Regulation of Middle Meningeal Artery Diameter by Pacap and ATP-Sensitive Potassium Channels Arsalan Urrab Syed University of Vermont

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Graduate College Dissertations and Theses Dissertations and Theses

2016 Regulation Of Middle Meningeal Artery Diameter by Pacap and ATP-Sensitive Potassium Channels Arsalan Urrab Syed University of Vermont

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This Dissertation 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]. REGULATION OF MIDDLE MENINGEAL ARTERY DIAMETER BY PACAP AND ATP-SENSITIVE POTASSIUM CHANNELS

A Dissertation Presented by Arsalan Syed to

The Faculty of the Graduate College of The University of Vermont

In Partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy Specializing in Pharmacology

January, 2016

Defense Date: October 2, 2015 Dissertation Examination Committee:

George C. Wellman Ph.D. Advisor Margaret A. Vizzard, Ph.D., Chairperson Mark T. Nelson, Ph.D. Joseph E. Brayden, Ph.D. Victor May, Ph.D. Cynthia J. Forehand, Dean of the Graduate College

ABSTRACT Migraine is one of the most prevalent contributors to the global burden of mental and neurological disorders. It is a complex episodic condition that presents as intense recurrent unilateral headaches lasting hours to days that can be accompanied by nausea, photophobia, phonophobia and other neurological symptoms. The causes of migraine appear multifactorial and are not fully understood. However, activation of the trigeminovascular system and sphenopalatine parasympathetic neurons and the resulting vasodilation of meningeal arteries have been associated with the development of migraine pain. Recently, the neurotransmitter and neurotrophic peptide pituitary adenylate cyclase activating polypeptide (PACAP) has been implicated in this migraine headache pathway. The effects of PACAP parallel those of other migraine inducing agents and notably PACAP induces vasodilation of the MMA concurrent with the genesis of migraine headache when administered to human subjects. The mechanisms by which PACAP induces dilation are presently unclear. The objective of this present work was to elucidate the signaling pathways linking PACAP to MMA dilation. To achieve this objective, we developed an ex vivo approach to study isolated MMA at physiologically relevant intravascular pressure. Using this preparation we found that PACAP dilates MMA at picomolar concentrations via PAC1 receptors. Further, in MMA, PACAP- induced dilation is mediated exclusively though activation of KATP channels. While investigating the mechanisms of PACAP-induced dilation of MMA we discovered that basal KATP channel activity influences MMA diameter.

Inhibition of KATP channels with glibenclamide or PNU37883 at physiological intravascular pressure resulted in a vasoconstriction of ≈ 20 %. Also consistent with basal KATP activity, glibenclamide induced a membrane potential depolarization of ≈ 14 mV. Further, in MMA loaded with the ratiometric Ca2+ indicator, Fura-2-AM, glibenclamide- induced MMA constriction was correlated with a simultaneous increase in the ratio of 340 nm/380 nm excited fura-2 fluorescence, consistent with an increase in intracellular Ca2+.

Vascular smooth muscle KATP channels can be phosphorylated and activated by PKA, resulting in membrane potential hyperpolarization. KT5720, a PKA inhibitor, induced a constriction in MMA similar to that of glibenclamide (≈ 25 %). Additional treatment with glibenclamide did not induce further constriction suggesting that PKA activity may underlie tonic KATP channel activation. Together these results suggest that tonic PKA activity underlies basal KATP channel activity and together play a key role in regulation of MMA diameter. In summary, results presented in this dissertation suggest that picomolar PACAP- induced dilation of MMA is via activation of the PAC1-Hop1 receptor splice variant and KATP channel activation. Furthermore, KATP channels are also involved in tonic regulation of MMA diameter due to basal PKA activity. These unique features of the MMA provide additional insight into potential therapeutic targets in the development of treatments for migraine.

CITATIONS

Material from this dissertation has been published in the following form:

Syed, A.U., Koide, M., Braas, K.M., May, V., Wellman, G.C.. (2012) Pituitary adenylate cyclase-activating polypeptide (PACAP) potently dilates middle meningeal arteries: implications for migraine. Journal of Molecular Neuroscience, 48(3):574.

Koide, M., Syed, A.U., Brass, K.M., May, V., Wellman, G.C.. (2014) Pituitary adenylate cyclase-activating polypeptide (PACAP) dilates cerebellar arteries through activation of 2+- + large-conductance Ca activated (BK) and ATP-sensitive (KATP) K channels. Journal of Molecular Neuroscience, April; 18

AND/OR

Material from this dissertation has been accepted for publication in PACAP handbook on (06/30/2015) in the following form:

Syed, A.U., Koide, M., May, V., Wellman, G.C.. (2016) PACAP regulation of vascular tone: differential mechanism among vascular beds. Book chapter, Springer.

AND/OR

Material from this dissertation will be submitted for publication to European Journal of Physiology on (11/30/2015) in the following form:

Syed, A.U., Koide, M., Brayden, J.E., Wellman, G.C.. Regulation of middle meningeal artery diameter (MMA) by ATP-sensitive potassium (KATP) channels. In preparation.

ACKNOWLEDGEMENTS

The function of education is to teach one to think intensively and to think critically.

Intelligence plus character - that is the goal of true education. Martin Luther King, Jr.

I would like to thank Dr. George Wellman, my mentor and a great scientist. George has always been a remarkable presence throughout my Ph.D. career and words cannot express my deep gratitude for his guidance. George has instill in me a question driven approach toward science. My past and future successes will always be attributed to his mentorship.

I will always be thankful to my committee members Dr. Mark Nelson, Dr. Joseph

Brayden, Dr. Margaret Vizzard, and Dr. Victor May for their excellent and continued support. To quote Patricia Cross “The task of the excellent teacher is to stimulate

‘apparently ordinary’ people to unusual effort. The tough problem is not in identifying winners. It is in making winners out of ordinary people”. I am truly grateful to have these outstanding scientists guiding me in my career.

My growth as a scientist would not be complete without the support of wonderful colleagues. Members of the Wellman lab, Nelson lab, Brayden lab and the Pharmacology department administrative staff have been instrumental in my success. They have always made me feel part of the pharmacology department family. Meeting each and every one of them has enriched my life experience at University of Vermont. I would also like to take this opportunity to thank Dr. Masayo Koide for all the help with my experiments and great scientific discussion. iii

I’d like to thank my parents Ehsan Urrab Syed and Farhat Ehsan for their continuous support and keeping me in their prayers. My parents have always encouraged me to pursue my dreams and have instilled in me the value of hard work.

Lastly, I would like to dedicate this dissertation to Dr. Neilia Gracias. My best friend and life partner. Neilia has always believed in my abilities even though there have been times where I failed to believe in myself. She has been a constant positive influence in my life.

Neilia your support, your friendship, your loyalty, and strength has always been the pillars I relied on. I am blessed to have you by my side. Thank you!

TABLE OF CONTENT

CITATIONS ...... ii ACKNOWLEDGEMENTS ...... iii LIST OF TABLES ...... viii LIST OF FIGURES ...... ix CHAPTER 1: LITERATURE REVIEW ...... 1 1.1 Introduction ...... 1 1.2 Section 1 ...... 4 1.21 Vascular components of migraine headache: meninges and the MMA ...... 4 1.22 Neural inputs to MMA ...... 5 1.23 PACAP and its receptors ...... 6 1.24 PACAP and nonvascular functions ...... 8 1.25 PACAP and vascular smooth muscle ...... 9 1.26 PACAP and disease ...... 12 1.3 Section 2 ...... 13

1.31 Role of potassium channels in regulation of arterial tone ...... 13

1.32 Discovery of KATP channels ...... 14

1.33 Structure of KATP channels ...... 15

1.34 Modulation of KATP channel gating ...... 16 1.35 Membrane trafficking ...... 17

1.36 Physiological relevance of KATP channels in pancreas, heart and brain ...... 17

1.37 Physiological relevance of KATP channels in vascular smooth muscle ...... 19

1.38 KATP channels and Pathophysiology ...... 22 1.39 Research Objectives of the Present Work ...... 23 1.4 Figures...... 25 1.5 Reference List ...... 29 CHAPTER 2: JOURNAL ARTICLE ...... 39 2.1 Abstract ...... 40 v

2.2 Introduction ...... 40 2.3 Methods...... 43 2.4 Results ...... 47 2.5 Discussion ...... 51 2.6 Reference List ...... 57 2.7 Figures...... 60 CHAPTER 3: JOURNAL ARTICLE ...... 67 3.1 Abstract ...... 68 3.2 Introduction ...... 69 3.3 Methods...... 72 3.4 Results ...... 76 3.5 Discussion ...... 80 3.6 Reference List ...... 85 3.7 Figures...... 90 CHAPTER 4: JOURNAL ARTICLE ...... 94 4.1 Abstract ...... 95 4.2 Introduction ...... 95 4.3 Methods...... 96 4.4 Results ...... 96 4.5 Discussion ...... 100 4.6 Reference List ...... 102 4.7 Figures...... 105 4.8 Supplemental Methods ...... 109 4.9 Reference List ...... 113 4.10 Supplemental Figures ...... 114 CHAPTER 5: DISCUSSION AND FUTURE DIRECTIONS ...... 117

5.1 Regulation of membrane potential and vascular diameter by KATP channels ...... 119 5.2 Unpublished observations ...... 122 5.3 Clinical relevance ...... 124 5.4 Conclusion ...... 125 5.5 Reference List ...... 126 vi

COMPREHENSIVE BIBLIOGRAPHY ...... 130 APPENDIX 1: BOOK CHAPTER ...... 152 1.1 Abstract ...... 153 1.2 Introduction ...... 155 1.21 PACAP and PACAP receptors in cerebral and cranial vessels ...... 157 1.22 PACAP mediates middle meningeal artery (MMA) vasodilation via PAC1 receptor activation of KATP channels ...... 159 1.23 PACAP and CGRP demonstrate differential potencies in stimulating MMA vasodilation ...... 162 1.24 PACAP mediates cerebellar artery vasodilation via different receptors and channels ...... 163 1.25 Alternate mechanism of PACAP-induced vasodilation ...... 164 1.26 Functional significance of PACAP regulation and pathophysiology ...... 165 1.3 Conclusion ...... 167 1.4 Reference List ...... 168 1.5 Figures...... 174 APPENDIX 2: UNPUBLISHED OBSERVATION ...... 180 2.1 Introduction ...... 180 2.2 Methods...... 181 2.3 Results ...... 183 2.4 Significance and future directions ...... 185 2.5 Figures...... 188 2.6 Reference List ...... 192

vii

LIST OF TABLES

Table 1: Primer information for PCR studies examining PACAP receptor transcript expression...... 66

viii

LIST OF FIGURES

CHAPTER 1: LITERATURE REVIEW Figure 1: Schematic representation of MMA involvement in migraine headache ...... 25 Figure 2: Location of middle meningeal artery ...... 26 Figure 3: Illustration of the proposed pathway of glibenclamide-induced MMA constriction ...... 27 Figure 4: Structure of ATP-sensitive potassium channel ...... 28 CHAPTER 2: JOURNAL ARTICLE Figure 1: Preparation of rat middle meningeal artery (MMA) for the arteriography studies ...... 60 Figure 2: Pressure-induced myogenic tone in isolated rat middle meningeal arteries (MMAs) ...... 61 Figure 3: Low picomolar concentrations of PACAP, but not VIP, dilate isolated pressurized rat middle meningeal arteries (MMAs) ...... 62 Figure 4: Block of picomolar PACAP-induced dilation of isolated pressurized middle meningeal artery (MMA) by the PAC1 receptor antagonist PACAP(6-38) ...... 63 Figure 5: Nanomolar, but not picomolar, concentrations of PACAP38 and VIP dilate pressurized cerebellar arteries ...... 64 Figure 6: PACAP receptor transcripts are differentially expressed in rat middle meningeal and cerebellar arteries ...... 65 CHAPTER 3: JOURNAL ARTICLE

Figure 1: KATP channel currents in rat cerebellar artery myocytes ...... 90 Figure 2: PACAP enhanced transient BK current activity in rat cerebellar artery ...... 91

Figure 3: PACAP-induced vasodilation is mediated via KATP and BK channels in isolated rat cerebellar arteries ...... 92 Figure 4: Proposed mechanisms of PACAP-induced vasodilation in rat cerebellar artery ...... 93 CHAPTER 4: JOURNAL ARTICLE

Figure 1: Tonic KATP channel activity contributes to MMA diameter regulation via membrane potential and intracellular calcium changes ...... 105

Figure 2: Basal PKA activity is responsible for tonic KATP channel activity in MMA ..107 Supplemental figure (S1): ...... 114

Supplemental figure (S2): ...... 116

APPENDIX 1: BOOK CHAPTER Figure 1: PACAP and CGRP immunoreactivities are colocalized in fibers innervating the MMA ...... 174

Figure 2: PACAP-induces MMA vasodilation through KATP channel activation ...... 175

Figure 3: PACAP-induced vasodilation is mediated via KATP and BK channels in isolated rat cerebellar arteries ...... 177 Figure 4: Schematic of PACAP mechansims in vascular smooth muscle vasodilation .178 APPENDIX 2: UNPUBLISHED OBSERVATION Figure 1: CGRP-induced dilation is not attenuated in the presence of glibenclamide and PNU37883 ...... 188 Figure 2: CGRP-induced dilation is independent of BK, Kv, eNOS, COX, IK and SK pathways ...... 189 Figure 3: CGRP induces a decrease in intracellular calcium paralleling MMA dilation ...... 190 Figure 4: CGRP-induced dilation is significantly attenuated in the presence of high extracellular potassium ...... 191

CHAPTER 1: LITERATURE REVIEW

1.1 Introduction

Migraine is defined as a debilitating neurological disorder characterized by episodes of severe, throbbing headache, which are frequently unilateral in nature. The duration of migraine headache ranges from 4 to 72 hours and is generally accompanied by one or all of the following symptoms: nausea, photophobia, phonophobia and allodynia (Goadsby et al., 2002). The causes of migraine are not clear. Two competing theories regarding the pathophysiology of migraine are the theory of central sensitization and the vascular theory of migraine. The theory of central sensitization postulates that abnormal neuronal excitability of the second order brain stem neurons of the trigeminal nucleus caudalis (TNC) results from a noxious stimulus causing hyperexcitibility of the peripheral trigeminal nerves. This central sensitization is prolonged and can last up to 10 hours corresponding to the duration of migraine headaches (Burstein et al., 1998, Dodick and Silberstein, 2006). Thus, as a result of central sensitization, the migraine patient will experience exaggerated and intensified pain in response to a stimulus that ordinarily would cause mild pain.

In the 1940’s Dr. Harold Wolff and his colleagues observed that mechanical distention and vasodilation of the cranial blood vessels and more specifically the middle meningeal artery (MMA) results in headache pain. They further postulated that this headache pain is similar to migraine headache. These experimental observations gave birth to the vascular theory of migraine headache (Ray BS, 1940). At the time, it was 1 already recognized that the dura mater containing the MMA is heavily innervated by sensory neurons (Schueler et al., 2013). Over time, the vascular theory of migraine has undergone several modifications, yet the vasodilation of MMA is still considered a key player in migraine headache. However the stimuli for the vasodilation has not been clearly established (Goadsby et al., 1990). In 1979, Moskowitz et al. proposed that neurogenic inflammation contributes to the pathophysiology of migraine. According to this theory, activation of the trigeminal sensory ganglion and/or the sphenopalatine ganglion nerve endings causes an increase in release of vasoactive neuropeptides such as calcitonin gene related peptide (CGRP) and Substance P (SP) from the nociceptive Aδ and C fibers respectively on to the cranial vessels resulting in vasodilation (Moskowitz et al., 1979). The resultant distention of the blood vessels is further sensed by the afferent sensory nerves innervating the arterial wall and is communicated to the brain where the signal is subsequently perceived as pain (Ray BS, 1940, Goadsby et al., 2002, Asghar et al., 2010, Asghar et al., 2011, Amin et al., 2012, Amin et al., 2014) (Figure 1).

Until recently “the vascular theory of migraine” had lost momentum. However, in

2011 Asghar et al. provided functional and visual evidence using magnetic resonance angiography (MRA) for the vascular involvement in migraine headache. In this clinical study, intravenous (IV) injection of CGRP (1.5 µg/min for 20 minutes) was given to migraine patients to induce and mimic migraine headache. CGRP has been previously reported to induce migraine–like symptoms when given to both healthy subjects and migraine patients (Asghar et al., 2011). Asghar et al. observed that injection of CGRP induced migraine like symptoms including headache in the subjects. Furthermore,

2 patients with unilateral migraine or headache had concomitant dilation of the MMA on the pain side but not on the non-headache side. Conversely, middle cerebral artery

(MCA) dilated on both the headache and non-headache sides. Additionally, when the patients were acutely given the anti-migraine drug sumatriptan (a 5-HT-1B/D receptor agonist), the attenuation of the headache coincided with the constriction of MMA on the headache side. However, MCA diameter remained unchanged. The evidence presented in this study highlights two key points; 1) there is a vascular component to migraine headache, and 2) MMA but not MCA dilation may be responsible for the onset of migraine headache (Asghar et al., 2010, Asghar et al., 2011). Moreover, in follow-up studies, Amin et al. presented a similar out come in both healthy volunteers and migraineurs with another potent vasodilator, pituitary adenylate cyclase activating polypeptide (PACAP) (Amin et al., 2012, Amin et al., 2014). In addition, recent studies also suggest that the levels of PACAP and CGRP in plasma and cerebrospinal fluid

(CSF) from migraineurs are significantly higher during migraine headaches (Cernuda-

Morollon et al., 2013, Tuka et al., 2013, Bukovics et al., 2014). These studies brought the spotlight back on to the physiological and pathophysiological role of MMA and the effect of the neuropeptide PACAP on MMA during migraine headache (Schytz et al., 2009,

Asghar et al., 2010, Schytz et al., 2010a, Schytz et al., 2010b).

The following literature review will provide an overview of the regulation of

MMA by PACAP. Section one will discuss the vasodilatory effects of PACAP on blood vessels followed by a comparison of the mechanisms of vasodilation in MMA vs. the cerebral vasculature. Additionally, during our attempts to decipher mechanism of

PACAP-induced dilation of MMA, we discovered that ATP-sensitive potassium (KATP) channels are tonically active in the MMA. Thus, section two of this review will focus on the role of KATP channels with emphasis on vascular smooth muscle.

1.2 Section 1 1.21 Vascular components of migraine headache: meninges and the MMA

Meninges consist of three membranous layers of connective tissue, which work together to form a protective barrier around the brain and spinal cord. The three layers are identified as dura mater, arachnoid mater and pia mater. Dura mater is the outer most layer apposed to the calvaria. Embryonically, dural tissue is derived from the mesoderm.

In constrast, the pia and arachnoid mater are derived from the ectoderm (Gil and Ratto,

1973). Dura mater is described as a pain sensitive tissue innervated by sensory nerves originating from the trigeminal ganglion. Structurally, dura mater consists mainly of connective tissue, but also has a population of dural mast cells. Mast cells are part of the immune system, containing granules rich in histamine and other inflammatory mediators that have been shown to play a key role in the inflammatory process (Dimitriadou et al.,

1997, Baun et al., 2012). The main artery supplying blood to the dura mater is the MMA

(Yoshida et al., 1966, Chmielewski et al., 2013). The MMA branches from the maxillary artery, passes through foramen spinosum to enter the middle cranial fossa and course with in the dura mater (Figure 2) (Chmielewski et al., 2013).

The MMA displays autoregulation, an intrinsic property of resistance arteries occurring in response to increasing intravascular pressure that is modulated by metabolic and endothelium-dependent mechanisms (Michalicek et al., 1996, Li et al., 2014).

Increases in intraluminal pressure induce membrane potential depolarization, activation 4 of L- type voltage gated calcium channels (L-VDCC), vascular smooth muscle contraction and vasoconstriction (Knot and Nelson, 1998). This mechanism allows resistance arteries to maintain a constant level of blood flow during fluctuations in systemic blood pressure. Interestingly, the dura mater does not display high metabolic demand, thus the physiological reason for MMA to display autoregulation is not clear.

1.22 Neural inputs to MMA

The German anatomist, Arnold F. Der Kopfteil first described the innervation of the dura mater with trigeminal sensory nerve fibers also known as the V cranial nerve in

1831. This observation later played a pivotal role in describing the generation of headache. Several studies have linked trigeminal sensory nerve activation in the dura mater to the sensation of pain (Yoshida et al., 1966, Schueler et al., 2013). Since then, a number of studies have demonstrated innervation of the meningeal blood vessels in the dura mater originating from different sources including sympathetic fibers from the superior cervical ganglion, trigeminal fibers from the trigeminal ganglion and parasympathetic nerve fibers from the sphenopalatine and otic ganglia (Edvinsson et al.,

2001, Goadsby, 2013, Schueler et al., 2013). Peptides released from these perivascular nerves can regulate the diameter of the meningeal vascular bed. For example, the sympathetic fibers release vasoconstriction-inducing neuropeptide Y (NPY) and norepinephrine (Uddman et al., 1993a, Goadsby, 2013, Tajti et al., 2015), trigeminal fibers release vasodilatory neuropeptides such as PACAP, vasoactive intestinal polypeptide (VIP), Substance P and CGRP (Uddman et al., 1993a, Edvinsson et al.,

2001), and the parasympathetic fibers originating from the sphenopalatine ganglion

5 release PACAP and CGRP (Csati et al., 2012a, Csati et al., 2012b). It is worth mentioning that the number of neurons demonstrating PACAP immunoreactivity differs between trigeminal and parasympathetic ganglia, with approximately 10% in the trigeminal population and 6-5% in the sphenopalatine and otic ganglia (Csati et al.,

2012a, Csati et al., 2012b).

Interestingly, there is co-localization of PACAP and CGRP in some of the nerves innervating the MMA although the identity of these nerves is not clear (Edvinsson et al.).

Both peptides can be simultaneously released upon neuronal stimulation to cause dilation of the MMA (Edvinsson et al., 2001). The requirement for two different peptides

(PACAP and CGRP) with overlapping functions within the same nerve fiber is unknown.

1.23 PACAP and its receptors

PACAP is classified as both a neuropeptide and a hormone. It is a member of the structurally related peptide hormone family that includes glucagon, secretin, vasoactive intestinal peptide (VIP), gastric inhibitory peptide and growth hormone-releasing hormone. PACAP was first isolated from ovine hypothalamus extract in 1989 and was named for its role in stimulating adenylyl cyclase (AC) and in turn increasing cyclic adenosine monophosphate (cAMP) levels (Miyata et al., 1989). PACAP has been evolutionarily conserved for over 700 million years, indicating that PACAP and its pleiotropic functions are important for survival. PACAP knockout mice display altered anxiety levels and depression behavior (Gaszner et al., 2012). PACAP is synthesized as a precursor peptide which is then cleaved by pro-hormone convertases including PC1, PC2,

PC4 and furin (Seidah et al., 1994, Seidah and Chretien, 1999) resulting in either 6

PACAP38 (38 amino acids) or PACAP27 (27 amino acids), the two endogenously active forms of PACAP (Miyata et al., 1990). While both isoforms have similar binding affinities for PACAP receptors, their potencies for second messenger activation appear dissimilar. PACAP 38 has been demonstrated to stimulate phospholipase C (PLC) with higher potency compared to PACAP 27 (15 nM vs. 1 µM respectively) (Journot et al.,

1995). Furthermore, Braas et al. have demonstrated that PACAP 38 is 10 times more potent than PACAP 27 in stimulating cAMP production in superior cervical ganglion

(SCG) neurons. Additionally, PACAP 38 is the more predominant isoform in brain tissue and nervous system (Sherwood et al., 2000).

PACAP 27 or 38 can bind to three distinct receptors, PAC1, VPAC1 and VPAC2, which have been divided into 2 classes based on their relative PACAP binding affinities

(Sherwood et al., 2000). PAC1 receptors have been traditionally identified as the type 1

PACAP selective receptor with a binding affinity to PACAP 1000 fold higher compared to its binding affinity for the structurally similar peptide vasoactive intestinal polypeptide

(VIP) with a Kd value of 0.5 nM for PACAP vs. 500 nM for VIP. In contrast, the type 2

VIP /PACAP receptors, VPAC1 and VPAC2, have equal binding affinities for PACAP and VIP with a Kd value of 1 nM (Sherwood et al., 2000, Vaudry et al., 2009).

The PACAP selective PAC1 receptor has several splice variants due to alternative splicing of the transcript from a single gene. The PAC1 receptor isoforms can have no additional inserts, a hip cassette, a hop cassette or both hip and hop cassettes incorporated in the third intracellular loop. Accordingly, the PAC1-R splice variants are characterized by the absence null or the presence of either one or two of the 28 amino acid cassettes

(hip or hop1) or the 27 amino acid cassette Hop2. Six splice variants have been identified for PAC1 receptors: PAC1-R-null, PAC1-R-hop1, PAC1-R-hop2, PAC1-R-hiphop1,

PAC1-R-hiphop2 and PAC1-R-hip (Journot et al., 1995). All splice variants of PAC1 receptors with the exception of PAC1-R-hip can couple to and activate Gs or Gq/11 with equal potencies but with varying degrees of efficacy. PAC1-R-hip can only activate AC

(Journot et al., 1995, Sherwood et al., 2000). Thus, alternative splicing of the PAC1 receptor mRNA confers differences in G protein coupling and downstream signaling pathways.

