Volume 59 · Supplement 1 · 2009

Volume 59 · Supplement 1 · 2009

The XXXVI International Congress of Volume 59 · Supplement 1 · 2009 · pp 1–XX · pp 1 · 2009 Volume 59 · Supplement Physiological Sciences (IUPS2009) International Scientific Program Committees (ISPC) ISPC Chair Yoshihisa Kurachi Vice Chair Ole Petersen ISPC from IUPS Council Akimichi Kaneko (IUPS President) Irene Schulz (IUPS Vice President) Pierre Magistretti (IUPS Vice President) Malcolm Gordon (IUPS Treasurer)

ISPC IUPS2009 Members and Associated Members Proceedings of the XXXVI International Congress of Physiological Sciences (IUPS2009) Commission I Locomotion Commission VII Comparative Physiology: Hans Hoppeler, Masato Konishi, Hiroshi Nose Evolution, Adaptation & Environment Function of Life: Elements and Integration Commission II Circulation/Respiration Malcolm Gordon, Ken-ichi Honma, July 27–August 1, 2009, Kyoto, Japan Yung Earm, Makoto Suematsu, Itsuo Kodama Kazuyuki Kanosue Commission III Endocrine, Reproduction & Commission VIII Genomics & Biodiversity Development David Cook, Hideyuki Okano, Gozoh Tsujimoto Caroline McMillen, Yasuo Sakuma, Toshihiko Yada Commission IX Others Commission IV Neurobiology Ann Sefton, Peter Hunter, Osamu Matsuo, Quentin Pittman, Harunori Ohmori, Fumihiko Kajiya, Tadashi Isa, Tadaharu Tsumoto, Megumu Yoshimura Jun Tanji Commission V Secretion & Absorption Local Executives Irene Schulz, Miyako Takaki, Yoshikatsu Kanai Yasuo Mori, Ryuji Inoue Commission VI Molecular & Cellular Biology Cecilia Hidalgo, Yoshihiro Kubo, Katsuhiko Mikoshiba, Masahiro Sokabe, Yukiko Gotoh Proceedings of the XXXVI International Congress of Physiological Sciences (IUPS2009) Function of Life: Elements and Integration

July 27–August 1, 2009, Kyoto, Japan

Hosted by the Physiological Society of Japan (PSJ) and the Science Council of Japan

Organizing Committee

Honorary Presidents: The Late Setsuro Ebashi Masao Ito Members: Toshinori Hongo Advisory Board: Ken-ichi Honma Akimichi Kaneko Katsumasa Kawahara President: Yoshio Maruyama Yasushi Miyashita Osamu Matsuo Katsuhiko Mikoshiba Vice Presidents: Harunori Ohmori Yoshihisa Kurachi (ISPC Chair) Hideyuki Okano Satoshi Kurihara (Treasurer) Taketoshi Ono Yasunobu Okada (Secretary) Tadaharu Tsumoto

Proceedings of the 86th Annual Meeting of the Physiological Society of Japan

President: Yoshinori Marunaka

IUPS2009  Pledged support for IUPS2009:

In addition to The Physiological Society of Japan, the following sources provide support for IUPS2009. Assembly for Myocardial Physiology Biophysical Society of Japan The Center for Advanced Medical Engineering and Infomatics, Osaka University Genomic Drug Discovery Forum Global COE Program "An in silico -oriented worldwide open platform, Osaka University" Global COE Program "Education and Research Center for Stem Cell Medicine, " Global COE Program "Formation of a strategic base for biodiversity and evolutionary research: from genome to ecosystem, Kyoto University" Grant-in-Aid for Creative Scientific Research, "Function and regulatory system of water transporting aquaporin channels" Grant-in-Aid for Scientific Research on Priority Area, "Cell signaling" Grant-in-Aid for Scientific Research on Priority Area, "Cellular sensor" Grant-in-Aid for Scientific Research on Priority Area, "G-protein signal" Grant-in-Aid for Scientific Research on Priority Area, "Mechanisms of sex differentiation" Grant-in-Aid for Scientific Research on Priority Area, "Transportsome on biomembrane systems: its molecular assembly and physiological function" ICORP/SORST Cell Mechanosensing Project International Academy of Cardiovascular Sciences, Japan Section International Cardiothoracic & Vascular Anesthesia Academy International Conference on Control and Disease of Sodium Dependent Transportation Proteins and Ion Channels (ICSDT) Japan Neuroendocrine Society The Japan Neuroscience Society Japan Society for Adaptation Medicine Japan Society for Cell Biology Japan Society of Pain Clinicians Japan Society of Research The Japanese Association of Anatomists Japanese Association for Study of Pain The Japanese Circulation Society Japanese College of Angiology The Japanese Pharmacological Society Japanese Society for Chronobiology Japanese Society for Behavioral Neuroendocrinology Japanese Society for Microcirculation Japanese Society of Aerospace and Environmental Medicine Japanese Society of Biometeorology Japanese Society of Clinical Neurophysiology Japanese Society of Clinical Physiology The Japanese Society of Electrocardiology The Japanese Society of Lymphology Japanese Society of Nephrology Japanese Society of Pathophysiology Japanese Society of Physical Fitness & Sports Medicine The Japanese Society of Sleep Research The Journal of Physiology The Pharmaceutical Society of Japan Credits

The societies listed below will provide medical credentials to their members upon their participation in IUPS2009. Japan Society of Obstetrics and Gynecology Japan Society of Pain Clinicians Japan Society of Ryodoraku Medicine The Japanese Circulation Society Japanese Society of Anesthesiologists The Japanese Society of Child Neurology Japanese Society of Clinical Neurophysiology Japanese Society of Nephrology Japanese Society of Neurology

IUPS2009  Wallace Fenn Lecture BIOPHYSICAL DISSECTION OF NEUROTRANSMITTER RELEASE Erwin Neher Named Lectures Department of Membrane Biophysics, Max Planck Institute for Biophysical Chemistry, Germany The ‘Calyx of Held', a glutamatergic presynaptic terminal in the auditory pathway is large enough that quantitative biophysical techniques, such as voltage clamp, Ca++ fluorimetry and Ca++ uncaging can be applied. Using these experimental tools, we have studied the role of Ca++ and other second messengers in neurotransmitter release and short-term synaptic plasticity (see E. Neher and T. Sakaba, 2008, Neuron 59, 861 for review). We found that the ++ concentration of free calcium ([Ca ]i) has at least three roles i) it triggers neurotransmitter release in a highly cooperative fashion ii) it accelerates linearly the recruitment of new releasable synaptic vesicles during ongoing stimulation and iii) it mediates short-term facilitation. Recruitment of vesicles is rate-limiting for neurotransmitter release during sustained high-frequency activity. Therefore, the acceleration of this process by Ca++ has a very important modulatory role. Surprisingly we found that this process is not limited by the availability of vesicles or the assembly of a molecular release machinery. Rather, new vesicles rapidly enter a state in which they can be released by caged Ca++. Another slower step is required to bring them near Ca++ channels, such that they may also be released by short action potentials.

IUPS President's Lecture PROTON-MEDIATED FEEDBACK FROM HORIZONTAL CELLS TO CONES IN THE RETINA: A MECHANISM OF RECEPTIVE FIELD SURROUND FORMATION Akimichi Kaneko School of Health Science, Kio University, Japan Feedback from horizontal cells (HCs) to cone photoreceptors plays a key role in the center-surround receptive field organization of retinal cells. In cone photoreceptors of the newt retinal slices the voltage-dependent calcium current (ICa) was enhanced by surround illumination. Enrichment of the pH buffering capacity of the extracellular solution increased cone ICa, and blocked any additional increase by surround illumination. Hyperpolarization of HCs by CNQX augmented cone ICa, whereas depolarization of HCs by kainate suppressed cone ICa. From these results, we propose that pH changes in the synaptic clefts, caused by the membrane voltage change of HCs, mediate the feedback from HCs to cone photoreceptors. When HCs, isolated from the goldfish retina, were depolarized by kainate or high-K+ medium, the pH of the immediate external surface (pHs) of HCs was lowered. The pH change was suppressed by 400 nM bafilomycin A1, an inhibitor of vacuolar type +H pump (V-ATPase). The V-ATPase was immunocytochemically identified on the plasma membrane of dissociated HCs. These results confirm that protons are released from HCs and caused the pH change around the cone terminals. [Supported by JSPS Grants.]

IUPS 2009 July 27 - August 1, 2009 in Kyoto  Named Lectures T. P. Feng Lecture THE COMPUTATIONAL LOGIC OF NETWORKS IN MOTION, FROM ION CHANNELS TO BEHAVIOUR Sten E Grillner Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden In all vertebrates from primates including man to fish and lamprey, the basic neuronal organization controlling patterns of motor behaviour such as locomotion, breathing, and posture are organized in a similar or identical way. These networks form a motor infrastructure used to initiate all the movements that each species can perform. These networks in turn can be selected through subpopulations of cells in the basal ganglia, which play an important role for the selection of motor programs. The networks generating the locomotor movements are located in the spinal cord. They are responsible for the sequential activation of the different limb muscle in each locomotor cycle. The activity level is controlled by locomotor command centres in the brainstem, conserved throughout the vertebrate phylum. To elucidate the intrinsic function of these pattern generating networks and command systems we have developed the lamprey nervous system as an experimental model. We now understand the cellular bases of the brainstem spinal cord networks generating goal-directed locomotion and control of body orientation. The detailed synaptic interaction has been identified, as well as the network contribution of subtypes of ion channels. A major tool has been biologically realistic large scale computing.

Robert Pitts Lecture A TR(I)P THROUGH THE WORLD OF EPITHELIAL CALCIUM AND MAGNESIUM CHANNELS René Bindels Department of Physiology, Radboud University of Nijmegen Medical Centre, The Netherlands Ca2+ and Mg2+ are of great physiological importance in their function in neural excitability, muscle contraction, blood coagulation, bone formation and hormone secretion. These divalents are maintained within a narrow range by the small intestine and kidney which both increase their fractional (re)absorption under conditions of deprivation. Rapid progress has recently been made in identification and characterization of the Ca2+ and Mg2+ transport proteins contributing to the delicate balance of divalent cations. Expression cloning approaches in combination with knockout mice models and genetic studies in families with a disturbed Mg2+ balance revealed novel gatekeeper proteins including those belonging to the super family of the transient receptor potential (TRP) channels. These epithelial Ca2+ (TRPV5 and TRPV6) and Mg2+ channels (TRPM6 and TRPM7) form prime targets for hormonal control of the active Ca2+ and Mg2+ flux from the urine space or intestinal lumen to the blood compartment. The characteristics of the newly identified transporters will be discussed and in particular the distinctive regulation of these new epithelial Ca2+ and Mg2+ channels in (patho)physiological situations will be highlighted.

 IUPS 2009 July 27 - August 1, 2009 in Kyoto Ernst Knobil Lecture SEX, STRESS AND THE BRAIN: HORMONE ACTIONS ABOVE THE HYPOTHALAMUS VIA NOVEL MECHANISMS Named Lectures Bruce S McEwen Laboratory of Neuroendocrinology, Rockefeller University, USA Neuroendocrine studies have revealed that the adult brain is much more resilient and adaptable than previously believed, and that adaptive structural plasticity involves growth and shrinkage of dendritic trees, turnover of synapses and limited amounts of neurogenesis in the forebrain. Stress and sex hormones mediate adaptive structural plasticity in hippocampus and in prefrontal cortex, amygdala and nigrostriatal and mesolimbic systems, all brain regions that are involved in cognitive and emotional functions. Stress and sex hormones exert their effects on brain structural remodeling through both classical genomic, as well as non-genomic mechanisms and they do so in collaboration with neurotransmitters and other intra- and extracellular mediators. This will be illustrated for estrogen actions on synapse formation and for stress-induced remodelling of dendrites and synapses. Because hormones influence so many aspects of brain structure and function and because hormone secretion is governed by the cognitive and emotional brain, the role of brain plasticity must be considered in understanding how the social environment “gets under the skin” to affect brain and body resilience and vulnerability to disease. Supported by NIH Grants NS07080, MH41256, 5P01 AG16765, 5P50MH58911.

Knut Schmidt-Nielsen Lecture OVERWINTERING STRATEGIES OF ARCTIC ANIMALS, OR TALES OF WHERE KNUT SCHMIDT-NIELSEN DIDN'T GO Brian M Barnes Institute of Arctic Biology, University of Alaska Fairbanks, USA Arctic animals that remain resident in winter either remain active by engaging with environment on a daily basis or they hibernate. Hibernating animals face different challenges in whether they must cope with above or below freezing body temperatures. This talk will contrast these strategies using examples of overwintering behavior and physiological mechanisms of freeze tolerance in amphibians and insects and of freeze resistance and supercooling in insects and mammals. Discussion will emphasize patterns and mechanisms of hibernation in arctic ground squirrels and black bears, with detail on thermoregulation and energetics and the functions of circadian and circannual rhythms in free-living and captive populations. We have also begun genomic and proteomic studies of the molecular basis of hibernation in ground squirrels and bears, with an intent towards discovering new therapies for ischemia and trauma in humans. When possible these topics will be discussed in reference to the life and discoveries of Professor Knut Schmidt-Nielsen, a leader in comparative and environmental physiology and past president of the IUPS.

IUPS 2009 July 27 - August 1, 2009 in Kyoto  Named Lectures August Krogh Lecture THE EVOLUTION OF HIGH BLOOD PRESSURE Tobias Wang Department of Zoophysiology, University of Aarhus, Denmark The evolution of the cardiovascular system can be viewed as a progression from a simple tube formed heart generating low pressures and flows to the double and completely divided heart of endothermic vertebrates that allows for a high cardiac output as well as a high systemic blood pressure while the lungs are protected by low blood pressures. The development of the fully divided heart seems to be correlated with the evolution of endothermy which required an efficient delivery of adequate amounts of oxygen, but the functional role of the undivided hearts of amphibians and reptiles remain largely unknown. I will discuss the functional anatomy of the undivided heart in amphibians and reptiles with emphasis on the functional consequences and using the giraffe as an example, I will discuss how very high blood pressures may be tolerated in mammals.

 IUPS 2009 July 27 - August 1, 2009 in Kyoto SL-1 PRINCIPLES OF SYSTEMS BIOLOGY AND THE FUTURE OF PHYSIOLOGY Denis Noble Department of Physiology, Anatomy & Genetics, University of Oxford, UK Physiology, the etymology of which is the study of the logic of life, is necessarily a form of Systems Biology. The new vogue for the systems approach is therefore a major opportunity for the physiological sciences. I will show work in progress on the IUPS Physiome Project (Bassingthwaighte, Hunter & Noble, Experimental Physiology,

2008, DOI: 10.1113/expphysiol.2008.044099) to illustrate the principles of Systems Biology from a physiological Special Lectures perspective (Noble, Experimental Physiology, 2008, 93, 16-26. Some of these principles have major implications for the nature of biology in general (Noble, The Music of Life, 2006, OUP). The lecture will outline some of these implications and indicate the ways in which physiology can re-connect with developmental and evolutionary biology.

SL-2 SPIKE TIMING-DEPENDENT PLASTICITY OF NEURAL CIRCUITS : HEBB POSTULATE REVISITED Mu-Ming Poo Molecular and Cell Biology, University of California, Berkeley, USA The year 2009 marks the 60th anniversary of the publication of “The Organization of Behavior”, a monumental treatise in which Donald Hebb proposed his physiological hypothesis of learning. i.e., correlated activity among an assembly of neurons could strengthen their synaptic connections, and this process represents the first step in perceptual learning in the brain. In this lecture, I will summarize our efforts in revisiting Hebb’s postulate by quantitative studies on the effects of correlated activities in modifying synaptic connections in various neural circuits. I will show that correlated spiking of pre- and postsynaptic neurons can result in synaptic strengthening or weakening, depending on the temporal order and the interval between the pre- and postsynaptic spiking, and these synaptic modifications are accompanied by persistent changes in the intrinsic excitability of the presynaptic neuron and postsynaptic dendrites. I will also demonstrate that such spike timing-dependent plasticity (STDP) may contribute to activity-dependent development and plasticity of retinotectal circuits and serve for perceptual memory of visual experience. These studies illustrate how Hebb’s postulate had inspired a fruitful line of studies on activity- induced neural circuit modifications.

IUPS 2009 July 27 - August 1, 2009 in Kyoto  SL-3 INHERITED DISORDERS OF NEUTRAL AMINO ACID TRANSPORT Stefan Bröer

Special Lectures School of Biochemistry & Molecular Biology, Australian National University, Australia Disorders of amino acid transport have been very influential in the elucidation of amino acid absorption in the kidney and intestine. Molecular identification of the transport systems mutated in these disorders highlights the complexity of amino acid transport and demonstrates its links to more complex pathologies such as blood pressure regulation and epithelial cell differentiation. Hartnup disorder is an autosomal recessive disorder. It is caused by mutations in the general neutral amino acid transporter SLC6A19. The transporter requires one of the two auxiliary proteins collectrin or ACE2 for surface expression. Collectrin is predominantly found in the kidney, whereas ACE2 is found predominantly in the intestine. Iminoglycinuria (IG) was first described fifty years ago as an autosomal recessive abnormality of renal transport of glycine and of the imino acids, proline and hydroxyproline. The disorder is caused by mutations in the common proline and glycine transporter PAT2 (SLC36A2). Hyperglycinuria (HG) is frequently observed in heterozygotes. In some individuals SLC36A2 mutants retained residual transport activity; in those cases the urinary phenotypes were modified by additional mutations in the proline transporter IMINO (SLC6A20).

SL-4 ATP-SENSITIVE POTASSIUM CHANNELS AND DIABETES: FROM MOLECULE TO MALADY Frances M Ashcroft Department of Physiology Anatomy and Genetics, University of Oxford, UK

ATP-sensitive potassium (KATP) channels act as key regulators of insulin secretion from pancreatic beta-cells by coupling glucose to the electrical activity of the plasma membrane. When these channels are open, the beta is hyperpolarised, inhibiting insulin release. KATP channel closure in response to metabolically generated ATP depolarizes the beta-cell, opening voltage-gated Ca2+ channels. The resulting Ca2+ influx triggers insulin release. The beta-cell KATP channel consists of pore-forming Kir6.2 (KCNJ11) and regulatory sulphonylurea receptor (SUR1; ABCC8) subunits. Gain-of-function mutations in either of these genes cause neonatal diabetes. Some mutations produce a severe clinical phenotype, characterized by developmental delay, epilepsy, muscle weakness and neonatal diabetes (DEND syndrome). In many patients, sulphonylureas can successfully be used to treat their diabetes, and in some individuals the neurological symptoms can also be alleviated. This lecture will discuss how mutations causing human disease alter KATP channel function, how alterations in KATP channel activity cause diabetes in man and mouse, novel mouse models of neonatal diabetes, and why some mutations are susceptible to sulphonylurea therapy and others are not.

 IUPS 2009 July 27 - August 1, 2009 in Kyoto SL-5 NEW FLUORESCENT PROBES AND NEW PERSPECTIVES IN BIOSCIENCE Atsushi Miyawaki Brain Science Institute / ERATO Miyawaki Life Function Dynamics, RIKEN / JST, Japan Many molecules involved in signal transduction have been identified, and the hierarchy among those molecules has also been elucidated. It is not uncommon to see a signal transduction diagram in which arrows are used to link molecules to show enzyme reactions and intermolecular interactions. To obtain a further understanding of a signal transduction system, however, the diagram must contain the three axes in space as well as a fourth dimension, time, Special Lectures because all events are controlled ingeniously in space and time. Since the isolation of GFP from the bioluminescent jellyfish in 1992 and later with its relatives, researchers have been awaiting the development of a tool, which enables the direct visualization of biological functions. This has been increasingly enhanced by the marriage of GFP with fluorescence resonance energy transfer, fluorescence cross-correlation spectroscopy, and is further expanded upon by the need for "post-genomic analyses." It is not my intent to discourage the trend seeking the visualization of biological function. I would like to propose that it is time to evaluate the true asset of "bio-imaging" or "in vivo imaging" for its potential and limitations in order to utilize and truly benefit from this novel technique.

SL-6 THE MACROMOLECULAR COMPLEX OF CALCIUM RELEASE UNITS IN CARDIAC AND SKELETAL MUSCLE: STRUCTURE FUNCTION CORRELATIONS Clara Franzini-Armstrong Department of Cell and Developmental Biology, University of Pennsylvania, USA Activation of contraction in muscle cells of all types requires a rapid surge in cytoplasmic calcium concentration. Most of the calcium is released from internal stores, the sarcoplasmic reticulum (SR) specifically at calcium release units (CRUs), or sites where a specialized domain of the SR, the junctional. jSR forms contacts with transverse (T) tubules and/or plasmalemma. SR calcium release channels, the ryanodine receptors (RyRs) are located at the sites of SR/T tubule interaction and are part of a large macromolecular complex that functionally and structurally connects T tubules and SR proteins. Four components of this complex can be visualized by electron microscopy. On the T tubule side are L type voltage sensitive calcium channels (CaV1.1 and CaV1.2 for skeletal and cardiac muscles). On the jSR side are the calcium buffering protein calsequestrin and two jSR membrane proteins (junctin and triadin) that act as links between calsequestrin and RyR. The contribution of these components of the protein complex to the function, maintenance and architecture of CRUs has been elucidated in experiments that take advantage of intrinsic differences in expression and function of protein isoforms between skeletal and cardiac muscle.

IUPS 2009 July 27 - August 1, 2009 in Kyoto  SL-7 GHRELIN: DISCOVERY AND PHYSIOLOGICAL SIGNIFICANCE Kenji Kangawa

Special Lectures Department of Biochemistry, National Cardiovascular Center Research Institute, Japan A complex network of cell-cell communication system by peptide hormones works for maintaining the mammalian homeostatic balance. To further clarify the intricate mechanisms of the regulation, it is important to discover unidentified bioactive peptides. By using our own methods, we discovered novel bioactive peptides such as neuromedins, three natriuretic peptides (ANP, BNP, CNP), and adrenomedullin. Moreover in 1999, we discovered ghrelin from stomach as an endogenous ligand for the growth hormone (GH) secretagogue receptor (GHS-R), an orphan G-protein coupled receptor. Ghrelin is a 28-amino acid peptide with a marvelous structure modified by fatty acid which is essential for its activity. Ghrelin is primarily produced in distinct endocrine cells, X/A-like cells, in the stomach. Beside the stimulatory effect of GH release, ghrelin is also involved in the stimulation of feeding and the regulation of energy metabolism. In fact, ghrelin stimulates feeding when administered centrally and peripherally, and its secretion is regulated under conditions of energy balance. In our studies, intravenous injection of ghrelin in heart failure patients showed beneficial effects via GH release and hemodynamics. Thus, ghrelin also has multifaceted roles in the cardiovascular and metabolic systems.

SL-8 GENETIC ANALYSIS OF CIRCADIAN CLOCKS IN MAMMALS Joseph S Takahashi Department of Neuroscience, HHMI, University of Texas Southwestern Medical Center, USA The molecular mechanism of circadian clocks has been uncovered by the use of forward genetic analysis in a number of model systems. Circadian oscillations are generated by a set of genes forming a transcriptional autoregulatory feedback loop involving: Clock, Bmal1, Per1, Per2, Cry1, Cry2 and Csnk1e. As part of a large-scale ENU mutagenesis screen, we have identified new circadian mutants in mice. These include new alleles of known circadian genes as well as novel circadian loci such Fbxl3 which interacts selectively with the CRY proteins to target them for degradation through the proteasome pathway. The discovery of ‘clock genes' also led to the realization that the capacity for circadian gene expression is widespread throughout the body. Using circadian gene reporter methods, one can demonstrate that most peripheral organs and tissues can express circadian oscillations in isolation. We have used tissue-specific, conditional gene expression methods to analyze the relative contributions of central and peripheral circadian oscillators to circadian organization. The cellular autonomy of circadian clocks has raised a number of questions concerning synchronization and coherence of rhythms at the cellular level as well as circadian organization at the systems level.

10 IUPS 2009 July 27 - August 1, 2009 in Kyoto SL-9 THE NEURAL NETWORK: INTEGRATION AND REGULATION OF SYNAPTIC TRANSMISSION Shigetada Nakanishi Osaka Bioscience Institute, Japan The cerebellar circuitry consists of two pathways: the pons - mossy fiber - granule cell - parallel fiber and the inferior olive - climbing fiber. The inputs of these two excitatory pathways converge on Purkinje cells and deep cerebellar nuclei. Golgi cells also participate in suppressing granule cell excitability in a feedback manner. We Special Lectures established a novel technique (reversible neurotransmission blocking), in which granule cell transmission to Purkinje cells was selectively and reversibly blocked. We revealed that Purkinje cells are essential for expression of eyeblink conditioning, but this memory is acquired and stored in deep cerebellar nuclei. We also developed a different technique (immunotoxin-mediated cell targeting), in which Golgi cells were selectively ablated from the cerebellar network. This study disclosed that the cooperative regulation by glutamate receptors and GABA receptors is indispensable for motor coordination and adaptation. The collaborative function by ionotropic and metabotropic glutamate receptors is also crucial in spatiotemporal regulation at granule cell - Golgi cell synapses. Neural information is thus hierarchically integrated and regulated at different levels of the cerebellar circuitry.

SL-10 STRUCTURAL PHYSIOLOGY OF MULTIFUNCTIONAL CHANNELS Yoshinori Fujiyoshi Graduate School of Science, Kyoto University, Japan Water channel, aquaporin-1 can permeate more than 300 million water molecules in a second without permeation of any ions and even proton. Structure analysis of the channel by electron crystallography revealed peculiar structural determinants as well as water selective mechanism [Nature 407 (2000) 599-605]. By analyzing structure of another water channel, aquaporin-0 at a resolution of 1.9Å, we discriminated water molecules in the channel [Nature 438 (2005) 633-638]. Aquaporin-4 is the predominant water channel in brain. Structure analyses showed weak but specific interactions suggesting a structural role for the water channel in adhesion of membrane layers. We named this type channel as "Adhennel" family [J. Mol. Biol. 355 (2006) 628-639]. Structure of an Adhennel, Gap junction channel, connexin-26 was also analyzed and we proposed a plug gating model [PNAS 104 10034-10039 (2007)]. By focusing on multifunctional channels, I would like to introduce recent results in structural biology of membrane proteins by utilizing our cryo-electron microscope with helium cooled specimen stage.

IUPS 2009 July 27 - August 1, 2009 in Kyoto 11 SL-11 ENHANCING MAMMALIAN REGENERATION Nadia Rosenthal

Special Lectures EMBL-Monterotondo, European Molecular Biology Laboratory, Italy The adult mammalian body does retain the robust repair capacity of other species and gradually loses its regenerative potential with age. Our approach has been to intervene in the mechanisms at work in the mammalian response to damage or disease by reducing the impediments to effective regeneration of skeletal and cardiac muscle. In one set of interventions, transgenic supplementation of a locally acting Insulin-like Growth Factor 1 isoforms or transient Notch activation both promote efficient tissue repair of damaged muscle without scar formation. A second set of interventions has been aimed at modulation of the inflammatory response to tissue damage. Genetic repression of the NFκB inflammatory pathway in injured skeletal or heart muscle produced dramatically different outcomes, highlighting the complexity of downstream responses in different tissues. The importance of inflammation in regeneration was also explored in a mouse model of impaired macrophage polarization where tissue repair was severely compromised. Taken together, these studies support the feasibility of modulating key pathways and immune cell activities to restore injured or degenerating mammalian tissues, providing new targets for clinical intervention and prospects for combination therapies.

SL-12 WHY REPLACE NEURONS IN ADULT BRAIN? Fernando Nottebohm The Rockefeller University, USA New neurons continue to be added to the forebrain of adult songbirds. I will describe three examples of this phenomenon, in canaries, zebra finches and black-capped chickadees, using data drawn from captive and free- ranging individuals. The observations to be presented suggest that neuronal replacement is associated with a space-limited memory system and the need to acquire, store and replace information. My laboratory is now ready to test these hypotheses using transgenic songbirds, which we have just started to produce. We use laser capture microdissection to harvest cells and their RNA. Comparisons of gene expression before and after learning identify candidate genes of particular interest. Expression of these genes can then be manipulated in transgenics, which we have just succeeded in producing. Since we know when a behavior such as song is learned and we can accurately quantify what is learned and the cells involved, we are now ready to test the hypothesis that neuronal replacement is driven by the need to acquire new memory space. Thus, I will describe work that is aimed at understanding the basic biology of memory as a way of explaining the function of neuronal replacement in adult vertebrate brain.

12 IUPS 2009 July 27 - August 1, 2009 in Kyoto SL-13 REMODELING OF CELL-CELL JUNCTIONS VIA CADHERIN-CYTOSKELETAL INTERACTIONS Masatoshi Takeichi RIKEN Center for Developmental Biology, Japan Cell-cell junctions are essential for the generation of multicellular systems. These junctions often need to be remodeled for allowing cells to move, relocate, or change shapes. The major junction in animal tissues is called

“adherens junction (AJ)”, which comprises cadherin and associated proteins, termed catenins. We have been Special Lectures studying whether the catenins can regulate AJ assembly. The AJs interact with F-actin as well as microtubules (MTs). For the interaction with F-actin, the cadherin-catenin complex further binds EPLIN, an acting-binding protein, becoming tethered to circumferential actin belts. Through these interactions, a typical epithelial junction, zonula adherens, is organized. When EPLIN is depleted, the junctions are converted to a mesenchymal type. Thus, the presence or absence of a linker between AJ and F-actin greatly affects junction morphology. The interaction of AJs with MTs is mediated via newly characterized proteins, Plekha7 and Nezha. This interaction occurs at the minus ends of MTs, providing a new anchoring site for MT minus ends. In the absence of this system, the zonula adherens loses its integrity. Thus, F-actin and MT have distinct roles in AJ assembly, and their coordinated interactions with the AJs likely play important roles in their remodeling.

SL-14 LEPTIN AND THE BIOLOGIC BASIS OF OBESITY Jeffrey M Friedman Department of Molecular Genetics, The Rockefeller University/Howard Hughes Medical Institute, USA The discovery of leptin has led to the elucidation of a robust physiologic system that maintains fat stores at a relatively constant level. Leptin is a peptide hormone secreted by adipose tissue in proportion to its mass. This hormone circulates in blood and acts on the hypothalamus to regulate food intake and energy expenditure. When fat mass falls, plasma leptin levels fall stimulating appetite and suppressing energy expenditure until fat mass is restored. When fat mass increases, leptin levels increase, suppressing appetite until weight is lost. By such a mechanism total energy stores are stably maintained within a relatively narrow range. Recessive mutations in the leptin gene are associated with massive obesity in mice and some humans. Treatment with recombinant leptin markedly reduces food intake and body weight. The low leptin levels in patients with leptin mutations are also associated with multiple abnormalities including infertility, diabetes and immune abnormalities all of which are corrected by leptin treatment.

IUPS 2009 July 27 - August 1, 2009 in Kyoto 13 SL-15

TRANSPLANTABLE SITES CONFER TEMPERATURE AND PIP2 SENSITIVITY TO THERMO TRANSIENT RECEPTOR POTENTIAL (TRP) CHANNELS

Special Lectures Ramón Latorre Centro de Neurociencia, Universidad de Valparaiso, Chile TRPV1 and TRPM8 are two thermoTRP channels able to sense both voltage and temperature. Although progress has been made in understanding the temperature-dependent properties of these channels the mechanisms by which temperature and voltage activate these channels are far from clear. Thus, a key question in understanding thermoTRP channel function is how do the protein domains in sensing stimuli (sensors) and opening the pore (gates) communicate. Taking advantage of our finding that the C-terminal domain of thermoTRPs is modular and determines the channel phenotype regarding temperature and sensitivity and PIP2 binding, I have investigated its role in thermosensation and PIP2 binding using wild type, chimeric channels, C-terminal fusion proteins of TRPM8 and TRPV1 and fluorescence techniques. The results indicate that thermoTRP channel-forming proteins are modular and certain structures confer temperature-dependent and PIP2 activation, whereas others confer voltage- dependent regulation. Supported by grant FONDECYT 1070049

SL-16 RECENT STUDIES OF POTASSIUM CHANNELS AND CALCIUM-ACTIVATED CHLORIDE CHANNELS Lily Y Jan Department of Physiology, University of California, San Francisco, USA Potassium channels and calcium-activated chloride channels are widely distributed and serve important physiological functions in both excitable and non-excitable cells. Whereas all cells have internal potassium concentration higher than external potassium concentration, the chloride concentration gradient across the cell membrane varies with the cell type and, in the case of neurons, may even vary from one subcellular compartment to the next. Molecular identification of these channels enables characterization of channel expression, which is critical to our understanding of channel function, as well as structure-function studies and analyses of activity-dependent channel regulation. Recent studies of both potassium channels and calcium-activated chloride channels will be presented.

14 IUPS 2009 July 27 - August 1, 2009 in Kyoto SL-17 SENSING WITH IONIC CHANNELS Michel Lazdunski Institut de Pharmacologie Moleculaire et Cellulaire, CNRS, France Ion channels are targets of numerous classes of drugs (antihypertensive, antidiabetic, antiepileptic, antiarythmic, etc …). The presentation will mainly deal with two relatively new families of ionic channels. The first one is the + 2 pore domain family of K channels (K2P), a family that comprises the background channels that impose the resting potentials in most cells. It also comprises an important class of mechano,osmo, and temperature-sensitive Special Lectures K+ channels also activated by polyunsaturated fatty acids that have interesting structure-function relationships and regulations. This later class of K+ channels has been associated with depression in mice but it also plays a central role in the action of volatile anesthetics, in neuroprotection (epilepsy and ischemia), in nociceptors to sense warm and cold, in osmo and in mechano-sensing. The second channel family to be described is that of the acid sensitive channels (ASICs). ASICs are important for acid sensing. These Na+ (and Ca++) permeable channels are esssential in all sorts of pain perceptions. One will explain how they work, how they are regulated particulary by inflammatory stimuli and describe the high-affinity pharmacology that has been developed and that can would used for analgesia.

IUPS 2009 July 27 - August 1, 2009 in Kyoto 15 Kyoto Memorial Lecture NEURAL CIRCUIT GENETICS OF HIPPOCAMPAL MEMORY Susumu Tonegawa Picower Institute for Learning and Memory, Massachusetts Institute of Technology (MIT), USA We have been studying the molecular, cellular and circuit mechanisms underlying memories of events and facts by applying genetic engineering to the hippocampus of a mouse. More specifically, we design and generate a variety of mutant mouse lines in which a specific gene encoding a receptor or enzyme is deleted or modified in a highly restricted type of neurons, or a specific neuronal circuit is blockable in a temporally controllable manner. These PSJ Named Lectures mutant mouse strains are then analyzed in comparison to normal counterparts by a variety of methods that permit an identification of impairments or deficits in the mutants at various levels of complexity: molecular, cellular, physiological, circuit and behavioral. For instance, we have demonstrated NMDA receptors in each of the three major sub-regions of the hippocampus, area CA1, CA3 or dentate gyrus, plays a distinct role in the acquisition and recall of hippocampus-dependent memory. Likewise, a mutant strain in which a blockade is present at only one of the two alternative excitatory circuits, the “trisynaptic pathway” and the monosynaptic pathway exhibits distinct deficits in the acquisition, consolidation and recall of memory. I will summarize these findings and discuss the exciting future of neural circuit genetics.

Congress Lecture TOWARD COMPLEX BIOLOGICAL FUNCTIONS - A REVIEW FROM CEREBELLAR PHYSIOLOGY Masao Ito Laboratory for Memory and Learning, RIKEN Brain Science Institute, Japan In studies of a complex biological system, we first divide the system to subsystems and identify individual unitary components consisting of each subsystem. Then, we turn to find local principles combining the components to constitute a subsystem and further to reconstruct the entire system by organizing the subsystems following some global principles. With this general scheme of physiology in mind, I shall review progresses in cerebellar research during the past four decades. Various cellular components and their interconnections have been analyzed, and local principles assorting them have been revealed: multilayered neural net, feedforward and feedback inhibition, neuromodulation, error learning, internal clock etc. Unique global principles in organizing a cerebellar system have also been extracted: 1) embedded computing for adaptation, 2) mode setting for environmental demand, 3) internal model control of voluntary movement, and 4) internal model control of mental activity. In the coming decade, reproduction of the entire cerebellar functions along these four global principles will be a major goal for our effort to understand how complex biological functions including human mental activities emerge from multicellular machinery of the body and brain.

16 IUPS 2009 July 27 - August 1, 2009 in Kyoto Hagiwara Lecture MAKING AN EFFORT TO LISTEN: MECHANICAL AMPLIFICATION BY MYOSIN MOLECULES AND ION CHANNELS IN HAIR CELLS OF THE INNER EAR Albert J Hudspeth HHMI and Laboratory of Sensory Neuroscience, The Rockefeller University, USA Our hearing is remarkable for its physical capabilities. We can discern sounds at frequencies up to 20 kHz, a bandwidth one-thousandfold that of our vision. At the auditory threshold, our ears detect vibrations of only ±0.3 nm, an atomic dimension. Finally, our hearing responds over twelve order of magnitude in intensity, a range unmatched by other sensory systems or indeed by manmade detectors. Uniquely among vertebrate sensory receptors, the ear's mechanoreceptive hair cells amplify their inputs by means of an active process that increases responsiveness to sound, sharpens frequency selectivity, and compresses the dynamic range of hearing. An overly exuberant active process can even cause the spontaneous emission of sound from an ear! In non-mammalian tetrapods― PSJ Named Lectures and perhaps in mammals as well―each hair cell uses its hair bundle, an elaborate array of tiny biological strain gauges, both to transduce mechanical stimuli and to implement the active process. Mechanical amplification is accomplished through the interaction of negative hair-bundle stiffness with the activity of the motor protein myosin-1c. The operation of the active process near a dynamical instability, the Hopf bifurcation, explains many of the characteristics of our hearing.

Tawara Lecture CARDIAC EXCITATION AND ARRHYTHMIAS IN THE HUMAN HEART : INSIGHTS FROM NONINVASIVE ECG IMAGING (ECGI) Yoram Rudy Cardiac Bioelectricity Research and Training Center (CBAC), Washington University, USA A noninvasive imaging modality for cardiac electrophysiology and arrhythmias is not yet available for clinical application. Such modality could be used to identify patients at risk, provide accurate diagnosis and guide arrhythmia therapy. Standard noninvasive diagnostic techniques, such as the electrocardiogram (ECG) provide only low-resolution reflection of cardiac electrical activity on the body surface. In my presentation, I will describe the successful application in humans of a new imaging modality called Electrocardiographic Imaging (ECGI) that noninvasively images cardiac electrical activity on the heart’s epicardial surface [Nature Medicine 2004; 10: 422-428]. In ECGI, a multi-electrode vest (or strips) records 250 body-surface electrocardiograms; then, using geometrical information from CT scans and a mathematical algorithm, electrical potentials, electrograms and isochrones are reconstructed on the heart‘s surface. In my presentation I will show examples of imaged atrial and ventricular activation and repolarization during normal sinus rhythm and selected arrhythmias.

IUPS 2009 July 27 - August 1, 2009 in Kyoto 17 Ishimori Lecture HUMORAL MECHANISMS OF SLEEP-WAKE REGULATION - COMMEMORATING THE CENTENNIAL ANNIVERSARY OF THE DISCOVERY OF ENDOGENOUS SLEEP SUBSTANCES Osamu Hayaishi Osaka Bioscience Institute, Japan Sleep is perhaps one of the most important and yet least understood of the physiological functions of the brain, even

PSJ Named Lectures though the number of sleep disorder patients now exceeds more than 30% of the total population in most countries. More than 88 different sleep disorders have been described; but in most instances, their aetiologies are not clearly understood. The humoral theory of sleep regulation, the concept that sleep is regulated by a hormone-like chemical substance rather than by a neural network, was initially proposed by K. Ishimori in Nagoya, Japan, and independently by H. Pieron, in Paris, in the first decade of the last century. Although these researchers became the first to demonstrate the existence of endogenous sleep substances, their chemical nature was not identified. Subsequently, more than 30 endogenous sleep substances were reported, but their physiological relevance remained uncertain in most instances. In 1982, we demonstrated that prostaglandin (PG) D2 induced physiological sleep in rats and monkeys, and in subsequent studies elucidated the molecular and genetic mechanisms underlying sleep-wake regulation and showed that PGD2 plays a crucial role in the homeostatic and circadian regulation of non-rapid eye movement sleep.

18 IUPS 2009 July 27 - August 1, 2009 in Kyoto WD I-1-1 WD I-1-2 HISTORICAL OVERVIEW OF STUDIES ON EXCITATION- RYANODINE RECEPTOR ISOFORMS IN EXCITATION- CONTRACTION COUPLING CONTRACTION COUPLING IN SKELETAL MUSCLE Makoto Endo Takashi Murayama Department of Pharmacology, Saitama Medical University, Japan Department of Pharmacology, Juntendo University, Japan Since Galvani's discovery that muscle contraction can be induced by Frog skeletal muscle fiber has been frequently used for muscle research. Two electrical stimulus in the 18th century, excitation-contraction coupling has brilliant discoveries in muscle excitation-contraction (E-C) coupling field, always been one of the most important subjects in physiology. Remarkable i.e., "Natori's skinned fiber" and "Endo's Ca2+-induced Ca2+ release (CICR)", progress in this field has been made in the latter half of the 20th century, were both done with frog muscle. On the other hand, mammalian skeletal studies being made following various methodologies in physiology, muscles were used for identification of key molecules and their function. In morphology, biochemistry and biophysics, and results integrated. Professors E-C coupling, depolarization of transverse tubules activates dihydropyridine Reiji Natori and Setsuro Ebashi, both of whom unfortunately passed away 2+ receptor/voltage sensor, and in turn it opens ryanodine receptor (RyR)/Ca in 2006, were distinguished pioneers and leaders among others in this 2+ release channel in the sarcoplasmic reticulum to release Ca . RyR releases field during this period, and greatly contributed to the progress. In this 2+ 2+ presentation, recollecting their achievement above all, historical overview Ca via two distinct modes: depolarization-induced Ca release (DICR) and of studies on excitation-contraction coupling will be made, which will CICR. Mammalian muscle has a single RyR isoform (RyR1) and it mediates unavoidably let us recognize the remaining problems. both DICR and CICR. In contrast, frog muscle expresses two RyR isoforms (RyR1 and RyR3) at almost equal amount. We have found that DICR is exclusively mediated by RyR1, whereas CICR is predominantly mediated by RyR3 in frog muscle, suggesting distinct roles in Ca2+ release. In addition, comparison of frog and rabbit RyR1 revealed that several significant differences in CICR. In this exciting symposium, I will talk about structure and function of the two RyR isoforms and discuss why the two isoforms are present in frog, but not in mammals.

WD I-1-3 WD I-1-4 CARDIOPROTECTION BY MG53 MEDIATED RYANODINE RECEPTOR-RELATED PROTEINS MEMBRANE REPAIR Do Han Kim Heping Cheng1, Xianhua Wang1, Wenjun Xie1, Yi Zhang1, Liang Han1, Whole-day symposia 1 1 2 2 1 Department of Life Science, Gwangju Institute of Science and Technology, Peidong Han , Yanru Wang , Zheng Chen , Guangju Ji , Ming Zheng , Korea Noah Weisleder3, Hiroshi Takeshima4, Jianjie Ma3 1Institute of Molecular Medicine, Peking University, China, 2Institute of In mammalian heart, activation of L-type Ca channel/dihydropyridine receptor Biophysics, Chinese Academy of Sciences, China, 3Department of Physiology (DHPR) in transverse tubules by membrane depolarization leads to Ca influx and Biophysics, Robert Wood Johnson Medical School, USA, 4Department through DHPR and Ca release from the sarcoplasmic reticulum (SR) through of Biological Chemistry, Kyoto University Graduate School of Pharmaceutical Ca release channel/ ryanodine receptor (RyR2). The consequently elevated Sciences, Japan cytoplasmic Ca induces muscle contraction. Muscle relaxation occurs Mitsugumin 53 (MG53) is a newly identified and striated muscle-specific tirpartite through reuptake of Ca into SR by SERCA2. The RyR in the junctional SR motif family protein (TRIM72), and constitutes a critical component of the membrane repair machinery in skeletal muscle (Cai et al. Nat Cell Biol. 11:56). Here we forms a quaternary protein complex with calsequestrin, triadin, and junction. demonstrate the cardio-protective function of MG53-mediated membrane repair. It has been postulated that calsequestrin is physically anchored to the RyR We found that MG53 translocates to membrane damage sites of cardiac myocytes by triadin and junction. We have also studied other RyR2 associated proteins in response to different acute membrane disruptions, including mechanical damage, which could affect the activity of muscle cells. In my presentation, recent electroporation and detergent treatment. Physiological level of stresses to cardiac evidence will be shown in regard to the interactions between RyR2 and other muscle, including membrane stretch and pacing-induced contraction, do not produce Ca binding proteins such as histidine-rich Ca binding protein (HRC) and MG53 translocation to the sarcolemmal membrane. Cholesterol within the membrane calumenin. The possible role of junctate (another Ca binding protein located is required for MG53 translocation to injury sites, as pretreatment with methyl-β in the SR lumen) in excitation-contraction coupling will be discussed. cyclodextran leads to impairment of MG53 function in cardiac myocytes. In the intact heart, MG53 participates in membrane repair process of cardiac muscle damage (This study was supported from the KMEST Systems Biology Research grant inflicted by two-photon radiation, and MG53 ablation exaggerates this injury response and GIST Systems Biology Infrastructure grant) to the heart. Our findings highlight the importance of membrane repair in heart function, which could be a novel target for therapy.

WD I-1-5 LS5 (WD I-1-related luncheon seminar) MEASUREMENT AND SIMULATION OF LOCAL AND NOVEL IMMAGING TECHNOLOGY & DISCOVERY IN 2+ GLOBAL MYOPLASMIC CALCIUM SIGNALS DURING DEVELOPMENT AND NEURAL FUNCTION (IP3-Ca EC COUPLING IN FROG AND MOUSE INTACT SIGNALING) SKELETAL MUSCLE FIBERS Katsuhiko Mikoshiba Stephen M Baylor1, Stephen Hollingworth1, Knox Chandler2 Laboratory for Developmental Neurobiology, RIKEN Brain Science Institute, 1 2 Japan Department of Physiology, University of Pennsylvania, USA, Department 2+ of Cellular and Molecular Physiology, Yale University, USA Ca is known to play various roles in cell function. We discovered IP3 receptor (IP R) and determined the structure and found that it is a Ca2+ Calcium (Ca) sparks were recorded with fluo-3 and a laser-scanning confocal 3 microscope; Ca signals elicited by action potentials (APs) were recorded with furaptra release channel located on endoplasmic reticulum. IP3R is essential for and standard fluorescence photometry. The sarcoplasmic reticulum (SR) Ca release fertilization, dorsoventral axis formation and cell division. IP3R is involved flux underlying the Ca signals was estimated with a multi-compartment reaction- in neurite extension and neural plasticity and IP3R1 knock out mouse diffusion Ca model that included troponin, ATP, parvalbumin, the SR Ca pump, and shows severe cerebellar ataxia. IP3R2 and IP3R3 are important for exocrine the Ca indicator at appropriate compartment concentrations. In frog twitch fibers in secretion. ERp44 works as a redox sensor in the ER and regulates IP3R1 activity suggesting that redox regulation is directly linked to Ca2+ signaling. 8 mM potassium Ringer’s, the release flux of an average Ca spark is estimated to be 2+ o IP releases not only Ca , but also releases IRBIT ( IP receptor binding 1.7 pA (units of Ca current) for 4.2 ms (18 C; peak dF/FR > 0.5); this flux likely arises 3 3 protein released with inositol trisphosphate). IRBIT regulates IP3 induced from the coordinated activity of 2-5 SR Ca release channels. In mouse fibers, sparks 2+ are difficult to measure, probably because the number of active channels is smaller. Ca release. In addition, IRBIT works as a third messenger to regulate pH In mouse fast-twitch fibers stimulated by an AP, the peak and FDHM of the spatially- inside cells. Since IP3R interacts with many molecules, IP3R works as a 2+ averaged release flux are estimated to be 200 μM/ms and 1.6 ms (16 oC). In slow- scaffold protein in addition to Ca releasing channel exhibiting diverse roles twitch fibers, the estimates are 80 μM/ms and 1.6 ms; here, however, a slowly reacting in cell function. Ca buffer (analogous to parvalbumin in fast-twitch fibers) must be included for the I will present molecular dynamics of IP3R and its associated molecules in decay of the simulated free Ca transient to match that of the measurement (cf., J. M. various normal and pathophysiological phenomena using various imaging Gillis, 1997). Supported by NIH. technology.

IUPS 2009 July 27 - August 1, 2009 in Kyoto 19 WD I-1-6 WD I-1-7 Ca-INDUCED STRUCTURAL CHANGES OF MOLECULAR MECHANISM OF MUSCLE REGULATORY PROTEINS IN SKELETAL MUSCLES CONTRACTION: FOCUSING ON THE DYNAMIC Naoto Yagi, Tatsuhito Matsuo PROPERTIES REVEALED AT INTERMEDIATE Research and Utilization Division, Japan Synchrotron Radiation Research ACTIVATION LEVELS Institute, Japan Shin'ichi Ishiwata It is well known that contraction of skeletal muscle is regulated by Ca- Department of Physics, Waseda University, Japan binding to troponin in the thin filament. Ca-binding induces a structural The contractile system of striated muscle possesses a hierarchy, which is, as change of troponin, which can be monitored by intensity changes in the clearly illustrated by "“:Natori’s staircase"”, composed of single molecules, X-ray diffraction pattern: the intensity of the meridional reflection from an assembly of the thick (myosin) and the thin (actin) filaments, sarcomeres, troponin at 1/38.5 nm-1 increases upon an electrical stimulus in live muscle, myofibrils, and their parallel assembly, i.e., muscle fibers. It is established indicating a narrower axial mass distribution. However, in a muscle with an that muscle cells take one of two states, either relaxation or contraction, in overlap between the thick and the thin filaments, this initial intensity increase an all-or-none fashion, depending on the presence or absence of electrical excitation. The contractile apparatus, prepared by removal of cell membrane, is followed by a large decrease, suggesting that attachment of myosin heads also takes either one of the two states depending on the concentration of free to actin affects the intensity of this reflection. Furthermore, this intensity 2+ Ca , which was established by Prof. Ebashi and his associates; however, decrease is smaller in tetanus than in twitch. To investigate the complicated it is not necessarily well recognized that the definition of relaxation is interaction between troponin and myosin heads, X-ray diffraction difficult because actin-myosin interaction is not completely inhibited even experiments were made on twitches of frog skeletal muscle elicited by an in the absence of Ca2+. In fact, we found that the contractile apparatus at either single or double stimulus at 16C. The peak tension was more than intermediate activation takes the third state in between the contraction and doubled by the second stimulus. The troponin meridional reflections was relaxation, which is the auto-oscillation of sarcomeres named SPOC. I would affected only slightly by the second stimulus, suggesting that attachment of like to stress that such dynamic properties are not realized in a stochastic

Whole-day symposia a small number of myosin heads to actin can cause a cooperative structural single-molecular system, but are intrinsic to the liquid crystalline-like, well- change in troponin. organized structures of sarcomeres and myofibrils.

WD I-1-8 WD I-1-9 HIGH RESOLUTION STRUCTURE AND BASIC ROLE OF EXCITATION-CONTRACTION COUPLING IN PROPERTIES OF F-ACTIN MUSCLE FATIGUE Yuichiro Maeda1, Toshiro Oda2, Akihiro Narita1, Mitsusada Iwasa3, David Grant Allen 3 4 Kayo Maeda , Tomoki Aihara School of Medical Sciences and Bosch Institute, University of Sydney, 1Structural Biology Research Center, Nagoya University Graduate School Australia 2 of Science, Japan, ERATO Actin filament dynamics project, JST and Muscle fatigue is the reversible decline in performance associated with RIKEN SPring-8 Center, Japan, 3ERATO Actin filament dynamics project, 4 intense activity. It provides an important limitation in many sporting JST, Japan, RIKEN SPring-8 Center, Japan activities and is a significant contributor to many diseases, for instance heart F-actin performs a variety of major cellular functions. In non-muscle cells, the actin failure. There are many mechanisms of fatigue and the mechanisms differ tread-milling drives motility of filopodia and lamelipodia. In muscle, calcium-ion according to intensity, duration and the type of movement or contraction binding to troponin must cause structural changes of the thin filament. Although the involved. The main identified causes of fatigue are changes in intracellular two appear to be distinct, either must originate from the basic properties of F-actin. metabolites, alterations in excitation-contraction coupling and accumulation We have obtained a high resolution structure of F-actin (Oda et al, 2009) and the of extracellular ions. Reactive oxygen species, protein modifications and structures of filament ends (Narita et al, 2006 and unpublished results). A newly structural changes are additional possibilities. Investigation of mechanisms established actin expression system (Iwasa et al, 2008) has allowed us to perform has proceeded most rapidly in isolated and simplified muscle preparations. mutation experiments, and to deduce the basic properties of F-actin from the structures. Actin consists of two halves on either side of the central Nd-binding groove. The An important focus for the future is the development of methods which can G-to-F transition is associated with a reduction of the twisting angle between two identify fatigue mechanism in intact muscles so that the fatigue in more halves from 20 degrees to null. The free energy to drive this flattening must be realistic activities, and especially in human diseases, can be understood. provided by the inter-molecular contacts in the F-actin structure. Therefore, F-actin structure is under the balance between the stabilizing and destabilizing factors. It is plausible that the calcium binding to troponin should alter the balance between the two. Structural basis for the treadmilling and the stable helical symmetry of subunits will also be discussed.

WD I-1-10 WD I-2-1 DISEASES RELATED TO CALCIUM RELEASE PHYSIOLOGICAL DYS-REGULATION WITH INACTIVITY CHANNELS IN SYSTEMS DESIGNED TO MOVE Robert Thomas Dirksen1, Ryan E. Loy1, Simona Apostol2, Murat 2 2 3 Frank W Booth Orynbayev , Zoita Andronache , Elena Zvaritch , Dept Biomedical Sciences, University of MIssouri, USA David H. MacLennan3, Susan L. Hamilton4, Werner Melzer2 1 2 To some, it is surprising that the body responds in a detrimental functional Pharmacology and Physiology, University of Rochester, USA, Institute of Applied Physiology, Ulm University, Germany, 3Banting and Best Department and health direction when used less than its historical norm. The classical of Medical Research, University of Toronto, Canada, 4Department of Molecular Dallas bed rest study in the 1960’s showed that 20-yr old, healthy men had Physiology and Biophysics, Baylor College of Medicine, USA 28%, 26%, and 29% decreases of for maximal O2 uptake, maximal cardiac The type 1 ryanodine receptor (RYR1) gene encodes the sarcoplasmic reticulum output, and maximal stroke volume, respectively, after 20 days of continuous calcium release channel of skeletal muscle. Mutations in the RYR1 gene result in bed rest. In the same era, it was first demonstrated that insulin sensitivity several distinct human congenital muscle disorders including Malignant Hyperthermia decreased with bed rest in healthy young subjects. More recent studies by (MH), Central Core Disease (CCD), and Multi-minicore Disease (MmD). More Pedersen of young, health men, who were not partaking in exercise programs recently, RYR1 mutations have also been linked to exercise-induced rhabdomyolysis prior to decreasing daily steps from either 6000 or 10,000 steps/day to 1300, and increased susceptibility to heat stroke. Knock-in mice genetically engineered to also decreased maximal O2 uptake and peripheral insulin sensitivity. Changes carry the human MH/CCD Y522S mutation in RyR1 exhibit increased MH and heat in insulin signaling with decreased muscle contractile activity will be shown. susceptibility and develop a myopathy associated with mitochondrial loss/dysfunction. These observations raise the question, what functional advantage might On the other hand, we find that knock-in mice harboring the human I4895T CCD have selected for physical inactivity in ancestral times to be so functionally mutation in the RyR1 pore region lack MH and heat susceptibility, but do exhibit in vivo muscle weakness and muscle fibers display a reduction in action potential- detrimental in the current world of low physical labor? Speculative answers induced Ca2+ transient amplitude. Finally, the frequency and calcium spark mass of to be discussed are ability to adapt to a new environment more rapidly local calcium release induced by osmotic shock is significantly reduced in acutely depends on short half lives of proteins, and thus improve survival percentages dissociated muscle fibers from I4895T/+ mice. Together, our findings indicate that the in ancestral era; and the low amount of carbohydrate stored for fuel. Y522S and I4895T mutations alter RyR1 channel activity and skeletal muscle function via fundamentally distinct mechanisms.

20 IUPS 2009 July 27 - August 1, 2009 in Kyoto WD I-2-2 WD I-2-3 THE ROLE OF MYOKINES IN MUSCLE-FAT CROSS- EXERCISE AND MUSCLE BLOOD FLOW TALK Michael J Joyner Bente Klarlund Pedersen Department of Anesthesiology, Mayo Clinic, USA Center of Inflammation and Metabolism, Rigshospitalet, University of This presentation will provide an overview of how blood flow to contracting Copenhagen, Denmark human muscles is regulated during exercise. Key elements of the overview It is now established that cytokines, besides their immunoregulatory roles, include: are important players in metabolism. Moreover, it has become evident that The link between muscle blood flow and metabolism during exercise skeletal muscles express several cytokines, which belong to distinct cytokine The magnitude of the rise in muscle blood flow with exercise classes. Recently, we suggested that cytokines and other peptides that are The factors that cause vasodilation in the contracting muscles Ideas about competition between local muscle blood flow and systemic blood produced, expressed and released by muscle fibers and exert either paracrine pressure regulation or endocrine effects should be classified as “myokines”. Muscle-derived How some of these factors change with aging cytokines appear to have important roles in metabolism, and exercise plays The basic idea is that blood flow to contracting muscles can be extremely high a role in orchestrating the interplay between cytokines and metabolism. as a result of "metabolic" dilation by substances that remain unknown. When Skeletal muscle can be identified as an endocrine organ, which by contraction a large mass of muscle is active this dilation is restrained by that sympathetic stimulates the production and release of myokines, which can influence nervous system to facilitate arterial pressure regulation. With aging there is metabolism and modify metabolism in muscle, fat tissue and other organs. evidence for blunted blood flow responses to contracting muscles as a result of both changes in local vasodilator responses and augmented restraint of vasodilation by the sympathetic nervous system. Supported by NIH HL-46493

LS17-1 (WD I-2-related luncheon seminar) LS17-2 (WD I-2-related luncheon seminar) PROTEIN AND CHO SUPPLEMENTATION INCREASES LACTATE: DARTH VADER OR SAMURAI HERO OF AEROBIC AND THERMO-REGULATORY CAPACITIES EXERCISE PHYSIOLOGY Kazunobu Okazaki, Hiroshi Nose, Masaki Goto George A. Brooks Whole-day symposia Department of Sports Medical Sciences, Shinshu University Graduate Integrative Biology, University of California, Berkeley, USA School of Medicine, Japan Rather than a 'dead-end metabolite', lactate is an important metabolic Increases in aerobic and thermoregulatory capacities after aerobic training intermediate and signaling molecule, a “Lactormone”. Lactate Shuttles are accompanied by plasma volume (PV) expansion. We examined whether (LS) play several key functions. As the product of glycolysis and main protein and carbohydrate (Pro-CHO) supplementation during training enhanced substrate for mitochondrial respiration, lactate links glycolytic and oxidative PV expansion and thereby these capacities in young (Y) and older (O) men. metabolism in anatomically close as well as distributed compartments. Subjects were divided into 2 groups: placebo [C; 0.9 kcal & 0 g protein/kg in Although most lactate is disposed of via oxidation, lactate is the most Y (n=9), 0.5 kcal & 0 g protein/kg in O (n=7)] and Pro-CHO [S; 3.6 kcal & important gluconeogenic precursor in all but starving individuals. As a 0.36 g protein/kg in Y (n=9), 3.2 kcal & 0.18 g protein/kg in O (n=7)]. Subjects reduced compound (compared to its keto-analog, pyruvate), millimolar exercised at ~70%VO2peak; 30 min/day for 5 days at 30°C in Y, 60 min/day for 8 exchanges of lactate and pyruvate have large effects on redox balance, both wks (3 days/wk) at 25°C in O; and took either placebo or Pro-CHO immediately within and across cellular compartments. Further, lactate accumulation results after exercise. After training, an increase in PV was greater in S than C for both in intracellular ROS production. As such, lactate affects not only metabolic age groups. The sensitivities of forearm skin vascular conductance and chest regulation, but also diverse adaptive processes such as gene expression, sweat rate responses to increased esophageal temperature during exercise at wound healing and vasculogenesis. Appreciation for the central role of lactate

60-65%VO2peak for 20-30 min at 30°C were enhanced more in S than C with in metabolic integration is augmented by discoveries that lactate modulates a greater increase in stroke volume. Thus, Pro-CHO supplementation during peroxisomal β-oxidation and adipocyte lipolysis. Translational aspects of training enhanced aerobic and thermoregulatory capacities through greater Lactate Shuttle understanding involve the use lactate salts an esters to support increases in PV and stroke volume in both age groups, which may provide a new cerebral function following TBI and as targets if intervention in tumor cell training regimen to improve these capacities more than exercise alone. metabolism.

WD I-2-4 WD I-2-5 EXERCISE TRAINING AND CARDIOVASCULAR PHYSIOLOGICAL EFFECT OF TRADITIONAL DISEASES CULTURE-BASED ELDERLY EXERCISE PROGRAM, Douglas R Seals WOORI-CHUM CHEJO Department of Integrative Physiology, University of Colorado, USA Sang Chul Park Cardiovascular diseases (CVD) remain the leading cause of morbidity and Department of Biochemistry and Molecular Biology, Seoul National University, Korea mortality in middle-aged and older adults. Approximately 80% of all CVD involve disorders of arteries. Two changes to arteries that contribute to The Institute on Aging of Seoul National University has developed a unique CVD are increased large elastic artery stiffness and vascular endothelial exercise program for the elderly people, based on traditional music and dance. dysfunction. Habitual physical activity and, particularly, regular aerobic The original purpose of the program was to encourage the old people to exercise exercise are associated with reduced large elastic artery stiffness, greater more, since traditionally Korean old people used to be sedentary because of traditional concept of exercise neglect. Secondly, it was intended to improve vascular endothelial function and a lower prevalence of CVD in middle-aged the mental health of the elderly people by motivation of participating in social and older adults. The mechanisms involved in reduced large elastic artery programs. For the elderly, the program has been emphasized with simplicity, stiffness with habitual exercise are largely unknown, but enhanced vascular rhythmic movement, emotion projection and dynamism in order to allow endothelial function is associated with reduced oxidative stress and greater the elderly to learn easily and participate anywhere and anytime, When the bioavailability of nitric oxide, possibly related to increased bioactivity physiological effects of the elderly exercise were tested on 1 year participated of tetrahydrobiopterin, an essential co-factor for nitric oxide production volunteers, the remarkable increases in balance and flexibility were observed by endothelial nitric oxide synthase. Suppression of oxidative stress in with some improvements in muscle power. Moreover in the psychosocial exercising adults may be mediated in part by down-regulation of the oxidant aspect, the improvements in motivation, social participation, and self satisfaction enzyme NADPH oxidase and an increase in antioxidant activity. Habitual were much more achieved than expected. Therefore, it can be concluded that exercise also may suppress oxidative stress via anti-inflammatory effects on the culture-based exercise program would be very effective for the physical arteries. AMPK, SIRT-1 and other novel molecular mechanisms currently are and mental health of the elderly people who are prone to be fragile not only under investigation. physically but also mentally.

IUPS 2009 July 27 - August 1, 2009 in Kyoto 21 WD I-2-6 WD I-2-7 THE EFFECTS OF PHYSICAL ACTIVITY AND MUSCLE BEYOND EPIDEMIOLOGY: FIELD STUDIES AND THE STRENGTH ON AGING AND AGE-RELATED DISEASES; PHYSIOLOGICAL LABORATORY AS THE WHOLE FROM THE NILS-LSA- WORLD Fujiko Ando1, Rumi Kozakai2, Hiroshi Shimokata2 Hiroshi Nose 1Faculty of Medical Welfare, Aichi Shukutoku University, Japan, Department of Sports Medical Sciences, Shinshu University Graduate 2Department of Epidemiology, National Institute for Longevity Sciences, School of Medicine, Jukunen Taiiku Daigaku Research Center, Japan National Center of Gerontology and Geriatrics, Japan There is no exercise training system broadly available in the field to improve Aging is characterized by progressive decline of muscle strength that lead to reduction physical fitness (PF) and lifestyle-associated diseases (LSD) based on individual in functional capacity and an increased risk of age-related diseases. However, the physical characteristics. We have developed a system for this purpose for effect of aging process and its associated factors are not uniform and very complicated. middle-aged and older people, composed of a handy calorie meter (JDM), high- The NILS-LSA (National Institute for Longevity Sciences - Longitudinal Studies of intensity interval walking training (IWT), and an IT network (IT). JDM is used Aging) has launched in 1997. The subjects were about 2300 males and females aged to determine peak aerobic capacity for walking (VO2peak) on individuals and 40 to 79 at baseline, sex and age-decade stratified random samples from neighborhood also their energy expenditure (EE) during IWT. IWT is a regimen to repeat ≥5 of the NILS, Aichi prefecture, Japan. They were invited to the examination center sets of 3-min slow walking at 40% VO2peak followed by a 3-min fast walking ≥ biannually and were inspected multidisciplinary examinations, including clinical 70% VO2peak/ day, for ≥4 days/wk, for ≥4 mos. IT is used to collect individual evaluation, body composition and anthropometry, physical functions, nutritional EE during daily IWT stored in JDM and to give them back their achievements analysis and psychological assessments, to assess usual aging process and the relative and advice from staff based on database (DB) through internet. From DB on factors of aging and age-related diseases. the effects of IWT on LSD and PF in more than 3,000 subjects, we found IWT We will introduce several outputs from the NILS-LSA, especially aging in physical improved most indices of LSD and PF more in those with lower PF, more serious activity and muscle strength, and its related factors including medical, psychological, LSD, or some single nucleotide polymorphisms. Thus, since this system is to be physical aspect. In latter half of the lecture, the theme will be focused on the run with low cost and less staff for more subjects, it would produce new research

Whole-day symposia relationship of physical activity and muscle strength with osteoporosis, with concerns fields for exercise physiology while exploring new exercise prescriptions more of the interaction of extrinsic and intrinsic factor (genetic polymorphism). fitted for individuals with varied genetic background.

WD I-2-8 WD II-3-1 GENETIC BASIS OF INTER-INDIVIDUAL VARIANCE DYNAMICS OF SPIRAL WAVE REENTRY IN THE EFFECTS OF EXERCISE ON PREVENTION OF Haruo Honjo1, Masatoshi Yamazaki1, Yuko S Ishiguro1, LIFESTYLE-RELATED DISEASES Masahide Harada1, Ichiro Sakuma2, Kaichiro Kamiya1, Masayuki Mori1, Keiichi Higuchi1, Shinshu University Genetic Research 1 2 Itsuo Kodama Consortium, Jukunen Taiiku Daigaku Research Center, Yasuharu Tabara , 1 3 4 Research Institute of Environmental Medicine, Nagoya University, Japan, Tetsuro Miki , Hiroshi Nose 2Graduate School of Engineering, University of , Japan 1Department of Aging Biology, Shinshu University Graduate School of Medicine, Japan, 2Department of Basic Medical Research and Education, Ehime University Spiral wave (SW) reentry is responsible for ventricular fibrillation/tachycardia Graduate School of Medicine, Japan, 3Department of Geriatric Medicine, Ehime (VF/VT). We characterized the dynamics of SW reentry induced in a University Graduate School of Medicine, Japan, 4Department of Sports Medical 2-dimensional ventricular myocardium of Langendorff-perfused rabbit hearts Sciences, Shinshu University Graduate School of Medicine, Japan by using optical mapping. Image analysis revealed that a SW during VT rotated The high-intensity interval walking exercise diminishes the risks of lifestyle-related diseases around a line of functional block (FBL), a narrow zone showing “double for middle-aged and older people. However, the extent of improvement differs considerably potentials”. Optical signals in the reentry circuit showed no isoelectric segments among individuals. Gene polymorphisms may underlie the inter-individual variance. We then between action potentials. These findings suggest that the rotation center is kept attempted to identify such “exercise-responsive genes”. Two hundred and one female (mean refractory due to electronic interactions. Around the pivot points of the SW, age = 64.8 yrs) and ninety-eight male (70.2 yrs) participants took high-intensity interval there was a conduction delay with slow action potential upstrokes as a result of walking exercise regimen for six months. The following parameters associated with lifestyle- high curvature of the returning wavefront. This localized conduction delay is related diseases were measured before and after the training program: body composition, responsible for terminal segments of the FBL parallel to the fiber orientation. blood pressure, ventilatory response, plasma LDL and HDL cholesterol, and triglycerides. Na+ channel blockade stabilized SW reentry with prolongation of the FBLs The participants were genotyped for 141 single nucleotide polymorphisms (SNPs) in 101 genes, which were supposed to be functionally associated with lifestyle-related and widening of excitable gaps to help their persistence, whereas IKr blockade diseases. Several genes showed genotype-dependent differential effects on improvements destabilized SW reentry by irregular meandering and frequent wave breakups of the parameters. It is important to elucidate the mechanisms by which the variations in to facilitate its early termination by collision with the anatomical boundaries. these genes interact with exercise to exert the genotype-dependent differential effects on These results may provide new insights on the mechanisms of ventricular improvement of lifestyle-related diseases. tachyarrhythmias and their treatment by control of SW reentry.

WD II-3-2 WD II-3-3 IN SITU CALCIUM IMAGING OF ARRHYTHMOGENIC GENETIC BACKGORUND OF CARDIAC ARRHYTHMIAS SUBSTRATES IN THE HEART Arthur Wilde Tetsuro Takamatsu Department of Cardiology, AMC, University of Amsterdam, The Department of Pathology and Cell Regulation, Kyoto Prefectural University Netherlands of Medicine, Japan Over the last decade, the molecular genetic substrate of both the primary While electrophysiological mechanisms for the genesis of ventricular arrhythmia syndromes, including the long and short QT syndromes (LQTS, tachyarrhythmias in myocardial infarct have been extensively clarified, SQTS), Brugada syndrome (BrS), and catecholaminergic polymorphic VT arrhythmogenic substrates that eventually lead to electrical derangements (CPVT), and the secondary arrhythmia syndromes, i.e. various inherited cardiomyopathies, has been unraveled to a great extend. are not fully understood. This paper focuses on the intracellular calcium ion 2+ In the former group ion channel genes are primarily involved. This includes (Ca ) dynamics that provide deeper insights into the upstream events of 2+ genes encoding for alpha- and Beta-subunits of ion channel complexes, but also life-threatening tachyarrhythmias. The Ca wave comprises heterogeneous chaperone proteins involved in proper trafficking and positioning of these ion wave-like elevations of the intracellular Ca2+ concentrations that develop 2+ channel proteins. In LQTS, SQTS and BrS 12, 3 and 6 genes have been identified under Ca -overloaded conditions of the injured myocardium. Previous respectively. Indeed, the underlying gene defect impacts on electrocardiographic electrophysiological studies in isolated ventricular myocytes suggested that and clinical features of the disease entity syndrome, including prognosis and Ca2+ waves could evoke abnormal depolarization via Na+-Ca2+ exchanger. 2+ preferred treatment modalities as has most clearly been shown in the Long QT Simultaneous fluorescence recording of intracellular Ca dynamics and syndrome. membrane potentials (Vm) of ventricular myocytes of Langendorff-perfused The inherited cardiomyopathies are subdivided into hypertrophic CM (HCM), rat hearts using in situ dual-view rapid-scanning confocal microscopy dilated CM (DCM), and arrhythmogenic RV cardiomyopathy. By and large, 2+ showed that burst emergence of Ca waves in individual myocytes evoked the genetic substrate is identified in the genes encoding for sarcomeric oscillatory Vm, which caused triggered activity. Burst emergence of Ca2+ proteins (HCM), in genes encoding for cytoskeletal proteins (DCM), and in waves in the heart actually produces arrhythmogenic membrane oscillation. genes encoding for cell adhesion molecules (desmosomes; ARVC). Overlap, This could be the basis for new therapeutic strategies for arrhythmias. however, is considerably.

22 IUPS 2009 July 27 - August 1, 2009 in Kyoto WD II-3-4 WD II-3-5 MECHANISM OF BRUGADA SYNDROME RECENT ADVANCES IN PHYSIOLOGY AND + Wataru Shimizu, Shiro Kamakura PHARMACOLOGY OF K CHANNELS 1 1 2 Division of Cardiology, Department of Internal Medicine, National Jacques Barhanin , Said Bendahhou , Florian Lesage , Cardiovascular Center, Japan Sophie Demolombe3 1CNRS-UNSA, France, 2IPMC-CNRS, University of Nice Sophia-Antipolis, Brugada syndrome is characterized by coved type ST-segment elevation in 3 V1-V3 leads and an episode of ventricular fibrillation (VF). Our experimental France, INSERM U533, Medicine faculty of Nantes University, France model of Brugada syndrome employing arterially perfused canine RV wedge Potassium channels form an extended and diverse class of ion channels, which preparations suggested that transient outward current (Ito)-mediated action in general serve to control and limit cell excitability. Their large heterogeneity potential (AP) notch and the loss of AP dome in the epicardial cells, but not originate in part from the large number of genes that code for pore forming in the endocardial cells, gives rise to a transmural voltage gradient, producing subunits (78 in mammals), heteromeric assembly (tetra- or dimerization) ST-segment elevation in the ECG. The heterogeneous loss of the AP dome and is further augmented by interactions with numerous accessory subunits (coexistence of loss of dome regions and restored dome regions) in the (approximately 15 genes). Understanding the subunits that generate the K epicardium creates a marked epicardial dispersion of repolarization, giving currents in the heart as well as the variety of factors that influence subunit assembly, trafficking and channel regulation is essential to understand how rise to premature beats due to phase 2 reentry, which precipitates VF. High- adverse genetic and pharmacologic factors cause cardiac arrhythmia. Many resolution optical mapping techniques in the wedges allowed us to record different K currents contributing to the control of the diastolic potential or to the transmembrane APs from 256 sites simultaneously at the epicardial surface, repolarization phase have been identified so far. They belong to the family of thus provided further advances of the cellular mechanism of VF. A steep voltage dependent channels (Kv) such as KCNH2 (IKr), KCNQ1 (IKs), KCND2 repolarization gradient between a loss of dome region and a restored dome and D3 (Ito) and to the family of inwardly rectifying channels (Kir) such as region in the epicardium is essential to produce phase 2 reentry-induced KCNJ2 (IK1), KCNJ5 (IKAch), KCNJ11 (IKATP). Recently, background K channels premature beats. Phase 2 reentry-induced premature beats induced a reentrant of the 2P-domain family have been described in the heart. Among them, TWK1 pathway rotated in the epicardium and finally involving the transmural and TASK1 are highly expressed in the human atria but their specific role myocardium, precipitating VF. remains elusive. The most recent data concerning this issue will be discussed.

LS 13 (WD II-3-related luncheon seminar) WD II-3-6 BENEFICIAL EFFECTS OF ANTIPLATELET AGENTS IN FUNCTIONAL CONSEQUENCES OF HYPERTROPHIC ATHEROSCLEROSIS, MYOCARDIAL INFARCTION AND CARDIOMYOPATHY MUTATIONS IN cMyBP-C HEART FAILURE Richard L Moss Whole-day symposia 1 2 Naranjan S Dhalla , Nobuakira Takeda Physiology, University of Wisconsin-Madison, USA 1St. Boniface General Hospital Research Centre, University of Manitoba, 2 Myosin binding protein C (MyBP-C) is a thick filament accessory protein Winnipeg, Canada, Aoto Hospital, Jikei University, Tokyo, Japan that has both structural and regulatory roles in striated muscle contraction. Although antiplatelet agents such as sarpogrelate (SAR) and cilostazol We are studying the roles of the cardiac isoform of MyBP-C in mouse (CIL) are used for the treatment of peripheral vascular disease, very little models in which the cMyBP-C gene has been disrupted, resulting in ablation information regarding their actions in ischemic heart disease is available. In of the protein, and in mice expressing mutant protein in which residues an experimental model of congestive heart failure (CHF) due to myocardial that are phosphorylated in vivo by PKA have been replaced with ala or infarction (MI), clinical signs of CHF and cardiac remodeling as well as asp. Ablation of cMyBP-C results in a cardiac phenotype similar to many depression in cardiac performance due to chronic coronary occlusion were inherited cardiomyopathies in humans, i.e., septal hypertrophy, increased attenuated by both SAR and CIL. Furthermore, alterations in the activities, protein content and gene expression of myofibrils and sarcoplasmic reticulum arrhythmic activity, and systolic and diastolic dysfunction. Studies of isolated were mitigated by SAR and CIL. However, unlike CIL, SAR reduced the myocytes from wild-type and null mice suggest that cMyBP-C regulates severity of ventricular trachycardia as well as mortality due to CHF. While the kinetics of cross-bridge interaction with actin, a mechanism that is treatment of control animals with SAR depressed whereas that with CIL lost in the null mouse. Studies of myocytes from mouse lines expressing augmented the incidence of arrhythmias due to acute MI. SAR was also phosphorylation mutants of cMyBP-C indicate that PKA stimulation of found to decrease 5-hydroxytryptamine induced vascular cell proliferation, an contraction kinetics in myocardium is in large part due to phosphorylation important component of events for the development of atherosclerosis. These of cMyBP-C, which appears to relieve a structural constraint on myosin and results suggest that some antiplatelet agents like SAR may be beneficial for increases the likelihood of myosin binding to actin. Studies involving EM of the treatment of atherosclerosis as well as MI induced arrhythmias and CHF. myofilaments and 3D reconstructions of cardiac thick filaments support our (Supported by a grant from the Canadian Institutes of Health Research) model of cMyBP-C function.

WD II-3-7 WD II-3-8 MULTI-SCALE APPROACH FOR THE UNDERSTANDING Ca2+ CHANNELS AND CARDIAC FUNCTIONS OF CARDIAC FUNCTION Masaki Kameyama1, Li-ying Hao2, Etsuko Minobe1, Jian-jun Xu1, Dong-yun Han3, Hong-guang Nie4, Zahangir A Saud5, Wu-yang Wang1, Hadhimulya Seiryo Sugiura, Asuka Hatano, Takumi Washio, Jun-ichi Okada, 1 Hiroshi Watanabe, Toshiaki Hisada Asmara 1Department of Physiology, Kagoshima University, Japan, 2Department Department of Human and Engineered Environmental Studies, The 3 University of Tokyo, Japan Pharmaceutical Toxicology, China Medical University, China, Department of Pharmacology, Shenyang Pharmaceutical University, China, 4Department of To elucidate the mechanisms underlying cardiac adaptation process, we Pharmacology, Chaina Medical University, China, 5Department of Biochemistry have studied the function of myosin molecules and single isolated myocytes and Molecular Biology, Rajshahi University, Bangladesh (Circ Res 2006, Nature Protocols 2006) obtained from normal and abnormal Voltage-dependent calcium channels play important roles in the regulation of heart animal models. Although these approaches have promoted our understanding 2+ functions. The T (Cav3.1 and 3.2) and L (Cav1.2 and 1.3) types of Ca channels in pathophysiology, complex structure and elaborate regulatory sytem of contribute, via intracellular Ca2+ signaling, to pacemaker activity (nodal cells), the cardiac myocyte as well as heart tissue make it difficult to establish formation of the plateau potentials, E-C coupling and excitation-transcription a causal link between the microscopic abnormality and the macroscopic coupling. The Cav1.2 channel is a predominant type in ventricular myocytes and is phenotype. In order to overcome this problem, we have developed a multi- known to be up-modulated by cAMP-protein kinase A signaling system. Calmodulin scale heart simulator using the finite element method, in which contraction (CaM) and Ca2+/CaM-dependent protein kinase II (CaMKII) also modulate activity 2+ and relaxation of the heart as well as the resulting blood flow are reproduced of Cav1.2 channel in Ca - dependent and independent manners. Facilitation and based on the dynamics of functional molecules. As a part of this project, we depression of channel activity induced by elevating [Ca2+]i is suppressed by CaM have also developed a finite element model of the cardiomyocyte with its inhibitors (calmidazolium and chlorpromazine) and modulated by CaMKII inhibitors sub-cellular structure and molecular mechanism. The model successfully (KN-62 and autocamtide 2-related inhibitory peptide). Direct application of CaM to reproduced experimental observations thus serving as a tool to evaluate our the channels in cell-free patches produces both facilitatory and inhibitory effects on experimental findings in the wide range of environment. channel activity, providing a bell-shaped curve in the concentration-activity relation.

Roles of CaM and CaMKII in the regulation of Cav1.2 channel activity and its relevance to heart functions will be discussed.

IUPS 2009 July 27 - August 1, 2009 in Kyoto 23 WD II-3-9 WD II-3-10 HOW DO CATECHOLAMINES PRODUCE ROLE OF SUBCELLULAR REMODELING IN THE ARRHYTHMOGENIC CALCIUM RELEASE? PATHOGENESIS OF CARDIAC DYSFUNCTION IN David Eisner, Takeshi Kashimura, Andrew W Trafford, HEART FAILURE Luigi A Venetucci Naranjan S. Dhalla1, Nobuakira Takeda2 Unit of Cardiac Physiology, University of Manchester, UK 1Department of Physiology, Institute of Cardiovascular Sciences, University 2 Beta-adrenergic stimulation can produce cardiac arrhythmias. In of Manitoba, Canada, Department of Medicine, Aoto Hospital, Jikei catecholaminergic polymorphic ventricular tachycardia (CPVT) mutations University, Tokyo, Japan in the sarcoplasmic reticulum Ca release channel (Ryanodine Receptor, Since both sarcolemma (SL) and sarcoplasmic reticulum regulate the intracellular RyR) result in lethal arrhythmias on exercise. These arrhythmias result concentration of Ca2+, whereas myofibrils (MF) are intimately involved in the from waves of Ca release from the SR during diastole activating delayed process of cardiac contraction and relaxation, these subcellular organelles are known after-depolarizations (DADs). Even in the absence of these mutations, high to participate in determining the status of cardiac function. Several studies have concentrations of beta-adrenergic agonists can result in DADs. Ca waves indicated that the activities of SL Na+-K+ ATPase and Na+-Ca2+ exchanger, SR Ca2+- occur when the SR Ca content exceeds a certain threshold level. Here we release channels and Ca2+-pump proteins, as well as MF Ca2+-stimulated ATPase were investigate the cellular mechanism by which beta adrenergic stimulation leads depressed in congestive heart failure (CHF) due to myocardial infarction. Alterations to Ca waves and DADs. There are two possibilities. (1) Phosphorylation of in these subcellular activities were observed to be associated with corresponding the L-type Ca current and/or phospholamban increasing the SR Ca content changes in SL, SR and MF protein content and mRNA levels in the failing hearts. or (2) phosphorylation of the RyR decreasing the threshold. In previous work Treatments of the heart failure animals with imidapril, an ACE inhibitor, or propanolol, (Venetucci et al, Circ Res 100, 105-111) we have shown that beta-adrenergic a β-adrenoceptor blocker, improved heart function and attenuated subcellular enzyme stimulation find that beta-adrenergic stimulation does not decrease threshold. activities as well as both the gene and protein expressions. These results indicate We conclude that the occurrence of arrhythmogenic Ca waves during beta- that remodeling of subcellular organelles with respect to changes in biochemical adrenergic stimulation results from an increase of SR Ca content and not composition and molecular structure plays a critical role in the genesis of cardiac

Whole-day symposia from changes of RyR phosphorylation. dysfunction during the development of CHF. (Supported by a grant from the Canadian Institutes of Health Research)

WD II-3-11 WD II-4-1 RENNIN-ANGIOTENSIN SYSTEM PLAYS AN PROF. MASAYOSHI GOTO, A DISTINGUISHED IMPORTANT ROLE IN THE PATHOGENESIS OF DCM IN CARDIAC PHYSIOLOGIST MOUSE Tsuguhisa Ehara Kenichi Hongo1, Satoshi Morimoto1, Jin O-Uchi2, Yoichiro Kusakari2, Department of Physiology and Anatomy, Saga University School of Kimiaki Komukai1, Makoto Kawai1, Michihiro Yoshimura1, Medicine, Japan Sachio Morimoto3, Iwao Ohtsuki2, Nobuakira Takeda4, Satoshi Kurihara2 Professor Masayoshi Goto (1921-2001), a respectable scientist in the field 1Department of Cardiology, The Jikei University School of Medicine, Japan, of heart physiology, was Professor and Chairman of the Department of 2Department of Cell Physiology, The Jikei University School of Medicine, Japan, Physiology, Faculty of Medicine, Kyushu University, Japan, from 1959 3Department of Clinical Pharmacology, Kyushu University Graduate School of 4 to 1985. He accomplished many electrophysiological studies on various Medicine, Japan, Department of Internal Medicine, Aoto Hospital, The Jikei aspects of cardiac function. In particular, he contributed a great deal to University School of Medicine, Japan the elucidation of the cardiac excitation-contraction coupling mechanism. To investigate the pathogenesis of dilated cardiomyopathy (DCM) associated with troponin Using double-sucrose gap voltage-clamp method, Prof. Goto analyzed the mutations, we used the mouse in which troponin T mutation of familiar DCM (delta K210) was knocked-in (DCM mouse). Echocardiography revealed chamber dilatation and depressed relationships among the membrane voltage, membrane current, and the contractility compatible with DCM. DCM mouse died suddenly during 6 months after birth. To contractile tension in bullfrog atrial muscle. With an elegant procedure he 2+ measure intracellular Ca transient (CaT) and tension, we applied the aequorin method to left first identified the ICa-dependent (phasic) and ICa-independent (tonic) tension ventricular papillary muscle. In CaT, the amplitude significantly increased and the time course components in the developed contraction. The latter component was clearly prolonged. In contrast, in tension, the amplitude significantly decreased and the time course a mechanical manifestation of the Na-Ca exchange across the cardiac shortened. Relationship between the peak CaT and the peak tension shifted to the right, suggesting membrane. In parallel with this work, Prof. Goto shed light on the ionic the decrease in the Ca2+ sensitivity of the myofilament. We applied angiotensin II type 1 receptor blockade (candesartan) to mouse by adding to drinking water (10mg/kg/day). Survival rate was mechanisms underlying the inotropic action of various neural and humoral significantly improved by candesartan. Candesartan reduced left ventricular chamber size and factors on heart. For example, the negative inotropic effect of acetylcholine partially improved cardiac functions measured by echocardiography. However, the altered Ca2+ was first analyzed by his team. As an introduction to this symposium, I would handling was not fully improved. Therefore, rennin-angiotensin system plays a significant role in like to revisit some of the works of Prof. Goto, which greatly facilitated our the pathogenesis of DCM in mouse possibly by manipulating left ventricular remodeling. understanding of the cardiac function.

WD II-4-2 WD II-4-3 CARDIAC L-TYPE Ca CHANNEL AS THE OXYGEN MOLECULAR REGULATION AND MANIPULATION OF SENSOR OF THE HEART CARDIAC CaV1.2 CHANNELS 1 2 Martin Morad , Shahrzad Movafagh Henry M Colecraft, Xianghua Xu, Tingting Yang 1 Department of Cell Biology and Anatomy, Medical University of South Physiology and Cellular Biophysics, Columbia University, USA Carolina, USA, 2Cardiac Signaling Center Of USC, MUSC and Clemson Universities, USA L-type calcium (CaV1.2) channels are essential for cardiac excitation- contraction coupling, excitability, and regulation of gene expression. Cardiac Ca2+ channels have evolved as multi-functional molecular units. In heart, Modulation of CaV1.2 channels by intracellular signaling molecules and hypoxia alters ionic currents through mitochondrial redox regulation. Here we proteins is a powerful method to regulate cardiac physiology. Conversely, report a novel O sensing mechanism mediated by the C-terminal of Ca2+ channel 2 aberrant regulation of Ca 1.2 channels contributes to cardiac patho- and regulated by phosphorylation in rat heart. In patch-clamped myocytes rapid V physiological conditions including arrhythmias, hypertrophy, and heart (>50ms) exposure to N2 solutions immediately suppressed (25%) Ica by ~40s. failure. I will discuss our work on the potent regulation of cardiac CaV1.2 The inhibition of ICa on removal of O2 was reversed by phosphorylation(0.2mM 2+ channels by three distinct proteins─ auxiliary CaVβ subunits, Rem GTPase, cAMP). Inhibiting CDI by Ba , sensitized the channel to O2 so that IBa decreased 2+ and protein kinase A─ which engage in a surprising degree of crosstalk. by 40% within first ~10s of O2 removal. However phosphorylation of Ba carrying channel did not desensitize it to O2. Inhibiting CDI and CICR (5uM thapsigargin) in phosphorylated channels did not protect it against loss of O2 in contrast to Ba2+, suggesting critical role for permeation of Ca2+. Recombinant 2+ 2+ Ca channels showed similar O2 sensing property directly implicating a Ca dependent motif of the channel. Mutating 80 aa’s in the CaM/CaM kinase binding domains of the C-terminal of alpha 1C that removes CDI abolished the 2+ O2 sensitivity of the channel. Our findings suggest that cardiac Ca channels may serve as O2 sensors in heart and that Ca/CaM and CaM kinase binding domains of the channel are critical sites for this regulation.

24 IUPS 2009 July 27 - August 1, 2009 in Kyoto WD II-4-4 WD II-4-5 CARDIAC Na/Ca EXCHANGE: A TALE OF TWO SITES EXCITATION-CONTRACTION COUPLING Kenneth Philipson, Debra Nicoll, Michela Ottolia, Xiaoyan Ren, Donald M. Bers Gabriel Mercado, Vincent Chaptal, Jeff Abramson Pharmacology, University of California, Davis, USA Department of Physiology, UCLA, USA Excitation-contraction coupling (ECC) in cardiac myocytes is initiated by The Na/Ca exchanger transports Ca but is also strongly regulated by Ca due activation of Ca influx via Ca current, which triggers release of additional Ca to two Ca binding domains (CBD) located within a large intracellular loop. from the sarcoplasmic reticulum (SR). The gain of ECC (SR Ca release/Ca Binding of Ca to these domains activates the exchanger and allows exchange current) increases at negative test voltages, which was attributed to increased transport to occur. We have determined the crystal structures of both of these single channel current. However, at negative voltages open probability (NPo) domains. The first of these domains (CBD1) binds 4 Ca ions. Mutation of declines as well. We separated the influence of each factor, and found that Ca-binding residues within CBD1 lowers the apparent affinity for regulatory reduction of either NPo or single channel current enhance gain and reduction Ca. In contrast, CBD2 binds 2 Ca ions. Mutation of Ca-binding residues of NPo is the cause of high gain at negative voltages (indicating a surplus within CBD2 eliminates Ca regulation. We are continuing these structural of Ca entry normally). SR Ca release terminates before complete depletion and mutational studies to better understand the roles of CBD1 and CBD2 of SR Ca (despite the positive feedback of Ca-induced Ca-release). We in regulation of the Na/Ca exchanger. In addition, we have two types of measured SR and cytosolic [Ca] and find that SR Ca release shuts off at a evidence that the Na/Ca exchanger forms a dimeric structure. First, exchanger specific free [Ca]SR, regardless of diastolic [Ca] SR or Ca release rate. This proteins can be crosslinked to each other through cysteine residues located at shows that low SR [Ca] terminates release via a regulatory effect (not due specific sites. Second, CFP- and YFP-labeled exchangers when coexpressed to exhaustion of local [Ca] or local [Ca]i-dependent inactivation). When display FRET signals indicating proximity. The magnitude of the FRET release rate is reduced, very long Ca sparks and blinks are observed (because signals is Ca dependent. The functional significance of the oligomerization is local SR [Ca] does not reach the shut-off level). Local SR sites refilled with being investigated. high variance, and we show by FRAP that this reflects variable junction connectivity.

WD II-4-6 LS16 (WD II-4-related luncheon seminar) CHANGES IN EC COUPLING IN HUMAN HEART THE MOLECULAR BASIS FOR HYPERCONTRACTILITY FAILURE AND PUMP DYSFUNCTION IN HYPERTROPHIC Mark B Cannell CARDIOMYOPATHY Whole-day symposia Department of Physiology, University of Auckland, New Zealand Richard L Moss Heart failure is a widespread problem and its study is complicated by the University of Wisconsin School of Medicine & Public Health, USA wide range of systems/mechanisms affected during disease progression. Hypertrophic cardiomyopathies (HCM) are the most common cause of While animal models are widely used, few match the protracted nature of unexpected sudden death in the young and are often due a point mutation human heart failure. We have examined the effect of changes in the human in a sarcomeric protein. HCMs are characterized by accelerated contraction action potential in a rat model. We find that the loss of the early "notch" in but cardiac function is paradoxically reduced. To study the basis for these repolarization has immediate effects on the efficiency of EC coupling. In phenomena, we developed a murine model of HCM due to disruption of addition, the resulting mismatch between trigger Ca and released Ca leads MYBPC3 encoding cardiac myosin binding protein C (MyBP-C), a thick to subsequent spontaneous SR Ca release which could contribute to the appearance of early after depolarizations. filament accessory protein that has structural and regulatory roles in striated Examination of structure in the human ventricular myocytes shows that it is muscle. Disruption results in ablation of cMyBP-C, which is similar to the quite different from that of the rat. Compared to rat, the number of couplons molecular phenotype in human patients with HCM due to C-terminal truncations is reduced and the degree of co-localization with L-type Ca channels of cMyBP-C. Hearts from these mice exhibit septal hypertrophy, increased is altered. In diseased myocytes, the reorganization of these critical EC chamber size, and reductions in stroke volume and ejection period. Myocardium coupling proteins is subtly altered with a preferential loss of what we term exhibits depressed twitch force-frequency relationships, accelerated relaxation, "close coupled" couplons. Our data suggest that in human EC coupling may and a loss of both frequency-dependent and β-agonist- induced acceleration be mediated by a combination of L-type Ca current triggered release plus a of relaxation. These results can be explained by acceleration of cross-bridge component due to the diffusion of Ca from adjacent release sites in a "fire- interaction kinetics due to ablation of cMyBP-C, which in turn accelerates twitch diffuse-fire" type mechanism. kinetics and reduces peak force or pressure and stroke volume.

WD II-4-7 WD II-4-8 NONLINEAR PROPERTIES OF CARDIAC SIGNALING IN SARCOMERES AND CARDIAC SARCOMERES: NOVEL INSIGHTS INTO THE ARRYTHYMIAS PHYSIOLOGY OF THE HEART R. John Solaro1, Yunbo Ke1, Katherine A Sheehan1, Norio Fukuda E. Eroume A Egom2, Ming Lei2 Department of Cell Physiology, The Jikei University School of Medicine, 1 2 Japan Physiology and Biophysics, University of Illinois at Chicago, USA, Division of Cardiovascular and Endocrine Sciences, University of Manchester, UK Cardiac muscle exhibits a variety of nonlinear properties. In this symposium, I will focus on two representative nonlinear properties that are consistently seen Studies of hearts expressing mutant sarcomeric proteins linked to hypertrophic in the supramolecular sarcomeric structure. First, cardiac sarcomeres produce cardiomyopathies provide strong evidence for an association of enhanced myofilament a greater magnitude of active force in response to stretch (length-dependent Ca-binding and arrhythmias. Thus, homeostasis requires fine control of intracellular activation, LDA), underlying the Frank-Starling mechanism of the heart. Our Ca release via Ca-release units (CRU) and sarcomeric Ca-binding. Well established analyses revealed that LDA is regulated via two different mechanisms; titin- adrenergic mechanisms enhance CRU activity via phosphorylation of channels and based interfilament lattice spacing reduction and “on-off” equilibrium of the phospholamban (PLB) and synchronously enhance power output of the sarcomeres thin filament state. Indeed, we found that tight coupling exists between titin- while depressing Ca-binding to the myofilaments. Our studies demonstrate that based lattice reduction and LDA, and that manipulation of the thin filament signaling through p21 activated kinase (Pak1), which activates protein phosphatase2A state by troponin exchange or change in the ionic condition modulates LDA. (PP2A), enhances sarcomeric response to Ca and depresses CRU activity via Second, under partial activation states, cardiac sarcomeres exhibit spontaneous dephosphorylation of sarcomeric proteins and channel proteins with no effect on PLB. oscillations (SPOC) with a saw-tooth waveform without changes in [Ca2+]. We We have now demonstrated that signaling through the sphingomyelin cascade and by found that the period of sarcomeric oscillations fell within the same range as the the S1P analogue, FTY720, to Pak1 serves as a mechanism to engage the Pak1/PP2A period of heartbeat of each animal species, suggesting that the auto-oscillatory module. In sino-atrial nodal cells, we have also developed evidence that signaling property of sarcomeres may contribute to myocardial beating in vivo. I will through Pak1/PP2A provides a novel mechanism for control of heart rate/ Overall discuss how these nonlinear properties are coordinately regulated by contractile these data demonstrate novel control of cardiac contractility and cardiac rhythm by proteins under physiologic conditions. Pak1 as well as potential new therapeutic targets.

IUPS 2009 July 27 - August 1, 2009 in Kyoto 25 WD II-4-9 WD II-4-10 KNOCK-IN MOUSE MODEL OF DILATED PATHOPHYSIOLOGY OF CARDIAC MUSCLES CARDIOMYOPATHY CAUSED BY TROPONIN Issei Komuro MUTATION Department of Cardiovascular Science and Medicine, Chiba University Sachio Morimoto Graduate School of Medicine, Japan Department of Clinical Pharmacology, Kyushu University Graduate School of Medicine, Japan Prolonged cardiac hypertrophy causes heart failure, but its mechanisms are largely unknown. Pressure overload, which is produced by constricting Dilated cardiomyopathy (DCM) is a cardiac muscle disorder characterized by transverse aorta of mice, induced cardiac hypertrophy without cardiac increased ventricular chamber volumes and systolic dysfunction, which often lead to sudden death or heart failure. Recent studies have revealed that mutations dysfunction until 14 days and initially promoted vascular growth in the heart in genes for sarcolemmal transmembrane protein, cytoskeletal proteins, nuclear by hypoxia-inducible factor-1 (Hif-1)-dependent induction of angiogenic envelope proteins and sarcomeric proteins are important causes of DCM. factors. After 14 days, however, there was no further cardiac hypertrophy Patients affected by a deletion mutation δK210 in the troponin T gene (TNNT2) but cardiac function was decreased, and the vascular density was reduced showed an early-onset severe phenotype with high incidence of sudden death with downregulation of Hif-1 and angiogenic growth factors. Inhibition of or heart failure death. We have created a knock-in mouse model of DCM, with angiogenesis prevented the development of cardiac hypertrophy and induced this mutation being introduced into the endogenous TNNT2 gene. Mutant mice systolic dysfunction. Sustained pressure overload induced an accumulation of developed marked cardiac enlargement (i.e., eccentric cardiac hypertrophy) with p53 that inhibited Hif-1 activity and thereby impaired cardiac angiogenesis systolic dysfunction and frequent sudden death, closely recapitulating the human 2+ and systolic function. Conversely, promoting cardiac angiogenesis by disease. Cardiac muscle fibers from mutant mice showed significantly lower Ca sensitivity in force generation than those from wild-type mice, suggesting that the introducing angiogenic factors or by inhibiting p53 accumulation developed mutation leads to eccentric cardiac hypertrophy to compensate for the reduced hypertrophy further and restored cardiac dysfunction under chronic pressure myocardial pump function caused by decreased myofilament Ca2+. Molecular overload. These results suggest that the anti-angiogenic property of p53 has

Whole-day symposia pathogenic mechanism and potential pharmacotherapy revealed through a crucial function in the transition from cardiac hypertrophy to heart failure extensive analyses of these mice will be discussed. and that angiogenic therapy is effective to treat heart failure.

WD II-4-11 WD II-5-1 ONTOGENETIC ASPECTS OF CARDIAC SENSITIVITY RENAL MEDULLARY VASA RECTA BLOOD FLOW TO ISCHEMIA; PROTECTIVE MECHANISMS IN THE REGULATION BY H2O2 AND NO IMMATURE HEART 1 1 2 Allen W. Cowley Jr. Bohuslav Ostadal , Ivana Ostadalova , Nobuakira Takeda , Zuzana Department of Physiology, Medical College of Wisconsin, USA Charvatova1, Libor Skarka1, Frantisek Kolar1, Jan Kopecky3 1Department of Developmental Cardiology, Institute of Physiology, Academy Salt-sensitive hypertension in Dahl S rats (SS) is accompanied by reductions of Sciences of the Czech Republic, Czech Republic, 2Jikei University, Tokyo, of renal medullary blood flow (MBF) and decreased nitric oxide (NO) 3 Japan, Departmernt of Adipose Tissue Biology, Institute of Physiology, production in the renal medulla (OM). Substitution of chr 13 of the Brown Academy of Sciences of the Czech Republic, Czech Republic Norway (BN) rat into the SS genome (SS.13BN) reduces medullary Immature mammalian heart is highly resistant to ischemia but the tolerance rapidly interstitial fibrosis and hypertension ~ 60%. OM of pre-hypertensive SS rats, decreases after birth. This phenomenon is probably associated with the switch from exhibited differentially expressed genes and proteins related to pathways of glycolytic to oxidative metabolism. Therefore, possible developmental changes in the oxidative stress and extracellular matrix formation. Other studies found that function of rat cardiac mitochondria have been analyzed. It has been found that the - specific content of cytochromes in mitochondria increased two-fold between birth and OM superoxide (O2 ) and H2O2 levels in pre-hypertensive SS rats were 2-3X day 30, similarly as the expression of adenine nucleotide translocase and peroxisome that found in SS.13BN rats with enhanced NADPH oxidase expression and proliferator-activated receptor α genes. In newborn animals, a single population of activity. Imaging studies of isolated thin tissue strips identified the medullary - mitochondria with a relatively high mitochondrial membrane potential (MMP) was thick ascending limb (mTAL) as the major site of excess O2 production. Pre- - observed; starting from the weaning period, a second population of mitochondria hypertensive SS rats exhibited a higher rate of O2 production and greater with a significantly lower MMP appeared. Moreover, in adult animals ischemia/ diffusion from mTAL to surrounding vasa recta pericytes and reduced MBF. reperfusion (I/R) involves the opening of a mitochondrial permeability transition pore In summary, gene and proteomic expression data together with a variety of (MPTP); this process can be prevented by sanglifehrin A (SfA), a potent inhibitor of - MPTP. However, the inhibition of MPTP has no protective effect on the I/R injury in physiological studies indicate that excess production of O2 and H2O2 in the the immature heart. These results support the idea that ontogenetic development of OM together with reduced bioavailability of NO contribute importantly to efficiency of mitochondrial energy conversion may be reflected in the mechanism of reduced MBF, sodium retention, hypertension and renal injury found in the cardiac resistance to oxygen deprivation. outer medulla of the SS rat.

