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Characterization of ai-adrenergic receptor mediated DNA and protein syntheses in primary cultured rat hepatocytes

Esbenshade, Timothy Allan, Ph.D.

The Ohio State University, 1991

U'M’I SOON. Zeeb Rd. Ann Arbor, MI 48106

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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 « -adrenergicreceptor .... 7 3. Subtypes of the«^-adrenergic re c e p t o r ...... 10 4. Hepatic «^-adrenergicreceptor ...... 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 of chirality at the 4-position of the imidazoline ring of catecholimidazolines on the stimulation of DNA synthesis mediated by the o^- adrenergic receptor in primary cultured rat hepatocytes ...... 54 4. Evaluation of the effects of B-carbon amino substitution in phenethylamines on DNA synthesis mediated by the a -adrenergic receptor in primary cultured rat hepatocytes ...... 55 D. D i s c u s s i o n ...... 56 1. Phenethylamines...... 56 2. 2-Substituted catecholimidazolines ...... 62 3. 4-Substituted catecholimidazolines ...... 65 4. 3/4-Dihydroxyphenylethylenediamines ...... 67 5. General Discussion ...... 68 CHAPTER III. PHARMACOLOGIC CHARACTERIZATION OF a -ADRENERGIC r e c e p t o r MEDIATED DNA AND PROTEIN SYNTHESES IN PRIMARY CULTURED RAT HEPATOCYTES ...... 86 A. Specific A i m ...... 86 B. M e t h o d s ...... 86 1. Materials ...... 86 2. Isolation and preparation of primary cultures of rat hepatocytes...... 87 3. Biochemical assays of cell lysates and data analysis 88 C. R e s u l t s ...... 89 1. Evaluation of the effect of modification of the substituent at the 4'-benzyl position of imidazoline analogs on stimulation and inhibition of a^-adrenergic receptor mediated DNA synthesis . 89 2. Effects of , and on DNA synthesis in 48 hr primary cultured rat hepatocytes...... 90 3. Characterization of the a -adrenergic receptor subtype(s) that modulates DNA synthesis in primary cultured rat hepatocytes ...... 93 4. Dete^ination of the possible role of extracellular Ca* and calmodulin in a -adrenergic receptor mediated DNA synthesis ...... 96 5. Evaluation of the effects of selected modifiers of DNA and protein syntheses in primary cultured rat hepatocytes...... 97 D. Discussion ...... 100 1. Effects of 4 '-benzyl substitution of selected imidazoline compounds ...... 100 2. Effects of tolazoline, cirazoline and idazoxan on DNA synthesis ...... 103 3. Effects of selective a-adrenergic receptor subtype antagonists on R-NE stimulated DNA synthesis . . . 107 4. Effects of verapamil and TFP on a -adrenergic receptor mediated DNA synthesis ...... Ill 5. Effects of selected modifiers of a^-adrenergic receptor mediated DNA and protein syntheses .... 113 CHAPTER IV. SUMMARY OF D A T A ...... 141 APPENDIX ...... 145 Specific A i m ...... 145 M e t h o d s ...... 146 1. Materials ...... 146 2. Isolation and preparation of primary cultures of rat hepatocytes...... 146 3. Preparation of autoradiographs ...... 147 4. Biochemical assays of cell lysates and data analysis 147 R e s u l t s ...... 148 1. Evaluation of the effects of growth factors on DNA synthesis in primary cultured rat hepatocytes . . . 148 2. Evaluation of the effects of media on DNA synthesis in primary cultured rat hepatocytes ...... 149 3. Determination of the effects of the culture dish surface on DNA synthesis in primary cultured rat hepatocytes ...... 149 4. Evaluation of the effects of nialamide treatment on R-NE stimulated DNA synthesis in primary cultured rat hepatocytes...... 150 5. Evaluation of the expression of [ H]-thymidine incorporation as DPM/culture and DPM/jjg protein . . 151 6. Evaluation of the effects of selected adrenergic antagonists on R-NE and DHT stimulated DNA synthesis in primary cultured rat hepatocytes . . . 152 7. Autoradiographs of primary cultured rat hepatocytes incubated with [ H]-thymidine...... 153 D i s c u s s i o n ...... 153 1. Growth factor effects ...... 153 2. Media effects ...... 155 3. Culture surface effects ...... 156 4. Nialamide effects ...... 157 5. Expression of data as DPM/culture and DPM/^ig protein 157 6. Effects of adrenergic antagonists ...... 158 7. Autoradiographs ...... 159 REFERENCES ...... 170 TABLE OF ABBREVIATIONS

ABBREVIATION NAME

ABI ...... 2-(4-Aminobenzyl)imidazoline

B SA ...... Bovine Serum Albumin

Ca " ...... Calcium

CEO ...... Chloroethylclonidine

CRZ ...... Cirazoline

DA ...... Dopamine

DAG ...... 1,2 Diacylglycerol

R-DBI ...... R-4-(3,4-Dihydroxybenzyl)imidazoline

S-DBI ...... S-4-(3,4-Dihydroxybenzyl)imidazoline

DHT ...... 2-(3,4-Dihydroxybenzyl)imidazoline [3,4-Dihydroxytolazoline]

DNA ...... Deoxyribonucleic Acid

DPM ...... Disintegrations Per Minute

R-DPDA ...... R-3,4-Dihydroxyphenylethylenediamine

S-DPDA ...... S-3,4-Dihydroxyphenylethylenediamine

EOF ...... Epidermal Growth Factor

IBI ...... 2-(4-Isothiocyanatobenzyl)imidazoline

IDZ ...... Idazoxan

Ins(l,4,5)P^ ...... Inositol 1,4,5-trisphosphate

G-protein ...... Guanine nucleotide binding protein

GTP ...... Guanosine Triphosphate

MAO ...... Monoamine Oxidase

M E M ...... Minimum Essential Media

R-NE ...... R-

S-NE ...... S-Norepinephrine

IX t a b l e o f ABBREVIATIONS (continued)

ABBREVIATION NAME

PIPj ...... Phosphatidylinositol-4,5-bisphcsphate PLC ...... Phospholipase C PRZ ...... S Z L - 4 9 ...... l-(4-Aniino-6,7-diinethoxy-2-quinazolinyl) 4-(2-bicyclo[2.2.2]octa-2,5-diene-2- carbonyl)- R-TBI ...... R-2-(3,4,a-Trihydroxybenzyl)imidazoline S-TBI ...... S-2-(3,4,a-Trihydroxybenzyl)imidazoline TCA ...... Trichloroacetic Acid TFP ...... T G F a ...... Trans foirming Growth Factor a TGFB ...... Transforming Growth Factor B T L 2 ...... Tolazoline TPA ...... 12-o-Tetradecanolylphorbol-13-acetate WB 4101 ...... 2-(2,6-Dimethoxyphenoxyethyl)-amino- methyl-1,4-benzodioxane WE M e d i a ...... Williams E Media LIST OF TABLES

TABLE PAGE

1. Summary of differences in the interaction of phenethylamines and imidazolines with a -adrenergic receptors [adapted from Ruffolo (1988)1 . 20

2. Effects of R-NE, S-NE and DA on total cellular protein in 48 hr primary cultured rat hepatocytes ...... 75

3. Effects of R-TBI, S-TBI and DHT on total cellular protein in 48 hr primary cultured rat hepatocytes ...... 79

4. Effects of R-DBI, S-DBI and R,S-DBI on total cellular protein in 48 hr primary cultured rat hepatocytes ...... 82

5. Effects of R-DPDA and S-DPDA on total cellular protein in 48 hr primary cultured rat hepatocytes ...... 84

6. Important parameters of the concentration response curves for the stimulation of [ H]-thymidine incorporation into the DNA of primary cultured rat hepatocytes by stereoisomers of phenethylamines and imidazolines ...... 85

7. Effects of tolazoline, ABI and IBI on total cellular protein in 48 hr primary cultured rat hepatocytes ...... 120

8. Effects of tolazoline and cirazoline alone and with R-NE and DHT stimulated cells on total cellular protein in 48 hr primary cultured rat hepatocytes ...... 124

9. Effects of idazoxan and R-NE on [^H]-thymidine incorporation 48 hr primary cultured rat hepatocytes ...... 126

10. Effects of idazoxan alone and with R-NE and DHT stimulated cells on total cellular protein in 48 hr primary cultured rat hepatocytes ...... 127

11. Effects of WB 4101, CEC, SZL-49 and PRZ alone on [^H]- thymidine incorporation in 48 hr primary cultured rat hepatocytes ...... 129

12. Effects of WB 4101, CEC, SZL-49 and PRZ alone and with R-NE stimulated cells on total cellular protein in 48 hr primary cultured rat hepatocytes ...... 130

13. Effects of TFP, verapamil and R-NE alone on [^H]-thymidine incorporation in 48 hr primary cultured rat hepatocytes . . 132

14. Effects of TFP, verapamil and PRZ alone and with R-NE stimulated cells on total cellular protein in 48 hr primary cultured rat hepatocytes ...... 133

XI TABLE PAGE 15. Comparative inhibition of R-NE stimulated [ ] -thymidine incorporation into the DNAof 48 hour primary cultured rat hepatocytes by TLZ, CRZ, IDZ, WB 4101, CEC,SZL-49 and PRZ . 134 16. Effects of PRZ, CEC, SZL-49, CRZ, YOH and IDZ on total cellular protein in 48 hr primary cultured rat hepatocytes stimulated by R-NE and DH T ...... 140 LIST OF FIGURES

FIGURE PAGE 1. Schematic representation of the Easson-Stedman hypothesis . 17 2. Chemical structures of R-norepinephrine, S-norepinephrine and dopamine ...... 71 3. Chemical structures of R-2-(3/4,a-trihydroxybenzyl)- imidazoline, S-2-(3,4,a-trihydroxybenzyl)imidazoline and 2-(3,4-dihydroxybenzyl)imidazoline ...... 72 4. Chemical structures of R-4-(3,4-dihydroxybenzyl)imidazoline, S-4-(3,4-dihydroxybenzyl)imidazoline, R-3,4-dihydroxyphenyl- ethylenediamine and S-3,4-dihydroxyphenylethylenediamine . . 73 5. Concentration response curves for the stimulation of [^H]- thymidine incorporation into the DNA of primary cultured rat hepatocytes by R-NE, S-NE and D A ...... 74 6. Effects of 1 pM prazosin on -thymidine incorporation stimulated by R-NE, S-NE and DA in primary cultured rat hepatocytes ...... 75 7. Concentration response curves for the stimulation of [^H]- thymidine incorporation into the DNA of primary cultured rat hepatocytes by R-TBI, S-TBI and D H T ...... 77 8. Effects of 1 prazosin on [^H]-thymidine incorporation stimulated by R-TBI, S-TBI and DHT in primary cultured rat hepatocytes ...... 78 9. Concentration response curves for the stimulation of [^H]- thymidine incorporation into the DNA of primary cultured rat hepatocytes by R-DBI, S-DBI andR,S-DBI ...... 80 10. Effects of 1 pM prazosin on [^H]-thymidine incorporation stimulated by R-DBI, S-DPDA and S-DPDA in primary cultured rat hepatocytes ...... 81 11. Concentration response curves for the stimulation of [^H]- thymidine incorporation into the DNA of primary cultured rat hepatocytes by R-DPDA and andS-DPDA ...... 83 12. Chemical structures of tolazoline, 2-(4-aminobenzyl)- imidazoline, 2-(4-isothiocyanatobenzyl)imidazoline, cirazoline and idazoxan ...... 116 13. Chemical structures of prazosin, WB 4101, chloroethyl­ and SZL-49 ...... 117 FIGURE PAGE 14. Concentration response curves for the stimulation of [^H]- thymidine incorporation into the DNA of primary cultured rat hepatocytes by tolazoline, ABI and IBI and the response to 100 pM D H T ...... 118 15. Effects of 1 f M prazosin, 100 ptH tolazoline, 30 ABI and 10 ]M IBI on [ H]-thymidine incorporation stimulated by 10 R-NE and 100 j M DHT in primary cultured rat hepatocytes ...... 119 16. Concentration response curves for the stimulation of [^H]- thymidine incorporation into the DNA of primary cultured rat hepatocytes by tolazoline and cirazoline and the response to 100 juM D H T ...... 121 17. Concentration dependent inhibition of 10 R-NE stimulated [ H]-thymidine incorporation by cirazoline and tolazoline in primary cultured rat hepatocytes ...... 122 18. Concentration dependent inhibition of 100 pM DHT stimulated [ H]-thymidine incorporation by cirazoline and tolazoline in primary cultured rat hepatocytes ...... 123 19. Concentration dependent inhibition of 10 pM R-NE and 100 pM DHT stimulated [ H]-thymidine incorporation by IDZ in primary cultured rat hepatocytes ...... 125 20. Concentration dependent inhibition of 10 pM R-NE stimulated [ H]-thymidine incorporation by WB 4101, CEC, SZL-49 and prazosin in primary cultured rat hepatocytes ...... 128 21. Concentration dependent inhibition of 10 pM R-NE stimulated [ H]-thymidine incorporation by TFP, verapamil and prazosin in primary cultured rat hepatocytes ...... 131 22. Time dependent effects of R-NE and DHT on [^H]-thymidine incorporation in primary culturedrat hepatocytes ...... 135 23. Time dependent effects of R-NE and DHT on [^^S]-methionine incorporation in primary cultured rathepatocytes ...... 136 24. Time dependent effects of R-NE and DHT on total cellular protein in primary cultured rathepatocytes ...... 137 25. Effect of PRZ, CEC, SZL-49, CRZ, YOH and IDZ on [^H]- thymidine incorporation stimulated by 10 pM R-NE and 100 pM DHT in 48 hr primary cultured rat hepatocytes co-incubated with [ S ]-methionine ...... 138 26. Effect of PRZ, CEC, SZL-49, CRZ, YOH and IDZ on [^^S]- methionine incorporation stimulated by 10 pM R-NE and 100 pM DHT in^48 hr primary cultured rat hepatocytes co-incubated with [ H]-thymidine ...... 139 27. Effects of growth stimulating hormones on [^H]-thymidine incorporation in control and 10 pM R-NE treated primary cultured rat hepatocytes ...... 160 FIGURE PAGE

28. Effects of Minimum Essential Medium and Williams E medium on [ H]-thymidine incorporation in control and R-NE treated primary cultured rat hepatocytes ...... 161

29. Effects of dish surfaces on -thymidine incorporation in control and 100 ]M R-NE treated primary cultured rat hepatocytes ...... 162

30. Effects of 50 pM nialamide treatment on [^H]-thymidine incorporation in control and R-NE treated primary cultured rat hepatocytes ...... 163

31. Concentration-dependent effects of R-NE on [^H]-thymidine incorporation in primary cultured rat hepatocytes: A comparison of expression of data as DPM/culture and DPM/pg p r o t e i n ...... 164

32. Concentration-dependent effects of R-NE on [^H]-thymidine incorporation (left ordinate) and total cellular protein (right ordinate) in primary cultured rat hepatocytes: A comparison of data expressed as DPM/culture and DPM/pg protein and transformed to percent of control ...... 165

33. Concentration-dependent effects of R-NE on [ ]-thymidine incorporation in primary cultured rat hepatocytes: A comparison of data expressed DPM/culture and DPM/jug protein and transformed to percent of maximal response ...... 166

34. Effects of the adrenergic receptor antagonists prazosin, yohimbine and on [ H]-thymidine incorporation stimulated by 10 pM R-NE and 100 pM DHT in primary cultured rat hepatocytes ...... 167

35. Autoradiographs of primary cultured rat hepatocytes incubated with [ H]-thymidine (5 pCi/ml) for 48 hr (magnification = lOOX) ...... 168

36. Autoradiographs of primary cultured rat hepatocytes incubated with [ H]-thymidine (5 pCi/ml) for 48 hr (magnification = 200X) ...... 169

XV CHAPTER I

1. Introduction. A. Historical Perspective of Adrenergic Pharmacology. Ahlguist (1948) first proposed that adrenergic receptors could be divided into two subtypes (designated a and J3) based upon the pharmacological properties of these receptors as opposed to the type of physiological response elicited upon stimulation of the adrenergic receptors. Thus, it was not the nature of the tissue response which characterized the receptor type but rather the ability of the receptor to recognize and respond to different drugs that characterized a receptor. This concept has led to additional classification of adrenergic receptors as well as other receptor types. The further subclassification of adrenergic receptors is reviewed below. The first adrenergic receptor class of receptors to be subtyped were the B-adrenergic receptors when it became apparent that these receptors possessed different pharmacological properties in different tissues. Furchgott (1967) and Lands (1967a,b) demonstrated the existence of two unique B-subtypes which have different pharmacologic properties, and these were defined as B^ and B^. These receptor subtypes exhibit a number of similarities but only a few differences. For example, the major signal transduction mechanism for both B^- and Bj-adrenergic receptors involves the formation of cyclic AMP via activation of adenylyl cyclase. Additionally, for most pharmacological agents, there is only a 20- to 50-fold difference in the potency of binding to the receptor subtypes (Minneman, 1988). More recently, it became apparent that the o-adrenergic receptor class was composed of a heterogenous population of receptors which did 2 not possess identical pharmacologic properties. Based upon the pharmacological, functional and anatomical differences of the pre- and post-synaptic a-adrenergic receptors, it was originally proposed by Langer (1974) that postsynaptic a-adrenergic receptors be referred to as a^-adrenergic receptors whereas those located presynaptically be referred to as a^-adrenergic receptors. Subsequent to this initial subclassification of the a-adrenergic receptors, it was proposed that these subtypes should be defined pharmacologically instead of anatomically since the a^-adrenergic receptor may have additional effects other than the presynaptic inhibition of catecholamine release (Berthelsen and Pettinger, 1977). The importance of this pharmacological means of subclassification, rather than a functional and/or anatomical classification, was emphasized upon the demonstration that not only a^-adrenergic receptors exist on smooth muscle cells, but that the a^-adrenergic receptor also exists as a postsynaptic receptor on these cells and can mediate contraction (Docherty and McGrath, 1980; Drew and Whiting, 1979; Langer et al., 1980; Starke, 1981; Timmermans and van Zwieten, 1981). Not only can a^-adrenergic receptors be located postsynaptically, but a^-adrenergic receptors can be found presynaptically on nerve terminals as well (Docherty, 1983; Kobinger and Pichler, 1982), demonstrating again the importance of a pharmacological classification scheme for adrenergic receptors (DeMarinis et al., 1987; Timmermans et al., 1988; Minneman, 1988). Recently, the introduction of new high affinity radioligands for binding competition studies and improved techniques for the measurement of signal transduction and ion fluxes has led to the proposal of further subclassification of the a^- and o^-adrenergic receptors. Evidence which supports this subtyping of a-adrenergic receptors has been gained from both pharmacological findings as well as characterization of these receptors on the molecular level (Bylund, 1988; McGrath and Wilson, 1988; Minneman, 1988). The first reported possible existence of heterogeneity of a- adrenergic receptors was in 1981 (Bylund, 1981) when the binding of

[ ] -clonidine and [^H]-yohimbine was compared in various tissues and species. After this initial finding, additional competition binding studies showed that prazosin was more potent in neonatal rat lung

(Latifpour et al., 1982) and rat cerebral cortex (Cheung et al., 1982) than in human platelets. Studies which compared the binding capabilities of a^-adrenergic receptors in various tissues and species showed that distinct differences in the pharmacological characteristics of these receptors existed (Neylon and Summers, 1985; Cheung et al,,

1986; Bylund, 1988). Two research groups independently proposed that two subtypes of the a^-adrenergic receptor, named as and a ^ , could be defined based on the differences in affinity these receptors display for prazosin (Bylund, 1985; Nahorski et al., 1985). The a^^-adrenergic receptor has a low affinity for prazosin (250 nM) and is the subtype present in human platelets whereas the a^^-adrenergic receptor demonstrates a high affinity for prazosin (5 nM) and is the subtype present in neonatal rat lung (Bylund, 1988). Additional binding assays utilizing tissues and cell lines showed that the affinities of various antagonists were highly correlated when compared in tissues and cell lines which possess the same a^-adrenergic receptor subtype. This further supports the existence of at least two subtypes of a^-adrenergic receptors. Additional evidence for the existence of these two a^- adrenergic receptor subtypes is provided by functional studies which are in agreement with the results from binding assays (Bylund, 1988).

Recently, a third subtype, termed present in an opossum kidney- derived cell line, has also been identified as a distinct a^-adrenergic receptor (Murphy and Bylund, 1988). The laboratory of Lefkowitz has cloned the genes that encode the a^-adrenergic receptor from human platelets (Kobilka et al., 1987) and human kidney (Regan et al., 1988).

The gene for the human platelet a^-adrenergic receptor is located in chromosome 10 and the receptor expressed from this gene has been designated a^-ClO, This receptor has been pharmacologically characterized as the a^^-adrenergic receptor. The human kidney a^- adrenergic receptor is encoded from a gene on chromosome 4 and is designated as 0^-04 and is pharmacologically distinct from the a^-ClO receptor. However, drug affinities for this receptor do not correlate with those for the a^ , or c^g-subtypes and may represent yet another subtype (Bylund, 1988). Arising from studies of the a^-adrenergic receptor, a receptor site that is not recognized by catecholamines but which selectively binds imidazolines and oxazolines has been identified and termed the imidazoline preferring receptor (Lehmann et al., 1989; Michel and Insel, 1989). Although this receptor is not an a-adrenergic receptor subtype, it is included in this section because of its possible importance in the mediation of effects elicited by imidazolines which also act at a^- and Oj-adrenergic receptors. Imidazoline preferring receptors have been identified in a number of tissues including brain, kidney and adipocytes in a variety of species including cow, rabbit, rat and man (Michel amd Insel, 1989). The exact physiological role of this receptor is not fully understood, but identification of imidazoline preferring receptors (sites where [^H]-clonidine was not displaced by catecholamines) in the nucleus reticularis lateralis of bovine and human brain suggest that perhaps these sites play a role in the central regulation of blood pressure (Bousquet et al., 1989). A partially purified brain extract called 'clonidine-displacing substance’ has been shown to displace [^H]- idazoxan from imidazoline preferring receptors and to have a number of physiological activities such as potentiation of ADP-stimulated aggregation in human platelets (Diamant et al., 1987), inhibition of the twitch response in rat vas deferens (Diamant and Atlas, 1986) and contraction of the smooth muscle in the rat gastric fundus (Felsen et , 1987). 5

It can therefore be seen that with the use of new agents which are

specific for the subtypes of a^-adrenergic receptors as well as the use

of molecular biological techniques, further subclassification of a-

adrenergic receptors can be realized. Both of these tools have also

recently been employed in an attempt to subclassify a^-adrenergic

receptors and will be discussed below. It is hoped that the knowledge

gained about the subtypes of a-adrenergic receptors will lead to an

increased understanding of the physiology and/or pathophysiology

associated with these receptors and to the development of agents with

selective therapeutic activity.

B. a^-Adrenergic Receptor.

A general definition for c^-adrenergic receptors is those

receptors which are selectively stimulated by agonists such as and , competitively antagonized by prazosin and and irreversibly blocked by the alkylating agent (Minneman, 1988). Almost all mammalian tissues contain o^-adrenergic receptors but these receptors perhaps serve their most

important function in smooth muscle where they are responsible for mediating the contractile state of the tissue. These receptors also have important functions in the central nervous system, heart and liver.

Much research has been conducted in an attempt to develop agonists and

antagonists of the a^-adrenergic receptor for the clinical treatment of

various disorders such as nasal congestion, hypertension, hypertensive

crisis, pheochromocytoma and paroxysmal atrial tachycardia (Hoffman,

1984; Minneman, 1988).

1. a^-Adrenergic receptor structure

The use of photoaffinity labelling and affinity labelling utilizing radiolabelled probes has enabled researchers to determine the

size and chemical composition of a^-adrenergic receptors. Venter and coworkers (1984) used [^H]-phenoxybenzamine to characterize the a^- adrenergic receptor from rat liver membranes as a single polypeptide with a molecular mass of 85 kDa. By utilizing target size analysis of rat liver c^-adrenergic receptors, it was determined that the intact membrane bound receptor has a molecular mass of 160 kDa, thus suggesting that the intact c^-adrenergic receptor exists in the membrane as a dimer consisting of two 85 kDa subunits (Venter et al., 1984). By using the radiolabelled probe [^^^I]-iodoazidoarylprazosin, the c^-adrenergic receptor from cultured smooth muscle cells has been characterized as a protein with a molecular mass of approximately 80 kDa (Terman et al.,

1988). Additional studies have shown that c^-adrenergic receptors are glycoproteins with a variety of carbohydrates attached to the protein portion of the receptor, most probably at arginine residues (Terman and

Insel, 1988; Terman et al., 1988). The addition of these carbohydrates to the protein portion of the c^-adrenergic receptor appears to play an important role in the insertion and the function of this receptor in the cell membrane (Terman and Insel, 1988; Insel, 1989).

With the advent of molecular biological approaches to the characterization of receptor structure, a number of important features of the c^-adrenergic receptor have now been described, Adrenergic receptors are members of a large family of plasma membrane receptors that possess a number of common features that include (Insel, 1989): seven membrane-spanning domains which are characterized by an intracellular carboxyl terminus, an extracellular amino terminus and three extracellular and three intracellular loops; asparagine sites for glycosylation on the extracellular amino terminal region; and phosphorylation sites on the third intracellular loop (which is the largest with 70 amino acid residues) and carboxyl terminal region

(Cotecchia et al., 1988,1990). This family of receptors also includes

and B^-adrenergic receptors, five muscarinic cholinergic receptor subtypes, two serotonin receptor subtypes, substance K receptor. 7 angiotensin receptor and the visual photoreceptor opsins in rods and cones (Insel, 1989). Since and a^-adrenergic receptors are members of the same family, there is a great deal of homology (44%) in the sequence of the proteins for these two receptors, especially in the transmembrane spanning regions (Cotecchia et al., 1988; Insel, 1989). The a^-adrenergic receptor is linked to a poorly defined guanine nucleotide-binding protein (G protein) which enables this receptor to mediate the effects of catecholamines by serving as an intermediate between the receptor and the intracellular message that is evoked by receptor occupation. Cotecchia et al. (1990) suggested that amino acids 252 to 259 of the third intracellular loop of the a^-adrenergic receptor serves as a recognition site for the G-proteins coupled to this receptor.

2. Signal transduction of a^-adrenergic receptors. The primary signal transduction mechanism employed by adrenergic receptors is the elevation of intracellular free Ca*^ levels (Minneman, 1988). Studies performed by Berridge (1983) demonstrated that Ca*^ mobilizing receptors cause the stimulation of a membrane bound enzyme called phospholipase C (PLC) which catalyzes the breakdown of phosphatidylinositol-4,5-bisphosphate (PIP^) resulting in the formation and release of inositol 1,4,5-trisphosphate [Ins(l,4,5)P^] and 1,2- diacylglycerol (DAG). It is the production of Ins(l,4,5)P^ which links stimulation of the receptor to the release of intracellular Ca*^ primarily from stores in the endoplasmic reticulum (Streb et al., 1983; Joseph, 1984). DAG also serves as a second messenger, causing the activation of protein kinase C by reducing its requirement for Ca*^ (Nishizuka, 1984). Activation of the c^-adrenergic receptor causes the metabolism of inositol phospholipids in almost all tissues in which this effect has been examined (Minneman, 1988), including the liver (Prpic et al.. 1982). Although it is fairly well accepted that both Ins(l,4,5)Pj and DAG play important roles mobilizing intracellular Ca*^ and stimulating protein kinase C following a^-adrenergic receptor activation, it is still not fully understood whether these are the only inositol phospholipid breakdown products which are capable of serving as second messengers for the a^-adrenergic receptor. The scheme described above is a simplified description of a very complex process of inositol phosphate metabolism in which a host of inositol phosphates are produced and may serve as second messengers (for reviews, see Minneman, 1988; Exton, 1988). The a^-adrenergic receptor is linked to one or more G-proteins which enable the receptor to convert extracellular signals such as catecholamines to intracellular signals that include increased levels of cytosolic Ca*^ and DAG. The exact identity of these G proteins is not well defined. The first evidence that G-proteins may be involved in the activity of a^-adrenergic receptors came from radioligand binding studies where it was demonstrated that stable nonhydrolyzable analogs of guanine triphosphate (GTP) modulated the affinity constants of agonists at R^-adrenergic receptors in rat liver membranes (Goodhardt, 1982). The reason for this is that in the absence of GTP, a stable high affinity ternary complex of agonist/receptor/G protein is formed. However, when GTP is introduced, the complex is destabilized, the 6- protein dissociates and only the low affinity agonist/receptor complex remains (Minneman, 1988). In addition, stable analogs of GTP also activate the metabolism of PIP^ in membrane preparations from liver (Wallace and Fain, 1985) suggesting that G-proteins regulate PLC activity. In addition to the metabolism of inositol phospholipids and the subsequent elevation of intracellular Ca^^ and activation of protein kinase C, a variety of other signal transduction mechanisms involving G- proteins may exist for the c^-adrenergic receptor. These include activation of phospholipase A^, an enzyme which releases arachidonic acid from membrane lipids which may then lead to the formation of prostaglandins, thromboxanes and leukotrienes; PLC mediated breakdown of phosphatidylcholine to choline phosphate and DAG; influx of extracellular Ca*^ via Ca*^ channels; activation of phospholipase D resulting in the formation of phosphatidic acid which may facilitate Ca*^ entry into cells; and activation of a cyclic nucleotide phosphodiesterase which would lower cellular levels of cyclic AMP. The liver represents a tissue where more than one signal transduction pathway for «j^-adrenergic receptors may exist. It has been shown by Morgan et al.(1983) that in isolated hepatocytes, increases in inositol phosphates caused by activation of cc^-adrenergic receptors are dependent upon extracellular Ca*^ but increases in cyclic AMP are independent of extracellular Ca*^, suggesting that the hepatic adrenergic receptor in mature male rats is coupled simultaneously to two separate signal transduction mechanisms. Substantial differences have been noted in the manner by which the a^-adrenergic receptor and receptors for the peptide hormones vasopressin and angiotensin II mobilize Ca*^ in hepatocytes. These include the findings that gluconeogenesis activated by a^-adrenergic receptor stimulation occurs in Ca*^-depleted cells whereas the effects of the peptides were eliminated (Garcia-Sainz and Hernandez-Sotomayor, 1985). Thus, these researchers proposed that there exist two signal transduction mechanisms for the a^-adrenergic receptor mediated metabolic effects in liver: one independent of extracellular Ca*^, inhibited by insulin and modulated by glucocorticoids whereas the other was dependent on extracellular Ca*^, was not inhibited by insulin and was modulated by thyroid hormones (Garcia-Sainz and Hernandez-Sotomayor, 1985). These mechanisms presumably corresponded to the cyclic AMP and inositol phosphate responses, respectively (Minneman, 1988). 10

3. Subtypes of the a^-adrenergic receptor.

It has become increasingly apparent that a^-adrenergic receptors do not possess the same pharmacological properties in all tissues

(Flavahan and van Houtte, 1986, Minneman, 1988, McGrath and Wilson,

1988). Morrow and Creese (1986) have subdivided a^-adrenergic receptor binding sites in rat brain based upon the potencies of competitive antagonists such as WB 4101. Before this subclassification was proposed, a number of pharmacological differences between adrenergic receptor mediated effects had been noted in a variety of tissues including the liver. Morgan and coworkers (1983) noted that ot^- adrenergic receptor modulated increases in cyclic AMP were more sensitive to WB 4101 and phentolamine than was the Ca*^ dependent a^- adrenergic receptor activation of phosphorylase activity.

Strong evidence has been obtained from both binding and functional studies that supports the existence of two subtypes of the c^-adrenergic receptor in mammalian tissues (Han et al., 1987a,b; Johnson and

Minneman, 1987; Minneman et al., 1988; Tsujimoto et al., 1989; Piascik et al., 1990). It has been demonstrated that chloroethylclonidine

(CEC), a site directed alkylating agent (LeClerc et al., 1980), inactivates a subset of a^-adrenergic receptor binding sites in different tissues (Han et al., 1987a; Johnson and Minneman, 1987).

These receptors (termed the a^^-adrenergic receptor) had a lower affinity for the competitive antagonists WB 4101 and benoxathian as compared to c^-adrenergic receptors that were not inactivated by CEC.

Therefore, it was concluded that CEC selectively inactivates the subtype whereas the a^^-subtype, which has a high affinity for WB 4101, is insensitive to CEC inactivation. Liver and spleen contain adrenergic receptors predominately of the a^^-subtype whereas heart, brain, kidney, vas deferens and vascular smooth muscle contain mixed populations of both subtypes (Han et al., 1987a,b; Johnson and Minneman,

1987; Tsujimoto et al., 1989). Both receptor subtypes can activate 11 formation of inositol phosphates (Han et al., 1990). Different pathways for the formation of [^H]-inositol phosphates mediated by the and a^^-receptor subtypes have been described by Wilson and Minneman (1990).

Activation of the a^^-adrenergic receptor in isolated hepatocytes with norepinephrine (NE) results in the rapid formation of [^H]Ins(l,4,5)P2 and [^H]-inositol (1,3,4)-trisphosphate and a slower formation of [^H]- inositol (1,4)-bisphosphate and [^H]-inositol (1)-monophosphate. stimulation of renal cells, which contain a mixed population of both subtypes, with NE results in a different pattern of [^H]-inositol phosphate formation, suggesting that cyclic inositol phosphates may have been the principle compounds formed. Additionally, it was suggested that the response to a^^-receptor activation may require the influx of extracellular Ca*^.

Both the and a^^-adrenergic receptor subtypes increase cytosolic Ca*^ levels, but the Ca*^ may originate from different sources since contractions modulated by the a^^-receptor are blocked by nifedipine or chelation of extracellular Ca*^ with EGTA, whereas a^^- adrenergic receptor mediated contractions were not affected by these treatments (Han et al., 1987b). Similarly, Tsujimoto et al. (1989) showed that the two subtypes of c^-adrenergic receptors utilize different mechanisms for increasing cytosolic Ca*^ required for the activation of glycogen phosphorylase activity in rat thoracic aorta.