The type 2 receptors VPAC1 and VPAC2 are also coupled with Gs protein and upon activation increase cAMP. Both PACAP and VIP can activate VPAC1 and VPAC2 with equal potency (Pisegna and Wank, 1993, Journot et al., 1995, Shioda et al., 1996,

McCulloch et al., 2001, Dickson and Finlayson, 2009). The distribution of the type 1 and type 2 PACAP receptors varies between tissues with all three receptors PAC1, VPAC1 and VPAC2 reported in arterial smooth muscle (Warren et al., 1991, Chan et al., 2011,

Syed et al., 2012).

1.24 PACAP and nonvascular functions

PACAP is a pleiotropic neuropeptide involved in multiple systems throughout the body. PACAP has been reported to play a role in cell cycle and development. For example, PACAP is reported to stimulate proliferation of chromaffin cells and primordial germ cells (Tischler et al., 1995). Not only does PACAP stimulate proliferation but it also regulates differentiation. PACAP activation of PAC1-R is known to stimulate neurite

8 growth. Additionally, PACAP also been described to initiate and inhibit apoptosis

(Sherwood et al., 2000).

One of the major roles of PACAP is as a regulator of the nervous system. For example PACAP levels during circadian rhythms decrease during daytime and increase during nighttime in the retinal ganglion cells that send axons to the suprachiasmatic nucleus, a region responsible for generating circadian rhythms (Fukuhara et al., 1997).

More recently, PACAP has been demonstrated to play a key role in anxiety behavior.

Hammack et al. have reported increased levels of PACAP and PAC1 receptor transcripts after chronic stress in the bed nucleus of the stria terminalis (BNST), a region known for modulating anxiety behavior. Furthermore, Missig et al. have reported an increase in

PACAP expression in neurons in the central nucleus of the amygdala. This region has been reported to represent a merging of pathways for pain, emotion and stress. Together, these results suggest PACAP is a regulator of behavior, anxiety, stress and pain (Missig et al., 2014).

1.25 PACAP and vascular smooth muscle

PACAP’s key function in the cardiovascular system is the modulator of vascular tone (Warren et al., 1991, Lenti et al., 2007, Schytz et al., 2009, Schytz et al., 2010b,

Chan et al., 2011, Syed et al., 2012, Erdling et al., 2013, Koide et al., 2014, Edvinsson,

2015). The general function of PACAP in the arterial blood vessels is to induce vasodilation.

1.25.1 PACAP and the cranial circulation: PACAP is released from the trigeminal sensory and parasympathetic nerves that innervate the walls of cranial arteries to produce 9 vasodilation. Additionally, PACAP has been shown to co-localize with other endogenous vasoactive neurotransmitters such as CGRP in trigeminal sensory nerve endings that innervate the cerebral and meningeal vascular bed (Baun et al., 2011). PACAP has been shown to potently regulate intra- and extra-cranial circulation and has been reported to dilate canine, cat, pig and rat (Tong et al., 1993, Uddman et al., 1993b, Anzai et al., 1995,

Syed et al., 2012, Koide et al., 2014) cerebral arteries. For example, PACAP induces dilation in both ex vivo pressurized rat MMA and cerebellar artery with an EC50 of 1 pM and 11 nM respectively, demonstrating a 100-fold increase in potency (Syed et al., 2012).

These observations suggest involvement of PACAP selective PAC1 receptor in MMA versus VPAC1 and VPAC2 receptors in the cerebral arteries.

1.25.2 Effect of PACAP on cardiac and mesenteric blood vessels: PACAP has been demonstrated to induce relaxation of systemic blood vessels. PACAP induces vasodilation in isolated, denuded rat and rabbit mesenteric arteries (Huang et al., 1993,

Wilson and Warren, 1993) indicating the presence of PACAP receptors in vascular smooth muscle (VSM) of these arteries.

PACAP immunoreactive nerve endings are localized in close proximity to vascular smooth muscles in cardiac tissue suggesting that PACAP plays a role in the coronary circulation. Indeed, there is evidence that PACAP has vasorelaxant effects in rabbit and human coronary arteries (Wilson and Warren, 1993, Chan et al., 2011).

Further, the EC50 of PACAP in rabbit mesenteric and aortic vascular beds has been reported to be ~ 100 nM suggesting involvement of VPAC1 and VPAC2 receptors

(Warren et al. 1991).

1.25.3 PACAP and the ophthalmic circulation: Retinoprotective effects of PACAP are well known and have been established in several disease models including diabetic retinopathy and ischemic retinal lesions. It is well established that decreased blood flow to the retinal layer can lead to apoptosis of the retinal cells subsequently leading to loss of vision. Interestingly, local application of PACAP has been shown to improve functional outcome in chronic retinal ischemic injury (Danyadi et al., 2014). Additionally, PACAP works synergistically with CGRP to relax ophthalmic arteries in humans supporting the vasodilatory role of PACAP in ocular vessels (Dorner et al., 1998). Not surprisingly

PACAP has been touted as a potential therapeutic target for diabetic retinopathy

(Marzagalli et al., 2015).

1.25.3 PACAP and the reproductive system: As the placental circulation has no autonomic innervation, hormonal signaling becomes essential in regulating placental blood flow (Y and S, 2010). PACAP is one hormone that has been demonstrated to play a key role in autocrine/paracrine signaling in placenta. The major source of PACAP in placental-tissue are the gonadotrophs and folliculostellate cells (Winters and Moore,

2011). By causing relaxation of utero-placental arteries and influencing maternal

(placental)-fetal blood circulation, PACAP plays an important role in fetal homeostasis

(Steenstrup et al., 1996). Furthermore, in adult males, PACAP has been shown to induce penile-erection due to its vasorelaxant properties, making it a potential therapeutic target in erectile dysfunction (Hedlund et al., 1995).

1.25.4 PACAP and the dermal circulation: The vasculature of the skin plays an important role in thermoregulation. PACAP contributes to the regulation of dermal blood flow by activation of PACAP receptors on arterial smooth muscle cells resulting in vasodilation (Warren et al., 1991). For example, the bite of the sand fly (genus: lutzomyia, family: Psychodidae) results in swelling of the affected area and vasodilation of the dermal blood vessel within the vicinity of the bite due to the action of the PAC1 selective agonist maxadilan present in the saliva of sand flies.

1.25.5 PACAP and the pancreatic circulation: PACAP has a biphasic effect on pancreatic blood vessels; low concentrations of PACAP (10-11 M to 10-9 M) induce vasodilation while concentrations above 10-7 M induce vasoconstriction. The mechanism by which PACAP is able to induce this biphasic effect is ambiguous and further studies are required, although Bertrand et al. suggest that PACAP selective PAC1 receptors may be involved in the vasoconstrictive response (Bertrand et al., 1996).

1.26 PACAP and disease

PACAP has been implicated in post-traumatic stress disorder (PTSD) and has been identified as a biomarker for PTSD in female patients (Ressler et al., 2010).

Furthermore, a strong correlation has been demonstrated between PTSD patients and migraine and PTSD has been implicated as a robust predictor of migraine (Smitherman and Kolivas, 2013). Elevated levels of PACAP are implicated in both migraine and

PTSD, however the mechanism by which PACAP effects vascular smooth muscle in

PTSD patients is an interesting question that requires further investigation.

PACAP is well known for its neuroprotective effects and activation of its receptors has been proposed to be therapeutically beneficial for cerebral vasospasm following subarachnoid hemorrhage (SAH) (Kaminuma et al., 1998). Moreover, PACAP levels in cerebral spinal fluid (CSF) and plasma are reported to be upregulated in patients suffering from traumatic brain injury (TBI). High PACAP levels in the CSF may be indicative of damage in blood brain barrier and this observation makes PACAP a potential biomarker to predict TBI mortality rate. (Bukovics et al., 2014).

In summary PACAP is a key vasoregulator of several vascular beds and is known to exert its effect via the cAMP-PKA pathway. Additionally, PACAP has been demonstrated to be a key player involved in several pathophysiological conditions including migraine and PTSD.

1.3 Section 2

1.31 Role of potassium channels in regulation of arterial tone

Potassium channels play a crucial role in regulation of arterial diameter via regulation of vascular smooth muscle membrane potential (Brayden, 2002). Under physiological conditions, activation of potassium channels results in efflux of K+ from the cell, driving the membrane potential to a more negative state towards EK resulting in membrane potential hyperpolarization of vascular smooth muscle (VSM) cells. This hyperpolarization decreases the open probability of L-type voltage-dependent calcium channels (L-VDCC) channels consequently decreasing intracellular Ca2+. The reduction in intracellular Ca2+ leads to smooth muscle relaxation and vasodilation (Nelson and

Quayle, 1995). Conversely, inactivation of potassium channels leads to membrane

13 potential depolarization and activation of L-VDCC and influx of Ca2+. Ca2+ binds to Ca2+- binding protein calmodulin (CaM) which in turn interacts and activates myosin light chain kinase (MLCK). MLCK then phosphorylates myosin light chain (MLC) resulting in increased myosin ATPase activity and activation of the contractile apparatus resulting in constriction (Walsh, 1981). Several potassium channels have been demonstrated to regulate arterial diameter via regulation of membrane potential including voltage gated potassium (Kv) channels, BK channels, inward rectifying potassium (Kir) channels and

KATP channels (Nelson and Quayle, 1995).

1.32 Discovery of KATP channels

In 1983, Noma et al. reported the presence of a potassium channel of previously unknown description in cardiac myocytes exposed to hypoxic conditions. These channels were characterized by inhibition to physiological ATP levels, suggesting a role of these channels in energy metabolism and earning them the name of ATP-sensitive potassium

(KATP) channels (Noma, 1983). Subsequently, KATP channels were discovered to be present and playing a key role in glucose homeostasis in pancreatic ß cells. KATP channels were determined to be a key regulator in insulin secretion from pancreatic ß cells

(Ashcroft et al., 1984). In 1989, Standen et al. first described the presence of KATP channels in vascular smooth muscle and its role in regulation of vascular smooth muscle membrane potential and myogenic tone (Standen et al., 1989).

1.33 Structure of KATP channels

KATP channels are a member of the ATP binding cassette (ABC) protein family that includes CFTR (cystic fibrosis transmembrane conductance regulator). As the name implies, KATP channels are sensitive to adenosine triphosphate (ATP). ATP (1-3 mM) inhibits the openings of these channels (Hayabuchi et al., 2001). Conversely, KATP channels are activated by nucleotide diphosphates such as adenosine diphosphate (ADP).

Thus KATP channels are described as metabolism sensing channels (Nichols, 2006).

KATP channels are comprised of four Kir6.x channel subunits, forming the ion channel pore and four auxiliary sulphonylurea subunits (SURx). Together Kir6x and

SURx subunits form a functional hetero-octameric channel. There are two Kir6 genes in vertebrates (KCNJ8, KIR6.1 and KCNJ11, KIR6.2) and two SUR genes (ABCC.8, SUR1 and ABCC.9, SUR2) and combinations of different Kir6.x and SURx subunits from distinct types of KATP channels. The Kir6x subunits consist of two transmembrane helices

M1 and M2 connected by an extracellular loop that generates the narrow portions of the channel and controls ion selectivity. The SUR subunits contain 17 transmembrane regions that are arranged into 3 transmembrane domains TMD0, TMD1, and TMD2.

SURs contain a NBF1 (nucleotide-binding fold) between TMD1 and TMD2 and a second

NBF2 after TMD2 for its cytoplasmic interaction with nucleotides (Nichols, 2006)

(Figure 4). The Kir subunit is responsible for ATP inhibition while the SUR protein is responsible for nucleotide diphosphate (NDP) activation. Exogenous KATP potassium channel openers (KCO) such as cromakalim and minoxidil sulphate also bind SUR

15 subunit and activate the channel (Nichols, 2006). Additionally, sulphonylurea drugs such as glibenclamide and tolbutamide can inhibit this channel (Nichols, 2006). Furthermore, the SURx subunits have also been observed to have different binding affinities for ATP,

ADP and sulphonylurea drugs. For example, it has been previously reported that glibenclamide binds to SUR1 subunit with a much higher affinity as opposed to SUR2A and SUR2B (Hibino et al., 2010). Hence splice variants introduce a diverse physiological function in different tissue types, consequently providing therapeutic opportunities to target specific tissue (Ashcroft et al., 1984, Hibino et al., 2010).

The molecular constituents for KATP channels differ in different tissue and the combination of Kir6x subunits and SURx proteins determines channel characteristics in a given tissue. For example, in pancreatic ß cells, Kir6.2 and SUR1 are the dominant isoforms, while in cardiac myocytes, Kir6.2 and SUR2A are the major isoforms. In vascular smooth muscle, the predominant isoforms are Kir6.1 and SUR2B (D'Arcy et al.,

1985, Nelson and Quayle, 1995, Quayle et al., 1995, Ploug et al., 2008, Hibino et al.,

2010, Ploug et al., 2012). KATP channels can also be modulated by a plethora of intracellular signaling molecules including pH and Ca2+, PKA and protein kinase C

(PKC) and the modulation varies with different compositions of Kir6.x and SURx subunits 89.

1.34 Modulation of KATP channel gating

ATP is an endogenous inhibitor of KATP channels and it exerts its effect by binding to a site located on the Kir6x subunit and stabilizing the closed stated of the channel (Kajioka et al., 1991). The activation site of the KATP channel is located on the 16

SURx subunit. Another important means of KATP channel regulation via Gs-coupled cAMP-PKA phosphorylation of the channels. The phosphorylation sites include S385 in

Kir6.1, T633 and S1465 in SUR2B, S1387 in NBF2 of SUR2B. Additionally, Kir6.2 can be phosphorylated at T224 by PKA and at T180 by PKC. The potassium channel openers (KCOs) such as cromakalim, pinacidil and diazoxide can also modulate KATP channel gating. In SUR2A and SUR2B the binding of the KCOs is between TMD1 and

TMD2. Intracellular nucleotides also affect the binding of KCOs. For example, with cardiac KATP channels, diazoxide cannot induce channel opening in the absence of ADP

(Hibino et al., 2010).

1.35 Membrane trafficking

Only functional hetero-octameric KATP channels are trafficked to the plasma membrane. However, Tucker et al., have shown that the truncated form of Kir 6.2ΔC26 can also produce functional KATP channels in the absence of SUR subunit that can be trafficked to the plasma membrane. In smooth muscle the trafficking of KATP channels involves the activity of PKCε as demonstrated by Jiao et al. They showed that PKCε dependent-inhibition of vascular KATP channels is via caveolin-dependent internalization resulting in reduction of surface expression of the channels both in HEK and native vascular smooth muscle cells (Jiao et al., 2008).

1.36 Physiological relevance of KATP channels in pancreas, heart and brain

Pancreas: In pancreas KATP channels are found in pancreatic ß cells, which are responsible for secretion of insulin. The molecular subunits of pancreatic KATP channels 17 consist of Kir6.2 and SUR1 (Inagaki et al., 1996). The critical role of KATP channels in pancreas is to couple blood glucose level to insulin secretion. As blood glucose levels increase in pancreatic ß cells, ATP also rise. Elevated ATP causes inhibition of KATP channel activity, which results in membrane potential depolarization, and opening of L-

VDCC. The Ca2+ influx initiates the fusion of the vesicles containing insulin to the membrane for release. Polymorphism in Kir6.2 and SUR1 has been associated with hypoglycemia and diabetes in humans. Furthermore, KATP channels are also present in the glucagon-secreting α-cell and may be involved in secretion of these hormones.

Additionally, over-activity of the KATP channels in the pancreatic ß cells can result in suppression of insulin release resulting in type 2 diabetes. Sulphonylurea drugs like glibenclamide are used to treat this condition. Glibenclamide inhibits KATP channels thus increasing the open probability of L-VDCC and increase in intracellular Ca2+ and consequently increase insulin secretion (Koster et al., 2005).

Heart: The molecular composition of KATP channels in the heart is Kir6.2 and SUR2A.

Cardiac KATP channels have been proposed to play a critical role in ischemia preconditioning, which is the phenomenon wherein transient ischemic events engage protective signaling pathways that involve a variety of second messengers including

PKC. This preconditioning event can protect the heart from prolonged ischemic insults and can be mimicked by openers of KATP channels (Tomai et al., 1999). However, the detailed mechanisms mediating ischemia preconditioning and cardioprotection is unclear.

(Cohen et al., 2000, Iliodromitis et al., 2007).

Brain: Like the heart, KATP channels in neurons have been proposed to play a neuroprotective role under pathophysiological conditions (Ballanyi, 2004). The brain represents approximately 2% of the body and yet it consumes 20% of the total energy

(Raichle and Gusnard, 2002). This high metabolic demand of the brain generally keeps neuronal KATP channels closed. However, during injurious events such as ischemia and hypoxia mammalian neurons are known to undergo depolarization, leading to excessive intracellular calcium and death. Several studies have demonstrated that during hypoxia

KATP channels are activated as a protective mechanism causing hyperpolarization and prevention of cell death (Ballanyi, 2004, Nichols, 2006).

1.37 Physiological relevance of KATP channels in vascular smooth muscle

In many vascular beds, KATP channels play an important role in regulation of the membrane potential in vascular smooth muscle and vascular diameter. Vascular KATP channels consist predominantly of Kir6.1 subunit and sulphonylurea receptor SUR2B isoforms (Hibino et al., 2010).

PKA and KATP channels: KATP channels are important targets for elevated cAMP and in turn PKA in vascular smooth muscle myocytes (Wellman et al., 1998). PKA phosphorylates serine 385 (S385) residue located on Kir 6.1 and two sites on SUR2B subunit, residue serine 1465 (S1465) and threonine 633 (T633) leading to channel activation (Quinn et al., 2004). Furthermore, Hayabuchi et al. have stipulated that basal

PKA activity is involved in tonic regulation of KATP channels in isolated rat mesenteric myocytes. They report that in the presence of physiological ATP concentration (1 mM) vascular KATP channels are active due to basal PKA activity. Using whole cell patch

19 clamp Hyabuchi et al. demonstrated that in the presence of low concentration of ATP (0.1 mM), pinacidil (KATP channels agonist)-induced currents were not inhibited by PKA inhibitor (Rp-cAMP). Conversely, at the physiological concentration of ATP (1 mM), pinacidil-induced currents become susceptible to inhibition by Rp-cAMP. Interestingly, this evidence suggests that tonic PKA activity is dependent on physiological ATP levels, which can affect the cAMP production in the cell (Hayabuchi et al., 2001). Additionally, in the presence of calyculin A (phosphatase 1 (PP1) and (PP2A) inhibitor), KATP channel currents are significantly increased, indicating a critical balance between phosphorylation and dephosphorylation in mesenteric myocytes. Dart et al. have shown that KATP channels are compartmentalized along with AC in a signaling pocket on the smooth muscle membrane by caveolae (a membrane invagination of the plasma membrane associated with cholesterol-binding protein caveolin) (Sampson et al., 2004, Sampson et al., 2007).

Epac and KATP channels: Exchange protein activated by adenylyl cyclase (Epac) is a novel protein activated by cAMP that has recently emerged as a regulator of vascular smooth muscle diameter independent of PKA activity (Purves et al., 2009). Epac has

+ been shown to regulate K channels in vascular smooth muscle; it inhibits KATP channels in a calcium dependent manner and activates BK, small-conductance calcium activated potassium (SK) channels, and intermediate-conductance calcium activated potassium

(IK) channels. Dart et al. have reported that Epac transiently increased intracellular Ca2+ levels in rat aortic myocytes resulting in activation of calcium-dependent phosphatase 2B

(PP2B, calcinuerin) and dephosphorylation of PKA-dependent phosphorylated KATP

20 channels (Roberts and Dart, 2014). In a separate study, Dart et al. further investigated the effect of this transient increase in Ca2+ by Epac. They found that the synthetic Epac agonist 8-pCPT acetocymethyl ester (8-pCPT-AM) increased the frequency of Ca2+ sparks from ryanodine receptors (RyRs) in vascular smooth muscle cells resulting in activation of BK channels and membrane potential hyperpolarization. The evidence presented by Dart et al. in these studies suggest that Epac may have two distinct roles in regulation of vascular tone depending on the vascular bed involved (Roberts et al., 2013,

Roberts and Dart, 2014).

PKC and KATP channels: In vascular smooth muscle cells, PKC is associated with vasoconstriction. Activation of PKC can result in phosphorylation and inhibition of KATP channels leading to depolarization and vasoconstriction (Aziz et al., 2014). Several vasoconstrictors can work via the PKC pathway to induce vasoconstriction in vascular smooth muscle cells including endothelin, neuropeptide Y, phenylephrine, histamine, serotonin (Wakatsuki et al., 1992, Aziz et al., 2014) and angiotensin-ІІ (Dart, 2014).

Angiotensin-ІІ is part of the renin-angiotensin system, which causes vasoconstriction and has been implicated in hypertension. Angiotensin receptors (AT1) are Gq/11 coupled and activate phospholipase C (PLC) that in turn activates PKC(Dart, 2014). Additionally, several studies suggest that the novel PKC isoform protein kinase C epsilon (PKCε) is responsible for the inhibition of KATP channels in vascular smooth muscle. Jiao et al. have demonstrated that activation of PKCε results in a caveolin-dependent internalization of the KATP channels (Jiao et al., 2008). Hayabuchi et al. proposed that not only is angiotensin-ІІ working via PKC to decrease KATP channel activity; they further suggest

21 that angiotensin-ІІ also inhibits PKA activity. AT1 receptors can also be Gi/o-coupled and their activation will decrease cAMP production hence effectively reducing PKA activation (Nelson and Brayden, 1993, Hayabuchi et al., 2001).

1.38 KATP channels and Pathophysiology

Excessive activation of KATP channels in vascular smooth muscle can result in hypotension and conversely, excessive KATP channel inhibition can lead to hypertension and ischemia (Nelson and Quayle, 1995, Brayden, 2002). In addition, during hypoxia there is dilation of coronary and small mesenteric arteries (Daut et al., 1990). Duat et al., suggest that under hypoxic conditions there is a decrease in ATP and an increase in ADP production resulting in opening of coronary KATP channels, triggering hyperpolarization and vascular smooth muscle relaxation and increased blood flow to the heart and cardioprotection. KATP channels have also been shown to play a role in acidosis. During hypoxic and ischemic events, the extracellular pH drops. Ishizaka et al. have demonstrated that this drop in pH causes activation of KATP channels in the coronary arteries resulting in vasodilation (Ishizaka and Kuo, 1996). However, the mechanism by which KATP channels are activated due to a drop in pH remains unclear. Ishzaka et al., suggested that it could be due to either decreased ATP levels arising from high metabolic demand or release of adenosine (Nelson and Brayden, 1993, Kleppisch and Nelson,

1995b, a).

Previous published studies using either global Kir6.1 or SUR2 knock out mice demonstrated that the mice were hypertensive and prone to sudden death due to coronary vasospasm (Chutkow et al., 2002, Miki et al., 2002, Kakkar et al., 2006). Yet, when

SUR2B was selectively reintroduced in smooth muscle of the coronary vasculature, the lethal phenotype persisted. Further, when SUR2A was reconstituted in cardiac myocytes the mortality rate decreased suggesting that it may not be vascular KATP channels that are critical in influencing the lethal phenotype in global knock out of either Kir6.1 or SUR2.

However, recently an extensive study done by Aziz et al. using Kir6.1 conditional knockout mice revealed that vascular KATP channels are essential for vascular reactivity and blood pressure (Aziz et al., 2014). This study provided evidence that mice with

Kir6.1 conditional knockout do not display sudden death due to coronary vasospasm yet, they still have a hypertension phenotype, indicating that Kir6.1 is the molecular pore- forming subunit in vascular smooth muscle (Kakkar et al., 2006).

In summary, KATP channels have been demonstrated to play crucial roles physiological and pathophysiologic conditions in vascular smooth muscle. Activation of

KATP channels has been demonstrated to relax vascular blood vessels and has been shown to be beneficial in pathophysiological conditions like hypertension (Aziz et al., 2014,

Aziz et al., 2015). Conversely, activation of this channel in the cranial vessels has been demonstrated to induce headache and to play a role in migraine headache (D'Arcy et al.,

1985, Sterndorff and Johansen, 1988, Ploug et al., 2008, Ploug et al., 2012).

1.39 Research Objectives of the Present Work

The objectives of the study were: 1) to decipher the mechanism involved in

PACAP-induced vasodilation of the MMA and 2) to determine the tonic regulation of

MMA diameter by basal KATP channel activity. We hypothesized that PACAP induces dilation in the MMA via activation of vascular KATP channels. Additionally, we show that 23

KATP channels are tonically active in the MMA and are contributing to MMA diameter regulation via regulation of vascular smooth muscle membrane potential.

1.4 Figures

Figure 1: Schematic representation of MMA involvement in migraine headache. 1)

Noxious stimuli the cause release of the neuropeptide PACAP and other vasodilators neuropeptides that bind to their respective receptors on MMA. 2) Binding of the neuropeptides to their respective receptors results in vasodilation. 3) Distention of the artery is proposed to activate afferent sensory neurons (mechanism unknown). 4) The signal is carried to the cortex and perceived as headache pain.

