentially represent efficient anticancer therapeutic tools, provided that specific approaches will be implemented for selective overexpression (under control of tissue/cell type specific promoter) or targeting of the channel of interest. Indeed, most of the TRP channels have broad expression patterns and thus are not cancer- specific. Therefore, selective targeting is vitally important to prevent toxicity to normal cells induced by the pharmacological impairment of channel function. Given that the expression level of certain TRP channels differs substantially between normal and cancerous tissues, these channels could be considered as potential diagnostic markers. For example, TRPV6 expression correlates with tumor grades in many tissues, which has led to the suggestion that TRPV6 represents a reliable tool to predict the clinical outcome of different cancers [100]. Nevertheless, further studies are needed to find out whether TRPV6 represents an oncogenic ion channel, or its expression is epigenetically altered during cancer progression. Furthermore, inverse correlation between TRPM1 mRNA expression and melanoma metastatic potential suggests that TRPM1 could serve as a diagnostic/prognostic marker for the development and progression of melanomas [27]. Expression levels of TRPM8 have been also reported to be useful when staging prostate cancer [70,72]. Other TRP channels, including TRPM7, TRPV1, TRPV2, TRPC1, and TRPC3, also show a great potential as diagnostic/prognostic markers in different cancers. However, it should be noted that the use of ion channels for diagnostic purposes involves tissue biopsies, as cellular ion channels normally cannot be detected in blood samples. Thus, the antibody-based approaches should be considered. Indeed, TRP channels localized on plasma membrane could be subjected to antibody-based targeting that can be particularly useful in the case of channel up-regulation in cancer. Fluorescently labeled antibodies could be used for tumor visualization, whereas radionuclide-labeled antibodies provide a powerful therapeutic tool [8]. Importantly, as the contribution of specific TRP channels to cancer-related processes could vary depending on cancer type and stage, the specific therapeutic/diagnostic approach should be implemented for each particular case. Thus, the targeting of TRP channels in cancer treatment should not be regarded as a general approach to treat cancer, but rather as a specific tool for individualized cancer therapy. Overall, TRP channels have emerged as promising anticancer therapeutic targets and diagnostic/prognostic markers. However, despite the essential progress achieved in this field, there is still a need in highly selective pharmacological modulators, novel drug delivery techniques, as well as siRNA transfection technologies to selectively target TRP channels in cancer cells without significant adverse effects.

References [1] Ferlay J, Soerjomataram I, Ervik M, Dikshit R, Eser S, Mathers C, et al. GLOBOCAN 2012 v1.0, cancer incidence and mortality worldwide: IARC Cancerbase no. 11 [internet]. Lyon, France: International Agency for Research on Cancer; 2013. Available from: http://globocan.iarc.fr, accessed on 23/02/2014. [2] American Cancer Society. Cancer facts & figures 2013. Atlanta: American Cancer Society; 2013. [3] Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011;144(5):646–74. [4] Prevarskaya N, Ouadid-Ahidouch H, Skryma R, Shuba Y. Remodelling of Ca2+ transport in cancer: how it contributes to cancer hallmarks? Philos Trans R Soc Lond B Biol Sci 2014;369(1638):20130097. [5] Prevarskaya N, Skryma R, Shuba Y. Targeting Ca(2)(+) transport in cancer: close reality or long perspective? Expert Opin Ther Targets 2013;17(3):225–41. 414 22. TRP Channels in Cancer

[6] Berridge MJ, Lipp P, Bootman MD. The versatility and universality of calcium signalling. Nat Rev Mol Cell Biol 2000;1(1):11–21. [7] Monteith GR, McAndrew D, Faddy HM, Roberts-Thomson SJ. Calcium and cancer: targeting Ca2+ transport. Nat Rev Cancer 2007;7(7):519–30. [8] Prevarskaya N, Skryma R, Shuba Y. Calcium in tumour metastasis: new roles for known actors. Nat Rev Cancer 2011;11(8):609–18. [9] Monteith GR, Davis FM, Roberts-Thomson SJ. Calcium channels and pumps in cancer: changes and consequences. J Biol Chem 2012;287(38):31666–73. [10] Berridge MJ, Bootman MD, Roderick HL. Calcium signalling: dynamics, homeostasis and remodelling. Nat Rev Mol Cell Biol 2003;4(7):517–29. [11] Rizzuto R, De Stefani D, Raffaello A, Mammucari C. Mitochondria as sensors and regulators of calcium signalling. Nat Rev Mol Cell Biol 2012;13(9):566–78. [12] Pedersen SF, Owsianik G, Nilius B. TRP channels: an overview. Cell Calcium 2005;38(3-4):233–52. [13] Bernardi P, von Stockum S. The permeability transition pore as a Ca(2+) release channel: new answers to an old question. Cell Calcium 2012;52(1):22–7. [14] Flourakis M, Prevarskaya N. Insights into Ca2+ homeostasis of advanced prostate cancer cells. Biochim Biophys Acta 2009;1793(6):1105–9. [15] Decuypere JP, Bultynck G, Parys JB. A dual role for Ca(2+) in autophagy regulation. Cell Calcium 2011;50(3):242–50. [16] Dubois C, Vanden Abeele F, Prevarskaya N. Targeting apoptosis by the remodelling of calcium-transporting proteins in cancerogenesis. FEBS J 2013;280(21):5430–40. [17] Prevarskaya N, Skryma R, Shuba Y. Ion channels and the hallmarks of cancer. Trends Mol Med 2010;16(3):107–21. [18] Shapovalov G, Lehen’kyi V, Skryma R, Prevarskaya N. TRP channels in cell survival and cell death in normal and transformed cells. Cell Calcium 2011;50(3):295–302. [19] Duncan LM, Deeds J, Hunter J, Shao J, Holmgren LM, Woolf EA, et al. Down-regulation of the novel gene melastatin correlates with potential for melanoma metastasis. Cancer Res 1998;58(7):1515–20. [20] Devi S, Kedlaya R, Maddodi N, Bhat KM, Weber CS, Valdivia H, et al. Calcium homeostasis in human melanocytes: role of transient receptor potential melastatin 1 (TRPM1) and its regulation by ultraviolet light. Am J Physiol Cell Physiol 2009;297(3):C679–87. [21] Oancea E, Vriens J, Brauchi S, Jun J, Splawski I, Clapham DE. TRPM1 forms ion channels associated with melanin content in melanocytes. Sci Signal 2009;2(70):ra21. [22] Fang D, Setaluri V. Expression and up-regulation of alternatively spliced transcripts of melastatin, a melanoma metastasis-related gene, in human melanoma cells. Biochem Biophys Res Commun 2000;279(1):53–61. [23] Zhiqi S, Soltani MH, Bhat KM, Sangha N, Fang D, Hunter JJ, et al. Human melastatin 1 (TRPM1) is regulated by MITF and produces multiple polypeptide isoforms in melanocytes and melanoma. Melanoma Res 2004;14(6):509–16. [24] Xu XZ, Moebius F, Gill DL, Montell C. Regulation of melastatin, a TRP-related protein, through interaction with a cytoplasmic isoform. Proc Natl Acad Sci USA 2001;98(19):10692–7. [25] Deeds J, Cronin F, Duncan LM. Patterns of melastatin mRNA expression in melanocytic tumors. Hum Pathol 2000;31(11):1346–56. [26] Duncan LM, Deeds J, Cronin FE, Donovan M, Sober AJ, Kauffman M, et al. Melastatin expression and prognosis in cutaneous malignant melanoma. J Clin Oncol 2001;19(2):568–76. [27] Hammock L, Cohen C, Carlson G, Murray D, Ross JS, Sheehan C, et al. Chromogenic in situ hybridization analysis of melastatin mRNA expression in melanomas from American Joint Committee on Cancer stage I and II patients with recurrent melanoma. J Cutan Pathol 2006;33(9):599–607. [28] Perraud AL, Fleig A, Dunn CA, Bagley LA, Launay P, Schmitz C, et al. ADP-ribose gating of the calcium- permeable LTRPC2 channel revealed by Nudix motif homology. Nature 2001;411(6837):595–9. [29] Olah ME, Jackson MF, Li H, Perez Y, Sun HS, Kiyonaka S, et al. Ca2 + -dependent induction of TRPM2 currents in hippocampal neurons. J Physiol 2009;587(Pt 5):965–79. [30] Sano Y, Inamura K, Miyake A, Mochizuki S, Yokoi H, Matsushime H, et al. Immunocyte Ca2+ influx system mediated by LTRPC2. Science 2001;293(5533):1327–30. [31] Togashi K, Hara Y, Tominaga T, Higashi T, Konishi Y, Mori Y, et al. TRPM2 activation by cyclic ADP-ribose at body temperature is involved in insulin secretion. EMBO J 2006;25(9):1804–15. [32] Heiner I, Eisfeld J, Halaszovich CR, Wehage E, Jungling E, Zitt C, et al. Expression profile of the transient receptor potential (TRP) family in neutrophil granulocytes: evidence for currents through long TRP channel 2 induced by ADP-ribose and NAD. Biochem J 2003;371(Pt 3):1045–53. REFERENCES 415

[33] Wehrhahn J, Kraft R, Harteneck C, Hauschildt S. Transient receptor potential melastatin 2 is required for lipopolysaccharide-induced cytokine production in human monocytes. J Immunol 2010;184(5):2386–93. [34] Hara Y, Wakamori M, Ishii M, Maeno E, Nishida M, Yoshida T, et al. LTRPC2 Ca2 + -permeable channel activated by changes in redox status confers susceptibility to cell death. Mol Cell 2002;9(1):163–73. [35] McHugh D, Flemming R, Xu SZ, Perraud AL, Beech DJ. Critical intracellular Ca2+ dependence of transient receptor potential melastatin 2 (TRPM2) cation channel activation. J Biol Chem 2003;278(13):11002–6. [36] Wehage E, Eisfeld J, Heiner I, Jungling E, Zitt C, Luckhoff A. Activation of the cation channel long transient receptor potential channel 2 (LTRPC2) by hydrogen peroxide. A splice variant reveals a mode of activation independent of ADP-ribose. J Biol Chem 2002;277(26):23150–6. [37] Ishii M, Oyama A, Hagiwara T, Miyazaki A, Mori Y, Kiuchi Y, et al. Facilitation of H2O2-induced A172 human glioblastoma cell death by insertion of oxidative stress-sensitive TRPM2 channels. Anticancer Res 2007;27(6B):3987–92. [38] Orfanelli U, Wenke AK, Doglioni C, Russo V, Bosserhoff AK, Lavorgna G. Identification of novel sense and antisense transcription at the TRPM2 locus in cancer. Cell Res 2008;18(11):1128–40. [39] Zeng X, Sikka SC, Huang L, Sun C, Xu C, Jia D, et al. Novel role for the transient receptor potential channel TRPM2 in prostate cancer cell proliferation. Prostate Cancer Prostatic Dis 2010;13(2):195–201. [40] Hofmann T, Chubanov V, Gudermann T, Montell C. TRPM5 is a voltage-modulated and Ca(2+)-activated monovalent selective cation channel. Curr Biol 2003;13(13):1153–8. [41] Launay P, Fleig A, Perraud AL, Scharenberg AM, Penner R, Kinet JP. TRPM4 is a Ca2+-activated nonselective cation channel mediating cell membrane depolarization. Cell 2002;109(3):397–407. [42] Ullrich ND, Voets T, Prenen J, Vennekens R, Talavera K, Droogmans G, et al. Comparison of functional properties of the Ca2 + -activated cation channels TRPM4 and TRPM5 from mice. Cell Calcium 2005;37(3):267–78. [43] Ashida S, Nakagawa H, Katagiri T, Furihata M, Iiizumi M, Anazawa Y, et al. Molecular features of the transition from prostatic intraepithelial neoplasia (PIN) to prostate cancer: genome-wide gene-expression profiles of prostate cancers and PINs. Cancer Res 2004;64(17):5963–72. [44] Narayan G, Bourdon V, Chaganti S, Arias-Pulido H, Nandula SV, Rao PH, et al. Gene dosage alterations revealed by cDNA microarray analysis in cervical cancer: identification of candidate amplified and overexpressed genes. Genes Chromosomes Cancer 2007;46(4):373–84. [45] Suguro M, Tagawa H, Kagami Y, Okamoto M, Ohshima K, Shiku H, et al. Expression profiling analysis of the CD5+ diffuse large B-cell lymphoma subgroup: development of a CD5 signature. Cancer Sci 2006;97(9):868–74. [46] Armisen R, Marcelain K, Simon F, Tapia JC, Toro J, Quest AF, et al. TRPM4 enhances cell proliferation through up-regulation of the beta-catenin signaling pathway. J Cell Physiol 2011;226(1):103–9. [47] Han S, Koo HH, Lan Q, Lee KM, Park AK, Park SK, et al. Common variation in genes related to immune response and risk of childhood leukemia. Hum Immunol 2012;73(3):316–19. [48] Monni O, Knuutila S. 11q deletions in hematological malignancies. Leuk Lymphoma 2001;40(3-4):259–66. [49] Prawitt D, Enklaar T, Klemm G, Gartner B, Spangenberg C, Winterpacht A, et al. Identification and characterization of MTR1, a novel gene with homology to melastatin (MLSN1) and the trp gene family located in the BWS-WT2 critical region on chromosome 11p15.5 and showing allele-specific expression. Hum Mol Genet 2000;9(2):203–16. [50] Stronach EA, Sellar GC, Blenkiron C, Rabiasz GJ, Taylor KJ, Miller EP, et al. Identification of clinically relevant genes on chromosome 11 in a functional model of ovarian cancer tumor suppression. Cancer Res 2003;63(24):8648–55. [51] Runnels LW, Yue L, Clapham DE. TRP-PLIK, a bifunctional protein with kinase and ion channel activities. Science 2001;291(5506):1043–7. [52] Ryazanova LV, Rondon LJ, Zierler S, Hu Z, Galli J, Yamaguchi TP, et al. TRPM7 is essential for Mg(2+) homeostasis in mammals. Nat Commun 2010;1:109. [53] Schmitz C, Perraud AL, Johnson CO, Inabe K, Smith MK, Penner R, et al. Regulation of vertebrate cellular Mg2+ homeostasis by TRPM7. Cell 2003;114(2):191–200. [54] Nadler MJ, Hermosura MC, Inabe K, Perraud AL, Zhu Q, Stokes AJ, et al. LTRPC7 is a Mg.ATP-regulated divalent cation channel required for cell viability. Nature 2001;411(6837):590–5. [55] Aarts M, Iihara K, Wei WL, Xiong ZG, Arundine M, Cerwinski W, et al. A key role for TRPM7 channels in anoxic neuronal death. Cell 2003;115(7):863–77. [56] Numata T, Shimizu T, Okada Y. TRPM7 is a stretch- and swelling-activated cation channel involved in volume regulation in human epithelial cells. Am J Physiol Cell Physiol 2007;292(1):C460–7. [57] Clark K, Langeslag M, van Leeuwen B, Ran L, Ryazanov AG, Figdor CG, et al. TRPM7, a novel regulator of actomyosin contractility and cell adhesion. EMBO J 2006;25(2):290–301. 416 22. TRP Channels in Cancer

[58] Abed E, Moreau R. Importance of melastatin-like transient receptor potential 7 and magnesium in the stimulation of osteoblast proliferation and migration by platelet-derived growth factor. Am J Physiol Cell Physiol 2009;297(2):C360–8. [59] Guilbert A, Gautier M, Dhennin-Duthille I, Haren N, Sevestre H, Ouadid-Ahidouch H. Evidence that TRPM7 is required for breast cancer cell proliferation. Am J Physiol Cell Physiol 2009;297(3):C493–502. [60] Jiang J, Li MH, Inoue K, Chu XP, Seeds J, Xiong ZG. Transient receptor potential melastatin 7-like current in human head and neck carcinoma cells: role in cell proliferation. Cancer Res 2007;67(22):10929–38. [61] Hanano T, Hara Y, Shi J, Morita H, Umebayashi C, Mori E, et al. Involvement of TRPM7 in cell growth as a spontaneously activated Ca2+ entry pathway in human retinoblastoma cells. J Pharmacol Sci 2004;95(4):403–19. [62] Middelbeek J, Kuipers AJ, Henneman L, Visser D, Eidhof I, van Horssen R, et al. TRPM7 is required for breast tumor cell metastasis. Cancer Res 2012;72(16):4250–61. [63] Meng X, Cai C, Wu J, Cai S, Ye C, Chen H, et al. TRPM7 mediates breast cancer cell migration and invasion through the MAPK pathway. Cancer Lett 2013;333(1):96–102. [64] Chen JP, Luan Y, You CX, Chen XH, Luo RC, Li R. TRPM7 regulates the migration of human nasopharyngeal carcinoma cell by mediating Ca(2+) influx. Cell Calcium 2010;47(5):425–32. [65] Yee NS, Chan AS, Yee JD, Yee RK. TRPM7 and TRPM8 ion channels in pancreatic adenocarcinoma: potential roles as cancer biomarkers and targets. Scientifica (Cairo) 2012;2012:415158. [66] Tsavaler L, Shapero MH, Morkowski S, Laus R. Trp-p8, a novel prostate-specific gene, is up-regulated in prostate cancer and other malignancies and shares high homology with transient receptor potential calcium channel proteins. Cancer Res 2001;61(9):3760–9. [67] McKemy DD, Neuhausser WM, Julius D. Identification of a cold receptor reveals a general role for TRP channels in thermosensation. Nature 2002;416(6876):52–8. [68] Peier AM, Moqrich A, Hergarden AC, Reeve AJ, Andersson DA, Story GM, et al. A TRP channel that senses cold stimuli and menthol. Cell 2002;108(5):705–15. [69] De Blas GA, Darszon A, Ocampo AY, Serrano CJ, Castellano LE, Hernandez-Gonzalez EO, et al. TRPM8, a ver- satile channel in human sperm. PLoS One 2009;4(6):e6095. [70] Fuessel S, Sickert D, Meye A, Klenk U, Schmidt U, Schmitz M, et al. Multiple tumor marker analyses (PSA, hK2, PSCA, trp-p8) in primary prostate cancers using quantitative RT-PCR. Int J Oncol 2003;23(1):221–8. [71] Stein RJ, Santos S, Nagatomi J, Hayashi Y, Minnery BS, Xavier M, et al. Cool (TRPM8) and hot (TRPV1) receptors in the bladder and male genital tract. J Urol 2004;172(3):1175–8. [72] Bidaux G, Flourakis M, Thebault S, Zholos A, Beck B, Gkika D, et al. Prostate cell differentiation status determines transient receptor potential melastatin member 8 channel subcellular localization and function. J Clin Invest 2007;117(6):1647–57. [73] Bidaux G, Roudbaraki M, Merle C, Crepin A, Delcourt P, Slomianny C, et al. Evidence for specific TRPM8 expression in human prostate secretory epithelial cells: functional androgen receptor requirement. Endocr Relat Cancer 2005;12(2):367–82. [74] Thebault S, Lemonnier L, Bidaux G, Flourakis M, Bavencoffe A, Gordienko D, et al. Novel role of cold/menthol- sensitive transient receptor potential melastatine family member 8 (TRPM8) in the activation of store-operated channels in LNCaP human prostate cancer epithelial cells. J Biol Chem 2005;280(47):39423–35. [75] Zhang L, Barritt GJ. Evidence that TRPM8 is an androgen-dependent Ca2+ channel required for the survival of prostate cancer cells. Cancer Res 2004;64(22):8365–73. [76] Henshall SM, Afar DE, Hiller J, Horvath LG, Quinn DI, Rasiah KK, et al. Survival analysis of genome-wide gene expression profiles of prostate cancers identifies new prognostic targets of disease relapse. Cancer Res 2003;63(14):4196–203. [77] Kiessling A, Fussel S, Schmitz M, Stevanovic S, Meye A, Weigle B, et al. Identification of an HLA-A*0201- restricted T-cell epitope derived from the prostate cancer-associated protein trp-p8. Prostate 2003;56(4):270–9. [78] Gkika D, Flourakis M, Lemonnier L, Prevarskaya N. PSA reduces prostate cancer cell motility by stimulating TRPM8 activity and plasma membrane expression. Oncogene 2010;29(32):4611–16. [79] Yamamura H, Ugawa S, Ueda T, Morita A, Shimada S. TRPM8 activation suppresses cellular viability in human melanoma. Am J Physiol Cell Physiol 2008;295(2):C296–301. [80] Chodon D, Guilbert A, Dhennin-Duthille I, Gautier M, Telliez MS, Sevestre H, et al. Estrogen regulation of TRPM8 expression in breast cancer cells. BMC Cancer 2010;10:212. [81] Li Q, Wang X, Yang Z, Wang B, Li S. Menthol induces cell death via the TRPM8 channel in the human bladder cancer cell line T24. Oncology 2009;77(6):335–41. REFERENCES 417

[82] Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, Julius D. The capsaicin receptor: a heat- activated ion channel in the pain pathway. Nature 1997;389(6653):816–24. [83] Domotor A, Peidl Z, Vincze A, Hunyady B, Szolcsanyi J, Kereskay L, et al. Immunohistochemical distribution of vanilloid receptor, calcitonin-gene related peptide and substance P in gastrointestinal mucosa of patients with different gastrointestinal disorders. Inflammopharmacology 2005;13(1-3):161–77. [84] Hartel M, di Mola FF, Selvaggi F, Mascetta G, Wente MN, Felix K, et al. Vanilloids in pancreatic cancer: potential for chemotherapy and pain management. Gut 2006;55(4):519–28. [85] Lazzeri M, Vannucchi MG, Spinelli M, Bizzoco E, Beneforti P, Turini D, et al. Transient receptor potential vanilloid type 1 (TRPV1) expression changes from normal urothelium to transitional cell carcinoma of human bladder. Eur Urol 2005;48(4):691–8. [86] Sanchez MG, Sanchez AM, Collado B, Malagarie-Cazenave S, Olea N, Carmena MJ, et al. Expression of the transient receptor potential vanilloid 1 (TRPV1) in LNCaP and PC-3 prostate cancer cells and in human prostate tissue. Eur J Pharmacol 2005;515(1-3):20–7. [87] Miao X, Liu G, Xu X, Xie C, Sun F, Yang Y, et al. High expression of vanilloid receptor-1 is associated with better prognosis of patients with hepatocellular carcinoma. Cancer Genet Cytogenet 2008;186(1):25–32. [88] Amantini C, Mosca M, Nabissi M, Lucciarini R, Caprodossi S, Arcella A, et al. Capsaicin-induced apoptosis of glioma cells is mediated by TRPV1 vanilloid receptor and requires p38 MAPK activation. J Neurochem 2007;102(3):977–90. [89] Bode AM, Cho YY, Zheng D, Zhu F, Ericson ME, Ma WY, et al. Transient receptor potential type vanilloid 1 suppresses skin carcinogenesis. Cancer Res 2009;69(3):905–13. [90] Ghilardi JR, Rohrich H, Lindsay TH, Sevcik MA, Schwei MJ, Kubota K, et al. Selective blockade of the capsaicin receptor TRPV1 attenuates bone cancer pain. J Neurosci 2005;25(12):3126–31. [91] Caterina MJ, Rosen TA, Tominaga M, Brake AJ, Julius D. A capsaicin-receptor homologue with a high threshold for noxious heat. Nature 1999;398(6726):436–41. [92] Lehen’kyi V, Prevarskaya N. TRPV2 (transient receptor potential cation channel, subfamily V, member 2). Atlas Genet Cytogenet Oncol Haematol 2012;16(8):563–7. [93] Monet M, Lehen’kyi V, Gackiere F, Firlej V, Vandenberghe M, Roudbaraki M, et al. Role of cationic channel TRPV2 in promoting prostate cancer migration and progression to androgen resistance. Cancer Res 2010;70(3):1225–35. [94] Vriens J, Janssens A, Prenen J, Nilius B, Wondergem R. TRPV channels and modulation by hepatocyte growth factor/scatter factor in human hepatoblastoma (HepG2) cells. Cell Calcium 2004;36(1):19–28. [95] Liu G, Xie C, Sun F, Xu X, Yang Y, Zhang T, et al. Clinical significance of transient receptor potential vanilloid 2 expression in human hepatocellular carcinoma. Cancer Genet Cytogenet 2010;197(1):54–9. [96] Caprodossi S, Lucciarini R, Amantini C, Nabissi M, Canesin G, Ballarini P, et al. Transient receptor potential vanilloid type 2 (TRPV2) expression in normal urothelium and in urothelial carcinoma of human bladder: correlation with the pathologic stage. Eur Urol 2008;54(3):612–20. [97] Yamada T, Ueda T, Shibata Y, Ikegami Y, Saito M, Ishida Y, et al. TRPV2 activation induces apoptotic cell death in human T24 bladder cancer cells: a potential therapeutic target for bladder cancer. Urology 2010;76(2):509e1–7. [98] Nabissi M, Morelli MB, Amantini C, Farfariello V, Ricci-Vitiani L, Caprodossi S, et al. TRPV2 channel negatively controls glioma cell proliferation and resistance to Fas-induced apoptosis in ERK-dependent manner. Carcinogenesis 2010;31(5):794–803. [99] Peng JB, Chen XZ, Berger UV, Vassilev PM, Tsukaguchi H, Brown EM, et al. Molecular cloning and characterization of a channel-like transporter mediating intestinal calcium absorption. J Biol Chem 1999;274(32):22739–46. [100] Lehen’kyi V, Raphael M, Prevarskaya N. The role of the TRPV6 channel in cancer. J Physiol 2012;590(Pt 6):1369–76. [101] Fixemer T, Wissenbach U, Flockerzi V, Bonkhoff H. Expression of the Ca2+-selective cation channel TRPV6 in human prostate cancer: a novel prognostic marker for tumor progression. Oncogene 2003;22(49):7858–61. [102] Peng JB, Zhuang L, Berger UV, Adam RM, Williams BJ, Brown EM, et al. CaT1 expression correlates with tumor grade in prostate cancer. Biochem Biophys Res Commun 2001;282(3):729–34. [103] Lehen’kyi V, Flourakis M, Skryma R, Prevarskaya N. TRPV6 channel controls prostate cancer cell proliferation via Ca(2+)/NFAT-dependent pathways. Oncogene 2007;26(52):7380–5. [104] Peleg S, Sellin JH, Wang Y, Freeman MR, Umar S. Suppression of aberrant transient receptor potential cation channel, subfamily V, member 6 expression in hyperproliferative colonic crypts by dietary calcium. Am J Physiol Gastrointest Liver Physiol 2010;299(3):G593–601. [105] Dhennin-Duthille I, Gautier M, Faouzi M, Guilbert A, Brevet M, Vaudry D, et al. High expression of transient receptor potential channels in human breast cancer epithelial cells and tissues: correlation with pathological parameters. Cell Physiol Biochem 2011;28(5):813–22. 418 22. TRP Channels in Cancer

[106] Bolanz KA, Kovacs GG, Landowski CP, Hediger MA. Tamoxifen inhibits TRPV6 activity via estrogen receptor-independent pathways in TRPV6-expressing MCF-7 breast cancer cells. Mol Cancer Res 2009;7(12):2000–10. [107] Chow J, Norng M, Zhang J, Chai J. TRPV6 mediates capsaicin-induced apoptosis in gastric cancer cells— mechanisms behind a possible new “hot” cancer treatment. Biochim Biophys Acta 2007;1773(4):565–76. [108] Hofmann T, Schaefer M, Schultz G, Gudermann T. Transient receptor potential channels as molecular substrates of receptor-mediated cation entry. J Mol Med (Berl) 2000;78(1):14–25. [109] Cheng KT, Liu X, Ong HL, Ambudkar IS. Functional requirement for Orai1 in store-operated TRPC1-STIM1 channels. J Biol Chem 2008;283(19):12935–40. [110] Kim MS, Zeng W, Yuan JP, Shin DM, Worley PF, Muallem S. Native store-operated Ca2+ influx requires the channel function of Orai1 and TRPC1. J Biol Chem 2009;284(15):9733–41. [111] Bomben VC, Turner KL, Barclay TT, Sontheimer H. Transient receptor potential canonical channels are essential for chemotactic migration of human malignant gliomas. J Cell Physiol 2011;226(7):1879–88. [112] Bomben VC, Sontheimer H. Disruption of transient receptor potential canonical channel 1 causes incomplete cytokinesis and slows the growth of human malignant gliomas. Glia 2010;58(10):1145–56. [113] El Hiani Y, Ahidouch A, Lehen’kyi V, Hague F, Gouilleux F, Mentaverri R, et al. Extracellular signal-regulated kinases 1 and 2 and TRPC1 channels are required for calcium-sensing receptor-stimulated MCF-7 breast cancer cell proliferation. Cell Physiol Biochem 2009;23(4–6):335–46. [114] Tajeddine N, Gailly P. TRPC1 protein channel is major regulator of epidermal growth factor receptor signaling. J Biol Chem 2012;287(20):16146–57. [115] Jiang HN, Zeng B, Zhang Y, Daskoulidou N, Fan H, Qu JM, et al. Involvement of TRPC channels in lung cancer cell differentiation and the correlation analysis in human non-small cell lung cancer. PLoS One 2013;8(6):e67637. [116] Zeng B, Yuan C, Yang X, Atkin SL, Xu SZ. TRPC channels and their splice variants are essential for promoting human ovarian cancer cell proliferation and tumorigenesis. Curr Cancer Drug Targets 2013;13(1):103–16. [117] Zhang H, Zhou L, Shi W, Song N, Yu K, Gu Y. A mechanism underlying the effects of polyunsaturated fatty acids on breast cancer. Int J Mol Med 2012;30(3):487–94. [118] Saito H, Minamiya Y, Watanabe H, Takahashi N, Ito M, Toda H, et al. Expression of the transient receptor potential channel c3 correlates with a favorable prognosis in patients with adenocarcinoma of the lung. Ann Surg Oncol 2011;18(12):3377–83. [119] Thebault S, Flourakis M, Vanoverberghe K, Vandermoere F, Roudbaraki M, Lehen’kyi V, et al. Differential role of transient receptor potential channels in Ca2+ entry and proliferation of prostate cancer epithelial cells. Cancer Res 2006;66(4):2038–47. [120] Yue D, Wang Y, Xiao JY, Wang P, Ren CS. Expression of TRPC6 in benign and malignant human prostate tissues. Asian J Androl 2009;11(5):541–7. [121] El Boustany C, Bidaux G, Enfissi A, Delcourt P, Prevarskaya N, Capiod T. Capacitative calcium entry and transient receptor potential canonical 6 expression control human hepatoma cell proliferation. Hepatology 2008;47(6):2068–77. [122] Song J, Wang Y, Li X, Shen Y, Yin M, Guo Y, et al. Critical role of TRPC6 channels in the development of human renal cell carcinoma. Mol Biol Rep 2013;40(8):5115–22. [123] Aydar E, Yeo S, Djamgoz M, Palmer C. Abnormal expression, localization and interaction of canonical transient receptor potential ion channels in human breast cancer cell lines and tissues: a potential target for breast cancer diagnosis and therapy. Cancer Cell Int 2009;9:23. [124] Guilbert A, Dhennin-Duthille I, Hiani YE, Haren N, Khorsi H, Sevestre H, et al. Expression of TRPC6 channels in human epithelial breast cancer cells. BMC Cancer 2008;8:125. [125] Bernaldo de Quiros S, Merlo A, Secades P, Zambrano I, de Santa Maria IS, Ugidos N, et al. Identification of TRPC6 as a possible candidate target gene within an amplicon at 11q21-q22.2 for migratory capacity in head and neck squamous cell carcinomas. BMC Cancer 2013;13:116. [126] Chigurupati S, Venkataraman R, Barrera D, Naganathan A, Madan M, Paul L, et al. Receptor channel TRPC6 is a key mediator of Notch-driven glioblastoma growth and invasiveness. Cancer Res 2010;70(1):418–27. [127] Stock K, Kumar J, Synowitz M, Petrosino S, Imperatore R, Smith ES, et al. Neural precursor cells induce cell death of high-grade astrocytomas through stimulation of TRPV1. Nat Med 2012;18(8):1232–8. [128] Beck B, Bidaux G, Bavencoffe A, Lemonnier L, Thebault S, Shuba Y, et al. Prospects for prostate cancer imaging and therapy using high-affinity TRPM8 activators. Cell Calcium 2007;41(3):285–94. [129] Zhang L, Brereton HM, Hahn M, Froscio M, Tilley WD, Brown MP, et al. Expression of Drosophila Ca2+ permeable transient receptor potential-like channel protein in a prostate cancer cell line decreases cell survival. Cancer Gene Ther 2003;10(8):611–25. CHAPTER 23 Are Brain TRPs Viable Targets for Curing Neurodegenerative Disorders and Improving Mental Health? Bernd Nilius,1 Arpad Szallasi2,* 1Katholieke Universiteit of Leuven, Department of Cellular and Molecular Medicine, Laboratory of Ion Channel Research and TRP Research Platform Leuven (TRPLe), Campus Gasthuisberg, Leuven, Belgium 2Department of Pathology, Monmouth Medical Center, Long Branch, NJ, USA *Corresponding author: [email protected]

OUTLINE

The Role of TRP Channels in Building the TRPM4 and M5 433 Brain and Regulating its Functions: TRPM7 and TRPM8 434 Introducing the Players 420 The Ankyrin TRPA1 Channel 434 Canonical TRP Channels 420 TRP Channel Dysfunction in Epilepsy 435 TRPC1 420 TRPC Channel and Epilepsy 435 TRPC3 424 TRPV1 as a Potential Target in TRPC4 and TRPC5 425 Epilepsy and Febrile Seizures 436 TRPC6 427 TRPM2 and Juvenile Myoclonic TRPC7 428 Epilepsy 437 Vanilloid TRP Channels 428 TRPV1 428 Cerebellar Ataxia as a “TRP TRPV2 and TRPV3 431 Channelopathy” 437 TRPV4 431 TRP Channels in Neuroprotection Melastatin TRPM Channels 432 and Their Dysfunction in TRPM2 432 Neurodegenerative Disorders 438 TRPM3 433

TRP Channels as Therapeutic Targets 419 © 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/B978-0-12-420024-1.00023-0 420 23. ARE BRAIN TRPs VIABLE TARGETS FOR CURING NEURODEGENERATIVE DISORDERS

Parkinson Disease 438 Rett Syndrome 443 Lou Gehrig’s Disease and Amyotrophic Bipolar Disorder 444 Lateral Sclerosis-Parkinson The “Addictive” TRP Channels 444 Dementia Complex 439 Cocaine Abuse 444 Alzheimer Disease 440 Opioid Addiction 444 Autoimmune Encephalomyelitis and Multiple Sclerosis 441 TRP Channels in Stroke and Traumatic Brain Injury 445 TRP Channels in Psychiatric Stroke 445 Disorders and Mental Retardation 442 Traumatic Brain Injury 446 Anxiety Disorders, Panic Attacks, Conclusions 447 and Depression 442 Schizophrenia 443 References 448

THE ROLE OF TRP CHANNELS IN BUILDING THE BRAIN AND REGULATING ITS FUNCTIONS: INTRODUCING THE PLAYERS

Canonical TRP Channels Every member of the TRPC subfamily is expressed in the brain (Figure 23.1), some (like TRPC1) ubiquitously, whereas others (e.g., TRPC4) in a highly restricted pattern [1]. Further details are out of the scope of this chapter (interested readers are referred to [2–4]). In general, TRPCs (a) regulate neural stem cell proliferation; (b) promote neuronal cell survival in response to neurotrophins such as BDNF; (c) are involved in chemoattraction of neurites, growth cone guidance and regulation of neurite outgrowth spine formation; and (d) are increasingly accepted as major players in synaptic plasticity and synaptogenesis (Figure 23.2; see 4 for a review]. These functions are especially prominent in the developing brain.

TRPC1 TRPC1 IN THE DEVELOPING BRAIN In the CNS, the expression pattern of TRPCs changes during embryogenesis, suggesting that coordinated alterations in TRPC gene expression are involved in brain development. One of the main functions of TRPC1 in the developing brain is thought to be the guidance of axonal growth by directing growth cone turning (see for a review [5–8]). Excitatory synapses in the vertebrate nervous system are characterized by an electron-dense structure, called the postsynaptic density (PSD). Homers are PSD proteins that (in concert with Shank and GRIP) play a pivotal role in building a complex submembrane platform for the anchorage of receptors and scaffolding proteins. By coupling extracellular receptors (such as TRPC1 and metabotropic glutamate receptors, mGluRs) to intracellular receptors (e.g., inositol triphosphate [IP3R] and ryanodine receptors) and signaling pathways, Homers are ideally placed to regulate Ca2+ dynamics within the neural growth cone. Spatiotemporal patterns of Ca2+ are believed to underpin growth cone motility [9–11]. Indeed, calcium imaging of motile growth THE ROLE OF TRP CHANNELS IN BUILDING THE BRAIN AND REGULATING ITS FUNCTIONS 421

Mouse brain Cortex (TRPC1 TRPC3 TRPC4 TRPC5 TRPM2 TRPV1 TRPV2 TRPV4 TRPC1 TRPC3 TRPC4 TRPM5 TRPV1 TRPV4) TRPC1 TRPC3 TRPC4 TRPV1 TRPV2 TRPC5 TRPM2 TRPM5 TRPC5 TRPC6 TRPM2 TRPV1 TRPV4 TRPV4 TRPC1 TRPM7 Hippocampus (TRPV1 Pineal gland TRPC3 TRPC4 TRPC5 TRPC6 TRPV2 TRPV4 TRPC1 TRPM2 TRPM3 TRPC3 TRPC4 TRPC5 TRPM5 TRPC6 TRPM2 TRPM7) Cerebral cortex

Hippocampus Thalamus (TRPV1 Olfactory Fornix Cerebellum TRPV4 TRPC4) bulb thalamus Septum Midbrain

Pons Medulla Amygdala (TRPV1 TRPV1 Hypothalamus Oblongata Spinal TRPC1 TRPC4 TRPC5) TRPV4 TRPC4 cord TRPV1 TRPV4 Hypothalamus TRPM3 Pituitary gland (TRPV1 TRPV4 TRPM3)

Human brain

Cortex (TRPV1 TRPV3 TRPM1 TRPM2 TRPM4 TRPM6 TRPM7 TRPC5)

Caudate nucleus (TRPC3 TRPC5 TRPC6 TRPC7)

Putamen (TRPC3 TRPC5 TRPC6 TRPC7)

Globus pallidus (TRPC6) Hypothalamus (TRPM3 TRPC7)

Amygdala (TRPC4 TRPC5) Substantia nigra Hippocampus (TRPM3 TRPC5 TRPC6) (TRPC4 TRPC5) Cerebellum (TRPM3 TRPC1 TRPC3 TRPC5)

FIGURE 23.1 Schematic representation of TRP channels distribution in the mouse and human brain. Reprinted with permission from Morelli, et al. CNS Neurol Disorder Drug Targets, 2013;12:274–93.

cones revealed that Homer-1 was required for the guidance-cue-induced rise in cytosolic Ca2+. Homer-1 knockdown reversed growth cone turning from attraction to repulsion in response to brain-derived neurotrophic factor (BDNF) and netrin-1 [12]. Of note, in the adult rat brain BDNF promotes neurogenesis and dendritic spine reorganization in the hippocampus [13,14]. BDNF is also an important regulator of striatal neuron survival, differentiation, and plasticity [15]. 422 23. ARE BRAIN TRPs VIABLE TARGETS FOR CURING NEURODEGENERATIVE DISORDERS

Neuronal survival

Trophic factors Neural stem cell proliferation Axon guidance NSCs bFGF TRPC1 BDNF TRPC3&6

BDNF TRPC3&6 (b) Netrin TRPC1

TRPCs AstrocyteNeuronOligodendrocyte (c) Synaptogenesis (a) Neuronal morphogenesis

TRPC5, TRPC6 (d) TRPC3, TRPC6 (e)

FIGURE 23.2 (a) TRPCs promote stem cell proliferation via basic fibroblast growth factor (bFGF). (b). Some TRPCs promote neuronal cell survival in response to brain derived neurotrophic factor (BDNF). (c) Neurotrophins (BDNF) induce chemoattraction, TRPCs are required for proper growth cone turning responses to microscopic gradients, such as netrin-1. (d) Some TRPCs regulate neurite outgrowth and dendrite patterning. (e) TRPCs are required for synaptogenesis and induce spine formation. Reprinted with permission from Tai et al. [5].

In different brain nuclei, distinct factors are thought to guide axon growth. Named after their role in building bone and cartilage, bone morphogenic proteins (BMPs) constitute a family of essential morphogenic agents that regulate tissue architecture throughout the body, including the CNS. BMP signaling involves the transcription factor, SMAD (a mammalian homolog of the C. elegans Small Body Size and the Drosophila Mothers Against Decapentaplegic). Disruption of BMP signaling prevents development of the neural plate. TRPC1 has been implicated in BMP signaling. BMP7 gradient causes a bi-directional turning response in the nerve growth cone. This effect is due to activation of the LIM kinase (so-called for the three transcription factors, Lin-1, Isl-1, and Mec-3, it activates) and the phosphatase, Slingshot. Both enzymes regulate actin dynamics via the actin-depolymerizing factor. This interaction requires the presence of TRPC1; apparently, TRPC1 initiates the Ca2+ signal that activates Slingshot via calcineurin, leading to growth cone repulsion [16]. In the corpus callosum, a Wnt5a (Wingless-type MMTV integration site family, member 5a) gradient guides axons after their midline crossing via RYK (Related to Tyrosine Kinase Receptor) proteins, and propels them into the spinal cord. In dissociated cortical neuron cultures, Wnt5a simultaneously promotes axon outgrowth and repulsion by Ca2+ signaling. The signaling pathway involves both Ca2+ release through IP3Rs and Ca2+ entry through TRPCs. Knock down of the RYK receptor reduced postcrossing (but not precrossing) axon growth by 50% and, importantly, led to misrouting [17]. In addition to RYK proteins, the repulsive growth-cone turning requires the protein, Frizzled (Fz) [18]. It should be mentioned here that (at least in expression systems) TRPC1 and TRPC5 exert opposite effects on neurite growth. In PC12 cells, topical expression of TRPC1 promotes, whereas TRPC5 suppresses, neurite outgrowth. Suppression of TRPC1-induced neurite outgrowth by TRPC5 was due to a marked reduction in the surface expression of TRPC1. THE ROLE OF TRP CHANNELS IN BUILDING THE BRAIN AND REGULATING ITS FUNCTIONS 423

These findings suggest that TRPC1 acts as a scaffold at the cell surface to assemble a signaling complex to stimulate neurite outgrowth [19]. Furthermore, in PC12 cells nerve growth factor (NGF) markedly up-regulates TRPC1 and, conversely, down-regulates TRPC5 expression while promoting neurite outgrowth. Knockdown by shRNA of TRPC1 decreases, whereas shRNA-mediated knockdown of TRPC5 increases NGF-induced neurite extension. Interestingly, overexpression of TRPC6 (which, similar to TRPC1, is up-regulated by NGF) counteracts TRPC1 effects: it slows down neuritogenesis. Confusingly, hyperforin, a specific TRPC6 activator, decreased TRPC6 expression in NGF-differentiated PC12 cells [20]. TRPC effects on axonal guidance likely depend on PIP3 [21]. In the developing brain, TRPC1 is also involved in the proliferation of oligodendrocytes. In oligodendrocyte progenitor cells, Golli proteins (alternative spliced products of the myelin basic protein gene) regulate migration by modulating intracellular Ca2+ levels. Experiments performed with acute brain slice preparations obtained from Golli knockout and Golli- overexpressing mice point to TRPC1 as an important downstream target for Golli [22].

THE ROLE OF TRPC1 IN DETERMINING THE EXCITATION STATE OF HIPPOCAMPAL PYRAMIDAL NEURONS In the hippocampus, the excitability of pyramidal neurons is regulated by an intricate interplay between coincident inputs and the intrinsic electrical activity of the cells. Following firing activity, afterdepolarizations determine the membrane potential of the neuron. There is accumulating evidence that TRPC1 is involved in the generation of small-amplitude (1 mV), long-lasting (for seconds) afterdepolarizations. Such depolarizations are thought to contribute to neural information processing during behavioral tasks [23]. In CA1 pyramidal neurons, mGluR activation leads to intracellular Ca2+ waves. Stimulation of mGluRs triggers a biphasic electrical response composed of a transient hyperpolarization mediated by SK (small conductance Ca2+-dependent) and KCNQ potassium channels, followed by a sustained depolarization attributed to TRPC1 activation [24]. Of note, TRPC1 has also been implicated in hippocampal neurogenesis. In mice, knockdown of Trpc1 markedly reduced adult neural progenitor cell proliferation. (As discussed later, this observation links TRPC1 to cognitive deficits [25]).

ROLE OF TRPC1 IN SYNAPTIC PLASTICITY AND LONG-TERM DEPRESSION (LTD) The evidence linking TRPC1 to synaptic plasticity and LTD is preliminary and speculative. In the brain, presynaptic firing rates are converted to postsynaptic membrane depolarizations by ion channels, especially by ionotropic NMDA and non-NMDA (e.g., AMPA) glutamate receptor. If the firing rate is low (below 20 Hz), ionotropic glutamate receptors may not produce sufficient summation, and other receptors may take over. It was speculated that under these conditions TRPC channels activated by mGluRs could be more effective owing to their slower kinetics [26]. Indeed, downstream of mGluR1 there are only two major sources of Ca2+: TRPC channels and IP3Rs. In rats, the mGluR1-evoked slow currents are mediated by TRPC channels whose inhibition blocks cerebellar LTD [27]. TRPC1 (along with TRPC4) is also expressed in the mouse olfactory bulb where it is believed to contribute to the central synaptic processing at the reciprocal mitral and tufted cell-granule cell microcircuits. Suprathreshold activation of these synapses causes long-lasting depolarization (LLD) in the granule cells, linked to a global dendritic postsynaptic calcium signal. 424 23. ARE BRAIN TRPs VIABLE TARGETS FOR CURING NEURODEGENERATIVE DISORDERS

The LLD is absent in granule cells deficient for the N-methyl-d-aspartate (NMDA) receptor subunit NR1. Interestingly, the LLD is also absent in granule cells from mice deficient for both TRPC1/C4 (double knockouts). The deletion of either TRPC1 or TRPC4 results in only a partial reduction of the LLD [28].

TRPC1 IN THERMOREGULATION AND FOOD INTAKE Histamine influences body temperature by interacting at H1 and H3 receptors expressed in distinct subpopulations of thermoregulatory neurons in the median preoptic nucleus. In these cells, single-cell RT-PCR revealed the expression of TRPC1 (and also TRPC5 and TRPC7) channels. Histamine activates a cationic inward current to increase the intracellular Ca2+ concentration. This current has two components, a transient current as well as a sustained one. The sustained component was blocked by intracellular application of a TRPC1-blocking antibody, identifying TRPC1 as a downstream target for histamine receptors [29]. Pro-opiomelanocortin (POMC) neurons of hypothalamic arcuate nucleus regulate food intake, energy homeostasis, and glucose metabolism. A subpopulation of POMC neurons (distinct from those activated by leptins) respond to meta-chlorophenylpiperazine (mCPP), a psychoactive 5-HT receptor agonist that suppresses food intake in rats. It was hypothesized that TRPC channels are a pivotal downstream target for 5-HT receptor activation. By contrast, in ventral premammillary nucleus neurons leptin-induced depolarization was dependent on TRPC channels [30].