WD II-5-2 WD II-5-3 PERIVASCULAR NITRIC OXIDE GRADIENTS SHEAR STRESS MECHANOTRANSDUCTION NORMALIZE TUMOR VASCULATURE VIA ENDOTHELIAL ATP RECEPTORS AND ITS Dai Fukumura, Satoshi Kashiwagi, Kosuke Tsukada, PHYSIOLOGICAL ROLE IN THE VASCULAR SYSTEM Rakesh K. Jain Joji Ando, Kimiko Yamamoto Laboratory of System Physiology, Department of Biomedical Engineering, Department of Radiation Oncology, Edwin L. Steele Laboraotry, Graduate School of Medicine, University of Tokyo, Japan Massachusetts General Hospiatal and Harvard Medical School, USA Endothelial cells (ECs) respond to shear stress generated by flowing blood and Normalization of tumor vasculature is an emerging strategy to improve change their cell function and gene expression. Previously, we demonstrated that cytotoxic therapies. Here we show that eliminating nitric oxide (NO) Ca2+ signaling plays an important role in the shear stress mechanotransduction. production from tumor cells via NO synthase silencing / blockade, Cultured human pulmonary artery ECs (HPAECs) showed Ca2+ influx via establishes perivascular NO gradients in tumors grown in various organ sites an ATP-operated cation channel, P2X4, in response to shear stress. We in mice and normalizes the tumor vasculature, resulting in improved tumor recently found that shear-induced activation of P2X4 requires endogenously oxygenation and response to radiation treatment. Creation of perivascular NO released ATP, and that shear stress induced HPAECs to release ATP, which gradients may be an effective strategy for normalizing abnormal vasculature. was mediated by cell-surface ATP synthase located in caveolae. To gain insight into the physiological role of this ATP receptor-meidated shear stress mechanotransduction, we generated P2X4-deficient mice. P2X4-/- mice do not exhibit normal EC responses to shear stress, such as Ca2+ influx and subsequent production of nitric oxide, a potent vasodilator. P2X4-/- mice show impaired vasodilatory responses to blood flow, and have higher blood pressure than wild-type mice. Adaptive vascular structural remodeling to chronic changes in blood flow is also impaired in P2X4-/- mice. Thus, P2X4-mediated shear stress mechanotransduction plays an important role in the vascular homeostasis, including the control of blood pressure and vascular remodeling.

26 IUPS 2009 July 27 - August 1, 2009 in Kyoto WD II-5-4 WD II-5-5 CO-MEDIATED MODULATION OF H2S GENERATION IT IS A SMELLY EDRF: HYDROGEN SULFIDE IN VIA CBS: A PUTATIVE TONIC MECHANISM FOR CIRCULATION NEUROVASCULAR UNITS Rui WANG Makoto Suematsu, Takayuki Morikawa, Katsuji Hattori, Department of Biology, Lakehead University, Canada Mayumi Kajimura Nitric oxide (NO) has been identified as an endothelium-derived relaxing Department of Biochemistry & Integrative Medical Biology, School of factor (EDRF) due to its endothelial origin and vasorelaxant effect. However, Medicine, Keio University, Japan not all endothelium-dependent vasorelaxations can be accounted by NO. Gases such as NO, CO and H2S account for unique mediators that target multiple rate- Recent studies have shown evidence that hydrogen sulfide (H2S) is another limiting enzymes in metabolic systems. We have recently discovered that CO inhibits EDRF. This gasotransmitter is generated in vascular endothelial cells as cystathionine beta-synthase (CBS) (Shintani, et al. Hepatology 2009), the rate-limiting enzyme regulating biosynthesis of thiols and H S, and exerts its ability to stimulate well as vascular smooth muscle cells catalysed by cystathionine gamma- 2 lyase (CSE) with L-cysteine as the substrate. Localization of CSE in vascular biliary fluid excretion via a mechanism involving 2H S modulation. Based on the fact that CO generated by heme oxygenase depends on amounts of O2 as a substrate, and endothelial cells is confirmed by both Western blot on isolated cells and that H2S serves as a vasodilatory gas, we hypothesize that hypoxia inhibits generation immunohistology staining on blood vessels. CSE in endothelial cells can of CO, and thereby alter responses in brain microcirculation. In brain slices of newborn be activated by the stimulation of muscarinic acetylcholine receptors and wild-type mice, suppression of CO by HO inhibitors causes marked microvascular the downstream calcium-calmodulin complex. Inhibition of endogenous vasodilation, while application of CO reverses it. This event did not occur in CBS-KO production of H2S leads to increased vascular contractile force whereas mice. In both mice, exogenous H2S causes vasodilation through mechanisms involving + supplement of exogenous H S relaxes vascular tissues in vitro. Targeted K channels. In this system, neural system constitutes a major CO generation, while 2 deletion of CSE in mice results in increased blood pressure and reduced astrocytes accounts for the H2S-generating site that might exert vasodilatory effects on pericytes in a paracrine manner. These results suggest that CO suppression might endothelium-dependent vasorelaxation. It is concluded that H2S, together cause compensatory vasodilation through mechanisms involving the CBS-H2S system. with other gasotransmitters, plays an important role in regulating vascular (supported by Global COE HMSB) contractility under both physiological and pathophysiological conditions.

WD II-5-6 WD II-5-7 ROLES OF TRANSCRIPTIONAL NETWORKS IN ATP DEVELOPS SUITABLE ENVIRONMENT FOR LYMPHATIC DEVELOPMENT METASTASIS OF CARCINOMA CELLS IN SENTINEL Tetsuro Watabe LYMPH NODE Whole-day symposia Department of Molecular Pathology, Graduate School of Medicine, Yoshiko Kawai, Toshio Ohhashi University of Tokyo, Japan Department of Physiology, Shinshu University School of Medicine, Japan During embryonic lymphatic development, Prox1 transcription factor We examined effects of supernatants of culture media of MDA-MB-231 plays important roles in sprouting and migration of a subpopulation of on the expression of adhesion molecules on human lymphatic endothelial blood vessel endothelial cells (BECs) towards VEGF-C expressing cells. cells (LECs), and evaluated whether the overexpression of adhesion We recently reported that Prox1-expressing BECs preferentially migrated molecules facilitated the attachment of carcinoma cells to LECs. The 48hr- toward VEGF-C via up-regulation of the expression of VEGF receptor 3 stimulation of MDA-MB-231 supernatant produced a significant expression (VEGFR3), which is preferentially expressed in lymphatic endothelial cells. of ICAM-1 on human LECs. On the other hand, ATP also caused a similar However, Prox1 is expressed in other tissues including liver and lens, and overexpression of ICAM-1 on human LECs to that produced by the molecular mechanisms that govern tissue-specific transcriptional activities supernatant of MDA-MB-231. The ATP- and MDA-MB-231 supernatant- of Prox1 remain to be elucidated. In the present study, we identified Ets-2 mediated responses were significantly reduced by treatment with suramin (a as a tissue specific co-regulator of Prox1. Ets-1 and -2 have been implicated purinergic P2X and P2Y receptor antagonist). In attachment assays, ATP or in angiogenesis, but not in lymphangiogenesis to date. We found that both MDA-MB-231 supernatant produced a significant increase in the attachment Ets-1 and -2 are expressed in human dermal LECs (HDLECs), and bind over- of carcinoma cells to human LECs. The treatment with suramin or anti- expressed Prox1 in human umbilical venous endothelial cells (HUVECs). ICAM-1 antibody caused a significant reduction of ATP- and the supernatant- Expression of Ets-1 and -2 synergistically induced Prox1-mediated up- mediated facilitation of the attachment responses. The findings suggest that regulation of VEGFR3 in HUVECs. Furthermore, Ets-1 and -2 induced MDA-MB-231 may release ATP, which produces the overexpression of Prox1-mediated chemotaxes of HUVECs towards VEGF-C. These results ICAM-1 on human LECs through activation of purinergic P2X and/or P2Y suggest that Ets-1 and -2, which are expressed in BEC and LEC, cooperate receptors, and then facilitates ICAM-1-mediated attachment of carcinoma with Prox1 to induce VEGFR3 to confer BECs with characteristics of LECs. cells to LECs.

WD II-5-8 WD II-5-9 INDIVIDUALIZED MANAGEMENT FOR GI CANCER LYMPHATICS REQUIRE TWO VALVE SYSTEMS: THE BASED ON LYMPHATIC MAPPING PRIMARY VALVES IN INITIAL LYMPHATICS AND Yuko Kitagawa, Hiroya Takeuchi FAILURE IN SECONDARY LYMPHEDEMA 1 2 Department of Surgery, Keio University, School of Medicine, Japan Geert W. Schmid-Schonbein , Walter L. Murfee 1 2 The first possible sites of metastases along the route of lymphatic drainage Bioengineering, University of California San Diego, USA, Dept. from the primary lesion are known as sentinel lymph nodes (SLN). Now, the Biomedical Engineering, Tulane University, New Orleans, LA, USA validity of the SLN concept has been actively investigated in various types Unidirectional lymph transport during periodic expansion and compression of solid tumors including gastrointestinal (GI) cancer.SLN detection in GI of initial and contractile lymphatics requires two valve systems. One valve is cancer is feasible by dye-guided and / or radio-guided method as already positioned upstream and one downstream of each fluid compartment subject reported in other organs A number of studies supporting the validity of to periodic compression. The primary (upstream) valves in initial lymphatics the SLN concept in early gastric cancer have been reported. A multi-center are at the junction between neighboring lymphatic endothelial cells while the prospective trial of SLN mapping for gastric cancer has been completed in secondary (downstream) valves are positioned in the lumen in form of funnel- Japan. A Clinical trial for individualized minimally invasive surgery for SLN shape bileaflets. The primary valves facilitate entry of interstitial fluid from the negative early gastric cancer has been started based on this result. There are tissue into the lymphatic lumen while secondary valves prevent reflow in the several studies demonstrating the feasibility and validity of the SLN concept lumen. PECAM and VE-Cadherin labeling shows a discontinuous distribution of in esophageal cancer. Definitive chemo-radiation using patient-specific junctions at primary valves with nonattached regions between endothelial cells irradiation field including SLNs detected by lympho-scintigram would be free to separate and form an open channel for fluid entry. Discontinuous junctions reasonable minimally invasive multimodal treatment for clinically node are most prevalent in initial lymphatics and change towards continuous junctions negative esophageal cancer. along the lymphatic tree. The initial lymphatics are highly susceptible to Using lymphatic mapping and SLN biopsy, management of clinically node endothelial apoptosis when bidirectional lymphatic endothelial pores are formed negative GI cancer would be changed from a uniform radical surgery to an and unidirectional transport from the tissue into the lumen fails. Supported by HL individualized minimally invasive and multimodal approaches. 10881.

IUPS 2009 July 27 - August 1, 2009 in Kyoto 27 WD II-5-10 WD III-6-1 TRANSPORT OF LYMPH: MECHANISMS AND GnRH NEURON MIGRATION: A LONG AND POTENTIAL FOR CORRECTION DANGEROUS ROAD? Anatoliy A. Gashev, David C. Zawieja Stuart Tobet1, Margaret Wierman2, Rao Veeramachaneni1, Department of Systems Biology & Translational Medicine, College of Gerald Schwarting3 Medicine, Texas A&M Health Science Center, USA 1 2 Biomedical Sciences, Colorado State University, USA, Department The transport of lymph is necessary for effective maintenance of the important of Endocrinology, University of Colorado Denver, Aurora, CO, USA, body functions - fluid and macromolecular homeostasis, gut lipid uptake and 3University of Massachusetts Medical School, Worcester, MA, USA immunity. The major units of lymphatic vessels (LV), lymphangions, pump Gonadotropin releasing-hormone (GnRH or LHRH) regulates the hypothalamo- lymph in self-regulatory mode by constantly adjusting their contractions to the pituitary-gonadal (HPG) axis in vertebrates. GnRH neurons that regulate the HPG complicated combinations of lymph pressures and flows. We demonstrated axis derive from progenitor cells in the nasal compartment and migrate along involvement of NO/cGMP/PKG-dependent pathways into adaptive vomeronasal nerve fibers across the cribriform plate into the forebrain. The long extrinsic/intrinsic flow-dependent reactions of LV. Aging severely alters these route provides diverse substrates to migrate upon and many secreted and diffusible mechanisms: degeneration of LV muscle cells occurs together with depletion of molecular factors for directional guidance. Experiments are showing potential roles eNOS and iNOS activation. New techniques for isolated LV provide possibilities for cytokines, particularly CXCL12, which is found along the migration route. to develop future measures to correct lymphatic dysfunctions. We optimized the GnRH neurons express the receptor for CXCL12 and when disrupted, migration organ culture technique to maintain the viability of isolated LV up to 10 days to was interrupted. Members of the TAM (Tyro 3/Axl/Mer) family of receptor tyrosine allow us to perform their monitoring under controlled low or high pressures. This kinases play important roles in the survival of GnRH neurons during migration. The model can be used as an accessible and reasonable simulation of the lymphatic diverse and prolonged migration of GnRH neurons make specific subpopulations of pump alterations during lymphedema. Using the adenovirus transfection GnRH neurons vulnerable to external stressors. Vinclozolin administered to pregnant we overexpressed SERCA2a gene in LV muscle cells; the functional tests rabbits decreased immunoreactive GnRH neuron numbers in selected brain regions in demonstrated diminished lymphatic tone (predicted lowered cytoplasmic Ca++) development and in adulthood. Either genetic defects or chemical pollutants affecting ++ Whole-day symposia and ~50% higher lymphatic phasic amplitude (predicted enhanced systolic Ca migration and/or the development of GnRH neurons could disrupt endocrine milieu release). causing significant anomalies.

WD III-6-2 WD III-6-3 BIOLOGY OF GnRH NEURON DEVELOPMENT IN THE ELECTROPHYSIOLOGICAL, ANATOMICAL, LIVING ZEBRAFISH EMBRYO AND MOLECULAR CHARACTERIZATION OF Nancy Lynne Wayne1, Siddharth Ramakrishnan1, GnRH NEURONS IN DWARF GOURAMI AND GFP David J Kozlowski2 TRANSGENIC MEDAKA 1Department of Physiology, University of California-Los Angeles School Yoshitaka Oka 2 of Medicine, USA, Department of Cellular Biology and Anatomy, Medical Department of Biological Sciences, Graduate School of Science, The College of Georgia, Augusta, GA, USA University of Tokyo, Japan Mechanisms that regulate the physiology of GnRH neurons during embryonic In general, the GnRH neuronal systems of vertebrates consist of one group of development are poorly understood because these neurons are difficult to identify hypothalamic neuroendocrine and one or two group(s) of extrahypothalamic in living tissue of wildtype animals. To overcome this obstacle, we generated a neuromodulatory GnRH neurons. By taking advantage of the brains of the dwarf stable line of transgenic zebrafish in which the GnRH promoter drives expression gourami and GnRH-GFP transgenic medaka expressing one of the three paralogous of green fluorescent protein (GFP). This new animal model provides us with the GnRH genes (H), we have characterized spontaneous electrical activities of all three unique opportunity to identify GnRH neurons for targeted electrophysiological different types of GnRH neurons. The hypophysiotropic POA-gnrh1 neurons show analysis in the intact, living embryo. The aim of this work is to reveal the irregular and episodic spontaneous electrical activities, which clearly show diurnal functional relationship between neuron development and spontaneous action changes in the overall frequencies of firing in the daily spawning medaka. Some potential firing in an intact neural network. We focused our attention on a subpopulations of gnrh1 neurons were identified by anatomy, gene expression, and population of GnRH neurons located in the terminal nerve (TN) associated with electrophysiology in the dwarf gourami POA. The extrahypothalamic midbrain gnrh2 the olfactory bulb, which is relatively easily accessible in the embryo. Results and terminal nerve (TN)-gnrh3 neurons show regular pacemaker potentials, which show that the probability of TN-GnRH neurons exhibiting spontaneous action depend on their intrinsic ion channel properties. Electrical coupling via gap junctions potential firing increased between 2 and 3 days post-fertilization, accompanied and synchronization of firing among TN-gnrh3 neurons were demonstrated by double by increased acquisition of an adult-like beating pattern of action potential firing. patch clamp recordings. It is suggested that these different properties are related to We will discuss these data in light of the effects of electrically silencing these their different functions, i.e., gnrh1 neurons as hypophysiotropic neuroendocrine neurons on axonal pathfinding during development. regulators, and gnrh2 and gnrh3 neurons as neuromodulators.

WD III-6-4 WD III-6-5 SLOW AFTERHYPERPOLARIZATION CURRENTS EXPRESSION OF ION CHANNELS IN GnRH NEURONS IN RAT GONADOTROPIN-RELEASING HORMONE AND THEIR ROLE IN BURST FIRING NEURONS Oline K Ronnekleiv, Chunguang Zhang, Martha A Bosch, Masakatsu Kato, Yasuo Sakuma Martin J Kelly Department of Physiology, Nippon Medical School, Japan Physiology and Pharmacology, Oregon Health and Science University, Gonadotropin-releasing hormone (GnRH) neurons constitute the final USA common pathway for the central regulation of reproduction. As for other GnRH is secreted in a pulsatile manner, and it is widely believed that the neurones, the discharge pattern of action potentials is closely related to pulsatility is the result of burst firing in GnRH neurons. The mechanism by the function of GnRH neurons. During the luteinizing surge, for example, which bursting activity is induced in GnRH neurons and modulated by estrogen GnRH neurons are believed to continuously fire. For this type of firing, is largely unknown. We have found that T-type calcium channel (T-channel) GnRH neurons must have a certain intrinsic property. To address this issue, subunits, which are important for regulating neuronal excitability, are expressed we have investigated the calcium-activated potassium currents underlying in GnRH neurons. The channel expression of Cav3.1 and 3.2 subunits are afterhyperpolarization (AHP), because these currents affect cell excitability. increased during the morning, but decreased during the evening by a surge- Dispersed GnRH neurons from adult GnRH-EGFP (enhanced green inducing treatment of 17beta-estradiol (E2). In concert with the mRNA fluorescent protein) transgenic rats were cultured overnight and then used expression, the T-current is also increased in the morning, yet remains increased for an electrophysiological experiment. The GnRH neurons show a slow during the evening with E2-treatment. Thus, the E2-induced mRNA expression AHP current but not a medium AHP current. The SK channel blocker apamin is transient, but the cellular function is maintained. Similarly, rebound excitation blocks this slow AHP current in GnRH neurons. In current clamp experiment, in GnRH neurons is increased with E2-treatment, whereas the activation/ apamin facilitates a generation of action potentials induced by a weak current inactivation kinetics are not altered. GnRH neurons also express HCN, GIRK (Kir pulse, while apamin facilitates the spike failure in relatively high current 3) and K-ATP (Kir 6) channels. Kir channels are regulated by E2 and closely pulse-induced train of action potentials. These results indicate that apamin- linked to the de-inactivation and recruitment of more T-channels for burst firing sensitive SK channels play dual roles in depolarization-evoked train of action of GnRH neurons. Therefore, T-channels may play a significant role in GnRH potentials depending on the stimulus intensity. excitability and burst firing. Supported by PHS grants NS43330, NS38809.

28 IUPS 2009 July 27 - August 1, 2009 in Kyoto WD III-6-6 WD III-6-7 DIFFERENTIAL REGULATION OF GnRH NEURONS BY KISSPEPTIN SIGNALING IN GnRH NEURONS ACUTELY APPLIED ESTRADIOL (E): DEPENDENCE ON Chunguang Zhang, Troy A Roepke, Oline K Ronnekleiv, DOSE AND ESTROGEN RECEPTOR SUBTYPE Martin J Kelly Sue Moenter, Zhiguo Chu Department of Physiology and Pharmacology, Oregon Health and Science Medicine, University of Virginia, USA University, USA In females, E inhibits or stimulates GnRH release depending on dose and Kisspeptin (KP) and its receptor, GPR54, are critical for reproductive cycle stage. We studied rapid effects of E using whole-cell recordings of development and for the regulation of GnRH secretion. It is well known that KP GnRH neurons in brain slices from ovariectomized mice. When ionotropic depolarizes GnRH neurons, but the underlying mechanism is not well known. GABA and glutamate receptors were blocked, high physiological doses of E We have found that KP, which is Gq-coupled, potently depolarizes GnRH increased action potential firing, slow afterdepolarization (sADP) amplitude neurons through a combination of inhibiting an inwardly rectifying potassium and I , and reduced afterhyperpolarizing potential (AHP) amplitude ADP (Kir) conductance and activating a nonselective cationic (TRPC) conductance. and I , with the latter appearing to involve calcium-activated potassium AHP Although GnRH neurons express nearly all of the TRPC channel subunits, channels. These effects were mimicked by an estrogen receptor (ER) B agonist and blocked by the antagonist ICI182780. These direct excitatory TRPC 4, 5 appear to be the most important in mediating the effects of KP based actions of E were via PKA activation. In contrast, low physiological doses of on the I-V relationship and pharmacology. We have further identified a robust E did not affect intrinsic properties. When ionotropic GABA and glutamate inhibitory effect of GABA on GnRH neurons via activation of the Gi,o-coupled receptors were not blocked, however, low E or ERa agonists reduced firing GABAB receptors that activate GIRK (Kir 3) channels to hyperpolarize the of GnRH neurons. ERa agonists decreased the frequency of potentially cell membrane. However, KP inhibits the GABAB-induced hyperpolarization excitatory GABA transmission to GnRH neurons, whereas ERB agonists in addition to activation of TRPC channels, leading to the robust activation of increased GABA transmission. Thus rapid intrinsic and network modulation GnRH neurons. Therefore, the activation of GABAB receptors in GnRH neurons of GnRH neurons by E depends on both dose and receptor subtype. Non- may provide increased inhibitory tone during estrogen negative feedback states genomic actions may provide a component of the overall response for both that is attenuated by kisspeptin during positive feedback. Supported by PHS negative and positive feedback regulation of GnRH neurons by E. grants NS43330, NS38809, DK68098.

WD III-6-8 WD III-6-9 DENDRITIC PROCESSING OF EXCITATORY SYNAPTIC PREPUBERTAL DEVELOPMENT OF ESTROGEN- INPUT IN HYPOTHALAMIC GONADOTROPIN KISSPEPTIN-GnRH NEURON POSITIVE FEEDBACK RELEASING HORMONE (GnRH) NEURONS MECHANISM Whole-day symposia Kelly J Suter Allan Herbison Department of Biology, University of Texas San Antonio, USA Physiology, University of Otago, New Zealand Recent studies have indicated that GnRH neurons have relatively long It is clear that kisspeptin and GPR54 are essential for normal reproductive dendrites. This raises the question of how synaptic inputs on distal dendrites functioning in mammals. In rodents, the kisspeptin neurons located in the contribute to action potential generation in these neurons. We used multi- rostral periventricular region of the third ventricle (RP3V) are likely to compartmental models of GnRH neurons to study the influence of synapses represent the principal kisspeptinergic input to the gonadotropin-releasing on GnRH dendrites. Simulations based on the assumption that dendrites were hormone (GnRH) neuron soma and dendrite. In mice, kisspeptin peptide passive indicated synaptic potentials induced by activation of distal dendritic is not apparent in RP3V cells until approximately postnatal day 15 and synapses were significantly attenuated at the soma when synapses were located at > 30% of the average dendritic length. Thus, passive integration thereafter increases to reach adult levels by the time of puberty onset of synaptic input was unlikely to be a suitable mechanism for much of the (approximately P30). Surprisingly, this prepubertal rise in kisspeptin peptide dendritic input. Based on dendritic recordings, we found GnRH dendrites levels in the RP3V is totally dependent upon circulating estradiol, suggesting initiate action potentials. Some GnRH neurons had exclusively dendritic sites that the hypothalamo-gonadal axis must be active in advance of the RP3V of action potential initiation. In other GnRH neurons, action potentials were kisspeptin neurons. As adults, Kiss1 and GPR54 knockout studies have initiated in both somata and dendrites. Based on simulations in model GnRH shown that both molecules are required for the estradiol-induced activation of neurons, synapses beyond 100 μm of dendrite length triggered dendritic GnRH neurons and consequent preovulatory LH surge. These studies suggest action potentials. The dual sites of spike initiation suggest a new view of the that an estradiol-kisspeptin-GnRH neuron positive feedback relationship control of GnRH neurons and control of reproductive hormone secretion, in develops prior to puberty and functions to enhance LH secretion leading which synaptic input to the dendrites of GnRH neurons plays a prominent to puberty onset and thereafter helps mediate estradiol's positive feedback role. HD-45436. actions upon GnRH neurons to generate the preovulatory LH surge.

WD III-6-10 WD III-6-11 KISSPEPTIN AND THE ONSET OF PUBERTY IN THE CENTRAL METASTIN/KISSPEPTIN IN THE RHESUS MONKEY REGULATION OF LUTEINIZING HORMONE Tony Plant SECRETION Cell Biology and Physiology, University of Pittsburgh School of Medicine, Hiroko Tsukamura, Kei-ichiro Maeda USA Graduate School of Agricultural Sciences, Nagoya University, Japan Puberty in monkeys is triggered by robust GnRH release after a protracted Kisspeptin (firstly named metastin) plays a critical role in regulating phase of juvenile development when pulsatility is arrested. This “up- reproductive function via stimulation of gonadotropin-releasing hormone down-up” pattern of GnRH unfolds in the absence of the gonad, and (GnRH) secretion in mammals including primates, ruminants and rodents. may be viewed to result from application of a neurobiological brake that We showed that endogenous kisspeptin is responsible for normal estrous restrains GnRH during juvenile development. Kisspeptin expression in the cyclicity and induction of luteinizing hormone (LH) surge in the rats. mediobasal hypothalamus (MBH) changes in parallel with GnRH release, Kisspeptin neurons are mainly located in the anteroventral periventricular and stimulation of GPR54 in the juvenile immediately induces a sustained nucleus (AVPV) and hypothalamic arcuate nucleus (ARC) in female rats. precocious pubertal pattern of GnRH release. Kisspeptin perikarya are It seems that the AVPV kisspeptin neuron is a target of positive feedback located in the arcuate nucleus (AN), while GnRH cells are found more action of estrogen, since the neurons contain estrogen receptor and estrogen laterally in the MBH. Extensive and intimate association between kisspeptin positively regulates kisspeptin expression. On the other hand, ARC kisspeptin and GnRH axonal projections in median eminence (ME) indicates that expression is negatively regulated by estrogen. In lactating rats, LH pulse and regulation of GnRH by kisspeptin may be exerted at the ME. It remains to be ARC kisspeptin expressions are profoundly suppressed, suggesting that ARC determined whether AN kisspeptin neurons are a component of a pubertal kisspeptin neurons may play a role in GnRH/LH pulse generation. Thus, the clock or growth tracking device, or serve as a link to relay information from two populations of kisspeptin neurons in the brain are closely involved in such a puberty control center to the GnRH neurons. Similarly, there is a need the generation of surge and pulse modes of GnRH/LH release and thereby to integrate kisspeptin signaling with that of other pathways (neurokinin controlling follicular development, steroidogenesis and estrous cyclicity in B, GABA, neuropeptide Y, glutamate, and glial derived growth factors) female rats. This research was in part supported by Grant-in-Aid from the implicated in the developmental regulation of the GnRH network. JSPS and PROBRAIN of Japan.

IUPS 2009 July 27 - August 1, 2009 in Kyoto 29 WD III-6-12 WD III-6-13 KISS1 AND REPRODUCTION: FOCUS ON ITS ROLE IN LIGAND-INDUCED-SELECTIVE-SIGNALLING (LISS) AT THE METABOLIC REGULATION OF FERTILITY THE GONADOTROPIN-RELEASING-HORMONE (GnRH) Manuel Tena-Sempere RECEPTOR Department of Cell Biology, Physiology and Immunology, University of Robert Peter Millar, Zhi-liang Lu, Kevin Morgan, Adam Pawson Cordoba, Spain Human Reproductive Sciences Unit, Medical Research Council, UK It is well known that reproductive maturation and function are gated by There is an emerging recognition that G protein coupled receptors can assume the state of body energy reserves. Such an impact of energy status on the different conformations which differentially couple to different intracellular reproductive axis is conveyed through a number of hormonal and metabolic signalling pathways. Different ligands are capable of differentially cues, whose nature is yet to be fully deciphered. Emergence of kisspeptins, stabilising these various receptor conformations. We have demonstrated this encoded by the Kiss1 gene, and their receptor, GPR54, as indispensable phenomenon at the GnRH receptor and termed it “Ligand-induced-selective- signals for normal pubertal maturation and gonadal function, has raised the signalling” (LiSS). We have determined the structure-activity relations of possibility that this system might participate in coupling of body energy GnRH analogues which induce differential signalling and the structural status and reproduction. Indeed, hypothalamic Kiss1 neurons seem to operate elements of the GnRH receptor which stabilise differentially signalling as a central conduit for conveying metabolic information onto the centers conformations through mutagenesis. Our findings have implications for the governing reproductive function, through a putative leptin-kisspeptin-GnRH differential signalling of GnRH in different tissues and the development of pathway. In addition, recent studies have disclosed the potential involvement more targeted GnRH therapeutics. of hypothalamic transcriptional regulators (e.g., Crtc-1) and intracellular energy sensors (e.g., mTOR) in this ‘metabolic’ regulation of Kiss1. Overall, it is proposed that such a network, involving peripheral afferents (such as leptin), central mediators (such as Crtc-1 and mTOR) and kisspeptin neurons,

Whole-day symposia is a key component of the physiological mechanisms coupling body energy status and reproduction.

WD III-6-14 WD III-6-15 KISSPEPTIN-GnRH: KEY REGULATORS OF DIFFERENT BEHAVIORAL ROLES FOR GnRH I AND II REPRODUCTION IN TELEOST Emilie F. Rissman Ishwar Parhar Biochemistry and Molecular Genetics, University of Virginia School of Brain Research Institute, Tan Sri Jeffrey Cheah School of Medicine, Medicine, USA Monash University Sunway Campus, Malaysia First I will review data which show that both reproductive behavior and The neuroendocrine brain integrates inputs from factors that stimulate and feeding in female musk shrews is affected exclusively by GnRH II given inhibit reproduction. Kisspeptin (product of Kiss1/kiss1 gene) was identified as an accelerator, a major gatekeeper of puberty through the regulation via intraventricular infusions. Next I will present new data assessing sex of gonadotropin-releasing hormone (GnRH) neurons. The availability differences in feeding responses to GnRH II. Adult males and females were of new tools for probing the genome has allowed us for the discovery of screened for eating style and classified as under- or over-eaters. Over a two genes encoding novel kisspeptins, kiss1 and kiss2 in non-mammalian series of trials we administered either the GnRH II specific analog, 135-18 vertebrates, the teleost. The structure of these molecules is highly conserved (provided by Dr. Robert Millar), or saline, via a subcutaneous injection prior during evolution. The kiss1 mRNA expression was observed in the ventro- to presenting the animal with a preferred food (mink chow) for one hour. In medial habenula and the periventricular hypothalamic nucleus, and the males a significant interaction between treatment and eating style was noted. kiss2 mRNA expression was observed in the posterior tuberal nucleus and Only males that over-ate were affected by the treatment. In over-eaters, the periventricular hypothalamic nucleus. During zebrafish development, pre-treated with the GnRH II analog reduced food intake. In females both kiss1, kiss2, gnrh2 and gnrh3 mRNA levels were significantly increased eating style and treatment had significant main effects but did not interact. at the start of the pubertal phase. In sexually mature female zebrafish, Kiss2 but not Kiss1 administration significantly increased gonadotropins All females treated with 135-18 prior to food presentation ate less during mRNA levels in the pituitary, which suggests that Kiss2 is the predominant the one hour feeding interval. This sex differences may be a dosage effect, regulator of gonadotropin synthesis in teleosts. This presentation will provide but regardless, these data show convincing that subcutaneous administration evidence for conserved multiple Kisspeptin-GnRH types and their roles in of 135-18 can prevent over eating in the musk shrew. These studies were reproduction. supported by NIH grant MH068729.

WD IV-7-1 WD IV-7-2 MOLECULAR PHYSIOLOGY OF THE HAIR CELL PRESYNAPTIC MOLECULAR REGULATION FOR RIBBON SYNAPSE TRANSMITTER RELEASE 1 1 1 1 Tobias Moser , Thomas Frank , Tina Pangsric , Jakob Neef , Sumiko Mochida1, Alexandra P Few2, William A Catterall2 1 1 1 2 Andreas Brandt , A C Meyer , Alexander Egner , Joerg Striessnig , 1Department of Physiology, Tokyo Medical University, Japan, 2Department 3 4 1 1 Ronald R Gregg , Christine Petit , Beat Schwaller , Darina Khimich of Pharmacology, University of Washington, USA 1 InnerEarLab, Dept. of Otolaryngology, University of Goettingen, Germany, 2+ 2 3 Transmitter release from presynaptic terminals is regulated by Ca sensor Dept. of Pharmacology, University of Innsbruck, Austria, University of 2+ Louisville, Louisville, USA, 4Pasteur Institute, Paris, France proteins (CaS). At the mouth of Ca channel a low affinity CaS triggers synaptic Sound coding at hair cell ribbon synapses is tightly regulated by Ca2+. Presynaptic Ca2+ vesicles fusion, while high affinity CaSs modulate transmitter release. The influx occurs at ~ 400 nm sized clusters of ~ 80 Cav1.3 channels and causes the rapid build- modulation should be related to the short-term synaptic plasticity that shapes the up of Ca2+ microdomains. Ca2+ microdomains vary in their voltage-dependence and maximal postsynaptic response to bursts of impulses. Activity-dependent modulation of amplitude. This presynaptic heterogeneity may serve to drive diverse spiking in postsynaptic presynaptic Cav2.1 channels mediated by a CaS, calmodulin, induces synaptic neurons. plasticity in cultured superior cervical ganglion neurons. A mutation of Cav2.1 Trafficking and function of Cav1.3 channels depend on Cavbeta subunits and the Cavbeta2 channels at the IQ-like motif in the C-terminus that blocks Ca2+/CaS-dependent 2+ subunit is essential for hearing. The Cav1.3 channels of hair cells show little Ca -dependent facilitation of the P/Q-type Ca2+ current markedly reduces facilitation of synaptic inactivation (CDI), probably due to efficient antagonism of CDI by Ca2+-binding proteins 2+ transmission. Deletion of the nearby calmodulin-binding domain, which inhibits such as CaBP1. Lack of the three cytosolic Ca binding proteins parvalbumin-alpha, CaS-dependent inactivation, substantially reduces depression of synaptic calbindin 28-k and calretinin speeds up CDI and augments sustained exocytosis. 2+ 2+ transmission. The depression and facilitation of synaptic transmission change Analysis of the apparent Ca dependence of exocytosis during Ca influx indicates that 2+ smaller nanodomains around one or few Cav1.3 channels, composing the observable Ca2+ along with time after Ca entry induced by an action potential, suggesting that the CaS modulation is dependent on the Ca2+ concentration at the mouth. These microdomains, regulate the exocytosis of a given synaptic vesicle. The molecular basis of 2+ Ca2+-dependent exocytosis in hair cells is far from being understood. The synaptic vesicle results demonstrate that residual Ca in presynaptic terminals can act through C2-domain protein otoferlin is essential for exocytosis and hearing and has been suggested CaS-dependent regulation of Cav2.1 channels to induce short-term synaptic as a candidate Ca2+ sensor. facilitation and rapid synaptic depression.

30 IUPS 2009 July 27 - August 1, 2009 in Kyoto WD IV-7-3 WD IV-7-4 GLYCINERGIC SYNAPTIC TRANSMISSION: CONTROL SOUND INTENSITY DEPENDENT COMPENSATION OF TIME COURSE AND IMPACT ON SYNAPTIC FOR THE SMALL ITD CUE IN THE CHICKEN PLASTICITY Harunori Ohmori Laurence Trussell, Veeramuthu Balakrishnan, Hai Huang, Department of Physiology, Kyoto University, Japan Tao Lu Interaural time difference (ITD) is a major cue for sound source localization. Oregon Hearing Research Center, Oregon Health and Science University, However, animals with small heads experience small ITDs, making ITD USA detection difficult, particularly for low-frequency sound. We describe a Rapid glycinergic transmission provides the major source of synaptic inhibition in sound-intensity-dependent mechanism for compensating for the small ITD auditory brainstem pathways of mammals. We have been interested in the mechanisms cues in the coincidence detector neurons in the nucleus laminaris (NL) of that determine the functional properties of these glycinergic synapses. Studies of IPSC the chicken. In vivo single-unit recordings revealed an improved contrast timecourse reveal that some of the briefest IPSCs in the CNS occur in the auditory of ITD tuning in low-best-frequency (<1kHz) NL neurons by suppressing system. Analysis of the gating kinetics of glycine receptors showed that fast IPSCs the firing activity at the worst ITD, whereas the firing rate was increased represents the convergence to two mechanisms: a very short lasting synaptic pulse of with increasing sound intensity at the best ITD. In contrast, level-dependent transmitter combined with the corelease of both GABA and glycine. Ironically, there suppression was so weak in the middle-and high-best-frequency (>1kHz) NL are also very long-lasting glycinergic IPSCs in the auditory system; these slow events neurons that loud sounds led to increases in firing rate at both the best and the seem to reflect glycine pooling between synaptic release sites. Glycine spilling out of synaptic clefts appears to act at both post- and presynaptic glycine receptors. Its action worst ITDs. The suppression of firing activity at the worst ITD in the low- on postsynaptic receptors leads to the slow IPSCs. Our studies show that activation best-frequency neurons required the activation of the superior olivary nucleus of presynaptic glycine receptors enhances transmitter release through a very indirect (SON). The frequency-dependent suppression reflected the dense projection route: Outward Cl- current depolarizes the terminal, an effect which is amplified by from the SON to the low-frequency region of NL. Thus, the small ITD cues persistent Na+ current. This larger depolarization activates Ca2+ channels; it is the Ca2+ available in low-frequency sounds were compensated by a sound-intensity- accumulation through these channels which then leads to potentiation of exocytosis. dependent inhibition from the SON.

WD IV-7-5 WD IV-7-6 BEHAVIORAL AND PHYSIOLOGICAL STUDIES OF HIERARCHICAL PROCESSING OF INTERAURAL TIME SOUND LOCALIZATION IN THE CAT DIFFERENCES IN THE HUMAN BRAIN

1 2 1 Whole-day symposia Tom Chi Tien Yin , Daniel J Tollin , Janet L Ruhland David McAlpine 1 2 Department of Physiology, University of Wisconsin, USA, Department of Ear Institute, University College London, UK Physiology and Biophysics, University of Colorado, USA Neural mechanism of binaural hearing, particularly those contributing Techniques to record the activity of single neurons from behaving animals to sensitivity to interaural time differences (ITDs), have been of interest have been exploited with great success in the visual and motor systems but to a wide range of investigators, and long-standing models suggest ITD less so in the auditory system. We have developed a preparation for studying sensitivity takes the form of a local code, in which small populations of behavior and physiology of sound localization in the cat. We found marked sharply-tuned (for auditory space) neurons encode source location. Here, I improvement in the accuracy and precision of localization when the head present data from recent neuro-imaging studies in the midbrain and auditory was free, as compared to the head restrained condition. With the head free we cortex indicating that this model of spatial hearing cannot hold, and that found a new reflex in the pinna muscles which we call the vestibulo-auricular reflex (VAR) because of its similarity to the well-known vestibulo-ocular even the broadly-accepted (although largely untested) view that each brain reflex (VOR). When the cat turned its head to localize an acoustic or visual hemisphere processes the opposite side of space is not consistent with the target, the VAR counter-rotated the pinna on the head to maintain pinna data. These data are consistent with data from in vivo recordings in the position in space to compensate for head movement. The VAR persisted ascending auditory pathway, including patch-clamp recordings from single during passive rotation of the head which ruled out efference copy as the neurons in auditory cortex to static and moving sounds, suggesting that the origin of the VAR, strongly implicating a vestibular origin. We also studied neural representation of auditory space in the cortex is ‘panoramic’, with the psychophysics of an auditory spatial illusion, the precedence effect, while neural responses to spatial cues depending on the context in which that cue is recording from single cells in the inferior colliculus. The temporal dynamics presented. and spatial features of inhibition of the lagging sound were similar to the cat’s behavior and to that experienced by human subjects.

WD IV-7-7 WD IV-7-8 RIDING THE RETINAL WAVE: THE ASSEMBLY OF RETINAL SIGNAL PROCESSING BY STARBURST FUNCTIONAL CIRCUITS IN THE RETINA AMACRINE CELLS 1 1 2 Marla Beth Feller Z. Jimmy Zhou , Seunghoon Lee , Kyongmin Kim 1Ophthalmology & Visual Science and Cellular & Molecular Physiology, Department of Molecular and Cell Biology and the Helen Wills 2 Neuroscience Institute, University of California Berkeley, USA Yale University School of Medicine, USA, Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, USA The retina is comprised of parallel neural circuits that are responsible for encoding all information about the visual scene. The results of the Since the introduction of Dale’s principle of “one neuron releases one computations performed by these retinal circuits converge onto a class of neurotransmitter”, an increasing number of exceptions to this rule have cells, called retinal ganglion cells, who send their axons to the brain. A major been discovered in many areas of the central nervous system, indicating that corelease of multiple neurotransmitters by a single neuron may represent goal of my research is to understand how these circuits are wired up during a significant mode of neurotransmission. However, the physiological development. Mice are born with their eyes closed and light responses are significance and synaptic mechanism of neurotransmitter corelease remain first detected at postnatal day 10 (P10) and their eyes open at P14. During poorly understood. Here, we show that the corelease of ACh and GABA by these first two postnatal weeks, immature retinal circuits spontaneously the starburst amacrine cell in the retina is controlled differentially by two generate propagating bursts of action potentials termed retinal waves. There distinct synaptic mechanisms, subserves two spatially selective synaptic is a critical period of retinal development during which retinal waves and circuits, and plays two complementary roles in detecting image motion and light-evoked responses coexist. I will present data describing the maturation motion direction. The results reveal a unique advantage of neurotransmitter of a few different RGC types during this period of development, including corelease in synaptic integration and multi-task computation by a minimal direction-selective RGCs, and the development of the mosaic organization neuronal circuit. of receptive fields. These data indicate that even though the light response properties of individual RGCs are not mature at eye-opening, cellular interactions have already ensured the formation of nearly mature receptive field organization.

IUPS 2009 July 27 - August 1, 2009 in Kyoto 31 WD IV-7-9 WD IV-7-10 FUNCTIONAL SIGNIFICANCE OF GAP JUNCTIONS VOMERONASAL PROCESSING AND PHEROMONAL AMONG RETINAL BIPOLAR CELLS LEARNING IN MICE Masao Tachibana, Itaru Arai, Masashi Tanaka, Mie Gangi Peter Anthony Brennan Department of Psychology, Graduate School of Humanities and Sociology, Department of Physiology and Pharmacology, University of Bristol, UK University of Tokyo, Japan The mouse vomeronasal system utilizes over 200 different receptor proteins Bipolar cells (BCs), the second order neurons, play an important role in retinal information processing. We performed dual whole-cell recordings to detect volatile molecules, peptides and proteins, which mediate important from an Mb1 (ON) BC and a ganglion cell (GC) in the goldfish retinal slice effects on mouse social and reproductive behaviour, and endocrine state. preparation. A short depolarizing pulse applied to the Mb1-BC generated a Vomeronasal sensory neurons provide a wealth of chemosensory information prolonged EPSC with two components in the GC. The fast component was that could potentially be used to provide information about sex, social status evoked soon after the pulse onset. The slow component lasted after the pulse and individual identity of con-specifics. But as yet little is know regarding termination, and it disappeared by application of gap junction blockers. how this information is processed by the accessory olfactory system. These results suggest that the slow component of the evoked EPSC may be Inhibitory feedback from local interneurons in the accessory olfactory generated by inputs from electrically-coupled Mb1-BCs. Dual whole-cell bulb (AOB) appears to play an important role in gating the transmission recordings from neighboring Mb1-BCs revealed that gap junctions existed of chemosensory information to central vomeronasal areas, conferring between their dendrites. Neither rectification nor voltage dependence was context-dependence on vomeronasal chemosensory responses. For instance, found between gap junctions, which showed the characteristic of a band-pass noradrenergic-dependent changes in inhibitory feedback following mating, filter. A depolarizing current pulse injected into an Mb1-BC could generate a Ca2+ spike, which triggered a Ca2+ spike in an electrically coupled Mb1- can account for the selective recognition of male individuality chemosignals BC with a delay (ca. 10 ms). This result suggests that Ca2+ spikes may spread in the block effect. Furthermore, recent findings have along the network of electrically-coupled Mb1-BCs with a delay. Such demonstrated the integrated roles of the main olfactory and vomeronasal

Whole-day symposia transmission through the BC network may contribute to evoking the slow systems, which can not only respond to the same stimuli, but also project to component of EPSCs in the postsynaptic GCs. the same brain areas, and influence the same chemosensory responses.

WD IV-7-11 WD IV-7-12 MOLECULAR BASIS OF ODOR PERCEPTION IN THE FUNCTIONAL COMPARTMENTALIZATION IN THE MOUSE MAMMALIAN OLFACTORY BULB Hitoshi Sakano Kensaku Mori, Kei Igarashi, Hideyuki Matsumoto, Nao Ieki, Department of Biophysics and Biochemistry, Graduate School of Science, Myonho An University of Tokyo, Japan Department of Physiology, Graduate School of Medicine, University of In mammals dorant receptors (ORs) are multifunctional signaling molecules Tokyo, Japan that are involved in odor recognition, singular OR gene choice, and Glomerular sheet of the mammalian olfactory bulb forms odorant receptor projection of olfactory sensory neurons (OSNs) at both global and local maps. Individual olfactory bulbs contain two maps, lateral map and levels. For axonal projection, two types of OR-derived cAMP signals regulate medial map, and each map is organized into zones, domains and clusters. the transcription of two distinct sets of axon-guidance/sorting molecules. Our Kobayakawa et al. (2007) showed that this compartmental organization is recent studies indicate that in immature OSNs, intrinsic OR signals regulate functionally related to specific odor-induced behavioral responses. A specific type-I genes (e.g., Neuropilin-1 and Sema3A) for global axon targeting along gradients, whereas in mature OSNs, extrinsic signals regulate type-II genes subset of glomeruli in DII domain mediates the innate fear response to (e.g., Kirrel2/3 and ephrin-A/EphA) for local sorting of axon termini. predator odors, while a subset of glomeruli in DI domain mediates aversive The topography of a neural map is shaped by target-derived guidance cues. response to spoiled food odors. To further understand the functional meaning Our recent study demonstrated that the axon-axon interaction also plays an of the compartmental organization of the olfactory bulb, we combined the important role in topographic map formation. It was found that Neuropilin-1 methods of optical imaging of glomerular activity, single unit recordings of and its repulsive ligand Sema3A are responsible for axon sorting within odor responses from mitral and tufted cells, and single cell electroporation the bundle well before targeting in the OB. We have previously reported with a tracer dye. With the methods, we labeled fox-odor TMT responsive that OSN axons are further sorted at the olfactory bulb (OB) to refine the mitral and tufted cells and fatty acid responsive mitral and tufted cells. We glomerular map in an activity-dependent manner. The step-wise regulation report the dendritic and axonal projection pattern of functionally identified of axon guidance/sorting appears to be a general feature of topographic map mitral and tufted cells in the mouse olfactory bulb and olfactory cortex. formation in the brain.

WD IV-8-1 WD IV-8-2 SENSITIZATION FOR PAIN AND ITCH MECHANICAL HYPERALGESIA AFTER EXERCISE- Martin Schmelz INVOLVEMENT OF BRADYKININ AND NERVE GROWTH Dept. Anesthesiology Mannheim, University Heidelberg, Germany FACTOR Itch and pain are distinct sensations provoking opposing reflex patters with Kazue Mizumura complex interactions between them. Recent data show astonishingly similar Department of Neuroscience II, Research Institute of Environmental patterns of neuronal peripheral and central sensitization in itch and pain. More Medicine, Nagoya University, Japan recently, the important role of nerve growth factor (NGF) has emerged both Unaccustomed strenuous exercise, especially lengthening contraction (LC), in painful and pruritic diseases: NGF expression is high in inflamed tissues often causes mechanical hyperalgesia (tenderness and movement related pain), and activation of the NGF receptor trkA, on nociceptive neurons triggers typically after some delay that is called delayed onset muscle soreness. Such and potentiates pain-signalling by multiple mechanisms, including axonal muscle often contains hardening with sensitive spot in it, therefore, can be sensitization. Histamine dependent itch has been the main experimental used for the study of myofascial pain syndrome. Until now the mechanism for generating and maintaining this hyperalgesia is not known. In a rat model of approach to induce pruritus in humans, but clinically histamine-independent DOMS, we found muscle mechanical withdrawal threshold was decreased 1-3 types are more important. Thus, currently itch research is focussing on days after LC, and corresponding to this, mechanical sensitivity of muscular distinctions between histamine dependent and histamine independent nerve C-fibers was facilitated. We found by administrating the B2 receptor antagonist, fibers, which could explain itch without accompanying axon reflex erythma. HOE140, that bradykinin released during LC initiated the process through B2 It can therefore be expected that also pruriceptive nerve fibers have different receptors leading to mechanical hyperalgesia. NGF mRNA and protein in the LC classes, as do the C nociceptors. Different classes of pruriceptors could also muscle were increased when the muscle was hyperalgesic, and intramuscularly account for the various submodalities of pruritus reported by patients. Based injected anti-NGF antibody reversed mechanical hyperalgesia. Injection of HOE on the striking similarities of sensitization processes in chronic pain and itch 140 inhibited increase of NGF mRNA/protein in the LC muscle. Intramuscularly analgesic therapies have already been successfully used in treating chronic injected NGF sensitized muscle C-fiber afferents to mechanical stimulation in itch. 10-20 min, raising a possibility that NGF sensitized nociceptors in periphery similarly to heat sensitivity and resulting in DOMS.

32 IUPS 2009 July 27 - August 1, 2009 in Kyoto WD IV-8-3 WD IV-8-4 VISCOSUPPLEMENTATION TO SCREEN KNEE JOINT PAIN SIGNALLING THROUGH PURINERGIC NOCICEPTORS AGAINST INFLAMMATORY AGENTS: RECEPTORS OF MICROGLIA LOCAL ANALGESIA WITHOUT SIDE EFFECTS Kazuhide Inoue Robert F. Schmidt Department of Molecular and System Pharmacology, Graduate School of Dept. of Physiology, University of Wuerzburg, Germany Pharmaceutical Sciences, Kyushu University, Japan Viscosupplementation consists in the supplementation of pathologically Neuropathic pain is a type of debilitating pain that sometimes occurs after altered synovial fluid with an insufficient concentration of hyaluronan and nerve injury and is generally resistant to currently available treatments. We average molecular weight resulting in a lower than normal elasticity and have shown that activating P2X4R upregulated in spinal microglia after nerve viscosity, by intraarticular injection of elastoviscous hyaluronan or of cross- injury contributes to neuropathic pain(Nature, 2003), and the stimulation of linked derivatives (hylans). This procedure has for some time been used to P2X4Rs evokes release of brain-derived neurotrophic factor (BDNF) (Nature, reduce articular pain when injected into human osteoarthritic knee joints but 2005) to cause a neuronal hyper-excitability. The mechanisms underlying the mode of action of this procedure has remained unknown. Meanwhile activation of microglia following nerve injury remains to be determined. it became feasible to explore the effects of elastoviscous solutions on the Here we demonstrated that interferon-gamma (IFN-γ) can be an important cellular and molecular mechanisms involved in the production of neural factor that causes microglia activation in the spinal cord after nerve injury. signals by nociceptor fibers innervating the joint that are the origin of to We found that a single intrathecal administration of IFN-γ to normal animals pain sensations. Various functional types of joint nociceptor fibers have produces a long-lasting tactile allodynia and activation of microglia in the been identified in the last decades, with specific patterns of nerve impulse spinal cord. The expression of IFN-γ receptor mRNA in the spinal cord is discharges in response to mechanical stimuli that change following tissue localized predominantly in microglia. Furthermore, IFN-γ receptor-deficient injury and inflammation. This knowledge opened up new ways to explore mice exhibited a striking reduction in nerve injury-induced tactile allodynia at the level of the identified individual nociceptors the mode of action of and in the increase in microglial cells with hypertrophic morphology. These hyaluronan solutions. This brief review summarizes the clarifying results findings suggest that IFN-γ signaling through IFN-γR plays as a crucial obtained with these experimental approaches. trigger of microglia activation after nerve injury.

WD IV-8-5 WD IV-8-6 SUPRASPINAL GLIAL-NEURONAL INTERACTIONS CORTICAL RESPONSES TO PAIN CONTRIBUTE TO DESCENDING PAIN FACILITATION Karen Deborah Davis Whole-day symposia AFTER NERVE INJURY Division of Brain, Imaging and Behaviour, Toronto Western Research Ronald Dubner Institute, Canada Department of Pain and Neural Sciences, University of Maryland, USA Understanding the cortical mechanisms underlying acute and chronic pain The present study investigated the role of supraspinal glial-neuronal in humans is a challenge given the complexities of the subjective experience interactions underlying descending facilitation of neuropathic pain. An early of pain. Individual factors may impact effective treatment in some chronic and transient reaction of microglia and prolonged reaction of astrocytes were found after chronic constriction injury (CCI) of the rat infraorbital nerve in pain patients, especially for the so -called “functional or idiopathic pain the rostral ventromedial medulla (RVM), a major component of brain stem disorders”. However the development of specific applications of functional descending pain modulatory circuitry. There were prolonged elevations of and structural MRI combined with assessment of individual factors can now cytokines, tumor necrosis factor (TNF)-α and interleukin (IL)-1β, expressed provide insight into the neurobiological mechanisms underlying pain. In in RVM astrocytes at 14 d after injury. Intra-RVM injection of glial inhibitors this presentation, I will provide data obtained with “percept-related fMRI”, attenuated behavioral mechanical hypersensitivity after CCI. TNFR1 and IL- an approach that can extract specific cortical responses related to specific 1R, receptors for TNF-α and IL-1β, respectively, were expressed primarily perceptions. I will also present new structural MRI data that corroborates in RVM neurons exhibiting immunoreactivity to the NMDA receptor our functional MRI data in patients with irritable bowel syndrome. Finally, I (NMDAR) subunit NR1. Neutralization of endogenous TNF-α and IL-1β in will present data that reveals how individual factors, such as personality and the RVM significantly reduced CCI-induced hypersensitivity and attenuated NR1 phosphorylation. Finally, intra-RVM administration of recombinant attention, can impact pain-evoked brain responses subjects. TNF-α or IL-1β upregulated NR1 phosphorylation and caused an NMDAR- dependent hypersensitivity in normal rats. These studies demonstrate a novel contribution of supraspinal astrocytes and cytokines to the enhancement of descending facilitation of orofacial pain.

WD IV-8-7 WD IV-8-8 MUSCULOSKELETAL PAIN AS A FUNCTION OF NEURAL MECHANISMS OF CHANGE IN CNS PAIN AGE AND GENDER: CLINICAL AND EXPERIMENTAL PATHWAYS WITH ADVANCING AGE STUDIES Koichi Iwata 1 2 Maria Adele Giamberardino , Raffaele Costantini Department of Physiology, Nihon University School of Dentistry, Japan 1Department of Medicine and Science of Aging, G. D'Annunzio University 2 To evaluate the neural mechanisms of change in CNS pain pathways of Chieti, Italy, Institute of Surgical Pathology, G. D'Annunzio University of Chieti, Italy with advancing age, nocifensive behavior, neuronal recording and immunohistochemical studies have been conducted in aged rats. The paw Most forms of musculoskeletal pain complaints increase with age. This presentation withdrawal latency was significantly shorter and paw-licking occurrence will discuss physiologic and pathologic factors contributing to changes in muscle pain sensitivity with aging in the two sexes. The largest psychophysical study so far was lower in aged rats. Dorsal horn (DH) nociceptive neuronal activity was conducted in healthy humans indicates a progressive physiologic decrease in pain significantly higher in aged rats. Blocking of descending pathways resulted threshold (pressure/electrical stimuli) with age, particularly in men, in parallel with in a dramatic increase in neuronal activity in young rats but not in aged rats. an increased oxidative damage to muscle fibers. The prevalence of most pathologic Following paw inflammation, the changes in foot temperature and thickness conditions potentially painful for the muscle increases with age. It is the case of following CFA injection were greater and lasted longer in aged rats, whereas primary muscle pain, e.g., from microtraumas, and referred muscle pain from deep DH nociceptive neuronal activity did not change. The number of pERK-LI somatic structures, e.g., from osteoarthritic joints. In contrast, referred muscle pain cells in trigeminal spinal nucleus caudalis was slightly, but not significantly from viscera declines with age, as also shown by animal experiments. Rats with an larger after face capsaicin injection in aged rats. Naloxone pretreatment artificial ureteral stone present a progressive decrease in their direct (ureteral crises) significantly increased the number of pERK-LI cells in adult rats but not in and referred (lumbar muscle hyperalgesia) pain behavior with age, more accentuated in females. The clinical experience of muscle pain in the elderly thus appears the result aged rats. These suggest that the descending modulation system is impaired of a complex interaction between the physiologic process of aging and the modified and the DH nociceptive system becomes sensitized with advancing age and prevalence with age of pathologic algogenic conditions, with a different impact in the its excitability cannot be further increased by inflammation, resulting in the two sexes. pain abnormality in aged rats.

IUPS 2009 July 27 - August 1, 2009 in Kyoto 33 WD IV-8-9 WD IV-8-10 ANOCTAMIN 1 (TMEM16A), A CLONED CALCIUM- INFLAMMATION-INDUCED SHIFT IN SPINAL GABA-A ACTIVATED CHLORIDE CHANNEL AND ITS ROLE IN RECEPTOR SIGNALING NOCICEPTION Michael Seth Gold Uhtaek Oh Anesthesiology, University of Pittsburgh, USA College of Pharmacy, Seoul National University, Korea GABA-A receptor mediated presynaptic inhibition of afferent input to the Ca2+-activated chloride channels (CaCCs) is one of Cl- channels that is 2+ - spinal cord is critical for the establishment of nociceptive threshold. In the activated by intracellular Ca and mediates vectorial transport of Cl in secretory epithelia. In other tissues, CaCCs are also known to control the absence of injury, spinal administration of GABA-A receptor antagonists excitability of muscles and neurons. Despite its roles in physiological produce hyperalgesia and allodynia whereas GABA-A receptor agonists functions, molecular species has not been resolved. Recently, we discovered are analgesic. We recently demonstrated, however, that in the presence that TMEM16A (also called Anoctamin 1) is activated by intracellular Ca2+ of persistent inflammation, there is a shift in GABA-A receptor signaling and Ca2+-mobilizing signals. When over-expressed in Xenopus oocytes or in such that activation of spinal GABA-A receptors actually contributes mammalian cells, biophysical and pharmacological profiles of anoctamin 1 to inflammatory hyperalgesia: spinal administration GABA-A receptor (ANO1) resemble those of native CaCCs. For example, ANO1 is activated antagonists reverse inflammatory hyperalgesia while agonists exacerbate by intracellular Ca2+ in a voltage-dependent way, permeable to anions with - - - - it. This change in GABA-A receptor signaling appears to reflect several permeability sequence, I > Br > Cl > F . Furthermore, ANO1 currents were changes in primary afferent neurons including: 1) a hyperpolarizing shift blocked by non-specific Cl- channel blockers as well as CaCC selective in the threshold for action potential generation; 2) a depolarizing shift in blockers such as tamoxifen, fluoxetine, and mefloquine. In addition, ANO1 is expressed in transport epithelia and other tissues where endogenous CaCC the anion equilibrium potential; and 3) several changes that contribute to a currents are found. Because ANO1 is expressed highly in a subset of dorsal dynamic collapse of the chloride gradient. The implications of these changes root ganglion neurons, its role in modulating pain signals would be suspected. in GABA-A receptor signaling will be discussed.

Whole-day symposia Therefore, in this symposium, a putative role of ANO1 in nociception is discussed in more detail.

WD IV-8-11 WD IV-8-12 DIFFERENTIAL ROLE OF THE NKCC1 CO- SECONDARY HYPERALGESIA AND PRESYNAPTIC TRANSPORTER IN PRIMARY AND SECONDARY INHIBITION: ROLE OF CATION-CHLORIDE ALLODYNIA AND HYPERALGESIA COTRANSPORTERS Donna L. Hammond, Elijah P. Frakes, Yang-Hsi Tsai Fernando Cervero Anesthesia and Pharmacology, The University of Iowa, USA Anesthesia Research, McGill University, Canada + + - The Na -K -2Cl (NKCC1) co-transporter enables GABA to depolarize the In neuronal tissues, cation-chloride cotransporters regulate Cl- homeostasis central terminals of primary afferent neurons (PAD) and inhibit synaptic and the activity and/or expression of these transporters has important transmission. If PAD is sufficiently large, dorsal root reflexes are generated implications for the direction and magnitude of anion flow through GABA-A that contribute to neurogenic inflammation in the periphery. However, loss channels. NKCC1 is expressed by both DRG and trigeminal neurons as of NKCC1 can also facilitate synaptic transmission. This study examined demonstrated by in situ hybridization and immunohistochemistry. NKCC1 the role of NKCC1 in primary and secondary hyperalgesia. Sprague-Dawley knockout mice have deficits in thermal nociceptive thresholds and display rats underwent ligation of the L5 spinal nerve (SNL), intraplantar injection of complete Freund’s adjuvant (CFA) or injection of kaolin-carrageenan a decrease in A-beta-fiber-mediated touch-evoked allodynia following (KC) in the knee joint. Western blot analysis after SNL demonstrated a large capsaicin injection into the hindpaw. These results are consistent with the decrease in NKCC1 in the dorsal horn. Intrathecal (i.t.) injection of the hypothesis that NKCC1 is involved in alterations in spinal processing NKCC1 inhibitor bumetanide in naive rats produced mechanical allodynia leading to A-beta-fiber-mediated touch-evoked allodynia. Intra-colonic and so replicated the effects of SNL. These data suggest that loss of NKCC1 capsaicin injection in mice induces a significant increase in dorsal, spinal contributes to mechanical allodynia. However, i.t. injection of bumetanide phosphorylated NKCC1 that was rapid and transient. Long lasting changes alleviated the secondary mechanical allodynia and thermal hyperalgesia were also observed in membrane delivery of NKCC1 in the dorsal spinal cord produced by KC in the knee joint. It did not alleviate the primary mechanical lasting for at least 180 min after the stimulus and were likely not dependent allodynia or thermal hyperalgesia produced by CFA in the hindpaw. These on transcriptional alterations. These results suggest that phosphorylation of findings suggest a differential involvement of NKCC1 in mechanisms of NKCC1 might play a role in the initiation of hyperalgesia whereas trafficking primary and secondary hyperalgesia. is involved in maintenance of the hyperalgesic state

WD V-9-1 WD V-9-2 CALCIUM SIGNALING AND CALCIUM-REGULATED INTEGRATION OF CELLULAR SIGNALING IN THE ION CHANNELS IN EPITHELIAL CELLS REGULATION OF ENaC ACTIVITY Ole Holger Petersen Douglas C. Eaton, Bela Malik, Hui-Fang Bao Physiology, University of Liverpool, UK Department of Physiology, Emory University School of Medicine, USA Since all Na-transporting epithelia contain epithelial Na channel (ENaC) Yoshio Maruyama and I summarized our identification and characterization subunits, α, β, and γ, it is generally assumed that Na transport is via some of Ca2+-activated ion channels in epithelial cells in a review article published 2+ form of ENaC. Functional ENaC are formed by assembly of the three 25 years ago (Petersen & Maruyama, Nature 307: 693-696, 1984). Ca - subunits likely into a trimeric structure. Assembly occurs within the + 2+ and voltage- activated high-conductance K channels as well as Ca - endoplasmic reticulum (ER) and is inefficient. One would expect that the activated non-selective monovalent cation channels were located in the ratios of subunit amounts at the surface membrane should be fixed by the baso-lateral membranes of exocrine acinar cells. Ca2+-activated Cl- channels stoichiometry of the channels and that when Na transport changes, subunits were proposed to be located in the apical (luminal) membrane, but we only in the plasma membrane should change in unison; but this is not the case: proved this many years later (Park et al PNAS 98: 10948-10953, 2001). In the different membrane subunits change in a non-coordinated manner when recent years, high-resolution confocal and two-photon studies of [Ca2+] in the transport changes, implying that some of the subunits must be present in the cytosol, all major organelles and the immediate extracellular environment membrane unaccompanied by other subunits. Assembly of trimeric channels prevents access of Golgi glycosidases so that functional channels arrive at of pancreatic acinar cells have provided a detailed understanding of the surface membrane with only core ER glycosylation. Functional channels physiological (Petersen & Tepikin Annu Rev Physiol 70:273-299, 2008) and 2+ are associated with lipid rafts and association with inositol-lipid-containing pathological (Petersen & Sutton TIPS 27: 113-120, 2006) Ca signaling rafts stabilizes channels. Trimers are poly-ubiquitinated and internalized, 2+ processes. New data on the specific Ca stores involved in normal stimulus- but are often de-ubiquitinated and recycled through a post-Golgi pool; but if secretion coupling as well as initiation of acute pancreatitis will be discussed. not recycled, then they are disassembled by specific chaperone proteins and degraded separately.

34 IUPS 2009 July 27 - August 1, 2009 in Kyoto WD V-9-3 WD V-9-4 - + ROLES OF CLC Cl CHANNELS AND Cl /H AQUAPORINS AND KIDNEY DISEASES: INSIGHTS EXCHANGERS IN PHYSIOLOGY AND PATHOLOGY INTO MOLECULAR PATHOGENESIS Thomas J. Jentsch Sei Sasaki Physiology and Pathology of Ion Transport, Max-Delbrueck-Centrum Fuer Department of Nephrology, Tokyo Medical and Dental University, Japan Molekulare Medizin (MDC), FMP, Germany Total 13 aquaporin water channels (AQP) have been identified in mammals. CLC chloride channels and transporters constitute a gene family with members in all phylae. Mammalian members of the first branch of CLC At least 7 AQPs exist in the kidney and contribute to water transport and other proteins funtion as plasma membrane chloride channels, with three of them functions. Of these, AQP1 in proximal tubules and AQP2, 3, 4 in collecting ducts (ClC-2, ClC-Ka and ClC-Kb) being involved in transepithelial transport. ClC- transport water and contribute to urine concentration. AQP7 in the S3 proximal Ka and -Kb need the small beta-subunit barttin for transport to the plasma tubules works as a route of glycerol reabsorption in addition to little role of water membrane and protein stability. ClC-Kb is mutated in Bartter syndrome type transport. AQP11 is a member of new AQP sub-family with an atypical NPA box III, which is asscociated with renal salt and fluid loss. The barttin-encoding and localizes at intracellular vesicles. AQP11 knockout mice show a phenotype BSND gene is mutated in Bartter IV, which combines severe renal salt loss of polycystic kidney disease with similar intracellular signaling abnormalities with deafness. ClC-K/barttin is expressed in certain nephron segments and in observed in other cystic kidney disease models. the stria vascularis of the inner ear. Inner ear-specific barttin KO mice display AQP2 is the vasopressin-regulated water channel and its gene mutations cause congenital deafness and show that ClC-K/barttin is needed for the generation human nephrogenic diabetes insipidus (NDI), a disease showing inability to of the endocochlear potential. Members of the other two branches function concentrate urine. We have done gene-analysis and found AQP2 mutations in rather as Cl/H exchangers. They are located mainly on endosomes and lysosomes and are assumed to be involved in their acidification. Disruption 7 NDI families. Heterologous expression studies and knock-in mice harboring of ClC-5 leads to an impairment of proximal tubular endocytosis and human disease-causing mutations clarified molecular pathogenesis of the disease. Dent's disease, whereas the loss of ClC-7 causes osteopetrosis. The loss of We further identified AQP2 binding protein complex including actin and the lysosomal ClC-7 also leads to lysosomal storage disease, although the pH tropomyosin-5b. Dynamic regulation of the complex by PKA phosphorylation of of lysosomes is not changed. AQP2 itself was shown to trigger AQP2 trafficking to the apical membrane.

WD V-9-5 LS10-1 (WD V-9-related luncheon seminar) ROLE OF GAS CHANNELS IN THE … TRANSPORTER PROTEIN QUANTIFICATION FOR THE AND BEYOND FUTURE OF TRANSPORTER SCIENCE Walter F. Boron, Yuehan Zhou, Raif Musa-Aziz Tetsuya Terasaki, Jun-ichi Kamiie, Sumio Ohtsuki Whole-day symposia Department of Physiology & Biophysics, Case Western Reserve University, Division of Membrane Transport and Drug Targeting, Graduate School of USA Pharmaceutical Sciences, Tohoku University, Japan The traditional view had been that gases cross membranes simply by dissolving For the true understanding of transport physiology, one of the key issues in/diffusing through membrane lipid. However, the discovery of gas- would be to know an absolute expression amount of the membrane transport impermeable membranes and gas-conducting channels (AQPs and Rh proteins) protein. We have employed a focused proteomics to establish the method. raises the possibility that gas movement through channels may sometimes After trypsin digestion of membrane proteins, a stable isotope-labeled be physiologically important. Proximal tubules (PTs) have high apical and peptide of the transporter protein to be quantified was added as an internal basolateral AQP1 levels. We find that, compared to isolated perfused PTs from reference. The sample was analyzed by LC tandem mass spectrometry with wild-type mice, those from AQP1-null mice have a substantially lower rate of - multiplexed Multiple Reaction Monitoring (Multiplexed MRM) mode. The HCO3 reabsorption, and a substantially lower transepithelial CO2 (but normal - highly selectivity resulting from the two stages of mass selection yields HCO3 ) permeability. To clarify the mechanism of CO2 movement through AQP1, we expressed AQP1 in oocytes and recorded the transient rise in surface a very specific and sensitive determination of the targeted peptide of the transporter protein in the sample. Moreover, we developed an in-silico design pH (pHs) caused by CO2 influx. pCMBS (blocks 4 H2O pores) reduces the AQP1-dependent ΔpHs by 40%, reduces Pf by 50%, and also eliminates AQP1- method to select an appropriate peptide in the LC/MS/MS quantification. Using 300 channels of MS/MS, a simultaneous (~30 transporters) and highly dependent NH3 permeability. The C189S mutant is resistant to these effects. DIDS reduces AQP1-ΔpHs(CO2) by 60%, but has no effect on Pf or NH3 sensitive (~1 fmol/assay) absolute quantification method of the membrane permeability. C189S is fully DIDS sensitive. The combination of pCMBS + proteins has been established (Pharm. Res., 25: 1469-1483 (2008)). Our DIDS eliminates AQP1’s CO2 permeability. Thus, H2O, NH3, and 40% of CO2 method would enable us to understand the differential transport function move through monomeric (pCMBS-sensitive) H2O pores, whereas 60% of CO2 in the various tissues, ages, diseases and animal, and should have many takes another (DIDS-sensitive) pathway, possibly the central pore. potential applications for the future of transporter science.

LS10-2 (WD V-9-related luncheon seminar) WD V-9-6 LARGE SCALE PURIFICATION AND ASSAY OF ACTIVE TRANSPORTSOME IN RENAL ORGANIC SOLUTE HUMAN TRANSPORTERS FOR THE FUTURE OF TRANSPORT TRANSPORTER SCIENCES Yoshikatsu Kanai Yoshinori Moriyama Department of Pharmacology, Graduate School of Medicine, Osaka Division of Membrane Biochemistry, Okayama University Graduate School University, Japan of Medicine, Dentistry and Pharmaceutical Sciences, Japan Transportsome is the multi-molecular assembly composed of transport Transporters play key roles in fundamental biological processes through proteins (transporters, channels or pumps), regulatory proteins, scaffold transport of ions, metabolites and drugs through cell membranes. proteins and other functional cellular components such as metabolizing Understanding the mechanisms of action of transporters is crucial for enzymes, which are assembled by means of multiple protein-protein development of basic and pharmacological research. However, studies interactions and/or protein-lipid interactions. Transportsome is proposed as on the structure and function of human transporters have been hampered a functional unit of membrane transport. It is responsible for the interplay mainly because of lack of technology for a large scale preparation and of transporters and the linkage to cellular functions. Transportsomes assay of recombinant transporters. Recently we have developed a procedure assembled by PDZ proteins such as PDZK1 and NHERF1 are found to overcome this issue, which includes large scale expression of any in the apical membrane of renal proximal tubules. Many organic anion human transporters in insect cells, their purification and reconstitution transporters including URAT1 urate transporter and OAT4 are involved in into liposomes. According to the procedure, we usually obtain mg order the transportsome. It is essential for the efficient functional coupling of the of active and functionally stable human transporters. Very recently, the transporters. In the basolateral membrane of proximal tubules, prostaglandin procedure using E. coli for expression of human transporters was developed. (PG) transporter OAT-PG forms a unique transportsome that involves an Consequently we can obtain more than 20-50 mg purified, active and stable enzyme degrading PGs. This transportsome enables efficient transport/ transporters from one liter E. coli culture. I will update our studies on metabolism coupling. The roles of this transportsome in the clearance of PGs vesicular neurotransmitter transporters and drug transporters, and discuss the from extracellular space and efficient inactivation of PGs were confirmed in impact of the technology for the future of transporters sciences. the OAT-PG knockout mice. The physiological significance of transportsome in the renal handling of organic solutes will be discussed.

IUPS 2009 July 27 - August 1, 2009 in Kyoto 35 WD V-9-7 WD V-9-8 WNK KINASES AND CATION-CHLORIDE PARTNERS AND PATHWAYS OF THE NEWLY COTRANSPORTERS, NOVEL TRANSPORTSOMES SYNTHESIZED Na+,K+-ATPase REGULATING BLOOD PRESSURE Michael J. Caplan, Glen Farr, Michael Hull, Michael Morton Shinichi Uchida, Tatemitsu Rai, Eisei Sohara, Akihito Ohta, Department of Cellular and Molecular Physiology, Yale University School Motoko Chiga, Sei Sasaki of Medicine, USA Department of Nephrology, Tokyo Medical and Dental University, Japan The Na,K-ATPase generates the ion gradients responsible for transport WNK1 and WNK4 mutations have been reported to cause pseudohypoaldosteronism processes in the kidney. To perform this function, it must be restricted to type II (PHAII), an autosomal-dominant disorder characterized by the basolateral surfaces of renal epithelial cells. Little is known about the hyperkalemia, metabolic acidosis, and hypertension. Although the genes partner proteins and trafficking pathways that determine its subcellular were identified, little was known about the molecular pathophysiology distribution and modulate its activity. We have adapted a novel labeling of this disease in vivo. To clarify this, we generated an ideal mouse methodology to investigate the attributes of temporally defined cohorts of model of PHAII, Wnk4D561A/+ knock-in mouse. In the knock-in mice, Na,K-ATPase. We can observe directly the trafficking itinerary pursued phosphorylation of thiazide-sensitive sodium-chloride cotransporter by newly synthesized Na,K-ATPase and isolate newly synthesized Na,K- (NCC) at the potential phosphorylation sites by OSR1/SPAK kinases was ATPase in association with its collections of partner proteins. We find that increased and phosphorylated NCC was concentrated in the apical plasma the basolateral delivery of newly synthesized Na,K-ATPase occurs via a membranes of distal tubules. In addition, phosphorylation of OSR1/SPAK at pathway distinct from that used by other basolateral membrane proteins. We the phosphorylation site by WNK kinases was also increased in the knock- also detect an interaction between the Na,K-ATPase α-subunit and the coat in mice. Thus, the activation of WNK-OSR1/SPAK-NCC signal cascade protein, β-COP, a component of the COP-I complex. The α-subunit associates by the mutant WNK4 was identified as the pathogenesis of PHAII. This with β-COP immediately after its synthesis and this interaction is rapidly lost signal cascade was also found to be regulated by dietary salt intake through in association with the assembly of the α and β-subunits to form the pump aldosterone secretion in the wild-type mice, indicating that WNK-OSR1/ holoenzyme. Thus, we have developed tools that permit us to evaluate the

Whole-day symposia SPAK-NCC signal cascade constitutes a novel transportsome regulating trafficking pathways and partner proteins that regulate the epithelial Na,K- NaCl homeostasis and blood pressure. ATPase.

WD V-9-9 WD V-9-10 ROLES OF ZO-1 AND ZO-2 IN THE FORMATION FUNCTION OF EPITHELIAL K+ TRANSPORT IN INNER OF EPITHELIAL CELL-CELL JUNCTIONS AND EAR AND ITS INTEGRATION WITH MODELING PARACELLULAR TRANSPORT Hiroshi Hibino1, Fumiaki Nin2, Yoshihisa Kurachi1 1 2 1 Yuji Yamazaki , Kazuaki Umeda , Masami Wada , Shoichiro 1 3 1 Division of Molecular and Cellular Pharmacology, Department of Tsukita , Sachiko Tsukita Pharmacology, Osaka University, Japan, 2Department of Otolaryngology- 1Graduate School of Frontier Biosciences and Graduate School Head and Neck Surgery, Kyoto Prefectural University School of Medicine, of Medicine, Osaka University, Japan, 2Department of Molecular Japan 3 Pharmacology, Kumamoto University, Japan, Department of Cell Biology, The endocochlear potential (EP) of +80 mV in cochlear endolymph is essential for Kyoto University, Japan + audition. Although K transport via an epithelial tissue, stria vascularis, seems crucial Epithelial cell sheets play a crucial role in the epithelial barrier function and selective for formation of the EP, the mechanism remains uncertain. We used multi barreled permeability in that ions and solutes selectively permeate through epithelial cell sheets by electrodes to measure the potential, K+ concentration ([K+]), and input resistance in the transcellular and paracellular transports. In contrast to transcellular transport, paracellular each compartment of the stria vascularis. The stria faces two fluids, perilymph and transport is established by the cell-cell adhesion which is generally characterized by the endolymph, and contains an extracelluar compartment, the intrastrial space (IS), pairing of the belt-like arranged adherens junctions and tight junctions. Tight junctions are surrounded by two epithelial layers, the marginal cell layer and that composed of required for the paracellular permselective barrier and among the tight junctional membrane intermediate and basal cells. The IS exhibits a low [K+] and a positive potential called proteins, claudins are the most critical components for the function of tight junctions. Here, + we have found that ZO-1and ZO-2, the membrane scaffolding proteins of tight junctions, the intrastrial potential (ISP). We proved that the ISP was formed by K diffusion across the apical membranes of intermediate cells and a major fraction of the EP. We play essential roles in polymerization of claudins to organize the barrier funtions of tight + junctions, by generating the ZO-1(ko)/ZO-2(kd) Eph4 cells in which the expressions of further found that the IS was electrically isolated from neighboring fluids and the K ZO-1/-2 are suppressed by the knockout and knockdown strategies. Furthermore, we have diffusion potential across the apical membranes of marginal cells also contributed to + found that for the tight junctional barrier function, the proper establishment of belt-like the EP. Thus, the EP depends on two K diffusion potentials and an electrical barrier adherens junction is required in a ZO-1/-2-dependent fashion. Thus, the cell-cell junctional in the stria vascularis. Computer simulation quantitatively demonstrates the process membrane-scaffolding ZO-1/-2 are the total organizer of epithelial paracellular permselective of formation of the EP and suggests particular ion transport pathway via the stria in barrier. pathological condition.

WD VII-10-1 WD VII-10-2 SYSTEMS BIOLOGY OF MAMMALIAN CIRCADIAN GENETIC INTERACTIONS IN THE MAMMALIAN CLOCKS CLOCKWORK Hiroki R Ueda Elizabeth Maywood1, Johanna E. Chesham1, Patrick M. Nolan2, Laboratory for Systems Biology, RIKEN, CDB, Japan Andrew S. Loudon3, Michael H. Hastings1 Mammalian circadian clock system is a complex and dynamic system 1Neurobiology Division, MRC Laboratory of Molecular Biology, Cambridge, consisting of complicatedly integrated regulatory loops and displaying the UK, 2MRC Mammalian Genetics Unit, Harwel, 3Faculty of Life Sciences, various dynamic behaviors including i) endogenous oscillation with about University of Manchester, UK 24-hour period, ii) entrainment to the external environmental changes At the cellular level the mammalian clockwork is viewed as a series of interlocked (temperature and light cycle), and iii) temperature compensation over the transcriptional/translational feedback loops. How this whole process takes ca. 24h is wide range of temperature. not fully understood; but for the clock to run effectively changes in protein stability, The logic of such biological networks such as circadian clocks is difficult to phosphorylation state and subcellular localization must be tightly regulated. A recently 1-3 elucidate without (1) comprehensive identification of network structure , identified mutation in an F-box protein Fbxl3 (Afterhours; Godhino et al Science (2) prediction and validation based on quantitative measurement and 2007) revealed that the period lengthening effect of this point mutation resulted from 4 perturbation of network behavior , and (3) design and implementation a selective preservation of CRY proteins. Current work is underway to determine 5 of artificial networks of identified structure and observed dynamics . In whether the endogenous target for Fbxl3 is CRY1or CRY2. A casein kinase 1 epsilon this symposium, we will report on the current progress in the analysis and mutation (CSK1e; Loudon et al Neuron 2008), had the effect of speeding up the clock. synthesis of mammalian circadian clocks. This mutation was found to facilitate the degradation of endogenous PER proteins. By Reference crossing Fbxl3 and CSK1e mutant mice, we demonstrated that the CSK1e mutation 1. Ueda, H.R. et al, Nature 418, 534-539 (2002). reversed the period lengthening effect of the Afterhours mutation in a dose-dependent 2. Ueda, H.R. et al, Nat. Genet. 37, 187-192 (2005). manner. This demonstrates the complexity of the genetic network in regulating clock 3. Sato T K, et al, Nat Genet. 38, 312-9 (2006). speed and that these two mutations have independent substrate targets for degradation. 4. Ukai H, et al, Nat Cell Biol. 9, 1327-34 (2007). These results will help inform on the precise timing and order of biochemical events in 5. Ukai-Tadenuma M, et al, Nat Cell Biol. 10, 1154-63 (2008). the molecular loop.

36 IUPS 2009 July 27 - August 1, 2009 in Kyoto WD VII-10-3 LS9 (WD VII-10-related luncheon seminar) CLOCK GENES AND ITS DISEASES CIRCUIT-BREAKERS: OPTICAL TECHNOLOGIES FOR Hitoshi Okamura PROBING NEURAL SIGNALS AND SYSTEMS Department of Systems Biology, Kyoto University, Japan Feng Zhang Departments of Bioengineering and Chemistry, Stanford University, USA In recent ten years, the molecular dissection of circadian clock is dramatically progressed, and now, it is known that circadian time is generated by an The integration of genetic, behavioral, and engineering techniques has enabled us to chart the heterogeneous cellular and circuit organization of the autoregulatory transcription-(post) translational feedback loop of clock brain with unprecedented resolution. Yet, the emerging brain atlas is both genes. This oscillation at the levels of genes reflects at cells, tissues, and illuminating and perplexing in its complexity. How do brain circuit elements system levels. Evidences are accumulating that mitosis of most cells of the interoperate and give rise to the panoply of behavioral and cognitive body, migration of stem cells from bone marrow to systemic circulation, functions? and calcification of osteoblasts occur at specific time of the day. Not only Recent developments in optogenetic technologies (channelrhodopsins and these, many of physiological and pathological symptoms show the daily halorhodopsins) are beginning to give researchers the ability to reverse rhythms. This timing is determined by the endogenous oscillatory system engineer intact neural circuits, by directly probing the necessity and called circadian system. Circadian system is consisted of central clock in the sufficiency of cellular and topological circuit characteristics with high-speed suprachiasmatic nucleus (SCN), and peripheral clocks. Moreover, various and cell type-specific perturbations, using light to directly activate or inhibit hormonal and neuronal signals connect with neuronal and hormonal routes. specific neurons. Our latest developments have focused on three directions: 1) expanding the repertoire of genetically-encoded neuromodulators, 2) Increasing evidence suggests that disruption of one or multiple sites of this developing better cell-type and circuit-specific gene targeting systems, and system is associated with various pathogenic conditions and may be highly 3) integrating optogenetics with a variety of circuit-level and behavioral relevant to various diseases including sleep-wake abnormality, metabolic readout methods. The emerging optogenetic platform enables deconstruction syndrome, osteoporosis, abnormal estrous cycle and cardiac disorder of previously inaccessible brain circuits, and will improve our understanding accompanied by hypertension. We present here the molecular evidences of the causal relationship between circuit activities and neuropsychiatric connecting clock genes and its diseases. diseases.

WD VII-10-4 WD VII-10-5 CIRCADIAN ENTRAINMENT BY DIFFERENT CLOCK MECHANISMS IN MAMMALS ENCODING DAYLENGTHS: THE ROLES OF DAWN AND DUSK PHOTOPERIODS Serge Daan, Marian Comas, Kamiel Spoelstra, Roelof A Hut, Sato Honma, Tomoko Yoshikawa, Ken-ichi Honma Whole-day symposia Domien G M Beersma Department of Physiology, Hokkaido University Graduate School of Dean, Faculty of Mathematics and Natural Sciences, University Groningen, Medicine, Japan The Netherlands To explain the mechanisms of photoperiodic time measurement by the In the early days of circadian rhythms research there were two competing circadian clock, a model of coupled evening (E) and morning (M) oscillators concepts of entrainment by light: through parametric action on the velocity has been proposed. We explored the localization of these oscillators by of the endogenous cycle versus non-parametric discrete phase shifts elicited analyzing clock gene expression rhythms in the Suprachiasmatic nucleus by the lights-on and off transitions. Although the phase shift model became (SCN) of mice and rats in different photoperiods. We cultured two coronal widely accepted, straightforward experimental tests of the alternatives are SCN slices from each transgenic mouse carrying a luciferase reporter for lacking. In recent years we performed a series of studies in mice to address Per1 expression (Per1-luc) and measured bioluminescence rhythms in single this issue, using protocols with single and double light pulses of different duration as well as single lights-on and lights-off signals. The results reveal cells as well as slices. The circadian Per1-luc rhythm of the posterior SCN no evidence for a specific role of the transitions per se. All features of the phase-locked to the behavioral activity-onset, while that of the anterior circadian system can be explained by velocity changes in response to light, SCN, to the activity-end, irrespective of photoperiods, suggesting the a response that is reduced under prolonged illumination and restored during localization of M and E oscillators, respectively. Mice exposed to the long prolonged darkness. This arrangement allows robust entrainment when the photoperiod exhibited two Per1-luc peaks a day in the anterior SCN, which signal is noisy and behavioural withdrawal from light adds to its variance. was composed of two different cell groups. We further analyzed Per1 and It leads to stable timing in species never observing dawn and dusk. It even Per2 expressing cell numbers in the entire rat SCN by in situ hybridization. keeps the clock entrained when mice must work for their food and show We confirmed differentially phased Per1 expression in the specific SCN diurnal (shiftwork) activity. A model for a neuronal pacer network in the SCN subregions. The results suggest cell specific roles of Period genes for the shows exactly these properties and predicts seasonal adaptation to daylength. photoperiodic clock in the SCN.

WD VII-10-6 WD VII-10-7 ON THE SOCIAL DIMENSION OF CIRCADIAN TIMING LIGHT AND THE HUMAN CIRCADIAN TIMING SYSTEM: IN MAMMALS AGE-RELATED CHANGES William Schwartz Debra Jean Skene, Tracey L Sletten, Katrin Ackermann, Mirela Department of Neurology, University of Massachusetts Medical School, Herljevic, Katharina A Lederle, Benita Middleton, Simon N Archer, USA Victoria L Revell Our knowledge of the circadian system of mammals at the molecular, cellular, Centre for Chronobiology, University of Surrey, UK tissue, and organismal levels is growing, mostly based on experiments using Ocular light is the primary time cue for entrainment of the human circadian timing singly-housed rodents in plastic cages with temperature, humidity, and access system to the 24 h light/dark cycle. Totally blind people with no conscious light to food rigidly controlled. Of course, in nature many species do not live out perception have a desynchronised circadian clock exhibiting free-running circadian their lives in seclusion but instead within communities with highly developed rhythms (melatonin, cortisol, sleep/wake cycle). In addition to the resetting effects of social structures. Social cues may play an important role in modifying light on the circadian clock, light induces other non-image forming (NIF) responses, activity rhythms in animals that live in colonies with a clear division of labor; such as suppression of nocturnal melatonin production, increased alertness, mood and or by allowing animals to synchronize their behaviors to achieve common improved performance. These responses have been shown to be maximally sensitive goals; or by enabling animals to avoid each other to lessen competition to short wavelength blue light (440-480 nm). Age-related changes in the eye result in a reduction in the transmission of short for a limited resource. Relatively little is known about the mechanisms for wavelength blue light and this has been shown to impair some acute NIF light such inter-individual temporal adaptations, or how the circadian system responses in older individuals (reduced melatonin suppression; reduced alertness). By might be involved. We have begun to study the conditions under which such contrast, the phase shifting response to blue light was not significantly impaired with cohabitation effects might be revealed in mice and hamsters in the laboratory. age; blue light phase advanced the rhythms in plasma melatonin and PERIOD3 (PER3) Societal regulation of the rhythmicity of individuals may be a critical factor clock gene expression in leukocytes more effectively than green light in both young for group adaptation to the natural habitat, and a better understanding of this and old subjects. level of biological organization will likely generate a more complex, but Supported by EU 5th Framework grant (QLK6-CT-2000-00499), Marie Curie grant ultimately more comprehensive, view of clocks and rhythms. (MCRTN-CT-2004-512362) and 6th Framework Project EUCLOCK (018471).

IUPS 2009 July 27 - August 1, 2009 in Kyoto 37 WD VII-10-8 WD VII-10-9 CIRCADIAN ORGANIZATION IN HUMANS: IS THERE HUMANS SUFFERING FROM DISORGANIZED ANY OSCILLATOR(S) FOR BEHAVIORAL RHYTHMS CIRCADIAN RHYTHMS INDEPENDENT OF THOSE IN THE SCN Makoto Uchiyama Ken-ichi Honma Department of Psychiatry, Nihon University School of Medicine, Japan Department of Physiology, Hokkaido University Graduate School of For socially successful achievement, one needs to synchronize one’s Medicine, Japan sleep timing with social requirements. For biologically optimal alertness It is still a matter of debate whether or not the behavioral rhythms in humans such and restful sleep, human activity and sleep need to be matched with the as sleep-wake cycles are separately regulated by the oscillator(s) which is different circadian pacemaker, which is normally synchronized with the 24-hour from the circadian pacemaker in the suprachiasmatic nucleus (SCN). The issue was raised by the observation of internal desynchronization in which the circadian rhythms light-dark cycle of the earth’s rotation. Circadian rhythm sleep disorders in rectal temperature and in behavioral sleep and wakefulness apparently free-ran are conditions in which sleep timing fails to be synchronized with internal with different periods. Two hypotheses have been advanced to explain the internal or external environment. Shift work and jet lag are frequent causes of sleep desynchronization; two-oscillator model and two-process model. However, neither the misalignment relative to the circadian pacemaker. Various chronic sleep mechanism nor the site of oscillation or of S process in terms of the two-process model misalignments have been found in totally blind subjects, who seem unable has been elucidated. to convey environmental light information to the circadian pacemaker. A Recent studies in animals as well as in humans suggest the existence of extra-SCN chronic sleep phase delay, or a free-running sleep-wake rhythm under a oscillators for behavioral rhythms. One is the non-photic entrainment of sleep-wake normal light-dark cycle is also observed in sighted subjects. Though these cycles in humans independent of the circadian rhythms, and the other is the peripheral circadian rhythm sleep disorders have been studied for over 20 years, there clocks located in the central nervous system, which are functional without the SCN and entrain to the external time cues. Methamphetamine (MAP)-treated animals are have been few hypotheses on their pathophysiology. Here, the author reviews a good model for the human circadian system and MAP treatment seems to drive current findings on circadian rhythm sleep disorders with special reference

Whole-day symposia the peripheral clocks for behavioral rhythms and uncouple the clock from the SCN to the phase relation between sleep and the circadian pacemaker and genetic circadian pacemaker. constitution of the disorders.

WD VII-11-1 WD VII-11-2 FUNCTIONAL REGULATION OF NEURONAL K-Cl ROLE OF CATION CHLORIDE COTRANSPORTERS IN TRANSPORTER BY TYROSINE PHOSPHORYLATION NEURONAL DEVELOPMENT AND PLASTICITY Junichi Nabekura, Miho Watanabe Kai Kaila Homeostatic Development, National Institute for Physiological Sciences, Department of Biosciences, University of Helsinki, Finland Japan The development of hyperpolarizing inhibition in hippocampal and K-Cl Cotrnasporter (KCC)2 is a principal molecule to exclude Cl- out of neocortical neurons is set by the up-regulation of KCC2, a neuron-specific the neurons and contributes to the maintenance of low [Cl-]i. In addition to K-Cl cotransporter (for review, see Blaesse et al. 2009, Neuron 61: 820-838.). an increasing the evidences of the long term regulation of KCC2 protein in The level of expression of total KCC2 protein is generally considered to be development and damaged neurons, much attention has been recently paid low and functionally insignificant in the early postnatal rodent hippocampus. to a rapid and dynamic alteration of KCC2 function. KCC2 contains one We have re-examined this idea by studying membrane surface expression consensus of a tyrosine protein kinase phosphorylation site located at the of KCC2 in the neonatal rat hippocampus using biochemical techniques. long carboxy terminal. Genistein, tyrosine kinase inhibitor, shifted EGABA Strikingly, we found that it is possible to induce a large increase in the to more depolarized values and induced translocation of KCC2 from punctate plasmalemmal KCC2 pool which is not based on increased synthesis of stainings to more uniform distribution in the hippocampal neurons. Mutation KCC2 protein. The enhanced surface expression leads to a dramatic increase of the tyrosine kinase amino acid residue (Y1087D) also reduced KCC2 in the efficacy of Cl- extrusion and to a consequent hyperpolarizing shift activity and abolished its punctate distribution. Sodium vanadate, a tyrosine in EGABA-A already during the first postnatal week. Indeed, under some phosphatase inhibitor, increased the proportion of KCC2 associated with experimental conditions, the net extrusion of Cl- by the immature pyramidal lipid raft Deletion of 28 amino acid residue in carboxy terminal of KCC2 neurons reaches a level of efficacy that is close to what is observed in adult did not show clustering and oligomer. Thus, KCC2 forms cluster by tyrosine neurons. The mechanisms and significance of the early functional activation phosphorylation via carboxy terminal, thereby extrude Cl- efficiently to of KCC2 will be discussed. maintain local Cl- low.

WD VII-11-3 WD VII-11-4 A [Na+] DEPENDENT METABOLOSTAT IN THE SENSING FUNCTION OF CL- CHANNELS. DOES IT SUBFORNICAL ORGAN TO CONTROL SALT INTAKE MAKE SENSE? Masaharu Noda Andres Stutzin, Diego Varela Division of Molecular Neurobiology, National Institute for Basic Biology, CEMC & ICBM Facultad de Medicina, Universidad de Chile, Chile Japan In verterbrate cells, swelling triggers the activation of an outwardly rectifying Sodium (Na) is a major electrolyte of the extracellular fluid and the main anion current allowing the passive loss of inorganic (and organic) anions as determinant of osmolarity. Na concentrations in plasma and CSF are part of the regulatory volume decrease process. The channel responsible for continuously monitored in the brain to maintain a physiological level of Na this current has been referred to by a rather unusually high number of names1, in body fluids. a clear indication of our (persistent) poor functional comprehension and We showed that the Nax channel expressed in the circumventricular organs complete lack of molecular information of this entitity. As a result, we should (CVOs) is the Na-level sensor with a threshold value of ~150 mM for the not be surprised when confronted to the task of elucidating fundamental extracellular Na ion. Nax channels are expressed in perineuronal processes questions related to its activation/modulation in several physiological and of specific astrocytes and ependymal cells in the CVOs including the pathophysiological processes. Here we will discuss the different intracellular subfornical organ and the organum vasculosum of the lamina terminalis. signaling pathways and second messengers described to be involved in the Nax-KO mice do not stop ingesting salt even under conditions that Na level activation/modulation of these channels and thus, try to define what are they in body fluids is highly elevated, while wild-type mice avoid salt at once really sensing. 1 under such conditions. We recently found that Nax channels stably interact As examples, we can find the names VSOAC, VRAC and VSOR. + + + with Na /K -ATPase to assist in the activation, and that Na influx by Nax Interestingly, all contain the letter V, volume. channels stimulates anaerobic glucose metabolism in the glial cells. The Supported by FONDAP 15010006 resultant lactate released from the glial cells plays a crucial role in the control of neuronal activities involved in the salt appetite. Glial cells thus take the initiative in the regulation of neural activity by using lactate. I will also show that autoimmunity to Nax induces hypernatremia in human.

38 IUPS 2009 July 27 - August 1, 2009 in Kyoto WD VII-11-5 LS20-1 (WD VII-11-related luncheon seminar) CHEMOSENSORY RECEPTORS FOR ODORS AND IMAGING MEMBRANE POTENTIAL DYNAMICS IN PHEROMONES FINE STRUCTURES USING THE SECOND HARMONIC Kazushige Touhara GENERATION IMAGING Integrated Biosciences, The University of Tokyo, Japan Mutsuo Nuriya Animals are attracted by general odorants derived from food or by Department of Pharmacology, Keio University, School of Medicine, Japan pheromones released from the opposite sex. The chemical information In neurons and other excitable cells, fast information processing is mainly is detected by chemosensory receptors expressed by peripheral olfactory performed electrically. Therefore, quantitative information on membrane sensory neurons (Touhara & Vosshall, Annu. Rev. Phy. 2009). In the potential dynamics is the key to the understanding of physiology of these silkmoth, the male-specific olfactory receptor BmOr1 in antenna is in charge cells. In neurons, however, this is challenged by their extremely elaborate and of the response to bombykol, the sex pheromone released by female moths fine structures including axons and dendrites, often below a micrometer in (Nakagawa et al. Science 2005). The larval BmOr56 plays a role in sensing diameter. To gain quantitative information on membrane potential dynamics cis-jasmone, a potent chemoattractant for silkworms that is emitted by their in these structures, second harmonic generation (SHG) imaging was applied food, mulberry leaves (Tanaka et al. Current Biol. 2009). Unlike vertebrate to neurons. Point-scan SHG imaging from dissociated cultured hippocampal olfactory receptors that couple with G proteins, insect olfactory receptors neurons allowed fast measurement of electrical signals from sub-micron consist of a heteromeric complex and comprise a novel class of ligand- structures. Furthermore, as was predicted from theoretical studies, SHG activated nonselective cation channels (Sato et al. Nature 2008). In mice, the signals showed a linear response to membrane potential changes, enabling vomeronasal receptor V2Rp5 is the functional receptor for a male-specific the quantitative imaging of membrane potential changes in any visible places peptide pheromone ESP1 that enhances female sexual behavior (Kimoto in neurons. In this presentation, application of this technique to membrane et al. Nature 2005; Haga et al. submitted). The neural circuitry activated potential imaging of fine neurites that were hardly accessible with other by these specialist chemosensory receptors appears to govern a distinct techniques will be introduced. In addition, potentials of SHG imaging for behavioral output of pheromone or biologically important odorant that is other applications will be discussed. crucial for survival or mating in various animal species.

LS20-2 (WD VII-11-related luncheon seminar) WD VII-11-6

NOVEL MICROSCOPE OBJECTIVE BY INNOVATIVE CALCIUM DYNAMICS THROUGH IP3 RECEPTORS DESIGN CONCEPT TARGETING TWO-PHOTON DEEP Katsuhiko Mikoshiba Whole-day symposia IMAGING RIKEN Brain Science Institute, JST, Japan Eiji Yokoi Ca2+ plays an important role in variety of physiological phenomena. We Olympus Corporation, MIS Development Department 1, Optical R&D., discovered IP3 receptor (IP3R) and determined the structure and found that it is Japan 2+ a Ca release channel located on endoplasmic reticulum. IP3R1 is essential for Two-photon (2PE) microscope has been known as one of the most powerful fertilization, dorsoventral axis formation, cell division, neurite extension and tool to observe inside the scattered living specimen like brains with sub-micron neural plasticity. ERp44. a redox sensor, in the ER and regulates IP3R1 activity. resolution, which contributed to the development of biological research such as 2+ IP3 releases not only Ca , but also releases IRBIT (IP3 receptor binding protein physiology and neuroscience. 2PE microscope has been in the market since in the released with inositol trisphosphate). IRBIT regulates IP induced Ca2+ release late 90s as well as self-build one, and whose demand is increasing every year. In 3 and regulates pH inside cells and importantly regulate protein synthesis. IP R1 any case, microscope objective is the most critical part, which determines overall 3 performance of the 2PE system. However, none of the commercial objectives are knock out mouse shows severe cerebellar ataxia and shows decreased BDNF designed for 2PE but for single-photon microscopy, which limits the potential secretion. Double knock out of IP3R2 and IP3R3 results in deficit of exocrine advantage of 2PE. That's why we decided to develop a special objective for secretion, such as saliva and pancreatic juice and furthermore nasal sensory system suffers from degeneration. Since IP3R interacts with many molecules like exclusive use of 2PE microscope. This special objective offers a super-wide field 2+ of view, flexibility for patch clamping and cover slip use, outstanding infrared a scaffold protein coupled to Ca releasing channel activity. IP3R associates with performance, and a deep-imaging correction collar for imaging far below the TRP channel directly or indirectly through 4.1N suggesting a strong connection surface of living specimens. In addition, new optical coatings allow the objective with sensation in which TRP channels may couple to various sensations. We to provide outstanding transmission in Vis-IR. It could perform up to 10 times found IP3R3 may work for taste sensation associating with TRPM5. better than the similar objective already in the market. As a result, we achieved the special objective for 2PE offering greater brightness, sensitivity and S/N ratio in deep living specimen.

WD VII-11-7 WD VII-11-8 THE GLUCOCORTICOID RECEPTOR: ONE GENE, MECHANISMS AND ROLES OF VOLTAGE-SENSING MANY PROTEINS - AN INTRACELLULAR SENSOR PROTEINS WITHOUT PORE STRUCTURE FOR GLUCOCORTICOIDS Yasushi Okamura John A. Cidlowski Department of Integrative Physiology, Osaka University, Japan Laboratory of Signal Transduction, National Institute of Environmental Membrane potentials are utilized in excitable cells for propagation of Health Sciences, NIH, USA information and in mitochondria for energy production. In cell membranes, Glucocorticoids are necessary for life after birth and regulate numerous changes in membrane voltages are transduced into chemical signals by biological processes in man, including glucose homeostasis, protein voltage-gated ion channels that contain “voltage sensors”. Identification of catabolism, skeletal growth, respiratory function, inflammation, development, novel voltage-sensor containing proteins that do not contain ion pore has behavior and apoptosis. They are also one of the most prescribed classes of led to an idea that cellular information based on membrane voltage could be drugs in the world. Our understanding of how one hormone regulates all of more widespread than previously thought. Voltage-sensing phosphatase (VSP) these diverse processes is limited, although most of these actions are thought contains voltage-sensor domain and enzyme region with significant homology to be mediated via the intracellular glucocorticoid receptors. Recent studies have shown that tissue specific glucocorticoid receptor isoforms are produced to a tumor suppressor protein, that is called PTEN. It shows depolarization- from one gene and each isoform is subject to post-translational modifications. activated phosphoinositide phosphatase activities dependent on the operation In this lecture, I will discuss how the glucocorticoid receptor isoforms and of the voltage sensor, and is abundantly expressed in testis throughout post-translational modifications selectively regulate diverse glucocorticoid many animal phyla. VSP differs from PTEN in its substrate specificity: it actions in man. dephosphorylates PI(4,5)P2 as well all PI(3,4,5)P3. VSOP (voltage-sensor only protein, or Hv1) is another voltage-sensor protein without pore structure, but it exhibits activities of voltage-gated proton channels that have been well described in mammalian phagocytes. Mechanisms of these proteins will be discussed in the physiological contexts.