These results suggest that the a^^-subtype may initially mobilize intracellular Ca*^ stores through formation of Ins(l,4,5)P^ and that the c^^-subtype promotes Ca*^ influx primarily through nifedipine-sensitive

Ca*^ channels.

Terman and coworkers (1990) recently isolated a^-adrenergic receptors from rat liver and brain membranes and showed that when the c^-adrenergic receptor obtained from the liver preparation was exposed to CEC, all [^H]-prazosin binding sites were eliminated, thus demonstrating that this receptor is the a^^-subtype. However, the 12

purified receptor preparation obtained from rat brain displayed two

binding sites with different affinities for [^H]-prazosin, one of which

was inactivated by CEC. The remaining a^-adrenergic receptor displayed

high affinity for WB 4101 and thus corresponded to the a^^-subtype. The

laboratory of Lefkowitz has cloned an c^-adrenergic receptor from the

Syrian hamster which has similar characteristics as those described for

the a^^-subtype (Cotecchia et al., 1988). A novel c^-adrenergic

receptor subtype was recently cloned from a bovine cDNA library by this

same laboratory (Schwinn et al., 1990) that demonstrates a higher

affinity for WB 4101 than the cloned a^^-subtype which indicates that

this receptor possesses some a^^-subtype characteristics. However, this

receptor is inactivated by CEC, which is not expected for the

subtype. Therefore, this receptor may represent a new c^-adrenergic

receptor (possibly an subtype) but additional work must be performed

in order to completely characterize this receptor.

4. Hepatic a^-adrenergic receptor.

Many of the important features of the hepatic a^-adrenergic

receptor such as its molecular properties and the cellular mechanisms

involved in a^-adrenergic receptor mediated responses in the liver are

discussed elsewhere in this chapter. This section will briefly describe

some of the effects mediated by the c^-adrenergic receptor in the liver

as well as regulation of this receptor.

Stimulation of the hepatic c^-adrenergic receptor can cause a

number of effects in the liver including increased amino acid transport,

increased respiration, changes in potassium fluxes, increased ureagenesis, inactivation of glycogen synthase, and increased glycolysis

and gluconeogenesis (Kunos, 1984). Additionally, activation of the

adrenergic receptor causes an increase in DNA synthesis in primary cultured rat hepatocytes, an effect associated with the heterologous downregulation of epidermal growth factor (EGF) receptors (see below). 13

The heterologous regulation of hepatic adrenergic receptors has received much attention. The relative contribution of the and a^- adrenergic receptors to glycogenolysis is species dependent, but even within species, certain conditions will cause a shift in the regulation of metabolic responses from an a^- to a G^-adrenergic receptor dominance. In rats, these conditions include hypothyroidism, adrenalectomy, partial hepatectomy, cholestasis, gender, age, treatment with chemical carcinogens, treatment with pertussis toxin, and primary culturing of hepatocytes (Kunos, 1984). A common feature among many of these conditions that cause a shift to a predominance of G^-adrenergic receptors is a decreased level of cellular differentiation. Primary cultures of rat hepatocytes provide a good model for the investigation of this phenomenon. It has been shown that conversion of the adrenergic activation of phosphorylase from an to a G-adrenergic receptor response occurs within 4 hr of the plating of the cells. This response is not accompanied with a decrease in the density or affinity of the adrenergic receptor. If these cells are preincubated for with lipomodulin, an endogenous inhibitor of phospholipase activity, the activation of phosphorylase is reversed from 6- to a^-adrenergic receptor control (Kunos et al., 1984). Mellitin, a phospholipase A^ activating agent, causes a rapid suppression of a^-adrenergic receptor activation of phosphorylase and the appearance of a response to 6- adrenergic activation within 30 min (Kunos, 1980). These results suggest that phospholipase A^ may play a role in the expression of and G-adrenergic receptor activation, perhaps by influencing the coupling of these receptors in an inverse, reciprocal manner (Kunos, 1984). In primary cultured hepatocytes incubated for up to 72 hr, the switch in the adrenergic control of glycogenolysis from a^- to G^- adrenergic receptor control was associated with progressive decreases and increases in a^- and G^-adrenergic receptor number, respectively, and little change in the affinity of these receptors (Schwarz, 1984). 14

C. Structure-Activity Relationships for a^-Adrenergic Receptor Agonists.

Much of the information about the geometry of the a^-adrenergic

receptor site and the ability of this receptor to modulate many

important physiological processes has been obtained though the study of

structure activity relationships of compounds which interact with this

receptor. Although the isolation and characterization of this receptor

by molecular biological techniques has provided important insights into

the actual chemical composition of the c^-adrenergic receptor, the use

of structure-activity studies has added considerably to the understanding of the manner by which these receptors recognize and

interact with c^-adrenergic agonists and antagonists. A brief discussion of the structure activity relationships of o^-adrenergic receptor agonists as reviewed by DeMarinis et al. (1987) and Ruffolo

(1988) is presented below.

There are two main classes of c^-adrenergic receptor agonists; 1) the phenethylamines, which are represented by agents such as NE, phenylephrine and methoxamine and 2) imidazolines, which include compounds such as , cirazoline and . There are many similarities in the structure activity relationships of these two classes of compounds but there exist several important differences between them and it is these differences which provide intriguing information concerning the manner in which the c^-adrenergic receptor interacts with these two classes of compounds. It has been suggested that these differences may be the result of 1) differences in the manner in which the phenethylamines and imidazolines bind to the same site of the o^-adrenergic receptor (Miller et al., 1983; DeMarinis et al., 1987) or 2) binding of phenethylamines and imidazolines to different parts of the active site of the receptor or different subtypes of the a^- adrenergic receptor (Ruffolo et al., 1977).

As a general rule, phenethylamines, with just a few exceptions, are often selective for the o^-adrenergic receptor and are full agonists 15 that can stimulate the maximal effect that a tissue can produce even when there is no large receptor reserve (Ruffolo and Waddell, 1983}. This is indicative of a high Level of efficacy for this category of agents (Ruffolo et al., 1979; Sevan, 1981; Besse and Furchgott, 1976). In contrast, imidazolines are often selective for e^-adrenergic receptors and are generally partial agonists, exhibiting a low efficacy for activation of the a^-adrenergic receptor (DeMarinis et al., 1987; Ruffolo, 1988). Because of this low efficacy of imidazolines, the potency of these compounds more closely predicts the affinity of these agents as opposed to the phenethylamines which have a high efficacy and thus can occupy a smaller proportion of «^-adrenergic receptors and still elicit a maximal response (Ruffolo, 1982). It is therefore important to use caution when potency alone is used to evaluate the effects of structural modifications of agonists with differing efficacies (Furchgott, 1972; DeMarinis, 1987).

I. Stereochemical requirements for «^-adrenergic receptor agonists. In a review of X-ray crystallographic studies by Carlstrom et al. (1973), five characteristics of direct acting phenethylamines were described as being important to the activity of these compounds. These include 1) a six-membered aromatic ring system, 2) an extended ethylamine side chain oriented approximately perpendicular to the aromatic ring system, 3) the presence of a positively charged (at physiological pH) nitrogen on the ethylamine side chain, 4) the orientation of the B-hydroxyl group on the same side of the molecule (cis) as the meta phenolic hydroxyl group of the aromatic ring thus creating hydrophilic and hydrophobic sides of the compound and 5) an R absolute configuration at the B-carbon atom (DeMarinis et al., 1987). When these five conditions are met, the amino, phenyl and B-hydroxyl groups will be oriented in the proper manner for optimal interaction with the adrenergic receptor. This agrees with the hypothesis presented 16 by Easson and Stedman (1933) which states that for optimal activity for biogenic amines, these three functionalities must be oriented so that a three point attachment with the receptor can be made.

The Easson-Stedman hypothesis (Easson and Stedman, 1933; reviewed by Patil et al., 1975) is the most important theory that predicts the relationship between the stereochemistry of optically active phenethylamines possessing one point of asymmetry at the B-carbon atom and c^-adrenergic receptor agonist activity. This hypothesis proposes a three point attachment of these phenethylamines with the adrenergic receptor. The groups which are responsible for this attachment to the adrenergic receptor include: 1) the basic nitrogen atom which is common to all sympathomimetic amines, 2) the phenyl ring, which upon the addition of a meta- or para-phenolic hydroxyl group was proposed to increase binding to the receptor and 3) the benzylic hydroxyl group present at the 13-carbon atom (see Fig. 1). This hypothesis states that only the R(-)-enantiomer of these optically active phenethylamines can provide the correct stereochemical orientation which will allow all three functional groups to interact with the adrenergic receptor. It has been suggested that the greater potency displayed by the R-isomers is attributable to the correct orientation of the 13-hydroxyl group which allows for possible hydrogen bonding to an interactive site on the receptor. For the corresponding S(+)-isomer and the 13-desoxy derivative of these phenethylamines, the B-hydroxyl group is either incorrectly oriented or absent, respectively, thus allowing only a less favorable two point interaction with the receptor. This then results in compounds which are much less active than the R(-)-isomer. Therefore, the Easson-

Stedman hypothesis predicts for phenethylamines possessing an asymmetric

B-carbon atom a rank order of potency of R(-) > S(+) > desoxy at the a^- adrenergic receptor.

A large number of optically active direct acting c^-adrenergic receptor agonists have been demonstrated to adhere to the Easson-Stedman 17

OH OH OH .OH .OH OH ---

HO H OH .

NH. ira.

Figure 1. Schematic representation of the Easson-Stedman hypothesis depicting the interaction of the R- and S-isomers and desoxy derivative (dopamine) of norepinephrine with the ct -adrenergic receptor. 18

hypothesis (Patil et al., 1967a,b). Additionally, this theory has been shown to be valid for the interaction of phenethylamines containing an asymmetric B-hydroxyl group with a^-, B^- and B^-adrenergic receptors as well (Ruffolo, 1983a,b; Ruffolo et al., 1983). In sharp contrast to the strict adherence of phenethylamines to the Easson-Stedman hypothesis, the a-adrenergic activity of optically active imidazolines are not predicted by this theory (Ruffolo et al., 1983). The rank order of potency for imidazolines at both a^- and a^-adrenergic receptors is commonly desoxy > R(-) > S(+) (Ruffolo et al., 1983; Banning et al., 1984; Sengupta et al., 1987). Results from structure-activity studies of imidazolines indicate that these «^-adrenergic receptor agonists may interact with the receptor by a two point attachment utilizing the nitrogen atom and phenyl ring (Ruffolo, 1983b). One useful means of characterizing the responses of optically active adrenergic agonists at the «^-adrenergic receptor is by the comparison of isomeric (enantiomeric) activity ratios. This ratio is defined as the potency ratio between the R- and S-isomers of these agonists which contain an asymmetric carbon atom. It has been demonstrated that for B-hydroxy-substituted phenethylamines tested in «^-adrenergic receptor systems utilizing guinea pig aorta (Ruffolo et al., 1983) and rat aorta (Rice et al., 1987), the isomeric activity ratios are in excess of 100-fold. However, for enantioraers of catecholimidazolines tested in the same systems, a marked reduction in the isomeric activity ratios to generally less than 10-fold are seen. The same trend is also seen in «^-adrenergic receptor systems utilizing field stimulated guinea pig ileum (Ruffolo et al., 1983) and human platelets (Ahn et al., 1986). By using the criterion of isomeric activity ratios, it is apparent that the «^-adrenergic receptor can better discriminate between enantiomers of phenethylamines which contain an asymmetric B-carbon atom than between isomers of optically active imidazolines. This suggests that the absolute steric requirements of 19

Oj^-adrenergic receptors for phenethylamines is much more stringent than for imidazolines (Ruffolo et al., 1983; Ruffolo, 1988).

2. Structural requirements of c^-adrenergic receptor agonists. A number of structural changes of phenethylamines such as aromatic hydroxyl substitution, aromatic halogénation, B-carbon hydroxyl substitution, substitution at the a-carbon atom and substitution at the nitrogen atom can markedly affect the activity of phenethylamines. Likewise, structural changes of imidazolines such as aromatic hydroxyl and methoxy substitutions, aromatic halogénation, benzylic hydroxyl substitution, addition of substituents to the imidazoline ring and different points of attachment of the imidazoline ring to the remainder of the molecule can also lead to large changes in the activities of these compounds. These structure-activity relationships will be described below. Table 1 summarizes the effects of structural changes on phenethylamine and imidazoline a-adrenergic activity.

a. Aromatic hydroxyl substitution. In a study which examined the effects of aromatic hydroxyl substitution of phenethylamines and imidazolines on a^-adrenergic receptor activity utilizing guinea pig aorta (Ruffolo and Waddell, 1983), it was discovered that this modification affects primarily the affinity of phenethylamines for a^-adrenergic receptors whereas there is little affect on the efficacy. All of the phenethylamines tested were full agonists, but the activity at the a^-adrenergic receptor declinedwith the following order of substitution: 3,4-dihydroxy (epinephrine) > 3-hydroxy > 4-hydroxy > nonphenolic. These differences in potency were accounted for by decreases in the ability of these compounds to bind to the a^-adrenergic receptor (affinity) and were not due to changes in the ability of the phenethylamines to stimulate the receptor (Ruffolo and Waddell, 1983; Ruffolo, 1988). 20

Table 1. Summary of differences in the interaction of phenethylamines and imidazolines with a-adrenergic receptors [Adapted from Ruffolo (1988)].

PARAMETER PHENETHYLAMINES IMIDAZOLINES

Stereochemical Requirements Easson-Stedman Hypothesis Applicable Not applicable Isomeric Activity Ratios Large Small Stereochemical Demands High Low structure—Activity Relationships Phenyl Ring Substitutions Hydroxyl t Affinity t Affinity Fluorine i Activity t & I Activity Iodine — I Activity General Demands Strict Low 8-Carbon Atom Substitution Hydroxyl f Affinity i Affinity Amino i Affinity a-Carbon Atom Substitution Methyl I Affinity N-methyl Substitution t Affinity * Affinity Imidazoline Ring Attachment 2-position t Activity 4-position t Activity N-substituted t Activity Imidazoline Bridge None No Activity Methylene I Activity Ethylene t Activity 21

In the same experimental system, it was demonstrated that aromatic hydroxyl substitution of the imidazolines also affects affinity for the c^-adrenergic receptor, but not to the same extent as phenethylamines

(Ruffolo and Waddell, 1983). However, a critical difference between phenethylamines and imidazolines was realized when it was shown that aromatic hydroxyl substitution of imidazolines, unlike that for phenethylamines, dramatically alters the efficacy of these compounds.

These researchers showed that the activity of imidazolines at the a^- adrenergic receptor declined with the same order of substitutions as with the phenethylamines: 3,4-dihydroxy [3,4-dihydroxytolazoline (DHT)]

> 3-hydroxy > 4-hydroxy > nonphenolic (tolazoline). However, this decline in activity was due primarily to decreases in efficacies of the compounds as opposed to a decline in affinities for phenethylamines.

With the exception of the full agonist DHT, these hydroxy-substituted imidazolines were all partial agonists.

b. Halogénation of the aromatic ring.

A reduction in a^-adrenergic activity occurs when the phenolic hydroxyl groups of the catechol moiety are replaced with 3,4-difluoro or

3,4-dichloro substituents (Ruffolo, 1983a). Aromatic fluorine substitution of NE affected a^-adrenergic receptor activity depending upon the position (2-, 5- or 6-position) at which the hydrogen atom was replaced with a fluorine atom (Kirk et al., 1979). It was shown that the a^/B-adrenergic receptor selectivity ratio could be altered by over

500-fold by these various substitutions. Substitution at the 2- position, producing 2-fluoronorepinephrine, created a compound which was much less potent than NE at the c^-adrenergic receptor thus conferring upon this agent a higher selectivity for B-adrenergic receptors. An a^- adrenergic selective agent (6-fluoronorepinephrine) was created upon fluoro substitution at the 6-position of the phenyl ring since this 22 compound was equiaotive as NE at the a^-adrenergic receptor but 100-fold less potent than NE at B-adrenergic receptors. When fluorine atoms are substituted at the same positions of the phenyl ring of DHT (a catechol-containing imidazoline) as in the phenethylamines, the resulting compounds still retain a^-adrenergic agonist activity in rat thoracic aorta (Lamba-Kanwal et al., 1988). The 2-fluoro and 5-fluoro analogs were full agonists in this system whereas the 6-fluoro analog was a partial agonist. The potencies of the 2- and 5-fluoro analogs were slightly greater than the parent nonfluorinated catecholimidazoline based upon ED^^ values.

c. Benzylic hydroxyl substitution. Differences in the way phenethylamines and imidazolines interact with a^-adrenergic receptors are seen when the effects of benzylic (B- carbon atom of phenethylamines) hydroxyl substitution in these compounds are examined. The substitution at the B-carbon atom does not produce any appreciable changes in the efficacies of either class of compounds but does cause distinctly opposite effects on the affinities of these agents. This benzylic hydroxyl substitution results in a significant increase in the affinity of phenethylamines for the a^-adrenergic receptor that is often more than 100-fold, which is in agreement with the Easson-Stedman hypothesis (1933). This effect has been demonstrated for both a series of NE (Ruffolo et al., 1983) and epinephrine derivatives (Ruffolo and Waddell, 1983) in guinea pig aorta. In contrast to the effects on phenethylamines, benzylic hydroxylat ion results in a decrease in affinity of imidazolines for the oc^-adrenergic receptor that are up to 10-fold in magnitude, again demonstrating the lack of adherence of this class of compounds to the Easson-Stedman hypothesis. The decrease in affinity caused by benzylic carbon hydroxylation of imidazolines has been demonstrated for catechol- containing imidazolines in guinea pig aorta (Ruffolo and Waddell, 1983) 23 and for noncatechol imidazolines (tolazoline derivatives) in rat aorta

(Sengupta et al., 1987). Thus it appears that the B-carbon atom and its substituents play a much greater role in the interaction of phenethylamines with the a^-adrenergic receptor than the corresponding benzylic carbon atom of imidazolines (Ruffolo and Waddell, 1983;

Ruffolo, 1988).

d. Benzylic amino substitution.

Relatively few studies have been performed that have examined the effects of substitution of an amino group in place of the hydroxyl group at the B-carbon atom of phenethylamines. Lehmann and Randall (1948) have investigated the effects of racemic 3,4-dihydroxyphenylethylene- diamine (DPDA) in vivo and showed that this B-carbon atom amino substitution results in a decrease in toxicity. Shams et al. (1990), using resolved isomers of DPDA, showed that the isomers of DPDA maintained the same rank order of potency (R-DPDA > S-DPDA) as the corresponding NE isomers for the stimulation of contraction of rat thoracic aorta via activation of a^-adrenergic receptors. However, the isomers of DPDA were less potent and selective than those of NE in this system. A similar pattern was noted for the aggregatory activity of these compounds mediated by the c^-adrenergic receptor in human platelets. However, S-DPDA was inactive as an agonist in this system, but did block the effect of NE induced aggregation (Shams et al., 1990).

e. a-Carbon atom substitution.

Only the phenethylamines can be substituted at the a-carbon atom and substituents at this position cause changes in the agonist activity of these compounds. The placement of a methyl group at the a-carbon atom causes a small reduction in the potency of the compound at the a^- adrenergic receptor, an effect due primarily to a decrease in affinity

(Patil et al., 1957a,b; Ruffolo and Waddell, 1982). When a methyl group 24

is placed on the a-carbon atom of NE, a compound with two asymmetric centers and thus four possible stereochemical configurations, is produced. When these stereoisomers were examined for a^-adrenergic receptor in rat vas deferens, it was determined that the lR,2S-(-)- erythro configuration (where the 1- and 2-positions correspond to the B- and a-carbon atoms, respectively) was preferred by the a^-adrenergic receptor (Patil et al., 1967a,b; Patil and Jacobowitz, 1968). Methyl substitution at the a-carbon atom also produces a substantial increase in activity at a^-adrenergic receptors and therefore these compounds are generally selective for these receptors (Ruffolo and Waddell, 1982; Ruffolo, 1988).

f. Substitution at the nitrogen atom. It has been proposed that the basic nitrogen atom of the phenethylamines is of importance in the binding of these agents to the a^-adrenergic receptor because of a possible ionic and/or hydrogen bonding between the agonist and receptor (DeMarinis et al., 1987). This proposal is supported by the findings that isosteric replacement of the nitrogen atom with carbon and oxygen in NE and epinephrine, respectively, results in the loss of a^-adrenergic activity (Ruffolo, 1983a). Creating a quaternary ammonium analog of NE also markedly reduces a^-adrenergic activity (Ruffolo, 1983a). N-Methyl substitution of phenethylamines generally causes slight increases in the activity at a^-adrenergic receptors but as increasingly larger alkyl substituents are added to the nitrogen atom, a^-adrenergic activity decreases (DeMarinis et al., 1987; Ruffolo, 1988).

g. Effect of connective bridge length and attachment position of imidazoline ring on a^-adrenergic activity of imidazolines. Varying the bridge length between the phenyl group and the imidazoline ring attached at the 2-position produces dramatic effects on 25

a^-adrenergic activity (Ruffolo et al., 1980; Hamada et al., 1985). When the bridge is removed, the ability of the resulting compound to stimulate a^-adrenergic receptor mediated contraction in rat thoracic aorta is completely lost (Ruffolo et al., 1980). The a^-adrenergic activity of imidazolines is also decreased significantly if the chain length is increased to two carbon atoms (ethylene bridge) when responses were measured in rat thoracic aorta (Ruffolo et al., 1980) and yohimbine treated pithed rats (Hamada et al., 1985). Therefore, it is apparent that the bridge chain length of imidazolines is critical for adrenergic activity. From the studies conducted to date, imidazolines which contain an imidazoline ring attached at the 2-position to the remaining portion of the molecule possess the optimal amount of a^-adrenergic activity. N- Substituted imidazolines (imidazolines which are linked to the bridge atom via a ring nitrogen atom) possess some a^-adrenergic activity but the EDgg for the pressor response in pithed rats is 10-fold greater than that for the corresponding 2-substituted imidazolines (Hamada et al., 1985). Attachment of the imidazoline ring at the 4-position to the remainder of the molecule introduces a chiral center into the compound and also causes a reduction in a^-adrenergic activity (Lamba-Kanwal et al., 1988). When comparing the concentrations for contraction of rat thoracic aorta, the 4-substituted imidazolines were over 10-fold less active than the corresponding 2-substituted imidazolines. In addition, no stereochemical differences in the c^-adrenergic activity of these 4-substituted imidazolines was seen (Lamba-Kanwal et al., 1988).

h. Miscellaneous structure-activity relationships of imidazolines. A series of imidazolines with aromatic methoxy-substitutions has also been evaluated for a^-adrenergic receptor activity in rat aorta (Ruffolo et al., 1979a). The greatest c^-adrenergic activity for this series of compounds was demonstrated by the 2,5- and 3,5-dimethoxy- 26

substituted analogs- Surprisingly, the 3,4-dimethoxy analog was only a poor «^-adrenergic agonist unlike its corresponding 3,4-dihydroxy- substituted derivative which is a potent agonist for «^-adrenergic receptors. This and other differences between the agonist activity of methoxy-substituted derivatives and their corresponding hydroxyl- substituted analogs have led to the suggestion that the methoxy compounds may activate the «^-adrenergic receptor, subsequent to binding, via a different molecular interaction with the receptor (DeMarinis et al-, 1987). Removal of the phenolic hydroxyl groups from imidazolines results in a large decrease in the activity of the compound, a result due primarily to a decrease in the efficacy of the agent (Ruffolo and Waddell, 1983) as discussed previously. Substitution of the noncatechol 2-substituted imidazoline (tolazoline) at the 4'-benzyl position with an amino group does not appreciably change «^-or «^-adrenergic receptor activity of the compound (Shams et al., 1990). However, substitution at this same position with an isothiocyanato group (in an attempt to create an affinity label for «-adrenergic receptors) creates a compound with unique pharmacologic characteristics. This compound [2-(4- isothiocyanatobenzyl)imidazoline (IBI)] has a reduced ability to antagonize the aggregatory activity of epinephrine mediated by the a^- adrenergic receptor in platelets when compared to the parent compound tolazoline. In addition to this effect, it was indicated that IBI may interact with other inhibitory sites in platelets based on Schild plot analysis of the data (Shams et al., 1990). In the rat thoracic aorta «^-adrenergic receptor system, tolazoline was more potent than IBI based on EDgg values and slope (Venkataraman, et al., 1989). However, IBI produced the same maximal contraction as tolazoline. The interesting aspect about the contraction stimulated by IBI is that this effect is not antagonized by either «^- or «^-adrenergic receptor antagonists, but is blocked by the Ca*^ channel blockers verapamil and nifedipine. 27

Therefore, these contractile effects of IBI are mediated by a nonadrenergic, Ca*^-dependent mechanism (Venkataraman et al., 1990). These results become of particular interest with the recent descriptions of 1) imidazoline preferring receptors found in various tissues including brain and kidney (Bricca et al., 1989; Lehmann et al., 1989) and 2) a non-a-adrenergic NE binding site in rat aorta (Oriowo and Sevan, 1990). Thus IBI may serve as a lead photoaffinity probe for the imidazoline preferring receptor or the as of yet fully characterized binding site in vascular tissues. For 2-substituted imidazolines, substitution of a methyl or benzyl group at the 4-position of the imidazoline ring causes a reduction in a^-adrenergic activity (Miller et al., 1983; Ruffolo, 1988). Substitution of a methyl or benzyl group at the 4-position of the imidazoline ring of noncatechol imidazolines such as naphazoline (an a^- adrenergic agonist) decreases the intrinsic activity of these compounds to the point where they now become competitive antagonists (DeMarinis et al., 1987). Although the placement of a substituent at the 4-position of the imidazoline ring introduces a chiral center into these molecules, no stereoselectivity was observed for the a^-adrenergic effects of either the noncatechol- or catecholimidazoline analogs (Miller et al., 1983; DeMarinis et al., 1987).

D. a^—Adrenergic Mediated DMA Synthesis and Liver Regeneration. 1. Liver regeneration. One of the best examples of controlled tissue growth is that of liver regeneration. This phenomenon is characterized by coordinated waves of DNA synthesis that halt upon the restoration of the original liver mass (Grisham, 1962; Rabes et al., 1976). Only recently have some of the factors which modulate liver regeneration and the manner in which they regulate this growth response been elucidated. Much of this new information has been provided through the study of growth regulation of 28

primary cultured hepatocytes as well as the study of alterations in liver gene expression following partial hepatectomy (Michalopoulos, 1990). Any acute insult, whether surgical or chemical, which removes or destroys a large percentage of the hepatic parenchymal cells will stimulate liver regeneration. While chemicals such as carbon tetrachloride can be used to destroy portions of the liver and thus cause liver regeneration, the preferred means of inducing this response is two-thirds partial hepatectomy (Michalopoulos, 1990). This surgical method involves the externalisation of two-thirds of the liver through an abdominal wall incision and the resection of this portion of the liver (Higgins and Anderson, 1931). For most vertebrate animals including humans, the liver regenerates in 6 to 8 days, obtaining the original weight of the liver before partial hepatectomy (Michalopoulos, 1990). Most studies of liver regeneration have focused primarily upon parenchymal hepatocytes (which comprise 80% to 90% of the liver mass), although other liver cells such as endothelial cells, Kupffer cells, lipid storage cells and bile ductule cells also participate in this phenomenon (Grisham, 1962? Michalopoulos, 1990). The kinetics of liver regeneration have been described (Grisham, 1962; Rabes et al., 1976) and will be discussed briefly below using the concept of the cell cycle of rat hepatocytes as a framework for this description (Leffert et al., 1988). The Gg phase, or quiescent state, of hepatocytes is not a condition of zero cell proliferation but instead is a state where from 0.005% to 0.05% of differentiated hepatocytes (as measured by [^H]- thymidine labelling indices) enter into the cell cycle to begin the process of cell division. This slow rate of entry into the regenerative cycle is balanced by an equivalently slow rate of hepatocyte aging and death (Leffert et al., 1988). 29

Upon exposure to a hepatoproliferogenic stimulus, such as a partial hepatectomy, the hepatocytes exit the phase and enter the or prereplicative phase, which lasts from 12 to 16 hr. When this phase is completed, the cells enter the S phase where they synthesize nuclear DNA. The rates of entry into the S phase often vary depending upon the age of the animal and the type and extent of the hepatoproliferogenic stimulus (Bucher and Malt, 1971; Leffert et al., 1988). However, regardless of when the hepatocyte enters the S-phase, the length of time this cell resides in this phase replicating its nuclear DNA is approximately 8 hr (Fabrikant, 1968; Rabes, 1978). After completion of the S-phase, the hepatocytes require 4 to 6 hr preparing for cell division in the phase. When these preparations are complete, the cell enters the M phase where within 30 to 60 min the cell divides to form two hepatocytes. Most hepatocytes will regenerate at least once within 24 to 35 hr of a growth stimulus (Leffert et al., 1988). DNA synthesis in nonparenchymal cells generally starts approximately 24 hr after that for parenchymal hepatocytes and thus proliferate later in time (Grisham, 1962). If there is a continuous proliferation of the hepatocytes, the progeny cells will enter into a G^ phase (as opposed to the Gg and subsequent phase) which is a stage between the M and S phases that lasts for only 6 to 8 hr and is much shorter in duration than the phase (Leffert et al., 1988). Therefore, when DNA synthesis is examined over this entire time frame, one sees the onset of DNA synthesis within 12 to 16 hr after the exposure to a hepatoproliferogenic stimulus. A peak in DNA synthesis is then first reached after 22 to 24 hr and a smaller peak is seen after 48 hr that corresponds to DNA synthesis in the centrilobular portion (inner third) of the hepatic lobules (Grisham, 1962; Michalopoulos, 1990). This regeneration sequence proceeds by the sequential division of other mature hepatocytes and does not involve stem cells as is the case with bone marrow cells (Michalopoulos, 1990). If [^H]-thymidine is 30

continuously administered during the course of liver regeneration, more than 95% of the hepatocytes will be labelled (Michalopoulos, 1990). Although stem cells are not normally involved in liver regeneration, they can appear when DNA synthesis is completely blocked in hepatocytes or when hepatocytes are destroyed in certain disease states such as fulminant hepatitis. In these cases, the stem cells can become functional and can produce hepatocytes (Gerber et al., 1983) or become involved in the formation of hepatocellular carcinomas (Sell and Dunsford, 1989; Michalopoulos, 1990). Although a number of characteristics of the regenerative liver response have been well defined, the question of what actually triggers this phenomenon still remains to be answered. Two hypotheses have been proposed to explain the initiation of liver regeneration (Michalopoulos, 1990). These include the concepts that 1) the mitogenic signals for hepatocellular proliferation come from outside the liver cells and 2) the signals responsible for initiating proliferation of the hepatocytes come from the hepatocytes themselves. The latter hypothesis states that metabolic changes that occur in the remaining hepatocytes due to the large decrease in liver mass following partial hepatectomy forces the cells to leave the quiescent state and enter whereupon the cells produce their own autocrine growth factors and eventually begin to synthesize DNA and ultimately regenerate. The first hypothesis states that the mitogenic stimuli originate from extrahepatic sites following partial hepatectomy and deliver a complete growth promoting signal to the hepatocytes. These two hypotheses are not mutually exclusive, and it may well be that the mitogenic signal to the hepatocytes is a combination of both processes.