TRPC3 TRPC3 AND MOTOR COORDINATION Cerebellar Purkinje cells play a pivotal role in motor coordination. In these cells, TRPC3 is highly expressed [31]. Cerebellar motor coordination is dependent on mGluR1 that partners with glutamate receptor δ2 (GluRδ2) and protein kinase C-γ (PKCγ). There is good evidence that TRPC3 is involved in the formation of mGluR1-dependent slow excitatory postsynaptic potentials (EPSCs). Mutations in GluRδ2 change the subcellular distribution of mGluR1 and TRPC3 to increase their surface expression. Loss of GluRδ2 disrupts mGluR1-dependent synaptic transmission at parallel fiber-Purkinje cells synapses; this will result in deficits in motor coordination [32]. TRPC3 also provides a negative feedback to cytosolic Ca2+ regulation via its C-terminal CIRB (calmodulin and I3PR) domain. Alternative splicing of the TRPC3 mRNA transcript results in a truncated TRPC3 protein referred to as TRPC3c that lacks approximately half of the CIRB domain. TRPC3c expression is brain region specific, with high prevalence reported in the cerebellum and brainstem. When expressed in HEK293 cells, the TRPC3c channel exhibits a high opening rate. Thus, TRPC3c appears to have enhanced efficacy as a neuronal Ca2+ signaling effector [33]. Of note, functional TRPC3 channels are required for BDNF to increase dendritic spine density in CA1 pyramidal neurons. TRPC3 blockade (by siRNA down-regulation or antagonist treatment) attenuated dendritic spine formation in CA1 hippocampal neurons after BDNF application [34]. Cerebellar LTD is induced by pairing the synaptic inputs provided by the climbing fibers with those of the parallel fibers. Cerebellar LTD is impaired by TRPC3-blocking antibodies, implying a pivotal role for TRPC3 in the induction of LTD [35]. In expression systems, TRPC3 THE ROLE OF TRP CHANNELS IN BUILDING THE BRAIN AND REGULATING ITS FUNCTIONS 425 activity is inhibited following phosphorylation by PKC. However, in native Purkinje cells the activation of TRPC3-dependent currents is not inhibited by PKC, implying that native TRPC3-dependent currents may differ significantly in their regulation from those studied in expression systems [36]. As discussed later, Trpc3 knockout mice exhibit impaired walking behavior. Also, disruption of mGluR signaling through TRPC3 is one of the major molecular defects in staggerer (sg/sg) mice, a model of human spinocerebellar ataxia [37]. Combined, these observations confirm the pivotal role of TRPC3 in motor coordination and establish TRPC3 as an important postsynaptic channel that mediates mGluR-dependent synaptic transmission in cerebellar Purkinje cells [38]. Of note, TRPC3 is also highly expressed in cerebellar granule neurons, where it is thought to exert a protective function against serum-deprivation cell death [39,40]. GABA projection neurons in the substantia nigra pars reticulata provide critical contribution to movement control by regulating the activity of basal ganglia. These neurons exhibit a sustained, spontaneous high-frequency spike firing that is believed to involve tonically active TRPC3 channels [41]. Moreover, large aspiny cholinergic interneurons provide the sole source for acetylcholine (Ach) in the striatum. These interneurons are important for keeping the balance between dopamine and GABA. ACh is released in response to corticostriatal glutaminergic afferents, which act mainly via mGluRs. The current activated by mGluR resembles TRPC3 (and also TRCP7). Indeed, in heterologous coexpression experiments TRPC3 is activated via mGluR1 and mGluR5 [42].

TRPC3 AS A NEURONAL ENERGY GENERATOR The brain uses more energy than any other human organ. Appropriate mitochondrial transport and distribution are essential for neurons because of the high energy and Ca2+ buff- ering requirements at synapses. In hippocampal neurons, BDNF halts mitochondrial transport via activation of TRPC3 and TRPC6; this results in the accumulation of mitochondria at the presynaptic sites. The Ca2+ sensor Miro1 plays an important role in this process. Mutant Miro1 (lacking the ability to bind Ca2+) prevents BDNF-induced presynaptic mitochondrial accumulation and synaptic transmission [43]. Of note, chronic exposure to elevated levels of manganese (Mn2+) causes neuronal injury. Astrocytes selectively accumulate Mn2+ which, in turn, inhibits mitochondrial respiration via TRPC3 [44].

TRPC4 and TRPC5 ROLE OF TRPC4 AND C5 IN NEUROTRANSMISSION AND MEMORY FORMATION Cholecystokinin (CCK) is one of the most abundant neuropeptides in the brain where it interacts with two G-protein coupled receptors (GPCRs), CCK-1 and CCK-2. Activation of both CCK receptors influences neurotransmission. For example, CCK-2 receptor activation attenuates dopamine release. The CCK effects are suppressed by the nonselectiveTRPC channel blockers, 2-APB and flufenamic acid; conversely, they are potentiated by lanthanides (Gd3+ and La3+). Importantly, the CCK-induced enhancement of neuronal excitability was significantly inhibited by intracellular application of a TRPC5-blocking antibody [45]. Persistent neuronal activity lasting seconds to minutes is thought to contribute to the transient storage of memory traces in the entorhinal cortex. In many cortical and subcortical structures, nonsynaptic plateau potentials induced by ACh account for the persistent firing. 426 23. ARE BRAIN TRPs VIABLE TARGETS FOR CURING NEURODEGENERATIVE DISORDERS

For example, in layer V of the rat medial entorhinal cortex, carbachol (a cholinomimetic drug that binds to muscarinic receptors) evokes persistent firing via phospholipase C (PLC) activation; this response was suppressed by the TRPC channel blocker, SKF-96365, but not the TRPV channel blocker, ruthenium red (RR). The diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol (OAG), a nonselective activator of TRPC3, C6, and C7 channels, did not alter the firing evoked by carbachol, implicating the involvement of TRPC1, C4, and/or C5 protein. However, the persistent firing was inhibited by intracellular application of the peptide EQVTTRL that disrupts interactions between the C-terminal domain of TRPC4 or C5 subunits and associated PDZ proteins [46]. Furthermore, a TRPC5 dominant negative construct inhibited, whereas the overexpression of wild-type TRPC5 (or TRPC6) enhanced the amplitude of the muscarinic receptor-induced inward after current. These results indicate that TRPC channels (most likely TRPC5 and C6) mediate the muscarinic receptor-induced slow afterdepolarization seen in pyramidal cells of the cerebral cortex and suggest a possible role for TRPC channels in mne- monic processes [47].

TRPC4 AND C5 IN AROUSAL, MOOD, AND REPRODUCTIVE FUNCTIONS The thalamic paraventricular nucleus contains abundant receptors for thyrotropin- releasing hormone (TRH), a neuropeptide known to modulate arousal and mood. In whole cell patch clamp recordings obtained in rat brain slice preparations, TRH induced two concurrent conductances. One conductance featured a K+-dependent current that was suppressed by the GIRK (G protein-coupled inwardly rectifying K+ channel) antagonists, tertiapin Q, and SCH 23390. The second conductance was attenuated by the nonselective TRPC channel blockers, 2-APB, flufenamic acid, and ML204. Based on these findings, it was speculated that TRH excites paraventricular nucleus neurons by a concurrent action on GIRK and a TRPC channel possibly involving TRPC4 and C5 subunits [48]. Hypothalamic kisspeptin-positive neurons are critical for reproductive functions by initiating the synthesis of gonadotropin-releasing hormone (GnRH) at puberty. In slice preparation from female guinea pig brain, arcuate kisspeptin neurons exhibit a negative resting membrane potential, with the majority (~80%) showing a pacemaker current. Leptins increase burst firing in kisspeptin-positive neurons, which was potentiated by lanthanum, a TRPC4/C5 channel activator. By contrast, the leptin-activated current was abrogated by the TRPC channel blocker, 2-APB. It was speculated that excitation by leptin of kisspeptin neurons may be involved in the regulation of GnRH-expressing neurons during different nutritional states [49]. Coordinated gene expression changes across the CNS are required to evoke maternal behavior. After giving birth, gene expression profiling revealed changes in a number of genes including Trpc4 in the lateral septum of female mice, a brain region implicated in maternal care [50]. In lateral septum neurons, TRPC4 is coexpressed with group I mGluRs. The group I mGluR agonist (S)-3,5-dihydrophenylglycine (DHPG) causes an immediate increase in firing rate, followed by a pause of firing; these DHPG actions can be correlated to below-threshold- depolarization (BTD) and above-threshold-plateau-depolarization (ATPD), respectively. In neurons obtained from Trpc4−/− mice the early phase of BTD and the entire ATPD are completely absent. These results suggest that TRPC4 integrates mGluR stimulation with intracellular Ca2+ signals to affect the excitability of lateral septum neurons [51]. THE ROLE OF TRP CHANNELS IN BUILDING THE BRAIN AND REGULATING ITS FUNCTIONS 427

TRPC5 IN GAIT AND MOTOR COORDINATION Trpc5 knockout mice harbor long, highly branched dendrites on their granule neurons with impaired dendritic claw differentiation in the cerebellar cortex. These animals also show deficits in gait and motor coordination. In the centrosome of cerebellar granule neuron, TRPC5 forms a complex with CaMKIIβ and thereby regulates the CaMKIIβ-dependent phosphorylation of the ubiquitin ligase, Cdc20-APC. Centrosomal CaMKIIβ signaling regulates dendrite morphogenesis. The role of TRPC5 is to couple Ca2+ signaling to the ubiquitin ligase pathway at the centrosome [52]. Of note, a similar signaling pathway regulates dendrite development in the hippocampus in response to neurotrophin-3 (NT-3) [53].

TRPC5 AND BRAIN DEVELOPMENT As discussed earlier, TRPC5 counteracts TRPC1 in regulating neurite outgrowth. TRPC5 also appears to be involved in growth cone collapse that occurs when the growth cone detects a repellant factor. Semaphorin 3A is a secreted guidance cue that keeps the growth cone away from inappropriate targets. Semaphorin 3A-mediated growth cone collapse is reduced in hippocampal neurons obtained from Trpc5 null mice. This effect is reproduced by inhibition of the Ca2+-sensitive protease, calpain, in wild-type, but not Trpc5−/−, neurons. Calpain-1 and calpain-2 cleave and activate TRPC5. Mutation of a critical threonine at position 857 in the TRPC5 protein inhibits calpain-2 cleavage. These findings identify TRPC5 as a downstream target for semaphorin signaling to cause changes in neuronal growth cone morphology and nervous system development [54]. In addition, Ca2+- influx via TRPC5 can activate CAMKIIβ, which, in turn, phosphorylates the ubiquitin-ligase complex Cdc20-APC and regulates dendrite patterning. TRPC5 knockdown causes ataxia due to defective dendrite patterning in the cerebellum [52].

TRPC6 TRPC6 IN LEARNING AND EXPLORATORY BEHAVIOR In the mouse brain, the peak of TRPC6 expression is between day 7 and 28 after birth [55]. TRPC6 has a postsynaptic location at excitatory synapses. Overexpression of TRPC6 increases the number of spines in hippocampal neurons. Indeed, these transgenic mice show improved spatial learning and memory in the Morris water maze [56]. The Trpc6−/− mice showed no significant differences in anxiety (as determined by the marble burying test), but demonstrated reduced exploration in the square open field and the elevated stair maze, suggesting an important role for TRPC6 in exploratory behavior [57].

TRPC6 AS A TARGET FOR HYPERFORIN Hyperforin, the principal bioactive ingredient in the medicinal plant Hypericum perforatum (St. John’s wort), is well known for its antidepressant action. The mechanism of action of 2+ hyperforin is, however, poorly understood. Hyperforin elevates [Ca ]I in cortical neurons [58]. In mouse brain cortex, chronic hyperforin treatment (daily injection of hyperforin at a dose of 4 mg/kg for 4 weeks) increases the expression of both TrkB (a receptor for BDNF) and TRPC6 [59]. It is not clear how hyperforin drives TRPC6 expression but it is believed to involve CREB (cAMP response element binding protein) phosphorylation [60]. In addition, hyperforin inhibits the reuptake of serotonin through the activation of TRPC6 channels. This is 428 23. ARE BRAIN TRPs VIABLE TARGETS FOR CURING NEURODEGENERATIVE DISORDERS important because selective serotonin reuptake inhibitors are clinically useful antidepressant drugs. Hyperforin by interacting at TRPC6 also modulates dendritic spine morphology in CA1 and CA3 hippocampal pyramidal neurons [61]. Last, hyperforin was shown to modify intracellular Zn2+ levels and to act as an NMDA receptor antagonist; there is some evidence that these actions may also be mediated by TRPC6 [62]. The physiological function of TRPC6 in cortical neurons remains unclear. Indeed, a variety of compounds that are known to block TRPC channels (including 2-ABP, flufenamic acid, lanthanum, SKF-96365, and Pyr-3) had little, if any, impact on cholinergic afterdepolarization potentials, nor were these potentials affected by genetic deletion of Trpc1, Trpc5, or Trpc6 (single knockouts), or both Trpc5 and Trpc6 together (double knockouts) [63].

TRPC7 Substance P (SP) plays an important role in modulating rhythmic activities driven by central pattern generators including respiration. In mouse brainstem slices containing the pre- Bötzinger complex, SP activates TRPC3 and C7 to enhance the respiratory rhythm regularity [64]. It was postulated that TRPC channel dysfunction may be responsible for the irregular respiratory rhythms in some central neuronal diseases [65].

Vanilloid TRP Channels Of the vanilloid TRP subfamily, the presence of TRPV1 to V4 has been reported in the brain (Figure 23.1). The pharmacology of these channels have been detailed elsewhere [2].

TRPV1 The literature on brain TRPV1 is highly controversial, ranging from widespread expression throughout the whole neuraxis (reviewed in [66,67]) to minimal expression in a few, discreet nuclei [68]. Clearly, a careful re-evaluation of the relevant findings is in order. Until it is done, the most one can do (especially if the author is biased by contributing data to this field) is a balanced review of the literature (pro and contra), allowing readers make up their own minds. Three lines of evidence support the existence of functional TRPV1 in the brain. First, capsaicin evokes responses in brain slices, neurons in culture, and in whole animal models. Second, both TRPV1 mRNA (by RT-PCR or ISH) and protein (by immunostaining or [3]RTX autoradiography) can be detected (albeit at lower levels than in sensory ganglia) in the brains of wild-type, but not Trpv1 null, mice. And third, Trpv1 (−/−) animals show distinct behavioral changes. Unfortunately, none of this proves the existence of brain TRPV1 unequivocally. Capsaicin (especially at high concentrations) is not selective for TRPV1; anti-TRP antibodies are known to detect proteins other than their expected target; and knockouts may have compensatory changes in the expression of other receptors. With these sobering thoughts in mind, let’s review some recent findings to suggest that brain TRPV1 not only exists but also serves crucial functions.

TRPV1 IN FEAR AND ANXIETY In mice, functional magnetic resonance imaging (fMRI) of brain activity revealed that intragastric infusion of capsaicin at doses at which it evokes nociceptive behavior activates several brain regions linked to fear and anxiety, including the amygdala and the periaqueductal gray THE ROLE OF TRP CHANNELS IN BUILDING THE BRAIN AND REGULATING ITS FUNCTIONS 429 matter. These brain nuclei were not activated in the Trpv1 knockout animals. Interestingly, capsiate (a nonirritant capsaicin congener) activated the same brain regions with the sole exception of the periaqueductal gray matter [69]. The endogenous compounds (so-called endovanilloids) that activate brain TRPV1 are subject to extensive research. Anandamide (an endocannabinoid that also activates TRPV1 at high concentrations) exerts a biphasic effect when injected into the dorsolateral periaqueductal gray matter in rats submitted to threatening situations. Lower doses of anandamide induce anxiolytic-like effects, presumably by activating cannabinoid CB1 receptors. This beneficial effect is, however, no longer observed at higher doses, possibly due to the simultaneous activation of TRPV1. Indeed, blockade of TRPV1 by capsazepine potentiates the anxiolytic-like effects of anandamide [70]. The marble-burying behavior is a useful murine model of obsessive-compulsive disorder. Anandamide (1-10 μg/mouse icv) inhibits marble-burying behavior, indicating an anticompulsive activity. Conversely, at higher doses (20 to 40 μg/ mouse) anandamide increased the marble-burying activity, and this effect was mimicked by capsaicin (100 μg/mouse) [71]. N-Arachidonoyl-serotonin (AA-5-HT) is a dual blocker of the endocannabinoid-inactivating enzyme, fatty acid amide hydrolase (FAAH), and the TRPV1 channel. Injection of AA-5-HT into the basolateral amygdala exerts a strong anxiolytic action. Indeed, in the elevated maze test, the AA-5-HT-treated animals spend significantly more time in the open arms than the controls. So, which target mediates the anxiolytic action of AA-5-HT, CB1 or TRPV1? Neither capsazepine, nor URB597 (a selective FAAH inhibitor) has a noticeable anxiolytic action on their own. Coadministration of capsazepine and URB597, however, mimicked the anxiolytic effect of AA-5-HT. Taken together, these findings imply that a simultaneous blockade of FAAH activity and TRPV1 activation are required for anxiolytic activity [72]. The periaqueductal gray and the amygdala (part of the limbic system) play complimentary roles in the innate and learned fear responses. A subset of lateral amygdala neurons express TRPV1: these cells are believed to store and recall established fear memory [73]. The amygdala is also involved in the stress modulation of learning and memory formation. In the mouse amygdala, the “endovanilloid” N-oleoyldopamine (OLDA) suppresses LTP in the lateral nucleus of wild-type, but not TRPV1-deficient mice. The specific TRPV1 receptor antagonist AMG 9810 also prevented the OLDA effect on LTP. At the behavioral level, OLDA enhanced LTP in mice subjected to the forced swim test [74]. Contextual fear is evoked by reexposing an experimental animal to an environment that has been previously paired with an aversive or unpleasant stimulus. A marked increase in neuronal activity is associated with contextual fear conditioning, especially in limbic structures involved with defense reactions such as the ventral portion of medial prefrontal cortex. Capsazepine microinjected into the medial prefrontal cortex reduces the freezing behavior and cardiovascular responses in the high aversive protocol. Conversely, capsaicin potentiates fear-associated responses [75]. By contrast, capsazepine microinjected in the ventral portion of the medial prefrontal increased exploration of open arms in the elevated plus maze test, suggesting an anxiolytic-like effect [76]. Of note, the anxiolytic-like response evoked by capsazepine is similar to that of diazepam. Desensitization to capsaicin attenuates the anxiolytic effect of diazepam, whereas coadministration of capsazepine and diazepam at subef- fective doses exhibit anxiolytic-like effect. These findings imply that the anxiolytic effect of diazepam, at least in part, involves TRPV1 [77], which appears to be tonically active. 430 23. ARE BRAIN TRPs VIABLE TARGETS FOR CURING NEURODEGENERATIVE DISORDERS

Of note, activation of TRPV1 in the ventrolateral periaqueductal gray also exerts antinociceptive effects (which are out of the scope of this chapter). Briefly, microinjection of capsaicin into the ventrolateral periaqueductal gray reduced noxious heat sensation in the rat (measured in the hot plate test); this effect was blocked by CB1-R and mGluR antagonists. These observations imply that capsaicin activates TRPV1 to release glutamate, which, in turn, activates postsynaptic mGlu5-R. The end result of this cascade activation of a descending pain inhibitory pathway [78].

TRPV1 AND SYNAPTIC PLASTICITY In amygdala neurons, 2-arachidonoylglycerol (2-AG) and anandamide mediate different forms of synaptic plasticity: 2-AG (acting on presynaptic CB1 receptors) triggers a retrograde short-term depression, whereas LTD is mediated by anandamide acting on postsynaptic TRPV1 receptors. This function sharing between CB1 and TRPV1 receptors is in contrast to the striatum where 2-AG (acting on CB1 receptors) mediates both forms of plasticity [79]. In the striatum, ACh controls both excitatory and inhibitory synaptic transmission by activating muscarinic M1 receptors. Capsaicin prevents the effects of M1 receptor activation on inhibitory postsynaptic potentials (IPSPs). Elevation of the anandamide tone by the FAAH inhibitor URB597 mimicked the effects of capsaicin, indicating that endogenous anandamide may act as the endovanilloid. In support of this model, URB597 effects were absent in mice lacking TRPV1 channels [80,81]. In the hippocampus and cortex of the mouse, TRPV1 expression was detected by a combination of real-time PCR and Western blot. The TRPV1 mRNA and protein expression levels showed dynamic changes during brain development, with a progressive increase between 4 and 8 weeks after birth [82]. Within the hippocampus, TRPV1 was predominantly expressed in the dentate molecular layer, an area linked to long-term synaptic plasticity. By high- resolution electron microscopy, TRPV1 immunoparticles were found to be highly concentrated in the postsynaptic dendritic spines; by contrast, TRPV1 was poorly expressed at the excitatory hilar mossy cell synapses. Importantly, this pattern of TRPV1 expression was completely absent in the Trpv1 null mice [83]. It was speculated that TRPV1 modulates synaptic plasticity (both LTP and LTD) in hippocampal CA1 pyramidal cells, possibly by changing the activity of CA1-inhibitory GABAergic interneurons. Indeed, the GABA antagonist picrotoxin eliminated the enhancement of LTP in CA1 neurons in response to TRPV1 agonists [84]. The calcineurin antagonists, cyclosporine and FK-506, also blocked TRPV1-dependent LTD [85]. In humans, a number of single-nucleotide polymorphisms (SNPs) have been described in the TRPV1 gene, some of which significantly alter the activity of the channel. In cell expression systems, the “G” allele of rs222747 was found to enhance the activity of the channel, whereas rs222749 had no functional effect. In a cohort of 77 healthy individuals, study subjects homozygous for the G allele in rs222747 exhibited larger short-interval intracortical facilitation (a measure of glutamate transmission) explored through paired-pulse transcranial magnetic stimulation of the primary motor cortex [86]. On a technical note, in brain slices that contain the basolateral complex of amygdala, capsaicin changes the magnitude of LTP in a manner that is determined by the anesthetic agent (ether or isoflurane) used before euthanasia. After ether anesthesia, capsaicin had a suppressive effect on LTP, which was completely blocked by the nitric oxide synthase (NOS) inhibitor L-NAME and was absent in neuronal NOS as well as in TRPV1-deficient mice. However, THE ROLE OF TRP CHANNELS IN BUILDING THE BRAIN AND REGULATING ITS FUNCTIONS 431 after isoflurane anesthesia capsaicin caused the opposite effect on LTD: a TRPV1-mediated increase in the magnitude of the response [87]. This may account for some of the discrepant findings in the literature.

TRPV1 IN APPETITE CONTROL AND GOAL-DIRECTED BEHAVIOR There is anecdotal evidence that capsaicin suppresses appetite, leading to weight loss. The phenotype of the Trpv1 null mouse is, however, confusing: it is lean when it is young, but it is overweight when it grows old. Gastrin-releasing peptide (GRP) is a bombesin-like peptide with a widespread distribution in the mammalian CNS, where it has been implicated in food intake. The paraventricular thalamic nucleus (that participates in arousal, motivational drives, and stress responses) has a dense network of GRP-positive fibers. GRP is thought to interact at postsynaptic bombesin type-2 (BB2) receptors that connect downstream to TRPV1. Indeed, the TRPV1 antagonists, capsazepine and SB-366791, ameliorate the GRP-induced membrane depolarization and rhythmic burst or tonic firing [88]. TRPV1 is also expressed in the nucleus accumbens, implying a role in goal-directed behavior and reward-dependent learning. Medium-size spiny neurons participate in two independent parallel circuits that likely subserve distinct behavioral functions: (1) direct pathway medium spiny neurons express D1 dopamine receptors and target midbrain dopamine centers, whereas (2) indirect pathway medium spiny neurons express D2 receptors and project to the ventral pallidum. In the indirect pathway, synaptic activation of group-I mGluRs leads to generation of endocannabinoids, which, in turn, activate postsynaptic TRPV1 channels [89].

TRPV2 and TRPV3 Compared to TRPV1, the literature on TRPV2 and TRPV3 is very limited (using TRPV1 and brain as key words, a recent Medline search has found 509 papers, whereas the combination of TRPV2 or TRPV3 with brain yielded only 30 and 24 hits, respectively). Cloned from rat brain as a “capsaicin receptor homolog with a high threshold for noxious heat,” TRPV2 is highly expressed in the brain, both in neurons and astrocytes. The functional role of brain TRPV2 is, however, puzzling because the Trpv2 knockout mouse has no relevant phenotype. Of note (cancer is discussed in a different chapter), TRPV2 was reported to negatively control glioma progression and increase survival. In spinal motor neurons, TRPV2 expression was first detected at embryonic day 10 when TRPV2 was localized in axon shafts and growth cones, implying a role in axon outgrowth regulation. It was suggested that TRPV2 in developing neurons is directly activated in a membrane stretch-dependent manner [90]. TRPV3 expression was reported in the rat brain [91], where it may be a target for incensole acetate (discussed later). Furthermore, in the hippocampus TRPV3 has been implicated in LTD at excitatory synapses on interneurons [92].

TRPV4 TRPV4 expression in the brain (including the cerebral cortex, hippocampus, thalamus, basal nuclei, cerebellum, and spinal cord) is increasing as the rats grow older. (As discussed later, it was speculated that TRPV4 may be involved in the pathogenesis of age-related neurodegenerative diseases [93].) TRPV4 is expressed both in neurons and astrocytes, where it may be activated by changes in osmolality. 432 23. ARE BRAIN TRPs VIABLE TARGETS FOR CURING NEURODEGENERATIVE DISORDERS

TRPV4 IN NEURONS In sensory and spinal motor neurons, both overexpression and chemical activation of TRPV4 promote the formation of neurites. Conversely, its knockdown and/or pharmacologic inhibition exerts the opposite effect. Furthermore, NGF up-regulates TRPV4 expression [94]. These findings imply a role for TRPV4 in brain development and neuroregeneration. In mature neurons, TRPV4 may function as an osmosensor. It is well documented that abrupt changes in the osmotic pressure of the CSF can alter the excitability of the brain. For example, exposure of hippocampal neurons to hypotonic solutions increases the field EPSPs; this response is prevented by the TRPV4 antagonist, HC-067047 [95]. Moreover, TRPV4 may be involved in the generation of miniature EPSPs in the paraventricular nucleus of the hypothalamus [96]. It is not completely clear how TRPV4 regulates synaptic transmission. According to a recent model, TRPV4 promotes presynaptic glutamate release and thereby increases postsynaptic AMPA receptor function [97].

TRPV4 IN ASTROCYTES Astrocytes are specialized glial cells in the brain with essential functions in maintaining cerebral homeostasis. Astrocytes give rise to a highly branched network of processes that form an "endfeet" around the cerebral blood vessels. These “endfeet” form the blood-brain barrier along with the endothelium and the pericytes. TRPV4 in astrocytes evokes Ca2+ oscillations that may lead to the release of gliotransmitters [98]. TRPV4-positive astrocytes may also play active roles in the regulation of synaptic transmission [99].

Melastatin TRPM Channels TRPM2 TRPM2 IS INVOLVED IN BRAIN DEVELOPMENT TRPM2 is highly expressed in the embryonic brain. Knockdown of Trpm2 markedly increased, whereas its overexpression, conversely, inhibited axonal growth. Furthermore, TRPM2 was a target for CSF rich in lysophosphatidic acid to induce neuronal retraction. Importantly, neurons isolated from the brain of Trpm2-deficient mice have significantly longer neurites with a greater number of spines than those obtained from the brain of wild-type mice. Combined, these observations imply an important role for TRPM2 in brain development [100].

IS TRPM2 A REDOX SENSOR IN MATURE NEURONS? In the rat substantia nigra, TRPM2 is expressed both in GABAergic and dopaminergic neurons. The pars reticulata of the substantia nigra contains GABAergic neurons that project to target brain nuclei. These neurons exhibit spontaneous regular firing, but also exhibit burst firing in the presence of excitatory glutamatergic input (parenthetically, an increase in burst firing is a seen in Parkinson disease). Both the spontaneous firing rate and the burst activity of the cells are modulated by the reactive oxygen species (ROS) acting via TRPM2 channels [101]. The pars compacta of the substantia nigra possesses dopaminergic neurons that are strongly inhibited by the nonselective TRPM2 channel blockers clotrimazole and flufenamic 2+ acid. In these cells, challenge with H2O2 initiates a rise in [Ca ]i which is partially blocked by clotrimazole, implying an involvement of TRPM2 [102]. THE ROLE OF TRP CHANNELS IN BUILDING THE BRAIN AND REGULATING ITS FUNCTIONS 433

TRPM2 channels are coactivated by intracellular ADP-ribose (a substance that is produced under conditions of oxidative stress) and Ca2+. Glutathione, a thiol redoxant, inhibits TRPM2 channel activity. In cultured hippocampal pyramidal neurons, l-buthionine-sulfoximine (a blocker of γ-glutamylcysteine synthetase, a key enzyme in glutathione biosynthesis) augments TRPM2-mediated currents [103]. In CA1 hippocampal pyramidal neurons, knockdown of Trpm2 by shRNA reduced the amplitude of the ADP-ribose-dependent current [104]. Moreover, in hippocampal slices obtained from Trpm2 null mice, a selective impairment of NMDA-R-dependent LTD was observed [105]. Combined, these findings create a strong case for TRPM2 being a neuronal redox sensor. The implications of this hypothesis for neurodegenerative disorders will be discussed later.

TRPM3 TRPM3 is thought of as a target for neuroactive steroids. Pregnenolone sulfate is an excitatory neurosteroid that acts as a negative allosteric modulator of GABA-A and a weak positive allosteric modulator of NMDA receptors. In the embryonic cerebellar cortex (where TRPM3 is abundantly expressed), pregnenolone sulfate is crucial for the normal development of Purkinje cells. In neonatal Purkinje cells, pregnenolone sulfate potentiates spontaneous glutamate release. This effect is mimicked by TRPM3 agonists and is blocked by the TRPM3 antagonist, mefenamic acid [106]. In mature neurons, as expected for a positive allosteric NMDA receptor modulator, pregnenolone sulfate increases the frequency of AMPA receptor-mediated miniature EPSPs, and this effect is blocked by the nonselective TRP channel antagonist, La3+ [107]. Of note, pregnenolone sulfate also increases glutamatergic-simulated EPSPs in acutely isolated dentate gyrus hilar neurons of the hippocampus. This increase was completely abolished by nonselective TRP channel blockers. It is unclear which TRP channel mediates this action but TRPM3 seems to be a good candidate [108].

TRPM4 and M5 Consistent with its role as a molecular pacemaker [109], TRPM4 is thought to regulate the activity of respiratory neurons in the pre-Bötzinger complex [110]. The pre-Bötzinger complex is located in the lower brainstem, where it generates the respiratory motor output. In functionally intact acute brainstem slices, brief hypoxia causes a biphasic response: a transient decrease in bursting activity followed by augmentation. These changes seem to be mediated by rhythmic Ca2+ transients in a TRPM4-dependent fashion [111]. The physiological role of TRPM4 in dopaminergic neurons is less clear. In the substancia nigra, dopaminergic neurons that respond to high-frequency glutamatergic inputs are believed to relay reward-associated information. These cells exhibit transient bursts of spikes that are absent after pretreatment with flufenamic acid and/or 9-phenanthrol, suggesting the involvement of TRPM2 and TRPM4 in the burst activity [112]. Recently, an intriguing role for TRPM4 and M5 has been suggested in mediating social interactions in rodents. Pheromones are secreted (or excreted) chemical agents that impact behavior including mate selection, aggression, and defense against predators. The main and accessory olfactory systems detect and process pheromonal stimuli. In mice, mitral cells isolated from the accessory (but not the main) olfactory bulb show a sustained firing activity (lasting up to several minutes), which is at least partially mediated by TRPM4 [113]. Mitral cells in 434 23. ARE BRAIN TRPs VIABLE TARGETS FOR CURING NEURODEGENERATIVE DISORDERS the main and accessory olfactory bulbs directly project to the medial amygdala. Neurons that connect the main olfactory bulb to the amygdala respond to volatile urine exposure by the opposite sex. These olfactory sensory neurons express TRPM5 [114]. Of note, TRPM4 and M5 are coexpressed in cerebellar Purkinje cells. Depolarization- induced slow currents are attenuated (but not abolished) in Purkinje cells derived from Trpm4 null, Trpm5 null, and double knockout mice, as well as in wild-type mice with Trpm4 shRNA knockdown. These findings imply a role for TRPM4 and M5 in the generation of cerebellar depolarization-induced slow currents [115].

TRPM7 and TRPM8 TRPM6 and M7 are closely related channels. Indeed, TRPM6 cannot efficiently form functional channels by itself and needs to assemble with TRPM7. TRPM6 has a highly restricted tissue expression pattern (mostly in intestines and kidney) that does not involve the brain; therefore, this channel is out of the scope of this chapter. TRPM7 is, however, expressed in neurons where it is believed to function as a cellular Mg2+ transporter [116]. TRPM8 is expressed in cultured hippocampal neurons, but little is known about its in vivo expression pattern in the brain. Under voltage-clamp conditions, TRPM8 activation seems to evoke an inward current that does not alter synaptic transmission [117]. Menthol (150 to 750 μM), a TRPM8 agonist, prolongs inhibitory postsynaptic currents in the hippocampus, but this effect is most likely mediated by a positive allosteric modulation of recombinant GABA-A receptors. In keeping with this hypothesis, menthol actions were unaffected by TRPM8 antagonists [118].

The Ankyrin TRPA1 Channel In the rat brain, TRPA1 is expressed in neurons, astrocytes, and cells lining the choroid plexus and the ventricles. In the hippocampus, TRPA1 has been implicated in neuronal cell death. The synthetic cannabinoid WIN 55,212-2 protects hippocampal neurons against ischemic damage. This protective action was suspended by the CB1 receptor antagonist AM251, but not the CB2 receptor antagonist AM630. The TRPA1 blocker HC-030031 enhanced the neuroprotective efficacy of WIN 55,212-2. In contrast, the TRPA1 agonist icilin or allyl isothiocyanate led to a stronger neurodegeneration. These data suggest that WIN 55,212-2 has a biphasic action: at low concentrations it protects neurons by activating CB1 receptors, and at high concentrations it additionally activates TRPA1 that interferes with the CB1 receptor- mediated neuroprotection [119].

Somewhat confusingly, TRPA1 plays a protective role in astrocytes. In these glial cells, H2S2 generates polysulfides that, in turn, induce Ca2+ influx. This effect is prevented by the TRPA1 antagonist HC-030031, as well as by Trpa1 gene silencing via siRNAs [120]. In, addition, astrocytic TRPA1 contributes to basal Ca2+ levels that are required for constitutive release of d-serine, a signaling molecule used to communicate with neurons [121]. TRPA1 is expressed in both the choroid plexus and ventricular lining epithelium [122], as well as in magnocellular neurosecretory cells of the supraoptic nucleus that produce vasopressin [123]. Given its role as a chemosensor, one might speculate that TRPA1 may respond to chemical stimuli to regulate CSF synthesis and vasopressin release. TRP Channel Dysfunction in Epilepsy 435 TRP CHANNEL DYSFUNCTION IN EPILEPSY

Epilepsy is one of the most common diseases seen in neurology between departments. It is caused by various perturbances that disturb the normal balance excitation and inhibition within the CNS. Current antiepileptic drugs target ion channels, neurotransmitter transporters, and neurotransmitter metabolic enzymes. They could control symptoms in the majority (70-80%) of the patients. A significant subset of patients (20-30%), however, develops intractable epilepsy. Even worse, existing antiepileptic drugs do not cure the disease, only alleviate symptoms, and possess significant dose-limiting adverse effects. Clearly, there is a dire need for novel antiepileptic drugs. Given their postulated role in modulating neuronal excitability, TRP channels are promising drug targets to explore. There is increasing evidence to implicate TRPC channels (in particular, TRPC1/C4, TRPC3, and TRPC5) and TRPV1 in the pathogenesis of epilepsy.

TRPC Channel and Epilepsy Unlike TRPC1, which is ubiquitously expressed in the brain, TRPC4 expression is highly restrictive, with the highest expression level reported in the lateral septum. A dysfunction of the septo-hippocampal network has been implicated in chronic epilepsy. In cats, injection of kainate (an agonist of a subset of AMPA receptors) evokes seizures and epileptiform EEG activity. The large depolarizing plateau potential that underlies the epileptiform burst firing was completely abolished in the Trpc1/c4 double knockout mice and was also absent in the majority (74%) of lateral septal neurons in Trpc1 null mice. The muscarinic agonist, pilocarpine, evokes seizures in experimental animals; this is a widely used model of temporal lobe epilepsy. Severe pilocarpine-induced seizures may cause neuronal cell death in the lateral septum; this effect is ameliorated in the Trpc1/c4 double knockout mouse. These results imply an essential role for TRPC1 and TRPC4 (likely as a heteromultimer) in forming the intrinsic membrane conductance that mediates the plateau potential in lateral septal neurons [124,125]. The Trpc5 knockout mouse also exhibits both significantly reduced seizure activity and attenuated seizure-induced neuronal cell death in the hippocampus. Yet, epileptiform bursting activity induced by mGluR agonists (which is normal in the Trpc1/c4 double knockout animals) is unaltered in Trpc5 null mice. By contrast, long-term potentiation is (which is normal in the Trpc1/c4 double knockout animals) is greatly reduced in TRPC5-deficient mice. The distinct phenotype of these knockout animals suggests that TRPC5 and TRPC1/C4 contribute to seizure activity by distinct cellular mechanisms [124]. In support of this model, muscarinic activation by carbachol in hippocampal neurons was shown to initiate a current through TRPC5 by promoting the membrane insertion of the channel. As expected, the muscarinic antagonist, atropine, prevented the increase in TRPC5 surface expression. TRPC5-like currents were also inhibited by the nonselective TRPC antagonists, 2-APB and SKF-96365, as well as the PI3K inhibitor, wortmannin, which blocks TRPC5 translocation from the cytosol to the plasma membrane. In conclusion, the rapid translocation of TRPC5 contributes to the generation of the cholinergic-induced plateau potentials through a Ca2+/calmodulin and PI3K-dependent pathway, providing new insights into the pathology of epilepsy [126]. 436 23. ARE BRAIN TRPs VIABLE TARGETS FOR CURING NEURODEGENERATIVE DISORDERS

In the motor cortex, TRPC3 expression is strong during embryogenesis but is weak to absent in the mature brain. In dysplastic cortex, TRPC3 expression, however, returns to the high levels seen in developing neurons. In pyramidal neurons, combinations of low Ca2+ and Mg2+ increases the amplitude of depolarization, which, in the dysplastic cortex, is sufficient to provoke epileptiform activity. Importantly, this activity was suppressed by the TRPC3 inhibitor, Pyrazole-3 (Pyr3) [127]. Following status epilepticus, TRPC3 expression is elevated (whereas TRPC6 is reduced) in pyramidal neurons and dentate granule cells. This increase in TRPC3 is blocked by Pyr3. Furthermore, hyperforin (a TRPC6 activator) prevents down-regulation of TRPC6 induced by epileptic seizures. Apparently, both increased TRPC3 and decreased TRPC6 are relevant because both Pyr3 and hyperforin (alone or in combination) protected neurons against damage by severe epileptic seizures [128]. Interestingly, status epilepticus induced TRPC3 expression in endothelial cells that did not contain this protein in control animals. The neo-expression of TRPC3 in endothelial cells was accompanied by a loss of SMI- 71, a blood-brain barrier marker, and correlated to the development of vasogenic edema and resultant neuronal damage. The vasogenic edema response was attenuated by Pyr3. It was speculated that TRPC3 neo-expression in endothelial cells may contribute to the neuronal damage during and after status epilepticus by disrupting the blood-brain barrier [129]. In summary, TRPC channels could represent shared downstream target for a number signaling pathways that may contribute to seizure and excitotoxicity, such as NMDA receptor-mediated Ca2+ influx, or mGluR activation [130]. Of TRPC channels, TRPC3 is particularly interesting given its preponderance in dysplastic (and low expression in normal) motor cortex and postulated role in disrupting the blood-brain barrier during status epilepticus.

TRPV1 as a Potential Target in Epilepsy and Febrile Seizures TRPV1 is an interesting, though highly controversial (the ongoing debate whether or not TRPV1 is expressed at all in the brain is discussed elsewhere), antiepileptogenic target. In rats, TRPV1 activation was reported to modulate activity-dependent synaptic efficacy in the hippocampus by facilitating LTP and suppressing LTD. In brain slices obtained from Trpv1 (−/−) mice, LTD was absent, and capsaicin was inefficient [131,132]. It was hypothesized that TRPV1 selectively inhibits excitatory synapses at hippocampal interneurons. 4-aminopyridine triggers epilepsy-like symptoms in the rat. In this model, the first-generation TRPV1 antagonist, capsazepine, suppresses epileptiform activity. By contrast, capsaicin potentiates the seizures [133]. To further strengthen the link between TRPV1 and epilepsy, it was pointed out that NGF (a known driver of TRPV1 expression) triggers epileptogenesis. Furthermore, the levels of the endocannabinoid anandamide (also an endogenous TRPV1 agonist) are increased in epilepsy. It was speculated that TRPV1 activation (possibly by high anandamide concentrations) may trigger apoptotic neuronal death (at least in the rat brain) that leads to chronic epilepsy [134]. The cannabinoid link to epilepsy is a hot topic. Those who favor legalization point out that medical marijuana (or its main active ingredient, tetrahydrocannabinol) can control seizures in experimental animals not responsive to other treatments. Also, there are anecdotal reports that medical marijuana can stop intractable seizures in children with Duvet syndrome, also known as myoclonic epilepsy of infancy. Boosting endogenous anandamide in the brain by blocking FAAH (the enzyme that hydrolyzes anandamide) has anticonvulsant effects, which Cerebellar Ataxia as a “TRP Channelopathy” 437 are mediated by CB1 receptors. The trick is to keep anandamide in the beneficial dose range because high anandamide concentrations can cause paradoxical seizures by activating TRPV1 [135,136]. One strategy to achieve this goal is by dual FAAH and TRPV1 blockade with N-arachidonoyl-serotonin (AA-5-HT). Indeed, seizures induced by pentylenetetrazole in mice were prevented by AA-5-HT [137]. Importantly, there is experimental evidence linking TRPV1 to epilepsy in humans. Both tuberous sclerosis complex and focal cortical dysplasia type IIb cause intractable epilepsy. In brain specimens removed during surgery from patients with tuberous sclerosis or focal cortical dysplasia IIb, TRPV1 was detected in the abnormal cell types, such as dysmorphic neurons, balloon cells, and giant cells. Interestingly, TRPV1 appeared to have both cytoplasmic and nuclear distribution, suggesting a potential nuclear role of TRPV1 [138]. Elevated TRPV1 was also detected in the brains of patients with mesial temporal lobe epilepsy compared to controls (brain surgery unrelated to epilepsy). TRPV1 was mainly present in the cell bodies and dendrites of glutaminergic and GABAergic neurons and GFAP-positive astrocytes [139]. At present, it is unclear if this abnormal TRPV1 expression is a cause or consequence of the disease. As a heat-activated channel, TRPV1 seems to be an interesting candidate to explore for mediating febrile seizures, the most common seizure type in children under the age of five. In mice, pentylene tetrazol induces clonic seizures that are made worse in febrile animals. In this model, Trpv1 gene deficiency decreased the intensity of experimental febrile seizures [140]. In rats treated with subconvulsive doses of pentylene tetrazol, the TRPV1 receptor agonist OLDA (injected intracerebroventricularly 30 min prior to pentylene tetrazol administration) accelerated the incidence of seizures, whereas the TRPV1 antagonist AMG-9810 ameliorated the seizure activity [141].

TRPM2 and Juvenile Myoclonic Epilepsy Mutations in the gene that encodes the EF-hand motif-containing protein EFHC1 have been linked to the pathobiology of juvenile myoclonic epilepsy (JME). In hippocampal neurons, TRPM2 and EFHC1 are coexpressed with a physical interaction between the N- and C-terminal cytoplasmic regions of TRPM2 with the EFHC1 protein. In recombinant TRPM2 expression systems, coexpression of EFHC1 was shown to potentiate the H2O2-induced currents with a resultant increase in cell death. Based on these findings it was speculated that TRPM2 mediates the disruptive effects of JME mutations of EFHC1 [142].

CEREBELLAR ATAXIA AS A “TRP CHANNELOPATHY”

As we saw earlier, TRPC3 is abundantly expressed in Purkinje cells, where it mediates slow mGluR-mediated synaptic responses. Heterozygous moonwalker mice represent a model of cerebellar ataxia. These mice carry a dominant gain-of-function mutation (T635A) in the Trpc3 gene [143]. This mutation leads to dysmorphism and/or loss of Purkinje cells, which have been suggested to cause the ataxia. Interestingly, the ataxic phenotype is present from a very early age (before weaning) although Purkinje cell loss does not appear until much later (several months of age). The intrinsic excitability of the mutant Purkinje cells is, however, 438 23. ARE BRAIN TRPs VIABLE TARGETS FOR CURING NEURODEGENERATIVE DISORDERS altered as early as 3 weeks after birth. To explain these seemingly contradictory findings, it was pointed out that (in addition to Purkinje cells) type-II unipolar brush cells in the cerebellum also express functional TRPC3 channels. These cells are ablated in moonwalker mice by 1 month of age, much earlier than the loss of Purkinje cells. Combined, these findings suggest that the ataxic phenotype of the moonwalker mouse reflects both the altered excitability of Purkinje cells (secondary to the gain-of-function TRPC3 mutant) and the TRPC3-mediated loss of type II unipolar brush cells [144]. If the overactivity of TRPC3 leads to ataxia, the loss of channel activity should not impair motor coordination. Apparently, this is not the case because animals carrying gain-of- function (moonwalker) and loss-of-function (Trpc3 null) mutations have a similar phenotype. For a detailed discussion on how opposing aspects of TRPC3 channel activation can lead to the same phenotype, see [145]. In cerebellar Purkinje cells, PKCγ is involved in the “pruning” of the climbing fiber synapses. Spinocerebellar ataxia type-14 (SCA14) is an autosomal dominant neurodegenerative disorder, caused by mutations in PKCγ. Interestingly, 18 of the 22 mutations described in SCA14 patients are concentrated in the C1 domain of the enzyme, which is responsible for the membrane binding of DAG that drives the translocation and regulation PKCγ. Wild-type, but not C1 domain mutant, PKCγ inhibits Ca2+ influx in response to muscarinic receptor stimulation. The C1 domain mutants are constitutively active, and they are unable to phosphorylate the TRPC3 protein, resulting in sustained Ca2+ entry. This alteration in Ca2+ homeostasis in Purkinje cells may contribute to neurodegeneration see in SCA14 patients [146]. The mutant PKCγ colocalizes with wild-type PKCγ, and the mutant PKCγ acts in a dominant-negative manner [147]. Hereditary cerebellar ataxia is rare (less than 1 in 100,000), but sporadic ataxia is somewhat more common (10 per 100,000). If a functionally overactive TRPC3 channel is responsible for SCA14 (a hereditary cerebellar ataxia), the TRPC3 gene could be a promising candidate for screening ataxic patients with unknown genetic etiology. Somewhat disappointingly, in a cohort of 98 patients with sporadic cerebellar ataxia a genetic screen for TRPC3 mutations did not reveal any potentially causative variants [148].