IUPS 2009 July 27 - August 1, 2009 in Kyoto 39 WD VII-11-9 WD VII-11-10 GATING MECHANISMS OF TRP CHANNELS: LINKING NOVEL INSIGHTS INTO NEURONAL OSMOSENSORY FUNCTION TO DISEASE TRANSDUCTION Bernd Nilius Charles Bourque Department Mol Cell Biol, Lab Ion Channel Research, KU Leuven, Centre for Research in Neuroscienc, McGill University, Canada Campus Gasthuisberg, Belgium Vasopressin (VP) is released during plasma hyperosmolality to increase Transient Receptor Potential (TRP) cation channels are unique cell sensors water reabsorption. This hormone is also released during hyperthermia, to responding to a plethora of gating stimuli. Three TRP channels, TRPM4, counter the impact of evaporative fluid loss during thermoregulatory cooling. TRPM5, TRPV4, will be described in the context of possible diseases. 2+ Previous studies have shown that VP neurons respond to local changes TRPM4 and TRPM5 are Ca activated non-selective cation channel in osmolality and temperature. Both types of stimuli activate calcium- modulated by phosphatidylinositol-phosphates. trpv4-/- mice develop a mast- permeable cation channels that can be blocked by gadolinium or ruthenium cell gain-of-function phenotype with altered migration. TRPM4 deficient red. The inward current resulting from this effect causes membrane mice show increased adrenergic inotropic responses and develop heart hypertrophy. TRPM5 seems to be critically involved in insulin release. depolarization, thereby increasing action potential discharge. We found that TRPV4, a mechano-activated channel, is highly expressed in bladder the responses of VP neurons to thermal and osmotic stimulation are impaired urothelium. The voiding pattern showed incontinence-like disturbances in in Trpv1-knockout (Trpv1-KO) mice. These animals also show impaired trpv4-/- mice, atypical cystometric pattern caused by a dysfunction of the VP release in response to hypertonicity and hyperthermia in vivo, indicating mechano-sensor affecting stretch-induced release of ATP. There is a clear that expression of the Trpv1 gene is required for normal homeostatic VP interaction with TRPV1. trpv4-/- mice develop a decreased shear stress release. However VP neurons do not generate an inward current in response induced endothelium dependent vasorelaxation. An osteoclast dysfunction to capsaicin, a selective agonist of the Trpv1 channel, and part of the amino causes in trpv4-/- mice an increased bone mass. TRPV4 is required for the terminal of Trpv1 is missing in these cells. These studies indicate that final differentiation of osteoclasts and is linked to osteoporosis. Finally, physiological osmosensory transduction and thermosensory transduction

Whole-day symposia TRPV4 bone channelopathies, including an autosomal dominant brachyolmia can be performed by single VP neurons and that both sensory mechanisms caused by a gain-of-function mutations of TRPV4 will be described. depend on a N-terminal variant of the Trpv1 channel.

WD VII-11-11 WD VII-12-1 PHYSIOLOGICAL SIGNIFICANCE OF THE GRAVITY OF CARDIOVASCULAR ADAPTATIONS THERMOSENSITIVE TRP CHANNELS Alan R Hargens Makoto Tominaga Department of Orthopaedic Surgery, University of California, San Diego, Section of Cell Signaling,Okazaki Institute for Integrative Bioscience USA (National Inst. for Physiol. Sciences), Japan Physiologic systems of animals have adapted to Earth’s gravity over millions TRP (transient receptor potential) channels were first described in Drosophila of years. In general, gravitational adaptations of the cardiovascular system in 1989, and in mammals, TRP channels comprise six related protein are more pronounced in terrestrial species with greater height (giraffe and families (TRPC, TRPV, TRPM, TRPA, TRPML, TRPP). TRP channels are dinosaurs) and thus, greater gradients of blood pressure from head to feet. In best recognized for their contributions to sensory transduction, responding this regard, tall animals have evolved sophisticated mechanisms to provide to temperature, nociceptive stimuli, touch, osmolarity, pheromones and adequate blood flow to their brains while restricting blood flow and tissue other stimuli from both within and outside the cell. Among the huge TRP swelling in their feet and legs. Structural and contractile tissues of the super family of ion channels, some have been proven to be involved in cardiovascular system respond, within physiologic limits, to increased (or thermosensation detecting ambient temperatures from cold to hot. There are now nine thermosensitive TRP channels (TRPV1, TRPV2, TRPV3, TRPV4, decreased) levels of gravitational stress by increasing (or decreasing) their TRPM2, TRPM4, TRPM5, TRPM8 and TRPA1) with distinct temperature functional capability and/or mass. Importantly, chronic changes in regional thresholds for their activation. I would like to talk about the physiological vascular transmural pressures alter macro- and microvascular structure and significance of some of the thermosensitive TRP channels (TRPV1, TRPA1, function. During microgravity exposure, altered pressures (blood and tissue) TRPV4 and TRPM2), especially focusing on channels activated by warm alter fluid balance in local tissues. Tissues of the lower body (e.g., foot) are temperatures. well adapted to local hypertension on Earth, whereas tissues of the upper body (e.g., head) are not as well adapted to increases in local blood pressure.

WD VII-12-2 WD VII-12-3 PHYSIOLOGIC LESSONS FROM ANIMALS (MAMMALS) VASCULAR ADAPTATION TO MICROGRAVITY AND ITS IN MICROGRAVITYS GRAVITY-BASED COUNTERMEASURE Charles M Tipton Li-Fan Zhang Department of Physiology, University of Arizona, USA Department of Aerospace Physiology, Fourth Military Medical University, China Since 1793, animals have been aloft within the atmosphere & after 1960, they have demonstrated they can live & function in microgravity before Vascular adaptation to microgravity is basically a process of vascular being safely returned to a 1-G- environment. But, what physiologic lessons autoregulation to the redistribution of transmural pressure caused by a loss or insights have been learned from animals that have biologic importance of hydrostatic pressure gradient due to the removal of gravity. Arterial tissue for humans? As "authorities" have attributed the physiologic consequences responds first by an immediate change in myogenic tone and vasoreactivity. When microgravity exposure persists for a longer time, vessels respond by of microgravity to be the single or collective effects of (a) alterations in a remodeling process to alter their geometrical dimensions. The underlying mechanical stress on the tissues, (b) alterations in afferent stimulation and mechanism involves, at least, vascular channel remodeling, activity of local in the integration of sensory information, (c) alterations in hydrostatic renin-angiotensin system (RAS) and NO-NOS system. The exact nature of pressures and the Starling forces & (d) alterations in the integrative functions the adaptation for a given arterial vessel may depend on the following factors: of biological systems, this topic is to be addressed by selectively examining its anatomic location (distance and direction from the hydrostatic indifference space flight animal results & relating them to the tissue or system involved. point), type and kind of the vessel (large elastic, medium-sized muscular, In addition, matters pertaining to growth and development & to data or small arteries, specificity within cerebral vasculature), and microgravity interpretation will be discussed. exposure time. Intermittent artificial gravity might be the most efficacious countermeasure for vascular deconditioning. A mechanotransduction mechanism involved is being studied with an ex-vivo culture of isolated vessels for several days. However, there remains a big challenge to translate these results from animals to humans in space.

40 IUPS 2009 July 27 - August 1, 2009 in Kyoto WD VII-12-4 WD VII-12-5 THE DIMENSIONS OF BRACHIOSAURUS BRANCAI PHYSIOLOGICAL ADAPTATIONS TO EXTREME AND THEIR PHYSIOLOGICAL IMPLICATIONS ALTITUDE Hanns-Christian Gunga John B. West Center of Space Medicine Berlin, Charite - University Medicine Berlin, Germany Department of Medicine, University of California San Diego School of INTRODUCTION Medicine, USA We re-evaluated 1) the metrical dimensions of this extinct dinosaur (body mass It is a remarkable coincidence that the highest point on earth is very close [Mb], volume [V], surface area [Sa]); 2) used then allometric equations to estimate to the human limits of tolerance to hypoxia. During the American Medical presumable organ sizes and physiological parameters, and 3) tested whether the organ Research Expedition to Everest, the first the physiological measurements dimensions found really fit into the thoracic and abdominal cavity of Brachiosaurus were obtained on the summit, altitude 8848 m. They showed that the most brancai [B. brancai]. critical feature of the adaptation to this extreme altitude was an enormous METHODS increase in ventilation which drove the alveolar PCO2 down to 8 mm Hg For the calculation of the V and the Sa classical photogrammetry and advanced (1.1 kPa), while the alveolar PO was only about 35 mm Hg (4.7 kPa). laser scanning were APPLIED. CAD techniques were used for the graphical three- 2 Calculations of the arterial PO showed that it was close to 30 mm Hg (4 kPa) dimensional reconstruction. The presumed shape of the dinosaur was divided into XI 2 parts, each consisting of a different number of elements. From the total V found, the because of diffusion limitation of oxygen across the blood-gas barrier. Recent Mb was calculated, assuming a density of 800 kg per cubic meter tissue. data from the British Extreme Everest expedition who took arterial blood RESULTS samples at altitude 8400 m confirm the very low PO2 values. The arterial pH We determined a Mb of 38,000 kg, Sa 119 qm, a length of the carotids of 9.8 m, a measured from the base excess and alveolar PCO2 exceeded 7.7. The very skeleton mass of 5,5 t, O2 consumption 0.05 l.h-1 . kg-1, basal metabolic rate of 915, high pH assists in the oxygenation of the blood in the lung because it shifts 000 kJ . 24 h-1, blood volume 1,880 l, heart weight 198 kg, and heart rate 17 min-1. the oxygen dissociation curve to the left thus facilitating oxygen loading in CONCLUSIONS the lung. Another consequence of the pH is that the arterial oxygen saturation Presumable skeleton mass, organ sizes, and derived physiological data show that is maintained near 70% in spite of the very low PO2 in the blood. In spite of the circulatory system, thermoregulation, metabolism, and nutrition must have been these adaptations, the maximal oxygen consumption on the summit is only extremely specialized in B. brancai. about one l/min.

WD VII-12-6 WD VII-12-7 WEIGHT SUPPORT SYSTEM USING LBPP: NEW COUNTERMEASURE FOR BONE LOSS AND PHYSIOLOGY AND CLINICAL USE MUSCLE ATROPHY DURING LONG DURATION HUMAN Whole-day symposia Yasuaki Kawai, Satoshi Matsuo SPACE FLIGHT Adaptation Physiology, Tottori University, Faculty of Medicine, Japan Hiroshi Ohshima, Shoichi Tachibana, Chiaki Mukai Space Biomedical Research Office, JAXA, Japan Rehabilitation is an important process through which adaptation to injury The Japanese Experiment Module, named "Kibo", is Japan's first human or disease is promoted. Early start of rehabilitation is encouraged because space facility and the Japan Aerospace Exploration Agency's (JAXA's) first immobilization causes muscle and bone atrophy in the lower extremities. contribution to the International Space Station (ISS) program. Kibo was Elderly patients with muscle pain or weakness, however, are often unable to developed for conducting scientific research activities on orbit, where a walk while supporting their body weight. Thus, we developed a lower body Japanese astronaut will stay aboard the ISS for several months from 2009. positive pressure (LBPP) apparatus which can reduce net ground reaction Bone loss and muscle atrophy are significant medical concerns for long- force (GRF), or apparent body weight. In the present study, we examined the duration human space flight. A two-hour exercise program is planned for each effects of LBPP on the net GRF and on the cardiovascular function during astronaut, who stays long-term in space. However, bone loss (1.5%/ month rest and walking. We also investigated the usefulness of walk training using at the femoral neck) and muscle weakness (20 to 30% loss) occurred after a LBPP as rehabilitation in patients after orthopedic surgery. During a graded six-month stay in ISS. To reduce the medical risk of space flight, JAXA has application of LBPP, the net GRF decreased in a linear manner. Exposure to been developing new physiological countermeasures for space bone loss and muscle atrophy. Bisphosphonate is widely used for treating osteoporosis, LBPP affected neither heart rate nor blood pressure during rest. The increase because it increases bone mineral density and reduces fracture rates. To in heart rate during walking, however, was smaller in the LBPP group than prevent bone loss and renal stone during space flight, a study of prophylactic in the control group. It was also shown that LBPP application improved the use of bisphosphonate for station astronaut has began this year through posture of patients during walking. We conclude that LBPP may provide a collaboration between JAXA and NASA. To improve the in-flight exercise useful tool as a weight support system during walk training in rehabilitation. training, new Japanese innovative training methods have been investigated in a ground validation study.

WD VII-12-8 WD VII-12-9 MARS EXPLORATION MISSION. SOME APPROACHES EFFECTIVE COUNTERMEASURE FOR MUSCLE AND TO THE COUNTERMEASURE SYSTEM BONE LOSS IN WEIGHTLESSNESS Inesa Kozlovskaya Dieter Felsenberg Department of sensory-motor physiology and countermeasure, RF SSC Centre Muscle & Bone Research, Charite-Campus Benjamin Franklin, - Institute of Biomedical Problems of the Russian Academy of Sciences, Free & Humboldt-University Berlin, Germany Russia Background: Prevention of muscle and bone loss in microgravity is one of the Care for the Martian crew health will be provided by a medically qualified most essential challenges in space medicine. crewmember with the highest degree of autonomy. The computer-based Objectives: Documentation of superiority of resistance vibration exercise (RVE) countermeasure system will generate the objective data about fitness and physical vs resistance exercise (RE). performance of crewmembers as a basis for elaboration of individual plans of training Subjects & Methods: 44 males participated in 2 bed rest studies, 19 controls and use of other countermeasures. To make this concept a reality, the available (CTR) and 25 subjects with RVE or RE. RVE and RE was performed with/ countermeasures need to undergo a fundamental revision and enhancement, the without ‘Galileo Vibration system’. BMD was measured with pQCT (tibia,radius). number of active and passive training facilities to increase, and a short-arm centrifuge Muscles were assessed with MRI and DXA. Bone markers (P1NP,CTX) were to be designed. It will be crucial for the autonomous crew to have reliable methods and measured weekly. tests to evaluate fitness and physical performance. The talk outlines the potentiality of Results: BMC loss of the tibia was about 3% (CTR), no loss in the RVE and an automated expert system for crew training management that has been built upon RE groups. Loss of the soleus muscle volume was 16% (CTR), no loss in the experimental grounds. Structuring of the training process will be guided by the the RVE group. CTX increased by 30% (CTR) during bedrest and decreased principles of the concept. As the mission proceeds, vehicle data bases and control after reambulation. 10% decrease of P1NP (CTR) and 45% increased after algorithms will be updated through the exploration vehicle-MCC-medical operations reambulation. In the RVE group P1NP increased by 45%. Exercise time was group network. 12-120 min. a week. The concluding part of the presentation will discuss physiological and methodical Conclusions: RVE is superior in some aspects to RE. RVE stimulates bone approaches to planning and designing the training process, and training requirements formation, prevents muscle and bone loss compared to CTR. from the perspective of an autonomous Mars mission. Keywords: Bone adaptation, muscle exercise, vibration training

IUPS 2009 July 27 - August 1, 2009 in Kyoto 41 WD VII-12-10 WD VII-12-11 HOW MUCH GRAVITATIONAL LOADING DOES EFFECTS OF MICROGRAVITY ON GAZE FIXATION THE NEUROMUSCULAR SYSTEM NEED FOR REACTION MAINTENANCE? Elena Tomilovskaya, Inesa B Kozlovskaya V. Reggie Edgerton1, Hui Zhong1, Jung Kim1, Kenneth Baldwin2, Department of sensory-motor physiology and countermeasure, RF SSC Roland R Roy1 - Institute of Biomedical Problems of the Russian Academy of Sciences, 1 Russia Physiological Science, University of California Los Angeles, USA, 2Department of Physiology and Biophysics, University of California Irvine, The study was performed to explore effects of long-duration space flights on USA the characteristics of gaze fixation reaction (predictable and nonpredictible Although it is apparent that some combination of activation and the generation of force target acquisition tasks). Sixteen cosmonauts, crew members of the “MIR” of skeletal muscles is necessary to maintain homeostasis, the amount and pattern of missions, took part in this study. The study was carried out 4 times before these events are only vaguely understood. It is particularly intriguing that the amount flight, 5 times in flight and 3-4 times after landing. Changes in gaze fixation and pattern of activation and force necessary to sustain skeletal muscle function varies reactions, such as increase of the task implementation time, increase of widely from muscle fiber to muscle fiber as reflected simply by the fact that the most coefficient of vestibular-ocular reflex, increase or decrease of head and infrequently activated muscle fibers (motor units) are the largest fibers and those eye movements’ velocity, observed in long-duration space flight and after motor units that are the most active, in general are the smallest fibers. Further, there its completion indicate serious disturbances of the vestibular-ocular reflex is a large difference in the amount of force that is generated by muscles of mammals system due to, apparently, altered vestibular activity. The changes of reaction’ that directly resist gravity during postural and locomotor tasks, i.e. the extensor s parameters were more expressed in nonpredictible target acquisition task musculature, compared to those which do not have a direct antigravity function (flexor compared to predictable one. musculature) in sustaining an upright position at 1G. In this presentation data will Two strategies of adaptation to microgravity conditions were discovered in be presented which demonstrate a level of activity that typically occurs in flexor and cosmonauts of civic occupations and pilots during space flight: the first one extensor muscles at 1G and in the microgravity environment. In addition, evidence of namely an increase of the head velocity and decrease of the saccade velocity

Whole-day symposia the minimum level and intervals of activation of skeletal muscle needed to maintain was dominated in cosmonauts-pilots; hypermetric gaze and delayed head homeostasis will be presented. movement were the feature of the strategy of all civic cosmonauts.

WD VII-12-12 WD VIII-13-1 GRAVITY STIMULATES POSTNATAL DEVELOPMENT FUNCTIONAL CROSSTALK BETWEEN CELL-SURFACE OF CARDIOVASCULAR REGULATION AND INTRACELLULAR CHANNELS MEDIATED BY Shunji Nagaoka1, Yuko Eno2, Tadayoshi Hata1, Yoshinobu Ohira3 JUNCTOPHILINS 1Department of Physiology, Fujita Health University Graduate School of 2 Hiroshi Takeshima Health Sciences, Japan, Nagoya City Univiversity Graduate School of Department of Biological Chemistry, Graduate School of Pharmaceutical Natural Sciences, Nagoya, Aichi, Japan, 3Applied Physiology, Graduate Sciences, Kyoto University, Japan School of Medicine, Osaka University, Osaka, Japan The junctional membrane complex (JMC) between the plasma membrane The respiration induced heart rate variability (RHRV) is only found in mammals and avian showing a remarkable postnatal development, whereas no RHRV in aquatic and the endoplasmic/sarcoplasmic reticulum (ER/SR) provides a structural species. To elucidate our hypothesis that gravity exposure stimulates the postnatal platform for channel communication in excitable cells. Junctophilins development of RHRV as well as its evolutional origin in the ground animals, we contribute to the formation of JCMs by interacting with the plasma membrane studied effects of hypergravity on the postnatal development of RHRV using rat. and spanning the ER/SR membrane. In muscle cells, junctophilin subtypes Pregnant Wister rats were kept in centrifugal cages system for 38 days from 6th are essential for channel communication during excitation-contraction days of pregnant mother to have neonates until 23 days old. ECG was recorded from coupling. In the brain, junctophilins also support functional communication neonates in 2 to 23 days old in 1G and 2G groups. RHRV analysis was performed by between cell-surface and intracellular channels, which modulates the calculating Fourier power spectral component coincide with the respiration frequency. excitability and synaptic plasticity of cerebellar and hippocampal neurons. In both groups, averaged resting heart rate gradually increase from 2 to 23 days old. The heart rate in 2G group was significantly lower than 1G control (p<0.001) in 2 days old, where as no significance in 23 days old. The RHRV of 2 days old neonates was very small but significantly lower in 2G group than 1G control (p<0.01). It steeply increased to reached 45 fold in 1G control, whereas 69 fold in 2G group. The results strongly suggested that the postnatal innervation from respiration to cardiovascular centers was gravity dependent.

WD VIII-13-2 WD VIII-13-3 CALSEQUESTRIN REGULATION OF SINGLE MODULATION OF SKELETAL MUSCLE EXCITATION- RYANODINE RECEPTOR CALCIUM RELEASE CONTRACTION COUPLING BY PROTEIN CHANNELS COMPONENTS OF THE SARCOPLASMIC RETICULUM Michael Fill JUNCTIONAL FACE MEMBRANE Francesco Zorzato1, Osvaldo Delbono2, Susan Treves3, Miyuki Nishi4, Molecular Biophysics and Physiology, Rush University Medical Center, 4 Hiroshi Takeshima USA 1Experimental and Diagnostic Medicine, University of Ferrara, Italy, 2Department The luminal calcium regulation of skeletal (RyR1) and cardiac ryanodine of Physiology and Pharmacology Wake Forest University School of Medicine, USA, 3Departments of Anaesthesia and Research, Basel University Hospital, receptor (RyR2) calcium release channels was measured at the single channel 4 level. Two luminal RyR2 calcium regulatory mechanism(s) were found, one Switzerland, Department of Biological Chemistry, Graduate School of Pharmacological Sciences, Kyoto University, Japan CSQ dependent and the other not. These mechanisms discriminate between Skeletal muscle contraction involves the transduction of an electrical signal, generated at the luminal calcium and magnesium sensitivity differently. To explore the CSQ 2+ neuromuscular junction, into a transient increase of [Ca ]i within skeletal muscle fibre via dependent mechanism, two cardiac CSQ mutants linked to catecholaminergic a mechanism which is referred to as Excitation-Contraction (EC) coupling. In the last few polymorphic ventricular tachycardia (CPVT) were tested. These mutants years we have identified few protein components of the EC-coupling machinery, namely support abnormal RyR2 regulation. Additionally, systematic CSQ and RyR JP-45, SRP27, SRP35 and junctate. JP-45 is (i) mainly expressed in skeletal muscle, (ii) isoform swapping studies were done and luminal calcium dependence of down regulated during ageing and (iii) a component interacting with the alfa1.1 subunit of RyR open probability defined for each of the four possible RyR:CSQ isoform the dihydropyridine receptor (Cav1) and calsequestrin, two key proteins of the EC-coupling pairings. Single RyR2 channels were regulated by either the skeletal or machinery. JP-45 appears to be important for the functional expression of the Cav1.1, voltage cardiac CSQ isoform. In contrast, neither CSQ isoform regulated single sensor. To examine the functional role of JP-45 we studied and compared the skeletal muscle function of 3, 12 and 18 months old JP-45 knock-out mice and age-matched wild-type litter RyR1 channels. We conclude that 1) CSQ regulation of single RyR2 channels mates. Our results show that in young mice, ablation of JP-45 reduces functional Cav1.1 is substantial and may underlie CPVT pathogenesis and 2) there is little or no protein expression on the t-tubule membrane, impairing the EC coupling signal between CSQ regulation of single RyR1 channels. The latter implies that the role CSQ Cav1.1 and RyR1, thereby reducing skeletal muscle force. However, ablation of JP-45 does in skeletal muscle may be limited to that of an intra-SR calcium buffer. not appear to accelerate the decay of EC coupling induced by ageing.

42 IUPS 2009 July 27 - August 1, 2009 in Kyoto WD VIII-13-4 WD VIII-13-5 MEMBRANE REPAIR - Ca2+ ENTRY AND VESICLE MG53, A MOLECULAR SENSOR OF THE ACUTE TRAFFICKING MEMBRANE REPAIR PROCESS IN STRIATED MUSCLE 1 2 1 Richard A Steinhardt Jianjie Ma , Hiroshi Takeshima , Chuanxi Cai , 1 3 4 Molecular and Cell Biology, University of California, Berkeley, USA Noah Weisleder , Shinji Komazaki , Yoshihide Sunada 1 When a cell loses its membrane integrity it dies within minutes unless it Department of Physiology and Biophysics, Robert Wood Johnson Medical School, USA, 2Department of Biological Chemistry, Kyoto University successfully reseals. There are a variety of mechanisms brought into play 3 Graduate School of Pharmaceutical Sciences, Japan, Saitama Medical depending on the size of the disruption. For breaks on the order of a micron School, Japan, 4Kawasaki Medical School, Japan and above, an active excytotic response is necessary for the membrane to reseal. The loss of this process is the chief cause of cell death during Defective membrane repair can contribute to the progression of muscular dystrophy. While mutations in caveolin-3 (Cav3) and dysferlin are linked to muscular dystrophy metabolic inhibition and in anoxia. in human patients, the molecular mechanism underlying the functional interplay I will describe the evidence leading to the hypothesis of membrane repair between Cav3 and dysferlin in membrane repair of muscle physiology and disease by exocytosis. Repeated breaks in cell membranes elicit a more rapid has not been fully resolved. We recently discovered that MG53, a muscle specific membrane repair response, using signaling pathways that parallel those TRIM-family protein (TRIM72), contributes to intracellular vesicle trafficking and in neurotransmission. I will attempt to clear up confusions about which is an essential component of the membrane repair machinery in striated muscle. We intracellular compartments are used at different scales of membrane breaks. show here that MG53 interacts with dysferlin and Cav3 to regulate membrane repair The process of exocytosis appears to be necessary in order to lower in skeletal muscle. MG53 is required for movement of dysferlin to sites of cell injury membrane tension to the point where the lipid bilayer is able to reseal. during repair-patch formation. Mutations in Cav3 (P104L, R26Q) that cause retention of Cav3 in Golgi apparatus result in aberrant localization of MG53 and dysferlin in Artificial treatments that lower the tension to the critical point can a dominant-negative fashion, leading to defective membrane repair. Our data reveal successfully reseal cells even the natural mechanism requiring exocytosis is that a functional complex formed by MG53, dysferlin and Cav3 is essential for repair blocked. For the artificial lowering of tension to be effective it has to be done of muscle membrane damage, and also provide a therapeutic target for treatment before the membrane breaks. The effective compound and concentration has of muscular and cardiovascular diseases that are linked to compromised membrane already been tested for safety in human volunteers for periods up to 48 hours. repair.

WD VIII-13-6 WD VIII-13-7 SYSTEMATIC ISOLATION OF MUTANTS DEFECTIVE IN PHARMACOGENOMICS NETWORKS IN HUMAN TRANSCRIPTION FACTORS IN C. ELEGANS DISEASE MODELS AND THERAPEUTIC TARGET Shohei Mitani, Keiko Gengyo-Ando, Sawako Yoshina, Eriko Kage- VALIDATION Whole-day symposia Nakadai, Muneyoshi Otori, Kenjiro Sakaki, Kazuko Fujisawa, Toshio Tanaka, Yuhei Nishimura, Yasuhito Shimada Hiroyuki Kobuna Pharmacogenomics and Pharmacoinformatics, Mie University, Japan Department of Physiology, Tokyo Women's Medical University School of The most important strategies in pharmacogenomics are gene expression Medicine, Japan profiling and the network analysis of human multifactorial disease models. Nematode C. elegans is an ideal model organism to study biological function of Delayed cerebral vasospasm after aneurysmal subarachnoid hemorrhage genes. C. elegans hermaphrodites have an invariable number (959) of somatic nuclei causes cerebral ischemia and infarction. Using a DNA microarray, a including 302 neurons. Detailed descriptions on cell lineage and morphology have prominant upregulation of heme oxygenase-1 (HO-1) and heat shock protein been completed. The genome of the C. elegans has been sequenced earliest among (HSP) 72 mRNAs were observed in the basilar artery of a rat vasospasm multicelluar animals. Together with the EST information, we now know what kinds of model. Antisense HO-1 and HSP 72 oligodeoxynucleotide inhibited HO-1 genes are expressed in the animals. and HSP 72 induction respectively, and significantly aggravated cerebral We have succeeded in the development of the high-efficient method to isolate deletion vasospasm. We found that gene induction of HO-1 and HSP 72 by clinical mutants. We mutagenize wild-type animals with trimethylpsoralen and ultraviolet. compounds reduced cerebral vasospasm, respectively. Recently, the zebrafish Because C. elegans can be stored as frozen stocks, we have made mutant banks consisting more than a million genomes. We have developed very sensitive screening is becoming an popular model organism to study network analysis of human techniques using PCR. disease models to validate potential drug targets. We have developed unique Using these techniques and frozen banks, we have isolated more than 3,000 alleles metabolic syndrome and heart failure models in zebrafish and identified deletion mutants comprising 2,500 genes. Among the mutants, we have been several candidate genes as novel drug targets. extensively trying to isolate deletion mutants of transcription factors in C. elegans. It is These results suggest that network analysis of human disease models has the said that about 900 transcription factors are assigned in the C. elegans genome. So far, potential to elucidate mechanisms of multifactorial diseases and to bridge we have isolated deletion mutants targeting more than 500 genes, more than half of the the gap between in vitro and in vivo studies, and could define strategies for whole transcription factors. identifying novel drug targets in various multifactorial diseases.

WD VIII-13-8 WD VIII-13-9 APPLICATION OF FUNCTIONAL GENOMICS NEW GENERATION SEQUENCING TECHNOLOGY TO PHYSIOLOGY AND PATHOPHYSIOLOGY: ENABLES REVOLUTION IN GENOMICS RESEARCH TRANSCRIPTOME ANALYSIS OF DISEASE MODEL Yafei Liu Gozoh Tsujimoto Sequencing Business, Asia Pacific and Japan, Illumina, China Department of Genomic Drug Discovery Science, Kyoto University, Japan 2008 and 2009 have been revolutionary for Genomics. The Illumina Genome We have recently succeeded in studying the molecular physiology/ Analyzer (Solexa Technology) platform, with its cutting edge advantage pathopysiology by using this functional genomics. Glomerulonephritis (GN) in data accuracy, assay throughput, automation, and cost effectiveness, has is a progressive inflammation that may be caused by a variety of underlying enabled a wide range of applications as well as nearly 300 peer-reveiwed disorders. DNA chip analysis identified CK2α, the catalytic subunit of CK2, genomics publications. Customer success is not only from Genome as a GN-related, differentially expressed gene. Overexpression of CK2α Centers, but also from smaller research laboratories. Cluster Generation and was noted in the proliferative glomerular lesions in rat GN models and in Reversible Terminator are two core technologies enabling unique Genome renal biopsy specimens from lupus nephritis or IgA nephropathy patients. Analyzer customer value and success, which will be reviewed. In past two Administration of either antisense oligodeoxynucleotide against CK2α or low molecular weight CK2-specific inhibitors effectively prevented years, the Genome Analyzer technology has improved from 1G data per run, the progression of renal pathology in the rat GN models. The resolution to nearly 20G per run. The platform remains to be highly scalable, and we of GN by CK2 inhibition may result from its suppression of extracellular will present a technology development roadmap targeting 95G per run by signal-regulated kinase-mediated cell proliferation, and its suppression of the end of 2009. Real customer experience on Genome Analyzer will also inflammatory and fibrotic processes that are enhanced in GN. Our results be reviewed. in a wide breadth of applications, such as re-sequencing, De show that CK2 plays a critical role in the progression of immunogenic renal Novo sequencing, ChIP-Seq, digital gene expression, mRNA-Seq, and DNA injury, and therefore, CK2 is a potential target for GN therapy. methylation.

IUPS 2009 July 27 - August 1, 2009 in Kyoto 43 WD VIII-13-10 WD VIII-14-1 THE SOLiD3 SYSTEM AND BEYOND: A TECHNOLOGY EVOLUTION OF THE VERTEBRATE PHARYNX ENABLING NEW APPLICATIONS Masataka Okabe Hideki Hanaoka Department of Anatomy, The Jikei University School of Medicine, Japan Next Generation Sequencing Business Unit, Applied Biosystems Japan, About 370 million years ago, in the late Devonian period, tetrapod ancestors Japan gradually evolved from fish-like aquatic animals to walking terrestrial SOLiD3 is Applied Biosystems next-generation genetic analysis plat form. SOLiD3 vertebrates. In order to move from water to land, they made a number of can produce 400 million tag sequences, 30 billion (30G) bp mappable sequence in modifications to their bodies: removal or transformation of existing organs a single run. Moreover, 2-bp encoding chemistry enables researchers to distinguish and invention of new organs. I am currently interested in understanding how between true SNPs and random/system errors and realized the accuracy of 99.9%. primitive tetrapods acquired new organs such as parathyroid and lungs. Our With such high-throughput and accuracy, SOLiD3 provides several applications which group integrates genomic information and embryology of various vertebrate were thought to be difficult to implement several years ago. species to elucidate genomic changes that allowed acquisition of new organs. With SOLiD3, each scientist can perform human whole genome analysis. Using mate- We compare major taxa of vertebrates using mice, chicks, frogs, zebrafish pair library, researchers can identify structural variations such as in/del and inversion and dogfish. We also use living transitory species: Polypterus and lungfish, in the genome. Also, target re-sequence provides identification of mutations in specific which retain many traits of the vertebrates living at the time of water-to- region of genome. With barcoding, SOLiD3 can analyze more than hundreds samples in a single run. land transition. In this talk, I will reveal the origin of the parathyroid gland Whole transcriptome analysis can provide enlarged dynamic range and identify new which regulates the concentration of calcium in blood, and show molecular splicing patterns. It establishes new standard in the gene expression study. Whole evidences suggesting that the lungs and the swimbladder evolved from a transcriptome analysis can be performed even with a single cell. Now ChIP analysis lung-like organ that was present in the common ancestor of teleosts and and methylation study also can be performed in genome-wide scale. tetrapods.

Whole-day symposia In this seminar, besides the latest data obtained from those applications, the future of SOLiD system will be presented.

WD VIII-14-2 WD VIII-14-3 ANIMAL MODELS TO STUDY KIDNEY DEVELOPMENT EVOLUTION AND DEVELOPMENT AT THE FISH- AND DISEASE TETRAPOD TRANSITION Tomoko Obara Per Erik Ahlberg Dept. of Medicine & Dept. of Genetics, MetroHealth Medical Center, Case Department of Physiology and Developmental Biology, Uppsala University, Western Reserve University, USA Sweden The vertebrate kidney is an important organ to maintain waste-free and The increasingly rich fossil record of lobe-finned fishes, early tetrapods and osmotically balanced blood in the circulation system. Experimental transitional forms is making it possible to reconstruct this morphological nephrology started over 100 years ago, when ex vivo culture systems transition in considerable detail. At the same time, developmental biology for amphibian and avian kidneys were first developed in 1920s. Next is giving us increasingly detailed insights into the molecular patterning milestone was in the 1950s when Grobstein demonstrated that inductive of fish and tetrapod anatomy. This creates opportunities for evolutionary- signaling events are essential for the formation of epithelial nephrons developmental investigation of specific transformations, where the molecular from mesenchyme. Since then, many different technologies and different animal model systems have emerged greatly expanding out understanding evidence helps to inform morphological homology judgements as well as of the cellular process involved in the kidney formation. The study of provide mechanistic explanations, while the fossils reveal transformational embryonic kidneys has been a paradigm for cell signaling, specification, morphologies that indirectly illuminate the evolution of the molecular and morphogenesis during organogenesis. My laboratory is using two non- patterning. I will discuss examples from the nasal region (the origin of the mammalian organisms, zebrafish and medaka. We have developed models internal nostril), neurocranium (the gain and loss of the intracranial joint) of polycystic kidney disease and glomerular disease and are studying the and appendages (the fin-to-limb transition), showing in each case how fossil underlying mechanisms. To better define the pathogenic mechanisms and to data add information to, and in some cases overturn, hypotheses based on identify new therapeutic targets, zebrafish and medaka have been a valuable comparisons of living animals. alternative to higher vertebrate. We believe that these studies have already changed how we view epithelial differentiation and will provide novel angles for therapeutic intervention for human kidney diseases.

LS14-1 (WD VIII-14-related luncheon seminar) LS14-2 (WD VIII-14-related luncheon seminar) STEM CELLS AS KEY CELLS FOR UNDERSTANDING CHARACTERIZATION OF STEM CELLS BY FLOW BOTH EVOLUTION AND REGENERATION CYTOMETRY; INTRODUCING THE NEXT GENERATION Kiyokazu Agata GUAVA EASYCYTE 8HT INSTRUMENT Department of Biophysics, Graduate School of Science, Kyoto University, Paul E. Cizdziel Japan Nihon Millipore K.K. Recently stem cells have attracted great interest in many fields of science, Understanding stem cell physiology and function is aided by flow cytometry particularly in medical areas, in which stem cells can be used for disease with fluorescent-tagged immunodetection reagents to identify the expression therapies. However, the fundamental cellular and molecular basis of stem of key biomarkers of pluripotency or differentiation. The advantages of cell systems still remains unclear. Here, I introduce both a stem cell system flow cytometry include qualitative and quantitative assessment of multiple and brain structure of planarian, and then propose several new insights to biomarkers simultaneously, speed in data acquisition, and high statistical investigate basic principles of stem cell systems. accuracy. Until Guava Technologies introduced its first benchtop instrument, flow cytometry was considered to be high maintenance, technically I propose that there are two different origins of stem cell systems. One challenging and expensive. Guava Technologies (now a part of Millipore) system basically evolved from adult pluripotent stem cells which support has recently released a next generation instrument series that enables up both asexual and sexual reproduction. The other system developed by to 6-color / 8-parameter testing. Called the EasyCyte 8HT, it is based on separating multiple functions of primitive cells into specialized cell the same unique capillary flow-cell and analytical only design, but with lineages. Specifically, the nervous and immune systems have achieved high improved performance and a new powerful data acquisition & analysis performance by evolving a variety of specialized cells. Such lineage-specific software. Also, similar to the prior model, new EasyCyte instruments have stem cell systems may evolve much later. These two different classes of stem the same low maintenance features, low running costs, and low price. cells may have different molecular properties. Positional cues provided by When used in combination with FlowCellect™ Stem Cell Characterization ERK and b-catenin signals may have a key role for handling pluripotent stem kits from Millipore, the analysis of human or mouse stem cells has never cells. On the contrary, to handle lineage-specific stem cells, it is important to been easier. In this seminar, we will present this and more about Millipore understand molecular mechanisms underlying asymmetric cell division. solutions for stem cell research and flow cytometry.

44 IUPS 2009 July 27 - August 1, 2009 in Kyoto WD VIII-14-4 WD VIII-14-5 USE OF iPSC TO STUDY HUMAN AGING ADVANTAGES OF HAIR FOLLICLE PLURIPOTENT ALAN COLMAN STEM (hfPS) CELLS OVER ES AND IPS CELLS FOR Wolfson Centre for Age related diseases, King's College London, UK, and REGENERATIVE MEDICINE 1 2 A*STAR Institute of Medical Biology, Singapore Robert M. Hoffman , Yasuyuki Amoh 1Department of Surgery, University of California San Diego, USA The process for generating induced pluripotent stem cells (iPSC) from 2 murine somatic cells was first reported in Kyoto in 2006. Since that time Department of Dermatology, Kitasato University School of Medicine, Japan this field has burgeoned, and it seems that this technology works in most mammalian species and with many somatic cell types. iPSC research has Embryonic stem (ES) cells and induced pluripotent cells (iPS) cells have critical permeated many different research areas. I will discuss its application to obstacles for clinical use including immune rejection and malignant potential. furthering our understanding of the early pathophysiology of specific human Hair follicle pluripotent stem cells (hfPS) cells can differentiate to neurons, diseases. In these circumstances, iPSC generated from patients displaying glial cells, and other cell types in vitro, and can promote nerve and spinal cord regeneration in mouse models. hfPS cells are located above the hair follicle bulge genetic disease are used as a surrogate model to understand some of the early in what we have termed the hfPS cell area (hfPSA) and are nestin positive and molecular correlates of that disease. There is an underlying assumption that keratin 15 (K-15) negative. Mouse and human hfPS cells when transplanted in a disease phenotype will be seen in the in vitro conditions. It is not clear that the severed sciatic nerve of the mouse, differentiate into glial fibrillary-acidic- this assumption is a valid one for many human diseases. In my talk I will protein (GFAP)-positive Schwann cells and promote the recovery of pre-existing describe our own research on iPSC to a premature aging disease. axons, leading to nerve generation. The regenerated nerve recovered function and, upon electrical stimulation, contracted the gastrocnemius muscle. Mouse hfPS can also functionally repair the severed spinal cord enabling the mouse to regain use of its hind linbs. Human hfPS cells can be readily isolated from the human scalp, thereby providing an accessible, autologous and safe source of stem cells for regeneration medicine that do not have immunological or malignant problems associated with ES or iPS cells.

WD VIII-14-6 WD VIII-14-7 NEURAL DIFFERENTIATION AND CELL THERAPY CELL THERAPY IN PARKINSON'S DISEASE AND USING PLURIPOTENT STEM CELLS STROKE Hideyuki Okano Olle Lindvall Whole-day symposia Department of Physiology, Keio University School of Medicine, Japan Clinical Sciences, Lund University, Sweden Although transplanted neural stem/progenitor cells (NS/PCs) differentiated Transplantation of stem cells and mobilization of endogenous stem cells into neuron, astrocyte, and oligodendrocyte and promoted functional have been proposed as future therapies for Parkinson’s disease and stroke. recovery after spinal cord injury (SCI), it remains to be resolved about the In support, neurons suitable for transplantation can be generated from stem differentiation potential and survival rate play a role in functional recovery cells in culture, and the adult brain produces new neurons from its own after transplantation. To address this, we took advantage of our original neural stem cells in response to injury. However, from a clinical perspective, in vitro differentiation of embryonic stem (ES) cells. We cultured mouse the development of stem cell-based therapies for brain diseases is still at an ES cells-derived neurogenic primary neurosphere (ES-PNS) and gliogenic early stage. We do not know what is the best stem cell source, and research secondary neurosphere (ES-SNS) in vitro and transplanted them into mouse on embryonic stem cells, induced pluripotent stem cells, and stem cells from SCI models. Surprisingly, transplantation of gliogenic ES-SNS, but not fetal or adult brain or from other tissues should be performed in parallel. We neurogenic ES-PNS, promoted axonal growth, remyelination, angiogenesis, need to understand how to control stem cell proliferation and differentiation and functional recovery after SCI. Thus, injured spinal cord required into specific cell types, induce their integration into existing synaptic and gliogenic ES-derived neurospheres for functional recovery and gave an neural networks, and optimize the functional recovery in animal models important cue for the establishment of the regenerative medicine for SCI closely resembling the human disease. Finally, before clinical application using ES or induced pluripotent stem cells (iPS cells) in the near future. we must know what is the biological mechanism underlying the functional improvements observed in animal models.

WD IX-15-1 WD IX-15-2 INTRODUCTION OF PHYSIOME AND SYSTEMS EXPECTATION FOR DEVELOPMENT OF PHYSIOME BIOLOGY SYMPOSIUM PROJECTS IN JAPAN 1 2 Yoshihisa Kurachi Fumihiko Kajiya , Satoshi Mohri 1Department of Medical Engineering, Kawasaki University of Medical Department of Pharmacology Graduate School of Medicine, Osaka 2 University, Japan Welfare, Japan, Department of Physiology, Kawasaki Medical School, Japan The integration of knowledge from many disciplines and the vast amount of biological data in the post-genome era, together with new techniques from Council for Science and Technology Policy (CSTP, Japanese government) is focusing on following four areas, “life science”, “information technology”, “environmental the mathematical and information sciences, are moving the world towards an science”, and “nanotechnology and materials". In CSTP, we have coordinated the new generation of life sciences where physiological and pathophysiological nanobiotechnology program to promote the fusion area between life science and information from the living human body can be quantitatively described in nanotechnology with several relevant ministries. Our prioritized fields are "bio- silico across multiple scales of time and size and through diverse hierarchies molecular imaging for fine structure and function", "diagnosis/ treatment using DDS of organization. The Physiome and Systems Biology represent such emerging with imaging guide", and "patient-friendly nano-based-biodevices". sciences. The challenge is to understand and quantitatively integrate not To make these practicable, it is crucially important to understand the living body only the structure and function of biological entities such as ion channels consisting of different scales from nano to macro, i.e., molecules, cells, tissues, and on a single spatio-temporal scale, but also functional relationships between organs by a synthetic framework: Physiome that covers entire hierarchical structure entities across multiple scales. It will allow us to understand the mechanisms and function. underlying biological functions that will emerge through the dynamics of As with CSTP, Medical Engineering Technology Industrial Strategy Consortium (METIS) with three ministries, i.e., MEXT, MHLW, and METI recognizes the each element and large aggregations of the elements. The main theme of the importance of Physiome for R&D of medical instruments. The "Next-Generation IUPS2009 congress is se to be 'Function of Life: Elements and Integration'. Integrated Simulation of Living Matter" is now being promoted, as a concrete example In this WDS, we will see the current status of this new emerging field all over of Physiome. the world and we wish to seek for the rode for the world wide cooperation in We expect that Physiome further accelerates our synthetic understanding of this grand challenge of new biological research field. hierarchical biological systems and improves the welfare of humankind.

IUPS 2009 July 27 - August 1, 2009 in Kyoto 45 WD IX-15-3 WD IX-15-4 EURO PHYSIOME AND THE LIVING HUMAN DIGITAL THE IUPS PHYSIOME PROJECT LIBRARY PROJECT Peter John Hunter Marco Viceconti Auckland Bioengineering Institute, University of Auckland, New Zealand Laboratorio di Tecnologia Medica (Medical Technology Lab), Istituto The Physiome Project1, an international effort to provide an integrative multi-scale Ortopedico Rizzoli, Italy modeling framework for computational physiology, has recently been boosted by a The paper summarises the most recent developments of the Living European initiative called the ‘Virtual Physiological Human’ (VPH) 2. The combined Human Project, an international initiative aimed to the physiome of the VPH/Physiome Project aims to link biochemical network systems biology models musculoskeletal system. We developed two team science tools that will be and biophysically and anatomically based bioengineering models to medical imaging essential to the collaborative effort necessary to realise a complete physiome and biomedical signal analysis. Achievements so far are the development of markup of the musculoskeletal system: Biomed Town, which is an on-line community languages (CellML, FieldML), freely accessible model repositories based on the where researchers can work together, and the PhysiomeSpace service, that markup languages, and open source computational tools for authoring, visualizing, makes possible to create large data collections, upload to a private sandbox, 3 executing and analyzing these models . The talk will discuss current efforts to develop fully annotate them with powerful and extensible ontologies, and share each metadata standards for annotating model components with terms from existing data resource only with the peers we choose. Data collections are formed 4 5 6 using PsBuilder, a dedicated software base don the MAF framework that ontologies such as GO , FMA and OPB that describe the biological, anatomical and biophysical meaning of these components. allow the creation of heterogeneous biomedical data collections including 1 medical imaging, biomedical signals, simulation results, etc., properly fuse Hunter, P.J., Crampin, E.J. and Nielsen, P.M.F. Bioinformatics, multiscale modelling and the IUPS Physiome Project. Briefings in Bioinformatics. 9 (4), 333-343, 2008. in time and space to provide a coherent and multiscale representation of 2 pathophysiological process of interest. The service is running in beta, and www.vph-noe.eu 3 already contain the LHDL data collection, the first multiscale data collection www.cellml.org; www.fieldml.org 4 where skeletal data from the whole body down to the molecule were www.geneontology.org 5 Whole-day symposia collected form a single donors. Some examples of early applications in the http://sig.biostr.washington.edu/projects/fm/AboutFM.html VPHOP project are provided. 6 http://bioportal.bioontology.org/ontologies/38990

WD IX-15-5 WD IX-15-6 A ROBUSTNESS-BASED APPROACH TO SYSTEMS- BIOPHYSICAL MODELS OF THE CORONARY ORIENTED DRUG DESIGN CIRCULATION MEET CLINICAL DECISION MAKING Hiroaki Kitano Jos Spaan1, Jeroen PHM van den Wijngaard1, Pepijn van The Systems Biology Institute, Japan Horssen1, Jan J Piek2, Maria Siebes1 Many potential drugs that specifically target a particular protein considered 1Dept. Biomedical Engineering and Physics, AMC, University of to underlie a given disease have been found to be less effective than hoped, Amsterdam, The Netherlands, 2Dept Cardiology, AMC, University of or to cause significant side effects. The intrinsic robustness of living systems Amsterdam, The Netherlands against various perturbations is a key factor that prevents such compounds Coronary blood supply, needed for heart function, can be limited by atherosclerosis from being successful. By studying complex network systems and of larger conduit arteries as well as by microvascular disease. Because of the multi- reformulating control and communication theories that are well established factorial control of coronary blood flow a more detailed model of the coronary in engineering, a theoretical foundation for a systems-oriented approach to circulation can facilitate the clinical decision making process for treatment and choice more effectively control the robustness of liing systems, particularly at the of treatment modality. cellular level, could be developed. Here, I use examples that are based on An anatomical based functional model of the coronary circulation is being developed existing drugs to illustrate the concept of robustness, and then discuss how based on measured 3D reconstruction of the vascular bed of the heart. For this a greater consideration of the importance of robustness could influence the intramural vessels, including the arteriolar microcirculation, is recontructed by a novel design of new drugs that will be intended to control complex systems. imaging cryomicrotome with voxel dimension of 20 microns. The model predicts the distribution of blood flow over the myocardial wall as well as the waveforms of pressure and flow velocity in the epicardial arteries under a wide variety of physiological conditions but also in the presence of disease. The validation data set for perfusion is obtained by MRI and for epicardial coronary signals by the combined measurement of pressure and flow velocity. The project is funded by the European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement n° 224495 (euHeart project).

WD IX-15-7 LS1 (WD IX-15-related luncheon seminar) SYSTEMS BIOLOGY OF ANGIOGENESIS: FROM REPRODUCIBLE MODELING, STANDARDS AND MOLECULES TO THERAPEUTICS MODULARITY Amina A. Qutub, Aleksander S Popel, Feilim Mac Gabhann, James B Bassingthwaighte Marianne O. Stefanini Department of Bioengineering, University of Washington, Seattle, USA Department of Biomedical Engineering, Johns Hopkins University School Scientific research is intended for dissemination and absorption into the of Medicine, USA generic knowledgebase of science, but reproducibility, in general, is difficult Angiogenesis is important under physiological and pathological conditions (e.g., to attain. For physiological modeling, the development of databases, the exercise, cancer, age-related macular degeneration, rheumatoid arthritis, myocardial adherence to standards, the use of modular design, and the automation ischemia, peripheral arterial disease). Angiogenesis involves numerous processes: of modular construction of multiscale models together can facilitate cell sensing of oxygen during hypoxia; upregulation of vascular endothelial growth reproducibility. Success in automating the construction of integrated models factor (VEGF), and of matrix metalloproteinases (MMPs); extracellular matrix (ECM) from databases of preconstructured modules (= modular model forms) proteolysis and release of matrix-binding growth factors; endothelial cell migration, would allow the extraction of thoroughly validated modules from well- proliferation and differentiation; tubulogenesis and network morphogenesis. We have curated databases, with assurance of clear documentation by acknowledged developed an array of molecular-level computational models that serve as modules experts. Competing modules should be available in similar form. To pull in a multiscale integrative model. These include a model of a transcription factor selected modules into a higher level models requires common ontologies, HIF1alpha; a model of interactions of VEGF isoforms with their receptors VEGFR, complete equations with boundary and initial conditions. completely defined Neuropilin-1 and ECM; a model of ECM proteolysis by MMPs, and MMP-mediated units, and running examples. Models stored in databases such as CellML, VEGF release from ECM. An agent-based computational framework integrates these SBML or ours at www.physiome.org or others need also to adhere to a models into multiscale rule-based models, spanning the levels from the molecular to common ontology. Achievement of this combination of capabilities requires organ and organism. The models are applied to several therapeutic interventions in serious collaborative planning by scientific societies and at the national and disease conditions including pro-angiogenic approaches to peripheral arterial disease international levels. and anti-angiogenic approaches to cancer.

46 IUPS 2009 July 27 - August 1, 2009 in Kyoto WD IX-15-8 WD IX-15-9 STRUCTURE BASED FUNCTIONAL MODELLING OF CARDIAC EXCITATION AND NEURAL CONTROL 2+ CELLULAR Ca DYNAMICS Shingo Murakami1, Shingo Suzuki1, Ian Findlay2, Yoshihisa 1 Taishin Nomura1, Yosuke Yumikura1, Rachid Ait-Haddou2, Shingo Kurachi 3 4 5 3 Murakami , Satomi Adachi-Akahane , Ian Findlay , Yoshihisa Kurachi 1Department of Pharmacology, Graduate School of Medicine, Osaka 1Graduate School of Engineering Science, Osaka University, Japan, 2The Center University, Japan, 2CNRS UMR 6542, Faculte des Sciences, Universite for Advanced Medical Engineering and Informatics, Osaka University, Japan, Francois-Rabelais de Tours, France 3Graduate School of Medicine, Osaka University, Japan, 4Graduate School of Medical Sciences, Toho University, Japan, 5Faculte des Sciences, Universite In response to physical and psychological demands, cardiac excitation is Francois-Rabelais de Tours, France dynamically controlled by the autonomic nervous system, i.e., sympathetic and 2+ parasympathetic nerves. These controls are achieved through G-protein coupled Ca dynamics underlying cardiac excitation-contraction coupling are essential for the 2+ receptor-dependent modulation of membrane ion channels. The signaling heart functions. Here we construct a microstructure based model of Ca dynamics to simulate Ca2+ influx through individual L type Ca2+ channels (LCCs), an effective Ca2+ mechanisms for each system are composed of cardiac GPCRs (β-adrenergic and diffusion within the cytoplasmic and dyadic spaces, and subsequent Ca2+ induced Ca2+ m2-muscarinic receptors), G proteins (Gs and PTX-sensitive GK), intracellular cell-signaling molecules and the target ion channels (L-type Ca2+ channel and G release (CICR) from the sarcoplasmic reticulum using an integrated modeling platform + (insilicoIDE and insilicoML). Calcium-dependent inactivation (CDI) of LCCs with protein-gated K channel). For quantitative understanding of the neural control mechanisms of local and global Ca2+ sensing is taken into account in the model. Multi- of cardiac excitation, it is essential to develop mathematical models for each Agent Simulations of the Ca2+ dynamics in the model are performed on the platform. control. For parasympathetic deceleration of heart beat, muscarinic activation of We first show that an appropriate set of parameter values of the model with effectively KG channels has been modeled based on our own experimental data. Our recent extra-slow Ca2+ diffusion enables the model to reproduce major features of the CDI finding on the role of RGS proteins in the control of GK activity has allowed process induced by the local and global sensing. We then examine how the local us to construct the models reasonably reproducing temporal behavior of ACh- 2+ and global CDIs affect the Ca2+ dynamics and CICR. The results suggests that the activation of KG channels. On the other hand, in cardiac L-type Ca channel, we 2+ effectively slow Ca2+ diffusion under Ca2+-CaM interaction, nanodomain structure of have succeeded in the modeling of the voltage-dependent and Ca -dependent LCCs for detailed CDI processes, and restricted narrow space of dyadic space play an inactivation processes to reproduce the Ca2+ channel current behaviors in the important role for controlling Ca2+ dynamics in the cytoplasmic and dyadic spaces. control and under β-modulation.

WD IX-15-10 WD IX-15-11 MULTI-SCALE MODELING AND SYSTEMS BIOLOGY UT-HEART - A MULTISCALE MULTIPHYSICS HEART OF THE HEART SIMULATOR Andrew D McCulloch1, Roy C Kerckhoffs1, Campbell G. Stuart1, Jun-ichi Okada, Takumi Washio, Seiryo Sugiura, Toshiaki Hisada Whole-day symposia 2 2 Jacob Feala , Giovanni Paternostro School of Frontier Sciences, University of Tokyo, Japan 1 2 Department of Bioengineering, UC San Diego, USA, The Burnham A multiscale multiphysics heart simulator has been under development at the Institute, La Jolla, USA University of Tokyo. At the macroscopic level, a fluid-structure interaction We review integrative Physiome models of the heart and their application finite element model of the human whole heart including aorta is combined to clinical diagnosis and therapy. Systems Biology approaches allow us with an electrophysiological bi-domain finite element model in which more to develop detailed functionally integrated models of cardiac myocyte than 20 million cell models, e.g., Noble model, are embedded. With this metabolism, signaling and excitation-contraction coupling. Recently we model, the pulsation of heart is realized by the contraction and relaxation have developed new multi-scale modeling techniques to solve fully coupled of each structural element driven by the molecular mechanism of excitation multiscale models of cardiac electromechanics in the normal and diseased contraction coupling. The finite element torso model surrounding the heart heart. We used these models to investigate the functional consequences in was also created to reproduce electrocardiogram. Clinical applications the whole ventricles of transmural cellular heterogeneities in excitation- have been attempted including the investigation of the mechanism of contraction coupling mechanisms. Finally, applying these models to arrhythmias in the LQT syndrome, the result of which is applicable to the dysynchronous human heart failure, may provide an opportunity to improve virtual drug discovery. At the microscopic level, the finite element cell patient selection and reduce non-responder rates for cardiac resynchronization models including intracellular structures were developed, where mechanical therapy. We show preliminary results from an ongoing clinical study. and electrochemical sub-cellular events are reproduced to simulate the contraction of myocyte. Finally, by bridging these two models with the use of the homogenization method, ultimate multiscale simulation is realized on a parallel computer. Some results will be exemplified to highlight the meanings of multiscale simulation.

WD IX-15-12 WD IX-15-13 STUDYING INTEGRATED FUNCTION IN THE LUNG IN BIOMECHANICAL MODELING OF MULTISCALE THE CLINICAL SETTING: APPLICATION OF IMAGING- MECHANICS IN THE LUNG BASED LUNG PHYSIOME MODELS Shigeo Wada Merryn Tawhai1, Kelly S Burrowes2, Eric A Hoffman3, Ching-Long Department of Mechanical Science & Bioengineering, Osaka University, Lin4, James Sneyd5 Japan 1Auckland Bioengineering Institute, University of Auckland, New Zealand, The lung is the primary organ to transport oxygen from external air into 2Oxford Computing laboratory, University of Oxford, UK, 3Departments bloodstream, and to release carbon dioxide in the opposite direction. This of Radiology and Biomedical Engineering, University of Iowa, USA, respiratory process includes various mechanical phenomena whose scale 4Department of Mechanical Engineering, University of Iowa, USA, diversely ranges from molecules to cells, tissues and organs. In order to 5Department of Mathematics, University of Auckland, New Zealand understand the underlying processes of how those mechanical phenomena Pathologic changes in the lung are typically slow to develop, are heterogeneous, and interact each other, how those link respiratory physiology, and how those multiple processes interact. The interpretive and predictive computational models that contribute to respiratory functions, it is essential to develop modeling and have been developed through collaborative effort in the international Lung Physiome simulation algorithms of the lung respiration. The talk will describe our are valuable tools to differentiate between interacting mechanisms in the lung. As recent work on biomechanical modeling of 3D airway anatomy based on the part of the Lung Physiome and the Human Lung Atlas projects we have established medical images, airflow and mass transfer in the branching airway, inflation methods to derive imaging-based, patient-specific models of the lung and its transport and deflation of the lung, microstructure of lung parenchyma, deformation of systems, combining high resolution imaging data and volume-filling branching the cell within lung tissue, blood flow in the pulmonary circulation, red blood algorithms beyond the limit of imaging resolution. More recent collaborative cell (RBC) behavior in the capillaries, gas exchange between alveolar gas and efforts have extended these models to integrate tissue and cellular function, and the RBC, and molecular dynamics of the gas transport across the alveolar liquid biochemistry of gas exchange. Several applications of these models in the clinical layer. Furthermore, we will show a framework to integrate those multiscale setting will be presented, in particular the study of airway hyper-responsiveness, the model toward the development of the in silico human lung. compromise of the bronchial during mechanical ventilation, and pathologic alterations to regional gas exchange.

IUPS 2009 July 27 - August 1, 2009 in Kyoto 47 WD IX-16-1 WD IX-16-2 NORMAL AND PATHOLOGICAL PATTERNING OF THE DOPAMINE-DEPENDENT DYNAMICAL STATE SUBTHALAMIC NUCLEUS CHANGES IN NETWORK MODELS OF SUBTHALAMIC Mark David Bevan NUCLEUS AND GLOBUS PALLIDUS 1 2 3 Physiology, Northwestern University, USA Tomoki Fukai , Katsunori Kitano , Atsushi Nambu 1 2 Under normal conditions subthalamic nucleus (STN) activity is asynchronous Brain Science Institute, RIKEN, Japan, Department of Human and Computer Intelligence, Ritsumeikan University, Japan, 3Division of System and arrhythmic. In Parkinson's disease (PD) STN activity is correlated and Neurophysiology, National Institute for Physiological Sciences, Japan rhythmic in the tremor (4-8 Hz) and beta (13-30 Hz) bands. Reversal of pathological STN activity by dopamine replacement and/or direct stimulation Basal ganglia (BG) are crucial for the coordination of cognitive motor behavior. ameliorates the motor symptoms of PD. The principles underlying the In particular, functional deficits in the excitatory-inhibitory loop circuit of transition from normal to pathological STN activity will be discussed. subthalamic nucleus (STN) and external globus pallidus (GPe) result in severe impairment of movement control in patients with Parkinson’s disease. STN neurons exhibit autonomous, rhythmic firing that is driven by Dopamine depletion in the Parkinsonian state causes several changes in neuronal axonal Nav channels and is regularized by Cav2.2-SKCa channel coupling. dynamics in the STN-GPe network. In particular, STN neurons change their The patterning of STN activity by GABAergic globus pallidus (GP) firing pattern from irregular single spikes to bursting, and GPe neurons show and glutamatergic cortical inputs is moderated by dopamine acting at enhanced synchrony in the Parkinsonian state. The biological mechanism of postsynaptic receptors, which depolarizes STN neurons and deregularizes these changes, however, remains elusive. To explore the possible mechanism, their autorhythmic activity and presynaptic receptors, which reduces the here we constructed network models of GPe and STN. Our model incorporates probability of synaptic transmission. In contrast, chronic dopamine depletion various known effects of dopamine on ionic channels, such as persistent sodium enhances the impact of GP and cortical inputs by reducing autonomous STN current and L-type calcium current on GPe neurons. Dopamine is also known to activity, downregulating dendritic HCN channels and upregulating synaptic modulate the efficacy of short-term depression at inhibitory synapses between receptors. Together, these studies suggest that the pathological patterning of GPe neurons. The effects of dopaminergic modulations on the spatiotemporal

Whole-day symposia STN activity is caused, in part, by the loss of direct dopaminergic modulation network activity pattern were examined with different patterns of neuronal wiring and maladaptive plasticity of STN neurons in PD. between GPe and STN as well as within GPe.

WD IX-16-3 WD IX-16-4 SYNAPTIC INTEGRATION AND BURST GENERATION BIASING, LEARNING, AND SWITCHING OF BEHAVIOR IN THE GLOBUS PALLIDUS BY THE BASAL GANGLIA Hitoshi Kita Okihide Hikosaka Department of Anatomy and Neurobiology, University of Tennessee, USA Laboratory of Sensorimotor Research, National Eye Institute, USA The globus pallidus consists of the external and internal segments. The It has been proposed that parallel circuits in the basal ganglia work in a external segment of the globus pallidus (GPe) has been a target of extensive coordinated manner for the selection of actions, but experimental evidence in vivo and in vitro studies for recent years. I like to discuss some of recent had been lacking. Using behavioral tasks in which saccadic eye movements exciting findings made by a number of research groups. The discussion will are initiated in various behavioral contexts, we investigated single neuron include 1) a brief description of the anatomical organization of the GPe, activity in the basal ganglia, and found that there are at least three ways in 2) the ionic currents that can support high frequency autonomous firing, which the basal ganglia could control motor actions: biasing, learning, and switching. The biasing function is achieved by pre-movement changes in irregular firing, and long pauses, 3) modulation of autonomous firing by activity which occur selectively in different parts in the striatum based on inhibitory inputs from the striatum and excitatory inputs from the subthalamic expected rewards. The signals are transmitted mainly through the direct nucleus, 4) alteration of firing activity by activation of dopamine receptors pathway to allow the animal to act on the location or object from which and loss of dopaminergic afferent inputs, and 5) possible link between altered a large reward is expected. The learning function is mainly mediated by firing activity and basal ganglia motor disorders. dopamine neurons in the substantia nigra which transmit reward prediction error signals to the direct and indirect pathway neurons in the striatum. The switching function is mainly achieved by the hyperdirect pathway, particularly the connection from the pre-supplmentary motor area to the subthalamic nucleus. This mechanism works when an automatic action needs to be switched to a controlled action.

WD IX-16-5 WD IX-16-6 INVOLVEMENT OF THE BASAL GANGLIA IN SYNAPTIC PLASTICITY IN THE STRIATUM: VALUATION AND SELECTION OF ACTIONS MODULATION BY DOPAMINE Minoru Kimura1, Hiroshi Yamada2, Kazuki Enomoto1, Naoyuki Jeffery Russell Wickens, Tomomi Shindou, Mayumi Ochi-Shindou Matsumoto1, Yukiko Hori1, Yasumasa Ueda1, Ko Nakayama1 Neurobiology Research Unit, Okinawa Institute of Science and Technology, 1Division of Neurophysiology, Kyoto Prefectural University of Medicine, Japan Japan, 2Center for Neural Science, New York University, USA The striatum is a site of convergence of glutamatergic afferents from the The values of benefit, reward, cost, and punishment influence decision making and cerebral cortex and thalamus with dopaminergic afferents from the midbrain. action selection. Mapping values of outcomes of external events and actions occurs These input pathways terminate on spiny projection neurons which are the mainly via the discharge rates of neurons in the cerebral cortex and the basal ganglia. principal output neurons of the striatum. We studied spike-timing dependent However, it remains unclear how the values encoded in the neuronal activity of the plasticity (STDP) in the corticostriatal pathway in adult mice using whole- basal ganglia are integrated into reward value-based control of limb-movement actions cell recording. Long-term depression (LTD) was induced by pre-post through the cortico-basal ganglia loops. We addressed this issue by recording neuronal protocols in which action potentials were induced after the EPSP, but post- activities of monkeys performing conditioned movement tasks in the striatum and pre protocols caused little change. The LTD was reversed by a D1-receptor midbrain dopamine neurons. We found that activity of neurons in the striatum encodes agonist, and was only observed in dopamine D1 receptor expressing neurons. 2+ decision variables for actions such as reward value of action options, direction of Dendritic spine Ca signals associated with the LTD-induction protocols actions and task strategy, before, during and even after individual steps of multi-step were measured using two-photon microscopy. Pre-post protocols that induced 2+ free choices and forced choices for rewards. Midbrain dopamine neurons responded to LTD were associated with larger spine Ca transients than those induced by conditioned (CS) and unconditioned stimuli (US) of single step and multi-step classical post-pre protocols that produced no LTD. Thus, increases in dendritic spine 2+ conditioning tasks for reward in which magnitude of CS responses represented values Ca concentration are associated with LTD in the corticostriatal pathway, expected in current trials and those in the future several steps. The results support our and dopamine D1 receptors modulate this form of STDP. Synaptic plasticity view that the basal ganglia are critically involved in adaptive action valuation and in the corticostriatal pathway may be important in the cellular mechanisms of selection of actions. learning.

48 IUPS 2009 July 27 - August 1, 2009 in Kyoto WD IX-16-7 WD IX-16-8 NEURAL CIRCUIT MECHANISMS UNDERLYING BRAINSTEM INTERACTIONS WITH THE BASAL DOPAMINE-DEPENDENT BEHAVIORS GANGLIA Kazuto Kobayashi Peter Redgrave Department of Molecular Genetics, Fukushima Medical University, Japan Psychology, University of Sheffield, UK Basal ganglia circuit mediates a variety of brain functions, such as motor An important component of the cortico-basal ganglia architecture is the control, motor learning, and reinforcement learning. Dopamine transmission parallel, closed-loop projections that originate and return to specific regions plays an essential role in the modulation of basal ganglia functions. of the cerebral cortex. It will be proposed that such loops are neither novel Immunotoxin cell targeting is a transgenic technology for conditional nor the first evolutionary example of closed-loop architecture involving the ablation of specific neuronal types based on the specificity of recombinant basal ganglia. A phylogenetically older series of subcortical connections can be shown to link the basal ganglia with a range of brainstem sensorimotor immunotoxins. This technology has been used for studying the behavioral structures. Anatomical and electrophysiological evidence suggests that and physiological roles of neuronal types consisting of basal ganglia circuit. functional connectivity between subcortical systems and all input/output First, ablation of the subthalamic nucleus neurons shows that these neurons nuclei of the basal ganglia has been retained in the mammalian brain. The are required for dopamine-induced motor behavior by regulating neuronal co-existence of cortically and subcortically based projections through the firing of the globus pallidus and output nuclei. The role of subthalamic basal ganglia has interesting functional implications. First, parallel projecting neurons in dopamine-induced behavior appears to be mediated through the loops, in conjunction with concept of selective disinhibition represents a projection originating from the medial prefrontal cortex. Second, elimination connectional architecture capable of selecting targeted regions on a winner- of the striatonigral pathway delays the reaction time of two-choice operant take-all basis. Secondly, the possibility would arise where parallel processing conditioning, suggesting a key role of this pathway in the excursion of sensorimotor, cognitive and affective systems represented at different levels reinforcement learning. Immunotoxin cell targeting provides the strategy to in the cerebral cortex and brainstem could compete directly for attention and elucidate the mechanisms through neural network underlying basal ganglia behavioural expression. functions.

WD IX-16-9 WD IX-16-10 PHYSIOLOGICAL STUDIES OF VALUE ENCODING IN DICHOTOMOUS DOPAMINERGIC CONTROL OF THE BASAL GANGLIA STRIATAL SYNAPTIC PLASTICITY Hagai Bergman Dalton James Surmeier Whole-day symposia Department of Physiology, The Hebrew University, Ireland Department of Physiology, Northwestern University, USA The basal ganglia (BG) are commonly viewed as two functionally related At synapses between cortical pyramidal neurons and principal striatal subsystems. These are the neuromodulators subsystem and the main-axis medium spiny neurons, postsynaptic D1 and D2 dopamine receptors are subsystem, in analogy with the critic-actor division of reinforcement machine postulated to be necessary for the induction of long-term potentiation and learning agents. depression, respectively, forms of plasticity thought to underlie associative We propose that the BG main axis is performing dimensionality reduction learning. Because these receptors are restricted to two distinct medium spiny of the cortical input leading to optimal trade-off between minimization populations, this postulate demands that synaptic plasticity be unidirectional of the complexity of the representation of the current state (cost and/or generalization of input space) while maximizing the expected (discounted) in each cell type. Using brain slices from dopamine receptor transgenic mice, future reward (information bottleneck). we show that this is not the case. Rather, dopamine plays complementary In line with the information bottleneck dimensionality reduction model, BG roles in these two types of medium spiny neuron to ensure that synaptic main axis neurons maintain flat spike cross-correlation functions, diverse plasticity is bidirectional and Hebbian. In models of Parkinson’s disease, and sustained responses to behavioral events, and broadly distributed values this system is thrown out of balance, leading to unidirectional changes in of signal and response correlations with zero population mean. On the other plasticity that could underlie network pathology and symptoms. hand, the spontaneous and the evoked activity of BG dopaminergic and cholinergic modulators (critics) are transient and significantly correlated. The neuronal responses of both BG systems encode better the positive domain of the emotional space. We conclude that BG plasticity and learning are controlled by homogenous modulators effects associated with local coincidences of cortico-striatal activity.

WD KO-17-1 WD KO-17-2 MECHANISM OF THE ACUPUNCTURE EFFECT ON INVOLVEMENT OF SPINAL ADRENERGIC PATHWAY IN CHRONIC VISCERAL HYPERALGESIA IN RATS ACUPUNCTURE ANALGESIA Weimin Li Sungtae Koo1, Jin Mo Chung2 SRCAM, Department of Mechanics and Engineering Science, Fudan 1Division of Meridian and Structural Medicine, School of Oriental Medicine, University, China Pusan National University, Korea, 2Department of Neuroscience and Cell The study examined the immediate and cumulative effects of Biology, University of Texas Medical Branch, USA electroacupuncture (EA) at Zu-san-li (ST-36) and Shang-ju-xu (ST-37) To show clear acupuncture analgesia in persistent pain, we developed a new rat acupoints on irritable bowel syndrome (IBS, a chronic visceral hyperalgesia) model of ankle sprain pain. The effect of electroacupuncture (EA) on this model was model rats. Behavioral abdominal withdrawal reflex (AWR) scores and examined. After sprained ankle, the rat subsequently showed swelling and a reduced electro-physiological rectus abdominis myograms (AEMG) recording stepping force of the affected limb for the next several days. EA was applied to points induced by colorectal distention (CRD) stimuli were used for estimating on the contralateral forelimb for 30 min. After the termination of EA, behavioral tests visceral pain intensity and EA effect. Results showed that a single EA but measuring stepping force were periodically conducted. EA applied to SI-6 significantly not sham EA significantly reduced both AWR scores and AEMG discharges improved the weight-bearing capacity, suggesting an analgesic effect. On the other in IBS rats. Repetitive EA for 14 days showed that effects on both abnormal hand, EA applied to a neighboring point (LI-4) was not effective. AWR and AEMG appeared 2-4 days after the start of EA, enhanced to its In addition, we tested the effects of various antagonists. The alpha-adrenoceptor maximum within 8-12 days. RT-PCR assay and western blot analysis showed antagonist phentolamine completely blocked the EA-induced analgesia, whereas that similar single EA but not sham EA could reverse significant increase opioid antagonists failed to block the effect. Further experiments showed that in mRNA and protein level of NMDA receptor NR1 subunit in the lumbar intrathecal administration of yohimbine, an α2-adrenergic antagonist, reduced the EA- enlargement of spinal cord of IBS rats. These results suggest that 1) EA can induced analgesia, whereas terazosin, an α1-adrenergic antagonist, did not produce relieve chronic visceral hyperalgesia in IBS rats; 2) repetitive EA treatment is any effect. more effective than single EA treatment; 3) visceral analgesia by EA may be These data suggest that EA produces an analgesic effect in a point-specific way, and caused by down-regulation of the increased mRNA and protein expression of the analgesic effect of EA is, at least in part, mediated by spinal α2-adrenoceptor NMDA receptor NR1 subunit at spinal level in IBS rats. mechanisms.

IUPS 2009 July 27 - August 1, 2009 in Kyoto 49 WD KO-17-3 WD KO-17-4 EFFECTS OF ACUPUNCTURE ON THE INHIBITORY THE MECHANISM OF NEURO-IMMUNE MODULATION TRANSMISSION IN THE SPINAL DORSAL HORN IN BY ELECTROACUPUNCTURE VIVO Hyunsu Bae 1 2 1 Megumu Yoshimura , Ji-Hoon Kim , Hidemasa Furue , Byung Il Department of Physiology, College of Oriental Medicine, KyungHee Min3 University, Korea 1 Department of Integrative Physiology, Graduate School of Medical Our previous study demonstrated that splenic NK cell activities were Sciences, Kyushu University, Japan, 2Department of East-West Medicine, 3 significantly higher in the EA-treated rats than in the non-acupunctured Graduate School, Kyung Hee University, Korea, Department of rats, and that lesion of the lateral hypothalamic area (LHA) abolished the Physiology, Kyung Hee University, Korea effects of EA on NK cell activity. We also examined EA-induced alterations To clarify the mechanisms of analgesic effect of acupuncture on the nociceptive of gene expression in splenic NK cells using microarray technique and real transmission in the spinal dorsal horn, patch-clamp recordings were made from time RT-PCR. Our data shows that EA treatment increased CD94, PTK and lamina II (substantia gelatinosa, SG) neurons in the spinal cord of CFA induced VCAM-1 expressions while decreased PTP and SHP-1. In additional sets of inflamed rats in vivo. Electical-acupuncture (EA) was applied to the Zusanli acupoint experiments, we showed that successive EA treatment reduces IgE production (ST36) enhanced spontaneous IPSCs in frequency and amplitude, while EPSCs were in BALB/c mice immunized with 2,4-dinitrophenylated keyhole limpet without effected. The enhanced IPSCs were largely depressed in the presence of TTX, protein (DNP-KLH) by suppression of the Th2 cell lineage development. suggesting that the some inhibitory interneurons are activated by EA stimulation. To test whether the effect of EA was mediated by the activation of the supraspinal Furthermore, pretreatment of phentolamine (α-adrenergic receptor system, spinalization was made at the level of Th12, resulting in disappearance of the antagonist) or Y-25130 (5-HT3 receptor antagonist), but not naloxone (general enhancement of IPSCs. These observations indicate that the EA stimulation causes the opioid receptor antagonist), completely blocks the EA-induced suppression activation of supraspinal system and then descends to the spinal cord, consequently of antigen-specific and total IgE levels in serum, and IL-4 production in anti- activates inhibitory interneurons. As we have reported using slice preparations of the CD3 mAb-activated splenocytes in DNP-KLH immunized mice. Therefore, it

Whole-day symposia spinal cord, noradrenaline and serotonin originating from the brainstem are the leading is suggested that EA modulate NK cell activities and Th1/Th2 cell responses candidates for the activation of inhibitory interneurons in the spinal dorsal horn. through the neuro-immune interaction.

WD KO-17-5 WD KO-17-6 THE EFFECT AND POSSIBLE MECHANISM OF NEURAL MECHANISM OF ACUPUNCTURE EFFECTS ACUPUNCTURE AND MOXIBUSTION ON THE ON CARDIAC FUNCTION COLLAGEN INDUCED ARTHRITIS IN RATS Sae Uchida Tadashi Hisamitsu Department of the Autonomic Nervous System, Tokyo Metropolitan Department of Physiology, Showa University, Japan Institute of Gerontology, Japan Acupuncture and moxibusution (Mox) is known to have beneficial effects Acupuncture has been used to alleviate cardiac dysfunction (e.g. on the human rheumatoid arthritis. To evaluate the effects and possible tachycardia). There have been several reports that acupuncture produces mechanism of these treatments on the arthritic disease, the influence of a decrease in heart rate in healthy human subjects. The neural mechanism electroacupuncture (EA) and Mox on collagen-induced arthritis (CIA) animal of this response is still, however, controversial. We studied the effects of was examined. DBA/1J mice were immunized with bovine type II collagen acupuncture-like stimulation on heart rate and cardiac sympathetic nerve (CII). The main incidence of arthritis started about on day 30 and lasted to activity in pentobarbital-anesthetized rats. Stimulation of forelimb, hindlimb, day 60 after the first immunization. EA stimulation or Mox, begun on day chest, and abdomen all produced significant heart rate decreases that were 21 simultaneously with the second immunization, was applied three times paralleled by decreases in cardiac sympathetic nerve activity. We found that a week for 3 weeks at the acupoint equivalent to GV4 (governor vessel 4, Ming Men, Meimon). The results showed that EA and Mox delayed the the reflex pathway to decrease heart rate by acupuncture-like stimulation onset, attenuated the severity of arthritis, and reduced the anti-collagen of a hindlimb consists mainly of group IV muscle afferent fibers whose antibody level. Furthermore, these stimulation significantly increased serum activity leads to the activation of GABAergic neurons in the brainstem IL-6 concentration and regulatory T cell (CD25+ CD4+ T cell) number, and and inhibition of the sympathetic outflow to the heart. The bradycardiac response by acupuncture-like stimulation of a hindlimb was less pronounced decreased splenic endogenous IL-1 β and serum prostaglandin E2 (PGE2) concentration. These data suggest that EA has an inhibitory effect on murine when cardiac sympathetic nerve activity was high (a condition produced by CIA, and the partial mechanism of its therapeutic result may be attributed to hypercapnia). It is suggested that inhibitory effects of acupuncture on cardiac inhibiting the productions of IL-1β and PGE2 and activation of regulatory T function are modest, best seen when the sympathetic tone is modest, and can cell. be overwhelmed by potent activators of sympathetic outflow.

WD KO-17-7 WD KO-17-8 MODULATION OF HYPERTENSION AND EFFECT OF ACUPUNCTURE ON NICOTINE ADDICTION HYPOTENSION WITH ELECTROACUPUNCTURE Younbyoung Chae, Hyejung Lee, Hi-Joon Park Peng Li Dept. Meridian and Acupoint, College of Korean Medicine, Kyung Hee Medicine, University of California, Irvine, USA University, Korea It was shown that EA inhibited experimental hypertension by activation Acupuncture has been demonstrated to have effects in several types of of opioid receptors in the rostral ventrolateral medulla (rVLM) and raise animal model of drug dependence. In the animal studies, we investigated the blood pressure (BP) in hypotensive animals via activation of cholinergic effect of acupuncture on anxiety-like behavior and corticotrophin-releasing receptors in the brain. Recently we observed that EA at P 5-6 & St 36-37 factor (CRF) and neuropeptide Y mRNA expression in the amygdala during on hypertensive patients once weekly for 4-8 weeks decrease systolic BP nicotine withdrawal. We observed that acupuncture at HT7 significantly significantly in 70% patients (n=13), while EA at G37-39 & LI 6-7 had no attenuated anxiety-like behavior and CRF mRNA levels in the amygdala. effect. In animal experiments EA inhibited gallbladder stimulation induced In the human studies, we examined whether acupuncture ameliorated the pressor response by activation of glutamate receptors in arcuate nucleus cigarette withdrawal symptoms (CWS) and the autonomic responses to (ARC) and ventrolateral periaqueductal gray (vlPAG), in turn activate smoking-related visual cues, using a power spectrum analysis of heart rate nuclei raphe and inhibited rVLM cardiovascular sympathetic neurons by variability (HRV). We found that the CWS of the real acupuncture (RA) endorphin, enkephalin & GABA,. The long-lasting EA inhibition is related group was significantly lower than that of the sham acupuncture (SA) group, to the activation of opioid & GABA receptors in rVLM, and the neural and that the increase in the low frequency/high frequency ratio of HRV circuit between ARC & vlPAG. Moreover, EA at P 5-6 in rat inhibited the induced by the smoking-related visual cues was significantly lower in the gastric distention-induced pressor response and decreased the depressor RA group, as compared to the SA group. Based on these animal and human response under different anesthetic condition. While bilateral stimulation studies, acupuncture not only ameliorated nicotine withdrawal symptoms, but with EA at LI 6-7 had no effect. These results demonstrate that stimulation also attenuated the autonomic responses to the smoking cues. These findings of certain acupoints can modulate hypertension and hypotension via different suggest that acupuncture might help in smoking cessation by attenuating neuropathways & nuerotransmitters. withdrawal symptoms and smoking cues-induced autonomic responses.

50 IUPS 2009 July 27 - August 1, 2009 in Kyoto WD KO-17-9 WD KO-17-10 EFFECTS OF ACUPUNCTURE ON THE CENTRAL THE NEUROPROTECTIVE MECHANISMS OF NERVOUS SYSTEM ACUPUNCTURE TREATMENT IN PARKINSON’S Etsuro Hori, Kouichi Takamoto, Natsuko Sakai, Susumu Urakawa, DISEASE 1 2 1 Taketoshi Ono, Hisao Nishijo Hi-Joon Park , Seung-Tae Kim , Woongjoon Moon , Seung-Nam 1 1 1 System Emotional Science, University of Toyama, Japan Kim , Younbyoung Chae , Hyejung Lee 1Department of Meridian & Acupoint, College of Korean Medicine, Kyung The autonomic and related central nervous systems are implicated in various 2 Hee University, Korea, School of Oriental Medicine, Pusan National pathological processes. We investigated the effects of acupuncture stimulation University, Korea (AS) on animal models of post-menopausal hot flash in ovarectomized mice Acupuncture frequently uses as an alternative therapy for PD, and it attenuates with high tail skin temperature (TST) due to hyperactivity of sympathetic dopaminergic neurodegeneration in the substantia nigra (SN) and striatum (ST) in PD vasodilator. The AS effectively reduced TST. In humans, AS at specific animal models. Using proteomic analysis, we investigated whether acupuncture alters points [trigger points (TPs)] elicits specific sensation (called "de-qi") that is protein expression in the SN and ST to favor attenuation of neuronal degeneration. related to clinical efficacy. We also investigated the relationships between de- In C57BL/6 mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, qi sensation induced by needling at TPs and non-TPs and brain hemodynamic 30 mg/kg/day, i.p.) for 5 days, electroacupuncture (EA) was applied at the effective responses using functional near infrared spectroscopy (fNIRS). The results and specific acupoint, GB34, once a day for 12 consecutive days from the first MPTP indicated that AS with de-qi sensation, regardless of the stimulated points treatment. This treatments in MPTP mice led to restoration of behavioral impairment (TPs vs. non-TPs), significantly decreased Oxy-Hb concentration in the and rescued tyrosine hydroxylase-positive DA neurodegeneration. Using peptide supplementary motor area (SMA), pre-supplementary motor area and anterior fingerprinting mass spectrometry, we identified changes in 24 and 13 proteins in the SN and the ST, respectively, following MPTP treatment, and 11 and 4 proteins dorsomedial prefrontal cortex. The results suggest that de-qi sensation is were normalized by EA. They were involved in cell death regulation, inflammation, essential for acupuncture therapy, and that effects of acupuncture stimulation restoration from damage, or cellular metabolism. In conclusion, these results suggest with de-qi sensation were mediated partly through the central nervous system that acupoint GB34-specific EA changes protein expression profiles in the SN and the including the SMA. These results might further provide physiological basis ST in favor of dopaminergic neuronal survival in MPTP-treated mice, and that EA of AS in treatment of chronic pain, and various psychiatric conditions. treatment may be an effective therapy for PD patients. Whole-day symposia

IUPS 2009 July 27 - August 1, 2009 in Kyoto 51 PSJ II-1-1 PSJ II-1-2 NETWORK MECHANISMS OF RESPIRATORY RHYTHM NEURONAL MECHANISMS OF RESPIRATORY GENERATION IN RODENTS MODULATION Hiroshi Onimaru, Ikuo Homma Akiko Arata, Morimitsu Fujii Department of Physiology, Showa University School of Medicine, Japan Laboratory for Memory and Learning, RIKEN Brain Science Institute The neural circuit that generates the respiratory rhythm in mammals is and Division of Physiome, Department of Physiology, Hyogo College of located in the medulla of the lower brainstem. The main groups of medullary Medicine, Japan respiratory neurons are distributed rostrocaudally in the ventrolateral parts Optical imaging using voltage sensitive dye is great tool for analysis network of the medulla, where they play a role in respiratory rhythm generation. dynamics. We can see the spacial and temporal respiratory network changes Over the past decade, optical imagings of respiratory neuron activity in using cycle triggering with C4 inspiratory activity. We investigated the effect the brainstem have contributed to provide new evidence for neuronal of catecholamine (dopamine, noradrenaline and adrenaline) on pre-inspiratory mechanisms of respiratory rhythm generation and its modulation. The (Pre-I) neurons and inspiratory (Insp) neurons using the combination of an brainstem and spinal cord of 0- to 4-day-old rats or mice isolated under deep optical recording technique and neuron recording. Adrenaline/NA strongly ether anaesthesia have been used for detection of respiratory neuron activity depressed on Pre-I neurons activity via GABAergic system. Optical signals by voltage-sensitive dye imagings. The optical recordings revealed two detected Pre-I neuron group even if rhythm slowed. On the other hand, DA predominant active regions in the ventral medulla; para-facial respiratory depressed respiratory rhythm mediated by a dopamine D4 receptor. However, group (pFRG), preceding the onset of inspiratory activity by about 500 optical signals of the Pre-I much decreased by DA since DA disperses Pre-I ms, and inspiratory activity in the more caudal ventrolateral medulla at the firing and desynchronized Pre-I phase. These results show that dopaminergic pre-Botzinger complex (preBotC) level. Coupling between the pFRG and depression is due to dispersion of synchronized Pre-I driving of Insp neurons. preBotC is important in the generation of regular respiratory rhythm. Recent DA is different mechanism from other catecholamine in respiratory rhythm studies further suggest that the pFRG is important in central chemoreception generator. Adrenaline (or NA) might regulate some reflex and tone of the as well as the primary respiratory rhythm generation during at least early rostral ventrolateral medulla. DA might modulate the switching mechanism postnatal period. between voluntary and involuntary respiration such as information from the cerebral cortex and the limbic system.

PSJ II-1-3 PSJ II-1-4 ADVANCES IN ANALYSES OF SPATIOTEMPORAL MULTIPHOTON LOOK AT THE ISOLATED NEWBORN RESPIRATORY NETWORK ACTIVITIES USING RAT INSPIRATORY PRE-BOTZINGER COMPLEX VOLTAGE-SENSITIVE DYES Klaus Ballanyi, Nicoleta Bobocea, Bogdan Panaitescu, Araya PSJ symposia Yoshitaka Oku Ruangkittisakul Department of Physiology, Hyogo College of Medicine, Japan Department of Physiology, University of Alberta, Canada Neuronal activities such as rhythm generation, pattern formation, Breathing is controlled by the inspiratory pre-Botzinger Complex (preBotC) and information processing involve large-scale networks of multiple in the lower brainstem. Generation of ‘calibrated’ transverse brainstem slices neurons. Here we show that voltage imaging is a powerful tool to analyze revealed that the newborn rat preBotC is centered 0.5mm caudal to the facial spatiotemporal network activities. We have been using the respiratory motor nucleus and extends less than 200μm rostrocaudally (Ruangkittisakul et al central pattern generator as a model system of large-scale networks. One of [2008] J Neurosci 28, 2447-2458). In physiological (3mM) potassium solution, successful applications of voltage imaging was the correlation coefficient 700μm thick slices with the preBotC kernel plus rostral tissue generated fictive imaging for the functional mapping of respiratory-related regions. This sighs plus intermingled fictive eupnea bursts, whereas sighs were not seen technique could visualize dynamic characteristics of respiratory-related regions, and revealed a novel neuronal group at the level of C1-C2 cervical in slices with the preBotC plus caudal tissue. Multiphoton calcium imaging spinal cord in the brainstem spinal cord preparation of the neonatal rat. indicated that preBotC neurons reconfigure between fictive sigh and eupnea Voltage imaging can be also applied to an amphibian preparation; we inspiratory patterns evoked by neurokinin-1 receptor agonist or thyrotropin- visualized respiratory neuronal activity in the frog brainstem, and identified releasing hormone, respectively. Inspiratory-related rhythm was revealed in pairs bilateral longitudinal distribution of respiratory neurons in the ventral of neighboring 700μm slices both with one boundary close to the preBotC center. medulla. More sophisticated approaches, e.g., autoregressive modeling, The density of preBotC neurons showing inspiratory-related calcium rises was nonlinear parameter estimation, and nonlinear dynamic analysis, have been notably lower in the rostral twin of such ‘preBotC sandwich’ slices. Findings made to analyze dynamic activities within a single breath without cycle- suggest that the preBotC is a distributed network with a rostrocaudally segregated triggered averaging. Limitations and future perspectives of voltage imaging chemical and structural organization. applications will be discussed. Supported by CIHR, AHFMR, CFI/ISRIP and Centre for Neuroscience (UofA).

PSJ II-1-5 PSJ II-1-6 DEGENERACY AS A SUBSTRATE FOR RESPIRATORY RESPIRATORY RHYTHM GENERATION IN THE MOUSE REGULATION EMBRYO : COMMISSURAL CONNECTIVITY IN THE Nicholas M Mellen PRE-BOTZINGER COMPLEX Pediatrics, University of Louisville, USA Gilles Fortin1, Julien Bouvier1, Muriel Thoby-Brisson1, Breathing is a unique motor pattern: on one hand it persists from birth until Alessandra Pierani2, Alain Chedotal3 death without pause; on the other, it adapts itself constantly to changes in 1Genetic and Integrative Neurobiology, Centre National de la Recherche metabolic demand and ambient conditions. Thus, the medullary networks Scientifique, France, 2Jacques Monod Institute, UMR7592, CNRS, Paris that mediate this behavior exemplify both a central pattern generator Diderot-Paris 7, France, 3Vision Institute, UMRS592, INSERM, Paris, (CPG), producing a rhythmic behavior in a feed-forward manner, and a France homeostatic network. Because this behavior is fundamental to survival, it is Breathing is a bilateral synchronous behaviour relying on a respiratory rhythm ubiquitous across the vertebrate lineage. Over evolutionary time, respiratory generator (RRG) located in the brainstem. An essential component of the RRG is the anatomy and biomechanics have undergone profound transformation. preBotzinger complex (preBotC) oscillator pacing inspirations. Little is known both This long history may explain the existence of functionally similar, but about the origin of interneuronal populations forming the preBotC oscillator, and anatomically and mechanistically distinct respiratory rhythm generating and the molecular signalings ensuring its bilateral synchronous activity. Using knock- homeostatic regulatory networks. Because these duplicative systems are in alleles of the homeobox gene Dbx1, we identify a population of glutamatergic mechanistically distinct the term “redundancy” is inaccurate; a more precise interneurons, deriving from Dbx1-expressing cells, required to generate the preBotC term is “degeneracy”, which has been defined formally to describe this type rhythm. We also show that a null mutation of the Robo3 gene leads to disrupted of duplicative organization. We present data from the tilted sagittal slab commissural connectivity in the hindbrain and to a “split breathing” behaviour. preparation supporting the hypothesis that respiratory rhythm is generated by Conditional invalidation of the Robo3 gene in Dbx1-derived cells induces left- more than one mechanism, in more than one location; further, we will argue right de-synchronisation of the preBotC oscillator. Together, these findings identify that the computational demands of robust homeostatic regulation may be met Dbx1-dependent interneurons as key components of the preBotC oscillator, and by respiratory network degeneracy. Robo3 expression therein as obligatory for the RRG to show characteristic bilateral synchronous activity.

52 IUPS 2009 July 27 - August 1, 2009 in Kyoto PSJ IV-2-1 PSJ IV-2-2 ROLE OF GUSTDUCIN, TASTE RECEPTORS AND ROLES OF VESICULAR NEUROTRANSMITTER TRANSIENT RECEPTOR POTENTIAL CHANNEL M5 IN TRANSPORTERS IN THE METABOLIC REGULATION GUT Yoshinori Moriyama Robert F Margolskee, Bedrich Mosinger, Zaza Kokrashvili Department of Biochemistry, Okayama Univ., Graduate School Medicine, Neuroscience, Mount Sinai School of Medicine, USA Dentistry, Pharm. Sci., Japan Taste receptors, gustducin, the transient receptor potential channel M5 Vesicular neurotransmitter transporters are membrane proteins of secretory (Trpm5) and other taste proteins are expressed in multiple types of gut vesicles and composed of six different members, which are vesicular cells. Human enteroendocrine L cells express GLP-1 along with many taste monoamine transporter (VMAT), vesicular acetylcholine transporter (VAchT), proteins. Mouse L cells also express gustducin, and gustducin knockout mice vesicular inhibitory amino acid transporter (VIAAT), vesicular glutamate have deficiencies in secretion of GLP-1, and in regulation of plasma insulin transporter (VGLUT), vesicular neucleotide transporter (VNUT), and and blood glucose. Isolated small intestine and intestinal villi from gustducin vesicular excitatory amino acid transporter (VEAT). These transporters are null mice display defective GLP-1 secretion in response to glucose. GLP-1 responsible for vesicular storage and subsequent exocytosis of the respective release from L cell lines is promoted by sugars and sweeteners, and blocked transmitters, and thus play an essential role in the chemical transmission. In by the sweet receptor antagonist lactisole or siRNA to gustducin. Thus, L cell-expressed gustducin and sweet receptor are implicated in sugar this symposium, I will focus VGLUT and VNUT and discuss their roles in sensing in gut. Other cells in mouse duodenum (a subset of brush cells) regulation of metabolism and signal transmission in digestive and endocrine express Trpm5, β-endorphin and Met-enkephalin. Wildtype mice secrete organs. β-endorphin into the gut lumen in response to high osmolarity stimuli. However, stimulated secretion of opioids is markedly attenuated in Trpm5 null mice. Trpm5-expressing brush cells of gut have chemosensory properties and appear to be a type of solitary chemosensory cell (SCC). We infer that brush cell-expressed Trpm5 is required for secretion of β-endorphin into the intestinal lumen in response to feeding.

PSJ IV-2-3 PSJ IV-2-4 USING THE INTESTINAL LYMPH FISTULA MODEL FREE DIETARY GLUTAMATE POTENTIATES GASTRIC TO STUDY THE REGULATION OF SECRETION OF SECRETION IN DOGS INCRETINS BY MACRONUTRIENTS Vasiliy A. Zolotarev1, Raisa P. Khropycheva1, Hisayuki Uneyama2, Patrick Tso, Tammy Kindel, Stephanie Yoder Kunio Torii2 Department of Pathology & Laboratory Medicine, University of Cincinnati, 1Department of Physiology of Digestion, Pavlov Institute of Physiology, USA Russia, 2Physiology and Nutrition Group, Institute of Life Sciences, Glucagon like peptide 1 (GLP-1) and glucose-dependent insulinotropic Ajinomoto Co., Inc., Japan polypeptide (GIP) are incretins secreted by enteroendocrine cells of the GI tract. Recent studies suggest that dietary amino acid, L-glutamate, is a specific stimulator GLP-1 is released by mucosal L cells located in the lower intestine while GIP is of the gastric vagal afferents and likely of chief cells of the gastric glands. The aim produced by K cells in the upper intestine. In rats, we examined portal plasma of the investigation was to evaluate a role of intra-gastrically applied glutamate in PSJ symposia and lymph sampled before and for 240 min after placing a liquid meal (Ensure, control of secretion of acid, pepsinogen and fluid. In mongrel dogs with surgically Abbott Laboratories, Chicago, IL) in the duodenum. Lymph contained detectable prepared small gastric pouches according to Pavlov (innervated) or Heidenhain (vagally concentrations of glucose, GLP-1 and GIP measured using chemical assays decentralized), secretion in a pouch was induced by infusion into the main stomach and immunoassay kits from Millipore (St. Charles, MI). Incretin levels were of an amino acid-rich diet lacking glutamate (Elental) alone or supplemented with significantly higher in lymph vs. portal plasma following nutrient administration. 100 mM monosodium glutamate (MSG). Having no effect alone, MSG potentiated This relative difference in incretin levels was even greater when we compared secretion induced by Elental both in Pavlov and Heidenhain models. In the Pavlov lymph to peripheral plasma. This marks the first time that incretin hormones pouch, the effect of MSG was reduced by antagonist of 5-HT3 receptors, granisetron. were observed to be disproportionately transported in lymph. We suggest In the Heidenhain model, MSG enhanced the stimulatory effect of pentagastrin (1 several possible explanations for the difference in lymph vs. plasma incretin μg/kg, s.c.). We conclude that free glutamate at doses not exceeding its common concentrations. First, portal blood flow is significantly higher than intestinal concentrations within dietary foods enhances gastric secretion induced by intra- lymph flow; thus, incretins are more diluted in portal blood than lymph. Second , luminal amino acids infusion. This effect of free glutamate is achieved predominantly DPP IV concentration (enzyme that degrades incretins) is significantly higher in by the potentiation of vago-vagal reflexes, and did not depend on certain allosteric blood than in lymph. interaction with other dietary amino acids.

PSJ IV-2-5 PSJ IV-2-6 ROLE OF GUT PEPTIDES IN SATIATION PHYSIOLOGICAL ROLES OF DIETARY GLUTAMATE Yvette Tache SIGNALING VIA GUT-BRAIN AXIS Medicine - Digestive Diseases, UCLA, USA Kunio Torii Short term regulation of food intake entails neural and endocrine signals Institute of Life Sciences, Ajinomoto Co., Inc., Japan for meal initiation and termination arising from the viscera (gut, pancreas, Gustatory and visceral stimulation of food regulates digestion and nutrient adipose tissue). We showed a synergistic interaction between leptin and utilization. Free glutamate (Glu) release from food induces the umami taste cholecystokin (CCK) to suppress food intake that is mediated by enhanced that increases food palatability. Dietary glutamate is the main source of vagal afferent signaling to the hindbrain. There is also a CCK-urocortin 1 energy for the intestinal mucosal absorption and metabolism, thus, only a synergistic interactions mediated by corticotropin releasing facto (CRF) 2 trace amount of Glu reaches the general circulation even after the intake receptor and vagal afferent in lean mice which is no longer observed in fat of dietary protein and Glu added in foods. In addition to these roles, we diet induced obesity or CRF2 knockout mice. In the stomach, ghrelin released demonstrated a unique gastric sensing system for Glu. Glu is the only during fasting stimulates food intakes through activation of NPY containing amino acid that activates rat gastric vagal afferents from the luminal side neurons in the hypothalamus (arcuate, paraventricular and dorsomedial). possibly via metabotropic Glu receptors on mucosal cells. Functional MRI Endotoxin (LPS), interleukin-1β and surgery inhibit ghrelin release and (4.7T) analysis revealed that luminal sensing with 1% Glu (most preferred the associated reduction of food intake and gastric emptying is restored concentration) in rat stomach activates the medial preoptic area and the by ghrelin injection. Ghrelin orexigenic action can be inhibited by CCK, dorsemedial hypothalamus, resulting in thermogenesis without changes bombesin but not amylin. Recently we showed nesfatin-1 is expressed in in food intake. Interestingly, rats fed a high fat and high sugar diet with gastric ghrelin cells and regulated by fasting and feeding and act in the brain free access to 1% Glu and water showed lower fat deposition, weight gain to inhibit dark phase food intake. There is a complex interaction between gut and blood leptin compared with those without Glu. From these results, we peptide signaling to modulate food intake. propose that dietary glutamate functions as a signal for the regulation of gastrointestinal tract via gut-brain axis, contributing to the maintenance of our healthy dietary life.

IUPS 2009 July 27 - August 1, 2009 in Kyoto 53 LS7-1 (PSJ IV-2-related luncheon seminar) LS7-2 (PSJ IV-2-related luncheon seminar) UMAMI AS THE FIFTH BASIC TASTE NUTRIENT-SENSING OF AMINO ACIDS BY THE Joseph G. Brand ABDOMINAL VAGUS NERVE AND THE GUT-BRAIN Monell Chemical Senses Center, USA FUNCTIONAL CHANGES Controversy over umami’s status as a basic taste continued unabated as Akira Niijima researchers argued over psychophysical and physiological data. Evidence that Niigata University School of Medicine, Japan umami is a basic taste came from the synergism of Glu and ribonucleotides. This report deals with effects of gastro-intestinal infusion of amino acids on the Only the identification of a specific L-Glu taste receptor would quell the activity of the vagal afferents in anesthetized rats. Intragastric infusion of isotonic arguments. Torii and Cagan (1980) published evidence of L-Glu-binding glutamate solution facilitated afferent activity of vagal gastric nerve, however, gastric to taste tissue whose affinity for Glu increased by co-incubation with 5’ infusion of other essential and non-essential amino acid showed no effect. On the other -ribonucleotides. Subsequently, evidence for several Glu receptor types hand, intraduodenal infusion of isotonic essential amino acid solutions such as valine, was presented. Continuing studies out of Chaudhari’s lab culminated in the leucine, isoleucine, lysine, tryptophane and phenylalanine and most of non-essential characterization of a taste cell - specific truncated form of the inhibitory amino acid including glutamate resulted in activation of vagal celiac afferents. glutamate receptor mGluR4. Zuker’s lab presented evidence (2002) that the On the contorary, infusion of two esential amino acid, methionine and threonine,and T1R1/T1R3 is an amino acid receptor for Glu in human. And within the past non-essential amino acids, glycine and histidine, suppressed afferent activity of vagal 6 years, a truncated metabotropic mGluR1 was reported by the Torii lab. celiac nerve. These observations indicate that amino acid sensors in the gastric wall With discovery of umami receptors in the gut, a new conceptualization of send glutamate information in the gastric canal to the cenral nervous system through the sense of taste is taking shape. Umami receptors are important for both vagal gastric afferents, and that amino acid sensors in the duodeno-intestinal wall sensory driven evaluation, and receptor-mediated signaling for digestion and send information on each essential and non-essential amino acid through vagal celiac metabolism. Disruption of one of these functions affects not only perception afferents. of umami, it also has digestive and metabolic consequences that can lead to Reflex effects from these sensors to autonomic outflow were also investigated. These changes in diet preference. observations suggest that amino acid sensors in the gastro-intestinal wall may play important roles in regulation of protein metabolsm by abdominal vagus.