Posterior branch Anterior branch piercing the bone

Posterior meningeal artery

Middle meningeal artery

Figure 2: Location of middle meningeal artery. Modified from: http://chestofbooks.com/ with permission:

Figure 3: Illustration of the proposed pathway of glibenclamide-induced MMA constriction. Tonic PKA-dependent activation of KATP channels results in membrane potential hyperpolarization and MMA dilation. Bottom: KATP inhibition by glibenclamide leads to membrane potential depolarization, enhanced Ca2+ influx via L-type VDCCs and vasoconstriction.

Figure 4: Schematic representation of the structural subunits of KATP channels.

Reproduced with permission from Quan et al., Acta Pharmacologica Sinica (2011) 32:

765–780.

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CHAPTER 2: JOURNAL ARTICLE

Title: Pituitary adenylate cyclase activating polypeptide (PACAP) potently dilates middle meningeal arteries: implications for migraine

Authors: Arsalan U. Syed1, Masayo Koide1, Karen M. Braas2, Victor May1,2, George C. Wellman1

Affiliations: Departments of Pharmacology1 and Neurological Sciences2, University of Vermont College of Medicine 149 Beaumont Avenue Burlington, Vermont 05405 USA

Correspondence: George C. Wellman, Ph.D. University of Vermont Department of Pharmacology 89 Beaumont Avenue Burlington, VT USA 05405-0068 e-mail: [email protected] Tel: 802-656-3470 Fax: 802-656-4523 39

2.1 Abstract

Migraine is a debilitating neurological disorder characterized by mild to severe headache that is often accompanied by aura and other neurological symptoms. Among proposed mechanisms, dilation of the dural vasculature especially the middle meningeal artery (MMA) has been implicated as one component underlying this disorder. Several regulatory peptides from trigeminal sensory and sphenopalatine postganglionic parasympathetic fibers innervating these vessels have been implicated in the process including pituitary adenylate cyclase activating polypeptide (PACAP). Although PACAP has been well described as a potent dilator in many vascular beds, the effects of PACAP on the dural vasculature are unclear. In the current study, we examined the ability of

PACAP to dilate MMAs that were isolated from rats and pressurized ex vivo. PACAP38 potently dilated pressurized MMAs with an EC50 of 1 pM. The PAC1 receptor antagonist, PACAP(6-38), abolished MMA dilation caused by picomolar concentrations of PACAP. In contrast, cerebellar arteries isolated from the brain surface were ~1000- fold less sensitive to PACAP than MMAs. Although cerebellar arteries expressed transcripts for all three PACAP receptor subtypes (PAC1, VPAC1 and VPAC2 receptors) by RT-PCR analyses, MMA demonstrated only PAC1 and VPAC2 receptor expression.

Further, multiple variants of the PAC1 receptor were identified in the MMA. The expression of PAC1 receptors and the high potency of PACAP to induce MMA vasodilation are consistent with their potential roles in the etiology of migraine.

2.2 Introduction

Migraine is one of the most prevalent contributors to the global burden of mental disorders (Collins et al. 2011). It is a complex episodic neurological disorder characterized by intense unilateral pulsating headaches that may last hours to days, which may be accompanied by aura, nausea, photophobia, phonophobia and other neurological symptoms (Asghar et al. 2011). Although the causes and mechanisms underlying migraine are multifactorial and remain poorly understood, one likely contributing factor, first proposed decades ago (Ray et al. 1940), involves dilation of the dural vasculature.

Subsequent investigations have implicated trigeminal sensory and/or sphenopalatine parasympathetic postganglionic neuronal signaling as a contributor to the experience of migraine pain (Mayberg et al. 1981; Brain et al. 1985; Lassen et al. 2002; Olesen et al.

2009; Schytz et al. 2009; Schytz et al. 2010; Asghar et al. 2011; Amin et al. 2012). In one current model, enhanced neurotransmitter/neuropeptide release causes MMA dilation leading to sensitization of the trigeminal sensory neuronal fibers innervating the MMA that contributes to the perception of pain (Goadsby et al. 1993). Bioactive neuropeptides may participate in all of these events and one prototypic neuroregulator in migraine is calcitonin gene-related peptide (CGRP). CGRP is a potent vasodilator (Brain et al.

1985), is highly expressed in dorsal root/trigeminal sensory and sphenopalatine autonomic ganglia neurons, has been localized to perivascular nerve terminals

(McCulloch et al. 1986), and has been shown to induce migraine-like headaches indistinguishable from spontaneous migraine attacks (Lassen et al. 2002). Notably, magnetic resonance angiography (MRA) imaging studies have correlated MMA

41 vasodilation following exogenous CGRP administration with headache onset to further implicate roles for neurally-mediated vasodilation in migraine (Asghar et al. 2011).

Similar to CGRP, more recent studies have also implicated pituitary adenylate cyclase activating polypeptide (PACAP) signaling in migraine. PACAP is a neurotransmitter and neurotrophic peptide in the secretin, glucagon and vasoactive intestinal peptide (VIP) superfamily and shares some G protein coupled receptor selectivity with VIP (Vaudry et al. 2009). PACAP binds potently and specifically to the

PAC1 receptor which may be coupled to multiple intracellular signaling cascades; by contrast, both PACAP and VIP bind with near equal affinity to VPAC1 and VPAC2 receptors coupled principally to adenylyl cyclase. Among its many functions, PACAP has prominent roles in nociception and like CGRP, causes relaxation of vascular and respiratory smooth muscle (Seki et al. 1995). The peptide is highly expressed in diverse central and peripheral nervous system tissues, including sensory and autonomic ganglia, and appears co-localized with CGRP for concomitant release upon neuronal stimulation

(Csati et al. 2012). Hence PACAP shares expression and regulatory parallels with

CGRP, and significantly, recent clinical studies have shown that intravenous infusion of

PACAP can produce MMA vasodilation and induce headache in both migraineurs and healthy subjects (Schytz et al. 2009; Amin et al. 2012). Further, the administration of the serotonin 5-HT1B and 5-HT1D receptor agonist sumatriptan, frequently used to treat migraine headaches, attenuated both PACAP-induced MMA vasodilation and headache pain. These studies indicate that PACAP signaling causes MMA dilation that may

42 contribute to the etiology of migraine (Amin et al. 2012) and represent an important therapeutic target to aid migraneurs.

Despite the immediate relevance to migraine, PACAP signaling in the MMA has not been well characterized. In the current study, we examined the effects of PACAP on isolated segments of MMA using pressurized artery myography. Compared to other intracranial vessels, the high potency of PACAP and the expression PACAP receptors in the MMA appear unique, suggesting that the MMA vasodilatory responses to

PACAP/PAC1 receptor signaling may be an important component of the complexities of migraine.

2.3 Methods

Isolation of middle meningeal and cerebellar arteries

All experiments and protocols were conducted in accordance with the Guide for the Care and Use of Laboratory animals (NIH Pub. No. 85-23, revised 1996) and approved by the

Institutional Animal Use and Care Committee of the University of Vermont. Sprague-

Dawley rats (males, 300-350 g; Charles River Laboratories, Saint Constant, QC, Canada) were euthanized by decapitation under deep isoflurane anesthesia. The brain and calvaria were carefully removed and placed in ice-cold 3-(N-morpholino) propanesulfonic acid

(MOPS)-buffered saline solution containing (in mM): 145 NaCl, 5 KCl, 1 Mg SO4, 2.5

CaCl2, 1 KH2PO4, 0.02 EDTA, 3 MOPS, 2 pyruvate, 5 glucose and 1% bovine serum albumin (pH 7.4). For MMA isolation, the dura mater was gently removed from the calvaria under a dissection microscope (Figure 1A). To enable a comparison of the

MMA to pial arteries, cerebellar arteries were dissected from the brain using iris scissors.

The MMA and cerebellar artery are both innervated by trigeminal sensory nerves

(Simons et al. 1988; Goadsby et al. 1993).

Pressurized artery myography

Middle meningeal and cerebellar arteries were cannulated onto glass micropipettes mounted in a 5 mL myograph chamber (Figure 1B) and intravascular pressure was adjusted using a pressure servo-controller and a peristaltic pump (Living Systems

Instruments, St, Albans, VT) as previously described (Ishiguro et al. 2002; Nystoriak et al. 2011). Arteries were continuously superfused with warmed artificial cerebral spinal fluid (aCSF; 37 °C, in mM: 125 NaCl, 3 KCl, 18 NaHCO3, 1.25 NaH2PO4, 1 MgCl2, 2

CaCl2 and 5 glucose, aerated with 5% CO2, 20% O2, 75% N2, pH: 7.35-7.40). Changes in vessel diameter were measured by video edge detection (Living Systems

Instrumentation, St Albans, VT) using Windaq data acquisition software (Dataq

Instruments; Akron, OH). Following equilibration (1 h), arterial viability was evaluated by brief exposure to 60 mM KCl (< 2 min; isosmotic replacement of NaCl with KCl in aCSF). Only arteries that demonstrated a constriction representing a decrease in diameter of 50 % or greater were used in subsequent studies. Pressure-induced constriction is expressed as a percent decrease of the fully dilated diameter of individual arteries at the same intravascular pressure. These values were obtained by using the following equation: % constriction = (DP-DA)/DP · 100 where DP = the (passive) diameter of the artery in Ca2+-free aCSF containing the vasodilators diltiazem (100 µM) and forskolin (1

µM), and DA = the (active) diameter of the artery in Ca2+-containing aCSF. Dilations in

44 response to PACAP38 and VIP are expressed as a percent reduction in the level of arterial constriction. The values were obtained using the following equation: % dilation =

(DV-DA)/(DP-DA) · 100 where DV = arterial diameter in the presence of vasodilator, DA

= the active diameter of the artery prior to addition of vasodilator, and DP = the passive diameter of the artery. The 38 amino acid form of PACAP, referred to as PACAP38

(Miyata et al. 1989), was used in all experimental series. The concentrations of

PACAP38 or VIP that caused half maximal vasodilation (EC50) were calculated from individual concentration response curves using Graphpad Prism 5 software (Graphpad software, Inc. La Jolla, CA). PACAP38, VIP and related peptides (American Peptide

Sunnyvale, CA) were prepared as 100 µM stock in deionized water; aliquots were stored at -80 °C. All aliquots were diluted to their final concentrations immediately before use and were administered to arteries by bath superfusion (5 mL/min flow rate) for a minimum of 20 minutes.

MMA and cerebellar artery PACAP receptor transcript expression

Total RNA extracted from isolated MMAs and cerebellar arteries using RNA STAT-60 total RNA isolation reagent (Tel-test, Friendswood, TX) was reverse-transcribed to cDNA using SuperScript First-Strand synthesis system (Invitrogen, Carlsbad, CA) as previously described (Nystoriak et al. 2009). To detect mRNA expression of PACAP receptor subtypes, semiquantitative RT-PCR was performed using primer sets for PAC1,

VPAC1 and VPAC2 receptor transcripts; amplification of the same templates for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as an internal control

(Table 1). To examine vessel expression of PAC1 receptor splice variants, amplification

45 using primers flanking the alternative splice site within the third cytoplasmic loop of the

PAC1 receptor was performed as previously described (Braas et al. 1999). Amplification of single-stranded cDNA was performed in a 13 µL reaction volume consisting of 15 mM

Tris-HCl, pH 8.0, containing 50 mM KCl, 2.5 mM MgCl2, 200 µM deoxynucleotide triphosphates, 0.5 µM primers, 0.5 µL of cDNA template and 0.3 U AmpliTaq Gold

DNA polymerase (Applied Biosystems, Norwalk, CT) with the following cycling parameters: (1) initial denaturation and enzyme activation 95 °C, 10 min; (2) denaturation and enzyme activation 94 °C,1 min; annealing, primer specific (Table 1), 1 min; extension 72 °C, 1 min (32 - 40 cycles); and (3) final extension, 72 °C; 10 min. For

PAC1 receptor variant identification, the amplified cDNA product was subjected to diagnostic restriction enzyme digestion as previously described (Braas et al. 1999). The amplified products were resolved on 2% agarose gel and visualized by UV illumination following ethidium bromide staining.

Statistics

Data are expressed as mean ± SEM and analyzed by Student’s unpaired t-test.

Statistical significance was considered at p < 0.05.

2.4 Results

Isolated middle meningeal arteries (MMA) exhibit pressure-induced myogenic tone.

Small diameter arteries and arterioles isolated from a variety of vascular beds, including those supplying blood to the heart and brain, constrict in response to increases in intravascular pressure (Bayliss 1902; Johnson 1980). In the cerebral circulation, pressure-induced constriction, also termed myogenic tone, plays an important role in the regulation of cerebral artery diameter (Nelson et al. 1995; Ishiguro et al. 2002; Nystoriak et al. 2011). To evaluate myogenic tone in freshly isolated MMA segments, changes in arterial diameter were examined in response to step-wise increases in intravascular pressure. At 10 mmHg, MMA diameter in aCSF was 45.8 ± 5.2 µm, representing a constriction of 39.1 ± 5.8 percent decrease from the maximally dilated diameter of 75.5 ±

4.6 µm obtained in Ca2+-free aCSF containing the vasodilators diltiazem (100 µM) and forskolin (1 µM). The level of myogenic tone increased as intravascular pressure was elevated, reaching a constriction of 62.9 ± 8.4 % at 120 mmHg (Figure 2). At the physiologically relevant pressure of 40 mmHg, myogenic tone represented a constriction of 49.1 ± 10 %. These data demonstrate that isolated MMA exhibit pressure-induced myogenic tone. Subsequent experimental series were performed using isolated MMAs held at 40 mmHg in the absence of exogenous vasoconstrictors.

Picomolar concentrations of PACAP38 dilate pressurized MMAs via activation of

PAC1 receptors.

The ability of PACAP to induce MMA dilation ex vivo, is unclear. To date, studies of PACAP-induced responses in isolated MMA segments have examined changes in isometric force generation in unpressurized arteries pre-contracted with exogenous vasoconstrictors (Baun et al. 2011). To examine the impact of PACAP on ex vivo pressurized MMAs exhibiting myogenic tone, increasing concentrations of PACAP38 or

VIP (0.1 pM to 100 nM) were added to the superfusate bathing MMAs held at an intravascular pressure of 40 mmHg (Figure 3A). PACAP38 potently dilated MMA with an EC50 of 1 pM (n = 4). In marked contrast, VIP was approximately 1000-fold less potent dilator of MMA (EC50: 1.4 nM, n = 4). As an example, 3 pM PACAP38 caused

MMA dilation of 60.3 ± 15.1 % of tissue maximum, whereas 3 pM VIP did not significantly alter arteriolar diameter (Figures 3B, C). Based on the differential potency of PACAP38 and VIP binding to PACAP receptor subtypes (Harmar et al. 2012), we hypothesized that MMA dilation to picomolar concentrations of PACAP38 are mediated via activation of PAC1 receptors. Consistent with this concept, MMA dilation to 3 pM

PACAP38 was abolished by the PAC1 receptor antagonist PACAP (6-38) (Harmar et al.

2012) (Figure 4A, B). Interestingly, MMA dilation to 1000-fold higher concentrations of

PACAP38 (i.e. 3 nM) were not abolished, but were reduced by approximately 50 %

(Figure 4C-E), consistent with higher concentrations of PACAP38 causing activation of

PAC1 receptors as well as VPAC1 and/or VPAC2 receptors. These data demonstrate that

PACAP38 potently dilates pressurized MMAs via activation of PAC1 receptors.

Nanomolar, but not picomolar, concentrations of PACAP38 dilate pressurized cerebellar arteries.

Intracranial cerebral arteries are innervated by sensory trigeminal nerves containing PACAP, but appear to contribute little to migraine symptoms (Asghar et al.

2011; Amin et al. 2012). To examine whether PACAP dilates pressurized cerebral arteries with an efficacy similar to that observed in MMA, cerebellar arteries were cannulated and studied ex vivo. In contrast to MMAs, 3 pM PACAP38 did not alter cerebellar artery diameter (n = 4, Figure 5 A, C). However, 1000-fold higher concentrations of PACAP38 (3 nM) did significantly dilate cerebellar arteries (34 ± 12

%, n = 4). The EC50 value of PACAP-induced cerebellar dilation was 11 nM (n = 6).

Similarly, VIP caused cerebellar artery dilation at nanomolar, but not picomolar, concentrations (Figure 5 B, C; EC50 = 6 nM, n = 3). These data demonstrate that PACAP is considerably more potent vasodilator of MMA compared to cerebellar arteries, suggesting that the PACAP- and VIP-induced vasodilation in cerebellar arteries is likely mediated by a VPAC receptor subtype.

MMA and cerebellar arteries express different PAC1 and VPAC receptor subtypes.

PACAP and VIP share receptor subtypes; the PAC1 receptor is specific for

PACAP peptides while VPAC1 and VPAC2 receptors bind VIP and PACAP with similar affinities. Differential PACAP receptor expression may contribute to the disparity in the sensitivity of MMA and cerebellar arteries to PACAP. To establish PACAP/VIP receptor

49 expression in MMA and cerebellar arteries, semiquantitative PCR studies were performed using primers unique to each receptor transcript, as described in previous work

(Braas et al. 1999). Interestingly, only PAC1 and VPAC2 receptor transcripts were detected in MMA; there was little to no VPAC1 receptor expression in MMA. By contrast, all three PACAP/VIP receptor subtypes (PAC1, VPAC1 and VPAC2 receptors) were identified in cerebellar arteries (Figure 6A).

There are multiple PAC1 receptor isoforms from alternative splicing of Hip and/or Hop cassettes in the receptor gene corresponding to the G protein interactive sites in the third cytoplasmic loop, which are important for downstream activation of signaling cascades (Spengler et al. 1993). When PAC1 receptor variant transcript analyses were performed using primers flanking the third cytoplasmic loop, MMA and cerebellar arteries not only expressed the PAC1null receptor (neither Hip nor Hop, Figure 6B, 303 bp band) but also the one cassette variant (Hip or Hop, Figure 6B, 387 bp band).

Furthermore, the relative abundance of the one cassette variant was much greater in the

MMA than the cerebellar artery. As the Hip and Hop cassettes are both 84 bp, the one cassette 387 bp amplified product could not distinguish the specific PAC1 receptor splice variant expressed in MMA. However, restriction enzyme digestion can be diagnostic

(Braas et al. 1999) and digestion of the 387 bp band with Blp I generated the 292 bp products consistent with the expression of the PAC1Hop receptor variant in MMA

(Figure 6C). While the PAC1null receptor is predominantly coupled to adenylyl cyclase, the PAC1Hop receptor variant can be potently coupled to multiple intracellular signaling cascades including adenylyl cyclase, phospholipase C, MAPK and Akt (Braas et al.

1999). The expression and coordinate signaling downstream of both PAC1 receptor variants in MMA may have engendered the potent PACAP vasodilatory responses; maladaptions of the same PACAP/PAC1 receptor signaling pathways in MMA, by contrast, may be a contributing factor to migraine.

2.5 Discussion

Migraine is one of the leading contributors to the global burden of mental and neurological disorders. The few therapeutic options are often unsatisfactory and hence an understanding of the underlying mechanisms and the identification of new clinical targets remain important goals for effective treatments. From the many proposed causes, the neurovascular component in migraine has remained a viable mediator especially in light of the recent data using high resolution magnetic resonance angiography (MRA) techniques (Asghar et al. 2011). In humans infused with the well studied sensory/autonomic vasodilator CGRP peptide, the resulting migraine attacks correlated temporally and spatially with middle meningeal and middle cerebral artery dilation. The arterial dilations were bilateral when the headaches were bilateral and unilateral on the corresponding side when uni-hemispheric headaches were presented. Further, treatments with sumatripans to ease the peptide-induced migraines always resulted in vessel constriction. These studies appear to supplant earlier Doppler studies which failed to correlate vascular changes with migraine (Zwetsloot et al. 1993). Although these studies do not address definitive mechanisms, the results do implicate cranial vascular dynamics in the presentation of migraine. Among intracranial and extracranial arteries, the meningeal artery vasodilatory responses were better correlated with headache onset and 51 treatments, and considered to be particularly significant as dural afferent nociceptor sensitization appears central to pain generation in migraine attacks (Burstein et al. 2005).

PACAP peptides behave as neurotransmitters and neurotrophic peptides in the central and peripheral nervous systems with diverse activities in neuroendocrine development and function. PACAP peptides are highly expressed in nociception sensory dorsal root and trigeminal ganglion neurons, and in autonomic systems regulating vasodilatory control; hence the effects of PACAP parallel those for CGRP in facilitating migraines. PACAP38 infusions in in vivo and ex vivo preparations cause intra- and extracranial artery vasodilation and can induce migraine-like attacks in normal subjects and migraineurs (Schytz et al. 2009; Amin et al. 2012). As in other physiological systems, the PACAP-elicited responses can be long lasting which may be one of the distinguishing characteristics for PACAP-induced headache pain (Amin et al. 2012).

Interestingly, while the related VIP peptide can also produce marked dilation in many arterial vessels, it induces only modest headache and no migraine-like attacks (Hansen et al. 2006; Rahmann et al. 2008) suggesting the selective expression and activation of the shared PAC1/VPAC receptors in the different cranial arterial systems.

Yet despite the unequivocal abilities for PACAP to instill migraine, the relative roles of PACAP and VIP in migraine, and whether specific PAC1/VPAC receptor subtype expression and function in the different cranial arteries can underlie the distinct

PACAP/VIP vasodilatory and migraine responses are still very much unclear. Some of the variability observed in the in vivo infusion studies may have stemmed from

PACAP/VIP-mediated endothelial cell activation of nitric oxide pathways, rapid peptide

52 degradation in the circulation, the poor ability for peptides in general to cross the blood brain barrier, and in the case of rodents, the presence of anesthetics during the course of the studies. The putative selectivity of the available receptor agonists and antagonists may have contributed to the uncertainties, and the ex vivo isometric force studies may have been complicated by the use of high concentrations of reagents including prostaglandin F2α and serotonin to precontract the unpressurized wire-mounted vessels.

From these considerations, the use of a pressurized arteriograph system offers a number of advantages in characterizing PACAP and VIP vasodilatory responses. The pressurized arterial vessels develop myogenic tone in the absence of exogenous vasoconstrictors, are extremely sensitive and responsive to regulatory dynamics (Dunn et al. 1994; Nelson et al. 1995), and obviate the need for drug-induced arterial precontraction, which can potentially dampen the peptide vasodilatory effects. The

MMA possesses the ability to autoregulate its blood flow and pressure intrinsically

(Michalicek et al. 1996); hence the pressurized MMA myography preparation presents a physiologically relevant means to examine PACAP-induced effects.

Using these preparations, the current studies demonstrated potent PACAP38 effects on MMA diameter. In contrast to previous isometric force myograph studies which suggested little or no PACAP effects on MMA contractility (Baun et al. 2011), subpicomolar concentrations of PACAP38 reproducibly dilated pressurized MMAs ex vivo. Picomolar PACAP concentrations maximally dilated the MMA to 50 - 60% of passive diameter; high nanomolar PACAP concentrations had little additional vasodilatory effects. VIP also dilated MMA but only at nanomolar peptide

53 concentrations. The picomolar potency of PACAP has been well described for several physiological systems including protection from ischemia in the brain and potentiation of glucose induced insulin release in pancreatic beta cells (Yamada et al. 2004; Dejda et al.

2011). These differential PACAP/VIP response profiles in the MMA differed from those observed for cerebellar arteries in which PACAP38 and VIP appeared equipotent in inducing vasodilations at nanomolar concentrations.

The higher potency of PACAP compared to VIP in the MMA suggest PAC1 receptor activation in the vasodilatory responses. Both PAC1 and VPAC2 receptor transcripts were identified in the MMA; interestingly, VPAC1 receptor expression in the

MMA was not detected, which varied with previous work (Boni et al. 2009). Diagnostic

PCR and restriction digests identified PAC1null and PAC1Hop1 receptor isoforms in the

MMA which may have allowed second messenger coupling not only to adenylyl cyclase but to other intracellular cascades to potently stimulate MMA dilation. PACAP(6-38) pretreatment blocked the PACAP38 responses in the MMA and although PACAP(6-38) may antagonize both PAC1 and VPAC2 receptors (Dickinson et al. 1997) to obfuscate clear identification of the relevant receptor subtype in the PACAP-induced responses, the apparent differential potencies observed for PACAP38 and VIP were consistent with

PAC1 receptor-mediated vasodilation in the MMA. These results appear consistent with previous interpretations implicating PAC1 receptors in migraine (Olesen et al. 2009;

Schytz et al. 2009; Schytz et al. 2010). Unlike the MMA, however, the cerebellar arteries exhibited expression for all three PACAP/VPAC receptor subtypes. The apparent equipotency of PACAP38 and VIP in eliciting cerebellar artery vasodilation

(Figure 5) implicate VPAC receptor signaling. These results were in good agreement with previous observations implicating VPAC1 receptors in cerebellar artery vasodilation

(Fahrenkrug et al. 2000).