TRP CHANNELS IN NEUROPROTECTION AND THEIR DYSFUNCTION IN NEURODEGENERATIVE DISORDERS

Parkinson Disease Parkinson disease (PD) is a devastating, relentlessly progressive neurodegenerative disorder characterized by motor impairment (such as bradykinesia and resting tremor) and dementia. Autopsy brain of PD patients show Lewy body formation (of note, Lewy bodies are not specific for PD because they can also be seen in Pick disease and other forms of dementia) and loss of dopaminergic neurons in the basal ganglia. The loss of dopaminergic neurons results in a chemical imbalance that affects the whole basal ganglia-thalamus-cortex circuit. The cause of PD is unknown but a number of environmental factors (e.g., pesticide exposure) have been associated with increased risk. In animal models, neurotoxins (e.g., rotenone, a pesticide) that kill dopaminergic neurons in a Ca2+ overload-dependent manner mimic some aspects of the human disease. The rise 2+ in [Ca ]i evoked by rotenone was attenuated by the TRPM2 blocker, N-(p-amylcinnamoyl) TRP CHANNELS IN NEUROPROTECTION AND THEIR DYSFUNCTION 439 anthranilic acid [149]. In SH-SY5Y cells, salsolinol (a toxic condensation product of dopamine and acetaldehyde) and 1-methyl-4-phenylpyridinium (MPP, a neurotoxin that causes parkinsonism in primates) induce apoptosis, which is reversed by TRPC1 activation. Exposure of SH-SY5Y cells to these toxins blocks thapsigargin-mediated Ca2+ influx and decreases TRPC1 in the plasma membrane by translocation to the cytoplasm. It was speculated that neurotoxins may alter Ca2+ homeostasis (in a way that involves TRPC1 possibly via PLC) and induce mitochondrial-mediated caspase-dependent cytotoxicity, an important characteristic of PD [150]. Unexpectedly, capsaicin microinjected into the substantia nigra provides partial protection against neuronal cell death induced by MPP by suppressing the production of microglia- derived ROS. These capsaicin effects are reversed by capsazepine. Bases on these findings, it was speculated that TRPV1 agonists may have a therapeutic value in PD [151]. (How one can safely deliver a TRPV1 agonist into the CNS is a completely different problem, not addressed in this article!) GABAergic interneurons that express TRPC3 control the activity of striatal projection neurons. Parkinsonian movement disorders are often associated with abnormalities in the firing intensity and/or pattern of these cells. TRPC3 channels expressed by GABAergic interneurons are tonically active and mediate an inward, Na+-dependent current, leading to a substantial depolarization. Conversely, inhibition of TRPC3 channels induces hyperpolarization and thereby decreases the firing frequency [152]. The mainstay of pharmacotherapy in PD is dopamine replacement. Unfortunately, the long-term use of levodopa (l-DOPA) as a side effect causes abnormal involuntary movements, called L-DOPA-induced dyskinesia. In a hemiparkinsonian model of PD (mice with 6-hydroxydopamine-induced striatal lesions), chronic L-DOPA treatment leads to the development of intense axial, forelimb, and orolingual dyskinetic symptoms, resembling the human adverse effect. Treatment with oleoylethanolamide (OEA) ameliorated these symptoms without altering the therapeutic motor effects of L-DOPA. The antidyskinetic action of OEA was most likely mediated by TRPV1 because it was absent in mice desensitized to capsaicin [153]. Excessive glutamate can cause neuronal dysfunction and degeneration. Glutamate excitotoxicity has been implicated in acute brain insults (such as ischemic and traumatic brain injury that are discussed later) and also in chronic neurodegenerative disorders including PD. 2-APB (a “universal” TRP channel blocker) reduces glutamate-induced cell death in hippocampal organotypic slice cultures presumably by inhibiting TRPC1 channels. In support of this hypothesis, knockdown by iRNA of Trpc1 in slice cultures prevents glutamate-induced cell death [154]. Apparently, in these cells TRPC1 is an important downstream target for mGluRs [155].

Lou Gehrig’s Disease and Amyotrophic Lateral Sclerosis-Parkinson Dementia Complex Amyotrophic lateral sclerosis (ALS, also known as Lou Gehrig’s disease) is a progressive neurodegenerative disorder that predominantly affects the spinal motor neurons, leading to muscle weakness and eventual paralysis. The etiology of ALS is unknown but a subset of cases (~10%) appears to be inherited. In 1993, mutations in the gene that encodes the SOD1 protein (superoxide dismutase-1, alternatively referred to as superoxide dismutase [Cu2+-Zn2+]), have 440 23. ARE BRAIN TRPs VIABLE TARGETS FOR CURING NEURODEGENERATIVE DISORDERS been linked to familial ALS. Sod1 (G93A) mutant transgenic mice represent an animal model of ALS. In these mice, increased expression of TRPV4 was noted in the cerebral cortex, hippocampal formation, thalamus, cerebellum, and spinal cord. Both in the cerebral cortex and the hippocampus, TRPV4 was especially increased in the pyramidal cells. It was speculated that TRPV4 may be involved in the pathogenesis of ALS, but the functional implications of increased TRPV4 remain unclear [156]. Amyotrophic lateral sclerosis-Parkinson dementia complex (ALS-PDC) is a mysterious neurodegenerative disorder that combines symptoms of ALS, Parkinsonism, and Alzheimer disease. ALS-PDC is prevalent in the Guam population but is a rarity elsewhere [157]. The etiology of ALS-PDC remains unknown, but the extremely limited geographic distribution of the disease strongly suggests an environmental factor specific to Guam. It was speculated that β-N-methyl-amino alanine (BMAA) may be the toxic agent that damages the neurons to cause ALS-PDC. BMAA is a mixed mGluR agonist present in the cycad plant, a traditional food source in Guam, which causes reversible membrane depolarization and initiates a rise 2+ in [Ca ]i in dopaminergic neurons. The inward current was mainly mediated by mGluR1 via TRPC channels. Indeed, the TRP channel blockers, SKF 96365 and ruthenium red (RR), reduced the BMAA-induced current. Prolonged exposure to BMAA leads to Ca2+ overload, cell shrinkage, massive cytochrome-c release into the cytosol, and ROS production; ultimately, these changes kill the treated neurons [158]. There is increasing evidence that L-BMAA per se is not sufficient to cause ALS-PDC; another environmental factor is needed. Recent studies have focused on drinking water, which has a unique mineral composition in these Pacific islands; it is extremely low in Ca2+ and Mg2+, but rich in metals like Mn2+, Al3+, and Fe3+. Indeed, rats fed diets that mimic the mineral composition of drinking water in Guam showed significant loss of nigral dopaminergic neurons. A TRPM7 missense mutation (T1482I) was also identified in a subset of ALS-PDC patients [159]. Recent clinical studies, however, have questioned the relevance of the TRPM7 T1482I variant in ALS-PDC. Parametric linkage analyzes of the TRPM7 locus in a large extended family with ALS-PDC patients did not reveal any evidence supporting the linkage to the TRPM7 locus. Resequencing of the entire coding region of TRPM7 did not reveal any pathogenic mutations in an affected individual in this family. The allele frequencies of the T1482I in affected individuals in this family or in those from other families are not significantly different from those in regional controls in Japan [160].

Alzheimer Disease Alzheimer disease (AD) is the most common form of dementia. Although some cases appear to be familial (and have an early onset), the most important risk factor is advanced age. Indeed, after age 85 the risk for developing AD approaches 50%. Hyperphosphorylated tau aggregated into neurofibrillary tangles is a hallmark lesion of AD. In animal models, cold water exposure causes reversible tau hyperphosphorylation, associated with cognitive deficits. In rats, intragastric capsaicin (10 mg/kg) was reported to mitigate the cognitive decline induced by cold water exposure. Furthermore, capsaicin attenuated the cold water stress-induced spatial memory impairment and prevented tau hyperphosphorylation [161]. These findings are puzzling because capsaicin absorbed from the GI tract undergoes extensive hepatic metabolism and is unlikely to reach pharmacologically meaningful concentrations in the CNS. TRP CHANNELS IN NEUROPROTECTION AND THEIR DYSFUNCTION 441

In the rat, TRPV4 is highly expressed in hippocampal astrocytes where it is thought to play a major role in oxidative stress-induced cell damage. In aged monkey, circulating amyloid- β40 peptide crosses the blood-brain barrier and is deposited in cerebrovascular endothelial cells, causing amyloid angiopathy. In rat hippocampal slices, synthetic amyloid-β40 peptide initiates cell death after cultures were preconditioned with sublethal concentrations of buthionine sulfoximine (1.5 μM), a compound that enhances endogenous ROS production. The damage is predominantly in the granule cell layer of the dentate gyrus, with additional cell loss in pyramidal neurons. The dying neurons evoke reactive gliosis with altered (accentuated) TRPV4 expression. In this model, neuronal damage is attenuated by the universal TRP blocker, RR [162]. Of note, the PARP (poly(ADP-ribose)polymerase) inhibitor SB-750139 also attenuates cell death initiated by amyloid β-peptide in rat striatum neurons [163]. PARP (in concert with Ca2+) activates endogenous TRPM2 channels, a neuronal “redox sensor.” Thus, it is entirely possible that TRPV4 up-regulation is coincidental and the real target for ROS- mediated cell death is TRPM2. Last, hyperforin (a TRPC6 activator) blocks the formation of amyloid-β aggregates in vitro and decreases astrogliosis and microglia activation in vivo. Importantly, hyperforin improves spatial memory formation in animal models of AD [164]. Indirectly, these findings implicate TRPV4, TRPM2, and TRPC6 in the pathogenesis of AD. If these observations hold true in humans, combined pharmacological blockade of TRPM2 and TRPV4 and stimulation of TRPC6 may be beneficial in AD patients.

Autoimmune Encephalomyelitis and Multiple Sclerosis Cholera toxin-B binds to the branched pentasaccharide moiety of the ganglioside, GM1. This protein-ganglioside interaction is instrumental in initiating the signal transduction pathway that is responsible for diarrhea. In murine experimental autoimmune encephalomyelitis (EAE), administration of the GM1 cross-linking unit of cholera toxin (the so-called B-toxin) is beneficial. Cholera toxin-B is a binding partner for the endogenous lectin, galectin-1. In these animals, both cholera toxin-B and galectin-1 caused symptomatic improvement. Conversely, mice lacking GM1 demonstrate enhanced susceptibility to EAE. Polyclonal activation of murine regulatory T (Treg) cells up-regulates galectin-1. Furthermore, activation of CD4+ and CD8+ effector T (Teff) cells elevates GM1. Of importance to the topic of this chapter, activation of Teff cells also up-regulates TRPC5, which, in turn, mediates the Ca2+ influx on GM1 cross-linking by either galectin-1 or cholera toxin-B. Knockdown by shRNA of Trpc5 in Teff cells blocks the contact-dependent proliferation inhibition by Treg cells. These observations suggest a role for TRPC5 in the negative control (suppression) of autoimmunity [165]. Multiple sclerosis (MS) is a chronic, relentlessly progressive demyelinating disorder. Although the pathogenesis of MS is unknown, increased glutamate production is thought to play a role in the inflammation-driven neurodegenerative process. In support of this hypothesis, CSF collected from MS patients potentiates glutamate-mediated neuronal swelling through a mechanism that seems to involve both IL-1β signaling and increased AMPA-R stimulation. Indeed, IL-1β is significantly higher in the CSF of patients with active MS. It was hypothesized that TRPV1 is an essential mediator for the synaptic action of IL-1β on central glutamatergic synapses [166]. In keeping with this hypothesis, in a mouse model of EAE Trpv1 null animals show reduced infiltration of the CNS by autoreactive T-cells [167]. In MS 442 23. ARE BRAIN TRPs VIABLE TARGETS FOR CURING NEURODEGENERATIVE DISORDERS patients, a missense SNP in the TRPV1 gene was correlated to the risk of progressive disease [167]. Taken together, these findings identify TRPV1 as a potential novel therapeutic target in MS patients.

TRP CHANNELS IN PSYCHIATRIC DISORDERS AND MENTAL RETARDATION

Anxiety Disorders, Panic Attacks, and Depression Anxiety disorders are the most common form of mental illness in the United States, affecting one out of six Americans age 18 and older. In mice, several TRP channels (including TRPC4, TRPC5, and TRPV1) are expressed in brain areas implicated in the control of fear and anxiety. In behavioral experiments, constitutive ablation of Trpc4 or Trpc5 was associated with diminished innate fear and anxiety levels. Selective knockdown of Trpc4 in the lateral amygdala via lentiviral-mediated gene delivery of RNAi mimicked the behavioral phenotype of the Trpc4 knockout mouse. It was speculated that TRPC4 is a crucial downstream target for two Gαq11 protein-coupled signaling pathways, activated via Group-I mGluRs and CCK2 receptors, respectively [168]. These observations imply that TRPC4 and/or TRPC5 blockers may constitute a new class of anxiolytic drugs. Several lines of experimental evidence implicate TRPV1 in anxiety and panic responses. In behavioral studies, TRPV1 activation by capsaicin (microinjected into the periaqueductal gray) initiates anxiety-like behavior, including less frequent entry into the open arm of the maze test, as well as reduced number of stretched-attend postures and head dippings. TRPV1 blockade by capsazepine did not change the behavior of the animals in the elevated maze test. However, when given before capsaicin, capsazepine completely blocked the anxiogenic-like effect. These findings imply that (1) TRPV1 activation in the periaqueductal gray causes anxiety-like behavior in the mouse, and (2) these TRPV1 receptors are not tonically active [169]. Other studies, however, are more consistent with a tonically active TRPV1. For example, capsazepine inhibits the escape response in the elevated T-maze test [170], and Trpv1 null animals exhibit diminished innate fear response and anxiety-like behavior [171]. In addition, it was shown that TRPV1 might be a functional tool to prevent the risks associated with the long-term use of benzodiazepines [77]. So, what is the endogenous substance that evokes anxiety by activating TRPV1? An interesting candidate molecule is anandamide. In the T-maze assay, microinjection of the selective FAAH inhibitor, URB 597, into the hippocampus produces anxiolytic-like effects, presumably by elevating endogenous anandamide levels. Further increase in anandamide levels, however, exerts the opposite effect. The TRPV1 antagonist AMG 9810 does not interfere with the anxiolytic effect of URB 597 but blocks the anxiogenic action. These findings suggest that the beneficial effect of anadamide (or URB 597) is mediated by CB1-Rs, whereas TRPV1 activation contributes to increased anxiety [172]. These findings add TRPV1 antagonists to the list of potential new anxiolytic agents. Unfortunately, the TRPV1 literature is confusing with some studies suggesting that it is TRPV1 activation that improves mood and reduces anxiety. In mice, nicotine induces depression-like behavioral alterations, similar to those seen in other murine models of TRP Channels in Psychiatric Disorders and Mental Retardation 443

depression such as the repeated immobilization stress. In both the nicotine and immobilization stress models, capsaicin and olvanil administered intraperitoneally exhibited significant antidepressant-like activity. By contrast, anandamide and N-arachidonyldopamine (NADA) lack antidepressant-like effects. In accord, the antidepressant-like effect of capsaicin and olvanil was reversed by capsazepine, but not the CB1R antagonist, AM 251 [173]. These observations imply that TRPV1 activation may improve mood, especially in “cold turkeys” suffering from nicotine withdrawal symptoms. Incensole acetate, the main active ingredient in Boswellia resin, activates TRPV3 in expression systems. In vivo, incensole acetate causes anxiolytic-like and antidepressive-like behavioral effects in wild-type, but not Trpv3 knockout, mice, with concomitant changes in c-Fos activation in the brain [174]. These findings imply a therapeutic potential for TRPV1 agonist in anxiety disorders and depression.

Schizophrenia Schizophrenia is a disabling mental disorder that affects ~1% of the population worldwide. It runs in families (identical twins have a ~50% chance of developing the disease), suggesting a genetic predisposition for neurochemical malfunction in the brain. Disrupted in schizophrenia-1 (DISC1) is a protein implicated in schizophrenia (also in bipolar disorder, major depressive disorder, and autism). The function of DISC1 is to modulate cAMP signaling by increased cAMP catabolism. DISC1 disruption by shRNA knockdown increases intracellular Ca2+ waves in response to mGluR activation. Furthermore, it decreases TRPC- mediated sustained depolarization. It was hypothesized that diminished DISC1 function disrupts the normal pattern of prefrontal cortex activity through the loss of cAMP regulation of mGluR-mediated intracellular Ca2+ waves; this eventually leads to perturbations in TRPC channel activity [175]. As of today, it is unclear which member of the TRPC subfamily is involved in this response. A link between TRPV1 and schizophrenia was postulated, but the experimental evidence is at best preliminary. In rats, capsaicin desensitization leads to behavioral changes (e.g., learning impairments in the novel object recognition test) that are somewhat reminiscent of those seen in patients with schizophrenia [176]. Furthermore, spontaneously hypertensive (SH) rats show schizophrenia-like deficits in social interactions that are ameliorated by atypical antipsychotics. Capsaicin (2.5 mg/kg) increases social interaction of in both SH and normotensive rats and decreases locomotion in SH rats [177]. These findings might be interpreted to imply that loss of TRPV1 function could contribute to the pathogenesis of schizophrenia, whereas TRPV1 activation may be beneficial in these patients.

Rett Syndrome Rett syndrome is a neurodevelopmental disorder that affects intellectual ability. Children with Rett syndrome exhibit autism-like behavior combined with impaired motor functions. In the hippocampus, BDNF activates TRPC3 to increase neuronal dendritic spine density. Indeed, knockdown of Trpc3 prevents the increase in spine density caused by BDNF application. It was hypothesized that dysfunction in the BDNF-TRPC3 interaction may contribute to the pathomechanism of Rett syndrome by causing abnormal dendritic spine density [178]. 444 23. ARE BRAIN TRPs VIABLE TARGETS FOR CURING NEURODEGENERATIVE DISORDERS

This model is supported by findings in mice that lack the methyl-CpG-binding protein-2 (Mecp2), a model of Rett syndrome. In symptomatic Mecp2 mutant mice, membrane currents and dendritic Ca2+ signals evoked by recombinant BDNF are impaired. In the hippocampus of Mecp2 mutants, both TRPC3 and TRPC6 are decreased. BDNF mRNA and protein levels are also lower in Mecp2 mutant hippocampus and dentate gyrus granule cells. Chromatin immunoprecipitation suggest that Trpc3 is a target of Mecp2 transcriptional regulation. According to these results, correction of the impaired BDNF-TRPC6 signaling is a potential therapeutic strategy in Rett syndrome [179].

Bipolar Disorder Two indirect findings implicate TRPM2 in the pathogenesis of bipolar disorder. First, B-lymphoblast cell lines established from bipolar disorder patients when exposed to rotenone (a broad-spectrum, neurotoxic pesticide and a known activator of TRPM2) exhibit reduced cell viability compared to healthy controls [180]. Second, in case-control studies, a number of SNPs in the TRPM2 gene were reported to increase the risk for developing bipolar disorder. Interestingly, the C-T-A haplotype of SNPs rs1618355, rs933151, and rs749909 was significantly associated with early age at onset of the disease [181].

THE “ADDICTIVE” TRP CHANNELS

Cocaine Abuse Two members of the canonical TRP subfamily, TRPC1 and TRPC6, have been linked to cocaine abuse and addiction. Conditional forebrain Trpc5 knockdown mice exhibit an increase in self-administration of cocaine without prior operant training. This observation was interpreted to imply a negative control of TRPC5 over addictive behavior [182]. Mice whose brain expresses the HIV-1 Tat protein show a heightened response to cocaine and appear to be vulnerable to relapse. This observation establishes a connection between HIV infection and drug abuse. Pretreatment of rat hippocampal neuronal progenitor cells with platelet- derived growth factor-BB (PDGF-BB) restores proliferation that had been impaired by HIV-1 Tat. TRPC1 appears to be a downstream target for PDGF-BB. These findings highlight TRPC1 as a novel target that regulates cell proliferation mediated by PDGF-BB with implications for therapeutic intervention in cocaine addiction [183].

Opioid Addiction In the rat, TRPC6 is expressed in CSF-contacting neurons, with increased levels found during morphine dependence and withdrawal [184]. In mice, repeated morphine administration up-regulates TRPV1 expression in the dorsal striatum. TRPV1 agonists potentiate, whereas TRPV1 antagonists attenuate, morphine reward in the conditioned place preference paradigm. TRPV1 antagonist treatment also suppresses morphine-induced increases in μ-opioid receptor binding. Thus, brain TRPV1 may represent a novel therapeutic target to treat morphine-addictive disorders [185]. TRP Channels in Stroke and Traumatic Brain Injury 445 TRP CHANNELS IN STROKE AND TRAUMATIC BRAIN INJURY

Stroke Intracerebral hemorrhage (hemorrhagic stroke) is a devastating event that stems from the rupture of blood vessels in the brain, with the subsequent accumulation of blood in the parenchyma. There is good evidence that blood-derived factors induce excessive inflammatory responses that, in turn, contribute to the progression of brain injury. When the blood-brain barrier is disrupted, thrombin leaks to the brain parenchyma to cause astrogliosis. Furthermore, thrombin up-regulates TRPC3. In mice, the TRPC3 blocker Pyr3 improves functional outcome and attenuates astrogliosis after hemorrhagic stroke. These findings highlight TRPC3 as a novel therapeutic target for the treatment of hemorrhagic brain injury [186]. Intracerebroventricular injection of hyperforin, a TRPC6 activator, ameliorates the brain damage that occurs after transient focal cerebral ischemia in rats. When applied immediately after middle cerebral artery (MCA) occlusion, hyperforin reduced the infarct volumes and increased neurologic scores at 24 hours after reperfusion. These beneficial effects were accompanied by elevated TRPC6 activity [187]. Resveratrol is another natural product that protects the brain from ischemia/reperfusion injury. In the transient MCA occlusion model, resveratrol protects neurons by inhibiting the proteolysis of TRPC6 by calpain. There are striking similarities between hyperforin and resveratrol actions, suggesting a shared biochemical pathway. For example, both hyperforin and resveratrol elevate TRPC6 and CREB (cAMP-response element binding protein) activities. When the MEK (MAPK/ERK kinase) or CaMK-IV activity was inhibited, the neuroprotective effect of resveratrol was lost. Taken together, these findings suggest that hyperforin and resveratrol protect the brain from ischemic injury through the TRPC6-MEK-CREB and TRPC6-CaMKIV-CREB pathways [188]. Excitotoxicity induced by NMDA receptor-mediated intracellular Ca2+ overload is a major cause of delayed neuronal death after ischemic stroke. As we saw earlier, TRPC6 protects neurons from ischemic brain damage. For example, the infarct volume in Trpc6 transgenic mice is much smaller than that in wild-type littermates after MCA occlusion. The Trpc6 transgenic mice also had better behavior performance and lower mortality than the control animals. These observations imply that increasing TRPC6 activity could be a potential strategy for stroke prevention and therapy [189]. Chronic cerebral hypoperfusion is a risk factor for the development of vascular dementia. In men, vascular dementia accounts for 20-40% of all dementia cases. In mice, bilateral ca- rotid artery occlusion is used to induce global chronic cerebral hypoperfusion. These animals show impairment in locomotion and motor coordination, as well as deficits in learning and memory formation. TRPV1 antagonists are protective against hypoperfusion-induced motor coordination impairment and cognitive decline [190]. Of note, following MCA occlusion the expression of TRPV1 is significantly increased in the hippocampus [191]. Inducing mild hypothermia is a promising therapeutic approach in stroke patients. In rodents, capsaicin evokes a rapidly developing and transient drop in body temperature. Rinvanil is a potent, synthetic capsaicin congener. Intraperitoneal rinvanil administration induces mild hypothermia and protects neurons from the ischemic damage that develops following transient MCA occlusion [192]. This observation highlights the therapeutic potential of TRPV1-mediated hypothermia to minimize brain damage in stroke patients. 446 23. ARE BRAIN TRPs VIABLE TARGETS FOR CURING NEURODEGENERATIVE DISORDERS

Incensole acetate protects against the neurological deficit caused by head trauma. In mice, incensole acetate also attenuates ischemic neuronal damage and reperfusion injury by limiting neuro-inflammation. The protective effects of incensole acetate were absent in Trpv3 deficient mice and were reversed by TRPV3 inhibitors [193]. In adult rat cortical and hippocampal astrocytes, TRPV4 expression is markedly increased 7 days after hypoxia-ischemia insult. The increase in TRPV4 expression coincides with the development of astrogliosis (astrogliosis is an abnormal astrocytic proliferation that occurs in response to neuronal death). In brain slices or cultured hippocampal astrocytes, the TRPV4 agonist 4αPDD elevates Ca2+ and activates a cationic current that is prevented by the universal TRP channel antagonist, RR. Importantly, hypoxic-ischemic injury augments the responses of astrocytes to 4αPDD. These observations imply a role for TRPV4 in the development of astrogliosis that follows ischemic insults [194]. Of note, microglial cells also express TRPM2 with increased levels following ischemic brain injury [195]. In addition to astrocytes, TRPV4 is expressed in hippocampal CA1 pyramidal neurons. These neurons are activated by both 4αPDD and hyposmotic insult; these responses were prevented by the TRPV4 antagonist, HC-067047, and the NMDA-R antagonist, AP-5, indicating that TRPV4 activation potentiates NMDA-R response. When given 60 minutes after MCA occlusion, HC-067047 reduced the size of the cerebral infarct. These findings indicate that activation of TRPV4 increases NMDA-R function, which in turn potentiates glutamate excitotoxicity. If this hypothesis holds true, TRPV4 antagonists may exert potent neuroprotection against cerebral ischemia injury [196]. In stroke models, TRPM2 shows a very unusual behavior. TRPM2 expression is similar in male and female control rats. During reperfusion following in vitro ischemia, TRPM2 channels get activated only in neurons of male animals [197]. In keeping with this finding, shRNA- mediated knockdown of Trpm2 expression protected male, but not female, neurons following in vitro oxygen-glucose deprivation. Importantly, the TRPM2 inhibitor, clotrimazole, reduced infarct volume in male mice, while having no effect in female animals [198]. If these observations hold true in humans, TRPM2 represents a potential target for protection against cerebral ischemia in male stroke patients.

Traumatic Brain Injury Traumatic brain injury initiates a cascade of complex biochemical changes, including oxidative stress, edema, inflammation, and excitotoxicity. Deferoxamine is a chelator that attenuates Fe2+-induced toxicity in rats with traumatic brain injury. These animals show a significant increase in brain Fe2+ on day 28, accompanied by a corresponding elevation in TRPC6 levels. Deferoxamine ameliorated the deficits in spatial learning and memory, supporting the notion that deferoxamine may reduce brain injury accentuated by Fe2+ overload by interfering with a biochemical pathway that involves TRPC6 [199]. In an impact-acceleration model of diffuse traumatic brain injury in rats, TRPM2 mRNA and protein levels are elevated in the cerebral cortex [200]. Microvascular failure exacerbates the damage caused by traumatic brain injury via a combination of cerebral edema formation and progressive secondary hemorrhage. In rat models of traumatic brain injury, two transport proteins have been identified in brain ConcluSIONS 447 endothelial cells as critical mediators of edema formation: the constitutively expressed Na+-K+-Cl− cotransporter, NKCC1, and the trauma/ischemia-induced SUR1/TRPM4 channel. Whereas NKCC1 function requires ATP, activation of SUR1/TRPM4 occurs only after ATP is depleted. With critical ATP depletion, sustained opening of SUR1/TRPM4 channels may result in the oncotic death of endothelial cells, leading to capillary fragmentation and progressive secondary hemorrhage. Bumetanide and glibenclamide are antidiabetic drugs that inhibit NKCC1 and the SUR1/TRPM4 channel, respectively. In animal models of traumatic brain injury, bumetanide and glibenclamide protects neurons by preventing injury-associated capillary failure [201–204]. Following spinal cord injury, Trpm4 mRNA and protein are up-regulated in capillaries, preceding their fragmentation and formation of petechial hemorrhages. Trpm4 gene suppression (by antisense) or deletion (Trpm4 knockout mice) preserved capillary structural integrity and eliminated secondary hemorrhage. In these TRPM4-deficient animals the lesion volume was reduced and the neurological function was improved compared to controls [205]. In cerebral arteries, TRPM4 might be a regulator of the myogenic constrictor response that occurs in response to increases in intravascular pressure, the so-called Bayliss effect. In rats, suppression of cerebrovascular TRPM4 expression by antisense oligonucleotides reduces myogenic constriction by 70% to 85% [206]. In mice, suppressing the expression of Trpm7 in hippocampal CA1 neurons confers resistance to ischemic cell death [207]. In these animals, hypoxia increases Mg2+ via TRPM7 in the hippocampus, and this contributes to cell death [208]. In men, conversely, depletion of intracellular Mg2+ is known to occur after stroke when it heralds poor neurological outcome. TRPM7 and TRPM6 play important roles in regulating Mg2+ homeostasis; whether or not these channels contribute to neuronal injury remains controversial [209]. To determine whether TRPM7 gene variants may increase (or decrease) the risk of ischemic stroke, a large cohort (14 916) of healthy American men were genotyped for 16 SNPs in the TRPM7 gene. Of these men, 245 subsequently suffered ischemic stroke. All SNPs were in Hardy-Weinberg equilibrium. Overall allele, genotype, and haplotype distributions were similar between cases and controls. Furthermore, marker-by-marker conditional logistic-regression analysis adjusted for potential risk factors showed no evidence for an association between any of the SNPs tested and ischemic stroke [210].

CONCLUSIONS

Brain TRP channels represent a challenging but potentially lucrative area of research. There are a number of hurdles for drug development. For example, some TRP channels (e.g., TRPC1) implicated in brain disorders are ubiquitously expressed and are involved in various physiological responses. It remains to be seen if these channels can be modulated without causing unacceptable adverse effects. Also, many TRP channels may exist both as homomeric and heteromeric channels. Thus, it will be necessary to search for compounds that are selective for channels composed of different sets of subunits. We hope that this review will encourage further studies into brain TRPs and lead to the synthesis of compounds that will find their way to the clinics. 448 23. ARE BRAIN TRPs VIABLE TARGETS FOR CURING NEURODEGENERATIVE DISORDERS References [1] Talavera K, Nilius B, Voets T. Neuronal TRP channels: thermometers, pathfinders and life-savers. Trends Neurosci 2008;31(6):287–95. [2] Nilius B, Szallasi A. Transient receptor potential channels as drug targets: from the science of basic research to the art of medicine. Pharmacol Rev 2014;66:676–814. [3] Vennekens R, Menigoz A, Nilius B. TRPs in the brain. Rev Physiol Biochem Pharmacol 2012;163:27–64. [4] Nilius B. Transient receptor potential (TRP) channels in the brain: the good and the ugly. Eur Rev 2012;20:343–55. [5] Tai Y, Feng S, Du W, Wang Y. Functional roles of TRPC channels in the developing brain. Pflugers Arch 2009;458(2):283–9. [6] Greka A, Navarro B, Oancea E, Duggan A, Clapham DE. TRPC5 is a regulator of hippocampal neurite length and growth cone morphology. Nat Neurosci 2003;6(8):837–45. [7] Moran MM, Xu H, Clapham DE. TRP ion channels in the nervous system. Curr Opin Neurobiol 2004;14(3):362–9. [8] Bezzerides VJ, Ramsey IS, Kotecha S, Greka A, Clapham DE. Rapid vesicular translocation and insertion of TRP channels. Nat Cell Biol 2004;6(8):709–20. [9] Hong K, Nishiyama M, Henley J, Tessier-Lavigne M, Poo M. Calcium signalling in the guidance of nerve growth by netrin-1. Nature 2000;403(6765):93–8. [10] Nishiyama M, Hoshino A, Tsai L, Henley JR, Goshima Y, Tessier-Lavigne M, et al. Cyclic AMP/GMP- dependent modulation of Ca2+ channels sets the polarity of nerve growth-cone turning. Nature 2003;423:990–5. [11] Henley J, Poo MM. Guiding neuronal growth cones using Ca2+ signals. Trends Cell Biol 2004;14:320–30. [12] Gasperini RJ, Choi-Lundberg D, Thompson MJ, Mitchell CB, Foa L. Homer regulates calcium signalling in growth cone turning. Neural Dev 2009;4:29. [13] Rex CS, Lin CY, Kramar EA, Chen LY, Gall CM, Lynch G. Brain-derived neurotrophic factor promotes long- term potentiation-related cytoskeletal changes in adult hippocampus. J Neurosci 2007;27:3017–29. [14] Kramar EA, Chen LY, Lauterborn JC, Simmons DA, Gall CM, Lynch G. BDNF upregulation rescues synaptic plasticity in middle-aged ovariectomized rats. Neurobiol Aging 2012;33:708–19. [15] Gokce O, Runne H, Kuhn A, Luthi-Carter R. Short-term striatal gene expression responses to brain-derived neurotrophic factor are dependent on MEK and ERK activation. PLoS One 2009;4(4):e5292. [16] Wen Z, Han L, Bamburg JR, Shim S, Ming GL, Zheng JQ. BMP gradients steer nerve growth cones by a balanc- ing act of LIM kinase and Slingshot phosphatase on ADF/cofilin. J Cell Biol 2007;178(1):107–19. [17] Hutchins BI, Li L, Kalil K. Wnt/calcium signaling mediates axon growth and guidance in the developing corpus callosum. Dev Neurobiol 2010;71:269–83. [18] Li L, Hutchins BI, Kalil K. Wnt5a induces simultaneous cortical axon outgrowth and repulsive turning through distinct signaling mechanisms. Sci Signal 2010;3(147):t2. [19] Heo DK, Chung WY, Park HW, Yuan JP, Lee MG, Kim JY. Opposite regulatory effects of TRPC1 and TRPC5 on neurite outgrowth in PC12 cells. Cell Signal 2012;24:899–906. [20] Kumar S, Chakraborty S, Barbosa C, Brustovetsky T, Brustovetsky N, Obukhov AG. Mechanisms controlling neurite outgrowth in a pheochromocytoma cell line: The role of TRPC channels. J Cell Physiol 2012;227:1408–19. [21] Henle SJ, Wang G, Liang E, Wu M, Poo MM, Henley JR. Asymmetric PI(3,4,5)P3 and akt signaling mediates chemotaxis of axonal growth cones. J Neurosci 2011;31:7016–27. [22] Paez PM, Fulton D, Spreuer V, Handley V, Campagnoni AT. Modulation of canonical transient receptor potential channel 1 in the proliferation of oligodendrocyte precursor cells by the golli products of the myelin basic protein gene. J Neurosci 2011;31:3625–37. [23] Petersson ME, Fransen E. Long-lasting small-amplitude TRP-mediated dendritic depolarizations in CA1 pyramidal neurons are intrinsically stable and originate from distal tuft regions. Eur J Neurosci 2012;36:2917–25. [24] El-Hassar L, Hagenston AM, Bertetto D’Angelo L, Yeckel M. Metabotropic glutamate receptors regulate hippocampal CA1 pyramidal neuron excitability via Ca2+ wave-dependent activation of SK and TRPC channels. J Physiol 2011;589:3211–29. [25] Li M, Chen C, Zhou Z, Xu S, Yu Z. A TRPC1-mediated increase in store-operated Ca(2+) entry is required for the proliferation of adult hippocampal neural progenitor cells. Cell Calcium 2012;51:486–96. [26] Petersson ME, Yoshida M, Fransen EA. Low-frequency summation of synaptically activated transient receptor potential channel-mediated depolarizations. Eur J Neurosci 2011;34:578–93. [27] Chae HG, Ahn SJ, Hong YH, Chang WS, Kim J, Kim SJ. Transient receptor potential canonical channels regulate the induction of cerebellar long-term depression. J Neurosci 2012;32:12909–14. REFERENCES 449

[28] Stroh O, Freichel M, Kretz O, Birnbaumer L, Hartmann J, Egger V. NMDA receptor-dependent synaptic activation of TRPC channels in olfactory bulb granule cells. J Neurosci 2012;32:5737–46. [29] Tabarean IV. Persistent histamine excitation of glutamatergic preoptic neurons. PLoS One 2012;7:e47700. [30] Williams KW, Sohn JW, Donato Jr J, Lee CE, Zhao JJ, Elmquist JK, et al. The acute effects of leptin require PI3K signaling in the hypothalamic ventral premammillary nucleus. J Neurosci 2011;31:13147–56. [31] Huang WC, Young JS, Glitsch MD. Changes in TRPC channel expression during postnatal development of cerebellar neurons. Cell Calcium 2007;42(1):1–10. [32] Kato AS, Knierman MD, Siuda ER, Isaac JT, Nisenbaum ES, Bredt DS. Glutamate receptor delta2 associates with metabotropic glutamate receptor 1 (mGluR1), protein kinase cgamma, and canonical transient receptor potential 3 and regulates mGluR1-mediated synaptic transmission in cerebellar purkinje neurons. J Neurosci 2012;32:15296–308. [33] Kim Y, Wong AC, Power JM, Tadros SF, Klugmann M, Moorhouse AJ, et al. Alternative splicing of the TRPC3 ion channel calmodulin/IP3 receptor-binding domain in the hindbrain enhances cation flux. J Neurosci 2012;32:11414–23. [34] Amaral MD, Pozzo-Miller L. TRPC3 channels are necessary for brain-derived neurotrophic factor to activate a nonselective cationic current and to induce dendritic spine formation. J Neurosci 2007;27(19):5179–89. [35] Kim SJ. TRPC3 channel underlies cerebellar long-term depression. Cerebellum 2013;12:334–7. [36] Nelson C, Glitsch MD. Lack of kinase regulation of canonical transient receptor potential (TRPC) 3 dependent currents in rat cerebellar Purkinje cells. J Biol Chem 2012;287:6326–35. [37] Mitsumura K, Hosoi N, Furuya N, Hirai H. Disruption of metabotropic glutamate receptor signalling is a major defect at cerebellar parallel fibre-Purkinje cell synapses in staggerer mutant mice. J Physiol 2011;589:3191–209. [38] Hartmann J, Dragicevic E, Adelsberger H, Henning HA, Sumser M, Abramowitz J, et al. TRPC3 channels are required for synaptic transmission and motor coordination. Neuron 2008;59(3):392–8. [39] Jia Y, Zhou J, Tai Y, Wang Y. TRPC channels promote cerebellar granule neuron survival. Nat Neurosci 2007;10:559–67. [40] Sossin WS, Barker PA. Something old, something new: BDNF-induced neuron survival requires TRPC channel function. Nat Neurosci 2007;10(5):537–8. [41] Zhou FM, Lee CR. Intrinsic and integrative properties of substantia nigra pars reticulata neurons. Neuroscience 2011;15:69–94. [42] Berg AP, Sen N, Bayliss DA. TRPC3/C7 and Slo2.1 are molecular targets for metabotropic glutamate receptor signaling in rat striatal cholinegrgic interneurons. J Neurosci 2007;27:8845–56. [43] Su B, Ji YS, Sun XL, Liu XH, Chen ZY. Brain-derived neurotrophic factor (BDNF)-induced mitochondrial motility arrest and presynaptic docking contribute to BDNF-enhanced synaptic transmission. J Biol Chem 2014;289:1213–26. [44] Streifel K, Miller J, Mouneimne R, Tjalkens RB. Manganese inhibits ATP-induced calcium entry through the transient receptor potential channel TRPC3 in astrocytes. Neurotoxicology 2013;34:160–6. [45] Wang S, Zhang AP, Kurada L, Matsui T, Lei S. Cholecystokinin facilitates neuronal excitability in the entorhinal cortex via activation of TRPC-like channels. J Neurophysiol 2011;106:1515–24. [46] Zhang Z, Reboreda A, Alonso A, Barker PA, Seguela P. TRPC channels underlie cholinergic plateau potentials and persistent activity in entorhinal cortex. Hippocampus 2011;21(4):386–97. [47] Yan HD, Villalobos C, Andrade R. TRPC channels mediate a muscarinic receptor-induced after depolarization in cerebral cortex. J Neurosci 2009;29(32):10038–46. [48] Zhang L, Kolaj M, Renaud LP. GIRK-like and TRPC-like conductances mediate thyrotropin-releasing hormone-induced increases in excitability in thalamic paraventricular nucleus neurons. Neuropharmacology 2013;72:106–15. [49] Qiu J, Fang Y, Bosch MA, Ronnekleiv OK, Kelly MJ. Guinea pig kisspeptin neurons are depolarized by leptin via activation of TRPC channels. Endocrinology 2011;152:1503–14. [50] Eisinger BE, Zhao C, Driessen TM, Saul MC, Gammie SC. Large scale expression changes of genes related to neuronal signaling and developmental processes found in lateral septum of postpartum outbred mice. PLoS One 2013;8:e63824. [51] Tian J, Thakur DP, Lu Y, Zhu Y, Freichel M, Flockerzi V, et al. Dual depolarization responses generated within the same lateral septal neurons by TRPC4-containing channels. Pflugers Arch 2014;466:1301–16, PMID: 24121765. 450 23. ARE BRAIN TRPs VIABLE TARGETS FOR CURING NEURODEGENERATIVE DISORDERS

[52] Puram SV, Riccio A, Koirala S, Ikeuchi Y, Kim AH, Corfas G, et al. A TRPC5-regulated calcium signaling pathway controls dendrite patterning in the mammalian brain. Genes Dev 2011;25:2659–73. [53] He Z, Jia C, Feng S, Zhou K, Tai Y, Bai X, et al. TRPC5 channel is the mediator of neurotrophin-3 in regulating dendritic growth via camkiialpha in rat hippocampal neurons. J Neurosci 2012;32:9383–95. [54] Kaczmarek JS, Riccio A, Clapham DE. Calpain cleaves and activates the TRPC5 channel to participate in semaphorin 3A-induced neuronal growth cone collapse. Proc Natl Acad Sci USA 2012;109:7888–92. [55] Xu P, Xu J, Li Z, Yang Z. Expression of TRPC6 in renal cortex and hippocampus of mouse during postnatal development. PLoS One 2012;7:e38503. [56] Zhou J, Du W, Zhou K, Tai Y, Yao H, Jia Y, et al. Critical role of TRPC6 channels in the formation of excitatory synapses. Nat Neurosci 2008;11:741–3. [57] Beis D, Schwarting RK, Dietrich A. Evidence for a supportive role of classical transient receptor potential 6 (TRPC6) in the exploration behavior of mice. Physiol Behav 2011;102:245–50. [58] Tu P, Kunert-Keil C, Lucke S, Brinkmeier H, Bouron A. Diacylglycerol analogues activate second messenger-operated calcium channels exhibiting TRPC-like properties in cortical neurons. J Neurochem 2009;108:126–38. [59] Gibon J, Deloulme JC, Chevallier T, Ladeveze E, Abrous DN, Bouron A. The antidepressant hyperforin increases the phosphorylation of CREB and the expression of TrkB in a tissue-specific manner. Int J Neuropsychopharmacol 2013;16:189–98. [60] Heiser JH, Schuwald AM, Sillani G, Ye L, Muller WE, Leuner K. TRPC6 channel-mediated neurite outgrowth in PC12 cells and hippocampal neurons involves activation of RAS/MEK/ERK, PI3K and CAMKIV signaling. J Neurochem 2013;127:303–13. [61] Leuner K, Li W, Amaral MD, Rudolph S, Calfa G, Schuwald AM, et al. Hyperforin modulates dendritic spine morphology in hippocampal pyramidal neurons by activating Ca(2+) -permeable TRPC6 channels. Hippocampus 2013;23:40–52. [62] Shen H, Pan J, Pan L, Zhang N. TRPC6 inhibited NMDA current in cultured hippocampal neurons. Neuromolecular Med 2013;15:389–95. [63] Dasari S, Abramowitz J, Birnbaumer L, Gulledge AT. Do canonical transient receptor potential channels mediate cholinergic excitation of cortical pyramidal neurons? Neuroreport 2013;24:550–4. [64] Ben-Mabrouk F, Tryba AK. Substance P modulation of TRPC3/7 channels improves respiratory rhythm regularity and ICAN-dependent pacemaker activity. Eur J Neurosci 2010;31:1219–32. [65] Ben-Mabrouk F, Amos LB, Tryba AK. Metabotropic glutamate receptors (mGluR5) activate transient receptor potential canonical channels to improve the regularity of the respiratory rhythm generated by the pre- Botzinger complex in mice. Eur J Neurosci 2012;35:1725–37. [66] Martins D, Tavares I, Morgado C. "Hotheaded": The role OF TRPV1 IN brain functions. Neuropharmacology 2014;85C:151–7. [67] Edwards JG. TRPV1 in the central nervous system: synaptic plasticity, function, and pharmacological implications. Prog Drug Res 2014;68:77–104. [68] Cavanaugh DJ, Chesler AT, Sigal YM, Yamanaka H, Grant R, et al. Trpv1 reporter mice reveal highly restricted brain distribution and functional expression in arteriolar smooth muscle cells. J Neurosci 2011;31:5067–77. [69] Tsurugizawa T, Nogusa Y, Ando Y, Uneyama H. Different TRPV1-mediated brain responses to intragastric infusion of capsaicin and capsiate. Eur J Neurosci 2013;38(11):3628–35. [70] Fogaca MV, Gomes FV, Moreira FA, Guimaraes FS, Aguiar DC. Effects of glutamate NMDA and TRPV1 receptor antagonists on the biphasic responses to anandamide injected into the dorsolateral periaqueductal grey of Wistar rats. Psychopharmacology (Berl) 2012;226:579–87. [71] Umathe SN, Manna SS, Jain NS. Endocannabinoid analogues exacerbate marble-burying behavior in mice via TRPV1 receptor. Neuropharmacology 2012;62:2024–33. [72] John CS, Currie PJ. N-Arachidonoyl-serotonin in the basolateral amygdala increases anxiolytic behavior in the elevated plus maze. Behav Brain Res 2012;233:382–8. [73] Kim J, Kwon JT, Kim HS, Josselyn SA, Han JH. Memory recall and modifications by activating neurons with elevated CREB. Nat Neurosci 2014;17:65–72. [74] Kulisch C, Albrecht D. Effects of single swim stress on changes in TRPV1-mediated plasticity in the amygdala. Behav Brain Res 2013;236:344–9. REFERENCES 451

