Activation of Trpv1 Channel Contributes to Serotonin-Induced Constriction of Mouse Facial Artery Bolu Zhou University of Vermont

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Graduate College Dissertations and Theses Dissertations and Theses

2017 Activation Of Trpv1 Channel Contributes To Serotonin-Induced Constriction Of Mouse Facial Artery Bolu Zhou University of Vermont

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Recommended Citation Zhou, Bolu, "Activation Of Trpv1 Channel Contributes To Serotonin-Induced Constriction Of Mouse Facial Artery" (2017). Graduate College Dissertations and Theses. 754. https://scholarworks.uvm.edu/graddis/754

This Thesis is brought to you for free and open access by the Dissertations and Theses at ScholarWorks @ UVM. It has been accepted for inclusion in Graduate College Dissertations and Theses by an authorized administrator of ScholarWorks @ UVM. For more information, please contact [email protected]. ACTIVATION OF TRPV1 CHANNEL CONTRIBUTES TO SEROTONIN-INDUCED CONSTRICTION OF MOUSE FACIAL ARTERY

A Thesis Presented

Bolu Zhou

The Faculty of the Graduate College

The University of Vermont

In Partial Fulfillment of the Requirements for the Degree of Master of Science Specializing in Pharmacology

May, 2017

Defense Date: March 28, 2017 Thesis Examination Committee:

George C. Wellman, Ph.D., Advisor Victor May, Ph.D., Chairperson Joseph E. Brayden, Ph.D. Karen M. Lounsbury, Ph.D. Cynthia J. Forehand, Ph.D., Dean of the Graduate College

ABSTRACT

Tight regulation of cephalic blood circulation is critical under normal physiological conditions, and dysregulation of blood flow to the head occurs in pathophysiological situations such as stroke and migraine headache. The facial artery is an extracranial artery which is one of branches from the external carotid artery territory and its extracranial position indicates its importance in regulating head hemodynamics. Transient receptor potential vanniloid type 1 (TRPV1) is a cation channel permeable to Ca 2+ and Na +. Intracellular Ca 2+ increase causes vasoconstriction. A previous study indicated the presence of TRPV1 in smooth muscle cells in the facial artery. Protein kinase C (PKC) is found to sensitize TRPV1 channels in neurons. Our lab’s preliminary data suggested PKC modulates TRPV1 in the middle meningeal artery. Serotonin (5-HT) is an endogenous vasoconstrictor, and the 5-HT 2 receptor is a Gq-protein-coupled receptor that activates PKC. In the present study, we found that 5-HT caused facial artery constriction. Thus, we studied whether TRPV1 channel acting as a Ca 2+ entry channel is involved in 5- HT induced facial artery constriction. We used a pressurized arteriography technique to examine the artery diameter. The results indicate that 1) TRPV1 antagonist blunted 30 nM 5-HT-induced mouse facial artery constriction. 5-HT constriction on the facial artery from TRPV1 knock out mice was significantly blunted compared to the constriction on the facial artery from wild type mice; 2) PKC, which is a downstream signaling molecule of 5-HT 2 receptor, is involved in capsaicin (TRPV1 agonist)-induced facial artery constriction; 3) 5-HT-induced facial artery constriction is mediated mostly by activation of 5-HT 1 and 5-HT 2 receptors; 4) 5-HT 2 but not 5-HT 1 receptor is involved in 5-HT- induced facial artery constriction via opening of TRPV1 channels; 5) PKC may be involved in 5-HT-induced facial artery constriction; 6) The L-type-voltage-dependent Ca 2+ channel is involved in 5-HT-induced facial artery constriction. We conclude that activation of TRPV1 channel contributes to serotonin-induced 5-HT 2 receptor-mediated constriction of the mouse facial artery.

ACKNOWLEDGEMENTS

I would like to thank Drs. George Wellman, Joseph Brayden and Masayo Koide for their advising support.

TABLE OF CONTENTS

ACKNOWLEDGEMENTS………………………………………………………………ii

LIST OF TABLES………………………………………………………………………..iv

LIST OF FIGURES……………………………………………………………………….v

CHAPTER 1: LITERATURE REVIEW………………………………………………….1

1.1. TRPV1 channels.…………………………………………...... 1

1.2. Serotonin…………………………………………………………………………….14

1.3. Relationship between 5-HT receptors and TRPV1………..………………………...23

CHAPTER 2: RESEARCH STUDY…………………………………………………….27

2.1. Introduction………………………………………………………………………….27

2.2. Methods and Material……………………………………………………………….32

2.3. Results……………………………………………………………………………….35

2.4. Conclusions and Discussion...…………………………………………………...... 49

2.5. Future directions…………………………………………………………………….58

2.6. References…………………………………………………………………………...61

iii

LIST OF TABLES

Table page

Table 1: …………………………………………………………………………………13

Table 2: …………………………………………………………………………………22

LIST OF FIGURES

Figure Page

Figure 1: …………………………………………………………………………………12

Figure 2: …………………………………………………………………………………36

Figure 3: …………………………………………………………………………………39

Figure 4: …………………………………………………………………………………41

Figure 5: …………………………………………………………………………………44

Figure 6: …………………………………………………………………………………46

Figure 7: …………………………………………………………………………………48

Figure 8: …………………………………………………………………………………57

CHAPTER 1: LITERATURE REVIEW

1.1 TRPV1 channels

1.1.1 General overview of TRPV1

The TRP family includes seven main subfamilies: TRPA (transient receptor potential ankyrin), TRPC (transient receptor potential canonical), TRPM (transient receptor potential melastatin), TRPP (transient receptor potential polycystin), TRPML (transient receptor potential mucolipin), TRPN (transient receptor potential NOMPC, referred to as no mechanoreceptor potential C), and TRPV (transient receptor potential vanilloid), among which TRPN is the only channel not expressed in mammalian cells (Pedersen et al., 2005). Many TRP channels were detected in vascular smooth muscles including

TRPC (1, 3, 4-6), TRPV (1-4), TRPA1, TRPM4, 8, TRPP2 (Earley and Brayden, 2015).

TRPV1 is a nonselective cation channel, however it exhibits approximately a tenfold preference for Ca 2+ over Na +. TRPV1 has also been shown to mediate the influx of

+ relatively large cations such as FM1-43, H 3O ions, tetraethylammonium and NMDG in response to agonist stimulation (Chung et al., 2008). The TRPV1 channel was first described in small nociceptive dorsal root ganglion (DRG) and trigeminal ganglion (TG) neurons. A single clone encoding the TRPV1 channel was first identified in 1997 by Dr.

David Julius’ research group in HEK293 cells transfected with pools of clones from a rodent dorsal root ganglion (Caterina et al., 1997). TRPV1 has an N-terminal intracellular 1

domain containing three ankyrin repeats, six transmembrane (TM) spanning regions interrupted by a pore loop domain (between TM 5 & 6) and a large C-terminal intracellular domain (Schumacher and Eilers, 2010). Tyr 511 and Ser 512, located at the transition between the second intracellular loop and the third TM domain has been suggested to interact with vanilloid ligands for example capsaicin at the intracellular face of the membrane (Tominaga and Tominaga, 2005). The distal half of the TRPV1 C- terminus is reportedly involved in the thermal sensitivity of the channel, most evidence suggesting that TRPV1 is activated by temperatures in the noxious range with an activation threshold of 43°C (Caterina et al., 1997; Vlachova et al., 2003). It can also be activated by some endogenous lipid-derived molecules such as lysophosphatidic acid, amines such as N-acyl-ethanolamines and N-acyl-dopamines, endogenous anandamide, acidic solutions (pH < 6.5) and some pungent chemicals such as capsaicin, as well as by toxins such as resiniferatoxin and vanillotoxins (neurotoxins found in the venom of the tarantula Psalmopoeus cambridgei) (Bevan et al., 2014). In addition to its capsaicin-, proton-, and temperature-dependent activation, TRPV1 can also be modulated by cell membrane and intra-cellular components. TRPV1 has been reported to be modulated by tyrosine kinases or G-protein-coupled receptors (Vellani et al., 2001). After TRPV1 channels are modulated by tyrosine kinases and G-protein-coupled receptors, the channel opens even at a normal body temperature (Vellani et al., 2001). TRPV1 is also modulated by protein kinase C (PKC), protein kinase A (PKA), calmodulin (CaM) (Numazaki et al.,

2003), CaMKII (Jung et al., 2004) and Src kinase (Jin et al., 2004). Stimulation of

TRPV1 activity can be achieved through inflammatory agents such as bradykinin, serotonin, histamine, or prostaglandins, which stimulate TRPV1 either by protein kinase 2

C–dependent pathways (Cesare et al., 1999; Premkumar and Ahern, 2000; Vellani et al.,

2001), by releasing the channel from phosphatidylinositol 4,5-bisphosphate (PIP 2)-

dependent inhibition (Chuang et al., 2001; Prescott and Julius, 2003), by a protein kinase

A–mediated recovery from inactivation (Bhave et al., 2002), or by formation of 12-

hydroperoxyeicosatetraenoic acid (12-HPETE) (Shin et al., 2002). Phosphorylation by

protein kinase A or protein kinase C reduces the threshold temperature for TRPV1

activation. Specifically, TRPV1 can be phosphorylated at S502A/S800A by PKC, leading

to temperature threshold decrease (Jordt et al., 2000; Numazaki et al., 2002). TRPV1

levels in DRG neurons were increased by nerve growth factor and the activation of p38

mitogen-activated protein kinase in inflamed skin (Chen et al., 2008).

Although TRPV1 is primarily restricted to nociceptors in primary sensory ganglia it has

minimal expression in a few discrete brain regions such as the caudal hypothalamus

(Cavanaugh et al., 2011). This channel is also present in a large portion of neurons of the

nodose and jugular sensory vagal ganglia (Lee and Gu, 2009). Prominent expression of

TRPV1 was found in sensory nerves and arteriolar smooth muscle (ASM) of bladder

(Phan et al., 2016). Measurements of capsaicin-evoked calcium in artery smooth muscle

(ASM) cells and constriction of bladder arterioles suggested TRPV1 has a functional role

in the overactive bladder (Nilius et al., 2007). Capsaicin or resiniferatoxin directly

activated capsaicin-sensitive primary sensory neurons in the sub-epithelial layer of the

bladder. This resulted in release of substance P that sensitized smooth muscle cells

causing increased contractility (Pinna et al., 1994). TRPV1 has also been shown to play a

role in heat hyperalgesia, motor disorders, skin disorders, gastrointestinal tract diseases, 3

airway hypersensitivity, and heart ischemia. Locally applied capsaicin reduced inflammation-evoked heat hyperalgesia, suggesting that TRPV1 plays an important role in the development of inflammatory heat hyperalgesia (Coderre et al., 1986). Capsaicin, through TRPV1 activating cyclooxygenase-2 in a Ca 2+ -dependent manner, induced the

release of interleukin-8 and prostaglandin E2, which are important signaling molecules

associated with skin disorders (Southall et al., 2003). Within the stomach and duodenum,

one of the most prominent functions of TRPV1-expressing sensory nerves is the

maintenance of tissue integrity against noxious compounds, such as protons and activated

enzymes (Holzer, 2002). In airway hypersensitivity, capsaicin-responsive afferents are

either superficial fibers terminating in the mucosa or deep pulmonary fibers located in the

alveolar septa. Superficial fibers monitor the chemical environment of the airway mucosa

and their activation results in cough, increased mucosal secretion and bronchoconstriction

(Nagy et al., 2004). The TRPV1 channel plays an imperative role in the activation in

remote ischemic post-conditioning, as denervation of TRPV1 channel positive afferent

nerve fibers or blockade of the TRPV1 receptor was shown to significantly attenuate

remote ischemic post-conditioning-induced cardio-protective effects in rat hearts

(Randhawa and Jaggi, 2015). TRPV1 in perivascular plexi activated by low pH of the

ischemic heart muscle during insufficient coronary circulation (Franco-Cereceda et al.,

1993) contributes to the development of chest pain and reflectory sympathetic activation

(Zahner et al., 2003).

