University of Vermont ScholarWorks @ UVM Graduate College Dissertations and Theses Dissertations and Theses 2010 Role of Voltage-Dependent Calcium Channels in Subarachnoid Hemorrhage-Induced Constriction of Intracerebral Arterioles Matthew ysN toriak University of Vermont Follow this and additional works at: https://scholarworks.uvm.edu/graddis Recommended Citation Nystoriak, Matthew, "Role of Voltage-Dependent Calcium Channels in Subarachnoid Hemorrhage-Induced Constriction of Intracerebral Arterioles" (2010). Graduate College Dissertations and Theses. 168. https://scholarworks.uvm.edu/graddis/168 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]. ROLE OF VOLTAGE-DEPENDENT CALCIUM CHANNELS IN SUBARACHNOID HEMORRHAGE-INDUCED CONSTRICTION OF INTRACEREBRAL ARTERIOLES A Dissertation Presented by Matthew A. Nystoriak 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 October, 2010 Accepted by the Faculty of the Graduate College, The University of Vermont, in partial fulfillment of the requirements for the degree of Doctor of Philosophy, specializing in Pharmacology. Dissertation Examination Committee: Mark T. Nelson, Ph.D. lJl_~ Chairperson =-----:-~",....-..........~7'f-------Dean, Graduate College Date: August 30, 2010 ABSTRACT Subarachnoid hemorrhage (SAH) following cerebral aneurysm rupture is associated with substantial morbidity and mortality. The ability of SAH to induce vasospasm in large diameter pial arteries has been extensively studied, although the contribution of this phenomenon to patient outcome is unclear. Conversely, little is known regarding the impact of SAH on intracerebral (parenchymal) arterioles, which are critical for regulation of cerebral blood flow. To assess the function of parenchymal 2+ 2+ arterioles following SAH, measurements of diameter, intracellular Ca ([Ca ]i) and membrane potential were performed in intact arterioles from unoperated (control), sham- operated and SAH model rats. At physiological intravascular pressure, parenchymal 2+ arterioles from SAH animals exhibited significantly elevated [Ca ]i and enhanced constriction compared with arterioles from control and sham-operated animals. Elevated 2+ [Ca ]i and enhanced tone following SAH were observed in the absence of vascular endothelium and were abolished by the L-type voltage-dependent Ca2+ channel (VDCC) inhibitor nimodipine. Molecular assessment of the L-type VDCC CaV1.2 indicated unchanged mRNA and protein expression in arterioles from SAH animals. Increased CaV1.2 activity following SAH may also reflect enhanced pressure-induced membrane potential depolarization of arteriolar smooth muscle. Membrane potential measurements in arteriolar myocytes using intracellular microelectrodes revealed approximately 7 mV depolarization at 40 mmHg in myocytes from SAH animals. Further, when membrane 2+ potential was adjusted to similar values, arteriolar [Ca ]i and tone were similar between groups. These results demonstrate that greater pressure-dependent membrane potential 2+ depolarization results in increased activity of CaV1.2 channels, elevated [Ca ]i and enhanced constriction of parenchymal arterioles from SAH animals. Thus, impaired 2+ regulation of parenchymal arteriolar [Ca ]i and diameter may restrict cerebral blood flow in SAH patients. Although nimodipine is used clinically to prevent delayed neurological deficits in SAH patients, the use of this drug has been limited by hypotension and treatment options remain inadequate. Therefore, our next objective was to explore strategies to selectively suppress CaV1.2 channels in the cerebral vasculature. To do so, we examined the physiological role of smooth muscle CaV1.2 splice variants containing the alternatively-spliced exon 9* in cerebral artery constriction. Using antisense oligonucleotides, we demonstrate that suppression of exon 9*-containing CaV1.2 splice variants results in substantially reduced cerebral artery constriction to elevated extracellular [K+]. In addition, no further reduction in constriction was observed following suppression of all Cav1.2 splice variants, suggesting that exon 9* splice variants are functionally dominant in cerebral artery constriction. In summary, results shown in this dissertation demonstrate that increased CaV1.2 activity following SAH results in enhanced constriction of parenchymal arterioles. Furthermore, evidence is provided supporting the concept that CaV1.2 splice variants with exon 9* are critical for cerebral artery constriction and may provide a novel target for the prevention of delayed ischemic deficits in SAH patients. CITATIONS Material