[75] Terzian AL, Dos Reis DG, Guimaraes FS, Correa FM, Resstel LB. Medial prefrontal cortex transient receptor potential vanilloid type 1 (TRPV1) in the expression of contextual fear conditioning in Wistar rats. Psychopharmacology (Berl) 2014;231:149–57. [76] Aguiar DC, Terzian AL, Guimaraes FS, Moreira FA. Anxiolytic-like effects induced by blockade of transient receptor potential vanilloid type 1 (TRPV1) channels in the medial prefrontal cortex of rats. Psychopharmacology (Berl) 2009;205:217–25. [77] Manna SS, Umathe SN. Transient receptor potential vanilloid 1 channels modulate the anxiolytic effect of diazepam. Brain Res 2011;1425:75–82. [78] Liao HT, Lee HJ, Ho YC, Chiou LC. Capsaicin in the periaqueductal gray induces analgesia via metabotropic glutamate receptor-mediated endocannabinoid retrograde disinhibition. Br J Pharmacol 2011;163:330–45. [79] Puente N, Cui Y, Lassalle O, Lafourcade M, Georges F, Venance L, et al. Polymodal activation of the endocannabinoid system in the extended amygdala. Nat Neurosci 2011;14:1542–7. [80] Musella A, De Chiara V, Rossi S, Cavasinni F, Castelli M, Cantarella C, et al. Transient receptor potential vanilloid 1 channels control acetylcholine/2-arachidonoylglicerol coupling in the striatum. Neuroscience 2010;167:864–71. [81] Musella A, De Chiara V, Rossi S, Prosperetti C, Bernardi G, Maccarrone M, et al. TRPV1 channels facilitate glutamate transmission in the striatum. Mol Cell Neurosci 2008;40:89–97. [82] Huang WX, Min JW, Liu YQ, He XH, Peng BW. Expression of TRPV1 in the C57BL/6 mice brain hippocampus and cortex during development. Neuroreport 2014;25:379–85. [83] Puente N, Reguero L, Elezgarai I, Canduela MJ, Mendizabal-Zubiaga J, Ramos-Uriarte A, et al. The transient receptor potential vanilloid-1 is localized at excitatory synapses in the mouse dentate gyrus. Brain Struct Funct 2014, PMID 24487914. [84] Bennion D, Jensen T, Walther C, Hamblin J, Wallmann A, Couch J, et al. Transient receptor potential vanilloid 1 agonists modulate hippocampal CA1 LTP via the GABAergic system. Neuropharmacology 2011;61:730–8. [85] Jensen T, Edwards JG. Calcineurin is required for TRPV1-induced long-term depression of hippocampal interneurons. Neurosci Lett 2012;510:82–7. [86] Mori F, Ribolsi M, Kusayanagi H, Monteleone F, Mantovani V, Buttari F, et al. TRPV1 channels regulate cortical excitability in humans. J Neurosci 2012;32:873–9. [87] Zschenderlein C, Gebhardt C, von Bohlen Und Halbach O, Kulisch C, Albrecht D. Capsaicin-induced changes in LTP in the lateral amygdala are mediated by TRPV1. PLoS One 2011;6(1):e16116. [88] Hermes ML, Kolaj M, Coderre EM, Renaud LP. Gastrin-releasing peptide acts via postsynaptic BB2 receptors to modulate inward rectifier K+ and TRPV1-like conductances in rat paraventricular thalamic neurons. J Physiol 2013;591:1823–39. [89] Grueter BA, Brasnjo G, Malenka RC. Postsynaptic TRPV1 triggers cell-type specific LTD in the nucleus accumbens. Nat Neurosci 2010;13:1519–25. [90] Shibasaki K, Murayama N, Ono K, Ishizaki Y, Tominaga M. TRPV2 enhances axon outgrowth through its activation by membrane stretch in developing sensory and motor neurons. J Neurosci 2010;30(13):4601–12. [91] Xu HX, Ramsey IS, Kotecha SA, Moran MM, Chong JA, Lawson D, et al. TRPV3 is a calcium-permeable temperature-sensitive cation channel. Nature 2002;418:181–6. [92] Brown TE, Chirila AM, Schrank BR, Kauer JA. Loss of interneuron LTD and attenuated pyramidal cell LTP in Trpv1 and Trpv3 KO mice. Hippocampus 2013;23:662–71. [93] Lee JC, Choe SY. Age-related changes in the distribution of transient receptor potential vanilloid 4 channel (TRPV4) in the central nervous system of rats. J Mol Histol 2014;45:497–505, PMID: 24917364. [94] Jang Y, Jung J, Kim H, Oh J, Jeon JH, Jung S, et al. Axonal neuropathy-associated TRPV4 regulates neurotrophic factor-derived axonal growth. J Biol Chem 2012;287:6014–24. [95] Li L, Yin J, Liu C, Chen L. Hypertonic stimulation inhibits synaptic transmission in hippocampal slices through decreasing pre-synaptic voltage-gated calcium current. Neurosci Lett 2012;507:106–11. [96] Boychuk CR, Zsombok A, Tasker JG, Smith BN. Rapid glucocorticoid-induced activation of TRP and CB1 receptors causes biphasic modulation of glutamate release in gastric-related hypothalamic preautonomic neurons. Front Neurosci 2013;7:3. [97] Li L, Yin J, Jie PH, Lu ZH, Zhou LB, Chen L. Transient receptor potential vanilloid 4 mediates hypotonicity- induced enhancement of synaptic transmission in hippocampal slices. CNS Neurosci Ther 2013;19:854–62. [98] Dunn KM, Hill-Eubanks DC, Liedtke WB, Nelson MT. TRPV4 channels stimulate Ca2+-induced Ca2+ release in astrocytic endfeet and amplify neurovascular coupling responses. Proc Natl Acad Sci USA 2013;110:6157–62. 452 23. ARE BRAIN TRPs VIABLE TARGETS FOR CURING NEURODEGENERATIVE DISORDERS

[99] Shibasaki K, Ikenaka K, Tamalu F, Tominaga M, Ishizaki Y. A novel subtype of astrocytes expressing TRPV4 regulates neuronal excitability via release of gliotransmitters. J Biol Chem 2014;289:14470–80, PMID: 24737318. [100] Jang Y, Lee MH, Lee J, Jung J, Lee SH, Yang DJ, et al. TRPM2 mediates the lysophosphatidic acid-induced neurite retraction in the developing brain. Pflugers Arch 2014;466:1987–98, PMID: 24413888. [101] Lee CR, Machold RP, Witkovsky P, Rice ME. TRPM2 channels are required for NMDA-induced burst fir-

ing and contribute to H2O2-dependent modulation in substantia nigra pars reticulata GABAergic neurons. J Neurosci 2013;33:1157–68. [102] Chung KKH, Freestone PS, Lipski J. Expression and functional properties of TRPM2 channels in dopaminergic neurons of the substantia nigra of the rat. J Neurophysiol 2011;106:2865–75. [103] Belrose JC, Xie YF, Gierszewski LJ, Macdonald JF, Jackson MF. Loss of glutathione homeostasis associated with neuronal senescence facilitates TRPM2 channel activation in cultured hippocampal pyramidal neurons. Mol Brain 2012;5:11. [104] Olah ME, Jackson MF, Li H, Perez Y, Sun HS, Kiyonaka S, et al. Ca2+-dependent induction of TRPM2 currents in hippocampal neurons. J Physiol 2009;587:965–79. [105] Xie YF, Belrose JC, Lei G, Tymianski M, Mori Y, MacDonald JF, et al. Dependence of NMDA/GSK3beta mediated metaplasticity on TRPM2 channels at hippocampal CA3-CA1 synapses. Mol Brain 2012;4:44. [106] Zamudio-Bulcock PA, Everett J, Harteneck C, Valenzuela CF. Activation of steroid-sensitive TRPM3 channels potentiates glutamatergic transmission at cerebellar Purkinje neurons from developing rats. J Neurochem 2011;119:474–85. [107] Zamudio-Bulcock PA, Valenzuela CF. Pregnenolone sulfate increases glutamate release at neonatal climbing fiber-to-purkinje cell synapses. Neuroscience 2011;175:24–36. [108] Lee KH, Cho JH, Choi IS, Park HM, Lee MG, Choi BJ, et al. Pregnenolone sulfate enhances spontaneous glutamate release by inducing presynaptic Ca2+-induced Ca2+ release. Neuroscience 2010;171:106–16. [109] Rubin JE, Hayes JA, Mendenhall JL, Del Negro CA. Calcium-activated nonspecific cation current and synaptic depression promote network-dependent burst oscillations. Proc Natl Acad Sci USA 2009;106:2939–44. [110] Mironov SL, Skorova EY. Stimulation of bursting in pre-Botzinger neurons by Epac through calcium release and modulation of TRPM4 and K-ATP channels. J Neurochem 2011;117:295–308. [111] Mironov SL. Calmodulin and CaMKII mediate emergent bursting activity in the brainstem respiratory network (preBotC). J Physiol 2013;591:1613–30. [112] Mrejeru A, Wei A, Ramirez JM. CAN currents are involved in generation of tonic and bursting activity in dopamine neurons of the substantia nigra pars compacta. J Physiol 2011;589:2497–514. [113] Shpak G, Zylbertal A, Yarom Y, Wagner S. Calcium-activated sustained firing responses distinguish accessory from main olfactory bulb mitral cells. J Neurosci 2012;32:6251–62. [114] Thompson JA, Salcedo E, Restrepo D, Finger TE. Second order input to the medial amygdala from olfactory ensory neurons expressing the transduction channel TRPM5. J Comp Neurol 2012;520:1819–30. [115] Kim YS, Kang E, Makino Y, Park S, Shin JH, Song H, et al. Characterizing the conductance underlying depolarization-induced slow current (DISC) in cerebellar Purkinje cells. J Neurophysiol 2013;109:1174–81. [116] Smirnov AV, Spasov AA, Shmidt MV, Snigur GL, Evsyukov OY, Zheltova AA. Patterns of TRPM7 expression in hypothalamic and hippocampal neurons in modeling of nutritional magnesium deficiency. Bull Exp Biol Med 2014;156(6):736–9. [117] Crawford DC, Moulder KL, Gereau RW, Story GM, Mennerick S. Comparative effects of heterologous TRPV1 and TRPM8 expression in rat hippocampal neurons. PLoS One 2009;4(12):e8166. [118] Lau BK, Karim S, Goodchild AK, Vaughan CW, Drew GM. Menthol enhances phasic and tonic GABA receptor-mediated currents in midbrain periaqueductal grey neurons. Br J Pharmacol 2014;171:2803–13. [119] Koch M, Kreutz S, Bottger C, Grabiec U, Ghadban C, Korf HW, et al. The cannabinoid WIN 55,212-2-mediated protection of dentate gyrus granule cells is driven by CB(1) receptors and modulated by TRPA1 and Ca(v)2.2 channels. Hippocampus 2010;21:554–64. [120] Kimura Y, Mikami Y, Osumi K, Tsugane M, Oka JI, Kimura H. Polysulfides are possible H2S-derived signaling molecules in rat brain. FASEB J 2013;27:2451–7. [121] Shigetomi E, Jackson-Weaver O, Huckstepp RT, O’Dell TJ, Khakh BS. TRPA1 channels are regulators of astrocyte basal calcium levels and long-term potentiation via constitutive d-serine release. J Neurosci 2013;33:10143–53. [122] Jo KD, Lee KS, Lee WT, Hur MS, Kim HJ. Expression of transient receptor potential channels in the ependymal cells of the developing rat brain. Anat Cell Biol 2013;46(1):68–78. REFERENCES 453

[123] Yokoyama T, Ohbuchi T, Saito T, Sudo Y, Fujihara H, Minami K, et al. Allyl isothiocyanates and cinnamaldehyde potentiate miniature excitatory postsynaptic inputs in the supraoptic nucleus in rats. Eur J Pharmacol 2011;655:31–7. [124] Phelan KD, Shwe UT, Abramowitz J, Wu H, Rhee SW, Howell MD, et al. TRPC5 and TRPC1/4 channels contribute to seizure and excitotoxicity by distinct cellular mechanisms. Mol Pharmacol 2013;83:429–38. [125] Phelan KD, Mock MM, Kretz O, Shwe UT, Kozhemyakin M, Greenfield LJ, et al. Heteromeric canonical transient receptor potential 1 and 4 channels play a critical role in epileptiform burst firing and seizure-induced neurodegeneration. Mol Pharmacol 2012;81:384–92. [126] Tai C, Himes DJ, MacVicar BA. Translocation of TRPC5 channels contributes to cholinergic-induced plateau potential in epilepsy. J Neurochem 2009;109(Suppl. 1):277–8. [127] Zhou FW, Roper SN. TRPC3 mediates hyperexcitability and epileptiform activity in immature cortex and experimental cortical dysplasia. J Neurophysiol 2014;111:1227–37. [128] Kim DS, Ryu HJ, Kim JE, Kang TC. The reverse roles of transient receptor potential canonical channel-3 and -6 in neuronal death following pilocarpine-induced status epilepticus. Cell Mol Neurobiol 2013;33:99–109. [129] Ryu HJ, Kim JE, Kim YJ, Kim JY, Kim WI, Choi SY, et al. Endothelial transient receptor potential conical channel (TRPC)-3 activation induces vasogenic edema formation in the rat piriform cortex following status epilepticus. Cell Mol Neurobiol 2013;33:575–85. [130] Zheng F, Phelan KD. The role of canonical transient receptor potential channels in seizure and excitotoxicity. Cells 2014;3:288–303. [131] Gibson HE, Edwards JG, Page RS, Van Hook MJ, Kauer JA. TRPV1 channels mediate long-term depression at synapses on hippocampal interneurons. Neuron 2008;57(5):746–59. [132] Alter BJ, Gereau 4th RW. Hotheaded: TRPV1 as mediator of hippocampal synaptic plasticity. Neuron 2008;57(5):629–31. [133] Gonzalez-Reyes LE, Ladas TP, Chiang CC, Durand DM. TRPV1 antagonist capsazepine suppresses 4-AP- induced epileptiform activity in vitro and electrographic seizures in vivo. Exp Neurol 2013;250:321–32. [134] Fu M, Xie Z, Zuo H. TRPV1: a potential target for antiepileptogenesis. Med Hypotheses 2009;73:100–2. [135] Manna SS, Umathe SN. Involvement of transient receptor potential vanilloid type 1 channels in the proconvulsant effect of anandamide in pentylenetetrazole-induced seizures. Epilepsy Res 2012;100:113–24. [136] Manna SS, Umathe SN. A possible participation of transient receptor potential vanilloid type 1 channels in the antidepressant effect of fluoxetine. Eur J Pharmacol 2012;685:81–9. [137] Vilela LR, Medeiros DC, de Oliveira AC, Moraes MF, Moreira FA. Anticonvulsant effects of N-arachidonoyl- serotonin, a dual FAAH enzyme and TRPV1 channel blocker, on experimental seizures: the roles of cannabinoid CB1 receptors and TRPV1 channels. Basic Clin Pharmacol Toxicol 2014;115:330–4, PMID: 24674273. [138] Shu HF, Yu SX, Zhang CQ, Liu SY, Wu KF, Zang ZL, et al. Expression of TRPV1 in cortical lesions from patients with tuberous sclerosis complex and focal cortical dysplasia type IIb. Brain Dev 2012;35:252–60. [139] Sun FJ, Guo W, Zheng DH, Zhang CQ, Li S, Liu SY, et al. Increased expression of TRPV1 in the cortex and hippocampus from patients with mesial temporal lobe epilepsy. J Mol Neurosci 2012;49:182–93. [140] Kong WL, Min JW, Liu YL, Li JX, He XH, Peng BW. Role of TRPV1 in susceptibility to PTZ-induced seizure following repeated hyperthermia challenges in neonatal mice. Epilepsy Behav 2014;31:276–80. [141] Shirazi M, Izadi M, Amin M, Rezvani ME, Roohbakhsh A, Shamsizadeh A. Involvement of central TRPV1 receptors in pentylenetetrazole and amygdala-induced kindling in male rats. Neurol Sci 2014;35:1235–41, PMID: 24577898. [142] Katano M, Numata T, Aguan K, Hara Y, Kiyonaka S, Yamamoto S, et al. The juvenile myoclonic epilepsy- related protein EFHC1 interacts with the redox-sensitive TRPM2 channel linked to cell death. Cell Calcium 2012;51:179–85. [143] Becker EBE, Oliver PL, Glitsch MD, Banks GT, Achilli F, Hardy A, et al. A point mutation in TRPC3 causes abnormal Purkinje cell development and cerebellar ataxia in moonwalker mice. Proc Natl Acad Sci USA 2009;106:6706–11. [144] Sekerkova G, Kim JA, Nigro MJ, Becker EB, Hartmann J, Birnbaumer L, et al. Early onset of ataxia in moonwalker mice is accompanied by complete ablation of type II unipolar brush cells and Purkinje cell dysfunction. J Neurosci 2013;33:19689–94. [145] Trebak M. The puzzling role of TRPC3 channels in motor coordination. Pflugers Arch 2010;459:369–75. [146] Adachi N, Kobayashi T, Takahashi H, Kawasaki T, Shirai Y, Ueyama T, et al. Enzymological analysis of mutant protein kinase Cgamma causing spinocerebellar ataxia type 14 and dysfunction in Ca2+ homeostasis. J Biol Chem 2008;283:19854–63. 454 23. ARE BRAIN TRPs VIABLE TARGETS FOR CURING NEURODEGENERATIVE DISORDERS

[147] Shuvaev AN, Horiuchi H, Seki T, Goenawan H, Irie T, Iizuka A, et al. Mutant PKC{gamma} in spinocerebellar ataxia type 14 disrupts synapse elimination and long-term depression in Purkinje cells in vivo. J Neurosci 2011;31:14324–34. [148] Becker EB, Fogel BL, Rajakulendran S, Dulneva A, Hanna MG, Perlman SL, et al. Candidate screening of the TRPC3 gene in cerebellar ataxia. Cerebellum 2011;10:296–9. [149] Freestone PS, Chung KK, Guatteo E, Mercuri NB, Nicholson LF, Lipski J. Acute action of rotenone on nigral dopaminergic neurons—involvement of reactive oxygen species and disruption of Ca homeostasis. Eur J Neurosci 2009;30:849–59. [150] Arshad A, Chen X, Cong Z, Qing H, Deng Y. TRPC1 protects dopaminergic SH-SY5Y cells from MPP+, salsolinol, and N-methyl-(R)-salsolinol-induced cytotoxicity. Acta Biochim Biophys Sin (Shanghai) 2014;46:22–30. [151] Park ES, Kim SR, Jin BK. Transient receptor potential vanilloid subtype 1 contributes to mesencephalic dopaminergic neuronal survival by inhibiting microglia-originated oxidative stress. Brain Res Bull 2012;89:92–6. [152] Zhou FW, Matta SG, Zhou FM. Constitutively active TRPC3 channels regulate basal ganglia output neurons. J Neurosci 2008;28(2):473–82. [153] Gonzalez-Aparicio R, Moratalla R. Oleoylethanolamide reduces L-DOPA-induced dyskinesia via TRPV1 receptor in a mouse model of Parkinson s disease. Neurobiol Dis 2013;62:416–25. [154] Narayanan KL, Irmady K, Subramaniam S, Unsicker K, von Bohlen Und Halbach O. Evidence that TRPC1 is involved in hippocampal glutamate-induced cell death. Neurosci Lett 2008;446:117–22. [155] Narayanan KL, Subramaniam S, Bengston CP, Irmady K, Unsicker K, von Bohlen Und Halbach O. Role of transient receptor potential channel 1 (TRPC1) in glutamate-induced cell death in the hippocampal cell line HT22. J Mol Neurosci 2013;52:425–33. [156] Lee JC, Joo KM, Choe SY, Cha CI. Region-specific changes in the immunoreactivity of TRPV4 expression in the central nervous system of SOD1(G93A) transgenic mice as an in vivo model of amyotrophic lateral sclerosis. J Mol Histol 2012;43:625–31. [157] Plato CC, Galasko D, Garruto RM, Plato M, Gamst A, Craig UK, et al. ALS and PDC of Guam: forty-year follow-up. Neurology 2002;58(5):765–73. [158] Cucchiaroni ML, Viscomi MT, Bernardi G, Molinari M, Guatteo E, Mercuri NB. Metabotropic glutamate receptor 1 mediates the electrophysiological and toxic actions of the cycad derivative beta-N-methylamino-l- alanine on substantia nigra pars compacta DAergic neurons. J Neurosci 2010;30(15):5176–88. [159] Hermosura MC, Nayakanti H, Dorovkov MV, Calderon FR, Ryazanov AG, Haymer DS, et al. A TRPM7 variant shows altered sensitivity to magnesium that may contribute to the pathogenesis of two Guamanian neurodegenerative disorders. Proc Natl Acad Sci USA 2005;102(32):11510–15. [160] Hara K, Kokubo Y, Ishiura H, Fukuda Y, Miyashita A, Kuwano R, et al. TRPM7 is not associated with amyotrophic lateral sclerosis-parkinsonism dementia complex in the Kii peninsula of Japan. Am J Med Genet B Neuropsychiatr Genet 2010;153B(1):310–13. [161] Jiang X, Jia LW, Li XH, Cheng XS, Xie JZ, Ma ZW, et al. Capsaicin ameliorates stress-induced Alzheimer’s disease-like pathological and cognitive impairments in rats. J Alzheimers Dis 2013;35:91–105. [162] Bai JZ, Lipski J. Involvement of TRPV4 channels in Abeta-induced hippocampal cell death and astrocytic Ca signalling. Neurotoxicology 2014;41:64–72. [163] Fonfria E, Marshall IC, Boyfield I, Skaper SD, Hughes JP, Owen DE, et al. Amyloid beta-peptide(1-42) and hydrogen peroxide-induced toxicity are mediated by TRPM2 in rat primary striatal cultures. J Neurochem 2005;95(3):715–23. [164] Griffith TN, Varela-Nallar L, Dinamarca MC, Inestrosa NC. Neurobiological effects of hyperforin and its potential in Alzheimer’s disease therapy. Curr Med Chem 2010;17:391–406. [165] Wang J, Lu ZH, Gabius HJ, Rohowsky-Kochan C, Ledeen RW, Wu G. Cross-linking of GM1 ganglioside by galectin-1 mediates regulatory T cell activity involving TRPC5 channel activation: possible role in suppressing experimental autoimmune encephalomyelitis. J Immunol 2009;182(7):4036–45. [166] Rossi S, Furlan R, De Chiara V, Motta C, Studer V, Mori F, et al. Interleukin-1beta causes synaptic hyperexcitability in multiple sclerosis. Ann Neurol 2012;71(1):76–83. [167] Paltser G, Liu XJ, Yantha J, Winer S, Tsui H, Wu P, et al. TRPV1 gates tissue access and sustains pathogenicity in autoimmune encephalitis. Mol Med 2013;19:149–59. [168] Riccio A, Li Y, Tsvetkov E, Gapon S, Yao GL, Smith KS, et al. Decreased anxiety-like behavior and galphaq/11-dependent responses in the amygdala of mice lacking TRPC4 channels. J Neurosci 2014;34:3653–67. REFERENCES 455

[169] Mascarenhas DC, Gomes KS, Nunes-de-Souza RL. Anxiogenic-like effect induced by TRPV1 receptor activation within the dorsal periaqueductal gray matter in mice. Behav Brain Res 2013;250:308–15. [170] Almeida-Santos AF, Moreira FA, Guimaraes FS, Aguiar DC. Role of TRPV1 receptors on panic-like behaviors mediated by the dorsolateral periaqueductal gray in rats. Pharmacol Biochem Behav 2013;105:166–72. [171] Marsch R, Foeller E, Rammes G, Bunck M, Kössl M, Holsboer F, et al. Reduced anxiety, conditioned fear, and hippocampal long-term potentiation in transient receptor potential vanilloid type 1 receptor-deficient mice. J Neurosci 2007;27(4):832–9. [172] Hakimizadeh E, Oryan S, Hajizadeh Moghaddam A, Shamsizadeh A, Roohbakhsh A. Endocannabinoid system and TRPV1 receptors in the dorsal hippocampus of the rats modulate anxiety-like behaviors. Iran J Basic Med Sci 2013;15(3):795–802. [173] Hayase T. Differential effects of TRPV1 receptor ligands against nicotine-induced depression-like behaviors. BMC Pharmacol 2011;11:6. [174] Moussaieff A, Rimmerman N, Bregman T, Straiker A, Felder CC, Shoham S, et al. Incensole acetate, an incense component, elicits psychoactivity by activating TRPV3 channels in the brain. Faseb J 2008;22:3024–34. [175] El-Hassar L, Simen AA, Duque A, Patel KD, Kaczmarek LK, Arnsten AF, et al. Disrupted in schizophrenia 1 modulates medial prefrontal cortex pyramidal neuron activity through cAMP regulation of transient receptor potential C and small-conductance K channels. Biol Psychiatry 2014;76:476–85, PMID: 24560582. [176] Petrovszki Z, Adam G, Kekesi G, Tuboly G, Morvay Z, Nagy E, et al. The effects of juvenile capsaicin desensitization in rats: behavioral impairments. Physiol Behav 2014;125:38–44. [177] Almeida V, Peres FF, Levin R, Suiama MA, Calzavara MB, Zuardi AW, et al. Effects of cannabinoid and vanilloid drugs on positive and negative-like symptoms on an animal model of schizophrenia: the SHR strain. Schizophr Res 2014;153:150–9. [178] Amaral MD, Chapleau CA, Pozzo-Miller L. Transient receptor potential channels as novel effectors of brain-derived neurotrophic factor signaling: potential implications for Rett syndrome. Pharmacol Ther 2007;113(2):394–409. [179] Li W, Calfa G, Larimore J, Pozzo-Miller L. Activity-dependent BDNF release and TRPC signaling is impaired in hippocampal neurons of Mecp2 mutant mice. Proc Natl Acad Sci USA 2012;109:17087–92. [180] Roedding AS, Gao AF, Au-Yeung W, Scarcelli T, Li PP, Warsh JJ. Effect of oxidative stress on TRPM2 and TRPC3 channels in B lymphoblast cells in bipolar disorder. Bipolar Disord 2012;14(2):151–61. [181] Xu C, Li PP, Cooke RG, Parikh SV, Wang K, Kennedy JL, et al. TRPM2 variants and bipolar disorder risk: con- firmation in a family-based association study. Bipolar Disord 2009;11(1):1–10. [182] Pomrenze MB, Baratta MV, Rasmus KC, Cadle BA, Nakamura S, Birnbaumer L, et al. Cocaine self- administration in mice with forebrain knock-down of, ion channels. F1000Res 2013;2:53. [183] Yao H, Duan M, Yang L, Buch S. Platelet-derived growth factor-BB restores human immunodeficiency virus Tat-Cocaine-mediated impairment of neurogenesis: role of TRPC1 channels. J Neurosci 2012;32:9835–47. [184] Wu TT, Zhao ZJ, Xu C, Zhang LC. Distribution of TRPC6 in the cerebrospinal fluid-contacting nucleus of rat brain parenchyma and its expression in morphine dependence and withdrawal. Neurochem Res 2011;36:2316–21. [185] Nguyen TL, Kown SH, Hong SI, Ma SX, Jung YH, Hwang JY, et al. Transient receptor potential vanilloid type 1 channel may modulate opioid reward. Neuropsychopharmacology 2014;39:2414–22, PMID: 24732880. [186] Munakata M, Shirakawa H, Nagayasu K, Miyanohara J, Miyake T, Nakagawa T, et al. Transient receptor potential canonical 3 inhibitor Pyr3 improves outcomes and attenuates astrogliosis after intracerebral hemorrhage in mice. Stroke 2013;44:1981–7. [187] Lin Y, Zhang JC, Fu J, Chen F, Wang J, Wu ZL, et al. Hyperforin attenuates brain damage induced by transient middle cerebral artery occlusion (MCAO) in rats via inhibition of TRPC6 channels degradation. J Cereb Blood Flow Metab 2013;33:253–62. [188] Lin Y, Chen F, Zhang J, Wang T, Wei X, Wu J, et al. Neuroprotective effect of resveratrol on ischemia/reperfusion injury in rats through TRPC6/CREB pathways. J Mol Neurosci 2013;50:504–13. [189] Li H, Huang J, Du W, Jia C, Yao H, Wang Y. TRPC6 inhibited NMDA receptor activities and protected neurons from ischemic excitotoxicity. J Neurochem 2012;123:1010–18. [190] Gupta S, Sharma B, Singh P, Sharma BM. Modulation of transient receptor potential vanilloid subtype 1 (TRPV1) and norepinephrine transporters (NET) protect against oxidative stress, cellular injury, and vascular dementia. Curr Neurovasc Res 2014;11:94–106, PMID: 24597602. 456 23. ARE BRAIN TRPs VIABLE TARGETS FOR CURING NEURODEGENERATIVE DISORDERS

[191] Lin YW, Hsieh CL. Electroacupuncture at baihui acupoint (gv20) reverses behavior deficit and long-term potentiation through N-methyl-d-aspartate and transient receptor potential vanilloid subtype 1 receptors in middle cerebral artery occlusion rats. J Integr Neurosci 2010;9(3):269–82. [192] Muzzi M, Felici R, Cavone L, Gerace E, Minassi A, Appendino G, et al. Ischemic neuroprotection by TRPV1 receptor-induced hypothermia. J Cereb Blood Flow Metab 2012;32:978–82. [193] Moussaieff A, Yu J, Zhu H, Gattoni-Celli S, Shohami E, Kindy MS. Protective effects of incensole acetate on cerebral ischemic injury. Brain Res 2012;1443:89–97. [194] Butenko O, Dzamba D, Benesova J, Honsa P, Benfenati V, Rusnakova V, et al. The increased activity of TRPV4 channel in the astrocytes of the adult rat hippocampus after cerebral hypoxia/ischemia. PLoS One 2012;7:e39959. [195] Fonfria E, Mattei C, Hill K, Brown JT, Randall A, Benham CD, et al. TRPM2 is elevated in the tMCAO stroke model, transcriptionally regulated, and functionally expressed in C13 microglia. J Recept Signal Transduct Res 2006;26(3):179–98. [196] Li L, Qu W, Zhou L, Lu Z, Jie P, Chen L. Activation of transient receptor potential vanilloid 4 increases NMDA- activated current in hippocampal pyramidal neurons. Front Cell Neurosci 2013;7:17. [197] Verma S, Quillinan N, Yang YF, Nakayama S, Cheng J, Kelley MH, et al. TRPM2 channel activation following in vitro ischemia contributes to male hippocampal cell death. Neurosci Lett 2012;530:41–6. [198] Jia J, Verma S, Nakayama S, Quillinan N, Grafe MR, Hurn PD, et al. Sex differences in neuroprotection provided by inhibition of TRPM2 channels following experimental stroke. J Cereb Blood Flow Metab 2011;318:2160–8. [199] Zhang L, Hu R, Li M, Li F, Meng H, Zhu G, et al. Deferoxamine attenuates iron-induced long-term neurotoxicity in rats with traumatic brain injury. Neurol Sci 2013;34:639–45. [200] Cook NL, Vink R, Helps SC, Manavis J, van den Heuvel C. Transient receptor potential melastatin 2 expression is increased following experimental traumatic brain injury in rats. J Mol Neurosci 2010;42:192–9. [201] Simard JM, Chen M, Tarasov KV, Bhatta S, Ivanova S, Melnitchenko L, et al. Newly expressed SUR1-regulated NC(Ca-ATP) channel mediates cerebral edema after ischemic stroke. Nat Med 2006;12(4):433–40. [202] Simard JM, Kahle KT, Gerzanich V. Molecular mechanisms of microvascular failure in central nervous system injury-synergistic roles of NKCC1 and SUR1/TRPM4. J Neurosurg 2010;113(3):622–9. [203] Simard JM, Sheth KN, Kimberly WT, Stern BJ, del Zoppo GJ, Jacobson S, et al. Glibenclamide in cerebral ischemia and stroke. Neurocrit Care 2013;20:319–33. [204] Simard JM, Woo SK, Aarabi B, Gerzanich V. The Sur1-Trpm4 channel in spinal cord injury. J Spine 2014; (Suppl 4), pii:002. [205] Gerzanich V, Woo KS, Vennekens R, Tsymbalyuk O, Ivanova S, Ivanov A, et al. De novo expression of TRPM4 initiates secondary hemorrhage in spinal cord injury. Nat Med 2009;15:185–91. [206] Reading SA, Brayden JE. Central role of TRPM4 channels in cerebral blood flow regulation. Stroke 2007;38(8):2322–8. [207] Rempe DA, Takano T, Nedergaard M. TR(I)Pping towards treatment for ischemia. Nat Neurosci 2009;12:1215–6. [208] Zhang J, Zhao F, Zhao Y, Wang J, Pei L, Sun N, et al. Hypoxia induces an increase in intracellular magnesium via TRPM7 channels in rat hippocampal neurons in vitro. J Biol Chem 2011;286:20194–207. [209] Cook NL, Van Den Heuvel C, Vink R. Are the transient receptor potential melastatin (TRPM) channels important in magnesium homeostasis following traumatic brain injury? Magnes Res 2009;22:225–34. [210] Romero JR, Ridker PM, Zee RY. Gene variation of the transient receptor potential cation channel, subfamily M, member 7 (TRPM7), and risk of incident ischemic stroke. Prospective, nested, case-control study. Stroke 2009;64:791–7. CHAPTER 24 Mucolipidosis Type IV: Part I Ehud Goldin* SENS Research Foundation, Mountain View, CA, USA *Corresponding author: [email protected]

OUTLINE

Introduction 457 The Mucolipin Channel and its Interacting Partners 463 Clinical Presentation 458 Animal Models for MLIV 464 Cellular Abnormalities in MLIV Patients 459 Summary and Future Goals 464 MCOLN1 And Mucolipin-1 459 References 465 Mutations in the MCOLN1 Gene 462

INTRODUCTION

Mucolipidosis IV MLIV (OMIM 252650) was first described by Berman in 1974 in an infant with eye abnormalities [1]. Already in the first patient pathological examination of various tissues revealed intracellular accumulation of large vacuoles, which seemed similar to those discovered in mucopolysaccharidosis and sphingolipidosis, leading to the categorization of the disease as mucolipidosis. Initial studies focused on identifying a distinct deficiency in lysosomal function [2–7] in various tissues including brain biopsy [8]. However, most of the lysosomal enzyme activities appeared normal, whereas a wide range of metabolites seemed to accumulate in patient’s cells. Because the first patients were discovered in Ashkenazi families, the disease was considered to be restricted to the Ashkenazi Jewish population [9]. Cultured cells obtained from MLIV patients revealed autofluorescence [10], a feature that is commonly seen in neuronal ceroid lipofuscinosis, a family of neurodegenerative genetic diseases caused by mutations in lysosomal peptidases and related proteins. Subsequently, several non-Jewish patients with MLIV were diagnosed [11], creating a large enough cohort to perform a linkage analysis study on MLIV that eventually led to the discovery of the mutant gene [12–15].

The natural history of MLIV was investigated at the National Institutes of Health. This study confirmed the developmental aspects of the disease [16,17]. In addition, it demonstrated a severe lack of myelin in the brains of the patients, resulting in a distinct loss of the corpus callosum and a small cerebellum [16,18]. A seminal discovery was initiated by the observation that MLIV patients have severe iron deficiency anemia secondary to hypochlorhydria, that is, they do not secrete acid in their stomach [19,20]. Importantly, the lack of gastric acid also causes a dramatic increase in the level of blood gastrin, a hormone that stimulates gastric acid secretion. This observation has provided a simple laboratory tool to diagnose the disease [16]. High serum gastrin remains the only biochemical abnormality specific to MLIV that can be utilized for drug discovery investigation. The discovery of the MCOLN1 gene has attracted investigators from the channel biology research field to join the investigation of MLIV. In addition, this gene was ablated in several species, including mice [21–25], C. elegans [23,24], drosophila [25], and Dictyostelium [26]. These model systems shed new light on the function of the gene whose defect is responsible for MLIV. Several studies have addressed the cellular localization of mucolipin-1 and other proteins that interact with it. Other research efforts, however, focused on the metabolites that accumulate in the large vesicles, although these entities are not specific to MLIV nor do they seem to affect cellular viability or specific cellular functions.

CLINICAL PRESENTATION

MLIV typically presents during the first 6 months of life with slow motor development and eye abnormalities [1,16,17,27]. However, the diagnosis is often missed (in many cases, MLIV is misdiagnosed as cerebral palsy) until the eye doctor recognizes the loss of retinal function. Skin and conjunctiva biopsies reveal large vacuoles that assist in making the correct diagnosis [28]. High levels of blood gastrin, followed by detection of mutations in MCOLN1, confirm the clinical diagnosis [28,29]. Most patients are small for their age and seem to have distinct facial features (although those are not too abnormal). Head MRI reveals an almost complete absence of corpus callosum and a small cerebellum [16,18]. The amount of gray matter in the brain is, however, not reduced; importantly, the gray matter is not lost with age either, an important feature that questions the possibility of progressive neurodegeneration [30]. MLIV seems to have variable clinical penetrance. The abnormalities in the brain correlate with the severity of the disease and are absent in mildly forms [31–34]. They also correlate with the predicted severity of the mutations in MCOLN1 [16]. Eye abnormalities are common to all the patients diagnosed so far [35]. The epithelial layers of cells in the cornea are opaque to some degree, presumably due to accumulation of lysosomal vacuoles [36–38]. Patients also suffer problems with eye movement. Retinal degeneration starts in the first 5 years and progresses more rapidly with age [27,39]. The central vision is affected first, and patients can rely on peripheral vision to some extent. The clinical presentation of the vision loss is, however, variable: For example, one patient with a mild form of MLIV had a ring pattern of blindness sparing the central and the most peripheral vision [34]. MCOLN1 and Mucolipin-1 459

The majority of the patients cannot speak and many use sign language to communicate [27]. This creates a huge problem when they lose their vision and sign language is not an option anymore. Hearing is not affected in MLIV, indicating that inability to speak is not caused by a hearing defect, and the sensory function loss is restricted to vision. The typical patient cannot walk independently but can use a walker to some extent. Patients also suffer from spasticity and other movement abnormalities, mostly in the area of fine motor skills. Muscles cells contain abnormal vacuoles; based on these findings, it was initially thought that the movement deficits are caused by a muscle disease [40]. About half of the patients show evidence of epilepsy detected by electroencephalogram (EEG) [41]. Many MLIV patients also suffer from episodic pain in half of their face [42]. Combined, these observations imply a defect in the activity of motor afferents in the central nervous system.

CELLULAR ABNORMALITIES IN MLIV PATIENTS

MLIV patients do not secrete acid in their stomach [19,20]. The defect is in the parietal cells of the stomach that are responsible for acid secretion. Upon stimulation, parietal cells normally undergo morphological changes to form long intracellular canaliculi (ducts). Hydrochloric acid is produced in these canaliculi by an interaction of the K+/H+ pump (that secretes hydro- nium ions) with the chloride channels. In MLIV parietal cells, the morphological change is not complete, indicating that the signal for acid secretion is impaired (Figure 24.1). The pump components are there but they do not seem to be organized around the canaliculi. There is also an abundance of vacuoles in the cells, some of them multilamellar and some seem to contain microvilli that were supposed to line the canaliculi (Figures 24.2 and 24.3). Pancreatic acinar cells, which secret digestion enzymes to the duodenum, are also highly vacuolated in MLIV. It is not known, however, whether or not these enzymes are secreted at a normal rate [43,44]. Digestion in MLIV patients is not optimal, but this seems to be a result of reduced intestinal motility. Iron absorption is deficient due to the lack of stomach acid, and the patients need iron supplementation [16,27]. Recently, it was discovered that mitochondria are fragmented in MLIV cells [45,46]. This may explain some of the clinical features of the disease. Many of the cells that require high oxidative metabolism are affected in patients, including brain cells, retinal cells, parietal cells of the stomach, and muscle cells. It is possible that dysfunctional mitochondria are directly responsible for the functional deficits in those cell types. Defects in autophagy are present in MLIV cells just like in any other disease associated with lysosomal dysfunction [47–51]. Slower rate of metabolism of certain components will slow the uptake of new material into the system and autophagy is delayed.

MCOLN1 AND MUCOLIPIN-1

MCOLN1 is a 12,000 bp gene with 14 exons that is located in human chromosome 19p13. The gene product, mucolipin-1, is a protein with six transmembrane domains, an outside loop between the first and the second transmembrane domain, and a cation channel pore between 460 24. MUCOLIPIDOSIS TYPE IV

FIGURE 24.1 Suggestions for the possible role of mucolipin1 in acid secretion from stomach parietal cell. A diagram describing the signaling process for acid secretion based on a model by John Forte, with the junctions in which mucolipin1 may be involved marked by red arrows.

FIGURE 24.2 Parietal cell line HGT1 stained with antimucolipin1. Wild-type and cells treated with shRNA against mucolipin1 (clone DDD) stained with antimucolipin1. Mucolipin1 appears to be localized in distinct round intracellular membranes, presumably the specialized compartments that contain the HCL secretion machinery in nonstimulated cells (tubulovesicles). Confocal images were taken with a X63 objective. the fifth and the sixth domains (Figure 24.4). The three mammalian mucolipins form the subfamily of TRPML channels and are close relatives of PKD2. They have the same distance between the different transmembrane domains and are probably of the same phylogenetic origin [15]. Invertebrates, unicellular organisms, and plants there have only one mucolipin, and it has a high homology with mucolipin-3. Generally speaking, TRPML channels are found mostly in intracellular membranes (rarely on the plasma membrane), many of them in the base of cilia. However, it is not clear exactly MCOLN1 and Mucolipin-1 461

FIGURE 24.3 MLIV stomach pathology. Parietal cells of the stomach are vacuolated containing lysosomal empty and multilamellar vesicles.

FIGURE 24.4 Mutations in MCOLN1. A schematic drawing of mucolipin1, the sections glowing in blue are highly similar to mucolipin3, the section glowing in red is the PKD channel domain pfam08016, the barrels are transmembrane domains, and the large arrow points to the channel pore. Mutations are labeled by the severity of the clinical phenotype: yellow-mild, orange-moderate, red-severe. 462 24. MUCOLIPIDOSIS TYPE IV where in the cell they function as channels. Some evidence points to the endolysosomal system as the site of action for mucolipin-1; this is supported by the abnormal lysosomal vacuoles in ML4 cells. TRP channels form functional tetramers, and it is believed that mucolipin-1 also forms functional oligomers. The composition of the oligomeric functional unit is still being investigated, with some studies indicating that different mucolipins may be situated in the same oligomer [49,52–55]. This complicates predicting the electrophysiological properties of the channel. Electrophysiology of mucolipin-1 in cell free system indicates a channel that permits various mono and divalent ions [56–61]. This channel is pH sensitive, and mutations that were found in patients result in reduced activity or mislocalization of the protein. The protein has a number of sequences in the N- and the C-terminals that are supposed to target it to the endolysosomal system [62]. However, two patients with frameshift mutations that modify the entire C-terminus were identified; surprisingly, these patients have a very mild phenotype that does not include any of the neurological symptoms [32,34]. On the N-terminus, no missense mutations have reported, yet. Interestingly, a mouse with a deletion of exon 1 had a normal length mRNA, indicating the use of an alternative first exon in the intron between exons 1 and 2. It is not known whether such an alternative exon exists in the human genomic DNA. Of note, mice with exon 1 deletion had variable phenotypes. Some of them had the stomach phenotype and minor brain abnormalities, whereas others looked completely normal (Kulkarni and Goldin, unpublished observations).

MUTATIONS IN THE MCOLN1 GENE

So far 31 different mutations have been discovered in MLIV patients. The disease is not considered embryonic lethal, and the most severe known mutation is a deletion of the first half of the gene from the 5’ UTR to exon 7. A patient who was homozygous for this mutation died in her late teens. Another patient who carried this mutation on one chromosome and an early frameshift mutation on the other survived into her late 20s. Other patients who were homozygous to a splice mutation in the 5’ of exon 4 lived to their third decade. The mutations discovered so far span the majority of the gene sequence, excluding exons 1 and 14. Mutations affecting the channel pore (AA 446–465 area) result in a severe clinical phenotype, indicating that the activity of the channel is the most critical property. A mutation in the lipase domain of the protein L106P also seems to cause a severe phenotype. Two mutations in the large external loop C166F and T232P also result in a severe phenotype; it may be speculated that the loss of cysteine 166 prevents the formation of a CC bond, which is crucial for the stability of the loop. The three frames of the 3’ end of the gene would make a transcript similar in length and frameshift mutations affecting exons 13 and 14; these mutants are associated with a very mild phenotype restricted to the eye [32] and acid secretion [34]. A 3-nucleotide deletion in exon 10 results in the loss of one of the phenylalanine residues: this causes a mild phenotype. This amino acid is situated at the end of transmembrane domain 4 and may destabilize the interaction between the protein and the head groups of lipids surrounding it. Mutation R403C results in a moderate phenotype and may affect a voltage-sensing domain [63]. The Mucolipin Channel and its Interacting Partners 463 THE MUCOLIPIN CHANNEL AND ITS INTERACTING PARTNERS

It was suspected for a long time that the molecular defect responsible for MLIV is in an ion channel because the cultured cells of MLIV patients are sensitive to chloroquine, a known inhibitor of ion channel activity [11]. Indeed, the MCOLN1 gene encodes a channel that permits the passage of various cations, including K+, H+, Na+, Ca2+, Fe2+, and Zn2+ [49,50,56–60,64,65]. A predicted structure of mucolipin was published recently [66]. One of the major technical difficulties with working with mucolipin is its strong association with the membrane, which makes isolating the protein and generating antibodies against it very challenging. Recently, an antibody was generated by the author of this chapter against a construct that contains the first 300 amino acids from the N-terminal: This antibody reacts with human and mouse tissue and cell preparations (Figure 24.5). Unfortunately, all the commercial antibodies that we tested so far were not specific as indicated by a 60 KD band that is apparent in the Western blots of null tissue. Using the antibody generated against the N-terminus construct, strong immunoreactivity was seen in brain tissue and in stomach parietal cells (Figure 24.5). Importantly, the immunoreactivity was very weak in human fibroblasts where many of the protein studies on mucolipin were performed. Indeed, all the findings based on overexpression of the protein (mostly tagged) were done in fibroblasts. This raises concerns about the conclusion from these studies, namely, that the protein is endolysosomal. One should keep in mind that many proteins

FIGURE 24.5 Expression of Mucolipin 1 in various mouse tissues. (a) Western blot of membranes prepared from tissues by centrifugation, with antimucolipin1. Lanes: 1, pancreas; 2, testis; 3, spleen; 4, liver; 5, kidney; 6, stomach; 7, lung; 8, heart; 9, brain cortex. Note the high level of expression in the Stomach. (b) Western blot of different parts of the mouse brain. Lanes: 1, cerebellum; 2, pons; 3, posterior cortex; 4, Lateral cortex; 5, thalamus; 6, superior cortex; 7, anterior cortex; 8, olfactory bulbs. 464 24. MUCOLIPIDOSIS TYPE IV will go to the endolysosomal system for degradation, especially if they are toxic in other locations. For example, human mucolipin is toxic in bacteria if the channel pore of the mutant protein is expressed (Stahl and Goldin, unpublished observations). The subcellular compartment where one may see the higher amount of a TRP protein is not necessarily site of action (Minke, personal communication). The representative examples of interactions of mucolipin in human cells should be read with these thoughts in mind [50,55,57,60,67–72].