1.1.2 TRPV1 and pain

The nociceptive sensors in DRG neurons play a critical role in the transmission of pain;

therefore, the DRG has become an important target for pain treatment. The TRPV1

channel is one of the nociceptive receptors in DRG neurons. Studies with TRPV1-

deficient mice demonstrate that the TRPV1 channel is essential for the thermal

hyperalgesia induced by tissue injury and inflammation, supporting the hypothesis that

TRPV1 is a molecular integrator of painful stimuli (Caterina et al., 1997). Protease-

activated receptor 2 (PAR 2)-induced thermal hyperalgesia depends on sensitization of

TRPV1. In addition, PAR 2 agonists and other inflammatory agents, including bradykinin, adenosine triphosphate (ATP), prostaglandin E2 (PGE2) and nerve growth factor (NGF) indirectly sensitize TRPV1, causing hyperalgesia. The mechanisms of this sensitization include activation of PKC and PKA, which phosphorylate TRPV1 to modify channel gating (Amadesi et al., 2006).

Capsaicin (the active ingredient of hot chili peppers and an agonist for the TRPV1 channel) and its vanilloid analogue, resiniferatoxin (RTX, from Euphorbia resinifera), elicit burning pain by activating TRPV1 expressed on the sensory nerve endings (Szallasi and Blumberg, 1999). TRPV1 antagonists alleviate pain behavior in rodent models of inflammation (Honore et al., 2005). Interestingly, peripheral delivery of capsaicin causes desensitization of sensory neurons (C fibers and A δ fibers), thus alleviating pain as well

(Rains and Bryson, 1995). Early work by Bodnar et al demonstrated that intracerebroventricular (ICV) capsaicin injection decreased the nociceptive threshold and reduced morphine-induced analgesia (Bodnar et al., 1982; Bodnar et al., 1983). In addition, the heat threshold of the tail, measured by an increasing-temperature water bath, 5

was significantly higher in TRPV1 knock out animals (Tekus et al., 2016). Modulation of

TRPV1 channels by dopamine in nociceptive neurons may represent a way for dopamine

to modulate incoming noxious stimuli (Chakraborty et al., 2016). One study showed that

diabetic thermal hyperalgesia was associated with a reduction in anti-nociceptive effects

of capsaicin (TRPV1 agonist) in the ventrolateral periaqueductal gray (VL-PAG) and

down-regulation of TRPV1 receptor expression in periaqueductal gray (PAG)

(Mohammadi-Farani et al., 2010). TRPV1 receptor stimulation by endocannabinoids or

by capsaicin in the PAG led to analgesia and this effect was associated with glutamate

increase and the activation of OFF cell population in the rostral ventromedial medulla

(Palazzo et al., 2008).

1.1.3 TRPV1 and inflammation

Experimentally, intradermal capsaicin injection induced neurogenic inflammation, and

was characterized by arteriolar vasodilation, plasma extravasation, and pain (hyperalgesia

and/or allodynia) (Jancso et al., 1967). The underlying mechanism is that capsaicin

sensitizes nociceptors by activating TRPV1 receptors distributed in small diameter

myelinated (A δ) and unmyelinated (C) primary afferent nociceptive fibers, which leads to the release of inflammatory peptides from these sensitized afferent terminals. In dorsal root ganglia, more than 50% of neurons express TRPV1, and the majority of TRPV1 positive neurons respond to nerve growth factor (NGF) and release substance P (SP) and

CGRP producing neurogenic inflammation by interacting with endothelial cells, mast cells, immune cells and arterioles. Bonnington et al reported that the early pathway in 6

TRPV1-mediated nociception activated by NGF involves phosphatidylinositol-3-kinase, as well as PKC and CaMKII. Activation of this pathway results in inflammatory heat hyperalgesia through TRPV1 sensitization (Bonnington and McNaughton, 2003). TRPV1 also plays a role in airway inflammation. Cough sensitivity to TRPV1 activators, such as capsaicin or citric acid aerosol, was markedly elevated in patients with asthma or airway inflammation (O'Connell et al., 1996). There was a significant correlation between the cough sensitivity to capsaicin inhalation challenge and the density of TRPV1-expressing nerves in the mucosa of patients with chronic cough (Groneberg et al., 2004). Some studies have been done on TRPV1’s role in joint diseases. Increased expression of

TRPV1 on unmyelinated nerves was demonstrated during Complete Freund's Adjuvant

(CFA)-induced hind paw inflammation (Carlton and Coggeshall, 2001). Keeble demonstrated the pathophysiological involvement of TRPV1 in a murine joint inflammation model, acutely and chronically. The study examined the vascular and hyperalgesic components of the joint inflammation in wild type mice and TRPV1 knockout mice after intra-articular injection of Complete Freund's Adjuvant (CFA), and demonstrated that knee swelling and vascular hyper-permeability were significantly higher in the CFA-treated joints of wild type mice than TRPV1 null mice, although leukocyte accumulation and cytokine production were not affected (Keeble et al., 2005).

By limiting neuro-inflammatory processes, TRPV1 exerted a protective role that restricts the initiation and progression of colon cancer (Vinuesa et al., 2012).

TRPV1 can be sensitized by many inflammatory mediators, indicating its important and broad role in inflammation. Cumulative evidence demonstrated that nociceptor 7

sensitization by inflammatory agents is primarily through potentiation of the TRPV1 channel activity. TRPV1 sensitization by pro-algesic agents occurs either through direct activation of the channel or its potentiation. Direct activation of TRPV1 by arachidonic acid metabolites including anandamide, N-arachidonoyl dopamine (NADA), N- oleyldopamine and 12-hydroperoxyeicosatetraenoic acid has been reported. They act as

2+ weak agonists, but potently increase TRPV1-mediated [Ca ]i in both heterologous expression systems and primary sensory neurons (Chu et al., 2003; Huang et al., 2002).

In addition, the acidosis that develops in inflamed tissues is also a direct activator of the

TRPV1 (Caterina and Julius, 2001). The potency and efficacy of each mediator is quite low, but in inflammatory conditions several of these modulators are simultaneously released and act synergistically.

1.1.4 TRPV1 and thermoregulation

Body temperature is regulated and maintained by several individual thermo-effectors such as vasomotor tone in subcutaneous tissue. Non-shivering thermogenesis is controlled by metabolic heat production in different tissues such as brown adipose tissue and in the liver, controlled by adrenaline and thyroid hormones respectively. In contrast, shivering thermogenesis is mediated by the skeletal muscle. Tonic TRPV1 activation modulates vasomotor tone and thermogenesis. TRPV1 agonists cause hypothermia via skin vasodilation and a reduction in metabolism. It was demonstrated that AMG0347 (a selective TRPV1 antagonist) caused hyperthermia by vasoconstriction and an increase in metabolism (Steiner et al., 2007). Accordingly, TRPV1 agonists cause hypothermia. 8

Capsaicin caused hypothermia when administered by a variety of routes including oral, intraperitoneal (i.p.) and intracerebral administration in a wide variety of animals such as mice, rats, guinea pigs, dogs, goats and ground squirrels. Additionally, the ability of capsaicin to cause hypothermia was not abolished or reversed in a hot environment (Hori,

1984), suggesting that ambient temperatures do not affect this response. Similar to capsaicin, other agonists of TRPV1, such as RTX 4 and arvanil, also caused hypothermia

(Di Marzo et al., 2000). These data support the central role of TRPV1 in mammalian thermoregulation.

There are cutaneous thermo-sensors and visceral thermo-sensors. Visceral thermoreceptors were considered to be within the walls of the rumen, intestine and around the mesenteric vessels. Several recent reports provide evidence that TRPV1- expressing capsaicin-sensitive visceral primary afferent neurons, their thoracic or abdominal receptors and central terminals could serve as thermoreceptors within the physiological range of thermoregulation (Szolcsanyi, 2015). Furthermore, the noxious heat-sensitive groups of polymodal nociceptors are the major targets of capsaicin. After treatment of rats with increasing doses of capsaicin within 2–4 days from 30–50 mg/kg up to a total dose of 120–300 mg/kg, the animals became insensitive to capsaicin and capsaicin did not induce hypothermia even after several months. Acute effects of capsaicin are mediated by TRPV1 activation but long-term effects are due to sensory desensitization of TRPV1-expressing neurons (Szikszay et al., 1982; Szolcsanyi, 2015).

1.1.5 TRPV1 and vasculature

TRPV1 activation in blood vessels causes constriction effects (Kark et al., 2008). RT–

PCR showed the expression of TRPV1 mRNA in the brain, aorta, and primary cultured

VSMC from TRPV1 reporter mice (Cavanaugh et al., 2011). Expression of TRPV1 in bladder was detected in arteriolar smooth muscle (ASM) in mice (Phan et al., 2016).

Expression of TRPV1 channels in aortic and skeletal muscle arteriolar myocytes have been reported (Kark et al., 2008; Lizanecz et al., 2006). The expression of TRPV1 was observed in human pulmonary artery smooth muscle cells (PASMCs) and is associated with hypoxia-induced cell proliferation (Wang et al., 2008). TRPV1 activator capsaicin increased hindlimb vascular resistance in vivo and caused substantial endothelium- independent vasoconstriction of skeletal muscle arterioles in vitro. Vasoconstriction of isolated canine mesenteric arteries following direct activation of TRPV1 channels has also been reported (Porszasz et al., 2002). However, capsaicin evoked constrictions in rats skeletal muscle arteries and carotid arteries, but had no effect on the femoral, mesenteric arteries or the aorta (Toth et al., 2014). The study showed capsaicin-induced vasodilation in the mesenteric arteries of normal rats (Zhang et al., 2015). Capsaicin alone did not cause constriction in the conduit canine coronary artery, however, pretreatment with pro-inflammatory prostaglandin-thromboxane agonists may unmask capsaicin's vasoconstrictive potential. Capsaicin first induced transient dilation that was followed by sustained constriction in the conduit canine coronary artery (Hiett et al.,

2014). Increased cation permeability associated with TRPV1 channel activation results in membrane depolarization and Ca 2+ influx, which are the primary mechanisms by which 10

this channel causes vasoconstriction (Song et al., 2017). However, some evidence suggest

TRPV1 may be involved in store operated Ca 2+ entry (SOCE) through functioning as

2+ Ca release channels in the sarcoplasmic reticulum (SR) in the RBL-2H3-M1 cells

(Turner et al., 2003). Calcium imaging of isolated arterioles from the ear showed that genetically marked SMCs respond to capsaicin application, leading to arteriole constriction. More recent work has revealed the presence of TRPV1 channels in murine arteriolar myocytes located in thermoregulatory tissues and has demonstrated a role for these channels as mediators of myocyte Ca 2+ entry and constriction in intact auricular

arterioles (Cavanaugh et al., 2011).

Besides constriction, TRPV1 activation also causes dilation, which was found to be

related to the perivascular sensory neuronal terminals, which release neurotransmitters

(CGRP) upon stimulation and mediate vasodilation (Zygmunt et al., 1999). Capsaicin

elicited concentration-dependent vasodilation of both rat aorta and porcine coronary

arteries (Hopps et al., 2012). TRPV1 activation has been recently reported to decrease

phosphorylation of endothelial NO synthase (eNOS) at threonine 497 (Thr497) in

cultured bovine aortic endothelial cells, leading to increased NO production and

vasodilation (Ching et al., 2013). Capsaicin also abolished the concentration-dependent

constriction of porcine coronary arteries induced by the L-type calcium activator, Bay-K

8644. These data suggested that capsaicin causes vascular dilation through the inhibition

of L-type Ca² ⁺ channels (Hopps et al., 2012).

TRPV1 mRNA distribution in dorsal root ganglia, spinal cord and arteries in a study

(Toth et al., 2014) is summarized in Fig.1. TRPV1 distribution in tissues and detection methods are summarized in Table.1.

Figure 1. TRPV1 mRNA in peripheral tissues of the rat. TRPV1 expression was examined with qPCR in peripheral tissues of the rat (Toth et al., 2014).

