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CHARACTERIZATION OF a^-ADRENERGIC RECEPTOR MEDIATED DNA AND PROTEIN SYNTHESES IN PRIMARY CDLTDRED RAT HEPATOCYTES A Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Timothy A. Esbenshade, B.S., M.S. ***** The Ohio State University 1991 Dissertation Committee: Approved by Dennis R. Feller, Ph.D. Duane D. Miller, Ph.D. Popat N. Patil, Ph.D. Lane J. Wallace, Ph.D. Dennis R. Feller, Advisor College of Pharmacy To Isabel and Mom and Dad 11 ACKNOWLEDGEMENTS I wish to express my sincere thanks to: My adviser. Dr. Dennis R. Feller, who in addition to his limitless patience and friendship, provided constant advice and support during the course of my graduate studies. The members of my dissertation committee: Dr. Duane D. Miller for providing the compounds necessary for completion of this dissertation work and assistance with the chemistiry questions associated with this project. Dr. Popat N. Patil for the interesting discussions concerning this research and Dr. Lane J. Wallace for his challenging questions and valuable assistance with computer associated problems. All of the graduate students, both past and present, that I have had the pleasure of becoming friends with both in and out of the laboratory. All have made my time here enjoyable and worthwhile. The National Institutes of Health (Grant GM-29358) for stipend support. My parents, Berlyn and Nedra, for their loving guidance through the years and their unending concern and support for my graduate education even when it meant time away from them. Finally, my thanks and love go to Isabel, whose understanding patience, help, encouragement and love during the course of this work meant everything to me. I anxiously await the day when we can start our lives together as one. I l l VITA October 31, 1959 ................... Born - Shelby, Ohio June, 1983 .......................... B.S. Pharmacy, The Ohio State University, Columbus, Ohio Sept., 1985 - Aug., 1987 ............. Graduate Teaching Associate, The Ohio State University Sept., 1987 - present .............. Graduate Research Associate, The Ohio State University June, 1988 .......................... M.S. Pharmacology, The Ohio State University PUBLICATIONS Romstedt K, Esbenshade T, Kocarek T, Loiodice F, Tortorella V, Witiak D, Newman H and Feller D. Effects of the isomers of 2-(4- chlorophenoxy)propionic acid (CPPA) in human platelets and rat hepatocytes. The Pharmacologist 28: 310, 1986. Esbenshade TA, Kocarek TA, Kamanna VJ, Loiodice F, Tortorella V, Newman HAI, Witiak DT and Feller DR. Hepatic proliferating actions of clofibric acid related analogs in vivo and in vitro; stereostructure activity relationships. Fed. Proc. 46: 6867, 1987. Esbenshade T, Loiodice F, Tortorella V, Newman HAI, Witiak D, Krishna G and Feller DR. Structural requirements of phenoxycarboxylic acid compounds for peroxisome proliferation in primary cultured rat hepatocytes. The Pharmacologist 30: 128.7, 1987. Esbenshade T, Hamada A., Miller D and Feller. Stereochemical dependent stimulation of DNA synthesis by isomers of catecholamines and imidazolines in primary cultured rat hepatocytes. FASEB J. 4: 354, 1990. Esbenshade T, Hamada A, Miller D and Feller D. Effects of a-adrenergic agents on DNA synthesis in primary cultured rat hepatocytes. The Pharmacologist 32: 378, 1990. Ordway GA, Esbenshade TA, Kolta MG, Gerald MC and Wallace LJ. Effect of age on cholinergic responsiveness and receptors in the rat urinary bladder. J. Urol. 136: 492-496, 1986. Witiak DT, Feller DR, Newman HAI, Kim SK, Tehim AK, Romstedt KJ, Kim SU, Esbenshade TA and Kamanna VJ. Medicinal chemistry aspects of ant ilipidemic drugs: aci-reductone antilipidemic and antiaggregatory agents. Actual. Chim. Ther. 15: 41-62, 1988. IV PUBLICATIONS (continued) Esbenshade TA, Kamanna vs, Newman HAI, Tortorella V, Witiak DT and Feller DR. In vivo and in vitro peroxisome proliferation properties of selected clofibrate analogues in rat: structure activity relationships. Biochem. Pharmacol. 40: 1263-1274, 1990. FIELD OF STUDY Major Field: Pharmacology TABLE OF CONTENTS DEDICATION . ................................................ ii ACKNOWLEDGEMENTS .............................................. iii VITA . ...................................................... iv TABLE OF ABBREVIATIONS......................................... ix LIST OF TABLES ............................................ xi LIST OF FIGURES .......................................... xiii CHAPTER I. INTRODUCTION ..................................... 1 A. Historical Perspective of Adrenergic Pharmacology .... 1 B. a^-Adrenergic Receptor ................................ 5 1. «^-Adrenergic receptor structure ................. 5 2. Signal transduction of « -adrenergic receptor .... 7 3. Subtypes of the «^-adrenergic re c e p t o r ............... 10 4. Hepatic «^-adrenergic receptor ..................... 12 C. Structure-Activity Relationships for «^-adrenergic receptor agonists ................................. 14 1. Stereochemical requirements for «^-adrenergic receptor agonists ............................ 15 2. structural requirements of «^-adrenergic receptor a g o n i s t s .................................. 19 a) Aromatic hydroxyl substitution ............... 19 b) Halogénation of the aromatic r i n g ........... 21 c) Benzylic hydroxyl substitution ............... 22 d) Benzylic amino substitution ................. 23 e) «-Carbon atom substitution................... 23 f) Substitution at the nitrogen a t o m ........... 24 g) Effect of connective bridge length and attachment position of imidazoline ring on « - adrenergic receptor activity of imidazolines . 24 h) Miscellaneous structure-activity relationships of imidazolines .......................... 25 D. «^-Adrenergic Receptor Mediated DNA Synthesis and Liver Régénérâtxon ...................... 27 1. Liver regeneration ................ .............. 27 2. Hepatocyte growth factors and growth factor r e c e p t o r s .................................. 30 a) Epidermal growth factor .................... 32 b) Epidermal growth factor receptor ............. 32 3. «^-Adrenergic receptor mediated DNA synthesis and liver regeneration ......................... 34 E. Statement of the P r o b l e m ........................... 41 1. Objectives and rationale ..................... 41 2. Significance ................................ 46 VI CHAPTER II. EFFECTS OF THE STEREOCHEMICAL ORIENTATIONOF A SERIES OF PHENETHYLAMINES AND IMIDAZOLINES ON a -ADRENERGIC RECEPTOR MEDIATED DNA SYNTHESIS IN PRIMARY CULTURED RAT HEPATOCYTES ............................................. 48 A. Specific A i m s ....................................... 48 B. M e t h o d s ............................................. 48 1. Materials ....................................... 48 2. A n i m a l s ......................................... 49 3. Isolation and preparation of primary cultures of adult rat hepatocytes ......................... 49 4. Biochemical assays of cell ly s a t e s ................. 50 5. Data analysis ................................... 51 C. R e s u l t s ............................................. 51 1. Evaluation of the stereochemical dependent stimulation of a^-adrenergic receptor mediated DNA synthesis by phenethylamines in primary cultured rat hepatocytes................................ 52 2. Determination of the stereochemical dependent stimulation of a -adrenergic receptor mediated DNA synthesis by 2-substituted catecholimidazolines in primary cultured rat hepatocytes ............... 53 3. Effect