2. Eepatocyte growth factors and growth factor receptors. Since the onset of liver regeneration studies, researchers have noted that blood-borne factors appear in animals which are responding to 31

a hepatoproliferogenic stimulus. The existence of these factors was proven by a variety of experimental methods including the measurement of the concomitant stimulation of DNA synthesis in both grafted and whole liver tissue (Grisham et al., 1964) and the stimulation of DNA synthesis in transplanted isolated hepatocytes (Jirtle and Michalopoulos, 1982; Michalopoulos, 1990). With the use of hepatocyte cultures in serum-free media, many of these factors involved in the regulation of this growth response have been defined. When cultured hepatocytes are kept in media that is either chemically defined or supplemented with fetal bovine serum, the cells do not enter into DNA synthesis (Michalopoulos, 1990). Insulin is a trophic hormone required for the maintenance of cultured hepatocytes which will degenerate in 24 to 48 hr in the absence of this peptide. However, insulin alone will not stimulate DNA synthesis in chemically defined media unless 1) the hepatocytes are plated at a low density (Michalopoulos et al., 1982) and 2) proline is present in the media (Houck and Michalopoulos, 1985; Michalopoulos, 1990). By using the primary cultured rat hepatocyte model, a number of factors have been identified which modulate DNA synthesis, and the effects of these factors have also been confirmed in vivo (Michalopoulos, 1990). Some of these factors will be briefly described below in order to provide some additional information concerning the complex control of hepatocellular regeneration. Michalopoulos (1990) has characterized these factors as 1) complete mitogens, substances which are capable of stimulating DNA synthesis by themselves in defined media; 2) growth inhibitors (e.g. transforming growth factor B, interleukin IB), substances which can inhibit epidermal growth factor-induced mitogenesis in vitro; and 3) co- mitogenic growth factors (NE, vasopressin, angiotensin II and III, estrogens, insulin and glucagon), substances which can influence cultured hepatocyte growth in a positive but indirect manner. 32

Examples of complete mitogens include epidermal growth factor (EGF)/ transforming growth factor a (TGFa), hepatopoietin A (or hepatocyte growth factor), heparin binding growth factor-1, hepatopoietin B and hepatic stimulatory substance. Many of these factors have only recently been identified and described (Michalopoulos, 1990). The role of EGF and its receptor in the regulation of DNA synthesis and liver regeneration will be briefly discussed because this growth factor is important for the expression of -adrenergic receptor mediated DNA synthesis and liver regeneration. For more complete reviews of EGF and the EGF receptor see Defize and deLaat (1986), Gill et al. (1987), and Cohen (1987).

a. Epidermal growth factor. EGF was the first peptide hormone to be shown to stimulate hepatic DNA synthesis in culture (Richman et al., 1975) and is perhaps the best characterized hepatic growth factor. This polypeptide consists of 53 amino acid residues and is synthesized in a number of tissues including the submaxillary gland, salivary glands and Brunner's glands of the duodenum (Defize and de Laat, 1985; Gill et al., 1987). The exact role of EGF in liver regeneration is not well understood, but it is the only hepatic mitogen that has been shown to cause hepatic DNA synthesis when injected into an animal (Bucher, 1982). In primary cultured rat hepatocytes exposed to EGF, synthesis of DNA starts at 24 hr and peaks at 48 to 72 hr (McGowan et al., 1981; Michalopoulos, 1990). This is in contrast to the time course of DNA synthesis described above following partial hepatectomy where DNA synthesis begins in 12 to 16 hr and peaks after 24 hr.

b. Epidermal growth factor receptor. EGF is specific for a 170 kDa transmembrane glycoprotein which has intrinsic protein kinase activity (Gill et al., 1987; Cohen, 33

1987). This receptor is capable of autophosphorylation upon stimulation, which is perhaps a means of regulating itself. In addition, this EGF receptor is a substrate for protein kinase C, thus providing another mechanism for interaction with other signal transduction pathways (Gill et al., 1987). EGF is capable of stimulating phopshatidyl inositol turnover and Ca*^ mobilization in the human epidermoid carcinoma cell line (Sawyer and Cohen, 1981; Pandiella et al., 1989) as well as stimulating the phosphorylation of endogenous and exogenously administered proteins (Cohen, 1987). Investigations into the identification of endogenous phosphoproteins that are formed by the tyrosine kinase activity of the EGF receptor and that are involved in the regulation of cell growth are just now underway. Phospholipase C-gamma (Wahl et al., 1989; Margolis et al., 1989) and calpactin and lipomodulin protein (Brugge, 1986) have been identified as substrates for the EGF receptor. Recently, a serine/threonine kinase (pp42) has also been identified as an EGF receptor substrate which when activated leads to the phosphorylation of a ribosomal protein (Rossomando et al., 1990). Autophosphorylation of three tyrosine residues located in the cytoplasmic C-terminal tail is also an important means of EGF receptor regulation and is necessary for both optimal receptor signalling and growth promotion (Pandiella et al., 1989). Receptors for EGF decrease in number and in affinity for EGF in cultured hepatocytes, a finding which is similar to the decline in EGF receptors observed in rats following a two-thirds partial hepatectomy (Francavill et al., 1986; Michalopoulos, 1990). The tyrosine kinase activity of this receptor also decreases during this time period (Rubin et al., 1982). EGF itself may be responsible for these effects and/or this may be the result of heterologous regulation by the a^-adrenergic receptor or the release of TGFa, a protein which also acts at the EGF receptor (Michalopoulos, 1990). Heterologous down-regu1ation (the down- regulation of one receptor type by another agent acting at a different 34

receptor type) of the EGF receptor by NE acting at the a^-adrenergic

receptor has been demonstrated in vitro (Cruise et al., 1985) and in

vivo (Cruise et al., 1987) where prazosin partially inhibited the

decrease in EGF receptors seen 8 hr after partial hepatectomy was

performed on the animals.

3. a^-Adrenergic receptor mediated DNA synthesis and liver regeneration.

Early work which led to the suggestion of a possible role of the

nervous system in liver regeneration included findings that a surgical

lesion of the spinal cord above the level innervating the liver causes a

suppression of DNA synthesis in regenerating liver (Maros et al., 1971;

Vaptzanova et al., 1973). Additional studies in vivo with selected

adrenergic receptor antagonists provided further evidence that

adrenergic mechanisms may be involved in liver regeneration (Morley and

Royse, 1981). Phenoxybenzamine and phentolamine were shown to alter the

level of DNA synthesis (Thrower and Ord, 1974; McManus et al., 1973) in regenerating liver, but tolazoline had no effect on DNA synthesis

(Ashrif et al., 1974). In addition, in animals treated with reserpine, which depletes catecholamine stores; and , which inhibits the release of neurotransmitters; [^H]-thymidine incorporation into the

DNA of regenerating liver was reduced (Ashrif et al., 1974; Morley and

Royse, 1981). In a more recent study in vivo, it was demonstrated that blockade of the c^-adrenergic receptor by prazosin eliminates the peak in DNA synthesis seen at 24 hr in regenerating liver (Cruise et al.,

1987). These results indicate a definite role of the a^-adrenergic receptor in the phenomenon of liver regeneration.

The first evidence for the involvement of the oc^-adrenergic receptor in the modulation of DNA synthesis came from in vitro studies where Cruise et al. (1985) demonstrated that NE when added to primary cultures of rat hepatocytes, stimulated the concentration-dependent incorporation of [^H]-thymidine into the DNA of these cells. This 35

effect was selectively blocked by the «^-adrenergic receptor antagonist prazosin but not by a^- or J3-adrenergic receptor antagonists, thus implicating the «^-adrenergic receptor as a modulator of growth in these cells. Subsequent studies (Cruise and Michalopoulos, 1985) by this group showed that this stimulation of DNA synthesis is maximal if the cells are exposed to NE for more than 12 hr, beginning 2 to 4 hr after the cells are first plated. If the hepatocytes are cultured for 24 hr and then exposed to NE, the cells will not respond with an increase in DNA synthesis. This group also demonstrated that NE and EGF act synergistically to increase DNA synthesis. For example, if the hepatocytes are first incubated with NE for 24 hr and then exposed to EGF for another 24 hr, the degree of DNA synthesis is greatly increased over those cells which had not been pretreated with NE before addition of EGF (Cruise and Michalopoulos, 1985). Additional work demonstrated that ^^^I-EGF binding to primary cultured rat hepatocytes was inhibited in a concentration dependent manner by NE (Cruise et al., 1986). This inhibition was the result of a decrease in the number of EGF receptors (approximate 40% decrease) rather than a change in receptor affinity. After 1 hr of incubation with NE the effect is maximal. Prazosin blocked the inhibition of binding, thus demonstrating that the c^- adrenergic receptor is involved in the heterologous regulation of the EGF receptor. This inhibition of EGF binding correlates with the ability of NE to stimulate DNA synthesis. A number of agents such as vasopressin, platelet derived growth factor and bombesin can stimulate phosphoinositide metabolism and reduce EGF binding (Rozengurt et al., 1981; Brown et al., 1984). NE can also stimulate phosphatidylinositol turnover and protein kinase C activation suggesting that phosphorylation, perhaps even that of the EGF receptor itself, may mediate the effects of these compounds (Brown et al., 1984; McCaffrey et al., 1984; Beguinot et al., 1985; Cruise et al., 1986). Several models have been proposed to explain the paradoxical 36

relationship between decreased EGF binding and increased EGF mediated

DNA synthesis that has been shown both in vitro (Cruise et al., 1986)

and in vivo during liver regeneration (Earp and O'Keefe, 1981) and

hepatocarcinogenesis (Harris et al., 1987). These include: 1) the

reduction of the number of EGF receptors increases the amount of

available EGF (since degradation via internalization with the EGF

receptor is decreased) to stimulate DNA synthesis (Magun et al., 1980);

and 2) different subtypes of EGF receptor may exist and down regulation

of one may be seen as a decrease in overall EGF binding but binding to

the other subtype still exists and produces the mitogenic signal

(Gregoriou and Rees, 1984; Rees et al., 1984). Much work needs to be

done in order to determine this relationship, but it does appear that

stimulation of the a^-adrenergic receptor down regulates EGF binding which in turn is correlated to increased DNA synthesis (Cruise et al.,

1986).

The a^-adrenergic receptor also modulates the inhibitory activity of TGFB in primary rat hepatocyte cultures (Houck et al., 1988). TGFJ3

is produced by a number of tissues, including the liver, and plays an

important role in the regulation of cell growth and differentiation of many cell types (for reviews see Assoian et al., 1985; Fausto and Meade,

1989). TGFB is a very potent polypeptide that inhibits EGF-stimulated

DNA synthesis (IC^^ = 2.8 pM) in primary cultured rat hepatocytes (Houck et al., 1988). NE caused a 5-fold increase in the IC^^ for TGFB to 14.4

pM. This modulatory effect of NE was concentration dependent,

significant at concentrations of 1 pM NE and greater, and blocked by

prazosin. These results suggest that NE may trigger the initial

proliferative response in regenerating liver by both increasing the

responsiveness of the hepatocytes to EGF as well as releasing the cells

from the negative control of TGFB (Houck et al., 1988).

In addition to these in vitro studies, the involvement of the

adrenergic receptor in liver regeneration in vivo has also been examined 37

(Cruise et al., 1987,1988,1989; Houck et al., 1989; Tsai et al., 1989). Investigations by Cruise et al. (1987,1988) in rats showed that plasma levels of NE and epinephrine increased in animals in which a partial hepatectomy had been performed in comparison to sham-operated animals. This increase was seen within 2 hr of the surgical procedure. [^H]- Thymidine incorporation into the DNA of the regenerating liver during the first wave of DNA synthesis following partial hepatectomy was reduced by surgical sympathectomy (severing of the nerve fibers along the hepatic artery) or injection of prazosin. Liver catecholamine levels were reduced by chronic treatment with guanethidine, but the ability of the liver to regenerate was not altered, thus demonstrating a lack of correlation between tissue catecholamine levels and the ability to regenerate. An alteration in the binding of EGF to its receptor on regenerating liver was caused by prazosin prior to the inhibition of DNA synthesis. Within 4 hr of partial hepatectomy, animals administered prazosin at the time of the operation showed an increase in ^^^I-EGF binding to the membranes prepared from the regenerating liver. This was the result of an increase in the number of EGF receptors in membranes prepared from regenerating liver relative to the number seen in the membranes obtained from control cells. Other hepatic proliferative responses in vivo have been shown to be modulated by the a^-adrenergic receptor. Cruise et al. (1989) demonstrated that in hepatocytes isolated from partially hepatectomized rats, the number of c^-adrenergic receptors (as measured by [^H]- prazosin binding) did not change for up to 24 hr after the time of the surgery. However, [^H]-prazosin binding decreased dramatically at the latter time points of 48 and 72 hr, indicating a down-regulation of the a^-adrenergic receptor in these regenerating livers. Additionally, it was shown that even though the number of a^-adrenergic receptors were maintained at early time points in the regenerating liver, the ability of NE to stimulate inositol phosphate production in isolated cells 38

preincubated with [^H]-myo-inositol was transiently decreased between

the 8 and 16 hr time periods after partial hepatectomy. After 24 hr,

the ability of NE to stimulate phosphoinos it ide metabolism was restored.

This uncoupling of phosphoinositide turnover from the occupation of the

a^-adrenergic receptor by NE was preceded by a decrease in hepatic

membrane ras p21 content. This is of interest since the a^-adrenergic

receptor is coupled to PLC through an uncharacterized G protein

(Goodhardt et al., 1982) and p21 (the product of the ras proto-oncogene)

is a membrane-associated G protein of unknown function that appears to

be involved in the regulation of cell growth (Cruise et al., 1989). It

has been shown that the initiation of hepatocyte DNA synthesis and the

primary wave of mitosis are associated with alterations in the

expression of ras proto-oncogenes (Fausto and Mead, 1989). Membrane

levels of p21 were significantly decreased by 2 hr after partial

hepatectomy, thus preceding the uncoupling of the a^-adrenergic receptor

from phosphoinositide turnover. However, the relationship (other than

the temporal association) between these two events has yet to be

determined but it does seem likely that both the a^-adrenergic receptor

and the ras proto-oncogene product (p21) are involved in the early

events of liver regeneration. Also unknown is the role that the

uncoupling of phosphoinositide turnover from the a^-adrenergic receptor

during the 8 to 16 hr time period after partial hepatectomy plays in the

regenerative response. If the growth mediating effects of the a^-

adrenergic receptor are transduced through this pathway, then the

stimulatory signal may have already been sent by this time. On the

other hand, the uncoupled a^-adrenergic receptor may be of importance

since it has been shown that activation of this receptor by NE 12 hr

after partial hepatectomy potently blocks the inhibitory effect of TGFB

(Houck and Michalopoulos, 1989).

Taken collectively, these findings suggest that DNA synthesis that occurs during liver regeneration is initiated through an enhanced 39

response to EGF in conjunction with a decreased sensitivity to TGFB by

stimulation of the a^-adrenergic receptor (Houck and Michalopoulos,

1989). Additionally, a number of other physiological processes that are

linked to hepatic oc^-adrenergic receptor activation have been demonstrated to occur very soon after partial hepatectomy has been performed. These events include glycogenolysis (Paloheimo et al.,

1984), the formation of DAG (Bocckino and Exton, 1989) and the hyperpolarization of the plasma membrane of hepatocytes (deHeptinne et al., 1985). Furthermore, since the a^-adrenergic receptor can be activated almost immediately by NE released from adrenergic neurons known to innervate the liver.(Burt et al., 1989), this receptor represents a good site for the generation of the signal that initiates

DNA synthesis and liver regeneration (Michalopoulos, 1990).

Investigations into some of the possible intracellular signal transduction mechanisms which may mediate the stimulation of DNA synthesis produced by the occupation of the hepatic ot^-adrenergic receptor by catecholamines have been pursued. Takai et al. (1988) demonstrated that epinephrine concentrâtion-dependently stimulated DNA synthesis in primary cultured rat hepatocytes. The Ca*^ ionophore A-

23187 and 12-o-tetradecanoylphorbol-13-acetate (TPA) were used to activate Ca*^ mobilization and protein kinase C, respectively, since both of these cellular events are known to be mediated by a^-adrenergic receptor activation (Fain and Garcia-Sainz, 1980; Nishizuka, 1984).

Neither of these agents induced DNA synthesis by themselves or in combination in primary cultured rat hepatocytes, although both compounds stimulated DNA synthesis in Swiss 3T3 cells. TPA caused the down regulation of epinephrine induced DNA synthesis within 15 min of exposure, suggesting that TPA may inactivate phosphoinositide metabolism ^

(Lynch et al., 1985) and thus inhibit a^-adrenergic receptor mediated

DNA synthesis. Therefore, if phosphoinositide turnover is involved in this mechanism, protein kinase C may play a regulatory role. The lack 40

of the ability of A-23187 to stimulate DNA synthesis could suggest that this compound did not mobilize the same compartments of the Ca*^ pool that are mobilized by stimulation of the «^-adrenergic receptor (Takai et al♦, 1988). Additional research is necessary in order to determine the exact cellular mechanisms that occur during these early phases of DNA synthesis and liver regeneration. A role of the «^-adrenergic receptor in augmentative hyperplasia activated by the xenobiotic hepatic tumor promoters phénobarbital and «- hexachlorocyclohexane and the peroxisome proliférâtor ciprofibrate was examined by Tsai et al. (1989). It was demonstrated that administration of prazosin to rats significantly inhibited DNA synthesis (but not liver weight gain) caused by phénobarbital and «-hexachlorocyclohexane but not ciprofibrate. This lack of involvement of the «^-adrenergic receptor in the mediation of DNA synthesis by ciprofibrate may be due to the different phenotype of the tumors promoted by peroxisome proliferators (Rao et al., 1988). The results obtained in phénobarbital and «- , hexachlorocyclohexane treated animals suggest that the «^-adrenergic receptor is involved in generating the mitogenic signal that leads to the stimulation of DNA synthesis by these xenobiotics and that the a^- adrenergic receptor may have a facilatory role in the promotion of hepatocyte proliferation and possible tumor formation by these and other xenobiotics. The involvement of the «^-adrenergic receptor in mediating DNA synthesis and liver regeneration fits into the hypothesis described previously where NE would serve as an extrahepatic mitogenic stimulus promoting its effects through activation of the «^-adrenergic receptor. To summarize, the evidence which supports the role of the hepatic «^~ adrenergic receptor in proliferative growth responses include the following findings (Michalopoulos, 1990); 1) Other «^-adrenergic receptor mediated responses such as glycogenolysis, DAG formation and hepatocyte membrane hyperpolarization occur immediately after partial 41

hepatectomy. 2) Blockade of the a^-adrenergic receptor inhibits the 24

hr peak in DNA synthesis. 3) Stimulation of the a^-adrenergic receptor

enhances the responsiveness of hepatocytes to EGF and decreases the

number of EGF receptors both in vitro and in vivo. 4) In regenerating

hepatocytes, a^-adrenergic receptor stimulation reduces the inhibitory

effect of TGFB. 5) Concentrations of NE available to act at the hepatic

a^-adrenergic receptor are increased in the plasma following partial

hepatectomy. These results show that the a^-adrenergic receptor plays

an integral role in the regulation of growth responses of hepatocytes,

and continued investigation of the properties of both the a^-adrenergic

receptor itself and the cellular events that occur following activation

of the receptor should provide new information about the regulation of

cell growth by the a^-adrenergic receptor.

E. Statement of the Problem.

1. Objectives and Rationale.

As previously described, the process of DNA synthesis and liver

regeneration is modulated by catecholamines through an interaction with

the hepatic a^-adrenergic receptor. Research in this area has primarily

focused upon the role of the hepatic a^-adrenergic receptor in 1) the

modulation of growth factor binding and activity and the subsequent

expression of growth related genes and DNA synthesis (Cruise and

Michalopoulos, 1985; Cruise et al., 1985,1986,1987,1988,1989; Houck et

al., 1988) and 2) the mediation of cellular mechanisms involved in this

growth response (Takai et al., 1988; Michalopoulos, 1990). Although

some of these studies have been performed in whole animals (Cruise et

al., 1987,1988,1989), the majority of the information concerning the

role of the hepatic a^-adrenergic receptor in growth responses has been

gained through the use of primary cultured rat hepatocytes (Cruise and

Michalopoulos, 1985; Cruise et al., 1985,1986,1988; Takai et al., 1988).

This system allows for the direct investigation of the effects of 42

various agents on o^-adrenergic receptor mediated regenerative DNA synthesis under defined conditions thus avoiding the complex responses caused by homeostatic control mechanisms in the in vivo situation. This culture system also offers the advantage of allowing for the pharmacologic evaluation of a large number of compounds at various concentrations on hepatic o^-adrenergic receptor mediated DNA synthesis, an investigation that if conducted in animals, would require much time and expense. A number of important findings have been made concerning the ability of NE to regulate DNA synthesis and cell growth through the activation of the hepatic c^-adrenergic receptor. These include the discovery that this receptor modulates the binding and activity of growth modulating peptides such as EGF and TGF-B and the expression of growth associated gene products (Cruise et al., 1986,1989; Houck et al., 1989). However, no studies have been performed to date which have examined the ability of other known a-adrenergic agonists besides R-NE and R-epinephrine to enhance DNA synthesis by activation of the a^- adrenergic receptor. Therefore, further pharmacological characterization of this growth regulating receptor by the utilization of known a-adrenergic agonists and antagonists is warranted in order that insight into the manner in which the a^-adrenergic receptor recognizes and interacts with compounds and eventually conveys this proliferogenic stimulus to the cell may be gained. In Chapter II, the ability of a series of phenethylamines and imidazolines to mediate DNA synthesis, as measured by [^H]-thymidine incorporation, via activation of the a^-adrenergic receptor in primary cultured rat hepatocytes was investigated. The reasons for conducting these experiments were two-fold: 1) to determine the effect of structural modification of these compounds on hepatic a^-adrenergic receptor mediated DNA synthesis and 2) to determine the influence of the stereochemical orientation of these agents on this response. No studies 43

have been performed to date which have examined the structural features

of these compounds that are required for the stimulation of DNA

synthesis mediated by the hepatic a^-adrenergic receptor. Additionally,

no research has been conducted that has examined the stereochemical

dependency of this hepatocyte growth phenomenon. An important property

possessed by adrenergic receptors is that of stereoselectivity; thus, it

is necessary to determine the manner in which the stereoisomers of

various adrenergic agents act at the receptor site in order to delineate

the selectivity of the receptor function (Patil et al., 1975).

Several investigations have been conducted which have determined the

stereoselectivity of the hepatic a^-adrenergic receptor for the isomers of NE and epinephrine using radioligand binding assays (Guellaen et al.,

1978; El-Refai et al., 1979; Aggerbeck et al., 1980; Schwarz et al.,

1984; Studer and Ganas, 1988) and functional studies (El-Refai et al.,

1979; Aggerbeck et al., 1980). Additionally, the stereoselective stimulation of a^-adrenergic receptor mediated growth has been evaluated to some extent in cultured heart cells using the R- and S-isomers of both NE and epinephrine. (Simpson, 1985). However, no studies have been performed that have evaluated the stereochemical requirements for and the applicability of the Easson-Stedman hypothesis (1933) to DNA synthesis stimulated by activation of the hepatic a^-adrenergic receptor by stereoisomers of a series of closely related phenethylamines and catecholimidazolines. Therefore, it was of great interest to determine both the structural and stereochemical requirements of agents which may be able to stimulate hepatic a^-adrenergic receptor mediated DNA

synthesis.

To further characterize the hepatic a^-adrenergic receptor mediated DNA and protein syntheses in primary cultured rat hepatocytes, a series of selected a-adrenergic receptor agonists and antagonists were evaluated for activity in this system (see Chapter III). Although it was demonstrated in Chapter II that the ability of phenethylamines and 4 4 catecholimidazolines to stimulate DNA synthesis was by activation of the a^-adrenergic receptor, it was still unknown whether imidazolines which do not possess a catechol moiety can stimulate DNA synthesis by the same receptor, and if not, can they block the response of R-NE and DHT.

Therefore, it was of interest to determine whether noncatechol imidazoline compounds can be recognized by the hepatic a^-adrenergic receptor and affect DNA synthesis either positively or negatively.

Tolazoline and tolazoline-derivatives which contain an amino group [2-

(4-aminobenzyl)imidazoline (ABI)] and an isothiocyanato group [2-(4- isothiocyanatobenzyl)imidazoline (IBI)] at the 4'-benzyl position are examples of imidazolines which do not contain a catechol functionality but still possess agonist activity in some a-adrenergic systems.

Since ABI demonstrates a^-adrenergic receptor agonist activity in rat thoracic aorta (Shams et al., 1990) and IBI has unique contractile activity in rat thoracic aorta that appears to be independent of the activation of classical a-adrenergic receptors (Shams et al., 1990;

Venkataraman et al., 1989), both compounds were evaluated in order to determine the effects of 4'-benzyl substitutions of imidazolines on DNA synthesis in primary cultured rat hepatocytes.

In order to further elucidate the effects of noncatechol imidazolines on DNA synthesis in primary cultured rat hepatocytes, the ability of the partial a-adrenergic receptor agonist tolazoline and the full a^-adrenergic receptor agonist cirazoline to stimulate DNA were compared. Additionally, if these compounds could not stimulate DNA synthesis in this system, it was of interest to determine whether these compounds could inhibit DNA synthesis stimulated by R-NE and DHT. The rationale for this is if tolazoline and/or cirazoline inhibited DNA synthesis caused by R-NE and DHT, then the receptor at which these two compounds were interacting would be most likely the a^-adrenergic receptor. However, if tolazoline and/or cirazoline did not inhibit DNA synthesis stimulated by R-NE but did inhibit that caused by DHT, this 45

could indicate that DHT may be eliciting part of its stimulatory effect through a non-adrenergic mechanism, perhaps through a site similar to that described for the imidazoline preferring receptor (Lehmann et al., 1989; Michel and Insel, 1989). The effect of idazoxan, an imidazoline preferring receptor ligand and c^-adrenergic receptor antagonist, on DNA synthesis elicited by R-NE and DHT was evaluated for the same reason previously discussed for tolazoline and cirazoline. Strong evidence has been provided by a number of laboratories (McGrath, 1982; Coates et al., 1982; Morrow and Creese, 1986; Han et al., 1987a,b; Piascik et al., 1990) that supports the existence of two subclasses of the a^-adrenergic receptor, designated and (Han et al., 1987a,b). The possible existence of two subtypes of the a^- adrenergic receptor in hepatocytes is supported by the work of Garcia- Sainz and Hernandez-Sotomayor (1985) who demonstrated that c^-adrenergic receptor mediated gluconeogenesis stimulated by epinephrine involved two pathways, one which was Ca*^-dependent, insulin-sensitive and modulated by glucocorticoids; and the other which was Ca*^-independent, insulin- insensitive and modulated by thyroid hormones. Radioligand binding assays performed in rat liver membranes demonstrate that the liver contains almost entirely the c^^-adrenergic receptor subtype (Han et al., 1987a, Han et al., 1990). Thus, based upon these findings and the availability of the selective antagonists of c^-adrenergic receptor subtypes (WB 4101, competitive c^^-subtype antagonist; SZL-49, a^^- subtype alkylating agent; and CEO, subtype alkylating agent), it was of interest to determine the subtype(s) of the a^-adrenergic receptor that modulates DNA synthesis. An increase in the cytosolic free Ca*^ level is one of many cellular events that occurs following both a^-adrenergic receptor and EGF receptor activation, and an involvement of a poorly understood Ca*^ channel has been shown to maintain these elevated Ca*^ levels over time (Exton, 1988). Calmodulin, a Ca*^ receptor protein, has been shown to 46

modulate a variety of Ca*^-dependent cellular processes. Both the elevation of intracellular levels and activation of calmodulin play a role in the regulation of cell proliferation (Whitfield et al., 1979; Alexander, 1988). Therefore, based on these findings, the effects of verapamil, a Ca.*^ channel blocker, and trifluoperazine (TFP), a calmodulin antagonist on a^-adrenergic receptor mediated DNA synthesis were evaluated. Hepatocyte cell number is restored rapidly in regenerating liver . following partial hepatectomy but the restoration of liver mass is slower and does not return to normal levels for 8 to 10 days (Gehard, 1975). This presents the question of whether cellular hypertrophy is regulated by the same factors as those involved in the regulation of DNA synthesis and cell proliferation. Although the effects of the various modifiers on total cellular protein levels in primary cultured rat hepatocytes often suggested that protein synthesis was modulated by the a^-adrenergic receptor in a similar manner as DNA synthesis, the effects on total cellular protein were inconsistent. Therefore, the effects of selected agents that affect a-adrenergic receptor mediated responses were examined for their ability to modify both DNA and protein syntheses in primary cultured rat hepatocytes, as determined by the incorporations of [^H]-thymidine and [ ]-methionine into DNA and protein, respectively.

2. Significance. Among the many physiological processes mediated by the o^- adrenergic receptor is the modulation of growth of a variety of cell types including myocardial cells (Simpson, 1985), endothelial cells (Sherline and Mascardo, 1984), vascular smooth muscle cells (Nakaki et al., 1990) and hepatocytes (Cruise et al., 1985,1987,1988). It has been suggested that a number of pathological conditions directly associated with several of these cell types is the result of abnormal a^-adrenergic 47

receptor mediated growth. Such disease states include atherosclerosis, where proliferation of smooth muscle cells is a critical event in the development of this vascular disease (Ross, 1986), and hypertrophic cardiomyopathy, a condition associated with the excessive hypertrophy of cardiac muscle cells (Simpson et al., 1985). In the liver, it is possible to propose an involvement of the hepatic a^-adrenergic receptor in the abnormal proliferation of hepatocytes that occurs during the development of hepatocellular carcinomas. Lending support to this hypothesis is the finding that blockade of the c^-adrenergic receptor with prazosin inhibits DNA synthesis stimulated by tumor promoters (Tsai et al., 1989). Since liver cancer is the eighth most common form of neoplasm in the world (Parkin et al., 1984), continued investigation of the role of the o^-adrenergic receptor in the regulation of DNA and protein syntheses in hepatocytes is warranted. In addition, the use of primary cultured rat hepatocytes, a well characterized system for the investigation of DNA synthesis and cell growth, for the continued study of a^-adrenergic receptor mediated DNA synthesis should contribute to our knowledge of the regulation of cellular processes that occur during cell growth. Therefore, it is important to pharmacologically characterize this hepatic «^-adrenergic receptor by 1) examining the structural and stereochemical aspects of phenethylamine and catecholimidazoline analogs required for stimulation of hepatic a^- adrenergic receptor mediated DNA synthesis and 2) determining the effects of selected agents that modify a-adrenergic receptor responses on DNA and protein syntheses stimulated by phenethylamines and catecholimidazolines in order to gain a better understanding of the role that the hepatic «^-adrenergic receptor plays in the stimulation of cell growth. CHAPTER II

EFFECTS OF THE STEREOCHEMICAL ORIENTATION OF A SERIES OF PHENETHYLAMINES AND IMIDAZOLINES ON a -ADRENERGIC RECEPTOR MEDIATED DNA SYNTHESIS IN PRIMARY COLTDRED RAT HEPATOCYTES

A. Specific Aims

The specific aims of this set of experiments were to evaluate the stereochemical dependent stimulation of a^-adrenergic receptor-mediated

DNA synthesis as determined by [^H]-thymidine incorporation in primary cultured rat hepatocytes by utilizing a series of isomers of 1) B- hydroxylated phenethylamines, 2) 2-substituted catecholimidazolines, 3)

4-substituted catecholimidazolines and 4) phenethylamines with a B-amino group (phenylethylenediamines).

B. Methods

1. Materials

The biochemicals and cell culture materials used and their sources were the following: antibiotic/antimycotic solution, bovine serum albumin [BSA] (fatty acid free), dopamine hydrochloride, epidermal growth factor (EGF), fetal bovine serum (heat inactivated), gentamicin sulfate, insulin, R-norepinephrine bitartrate, Percoll, prazosin hydrochloride, 1-proline, 1-pyruvic acid, 1-serine, trypan blue (Sigma

Chemical Company, St. Louis, MO); s-norepinephrine bitartrate (Sterling-

Winthrop, Rennsaeler, NY); R-2-(3,4,a-trihydroxybenzyl)imidazoline hydrochloride, S-2-(3,4,a-trihydroxybenzyl)imidazoline hydrochloride,

3,4-dihydroxybenzylimidazoline hydrochloride, R-4-(3,4-dihydroxybenzyl) imidazoline oxalate, S-4-(3,4-dihydroxybenzyl)imidazoline oxalate, R,S-

4-(3,4-dihydroxybenzyl)imidazoline oxalate, R-3,4-dihydroxyphenyl- ethylenediamine hydrochloride, S-3,4-dihydroxyphenylethylenediamine hydrochloride (synthesized in the laboratory of Dr. Duane D. Miller,

48 49

Department of Medicinal Chemistry, College of Pharmacy, The Ohio State University); collagenase type IV (Worthington Biochemical Corp., Freehold, NJ); Williams Medium E (Gibco, Grand Island, NY); and [methyl- ]-thymidine (40-60 Ci/mmol) (ICN Biochemicals, Inc., Costa Mesa, CA); Primaria tissue culture plates (10 x 35 mm). Spectra Mesh (111 and 202 pm) and Scintiverse E (Fisher Scientific, Cincinnati, OH) and Millex-GV filters (0.22 pm) (Millipore Corp., Medford, MA). Other reagents and organic solvents were of the highest purity available.

2. Animals. Female Fisher F-344 rats (120-180 gm) were obtained from Harlan Sprague-Dawley, Inc. (Indianapolis, IN). The animals were housed in a vivarium at 25-26 ®C with an alternate 12 hr light and dark cycle and with free access to Purina rat chow and water.

3. Isolation and preparation of primary cultures of adult rat hepatocytes. Hepatocytes were isolated from female Fisher F-344 rats by the recirculating collagenase perfusion method described by Seglen (1976) and modified by Cruise et al. (1985) with some minor alterations. Immediately following a 10-15 min perfusion of the liver with collagenase buffer (the perfusion was terminated when the liver was sufficiently swollen and furrowed in appearance), the cells were placed into a sterile dish with approximately 25 ml of ice cold HEPES buffer containing 0.5% bovine serum albumin (BSA). This cell suspension was then filtered twice, first through a sterile 202 pm polyethylene Spectra/Mesh and then through a sterile 111 pm Spectra/Mesh. This crude cell suspension was washed three times by centrifugation for 5 min at 50 X 2" Following the third low speed centrifugation, the cells were resuspended in HEPES buffer with 0.5% BSA, and these cells were added to Percoll (pH adjusted to 7.2-7.4), and this cell suspension was then 50

centrifuged at 38,000 x g at 4°C for 40 min. The utilization of this

Percoll gradient technique to separate live from dead cells greatly

enhanced the viability of the final cell preparation. Following

centrifugation in the Percoll gradient, the upper dead cell layer was

aspirated and the remaining viable cells were resuspended in HEPES

buffer with 0.5% BSA, and the cells centrifuged at 110 x g for 15 min.

After this centrifugation, the supernatant was discarded and the cells

resuspended in a given volume of HEPES buffer with 0.5% BSA. The trypan

blue exclusion method was used to determine cell viability (usually

greater than 85%) and cell yield (a yield of 100 million cells or more

was considered a good yield). The cell suspension was then diluted to

50,000 cells/ml with Williams Medium E supplemented with 5% fetal bovine

serum, pyruvate (1 itiM), serine (0.2 mM), proline (1 mM), insulin (0.1 piK), penicillin (100 U/ml), streptomycin (100 pg/ml), amphotericin B

(0.25 pg/ml) and gentamicin sulfate (50 pg/ml). These isolated hepatocytes were plated at a density of 100,000 cells/2 ml medium onto

Primaria 35 mm dishes and placed in a humidified 37°C, 95%/5% CO^

incubator. The cells were then allowed to settle and adhere for 2 hr.

Following this attachment period, the plating medium was aspirated and replaced with serum-free media (supplemented Williams Medium E minus

fetal bovine serum). In addition, this medium is further supplemented with EGF (10 ng/ml) and [^H]-thymidine (5 uCi/ml). Drugs (dissolved in

10 pi of distilled water) were also added at this time and the cultures

incubated for 48 hr at 37®C in 5% CO^.

