EXPRESSION OF MULTIPLE POPULATIONS OF NICOTINIC ACETYLCHOLINE RECEPTORS IN BOVINE ADRENAL CHROMAFFIN CELLS DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Graduate School of The Ohio State University By Bryan W. Wenger, B.A. The Ohio State University 2003 Dissertation Committee: Approved by: Professor Dennis B. McKay, Advisor Professor R. Thomas Boyd ________________________ Advisor Professor Popat N. Patil College of Pharmacy Professor Lane Wallace, Ph.D. ABSTRACT The importance of the role of nAChRs in physiological and pathological states is becoming increasingly clear. It is apparent that there are multitudes of nAChR subtypes with different expression patterns, pharmacologies and functions that may be important in various disease states. Therefore, a greater understanding of nAChR subtypes is essential for potential pharmacological intervention in nAChR systems. Bovine adrenal chromaffin cells are a primary culture of a neuronal type cell that express ganglionic types of nAChRs whose activation can be related to a functional response. While much is known about the outcome of functional activation of adrenal nAChRs, little work has been done in characterizing populations of nAChRs in adrenal chromaffin cells. These studies characterize the pharmacology and regulation of populations of nAChRs found in bovine adrenal chromaffin cells. The primary findings of this research include 1) the characterization of an irreversible antagonist of adrenal nAChRs, 2) the discovery of spare nAChRs in bovine adrenal chromaffin cells, 3) the pharmacological characterization of mAb35 insensitive nAChRs which may account for as many as 45% of nAChRs in bovine adrenal chromaffin cells, 4) the finding that disulfide bonds do not play a critical role in the function of adrenal nAChRs which is contrary to their importance in other tissues, 5) the use of (+)-tubocurarine in a receptor protection assay ii to identify a subpopulation of nAChRs, 6) the characterization of the role of protein synthesis, glycosylation, and phosphorylation in the turnover of nAChRs, and 7) the identification and partial sequencing of a bovine β4 nAChR transcript expressed in adrenal chromaffin cells. iii This work is dedicated to Amelia Elisabeth Wenger, whose mere possibility allowed me to finish what I had started. iv ACKNOWLEDGMENTS I wish to sincerely thank Dr. Dennis McKay who guided me through this long process and never gave up on me even when I disappeared into another profession. I can not overstate the importance of the love of my wonderful wife Jane and my family in the completion of this long task. Their interest, support, understanding, and, yes, even nagging pushed me the rest of the way through. Finally, I would also like to thank the excellent staff of the College of Pharmacy and my dissertation committee, Dr. R. Thomas Boyd, Dr. Popat N. Patil, and Dr. Lane J. Wallace, for teaching me the nuances of this amazing science and for allowing me to complete my studies. v VITA December 28, 1966……………………….. Born – Tiffin, Ohio December, 1989…………………………... BA. Chemistry, Miami University, Oxford, Ohio September 1993 – September 1996………. Graduate Teaching Assistant, College of Pharmacy, The Ohio State University, Columbus, Ohio September 1996 – September 1997 Presidential Fellow, The Ohio State University, Columbus, Ohio August 1999 – present Chemistry Teacher, Westland High School, South Western City Schools, Columbus, Ohio PUBLICATIONS 1. Maurer, J. A., Wenger, B. W., Guan, Z., Stokes, B. T. and McKay, D. B.: Staurosporine affects calcium homeostasis in cultured bovine adrenal chromaffin cells. Eur. J. Pharmacol. 288: 163-172, 1995. 2. Maurer, J. A., Wenger, B. W. and McKay, D. B.: Effects of protein kinase inhibitors on morphology and function of cultured bovine adrenal chromaffin cells: KN- 62 inhibits secretory function by blocking stimulated Ca2+ entry. J. Neurochem. 66: 105- 113, 1996. 3. Gu, H., Wenger, B. W., Lopez, I., McKay, S. B., Boyd, R. T. and McKay, D. B.: Characterization and localization of adrenal nicotinic acetylcholine receptors: evidence that mAb35-nicotinic receptors are the principle receptors mediating adrenal catecholamine secretion. J. Neurochem. 66: 1454-1461, 1996. 4. Wenger, B. W., Bryant, D. L., Boyd, R. T. and McKay, D. B.: Evidence for spare nicotinic acetylcholine receptors and a ß4 subunit in bovine adrenal chromaffin cells: studies using bromoacetylcholine, epibatidine, cytisine, and mAb35. J. Pharmacol. Exp. Ther. 281: 905-913, 1997. 5. Free, R. B., Wenger, B. W., McKay, D. B.: Effects of sulfhydryl modifications of adrenal nicotinic acetylcholine receptors. Disulfide integrity is not essential for activation. Life Sciences 68: 373-385, 2000. vi 6. Free, R. B., and McKay, D. B.: Receptor protection studies to characterize neuronal nicotinic receptors: tubocurarine prevents alkylation of adrenal nicotinic receptors. Brain Research 891: 176-184, 2001. 