Table.1. TRPV1 distribution in various tissues and the identification methods

TRPV1 containing tissues Methods Human dorsal root ganglia Immunostain (Anand et al., 2006) Human kidney, pancreas, stomach, small RT-PCR, Radioimmunoassay, intestine, lung immunohistochemistry (Sharrad et al., 2015; Wick et al., 2006; Zhao et al., 2016; Zhu et al., 2005) Human skin and mouse ear Immunohistochemistry, histology (Denda et al., 2001) Rat dorsal root ganglia Electrophysiology (McDonald et al., 2008) Rat cerebral cortex, striatum, RT-PCR (Cavanaugh et al., 2011; hippocampus, central amygdala, Huang et al., 2014; Liapi and Wood, cerebellum, locus, cerulean, cochlear 2005; Toth et al., 2005) nuclei, spinal nucleus of the trigeminal nerve, inferior olive and spinal cord Rat kidney, pancreas, placenta and RT-PCR, isolated perfused rat urinary bladder kidney, electrophysiology (Li and Wang, 2008; Zhu et al., 2011) Rat astrocytes Immunohistochemistry (Sharrad et al., 2015) Rat renal artery RT-PCR (Ueda et al., 2008) Mouse esphagus Immunohistochemistry (Matsumoto et al., 2014a) Mouse asymmetric synapses in mouse Immunolocalization (Puente et al., dentate molecular layer 2015) Mouse trigeminal ganglion dura afferents Immunohistochemistry (Huang et al., 2012)

1.2 Serotonin

1.2.1 General overview of serotonin

Serotonin, also known as 5-hydroxytryptamine (5-HT), was initially isolated from bovine serum as a substance with vasoconstriction effects (Cavanaugh et al., 2011).

Serotonin is synthesized from dietary L-tryptophan via an intermediate compound, 5- hydroxytryptophan (5-HTP) by the enzyme tryptophan hydroxylase (TPH). At least 15 distinct 5-HT receptor subtypes have been found, which are grouped into 7 families with distinct downstream signaling pathways (Kroeze et al., 2002). More than 90 % of 5-HT found in the body is synthesized and stored in enterochromaffin cells in the gut. In the gastrointestinal (GI) tract, 5-HT acts as a critical signaling molecule participating in intestinal secretion, sensation, and peristalsis. These effects predominantly signal through

5-HT (3) and 5-HT (4) receptors (Gershon, 1999). Enteric neuronal 5-HT has a vital role in the growth and maintenance of intestinal mucosa and 5-HT promotes development of enteric neurons and proliferation of central nervous system neuronal precursors, and induces adult stem cells to undergo neurogenesis (Gross et al., 2012). Platelets actively take up 5-HT as platelets pass through the GI tract and provide a circulating reservoir

(Bertrand and Bertrand, 2010). In healthy human subjects, the mean level of 5-HT is approximately 1 nM in plasma, 1 µM in serum, and 400 µM in platelets (Flachaire et al.,

1990). When platelets bind to a clot, they release serotonin, which serves as a vasoconstrictor and helps to regulate hemostasis and blood clotting. 5-HT is also a monoamine neurotransmitter. A small proportion of 5-HT is enclosed within synaptic 14

vesicles of central serotonergic neurons as a neurotransmitter, which, after releasing, will display its major physiological function in nerve terminals (Gamoh et al., 2013). 5-HT is found extensively in neurons in the central and peripheral nervous systems and following release, its actions are terminated by re-uptake and/or metabolism by monoamine oxidase

(MAO). Serotonin in the central nervous system (CNS) has long been associated with pain processing and modulation (Eide and Hole, 1993). 5-HT from the rostral ventromedial medulla (RVM) is an important contributor to pain facilitation during the development of persistent pain (Wei et al., 2010). Besides modulation in pain, 5-HT is participating in many kinds of ischemia. During myocardial ischemia-reperfusion, serotonin accumulates in the myocardial interstitium and plays an important role in the progression of myocardial cell injury (Naumenko et al., 2014).

1.2.2 Serotonin receptors

5-HT functions via 15 classes of receptors that are present (and differentially distributed) in the cardiovascular system, peripheral nervous and central nervous systems (CNS), including the frontal cortex, hippocampus, amygdala, striatum, hypothalamus and dorsal horn. All (intronless) 5-HT 1 receptors negatively couple to adenylyl cyclase (AC) via G i/o , whereas 5-HT 4, 5-HT 6 and 5-HT 7 receptors stimulate AC through G s. 5-HT 2 triplets (A,

B and C) share recruitment of phospholipase C (PLC) via G q/11 as their modes of

signaling (Nakajima et al., 2009). Previous reports have indicated that 5-HT 1-5 receptors

are involved in the modulation of nociception in the spinal cord (Jeong et al., 2004). 5-

HT receptor types on C-fibers, including 5-HT 2A , 5-HT 2B , 5-HT 2C , 5-HT 3, 5-HT 4, 5-HT 6 15

and 5-HT 7 receptors, have been reported to mediate 5-HT-induced pro-nociceptive effects at the periphery nervous system (Godinez-Chaparro et al., 2011; Sasaki et al.,

2006; Sommer, 2004). Besides central nervous system and peripheral nervous system, the expression of mRNA of 5-HT 1, 5-HT 2 and 5-HT 7 receptors has been reported in vascular

smooth muscle cells (VSMCs) (Machida et al., 2013). 5-HT 2A and 5-HT 1 receptor

subtypes are almost exclusively responsible for vasoconstriction (Yildiz et al., 1998).

Specifically, activation of 5-HT 1B receptors produces vasoconstriction of large arteries and veins, whereas 5-HT 2B activation induces vasodilation of the microcirculation

(Arreola-Ramirez et al., 2015).

1.2.3 Role of serotonin in the gut

Serotonin receptors are widely expressed within the GI tract, and five of the seven known families, 5-HT 1, 5-HT 2, 5-HT 3, 5-HT 4, and 5-HT 7 receptors, are expressed in the

gut and can affect gut functions (Hoyer et al., 2002). Three subtypes of 5-HT receptor (5-

HT 1p , 5-HT 3, and 5-HT 1A ) have been found on enteric neurons. 5-HT 1P receptors and

perhaps 5-HT 3 receptors in neurons are involved in initiating the peristaltic reflex and regulating gastric emptying (Gershon et al., 1990). Antagonists of 5-HT 3 and/or 5-HT 4 receptors slowed intestinal motility and a study demonstrated that corticotropin-releasing hormone-induced defecation was blocked by the peripheral action of the 5-HT 3 receptor

antagonist, ramosetron (Miyata et al., 1998). 5-HT 3 receptor antagonists and 5-HT 4 receptor agonists have been used to treat functional disorders such as diarrhea and constipation (Camilleri, 2009). Other functions of 5-HT involving intrinsic reflexes 16

include stimulation of propulsive and segmentation motility patterns, epithelial secretion and vasodilation. A unique feature of the gut is the ability of intrinsic reflex circuitry to locally regulate blood vessel diameter that is achieved by mucosal stimulation. The intrinsic reflex circuitry was blocked by inhibition of 5-HT 3 and 5-HT 4 receptors (Vanner

and Macnaughton, 2004). 5-HT 3-mediated synaptic responses participate in descending pathways in the myenteric plexus. However, functional studies examining the polarity of intestinal reflexes demonstrated that ascending but not descending reflexes were inhibited by 5-HT 3 receptor antagonists (Yuan et al., 1994). Mechanical stimulation of the mucosal

surface leads to 5-HT release, which activates neural mediated Cl - and bicarbonate

secretion. Infusion of hyperosmotic solutions or carbohydrate breakdown products into

the lumen of the duodenum evokes release of 5-HT from EC cells and 5-HT acts on 5-

HT 3 receptors to stimulate vagal afferent neurons (Li, 2007). Serotonin released from EC cells is an important activator of the reflex-mediated secretion like neurotransmitters such as acetylcholine and vasoactive intestinal peptide (VIP) (Kim et al., 2001). Within the gut,

5-HT also exerts actions such as promoting inflammation and serves as a trophic factor to promote the development and maintenance of neurons and interstitial cells of Cajal

(Furness and Costa, 1982; Galligan et al., 2000).

1.2.4 Role of serotonin in central nervous system (CNS)

5-HT receptor family including different subtypes is involved in the functional pathways in the brain and their balance in activity helps to maintain the mental stability.

In the CNS, serotonergic cell bodies are clustered in the midbrain and brainstem raphe 17

nuclei from which they project locally, as well as to spinal cord and many forebrain targets (Moore et al., 1978). The contributions of 5-HT include modulation of a broad range of targets, most notably, hypothalamic, limbic and cortical circuits linked to the control of mood and mood disorders. Dysregulation of 5-HT signaling has been implicated in multiple neurobehavioral disorders, including anxiety, depression, obsessive-compulsive disorder (OCD) and addiction (Moore et al., 1978). 5-HT 3 receptor

is ubiquitously expressed in the central nervous system. Sites of expression include

several brain stem nuclei and higher cortical areas such as the amygdala, hippocampus,

and cortex. On the subcellular level, both presynaptic and postsynaptic 5-HT 3 receptors can be found. Presynaptic 5-HT 3 receptors are involved in mediating or modulating

neurotransmitter release (Chameau and van Hooft, 2006). Serotonin transporter (SERT)

is antagonized by the 5-HT selective reuptake inhibitors (SSRI) that are used as

antidepressant medications that continue to represent the most common pharmacological

treatment for affective disorders (Blakely et al., 1998).

1.2.5 Role of serotonin in pain sensation and inflammation

Most studies of 5-HT have been focused on two central levels of pain control: the

dorsal horn of the spinal cord and the midbrain, which are anatomically and functionally

interconnected by a spino-bulbo-spinal loop (the brain's feedback to the spinal cord)

(Saade and Jabbur, 2008). Systemic (subcutaneous or intravenous) injections of 5-HT

trigger excitation and sensitization of primary nociceptive afferent fibers (A δ and C fibers) as well as nociceptive neurons originated in dorsal root ganglia, thereby contributing to 18

peripheral sensitization and hyperalgesia (Sommer, 2004). Relevant studies in various animal models showed that most if not all 5-HT receptor types, namely 5-HT 1A , 5-HT 1B ,

5-HT 1D , 5-HT 1F , 5-HT 2A , 5-HT 2B , 5-HT 2C , 5-HT 3, 5-HT 4, 5-HT 5, 5-HT 6 and 5-HT 7, are involved in modulatory mechanisms of nociception (Kayser et al., 2011). Indeed, spinal

5-HT, released from the serotonergic projections in response to formalin injection, activates pre- or post-synaptic 5-HT (4/6/7) receptors at the dorsal root ganglion/spinal cord, promoting the development and maintenance of secondary allodynia and hyperalgesia (Godinez-Chaparro et al., 2012).

5-HT, a potent immune modulator, plays a role in inflammation pain sensation. 5-HT is released from platelets and mast cells during inflammation or injury (Dray, 1995).