ANIMAL MODELS FOR MLIV

As soon as the MLIV gene was discovered in humans, the homologous gene in Drosophila was cloned (CG8743). Soon after the C. elegans gene CED9 or CUP-5 was reported. Interestingly, in C. elegans the more severe mutations cause death during larval stages, whereas the less severe mutations cause an endolysosomal dysfunction in Coelomocytes [23,24]. This phenotype can be corrected using the human mucolipin constructs for gene therapy [73]. The mucolipin-deficient phenotype can also be rescued by knocking out an ABC transporter in C. elegans, which may point to a target for therapeutic intervention [74]. The Drosophila mutant has a variety of abnormalities, the most interesting of which is a defect in inflation of the wings [25,75]. Both animals are small enough for high throughput screening studies; however, their use is limited by the lack of our understanding of the role of mucolipins in these species. In 2003, a mouse knockout model was generated by eliminating exon 1 of MCOLN1. However, this mouse had an unstable phenotype. A better mouse model was subsequently produced by eliminating exons 3 and 4 [21]. A third knockout mouse was generated by eliminating exons 1 and 2 [22]. These mouse models have a clear stomach phenotype and eye abnormalities. The neurological phenotype, however, does not replicate the human disease [21,76]. The mice have normal motor function for several months and then they develop a severe neurological phenotype and die. Furthermore, the mouse strain with the deleted exon 3 and 4 does not have an appreciable change in corpus callosum. A comprehensive study of the eye phenotype in the mouse is yet to be published. Unfortunately, we still do not have a good animal model to study the patients’ brain abnormalities.

SUMMARY AND FUTURE GOALS

The study of MLIV so far has yielded some very important discoveries that are directly relevant to the care for the patients and their families. Such are the natural history study of typical patients and patients with a mild disease phenotype with only eye involvement, discovery of mutations in MCOLN1, the loss of acid secretion, and the description of vacuoles in various cell types in MLIV. The defects in brain development associated with the disease, in particular the underdeveloped white matter in the corpus callosum and other areas, indicate a critical role for the gene in signaling in early embryonic development. The retinal degeneration causing loss of vision, provide the evidence on how MCOLN1 is required for maintenance of this highly active tissue. Delineating the mechanism that causes these deficiencies is the challenge facing us. Why can’t the brain axon myelination proceed? How is MCOLN1 involved in stomach acid secretion or in maintenance of the retina? Answering these questions References 465 requires a better understanding of the channel function, the regulation on its activity, and the signaling pathway that requires it. Probably the biggest hint on MCLON1 function can be obtained from studying its role in regulated secretion; hopefully this will open the door to understanding the pathogenesis of MLIV and enable the development of a specific treatment for the patients.

References [1] Berman ER, Livni N, Shapira E, Merin S, Levij IS. Congenital corneal clouding with abnormal systemic storage bodies: a new variant of mucolipidosis. J Pediatr 1974;84(4):519–26. [2] Bach G, Cohen MM, Kohn G. Abnormal ganglioside accumulation in cultured fibroblasts from patients with mucolipidosis IV. Biochem Biophys Res Commun 1975;66(4):1483–90. [3] Bach G, Desnick RJ. Lysosomal accumulation of phospholipids in mucolipidosis IV cultured fibroblasts. Enzyme 1988;40(1):40–4. [4] Bach G, Zeigler M, Schaap T, Kohn G. Mucolipidosis type IV: ganglioside sialidase deficiency. Biochem Biophys Res Commun 1979;90(4):1341–7. [5] Bach G, Ziegler M, Kohn G, Cohen MM. Mucopolysaccharide accumulation in cultured skin fibroblasts derived from patients with mucolipidosis IV. Am J Hum Genet 1977;29(6):610–18. [6] Bargal R, Bach G. Phosphatidylcholine storage in mucolipidosis IV. Clin Chim Acta 1989;181(2):167–74. [7] Crandall BF, Philippart M, Brown WJ, Bluestone DA. Review article: mucolipidosis IV. Am J Med Genet 1982;12(3):301–8. [8] Tellez-Nagel I, Rapin I, Iwamoto T, Johnson AB, Norton WT, Nitowsky H, Mucolipidosis IV. Clinical, ultrastructural, histochemical, and chemical studies of a case, including a brain biopsy. Arch Neurol 1976;33(12):828–35. [9] Bach G. Mucolipidosis type IV. Mol Genet Metab 2001;73(3):197–203. [10] Goldin E, Blanchette-Mackie EJ, Dwyer NK, Pentchev PG, Brady RO. Cultured skin fibroblasts derived from patients with mucolipidosis 4 are auto-fluorescent. Pediatr Res 1995;37(6):687–92. [11] Goldin E, Cooney A, Kaneski CR, Brady RO, Schiffmann R. Mucolipidosis IV consists of one complementation group. Proc Natl Acad Sci USA 1999;96(15):8562–6. [12] Slaugenhaupt SA, Acierno Jr JS, Helbling LA, Bove C, Goldin E, Bach G, et al. Mapping of the mucolipidosis type IV gene to chromosome 19p and definition of founder haplotypes. Am J Hum Genet 1999;65(3):773–8. [13] Bargal R, Avidan N, Ben-Asher E, Olender Z, Zeigler M, Frumkin A, et al. Identification of the gene causing mucolipidosis type IV. Nat Genet 2000;26(1):118–23. [14] Bassi MT, Manzoni M, Monti E, Pizzo MT, Ballabio A, Borsani G. Cloning of the gene encoding a novel integral membrane protein, mucolipidin-and identification of the two major founder mutations causing mucolipidosis type IV. Am J Hum Genet 2000;67(5):1110–20. [15] Sun M, Goldin E, Stahl S, Falardeau JL, Kennedy JC, Acierno Jr JS, et al. Mucolipidosis type IV is caused by mutations in a gene encoding a novel transient receptor potential channel. Hum Mol Genet 2000;9(17):2471–8. [16] Altarescu G, Sun M, Moore DF, Smith JA, Wiggs EA, Solomon BI, et al. The neurogenetics of mucolipidosis type IV. Neurology 2002;59(3):306–13. [17] Amir N, Zlotogora J, Bach G. Mucolipidosis type IV: clinical spectrum and natural history. Pediatrics 1987;79(6):953–9. [18] Frei KP, Patronas NJ, Crutchfield KE, Altarescu G, Schiffmann R. Mucolipidosis type IV: characteristic MRI findings. Neurology 1998;51(2):565–9. [19] Schiffmann R, Dwyer NK, Lubensky IA, Tsokos M, Sutliff VE, Latimer JS, et al. Constitutive achlorhydria in mucolipidosis type IV. Proc Natl Acad Sci USA 1998;95(3):1207–12. [20] Lubensky IA, Schiffmann R, Goldin E, Tsokos M. Lysosomal inclusions in gastric parietal cells in mucolipidosis type IV: a novel cause of achlorhydria and hypergastrinemia. Am J Surg Pathol 1999;23(12):1527–31. [21] Venugopal B, Browning MF, Curcio-Morelli C, Varro A, Michaud N, Nanthakumar N, et al. Neurologic, gastric, and opthalmologic pathologies in a murine model of mucolipidosis type IV. Am J Hum Genet 2007;81(5):1070–83. [22] Chandra M, Zhou H, Li Q, Muallem S, Hofmann SL, Soyombo AA. A role for the Ca2+ channel TRPML1 in gastric acid secretion, based on analysis of knockout mice. Gastroenterology 2011;140(3):857–67. [23] Hersh BM, Hartwieg E, Horvitz HR. The Caenorhabditis elegans mucolipin-like gene cup-5 is essential for viability and regulates lysosomes in multiple cell types. Proc Natl Acad Sci USA 2002;99(7):4355–60. 466 24. MUCOLIPIDOSIS TYPE IV

[24] Fares H, Greenwald I. Regulation of endocytosis by CUP-5, the Caenorhabditis elegans mucolipin-1 homolog. Nat Genet 2001;28(1):64–8. [25] Venkatachalam K, Long AA, Elsaesser R, Nikolaeva D, Broadie K, Montell C. Motor deficit in a Drosophila model of mucolipidosis type IV due to defective clearance of apoptotic cells. Cell 2008;135(5):838–51. [26] Lima WC, Leuba F, Soldati T, Cosson P. Mucolipin controls lysosome exocytosis in Dictyostelium. J Cell Sci 2012;125(Pt 9):2315–22. [27] Goldin E, Slaugenhaupt SA, Smith JA, Schiffmann R. Mucolipidosis type IV. In: Valle D, Beaudet AL, Vogelstein B, Kinzler KW, Antonarakis SE, Ballabio A, editors. The online metabolic and molecular bases of inherited disease. New York, USA: McGraw-Hill Medical; 2012. [28] Alroy J, Ucci AA. Skin biopsy: a useful tool in the diagnosis of lysosomal storage diseases. Ultrastruct Pathol 2006;30(6):489–503. [29] Livni N, Merin S. Mucolipidosis IV: ultrastructural diagnosis of a recently defined genetic disorder. Arch Pathol Lab Med 1978;102(11):600–4. [30] Schiffmann R, Mayfield J, Swift C, Nestrasil I. Quantitative neuroimaging in mucolipidosis type IV. Mol Genet Metab 2014;111(2):147–51. [31] Casteels I, Taylor DS, Lake BD, Spalton DJ, Bach G. Mucolipidosis type IV. Presentation of a mild variant. Ophthalmic Genet 1992;13(4):205–10. [32] Dobrovolny R, Liskova P, Ledvinova J, Poupetova H, Asfaw B, Filipec M, et al. Mucolipidosis IV: report of a case with ocular restricted phenotype caused by leaky splice mutation. Am J Ophthalmol 2007;143(4):663–71. [33] Bonavita S, Virta A, Jeffries N, Goldin E, Tedeschi G, Schiffmann R. Diffuse neuroaxonal involvement in mucolipidosis IV as assessed by proton magnetic resonance spectroscopic imaging. J Child Neurol 2003;18(7):443–9. [34] Goldin E, Caruso RC, Benko W, Kaneski CR, Stahl S, Schiffmann R. Isolated ocular disease is associated with decreased mucolipin-1 channel conductance. Invest Ophthalmol Vis Sci 2008;49(7):3134–42. [35] Merin S, Livni N, Berman ER, Yatziv S. Mucolipidosis IV: ocular, systemic, and ultrastructural findings. Invest Ophthalmol 1975;14(6):437–48. [36] Dangel ME, Bremer DL, Rogers GL. Treatment of corneal opacification in mucolipidosis IV with conjunctival transplantation. Am J Ophthalmol 1985;99(2):137–41. [37] Merin S, Nemet P, Livni N, Lazar M. The cornea in mucolipidosis IV. J Pediatr Ophthalmol 1976;13(5):289–95. [38] Smith JA, Chan CC, Goldin E, Schiffmann R. Noninvasive diagnosis and ophthalmic features of mucolipidosis type IV. Ophthalmology 2002;109(3):588–94. [39] Pradhan SM, Atchaneeyasakul LO, Appukuttan B, Mixon RN, McFarland TJ, Billingslea AM, et al. Electronegative electroretinogram in mucolipidosis IV. Arch Ophthalmol 2002;120(1):45–50. [40] Weitz R, Kramer I, Nissenkorn I, Shapira Y, Ben-David E, Kohn G. Muscle involvement in mucolipidosis IV. Brain Dev 1990;12(5):524–8. [41] Siegel H, Frei K, Greenfield J, Schiffmann R, Sato S. Electroencephalographic findings in patients with mucolipidosis type IV. Electroencephalogr Clin Neurophysiol 1998;106(5):400–3. [42] Newman NJ, Starck T, Kenyon KR, Lessell S, Fish I, Kolodny EH. Corneal surface irregularities and episodic pain in a patient with mucolipidosis IV. Arch Ophthalmol 1990;108(2):251–4. [43] Folkerth RD, Alroy J, Lomakina I, Skutelsky E, Raghavan SS, Kolodny EH. Mucolipidosis IV: morphology and histochemistry of an autopsy case. J Neuropathol Exp Neurol 1995;54(2):154–64. [44] Hammel I, Alroy J. The effect of lysosomal storage diseases on secretory cells: an ultrastructural study of pancreas as an example. J Submicrosc Cytol Pathol 1995;27(2):143–60. [45] Coblentz J, St Croix C, Kiselyov K. Loss of TRPML1 promotes production of reactive oxygen species: is oxidative damage a factor in mucolipidosis type IV? Biochem J 2014;457(2):361–8. [46] Jennings Jr JJ, Zhu JH, Rbaibi Y, Luo X, Chu CT, Kiselyov K. Mitochondrial aberrations in mucolipidosis type IV. J Biol Chem 2006;281(51):39041–50. [47] Curcio-Morelli C, Charles FA, Micsenyi MC, Cao Y, Venugopal B, Browning MF, et al. Macroautophagy is defective in mucolipin-1-deficient mouse neurons. Neurobiol Dis 2010;40(2):370–7. [48] Venugopal B, Mesires NT, Kennedy JC, Curcio-Morelli C, Laplante JM, Dice JF, et al. Chaperone-mediated autophagy is defective in mucolipidosis type IV. J Cell Physiol 2009;219(2):344–53. [49] Zeevi DA, Lev S, Frumkin A, Minke B, Bach G. Heteromultimeric TRPML channel assemblies play a crucial role in the regulation of cell viability models and starvation-induced autophagy. J Cell Sci 2010;123(Pt 18):3112–24. References 467

[50] Soyombo AA, Tjon-Kon-Sang S, Rbaibi Y, Bashllari E, Bisceglia J, Muallem S, et al. TRP-ML1 regulates lysosomal pH and acidic lysosomal lipid hydrolytic activity. J Biol Chem 2006;281(11):7294–301. [51] Vergarajauregui S, Connelly PS, Daniels MP, Puertollano R. Autophagic dysfunction in mucolipidosis type IV patients. Hum Mol Genet 2008;17(17):2723–37. [52] Kiselyov K, Colletti GA, Terwilliger A, Ketchum K, Lyons CW, Quinn J, et al. TRPML: transporters of metals in lysosomes essential for cell survival? Cell Calcium 2011;50(3):288–94. [53] Samie MA, Grimm C, Evans JA, Curcio-Morelli C, Heller S, Slaugenhaupt SA, et al. The tissue-specific expression of TRPML2 (MCOLN-2) gene is influenced by the presence of TRPML1. Pflugers Arch 2009;459(1):79–91. [54] Venkatachalam K, Hofmann T, Montell C. Lysosomal localization of TRPML3 depends on TRPML2 and the mucolipidosis-associated protein TRPML1. J Biol Chem 2006;281(25):17517–27. [55] Zeevi DA, Frumkin A, Offen-Glasner V, Kogot-Levin A, Bach G. A potentially dynamic lysosomal role for the endogenous TRPML proteins. J Pathol 2009;219(2):153–62. [56] Raychowdhury MK, Gonzalez-Perrett S, Montalbetti N, Timpanaro GA, Chasan B, Goldmann WH, et al. Molecular pathophysiology of mucolipidosis type IV: pH dysregulation of the mucolipin-1 cation channel. Hum Mol Genet 2004;13(6):617–27. [57] Dong XP, Cheng X, Mills E, Delling M, Wang F, Kurz T, et al. The type IV mucolipidosis-associated protein TRPML1 is an endolysosomal iron release channel. Nature 2008;455(7215):992–6. [58] Dong XP, Shen D, Wang X, Dawson T, Li X, Zhang Q, et al. PI(3,5)P(2) controls membrane trafficking by direct activation of mucolipin Ca(2+) release channels in the endolysosome. Nat Commun 2010;1:38. [59] Eichelsdoerfer JL, Evans JA, Slaugenhaupt SA, Cuajungco MP. Zinc dyshomeostasis is linked with the loss of mucolipidosis IV-associated TRPML1 ion channel. J Biol Chem 2010;285(45):34304–8. [60] Kiselyov K, Chen J, Rbaibi Y, Oberdick D, Tjon-Kon-Sang S, Shcheynikov N, et al. TRP-ML1 is a lysosomal monovalent cation channel that undergoes proteolytic cleavage. J Biol Chem 2005;280(52):43218–23. [61] LaPlante JM, Ye CP, Quinn SJ, Goldin E, Brown EM, Slaugenhaupt SA, et al. Functional links between mucolipin-1 and Ca2+-dependent membrane trafficking in mucolipidosis IV. Biochem Biophys Res Commun 2004;322(4):1384–91. [62] Vergarajauregui S, Puertollano R. Two di-leucine motifs regulate trafficking of mucolipin-1 to lysosomes. Traffic 2006;7(3):337–53. [63] Goldin E, Stahl S, Cooney AM, Kaneski CR, Gupta S, Brady RO, et al. Transfer of a mitochondrial DNA fragment to MCOLN1 causes an inherited case of mucolipidosis IV. Hum Mutat 2004;24(6):460–5. [64] Feng X, Huang Y, Lu Y, Xiong J, Wong CO, Yang P, et al. Drosophila TRPML forms PI(3,5)P2-activated cation channels in both endolysosomes and plasma membrane. J Biol Chem 2014;289(7):4262–72. [65] LaPlante JM, Falardeau J, Sun M, Kanazirska M, Brown EM, Slaugenhaupt SA, et al. Identification and characterization of the single channel function of human mucolipin-1 implicated in mucolipidosis type IV, a disorder affecting the lysosomal pathway. FEBS Lett 2002;532(1–2):183–7. [66] AlBakheet A, Qari A, Colak D, Rasheed A, Kaya N, Al-Sayed M. A novel mutation in a large family causes a unique phenotype of Mucolipidosis IV. Gene 2013;526(2):464–6. [67] Manzoni M, Monti E, Bresciani R, Bozzato A, Barlati S, Bassi MT, et al. Overexpression of wild-type and mutant mucolipin proteins in mammalian cells: effects on the late endocytic compartment organization. FEBS Lett 2004;567(2–3):219–24. [68] Grimm C, Hassan S, Wahl-Schott C, Biel M. Role of TRPML and two-pore channels in endolysosomal cation homeostasis. J Pharmacol Exp Ther 2012;342(2):236–44. [69] LaPlante JM, Falardeau JL, Brown EM, Slaugenhaupt SA, Vassilev PM. The cation channel mucolipin-1 is a bifunctional protein that facilitates membrane remodeling via its serine lipase domain. Exp Cell Res 2011;317(6):691–705. [70] Spooner E, McLaughlin BM, Lepow T, Durns TA, Randall J, Upchurch C, et al. Systematic screens for proteins that interact with the mucolipidosis type IV protein TRPML1. PLoS One 2013;8(2):e56780. [71] Vergarajauregui S, Martina JA, Puertollano R. Identification of the penta-EF-hand protein ALG-2 as a Ca2+- dependent interactor of mucolipin-1. J Biol Chem 2009;284(52):36357–66. [72] Vergarajauregui S, Martina JA, Puertollano R. LAPTMs regulate lysosomal function and interact with mucolipin 1: new clues for understanding mucolipidosis type IV. J Cell Sci 2011;124(Pt 3):459–68. 468 24. MUCOLIPIDOSIS TYPE IV

[73] Treusch S, Knuth S, Slaugenhaupt SA, Goldin E, Grant BD, Fares H. Caenorhabditis elegans functional or- thologue of human protein h-mucolipin-1 is required for lysosome biogenesis. Proc Natl Acad Sci USA 2004;101(13):4483–8. [74] Schaheen L, Patton G, Fares H. Suppression of the cup-5 mucolipidosis type IV-related lysosomal dysfunction by the inactivation of an ABC transporter in C. elegans. Development 2006;133(19):3939–48. [75] Sandstrom DJ. Extracellular protons reduce quantal content and prolong synaptic currents at the Drosophila larval neuromuscular junction. J Neurogenet 2011;25(3):104–14. [76] Micsenyi MC, Dobrenis K, Stephney G, Pickel J, Vanier MT, Slaugenhaupt SA, et al. Neuropathology of the Mcoln1(-/-) knockout mouse model of mucolipidosis type IV. J Neuropathol Exp Neurol 2009;68(2):125–35. CHAPTER 25 TRPML1-Dependent Processes as Therapeutic Targets Kartik Venkatachalam,1,* Kirill Kiselyov2,* 1Department of Integrative Biology and Pharmacology, University of Texas School of Medicine, Houston, TX, USA 2Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA, USA *Corresponding authors: [email protected]; [email protected]

OUTLINE

Introduction 469 Conclusions 477 TRPML1 Features and Properties 470 Acknowledgments 478 TRPML1-Dependent Processes, References 478 Shown and Discussed 472 TRPML1-Dependent Processes Inferred from its Functional Context 475

INTRODUCTION

Although MLIV is considered a developmental brain disorder with a slowly progressing degenerative component [1–10], the fact that TRPML1 is ubiquitously expressed suggests a role outside the central nervous system (CNS), which is further supported by the findings of gastric and pancreatic involvement in human MLIV patients and mouse models [11–14]. Beyond its wide tissue distribution, TRPML1 appears to regulate an array of cellular processes pertaining to the function of late endosomes and lysosomes. The latter notion reflects our growing realization that far from being just a cellular digestion and absorption center, lysosomes are important cellular signaling and cell-fate hubs. Recent evidence linked lysosomes

TRP Channels as Therapeutic Targets 469 © 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/B978-0-12-420024-1.00025-4 470 25. TRPML1-Dependent Processes as Therapeutic Targets to regulation of transition metal toxicity and revealed their role in key physiological processes such as aging, antigen processing, and mitophagy, as well as autophagy in general [15–19]. Furthermore, the findings that lysosomal gene network responds to stress and amino acid scarcity via mechanistic target of rapamycin (MTOR), and transcription factors such as transcription factor EB (TFEB) suggest that lysosome-dependent processes are actively regulated depending on the physiological state of cells [20–24]. Previously published results suggest that TRPML1 regulates endocytic membrane traffic, lysosomal exocytosis, metal distribution, phagocytosis, and autophagy [25–29]. Drugs that affect TRPML1 function may provide new possibilities for treating conditions in which lysosomal function is a factor. With this in mind, here we will focus on TRPML1-dependent processes outside neurodevelopment and neurodegeneration. Such processes have been inferred based on the different aspects of TRPML1 function. We aim to delineate TRPML1 activators and inhibitors as potential treatments for various pathophysiological conditions.

TRPML1 FEATURES AND PROPERTIES

TRPML1 (Mucolipin 1) is a six-transmembrane-domain containing ion channel, a member of the TRPML family of the TRP channels [25,27]. It is coded by the MCOLN1 gene, which in chordates has two additional isoforms encoding TRPML2 and TRPML3 [30,31]. Although both TRPML2 and TRPML3 appear to function in the endocytic pathway, there are no known human diseases related to mutations in genes encoding either TRPML2 or TRPML3. Invertebrates have a single trpml gene, with closest homology to MCOLN1 [28,30]. The trpml gene family evolution has been recently discussed in a series of excellent reviews [28,30]. Although the different TRPMLs form heteromultimers [32], the range of tissues expressing TRPML2 and TRPML3 is reported to be very narrow [33,34], and thus the functional significance of such multimerization remains elusive. It should be noted that our knowledge of signals regulating TRPML2 and TRPML3 expression is very limited, and it is indeed possible that heteromultimers exist under certain conditions of cell stimulation or developmental stages. In humans, TRPML1 is a 540-amino-acid protein, with a putative pore region between the fifth and sixth transmembrane domains [25,27]. Additional splice variants of TRPML1 have not been described. Some lysosomal proteins that conduct ions appear to combine the functions of channels and transporters (e.g., Cl− permeation coupled with H+ transport by the ClC channels/transporters) [35]. Transporter function of TRPML1 has not been described and it is treated a channel. Mammalian TRPML1 has lysosomal localization signals in both its C- and N-termini that have been shown to be critical for its lysosomal localization via an AP-1-dependent mechanism [36,37]. These domains are poorly conserved, as they appear to be absent in the invertebrate or even fish TRPMLs, albeit the evidence pinning invertebrate TRPML1 to the endolysosomes is overwhelming. These differences likely reflect the evolu- tionary constraints driving the functional/spatial specialization within this family. The N-terminus of TRPML1 contains a domain that binds phosphoinositides (PI) [38,39], and this domain is evolutionarily extremely well conserved (Figure 25.1). The PI-binding domain was shown to bind the lysosomal lipid marker PI(3,5)P2 leading to TRPML1 activation, whereas its binding to the plasma membrane lipid PI(4,5)P2 was shown to inhibit the channel activity [38]. This provides a nearly perfect structural reflection of the TRPML1 functional TRPML1 Features and Properties 471

FIGURE 25.1 Amino acid alignment of the phosphoinositide-binding sequences of vertebrate TRPML1 and invertebrate TRPML. Large positively charged amino acid clusters previously implicated in phosphoinositide binding are shown in red. context as TRPML1 travels to the lysosomes via the plasma membrane; its premature activation is inhibited by the PI(4,5)P2 binding until it is delivered to the PI(3,5)P2-rich lysosomes. Conversely, the inhibition by PI(4,5)P2 serves as a mechanism to abrogate channel activity following lysosomal exocytosis—a process driven by TRPML1 channel activity. The

PI-dependence of TRPML1 raises some interesting questions. First, if PI(3,5)P2 is the TRPML1 activator and its lysosomal levels are fairly stable, then is TRPML1 a lysosomal leak channel activated on its delivery to the lysosomes? Or do lysosomal PI(3,5)P2 levels and/or spatial distribution change, and if so, what are the signals and machinery actuating these changes? Finally, the PI-binding domains are very similar within the TRPML family, yet both localization and function of TRPML3 was shown to be different from that of TRPML1 due to wider distribution in the endocytic pathway, including early endosomes [40,41]. Whether or not

TRPMLs respond to PI species other than PI(3,5)P2 is currently unknown. Another unique feature of TRPML1 is a large extracytosolic loop between its first and second transmembrane domains. The loop is cleaved by a protease that is sensitive to cathepsin B inhibitors, but neither the identity of the cleavage site nor its functional significance has been unequivocally established [42]. The loop contains large numbers of histidine residues: 75% of the TRPML1 histidines are in that loop, whereas the loop is only 25% of the molecule’s length. In TRPML3 these histidines were shown to participate in this channel’s inhibition by low pH but their functional significance for TRPML1 is unknown. It is worth noting that the Zn transporters, ZnT (Slc30) and Zip (Slc39), also contain similar structures, which appear to be essential for Zn transport [43,44]. It is tempting to speculate that the TRPML1 histidine stretch is important in the context of Zn regulation by TRPML1 (or perhaps TRPML1 regulation by Zn), which will be discussed later. Electrophysiological inquiry into TRPML1 permeability properties began with analysis of human recombinant proteins in planar lipid bilayers and in whole cell patch clamp, but the field only matured after the whole-lysosome recording technique was established [45]. The latter approach revealed inwardly rectifying (from lysosomal lumen into the cytoplasm) ion channel permeable to monovalent and divalent cations including K+, Na+, Ca2+, Fe2+ and Zn2+ [45]. The wild type channel is about equally selective to Na+ and Ca2+, which means that at the presumed lysosomal Na+ and Ca2+ concentrations (about 50:1 ratio), TRPML1 efficacy of releasing lysosomal Ca2+ is about the same as the efficacy of a plasma membrane TRPC 472 25. TRPML1-Dependent Processes as Therapeutic Targets

channel maintaining receptor-mediated Ca2+ influx. Genetically engineered Ca2+ probes report increased cytoplasmic Ca2+ in the immediate proximity of TRPML1 [39]. In one report TRPML1 overexpression and loss failed to appreciably change total lysosomal Ca2+, suggesting that TRPML1 deals with a small amount of lysosomal Ca2+, probably effecting changes in its immediate proximity [46]. Indeed, localized Ca2+ signaling appears to be the theme of this aspect of its proposed function. That being said, the loss of Drosophila TRPML does result in elevated levels of lysosomal Ca2+ [47]. These differences could reflect the fact that Drosophila expresses a single TRPML homolog in contrast to the three genes in mammalian cells. Electrophysiological recordings of the sole TRPML gene in Drosophila reveal a channel re- producing most physiological features of human TRPML1, including its regulation by pH and Ca2+ permeability [48]. At present, the functional overlap between Drosophila TRPML and the three mammalian TRPMLs remains unknown. TRPML1 activity is increased by low pH, in agreement with its function in the lysosomes [45]. The development of TRPML1 patch clamp enabled screening for additional modulators of its activity, leading to identification of SF-22 and ML-SA1 as its activators and sphingomy- elin as an inhibitor [49,50]. It is clear that delineating the wider range of TRPML1-dependent cellular functions will stimulate the search for additional compounds modulating TRPML1 activity as such compounds could serve as drug candidates for a range of conditions.

TRPML1-DEPENDENT PROCESSES, SHOWN AND DISCUSSED

Identification of TRPMLs as members of TRP superfamily, the majority of which are Ca2+- permeable channels, narrowed the focus of the search for TRPML1 function to Ca2+-dependent events driving the SNARE-mediated fusion of the endocytic membranes. The current paradigm of the TRPML1-dependent aspects of membrane traffic suggests that Ca2+ release from the lysosomes via TRPML1 actuates conformational change in SNARE driving the homotypic and hetero- typic membrane fusion events. Membrane traffic delays at the prelysosomal stage and suppressed fusion of the lysosomal and autophagosomal/amphisomal compartments have been shown in human MLIV fibroblasts, murine macrophages, and Drosophila cells lacking trpml [49,51–53]. The membrane traffic model has recently been developed into the idea that TRPML1 participates in lysosomal exocytosis by promoting the fusion of lysosomes with the plasma membrane [54–56]. This process emerged as an important mechanism for plasma membrane repair [57]. Lysosomal exocytosis has also been implicated in transition metal detoxification including the excretion of Zn2+ and Cu2+ from cells [18,19]. Up-regulation of lysosomal exocytosis via TFEB overexpression has been used to clear storage material and suppress cell death in several lysosomal storage disease models [16,58,59]. From all these findings, it is safe to conclude that lysosomal exocytosis is an important cytoprotective mechanism. The proposed role of TRPML1 in the lysosomal membrane fusion events raises several questions including the mechanism triggering Ca2+ release from the lysosomes, lysosomal maturation before fusion/exocytosis, and indispensability of specifically TRPML1-mediated Ca2+ release for this process. At the same time, a definitive role of TRPML1 in this process means that TRPML1 activators and inhibitors may be used to modulate cell repair and detoxification. In the C. elegans MLIV model, deletion of TRPML1 ortholog CUP-5 delayed reformation of the lysosomes from hybrid organelles [60–62], which is somewhat similar to postlysosomal TRPML1-Dependent Processes, Shown and Discussed 473 lipid traffic deficits reported in an siRNA-driven acute human TRPML1 knockdown model [63]. A role of Ca2+ channels in membrane fission has not been shown, and thus it remains unclear what role TRPML1 would play in this process. Postlysosomal lipid traffic delays can be explained by incomplete processing of the endocytosed material resulting in unsuitabil- ity of the degradation products in TRPML1-deficient cells with the lysosomal transporters. Although diminished enzymatic activity was shown in MLIV fibroblasts [46,64], biochemical characterization of storage material in MLIV cells has not been consistent. The lack of a comprehensive biochemical account of storage material and lysosomal enzymatic profiling of TRPML1-deficient cells is a serious impediment to understanding the function of this ion channel in lysosomes. As TRPML1 regulates the endolysosomal membrane fusion, it may modulate the degradative function of lysosomes by driving the delivery of enzymes and transporters to lysosomes (Figure 25.2). Some initial experiments suggested lysosomal enzyme mislocalization in MLIV cells, but this model was not developed further [64], and to this day no comprehensive analysis of the lysosomal enzymatic and transporter profile as a function of TRPML1 status has been published. Macroautophagy deficits were shown in MLIV patients’ fibroblasts and Drosophila models [47,53,65–67]; because macroautophagy relies on membrane fusion events its suppression has been explained by delayed fusion of autophagosomes with lysosomes in TRPML1-deficient cells. Interestingly, chaperone-mediated autophagy appears to

Demonstrated

Predicted

FIGURE 25.2 Predicted and demonstrated role of TRPML1. Numbers denote, in “demonstrated”: (1) lysosomal exocytosis, (2) autophagy, (3) postlysosomal lipid traffic, (4) lysosomal enzymatic activity, (5) lysosomal-endosomal fission, (6) lysosomal-endosomal fusion, and (7) endocytosis. In “predicted”: (1) exosome secretion, (2) lysosomal antigen, growth factor and metal processing, (3) metal absorption, (4) antigen presentation, (5) toxin traffic to Golgi, and (6) parasite invasion. 474 25. TRPML1-Dependent Processes as Therapeutic Targets be defective in human MLIV patients’ fibroblasts as well [68]. In contrast to macroautophagy, chaperone-mediated autophagy involves chaperone-dependent translocation of proteins from cytoplasm into the lysosomes [69]. In addition to chaperones, it requires a translocator complex comprising several proteins including lysosomal associated membrane protein-2 (LAMP-2A). It will be interesting to answer whether or not localization of the translocator complex depends on TRPML1 and whether it is abnormal in cells affected by MLIV. Whereas the role of ion channels in membrane traffic is widely discussed, direct evidence linking ion channel activity with intracellular localization of a specific transporter or another transmembrane protein is lacking. A finding that TRPML1-deficient cells have abnormal distribution of lysosomal enzymes or transporters would set a precedent for a novel function of ion channels. An alternative mechanism involving TRPML1 in lysosomal degradation is via regulation of the activities of lysosomal enzymes and transporters. The initial findings that MLIV is associated with suppressed lysosomal enzymatic activity and the subsequent findings that lysosomal acid lipase activity is suppressed in MLIV fibroblasts [46,64] were not followed up, and thus the extent of the lysosomal malfunction in MLIV is not really known. TRPML1 may regulate the lysosomal enzymatic activity by regulating different aspects of the lysosomal ion homeostasis such as pH or metal content. TRPML1 was suggested to prevent lysosomal acidification [46,63], a notion that is indirectly supported by the recent evidence that proper maintenance of the lysosomal pH requires H+ leak or a monovalent cation channel [70,71]. Lysosomes are critical for Fe absorption in the majority of tissues as lysosomes are involved in processing the protein-bound Fe, and its subsequent extrusion into the cytoplasm [72]. Furthermore, recent evidence shows that lysosomes are also indispensable for seques- tration and exocytosis of cytoplasmic Zn2+ and Cu2+ [18,19]. Indeed, loss of lysosomal metal uptake and exocytosis significantly increases metal toxicity. The idea that TRPML1 regulates lysosomal metal content emerged with the evidence of TRPML1 permeability to Fe and, therefore, Fe buildup in MLIV fibroblasts [45]. In subsequent studies, it was shown that TRPML1-deficient cells exposed to Fe have higher levels of oxidative stress and mitochondrial damage than normal cells treated similarly [73]. This too can be explained by the lysosomal Fe buildup in TRPML1-deficient cells. In addition to damaging lysosomal membrane, which is a likely contributor to the leak of the lysosomal digestive enzymes into the cytoplasm in TRPML1-deficient cells, reactive oxygen species generated by the excess lysosomal Fe may damage lysosomal digestive enzymes or render endocytosed material and products of its digestion unsuitable for degradation and absorption. Zn is another transition metal whose handling appears to depend on TRPML1, as two groups demonstrated its buildup in TRPML1-deficient cells [74,75]. Although Zn may enter lysosomes via endocytosis or by autophagy of Zn-rich organelles such as mitochondria and secretory granules, the evidence that TRPML1-deficient cells in which Zn transporter ZnT4 was knocked down do not accumulate lysosomal Zn suggest that lysosomes take up Zn from the cytoplasm and TRPML1 is involved in dissipation of the lysosomal Zn via exocytosis or its release into the cytoplasm [74]. The mechanisms underlying the regulation of lysosomal metals by TRPML1 are unclear. On one hand, TRPML1 has been shown to be permeable to Fe and Zn [45]. Although brain Fe was not consistently surveyed in MLIV patients or mouse model, a deficient brain Fe delivery would be compatible with the hypomyelination suggested in MLIV [10]. Fe is required for oligodendrocyte maturation and for myelin production by these cells [76,77]. Although TRPML1-Dependent Processes Inferred from its Functional Context 475 hypomyelination is not a defining feature of MLIV and, indeed, it has been shown in several lysosomal storage diseases, the recent evidence points that TRPML1 is inhibited by sphin- gomyelin [49]. This lipid is a common secondary storage component in lysosomal storage diseases; therefore, it is possible that the loss or suppression of TRPML1 activity followed by drop in brain Fe uptake is a factor in other storage diseases. Accordingly, TRPML1 activation by chemical means alleviated the key aspects of Niemann-Pick C1 phenotype, a disease not directly caused by the TRPML1 loss [49]. A detailed survey of brain Fe dynamics in MLIV human patients as well as human patients and mouse models of other storage diseases would be very helpful in elucidating the role if TRPML1 in handling this vital ion at the organism level. Regarding the mechanism of TRPML1-dependent brain Fe uptake, TRPML1 Fe permeability could be particularly important in cells that appear to lack the divalent metal transporter 1 (DMT1) transporter—another component of Fe-handling machinery [72]. Among such cells are brain capillary endothelial cells, which are essential for brain Fe uptake. On the other hand, brain capillary endothelial cells were proposed to move Fe via transcytosis: capture of the extracellular material at the apical surface of the cell, followed by its transport across the cell and by the release of endocytic content via endo/lysosomal fusion with the basal membrane [78,79]. This is compatible with the TRPML1 role in the lysosomal secretion. It could be argued that intracellular and lysosomal-free Zn levels are too low (estimated to be in the picomolar range) for these cations to pass through an ion channel. However, the presence of the histidine-rich loop between the first and second transmembrane domains on TRPML1 could play a role in conferring Zn permeability to these channels. Interestingly, these regions exhibit similarity with similar domains in ZnT and Zip transporters, in which such loops were suggested to assist in Zn permeation by assisting in the transfer of Zn from other Zn-binding proteins to the transporters [43,44]. However, a role of TRPML1 in localization of other Zn transporters, such as a lysosomal Zip (which would export Zn from the lysosomes into the cytoplasm) or DMT1 cannot be ruled out at this moment. This, and an analysis of brain Zn as a function of TRPML1 status awaits further investigation.

TRPML1-DEPENDENT PROCESSES INFERRED FROM ITS FUNCTIONAL CONTEXT

It should be noted that some of the most fascinating aspects of the lysosomal function lie beyond degradation and absorption of the endocytosed material, and thus modulation of TRPML1 activity using pharmacological means could be key to a range of processes and conditions. Next we outline some of the processes in which TRPML1 involvements has not been demonstrated, but can be predicted using the present models of the TRPML1 function and the current knowledge of the endocytic pathway. In addition to nutrient uptake and plasma membrane repair/remodeling, the endocytic pathway modulates the processing of growth factor receptors via its endocytosis followed by ligand dissociation and receptor degradation or recycling [80,81]. Beyond a possible role of TRPML1 in the membrane fusion in the latter endocytic pathway, the recent evidence that TRPML1 regulates lysosomal exocytosis and endo/phagocytosis [54–56] suggests that it may be involved in growth factor receptor handling. Whether or not TRPML1 activators and inhibitors affect cell growth and proliferation is an open question. 476 25. TRPML1-Dependent Processes as Therapeutic Targets

Similarly, antigen processing and presentation depends on both degradative and sorting/ trafficking functions of the lysosomes [82]. Based on the predicted function of TRPML1 in the endocytic membrane traffic, this suggests immune system involvement in MLIV. No consistent information on such an involvement in MLIV patients is available, and thus it would be important to establish whether or not it is a factor in MLIV patients and model mice. Interestingly, genome-wide RNAi screen in S2 cells for microbial clearance revealed fly trpml as one of the few hits for diminished degradation of bacteria [83]. ESCRT family members (proteins responsible for formation of intraluminal vesicles in multivesicular bodies [84]) were also involved, suggesting an interaction between TRPML function and the formation of multivesicular bodies. Directly showing that TRPML1 is involved in antigen processing and presentation would suggest that its activators and inhibitors could be novel candidates for treating immune and autoimmune diseases. Exosomes are extracellular vesicles secreted by some cell types. They have been implicated in establishment of the metastatic loci and in cell death in neurodegeneration [85–88]. Evidence suggests that exosomes are luminal vesicles of the multivesicular bodies secreted due to their fusion with the plasma membrane [89–91]. If TRPML1 is involved in formation of multivesicular bodies and/or their exocytosis, then it is possible that TRPML1 regulates exosome secretion. Keeping in mind their significance in cancer and neurodegenerative diseases, it would be important to establish whether not there’s a role of TRPML1 in exosomal secretion. Beyond that, a suggestion of non-cell-autonomous component of cell loss in storage diseases is very appealing because it would justify completely novel approaches to them. Beyond the digestive and sorting function of the endocytic pathway, TRPML1 appears to be an important component of the energy-sensing signaling circuit actuated by MTOR (mechanistic target or rapamycin). MTOR is a kinase responsible for translating the cellular energy status into activation of cellular circuits regulating metabolism, growth, and motility [24,92–94]. The recently demonstrated requirement for TRPML1 function for MTOR [47] suggests that the aspects of the endocytic function regulated by TRPML1 are read by MTOR suggesting that both molecules are involved in the same signaling pathway regulating key aspect of cellular function. It should be noted that MTOR dysregulation has been linked to several forms of cancer, potentially linking TRPML1 to this condition [95–97]. Further highlighting the potential role of TRPML1 in cancer is its role in regulation of lipid traffic and metabolism. Several types of lipids have been implicated in regulating cell proliferation, and thus regulation of lipid handling by TRPML1 is likely an important aspect of cell cycle regulation and, potentially, cancer research. The processing of several toxins such as shiga, cholera, botulinum, and diphtheria toxins depends on trafficking along the endocytic pathway [98–103]. Parasite invasion itself depends on membrane traffic as well. Indeed a role of TRPML1 activator PI(3,5)P2 in Plasmodium berghei invasion has been recently shown [104]. Although we still need to establish whether or not TRPML1 function affects parasite invasion or microbial toxicity, a role of TRPML1 in membrane traffic suggests that modulators of its activity could serve as antiparasitic drugs. As discussed earlier, roles of TRPML1 in both transition metal permeability and membrane traffic may underlie its utility in metal handling. Fe uptake depends on its dissociation from transferrin in late-endosomes/lysosomes, which in turn is a function of membrane traffic and organellar pH [72]. If TRPML1 regulates either of them, it may play key role in the Fe absorption process. The transfer of Fe across the lysosomal membrane depends on the conversion Conclusions 477 of ferric iron to divalent ferrous Fe. Interestingly, TRPML1 is permeable to Fe2+ but not to Fe3+ [45]. Although in the majority of tissues DMT1 is presumed to be the lysosomal Fe exporter, some tissues lack DMT1 [78,79]. Furthermore, DMT1 and TRPML1 appear to be regulated by different signaling circuits, as DMT1 does not appear to belong to the lysosomal gene network. It is possible that TRPML1 substitutes DMT1 in some tissues or under certain conditions such as during oxidative stress or during phases of development requiring Fe, such as myelination in the CNS. Whether TRPML1 regulates Fe by conducting it or by ensuring its delivery via membrane traffic, modulating its activity could be an important tool for modulating developmental processes. Fe excess is toxic due to the role of Fe in catalysis of ROS production via Fenton-type reactions. Indeed, Fe toxicity is a factor in neurodegenerative diseases, cell death during stroke, and the aging-related suppression of the lysosomal/autophagic function [105–110]. It was shown that, in aged cells, suppression of autophagy due to Fe-dependent accumulation of lipofuscin is associated with the toxic buildup of aged, dysfunctional mitochondria [111]. Accordingly, autophagy and mitophagy deficits were shown in MLIV, including human fibroblasts and mouse and Drosophila models [47,53,65–67], and a link between autophagic deficits and cell death has been proposed [112]. Beyond autophagic deficits, Fe buildup in TRPML1-deficient cells appears to be associated with oxidative stress and mitochondrial damage [73]. It is, therefore tempting to suggest that TRPML1 activators or increased expression may alleviate some of the effects of aging and oxidative stress. Zn2+ is a key element in numerous aspects of cellular and systems processes including gene expression, enzymatic activity, and neurotransmission [113,114]. The importance of this ion is emphasized by the fact that about twice as many genes code for proteins dedicated to regulation of Zn2+ than Ca2+. The previously published and recent evidence link lysosomes to the regulation of cellular Zn, and indeed key biological processes such as cell death during mammary gland involution appear to depend on Zn uptake by the lysosomes. The role of Zn in the mammary gland involution has been recently reviewed [114]; it will be interesting to establish whether or not TRPML1 is involved in regulation of Zn in other involuting tissues such as ovaries and uterus. Whether TRPML1 is involved in the dissipation of the lysosomal Zn, its exocytosis, or localization of the lysosomal transporters, it is important to note that involution was suggested to clear premalignant cells, and thus TRPML1 activators and inhibitors may play a role in defense against cancer.

CONCLUSIONS

Since its discovery, the search for TRPML1 function has been focused on the CNS. However, the examples outlined earlier are just a glance at the entire range of processes predicted to be regulated by TRPML1. Although TRPML1 has been shown to play a role in neuronal, gastric, and muscle function, it is clear that its repertoire is much wider. Its central role in the lysosomal biogenesis, traffic, or ion homeostasis makes it an almost inescapable conclusion that those processes that depend on lysosomal membrane traffic or ion homeostasis are regulated by TRPML1. Such processes include growth factor processing, transition metal uptake, and recycling of aged organelles. These processes are solidly established to drive neurodevelopment, aging, and cancer, and thus, based on the aspects of the cellular function in which 478 25. TRPML1-Dependent Processes as Therapeutic Targets

TRPML1 has been previously implicated, we propose that TRPML1 activators and inhibitors promise new approaches to a range of conditions likely including such key processes as development, cancer, and aging.

Acknowledgments This work was supported by the National Institutes of Health grant RO1HD058577 to KK, and R01NS081301 to KV.

