University of Vermont ScholarWorks @ UVM 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 Follow this and additional works at: https://scholarworks.uvm.edu/graddis Part of the Medical Sciences Commons, and the Pharmacology Commons Recommended Citation Syed, Arsalan Urrab, "Regulation Of Middle Meningeal Artery Diameter by Pacap and ATP-Sensitive Potassium Channels" (2016). Graduate College Dissertations and Theses. 486. https://scholarworks.uvm.edu/graddis/486 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. ii 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! iv 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 .................................................................................................