In summary, the pressurized myograph preparations in the current study demonstrated differences between PACAP and VIP in eliciting MMA and cerebellar artery vasodilation which may be notable with respect to migraine physiology. The very high potency for PACAP38 to elicit and sustain MMA vasodilation suggested that even very modest levels of neuropeptide release and receptor activation may be sufficient to mediate migraine pain. As suggested in previous studies, the physiological and molecular analyses demonstrated differential PAC1/VPAC receptor expression profiles among the cranial vessels examined. The high potency of PACAP38, the expression of

PAC1 receptor subtype and isoforms, and the abilities for the antagonist PACAP(6-38) to block PACAP-induced MMA dilation all implicated PAC1 receptor signaling in MMA vasodilation. As MMA vasodilatory dynamics well correlated with migraine onset and abatement in humans, PACAP/PAC1 receptor regulation of the MMA may be a crucial component to the development of migraine attacks. This action of PACAP on MMA contrasts with PACAP/VIP dilation of cerebellar arteries which appeared to be mediated via VPAC1 receptor activation. Together with the human PACAP infusion studies, these observations in sum suggest that PACAP and PAC1 receptor signaling may be a potential therapeutic target for the treatment of migraine.

Acknowledgements

The authors wish to thank Mr. Kevin O’ Connor for his assistance and acknowledge the

University of Vermont Neuroscience COBRE molecular biology core facility. This work was supported by the Totman Medical Research Trust and National Institutes of Health

Grants P01-HL-2095488, P20-RR-16435 and R01-HL-078983.

Disclosures

No conflicts of interest, financial or otherwise, are declared by the authors.

2.6 Reference List

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2.7 Figures

Figure 1: Preparation of rat middle meningeal artery (MMA) for arteriograph studies. A) Rat MMA embedded in the dura matter prior to dissection. Arrows indicate the MMA region typically used for ex vivo studies. B) Isolated rat MMA cannulated on glass micropipettes mounted in an arteriograph chamber. Both ends of MMA were tied by suture thread, and the artery pressurized to 40 mmHg. Arterial diameter was measured using video edge detection and recorded using data acquisition software

(details in Materials and Methods). One graduation of scale (e.g. 0 to 1) represents 100

µm.

Figure 2: Pressure-induced myogenic tone in isolated rat middle meningeal arteries

(MMAs). A) Diameter measurements of isolated rat MMA obtained in response to step- wise increases in intravascular pressure from 10 mmHg to 120 mmHg. Squares with solid line represent active diameter measurements obtained in artificial cerebral spinal fluid (aCSF) (n = 4). Circles with dashed line represent maximally dilated (passive) diameter measurements obtained from the same arteries in Ca2+-free aCSF containing the vasodilators diltiazem (100 µM) and forskolin (1 µM). B) Pressure-induced constriction

(myogenic tone) in rat MMAs superfused with aCSF ex vivo. Constriction is expressed as a percent decrease from maximally dilated diameter obtained at the same intravascular pressure (n=4).

Figure 3: Low picomolar concentrations of PACAP, but not VIP, dilate isolated pressurized rat middle meningeal arteries (MMAs). A) Cumulative concentration response curves of PACAP and VIP obtained from rat MMAs pressurized to 40 mmHg ex vivo. Arteries were exposed to aCSF containing each concentration of PACAP38 or

VIP for 20 minutes. Dilation to PACAP38 or VIP are expressed as percentage of maximum dilation obtained in the presence of Ca2+-free aCSF containing 100 µM diltiazem and 1 µM forskolin. P<0.05 by unpaired t-test, n = 4. B, C) Diameter recordings of an isolated MMA treated with aCSF containing a single concentration (3 pM) of PACAP38 (B) and VIP (C). At the end of the experiment, maximally dilated diameter was obtained in the presence of Ca2+-free aCSF containing 100 µM diltiazem and 1 µM forskolin (shown as “passive”).

Figure 4: Block of picomolar PACAP-induced dilation of isolated pressurized middle menigeal artery (MMA) by the PAC1 receptor antagonist PACAP(6-38). A-

D) Diameter recordings from isolated MMAs obtained in the absence (A, C) and presence (B, D) of the PAC1 antagonist, PACAP(6-38). Arteries, pressurized to 40 mmHg, were treated with either 3 pM PACAP38 (A, B) or 3 nM PACAP38 (C, D).

Maximum dilation (passive) was obtained by treatment with 100 µM diltiazem and 1 µM forskolin in Ca2+-free aCSF. E) Summary data of PACAP38-induced dilation in the absence or presence of PACAP(6-38). PACAP(6-38) abolished 3 pM PACAP38-induced vasodilation, but caused only partial inhibition of 3 nM PACAP38-induced vasodilation in rat MMAs (n = 4 each). Diameter changes by PACAP38 treatment were expressed as percentage of maximum dilation. * P<0.05 vs PACAP-induced vasodilation in the absence of PACAP(6-38).

Figure 5: Nanomolar, but not picomolar, concentrations of PACAP38 and VIP dilate pressurized cerebellar arteries. A) Diameter recording obtained from pressurized rat cerebellar artery treated with PACAP38. PACAP38 induced dilation at 3 nM, but not

3 pM. Maximum dilation was obtained using a combination of 100 µM diltiazem and 1

µM forskolin in Ca2+-free aCSF (passive). B) Diameter recording obtained from pressurized rat cerebellar artery treated with VIP. VIP induced dilation at 3 nM, but not 3 pM. Maximum dilation was obtained using a combination of 100 µM diltiazem and 1

µM forskolin in Ca2+-free aCSF (passive). C) Summarized data of PACAP38 and VIP treatment in rat cerebellar arteries. PACAP38-induced diameter changes are expressed as percentage of maximum dilation (n = 4 each). * P<0.05 by paired t-test.

Figure 6: PACAP receptor transcripts are differentially expressed in rat middle meningeal and cerebellar arteries. Total RNA from rat MMA and cerebellar arteries were reverse transcribed for semiquantitative PCR analyses of PAC1 (413 bp), VPAC1

(323 bp) and VPAC2 (396 bp) receptor transcript expression. A) Unlike cerebellar artery preparations which demonstrated mRNA for all three receptor subtypes (right panel, n =

5), the MMA appeared to express only PAC1 and VPAC2 receptor transcripts (left panel, n = 5). B) To examine whether there were differences in PAC1 receptor isoform expression levels between the two arteries, primers flanking the third cytoplasmic loop were employed in the PCR studies. While cerebellar arteries appeared to possess predominantly the PAC1null receptor isoform (right panel, neither Hip nor Hop; 303 bp), the MMA expressed near equal levels of the PAC1null (303 bp) and the one-cassette

PAC1 receptor variant (387 bp; left panel). Each lane represents an independent sample replicate. Amplification of the same templates for GAPDH demonstrates approximate equal sample input. C) The specific one-cassette PAC1 receptor variant could be identified by diagnostic restriction digest; isolation of the MMA 387 bp PAC1 receptor band followed by Blp I restriction enzyme incubation resulted in amplicon digestion to a smaller 292 bp product diagnostic of the Hop receptor variant. 65

Table 1: Primer information for PCR studies examining PACAP receptor transcript expression.

CHAPTER 3: JOURNAL ARTICLE

Title: Pituitary adenylate cyclase activating polypeptide (PACAP) dilates cerebellar arteries through activation of large-conductance Ca2+-activated + (BK) and ATP-sensitive (KATP) K channels

Authors: Masayo Koide1*, Arsalan U. Syed1*, Karen M. Braas2, Victor May1,2, George C. Wellman1

Affiliations: Departments of Pharmacology1 and Neurological Sciences2, University of Vermont College of Medicine 149 Beaumont Avenue Burlington, Vermont 05405 USA

* Both authors contributed to this project equally.

3.1 Abstract

Pituitary adenylate cyclase activating polypeptide (PACAP) is a potent vasodilator of numerous vascular beds, including cerebral arteries. Although PACAP-induced cerebral artery dilation is suggested to be cyclic AMP (cAMP)-dependent, the downstream intracellular signaling pathways are still not fully understood. In this study, we examined the role of smooth muscle K+ channels and hypothesized that PACAP-mediated increases in cAMP levels and protein kinase A (PKA) activity result in the coordinate activation of

+ 2+ + ATP-sensitive K (KATP) and large-conductance Ca -activated K (BK) channels for cerebral artery dilation. Using patch-clamp electrophysiology, we observed that PACAP enhanced whole-cell KATP channel activity and transient BK channel currents in freshly isolated rat cerebellar artery myocytes. The increased frequency of transient BK currents following PACAP treatment is indicative of increased intracellular Ca2+ release events termed Ca2+ sparks. Consistent with the electrophysiology data, the PACAP-induced vasodilations of cannulated cerebellar artery preparations were attenuated by approximately 50% in the presence of glibenclamide (a KATP channel blocker) or paxilline (a BK channel blocker). Further, in the presence of both blockers, PACAP failed to cause vasodilation. In conclusion, our results indicate that PACAP causes cerebellar artery dilation through two mechanisms: 1) KATP channel activation and 2) enhanced BK channel activity, likely through increased Ca2+ spark frequency.

3.2 Introduction

Pituitary adenylate cyclase activating polypeptide (PACAP) is a peptide that is highly conserved over species and widely distributed in the brain and peripheral organs.

While acting as a neurotransmitter and neurotrophic peptide in the central and peripheral nervous systems (reviewed in Vaudry et al. 2009), PACAP is also a potent vasodilator

(Warren et al. 1992; Vaudry et al. 2009; Syed et al. 2012). PACAP-induced vasodilation has been observed in various vascular beds including coronary arteries (Bruch et al. 1997;

Dalsgaard et al. 2003), pulmonary arteries (Cheng et al. 1993), mesenteric arteries

(Wilson et al. 1993) and cerebral arteries (Tong et al. 1993; Anzai et al. 1995). For example, in vivo treatment of PACAP via an open cranial window resulted in brain pial artery dilation in newborn pigs (Tong et al. 1993), and intracisternal administration of

PACAP caused dose-dependent canine basilar artery dilation detected by angiography

(Seki et al. 1995). Further, in ex vivo studies, PACAP caused concentration-dependent relaxation of isolated canine basilar arteries (Anzai et al. 1995; Seki et al. 1995), cat cerebral arteries (Uddman et al. 1993), rabbit posterior cerebral arteries (Dalsgaard et al.

2003), rat middle cerebral arteries (Erdling et al. 2013) and rat intracerebral arterioles

(Anzai et al. 1995). This vasodilatory effect is, at least in part, a direct effect of PACAP on arterial smooth muscle cells. PACAP binds to three types of receptors; PAC1,

VPAC1 and VPAC2 (Vaudry et al. 2009; Harmar et al. 2012), and all three receptors are expressed in cerebral artery smooth muscle cells (Syed et al. 2012; Erdling et al. 2013).

Binding of PACAP to PACAP receptors increases production of adenosine 3’, 5’-cyclic

69 monophosphate (cAMP) (Tong et al. 1993; Vaudry et al. 2009) in arterial myocytes which likely accounts for the mechanism of PACAP-induced cerebral artery dilation.

Increased cAMP production and subsequent activation of cAMP-dependent protein kinase (protein kinase A, PKA) can produce vasodilation by several mechanisms including activation of smooth muscle K+ channels (Nelson et al. 1995b; Wellman et al.

1998). Potassium channels play an important role in the regulation of smooth muscle membrane potential, intracellular Ca2+ concentration and arterial diameter; activation of smooth muscle K+ channels causes membrane potential hyperpolarization, decreased open-state probability of voltage-dependent Ca2+ channels and reduced intracellular Ca2+ concentration resulting in vasodilation (Standen et al. 1989; Nelson et al. 1995b; Standen et al. 1998). Dilations induced by a number of endogenous vasoactive substances such as calcitonin gene-related peptide (CGRP) have been shown to result from increased ATP-

+ sensitive K (KATP) channel currents via mechanisms involving adenylyl cyclase activation, increased cAMP production and PKA activation (Nelson et al. 1990; Quayle et al. 1994; Kleppisch et al. 1995a; Wellman et al. 1998). Similarly, KATP channel blockers were able to partially inhibit PACAP-induced vasodilation in coronary and pulmonary arteries, suggesting that PACAP may also activate KATP channels (Bruch et al.

1997; Bruch et al. 1998).

Stimulated cAMP/PKA activity however, can also augment the frequency of transient large-conductance Ca2+-activated K+ (BK) channel currents (previously referred to as spontaneous transient outward currents or STOCs) following intracellular Ca2+ spark events (Nelson et al. 1995a; Wellman et al. 2003). Transient outward BK currents

70 occur as a result of increases in intracellular Ca2+, called Ca2+ sparks, and represent an important physiological vasodilatory pathway in cerebral arteries (Nelson et al. 1995a;

Wellman et al. 2003). Ca2+ sparks are transient and localized Ca2+ increases resulting from the opening of a small cluster of ryanodine-sensitive Ca2+ channels (ryanodine receptors, RyRs) located on the sarcoplasmic reticulum (SR). These localized increases in intracellular Ca2+ lead to activation of nearby BK channels on the plasma membrane

(transient outward BK currents), resulting in smooth muscle membrane potential hyperpolarization and vasodilation (Nelson et al. 1995a; Wellman et al. 2003).

Considering that the treatment of cerebral artery myocytes with cAMP analogues or adenylyl cyclase activators (to elevate intracellular cAMP levels) can increase the frequency and amplitude of transient outward BK currents (Porter et al. 1998; Wellman et al. 2001; Hayoz et al. 2007), we hypothesized that PACAP-induced increases in cAMP can also activate the Ca2+ spark/transient outward BK current pathway to promote cerebral vasodilation.

Because of its vasodilatory, neurotrophic and neuroprotective effects, PACAP has been examined in animal models as possible treatments for cerebral vasospasm after subarachnoid hemorrhage (Kaminuma et al. 1998) and ischemic stroke (Reglodi et al.

2000; Ohtaki et al. 2006). However, despite its therapeutic potential, the detailed intracellular signaling mechanisms involved in PACAP-induced cerebral artery dilation are not fully understood. In this study, we have examined the vasodilatory mechanisms of PACAP, focusing specifically on the role of smooth muscle K+ channels in freshly

71 isolated rat superior cerebellar arteries. Our results indicate that activation of both KATP and BK channels contribute to PACAP-induced cerebral artery dilation.

3.3 Methods

Measurements of KATP currents and transient outward BK currents

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 Sprague-

Dawley rats (300-350g; Charles River Laboratories, Saint Constant, QC, Canada) were euthanized by decapitation under deep pentobarbital anesthesia. The brain was carefully removed and superior cerebellar arteries were isolated in ice cold artificial cerebrospinal fluid (aCSF: 125 mM NaCl, 3 mM KCl, 18 mM NaHCO3, 1.25 mM NaH2PO4, 1 mM

MgCl2, 2 mM CaCl2, and 5 mM glucose, aerated with 5 % CO2, 20 % O2, 75 % N2, pH

7.35–7.40).

Arterial myocytes were enzymatically isolated using a previously described protocol (Ishiguro et al. 2005; Koide et al. 2007; Koide et al. 2011). Briefly, arteries were incubated in a solution containing papain (0.3 mg/mL papain, 0.7 mg/mL 1,4- dithioerythriol) for 17 min at 37 °C, and then in collagenase solution (0.7 mg/mL collagenase type F, 0.3 mg/mL collagenase type H, 0.1 mM CaCl2) for 20 min at 37 °C.

Enzyme solutions were made using a glutamate-containing isolation solution (GIS: 55 mM NaCl, 5.6 mM KCl, 80 mM L-glutamic acid-Na, 2 mM MgCl2, 2 mM CaCl2, 10 mM

HEPES, and 10 mM glucose; pH 7.3). Finally, arteries were washed three times (10

72 minutes each wash) in GIS containing 2 mM CaCl2 on ice then gently triturated into individual myocytes using a fire-polished Pasteur pipette.

Using the conventional whole cell patch clamp configuration, KATP currents were recorded in freshly isolated cerebellar arterial myocyte at room temperature (Wellman et al. 1999). The bath solution contained 134 mM NaCl, 6 mM KCl, 1 mM MgCl2, 1.8 mM

+ CaCl2, 10 mM glucose and 10 mM HEPES (pH 7.4). The “140 mM K ” bath solution was made by iso-osmotic replacement of NaCl with KCl. The pipette solution contained

107 mM KCl, 1.0 mM MgCl2, 1.9 mM CaCl2, 10 mM HEPES, 5 mM EGTA, 25 mM

KOH (pH 7.2 adjusted with KOH, free Ca2+ ~100 nM). On the day of the experiment,

0.1 mM Na2ATP, 0.1 mM NaADP and 0.1 mM LiGTP were freshly added into an aliquot of pipette solution. KATP currents were recorded at -60 mV. After a stable baseline was obtained, the extracellular solution was changed from “6 mM K+” bath solution to “140 mM K+” solution. PACAP was treated by superfusation into the bath in the presence of

140 mM K+.

Transient outward BK currents were measured using the perforated whole-cell patch-clamp technique at room temperature (Wellman et al. 2002; Koide et al. 2011). The bath solution contained 134 mM NaCl, 6 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 10 mM glucose and 10 mM HEPES (pH 7.4). Patch pipettes (8-10 MΩ) were filled with a pipette solution that contained 110 mM K+-aspartate, 30 mM KCl, 10 mM NaCl, 1 mM

MgCl2, 10 mM HEPEs, 0.05 mM EGTA and 0.2 mM amphotericin B (pH 7.2).

Transient outward currents were recorded at -40 mV. PACAP was treated by bath superfusation. Recordings were analyzed using Mini Analysis 6.0.3 software

(Synaptosoft, Inc., Decatur, GA, USA) to determine transient BK current amplitude, frequency, rise time (time from 10 % to 90 % of the peak), decay time (time from 90% to

37 % of the peak) and half-width (time at 50% of the peak). The threshold for current peak detection was set at two and half times the single channel amplitude of the BK channel; 5.0 pA at -40 mV (Perez et al. 2001).

Diameter measurements of isolated cerebellar arteries

Superior cerebellar arteries were cannulated onto glass micropipettes mounted in

5 ml myograph chamber and the intravascular pressure was increased using a servo controller and a peristaltic pump (Living Systems Instruments, St. Albans, VT) as previously described (Ishiguro et al. 2002; Nystoriak et al. 2011; Koide et al. 2012). The arteries were continuously superfused with aCSF warmed at 37°C. Luminal diameter was measured and recorded using video edge detection (Living Systems Instrumentation,

St Albans, VT) and WinDaq data acquisition software (Dataq Instruments; Akron, OH).

Following a 30 minute equilibration period at 20 mmHg, arteries were briefly exposed to

60 mM KCl-aCSF (<2 min; iso-osmotic replacement of NaCl with KCl) to test their viability. Only arteries that exhibited constriction of 50 % or more to 60 mM KCl-aCSF were included for study. Following an additional 30 minutes, intraluminal pressure was increased to 60 mmHg. Over the course of approximately 1 hour, arteries constricted to the increase in intravascular pressure, i.e. developed pressure-induced myogenic tone.

Once arterial diameter was stabilized at 60 mmHg, PACAP (3 nM) was superfused into the chamber in the absence and presence of K+ channel blocker(s). Percent constriction in response to elevation of intravascular pressure was determined using the following

74 equation: [(DP−DA)/DP]×100; where DP = the (passive) diameter of the artery in Ca2+- free aCSF containing the vasodilators diltiazem (100 µM) and forskolin (1 µM), and DA

= the (active) diameter of the artery in Ca2+-containing aCSF. Dilation responses to

PACAP are expressed as a reduction in arterial constriction using the following equation:

% dilation = [(DV–DA)/(DP–DA)]×100, where DV = arterial diameter in the presence of vasodilator, DA = the active diameter of the artery prior to addition of vasodilator, and

DP = the passive diameter of the artery. All aliquots of compounds used in the study were diluted to their final concentrations immediately before use and were administered to arteries by bath superfusion (5 mL/min flow rate) for a minimum of 10 min.

Chemicals

PACAP was obtained from American Peptide Company (Sunnyvale, CA). All other compounds were purchased from Sigma-Aldrich (St. Louis, MO).

Statistics

Data are expressed as mean ± SEM. Comparisons between multiple groups were analyzed by one-way ANOVA followed by post hoc comparison of means using Tukey test. Student’s paired t-test was used to compare data between two groups. Statistical significance was considered at p<0.05.

3.4 Results

PACAP activates KATP channels in freshly isolated rat cerebellar artery myocytes.

Several endogenous vasodilators (e.g. CGRP, adenosine) linked to increased adenylyl cyclase activity have been shown to activate KATP channels in arterial smooth muscle cells (Nelson et al. 1990; Quayle et al. 1994; Kleppisch et al. 1995a; Kleppisch et al.

1995b; Wellman et al. 1998). To examine whether PACAP can increase KATP channel activity in freshly isolated cerebellar artery myocytes, membrane K+ currents were measured by patch clamp electrophysiology. Here, recording conditions using the conventional whole-cell configuration were designed to optimize KATP currents and

+ minimize BK and voltage-dependent K (KV) channel activity (e.g. 0.1 mM ATP, 0.1 mM ADP and 5 mM EGTA in intracellular pipette solution; holding potential of -60 mV)

(Wellman et al. 1998). In symmetrical (intracellular and extracellular) 140 mM K+, the

+ K equilibrium potential (EK) predicted by the Nerst equation is 0 mV. Thus, at a holding

+ potential negative to EK, e.g. -60 mV, KATP channel activation will cause K ions to move into the cell creating an inward electrical current (Quayle et al. 1994; Kleppisch et al.

1995b; Wellman et al. 1998). As shown in figure 1A, treatment of a cerebral artery myocyte with the synthetic KATP channel opener, pinacidil (10 µM), caused a significant

+ increase in inward K current that was inhibited by the KATP channel blocker, glibenclamide (10 µM). Using similar recording conditions, PACAP (3 nM) treatment also caused an increase in inward K+ current (figure 1B). This data suggests that PACAP can activate KATP channels in rat cerebellar artery myocytes.

PACAP increases the frequency of transient outward BK currents in rat cerebellar artery myocytes

An increase in the frequency of smooth muscle transient outward BK currents represents another potential mechanism involved in PACAP-induced cerebral artery dilation.

Transient BK currents represent the coordinated opening of up to 100 individual BK channels in regions of the cell membrane (≈ 1 % of the total cell surface area) exposed to brief local increases in Ca2+ sparks (Wellman et al. 2003). Treatment of vascular smooth muscle cells with either a cAMP analogue, an adenylyl cyclase activator (e.g. forskolin) or endogenous vasodilators that elevate intracellular cAMP levels causes an increase in the frequency of Ca2+ spark-induced transient BK currents triggered by Ca2+ sparks

(Porter et al. 1998; Wellman et al. 2001). Considering that PACAP can potently increase intracellular cAMP levels, activation of the Ca2+ spark/transient BK current pathway may also contribute to PACAP-induced cerebral artery dilation. To examine this hypothesis, we directly measured transient BK currents in freshly isolated cerebellar artery myocytes using the whole-cell perforated patch configuration of the patch clamp technique. In this experimental series, cells were held at the physiological membrane potential of -40 mV in an extracellular (bath) solution containing 6 mM K+. Spontaneous transient outward BK currents were observed in these cells and PACAP (3 nM) significantly increased the frequency of these events by approximately 50% (from 1.38 ± 0.47 Hz to 2.02 ± 0.51 Hz; n = 6 cells from 3 animals, figure 2A and 2B). Although PACAP markedly increased the transient BK current frequency, the amplitude of these events was not dramatically altered by PACAP treatment (before treatment: 14.71 ± 2.11 pA, after PACAP treatment:

17.49 ± 2.98 pA, figure 2C). Further, the temporal characteristics of transient BK currents were similar before and after PACAP treatment: rise-time10-90% (msec): 7.94 ±

0.85 / 7.28 ± 0.30; decay time90-37% (msec): 25.03 ± 3.84 / 23.57 ± 4.16; half-width

(msec): 30.33 ± 3.21 / 31.33 ± 3.34 (n = 6 cells from 3 animals, before / after PACAP treatment, respectively). In summary, these data demonstrate that PACAP increased the frequency of Ca2+ spark-mediated transient BK currents in isolated rat cerebellar artery myocytes.

PACAP-induced cerebellar artery dilation involves activation of both BK and KATP channels

The above studies indicate that PACAP can increase the activity of both BK and KATP channels in freshly isolated cerebellar artery myocytes. Next, the functional contribution of these K+ channels in PACAP-induced dilation was examined in isolated and pressurized rat superior cerebellar arteries. At a physiological intravascular pressure of

60 mmHg, arteries superfused with aCSF developed pressure-induced constriction

(myogenic tone) representing a 23.0 ± 1.3 % decrease in diameter (n = 19 from 19 animals). At 60 mmHg, the average diameter of cerebellar arteries in aCSF with myogenic tone was 191.1 ± 7.5 µm compared to a fully-dilated diameter of 247.2 ± 7.5

µm obtained in Ca2+-free aCSF containing diltiazem (100 µM, a voltage-dependent Ca2+ channel blocker) and forskolin (1 µM). PACAP (3 nM) caused significant dilation (52.2

± 10.7% of maximal dilation), increasing the average arterial diameter from 189.8 ± 9.1

µm to 220.8 ± 14.0 µm (n = 4 from 4 animals, figure 3A and 3E). To examine whether

KATP channel activation (shown in figure 1B) contributes to PACAP-induced cerebellar

78 artery dilation, arteries were treated with the KATP channel blocker, glibenclamide (10

µM). Gibenclamide treatment alone, did not change arterial diameter (187.0 ± 14.7 µm and 185.8 ± 16.4 µm, in the absence and presence of glibenclamide, respectively, n = 4 from 4 animals), suggesting minimal basal KATP channel activity in these arteries.