PSJ V-3-1 PSJ V-3-2 CALCIUM AND ELECTRIC ACTIVITIES IN GUT MECHANISM OF INITIATION OF CALCIUM WAVES IN PACEMAKER CELLS ISOLATED URETHRAL PACEMAKER CELLS OF THE Shinsuke Nakayama1, Hong-Nian Liu1, Kazunori Goto2, Miyako Takaki3, RABBIT 4 PSJ symposia Shunichi Kajioka 1 Noel McHale, Mark A Hollywood, Gerard P Sergeant, Keith D Department of Cell Physiology, Nagoya University Graduate School of Thornbury Medicine, Japan, 2Department of Neuropsychiatry, Graduate School of Medicine, Kyoto University, Japan, 3Department of Physiology II, Nara Medical Smooth Muscle Research Centre, Dundalk Institute of Technology, Ireland University, Japan, 4Department of Urology, Graduate School of Medicine, Kushu The rabbit urethra contains a sub-population of cells which are distinct, both University, Japan functionally and morphologically, from the bulk smooth muscle cells. These Interstitial cells of Cajal (ICC), identified with c-Kit-immunoreactivity, act as gut pacemaker non-contractile cells are spontaneously active showing regular spontaneous 2+ cells, with spontaneous Ca activity in ICC as the probable primary mechanism. Namely, transient depolarisations (STDs) under current clamp conditions. The 2+ 2+ cytosolic Ca oscillations in ICC periodically activate plasmalemmal Ca -dependent ion spontaneous transient inward currents (STICs) which underlie the STDs, channels and thereby generate pacemaker potentials. are believed to result from activation of Ca2+-dependent Cl- channels by In this presentation, we first show evidence that spontaneous electrical activity in ICC is Ca2+ released from intracellular stores. We have shown that interstitial cells linked with cytosolic Ca2+ oscillations, and that this Ca2+ activity requires co-contribution 2+ 2+ fire large STICs and two types of STOCs (spontaneous transient outward of Ca -permeable channels across the plasma membrane and intracellular Ca release currents), “fast” (<100 ms in duration) and “slow” (>1 s in duration). The channels. Also, some endogenous factors, such as ATP and 5-HT modulate their activities. The modulation mechanisms on ICC may play an important role in the brain-gut axis. latter are coupled to STICs, suggesting that they share the same mechanism, Next, we show microelectrode array mapping of ICC electrical activity. This technology while the former occur independently at faster rates. All of these currents were abolished by cyclopiazonic acid, caffeine, or ryanodine, suggesting that revealed that ICC network produces propagating electric waves in both oral-to-anal and anal- 2+ to-oral directions even in the presence of TTX. In addition to the enteric nervous system, they are activated by Ca release. When D-myo-inositol 1,4,5-trisphosphate ICC appear to make an essential contribution to the co-ordinated actions of gut motility, e.g. (IP3)-sensitive stores were blocked with 2-aminoethoxydiphenyl borate segmentation and peristalsis. (2-APB), the STICs and slow STOCs were abolished, but the fast STOCs Lastly, we wish to introduce a brand new technology for non-invasive detection of ICC remained. The talk will report recent work suggesting that, despite apparent pacemaking. differences, all of the above phenomena share a common initiating event.

PSJ V-3-3 PSJ V-3-4 EXCITABILITY, PACEMAKING AND CALCIUM SIGNALS VOLTAGE-DEPENDENT SENSOR THAT COORDINATES IN UTERINE CELLS PACEMAKER ACTIVITY IN COUPLED NETWORKS OF Susan Wray, Theodor Burdyga, Ludmylla Borysova, Karen Noble INTERSTITIAL CELLS OF CAJAL Physiology, School of Biomedical Sciencs, University of Liverpool, UK Kenton M Sanders There is general agreement that the mechanism underlying spontaneous Physiology and Cell Biology, University of Nevada School of Medicine, USA activity in the myometrium is not well understood. Thus, the details of the channels and currents underlying the initiation of electrical activity Pacemaker activity in gastrointestinal muscles results from spontaneous generation of inward currents in interstitial cells of Cajal (ICC). Pacemaker and the action potential in human myometrium during gestation and in- 2+ - labour, the nature of any pacemaker and its relation to uterine myocytes, currents are due to non-selective cation channels and/or Ca -activated Cl and the mechanism of transition from non-synchronized to synchronized channels. These conductances are not voltage-dependent. We have asked the question of how pacemaker activity can be coordinated in a network of thousands electrical and mechanical activity at tem, are all little understood. In this 2+ of cells. Generation of pacemaker currents is linked to release of Ca from presentation I will examine recent data investigating, the presence of KIT- intracellular stores through IP3 receptors. Propagation rates in ICC networks positive interstitial cells in the , the nature of electrical activity in suggest a voltage-dependent process is responsible for entraining activation cell- intact myometrium, and the Ca signalling present in in situ cells revealed to-cell. Some have proposed that voltage-dependent regulation of IP3 production by confocal microscopy. I will discuss what direction current findings are may explain r entrainment; others suggest that sensitivity of IP3 receptors or Ca2+ taking us, for example are pacemaking cells myocytes or interstitial cells? -induced Ca2+ release may be responsible for entrainment. Voltage-dependent Do neighbouring cells electronically suppress the spread of excitation? What regulation of IP3 production has not been demonstrated in ICC, but ICC express have gene array studies and modelling approaches suggested? How similar or voltage-dependent Ca2+ channels (possible T-type channels). Both Ni2+ and different is the myometrium from other smooth muscles? Finally, what tools mibefradil, blockers of T-type Ca2+ channels, reduce the frequency of slow waves, and experiments do we need to address the questions raised and to help us to slow propagation velocity, and reduce the velocity of the upstroke depolarization. become better able to control the activity of the myometrium. Thus, Ca2+ entry may initiate IP3 release and organization of pacemaker activity in networks of ICC.

54 IUPS 2009 July 27 - August 1, 2009 in Kyoto PSJ V-3-5 PSJ V-3-6 PHARMACOLOGICAL ACTION ON GUT MOTOR SPONTANEOUS ACTIVITY IN GUT-LIKE ORGAN ACTIVITY THROUGH INTERSTITIAL CELLS OF CAJAL FORMED FROM ES CELLS 1 1 1 Jan D Huizinga Miyako Takaki , Hiromi Misawa , Hiroko Matsuyoshi , Hiroki 2 Department of Medicine, McMaster University, Canada Kuniyasu 1 2 Unique motor patterns are intrinsic to every organ of the gastrointestinal Department of Physiology II, Nara Medical University, Japan, Department of Molecular Pathology, Nara Medical University, Japan tract which suit their functions related to mixing, absorption and anally directed movement. Interstitial cells of Cajal (ICC) are an integral part of the Using an embryoid body (EB) culture system, we developed a functional control of motor activity. The best understood function is that of pacemaker organ-like cluster, a "gut", from mouse embryonic stem (ES) cells (ES gut). activity to the stomach and small intestine. In these organs, distention or Each ES gut exhibited various types of spontaneous movements. In these neural excitation evoke characteristic motor patterns that have the unique spontaneously contracting ES guts, dense distributions of interstitial cells of Cajal (ICC) (c-kit, a transmembrane receptor that has tyrosine kinase activity, frequency and propagation characteristics of the ICC pacemaker system. positive cells; gut pacemaker cells) and smooth muscle cells were discernibly In the antrum this results in peristalsis which serves to mix and grind the identified. By adding Glivec 10 μM, a tyrosine kinase receptor c-kit inhibitor, stomach content. In the small intestine this results in short distance peristaltic only during EB formation, we for the first time succeeded in suppressing in activity and segmentation motor patterns. Pharmacologically affecting ICC vitro formation of ICC in the ES gut. The ES gut without ICC did not exhibit excitability can be demonstrated through actions of the ERG K channels; it any movements. However, it appeared that Glivec 0.1-1 μM rather increased can be beautifully demonstrated through their action on calcium influx. Other number of ICC in ES guts and spontaneously moving ES guts associated with pathways to pacemaker modulation are the unique chloride and non-selective 2+ 2+ increase of intracellular Ca concentration ([Ca ]i). By adding 5-HT4 agonist cation pacemaker ion channels. The innervation of ICC serves to connect 10 μM only during EB formation, we succeeded in forming more dense nerve ICC to enteric sensory nerves as well as affect ICC pacemaker activity. fibers. However, by adding 5-HT4 agonist 1 μM, we succeeded in forming Better understanding of the communications between ICC and enteric nerves more dense networks of ICC and inducing more active spontaneous motility will provide unique pathways for pharmacological modulation of gut motor in the ES gut. These results suggest ICC is critical for in vitro formation of activity. ES guts with spontaneous movements.

PSJ VI-4-1 PSJ VI-4-2 SINGLE MOLECULE ANALYSIS AND THE MYOSIN SINGLE MOLECULE IMAGING AND NANO- FAMILY OF MOLECULAR MOTORS BIOTECHNOLOGY James A. Spudich, Sivaraj Sivaramakrishnan Toshio Yanagida Department of Biochemistry, Stanford University, USA Graduate School of Frontier Biosciences Osaka University, Japan The molecular basis of how myosin motors work has been significantly Biomolecules assemble to form molecular machines such as molecular advanced by studies of myosins V and VI. Myosin V moves processively, motors, cell signal processors, DNA transcription processors and protein walking along the 36-nm pseudo-repeat of an actin filament by swinging its synthesizers to fulfill their functions. The reactions and behaviors of long lever arms through an angle of ~70 deg. We have used single-molecule molecular machines respond to their surroundings with great flexibility. high resolution co-localization (SHREC) to directly observe myosin V This flexibility is essential for biological organisms and biological systems. PSJ symposia molecules walking hand-over-hand. Myosin VI has a very short conventional In recent years, single molecule imaging and nano-technologies have lever arm, but steps 36 nm along actin. Our most recent work shows how rapidly been expanding to include a wide range of life science applications. this unusual motor achieves this feat. Part of the answer is that the medial tail, which had been thought to be a coiled coil, forms a rigid stable α-helix The dynamic properties of biomolecules and the unique operations of ~70 residues long, extending the lever arm considerably. Using molecular molecular machines, which were previously hidden in averaged ensemble dynamics simulations, we characterize a dynamic pattern of side chain measurements, are now being unveiled. The aim of our research is to interactions that extends along the backbone of this α-helix. A simplified approach the engineering principle of adaptive biological systems by model predicts that side chain interactions alone contribute substantial uncovering the unique operation of biological molecular machines. Here, I bending rigidity to this α-helix. Results of small angle X-ray scattering review our single molecule experiments designed to investigate molecular (SAXS) and single molecule optical trap analyses are consistent with the high motors, enzyme reactions, protein dynamics and cell signaling, and discuss bending rigidity predicted by our model. Myosin VI is now a paradigm of how Brownian motions (noise) play a positive role in the unique operation of how intermolecular tension sensing is translated into biologically important biological molecular machines allowing for flexible and adaptive biological trafficking regulation. systems. http://www.phys1.med.osaka-u.ac.jp/

PSJ VI-4-3 PSJ VI-4-4 SINGLE MOLECULE ANALYSES ON THE GATING OF A REAL TIME MONITORING OF STRUCTURAL CHANGES MECHANOSENSITIVE ION CHANNEL: EXPERIMENTS IN K-CHANNEL GATING AND MOLECULAR SIMULATIONS Shigetoshi Oiki Masahiro Sokabe, Yasuyuki Sawada Molecular Physiology and Biophysics, University of Fukui Faculty of Department of Physiology, Nagoya University Graduate School of Medical Sciences, Japan Medicine, Japan Ion channels perform their function through opening and closing of the Mechanosesnitive (MS) channels are the only established molecular class of gate under control of various stimuli. Crystal structures of several types cell mechanosensors. Among them the bacterial MS channel MscL is the best of potassium channels in closed and open states of the gate have been studied one owing to the resolved 3D structure of its closed state. MscL forms a elucidated. However, trajectories of conformational changes during the homopentamer with each subunit having two (inner and outer) transmembrane gating were hardly predicted from the still pictures of open and closed helices, displaying fivefold radial symmetry around a central pore. The conformations. We applied a method named as the diffracted X-ray tracking neighboring inner helices cross each other near the cytoplasmic surface, thus to trace structural changes of single channel molecules in video rate. The forming the most constricted and strongly interacting part (gate) of the pore. The issue is to understand the underlying mechanism during the gating process driven KcsA potassium channel from Streptomyces lividans was attached to a by tension in the membrane; more concretely where and how the channel protein glass surface and a gold nano-crystal was labeled to the free end of the receives forces and how the forces lead to gate opening. To identify the tension- channel molecule. Irradiations of synchrotron white X-rays (SPring-8) to the sensing site, we performed molecular dynamics (MD) simulations and site- nanocrystals elicited diffraction spots, whose motions gave conformational directed mutagenesis at each hydrophobic residue in the outer helix facing lipid changes of the channel molecule. At acidic pH, where the channel shows moiety and found that the hydrophobic interaction between Phe78 and lipids near active gating, twisting conformational changes in the range of several tens of the periplasmic surface is essential to tension sensing. Upon tension increase, the degrees around the pore axis were detected. This twisting motion originated tension sensor Phe78s were dragged radially by lipids followed by a tilting down from the transmembrane domain and propagated towards the cytoplasmic of the helices and an outward sliding of the cross points (gate) between inner domain. This mode of motion may prevail for channel molecules having helices, leading to the gate opening. symmetrical structure around the pore axis.

IUPS 2009 July 27 - August 1, 2009 in Kyoto 55 PSJ VI-4-5 PSJ VI-5-1 MECHANISMS OF ACETYLCHOLINE RECEPTOR REGULATION OF cAMP SIGNAL BY G PROTEINS: A GATING NEW PATHWAY AFTER THREE DECADES Anthony Auerbach Yoshihiro Ishikawa Department of Physiology and Biophysics, State University of New York at Cardiovascular Research Institute, Yokohama City University Graduate Buffalo, USA School of Medicine, Japan Nicotinic acetylcholine receptor-channels (AChRs) mediate synaptic It was in 1971 when Earl Wilbur Sutherland received the Novel Prize for transmission between vertebrate nerve and muscle. The C(losed)-O(pen) identifying cAMP as a second messenger for hormone action. Thirty years ‘gating’ isomerization of the AChR involves both a low-high affinity change later, we now know that cAMP is produced by adenylyl cyclase upon at two transmitter binding sites and a low-high conductance change in the hormonal receptor stimulation and thus G protein activation, and that there pore. We use single-channel kinetics to probe the changes in structure and are at least nine isoforms of adenylyl cyclase that show distinct tissue energy that couple agonist affinity and conductance in this ~300 kD, five- distribution and biochemical properties. Adenylyl cyclase may be stimulated subunit protein. 1) The correlation between the opening rate- and the gating or inhibited by the same receptor stimulation depending upon its isoform. equilibrium-constant on a log-log scale (phi) can reflect the relative time The heart expresses multiple isoforms including the type 5 isoform, which when a residue flips, instantaneously and in an all-or-none fashion, between is dominant in adults, while type 6 in neonates. Various studies employing its C and O conformations. 2) The map of phi in the AChR reveals a coarse- transgenic techniques have revealed that the type 5 isoform, which possess grained, decreasing gradient between the transmitter binding sites (‘early’) the highest catalytic activity among all the isoforms, does not play a major and the gate (‘late’). 3) The end state structures, the conformational pathway role in maintaining basal cardiac function, but may play a important role and the transition state are similar for unliganded vs. diliganded gating. 4) in the parasympathetic regulation of the heart. Disruption of this isoform Evidence suggests that a change in the energy of water at the pore equator does not impair basal cardiac function, but it may play a protective role makes a late and large contribution to the gating equilibrium constant. The against various stresses. Further, longevity was observed. Accordingly, AChR gating reaction is extraordinarily robust and occurs by the same pharmacological compounds that regulate this isoform have been studied. Brownian conformational cascade over almost a billion-fold range of Various techniques including computer simulation have been employed with equilibrium constant. promising results for drug development.

PSJ VI-5-2 PSJ VI-5-3 MECHANISMS FOR INHIBITION OF GBETA/GAMMA THE ARF-LIKE GTPase ARL8 IS REQUIRED FOR SIGNALING BY PROTEINS, PEPTIDES AND SMALL LYSOSOME BIOGENESIS IN C. ELEGANS MOLECULES Toshiaki Katada, Kenji Kontani PSJ symposia 1 1 2 Alan Victor Smrcka , Sundeep Malik , Pramodh Seneviratne , Department of Physiological Chemistry, Graduate School of Axel Dessal1, Nessim Kichik3, Teresa Tarrago3, Ernest Giralt3 Pharmaceutical Sciences, University of Tokyo, Japan 1 Pharmacology and Physiology, University of Rochester School of Lysosomes are fundamental organelles responsible for the degradation Medicine, USA, 2Pathology, University of Rochester School of Medicine, 3 of macromolecules derived from endocytic, phagocytic and autophagic USA, Chemistry and Molecular Pharmacology Program, Institute for pathways. It is still not fully understood how functional lysosomes are Research in Biomedicine, Barcelona, Spain generated and maintained. Here we show that the C. elegans ARL-8 GTPase The G protein βγ (G βγ) subunit complex performs a central function transducing is localized primarily to lysosomes and essential for lysosome biogenesis in signals from G protein coupled receptors to changes in cellular physiology through a the macrophage-like coelomocytes. Loss of arl-8 results in an increase in series of highly regulated protein-protein interactions. We proposed that Gβγ subunits number of lysosomes, which are small in size and non-acidic. Localization have a dynamic protein interaction “hot spot” mediating interactions between Gβγ of V-ATPase subunits to lysosomes is disorganized in arl-8 mutants. The and multiple downstream signaling molecules, that can also be targeted by selective lysosomal aspartic protease ASP-1 is enriched in a limited population inhibitors. Natural Gβγ inhibitors such as GαGDP and phosducin, as well as artificial of lysosomes in arl-8 mutants, whereas endocytosed macromolecules peptide and small molecule inhibitors alter effector binding to this “hot spot” and thus regulate Gβγ-dependent in physiology. Small molecule inhibitors, thought to interact accumulate in another subpopulation of lysosomes not containing ASP-1. with the Gβγ “hot spot”, are beneficial in several animal models of disease. Recent Furthermore, arl-8 epistatic to cup-5, which is the orthologue of human studies from our laboratory have combined biophysical, biochemical and physiological mucolipin-1 implicated in mucolipidosis type IV: loss of arl-8 strongly methods to obtain information about how ligands bind to Gβγ to result in selective suppresses formation of enlarged lysosome-like vacuoles in cup-5 mutants. effects on cell physiology. Results from these studies have implications with respect These findings suggest that ARL-8 acts as an essential node that tightly links to development of novel specific small molecule inhibitors as well as to understanding appropriate maintenance of number and size of lysosomes with their intrinsic basic mechanisms in the heterotrimeric G protein signaling system. functions.

PSJ VI-5-4 PSJ VI-5-5 REGULATION OF PIP5K ISOZYMES BY ARF6 G PROTEINS IN ENDOSOMAL TRAFFICKING Yasunori Kanaho PATHWAYS Department of Physiological Chemistry, University of Tsukuba, Japan Julie G. Donaldson Laboratory of Cell Biology, NHLBI, NIH, USA Phosphatidylinositol 4-phosphate 5-kinase (PIP5K), which catalyzes phosphorylation of PI4P to produce the versatile phospholipid, Cells internalize plasma membrane (PM), ligands, and extracellular fluid through the process of endocytosis. The endocytosed material is subsequently phosphatidylinositol 4,5-bisphosphate (PI4,5P .), regulates a wide variety 2 sorted and routed to lysosomes for degradation, to other destinations, of cellular events and functions through its product PI4,5P2. To date, three or recycled back to the PM. Although much is known about clathrin- mammalian PIP5K isozymes, α, β and γ, and three splicing variants of dependent endocytosis (CDE), little is known about clathrin-independent PIP5Kγ, γ635, γ661 and γ687, have been identified. We have previously endocytosis (CIE) and the subsequent trafficking of CIE cargo proteins. reported that the small G protein ARF6 directly activates bacterially We and others have been studying a CIE pathway associated with the Arf6 expressed PIP5K in vitro. Recently, we found that of three PIP5K isozymes, GTPase that brings a variety of PM proteins into cells including the major which were expressed in HEK293T cells, only PIP5Kβ was activated by histocompatibility complex Class I protein (MHCI), integrins and some G ARF6. Furthermore, the kinase core domain (KCD) of PIP5Kγ661, which protein coupled receptors in the absence of ligand. Arf6 activities, including lacks N- and C-termini, interacted with and activated by ARF6, while the the activation of phosphatidylinositol kinases and phospholipase D, mediate the movement of membrane through the pathway and are required for the full length of PIP5Kγ661 interacted very weekly, if any, with ARF6. These recycling of many of these endocytosed cargo proteins back to the PM. results suggest that in mammalian cells PIP5Kγ661 is posttranslationally Rab11 and Rab22 are also required for recycling of CIE cargo proteins modified to form a conformation, which prevents the binding of ARF6. In back to the PM. A number of signaling proteins, such as H-Ras, src, and addition these results led us to speculate that under physiological conditions heterotrimeric G proteins, are associated with the CIE pathway suggesting PIP5Kγ661 changes its conformation to interact with ARF6 in response to that these endosomes could serve as platforms for signaling or mechanisms agonist stimulation. to route signaling molecules to other PM domains.

56 IUPS 2009 July 27 - August 1, 2009 in Kyoto PSJ VI-5-6 PSJ VII-6-1 THE REGULATION AND FUNCTION OF EPAC WHETHER AND HOW DOES BLOOD-BORNE PROTEINS PROSTAGLANDIN CONTRIBUTE TO FEVER? Johannes L. Bos Andrej A. Romanovsky Physiological Chemistry, University Medical Centre, The Netherlands Systemic Inflammation Laboratory, Trauma Research, St. Joseph's Hospital, USA Epac proteins are guanine nucleotide exchange factors for Rap directly All phases of lipopolysaccharide (LPS)-induced fever are mediated by activated by cAMP that function among others in the control of integrin- prostaglandin (PG) E . It is known that the second (starts at 1.5 h post-LPS) mediated cell adhesion, cell-cell junction formation and secretion. We 2 and later febrile phases are mediated by PGE2 originated in the brain. We have previously determined the crystal structure of Epac2 in the inactive attempted to identify the location and phenotypes of cells that trigger the first conformation, revealing the auto-inhibited conformation of the protein. We phase (starts at 0.5 h) of LPS fever in rats. We found that, during the first have now determined the active conformation of Epac2 in the presence of phase, the enzymes of PGE2 synthesis were activated in the lung and liver, cAMP and Rap1, showing in atomic detail the mechanism of activation. but not in the brain. This activation involved phosphorylation of cytosolic In addition, we found that Epac1, but not Epac2, has an additional level phospholipase (cPL) A2 and transcriptional upregulation of cyclooxygenase of regulation by cAMP, which is its translocation from the cytosol to the (COX)-2. The number of COX-2-positive cells surged in the lung and liver plasma membrane. This is driven by diffusion and requires the DEP domain at the onset of fever; most of these cells were macrophages. When PGE2 in the open, cAMP-bound, conformation of the protein. The translocation is synthesis in the periphery was activated, the concentration of PGE2 increased both in the venous (collects PGE from tissues) and arterial (delivers PGE essential for proper activation of Rap1 at the plasma membrane by Epac1 and 2 2 to the brain) blood. Neutralization of circulating PGE2 with an anti-PGE2 for downstream biological effects including integrin-mediated cell adhesion. antibody delayed and attenuated LPS fever. We conclude that fever is Independently, signalling through the small GTPase Rho induces a similar initiated by PGE2 synthesized by macrophages of the LPS-processing organs translocation of Epac1, although to a more polarized plasma membrane (lung and liver) via phosphorylation of cPLA2 and upregulation of COX-2. localization. How peripherally produced PGE2 causes the brain-mediated fever is a subject of studies in progress.

PSJ VII-6-2 PSJ VII-6-3 HOW IS THE ARACHIDONIC ACID CASCADE WHAT ARE THE ROLES OF THE DIFFERENT REGULATED IN THE BRAIN DURING FEVER? PROSTAGLANDIN E2 RECEPTORS IN FEVER? Kiyoshi Matsumura1, Aiko Hori2, Tomoko Yamamoto2, Hiroshi Clifford B Saper1, `Michael Lazarus2, Kyoko Yoshida1 2 2 Hosokawa , Shigeo Kobayashi 1Neurology, Harvard Medical School/Beth Israel Deaconess Med Ctr, USA, 2 1Information Science and Technology, Osaka Institute of Technology, Osaka Bioscience Institute, Osaka, Japan Japan, 2Graduate School of Informatics, Kyoto University, Japan A key step in fever response is the action of prostaglandin E2 (PGE2) on Undoubtedly arachidonic acid (AA) is a key molecule in fever because it neurons in the preoptic area. Three PGE2 (EP) receptors, 1, 3, and 4, are is the sole precursor of prostaglandin E2 (PGE2). We here propose another found in this region. Inhibitory EP3 receptors are particularly concentrated possible significance of AA in fever. Intracerebroventricular injection of AA in the median preoptic nucleus (MnPO), on neurons that are thought to PSJ symposia evoked biphasic fever in rats. The first phase started immediately after the inhibit the brain's thermogenic system, thus PGE2 action on these neurons injection, and was suppressed by diclofenac, a nonselective cyclooxygenase would release thermogenesis. We produced EP3 conditional knockout (COX) inhibitor, but not by NS398, a COX-2 inhibitor. The second phase mice, and used an adeno-associated viral vector containing the gene for started 60 min after the injection and was suppressed by NS398. During the Cre recombinase to cause deletion of the EP3 gene limited to the MnPO. second phase, COX-2 protein was expressed in brain endothelial cells. The These mice showed only a hypothermic response, instead of a fever, to i.p. induction of COX-2 by AA was not completely suppressed by an inhibitor lipopolysaccharide (LPS). One strain of EP1 knockout mice showed a modest of COX, lipoxygenase or epoxygenase. Other unsaturated fatty acids evoked reduction in LPS fever, but a second strain showed little if any effect. Hence, a monophasic fever, which temporally corresponded to the second phase of EP3 receptors in the MnPO are the primary pyrogenic PGE2 receptors, AA fever, and also induced COX-2 in brain endothelial cells. These results with at most a modest contribution by EP1 receptors. The hypothermic suggest a dual role of AA in fever being an inducer of COX-2 as well as the response unmasked by removing the EP3 receptors was evaluated in mice precursor of PGE2. One possible occasion in which AA plays such a dual with deletion of the EP3 receptor, crossed with mice with a deletion of the role is brain stroke, which is often accompanied by an increase in AA in the EP4 receptor in the CNS. These mice showed absence of the hypothermic extracellular space. In fact, we demonstrate that hypothalamic hemorrhage response to LPS. Thus the EP3 receptor causes Tb elevation and EP4 causes evoked biphasic fever, the second phase of which was COX-2-mediated. hypothermic responses to LPS.

PSJ VII-6-4 PSJ VII-6-5 DIFFERENTIAL CONTROL OF THERMAL EFFECTOR HOW ARE FEVER AND SICKNESS BEHAVIOR PATHWAYS CONTRIBUTING TO FEVER DIFFERENTIALLY CONTROLLED? Kazuhiro Nakamura, Yoshiko Nakamura, Shaun F Morrison Marta Balasko, Miklos Szekely Oregon National Primate Research Center, Oregon Health & Science Department of Pathophysiology and Gerontology, Medical School, University, USA University of Pecs, Hungary The action of the febrile mediator, prostaglandin E (PGE ), on preoptic area 2 2 Sickness behavior (SB), the individual’s complex response to systemic inflammation (POA) neurons through prostaglandin EP3 receptors (EP3R) triggers a variety involves fever, anorexia, somnolence, allodynia and a variety of other somatic, of febrile responses, such as shivering thermogenesis in skeletal muscles, non-shivering thermogenesis in brown adipose tissue and vasoconstriction cognitive, or affective alterations. Central and peripheral mechanisms were shown to in the skin. Experimental results support a model in which febrile shivering overlap in the pathogenesis of these adaptive responses. However, fever and other symptoms of SB show differential control. Inflammatory and non-shivering thermogenesis are stimulated when PGE2 attenuates the tonic inhibition from EP3R-expressing POA neurons to neurons in the cytokines have long been considered to be important in the pathogenesis of SB. dorsomedial hypothalamus (DMH) that activate somatic and sympathetic However, IL-6, a pyrogenic cytokine fails to elicit anorexigenic and cognitive/affective premotor neurons in the rostral raphe pallidus nucleus (rRPa). In contrast, responses. The dominant role of IL-1 has been questioned in the central phase of the induction of endotoxin fever, whereas its contribution to the anorexigenic or cognitive/ PGE2-mediated increases in cutaneous vasoconstrictor tone arise from a reduced tonic inhibition from EP3R-expressing POA neurons to sympathetic affective effects of SB has been confirmed. Among potential peripheral and central premotor neurons in the rRPa. Separate populations of EP3R-expressing POA mediators of SB we find examples where mediators inducing some other symptoms neurons directly innervate neurons in the DMH and those in the rRPa. These of SB fail to induce fever or even show antipyretic effects. Leptin, a major catabolic results, together with the different threshold temperatures at which these peptide of peripheral origin, failed to elicit any elevation of Tc in rats even at high thermal effectors are activated during cooling challenges, suggest that the doses. Alpha-MSH an endogeneous agonist of the anorexigenic melanocortin system thermal effector responses contributing to fever are differentially controlled reportedly has antipyretic effects. by distinct outflow pathways from separate sets of EP3R-expressing neurons Investigations of the differential pathomechanisms of SB may yield relevant new in the POA. information in the future. (OTKA 49321)

IUPS 2009 July 27 - August 1, 2009 in Kyoto 57 PSJ II-7-1 PSJ II-7-2 MULTISCALE MODELING OF MICROCIRCULATORY MULTI-SCALE MODELING OF BLOOD FLOW FOR THE EXCHANGE PROCESSES ANALYSIS OF RED BLOOD CELL BEHAVIORS IN A James B Bassingthwaighte MICRO-CIRCULATION Department of Bioengineering, University of Washington, Seattle, USA Masanori Nakamura Models of human physiology integrate knowledge at increasing levels The Center for Advanced Medical Engineering and Medical Informatics, of biological organization from genomics, molecular biology and the Osaka University, Japan environment, to cells, tissues and organs, and ultimately to the integrated Blood is essentially mixture of solid (cells) and liquid (plasma). By volume, most oranism. Large-scale top-down models of the cardiorespiratory system for of cells in blood are red blood cells (RBCs). While flowing, RBCs agglomerate, solute exchange can serve as phenotypic targets for understanding genomic forming rouleaux and aggregations which can be called a kind of cluster. The and proteomic-based modeling and be predictive of physiological responses. particulate nature of RBCs, their dynamic deformations, and physical interactions These models are hierarchical, with representation at levels of (1) cell significantly contribute to behaving as a multiphase suspension. Here we describe processes, (2) tissues and organs, and (3) system interactions. To model our recent works on the analysis of hemorheology in microcirculations by means respiratory gas and solute exchanges we use convection diffusion reaction of multi-scale modleing of blood flow. We modeled blood flow at two different models (in JSim, a Java-based simulation system) giving spatial profiles. scales and coupled them to investigate mesoscopic blood flow phenomena. Metabolic modules link cellular fluxes with cardiac contractile function We represented a macroscopic blood flow by a continuum model described and coronary flow regulation or link alveolar-blood exchange of gases with by the Navier-Stokes equation and equation of continuity. On the other hand, ventilation-perfusion heterogeneity. The robustness of responses to changing a microscopic blood flow was represented by RBCs. An RBC was modelled conditions is usually at the cellular level, so oversimplified models fail. as a closed shell membrane of spring networks and its dynamic behavior was Linking finite element modeling to biochemical and transport modeling determined on the basis of the minimum energy principle. We present that RBC and simultaneously to circulatory pressure-flow modeling is critical in such show variouis shapes and flowing behaviors depending on its stiffness and a systems. See http://www.physiome.org. (Supported by NSF grant 0506477, shape of flowing RBC is determined not only by instantaneous fluid force acting NIH grants T15-HL088516, and R01-HL073598.) on it but also its deformation history. We also discuss importance of individual RBC behaviors in mesocopic blood flow phenomena.

PSJ II-7-3 PSJ II-7-4 MOLECULAR MECHANISTIC INSIGHTS INTO THE COMPUTATIONAL BIOMECHANICS OF MALARIA AND ADHESION OF MALARIA INFECTED ERYTHROCYTES ARTERIAL DISEASES WITH ENDOTHELIUM IN CIRCULATION Takuji Ishikawa PSJ symposia Chwee Teck Lim1, Ang Li1, Kevin Shyong Wei Tan2 Department of Bioengineering and Robotics, Tohoku University, Japan 1Division of Bioengineering, National University of Singapore, Singapore, We have been investigating the cardiovascular system over micro to macro 2Department of Microbiology, National University of Singapore, Singapore levels by using conjugated computational mechanics analyzing fluid, solid Plasmodium falciparum is the most deadly species of malaria parasites. When and bio-chemical interactions. In this paper, I introduce our recent research the parasite invades and matures within an erythrocyte, parasite induced on malaria-infected red blood cell (IRBC) mechanics using a particle method. proteins are secreted and embedded in the cell membrane. This gives rise to We performed the stretch test of IRBCs, and the numerical results agreed well with experimental results. Our model also successfully simulated flow of cytoadherence where infected red blood cells (IRBCs) are found to adhere to IRBCs into narrow channels. We think that the computational biomechanics endothelial cells that line the blood vessel walls. Often, this phenomenon causes can be a useful tool for investigating malaria disease. severe consequences such as blood clogging. Here, we quantify the molecular Moreover, I introduce our recent researches on a novel hemodynamic index interactions between TSP and CD36 (receptor proteins on endothelial cell for the initiation of cerebral aneurysms focusing on temporal variation surface) and that of the parasite exported proteins PfEMP1 secreted to the surface of spatial wall shear stress gradient, and primary thrombus formation in of the IRBCs using an atomic force microscope (AFM). Although the binding microcirculation. kinetics of CD36 and TSP molecules were previously studied using flow based assay, they were performed on a population of cells and there is no quantification of binding forces. Also, there was uncertainty in the number of bonds formed in cell-cell or cell-molecule contact in these assays. Our study seek to help better understand quantitatively the molecular interactions involved in malaria parasitology and suggest strategies in quantitatively evaluating the effectiveness of drugs developed to inhibit cytoadherence.

PSJ II-7-5 PSJ II-7-6 REGULATION OF CEREBRAL VASCULAR TONE BY MULTI-LEVEL SYSTEMS APPROACH TO ANP GASEOUS MEDIATORS; INTERACTIONS OF MULTIPLE DEPENDENT REGULATION OF VASCULAR GAS-TRANSDUCING SYSTEMS PERMEABILITY AND PLASMA VOLUME IN MICE Mayumi Kajimura1, Katsuji Hattori1, Yakayuki Morikawa1, Fitzroy Edward Curry1, Haris Samardizc1, Joyce F Lenz1, Mami Ishikawa2, Makoto Suematsu1 Roger H Adamson1, Y-C Lin1, Tine Karlsen2, Cecilie Rygh3, Rolf Reed3, 4 1Department of Biochemistry and Integrative Medical Biology, Keio Michaeal Kuhn 2 1Physiology and Membrane Biology, University of California, Davis, USA, University School of Medicine, Japan, Department of Neurosurgery, Jichi 2 3 Medical University, Saitama, Japan Department of Biomedicine, University of Bergen,Norway, Department of Biomedicine, University of Bergen, Norway, 4Institutes of Physiology, University Although the brain possesses the ability to generate gaseous mediators such as CO, of Wuerzburg,Germany NO and H2S, it is unknown how these gases interact with one another to regulate cerebrovascular function. We show that CO acts as a tonic regulator against NO- Mice with endothelial-restricted deletion of the guanylyl cyclase-A (GC-A) receptor for ANP (EC GC-A KO) have expanded intravascular volumes and reduced tissue albumin content dependent vasodilatation. We found that (i) suppressing endogenous CO caused in spite of normal renal function (Sabrane et al. J Clin Invest 115:1666). Investigations an increase in arteriolar diameter that was accompanied by an increase in local NO in cultured endothelium, perfused microvessels, and whole organs suggest ANP may generation and (ii) CO- and NO- generating sites around subarachnoid space were co- regulate plasma volume by modulating vascular permeability. We are expanding the localized in the rat pial circulation. We tested further the hypothesis that, in the cortical multilevel systems approach to test the hypothesis that loss of physiological regulation circulation, CO modulates the generation and the action of H2S. Immunohistochemical of macromolecular permeability by ANP when plasma volume is expanded maintains analyses of the murine cortex indicated that heme oxygenase (HO)-2, a CO-producing fluid shifts from interstitium to plasma. We used non-invasive MRI methods to repeatedly enzyme, occurred in neurons whereas cystathionine β -synthase (CBS), the enzyme measure blood-tissue transport of high MW contrast agents, and modified two tracer responsible for the H2S production, was expressed in astrocytes. Cerebellum-slice methods to measure albumin clearance and microvascular plasma volume in EC GA-A KO preparation revealed that blockade of the HO activity dilated arterioles. By contrast, mice, floxed littermates, and C57 control mice. ANP-increased macromolecule clearances slices prepared from the CBS-deficient mice did not exhibit such a vasodilatory in muscle and skin were attenuated in KO mice relative to controls. Normalization of response caused by suppression of endogenous CO. These results raise the possibility clearance to local plasma volume confirmed real reductions in permeability and tested for that CO derived from HO-2 limits H2S generation by CBS in the murine cerebellar ANP modulation of microvascular surface area for exchange. Similar multilevel approaches circulation. can be used for all mechanisms regulating microvascular exchange.

58 IUPS 2009 July 27 - August 1, 2009 in Kyoto RS I-1-1 RS I-1-2 EVIDENCE FOR CELL-CELL AND INTRACELLULAR LACTATE AS A FUEL FOR EXERCISE LACTATE SHUTTLES Hideo Hatta George A. Brooks Department of Sports Sciences, The University of Tokyo, Japan Integrative Biology, University of California, Berkeley, USA Lactate is not a waste but an oxidizable fuel particularly during exercise. Recognition of a Cell-Cell Lactate Shuttle (CCLS) came from comparisons Ingestion of lactate before prolonged exercise can prevent hypoglycemia by of glucose and lactate turnover measurements. Subsequently, measurements the increased utilization of ingested lactate. Lactate is transported through of blood and tissue lactate contents showed arterial levels to be intermediate monocarboxylate transporter (MCT). MCT1, which is abundant in slow between lactate releasing glycolytic and oxidative consuming tissues and type muscle fibers and heart, is related to incorporation and oxidation of determinations of lactate exchange across working muscle beds showed lactate. Significant negative relation was found between MCT1 and blood simultaneous net muscle lactate release and tracer-measured lactate oxidation lactate concentration during exercise possibly because of utilization of blood and uptake. Studies on isolated sarcolemmal vesicles showed carrier- lactate through MCT1. MCT4, which is abundant in fast fibers, is related to mediated lactate transport. Most recent evidence for a CCLS comes from extrusion of lactate out of working muscle. In Thoroughbred horses, whose studies using Lactate Clamp technology. Observations of whole-body and working muscle and heart lactate uptake and oxidation led to discoveries muscles have high type IIa fiber, not only MCT1 but also MCT4 can be an of an Intracellular Lactate Shuttle (ICLS) and the Mitochondrial Lactate important factor for sustaining maximal running. Developmental increase Oxidation Complex (MLOC) minimally involving a monocarboxylate of MCT1 occurs during 2 to 24 months age of horses with increase in PGC- transporter (MCT), its glycoprotein scaffold (CD147), lactate dehydrogenase 1alpha and mitochondrial enzyme activity. Increase in the concentration of (LDH) and cytochrome oxidase (COX). The presence of a MLOC is lactate in the blood and muscle by ingestion of lactate or by production of supported by data on adult rat muscle and brain, human skeletal muscles, and lactate in the working muscle relates to mitochondrial biogenesis. Endurance cultured rat myocytes and neurons. The central role of lactate in metabolic training with ingestion of lactate increased mitochondrial enzyme activity. integration was augmented by discovery of peroxisomal MCTs and LDH that High intensity repetition training increases mitochondria with production of permit redox control of peroxisomal β-oxidation. lactate.

RS I-1-3 RS I-1-4 SIGNALING EFFECTS OF LACTATE IN CULTURED L6 FUELLING CEREBRAL ACTIVITY IN HUMANS SKELETAL MUSCLE CELLS Niels Henry Secher Takeshi Hashimoto Department of Anesthesiology, Rigshospitalet, Denmark Department of Life Science, University of Hyogo, Japan The brain relies mainly on glucose as substrate and releases little lactate, Functional adaptations of muscle cells to exercise involve alterations in cell but during exercise it takes up lactate and, as determined by labeled lactate, structure, mitochondrial density and respiratory capacity, solute transport, virtually all lactate taken up is metabolized. Furthermore, cerebral activation and energy substrate partitioning. Our interest was to define the role of lactate is associated with a larger uptake of carbohydrate than of oxygen and the as a metabolic signal that affects gene transcription. We hypothesized that cerebral metabolic ratio (uptake of oxygen x 6/(glucose + half lactate); CMR) lactate anion is a signaling molecule whose actions affect the transcription of decreases from a resting value close to 6 to a nadir of 1.7. Thus, the brain’ specific genes. Therefore, we screened genome-wide responses of L6 cells s nonoxidative carbohydrate consumption may amount to approximately to elevated (10 and 20 mM) concentrations of sodium lactate. Analysis of 20 mmol. Since the decrease in CMR is eliminated by propranolol, while ~15,000 L6 genes suggested that lactate anion signaling cascade involves it is maintained during exercise with metroprolol, a beta-2 adrenergic ROS production and converges on transcription factors that may affect mechanism appears to be important. In support, CMR decreases with expression of the mitochondrial lactate oxidation complex (lactate transporter infusion of epinephrine but not of norepinephrine at rest. It is suggested that MCT1, its chaperon protein CD147, cytochrome c oxidase, and lactate what is described as the brain‘s nonoxidative carbohydrate consumption dehydrogenase), mitochondrial biogenesis, and upregulation of antioxidant reflects glycolysis driven by epinephrine. An important role for glycolysis in Regular Symposia enzymes and moieties of calcium signaling. As well data provide insight into accelerated brain metabolism may implicate fatigue. Fatigue could develop how lactate may serve as a signaling molecule (a “lactormone”) eliciting not following depletion of the brain’s glycogen deposit, as it is known for the only adaptation of pathways of lactate removal (lactate oxidation complex), muscle glycogen level during prolonged exercise. but also signals many of the adaptations in muscle found in response to exercise training.

RS I-1-5 RS I-1-6 DEVELOPMENTAL CHANGES IN MCTs AND PGC-1 EXERCISE-MODULATED TRANSCRIPTS IN THE ALPHA IN THOROUGHBREDS SKELETAL MUSCLE AND THE RELATIONSHIP WITH Yu Kitaoka1, Daisuke Hoshino1, Kazutaka Mukai2, Atsushi Hiraga2, METABOLIC SYNDROME, AGING AND MUSCLE Hideo Hatta1 ATROPHY 1Department of Life Science, University of Tokyo, Japan, 2Equine Reserch Mayumi Yoshioka1, Hiroaki Tanaka2, Yuichiro Nishida3, Takuro Tobina2, Institute, Japan Racing Association, Japan Kiyohide Murakami2, Naoko Shono4, Munehiro Shindo2, Jonny St-Amand1 1 2 The purpose of this study was to investigate the longitudinal changes CREMO, CRCHUL and Laval University, Canada, Faculty of Health and Sports Science, Fukuoka University, Japan, 3Department of Preventive Medicine, Saga in monocarboxylate transporters (MCTs) and PGC-1alpha proteins in 4 Thoroughbreds. We previously suggested that MCT1, which is involved with Medical School, Japan, Institute of Lifestyle Medical Science, Japan taking up lactate into myocyte and MCT4, which is involved with extruding Regularly performed aerobic exercise improves metabolic syndrome. Exercise at lactate lactate, play key roles in sustaining high intensity exercise in Thoroughbreds. threshold (LT) is the minimum intensity which can promote health benefits. However, the Recently, it is suggested that PGC-1alpha might regulate the expression of molecular mechanisms are largely unknown. Thus, we performed the serial analysis of MCT1. It has been reported that MCTs and PGC-1alpha proteins can be changed gene expression (SAGE) strategy in human skeletal muscle before and after LT training and detraining. The LT training induced 274 transcripts whereas suppressed 212 transcripts, by training or chronic muscle stimulation, but it is unknown about developmental in which those encoding proteins with known functions accounted for 77% and 5%, changes. Six Thoroughbred horses were used for the analysis. Gluteus medius respectively. A total of 23 transcripts were quickly regulated by LT training (5 days) and muscles were obtained at the age of 2, 6, 12, and 24 months old. MCT1 and were the most induced transcripts (6 and/or 12 wks), in which 19 transcripts kept the effect PGC-1alpha protein contents were significantly increased from 2 to 24 months even after detraining (12 wks). They are related to oxidative capacity, muscle contraction, old longitudinally and MCT2 also showed a tendency to increase. On the other protein synthesis and creatine metabolism, as well as a novel transcript. Moreover, many hand, MCT4 was not changed through 2 to 24 months old. Citrate synthase of the induced transcripts have been reported to be down-regulated by diabetes, aging and (CS) activity was significantly increased from 6 to 24 months old investigated muscle atrophy, whereas 4 out of 10 functionally-known and repressed transcripts are the in a crosssectional manner, while phosphofructokinase (PFK) activity was not opposite. These results support the health benefits of LT training at gene expression levels. altered. These results show that Thoroughbreds would get oxidative capacity as The characterizations of novel and most of repressed transcripts by LT training will be they grow up, with keeping glycolytic capacity unchanged. needed.

IUPS 2009 July 27 - August 1, 2009 in Kyoto 59 RS I-2-1 RS I-2-2 MATRIX METALLOPROTEINASES REMODELING HIGH-SPEED ATOMIC FORCE MICROSCOPY FOR EXTRACELLULAR MATRICES OF TISSUE VISUALIZING DYNAMIC BIOMOLECULAR PROCESSES CONSTRUCTS Toshio Ando 1 2 3 4 Elliot L. Elson , Tetsuro Wakatsuki , Saveez Saffarian , Guy Genin , Department of Physics, Kanazawa University, Japan Gregory Goldberg5 1 Life is dynamic in nature. The biological function of proteins is closely Department of Biochemistry and Molecular Biophysics, Washington University in St. Louis, USA, 2Department of Physiology, Medical College of Wisconsin, associated with their ability to undergo structural changes. Nevertheless, USA, 3Department of Cell Biology, Harvard Medical School, USA, 4Department lack of suitable techniques has never allowed direct visualization of the of Mechanical and Aerospace Engineering, Washington University in St. Louis, nanometer-scale dynamics under physiological condition, and hence, minute USA, 5Division of Dermatology, Washington University School of Medicine, USA biomolecular processes have been hardly revealed. Here, I report direct and While forming a tissue construct, cells compress and remodel the collagen-based real-space visualization of lively changing structure of stimulated proteins. extracellular matrix (ECM) in which they are embedded, providing a model for tissue This is accomplished using the state-of-the-art high-speed atomic force development and wound healing. Depending on the cell concentration, the compression can microscopy developed by us. The real-time and high-resolution movies be 10-fold or more and depends on the ability of the cells to adhere to the ECM collagen and reveal elaborate biomolecular processes impossible to be found with other to exert a myosin-dependent contractile force (Wakatsuki, 2000). Matrix Metalloproteinases (MMPs) that degrade collagen and other ECM components, also play a role in remodeling. approaches. Thus, this study will inspire a fundamental change in approaches It has been observed that the collagenase MMP-1 is an extracellular molecular “motor” to studying the functional mechanisms of proteins and other biological operating as a “burnt bridge” Brownian ratchet (Saffarian, 2004). As a pure Brownian macromolecules, and consequently, will have a great impact on a wide range ratchet the MMP-1 motor can exert negligible force. A mechanism has been suggested, of biological sciences. however, for conversion of the large free energy change produced by cleavage of collagen fibrils into substantial force by MMP-1 dimers bound to cell surfaces (Saffarian, 2006). This mechanism can be tested experimentally to determine whether the MMP-1 ratchet can exert significant force and what is the role of this MMP and others in remodeling tissue constructs. Saffarian, S. et al. (2004). Science 306:108. Saffarian, S. et al. (2006). Phys Rev E 73:041909. Wakatsuki, T. et al. (2000). Biophys J 79:2353.

RS I-2-3 RS I-2-4 TRANSLATING MULTI-SCALE CARDIAC MODELLING THE MOLECULAR MECHANISM OF INTO THE CLINIC CARDIOMYOPATHY CAUSED BY MUTANT TROPONIN: Steven Alexander Niederer1, Gernot Plank2, Kawal Rhode3, A MOLECULAR DYNAMICS STUDY TESTED BY AN 3 1 X-RAY DIFFCACTION EXPERIMENT Reza Razavi , Nic P Smith 1 1 1 1 1 2 Maki Yamaguchi , Masako Kimura , Shigeru Takemori , Tetsuo Ohno , Computational Biology, University of Oxford, UK, Oxford e-Research 2 3 4 3 Yumiko Otsuka , Nobutake Akiyama , Naoto Yagi Centre, University of Oxford, UK, Division of Imaging Sciences, King's 1 College London, UK Department of Physiology, The Jikei University School of Medicine, Japan, 2Teine Keijinkai Hospital, Japan, 3Department of Immunology, The Jikei The application of multiscale computational cardiac models in the clinic poses a University School of Medicine, Japan, 4SPring8/JASRI, Japan number of new challenges for existing frameworks largely parameterised from animal Several single mutations on troponin molecules have been reported to cause familial data. In many pathologies the already significant physiological variations between cardiomyopathies through the altered contractility of cardiac cells. Since the x-ray Regular Symposia normal individuals is amplified, necessitating the need for patient specific models to crystallographic structure of human cardiac troponin has been determined (PDB 1J1E), we provide clinically relevant predictions of function and insight into disease mechanisms. are now ready to elucidate the molecular mechanism of the altered contractility. We first To provide a proof of concept for clinical applications of multiscale cardiac models studied the molecular dynamics of troponin mutants; E244D/K247R in troponin T, P82S in we have developed a coupled electromechanics model of a human heart. The model troponin I, and G159K in troponin C. Their dynamics in water was simulated with periodic uses a human anatomical geometry fitted from failing heart MRI data. Deformation condition at constant temperature and pressure with Amber 9 software. Introducing each is driven by a novel model of tension generation and electrical activation is calculated mutation to the determined structure of human cardiac troponin, we constructed the structure using an existing model of cellular electrophysiology, both models are fitted to human of the mutant troponin. All the mutations significantly affected electrostatic interactions experimental data. These cellular models are embedded within an aniosotropic model in troponin T. In K247R mutation, an electrostatic interaction between troponin subunits of the mechanical and electrical properties of the myocardial tissue. was also decreased. These changes are considered to alter troponin T movement on muscle Perturbing model parameters, fibre orientations and boundary conditions allows for activation. To verify this prediction of our simulation, we secondly carried out an x-ray the prediction of metrics of cardiac function that are sensitive to different parameters diffraction experiment, where native or mutant troponin subunits were introduced into or boundary conditions. This allows for the identification of parameters that can be skinned muscle specimens. With these specimens, the differences in the x-ray diffraction determined from different diagnostic modalities. patterns of resting and activated conditions were analyzed.

RS I-2-5 RS I-2-6 INTEGRATING ANALYSIS OF CELL AND TISSUE COUPLING COMPUTATIONAL MODELS ACROSS MECHANICS FOR DEVELOPING AN EQUIVALENT OF SCALES AND PHYSICS IN THE HEART HEART MUSCLE IN VITRO Nicolas Peter Smith, Jack Lee, David A Nordsletten, Steven A 1 2 2 Tetsuro Wakatsuki , Jozef Lazar , Howard J Jacob Niederer 1 2 Physiology, Medical College of Wisconsin, USA, Human Molecular Computing Laboratory, University of Oxford, UK Genetics Center The development and multi-scale coupling in the construction of whole- Cell Biology is rapidly advancing to integrate molecular and biochemical heart physiological models now underpins the simulation of a wide range studies to understand, predict, and control cell function. This advancement of cardiac function in a number of specific areas: (1) Coronary blood flow is to a large extent enabled by new technologies, including cell mechanics dynamics: the development of anatomically accurate vascular models of the assays, developed for analyzing cells cultured in 2D environment. Despite coronary tree from imaging data, the application of scale specific coronary this progression, the paradigm that defines the cell as the fundamental unit of hemodynamic models and their coupling with the contractile dynamics of life has significant limitations, including an artificial growth environment and lack of integration at the tissue/organ level. There are very few tools and little the myocardium. (2) Whole heart excitation-contraction: the integration of conceptual framework that can integrate cell biological analysis with data excitation-contraction cellular mechanisms in whole organ models where the obtained at the tissue/organ level. Due to this lack of the integration, studies regional mechanical environment feeds back on cell electrophysiology and, at the molecular and cellular level often incorrectly predict physiology of in particular, the intracellular calcium handling. (3) Whole chamber fluid- whole animals and humans. This discrepancy often delays discovery of solid mechanics: the coupling of three-dimensional, computational fluid, novel drugs. To overcome these challenges, we are developing tools and dynamics models of ventricular blood flow to cardiac contraction and the conceptual frameworks that describe regulation of mechanical systems, such interaction of wall stress, fluid movement and cardiac output. Through an as heart, by analyzing cells growing in 3D environments that mimic nature’ examination of recent computational modelling developments, we analyse s complexity. We will present a plan on developing a myocardium in vitro the significance of coupling mechanisms for the increased understanding of by precisely reproducing the complex distribution of various cell-types. cardiac function in each of these physiological domains. Challenges and solutions to achieve this ambitious goal will be discussed.

60 IUPS 2009 July 27 - August 1, 2009 in Kyoto RS I-3-1 RS I-3-2 SENSING OF ENERGY AVAILABILITY BY THE AMP- ENERGY BALANCE IN REGULATION OF INSULIN ACTIVATED PROTEIN KINASE SENSITIVITY IN MUSCLE: GENDER ASPECTS David Grahame Hardie, Andrew McBride Bente Kiens Division of Molecular Physiology, University of Dundee, UK Department of Human Physiology, Exercise and Sports Science, University AMP-activated protein kinase (AMPK) has a catalytic α subunit and of Copenhagen, Denmark regulatory β and γ subunits. It acts as an energy sensor that regulates energy Intralipid infusion in matched female and male volunteers resulted in a balance at the cellular and whole body levels. Metabolic stresses such as similar decrease in insulin sensitivity in both sexes on a whole body level muscle contraction cause an increase in ADP:ATP that is amplified by which largely could be ascribed to a decrease in insulin stimulated glucose adenylate kinase into a larger increase in AMP:ATP. AMP binds to two sites uptake (ISGU) in skeletal muscle. The decrease in ISGU in muscle was not on the γ subunit of AMPK, displacing ATP. This causes kinase activation, associated with changes in activity or phosphorylation of key-proteins in both by allosteric activation and by inhibiting dephosphorylation at the the muscle insulin signalling cascade (PI3K, Akt, AS160) or in muscle lipid critical phosphorylation site on the α subunit (Thr-172), which in muscle is intermediates during a hyperinsulinaemic, euglycemic clamp compared constitutively phosphorylated by LKB1. Thr-172 can also be phosphorylated to the control situation. It was speculated if the decrease in insulin action by the Ca2+/calmodulin-dependent kinases (CaMKKs), of which the α after intralipid infusion was due to the lipids infused or to the energy excess isoform is expressed in skeletal muscle. Thus, AMPK may be activated during the infusion. In a follow up study, the influence of types of nutrients during muscle contraction via two mechanisms, a feed-forward effect (unsaturated, saturated and carbohydrates, respectively) as opposed to energy mediated by Ca2+ and a feedback effect mediated by AMP. It then triggers surplus were evaluated in terms of insulin sensitivity in whole body as well many of the metabolic changes and adaptations induced by exercise. The β as in skeletal muscle. Our studies reveal that the decrease in whole body and subunit of AMPK contains a glycogen-binding domain, and we have recently in skeletal muscle insulin sensitivity on various diets seems to be due to the provided evidence that AMPK can sense the structural state of glycogen. This type of dietary lipids rather than energy surplus and the decreased insulin mechanism may ensure that glycogen is rapidly resynthesized following its action induced by lipids can not be ascribed to a change skeletal muscle depletion during exercise. insulin signalling.

RS I-3-3 RS I-3-4 METABOLIC SIGNALING IN MUSCLE INSULIN CROSS-TALK BETWEEN Ca++ SIGNALLING AND RESISTANCE; IMPLICATIONS OF AMPK AMPK IN SKELETAL MUSCLE Sebastian Beck Jorgensen Erik A. Richter Molecular Physiology Group, Section of Human Physiology, Department of Department of Exercise and Sport Sciences, University of Copenhagen, Exercise and Sport Sciences, University of Copenhagen, Denmark Denmark The AMP-dependent protein kinase (AMPK) is activated by metabolic AMP-activated protein kinase (AMPK) is a phylogenetically conserved fuel- stress and aids in maintaining the cellular energy homeostasis by regulating sensing enzyme that is present in all mammalian cells. During exercise, it is several metabolic pathways including lipid β-oxidation. AMPK is in addition activated in skeletal muscle at least in part by increase in the AMP/ATP ratio. activated by the hormones leptin and adiponectin suggesting that AMPK is a As well, during muscle contractions, cytosolic Ca++-levels are increased and potential key player in leptin and adiponectin controlled metabolic events in are thought to regulate muscle metabolism as well in particular by activation muscle. As accumulation of bioactive lipid species in insulin sensitive tissues of the Ca/CaM dependent protein kinase family (CaMK). Data support a has been linked to impaired insulin signaling and type 2 diabetes, AMPK prominent role of Ca++-signalling in regulation of protein synthesis and may modulate insulin sensitivity by promoting lipid oxidation and protect fatty acid oxidation during muscle contractions and to a lesser extent glucose against lipid accumulation. In addition, AMPK has also been reported to uptake. In addition CaMKK may act as an upstream AMPK kinase in muscle. interact directly with the insulin signaling pathway by phosphorylating IRS-1 In mice over-expressing a dominant negative AMPK construct, glucose and by impairing mTOR signaling towards the IRS-1 kinase p70S6K. I will transport during muscle contractions is decreased suggesting a role for Regular Symposia in the presentation focus on whether AMPK signaling is compromised with AMPK in regulation of glucose uptake in vitro whereas there is no apparent obesity and if a mouse model with defects in muscle AMPK are more prone effect on protein synthesis or fatty acid oxidation during contractions. It is to develop insulin resistance with obesity. The more directs interactions concluded that there is some, albeit limited, cross-talk between Ca++ and between AMPK, IRS-1 and the insulin signaling pathway will also be AMPK signalling in skeletal muscle but that each signalling pathway has addressed. significant impact on muscle metabolism.

RS I-3-5 RS I-4-1 CONTROL OF MUSCLE PROTEIN DEGRADATION MECHANISMS UNDERLYING AGE AND IMMOBILITY INDUCED BY STARVATION VIA THE PROTEASOME ASSOCIATED HUMAN MUSCLE WASTING AND LYSOSOME PATHWAYS Michael John Rennie Stefano Schiaffino, Eva Masiero, Cristina Mammucari, Marco School of Graduate Entry Medicine and Health, University of Nottingham, Sandri UK Department of Biomedical Sciences, Padova University, Italy In healthy active older people, there are few signs of derangements of basal muscle protein turnover but there are deficits in their ability to regulate Energy deficiency due to starvation stimulates protein degradation in skeletal the maintenance of muscle during feeding. The relationship between muscle that is essential to maintain glycemia via gluconeogenesis in the myofibrillar protein synthesis (MPS) and availability of essential amino liver. Starvation leads to activation of the two major pathways of muscle acids (EAA) is shifted down and to the right; giving large amounts of EAA protein degradation, the ubiquitin-proteasome and the autophagy-lysosome does not overcome this "anabolic resistance"(AR) . This is also shown by pathway. Experiments in cultured muscle cells and in vivo have shown that the decreased phosphorylation in signalling molecules in response to EAA the transcription factor FoxO3, which was found to control the ubiquitin- availability; this system is refractory to EAA provision irrespective of proteasome pathway (Sandri et al, 2004), has also a central role in the the availability of insulin, IGF-1 and growth hormone. However there is activation of the autophagy-lysosome pathway (Mammucari et al, 2007; Zhao blunting of the ability of older muscle to decrease proteolysis in response to et al, 2007). The effect of FoxO3 on autophagy is in part mediated by BNIP3 insulin. Recent N-balance data confirms that older persons’ dietary protein and is not dependent on proteasome activation. FoxO factors also control requirements are not increased so a retuning of the muscles' response is gluconeogenesis in the liver, thus appear to be master genes orchestrating the needed. One way might be resistance exercise. We have shown that the adaptation of the organism to starvation. To establish the role of autophagy dose response between MPS and resistance exercise intensity is also down in muscle protein turnover during starvation, we have generated a muscle- shifted with age. Decreased physical activity itself produces AR. A feature specific knockout of the autophagy gene Atg7. The mice are viable and of AR may be decrements in nutritive muscle blood. Such flow responses to preliminary observations indicate that the response to starvation is altered in feeding/exercise decrease with age and are restored on training. This may be these mice. associated with better muscle maintenance.

IUPS 2009 July 27 - August 1, 2009 in Kyoto 61 RS I-4-2 RS I-4-3 MUSCLE AND TENDON PLASTICITY TO LOADING, THE EXTRACELLULAR MATRIX AND ITS UNLOADING AND AGEING ADAPTATIONS TO CHANGE IN CHRONIC LOADING Marco Vincenzo Narici Michael Kjaer, Peter Magnusson, Charlotte Suetta, Simon Institute for Biomedical Research into Human Movement and Health (IRM), Dossing, Katja Heinemeier, Peter Schjerling, Mette Hansen, Manchester Metropolitan University, UK Mads Kongsgaard, Abigail Mackey, Jesper L Andersen, Henning Muscles and tendons show a remarkable plasticity in response to chronic Langberg loading, unloading and ageing. In recent unloading studies we showed Institute of Sports Medicine, Bispebjerg Hospital, University of that bed rest (BR) and unilateral lower limb suspension (ULLS) induce a Copenhagen, Denmark significant decrease in muscle fibre length (-8% after 23 day ULLS and 9% The adaptive response of connective tissue to loading requires increased synthesis and after 35 day BR). Concomitantly, tendon stiffness drastically decreased (30% turnover of matrix proteins, with special emphasis on collagen. Collagen formation after 23 days ULLS, De Boer et al. J Physiol 583, 2007). These changes are and degradation in the tendon rises with both acute and chronic loading, and a gender very similar to those found in ageing since both lower limb fibre length and difference exists, in that females respond less than males. Estrogen may contribute tendon stiffness are reduced in older humans (Narici & Maganaris, Exerc to a diminished collagen synthesis response in females. Conversely, stimulation of Sport Sci Rev 35, 2007). Instead, with increased loading, these myotendinous collagen synthesis by other growth factors can be shown in both animal and human changes are reversed (Reeves et al. Exp Physiol 91, 2006). The rapid muscle models where IGF-1 and TGF-b1 expression increases accompanying or preceding remodeling produced by these experimental paradigms (occurring within a rise in procollagen expression and collagen synthesis. The rise in tendon IGF-1 10 days of loading or unloading) seems regulated by changes in mechano- and procollagen expression showed similar response independent of the muscle sensitive proteins such as focal adhesion kinase whose content and activity contraction type, suggesting that strain rather that stress/torque determines the collagen increase with loading (Flueck et al. Am J Physiol, 1999) and decreases synthesis. The adaptation time to chronic loading is longer in tendon tissue compared with unloading (De Boer et al. J Physiol 585, 2007). Since skeletal muscle to contractile elements of skeletal muscle, and only with prolonged loading changes in mechanical behaviour notoriously depends on tendon stiffness, muscle gross dimensions are observed. An intimate interplay between mechanical signalling weakness in disuse and ageing and its reversal by training, likely reflects not and biochemical changes in the matrix is required, so that chemical changes can be only muscular but also tendinous adaptations. converted into adaptations in morphology, structure and material properties.

RS I-4-4 RS I-4-5 USE IT OR LOSE IT: DOES ACTIVITY MITIGATE EXERCISE AND HISTONE MODIFICATIONS IN HUMAN NEUROMUSCULAR SYNAPTIC DEGENERATION? SKELETAL MUSCLE 1 1 1 2 Richard R Ribchester Mark Hargreaves , Sean McGee , Erin Fairlie , Andrew Garnham 1 2 School of Biomedical Sciences, University of Edinburgh, UK Department of Physiology, The University of Melbourne, Australia, School of Exercise and Nutrition Sciences, Deakin University, Australia Some individuals diagnosed with amyotrophic lateral sclerosis (ALS) perceive benefits of increased exercise at early stages in their disease Exercise increases transcription of various genes in skeletal muscle. Gene progression. This raises a potentially important role for activity in transcription is highly dependent on local chromatin structure, which is remodelling, sustaining or strengthening neuromuscular synapses, since related to post-translational modifications of the histone proteins that form these compartments of the motor neurone show early signs of vulnerability the nucleosome core. Many histone modifications have been characterised, notably acetylation, methylation, ubiquitination and phosphorylation and degeneration in humans with ALS and in animal models. However,

Regular Symposia (Berger. Nature. 447: 407-412, 2007); however, it is generally recognised some reports suggest that exercise may be a risk factor for ALS; and that that acetylation of lysine residues within histone 3 (H3) is required for excitotoxicity, induced by synaptic activity, could exacerbate the vulnerability transcription initiation. In the present study, muscle (v. lateralis) samples of some motor neurones and accelerate their degeneration. We are testing were obtained before and after exercise (60 min, 77 + 2% peak pulmonary the potential benefits versus penalties of neuromuscular activity on motor oxygen uptake) to examine effects of exercise on H3 lysine acetylation. No nerve terminal degeneration, when triggered either surgically or by disease, change in acetyl-H3 (K9/K14) was observed (1.16 + 0.15 vs. 1.0 + 0.0 arb. in two genetically-modified animal models: WldS mice which express a units); however, there was a 64% increase (P<0.05) in H3K36 acetylation mutant protein that delays Wallerian degeneration; and SOD1G93A mice, after exercise (1.64 + 0.29 vs. 1.0 + 0.0 arb. units). It has been shown that a transgenic model of ALS. Our physiological assays are complemented H3K36 acetylation is a highly conserved histone modification, localised to by in vivo imaging using fibre-optic confocal microendoscopy. The data RNA polymerase II-transcribed genes (Morris et al. J. Biol. Chem. 282: thus far suggest that synaptic degeneration is modifiable by various point 7632-7640, 2007). Our results suggest that this H3 modification may also mutations and some of these add to the protective benefits of WldS; but a play a role in the exercise-induced increase in gene transcription. complementary protective role for activity remains uncertain.

RS I-4-6 RS II-5-1 TOWARDS A MOLECULAR MECHANISM FOR POSSIBLE INTEGRATING MECHANISM FOR 2+ CALCIUM-DEPENDENT EXCITATION-TRANSCRIPTION ACTIVATION OF CARDIOVASCULAR Ca ENTRY COUPLING IN SKELETAL MUSCLE TRPC6 CHANNEL BY LIPID MEDIATORS AND Enrique Jaimovich, Sonja Buvinic, Mariana Casas, Reinaldo PHOSPHORYLATION Figueroa, Isaac Garcia Ryuji Inoue Centro de Estudios Moleculares de la Celula, ICBM, Facultad de Medicina, Department of Physiology, Fukuoka University, Japan Universidad de Chile, Chile TRPC6 is a predominant isoform expressed in vascular smooth muscle cells Membrane depolarization of skeletal muscle cells induces, besides contraction, a (VSMCs) being implicated in regulation of vascular tone and remodeling 2+ slow Ca signal that depends on IP3 and regulates gene expression. In rat myotubes, via neurohormonal and mechanosensitive mechanisms. Although these a tetanic stimulus rapidly increased extra-cellular levels of ATP, ADP and AMP. two mechanisms are generally thought to work independently, our recent Exogenous ATP induced a dose-dependent increase in intracellular Ca2+, (EC50 of 7.8 investigation has revealed that TRPC6 channel can be activated synergistically

± 3.1 μM). We detected mRNA expression for P2X1-7 and P2Y1,2,4,6,11 in these cells. by receptor stimulation and mechanical forces, allowing very low concentrations Both fast and slow Ca2+ signals were partly inhibited by either 10-100 μM suramin of vasoconstrictors to effectively enhance the vascular reactivity in concert with or 2U/ml apyrase. Similar Ca2+ signals were observed in stimulated adult fibers; the mechanical stress via generation of 20-hydroxyeicosatetraenoic acid, a reportedly

slow signal was inhibited by 25 μM nifedipine, by the IP3R inhibitor Xestospongin-C potent vasoconstrictive lipid messenger. Furthermore, TRPC6 channels are 2+ (5 μM) and by 2U/ml apyrase. The amplitude of post-tetanic Ca2+ transients depended subject to both positive and negative regulations mediated by Ca /calmodulin-

on both tetanus frequency and duration. Immunofluorescence showed all three IP3R dependent kinase, and protein kinases C and G (PKG), respectively. Interestingly, isoforms in adult muscle and IP3R-1 was expressed with a mosaic pattern, being higher activation of PKG not only inhibited receptor-activation of TRPC6 channel but in a subset of fast-type fibers. ERK 1/2 phosphorylation after stimulation appears to also greatly attenuated its mechanosensitivity, and induced spontaneous opening. relate slow Ca2+ signals to transcription-related events. Nucleotides released during Disruption of actin cytoskeleton by cytochalasin D treatment mimicked these skeletal muscle activity may be acting through P2Y (and P2X) receptors to modulate consequences. It can be speculated that phosphorylation of TRPC6 channel by both Ca2+ homeostasis and muscle plasticity. PKG may regulate the transitions between differential activation modes. This FONDAP 15010006, Fondecyt 3080016, 1080120, PSD24 mechanism may act as an effective control for VSMC phenotype switching.

62 IUPS 2009 July 27 - August 1, 2009 in Kyoto RS II-5-2 RS II-5-3 2+ MOLECULAR IMAGING ANALYSES OF Ca SIGNALING PATHOGENIC ROLE OF ION CHANNELS IN UNITS REGULATING THE EXCITABILITY AND TONE PULMONARY HYPERTENSION OF VASCULAR SMOOTH MUSCLE CELLS Jason X.-J. Yuan, Amy L. Firth Yuji Imaizumi Department of Medicine, University of California, San Diego, USA Department of Molecular & Cellular Pharmacology, Nagoya City University, Japan Pulmonary vasoconstriction, vascular remodeling and in situ thrombosis are 2+ the major causes for the elevated pulmonary vascular resistance in patients The molecular imaging of Ca signaling units, which contribute to the regulation with idiopathic pulmonary arterial hypertension (IPAH). A rise in cytosolic of resting membrane potential, muscle tone and excitation-contraction coupling 2+ 2+ was obtained using a total internal reflection fluorescence (TIRF) microscope Ca ([Ca ]cyt) in pulmonary artery smooth muscle cells (PASMC) is a major in vascular smooth muscle cells (VSMCs) under whole cell voltage-clamp. trigger for vasoconstriction and an important stimulus for PASMC proliferation. Molecules in the Ca2+ signaling unit include ryanodine receptor (RyR), large Inhibited PASMC apoptosis also contributes to pulmonary vascular remodeling. 2+ + + conductance Ca activated K (BK) channel, caveolin-1, L-type voltage- In PASMC from IPAH patients, a downregulation of the voltage-gated K (Kv) 2+ dependent Ca channels and other associated components. Some of these channels (e.g., Kv1.5) with a correlating decreased Kv current amplitude has been molecules labeled with fluorescent proteins (YFP or CFP) were co- expressed in observed causing membrane depolarization and activation of voltage-dependent VSMCs. Other molecules were visualized by their BODIPY-ligands/fluorescent 2+ 2+ Ca channels. [Ca ]cyt is further elevated by increased store-operated channel dyes. Direct molecular interactions between some of them were clearly shown activity due to an upregulation of the transient receptor potential (TRP) channels. by FRET analyses with acceptor-bleaching methods. The significance of RyR Furthermore, we identified a single nucleotide polymorphism in the TRPC6 subtypes including type3 isoforms in VSMCs was also revealed. Taken together, geometric analyses based on TIRF imaging of the molecular assembly and Ca2+ gene in IPAH patients which converts to an NF-κB binding site in the TRPC6 sparks with concomitant measurement of spontaneous transient outward currents promoter region and increases the susceptibility to develop IPAH. These data 2+ give new insights into the physiological impact and the efficacy of the Ca indicate that decreased Kv channel function and expression and increased TRPC signaling units in the regulation of membrane excitability, tone and E-C coupling channel function and expression both contribute to sustained vasoconstriction in VSMCs. and excessive vascular remodeling in IPAH patients.

RS II-5-4 RS II-5-5 A KEY ROLE FOR KCNQ (KV7) POTASSIUM CHANNELS NON-SELECTIVE CATIONIC CHANNELS OF IN THE PULMONARY CIRCULATION VASCULAR DISEASE 1 2 Alison M Gurney , Shreena Joshi David John Beech 1 2 Faculty of Life Sciences, University of Manchester, UK, Faculty of Institute of Membrane and Systems Biology, University of Leeds, UK Medical and Human Sciences, University of Manchester, UK Vascular cells contain a variety of non-selective cationic channels that are The membrane potential of smooth muscle cells is an important factor in regulating Ca2+ influx and the contractile behaviour of vascular muscle. permeable to the key signalling ion, calcium. In recent years there has been Potassium channels are key determinants of the membrane potential and in expansion in knowledge of the molecular components of the channels and pulmonary arteries they have been implicated in the regulation of intrinsic recognition of their roles in the adaptive processes of vascular disease. Our tone, the vasoconstrictor response to hypoxia and the development of studies in this area began with E3-targeted antibodies, which can provide pulmonary arterial hypertension in both humans and animal models. The externally-acting and isoform-specific channel inhibitors. Antibody targeted + KCNQ (Kv7) family of voltage-gated K channels has recently attracted to TRPC1 protein partially inhibited calcium entry and suppressed neointimal interest as possible contributors to the membrane potential of vascular formation. Similarly, inhibition of TRPC5 suppressed cell migration evoked smooth muscle cells. In pulmonary arteries, drugs that block Kv7 channels are by the signalling lipid, sphingosine-1-phosphate. Further studies have shown potent vasoconstrictors while Kv7 channel activators have the opposite effect. TRPC channels are stimulated by a variety of lipid and redox factors. The In each case the effects can be explained by modulation of a background TRPC channels do not act alone, however. Auxiliary protein partners include K+ current, which changes the membrane potential recorded from isolated STIM1 and there are additional ion pore-forming proteins. In some cases, pulmonary artery smooth muscle cells. Furthermore, pulmonary arteries Regular Symposia express mRNA for KCNQ4, along with smaller amounts of KCNQ1 and ion channels are formed by the related TRPM proteins, but other classes of membrane protein may also be involved. Orai1, a member of the tetraspanin KCNQ5. This presentation will consider the evidence that Kv7 channels are key determinants of pulmonary vascular tone and that KCNQ activators family, is emerging as a previously unrecognised player in calcium entry of could provide useful pulmonary vasodilators for the treatment of pulmonary vascular cells. Recent progress in these areas will be described. Supported by hypertension. the Wellcome Trust and British Heart Foundation.

LS6 (RS II-5-related luncheon seminar) RS II-6-1 TRPC CHANNELS AS A NEW THERAPEUTIC TARGET THE HYPERTENSIVE BRAINSTEM FOR HEART FAILURE Julian F.R. Paton Motohiro Nishida Physiology & Pharmacology, University of Bristol, UK Department of Pharmacology and Toxicology, Graduate School of There is now substantial information from animal models of hypertension Pharmaceutical Sciences, Kyushu University, Japan (e.g. the spontaneously hypertensive rat; SHR) and humans with essential Structual remodeling of the heart, including myocardial hypertrophy, is hypertension that implicate an association with elevated sympathetic nerve a key determinant for the clinical outcome of heart failure. Activation of 2+ activity. Intriguingly, this occurs prior to the on start of hypertension in both Ca -dependent signaling pathways in cardiomyocytes by neurohumoral the SHR and man indicating a potential causal link. Based on physiological factors play a role in the development of pathological hypertrophy. It is and genomic results new insights into potential mechanisms within the believed that the sustained increase in intracellular Ca2+ concentration 2+ brainstem for generating excessive sympathetic nerve activity will be ([Ca ]i) for hypertrophy occurs via Gq-stimulated production of inositol- 2+ presented. I will show altered respiratory-sympathetic coupling in SHR 1,4,5-trisphosphate (IP ) and IP -meditaed release of Ca from intracellular 3 3 and how this can raise peripheral vascular resistance. In the SHR, I will store. However, we demonstrated that diacylglycerol (DAG)-sensitive transient receptor potential canonical channels (TRPC3 and TRPC6) are describe increases in brainstem vascular resistance, brainstem hypoperfusion 2+ resulting in borderline hypoxia. Based on differential gene expression responsible for agonist-induced increase in [Ca ]i, activation of nuclear factor of activated T cells (NFAT). As inhibition of either TRPC3 or TRPC6 between SHR and normotensive rats, I will show a shift from aerobic to completely suppressed agonist-induced hypertrophic responses of rat anaerobic metabolism and vascular inflammation within the brainstem. Some cardiomyocytes, TRPC3 and TRPC6 may form a heterotetramer channel. examples of the functional role of these differentially expressed genes will Furthermore, treatment with a TRPC3-selective blocker, Pyr-3, attenuates be demonstrated using in vivo gene transfer. Finally, I will introduce the pressure overload-induced cardiac hypertrophy in mice. As TRPC channels Cushing Mechanism as a plausible way to account for hypertension in the participate in pathological hypertrophy but not physiological contraction and SHR and man. the relaxation cycle, we suggest that DAG-sensitive TPRC channels are a new target for the treatment of hypertrophy.

IUPS 2009 July 27 - August 1, 2009 in Kyoto 63 RS II-6-2 RS II-6-3 CENTRAL ANGIOTENSIN TYPE 2 RECEPTOR: AN THE “SYMPATHETIC SIGNATURE" OF ANGII-SALT INHIBITORY MEDIATOR OF SYMPATHETIC DRIVE IN HYPERTENSION HEART FAILURE John W. Osborn 1 1 2 2 Lie Gao , Wei Wang , Hongwei Li , Colin Sumners , Integrative Biology and Physiology, University of Minnesota, USA 1 Irving H Zucker Hypertension caused by administration of angiotensin II (AngII) is 1Department of Cellular and Integrative Physiology, University of 2 exacerbated by a high salt (HiSalt) diet. Our studies suggest that this form Nebraska Medical Center, USA, Department of Physiology and Functional of salt-sensitive hypertension is, in part, neurogenically mediated. Whole Genomics, University of Florida, Gainesville, USA body norepinephrine (NE) spillover and arterial pressure (AP) are increased Elevated central AngII and AT1R signaling have been demonstrated as critical after 14 days of AngII administration in rats consuming a HiSalt diet mediators of sympatho-excitation in heart failure (HF). However, the significance compared to rats with a normal salt intake. This increase in sympathetic of central AT2R is unknown. Electrophysiolgical studies have shown that activation of AT2R’s increased potassium current. We hypothesized that AT2R’s in the RVLM nerve activity (SNA) appears to be targeted to the splanchnic vascular bed. exhibit inhibitory influences on sympathetic outflow. These experiments were Celiac ganglionectomy attenuates AngII induced increases in AP, peripheral carried out in HF rats induced by coronary ligation. We found that (1) AT2R protein resistance and mean circulatory filling pressure in HiSalt rats, consistent with expression in the RVLM was significantly downregulated in HF rats compared with sympathetically mediated increases in splanchnic arteriolar and venomotor sham. Activating RVLM AT2R’s with CGP42112 evoked sympatho-inhibition only tone. In contrast, renal SNA is decreased 30-40%, and renal denervation does in sham rats, but not in HF. Simultaneously stimulating RVLM AT1R and AT2R’s by not alter the hypertensive response to AngII in HiSalt rats. Finally, skeletal AngII induced sympatho-excitation in both sham and HF rats with a greater response muscle SNA is not affected by AngII in HiSalt rats as determined by direct in HF; (2) Overexpression of AT2R in the RVLM by AT2R adenovirus significantly recording of lumbar SNA and hindlimb NE spillover. We conclude that decreased blood pressure and urinary norepinephrine excretion in normal rats. (3) Chronic microinfusion of Compound 21, a newly created non-peptide AT2R agonist, AngII-salt hypertension in the rat is, in part, sympathetically mediated and is into lateral ventricle also evoked hypotension in normal rats. These results suggest that associated with a specific “sympathetic signature”: an increase in SNA to the activation of central AT2R’s inhibits sympathetic outflow and that an attenuation of splanchnic vascular bed, a decrease in SNA to the kidney and no change to central AT2R signaling contributes to the sympatho-excitation in the HF state. skeletal muscle.

RS II-6-4 RS II-6-5 SELECTIVE RENAL SYMPATHETIC ABLATION IN ACE2: A NEW TARGET FOR NEUROGENIC RESISTANT HYPERTENSION WITH A RENAL ARTERY HYPERTENSION RADIOFREQUENCY CATHETER Eric Lazartigues 1 1 2 Murray David Esler , Markus Schlaich , Rob Whitbourn , Henry Pharmacology & Experimental Therapeutics, Louisiana State University Krum3 HealthSciences Center- N.O., USA 1Cardiovascular Neuroscience Division, Baker IDI Heart and Diabetes Overactivity of the renin-angiotensin system (RAS) is involved in the 2 3 Institute, Australia, St. Vincents Hospital, Melbourne, Australia, Monash pathogenesis of Hypertension and a hyper-functioning brain RAS has University and Alfred Hospital, Melbourne, Australia been highlighted in several genetic and experimental models. Until now, The application of radioisotope dilution methodology, using infusion of tritiated Angiotensin (Ang)-II was thought to be the main actor of this system and noradrenaline and renal venous sampling demonstrates marked activation of the the main target for antihypertensive therapies. A new member of the RAS,

Regular Symposia renal sympathetic outflow in patients with essential hypertension. This is important in ACE2 (angiotensin converting enzyme type 2) has been identified in organs initiating and sustaining the blood pressure elevation. Renal sympathetic activation is and tissues related to cardiovascular function (e.g. heart, kidney, vessels) also a feature of some experimental forms of hypertension, in which selective surgical and appears to be part of a counter-regulatory pathway buffering the denervation of the kidneys ameliorates or abolishes the hypertension. Radical, non- excess of Ang-II. We recently identified the ACE2 protein in brain regions selective surgical sympathectomy (e.g. thoracolumbar sympathetectomy) antedated involved in the central regulation of blood pressure (BP) and showed that it pharmacological therapies, but was associated with an adverse side effect profile, regulates, and is regulated by, other components of the RAS. Here, we will (postural hypotension, bladder and sexual dysfunction). We have applied a selective renal denervation procedure in 45 patients with resistant hypertension (systolic present evidence for brain ACE2’s involvement in the central regulation BP>160 mm Hg on 3 or more drugs). A purpose-made radiofrequency catheter of BP, autonomic and cardiac function. We will show that lack of ACE2 is (ARDIAN Corporation, USA) delivered radiofrequency energy in the lumen of deleterious for the central regulation of BP and that brain ACE2 gene therapy both renal arteries (total 16-40 watts bilaterally). Efferent sympathetic denervation can restore baroreflex and autonomic functions and prevent the development was confirmed by substantial reductions in renal noradrenaline spillover (typically of Hypertension. Additionally and independently of Ang-II levels reduction, 25-85%). At 12 months follow up no renal artery damage was demonstrable with we will highlight some of the mechanisms responsible for the beneficial angiography or MRI. Mean BP reduction at 12 months was 27/17 mm Hg. effects of central ACE2 in cardiovascular function.

RS II-6-6 RS II-7-1 TRACTUS SOLITARIUS (TS) EVOKED THE ROLE OF REGULATORY PROTEINS IN THE NEUROTRANSMISION IN SECOND- BUT NOT IN CROSS-BRIDGE CYCLE IN THIN-FILAMENT HIGHER-ORDER NTS NEURONS IS DEPRESSED BY RECONSTITUTED SYSTEMS INTERMITTENT HYPOXIA IN RATS Masataka Kawai1, Xiaoying Lu1, Yusuke Oguchi2, Kristen J Stanton1, 1 2 3 Carlos Eduardo Lopes Almado, Benedito H. Machado, Ricardo M. Leao Michael W Wandling , Junji Ishizuka , Sarah E Hitchcock-DeGregori , Shin'ichi Ishiwata2 Department of Physiology, School of Medicine of Ribeirao Preto, University of 1 2 Sao Paulo, Brazil Anatomy and Cell Biology, University of Iowa, USA, Dept of Physics, Faculty of Science and Engineering, Waseda University, Tokyo, Japan, 3Dept Chronic intermittent hypoxia (CIH) induces several changes in the control of of Neuroscience and Cell Biology, Robert Wood Johnson Medical School, cardiovascular and respiratory systems. In the present study we evaluated possible Piscataway, NJ, USA alterations in synaptic transmission in the nucleus tractus solitarius (NTS) neurons The role of regulatory proteins in the cross-bridge cycle was assessed using the thin- of juvenile male rats submitted to CIH. Rats (21 days old) were exposed to 10 days filament reconstituted system in bovine skinned myocardium and an in-vitro motility of CIH, and after this period electrophysiological experiments were performed in assay. With myocardium, tension increased with addition of regulatory proteins Tm brainstem slices containing the NTS. TS-evoked glutamatergic post-synaptic currents and Tn, but the number of force-generating cross-bridges did not increase, implying (eEPSCs) were obtained by TS electrical stimulation. CIH selectively reduced the that force/cross-bridge is increased. With the in-vitro assay, both force/cross-bridge amplitude of the eEPSCs in second-order neurons (Con=284±34 pA, n=22; CIH=182 and the sliding velocity increased with Tm and Tn. This positive allosteric effect was ±18 pA, n=23; p<0.05) but did not affect the eEPSCs amplitude of higher-order NTS enhanced at higher temperatures without changing the force/cross-bridge, indicating neurons (Con=102±19 pA, n=27; CIH=113±16 pA, n=20). However, CIH produced that the hydrophobic interaction underlies the mechanism of force generation. With a no changes in the frequency-dependent depression or in the number of failures in Tm mutant d2Tm, in which the 2nd (res 47-88) of 7 periodic repeats was deleted, force second-order neurons. The amplitude of miniature eEPSCs in the presence of 25-75 did not change significantly (myocardium) or force and velocity diminished slightly μM CdCl2 in second-order neurons was not affected by CIH. The present findings (in-vitro assay). With another Tm mutant, d3Tm, which lacks period 3 (res 89-123), show that CIH selectively affects TS glutamatergic synaptic transmission on second- these parameters were reduced below the values with unregulated actin filaments. order NTS neurons by a pre-synaptic mechanism independent of changes in the The cooperativity of the pCa-tension curve was reduced in d3Tm, but the change was vesicular release probability. small in d2Tm. These results imply that the period 3 is essential for the allosteric effect Supported by FAPESP and CNPq. of Tm on the actomyosin interaction, but period 2 is not as important.

64 IUPS 2009 July 27 - August 1, 2009 in Kyoto RS II-7-2 RS II-7-3 THE REGULATION OF THIN-FILAMENT ACTIVATION THE REGULATION OF CROSS-BRIDGE KINETICS BY AND CROSS-BRIDGE CYCLE KINETICS BY THE SARCOMERE LENGTH PHOSPHORYLATION OF MYOFILAMENT PROTEINS Pieter P de Tombe Yasutake Saeki, Yoshiki Ohnuki Cell and Molecular Physiology, Loyola University Chicago, Stritch School Department of Physiology, Tsurumi University School of Dental Medicine, of Medicine, USA Japan The Frank-Starling law of the heart describes the interrelationship between The kinetics of thin-filament activation/inactivation, including the rates of end-diastolic volume and cardiac ejection volume, a regulatory system that Ca2+ binding and release from Tn, and the rate of cross-bridge cycling are now operates on a beat to beat basis. At the cellular level, sarcomere length (SL) recognized to be two major factors in determinaning the contraction profile of dependent myofilament Ca2+ sensitivity underlies this phenomenon (length cardiac muscle. The functional changes associated with the phosphorylation dependent activation-LDA). How information concerning SL is transduced of myofilament proteins by PKA and PKC have also been identified, yet the by the contractile apparatus of muscle is not known. A unifying theory has underlying mechanism remains poorly understood. We analyzed (1) the rate of recently gained acceptance that states that LDA is due to SL induced changes tension development and relaxation following laser flash photolysis of caged in inter-filament spacing (IFS); our recent data, however, indicates that compounds at various pre- and post-photolysis tension levels, and (2) the Ca2+- IFS does not underlie LDA. In contrast, we found that troponin (Tn), and dependence of tension and ATPase activity and the rate constant of delayed particularly cardiac TnI, plays a pivotal role in LDA. We now identify the tension response to step change in length before and after treatment with the critical region of TnI to be threonine-144. In addition, phosphorylation of the catalytic subunit of PKA in skinned ventricular trabeculae prepared from normal, serines 22,23 in TnI modulates LDA, while phosphorylation of threonine-144 chelerythrine (PKC inhibitor)-pretreated and 14-22 amide (PKA inhibitor)- does not affect LDA. Finally, changes in SL affect the disposition and pretreated rats. Our results can be well explained in terms of the changes orientation of relaxed, weakly bound cross-bridges as determined by x-ray in number of tension-generating cross-bridges through the Ca2+-dependent diffraction. Although the Frank-Starling Law of the Heart constitutes a cooperative thin-filament activation/inactivation kinetics. The thin-filament fundamental property of the heart that has been appreciated for well over a inactivation kinetics (i.e., relaxation phase) seems to be faster and more affected century, the molecular mechanisms that underlie this phenomenon are still by the phosphorylation of TnI and MyBP-C than its activation. incompletely understood.

RS II-7-4 RS II-7-5 MOLECULAR MECHANISMS OF MYOFILAMENT THE REGULATION OF CROSS-BRIDGE ACTIVATION- DYSFUNCTION IN HEART FAILURE RELAXATION KINETICS BY CALCIUM Ger Stienen, Jolanda van der Velden Corrado Poggesi Department of Physiology, VU University Medical Center, The Netherlands Department of Physiological Sciences, University of Florence, Italy Three major sources of contractile dysfunction can be distinguished in failing 2+ A major goal of striated muscle research is to understand which sarcomeric human myocardium: passive stiffness, Ca -sensitivity of force and the processes rate-limit or modulate the dynamics of contraction and relaxation maximum force generating capacity. under physiological and pathophysiological conditions. Previously, models In failing myocardium, a reduction in passive stiffness has been observed, of regulation and contraction kinetics were tested with pooled data from which could be attributed to a change in the distribution from the stiff biochemical studies on isolated proteins and mechanical studies on skinned (N2A) to the more compliant (N2BA) titin isoform. Passive stiffness in muscle fibres. The gap between the protein and fibre level is now bridged by cardiomyocytes from patients with diastolic dysfunction was increased, but recent techniques that allow kinetic analysis in single myofibrils. Myofibrils, could be corrected by protein kinase A (PKA) treatment. This indicates that formed of serially stacked sarcomeres, are easily isolated from skeletal hypophosphorylation of titin may cause an increase in passive stiffness. 2+ An increase in Ca2+-sensitivity of force development in end stage failing and cardiac muscle. They contain all essential proteins for Ca regulated contraction while their thin diameter enables us to abruptly change [Ca2+] myocardium also could be corrected by PKA. The disbalance of PKA- and 2+ PKC-mediated signal transduction in failing hearts observed, indicates that and investigate the kinetics of contraction and relaxation and their Ca altered PKC-mediated phosphorylation could be involved as well. regulation. Myofibrils are also suitable for experiments aimed at evaluating

In hypertrophic cardiomyopathy patients carrying truncation mutations in the impact of replacing thin filament regulatory proteins with exogenous, Regular Symposia cardiac myosin binding protein C (cMyBP-C), a reduction in the maximum genetically engineered, proteins. Myofibrils are a unique experimental force generating capacity and a deranged phosphorylation status of cTnI and model to dissect the cascade of dynamic processes determining sarcomere cMyBP-C have been observed. contraction and relaxation, including the Ca2+ controlled switch kinetics of These data show that altered phosphorylation of contractile proteins is an thin filaments and the crossbridge turnover kinetics with their apparent Ca2+ important cause of myofilament dysfunction in human heart failure. regulation.

RS II-7-6 RS II-8-1 THE IMPORTANCE OF INTERFILAMENT SPACING IN FUNNY CHANNEL-BASED PACEMAKING: FROM THE REGULATION OF CROSS-BRIDGE KINETICS IN CONCEPT TO CLINICAL APPLICATIONS THE IN SITU HEART Dario DiFrancesco 1 2 3 James Todd Pearson , Naoto Yagi , Daryl O Schwenke , Dept Biomolecular Sciences & Biotechnology, University of Milano, Italy Mikiyasu Shirai4 1 2 The “funny” (pacemaker, If) current is activated during the diastolic Department of Physiology, Monash University, Australia, Japan depolarization phase of an action potential and underlies spontaneous Synchrotron Radiation Research Institute, Japan, 3Department of 4 activity of cardiac pacemaker cells. In the three decades following its Physiology, Otago University, New Zealand, Department of Clinical Radiology, Hiroshima International University, Japan original discovery in 1979, a wealth of evidence elucidating the function of If in pacemaking has been gathered. The slope of diastolic depolarization, Under steady-state contractions the myosin interfilament spacing (IS) of cardiac muscle hence heart rate, is controlled precisely by the degree of If activation. If is in situ has been found to undergo cyclic increases of 2-3 nm during systole (Pearson et al. Circulation 109:2976-9, 2004). Using this same approach in combination with also efficiently modulated by intracellular cAMP, according to a mechanism ventricular pressure-volumetry we have shown that myosin IS plays a significant role mediating heart rate control by the autonomic nervous system. in modulating cross-bridge (CB) formation and the force developed before shortening The concept of f-channel based pacemaking has recently raised a growing in length-dependent activation. Now in this study we demonstrate that IS also plays a interest because of developments of clinical relevance. For example, role in positive inotropy, and that X-ray diffraction can be used to investigate regional some inheritable forms of sinus bradycardia and more complex types of heterogeneity in CB kinetics in hypertensive hypertrophy and chronic heart failure. arrythmias are associated with mutations of hHCN4 (the molecular correlate CB cycling was investigated during β-adrenoceptor stimulation with dobutamine. We of f-channels). Also, transfer of HCN channels by viral transfection, or of found that an increase in CB formation was correlated with a delayed IS increase, until spontaneously active HCN-expressing stem cells, to silent tissue is a valid after systole. Further, topical application of ryanodine completely inhibited the delay in IS change. Therefore, we speculate that enhanced CB formation and IS changes are protocol for development of "biological pacemakers". Finally, drugs acting modulated by Ca2+-regulated thick filament structural changes. We conclude that our as specific blockers of f-channels, such as ivabradine (now prescribed against in vivo approach has potential for investigating the roles of regulatory proteins in CB stable angina), are innovative tools for a pharmacological control of heart kinetics. rate devoid of cardiovascular side-effects.

IUPS 2009 July 27 - August 1, 2009 in Kyoto 65 RS II-8-2 RS II-8-3 NOVEL PERSPECTIVES ON CARDIAC PACEMAKER ION CHANNEL EXPRESSION AND PACEMAKER REGULATION BASED ON NUMERICAL MODELING ACTIVITY IN THE SINOATRIAL AND OF COUPLED SUBCELLULAR Ca AND MEMBRANE ATRIOVENTRICULAR NODES VOLTAGE OSCILLATORS Mark R. Boyett1, Joseph Yanni1, Tomoko Yamanushi2, 1 Victor A Maltsev, Edward G Lakatta Halina Dobrzynski 1 2 Laboratory of Cardiovascular Science, National Institute on Aging, NIH, Cardiovascular Medicine, University of Manchester, UK, Kagawa USA Prefectural College of Health Sciences, Japan Classical numerical models attribute normal pacemaker automaticity largely to We and others have studied ion channel expression (at the mRNA and protein interactions within an ensemble of sarcolemmal ion currents (membrane clock). levels) in the sinoatrial and atrioventricular nodes in various species including Recent experimental evidence, however, indicates that spontaneous rhythmic, local the rabbit, rat, human and mouse using techniques such as quantitative PCR, Ca releases (Ca clock) from sarcoplasmic reticulum (SR) accelerate the diastolic in situ hybridisation and immunohistochemistry. Ion channel expression in the

depolarization (DD) via activation of Na/Ca exchanger current (INCX) to ignite two nodes is similar, but it is specialised and different from that in the working membrane clock. We have explored the coupling of Ca and membrane clocks in a myocardium. For example, unlike in the working myocardium, in the nodal new numerical model of rabbit sinoatrial node cell that simulates known regulation tissues, there is evidence that expression of Kir2.1 is poor, whereas expression of of both clocks, including effects of cAMP/PKA-dependent phosphorylation. When HCN1, HCN4 and Cav1.3 is high, and this may explain the pacemaker activity experimentally documented shifts in G protein-dependent cAMP/PKA signaling are of the nodal tissues. During ageing and heart failure there is a decrease in the graded up/down, respective diastolic Ca releases occur earlier/later in the model. intrinsic heart rate and this can be explained by changes in the expression of ion

This leads to earlier/later activation of diastolic INCX, which leads to earlier/later DD channels and related proteins in the sinoatrial node. For example, in a rat model acceleration and, in turn, to faster/slower beating rate. Concomitantly, more/less of heart failure there is a decrease in the expression of transcripts for HCN4,

frequent ICaL activation that occur with rate changes, shifts cell Ca balance to increase/ Cav1.3 and NCX1 and this can potentially explain the bradycardia. The heart rate decrease the amplitude of the releases and of INCX, resulting in concomitant stronger/ of the human is ~10x lower than that of the mouse. Comparison of ion channel weaker DD acceleration. The model thus suggests a new paradigm for heart rate expression in the sinoatrial node of the two species suggests that this is the result regulation based on the coupled function of Ca and membrane clocks. of a lower expression of many ion channels in the human.

RS II-8-4 RS II-8-5 ABNORMAL IMPULSE GENERATION IN CANINE CONSTITUTIVE SUMOYLATION OF TRPM4 CHANNEL PURKINJE CELLS THAT HAVE SURVIVED IN THE ASSOCIATED WITH CARDIAC CONDUCTION DISEASE INFARCTED HEART Olaf Pongs, Martin Kruse Penelope A Boyden Institut for neuronal signal transduction, Center for Molecular Neurobiology, Pharmacology, Columbia Univ., USA Germany Triggered Purkinje ectopy can lead to initiation of serious storms of TRPM4, a member of the transient receptor potential melastatin (TRPM) ventricular arrhythmias in post myocardial infarction patients. We have family, encodes a calcium-activated non-selective cation (CAN) channel. developed the model of the isolated Purkinje cell aggregate from both the The TRPM4 channel matches the native cardiac CAN channel NSCCa, for 2+ normal (NZPC) and 48hr infarcted canine myocardium (IZPCs). These latter example ion selectivity, Ca -dependence, single channel conductance and Purkinje myocytes isolated from the subendocardial border zone are the open probability. The CAN-channel has been suggested to contribute to the Regular Symposia cells that initiate spontaneous ventricular tachycardias. While our original transient inward current (Iti) initiated by Ca-waves and to limit the driving studies defining the intense electrical remodeling in IZPCs revealed new force for Ca2+ entry in cardiac and other excitable cells. Analyzing gain-of- information, it was not sufficient to understand the enhanced abnormal function TRPM4-mutants causing isolated cardiac conduction disorders, pacemaking function of IZPCs. Our recent data on the fundamental changes our study identifies an essential role for CAN-channel activity in the cardiac in Ca2+ event activity in IZPCs will be discussed. Here we found that conduction system. Cellular expression studies showed that the mutations increased spontaneous Ca2+ release events in IZPCs are due to uniform attenuate deSUMOylation of the TRPM4 channel. The resulting constitutive regionally increased Ca2+ release channel sensitivity to Ca2+ without a change SUMOylation of the channel impairs endocytosis and leads to elevated in SR content. In addition, Ca2+ reuptake in IZPCs is accelerated. We suggest TRPM4 channel density at the cell surface. Increased cardiac CAN-channel that these properties lower the threshold of Ca2+ release channels, setting the activity is likely to slow or even blunt cardiac conduction because of its stage for the highly frequent arrhythmogenic cell wide Ca2+ waves observed effects on membrane depolarization and Ca2+ influx. This study thus defines a in IZPCs. The cell wide Ca2+ wave is arrhythmogenic in IZPCs. We will also previously unknown mechanism in heart block pathogenesis. discuss the pharmacology of these events.