4. Biochemical assays of cell lysates.

Following 48 hr of incubation, the medium was aspirated from the

cultured hepatocytes, and the cells solubilized with 0.33 N NaOH. The

resulting lysates were precipitated by adding ice-cold TCA to a final

concentration of 10%. The precipitate was centrifuged at 380 x g for 10 min and the supernatant discarded. The pellet was washed by repeating 51

the dissolution, precipitation and centrifugation steps two additional times. Following the final dissolution, an aliquot of the final lysate was neutralized with TCA and analyzed by.liquid scintillation counting. Protein concentrations of the cell lysates were determined by the method of Lowry et al (1951) utilizing BSA as the standard.

5. Data analysis. All dishes of hepatocytes receiving a given treatment of the same compound and its respective concentration were grouped together over all the experiments for the calculations of the results. In each experiment, the number of samples for control or drug concentration was 3-4 dishes. The amount of [^H]-thymidine incorporated into TCA precipitable material in both control and treated cells was calculated as DPM [ ] -thymidine/culture. These results were then routinely normalized by expressing the treatment DPM/culture (minus control DPM/culture) as a percentage of the maximal [^H]-thymidine incorporation (100% maximal response) elicited by either 30 jM or 100 }M R-NE (minus control DPM/culture). Because of the low potency of some compounds, EC^g rather than EC^g values were calculated from concentration response curves constructed from individual experiments for comparisons of the relative potencies of the compounds. The EC^g value was defined as the concentration of a treatment required to produce a response equivalent to 30% of the maximal response by either 30 ;^M or 100 pM R-NE. Protein data are expressed as the pg protein/dish. Comparisons among the means were made using one-way analysis of variance followed by a Student's unpaired t-test for direct comparisons of means or by Student-Newman- Keuls test for multiple comparison of means (Zar, 1984).

C. Results. The chemical structures and abbreviations of the compounds that were used for the determination of the stereochemical requirements for 52 the stimulation of hepatic u^-adrenergic receptor mediated DNA synthesis are shown in Figs. 2-4.

1. Evaluation of the stereochemical dependent stimulation of adrenergic receptor mediated DNA synthesis by phenethylamines in primary cultured rat hepatocytes. The agents used to evaluate the effect of the stereochemical orientation at the B-carbon atom of phenethylamines on [^HJ-thymidine incorporation modulated by the hepatic «^-adrenergic receptor in primary cultured cells were R-norepinephrine (R-NE), S-norepinephrine (S-NE) and its desoxy analog dopamine (DA) (see Fig. 2). The amount of [^H]- thymidine incorporation stimulated by 30 pM R-NE was used as the maximal response. Both the R- and S-isomers of NE produced concentration dependent increases in -thymidine incorporation in primary cultured rat hepatocytes incubated for 48 hr (see Fig. 5). R-NE produced a significant response (28% of maximal response) at a concentration of 0.3 pM whereas a much higher concentration of S-NE (10 pM) was required in order to stimulate DNA synthesis to a level (24% of maximal response) significantly greater than control activity. DA did not elicit a DNA synthetic response at concentrations up to 30 pM. Concentrations of DA greater than 30 pM caused cell detachment and cell death. The respective EC^^ values for the activation of [^H]-thymidine incorporation by R- and S-NE are 0.34 and 8.24 pM (pEC^^s = 6.54 and 5.15, respectively) (see Teible 6). Thus, based on a comparison of the EC^g values of these two isomers of NE, the relative potency of S-NE is only 4% that of R-NE (see Table 6). The EC^^ for DA is greater than 30 pM. The maximal responses for S-NE and DA were produced at 30 pM for both agents and were 53% and 15%, respectively, of that for 30 pM R-NE. ECgg values were also calculated for the R- and S-isomers of NE for the determination of the isomeric activity ratio (log of the difference 53

between the -log EC^g values for R-NE and S-NE) and comparison to EC^^ values determined for these two enantiomers in other a^-adrenergic receptor systems. The EC^^ (± S.E.M. ) for R-NE was determined to be 0.90 ± 0.52 [-log EC^g (± S.E.M.) = 6.18 ± 0.24] whereas that for S- NE was 26.8 ± 9.12 juH (-log EC^^ = 4.64 ± 0.20). The resulting isomeric activity (mean ± S.E.M.) was 34.7 ± 2.69. Prazosin (1 pM) completely inhibited [^H]-thymidine incorporation stimulated by R-NE, S-NE and DA (see Fig. 6). R-NE, S-NE and DA also increased total cellular protein in cells incubated for 48 hr with these compounds producing maximal increases of 140%, 144% and 135% of control levels, respectively (see Table 2). Prazosin (1 juM) did not significantly block this increase in total cellular protein.

2. Determination of the stereochemical dependent stimulation of a^— adrenergic receptor mediated DNA synthesis by 2-substituted catecholimidazolines in primary cultured rat hepatocytes. The compounds utilized for the determination of the effect of stereoisomerism of 2-substituted catecholimidazolines on hepatic a^- adrenergic receptor mediated [^H]-thymidine incorporation were R-2- (3,4,a-trihydroxybenzyl)imidazoline (R-TBI), S-2-(3,4,a-trihydroxy- benzyl )imidazoline (S-TBI) and 2-(3,4-dihydroxybenzyl)imidazoline (DHT) (see Fig. 3). All three compounds produced concentration-dependent increases in [^H]-thymidine incorporation into primary cultures of rat hepatocytes incubated with these agents for 48 hr (see Fig. 7). The threshold concentration for the significant stimulation of DNA synthesis for both R-TBI and DHT (18% and 26% of maximal response, respectively) was 10 pM whereas that for S-TBI (19% of maximal response) was 30 pM. EC^^ determinations for the stimulation of [^H]-thymidine incorporation were 15.0, 61.6 and 10.8 pM for R-TBI, S-TBI and DHT, respectively (see Table 54

6). The maximal responses for R-TBI, S-TBI and DHT were produced at 100 fjM and were 45%, 39% and 80%, respectively, of that for 100 pM R-NE. Prazosin (1 ptH) inhibited DNA synthesis stimulated by all three compounds (see Fig. 8). Total cellular protein was also increased significantly in cells incubated for 48 hr with these catecholimidazolines (see Table 3). When primary cultured hepatocytes were exposed to 100 pM concentrations of R- TBI, S-TBI and DHT, total cellular protein was increased to 139%, 140% and 155% of the control protein content, respectively. As was the case with the phenethylamines tested above (see Section II.C.l above), 1 pM prazosin failed to significantly inhibit this increase in protein content caused by these compounds.

3. Effect of chirality at the 4—position of the imidazoline ring of catecholimidazolines on the stimulation of DNA synthesis mediated by the a^-adrenergic receptor in primary cultured rat hepatocytes. The compounds R-4-(3,4-dihydroxybenzyl)imidazoline (R-DBI), S-4- (3,4-dihydroxybenzyl)imidazoline (S-DBI) and R,S-(3,4-dihydroxybenzyl) imidazoline (R,S-DBI), shown in Pig. 4, were used to evaluate the effect of the presence of a site of asymmetry in the imidazoline ring of a 4- substituted catecholimidazoline on hepatic [^H]-thymidine incorporation. Only the R-isomer of DBI demonstrated any appreciable concentration-dependent stimulation of DNA synthesis whereas the S- isomer was inactive in concentrations up to 30 pM (see Fig. 9). At concentrations above 30 pM, S-DBI caused cell death. Incubation of the hepatocytes with 30 pM R-DBI produced a significant increase (49% of maximal response) in [^H]-thymidine incorporation, and the EC^^ for R- DBI was determined to be 17.3 pM (see Table 6). The maximal response elicited by 100 pM R-DBI was 61% of that produced by 30 pM R-NE. The racemate (R,S-DBI) exhibited activity intermediate between the R- and S- isomer of DBI with a significant increase in DNA synthesis (29% of 55 maximal response) seen at 30 pM and a decrease in stimulatory activity at 100 ptK. Prazosin (1 pM) blocked the stimulation of DNA synthesis produced by R-TBI (see Fig. 10).

Total cellular protein was increased in cultured hepatocytes incubated for 48 hr with the isomers and racemate of DBI (see Table 4).

Total cellular protein was 151%, 128% and 142% of control levels for 100 pM R-DBI and 30 pM S-DBI and 30 pM R,S-DBI respectively. Prazosin (1 pM) did not significantly block this increase in protein content.

4. Evaluation of the effects of S-carbon atcan amino substitution in

phenethylamines on DNA synthesis mediated by the a^-adrenergic

receptor in primary cultured rat hepatocytes.

The R- and S-isomers of 3,4-dihydroxyphenylethylenediamine (DPDA) used in these experiments are shown in Fig 4. The DPDA isomers stimulated DNA synthesis in a concentration dependent manner in primary cultured rat hepatocytes incubated for 48 hr in the presence of the compounds (see Fig. 11). At a concentration of 10 iM, S-DPDA caused a significant increase in [^H]-thymidine incorporation (30% of maximal response) whereas exposure of the cells to 30 R-DPDA was required for a significant stimulation in DNA synthesis (42% of maximal response).

The EC^g values for R- and S-DPDA were determined to be 22.5 and 10.0 pJA, respectively (see Table 6). Maximal responses to both enantiomers were 48% and 70% of the 30 pM R-NE response. Exposure of the cells to 1 j M prazosin completely blocked the stimulation of DNA synthesis by 100

]M R-DPDA and also decreased the amount of DNA synthesis caused by S-

DPDA, but not to the same degree as with R-DPDA (see Fig. 10).

Total cellular protein was not increased significantly by treatment with either isomer of DPDA, although the amount of protein content was elevated somewhat by R-DPDA (see Table 5). R-NE (10 and 30

}M) significantly increased total cellular protein in cells obtained from the same isolations as those tested with the isomers of DPDA. 56

Prazosin (1 ]M) blocked the small increase in protein content caused by 100 pM R-DPDA and also inhibited the increase in total cellular protein caused by 10 pM R-NE.

D. Discussion. Information garnered from studies of liver growth responses (e.g. regeneration, maturation and carcinogenesis) has provided much of the understanding about the regulation of mammalian cell growth (Koch and Leffert, 1980; Michalopoulos, 1990). An increase in DNA synthesis (as determined by [^H]-thymidine incorporation into DNA) is often measured as an indicator of stimulation of hepatocyte growth both in vivo in regenerating liver of rats subjected to partial hepatectomy (Grisham et al., 1962; Rabes et al., 1976; Cruise et al., 1987) and in vitro in primary cultures of rat hepatocytes which simulate proliferative growth events occurring in the intact animal (Leffert et al., 1977; Koch and Leffert, 1980; McGowan et al., 1981; Michalopoulos et al., 1982; Cruise et al., 1985). The use of serum-free primary cultured rat hepatocytes provides a very useful and convenient model system for examining cell growth and its regulation as has been demonstrated by several research groups (Richman et al., 1976; Koch and Leffert, 1980; McGowan et al., 1981; Michalopoulos et al., 1982 and Cruise et al., 1988). Additionally, this system is used to investigate the modulatory role of catecholamines, via activation of the oc^-adrenergic receptor, on hepatic growth (Cruise and Michalopoulos, 1985; Cruise et al., 1985,1986,1987,1989; Houck et al., 1988; Takai et al., 1988; Tsai et al., 1989; Michalopoulos, 1990).

1. Pbenethylamines. The stimulation of [^H]-thymidine incorporation into DNA of primary cultured rat hepatocytes by R-norepinephrine has been characterized as an o^-adrenergic receptor mediated event by several 57 laboratories (Cruise et al., 1985; Takai et al., 1988) and the results presented in this work are in basic agreement with these previous findings.

The compatibility of the Easson-Stedman hypothesis for the interaction of phenethylamines with the hepatic c^-adrenergic receptor that modulates [^H]-thymidine incorporation into DNA was tested by utilizing the R- and S-isomers of NE along with its desoxy derivative

DA. From the concentration response curves generated by this set of compounds, it can be seen that the rank order of potency for the stimulation of [^H]-thymidine incorporation is R-NE > S-NE > DA. DA was completely inactive in this particular system and was toxic to the cells at 100 pM causing cell detachment and death. There was also a large decrease in the amount of [^H]-thymidine incorporated into DNA at 100 pM concentrations of R- and S-NE relative to the amount stimulated at 30 pM. This may have been due in part to toxicity to the cells. When compared at the EC^^ concentrations, the activity of R-NE (EC^g = 0.90 pM) was greater than that of its corresponding S-isomer (ECg^ = 26.8 pM) giving an isomeric activity ratio of 38. The c^-adrenergic selective antagonist prazosin completely abolished the DNA synthesis stimulated by the isomers of NE. Taken collectively, these results indicate that

[^H]-thymidine incorporation stimulated by R- and S-NE in cultured rat hepatocytes is mediated by the o^-adrenergic receptor and that these phenethylamines follow the Easson-Stedman hypothesis with respect to the activity of the isomers of NE.

The finding that R-NE is more potent than its S-isomer in stimulating a^-adrenergic receptor mediated DNA synthesis is in general agreement with the results from experiments comparing catecholamine isomeric activity in other adrenergic systems (Patil et al., 1972,1975;

Ruffolo et al., 1983; Banning et al., 1984). It has been demonstrated that the R-enantiomer of NE is more active than the S-enantiomer in a vcuriety of tissue preparations including rat vas deferens (Patil et al.. 58

1967a,b, 1971), rabbit aorta (Patil et al., 1971,1972), guinea pig aorta (Patil et al., 1971,1972; Ruffolo et al., 1983), rat aorta (Patil et al., 1971,1972), cat aorta (Patil et al., 1972), rat seminal vesicle, rabbit ileum and rabbit spleen (Patil et al., 1971). The negative log EDgg values for R-NE varied from 6.1 to 8.9 for rat aorta and rabbit ileum, respectively; whereas those for S-NE varied from 3.6 to 6.5 for the same tissue preparations. This produced isomeric activity ratios that ranged from 170 for rat aorta (Patil et al., 1972) to 680 for the rabbit ileum (Patil et al., 1971). These tissues had been treated with cocaine (to block neuronal uptake), tropolone (to block degradation by catechol O-methyltransferase) and sotalol (to block B-receptors) in order to remove any interference these parameters may have on a- adrenergic effects. None of these agents were used in the cultured rat hepatocyte system. Despite these differences in the manner in which the systems were treated, the negative log EC^g values obtained for the stimulation of hepatic [^H]-thymidine incorporation by R-NE and S-NE (6.18 and 4.64, respectively) fell in the same range for those obtained in the aforementioned o-adrenergic systems. However, the isomeric activity ratio for R-NE versus S-NE stimulation of a^-adrenergic mediated DNA synthesis was much less than that for the other c^- adrenergic systems. The stereoselective stimulation of hepatic [^H]-thymidine incorporation by the isomers of NE is also consistent with previous studies that have examined the stereochemical requirements of phenethylamines for interaction with a-adrenergic receptors in liver preparations. Stereoselectivity by a-adrenergic receptors has been demonstrated by radioligand binding assays and functional studies with the isomers of NE in rat liver membranes and in isolated rat liver cells, respectively (El-Refai et al., 1979; Aggerbeck et al., 1980). R- NE inhibited 50% of [^H]-epinephrine binding in rat liver membranes at a concentration of 44 nM whereas 4500 nM S-NE was required for the same 59 degree of radioligand displacement (El-Refai et al., 1979). In the same study, the EC^g values for the activation of phosphorylase and stimulation of ^^Ca efflux in isolated hepatocytes were 51 nM and 87 nM respectively for R-NE whereas those for the S-isomer were 3900 nM and 4300 nM, respectively. Thus, R-NE is approximately 100-fold more potent than S-NE in displacing [^H]-epinephrine binding in rat liver membranes. Likewise, in functional studies, R-NE is 75-fold and 50-fold more potent than its corresponding S-enantiomer in stimulating phosphorylase activity and '*^Ca efflux (El-Refai et al., 1979). In a similar series of experiments, Aggerbeck et al. (1980) also showed that the for displacement of [^H]-dihydroergocryptine (an a-adrenergic receptor antagonist) in rat liver membranes was 0.31 pM whereas that for S-NE was 3.3 pM, a resultant 10-fold difference in these values. Similar results with isomers of epinephrine have also been obtained (Guellaen et al., 1978; El-Refai et al., 1979; Aggerbeck et al., 1980 and Studer and Ganas, 1988). Thus the EC^g values obtained for R-NE (0.9 pM) and S-NE (26.8 pM) and the 38-fold difference in the ability of these isomers to stimulate a^-adrenergic receptor mediated DNA synthesis are comparable to the stereoselective effects of these NE isomers on other hepatocyte functions. The slightly higher EC^^ values required for the stimulation of [ ] -thymidine incorporation relative to the activation of phosphorylase or stimulation of ^^Ca efflux may perhaps be explained by the length of exposure of the isomers to the hepatocytes (48 hr versus min) whereupon increased degradation of the compounds may occur in the 48 hr cultured hepatocytes. In addition, the contribution of a small population of a^-adrenergic receptors (Hoffman et al., 1981) to the effects produced by the isomers of NE was not accounted for in the above binding and functional studies performed by El-Refai et al. (1979) and Aggerbeck et al. (1980). It still remains, however; that the demonstration of stereoselective stimulation of a^-adrenergic receptor mediated [^H]-thymidine incorporation provides further evidence that the 60 a^-adrenergic receptor plays a modulatory role in the growth response of hepatocytes. The ability of hepatocytes to take up and metabolize compounds often adds difficulty to the quantitative analysis of drug-receptor interactions (Kunos, 1984). Thus, these and other factors such as agonist binding to and interaction with other receptor types and differences in the diffusion of agonists and antagonists to the receptor site may account in part for some of the concentration differences required for stimulating a^-adrenergic receptor mediated DNA synthesis and effects seen in other a^-adrenergic systems. Some of the effects of these parameters were evaluated early in the development of this system for use as a model to determine growth effects of catecholamines and catecholimidazolines (see Appendix). The monoamine oxidase (MAO) inhibitor nialamide (50 pM) failed to shift the concentration-response curve by R-NE (see Fig. 30) after a 48 hr incubation with the cells. In addition, Giachetti and Shore (1966) demonstrated that in rat liver preparations, MAO activity was stereoselective for R-NE, making it unlikely that the lower activity produced by S-NE was the result of metabolism by MAO. Another route of degradation which may alter the steric effects of NE is méthylation of the catechol moiety by catechol O-methyltransferase (COMT) (Iversen et al., 1971; Woodard et al., 1980). However, it appears that there is no stereoselectivity of the COMT enzyme towards NE (Woodard et al., 1980). Additionally, Iversen et al. (1971) showed that differences in metabolite formation in vivo were most likely due to differences in the uptake of the isomers of NE and that these differences in uptake, storage and metabolism of NE were small in relation to the ability of adrenergic receptors to discriminate between the isomers. It has been suggested that in order to minimize uptake effects, low concentrations of the test agents should be utilized (Kunos, 1984) but, due to the long term (48 hr) nature of these studies, this was impractical. Differences in the ability of agonists and/or 61 antagonists to diffuse to the site of action can also complicate the results obtained in some systems such as isolated tissue preparations. However, the 48 hr exposure of the compounds to the cultured cells should allow sufficient time for all of the tested agents to achieve equilibrium. Since hepatocytes also contain a^~ (Hoffman et al., 1981) and 8^- adrenergic receptors (Kunos, 1980; Okajima and Ui, 1982), the effect of agonist binding and interaction with other adrenergic receptor subtypes may confuse the interpretation of the manner in which the agonists stimulate a^-adrenergic receptor mediated [^Hj-thymidine incorporation. However, Cruise et al. (1985) demonstrated that the a^-adrenergic antagonist yohimbine and the B-adrenergic receptor antagonist propranolol did not inhibit R-NB stimulated [^H]-thymidine incorporation. Additionally, in preliminary studies, neither yohimbine nor propranolol blocked DNA synthesis stimulated by 10 pM R-NE until high concentrations were reached (see Appendix, Fig. 34). Nakaki et al. (1989) recently demonstrated that blockade of B^-adrenergic receptors of cultured rat vascular smooth muscle cells resulted in an increase or potentiation of the stimulatory effect of R-NE, thus indicating an inhibitory role of the B^-adrenergic receptor on cell growth. In light of the fact that there is a shift in the expression of the adrenergic receptor subtype from to B^ under certain conditions including partial hepatectomy (Aggerbeck et al., 1983) and primary hepatocyte cultures (Kunos, 1980; Okajima and Ui, 1982; Kunos, 1984), it would be of great interest to determine if the B^-adrenergic receptor has a similar inhibitory effect on cell growth in hepatocytes. This could be examined by determining whether blockade of the B^-adrenergic receptor results in the leftward shift of the R-NE concentration response curve for [^H]-thymidine incorporation. 62

2. 2-Substituted catecholimidazolines.

It can be seen from the concentration response curves of catecholimidazolines for the stimulation of [^H]-thymidine incorporation into DNA that the rank order of potency is DHT > R-TBI > S-TBI, unlike the order produced by the NE isomers and DA which follow the predicted order of activity. The EC^g values obtained from these concentration response curves for DHT and R-TBI were approximately the same (10.8 and

15.0 pM, respectively), whereas the EC^^ for S-TBI (61.6 pM) was approximately 4-fold greater than that for its corresponding R-isomer.

This difference between the activities of the catecholimidazoline enantiomers is much less than that seen between the phenethylamine enantiomers. Additionally, the DNA synthesis stimulated by all three compounds (at 100 pM concentrations) was completely inhibited by prazosin (1 pM), providing evidence that these compounds act via an a^- adrenergic mechanism. These results indicate that the hepatic adrenergic receptor [^H]-thymidine incorporation stimulated by the catecholimidazolines does not adhere to the Easson-Stedman hypothesis, despite the fact that phenethylamines do (see above). The results obtained with this set of catecholimidazolines in the cultured hepatocyte c^-adrenergic receptor system are in agreement with those obtained in other a^-adrenergic receptor systems. Ruffolo et al. (1983) showed that in an c^-adrenergic test system utilizing guinea pig aorta, the catecholimidazolines followed a rank order of potency of DHT > R-TBI

> S-TBI and thus did not fit the Easson-Stedman hypothesis. The approximate EC^^ values obtained from the guinea pig aorta dose response curves were 0.1 piK and 0.8 f/M for R- and S-TBI, respectively, for an approximate eight-fold difference in the relative potency. Similarly,

Miller et al. (1983) showed that for contraction of rat aorta, DHT and the R-isomer of TBI (-log molar ED^^ = 8.6 for both) are ten-fold more active than the S-isomer (-log molar ED^^ = 7.7). Likewise, in rat aorta and rabbit fundus, spleen and ileum (Banning et al., 1983), the 63

same rank order of potency was demonstrated for these compounds and similar isomeric activity ratios (from 5.6 to 9.8) for the enantiomers were obtained in all four tissues. Rice et al. (1987) showed that in rat aorta, this series of compounds displayed the same order of potency as described previously (Miller et al., 1983; Banning et al., 1984) and that these differences in activity were the result of changes in affinities and not due to changes in efficacies. These compounds also exhibit «^-adrenergic activity in field stimulated guinea pig ileum (Ruffolo et al., 1983) and in human platelets (Ahn et al., 1986). In both of these «^-adrenergic receptor systems, R-TBI is more active than the corresponding S-isomer as is the case in the «^-adrenergic test systems. However, removing the 8- hydroxyl group from the catecholimidazoline (producing DHT) markedly reduces the «^-adrenergic activity so that DHT is only a partial agonist, unlike the «^-adrenergic receptor systems where DHT is eguipotent to R-TBI. This provides further evidence that the effects of these catecholimidazolines are stimulating hepatic [^H]-thymidine incorporation via an «^-adrenergic receptor mediated pathway since these compounds act similarly to other «^-adrenergic receptor systems (DHT > R-TBI > S-TBI) as compared to «^-adrenergic systems (R-TBI > S-TBI > DHT). The concentrations of the catecholimidazolines required to elicit hepatic «^-adrenergic receptor mediated DNA synthesis and the values obtained from the concentration-response curves are lower by approximately two orders of magnitude from those obtained in other adrenergic receptor systems. As described above for the phenethylamines, this may possibly be the result of uptake, metabolism or other hepatocyte mechanisms that differ from other tissue systems. However, demonstration that 1) the stimulation of «^-adrenergic receptor mediated [^H]-thymidine incorporation by all three of the 2-substituted catecholimidazolines is blocked by prazosin and 2) the rank order of 64 potency and relative potency obtained for these compounds were similar to those observed in other c^-adrenergic receptor systems strongly suggests that the 2-substituted catecholimidazolines stimulate a^- adrenergic receptor modulated DNA synthesis in cultured rat hepatocytes in a stereoselective manner.

Several similarities in the stimulation of c^-adrenergic receptor modulated DNA synthesis by phenethylamines and 2-substituted catecholimidazolines were noted. These include 1) the steric requirement for the R-isomer of both phenethylamines and catecholimidazolines for maximal receptor activation and 2) the inhibition of the stimulatory effect of both classes of compounds by the c^-adrenergic receptor selective antagonist prazosin.

Phenethylamines and 2-substituted catecholimidazolines also exhibit a number of dissimilarities in the manner in which they stimulate c^-adrenergic receptor mediated [^H]-thymidine incorporation.

These include the findings that 1) for phenethylamines, the desoxy form

(DA) is inactive whereas for catecholimidazolines, the desoxy form (DHT) is slightly more active than the R-isomer which is more active than the

S-isomer and 2) the relative potency differences between the values for the enantiomers is greater for the phenethylamines (25-fold) than for the catecholimidazolines (4-fold).

The differences in the stereoselective stimulation of a^- adrenergic receptor responses indicate that these compounds may interact with 1) different sites on the c^-adrenergic receptor or 2) different subtypes of o^-adrenergic receptors. Ruffolo et al. (1977) provided evidence that imidazoline-type agonists may be interacting with different attachment sites on the same receptor or with separate types of a^-adrenergic receptors by demonstrating that in rat vas deferens, imidazolines would cause a desensitization to this class of agonists but no loss of responsiveness to phenethylamines. However, factors such as the lower efficacy of imidazolines (DeMarinis et al., 1987) and receptor 65 reserve (which may have a greater influence on the responsiveness of low efficacy agonists such as imidazolines) complicate these findings

(Minneman, 1988). McGrath (1982) proposed that there may exist two subclasses of c^-adrenergic receptors (a^^ and c^^). Research conducted in the laboratories of Minneman (Han et al., 1987a,b; Johnson et al.,

1987; Minneman et al., 1988) and Morrow and Creese (Morrow et al., 1985;

Morrow and Creese, 1986) which utilized selective antagonists of the a^- adrenergic subtypes to characterize the receptors; and of Lefkowitz

(Cotecchia et al., 1988; Schwinn et al., 1990), which employed molecular biological techniques to clone and characterize these two subsets and possibly a third (a^^), has added considerable support to this subtype classification of the c^-adrenergic receptor. [See also the Introduction

(Section I.B.3) for additional information in this area.] Thus the differences found in the stereochemical stimulation of c^-adrenergic receptor mediated DNA synthesis by both the phenethylamines and catecholimidazolines may be due to differences in interactions of these agonists with one or more a^-adrenergic receptor subtypes. These possible interactions with hepatic c^-adrenergic receptor subtypes will be presented and discussed in greater detail in Chapter 3.

3. 4—Substituted catecholimidazolines.

When cultured rat hepatocytes are exposed to the isomers of the

4-substituted catecholimidazolines (R-DBI and S-DBI) and examined for

[^H]-thymidine incorporation into DNA, it can be seen that only the R- isomer is capable of stimulating DNA synthesis. Prazosin (1 pM) completely abolishes the response to 100 pM R-DBI establishing the a^- adrenergic receptor as the means by which this isomer elicits its effects. It also appears that substitution at the 4-position of the imidazoline ring creates a compound that is slightly less potent than the 2-substituted catecholimidazoline, DHT. This is consistent with the greater potency of DHT relative to the isomers of DBI in stimulating 66 contraction of rat aorta (Lamba-Kanwal et al., 1988). However, in the cultured rat hepatocyte system, only the R-isomer of DBI stimulated a^- adrenergic receptor mediated [^H]-thymidine incorporation whereas in the rat aorta, both isomers were essentially eguiactive over the entire range of concentrations used (Lamba-Kanwal et al., 1988). In the human platelet a^-adrenergic system, only R-DBI was capable of stimulating primary wave platelet aggregation (Miller et al., 1990). In contrast, the S-isomer was over 100-fold more potent than its corresponding R-isomer for the inhibition of epinephrine induced primary wave aggregation. Thus, the stereochemical requirements for stimulation of a^-adrenergic receptor mediated DNA synthesis by 4-substituted catecholimidazolines (DBI) more closely resembles the effects seen in the platelet a^-adrenergic system as opposed to the rat thoracic aorta a^-adrenergic system. A possible explanation for the activity of these compounds on DNA synthesis is that these 4-substituted catecholimidazolines may be exerting their effects through a small population of «^-adrenergic receptors which are present in hepatocytes (Hoffman et al., 1981) or through an as of yet unidentified imidazoline preferring receptor which has been identified in other tissues such as brain and kidney (Lehmann et al., 1989; Michel and Insel, et al., 1989; Bricca et al., 1989; Parini et al., 1989). However, the finding that prazosin, an adrenergic receptor selective antagonist, blocks the effect of R-DBI argues against this point. A more plausible explanation is that hepatic a^-adrenergic receptor mediated DNA synthesis is mediated by an adrenergic receptor subtype(s) which is capable of recognizing the 4- substituted catecholimidazolines in a manner analogous to the platelet Qg-adrenergic receptor system. Further characterization of the subtype(s) of hepatic «^-adrenergic receptor which may be involved in the stimulation of [^H]-thymidine incorporation is presented below in Chapter III. Additional research including radioligand binding analysis 67

of phenethylamine and catecholimidazoline displacement of ligands

specific for o^-adrenergic receptor subtypes, a^-adrenergic receptors

and imidazoline preferring receptors in rat liver membrane preparations

will be necessary in order to establish whether the imidazoline

preferring receptor actually exists in the liver and the binding

characteristics of these receptor sites which recognize phenethylamines

and catecholimidazolines.

4. 3 , 4-Dihydroxyphenylethylenediamines.

It has been shown that agents with similar pharmacological

properties can be obtained by the exchange of isosteric groups such as

methyl, hydroxyl or amine moieties (Lehmann and Randall, 1948). These

researchers reported that substitution of the hydroxyl group of

catecholamines with an amino group at the 8-carbon atom decreases the

toxicity of these compounds. Thus it was of interest to determine the

ability of the isomers of DPDA, a compound identical to NE with the lone

exception of an amino group in the place of the hydroxyl group at the

benzylic carbon position, to stimulate c^-adrenergic mediated DNA

synthesis.

Both isomers of DPDA produced concentration dependent increases in

[^H]-thymidine incorporation in cultured rat hepatocytes. However,

there appeared to be little, if any, stereoselectivity exhibited for the

stimulation of DNA synthesis by these enantiomers as only a two-fold

difference exists in their relative potencies (R-DPDA EC^^ =22.5 pM, S-

DPDA EC^g = 10.0 pM). Additionally, prazosin completely inhibited [^H]-

thymidine incorporation by R-DPDA but only partially inhibited

(approximately 50% blockade) the response to S-DPDA. This possibly

suggests that part of the effect of the S-isomer may be expected via

growth promoting mechanism not associated with the c^-adrenergic receptor. The important contribution of the 8-hydroxyl group to the activity of phenethylamines is demonstrated when the degree of 6 8

stimulation of [^H]-thymidine incorporation by R-NE and R-DPDA are compared. The B-hydroxylated phenethylamine, R-NE, has an EC^^ of 0.34 pM for the stimulation of [^H]-thymidine incorporation whereas for the corresponding R-DPDA, which contains an amino group at the B-oarbon, the EC^g is 22.5 pM for a 66-fold difference in relative activity. This difference in activity between compounds containing a B-hydroxyl group and B-amino group is similar to that found in rat aorta (a^-adrenergic system) and human platelets (a^-adrenergic system) (Miller et al., 1990). However, while little or no isomeric stereoselectivity was observed for the interactions of the DPDA isomers with the hepatic a^- adrenergic receptor stimulating DNA synthesis, contraction of rat aorta was elicited in a stereoselective [R-DPDA (EC^^ = 0.95 pM) > S-DPDA (EC^g = 21.4 pM)] manner. R-DPDA was also more potent than S-DPDA in stimulating platelet aggregation. Therefore, it appears that upon replacing the B-hydroxyl group of R-NE with an amino group, the ability of the resulting compound (R-DPDA) to stimulate a^-adrenergic receptor mediated DNA synthesis is markedly reduced. Additionally, this same substitution results in an almost complete elimination of the stereoselective stimulation of [^H]-thymidine incorporation which characterizes the responses to the isomers of NE, thus emphasizing the importance of the B-hydroxyl group to the agonist activity in this system.