Enterochromaffin cells (EC) isolated from patient’s mucosa with the inflammatory disease such as Crohn’s disease have a greater production of 5-HT (mediated in part by

Toll-like receptor 4). Patients with another inflammatory disease ulcerative colitis have reduced expression of serotonin transporter (SERT), and it is also observed in another form of intestinal inflammation, diverticulitis (Coates et al., 2004; Costedio et al., 2008;

Kidd et al., 2009). Rheumatoid arthritis (RA) is a systemic inflammatory disorder that principally attacks synovial joints, characterized by cartilage erosion and ankylosis, and is associated with increased levels of circulating 5-HT (Voog et al., 2004). In inflammation-induced pain, both peripheral 5-HT 2A receptors and 5-HT 2C receptors are

involved. Local application of a selective 5-HT 2A/2C receptor antagonist may be effective to treat pain provoked by 5-HT release, which commonly occurs with inflammation

(Nakajima et al., 2009) . 19

1.2.6 Role of serotonin in the cardiovascular system

Serotonin induces a wide spectrum of effects in the cardiovascular system, mediated through a variety of receptor subtypes. 5-HT is taken up by serotonin transporter (SERT) in platelets, as platelets do not synthesize 5-HT. The function of 5-HT in the blood is to promote platelet aggregation and blood clotting (Mauler et al., 2016). Although platelets contain 5-HT, they also possess 5-HT 2A receptors which, when activated, promote further platelet activation and aggregation (Watts, 2005). Thus 5-HT 2A receptor antagonists such as ketanserin and sarpogrelate have been proved effective as anti-platelet/anti-thrombotic agents. Interestingly, reports have described platelets in hypertensive subjects as being hyper-aggregatable or more fragile. Aspirin (a platelet aggregation inhibitor inhibiting 5-

HT release from platelets) attenuates pulmonary arterial hypertension in rats by reducing

the plasma 5-HT level (Shen et al., 2011). In several models of chronic hypertension,

there are a specific increase in the responsiveness of the blood vessel wall to the

vasoconstrictor property of 5-HT and a delayed desensitization to the monoamine. The

ability of hypertensive animals to clear 5-HT from the blood is reduced, and the platelets

of hypertensive patients take up less 5-HT than those of normotensive humans; their

activation to release 5-HT is accelerated (Vanhoutte, 1983). After 5-HT is released from

platelets, vasoconstrictive effect of 5-HT is mediated by 5-HT 1B and 5-HT 2A receptors

located on the membrane of smooth muscle cells, except in the intracranial arteries whose

constriction are only mediated by 5-HT 1B receptors (Gamoh et al., 2013). Constriction of mice pulmonary arteries by 5-HT was attenuated by applying GR-127935 (a 5-HT 1B/1D receptor inhibitor) (Liu and Folz, 2004). 5-HT 1D -like receptor mediates constriction in the 20

canine coronary artery (Cushing et al., 1994). 5-HT-induced canine internal carotid arteries and rat middle cerebral arteries constriction is predominantly mediated by 5-

HT 1B/1D and 5-HT 2A receptors (Centurion et al., 2001; Kovacs et al., 2012). 5-HT 2A receptors mediate the serotonin-induced constriction in the rabbit aorta (Clancy and

Maayani, 1985), dog femoral arteries (Apperley et al., 1980), and many other arteries from several species (Martin, 1994). Receptor-coupled stimulation of artery constriction involves elevating smooth muscle intracellular Ca 2+ levels and also sensitizing the

2+ myofilaments to the [Ca ]i increase (MacDonald et al., 2000).

There are limited studies about 5-HT effect on carotid artery tone and the facial artery is an important extracranial artery from the external carotid artery territory that is involved in the head hemodynamic. Thus, studying the 5-HT’s constriction role in the facial artery

improves our understanding of the head blood flow regulation under common

endogenous substances such as 5-HT, which may provide a potential drug target for head

blood dysregulation diseases such as stroke and migraine headache. Common carotid

artery vasoconstriction response to 5-HT in vagosympathectomized dogs is mainly

mediated by activation of sumatriptan-sensitive 5-HT 1-like receptors (Villalon et al.,

1995). 5-HT can produce vasodilatation or vasoconstriction of the canine external carotid

bed depending upon the degree of carotid sympathetic tone. 5-HT 1B receptors and alpha

2A/2C-adrenoceptors mediate external carotid artery vasoconstriction to

dihydroergotamine (a 5-HT 1D receptor agonist used to treat migraine) (Villalon et al.,

2004). Serotonin elicited a biphasic concentration response curve (CRC) in the rat femoral artery, the first phase of the serotonin CRC in rabbit femoral artery is mediated 21

predominantly by 5-HT 2 receptors and that the second phase is mediated by alpha 1-

adrenoceptors (Grandaw and Purdy, 1996). 5-HT-mediated pulmonary arterial

vasoconstriction occurs largely due to the activation of 5-HT 2 receptors, which then

activates Gq-coupled pathways and increases cytosolic calcium leading to arterial

constriction. 5-HT also acts as a vasodilator because it releases nitric oxide from

endothelial cells. This response is mediated by 5-HT 1B receptors but not by 5-HT 2A receptors (Gamoh et al., 2013). Our study mainly focuses on 5-HT constriction mechanism by studying serotonin receptor subtypes and downstream signaling pathways in the facial artery constriction. 5-HT receptors distribution within the vasculature is listed in Table.2.

Table.2 5-HT receptors distribution in the vasculature

Receptor Distribution and effect subtype 5-HT (1A) Rat renal artery relaxation (Nakajima et al., 2009) 5-HT (1B) Human coronary artery constriction (Bopp et al., 2016) Human pulmonary artery constriction (Bopp et al., 2016) Human cerebral artery constriction (Comer, 2002) Human mesenteric artery constriction (Gul et al., 2003) Human temporal artery constriction (van den Broek et al., 2002a) Human middle meningeal artery constriction (van den Broek et al., 2002a) Human coronary artery dilation via endothelium cells (Ishida et al., 1998) Rat pulmonary artery constriction (Wang et al., 2001) Rat middle cerebral artery constriction (Kovacs et al., 2012) 5-HT (1D) Human coronary artery and middle meningeal artery constriction (van den Broek et al., 2002b) Human temporal artery constriction (Verheggen et al., 1996) Human pulmonary artery constriction (MacLean et al., 1996) Human internal thoracic artery constriction (Chester et al., 2000) Endothelium cells of human coronary artery causing dilation (Ullmer et al., 1995) 22

Relaxation through endothelium cells of pig coronary artery (Chan et al., 2013) Canine denuded coronary artery constriction (Egashira et al., 1992) 5-HT (1F) Human middle meningeal artery constriction (Razzaque et al., 1999) Human coronary arteries (Nilsson et al., 1999) 5-HT (2A) Human temporal artery constriction (Verheggen et al., 1996) Human pulmonary artery constriction (Cogolludo et al., 2006) Human internal thoracic artery constriction (Tanaka et al., 2008) Pig coronary artery constriction (Miyata et al., 2000) Rats mesenteric arteries constriction (Sung et al., 2013) Rats middle cerebral artery (Back et al., 1998) Rats common carotid artery constriction (Radenkovic et al., 2010) Aorta of larval (Majeed et al., 2014) 5-HT (2B) Endothelium cells of pig meningeal artery causing dilation (Schmuck et al., 1996) Pulmonary artery in weaned pigs dilation (Jahnichen et al., 2005) Endothelium cells of human coronary artery causing dilation (Ishida et al., 1998) Human pulmonary endothelium cells (Ullmer et al., 1996) 5-HT (4) Endothelium cells of human coronary artery (Ullmer et al., 1995) 5-HT (7) Human coronary artery (Nilsson et al., 1999) Human pulmonary artery (Ullmer et al., 1995) Middle meningeal artery of rats dilation (Terron and Martinez-Garcia, 2007)

1.3. Relationship between 5-HT receptors and TRPV1

5-HT receptors can regulate TRPV1 function in various tissues that are associated with

many physiological processes. Sumatriptan (10 µM), a selective antimigraine drug (5-

HT 1 receptor agonist), inhibited TRPV1-mediated inward currents in trigeminal ganglia

(TG) and capsaicin-elicited spontaneous excitatory postsynaptic currents in trigeminal nucleus caudalis (TNC) slices (Evans et al., 2012). 5-HT 2B receptor mediates 5-HT- induced mechanical hyperalgesia by upregulating TRPV1 function (Su et al., 2016).

Studies demonstrate that 5-HT 2 and 5-HT 7 receptors in nervous system are modulating

TRPV1 channel. Stimulation of spinal 5-HT (2A/2C) receptors potentiates the capsaicin- induced release of substance P-like immunoreactivity in the rat dorsal horn (Kjorsvik

Bertelsen et al., 2003). PKC and PKA-mediated signaling pathways are involved in the potentiating effect of 5-HT on TRPV1 functions through the activation of 5-HT 2A and 5-

HT 7 receptors respectively in cultured dorsal root ganglion (DRG) neurons isolated from

neonatal rats (Ohta et al., 2006). 5-HT-induced facilitation of TRPV1 function was

significantly reversed by SB204070 (a selective 5-HT 4 receptor antagonist) and this

facilitation is probably due to activation of 5-HT 4 receptors. Activation of both 5-HT 2 and

5-HT 4 receptors similarly enhanced TRPV1 function in colon sensory neurons. 5-HT 2 receptor agonists mimicked the facilitating action of 5-HT on TRPV1 function in response to acid (Sugiuar et al., 2004).

Protein kinase C (PKC) can be regarded as a molecule that links 5-HT receptors

(specifically 5-HT 2 receptor) and TRPV1 channels. TRPV1 was sensitized at the knee joint and at DRG neurons of mono-iodoacetate-induced joint pain in rats through PKC activation (Koda et al., 2016). PKC activation has also been shown to increase TRPV1 activity by inflammatory mediators such as bradykinin (Wang et al., 2015). 5-HT 2 receptor activation leads to activation of Gq/11 , resulting in activation of phospholipase C

(PLC) and PKC (Conn and Sanders-Bush, 1984; Roth et al., 1986). 5-HT-induced

mechanical hyperalgesia may be mainly mediated by 5-HT 2B -Gq-PLC β-PKC ε signaling

by regulating TRPV1 function (Su et al., 2016). Besides PKC and TRPV1, 5-HT-induced

contraction (also 5-HT 2A -mediated contraction) is coupled with various kinase pathways 24

including mitogen-activated protein kinase (MAPK), Phosphatidylinositol-4, 5- bisphosphate 3-kinase (PI3K), and Rho kinase in smooth muscle cells (SMCs)

(Matsumoto et al., 2014b). Caldesmon is a physiological substrate of p44/42 MAPKs and inhibits the ATPase activity of actin-myosin complex in smooth muscles. Phosphorylated caldesmon positively regulates smooth muscle contraction by relieving the inhibition of actin-myosin ATPase activity and, consequently, promoting smooth muscle contraction

(Gerthoffer et al., 1996; Matsumura and Yamashiro, 1993). PI3K γ isoform generates PIP 3, which eventually stimulates L-type calcium channels in vascular myocytes (Le Blanc et al., 2004). RhoA/Rho kinase-mediated Ca 2+ sensitization is generally attributed to the inhibition of myosin light chain phosphatase, increased myosin light chain phosphorylation and increased vascular tone (Broughton et al., 2008).

Preliminary studies in our laboratory indicated that PKC is involved in capsaicin- induced mouse middle meningeal artery constriction and is a necessary component in sustained constriction (unpublished observations by Hughes, Wellman et al.). In the present study, serotonin induced facial artery constriction. PKC, a downstream molecule for 5-HT 2 receptors activation (Zhang et al., 2003), clues to a possible link between the

TRPV1 channel and 5-HT receptors in arteries. Therefore, we hypothesized that TRPV1 is involved in 5-HT-induced facial artery constriction. We further propose the constriction effect of serotonin on the facial artery via opening of TRPV1 channels is mediated by 5-HT 2 but not 5-HT 1 receptors due to PKC’s potential role in modulating

both TRPV1 and 5-HT 2 receptors. In the present study, we found that TRPV1 inhibition cannot suppress sumatriptan (5-HT 1 receptor agonist) constriction, suggesting that 5-HT- 25

induced facial artery constriction via opening of TRPV1 may signal through 5-HT 2 receptors, not 5-HT 1 receptors.

CHAPTER 2: RESEARCH STUDY

2.1. Introduction

TRPV1, a member of the TRP family of ion channels, is a non-selective cation channel

(high calcium permeability), which is activated or modulated by diverse exogenous

noxious stimulus such as high temperatures, irritant and pungent compounds, and by

selected molecules released during tissue damage and inflammatory processes.

Endogenous regulators of TRPV1's activity include acidic pH and anandamide which

activate TRPV1, lipids and lipids metabolites that negatively regulate TRPV1, for

example cholesterol and phosphatidylinositol 4, 5-biphosphate (PIP 2) (Bevan et al., 2014).

TRPV1 was found in the central nervous system (Toth et al., 2005) and peripheral blood vessels (Lizanecz et al., 2006). Later research on the effects of TRPV1 stimulation in blood vessels suggested both dilation and constriction (Kark et al., 2008). TRPV1- mediated dilation was found to be related to the perivascular sensory neuronal terminals, which released neurotransmitters (calcitonin gene-related peptide CGRP, substance P) upon stimulation and mediated vasodilation (Rosenbaum and Simon, 2007). The vasoconstrictive properties of TRPV1, however, were much less well characterized.