References [1] Bonavita S, Virta A, Jeffries N, Goldin E, Tedeschi G, Schiffmann R. Diffuse neuroaxonal involvement in mucolipidosis IV as assessed by proton magnetic resonance spectroscopic imaging. J Child Neurol 2003;18(7):443–9. [2] Slaugenhaupt SA. The molecular basis of mucolipidosis type IV. Curr Mol Med 2002;2(5):445–50. [3] Pradhan SM, Atchaneeyasakul LO, Appukuttan B, Mixon RN, McFarland TJ, Billingslea AM, et al. Electronegative electroretinogram in mucolipidosis IV. Arch Ophthalmol 2002;120(1):45–50. [4] Bach G. Mucolipidosis type IV. Mol Genet Metab 2001;73(3):197–203. [5] Frei KP, Patronas NJ, Crutchfield KE, Altarescu G, Schiffmann R. Mucolipidosis type IV: characteristic MRI findings. Neurology 1998;51(2):565–9. [6] Amir N, Zlotogora J, Bach G. Mucolipidosis type IV: clinical spectrum and natural history. Pediatrics 1987;79(6):953–9. [7] Cantani A. Mucolipidosis IV. Am J Dis Child 1983;137(1):88. [8] Tellez-Nagel I, Rapin I, Iwamoto T, Johnson AB, Norton WT, Nitowsky H. Mucolipidosis IV. Clinical, ultrastructural, histochemical, and chemical studies of a case, including a brain biopsy. Arch Neurol 1976;33(12):828–35. [9] Newell FW, Matalon R, Meyer S. A new mucolipidosis with psychomotor retardation, corneal clouding, and retinal degeneration. Am J Ophthalmol 1975;80(3):440–9. [10] Micsenyi MC, Dobrenis K, Stephney G, Pickel J, Vanier MT, Slaugenhaupt SA, et al. Neuropathology of the Mcoln1(-/-) knockout mouse model of mucolipidosis type IV. J Neuropathol Exp Neurol 2009;68(2):125–35. [11] Venugopal B, Browning MF, Curcio-Morelli C, Varro A, Michaud N, Nanthakumar N, et al. Neurologic, gastric, and opthalmologic pathologies in a murine model of mucolipidosis type IV. Am J Hum Genet 2007;81(5):1070–83. [12] Chandra M, Zhou H, Li Q, Muallem S, Hofmann SL, Soyombo AA. A role for the Ca2+ channel TRPML1 in gastric acid secretion, based on analysis of knockout mice. Gastroenterology 2011;140(3):857–67. [13] Lubensky IA, Schiffmann R, Goldin E, Tsokos M. Lysosomal inclusions in gastric parietal cells in mucolipidosis type IV: a novel cause of achlorhydria and hypergastrinemia. Am J Surg Pathol 1999;23(12):1527–31. [14] Schiffmann R, Dwyer NK, Lubensky IA, Tsokos M, Sutliff VE, Latimer JS, et al. Constitutive achlorhydria in mucolipidosis type IV. Proc Natl Acad Sci USA 1998;95(3):1207–12. [15] Giordano S, Darley-Usmar V, Zhang J. Autophagy as an essential cellular antioxidant pathway in neurodegenerative disease. Redox Biol 2014;2:82–90. [16] Feeney EJ, Spampanato C, Puertollano R, Ballabio A, Parenti G, Raben N. What else is in store for autophagy? Exocytosis of autolysosomes as a mechanism of TFEB-mediated cellular clearance in Pompe disease. Autophagy 2013;9(7):1117–18. [17] Settembre C, Di Malta C, Polito VA, Garcia Arencibia M, Vetrini F, Erdin S, et al. TFEB links autophagy to lysosomal biogenesis. Science 2011;332(6036):1429–33. [18] Polishchuk EV, Concilli M, Iacobacci S, Chesi G, Pastore N, Piccolo P, et al. Wilson disease protein ATP7B uti- lizes lysosomal exocytosis to maintain copper homeostasis. Dev Cell 2014;29(6):686–700. [19] Kukic I, Kelleher SL, Kiselyov K. Zinc efflux through lysosomal exocytosis prevents zinc-induced toxicity. J Cell Sci 2014;127(Pt 14):3094–103. [20] Martina JA, Puertollano R. RRAG GTPases link nutrient availability to gene expression, autophagy and lysosomal biogenesis. Autophagy 2013;9(6):928–30. [21] Settembre C, Zoncu R, Medina DL, Vetrini F, Erdin S, Huynh T, et al. A lysosome-to-nucleus signalling mechanism senses and regulates the lysosome via mTOR and TFEB. EMBO J 2012;31(5):1095–108. [22] Roczniak-Ferguson A, Petit CS, Froehlich F, Qian S, Ky J, Angarola B, et al. The transcription factor TFEB links mTORC1 signaling to transcriptional control of lysosome homeostasis. Sci Signal 2012;5(228):ra42. REFERENCES 479

[23] Martina JA, Chen Y, Gucek M, Puertollano R. MTORC1 functions as a transcriptional regulator of autophagy by preventing nuclear transport of TFEB. Autophagy 2012;8(6):903–14. [24] Zoncu R, Bar-Peled L, Efeyan A, Wang S, Sancak Y, Sabatini DM. mTORC1 senses lysosomal amino acids through an inside-out mechanism that requires the vacuolar H(+)-ATPase. Science 2011;334(6056):678–83. [25] Wang W, Zhang X, Gao Q, Xu H. TRPML1: an ion channel in the lysosome. Handb Exp Pharmacol 2014;222:631–45. [26] Weiss N. Cross-talk between TRPML1 channel, lipids and lysosomal storage diseases. Commun Integr Biol 2012;5(2):111–13. [27] Colletti GA, Kiselyov K. Trpml1. Adv Exp Med Biol 2011;704:209–19. [28] Venkatachalam K, Luo J, Montell C. Evolutionarily conserved, multitasking TRP channels: lessons from worms and flies. Handb Exp Pharmacol 2014;223:937–62. [29] Venkatachalam K, Wong CO, Montell C. Feast or famine: role of TRPML in preventing cellular amino acid starvation. Autophagy 2013;9(1):98–100. [30] Garcia-Anoveros J, Wiwatpanit T. TRPML2 and mucolipin evolution. Handb Exp Pharmacol 2014;222:647–58. [31] Grimm C, Barthmes M, Wahl-Schott C. Trpml3. Handb Exp Pharmacol 2014;222:659–74. [32] Venkatachalam K, Hofmann T, Montell C. Lysosomal localization of TRPML3 depends on TRPML2 and the mucolipidosis-associated protein TRPML1. J Biol Chem 2006;281(25):17517–27. [33] Castiglioni AJ, Remis NN, Flores EN, García-Añoveros J. Expression and vesicular localization of mouse Trpml3 in stria vascularis, hair cells, and vomeronasal and olfactory receptor neurons. J Comp Neurol 2011;519(6):1095–114. [34] Samie MA, Grimm C, Evans JA, Curcio-Morelli C, Heller S, Slaugenhaupt SA, et al. The tissue-specific expression of TRPML2 (MCOLN-2) gene is influenced by the presence of TRPML1. Pflugers Arch 2009; 459(1):79–91. [35] Graves AR, Curran PK, Smith CL, Mindell JA. The Cl-/H+ antiporter ClC-7 is the primary chloride permeation pathway in lysosomes. Nature 2008;453(7196):788–92. [36] Miedel MT, Weixel KM, Bruns JR, Traub LM, Weisz OA. Posttranslational cleavage and adaptor protein complex-dependent trafficking of mucolipin-1. J Biol Chem 2006;281(18):12751–9. [37] Vergarajauregui S, Puertollano R. Two di-leucine motifs regulate trafficking of mucolipin-1 to lysosomes. Traffic 2006;7(3):337–53. [38] Zhang X, Li X, Xu H. Phosphoinositide isoforms determine compartment-specific ion channel activity. Proc Natl Acad Sci USA 2012;109(28):11384–9. [39] Dong XP, Shen D, Wang X, Dawson T, Li X, Zhang Q, et al. PI(3,5)P(2) controls membrane trafficking by direct activation of mucolipin Ca(2+) release channels in the endolysosome. Nat Commun 2010;1(4):38. [40] Martina JA, Lelouvier B, Puertollano R. The calcium channel mucolipin-3 is a novel regulator of trafficking along the endosomal pathway. Traffic 2009;10(8):1143–56. [41] Kim HJ, Soyombo AA, Tjon-Kon-Sang S, So I, Muallem S. The Ca(2+) channel TRPML3 regulates membrane trafficking and autophagy. Traffic 2009;10(8):1157–67. [42] Kiselyov K, Chen J, Rbaibi Y, Oberdick D, Tjon-Kon-Sang S, Shcheynikov N, et al. TRP-ML1 is a lysosomal monovalent cation channel that undergoes proteolytic cleavage. J Biol Chem 2005;280(52):43218–23. [43] Costello LC, Fenselau CC, Franklin RB. Evidence for operation of the direct zinc ligand exchange mechanism for trafficking, transport, and reactivity of zinc in mammalian cells. J Inorg Biochem 2011;105(5):589–99. [44] Milon B, Wu Q, Zou J, Costello LC, Franklin RB. Histidine residues in the region between transmembrane domains III and IV of hZip1 are required for zinc transport across the plasma membrane in PC-3 cells. Biochim Biophys Acta 2006;1758(10):1696–701. [45] Dong X, Cheng X, Mills E, Delling M, Wang F, Kurz T, et al. The type IV mucolipidosis-associated protein TRPML1 is an endolysosomal iron release channel. Nature 2008;455(7215):992–6. [46] Soyombo AA, Tjon-Kon-Sang S, Rbaibi Y, Bashllari E, Bisceglia J, Muallem S, et al. TRP-ML1 regulates lysosomal pH and acidic lysosomal lipid hydrolytic activity. J Biol Chem 2006;281(11):7294–301. [47] Wong CO, Li R, Montell C, Venkatachalam K. Drosophila TRPML is required for TORC1 activation. Curr Biol 2012;22(17):1616–21. [48] Feng X, Huang Y, Lu Y, Xiong J, Wong CO, Yang P, et al. Drosophila TRPML forms PI(3,5)P2-activated cation channels in both endolysosomes and plasma membrane. J Biol Chem 2014;289(7):4262–72. [49] Shen D, Wang X, Li X, Zhang X, Yao Z, Dibble S, et al. Lipid storage disorders block lysosomal trafficking by inhibiting a TRP channel and lysosomal calcium release. Nat Commun 2012;3:731. 480 25. TRPML1-Dependent Processes as Therapeutic Targets

[50] Chen CC, Keller M, Hess M, Schiffmann R, Urban N, Wolfgardt A, et al. A small molecule restores function to TRPML1 mutant isoforms responsible for mucolipidosis type IV. Nat Commun 2014;5:4681. [51] Thompson EG, Schaheen L, Dang H, Fares H. Lysosomal trafficking functions of mucolipin-1 in murine macrophages. BMC Cell Biol 2007;8:54. [52] Pryor PR, Reimann F, Gribble FM, Luzio JP. Mucolipin-1 Is a lysosomal membrane protein required for intracellular lactosylceramide traffic. Traffic 2006;7(10):1388–98. [53] Venkatachalam K, Long AA, Elsaesser R, Nikolaeva D, Broadie K, Montell C. Motor deficit in a Drosophila model of mucolipidosis type IV due to defective clearance of apoptotic cells. Cell 2008;135(5):838–51. [54] Laplante JM, Sun M, Falardeau J, Dai D, Brown EM, Slaugenhaupt SA, et al. Lysosomal exocytosis is impaired in mucolipidosis type IV. Mol Genet Metab 2006;89(4):339–48. [55] Samie M, Wang X, Zhang X, Goschka A, Li X, Cheng X, et al. A TRP channel in the lysosome regulates large particle phagocytosis via focal exocytosis. Dev Cell 2013;26(5):511–24. [56] Samie MA, Xu H. Lysosomal exocytosis and lipid storage disorders. J Lipid Res 2014;55(6):995–1009. [57] Reddy A, Caler EV, Andrews NW. Plasma membrane repair is mediated by Ca(2+)-regulated exocytosis of lysosomes. Cell 2001;106(2):157–69. [58] Spampanato C, Feeney E, Li L, Cardone M, Lim JA, Annunziata F, et al. Transcription factor EB (TFEB) is a new therapeutic target for Pompe disease. EMBO Mol Med 2013;5(5):691–706. [59] Medina DL, Fraldi A, Bouche V, Annunziata F, Mansueto G, Spampanato C, et al. Transcriptional activation of lysosomal exocytosis promotes cellular clearance. Dev Cell 2011;21(3):421–30. [60] Treusch S, Knuth S, Slaugenhaupt SA, Goldin E, Grant BD, Fares H. Caenorhabditis elegans functional or- thologue of human protein h-mucolipin-1 is required for lysosome biogenesis. Proc Natl Acad Sci USA 2004;101(13):4483–8. [61] Hersh BM, Hartwieg E, Horvitz HR. The Caenorhabditis elegans mucolipin-like gene cup-5 is essential for viability and regulates lysosomes in multiple cell types. Proc Natl Acad Sci USA 2002;99(7):4355–60. [62] Fares H, Greenwald I. Regulation of endocytosis by CUP-5, the Caenorhabditis elegans mucolipin-1 homolog. Nat Genet 2001;28(1):64–8. [63] Miedel MT, Rbaibi Y, Guerriero CJ, Colletti G, Weixel KM, Weisz OA, et al. Membrane traffic and turnover in TRP- ML1-deficient cells: a revised model for mucolipidosis type IV pathogenesis. J Exp Med 2008;205(6):1477–90. [64] Bach G, Zeigler M, Schaap T, Kohn G. Mucolipidosis type IV: ganglioside sialidase deficiency. Biochem Biophys Res Commun 1979;90(4):1341–7. [65] Jennings Jr JJ, Zhu JH, Rbaibi Y, Luo X, Chu CT, Kiselyov K. Mitochondrial aberrations in mucolipidosis type IV. J Biol Chem 2006;281(51):39041–50. [66] Curcio-Morelli C, Charles FA, Micsenyi MC, Cao Y, Venugopal B, Browning MF, et al. Macroautophagy is defective in mucolipin-1-deficient mouse neurons. Neurobiol Dis 2010;40(2):370–7. [67] Vergarajauregui S, Connelly PS, Daniels MP, Puertollano R. Autophagic dysfunction in mucolipidosis type IV patients. Hum Mol Genet 2008;17(17):2723–37. [68] Venugopal B, Mesires NT, Kennedy JC, Curcio-Morelli C, Laplante JM, Dice JF, et al. Chaperone-mediated autophagy is defective in mucolipidosis type IV. J Cell Physiol 2009;219(2):344–53. [69] Massey A, Kiffin R, Cuervo AM. Pathophysiology of chaperone-mediated autophagy. Int J Biochem Cell Biol 2004;36(12):2420–34. [70] Ishida Y, Nayak S, Mindell JA, Grabe M. A model of lysosomal pH regulation. J Gen Physiol 2013;141(6):705–20. [71] Steinberg BE, Huynh KK, Brodovitch A, Jabs S, Stauber T, Jentsch TJ, et al. A cation counterflux supports lysosomal acidification. J Cell Biol 2010;189(7):1171–86. [72] Valerio LG. Mammalian iron metabolism. Toxicol Mech Methods 2007;17(9):497–517. [73] Coblentz J, St Croix C, Kiselyov K. Loss of TRPML1 promotes production of reactive oxygen species: is oxidative damage a factor in mucolipidosis type IV? Biochem J 2014;457(2):361–8. [74] Kukic I, Lee JK, Coblentz J, Kelleher SL, Kiselyov K. Zinc-dependent lysosomal enlargement in TRPML1- deficient cells involves MTF-1 transcription factor and ZnT4 (Slc30a4) transporter. Biochem J 2013;451(2):155–63. [75] Eichelsdoerfer JL, Evans JA, Slaugenhaupt SA, Cuajungco MP. Zinc dyshomeostasis is linked with the loss of mucolipidosis IV-associated TRPML1 ion channel. J Biol Chem 2010;285(45):34304–8. [76] Perez MJ, Fernandez N, Pasquini JM. Oligodendrocyte differentiation and signaling after transferrin internalization: a mechanism of action. Exp Neurol 2013;248:262–74. [77] Todorich B, Pasquini JM, Garcia CI, Paez PM, Connor JR. Oligodendrocytes and myelination: the role of iron. Glia 2009;57(5):467–78. REFERENCES 481

[78] Moos T, Skjoerringe T, Gosk S, Morgan EH. Brain capillary endothelial cells mediate iron transport into the brain by segregating iron from transferrin without the involvement of divalent metal transporter 1. J Neurochem 2006;98(6):1946–58. [79] Moos T, Morgan EH. The significance of the mutated divalent metal transporter (DMT1) on iron transport into the Belgrade rat brain. J Neurochem 2004;88(1):233–45. [80] Haglund K, Dikic I. The role of ubiquitylation in receptor endocytosis and endosomal sorting. J Cell Sci 2012;125(Pt 2):265–75. [81] Tomas A, Futter CE, Eden ER. EGF receptor trafficking: consequences for signaling and cancer. Trends Cell Biol 2014;24(1):26–34. [82] Watts C. The endosome-lysosome pathway and information generation in the immune system. Biochim Biophys Acta 2012;1824(1):14–21. [83] Philips JA, Porto MC, Wang H, Rubin EJ, Perrimon N. ESCRT factors restrict mycobacterial growth. Proc Natl Acad Sci USA 2008;105(8):3070–5. [84] Schuh AL, Audhya A. The ESCRT machinery: from the plasma membrane to endosomes and back again. Crit Rev Biochem Mol Biol 2014;49(3):242–61. [85] Atay S, Godwin AK. Tumor-derived exosomes: a message delivery system for tumor progression. Commun Integr Biol 2014;7(1):e28231. [86] Roma-Rodrigues C, Fernandes AR, Baptista PV. Exosome in tumour microenvironment: overview of the cross- talk between normal and cancer cells. Biomed Res Int 2014;2014:179486. [87] Kalani A, Tyagi A, Tyagi N. Exosomes: mediators of neurodegeneration, neuroprotection and therapeutics. Mol Neurobiol 2014;49(1):590–600. [88] Russo I, Bubacco L, Greggio E. Exosomes-associated neurodegeneration and progression of Parkinson’s disease. Am J Neurodegener Dis 2012;1(3):217–25. [89] Stahl PD, Barbieri MA. Multivesicular bodies and multivesicular endosomes: the "ins and outs" of endosomal traffic. Sci STKE 2002;2002(141):PE32. [90] Kowal J, Tkach M, Thery C. Biogenesis and secretion of exosomes. Curr Opin Cell Biol 2014;29C:116–25. [91] Tsilioni I, Panagiotidou S, Theoharides TC. Exosomes in neurologic and psychiatric disorders. Clin Ther 2014;36(6):882–8. [92] Bar-Peled L, Sabatini DM. Regulation of mTORC1 by amino acids. Trends Cell Biol 2014;24(7):400–6. [93] Haissaguerre M, Saucisse N, Cota D. Influence of mTOR in energy and metabolic homeostasis. Mol Cell Endocrinol 2014; in print. [94] Howell JJ, Manning BD. mTOR couples cellular nutrient sensing to organismal metabolic homeostasis. Trends Endocrinol Metab 2011;22(3):94–102. [95] Chan J, Kulke M. Targeting the mTOR signaling pathway in neuroendocrine tumors. Curr Treat Options Oncol 2014;15(3):365–79. [96] Fasolo A, Sessa C. Targeting mTOR pathways in human malignancies. Curr Pharm Des 2012;18(19):2766–77. [97] Wazir U, Wazir A, Khanzada ZS, Jiang WG, Sharma AK, Mokbel K. Current state of mTOR targeting in human breast cancer. Cancer Genomics Proteomics 2014;11(4):167–74. [98] Stein MP, Muller MP, Wandinger-Ness A. Bacterial pathogens commandeer Rab GTPases to establish intracellular niches. Traffic 2012;13(12):1565–88. [99] Bergan J, Dyve Lingelem AB, Simm R, Skotland T, Sandvig K. Shiga toxins. Toxicon 2012;60(6):1085–107. [100] Spooner RA, Lord JM. How ricin and Shiga toxin reach the cytosol of target cells: retrotranslocation from the endoplasmic reticulum. Curr Top Microbiol Immunol 2012;357:19–40. [101] Murphy JR. Mechanism of diphtheria toxin catalytic domain delivery to the eukaryotic cell cytosol and the cellular factors that directly participate in the process. Toxins (Basel) 2011;3(3):294–308. [102] Abrami L, Liu S, Cosson P, Leppla SH, van der Goot FG. Anthrax toxin triggers endocytosis of its receptor via a lipid raft-mediated clathrin-dependent process. J Cell Biol 2003;160(3):321–8. [103] Leka O, Vallese F, Pirazzini M, Berto P, Montecucco C, Zanotti G. Diphtheria toxin conformational switching at acidic pH. FEBS J 2014;281(9):2115–22. [104] Thieleke-Matos C, da Silva ML, Cabrita-Santos L, Pires CF, Ramalho JS, Ikonomov O, et al. Host PI(3,5)P activity is required for Plasmodium berghei growth during liver stage infection. Traffic 2014; 15(10):1066–82. [105] Ward RJ, Dexter DT, Crichton RR. Chelating agents for neurodegenerative diseases. Curr Med Chem 2012;19(17):2760–72. 482 25. TRPML1-Dependent Processes as Therapeutic Targets

[106] Schrag M, Mueller C, Oyoyo U, Smith MA, Kirsch WM. Iron, zinc and copper in the Alzheimer’s disease brain: a quantitative meta-analysis. Some insight on the influence of citation bias on scientific opinion. Prog Neurobiol 2011;94(3):296–306. [107] Hegde ML, Hegde PM, Rao KS, Mitra S. Oxidative genome damage and its repair in neurodegenerative diseases: function of transition metals as a double-edged sword. J Alzheimers Dis 2011;24(Suppl 2):183–98. [108] Brewer GJ. Risks of copper and iron toxicity during aging in humans. Chem Res Toxicol 2010;23(2):319–26. [109] Wright RO, Baccarelli A. Metals and neurotoxicology. J Nutr 2007;137(12):2809–13. [110] Kurz T, Terman A, Gustafsson B, Brunk UT. Lysosomes in iron metabolism, ageing and apoptosis. Histochem Cell Biol 2008;129(4):389–406. [111] Zhao M, Antunes F, Eaton JW, Brunk UT. Lysosomal enzymes promote mitochondrial oxidant production, cytochrome c release and apoptosis. Eur J Biochem 2003;270(18):3778–86. [112] Kiselyov K, Jennigs Jr JJ, Rbaibi Y, Chu CT. Autophagy, mitochondria and cell death in lysosomal storage diseases. Autophagy 2007;3(3):259–62. [113] Stefanidou M, Maravelias C, Dona A, Spiliopoulou C. Zinc: a multipurpose trace element. Arch Toxicol 2006;80(1):1–9. [114] McCormick NH, Hennigar SR, Kiselyov K, Kelleher SL. The biology of zinc transport in mammary epithelial cells: implications for mammary gland development, lactation, and involution. J Mammary Gland Biol Neoplasia 2014;19(1):59–71. CHAPTER 26 TRPs in Respiratory Disorders: Opportunities Beyond TRPA1 Alexander Zholos,1,* Lorcan McGarvey,2 Madeleine Ennis2 1Department of Biophysics, Educational and Scientific Centre “Institute of Biology”, Taras Shevchenko Kiev National University, Kiev, Ukraine 2Centre for Infection and Immunity, School of Medicine, Dentistry and Biomedical Sciences, Queen’s University Belfast, Belfast, Northern Ireland, UK *Corresponding author: [email protected]

OUTLINE

Introduction 484 The Vanilloid TRPs 488 The Melastatin TRPs 492 Overview of TRP Channels and Their Expression and Function in the Summary and Perspectives 493 Respiratory System 485 The Canonical TRPs 486 References 495

Abbreviations 4α-PDD 4α-phorbol-12,13-didecanoate ASM airway smooth muscle 2+ [Ca ]i intracellular concentration of free calcium ions CaMKII Ca2+/calmodulin-dependent kinase II CF cystic fibrosis CFTR cystic fibrosis transmembrane conductance regulator CGPR calcitonin gene-related peptide COPD chronic obstructive pulmonary disease mICAT muscarinic cation current Orai ORAI Ca2+ release-activated Ca2+ modulator 1 PI3K phosphatidylinositol 3-kinase

PKA protein kinase A PKC protein kinase C PLC phospholipase C PM airborne particulate matter ROS reactive oxygen species RVD regulatory volume decrease SOC store-operated channel TRP transient receptor potential TM transmembrane domain TNF-α tumor necrosis factor alpha

INTRODUCTION

Many physical and chemical environmental factors affect the respiratory system by virtue of its large surface area, estimated at 100 m2, which is needed for an efficient gas exchange and which can be, at the same time, relatively easily exposed to various harmful airborne factors. The American Lung Association in its State of the Air 2014 report (http://www.stateoftheair. org) shows, that air quality in the United States worsened in 2010-2012 and that almost half of the population lives in areas where pollution levels are too often dangerous to breathe. Ozone

(O3) and airborne particulate matter (PM; e.g., exhaust smoke) are the two most common categories of air pollutants, which can cause many adverse clinical effects, such as chronic cough, wheezing, airway inflammation, and exacerbations of chronic obstructive pulmonary disease (COPD) and asthma. Airway parasympathetic reflex responses, such as cough, sneezing, apnea, mucus secretion, and bronchoconstriction, which are triggered by various types of peripheral sensory nerve endings located in or below the epithelial layers throughout the upper and lower respiratory tract, provide natural defenses against these airborne chemical irritants, primarily aiming to clear or neutralize these harmful irritants [1–8]. These reflexes can be modified by various factors, whereas both their suppression and their sensitization can present clinical problems [1,7,9,10]. The airways are innervated by chemo-, thermo- and mechanosensory nerves (e.g., thinly myelinated Aδ-type and polymodal unmyelinated C fibers), which are capable of detecting a large variety of noxious physical and chemical stimuli, such as cold air, hypoxia, osmolarity, oxidative stress, pressure and mechanical stress, aldehydes (e.g., acrolein, formaldehyde, acetaldehyde), ammonia, chlorine, nicotine, and capsaicin [1,2,4,11]. Persistent nerve activation and epithelial injury can also lead to inflammatory responses known as neurogenic inflammation, which is associated with the release of neuropeptides, such as tachykinins and calcitonin gene-related peptide (CGRP), from peripheral nerve endings as a response of sensory neurons to noxious stimuli and/or inflammatory mediators [12]. These neuropeptides trigger vascular (e.g., an increase in vascular permeability, arterial vasodilatation, extravasations of plasma protein, leukocyte adhesion to the endothelium), and nonvascular responses (e.g., airway smooth muscle (ASM) constriction, and mucus hypersecretion), as well as further release of inflammatory mediators [13,14]. Neurogenic inflammation may be a key component of respiratory diseases, such as asthma and COPD, and it is likely to be involved in the sensitization of the cough response in chronic cough [15]. TRP CHANNELS IN THE RESPIRATORY SYSTEM 485

Until recently it was generally assumed that the adverse effects of various environmental insults are rather nonspecific, but with the discovery of one of the largest superfamilies of ion channels—sensory transient receptor potential (TRP) channels—there has been a large shift of the paradigm toward identification of the specific roles of these channels in the physiology and pathology of the respiratory system. In this context, in recent years much interest and effort has been directed to the identification of the specific pathophysiological roles of individual TRP subtypes in the respiratory system because (i) TRPs are well recognized as ubiquitous cell sensors responding to a wide range of physical and chemical stimuli, and (ii) various members of this large group of Ca2+-permeable cation channels are widely expressed not only in sensory neurons, but also in other tissues and cell types, which are relevant to respiratory disorders, such as airway epithelial cells, mast cells, macrophages, lymphocytes, neutrophils, eosinophils, and ASM [16–20]. Identification of specific molecules capable of selective detection of various environmental chemical and physical stimuli indeed opens up a new prospect for our better understanding of relevant respiratory symptoms and disorders and, subsequently, for the development of informed and purposeful strategies for their treatment. In this chapter, the current knowledge of TRP roles in various cell types within the respiratory system will be discussed focusing on several well-studied TRP subtypes (but not TRPA1, which is discussed in-depth by McAlexander in this volume), their expression patterns in cell types relevant to respiratory disorders, and their responsiveness to noxious stimuli. Within this broad context, we discuss the rationale of pharmacological targeting of TRPs in respiratory pathology. It should be noted that central mechanisms of airway responses are outside the scope of this review. We aimed to group the accumulating evidence for TRP roles in respiratory disorders according to channel subtypes, rather than specific pathology, in order to highlight multiple and widespread roles of individual TRPs. The interested reader is referred to several excellent reviews discussing in detail the important roles of various TRPs in specific respiratory diseases [3,4,8,9,11,21–32].

OVERVIEW OF TRP CHANNELS AND THEIR EXPRESSION AND FUNCTION IN THE RESPIRATORY SYSTEM

In mammals, the TRP superfamily consists of 28 structurally related proteins, which are classified into six groups: the TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPP (polycystin), TRPML (mucolipin), and TRPA (ankyrin) subfamilies. This categorization is based on their structural homology rather than any similarities in biophysical, pharmacological, or physiological properties [16–19]. All TRPs have cytoplasmic N- and C-terminals separated by six putative transmembrane (TM) domains with the pore-forming region found in the loop between TM5 and TM6. There are also additional protein domains specific to certain TRP subfamilies (e.g., N-terminus ankyrin repeats are found in TRPCs and TRPVs, but lacking in TRPMs) or even individual TRP subtypes (e.g., a C-terminal atypical serine/threonine kinase domain is present only in TRPM6 and TRPM7 channels). These domains contribute to large differences in protein length, ranging from 553 amino acid residues in hTRPML3 to 2022 residues in hTRPM6. TRP channels are widely expressed in excitable and nonexcitable cells, where they perform diverse functions ranging from detection of temperature change, osmolarity, redox 486 26. PATHOPHYSIOLOGICAL ROLES OF RESPIRATORY TRPs state, and pH to control of cell proliferation or death. Functional TRP channels are cation- selective homo- or heterotetramers, and most are permeable to Ca2+, although Ca2+ permeability can differ very significantly between TRP isoforms, from impermeable to Ca2+ TRPM4 and 2+ TRPM5 to highly Ca permeable (PCa/PNa > 100) TRPV5 and TRPV6 [19]. TRPM6 and TRPM7 2+ 2+ are moderately permeable to Ca (PCa/PNa < 1), but they are more permeable to Mg . Thus, when activated, these channels invariably cause two main effects—a rise in intracel- 2+ 2+ lular free Ca concentration ([Ca ]i) and membrane depolarization that affects voltage-gated ion channels and other membrane potential sensitive pathways. The former can be achieved not only via Ca2+ influx mediated by TRPs and voltage-gated Ca2+ channels, if these are present, but often via Ca2+ release as well because TRPs can be expressed both in the plasma membrane and in various intracellular organelles. Thus, it can be easily envisaged, that biological and pathophysiological roles of each individual TRP subtype can be diverse, depending on the cell type (e.g., excitable or nonexcitable cells), cellular environment, and cell type-specific sets of voltage-gated ion channels and Ca2+-mediated responses. Moreover, the same or different TRP isoforms present in sensory neurons and effector cells can mediate complex positive and negative feedback controls in cell signaling within disease-specific tissue microenvironment (neuropeptides, various epithelial factors, cytokines, histamine, bradykinin, prostaglandins, leukotrienes) and as such may be responsible for the heightened airway responses associated with cough reflex hypersensitization and bronchial hyperreactivity.

The Canonical TRPs The mammalian “canonical” TRPCs are structurally most closely related to Drosophila TRP and include seven proteins (TRPC1-7), although in humans TRPC2 is a pseudogene [17,18,33–35]. All TRPCs are activated by stimulation of G-protein-coupled receptor and receptor tyrosine kinases, commonly downstream of phospholipase C (PLC) activation. These channels play important roles in calcium homeostasis by admitting external Ca2+ in a receptor- and/or store-operated manner, in the latter case likely in heteromeric complexes with Orai proteins [36]. ASM which controls airway caliber, plays a central role in the pathogenesis of airway hy- 2+ perresponsiveness in asthma. Increase in [Ca ]i in ASM causes contraction leading to airway 2+ narrowing, and these [Ca ]i signals are also important for smooth muscle proliferation, hyperplasia, or hypertrophy, the defining characteristics of asthma [37–40]. In addition, ASM can secrete inflammatory mediators, which recruit and activate inflammatory cells, such as mast cells and T-lymphocytes. In turn, inflammatory mediators can alter Ca2+ homeostasis in ASM and sensitize them to agonists [39]. There is growing evidence for the roles of TRPCs in ASM calcium regulation relevant to these pathogenic events. The airways are innervated by cholinergic nerve fibers, and the major parasympathetic transmitter acetylcholine, when released, induces excitation and contraction of ASM. Although numerous ion channel mechanisms mediate acetylcholine-induced membrane depolarization, opening of receptor-operated cation channels is one common mechanism present in ASM and other types of visceral smooth muscles [41–43]. Studies of muscarinic receptor and TRPC knockout mice, as well as the intracellular signal transduction pathways, showed that in ileal myocytes both M2 and M3 acetylcholine receptor subtypes, which are commonly coexpressed in visceral smooth muscles, converge to activate two main contributors to the TRP CHANNELS IN THE RESPIRATORY SYSTEM 487

muscarinic cation current termed mICAT—TRPC4 (which mediates about 80% of mICAT) and TRPC6 [44–46]. Although similar studies have not yet been performed on ASM, it should be noted that histamine, which is intimately associated with the pathology of allergy and inflammation of the airways, acting at H1 receptor activates cation currents in ASM, that re- 2+ quire [Ca ]i rise and simultaneous activation of Gi/o and Gq/11 proteins, but not diacylglycerol formation [43]. These signal transduction pathways are similar to those leading to the activation of mICAT by costimulation of M2 and M3 receptors [47]. However, I-V relationships of this cation current in ASM differ from mICAT I-V curves [43]. It thus appears that receptor-operated cation channels are similar, but not identical, in ASM and gastrointestinal myocytes. Indeed, expression of TRPC4 and -C6, as well as other TRPC channels (TRPC1, -3, -4, and -6) has been described in human ASM [48,49]. In guinea-pig ASM, mRNA encoding TRPC1, -3, -4, -5, and -6 was also detected at levels comparable to the brain, whereas mRNA encoding TRPC2 and TRPC5 was even more abundant [50]. These studies implicate TRPCs as strong candidates for the Ca2+ influx pathways required for sustained ASM contraction. Indeed, more recently in freshly isolated mouse ASM myocytes, constitutively active single cation channels were described, which could be inhibited by TRPC3, but not TRPC1, antibodies, and siRNAs [51]. Gene silencing experiments revealed that TRPC3 plays an important role in the regulation 2+ of the resting membrane potential and [Ca ]i in ASM, as well as in methacholine-evoked in- 2+ crease in [Ca ]i [51]. Importantly, in ovalbumin-sensitized mice, a model for airway hyperresponsiveness, ASM cells’ TRPC3 protein expression was increased, accompanied by increased cation channel activity, membrane depolarization, and enhanced contractile responses to methacholine. Although TRPC1 expression was not altered, it was also involved in these cellular responses in asthmatic, but not in normal, ASM [51]. It is interesting to note that similar contributions of TRPC1, -3, and -6 proteins to the formation of native constitutively active, store-, and receptor-activated cation channels have been described in vascular smooth muscles [52]. However, the role of TRPC3 in ASM appears to be more pronounced; for example, TRPC6 gene silencing in guinea-pig primary ASM cells does not affect OAG-induced Ca2+ signaling [53]. In contrast, TRPC3 gene silencing by siRNA treatment significantly inhibits tumor necrosis factor alpha (TNF-α)- and acetylcholine-induced Ca2+ influx in cultured human ASM cells [49]. Because TNF-α, the proinflammatory cytokine, 2+ contributes to airway hyperresponsiveness by altering ASM [Ca ]i homeostasis, these results suggest the important role of TRPC3 in inflammatory airway diseases, such as asthma and COPD [54]. In addition, there is evidence that up-regulated TRPC1, which is associated with 2+ 2+ an enhanced Ca entry and elevated [Ca ]i, may play an important role in bronchial constriction and ASM proliferation in asthma [54,55]. Cystic fibrosis (CF), one of the most common severe life-threatening inherited diseases, is caused by an autosomal recessive mutation of the CF transmembrane conductance regulator (CFTR) gene. The most common phenylalanine deletion mutant F508del causes the most severe phenotype—a dehydrated airway surface and impaired mucus clearance due to the lack of chloride secretion and excessive sodium absorption [56]. CFTR regulates various − apical epithelial ion channels (e.g., outwardly rectifying Cl channels, ENaC, KATP), but very little is known about its interaction with TRP channels. In aortic endothelial cells isolated from TRPC4−/− mice functional interaction between TRPC4 and CFTR has been established by showing that, although the expression of CFTR was unchanged, the current was suppressed in TRPC4-deficient cells [57]. It was thus suggested that TRPC4 may provide a scaffold for 488 26. PATHOPHYSIOLOGICAL ROLES OF RESPIRATORY TRPs the formation of functional CFTR channels. Such multiprotein complexes have been recently revealed by coimmunoprecipitation analysis of CFTR, F508del-CFTR, and TRPC6 interactions in human tracheal epithelial cells. Functionally, CFTR down-regulates TRPC6-mediated Ca2+ influx, but TRPC6 up-regulates CFTR-dependent Cl− transport in airway epithelial cells. Because in CF this functional coupling is lost, TRPC6-mediated Ca2+ influx is abnormally increased in CF compared with non-CF cells [58]. There is also substantial evidence for the involvement of TRPCs, and TRPC6 in particular, in the regulation of hypoxic pulmonary vasoconstriction, which is responsible for ensuring the physiological ventilation/perfusion matching during acute episodes of local alveolar hypoxia, but with no such role in chronic generalized hypoxia associated with vascular remodeling and pulmonary hypertension [59,60]. TRPC6 may also contribute to the hypersecretion of mucus in chronic bronchitis [61], migration of neutrophils [62], and allergic airway inflammation [63], making it a potential new drug target in asthma and COPD [54]. Compared with WT mice, TRPC6−/− mice exhibited reduced allergic responses after allergen challenge (e.g., reduced airway eosinophilia and blood IgE levels, decreased levels of Th2 cytokines IL-5 and IL-13), but unaltered lung mucus production [63]. Intriguingly, agonist-induced contractility of tracheal rings was increased in TRPC6-deficient mice, contrary to the expectations based on the previously discussed role of TRPC6 in ASM. However, compensatory up-regulation of TRPC3 in ASM could explain this controversy. TRPC1 also plays an essential role in allergic reactions by negatively regulating TNF-α production by mast cells, as recent comparison of antigen-mediated anaphylaxis in Trpc1−/− and WT mice has revealed [64]. Macrophages play key roles in the pathophysiology of COPD. Analysis of TRPC expression in human lung macrophages revealed that TRPC6 mRNA expression was significantly elevated in alveolar macrophages from patients with COPD compared with control subjects, whereas there was no difference in the expression levels of TRPC3 and -7 [65]. This increased molecular and functional expression, as confirmed by patch-clamp analysis, was particularly pronounced in small macrophages, and it correlated with COPD diagnosis [65].

The Vanilloid TRPs This TRP subfamily contains six mammalian members, TRPV1-6. TRPV1-4 are activated by heat, as well as a broad array of chemicals or mechanical stimuli. As already noted, 2+ 2+ TRPV5 and -6 are highly Ca permeable and are tightly controlled by [Ca ]i, which is important for the regulated Ca2+ uptake in epithelial cells [17,18,34,35,66,67]. There have been many exciting developments regarding the roles of TRPV channels in the respiratory system. These roles often conformed to the expectations based on the knowledge of their diverse expression patterns and activation mechanisms, as well as functional properties of heterologously expressed TRPV channels, but in native cells there have been also many unexpected findings. Among TRPVs, TRPV1 (capsaicin receptor) remains the most extensively studied channel. It is truly a polymodal sensor, that can be activated by agonists (capsaicin and resiniferatoxin), heat (> 42 °C), protons, endocannabinoid lipids such as anandamide, and eicosanoids [17,18,34,35,66–69]. Furthermore, TRPV1 is sensitized by protein kinase A (PKA), protein kinase C (PKC), Ca2+/calmodulin-dependent kinase II (CaMKII), phosphatidylinositol 3-kinase

(PI3K), and PLC activation (likely in a PIP2-dependent manner). TRPV1 is widely expressed TRP CHANNELS IN THE RESPIRATORY SYSTEM 489 in neuronal, nociceptive neurons in particular and nonneuronal cells, and thus it is often considered as a focal point of signal transduction and cell communication. Heterogeneous groups of TRPV1-positive afferent fibers (some colocalized with substance P and CGRP) are found throughout the entire respiratory tract—within and beneath the epithelium, around blood vessels, within ASM and alveoli, and their number increases under allergic inflammatory conditions [70,71]. It is thus not surprising that the majority of studies have focused on TRPV1 roles in sensory nerves and in particular on its role in airway inflammation and sensitization of cough reflex in chronic cough [3,9,11,21,22,24,32,54,72–76]. Indeed, the TRPV1 agonist capsaicin readily provokes cough in human and animal challenge testing. This response is enhanced in asthma and COPD, and it can be inhibited by TRPV1 antagonists, strongly suggesting TRPV1 as a putative molecular target for the development of novel antitussive drugs [21,24,32,75,76]. Endogenous ligands, such as prostaglandin E2 and bradykinin, are known to sensitize the response to tussive stimuli (via EP3 and B2 receptors, respectively), and they both have been shown to mediate their effects though TRPV1 (and TRPA1) channels [77]. It should be noted that TRPV1 gene polymorphisms can further increase the risk of cough due to irritant exposures, such as cigarette smoke and occupational exposures [27]. In contrast, one genetic variant (SNP) of TRPV1 (TRPV1-I585V) shows decreased channel activity in response to capsaicin and heat, and this loss-of-function is associated with a reduced risk of childhood asthma [78]. Interestingly, comparative analysis of TRPV1- and TRPA1-mediated cough responses in guinea pigs showed higher efficacy of TRPV1 agonists, likely due to higher potency of TRPV1 in the sustained activation of airway C fibers [79]. Our recent study showed that TRPV1 is also functionally expressed in human bronchial epithelial cells, where it can mediate capsaicin- and low pH-induced cation currents [20]. In these cells the TRPV1 agonist capsaicin induced dose-dependent IL-8 release, which could be blocked by the TRPV1 antagonist capsazepine. Moreover, TRPV1 was overexpressed in patients with refractory asthma, suggesting its contribution to airway hypersensitivity in severe asthma [20]. We have also detected molecular expression of TRPV2, -3, -4, and V6 in human bronchial epithelial cells, but their functional and pathophysiological roles in these cells remain to be studied. In the context of lung inflammatory diseases it is an important finding that TRPV1 activation can lead to lung cell death [80]. Moreover, in animal models of asthma, TRPV1 knockdown attenuated airway hyperresponsiveness, airway inflammation, goblet cell metaplasia, and subepithelial fibrosis induced by IL-13 in BALB/c mice, suggesting that up-regulation of TRPV1 by mediators of allergic inflammation in bronchial epithelia could lead to epithelial injury [81]. TRPV1 was also implicated in a formaldehyde-induced model of asthma in mice [82]. TRPV1 can be selectively activated by some types of PM, such as coal fly ash, the most potent TRPV1 activator [83]. Exposure of human airway and epithelial cells and mouse sensory neurons to PM evoked apoptosis associated with sustained calcium influx through TRPV1 channels, which was completely prevented by capsazepine, the TRPV1 antagonist, or in sensory neurons from TRPV1−/− mice [84]. In human bronchial epithelial cells, exposure to 2+ PM induces [Ca ]i-dependent production of cytokines IL-1β and IL-8, although the role of TRP channels in this process has not been elucidated [85]. Sepsis-evoked acute lung injury induced by hydrogen sulfide (H2S) has been shown to enhance neurogenic inflammation, 490 26. PATHOPHYSIOLOGICAL ROLES OF RESPIRATORY TRPs

COX-2 and PGE2 via TRPV1 activation [86]. Consistent with these findings, capsazepine can protect against H2S-induced lung inflammation [87]. Similarly, sulfur dioxide (SO2) exposure also causes TRPV1 up-regulation, paralleled by an increased sensitivity of the cough response to capsaicin [88]. Inflammatory cells, such as mast cells, by releasing inflammatory proteases (mast cell tryptase and trypsin) cleave protease-activated receptor 2 (PAR2), which is coexpressed with TRPV1 in small- to medium-diameter neurons and thus sensitize neuronal TRPV1 through PKC [89]. This cascade of events can reduce the temperature threshold for TRPV1 activation from 42 °C to well below body temperature [90]. In addition, endogenous agonists of TRPV1 (endovanilloids) also appear to be involved in lung injury during inflammation [91]. Based on the preceding studies, it is an attractive hypothesis that in neurogenic inflammation enhanced release of pro-tussive inflammatory mediators and inflammatory proteases from nerve endings, epithelial cells, and mast cells could explain TRPV1 sensitization and heightened cough reflex. In this scenario, TRPV1 can function at multiple points, from nerve activation and release of sensory neuropeptides to the secretion of pro-inflammatory cytokines by airway epithelial cells [14,92]. Similar mechanisms acting via up-regulation of neuronal TRPV1 (as well as TRPA1 and TRPM8) channels are also involved in respiratory virus-induced cough hypersensitivity [93] and in bronchoconstriction, although the role of TRPV1 in bronchospasm in humans remains somewhat controversial [26,32]. About two- thirds of the increase in bronchoconstriction induced by hyperventilation with humidified hot air in guinea pigs could be attributed to the cholinergic reflex, likely elicited by the activation of TRPV1-expressing airway afferents, but the remaining bronchoconstriction is caused by other, as yet unidentified, mechanisms [94]. Similar to bronchial epithelial cells, TRPV1 shows increased expression in ASM in patients suffering from chronic cough, but it is present in intracellular compartments, and its exact role in ASM contractility remains unclear [95]. There is recent evidence for overexpression of TRPV1 in asthmatic ASM, whereby TRPV1 channel involved in the regulation of proliferation and apoptosis in asthmatic ASM cells [96]. Interestingly, bronchial hyperresponsiveness was enhanced in TRPV1−/− mice, whereas inflammation elevated somatostatin concentrations in wild-type (WT), but not in TRPV1−/− mice. Thus, it is possible that TRPV1 activation can also counteract inflammation via somatostatin release [97]. There is also recent evidence for a TRPV1 role in airway sensory nerves in connection with the action of tiotropium, the long-acting muscarinic receptor antagonist prescribed for its bronchodilator activity in COPD and asthma. However, tiotropium also attenuates cough 2+ through its direct inhibition of (i) TRPV1-triggered [Ca ]i rises, (ii) action potential discharge in airway-specific sensory afferent nerves, and (iii) capsaicin-induced bronchospasm [98]. TRPV1 sensitization in respiratory inflammation is mediated by the PI3K and PKC signaling pathways, causing hypersecretion of mucus and inflammatory cytokines [99]. Nerve growth factor, which itself does not induce a cough response, nevertheless causes a significant increase in the citric acid-induced cough and airway obstruction via TrkA receptor and TRPV1 activation, possibly downstream of PI3K and PLCγ activation [100]. TRPV1 has also been recently implicated in Cl− secretion. Formaldehyde is an environmental pollutant and strong stimulant, which can also be formed endogenously from me- thylamine. Formaldehyde-induced activation of TRPV1 expressed in the intraepithelial nerve endings stimulated Cl− secretion in rat tracheal epithelium. This secretion was mediated by TRP CHANNELS IN THE RESPIRATORY SYSTEM 491 the release of adrenaline and substance P, and it could be blocked by the CFTR specific inhibitor CFTRi-172 [101]. Thus, there is substantial evidence implicating TRPV1 in chronic cough, asthma, and COPD, whereas successful use of TRPV1 antagonists for the alleviation of cough and airway hyperresponsiveness in animal models further highlights the promise such drugs hold for treating human respiratory disorders [102,103]. TRPV4 is another member of this subfamily that attracts growing interest in the context of respiratory disorders. TRPV4 channels are widely expressed in neuronal and nonneuronal cells within the respiratory tract, including macrophages and neutrophils, suggesting their relevance to airway inflammation [32]. Jia et al. [104] reported, that TRPV4 is expressed in 2+ ASM, where it acts as an osmolarity sensor directly causing hypotonicity-induced [Ca ]i rise and airway contraction. These findings are relevant to asthma because bronchial fluid in asthmatics can be hypotonic, and inhalation of hypotonic aerosols induces airway narrowing in people with asthma. TRPV4 present in airway macrophages can also be activated by mechani- 2+ cal stimuli. Thus, TRPV4 agonist 4-alpha-phorbol didecanoate (4α-PDD) increased [Ca ]i and reactive oxygen and nitrogen species in lung WT, but not TRPV4−/−, macrophages, suggesting a role for TRPV4 in the development of ventilator-induced injury [105]. In addition, activation of TRPV4 by 4α-PDD and 5,6- or 14,15-EET, as well as thapsigargin treatment increased lung endothelial permeability measured by the filtration coefficient. The response to 4α-PDD, but not to thapsigargin, was absent in TRPV4−/− mice, thus implicating TRPV4 in disruption of the alveolar septal barrier and in acute lung injury [106]. P450-derived epoxyeicosatrienoic acid-dependent regulation of calcium entry via TRPV4 has also been implicated in the endothelial permeability increase in acute lung injury caused by high vascular pressure [107]. Under hypotonic conditions regulatory volume decrease (RVD) occurs in human airway epithelial cells, but RVD is lost in CF airways [108]. Using TRPV4 antisense oligonucleotides treatment, Arniges et al. [109] showed that TRPV4 is uniquely required for the swelling-induced Ca2+ entry, which is needed for a full RVD in human tracheal epithelial cells. Furthermore, the impaired RVD response in CF airway epithelia is due to the specific lack of TRPV4 activation in response to cell swelling; notably, activation of TRPV4 by 4α-PDD was unimpaired in CF. These data suggest that hypotonic activation of TRPV4 channels depends on CFTR. However, it should be noted that CFTR requirement for TRPV4 activation in airway epithelia is stimulus-dependent because the functional coupling of TRPV4 and large conductance Ca2+-activated K+ channel in response to high-viscous solutions is preserved in CF [110]. TRPV4 is functionally expressed in ASM cells, and it is likely associated with airflow obstruction in COPD patients. There is genetic evidence that polymorphisms in the TRPV4 gene are associated with COPD [111], but not with asthma [78]. However, McAlexander et al. [112] have provided compelling pharmacological evidence with the use of TRPV4 agonists and antagonists, that, unexpectedly, TRPV4 activation causes human airway constriction entirely due to the production of cysteinyl leukotrienes. The authors propose cysteinyl leukotriene synthesis as a novel mechanism for the involvement of TRPV4 in airway inflammation and obstruction. Although the source of cysteinyl leukotrienes has not been yet elucidated, one possibility is that mast cells in the immediate vicinity to bronchial smooth muscle are involved in leukotriene production [112]. There is also recent evidence that, similar to TRPC6 and other TRPCs, up-regulation of TRPV4 by chronic hypoxia is associated with enhanced myogenic tone, whereas TRPV4 492 26. PATHOPHYSIOLOGICAL ROLES OF RESPIRATORY TRPs

knockout suppresses the development of chronic hypoxic pulmonary hypertension. Specifically, TRPV4 plays an essential role in serotonin-induced pulmonary vasoconstriction and the enhanced vascular reactivity in chronic hypoxic pulmonary hypertension [113].