5. General discussion. The effects of these compounds on total cellular protein are difficult to interpret. For all of the experiments except those involving the DPDA isomers, increases in total cellular protein content were seen regardless of whether or not the compounds stimulated DNA synthesis. For example, both dopamine and S-DBI failed to significantly stimulate DNA synthesis but both compounds produced significant increases in total cellular protein. In addition, for most of the 69 experiments (again with the exception of the experiments examining the effects of the DPDA isomers) prazosin failed to inhibit the increases in total cellular protein caused by the tested compounds, including R-NE. This could indicate that perhaps there is a dissociation of the abilities of these compounds to stimulate DNA and protein syntheses; that is, these effects are regulated by different receptors. This is unlikely since it has been shown that the stimulation of both DNA and protein syntheses, when measured by [^H]-thymidine and [ ]-methionine incorporations, respectively, into TCA-precipitable material, by R-NE and DHT are both inhibited by prazosin (see Chapter III). Another explanation may be that the increases in total cellular protein are nonspecific effects which augment the ability of the growth factors (EGF and insulin) present in the media to increase DNA synthesis but have little influence on the ability of these growth factors to stimulate DNA synthesis. It is interesting to note that although TGFB inhibits DNA synthesis caused by EGF, it does not inhibit the increase in protein synthesis produced by EGF (Houck and Michalopoulos, 1989). These researchers suggested that TGFB shifted the hepatocyte response to EGF from proliferation to primarily protein synthesis. However, the variable effects of the phenethylamines and catecholimidazolines on total cellular protein levels make interpretation of the effects of these compounds on protein synthesis based on total celluleir protein levels difficult. A major goal of these studies was to analyze the structural and stereochemical requirements of a series of closely related phenethylamine and catecholimidazoline compounds on o^-adrenergic receptor mediated DNA synthesis in cultured rat hepatocytes. From these results a number of conclusions can be made concerning the manner in which these compounds stimulate [^H]-thymidine incorporation. Similarities in the ability of these two classes of compounds to stimulate hepatic DNA synthesis were demonstrated and include findings 70

that 1) the effects of both phenethylamines and catecholimidazolines are

blocked by the a^-adrenergic receptor antagonist prazosin and 2) the R-

enantiomer is more potent than the S-enantiomer. Differences in the

ability of these classes of agents to stimulate a^-adrenergic receptor

mediated DNA synthesis were also noted and include the findings that 1)

the desoxy form (DA) of the phenethylamine series tested is inactive

whereas the desoxy form (DHT) of the catecholimidazolines is the most

active form for this series of compounds and 2) the relative potency

differences between the values for the enantiomers is greater for the phenethylamines (25-fold) than for the catecholimidazolines (4-

fold). These series of experiments also showed that substitution of the imidazoline ring at the 4-position decreases the ability of the resultant compound to stimulate c^-adrenergic receptor mediated DNA synthesis relative to 2-substituted catecholimidazolines. In experiments in which the effect of isosteric substitution of the B- hydroxyl group with an amino moiety were examined, it was shown that in compounds containing a B-amino group (DPDA series), the agonist activity and stereoselectivity were decreased relative to the B-hydroxylated agents (NE series). 71

OH HO ,NH,

HO

R-NOREPINEPHRIHB (R-HE]

HO HO NH

HO

S-HOREPIHEPHRIHB (S-NE)

HO

DOPAMINE (DA)

Figure 2. Chemical structures of R-norepinephrine, s-norepinephrine and dopamine. 72

OH

HO NH

HO

R-2-(3,4,ot-TRIHYDROXÏBBNZYI.)IMIDA20LIHB (R-TBI)

OH

HO NH

HO

S-2-(3,4,ec-TRIEZI>R0ZZBEHZYL)ZMI0&Z0LZIiE (S-TBI)

2-( 3 , 4-0IHZDRQZÏBEHZTI. ) IMIDAZOU31E

[3,4-DIHZDROZrrOL&ZOLIHE] (DBI)

Figure 3. Chemical structures of R-2-(3,4, ot-trihydroxybenzyl)- imidazoline, S-2-(3,4,o-trihydroxybenzyl)imidazoline and 2-(3,4- dihydroxybenzyl)imidazoline. 73

R-4-(3,4-DIHÏDROXYBENZÏL) S-4- ( 3 , 4-DIHÏDROXXBENZYL) IMIDAZOLINE IMIDAZOLINE (R-DBI) (S-DBI)

HO HO

NH. NH. HO HO

NH. NH

R-3,4-DIEYDROZZPaENYLETHYL- S-3,4-DIHYDROXYPHENYLSTHYL- ENEDIAMINE (R-DPDA)(S-DPDA)

Figure 4. Chemical structures for R-4-(3,4-dihydroxybenzyl)imidazoline, S-4-(3,4-dihydroxybenzyl)imidazoline, R-3,4-dihydroxyphenylethylene— diamine and S-3,4-dihydroxyphenylethylenediamine. 74

120 r — : LU LU O — o R -N E CO CH z Z 3 100- # @ S—NE o 5 Q_ in A A DA LU

CL Q X <

o 4-0 -- ro z LU u LU z z LU Q.

—8 - 7 —6 - 5 —Æ - 3 LOG DRUG CONCENTRATION (M)

Figure 5. concentration response curves for the stimulation of [H]- thymidine incorporation into DNA of primary cultured rat hepatocytes by R-norepinephrine (R-NE), S-norepinephrine (S-NE) and dopamine (DA). Results are expressed as a percentage of the maximum response produced by 30 pM R-NE. Each point represents the mean ± S.E.M. of determinations from 8-12 dishes. Points with letters indicate levels of [ H]-thymidine incorporation that were significantly greater than control and points with different letters indicate degrees of DNA synthesis which were significantly different from each other (p < 0.05, Student-Newman-Keuls test). Control DNA synthesis was 525519 ± 52280 DPM/culture (mean ± S.E.M.) and cells treated with 30 pH R-NE incorporated [ H]-thymidine at a level of 335.4 ± 17.8% (mean ± S.E.M.) of control. 75

CZ3 DRUG ALONE 1 2 0 t r a DRUG + PRZ (1 /^M)

P 100" O ü_ c/0 LU 80 -- DC

5 0 "

4 0 "

2 0 " *** LU I *** ilCL DC *** m ■r R -N E S -N E DA 30 /j,U 30 jüM 30 jjM

Figure 6. Effects of 1 ;l/M prazosin (PRZ) on [ H]-thymidine incorporation stimulated by R-norepinephrine (R-NE), S-norepinephrine (S-NE) éind dopamine (DA) in primary cultured rat hepatocytes. Results are expressed as a percentage of the meucimum response produced by 30 j M R-NE and each bar represents the mean ± S.E.M. of determinations from 4 dishes. Asterisks (***) indicate that the mean of drug + PRZ treated dishes is significantly different (p < 0.001, Student's unpaired t-test) from the mean of the drug only treated dishes. Control DNA synthesis was 696113 ± 20084 DPM/culture and cells treated with 30 }M R-NE incorporated [ H]-thymidine at a level of 290.0 ± 5.0% (meeui ± S.E.M.) of control. 76

Table 2. Effects of R-norepinephrine (R-NE), S-norepinephrine (S-NE) and dopamine (DA) on total cellular protein in 48 hr primary cultured rat hepatocytes.

TREATMENTCONC. CELLULAR PROTEIN® (pM) PERCENT CONTROL (%)

R-NE 10 139.0 ± 6.si 30 137.7 ± 4.9 S-NE 10 128.4 ± 8.2» 30 144.1 ± 9.1» DA 10 134.2 ± 5.8» 30 134.6 ± 4.9» PRAZOSIN (1 pM) + R-NE 30 140.6 ± 11.4 S-NE 30 136.5 ± 3.8» DA 30 150.0 ± 7.1»

“ Values are expressed as mean ± S.E.M. of the percentage of control total cellular protein (69.2 ± 4.3 pg protein/dish); n = 12 dishes for all treatments except for the prazosin treated cells where n = 4 dishes. ^ Means are significantly greater than control (p < 0.05, Student- Newman-Keuls test). 77

1 00 LU a: O ------O R-TBI to Z 80 -• o # ------9 S—TBI û_ 3 ü A ------A DHT 5 6 0 -- q : CL Q 4 0 -

8 2 0 - LU ü LU cc Z 0 - LU CL -20 —8 - 7 -6 - 5 - 4 - 3 LOG DRUG CONCENTRATION (M)

Figure 7. Concentration response curves for the stimulation of [ H]- thymidine incorporation into DNA of primary cultured rat hepatocytes by R-2-(3,4,a-trihydroxybenzyl)imidazoline (R-TBI), S-2-(3,4,a- trihydroxybenzyl)imidazoline (S-TBI) and 2-(3,4-dihydroxybenzyl)- imidazoline (DHT). Results are expressed as a percentage of the maximum response produced by 100 }M R-norepinephrine (R-NE). Each point represents the mean ± S.E.M. of determinations from 7-8 dishes. Points with letters indicate levels of [ H]-thymidine incorporation that were significantly greater than control and points with different letters indicate degrees of DNA synthesis which were significantly different from each other (p < 0.05, Student-Newman-Keuls test). Control DNA synthesis was 593604 ± 37610 DPM/culture (mean ± S.E.M.) and cells treated with 100 jjK R-NE incorporated [ E]-thymidine at a level of 390.2 ± 17.8% (mean ± S.E.M.) of control. 78

IOOt

LU a DRUG ALONE ce ~Z. z> 80 -■ ES] DRUG + PRZ (1 iM) O CL t: in 3 LU ü ce £L Q 4 0 --

LU O LU ce Z LU 3. -20 S-TBIR-TBl DHT 100 fj,U 100 f M 100 fj.U

Figure 8. Effects of 1 jM prazosin (PRZ) on [ H]-thymidine incorporation stimulated by R-2— (3,4,a-trihydroxybenzyl)imidazoline (R— TBI), S-2-(3,4,o-trihydroxybenzyl)imidazoline (S-TBI) and 2-(3,4- dihydroxybenzyl)imidazoline (DHT) in primary cultured rat hepatocytes. Results are expressed as a percentage of the maximum response produced by 100 R-norepinephrine (R-NE) and each bar represents the mean ± S.E.M. of determinations from 8 dishes. Asterislcs (***) indicate that the mean of drug + PRZ treated dishes is significantly different (p < 0.001, Student-Newman-Keuls test) from the mean of the drug only treated dishes. Control DNA synthesis was 593604 ± 37610 DPM/culture (mean ± S.E.M.) and cells treated with 100 jjH R-NE incorporated [ H]-thymidine at a level of 390.2 ± 17.7% (mean ± S.E.M.) of control. 79

Table 3. Effects of R-2-(3,4,a-trihydroxybenzyl)imidazoline (R-TBI), S-2-(3,4,o-trihydroxybenzyl)imidazoline (S-TBI) and 2-(3,4-dihydroxy- benzyl)imidazoline (DHT) on total cellular protein in 48 hr primary cultured rat hepatocytes.

TREATMENT CONC. CELLULAR PROTEIN® (pM) PERCENT CONTROL (%)

R-TBI 10 113.3 + 4.2* 30 127.4 ± 3.9* 100 138.6 ± 5.9* S-TBI 10 120.6 ± 5.3* 30 125.2 ± 6.9* 100 139.5 ± 5.3* DHT 10 130.0 ± 3.9* 30 140.6 ± 6.5* 100 156.1 ± 2.8*

R-NE 10 143.1 ± 11. 100 169.5 ± 3.7* PRAZOSIN (1 pM) + R-TBI 100 119.7 ± 4.8* S-TBI 100 137.2 ± 4.3* DHT 100 136.7 ± 5.8* R-NE 100 145.9 ± 5.2*

Values are expressed as mean ± S.E.M. of the percentage of control total cellular protein (111.6 ± 5.6 pg protein/dish); n = 7-8 dishes. Means are significantly greater than control (p < 0.05, Student- Newman-Keuls test). 80

1 0 0

LU LU O- -O R -D BI CO 0: s ^ 8 0 -- ® — e S-D BI & Z) A- •A R.S-DBI LU ü cc 60-- Q. Q

^ 4 0 -

20

— e —8 —7 —6 —5 —4 - 3

LOG DRUG CONCENTRATION (M)

Figure 9. Concentration response curves for the stimulation of [ H]- thymidine incorporation into DNA of primary cultured rat hepatocytes by R-4-(3,4-dihydroxybenzyl)imidazoline (R-DBI), S-4-(3,4-dihydroxybenzyl)- imidazoline (S-DBI) and R,S-4-(3,4-dihydroxybenzyl)imidazoline (R,S- DBI). Results are expressed as a percentage of the maximum response produced by 30 pM R-norepinephrine (R-NE). Each point represents the mean i S.E.M. of determinations from 8-12 dishes. Points with letters indicate levels of [ H]-thymidine incorporation that were significantly greater than control and points with different letters indicate degrees of DNA synthesis which were significantly different from each other (p < 0.05, Student-Newman-Keuls test). Control DNA synthesis was 1084356 ± 85063 DPM/culture (mean ± S.E.M.) and cells treated with 30 pM R-NE incorporated [ H]-thymidine at a level of 203.6 ± 10.4% (mean ± S.E.M.) of control. 81

IOOt C 3 DRUG ALONE K % 80 Z P C S DRUG + PRZ (1 fM) O 0_ CO LU 60 1 O' I 4 0 --

2 0 " i W I o_ 0: -20 R-DBI R-DPDA S-DPDA lOO^M 100 jCiM 100/iM

Figure 10. Effects of 1 jiM prazosin (PRZ) on [ H]-thymidine incorporation stimulated by R-4-(3,4-dihydroxybenzyl)imidazoline (R- DBI), R-3,4-dihydroxyphenylethylenediamine (S-DPDA) auid S-3,4- dihydroxyphenylethylenediamine (S-DPDA) in primary cultured rat hepatocytes. Results are expressed as a percentage of the maximum response produced by 30 jiM R-norepinephrine (R-NE) and each baur represents the mean ± S.E.M. of determinations from 8 dishes. Asterisks (••♦) indicate that the mean of drug + PRZ treated dishes is significantly different (p < 0.001, Student's unpaired t-test) from the mean of the drug only treated dishes. Control DNA synthesis was 1084356 ± 85063 DPM/culture (mean ± S.E.M.) and cells treated with 30 jM R-NE incorporated [ a]-thymidine at a level of 203.6 ± 10.4% (mean ± S.E.M.) of control. 82

Table 4. Effects of R-4-(3,4-dihydroxybenzyl)imidazoline (R-DBI), S-4-(3,4-dihydroxybenzyl)imidazoline (S-DBI) and R,S-4-(3,4-dihydroxy­ benzyl ) imidazoline (R,S-DBI) on total cellular protein in 48 hr primary cultured rat hepatocytes.

TREATMENTCONC. CELLULAR PROTEIN* (^M) PERCENT CONTROL (%)

R-DBI 10 130.3 ± 3.7^ 30 142.0 ± 4.4^ 100 151.1 ± 7.6" S-DBI 10 122.8 ± 2.6" 30 128.4 ± 2.8" 100 129.5 + 3 .2" R, S-DBI 10 134.8 + 4.7^ 30 141.6 ± 2 .2" 100 137.4 ± 5.5 R-NE 10 147.3 ± 4 .7" 100 153.6 4 .9" PRAZOSIN (1 pM) + R-DBI 100 130.2 ± 7 .0" S-DBI 100 133.2 ± 2 .5" R,S-DBI 100 131.0 ± 4 .0" R-NE 10 125.7 ± 9.6" R-NE 100 152.2 ± 7.6"

“ Values are expressed as mean ± S.E.M. of the percentage of control total cellular protein (69.2 ± 5.4 pg protein/dish); n = 7-12 dishes. ^ Means are significantly greater than control (p < 0.05, Student- Newman-Keuls test). 83

100 LU Lü O R-DPDA CO ce Z =) O 5 • S-DPDA û. en a,b ui ce a,b Q. Q

X < î a,b

LU O z ÙC I LU z CL 1—J - 2 0 4 7 —6 - 5 •3-4

LOG DRUG CONCENTRATION (M)

Figure 11. Concentration response curves for the stimulation of [ H]- thymidine incorporation into DNA of primary cultured rat hepatocytes by R-3,4-dihydroxyphenylethylenediamine (R-DPDA) and S-3,4-dihydroxy- phenylethylenediamine (S-DPDA). Results are expressed as a percentage of the maximum response produced by 30 R-norepinephrine (R-NE). Each point represents the mean ± S.E.M. of determinations from 6—9 dishes. Points with letters indicate levels of [ H]-thymidine incorporation that were significantly greater than control and points with different letters indicate degrees of DNA synthesis which were significantly different from each other (p < 0.05, Student-Newman-Keuls test). Control DNA synthesis was 422578 ± 33629 DPM/culture (mean ± S.E.M.) and cells treated with 30 pK R-NE incorporated [ H]-thymidine at a level of 276.6 ± 22.5% (mean ± S.E.M.) of control. 84

Table 5. Effects of R-3,4-àihydroxyphenylethylenediamine (R-DPDA) and S-3,4-dihydroxyphenylethylenediainine (S-DPDA) on total cellular protein in 48 hr primary cultured rat hepatocytes.

TREATMENTCONC. CELLULAR PROTEIN® (MM) PERCENT CONTROL (%)

R-DPDA 30 112.1 ± 14.4 100 126.0 ± 17.6 S-DPDA 30 109.4 ± 11.5 100 103.5 ± 15.2 R-NE 10 156.7 ± 24.5^ 30 162.2 ± 31.0^ PRAZOSIN (1 ^M) + R-DPDA 100 95.7 ± 17.0 S-DPDA 100 104.2 ± 17.2 R-NE 10 109.9 ± 15.6

* Values are expressed as mean ± S.E.M. of the percentage of control total cellular protein (68.2 ± 8.9 jLig protein/dish); n = 8-9 dishes. ^ Means are significantly greater than control (p < 0.05, Student- Newman-Keuls test). 85

Table 6. Important parameters of the concentration response curves for the stimulation of [ H]-thymidine incorporation into the DNA of primary cultured rat hepatocytes by stereoisomers of phenethylamines and imidazolines.

c COMPOUND n E REL.‘* ( 5 6 POT.

R-NE 3 0.34 ± 0.14 6.54 ± 0.17 1.1DO 1.00 S-NE 3 15.49 ± 7.64 4.94 ± 0.25 0.53 ± 0.09 0.022 DA 3 > 30 < 4.5 0.15 ± 0.02 — R-TBI 2 15.05 ± 0.05 4.82 ±0.01 0.45 ± 0.10 0.022 S-TBI 2 61.60 ± 0.28 4.26 ± 0.21 0.39 ± 0.07 0.0055 DHT 2 10.77 ± 0.12 4.97 ± 0.05 0.80 ± 0.05 0.032 R-DBI 3 17.34 ± 0.58 4.81 ± 0.14 0.61 ± 0.09 0.020 S-DBI 3 > 30 < 4.5 0.05 ± 0.02 — R, S-DBI 3 > 30 < 4.5 0.29 ± 0.06 — R-DPDA 3 22.52 ± 4.74 4.66 ± 0.08 0.48 ± 0.08 0.015 S-DPDA 2 10.02 ±2.58 5.01 ± 0.12 0.70 ± 0.22 0.034

Values are expressed as the mean ± S.E.M. of the EC determinations from individual (n) experiments. EC^g is defined as the effective concentration of a compound which produces a 30% level of response relative to the maximal response of R-NE. For each concentration response curve, the response from 3-4 dishes of cells were used for each concentration of the tested compound.

PEC30 = -log ECgg ' E is the maximum response obtained with each compound relative to the maximum for R-NE which equals 1.00. Relative potency calculated by: (EC^^ of R-NE/EC^g of tested compound). CHAPTER III

PHARMACOLOGIC CHARACTERIZATION OF a -ADRENERGIC RECEPTOR MEDIATED DNA AND PROTEIN SYNTHESES IN PRIMARY COLTORED RAT HEPATOCYTES

A. Specific Aim

The specific aim of this set of experiments was to evaluate the

effects of a series of compounds that may modify a^-adrenergic receptor

mediated DNA and protein syntheses in primary cultured rat hepatocytes.

In these experiments 1} the effect of modification of the substituent at

the 4 '-benzyl position imidazoline analogs on stimulation and inhibition

of ct^-adrenergic receptor mediated [^H]-thymidine incorporation into DNA

was examined, 2) the effects of cirazoline and idazoxan on hepatic DNA

synthesis were examined in order to determine whether an c^-adrenergic

receptor and/or non-adrenergic receptor-mediated mechanism is involved,

3) characterization of the a^-adrenergic receptor subtype(s) responsible

for modulating DNA synthesis was evaluated utilizing selective a^- receptor subtype antagonists, 4) the possible role of extracellular Ca*^

and calmodulin in a^-adrenergic receptor mediated DNA synthesis was examined and 5) the effect of selected a^-adrenergic receptor agonists and antagonists on hepatic [ ]-methionine incorporation was determined.

B. Methods

1. Materials

The biochemicals and their sources were: trifluoperazine dihydrochloride (Sigma Chemical Company, St. Louis, MO); verapamil hydrochloride (Knoll Pharmaceutical Co., Whippany, NJ); chloroethyIclonidine dihydrochloride, idazoxan hydrochloride and WB-4101

(Research Biochemicals Inc., Natick, MA); cirazoline hydrochloride (gift

86 87 from Synthelabo Recherche, Paris, France); tolazoline hydrochloride

(Ciba Pharmaceutical Co., Summit, NJ); SZL-49 (gift from Dr. Michael

Piascik, Department of Pharmacology, College of Medicine, University of

Kentucky, Lexington, KY); [^^S]-1-methionine (specific activity = 1168

Ci/mmole) (ICN Biomedicals, Inc., Irvine, CA). Other materials used were the same as those described in Chapter II.

2. Isolation and preparation of primary cultures of rat hepatocytes.

The animals and procedures used were the same as those described in Chapter II. Cells were treated with the compounds in the same manner as that described in Chapter II with the following exceptions. After a

90 min attachment period, cells were preincubated with either chloroethyIclonidine (CEC) or SZL-49 for 30 min. Following this preincubation period, the media was aspirated and replaced with fresh serum free supplemented Williams Medium E. At this time agonists (R-NE or DHT) were added to hepatocytes. The only difference from CEC treatment was that the original stock solution of SZL-49 was prepared by dissolving this agent in 100% ethanol. From this, appropriate dilutions were made in double distilled water and aliquots were added to the cells. The amount of [^H]-thymidine incorporation produced by cells exposed to the greatest amount of 100% ethanol (20 pil, 1% of total volume) for 30 min did not differ from control cells to which 20 jul of double distilled water had been added for 30 min.

The effects of a^-adrenergic receptor agonists and antagonists on protein synthesis were measured by determining the incorporation of

[^^H]-methionine into TCA precipitable material. For these experiments, the hepatocytes were isolated and plated in the same manner as that described in Chapter II. In experiments in which [^^S]- methionine incorporation was determined, both protein and DNA syntheses were measured. ]by incubating the hepatocytes with [ ] -methionine (2 pCi/ml) and [^H]-thymidine (5 ^Ci/ml). 8 8

3. Biochemical assays of cell lysates and data analysis. The assays for [ ] -thymidine incorporation and total cellular protein utilized in these experiments are the same as those described in Chapter II. In experiments in which both DNA and protein syntheses were measured, the hepatocytes were lysed, the lysates washed and aliquots taken as described in Chapter II. The radioactivity resulting from [ ]-methionine and [^H]-thymidine was distinguished from each other by liquid scintillation counting utilizing a Beckman 6800 liquid scintillation counter and measuring activities in a selected range of the respective energy spectra (0-400 and 400-670 for and respectively) using the exclusion method. The amount of [^H]-thymidine and [ ]-methionine incorporated into TCA precipitable material was calculated as DPM ( ]-thymidine/culture and DPM methionine/culture, respectively. These results were then routinely normalized by expressing the treatment DPM/culture (minus control DPM/culture) as a percentage of the respective maximal amount of radiolabel DPM/culture elicited by either 30 pM R-NE or 100 pM DHT (minus control DPM/culture). The results are also expressed as a percentage of the control response for the time course studies. Total cellular protein was expressed as pg protein/dish and presented as a percentage of the control response. Inhibitory concentration 50 (IC^^) values were calculated from inhibition response curves constructed from individual experiments for comparisons of the relative inhibitory potencies of various antagonists. The IC^g value was defined as the concentration of an antagonist required to reduce the response of 10 pM R-NE by 50%. The data were statistically analyzed by comparisons among the means using Student's unpaired t-test for direct comparisons of means or by one-way analysis of variance followed by the Student-Newman-Keuls test for multiple comparison of means. 89

C. Results.

The chemical structures of the compounds that were used in this

series of experiments are shown in Figs. 12 and 13. In experiments in

which the antagonist action of a compound was investigated, the

inhibitory effect of the a^-adrenergic receptor selective antagonist

prazosin on R-NE and DHT stimulated [^H]-thymidine incorporation in

cells from the same isolation was also determined for the purpose of

comparison to a positive response.

1. Evaluation of the effect of modification of the 4'-benzy1-substituent

of imidcusoline analogs on stimulation and inhibition of a^-

adrenergic receptor mediated DNA synthesis.

The agents used to determine the effect that selected substituents

at the 4'-benzyl position of imidazoline analogs have on a^-adrenergic

receptor mediated DNA synthesis of 48 hr primary cultured hepatocytes

are shown in Fig. 12. Tolazoline, 2-(4-aminobenzyl)imidazoline (ABI)

and 2-(4-isothiocyanatobenzyl)imidazoline (IBI) differ structurally from

each other only by the presence of a hydrogen atom, an amine group and

an isothiocyanate moiety, respectively, at the 4'-benzyl position of these 2-substituted benzylimidazolines. All three of these compounds

failed to stimulate [^H]-thymidine incorporation in primary cultured rat

hepatocytes exposed to these agents for 48 hr (see Fig. 14).

Comparatively, [^H]-thymidine incorporation was significantly increased

(68% of the maximal response) in cells exposed to 100 pM DHT for 48 hr.

At concentrations greater than 10 pM, IBI was toxic to the cells causing

cell death and detachment upon 48 hr exposure. Tolazoline, ABI and IBI

did not significantly increase total cellular protein relative to

control cells (see Table 7) whereas 30 pta R-NE significantly increased

protein levels and 100 pM DHT produced a small increase in total

cellular protein that was not statistically significant. 90

The effect of these three compounds on DNA synthesis stimulated by 10 ]Æ R-NE and 100 pM DHT (for tolazoline only) was also evaluated (Fig. 15). A more complete analysis of the concentration dependent inhibitory effects of tolazoline on R-NE and DHT stimulated [^H]-thymidine incorporation is presented in the next section (Section III.C.2). When compared to the stimulated level of DNA synthesis, 100 tolazoline significantly reduced the level of [^H]-thymidine incorporation produced by 10 R-NE and 100 pM DHT to 48% and 69% of the stimulated response. Similarly, ABI significantly decreased the amount of [^H]-thymidine incorporation (69% of the R-NE stimulated response) in cells exposed to 10 pM R-NE relative to the cells incubated with agonist alone. [^H]- Thymidine incorporation stimulated by 10 ptK R-NE was not inhibited (107% of stimulated response) in hepatocytes preincubated with 10 IBI for 30 min prior to agonist exposure. Prazosin (1 pM) completely abolished the stimulation of [^H]-thymidine incorporation by both 10 pM R-NE and 100 fiM DHT (-11% and -15% of stimulated responses, respectively) [Fig. 15]. Although the effects of these compounds on total cellular protein are more variable than the effects on DNA synthesis, it can be seen that 100 pM tolazoline does not block the increases in protein by R-NE and DHT (150% and 135% of control, respectively) whereas prazosin completely inhibits these increases (89% and 93% of control, respectively) [Table 7]. Additionally, ABI appears to block the increase in protein caused by 10 )M R-NE (93% of control) whereas in cells preincubated with 10 pM IBI, the total cellular protein is unchanged (118% of control) from cells incubated only with 10 pM R-NE.

2. Effects of tolazoline, cirazoline and idazoxan on DNA synthesis in 48 hr primary cultured rat hepatocytes. The chemical structures for tolazoline, cirazoline and idazoxan are shown in Fig. 12. As discussed above in Section III.C.l, tolazoline did not stimulate DNA synthesis in concentrations up to 100 fM. 91

Likewise, the a^-adrenergic receptor agonist cirazoline failed to cause an increase in hepatic [^H]-thymidine incorporation into DNA of cultured hepatocytes incubated with this agent in concentrations up to 30 fM for 48 hr (see Fig. 16). At a concentration of 100 fM, cirazoline caused cell death and detachment from the culture dishes. In comparison, 100 fM DHT, the 3,4-dihydroxybenzyl derivative of tolazoline, produced a significant increase in [ ] -thymidine incorporation that was 62% of the maximal response. The effects of these compounds on total cellular protein is similar to that seen on [^H]-thymidine incorporation (see Table 8). Neither tolazoline nor cirazoline caused significant increases in protein levels of the primary cultured rat hepatocytes. However, 10 fM R-NE increases total cellular protein to a level that is 141 % of the control response and a further significant increase to 147% of control is seen when cells are exposed to 30 pK R-NE. Similairly, 100 pM DHT causes an increase in the protein content to 129% of control. The concentration dependent inhibitory effect of these compounds against R-NE (10 pM) stimulated [^H]-thymidine incorporation was also determined (Fig. 17). Tolazoline caused a small concentration dependent inhibition of R-NE stimulated DNA synthesis. From concentrations of 1 to 10 pK, tolazoline produced a slight inhibition of the response to R- NE (10 fM) that ranged from 68% to 72% of the stimulated level of [^H]- thymidine incorporation. Tolazoline elicited a significant inhibition of [^H]-thymidine incorporation stimulated by R-NE at concentrations of 30 pK and 100 fM that were 50% and 45% of the stimulated response, respectively. The value for tolazoline for the inhibition of R-NE stimulated DNA synthesis was 45.4 fM (Table 15). Cirazoline also caused a concentration dependent inhibition of R-NE stimulated DNA synthesis. In a similar manner as tolazoline, low concentrations (1 and 3 pM) of cirazoline produced small decreases in the level of [^H]-thymidine incorporation evoked by R-NE that were 69% to 85% of the stimulated response. At concentrations of 10 pH and 30 pH, cirazoline 92

significantly lowered 10 ]M R-NE stimulated [^H]-thymidine incorporation to levels that were 48% and 24% of the stimulated response, respectively. The value for the inhibition of R-NE stimulated DNA synthesis by cirazoline was 15.7 juM. Prazosin (1 pM) significantly decreased DNA synthesis by 10 pM R-NE to 6% of the stimulated response. The concentration dependent inhibition of DHT (100 pM) stimulated [^H]-thymidine incorporation by tolazoline and cirazoline was also examined in primary cultured rat hepatocytes (Fig. 18). Tolazoline, in concentrations up to 100 pM, did not cause a significant inhibition of the amount of [^H]-thymidine incorporation stimulated by DHT. On the other hand, 10 pM cirazoline produced a small, although not statistically significant, decrease in the amount of DHT-stimulated [^H]-thymidine incorporation to a level of 69% of the stimulated response. When exposed to 30 pM cirazoline, the level of [^H]-thymidine incorporation in DHT-stimulated cells was significantly reduced to 34% of the 100 pM DHT response. The value of cirazoline for the inhibition of DHT stimulated DNA synthesis is 16.5 ±0.8 pH. Prazosin (1 pM) significantly inhibited DHT-stimulated DNA synthesis and lowered the amount of [^H]-thymidine incorporation to 7% of the stimulated response. Both tolazoline and cirazoline at concentrations up to 100 pM and 30 pM respectively, failed to significantly reduce the increase in total cellular protein produced by R-NE and DHT (see Table 8). Prazosin (1 pM) lowered the amount of total cellular protein in 10 pM R-NE and 100 pM DHT treated primary cultured hepatocytes to near control levels (102% and 111% of control protein content, respectively). The level of [^H]-thymidine incorporation in primary cultured rat hepatocytes exposed to idazoxan alone in concentrations up to 30 pM did not vary significantly from that in control cells (see Table 9). Likewise, in cells exposed to concentrations up to 30 pM of idazoxan, the total cellular protein was not significantly different from control levels. Although there was a slight elevation in the protein content of 93

these cells (see Table 10), these small increases (113% to 126% of the control level) were much less than that caused by 10 juM R-NE (155%). Idazoxan failed to inhibit the stimulation of [^H]-thymidine incorporation caused by both 10 pM R-NE and 100 pM DHT in concentrations up to 10 }M (see Fig. 19). At 30 jiM, idazoxan significantly decreased the amount of [^H]-thymidine incorporation by R-NE and DHT to 14% and 38% of the stimulated responses, respectively. In R-NE and DHT treated cells exposed to 1 }M prazosin, [^H]-thymidine incorporation was reduced to 8% and 0% of the R-NE and DHT stimulated responses, respectively. Additionally, idazoxan did not inhibit the increases in total cellular protein caused by either 10 pM R-NE or 100 pM DHT (155% and 146% of control protein content, respectively) (see Table 10). Prazosin (1 fM) decreased the amount of total cellular protein in 10 ]M R-NE stimulated cells to 115% of the control value but had little effect on the total cellular protein in cells exposed to 100 juM DHT (137% of control).

3. Characterization of the a^-adrenergic receptor subtype(s) that modulates DNA synthesis in primary cultured rat hepatocytes. The chemical structures of the selective competitive c^-adrenergic receptor antagonist prazosin, selective competitive c^^-adrenergic receptor subtype antagonist WB 4101, selective a^^^-adrenergic receptor subtype alkylating agent chloroethyIclonidine (CEC) and the adrenergic receptor alkylating agent SZL-49 are shown in Fig. 13. Each of these compounds, with the exception of WB 4101, produced concentration dependent inhibition of [^H]-thymidine incorporation stimulated by 10 fM R-NE (see Fig. 20). Prazosin caused a concentration dependent inhibition of R-NE stimulated [^H]-thymidine incorporation. A significant decrease to 47% of the stimulated response was caused by 10 nM prazosin and further decreases to 10% and 14% of the stimulated level of [^H]-thymidine incorporation was produced by 0.1 fM and 1.0 pM prazosin, respectively. 94

The ICgg value for the inhibition of R-NE stimulated DNA synthesis by prazosin was 8.7 nM (see Table 15). In cultured hepatocytes exposed only to 1 pM prazosin, the level of -thymidine incorporation was not significantly different from that in control cells (see Table 11) and although the total cellular protein content in these cells was slightly elevated, the protein content was not significantly different from the control level of protein (see Table 12). Unlike the results discussed previously for the effects of 1 pM prazosin on the total cellular protein content of 10 f/M R-NE stimulated cells (see Sections III.C.l and

III.C.2), prazosin, in concentrations up to 1 pM, did not inhibit the increase in protein levels caused by R-NE (see Table 12).