However, in the presence of glibenclamide, the PACAP-induced dilations were significantly attenuated (approximately 50% of maximal dilation with PACAP alone vs

22.6 ± 11.8 % of maximal dilation in PACAP + glibenclamide; n = 4 from 4 animals, figure 3B). This data indicates that PACAP-induced vaosodilation is partially caused by

KATP channel activation.

We next examined if activation of the Ca2+ spark/transient BK current pathway also plays a role in PACAP-induced cerebellar artery dilation. To inhibit this pathway, arteries were treated with the BK channel blocker, paxilline (1 µM), which alone constricted arteries in aCSF from 204.0 ± 13.4 µm to 174.4 ± 14.7 µm, an approximate

15% decrease in diameter on top of myogenic tone (fig. 3C, note paxilline-induced vasoconstriction before PACAP application; fully-dilated diameter: 262.6 ± 11.1 µm, n =

7 from 7 animals). This paxilline-induced vasoconstriction is consistent with previous studies demonstrating that the Ca2+ spark/transient BK current pathway plays an important negative-feedback role in the regulation of cerebral artery myogenic tone

(Nelson et al. 1995a; Koide et al. 2011; Nystoriak et al. 2014). The PACAP-induced vasodilation was significantly attenuated in the presence of paxilline (approximately 50% maximal dilation with PACAP alone vs 14.8 ± 2.6% of maximal dilation in PACAP + paxilline; n = 7 from 7 animals), suggesting that the Ca2+ spark/transient BK current

79 pathway is also involved in PACAP-induced dilation (figure 3C and 3E). Thus, when applied separately, blockers of KATP channels (glibenclamide) and BK channels

(paxilline) both decreased PACAP-induced dilations by approximately 50%.

Interestingly, the combined treatment of arteries with both glibenclamide and paxilline abolished PACAP-induced cerebellar artery dilation. In the presence of glibenclamide and paxilline, arterial diameter before and after PACAP treatment was 174.0 ± 21.9 µm and 163.5 ± 23.7 µm, respectively (n = 4 from 4 animals, figure 3D and 3E). In summary, these data indicate that PACAP causes cerebellar artery dilation through activation of both KATP and BK channels.

3.5 Discussion

Here we demonstrate that two distinct mechanisms contribute to PACAP-induced cerebellar artery dilation; activation of KATP channels and BK channels. The involvement of smooth muscle KATP and BK channels in cerebral artery dilations caused by PACAP

(depicted in figure 4) is supported by our following observations: 1) PACAP increased

KATP channel activity in isolated rat cerebellar artery myocytes, 2) PACAP also increased the frequency of cerebral artery smooth muscle transient BK currents, 3) Individual application of either the KATP channel blocker (glibenclamide) or the BK channel blocker

(paxilline) significantly attenuated PACAP-induced cerebral artery dilation (~ 50 % attenuation in the presence of either blocker) and 4) the combined treatment of arteries with glibenclamide and paxilline abolished PACAP-induced dilation of pressurized rat cerebellar arteries.

In vascular smooth muscle, functional KATP channels are thought to be composed of four Kir 6.1 pore-forming subunits encoded by the gene KCNJ8 and four SUR2B accessory subunits encoded by the gene ABCC9 (Flagg et al. 2010). KATP channels of arterial myocytes are activated by a variety of endogenous vasodilators such as CGRP

(Nelson et al. 1990; Quayle et al. 1994; Kleppisch et al. 1995a; Kleppisch et al. 1995b;

Wellman et al. 1998) as well as cAMP analogs and cell dialysis with the catalytic subunit of PKA (Quayle et al. 1994; Wellman et al. 1998). PKA-mediated phosphorylation of serine and threonine residues within the nucleotide binding folds of SUR2B subunits is thought to promote ADP binding to increase channel activity (Quinn et al. 2004; Shi et al.

2007; Flagg et al. 2010). In this study, we demonstrate that PACAP, another endogenous vasodilator known to stimulate adenylyl cyclase activity (Tong et al. 1993; Vaudry et al.

2009), can activate KATP channels in rat cerebral artery smooth muscle. As shown Figure

1, activation of KATP currents by pinacidil and PACAP evoked inward membrane currents at -60 mV using intracellular and extracellular solutions that both contained 140 mM K+. However, with physiological K+ concentrations (e.g. extracellular K+ ≈ 5.4 mM;

+ intracellular K ≈ 140 mM), KATP channel activation causes outward membrane currents in isolated arterial smooth muscle cells and membrane potential hyperpolarization and vasodilation of intact arteries (Standen et al. 1989; Masuzawa et al. 1990; Quayle et al.

1994; Kleppisch et al. 1995b; Nelson et al. 1995b; Bruch et al. 1997; Bruch et al. 1998).

Consistent with our electrophysiological recordings, PACAP-induced cerebellar artery dilation was significantly attenuated by the KATP channel blocker glibenclamide (Figure

3). Together, these data indicate that KATP channel activation contributes to PACAP-

81 induced vasodilation in rat cerebellar arteries likely via activation of the cAMP/ PKA pathway.

In addition to activation of KATP channels, increased intracellular cAMP and PKA activation can also enhance the Ca2+ spark/transient BK current signaling pathway to promote cerebral artery dilation (Porter et al. 1998; Wellman et al. 2001). Our data now shows that PACAP increases the frequency of transient BK currents in rat cerebellar artery myocytes (figure 2) and that BK channel activity is involved in PACAP-induced cerebral artery dilation (figure 3). Previous investigations have revealed three mechanisms that can contribute to PKA-mediated increased Ca2+ spark/BK channel activity. First, PKA can phosphorylate ryanodine receptors (RyRs), which are the sarcoplasmic reticulum (SR) Ca2+ release channels responsible for Ca2+ spark generation

(Marx et al. 2000; Wehrens et al. 2006). Phosphorylation of RyRs by PKA increases their Ca2+ sensitivity resulting in elevated frequencies of Ca2+ sparks and Ca2+-spark induced transient BK currents. Secondly, phosphorylation of phospholamban (PLB) can also contribute to PKA-mediated cerebral artery dilation (Simmerman et al. 1998;

Wellman et al. 2001). Phospholamban is a regulatory protein that when bound to SR

Ca2+-ATPase (SERCA) causes a decrease in SERCA activity and reduced filling of the

SR with Ca2+. Phosphorylation by PKA causes PLB to dissociate from SERCA, leading to disinhibition of SERCA activity and elevation of SR Ca2+ load (Simmerman et al.

1998; Wellman et al. 2001) that can increase Ca2+ spark/BK current frequency and amplitude (ZhuGe et al. 1999). Lastly, PKA can increase BK channel activity via direct channel phosphorylation (Minami et al. 1993; Perez et al. 1994; Porter et al. 1998).

Phosphorylation of BK channels by PKA causes a leftward-shift in the voltage-activation relationship (toward more negative membrane potential) without changing single channel conductance (Perez et al. 1994). Further, it has been suggested that cAMP can directly increase the open-state probability of BK channels based on the observation of increased single BK channel activity by application of cAMP to the intracellular side of excised membrane patches (Minami et al. 1993). A direct cAMP/PKA-mediated increase in BK channel activity would likely cause an increase in the amplitude of transient BK currents without altering Ca2+ spark frequency. In the present study, we demonstrate that PACAP increases the frequency, but not amplitude, of transient BK currents in isolated rat cerebellar artery myocytes. This observation is consistent with PACAP causing an increase in Ca2+ spark frequency rather than directly altering the voltage-sensitivity of plasma membrane BK channels. Future studies are required to confirm the involvement of the cAMP/PKA pathway in PACAP-induced cerebral artery dilation, as well as the specific proteins involved in the observed increase in Ca2+ spark/transient BK current frequency. However, we clearly demonstrate the functional importance of the Ca2+ spark/BK channel pathway in PACAP-induced cerebellar artery dilation. As illustrated in figure 3, the BK channel blocker, paxilline, attenuated PACAP-induced vasodilation by approximately ~50%. Furthermore, the combined treatment of cerebral arteries with paxilline and the KATP channel blocker, glibenclamide, completely inhibited cerebral artery responses to PACAP. These data suggested that two pathways, activation of KATP and BK channels, are independent and both play an important role in PACAP-induced cerebellar artery dilation.

In summary, here we demonstrate that two distinct cell signaling mechanisms contribute to PACAP-induced dilation in rat cerebellar arteries; KATP channel activation and enhanced BK channel activity. Both mechanisms likely involve activation of the cAMP/PKA pathway, however further studies are needed to determine additional details of the intracellular signaling pathways linking PACAP receptor activation to these ion channels.

Acknowledgements

This work was supported by the Totman Medical Research Trust Fund, the Peter Martin

Aneurysm Endowment and National Institutes of Health Grants P01-HL-2095488 and

NCRR P30RR032135/NIGMS P30GM103498.

Disclosures

No conflicts of interest, financial or otherwise, are declared by the author(s).

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3.7 Figures

Figure 1: KATP channel currents in rat cerebellar artery myocytes. A) KATP current measurement obtained from an isolated cerebellar artery myocyte. Whole-cell patch clamp recording conditions were used to optimize KATP currents and minimize other BK

+ and KV channel currents (e.g. 140 mM K in extracellular bath solutions, 0.1 mM ATP and 0.1 mM ADP in intracellular pipette solution, -60 mV of holding potential). The synthetic KATP channel opener, pinacidil (10 µM), caused an increase in inward current that was inhibited by the KATP channel blocker, glibenclamide (10 µM). B) Original recording of PACAP treatment on inward K+ current using the same recording conditions described panel A. PACAP (3 nM) increased inward K+ current, suggesting increased

KATP current.

Figure 2: PACAP enhanced transient BK current activity in rat cerebellar artery myocytes. A) Effect of PACAP (3 nM) on transient outward BK currents, showing increased frequency, but not amplitude, in rat cerebellar artery myocytes. Transient BK currents were measured at -40 mV. Expanded traces of transient outward BK currents are shown in lower panels. B and C) Summary data of transient outward BK current frequency (B) and amplitude (C). n = 6 cells from 3 animals. ** P < 0.01 vs before

PACAP treatment (-PACAP). 91

Figure 3: PACAP induced vasodilation is mediated via both KATP and BK channels in isolated rat cerebellar arteries. A) Representative trace of PACAP-induced dilation of a cerebellar artery. The artery was pressurized to 60 mmHg and treated with PACAP

(3 nM) by bath superfusion. B and C) PACAP-induced vasodilation was significantly attenuated in the presence of the KATP channel blocker, glibenclamide (10 µM, panel B) or the BK channel blocker, paxilline (1 µM, panel C). D) The combined treatment of glibenclamide (Glib) and paxilline (Pax) abolished PACAP-induced cerebellar artery dilation. E) Summary data of PACAP-induced dilations in the absence and presence of glibenclamide and paxilline (n = 4-7). Data are expressed as mean ± SE. * P < 0.05, **

P < 0.01 vs PACAP-induced dilation in the absence of K+ channel blockers (Control).

Figure 4: Proposed mechanisms of PACAP-induced vasodilation in rat cerebellar artery. AC: adenylyl cyclase, ATP: adenosine tri-phosphate, BK: large-conductance

2+ + Ca -activated potassium (K ) channels, cAMP: cyclic adenosine mono-phosphate, KATP:

ATP-sensitive potassium (K+) channels, PACAP: pituitary adenylate cyclase activating polypeptide, PKA: protein kinase A, PLB: phospholamban, RyR: ryanodine receptors,

SERCA: sarcoplasmic reticulum calcium transport ATPase, SR: sarcoplasmic reticulum,

VDCC: voltage-dependent Ca2+ channels.

CHAPTER 4: JOURNAL ARTICLE

Tonic Regulation of Middle Meningeal Artery Diameter by ATP-sensitive Potassium Channels

Authors: Arsalan U. Syed, Masayo Koide, Joseph E. Brayden, George C. Wellman*

Institutions: Department of Pharmacology University of Vermont College of Medicine 149 Beaumont Avenue Burlington, Vermont 05405 USA * Corresponding author

4.1 Abstract

ATP-sensitive potassium (KATP) channels have been suggested to play a crucial role in the regulation of membrane potential and diameter in several vascular beds. The objective of the current study was to determine if KATP channels are tonically regulating middle meningeal artery (MMA) diameter, a vascular bed suggested to be involved in migraine headache. Using ex vivo diameter measurements, smooth muscle membrane potential measurements and ratiometric calcium measurements, we demonstrate that basal KATP channel activity, due to ongoing cyclic AMP-dependent protein kinase (PKA) activity, profoundly impacts MMA diameter.

4.2 Introduction

Potassium channels play a crucial role in regulation of vascular diameter.

Activation of these channels results in hyperpolarization of the membrane potential (EM)

+ towards the equilibrium potential of K (EK). The resulting hyperpolarization reduces the open-state probability of L-type voltage gated calcium channels, reducing the influx of calcium and causing relaxation of vascular smooth muscle. One key potassium channel in vascular smooth muscle is the ATP-sensitive potassium (KATP) channel (Nelson and

Quayle, 1995). In vascular smooth muscle the predominant KATP channel isoform consists of four pore forming Kir 6.1 subunits and four sulphonylurea receptor SUR2B subunits (Nichols, 2006). KATP channels are important targets for protein kinase A (PKA) in response to increased adenylate cyclase activity in vascular smooth muscle myocytes 95

(Shi et al., 2007). PKA can phosphorylate serine 385 (S385) residue located on Kir 6.1 subunits and two sites on SUR2B subunits, residue serine 1465 (S1465) and threonine

633 (T633) (Quinn et al., 2004). Phosphorylation of these sites have been proposed to enhance binding of the stimulatory Mg-ADP to the nucleotide binding folds (NBFs) leading to activation (Nichols, 2006). Under physiological conditions, treatment with the

KATP channel inhibitor, glibenclamide induces constriction in the meningeal vascular bed while it induces no effect in mesenteric or brain surface arteries (pial) (Quayle et al.,

1995, Koide et al., 2014).

In the present study we investigate basal regulation of MMA diameter by KATP channels. Our findings indicate that KATP channels are tonically regulating MMA diameter. Furthermore, this tonic regulation is mediated by protein kinase A (PKA).

4.3 Methods

Material, methods and references associated with them are available in supplemental information on the JCBFM website.

4.4 Results

Inhibition of basal KATP channel activity induces MMA constriction

Involved in setting membrane potential of vascular smooth muscle cells, potassium channels, including KATP channels have been described as key regulators of resistance artery diameter (Quayle et al., 1997). To explore and establish the role of KATP channels in the regulation of MMA diameter, we utilized a pressurized ex-vivo diameter measurement technique that has been previously described (Syed et al., 2012). At an 96 intravascular pressure of 40 mm Hg, MMA diameter was 54.3 ± 2.4 µm in artificial cerebral spinal fluid (aCSF), representing a 40 ± 2 % (n = 30 arteries) constriction compared to the fully dilated diameter of 90 ± 3 µm. Treatment with the KATP channel inhibitor, glibenclamide (10 µM), caused an additional constriction representing a further

20 ± 2 % decrease in lumen diameter (Figure 1 A, n = 5). Furthermore, treatment with another KATP channel inhibitor, PNU37883 (10 µM), resulted in a similar vasoconstriction (20 ± 3 % decrease in diameter, Figure 1 A). In contrast, treatment of cerebellar arteries with glibenclamide (10 µM) or PNU37883 (10 µM) did not significantly affect diameter (Figure 1 A n = 5). These results suggest that there is basal

KATP channel activity in MMA, but not in cerebral arteries.

Inhibition of KATP channels leads to membrane potential depolarization in MMA

Inhibition of potassium channels leads to membrane potential depolarization and vasoconstriction (Brayden et al., 1991). As inhibition of KATP channels causes MMA vasoconstriction, we hypothesized that KATP channels are contributing to MMA smooth muscle membrane potential. At 40 mmHg, MMA smooth muscle membrane potential was -45.4 ± 1.4 mV. Treatment with 10 µM glibenclamide resulted in significant membrane potential depolarization to -31.3 ± 2.7 mV (Figure 1 B, n= 4 each), representing a change in membrane potential of approximately 14 mV. In comparison, cerebellar artery smooth muscle membrane potential was not significantly different in the absence (-37.1 ± 0.9 mV) and presence (-36.8 ± 0.8 mV) of glibenclamide (Fig. S 1 A, n

= 4). This data supports our hypothesis that KATP channels contribute a tonic hyperpolarizing influence to MMA smooth muscle membrane potential.

Inhibition of KATP channels increases intravascular calcium in MMA smooth muscle via L-VDCC

Given that inhibition of KATP channels induced vasoconstriction and membrane potential depolarization, we hypothesized that glibenclamide causes increased Ca2+ influx via enhanced activity of L-type VDCCs. To first test this hypothesis, we examined glibenclamide-induced changes in arterial wall calcium using the ratiometric calcium indicator, Fura 2. Arteries were initially equilibrated at 10 mmHg where the diameter was

37.8 ± 5.6 µm with an average vascular smooth muscle calcium level of 179 ± 28 nM

(n=18). At 40 mmHg MMA developed myogenic tone of 18.3 ± 2.9 %, accompanied with an increase of arterial wall calcium to 470 ± 50 nM. Treatment with 10 µM glibenclamide evoked an average vasoconstriction of 23.4 ± 1.0 % with an increase of

187.5 ± 77 nM in intracellular calcium corresponding to an increase in Fura-2

F340/F380-ratio of 0.24 ± 0.03 (Figure 1 C, n = 4). These data demonstrate that inhibition of KATP leads to an increase smooth muscle intracellular calcium.

As L-VDCCs play a critical role in development of myogenic tone, the following experiments were performed at 10 mmHg to minimize the potentially confounding effects of the L-type VDCC inhibitor, diltiazem, on myogenic tone. In the absence of diltiazem (100 µM), glibenclamide (10 µM) resulted in vasoconstriction of 21.2 ± 2.6 %

(Fig. S 2 A and C, n = 13) representing a change in diameter of 14.7 ± 2.1 µm. However, in the presence of diltiazem, the glibenclamide-induced vasoconstriction was abolished

(Fig. S 2B, n = 5). Together, these results support our hypothesis that increased cytosolic

98 calcium following KATP channel inhibition is due to enhanced activity of L-VDCC in

MMA smooth muscle.

Regulation of KATP channels by basal PKA activity in MMA smooth muscle

Previous studies using the patch clamp technique and isolated rat mesenteric artery myocytes have shown that basal KATP activity is dependent on tonic PKA activity

(Hayabuchi et al., 2001, Sampson et al., 2004, Dart, 2014). We therefore hypothesized that KATP channels in the MMA are being tonically regulated by basal PKA activity. We tested this hypothesis by treating MMAs pressurized to 40 mmHg with the PKA inhibitor

KT5720 (1 µM); this resulted in a vasoconstriction of 25.2 ± 5.2 % (Figure 2 A and B, n

= 4). In contrast, KT5720 induced no significant vasoconstriction in cerebellar arteries

(1.9 ± 2.3 %). Furthermore, in the presence of KT5720, glibenclamide (10 µM) did not induce any further constriction of MMA (Figure 2 A). These findings suggest that basal

PKA activity contributes to opening of KATP channels in MMA. Additionally, treatment with another PKA inhibitor, H89 (1 µM) resulted in significant membrane potential depolarization from -46.8 ± 0.5 mV to 33.5 ± 0.6 mV and no further depolarization was observed with addition of 10 µM glibenclamide (Fig. 1S C, n = 4).

Our data indicate that KATP channels are endogenously active in MMA and that

PKA is responsible for this phenomenon. These findings are consistent with the concept that basal PKA activity is contributing to sensitization of KATP channels (Quinn et al.,

2004). To further explore this possibility, cumulative concentration-response curves to the KATP channel agonist, cromakalim, were obtained using both MMA and cerebellar arteries. We observed that MMAs were 10-fold more sensitive to cromakalim (EC50 of 99

100 nM) compared to cerebellar arteries (EC50 of 1 µM, Figure 2 C n = 4-5). However, in

MMA, PKA inhibition with KT5720 (1 µM) resulted in a decrease in cromakalim sensitivity (EC50 of 300 nM, Figure 2 C).

4.5 Discussion

The results presented in this study provide strong evidence of tonic regulation of

MMA diameter by KATP channels. Inhibition of these channels leads to membrane potential depolarization, increased intracellular calcium and vasoconstriction of MMA

(Figure 1-2). In marked contrast, KATP inhibition did not alter cerebral artery diameter.

Our data also indicate that PKA activity underlies basal KATP channel activity observed in the MMA.

Previous studies have suggested that KATP channels can be activated through a signal transduction pathway involving increased adenylyl cyclase activity, enhanced levels of cAMP and activation of PKA (Hayabuchi et al., 2001, Sampson et al., 2004).

KATP channels can be phosphorylated and activated by PKA (Quinn et al., 2004, Shi et al., 2007) and exogenous agonists of cAMP such as forskolin can elicit KATP currents in isolated smooth muscle myocytes (Wellman et al., 1998). Our results provide strong evidence of ongoing PKA regulation of KATP channels in isolated pressurized MMA.

However, the question still remains as to why PKA is basally active in this tissue? One possibility could be basal release of neuropeptides such as calcitonin gene related peptide

(CGRP) and pituitary adenylate cyclase activating polypeptide (PACAP) from trigeminal nerves, which heavily innervate the MMA (Ploug et al., 2008, Ploug et al., 2012).

Receptors of these neuropeptides are Gs-cAMP coupled leading to increased PKA 100 activity and are present in MMA (Edvinsson et al., 1987, Edvinsson, 2015b, a).

Additionally, it is plausible that protein phosphatase 1 (PP1) and/or protein phosphatase

2A (PP2A) that are responsible for dephosphorylation, may have lower activity in MMA smooth muscle myoctes (Quinn et al., 2004, Brignell et al., 2015). Similarly, we can speculate that the activity of phosphodiesterase 3 (PDE3) may be attenuated in the MMA smooth muscle myocytes. PDE3 is the dominant isoform in vascular smooth muscle responsible for metabolizing and inactivating cAMP and this process is independent of physiological pressure (Elvebak et al., 2010). However, it is also possible that the apparent increase in basal KATP activity in MMA compared to cerebral arteries represents a higher relative membrane resistance in MMA smooth muscle independent of KATP channel activity (Zimmermann et al., 1997).

In conclusion, our data suggests that KATP channels may be playing a fundamental role in tonic regulation of MMA diameter. The MMA has been implicated in the pathophysiology of migraine headache and several vasodilators have been suggested to induce migraine like symptoms including KATP channel openers (D'Arcy et al., 1985,

Goldberg, 1988, Sterndorff and Johansen, 1988, Ploug et al., 2008, Ploug et al., 2012).

Thus, it is conceivable that aberrant signaling in KATP channels may contribute to the onset of migraine headache.

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4.6 Reference List

Brayden JE, Quayle JM, Standen NB, Nelson MT (1991) Role of potassium channels in the vascular response to endogenous and pharmacological vasodilators. Blood Vessels 28:147-153.

Brignell JL, Perry MD, Nelson CP, Willets JM, Challiss RA, Davies NW (2015) Steady- state modulation of voltage-gated K+ channels in rat arterial smooth muscle by cyclic AMP-dependent protein kinase and protein phosphatase 2B. PLoS One 10:e0121285.

D'Arcy V, Laher M, McCoy D, Sullivan P, Walsh CH, Hickey MP (1985) Pinacidil, a new vasodilator, in the treatment of mild to moderate essential hypertension. Eur J Clin Pharmacol 28:347-349.

Dart C (2014) Verdict in the smooth muscle KATP channel case: guilty of blood pressure control but innocent of sudden death phenotype. Hypertension 64:457-458.

Edvinsson L (2015a) CGRP receptor antagonists and antibodies against CGRP and its receptor in migraine treatment. Br J Clin Pharmacol 80:193-199.

Edvinsson L (2015b) PACAP and its receptors in migraine pathophysiology: Commentary on Walker et al., Br J Pharmacol 171: 1521-1533. British journal of pharmacology 172:4782-4784.

Edvinsson L, Ekman R, Jansen I, McCulloch J, Uddman R (1987) Calcitonin gene- related peptide and cerebral blood vessels: distribution and vasomotor effects. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism 7:720-728.

Elvebak RL, Eisenach JH, Joyner MJ, Nicholson WT (2010) The function of vascular smooth muscle phosphodiesterase III is preserved in healthy human aging. ClinTranslSci 3:239-242.

Goldberg MR (1988) Clinical pharmacology of pinacidil, a prototype for drugs that affect potassium channels. J CardiovascPharmacol 12 Suppl 2:S41-S47.