The Melastatin TRPs Eight mammalian members of the TRPM subfamily, TRPM1-8, are structurally and functionally diverse cation channels, which are involved in processes ranging from detection of cold, taste, osmolarity, redox state, and pH to control of Mg2+ homeostasis and cell proliferation or death [17,18,34,35,66,67]. TRPMs have a TRP box in the C-terminus, similarly to TRPC channels, but their N-terminus lacks the ankyrin repeats found in TRPCs and TRPVs; instead it has a common large TRPM homology domain. Remarkably, three out of eight TRPM members have connections with cancer development. Thus, the founding member melastatin (TRPM1) as well as TRPM5 and TRPM8 were identified by analysis of gene expression in several carcinomas [114]. The roles of these multifunctional cation channels in respiratory disorders are becoming increasingly apparent in connection with the effects produced by air pollution (e.g., oxidative stress, inflammation) and thermal irritants (e.g., cold). Ozone, particulate matter, nitrogen oxides, and some metals are potent oxidants. In addition, these airborne pollutants can generate reactive oxygen species (ROS), which may also be produced endogenously under hypoxic and ischemia/reperfusion conditions. Oxidative stress through the production of ROS causes damage to all cellular macromolecules, including proteins, lipids, and DNA, thus disrupting normal cell signaling. Oxidative stress plays a role in many diseases, including asthma and COPD. Various nonselective Ca2+-permeable cation channels have been implicated in cell damage 2+ or death due to oxidative stress, ischemia, and associated [Ca ]i overload [115]. TRPM2 is activated by ROS [116–118]. Ca2+ entry via TRPM2 induces chemokine production in monocytes, followed by inflammatory neutrophil infiltration [119]. Thus, ROS can lead to inflammation and tissue injury with the involvement of TRPM2—a scenario, which is well established in endothelial injury and endothelial hyperpermeability [120–122]. However, in the airways this hypothesis was not confirmed in TRPM2−/− mice, as no difference in the development of airway inflammation or cell activation between WT and TRPM2−/− mice could be found, implying that inhibition of TRPM2 activity in COPD would have no anti-inflammatory effect [123]. Moreover, TRPM2 turned out to be protective in endotoxin-induced lung inflammation in mice. Somewhat unexpectedly, in this scenario ROS-induced activation of TRPM2 causes negative feedback inactivation of ROS production in phagocytic cells mediated by the inhibition of the membrane potential-sensitive NADPH oxidase, which prevents lung inflammation [124]. The inhibitory role of TRPM2 in ROS production was also shown in poly- morphonuclear leukocytes and macrophages derived from TRPM2−/− mice [124]. In contrast, 2+ deletion of TRPM4, which is a [Ca ]i-activated channel, results in an enhanced release of histamine, leukotrienes, and tumor necrosis factor from mast cells [125]. Thus, it is attractive to speculate that the balance between the activities of TRPM2 and TRPM4 channels is an important factor in airway inflammation. Thermal irritants, such as cold air, can provoke bouts of coughing, especially in respiratory virus-induced cough hypersensitivity [75,93]. Other autonomic responses include airway Summary and Perspectives 493 constriction and mucosal secretion, which can trigger asthma attacks [32]. In connection with these well-known cold-induced airway symptoms, the expression and function of the cold- and menthol-receptor TRPM8 in the airways has been examined in several studies, but molecular and functional data remain somewhat contradictory, whereas conclusive evidence for the role of neuronal TRPM8 in these symptoms is still lacking (see [32,126] for recent reviews). The problems of interpreting these data relate to the difficulties with differentiation between TRPA1- and TRPM8-mediated effects and the lack of highly selective ligands. However, as already noted, up-regulation of neuronal TRPM8 clearly contributes to respiratory virus- induced cough hypersensitivity [93]. Epithelial TRPM8 present in the respiratory tract is comparatively better studied. Functional expression of TRPM8 was shown in human lung epithelial cells, whereby its activation causes the release of inflammatory cytokines IL-6 and IL-8 [127]. Interestingly, lung epithelial TRPM8 is represented by its truncated splice variant, localized mainly in the endoplasmic reticulum, which seems similar to prostate cancer epithelial cells [128]. Using immunohistochemistry, RT-PCR, and Western blotting enhanced expression of TRPM8 was found in bronchial epithelial cells in patients with COPD compared to healthy subjects. Functionally, activation of 2+ the epithelial TRPM8 isoform causes [Ca ]i rises, which mediate cold-induced mucus hypersecretion [129]. In addition, TRPM8 expressed in mast cells has been implicated in cold- and menthol-induced histamine release, likely explaining the role of TRPM8 channels in the menthol- and cold-induced allergic responses [130]. TRPM8 immunoreactive nerve fibers are abundant in nasal mucosa, especially around blood vessels, where TRPM8 may mediate neurovascular reflexes. These findings led to the suggestion of a role for TRPM8 in rhinitis [131]. We have recently developed a new method for short-term culture of human nasal epithelial cells (HNEC) for the study of CF pathophysiology [132]. TRPM8 gene transcripts and protein expression were revealed in these cells. Application of the TRPM8 agonist menthol-induced robust membrane current responses, as 2+ well as [Ca ]i rises ablated by the TRPM8 antagonist BCTC [133]. Taken together, these studies show that functional TRPM8 channels are expressed in the upper airway, both in sensory fibers and in airway epithelial cells. Finally, it should be noted that although TRPM8 is expressed in vascular smooth muscle cells [134,135], its expression in ASM and any direct role in airway constriction remain to be studied.

SUMMARY AND PERSPECTIVES

As summarized in this chapter, multifunctional TRP channels are clearly the important players regulating many aspects of respiratory function (Table 26.1). In recent years there have been many exciting developments in this area of research, which is undergoing a rapid expansion. Some areas of major importance have received particular attention, such as the role of TRPV1 in airway inflammation and hypersensitization of cough reflex. Other areas are just beginning to be exploited, for example, relatively little is known about the roles of TRP channels in ASM and agonist-induced airway constriction. Somewhat intriguingly, airway epithelial cells of the respiratory tract, which represent an important interface with the environment, are a site where several classical “neuronal sensors,” including TRPV1 and TRPM8 494 26. PATHOPHYSIOLOGICAL ROLES OF RESPIRATORY TRPs

TABLE 26.1 TRPs in respiratory disorders at a glance

Subtype Localization Proposed functions and relevance to airway disease

2+ TRPC1 Airway smooth muscle Involved in [Ca ]i rise in asthmatic ASM, as well as in bronchial constriction and ASM proliferation1

Mast cells Inhibits TNF-α production1, a TRPC3 Airway smooth muscle Increased expression and activity leading to membrane depolarization and enhanced agonist-induced contractile responses in asthmatic ASM1

Initiates TNF-α- and acetylcholine-induced Ca2+ influx1,2 TRPC4 Endothelial cells Required for CFTR function4, a TRPC6 Airway smooth muscle Involved in hypoxic pulmonary vasoconstriction leading to pulmonary hypertension Human tracheal epithelial cells Reciprocal interactions with CFTR leading to abnormally increased Ca2+ influx in CF4 Multiple cell types Involved in hypersecretion of mucus in chronic bronchitis and in allergic responses1,2, a Alveolar macrophages Shows increased molecular and functional expression in patients with COPD2 TRPV1 Multiple cells types, including Expression increases under allergic inflammatory conditions sensory neurones Tracheal epithelial cells Plays important roles in airway inflammation and hyperresponsiveness, sensitization of cough reflex in chronic cough, proliferation and apoptosis in asthmatic ASM cells, epithelial injury, and lung cell apoptosis1,2,3 Stimulates CFTR-mediated chloride secretion

2+ 1 TRPV4 Airway smooth muscle Initiates hypotonicity-induced [Ca ]i rise and airway contraction Associated with airflow obstruction in COPD patients2 Involved in the enhanced vascular reactivity in chronic hypoxic pulmonary hypertension

2+ Macrophages Causes increased [Ca ]i and reactive oxygen and nitrogen species, plays a role in the development of ventilator-induced injurya Endothelial cells Involved in disruption of the alveolar septal barrier and in acute lung injurya Tracheal epithelial cells Required for the swelling-induced Ca2+ entry needed for a full RVD, which is reduced in CF4 Possibly mast cells Airway inflammation and obstruction mediated by cysteinyl leukotriene synthesis1,2 TRPM2 Phagocytes, leukocytes, Involved in protection against lung inflammation via inactivation macrophages of ROS productiona REFERENCES 495

TABLE 26.1 TRPs in respiratory disorders at a glance—Cont’d

Subtype Localization Proposed functions and relevance to airway disease TRPM4 Mast cells Controls release of histamine, leukotrienes, and tumor necrosis factora TRPM8 Sensory neurons Virus-induced up-regulation contributes to cough hypersensitivity3

2+ Lung epithelial cells Activation causes [Ca ]i rise, the release of inflammatory cytokines and mucus hypersecretion Mast cells Cold- and menthol-induced histamine release, likely contributing to allergic responses Nerve fibers Involved in neurovascular reflexes, suggesting a role in rhinitis

Cellular pathophysiological functions relevant to major disease conditions are indicated as following: (1) asthma, (2) COPD, (3) chronic cough, (4) CF. aDenotes mouse knockout phenotype. Further details are found in the body of this chapter. channels, are functionally expressed. At this stage it is clear that various TRP subtypes are present in all major cell types constituting the respiratory tract and that the total pool of these Ca2+-permeable channels is considerable, making them important determinants of calcium signaling and related cell-specific functions. At the same time, this makes it difficult to dis- sect the pathophysiological roles of individual TRP subtypes, and hence target these in the specific disease conditions. Thus, although TRP channels represent generally very attractive targets for novel therapeutics in a wide range of respiratory disorders, much further research is needed to fully realize this potential.

References [1] Nishino T. Physiological and pathophysiological implications of upper airway reflexes in humans. Jpn J Physiol 2000;50:3–14. [2] Widdicombe J, Lee LY. Airway reflexes, autonomic function, and cardiovascular responses. Environ Health Perspect 2001;109:579–84. [3] Bessac BF, Jordt SE. Breathtaking TRP channels: TRPA1 and TRPV1 in airway chemosensation and reflex control. Physiol 2008;23:360–70. [4] Undem BJ, Carr MJ. Targeting primary afferent nerves for novel antitussive therapy. Chest 2010;137:177–84. [5] Canning BJ, Spina D. Sensory nerves and airway irritability. Handb Exp Pharmacol 2009;194:139–83. [6] Yu J. Airway receptors and their reflex function. Adv Exp Med Biol 2009;648:411–20. [7] Mazzone SB, McGovern AE. Sensory neural targets for the treatment of cough. Clin Exp Pharmacol Physiol 2007;34:955–62. [8] Bessac BF, Jordt SE. Sensory detection and responses to toxic gases: mechanisms, health effects, and counter- measures. Proc Am Thorac Soc 2010;7:269–77. [9] McGarvey L, McKeagney P, Polley L, MacMahon J, Costello RW. Are there clinical features of a sensitized cough reflex? Pulm Pharmacol Ther 2009;22:59–64. [10] Nasra J, Belvisi MG. Modulation of sensory nerve function and the cough reflex: understanding disease pathogenesis. Pharmacol Ther 2009;124:354–75. [11] Grace MS, Dubuis E, Birrell MA, Belvisi MG. Pre-clinical studies in cough research: role of transient receptor potential (TRP) channels. Pulm Pharmacol Ther 2013;26:498–507. [12] Groneberg DA, Quarcoo D, Frossard N, Fischer A. Neurogenic mechanisms in bronchial inflammatory diseases. Allergy 2004;59:1139–52. [13] Chung KF, Widdicombe JG. Cough: setting the scene. Handb Exp Pharmacol 2009;187:1–21. 496 26. PATHOPHYSIOLOGICAL ROLES OF RESPIRATORY TRPs

[14] Couto M, de, D. A, Perpini M, Delgado L, Moreira A. Cough reflex testing with inhaled capsaicin and TRPV1 activation in asthma and comorbid conditions. J Investig Allergol Clin Immunol 2013;23:289–301. [15] Patterson RN, Johnston BT, Ardill JE, Heaney LG, McGarvey LP. Increased tachykinin levels in induced sputum from asthmatic and cough patients with acid reflux. Thorax 2007;62:491–5. [16] Clapham DE. TRP channels as cellular sensors. Nature 2003;426:517–24. [17] Venkatachalam K, Montell C. TRP channels. Annu Rev Biochem 2007;76:387–417. [18] Wu LJ, Sweet TB, Clapham DE. International union of basic and clinical pharmacology. LXXVI. Current progress in the mammalian TRP ion channel family. Pharmacol Rev 2010;62:381–404. [19] Gees M, Colsoul B, Nilius B. The role of transient receptor potential cation channels in Ca2+ signaling. Cold Spring Harb Perspect Biol 2010;2:1–31. [20] McGarvey LP, Butler CA, Stokesberry S, Polley L, McQuaid S, Abdullah H, et al. Increased expression of bronchial epithelial transient receptor potential vanilloid 1 channels in patients with severe asthma. J Allergy Clin Immunol 2014;133:704–12. [21] Geppetti P, Materazzi S, Nicoletti P. The transient receptor potential vanilloid 1: role in airway inflammation and disease. Eur J Pharmacol 2006;533:207–14. [22] Adcock JJ. TRPV1 receptors in sensitisation of cough and pain reflexes. Pulm Pharmacol Ther 2009;22:65–70. [23] Andre E, Gatti R, Trevisani M, Preti D, Baraldi PG, Patacchini R, et al. Transient receptor potential ankyrin receptor 1 is a novel target for pro-tussive agents. Br J Pharmacol 2009;158:1621–8. [24] Materazzi S, Nassini R, Gatti R, Trevisani M, Geppetti P. Cough sensors. II. Transient receptor potential membrane receptors on cough sensors. Handb Exp Pharmacol 2009;187:49–61. [25] Banner KH, Igney F, Poll C. TRP channels: emerging targets for respiratory disease. Pharmacol Ther 2011;130:371–84. [26] Preti D, Szallasi A, Patacchini R. TRP channels as therapeutic targets in airway disorders: a patent review. Expert Opin Ther Pat 2012;22:663–95. [27] Smit LA, Kogevinas M, Anto JM, Bouzigon E, Gonzalez JR, Le MN, et al. Transient receptor potential genes, smoking, occupational exposures and cough in adults. Respir Res 2012;13:13–26. [28] Abbott-Banner K, Poll C, Verkuyl JM. Targeting TRP channels in airway disorders. Curr Top Med Chem 2013;13:310–21. [29] Buch T, Schafer E, Steinritz D, Dietrich A, Gudermann T. Chemosensory TRP channels in the respiratory tract: role in toxic lung injury and potential as "sweet spots" for targeted therapies. Rev Physiol Biochem Pharmacol 2013;165:31–65. [30] Chung KF, McGarvey L, Mazzone SB. Chronic cough as a neuropathic disorder. Lancet Respir Med 2013;1:414–22. [31] Geppetti P, Patacchini R, Nassini R. Transient receptor potential channels and occupational exposure. Curr Opin Allergy Clin Immunol 2014;14:77–83. [32] Grace MS, Baxter M, Dubuis E, Birrell MA, Belvisi MG. Transient receptor potential (TRP) channels in the airway: role in airway disease. Br J Pharmacol 2014;171:2593–607. [33] Vazquez G, Wedel BJ, Aziz O, Trebak M, Putney J. The mammalian TRPC cation channels. Biochim Biophys Acta 2004;1742:21–36. [34] Clapham DE, Julius D, Montell C, Schultz G. International union of pharmacology. XLIX. Nomenclature and structure-function relationships of transient receptor potential channels. Pharmacol Rev 2005;57:427–50. [35] Ramsey IS, Delling M, Clapham DE. An introduction to TRP channels. Annu Rev Physiol 2006;68:619–47. [36] Liao Y, Erxleben C, Abramowitz J, Flockerzi V, Zhu MX, Armstrong DL, et al. Functional interactions among Orai1, TRPCs, and STIM1 suggest a STIM-regulated heteromeric Orai/TRPC model for SOCE/Icrac channels. Proc Natl Acad Sci USA 2008;105:2895–900. [37] Amrani Y, Panettieri Jr RA. Modulation of calcium homeostasis as a mechanism for altering smooth muscle responsiveness in asthma. Curr Opin Allergy Clin Immunol 2002;2:39–45. [38] Janssen LJ, Killian K. Airway smooth muscle as a target of asthma therapy: history and new directions. Respir Res 2006;7:123. [39] Bara I, Ozier A, Tunon de Lara JM, Marthan R, Berger P. Pathophysiology of bronchial smooth muscle remodelling in asthma. Eur Respir J 2010;36:1174–84. [40] James AL, Elliot JG, Jones RL, Carroll ML, Mauad T, Bai TR, et al. Airway smooth muscle hypertrophy and hyperplasia in asthma. Am J Respir Crit Care Med 2012;185:1058–64. [41] Janssen LJ, Sims SM. Acetylcholine activates non-selective cation and chloride conductances in canine and guinea-pig tracheal myocytes. J Physiol 1992;453:197–218. REFERENCES 497

[42] Wang YX, Kotlikoff MI. Muscarinic signaling pathway for calcium release and calcium-activated chloride current in smooth muscle. Am J Physiol 1997;273:C509–19. [43] Wang YX, Kotlikoff MI. Signalling pathway for histamine activation of non-selective cation channels in equine tracheal myocytes. J Physiol 2000;523:131–8. [44] Zholos AV, Bolton TB. Muscarinic receptor subtypes controlling the cationic current in guinea-pig ileal smooth muscle. Br J Pharmacol 1997;122:885–93. [45] Sakamoto T, Unno T, Kitazawa T, Taneike T, Yamada M, Wess J, et al. Three distinct muscarinic signalling pathways for cationic channel activation in mouse gut smooth muscle cells. J Physiol 2007;582:41–61. [46] Tsvilovskyy VV, Zholos AV, Aberle T, Philipp SE, Dietrich A, Zhu MX, et al. Deletion of TRPC4 and TRPC6 in mice impairs smooth muscle contraction and intestinal motility in vivo. Gastroenterology 2009;137:1415–24. [47] Zholos AV. Regulation of TRP-like muscarinic cation current in gastrointestinal smooth muscle with special 2+ reference to PLC/InsP3/Ca system. Acta Pharmacol Sin 2006;27:833–42. [48] Corteling RL, Li S, Giddings J, Westwick J, Poll C, Hall IP. Expression of transient receptor potential C6 and related transient receptor potential family members in human airway smooth muscle and lung tissue. Am J Respir Cell Mol Biol 2004;30:145–54. [49] White TA, Xue A, Chini EN, Thompson M, Sieck GC, Wylam ME. Role of transient receptor potential C3 in TNF-α-enhanced calcium influx in human airway myocytes. Am J Respir Cell Mol Biol 2006;35:243–51. [50] Ong HL, Brereton HM, Harland ML, Barritt GJ. Evidence for the expression of transient receptor potential proteins in guinea pig airway smooth muscle cells. Respirology 2003;8:23–32. [51] Xiao JH, Zheng YM, Liao B, Wang YX. Functional role of canonical transient receptor potential 1 and canonical transient receptor potential 3 in normal and asthmatic airway smooth muscle cells. Am J Respir Cell Mol Biol 2010;43:17–25. [52] Albert AP, Large WA. Signal transduction pathways and gating mechanisms of native TRP-like cation channels in vascular myocytes. J Physiol 2006;570:45–51. [53] Godin N, Rousseau E. TRPC6 silencing in primary airway smooth muscle cells inhibits protein expression without affecting OAG-induced calcium entry. Mol Cell Biochem 2007;296:193–201. [54] Colsoul B, Nilius B, Vennekens R. On the putative role of transient receptor potential cation channels in asthma. Clin Exp Allergy 2009;39:1456–66. [55] Sweeney M, McDaniel SS, Platoshyn O, Zhang S, Yu Y, Lapp BR, et al. Role of capacitative Ca2+ entry in bronchial contraction and remodeling. J Appl Physiol 2002;92:1594–602. [56] Davies JC, Alton EWFW, Bush A. Cystic fibrosis. BMJ 2007;335:1255–9. [57] Wei L, Freichel M, Jaspers M, Cuppens H, Cassiman JJ, Droogmans G, et al. Functional interaction between TRP4 and CFTR in mouse aorta endothelial cells. BMC Physiol 2001;1:3. [58] Antigny F, Norez C, Dannhoffer L, Bertrand J, Raveau D, Corbi P, et al. Transient receptor potential canonical channel 6 links Ca2+ mishandling to cystic fibrosis transmembrane conductance regulator channel dysfunction in cystic fibrosis. Am J Respir Cell Mol Biol 2011;44:83–90. [59] Fuchs B, Dietrich A, Gudermann T, Kalwa H, Grimminger F, Weissmann N. The role of classical transient receptor potential channels in the regulation of hypoxic pulmonary vasoconstriction. Adv Exp Med Biol 2010;661:187–200. [60] Fuchs B, Rupp M, Ghofrani HA, Schermuly RT, Seeger W, Grimminger F, et al. Diacylglycerol regulates acute hypoxic pulmonary vasoconstriction via TRPC6. Respir Res 2011;12:12–20. [61] Li S, Westwick J, Poll C. Transient receptor potential (TRP) channels as potential drug targets in respiratory disease. Cell Calcium 2003;33:551–8. [62] Damann N, Owsianik G, Li S, Poll C, Nilius B. The calcium-conducting ion channel transient receptor potential canonical 6 is involved in macrophage inflammatory protein-2-induced migration of mouse neutrophils. Acta Physiol 2009;195:3–11. [63] Sel S, Rost BR, Yildirim AO, Sel B, Kalwa H, Fehrenbach H, et al. Loss of classical transient receptor potential 6 channel reduces allergic airway response. Clin Exp Allergy 2008;38:1548–58. [64] Medic N, Desai A, Olivera A, Abramowitz J, Birnbaumer L, Beaven MA, et al. Knockout of the Trpc1 gene reveals that TRPC1 can promote recovery from anaphylaxis by negatively regulating mast cell TNF-α production. Cell Calcium 2013;53:315–26. [65] Finney-Hayward TK, Popa MO, Bahra P, Li S, Poll CT, Gosling M, et al. Expression of transient receptor potential C6 channels in human lung macrophages. Am J Respir Cell Mol Biol 2010;43:296–304. [66] Pedersen SF, Owsianik G, Nilius B. TRP channels: an overview. Cell Calcium 2005;38:233–52. 498 26. PATHOPHYSIOLOGICAL ROLES OF RESPIRATORY TRPs

[67] Nilius B, Owsianik G, Voets T, Peters JA. Transient receptor potential cation channels in disease. Physiol Rev 2007;87:165–217. [68] Szallasi A, Blumberg PM. Complex regulation of TRPV1 by vanilloids. In: Liedtke WB, Heller S, editors. TRP ion channel function in sensory transduction and cellular signaling cascades. Boca Raton (FL): CRC Press; 2007, chapter 6. [69] Bevan S, Quallo T, Andersson DA. TRPV1. Handb Exp Pharmacol 2014;222:207–45. [70] Watanabe N, Horie S, Michael GJ, Keir S, Spina D, Page CP, et al. Immunohistochemical co-localization of transient receptor potential vanilloid (TRPV)1 and sensory neuropeptides in the guinea-pig respiratory system. Neuroscience 2006;141:1533–43. [71] Watanabe N, Horie S, Spina D, Michael GJ, Page CP, Priestley JV. Immunohistochemical localization of transient receptor potential vanilloid subtype 1 in the trachea of ovalbumin-sensitized guinea pigs. Int Arch Allergy Immunol 2008;146(Suppl. 1):28–32. [72] Groneberg DA, Niimi A, Dinh QT, Cosio B, Hew M, Fischer A, et al. Increased expression of transient receptor potential vanilloid-1 in airway nerves of chronic cough. Am J Respir Crit Care Med 2004;170:1276–80. [73] Morice AH, Geppetti P. Cough. 5: the type 1 vanilloid receptor: a sensory receptor for cough. Thorax 2004;59:257–8. [74] Zhang G, Lin RL, Wiggers M, Snow DM, Lee LY. Altered expression of TRPV1 and sensitivity to capsaicin in pulmonary myelinated afferents following chronic airway inflammation in the rat. J Physiol 2008;586:5771–86. [75] McGarvey LP, Elder J. Future directions in treating cough. Otolaryngol Clin North Am 2010;43:199–211. [76] Lee LY, Ni D, Hayes Jr D, Lin RL. TRPV1 as a cough sensor and its temperature-sensitive properties. Pulm Pharmacol Ther 2011;24:280–5. [77] Maher SA, Dubuis ED, Belvisi MG. G-protein coupled receptors regulating cough. Curr Opin Pharmacol 2011;11:248–53. [78] Cantero-Recasens G, Gonzalez JR, Fandos C, Duran-Tauleria E, Smit LA, Kauffmann F, et al. Loss of function of transient receptor potential vanilloid 1 (TRPV1) genetic variant is associated with lower risk of active childhood asthma. J Biol Chem 2010;285:27532–5. [79] Brozmanova M, Mazurova L, Ru F, Tatar M, Kollarik M. Comparison of TRPA1-versus TRPV1-mediated cough in guinea pigs. Eur J Pharmacol 2012;689:211–18. [80] Thomas KC, Sabnis AS, Johansen ME, Lanza DL, Moos PJ, Yost GS, et al. Transient receptor potential vanilloid 1 agonists cause endoplasmic reticulum stress and cell death in human lung cells. J Pharmacol Exp Ther 2007;321:830–8. [81] Rehman R, Bhat YA, Panda L, Mabalirajan U. TRPV1 inhibition attenuates IL-13 mediated asthma features in mice by reducing airway epithelial injury. Int Immunopharmacol 2013;15:597–605. [82] Wu Y, You H, Ma P, Li L, Yuan Y, Li J, et al. Role of transient receptor potential ion channels and evoked levels of neuropeptides in a formaldehyde-induced model of asthma in BALB/c mice. PLoS One 2013;8:e62827. [83] Deering-Rice CE, Johansen ME, Roberts JK, Thomas KC, Romero EG, Lee J, et al. Transient receptor potential vanilloid-1 (TRPV1) is a mediator of lung toxicity for coal fly ash particulate material. Mol Pharmacol 2012;81:411–19. [84] Agopyan N, Head J, Yu S, Simon SA. TRPV1 receptors mediate particulate matter-induced apoptosis. Am J Physiol 2004;286:L563–72. [85] Sakamoto N, Hayashi S, Gosselink J, Ishii H, Ishimatsu Y, Mukae H, et al. Calcium dependent and independent cytokine synthesis by air pollution particle-exposed human bronchial epithelial cells. Toxicol Appl Pharmacol 2007;225:134–41. [86] Ang SF, Sio SW, Moochhala SM, MacAry PA, Bhatia M. Hydrogen sulfide upregulates cyclooxygenase-2 and prostaglandin E metabolite in sepsis-evoked acute lung injury via transient receptor potential vanilloid type 1 channel activation. J Immunol 2011;187:4778–87. [87] Bhatia M, Zhi L, Zhang H, Ng SW, Moore PK. Role of substance P in hydrogen sulfide-induced pulmonary inflammation in mice. Am J Physiol 2006;291:L896–904. [88] McLeod RL, Jia Y, McHugh NA, Fernandez X, Mingo GG, Wang X, et al. Sulfur-dioxide exposure increases TRPV1-mediated responses in nodose ganglia cells and augments cough in guinea pigs. Pulm Pharmacol Ther 2007;20:750–7. [89] Amadesi S, Nie J, Vergnolle N, Cottrell GS, Grady EF, Trevisani M, et al. Protease-activated receptor 2 sensitizes the capsaicin receptor transient receptor potential vanilloid receptor 1 to induce hyperalgesia. J Neurosci 2004;24:4300–12. REFERENCES 499

[90] Dai Y, Moriyama T, Higashi T, Togashi K, Kobayashi K, Yamanaka H, et al. Proteinase-activated receptor 2-mediated potentiation of transient receptor potential vanilloid subfamily 1 activity reveals a mechanism for proteinase-induced inflammatory pain. J Neurosci 2004;24:4293–9. [91] Thomas KC, Roberts JK, Deering-Rice CE, Romero EG, Dull RO, Lee J, et al. Contributions of TRPV1, endovanilloids, and endoplasmic reticulum stress in lung cell death in vitro and lung injury. Am J Physiol 2012;302:L111–19. [92] Reilly CA, Johansen ME, Lanza DL, Lee J, Lim JO, Yost GS. Calcium-dependent and independent mechanisms of capsaicin receptor (TRPV1)-mediated cytokine production and cell death in human bronchial epithelial cells. J Biochem Mol Toxicol 2005;19:266–75. [93] Abdullah H, Heaney LG, Cosby SL, McGarvey LPA. Rhinovirus upregulates transient receptor potential channels in a human neuronal cell line: implications for respiratory virus-induced cough reflex sensitivity. Thorax 2014;69:46–54. [94] Lin RL, Hayes Jr D, Lee LY. Bronchoconstriction induced by hyperventilation with humidified hot air: role of TRPV1-expressing airway afferents. J Appl Physiol 2009;106:1917–24. [95] Mitchell JE, Campbell AP, New NE, Sadofsky LR, Kastelik JA, Mulrennan SA, et al. Expression and characterization of the intracellular vanilloid receptor (TRPV1) in bronchi from patients with chronic cough. Exp Lung Res 2005;31:295–306. [96] Zhao L, Zhang X, Kuang H, Wu J, Guo Y, Ma L. Effect of TRPV1 channel on the proliferation and apoptosis in asthmatic rat airway smooth muscle cells. Exp Lung Res 2013;39:283–94. [97] Helyes Z, Elekes K, Nemeth J, Pozsgai G, Sandor K, Kereskai L, et al. Role of transient receptor potential vanilloid 1 receptors in endotoxin-induced airway inflammation in the mouse. Am J Physiol 2007;292:L1173–81. [98] Birrell MA, Bonvini SJ, Dubuis E, Maher SA, Wortley MA, Grace MS, et al. Tiotropium modulates transient receptor potential V1 (TRPV1) in airway sensory nerves: a beneficial off-target effect? J Allergy Clin Immunol 2014;133:679–87. [99] Yang J, Yu HM, Zhou XD, Kolosov VP, Perelman JM. Study on TRPV1-mediated mechanism for the hypersecretion of mucus in respiratory inflammation. Mol Immunol 2013;53:161–71. [100] El-Hashim AZ, Jaffal SM. Nerve growth factor enhances cough and airway obstruction via TrkA receptor- and TRPV1-dependent mechanisms. Thorax 2009;64:791–7. [101] Luo YL, Guo HM, Zhang YL, Chen PX, Zhu YX, Huang JH, et al. Cellular mechanism underlying formaldehyde-stimulated Cl- secretion in rat airway epithelium. PLoS One 2013;8:e54494. [102] Trevisani M, Milan A, Gatti R, Zanasi A, Harrison S, Fontana G, et al. Antitussive activity of iodo-resiniferatoxin in guinea pigs. Thorax 2004;59:769–72. [103] Delescluse I, Mace H, Adcock JJ. Inhibition of airway hyper-responsiveness by TRPV1 antagonists (SB-705498 and PF-04065463) in the unanaesthetized, ovalbumin-sensitized guinea pig. Br J Pharmacol 2012;166:1822–32. [104] Jia Y, Wang X, Varty L, Rizzo CA, Yang R, Correll CC, et al. Functional TRPV4 channels are expressed in human airway smooth muscle cells. Am J Physiol 2004;287:L272–8. [105] Hamanaka K, Jian MY, Townsley MI, King JA, Liedtke W, Weber DS, et al. TRPV4 channels augment macrophage activation and ventilator-induced lung injury. Am J Physiol 2010;299:L353–62. [106] Alvarez DF, King JA, Weber D, Addison E, Liedtke W, Townsley MI. Transient receptor potential vanilloid 4-mediated disruption of the alveolar septal barrier: a novel mechanism of acute lung injury. Circ Res 2006;99:988–95. [107] Jian MY, King JA, Al-Mehdi AB, Liedtke W, Townsley MI. High vascular pressure-induced lung injury requires P450 epoxygenase-dependent activation of TRPV4. Am J Respir Cell Mol Biol 2008;38:386–92. [108] Vazquez E, Nobles M, Valverde MA. Defective regulatory volume decrease in human cystic fibrosis tracheal cells because of altered regulation of intermediate conductance Ca2+-dependent potassium channels. Proc Natl Acad Sci USA 2001;98:5329–34. [109] Arniges M, Vazquez E, Fernandez-Fernandez JM, Valverde MA. Swelling-activated Ca2+ entry via TRPV4 channel is defective in cystic fibrosis airway epithelia. J Biol Chem 2004;279:54062–8. [110] Fernández-Fernández JM, Andrade YN, Arniges M, Fernandes J, Plata C, Rubio-Moscardo F, et al. Functional coupling of TRPV4 cationic channel and large conductance, calcium-dependent potassium channel in human bronchial epithelial cell lines. Pflugers Arch 2008;457:149–59. [111] Zhu G, Gulsvik A, Bakke P, Ghatta S, Anderson W, Lomas DA, et al. Association of TRPV4 gene polymorphisms with chronic obstructive pulmonary disease. Hum Mol Genet 2009;18:2053–62. [112] McAlexander MA, Luttmann MA, Hunsberger GE, Undem BJ. Transient receptor potential vanilloid 4 activation constricts the human bronchus via the release of cysteinyl leukotrienes. J Pharmacol Exp Ther 2014;349:118–25. 500 26. PATHOPHYSIOLOGICAL ROLES OF RESPIRATORY TRPs

[113] Xia Y, Fu Z, Hu J, Huang C, Paudel O, Cai S, et al. TRPV4 channel contributes to serotonin-induced pulmonary vasoconstriction and the enhanced vascular reactivity in chronic hypoxic pulmonary hypertension. Am J Physiol 2013;305:C704–15. [114] Kraft R, Harteneck C. The mammalian melastatin-related transient receptor potential cation channels: an overview. Pflugers Arch 2005;451:204–11. [115] Simard JM, Tarasov KV, Gerzanich V. Non-selective cation channels, transient receptor potential channels and ischemic stroke. Biochim Biophys Acta 2007;1772:947–57. [116] Yamamoto S, Takahashi N, Mori Y. Chemical physiology of oxidative stress-activated TRPM2 and TRPC5 channels. Prog Biophys Mol Biol 2010;103:18–27. [117] Takahashi N, Kozai D, Kobayashi R, Ebert M, Mori Y. Roles of TRPM2 in oxidative stress. Cell Calcium 2011;50:279–87. [118] Faouzi M, Penner R. TRPM2. Handb Exp Pharmacol 2014;222:403–26. [119] Yamamoto S, Shimizu S, Kiyonaka S, Takahashi N, Wajima T, Hara Y, et al. TRPM2-mediated Ca2+ influx induces chemokine production in monocytes that aggravates inflammatory neutrophil infiltration. Nat Med 2008;14:738–47. 2+ [120] Hecquet CM, Ahmmed GU, Vogel SM, Malik AB. Role of TRPM2 channel in mediating H2O2-induced Ca entry and endothelial hyperpermeability. Circ Res 2008;102:347–55.

[121] Hecquet CM, Malik AB. Role of H2O2-activated TRPM2 calcium channel in oxidant-induced endothelial injury. Thromb Haemost 2009;101:619–25. [122] Hecquet CM, Ahmmed GU, Malik AB. TRPM2 channel regulates endothelial barrier function. Adv Exp Med Biol 2010;661:155–67. [123] Hardaker L, Bahra P, de Billy BC, Freeman M, Kupfer N, Wyss D, et al. The ion channel transient receptor potential melastatin-2 does not play a role in inflammatory mouse models of chronic obstructive pulmonary diseases. Respir Res 2012;13:13–30. [124] Di A, Gao XP, Qian F, Kawamura T, Han J, Hecquet C, et al. The redox-sensitive cation channel TRPM2 modulates phagocyte ROS production and inflammation. Nat Immunol 2011;13:29–34. [125] Vennekens R, Olausson J, Meissner M, Bloch W, Mathar I, Philipp SE, et al. Increased IgE-dependent mast cell activation and anaphylactic responses in mice lacking the calcium-activated nonselective cation channel TRPM4. Nat Immunol 2007;8:312–20. [126] Buday T, Brozmanova M, Biringerova Z, Gavliakova S, Poliacek I, Calkovsky V, et al. Modulation of cough response by sensory inputs from the nose—role of trigeminal TRPA1 versus TRPM8 channels. Cough 2012;8:11–18. [127] Sabnis AS, Shadid M, Yost GS, Reilly CA. Human lung epithelial cells express a functional cold-sensing TRPM8 variant. Am J Respir Cell Mol Biol 2008;39:466–74. [128] Bidaux G, Flourakis M, Thebault S, Zholos A, Beck B, Gkika D, et al. Prostate cell differentiation status determines transient receptor potential melastatin member 8 channel subcellular localization and function. J Clin Invest 2007;117:1647–57. [129] Li M, Li Q, Yang G, Kolosov VP, Perelman JM, Zhou XD. Cold temperature induces mucin hypersecretion from normal human bronchial epithelial cells in vitro through a transient receptor potential melastatin 8 (TRPM8)- mediated mechanism. J Allergy Clin Immunol 2011;128:626–34. [130] Cho Y, Jang Y, Yang YD, Lee CH, Lee Y, Oh U. TRPM8 mediates cold and menthol allergies associated with mast cell activation. Cell Calcium 2010;48:202–8. [131] Keh SM, Facer P, Yehia A, Sandhu G, Saleh HA, Anand P. The menthol and cold sensation receptor TRPM8 in normal human nasal mucosa and rhinitis. Rhinology 2011;49:453–7. [132] de Courcey F, Zholos AV, Atherton-Watson H, Williams MTS, Canning P, Danahay HL, et al. Development of primary human nasal epithelial cell cultures for the study of cystic fibrosis pathophysiology. Am J Physiol 2012;303:C1173–9. [133] Hanrahan S, Stokesberry S, McGarvey L, Elborn JS, Zholos A, Ennis M. Expression and functional role of TRPM8 in primary human nasal epithelial cells. Pulm Pharmacol Ther 2011;24:e6. [134] Yang XR, Lin MJ, McIntosh LS, Sham JSK. Functional expression of transient receptor potential melastatin- and vanilloid-related channels in pulmonary arterial and aortic smooth muscle. Am J Physiol 2006;290:L1267–76. [135] Johnson CD, Melanaphy D, Purse A, Stokesberry SA, Dickson P, Zholos AV. Transient receptor potential melastatin 8 channel involvement in the regulation of vascular tone. Am J Physiol 2009;296:H1868–77. CHAPTER 27 Conclusions Magdalene Moran* Hydra Biosciences Cambridge, MA, USA *Corresponding author: [email protected]

As you have read, it is a very exciting time for transient receptor potential (TRP) research. Studies involving TRPs continue to proliferate; a recent PubMed search on TRP channels uncovered over 1000 papers published in 2013 alone (see Figure 27.1). With this continuing surge in research, the diversity in TRP channel function becomes even more apparent, and we find we can make fewer generalizations about this superfamily. These fascinating cation channels are involved in multiple cellular processes and contribute to numerous disease states. Hopefully our increasing knowledge about TRP channel function will translate into new medicines that will address underserved patient populations. So far, few TRP channel modulators have been used clinically; the true clinical value of the TRPs remains to be determined. TRPV1 remains the best-studied family member. Both agonists and antagonists of TRPV1 have been tested in human clinical trials. Although the TRPV1 antagonists have thus far been plagued with issues of impaired thermoregulation and thermosensation that remain to be overcome, TRPV1 agonists have been shown to be efficacious analgesics. Topical capsaicin rubs remain popular, and the Qutenza patch received FDA approval for the treatment of postherpetic neuralgia [1]. In addition, intrathecal and intravesicular TRPV1 agonists also show promise in cancer pain and bladder disorders. Significant excitement also surrounds TRPA1. TRPA1 antagonists show efficacy in multiple preclinical models of both inflammatory [2–4] and peripheral neuropathic pain. Recent discoveries in models of painful diabetic [5,6] and chemotherapy-induced peripheral neuropathies [7,8] suggest that TRPA1 antagonists might be able to prevent damage in addition to reducing pain [5,7,8]. A trial of a TRPA1 antagonist in painful diabetic neuropathy (Glenmark, see http://clinicaltrials.gov/ct2/show/NCT01556152) recently completed. According to a Glenmark press release, in a double-blind, placebo controlled trial of 138 patients in Europe and India, GRC 17536 demonstrated a statistically and clinically significant pain reduction in a pre-specified subgroup of patients with moderate to severe pain due to diabetic neuropathy. Clinical trials in chronic cough and asthma are also underway, highlighting the excitement around recent discoveries of a role for TRPA1 in the pulmonary field. TRPV4 is also a potentially interesting pulmonary target. Single nucleotide polymorphisms in TRPV4 are associated with airflow obstruction in chronic obstructive pulmonary disease (COPD) patients [9]. Ex vivo studies on the human bronchus suggest that TRPV4 activation

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0 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014

FIGURE 27.1 Number of TRP publications in pubmed by year (1994–2013) leads to bronchiole constriction that is dependent on cysteine leukotrienes [10]. Studies in laboratory animals from multiple groups also implicate TRPV4 in both cardiogenic and noncardiogenic pulmonary edema [11–13]. Antagonizing murine TRPV4 reduces pulmonary edema and improves arterial oxygen tension after aortic banding [12]. Similarly, blocking TRPV4 in mice attenuates edema after myocardial infarction [12]. TRPV4 is one of several TRP channels implicated in metabolic syndromes. Inhibition of TRPV4 increases markers of brown fat in cultured adipocytes and increases glucose tolerance in mice [14]. Similarly, the cold-activated channel, TRPM8, has been found in brown fat. Activation of TRPM8 with menthol increases brown adipose tissue thermogenesis and reduces dietary obesity in mice [15]. In addition to their potential role in brown fat, TRPs, including TRPM5 and TRPV1, have also been implicated in diabetes [16]. One of the most intriguing areas of recent TRP channels research has been in dermatology. Both TRPA1 and TRPV1 have been shown to play significant roles in pruritus (itch) [17–19]. Genetic deletion or pharmacological inhibition of TRPA1 also attenuates the severity of atopic dermatitis symptoms in a murine model [20]. In addition, human and rodent genetic data implicate overactive TRPV3 in keratinocyte dysfunction and itch [21–25]. Mutations in TRP channels lead to a variety of heritable disorders. These include skeletal dysplasias and neuropathies (TRPV4) [26], kidney diseases (TRPC6) [27], mucolipidosis type REFERENCES 503

IV (TRPML1) [28,29], dermatologic disorders (TRPV3) [23], and pain syndromes (TRPA1) [30]. Most, though not all, of the disease-causing mutations appear to be gain-of-function mutations that lead to channel hyperexcitability and subsequent toxicity. In addition, polymorphisms in TRP channels have also been associated with susceptibility to disease. For example, TRPV1 polymorphisms have been associated with multiple pathologies including cough [31], migraine [32], and type I diabetes [33]. Most recently, epigenetic studies revealed a potential link between heat pain sensitivity and methylation of the TRPA1 promoter [34], though the significance of this remains to be determined. As TRPs are implicated in more disease states and the pace of research accelerates, questions regarding the clinical utility of TRP channel modulators take center stage. With any new mechanism one wonders whether preclinical efficacy will translate into humans. Which will be a more fruitful approach—agonizing a channel to induce a persistent desensitization or antagonizing it? Will it depend on the channel? Are TRP channels performing functions we have yet to appreciate that will impact the tolerability of modulators? Although speculation is rampant, data remains scarce. As in the preclinical arena, the inherent diversity of TRP channel structure and function will make it difficult to generalize about the family from early results, and each mechanism will require independent testing. In the next few years, clinical studies involving a number of mechanisms should help us answer these questions. Meanwhile, the rapidly emerging stories around TRP channels make us optimistic that this diverse superfamily will yield useful therapies for human disease.