from this dissertation has been published in the following form: Nystoriak, M.A., Murakami, K., Penar, P.L., Wellman, G.C.. (2009) Cav1.2 splice variant with exon 9* is critical for regulation of cerebral artery diameter. American Journal of Physiology, Heart and Circulatory Physiology, 297(5):H1820-8. Material from this dissertation has been accepted for publication in the following form: Koide, M., Nystoriak, M.A., Brayden, J.E., Wellman, G.C.. (December, 2009) Impact of subarachnoid hemorrhage on local and global calcium signaling in cerebral artery myocytes. Early Brain Injury or Cerebral Vasospasm, Vol. 1: Pathophysiology, Springer Press, Inc. Nystoriak, M.A., Koide, M., Wellman, G.C.. (February, 2010) Assessment of intracellular calcium in cerebral artery myocytes. Animal Models of Acute Neurological Injuries II: Injury and Mechanistic Assessments, Springer Press, Inc. Koide, M., Nystoriak, M.A., Krishnamoorthy, G., O’Connor, K.P., Bonev, A.D., Nelson, M.T., Wellman, G.C.. (July, 2010) Reduced Ca2+ spark activity following subarachnoid hemorrhage disables BK channel control of cerebral artery tone. Journal of Cerebral Blood Flow and Metabolism. Material from this dissertation has been submitted for publication to American Journal of Physiology, Heart and Circulatory Physiology on August 2, 2010 in the following form: Nystoriak, M.A., Sonkusare, S.K., Brayden, J.E., Nelson, M.T., Wellman, G.C.. Fundamental increase in pressure-dependent constriction of brain parenchymal arterioles from subarachnoid hemorrhage model rats due to membrane depolarization. American Journal of Physiology, Heart and Circulatory Physiology. ii ACKNOWLEDGEMENTS I believe that the success of one in pursuit of a doctoral degree is determined not only by the effort put forth by the student, but also that put forth by the mentor. With that said, I am extremely fortunate to have worked with my advisor, Dr. George Wellman. From day one, George has been a truly great mentor and the experience that I have had working with him has been invaluable. I would like to thank George for everything, including help with wrting manuscripts and proposals, advice with designing and performing experiments, as well as encouraging me to attend scientific meetings and work on my golf swing. In addition, this dissertation is physical proof that a Yankees fan can peacefully and productively coexist with a Red Sox fan. I also feel very fortunate to have had a dissertation committee consisting of faculty members who are internationally recognized as experts in their respective fields. For their time and effort in helping to guide my work, I would like to thank: Drs. Joseph Brayden, Victor May, Mark Nelson, and Margaret Vizzard. I believe that the willingness of these outstanding scientists to share their expertise is very much reflected in my research. I must thank everyone working in the Wellman laboratory for all of their help: Drs. Masayo Koide and Kentaro Murakami, Mr. Micah Been-Miller, Mr. Colby Cantu, iii Ms. Jaqueline Hubbard, Mr. Kevin O’Connor, Ms. Ashley Ross, and Ms. Sheila Russell. Working alongside you every day in the lab has been a very enjoyable experience. I would also like to express gratitude toward Dr. Natalia Gokina for taking time to share expertise regarding the use of fura-2 to measure intracellular calcium in intact arteries. Members of the Center of Biomedical Research Excellence Program, particularly Thomm Buttolph and Edward Zelazny, were also instrumental in my training and understanding of several techniques. As an undergraduate at the Unversity of Vermont, I spent time as a work-study student in the department of Phamacology in Dr. Deborah Damon’s laboratory. Initially, most time was spent washing dishes, but later I began to perform various molecular biology and cell culture techniques. It was during this time that I first became excited by research. I would like to thank Dr. Damon for allowing me to work in her laboratory, as I would not have chosen this career path otherwise. This dissertation is dedicated to my parents, Barbara and David Nystoriak. From a very early age, my parents taught me the importance of working hard and receiving a high-quality education. Without their constant encouragement, I never would have dreamed of pursuing a career in science. I would also like to thank my friends and family in New York for their love and support. iv Finally, I am deeply indepted to my wife and best friend Jessica. On days when experiments failed, she would see my frustration and remind me to bear in mind how good the good days feel. I couldn’t