Nelson MT, Quayle JM (1995) Physiological roles and properties of potassium channels in arterial smooth muscle. AmJ Physiol 268:C799-C822.

Nichols CG (2006) KATP channels as molecular sensors of cellular metabolism. Nature 440:470-476.

Quayle JM, Bonev AD, Brayden JE, Nelson MT (1995) Pharmacology of ATP-sensitive K+ currents in smooth muscle cells from rabbit mesenteric artery. AmJ Physiol 269:C1112-C1118.

Quayle JM, Nelson MT, Standen NB (1997) ATP-sensitive and inwardly rectifying potassium channels in smooth muscle. Physiol Rev 77:1165-1232.

Quinn KV, Giblin JP, Tinker A (2004) Multisite phosphorylation mechanism for protein kinase A activation of the smooth muscle ATP-sensitive K+ channel. Circulation research 94:1359-1366.

Sampson LJ, Hayabuchi Y, Standen NB, Dart C (2004) Caveolae localize protein kinase A signaling to arterial ATP-sensitive potassium channels. Circulation research 95:1012- 1018.

Shi Y, Wu Z, Cui N, Shi W, Yang Y, Zhang X, Rojas A, Ha BT, Jiang C (2007) PKA phosphorylation of SUR2B subunit underscores vascular KATP channel activation by beta-adrenergic receptors. Am J Physiol Regul Integr Comp Physiol 293:R1205-1214.

Sterndorff B, Johansen P (1988) The antihypertensive effect of pinacidil versus prazosin in mild to moderate hypertensive patients seen in general practice. Acta MedScand 224:329-336.

103

Zimmermann PA, Knot HJ, Stevenson AS, Nelson MT (1997) Increased myogenic tone and diminished responsiveness to ATP-sensitive K+ channel openers in cerebral arteries from diabetic rats. Circulation research 81:996-1004.

104

4.7 Figures

MMA MMA

Figure 1: Tonic KATP channel activity contributes to MMA diameter regulation via membrane potential depolarization and increased intracellular calcium. (A)

Representative traces demonstrating the effect of 10 µM glibenclamide on MMA at 40 mmHg and cerebellar arteries at 60 mmHg. Bar graph represents summary data of glibenclamide and PNU37883-induced constriction in MMA vs cerebellar artery. Passive diameter (maximally dilatation) was induced by Ca2+-free aCSF containing 100 µM diltiazem and 1 µM forskolin. P < 0.01 for glibenclamide and P < 0.001 for PNU37883 in

MMA vs cerebellar artery (B) Representative traces of smooth muscle membrane potential measurements in isolated MMA at 40 mmHg in the absence and presence of 10

µM glibenclamide. Bar graph represents summary data of smooth muscle membrane

105 potential measurements obtained in the presence of 10 µM glibenclamide in MMAs.

(n=5) P < 0.01 before treatment vs MMA, using paired t-test. (C) Representative trace of increased fura-2 340/380 nm emission ratio caused by 10 µM glibenclamide. Bar graph represents summary data of 10 µM glibenclamide-induced fura-2 calcium change (n=5).

P < 0.05 glibenclamide vs control using paired t-test.

106

Figure 2: Basal PKA activity underlies tonic KATP channel activity in MMA. (A)

Representative trace of 1 µM KT5720-induced vasoconstriction in the presence or absence of glibenclamide (10 µM). Bar graph represents summary data of glibenclmaide- induced vasoconstriction in the presence and absence of 1 µM KT5720 (n=4) P < 0.01 unpaired t-test. (B) Bar graph representing summary data of 1 µM KT5720-induced constriction in MMA vs. cerebellar artery (n=4 each) P < 0.01 unpaired t-test. (C)

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Summary of cromakalim concentration-response curves of MMA and cerebellar arteries.

EC50 values were 100 nM and 300 nM for cromakalim-induced concentration-dependent vasodialtion in the absence and presence of 1 µM KT5720 in MMA (n = 4 each). EC50 value was 1 µM for cromakalim-induced concentration-dependent vasodilation in cerebellar arteries (n = 5).

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4.8 Supplemental Methods Methods

All experiments were conducted in accordance with the Guide for the Care and Use of

Laboratory animals (eighth edition, 2011) and approved by the Institutional Animal Use and Care Committee of the University of Vermont. Sprague–Dawley rats (males, 300–

350 g; Charles River Laboratories, Saint Constant, QC, Canada) were euthanized by decapitation under deep pentobarbital anesthesia. The brain and calvaria were carefully dissected and placed in ice-cold artificial cerebral spinal fluid (aCSF) in millimolar: 125

NaCl, 3 KCl, 18 NaHCO3, 1.25 NaH2PO4, 1 MgCl2, 2 CaCl2, and 5 glucose (pH 7.4).

Dura mater was removed from the calvaria and MMA dissected using iris scissors.

Similarly cerebellar artery was dissected from the brain using iris scissors.

Ex vivo diameter measurements: MMAs were cannulated onto glass micropipettes mounted in a 5-mL myograph chamber perfused with aCSF at 37 °C. The vessel was equilibrated for 1 hr at an intravascular pressure of 10 mmHg using a pressure servo- controller and a peristaltic pump as previously described 8. Throughout the experiment, changes in vessel diameter were measured by video edge detection (Living Systems

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

Instruments; Akron, OH). After allowing the arteries to equilibrate for 1 hour, viability of the artery was determined by brief superfusion with 60 mM KCl in artificial cerebrospinal fluid (aCSF; isosmotic replacement of NaCl with KCl). Only those arteries that constricted to at least 50% of its initial diameter in the presence of 60 mM KCl were used in subsequent studies. After initial viability tests the vessels were subjected to an

109 increase in intravascular pressure to 40 mmHg and allowed to develop myogenic tone

(pressure-induced constriction). Myogenic tone is expressed as a percent decrease of the fully dilated diameter of individual arteries at the same intravascular pressure. The values were obtained by using the following equation:% myogenic tone= (DP-DA)/DP, where

DP is the passive diameter of the artery in Ca2+-free aCSF containing the vasodilators diltiazem (100 µM) and forskolin (1 µM) and DA is the active diameter of the artery in

Ca2+-containing aCSF . Vasoconstriction induced by a drug treatment was calculated using the following equation % constriction = [(DA−DC)/DA]×100, where DA is the active diameter of the artery in Ca2+-containing aCSF and DC is the active diameter of the artery in Ca2+-containing aCSF with the drug during vasoconstriction. Similarly % dilation were obtained using the following equation: % dilation = [(DV-DA)/(DP-

DA)]x100, where DV is the diameter with vasodilator treatment, DA is the active diameter of the artery in Ca2+-containing aCSF and DP is the passive diameter of the artery in Ca2+-free aCSF containing the vasodilators diltiazem (100 µM) and forskolin (1

µM). For experiments done in the presence of VDCC channels blocker (diltiazem), pressure was kept at 10 mmHg. This was done in order avoid complication due to pressure-induced constriction caused by myogenic tone.

Ratiometric calcium measurments: Freshly isolated MMA were cannulated on glass micropipettes mounted in a 5-ml myograph chamber as detailed above. Arteries were then loaded with 5 µM of the ratiometric Ca2+-sensitive dye fura-2 acetoxymethyl ester

(fura-2 AM, Invitrogen, Carlsbad, CA) in aCSF solution containing pluronic acid (0.1%) at room temperature (~22°C) for 1 hour. Next, the myograph chamber was mounted on a

110

Nikon TE2000-S inverted fluorescence microscope coupled with IonOptix calcium, diameter and flow system in pressurized vessels (Milton, MA). To allow for equilibration and de-esterification of fura-2 AM. Isolated MMA arteries were pressurized to 40 mmHg and continuously superfused (37°C, 30 min). Fura-2 was excited simultaneously by two separate wavelengths (340 and 380 nm) and fluorescence emission was measured at 510 nm, the background signal was subtracted and the ratio between the intensity levels were

2+ used to calculate the free calcium concentration using the following equation: [Ca ] = Kd

28 (R-Rmin)/(Rmax-R)×(Sf2/Sb2) . Rmax and Rmin are the ratio values (F340/F380) measured under conditions of saturating calcium levels and in the absence of calcium respectively.

Sb2 and Sf2 are proportional to the fluorescence excited by the denominator wavelength

(380 nm) under the saturated calcium condition (Sb2) and in the absence of calcium (Sf2).

2+ Sb2 and Sf2 values were calculated in the presence of ionomycin with Ca - free and

2+ saturated Ca condition. The Kd value used is 282 nM as estimated previously by Knot et al., for fura-2 calcium binding 29.

Membrane potential measurements: Arteries were cannulated as described above and pressurized to 40 mmHg (MMA) and 60 mmHg (cerebellar artery). Smooth muscle membrane potential was measured by insertion of a sharp glass microelectrode with resistance (~200 -MΩ) containing 0.5 M KCl into the vessel wall. The criteria for successful impalement are 1) an abrupt negative potential deflection upon entry, 2) a stable membrane potential for ≥30 s, and 3) an abrupt positive potential deflection upon removal 29. Measurements were made with an electrometer (World Precision

111

Instruments) and recorded via computer with Axotape and Dataq software. All recordings were done in the presence of 300 nM nimodipine.

Statistics: Data are expressed as mean ± SEM and analyzed by Student’s paired and unpaired t test. Statistical significance was considered at p<0.05.

Chemicals: PNU37883, KT5720 and H89 were purchased from Tocris Bioscience

(USA). Glibenclamide and rest of the chemicals were ordered from Sigma-Aldrich (St.

Louis MO, USA).

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4.9 Reference List

Kao,J.P., Li,G., & Auston,D.A. Practical aspects of measuring intracellular calcium signals with fluorescent indicators. Methods Cell Biol. 99, 113-152 (2010). Knot,H.J. & Nelson,M.T. Regulation of arterial diameter and wall [Ca2+] in cerebral arteries of rat by membrane potential and intravascular pressure. J Physiol 508 ( Pt 1), 199-209 (1998). Nystoriak,M.A. et al. Fundamental increase in pressure-dependent constriction of brain parenchymal arterioles from subarachnoid hemorrhage model rats due to membrane depolarization. Am. J Physiol Heart Circ. Physiol 300, H803-H812 (2011). Syed,A.U., Koide,M., Braas,K.M., May,V., & Wellman,G.C. Pituitary adenylate cyclase- activating polypeptide (PACAP) potently dilates middle meningeal arteries: implications for migraine. J Mol. Neurosci. 48, 574-583 (2012).

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4.10 Supplemental Figures

Supplemental Figure 1 (S 1): (A) Representative traces of smooth muscle membrane potential measurements in cerebellar artery at 60 mmHg in the absence and presence of

10 µM glibenclamide. Bar graph represents summary of smooth muscle membrane potential measurements obtained in the presence of 10 µM glibenclamide in cerebellar arteries. (n=5) (B) Representative trace of membrane potential in the absence and presence of vehicle (DMSO) used to solubilizing KATP channel inhibitor glibenclamide and PKA inhibitor H89 (1 µM). Bar graph represents summary data for effect of DMSO before and after treatment (n=4). (C) Representative trace of 10 µM glibenclamide induced membrane potential depolarization in the absence and presence of PKA inhibitor 114

H89. Bar graph represents summary data of 10 µM glibenclamide-induced depolarization in the presence and absence of PKA inhibitor H89 (1 µM) (n = 4).

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Supplemental Figure 2 (S 2): (A-B) Representative trace of 10 µM glibenclamide- induced diameter change at 10 mmHg in the absence (A) and presence (B) of the L-

VDCC blocker diltiazem (100 µM). (C) Bar graph representing summary data of glibenclamide-induced diameter change of rat MMA in the presence and absence of diltiazem. P < 0.002 absence vs presence of 100 µM diltiazem (n=13 and 5).

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CHAPTER 5: DISCUSSION AND FUTURE DIRECTIONS

The dura mater is heavily innervated by sensory nerves and acts not only as a protective barrier for the brain but also plays a key role in migraine headache (Asghar et al., 2010, Asghar et al., 2011, Amin et al., 2012, Chmielewski et al., 2013, Schueler et al.,

2013, Amin et al., 2014). The MMA is the main arterial branch supplying blood to the dura mater and vasodilation of this vascular bed has been hypothesized to play a critical role in migraine headache genesis (Asghar et al., 2011, Amin et al., 2012). Furthermore, several researchers have hypothesized that the neuropeptide PACAP may be a key player in the generation of migraine headache; however, the reported effects of PACAP on

MMA have been ambiguous (Schytz et al., 2009, Schytz et al., 2010a, Schytz et al.,

2010b, Baun et al., 2011, Baun et al., 2012). The research focus of my dissertation was to decipher the role of PACAP in the regulation of MMA diameter. During the course of this study, we also discovered that basal KATP channel activity contributes to the regulation of MMA diameter.

In chapter two, we evaluated the effect of PACAP and VIP on MMA and cerebral arteries. Our findings demonstrated that PACAP is a potent vasodilator of the MMA, inducing vasodilation in MMA at picomolar concentrations as opposed to nanomolar concentrations in cerebral arteries. We further established that the PAC1-R-Hop splice variant of the PAC1 receptor is present in the MMA (Syed et al., 2012). However, although we detected PACAP receptors PAC1-R-Hop and VPAC2 transcripts in the

MMA whole artery lysate, we did not determine the cellular location of these receptors and this remains to be resolved. PACAP receptors have been reported to be present on 117 vascular smooth muscle cells of MMA but have also been demonstrated to be present on dural mast cells encasing the MMA. Baun et al. have reported that activation of the PAC1 receptor on dural mast cells induces degranulation invoking an inflammatory response by releasing histamine and cytokines such as TNF-α (Baun et al., 2012). It is possible that these inflammatory mediators along with PACAP act on vascular smooth muscle to induce a prolonged dilation of MMA. One possible approach to examine this would be to use laser capture micro-dissection to specifically isolate MMA smooth muscle cells.

RNA extraction and polymer chain reaction (PCR) could then confirm the presence of

PAC1 receptors in MMA myocytes.

Clinical studies have demonstrated that PACAP-induced dilation of MMA but not of the cerebral arteries is thought to be responsible for the onset of migraine headache

(Asghar et al., 2010, Asghar et al., 2011, Amin et al., 2012, Amin et al., 2014). The mechanism by which PACAP is inducing dilation in the MMA and cerebral artery is not known. Therefore, in appendix one we explored the mechanism that PACAP employs in order to induce dilation in the MMA. We discovered that in the presence of KATP channel inhibitors PACAP-induced vasodilation of the MMA was abolished. This data suggested that PACAP works via KATP channels to induce vasodilation in the MMA (AU et al.,

2016). Further, BK channel inhibition and eNOS inhibition did not affect the PACAP- induced vasodilation of MMA, suggesting that PACAP is working exclusively via KATP channels in the MMA vascular smooth muscle cells. In contrast, in chapter 3 we found that PACAP engaged both KATP and BK channels to induce vasodilation in cerebral arteries. The spatial and temporal regulation of KATP channels by PKA is not clear and

118 requires further investigation. However, A-kinase anchoring protein (AKAP) has been postulated to play a crucial role in specific PKA interactions providing higher fidelity and compartmentalization in cellular signaling (Marx et al., 2000, Nystoriak et al., 2014).

Thus, PACAP receptors may be compartmentalized with KATP channels to form a complex in MMA smooth muscle cells. On the other hand, in cerebral artery myocytes,

PACAP receptors may compartmentalize with both KATP and BK channels.

Another future direction of this project would be the investigation of the interactions between PACAP, AKAP and PKA. Here, the first step would be to decipher the identity of the AKAP associated with PACAP receptors and PKA using co- immunoprecipitation experiments. This technique utilizes antibodies that specifically bind to the protein of interest, in this case PACAP receptors, to precipitate the protein along with any proteins that exist with the receptor in a complex. This can then be followed by mass spectrometry to identify the proteins associating with the PACAP receptors, including AKAPs. Further, super resolution microscopy would enable better visualization of the co-localization of the protein in question with PACAP receptors.

Identification of the AKAP involved with PACAP signaling in vascular smooth muscle could potentially provide additional down-stream therapeutic targets for migraine headache.

5.1 Regulation of membrane potential and vascular diameter by KATP channels

Membrane potential is defined as the electrical potential difference due to separation of charges across the cell membrane. For an artery pressurized at physiological pressure, resting membrane potential is approximately -40 mV (Knot and Nelson, 1998). 119

Two major factors contribute to the membrane potential of the cell: 1) the concentration of the ions inside and outside the cell and 2) the permeability of these ions through the lipid bilayer of the cell. In order to calculate the membrane potential for a given condition we can utilize the Goldman Hodgkin-Katz (GHK) equation:

! ! ! �� + �� + �� = ��( ! ! ! ) � �� + �� + ��

Where Em = membrane potential, P = permeability of the specific ion, R = ideal gas constant T= temperature and F= Faraday’s constant.

As evident from the GHK equation, the membrane potential is regulated by different ionic species and therefore ion channels and plasma membrane pumps play a crucial role in the regulation of membrane potential. In vascular smooth muscle, a tight relationship exists between membrane potential and vasodilation i.e., a small change in membrane potential (~1 mV) will induce a significant change in the diameter of the artery (~7.5 µm) by increasing or decreasing of calcium entry via L-voltage gated calcium channels (L-VDCC) (Nelson and Quayle, 1995, Knot and Nelson, 1998).

Potassium channels play a key role in regulation of vascular diameter by regulating membrane potential (Brayden et al., 1991, Brayden and Nelson, 1992, Nelson and Quayle, 1995, Brayden, 2002). In chapter four, we present evidence that KATP channels are involved in regulating MMA membrane potential and diameter. In contrast, pial arteries do not demonstrate tonic KATP channel activity. Our data not only suggest that KATP channels are tonically regulating MMA diameter but the reason for their tonic activation is due to basal activity of PKA. There are several possibilities to account for 120 basal PKA activity including but not limited to basal release of neuropeptides such as

PACAP or CGRP from the trigeminal sensory nerve innervating the MMA.

Alternatively, protein phosphatase 2B (PP2B) and or phosphodiestarase 3 (PDE3) activity may be low in the MMA smooth muscle cells (Quinn et al., 2004, Shi et al.,

2007, Elvebak et al., 2010). Therefore, the dephosphorylation of KATP channel or metabolism of cAMP maybe lower compared to cerebral artery smooth muscle. Future experiments for this project will aim to determine the source of PKA activation.

Additionally, depolarization induced by KATP channel inhibition may further activate BK channels, which are functionally present in the MMA smooth muscle, thus the mV change that we have observed may be an effect of multiple channels being regulated rather than just the KATP channels alone. Furthermore, we cannot state unequivocally that the increased sensitivity to KATP channel inhibition represents a specific increase in KATP channel activity. For example, Zimmerman et al., have demonstrated that in diabetic rat cerebral arteries, KATP channel agonist sensitivity is diminished due to decrease in tonic nitric oxide (NO) release (Zimmermann et al., 1997). One possible future direction for this project could be to test the effect of KATP channel agonist (cromakalim) in the presence of NO inhibitors. The prediction would be that if basal NO activity is increasing

KATP channel sensitivity in MMA, then in the presence of NO inhibitor, cromakalim EC50 value would shift from 100 nM to 1 µM, similar to that of cerebral arteries. If this is indeed the case, the next step would be to explore how NO is regulating KATP channel sensitivity in the MMA myocytes. Further, it is possible that factors other than enhanced

121 basal KATP channel activity are causing the apparent increase in sensitivity to KATP channel inhibitors in this tissue.

Another possible future direction for this project would be to examine KATP channel activity in isolated MMA smooth muscle cells. Using the whole cell patch clamp configuration, we can record KATP channel currents from isolated smooth muscle myocytes (Wellman et al., 1998, Hayabuchi et al., 2001). This would enable us to decipher if channel activity is higher in MMA myocytes compared to pial artery myocytes. Furthermore, this would also enable us to determine KATP channel density in

MMA myocytes. Additionally, using the whole cell patch clamp configuration we can also test PKA and NO regulation of KATP channels in MMA myocytes (Wellman et al.,

1998, Hayabuchi et al., 2001). Inhibition of PKA using PKA inhibitor KT5720 should result in inhibition of KATP channel currents, confirming tonic KATP channel regulation by basal PKA activity. Furthermore, we can test effect of NO on KATP channel activity and sensitivity.

Information gained from this research will not only help in providing a better understanding of the role of KATP channels in MMA diameter regulation but also help in identifying potential therapeutic targets for migraine headache.

5.2 Unpublished observations

Like PACAP, CGRP is endogenously expressed throughout the body and has pleiotropic effects (1992). Interestingly both PACAP and CGRP co-localize in several types of nerve fibers including sensory neurons (Edvinsson et al., 2001). CGRP binds to

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Gs-coupled calcitonin receptor like receptor (CRLR). Accessory CRLR subunits, receptor activity modifying proteins (RAMPs), modulate the specificity and cell surface expression of the receptor. CGRP receptors have been shown to be present in vascular smooth muscle (Edvinsson et al., 1987)and in the endothelium (Brain and Grant, 2004).

CGRP has been proposed to induce dilation via several different mechanisms. In mesenteric and coronary smooth muscle, CGRP has been shown to induce dilation via direct activation of KATP channels (Nelson et al., 1990, Wellman et al., 1998). In contrast, in vascular endothelium CGRP has been reported to work by activating endothelium nitric oxide synthase (eNOS) via PKA resulting in the production of nitric oxide, which in turn activates BK channels via cGMP and protein kinase G pathway in mice cerebral arteries (Brain and Grant, 2004). Our preliminary data indicate that KATP channels are not involved in CGRP-induced dilation in the MMA nor is CGRP working via activation of the NOS pathway in the vascular endothelium (appendix 2, Figure 1-2). CGRP receptors activate the cAMP-PKA signaling cascade (Brain and Grant, 2004). Therefore, it is possible that activation of PKA by CGRP mediated signaling results in activation of several vasodilatory pathways including activation of KATP, BK, and voltage gated potassium (Kv) channels and the sarcoplasmic/endoplasmic reticulum calcium ATPase

(SERCA) pump (Perez and Toro, 1994, Lin et al., 2000, Hayabuchi et al., 2001, Brignell et al., 2015). A possible future direction for this project would be to test if CGRP-induced dilation is abolished in the presence of a combination of BK, KATP channel and SERCA pump inhibitors in endothelium denuded MMA. Our preliminary data demonstrate

(Figure 4, appendix 2) that CGRP-induced vasodilation is inhibited in the presence of

123 high K+; suggesting that CGRP-induced vasodilation works via activation of K+ channels inducing membrane potential hyperpolarization.

These preliminary findings are novel and potentially important, as both neuropeptides PACAP and CGRP have been implicated in migraine headache and reported to potently dilate MMA. Even though these peptides have been reported to work via cAMP-PKA pathway our preliminary data demonstrates that they seem to be evoking two distinct mechanisms to induce MMA vasodilation. The logical question that comes to mind is: are CGRP and PACAP working in synergy to induce vasodilation in

MMA? Elas et al., have demonstrated that in porcine ophthalmic artery low concentration of CGRP and PACAP together result in significantly increased vasorelaxation compared to individual effects of the peptides (Elsas and White, 1997). Additionally, our preliminary data suggest that PACAP and CGRP are working via two separate mechanisms in the MMA. Thus, the spatial regulation of PKA by the CGRP and PACAP receptors seems to be different in the MMA. Hence targeting both vasodilatory mechanisms of both peptides may lead to development of better therapeutic strategies.

5.3 Clinical relevance

PACAP and CGRP are being heavily investigated in the field of migraine headache (Kaiser and Russo, 2013, Tajti et al., 2014). CGRP antagonists have been tested in clinical trails with disappointing results due to liver toxicity as a side effect (Ho et al., 2014). Currently, there is push to develop antagonists for PACAP receptors and more specifically PAC1 receptor antagonists (Schytz et al., 2009, Schytz et al., 2010a,

Schytz et al., 2010b). Consistent with this line of investigation, previous studies have 124 shown that activation of KATP channels in the meningeal vessels may result in mild to moderate headache (Ploug et al., 2008, Ploug et al., 2012). Indeed, several clinical studies have reported side effects of KATP channel agonist as mild to severe headache (Goldberg,

1988, Sterndorff and Johansen, 1988). Thus, this research may shed some light on the mechanism by which the MMA is being regulated by CGRP, PACAP and KATP channels.

5.4 Conclusion

In conclusion, the MMA is a unique blood vessel. Vasodilation of this blood vessel by PACAP and CGRP has been suggested to induce migraine headache. PACAP antagonists and more specifically PAC1 receptor antagonists may be a potential therapeutic target for migraine headache. Furthermore, targeting both PACAP and CGRP together may result in an effective therapeutic strategy for alleviating the symptoms of migraine headache. Additionally, KATP channels in the MMA are responsible for in part setting the membrane potential and regulating MMA diameter. It is conceivable that aberrant signaling of KATP channels or the release of neuropeptides from the sensory neurons innervating the MMA smooth muscle can result in excessive prolong vasodilation and migraine headache. Our research has revealed a number of unique characteristics of the MMA. Our hope is that these findings will lead to better understanding of the role of the MMA in the genesis of migraine headache.