References [1] Moran MM, McAlexander MA, Biro T, Szallasi A. Transient receptor potential channels as therapeutic targets. Nat Rev Drug Discov 2011;10(8):601–20. [2] Eid SR, Crown ED, Moore EL, Liang HA, Choong KC, Dima S, et al. HC-030031, a TRPA1 selective antagonist, attenuates inflammatory- and neuropathy-induced mechanical hypersensitivity. Mol Pain 2008;4:48. [3] del Camino D, Murphy S, Heiry M, Barrett LB, Earley TJ, Cook CA, et al. TRPA1 contributes to cold hypersensitivity. J Neurosci 2010;30(45):15165–74. [4] Chen J, Joshi SK, DiDomenico S, Perner RJ, Mikusa JP, Gauvin DM, et al. Selective blockade of TRPA1 channel attenuates pathological pain without altering noxious cold sensation or body temperature regulation. Pain 2011;152(5):1165–72. [5] Koivisto A, Hukkanen M, Saarnilehto M, Chapman H, Kuokkanen K, Wei H, et al. Inhibiting TRPA1 ion channel reduces loss of cutaneous nerve fiber function in diabetic animals: sustained activation of the TRPA1 channel contributes to the pathogenesis of peripheral diabetic neuropathy. Pharmacol Res 2012;65(1):149–58. [6] Wei H, Hamalainen MM, Saarnilehto M, Koivisto A, Pertovaara A. Attenuation of mechanical hypersensitivity by an antagonist of the TRPA1 ion channel in diabetic animals. Anesthesiology 2009;111(1):147–54. [7] Nassini R, Gees M, Harrison S, De Siena G, Materazzi S, Moretto N, et al. Oxaliplatin elicits mechanical and cold allodynia in rodents via TRPA1 receptor stimulation. Pain 2011;152(7):1621–31. [8] Trevisan G, Materazzi S, Fusi C, Altomare A, Aldini G, Lodovici M, et al. Novel therapeutic strategy to prevent chemotherapy-induced persistent sensory neuropathy by TRPA1 blockade. Cancer Res 2013;73(10):3120–31. [9] Zhu G, Gulsvik A, Bakke P, Ghatta S, Anderson W, Lomas DA, et al. Association of TRPV4 gene polymorphisms with chronic obstructive pulmonary disease. Hum Mol Genet 2009;18(11):2053–62. [10] McAlexander MA, Luttman MA, Hunsberger GE, Undem BJ. Transient receptor potential vanilloid 4 (TRPV4) activation constricts the human bronchus via the release of cysteinyl leukotrienes. J Pharmacol Exp Ther 2014;349(1):118–25. [11] Hamanaka K, Jian MY, Weber DS, Alvarez DF, Townsley MI, Al-Mehdi AB, et al. TRPV4 initiates the acute calcium-dependent permeability increase during ventilator-induced lung injury in isolated mouse lungs. Am J Physiol Lung Cell Mol Physiol 2007;293(4):L923–32. 504 27. Conclusions

[12] Thorneloe KS, Cheung M, Bao W, Alsaid H, Lenhard S, Jian MY, et al. An orally active TRPV4 channel blocker prevents and resolves pulmonary edema induced by heart failure. Sci Transl Med 2012;4(159):159ra48. [13] Yin J, Hoffmann J, Kaestle SM, Neye N, Wang L, Baeurle J, et al. Negative-feedback loop attenuates hydrostatic lung edema via a cGMP-dependent regulation of transient receptor potential vanilloid 4. Circ Res 2008;102(8):966–74. [14] Ye L, Kleiner S, Wu J, Sah R, Gupta RK, Banks AS, et al. TRPV4 is a regulator of adipose oxidative metabolism, inflammation, and energy homeostasis. Cell 2012;151(1):96–110. [15] Ma S, Yu H, Zhao Z, Luo Z, Chen J, Ni Y, et al. Activation of the cold-sensing TRPM8 channel triggers UCP1- dependent thermogenesis and prevents obesity. J Mol Cell Biol 2012;4(2):88–96. [16] Uchida K, Tominaga M. The role of thermosensitive TRP (transient receptor potential) channels in insulin secretion. Endocr J 2011;58(12):1021–8. [17] Wilson SR, Gerhold KA, Bifolck-Fisher A, Liu Q, Patel KN, Dong X, et al. TRPA1 is required for histamine- independent, Mas-related G protein-coupled receptor-mediated itch. Nat Neurosci 2011;14(5):595–602. [18] Wilson SR, Nelson AM, Batia L, Morita T, Estandian D, Owens DM, et al. The ion channel TRPA1 is required for chronic itch. J Neurosci 2013;33(22):9283–94. [19] Imamachi N, Park GH, Lee H, Anderson DJ, Simon MI, Basbaum AI, et al. TRPV1-expressing primary afferents generate behavioral responses to pruritogens via multiple mechanisms. Proc Natl Acad Sci USA 2009;106(27):11330–5. [20] Liu B, Escalera J, Balakrishna S, Fan L, Caceres AI, Robinson E, et al. TRPA1 controls inflammation and pruritogen responses in allergic contact dermatitis. FASEB J 2013;27(9):3549–63. [21] Duchatelet S, Pruvost S, de Veer S, Fraitag S, Nitschke P, Bole-Feysot C, et al. A new TRPV3 missense mutation in a patient with Olmsted syndrome and erythromelalgia. JAMA Dermatol 2014;150(3):303–6. [22] Eytan O, Fuchs-Telem D, Mevorach B, Indelman M, Bergman R, Sarig O, et al. Olmsted syndrome caused by a homozygous recessive mutation in TRPV3. J Invest Dermatol 2014;134(6):1752–4. [23] Lin Z, Chen Q, Lee M, Cao X, Zhang J, Ma D, et al. Exome sequencing reveals mutations in TRPV3 as a cause of Olmsted syndrome. Am J Hum Genet 2012;90(3):558–64. [24] Yamamoto-Kasai E, Imura K, Yasui K, Shichijou M, Oshima I, Hirasawa T, et al. TRPV3 as a therapeutic target for itch. J Invest Dermatol 2012;132(8):2109–12. [25] Yoshioka T, Imura K, Asakawa M, Suzuki M, Oshima I, Hirasawa T, et al. Impact of the Gly573Ser substitution in TRPV3 on the development of allergic and pruritic dermatitis in mice. J Invest Dermatol 2009;129(3):714–22. [26] Cho TJ, Matsumoto K, Fano V, Dai J, Kim OH, Chae JH, et al. TRPV4-pathy manifesting both skeletal dysplasia and peripheral neuropathy: a report of three patients. Am J Med Genet A 2012;158A(4):795–802. [27] Winn MP, Conlon PJ, Lynn KL, Farrington MK, Creazzo T, Hawkins AF, et al. A mutation in the TRPC6 cation channel causes familial focal segmental glomerulosclerosis. Science 2005;308(5729):1801–4. [28] Sun M, Goldin E, Stahl S, Falardeau JL, Kennedy JC, Acierno Jr JS, et al. Mucolipidosis type IV is caused by mutations in a gene encoding a novel transient receptor potential channel. Hum Mol Genet 2000;9(17):2471–8. [29] Bassi MT, Manzoni M, Monti E, Pizzo MT, Ballabio A, Borsani G. Cloning of the gene encoding a novel integral membrane protein, mucolipidin-and identification of the two major founder mutations causing mucolipidosis type IV. Am J Hum Genet 2000;67(5):1110–20. [30] Kremeyer B, Lopera F, Cox JJ, Momin A, Rugiero F, Marsh S, et al. A gain-of-function mutation in TRPA1 causes familial episodic pain syndrome. Neuron 2010;66(5):671–80. [31] Smit LA, Kogevinas M, Anto JM, Bouzigon E, Gonzalez JR, Le Moual N, et al. Transient receptor potential genes, smoking, occupational exposures and cough in adults. Respir Res 2012;13:26. [32] Carreno O, Corominas R, Fernandez-Morales J, Camina M, Sobrido MJ, Fernandez-Fernandez JM, et al. SNP variants within the vanilloid TRPV1 and TRPV3 receptor genes are associated with migraine in the Spanish population. Am J Med Genet Part B 2012;159B(1):94–103. [33] Sadeh M, Glazer B, Landau Z, Wainstein J, Bezaleli T, Dabby R, et al. Association of the M3151 variant in the transient receptor potential vanilloid receptor-1 (TRPV1) gene with type 1 diabetes in an Ashkenazi Jewish population. IMAJ, Isr Med Assoc J 2013;15(9):477–80. [34] Bell JT, Loomis AK, Butcher LM, Gao F, Zhang B, Hyde CL, et al. Differential methylation of the TRPA1 promoter in pain sensitivity. Nat Commun 2014;5:2978. Index

Note: Page numbers followed by f indicate figures and t indicate tables.

A skin ageing effects, 306 Above-threshold-plateau-depolarization (ATPD), 426 skin inflammation, 307–308 α,β-unsaturated aldehydes, 169–170 skin sensitizers, 306 Acetaminophen/paracetamol, 168–169 thermosensation, 306 Acute lung injury (ALI), 222 Ankyrin 1 (TRPA1) antagonists Airway disease, 232–233 pain relief (see Pain transduction and amplification) Airway smooth muscle (ASM) preclinical models, 501 apoptosis, 490 respiratory diseases (see Respiratory tracts) structure of, 154–156 155f H1 receptor activates cation currents, 486–487 pathogenesis, 486 Anxiety disorders, 442 proliferation regulation, 487, 490 Appaloosa horse, 20–21, 312 TRPC3, 488 Arachidonic acid (AA), 173–174, 197–198, 207–208, 208f TRPC6, 488 2-Arachidonoylglycerol (2-AG), 207–208, 208f, 430 TRPV1, 490 Atherosclerosis, 335–336, 336f TRPV4, 491 Atopy (allergy), 164–166 A-kinase anchoring protein (AKAP), 9 Atrial fibrillation (AF), 374–375 ALI. See Acute lung injury (ALI) Autosomal-dominant hypercalciuria, 20 Allicin, 268, 306, 351 Autosomal-dominant polycystic kidney disease, 24–25 Allodynia, 244–246 Allyl isothiocyanate (AITC), 167–168, 177, 268 B Alveolar macrophages, 225–227, 227f BAA. See Bisandrographolide A (BAA) Alzheimer disease (AD), 440–441 Basal cell carcinomas (BCCs), 14 Animal pain models, 114 Below-threshold-depolarization (BTD), 426 Ankyrin 1 (TRPA1) Bipolar disorder (BD), 444 antagonists, 501 Bipolar disorder type I (BD-I), 21 clinical trials, 501 Bisandrographolide A (BAA) 21, 210, 211f diabetic nephropathy, 355–356 Bladder disorders. See Urinary bladder digestive disease, 389 Bladder pain syndrome/interstitial cystitis (BPS/IC), familial episodic pain syndrome, 306 123–124 genetic deletion, 502 Blood-brain barrier (BBB), 267–268 IBD, 392–393 Bone cancer, companion dog models, 108–111, 109f itch, 284–286, 284f Bone morphogenic proteins (BMPs), 422 in itching, 306 Boswellia cerrata resin, 197 keratinocytes, 307 Botulinum toxin, 122–123, 125 mechanosensation, 306 Brachyolmia type 3, 18–19 melanocytes, 308 Bradykinin (BK), 174–175 nonneuronal expression, skin, 306–307 Brain-derived neurotrophic factor (BDNF), 420–421 noxious cold, 306 Brugada syndrome, 22 pain, 306 pain syndromes, 502–503 C pain transduction (see Pain transduction and Ca2+ channel amplification) L-type activation, 350–351 pancreatic β cells, 351 L-type inhibition, 230 pharmacological inhibition, 502 TRPV4, 224, 225–227, 228–229, 229f, 230, 232–233 pruritus, 502 voltage-dependent L-type, 347

505 506 INDEX

Cadherin-8, 252 patch–practical aspects, 94–96 Cadmium, 170 patients postprocedural, 91 Calcineurin-NFAT signaling pathway, 368–370 pharmacodynamics, 90–91 Calcitonin gene-related peptide (CGRP), 329 pharmacokinetics, 91 Camphor, 196–197 postherpetic neuralgia, 91–92 Cancer safety and tolerability, 93–94 clinical implications, 412–413 TRPV1, 103–104 TRPC channels, 411–412 xenobiotic sensor, 167–168 TRPM1, 407 Capsazepine 32, 215, 215f TRPM2, 407 Cardiac arrhythmias, 372–373 TRPM4, 408 Cardiac hypertrophy TRPM5, 408 adverse cardiac events, 367 TRPM7, 408 ANP-GC-A-cGMP-PKG-TRPC pathway, 372 TRPM8, 408–410 BTP2, 370, 371 TRPV1, 42, 410 calcineurin-NFAT signaling pathways, 369–370 TRPV2, 410 definition, 367 TRPV6, 410–411 eNOS-/- mouse line, 372 types, 405–406 GSK503A and GSK255B compounds, 371–372 Canine osteoarthritis, 112, 112f hypertension, 330–331 Canonical TRP (TRPC) Klotho, 372 ASM, 486–487 PE and angiotensin II (ATII) infusions, in brain, 420, 421f 367–368 calcium homeostasis, 486 piperidine and isoquinolone analogs, cancer, 411–412 371–372 CFTR, 487–488 Pyr3, 371 clinical diagnosis, 81–82 TRPC3/TRPC6 transgenic mice, 367–368 diabetic nephropathy, 354 Cardiovascular disease diabetic vasculopathy, 353–354 atrial fibrillation, 374–375 disease risk, 81–82 calcium signaling, 365–366 gene polymorphism, 74–75 cardiac arrhythmias, 372–373 keratinocyte differentiation and proliferation, cardiac conduction disease, 373–374 309–310 cell types, 366 knockout mice, 486–487 hypertrophy (see Cardiac hypertrophy) macrophages, 488 PAH (see Pulmonary hypertension (PAH)) muscarinic receptor, 486–487 ROCE, 367 numerous ion channel mechanisms, 486–487 SOCE, 367 pancreatic β cells, 345 TRPM4, 373–374 physiological ventilation/perfusion matching, 488 TRPM7 channels, 374–375 psoriasis, 310 Carvacrol, 197, 268, 300 skin cells, 308–309 Central nervous system (CNS), 240–241 skin-innervating nerves, 308–309 Cerebellar ataxia, 437–438 synaptic plasticity and synaptogenesis, 420, 422f Cerebrovascular diseases TRPC1, 420–424 angiogenesis, 268–269 TRPC3, 424–425 cerebral blood flow, 266 TRPC4, 425–426 channelosomes, 265 TRPC5, 427 distribution, 264–265, 264f TRPC6, 427–428 endothelial proliferation, 268–269 TRPC7, 428 function, 264–265 TRPC3 gene silencing, 487–488 hereditary diseases, 265 Capsaicin, 282, 295 hypertension, 269–271 application procedure, 96 myogenic tone, 266–267 HIV-associated distal sensory polyneuropathy, 92–93 physiological processes, 265 neuropathic pain states, 93 smooth muscle proliferation, 266 NGX-4010, 90 stroke, 272 INDEX 507

TRP channelopathies, 265 Defunctionalization, 282 types, 264–265, 264f Dermatologic disorders (TRPV3), 502–503 vascular barrier function, 267–268 Diabetes mellitus (DM) vascular remodeling, 268–269 capsaicin, 334 vasodilatation, 268 drug target, 356 Channelopathy insulin secretion/sensitivity, 334 definition, 13–14 TRPM5, 334–335 TRPA, 23 TRPV1, 334, 351–352 TRPC (see Transient receptor potential canonical TRPV5, 334–335 (TRPC) channelopathies) Diabetic nephropathy TRPM (see Transient receptor potential melastatin TRPA1, 355–356 (TRPM) channelopathies) TRPCs, 354 TRPML, 23–24 TRPV1, 354–355 TRPP, 24–25 Diabetic nephropathy in type 1 (T1D), 74 TRPV (see Transient receptor potential vanilloid Diabetic vasculopathy, 353–354 (TRPV) channelopathies) Digestive disease Channelosomes, 265 etiology, 386 Charcot-Marie-Tooth neuropathy type 2C (CMT2C), myenteric plexus, 386–387 19–20 plant extracts, 386–387 Chemotherapy-induced neuropathy, 149 regulating GI function, 389, 396t Chiral piperazine amides, 184 selective pharmacological tools, 386–387 Cholecystokinin (CCK), 425 smooth muscle and ICC, 390–391 Chronic hypoxia, 376–377 submucosal plexus, 386–387 Chronic obstructive pulmonary disease (COPD), 20 TRPA1, 389 Chronic pain TRPM8, 390 analgesic compounds, 100 TRPV1, 389–390 companion dog (see Companion dog models) TRPV2, 390 RTX (see Resiniferatoxin (RTX)) TRPV3, 390 therapeutic agents, 101, 102t visceral hyperalgesia and inflammation, TRPV1, 17 386, 387f TRPV3, 200, 202 (S)-3,5-Dihydrophenylglycine (DHPG), 426 Cigarette smoke, 169–170 Dimethylallyl pyrophosphate (DMAPP) 14, Citral 34, 215, 215f 208, 208f Clotrimazole, 246–247, 295 DMAPP. See Dimethylallyl pyrophosphate Companion dog models (DMAPP) bone cancer, 108–111, 109f Dorsal root ganglia (DRG) chronic pain, 108 itch, 280–281 osteoarthritis, 111–113, 112f, 113f neurons, 196 osteosarcoma and osteoarthritis pain, 107–108 TRPM8, 240–242, 241t Complete Freund’s Adjuvant (CFA), 244 TRPV1, 101, 240–241, 241t, 242 Congenital distal spinal muscle atrophy (CDSMA), Drug target 19–20 T1DM, 356 Congenital hyperinsulinism of infancy, 17–18 T2DM, 356 Congenital stationary night blindness type 1C Dry eye syndrome, 23 (CSNB1C), 20–21 Duchenne muscular myopathy, 17 C-type bladder afferents, 120 Duvet syndrome, 436–437 Cystic fibrosis (CF), 232–233, 487–488 Cystic fibrosis transmembrane conductance regulator E (CFTR) gene, 487–488 EET. See Epoxyeicosatrienoic acids (EETs) Endogenous 3-S-hydroxyoctadecadienoic D acid (13-S-HODE), 41 d-Amino acid oxidase (DAAO), 148 Endothelial nitric oxide synthase Darier(-White) disease (DD), 14 (eNOS), 372 Darier’s disease, 310 Endovanilloids, 429 508 INDEX

Epilepsy Green fluorescent protein (GFP), 240–241 epileptic seizures, 436 GSK. See GlaxoSmithKline (GSK) febrile seizures, 436–437 GSK2193874, 212–214, 213f, 230–231, 231f juvenile myoclonic epilepsy, 437 GSK1016790A, 208–210 TRPC3, 436 Guamanian amyotrophic lateral sclerosis (ALS-G), 76 Trpc1/c4 double knockout mouse, 435 Gut Trpc5 knockout mouse, 435 ICC, 390–391 TRPM2, 437 smooth muscle, 390–391 TRPV1, 436–437 therapeutics targets, 387–388, 387f, 388f Epoxyeicosatrienoic acids (EETs), 221–222, 224, TPV1, 389–390 228–229, 230 TRPA1, 389 Ethyl-1-(4-(2,3,3-trichloroacrylamide)phenyl)-5- TRPM8, 390 (trifluoromethyl)-1H-pyrazole-4-carboxylate, 371 TRPV2, 390 Eugenol, 197 TRPV3, 390 Extracellular signal regulated kinase (ERK), 269 H F Hazardous air pollutants, TRPA1 Familial digital arthropathy-brachydactyly, 18–19 isocyanates, 171 Familial episodic pain syndrome 1 (FEPS1), 23 ozone, 170–171 Febrile seizures, 436–437 volatile gas anesthetics, 171–172 Filtration coefficient (Kf), 222, 224, 225f, 226f, 228f HC-030031, 176–177 Focal and segmental glomerulosclerosis (FSGS), 16–17 HC-067047, 211–212 Focal and segmental glomerulosclerosis type 2 Head and neck squamous cell carcinoma, 16–17 (FSGS2), 75 Heart failure, 229–231 Formaldehyde, 169, 297, 490–491 Heat-induced TRPV3 signals, 196 Functional dyspepsia, 17 Herbal extracts, natural products, 210, 211f Hereditary motor sensory neuropathy type IIc (HMSN G IIc), 19–20 Gastrin-releasing peptide (GRP), 280–281, 431 Hypocalciuria, 20 Gastrointestinal (GI) motility High vascular pressure-induced lung injury, 228–229 ICC, 390–391 Histamine, 280 smooth muscle, 390–391 Human umbilical vein endothelial cells (HUVECs), 269 therapeutics targets, 387–388, 387f, 388f Hutchinson-Gillford progeria syndrome, 17 TPV1, 389–390 Hydra biosciences, 211–212 TRPA1, 389 Hyperinsulinism of infancy, 17–18 TRPM8, 390 Hypertension TRPV2, 390 in animal models, 327–329 TRPV3, 390 cardiac hypertrophy, 330–331 Gene polymorphism cerebrovascular dysfunction, 330 genetic variances, 59, 60t definition, 326 TRPA1, 74 endothelial cells, 326 TRPC, 74–75 functional balance, 331–332, 332f TRPM, 76–78 20-HETE activation, 270 TRPML, 78–79 in human, 327, 328t TRPV, 79–81 mechanosensitive Ca2+ permeable channels, 271 Gene therapy, 10 Mg2+, 271 Ghrelin-secreting ε cells, 344–345 primary risk factor, 269–270 GlaxoSmithKline (GSK), 134–136, 208–210, 212–214 renal dysfunction, 331 Glioblastoma multiforme (GBM), 269 vascular function, 326–327 Glomerular diseases, TRPC6, 16–17 VDCC Ca2+ entry, 269–270 Glucagon-releasing α cells, 344–345 Hypomagnesemia with secondary hypocalcemia 1 Glutamate receptor δ2 (GluRδ2), 424 (HSH1/HOMG1), 22–23 Gorlin/Gorlin-Goltz syndrome, 14 Hypothalamic kisspeptin-positive neurons, 426 G protein-coupled receptor kinase 2 (GRK2), 250–251 Hypoxic pulmonary vasoconstriction (HPV), 376–377 INDEX 509

I TRPM, nonneuronal expression, 311 IBD. See Inflammatory bowel disease (IBD) TRPV1, 298–299 Icilin, 240, 243, 246, 248–249 TRPV3, 300, 302 Idiopathic detrusor overactivity (IDO), 122 Keratosis follicularis, 14 Idiopathic pulmonary arterial hypertension, 16–17 Kidney diseases (TRPC6), 502–503 Immunohistochemistry, 222, 223f Infantile hypertrophic pyloric stenosis (IHPS), 15 L Inflammatory bowel disease (IBD) Lidocaine, 172 clinical trails, 393 Lipid metabolism, 335 complementary medicines, 393–394 Lipid peroxidation, 172 neurogenic inflammation, 392 Lipopolysaccharide (LPS), 170 therapeutic targets, 388f, 394–395 Long-lasting depolarization (LLD), 423–424 TRPA1, 392–393 Long-term depression (LTD), 423–424 TRPM2, 393 Lou Gehrig’s disease, 439–440 TRPM8, 393 Lung disease, TRPV4 antagonists TRPV1, 392 airway disease and cough, 232–233 TRPV4, 392 direct channel activation, ALI, 222 visceral hypersensitivity, 392 heart failure and pulmonary edema, 229–231 Insulin-secreting β cells, 344–345 high vascular pressure-induced lung injury, Interstitial cells of Cajal (ICC), 387–388, 390–391 228–229 Intracellular Mg2+ depletion, 329 localization, 222 Intranasal capsaicin desensitization PAH, 231–232 protocols, 166 VILI, 223–227 Intraplantar injections, 243 Lysosomal associated membrane protein-2 (LAMP-2A), Irritable bowel syndrome (IBS) 473–474 constipation and symptoms, 386, 387f etiology, 394 M hyperexcitability, 394 Mas-related G-protein receptor A3 (MrgprA3), 280–281 therapeutic targets, 388f, 394–395 MCOLN1 gene Isolated cardiac conduction disease (ICCD), 12,000 bp, 459–460 373, 374 clinical diagnosis, 458 Isoxazole, 248–249 discovery, 458 Itch location, 459–460 capsaicin, 282, 283f mouse knockout model, 464 chronic condition, 280 mutation, 458, 461f, 462 definition, 279–280 Meckel syndrome, 24–25 highjacking TRP channels, 286–287, 287f Melanocytes inhibiting TRP channels, 284–286 TRPA1, 308 menthol, 282–283 TRPM channels, 311–313 sensory neurons signal, 280–281 TRPML3, 313 skin diseases, 198, 199 TRPV1, 296 TRPM8 activation, 282–283 Melanoma TRPV1 defunctionalization, 282, 283f TRPM1, 311–312 TRPV3, Olmsted syndrome, 287–288 TRPM2, 312 TRPM7, 312 J TRPM8, 312–313 Joubert syndrome, 24–25 Melastatin TRP (TRPM) Juvenile myoclonic epilepsy (JME), 437 cold sensor, 310–311 epithelial TRPM8, 493 K gene polymorphism, 76–78 Kabuki syndrome, 21–22 immunoreactive nerve fibers, 493 Keratinocytes, 287–288 malignant melanoma, 311–313 TRPA1, 307 melanocyte function, 311–313 TRPC, differentiation and proliferation, 298–299 neuronal expression, 310–311 510 INDEX

Melastatin TRP (TRPM) (Continued) N nitrogen oxides, 492 N-acetyl-p-benzo-quinoneimine (NAPQI), 168–169 nonneuronal expression, 311 N-Arachidonoyl-serotonin (AA-5-HT), 429 ozone, 492 Natural products, 210, 211f particulate matter, 492 Naturietic Polypeptide b (Nppb), 280–281 thermal irritants, 492–493 N-[4-(3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl)phenyl]- TRPM2, 432–433 4-methyl-1,2,3-thiadiazole-5-carboxamide TRPM3, 433 (BTP2), 370, 371 TRPM4, 433–434 Nephrin, 16–17 TRPM5, 433–434 Netrin-1, 420–421 TRPM7, 434 Neurodegenerative disorders TRPM8, 434 Alzheimer disease, 440–441 Menthol, 196–197, 240, 242, 243, 246, 248–249 amyotrophic lateral sclerosis, 439–440 Metabolic syndrome autoimmune encephalomyeli, 441–442 atherosclerosis, 335–336, 336f cerebellar ataxia, 437–438 clinical trials, 332 cocaine abuse, 444 diabetes mellitus, 334–335 epilepsy (see Epilepsy) lipid metabolism, 335 multiple sclerosis, 441–442 obesity, 333–334 opioid addiction, 444 risk, 332 Parkinson disease, 438–439 Metabotropic receptors, 284 Neurogenic detrusor overactivity (NDO), 121–122 Metatropic dysplasia, 18–19 Neurogenic inflammation, 285, 286f Micturition, 120 Neurokinin-1 receptor (NK1R), 285 Migraine, 151–153 Neurovascular headache, 151 Miller-Dieker lissencephaly syndrome, 17 NGX-4010, 90 Mucolipidosis type IV (MLIV) Nicotine, 169–170 cellular abnormalities, 459, 460f, 461f Niemann-Pick C1 phenotype, 474–475 clinical presentation, 458–459 Niemann-Pick type C disease (NPC), 23–24 history, 458 Nonerosive gastroesophageal reflux disease (NERD), mouse knockout model, 464 138–139 seminal discovery, 458 Nonobese diabetic (NOD), 352 Mucolipin 1 (TRPML1) Nonpeptidergic classes, 281 cellular localization, 458 Nuclear factor of activated T-cells (NFAT), 16–17 chaperone-mediated autophagy, 473–474 degradative and sorting/trafficking functions, 476 O DMT1, 476–477 Obesity, 333–334 electrophysiology, 462, 471–472 1-oleoyl-2-acetyl-sn-glycerol (OAG), 425–426 endocytic pathway, 475 Olmsted’s syndrome (OS), 6, 17–18, 287–288, endolysosomal system, 460–462 302–303 expression, 463–464, 463f Osteoarthritis (OA) features, 470–471, 471f causes, 149–150 Fe toxicity, 477 companion dog model, 111–113, 112f, 113f identification, 472 TRPV3 channel, 197–198, 202 lysosomal exocytosis, 472 Overactive bladder (OAB) patients, 122 lysosomes, 474 macroautophagy deficits, 473–474 P membrane traffic model, 472 PAH. See Pulmonary arterial hypertension (PAH) MLIV model, 472–473 Pain transduction and amplification MTOR, 476 acetaminophen, 153 mutations, 459–460, 461f bacterial infection, 150–151 Niemann-Pick C1 phenotype, 474–475 biomarkers, 156–158 predicted and demonstrated role, 473–474, 473f chemotherapy-induced neuropathy, 149 Zn, 474, 475, 477 mechanical nociception and primary hyperalgesia, Myoclonic epilepsy, 436–437 146–147 INDEX 511

migraine, 151–153 Primary open-angle glaucoma, 16–17 osteoarthritis, 149–150 Progressive familial heart block type I (PFHBI), patient clinical trails, 156–158 22, 373, 374 PDN, 148–149 Prokinetic drugs peripheral traumatic neuropathy, 149 Dai Kenchu To, 391 postoperative pain, 150 peppermint oil, 392 secondary hyperalgesia, 147 TRPA1 agonists, 391 sleep deprivation-induced pain hypersensitivity, 148 Proline sulfonamides, 184–186 thermal and chemical sensation, 146 Pro-opiomelanocortin (POMC) neurons, 424 Pancreatic β cells Prostaglandins, 173–174 cell types, 344–345 Protein kinase C-γ (PKCγ), 424 electrical activity and oscillations, 344–345 Pruritus, 280, 282–283, 285 GLUT-2, 344–345 Psychiatric disorders T2DM, 345 anxiety disorders, 442 TRPA1, 351 bipolar disorder, 444 TRPCs, 345 depression, 442–443 TRPM2, 345–347 panic attacks, 442 TRPM3, 347 Rett syndrome, 443–444 TRPM4, 348 Schizophrenia, 443 TRPM5, 348–349 Psychosis maniaco-depressiva, 21 TRPM8, 349 PubMed, 501, 502f TRPV1, 349–350 Pulmonary arterial hypertension (PAH) TRPV2, 350 chronic hypoxia, 376–377 TRPV4, 350–351 epigenetic mechanisms, 376 Paradoxical heat sensation, 23 histological findings, 375–376 Parastremmic dysplasia (PD), 18–19 in mouse model, 327–329 Parkinson disease (PD), 438–439 NF-kB regulates cellular responses, 376 Parkinsonism dementia complex (PDC), 21 therapeutic strategy, 376–377, 377t Peak inflation pressure (PIP) ventilation, 224, 225f TRPC1 and TRPC6, 376 Pendred syndrome, 20 Pulmonary artery smooth muscle cells (PASMC), Periaqueductal gray matter (PAG), 120 375–377 Peripheral diabetic neuropathy (PDN), 148–149 Pulmonary edema, 229–231 Peripheral traumatic neuropathy, 149 Pfizer, 178–184, 214–215 Q 4α-Phorbol 12,13-didecanoate (4α-PDD), 206, 222, QX-314, 287 223f, 232–233 Phorbol esters, 206–207 R

Phospholipase A2 (PLA2), 221–222, 224, 249–250 Rat gastric epithelial cell lines (RGM-1), 37 Photoparoxysmal response (PPR), 75 Receptor-operated Ca2+ entry (ROCE), 367 Physiology Renal cell carcinoma (RCC), 269 brain, 5 Renovis cardiovascular function, 5 RN-1747, 210 gastrointestinal function, 5 RN-9893, 212 immune function, 5–6 Resiniferatoxin (RTX) reproduction and embryonic development, 6 cellular level selectivity, 101–104, 102f sensory physiology, 4–5 chemical structure, 206 urological function, 5 DRG neurons, 105–106, 106f PIP. See Peak inflation pressure (PIP) extracellular calcium concentration,

PLA2. See Phospholipase A2 (PLA2) 104–105 Polypeptide-containing PP cells, 344–345 intrathecal administration, 36–37, 38 Pontine micturition center (PMC), 120 local administration, 106–107 Positive and negative syndrome scale, 76 NDO patients, 121–123 Postherpetic neuralgia, 91–92 prolonged channel opening, 104–105 Postsynaptic density (PSD), 420–421 urinary bladder, 120 512 INDEX

Respiratory disorders TRPV1, 295–299 airway parasympathetic reflex responses, 484 TRPV2, 299–300 canonical TRP, 486–488 TRPV3, 300–303 identification, 485 TRPV4, 303–305 localization, 485, 494t TRPV5, 305 melastatin TRP, 492–493 TRPV6, 305 neurogenic inflammation, 484 Skin keratinocytes, 196 ozone and airborne particulate matter, 484 Small cell lung carcinoma (SCLC), 176 positive and negative feedback controls, 486 Somatostatin-producing δ cells, 344–345 specific pathophysiological roles, 485 Sphingomyelins (SMs), 23–24 vanilloid TRP, 488–492 Spinal nerve ligation (SNL) model, 132–133 Respiratory tracts Spinocerebellar ataxia type 14 (SCA14), 15 α, β-unsaturated oxime, 178, 183t S682P mutation, 20 airway pathophysiology, 164 Spondyloepimetaphyseal dysplasia Maroteaux pseudo- amplifies neuronal signals, 174–175 Morquio type 2 (SEDM-PM2), 18–19 capsaicin-sensitive sensory neurons, 167–168 Spondylometaphyseal dysplasia Kozlowski type, 18–19 chiral piperazine amides, 183t, 184 Staphylococcus aureus, 150–151, 287–288 cigarette smoke, 169–170 Steroid-resistant nephrotic syndrome (SRNS), 16–17 cyclopentenone prostaglandins, 173–174 Store-operated Ca2+ entry (SOCE), 367 decalin-based compounds, 183t, 184 Store-operated channels (SOC), 265–266 exogenous agonists, 168–169 Stroke, 272, 445–446 function of, 168 Structure-activity relationships (SARs), 206–207 hazardous air pollutants, 170–172 Substance P (SP), 428 heteroatom, 178, 179t isoprostane, 173–174 T neurogenic inflammation, 175 Tacrolimus, 295 nitro-containing analogs, 183t, 184 Therapeutic strategy development oxidative tissue damage, 172–173 agonists, 9–10 phenoxypyrrolidine amide, 178–184, 183t antagonists, 9 proline sulfonamides, 184–186, 184t cargos, 10 sensory innervation, 164, 165f gene therapy, 10 small cell lung carcinoma, 176 inhibiting compound, 377t thioureas, 186, 186t Thyrotropin-releasing hormone (TRH), 426 trichloro thiophenyl benzamides, 178, 183t Toluene diisocyanate (TDI), 171 tricyclic ureas, 186, 186t Transient receptor potential canonical 1 (TRPC1) in vivo preclinical pharmacology of, 176–177 in brain, 420–423 xanthine (caffeine)-based core, 177, 177f, 178f cancer, 411 Retinoids, 295 Darier’s disease, 310 Rett syndrome (RTT), 15, 443–444 hippocampal pyramidal neurons, 423 RN-1747, 210, 210f LTD, 423–424 RTX. See Resiniferatoxin (RTX) synaptic plasticity, 423–424 Ruthenium red, 210–211, 211f thermoregulation and food intake, 424 Transient receptor potential canonical 3 (TRPC3) S arterial vascular smooth muscle, 330 SARs. See Structure-activity relationships (SARs) cancer, 411 Scapuloperoneal spinal muscle atrophy (SPSMA), cerebellar purkinje cells, 424 19–20 GABA projection neurons, 425 Schizophrenia, 443 neuronal energy generator, 425 Secondary hyperalgesia, 147 Transient receptor potential canonical 4 (TRPC4) Sensorimotor axonopathies, 6 CCK, 425 Sjögren syndrome, 15 gene expression, 426 Skin disease hypothalamic kisspeptin-positive neurons, 426 TRPA1, 305–308 keratinocyte differentiation, 309 TRPC, 308–310 persistent neuronal activity, 425–426 TRPMs, 310–313 TRH, 426 INDEX 513

Transient receptor potential canonical 5 (TRPC5) CNS processes, 252–253 brain development, 427 digestive disease, 390 CaMKIIβ-dependent phosphorylation, 427 DRG and TG neurons, 240–241, 241t, 242 CCK, 425 G protein-coupled receptor signaling, 250–251, 252f gene expression, 426 IBD, 393 hypothalamic kisspeptin-positive neurons, 426 itch, 282–283 persistent neuronal activity, 425–426 kinases, 250 TRH, 426 lipids, 249–250 Transient receptor potential canonical 6 (TRPC6) melanoma, 312–313 cancer, 412 mouse-keratinocytes, 311 hyperforin, 427–428 pancreatic β cells, 349 keratinocyte differentiation, 309–310 in sensing cool/cold temperatures, 241–243 learning and exploratory behavior, 427 structure, 240 Transient receptor potential canonical 7 (TRPC7) thermal sensitivity, sensory neurons, 243–244 DAG-evoked calcium signaling, 310 thermoregulation, 246 Darier’s disease, 309 wound healing, 311 in vitro and ex vivo, 309–310 Transient receptor potential melastatin (TRPM) Transient receptor potential canonical (TRPC) channelopathies channelopathies TRPM1, 20–21 TRPC1, 14 TRPM2, 21 TRPC3, 15 TRPM3, 21–22 TRPC4, 15 TRPM4, 22 TRPC6, 15, 16–17 TRPM5, 22 Transient receptor potential melastatin 1 (TRPM1) TRPM6, 22–23 Appaloosa horse, 312 TRPM7, 21 cancer, 407 TRPM8, 23 MiR-211, 312 TRPM2 and TRPM7, 21 prognostic marker, melanoma, 311–312 Transient receptor potential mucolipin (TRPML) Transient receptor potential melastatin 2 (TRPM2) channels cancer, 407 channelopathy, 23–24 IBD, 393 gene polymorphism, 78–79 melanoma, 312 intracellular endosomes and lysosomes, 5, 78–79 pancreatic β cells, 345–347 melanoma, 311–313 in T cells, 311 subunits, 264–265, 264f Transient receptor potential melastatin 3 (TRPM3), 347 Transient receptor potential mucolipin 3 (TRPML3) Transient receptor potential melastatin 4 (TRPM4) channels cancer, 408 melanocytes, 313 cardiovascular disease, 373–374 melanoma, 313 in immune cells, 311 mutations, 6, 24 pancreatic β cells, 348 skin disease, 313 vascular endothelial cells, 326 Transient receptor potential vanilloid 1 (TRPV1) Transient receptor potential melastatin 5 (TRPM5) acute inflammatory conditions, 298 cancer, 408 agonists, 501 pancreatic β cells, 348–349 allergy, 298 Transient receptor potential melastatin 7 (TRPM7) amygdala, 428–429 cancer, 408 anandamide, 429 cardiovascular disease, 374–375 antagonists, 501 in immune cells, 311 antiproliferative effects of, 298–299 melanoma, 312 apocrine chromhidrosis, 296 Transient receptor potential melastatin 8 (TRPM8) appetite control, 431 agonists, 248–249, 248f cancer, 42, 410 analgesia, 244–246 capsaicin, 295 antagonists, 246–247, 247f capsaicin evokes responses, 428 cancer, 408–410 cardiovascular system-related diseases, 42–44 chronic pain states, 244 central nervous system-related diseases, 39–41 514 INDEX

Transient receptor potential vanilloid 1 (TRPV1) atopic dermatitis, 303 (Continued) camphor, 196–197 chronic inflammatory skin diseases, 298 cancer, 410–411 chronic pain relief (see Chronic pain) carvacrol and eugenol, 197 diabetic nephropathy, 354–355 cosmetic products, 301 digestive disease, 389–390 digestive disease, 390 DRG neurons, 240–241, 241t Gly573Ser, 300 endogenous compounds, 429 hair growth, 301–302 epidermal barrier formation, 299 itch, 300 epilepsy, 436–437 keratinocytes, 300 fear, 429 menthol, 196–197 febrile seizures, 436–437 neuronal and nonneuronal expression, 196 formaldehyde, 297 neuropathic pain, 200 gastrointestinal system-related disease, 37–38 nitric oxide (NO), 197 gene polymorphism, 79–81 Olmsted syndrome, 302–303 goal-directed behavior, 431 osteoarthritic pain, 200–201, 201f hair follicles, 298–299 pharma efforts, 202 histamine-induced itch, 295 protein kinase C (PKC), 197 IBD, 392 sensitizers, 300 immune cells, 296 skin barrier function, 301 immune cells express, 45 skin disease, 198–199, 199f itch, 282–283, 284–286, 284f skin homeostasis, 301 keratinocytes, 298–299 temperature sensor, 300 marble-burying behavior, 429 thermosensation and nociceptive signaling mediating pruritic stimuli, 295 pathway, 198 melanocytes and fibroblasts, 296 in vitro, 302 metabolic diseases, 44–45 in vivo, 302 mRNA and protein, 428 wound healing, 301 N-Arachidonoyl-serotonin (AA-5-HT), 429 Transient receptor potential vanilloid 4 (TRPV4), neurogenic inflammation, 297 205–206 pain, 36–37 in astrocytes, 432 pancreatic β cells, 349–350 cardiogenic and noncardiogenic pulmonary periaqueductal gray matter, 428–429 edema, 501–502 primary sensory neurons, 351–352 COPD, 501–502 pruritus, 502 IBD, 392 respiratory system-related diseases, 41 inflammation, 304 reward-dependent learning, 431 in neurons, 432 rosacea, 295 NMS cancer, 305 sensitive skin, 295 pancreatic β cells, 350–351 synaptic plasticity, 430–431 single nucleotide polymorphisms, 501–502 thermosensation and thermal hyperalgesia, 295 skeletal dysplasias and neuropathies, 502–503 Trpv1 (-/-) animals, 428 skin barrier, 304–305 type 1 diabetes mellitus, 352 temperature sensor, 303–304 type 2 diabetes mellitus, 352 vascular endothelial cells, 326 urinary system-related diseases, 38–39 Transient receptor potential vanilloid 5 in vitro study, 298 (TRPV5), 305 in vivo study, 297, 298 Transient receptor potential vanilloid 6 Transient receptor potential vanilloid 2 (TRPV2) (TRPV6), 305 cancer, 410 Transient receptor potential vanilloid 4 (TRPV4) digestive disease, 390 agonists pancreatic β cells, 350 4α-PDD and phorbol esters, 206–207 skin diseases, 299–300 endogenous, 207–208 Transient receptor potential vanilloid 3 (TRPV3) GSK1016790A, 208–210 antagonists, 202 natural products, 210 arachidonic acid, 197–198 renovis, RN-1747, 210 INDEX 515

Transient receptor potential vanilloid 4 (TRPV4) antagonists U GSK, 212–214 Unsaturated aldehydes, 169–170 hydra biosciences, HC-067047, 211–212 Urinary bladder Pfizer, 214–215 capsaicin, 120 renovis, RN-1734 and RN-9893, 212 resiniferatoxin (RTX), 120 ruthenium red, 210–211 vanilloids, 120–121 vanilloid and terpene, 215, 215f Transient receptor potential vanilloid (TRPV) V channelopathies Vanilloid TRP (TRPV) TRPV1, 17 botulinum toxin type-A, 125 TRPV2, 17 BPS/IC, 123–124 TRPV3, 17–18 in brain, 421f, 428 TRPV4, 18–20 cancer, 410–411 TRPV5, 20 capsaicin instillation, 124 TRPV6, 20 Cl− secretion, 490–491 Transient receptor potential vanilloid type 1 (TRPV1) endogenous ligands, 489 antagonist formaldehyde-induced model, 489 A-1106625, 132f, 134 functionally expression, 489 A-1165442, 132f, 134 gene polymorphisms, 489 ABT-102, 135f, 136–137 heterogeneous groups, 488–489 alopecia, 298–299 IDO, 122 AMG-517, 135f, 136 micturition, 120 AMG-8562, 131–132, 132f NDO, 121–122 AS-1928370, 132–133, 132f neuronal, nociceptive neurons, 488–489 AZD-1386, 135f, 138–139 OAB patients, 122–123 barrier recovery, 299 PI3K and PKC signaling pathways, 490 capsaicin and capsazepine, 130–131 polymodal sensor, 488–489 chemotype-independent hyperthermia, 131 sulfur dioxide exposure, 489–490 Compound 6, 132f, 133–134 tiotropium, 490 Compound 10, 132f, 133–134 TRPV1, 428–431 Compound 41, 132f, 133 TRPV2, 431 compounds 12 and 15, 132f, 133 TRPV3, 431 DWP05195, 135f, 139 TRPV4, 215, 215f, 431–432, 491–492 GRC-6211, 135f, 139 Vascular endothelial growth factor (VEGF), 267–268 hirsutism, 298–299 Vascular smooth muscle cells (VSMCs) hypothermia-inducing properties, 131 cerebral blood flow, 266 MK-2295, 135f, 137–138 myogenic tone, 266–267 PHE377, 139 proliferation, 266 SB-705498, 134–136, 135f voltage-independent calcium channels, 265–266 skin cancer, 299 Vasodilatation, 268 thermoregulatory effects, 131 Ventilator-induced lung injury (VILI), 223–227 TS-653, 135f, 139 VILI. See Ventilator-induced lung injury (VILI) Traumatic brain injury (TBI), 446–447 Volatile gas anesthetics, 171–172 Trichloro thiophenyl benzamides, 178, 183t Von Hippel Lindau tumor suppressor gene, 14 Trigeminal gangilia (TG), 240–241 Triptans, 153 W TRP channel regulation, 2–3, 3f Williams-Beuren syndrome, 15 Tryptamine, 246–247 Type 2C Charcot-Marie-Tooth disease, 6 X Type 1 diabetes mellitus (T1DM), 352, 356 Xanthine-based TRPA1 blocker, 177, 177f Type 2 diabetes mellitus (T2DM) Xenobiotic sensor, 167–168 drug target, 356 pancreatic β cells, 345 Z TRPC1 gene, 74 Zucker diabetic fatty (ZDF) rats, 334 TRPV1, 352