TRPV1 is expressed in a subset of arteriolar smooth muscle cells within thermoregulatory tissues and capsaicin (a TRPV1 agonist) increases calcium influx and induces vasoconstriction (Cavanaugh et al., 2011). Capsaicin evoked constrictions in skeletal muscle arteries, carotid arteries and femoral arteries, but had no effect on mesenteric arteries or the aorta (Toth et al., 2014). However, constriction of isolated canine

mesenteric arteries following direct activation of TRPV1 channels has been demonstrated

(Porszasz et al., 2002). Expression of TRPV1 channels in aortic and skeletal muscle arteriolar myocytes from rats has been reported (Lizanecz et al., 2006; Toth et al., 2014).

We previously found functional expression of TRPV1 channels in ASM of arteries and arterioles of the cephalic, cutaneous and skeletal muscle circulations (unpublished observations by Koide, Wellman et al.). Using our TRPV1-td Tomato reporter mice

(using the Cre/loxP system to express the red fluorescent protein, tdTomato, under the control of the entire TRPV1 gene promoter region, yielding a staining pattern that recapitulates endogenous TRPV1 expression), we have established the presence of

TRPV1 throughout the external carotid artery (ECA) territory and demonstrated its complete absence in the cerebral vasculature. Capsaicin constricted facial arteries (FA) and middle meningeal arteries (MMA) that are from the external carotid artery. In significant contrast, capsaicin did not constrict cerebral arteries from wild type mice or

MMA)/FA from TRPV1-knock out mice (unpublished observations by Koide, Wellman and Brayden et al.). PKC is involved in endothelin-1 potentiating effect on TRPV1 and this plays a major role in hyperalgesic effects of endothelin-1 in sensory neurons (Plant et al., 2007). PKC ε phosphorylates TRPV1 at Ser800 and sensitizes nociceptors (Aley et al.,

2000). PKC is the downstream molecule for G q\11 -protein-coupled receptors (Mironneau

and Macrez-Lepretre, 1995). The present study showed 5-HT caused facial artery

constriction, which is probably through binding with 5-HT receptors. Specifically, 5-HT 2 receptor is G q\11 -protein-coupled receptor that can activate PKC through generating diacylglycerol (DAG). Thus we speculated TRPV1 is involved in 5-HT-induced facial artery constriction. 28

Serotonin (5-HT) is a bioamine derived from the amino acid tryptophan. Seven classes of serotonin receptors have been identified (5-HTR1 to 5-HTR7). Among them, 5-HT 3 receptor is the only ligand-gated ion channel receptor. All other serotonin receptors belong to the G-protein-coupled receptor superfamily. The 5-HT 1A , 5-HT 1B/1D , 5-HT 2 receptor family (5-HT 2A and 5-HT 2B ), 5-HT 3, 5-HT 4 and 5-HT 7 receptors are found in cardiovascular tissues (Cote et al., 2004; Doggrell, 2003; Marwood and Stokes, 1984;

Ramage and Villalon, 2008). 5-HT 2A and 5-HT 1 receptor subtypes are almost exclusively

responsible for vasoconstriction (Yildiz et al., 1998). Vasoconstriction of the external

carotid artery induced by 5-HT in vagosympathectomized dogs is mainly mediated by

activation of sumatriptan-sensitive 5-HT 1 receptors (Villalon et al., 1995). 5-HT caused a constriction response both in the external and internal carotid vascular beds, ketanserine

(10 nM) (5-HT 2 receptor antagonist) antagonized both responses, indicating the involvement of 5-HT 2 receptors (Vasquez and Pinardi, 1992). The mesenteric artery constriction to 5-HT (seconds-minutes) from normotensive rats is mediated primarily by

5-HT 2A receptors (Watts et al., 1996). 5-HT 1 receptor family is mostly linked to G i/o protein, which inhibits cAMP formation, whereas 5-HT 2 receptor is coupled to G q/11

2+ protein leading to the activation of IP 3/PKC/cytosolic [Ca ] signaling pathway (Martin et al., 1998).

importance in regulating hemodynamics in the head. In the systemic circulation, serotonin activates a variety of direct and reflex responses including vasospasm. TRPV1 is found to have vasoconstrictive effect in many vascular beds. In the present study, we studied the endogenous substance serotonin (5-HT) constriction in the facial artery from the external carotid artery territory and its relation with TRPV1 channels. In terms of 5-

HT-induced constriction, 30 nM 5-HT induced mouse facial artery constriction and this constriction was not desensitized (data not shown). We studied TRPV1 channel involvement in 5-HT-induced facial artery constriction by examining blocking of TRPV1 channel on 5-HT constriction, and 5-HT receptor subtypes in mediating 5-HT-induced constriction via opening of TRPV1 channels. Using pressurized arteriography measurements, our ex vivo data firstly showed that 5-HT-induced constriction of the facial artery can be partly inhibited by applying the TRPV1 antagonist capsazepine or L- type-voltage–dependent Ca 2+ channels (L-VDCC) inhibitor nifedipine. We found that

protein kinase C (PKC) may involve in capsaicin-induced constriction in the facial artery,

which suggests that 5-HT receptor subtype 2 is probably involved in serotonin-induced

facial artery constriction via opening of TRPV1 channels. We found 5-HT 1 and 5-HT 2 receptors are the main 5-HT receptor subtypes that mediate 5-HT-induced facial artery constriction. We used 5-HT + GR55562 (5-HT 1 receptor antagonist) to activate 5-HT 2 receptor in the facial artery and found that capsazepine significantly blunted this constriction. Furthermore, we demonstrated that 10 µM sumatriptan (5-HT 1 receptor

agonist) caused facial artery constriction, that was unaffected by capsazepine. This

indicated that 5-HT 1 receptor is not involved in serotonin-induced facial artery

constriction via opening of TRPV1 channels. Thus, activation of TRPV1 channels is involved in 5-HT-induced 5-HT 2 receptor-mediated mouse facial artery constriction.

2.2. Methods and Material

Dissection of the facial artery and the arteriography diameter measurement

All experiments and protocols were performed in accordance with the Guide for the

Care and Use of Laboratory Animals (eighth edition, 2011) and were approved by the

Institutional Animal Care Committee of the University of Vermont. Male C57BL/6J mice

(20 to 23 g body weight) and TRPV1-knock out (KO) mice were used in this study (from

Jackson Labs). Specifically, TRPV1 knock out (KO) mice were generated by targeted

deletion of exons encoding the fifth and sixth transmembrane domains of the channel,

including the pore-loop domain (Caterina et al., 2000). Mice were anesthetized using

pentobarbital salt solution (0.02 ml per mouse, pentobarbital sodium 390 mg/ml) and

sacrificed by decapitation. The head was preserved in 4°C physiological salt solution

(PSS; NaCl 119 mM, KCl 4.69 mM, KH 2PO 4 1.69 mM, MgSO 4 1.18 mM, EDTA 0.033

mM, NaHCO 3 23.81 mM, Glucose 10.55 mM, CaCl 2 1.6 mM). Facial arteries from

C57BL/6J mice were dissected using a light microscope (Fiber-Lite, Dolan-Jenner

Industries) in cold physiological salt solution (PSS). Then the artery was pinned down in

a dissection dish with PSS and cleaned to remove all connective tissues. For endothelial

cell removal, arteries were cannulated on a system in which the proximal cannula and

perfusion tubing were pre-filled with 1–2 ml of air using a syringe attached to the Luer-

Lok inflow port. Air bubbles were run slowly through the lumen to remove the

endothelial cells. A 3-4 mm piece of artery was cannulated in a chamber filled with PSS 32

and intravascular pressure was adjusted to 20 mmHg using a pressure servo-controller and a peristaltic pump (Living Systems Instruments, St. Albans, VT) as previously described (Ishiguro et al., 2002). PSS was bubbled with mixed gas (20% O 2 / 5% CO 2 / 75%

N2) to maintain physiological pH (7.35-7.4). Arteries were continuously superfused with

warmed PSS (37°C). Vessel diameters were measured by video edge detection (Living

Systems Instrumentation, St Albans, VT) and WinDaq data acquisition software (Dataq

Instruments, Akron, OH). Following equilibration for 30 minutes, arterial viability was

evaluated by brief exposure to 60 mM KCl (< 2 min; isosmotic replacement of NaCl with

KCl in PSS). Only arteries that demonstrated a constriction represented by decrease in

diameter of 50% or greater were used in subsequent studies. Perfused drugs for all

subsequent studies were dissolved in PSS.

Calculation of artery constriction

Baseline was defined as the pressurized arterial diameter at 80 mmHg with or without

addition of antagonists. Arterial constriction induced by agonists was calculated as a

percentage of decrease in diameter from the baseline {percent of constriction =

[(diameter before adding the drug – average diameter after adding the drug) / diameter

before adding the drug] × 100%}. The fully dilated (passive) arterial diameter was

determined at the end of each experiment by exposing arteries to Ca 2+ -free PSS

containing nondihydropyridines (non-DHP) calcium channel blocker, diltiazem (100

µmol/L) and the adenylyl cyclase activator, forskolin (1 µmol/L).

Statistical analysis

The data are presented as mean ± S.E.M of at least three independent experiments. One-

way analysis of variance followed by Tukey multiple comparison test was used to

compare groups. P values less than 0.05 were considered statistically significant (*P ˂

0.05). Power calculations indicated the power possibility (%) to get statistical significance (*P < 0.05) under certain repetitive experiments times (N).

Reagents

5-HT and nifedipine were purchased from Sigma. Capsaicin, capsazepine, GR55562

dihydrochloride and ritanserin were purchased from Tocris. Sumatriptan, 2, 5-dimethoxy-

4-Iodoamphetamine (DOI hydrochloride), forskolin, chelerythrine and phorbol 12-

myristate 13-acetate (PMA) were purchased from Cayman.

2.3. Results

Inhibition of TRPV1 channel blunted serotonin-induced facial artery constriction.

Using TRPV1–tdTomato–reporter mouse, our lab found there is TRPV1 in smooth muscle cells but no TRPV1 in endothelial cells or perivascular nerves in the facial artery bed (unpublished observations by Wellman, Brayden and Koide et al.). We found that 5-

HT (30 nM) evoked facial artery constriction by the arteriography diameter measurement

(Fig. 2A). TRPV1 is a cation channel that has high permeability for Ca 2+ and increase of intracellular Ca 2+ causes smooth muscle cell contraction. Thus we speculated 5-HT- induced facial artery constriction is via opening of TRPV1 channels. 5-HT (30 nM) constricted facial arteries to 40.98 ± 3.02% (Fig. 2B). Percent of constriction of the facial artery with 30 nM 5-HT was reduced to 24.94 ± 5.71% in the presence of TRPV1 antagonist capsazepine (1 µM) compared to 30 nM 5-HT alone (Fig. 2B, P = 0.038). In other words, 5-HT-induced constriction was reduced by 39.14% by capsazepine (Fig. 2B).

The data indicated that TRPV1 is probably involved in 5-HT-induced facial artery constriction. To examine the role of TRPV1 channel in 5-HT-induced facial artery constriction, concentration response curves of 5-HT (1 nM-1 µM) have been done on

endothelium intact and endothelium denuded facial arteries from wild type mice and

endothelium intact facial arteries from TRPV1-KO mice. To rule out endothelial cells

involvement in 5-HT-induced facial artery constriction, we ran air bubbles slowly

through artery lumen to denude endothelial cells and found 5-HT (1 nM–1 µM)

constrictions between endothelium intact and endothelium denuded arteries are not 35

significantly different (Fig. 2E, P > 0.05). EC 50 of 5-HT in the endothelium denuded artery is 85.6 nM, which is similar with EC 50 of 5-HT in the endothelium intact artery from the wild type mouse (Fig. 2E). These data suggested that endothelial cells are not involved in 5-HT-induced facial artery constriction. The constriction caused by 5-HT (30 nM, 100 nM, 300 nM) in TRPV1-KO mice were significantly decreased compared to the constriction in the facial artery from wild type, suggesting that TRPV1 is involved in 5-

HT-induced facial artery constriction (Fig. 2E, P = 0.004, P = 0.009, P = 0.01 respectively). However, 5-HT (1 µM)-induced constriction was not significantly different between facial arteries from wild type mice and TRPV1-KO mice (Fig. 2E, P = 0.08).