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APPENDICES

151

APPENDIX 1: BOOK CHAPTER

Title: PACAP regulation of vascular tone: differential mechanism among

vascular beds

Authors: Arsalan U. Syed, Masayo Koide, Victor May1, George C. Wellman*

Institutions: Department of Pharmacology

University of Vermont College of Medicine

149 Beaumont Avenue

Burlington, Vermont 05405 USA

1Department of Neurological Sciences

University of Vermont College of Medicine

149 Beaumont Avenue

Burlington, Vermont 05405 USA

* Corresponding author

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1.1 Abstract

The homeostatic control of cranial and cerebral blood flow is essential for normal brain function. Brain surface (cerebral) and meningeal (cranial) arteries/arterioles are densely innervated by sensory and autonomic fibers containing a variety of vasoactive peptides including pituitary adenylate cyclase activating polypeptide (PACAP, Adcyap1) which has been shown to exert potent vasodilatory effects in a variety of vascular beds.

Recent studies have demonstrated that PACAP potency, activation of PACAP receptor subtypes and signaling to potassium channels to mediate vasodilatory responses are different between cerebral and cranial arteries, such as the cerebellar artery and the middle meningeal artery (MMA). PACAP demonstrates exquisite picomolar potency at the PACAP-selective PAC1 receptor (Adcyap1r1) to activate ATP-sensitive potassium

(KATP) channels of the MMA causing vasodilation. Although PACAP frequently colocalizes with the vasodilatory calcitonin gene related peptide (CGRP) in the fiber networks innervating the MMA, PACAP potency is nearly 3-orders of magnitude greater than that of CGRP. CGRP is equipotent in the MMA and cerebral arteries. In contrast,

PACAP is a less potent vasodilator of cerebral arteries compared to the MMA, with nanomolar concentrations required to activate VPAC receptors leading to KATP and calcium-dependent potassium (BK) channel activation and cerebellar artery dilation.

Further, PACAP’s effects on the brain are multidimensional. PACAP signaling also exerts direct neurotrophic and neuroprotective effects in the central nervous system against degenerative processes or physiological insults. Thus, PACAP regulation of cerebral blood flow helps sustains brain metabolic demands, maintains neural

153 physiological functions— including learning and memory, and protects neurons from neurodegeneration in disease and trauma. Coherent with CGRP vascular responses and functions, the vasodilatory effects of PACAP in the MMA also implicate a role in genesis of migraine disorders.

Keywords: PACAP, PAC1 receptor, middle meningeal artery (MMA), cerebral arteries, cranial vessels, ATP-sensitive potassium channels (KATP), large conductance calcium activated potassium channels (BK), vascular smooth muscle, migraine.

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1.2 Introduction

Many regulatory peptides, including vasopressin, endothelin, calcitonin gene related peptide (CGRP), angiotensin peptides, natriuretic peptides, substance P, cholecystokinin, neuropeptide Y (NPY) and the vasoactive intestinal peptide

(VIP)/pituitary adenylate cyclase activating polypeptide (PACAP) family of related peptides, have central roles in maintaining systemic and cerebrovascular homeostasis

(Edvinsson et al., 2001, Goadsby, 2013). Of these peptides, PACAP has been identified in sensory and autonomic neuronal fibers along diverse arterial beds in numerous species

(Mulder et al., 1994, Mulder et al., 1995, Sherwood et al., 2000, Baeres and Moller, 2004,

Goadsby, 2013). Direct PACAP G protein-coupled PAC1 and VPAC receptor signaling within vascular smooth muscle cells has been shown to unvaryingly induce vasodilation with high potency and efficacy (Wilson and Warren, 1993, Anzai et al., 1995, Sherwood et al., 2000, Baun et al., 2011, Syed et al., 2012, Koide et al., 2014). Among peripheral organs, PACAP fibers have been found to innervate vascular beds of the skin, eye, salivary glands, respiratory airways, cardiac tissues, gastrointestinal tract, mesentery, pancreas, kidney, testis, corpus cavernosum, ovary and placenta (Lerner et al., 1991,

Uddman et al., 1991, Warren et al., 1992, Uddman et al., 1993, Wilson and Warren,

1993, Graf et al., 1995, Hedlund et al., 1995, Steenstrup et al., 1996, Yao et al., 1996,

Bruch et al., 1997, Elsas and White, 1997, Bruch et al., 1998, Dorner et al., 1998,

Vuckovic et al., 2009, Banki et al., 2014a, Danyadi et al., 2014). From this wide distribution, it is clear that PACAP plays an important role in maintaining vascular function throughout a broad spectrum of physiological systems.

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In addition, there is an increasing appreciation for the roles of PACAP in the regulation of cerebral hemodynamics. Due to the high metabolic demands coupled with limited energy storage within the CNS, the blood supply to the brain must be continuous and operate within a narrow range to maintain structural and functional integrity of the brain (Brayden and Nelson, 1992, Nelson and Quayle, 1995, Li et al., 2014). In this regard, the brain is like no other organ and highly susceptible to irreversible damage even upon transient ischemic events. Hence, a series of complex regulatory mechanisms preserve cerebrovascular homeostasis. Upon systemic vascular challenges, the brain, through neural and humoral control, can redirect and redistribute blood flow from the peripheral circulatory system to cerebral circulation. Complementary to these changes in blood flow dynamics, the cerebrovasculature also has autoregulatory mechanisms that can respond to the perturbations in arterial pressure that typically occur in the course of normal physiological activities. In addition, cerebral blood flow is differentially modulated by sensory/autonomic innervation, and astrocytic signaling along the length of the cerebral artery (Dunn and Nelson, 2014, Filosa et al., 2015). Pial, or brain surface arteries are densely innervated by sensory and autonomic sympathetic and parasympathetic fibers containing diverse regulatory transmitters and peptides for vasodilation (i.e., acetylcholine, CGRP, VIP, PACAP) and vasoconstriction

(norepinehphrine, NPY) activities (Uddman et al., 1993, Edvinsson et al., 2001, Dunn and Nelson, 2014). However, as vessels pass through the Virchow-Robin space and enter the brain parenchyma, extrinsic perivascular nerves disappear and intracerebral arterioles come under the regulatory control of astrocytic endfeet (Dunn and Nelson, 2014, Filosa

156 et al., 2015). Hence, cerebral arterial tone is regulated in part by sensory and autonomic peptides. The homeostatic balance of cerebral blood flow is delicate but critical for normal brain function, and altered cerebrovascular activities from dysregulation, disease or trauma have been shown not only to precipitate stroke but contribute in other pathologies including neurodegenerative disorders, behavioral abnormalities and migraine (Asghar et al., 2011, Longden et al., 2014). The following sections highlight

PACAP signaling mechanisms drawing data from our work in the middle meningeal and cerebellar arteries to provide insights into their potential roles in the regulation of cerebral and cranial blood flow (Syed et al., 2012, Koide et al., 2014).

1.21 PACAP and PACAP receptors in cerebral and cranial vessels

The PACAP precursor molecule is endoproteolytically processed to PACAP27 or

PACAP38 bioactive peptides, which share significant homology with VIP (Seidah et al.,

1994, Seidah and Chretien, 1999, Sherwood et al., 2000, Vaudry et al., 2009).

Accordingly, PACAP and VIP share receptor subtypes. PACAP peptides bind selectively at PAC1 receptors, but both VIP and PACAP bind with near equal affinity at

VPAC1 and VPAC2 receptors. PAC1 and VPAC receptors have been identified in cerebral and cranial arteries by immunocytochemistry, ligand binding studies, transcript analyses and physiological assays (Edvinsson et al., 2001, Baeres and Moller, 2004, Baun et al., 2011, Chan et al., 2011, Erdling et al., 2013). Further, PACAP infusions have been shown to facilitate cerebral artery vasodilation in cranial window (Tong et al., 1993) and isolated vessel preparations (Anzai et al., 1995). However, PACAP signaling mechanisms underlying vasodilation can differ among cerebral and cranial vascular beds 157

(see below). There are several sources that can supply PACAP to both cerebral and cranial vessels. One is the trigeminal ganglion in which subpopulations of sensory neurons express a variety of vasoregulatory peptides including PACAP, VIP or CGRP

(Uddman et al., 1993, Edvinsson et al., 2001). As in dorsal root ganglion (DRG),

PACAP is expressed in approximately 10% of the trigeminal neurons and frequently colocalized with CGRP for synergistic functions. The other potential sources of PACAP include the parasympathetic sphenopalatine and otic ganglia in the head; but the relative abundance of PACAP neurons in these ganglia has been highly variable ranging from 5 -

6% to nearly all neurons in the population (Mulder et al., 1994, Mulder et al., 1995,

Baeres and Moller, 2004). The reasons underlying the discrepancy are unclear but may reflect immunocytochemical vs in situ hybridization methods and detection of low

PACAP-expressing neurons. Retrograde tracing studies have suggested that the majority of the PACAP fibers in cerebral arteries are derived from peripheral trigeminal sensory axons (Baeres and Moller, 2004). Our immunocytochemical studies demonstrating prevalent dual PACAP and CGRP colocalization in neuronal fibers along the middle meningeal artery are consistent with that interpretation (Figure 1), but many fibers also demonstrate PACAP-immunoreactivity alone suggesting that some may also be of postganglionic parasympathetic origins.

Both PAC1 and VPAC receptors have been described in cerebral and cranial vessels based on ligand binding, transcript analyses and pharmacological assessments.

The relative abundance of each receptor subtypes in cerebral and cranial arteries has not been well characterized but these blood vessels undoubtedly express multiple PACAP

158 receptor classes and all are likely to have physiological roles (Tong et al., 1993, Uddman et al., 1993, Edvinsson et al., 2001, Baun et al., 2011, Syed et al., 2012, Koide et al.,

2014). PACAP peptides bind to all three receptors with high affinity and hence are relevant ligands regardless of receptor subtype expression. The vasodilatory effects of

PACAP appear to be direct on arterial smooth muscle and largely endothelium- independent (Huang et al., 1993, Wilson and Warren, 1993, Chan et al., 2011).

1.22 PACAP mediates middle meningeal artery (MMA) vasodilation via PAC1 receptor activation of KATP channels

Unlike many previous studies, we used an ex-vivo pressurized arteriograph system to examine the vasodilatory effects of PACAP on the MMA to obviate pharmacological approaches to pre-constrict the arteries. Myogenic tone (pressure induced constriction) is an intrinsic property of resistance arteries and largely dependent on the intraluminal pressure on the arterial walls. Hence, ex-vivo pressurized arteriography utilizes this intrinsic property of the resistance arteries to mimic physiological conditions. For these studies, the MMA was dissected from adult male rats and cannulated onto glass micropipettes as described previously (Syed et al., 2012). The artery was bathed continuously with artificial cerebral spinal fluid (aCSF) at 37 °C and the intraluminal pressure increased to 40 mmHg to allow development of myogenic tone.

Upon artery constriction and stabilization, the peptides and drugs were added to the bathing solution to assess changes in vessel diameter, and at the end of the treatments, the maximum passive diameter of the artery was assessed in Ca2+-free aCSF containing vasodilator diltiazem (100 µM) and forskolin (1 µM).

159

Using this approach, we found that PACAP38 potently dilated the MMA in a concentration dependent manner with an EC50 of 1 pM; VIP by contrast, was approximately 1000 - fold less potent and induced dilation in MMA with EC50 of 1.4 nM

(Syed et al., 2012), which suggested that the PACAP effects were mediated by the

PACAP selective PAC1 receptor. MMA pretreatments with a PAC1/VPAC2 receptor antagonist PACAP6-38 (100 nM) completely abolished the vasodilation induced by 3 pM

PACAP38. However, 100 nM PACAP6-38 attenuated the vasodilatory effects of 3 nM

PACAP38 by 50%, which may have reflected pharmacological mass action effects or potential roles for VPAC1 receptor signaling in the MMA. Even though VPAC1 receptors have been described in cerebral arteries (Baun et al., 2011), semi quantitative

PCR analyses of rat MMA cDNA only detected prominent PAC1 and VPAC2 receptor transcript expression. These PCR results were consistent with the peptide potency studies in our pressurized vessel preparations, but whether MMA also demonstrates very low levels of VPAC1 receptor remains to be evaluated. Further, more detailed diagnostic restriction digest analyses of the PAC1 amplicon revealed that the MMA PAC1 receptor was predominantly the PAC1Hop1 receptor variant (Syed et al., 2012). This observation is notable and suggests that the MMA PAC1 receptor signaling is capable of engaging multiple intracellular second messenger cascades to elicit vascular responses.

Potassium channels play a crucial role in arterial tone via regulation of vascular smooth muscle membrane potential. Potassium channel activation causes vascular smooth muscle membrane hyperpolarization, leading to decreased L-type voltage-gated calcium channel (VDCC) open probability and a reduction of intracellular calcium to

160 facilitate smooth muscle relaxation and vasodilation. (Figure 4, modified from Koide et al., 2014) (Koide et al., 2014). From many studies, the PKA-dependent phosphorylation and activation of several potassium channels, including ATP-sensitive potassium (KATP) channels (Wellman et al., 1998), large conductance calcium-activated potassium (BK) channels (Standen and Quayle, 1998) and voltage gated potassium (KV) channels

(Brignell et al., 2015) can participate in the cerebral vasodilatory process. Accordingly, the ability for PAC1Hop1 receptors to engage the adenylyl cyclase/cAMP/PKA pathway for potassium channel phosphorylation and activation represents a principal mechanism to stimulate MMA dilation.

Among the different potassium channels, our studies have shown that PACAP and

PAC1 receptor activation of KATP channels is sufficient to induce MMA vasodilation

(Figure 2 A-C, F). For these studies, the isolated MMA segments were again pressurized to 40 mmHg and allowed to develop myogenic tone, and 3 nM PACAP produced a 56.2

± 7.6% increase in dilation. However, in the presence of the KATP channel inhibitor glibenclamide (10 µM) or a vascular selective KATP channel inhibitor PNU37883 (10

µM) (Wellman et al., 1999), the PACAP effects were completely abolished. By contrast,

MMA treatment with a BK channel inhibitor, paxilline (1 µM), failed to attenuate the

PACAP vasodilatory responses (Figure 2 D, F). Furthermore, pretreatment with the competitive nitric oxide synthase inhibitor nitro-L-arginine (L-NNA, 100 µM) had no effect on PACAP-mediated MMA vasodilation (Figure 2 E, F). Together, these results suggest that PACAP/PAC1 receptor-mediated activation of cAMP/PKA and KATP channels can regulate MMA tone exclusively without engaging BK channel functionor

161 nitric oxide (NO) production. The spatial and temporal regulation of KATP channels by

PKA requires further investigation but A-kinase anchoring proteins (AKAPs) have been postulated to play crucial roles in conferring the specific PKA interactions needed for channel regulatory fidelity (Kapiloff and Chandrasekhar, 2011).

1.23 PACAP and CGRP demonstrate differential potencies in stimulating MMA vasodilation

Described earlier, PACAP and CGRP are frequently colocalized in the fiber networks around the cerebral arteries including the MMA (Figure 1). CGRP also has vasodilatory functions via G protein-coupled calcitonin receptor-like receptors (CALCR).

We have begun to examine whether there are differences between PACAP and CGRP in their vasodilatory activities with respect to potency, efficacy or signaling mechanisms.

Using the same ex-vivo pressurized MMA vessel preparation, concentration-dependence studies demonstrated that the EC50 for CGRP-induced MMA dilation was 1 nM, which was comparable to the potency of VIP, but again 1000-fold lower than that for PACAP- mediated vasodilation (EC50 1 pM). These results appeared to agree with previously published data in which PAC1 receptor-stimulated increase in cAMP production was

500-fold more potent than that of CGRP (Lerner et al., 1991). The efficacy of PACAP and CGRP in MMA vasodilation appeared comparable; the mechanisms underlying the differences in potency are under study.

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1.24 PACAP mediates cerebellar artery vasodilation via different receptors and channels

Unlike the responses in MMA, PACAP and VIP dilated cerebellar arteries with equal potency. Using the same ex-vivo procedures to perfuse the cannulated cerebellar arterial segments, PACAP38 and VIP induced vasodilation with an EC50 of 11 nM and 6 nM, respectively (Syed et al., 2012, Koide et al., 2014). All three VIP/PACAP receptor subtypes have been identified in cerebral arteries but distinct from the MMA in which

PAC1 receptor signaling was implicated, the near equal potency of VIP and PACAP in eliciting cerebellar arterial dilation indicated that the responses were largely mediated by

VPAC receptors.

In cerebellar arteries, PACAP-mediated stimulation of the cAMP/PKA pathway not only activated KATP channels but also BK channels to increase spontaneous transient outward currents (STOCs) for vasodilation (Jaggar et al., 1998, Wellman et al., 2001,

Wellman and Nelson, 2003, Brayden et al., 2013, Koide et al., 2014). The BK channels, like KATP channels, can be activated by direct PKA phosphorylation (Wellman et al.,

2001, Wellman and Nelson, 2003), but characteristically, BK channels are regulated by local increases in intracellular calcium (Ca2+ sparks). PACAP signaling and stimulation of PKA can potentially activate ryanodine receptors (RyRs) on the sarcoplasmic reticulum (SR) for intracellular calcium release, resulting in a localized transient increase in cytosolic calcium up to 10 µM, termed a calcium spark (Brayden and Nelson, 1992,

Jaggar et al., 1998, Wellman et al., 2001). These calcium spark events in turn can lead to localized BK channel activation on the plasma membrane (Figure 4). Additionally, PKA

163 can also phosphorylate phospholamban (PLB), a pentameric protein that regulates the sarcoplasmic reticulum calcium-ATPase (SERCA) pump, critical for the management of intracellular calcium. PLB in the unphosphorylated state inhibits SERCA activity;

PACAP activation of PKA and downstream phosphorylation of PLB relieves that inhibition to increase SERCA activity and sarcoplasmic reticulum calcium load, resulting in increased calcium spark frequency and BK channel activity (Brayden and Nelson,

1992, Jaggar et al., 1998, Wellman et al., 2001). Unlike the MMA studies, the addition of the KATP channel blocker glibenclamide or the BK channel blocker paxilline only attenuated and did not abolish the PACAP-induced vasodilation in cerebellar arteries

(Figure 3 B-C, E, modified from Koide et al., 2014). However, the concurrent addition of glibenclamide and paxilline fully eliminated the PACAP vasodilatory responses

(Figure 3 D, E, modified from Koide et al 2014) (Koide et al., 2014). These findings suggest that similar to coronary and pulmonary arteries (Bruch et al., 1997, Bruch et al.,

1998), PACAP regulates cerebral artery tone via both KATP and BK channels, but in contradistinction, PACAP only regulates KATP channels for MMA vasodilation.

1.25 Alternate mechanism of PACAP-induced vasodilation

There is evidence suggesting PACAP-induced dilation may also be endothelium- dependent in certain species. For example in newborn pigs, studies using an in vivo closed cranial window system suggested that the PACAP-induced vasodilation may be mediated partially through cyclooxygenase (COX). Intravenous injections of COX1 inhibitor SC-560 (1 mg/kg) appeared to significantly decrease PACAP-induced vasodilation whereas treatments with a COX2 specific inhibitor NS-398 (1 mg/kg) had no 164 apparent effects (Lenti et al., 2007). The mechanisms by which PACAP signaling can engage COX1 are unclear.

1.26 Functional significance of PACAP regulation and pathophysiology

Cerebrovascular homeostasis to maintain blood flow is essential for normal brain activities. Pial arteries present the greatest resistance to cerebral blood flow and cerebral artery relaxation after neurotransmitter/neuropeptide release from sensory and autonomic nerve terminals can contribute significantly to vasodilation in downstream branches

(Iadecola, 2004). The dynamic homeostatic changes in cerebral blood flow is essential to meet metabolic demands, and failures to meet those demands from cerebrovascular dysfunction have been associated with impairments in learning and memory which may lead to vascular dementia, and increased risks for Alzheimer’s and related neurodegenerative diseases (Claassen, 2015). PACAP receptor activation can engage neurotrophic signaling pathways for neuroprotection against injuries, but the abilities for

PACAP to regulate vasodilation and cerebral blood flow may offer means to mitigate ischemic damage (Elsas and White, 1997, Banki et al., 2014b, Danyadi et al., 2014).

Recently, there has been interest in PACAP signaling in the MMA and migraine.

Migraine is a complex and often debilitating neurological disorder characterized by intense unilateral pulsating headache, frequently accompanied with aura, photophobia, phonophobia, nausea and related symptoms (Asghar et al., 2011, Edvinsson, 2015). The causes of migraine are not understood and are likely to be multifactorial. One mechanism that has been debated for decades implicates MMA vasodilation from trigeminal sensory and/or sphenopalatine parasympathetic fiber innervation and 165 signaling. A different and more recent concept suggests that migraine results from cortical spreading depression (Ayata, 2010, Cui et al., 2014). But regardless whether

MMA vasodilation plays a causal or contributory role in the pathophysiology of migraine, there is good evidence that vasodilatory peptides participate in migraine development. As described in previous work, CGRP is localized to sensory fibers innervating the MMA, facilitates MMA vasodilation, and can induce migraine-like headaches similar to spontaneous migraine attacks. Magnetic resonance angiography imaging correlated MMA vasodilation with migraine onset upon exogenous CGRP infusions and notably, CGRP receptor antagonists can ameliorate migraine headaches

(Asghar et al., 2010, Asghar et al., 2011). However, clinical trials examining the efficacy of the CGRP receptor antagonists were halted because of liver toxicity (Bigal and Walter,

2014). More recently, plasma PACAP levels have been reported to be elevated in patients with migraine (Tuka et al., 2013). Further, PACAP is associated with post-traumatic stress disorder (PTSD), which is highly comorbid with migraine, suggesting an intersection of PACAP mechanisms and neural pathways (Bukovics et al., 2014). As described above, PACAP is frequently colocalized with CGRP in sensory fibers innervation the MMA and similar to CGRP, exogenous PACAP administration can initiate migraine ipsilateral to the infusion site coincident with MMA vasodilation (Amin et al., 2012, Amin et al., 2014). Specific small molecule PACAP receptor antagonists have yet to be identified but blocking MMA PACAP signaling, either alone or in combination with CGRP receptor antagonists, may offer means for migraine therapeutics.

From these examples, a better understanding of PACAP and VIP neurocircuits and

166 signaling mechanisms regulating cerebral arterial tone may be clinically relevant for a number of neurophysiological disorders and states.

1.3 Conclusion

The regulation of cerebrovascular resistance is essential to meet brain metabolic demands and function. PACAP has potent vasodilatory activities in a variety of vascular systems including the cerebral and cranial arteries. The PACAP fibers innervating the cerebral arteries are sensory or autonomic in origin, and interestingly, PACAP potency, the PACAP receptor subtypes and the downstream signaling mechanisms mediating the vasodilatory effects appear to vary for cranial blood vessels. PACAP signaling via the

PAC1 and VPAC receptors in the MMA activates exclusively KATP channels for vasodilation; the PACAP effects on cerebellar arteries appear to be primarily mediated by

VPAC receptor regulation of KATP and BK channels. Thus the roles of PACAP in the brain are multidimensional. In addition to its direct trophic and protective activities on neurons, the roles of PACAP in regulating blood vessel diameter may be important in maintaining learning and memory, and mitigating brain damage from ischemic events.

Additionally, the vasodilatory effects of PACAP on the MMA may have implications in migraine disorders.

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1.4 Reference List

Asghar MS, Hansen AE, Amin FM, van der Geest RJ, Koning P, Larsson HB, Olesen J, Ashina M (2011) Evidence for a vascular factor in migraine. AnnNeurol 69:635-645.

Ayata C (2010) Cortical spreading depression triggers migraine attack: pro. Headache 50:725-730.

168

Bigal ME, Walter S (2014) Monoclonal antibodies for migraine: preventing calcitonin gene-related peptide activity. CNS drugs 28:389-399.

Brayden JE, Nelson MT (1992) Regulation of arterial tone by activation of calcium-dependent potassium channels. Science 256:532-535.

Brignell JL, Perry MD, Nelson CP, Willets JM, Challiss RA, Davies NW (2015) Steady-state modulation of voltage-gated K+ channels in rat arterial smooth muscle by cyclic AMP-dependent protein kinase and protein phosphatase 2B. PLoS One 10:e0121285.

Chan KY, Baun M, de Vries R, van den Bogaerdt AJ, Dirven CM, Danser AH, Jansen-Olesen I, Olesen J, Villalon CM, MaassenVanDenBrink A, Gupta S (2011) Pharmacological characterization of VIP and PACAP receptors in the human meningeal and coronary artery. Cephalalgia 31:181-189.

Claassen JA (2015) New cardiovascular targets to prevent late onset Alzheimer disease. Eur J Pharmacol 763:131-134.

Cui Y, Kataoka Y, Watanabe Y (2014) Role of cortical spreading depression in the pathophysiology of migraine. Neuroscience bulletin 30:812-822.

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Dunn KM, Nelson MT (2014) Neurovascular signaling in the brain and the pathological consequences of hypertension. Am J Physiol Heart Circ Physiol 306:H1-14.

Edvinsson L (2015) PACAP and its receptors in migraine pathophysiology: Commentary on Walker et al., Br J Pharmacol 171: 1521-1533. British journal of pharmacology 172:4782-4784.