RS II-8-6 LS11-1 (RS II-8-related luncheon seminar) SR Ca2+ OSCILLATIONS ARE REQUIRED FOR PHARMACOLOGICAL CONTROL OF HEART RATE BY "FUNNY” CHANNELS DIFFERENTIATION OF EMBRYONIC MOUSE 1 2 3 CARDIOMYOCYTES Dario DiFrancesco , Martin Biel , Richard B. Robinson , Jeffrey S. Borer4 Sandra Lynn Hanninen, Risto Rapila, Pasi Tavi 1Dept Biomolecular Sciences & Biotechnology, University of Milano, Italy, Department of Physiology, Institute of Biomedicine, University of Oulu, 2Department of Pharmacy, University of Munich, Germany, 3Department 4 Finland of Pharmacology, Columbia University medical Center, USA, Division of 2+ Cardiovascular Medicine and The Howard Gilman Institute, SUNY Downstate Sarcoplasmic reticulum (SR)-derived spontaneous Ca oscillations in developing Health Sciences Center and Medical School, Brooklyn and New York, USA cardiomyocytes generate the initial contractions that guide cell signaling pathways This seminar will address novel findings concerning cardiac automaticity/heart rate control, leading to the development of the functional, beating heart. To determine the to highlight the potential for translating basic knowledge to clinical therapy. contribution of SR Ca2+ signals to cardiomyocyte differentiation, pharmacological Formal discussions will include: inhibitors were used to block Ca2+ signals originating from the SR and sarcolemma. a) overview of basic mechanisms underlying rhythm generation, focusing on properties of Thapsigargin, cyclopiazonic acid, and tBu-BMQ (blockers of the SR Ca2+ ATPase, the “funny” (f)-channels, their involvement in the control of pacemaker activity, and the pharmacological control of heart rate by ivabradine by selective f-channel blockade without SERCA) significantly altered the genetic program of cardiomyocytes, with 2+ other cardiovascular effects; upregulation of calreticulin (embryonic SR Ca buffering protein) and downregulation b) regulation of HCN channels (the molecular components of f-channels) by cellular factors; 2+ of calsequestrin 2 (adult SR Ca buffering protein). Other cardiac-specific genes were use of mouse models to dissect the role of individual HCN subunits for pacemaking and their downregulated, including transcription factors necessary for cardiac development interactions with drugs; (GATA4, Nkx2.5, MEF2C), indicating a reversal of the normal gene expression c) development of a biological pacemaker by applying/organizing the HCN gene family, to pattern of developing embryonic cardiomyocytes. The Ca2+-handling of these cells create a durable cell based system operating at physiologically useful rates and sensitive to 2+ changes in autonomic tone and pharmacological intervention; was severely compromised, with little or no spontaneous Ca activity. The induction d) review of ivabradine application in randomized clinical trials and observational studies for of sarcolemmal calcium influx by electrical pacing of cells exposed to the inhibitors mitigation of angina, myocardial infarction and death in patients with CAD (including more did not rescue gene expression or Ca2+-handling, indicating that SR Ca2+ oscillations than 15,000 patients), for relief of heart failure and its sequelae (more than 6500 patients), are essential for cardiomyocyte differentiation. and planned studies to support additional clinical indications.

66 IUPS 2009 July 27 - August 1, 2009 in Kyoto RS II-9-1 RS II-9-2 CARDIAC TISSUE-A HETEROGENOUS SUBSTRATE ELECTRICAL INTEGRATION OF ENGINEERED HEART FOR ELECTRO-MECHANICAL COUPLING TISSUE Troy Baudino1, Thomas K Borg2 Wolfram Hubertus Zimmermann 1Dept. Cell Biology and Anatomy, University of South Carolina, Columbia, Department of Pharmacology, University Medical Center Goettingen, USA, 2Dept of Cell Biology and Anatomy, Medical University of South Germany Carolina, USA Myocardial tissue engineering aims at providing heart muscle equivalents for The development of the heart, from inception to death, involves dynamic cardiac repair, in vitro disease modelling, and drug-screening applications. interactions between the components of the extracellular matrix (ECM) Tissue engineered myocardium must resemble a functional syncytium and and the cellular components of the heart. A combination of outside-in and generate active systolic force to be of use for either of these applications. inside-out signaling between the ECM and the cellular components serves Engineered heart tissues (EHTs) can be generated by entrapping immature to coordinate the development of the myocardium, the fibroblast-connective myocytes and non-myocytes at a high cell density in collagen-hydrogels. tissue network, as well as angiogenesis. This dynamic communication takes This process can be facilitated by mechanical and electrical stimulation. place by a combination of mechanical, chemical and electrophysiological Eventually, EHTs resemble an anisotropically conducting syncytium capable signals via different families of receptors that regulate cell-cell and cell- of systolic force-development. In addition, EHTs have been exploited in ECM interactions. Cardiac myocytes vary in size and shape depending on experimental cardiac repair. This was so far mainly tested in a rat model their location in the heart, as well as under pathophysiological conditions. of myocardial infarction. More recent studies focus on the application of Closely associated with these changes are both the number and arrangement non-immunogenic/non-embryonic stem cells in cardiac tissue engineering. of cardiac fibroblasts as a network associated with the ECM and the myocyte. Ultimately, EHTs have to not only form a syncytium in vitro, but also be Angiogenesis is associated with the metabolic demands of the heart and is capable of electrical integration in vivo. Here I will discuss key prerequisites also intimately associated via cell-cell contacts with myocytes, fibroblasts for a therapeutic application of tissue engineered myocardium and speculate and ECM. The sum total of these heterogenous interactions is critical in on the advantages, but also potential disadvantages of electrical coupling of determining the electro-mechanical properties of the heart. tissue engineered heart muscle to diseased hearts.

RS II-9-3 RS II-9-4 EFFECTS OF PULSATILE STRETCH ON CELL-TO- MECHANICAL MODULATION OF CARDIAC CALCIUM CELL COUPLING IN CARDIOMYOCYTES IN VITRO HANDLING 1 2 Andre G Kleber , Jeffrey E Saffitz Gentaro Iribe 1 2 Physiology, University of Bern, Switzerland, Department of Pathology, Department of Cardiovascular Physiology, Okayama University, Japan Beth Israel and Diaconess Medical Center, Harvard Medical School, 2+ Boston, USA Cardiac mechanics influences Ca dynamics and electrophysiology via mechano-electric feedback. In myofilament level, stretch immediately Signaling mediated by adhesion of cells to the extracellular matrix regulates increases myofilament Ca2+ sensitivity, and muscle shortening decreases cell-to-cell coupling during the early phase of cardiac hypertrophy. Ca2+ sensitivity. Myocardial stretch also alters sarcoplasmic reticulum (SR) Modification of electrical coupling, connexin43 (Cx43), and adherence 2+ 2+ Ca handling. We investigated the effects of diastolic stretch on SR Ca proteins were assessed in cultures of neonatal rat cardiomyocytes exposed to handling, using carbon fiber technique. We found that diastolic stretch linear pulsatile stretch (LPS). Short periods of LPS produce an upregulation increases SR Ca2+ leak and uptake. The observation was also reproduced of Cx43, N-cadherin, plakoglobin and desmoplakin. Vascular endothelial 2+ growth factor (VEGF) and TGF-β released in response to LPS stimulates using our mathematical model of single cell activity. An increase in Ca leak would be expected to correspond to an increase in Ca2+ spark rate, which Cx43 expression in cardiac myocytes. Addition of either exogenous TGF-β 2+ or VEGF to unstretched neonatal rat ventricular myocytes produces effects we confirmed experimentally. This stretch-induced increase in Ca spark comparable to LPS. These results taken together show that very early rate requires an intact cytoskeleton. In membrane channel level, stretch modifies Ca2+ influx via stretch-activated ion channels, and then the altered (within 1 hour) stretch-induced up-regulation of Cx43 expression is strongly 2+ 2+ modulated by VEGF which acts downstream of TGF-β. Ca influx and/or membrane potential modify other Ca - and/or voltage- Regular Symposia In contrast to regulation of Cx43, acute LPS upregulates mechanical junction regulated channels. We have previously reported that a splicing variant of 2+ + proteins directly via integrin-mediated activation of focal adhesion kinases. A Ca -regulated K channels (SAKCA), cloned from cultured chick embryonic complex regulatory interaction between mechanical and electrical cell-to-cell ventricular myoycte and human cDNA library, also have stretch-sensitivity. coupling is suggested by recent findings in arrhythmogenic right ventricular As a whole, cardiac mechanics modulates excitation-contraction coupling in cardiomyopathies in humans. a complicated manner.

RS II-9-5 RS II-10-1 STRETCH AND ARRHYTHMIA CELLULAR AND MOLECULAR MECHANISMS OF Peter Kohl PERIPHERAL CHEMORECEPTORS Department of Physiology, Anatomy & Genetics, University of Oxford, UK Constancio Gonzalez, Teresa Agapito, Ana Obeso, Cardiac electrical and mechanical activity are linked by multiple feed- Ricardo Rigual, Asuncion Rocher forward and feed-back pathways. This talk will focus on the propensity of Biochemistry and Molecular Biology and Physiology, University of mechanical stimuli to cause acute alterations in the origin and spread of Valladolid Schoool of Medicine, Spain electrical excitation. Key mediators include mechanically-induced changes in The carotid body (CB) is formed by clusters of chemoreceptor cells connected ion channel activity and ion handling, both in myocytes and non-myocytes of synaptically with afferent sensory fibers of the carotid sinus nerve (CSN). the heart. Effects of mechanical stimulation on electrophysiology can range Chemoreceptor cells (ChC) sense variations of the arterial blood PO2 , PCO2 and [H]. from physiologically relevant adaptation in heart rate to the mechanical In hypoxia and hypercapnia/acidosis ChC increase their release of neurotransmitters induction of arrhythmias, and their mechanical termination. Experimental augmenting the activity in the CSN and, hyperventilation aimed to restore blood gases/ and clinical observations, from ion channel to whole organism, obtained and pH, ensues. analysed using wet (laboratory) and dry (computational) techniques will be Stimuli transduction cascades possess some links that remain unidentified at the discussed, and directions for future investigation highlighted. molecular level. First element in low PO2 transduction, the O2 -sensor remains unknown, existing several candidates to play the role of O2 -sensor (plasma membrane hemeprotein, reactive oxygen species, ion channels, hemeoxygenases). O2 -sensor(s) must be coupled to K channels, as it is known that low PO2 inhibits a variety of K channels in ChC; beta regulatory subunits are needed to couple the O2 -sensor to some K channels. Inhibition of K channels leads to ChC depolarization, activation of Na and Ca channels, entry of Ca and release of neurotransmitters. Several systems of second

messengers modulate the gain of the transduction cascade. CO2 /H-sensing would involve the same elements, although alternative models have been proposed. Grants: BFU2007-61848, CIBER CB06/06/0050 and GR242.

IUPS 2009 July 27 - August 1, 2009 in Kyoto 67 RS II-10-2 RS II-10-3 IDENTIFICATION OF SUBDOMAINS IN NADPH PERIPHERAL CHEMORECEPTOR REFLEX OXIDASE-4 (NOX-4) CRITICAL FOR THE OXYGEN- RESPONSES + DEPENDENT REGULATION OF TASK-1 K CHANNELS Prem Kumar 1 1 1 2 Sung Joon Kim , Su Jung Park , Yang Sook Chun , Si-On Choi , Department of Physiology, University of Birmingham, UK 2 2 Hye-Lim Kim , Jong Wan Park Peripheral chemoreceptor discharge is modulated by blood borne stimuli 1 2 Department of Physiology, Seoul National University, Korea, Department that include, not only, blood gases and pH, but also blood potassium, of Pharmacology, Seoul National University, Korea osmolarity and temperature as well as a number of hormones. Of these + Hypoxic inhibition of K current is a critical O2-sensing mechanism. Previously, it was stimuli, only changes in blood gases and pH are involved in the direct demonstrated that NOX4 mediates the hypoxic inhibition of TASK1. Here we further feedback modulation of ventilation, which seemingly precludes their having investigated the mechanism. In TASK1 and NOX4-coexpressing HEK293 cells, a role in the mediation of exercise hyperpnoea. Recent evidence, however, hypoxia (5% O2) decreased the current amplitude (hypoxia-ITASK1). To examine whether demonstrates how changes in reflex sensitivity to blood gas tensions, ROS mediate the response, we treated the cells with carbon monoxide (CO) to inhibit particularly carbon dioxide, may underlie the almost perfect matching the heme-containing NOX4. However, CO did not inhibit TASK but blocked the of ventilation to metabolism during exercise. Of greater pathological hypoxia-ITASK1. Hypoxia-ITASK1 was not recovered by H2O2 treatment. Next, we tested significance are the cardiovascular reflexes arising from chemoreceptor the possibility that the binding of O to heme per se controls TASK1. A heme synthase 2 stimulation. These appear dependent upon the concomitant ventilatory effort inhibitor, succinyl acetone noticeably attenuated the hypoxia-I , suggesting the TASK1 but may, for example, account for the hypertension of sleep apnoea and the participation of heme in the O2 sensing of TASK1. Also, we constructed and expressed various NOX4 mutants, such as HBD(-) lacking the heme binding domain, NBD(-) augmented sympathetic discharge associated with heart failure. In addition, lacking the NADPH binding domain, FBD(-) lacking the FAD binding domain, and chemoreceptor discharge and reflex chemosensitivity may be modulated in the adult by pre-term, in uterine events leading to the symptoms associated HFBD(-) lacking both heme and FAD domains. The hypoxia-ITASK1 was significantly reduced in HBD(-), FBD(-), or HFBD(-) expressing cells. However, NBD(-) did not with cardiovascular disease and diabetes. An important question that affect the TASK1 response to hypoxia. These results suggest that the heme moiety and remains to be answered is whether these various pathologies arise as a direct FBD, rather than NBD, are responsible for the NOX4 regulation of TASK1. consequence of carotid body dysfunction.

RS II-10-4 RS II-10-5 LOCALIZATION AND MECHANISIMS OF CENTRAL HYPOTHALAMIC CONTROL OF BREATHING CHEMORECEPTION Tomoyuki Kuwaki 1 2 3 4 Yasumasa Okada , Shun-ichi Kuwana , Shigefumi Yokota , Zibin Chen , Department of Physiology, Kagoshima University, Japan Yoshitaka Oku5 1Department of Medicine, Keio University Tsukigase Rehabilitation Center, Basal respiration and respiratory reflex regulations are considerably different Japan, 2Department of Physiology, Faculty of Health Sciences, Uekusa Gakuen during the awake and sleep states. Tidal volume and respiratory frequency University, Japan, 3Department of Anatomy and Morphological Neuroscience, diminish during sleep, and hypoxic and hypercapnic ventilatory responses 4 Shimane University School of Medicine, Japan, Department of Biochemical and also decline during sleep. Reduced metabolic demand during sleep cannot Analytical Pharmacology,GlaxoSmithKline, USA, 5Department of Physiology, Hyogo College of Medicine, Japan completely explain these phenomena because PaCO2 increases during The exact localization of the central respiratory chemoreceptor has not been determined, sleep. I will summarize our recent discovery of the possible contribution

Regular Symposia specific chemoreceptor cells have not been identified, and chemosensing mechansims have of orexin, a hypothalamic neuropeptide, to the vigilance-state-dependent not been elucidated. We conducted electrophysiological and optical recordings, and lesioning adjustment of central respiratory regulation. Orexin-deficient mice show an experiments to identify the chemoreceptive sites. We conducted immunohistological attenuated hypercapnic ventilatory response during the awake but not during examination of neurotransmitters and ion channels involved in chemoreception. We found the sleep period, whereas basal ventilation remained normal, irrespective that the central chemoreceptor is located mainly in the most superficial layer of the rostral ventral medulla. We propose a cell-vessel architecture model for the central chemoreceptor. of the vigilance state. Orexin supplementation remedied the defect, and the Primary chemoreceptor cells are glia that are located beneath large surface vessels within administration of an orexin receptor antagonist to wild-type mice mimicked the marginal glial layer, and surround fine penetrating vessels that branch from a large the abnormality. Orexin-deficient mice also showed frequent sleep apneas surface vessel. Some respiratory neurons in the rostral medulla are chemosensitive, but and loss of repetitive intermittent hypoxia-induced ventilatory long-term the preBotzinger complex is not chemosensitive in itself. Various potassium channels facilitation. Hence, it is possible that the orexin system is one of the essential are involved as chemosensing mechanisms, and acetylcholine plays an important role in modulators required for coordinating the circuits controlling respiration and information transmission. Changes in the perivascular PCO2/pH level affect the functional connectivity between the parafacial respiratory group and the preBotzinger complex, and behavior. even alter the mechanism of respiratory rhythm generation.

RS II-10-6 RS III-11-1 CENTRAL CHEMORECEPTION IN HEALTH AND IMPRINTED GENES AND PLACENTAL DEVELOPMENT DISEASE Abigail Lesley Fowden1, Philip M Coan1, Miguel Constancia2 Ronald M. Harper 1Department of Physiology, Development and Neuroscience, University of 2 Department of Neurobiology, David Geffen School of Medicine at UCLA, Cambridge, UK, Department of Obstetrics and Gynecology, University of USA Cambridge, UK Most of the imprinted genes identified to date are expressed in the placenta. Central structures responding to CO2 and O2 are located in multiple sites. Functional magnetic resonance imaging reveals that the deep cerebellar Generally, genes expressed solely from the paternal allele enhance placental size whereas, genes expressed solely from the maternal allele retard nuclei respond vigorously to CO and O . Other CO areas include the 2 2 2 placental growth. Dysregulation of imprinted gene expression by deletion, putamen and right insula, posterior thalamus and hypothalamus, the inappropriate silencing or loss of imprinting is implicated in sporadic, hippocampus, midline raphe, and dorsal pons. Cerebellar, thalamic and basal inherited and environmentally induced intrauterine growth disorders. In mice, ganglia sites, as well as limbic areas also respond to low O2. Patients with these growth abnormalities can be attributed, in part, to changes in placental Congenital Central Hypoventilation Syndrome (CCHS), a condition with development, particularly of the labyrinthine trophoblast responsible for reduced ventilatory responses to CO2 or O2, typically lose the drive to breathe materno-fetal nutrient transfer. One of the key imprinted genes involved in during sleep, but not to exercise or volition. Functionally impaired responses placental development is Igf2 which regulates growth of both the spongio- or injury appear in the cerebellum, posterior thalamus, hippocampus, basal and labyrinthine trophoblast. This gene also affects passive diffusion across ganglia, insula, ventral and medial midbrain, dorsolateral medulla and dorsal the placenta, in part, by altering placental morphology. The labyrinthine pons to CO2 challenges; impaired functional responses overlap structural specific transcript of the Igf2 gene is also involved in regulating glucose deficits. The cerebellum and basal ganglia likely mediate respiratory muscle and amino acid transport across the placenta by altering abundance of the coordination, while limbic sites may induce breathlessness to low O2 or high glucose and System A amino acid transporters. This transcript also mediates CO2, enhancing breathing, a perception lost in CCHS. Obstructive sleep the adaptative responses of the placenta to maternal undernutrition and to apnea patients also show cerebellar, insular cortex, and basal ganglia injury, mismatches between the placental nutrient supply capacity and the fetal which may underlie CO2 dysregulation in the condition. nutrient demands

68 IUPS 2009 July 27 - August 1, 2009 in Kyoto RS III-11-2 RS III-11-3 EARLY PATTERNS OF NUTRITION, GROWTH AND EPIGENETIC COMPONENTS OF PRE- AND METABOLIC HEALTH POSTNATAL MISMATCH 1 2 1 Caroline McMillen , Jaime A Duffield , Beverly S Muhlhausler , Mark Hanson1, Peter D Gluckman2 1 1 1 Leewen Rattanatray , Sheridan Gentili , Janna L Morrison 1Developmental Origins of Health and Disease Division - MAH is supported 1Early Origins of Adult Health Research Group, Sansom Institute,University by British Heart Foundation, University of Southampton, UK, 2Liggins of South Australia, Australia Institute, University of Auckland - PDG is supported by the National 2University of Adelaide, South Australia, Australia Research Centre of Growth and Development, New Zealand An extensive series of studies has shown that there is a U shaped relationship Epidemiological and animal studies show that small changes in the developmental between birth weight and adult fat mass, with a higher prevalence of adult environment, e.g. in nutrient balance, induce phenotypic changes affecting an obesity occurring in individuals with birth weights at the low and at the high end individual’s responses to their later environment and so risk of chronic disease, e.g. of the birth weight distribution. Hales and Barker coined the term the "thrifty metabolic syndrome or cardiovascular disease. Epigenetic processes mediate such phenotype" hypothesis, which suggested that when the fetal environment is poor, effects, through changes in DNA methylation targeted to promoter regions of specific there is a series of adaptive responses, which optimize the growth of key body genes in specific tissues, histone structure and miRNAs. The mechanisms may have organs to the detriment of others and lead to an altered postnatal metabolism, evolved through the adaptive advantage which they confer, rather than representing which is designed to enhance postnatal survival under conditions of intermittent extreme effects of developmental disruption. The advantage of a developmental cue or poor nutrition. It was proposed that these adaptations lead to metabolic inducing epigenetic effects and phenotypic change may extend to generations beyond dysfunction when postnatal nutrition is more abundant than in the prenatal the immediately affected pregnancy, and human and animal data support this concept. environment. During the past decade there has been a growing interest on the Such non-genomic inheritance can also be mediated via alterations in maternal effects of fetal overnutrition on the offspring in light of the increasing prevalence adaptations to pregnancy or behavioral influences. Epigenetic marks measured in early of obesity in women entering pregnancy. This presentation will focus on studies life could indicate an individual’s potential to be mismatched and the opportunity which have investigated the effects of exposure of the sheep embryo or fetus to for interventions before plasticity ceases. Research in this area is important for either low or excess nutrition on tissue growth and expression of metabolic target mechanistic understanding and for devising interventions to reduce the risk of chronic genes in the liver, muscle and adipose tissue. disease in subsequent generations.

RS III-11-4 RS III-11-5 SUPPRESSION OF FETAL CARDIOMYOCYTE INTERACTIONS BETWEEN PRE AND POSTNATAL PROLIFERATION NUTRITION AND THE EARLY ORIGINS OF OBESITY Kent L. Thornburg, Perrie O'Tierney, Natasha Chattergoon, RELATED DISEASE Samantha Louey, George D Giraud Michael Symonds, Sylvain P Sebert, D Sharkey, H Budge Heart Research Center, Oregon Health & Science University, USA Clinical Sciences, The University of Nottingham, UK In late gestation, fetal ovine cardiomyocytes go through a maturation process Global changes in nutrient intake either pre or postnatally have the potential called terminal differentiation, yielding a binucleated cell. Binucleated cells to reset an array of key regulatory mechanisms that markedly affect adipose cannot divide and once all cells are terminally differentiated, the heart loses tissue development through the life cycle. This includes the unfolding protein generative capacity. Excess diastolic and systolic pressure loads increase response that underpins the adipocytes response to stress and inflammation. proliferation and terminal differentiation rates and these hearts lose generative One critical window is the time at which adipoctye precursor cells first appear capacity before birth. While there are several hormones like IGF-1, cortisol in the fetus (i.e. early-to-mid gestation) that act to reset fat cell number in and Ang II that stimulate fetal cardiomyocyte proliferation, only two known early life. The adverse consequences do not then become apparent until the hormonal signals down-regulate cardiomyocyte growth―atrial natriuretic offspring become obese when they exhibit all the primary markers of the peptide (ANP) and L-triiodothyronine (T3). ANP is known to suppress run- metabolic syndrome, including central obesity, with pronounced appearance away hypertrophy in adult cardiomyoctes under stress conditions. We show of crown-like structures within adipose tissue, together with intra-organ that ANP also suppresses proliferation of myocytes that have been stimulated triglyceride accumulation in the heart and liver, responses not seen in obese by Ang II in vitro. T3 suppresses cardiomyocyte proliferation in vitro and controls. In contrast, increasing maternal food intake at specific stages of Regular Symposia in vivo and may stimulate the progression of maturation of cardiomyocytes gestation has little, if any, additional long term adverse outcomes to obesity. as well. The integration of the load and hormone regulation determines the Accelerated growth after birth, moreover, magnifies any fetal adaptation final heart cell mass and cardiomyocyte numbers at the time of birth. In rat resulting in hypertension, increased adult fat mass and leptin resistance on a models, the number of myocytes at birth determines the vulnerability for gender specific basis. The differential mechanisms mediating these responses infarct damage in adulthood and thus, the vulnerability for cardiac disease. will be discussed together with new opportunities to combat obesity.

RS III-12-1 RS III-12-2 MOLECULAR MECHANISMS UNDERLYING THE EARLY INTERGENERATIONAL TRANSMISSION OF INSULIN ORIGINS OF INSULIN RESISTANCE RESISTANCE Susan Ozanne Lucilla Poston Department of Clinical Biochemistry, University of Cambridge, UK Division of Reproduction and Endocrinology, King's College London, UK Epidemiological studies revealed relationships between poor early growth, Children born to women who have gestational diabetes are known to have type 2 diabetes and insulin resistance. Evidence suggests that early a higher risk of type 2 diabetes in later life. This appears not to be entirely environmental factors such as nutrition mediate these relationships. Studies due to an hereditary trait but also, at least in part, to the consequences of a of individuals in utero during famine showed a direct relationship between disturbed metabolic environment in utero or in early post natal life. Women maternal nutrition and offspring glucose tolerance. Studies of monozygotic who are obese are also often insulin resistant and there is growing evidence twins, discordant for type 2 diabetes, provide further support, showing to suggest their children are also at risk of obesity and insulin resistance in that diabetic twins had lower birth weights than non-diabetic co-twins. adulthood. Separating effects of acquired and genetically determined risk Animal models have further defined mechanisms linking early growth to adult disease. The most extensively studied is the maternal low protein of disease is always difficult in human cohort studies, and in this lecture model where rats are fed a low protein diet during pregnancy and lactation. the role that animal models have played in elucidating transgenerational Offspring have a low birth weight and develop insulin resistance and type influences of insulin resistance will be reviewed. In our laboratory we 2 diabetes. The insulin resistance is associated with changes in expression have induced maternal insulin resistance in rodents by dietary intervention of insulin-signalling proteins (e.g. protein kinase C zeta and the p110 beta with fat, or sugar and fat rich diets and have shown that offspring become subunit of PI 3-kinase). Strikingly similar changes are observed in tissue insulin resistant in later life, as well as developing obesity and raised blood from young men with a low birth weight. The molecular mechanisms by pressure. Expression of insulin signalling pathways is abnormal in the liver which a phenomenon that occurs in utero has a phenotypic consequence of the young offspring and may play a primary role in insulin resistance. To many years later are starting to emerge. These include epigenetic changes at conclude, there is increasing evidence that insulin resistance may be acquired the transcriptional and post-transcriptional levels. through an imbalanced metabolic environment in early life.

IUPS 2009 July 27 - August 1, 2009 in Kyoto 69 RS III-12-3 RS III-12-4 MEAL-INDUCED INSULIN SENSITIZATION: ROLE ROLE OF THE RENAL INSULIN RECEPTOR IN BLOOD IN THE PATHOPHYSIOLOGY AND PREVENTION OF PRESSURE CONTROL DIABETES Carolyn Mary Ecelbarger, Swasti Tiwari W. Wayne Lautt Department of Medicine, Georgetown University, USA Department of Pharmacology & Therapeutics, University of Manitoba, Insulin has been shown to be anti-natriuretic and increase sodium Canada reabsorption via the activation of a variety of renal sodium transporters and Meal-induced insulin sensitization (MIS) is demonstrated by the dramatic channels. Moreover, endogenous hyperinsulinemia and insulin infusion increase in insulin response that occurs following a meal. Absence of meal- have been correlated to increased blood pressure (BP) in humans and animal induced insulin sensitization (AMIS) leads to postprandial hyperglycemia, models. Insulin increases the activity of ENaC and apical localization hyperinsulinemia, hyperlipidemia and oxidative stress. AMIS represents of all 3 subunits (α-, β-, and γ-) in the collecting duct of mice and rats the earliest metabolic defect in the prediabetic state that leads to a in response to acute or chronic insulin. Nonetheless, the renal insulin progressive predictable series of dysfunctions, including generalized receptor (IR) is also coupled to vasodilatory signaling cascades including adiposity. AMIS increases progressively with age and is potentiated by a low dose sucrose supplement. A synergistic antioxidant cocktail, consisting Akt activation of endothelial nitric oxide (NO) synthase. We’ve shown of S-adenosylmethionine, vitamin E, and vitamin C, protects against the reduced renal IR protein, as well as, a reduction in the first step in signaling development of AMIS. Further, if a full state of AMIS is allowed to develop (IR-β phosphorylation) in the insulin-resistant, obese Zucker rat. This may as a result of a high sucrose supplemented diet, MIS can be restored to normal contribute to increased BP in this model. In support of this, we’ve also shown levels by targeting the signaling pathway that regulates the MIS process. In increased BP and impaired natriuresis in mice with knockout of the IR in order for a meal to lead to MIS, two feeding signals must be delivered to the the renal tubule from the thick ascending limb through the collecting duct. liver, the first being a parasympathetic reflex and the second being an elevation These mice also had reduced basal and insulin-stimulated NO excretion. of hepatic glutathione levels. Neither signal alone is sufficient to allow MIS to In aggregate, insulin in the kidney may be an important determinant of BP occur whereas mimicking both the parasympathetic signal (bethanechol) and the and sodium balance via a complex balance of anti-natriuretic and natriuretic elevated hepatic GSH signal (n-acetylcysteine) restores MIS. actions.

RS III-12-5 RS III-12-6 EFFECTS OF INHIBITING NONPROTEOLYTIC GLYCATION OF AKT1 BY METHYLGLYOXAL AND ACTIVATION OF INSULIN RESISTANCE IN FRUCTOSE- PROLIFERATION OF VASCULAR SMOOTH MUSCLE FED RATS CELLS Daisuke Nakano1, Yukiko Nagai2, Atsuhiro Ichihara3, Nicolas Pelisch4, Lingyun Wu, Tuanjie Chang 2 5 4 Yoshihide Fujisawa , Hirofumi Hitomi , Masakazu Kohno , Department of Pharmacology, University of Saskatchewan, Canada 4 5 3 5 Hideyasu Kiyomoto , Shoji Kimura , Hiroshi Ito , Akira Nishiyama The serine/threonine kinase B, also known as Akt, is an important 1Department of Radiology, Kagawa University, Japan, 2Life Sciences Research 3 stimulatory regulator of cell proliferation. Methylglyoxal (MG) is a highly Center, Kagawa University, Japan, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Japan, 4Department of Cardiorenal reactive dicarbonyl molecule, having been suspected as a causing factor for and Cerebrovascular Medicine, Kagawa University, Japan, 5Department of insulin resistance syndrome. In the present study, we found that proliferation Pharmacology, Kagawa University, Japan and DNA synthesis in cultured VSMC were significantly increased by MG Regular Symposia Recent studies have shown that a decoy peptide for prorenin/(pro)renin receptor binding (HRP) treatment (P<0.05). Application of SH-6, an Akt inhibitor, or silencing Akt1 inhibits the development of microvascular complications in type 2 diabetic animals; thus, we expression with siRNA abolished the effects of MG on cell proliferation investigated whether nonproteolytic activation of prorenin contributes to the development of and DNA synthesis. Phospho-Akt, phospho-Akt1 and Akt1 kinase activities fructose-induced insulin resistance. Rats were fed a standard diet (n=10), a 60% high-fructose diet were significantly increased by MG treatment, which was reversed by (n=16), or a high-fructose diet+HRP (n=16) for 10 weeks. Responses of plasma glucose and insulin levels to oral glucose loading were significantly greater in fructose-fed rats (189+/-25 mg/dL and SH-6 (P<0.05). Total p21 levels were decreased but CDK2 kinase activity 8.0+/-1.1 ng/mL; 10 min after glucose administration, respectively) than in standard diet-fed rats was increased in MG-treated cells, which were all antagonized by SH-6. (161+/-15 mg/dL and 4.8+/-1.1 ng/mL, respectively). HRP normalized this enhanced responses MALDI-TOF MS and MS/MS analysis revealed MG glycated a cysteine (151+/-13 mg/dL and 4.2+/-1.4 ng/mL, respectively, p<0.05). Moreover, HRP suppressed the residue located on the side chain of Akt1 protein in pleckstrin homology enhancement of prorenin activation and angiotensin II formation in the soleus muscle of fructose- domain. In conclusion, MG enhances VSMC proliferation by increasing fed rats (angiotensin II formation: standard diet; 25.8+/-4.2 fmol/g, fructose; 38.9+/-4.7 fmol/g, fructose+HRP; 19.6+/-2.0 fmol/g). These data suggest that local angiotensin II generation in skeletal Akt1 activity and subsequently altering p21 and CDK2 activity. Glycation muscle induced by nonproteolytic activation of prorenin contributes to the development of fructose- of Akt1 by MG constitutes a molecular mechanism for the pathogenesis of induced insulin resistance. vasculoproliferative diseases related to insulin resistance.

RS III-13-1 RS III-13-2 PRIMORDIAL GERM CELLS CONTAIN CELLULAR MECHANISMS OF REPRODUCTIVE SUBPOPULATIONS THAT HAVE GREATER ABILITY TO ORGAN FORMATION DEVELOP INTO PLURIPOTENTIAL STEM CELLS Richard R Behringer Yasuhisa Matsui, Yuko Tokitake Department of Genetics, University of Texas M.D. Anderson Cancer Cell Resource Center for Biomedical Research, Institute of Development, Center, USA Aging and Cencer, Tohoku University, Japan The gonads and reproductive tract of the mouse develop from the urogenital Primordial germ cells (PGCs) are undifferentiated germ cells in embryos. ridge. Initially, the fetal gonads are composed of a homogeneous mixture Previous studies indicated that a small portion of PGCs developed to of somatic and germ cells connected to the mesonephros. Testes will form pluripotential stem cells (EG cells) when they were cultured on a feeder layer cords composed of Sertoli and germ cells separated by an interstitium. Less expressing Steel factor with the culture medium containing LIF and bFGF. is known about ovarian differentiation. The Wolffian and Mullerian ducts We investigated a possibility that PGCs contained subpopulations that had the form within the mesonephros in both sexes but later differentiate into male greater ability to develop into EG cells, and we found that efficiency of EG or female reproductive organs, respectively. To visualize how these complex cell formation was higher in the PGCs showing negative or low expression organs form, we have devised a binary color code using red, green, and blue of the Integrin α6 than that in the cells with its high expression. To confirm fluorescent proteins to uniquely label specific cell types of the developing a possibility that a part of PGCs has higher ability to develop into EG cells, urogenital system. Our system visualizes nuclei, mitotic chromosomes, and we examine side polupation (SP) cells in PGCs. SP cells are detected as a the plasma membrane to assess cell position, proliferation, and shape. Tissue- cell population showing weaker fluorescence after staining by the Hoechst specific regulatory sequences are used to express these fluorescent reporters 33342 dye, and contain stem cells in various tissues. We found that about in various cell types of the developing urogenital system of transgenic mice. 60 % and 30 % of PGCs at E (embryonic day) 10.5 and E12.5, respectively Culture of these transgenic urogenital ridges combined with time-lapse showed characteristics of SP cells, and that efficiencies of EG cell formation microscopy has allowed visualization of live cells as they differentiate and of SP-PGCs at both stages were about two-fold higher than those of non-SP- interact to form the reproductive organs. These studies should lead to an PGCs. The results strongly suggest that PGCs contain subpopulations of cells understanding of the cellular behaviors and morphogenetic processes during having higher developmental potential for changing to EG cells. organogenesis.

70 IUPS 2009 July 27 - August 1, 2009 in Kyoto RS III-13-3 RS III-13-4 MOLECULAR PATHWAYS REGULATING GONADAL ROLE OF CAPILLARY BLOOD VESSELS AND DEVELOPMENT IN MICE MACROPHAGES IN FOLLICULAR DEVELOPMENT Peter Koopman Eimei Sato Genetics and Developmental Biology, Institute for Molecular Biosciences, Department of Animal Science, Tohoku University, Japan University of Queensland, Australia The genetic and molecular mechanisms that control the development of Different patterns of gene activity cause development of either testes or capillary blood vessels during follicular development are beginning to be ovaries from the same primitive tissue in the embryo. The cells of this tissue elucidated. Ovarian follicles contain and produce angiogenic factors that must be able to specialize for both male or female roles, depending on genetic may act alone or in concert to regulate the process of thecal angiogenesis. and hormonal signals received. These signals are easily upset, resulting in These factors are ultimately controlled by endocrine, paracrine and autocrine regulation. A recent study indicated that vascular endothelial growth factor either sexual ambiguity or complete sex reversal in some individuals. plays an important role in the process of thecal angiogenesis during follicular In mammals, Sry (sex-determining region Y gene) is the master regulator of development. Moreover, we are developing a novel technology for the male sex determination. Intriguingly, the mechanisms by which it mediates induction of follicular development using the technique of in vivo gene its developmental functions are still elusive, but it appears that Sry function administration. In addition, a few selected follicles complete growth and and hence the mechanisms controlling male sexual development are development for ovulation, whereas most undergo a degenerative process fragile, a situation that has profound biological, medical and evolutionary known as atresia at some stage in their development. Follicular atresia is implications. We find that in addition to pathways that promote male sex a key phenomenon by which the ovary eliminates follicles that will not differentiation, other pathways in the developing testis antagonize ovarian ovulate. Recently, CD 44 on macrophages is recognized as a key molecule differentiation. Other pathways active in the developing ovary antagonize for phagocytosis of apoptotic cells in the atretic follicles. These findings testis differentiation. The nature of these pathways, and the way they break may offer an innovative technique for enhanced induction of follicular down in disorders of sex development, will be described in this paper. development in the ovary, which may lead to prevention of infertility caused by ovarian dysfunction.

RS III-13-5 RS III-13-6 MATURATION OF HUMAN OVARIAN FOLLICLES IN HERITABLE IMPRINTING DEFECT CAUSED BY FETAL VITRO MALE GERM CELL CULTURE IN MICE Outi Hovatta Takashi Shinohara, Mito Kanatsu-Shinohara, Jiyoung Lee Department of Clinical Science, Technology and Intervention, Karolinska Molecular Genetics, Kyoto University, Graduate School of Medicine, Japan Institutet, Sweden Male germ cells undergo dynamic epigenetic reprogramming during fetal Human ovarian follicle culture would help women who have ovarian cortical development, eventually establishing spermatogonial stem cells that can tissue cryopreserved before cancer treatment. Orthotopic transplantation is convert into pluripotent stem cells. However, little is known about the one method to get mature oocytes from such tissue, but it has high risk of developmental potential of fetal germ cells and how they mature into re-transplantation of the malignancy in certain cases. In vitro maturation of spermatogonial stem cells. We herein developed a culture system for fetal oocytes from the cryostored tissue would be a safer option. Live offspring germ cells that proliferate for long periods of time. Male germ cells from have been obtained in mice, but not yet in human. Follicle cultures have embryos 12.5 to 18.5 days post-coitum could expand by glial cell line- also been an excellent model to study the regulation of early human folliculogenesis. We can activate follicular growth within slices of ovarian derived neurotrophic factor, a self-renewal factor for spermatogonial stem cortical tissue within one week, and within two weeks, the majority of the cells. These cells did not form teratomas, but repopulated seminiferous follicles have reached secondary, sometimes early antral stage. FSH, insulin, tubules and produced spermatogenesis, exhibiting spermatogonia potential. growth and development promoting factor (GDF9) and activin promote the However, the offspring from cultured cells showed growth abnormalities and were defective in genomic imprinting. The imprinting defect persisted in maturation and survival of the follicles, but antimullerian hormone inhibits Regular Symposia the initiation of growth. By isolating the follicles from the tissue after one both the male and female germlines for at least four generations. Moreover, week, the growth can be further improved, and formation of antral follicles germ cells in the offspring showed abnormal histone modifications and promoted. Up to eight week cultures have been feasible, but it seems that DNA methylation patterns. These results indicate that fetal germ cells have such long cultures are not needed if the cumulus-oocyte complex is removed a limited ability to become pluripotent cells and lose the ability to undergo at early antral stage for a third step culture. epigenetic reprogramming by in vitro culture.

RS IV-14-1 RS IV-14-2 IONOTROPIC GABA RECEPTORS CONTROL Cl ION MEDIATED GABA RELEASE FROM BERGMANN NEURONAL MIGRATION IN NEONATAL RAT GLIA IN DEVELOPING CEREBELLAR CORTEX CEREBRAL CORTEX Sachiko Yoshida 1 1 2 Heiko J. Luhmann , Werner Kilb , Atsuo Fukuda Department of Material Science, Toyohashi University of Technology, 1 Institute of Physiology and Pathophysiology, University of Mainz, Germany, Japan 2Department of Physiology, Hamamatsu University School of Medicine, Japan We observed GABA release from developing cerebellar cortex using GABase, We have previously shown that neuronal migration in neonatal rat somatosensory GABA degenerating enzyme, -linked and UV-LED illuminating GABA assay system. cortex can be influenced by the release of drugs acting on GABA-A receptors (Cerebral GABA was released in the external germinal layer (EGL) from the newborn cortex Cortex 17: 138-148, 2007). Elvax polymer implants positioned on the neocortex of to postnatal day 7 (P7). The cells in the EGL expressed GABA receptor (GABA R), newborn rats released the GABA-A receptor antagonist bicuculline or the agonist A A and were tonically activated by the ambient GABA with Patch-clamp recordings. After muscimol and both drugs caused severe neuronal migration disorders in vivo (i.e. P8, GABA observation was localized in the layers of GAD-expressing neurons, that heterotopia in upper cortical layers). Application of both drugs to organotypic neocortical slice preparations induced an increase in the migration speed in vitro. is, in both the molecular layer containing many GABAergic neurons and the upper the Blockade as well as desensitization of GABA-A receptors on migrating neurons internal granular layer corresponding to Golgi cells. profoundly influenced the migration process. GABA observation in the developing EGL was in accord with the distribution of Recently we investigated the role of GABA-C receptors in neocortical neuronal Bergmann glia which expressed vesicular GABA transporter, VGAT. This expression migration. The rt-PCR analysis of E18 mouse somatosensory neocortices was eliminated and localized in GABAergic neurons after P8. - - demonstrated the expression of the rho1 and rho2 subunits of the GABA-C receptor. GABA release was inhibited by low outer Cl concentration ([Cl ]o), NKCC1 inhibitor: Furthermore, calcium imaging experiments as well as patch-clamp recordings revealed bumetanide, and the anion transport inhibitor: DIDS. Especially the effects of low - a functional expression of GABA-C receptors in the intermediate zone. In organotypic [Cl ]o and DIDS were specific in developing period. In addition, GABA release was slices, blockade of the GABA-C receptors with TPMPA slowed down migration. controlled by GAT inhibitors, whereas a few GATs express in the developing EGL. Our results indicate that GABA-A and -C receptors control different stages of neuronal These findings suggest that extracellular GABA released from the Bergman glia may migration. be controlled Cl- and accelerate the cerebellar development.

IUPS 2009 July 27 - August 1, 2009 in Kyoto 71 RS IV-14-3 RS IV-14-4 MATERNAL TRIGGERS INHIBITORY KCC2 A SYNCHRONIZING FACTOR IN SYNAPTIC SWITCH IN FETAL NEURONS DURING THE BIRTH MATURATION Rustem Khazipov, Roman Tyzio, Yezekel Ben-Ari Claudio Rivera Inmed, Inserm U901, France Institute of Biotechnology, University of Helsinki, Finland Oxytocin exerts multiple effects in the adult central nervous system. A common assumption is that the maturation of both inhibitory and excitatory However, little is known about the effects of oxytocin on fetal neurons during delivery, at the time when a surge of oxytocin occurs. In a recent neurotransmission is tightly synchronised during development. However, study, the effects of oxytocin on GABA signalling have been reported in fetal little is known about the molecular mechanism involved. The neuron and newborn rats (Tyzio et al. 2006). In the immature rat hippocampal and specific K-Cl cotransporter KCC2 plays a pivotal role in the maturation neocortical neurons at birth, endogenous oxytocin induced a switch in the of GABAergic synapses. Its developmental expression sets the gradual action of GABA from excitatory to inhibitory. This excitatory-to-inhibitory shift in GABAA mediated responses from depolarizing to hyperpolarizing. switch was caused by a switch in the polarity of the GABAergic responses Interestingly, it is accumulated in dendritc spines which are the major targets from depolarizing to hyperpolarizing, reflecting a decrease in the intracellular for excitatory glutamatergic synapses. Our recent results show that KCC2 chloride concentration. The effects of oxytocin were mimicked and occluded plays a crucial role in the formation of drendritic spines and functional by bumetanide, a selective blocker of the chloride co-transporter NKCC1, glutamatergic synapses. Here, new results will be presented on the interaction suggesting that the effects of oxytocin involve inhibition of NKCC1. of KCC2 with the dendritic spine cytoskeleton as well as with important Neuronal death caused by anoxic-aglycaemic episodes was substantially intra-spine proteins with established functions in spine morphology and delayed in the fetal hippocampus by endogenous oxytocin. These findings suggest that oxytocin plays important role in the preparation of the fetal brain stability of glutamatergic synapses. These results further suggest that KCC2 to delivery. plays a key role in the developmental synchronization of GABAergic and Tyzio R. et al., (2006) Maternal Oxytocin Triggers a Transient Inhibitory glutamatergic neurotransmission. Switch in GABA Signaling in the Fetal Brain During Delivery. Science. 314, pp. 1788-1792.

RS IV-14-5 RS IV-15-1 PHYSIOLOGICAL SIGNIFICANCE OF TAURINE- CHARACTERIZATION OF P2X RECEPTORS IN SPINAL - MEDIATED Cl CONDUCTANCES DURING (TRIGEMINAL) MICROGLIA AND MACROPHAGES CORTICOGENESIS Seog Bae Oh1, Yong Ho Kim1, Joan A Sim2, Chul-kyu Park1, Atsuo Fukuda Alan North2 Department of Physiology, Hamamatsu University School of Medicine, 1National Research Laboratory for Pain, Department of Physiology, School Japan 2 - of Dentistry, Seoul National University, Korea, Faculty of Medical and GABA (γ-aminobutyric acid) depolarizes (Cl efflux) immature cortical neurons Human Sciences, University of Manchester, Manchester, Lancashire, UK even before synaptogenesis. It has been reported that continuous activation of Macrophages, including microglia in the central nervous system, play the GABAA receptor (-R) contributes to a tonic depolarization of the embryonic neurons, however, physiological details and roles are not well known. GABAA-R a crucial role in surveillance as the primary innate immune cells. P2X

Regular Symposia mediated tonic currents in embryonic cortical cells were GABA transporter- receptors are ionotropic receptors for ATP that acts as an extracellular insensitive, paradoxically, which could be recorded even in a deficiency of signalling molecule in inflammatory response and neuropathic pain, and ambient GABA (homozygous GAD67-GFP knock-in mice). Taurine transporter the expression of P2X receptor subtypes in macrophages and microglia is inhibitor induced inward shift of holding currents, which was reversed by known to markedly depend on their activation level. However, little is known a GABAA-R antagonist. When cerebral ambient taurine concentration in about functional expression of P2X receptors in resident macrophages and homozygous embryos was reduced to 50% by maternally administering microglia at their ‘resting' state. We have demonstrated functional expression D-cysteine sulfinic acid, GABA -R-mediated tonic currents were disappeared A of P2X receptors in macrophages (BJP, 2007 Dec;152(8):1283-90). We are and radial migration of cortical plate cells was accelerated. The results indicate currently studying microglia of medullary dorsal horn (MDH, trigeminal taurine may be an endogenous agonist for embryonic tonic GABAA-R in the spinal nucleus subnucleus caudalis) at ‘resting conditions’ by using brain neocortex. Considering striking difference in distributions of GABA and taurine GFP/+ and indifferent tonic GABAA-R currents among genotypes of GAD67-GFP slices from CX3CR1 mice. After electrophysiological properties of knock-in mice, taurine of maternal origin might serve as a stop signal for radially MDH microglia are reviewed, characteristics of P2X receptors in MDH are migrating cortical plate cells. discussed by comparison with those of macrophages.

RS IV-15-2 RS IV-15-3 PURINERGIC SIGNALLING AND MICROGLIA NEURON-MICROGLIA INTERACTION MEDIATED BY Helmut Kettenmann MULTIPLE P2Y RECEPTORS Cellular Neurosciences, Max Delbrueck Center for Molecular Medicine Schuichi Koizumi1, Kayoko Fujishita1, Atsuhito Nakao2, Kazuhide Inoue3 (MDC), Germany 1Department of Pharmacology, University of Yamanashi, Faculty of Medicine, Microglial cells are the major immunocompetent cells in the brain and Japan, 2Department Immunology, University of Yamanashi, Faculty of Medicine, 3 express many features of monocytes. This includes signalling cascades well Japan, Department of Molecular and System Pharmacology, Graduate School described in the immune system involving chemokines and cytokines and of Pharmaceutical Sciences, Kyushu University, Japan their receptor systems. In the last years, we have provided evidence that ATP Microglia express multiple P2 receptors. We focused on P2Y receptors and is an important signalling molecule for microglial cells. We have described investigated their functional roles in regulation of microglial functions. With regard to functional purinergic receptors in microglia in cell culture and in microglia P2Y12 receptors, they were already reported to be involved in chemotaxis (Honda et from brain slices both adult and during development. ATP can be degraded al., J. Neurosci. 2001) and neuropathic pain (Tozaki-Saito et al., J. Neurosci., 2008). by specific ectoenzymes. Two types are prominently expressed by microglia, When neurons are damaged and leak ATP, microglia sense it by P2Y12 receptors to CD39 which degrades ATP to AMP and CD73 which degrades AMP to migrate. However, functional roles for P2Y6 and P2Y2 receptors in microglia remain adenosine. We have recently shown that microglial migration requires the to be clarified. Here, we show their roles in relation to neuronal damage. We found that costimulation of ATP and adenosine receptors and that CD39 is essential when microglia are activated and surrounded the damaged neurons, they upregulate for generating adenosine from ATP (Faerber et al., 2008). We are currently P2Y6 receptors, by which microglia sense UDP, a ligand for P2Y6 receptors, leaked exploring the importance of these two enzymes for ATP mediated functions from dying neurons and start to phagocyte to remove damaged neurons or debris. We in microglial cells. also investigated the mechanisms underlying upregulation of P2Y6 receptors, and found that induction of TGFbeta is responsible. We further asked the roles of P2Y2 receptors, and found that they are also upregulated when neurons are damages and are involved in their proliferation. Taken together, multiple P2Y receptors in microglia control different functions independently, thereby leading to regulation of brain functions or repairs when neurons are damaged.

72 IUPS 2009 July 27 - August 1, 2009 in Kyoto RS IV-15-4 RS IV-15-5 MICROGLIAL PURINOCEPTORS IN THE SPINAL CORD PURINOCEPTORS IN MICROGLIA-NEURON AND PATHOLOGICAL PAIN SIGNALING IN NEUROPATHIC PAIN Makoto Tsuda, Hidetoshi Tozaki-Saitoh, Kazuhide Inoue Michael Salter Department of Molecular and System Pharmacology, Graduate School of Neurosciences & Mental Health, Hospital for Sick Children, Canada Pharmaceutical Sciences, Kyushu University, Japan Microglia in the dorsal horn of the spinal cord are increasingly recognized Neuropathic pain is a debilitating pain that occurs after nerve injury and as being crucial in the pathogenesis of pain hypersensitivity following injury is generally resistant to currently available treatments. Such pain involves to a peripheral nerve through activation of the P2X4 purinoceptor (Tsuda et aberrant excitability in dorsal horn neurons after nerve injury. Emerging al, Nature 424:778-783, 2003). PNI leads to increased intracellular chloride evidence indicate that the enhanced activity of dorsal horn neurons level and disinhibition of nociceptive neurons in lamina I of the dorsal horn requires a communication with microglia. Recent findings have shown (Coull et al, Nature 424:938-942, 2003). This increase in chloride is due to that activating P2X4R upregulated in spinal microglia after nerve injury brain-derived neurotrophic factor (BDNF), released upon P2X4 receptor contributes to neuropathic pain through a release of BDNF from microglia, stimulation from spinal microglia, acting via TrkB receptors on the lamina which is a crucial factor to signal to dorsal horn neurons to cause neuronal I neurons (Coull et al, Nature 438:1017-1021, 2005). Recently, we have hyperexcitability. Activated spinal microglia also express P2Y12R, and identified a unifying mechanism for P2X4-stimulated BDNF release through P2Y12R-KO mice display impaired neuropathic pain. The mechanisms an increase in Ca2+ and activation of p38-MAPK leading to the synthesis and of microglia activation are unknown, but our recent study shows that SNARE-mediated exocytotic release of BDNF from microglia. Targeting interferon-γ (IFN-γ) can be an important factor that causes spinal microglia this release pathway may open new therapeutic opportunities for neuropathic activation after nerve injury. IFN-γ upregulates P2X4R in microglia pain. and causes P2X4R-dependent allodynia. These findings suggest that Supported by the Canadian Institutes of Health Research, Neuroscience purinoceptors in spinal microglia is crucial for pathological intractable pain. Canada and the Howard Hughes Medical Institute.

RS IV-15-6 RS IV-15-7 NOVEL MECHANISMS OF CENTRAL PRESYNAPTIC P2X RECEPTORS AS ASTROCYTE- CHEMOSENSITIVITY NEURON INTERFACE Nicholas Dale Fusao Kato Biological Sciences, Warwick University, UK Department of Neuroscience, Jikei University School of Medicine, Japan Chemosensory transduction, allows the brain to sense and regulate the levels Physiological roles of the ATP-gated receptor channels (P2X receptors) in the 2+ of blood gasses such as CO2. At least one class of chemosensory cell at the brain remain undetermined. Their high Ca permeability makes it likely to ventral surface of the medulla releases ATP as a key step in the detection of be a presynaptic source of Ca2+ entry that triggers exocytosis. For example,

CO2. We have studied the mechanisms of CO2-dependent ATP release at the in the nucleus of the solitary tract (NTS) of the brainstem, space- and time- ventral surface of the medulla and have discovered a new principle of CO2 delimited ATP application (3-μm diameter; a few hundreds ms) onto dendritic chemosensory transduction. ATP is released via the direct CO2-dependent regions with laser photoactivation of caged ATP (Imura et al., 2007) triggers gating of connexin 26 (Cx26) hemichannels to mediate the adaptive action potential-independent glutamate release. The question here is the ventilatory response to elevated CO2. The Cx26 channels are located only intrinsic source of ATP that activates these receptors. Electronmicroscopic in the pia mater, the marginal glial layer and the walls of the superficial observation revealed a close apposition between excitatory presynaptic blood vessels. Interestingly there is a tone of ATP at the ventral surface of terminals and astrocyte processes. Pharmacological activation of P2Y1 2+ the medulla under physiological levels of PCO2 that will provide drive to the receptors increased intraglial Ca concentration especially in thin processes respiratory network. This tone comes from release through the Cx26 channel in GFAP-GFP mice and facilitated glutamate release in a manner sensitive which is sensitive to variations in PCO2 from 19 mmHg to 72 mmHg. The to P2Y1 receptor antagonist, P2X receptor antagonist and fluoroacetate Regular Symposia gating of this channel and subsequent release of ATP can thus underlie both in rat slices, suggesting implication of astrocytes. We propose that these responses to hypercapnia and hypocapnia. presynaptic P2X receptors in the NTS interface astrocytic syncytium and neuron network through intermediary of ATP release, thus enabling glial control of neuronal excitability in response to various humoral influences affecting astrocytes.

RS IV-15-8 RS IV-16-1 ASTROCYTE CALCIUM TRANSIENTS: NEURONAL ROLE OF SYNAPTIC ACTIVITY IN SPINE EFFECTS, DIVERSITY AND COMPARTMENTALIZATION MORPHOGENESIS Baljit Khakh, Eiji Shigetomi Tomoaki Shirao Physiology, University of California Los Angeles, USA Department of Neurobiology and Behavior, Gunma University Graduate School of Medicine, Japan The brain contains glial cells, which out number neurons by ten-fold. Long considered to provide only a passive role, increasing evidence now suggests Activity-dependent morphological changes in dendritic spines in the that specialized glial cells, called astrocytes, actively participate in normal mammalian brains underlie synaptic plasticity which is involved in higher brain function through interactions with neurons, and that these interactions brain functions such as learning and memory. Synaptic activity regulates are involved in the initiation or duration of epileptiform activity. However, spine morphogenesis, by modifying the actin cytoskeleton and PSD. Although multiple subtypes of the glutamate receptor play differential many fundamental and important aspects of astrocyte-neuron interactions roles in excitatory synaptic transmission, subtype-specific involvement remain unclear and/or experimentally unexplored. Astrocytes have the of glutamate receptors in spine morphogenesis is unclear. We show that capability to release neurotransmitters such as ATP and glutamate via glutamate-receptor subtypes differentially regulate spine morphogenesis exocytosis, but how astrocytic exocytosis is regulated and precisely how it during development by modulating the dynamics of an actin-binding protein, affects neuronal synaptic transmission is not fully understood. In this talk I drebrin. Spontaneous activation of AMPARs increased the stable fraction will present recent data on the neuronal impact of increasing calcium levels of drebrin within spines. Consequently, AMPAR activation converted long in astrocytes for the hippocampal CA1 region. In addition I will present headless filopodia into bulbous spines through facilitation of synaptic drebrin some of our recent data on near membrane and global calcium transients clustering. This shows a novel activity-dependent effect of AMPARs on in hippocampal astrocytes in culture and their relation to astrocyte calcium spine morphogenesis. In contrast, spontaneous activation of NMDARs transients in acute brain slices. decelerated the turnover of drebrin within spines and elongated spines. Our findings indicate that glutamate receptor subtype-specific regulatory mechanism of drebrin dynamics is important for the structural basis of synaptic plasticity.

IUPS 2009 July 27 - August 1, 2009 in Kyoto 73 RS IV-16-2 RS IV-16-3 A NOVEL NEUROTRANSMITTER PHENOTYPIC LOCAL TRANSLATIONAL CONTROL IN NEURONS PLASTICITY INDUCED BY ALTERED POSTSYNAPTIC AND ITS IMPLICATION FOR NEURAL PLASTICITY ORGANIZATION Nobuyuki Takei, Yuta Ishizuka, Hiroyuki Nawa Sebnem Tuncdemir1, Wu Xia1, Antoine Triller2, Gong Chen1 Department of Molecular Neurobiology, Brain Research Institute, Niigata 1Biology, Pennsylvania State University, USA, 2Biologie Cellulaire de la Synapse, University, Japan INSERM U789, Ecole Normale Superieure, France Activity-dependent local protein synthesis at the perisynaptic area is Neuronal transmitter phenotype is determined mainly by intrinsic transcription factors implicated in synapse-wide alterations of function and morphology. The during development. Recent studies suggest that extrinsic factors may also play a role molecular mechanisms of local protein synthesis, however, have yet to be but the underlying molecular mechanism remains largely unknown. In embryonic illuminated. We have shown that local protein synthesis and underlying hypothalamic and hippocampal cultures, inhibitory neurotransmission is mediated translational activation in neuronal dendrites were upregulated by brain- by GABAergic neurons only, although most neurons do express high level of GlyRs derived neurotrophic factor (BDNF), a most prominent trophic factor in the in addition to GABAA-Rs. Despite that no glycinergic currents can be detected in brain. In parallel, BDNF activates mammalian target of rapamycin (mTOR) pure neuronal cultures, functional glycinergic synapses are inducible in cocultured signaling pathways that leads to the enhancement of translation initiation HEK 293T cells expressing glycine receptors (GlyRs) and a cell adhesion molecule and elongation in dendrites. Inhibition of mTOR activity by rapamycin and neuroligin-2 (NL-2). Ectopic expression of NL-2 or GlyRs alone in hypothalamic knockdown of mTOR by siRNA completely suppresses the action of BDNF neurons cannot change GABAergic transmitter phenotype. However, coexpression on both translation signaling and actual protein synthesis rate. of NL-2 and GlyRs in hypothalamic neurons induces robust functional glycinergic The present results indicate that mTOR-dependent translation activation at synapses in hypothalamic cultures. Our data reveal that molecular manipulation of multiple processes is essential for the up-regulation of local protein synthesis postsynaptic receptor organization by cell adhesion molecules, not receptor expression in neuronal dendrites. We further showed the evidences that mTOR- per se, is critical in altering the neurotransmission phenotype. Such inducible plasticity dependent translational activation occurred during plastic event, such as LTP in central neurons also suggests that brain function is much flexible in adapting to and spatial memory paradigms in the CNS. As rapamycin canceled these extrinsic signals. effects, mTOR-dependent translational activation is essential for the certain Supported by NIH, NSF, and AHA to G.C. types of neural plasticity.

RS IV-16-4 RS IV-16-5 ATP (P2X1-3) AND α3β2 NICOTINIC ACETYLCHOLINE DREBRIN-MEDIATED ESTABLISHMENT OF CELL-CELL RECEPTORS INTERACT PHYSICALLY AND CONTACTS BY NEURONAL CONNEXINS FUNCTIONALLY IN HIPPOCAMPAL TERMINALS Irina Majoul1, Daria Onichtchouk2, Tomoaki Shirao3, Rainer Duden1 1 1 1 Rodrigo A Cunha , Teresa Almeida , Ricardo J Rodrigues , 1Institute of Biology, Centre for Structural and Cell Biology in Medicine, 2 3 4 3 University of Luebeck, Germany, 2Developmental Biology Unit, Department of Miguel Diaz-Hernandez , Jordi Aleu , Rafael Franco , Carles Solsona , 3 Maria-Teresa Miras-Portugal2, Francisco Ciruela4 Biology I, University of Freiburg, Germany, Department of Neurobiology and 1 Behavior, Gunma University Graduate School of Medicine, Maebashi, Gunma, Center for Neuroscience of Coimbra, Faculty of Medicine, , Portugal, 2Department of Biochemistry, Faculty of Veterinary, Japan Complutense University of Madrid, Spain, 3Laboratory of Molecular and Cellular During cellular migration in early development neurons and astrocytes assemble and Neurobiology, Department of Pathology and Experimental Therapeutics, dismiss their gap junction contacts. Earlier we showed that Drebrin (Developmentally 4 IDIBELL-Medical School, University of Barcelona, Spain, Department of REgulated BRain ProteIN) is a direct binding partner of the cytosolic part of Regular Symposia Biochemistry and Molecular Biology of the University of Barcelona, Barcelona, Connexin-43, (Cx43). Connexins form gap junction channels directly permeable Spain ++ to cAMP, cGMP, ATP, Ca , IP3, glutathione and other second messengers. Cx43 In the CNS, ATP and acetylcholine (ACh) are coreleased from nerve terminals. P2X and nicotinic together with other connexins (e.g. Cx36, Cx45) expressed in brain participate in the ACh receptors (nAChR) are present in the brain but they fail to mediated transmission, suggesting that they might function as modulation systems. P2XR and nAChR were enriched in hippocampal formation of neuronal circuits. Mobile astrocytes with their Cx43 are permeable to terminals, where both P2X1-3 and α3, but not α4, subunits are located in the active zone and co- dyes < 1kDa. Using interdisciplinary approach and GFP-transfected cells we show localized with dopamine-β-hydroxylase. Co-activation of P2XRs and nAChRs triggers a lower that actin-dependent clustering of Drebrin regulates lateral mobility of Cx43. We current than each receptor alone when Xenopus oocytes are transfected with P2X1-3R and α3β2 hypothesize that the connexin-drebrin-actin complex transduces signals to other (but not α4β2) nAChR. In HEK cells, P2X2R coimmunoprecipitate with α3β2 (but not α4β2) nAChR and the same occurs in the hippocampus. Furthermore, P2XR ligands displace nAChR components e.g. myosin-V motors to coordinate morphological changes at the cell-cell binding and nAChR ligands displace P2XR binding to hippocampal nerve terminal membranes. interface. We show that individual gap junction channels are not strictly autonomous Noradrenaline release from superfused hippocampal synaptosomes was enhanced by epibatidine and that coordination between opposite Cx43 hemi-channels is supported by Drebrin. (10-300nM, sensitive to α-conotonin MII) or β,γ-imido ATP (3-60μM, prevented by suramin but not In summary, actin-Drebrin-Connexin interactions are not only important for shaping reactive blue-2) and, again, P2XR antagonists prevent niconitic responses and, conversely, nAChR antagonists prevent P2XR responses. This indicates that P2X1-3R and α3β2 nAChR interact cellular membranes, but a mechanism by which neighboring cells can regulate strength physically and functionally to control transmitter release. (Supported by FCT) of gap junction permeability in connexin-mediated cellular networks.

RS IV-16-6 RS IV-17-1 MOLECULAR MECHANISMS OF SYNAPTIC A DECADE OF RESEARCH ON OREXIN STRUCTURAL PLASTICITY AND PATHOLOGY Takeshi Sakurai 1 2 2 Peter Penzes , Michael Cahill , Zhong Xie Molecular Neuroscience and Integrative Physiology, Kanazawa University, 1Physiology, Northwestern University, USA, 2Department of Physiology, School of Medicine, Japan Northwestern University, Chicago, USA Hypothalamic neuropeptides orexin-A and -B are critical regulators of Most excitatory synapses in the mammalian forebrain are located on dendritic sleep and wakefulness. They were initially recognized as regulators of spines. Spiny synapses undergo structural and functional modifications, feeding behavior, firstly because of their exclusive production in the which are crucial for brain development and function. Spine structural lateral hypothalamic area (LHA), a region known as the feeding center, changes have been observed during the induction of synaptic plasticity in and secondly owing to their pharmacological activity. Subsequently, the brain slices, but also in vivo during learning, in many behavioral situations, finding that an orexin deficiency causes narcolepsy suggested that these and in mental disorders. Indeed, spine morphology and number are altered hypothalamic neuropeptides also play a critical role in regulating sleep in many types of neuropsychiatric diseases, including schizophrenia and and wakefulness. Recent studies of orexin-producing neurons’ efferent and AD. Kalirin-7 is a GEF for small GTPase Rac1, and regulates postsynaptic actin cytoskeletal remodeling. Kalirin is a brain specific protein, enriched afferent systems, as well as phenotypic characterizations of mice with genetic in the forebrain, mainly cortex and hippocampus. In pyramidal neurons, alterations in the orexin system, have suggested further roles for orexin in the it is enriched in dendritic spines, and regulates spine morphology: its coordination of emotion, energy homeostasis, reward, drug addiction, and overexpression causes spine growth, its knockdown causes spine loss. arousal, suggesting that orexin neurons might provide a link between energy Kalirin-7 functions in several important pathways that regulate spine homeostasis and vigilance states. Orexin neurons are involved in sensing the development, maintenance, and activity-dependent plasticity. We will present body’s external and internal environments, and regulate states of sleep and data that support a key role for kalirin in multiple signaling pathways. Our wakefulness accordingly, which is beneficial for survival. I will discuss the new data support a role for a signaling pathway that includes NRG1, ErbB4, mechanisms by which the orexin system maintains sleep and wakefulness, and kalirin in controlling normal spine morphology in the cortex, which is and how this mechanism relates to other systems that regulate emotion, important for normal cognitive functions. reward, and energy homeostasis.

74 IUPS 2009 July 27 - August 1, 2009 in Kyoto RS IV-17-2 RS IV-17-3

SIGNAL COUPLING OF THE OX1 OREXIN/HYPOCRETIN THE OREXIN/HYPOCRETIN SYSTEM AS A NUTRIENT RECEPTOR SENSOR Jyrki P. Kukkonen Denis Burdakov Department of Basic Veterinary Sciences, University of Helsinki, Finland Department of Pharmacology, Cambridge University, UK

We, together with other groups, have shown primary coupling of the OX1 The electrical activity of central orexin/ypocretin neurons is directly receptor to three families of G-proteins. Downstream, diverse responses, silenced by glucose, providing a potentially important link between energy such as regulation of ion fluxes and adenylyl cyclase and activation of state and behavioural coordination. The underlying mechanisms are not phospholipases PLA2, PLC and PLD, are seen. Interestingly, also effects on well understood. Our new experiments show that the orexin/hypocretin cell fate and plasticity have been observed. glucosensors d isplay a novel sugar selectivity, detecting mannose, D-glucose, Ca2+ influx appears to be a central orexin receptor signalling pathway. and 2-deoxyglucose, but not galactose, L-glucose, fruc- tose or alpha- Though Ca2+ influx may be mediated by processes such as depolarisation methyl-D-glucoside. Furthermore, conventional glucose metabolism does or Ca2+ store depletion, there also appears to be a "direct" connection to not appear critical for orexin cell glucosensing: the effects of extracellular non-selective cation channels. The recently observed very high potency glucose on orexin cells are not mimicked by intracellular glucose or lactate, diacylglycerol production by orexin receptor-activated PLD and PLC may and are not affected by glucokinase inhibitors. We also show that orexin offer a possible route to the channel regulation. Another mechanism for Ca2+ cell glucosensing also exhibits an unusual temporal profile. About 70% of influx utilised by orexin receptors is reverse function of Na+/Ca2+-exchanger, orexin neurons are inhibited by glucose only transiently, self-restoring their + triggered by Na influx. PLA2-mediated arachidonic acid release may offer firing despite high sugar levels. The remaining 30% of cells display sustained 2+ yet a way for regulation of Ca influx and release by OX1 receptors. inhibition that follows the time-course of glucose application. The transient Though most of these discoveries have been made in recombinant expression and sustained glucosensor cells display significant differences in their ion systems, studies on native tissues show similar signaling. One major question channel expression. The implications of these findings for the control of for orexin receptor research therefore is, which of all the possible responses feeding and cognitive arousal, and for brain sensing of other indicators of are utilized in which tissues. body energy balance, will be discussed.

RS IV-17-4 RS IV-17-5 EFFECT OF OREXINS ON DUODENAL BICARBONATE THE NEUROBIOLOGY OF OREXINS (HYPOCRETINS) SECRETION AND ITS REGULATION AND NARCOLEPSY 1 1 2 Gunnar Flemstrom , Magnus W Bengtsson , Kari Makela , Seiji Nishino 2 Karl-Heinz Herzig Department of Psychiatry, Stanford University School of Medicine, USA 1 2 Department of Neuroscienc, Uppsala University, Sweden, Department of Using forward (i.e., positional cloning) and reverse genetics (i.e., mouse Physiology, Oulu University, Finland gene knockout), genes (i.e., prepro-hypocretin/orexin and hypocretin/orexin Orexins and receptors are expressed in peripheral tissues, including intestinal receptor genes) involved in the pathogenesis of narcolepsy in animals has mucosa. We have characterized effects of orexins on the bicarbonate secretion by been identified. Human narcolepsy, characterized by excessive daytime duodenal mucosa in anaesthetized rats. Close intra-arterial infusion of orexin-A sleepiness and abnormal manifestations of REM sleep, is a chronic sleep (60-600 pmol/kg,h) stimulated the secretion and, interestingly, over-night food disorder affecting 1:2000 individuals. The disease in humans is tightly deprivation abolished secretory responses. Food deprivation also decreased associated with HLA DR2 and DQB1*0602. Contrary to findings in animals, mucosal OX and OX mRNA expression as well as OX protein expression. To 1 2 1 mutations in hypocretin related-genes are extremely rare in humans, but further elucidate the action of orexins, we studied intracellular calcium signaling hypocretin-ligand deficiency (possibly due to the acquired cell death of in acutely isolated duodenal enterocytes. Orexin-A (1-100 nM) induced calcium signaling in enterocytes from continuously fed rats, and similar responses hypocretin containing neurons) is found in most narcolepsy-cataplexy cases. This discovery led to an establishment of new diagnostic tests (i.e. were observed in human enterocytes. The OX1R-antagonist SB-334867 (10 nM) attenuated the response. No responses occur in enterocytes from food- CSF hypocretin-1 measures) and is likely to lead to the development of 11 15 new treatments in human narcolepsy. Hypocretins are also involved in Regular Symposia deprived animals and the OX1R agonist (Ala , D-Leu )-orexin-B (1-10 nM) did not induce calcium signaling. In line with the findings in intact animals, various fundamental hypothalamic functions such as energy homeostasis, food deprivation decreased enterocyte OX1R and OX2R mRNA. Induction of neuroendocrine functions and stress responses. Thus, hypocretin-deficient duodenal electrolyte secretion and enterocyte calcium signaling is thus mediated narcolepsy now appears to be a more complex condition than a simple sleep primarily by OX1R receptors. Short food deprivation depresses receptor disorder. The progress in narcolepsy research, along with pathophysiological expression and markedly inhibits orexin-A induced secretion. aspects of hypocretin-deficient narcolepsy will be discussed.

RS IV-17-6 RS IV-18-1 IMPLICATION OF OREXIN (HYPOCRETIN) SYNAPTIC SYNAPTIC PHYSIOLOGY OF COCHLEAR HAIR CELLS TRANSMISSION ON SLEEP-WAKE CYCLES: A Paul Albert Fuchs COMPUTATIONAL STUDY Otolaryngology, Johns Hopkins University School of Medicine, USA Svetlana Postnova, Karlheinz Voigt, Hans A Braun Mechanosensory hair cells are electrically-compact and non-excitable Institute of Physiology, Philipps University of Marburg, Germany (in the sense of not generating overshooting action potentials), producing A generally accepted concept of sleep regulation postulates that sleep-wake transitions graded neurotransmitter release onto primary afferent neurons. The receptor result from the interaction between circadian and homeostatic processes. The circadian potentials of mechanosensory hair cells of the inner ear directly gate calcium process is ascribed to the activity of the suprachiasmatic nucleus of the hypothalamus, channels to alter the rate of neurotransmitter release. In return, many hair while the mechanism of the homeostatic process are still unclear. cells are subject to inhibitory synaptic regulation by efferent cholinergic In this study we present a concept of orexin (hypocretin)-based control of sleep neurons originating in the brainstem. Patch-clamp recording methods in ‘ex homeostasis. We propose that 1) high frequent impulse activity of the orexin neurons vivo’ inner ear preparations have revealed novel details of these synaptic during the wake state is sustained by reciprocal excitatory connections with other, mechanisms. For example, cholinergic inhibition of hair cells is mediated e.g. local glutamate neurons; 2) the transition to a silent state is going along with by ligand-gated cation channels related to nicotinic receptors of muscle. a weakening of the orexin synaptic efficacy. These mechanisms constitute a state- Inhibition results through the activation of associated calcium-dependent dependent, i.e. homeostatic process which can be synchronized with the circadian potassium channels that hyperpolarize and shunt the hair cell membrane. pacemaker. This concept has been realized in a mathematical model with Hodgkin-Huxley-type Afferent transmission has provided some surprises as well, with the hair neurons and physiology-based synapses. The model offers a new approach for further cell’s ribbon synapse capable of spontaneous multivesicular release whose evaluation of the physiological, especially homeostatic mechanisms of sleep-wake underlying mechanism remains unclear. This presentation will highlight new cycles on the basis of neuronal activity and synaptic transmission. findings that address the unusual synaptic mechanisms of hair cells. The work was supported by the through the Network of Excellence BioSim contract No LSHB-CT-2004-005137.

IUPS 2009 July 27 - August 1, 2009 in Kyoto 75 RS IV-18-2 RS IV-18-3 LOCALIZING HAIR CELL MECHANOTRANSDUCER TEAMING UP TO BOOST THE HAIR BUNDLE CHANNELS USING HIGH SPEED CALCIUM IMAGING AMPLIFIER IN SENSORY HAIR CELLS 1 1 2 2 Anthony John Ricci1, Maryline Beurg2, Jong-Hoon Nam3, Pascal Martin , Jeremie Barral , Kai Dierkes , Benjamin Lindner , 2 3 Frank Juelicher Robert Fettiplace 1 1 2 Laboratoire Physico-Chimie Curie, CNRS /Institut Curie / UMPC, France, Otolaryngology, Stanford University, USA, Inserm, Universite Victor 2 Segalen Bordeaux, Hopital Pellegrin, Bordeaux, France, 3Department of Max-Planck-Institute for the Physics of Complex Systems, Germany Physiology, University of Wisconsin, USA The dazzling sensitivity and frequency selectivity of the vertebrate ear rely Hair cells are the inner ear mechansensors that respond to vibration of their senory on mechanical amplification of small sound stimuli by hair cells. In an acute hair bundle by activating mechanically-gated channels (MT). Evidence suggests preparation of the bullfrog’s sacculus, the mechano-sensory hair bundle that the MT channels are near stereociliary tops and are opened by force imparted by tip adorns each hair cell can oscillate spontaneously and harness these active links connecting contiguous stereocilia, however, the exact locations remains to be movements to amplify its response to sinusoidal stimuli at frequencies near discerned. Fast confocal imaging of fluorescent signals associated with calcium entry that of the oscillation. Frequency-selective amplification relies on negative hair-bundle stiffness, myosin-based adaptation of the transduction process during bundle deflection was used to localize MT channels. Calcium signals were 2+ visible in single inner hair cell stereocilia and were up to ten times larger and faster and electro-mechanical feedback by the Ca component of the transduction in the second and third stereociliary rows than in the tallest first row. The number current. We have developed a physical description of active hair-bundle of functional stereocilia was proportional to MT current amplitude indicating about motility which provides quantitative agreement with experiments. Because two channels/ cilium. Comparable results were obtained in outer hair cells. The hair-bundle oscillations are noisy, a single hair bundle can afford only observations, supported by theoretical simulations, suggest there are no functional limited frequency selectivity and gain in sensitivity. In vivo, however, tens MT channels in first row stereocilia and imply the MT channels are present only at the of hair cells of similar characteristic frequencies are mechanically coupled bottom of the tip links. by accessory structures. By combining mechanical stimulation with a micro- This work was supported by NIDCD grants RO1 DC03896 to Tony Ricci and fiber and a dynamical-clamp technique, we demonstrate experimentally that DC01362 to Robert Fettiplace. Authors are in alphabetical order. We thank Redshirt both sensitivity and frequency selectivity of a single hair bundle are enhanced Imaging and Prairie Technologies for help in optimizing the confocal system. by elastic coupling to "cyber bundles" of similar characteristics.

RS IV-18-4 RS IV-18-5 LINKING DEAFNESS GENES TO HAIR-BUNDLE MAMMALIAN AUDITORY HAIR CELL REGENERATION DEVELOPMENT AND PHYSIOLOGY: THE ROLE OF AND COCHLEAR REPAIR HAIR-BUNDLE LINKS AND ASSOCIATED PROTEINS Stefan Heller 1 1 1 1 Christine Petit , Nicolas Michalski , Vincent Michel , Elisabeth Verpy , Elisa Departments of Otolaryngology and Molecular & Cellular Physiology, 1 2 1 1 Caberlotto , Lefevre Gaelle , Michel Leibovici , Dominique Weil , Stanford University School of Medicine, USA 1 3 4 5 Jean-Pierre Hardelin , Guy P. Richardson , Pascal Martin , Paul Avan Regeneration of cochlear hair cells is considered the ultimate remedy 1 2 Neuroscience, Institut Pasteur, France, Institut Pasteur, Unite de Genetique for mammalian hearing loss. Nevertheless, what appears to be a simple et Physiologie de l’Audition, Inserm UMRS587-UPMC, France / NIDKK, NIH, 3 replacement of a single cell type turns out to be a remarkably complex Bethesda, USA, University of Sussex, School of Life Sciences, Falmer, Brighton, UK, 4Institut Curie, Laboratoire Physico-Chimie, CNRS UMR 168, endeavor when one puts into account the very different functions of inner France, 5Universite d’Auvergne, Laboratoire de Biophysique Sensorielle, France and outer hair cells, as well as their precise integration into accessory Regular Symposia The identification of the genes underlying monogenic, early-onset forms of deafness in humans structures, such as the tectorial membrane, and the restoration of organ of has provided unprecedented insight into the molecular mechanisms of hearing. Most of the known Corti micromechanics. This situation is aggravated by the morphological de- genes enlighten the way the hair-bundle, the mechanotransduction (MET) device of the sensory differentiation of the cellular components of the organ of Corti, particularly in hair cell, is formed and operates. For each subsets of hair-bundle links, at least one component has cases of progressive hearing loss. Consequently, hair cell replacement cannot been identified thanks to deafness genes, in particular the genes defective in Usher syndrome type I & II. Evidence for colocalisation, direct in vitro interactions, and interdependence of localisation be viewed as simply seeding of new hair cells and getting them connected in the hair-bundle allowed us to include all the proteins (cadherin23, protocadherin15, harmonin, to the afferent auditory nerve. For proper restoration, hair cell regeneration sans and myosinVIIa) encoded by the Usher I genes in the same molecular complex underlying the needs to be conducted in context with extensive cochlear restoration, either formation and the anchoring to the stereocilia actin filaments of the early transient hair-bundle links. back to the original morphological configuration, or into an alternative design By morphofunctional analyses of mouse mutants, we showed the critical roles of these links in the featuring sensitivity and tonotopy. I will discuss the limitations of different early cohesion of the hair-bundle. Other links, the top connectors, revealed to be essential for hair- bundle production of sound waveform distortion. Finally, harmonin-b, a PDZ protein anchoring the approaches to repair the mammalian organ of Corti using transcriptional tip-link to the actin filaments, was shown to be involved in the MET adaptation process. regulators such as Atoh 1, abrogation of cell cycle inhibitors, stem cell Supported by EuroHear FP6 program approaches, as well as potential entry points for drug development.

RS IV-18-6 LS8 (RS IV-18-related luncheon seminar) THE MOLECULAR ASSEMBLY OF THE TIP-LINK ANIMAL MODELS OF NON-SYNDROMIC HEREDITARY FILAMENTS IN SENSORY HAIR CELLS DEAFNESS Hirofumi Sakaguchi1, Joshua Tokita2, Tomoki Fujita1, Katsuhisa Ikeda Toshihiro Suzuki1, Bechara Kachar2, Yasuo Hisa1 Department of Otorhinolaryngology, Juntendo University School of 1Department of Otolaryngology-Head and Neck Surgery, Kyoto Prefectural Medicine, Japan University of Medicine, Japan, 2Laboratory of Cell Structure and Dynamics, Recent studies of human deafness genes gave a great impact on auditory NIDCD, NIH, USA medicine. An animal model for hereditary deafness is quite useful for a Hair cells of the inner ear are mechanosensors that transduce mechanical better understanding of pathogenesis. Mutations in the Gjb2 gene, which stimulation arising from sound and head movement into electrochemical signals encodes gap junction protein connexin26, are the major cause of deafness to provide our sense of hearing and balance. Each hair cell contains a bundle in various ethnic groups. However, the pathogenesis of the hearing loss of stereocilia at the apical surface. Mechanoelectrical transduction (MET) caused by the Gjb2 mutations remains obscure. The characteristic phenotype takes place close to the tips of stereocilia in proximity to extracellular tip-link observed in the R75W transgenic mice was incomplete development of filaments that connect the stereocilia and are thought to gate the MET channel. the cochlear supporting cells, resulting in profound deafness from birth We demonstrate that two cadherins that are linked to inherited forms of deafness (Kudo et al., 2003; Inoshita et al., 2008). These characteristic features were in humans interact to form tip links. Immunohistochemical studies show that confirmed by a recessive form of Gjb2 mutation. No detectable responses cadherin 23 (CDH23) and protocadherin 15 (PCDH15) localize to the upper of distortion product otoacoustic emission were observed throughout the and lower part of tip links, respectively. The N-termini of the two cadherins co- postnatal days. On the other hand, the OHC developed normally with the localize on tip-link filaments. These findings are consistent with biochemical fine structures of the lateral wall and the presence of prestin motor protein. study performed in parallel with ours, showing that CDH23 homodimers interact The developmental change regarding the electromotility of the isolated in trans with PCDH15 homodimers to form a filament with structural similarity OHCs was observed. Thus, normal development of the supporting cells is to tip links. Our studies define the molecular composition of tip links and indispensable for the cellular function of the OHC. Development of animal provide a conceptual base for exploring the mechanisms of sensory impairment models of non-syndromic deafness will provide us with a promising tool to associated with mutations in CDH23 and PCDH15. create fundamental therapies.

76 IUPS 2009 July 27 - August 1, 2009 in Kyoto RS IV-19-1 RS IV-19-2 MECHANISMS OF DENDRITIC PEPTIDE RELEASE DENDRITIC SYNAPTIC CALCIUM SIGNALS IN THE Mike Ludwig AXONLESS OLFACTORY BULB GRANULE CELLS Centre for Integrative Physiology, University of Edinburgh, UK Veronica Egger The neuropeptides vasopressin and oxytocin are released directly into the Physiology, Ludwig-Maximilians-Universitaet Munich, Germany hypothalamus, and the major source of this central release is the dendrites of In the mammalian olfactory bulb, axonless granule cells (GCs) mediate the magnocellular neurones. Dendritic release within the hypothalamic nuclei self- and lateral inhibitory interactions between mitral/tufted cells via a does not necessarily parallel axon terminal release in the pituitary, and occurs dendrodendritic reciprocal synapse. Using two-photon imaging in acute semi-independently of spike activity in the soma and axons. Investigation rat brain slices and sensory-like stimulation, i.e. extracellular activation into this phenomenon resulted in the description of a novel mechanism of a glomerulus, we found direct evidence for three distinct forms of (priming) involving activation of intracellular calcium stores to trigger synaptic dendritic Ca2+ signals in GCs and identified molecular components activity-independent dendritic peptide release. Priming can contribute to a underlying these responses. form of functional plasticity by changing the nature of interactions between Weak stimulation generates synaptic input to isolated reciprocal GC spines, neurons and their inputs over a long time period. Many neuropeptides have which results in robust Ca2+ transients localized to the spine due to activation profound effects on behaviours that are exerted at sites that, in some cases, of NMDA receptors, voltage-dependent Ca2+ channels and Ca2+-induced express peptide receptors but are innervated by few peptidergic projections. Ca2+ release. Stronger synaptic input triggers a low-threshold spike (LTS) We believe that dendrites are a major source of peptides released in the brain; mediating Ca2+ influx via T-type Ca2+ channels throughout the GC dendrite, the abundance of peptides in the extracellular fluid mean that, after release, a candidate mechanism for subthreshold lateral inhibition in the olfactory they can diffuse to distant targets. At their targets, the process of priming bulb. Upon a synaptic action potential (AP), substantial Ca2+ influx is also allows peptides to functionally reorganize neuronal networks, providing a seen throughout the dendrite. The AP is followed by a plateau current that substrate for prolonged behavioural effects. provides further Ca2+ entry. In coincidentally activated spines, we observed summation of local and global Ca2+ signals for both LTS and AP. The variety of signals reflects the multifunctional nature of the reciprocal GC spine.

RS IV-19-3 RS IV-19-4 DENDRITIC LOCATION AFFECTS SYNAPTIC PROPERTIES OF TUFT DENDRITES FROM LAYER 5 INTEGRATION AND PLASTICITY VIA MULTIPLE PYRAMIDAL NEURONS MECHANISMS IN HIPPOCAMPAL CA1 PYRAMIDAL Jackie Schiller NEURONS Physiology, Technion Medical School, Israel Nelson Spruston, Dan Nicholson, Yael Katz, Vilas Menon, The layer-5 pyramidal neuron receives information from all cortical William L Kath, Jason Hardie layers through its elaborated dendritic arborization and conveys the main Neurobiology & Physiology, Northwestern University, USA output of the cortical hemisphere. Understanding the way this neuron is Excitatory synapses contact the dendrites of hippocampal CA1 pyramidal integrating its vast synaptic inputs is fundamental to understanding sensory- neurons at a variety of locations. We have used a combination of patch-clamp motor processing in the cortex. In the past decade direct patch-clamp recording in hippocampal slices, serial-section electron microscopy, and recordings were routinely performed from thicker portions of the dendritic immunogold localization of AMPA and NMDA receptors to study the properties tree. However, thin basal and tuft dendrites, which receive the majority of of these synapses in different dendritic domains of CA1 neurons. We have also synaptic inputs, were inaccessible to direct voltage recording and have only used computational modeling to develop testable predictions concerning how been investigated using indirect imaging methods. Recently, we used a novel inputs onto these dendritic domains are integrated and converted to output (action technique to record from the finest basal dendrites (<1 micrometer thick). potential firing in the axon) and used patch-clamp recording to understand This method which consists of using two-photon excitation fluorescence the mechanisms responsible for dendritic integration and plasticity in these microscopy with infrared-scanning gradient contrast opens now the way neurons. This lecture will describe the results of several studies that have led to a to study the yet unexplored tuft dendrites. In this lecture I will describe the working model of synaptic integration and plasticity in CA1 pyramidal neurons. passive and active properties of tuft dendrites recorded from neocortical layer Regular Symposia Specifically, we will present evidence that synaptic specializations and voltage- 5 pyramidal neurons. gated channels in CA1 dendrites are critical for ensuring that synapses on disparate dendritic domains can contribute to output via the axon and that some combinations of inputs are particularly effective for driving action potential firing or synaptic plasticity in these neurons.

RS IV-19-5 RS IV-19-6 DENDRITIC MORPHOLOGIY AND SIGNAL IMAGING DENDRITIC EXCITATION OF NEOCORTICAL CONDUCTION PROPERTY OF CORTICAL PYRAMIDAL CELL DENDRITES IN VIVO NONPYRAMIDAL CELLS Fritjof Helmchen, Narimane Benhassine 1 1 2 Yoshiyuki Kubota , Fuyuki Karube , Masaki Nomura , Department of Neurophysiology, Brain Research Institute, University of Toshio Aoyagi2, Yasuo Kawaguchi1 Zurich, Switzerland 1Division of Cerebral Circuitry, National Institute for Physiological Sciences, The apical dendrite of layer 5 pyramidal neurons can produce calcium based Japan, 2Kyoto Univ. Grad. School of Informatics, Kyoto, Japan regenerative electrical signals. We have developed two complementary The dendritic morphology of neurons greatly influences synaptic signal approaches for tracing dendritic calcium electrogenesis in layer 5 pyramidal integration. We analysed dendritic arborization and dimensions from four cortical neurons in vivo. First, we use electroporation of fluorescent calcium nonpyramidal (NP) cell subtypes: FS basket cell, Martinotti cell, double bouquet indicator in deep cortical layers in the somatosensory cortex of anestheszied cell, and large basket cell. The NPs were identified in isolated slices of young adult Wistar rats. This approach stains few layer 5 pyramidal neurons. rat frontal cortex by whole cell, current-clamp recording, and the subtypes by Using in vivo 2-photon microscopy we could reveal spontaneous calcium firing patterns, morphology and innervation characteristics. We analyzed the transients. To further characterize dendritic calcium transients we developed dendritic arborization with Neurolucida. Many dendritic segments from each a second approach for direct whole-cell patch clamp recording from apical subtype were reconstructed three-dimensionally from serial ultra-thin sections dendrites. Targeted recordings were achieved by staining the surrounding and the dimension of the dendrite was measured. We found rules to determine tissue with a red marker. The patch pipette could then be visually guided to the dendritic size. The cross sectional areas of dendrites were well correlated negatively stained apical dendrites. While backpropagating action potentials with summated length of the dendrites located between the measured point and recorded electrophysiologically did not gave rise to resolvable calcium end tips. At branch points, the cross-sectional area of the mother dendrite was transients, spontaneous dendritic calcium spikes could be identified from almost equal to the sum of those of the two daughter branches. We also analyzed the electrophysiological recording and two-photon imaging. At present, conduction property of synapse signal using computational model neurons got we use these approaches in identified columns of barrel cortex to elucidate into line with the morphological rules. Our simulation studies suggested that most the likelihood of layer 5 dendritic calcium action potentials under different synaptic inputs to these neurons should generate similar somatic depolarization. whiskers stimulus conditions.

IUPS 2009 July 27 - August 1, 2009 in Kyoto 77 RS IV-20-1 RS IV-20-2 CALCIUM CHANNEL REGULATION AND SHORT-TERM MODULATION OF PRESYNAPTIC CALCIUM SYNAPTIC PLASTICITY CHANNELS AT THE CALYX OF HELD William A. Catterall Tomoyuki Takahashi Pharmacology, University of Washington School of Medicine, USA Department of Neurophysiology, Doshisha University Faculty of Life and Medical Sciences, Japan P/Q-type calcium (Ca) currents conducted by Ca 2.1 channels initiate 2+ v In the nerve terminal, Ca entered through VGCCs triggers neurotransmitter synaptic transmission. These channels are regulated by binding of calmodulin release, and bi-directionally modulates synaptic transmission. At the calyx (CaM) to a bipartite site in the C-terminal domain composed of an IQ-like of Held synapse in rodent brainstem, Ca2+ inactivates VGCCs during its motif and a CaM binding domain (CBD). Brief local increases in Ca support influx, thereby contributing to synaptic depression during tetanic stimulation facilitation of Ca channel activity through Ca binding to CaM EF-hands 3 and (Forsythe et al, 1998). Ca2+ can also inactivate VGCCs via binding to 4 and interaction with the IQ-like motif of Cav2.1 channels. Sustained global calmodulin (CaM), thereby causing paired-pulse synaptic depression increases in Ca cause Ca-dependent inactivation through Ca binding to CaM at second order intervals (Xu & Wu, 2005). However, this mechanism EF-hands 1 and 2 and interaction with the CBD. Neuronal Ca sensor proteins operates only at immature synapses, or during tetanic stimulation, where 2+ 2+ CaBP1 and VILIP-2 bind to the same site and cause enhanced inactivation or Ca can sum up to high concentrations (Nakamura et al, 2008). Ca also facilitates presynaptic Ca2+ currents (Cuttle et al, 1998), by binding to enhanced facilitation, respectively. Cav2.1 channels expressed exogenously in sympathetic ganglion neurons reconstitute synaptic transmission initiated by NCS-1 (Tsujimoto et al, 2002), or CaM (Nakamura et al, 2008), as reported for recombinant VGCCs (DeMaria et al, 2001). This mechanism operates P/Q-type Ca currents with typical synaptic facilitation and depression. Both specifically for Cav2.1 (P/Q-type), with its knockout mice showing reduced facilitation and depression of synaptic transmission are markedly reduced synaptic facilitation (Ishikawa et al, 2005). The Ca2+-dependent Ca2+ current by mutations that prevent regulation of Cav2.1 channels by CaM and the facilitation accounts for 50% of synaptic facilitation, caused by presynaptic Ca sensor proteins. Our results show that, at this model synapse, Ca- and depolarization, irrespective of intra-terminal Ca2+ buffer conditions. Thus, 2+ 2+ voltage-dependent regulation of Cav2.1 channels through interaction with Ca Ca -dependent Ca channel modulations play essential roles in short-term sensor proteins is responsible for short-term synaptic plasticity. presynaptic plasticity.

RS IV-20-3 RS IV-20-4 ACTIVITY-DEPENDENT REGULATION OF VOLTAGE- POSTSYNAPTIC REGULATION OF IH AND IA IN LONG GATED POTASSIUM CHANNELS BY NITRIC OXIDE TERM PLASTICITY Ian D Forsythe Daniel Johnston, Rishi Narayanan, Darrin Brager MRC Toxicology Unit, University of Leicester, UK Center for Learning and Memory, University of Texas at Austin, USA Principal neurons of the medial nucleus of the trapezoid body (MNTB) The dendrites of hippocampal pyramidal neurons express numerous types receive a large glutamatergic input from the calyx of Held synapse. This of voltage-gated ion channels. The properties and/or distributions of these relay synapse forms part of the circuitry for sound-source localization, and channels in the dendrites are very non-uniform and highly regulated. We have its large size guarantees accurately timed action potentials (AP). Rather than investigated long-term changes in voltage-gated channels in hippocampal changes in synaptic strength there is increasing evidence that information neurons following the induction of long-term potentiation (LTP) and long- transmission is regulated by changes in postsynaptic excitability. Here we term depression (LTD). We have found that there are activity-dependent, Regular Symposia show that the MNTB utilizes neuronal nitric acid synthase (nNOS) and nitric and bi-directional, changes in the intrinsic excitability of these neurons with oxide (NO) signalling for control postsynaptic excitability. LTP and LTD. The changes in ion channels occur in parallel to those at the Stimulation of the calyx of Held caused NMDAR-mediated NO generation synapse and affect both the local and overall excitability of the neuron. The as assessed by DAR-4M imaging and increased cGMP concentrations. Patch- change in local excitability can increase the probability that a given synaptic clamp recording showed a reduction in synaptic strength, via an action of input fires the cell while a change in the overall excitability of the neuron NO at AMPAR, however NO induced a profound change in post¬synaptic may act to stabilize its firing rate following experience-dependent plasticity. excitability by inhibition of Kv3 high voltage-activated potassium currents The underlying mechanisms for these changes, and their implications with over a time-course of 10-20 minutes. This causes activity-dependent slowing respect to certain learning rules and disease states, will be discussed. of AP time-course and increased transmission failure in both innervated and non-innervated neurons. We demonstrate that NO is serving as a volume transmitter, tuning postsynaptic excitability and gain in both active and inactive neurons following sound stimulation.

RS IV-20-5 RS IV-21-1 REGULATION OF NEURONAL RESPONSIVENESS BY CONTRIBUTION OF THE VENTRAL VISUAL PATHWAY AXONAL INITIAL SEGMENT KCNQ CHANNELS TO STEREOPSIS Edward C. Cooper, Stephen Cranstoun, Zongming Pan Ichiro Fujita Neurology, University of Pennsylvania, USA Graduate School of Frontier Biosciences, Osaka University, Japan Human mutations in KCNQ2 and KCNQ3 cause epileptic seizures and The visual system has a remarkable capability of deriving 3-D surface myokymia, indicating that these voltage-gated K+ channels regulate structure from two retinal images. The computation for stereopsi starts excitability in brain circuits and motoneuron axons. KCNQ2 and KCNQ3 are in the striate cortex (V1), where signals from the two eyes converge onto

colocalized with NaV channels at axonal initial segments (AISs) and nodes single neurons and binocular disparity information is encoded. Properties of Ranvier, axonal subdomains that generate and regenerate action potentials of V1 cells, however, do not account for a number of aspects of stereo

(APs). This axonal colocalization of NaV and KCNQ channels depends on perception, suggesting that subsequent processing in cortical areas beyond evolutionarily conserved anchor motifs that coordinately link the channels V1 is responsible for conscious perception of stereoscopic depth. It has long to the cytoskeleton. We used NEURON modeling and electrophysiology been believed that binocular disparity information is routed exclusively to analyze how AIS localization contributes to the ability of KCNQ2 and to the dorsal visual pathway. In fact, binocular signals for stereopsis are KCNQ3 to regulate neuronal responsiveness. In the model, a relatively processed both along the ventral and dorsal pathways. Cells in the ventral small amplitude KCNQ conductance placed in the AIS near the site of AP pathway areas solve the binocular correspondence problem, compute relative initiation exerts considerable influence over neuronal threshold and gain. The disparity between visual features, and exhibit activities correlated with same amplitude of KCNQ conductance placed at the soma and/or dendrites behavioral judgment of fine disparity discrimination, whereas those in the exerts minimal influence on excitability. We tested this model using acutely dorsal pathway areas encode binocular correlation, signal local absolute- dissociated Purkinje cells (adPCs), which fire spontaneously (50-100 Hz) in disparity, and are involved in judgment of coarse disparity. Psychophysics vitro. When prepared with procedures that preserve their AISs, adPC firing in human subjects suggests that perception of plane-in-depth depends on the is strongly sensitive to KCNQ channel modulators. AIS removal markedly ventral pathway mechanisms. Thus, the two cortical pathways contribute to diminishes the influence KCNQ modulators on firing. stereopsis in a complimentary manner.

78 IUPS 2009 July 27 - August 1, 2009 in Kyoto RS IV-21-2 RS IV-21-3 HOW THE BRAIN HANDLES VERTICAL DISPARITY HOW CORTICAL NEURONS REPRESENT NATURALLY Jenny C. A. Read OCCURRING DISPARITIES Institute of Neuroscience, Newcastle University, UK Bruce Gordon Cumming, Ralf Haefner, Seiji Tanabe Because the eyes are set apart horizontally in the head, disparities between Laboratory of Sensorimotor Research, National Eye Institute, NIH, USA the two eyes' images are mainly horizontal. However, when the eyes Many neurons in the striate cortex are sensitive to binocular disparity. The converge and/or look to one side, vertical disparities (VDs) also occur. VD binocular energy model (BEM) explains many of their properties. Notably, is problematic in that it complicates stereo correspondence, i.e. the process the model predicts responses to “unnatural” disparities: e.g. when the left of finding pairs of retinal points which are viewing the same object in space. eye is shown a photographic negative of the image shown to the right eye However, it also represents an independent source information about eye (anticorrelated). Although real neurons also respond to these disparities, position, and there is psychophysical evidence that this information has their responses are attenuated, indicating a degree of specialization for naturally occurring stimuli. We propose that this can be explained by a small perceptual consequences. Yet, at the moment almost nothing is known about modification of the BEM, in which model simple cells are more diverse how the brain detects retinal VD. Physiologists have reported neurons tuned than originally proposed. We show that this generalized BEM can be used to non-zero stimulus VD, but what this means on the retina is unclear. In to explain other properties of real neurons that deviate from the predictions natural viewing, VD varies only slowly across an image, suggesting that a of the BEM. To test this hypothesis without model fitting, we used a spike- very sparse encoding would suffice. Yet recent results from my lab show that triggered analysis of responses to white noise stimuli. Principal component at least some individuals apparently encode VD on much finer scales. Finally, analysis is used to summarize the features of images associated with spikes. it seems likely that the brain concentrates on encoding the VDs associated This analysis of spike-triggered covariance reveals that most neurons are with the most useful eye positions, analogous to the concentration on zero indeed composed of multiple functional subunits, and that these are more horizontal disparity which is manifest in both physiology and psychophysics. diverse than originally supposed in the BEM. The organization of these This is why stereopsis fails for extreme eye positions. subunits allows these neurons to devote their full dynamic range to naturally occurring disparities.

RS IV-21-4 RS IV-21-5 ORGANIZATION OF 3-d BINOCULAR RECEPTIVE NEURONS IN VISUAL AREA V5/MT IN THE AWAKE FIELDS OF NEURONS IN THE EARLY VISUAL CORTEX RHESUS MONKEY ARE SELECTIVE FOR THE Izumi Ohzawa, Kota S Sasaki, Yuka Tabuchi RELATIVE DISPARITY BETWEEN TWO TRANSPARENT Graduate School of Frontier Biosciences, Osaka University, Japan PLANES Kristine Krug1, Bruce G Cumming2, Jon V Dodd1, Binocular disparity selectivity of a visual neuron is defined in the most 1 complete form by its binocular receptive field (RF), which is inherently a 3-d Andrew J Parker 1Department of Physiology, Anatomy and Genetics, Oxford University, UK, entity in space defined as a function of visual direction (X, Y) and distance 2 Z. We have examined binocular interaction in the 3D space to elucidate Lab Sensorimotor Res, NEI, NIH, USA mechanisms to detect binocular disparity signals. Binocularly uncorrelated Dorsal stream neurons are thought to represent absolute disparity (Uka, DeAngelis 2-d dynamic dense noise stimuli allow examination of 3-d binocular RFs. We 2003), whereas ventral stream neurons signal the relative disparity between adjacent planes (Thomas et al 2002; Umeda et al 2007). We recorded from 53 single neurons used a spike-triggered analysis to examine structure of the 3D binocular RFs. in dorsal visual area V5/MT in three macaques. We presented two superimposed The degree of spatial pooling along the Y dimension is examined by transparent planes: one moving in the neuron’s preferred direction, the other in the comparing the spatial extent of single subunits internal to the 3-d binocular null direction. We varied both the relative disparity between the planes and an absolute RF of the neurons. A single subunit often did not cover the entire length-wise disparity pedestal added to both planes. We compared the tuning curves for relative extent of the RF for some complex cells. For these complex cells, multiple disparity obtained at different absolute disparities by fitting simultaneously pairs of subunits must therefore be pooled length-wise to comprise a large RF, and Gabor functions, differing only in horizontal offset. Horizontal offsets quantified the shift in the disparity tuning function induced by the pedestal. Some V5/MT neurons the tuning curves to binocular disparity were invariant along the Y axis. Regular Symposia These results indicate that a proportion of complex cells detect constant signalled absolute (shift 0), some perfectly signalled relative disparity (1) and others were intermediate. The population shift was highly significant (median 0.31, p<0.0001 binocular disparities in the receptive fields by collecting inputs from many Wilcoxon test). The results reveal a representation of relative disparity between antecedent neurons that prefer identical disparities at different positions. transparent planes in V5/MT, not found for adjacent planes (Uka, DeAngelis 2003). Support by: MEXT(18020017), Global COE, CREST Both dorsal and ventral streams signal relative disparity but differ in the spatial configurations they represent.