A concentration dependent inhibition of R-NE stimulated [^H]- thymidine incorporation was also elicited in primary cultured rat hepatocytes that were preincubated for 30 min with CEC prior to agonist exposure. This inhibition curve lies to the right of those for prazosin and SZL-49 (see Fig. 20). The first significant decreases in the level of R-NE stimulated [^H]-thymidine incorporation were produced by 0.3 pM and 1.0 pM CEC which produced approximately equal amounts of inhibition to 60% of the stimulated response. Further increases in the concentration of CEC resulted in greater blockade of R-NE stimulated DNA synthesis as evidenced by reductions to 12% and -3% of the stimulated response by 10 pM and 30 pM CEC, respectively. The IC^^ value of CEC for the inhibition of R-NE stimulated DNA synthesis was determined to be

2.3 pM (see Table 15). In cells preincubated for 30 min with CEC and not exposed to R-NE, the level of [^H]-thymidine incorporation was not significantly different from control levels for concentrations of CEC up to 30 pM (see Table 11). However, a decrease, which was not statistically significant, in the amount of [^H]-thymidine incorporation to 78% of the control response was produced by 30 pM CEC. The protein content of cells preincubated with concentrations of CEC up to 30 pM were not significantly different from control total cellular protein 95

(Table 12). In cultured hepatocytes preincubated with CEC and then exposed to 10 pVL R-NE, the total cellular protein levels were not significantly different from control levels and were less than that caused by 10 R-NE (131% of control). Preincubation of cells with 1, 10 and 30 }M CEC before exposure to R-NE resulted in reductions in the protein contents to 115%, 103% and 105% of control level, respectively. WB 4101 did not inhibit 10 fjK R-NE induced [^H]-thymidine incorporation at concentrations up to 1 pH. A small reduction in the level of [^H]-thymidine incorporation to 73% of the stimulated response was produced by 10 pH WB 4101 but this was not statistically significant. WB 4101 caused cell detachment and death at a concentration of 100 pH. In cultured primary rat hepatocytes exposed to only WB 4101, the amount of [^H]-thymidine incorporation was equal to that of the control cells (see Table 11). similarly, the amount of total cellular protein in cells treated with 1 pH and 10 pH WB 4101 was slightly elevated (127% and 118% of control levels, respectively) but not significantly different from the control protein content (see Table 12). In addition, 1 and 10 pH WB 4101 did not cause any inhibition (147% and 141% of control levels, respectively) of the increase in total cellular protein caused by 10 pH R-NE (131% of control). In cultured hepatocytes preincubated with SZL-49 for 30 min before administration of 10 pH R-NE, a concentration dependent inhibition of R- NE stimulated [ ] -thymidine incorporation was demonstrated. This inhibition curve lies between those for prazosin and CEC (see Pig. 20). A small, although not statistically significant decrease in the level of [ ] -thymidine incorporation to 76% of the stimulated response was produced by 0.03 pH SZL-49 and significant reductions to 51% and 20% of the 10 pH R-NE response were caused by 0.1 pH and 1.0 pM SZL-49, respectively. Further blockades of R-NE activated [^H]-thymidine incorporation to -3% and 10% of the stimulated response were elicited by 3 pH and 10 pH SZL-49, respectively. The ICj^ value of SZL-49 for the 96 inhibition of 10 pM R-NE stimulated DNA synthesis was 0.24 juM (see Table

15). SZL-49 did not significantly affect either [^H]-thymidine incorporation (see Table 11) or total cellular protein (see Table 12) in cells preincubated with concentrations up to 10 pM of SZL-49.

Pre incubât ion of cells with 30 pM SZL-49 resulted in cell detachment and death. Total cellular protein levels were not significantly different from control levels in cultured hepatocytes first preincubated with 1 pM and 10 pM SZL-49 and then exposed to 10 pM R-NE (117% and 118% of control, respectively), but these differed only slightly from the amount of total cellular protein in R-NE stimulated cells.

4. Determination of the possible role of extracellular Ca*^ and

calmodulin in a^-adrenergic receptor mediated DNA synthesis.

The concentration dependent effects of the Ca*^ channel blocker verapamil and the calmodulin antagonist trifluoperazine (TFP) on 10 pM

R-NE stimulated -thymidine incorporation in primary cultured rat hepatocytes are shown in Fig. 21. TFP, at concentrations of 2 pM and 3 pM, produced significant inhibitions of R-NE stimulated [^H]-thymidine incorporation that were 27% and 9% of the stimulated response, respectively. When cells were exposed to 6 pM and 10 pM verapamil, significant reductions of the level of [^H]-thymidine incorporation to

72% and 45% of the 10 pM R-NE response were produced. In these same experiments, prazosin (1 pM) significantly inhibited -thymidine incorporation by R-NE to a level of -9% of the control response.

The amount of [ ]-thymidine incorporation in cells exposed only to TFP was not altered significantly at concentrations of 1 pM and 2 pM

(see Table 13). However, at 3 pM, there is a moderate degree of inhibition of [^H]-thymidine incorporation (64% of control) by TFP.

Similarly, verapamil at lower concentrations did not significantly affect DNA synthesis but at the highest concentration tolerated by the 97 cultured cells (10 p K ) , a moderate decrease in the amount of [^H]- thymidine incorporation to a level of 70% of control was seen.

As was the situation for the degree of -thymidine incorporation in cells exposed to TFP alone, total cellular protein was significantly decreased in cultured rat hepatocytes by 3 ]M TFP to a level of 64% of the control protein content (see Table 14). Verapamil, at concentrations up to 10 pM, did not significantly alter the total cellular protein in cells exposed to this agent alone. The protein content of R-NE stimulated hepatocytes exposed to 1 pM TFP was 126% of the control level which was similar to the amount of total cellular protein in cells stimulated by 10 pM R-NE in the absence of TFP (134%).

Upon increasing the concentration of TFP to 3 pM, the protein content in these cells which were also exposed to R-NE was reduced to control levels (98%). Verapamil, at concentrations up to 10 pM, did not significantly lower the protein content of cells stimulated with R-NE.

Prazosin (1 pM) lowered the total cellular protein in cells exposed to

10 pM to 111% of the control response.

5. Evaluation of the effects of selected modifiers of DNA and protein

syntheses in primary cultured rat hepatocytes.

The time dependent increases in [^H]-thymidine incorporation in both control and stimulated primary cultured rat hepatocytes co­ incubated with [^H]-thymidine (5 pCi/ml) and [^^S]-methionine (2 pCi/ml) are shown in Fig. 22. After 12 hr of incubation, there is little difference in the amount of [^H]-thymidine incorporation between the stimulated and control cells, although a significant increase to 113% of the control level was caused by 30 pM R-NE. The amount of [^H]- thymidine incorporation continued to increase with time in the control cultured hepatocytes as well as the cells stimulated by 30 pM R-NE and

100 pM DHT up to an incubation period of 48 hr. Significant increases of 173%, 210% and 198% of control amounts of [^H]-thymidine 98

incorporation were produced by 30 pM R-NE after 24, 36 and 48 hr of incubation, respectively, similarly, ICO pM DHT evoked significant increases in [^H]-thymidine incorporation at these same time points that were 149%, 204% and 179% of the control response, respectively. After 72 hr, the amount of [^H]-thymidine incorporation decreases in relation to the 48 hr level for the control and treated hepatocytes. However, both R-NE and DHT continued to significantly increase the levels of [^H]-thymidine incorporation after this incubation period with responses that were 235% and 178% of control, respectively. The time dependent increases in [^^S]-methionine incorporation in control and in 30 pM R-NE and 100 pM DHT treated primary cultured rat hepatocytes co-incubated with [^H]-thymidine and [^^S]-methionine followed a similar pattern as that demonstrated for the incorporation of [^H]-thymidine (see Fig. 23). A more discernible increase relative to control cells in [^^S]-methionine incorporation by R-NE and DHT after a 12 hr incubation period was seen in comparison to the increase in [^H]- thymidine incorporation produced at 12 hr. Significant increases of 122% and 121% of the control levels were caused by R-NE and DHT, respectively, at this time point. Further increases in [ ] -methionine incorporation were seen at the next three time intervals (24, 36 and 48 hr) for the control and stimulated cells. Stimulation of the cultured hepatocytes with 30 pM R-NE elicited significant increases in [^^S]- methionine incorporation that were 142%, 138% and 142% of the control level at 24, 36 and 48 hr, respectively. Likewise, 100 pM DHT produced significant increases that were 134%, 136% and 146% of the control response at 24, 36 and 48 hr, respectively. As was the situation with [^H]-thymidine incorporation, [^^S]-methionine incorporation declined after 48 hr for control and stimulated cells. After a 72 hr incubation period, the R-NE stimulated cultured hepatocytes significantly incorporated [^^S]-methionine at a level that was 174% of the control 99

response and DHT produced a significant increase that was 168% of the

control level.

The effect of incubation time on total cellular protein in both

control and stimulated primary cultured hepatocytes used for the

investigation of time dependent DNA and protein synthesis is shown in

Fig. 24. Protein content in control cells declined steadily with

increasing time. Stimulation of the hepatocytes with 30 pM R-NE

maintained the total cellular protein content at a fairly constant level

of approximately 90 pg protein/dish until 48 hr at which time the

protein content declines. Significant increases in total cellular

protein of 132%, 116%, 127% and 139% of control were produced by 30 pM

R-NE at 24, 36, 48 and 72 hr, respectively. Treatment of the cells with

100 DHT also increased the protein content of the cultured cells with

respect to control. The amount of total cellular protein in DHT

stimulated cells declined in an almost parallel manner with the control cells after 24 hr of incubation. Like R-NE, DHT also caused significant

increases in protein content after 24, 36, 48 and 72 hr incubation periods that were 122%, 127%, 112% and 150% of the control response.

The effects of various modifiers of DNA synthesis stimulated by 10 jljM R-NE and 100 piU DHT in 48 hr primary cultured rat hepatocytes co­ incubated with [^H]-thymidine and [ ]-methionine are shown in Fig. 25.

The amount of [^H]-thymidine incorporation in 10 pM R-NE treated cells was significantly reduced to 3% and 12% of the stimulated response by 1 piK prazosin and 10 pM CEC. These reductions were significantly less than those produced by 1 pM SZL-49, 30 piti cirazoline and 1 pM yohimbine which inhibited the R-NE response to 35%, 48% and 56%, respectively, of

the response produced by 10 piK R-NE alone. Idazoxan (10 ;^M) did not

significantly inhibit the response to R-NE. In cells exposed to 100 pM

DHT, the stimulatory effect of DHT on [^H]-thymidine incorporation was

completely reduced by 1 piM. prazosin to control levels (0% of the 100 pM

DHT response). This total blockade was significantly less than the 100

inhibitions caused by 10 pM CEC, 1 SZL-49 and 30 yK cirazoline which reduced the amount of [^H]-thymidine incorporation to 30%, 32% and 38%

of the DHT stimulated response. Yohimbine (1 yH ) and idazoxan (10 yJA)

did not significantly inhibit DNA synthesis caused by 100 yU DHT although yohimbine did reduce the response somewhat to 78% of the [^H]- thymidine incorporation caused by DHT. The effects of the modifiers described above on protein synthesis stimulated by R-NE and DHT were also examined in 48 hr primary cultured rat hepatocytes co-incubated with [ ^H]-thymidine and [ ]-methionine

(see Fig. 26). Prazosin (1 yH ) and CEC (10 y K ) significantly inhibited [ ]-methionine incorporation stimulated by R-NE to levels of 14% and

21%, respectively, of the 10 y K R-NE response. Although the decreases were not significant, 1 yohimbine, 30 yH cirazoline, 10 }M SZL-49 and 10 }M idazoxan did produce small reductions in the amount of [^^S]- methionine incorporation to 61%, 66%, 80% and 80% of the 10 fiM R-NE response, respectively. Similarly, only 1 yK prazosin and 10 jiM CEC caused significant inhibitions of DHT stimulated [ ^^S]-methionine

incorporation to 5% and 47% of the 100 y K DHT response. In the concentrations tested, idazoxan, yohimbine, cirazoline and SZL-49 did not cause significant inhibitions of DHT stimulated [^^S]-methionine

incorporation. However, 1 y K SZL-49, 30 y K cirazoline and 1 y K yohimbine did produce small decreases in [^^S]-methionine incorporation to 64%, 79% and 91% of the DHT response. None of these treatments produced a significant change on total cellular protein (see Table 16).

D. Discussion 1. Effects of 4'—benzyl substitution of selected imidazoline compounds. The primary objective for the development of ABI and IBI was to evaluate whether 4'-benzyl substitution with amino or isothiocyanato groups on tolazoline has an effect on the a^-adrenergic receptor activities of these compounds, and to determine whether IBI is an 101

appropriate affinity probe for a-adrenergic and/or imidazoline preferring receptors. ABI possesses a^-adrenergic receptor agonist activity in rat thoracic aorta (Shams et al., 1990) and IBI produces contraction in the same tissue that is nonadrenergic and dependent upon Ca*^ (Shams et al., 1990; Venkataraman et al., 1989). Therefore, ABI, IBI and the parent compound tolazoline were used to evaluate the effects of selected substituents at the 4 '-benzyl position on a^-adrenergic receptor mediated DNA synthesis. All three compounds failed to stimulate DNA synthesis in primary cultured rat hepatocytes. The lack of activity of tolazoline, a noncatechol imidazoline, is in marked contrast to the activity demonstrated by its catechol-containing derivative, DHT (see Chapter II). This emphasizes the importance of the catechol moiety to the ability of these compounds to stimulate DNA synthesis. These results are in agreement with those of Ruffolo and Waddell (1983) who demonstrated that the a^-adrenergic receptor activity of imidazolines declined with the removal of the phenolic hydroxyl groups, an effect due primarily to a decrease in the efficacies of these compounds. Further discussion of the effects of tolazoline will be presented in the next section (see Section III.D.2). The addition of an amino group at the 4 ’-benzyl position of tolazoline did not increase the ability of resultant compound (ABI) to stimulate [ ]-thymidine incorporation into the DNA of the cultured cells. The absence of a catechol moiety on this molecule, as is the situation with tolazoline, may contribute to the lack of agonist activity in this system. In the rat thoracic aorta a^-adrenergic receptor system, the addition of the amino group at the para-position of the phenyl ring of tolazoline did not appreciably change the EC^g value of ABI [0.2 pM] (Sengupta et al., 1987) from the parent compound [EC^g = 0.1 pM] (Shams et al., 1990). Additionally, in the human platelet a^- adrenergic receptor system, both ABI and tolazoline possess approximately equal ability to antagonize the aggregatory effects of 102 epinephrine. Therefore, it is not surprising that ABI does not stimulate DNA synthesis, as is the case with tolazoline. However, ABI significantly decreased the amount of [^H]-thymidine incorporation produced by R-NE, although notas potently as prazosin, indicating that ABI can recognize the a^-adrenergic receptor that mediates DNA synthesis stimulated by R-NE. This is not unexpected, given the fact that tolazoline is a partial agonist (Sanders et al., 1975) with low efficacy and most likely ABI behaves in the same manner (Shams et al., 1990). The finding that ABI effectively lowered the amount of total cellular protein in cells treated with R-NE whereas tolazoline does not is difficult to interpret in light of their similar effects on DNA synthesis, but this finding again indicates that there may be a dissociation of the ability of these compounds to affect DNA synthesis and total cellular protein, an effect that may be nonspecific or mediated through a different receptor type. Additionally, 30 pM ABI alone caused an approximate 20% decrease in total cellular protein from control levels which indicates that at this concentration ABI may suppress protein accumulation or may be slightly toxic to the cells. In view of this, a more complete concentration response study of the effects of ABI on R-NE and DHT stimulated DNA and protein syntheses should be performed. An isothiocyanate moiety was originally placed at the 4 '-benzyl position of tolazoline in an attempt to synthesize a photoaffinity probe for a-adrenergic receptors. However, this compound (IBI) demonstrates a lack of specificity for the a^-adrenergic binding site in human platelets where it also binds to other aggregation inhibitory sites (Shams et al., 1990). Additionally, IBI stimulates contraction of rat thoracic aorta by a nonadrenergic, Ca*^-dependent mechanism. Based upon the unique characteristics of this compound, IBI was evaluated for the ability to stimulate the incorporation of [^H]-thymidine into the DNA of 103 primary cultured rat hepatocytes in an attempt to determine if DNA synthesis can also be stimulated by a nonadrenergic mechanism.

IBI did not stimulate DNA synthesis in this cell system at concentrations up to 10 pM. At concentrations of 20 pM and greater, IBI was toxic to the cells causing cell detachment and death. Similarly,

IBI had no effect on total cellular protein in concentrations up to 10 pM. R-NE stimulated DNA synthesis was not antagonized in cells preincubated with IBI for 30 min prior to R-NE exposure. The rationale for the 30 rain preincubation of IBI as opposed to a 48 hr exposure to the cells was two-fold; 1) IBI has a long duration of action which may be attributable to the isothiocyanate substitution (Venkataraman et al.,

1989) and 2) to decrease cell toxicity since even preincubation for only

30 min with concentrations of IBI greater than 10 pM were toxic to the cells. From these results, it appears that IBI neither stimulates nor inhibits DNA synthesis in cultured rat hepatocytes in concentrations that are tolerable to the cells.

2. Effects of tolazoline, cirazoline and idazoxan on DNA synthesis.

Imidazoline agonists have been used to obtain evidence for the existence of recognition sites on the a^-adrenergic receptor that differ from those for phenethylamines or for the existence of discrete subtypes of this receptor. The first clues that there may exist different subtypes of the a^-adrenergic receptor were provided by Ruffolo and coworkers (1977) who demonstrated that there was a lack of cross­ desensitization between the agonist activities of phenethylamines and imidazolines in the rat vas deferens a^-adrenergic receptor system.

This evidence suggests that these two classes of compounds may be interacting with a different site on the same receptor or with two different types of a^-adrenergic receptors. As part of his argument for dividing a^-adrenergic receptors into two subtypes, a^- and ot^-. 104

McGrath discussed the differential effects of imidazolines and phenethylamines (McGrath, 1982). In addition to agonist activity at the a^-adrenergic receptor, imidazolines also demonstrate activity at a newly described receptor site termed the imidazoline preferring receptor (or the imidazoline/guanidinium receptor site). This receptor resembles an a^- adrenergic receptor, but is virtually insensitive to catecholamines and selectively binds imidazolines such as idazoxan and cirazoline (Lehmann et al., 1989; Bricca et al., 1989). Most of the studies of this receptor to date have been in vitro physiological studies and radioligand binding determinations. Tolazoline is a well known partial agonist in the a-adrenergic receptor system (Sanders et al., 1975) that is a member of the imidazoline class. It served as the parent compound for the development of ABI and IBI discussed in the previous section. Cirazoline is also an imidazoline with a unique structure in that at an oxygen atom is inserted into the bridge between the phenyl and imidazoline rings. Additionally, this compound contains no phenolic hydroxyl groups but instead possesses an ortho-cyclopropyl group on the phenyl ring. Despite these differences from the more classical imidazolines, cirazoline is a potent full agonist at the a^-adrenergic receptor (Van Meel et al., 1981; Ruffolo and Waddell, 1982). Ruffolo and Waddell (1982) demonstrated that the affinity and efficacy at the a-adrenergic receptors of guinea pig aorta were almost identical for cirazoline and R-NE. They also showed that cirazoline potently and competitively antagonized the a^-adrenergic receptor mediated inhibition of the twitch response in field stimulated guinea pig ileum by R-NE. In addition, cirazoline has been shown to be one of the most potent ligands known for the imidazoline preferring receptor (Parini et al., 1989). This combination of effects of cirazoline make it an interesting compound for 1 0 5 the further investigation of DNA synthesis stimulated by R-NE and the catecholimidazoline DHT.

Although the effects of tolazoline were presented in the preceding section, they are also included here for a comparison with the effects of cirazoline and because they were evaluated in conjunction with cirazoline in cultured hepatocytes obtained from the same isolation.

Neither tolazoline (as demonstrated in the preceding section) nor cirazoline stimulated DNA synthesis in primary cultured rat hepatocytes.

Since tolazoline is a partial agonist with very low efficacy in rat aorta strips (Ruffolo et al., 1979), little or no stin.ulation of DNA synthesis by tolazoline was anticipated. However, cirazoline also did not stimulate DNA synthesis in primary cultured rat hepatocytes despite the fact that this compound is a potent a^-adrenergic receptor agonist in other tissues. These results again underscore the importance of the catechol moiety to the agonist activity of imidazoline compounds in this system. Morgan and coworkers (1983) obtained qualitatively similar results in isolated hepatocytes when it was demonstrated that only hydroxylated phenethylamines caused a Ca*^ mediated activation of phosphorylase but other nonhydroxylated a^-adrenergic receptor agonists such as methoxamine and oxymetazoline did not produce this response.

Thus, these results suggest that the hepatic c^-adrenergic receptor may recognize agonists in a different manner than c^-adrenergic receptors in other tissues and thus have a more stringent requirement for certain structural elements such as the phenolic hydroxyl groups that must be met before the receptor is activated, the signal transduced and DNA synthesis increased.

Both tolazoline and cirazoline produced concentration dependent inhibitions of R-NE stimulated DNA synthesis in primary cultured rat hepatocytes. Cirazoline (IC^^ = 16.7 pM) was more potent than tolazoline (IC^^ = 45.-4 pM) in this inhibition although neither are as potent as prazosin which abolished the R-NE response at a concentration 106

of 1 fjM. Since the response of R-NS has been characterized as an a^- adrenergic receptor mediated phenomenon, the ability of both tolazoline and cirazoline to inhibit R-NE stimulated DNA synthesis indicates that both compounds are able to interact with but cannot activate the a^- adrenergic receptor and thus are antagonists in this system. Cirazoline concentration dependently inhibited DNA synthesis stimulated by DHT in primary cultured rat hepatocytes with an value of 16.5 pM, which is the same for that determined against R-NE stimulated cells. This provides further evidence that DHT is producing its effects through activation of the a^-adrenergic receptor since cirazoline blocks the effects of R-NE and DHT in an equal fashion. Tolazoline failed to block DHT-stimulated DNA synthesis at concentrations up to 100 pM. This may be the result of a combination of the low activity of tolazoline and the high concentration of DHT (100 pM) which may make it difficult for tolazoline to displace DHT from the receptor site. Prazosin was much more potent than cirazoline at blocking the stimulation of DNA synthesis caused by DHT, adding additional evidence that the effects of DHT are mediated through the o^- adrenergic receptor and not through a nonadrenergic modulated mechanism. Neither cirazoline nor tolazoline effectively blocked the increase in total cellular protein caused by both R-NE and DHT, whereas prazosin did. It is difficult to make definitive interpretations of these data because of the variability in responses seen between groups of experiments. However, in this case, the lack of effects of cirazoline and tolazoline on levels of total cellular protein in R-NE and DHT stimulated cells correlates to some extent with their relatively low (with respect to prazosin) inhibitory activity on R-NE and DHT- stimulated DNA synthesis. To further test the for the possible existence of a nonadrenergic mechanism involved in the stimulation of DNA synthesis by DHT, the effect of idazoxan on R-NE and DHT stimulated DNA synthesis was 107 investigated. Idazoxan is a benzodioxan which possesses potent a^- adrenergic antagonist activity (Clark et al., 1986). In addition, this compound is often used as a radiolabelled ligand to describe the imidazoline preferring receptor (Michel and Insel, 1989; Parini et al., 1989). In cells incubated with idazoxan alone, the level of [^H]- thymidine incorporation did not change significantly from control in concentrations up to 30 pM. This indicates that the effects due to idazoxan over the range of the concentrations used are not the result of a change in the basal level of DNA synthesis. Idazoxan is a weak antagonist of R-NE and DHT stimulated DNA synthesis. This compound did not inhibit either R-NE or DHT stimulated DNA synthesis until a concentration of 30 pM was reached. At this high concentration, the selectivity of idazoxan for either the imidazoline preferring receptor or the Cj-adrenergic receptor is probably lost since the pA^ value for idazoxan at the a^-adrenergic receptor (as determined by inhibition of R-NE induced contraction of rat anococcygeus muscle) is 6.3 (Clark et al., 1986). In addition, idazoxan may be metabolized by hepatocytes since WB 4101, which contains a similar benzodioxan structure, is rapidly metabolized in hepatocytes (Han et al., 1990). Additional studies would have to be conducted to verify this suggestion. Idazoxan also did not block the increase in total cellular protein caused by both R-NE and DHT which in agreement with the effects of idazoxan on DNA synthesis.

3. Effects of selective a^-adrenergic receptor subtype antagonists on R- NE stimulated DNA synthesis. There is much strong evidence for the existence of two subtypes («^g and of the a^-adrenergic receptor (Minneman, 1988). The hepatic a^-adrenergic receptor population has been described as being composed almost entirely of the a^^-subtype (Han et al., 1987a). The 108 availability of selective antagonists for these a^-adrenergic receptor subtypes allowed for the evaluation of the involvement of these receptor subtypes in hepatic a^-adrenergic receptor mediated DNA synthesis. Concentration dependent inhibitions of DNA synthesis were produced by each of the antagonists tested with the exception of WB 4101. In addition, none of the compounds when incubated alone with primary cultured hepatocytes in concentrations used to inhibit R-NE stimulated DNA synthesis caused significant changes from control levels in either the amount of -thymidine incorporation or in the amount of total cellular protein in the cells. This indicates that the effects produced by these antagonists in R-NE treated cells were the result of antagonism of the R-NE response and not a nonselective effect. Prazosin, the prototype a^-adrenergic receptor antagonist, was the most potent inhibitor (10^^ = 8.7 nM) of R-NE stimulated DNA synthesis in this selected series of antagonists, again emphasizing the role of the a^-adrenergic receptor in the modulation of DNA synthesis. WB 4101, a selective a^^-adrenergic receptor competitive antagonist, did not significantly inhibit R-NE stimulated DNA synthesis in concentrations up to 10 pK. Concentrations above this caused cell detachment and death. These experiments were conducted before it was shown that WB 4101 is rapidly metabolized by liver cells (Minneman, personal communication; Han et al., 1990). With just a 30 min exposure to isolated hepatocytes, over 99% of WB 4101 was destroyed as quantitated by radioreceptor assay (Han et al., 1990). Therefore, the inability of WB 4101 to inhibit R-NE stimulated DNA synthesis may be expected. Since WB 4101 is rapidly metabolized by hepatocytes, another selective a^^-adrenergic receptor antagonist was needed in order to determine if this receptor subtype is involved in the mediation of DNA synthesis. SZL-49 was chosen since this compound has been shown to possess the ability to alkylate an u^-adrenergic receptor subtype with 1 0 9

high affinity for WB 4101 and phenylephrine (Piascik, et al., 1990) which correlates well with the a^^-subtype described by the Minneman group (Han et al., 1987a,b; Minneman, 1988).

SZL-49 produced a concentration-dependent inhibition of R-NE

stimulated DNA synthesis in primary cultured rat hepatocytes preincubated with this agent 30 min before exposure to R-NE. This

selective a^^-receptor subtype antagonist was less effective than prazosin at inhibiting the DNA synthesis stimulated by R-NE (ICg^ = 0.24 pM) but more effective than CEC by approximately one order of magnitude.

SZL-49 also caused a small decrease in the amount of total cellular protein in cells treated with R-NE.

CEC, a selective a.^^adrenergic receptor alkylating agent, caused a concentration dependent inhibition of DNA synthesis in R-NE treated cells preincubated with this compound for 30 min prior to R-NE exposure.

CEC also blocked the increase in total cellular protein content produced b y R-NE. CEC was less potent (ICg^ = 2.3 pM) as an antagonist of oc^- adrenergic receptor mediated DNA synthesis than either prazosin or SZL-

49. The fact that CEC is capable of inhibiting DNA synthesis caused by the activation of the a^-adrenergic receptor by R-NE was expected since it has been shown that the liver contains predominately the CEC- sensitive a^^-adrenergic receptor subtype (Han et al., 1987a, Han et al., 1990). However, it is surprising that SZL-49 is apparently more potent (when IC^^ values are compared) than CEC since the number of a^^- receptor subtypes in liver as calculated by radioligand binding assays is much less than those for the a^^-subtype. Several possible explanations for this exist: 1) SZL-49 is no longer selective for just the oc^^-subtype at the concentrations which begin to inhibit R-NE stimulated DNA synthesis (1 x 10 ^ M) and thus inhibits the adrenergic receptor subtype as well. SZL-49 causes the complete displacement of [^H]-prazosin binding from both high and low affinity receptor sites for [^H]-prazosin at concentrations of 1 x 10’^ M 110

(Piascik et al., 1988), but at concentrations up to 1 jM, still leaves a population of a^-adrenergic receptors in in vitro tissue preparations that are not alkylated (Piascik et al.,1990). Therefore, additional work including the use of radioligand binding assays would be needed in order to determine the amount of inactivation of the hepatic adrenergic receptor subtypes by SZL-49. 2) Piascik and coworkers (1990) showed that CEC was less effective than SZL-49 at inhibiting the actions of phenethylamines both in vivo for the inhibition of the pressor response of phenylephrine and in vitro for the blockade of inositol phosphate formation by R-NE. The ability of SZL-49 to more effectively inhibit R-NE stimulated DNA synthesis is in agreement with these results. 3) A small population of a^^-adrenergic receptors which may be present in the liver are involved with the regulation of DNA synthesis, and are coupled to the response in such a manner that these relatively few subtypes (in comparison to the number of a^^^-subtypes) can mediate the phenomenon of a^-adrenergic receptor mediated DNA synthesis in conjunction with the subtype. The hypothesis that both adrenergic receptor subtypes may be involved in the modulation of DNA synthesis is supported by the findings that there exists two signalling pathways for hepatic c^-adrenergic receptor mediated gluconeogenesis which differ in their dependence upon extracellular Ca*^ (Garcia-Sainz and Hernandez-Sotomayor, 1985). The two receptor subtypes have been shown to increase intracellular Ca*^ levels by different mechanisms (Tsujimoto et al., 1989). Activation of the subtype causes influx of extracellular Ca*^ and c^^-subtype stimulation produces mobilization of intracellular Ca*^ stores in cultured rat thoracic aorta smooth muscle cells. Therefore, it is conceivable that the and subtypes may modulate the extracellular Ca*^-dependent and -independent transductory pathways, respectively, in the hepatocyte. Additional biochemical studies will be needed in order to delineate the exact role 11 1 that each subtype plays in the phenomenon of a^-adrenergic receptor mediated DNA synthesis.

4. Effects of verapamil and TFP on c^-adrenergic receptor mediated DNA

synthesis.

Increases in cytosolic Ca*^ levels are one of many events that occur in cells following activation of the EGF and a^-adrenergic receptor with their respective hormones. The activation of both the EGF receptor as well as the o^-adrenergic receptor results in the activation of PLC and the breakdown of PIPg to Ins(l,4,5)P^ and DAG (Chen et al.,

1987; Pandiella et al., 1988,1989; Creba et al., 1983; Exton, 1988).

Intracellularir Ca*^ stores are then mobilized by Ins(l,4,5)P2 to elevate cytosolic Ca*^ levels (Charest et al., 1985) but in order to maintain these elevated Ca*^ levels over an extended period of time, changes in

Ca*^ fluxes across the plasma membrane must occur (Exton, 1988). Little is known about the mechanisms which control this channel. Thus, the effects of verapamil, a Ca*^ channel blocker, on DNA synthesis that is stimulated by EGF and modulated by the a^-adrenergic receptor was evaluated. In addition, calmodulin, a Ca^^ receptor protein, appears to modulate a variety of Ca*^ dependent processes such as contraction, secretion and nerve function (Klee et al., 1980) and may also be involved in the mediation of cellular proliferation (Whitfield et al.,

1979). Studies by Alexander et al. (1988) have shown that the TFP, a calmodulin antagonist, inhibits polyamine biosynthesis and liver regeneration. In addition, Pujol and coworkers (1989) demonstrated that calmodulin levels are increased in the cytosol during the prereplicative phase of rat hepatocyte proliferation following partial hepatectomy.

Following this accumulation, the calmodulin translocated to the nuclei where it associated with the nuclear matrix. The translocation but not the increase in calmodulin was blocked by the administration of prazosin, suggesting that the release of Ca*^ from the endoplasmic 112 reticulum stores may mediate calmodulin association with the nuclear matrix. Because of the involvement of Ca*^ and calmodulin in both EGF and a^-adrenergic receptor mediated events, the effects of inhibitors of

Ca*^ influx and calmodulin activity on a^-adrenergic receptor mediated

DNA synthesis were evaluated.

Verapamil, when exposed alone to cultured hepatocytes in concentrations up to 1 pM, did not significantly affect the amount of

[^H]-thymidine incorporation but at 10 pM did lower the amount of [^H]- thymidine incorporation to 70% of the control value, suggesting that perhaps the basal level of DNA synthesis produced by EGF in these control cells has been reduced. In R-NE treated cells, 6 and 10 pM verapamil significantly reduced the level of DNA synthesis, suggesting that blockade of Ca*^ channels with verapamil may interfere with DNA synthesis in R-NE stimulated cells. However, these results should be interpreted with caution for a number of reasons including; 1) The range of activity for verapamil is very narrow with cellular toxicity seen at concentrations greater than 10 ]M. 2) The small amount of inhibition of

[^H]-thymidine incorporation in cells exposed only to 10 pM verapamil suggests that verapamil may be interfering with the control level of DNA synthesis produced by EGF. 3) Verapamil has been shown to possess a- adrenergic receptor antagonist activity so that the effect of verapamil may be due to the displacement of R-NE from the c^-adrenergic receptor as opposed to blockade of Ca*^ channels. Therefore, although verapamil interferes to some extent with a^-adrenergic receptor mediated DNA synthesis, the exact mechanism by which the compound is acting is far from clear, and additional research is necessary in order to determine the role of extracellular Ca*^ in growth responses of hepatocytes.