This is probably due to multiple mechanisms participating when high concentration of 5-

HT is applied on the artery. But EC 50 of 5-HT on the facial artery in TRPV1-KO is 202 nM, which is significantly greater than EC 50 (86.8 nM) in the wild type mouse (Fig. 2E).

A B *

percent of 10constriction(%)

0 0.5 1.0 1.5 2.0 2.5 5-HT 5-HT+capZ

D CE

Figure 2. 5-HT constricted mouse facial artery and blocking of TRPV1 channel blunted 5-HT-induced facial artery constriction. A: Diameter recordings of an isolated facial artery (FA) treated with PSS containing serotonin (30 nM), serotonin (30 nM) + capsazepine (1 µM). Diameter changes by serotonin and serotonin + capsazepin were expressed as percentage of diameter change over diameter before drugs were added. B: Summary data obtained from A (mean ± S.E.M) (N = 5). C: Cumulative concentration response curve of 5-HT obtained from endothelial cells intact facial artery in wild type mouse pressurized to 80 mmHg ex vivo. D: Cumulative concentration response curve of 5-HT obtained from endothelial cells intact facial artery in TRPV1 knock out mouse. Arteries were exposed to PSS containing each concentration of 5-HT and diameters were recorded when the diameters were stable. E: Summary data obtained from C, D and 5-HT concentration response curve from endothelial cells denuded facial artery from wild type mouse (mean ± S.E.M) (N = 4). Each artery is from a different animal.*P<0.05

PKC is involved in capsaicin (TRPV1 agonist)-induced facial artery constriction

During inflammation, TRPV1 is reported to be sensitized by protein kinase C (PKC) in dorsal root ganglia (DRG) neurons, which leads to reduction in the threshold of the temperature for TRPV1 activation to body temperature (Li et al., 2014). 5-HT 2 receptor is a G q-protein-coupled receptor that activates phospholipase C (PLC) generating

diacylglycerol (DAG). DAG is a necessary component in activating PKC. Therefore, we

studied the TRPV1 and 5-HT 2 receptor downstream signalling molecule PKC

relationship in artery constriction by studying PKC’s effect on capsaicin (TRPV1

agonist)-induced constriction. Vasoconstriction to capsaicin (100 nM) were examined in

the presence or absence of PKC inhibitor chelerythrine (1 µM) or PKC activator phorbol

12-myristate 13-acetate (PMA, 100 nM) on the facial artery from wild type mice.

Capsaicin (100 nM) constricted the mouse facial artery to 74.3 ± 1.14%. Percent of constriction of the facial artery to capsaicin was reduced to 58.6 ± 4.89% in the presence of PKC inhibitor chelerythrine (1 µM) compared to capsaicin alone 74.3 ± 1.14% (Fig.

3B, P = 0.048). Washing 30 min with PSS restored capsaicin constriction to previous level 72.62 ± 3.59% (P = 0.62). There was a trend that percent of constriction of the facial artery to capsaicin was slightly increased to 81.04 ± 4.93% in the presence of PKC activator PMA (100 nM) compared to capsaicin alone 67.58 ± 3.14% without statistical significance (Fig. 3B, P = 0.22). This lack of effect may be because PMA (100 nM) is a potent PKC activator that saturates PKC activity prior to the addition of capsaicin. 5-HT 2 receptor downstream signaling pathway involves PKC and PKC is involved in capsaicin- 38

induced facial artery constriction. Thus, these data indicated that 5-HT 2 receptor is the potential subtype in serotonin-induced facial artery constriction through opening of

TRPV1 channels.

Figure 3. Capsaicin constricted mouse facial artery and PKC is involved in capsaicin-induced facial artery constriction. A: Diameter recordings of an isolated facial artery (FA) treated with PSS containing capsaicin (100 nM), capsaicin (100 nM) + PKC inhibitor chelerythrine (1 µM), capsaicin (100 nM) + PKC activator PMA (100 nM). Diameter changes by capsaicin, capsaicin + PKC inhibitor chelerythrine and capsaicin + PKC activator PMA were expressed as percentage of diameter change over diameter before drugs were added. B: Summary data obtained from A (mean ± S.E.M) (N = 5). Each artery is from a different animal.*P<0.05 39

5-HT-induced facial artery constriction is mediated mostly by activation of 5-HT 1 and 5-HT 2 receptors

We applied 5-HT 1 or 5-HT 2 receptor antagonists separately or in combination to examine 5-HT (100 nM)-induced facial artery constriction. Applying 5-HT 1 or 5-HT 2 receptor subtype antagonists after serotonin constriction respectively to examine whether

5-HT 1 and 5-HT 2 receptors are involved in 5-HT-induced facial artery constriction. 5-

HT 1 receptor antagonist GR55562 (1 µM) decreased the constriction caused by 100 nM

- 4 5-HT from 29.63 ± 2.15% to 10.08 ± 0.66% (Fig. 4B, P = 1.28 × 10 ). 5-HT 2 receptor antagonist ritanserin (1 µM) decreased 100 nM 5-HT-induced facial artery constriction from 29.5 ± 2.24% to 12.08 ± 0.82% (Fig. 4D, P = 3.35 × 10 - 4). To study how much of

5-HT-induced facial artery constriction is caused by the activation of 5-HT 1 and 5-HT 2 receptors, we preincubated 1 µM GR55562 and 1 µM ritanserin to investigate the 100 nM

5-HT-induced facial artery constriciton. The data indicated that 92.4 % of 5-HT-induced facial artery constriction is caused by the activation of 5-HT 1 and 5-HT 2 receptors (Fig.

4F, P = 0.001). This enabled us to use 5-HT plus 5-HT 1 receptor antagonist GR55562 to activate 5-HT 2 receptor and 5-HT + 5-HT 2 receptor antagonist ritanserin to activate 5-

HT 1 receptor in the facial artery constriction in the subsequent experiments. These data demonstrated that both 5-HT 1 and 5-HT 2 receptors are involved in 5-HT-induced facial artery constriction with different contributions.

B A

C D

E F

Figure 4. Both 5-HT 1 and 5-HT 2 receptor antagonists can partly decrease 5-HT- induced facial artery constriction. 5-HT-induced facial artery constriction is mediated mostly by activation of 5-HT 1 and 5-HT 2 receptors. A: Diameter recordings of an isolated facial artery (FA) treated with PSS containing 5-HT (100 nM), 5-HT (100 nM) + GR55562 (1 µM). Diameter changes by 5-HT and 5-HT + GR55562 were expressed as percentage of diameter change over the diameter before drugs were added. B: Summary data obtained from A (mean ± S.E.M) (N = 4). C: Diameter recordings of an isolated facial artery (FA) treated with PSS contai ning 5-HT (100 nM), 5-HT (100 nM) + ritanserin (1 µM). Diameter changes by 5-HT and 5-HT + ritanserin were expressed as percentage of diameter change over the diameter before drugs were added. D: Summary data obtained from C (mean ± S.E.M) (N = 4). E: Diameter recordings of an isolated facial artery (FA) treated with PSS containing GR55562 (1 µM) + ritanserin (1 µM), GR55562 (1 µM) + ritanserin (1 µM) + 5-HT (100 nM). Diameter changes by GR55562 + ritanserin and GR55562 + ritanserin + 5-HT treatments were expressed as percentage of diameter change over the diameter before drugs were added. F: Summary data obtained from E (mean ± S.E.M) (N = 4). Each artery is from a differ ent animal.*P<0.05

5-HT 2 but not 5-HT 1 receptor is involved in 5-HT-induced facial artery constriction

via TRPV1 activation

We next examined the relative role of TRPV1 channels in facial artery constrictions

induced by 5HT 1 and 5HT 2 receptor activation. From Fig. 3, PKC is involved in

capsaicin-induced constriction. Thus we speculated 5-HT 2 is the potential 5-HT receptor

subtype in 5-HT-induced constriction via opening of TRPV1 channels. We used 5-HT 1 receptor agonist (sumatriptan, 10 µM) or 5-HT 2 receptor agonist (DOI, 1 µM) in the presence or absence TRPV1 antagonist capsazepine to examine TRPV1’s involvement in

5-HT 1 receptor- or 5-HT 2 receptor-induced constriction to further demonstrate that 5-HT 2 but not 5-HT 1 receptor is involved in the TRPV1 mechanism in the facial artery constriciton. Sumatriptan (5-HT 1 receptor agonist, 10 µM) constricted mice facial arteries

to 18.35 ± 1.74%. Percent of constriction of the facial artery with 10 µM sumatriptan was 42

not reduced significantly in the presence of TRPV1 antagonist 1 µM capsazepine (14.1 ±

1.52%) compared to sumatriptan alone (Fig. 5B, P = 0.9). TRPV1 is not involved in sumatriptan-induced facial artery constriction. To further elucidate the role of TRPV1 in

5-HT receptor subtype-induced constriction in the facial artery, vasoconstriction to 100 nM 5-HT + 1 µM GR55562 (activating 5-HT 2 receptor) was examined in the absence and presence of TRPV1 antagonist capsazepine (1 µM) using facial arteries obtained from wild type mice. Similarly, we used 5-HT (100 nM) + 1 µM ritanserin (5-HT 2 receptor antagonist) to activate 5-HT 1 receptor in the facial artery as control. The results

demonstrated that 5-HT (100 nM) + GR55562 (1 µM) (5-HT 2 receptor activation) caused

12.63 ± 0.41% constriction and 1 µM capsazepine decreased this constriction to 6.83 ±

0.46% significantly (Fig. 5D, P = 0.002). Washing 30 min with PSS restored 5-HT +

GR55562-induced constriction to previous level 14.68 ± 1.33% (P = 0.25). However, 1

µM capsazepine cannot blunt constriction induced by 5-HT (100 nM) + ritanserin (1 µM)

(5-HT 1 receptor activation) in the facial artery from the wild type mouse significantly

(Fig. 5F, P = 0.77) indicating that TRPV1 is not involved in 5-HT 1 receptor activation- induced facial artery constriction. TRPV1 inhibition by capsazepine can significantly blunt 5-HT-induced 5-HT 2 receptor-mediated facial artery constriction but not 5-HT 1 receptor-mediated facial artery constriction. These data demonstrated that 5-HT 2 receptor

but not 5-HT 1 receptor is involved in 5-HT-induced facial artery constriction through opening of TRPV1 channels.

A B

C D

E F

Figure 5. 5-HT 2 but not 5-HT 1 receptor is involved in 5-HT-induced facial artery constriction via TRPV1 activation. A: Diameter recordings of an isolated facial artery (FA) treated with PSS containing sumatriptan (10 µM), sumatriptan (10 µM) + capsazepine (1 µM). Diameter changes by sumatriptan and sumatriptan + capsazepine were expressed as percentage of diameter change over the diameter before drugs were added. B: Summary data obtained from A (mean ± S.E.M) (N = 4). C: Diameter recordings of an isolated facial artery (FA) treated with PSS containing 5-HT (100 nM) + GR55562 (1 µM), 5-HT (100 nM) + GR55562 (1 µM) + capsazepine (1 µM). Diameter changes by 5-HT + GR55562 and 5-HT + GR55562 + capsazepine were expressed as percentage of diameter change over the diameter before drugs were added. D: Summary data obtained from C (mean ± S.E.M) (N = 4). E: Diameter recordings of an isolated facial artery (FA) treated with PSS containing 5-HT (100 nM) + ritanserin (1 µM), 5-HT (100 nM) + ritanserin (1 µM) + capsazepine (1 µM). Diameter changes by 5- HT + ritanserin and 5-HT + ritanserin + capsazepine were expressed as percentage of diameter change over the diameter before drugs were added. F: Summary data obtained from E (mean ± S.E.M) (N = 4). Each artery is from a different animal.*P<0.05

Possible PKC involvement in 5-HT-induced facial artery constriction.