RS IV-21-6 RS V-22-1 BINOCULAR VISION AND THE CEREBRAL CORTEX EVIDENCE FOR ClC-2 Cl- CHANNELS IN ACID Andrew J Parker SECRETION Physiology, Anatomy & Genetics, Oxford University, UK John Cuppoletti Neurophysiological studies in macaque monkeys show that important Department of Cellular and Molecular Physiology, University of Cincinnati, perceptual signals about binocular stereopsis pass through both the dorsal and USA ventral visual pathways. Dorsal visual pathways exploit information about Apical membranes from gastric parietal cells of histamine stimulated (but binocular depth, particularly towards the perception of extended surfaces, not cimetidine treated) rabbits exhibit a Cl- conductance as first measured by responding to properties such as slant, tilt and segregation of overlapping by potential sensitive dyes and contain a Cl- selective channel which can movements. Ventral visual pathways respond to small depth differences be activated by PKA and low external pH (pH 3). ClC-2 channels cloned between nearby binocular features and, in higher areas, respond selectively to o from the gastric mucosa of rabbits also exhibit PKA and pH 3 activation. 3-D shape of objects. Reconciling these data with the pattern of results from o PKA activation was also found in ClC-2 from humans, and the unique sites human visual cortex, gained from imaging data and studies of human neuro- ophthalmological subjects, is an important long-term goal. Comparison of of phosphorylation responsible for this were identified. Finally, histamine human and macaque visual cortex raises questions about the structural and plus carbachol stimulated acid secretion was studied in ClC-2 knockout functional correspondences between identified visual areas. Evidence will be mice using an in vivo perfused stomach. Acid secretion was measured by presented from recent studies of binocular vision that addresses the question titration every 15 min over 1.5 hours. Acid secretion in KO ClC-2 (-/-) of functional correspondence and shows how these studies would be better mice was significantly lower at 30 min (peak secretion) compared to WT informed with more evidence about structural correspondences. ClC-2 (+/+) mice, while pepsinogen secretion was similar in both KO and WT mice. These studies demonstrate that the ClC-2 Cl- channel is required for maximum acid secretion stimulated by histamine plus carbachol in the mouse.

IUPS 2009 July 27 - August 1, 2009 in Kyoto 79 RS V-22-2 RS V-22-3 K+ CHANNELS IN PARIETAL CELLS GASTRIC KNCQ1 CHANNELS PROVIDE LUMINAL K+ + + Richard Warth TO THE H K ATPase, WHEREAS Kir 4.1 CHANNELS Department of Physiology, University of Regensburg, Germany ARE INVOLVED IN SECRETORY MEMBRANE + + + RECYCLING In gastric parietal cells the H , K -ATPase pumps H into the lumen and takes up 1 1 2 1 + + Ursula Seidler , Penghong Song , Stephanie Groos , Brigitte Riederer , K in parallel. K channels localized in the luminal membrane play a pivotal role 1 1 1 3 + + Zhe Feng , Anja Krabbenhoeft , Michael P. Manns , Adam Smolka , in replenishing K in the luminal fluid. Over the last years, several K channels 4 5 + Susan Hagen , Clemens Neusch have been proposed to act as luminal recycling pathway for K . Today, the 1 + Department of Gastroenterology, Hepatology and Endocrinology, Hannover evidence for KCNE2/KCNQ1 acting as a K channel of the luminal membrane Medical School, Germany, 2Department of Anatomy I, Hannover Medical School, compartment is compelling. This channel complex is a major component for Germany, 3Department of Medicine, Medical University of South Carolina, USA, 4Department of Surgery, Beth Israel Deaconess Medical Center, Boston, USA, gastric acid secretion under stimulated conditions and for the maintenance of 5 normal parietal cell morphology. One could speculate that KCNE2/KCNQ1 Department of Neurology, University of Goettingen, Germany channels act in concert with inward rectifiers such as KCNJ10 and/or other +K Aim: This study was undertaken to explore the functional role of KCNQ1 and Kir4.1 channels in + gastric acid secretion. Results: Forskolin-stimulated acid secretory rates were strongly reduced in channels. On the other hand, the channel proteins underlying the luminal K KCNQ1-/- gastric mucosa of 7 day-old mice in mini-Ussing chamber setups. Application of a high conductance could vary dependent on the developmental status of the parietal K+ concentration to the luminal membrane restored normal acid secretory rates in the KNCQ1-/- + cells. Better knowledge of the molecular nature of the luminal K+ conductance mucosa, indicating that the primary function of KCNQ1 is the supply of K to the extracellular K+ binding site of the H+K+-ATPase. Surprisingly, Kir4.1-/- mucosa secreted significantly more will ameliorate our understanding of the mechanisms of gastric acid secretion, acid and initiated secretion more rapidly. Electrone microscopy revealed the presence of fully parietal cell biology, and functional morphogenesis of gastric mucosa. It is elaborate but collapsed canalicular membranes and a lack of tubulovesicles in resting state Kir4.1-/- envisioned, that in the future, this information could offer new perspectives for parietal cells. After in vitro stimulation by forskolin, massive membrane fusion and canalicular the treatment of diseases caused by inappropriate acid secretion. enlargement was seen in the WT but not in the Kir4.1-/- mucosae, suggesting that Kir4.1 is essential for the membrane recycling events that accompany the acid secretory cycle. Conclusions: KCNQ1 channels are the major pathway to provide K+ as substrate for H+K+ ATPase. Kir4.1 channels are involved in the control of tubulovesicular fusion and/ or retrieval.

RS V-22-4 RS V-22-5 PHOSPHO-REGULATED ACAP4-EZRIN INTERACTION PARIETAL CELL FUNCTION AND GASTRIC IN GASTRIC PARIETAL CELL ACTIVATION EPITHELIAL CELL HOMEOSTASIS Fengsong Wang1,3, Xia Ding1,2, Hui Deng1, Peng Xia1, Xuebiao Yao1,3 Linda C Samuelson 1 Molecular and Integrative Physiology, The University of Michigan, USA Anhui Key Laboratory for Cellular Dynamics and Chemical Biology, China, 2Department of Internal Medicine, Beijing University of Chinese Medicine, China, Gastric parietal cells have complex physiologic functions. In addition to 3Department of Physiology, Morehouse School of Medicine, USA the well-studied process of acid secretion, parietal cells secrete numerous The ezrin-radixin-moesin proteins provide a regulated linkage between membrane growth factors and provide a critical, but less well defined function proteins and the cortical cytoskeleton. Our recent proteomic study revealed ezrin- regulating the differentiation and proliferation of epithelial cells in the gastric glands. We have been using genetically engineered mouse models to define ACAP4-ARF6 protein complex essential for membrane remodeling (Mol. Cell pathways regulating parietal cell function and to characterize epithelial cell Proteomics 5, 1437-1448). However, it has remained elusive as how their interaction is

Regular Symposia transformation in response to parietal cell dysfunction or loss. Our studies integrated into membrane remodeling. Here we report that ezrin interacts with ACAP4 have identified the gastrointestinal hormone gastrin as a key regulator of acid in PKA-phosphorylation-dependent manner. ACAP4 locates in the cytoplasmic secretion and parietal cell function. The critical importance of parietal cells membrane in resting parietal cells but translocates to the apical membrane upon for gastric epithelial cell homeostasis was revealed by analysis of a number histame stimulation. ACAP4 was precipitated with ezrin from secreting but not resting of mouse mutants with parietal cell dysfunction. These studies demonstrated parietal cell lysates, suggesting a phospho-regulated interaction. Indeed, this interaction that parietal cell loss leads to glandular hypertrophy coupled with disruption is abolished by phosphatase treatment and validated by an in vitro reconstitution assay. of differentiation of the mucous neck cell/zymogenic cell lineage. Our studies This phospho-mediated interaction was mapped to the N-terminal 400 amino acids demonstrate that this complex cellular transformation is a general response to of ACAP4. Importantly, ezrin specifies the apical localization of ACAP4 in secreting parietal cell dysfunction and that the cellular changes are associated with the cells as ezrin repression prevents the distribution. In addition, over-expressing GAP- development of gastric cancer. deficient ACAP4 results in an inhibition of apical membrane remodeling and parietal cell activation. These results define a novel molecular mechanism linking ACAP4- ezrin interaction to regulated parietal cell secretion.

RS V-22-6 RS V-22-7 EXFOLIATION OF PARIETAL CELLS AT GASTRIC PITS SULFORAPHANE MODULATES ON GASTRIC OXYNTIC ASSOCIATED WITH ACID SECRETION IN VITRO CELL FUNCTIONS BY INCREASING PRODUCTION OF Akira Sawaguchi REACTIVE OXYGEN SPECIES Department of Anatomy, University of Miyazaki, Faculty of Medicine, Japan Akinori Yanaka It is clinicopathologically important to elucidate the cell kinetics for Department of Clinical Pharmacology, Tokyo University of Science, Japan the maintenance of normal gastric epithelium. To elucidate a functional Sulforaphane (SFN), a substance abundantly included in broccoli sprouts, affords transformation of gastric parietal cells, we have newly developed an in vitro chemoprevention against cancer via nrf2-dependent mechanisms. In the present study, we determined if SFN modulates gastric oxyntic cell functions. experimental model, named as isolated gastric mucosa. Interestingly, in a rat Methods: Study A. The effects of SFN on isolated guinea pig gastric glands were examined. gastric mucosa isolated after stimulation, a number of pit-parietal cells were 1) ROS production was evaluated using ROS-sensitive fluorescent dye, CM-H2-DCFDA. exfoliated into the gastric lumen at 60 min after incubation. Quantitative 2) Histamine release was measured by ELISA. 3) Acid production was estimated from 14C-aminopyrine (AP) accumulation. 4) Cell death was evaluated by LDH release. Study B. analysis verified a time-dependent increase in the number of exfoliated +/+ -/- pit-parietal cells after stimulation of acid secretion. Ultrastructurally, Effects of SFN on gastric mucosal functions were compared between nrf2 and nrf2 C57/ + + BL6 mice. autophagosome-like structures consisting of H /K -ATPase positive Results: Study A: 1) SFN enhanced ROS production, effects prevented by a ROS scavenger, membranes were frequently seen in the exfoliated pit-parietal cells. In N-acetyl-l-cysteine (NAC). 2) SFN, < 10 μM, stimulated 14C-AP accumulation, effects addition, the pit-parietal cell exfoliation was accompanied by sealing of their prevented by NAC or famotidine. 3) SFN increased histamine release, an effect prevented basal portion with the cytoplasmic processes of adjacent surface mucous by NAC. 4) SFN, > 100 μM, inhibited 14C-AP accumulation, increased LDH release, effects +/+ -/- cells. The present morphological findings provide a new insight into the cell attenuated by NAC. Study B: 1) In nrf2 , but not in nrf2 mice, SFN up-regulated nrf- 2-depdendent anti-oxidant enzymes and protected gastric mucosa from oxidative injury. kinetics in the gastric epithelium in vitro +/+ -/- 2) In both nrf2 and nrf2 mice, low doses of SFN stimulated histamine release and acid secretion, while high doses of SFN decreased acid secretion, and increased LDH release. Conclusion: SFN modulates acid secretion by gastric oxyntic cells. These effects of SFN appear to be related to SFN-induced ROS production.

80 IUPS 2009 July 27 - August 1, 2009 in Kyoto RS V-23-1 RS V-23-2 CELLULAR MECHANISMS OF ALCOHOLIC AUTOPHAGY IN PANCREATITIS PANCREATITIS INDUCED BY NON-OXIDATIVE Anna Gukovskaya ETHANOL METABOLITES Medicine, VA Greater Los Angeles medical Center/UCLA School of Robert Sutton Medicine, USA Liverpool NIHR Pancreatic Biomedical Research Unit, University of Pancreatitis is a potentially lethal inflammatory disease of the pancreas, Liverpool, UK pathogenesis of which remains obscure. We provide evidence that acute Ethanol alone causes minimal experimental pancreatic injury, but ethanol pancreatitis causes a profound autophagy impairment, which is responsible with a high fat diet causes pancreatitis. Fatty acid ethyl esters (FAEEs) form for two key manifestations of this disease: the accumulation of large vacuoles via a non-oxidative metabolic shuttle, accumulate in mitochondria, and cause in acinar cells and the intra-acinar trypsinogen activation. During autophagy pancreatitis. FAEEs are hydrolysed to ethanol and fatty acids (FAs), which are cytoplasmic organelles destined for degradation are sequestered in vacuoles oxidised. Acetaldehyde does not cause pancreatic acinar injury, but FAEEs 2+ which then fuse with lysosomes, where cargo is degraded by cathepsins induce cytosolic calcium (Ca ) overload, intracellular enzyme activation, family of proteases. Our results suggest that one mechanism mediating the mitochondrial impairment and cellular necrosis. FAEEs release cytosolic 2+ autophagic flux retardation in pancreatitis is the impaired processing and Ca from endoplasmic reticulum (ER) and acid secretory stores via inositol trisphosphate receptors (IP3Rs). With low levels of agonist, low levels of FAEEs activity of cathepsins resulting in pathologic accumulation of vacuoles in transform apical Ca2+ signals into prolonged, global elevations. Cytosolic Ca2+ acinar cells. Trypsinogen activation in pancreatitis is mediated by cathepsins. overload from FAEEs is associated with a failure of Ca2+ clearance from a lack Pancreatitis causes an imbalance between cathepsin (Cat) B, which converts of mitochondrial ATP generation to supply Ca2+ pumps. Supplementary ATP or trypsinogen into trypsin and CatL, which degrades trypsinogen and trypsin, inhibition of FAEE hydrolysis prevents failure of Ca2+ clearance, indicating that resulting in intra-acinar accumulation of active trypsin. Analysis indicates FAs released from FAEEs cause mitochondrial impairment. FAs do not induce that a number of characteristics of pancreatitis are similar to those in calcium release via IP3Rs, but inhibit mitochondria directly, causing necrosis disorders which are caused by inactivation of lysosomal hydrolases. This due to an uncompensated leak of Ca2+ from the ER. These mechanisms have should be taken into consideration in designing strategies to treat or mitigate implications for pancreatitis induced by alcohol and hyperlipidaemia. pancreatitis

RS V-23-3 RS V-23-4 PANCREATIC PROTEASE ACTIVATION BY ALCOHOL GASTROINTESTINAL LIPOLYSIS IN CHRONIC METABOLITE DEPENDS ON Ca2+ RELEASE VIA ACID PANCREATITIS STORE IP3 RECEPTORS Frederic Carriere Julia Gerasimenko, Gyorgy Lur, Alexei Tepikin, Enzymology at Interfaces and Physiology of Lipolysis UPR9025, CNRS, Oleg Gerasimenko, Ole H. Petersen France Department of Physiology, School of Biomedical Sciences, Liverpool Lipolytic enzymes in healthy subjects and patients with severe chronic University, UK pancreatitis (CP) were better characterized in recent years. Human pancreatic Fatty acid ethyl esters induce excessive Ca2+ liberation from the internal stores lipase (HPL) is the major lipase involved in fat digestion under normal which leads to Ca2+-dependent necrosis due to intracellular trypsin activation conditions but its secretion is drastically reduced in CP patients. These and causes acute pancreatitis. We have identified the specific source of the patients are still able to absorb significant amounts of fat because human Ca2+ release linked to the fatal intracellular protease activation by monitoring gastric lipase (HGL) compensates partly for the loss of HPL. HGL secretion changes of the Ca2+ concentration in the endoplasmic reticulum (ER) as well is increased 3 to 4-fold in CP and HGL alone can achieve 30% of dietary as in an acid compartment, localized in the apical granular pole of two-photon fat lipolysis. Two novel pancreatic lipase-related proteins (PLRP1 and permeabilized mouse pancreatic acinar cells. The acid store has a bafilomycin- PLRP2) are also produced by the exocrine pancreas. No enzyme activity sensitive vacuolar proton pump, but does not contain the thapsigargin-sensitive was found for PLRP1 so far and PLRP2 hydrolyses various acylglycerols, ER Ca2+ pump. Palmitoleic acid ethyl ester (POAEE) elicits Ca2+ release from phospholipids, vitamin esters and galactolipids, whereas HPL is specific both the thapsigargin-sensitive ER as well as the bafilomycin-sensitive acid pool, for triglycerides. In humans, PLRP2 is mainly involved in the digestion of 2+ but trypsin activation depends specifically on Ca release from the acid pool and galactolipids, the major lipids from plants. PLRP2 was however found in Regular Symposia is mainly mediated by functional inositol 1,4,5- trisphosphate receptors (IP3Rs) various tissues and cellular types and might play other roles depending on of types 2 and 3. Antibodies against IP3Rs of type 2 and 3, but not of type 1, species. PLRP2 displays a phospholipase A1 activity and is directly produced markedly inhibited POAEE-elicited trypsin activation. We propose that Ca2+ as an active enzyme, unlike PLA2. In pathological situations, PLRP2 might release from an acid granular store induces intracellular protease activation and act on membrane phospholipids and play a role in intracellular lipolysis and that this is a critical process in the initiation of alcohol-related acute pancreatitis. the destruction of pancreatic acinar cells.

RS V-23-5 RS V-23-6 CHARACTERISATION OF pH-DEPENDENT ATP INTRACELLULAR ALKALIZATION CAUSES PAIN UPTAKE INTO ISOLATED PANCREATIC ZYMOGEN SENSATION THROUGH ACTIVATION OF TRPA1 GRANULES Fumitaka Fujita1, Kunitoshi Uchida2, Tomoko Moriyama3, Asako Shima4, 2 5 5 2 Kristian Agmund Haanes, Ivana Novak Koji Shibasaki , Hitoshi Inada , Takaaki Sokabe , Makoto Tominaga 1Central Research Laboratories, Mandom corp., Japan, 2Section of Cell Department of Biology, University of Copenhagen, Denmark Signaling, Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, National Institute for Physiological Sciences, Okazaki, Japan, Imaging studies at our lab indicated that pancreatic acinar cells store ATP in granules. 3 Present study aimed to quantify the ATP content and characterise ATP uptake into Department of Rehabilitation Medicine, Institute of Brain Science, Hirosaki University School of Medicine, Hirosaki, Japan, 4Department of Physiology, zymogen granules (ZG). ZGs were isolated using a method of Chen et al (2006). ATP 5 School of Dentistry, Nihon University, Tokyo, Japan, Section of Cell Signaling, content was measured using Luciferin/Luciferase and related to protein concentration Okazaki Institute for Integrative Bioscience, National Institutes of Natural in the sample. Freshly isolated granules contained 55 ± 7 μM of ATP (n=6), based on a Sciences, Okazaki, Japan protein content of 0.31 pg per granule of 1 μm diameter. ATP uptake was characterised Vertebrate cells require a very narrow pH range for their survival. Cells accordingly possess by incubating the granules in pH 6 buffer containing sucrose, MES, EGTA, 4 mM KCl sensory and defense mechanisms for situations where the pH deviates from the viable range. and trypsin inhibitor. A Km of 3.0 ± 0.9 mM and a Vmax of 540 ± 110 pg/mg/30min was Although the monitoring of acidic pH by sensory neurons has been attributed to several ion calculated from Michaelis-Menten plots (n=6). channels, the mechanisms by which these cells detect alkaline pH are not well understood. 2+ DIDS and Evans blue both at 100 μM completely blocked ATP uptake. For DIDS the Here, using Ca -imaging and patch-clamp methods, we show that intracellular alkalization activates transient receptor potential cation channel, subfamily A, member 1 (TRPA1) and IC50 was 7.5 ± 1.1 μM. Bafilomycin, CCCP and valinomycin did not have significant effect at pH 6. However in pH 7.2 or at 130 mM NMDG-Cl buffer, CCCP had a that activation of this ion channel is involved in nociception. In addition, analyses of mutants suggested that the two N-terminal cysteine residues in TRPA1 were involved in activation by significant inhibitory effect. In addition, there was a strong pH dependence of the ATP intracellular alkalization. Furthermore, intraplantar injection of ammonium chloride into the uptake in ZG. At pH 7.2, ATP content was more than 2-fold higher than at pH 6 (n=4; mouse hind paw caused pain-related behaviors that were not observed in TRPA1-deficient P<0.05). In conclusion, these data show that the pH gradient is important for the ATP mice. These results suggest that alkaline pH causes pain sensation through activation of uptake mechanism into ZGs. TRPA1 and may provide a molecular explanation for some of the human alkaline pH-related Support: DNSRC 272-05-0420 sensory disorders whose mechanisms are largely unknown.

IUPS 2009 July 27 - August 1, 2009 in Kyoto 81 RS V-24-1 RS V-24-2 THE MOLECULAR PHYSIOLOGY OF FLOW- SRC FAMILY PROTEIN TYROSINE KINASE( PTK) STIMULATED K SECRETION IN THE DISTAL NEPHRON MODULATES THE EFFECT OF SGK1 AND WNK4 ON Lisa Michelle Satlin1, Wen Liu1, Yuan Wei1, Tetsuji Morimoto2, ROMK CHANNELS 3 Thomas R. Kleyman Wen-Hui Wang1, Peng Yue1, Qiang Leng2, DaoHong Lin1, 1Departments of Pediatrics and Medicine, Mount Sinai School of Medicine, 1 2 ChuYang Pan USA, Department of Pediatrics, Tohoku University Graduate School 1 2 3 Pharmacology, New York Medical College, USA, Department of Cell. & of Medicine, Japan, Medicine/Renal-Electrolyte Division, University of Mol. Physiology, Yale Univ.Sch.Med. New Haven, CT, USA Pittsburgh, Pittsburgh, Pennsylvania, USA We used the whole cell patch clamp technique to study the role of c-Src in modulating The final regulation of urinary K excretion is accomplished in the distal nephron the effect of WNK4 and SGK1 on ROMK channels. Expression of c-Src decreased K including the cortical collecting duct (CCD). In this segment, cell K passively diffuses current from 1080 pA to 520 pA in HEK293T cells transfected with GFP-ROMK1+c- into the urinary space through apical K selective channels. Both SK/ROMK and BK channels have been functionally identified therein. Apical BK channels are activated Src. The inhibitory effect of PTK was abolished either by treatment of cells with 2+ herbimycin A or in cells transfected with R1Y337A, in which tyrosine residue 337, a by membrane depolarization, elevation of [Ca ]i, and/or membrane stretch, and can be selectively blocked by iberiotoxin (IBX). We have previously reported that an PTK phosphorylation site of ROMK1, was mutated to alanine. Coexpression of WNK4 2+ decreased K current by 50% in cells transfected with ROMK1 (from 1050 to 560 pA) increase in luminal flow rate in the rabbit CCD leads to a transient increase in [Ca ]i and sustained increases in net Na absorption and IBX-sensitive and thus BK channel- or R1Y337A (from 1790 to 800 pA). The inhibitory effect of WNK4 on ROMK1 mediated net K secretion (JK). These data and studies by others showing that mice or ROMK mutant was abolished when SGK1 is coexpressed (R1+WNK4+SGK1, lacking ROMK secrete K by a process that is, at least in the late distal tubule, IBX- 1040 pA; R1Y337A+WNK4+SGK1, 1740 pA), suggesting that SGK1 antagonizing sensitive have led to the conclusion that the BK channel mediates flow-stimulated the WNK4-induced inhibition of ROMK. However, expression of c-Src significantly JK. Whereas the SK/ROMK channel is restricted to principal cells, conducting BK decreased K current again in cells transfected with R1+WNK4+SGK1 (560 pA) channels are detected in both principal and intercalated cells. The focus of this talk or with R1Y337A+WNK4+SGK1 (980 pA). In contrast, expression of c-Src had will be to address two unanswered questions: (i) how does a flow-induced transient no effect on K current in cells transfected with R1337A or R1Y337A+SGK1. We 2+ increase in [Ca ]i lead to a sustained increase in JK, and (ii) is flow-mediated JK conclude that c-Src inhibits ROMK channels by phosphorylating ROMK channels and mediated by intercalated or principal cells. by antagonizing the SGK1-mediated inhibition of WNK4.

RS V-24-3 RS V-24-4 MOLECULAR MECHANISMS OF ROMK CHANNEL ROLE OF MAXI-K AND IK CHANNELS IN SALIVARY TRAFFICKING IN THE KIDNEY GLAND FLUID AND POTASSIUM SECRETION Paul A. Welling, Liang Fang, Bo-Young Kim, James B. Wade James Edward Melvin1, Marcelo A Catalan1, Physiology, University of Maryland Medical School, USA Mireya Gonzalez-Begne1, Tetsuji Nakamoto2, 1 1 The density of ROMK channels in the distal nephron is exquisitely regulated Victor G Romanenko , Ted Begenisich to adjust renal potassium excretion for potassium balance. Recent discoveries 1Oral Biology, University of Rochester Medical Center, USA, 2Kyushu of regulated trafficking, involving ER retention and phosphorylation- Dental College, Japan dependent Golgi export, will be discussed. The talk will highlight a novel Salivary glands express two types of Ca2+-activated K channels: intermediate endocytotic mechanism, which limits urinary potassium loss in states of conductance IK1 and maxi-K channels. We confirmed that the IK1 and maxi-K

potassium deficiency and may contribute to potassium retention in renal channels are encoded by the KCa3.1 and KCa1.1 genes, respectively. Fluid secretion was Regular Symposia disease. Available evidence indicates ROMK endocytosis is stimulated normal in both KCa3.1 and KCa1.1 null mice, and consistent with these observations, by WNK kinases but the precise nature of the endocytotic signal and the the membrane potential of acinar cells from KCa3.1 and KCa1.1 null mice remained - internalization machinery that decodes it has been a mystery. Here, we show hyperpolarized during muscarinic stimulation (about -50 mV) relative to the Cl ROMK contains an unusual variant of the canonical “NPxY” endocytotic equilibrium potential (-24 mV). In contrast, fluid secretion from double KCa3.1/KCa1.1 signal. Characterized by the transplantable sequence, YxNPxFV, the signal null mice was severely reduced and acinar cells were only slightly hyperpolarized (-35±2 mV). The K+ content of the secreted fluid was reduced more than 75% in in ROMK serves as a recognition site for ARH, a member of a new class of KCa1.1 null mice but was unchanged in KCa3.1 null mice. KCa1.1 channels localized clathrin adaptors. ARH is predominantly expressed in the distal nephron, to the apical membranes of both acinar and duct cells. We also found that the [K+] where it interacts with ROMK. The ROMK internalization signal directly -/- of submandibular saliva increased nearly 2-fold in Aqp5 mice. Paxilline, a KCa1.1- binds to the phosphotyrosine binding domain of ARH while other domains + specific channel blocker, inhibited the enhanced K secretion while KCa1.1 protein of ARH simultaneously recruit clathrin and the AP-2 adaptin. Consequently, expression increased dramatically in the plasma membranes of Aqp5-/- mice. These ARH recruits ROMK to clathrin-coated pits for constitutive and WNK1- findings show that both IK1 and maxi-K channels can support fluid secretion, but only stimuated endocytosis. maxi-K channels are critical for K+ secretion in salivary glands.

RS V-24-5 RS V-24-6 + THE ESSENTIAL ROLE OF LUMINAL BK CHANNELS COLONIC K CHANNELS IN HEALTH & DISEASE + IN DISTAL COLONIC K SECRETION Malcolm Hunter1, Adel Al-Hazza1, John Linley1, Geoffrey I Sandle2 1 1 1 1Institute of Membrane & Systems Biology, University of Leeds, UK, Jens G. Leipziger , Mads V Sorensen , Helle A Praetorius , 2 Matthias Sausbier2, Peter Ruth2 Institute of Molecular Medicine, St James's University Hospital, Leeds, UK 1Physiology and Biophysics, Aarhus University, Denmark, 2Pharmacology Changes in ion transport are a common symptom of colonic disease. Two K and Toxicology, Institute of Pharmacy, University of Tuebingen, Germany channels of physiological importance have been identified in normal human + + + colon: apical BK (220 pS) channels whose expression in normal individuals Distal colonic K excretion is determined by the balance of K absorption and K is restricted to surface cells and cells in the upper 25% of the crypt, and IK secretion by the enterocytes. This presentation summarizes the current knowledge (28 pS) channels which are the dominant basolateral K channel and present of the cellular mechanism for colonic K+ secretion, with focus on the luminal + throughout the surface-crypt axis. Apical BK channel expression is increased secretory K channel. Several recent observations highlight a pivotal role of the large along the surface-crypt axis in patients with end-stage renal disease, where 2+ conductance, Ca -activated KCa1.1 (BK, KCNM) channel as the functionally relevant the colon is transformed into an accessory K secretory organ, and apical + luminal K efflux pathway in mouse distal colon. This conclusion was based on clear BK channel expression is also enhanced in crypt cells in patients with cut results from BK knock-out mice. Several relevant issues will be presented: 1. BK ulcerative colitis, a disease often associated with excessive colonic K losses. + channels mediate the resting distal colonic K secretion, 2. they are acutely stimulated By contrast, basolateral IK channel expression and activity is markedly 2+ by luminal nucleotide receptor activation and an intracellular Ca increase, 3. BK decreased in ulcerative colitis; these changes will contribute to the enhanced channels are regulated by aldosterone, 4. the cAMP-stimulated distal colonic K+ pathogenesis of impaired Na and water absorption, the resultant diarrhoea secretion is apparently mediated via BK channels, 5. and finally aldosterone was found and enhanced colonic K loss which typifies this disease. to specifically up-regulate the ZERO (e.g. cAMP activated) C-terminal splice variant of the BK channel. In summary we suggest that the sole exit pathway for transcellular K+ secretion in mammalian distal colon is the BK channel, which is the target for short term intracellular Ca2+ and cAMP activation and long term aldosterone regulation.

82 IUPS 2009 July 27 - August 1, 2009 in Kyoto RS V-25-1 RS V-25-2 REGULATION OF EPITHELIAL SODIUM CHANNELS IN THE EPITHELIAL SODIUM CHANNEL (ENaC) AS PATHOLOGICAL AND PHYSIOLOGICAL STATES A MOLECULAR TARGET FOR BLOOD PRESSURE David Ian Cook, Il-Ha Lee, Sung_he Song, Craig R Campbell, CONTROL Anuwat Dinudom Christoph Korbmacher School of Medical Sciences K25, University of Sydney, Australia Department of Cellular and Molecular Physiology, University of Erlangen- Nuremberg, Germany Epithelial Na channels are expressed in the apical membranes of the epithelia of the respiratory tract, the colon, the distal nephron and the excretory ducts of The appropriate regulation of the epithelial sodium channel (ENaC) in the aldosterone sensitive distal nephron is critical for the maintenance of body the exocrine glands. Their activity plays a key role in the regulation of blood sodium balance and hence for blood pressure control. This is evidenced by 'gain pressure, extracellular fluid volume and the thickness of the surface fluid coating of function' mutations of ENaC which cause a rare form of severe salt-sensitive the respiratory tract. Our and other laboratories have shown that a number of arterial hypertension (Liddle's syndrome). common respiratory pathogens affect the activity of ENaC. The molecular mechanisms that contribute to ENaC regulation involve a complex The activity of the channels is regulated by the ubiquitin-protein ligase, Nedd4-2, network of signaling pathways. We use a combination of electrophysiological which ubiquitinates the channels leading to their endocytotic removal from the and molecular biological techniques in expression systems and microdissected apical membrane, as well as by a number of serine-threonine kinases. Of these mouse tubules to study ENaC regulation, to characterize the functional effects kinases, some, for example Sgk and Akt, phosphorylate Nedd4-2 leading to its of regulatory proteins, and to identify channel regions that are relevant for sequestration by 14-3-3 proteins, whereas others, for example ERK, Grk2 and ENaC regulation by kinases and proteases. Proteolytic activation of ENaC CK2, phosphorylate the channel leading to changes in the affinity of Nedd4-2 may contribute to renal sodium retention under pathophysiological conditions. for the channel. In addition, ENaC activity is regulated by the level of PIP2 n the We also investigate the role of lipid microdomains (‘lipid rafts’) in the plasma inner leaflet of the apical membrane. membrane for ENaC function and its association with regulatory proteins. This presentation will describe our recent work on the impact of pathogenic A better understanding of the molecular mechanisms involved in ENaC regulation mycobacteria and of malaria parasites on ENaC activity, as well as on our recent will hopefully provide novel insights into the pathophysiology of arterial findings on the regulation of ENaC by caveolin and by tyrosine kinases. hypertension. This ultimately may lead to new diagnostic and therapeutic concepts.

RS V-25-3 RS V-25-4 THE PHYSIOLOGICAL ROLES OF SUBCELLULAR PROCESSING AND TRAFFICKING OF MUTANT CFTR AQUAPORINS AND SLC 26 TRANSPORTERS Kenichi Ishibashi Min Goo Lee Medical Physiology, Meiji Pharmaceutical University, Japan Department of Pharmacology, Yonsei University College of Medicine, AQP11 is a subcellular AQP with unusual pore forming sequences, NPA Korea boxes highly conserved in AQPs. Such AQPs are found only in multicellular The cystic fibrosis transmembrane conductance regulator (CFTR) and solute- organisms with a vital cysteine residue. This new subfamily, AQP11 and 12 linked carrier 26 transporters play an important role in epithelial physiology, in mammals, is group III AQPs following water selective AQPs (group I) especially in the transepithelial anion transport. Genetic defects in these and aquaglyceroporins (group II). AQP11 is indeed a water channel when transporter genes are known to cause human diseases, such as cystic fibrosis expressed in liposomes. In the kidney, AQP11 is expressed intracellularly and Pendred sysdrome. Most of the disease-causing mutations in the CFTR at the proximal tubule, mostly at the endoplasmic reticulum (ER). AQP11 and SLC26A4 (pendrin) genes induce processing defects of their gene null mice survived only for two months after birth with a kidney problem, product. This, in turn, impairs the normal trafficking of CFTR and pendrin polycystic kidneys. The mechanism for the development of polycysts is unknown but is preceded by the formation of intracellular vacuoles which to the plasma membrane and evokes a loss-of-function type human disease. may represent cellular damage or accumulation of specific substances. There has been an intensive search for therapeutic strategies to rescue the Indeed, apoptotic activities are stimulated in the kidney and testis in the conformation or trafficking defects of the mutant proteins. Recently, we and others have reported that ER stress or alterations in the ER environment can absence of AQP11 suggesting that vacuoles will be the sign of cell damage Regular Symposia like ER stress. On the other hand, the primary culture cells of the proximal partially rescue the processing defect possibly by disturbing the function of tubule revealed dysfunction of endosomal acidification in the absence of ER-quality control system. In addition, we found that these situations can AQP11. The vacuoles observed in the proximal tubule in AQP11 null mice induce the non-conventional trafficking of the mutant proteins. This unknown may indicate the accumulation of dysfunctional endosomes. The research on pathway might be a new therapeutic target for the loss-of-function type subcellular AQPs will lead to a new field of AQP research. conformation disease caused by mutations in the membrane transporter gene.

RS V-25-5 RS V-25-6 PURINERGIC REGULATION OF PANCREATIC DUCTAL NEW INSIGHTS INTO BICARBONATE SECRETION BY SECRETION THE HUMAN AIRWAY CELL LINE CALU-3 Toan D Nguyen John William Hanrahan1, Jiajie Shan1, Junwei Huang1, Jie Liao1, Gastroenterology Section and Division, VA Puget Sound Health Care Renaud Robert1, Melissa L Palmer2, Scott C Fahrenkrug2, System and University of Washington School of Medicine, USA Scott M O'Grady2 ATP is not only an energy source but also an extracellular messenger acting 1Department of Physiology, McGill University, Canada, 2Departments of through purinergic receptors (P-R). Using I and Rb efflux (to assess Cl and K Physiology and Animal Science, University of Minnesota, USA channels) and Ussing chamber studies, we demonstrated that, in monolayers The Calu-3 epithelial cell line is widely used as a model for human airway submucosal of cultured non-transformed dog pancreatic duct epithelial cells (PDEC), glands but little is known about its transport properties under open circuit conditions. apical and basolateral P2Y2-R stimulate Cl/HCO3 exchange and Ca-activated Cell monolayers were cultured on porous supports at the air-liquid interface and K and Cl channels through increased intracellular Ca and CaM kinase II, studied under current clamp with apical pH-stat. Forskolin stimulated net secretion while basolateral P2Y11-R stimulate CFTR through increased cAMP. ATP - of HCO3 and increased the equivalent short-circuit current (eqIsc). eqIsc declined (> 10 μM) also stimulates exocytosis. With RT-PCR, western blotting, and - - in Cl -free solution to a level similar to that of net HCO3 flux. Forskolin stimulation immunostaining, we also established the expression of P2X1-R, P2X4-R, - + of eqIsc was nearly abolished by removing HCO3 or Na and was partially inhibited P2X5-R, and P2X7-R on human and dog PDEC. Through Ca photometry and - by adding basolateral bumetanide, indicating >66% of eqIsc was electrogenic HCO3 Ussing chamber studies, we also verified that these P2X-R function as ATP- secretion as reported previously. The prevailing model for Calu-3 can account for - gated cation channels. Based on luciferin-luciferase bioluminescence, we also HCO3 flux under Isc conditions but not the composition of secreted fluid, which - - established that certain bile acids (e.g. the conjugated dihydroxy bile acids, contained five times more Cl than HCO3 . Fluid transport was resistant to basolateral taurodeoxycholate and taurochenodeoxycholate), as well as hypo-osmolar bumetanide indicating NKCC1 plays little role. We propose a scheme in which the - - challenges (≥ 20%), stimulated ATP release from dog and human PDEC. This secretion of Cl -rich fluid is driven by electrogenic HCO3 transport. It features CFTR - - release is mediated through transport process(es) inhibited by ethanol and as the predominant apical exit pathway for both Cl and HCO3 and a crucial role for gadolinium. Thus, purinergic signaling is an autocrine/ pacracrine mechanism basolateral anion exchange under physiological conditions. Support: CCFF, CIHR, for the regulation of PDEC secretion. NIDDK(NIH)

IUPS 2009 July 27 - August 1, 2009 in Kyoto 83 RS V-26-1 RS V-26-2 HETEROMERIC AMINO ACID TRANSPORTERS: AMINO ACID TRANSPORT ACROSS CELLULAR PATHOLOGY AND STRUCTURE BARRIERS Manuel Palacin, Joana Fort, Merce Ratera, Nuria Reig, Francois Verrey Cesar del Rio, Paola Bartoccioni, Eva Valencia, Institute of Physiology and Zurich Center for Integrative Human Physiology, Lukasz Kowalczyk, Jose Luis Vazquez University of Zurich, Switzerland Molecular Medicine, Institute for Research in Biomedicine Barcelona, Small intestine and kidney proximal tubule epithelia are important cellular Spain barriers where transcellular (re)absorption of amino acid takes place. The defect of the luminal sodium-dependent amino acid transporter B0AT1 HAT are composed of two subunits (heavy and light) linked by a disulphide bridge. (SLC6A19) causes Hartnup disorder. This transporter and other members HAT are plasma membrane exchangers expressed in different tissues. In kidney, 0,+ + + of the SLC6 family require for their efficient surface expression in kidney systems b (rBAT/4F2hc) and y L (4F2hc/y LAT1) participate in renal reabsorption and intestine association with collectrin (Tmem27) or ACE2, respectively. of amino acids as revealed by its role in the inherited aminoacidurias cystinuria and 0 lysinuric protein intolerance. The structural information on HAT is limited. The Since some mutations of B AT1 differentially interact with collectrin heavy subunits are type II membrane glycoproteins, whereas the light subunits have and ACE2, a new mechanism leading the variable phenotype of Hartnup 12 transmembrane segments and are the catalytic part of the transporter. The atomic disorder is proposed. The closely related and previously orphan transporter structure of the ectodomain of the heavy subunit 4F2hc (CD98) revealed a [βα]8 XT2 (SC6A18) is shown to function as a higher affinity neutral amino acid transporter and to localize to the late kidney proximal tubule. We thus domain (TIM Barrel) and a domain C (β8) similar to bacterial amylases. Structural 0 information of the light subunits is scarce. To address this question we studied propose to rename it B AT3. The basolateral amino acid transport machinery prokaryotic homologues within their common APC superfamily of transporters: SteT of these epithelia is less well understood and involves the interplay of (Ser/Thr exchanger) and AdiC (arginine/agmatine exchanger). Recently, we obtained exchangers and facilitated diffusion pathways such that it can fulfil, next a projection map of the electronic density of AdiC from E. coli at 6.5 Å. Interestingly to the efflux function, a role for specialized metabolic tasks and cellular this projection is similar to that of other apparently unrelated transporters with the housekeeping. A similarly complex task is that of the brain microvascular “double inverted repeat” (LeuT, vSGLT, Mph1). Recent structural data and structure- endothelial cells of the blood brain barrier across which an amino acid function relationship studies will be presented. concentration gradient is also maintained.

RS V-26-3 RS V-26-4 TOWARDS THE SYTSEMS LEVEL OF PEPTIDE GLUTAMINE TRANSPORT AND AMMONIAGENESIS TRANSPORTER PHYSIOLOGY Carsten Alexander Wagner, Soline Bourgeois, Stephanie Busque Hannelore Daniel, Britta Spanier, Isabel Rubio-Aliaga, Institute of Physiology, University of Zurich, Switzerland Isabelle Frey The kidneys excrete acid in part by synthesizing and excreting NH binding Nutrition and Food Science, Technische Universitaet Muenchen, Germany 3 protons. NH3 synthesis in the proximal tubule requires uptake of glutamine Uptake of di- and tripeptides into epithelial cells is mediated by either and its subsequent metabolism. The Na-dependent glutamine transporter PEPT1, found in the intestine or by PEPT2 expressed kidney, lung and SNAT3 (Slc38a3) is expressed on the basolateral membrane of late proximal choroid plexus. Loss of the intestinal peptide transporter in C. elegans is accompanied by an increased expression of amino acid transporters and tubules and is induced during metabolic acidosis. Regulation of SNAT3 increased amino acid uptake capacity. However, body amino acid status is occurs in situations that are paralleled by increased ammoniagenesis and

Regular Symposia reduced whereas animals accumulate fat and have much larger intestinal urinary ammonium excretion (metabolic acidosis, potassium depletion, or fat droplets. We performed microarray, proteome and metabolite profiling high dietary protein intake). Transcriptome and proteome analysis indicates to understand the course of these metabolic pertubations. Major changes that increased SNAT3 expression occurs as part of a concerted regulation of in insulin-IGF signalling and in lipid, amino acid and glutathion (GSH) transport and metabolic pathways. We also report a mouse model carrying metabolism were observed. A mouse line lacking PEPT1 has no obvious an stop codon in the slc38a3 gene (Slc38a3-Q263X) leading to a truncated phenotypic alterations despite the fact that animals also have altered transporter. Homozygous SNAT3 mutant mice die about 16-18 days after metabolic parameters. In a mouse line deficient of the high-affinity peptide birth and show multiple abnormalities in ammoniagenesis and urea cycle. transporter PEPT2, analysis of kidney tissues revealed also changes in amino aicd status, a reduced capacity for reabsorption of cysteinyl-glycine Ammonium excretion into urine occurs as a multiple step process requiring produced by the breakdown of GSH via gamma-GT and changes in proteins the Rhesus protein Rhcg in the collecting duct. Ablation of Rhcg reduces and transcripts related to detoxification or reduction of oxidised proteins. The transepithelial NH3 permeability dramatically and causes metabolic acidosis + findings are the basis for describing on a systems level peptide transporter in mice. Thus, transporters for glutamine and NH3/NH4 may play a crucial biology in multicellular organisms. role in renal ammoniagenesis and final excretion.

RS V-26-5 RS V-26-6 DUAL ROLE OF THE Na+/H+ EXCHANGER ISOFORM 3 MOLECULAR ONTOLOGY OF ESSENTIAL AMINO ACID FOR PEPT1-MEDIATED H+/DIPEPTIDE COTRANSPORT TRANSPORT IN NATIVE MURINE INTESTINE Dmitri Y Boudko Mingmin Chen1, Anurag Singh1, Ulrike Dringenberg1, Sunil Yeruva1, Physiology and Biophysics, Rosalind Franklin University, Chicago Medical Jian Wang1, Regina Engelhardt1, Brigitte Riederer1, Michael P Manns1, School, USA Isabel Rubio-Aliaga2, Anna-Maria Naassl2, Manoocher Soleimani3, 18 L-amino acids, ahiral glycine, and imino proline provide the Gary Shull4, Hannelore Daniel2, Ursula Seidler1 proteinogenic foundation of extant Life and flux fixed nitrogen between 1Clinic for Gastroenterology, Hepatology and Endocrinology, Medical School cells in organism and between organisms in nutrient chains of the Biosphere. Hannover, Germany, 2Institute of Nutritional Sciences, Technical University of Small amino acids are ubiquitously biogenic, but the ~10 largest and most Munich, Germany, 3Departments of Medicine, University of Cincinnati, USA, 4 metabolically expensive in bacteria and plants became essential in animals. Departments of Cellular and Molecular Biology, University of Cincinnati, USA Hence, a comprehensive membrane transport network evolved to assure a To investigate NHE3 and PEPT1 coupling role in the native intestine. Luminal application of the thermodynamically and lipid phase unfavorable delivery of essential amino dipeptide Gly-Sar resulted in increased murine small intestinal fluid absorption in vivo, Isc response acids from nutrient sources to the metabolic framework of individual cells. in chambered jejunal mucosa, and a decrease in pHi in BCECF-loaded enterocytes in isolated However, the putative mechanism for comprehensive uphill delivery of jejunal villi. Genetic ablation of PEPT1 abolished Gly-Sar-induced fluid absorption, Isc response, and enterocyte acidification. Genetic ablation, pharmacological or second-messenger-mediated all essential amino acids remains enigmatic. We analyzed phylogenetic inhibition of NHE3 also reduced Gly-Sar-induced fluid absorption in vivo and Isc response in vitro, and physiological traits of the emerging Nutrient Amino acid Transporters - - but enhanced enterocyte acidification. Genetic ablation of the apical Cl /HCO3 exchanger Slc26a6 subfamily of the SLC6 family (a.k.a. NSS & SNF). The biological consensus also enhanced enterocyte acidification, but did not alter either Gly-Sar induced fluid absorption of identified and a few characterized mammalian, insect, and bacterial or Isc response. In native jejunal epithelium, the transmembrane proton gradient was irrelevant + + + + model NATs implies that they evolved and specialized to resolve raising for sustained H /dipeptide transport rates, but luminal Na concentration, enterocyte Na /K demands for conditionally essential and essential amino acids. Frequent gene ATPase activity, and luminal membrane potential were essential determinants. PEPT1 is the only H+/dipeptide uptake pathway, and we speculate that NHE3 is the major Na+-uptake pathway into duplications bound to a high plasticity of NAT phenotypes are reciprocal to enterocytes during dipeptide-induced volume increase, and therefore essential to the maintenance of the essential amino acids homeostasis. Expansions and extinctions of NATs membrane potential negativity. shaping metazoan transport and metabolism will be discussed.

84 IUPS 2009 July 27 - August 1, 2009 in Kyoto RS V-27-1 RS V-27-2 AT1 ANGIOTENSIN RECEPTORS IN THE KIDNEY: A CELL TO CELL COMMUNICATION IN RENIN KEY TO BLOOD PRESSURE CONTROL SECRETION 1 1 2 1 Thomas M Coffman Armin Kurtz , Lisa Kurtz , Cor deWit , Frank Schweda , 1 Medicine/Nephrology, Duke University Medical Center, USA Charlotte Wagner 1 2 Control of blood pressure by the renin-angiotensin system is dependent Physiology, University of Regensburg, Germany, Department of upon the AT1 angiotensin receptor. AT1 receptors are highly expressed in Physiology, University of Luebeck, Germany epithelium along various nephron segments, where they modulate solute and Renin producing cells (RC) in the kidney are coupled with their neighbouring fluid flux. Using renal cross-transplantation, our laboratory has previously cells by gap junctions. Gap junctions are formed by connexin proteins. demonstrated a non-redundant role for AT1A receptors in the kidney to RC in adult kidneys express connexin 37, 40 and 43, amongst which Cx40 regulate blood pressure and in the pathogenesis of hypertension. Along with appears to the predominant one. Cx40 expression is also associated with renin expression during kidney development and during recruitment of its actions in renal epithelia, angiotensin II is a powerful vasoconstrictor renin producing cells upon chronic challenge of the renin system. Genetic and its actions to increase vascular resistance are also mediated by AT1 deletion of Cx40 leads to massive hyperrenemia due to a defective negative receptors. However, the individual contributions of AT1A receptor actions feeback regulation of renin secretion by blood pressure and by angiotensin in specific cell lineages, such as renal epithelia or vascular smooth muscle, II. In addition, renin producing cells are dislocated from the walls of afferent to blood pressure regulation are difficult to assess in the intact organism. In arterioles into the periglomerular interstitium. Replacement of Cx40 by order to address this question, we generated a mouse line with a conditional Cx45, which forms smaller pores than Cx40, produces moderate changes of Agtr1a allele. In the presence of Cre recombinase, the Agtr1a locus is the renin system in adult mice, suggesting that Cx45 can substitute for the excised generating a null mutation. These mice have now been crossed with function of Cx40 in the control of the renin system at least under normal other transgenic lines expressing Cre recombinase in smooth muscle or in conditions. renal epithelial cell lineages. The phenotypes of these animals and their Ongoing work in our lab aims to identify the signals that pass through the physiological implications will be discussed. gap junctions of RC in order to make the renin system work properly.

RS V-27-3 RS V-27-4 ROLE OF ALDOSTERONE IN THE PATHOGENESIS OF THE (PRO)RENIN RECEPTOR:FACTS AND MYTHS RENAL INJURY Genevieve Nguyen Akira Nishiyama, Daisuke Nakano, Hirofumi Hitomi Inserm Unit 833 and College de France, Institut National de la Sante et de Department of Pharmacology, Kagawa University Medical School, Japan la Recherche Medicale, France Recent studies have indicated the role of aldosterone and mineralocorticoid The (pro)renin receptor [P)RR] binds renin and its proenzyme inactive form, receptor (MR) in the pathogenesis of renal injury. We have demonstrated in prorenin. Receptor-bound prorenin becomes enzymatically active and the binding activates the MAP kinases ERK1/2 and p38 pathways leading to rats that chronic treatment with aldosterone results in severe proteinuria and the up-regulation of profibrotic and of cyclo-oxygenase 2 genes expressions renal injury, characterized by glomerular changes, tubulointerstitial fibrosis independently of angiotensin II generation. There is a lot of interest for the and collagen accumulation. We also observed increased reactive oxygen potential role of (P)RR in organ damage in particular in hypertension and species (ROS) generation and activities of mitogen-activated protein kinases in diabetes. But if it is admitted that (P)RR has improved our understanding (MAPKs) and Rho-kinase in renal tissues. Treatment with a selective MR of the physiology of the tissue RAS, its role in pathology is far from being antagonist, eplerenone, prevented elevation of ROS levels and activities of demonstrated. Transgenic animals overexpressing (P)RR in smooth-muscle MAPK and Rho-kinase, as well as ameliorating renal injury. In vitro studies cells or ubiquitously develop high blood pressure or glomerulosclerosis and revealed that MRs are highly expressed in cultured glomerular mesangial increased (P)RR expression was reported in models of hypertension or kidney cells and renal fibroblasts. Aldosterone induces renal cellular injuries through damage. However, the definite proof of the role of (P)RR in disease such NADPH oxidase-dependent ROS production and/or MAPK and Rho-kinase as improvement of the disease by tissue- specific ablation of (P)RR or by Regular Symposia activation. Aldosterone-induced cellular injuries were markedly attenuated administration of a specific (P)RR antagonist, is still missing. Furthermore, by treatment with eplerenone. These data suggest that aldosterone induces the difficulty in generating (P)RR floxed mice and the impossibility of renal injury through activation of MRs, and support the notion that MR generating (P)RR null mice, a situation that has never happened before for blockade has beneficial effects on aldosterone-dependent renal injury through other components of the RAS, suggest that (P)RR has additional essential mechanisms that cannot be simply explained by hemodynamic changes. cellular functions.

RS V-27-5 RS V-27-6 IN VIVO RENIN IMAGING AND METABOLIC CONTROL PATCH CLAMP ANALYSIS OF ION CHANNELS IN OF RENIN RELEASE JUXTAGLOMERULAR (JG) GRANULAR CELLS Janos Peti-Peterdi, Ildiko Toma, Jung Julie Kang, Ole Skott, Boye L. Jensen, Ulla G. Friis Sarah Laurin Vargas Department of Physiology and Pharmacology, University of Southern Physiology and Biophysics, University of Southern California, USA Denmark, Denmark Renin release from the juxtaglomerular apparatus (JGA) is the rate-limiting Current-voltage (I-V) relationships of rat and mouse JG cells demonstrate step of the activation of the renin-angiotensin system (RAS). We applied outward rectification at positive potentials and limited net currents between multiphoton fluorescence imaging to visualize renin content, release and -30 mV and +10 mV. Pharmacological and molecular characterizations show tissue activity in the intact Munich-Wistar rat kidney in metabolic diseases. In that the outward currents are mainly carried through outwardly rectifying addition to the JGA in diabetes, renin was also found in the renal collecting potassium channels of the calcium and cAMP-sensitive zero-splice variant duct (CD) mainly in the form of prorenin. Angiotensin II differentially of BKCa. These channels are important for the resting membrane potential, regulated the synthesis of JGA (inhibition) and CD (stimulation) renin. and they hyperpolarize JG cells after stimulating renin release by increases + GPR91, a novel metabolic receptor that binds the TCA-cycle intermediate in intracellular cAMP. Voltage-dependent K channels play a minor role succinate was found in the JGA, in cells of the macula densa and in for the membrane potential. There is evidence in JG cells for hormonally glomerular endothelial cells. GPR91 activation by either vascular or tubular regulated inward rectifier (Kir) K-channels, and for KATP channels that may succinate caused renin release via a novel paracrine signaling mechanism couple cellular metabolism to membrane potential. The JG cells also exhibit 2+ which involves [Ca ]i, MAP kinases, COX and NOS activation, release voltage-dependent calcium currents that can be pharmacologically and and action of PGE2 and NO on JGA cells. Similar to succinate, uric acid, molecularly characterized to be of the L-type (Cav 1.2) but their functional another major metabolic end product directly caused JGA renin release via role is yet uncertain. In addition, renin-producing cells possess calcium- a macula densa-dependent mechanism. Our studies showed that metabolic activated chloride channels whose activation may lead chloride efflux and intermediates that accumulate in diabetes and hyperuricemia modulate JGA depolarization after exposure JG cells to calcium-mobilizing hormones. functions, regulate GFR and renin release, through their direct effects on the While much is known about ion channels in JG cells, much still remains to vascular endothelium and the macula densa. be learnt about their functional roles.

IUPS 2009 July 27 - August 1, 2009 in Kyoto 85 RS V-28-1 RS V-28-2 SIGNALLING AT TIGHT JUNCTIONS AND REGULATION EPITHELIAL INTERCELLULAR JUNCTION PROTEINS: OF GENE EXPRESSION IN EPITHELIAL CELLS TO SERVE AND PROTECT Karl Matter, Maria Susana Balda Asma Nusrat, Eric A Severson, Charles A Parkos Department of Cell Biology, UCL Institute of Ophthalmology, University Pathology & Lab. Medicine, Cell Biology, Emory University, USA College London, UK The gastrointestinal epithelium interfaces luminal contents and underlying Tight junctions crucial for the formation of epithelial barriers and recruit tissue compartments. Pivotal epithelial barrier properties are achieved by signalling proteins that participate in the regulation of cell proliferation and intercellular tight junctions (TJs) that do not merely regulate paracellular differentiation. These signalling proteins include components that regulate permeability, but also posses signaling properties that determine cell established signalling cascades as well as dual localisation proteins that can fate. A classic example of a TJ protein with such diverse functions is associate with junctions as well as travel to the nucleus where they regulate Junctional Adhesion Molecule-A (JAM-A). JAM-A knockout mice have gene expression. The latter class of proteins and their partners participate in increased intestinal epithelial permeability and mucosal inflammation. various steps of gene expression, ranging from regulation of transcription to mRNA processing. The Y-box transcription factor ZONAB/DbpA is one such Additionally, JAM-A regulates epithelial cell migration that is dependent protein and interacts with the junctional adaptor ZO-1. ZONAB promotes on cis-dimerization of its membrane-distal immunoglobulin-like loop and proliferation, and binding to ZO-1 results in cytoplasmic sequestration interactions of a C-terminal PDZ binding motif with scaffolding proteins. and, hence, inactivation of its transcriptional activity and inhibition of Studies to define the mechanisms of JAM-A function have revealed that proliferation. To understand how ZONAB functions, we employed different it physically and functionally associates with the PDZ domain containing approaches to identify interaction partners, regulatory mechanisms and target scaffolding proteins, Afadin and the guanine nucleotide exchange factor, genes, as well as assays to monitor epithelial proliferation and migration, PDZ-GEF2. In a dimerization-dependent fashion, we have determined gene expression and differentiation, as well as responses to different stimuli. that JAM-A interactions with afadin and PDZ-GEF2 regulate Rap1A Our data point to molecular mechanisms by which the ZONAB pathway and cell surface levels of βintegrin which play key roles in epithelial cell contributes to epithelial differentiation, homeostasis and the cellular stress differentiation and migration. This study further emphasizes the critical response. pluripotent function of epithelial TJ proteins.

RS V-28-3 RS V-28-4 MANNER OF CLAUDIN ASSEMBLY WITHIN TIGHT TIGHT JUNCTION REMODELING PARTICIPATES JUNCTION STRANDS IN CYTOSKELETALLY-MEDIATED BARRIER Mikio Furuse REGULATION: THE UNIQUE ROLE OF ZONULA Division of Cell Biology, Kobe University Graduate School of Medicine, OCCLUDENS-1 (ZO-1) Japan Jerrold Turner Claudin family membrane proteins form tight junction (TJ) strands, the core Department of Pathology, The University of Chicago, USA structure of TJs within the plasma membrane, and determine the barrier The tight junction is composed of transmembrane and membrane-associated properties in the paracellular pathway. In general, multiple claudin types are proteins previously thought to form stable complexes. Our recent data (Shen et co-expressed in individual cells and the combinations and proportions of al., J Cell Biol, 2008) indicates that this is not the case. While claudin-1 is stably claudin types vary among cell types. Recent gain-of-function and loss-of- anchored at the tight junction, the majority of occludin diffuses rapidly within Regular Symposia function studies have revealed that each claudin has its unique characteristic cell membranes. ZO-1 is also highly dynamic and exchanges between membrane as a barrier or a channel with charge selectivity within TJs. Therefore the and intracellular pools by an energy-dependent process. To determine whether this continuous molecular remodeling contributes to function, we assessed the complex expression pattern of claudins creates the functional diversity in effects myosin light chain kinase (MLCK) inhibition, which enhances barrier the barrier/channel properties of TJs depending on cell types. Many claudins function, on tight junction protein dynamics. MLCK inhibition had no effect reconstitute TJ strands in TJ-negative fibroblastic cells when overexpressed. on exchange of tight junction-associated actin, occludin, or claudin-1, but the Utilizing this experimental system, we demonstrated that claudins assemble ZO-1 immobile fraction was increased by MLCK inhibition. In contrast, a ZO-1 in a homotypic and heterotypic manner between cells, and polymerize in a mutant lacking the actin binding region (ABR) was not immobilized by MLCK homomeric and heteromeric manner within a cell, although the compatibility inhibition. Moreover, expression of the free ABR limited exchange of full-length depends of claudin types. Furthermore, some claudin types are unable to ZO-1 (in the absence of MLCK inhibitors) and free ABR expression prevented form TJ strands by themselves but appear to be recruited to TJs by the lateral barrier enhancement induced by MLCK inhibition. The data demonstrate that interaction with other claudin types. Based on our cell biological studies, I ZO-1 stabilization at the tight junction is dependent on the ABR and contributes will overview the structure of TJ strands in terms of claudin assembly. to cytoskeletally-mediated barrier regulation.

RS V-28-5 RS V-28-6 PARACELLULAR CHANNEL-LIKE ROLE OF CLAUDINS STRUCTURE-FUNCTION STUDIES OF THE CLAUDIN- : KNOCKOUT-MICE ANALYSES BASED ION PORE 1 1 2 Sachiko Tsukita , Atsushi Tamura , Shoichiro Tsukita Alan S. L. Yu 1Lab. of Biological Science,Graduate School of Frontier Biosciences and 2 Departments of Medicine and Physiology, University of Southern California Graduate School of Medicine, Osaka University, Japan, Department of Keck School of Medicine, USA Cell Biology, Graduate School of Medicine, Kyoto University, Japan Paracellular ion transport in epithelia is mediated by pores formed by In the epithelial cell sheets, evidence has accumulated that the belt-like tight junction, zonula occludens, acts as a paracellular permselective barrier. members of the claudin family. The degree of selectivity and the molecular Among the tight junctional integral membrane proteins, claudins are a protein mechanism of ion permeation through claudin pores are poorly understood. family composed of at least 24 members and are regarded as essential factors By expressing a high-conductance claudin isoform, claudin-2, in high- for the formation of tight junctions in a way that paracellular barrier is not resistance MDCK cells under the control of an inducible promoter, we were formed without claudins. It is supposed that the claudin-based paracellular able to quantitate claudin pore permeability. Claudin-2 pores were found to barrier is not a simple barrier that inhibits paracellular permeability but is be narrow, fluid-filled, and cation-selective. Charge selectivity was mediated a sophisticated barrier that allows the selective permeation of specific ions by the electrostatic interaction of partially dehydrated permeating cations and solutes in a claudin-dependent way. However, the critical molecular with a negatively charged site within the pore that is formed by the side mechanism for in vivo permselectivity of the claudin-based paracellular chain carboxyl group of aspartate-65. We have now performed cysteine barrier and its in vivo physiological meaning are still the subject to be mutagenesis of residues in the 1st extracellular loop and investigated their clarified. In this respect, the functional analyses on the knockout mice of accessibility to modification and block by methanethiosulfonate reagents. various kinds of claudin-family members are highly potent. Here, we found This technique is allowing us to map the location of these residues to identify the in vivo role of claudin-15 and related claudins in paracellular channel- like activity of tight junctions to organize the microenvironment of small those that line the claudin-2 pore and to develop pore-blocking reagents to intestinal mucosa, possibly regulating cell proliferation. probe paracellular ion pore function.

86 IUPS 2009 July 27 - August 1, 2009 in Kyoto RS V-29-1 RS V-29-2 INCRETIN HORMONES GIP AND GLP-1 DISTRIBUTIONS OF SHORT-CHAIN FATTY ACID Timothy James Kieffer1, Yukihiro Fujita2, Michael Riedel1, RECEPTORS GPR41 AND GPR43 IN THE HUMAN Rhonda Wideman1 COLON 1Departments of Cellular & Physiological Sciences and Surgery, University Hideaki Tazoe, Yasuko Otomo, Izumi Kaji, Ryo Tanaka, 2 of British Columbia, Canada, Asahikawa Medical College, Japan Shin-ichiro Karaki, Atsukazu Kuwahara Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like Department of Environmental Health Sciences, Institute for Environmental peptide-1 (GLP-1) are gut hormones that promote brisk secretion of insulin Sciences, Japan following ingestion of a meal. Interestingly, both GIP and GLP-1 are also Short-chain fatty acids (SCFAs), including acetate, propionate and butyrate, are the produced in islets, perhaps functioning to improve islet survival and function. major anion in the monogastric mammalian large intestine, and we have previously We are exploring various methods to harness the therapeutic potential of reported that SCFA controls colonic motility [Neurogastroenterol Motil 17:585-94 these hormones. By viral delivery of prohormone convertase 1 (PC1) to islet :2005]. However, we could not define which types of receptors are involved in the alpha-cells, we have been able to increase islet GLP-1 production and further regulation of intestinal motility. Recently, SCFA receptors GPR41 and GPR43 were enhance islet survival and function in transplant models. Mice treated with identified from orphan G protein coupled receptors. These receptors are considered an adeno-associated virus engineered to express GLP-1 specifically in beta- to be chemical sensors to detect/monitor SCFAs in intestinal lumen. In the present cells displayed protection from streptozotocin-induced diabetes and improved study, therefore, we investigated GPR41 mRNA and protein expressions in human colon by reverse transcriptase-polymerase chain reaction, Western blot analysis, glucose tolerance. Based upon the similar circulating profiles of GIP and and immunohistochemistry. As results, GPR41 mRNA and protein were expressed insulin, we investigated the potential of harnessing the GIP producing gut in intestinal mucosa, and GPR41 protein was expressed in enteroendocrine cells K-cells as surrogate beta-cells for insulin replacement therapy. Insulin containing peptide YY but not serotonin. Moreover, we investigated the quantitative expression in K-cells of transgenic mice protected mice from diabetes when analysis for distributions of GPR41 and GPR43 proteins in human intestinal mucosa. beta-cells were destroyed. Notably, the insulin-secreting K-cells were able The results showed that GPR41 and GPR43 expressions were different among the to escape the autoimmune-mediated destruction that targeted beta-cells in the intestinal regions. In conclusion, GPR41- and GPR43-immunoreactive L cells may act NOD mouse model of type 1 diabetes. as luminal SCFA sensors, and their expressions differ in the intestinal regions.

RS V-29-3 RS V-29-4 INTESTINAL GLUCOSE SENSING AND REGULATION ROLE OF GHRELIN IN THE CENTRAL REGULATION OF GLUCOSE TRANSPORT OF FEEDING Soraya P Shirazi-Beechey Masamitsu Nakazato Department of Veterinary Preclinical Sciences, University of Liverpool, UK Division of Neurology,Respirology, Endocrinology and Metabolism + Department of Internal Medicine, Miyazaki Medical College, University of The Na /glucose cotransporter (SGLT1) is the major route for the transport Miyazaki, Japan of dietary sugars from the lumen of the intestine into enterocytes. Regulation The molecular mechanisms of enegy balance are coming to light by the of this protein is essential for the provision of glucose to the body and thus recent robust progresses in the molecular biology and neuroscience. The is important for maintenance of glucose homeostasis. We have shown that gastrointestinal tract produces a large array of substances to regulate feeding. dietary sugars and artificial sweeteners increase SGLT1 expression and Hypothalamus, the center of energy homeostasis, receives information related glucose absorptive capacity in wild type mice, but not in T1R3-, a subunit of to satiety and fast from the body and other brain regions, and integrate them. the sweet taste receptor, and α-gustducin-, the partner G-protein, knockout Ghrelin, a 28-amino acid peptide, is produced mainly by stomach cells and mice. We proposed that T1R2+T1R3, the sweet taste receptor expressed on stimulates hunger centers in the hypothalamus. Peripherally administered the luminal membrane of villus enteroendocrine cells, senses the lumenal ghrelin preferentially enhanced carbohydrate intake. The plasma ghrelin glucose concentration. Lumenal glucose above a threshold level activates, in concentration is upregulated before meal and decreases after feeding. At present ghrelin is the only peptide to transmit hunger information from enteroendocrine cells, a signalling pathway involving T1R2+T1R3, gustducin the periphery to the brain. We have clarified that the vagal afferent is the and other signalling elements. This results in secretion of candidate gut major pathway conveying signals of gut hormones to the brain. Clinical Regular Symposia hormones. These hormones bind to receptors on target cells and, through a trials to investigate therapeutic potentials of ghrelin have started for patients paracrine mechanism, enhance SGLT1 expression. In my talk I shall describe with anorexia nervosa, cancer, chronic heart failure, chronic obstructive our recent findings on kinetics of SGLT1 upregulation in terms of time and pulmonary disease, or chronic respiratory infections. The effects of ghrelin threshold glucose concentration. I will also discuss the pathway by which the on energy balance have implications in its potential use as a therapeutic candidate gut hormone affects the expression of SGLT1 in enterocytes. target associated with cachexic and/or inflammatory diseases.

RS V-29-5 RS V-29-6 LUMINAL L-GLUTAMATE SENSING VIA MULTI GASTROINTESTINAL CHEMOSENSATION: ROLES OF GLUTAMATE RECEPTORS ENHANCES MUCOSAL TASTE CELLS OF THE GUT AND ENDOCRINE CELLS DEFENSES IN RAT DUODENUM OF THE TONGUE Yasutada Akiba1, Chikako Watanabe2, Jonathan D Kaunitz1 Robert F Margolskee, Zaza Kokrashvili, Bedrich Mosinger 1Medicine, VAGLAHS, CURE/UCLA, Brentwood Biomed Res Inst, USA, Neuroscience, Mount Sinai School of Medicine, USA 2 UCLA Med, USA We previously observed that gustducin, T1r taste receptors and other taste Expression of taste receptor families in the gut mucosa suggests a physiologic basis signalling proteins are expressed in duodenal enteroendocrine L cells that for local and early detection of a meal. Luminal L-glutamate (L-Glu), the primary express glucagon-like peptide 1 (GLP-1). Knockout mice lacking gustducin nutrient conferring umami taste, increases epithelial intracellular pH (pHi) and mucus or T1r3 have deficiencies in secretion of GLP-1, and in regulation of plasma gel thickness (MGT) in rat gastroduodenum. We further investigated the mechanisms insulin and blood glucose. Results from in vivo and ex vivo preparations in of L-Glu effects on mucosal defenses. mice implicate L cell-expressed gustducin and the sweet receptor in sugar We measured pHi and MGT in anesthetized rat duodenum perfused with L-Glu (10 sensing in gut. We speculated that if the gut’s L cells expressed taste elements mM). Duodenal bicarbonate secretion (DBS) was measured with flow-through pH/ then taste cells might in turn express GLP-1 and other L cell hormones. CO2 electrodes. Taste cells were found to express GLP-1, glucagon, PYY and other gut L-Glu-induced pHi increase and mucus secretion were inhibited by phospholipase C inhibitor U73122. L-Glu effects on pH and MGT were mimicked by metabotropic hormones. Patterns of expression indicated that gustducin-expressing type i II taste cells and other subtypes of taste cells express GLP-1. In wild-type Glu receptor 4 (mGluR4) agonist L-AP-4 and inhibited by mGluR4 antagonist MAP4. Luminal high Ca2+ or spermine acidified the cells and increased MGT, unlike L-Glu. In mice application of glucose to the tongue induced a rapid elevation of blood contrast, L-Glu + IMP enhanced DBS only in combination, suggesting the synergism, GLP-1. Stimulation of taste cell explants with glucose led to release of GLP-1 into the medium. However, glucose stimulation of gustducin-null likely via T1Rs. L-AP-4 or mGluR1 agonist S-DHPG had no effect on DBS. Pre- perfusion of L-Glu prevented acid-induced cellular injury. mice did not lead to significant release of GLP-1 from taste cells in vivo or Luminal L-Glu differentially activates duodenal defense mechanisms via mGluR4 and in explants. We conclude that the cephalic phase rise in circulating GLP-1 T1Rs. L-Glu receptors may thus serve as ‘early warning’ system for subsequent acid depends on direct release of GLP-1 from gustducin-expressing taste cells into exposure and protein digestion in the duodenum. the bloodstream.

IUPS 2009 July 27 - August 1, 2009 in Kyoto 87 RS V-30-1 RS V-30-2 BLOCKING THE PORE OF THE CLC-0 CHLORIDE DRUG THERAPY FOR CYSTIC FIBROSIS BASED CHANNEL WITH AMPHIPHILIC BLOCKERS ON A RATIONAL UNDERSTANDING OF THE CFTR Tsung-Yu Chen, Xiao-Dong Zhang, Pang-Yen Tseng, CHLORIDE CHANNEL Wei-Ping Yu David Noel Sheppard Ctr for Neuroscience and Dept of Neurology, University of California, Department of Physiology & Pharmacology, University of Bristol, UK Davis, USA The common genetic disease cystic fibrosis (CF) is caused by mutations CLC-0 is the prototype molecule of the CLC family, which consists of epithelial in the cystic fibrosis transmembrane conductance regulator (CFTR), an chloride channels and transporters. Recently, clofibric-acid compounds, such as epithelial Cl- channel with complex regulation. The most common CF parachlorophenoxy acetate (CPA), were found to block the pore of CLC-0. We mutation, F508del-CFTR, disrupts the expression and function of CFTR in examined the mechanism of CPA blocking in pore-open mutants of CLC-0 in at least two ways. First, F508del causes misfolding of CFTR protein, leading which two critical positions in the pore were altered. When a deep pore residue, to the retention of the vast majority of the mutant protein in the ER and its E166, was mutated, the CPA affinities in various E166 mutants could vary by 3 degradation by the proteasome. Second, F508del-CFTR Cl- channels exhibit orders of magnitude, depending on the side-chain volume and the hydrophobicity a profound defect in channel gating. Thus, restoration of channel function of the introduced residue. On the other hand, mutations at the intracellular pore to F508del-CFTR requires the use of drugs to deliver the mutant protein to entrance generated only a modest effect on the blocking affinity, although the its correct cellular location (termed CFTR correctors) and rescue defective mutations affect the association and dissociation rates of the block. In addition, channel gating (termed CFTR potentiators). A number of small molecules various amphiphilic compounds, including fatty acids and alkyl sulfonates, can have been identified (e.g. VRT-325) that rescue the cell surface expression also block the pore-open mutants of CLC-0, and the blocking affinity increases of F508del-CFTR. By contrast, large numbers of small molecules have with the length of these amphiphilic blockers. These results suggest that the CPA been identified (e.g. genistein), which act as CFTR potentiators, rescuing block of CLC-0 is similar to the block of voltage-gated K+ channels by long- the gating defect of F508del-CFTR. Many of these agents interact directly chain quaternary ammoniums or by the inactivation ball peptide: the blocker with CFTR to modulate channel gating. This presentation will highlight first “dock” at the pore entrance, and then enters the pore to interact with a how knowledge of the molecular pharmacology of CFTR is leading to the hydrophobic region of the pore. development of new therapies for CF.

RS V-30-3 RS V-30-4 DISTINCT FUNCTIONAL ROLES OF CFTR’S TWO ATP PROSTONES AS CLC-2 CHANNEL ACTIVATORS FOR BINDING SITES: WHY DO WE CARE? TREATMENT OF DISEASES AND DISORDERS Tzyh-Chang Hwang John Cuppoletti1, Ryuji Ueno2 Department of Medical Pharmacology and Physiology, University of 1Department of Cellular and Molecular Physiology, University of Cincinnati, Missouri-Columbia, USA USA, 2Sucampo Pharmaceuticals, Inc., Bethesda, MD, USA As a member of the ABC Transporter Family, CFTR contains two nucleotide Prostones are a class of bicyclic fatty acids. Lubiprostone is a prostone binding domains (NBD1 and NBD2). The Walker A and B motifs of one approved for the treatment of chronic idiopathic constipation (CIC) and NBD and the signature sequence of the partner NBD form the ATP binding irritable bowel syndrome with constipation (IBS-C) in the United States. pocket ATP upon dimerization of two NBDs in a head-to-tail configuration. Lubiprostone activated apical Cl- currents in T84 cells at <100 nM and these We have identified a critical role of the NBD2 binding site in channel are ablated with siRNA to ClC-2. Recombinant human ClC-2 is activated

Regular Symposia opening by ATP. This idea is further supported by the observation that the by lubiprostone (EC50 = 20 nM). CFTR was not activated by 1 micromolar G551D mutation, a disease-associated mutation at the signature sequence lubiprostone. Lubiprostone increased single 3-4 pS Cl<- (ClC-2) channels of NBD1, completely eliminates ATP-dependent gating. Interestingly, the in ClC-2 expressing HEK293 cells and the apical membrane of Xenopus activity of G551D can be enhanced by N6-phenylethyl-ATP, a high affinity A6 cells, at concentrations <100 nM. Lubiprostone showed increased ATP analog, and related analogs via the NBD1 site. Fortuitously, we found salt and water transport into the intestinal lumen in animal studies and that cadmium can also increase G551D activity by forming a metal bridge from basolateral to apical surfaces of T84 cultures. ClC-2 activation by with endogenous cysteine(s). Cadmium can serve as an effective ligand lubiprostone does not involve increases in intracellular cAMP or Ca2+, when a cysteine residue is engineered in the signature sequence of NBD1, activation of prostaglandin receptors or PKA. Prostones protect against suggesting that this signature sequence acts as a switch that transduces indomethacin-induced cellular injury. In Caco-2 cells and porcine ileum the signal of ligand binding to the channel gate. Unexpectedly, removing ClC-2 is present at the tight junctions. In porcine ileum, lubiprostone all cysteines in NBD2 does not affect the action of cadmium on G551D. promotes repair of epithelial barrier function impaired by ischemia, Identification of endogenous cysteine(s) will provide mechanistic insights accompanied by movement of ClC-2 to the tight junctions, a mechanism into the precise mechanism of ligand-dependent gating for CFTR. which may contribute to the treatment of IBS-C.

RS V-30-5 RS V-30-6 EFFECTS OF PROSTONES ON TIGHT JUNCTIONS CLC-2 KNOCKOUT MICE HAVE IMPAIRED POST- Atsushi Tamura1, Ryuji Ueno2, Sachiko Tsukita1 ISCHEMIC INTESTINAL BARRIER RECOVERY 1Lab. of Biological Science, Graduate School of Frontier Biosciences/ Anthony Blikslager 2 Graduate School of Medicine, Osaka University, Japan, Sucampo Department of Clinical Sciences, North Carolina State University, USA Pharmaceuticals, Incorporated, Bethesda, Maryland, USA We have previously shown that inhibition of ClC-2 retards recovery of The tight junction is an essential structure for maintaining homeostasis in barrier function in ischemic-injured intestine. We studied barrier function multicellular organisms, forming a paracellular permselective barrier between in ischemic-injured intestine of ClC-2 knockout mice. Intestinal segments epithelial cells. CLC2 chloride channels which are reportedly localized in from wild type (WT) and knockout mice siblings were subjected to in vivo tight junctions as well as basolateral or apical cell membranes, although mesenteric ischemia for 45-minutes, after which tissues were recovered for

the function of CLC2 in tight junctions is unclear at present. It is reported up to 3 hours and blood-to-lumen clearance of 3H-mannitol was performed. that several other ion channels, such as CFTR, also exist in tight junctions, Histological, electron and confocal microscopic examination was performed. suggesting that chloride channels may have some important biological role In WT mice, mannitol flux did not differ significantly between control and within them. Based on recent studies on molecular architecture of tight ischemic-injured intestine. Alternatively, in ClC-2 knockout mice, mannitol junctions, the multi-gene family of claudins are likely the most essential flux was significantly higher at 1.5 and 3 hours recovery compared to that of components. Based on the knockout mice study of claudins, such as control intestine by 226% and 122% respectively. Histological examination claudin-15, we believe that the ion microenvironment around tight junctions of intestinal tissues revealed near complete epithelial restitution. However, plays an important role in cell proliferation and/or morphogenesis. Regarding electron microscopic and confocal microscopic examination revealed dilated this, the channels which are correlated with the tight junctional selective tight junctions in ischemic intestinal tissues from ClC-2 knockout mice as permeability could create organ-specific microenvironments around tight compared with WT ischemic loops after 3 hours of recovery. In vivo blood- junctions. Herein, we investigated the physiological and cell biological to-lumen mannitol clearance data demonstrated delayed recovery of intestinal correlation between CLC2 and tight junctions, and the effects of an activator barrier function in ClC-2 knockout mice after ischemic injury, associated of CLC2 for some pathological state,such as DSS colitis. with dilated tight junctions.

88 IUPS 2009 July 27 - August 1, 2009 in Kyoto RS VI-31-1 RS VI-31-2 TRANSLATIONAL APPROACH TO THE ROLE OF T-TYPE CALCIUM CHANNELS IN NEURONAL IDENTIFICATION OF T-TYPE CALCIUM CHANNEL FIRING AND EPILEPSY BLOCKERS TARGETING EPILEPSY 1 1 2 1 Gerald W Zamponi Terrance P. Snutch , Stuart M. Cain , Kim L. Powell , Esperanza Garcia , Physiology and Pharmacology, University of Calgary, Canada Elizabeth Tringham3, Hassan Pajouhesh3, Terence J. O'Brien2 1Michael Smith Laboratories, University of British Columbia, Canada, T-type channels are involved in the genesis of spike-and- wave discharges in 2Departments of Medicine, Surgery and Neurology, University of Melbourne, the thalamo-cortical network. The nervous system expresses 3 types of T-type 3 Melbourne, Australia, Neuromed Pharmaceuticals Inc, Vancouver, BC, Canada channels (Cav3.1, Cav3.2 and Cav3.3) with distinct cellular and subcellular Genetic Absence Epilepsy Rats from Strasbourg (GAERS) are a well validated distributions and functions in the CNS. Cav3.1 channels are important for model of absence epilepsy used in epilepsy research for more than two decades. burst firing, and their genetic ablation protects mice from absence seizures. Electroencephalographic recordings during seizures in GAERS show generalized Point mutations in Cav3.2 have been linked to the development of childhood spike-and-wave discharges closely resembling those seen during human absence seizures. We describe a homozygous missense mutation in the Cav3.2 T-type calcium absence epilepsy and other forms of idiopathic generalized epilepsies. We channel gene in the GAERS model. The GAERS Cav3.2 mutation (gcm) segregates functionally characterized a series of these mutations, revealing that many co-dominantly with the number of seizures and time in seizure activity. Exon of them cause gain of function effects on channel gating. We identified a scanning of thalamic cDNA identified a number of thalamic Cav3.2 splice variants. novel pathway for regulation of T-type channel activity. Activation of LPA Gcm introduced into the splice variants acts epistatically requiring the presence of receptors inhibits Cav3.1 and Cav3.3 via Rho-associated kinase (ROCK), via exon 25 to produce an apparent gain-of-function phenotype. In order to translate this a G12/13 linked pathway. This in turn results in the inhibition of burst firing molecular genetic information into new therapeutic approaches targeting epilepsy, a of habenular neurons which rely largely on Cav3.1 for their burst activity. In high-throughput fluorescent-based screening assay was developed towards identifying T-type calcium channel blockers. Combining this functional cell-based assay with a contrast, there was a functional upregulation of Cav3.2 channels. Together rationale drug design approach, we have identified novel, high affinity T-type calcium with recent reports showing that inhibition of ROCK reduces seizure activity blockers. The effects of administration of some of the novel T-type calcium channel in mice, this latter finding may implicate ROCK mediated T-type channel antagonists on GAERS and other animal epilepsy models will be described. modulation as a possible factor in seizure genesis.

RS VI-31-3 RS VI-31-4 T-TYPE CALCIUM CHANNELS IN THE GENERATION T-TYPE CALCIUM CHANNEL CONTROL OF OF SLEEP RHYTHMS CARDIOVASCULAR FUNCTION Hee-Sup Shin Philippe LORY, Joel NARGEOT, Matteo MANGONI Center for Neural Science, Korea Institute of Science and Technology, Physiology, IGF / CNRS UMR 5203, France Korea T-type calcium channels (T-channels) contribute to a wide variety of T-type calcium channels have been implicated as a pacemaker for brain physiological functions, especially in the nervous and cardiovascular rhythms during sleep. Mice lacking a1G T-type Ca2+ channels show a loss systems. Studies using knock-out mouse models have been instrumental of the thalamic delta (1-4 Hz) waves and, while slow (<1 Hz) rhythms were in documenting further the role of T-channels in heartbeat, vascular tone relatively intact, when compared to the wildtype during urethane anesthesia and cardiovascular diseases. Our recent studies have pointed out that and physiological NREM sleep. These results are consistent with the fact that genetic inactivation of the Cav3.1 T-channels in mice results in a significant a1G is the major T-type channels in the thalamocortical neurons where delta slowing of the heart rate and atrio-ventricular (AV) conduction. The lack of rhythms are believed to be generated. The results of analysis of the status of Cav3.1 channels in isolated sino-atrial node (SAN) cells induces slowing sleep spindles (7-14 Hz) in this mutant mouse have been less straightforward. of pacemaker activity through a reduction of the slope of the diastolic Based on the conventional idea on the generation and propagation of sleep depolarisation. This provides evidence for a direct contribution of Cav3.1 spindles, which counts on the T-type channel-mediated low threshold burst channels in the setting of the mammalian heartbeat by contributing to both firings of thalamocortical relay neurons, it may be predicted that sleep the diastolic depolarisation in the SAN and impulse conduction through the

spindles may not be observed on the cortical EEG in the a1G mutant mouse. AV node. In addition, several novel aspects of the regulation of T-channels Regular Symposia The EEG recording, however, showed that sleep spindles are observed on were identified these last years, including modulation by various endogenous the cortical EEG, albeit reduced in strength, in the mutant. We have analyzed ligands such as bioactive lipids, zinc, as well as G-protein and protein the thalamocortical circuit of the mutant with regard to spindle rhythms, kinases pathways. This provides new insights into their physiological and using various tools in vivo and in vitro. I will present those results and try to pathophysiological roles and opens new avenues for the design of novel discuss their physiological implications. therapeutic strategies targeting these channels.

RS VI-31-5 RS VI-31-6 REGULATION OF Cav1.2 BY PROTEIN KINASES Cav1 CHANNEL MODULATORY COMPLEXES IN Franz Hofmann1, Stefanie Fischer2, NEURONS Anne Blaich2, Katrin Domes2, Sven Moosmang2, Andrea Welling2 Amy Lee 1Department of Pharmacology, Tu Muenchen, Germany, 2Department of Molecular Physiology and Biophysics, University of Iowa, USA Pharmacology, Tu Muenchen, Germany Cav1 L-type Ca2+ channels play essential and diverse roles in the nervous Classical inactivation of the L-type Cav1.2 (a1C) calcium channel gene is system. In the brain, postsynaptic Cav1 channels participate in synaptic embryonal lethal before day 14.5 pc. We have generated a mouse line in plasticity and Ca2+-dependent gene expression while in the retina and which exon 14 and 15 of Cav1.2 is flanked by two loxP sites. These mice inner ear, presynaptic Cav1 channels mediate Ca2+ signals controlling were crossed with various lines expressing tissue-specific Cre recombinase. neurotransmitter release. These functions of Cav1 channels not only depend The off-springs of these mice had severe defects in insulin secretion, blood on their intrinsic biophysical properties but also their dynamic regulation by pressure regulation, intestinal muscle contraction, hippocampal memory an array of cellular influences including protein kinases, scaffolding proteins, acquisition. These mice lines showed that the putative T-type calcium and Ca2+-binding proteins. The cytoplasmic domains of the main pore channel blocker mibefradil lowers blood pressure by inhibition of the forming Cav1 alpha1 subunit provide binding sites for these modulators, Cav1.2 channel. Mutation of Ser1928 to Ala has no effect on beta-adrenergic permitting fast and localized regulation of Ca2+ entry. Through effects on stimulation of the cardiac Cav1.2 channel. We analysed further the coupling Cav1 gating, localization, and coupling to effectors, protein modulators between Cav1.2 and the CaM-kinase II. Facilitation of the Cav1.2 channel are efficiently positioned to adjust Cav1 Ca2+ signals. Evidence for protein by CaM-KII involves the phosphorylation of serine 1512 and 1570 in the interactions regulating Cav1 channels will be discussed in addition to the C-terminus of Cav1.2. Mutation of S1512/1570A results in viable off spring significance of these interactions for neuronal function. with no obvious phenotype. The mutated cardiac Cav1.2 shows no voltage- dependent but still frequency-dependent facilitation. The electrophysiological consequences of these mutations will be discussed.

IUPS 2009 July 27 - August 1, 2009 in Kyoto 89 RS VI-32-1 RS VI-32-2 VOLTAGE- AND [ATP]- DEPENDENT GATING OF THE VARIATIONS ON THE MELODIES OF MEMBRANE P2X2 ATP RECEPTOR CHANNEL VOLTAGE 1 2 1 1 Yoshihiro Kubo , Yuichiro Fujiwara , Batu Keceli , Koichi Nakajo Ehud Isacoff 1 Division of Biophysics and Neurobiology, National Institute for MCB, UC Berkeley, USA Physiological Sciences, Japan, 2Department of Integrative Physiology, Osaka University Graduate School of Medicine, Japan Voltage-sensing domains (VSDs) confer voltage dependence onto ion channel pores. In the classical Na+, K+ and Ca++ channels 4 VSDs work in concert P2X2 receptor is a ligand- gated cation channel activated by extracellular ATP. It to control gates in a pore located at the interface between the pore domains is known to be a trimer of 2-transmembrane type subunits. In spite of the absence of the four subunits. We have discovered that the situation is different in two of putative voltage sensor domain, it shows voltage- dependent activation upon recently isolated proteins: the sea squirt voltage-sensitive phosphatase, Ci- hyperpolarization at the steady- state after ATP application. We analyzed the VSP and the human voltage-gated proton channel, Hv1. Single molecule voltage- dependent gating of P2X2 quantitatively in Xenopus oocytes expression system, and observed that the voltage- dependent gating depends also on [ATP]; microscopy to count subunits combined with electrophysiology to count with the increase in [ATP], the conductance- voltage relationship shifted to the pores and voltage clamp fluorometry to detect protein motions associated depolarized potential and the activation speed was accelerated. We assumed a with functional transitions were used to study these proteins. Ci-VSP was three state model which consists of an ATP binding step and a following gating found to have broken with the “rule of four” and is a monomer, while step, and calculated the rate constants of the gating step at various membrane maintaining multi-step structural rearrangements of voltage sensing of potentials and in various [ATP] based on our experimental data and reported the tetrameric channels. Current experiments are aimed at understanding Kd values of ATP binding. We then carried out simulation with no voltage- coupling between the VSD and enzyme. In contrast to both the classical dependency in the parameters of the ATP binding step, and successfully tetrameric channels and the maverick Ci-VSP, Hv1 is a dimer, which reproduced [ATP] dependency of the voltage- dependent activation. Furthermore, contains two pores, one in the heart of each VSD. The two pores are separate by systematic mutagenesis study, we identified Gly344 in the middle of the in each dimer, and, indeed, single subunits can function on their own. Why 2nd transmembrane region as a gating hinge which is critical at the final step of then would Hv1 be a dimer? We have found that cooperativity between the voltage- dependent gating. subunits sculpts gating.

RS VI-32-3 RS VI-32-4 VOLTAGE SENSOR: FUNCTION AND STRUCTURE MOLECULAR MECHANISMS OF K+ CHANNEL Francisco Bezanilla ACTIVATION AND INACTIVATION GATING Biochemistry and Molecular Biology, University of Chicago, USA Eduardo Perozo The most studied sensor is in voltage-gated channels such as Na, Ca or K Biochemistry and Molecular Biology, University of Chicago, USA channels. The sensor resides in the first four transmembrane segments of We aim to understand the molecular mechanisms of gating in voltage- each of the subunits (or domains) of these proteins where the fourth segment dependent channels, by focusing on the analysis of K+ channel gating (S4) has a series of basic residues known to constitute most of the sensing in prokaryotic systems. Specifically we will address the following key charges. The movement of the sensing charges by an applied electric field questions: What are the molecular entities determining channel activity? How produces the gating (or sensing) currents that, through conformational energy is transduced into protein motion? How different parts of the channel

Regular Symposia changes, open or close the conduction pore in the channels or activate a interact to define open channel activity? We plan to study these problems by phosphatase in the case of Ci-VSP. The study of the function, using gating combining spectroscopic techniques (EPR NMR and Fluorescence), X-ray currents, is complemented with techniques including site-specific quenching crystallography and electrophysiological methods. changes of fluorescent probes, fluorescence resonance energy transfer, and Here we will discuss a series of new crystal structures of the K+ channel cysteine and histidine scanning of the residues of the sensor. These multiple KcsA, trapped in a series of functional conformations that include the open/ techniques, combined with the static picture of one particular conformation conductive and open/inactivated states, as well as a number of partial opening provided by X-ray crystallography has delineated a basis picture of the conformations. These structures, together with functional spectroscopic conformational changes that give origin to the function of the sensor. The and computational data have defined the molecular basis of activation and sensor has at least three major states that are voltage and time dependent. The inactivation gating in KcsA, and the coupling between the inner bundle gate conversion between states includes rotation, translation and tilt changes of and the selectivity filter. Our results clearly demonstrate that in KcsA, and the S4 segment and possibly a change of its secondary structure. Supported most likely other K+ channels, a variety of gating transitions are determined by NIH grant GM30376. by the conformational dynamics at the selectivity filter.

RS VI-32-5 RS VI-32-6 G PROTEIN-MEDIATED GATING OF POTASSIUM GPCR OLIGOMERS, TWO OR MORE FOR WHAT? THE CHANNELS: A SPECTROSCOPIC VIEW CASE OF THE GABAB RECEPTOR Eitan Reuveny Jean-Philippe R Pin1, Damien Maurel1, Laetitia Comps-Agrar1, 2 1 1 Biological Chemistry, Weizmann Institute of Science, Israel Eric Trinquet , Laurent Prezeau , Philippe Rondard 1Institute of Functional Genomics, Department of Molecular Pharmacology, Regulation of cellular excitability is mediated, in part, by the activation of 2 G protein coupled receptors. These receptors, through specific activation of CNRS, INSERM, University of Montpellier, France, CisBio International, associated G proteins, directly and/or indirectly modulate the activity of ion Bagnols sur Ceze, France channels and various transporters. One of the classical examples for such Cell surface proteins play key roles in cell-cell communication. They regulation is the activation of the G protein coupled potassium channels assemble into hetero-complexes that include different receptors and effectors. (GIRK) by G proteins. This activation involves the intimate association of Demonstrating and manipulating such protein complexes will certainly the G protein with the channels in concert with other intracellular factor to offer new therapeutic possibilities. Here we have developed reagents to stabilize the channel’s open conformation. To understand such interactions quantitatively analyze protein-protein interactions at the surface of living it is necessary to develop sensitive means for the detection of intrinsic cells, in a high throughput format. Using this approach we examined subtle motions of the channel molecule during activation, which can be whether G-protein coupled receptors (GPCRs) are monomers or assemble acomplished using FRET as a molecular ruler for such motions. One of into dimers or larger oligomers, a matter of intense debate. We bring new the limitations of such an approach is the fact that it is rather difficult to evidence for the oligomeric state of both class A and class C GPCRs. We specifically fluorescently label the cytosolic face of membrane proteins in also report a different quaternary structure of the GPCRs for the two major situ. To overcome this limitation, genetically encoded labeling is used in neurotransmitters. Whereas metabotropic glutamate receptors assemble conjunction with membrane restricted FRET measurements. An example into strict dimers, the GABAB receptors spontaneously form dimers of will be given using the GIRK channel as a model for a G protein effector, heterodimers, offering a way to modulate G-protein coupling efficacy. its intrinsic rearrangements and its mode of association with G proteins and This approach will be useful in systematic analysis of cell surface protein other signaling molecules. interaction in living cells.

90 IUPS 2009 July 27 - August 1, 2009 in Kyoto RS VI-33-1 RS VI-33-2 REGULATION OF STORE-OPERATED CALCIUM ENTRY CALCIUM SIGNALLING IN LYMPHOCYTES: STIM AND James W Putney ORAI Laboratory of Signal Transduction, National Institute of Environmental Anjana Rao, Sonia Sharma, Yubin Zhou, Paul Meraner, Health Sciences - NIH, USA Hyoung Kwon, Patrick G. Hogan Activation of phospholipase C results in release of intracellular Ca2+ and Department of Pathology, Harvard Medical School, USA activation of Ca2+ entry. Plasma membrane Ca2+ entry most commonly is Store-operated CRAC channels constitute the major mechanism of calcium signaled by the depletion of intracellular Ca2+ stores, a mechanism referred entry in cells of the immune system. Two key players in this pathway, to as capacitative calcium entry or store-operated calcium entry. Recent Stim and Orai, were recently identified through limited or whole-genome work from a number of laboratories has highlighted the roles of Ca2+ sensor RNAi screens in Drosophila cells. Moreover, Orai1 has been identified proteins, STIM1 and 2, and a channel subunit, Orai1 (CRACM1). In addition as the gene mutated in at least two cases of hereditary severe combined to Orai1, two other related mammalian proteins, Orai2 and 3, are capable of immunodeficiency disease. We are conducting additional RNAi and forming store-operated channels with pore properties similar to Orai1. STIM1 proteomic screens in mammalian cells to identify other regulators of this pathway. activates Orai channels by a mechanism that depends upon its co-localization Sustained Ca influx causes sustained nuclear translocation of NFAT with Orai at endoplasmic reticulum - plasma membrane junctions. STIM1 is transcription factors, which associate with a variety of transcriptional organized within the endoplasmic reticulum by mechanisms dependent on partners to control transcriptional programmes in many cell types. NFAT the microtubular cytoskeleton and collects upon store depletion at specific is heavily phosphorylated and resides in the cytoplasm of resting cells; sites. During mitosis, store-operated entry is down-regulated by complex when cells are stimulated, NFAT is dephosphorylated by the calmodulin- mechanisms involving changes in protein expression as well as co-valent dependent phosphatase calcineurin and translocates to the nucleus. In T cells, modification of signaling proteins. In some cell types, mitogens cause NFAT controls chromatin reorganisation during differentiation into different remodeling of the Orai channel composition resulting in altered physiological effector cell types, the acute transcription of activation-associated genes, and and pharmacological profiles of Ca2+ signaling. regulatory T cell function.

RS VI-33-3 RS VI-33-4 FUNCTIONAL CONSEQUENCES OF CRAC CHANNEL MOLECULAR COMPONENTS OF STORE-OPERATED 2+ ACTIVATION IN HEALTH AND DISEASE Ca ENTRY Anant B Parekh, Joseph L Di Capite Reinhold Penner Physiology, Oxford University, UK Center for Biomedical Research, The Queen's Medical Center and University of Hawaii, USA CRAC channels are activated by emptying the intracellular Ca2+ stores 2+ In immune cells such as lymphocytes and mast cells, the major route of Ca and drive a wide array of cellular responses. Functional CRAC channels influx is through store-operated calcium entry (SOCE) mediated by Calcium are important in the immune system. Knockdown of Orai, which comprises Release-Activated Calcium (CRAC) channels. Recent work has identified the the CRAC channel pore, prevents mast cell activation. Our recent work has molecular components of SOCE. STIM1 in the endoplasmic reticulum (ER) revealed that Ca2+ microdomains arising from open CRAC channels drives and CRACM1 (or Orai1) in the plasma membrane are essential molecular the synthesis and release of pro-inflammatory leukotrienes like leukotriene components for controlling SOCE in lymphocytes. Combined overexpression C4 (LTC4). Here, we describe a positive feedback loop whereby secreted of both proteins reconstitutes amplified CRAC currents and all three LTC4, acting on cysteinyl leukotriene receptors, activates CRAC channels mammalian CRAC channel homologs (CRACM1, CRACM2 and CRACM3) and the rise in cytoplasmic Ca2+ then drives the secretion of more LTC4. represent functional store-operated channels with distinctive properties in This results in the propagation of an intercellular Ca2+ wave through the terms of kinetics of activation, selectivity for cations, feedback regulation by intracellular Ca2+, and pharmacological effects of 2-aminoethoxydiphenyl entire cell population, co-ordinating mast cell activity. This form of paracrine

borate (2-APB). More recently, STIM2 has also been proposed to mediate Regular Symposia signalling is seen in mast cells from patients with severe allergic rhinitis and SOCE and additionally regulate basal cytosolic Ca2+ levels in heterologous nasal polyposis. Blocking CRAC channels with a novel channel inhibitor expression systems, but the functional role of STIM2 in native cells is not combined with cysteinyl leukotriene receptor antagonists effectively understood. In my presentation I will focus on store-operated and store- suppresses mast cell activation, identifying a new approach for treating nasal independent activation of CRAC channels and STIM2 function in native polyposis and related disorders. lymphocytes and mast cells.

RS VI-33-6 RS VI-34-1 SELECTIVE AND DIRECT INHIBITION OF TRPC3 STRUCTURAL INSIGHTS INTO THE FUNCTION OF CHANNELS UNDERLIES BIOLOGICAL ACTIVITIES OF CYTOPLASMIC REGION OF G PROTEIN-GATED A PYRAZOLE COMPOUND INWARD RECTIFIER K+ CHANNEL Yasuo Mori Atsushi Inanobe Department of Synthetic Chemistry and Biological Chemistry, Graduate Department of Pharmacology, Osaka University Graduate School of School of Engineering, Kyoto University, Japan Medicine, Japan Canonical transient receptor potential (TRPC) channels control influxes of The G protein-gated inward rectifier +K (K ) channel regulates the cell excitability Ca2+ and other cations that induce diverse cellular processes upon stimulation G in response to G protein-coupled receptor stimulations. The KG channels are a of plasma membrane receptors coupled to phospholipase C (PLC). Here, we tetrameric assembly of Kir3.1-Kir3.4 subunits. Like the other inward rectifiers, identify a novel pyrazole compound (Pyr3) which selectively inhibits TRPC3 the K channel possesses two distinct domains: transmembrane and cytoplasmic channels. Structure-function relationship studies of pyrazole compounds showed G domains. The former is involved in ion selectivity, ion permeation and gating. that the trichloroacrylic amide group is important for the TRPC3 selectivity of Pyr3. Electrophysiological and photoaffinity labeling experiments reveal a direct The latter controls the gating via conformational changes in the region evoked by action of Pyr3 on the TRPC3 protein. In DT40 B lymphocytes, Pyr3 potently the association with regulatory factors. To reveal structural elements responsible 2+ for the gating of KG channel, the cytoplasmic region of KG channel subunit Kir3.2 eliminated the Ca influx-dependent PLC translocation to the plasma membrane + and late oscillatory phase of B cell receptor-induced Ca2+ response. Moreover, was crystallized in the presence or absence of its channel activator Na . The two Pyr3 attenuated activation of NFAT, a Ca2+-dependent transcription factor, structures revealed a change in the conformation of CD loop. The change affected and hypertrophic growth in rat neonatal cardiomyocytes, and in vivo pressure on the interaction with PIP2, presumably reflecting the conformational transition overload-induced cardiac hypertrophy in mice. These findings on important roles between closed states. Furthermore, a divalent cation was identified to be docked of native TRPC3 channels are strikingly consistent with previous genetic studies. at a site surrounded by amino acids from four subunits. Since mutagenesis at Thus, the TRPC3-selective inhibitor Pyr3 is a powerful tool to study in vivo these residues caused the alteration in the permeation properties, the site would function of TRPC3, suggesting a pharmaceutical potential of Pyr3 in treatments be crucial for the ion conduction through the cytoplasmic region. These structural of TRPC3-related diseases. insights into the function of cytoplasmic region of Kir3.2 will be discussed.