Like verapamil, 3 pM TFP decreased [^H]-thymidine incorporation in cells incubated alone with this compound, suggesting that TFP reduces the level of DNA synthesis produced by EGF in these control cells. R-NE stimulated DNA synthesis is significantly reduced in cells exposed to 2 113 and 3 }Jîi TFP. These results suggest that calmodulin may play a role in a^-adrenergic receptor mediated DNA synthesis but as was the case with verapamil, the interpretation of these results requires much caution for the following reasons: 1) In this system, TFP demonstrated an even narrower range of activity than verapamil with toxic effects consistently seen at concentrations greater than 3 fuM, This indicates that the concentrations used are the maximal levels tolerated by the hepatocytes and thus more nonspecific effects unrelated to «^-adrenergic receptor mediated mechanisms (such as inhibition of the effects of EGF) may be occurring as suggested by the results obtained from cells treated with 3 yiK TFP alone. 2) TFP has been demonstrated to possess «- adrenergic receptor blocking activity so that the results seen in the presence of R-NE may be due in part to antagonism of R-NB at its receptor site. Therefore, although it appears that TFP can inhibit DNA synthesis mediated by the «^-adrenergic receptor, an exact interpretation of the mechanism by which TFP elicits its effects cannot be made from these experiments.

5. Effects of selected modifiers of «^-adrenergic mediated DNA and protein syntheses. It has been shown that while the number of hepatocytes is restored to normal fairly quickly during liver regeneration, the restoration of cell mass requires a longer amount of time, up to 8 to 10 days (Gehard, 1975). Therefore, to gain a better understanding of the manner in which protein and DNA syntheses are modulated by the hepatic «^-adrenergic receptor, time courses for the stimulation of both processes were performed. In addition, selected modifiers of «-adrenergic receptor responses were evaluated for their effects on DNA and protein syntheses. The stimulation of both DNA and protein syntheses by R-NE and DHT followed approximately the same time course. Maximal incorporation of both [ ]-thymidine and [ ]-methionine was seen at 48 hr for both R-NE 114 and DHT. At 12 hr, the two agonists may stimulate slightly more protein synthesis in comparison to DNA synthesis, which may indicate a gearing up of the cells for DNA synthesis, but no definitive conclusions about this aspect of cell growth can be made from these studies. At 72 hr, there is a large decline in the level of both [ ]-thymidine and [^^S]- methionine incorporation, which most probably indicates an exhaustion of the nutrients in the media (which was incubated unchanged with the cells for 72 hr) and a degeneration of the cells. At 24, 36, 48 and 72 hr, both R-NE and DHT produced significantly greater levels of DNA and protein syntheses than the corresponding control values. Therefore, from these results it appears that similar time courses for the stimulation of both protein and DNA syntheses are elicited by R-NE and DHT. Total cellular protein declined with time for both control and R-NE and DHT treated cells. However, at 24 hr and beyond, the stimulated cells maintained a higher level of total cellular protein in comparison to the respective controls, suggesting that these agonists stimulate the growth of the cells or perhaps better maintain the status of the cells over the course of the incubation. The effects of selected «-adrenergic inhibitors (cirazoline behaves as an antagonist in this system) were examined in R-NE eind DHT stimulated cells co-incubated with [^H]-thymidine and [ ]-methionine so that the effects of these agents could be examined in the same hepatocytes. Prazosin (1 pM) produced the greatest inhibition of both DNA and protein syntheses stimulated by R-NE and DHT than any other the other selected treatments. CEC (10 pM) also produced significant inhibition of both types of growth. SZL-49 (1 pM) and cirazoline (30 pM) produced significant inhibitions of DNA synthesis stimulated by R-NE and DHT but failed to significantly inhibit protein synthesis. Perhaps a higher concentration of SZL-49 should have been employed in order to determine whether this compound can antagonize protein synthesis. Yohimbine (1 pM) produced a significant reduction, although not as great 115 as prazosin, of R-NE stimulated DNA synthesis; a result similar to that described by Takai et al. (1988) who demonstrated that 1 jjM yohimbine caused a significant decrease in the level of DNA synthesis stimulated by epinephrine. Yohimbine failed to significantly lower the level of DNA synthesis in DHT treated cells nor did it significantly block protein synthesis stimulated by both R-NE and DHT. Idazoxan failed to inhibit both DNA and protein syntheses stimulated by the two agonists. Since these experiments were performed with single concentrations, it is difficult to compare the results. However, based upon the time course data and the inhibitory effects of the a^-adrenergic receptor antagonist prazosin and the a^^-adrenergic receptor alkylating agent CEC, it appears that the syntheses of DNA and protein are associated with each other through the activation of the hepatic a^-adrenergic receptor. 116

IQLAZOLZKB (TLZ) 2-{4-AMIHOBKNZYL) IMIDAZOLIHE (ABI)

SCN

2- ( 4-ISOiaiOCyaNaTOBENZYL) IMIDaZOLIRE (XBX)

.0 NH NH

0

CIRAZOLIRE (CRZ) XOAZOZAH (H>Z)

Figure 12. Chemical structures of tolazoline, 2-(4-aminobenzyl)- imidazoline, 2-(4-isothiocyanatobenzyl imidazoline, cirazoline and idazoxan. 117

CHLO CH,0

NH

NH; OCH3

PRAZOSIN WB 4101

CH.O H,C — N, NH NH

NH;

CHLQROBTHYIiCLCRIIDINB (CEC) SZL-49

Figure 13. Chemical structures of prazosin, WB 4101, chloroethylclonidine and SZL-49. 1 1 8

100 LU o DHT LU OC C/D Z 3 80 - - • TOLAZOLINE o û_ A ABl C/D LU ▲ ------▲ IBI q : 0_ s 4 0 - X <

o 2 0 - z rO LU LU ü Z O' 0 - LU D_ -20 - 7 -6 - 5 - 4 - 3

LOG DRUG CONCENTRATION (M )

Figure 14. Concentration response curves for the stimulation of [ H]- thymidine incorporation into DNA of primary cultured rat hepatocytes by tolazoline, 2-(4-aminobenzyl) imidazoline (ABI), 2-(4-isothiocyanato- benzyl)imidazoline (IBI) auid 2-(3,4-dihydroxybenzyl)imidazoline (DHT). Results are expressed as a percentage of the maximum response produced b y 30 }M R-NE. Each point represents the mean ± S.E.M. of determinations from 3-12 dishes. Points with letters indicate levels of [ H]-thymidine incorporation that were significantly greater them control and points with different letters indicate degrees of DNA synthesis which were significauitly different from each other (p < 0.05, Student-Newman-Keuls test). In the experiments in which the effects of tolazoline, ABI and IBI were evaluated, control DNA synthesis was 867150 ± 118726 DPM/culture (mean ± S.E.M.) and cells treated with 30 j M R-NE stimulated [ E]-thymidine incorporation at a level that was 204.1 ± 7.8% (mean ± S.E.M.) of control. 119

1 2 0 t W 00 HD COMPOUND + R-NE (10 /iM) z o 100 " m COMPOUND + DHT (100 ptM) û. c/7 üJ Z 80 " û LU 60 *■ S Z) 4 0 " co 2 0 " LU *** *** O q : LU o_ a -20 PRZ TL2 ABl IBI 1 /M 100 uM 30 jCiM 10 fM

Figure 15. Effects of 1 f/M prazosin (PRZ), 100 tolazoline, 30 fuM 2- (4-aminobenzyl)imidazoline (ABI) and 10 ]jM 2-(4-isothiocyanatobenzyl)- imidazoline (IBI) on [ H]-thymidine incorporation stimulated by 10 f M R- norepinephrine (R-NE) and 100 f M 2-(3,4-dihydroxybenzyl)imidazoline (DHT), in primary cultured rat hepatocytes. Results are expressed as a percentage of the response produced by 10 f M R-NE or 100 f M DHT and each point represents the mean ± S.E.M. of determinations from 4-9 dishes. Asterisks (*) indicate that the mean of the stimulated + treated dishes is significantly different (p < 0.05, Student's unpaired t-test) from the mean of the stimulated only dishes. Control DNA synthesis was 864876 ± 110065 pPM/culture and cells treated with 10 f M R-NE and 100 fM DHT stimulated [ H]-thymidine incorporation at a level that was 205.5 ± 11.0% and 207.6 ± 10.5% of control, respectively. 120

Table 7. Effects of tolazoline, 2-(4-aminobenzyl)imidazoline (ABI) and 2-(4-isothiocyanatobenzyl)imidazoline (IBI) on total cellular protein in 48 hr primary cultured rat hepatocytes.

TREATMENTCONC. CELLULAR PROTEIN® (jiM) PERCENT CONTROL (%)

ABI 30 83.7 ± 3.7 100 81.2 ± 4.1 IBI 1 92.3 ± 2.3 3 99.1 ± 6.7 10 90.3 ± 5.0 TOLAZOLINE 30 95.6 ± 5.4 100 100.1 ± 5.0 R-NE 10 118.0 ± 5.8. 30 128.7 ± 7.8^ DHT 100 116.5 ± 14.9 R-NE (10 pM) + ABI 30 92.6 ± 10.7 IBI 10 118.4 ± 2.2^ TOLAZOLINE 100 150.4 ± 7.4” PRZ , 1 89.5 ± 4.7

DHT (100 }jK) +

TOLAZOLINE 100 135.0 ± 8.4” PRZ 1 93.3 ± 6.4 Values are expressed as mean ± S.E.M. of the percentage of control total cellular protein (72.0 ± 5.0 jug protein/dish); n = 4-25 dishes. ^ Means are significantly greater than control (p < 0.05, Student- Newman-Keuls test). 121

1 0 0 O DHT N I i i 80--- 9 -----• TOLAZOLINE a 0- =) A — A CIRAZOUNE T ^ 50-- O iî !î ÜJ o LU c: —— Lü ü_ DC -20 ______1------:------!------— 1------—8 —7 —6 —5 —4 - 3 LOG DRUG CONCENTRATION (M)

Figure 16. Concentration response curves for the stimulation of [ H]- thymidine incorporation into DNA of primary cultured rat hepatocytes by tolazoline and cirazoline and the response to 100 fOi 2-(3,4-dihydroxy­ benzyl ) imidazoline (DHT). Results are expressed as a percentage of the maximum response produced by 30 ;uM R-norepinephrine (R-NE). Each point represents the mean ± S.E.M. of determinations from 3-21 dishes. Points with letters indicate levels of [ H]-thymidine incorporation that were significantly greater than control and points with different letters indicate degrees of DNA synthesis which were significantly different from each other (p < 0.05, Student-Newman-Keuls test). Control DNA synthesis was 727577 ± 140089 DPM/culture (mean ± S.E.M.) and cells treated with 10 pM R-NE stimulated [ H]-thymidine incorporation at a level that was 245.9 ± 19.8% (mean ± S.E.M.) of control. 122

Lü if) o CIRAZOUNE Z O • TOLAZOLINE CL E U PRAZOSIN ÛC Lü Z 60--

o a,b

L i _ O t— z 2 0 - Lü O g CL - 7 — 6 - 5 - 4 - 3

LOG DRUG CONCENTRATION (M)

Figure 17. Concentration dependent inhibition of 10 pM R-norepinephrine (R-NE) stimulated [ H]-thymidine incorporation by cirazoline and tolazoline in primary cultured rat hepatocytes. Results are expressed as a percentage of the response produced by 10 pM R-NE and each point represents the mean ± S.E.M. of determinations from 3-6 dishes. Points with letters indicate levels of [ H]-thymidine incorporation that were significantly less than that produced by 10 pM R-NE and points with different letters indicate degrees of DNA synthesis which were significantly different from each other (p < 0.05, Student-Newman-Keuls test). The inhibitory effect of 1 prazosin (PRZ) on [ H]-thymidine incorporation stimulated by R-NE in these experiments is included for comparison. Control DNA synthesis was 1046687 ± 220228 DPM/culture (mean ± S.E.M.) and cells treated with 10 jyM R-NE stimulated [ H]- thymidine incorporation at a level that was 216.3 ± 8.3% (mean ± S.E.M.) of control. X23

-O CIRAZOLINE • ----- • TOLAZOLINE n H PRAZOSIN T00-- Lü a;

X 80 - ■ Q t

t- Z

Lü 0. - 7 —6 -5 - 4 - 3 LOG DRUG CONCENTRATION (M)

Figure 18. Concentration dependent inhibition of 100 2-(3,4- dihydroxybenzyl)imidazoline (DHT) stimulated [ H]-thymidine incorporation by cirazoline and tolazoline in primary cultured rat hepatocytes. Results are expressed as a percentage of the response produced by 100 }M DHT and each point represents the mean ± S.E.M. of determinations from 3-6 dishes. Points with letters indicate levels of [ H]-thymidine incorporation that were significantly less than that produced by 100 DHT and points with different letters indicate degrees of DNA synthesis which were significantly different from each other (p < 0.05, Student-Newman-Keuls test). The inhibitory effect of 1 juM prazosin (PRZ) on [ H]-thymidine incorporation stimulated by DHT in these experiments is included for comparison, control DNA synthesis was 1046687 ± 220228 DP^culture (mean ± S.E.M.) and cells treated with 100 pM DHT stimulated [ H]-thymidine incorporation at a level that was 179.8 ± 15.2% (mean ± S.E.M.) of control. 124

Table 8. Effects of tolazoline and cirazoline alone and with R- norepinephrine (R-NE) and 2-(3,4-dihydroxybenzyl)imidazoline (DHT) stimulated cells on total cellular protein in 48 hr primary cultured rat hepatocytes.

TREATMENTCONC. CELLULAR PROTEIN® (pM) PERCENT CONTROL (%)

TOLAZOLINE 10 89.0 ± 1.9 30 95.6 ± 5.4 100 100.1 ± 5.0 CIRAZOLINE 10 100.1 ± 5.6 30 108.9 ± 7.6 R-NE 10 140.7 ± 7.6. 30 147.4 ± 7.4” DHT 100 129.0 ± 9.8

R-NE (10 pM) + TOLAZOLINE 10 139.6 ± 2.7. 30 142.8 ± 6.6” 100 150.4 ± 7.4” CIRAZOLINE 10 129.2 ± 6.8 30 126.4 ± 3.1 PRAZOSIN 1 101.6 ± 5.8

DHT (100 pK) + TOLAZOLINE 10 147.2 ± 3.6” 30 136.7 ± 11.6 100 135.0 ± 8.4 CIRAZOLINE 10 146.9 ± 13.8” 30 135.2 ± 4.0 PRZ 1 110.9 ± 8.6

Values are expressed as mean ± S.E.M. of the percentage of control total cellular protein (49.0 ± 5.5 pg protein/dish); n = 3-21 dishes. ^ Means are significantly greater than control (p < 0.05, Student- Newman-Keuls test). 12 5

b_ 1 2 0 O LU 2 en 3. 1 0 0 T z O -- o O CL i en 80 IDZ + R-NE (10 /iM) LU - 0— 0 \ oc X @— 9 IDZ + DHT (100 /zM) û en 60 LU n u PRZ (1 /iM) + R-NE (10/iM) § E3 PRZ (1 /iM) 4- DHT(l0/tiM) 3 2 40 3. \* O C A en 2 0 + 0 LU + c LU Z 1 ü oc I i a LU oc ÛL -20 --- _!------1------1------1------9 -8 -7 -6 -5 - 4

LOG DRUG CONCENTRATION (M)

Figure 19. Concentration dependent inhibition of 10 f M R-norepinephrine (|l-N E ) and 100 ^ 2-(3,4-dihydroxybenzyl)imidazoline (DHT) stimulated [ H]-thymidine incorporation by idazoxan (IDZ) in primary cultured rat hepatocytes. Results are expressed as a percentage of the response produced by either 10 j M R-NE or 100 pM DHT and each point represents the mean t S.E.M. of determinations from 3-9 dishes. Points with letters indicate levels of [ H]-thymidine incorporation that were significantly less than that produced by either 10 juM R-NE or 100 f M DHT and points with different letters indicate degrees of DNA synthesis which were significantly different from each other (p < 0.05, Student- Newman-Keuls test). The inhibitory effect of 1 fM prazosin (PRZ) on [ H]-thymidine incorporation stimulated by R-NE and DHT in these experiments is included for comparison. Control DNA synthesis was 1125404 ± 191440 DPM/culture and 799672 ± 155262 (mean ± S.E.M.) in the experiments that examined the effects of IDZ on R-NE- and DHT-stimulated cells, respectively. Hepatocytes treated with 10 f M R-NE and 100 DHT stimulated [ H]-thymidine incorporation at a level that was 272.1 ± 34.6% and 191.2 ± 15.2% (mean ± S.E.M.) of control, respectively. 126

Table 9. Effects of idazoxan and R-norepinephrine (R-NE) alone on [^H]- thymidine incorporation in 48 hr primary cultured rat hepatocytes.

TREATMENT CONC. DPM/CULTURE* (MM) PERCENT CONTROL (%)

IDAZOXAN 0.1 106.2 ± 3.3 1 124.9 + 10.0 10 120.9 ± 9.4 30 116.3 ± 3.8 R-NE 10 307.7 ± 45.6^ 30 309.4 ± 47.3*

® Values are expressed as mean ± S.E.M. of the percentage of control DPM/culture (476864 ± 168754 DPM/culture); n = 3-6 dishes. ** Means are significantly greater than control (p < 0.05, Student- Newman-Keuls test). 127

Table 10. Effects of idazoxan alone and with R-norepinephrine (R-NE) and 2-(3,4-dihydroxybenzyl) imidazoline (DHT) stimulated cells on total cellular protein in 48 hr primary cultured rat hepatocytes.

TREATMENT CONC. CELLULAR PROTEIN® (pM) PERCENT CONTROL (%)

IDAZOXAN 1 126.4 ± 7.2 10 112.9 ± 8.5 30 116.1 ± 1.8 R-NE 10 154.6 ± 12.2* 30 148.7 + 12.1 DHT 100 145.8 ± 8.2* R-NE (10 pM) + IDAZOXAN 1 163.4 ± 13.3* 10 158.4 ± 18.4* 30 150.4 ± 24.8 PRAZOSIN 1 115.2 ± 10.6 DHT (100 pM) + IDAZOXAN 1 167.8 ± 21.4* 10 158.4 ± 18.4* 30 154.8 ± 15.5 PRAZOSIN 1 137.4 ± 13.5

“ Values are expressed as mean ± S.E.M. of the percentage of control total cellular protein (35.2 ± 4.5 pig protein/dish); n = 3-9 dishes. ^ Means are significantly greater than control (p < 0.05, Student- Newman-Keuls test). 128

üJ en 120 z 0 a. 100

ü: LU 80 Z 1 CL 60

a. O— O PRZ o 40 e — • C E C ü_ O 2 0 A A WB—4101 ▲ A SZL-49 LU ü CL LU CL -20 4 - î- —10 —9 —8 —7 —8 —5 —4 - 3

LOG DRUG CONCENTRATION (M)

Figure 20. Concentration dependent inhibition of 10 pK R-norepinephrine (R-NE) stimulated [ H]-thymidine incorporation by WB 4101, chloroethylclonidine (CEC), SZL-49 and prazosin (PRZ) in primary cultured rat hepatocytes. Results are expressed as a percentage of the response produced by 10 jM R-NE and each point represents the mean ± S.E.M. of determinations from 3-9 dishes. Points with letters indicate levels of [ H]-thymidine incorporation that were significantly less than that produced by 10 pK R-NE and points with different letters indicate degrees of DNA synthesis which were significantly different from each other (p < 0.05, Student-Newmcui-Keuls test). Control DNA synthesis was 1245182 ± 182684 DPM/culture (mean ± S.E.M.) and cells treated with 10 pa R-NE stimulated [ H]-thymidine incorporation at a level that was 203.5 ± 9.2% (mean ± S.E.M.) of control. 129

Table 11. Effects of WB 4101, chloroethylclonidine (CEC), SZL-49 and prazosin (PRZ) alone on [ H]-thymidine incorporation in 48 hr primary cultured rat hepatocytes.

TREATMENT CONC. DPM/CULTURE® ( m PERCENT CONTROL (%)

WB 4101 1 104.6 ± 4.2 10 98.1 ± 3.4 CEC 1 101.6 ± 9.4 10 98.4 ± 9.1 30 78.3 ± 2.1 SZL-49 1 113.9 ± 8.6 10 107.6 ± 8.8 PRZ 1 97.4 ± 7.7 R-NE 10 212.6 ± 8.9*’ 30 218.0 ± 10.4b

Values are expressed as mean ± S.E.M. of the percentage of control DPM/culture (1159357 ± 171735 DPM/culture); n = 3-15 dishes. ” Means are significantly greater than control (p < 0.05, Student- Newman-Keuls test). 130

Table 12. Effects of MB 4101, chloroethylclonidine (CEC), SZL-49 and prazosin (PRZ) alone and with R-norepinephrine (R-NE) stimulated cells on total cellular protein in 48 hr primary cultured rat hepatocytes.

TREATMENTCONC. CELLULAR PROTEIN® (MM) PERCENT CONTROL (%)

MB 4101 1 126.8 ± 3.4 10 118.4 ± 3.4 CEC 1 104.7 ± 3.2 10 103.2 ± 5.5 30 107.1 ± 4.0 SZL-49 1 97.4 ± 3.8 10 94.9 ± 3.5 PRZ 1 123.7 ± 5.8 R-NE 10 130.6 ± 7.5^ 30 138.1 ± 8.0^ R-NE (10 pM) + MB 4101 1 147.2 ± 5.5^ 10 141.3 ± 5.6” CEC 1 115.1 ± 5.0 10 103.1 ± 6.1 30 105.4 ± 5.1 SZL-49 1 117.4 ± 11.3 10 118.4 ± 7.4 PRZ 0.01 131.8 ± 9.8” 0.1 126.9 ± 12.2, 1 135.4 ± 14.6

“ Values are expressed as mean ± S.E.M. of the percentage of control total cellular protein (67.2 ± 4.9 pg protein/dish); n = 3-18 dishes. ^ Means are significantly greater than control (p < 0.05, Student- Newman-Keuls test). 1 3 1

Zo O- {/) u QL 100 " zUI

t o 4 0 " b_ c,d O 2 0 " zI— LÜ 0 Ü OL ^ -20 ,3123 1 3 6 10 1 TFP O^M) VERAPAMIL (jciM) PRZ Ou-M)

Figure 21. Concentration dependent inhibition of 10 R-norepinephrine (R-NE) stimulated [ H]-thymidine incorporation by trifluoperazine (TFP), verapamil and prazosin (PRZ)in 48 hr primary cultured rat hepatocytes. Results are expressed as a percentage of the response produced by 10 j M R-NE and each bar represents the mean ± S.E.M. of determinations from 3- 12 dishes. Bars with letters indicate levels of [ H]-thymidine incorporation that were significantly less than that produced by 10 }M R-NE and bars with different letters were significantly different from each other (p < 0.05, Student-Newman-Keuls test). Control DNA synthesis was 744040 ± 60307 DPM/culture (mean ± S.E.M.) and cells treated with 10 ]M R-NE stimulated ( H]-thymidine incorporation at a level that was 215.6 ± 10.2% (mean ± S.E.M.) of control. 132

Table 13. Effects of trifluoperazine (TFP), verapamil and R- norepinephrine (R-NE) alone on [ H]-thymidine incorporation in 48 hr primary cultured rat hepatocytes.

TREATMENT CONC. DPM/COLTURE® (juM) PERCENT CONTROL (%)

TFP 1 92.5 ± 8.9 2 91.0 ± 4.9 3 64.0 ± 2.8 VERAPAMIL 1 98.6 ± 5.1 10 69.6 ± 4.0 R-NE 10 192.5 ± 6.7^ 30 205.2 ± 9.9

Values are expressed as mean ± S.E.M. of the percentage of control DPM/culture (744040 ± 60307 DPM/culture); n = 2-15 dishes. ** Means are significantly greater than control (p < 0.05, Student- Newman-Keuls test). 133

Table 14. Effects of trifluoperazine (TFP), verapamil and prazosin (PRZ) alone and with R-norepinephrine (R-NE) stimulated cells on total cellular protein in 48 hr primary cultured rat hepatocytes.

TREATMENTCONC. CELLULAR PROTEIN* (pM) PERCENT CONTROL (%)

TFP 1 84.6 ± 0.5 3 64.0 ± 3.0° VERAPAMIL 1 113.8 ± 4.1 3 112.2 ± 8.0 10 116.1 ± 3.8 R-NE 10 133.8 ± 6.6^ 30 141.8 ± 8.1 R-NE (10 ^M) + TFP 1 125.6 ± 7.1 3 98.4 ± 4.8 VERAPAMIL 3 157.1 ± 8.5^ 10 146.5 ± 6.8 PRZ 1 110.7 ± 5.0

Values are expressed as mean ± S.E.M. of the percentage of control total cellular protein (60.7 ± 5.8 pg protein/dish); n = 3-15 dishes. ^ Means are significantly greater than control (p < 0.05, Student- Newman-Keuls test), ° Means are significantly less than control (p < 0.05, Student-Newman- Keuls test). 1 3 4

T^le 15. Comparative inhibition of R-norepinephrine (R-NE) stimulated [ H]-thymidine incorporation into the DNA of 48 hr primary cultured rat hepatocytes by tolazoline (TLZ), cirazoline (CRZ), idazoxan (IDZ), WB 4101, chloroethylclonidine (CEC), SZL-49 and prazosin (PRZ).

DRUG n REL. ' ( m INBIB.

TLZ 2 4.54 ± 3.57 X 10"^ 4.55 ± 0.46 1.91 X 10'^

CRZ 2 1.67 ± 1.11 X 10"^ 4.90 ± 0.35 5.18 X 10"*

IDZ 2 2.32 ± 0.52 X 10'® 4.65 ± 0.10 3.73 X 10"* 4 W B 4101 2 > 1 X 10' < 4.0 . -

CEC 3 2.28 ± 0.48 X 10-' 5.66 ± 0.10 3.80 X 10"^

SZL-49 3 2.45 ± 1.10 X 10'’ 6.70 ± 0.21 3.53 X 10"^

PRZ 3 8.66 ± 1.18 X 10’® 8.07 ± 0.06 1.00

° Values are expressed as the mean ± S.E.M. of the IC^^ determinations from individual (n) experiments. IC is equal to the molar concentration of a compound which inhibits 50% of the 10 pM R-NE response. For each inhibition response curve, the response from 3 dishes of cells were used for each concentration of the tested compound.

" PlCgo = -log ^ Relative potency calculated by: (ICg^ of PRZ/lCg^ of tested compound) 135 EZI 30 R-NE 250 ESI 100;Ai DHT

I 200 IO I JL 150 l!100 ii 50 t 12t 24 36 48 72 TIME OF INCUBATION (HOURS)

3250 j O O CONIROL (R-NE) § 3000i • R-NE (30 /*M) ’ 5 ^2750-- I 2500" A' A CONTROL (DHT) o 2250" ▲ DHT (100 /*U) X 2 0 0 0 " I tt17S0" 1 1500" g 1250" ^ 1 0 0 0 " 750" 500" 250" -i------H - i------f- —! 12 24 36 48 60 72 84 TIME OF INCUBATION (HOURS)

Figure 22. Time dependent effects of R-norepinephrine (R-NE) auid 2- (3,4-dihydroxybenzyl)imidazoline (DHT) on [ H]-thymidine incorporation. Bottom panel: Effects of 30 pM R-NE and DHT on [ H]-thymidine incorporation over a 72 hr time course study. The control responses obtained in experiments in which the effects of R-NE and DHT were examined are depicted as CONTROL (R-NE) and CONTROL (DHT), respectively. Results are expressed as the mean ± S.E.M. of the DPM/culture determined from 6 dishes for each treatment and time point. Top panel: Time dependent effects of R-NE and DHT on [ H]-thymidine incorporation relative to the control responses. Results are expressed as the mean ± S.E.M. of the percent of the control level of [ H]-thymidine incorporation obtained from 6 dishes. The dashed line indicates the control (100%) response. Bars with asterisks indicate levels of [ H]- thymidine incorporation that were significantly greater,than that produced by the respective control cells ( ’*p < 0.001, p < 0.05; Student's unpaired t-test). 136

C3 30^ R-NE il d 100 fM DHT 11 " i g -

100 -I— — ■ Eii o 50 J-

nÎI D. 12 24 36 48 72 TIME OF INCUBATIONII (HOURS) 1 300 T i O— O CONTROL (R-NE) f= •-• R-NE (30/iM)- i ^ 250" II o »- 200 Ë: z S '50 +

ÜE \ 100 X+ A — A CONTROL (DHT) î i a o . A— A DHT(IOOAtM) n 4- 4- 4- 12 24 36 48 60 72 84 TIME OF INCUBATION (HOURS)

Figure 23. Time dependent effects of R-norepinephrine (R-NE) and 2- (3,4-dihydroxybenzyl)imidazoline (DHT) on ( S]-methionine incorporation. Bottom panel; Effects of 30 pM R-NE and DHT on [ S]- methionine incorporation over a 72 hr time course study. The control responses obtained in experiments in which the effects of R-NE and DHT were examined are depicted as CONTROL (R-NE) and CONTROL (DHT), respectively. Results are expressed as the mean ± S.E.M. of the DPM/culture determined from 6 dishes for each treatment^d time point. Top panel: Time dependent effects of R-NE and DHT on [ S]-methionine incorporation relative to the control responses. Results are expressed as the mean ± S.E.M. of the percent of the control level of [ S]- methionine incorporation obtained from 6 dishes. The dashed line indicates tte control (100%) response. Bars with asterisks indicate levels of [ S]-methionine incorporation that were, significantly ggeater than tjiat produced by the respective control cells ( *p < 0.001, p < 0.01, p < 0.05, Student's unpaired t-test). 137 250 rZ3 30 /iU R-NE m 100 juM DHT I £g 200 -• o 1S0--

o « 100--T i o i g 50-. Ë a. 12 24 36 48 72 TIME OF INCUBATION (HOURS)

125t

100

a.iî a 75

50 O O CONmOL (R-NE) ® • R-NE (30 ftU) $ 25 A— A CONTOOL (DHT) A— A DHT(100jAO

12 24 36 48 60 72 84 TTME OF INCUBATION (HOURS)

Figure 24. Time dependent effects of R-norepinephrine (R-HE) and 2- (3,4-dihydroxybenzyl)imidazoline (DHT) on total cellular protein. Bottom panel: Effects of 30 pM R-NE and DHT on total cellular protein in cells examined for [ H]-thymidine and [ S]-methionine incorporation (see Figs. 22 and 23) over a 72 hr time course study. The control responses obtained in experiments in which the effects of R-NE and DHT were examined are depicted as CONTROL (R-NE) and CONTROL (DHT), respectively. Results are expressed as the mean ± S.E.M. of the pg protein/dish determined from 6 dishes for each treatment and time point. Top panel: Time dependent effects of R-NE and DHT on total cellular protein relative to the control levels. Results are expressed as the mean ± S.E.M. of the percent of the control level total cellular protein obtained from 6 dishes. The dashed line indicates the control (100%) response. Bars with asterisks indicate levels of total cellular protein that were s ignif^cantly greater than thcit produced by the respective control cells (’ p < 0.001, "p < 0.01, p < 0.05; Student's unpaired t- test ). 138

UJ co 120t z O o n z i + 10 /iM R -N E i< X 1 0 0 û : en ç a -t- 100 /zM DHT JL o LÜ ü_ £X à\ DC O 80 \ O UJ O A N \ LÜ 6 0 " \ Z \ \ a en \ 2 u. 40-- \ >• O \ N ï z N \ 1— 1 LÜ 2 0 -- \ \ \ \ X a a ro K N LÜ \ X 0 -fh -T - \ PRZ CEC S Z L -4 9 CRZ YOH IDZ 1 ytiM 10/iM 1/iM 30/iM 1 jCiM 10/iM

Figure 25. Effect of prazosin (PRZ), chloroethylclonidine (CEC), SZL- 49, cirazoline (C»Z), yohimbine (YOH) and idazoxan (IDZ) on [ H]- thymidine incorporation stimulated by 10 R-norepinephrine (R-NE) and 100 jjM 2-(3,4-dihydroxybenzyl)imidazoline (DffiC) in 48 hr primary cultured rat hepatocytes co-incubated with [ S]-methionine for determinations of effects on protein synthesis (see Fig. 26). Results are expressed as a percentage of the stimulated response produced by either 10 pM R-NE or 100 ;jM DHT and each bar represents the mean ± S.E.M. of determinations from 6 dishes. Bars with letters indicate levels of [ H]-thymidine incorporation that were significantly less them that produced by the respective stimulating agents (10 pM R-NE or 100 fjM DHT) emd bars with different letters indicate degrees of [ H]-thymidine incorporation which are significantly different from each other (p < 0.05, Student-Newman-Keuls test). The control level of [ H]-thymidine incorporation was 977870 ± 200222 DPM/culture (mean ± S.E.M.). Cells treated with 10 fM R-NE and 100 jjM DHT stimulated [ H]-thymidine incorporation at levels that were 181.8 ± 4.6% and 177.4 ± 9.7% (mean ± S.E.M.) of control, respectively. 139

LÜ ÜO 1 2 0 t z [!□ + 10 /iM R-NE < O CC CL 100 O co a + 100 jjM DHT 0_ LÜ ai ÛC o û 80 i é o LÜ \ b \ LÜr: 3 \ 60 N \ N N en N ü_ 40 N 1\ \ E O \ z !- \ I a a LÜ 20 \ (n ü ri, in OC \ n LÜ \ \ \ CL 0 l i . I PRZ CEC SZL-49 CRZ YOH IDZ 1 jttM 10 /iM ^ } M 3 0 jtiM 1 !jM 1 0 i M

Figure 26. Effect of prazosin (PRZ), chloroethylclonidine (C£^), SZL- 49, cirazoline (CRZ), yohimbine (YOH) and idazoxan (IDZ) on ( S]- methionine incorporation stimulated by 10 yiSA R-norepinephrine (R-NE) and 100 }M 2-(3,4-dihydroxybenzyl)imidazoline (DOT) in 48 hr primaury cultured rat hepatocytes co-incubated with [ H]-thymidine for determinations of effects on protein synthesis (see Fig. 25). Results are expressed as a percentage of the stimulated response produced by either 10 ]M R-NE or 100 y M DOT and each bar represents the mean ± S.E.M. of determinations from 6 dishes. Bars with letters indicate levels of [ S ]-methionine incorporation that were significantly less than that produced by the respective stimulating agents (10 f M R - Œ or 100 yUA DOT) and bars with different letters indicate degrees of [ S]- methionine incorporation which were significantly different from each other (p < 0.05, Student-Newman-Keuls test). The control level of [ S]-methionine incorporation was 168542 ± 12111 DPM/culture (mean ± S.E.M.). Cells treated with 10 y M R-NE and 100 ;^M DOT stimulated [ S]- methionine incorporation at levels that were 141.1 ± 5.9% and 143.2 ± 6.4% (mean ± S.E.M.) of control, respectively. 140

Table 16. Effects of prazosin (PRZ), chloroethylclonidine (CEC), SZL- 49, cirazoline (CRZ), yohimbine (YOH) and idazoxan (IDZ) on total cellular protein in 48 hr primary cultured rat hepatocytes stimulated by R-norepinephrine (R-NE) and 2-(3,4-dihydroxybenzyl)imidazoline (DHT).