PKC is the downstream molecule for 5-HT 2 receptor activation and may be involved in

5-HT-induced facial artery constriction if 5-HT binds with 5-HT 2 receptor. There was a trend that percent of constriction of the facial artery with 30 nM 5-HT was decreased to

24.56 ± 4.81% in the presence of PKC inhibitor chelerythrine (1 µM) compared to 5-HT alone (36.46 ± 2.70%) without statistical significance (Fig. 6B, P = 0.32). Washing 30 min with PSS restored 5-HT-induced constriction to previous level 31.06 ± 5.07% (P =

0.55). Fig. 6B showed a trend that percent of constriction in the facial artery with 30 nM

5-HT was increased to 38.3 ± 6.89% in the presence of PKC activator PMA (100 nM) compared to 5-HT alone (31.06 ± 5.07%) without statistical significance (Fig. 6B P =

0.41). This may be due to the fact that 5-HT has multiple intracellular second messengers that mediate artery constriction besides PKC. PKC’s contribution is not strong enough to

influence 30 nM 5-HT constriction and PKC’s role is taken over by other molecules after

PKC activity is changed. Since PKC (activated upon 5-HT 2 receptor activation) activator and inhibitor had the trend of increasing and decreasing 5-HT-induced facial artery constriction respectively, we conclude that 5-HT 2 receptor activation may be involved in

5-HT-induced facial artery constriction.

Fig ure 6. PKC is probably involved in 5 -HT -induced facial artery constriction. A: Diameter recordings of an isolated facial artery (FA) treated with PSS containing 5-HT (30 nM), 5-HT (30 nM) + chelerythrine (1 µM), 5-HT (30 nM) + PMA (100 nM). Diameter changes by 5-HT, 5-HT + chelerythrine and 5-HT + PMA were expressed as percentage of diameter change over the diameter before drugs were added. B: Summary data obtained from A (mean ± S.E.M) (N = 5). Each artery is from a different animal.*P<0.05

L-type-voltage-dependent Ca 2+ channel (L-VDCC) is involved in 5-HT-induced facial artery constriction.

Since TRPV1 is a non selective cation channel that has high permeability for Ca 2+ , we studied L-type-voltage-dependent Ca 2+ channel involvement in 5-HT-induced facial artery constriction to provide further evidence that Ca 2+ entry channel, for example

TRPV1, is involved in 5-HT constriction. 5-HT (30 nM) constricted mouse facial artery to 32.12 ± 2.68% (Fig. 7B). The L-type-VDCC inhibitor nifedipine (1 µM) partly decreased facial artery constriction caused by 30 nM serotonin from 32.12 ± 2.68% to

12.36 ± 2.57% significantly (Fig. 7B, P = 0.001). In other words, 5-HT-induced constriction was decreased by 61.5% by nifedipine. Percent of constriction in the facial artery with 30 nM 5-HT was reduced significantly to 8.26 ± 1.19% in the presence of

TRPV1 antagonist capsazepine (1 µM) and nifedipine (1 µM) compared to 30 nM 5-HT

alone (Fig. 7B, P = 4.36 × 10 -5). These data suggested that L-type-voltage-dependent

Ca 2+ channel (L-VDCC) is involved in 5-HT-induced facial artery constriction. However, constriction in the presence of 5-HT + capsazepine + nifedipine (8.26 ± 1.19%) was not significantly reduced compared with 5-HT + nifedipine constriction (12.36 ± 2.57%) (Fig.

7B, P = 0.75), although we saw a greater reduction (76.3%) of 5-HT constriction by

nifedipine + capsazepine compared with by nifedipine alone (61.5%) (Fig. 7B). This may be due to the possibility that after L-VDCCs are blocked, the contribution of TRPV1 is masked by other cellular pathways. In other words, TRPV1’s involvement in 5-HT- induced constriction is weakened when L-VDCC is blocked.

A B

Fig ure 7. Blocking of L -VDCC blunted 5 -HT -induced facial artery constriction. A: Diameter recordings of an isolated facial artery (FA) treated with PSS containing serotonin (30 nM), serotonin (30 nM) + nifedipine (1 µM) and serotonin (30 nM) + nifedipine (1 µM) + capsazepine (1 µM). Diameter changes by serotonin, serotonin + nifedipine and serotonin + nifedipine + capsazepine were expressed as percentage of diameter change over the diameter before drugs were added. B: Summary data obtained from A (mean ± S.E.M) (N = 5). Each artery is from a different animal.*P<0.05

2.4. Conclusions and Discussion

Functional TRPV1 was expressed in the mouse facial artery and activation of TRPV1 by capsaicin caused the artery constriction (unpublished observations by the Wellman laboratory). In the present study, 5-HT, an endogenous vasoactive monoamine, was found to cause the mouse facial artery constriction. In the peripheral system, 5-HT is mostly stored in platelets (Mercado and Kilic, 2010). When activated platelets bind with a clot, platelets release 5-HT and 5-HT serves as a vasoconstrictor and helps to regulate hemostasis and blood clotting (Ziu et al., 2012). Tight regulation of cephalic blood circulation is critical under normal physiological conditions, and dysregulation of blood flow to the head occurs in pathophysiological situations such as stroke and migraine headache. External carotid artery mainly branches into the superficial temporal artery, maxillary artery and facial artery. The facial artery (external maxillary artery in older texts) supplies blood of the superficial face which is important in the head hemodynamic regulation. Additionally, if arteries in external carotid artery territory are constricted, the blood will be shunted to the internal carotid artery which has no TRPV1 expression

(unpublished observations by the Wellman laboratory) providing intracranial blood. The increase of intracranial blood is beneficial to many CNS diseases that are due to shortage of brain blood supply. Thus studying how TRPV1 channel activation-induced artery constriction is regulated by endogenous substance such as 5-HT in the facial artery is important in understanding TRPV1’s vasoactive effect in the vasculature. Our findings suggested that inhibition of the TRPV1 channel blunted serotonin-induced mouse facial artery constriction. We found PKC was involved in TRPV1 activation-induced facial 49

artery constriction. 5-HT-induced facial artery constriction was caused mainly by 5-HT 1 and 5-HT 2 receptors activation. Capsazepine can not blunt sumatriptan (5-HT 1 receptor

agonist)-induced facial artery constriction indicating that TRPV1 is not involved in

sumatriptan-induced facial artery constriction. We used 5-HT + 5-HT 1 receptor antagonist or 5-HT 2 receptor antagonist to activate 5-HT 2 receptor or 5-HT 1 receptor

respectively. Capsazepine can blunt constriction induced by 5-HT + 5-HT 1 receptor antagonist but not constriction induced by 5-HT + 5-HT 2 receptor antagonist. DOI (5-

HT 2 receptor agonist) did not constrict the facial artery (data not shown). DOI is a partial

agonist for 5-HT 2A receptor (EC 50 = 0.03 µM), which constricted the endothelial denuded mouse aorta, dog internal carotid artery and rat mesenteric artery (Centurion et al., 2001;

Fernandez et al., 2000; McKune and Watts, 2001). DOI has 30 times higher affinity for

5-HT (2A) receptor than 5-HT (2B) receptor (Nelson et al., 1999). 5-HT (2B) receptors agonists produced concentration-dependent constriction of mouse aorta (Nelson et al.,

2012). Thus the facial artery constriction is possibly through both 5-HT (2A) and 5-HT

(2B) receptors activation and 5-HT (2B) receptor contributes more to the constriction than 5-HT (2A) receptor. Therefore 1 µM DOI is not potent enough to cause constriciton in the facial artery. This work represents the first study examining TRPV1 potential involvement in 5-HT-induced 5-HT 2 receptor-mediated facial artery constriction.

Capsaicin (100 nM) constricted the facial artery from C57BL/6J mouse indicating

functional TRPV1 channel is expressed in the facial artery (Fig. 3A). TRPV1 is a cation

channel on the cell membrane, which has a high permeability for Ca 2+ . Ca 2+ -calmodulin-

dependent activation of myosin light chain kinase (MLCK) is a requisite step for 50

myogenic constriction (Raina et al., 2009). 5-HT constricts arteries by inhibiting Kv channels via 5-HT 2A receptor and Src tyrosine kinase pathway (Sung et al., 2013). Kv

channel inhibition induces cell membrane depolarization (Waldron and Cole, 1999).

TRPV1 channel opening induces Ca 2+ influx, which leads to membrane depolarization as

well (Boillat et al., 2014). Since both 5-HT and capsaicin constricted the mouse facial

artery, 5-HT constriction on the facial artery is probably via opening of TRPV1 channels.

During inflammation, TRPV1 is reported to be sensitized by protein kinase C (PKC) in

dorsal root ganglia (DRG) neurons, which leads to reduction in the threshold of the

temperature for TRPV1 activation to body temperature (Sugimoto et al., 2013). PKC

activation results in direct phosphorylation of TRPV1(Li et al., 2014). Our lab found

PKC is involved in capsaicin-induced mouse middle meningeal artery constriction and is

a necessary component in sustaining capsaicin constriction on the artery (unpublished

observations by the Wellman laboratory). By using in vitro phosphorylation and protein

sequencing techniques, people identified several potential PKC phosphorylation sites on

TRPV1 intracellular domains (Numazaki et al., 2002). Functional studies suggested that

activation of TRPV1 by phorbol esters is phosphorylation-independent but modulated by

PKC-mediated phosphorylation (Bhave et al., 2003). 5-HT exerts vasoconstrictive and

2+ mitogenic effects and both of them are dependent on an increase of cytosolic [Ca ]i in

pulmonary artery smooth muscle cells (PASMCs) (Goyal et al., 2011). Stimulation of 5-

HT receptor may activate a novel type of choleratoxin-sensitive G-protein and

subsequent PKC, and the phosphorylation of voltage-dependent Ca 2+ channels by PKC

may result in the increase in Ca 2+ entry and subsequent Ca 2+ -activated K + current

(Kawasaki et al., 2007). Thus, we supposed PKC links the 5-HT receptor and the TRPV1 51

channel, which leads to our hypothesis of TRPV1’s involvement in 5-HT-induced constriction. Although we found PKC inhibitor chelerythrine blunted capsaicin-induced constriction, However, we can only see the trend of PKC activator (PMA)’s potentiating effect on capsaicin-induced constriction without statistical significance. We used power calculation to calculate the possibility of getting P < 0.05 significance and found with N

= 5 (repetitive experiments times), the possibility of getting statistical significance is 70% between capsaicin and capsaicin + PMA. Thus, to increase the power of the statistical analysis, the observed trend may turn into a significant difference (P ˂ 0.05). PKC is the downstream signalling molecule of 5-HT 2 receptor activation (Blank et al., 1996). 5-HT

augmented voltage-dependent Ca 2+ channels currents in Aplysia ganglia (Kawasaki et al.,

2007), which enables us to propose the TRPV1 involvement in 5-HT-induced facial artery constriction acting as a Ca 2+ entry channel. Additionally, there is evidence demonstrating direct correlation between 5-HT receptor and TRPV1 channel. Facilitation of TRPV1 by metabotropic 5-HT receptor activation may contribute to hypersensitivity of primary afferent neurons in Irritable Bowel Syndrome patients (Sugiuar et al., 2004).

Endothelial cells play a significant role in vascular tone, and have been demonstrated to be involved in many diseases (Veerasamy et al., 2015). Serotonin stimulates release of vasodilator substances such as nitric oxide by activating 5HT 1B and 5-HT 2B receptors on

endothelial cells to cause artery dilation (Ishida et al., 1998). We used a series of different

5-HT concentrations (1 nM-1 µM) to examine the constrictions in endothelial cells

denuded and endothelial cells intact facial arteries. There was no significant difference

between those two groups, indicating that endothelial cells are not involved in 5-HT- 52

induced facial artery constriction. Our lab used TRPV1-tdTomato reporter mice to

examine the TRPV1 channel of the facial artery and found that TRPV1 is only expressed

in smooth muscle cells of the facial artery but not in endothelial cells or perivascular

nerves (unpublished observations by the Wellman laboratory). However, capsaicin is

reported to increase nitric oxide (NO) metabolites production in human vein endothelial

cells (ECs) (Lo et al., 2003). In vivo and in vitro experimental evidence demonstrated

that long-term activation of TRPV1 can actually increase the phosphorylated levels of

protein kinase A (PKA) and endothelial NO synthase (eNOS) in mesenteric arteries and

plasma levels of NO metabolites. Phosphorylated eNOS augmented endothelium-

dependent relaxation and lower arterial pressure in hypertensive rats (Yang et al., 2010).