7. Wenger, B. W., Boyd, R. T. and McKay, D. B.: Evidence that phosphorylation events and protein synthesis are important in nAChR turnover in bovine adrenal chromaffin cells. Soc. Neurosci. Abstr. 22: 1523, 1996. 8. Wenger, B.W., Boyd, R. T., and McKay, D. B.: Evidence for a nicotinic acetycholine receptor reserve in bovine adrenal chromaffin cells. Pharmacologist 39: 55, 1997. FIELDS OF STUDY Major Field: Pharmacy vii TABLE OF CONTENTS ABSTRACT…………………………………………………………………………….ii. ACKNOWLEDGMENTS……………………………………………………………....v. VITA……………………………………………………………………………………vi. LIST OF TABLES………………………………………………………………………x. LIST OF FIGURES……………………………………………………………………..xi. STATEMENT OF THE PROBLEM……………………………………………………1 CHAPTERS 1. Introduction 1.1 Nicotinic acetylcholine receptors in health and disease………………………..4 1.2 nAChR diversity………………………………………………………………..6 1.3 nAChR Subunit Structure………………………………………………………7 1.4 Neuronal nAChR Subtypes…………………………………………………….9 1.5 Heterogeneous Population of Neuronal nAChRs……………………………..13 1.6 Biochemical Regulation of nAChRs………………………………………….13 1.7 Adrenal nAChRs………………………………………………………………20 2. Materials and methods 2.1 Materials………………………………………………………………………23 2.2 Isolation and primary culture of bovine adrenal chromaffin cells……………24 2.3 Catecholamine secretion studies……………………………………………..27 viii 2.4 Alkylation of nAChRs………………………………………………………..28 2.5 Isolation and purification of mAb35…………………………………………29 2.6 Reverse transcription/polymerase chain reaction (RT/PCR), cloning, sequencing and Northern analysis………………………………………………………..29 2.7 Calculations and Statistics…………………………………………………..32 3. Results 3.1 Alkylation of adrenal nAChRs………………………………………………33 3.2 Identification of a nAChR reserve…………………………………………..38 3.3 Identification of nAChR subpopulations using monoclonal antibodies…….44 3.4 Identification of nAChR subpopulations using receptor protection assays…53 3.5 Effects of disulfide bond reduction on nAChR pharmacology……………..65 3.6 Biochemical regulation of nAChR turnover………………………………..75 3.7 Identification of nAChR subunits…………………………………………..85 4. Discussion 4.1 Adrenal chromaffin cells contain spare receptors………………………….89 4.2 Receptor protection assays detect a subpopulation of nAChRs……………96 4.3 Disulfide integrity is not required for nAChR function…………………..100 4.4 Protein synthesis, glycosylation and phosphorylation are important in nAChR turnover…………………………………………………………………...103 4.5 β4 subunits are expresses in bovine adrenal chromaffin cells……………108 BIBLIOGRAPHY…………………………………………………………………110 ix LIST OF TABLES Table 1. Changes in EC50 values and Emax values of several nAChR agonists after brACh treatment……………………………………………………….43 Table 2. Changes in EC50 values and Emax values of several nAChR agonists after mAb35 treatment………………………………………………………50 Table 3. Sensitivity of residual adrenal nAChRs to nAChR antagonists after mAb35-induced nAChR modulation………………………………….51 Table 4. Ability of nAChR inhibitors to protect nAChRs from alkylation: studies using intermediate and low concentrations of brACh for alkylation….61 Table 5. Pharmacology of tubocurarine-protected nAChRs……………………64 Table 6. Effects of DTT pretreatment on the affinity of nAChR inhibitors……67 Table 7. EC50 and Emax values for agonists after treatment with DTT…………74 x LIST OF FIGURES Figure 1. BrACh-induced stimulation of bovine adrenal nAChRs: concentration- dependent effects………………………………………………………...35 Figure 2. BrACh-induced inactivation of bovine adrenal nAChRs: concentration- dependent effects………………………………………………………..36 Figure 3. Time course for recovery from the inhibitory effects of brACh on adrenal catecholamine release……………………………………………………37 Figure 4. The concentration-response effects of nicotine after brACh-induced nAChR inactivation……………………………………………………...40 Figure 5. The concentration-response effects of epibatidine after brACh-induced nAChR inactivation……………………………………………………...41 Figure 6. The concentration-response effects of cytisine after brACh-induced nAChR inactivation……………………………………………………...42 Figure 7. The concentration-response effects of nicotine after mAb35-induced nAChR modulation………………………………………………………47 Figure 8. The concentration-response effects of cytisine after mAb35-induced nAChR modulation………………………………………………………48 Figure 9. The concentration-response effects of cytisine after mAb35-induced nAChR modulation………………………………………………………49 Figure 10. Effects of submaximal concentrations of brACh on mAb35 insensitive nAChRs………………………………………………………………….52 Figure 11. Ability of nAChR inhibitors to protect nAChRs from alkylation using 30 µM brACh………………………………………………………………..57