Edvinsson L, Elsas T, Suzuki N, Shimizu T, Lee TJ (2001) Origin and Co- localization of nitric oxide synthase, CGRP, PACAP, and VIP in the cerebral circulation of the rat. Microsc Res Tech 53:221-228.

Filosa JA, Morrison HW, Iddings JA, Du W, Kim KJ (2015) Beyond neurovascular coupling, role of astrocytes in the regulation of vascular tone. Neuroscience.

Goadsby PJ (2013) Autonomic nervous system control of the cerebral circulation. Handb Clin Neurol 117:193-201.

Hedlund P, Alm P, Ekstrom P, Fahrenkrug J, Hannibal J, Hedlund H, Larsson B, Andersson KE (1995) Pituitary adenylate cyclase-activating polypeptide,

170 helospectin, and vasoactive intestinal polypeptide in human corpus cavernosum. British journal of pharmacology 116:2258-2266.

Huang M, Shirahase H, Rorstad OP (1993) Comparative study of vascular relaxation and receptor binding by PACAP and VIP. Peptides 14:755-762.

Iadecola C (2004) Neurovascular regulation in the normal brain and in Alzheimer's disease. Nature reviews Neuroscience 5:347-360.

Kapiloff MS, Chandrasekhar KD (2011) A-kinase anchoring proteins: temporal and spatial regulation of intracellular signal transduction in the cardiovascular system. J Cardiovasc Pharmacol 58:337-338.

Koide M, Syed AU, Braas KM, May V, Wellman GC (2014) Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) Dilates Cerebellar Arteries Through Activation of Large-Conductance Ca-Activated (BK) and ATP-Sensitive (KATP ) K+ Channels. J MolNeurosci.

Lenti L, Domoki F, Kis D, Hegyi O, Toth GK, Busija DW, Bari F (2007) Pituitary adenylate cyclase-activating polypeptide induces pial arteriolar vasodilation through cyclooxygenase-dependent and independent mechanisms in newborn pigs. Brain Research 1165:81-88. Lerner EA, Ribeiro JM, Nelson RJ, Lerner MR (1991) Isolation of maxadilan, a potent vasodilatory peptide from the salivary glands of the sand fly Lutzomyia longipalpis. The Journal of biological chemistry 266:11234-11236.

Longden TA, Dabertrand F, Hill-Eubanks DC, Hammack SE, Nelson MT (2014) Stress-induced glucocorticoid signaling remodels neurovascular coupling through impairment of cerebrovascular inwardly rectifying K+ channel function. Proceedings of the National Academy of Sciences of the United States of America 111:7462-7467.

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Mulder H, Uddman R, Moller K, Elsas T, Ekblad E, Alumets J, Sundler F (1995) Pituitary Adenylate-Cyclase Activating Polypeptide Is Expressed in Autonomic Neurons. Regulatory Peptides 59:121-128.

Mulder H, Uddman R, Moller K, Zhang YZ, Ekblad E, Alumets J, Sundler F (1994) Pituitary adenylate cyclase activating polypeptide expression in sensory neurons. Neuroscience 63:307-312.

Nelson MT, Quayle JM (1995) Physiological roles and properties of potassium channels in arterial smooth muscle. AmJ Physiol 268:C799-C822.

Seidah NG, Chretien M (1999) Proprotein and prohormone convertases: a family of subtilases generating diverse bioactive polypeptides. Brain Res 848:45-62.

Seidah NG, Chretien M, Day R (1994) The family of subtilisin/kexin like proprotein and pro-hormone convertases: divergent or shared functions. Biochimie 76:197-209.

Sherwood NM, Krueckl SL, McRory JE (2000) The origin and function of the pituitary adenylate cyclase-activating polypeptide (PACAP)/glucagon superfamily. Endocrine Reviews 21:619-670.

Standen NB, Quayle JM (1998) K+ channel modulation in arterial smooth muscle. Acta Physiol Scand 164:549-557.

Syed AU, Koide M, Braas KM, May V, Wellman GC (2012) Pituitary adenylate cyclase-activating polypeptide (PACAP) potently dilates middle meningeal arteries: implications for migraine. J MolNeurosci 48:574-583.

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Uddman R, Luts A, Arimura A, Sundler F (1991) Pituitary adenylate cyclase- activating peptide (PACAP), a new vasoactive intestinal peptide (VIP)-like peptide in the respiratory tract. Cell Tissue Res 265:197-201.

Vaudry D, Falluel-Morel A, Bourgault S, Basille M, Burel D, Wurtz O, Fournier A, Chow BK, Hashimoto H, Galas L, Vaudry H (2009) Pituitary adenylate cyclase-activating polypeptide and its receptors: 20 years after the discovery. PharmacolRev 61:283-357.

Warren JB, Larkin SW, Coughlan M, Kajekar R, Williams TJ (1992) Pituitary adenylate cyclase activating polypeptide is a potent vasodilator and oedema potentiator in rabbit skin in vivo. British journal of pharmacology 106:331-334.

Wellman GC, Nelson MT (2003) Signaling between SR and plasmalemma in smooth muscle: sparks and the activation of Ca2+-sensitive ion channels. Cell Calcium 34:211-229.

Wellman GC, Santana LF, Bonev AD, Nelson MT (2001) Role of phospholamban in the modulation of arterial Ca2+ sparks and Ca2+-activated K+ channels by cAMP. Am J Physiol Cell Physiol 281:C1029-1037.

Wilson AJ, Warren JB (1993) Adenylate cyclase-mediated vascular responses of rabbit aorta, mesenteric artery and skin microcirculation. BrJ Pharmacol 110:633- 638.

Yao W, Sheikh SP, Ottesen B, Jorgensen JC (1996) Vascular effects and cyclic AMP production produced by VIP, PHM, PHV, PACAP-27, PACAP-38, and NPY on rabbit ovarian artery. Peptides 17:809-815.

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1.5 Figures

Figure 1: PACAP and CGRP immunoreactivities are colocalized in fibers innervating the MMA. Rat MMA was dissected from the dura, perfusion fixed in 4% paraformaldehyde and immunocytochemically dually processed for PACAP (1:10, Jen

Hannibal, Bisperg Hospital, Copenhagen Denmark) and CGRP (1-17) (1:1500, Ian

Dickerson), University of Rochester) for visualization with Cy3 (PACAP, red, left panel) and AlexaFluor 488 (CGRP, green, middle panel), respectively. The fluorescent signals were merged (yellow, left panel) to illustrate high levels of colocalization; the distribution patterns suggested that the fibers were sensory in origin from the trigeminal ganglion. Occasional fibers expressed only PACAP or CGRP.

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Figure 2: PACAP-induces MMA vasodilation through KATP channel activation.

Segments of the MMA were cannulated and pressurized for superfusion experiments as described previously (Syed et al.). MMA perfusions with 3 nM PACAP-induced produced maximal vasodilation (A; n = 8). The addition of KATP channel inhibitor glibenclamide (B; GLIB, 10 µM, N = 4) or the vascular selective KATP channel inhibitor

PNU37883 (C; PNU, 10 µM, n=4) blocked the PACAP vasodilatory effects. By contrast,

175 treatments with the BK channel inhibitor paxilline (D; PAX, 1 µM, n = 4) or the nitric oxide synthase inhibitor L-NNA (E; 100 µM, n = 4) had no effects. The data are summarized in (F) represent as mean ± SE. ***, significantly different from PACAP alone (CTRL, control) at p < 0.001. One-way ANOVA followed by post hoc comparisons of the means using Tukey test.

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Figure 3: PACAP-induced dilation in isolated cerebellar artery is mediated via both

KATP and BK channels. (A) Representative trace of 3 nM PACAP-induced vasodilation of cerebellar artery. (B-C) Representative traces of 3 nM PACAP-induced vasodilation in the presence of KATP channel inhibitor, glibenclamide (Glib) (10 µM) (B) and BK channel inhibitor, paxilline (Pax) (1 µM) (C). The vasodilation was significantly attenuated but not abolished. (D) Representative trace of 3 nM PACAP-induced vasodilation in the presence of 10 µM glibenclamide and 1 µM paxilline combined. The combined treatment with glibenclamide and paxilline abolished 3 nM PACAP-induced vasodilation in cerebellar artery. (E) Summary data of PACAP-induced vasodilation of cerebellar artery in the absence and presence of glibenclamide and paxilline (n = 4-7).

Data are expressed as mean ± SE. * P<0.05, **P<0.01 vs PACAP-induced dilation in the absence of K+ channel blockers (control). Reproduced and modified with permission from the Journal of Molecular Neuroscience (Koide et al., 2014).

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Figure 4: Schematic of PACAP mechanisms in vascular smooth muscle vasodilation.

(A) PACAP binding to PAC1 G protein-coupled receptors stimulates cAMP/PKA pathways, resulting in KATP channel phosphorylation and activation. The ensuing hyperpolarization closes voltage-dependent calcium channels (VDCC) and reduces intracellular calcium to facilitate smooth muscle relaxation and vasodilation. This is the principal PACAP vasodilatory mechanism in MMA. (B) In cerebellar arteries, PACAP- mediated cAMP/PKA signaling by binding to VPAC1/2-R, can also activate calcium- sensitive potassium (BK) channels by direct channel phosphorylation (not shown), activation of ryanodine receptors (RyR) or disinhibition of phospholamban (PLB) on the 178 sarcoplasmic reticulum ATPase pump (SERCA). The latter mechanisms increase sarcoplasm reticulum calcium release and spark generation to activate BK channels. The

BK mechanisms are synergistic with those via KATP for cerebellar artery vasodilation.

Reproduced and modified with permission from the Journal of Molecular Neuroscience

(Koide et al., 2014).

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APPENDIX 2: UNPUBLISHED OBSERVATION

TITLE: Mechanism of CGRP-induced vasodilation of the middle meningeal artery

2.1 Introduction Migraine is the most common incapacitating neurological disorder, characterized by an intense pulsating headache (Goadsby et al., 2002). It accounts for 1 % of total disability and has an economic burden of $20 billion in missed work and loss of productivity per year in the United States (Shapiro, 2007). Currently, treatments for migraine headache are relatively ineffective and fraught with adverse effects.

Approximately 50 % of migraine suffers take over-the-counter medications, or nothing at all. The cellular mechanisms contributing to migraine headache are poorly understood, but a leading hypothesis is that prolonged dilation of cranial arteries, and specifically the middle meningeal artery (MMA), is involved in the sensation of headache pain. Recent clinical studies have shown that administration of pituitary adenylate cyclase activating polypeptide (PACAP) or calcitonin gene related peptide (CGRP) induce migraine-like headaches that coincide with unilateral MMA vasodilation. Furthermore, following treatment with sumatriptan, a 5-HT-1B/D receptor agonist, PACAP and CGRP-induced headaches are attenuated corresponding to constriction of the MMA (Schytz, 2010,

Asghar et al., 2011, Amin et al., 2012).

Our lab has recently demonstrated that PACAP is a potent vasodilator of rat

MMA exclusively via activation of ATP-sensitive potassium (KATP) channels (AU et al.,

2016). The goal of this study was to compare and contrast the mechanism of neuropeptides PACAP and CGRP-induced vasodilation implicated in migraine headache.

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Our novel data indicate that although PACAP and CGRP have both traditionally been linked to increased adenylyl cyclase activity, the downstream mechanisms leading to

MMA vasodilation are distinct for these neuropeptides. Considering the evidence indicating that PACAP- and CGRP-induced MMA dilation contribute to migraine headache (Asghar et al., 2010, Asghar et al., 2011, Amin et al., 2012, Amin et al., 2014), targeting both of these pathways is likely to provide added benefit to migraine patients.

2.2 Methods

MMAs were cannulated onto glass micropipettes mounted in a 5-mL myograph chamber perfused with aCSF at 37 °C, (aCSF in millimolar:125 NaCl, 3 KCl, 18

NaHCO3, 1.25 NaH2PO4, 1 MgCl2, 2 CaCl2, and 5 glucose, aerated with 5 % CO2, 20 %

O2, 75 %N2, pH 7.35–7.40). Intravascular pressure was adjusted using a pressure servo- controller and a peristaltic pump as previously described (Ishiguro et al., 2002, Nystoriak et al., 2011). Throughout the experiment, changes in vessel diameter were measured by video edge detection (Living Systems Instrumentation, St Albans, VT) using WinDaq data acquisition software (Dataq Instruments; Akron, OH). After initially allowing the arteries to equilibrate for 1 hour, viability and integrity of the artery was determined by brief superfusion with 60 mM KCl in artificial cerebrospinal fluid (aCSF; isosmotic replacement of NaCl with KCl). Only arteries that constricted by 50% or more

(calculated from % decrease of resting diameter) were used in subsequent studies.

Pressure-induced constriction is expressed as a percent decrease of the fully dilated diameter of individual arteries at the same intravascular pressure. These values will be obtained by using the following equation: % constriction = [(DP−DA)/DP]×100, where 181

DP is the passive diameter of the artery in Ca2+-free aCSF containing the vasodilators diltiazem (100 µM) and forskolin (1 µM), and DA is the active diameter of the artery in

Ca2+-containing aCSF. Dilation in response to PACAP38 and CGRP are expressed as a percent reduction in the level of arterial constriction. The values are obtained using the following equation: % dilation [(DV−DA)/(DP−DA)]×100, where DV is arterial diameter in the presence of vasodilator, DA is the active diameter of the artery prior to addition of vasodilator, and DP is the passive diameter of the artery.

Ratiometric calcium measurements: Freshly isolated MMA were cannulated on glass micropipettes mounted in a 5-ml myograph chamber as described above. After cannulation, arteries were loaded with 5 mM of the ratiometric Ca2+-sensitive dye fura-2 acetoxymethyl ester (fura-2 AM, Invitrogen, Carlsbad, CA) in HEPES-buffered saline solution containing pluronic acid (0.1%) at room temperature (~22°C) for 1 hour. Next, the myograph chamber was mounted on a Nikon TE2000-S inverted fluorescence microscope. To allow for equilibration and de-esterification of fura-2 AM, isolated MMA sections were pressurized to 10 mmHg, were continuously superfused with aCSF (37°C, for 30 min). Fura-2 was excited by two separate wavelengths (340 nm and 380 nm) and fluorescence emission at 510 nm was measured; the background signal was subtracted and the ratio between the intensity levels was used to calculate the free calcium

2+ concentration using the following equation: [Ca ] = Kd (R-Rmin)/(Rmax-R)×(Sf2/Sb2).

Rmax and Rmin are the ratio values measured under conditions of saturating calcium levels and in the absence of calcium respectively. Sb2 and Sf2 are proportional to the fluorescence excited by the denominator wavelength (380 nm) under the saturated

182 calcium condition (Sb2) and in the absence of calcium (Sf2). These values were measured from a separate set of ionomycin treated arteries under Ca2+-free and Ca2+-saturated conditions. The Kd value used is 282 nM for fura-2 calcium binding.

2.3 Results

CGRP-induced dilation of MMA is independent of KATP channels

CGRP has been shown to induce dilation in vascular smooth muscle through KATP channels (Quayle et al., 1994a, Wellman et al., 1998) via the cAMP-PKA pathway

(Wellman et al., 1998). However, in the presence of the KATP channel inhibitors, glibenclamide (10 µM) or PNU37883 (10 µM), CGRP-induced dilation of MMA was unaffected (Figure 1 B-D). These results indicate that KATP channels are not involved in

CGRP mediated dilation of MMA.

BK and KV channels are not involved in CGRP induced dilation

PKA has been shown to phosphorylate and activate BK channels and voltage gated potassium (KV) channels in vascular smooth muscle (Nelson et al., 1995, Ko et al.,

2010). Interestingly, CGRP-induced MMA dilation was not altered by the BK channel inhibitor (Bol et al., 2012) paxilline (1 µM) or the KV channel inhibitor 4-aminopyridine

(4-AP, 5 mM) (Figure 2 B-C).

Nitric oxide, cyclooxygenase and SK/IK channels inhibition do not alter CGRP- induced dilation of MMA

CGRP has been proposed to induce vasodilation via endothelial-dependent nitric oxide synthase in mice pial arteries (Rosenblum et al., 1993). However, our data indicate

183 that inhibition of eNOS with L-NNA (100 µM) did not reduce CGRP-induced dilation of

MMA (Figure 2 D). Additionally, we also tested other endothelium-dependent vasodilatory pathways including cyclooxygenase (COX). In the presence of COX inhibitor indomethacin (10 µM), CGRP-induced vasodilation remained unaffected.

Furthermore, activation of calcium activated intermediate conductance potassium (IK) channel and calcium activated small conductance potassium channel (SK) in the vascular endothelium induce vasodilation via membrane potential hyperpolarization (Hannah et al., 2011). However, the IK inhibitor TRAM34 (1µM) and the SK inhibitor apamin (300 nM) also did not alter CGRP-induced vasodilation of MMA (Figure 2 E-G).

CGRP induces a decrease in arterial wall calcium

To examine whether a decrease in arterial wall calcium underlies CGRP-induced

MMA dilation, ratiometric calcium measurements were made using Fura-2 in isolated pressurized MMA segments. As illustrated in Figure 3, CGRP-induced dilation was accompanied by a decrease in calcium that was comparable to the decrease in calcium caused by the KATP channel opener cromakalim. These data demonstrate that CGRP- induced dilation in MMA results from a decrease in intracellular calcium.

CGRP-induced dilation is abolished by increased extracellular K+

CGRP has been shown to induce membrane potential hyperpolarization via potassium channel activation (Quayle et al., 1994a) resulting in decreased open probability of VDCC and decreased intracellular Ca2+ in vascular smooth muscle. To

+ examine whether shifting the K equilibrium potential (EK) to a more depolarized

184 membrane potential influenced CGRP-induced dilations, arteries were pre-constricted with either 30 mM KCl or 10 nM U46619 (thromboxane analog). In the presence of 30

+ mM potassium, EK is calculated to be -38mV with no net K movement into or out of the cells. CGRP-induced dilation was markedly reduced in the presence of high extracellular potassium compared to 10 nM U46619, suggesting that potassium channel activation and smooth muscle membrane potential hyperpolarization play a role in CGRP-induced dilation of MMA (Figure 9A-B).

2.4 Significance and future directions

Previous studies have demonstrated that CGRP-induced relaxation of vascular smooth muscle is primarily through the activation of KATP channels (Nelson et al., 1990).

However, data presented in the following study argues that CGRP-induced dilation of

MMA is working via an alternative mechanisms. Conversely, PACAP is working through the activation of KATP channels in the MMA, while in cerebral artery PACAP utilizes both KATP and BK channels (Syed et al., 2012, Koide et al., 2014). Both PACAP and

CGRP have been shown to co-localize and released from the trigeminal sensory nerve during migraine. Furthermore, PACAP and CGRP induced relaxation in vascular smooth muscle has been traditionally linked to increased adenylyl cyclase activity and PKA activation (Quayle et al., 1994b, Vaudry et al., 2009). However, we have not tested if

CGRP is working via PKA pathway in the MMA to induce vasodilation. Additionally, we have not ruled out the involvement of vascular endothelium in CGRP-induced dilation of the MMA. In the future, deciphering the involvement of PKA and endothelium will help focus on the mechanism of CGRP-induced dilation of MMA. If CGRP does indeed 185 signal via PKA activation to cause dilation in the MMA, then it becomes important to examine the spatial regulation of PKA by CGRP and PACAP. A-Kinase anchoring proteins (AKAPs) have been suggested to play a crucial role in temporal and spatial regulation of PKA (Marx et al., 2000, Nystoriak et al., 2014). Therefore, additional experiments are required to decipher specific AKAP association with CGRP or PACAP receptors so we can better understand downstream signaling cascades.

Our preliminary data demonstrate that CGRP is working via membrane potential hyperpolarization. This hyperpolarization is due to the activation of potassium channels as in the presence of high potassium (30 mM) CGRP-induced vasodilation is completely abolished (Figure 4). Furthermore, our Fura 2 data show that CGRP-induced vasodilation is calcium dependent as there is a decrease in intracellular smooth muscle calcium following CGRP treatment (Figure 3).

CGRP can utilize multiple pathways to induce vasodilation in the MMA. For example, PKA has been demonstrated to regulate not only KATP channels but also BK and Kv channels in the vascular smooth muscle (Brignell et al., Perez and Toro, 1994,

Marx et al., 2000, Hayabuchi et al., 2001, Shi et al., 2007). PKA can also regulate sarcoplasm/endoplasmic reticulum calcium ATPase (SERCA) pump in vascular smooth muscle cells, resulting in increased SERCA activity and decreased cytosolic calcium

(Wellman and Nelson, 2003). Therefore in future, we must examine CGRP-induced dilation in the presence of a combination of pharmacological inhibitors of the KATP, BK and Kv channels in conjunction with SERCA pump inhibitors.

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By understanding the distinct mechanisms involved in MMA dilation caused by

CGRP and PACAP, it may be possible to develop new combination therapies for migraine that target both of these important pathways.

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2.5 Figures

Figure 1: CGRP-induced dilation is not attenuated in the presence of glibenclamide and PNU37883. A-C) Representative traces of 1 nM CGRP-induced dilation in the presence or absence of KATP channel inhibitor glibenclamide (10 µM) and PNU37883 (10

µM). D) Summary data for CGRP-induced dilation in presence or absence of glibenclamide and PNU37883, n = 5 each. Unpaired student t-test. Glibenclamide vs control and PNU37883 vs control.

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Combination- Apamin TRAM34 Indomethacin H L-NNA 100

4-AP L-NNA % CGRP-induced dilation ControlPaxilline

Indomethacin Combination Apamin/TRAM34 Figure 2: CGRP-induced dilation is independent of BK, Kv, eNOS, COX, IK and

SK pathways. A-G) Representative traces of 1 nM CGRP-indiced dilation in the absence

(A) and presence of BK channel inhibitor paxilline (1 µM, n = 4)) (B), Kv channel inhibitor 4-AP (5 mM, n = 1)) (C), eNOS inhibitor L-NNA (100 µM, n = 1) (D), cyclooxygenase inhibitor indomethacin (10 µM, n=3) (E), IK/SK channel inhibitors

Apamin and TRAM34 (300 nM and 1 µM respectively, n = 4) (F), combination of L-

NNA (100 µM), Indomethacin (10 µM), Apamin (300 nM) and TRAM34 (1 µM) (G).

(H) Summary data of 1 nM CGRP-induced vasodilation in the presence of inhibitors.

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Figure 3: CGRP induces a decrease in intracellular calcium paralleling MMA dilation. A-B) Representative traces of CGRP and cromakalim-induced dilation. C-D)

Representative traces of ratiometric calcium decrease following treatment of 1 nM CGRP

(C) and 1µM cromakalim (D). Summary data of ratiometric calcium measurements in presence or absence of CGRP and cromakalim n = 5.Paired Student t-test P<0.001 vs control.

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C *** 80 60 40 20 0 + % CGRP-induced dilation 30 mM K 10 nM U46619 Figure 4: CGRP-induced dilation is significantly attenuated in the presence of high extracellular potassium. A) Representative trace of 1 nM CGRP response in the presence of 30 mM KCl. B) Representative trace of 1 nM CGRP response in the presence of 10 nM U46619 (n = 4). C) Summary data for CGRP-induced dilation in MMA in the presence of U46619 (10nM) and KCl (30 mM). Paired t-test P<0.001 U46619 vs 30 mM

K+.

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2.6 Reference List

Asghar MS, Hansen AE, Amin FM, van der Geest RJ, Koning P, Larsson HB, Olesen J, Ashina M (2011) Evidence for a vascular factor in migraine. AnnNeurol 69:635-645.

AU Syed, M Koide, V May, GC Wellman (2016) PACAP regulation of vascular tone: differential mechanism among vascular beds. In: PACAP handbook: Springer.

Brignell JL, Perry MD, Nelson CP, Willets JM, Challiss RA, Davies NW Steady-State Modulation of Voltage-Gated K+ Channels in Rat Arterial Smooth Muscle by Cyclic AMP-Dependent Protein Kinase and Protein Phosphatase 2B.

Goadsby PJ, Lipton RB, Ferrari MD (2002) Migraine--current understanding and treatment. NEnglJ Med 346:257-270.

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Ko EA, Park WS, Firth AL, Kim N, Yuan JX, Han J (2010) Pathophysiology of voltage- gated K+ channels in vascular smooth muscle cells: modulation by protein kinases. ProgBiophysMolBiol 103:95-101.

Nelson MT, Cheng H, Rubart M, Santana LF, Bonev AD, Knot HJ, Lederer WJ (1995) Relaxation of arterial smooth muscle by calcium sparks. Science 270:633-637.

Nelson MT, Huang Y, Brayden JE, Hescheler J, Standen NB (1990) Arterial dilations in response to calcitonin gene-related peptide involve activation of K+ channels. Nature 344:770-773.

Nystoriak MA, O'Connor KP, Sonkusare SK, Brayden JE, Nelson MT, Wellman GC (2011) Fundamental increase in pressure-dependent constriction of brain parenchymal arterioles from subarachnoid hemorrhage model rats due to membrane depolarization. AmJ Physiol Heart CircPhysiol 300:H803-H812.

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