Vasodilation induced by electrical field stimulation (EFS) of perivascular sensory nerves

is mediated by the TRPV1 via releasing of sensory neurotransmitters (CGRP) in wild

type mice (Wang et al., 2006). Thus TRPV1-mediated artery dilation is mainly mediated

by two pathways: Activation of TRPV1 in endothelial cells causing eNOS activation and

NO increase, and activation of TRPV1 in perivascular nerves leading to CGRP release.

Both NO and CGRP dilate the artery. In the facial artery, our data indicate that TRPV1

activation is associated with vasoconstriction rather than vasodilation.

5-HT-evoked constriction of human coronary arteries is mostly mediated via activation

of both 5-HT 1B and 5-HT 2A receptors (Nilsson et al., 1999). Sumatriptan or eletriptan produced vasocontriction in the common carotid artery (CCA) by stimulating 5HT 1B receptors in rabbits (Akin et al., 2002). However, ketanserin (5-HT 2A receptor antagonist) inhibited 5-HT-induced constriction of canine pulmonary arteries, indicating that 5-HT 2A 53

receptor activation is important to arterial constriction (Wilson et al., 2005). Thus, we focused on 5-HT 1 and 5-HT 2 receptor subtypes when studying 5-HT-induced facial artery

constriction. Studies showed that 80-86% of the maximum 5-HT-evoked constriction of

the human temporal artery is mediated through 5-HT 2A receptors, the remainder through

5-HT 1-like-receptors, regardless of whether or not endothelium is functional (Verheggen

et al., 1996). However, our data indicated that 5-HT 1 and 5-HT 2 receptor play an even role (around 34 % and 41 % respectively) in 5-HT-induced facial artery constriction.

Based on the different ratios of 5-HT 1 and 5-HT 2 receptor involvement in 5-HT-induced constriction between the temporal artery and facial artery, thus, although temporal artery and facial artery are all branches from the external carotid artery, 5-HT 1 and 5-HT 2 receptor’s involvement in 5-HT-induced arteries constriction is different. Our data suggested that 5-HT-induced facial artery constriction is mostly mediated by 5-HT 1 and

5-HT 2 receptors activation. Either 5-HT 1 or 5-HT 2 antagonist can partly decrease constriction caused by 100 nM 5-HT (Fig. 4B, D). By preincubation of both 5-HT 1 and 5-

HT 2 receptors antagonists, 5-HT constriction on the facial artery was mostly reversed although there was still little constriction that is probably caused by other mechanisms

(Fig. 4F). Some studies demonstrated that there are other receptors involved in 5-HT- induced artery cosntriction. At low concentrations of 5-HT (≤ 10 -5 M), 5-HT-induced dog

mesenteric artery constriction is mediated by 5-HT 2 receptors, but with higher

concentration, it is mediated by α-adrenergic receptors (Shimamoto et al., 1994). This indicates that different 5-HT concentrations can act on different receptors besides 5-HT receptors, which makes studying 5-HT vasoactive role on the artery more complicated.

Downstream signalling molecules that are involved in 5-HT-induced facial artery 54

constriction through 5-HT 1 and 5-HT 2 receptor activation have not been studied either. 5-

HT constricts rat mesenteric artery by inhibiting 4-aminopyridine-sensitive Kv channels

via the 5-HT (2A) receptor (Sung et al., 2013). Kv channel inhibition leads to smooth

muscle cell membrane depolarization and L-VDCC opening (Sung et al., 2013). L-VDCC

is a major Ca 2+ entry channel to induce smooth muscle contraction (Amberg and Navedo,

2013). We found blocking of TRPV1 channels and L-VDCC blunted 5-HT-induced facial

artery constriction, indicating TRPV1 may represent an additional Ca 2+ entry pathway.

PKC is involved in capsaicin-induced facial artery constriction (Fig. 3). Therefore, we examined PKC’s involvement in 5-HT-induced constriction to determine potential 5-HT receptor subtype that is involved in 5-HT-induced constriction via opening of TRPV1 channels. However, we can only see the trend of PKC’s involvement in 5-HT-induced constriction without statistical significance. We used power calculation to calculate the possibility of getting P < 0.05 significance and found with N = 5 (repetitive experiments times), the possibility of getting statistical significance are 70% between 5-HT and 5-HT

+ chelerythrine, 40% between 5-HT and 5-HT + PMA. If we increase the sample number, the trend may turn to significance (P ˂ 0.05). Our finding suggests serotonin binds with

2+ 5-HT 2 receptor leading to PKC activation. PKC activates TRPV1 channel causing Ca entry leading to smooth muscle cell contraction in the facial artery. Intracellular Ca 2+ and

Na + increase causes cell membrane depolarization and subsequently opening of L-VDCC

channels that leads to more constriction. However, capsazepine cannot decrease 5-HT +

nifedipine induced constriction significantly (Fig. 7). There was a trend of capsazepine’s

reduction on 5-HT + nifedipine constriction because nifedipine blunted 5-HT constriction 55

by 61.5% and capsazepine + nifedipine blunted 5-HT constriction by 76.3% (Fig. 7).

Using power calculation, we found with 5 repetitive experiments, power possiblity to get statistical significance (P < 0.05) is 50%. Thus more repetitive experiments are required to get statistical significance. After both L-VDCC and TRPV1 channels were blocked, there was still around 8% 5-HT constriction left. Therefore, besides Kv channel inhibition,

L-VDCC activation and TRPV1 activation, there are other signalling pathways that are involved in 5-HT-induced artery constriction. For example, by activating rho/rho kinase

2+ pathway, c-Src pathway or releasing of IP 3 to cause sarcoplasmic reticulum Ca release,

5-HT causes arteries constriction. Serotonin-induced contrictions are mediated by 5HT 2A receptors on vascular smooth muscle, which activates the rho/rho kinase pathway to regulate phosphorylation of myosin light chain phosphatase and myosin light chain

(Dreja et al., 2002; Nuno et al., 2007). Inhibition of c-Src tyrosine kinase activity prior to constriction resulted in approximately 90 - 99% inhibition of constrictions induced by 5-

HT or 5-HT (2) recptor agonist in rats aortic arteries (Lu et al., 2008). 5-HT-mediated constriction in canine pulmonary arteries is reliant on release of IP 3 but not ryanodine- sensitive Ca 2+ stores and cytosolic [Ca 2+ ] increases were partially inhibited by nisoldipine,

a voltage-dependent Ca 2+ channel blocker (Wilson et al., 2005). Ca 2+ entry upon exposure

to 5-HT through L-VDCC versus TRPV1 channels will require further studies. But in

addition to L-VDCC opening, 5-HT 1 receptor activation, and 5-HT 2 receptor indirectly

activating TRPV1, whether there are other mechanisms (for example by activating other

Ca 2+ entry channels) are involved in 5-HT-induced facial artery constriction will require further studies.

The proposed pathway showing the contribution of TRPV1 to 5-HT-induced mouse facial artery constriction is described as below (Fig. 8).

Figure 8. : The Scheme of TRPV1 involvement in 5-HT-induced facial artery constriction.

2.5. Future directions

In the present study, we found activation of TRPV1 channel contributes to 5-HT-

induced 5-HT 2 receptor-mediated mouse facial artery constriction. Our lab used TRPV1-

tdTomato mouse demonstrated TRPV1 expression in external carotid artery territory.

However, we did not find TRPV1 expression in internal carotid artery including cerebral

artery or common carotid artery (unpublished observations by Wellman laboratory).

Since TRPV1 channel is a cation channel permeable to Ca 2+ and Na + causing cell membrane depolarization (Boillat et al., 2014). Thus the TRPV1 channel opening contributes to two pathways that lead to smooth muscle contraction. One is by increasing cytoplasmic Ca 2+ , which binds with calmodulin to activate myosin light chain kinase

(Webb, 2003); the other one is the cation entry inducing cell membrane depolarization and L-VDCC opening to cause more Ca 2+ entry and constriction. Since the cerebral artery

is TRPV1 negative, it has less cation influx compared with the facial artery that has

TRPV1 expression when being exposed to vasoactive G-protein-coupled receptors

agonists for instance 5-HT. 5-HT constricts cerebral arteries via activation of 5-HT (1B)

and 5-HT (2A) receptors (Kovacs et al., 2012). Further study is required to compare

concentration response curves of 5-HT on the facial artery and cerebral artery. Without

TRPV1 channel involvement, cerebral artery constriction to 5-HT will be smaller than

the facial artery constriction.

Using G-CaMP5 (green fluorescent protein, Ca 2+ -sensitive) mice, we examined Ca 2+ image in the facial artery with addition of α-adrenergic receptor (G q–protein–coupled 58

receptor, GPCR) agonist phenylephrine and capsazepine. We found capsazepine significantly blunted phenylephrine-induced Ca 2+ increase in the facial artery and caused artery dilation after phenylephrine-induced constriction (unpublished observations by the

Wellman laboratory). It suggests the GPCR–Gq-PLC–PKC–TRPV1 signaling in

mediating phenylephrine-induced facial artery constriction. Similarly, since 5-HT (2)

receptor is also a G q–protein–coupled receptor, blocking of TRPV1 channel may reduce

5-HT-induced Ca 2+ increase in the facial artery. Using G-CaMP5 mice, we will apply 5-

HT and capsazepine to examine TRPV1’s involvement in 5-HT-induced Ca 2+ increase and constriction in the facial artery.

Thromboxane, angiotensin II and endothelin are endogenous vasoconstrictors that bind with GPCR. Thromboxane is an endogenous vasoconstrictor and induces rabbit pulmonary artery constriction and PKC inhibitors blunt thromboxane-induced constriction (Murtha et al., 1999). Thromboxane A2 is one of the major thromboxanes.

The thromboxane A2 receptor is a G q–protein–coupled receptor that activates PLC-β which mediates the conversion of phosphatidylinositol 4,5-bisphosphate (PIP 2) to IP 3 and

DAG. The second messenger IP 3 serves to release intracellular calcium stores and DAG

activates protein kinase C (Ting et al., 2012). PKC can activate TRPV1 causing Ca 2+ entry. Thus, we suppose that the activation of TRPV1 constributes to thromboxane- induced facial artery constriction. Angiotensin II (AngII), the major bioactive peptide of the renin–angiotensin system, plays a critical role in controlling cardiovascular homoeostasis (Li et al., 2012). AngII type-1 receptor (AT 1) has been shown to be predominantly expressed in cardiovascular cells, such as vascular smooth muscle cells 59

mediating artery constriction, for example, skeletal muscle artery, renal artery, coronary artery, aortic artery and cerebral artery, etc (Czikora et al., 2015; Guo et al., 2013; Huang et al., 1994; Li et al., 2016). The AT 1 receptor interacts with multiple heterotrimeric G–

proteins including G q, which activates PKC (Higuchi et al., 2007) which potentially leads

to TRPV1 activation. Thus, TRPV1 is probably involved in AngII-induced artery

constriction. Similarly, endothelin constricts a variety of arteries, such as, pulmonary

artery, coronary artery, cerebral artery and mesenteric artery (Assenzio et al., 2015;

Compeer et al., 2013; Liang et al., 2012; Skovsted et al., 2015). In human coronary

microvasculature, the constriction response to endothelin-1 is in part through the

activation of endothelin-A receptor and PKC α (Feng et al., 2010). Endothelin, acting

through endothelin-B receptors, induces mechanical hypernociception in the rat hindpaw

via cAMP formation and activation of the PKC-dependent phosphorylation cascade (da

Cunha et al., 2004). Thus endothelin also has the potential of sensitizing TRPV1 through

PKC activation. We propose that activation of TRPV1 contributes to endothelin-induced

artery constriction.

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