IUPS 2009 July 27 - August 1, 2009 in Kyoto 91 RS VI-34-2 RS VI-34-3 CELL-SPECIFIC ROLE OF PIP2 IN GIRK CHANNEL RECEPTOR-SPECIFIC MODULATION OF POTASSIUM REGULATION BY RECEPTORS AND CALCIUM CHANNELS BY PHOSPHOINOSITIDE Won-Kyung Ho1, Suk-Ho Lee1, Jong-Woo Sohn1, Hana Cho2 SIGNALS 1Department of Physiology, Seoul National University, Korea, 2Department Mark S. Shapiro, Oleg Zaika, Ciria C. Hernandez, Manjot Bal, of Physiology, Sungkyunkwan University, Korea Jie Zhang Phosphatidylinositol 4,5-bisphosphate (PIP2) is the precursor of important Department of Physiology, University of Texas Health Science Center at signaling molecules such as IP3 and DAG. Recently, it was shown that PIP2 San Antonio, USA itself exerts a direct role in the regulation of various ion channels, including + Many ion channels are regulated by phosphoinositides, including voltage-gated K and G protein-gated inward rectifying K+ (GIRK) channels. We have investigated 2+ Ca types, sensory channels involved in sensing pain and heat, and those in epithelia how PIP2 plays its signaling roles in Gq protein-coupled receptor (GqPCR)- (Gamper & Shapiro, Nat. Rev. Neurosci., 2007, 8:921-34). We focus on modulation mediated signaling, with special reference to GIRK channel regulation. In of M-type (KCNQ) K+ and N-type (Ca 2.2) Ca2+ channels by phosphatidylinositol atrial myocytes, we found that phenyleprine, endothelin-1 and prostaglandin- V 4,5-bisphosphate (PIP2), which is necessary for their activity. Our work probes F2a caused inhibition of GIRK currents via PIP2 depletion, whereas 2+ the determinants of PIP2 action on KCNQ channels, and the mechanisms of Ca / bradykinin receptors or M1/M3 muscarinic receptors had no effect. We calmodulin (CaM) and AKAP79/150, which also act on the channels in concert provided supporting evidence that lateral mobility of PIP2 is extremely low with lipid signals. We identified an inter-helical domain in the carboxy-terminus as and that GIRK channels are co-localized with specific receptors based on a probable binding site of PIP2, and are now probing the biochemical affinities of caveola, so that GIRK channels are inhibited by PIP2 depletion induced by PIP2 for this domain using two advanced methods. In sympathetic neurons of the the co-localized GqPCR. In hippocampal neurons, however, GqPCR (M1/M3 superior cervical ganglion (SCG), stimulation of muscarinic M1 receptors depresses muscarinic receptor and group I metabotropic glutamate receptor) inhibited IM and N-type ICa via Gq/11 G proteins, phospholipase C activity and depletion of PIP2. GIRK channels, not via PIP2 depletion, but via PKC and arachidonic acid, However, this mechanism is receptor specific, as stimulation of Gq/11-coupled B2 2+ respectively, which in turn induced the decrease in PIP2-channel interactions. bradykinin or P2Y purinergic receptors do not deplete PIP2, depress IM via Ca /CaM Taken together, signaling roles of PIP2 in GqPCR-mediated regulation are binding to the channels, and neither depresses ICa due to concurrent simulation of lipid receptor-specific and cell-specific. kinases. We will discuss current progress on these topics.

RS VI-34-4 RS VI-34-5 CELL SURFACE REGULATION OF GIRK CHANNELS G-PROTEIN COUPLED INWARDLY RECTIFYING Paul A. Slesinger POTASSIUM (GIRK) CHANNELS AS EFFECTORS OF Peptide Biology Laboratory, The Salk Institute, USA ADDICTIVE DRUGS GIRK channels are part of a macromolecular signaling complex that Christian Luscher contains GPCRs, G proteins, RGS proteins and other signaling proteins. Dept of Basic Neurosciences, University of Geneva, Switzerland We hypothesized that cytoplasmic proteins might regulate the GPCR/GIRK Several addictive drugs activate GIRK currents, which may contribute to signaling complex and searched for new GIRK regulatory proteins using the increase of mesolimbic dopamine levels. Opioids and the club drug tandem mass spectrometry. We discovered that sorting nexin 27 (SNX27) gamma-hydroxy butyrate (GHB) for example increase dopamine in the associates with GIRK2c/GIRK3 channels. As a member of the sorting nexin ventral tegmental area (VTA) via GPCR/GIRK-mediated disinhibition of family, SNX27 has a PX domain that selectively binds to phosphatidylinositol the dopamine neurons. In the VTA, mu-opioid receptors are selectively Regular Symposia 3-phosphate (PI(3)P) and is important for localization to the early endosome. expressed on GABA neurons, which explains disinhibition. For GHB the SNX27 also contains a PDZ domain that distinguishes between channels situation is more complicated since GABAB receptors are expressed on both, GABA neurons as well as dopamine neurons. A low concentration of GHB with similar class I PDZ binding motifs, binding to GIRK2c/GIRK3 (-SKV) still causes disinhibition because the GABAB receptor to GIRK coupling channels but not to IRK1 (-SEI) or Kv1.4 (-TDV) channels. We examined the in the dopamine neurons is very weak. Our studies suggest that such weak functional consequence of up-regulated SNX27. In HEK293 cells, SNX27 coupling is mediated by the cell-type specific GIRK subunit expression. and GIRK2c/GIRK3 channels colocalize in early endosomal compartments. In fact GIRK1 is absent from dopamine neurons. We will also provide Coexpression of SNX27 with GIRK3-containing channels reduces GABAB evidence that regulator of G-protein signaling (RGS) proteins dynamically receptor-activated currents and increases GIRK degradation. Similarly, modulate this coupling efficiency in dopamine neurons and show that GABAB receptor-activated GIRK currents are smaller in hippocampal chronic drug exposure can dynamically regulate the expression levels of neurons coexpressing SNX27. These data demonstrate SNX27 controls RGS. We conclude that the GPCR to GIRK coupling efficiency determines surface expression of GIRKs, providing a novel mechanism for regulating disinhibition in the VTA and that regulation of RGS proteins may underlie neuronal excitability. adaptation to addictive drugs.

RS VI-34-6 RS VI-35-1 DIRECT VERSUS INDIRECT REGULATION OF REGULATION AND PHYSIOLOGY OF THE UBIQUITOUS IONOTROPIC GLUTAMATE RECEPTORS BY PLASMA MEMBRANE Na+/H+ EXCHANGER, Nhe1 PHOSPHOINOSITIDES Stine Falsig Pedersen Diomedes Elias Logothetis, Vasileios I Petrou Department of Biology, University of Copenhagen, Denmark Physiology and Biophysics, Virginia Commonwealth University School of The Na+/H+ exchanger NHE1 (SLC9A1) is nearly ubiquitously expressed in Medicine, USA the plasma membrane of cells from vertebrate organisms. The involvement NMDA receptor (NMDAR) activity is supported by phosphatidylinositol-bisphosphate of NHE1 in cellular pH and volume homeostasis is well established, although (PIP2), as wortmannin treatment, agonist-mediated stimulation of phospholipases the mechanisms by which cell volume perturbations control NHE1 activity (PLCs) or overexpression of PLC reduces NMDA currents. The coupling of have yet to be fully understood. A plethora of physiological functions NMDARs to PIP2 is achieved through the cytoskeletal protein alpha (a)-actinin. of NHE1 have been proposed, including pivotal roles in control of cell Mutations that uncouple a-actinin from the NMDAR subunits or mutations that disable morphology, migration, proliferation, and survival. Nonetheless, the precise a-actinin from interacting with PIP2 disrupt PLC-dependent regulation of NMDA contribution of NHE1 to these processes in vivo remains controversial. currents and accelerate their inactivation. These effects suggest that NMDARs are This is likely in part due to the complex network of binding partners and regulated indirectly by PIP2, through a-actinin (Michailidis et al., 2007 27:5523-5532). posttranslational regulatory events controlling NHE1, rendering the outcome We have also examined the dependence of the delta2 glutamate receptor (d2) activity of a given stimulus context-specific. NHE1 is implicated in important on phosphoinositides. d2 has no known ligand that has been shown to activate it but a naturally-occurring single point mutant (A654T), named Lurcher (d2-LC), pathological conditions including cancer and ischemia-reperfusion damage; exhibits constitutive activity. d2-LC currents were directly inhibited by PIP2 and thus, further understanding of NHE1 structure, regulation, and physiology stimulated by a decrease in PIP2 levels. This inhibitory effect of PIP2 on the d2-LC is of major clinical interest. An up-to-date overview of NHE1 regulation currents contrasts with that of most other channels that are usually activated by PIP2. and function will be provided, and some major controversies discussed. Moreover, the direct effects of PIP2 on d2-LC contrast with the indirect effects seen Finally, recent evidence implicating NHE1 in control of tumor cell survival, with the NMDAR. migration, and invasion will be presented, as will very recent findings shedding new light on NHE1 structure and its dynamics.

92 IUPS 2009 July 27 - August 1, 2009 in Kyoto RS VI-35-2 RS VI-35-3 ROLES OF VOLUME-SENSITIVE ANION CHANNELS AN UPDATE ON TRPV4-MEDIATED SIGNALING IN IN CELL DEATH INDUCTION AND GLIA-TO-NEURON HEALTH AND DISEASE SIGNALING Wolfgang B Liedtke Yasunobu Okada Department of Medicine / Division of Neurology, Duke University, USA National Institute for Physiological Sciences, Japan In signal transduction of metazoan cells, ion channels of the family of The volume-sensitive outwardly rectifying anion channel (VSOR) is involved transient receptor potential (TRP) have been identified to respond to diverse in cell volume regulation (CVR) and CVR-coupled cell functions such as external and internal stimuli, amongst them osmotic and mechanical cell proliferation and migration. Also, VSOR activation was shown to play stimuli. This presentation will highlight the TRPV4 channel, initially named a key role in induction of the apoptotic volume decrease (AVD). Inhibition vanilloid-receptor related osmotically activated channel (VR-OAC) or of VSOR activation prevented AVD induction and thereby rescued cells OTRPC4. Illustrating evolutionary conservation of function, mammalian from apoptotic death in a variety of cell types stimulated by staurosporine, TRPV4 has been found to rescue osmo- and mechano-sensory deficits of the Fas ligand or TNFα, and in cancer cells treated with an anticancer drug TRPV mutant osm-9 in Caenorhabditis elegans, despite < 26% orthology cisplatin as well as in cardiomyocytes and hippocampal neurons subjected to ischemia-reperfusion. Furthermore, loss of VSOR activity was found to of the respective proteins. Recent findings pertaining to TRPV4’s function be associated with acquisition of cisplatin resistance by human epidermoid in endothelia, in the lung, in intestinal sensory innervation and in skeletal cancer cells. Impaired VSOR activity was also shown to be a causal factor structure and function will be reviewed. TRPV4 has been found to function for the necrotic volume increase (NVI) and death in glial cells subjected to in transduction of mechanical shear stress, airway epithelia ciliary beat lactacidosis. Under excitotoxic conditions, on the other hand, VSOR activity regulation, maintenance of alveolar integrity, intestinal mechanosensation of rather resulted in NVI due to Cl- influx driven by depolarization induced by noxious stimuli, sensitized by activation of the proteinase-activated receptor glutamate receptor activation in cortical neurons. Moreover, when stimulated 2 (PAR2), and function of articular chondrocytes and osteoclasts. Tyrosine by bradykinin, an initial mediator of inflammation, VSOR was found to phosphorylation of TRPV4 has been identified as an important regulatory serve as a pathway for glutamate release from astrocytes, thereby mediating mechanism, a finding also true for OSM-9, a subject currently under astrocyte-neuron communication. investigation.

RS VI-35-4 RS VI-35-5 CELL VOLUME IN REGULATION OF CELL K+, Cl- AND WATER CHANNELS IN REGULATION OF PROLIFERATION AND SUICIDAL CELL DEATH CELL VOLUME AND CELL SURVIVAL 1 2 3 4 Florian Lang , Markus Ritter , Ildiko Szabo , Erich Gulbins , Else Kay Hoffmann 1 Michael Foeller Department of Biology, University of Copenhagen, Denmark 1Department of Physiology, Eberhard-Karls-University of Tuebingen, Germany, 2Dept. of Physiology and Pathophysiology, Paracelsus Medical University, Using Ehrlich ascites tumor cells (EATC) as an experimental model we have Austria, 3Dept. of Biology, University of Padua, Italy, 4Dept. of Molecularbiology, shown that the volume sensitive potassium channel TASK-2, the volume University Clinic Essen, Germany regulated anion channel (VRAC) and the water channel AQP5 all play Cell shrinkage is a hallmark of suicidal cell death and cell proliferation eventually essential roles not only in cell volume control but also in cell proliferation requires increase in cell volume. The interplay between cell volume regulatory and programmed cell death (PCD). The role of TASK-2 was studied mechanisms and cell proliferation or suicidal cell death is described. Stimulation of cell by over-expression of TASK-2 and by inhibitor studies using clofilium. 2+ proliferation by ras oncogene leads to ICRAC activation, oscillations of cytosolic Ca Tyrosine phosphorylation of TASK-2 during swelling was studied by with early activation of K+ channels, depolymerization of actin filaments, disinhibition immunoprecipitation and mass spectroscopy. The roles of VRAC in PCD + + - + + of Na ,K ,2Cl -cotransport and Na /H exchanger eventually leading to cell volume and in cell proliferation were studied using a high affinity anion channel increase. Triggering of lymphocyte apoptosis by CD95 leads to ICRAC inhibition, early + - - inhibitor, the acidic di-aryl-urea NS3728. In an attempt to find the molecular inhibition of K channels (Kv1.3), activation of Cl ,HCO3 channels and late osmolyte identity of VRAC we have cloned and expressed some members of the release with cell volume decrease. Excessive hyperosmotic (addition of sucrose) or - + recently identified TMEM16 family and analyzed their biophysical channel isosmotic (Cl -replacement by gluconate or treatment with K ionophore valinomycin) shock leads to apoptosis of nucleated cells and suicidal death of erythrocytes (eryptosis). properties and possible volume sensitivity. Furthermore, by miRNA mediated Regular Symposia Eryptosis is at least in part due to stimulation of Ca2+-permeable cation channels. As knockdown and over-expression, respectively, we have evaluated their roles shown for eryptosis, suicidal cell death may be regulated by a variety of signaling in PCD. Finally, we have generated stable EATC cell lines with constitutive pathways including platelet activating factor, sphingomyelinase and ceramide, miRNA mediated knockdown (miR-AQP5) or over-expression (AQP5ex) of eicosanoids, PI3 kinase pathway, protein kinase C, AMP kinase, G kinase, and nitric AQP5, respectively, and studied them with regard to water permeability, cell oxide. volume regulation, growth rates and drug-induced PCD.

RS VI-35-6 RS VI-36-1 α-ENaC IS A FUNCTIONAL ELEMENT OF THE CALCIUM SIGNALS IN CELL DEATH AND DISEASE HYPERTONICITY-INDUCED CATION CHANNEL IN Pierluigi Nicotera1, Alexei Verkhratsky2 HepG2 CELLS AND IT MEDIATES PROLIFERATION 1MRC Toxicology Unit, Leicester University, UK, 2Faculty of Life Sciences, 1 1 2 3 Frank Wehner , Maryna Bondarava , Tongju Li , Elmar Endl The University of Manchester, Manchester, UK 1 Systemic Cell Biology, Max-Planck-Institute of Molecular Physiology, Most leaving creatures have one thing in common: they all undergo Germany, 2Institute of Physiological Chemistry, University of Essen, 3 the process of death. Evolution based on simple cell division required Germany, Institute of Molecular Physiology and Experimental Immunology, extinction of unfit cells and selection of those endowed with environmental University of Bonn, Germany adaptation. The most obvious example of a primordial death mechanism In HepG2 cells, hypertonic stress (340 → 450 mosM) reversibly increased is the failure of ion homeostasis when the environmental changes strained whole-cell Na+ conductance from 0.8 to 5.8 nS. The effect was inhibited by ion compartmentalisation between the intra- and extracellular space. These flufenamate and amiloride, blockers of hypertonicity-induced cation channels mechanisms of cell and tissue damage are universally operative among all (HICCs), with some 50% efficiency at 300 μM. In parallel, both drugs forms of life. In mammalian cells both Ca2+ and Na+ overloads are one decreased the rate of cell proliferation. siRNA silencing of the α-subunit of the epithelial Na+ channel (ENaC) reduced hypertonicity-induced Na+ of the major causes of tissue damage. Imbalances in Ca2+ are the most currents to 60% whereas the rate of cell proliferation was approx. half common examples of death routines using physiological signalling systems. of control. Moreover, α-ENaC siRNA inhibited the regulatory volume Deregulation of Ca2+ signalling is involved in many types of cell death: it increase of cells by 60%. In cell-cycle analysis, silencing of α-ENaC led to triggers excitotoxicity in neurones and muscle cells, it initiates apoptosis a significant decrease in the G1 and an increase in the G2/M phase whereas in many excitable and non-excitable tissues, and it acts as activator and the S phase was not changing. Finally, HICC inhibition by flufenamate and executor of necrotic cell death. All these death subroutines use existing Gd3+ as well as siRNA silencing of α-ENaC increased the rate of apoptosis. molecular systems, which are responsible for physiological Ca2+ signalling. It is concluded that α-ENaC is one functional element of the HICC in HepG2 As tissue plasticity and remodelling became a fundamental step in evolution cells and that the channel is a mediator of cell proliferation. Likewise, HICC of complex organisms, biochemical programmes involving complex cascades blockage shifts the system from a proliferative into an apoptotic one. leading to cell disassembly have also developed.

IUPS 2009 July 27 - August 1, 2009 in Kyoto 93 RS VI-36-2 RS VI-36-3 2+ IP3R Ca SIGNALING REGULATION OF APOPTOSIS, INTRACELLULAR CALCIUM RELEASE MECHANISMS AUTOPHAGY AND ALZHEIMER’S DISEASE IN CETRAL NERVOUS SYSTEM FUNCTIONS Kevin Foskett, King-Ho Cheung, Cardenas Cesar, Carl White Masamitsu Iino Department of Physiology, University of Pennsylvania, USA Department of Pharmacology, Graduate School of Medicine, The University of Tokyo, Japan The activity of the inositol trisphosphate receptor (IP3R) endoplasmic 2+ reticulum Ca2+ release channel is regulated by interactions with proteins Intracellular Ca release via the ryanodine receptor and the inositol 1,4,5-trisphosphate (IP3) receptor regulates a vast array of important cell involved in apoptosis and Alzheimer’s disease. Interactions with anti- 2+ apoptotic Bcl-2 proteins provide apoptosis resistance, whereas interactions functions. Although these Ca release channels are widely expressed in with Alzheimer’s disease causing presenilins may play a role in the central nervous system (CNS), their (patho)physiological functions are not fully understood. We have been trying to elucidate the roles of Ca2+ neurodegeneration. Paradoxically, these proteins interactions have similar release mechanisms in CNS to shed new light on the CNS functions. The effects on release channel activity but lead to very divergent phenotypes - cerebellar parallel fiber-Purkinje cell synapse is one of the most extensively cell survival or death. IP3R activity is tightly coupled to the regulation of studied central synapses and is important for motor learning. Imaging of macroautophagy, providing clues to a fundamental constitutive role of IP3R 2+ both extrasynaptic glutamate and postsynaptic IP3 dynamics at this synapse 2+ Ca release in the maintenance of optimal cell bioenergetics. The IP3R suggested that physiological sensory input generates postsynaptic IP -Ca may therefore play a central role in providing Ca2+ signals that regulate cell 3 2+ signaling in Purkinje cells. Furthermore, we found that the postsynaptic IP3- life and death decisions, possibly linked to Ca dependent mitochondrial Ca2+ signaling is required for the maintenance of glutamate release function activities. from the presynaptic terminals. This retrograde signaling is mediated by the brain-derived neurotrophic factor (BDNF). These results indicate that constant sensory input is required to maintain the synaptic strength, and 2+ that the IP3-Ca -BDNF signaling plays an essential role in the synaptic maintenance. I would like to discuss our recent results along these lines of study.

RS VI-36-4 RS VI-36-5 TRPP2 CHANNELS AND POLYCYSTIC KIDNEY ENDOPLASMIC RETICULUM Ca2+ HOMEOSTASIS AND DISEASE: CALCIUM SIGNALING AND CELL DEATH NEURAL CELL DISEASE AND DEATH Michael Kottgen1, Tomasz Wegierski2, Daniel Steffl2, Alexei Verkhratsky1, Pierluigi Nicotera2 Christoph Kopp2, Bjorn Buchholz2, Robert Tauber2, Gerd Walz2, 1Faculty of Life Sciences, The University of Manchester, UK, 2MRC Terry Watnick3, Craig Montell3 Toxicology Unit, Leicester University, UK 1Department of Biological Chemistry, Johns Hopkins University, USA, Most leaving creatures have one thing in common: they all undergo 2Renal Division, University Hospital Freiburg, Germany, 3Johns Hopkins the process of death. Evolution based on simple cell division required University, USA extinction of unfit cells and selection of those endowed with environmental Mutations in Transient Receptor Potential Channel P2 (TRPP2) cause autosomal adaptation. The most obvious example of a primordial death mechanism dominant polycystic kidney disease (ADPKD), a common genetic disorder is the failure of ion homeostasis when the environmental changes strained

Regular Symposia characterized by progressive development of fluid-filled cysts in the kidney. TRPP2 is ion compartmentalisation between the intra- and extracellular space. These 2+ a Ca -permeable nonselective cation channel that has been implicated in the regulation mechanisms of cell and tissue damage are universally operative among of diverse physiological functions including mechanosensation, cell proliferation, all forms of life. In mammalian cells both Ca2+ and Na+ overloads are one polarity, and apoptosis. TRPP2 localizes to different subcellular compartments, such of the major causes of tissue damage. Imbalances in Ca2+ are the most as the endoplasmic reticulum (ER), the plasma membrane and the primary cilium, and common examples of death routines using physiological signalling systems. the channel appears to have distinct functions in different subcellular compartments. Deregulation of Ca2+ signalling is involved in many types of cell death: it We have recently shown that ER-resident TRPP2 protects cells from apoptosis by lowering the Ca2+ concentration in the ER. In the primary cilium, TRPP2 acts as triggers excitotoxicity in neurones and muscle cells, it initiates apoptosis a molecular sensor that translates extracellular signals into programs that regulate in many excitable and non-excitable tissues, and it acts as activator and executor of necrotic cell death. All these death subroutines use existing tubular morphology. Despite extensive studies, the upstream and downstream 2+ components in the TRPP2 signaling pathway are unknown. To gain insights into how molecular systems, which are responsible for physiological Ca signalling. channel function translates into complex morphogenetic programs that regulate tubular As tissue plasticity and remodelling became a fundamental step in evolution structure, we performed an unbiased forward genetic screen in Drosophila to identify of complex organisms, biochemical programmes involving complex cascades evolutionarily conserved components in the TRPP2 signaling pathway. leading to cell disassembly have also developed.

RS VI-36-6 RS VI-37-1 Ca2+- RELATED MECHANISMS OF APOPTOSIS THE SODIUM/IODIDE SYMPORTER (NIS): AN RESISTANCE IN CANCER CELLS UNENDING SOURCE OF SURPRISES Natalia Prevarskaya Nancy Carrasco Department of Physiology, University of Lille 1, INSERM U800, France Department of Molecular Pharmacology, Albert Einstein College of Prostate cancer (PCa) is one of the leading threats to men’s health. The Medicine, USA progression to androgen independence is associated with the appearance of The Na+I- symporter (NIS) is a plasma membrane protein that mediates active new cell phenotypes, characterized by a low rate of apoptosis. It is, therefore, I- uptake in the thyroid, lactating breast, and other tissues with an electrogenic vital to understand what drives PCa to apoptosis resistance. stoichiometry of 2 Na+ per I-. In the thyroid, NIS-mediated I- uptake is the We have previously shown that the apoptosis inhibition in androgen- first step in the biosynthesis of the thyroid hormones, which are essential independent PCa cells is associated with the downregulation of Store early in life for CNS development. In the lactating breast, NIS mediates Operated Calcium Entry (SOCE) due to the decrease in the number of - the translocation of I to the milk, thus supplying it to the nursing newborn. functional channels (Cancer Cell, 2002). However, the molecular nature - Perchlorate (ClO4 ) is a competitive inhibitor of NIS and an environmental of channels playing major role in PCa cells apoptosis induction remains - - unknown. pollutant. We show that NIS actively transports ClO4 , including ClO4 translocation to the milk, suggesting that exposure of newborns to high levels Now, we show that the recently identified Orai1 protein is a store-operated - of ClO4 may pose a greater health risk than previously thought. A simple calcium channel in human prostate cancer cells. The expression of Orai1 - mathematical fluxes model accurately predicts the effect of ClO4 transport and the amplitude of store-operated current were dramatically decreased in - + - - androgen-deprived cells. Such downregulation of the Orai1 channels leads on the rate and extent of I accumulation. Strikingly, the Na /ClO4 (or ReO4 ) to the inhibition of sustained SOCE and consecutively to the decrease in transport stoichiometry is electroneutral, uncovering that NIS translocates apoptosis. Orai1 knockdown protects LNCaP cells against apoptosis. Thus, different substrates with different stoichiometries. Substitutions in TMS 3 + - - our work demonstrates that Orai1 plays a pivotal role in the progression result in a change in stoichiometry from 1 Na :1 ClO4 (or ReO4 ) in WT + - - of prostate cancer to apoptosis-resistant phenotype and makes this channel NIS to 2Na :1 ClO4 (or ReO4 ) in the mutants, an observation that may have potentially attractive target for therapeutic interventions. clinical significance in gene transfer studies. [DK-41544 and CA-098390].

94 IUPS 2009 July 27 - August 1, 2009 in Kyoto RS VI-37-2 RS VI-37-4 + - + + NBCn1 IS THE ONLY Na ,HCO3 COTRANSPORTER IN STRUCTURAL MODELING OF THE HUMAN Na /H VASCULAR SMOOTH MUSCLE AND ENDOTHELIAL EXCHANGER ISOFORM 1: STUDIES OF THE PUTATIVE CELLS IN SITU: IMPORTANCE FOR VASCULAR TONE ION TRANSLOCATION SITE BY EPR SPECTROSCOPY REGULATION Eva Byskov Nygaard1, Jens O Lagerstedt2, Robert R Rigor3, 1 2 1 Ebbe Boedtkjer , Jeppe Praetorius , Susie Mogensen , Gabriel P Bjerre1, Kristian A Poulsen1, Stine F Pedersen1, 3 1 Ernst-Martin Fuchtbauer , Christian Aalkjaer John C Voss4, Peter M Cala3 1 Department of Physiology and Biophysics, University of Aarhus, Denmark, 1 2 2 3 Department of Biology, University of Copenhagen, Denmark, Sahlgrenska Department of Anatomy, University of Aarhus, Denmark, Department of Center for Cardiovascular & Metabolic Research, Wallenberg Laboratory, Molecular Biology, University of Aarhus, Denmark University of Gothenburg, Sweden, 3Department of Physiology & Membrane 4 We investigated the regulation of intracellular pH (pHi) in vascular smooth muscle (VSMCs) Biology, University of California, Davis, USA, Department of Biochemistry & and endothelial cells (ECs) of mouse mesenteric arteries using fluorescence microscopy. The Molecular Medicine, University of California, Davis, USA + - importance of Na ,HCO3 cotransport for arterial tone regulation was studied. The Na+/H+ exchanger NHE1 is fundamental to normal cell function. We recently presented In arteries from NBCn1-/- mice, intracellular acidification of ECs after extracellular Na+ - functional evidence that transmembrane (TM) domain IV and TM X-XI are important for inhibitor removal in the presence of CO2/HCO3 and amiloride was abolished. In VSMCs from -/- + - + binding and ion transport by human (h)NHE1. Here, we present a structural model of the TM region NBCn1 mice, the Na - and HCO3 -dependent pHi recovery after an NH4 -prepulse was of hNHE1 further supporting this conclusion. The model was based on the crystal structure of E. abolished. In ECs and VSMCs, NBCn1 knockout reduced steady-state pHi (0.1-0.3 units). coli NhaA and validated by electron paramagnetic resonance (EPR) spectroscopy and functional The response of isolated arteries to norepinephrine (NE) was unaffected by NBCn1 knockout studies of hNHE1 mutants. Cys-less hNHE1 with Ala173Cys (TM IV) and/or Ile461Cys (TM XI) but the decrease in EC50 for NE upon treatment with L-NAME was reduced in arteries from mutations were functional after expression in mammalian cells. The distance between the TM IV -/- -/- NBCn1 mice. Relaxation to acetylcholine (ACh) was smaller in arteries from NBCn1 and TM XI spin-labels was 15±2A, confirming the close proximity of these domains predicted by mice. The difference in ACh-response disappeared after treatment with L-NAME. The the model. This distance was decreased at pH 5.1 (NHE1 activation) and increased by the NHE1 response to the NO-donor SNAP, the expression of endothelial NO-synthase (eNOS) and the inhibitor amiloride, consistent with a role for TM IV-TM XI rearrangement in ion translocation 2+ EC Ca -response to ACh were all unaffected by NBCn1 knockout. and inhibitor binding. The model predicts that Arg425 in TM X could stabilize an energetically + - 425 We conclude that NBCn1 is the only Na ,HCO3 cotransporter in ECs and VSMCs in situ. unfavorable dipole-dipole arrangement in the TM IV/XI crossover. Accordingly, an Arg Ala We propose that NBCn1 is important for normal NO-mediated vasorelaxation through Ca2+- mutation resulted in failure of hNHE1 membrane targeting and reduced function. EPR studies of independent modulation of eNOS. other spin-label sites and NHE homologs are ongoing to further refine the model.

RS VI-37-5 RS VI-37-6 STRUCTURAL ASPECTS OF CATION PUMPING BY THE ION PATHWAY THROUGH THE Na,K-ATPase P-TYPE ATPASES PUMP Chikashi Toyoshima1, Takehiro Shinoda1, Haruo Ogawa1, David C Gadsby1, Ayako Takeuchi1, Nicolas Reyes2, Pablo Artigas3 2 Flemming Cornelius 1Laboratory of Cardiac/Membrane Physiology, Rockefeller University, USA, 1 2Department of Physiology and Biophysics, Weill Medical College of Cornell Institute of Molecular and Cellular Biosciences, The Univ of Tokyo, Japan, 3 2Department of Physiology and Biophysics, Aarhus University, Denmark University, New York, NY, USA, Department of Cell Physiology and Molecular Biophysics, Texas Tech University Health Science Center, Lubbock, Texas, USA P-type ion transporting ATPases play a vital role in establishing ion Though a channel needs only a single gate to control ion flow, an ion pump needs at concentration gradients across cell and organelle membranes. Structurally least two gates that must never both be open. The two gates are constrained to open the best studied member is the Ca2+-ATPase of skeletal muscle sarcoplasmic and close alternately to allow access to ion-binding sites from only one side of the reticulum (SERCA1a), of which more than 25 structures are registered in + + membrane at a time. In the Na,K-ATPase pump this alternating gating, coupled to ATP the PDB. We are also working on the Na ,K -ATPase, another principal hydrolysis, results in transport of 3 Na ions out and 2 K ions in across the membrane member of P-type ATPases, and obtained a 2.4 A resolution structure of per ATP, up to 100 times a second. Binding of the marine toxin, palytoxin, to a Na,K- this important pump from shark rectal gland consisting of α- and β-subunits ATPase pump uncouples its two gates so that, although each gate still responds to its and a regulatory FXYD protein. The ATPase was fixed in a state analogous physiological ligand, they are no longer constrained to open and close alternately. The to E2.2K+.Pi, in which the ATPase has a high affinity to K+ and still binds + palytoxin-bound Na,K-ATPase pump is thus transformed into a gated cation channel. Pi. Clearly visualised now are co-ordination of K and associated water Whenever both its gates happen to be open, millions of Na or K ions per second can molecules in the transmembrane binding sites and a phosphate analogue in flow energetically downhill through the resulting open pump-channel. This permits Regular Symposia the phosphorylation site. The crystal structure shows that the β-subunit plays study of single Na,K-ATPase molecules and allows unprecedented real-time access to + an unexpectedly critical role in K binding and explains, at least partially why their ion transport pathway. Use of variously-charged small hydrophilic thiol-specific 2+ + + the homologous Ca -ATPase countertransports H rather than K , although reagents to probe cysteine targets introduced one at a time throughout the Na,K- the co-ordinating residues are almost identical. ATPase’s transmembrane helices allows mapping and characterization of the route traversed by transported ions.[HL36783]

RS VI-38-1 RS VI-38-2 MAPPING FEATURE-SELECTIVE NEURONS IN THE SYNAPTIC MECHANISMS OF SENSORY PERCEPTION MOUSE VISUAL CORTEX IN VIVO Carl CH Petersen Arthur Konnerth Brain Mind Institute, Ecole Polytechnique Federale de Lausanne (EPFL), Institute of Neuroscience, Technical University Munich, Germany Switzerland Neurons in the primary visual cortex of mammals respond with a high A key goal of modern neuroscience is to understand the neural circuits and degree of specificity to visual stimuli. For example, drifting gratings of the synaptic mechanisms underlying simple forms of sensory perception and associative learning. Here, I will discuss our efforts to characterise tactile given direction activate specifically a distinct subset of neurons, while other processing in the primary somatosensory barrel cortex of awake behaving directions activate a different subset of neurons. By using in vivo two-photon mice. Using reward-based learning paradigms, viral manipulations, whole- calcium imaging (Stosiek et al., PNAS, 2003; Busche et al., Science, 2003), cell recordings, voltage-sensitive dye imaging and two-photon microscopy we mapped feature-selective neurons in layer 2/3 of the mouse primary we find evidence for dynamic state-dependent processing of sensory visual cortex. First, we established cortical maps of direction-selective and information within defined synaptically connected networks of neurons. We of orientation selective neurons in adult mice. We found that in nearly 50% think that these combined approaches will lead to significant advances in our of all layer 2/3 neurons are direction- and/or orientation-selective. Next, we understanding of sensory perception. explored direction- and orientation selectivity at the onset of vision, which starts in mice around postnatal day 14 (P14). Direction- and orientation- selective cells were found within 1-2 days after eye-opening, indicating that feature-selectivity develops in the absence of visual stimuli. We found that layer 2/3 neurons in dark-reared mice are highly similar to neurons from control mice in terms of direction- and orientation selectivity. Together, our findings firmly establish that innate mechanisms, but not visual experience underlie direction- and orientation selectivity in the mouse visual cortex.

IUPS 2009 July 27 - August 1, 2009 in Kyoto 95 RS VI-38-3 RS VI-38-4 THE SPATIAL ORGANIZATION OF PALMITOYLATED SECRETAGOGUE-INDUCED MULTIPLE SIGNAL RAS SIGNALING CROSS-TALK IN INSULIN SECRETING CELLS 1 1 2 3 Philippe Bastiaens Hideo Mogami , Eisuke Adachi , Itaru Kojima , Naoaki Saito , 1 1 Department of Systemic Cell Biology, MPI of Molecular Physiology, Yuko Suzuki , Tetsumei Urano 1 Germany Department of Physiology, Hamamatsu University School of Medicine, Japan, 2Institute for Molecular and Cellular Regulation, Gunma University, In a chemical-biological approach that enables the direct kinetic evaluation Japan, 3Labolatory of Molecular Pharmacology, Biosignal Research of reversible protein acylation in single living cells, we investigated the Center, Kobe University, Japan localization, efficiency and specificity of the mammalian palmitoylation 2+ Ca , cAMP/PKA and diacylglycerol (DAG)/PKC are components of ubiquitous machinery in situ, by monitoring the turnover kinetics of semi-synthetic signaling cascades that orchestrate diverse physiological phenomena in living cells Ras proteins. We thereby showed that this spatially organizing system upon receptor stimulation. Recent advances in the use of fluorescent proteins and differs from conventional receptor-mediated targeting mechanisms and optical instrumentation now allow us to simultaneously monitor these signal cascades efficiently counteracts the tendency of the system to increase its entropy and analyze their interconnections in intact living cells. Using such fluorescence by redistribution of palmitoylated peripheral membrane proteins over all imaging in insulin-secreting cells, we have demonstrated that depolarization-evoked membranes. We also showed that thioacylation is not stereo-selective and Ca2+ influx generates DAG, thereby activating PKC. We also documented that a does not require any recognition signature on the substrate other than a cAMP-generating agonist activates the two different isoforms of PKC and that the stabilized thiol in transient proximity to Golgi membranes. With this in contribution of the PKC signaling pathway to such agonist-induced insulin secretion mind, I will discuss strategies of dynamic acylation interference via small is substantial. Finally, using a combination of prism-based total internal reflection fluorescence microscopy and epifluorescence microscopy, we provide direct evidence molecules to modulate localization and thereby oncogenic Ras activity. I will that forskolin simultaneously activates Ca2+, cAMP/PKA and PKC signals, whereas show how inhibition of thioesterase activity leads to entropy driven loss of acetylcholine activates Ca2+and PKC signals as well as inhibiting cAMP generation. specific plasma membrane localization and thus has the potential to modulate Thus, insulin secretagogues interconnect the ternary signaling of a Ca2+-cAMP/PKA- rather than to impair protein activity and function, thereby allowing cell DAG/PKC pathway; these intersecting pathways controlling insulin secretion cannot viability. be separated.

RS VI-38-5 RS VI-38-6 COMPUTATIONAL MODELS OF CALCIUM MAPPING THE INTRACELLULAR NETWORK OF SIGNALLING IN PANCREATIC ACINAR CELLS SHOW Ca2+ SIGNALLING, CALMODULIN MOVEMENT, NO UNIQUE ROLES FOR CALCIUM DIFFUSION IN THE CHANGES AND MITOCHONDRIA RESPONSES ENDOPLASMIC RETICULUM Alexei V Tepikin Kojiro Yano1, Alexei V Tepikin2, Ole H Petersen2 Department of Physiology, School of Biomedical Sciences, The University 1Department of Physiology, Development and Neuroscience, University of of Liverpool, UK Cambridge, UK, 2Department of Physiology, University of Liverpool, UK Local calcium signals are formed in the apical (secretory) region of pancreatic acinar cells. The mitochondria that surround the apical region play an important role in The endoplasmic reticulum (ER) in pancreatic acinar cells forms a continuous network 2+ 2+ in which Ca2+ can diffuse across the cell. This is important for replenishing the apical restricting Ca responses to the apical part of the cell. Mitochondria also import Ca 2+ 2+ and upregulate energy production to match the enhanced energy demands of the cell.

Regular Symposia part of the ER Ca store during local apical Ca spiking, but it is unclear whether 2+ changes in intra-ER diffusion rates significantly alter the dynamics of Ca2+ spiking. In addition to local Ca responses physiological doses of secretagogues trigger 2+ 2+ Using a computational model of IP3-induced local Ca2+ spiking, we found that, in Ca oscillations involving global Ca transients. These type of responses initiate 2+ self-sustained (fixed elevated [IP3]) Ca2+ oscillations, faster Ca2+ diffusion in the ER oscillations of mitochondrial NAD(P)H. Global Ca oscillations also cause profound significantly increased the spike amplitude over a wide range of diffusion coefficients effect on the distribution of Calmodulin, triggering its redistribution from the basal to (Der=0.056-200μm2/s). When Der was at its lowest range (Der=0.056-1.78μm2/s), the apical part of the cell. During these responses the nucleus serves as an integrator faster diffusion increased the spike frequency as well. On the other hand, when Ca2+ of Calmodulin. An elevated plateau of Calmodulin concentration is produced in the 2+ spiking was driven by IP3 oscillations, enhanced ER Ca2+ diffusion dramatically nucleus during Ca oscillations. Finally bile acids and supramaximal concentrations 2+ improved the encoding efficiency, i.e. the degree to which the change in the amplitude of secretagogues produce prolonged elevation of cytosolic Ca . One of the recently of IP3 oscillations is reflected in the amplitude of the Ca2+ spikes. Other Ca2+ transport discovered consequences of such long-lasting global Ca2+ elevations is Ca2+ - the parameters, i.e. cytosolic Ca2+ diffusion, ER Ca2+ release or uptake rate had little dependent release of NO from intracellular NO buffers. Pancreatic acinar cells provide influence on the encoding efficiency. These results suggest that effective ER Ca2+ an interesting example of how amplitude, temporal and spatial properties of Ca2+ diffusion plays a unique role in ensuring robust localised Ca2+ spikes in the acinar cells. signals are decoded.

RS VII-39-1 RS VII-39-2 MOLECULAR MECHANISM OF COLD-INDUCED COLD STRESS ON NEUROPEPTIDE Y AND HYPERTENSION NOREPINEPHRINE NEUROTRANSMISSION AND Zhongjie Sun HYPERTENSION Department of Physiology, University of Oklahoma Health Sciences Thomas C Westfall, Chun-lian Yang, Heather Macarthur, Center, USA Xiaoli Chen, Song-Ping Han Numerous studies have shown that chronic exposure of animals to moderate Department of Pharmacological and Physiological Science, Saint Louis cold (5°C) causes hypertension and cardiovascular hypertrophy within 3 weeks. University School of Medicine, USA o Cold exposure upregulates expression of genes of angiotensinogen (AGT), Chronic cold stress of rats (4 C 1-3 weeks) induced a marked increase in gene AT1 receptors and mineralocorticoid (MR) receptors. Knockout of AGT or AT1 expression (adrenal medulla, superior cervical gangia); tissue content (mesenteric receptor genes or RNAi silence of MR prevents the development of cold-induced arterial bed) and nerve stimulation (NS) induced overflow of NPYir from the hypertension (CIH) but does not attenuate cold-induced cardiac hypertrophy perfused mesenteric arterial bed. In contrast, increased NPY neurotransmission (CICH). In contrast, eNOS gene expression was suppressed by cold exposure, was offset by an apparent decrease in the evoked overflow of NE due to a which is likely mediated by upregulation of AT1 receptors because AT1 receptor presumed deactivation of NE by nitric oxide despite increased sympathetic nerve activity. There was an increase in tyrosine hydroxylase (TH) gene expression knockout abolishes cold-induced suppression of eNOS. In vivo eNOS gene measured in the superior cervical ganglia and NE content measured in the delivery prevents CIH. Cold exposure also increases Nox2 gene expression and mesenteric arterial bed. Sodium nitroprusside (SNP) produced attenuation of oxidative stress in arteries and kidneys. RNAi inhibition of gp91phox attenuates the NS-induced overflow of NE and perfusion pressure. L-NAME placed in the CIH and vascular dysfunction and abolishes cold-induced renal damage. Cold drinking water prevented the attenuation of the pre- and post-junctional response exposure upregulates protooncogene c-myc expression in the heart. Heat-specific to NS-stimulation of the mesenteric bed from cold exposed rats. L-NAME also inhibition of c-myc expression abolishes CICH, suggesting that up-regulation increased the cold-induced elevation of blood pressure in whole animals. of c-myc may be involved in the pathogenesis of CICH. Therefore, cold It is concluded that differences in NPY and NE transmission act as an important temperatures activate genes of the renin-angiotensin system, up-regulates Nox2 compensatory mechanism preventing dramatic changes in arterial pressure when and c-myc genes, and downregulates eNOS gene, contributing to CIH and CICH. sympathetic nerve activity is high during cold stress.

96 IUPS 2009 July 27 - August 1, 2009 in Kyoto RS VII-39-3 RS VII-39-4 THERMAL PLASTICITY OF CELLULAR CALCIUM COLD PAWS, LARGE HEART: CHRONIC COLD CYCLING IN BLUEFIN TUNA MYOCYTES EXPOSURE AND CARDIAC HYPERTROPHY Holly Shiels1, Barbara A Block2 David Hauton 1Faculty of Life Sciences, University of Manchester, UK, 2Hopkins Marine Department of Physiology, School of Clinical and Experimental Medicine, Station, Stanford University, Pacific Grove, Ca, USA University of Birmingham, UK Bluefin Tuna (Thunnus orientalis) move between warm surface waters and Cold exposure places significant demand on the cardiovascular system of cold deep waters as they dive to depth in search of prey, or during seasonal rodents. Cold acclimation (CA) led to cardiac hypertrophy (P<0.01) in rats. migrations into colder northern latitudes. To understand cardiac thermal CA increased mean arterial pressure in vivo (P<0.01) and performance tolerance we used confocal microscopy and Ca2+ imaging to investigate estimated as rate-pressure product (RPP) was preserved. For ex vivo perfused the temporal and spatial characteristics of cellular Ca2+ flux in bluefin tuna hearts, developed pressure at end diastolic volume (EDV) was maintained and CA increased EDV 20% in hearts (P<0.01) however peak RPP was cardiac myocytes. Tuna were acclimated to either 14°C or 24°C and isolated decreased 40% (P<0.01). Challenge with β-agonists revealed a loss of myocytes were loaded with Fluo-4 and imaged during field-stimulation at 2+ responsiveness to dobutamine (300nM), potentially through β-receptor down- 14, 19 and 24°C. We show cellular Ca flux in the bluefin tuna ventricle regulation. CA increased the uptake of triacylglycerol emulsions (P<0.01) demonstrates thermal compensation including an increased cell size, mediated through increased lipoprotein lipase (P<0.05) and β-oxidation of increased transient amplitude and faster contraction and relaxation kinetics. intralipid-derived fatty acid (FA) was preserved. Excess lipid was diverted This remodeling occurs to a greater extent in the cell periphery than the to tissue storage. Estimation of AMPK activation suggested that oxygenation cell centre which may involve an increase in peripheral couplings between of CA hearts is adequate to meet the metabolic demands of the myocardium. 2+ the sarcoplasmic reticulum (SR) and the sarcolemmal membrane. SR Ca Supplemention with oxfenicine increased cardiac performance (estimated 2+ cycling contributes to the kinetics of Δ[Ca ]i at all temperatures but acute as developed pressure and RPP), regressed the cardiac hypertrophy and 2+ 2+ cooling recruits SR Ca cycling to larger extent than acute warming. Δ[Ca ]i also restored response to β-agonists. Taken together, CA led to cardiac is resistant to acute temperature changes (over 5 °C) suggesting cellular Ca2+ hypertrophy and a mechanically inefficient heart with normal metabolism, cycling pathways compensate for Q10 effects to maintain contractility. yet inhibition of FA metabolism restored cardiac performance.

RS VII-39-5 RS VII-39-6 SYSTEMS BIOLOGY APPROACH TO MAMMALIAN HYPERTROPHIC HAMSTER HEARTS - A COMPROMISE HIBERNATION FOR COLD ACCLIMATION? 1 2 1 1 Jun Yan , Brian M Barnes , Haifang Wang , Chunxuan Shao , Stuart Egginton1, Shaun May1, Durmus Deveci2, David Hauton1 1 1 1 2 Yuting Liu , Ying Li , Wenchao Hu , Vadim Fedorov 1Department of Physiology, University of Birmingham, UK, 2Cumhuriyet 1 CAS-MPG Partner Institute for Computational Biology, Shanghai Institutes University, Turkey for Biological Sciences, China, 2Institute of Arctic Biology, University of Cold acclimation (CA) reveals intrinsic cardiovascular differences, and adaptive Alaska, USA capacity, among rodents exposed to acute reduction in core temperature with Hibernation is an energy saving strategy adopted by a wide range of mammalian or without prior exposure to a simulated onset of winter. Hamsters display species to survive extreme environments through regulated metabolic a baroreceptor reflex that is less sensitive to cold impairment than in the rat, suppression. Mammalian hibernators also evolved the abilities to sustain the low and power spectral analysis of heart rate variability suggests a more plastic body temperature, metabolic rate, and oxygen consumption through complex autonomic control. Cardiac hypertrophy (heart:body mass ratio increased 33% cellular and molecular reorganization. The arctic ground squirrels living in Alaska is an extremely deep hibernator reaching sub-zero body temperature P<0.001) serves to maintain blood pressure, stoke volume and coronary flow while hibernating in the wild. Hibernation is a highly regulated physiological in vivo. However, cardiac work estimates suggest a hypodynamic heart after process. On the molecular level, the regulations are mainly at the transcriptional remodelling. An important aspect of this remodelling is a significant angiogenesis and post-transcriptional levels. On the mRNA level, our and other laboratories (55% increase after 4 weeks, P<0.05), but a reduced capacity for fatty acid have utilized large-scale genomic approaches to investigate differential gene oxidation. Ex vivo (Langendorff) preparations confirm that hypertrophied expression during hibernation in several ground squirrel species. On the protein hearts are severely compromised in function, developed pressure is decreased Regular Symposia levels, we recently have conducted a global high-throughput proteomic study 66% in CA hearts (P<0.05) and rate-pressure product is correspondingly in the liver of arctic ground squirrels using an unbiased high-throughput LC- poor (P<0.05) irrespective of hypo- or normothermia. Thus, maintenance of MS/MS approach . On the miRNA level, we systematically assessed the global adequate performance on cold exposure relies on a significant autonomic drive, expression profile of miRNAs in the arctic ground squirrels by massively parallel suggesting that cardiac remodelling in hibernators is a compromise between the illumina sequencing and Agilent microarray technologies. requirements for hypometabolic periods and intense demands of arousal.

RS VII-40-1 RS VII-40-2 AN OVERVIEW OF PHYLOGENETICALLY BASED PHYLOGENTICALLY-INFORMED ANALYSIS OF THE STATISTICAL METHODS FOR COMPARATIVE DATA UNIVERSALITY OF METABOLIC ALLOMETRY Theodore Garland Craig White Department of Biology, University of California, Riverside, USA School of Biological Sciences, The University of Queensland, Australia Felsenstein's (1985 Am. Nat. 125:1-15) paper on phylogenetically The relationship between the metabolic rate (MR) and body mass (M) independent contrasts ushered in a revolution in the way interspecific of animals has been debated for almost seven decades, focusing on the comparative data are analyzed. Phylogenetic information is generally value of the scaling exponent (b, where MR is proportional to M b) and the required to allow proper statistical hypothesis testing and estimation of the relative merits of b = 0.67 (geometric scaling) and b = 0.75 (quarter-power error associated with such parameters as the slopes of allometric scaling scaling). However, most analyses of the allometry of metabolic rate are relations. In addition, incorporation of phylogenetic information allows one not phylogenetically informed and therefore fail to account for the shared to address such topics as the ancestral states of traits, the sequence of trait evolutionary history of the species they consider. Here, we present the evolution, whether rates of evolution vary among clades, and whether the amount of "phylogenetic signal" (evolutionary lability) varies among types results of phylogentically-informed comparative analyses of the allometry of of traits (e.g. physiology vs. morphology). Finally, due consideration of metabolic rate in insects, birds, and mammals, as well as a quantitative meta- phylogenetic relationships can guide choice of species to be compared so analysis of published scaling exponents. While the results of conventional that the power to detect adaptive (ecologically related) variation or functional non-phylogenetic and phylogenetically informed analyses produce different relations is maximized. Existing phylogenetic methods can be applied to a estimates of the scaling exponent of metabolic rate, it is nevertheless clear wide range of statistical models, including multiple regression, ANCOVA, that the value of b can differ from both 0.67 and 0.75, differs between activity and principal components analysis (Syst. Biol. 1993 42:265-292; J. Exp. states, and differs between ectotherms and endotherms. Thus, we conclude Biol. 2005 208:3015-3035; Physiol. Biochem. Zool. 2008 81:526-550). that there is no single value of b, and no universal metabolic allometry. This Phylogenetic analyses often lead to different conclusions than non- finding represents a significant challenge to models that accomodate only a phylogenetic analyses, as illustrated by our speakers. single value of b.

IUPS 2009 July 27 - August 1, 2009 in Kyoto 97 RS VII-40-3 RS VII-40-4 PHYLOGENETIC AND ECOLOGICAL CORRELATES OF THE SCALING OF MAXIMUM FLIGHT PERFORMANCE RODENT METABOLIC RATES IN HUMMINGBIRDS Enrico L. Rezende Douglas Altshuler1, Robert Dudley2, Sylvia Heredia1, Departament de Genetica i de Microbiologia, Universitat Autonoma de Jimmy McGuire2 Barcelona, Spain 1Department of Biology, University of California, Riverside, USA, Rodents are one of the most widespread group of animals in the world, 2University of California, Berkeley, USA encompassing over 2,000 species that, at present, can be found natively Muscle mechanical theory predicts that mass-specific power output should in all continents except for Antarctica. Their small body size and fast decline with increasing mass. For over fifty years, investigators have recognized generation times make this group a very suitable model to study evolutionary that the study of load-lifting by flying animals should lead to an unbiased metric physiological adaptations to local conditions from a phylogenetic of maximum performance. Prior work indicated that whole body forces actually perspective. Comparative studies of rodent species have focused on a variety exhibit isometry with respect to muscle mass. Here, we focus on the load-lifting of estimates of aerobic performance, including basal metabolism, maximum performance of hummingbirds, which represent an extreme in mechanical metabolic rates, nonshivering thermogenesis, and field metabolic rates. specialization. Our analysis included direct measurements of lifted load and These rates are intrinsically related to some extent, but potentially subject wingbeat kinematics for 62 species represented in a modern phylogenetic to different types of selection. In this talk, I will focus on: (i) the ecological framework. Maximum lifting capacity scaled isometrically with respect to factors associated with the variability in metabolic rates of rodents, including muscle mass, in agreement with previous studies. However, the relative capacity such environmental factors as temperature, habitat, primary productivity, of hummingbird flight muscle to produce power exhibited negative allometry, and food habits; (ii) how these factors may affect different estimates of in support of theoretical predictions from muscle mechanics. We further aerobic performance and result in correlated responses at other levels; (iii) examined allometric relationships across elevations because these gradients how a phylogenetic comparative approach can shed light on the relevance of represent natural experiments for varying muscle capacity. The log-log slopes plasticity as a determinant of phenotypic variation; and (iv) future venues for declined progressively at higher elevations. These reductions were mediated comparative studies on energetics in rodents in particular and endothermic mechanistically through systematic increases in wing stroke amplitude with species in general. elevation, combined with an upper angular limit to wing motions.

RS VII-40-5 RS VII-40-6 ENERGETICS AND THERMOREGULATION OF CROSS-TALK BETWEEN THE METAZOAN FAMILY 2 THE BLACK-CHEEKED LOVEBIRD, AGAPORNIS GPCR SYSTEM NIGRIGENIS: ASPECTS OF PHYLOGENY AND Joao Carlos Cardoso, Nelson Alexandre Coelho, ECOLOGY Deborah Mary Power Jenny Richber, Elke Schleucher Centre of Marine Sciences, Universidade do Algarve, Portugal Dept. of Vegetative Physiology, Institute of Ecology, Evolution and Family 2 G-protein coupled receptors (GPCRs) are one of the largest groups Diversity, Goethe-University Frankfurt, Germany of seven transmembrane receptors with established ligand-receptor pairs in The lovebirds Agapornis are a prominent, well-known genus within the family vertebrates. They are activated by small pleotropic hormones that have conserved Psittacidae, the parrots. This family, in turn, contains the majority of species within the physiological, developmental and metabolic functions from teleosts to tetrapods. order Psittaciformes (332 out of 353 species). The Psittaciformes are a very diverse Recently, in silico comparative studies characterised homologues of family 2

Regular Symposia bird order, with members occupying a vast range of habitats, exploiting various food GPCR in invertebrates but failed to identify potential ligands raising intriguing niches and covering a wide body mass range (10 g in the smallest species of pygmy- question about the evolution and physiological profile of the family 2 GPCR parrots to over 3 kg in the flightless New Zealand Kakapos). Thus, the order is ligand-receptor system. In this study three C. elegans family 2 GPCRs were extremely interesting for allometric studies considering taxonomical and ecological isolated and functionally characterised. The physiological role of the receptors variables. was compared to the vertebrate system using “cross-talk” strategies. The The 9 species of Agapornis are confined to sub-Saharan regions of the African continent, in fact, the Black-cheeked Lovebird is considered endangered due to response of nematode receptor signalling, physiology and gene expression to its extremely restricted range in Zambia. We investigated the metabolic rate and challenge with human family 2 GPCR ligands were all assessed. Results suggest body temperatures of A. nigrigenis (body mass: 38 ± 1.6 g) with regard to various that during nematode life cycle family 2 GPCRs probably regulate different environmental and behavioural factors. Data on gaseous metabolism and rest phase functions. Moreover, differential activation of receptor signalling by human body temperatures are discussed against the ecological background of the species peptides suggests structural pre-adaptation of the receptor and the underlying and its taxonomic affiliation particularly in the light of a recent phylogenomic study mechanisms are currently under study. identifying the Psittaciformes as sister group to the Passeriformes, the "songbirds“ Acknowledgements: Work co-financed by CCMAR Pluriannual project and FCT (Hackett et al., Science 320:1763-8, 2008). project POCI/CVT/61052/2004

RS VII-41-1 RS VII-41-2 GRAVITY AND CELLS GRAVITATIONAL PHYSIOLOGY OF CRABS Millie Hughes-Fulford Peter John Fraser Dept. of Medicine, University of California San Francisco, USA Institute of Biological and Environmental Sciences, Integrative Physiology, Mechanotransduction is the process of translating mechanical force into University of Aberdeen, UK a biological response. In Earth’s gravity, signal transduction affects body Crabs have angular acceleration detectors similar in size and construction function through a wide array of receptors and ligands that signal induction to the analogous vertebrate semicircular canals. They have a row of slender of gene expression. The most common signaling pathways include thread instead of a gelatinous cupula embedded on a bed of hair cells. These receptor tyrosine kinase (RTK), G-Protein coupled receptors (GPCR) and thread hairs are hinged to their bases on one side and linked via a cuticular extracellular matrix components (integrins). During the last decades, space rod or chorda to two bipolar sensory neurones contained in a specialized flight experiments have demonstrated that gravity has profound effects on chordotonal organ. Linear accelerations affect the angular acceleration astronauts resulting in bone loss, immuno-suppression and several other receptors in crabs as in vertebrates, so tilt in a gravitational field influences aspects of clinical medicine. Interestingly, microgravity has profound effects resting nerve spike frequencies and the relative sensitivity of different on the cellular and molecular level, including changes in cell morphology, directional classes of angular acceleration afferents. A recent discovery is that collapse of the actin cytoskeleton, modification of gene expression, changes the angular acceleration afferents in crabs are modulated by small changes in signal transduction cascades and changes in the polymerization of tubulin. in hydrostatic pressure allowing these organs to function as depth receptors. The effects of mechanical stress (e.g. gravity) or lack of stress (microgravity) Using a comparative approach, this discovery prompted a similar finding for on cell and molecular properties is discussed with an emphasis on the the isolated semicircular canal system of a shark. Crabs also have a brain involvement of signal transduction cascades of RTK, integrins and FasR as with a small number of large identified cells. A small set of equilibrium well as their role in cytoskeleton perception of gravity in mammalian cells. interneurones with direct input from the statocyst are known and their activity These changes in signal transduction in microgravity suggest a role of gravity under gravity may be sampled using implanted Teflon coated electrodes for in cell function. long periods including during oscillation and freefall in Parabolic flight.

98 IUPS 2009 July 27 - August 1, 2009 in Kyoto RS VII-41-3 RS VII-41-4 A GENETIC APPROACH TO UNDERSTANDING GRAVITY SENSING ACROSS VERTEBRATE SPECIES GRAVITY RESPONSES IN DROSOPHILA Dmitry Vitalevich Lychakov 1 2 Kathleen Mary Beckingham , James D. Armstrong , Laboratory of Sense Organs, Sechenov Institute of Evolutionary Michael J. Texada3, Ravi Munjaal1, Rebecca A. Simonette1, Physiology and Biochemistry of Russian Academy of Sciences, Russia Cassidy B. Johnson1, Dean A. Baker4, Sonia Bjorum1 The structural and functional organization of the otolithic membrane remains 1Biochemistry and Cell Biology, Rice University, USA, 2School of one of the challenging problems of vestibulology. For example, despite Informatics, University of Edinburgh, Edinburgh, UK, 3HHMI Janelia Farm similar functions of the otolithic membranes in different groups of animals, Research Campus, Ashburn, VA, USA, 4Dept. Genetics, University of otolithic membrane mass, growth etc vary considerably. It is not clear are the Cambridge, Cambridge, UK different otolithic membrane distinguished by their resistance to the changed The fruit fly Drosophila melanogaster offers unique opportunities to gravity etc.? Otolithic organs of 37 species of seven vertebrate classes understand physiology and behavior through genetic approaches. Working were examined with light, transmission and scanning electron microscopy, with this organism we performed a large behavioral screen to identify morphometry, and mathematical modeling. Vestibular apparatus was studied mutants with altered gravity responses. A vertical maze with eight choice experimentally under condition of microG (Xenopus embryos) and hyperG points at which flies could walk up or down was used as our main assay. (guppy larvae). The otolith mass asymmetry was evaluated in rays, fishes Mutant lines with aberrant maze exit profiles were further tested in additional and pigeon. Based on the present and previous investigations, four otolith behavioral paradigms to identify lines specifically defective in gravity regularities were derived. Otolithic membrane response to a stepwise force responses. A total of 18 genes that affect gravity behavior were identified. and gravity was modeled and phylogenetical and functional explanations for Molecular characterization of a subset of these genes has identified the observed otolithic membrane variability were suggested. The results of mechanosense organs that play roles in gravity responses and indicated both the microG and hyperG experiments are now discussed. molecular and neuronal components of the gravity signaling pathways.

RS VII-41-5 RS VII-41-6 REGULATION OF BLOOD PRESSURE DURING HEAD NON-PHOTIC ENTRAINMENT: GRAVITY AND THE MOVEMENT IN THE ANESTHETIZED GIRAFFE NEUROVESTIBULAR SYSTEM Emil T Brondum1, Tobias Wang2, Michael Hasenkam3, Niels H Secher4, Kristyn Michelle Ringgold, Charles A. Fuller Hans Nygaard3, Karin Kastberg5, Rikke Buhl6, Christian Aalkjaer1 Department of Neurobiology, Physiology and Behavior, University of 1Institute of Physiology and Biophysics, Aarhus University, Denmark, California, Davis, USA 2Department of Zoophysiology, Aarhus University, Denmark, 3Institute of 4 Gravitational load can have a direct influence on the period or phase of Clinical Science, Aarhus University Hospital, Skejby, Denmark, Department the circadian timing system (CTS), however the mechanisms involved Anesthesiology, Rigshospitalet, Copenhagen, Denmark, 5Department of 6 remain poorly understood. Work in our lab has a suggested a role for Radiology, Aarhus University Hospital, Skejby, Denmark, Department of Large Animal Science, Copenhagen University, Denmark the neurovestibular system in gravity-induced changes in the CTS. For example, we have shown that changes in circadian period and phase in During daily life the giraffe experiences great cardiovascular challenges, e.g., when it lowers the response to locomotion (wheel running) require functional macular gravity head to drink. This study investigated how blood pressure is regulated when lowering the head of the anaesthetized giraffe. receptors. This study tested the hypothesis that daily, timed centrifugation METHODS: We measured pressure and flow by insertion of catheters and flow-probes in the (2G) will entrain the CTS in animals with functional vestibular organs, but carotid arteries and jugular veins of 5 anaesthetized spontaneously breathing giraffes, suspended in not animals lacking vestibular inputs. To test this hypothesis, chemically upright position. Changes in jugular vein cross sectional area were visualized by ultrasound (US). labyrinthectomized (Labx) mice and wildtype (WT) littermates were RESULTS: When the giraffes head was lowered to heart level, mean arterial pressure (MAP) implanted i.p. with biotelemetry and individually housed in a 4-meter decreased from 205 + 14 mmHg to 139 + 18 mmHg, and central venous pressure (CVP) fell 2.9 + 1.2 diameter centrifuge in constant darkness (DD). After 2 weeks in DD, the mice

mmHg. US images revealed significant increase of the cross sectional area of the jugular vein from Regular Symposia 2 2 were exposed daily to 2G via centrifugation from 1000-1200 for 9 weeks. 0.12 + 0.04 cm to 3.16 + 0.59 cm , and thus an accumulation of estimated 2.5L of blood. Only WT mice showed entrainment to the daily 2G pulse. The 2G pulse was CONCLUSION: When the giraffe lowers its head, central blood volume falls and the associated reduction in cardiac filling lowers MAP by 60 mmHg. When the giraffe lifts the head, the vein then re-set to occur at 1200-1400 for 4 weeks. Only WT mice demonstrated a collapses and the accumulated blood is returned to the central circulation, increasing preload. Our phase shift in response to the re-setting of the 2G pulse. These results provide study shows that the Starling mechanism may be involved in protecting the giraffe’s brain when further evidence that gravitoinertial stimuli require a functional vestibular lowering the head. system to both entrain and phase shift the CTS.

RS Ed-43-1 RS Ed-43-2 INTRODUCTION: THE IMPORTANCE OF ACTIVE PROMOTING ACTIVE LEARNING IN LARGE LECTURE LEARNING AND ITS FACILITATION THROUGH GROUPS WITH A PERSONAL RESPONSE SYSTEM CURRICULUM DESIGN David Alan Williams Jonathan David Kibble Department of Physiology, The University of Melbourne, Australia Department of Medical Education, University of Central Florida, USA We report on two different long term programs that have aimed to improve Since the last IUPS meeting the literature has continued to emphasize the student engagement in large classes (up to ~300 students) and student importance of learner-centered approaches to education. The global trend achievements in subsequent assessment tasks. Over the last 5 semesters of is towards curriculum design that facilitates construction of knowledge 2nd year Physiology (2006-08) we have progressively evolved a program and skills through the application of active learning methods. There are active learning by using a personal response system (PRS). Concurrently, many definitions of active learning and now many published examples we used PRS more disparately in small blocks of Physiology lectures (2-5 of approaches that successfully engage our students. This symposium lectures) presented in multi-disciplinary 1st and 2nd year Medical and showcases examples of active learning pedagogy from around the world and Physiotherapy courses (100 to 300 students). from different educational settings. Physiology students leased personal units for use throughout the semester. Lecture focus and assessment of the course was totally under control of the Physiology Department. In multidisciplinary Medical courses we contributed a small number of topic-focussed lecture blocks amongst a variety of topics/ styles presented by other disciplines. With less control over philosophy and structure we distributed smaller numbers of PRS units, to small competitive student teams (8-12), each working with a single PRS unit. Detailed evaluations noted significant improvements in student engagement and enjoyment with both approaches, and problem-solving ability and assessment outcomes where a more concerted approach was possible.

IUPS 2009 July 27 - August 1, 2009 in Kyoto 99 RS Ed-43-3 RS Ed-43-4 ACTIVE LEARNING IN THE TEACHING LABORATORY: ACTIVE LEARNING THROUGH FORMATIVE A MEANS FOR IMPROVING STUDENT ATTITUDES ASSESSMENTS IN THE INTRODUCTORY BASIC SCIENCES STREAM OF THE MEDICAL CURRICULUM Kim Henige Mangala Gunatilake Department of Kinesiology, California State University, Northridge, USA Department of Physiology, Faculty of Medicine, University of Colombo, Sri An important element of science teaching and learning takes place in the Lanka teaching laboratory. The National Research Council describes typical Formative Assessment is a novel concept introduced to the Introductory laboratory experiences in the US as, "poor for most students". The traditional Basic Sciences Stream of the Medical Curriculum in 2006, for the AL/2005 science laboratory assignment is a cookbook investigation where teachers or batch of medical students. Prior to the introduction of formative assessments lab manuals carefully guide students through experiments, like the steps of a to the curriculum, sessions were held with students to discuss examination papers after “Continuous Assessments (CA)”. When compared to a session recipe, in order to verify something the students already know. There is little held to discuss an examination paper after a CA, formative assessments are incentive to think or be creative, minimizing anticipation and the stimulation beneficial to both the students and teachers as the same is held while the of curiosity. Lab in this traditional form is often viewed by students as boring teaching activities are continued in a particular term. and tedious. Humans have a natural desire to make sense of the world around Formative assessments provide an opportunity for students to clarify their them; they are naturally curious. Active learning takes advantage of this problems in understanding subject contents, to learn about type of questions natural desire and uses curiosity to drive the curriculum. A switch to active given at Continuous and Summative assessments, to learn how to write a learning labs, where students are in the active pursuit of answers to questions suitable answer for a structured essay question and also how to do a self assessment as most of the time students answer scripts are marked by or solutions to problems could be the key to improving student attitudes themselves at the end of the formative assessment. toward the subject matter and science in general. Improved attitudes may Students often request to increase the number of formative assessments per then lead to higher science achievement, since achievement in science has discipline per term and the higher rate of attendance of students at formative been shown to be positively correlated with the attitude toward science. assessments are clear indications of its usefulness in student learning.

RS Ed-43-5 RS Ed-43-6 ACTIVE LEARNING AND CURRICULUM REFORM IN CLOSING REMARKS JAPAN Maria Jose Alves Rocha Osamu Matsuo Department of Morphology, Stomatology and Physiology, Sao Paulo Department of Physiology, Kinki University School of Medicine, Japan University, Brazil In the traditional curriculum in Japan, most of the time had been used for Active learning can improves student’s understanding and retention of lecture, and no small group learning was implemented. At Kinki University, information and be very effective in developing higher order cognitive skills we have introduced small group learning session with a tutor for problem- such as problem solving and critical thinking. However, using active learning solving learning in 1998. This new curriculum with PBL-tutorial encouraged is not without its challenges as sometimes we face disinterested students, students to learn by themselves, as a group or alone. In 2001, our government time pressures, and classroom management issues. Several strategies gave an alarm to declare that reform of medical education is necessary, in are used in classes both large and small. Active learning in the teaching Regular Symposia which active learning is recommended. Since then, most of medical school laboratory where students are in the active pursuit of answers to questions or (total 80 in Japan) started curriculum reform. Further, the government set up solutions to problems is a mean for improving student’s attitudes. Formative new residency program after graduation. According to his program, more assessment instead the summative assessment can be beneficial to both the than 60% of gradates left the university. Thus, shortage of human resource is students and teachers as can be held while teaching activities are continuing. currently serious problem for clinical situation as well as medical education PBL-tutorial is another example of strategy used to engage students in their including physiology. own learning. As the students nowadays are constantly connected to internet, For PBL-tutorial, students enjoy the process how to find the problem and learning in real time is not recommendable to have anymore the traditional how to approach to required resource with discussion among the group way to teaching in which the teachers talk and students listen, and we have to members. After the course (3-5 tutorial class), students felt they did it by look for other learning strategies. themselves, feel happy, but still something unresolved remains. This point further stimulates students to learn by themselves. We have developing integrated laboratory practice curriculum based on PBL

RS Ed-44-1 RS Ed-44-2 TEN YEARS OF ACTIVITY ON THE IUPS EDUCATION INTERNATIONAL PARTICIPATION IN THE JOURNAL COMMITEE LIST SERVE ADVANCES IN PHYSIOLOGY EDUCATION Adrianta Surjadhana Robert Graham Carroll Department of Physiology, Brody School of Medicine, East Carolina Dept Physiology, Airlangga Univ and Widya Mandala Univ, Indonesia University, USA Contacts developed through IUPS meetings have allowed physiologists to The journal Advances in Physiology Education promotes and disseminates build international communities. Following the IUPS teaching workshop educational scholarship in order to enhance the teaching of physiology, 1983 in Australia (Jenolan Caves), the community was maintained through neuroscience and pathophysiology. From its inception in 1989, Advances has postal mail and telephones. In 1999, a mailing list serve was built and named reduced the barriers to international participation. The costs associated with “IUPS-TEACHING”. This forum distributing messages, allowing participants journal publication are underwritten by the American Physiological Society, including submission fees and page charges. Journal leadership extends to share instructor and faculty generated teaching materials, important links, beyond the USA, including a previous Editor in Chief, multiple associate files of programs of IUPS Teaching workshops and PowerPoint slides of editors, and members of the editorial board. The journal is freely available lectures. Members could conduct surveys and distribute the results of their online, a particular advantage for individuals in developing countries. Over teaching projects. The list serve functions even in environments where half of the submitted manuscripts originate from outside of the United States access to technology is limited, allowing participation from both developed (56% in both 2007 and 2008), representing over 20 countries. For the 2008 and developing countries. The IUPS Education Committee maintains a list- calendar year, submissions from the USA had a greater acceptance rate serve that allows interested educators to share ideas and to remain in contact. (89%) than did submissions from other English speaking countries (42%) or from countries where English is not the primary language (26%). Advances Similar list-serves are maintained for the FAOPS (Federation of Asian and in Physiology Education serves the international physiology teaching Oceania Physiological Societies) and the Indonesian Physiology Society. community through the solicitation, peer review and publication of quality Important meetings connected to physiology teaching and learning could be educational scholarship. announced by the mailing list serve which will benefit its members.

100 IUPS 2009 July 27 - August 1, 2009 in Kyoto RS Ed-44-3 RS Ed-44-4 THE SPREAD OF COMPETENCY-BASED EDUCATION INTRODUCTION Beatriz Unwin Ramirez Ann Elizabeth Sefton Escuela de Medicina, Universidad de Santiago de Chile (USACH), Chile Physiology, University of Sydney, Australia Globalization initially affected mainly politics and commerce but has also For more than 30 years, IUPS has supported the development of quality produced profound changes in education. It is promoting modifications of education in physiology through symposia and poster sessions associated programs and organization of higher education and professional training. with each International Congress. In addition, advances in teaching and After the Bologna process in the 1990´s, competency-based curriculum learning physiology have been developed in regional or local meetings, and supported by national vocational qualification systems in England, Wales, through an active email discussion group. Over that time, not only has there New Zealand, and others, has spread to the world. Latin America, Caribbean been a substantial increase in our understanding of physiology, but evidence- countries, South Africa, Asia and Australia joined to Europe, EEUU and based educational practice has advanced rapidly. There is now a substantial Canada, trying to solve the challenges imposed by economic competition and growing literature in many aspects of teaching and learning physiology onto the development of vocational education. and related sciences. The new information technologies have offered both Oriented toward outcomes, competency-based education should produce challenges and solutions to many aspects of teaching and learning. With the authentic learning and a lifelong learning attitude, aligned with pertinence growth of the new educational approaches, the new knowledge is rigorously and relevance, since one of the main objectives of education is the promotion evaluated and applied on the basis of evidence. A brief summary of the of social sustainable development. A wide range of approaches to get these development of teaching workshops and related activities will be presented targets have been developed by different countries and universities. and discussed. Despite there is general agreement that globalization has improved the quality of education, there are still many problems to solve, like national versus regional context, valid assessment, quality control mechanisms and curriculum equivalence among others.

RS Ed-44-5 RS Ed-44-6 ANALYSES OF THE EFFECT OF INTERNATIONAL AND BREAKING BARRIERS: TEACHING RESOURCE REGIONAL TEACHING WORKSHOPS SHARING IN A GLOBAL COMMUNITY Arif Siddiqui1, Ann Sefton2 Marsha Lakes Matyas 1Department of Biological and Biomedical Sciences, The Aga Khan Education Office, American Physiological Society, USA 2 University, Pakistan, Physiology, University of Sydney, Australia The American Physiological Society (APS) Archive of Teaching Resources Physiology Teaching since past decade has been subjected to new challenges. (www.apsarchive.org) launched in April 2002 as a venue for colleague- Integrated approach to learning of basic sciences, problem based learning to-colleague sharing of educational materials. Since then, the Archive has and increasing use of information technology were regarded as major grown into a collaborative digital library with resources catalogued by APS interventions to review the approaches to physiology learning. Physiology and other scientific societies that shares its metadata with both the U.S. teachers generally remained vigilant and were taking initiatives in order National Science Foundation Life Sciences Portal (www.biosciednet.org) to develop understanding towards the challenges and devise strategies to and the U.S. National Science Digital Library (www.nsdl.org). In 2008, the meet the changed needs. IUPS and several other regional fora since the Archive was redesigned with new features for partner customization, easier past decade or so found generously supportive to the initiatives. Teaching submission and review, and user collaboration tools. While digital libraries workshops organized in different parts of the world in recent years have are often simply distribution points for published materials, the Archive been continuously addressing emerging challenges. Vigorous debates at offers opportunities for collaboration, sharing, and improvement of teaching these workshops provided opportunities to raise issues in the context of local materials among educators around the world. This presentation will provide

difficulties. Valuable interaction occurred between less privileged participants both examples of how the Archive has been used for colleague-to-colleague Regular Symposia and the more enlightened colleagues. One specific observation noted was sharing in the past and will outline future opportunities for international the openness with which individuals listened as they felt encouraged to join collaboration and use. discussion. Plethora of ideas generated and the variety of experiences learnt and sense of sharing common purpose of educating students in physiology with an even higher standard served a common purpose.

RS Ph-45-1 RS Ph-45-2 IN SILICO ML AND INTEGRATED ENVIRONMENT TRANSLATING THE CARDIAC PHYSIOME INTO THE Yoshiyuki Asai1, Yasuyuki Suzuki2, Yoshiyuki Kido1, Hideki Oka3, CLINIC Eric Heien4, Takahito Urai5, Kenichi Hagihara4, Yoshihisa Kurachi6, Nicolas Peter Smith1, Steven A Niederer1, David A Nordsleten1, Taishin Nomura2 Mathew McCormick1, Kawal Rhode2, Reza Razavi2 1The Center for Advanced Medical Engineering and Informatics, Osaka 1 2 2 Computing Laboratory, University of Oxford, UK, Cardiovascular Medicine University, Japan, Department of Mechanical Science and Bioengineering. Kings College London, UK Graduate School of Engineering Science, Osaka University, Japan, 3Fujitsu Limited, Japan, 4Department of Computer Science, Graduate School of The significance of heart disease has motivated the application of state of Information Science and Technology, Osaka University, Japan, 5Intasect the art clinical imaging techniques to aid diagnosis and clinical planning. 6 Communications, Japan, Department of Pathophysiology and Therapeutics, However to exploit the full value of such imaging technologies, and the Graduate School of Medicine, Osaka University, Japan combined information content they produce, requires the ability to integrate We have developed the specifications of insilicoML (ISML) version 1.0, which is an multiple types of functional data into a consistent framework. An exciting extensible markup language format for describing multi-level biophysical models, as an and highly promising strategy for underpinning this integration is the extension from the previously released version ISML 0.1 available in the public domain (http://www.physiome.jp). ISML 1.0 can describe mathematical models of physiological assimilation of multiple image sets into personalised and biophysically functions by ordinary/partial differential/difference equations and the geometry of living consistent mathematical models. The development of such models provides organisms underlying the functions. ISML is fully compatible with CellML 1.0, a pioneering the ability to capture the multi-factorial cause and effect relationships descriptive language of models developed by the IUPS Physiome Project. In ISML, a model which link the underlying pathophysiological mechanisms. Applying this is described as an aggregation of functional elements (modules) connected to each other approach we report on the development and application of a human cardiac by edges representing structural, logical and functional relationships, allowing modular, framework representing the cardiac electrical, mechanical and fluid systems hierarchical, and/or network representations of the model. This module-edge-relationships to optimize clinical outcomes. Specifically these include cardiovascular labeled by keywords provide rich information for constructing a physiological ontology. ISML 1.0 is designed to operate with a model database, morphology database and integrated resynchronization cases, implantation of left ventricular assist devices and graphical environments (called insilicoIDE) for model development and simulations for analysis of coronary perfusion. knowledge integration and discovery. For large scale models, ISIDE uses parallel computing for effective simulations.

IUPS 2009 July 27 - August 1, 2009 in Kyoto 101 RS Ph-45-3 RS Ph-45-4 AN OVERVIEW OF SEVERAL STANDARDIZATION VPH TOOLS EFFORTS FOR SYSTEMS BIOLOGY S. Randall Thomas 1 2 Michael Hucka , BioModels.net Participants IBISC (Informatics, Integrative Biology and Complex Systems), CNRS 1Engineering and Applied Science, California Institute of Technology, USA, FRE 3190, France 2 Worldwide Within the European Virtual Physiological Human (VPH) Network of A computational model represents a modeler's understanding of the structure and Excellence (NoE), a key deliverable is the “VPH ToolKit”, defined as "a function of parts of a biological system. As the overall number of quantitative models technical and methodological framework that will support and enable VPH continues to grow, and they become ever more sophisticated, they collectively Research through the creation, accumulation, and curation of VPH research- represent a significant accumulation of knowledge about the structural and functional organization of biological systems. Enabling effective sharing of such quantitative related 'capacities'." In particular, the VPH ToolKit consists of markup models is the driving vision behind SBML and several related efforts that I will languages for models and for data, software for signal and image analysis, describe in this presentation. infrastructure for grid access, and various other elements grouped under •SBML (the Systems Biology Markup Language) is a machine-readable exchange the heading "VPH Tools". I will present progress within the VPH toolkit format for computational models in systems biology. development team on adaptation of a number of such tools for the VPH •MIRIAM (Minimum Information Requested in the Annotation of Models) is a set of framework, in particular, tools for collaborative development of multi- guidelines for model annotation scale models, robust multi-formalism numerical solvers, visualization of •SBO (Systems Biology Ontology) is an ontology of mathematical concepts used in simulation results, interactive model repositories, and others. computational models •MIASE (Minimum Information About a Simulation Experiment) is a set of guidelines for making simulation results reproducible. •SBGN (Systems Biology Graphical Notation) is a standard for graphical drawings of biological networks. All of these efforts are, at their core, a means of improving our ability to communicate our discoveries and understanding.

RS Ph-45-5 RS Et-46-1 MULTISCALE, MULTI-PARADIGM MODELLING OF ANIMALS IN MEDICAL RESEARCH: MAGIC OR EPITHELIAL TISSUE TRAGIC? Rod Smallwood Colin Blakemore Department of Computer Science, University of Sheffield, UK Department of Physiology, Anatomy and Genetics, University of Oxford, UK The understanding of development, homeostasis and wound healing in epithelial tissues (the skin and lining of body cavities) has to be centred There is an abiding tension between the desire of reasonable people to avoid around the behaviour of individual cells. An individual-based modelling deliberate harm and their wish to benefit from advances that accrue from the approach is used, with each cell represented by a finite-state machine (a use of animals in research. communicating stream x-machine, originally formulated by Eilenberg as a Many opponents of animal research argue that ‘alternative’ approaches make the use of animals unnecessary. Moreover, some claim that animal research Turing-complete computational machine). State transitions are controlled by a

Regular Symposia is unreliable as a guide to understanding human function and developing and set of functions, modulated by information from the state machine's memory testing new treatments. Politicians and the public face powerful arguments and the input data stream. At the end of each time step, information is written that animal research is unethical, unnecessary and even dangerous. to the output data stream. The functions describe both the biochemical and What should be done in response? A strong regulatory framework; strict the physical behaviour of each cell [Walker 2004, Sun 2007]. Three aspects requirements for welfare and husbandry; certification of researchers and will be discussed - the cell-level model; incorporating signalling models projects; good record-keeping. These all help to maintain public confidence. (sub-cellular); and embedding the cell in a physical environment in which it But clear, informative communication, both proactive and reactive, is can grow, divide and exert forces on other cells (tissue-level) - with examples also essential. Recent experience in Europe, and especially Britain, shows drawn from work on wound healing. how important it is that the scientific community should take the lead in Walker D C, Hill G, Wood S M, Smallwood R H, Southgate J (2004) IEEE promoting not only impeccable standards of practice in animal research but Trans Nanobioscience 3:153-163 also openness, honesty and rational debate. The willingness of researchers to Sun T, McMinn P, Coakley S, Holcombe ,M Smallwood R, MacNeil S (2007) speak openly about their work has done a great deal to secure the increased J Roy Soc Interface 4:1077-1092 trust of the media and the public in Britain.

RS Et-46-2 RS Et-46-3 GUIDELINES FOR PROPER TREATMENT OF ANIMALS STANDARDS FOR GOOD RESEARCH PRACTICE: IN RESEARCH SCIENTIFIC INTEGRITY AND DEALING WITH Naoko Kagiyama1, Shigeo Ito1, Gilles Demers2 MISCONDUCT 1Graduate School of Veterinary Medicine, Hokkaido University, Japan, Matthias Kaiser 2International Council for Laboratory Animal Science, Canadian Council for National Committees for Research Ethics, NENT, Norway Animal Care, Canada While some countries, notably the USA, have been aware of the problem of The International Council for Laboratory Animal Science (ICLAS), as an international scientific misconduct for some time, and have indeed established bodies to umbrella organization, published harmonization guidance in Science in 2006 focusing deal with allegations of misconduct professionally, most countries have only on the principles for humane endpoints & animal euthanasia. The second guidance recently realized their vulnerability and lack of preparedness in this regard. addressed animal user training programs & experimental protocol review. The latest Some well publicized scandals during the last years initiated a number of manuscript under discussion is regarding the care & use of genetically engineered national and international activities with the aim to deal with scientific animals. misconduct and to foster scientific integrity. The Global Science Forum of Based on an inquiry from the related ministries, the Science Council of Japan (SCJ), the OECD has looked at the need of international cooperation and standards as the ICLAS National Member, established the SCJ Guidelines for Proper Conduct for best practice in dealing with scientific misconduct. The first World of Animal Experiments in 2006. These Guidelines were elaborated by collaboration Conference on Research Integrity was held in Lisbon 16-19 September of representatives from all scientific fields related to animal experiments including 2007. One has come to realize that the hard-core of scientific misconduct, so the biological, pharmaceutical, agricultural, veterinary & medical sciences. The SCJ called FFP (fraud, fabrication, plagiarism), needs to be understood within the considers that animal experimentation ethics should agree with scientific rationale & wider framework of so called QRPs (questionable research practices, such motivates scientists to conduct animal experiments with creativeness and flexibility. as e.g. honorary authorship etc). Episodes of FFP are in general rare, though To implement the Guidelines, Hokkaido University School of Veterinary Medicine they are disturbing for the progress of research and undermining the trust will develop a 3R-based high-level educational program. The program includes in science in general. Episodes of QRP are more wide-spread and can serve quantitative measurement of pain using isolated neonatal rat spinal cords & functional as indicators of a lack of awareness about normative constraints for good MRI technology on anesthetized rats. research practices.

102 IUPS 2009 July 27 - August 1, 2009 in Kyoto RS Et-46-4 RS A-47-1 PUBLISHING PHYSIOLOGICAL RESEARCH: SIMULTANEOUS MR-PET ENABLES MECHANISTIC INTEGRITY AND MISCONDUCT HUMAN NEUROIMAGING Kim Elaine Barrett A. Gregory Sorensen Department of Medicine and Office of Graduate Studies, University of AA Martinos Center for Biomedical Imaging, Department of Radiology, California, San Diego, USA Massachusetts General Hospital / Harvard Medical School, USA Publication is critical in physiological research, and carries much weight in There is tremendous interest in using imaging as a biomarker for funding and promotion decisions. Moreover, the scientific enterprise rests understanding disease mechanisms, therapeutic effect, pharmacodynamics, heavily on our ability to trust the data and conclusions of our colleagues. and as surrogate endpoint. Mechanistic neuroimaging is an approach The majority of scientists are entirely ethical in publishing their findings. to utilizing advanced or mainstream imaging methods to identify However, some studies suggest that a significant proportion of scientists will pathophysiological pathways and to image specific events along that admit to occasional questionable practices if surveyed anonymously. Further, pathway. One of the newest brain imaging methods used for this purpose the trends towards multiple authors, image manipulation, and increased combines simultaneous magnetic resonance imaging and positron emission competition for positions and funds are all risk factors for misconduct. As tomographic imaging. This presentation will review the use of mechanistic Chair of the APS Publications, I am responsible for review and disposition neuroimaging with MRI and with simultaneous MRI and PET in the quest to of ethical complaints in our 14 journals. During my tenure, the number of identify biomarkers to understand human disease and novel interventions. instances has increased steadily. There are a wide range of transgressions, including redundant or duplicate publication, animal and human subject concerns, authorship disputes, data fabrication/falsification, plagiarism, conflicts of interest, and others. My presentation will define these unethical practices and how one might avoid them. I will also discuss the Society’s approach when our ethical policies are breached, and some of the challenges to resolving allegations of misconduct, particularly in the international arena.

RS A-47-2 RS A-47-3 DIFFUSION fMRI AND POTENTIAL FOR VISUALIZING COLUMNAR ARCHITECTURES USING NEUROIMAGING HIGH-FIELD FUNCTIONAL MAGNETIC RESONANCE Denis LE BIHAN IMAGING NEUROSPIN, DSV/CEA, France Kang Cheng During their diffusion-driven displacements water molecules probe tissue Laboratory for Cognitive Brain Mapping, RIKEN Brain Science Institute, Japan structure at a microscopic scale, interacting with cell membranes and, thus, providing unique information on the functional architecture of tissues. In the Since its inception early in 1990s, functional magnetic resonance imaging mid 1980s it was shown that water diffusion in the brain could be imaged (fMRI) has become one of the most dominant neuroimaging tools for using MRI, and diffusion MRI has been used as an exquisite marker of studying human brain fucntions. High-resolution fMRI, with its improved acute brain ischemia and cell swelling through cytotoxic edema. Also, water signal-to-noise ratio and spatial specificity, has strengthened the capability of fMRI and allowed mapping of functional architectures in human brains. diffusion is anisotropic in white matter, allowing to map out the orientation in In this talk, I will first explain the principle of the blood oxygenation level- space of the white matter tracks and image brain connections. More recently, dependent (BOLD) effect, upon which most of fMRI experiments are it has been shown that diffusion MRI is sensitive to cortical activation. This conducted, factors restricting the spatial specificity of the BOLD signal, and discovery could represent a significant breakthrough, allowing non invasive measures dealing with these factors. I will then briefly introduce several access to a direct physiological marker of brain activation, while current practical techniques developed using high-field MRI systems for revealing functional neuroimaging approaches in humans are based on the indirect and functional architectures in human visual cortices. remote monitoring of blood flow changes. Regular Symposia

RS A-47-4 RS A-47-5 ON ARTERIAL AND CEREBRAL BLOOD FLOW UTERINE CONTRACTILITY EVALUATED WITH CINE RESPONSES TO FUNCTIONAL CHALLENGES MR IMAGING Xavier Golay1, Yi-Ching L. Ho2, Esben T. Petersen2 Kaori Togashi 1Department of Brain Repair and Rehabilitation, UCL Institute of Neurology, Department of Diagnostic Imaging and Nuclear Medicine, Kyoto University UK, 2Department of Neuroradiology, National Neuroscience Institute, Graduate School of Medicine, Japan Singapore Cine MR images obtained with use of ultra-fast MR imaging technique Upon functional activation, a complex series of events happen in the brain. has enabled us evaluate uterine inherent contractility directly and non The first event, and the one we are interested in is a local increase in cerebral invasively. The observation of dysmenorrheal patients revealed clinical blood flow (CBF) or hyperaemia. This local increase in perfusion is believed to relevance of sustained contraction with pain scale, which was alleviated be directly related to the raise in electrical and metabolic activity in the brain, by the oral contraceptive. Another type of contraction that is called uterine realized through an elaborated mechanism involving pericytes, endothelial and peristalsis is clearly shown as rhythmic and subtle stripping movement in smooth muscle cells. the subendometrial myometrium. The uterine peristalsis is different in its The measurement of CBF and other vascular parameters in functional MRI frequency and direction depending on the menstrual cycle. The direction studies has been a challenge so far. First, typical techniques, based on arterial spin of the peristalsis is cervico-fundal in periovulatory phase, whereas fundo- labelling (ASL) preparation of the arterial magnetization, have a relatively low cervical in t menstrual phase. The former wave direction may help to carry SNR. Then, they are based on assumption usually not fulfilled in fMRI. Here, sperm to the fundus and the fallopian tube, where fertilization occurs, and we will demonstrate the capacity of a new ASL technique to study physiological the latter may facilitate discharge of the menstrual hematoma. In a variety of changes upon functional activation. Using graded visual stimulation, in certain conditions where the fertilization is disturbed (i.e. subendometrial myoma, cases combined with hypercarbic or hypoxic challenges, we aimed at studying endometriosis, and intrauterine device), uterine peristalsis was found to the fine local and regional regulations of perfusion at several arteriolar and tissue be abnormal on cine MR. Cine MR imaging can be applied for directly levels in humans. These measurements indicate that coupling between CBF and monitoring changes of the uterus related to important uterine functions such metabolism is regulated at a regional level, with flow increase already present in as fertility and menstrual problems. the larger arteries, while largest proportional changes happen at capillary levels.

IUPS 2009 July 27 - August 1, 2009 in Kyoto 103 RS A-48-1 RS A-48-2 AUTOPHAGY: A MOLECULAR SYSTEM FOR TRPMLs REGULATE TRAFFICKING OF LIPIDS AND DEGRADATION OF PROTEINS AND ORGANELLES PROTEINS ALONG THE ENDOSOMAL/LYSOSOMAL UNDER PHOSPHOINOSITIDE SIGNALING PATHWAY Yasuyoshi Sakai Rosa Puertollano, Jose Martina, Silvia Vergarajauregui, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto Benjamin Lelouvier University, Japan Laboratory of Cell Biology, National Institutes of Health, USA Autophagy is the membrane traffic system to lysosome/vacuole of intracellular TRPMLs constitute a family of cation channels with homology to the transient

protein and organelle degradation. The yeast studies have revealed the molecular receptor potential (TRP) superfamily. Mutations in TRPML1 have been linked to machineries called Atg proteins responsible for autophagy, conserved from mucolipidosis type IV (MLIV). We found that autophagy is impaired in MLIV yeast to higher eukaryotes. The autophagic system contributes to various aspects fibroblasts, causing an abnormal accumulation of ubiquitinated aggregates and protein of cellular quality control. Autophagy functions not only in bulk intracellular inclusions that may contribute to the neurodegenerative phenotype observed in degradation but also in selective degradation of organelles, elimination of MLIV patients. Mutations in TRPML3, a Ca2+ permeable channel regulated by pH, aggregated proteins, a defense system against infected bacteria, maintenance are responsible for the varitint-waddler phenotype in mice. We found that TRPML3 of neuronal homeostasis. Alteration of autophagic activity could lead to major primarily localizes to early and late endosomes in human epithelial cells. TRPML3 neurodegenerative disorders, e.g., Alzheimer’s disease, Parkinson’s disease, over-expression caused dramatic alterations in the endosomal pathway, including and Huntington’s disease. We are studying the molecular basis of autophagy in enlargement of Hrs-positive endosomes, delayed degradation of EGF and EGFR, and relation to phosphoinositide signaling in the yeast Pichia pastoris, which conduct defective autophagosome maturation, indicating that TRPML3 is a novel component both microautophagic degradation of peroxisomes. During micropexophagy, of the machinery implicated in the biogenesis and regulation of the endosomal vacuoles engulfs a cluster of peroxisomes, and concomitant with transient function. Finally, we found that TRPML2 traffics through the Arf6-associated pathway formation of the micropexophagy-specific membrane apparatus (MIPA) on the and regulates recycling of certain GPI-AP proteins to the plasma membrane. cytosolic side of the peroxisome surface. Our recent findings on the molecular In conclusion, we found that TRPMLs distribute to specific locations along the functions of PI(3)P, PI(4)P and PI(3,5)P2-signaling together with their interacting endosomal pathway and play an important regulatory role in the sorting of lipids and Atg proteins will be introduced . proteins.

RS A-48-3 RS A-48-4 THE Ca2+ CHANNEL TRPML3 REGULATES MEMBRANE DROSOPHILA AS A MODEL SYSTEM FOR TRAFFICKING AND AUTOPHAGY MUCOLIPIDOSIS TYPE IV Hyun-Jin Kim1, Abigail A Soyombo1, Sandra Tjon-Kon-Sang1, Kartik Venkatachalam, Craig Montell Insuk So2, Shmuel Muallem1 Biological Chemistry, The Johns Hopkins University School of Medicine, 1Department of Physiology, University of Texas Southwestern Medical USA Center at Dallas, USA, 2Department of Physiology and Biophysics, Seoul Disruption of TRPML1 results in mucolipidosis type IV (MLIV), a National University College of Medicine, Korea lysosomal storage disease leading to neurodegeneration and motor TRPML3 is an inward rectifying Ca2+ channel that is regulated by extracytosolic impairments. The mechanisms underlying MLIV are poorly understood and H+. Although gain-of-function mutation in TRPML3 causes the varitint-waddler there is no treatment. We developed a Drosophila model of MLIV which phenotype, the role of TRPML3 in cellular physiology is not known. Here, we report recapitulates the key disease features, including accumulation of intracellular

Regular Symposia that TRPML3 is a prominent regulator of endocytosis, membrane trafficking and macromolecules, motor defects and neurodegeneration. The basis for the autophagy. Gradient fractionation and confocal localization reveal that TRPML3 buildup of macromolecules was defective autophagy, resulting in oxidative is expressed in the plasma membrane and multiple intracellular compartments. stress and impaired synaptic transmission. These phenotypes were suppressed + However, expression of TRPML3 is dynamic, with accumulation of TRPML3 in by expression of wild-type trpml in neurons. Surprisingly, introduction + the plasma membrane upon inhibition of endocytosis, and recruitment of TRPML3 of trpml in phagocytic cells such as glia and blood macrophages, also to autophagosomes upon induction of autophagy. Accordingly, overexpression of suppressed the trpml mutant phenotypes. This surprising result was explained TRPML3 leads to reduced constitutive and regulated endocytosis, increased autophagy by the observation that late-apoptotic cells accumulated in trpml mutant and marked exacerbation of autophagy evoked by various cell stressors. Importantly, brains as a result of diminished phagocytic clearance of dying cells. Since both knock-down of TRPML3 by siRNA and expression of the channel-dead apoptotic cell clearance is the responsibility of the cellular components dominant negative TRPML3 (D458K) have a reciprocal effect, reducing endocytosis of innate immunity, we propose that the severity and early onset of motor and autophagy. These findings reveal a prominent role for TRPML3 in regulating deficits in MLIV are due to defects in innate immunity. Our results also raise endocytosis, membrane trafficking and autophagy, perhaps by controlling the Ca2+ in the possibility that introduction of functional macrophages by bone marrow the vicinity of cellular organelles that is necessary to regulate these cellular events. transplantation may limit the progression of MLIV.

RS A-48-5 RS A-49-1 MACROAUTOPHAGY IS DEFECTIVE IN MUCOLIPIN MITOCHONDRIAL CALCIUM AND ROS CROSSTALK 1-DEFICIENT MOUSE NEURONS SIGNALING 1 1 1 Susan Slaugenhaupt , Cyntia Curcio-Morelli , Florie Charles , Shey-Shing Sheu, Jennifer Hom, Gisela Beutner, Virendra Sharma, 2 1 1 Matthew C Micsenyi , Yi Cao , Bhuvarahamurthy Venugopal , Robert Feissner, Alina Ainbinder, Yawen Ju, Yisang Yoon 1 2 1 Marsha F Browning , Konstantin Dobrenis , Susan L Cotman , Pharmacology and Physiology, University of Rochester Medical Center, USA Steven U Walkley2 1 The long-term objective of this study is to establish a unified theory to describe the Center for Human Genetic Research, Massachusetts General Hospital, 2+ 2+ 2 mechanisms of crosstalk signaling between mitochondrial Ca concentrations ([Ca ]m) USA, Department of Neuroscience, Albert Einstein College of Medicine, and reactive oxygen species (ROS) in heart cells. Bronx, New York, USA Two questions are addressed: 1) what mechanisms are responsible for Ca2+-mediated Mucolipidosis Type IV is a neurodegenerative lysosomal disease that is caused by mitochondrial ROS generation? 2) Do redox environments modulate mitochondrial mutations in the Mcoln1 gene, which encodes the protein TRPML1. The disease is Ca2+ uptake? characterized by severe psychomotor retardation, visual impairment and achlorhydria. We use fluorescence confocal microscopy, Western blotting, gene knockout or We previously generated the first murine model for MLIV. Here, we describe for the overexpression, and inhibitors for mitochondrial fission protein DLP1, to investigate first time the establishment and characterization of neuronal cultures derived from these questions. The results show that [Ca2+] increases ROS generation. This increase cerebrum of 17 day Mcoln1-/- mouse embryos. A large number of membranous m in ROS is diminished by blocking 1) mitochondrial uniporter, 2) mitochondrial intracytoplasmic storage bodies are readily evident by 10 days in vitro in these permeability transition pores (mPTP), or 3) DLP1. The ROS increases in turn embryonic neuronal cultures. As recently reported in human fibroblast cultures, 2+ macroautophagy is altered in this MLIV neuronal cell model: LC3-II levels were could feedback to modulate further [Ca ]m increases. Indeed, exposure of isolated mitochondria to oxidized solutions augments mitochondrial Ca2+ uptake. increased; Beclin-1, which is involved in autophagosome formation, is increased, 2+ and the mammalian target of rapamycin activity, analyzed via p70S6 kinase In conclusion, an increased [Ca ]m promotes mitochondrial dynamics towards fission phosphorylation, was slightly decreased. The characterization of this neuronal cellular and mPTP flickering that favor ROS generation. The resulting oxidized environment 2+ model for MLIV provides us with the means to test, in the appropriate cell type, causes additional [Ca ]m increases. Eventually, this high-gain positive feedback loop future therapies aimed at preventing or reversing the abnormal lysosomal storage and is counter balanced by Ca2+ and ROS activated various mitochondrial Ca2+ efflux associated cellular pathology. mechanisms.

104 IUPS 2009 July 27 - August 1, 2009 in Kyoto RS A-49-2 RS A-49-3 MOLECULAR AND FUNCTIONAL ALTERATIONS ROLE OF MITOCHONDRIAL DNA IN CALCIUM OF ORGANELLE CROSSTALK DURING CELLULAR SIGNALING ABNORMALITY IN BIPOLAR DISORDER STRESS AND TUMORIGENESIS Tadafumi Kato 1 2 3 3 Laboratory for Molecular Dynamics of Mental Disorders, RIKEN Brain Gyorgy Szabadkai , Katiuscia Bianchi , Mounia Chami , Oules Benedicte , Science Institute, Japan Paterlini-Brechot Patrizia3, Jose Miguel Vicenzio4, Guido Kroemer4 1 Bipolar disorder is a major mental disorder causing severe psychosocial Department of Cell and Developmental Biology, University College London, 2 3 impairment. Altered intracellular calcium signaling in blood cells has UK, Institute of Cancer Research, London, UK, INSERM U807, Paris, France, 4 been reported. A recent genetic study indicated that calcium channel genes INSERM U848, Villejuif, France may be associated with bipolar disorder. Studies of magnetic resonance We have recently shown that protein complexes at the ER-mitochondria interface, involving spectroscopy showed that patients with bipolar disorder have altered brain 2+ 2+ Ca channels (VDAC and IP3R) and molecular chaperones (grp75), contribute to Ca energy metabolism similar to mitochondrial diseases. Comorbidity of mood signal transmission between the organelles, and that ER stress induces mitochondria- disorders with mitochondrial diseases is reported. Accumulation of mutant mediated cell death through ER-mitochondrial crosstalk. Here we used a chemically induced mitochondrial DNA (mtDNA) was detected in the postmortem brains of hepatocellular carcinoma model to study the stress response, ER-mitochondrial interactions several patients with bipolar disorder. We reported that mtDNA variations and ER stress induced apoptosis during the tumorigenesis process. affect the intracellular calcium signaling using transmitochondrial cybrids. We found significant down-regulation of mitochondrial and decrease of the expression of the We developed mutant polymerase gamma transgenic mice with neuron- transcriptional co-activator of PPAR-γ (PGC-1), consistent with a principal role of PGC-1 specific accumulation of mtDNA mutations. The mice showed bipolar in deregulation of mitochondrial biogenesis. Furthermore, our data indicated changes in disorder-like phenotypes and this was improved by a mood stabilizer. several pathways associated with tumorigenesis and regulation of mitochondrial biogenesis: Calcium uptake rate was altered in mitochondria of the transgenic mice. This (i) mTOR complex 1 activity was down-regulated, (ii) phosphorylation of p38MAPK was was found to be mediated by downregulation of cyclophilin D, a component decreased and (iii) The ER stress marker CHOP was strikingly upregulated. Further studies of the mitochondrial permeability transition pore. These findings suggest that will investigate whether tumor-associated changes of mitochondrial biogenesis can be mitochondrial calcium dysregulation may be involved in the pathophysiology responsible for reduced sensitivity to stress induced apoptosis in liver carcinogenesis. of bipolar disorder.

RS A-49-4 RS A-49-5 DIET, MITOCHONDRIAL UNCOUPLING AND CONTROL ROLE OF THE PERMEABILITY TRANSITION PORE IN OF REDOX STATE THE PATHOGENESIS OF COMPLEX I DYSFUNCTION Alicia J. Kowaltowski DUE TO mtDNA MUTATIONS 1 1 2 2 Departamento de Bioquimica, Universidade de Sao Paulo, Brazil Paolo Bernardi , Alessia Angelin , Anna Maria Porcelli , Anna Ghelli , 2 3 4 5 Mitochondria are the most significant source of reactive oxygen species Elisa Mariani , Andrea Martinuzzi , Valerio Carelli , Valeria Petronilli , Michela Rugolo2 (ROS) in most tissues. While most antioxidant supplementation regimens 1 2 have proven ineffective in preventing damage induced by mitochondrial Department of Biomedical Sciences, Padova University, Italy, Department of Evolutionary and Experimental Biology, Bologna University, Italy, 3IRCCS ROS, calorie restriction diets are effective [1]. We found [2] that systemic “E. Medea”, Conegliano Veneto, Italy, 4Department of Neurological Sciences, mild uncoupling is also a highly effective antioxidant strategy, a finding in Bologna University, Bologna, Italy, 5CNR Institute of Neuroscience at the keeping with the idea that uncoupling prevents mitochondrial ROS formation Department of Biomedical Sciences, Padova University, Italy [3]. Interestingly, we also find that high calorie diets increase mitochondrial We have studied HL180 cells (a cybrid line harboring the T14484C/ND6 and G14279A/ND6 ROS release and activate uncoupling pathways. Indeed, uncoupling proteins mtDNA mutations of LHON) and XTC.UC1 cells (bearing a disruptive frameshift mutation in the and ATP-sensitive K+ channels, that promote uncoupling, are redox sensitive MT-ND1 gene). Addition of rotenone to HL180 cells and of antimycin A to XTC.UC1 cells caused [4, 5]. High and low calorie diets also promote significant changes in mitochondrial membrane depolarization that was prevented by treatment with cyclosporin A, intracellular Ca2+ chelators, and antioxidant. Both cell lines also displayed an anomalous response mitochondrial content and redox state. Altogether, our data support the idea to oligomycin, with rapid onset of depolarization that was prevented by cyclosporin A and by that mild mitochondrial uncoupling pathways are important regulators of overexpression of Bcl-2. These findings indicate that depolarization by respiratory chain inhibitors cellular redox state and energy metabolism, with activities that can be altered and oligomycin was due to opening of the mitochondrial permeability transition pore (PTP). A Regular Symposia by specific dietary regimens. shift of the threshold voltage for PTP opening close to the resting potential may therefore be the Supported by FAPESP, CNPq, INCT Redoxoma. underlying cause facilitating cell death in diseases affecting complex I activity. This study provides a unifying reading frame for previous observations on mitochondrial dysfunction, bioenergetic [1] Cell 2005; 120:483-95 [2] Aging Cell 2008; 7:552-60 [3] FEBS Lett. defects and Ca2+ deregulation in mitochondrial diseases; and indicates that therapeutic strategies 1997; 416:15-8 [4] Nature 2002; 415:96-9 [5] Free Radic Biol Med. 2007; aimed at normalizing the PTP voltage threshold may be useful in ameliorating the course of 42:1039-48 complex I-dependent mitochondrial diseases.

RS A-49-6 RS A-50-1 MITOCHONDRIA MODULATE INTRACELLULAR SK2 CHANNELS IN CA1 PYRAMIDAL NEURONS: CALCIUM SIGNALS THAT CONTROL ATP TRANSIENTS MODULATION OF NEUROTRANSMISSION AND INDUCED BY ELECTRICAL STIMULATION OF CONTRIBUTIONS TO LTP SKELETAL MYOTUBES John Adelman1, Mike T Lin1, Rafael Lujan2, Masahiko Watanabe3, Veronica Raquel Eisner, Valentina Parra, Sergio Lavandero, James Maylie4 Cecilia Hidalgo, Enrique Jaimovich 1Vollum Institute, Oregon Health & Science University, USA, 2Departamento de 3 Center for Molecular Studies of the Cell, Universidad de Chile, Chile Ciencias, Universidad de Castilla-La Mancha, Albacete, Spain, Department of Anatomy, Hokkaido University School of Medicine, Sapporo, Japan, Depolarizing stimuli induce two calcium signals in skeletal myotubes: a fast signal 4Department of Obstetrics and Gynecology, Oregon Health & Science University, associated with excitation-contraction coupling and a slow signal that regulates gene Portland, Oregon, USA expression. Components of mitochondrial calcium transients induced by electrical 2+ + In many central neurons, small conductance Ca -activated K channels, SK channels, field stimulation (ES, 400 pulses, 45 Hz), as well as their role on cytoplasm and nuclei contribute to the afterhyperpolarization (AHP) that follows an action potential and thereby slow calcium transients and ATP levels were analyzed. regulate somatic excitability. In CA1 pyramidal neurons of the hippocampus, SK2 channels In skeletal myotubes, ES induced two mitochondria calcium transients; the are expressed throughout the dendrites and in dendritic spines. In spines, SK2 channels first was blocked by Ryanodine and the slow component was inhibited by PLC form a Ca2+-mediated feedback loop with NMDA receptors; Ca2+ entering the spine through inhibitor U73122. The slow calcium signal is still present after either uncoupling synaptically activated NMDA receptors activates SK2 channels that are co-localized in of mitochondria with CCCP, blockage of mitochondria calcium uptake using the postsynaptic density. The SK2 channel activity shunts the AMPA receptor mediated Ru360 or mitochondria calcium release with CGP37157. Nevertheless, under these depolarization of the spine membrane potential and reduces the EPSP. In addition SK2 conditions larger and longer slow transients were evidenced. CGP37157 decreased channel activity in CA1 spines in plastic. Upon the induction of LTP, SK2 channels are nuclear calcium accumulation and overexpression of mitochondria fission GTPase phosphorylated at three consecutive serine residues in their intracellular C-terminal domain. Drp-1, increased myotubes slow calcium signal. ES prompted a transient increase in This phosphorylation results in SK2 channel endocytosis. Therefore the increased EPSP intracellular ATP that was blocked by both CCCP and Ryanodine. that results from the induction of LTP at Shaffer collateral to CA1 synapses results from the We conclude that slow calcium transients in skeletal myotubes are modulated by concerted increase in the deploarizing AMPA contribution and decrease in the repolarizing mitochondria. Mitochondria fast calcium component controls ATP synthesis. SK2 contribution to the EPSP. Further results concerning the cell biologic mechanisms and FONDECYT 3070043, FONDAP 15010006. regulation of SK2 channels in CA1 spines will be discussed.

IUPS 2009 July 27 - August 1, 2009 in Kyoto 105 RS A-50-2 RS A-50-3

SODIUM-ACTIVATED POTASSIUM CHANNELS AND K2P CHANNEL AND ASSOCIATED PROTEINS: THE CONTROL OF NEURONAL ACCURACY MOLECULAR AND FUNCTIONAL PROPERTIES Leonard K. Kaczmarek Florian Lesage Department of Pharmacology, Yale University, USA IPMC, CNRS, France The molecular basis of potassium channels activated by intracellular Potassium channels regulate neuronal signalling, heart rate, vascular tone, sodium has been found only relatively recently. There are two genes Slick and hormone secretion. They also regulate cell volume and K+ uptake, and and Slack that encode such channels, and alternative splicing of mRNA for control the flow of salt across epithelia. We have identified a particular + + Slack channels has been found to contribute to the diversity in properties of class of K channels, called two-P-domain K channels (K2P). They produce these channels in different tissues. Physiological roles proposed for these background conductances that are a major determinant of resting potential

channels are to produce an adaptation of firing rate during repetitive neuronal and input resistance, two key components of neuronal excitability. K2P stimulation, and to protect neurons from hypoxic insults. In addition, channels are targets of many physiological stimuli, including pH and activation of these channels in the auditory brainstem improves the ability of temperature variations, hypoxia, bioactive lipids, and neurotransmitter neurons to follow rapid synaptic stimulation with high temporal precision. modulation. They are also activated by clinically-relevant drugs such as

Both Slack and Slick channels have unusually large C-terminal cytoplasmic volatile anesthetics and neuroprotective agents. K2P channels interact with domains. Recent findings indicate that the large C-terminal domain of Slack trafficking, addressing and scaffolding proteins. The identification of such interacts with Fragile X Mental Retardation Protein (FMRP), an mRNA proteins provides useful information on the cellular and molecular context in binding protein that has been proposed to regulate local translation of a which these channels act. I will discuss two different examples of physical

subset of neuronal proteins. Thus, in addition to their function in regulating and functional coupling between K2P channels and protein partners: TREK neuronal excitability, the activation of these channels by sodium during channels / AKAP150 and Mtap2 and TWIK1 channel / EFA6-ARF6GDP. neuronal firing may provide a mechanism for linking neuronal activity to changes in intracellular signaling pathways that control development and learning.

RS A-50-4 RS A-50-5 ACTIVATION MECHANISM OF BK CHANNEL BY DIRECT ACTIONS OF ESTROGEN-RECEPTOR INTRACELLULAR CALCIUM MODULATORS ON THE ACTIVITY OF LARGE- Chul-Seung Park CONDUCTANCE CALCIUM-ACTIVATED POTASSIUM Department of Life Science, GIST, Korea CHANNELS Sheng-Nan Wu Large-conductance calcium-activated potassium (BKCa) channels play an important role in modulating a number of physiological processes, such Department of Physiology, National Cheng Kung University Medical as neuronal excitability, smooth-muscle contraction, frequency tuning of College, Taiwan 2+ + hair cells, and immunity. The opening of these channels is promoted by The large-conductance Ca -activated K (BKCa) channels, which are formed by membrane depolarization and elevated cytosolic free calcium via separate α-subunit tetramers, are encoded by a nearly ubiquitous, alternatively spliced gene,

regions of the alpha subunit. Calcium-dependent gating of the BKCa channel SLO1 (KCa1.1 or KCNMA1). Their activation is allosterically switched on either by

Regular Symposia 2+ is conferred by the large cytosolic carboxyl terminus containing two domains membrane depolarization or by increased intracellular Ca . Recent work suggested of the regulator of K+-conductance (RCK) and the high-affinity Ca2+-binding that in addition to the bindings to estrogen receptors (ERs), these modulators may site. Based on a series of recent studies, we now can envision the activation modify the activity of these channels in a non-genomic pathway. Effects of a series mechanism of this channel by intracellular Ca2+. A conformational change of compounds, known to bind to ERs, on ion currents were investigated. Resveratrol directly stimulates the activity of BK channels in vascular endothelial cells. PPT and in the 'gating ring' composed of hetro-octameric RCK domains seems to Ca 2+ DPN are ER agonists selective for ER-α and ER-β, respectively. In human cardiac underlie the Ca -dependent activation of the BKCa channel. fibroblasts, PPT or DPN applied to the intracellular face of the membrane enhanced

BKCa-channel activity with no change in single-channel conductance. In addition, PPT and DPN caused a stimulation of BKCa channels in vascular myocytes. These two compounds mediated vascular contractile responses in isolated rat aorta. The inhibition by these two compounds of vascular contractility is be mediated through a direct

stimulation of BKCa channel. Their direct activation of these channels may contribute to cellular function.

RS A-50-6 RS LB-51-1 POTASSIUM BINDING AND COUPLED MOVEMENT PURINERGIC CO-TRANSMISSION IN THE PERIPHERAL IN THE G-LOOP AND WATER CAVITY FACILITATE AND CENTRAL NERVOUS SYSTEM BARIUM FLOW IN THE Kir2.1 CHANNEL Peter Paul Illes 1 1 2 Ru-Chi Shieh , Hsueh-Kai Chang , Laurence J. Marton , Department of Pharmacology, University of Leipzig, Germany 3 Kuo Kan Liang It was proposed for the first time in the early seventies of the last century 1Institute of Biomedical Sciences, Academia Sinica, Taiwan, 2Progen (G. Burnstock, J. Theor. Biol., 62: 491-501, 1976) that nerve terminals store 3 Pharmaceuticals, Inc., USA, Research Center for Applied Sciences, and release their main transmitter together with several co-transmitters, the Academia Sinica, Taiwan most important of which appears to be the purine molecule ATP. In effector K ions are selectively coordinated in the selectivity filter and concerted +K and water organs innervated by postganglionic sympathetic/parasympathetic neurons, movements in this region ensure high conduction rates in K+ channels. In channels co-released ATP modulates both the postsynaptic effect of noradrenaline/ + with long pores many K binding sites are located intracellular to the selectivity filter acetylcholine and their exocytotic release via presynaptic receptors. In the (inner vestibule), but their contribution to permeation has not been well studied. We guinea-pig vas deferens, electrical nerve stimulation evoked a biphasic investigated this phenomenon by slowing the ion permeation process via blocking 2+ contractile effect, an early twitch response followed by a more sustained slow inwardly rectifying Kir2.1 channels with Ba in the selectivity filter and observing contraction. These effects were due to ATPergic rapid excitatory junction the effect of K+ ions in the inner vestibule on Ba2+ exit. The dose-response effect of + + 2+ potentials (EJPs) and a simultaneous, slowly developing noradrenergic the intracellular K concentration ([K ]i) on Ba exit was recorded with and without + 2+ depolarization of the smooth muscle membrane. Noradrenaline-ATP co- intracellular polyamines, which compete with K for binding sites. Ba exit to the extracellular side was facilitated by the binding of at least three K ions. Mutations transmission has been shown to take also place in central noradrenergic in water cavity and G-loop altered the effects of K+ and polyamines on Ba2+ exit, neurons of the locus coeruleus (LC) e.g. by a co-release of these molecules suggesting that K+ and polyamines compete for binding in these regions. A sequential from the recurrent axon collaterals of the LC neurons terminating at their + 2+ model could not completely explain the effects of the [K ]i on Ba exit. We propose own somata. In the CNS, ATP is released with acetylcholine, glutamate, that in addition to K+ binding, other mechanism such as K+ coupling interaction in the 5-hydroxytryptamine and dopamine from different subpopulations of inner vestibule is required for high ion conductance in the Kir2.1 channel. neurons.

106 IUPS 2009 July 27 - August 1, 2009 in Kyoto RS LB-51-2 RS LB-51-3 PRESYNAPTIC RECEPTORS MODULATING AUTO- VERSUS HETERORECEPTOR FUNCTIONS OF TRANSMITTER RELEASE: THERAPEUTIC POTENTIAL ALPHA2-ADRENOCEPTORS Salomon Z Langer Lutz Hein, Ralf Gilsbach, Julian Albarran, Johanna Schneider, Research and Development, Euthymia ,Tel Aviv, Israel Nadine Beetz Presynaptic nerve terminals possess receptors that modulate the release Institute of Pharmacology, University of Freiburg, Germany of neurotransmitters (NT's).Autoreceptors are the presynaptic receptors α2-adrenoceptors mediate diverse functions of the sympathetic system. acted upon by the endogenous NT of the neuron.For noradrenaline To determine whether α2-adrenoceptors on adrenergic neurons or α2- (NA) the presynaptic autoreceptor is of the alpha-2 subtype.The term adrenoceptors on non-adrenergic neurons mediate the physiological heteroreceptors identifies presynaptic receptors that modulate NT release and pharmacological responses of α2-agonists, we used the dopamine in response to chemical signals that are different from the neuron's own β-hydroxylase (Dbh) promoter to drive expression of α2A-adrenoceptors NT.Presynaptic receptors are targets for pharmacological intervention by exclusively in (nor)adrenergic cells of transgenic mice. Dbh-α2A transgenic exogenous compounds acting as agonists,partial agonists or antagonists. mice were crossed with double knockout mice lacking both α2A- and α2C- Alpha-2 adrenoceptor antagonists,are useful as add-on therapy in depressed receptors to generate lines with selective expression of α2A-autoreceptors in patients non responders to selective serotonin reuptake inhibitors (SSRI's). adrenergic cells. Surprisingly, only few functions previously ascribed to α2- Compounds which combine SSRI's properties and alpha-2 adrenoceptor adrenoceptors were mediated by receptors on adrenergic neurons, including antagonist actions,like R226161,may have a short latency period.Three feedback inhibition of norepinephrine release from sympathetic nerves and marketed drugs act - at least partly through presynaptic receptors:1) the spontaneous locomotor activity. Other α2-agonist effects including analgesia, antidepressant mirtazapine,antagonist of central alpha-2 adrenoceptors hypothermia, sedation and anesthetic-sparing were mediated by α2-receptors modulating the release of NA and serotonin;2) Aripiprazole,a presynaptic in non-adrenergic cells. Dbh-deficient mice were used to confirm that the dopamine autoreceptor partial agonist for the treatment of schizophrenia.3) sedative effect of α2-agonists is not mediated via inhibition of norepinephrine sumatriptan for the treatment of migraine,is a selective 5-HT 1D agonist release by α2-autoreceptors. The present study paves the way for a revision which inhibits presynaptically the release of substance P and CGRP. of the current view of the α2-adrenergic receptors.

RS LB-51-4 RS LB-52-1 PRESYNAPTIC ADENOSINE AND P2Y RECEPTORS IDENTIFICATION OF A NOVEL MOLECULAR AND THEIR ROLE ON MODULATION OF MECHANISM AND A MAJOR CAUSE OF FATIGUE NEUROTRANSMITTER RELEASE Kazuhiro Kondo 1,2 2 Gloria Queiroz , Jorge Gonclaves Department of Virology,The Jikei University School of Medicine, Japan 1Pharmacology, ICETA, Instituto de Ciencias e Tecnologias Agrarias e 2 Fatigue is an indispensable bio-alarm to avoid the exhaustive state caused by Agro-Alimentares, Portugal, Department of Pharmacology, REQUIMTE, Faculty of Pharmacy, University of Porto, Portugal severe stresses and overwork, which may also induce a variety of diseases. There may be commonalty in the mechanisms of different types of fatigue. Adenosine and ATP act as modulators of neurotransmitter release through activation For years many scientists thought lactic acid caused fatigue. However, now it's of adenosine and P2 receptors. The adenosine receptor family comprises the A1, A2A, A2B and A3 subtypes; the P2Y receptor family includes eight subtypes: understood that in itself it does not cause fatigue, because lactic acid is a key P2Y1,2,4,6,11,12,13,14. Presynaptic receptors that depress neurotransmitter release substance used to provide energy and because the acidity through a build up of include mainly the A1 receptor and P2Y1,2,4,12 subtypes whereas A2A receptors lactic acid helps prevent muscle fatigue. Thus, the molecular mechanisms of facilitate neurotransmitter release. Activation of A1 or the P2Y1,2,4,12 receptor fatigue have come to be unclear. subtypes may inhibit Ca2+ entry through voltage-sensitive N-type calcium channels We have investigated the molecular mechanism of herpesvirus reactivation that whereas facilitation caused by A2A receptors involves activation of protein kinases was known to be stimulated by fatigue, and identified the molecule that can 2+ A and C that may directly or indirectly act on Ca -currents or at the exocytotic induce viral reactivation during fatigue. The molecule was up-regulated more machinery. than ten fold with fatigue that was induced by forced swim or shortened sleep. Since different types of receptors may be present in the same nerve terminal, the effect In this symposium, we are going to talk about the newly found molecular Regular Symposia that prevails may be determined by the functional interaction, in the receptor biophase, between purine release, uptake and metabolism. mechanism and major cause of fatigue. Our talk includes the novel signal The adenosine modulation exceeds the simple direct control of release. It may transduction pathway of fatigue and its relationship with the reported candidates have a permissive effect on responses to other modulators, may regulate its rate of of fatigue-causing substance such as cytokines and oxidative stress. desensitization, or prevent an excessive influence of inhibitory receptors. In this We also talk about the mechanisms how fatigue can cause several diseases respect it is often viewed as a “modulator of modulators” of neurotransmitter release. including heart failure, diabetes, etc..

RS LB-52-2 RS LB-52-3 PROLONGED FATIGUE ALTERS BRAIN FUNCTIONS NEURAL AND MOLECULAR MECHANISMS OF AND AFFECTS HOMEOSTASIS SYSTEMS IN RAT CENTRAL FATIGUE IN THE ANIMAL MODELS Hiroshi Kiyama Yilong Cui1, Yasuyoshi Watanabe2, Yosky Kataoka1 Department of Anatomy & Neurobiology, Osaka City University, Japan 1Cellular Function Imaging Laboratory, RIKEN Center for Molecular 2 Accumulation of fatigue is supposed to impair the homeostasis systems and Imaging Science, Japan, Molecular Probe Dynamics Laboratory, RIKEN causes a prolonged illness and chronic fatigue symptoms. We have recently Center for Molecular Imaging Science, Japan revealed that prolonged stress brought about various alterations in the brain Central fatigue is caused by excessive brain activity such as concentrating and subsequent changes in the endocrine and immune organs using a rat attention or feeling stress, and is thought to be relieved by taking rest or fatigue model. The most intriguing change was seen in pituitary gland. sleep. Two animal models mimicking overtaxed brain function have been The prolonged fatigue stress caused over-activation of the melanotroph developed in the rats: generation of cortical spreading depression, the in pituitary gland and their eventual cell death. The over-activation and excitatory waves of depolarization spreading throughout the cerebral cortex, subsequent cell death were caused by the alteration of dopamine expression and long-term intracranial self-stimulation (ICSS). Following such excessive in the specific region of hypothalamus, suggesting that the molecular activations of the brain, the amount of non-REM sleep increased for several alteration in the brain leads to the dysfunction and even death of pituitary hours via dramatic up-regulation of neuronal COX-2 expression in the cells. The similar change may occur in human under chronic stress, because cerebral cortex and subsequent production of prostaglandins. In the long-term our recent study revealed the increases of alpha-MSH level in the chronic ICSS study, resting behavior for dozens of minutes started to be observed a fatigue syndrome (CFS) patients. The immune system was also perturbed few hours after the start of stimulation. The amount of resting behavior was in the rats. Both spleen and thymus showed atrophic features, and strikingly modulated by the dorsal raphe serotonergic nervous system. These results some inflammatory responses in this animal were suppressed. These results indicate that arachidonic acid cascade and serotonergic system would be suggest that an accumulated fatigue affects brain functions and elicits involved in central fatigue induced by overtaxing brain functions. subsequent dysfunctions in essential machineries for homeostasis.

IUPS 2009 July 27 - August 1, 2009 in Kyoto 107 RS LB-52-4 RS LB-52-5 FATIGUE IN INFLAMMATORY DISORDERS CHANGES IN AUTONOMIC NERVE FUNCTION IN THE Mark Gordon Swain MENTAL FATIGUE STATE CAUSED BY LONG-TERM Department of Medicine, University of Calgary, Canada COMPUTERIZED KRAEPELIN TEST WORKLOAD Hirohiko Kuratsune Changes in behavior (termed sickness behavior), which include fatigue and Faculty of Health Science for Welfare, Kansai University of Welfare malaise, are often encountered in the setting of peripheral inflammatory Sciences, Japan diseases. Despite their prevalence, the etiology of such symptoms is Here we describe a new mental fatigue model of healthy volunteers with the poorly understood. In order to mediate behavioral changes, pathways of fatigue caused by long-term computerized Kraepelin test (CKT) workload. communication must exist between the periphery and the CNS. The periphery CKT is our new software for automatically checking the calculation capability, can communicate with the CNS via humoral and/or neural routes. However, with which it is easy to determine the reaction time (RT), coefficient of variance we have recently observed that an immune-to-brain communication pathway of reaction time (CV), and accuracy of the answers (AC) during tasks. We put also exists in mice with peripheral organ specific inflammatory injury. 24 normal healthy volunteers into the fatigue state by subjecting them to 120 Specifically, using a model of hepatic inflammation in which fatigue-like minutes’ CKT workload, and then studied the changes in fatigue sensation, RT, sickness behaviors have been well characterized, we found a marked increase CV, and AC before and after the CKT workload. The fatigue sensation, RT, in the number of leukocytes rolling/adhering along the cerebral endothelium, and CV were clearly increased by the fatigue task and recovered during the paralleled by the recruitment of activated monocytes (producing TNFalpha) resting period. Furthermore, when we studied the changes in autonomic nerve activity by using heart rate variability analysis, the LF/HF ratio was significantly into the brain of these mice. Importantly, inhibition of the cerebral increased by the fatigue task and decreased by resting, suggesting that mental recruitment of activated monocytes markedly attenuated the development of stress causes a relatively sympathetic nerve activity-dominant state. Therefore, fatigue-like sickness behaviors in this model. These observations suggest that our new long-term CKT workload is a good mental fatigue model to provide the recruitment of circulating immune cells into the brain plays a significant much information about the fatigue state and should be useful for evaluating role in the development of peripheral inflammation associated sickness the anti-fatigue effects of various therapies such as nutrient supplements, herbal behaviors, including fatigue. , and aromatherapies.

RS LB-52-6 RS N-53-1 MECHANISMS AND SCALES OF FATIGUE AND MULTIPLE BRAIN CIRCUITS FOR DECISION-MAKING DEVELOPMENT OF ANTI-FATIGUE PRODUCTS Masamichi Sakagami Yasuyoshi Watanabe Brain Science Institute, Tamagawa University, Japan Center for Molecular Imaging Science, RIKEN, Japan To survive in changeable circumstances, we have to make appropriate Fatigue is really an important bio-alarm, without which we might drop into an decisions on our behavior. For the purpose, the brain should recognize unrecoverable exhaustive state and in the most severe case even die, referred reward information from objects in the circumstances. Recent experimental to in Japanese as “Karoshi.” It is of great value to our modern society for and theoretical studies have suggested that primates, including human scientists to extensively analyze the causes of fatigue and to develop methods beings, have two brain processes to calculate reward values of objects. to quantify fatigue, with the goal of developing methods or therapies to afford One is the process coding a specific reward value of a stimulus or event better recovery from and perhaps even avoidance of severe chronic fatigue.

Regular Symposia dependent on direct experience (e.g. classical conditioning and TD learning). We organized an integrated research project under the MEXT, Japanese The other enables us to predict reward based on the internal model of Government and carried it out from 1999 to 2005, which was succeeded by given circumstances without direct experience (e.g. categorization and the 21st Century COE program “Base to overcome fatigue” also sponsored by MEXT. The following major topics will be introduced in this talk as inference). To clarify neuronal correlates of the multiple processes on reward a summarized achievement of these projects: 1) Elucidation of the brain prediction, we have executed three experiments. 1) Human fMRI imaging regions and their neurotransmitter systems responsible for fatigue sensation during random-dot discrimination with asymmetric reward condition. 2) and chronic fatigue; 2) Development of a variety of methods and scales Single unit recording from the monkey dopamine neuron in the random-dot to quantitatively evaluate the extent of fatigue; 3) Development of animal discrimination task with asymmetric reward schedule. 3) Simultaneous single models by different causes of fatigue; 4) Elucidation of molecular/neural unit recording from the monkey caudate and lateral prefrontal cortex in the mechanisms of fatigue in humans and animals; and 5) Invention of various reward inference task. Results suggest that the nigro-striatal network works methods or therapies to treat chronic fatigue and chronic fatigue syndrome. for the model-free reward prediction and the prefrontal network contributes to the model-based reward prediction.

RS N-53-2 RS N-53-3 MECHANISMS FOR DECISION MAKING IN THE THE NEURAL BASIS OF GENE-ENVIRONMENT PRIMATE PREFRONTAL CORTEX INTERACTIONS Daeyeol Lee, Soyoun Kim, Hyojung Seo Turhan Canli Department of Neurobiology, Yale University School of Medicine, USA Psychology, Stony Brook University, USA To investigate the role of the primate frontal cortex in combining multiple Personality traits have a high degree of heritability, but are also influenced reward parameters, we trained monkeys in an inter-temporal choice task by the environment. In this presentation, I will focus on the personality trait and a computer simulated competitive game. Consistent with the previous of neuroticism to summarize our current thinking on the role of genes and findings in humans and other animals, monkeys discounted the value of environment in personality research. Neuroticism, a risk factor for depression, delayed rewards according to a hyperbolic discount function, whereas their is associated with a common variation in the serotonin transporter gene, behaviors during the competitive game were well described by a simple which renders carriers of the short variant vulnerable for depression when reinforcement learning algorithm. Activity of individual neurons in the lateral exposed to life stress. We investigated the underlying neural mechanisms in prefrontal cortex revealed that this cortical area processes an array of signals individuals with no history of psychopathology, using magnetic resonance- essential for decision making during inter-temporal choice and reinforcement based imaging, genotyping, and self-reported life stress and rumination. learning. During inter-temporal choice, prefrontal neurons often encoded the We found support for a model in which life stress interacts with the effect subjective value of a delayed reward by combining the information about its of serotonin transporter genotype on amygdala and hippocampal resting magnitude and delay. During the competitive game, these neurons changed activation, two regions involved in depression and stress. Life stress also their activity according to the value functions of alternative targets as well differentially affected, as a function of serotonin transporter genotype, as the memory signals related to previous choices and their outcomes. Thus, individuals’ level of rumination. Thus, individual differences in vulnerability the lateral prefrontal cortex might play a key role in evaluating the expected for mood disorders may be mediated by a gene x environment interaction outcomes for alternative actions and monitoring the actual outcomes to in brain regions previously associated with affective processing and brain improve the animal’s decision-making strategies. response to stress.

108 IUPS 2009 July 27 - August 1, 2009 in Kyoto RS N-53-4 RS N-53-5 NEURAL BASIS OF EXPECTED UTILITY AND MEAN- SOCIAL NEUROECONOMICS: DECISIONS AND GAMES VARIANCE MODELS OF RISK Ming Hsu Mathieu d'Acremont, Peter Bossaerts Economics, University of Illinois at Urbana-Champaign, USA Laboratory for Decision-Making Under Uncertainty, Swiss Federal Institute Distributive justice concerns how individuals and societies distribute benefits of Technology, Lausanne, Switzerland and burdens in a just or moral manner, and is central to economics, political When modeling valuation under uncertainty, economists generally favor philosophy, and moral psychology. Using a novel series of distribution tasks Expected Utility (EU) theory. Finance academics and professionals, however, with real outcomes and functional magnetic resonance imaging (fMRI), we prefer to value risky prospects with Mean-Variance (MV) model. Many investigate the central problem of the distributive justice calculus: the tradeoff articles have examined the conditions under which MV and EU models between equity and efficiency. We find that the putamen responds solely to coincide, focusing on the shape of the utility function or subtle measures efficiency, whereas inequity is encoded in the insula, and a unified measure of risk such as semi-variance. Yet, there is a fundamental difference in the of efficiency and inequity (utility) is encoded in the caudate. Strikingly, two approaches: EU requires the explicit learning of probabilities using, individual differences in inequity aversion correlate with differences in for instance, Bayesian updating, with the drawback that, as the number of insula and caudate activation. Against utilitarianism, our results support the states increases, accurate estimation of probabilities becomes impossible. deontological intuition that distributive justice is based on a sense of fairness, This learning limitation does not hold for the MV optimizer, who can simply but, as suggested by moral sentimentalists, is rooted in emotional processing. learn the mean and variance using a reinforcement learning algorithm. More generally, negative emotions related to norm violations may underlie Taking advantage of this fundamental difference, we present behavioral and individual differences in equity considerations and adherence to ethical rules. neuroimaging data suggesting that humans use both approaches, but favor one of the model in certain conditions.

RS N-54-1 RS N-54-2 THALAMIC SYNAPSES; THEIR PROPERTIES AND TONIC AND BURST FIRING OF THALAMOCORTICAL MODULATIONS NEURONS: A CONSTRAINED VIEW OF THALAMIC Mariko Miyata PHYSIOLOGY Department of Physiology, School of Medicine, Tokyo Women's Medical Vincenzo Crunelli University, Japan School of Bioscience, Cardiff University, UK Most of sensory information is relayed by thalamic nuclei before reaching The classical view of thalamocortical (TC) neuron responsiveness describes the cortex. Thalamic relay neurons commonly receive two glutamatergic their activity as being characterized by two firing modes. The first, called synapses: primary sensory synapses and massive feedback synapses from the ‘the tonic firing mode’, includes firing patterns made up of single action cortex (corticothalamic synapses; CT synapses). Despite extensive studies potentials at < 50-100Hz, that are elicited from membrane potentials >-60mV over past decades on thalamic neurons, differences of synaptic features in and are associated with alert and conscious states. The second, or ‘burst these two synapses are still not fully understood. In particular, little is known firing mode’, consists of (single or repetitive) short bursts of high frequency (> 200Hz) action potential firing generated by low threshold, T-type calcium regarding the differences in the composition of glutamate receptors of the two channel-mediated potentials: this occurs when the membrane potential is synapses. In this talk, I will introduce our work on the receptor composition < -65mV and is associated with different stages of non-REM sleep. I will at lemniscal synapses (primary sensory synapse) and CT synapses in discuss recent data which indicate that TC neurons can elicit a far more the ventrobasal thalamic complex (VB). NMDA receptors receptor- complex repertoire of intrinsic electrical behaviours. In particular, I will mediated EPSCs are dominant in CT synapses, whereas AMPA receptor- show how these neurons are capable of generating high threshold burst and mediated EPSCs are dominant in lemniscal synapses. This difference in the complex oscillations at low frequency, which are relevant to physiological Regular Symposia composition of glutamate receptors between corticothalamic and lemniscal and pathological conditions. Thus, although the simple ‘tonic versus burst synapses plays an important role for the formation of distinct temporal firing firing’ approach to TC neuron behaviour has been extremely valuable for patterns in VB relay neurons. I will also introduce our recent work on the many years, it should now be substituted with a more comprehensive view presynaptic modulation mediated by kainate receptors and nicotinic ACh that encompasses the variety of pacemaker activities that these neurons are receptors at synaptic transmissions in VB. capable of expressing.

RS N-54-3 RS N-54-4 SYNAPTIC INTEGRATION: A TIPPING POINT IN CORTICOTHALAMIC GATING OF AUDITORY EPILEPSY INFORMATION John Huguenard Ying Shing Chan1, Ju Fang He2 Department of Neurology and Neurological Sciences, Stanford University, 1Department of Physiology, The University of Hong Kong, China, USA 2Department of Rehabilitation Sciences, The Hong Kong Polytechnic The thalamocortical circuit is comprised of a large scale network containing University, Hung Hom, Hong Kong, China multiple subcortical nuclei reciprocally connected with various regions of Corticofugal projection of the auditory system is believed to play a crucial role neocortex. This network creates coordinated widespread oscillatory activity in tuning our auditory attention while filtering unwanted noise. Our in vivo during sleep and in some forms of epilepsy. Thalamocortical oscillations data from medial geniculate body (MGB) neurons of anesthetized guinea pigs depend in large part on the activity of the specialized thalamic reticular showed that both their temporal firing pattern and onset responses to sound were nucleus (nRt), which through its inhibitory output to thalamocortical relay modified by electrical stimulation of the auditory cortex (AC). The majority neurons, pace the activity in the larger network. Neurons within the nRt of neurons in the lemniscal MGB showed acoustic-evoked depolarization and receive 5 major types of synaptic input, excitatory inputs from neocortex facilitatory response to electrical stimulation of AC. However, the majority of and dorsal thalamus, two forms of synaptic connection from other nRt cells non-lemniscal MGB neurons showed acoustic-evoked hyperpolarization and - chemical inhibitory and electrotonic excitatory, and neuromodulatory received inhibitory corticofugal input via the thalamic reticular nucleus (TRN). inputs. nRt neurons normally integrate these varied synaptic inputs in order These suggest that corticofugal projections amplified the matched ascending to regulate sensory throughput and generate sleep-related cortical activities. auditory information via the lemniscal MGB but switched off the non-lemniscal Under pathological conditions, excessive activation of excitatory inputs MGB so as to prepare the AC for sole processing of auditory information via the from cortex and/or thalamus converts the activity of nRt neurons such that lemniscal route. Using c-fos expression, we further demonstrated that activation abnormal synchrony develops suddenly and catastrophically to produce of rat MGB neurons was triggered by synchronized corticothalamic oscillation absence seizures. Recent findings in mice with mutations in excitatory that did not involve TRN. Our results thus suggest that the transmission of synaptic receptors provide insight into the mechanisms through which ascending auditory information, especially for attentive purpose, is critically abnormal synchrony develops. governed by corticothalamic modulation.

IUPS 2009 July 27 - August 1, 2009 in Kyoto 109 RS G-55-1 RS G-55-2 NETWORK OF WOMAN PHYSIOLOGISTS IN JAPAN GROWING UP TOGETHER WITH THE CHILDREN; -IT’S AN EXPERIENCE!- WOMEN SCIENTISTS IN CHINA Kazue Mizumura Li-Ying Hao Department of Neuroscience II, Research Institute of Environmental Dept. of Pharmaceutical Toxicology, School of Pharmaceutical Sciences, Medicine, Nagoya University, Japan China Medical University, China The number of woman physiologists in Japan is still small (c.a.13 % of all members of Physiological Society of Japan (PSJ)), and often only one woman Women have constituted more than one third of total scientists in China. exists in her working place or research group. In such instances, she finds In some research field such as information technology and life science, the no woman researchers to talk with and no role model in her surroundings. constitution of women scientists is even near to fifty percent. However, the In addition, lower positions of woman researchers and heavy duty of female proportion declined significantly in the top-level scientists. Women housekeeping and upbringing children make it difficult to get information take more responsibilities in taking care of family and educating children. on new research techniques, grants, job offers, etc. To break through this It is not easy for women scientists to get well balanced and fulfill both roles situation, I found making a network of woman researchers is important. Here in work and family. Therefore, we usually have to do much more efforts to I introduce my experience with the network of woman physiologists. The achieve success. The most difficult time is the period after a baby is born. network ‘Women in Physiology of Japan (WPJ)’ was founded in 1995 with Being positive will help to persist and pass the hard time. Being honest to only about 20 members. Now more than 80 woman physiologists (about 20 admit that we can not handle all of those things by ourselves will make it % of woman members of Physiological Society of Japan) are members. The easy to ask for helps. Grandparents are usually the big helping hands in most important activities of WPJ are publishing newsletters and having the annual meeting. Through these activities many WPJ members (including China. Growing children apparently costs time and energy, but we ourselves I) have become more active and felt that attending the annual meeting of grow up meanwhile. Children inspire us and enrich our life. We became more PSJ has become more enjoyable than before. Based on WPJ’s proposal the optimistic, braver, stronger, and arrange things more effectively. Women official committee for Gender Equality was organized in the PSJ. WPJ and scientists are serious, attentive, meticulous, persistent, easy to cooperate and the committee collaborate to attain gender equality in PSJ. creative, and they are invaluable intelligence resources in scientific research.

RS G-55-3 RS G-55-4 INDIAN WOMEN'S PROSPECTIVE ON GENDER WE DO IT OUR WAY: A PERSONAL AND UK EQUALITY: FIGHT OR FLIGHT! PERSPECTIVE ON GENDER EQUALITY AND Kamalesh K Gulia PHYSIOLOGY Systems Neuroscience, National Brain Research Centre, India Susan Wray The Indian woman in the 21st century is evolving into a strong, innovative, Physiology, School of Biomedical Sciencs, University of Liverpool, UK revolutionary, multifaceted icon, to take up demanding research, to contribute My title is chosen to reflect two points about women in physiology and equally to science and make a mark for herself. In modern times, she has free gender equality; firstly what works in one environment/country may not access to higher learning in all the fields without any gender discrimination, be the most effective solution in others and secondly, what works for one but subtle biases surface at the time of placement. The glass ceiling which woman may not for another. By having symposia such as this we can generally seems to appear at a higher level for women in general, becomes share strategies and experiences so that we build on success and increase Regular Symposia more evident for these women who face break in career due to social dissemination of helpful mechanisms and processes. This is useful as there is constraints. These women have to undergo relatively harder experiences much that still needs to be done worldwide. to match global competition. The trials and tribulations are compounded In the UK my view is that the biggest issue remains combining life as a by having to prove her credibility and capability, face age bar restrictions, physiologist and a mother, rather than discrimination. Many women want to recover lost time, enhance scientific productivity, and struggle for higher achieve at the highest possible level, but not if it is always at the cost of their position, for more independence in work and policies, even while she family. This means we need to hear more from the voices of mothers who remains a strong pillar of society and at the home front. are also successful physiologists (or scientists). It also means that we have to Since 2003, Department of Science and Technology, the Government of India consider what we mean by success. I have more questions than answers, but has initiated one scheme that is emerging as catalytic force in bringing these that’s also why I am a physiologist! highly qualified potential women scientists to bring back into mainstream of active research. Recently, a National Task Force on Women in Science is also constituted to encounter these issues.

RS G-55-5 RS G-55-6 SUSTAINABLE SHOULD BE FEMALE RESEARCHERS’ BARRIERS TO GENDER EQUITY IN US BIOMEDICAL CAREER: INTEGRATED MASTER PLAN FOR THE SCIENCE: THE SLOW DRIP OF THE LEAKY PIPELINE SUPPORT OF FEMALE RESEARCHERS AT HOKKAIDO Kim Elaine Barrett UNIVERSITY Department of Medicine and Office of Graduate Studies, University of Sanae Maria Margherita Iguchi-Ariga California, San Diego, USA Graduate School of Agriculture/Life Science, Hokkaido University, Japan Women are substantially under-represented among biomedical faculty in the A big tide of promotion for female researchers has risen in 2006, called the US, particularly at senior levels. However, women have reached - or in some First Year or “Dawn” of Promotion for Female Researchers in Japan: Japan’ cases, exceeded - parity in both graduate and clinical biomedical training s 3rd Science and Technology Basic Plan has set the target to increase the programs. Thus, the lack of female academic leaders can no longer be ratio of female researchers to 25% by fiscal year 2010. Several funds and attributed to a lack of women entering the field, but rather, disproportionate grants have started relevant to female researchers promotion, including the attrition - the so-called “leaky pipeline”. Factors suggested to contribute fund for model programs to support female scientists. Hokkaido University, include family responsibilities, the challenges of managing dual-career one of the first 10 recipient institutions of the fund, has been driving forward life-styles, systematic inequities in pay and conditions of employment on various supporting activities. The Support Office for Female Researchers in the basis of gender, conscious and unconscious biases that reduce female Hokkaido University (FResHU) has been established within the framework hiring, a hostile climate, and likely others. However, many US educational of Hokkaido University’s policy of gender equality promotion and been institutions as well as businesses are realizing that they cannot be competitive functioning a policy making role as well as a one-stop contact point for without tapping fully into the scientific talent pool. My presentation will female researchers in need. Hokkaido University has committed itself to a discuss evidence for inequity and bias in the involvement of women in US “Triple Twenties Policy”: by 2020, 20% of all staff in teaching and research science, based on national data as well as studies that have been conducted positions should be women. To achieve this goal, the University has devised at my own institution, and what might be done to correct the situation. While a “positive action scheme”, which will be discussed as well as other issues gains have been made, the leaky pipeline has persisted for more than 30 years for female researchers’ career. and the scientific enterprise will be the poorer if solutions are not identified.

110 IUPS 2009 July 27 - August 1, 2009 in Kyoto RS G-55-7 RS LB-56-1 PROMOTION OF GENDER EQUALITY AND WORK/LIFE A ROLE FOR FOXO1 IN THE EFFECT OF CALORIE BALANCE AT SHISEIDO RESTRICTION 1 1 1 Chika Katagiri Isao Shimokawa , Haruyoshi Yamaza , Takuya Chiba 2 Shiseido Research Center, SHISEIDO Co., LTD, Japan Tatsuo Furuyama 1 Of all the scientists in Japan, the female scientists consist of only 12.4% in Department of Investigative Pathology, Nagasaki University Graduate School of Biomedical Sciences, Japan, 2Department of Foods and 2008. This ratio is significantly less than other major developed countries. Nutrition, Sonoda Women's University, Japan At Shiseido, the percentage of female scientists is 40%, which is higher than the national average. As the prime target customers for a cosmetic company Extension of lifespan by the reduced insulin-like signaling in nematodes required a transcription factor, Daf-16. Calorie restriction (CR) reduces plasma concentrations of are female, all employees are expected to have understanding of female's insulin and IGF-1 in mammals. Thus, FoxO transcription factors (FoxO1, 3a, and 4), behaviors. At Shiseido, we do have various programs to promote gender mammalian orthologues of Daf-16, may play a role in the effect of CR. The present equality such as framework for promoting gender equality, gender equality study confirmed up-regulation of FoxO1-mRNA in the mouse liver and skeletal promotion system, and various action plans. According to the Shiseido case muscle by CR, while an increment of FoxO4 in the skeletal muscle and fat tissue; study, the workplace equality has significantly improved according to these there was no up-regulation of FoxO3a-mRNA in the tissues examined. Using FoxO1- action plans to assist employees for child-rearing and nursing care. However, knockout (+/-) mice, effects of CR on stress resistance, glucose-insulin homeostasis, percentage of females in leadership position remains to be still low. We also and longevity were evaluated. In CR mice, up-regulation of cell cycle arrest or DNA recently introduced efforts in work/life balance, which is a way of working repair genes in response to experimentally-induced oxidative stress was augmented, whereas the CR effect was attenuated in FoxO1-KO mice, suggesting a role for FoxO1 efficiently that enables employees to enhance and balance other aspects of life in the oxidative stress response. There was no significant change in the glucose-insulin outside of work. To accomplish the action plans to support the development homeostasis in FoxO1-KO mice. Lifespan in wild-type mice was increased by CR, of the next generation, we try to achieve work/life balance included in gender while the effect was maintained in FoxO1-KO mice. The present study suggests a equality action plan. Shiseido also offers its female employees, ''Shiseido role for Foxo1 in the effect of CR on stress response, although it may be minor for Grants for Women Scientists''. Let's participate in this female network. longevity.

RS LB-56-2 RS LB-56-3 CALORIE RESTRICTION MIMETICS: DEVELOPING MOLECULAR INFLAMMATION AND LIPID INTERVENTIONS FOR HEALTHY AGING AND ACCUMULATION AS UNDERLYING CAUSES OF LONGEVITY AGING AND THEIR INTERVENTION BY CALORIE Joanne Stacey Allard1, Rafael de Cabo1, Julie Mattison1, RESTRICTION Kevin Pearson2, Robin Minor1, Donald Ingram3 Hae Young Chung, Jaewon Lee Pharmacy, Pusan National University, Korea 1Laboratory of Experimental Gerontology, National Institute on Aging, USA, 2University of Kentucky, Lexington, Kentucky, USA, 3Pennington The purpose of this study was to stimulate research endeavor toward a better Biomedical Research Center, Baton Rouge, Louisiana, USA understanding of the molecular bases of age-related degenerative process and oxidatively stressed condition, i.e. aging. The basic tenet of aging is under Long term calorie restriction (CR) is the most effective and reproducible intervention for incessant redox imbalance that various inflammatory proteins are induced to mitigating diseases of aging and extending lifespan in several animal models. Currently make cells and tissues to be pro-inflammatory conditions. The key players there is an effort to develop CR mimetics (CRM). The objective of this strategy is to involved in the proposed mechanism are the age-related up-regulation of NF-κB, identify compounds or interventions that mimic CR effects by targeting metabolic and stress response pathways evoked by CR, but without the need of restricting caloric intake. There IL-1β, IL-6, TNF-α, cyclooxygenase-2 and inducible NO synthase. The strongest are a growing number of mediators of calorie restriction being identified, including the evidence supporting the proposal came from the ant-oxidative actions of calorie nicotinamide adenine dinucleotide (NAD)-dependent deacetylase, SIRT1. SIRT1 has been restriction (CR) paradigm, antioxidative, anti-inflammatory agents, and exercise found to mediate a wide range of physiological effects including improved cardiovascular by which all the proinflammatory activities were attenuated. In addition, the health, decreased cancer incidence and neuroprotection. Researchers have begun targeting biological systems and regulatory network in aging and CR were investigated molecules that modulate the enzymatic activity of SIRT1 as potential CRMs. Resveratrol, a via systems biological approach based on the large-scale data compendium. Regular Symposia phytoalexin found in red wine, is one such molecules that have been shown to increase the Functional modules analysis also implied that lipid metabolism and immune lifespan of several species including mice fed on a high-fat diet. In addition, synthetic SIRT1 response are closely related in aging and CR with the opposite manner. These activating agents have been developed and are currently being tested in rodent models. These data suggest that the molecular inflammation and lipid accumulation are initial steps in the discovery of CRMs illustrate their strong possibilities as health enhancing, intricately involved in the oxidatively stressed aging process and anti-aging life-extending interventions. intervention from biological and bioinformatics data.

RS LB-56-4 RS LB-56-5 WOULD MOSES (MICE OVEREXPRESSING CALORIC RESTRICTION AND ADIPONECTIN IN EXOGENOUS SIRT6) REACH 120? AND IF SO, HOW? CARDIOPROTECTION Haim Cohen, Yariv Kanfi, Victoria Peshti Ken Shinmura, Kayoko Tamaki Faculty of Life Sciences, Bar-Ilan University, Israel Internal Medicine, Keio University School of Medicine, Japan The Sirtuins family of proteins consist of NAD+ protein deacetylases/mono- Hearts from senescent animals are more susceptible to ischemia than those ADP_ribosyltransferases that can regulates diverse biological processes, from young animals. Clinical studies have demonstrated that morbidity and including gene silencing, metabolism and aging. Mice deficient for SIRT6 , mortality after myocardial infarction are higher in the elderly. These facts one of the seven members mammalian Sirtuins (SIRT1-7), die prematurely might reflect a generalized deterioration in the innate adaptive response of and show a range of aging associated abnormalities including defects in tissues against various stresses with ageing. We had speculated that caloric restriction (CR), of which anti-ageing effects are scientifically proven, could glucose homeostasis. A significant body of data demonstrates that fat and improve myocardial ischemic tolerance. We have demonstrated that short- glucose homeostasis are key factors in modulating age related diseases and term (4 weeks) CR confers cardioprotection in both young and aged rats. life span. In order to evaluate SIRT6 involvement in metabolic homeostasis Using adiponectin antisense transgenic mice, we have shown that the increase and aging processes, we generated lines of transgenic mice over expressing in circulating adiponectin levels, and subsequent activation of AMPK, are exogenous SIRT6 (MOSES) showing an improved an improved fat and necessary for this cardioprotection. In contrast, the mechanism(s) by which glucose homeostasis. These observations together with our findings that prolonged (6 months) CR confers cardioprotection seems more complicated. SIRT6 levels are increased under calorie restricted diet, strongly demonstrate Recently we have found that the interaction among adiponectin, nitric oxide that SIRT6 is a key factor in regulating glucose and fat homeostasis and synthase, and sirt1 plays a key role in the cardioprotective effect of prolonged potentially might also regulate longevity. CR. In addition, CR might retard cardiovascular senescence and prevent the progression of atherosclerosis. Although it has not been proven yet, we are expecting that CR can reduce morbidity and mortality of cardiovascular events in humans.

IUPS 2009 July 27 - August 1, 2009 in Kyoto 111 RS LB-56-6 RS LB-57-1 CALORIC RESTRICTION DELAYS AGING CHANGES IN FUNCTIONAL ALTERATIONS OF CORTICES AFTER CELLS OF MANY TISSUES NEUROSURGICAL TREATMENT FOR SENSORIMOTOR Norman S. Wolf, William R. Pendergrass LESIONS 1 2 3 3 Pathology, University of Washington, USA Amami Kato , Yoshinobu Hara , Masayuki Hirata , Satoru Oshino , Haruhiko Kishima3, Naokii Nakano1, Takufumi Yanagisawa3, Caloric restriction (CR) is the oldest and, so far, the most robust method Takeshi Okinaga3, Masaaki Taniguchi3, Toshiki Yoshimine3, known to extend the lifespan of wild type (non-mutant) mice, as well as that Sadao Shiosaka2, Jun Ohta2 of invertebrates and fish. Recent data suggests that it will do so in dogs and 1 non-human primates, as well. Our experiments indicate that long term CR Neurosurgery, Kinki Unniversity, School of Medicine, CREST JST, Japan, 2Nara Institute of Science and Technology, CREST JST, Japan, 3Department of conserves the cellular replicative capacity and cellular replicative potential Neurosurgery, Osaka University, Japan as measured in vivo and in vitro, respectively. Thus, a given tissue’s cell Electrical rhythm change of the cortex evoked by task performance (ERRC), which is referred as replication capacity becomes reduced as an animal (mouse) ages, but is event related synchro-/desynchronization, closely correlates with the activation of brain function. significantly preserved in long term CR mice of the same age. The data for Less invasive functional mapping or high-performance brain-machine interface may be developed several tissue types in mice will be presented here. The system by which CR by applying ERRC. accomplishes this appears to be complex. However, present evidence is that Eighteen patients underwent epilepsy surgery and nine glioma patients underwent tumor removal involving or around the sensorimotor areas were enrolled in this study. ERRC either by motor the pathway includes FOXO3 and SirT1 and may involve reduced oxidative task or by sensory stimulation were examined with magnetoencephalography (MEG) in both damage at the cellular level. preoperative and post operative period. The patterns of sensorimotor activations were compared with the pre- and post operative sensorimotor deficits and the surgical outcome. The most prominent finding was the beta band ERD evoked by motor task was usually wider in the contralateral sensorimotor cortex when the lesions or surgical wound around the central sulcus existed. And this impairment could be normalized in the epilepsy patients with the seizure cessation, but, not in the tumor patients even after symptoms ameliorated. Functional migration disclosed by ERRC would be important for designing BMI for diseased brain.

RS LB-57-2 RS LB-57-3 WHAT’S REQUIRED FOR BRAIN-MACHINE BIDIRECTIONAL BRAIN-MACHINE INTERFACES FOR INTERFACES FOR NEURON-REHABILITATION? RESTORING MOTOR FUNCTION 1 2 Tadashi Isa Andrew Jackson , Kazuhiko Seki 1 2 Department of Developmental Physiology, National Institute for Institute of Neuroscience, Newcastle University, UK, Department of Developmental Physiology, National Institute for Physiological Sciences, Physiological Sciences, Japan Japan To develop brain-machine interfaces for restoring the motor function of New technological developments in neuroprosthetics including chronic the disabled due to the spinal cord injury or brain stroke, it is necessary to electrodes and low-power, implantable electronics are allowing long-term know where the appropriate signal for the control of limb movements are monitoring and manipulation of activity in the nervous system. At the same represented. We have studied the neuronal activity for the control of reach time, an increasing scientific understanding of activity-dependent plasticity

Regular Symposia and grasp movements in monkeys after the lesion of the spinal cord lesion and its importance for rehabilitation following injury is informing new and primary motor cortex (M1) by using positron emission tomography closed-loop neurostimulation paradigms aimed at restoring nervous system (PET). It was clarified that during the early stage of recovery (1 mo post- function. I will review progress in this area with a focus on spinal cord injury. lesion) after lesion of the corticospinal tract at C5 cervical segment, bilateral Microstimulation through electrodes implanted in the spinal cord of non- M1 were activated. Then, during the late recovery stage (3-4 mo post-lesion), human primates evokes upper-limb movements involving multiple muscles, contra-lesional M1 and bilateral ventral premotor cortex were activated. In possibly via recruitment of local spinal circuitry. Control of such stimulation by Brain-Machine Interfaces provides a communication channel allowing contrast, after the unilateral M1 lesion, bilateral PMv and the primary and volitional access to spinal motor circuits after injury. Furthermore, evidence secondary sensory cortex were activated during both early and late stages. from human and animal studies suggests that precise temporal correlations Thus the pattern of brain activation varies depending on the lesion site introduced by such stimulation can induce lasting reorganisation in surviving and the recovery stages, and such information might be important for the motor pathways. In future this could lead to new BMI-based approaches to appropriate control of brain machine interfaces. rehabilitation following motor injury.

RS LB-57-4 RS LB-57-5 RESTORING MOTOR AND SENSORY FUNCTION WITH ANALOG VLSI VISION DEVICE FOR CORTICAL NEUROPROSTHESES IMPLANTS 1 1 1 Gregory A. Clark , David J Warren , Noah M Ledbetter , 1 2 2 2 1 3 Tetsuya Yagi , Kazuhiro Shimonomura , Yuka OKazaki , Hirotsugu Andrew M Wilder , Brett R Dowden , Florian Solzbacher , 1 1 3 3 4 1 Okuno , Makoto Osanai , Sawai Hajime Reid R Harrison , Lee E Miller , Richard A Normann 1 2 1 2 Graduate School of Engineering, Osaka University, Japan, The center for Department of Bioengineering, University of Utah, USA, School of Computing, 3 Advanced Medical Engineering and Informatics, Osaka University, Japan, Univ. of Utah, USA, Dept. of Electrical and Computer Engineering, Univ. of 3 Utah, USA, 4Feinberd School of Medicine, Northwestern Univ., USA Graduate School of Medicine, Osaka University, Japan Our long-term research goals are to develop and use neural interfaces to 1) restore Electrical stimulation of visual cortex can evoke the sensation of light (phosphenes) sensorimotor function after spinal cord injury, and 2) provide natural, intuitive in blind patients, demonstrating the feasibility of a cortical prosthesis. The visual motor control of, and sensory feedback from, an advanced, dexterous prosthetic system of the brain is composed of hierarchically arranged parallel networks that limb. Key to this effort are the Utah Electrode Array (UEA) and the Utah Slanted carry different levels of image features, from simple to abstract. Thus, effective real- Electrode Array (USEA). A single USEA implanted in cat sciatic nerve can time preprocessing of the image with compact hardware is crucial for mapping visual access multiple hindlimb muscles at low stimulus currents, and can produce scenes onto the cortical networks where the stimulation electrodes are implanted. A fatigue-resistant, finely-graded control of muscle forces. USEAs can also record neuromorphic vision device, namely, a very large scale integrated (VLSI) circuit that both cutaneous and proprioceptive sensory signals, and these nerve discharges emulates the structure and function of cortical networks, is the most feasible candidate can be decoded to predict limb position. Similarly, USEAs in arm nerves of non- for this purpose. We have designed a mixed analog/digital neuromorphic vision device human primates can evoke discrete hand movements and can record nerve fiber for such a prosthesis, implantable in different areas of visual cortex. This device can discharges. USEAs may also serve as effective neural interfaces to a dexterous emulate neuronal responses of different stages in the visual pathway, e.g., orientation- prosthetic arm and hand, recording spikes from efferent nerve fibers for intuitive selective or direction-selective responses, in real time. Using the device, we conducted motor control, and stimulating sensory fibers to provide sensory feedback. a simulation study of possible visual perceptions evoked by the cortical prosthesis, A major emerging development is wireless Utah arrays for recording and based on our measurements of spatiotemporal properties of electrically induced stimulation. Taken together, these and related approaches hold great promise for responses in mouse visual cortex and previous clinical observations obtained in other greatly improved restoration of sensory and motor function. laboratories.

112 IUPS 2009 July 27 - August 1, 2009 in Kyoto RS LB-57-6 VISUAL IMAGE RECONSTRUCTION FROM HUMAN BRAIN ACTIVITY: A MODULAR DECODING APPROACH Yukiyasu Kamitani Department of Neuroinformatics, ATR Computational Neuroscience Laboratories, Japan Perceptual experience consists of an enormous number of possible states. Previous fMRI studies have predicted a perceptual state by classifying brain activity into pre-specified categories. Constraint-free visual image reconstruction is more challenging, as it is impractical to specify brain activity for all possible images. Here, we present a modular decoding approach in which a visual image was assumed to consist of multiscale local image bases (modules). The contrasts of the local image bases were independently decoded from fMRI activity, and then combined to create a reconstructed image. We show that arbitrary binary-contrast images were accurately reconstructed by the decoding model trained with fMRI activity patterns only for several hundred random images. Reconstruction was also used to identify the presented image among millions of candidates. The results suggest that our approach provides an effective means to read out complex perceptual states from brain activity. Regular Symposia

IUPS 2009 July 27 - August 1, 2009 in Kyoto 113 WS2-1 WS2-2 BIOLUMINESCENCE IMAGING OF CIRCADIAN CLOCK OVEREXPRESSING CLOCK GENES IN CIRCADIAN GENE EXPRESSION IN SINGLE CELLS RHYTHMS HAVE VARIOUS EFFECTS ON CELL CYCLE David K Welsh1, Steve A Kay2 Rika Numano, Atsushi Miyawaki 1Psychiatry, University of California, San Diego, USA, 2Cell & ERATO, CFDS, Japan Science and Technology Agency, RIKEN, Japan Developmental Biology, University of California, San Diego, USA Recent publications have reported that Peiodr2 (Per2) mutant mice increased Since pioneering studies over 50 years ago in the bioluminescent marine tumor occurrence and reduced apoptotic response in thymocytes after gamma dinoflagellate Gonyaulax, bioluminescence has been used to monitor radiation and that circadian rhythm was concerned with cell cycle gating. circadian clock function. In recent years, exogenous luciferase genes have Relation between circadian rhythm and cell cycle is regarded as important to been introduced into a wide variety of organisms, including cyanobacteria, understand various biological phenomena, regeneration and differentiation. plants, fruit flies, zebra fish, and mice. Luciferase enzymes catalyze emission Then we established mammalian stable cell line with fluorescence and bioluminescence (BL) emission during S/G2/M phase by new optical of photons from a substrate. When expressed under control of a circadian indicator (Bucci). Cell cycle timing could be monitored in microscope by promoter, they can report circadian clock gene expression in vivo, in cultured oscillatory emission of each cell. We elucidated effects of overexpressed tissues, or in cultured cells or cell lines. Luminescent reporters are more clock gene on cell cycle using Bucci cell system. Three clock genes, Timeless sensitive and less toxic than fluorescent reporters, making them ideal for (Tim), Per2: one of central oscillator in circadian rhythms and Clock: the long-term, longitudinal studies of circadian clock function. They are also transcriptional factor, were cheesed and overexpressed in Bucci cell by CMV exceedingly dim, and so most studies have used photomultiplier tubes to promoter by means of stampolation technique and lipofection method. Cell collect photons from many cells at once. Because circadian clocks function cycle was observed by time lapse imaging of BL and bright field for 3-4 at the level of single cells, however, we have optimized bioluminescence days. imaging of circadian clock gene expression in mammalian cells, and used this Cells overexpressed Tim or Per2 had low BL and blocked mitosis before S technique to examine single cell phenotypes of various genetic perturbations phase, while those overexpressed Clock kept on cell cycle as well as normal of clock function. [Supported in part by NIH grant R01 MH082945 and a V.A. cells. Therefore, up-regulation of clock gene, Tim, Per2 or Clock, has various Career Development Award.] effects on cell cycle.

WS2-3 WS2-4 DEVELOPMENT OF MULTICOLOR LUCIFERASE CLOSE INSPECTION OF REAL-TIME ASSAY SYSTEM USING DIFFERENT COLOR EMITTING BIOLUMINESCENCE RHYTHMS REVEALED A NEW LUCIFERASES FOR MONITORING MULTIPLE GENE RESETTING MECHANISM OF THE FIBROBLAST EXPRESSIONS CLOCK Yoshihiro Nakajima Yoshitaka Fukada Research Institute for Cell Engineering, National Institute of Advanced Department of Biophysics & Biochemistry, Graduate School of Science, Industrial Science and Technology, Japan The University of Tokyo, Japan Luciferases have been widely used as reporters of gene expression in the real- Real-time bioluminescence monitoring system using luciferase reporters enables us time monitoring in living cells, and real-time imaging at single cell level and to study biochemical properties of circadian clocks with high temporal resolution. in vivo. To enable the long-term, quantitative and simultaneous monitoring of By using Rat-1 fibroblasts stably expressing Bmal1-luciferase reporter, we found that multiple gene expressions, we previously developed dual-color luciferase assay extracellular pH level (pHo) markedly affects both the period length and phase of the system using green- and red-emitting beetle luciferases with single luminescent cellular rhythm: Upon acidification of the culture medium, the period of the rhythm substrate. Based on this technique, we generated double transgenic mice that became shorter, and the oscillation damped more rapidly when culture was started at

report simultaneously the expressions of two clock genes, Bmal1 and Per2, from lower pHo. We found that acute alkalization of pHo resets the cellular clock through a single tissue, in which expressions of two genes are monitored using green- and activation of TGF-β signaling pathway. Importantly, the resetting accompanied no red-emitting beetle luciferases with a single luminescent substrate. Antiphasic significant Per induction but evoked instead immediate-early induction of Dec1, a oscillations of Bmal1 and Per2 were clearly monitored in suprachiasmatic gene whose physiological role in clockwork has been enigmatic. The Dec1 induction nucleus (SCN), the master circadian pacemaker, and peripheral tissues, indicating was prerequisite for the resetting, and its induction was dependent on SMAD3 which that the system enable long-term, quantitative, and simultaneous monitoring of was phosphorylated for activation in response to TGF-β treatment. Intraperitoneal Workshops two gene expressions. We also show that circadian phases and periods of Bmal1 injection of TGF-β into mice demonstrated its physiological role for phase-shift of and Per2 in each organ are unique, but expressions of both genes are strictly clock gene expression in peripheral tissues such as the kidney and adrenal gland. anthiphasic. These results suggest that the system can provide a simple solution These results highlight TGFβ-SMAD3-Dec1 signaling as a novel input pathway in the to unravel complex interactions of two genes in the body. mammalian clock system.

WS2-5 WS2-6 CIRCADIAN BIOLOGY OF SCN CONTINUOUS AND DISCONTINUOUS MEASUREMENT Toru Takumi OF BRAIN OSCILLATORS Hiroshima University, Japan Hugh D Piggins, Alun T Hughes, Clare Guilding The circadian rhythm of about 24 hours is a fundamental physiological Faculty of Life Sciences, University of Manchester, UK function observed in almost all organisms from prokaryotes to humans. Recent progress in understanding the molecular and cellular basis for Although we know much about of molecular clock mechanism, we still circadian timekeeping has revealed intercellular communication to be don’t fully understand how the molecular clock is coupled to the output important for coordination of suprachiasmatic (SCN) neuronal oscillators and pathways which controls circadian phenotypes. The neural output of the that cells in extraSCN brain sites may also oscillate. Typically, discontinuous suprachiasmatic nucleus (SCN) in the mammalian hypothalamus is essential sampling methods are used to assess oscillations in the SCN and beyond, in the circadian regulation of behavioral activity. We have examined the but these may not be appropriate. For example, determination of SCN function in Vipr2 -/- mice by discontinuous measurement of core clock gene downstream pathway of the central clock by means of real-time monitoring expression or neurophysiogical activity indicates abnormally low molecular of multi-unit neural activity in feely moving mice. The results demonstrate and cellular activities and the absence of intrinsic oscillations. However, that SCN neural activity is tightly coupled to environmental photic input that continuous measurement of neurophysiological activity or clock gene is relayed to the subparaventricular zone (SPZ), the neural activity rhythm in expression via reporter constructs demonstrates that many Vipr2-/- SCN cells the SPZ being reversed in phase compared in the SCN. In Clock mutant mice sustain low amplitude circadian oscillations. Similarly in wildtype rodents, exhibiting attenuated circadian locomotor rhythmicity, neural rhythmicity in discontinous measurements suggest that extra SCN sites lack circadian the SCN and SPZ is affected in a similar manner. These results suggest that rhythms, while continuous recordings indicate that areas such as the the SPZ plays a functional role in relaying circadian and photic signals from mediobasal hypothalamus contain a range of cellular oscillators. Collectively, the SCN to centers involved in generating behavioral activity. The real-time these studies indicate that discontinuous sampling methods are not sensitive monitoring of circadian genes in the SCN will be presented. enough to detect to low amplitude circadian oscillations in SCN and extra SCN brain sites.

114 IUPS 2009 July 27 - August 1, 2009 in Kyoto WS3-1 WS3-2 SOME LIKE IT HOT: TEMPERATURE PROFILES OF DOMINANT PARASYMPATHETIC NERVOUS ACTIVITY DIVING ELEPHANT SEALS OF GREAT CORMORANTS Phalacrocorax carbo Jessica Ulrika Meir, Paul J Ponganis ASSESSED BY POWER SPECTRUM ANALYSIS OF HR Center for Marine Biotechnology and Biomedicine, Scripps Institution of VARIABILITY 1 2 3 Oceanography (UCSD), USA Maki Yamamoto , Akiko Kato , Yan Ropert-Coudert , 4 5 2 To account for the repetitive, long duration dives of the northern elephant Masayoshi Kuwahara , Shinichi Hayama , Yasuhiko Naito 1Department of Bioengineering, Nagaoka University of Technology, Japan, seal, Mirounga angustirostris, it has been hypothesized that this species must 2 3 National Institute of Polar Research, Japan, Institut Pluridisciplinaire Hubert either become hypometabolic, or, contrary to concepts of aerobic dive limits, Curien UMR, France, 4Department of Comparative Pathophysiology, Graduate resort to glycolysis and lactate accumulation during dives. As recent studies School of Agriculture and Life Sciences, The University of Tokyo, Japan, have revealed reductions in regional temperature during diving, it is possible 5Department to Veterinary Science, Nippon Veterinary and Life Science that hypothermia may serve as a mechanism of reducing oxygen consumption University, Japan and extending aerobic dive time. To document core body temperature during The characteristics of autonomic nervous activity were examined on captive great cormorants Phalacrocorax carbo hanedae, using a power spectral analysis of heart rate variability. Heart diving in this species, a thermistor was inserted into extradural vein, hepatic rates were calculated from recordings of the electrocardiograms of the birds via embarked data sinus, or aortic sites and a backpack recorder attached to translocated, loggers. We investigated the effects of autonomic nervous blockade using the indices of autonomic juvenile elephant seals. Venous temperature during routine dives remained nervous activity such as high frequency (0.061-1.5 Hz) component, low frequency (0.02-0.060 Hz) near 37°C. Arterial temperature profiles often revealed increases during component and the low frequency power component to high frequency power component ratio. Resting heart rate (85.5 ± 6.1 bpm) was lower than the intrinsic heart rate (259.2 ± 15.3 bpm). The diving (similar to that of exercising mammals), even to greater than 40°C, heart rate drastically increased after the injection of parasympathetic nervous blockade, on the other with subsequent decreases during each surface interval. Decreases in venous hand it slightly decreased after the injection of sympathetic nervous blockade. The sympathetic, and arterial temperature to as low as 31-34°C did occur in long (>30 min) parasympathetic and net autonomic nervous tones calculated from heart rate with and without dives, though these drops were transient and re-warming occurred prior to the autonomic nervous blockade were 40.9 ± 27.6%, -44.5 ± 7.4% and -29.5 ± 9.0%, respectively. The effect of the parasympathetic nervous blockade on low frequency and high frequency end of dive. These data suggest that hypothermia-induced hypometabolism is power was greater than that of the sympathetic nervous blockade. Those data suggested that the an unlikely contributor to metabolic suppression while diving. parasympathetic nervous activity was dominant for great cormorants.

WS3-3 WS3-4 CARDIAC ARREST OF SPAWNING CHUM SALMON MUSCLE OXYGEN DEPLETION IN DIVING EMPEROR RECORDED BY ELECTROCARDIOGRAM (ECG) PENGUINS DATALOGGER Cassondra L. Williams, Paul J. Ponganis 1 1 2 Yuya Makiguchi , Yoshifumi Konno , Masaki Ichimura , Scripps Institution of Oceanography, University of California, San Diego, 3 3 4 5 Shinya Nagata , Hideki Murata , Takahito Kojima , Hiroshi Ueda USA 1Graduate School of Environmental Science, Hokkaido University, Japan, 2 3 A dual-wavelength near-infrared spectrophotometer was developed to Shibetsu Salmon Museum, Japan, Dainippon Sumitomo Pharmacy Co., measure myoglobin oxygen saturation levels in working muscle during Ltd, Japan, 4College of Bioresources Sciences, Nihon University, Japan, 5Field Science Center for Northern Biosphere, Hokkaido University, Japan dives of emperor penguins (Aptenodytes forsteri). The spectrophotometer probe consists of two surface-mount LEDs with wavelengths of 760 nm Cardiac performance is a sensitive indicator of various metabolic activities, and can and 810 nm and two photodiodes. The probe is connected to a data logger be precisely evaluated fish stress in detail. To investigate cardiac performance of chum salmon (Oncorhynchus keta) during spawning behaviour, electrocardiogram (ECG) which provides power to the LEDs at 50 Hz and then records the reflectance were examined using ECG dataloggers (W400-ECG, Little Leonard) recording ECG received by the photodiodes. During the 2007 and 2008 austral spring in from free-swimming fish in the spawning channel. In this study, 13 adult chum salmon Antarctica, the spectrophotometer probe was implanted on the pectoralis (5 males and 8 females) were tagged with ECG dataloggers, and their spawning muscle of emperor penguins. The penguins dove freely at the isolated dive behaviour was monitored using a video camera. The heart rate of both sexes increased hole on the sea ice in McMurdo Sound. During removal of the probe, muscle just prior to spawning, decreased immediately after spawning, and rapidly increased tissue below the probe was excised for a zero-calibration experiment. Initial again only in females, showing clear sex differences of chum salmon spawning results demonstrate a monotonic decline in oxygen saturation during emperor behaviour. Further, cardiac arrest for 7.39±1.61 sec in females and for 5.20±0.97 penguin dives of five to ten minutes. Based on myoglobin concentration in sec in males was observed at the moment of releasing eggs and ejaculating sperm. the pectoralis muscle, this corresponds to an oxygen consumption rate of six Development of T-waves was also observed during the spawning behaviour and -1 -1 peaked at the moment of releasing eggs and ejaculating sperm. Our results suggested to twelve ml O2 kg muscle min . that cardiac arrest of chum salmon during spawning behaviour might be controlled by the autonomic nerve system. Workshops WS3-5 WS3-6 COMPARISON OF DIVING PATTERNS AMONG THREE BIO-LOGGING THE EFFECTS OF CLIMATE ON FREE- EMPEROR PENGUINS FORAGING UNDER THE SAME LIVING LARGE TERRESTRIAL MAMMALS ENVIRONMENTAL CONDITIONS Duncan Mitchell1, Shane K Maloney2, Robyn S Hetem1, Kozue Shiomi1, Katsufumi Sato2, Nobuaki Arai1, Paul J Ponganis3 Andrea Fuller1 1Graduate school of Informatics, Kyoto University, Japan, 2Ocean Research 1School of Physiology, University of the Witwatersrand, South Africa, Institute, University of Tokyo, Japan, 3Scripps Institution of Oceanography, 2Physiology, University of Western Australia, Perth, Australia University of California San Diego, USA The complex effects of climate can be measured properly only in animals living What determines dive depths of diving animals is an important question. free in natural habitats. We have measured arterial and venous blood, abdominal, Determinants of dive depths can be divided into three categories; (1) and limb muscle temperature, continuously and for up to a year, in African, characteristics of individuals (e.g. physiological and morphological property, Australian and American mammals. We also have measured brain temperature in experience), (2) purpose of a dive (e.g. foraging, transit), and (3) environment (e.g. many species. Simultaneous bio-logging of subcutaneous and core temperatures prey distribution and quality, light intensity). For emperor penguins Aptenodytes has allowed us to detect the vasomotor state of free-living mammals, and bio- forsteri diving at the isolated hole of ‘Penguin Ranch’, all dives are foraging logging of stomach temperature to detect when they drink. A miniature globe dives and environmental conditions are the same for all animals. This allows thermometer attached to a collar has allowed bio-logging of microclimate evaluation of the effects of individual characteristics on dive patterns of different selection, and implanted accelerometers have allowed continuous measurement birds. We deployed 3-D dive path data loggers simultaneously on three emperor of activity. We initially captured data with radiotelemetry, but observers disturb penguins. Dive depth of each bird (avg. ± S.D.) was 36.3 ± 27.4 m (n = 139, the behaviour of the animals. That problem was solved by implanted data loggers max. 107.8 m), 51.2 ± 25.9 m (n = 127, max. 158.8 m), and 177.8 ± 94.3 m (n but they preclude real-time monitoring of the data. We now use dual data capture, = 82, max. 289.8 m) respectively. Dive duration, swimming speed, and stroke with an on-board back-up data logger and a radio repeater on a collar, which frequency were not different among the three birds, and horizontal distance transmits data to cellphone networks. Using bio-logging, we have discovered, traveled was greater in shallower dives. It is concluded that these birds selected for example, that selective brain cooling does not serve to protect the brain from different prey items in the same environment at the same time, and that the hyperthermia, that adaptive heterothermy is driven by water shortage and not individual choice of prey influenced dive depths. environmental heat load.

IUPS 2009 July 27 - August 1, 2009 in Kyoto 115 WS4-1 WS4-2 STRUCTURE OF THE LEUCINE TRANSPORTER LeuT ALTERNATING ACCESS IN THE MALTOSE ABC IN COMPLEX WITH DESIPRAMINE SUGGESTS HOW TRANSPORTER ANTIDEPRESSANTS INHIBIT NEUROTRANSMITTER Jue CHEN, Michael Oldham, Dheeraj Khare, Amy L Davidson REUPTAKE Department of Biological Sciences, Purdue University, Howard Hughes Da-Neng Wang Medical Institute, USA Skirball Institute, New York University School of Medicine, USA ATP-binding cassette transporters couple ATP hydrolysis to substrate Tricyclic antidepressants exert their pharmacological effect-inhibiting the reuptake translocation through an alternating access mechanism, but the nature of the of serotonin, norepinephrine and dopamine - by directly blocking neurotransmitter conformational changes in a transport cycle remains elusive. Previously we transporters (SERT, NET and DAT, respectively) in the presynaptic membrane. The reported the structure of the maltose transporter MalFGK2 in an outward- drug-binding site and the mechanism of this inhibition are poorly understood. We facing conformation where the transmembrane (TM) helices outline a determined the crystal structure at 2.9 Aº of the bacterial leucine transporter (LeuT), substrate-binding pocket open to the periplasmic surface an d closed to the a homolog of SERT, NET and DAT, in complex with leucine and the antidepressant cytoplasm. In this conformation, ATP is poised for hydrolysis along the desipramine [1]. Desipramine binds at the inner end of the extracellular cavity of the closed nucleotide-binding dimer interface. Most recently we determined the transporter and is held in place by a hairpin loop and by a salt bridge. This binding site structure of the nucleotide-free maltose transporter in which the substrate is separated from the leucine-binding site by the extracellular gate of the transporter. translocation pathway is only accessible from the cytoplasm and the By directly locking the gate, desipramine prevents conformational changes and nucleotide-binding interface is open. Comparison of the same transporter blocks the release of the substrate. Mutagenesis experiments on human SERT and crystallized in two different conformations shows that alternating access DAT indicate that both the desipramine-binding site and its inhibition mechanism are involves rigid-body rotations of the TM subunits coupled to the closure and probably conserved in the human neurotransmitter transporters. opening of the nucleotide-binding domain interface. The comparison also Reference: [1] Z. Zhou, J. Zhen, N.K. Karpowich, R.M. Goetz, C.J. Law, M.E.A. reveals that point mutations enabling binding-protein independent transport Reith and D.N. Wang (2007) LeuT-desipramine structure suggests how antidepressants may alter dynamic interfaces in the TM region. inhibit human neurotransmitter transporters. Science, 317, 1390-1393.

WS4-3 WS4-4 MOLECULAR MECHANISMS OF SELF-ASSEMBLY, ELECTRON MICROSCOPY OF AQP0-MEDIATED SWITCHING AND MOTION OF THE BACTERIAL MEMBRANE JUNCTIONS FLAGELLUM Thomas Walz1, Richard K. Hite1, Zongli Li1, Tamir Gonen2, Keiichi Namba Yifan Cheng3, Yoko Hiroaki4, Yoshinori Fujiyoshi4, Graduate School of Frontier Biosciences, Osaka University, Japan Stephen C. Harrison1 Bacterial motility is mainly driven by high-speed rotation of the flagellum 1Department of Cell Biology, Harvard Medical School, USA, 2Department made of a rotary motor and a long helical propeller. The motor rotates of Biochemistry, University of Washington, Seattle, USA, 3Department of at around 300 Hz by the energy of proton motive force (pmf) across the Biochemistry and Biophysics, University of California, San Francisco, USA, cell membrane. The propeller is a relatively rigid, supercoiled filament 4Department of Biophysics, Kyoto University, Japan that switches between left- and right-handed helical forms in response to reversal of the motor rotation, allowing bacteria to alternate their swimming Lens-specific aquaporin-0 (AQP0) functions as a specific water pore and pattern between run and tumble for taxis. A short, highly curved segment forms the thin junctions between fiber cells in the lens. We describe a called hook connects the motor with the helical propeller, and its bending 1.9 Aº resolution structure of junctional AQP0, determined by electron flexibility makes it work as a universal joint. The axial structure of the crystallography of double-layered two-dimensional crystals. Comparison of flagellum is constructed by self-assembly of proteins translocated from the junctional and non-junctional AQP0 structures shows that junction formation cytoplasm to the distal end of the growing structure through its 2 nm wide depends on a conformational switch in an extracellular loop, which may central channel. One of the three cap complexes is always attached at the result from cleavage of the cytoplasmic N- and C-termini. In the center of the tip to promote efficient self-assembly. The flagellar type III protein export water pathway, the closed pore in junctional AQP0 retains only three water apparatus also uses pmf to drive the export process by successive unfolding molecules, which are too widely spaced to form hydrogen bonds with each

Workshops and translocation of export substrates. We combine electron cryomicroscopy other. Packing interactions between AQP0 tetramers in the crystalline array and X-ray crystallography to visualize flagellar structures at nearly atomic are mediated by lipid molecules, which assume preferred conformations, resolution to understand molecular mechanisms of protein export, self- making it possible to build an atomic model for the lipid bilayer surrounding assembly, and rotation. the AQP0 tetramers and to describe non-specific lipid-protein interactions.

WS4-5 WS5-1 ROLE OF THE N-TERMINUS OF CX26 IN THE GATING pH DEPENDENCE OF GROWTH REGULATORY MECHANISM OF GAP JUNCTION CHANNELS SIGNALS IN NEURAL STEM CELLS 1 1 2 1 Atsunori Oshima , Kazutoshi Tani , Masoud M Toloue , Yoko Hiroaki , Ying-Mei Wong, Thilina Jayatilleke, Kojiro Yano 3 2 3 1 Amy Smock , Bruce J Nicholson , Gina E Sosinsky , Yoshinori Fujiyoshi Department of Physiology, Development and Neuroscience, University of 1Department of Biophysics, Faculty of Science, Kyoto University, Japan, Cambridge, UK 2Department of Biochemistry, University of Texas Health Science Center at San Antonio, USA, 3National Center for Microscopy and Imaging Research, Neural stem cells (NSCs) have the remarkable ability of self renewal Department of Neurosciences, University of California San Diego, USA and differentiation into a wide variety of neural cells. The signaling Two connexin26 constructs were used to assess gap junction channel structure/ pathways that regulate these processes are influenced by intra- and extra- function: a full length connexin26 mutant (Cx26M34A) and one with an N-terminal cellular environment. We investigated how intracellular and extracellular deletion (Cx26M34Adel2-7). Purified proteins expressed in Sf9 cells were pH affect the growth and motility of NSCs. Cell growth was determined reconstituted into the lipid bilayers that formed 2D crystals. The 3D cryo-electron by cell proliferation assays while motility and lysosomal integrity were crystallography maps at 10 angstrom resolution revealed that crystals obtained from investigated using a neutral red assay and time-lapse fluorescent imaging. both constructs were composed of three lipid bilayers. The structure of Cx26M34A We found that raising ambient CO2 from 5% to 8% significantly enhanced clearly showed a prominent density we refer to as a “plug”, which resides in each cell proliferation and motility, while increased cytosolic acidity by Na/H hemichannel pore (Oshima et al., 2007). The 3D structure of Cx26M34Adel2-7 exchanger (NHE) inhibitor amiloride caused cell death. The use of other contained a reduced plug and a partially reduced density in the cytoplasmic domain NHE inhibitors (5-ethylisopropyl amiloride and Zoniporide dihydrochloride) that link the four helix bundle of each subunit, suggesting that the N-terminus of as well as H+-ATPase inhibitor Bafilomycin and anion transport inhibitors Cx26 has an important role in both formation of the pore plug and configuration of the cytoplasmic domains. Cx26M34Adel2-7 exhibited no electrical functionality. (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid & 4,4'-dinitrostilbene-2, Cx26M34A channels showed minimal dye transfer and a dramatically reduced 2'-disulfonic acid) demonstrated that the inhibition of cell growth and conductance, although the voltage gating characteristics of the residual conductance motility was correlated to the loss of lysosomal integrity. Taken together, were normal. Physical blockage by the N-terminus may be one of the gating these results suggest that lysosomal pH homeostasis has significant influence mechanisms of Cx26 channels, however, this may represent only one of multiple on NSC and the reagents which increase its acidity may encourage brain gating configurations. repair by stimulating NSC growth and migration.

116 IUPS 2009 July 27 - August 1, 2009 in Kyoto WS5-2 WS5-3 TRPV2 EHHANCES AXON OUTGROWTH THROUGH MICRORNA LET-7F AUGMENTS ANGIOGENESIS MEMBRANE STRETCH ACTIVATED PROPERTY IN OF ENDOTHELIAL PROGENITOR CELLS VIA AMPK DEVELOPING SENSORY AND MOTOR NEURONS ACTIVATION AND MNSOD INDUCTION IN TYPE 1 Koji Shibasaki1, Namie Murayama1, Katsuhiko Ono2, DIABETES 1 Makoto Tominaga Xiao-Rong Wang, Alex F. Chen 1 Section of Cell Signaling, Okazaki Institute for Integrative Bioscience, Department of Surgery, University of Pittsburgh School of Medicine, USA Japan, 2Department of Biology, Kyoto Prefectural University of Medicine, EPCs play an essential role in angiogenesis, but are dysfunctional in diabetes. Japan AMPK activation improves angiogenesis. We tested the hypothesis that miRNA let- Thermosensitive TRP (thermo TRP) channels are well recognized for their 7f improves EPC-mediated angiogenesis via AMPK activation and mitochondrial contributions to sensory transduction, responding to a wide variety of stimuli including MnSOD induction. Bone marrow-derived EPCs from STZ diabetic mice were temperature, nociceptive stimuli, touch, and osmolarity. However, the precise roles used. Endogenous let-7f expression was decreased by >60% in diabetic vs. normal for the thermo TRP channels during development have not been determined. To EPCs. Transfection of diabetic EPCs with let-7f mimic significantly improved their explore the functional importance of thermo TRP channels during neural development angiogenesis and adhesion, both were retarded by the selective AMPK inhibitor in DRG, the temporal expression of TRPV1, TRPV2 and TRPM8 was determined (TRPV2, E10.5; TRPV1, E13.5; TRPM8, E16.5). Interestingly, TRPV2 expression compound C. Let-7f mimic transfection also increased AMPK phosphorylation was detected in spinal motor neurons in addition to the DRG. Furthermore, TRPV2 and MnSOD in diabetic EPCs. Compound C pretreatment decreased AMPK was localized in axon shafts and growth cones in the developing DRG and spinal phosphorylation by 37% and MnSOD induction by >50%, induced by let-7f mimic motor neurons, suggesting that the channel is important for axon outgrowth regulation. transfection. Furthermore, elevated mitochondrial ROS level in diabetic EPCs was Endogenous TRPV2 was activated in a membrane stretch dependent manner in significantly reduced after let-7f mimic transfection, preventable by compound C. developing neurons, and significantly promoted axon outgrowth. We also confirmed Protein phosphatase 2A (PP2A), a key enzyme for AMPK inactivation, was increased by an in ovo electroporation method that ectopic TRPV2 expression promoted axon in diabetic EPCs, which was inhibited by let-7f mimic transfection. Finally, let-7f outgrowth in chick embryos. Thus, for the first time we revealed that TRPV2 is an inhibitor transfection significantly impaired normal EPC angiogenic and adhesion important regulator for axon outgrowth through activation by membrane stretch during functions. Thus, microRNA let-7f improves EPC angiogenesis through AMPK development, and is a thermo-sensor for noxious heat in postnatal animals. activation and MnSOD induction in type 1 diabetes.

WS5-4 A NEW SYNTHETIC ISOFLAVONOID, TCH2972, ENHANCES OSTEOGENESIS IN CULTURED HUMAN ADIPOSE DERIVED STEM CELLS Yu-Fang Chang1,3,4, Chien-Fu Chen2, Shin-Mon Chen1,2,4, Je-Ken Chang2,4, Gwo-Jaw Wang2,4, Mei-Ling Ho1,3,4 1Department of Physiology, Kaohsiung Medical University, Kaohsiung, Taiwan, 2Department of Orthopaedics, Kaohsiung Medical University, Kaohsiung, Taiwan, 3Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan, 4Orthopaedic Research Center, Kaohsiung Medical University, Kaohsiung, Taiwan, 5College of Medicine, Department of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung, Taiwan Nature products and their components have been used as the food factor for improving osteoporosis [1,2]. Isoflavones, such as daidzin, daidzein, genistin and genistein, are nature products and useful for preventing and treating osteoporosis [3]. Ipriflavone (7-isopropoxyisoflavone), one kind of isoflavone which synthesized from soy isoflavone daidzein, has been approved for treating involutional osteoporosis in European and in Japan [4,5]. We synthesized several new isoflavone derivatives and screened their potential for promoting osteogenesis. We found that one potent compound, TCH2972, significantly enhanced the osteogenesis of osteogenic cells. Here, we investigated the effect of TCH2972, compared with ipriflavone and raloxifene, on cell viability, cytotoxicity, and osteogenesis in human adipose derived stem cells (hADSC) . Workshops

IUPS 2009 July 27 - August 1, 2009 in Kyoto 117 TU1 (outline) TU2-1 PHYSIOME/VPH PROJECTS TOOLS (CELLML PET MOLECULAR IMAGING FOR INTEGRATED TUTORIAL) PHYSIOLOGICAL AND MEDICAL SCIENCES Peter Hunter, Poul Nielsen Yasuyoshi Watanabe Auckland Bioengineering Institute, University of Auckland, New Zealand RIKEN Center for Molecular Imaging Science, Japan Both CellML and cmgui are core elements of the Virtual Physiological In vivo molecular imaging has become a key technology for drug Human ToolKit. CellML (http://www.cellml.org/) is an XML-based language for description of quantitative mathematical models. The primary goal of the development and pathophysiological science. We are mostly utilizing CellML project is to provide a lingua franca for such models to facilitate their PET (Positron Emission Tomography) as a first-choice modality, because distribution and reuse by the research community. cmgui (http://www.cmiss. of its ultra-high sensitivity for molecules, adequate temporal and spatial org/cmgui) is a application for visualizing complex multidimensional data, resolution, and especially broad spectrum of target molecules. The present for example, the kinds of detailed functional models of human organ systems status for development of PET molecular probes, instrumentations including developed within the Physiome Project. microPET, and the methods for quantitative analyses will be introduced in This is a combined tutorial that will consist of a one hour lecture style this tutorial. Molecular imaging could bring the high-quality information introduction, followed by 90 minutes of hands-on exposure to either CellML about key molecules as disease markers for diagnosis, direct follow-up, and or cmgui, (according to the attendee's choice) supervised by senior members demonstration of drug effect in living patients with symptoms. Molecular of both projects. The CellML tutorial will show attendees how to build and imaging is useful for drug development; especially, drug delivery system, simulate a well known model in the OpenCell software, while the cmgui pharmacokinetics and pharmacodynamics, dose finding information for tutorial will give an introduction to cmgui, demonstrating how to create a custom visualisations of 3D organ models and data. Attendees should bring individuals corresponding to SNP’s, direct evidence for accumulation in non- their own computers if possible, but workstations will be provided. target organs related to the adverse effects, and evidence for drug effects with surrogate markers.

TU2-2 TU3-1 PET MEASUREMENT: PRINCIPLES, THEORIES, AND FINDING PROTEIN SEQUENCE(S) OF YOUR INTEREST PRACTICE FROM PUBLIC DATABASES Yasuhiro Wada Kenta Nakai RIKEN Center for Molecular Imaging Science, Japan Institute of Medical Science, The University of Tokyo, Japan In general, PET can provide highly quantitative images. To produce voxel Since there are many useful databases publicly available for biologists, it value in activity concentration, a number of corrections are required. could be fatal if you are unable to access to their latest information. However, And also, the optimal settings of image reconstruction algorithm and due to mostly historical reasons, these resources exist in dispersed web sites parameters are necessary. In this lecture, principles of PET measurement, and their usages may not look very intuitive. Therefore, in this tutorial, I data processing and intrinsic problems underlying misreading of data will will briefly explain about a few typical sequence/genome databases so that be introduced. Then, in practice, how to convey efficient PET measurement you will be able to find amino acid sequences by yourself in a variety of for due extraction of quantitative physiological and pathological parameters, situations, such as finding a homologous sequence in a certain organism or how to decrease noises, and how to evaluate the quality of data will also be finding sequences of potential membrane proteins existing near a certain gene suggested through our long-term experience. in the genome.

TU3-2 TU3-3 STRUCTURAL MODELING OF PROTEINS: PRINCIPLE COXPRESDB: GENE CO-EXPRESSION DATABASE Tutorials AND APPLICATION TO AN ION CHANNEL FOR HUMAN, MOUSE, AND RAT Haruki Nakamura Kengo Kinoshita Institute for Protein Research, Osaka University, Japan Institute of Medical Science, The University of Tokyo, Japan With the rapidly increasing structural data in the Protein Data Bank (PDB), As the vast accumulation of gene expression data in public database such as many physiological phenomena are now understood based on the tertiary NCBI/GEO, gene coexpression is becoming powerful and useful information structures of the target or their homologous proteins. In this tutorial, we to infer genetic interactions for a biological function of the genes, because will introduce several Web services associated with the structural database coexistence is a necessary key condition for genes to interact with each other. search, not only searching particular texts, but also searching several similar In this tutorial, we will briefly introduce the contents and usages of our gene "shapes" such as the protein folds, the local atomic architecture, and the coexpression databases, COXPRESdb, which provides coexpressed gene local molecular surfaces with electrostatic potentials. We may also prepare a lists, coexpressed gene networks, expression patterns among different tissues, tutorial using a PC for homology modeling of an ion channel protein, whose gene annotations and some other useful information for Human, Mouse and homologous proteins are registered in the PDB database. Rat genes.

118 IUPS 2009 July 27 - August 1, 2009 in Kyoto LS4-1 ADVANCED PROTEOMICS TECHNOLOGIES BY MASS SPECTROMETRY -A NEW METHOD OF SAMPLE PREPARATION FOR Abstracts of luncheon seminars MASS SPECTROMETRY- related with whole-day symposia, Nathan P. Lawrence Pressure BioSciences Inc., USA PSJ symposia or regular symposia Data will be presented on PBI’s ProteoSolve-SB Kit, a Pressure-dependant system for the simultaneous extraction of proteins, lipids, and nucleic acids are placed in each symposium from samples. Protein and lipids extracted by this method are suitable for section. analysis by MS. The use of the PCT significantly reduced sample preparation and digestion time from hours to minutes, while improving the quality of protein identification.

LS4-2 LS12-1 ADVANCED PROTEOMICS TECHNOLOGIES BY MASS UNFINISHED DREAM FOR 50 YEARS OF BRAIN SPECTROMETRY RESEARCH -MOLECULAR IMAGING BY MASS SPECTROMETRY- Keisuke Toyama Taka-Aki Sato Kyoto Prefectural University of Medicine, Japan Life Science Res. CTR., Shimadzu Corporation, Japan My dream was triggered by Hubel and Wiesel’s work of the simple and Significant advances have been made in the past decade in the field of MS complex cell. The first step was to demonstrate the structures of visual imaging. There is tremendous potential in its application especially in clinical cortical circuitry using intracellular recording, and we found the layer- field, such as biomarker discovery or pharmacokinetic study. However, vast specific cortical circuitry. The second step was to demonstrate the working majority of the work has been performed on frozen tissue sections, while of the cortical circuitry using crosscorrelation analysis, and we found serial it remains generally unpractical to produce frozen sections with clinically excitation and inhibition and common inputs. Later works reported stimulus- resected tumor samples. Here we report our novel sample preparation dependent common inputs of broader coincidence probably due to recursive technique that enabled MS imaging from FFPE tissue section, including activation through recurrent axon collaterals. The third step started rather retrospective archive as old as 11 years. Furthermore, we have developed recently and still continues. That is to study relevance of coincident visual a new MS Microscope, which the effective laser spot size is 10 micro m or cortical firing with human visual perception using fMRI-constrained MEG. less. We opened the door to retrospective study of past clinical cases in aim We found strong enhancement of firing coincidence between V2/3 and V5 to discover molecular biomarker. during perception of contour from motion. Autoregression analysis showed that it is due to recursive activation between the two cortical areas. Similar increase of firing coincidence was found lately in other cortical areas during tasks demanding problem solution. Evidence is accumulating for recursive computation of problem solution. The day is coming up, and I encourage young neuroscientists to join this field. Approach may still be uneasy, but the goal is extremely rewarding.

LS12-2 LS15 TOWARD THE FUTURE OF NEUROPHYSIOLOGY NEW HORIZONS IN SCIENCE EDUCATION Yuji Ikegaya Eva-Maria Neher Department of Medical Pharmaceutics, The University of Tokyo Graduate XLAB Goettingen Experimental Laboratory for the Youth, Goettingen, School of Pharmaceutical Science, Japan Germany XLAB provides science education, which bridges the gap between high school and To understand the normal and pathological brain function, capitalizing on the university. One of our principle aims is to raise student’s interest in science and full potential of recent advances in monitoring and controlling neural circuits encourage students to pursue scientific careers. At XLAB, school classes or individual will depend crucially on the emergence of new technologies. In this scope, students can do hands-on experiments in Biology, Chemistry, Computer Science, and neurophysiology has a tremendous potential, as it covers the entire span from Physics with state-of-the-art-equipment under professional guidance. XLAB`s general the individual neuronal activity to interactions in large neuronal assemblies, aims are promoting the attractiveness of the science education, promoting the mobility and functional networks underlying the dynamics of complex higher-order of the students, and encouraging them to take up university studies abroad. behaviors. My talk aims at extrapolating its future by covering the advanced Our missions are: Professionalism technologies and challenges such as Blue Brain project, connectome, The XLAB tries to provide an atmosphere of genuine research laboratories with brain machine interface, multineuronal calcium imaging, and functional authentic tools and equipment and, most importantly, with lecturers, who are Luncheon Seminars mapping of microcircuit connections, catFISH with immediate early gene, experienced scientists. and photostimulation with uncaged compounds and channelrhodopsins. Intensity Obviously, they provide massively parallel data of neuronal activity and Students concentrate on one subject, without interruptions by other activities as during connectivity, and thus, the efficient extraction of useful information from the an ordinary day at school. This is prerequisite for intensive learning comparable with university teaching. flood of data will also become an increasing scientific demand. All in all, can Internationality these methods reevaluate the classically designed experiments? Is it possible Science is international, and the scientific community resembles a worldwide family. to evaluate which neuronal population is relevant to a behavior? Is there a The XLAB International Science Camp conveys this experience to our future new road in degenerative and mental disorders research? scientists.

IUPS 2009 July 27 - August 1, 2009 in Kyoto 119 LS21-1 LS21-2 NANO-MANIPULATION OF OLFACTORY SENSATION FUNCTIONAL ROLE OF SUBUNIT ASSEMBLY OF CNG Hiroko Takeuchi CHANNELS IN SENSORY TRANSDUCTIONS Graduate School of Frontier Biosciences, Osaka University, Japan Tsung-Yu Chen Center for Neuroscience and Development of Neurology, University of Olfactory masking has been employed to erase the unpleasant sensation in California, USA human cultures for a long period of history. In the present lecture we show a The conversion of olfactory stimuli to electric signals in vertebrate olfactory positive correlation between the human masking and the odorant suppression receptor neurons relies on cyclic nucleotide-gated (CNG) channels. of the transduction current through the CNG and Cl(Ca) channels. Channels in The binding of odorants to their receptors leads to an increase of intracellular the olfactory cilia were activated with the cytoplasmic photolysis of caged cAMP that opens the olfactory CNG channels. Odorants, however, also compounds, and their sensitiveness to odorant suppression was measured directly inhibit olfactory CNG channels. This inhibition is thought to be the with the WC patch clamp. When 16 different types of chemicals were mechanism underlying the odorant masking effect. We used a heterologous applied to cells, cAMP-induced responses were suppressed widely with these expression system, Xenopus oocytes, to examine the effect of structurally substances, but with different sensitivities. Using the same chemicals, in un-related odorant molecules on the cloned olfactory CNG channels. We parallel, we measured human olfactory masking with 6 rate scoring tests, and found that the homomeric channel formed by subunit CNGA2 alone is less saw a correlation coefficient of 0.81 with the channel block. Furthermore, we sensitive to the odorant inhibition than heteromeric channels formed by co- investigated the contribution of Cl and confirmed that Cl channels were expressing CNGA2 with CNGA4, CNGB1, or both. Our results suggest that (Ca) (Ca) odorants inhibit CNG channels by altering the lipid membrane structure. less sensitive to the odorant suppression. We conclude that odorants regulate Furthermore, the direct odorant inhibition of the expressed CNG channels CNG level to express masking, and Cl(Ca) in the cilia carries out the signal appears to be less potent than the inhibition of the odorant-induced current in amplification and reduction evenly spanning the entire cilia. The present native olfactory neurons, suggesting that the inhibition on the CNG channels findings may serve possible molecular architectures to design effective in native neurons may be amplified by the reduction of the calcium-activated masking agents, targeting olfactory manipulation at the nano-scale ciliary chloride current, the activation of which relies on the influx of calcium ions membrane. through the olfactory CNG channels. Luncheon Seminars

120 IUPS 2009 July 27 - August 1, 2009 in Kyoto Volume 59 · Supplement 1 · 2009

Volume 59 · Supplement 1 · 2009

The XXXVI International Congress of Volume 59 · Supplement 1 · 2009 · pp 1–XX · pp 1 · 2009 Volume 59 · Supplement Physiological Sciences (IUPS2009) International Scientific Program Committees (ISPC) ISPC Chair Yoshihisa Kurachi Vice Chair Ole Petersen ISPC from IUPS Council Akimichi Kaneko (IUPS President) Irene Schulz (IUPS Vice President) Pierre Magistretti (IUPS Vice President) Malcolm Gordon (IUPS Treasurer)

ISPC IUPS2009 Members and Associated Members Proceedings of the XXXVI International Congress of Physiological Sciences (IUPS2009) Commission I Locomotion Commission VII Comparative Physiology: Hans Hoppeler, Masato Konishi, Hiroshi Nose Evolution, Adaptation & Environment Function of Life: Elements and Integration Commission II Circulation/Respiration Malcolm Gordon, Ken-ichi Honma, July 27–August 1, 2009, Kyoto, Japan Yung Earm, Makoto Suematsu, Itsuo Kodama Kazuyuki Kanosue Commission III Endocrine, Reproduction & Commission VIII Genomics & Biodiversity Development David Cook, Hideyuki Okano, Gozoh Tsujimoto Caroline McMillen, Yasuo Sakuma, Toshihiko Yada Commission IX Others Commission IV Neurobiology Ann Sefton, Peter Hunter, Osamu Matsuo, Quentin Pittman, Harunori Ohmori, Fumihiko Kajiya, Tadashi Isa, Tadaharu Tsumoto, Megumu Yoshimura Jun Tanji Commission V Secretion & Absorption Local Executives Irene Schulz, Miyako Takaki, Yoshikatsu Kanai Yasuo Mori, Ryuji Inoue Commission VI Molecular & Cellular Biology Cecilia Hidalgo, Yoshihiro Kubo, Katsuhiko Mikoshiba, Masahiro Sokabe, Yukiko Gotoh