TREATMENT CONC. CELLULAR PROTEIN® (pM) PERCENT CONTROL (%)

R-NE 10 123.3 ± 7.9 DHT 100 111.6 ± 3.8 R-NE (10 pM) + PRZ 1 97.4 + 5.2 CEC 10 97.4 ± 1.6 SZL-49 1 123.4 ± 7.2 CRZ 30 101.0 ± 4.8 YOH 1 108.7 ± 3.9 IDZ 10 112.4 ± 4.7 DHT (100 pM) + PRZ 1 98.9 ± 2.7 CEC 10 97.4 ± 1.6 SZL-49 1 120.4 ± 7.3 CRZ 30 116.7 ± 5.5 YOH 1 122.2 ± 3.2 IDZ 10 123.5 ± 5.2

* Values are expressed as mean ± S.E.M. of the percentage of control total cellular protein (78.0 ± 5.6 jjg protein/dish); n = 5-6 dishes. ^ Means are significantly greater than control (p < 0.05, Student- Newman-Keuls test). CHaPTER IV SDMHARX OF DATA

The primary cultured rat hepatccyte system provides a useful tool for the pharmacological characterization of a^-adrenergic receptor mediated DNA and protein syntheses. By using this system, a number of structural and stereochemical features of phenethylamine and imidazoline compounds important for the stimulation of a^-adrenergic receptor modulated DNA synthesis were investigated. In addition, the abilities of selected antagonists of the imidazoline preferring receptor and subtypes of the a^-adrenergic receptor to block DNA and protein syntheses in primary cultured rat hepatocytes were evaluated. The major findings of these experiments are listed below.

1. Concentration-dependent increases in [^H]-thymidine incorporation were elicited by the R- and S-isomers of NE (EC^^ = 0.34 and 8-24 pH, respectively) whereas DA (the desoxy derivative) was inactive in this system. The rank order of potency was R-NE > S-NE > DA. Thus, the Easson-Stedman hypothesis (1933) is valid for the stimulation of DNA synthesis by these compounds. Prazosin (1 pM) completely inhibited the effects of the NE isomers.

2. Concentration-dependent increases in [^H]-thymidine incorporation were elicited by the 2-substituted catecholimidazoline compounds tested. The R- and S-isomers of TBI (EC^^ = 15.0 and 61.5 pH, respectively) were less active than the desoxy derivative, DHT (EC^^ = 10.8 pH) for a rank order of potency of DHT > R-TBI > S-TBI. Thus these catecholimidazolines do not adhere to the Easson-Stedman hypothesis

141 142

(1933). Prazosin (1 }M) potently blocked the effects of all three compounds.

3. Only the R-isomer of DBI (EC^^ = 17.3 pM), a 4-substituted catecholimidazoline, was capable of stimulating DNA synthesis in primary cultured rat hepatocytes. The racemate demonstrated intermediate activity. Prazosin blocked the stimulation of [^H]-thymidine incorporation caused by R-DBI.

4. Both the R- and S-isomers of DPDA stimulated DNA synthesis in a concentration dependent manner (EC^^ =22.5 and 10.0 pM, respectively). Little stereoselectivity was demonstrated by the isomers of DPDA for this effect. Prazosin (1 pM.) potently blocked the effect of R-DPDA but only partly inhibited the effect of S-DPDA.

5. Removal of the phenolic hydroxyl groups from DHT (creating tolazoline) eliminates the ability of imidazolines to stimulate a^- adrenergic receptor mediated DNA synthesis. Placement of amino or isothiocyanato groups at the 4'-benzyl position of tolazoline does not increase the agonist activity of the resultant compounds. IBI, which stimulates contraction of rat thoracic aorta by a unique nonadrenergic, Ca*^-dependent mechanism, is inactive as either an agonist or antagonist in this cell system.

6. Two noncatechol imidazolines, tolazoline and cirazoline, did not stimulate c^-adrenergic receptor mediated DNA synthesis. However, both compounds blocked DNA synthesis in a concentration dependent manner. Cirazoline (10^^ = 16.7 pM) blocked R-NE stimulated DNA synthesis more potently than tolazoline (IC^g = 45.4 pM). Cirazoline also blocked DHT stimulated DNA synthesis (IC^^ = 16.5 pM) but tolazoline did not. Idazoxan, an a^-adrenergic receptor antagonist and imidazoline 143

preferring receptor ligand, only inhibited R-NE and DHT stimulated DNA synthesis at the maximum concentration (30 pM) tolerated by the primary cultured rat hepatocytes.

7. The ability of a selected series of a^-adrenergic receptor antagonists, including selective antagonists of the and a^^- subtypes, to inhibit R-NE stimulated DNA synthesis in primary cultured rat hepatocytes was examined. Prazosin (IC^g = 8.7 nM) was the most potent antagonist of those examined. WB 4101, a selective competitive antagonist of the a^^-receptor subtype, was essentially inactive as an antagonist in this system. SZI.-49, a selective alkylating agent of the subtype, concentration dependently inhibited (10^^ = 0.24 pM) R-NE stimulated DNA synthesis. CEC, a selective a^^-subtype alkylating agent, also inhibited R-NE stimulated DNA synthesis (IC^g = 2.3 pM) in a concentration dependent manner but was less potent than either prazosin or SZL-49. These results indicate that perhaps both subtypes and of the a^-adrenergic receptor modulate DNA synthesis in primary cultured rat hepatocytes.

8. Both verapamil, a Ca*^ channel blocker, and TFP, a calmodulin antagonist, inhibited R-NE stimulated DNA synthesis at the maximal concentrations of these compounds tolerated by primary cultured rat hepatocytes. Although these results suggest a role of Ca*^ influx and calmodulin activity in o^-adrenergic receptor mediated DNA synthesis, the effects of verapamil and TFP may not be specific for their intended use and may reflect blockade of the o^-adrenergic receptor instead.

9. R-NE and DHT Stimulated DNA and protein syntheses in a similar time dependent fashion with peak incorporation of both [^H]-thymidine and [^^S]-methionine occurring at 48 hr. Prazosin and CEC inhibited both DNA and protein syntheses stimulated by R-NE and DHT. SZL-49 and 1 4 4 cirazoline inhibited DNA synthesis caused by R-NE and DHT but failed to inhibit protein synthesis and yohimbine inhibited DNA synthesis stimulated by R-NE. These results show that the time courses for the stimulation of DNA and protein syntheses closely parallel each other and that both DNA and protein syntheses stimulated by R-NE and DHT are sensitive to inhibition by the selected concentrations of prazosin and

CEC used in these experiments.

In conclusion, these results demonstrate several important features concerning a^-adrenergic receptor mediated DNA synthesis.

These include the findings that: 1) the structural and stereochemical requirements of phenethylamines and imidazolines for the activation of hepatic a^-adrenergic receptor mediated DNA synthesis are similar in many respects to those required for stimulation of other a^-adrenergic receptor systems, 2) both the and a^^^-adrenergic receptor subtypes may be involved in the modulation of DNA synthesis by R-NE, and 3) DNA and protein syntheses are modulated by the a^-adrenergic receptor in a similar manner. These studies have provided new insights into the structural and stereochemical features of phenethylamine and imidazolines required for stimulation of hepatic a^-adrenergic receptor mediated DNA synthesis and have further characterized this response pharmacologically. This increased understanding of the nature of the stimulation and inhibition of a^-adrenergic receptor mediated DNA synthesis may eventually lead to the development of other more selective and active agents that will allow for the continued investigation of both molecular and cellular mechanisms involved in a^-adrenergic receptor mediated hepatocellular growth responses. Thus, the potential for the development of drugs that may be effective in the treatment of disorders of hepatocyte proliferation such as liver cancer is increased by our understanding of the pharmacological characterization of a^- adrenergic receptor mediated liver cell growth. APPENDIX

SPECIFIC AIM.

These studies were performed in order to establish and test the effects of certain experimental conditions on DNA synthesis in primary cultured rat hepatocytes. These studies are included as an appendix because they do not directly contribute new information concerning the role of the a^-adrenergic receptor in mediating growth responses in primary cultured rat hepatocytes. However, these studies do provide additional insight into the use of this experimental model as a tool for the examination of a^-adrenergic receptor modulated cell growth.

In these experiments 1) the effects of growth factors on [^H]- thymidine incorporation in both control and R-NE stimulated cells were examined, 2) the effects of different types of media on DNA synthesis in control and R-NE treated primary cultured hepatocytes were studied, 3) two types of culture dish surfaces (surface matrices) were examined for their effect on [ ] -thymidine incorporation in control and stimulated cells, 4) the influence of nialamide (an MAO inhibitor) on R-NE stimulated DNA synthesis was examined, 5) the effect that expression of the data as DPM/culture or DPM/pg protein has on the appearance and interpretation of the data was determined, 6) the effects of a series of adrenergic antagonists on [^H]-thymidine incorporation in R-NE and DHT treated primary cultured rat hepatocytes were examined and 7) autoradiographs of hepatocytes incubated with [^H]-thymidine were prepared in order to determine the localization of the radiolabel in the cell.

145 146

METHODS. 1. Materials. The biochemicals and their sources were: nialamide (Sigma Chemical Company, St. Louis, MO) and propranolol hydrochloride (Ayerst Laboratories, Inc., New York, NY). Other materials and their sources include Minimum Essential Medium [MEM] (Gibco, Grand Island, NY); Vitrogen (Collagen Corporation, Palo Alto, CA); and NTB-2 emulsion, high contrast developer (Formula D-19) and acid hardening fixing bath (Formula F-5) (Eastman-Kodak, Rochester, NY). Other materials used were the same as those described in Chapters II and III.

2. Isolation and preparation of primary cultures of rat hepatocytes. The animals and procedures used were the same as those described in Chapters II and III with one exception. In most of the experiments described in this section, the use of a Percoll gradient was not used during the isolation procedure. In the earlier work performed during the development of this project, a rather consistent lower viability of the cells (from 80% to 90% viable cells as determined by trypan blue exclusion) obtained after the isolation procedure was noted. Because of this, a step involving the use of a Percoll gradient during the cell isolation was included in order to remove dead cells and viability was increased to levels usually greater than 90%. Since most of the results presented in this section were obtained prior to the introduction of the Percoll gradient to the hepatocyte isolation procedure, the cultured hepatocytes differ in this respect from those used in Chapters II and III. Only the hepatocytes used for the autoradiographs and those used for the determinations of the effects of various adrenergic antagonists on R-NE and DHT stimulated [^H]-thymidine incorporation were isolated using a Percoll gradient. In all experiments except those in which the effects of media were examined, the primary cultured rat hepatocytes 147

were incubated with the supplemented Williams E (WE) media described in Chapters II and III.

3. Preparation of autoradiographs. Autoradiographs were prepared according to a method adapted from Richman et al. (1976). In experiments in which a^-adrenergic receptor modulated DNA synthesis in cultured rat hepatocytes was assessed by autoradiography, [^H]-thymidine was added at a concentration of 5 fiCL/ml and the cells incubated under the same conditions as those indicated in Chapters II and III for 48 hr. After this incubation period, the cells were rinsed once with ice-cold isotonic NaCl and then incubated for 10 min at 4° with 7% TCA. Following 2 rinses with 7% TCA, the cells were washed sequentially with 70% and then 95% ethanol for 10 min each at 4°. The cells were allowed to dry in the culture dishes overnight. NTB-2 photographic emulsion (diluted 1:1 with double distilled water) was then added to the plates, the excess drained and the cells exposed for two days prior to development. Best results for the development of these autoradiographs were obtained when the cells were developed in high contrast developer (Formula D-19) for 1 min followed by a 12 to 15 min fixing period in acid hardening fixing bath (Formula F-5). Photographs of the autoradiographs were then taken at lOOX and 200X utilizing an inverted microscope (Nikon Diaphot-TMD).

4. Biochemical assays of cell lysates and data analysis. The assays for -thymidine incorporation and total cellular protein utilized in these experiments are the same as those described in Chapters II and III. The results for the measurement of [^H]-thymidine incorporation are expressed as DPM/culture (and in one experiment as DPM/pg protein) and normalized as a percentage of control and as a percentage of the maximal response as described in Chapter II and III. 148

Total cellular protein was expressed as pg protein/dish and presented as a percentage of the control response. As described in Chapters II and III, the data were statistically analyzed by comparisons among the means using the unpaired t-test for direct comparisons of means or by one-way analysis of variance followed by the Student-Newman-Keuls test for multiple comparison of means.

BESDLTS. 1. Evaluation of the effects of growth factors on DNA synthesis in primary cultured rat hepatocytes. The effects of two growth factors (insulin and EGF) on DNA synthesis in primary cultured rat hepatocytes are shown in Fig. 27. In the absence of insulin and EGF, the amount of [ ] -thymidine incorporated into the DNA of both the control and 10 pM R-NE treated cells (7.56 X 10* and 2.22 x 10® DPM/culture, respectively) is less than that incorporated in the cells incubated with insulin, EGF or a combination of both. The control cells incubated with either insulin (0.1 pM) or EGF (10 ng/ml) had similar levels of [®H]-thymidine incorporation (2.98 x 10® and 3.61 x 10® DPM/culture, respectively). Likewise, the R-NE stimulated cells incubated with either insulin or EGF displayed similar levels of [®H]-thymidine incorporation that were 8.20

X 10® and 8.33 x 10® DPM/culture, respectively. Cells incubated with both insulin (0.1 pM) and EGF (10 ng/ml) had higher levels of thymidine incorporation than the other incubation conditions for both the control (9.23 x 10® DPM/culture) and R-NE treated cells (2.08 x 10® DPM/culture). Exposure of the cells to 10 pM R-NE caused similar significant increases in [®H]-thymidine incorporation that were 2.9-, 2.1-, 2.8- and 2.3-fold greater than the respective controls for all four experimental conditions tested (no insulin or EGF, insulin only, EGF only and both insulin and EGF, respectively). 149

2. Evaluation of the effects of media on DH2V synthesis in primary cultured rat hepatocytes. The effects that MEM and WE media have on DNA synthesis in R-NE stimulated primary cultured rat hepatocytes are shown in Fig. 28. The results from this experiment are representative of an additional experiment which also examined the effects of these two types of media on [ ] -thymidine incorporation in cultured hepatocytes. When results are expressed as DPM/culture (Fig. 28, lower panel) and percent of control of DPM/culture (Fig. 28, upper left panel), the levels of DNA synthesis in control cells incubated with MEM or WE media were not significantly different from each other. However, in cells incubated with WE medium, 1, 10 and 100 jkM R-NE caused increases in -thymidine incorporation that were significantly greater than those caused by the same concentrations of R-NE in cultured hepatocytes incubated with MEM medium. When results are expressed as a percent of the maximal response obtained for each type of media (Fig. 28, upper right panel), the results are fairly similar with only the 1 R-NE treatment in WE medium causing a significantly greater increase in [^H]-thymidine incorporation than the corresponding treatment in MEM medium. Additionally, similar EC^g values of 23 pH and 16 were obtained for R-NE in cells incubated with MEM and WE media, respectively. Prazosin (1 ]MA) significantly inhibited [^H]-thymidine incorporation caused by 100 }u}A R-NE to 31% and 57% of the maximal response in primary cultured rat hepatocytes incubated with WE and MEM medium, respectively.

3. Determination of the effects of the culture dish surface on DNA synthesis in primary cultured rat hepatocytes. [^H]-Thymidine incorporation in primary cultured rat hepatocytes plated onto collagen-coated culture dishes and Primaria culture dishes was evaluated, and the results shown in Fig. 29. On collagen coated plates, the amount of [^H]-thymidine incorporation in control and 100 pM 150

R-NE treated cells was 2.52 x 10^ and 5.93 x 10^ DPM/culture, respectively. When cells were plated on Primaria culture dishes, the levels of [^H]-thymidine incorporation were 4.36 x 10^ and 1.04 x 10® DPM/culture, respectively. The increases in DNA synthesis caused by R- NE in these cells attached to collagen-coated and Primaria dishes are similar (236% and 239%, respectively) when expressed as a percentage of the respective control values. Prazosin (1 pM) significantly inhibited the 100 pM response to levels of [^H]-thymidine incorporation (2.85 x 10® and 3.99 x 10®) that were not significantly different from control values in cells plated on collagen-coated and Primaria culture dishes, respectively.

4. Evaluation of the effects of nialamide treatment on R-NE stimulated DNA synthesis in primary cultured rat hepatocytes. The effects of exposure of the primary cultured rat hepatocytes to 50 pM nialamide on R-NE stimulated [®H]-thymidine incorporation are shown in Fig. 30. When results are expressed as DPM/culture and percent of control of DPM/culture, the levels of DNA synthesis in control cells and cells treated with nialamide were not significantly different from each other. The concentration-response curves for R-NE stimulated DNA synthesis are similar to each other and levels of [ ®H]-thymidine incorporation significantly greater than control levels were seen for the three types of data presentation (DPM/culture, Fig. 30, lower panel; percent of control. Fig. 30, upper left panel; and percent of maximal response. Fig. 30, upper right panel). At several concentrations of R- NE, there are significant differences in the level of [®H]-thymidine incorporation in nialamide treated and untreated (control) cells. The most notable difference is noted for the concentration of R-NE required to evoke a maximum response in control and nialamide treated cells. In control cells, a maximal level of [®H]-thymidine incorporation is produced by 30 pM R-NE whereas in nialamide treated cells a maximal 151 response is elicited by 10 ]M R-NE. However, despite these differences, similar EC^g values (1.2 pM and 0.9 jjM) for R-NE were obtained in control and nialamide treated cells, respectively. Prazosin (1 ^M) significantly inhibited [ ] -thymidine incorporation caused by 100 pM R- NE to -1.1% and -3.7% of the 100 pM R-NE response in control and nialamide treated primary cultured rat hepatocytes, respectively.

5. Evaluation of the expression of [^H]-thymidine incorporation as DPM/culture and DPM/pg protein. The concentration-dependent stimulation of DNA synthesis caused by R-NE and expressed as DPM/culture and DPM/pg protein for cells from the same experiment is shown in Fig. 31. Control levels of [^H]-thymidine incorporation were 4.36 x 10^ DPM/culture and 5.25 x 10^ DPM/pg protein. R-NE, at concentrations of 3 }jM and higher, significantly increased the level of [^H]-thymidine incorporation (expressed as either DPM/culture or DPM/pg protein) to levels greater than control. For both means of data expression, prazosin (1 pM) significantly inhibited [^H]-thymidine incorporation caused by 100 pM R-NE. When the same data shown in Fig. 31 are transformed into the respective percent of control (either DPM/culture or DPM/pg protein), the concentration-response curves depicted in Fig. 32 result. As was the situation for the expression of the data as DPM/culture and DPM/pg protein, when the data are expressed as a percentage of the respective controls, concentrations of R-NE that were 3 pM and higher significantly increased the level of [^H]-thymidine incorporation to levels greater than control. However, when the data expressed originally as DPM/culture are transformed to percent of control, the levels of [^H]- thymidine incorporation produced by 3, 10, 30 and 100 pM R-NE are significantly greater than the responses produced for these same concentrations when the data expressed as DPM/pg protein is transformed to percent of control. When expressed as a percentage of the control 152 response for data calculated as DPM/culture and DPM/;/g protein, prazosin (1 pK) significantly inhibited [^H]-thymidine incorporation caused by 100 pH R-NE. The concentration dependent increase in total cellular protein is also shown in Pig. 32. In a manner similar to the increase in DNA synthesis, concentrations of R-NE of 3 pK and higher significantly increased total cellular protein content to levels greater than control (84 pg protein/dish). Transformation of the data expressed as DPM/culture and DPM/ng protein to a percent of the maximal response creates the concentration- response curves shown in Fig. 33. Again, concentrations of R-NE grater than 3 pH produced increases in [^H]-thymidine incorporation that were significantly greater than control. When the data expressed as either DPM/culture or DPM/pg protein are transformed to percent of the maximal response, no significant differences in the level of [ ] -thymidine incorporation were seen between the transformed data for each concentration of R-NE tested. In addition, similar EC^g values for R-NE (10 pH and 14 pH) were obtained from the concentration response curves generated from data expressed as DPM/culture and DPM/pg protein, respectively. Prazosin (1 pM) significantly inhibited [^H]-thymidine incorporation to -10% and -27% of the transformed DPM/culture and DPM/pg protein data, respectively.

6. Evaluation of the effects of selected adrenergic antagonists on R-NE and DHT stimulated DNA synthesis in primary cultured rat hepatocytes. The effects of the «^-adrenergic receptor antagonist prazosin, the «g-adrenergic receptor antagonist yohimbine and E-adrenergic antagonist propranolol on DNA synthesis stimulated by 10 pH R-NE and 100 pM DHT in primary cultured rat hepatocytes are shown in Fig. 34. Prazosin produced significant inhibitions of R-NE stimulated [^H]-thymidine incorporation at concentrations of 10 nM and above. Yohimbine did not 153 cause a significant inhibition of R-NE stimulated [ ]-thymidine incorporation until a concentration of 10 jdîA was reached and propranolol did not inhibit R-NE stimulated DNA synthesis in concentrations up to 10 pM. In DHT stimulated cells, prazosin caused a significant inhibition (14% of 100 pM DHT response) of DNA synthesis at a concentration of 0.1

}jM whereas 1 }M yohimbine produced a significant inhibition (43% of the 100 }M DHT response) of [^H]-thymidine incorporation.

7. Autoradiographs of primary cultured rat hepatocytes incubated with [ ^H]-thymidine. Autoradiographs of cultured hepatocytes incubated with [^H]- thymidine (5 pCi/ml) and exposed to selected treatment conditions are shown in Figs. 35 and 36. It can be noted that in the 10 pM R-NE treated cells, there is a larger number of labelled nuclei than inthe control cells or in R-NE treated cells exposed to 1 pM prazosin.

DISCUSSION. 1. Growth factor effects. A number of studies have been conducted that have evaluated the effects of growth regulating hormones such as insulin and EGF on DNA synthesis in primary cultured rat hepatocytes (Richman et al., 1976; McGowan et al., 1981; Cruise and Michalopoulos, 1985; Wollenberg et al., 1989). In the absence of insulin and EGF, there is a very low level of DNA synthesis in relation to cells exposed to the other growth stimulating conditions. Treatment of the hepatocytes not exposed to EGF or insulin with 10 pM R-NE causes an increase in DNA synthesis. This result differs from that described by Cruise and Michalopoulos (1985) where R-NE had no effect on DNA synthesis in defined media lacking the two growth factors. However, differences in the media used, the substratum to which the cells were attached and possibly other differences in the experimental conditions may account for these 154 variances. The important issue is that there are very low levels of DNA synthesis occurring in both the control and R-NE stimulated cells when EGF and insulin are absent from the incubation media. Insulin has been reported to exert a strong trophic influence on hepatocytes (Michalopoulos, 1990) and will increase DNA synthesis in hepatocytes incubated with fetal calf serum-supplemented media (Richman et al., 1976). However, insulin by itself will demonstrate little DNA stimulating activity in cultured rat hepatocytes incubated under defined media conditions (McGowan et al., 1981). Incubation of the cells with 0.1 f/M insulin in the cultured rat hepatocyte system used in these experiments resulted in an almost 4-fold increase in the amount of [^H]- thymidine incorporation in comparison to cells exposed to no insulin or EGF. Exposure of insulin treated cells to R-NE causes a further increase in [^H]-thymidine incorporation. These effects are similar in some respects to those described by Cruise and Michalopoulos (1985) who demonstrated that insulin increased (^H]-thymidine incorporation in both control and R-NE treated cells. However, these increases were not accompanied by corresponding increases in labeling indices and thus suggest that insulin is nonmitogenic by itself but that this hormone plays a permissive role in the growth promoting effects of EGF and R-NE. The increase in [^H]-thymidine incorporation when expressed as DPM/culture produced by insulin may be the result of the trophic influences possessed by this peptide which may assist and facilitate the repair and restoration of hepatocellular functions following cell isolation. Results seen in cells incubated with EGF alone are similar to those exhibited by those treated with insulin alone. EGF produces a 5- fold increase in DNA synthesis in relation to cells exposed to neither insulin or EGF. Exposure to R-NE produces an additional increase in I ^H]-thymidine incorporation. These findings are similar to those 15 5

described by Cruise and Michalopoulos (1985) who also reported that

labeling indices were correspondingly increased by these treatments.

Exposure of cultured cells to both EGF and insulin stimulates the

greatest amount of DNA synthesis in both control and R-NE treated cells

relative to the other tested incubation conditions. These results are

consistent with those described by Cruise and Michalopoulos (1985). An

approximately 12-fold increase in [^H]-thymidine incorporation was seen

in cells incubated with both insulin and EGF when compared to cells

exposed to neither growth factor. Stimulation with R-NE caused a

further increase in [^H]-thymidine incorporation, indicating that these

three agents interact with each other in such a way as to greatly

enhance DNA synthesis. Therefore, based upon these findings as well as

those reported by others (McGowan et al., 1981; Cruise and

Michalopoulos, 1985), the decision was made to include both insulin (0.1

;iM) and EGF (10 ng/ml) in the defined media used for the incubation of

the primary cultured rat hepatocytes to be used for the evaluation of

the pharmacological characterization of c^-adrenergic receptor mediated

growth responses.

2. Media effects.

Studies of hepatic a^-adrenergic receptor mediated DNA synthesis conducted in the laboratory of Michalopoulos utilize MEM (Cruise et al.,

1985,1986) whereas the laboratory of Ichihara (Takai et al., 1988) uses

WE medium for the incubation of the primary cultured rat hepatocytes.

Our laboratory has had good success with WE medium in previous studies

involving the evaluation of peroxisome proliferation in cultured

hepatocytes (Feller et al., 1986; Esbenshade et al., 1990); and therefore, continued use of this medium in studies involving

adrenergic receptor mediated DNA synthesis was warranted. However, it was of interest to determine if the type of media used would have any effect on the stimulation of DNA synthesis by R-NE in this cell system. 156

Both the absolute and relative percent of control responses were greater for R-NE stimulated cells incubated in WE media as opposed to MEM. Although similar EC^g values were obtained for the stimulation of DNA synthesis by R-NE in these two types of media, the decision to use WE medium for the studies presented in this work was based upon the greater percent of control increase in [ ]-thymidine incorporation caused by R- NE in cells incubated with WE media. The greater absolute activity exhibited in cells incubated with supplemented WE medium may be the result of the composition of this medium which is much more fortified with amino acids and vitamins.

3. Culture surface effects. Most of the studies that have investigated the regulation of cell growth and DNA synthesis in vitro have utilized rat tail collagen as the substratum to which the cells attach (Strom and Michalopoulos, 1982; Cruise et al., 1985). However, because of the expense involved in the use of collagen, it was decided upon that Primaria culture dishes (Fisher Scientific, Cincinnati, OH) should be tested to determine if this culture surface is suitable for use in the investigation of u^- adrenergic receptor mediated DNA synthesis. These plates incorporate amide and amino functional groups into the polystyrene cell surface thus providing a positively charged substratum for cell attachment. Although the absolute amount of [^H]-thymidine incorporated into the DNA of cells plated on Primaria plates was greater than that in cells plated on rat tail collagen-coated plates, the same percent increase in DNA synthesis stimulated by 100 pM R-NE was obtained for cells plated on both surface types. Additionally, upon morphologic examination, the hepatocytes on both surfaces flattened out and had similar appearances over the course of the incubation period. Based on these findings, Primaria plates were utilized for the investigations conducted in this work. 157

4. Nialamide effects. Monoamine oxidase (MAO) is an integral membrane protein located in the outer mitochondrial membrane (Ziegler, 1988). The primary degradative pathway for catecholamines in rat and man is by oxidative deamination by MAO (Pletscher, 1966). Activity of this enzyme is highest in the liver, heart and brain tissues of man and rats (Pletscher, 1966; Prange et al., 1967). This flavoenzyme exists as two forms, termed MAO A and MAO B, designations which are based upon substrate specificity auid the selective inhibition of theses isoenzymes by clorgyline and deprenyl, respectively (Tipton, 1980). Rat liver possess both forms of MAO (Ziegler, 1988). Since this enzyme is present in high concentrations in rat liver, it was suspected that perhaps blockade of MAO with nialamide, a nonselective, irreversible MAO inhibitor (Neff and Young, 1974; Cousins, 1985), would shift the R-NE concentration response curve to the left since less Of the R-NE exposed to the cells would be metabolized. However, this was not the case and R-NE produced similar concentration- dependent increases in DNA synthesis in both control and nialamide treated cells. One possible explanation for these results may be that MAO enzyme activity decreases under these culture conditions and thus does not play a significant role in the degradation of R-NE. Since there was no appreciable difference between the R-NE responses in control and nialamide treated cells, nialamide was not included in any of the experiments in which the effects of R-NE were monitored.

5. Expression of data as DPM/culture and OPM/^g protein. The data from the same treatments in the same dishes of cells were expressed as DPM/culture and DPM/pg protein in order to gain information into the most appropriate manner of presenting the results. As can be seen from Figs. 32-34, similar shaped concentration response curves for the stimulation of DNA synthesis by R-NE were obtained. However, upon 158

examination of these results and those in other experiments, it was decided upon to express the raw data as DPM/culture for the following reasons; 1) Data calculated as DPM/culture consistently provided greater ,increases (generally 2- to 3-fold) when the maximal effect was transformed into percent of the control response as compared to the data calculated as DPK/pg protein, which when the maximal effect was transformed into percent of control response, resulted in usually a less than 2-fold increase in the response. Thus, a larger response range in which to measure concentration-dependent effects of agonists and antagonists is provided when the data are presented as DPM/culture. 2) The increases in total cellular protein often obscure the increases in [^H]-thymidine incorporation when the results are expressed as DPM/j^g protein (this is the reason for the lower percent of control response seen with the data presented as DPK/jug protein discussed in the first reason above). 3) When data are transformed into percent of maximal response, similar results, includingvalues, are obtained for both manners of data expression. 4) DNA synthesis measured in vitro is often expressed as DPM/ culture (Cruise et al., 1985).

6. Effects of adrenergic antagonists. The ability of prazosin, yohimbine and propranolol to block DNA synthesis induced by R-NE are similar to those described by Cruise et al. (1985). Prazosin, at concentrations of 1 pM and less, potently blocked the stimulation of DNA synthesis caused by 10 pM R-NE and 100 ^/M DHT whereas the «^-adrenergic receptor antagonist yohimbine did not. However, at higher concentrations of yohimbine (1 pH and particularly 10 pM), blockade of DNA synthesis in cells exposed to R-NE and DHT was seen. These results are similar to those described by Takai et al. (1988) who demonstrated that 1 pM yohimbine partially blocked DNA synthesis induced by 10 pM epinephrine in primary cultured rat 159

hepatocytes. Further discussion of the effects of these two antagonists can be found in Chapter III. There does not appear to be a 3-adrenergic receptor component involved in the stimulation of DNA synthesis in primary cultured rat hepatocytes. However, small increases in DNA synthesis caused by increasing concentrations of propranolol (which fall to control level at 10 pM propranolol) does suggest that perhaps the B-adrenergic receptor may inhibit DNA synthesis as seen in cultured vascular smooth muscle cells (Nakaki et al., 1990). Additionally, Houck et al. (1989) reported that propranolol enhanced the stimulatory effect of R-NE in cultured hepatocytes exposed to TGFB, a potent inhibitor of EGF-mediated DNA synthesis, suggesting a role for the B-adrenergic receptor in the regulation of hepatocyte growth. The shift in the regulation of hepatocellular mechanisms from c^- to B^- adrenergic receptor control that is seen upon the primary culturing of hepatocytes (Schwarz et al., 1985; Kunos et al., 1984) may influence the response caused by R-NE. Thus, further experimentation is required in order to determine the exact role, if any, the B^-adrenergic receptor plays in the phenomenon of DNA synthesis in primary rat hepatocytes.

7. Autoradiographs. From the autoradiographs of cells incubated with [^H]-thymidine and exposed to selected treatments, it can be seen that the number of Icibelled nuclei in cell treated with 10 pM R-NE are much greater than in the control cells. Treatment of cells with both 10 pM R-NE and 1 pM prazosin produces a decrease in the number of cells containing labelled nuclei. These autoradiographs provide additional evidence that the [^H]-thymidine is incorporated into the DNA of the nucleus of the cells and that the measurement of DNA synthesis by radiometric means (DPM/culture) is a valid representation of DNA synthesis. 160

2500 T *** I e 7 2000+ HD CONTROL

O' ^ nSJ R-NE (10 /iM) O O LU 1 5 0 0 -- CC LU X» 1 0 0 0 " *** >- X o_ Q 5 0 0 " ** X ro S NO INSUUN INSUUN EGF INSUUN NO EGF (0.1 /ilvl) (10 ng/ml) + EGF

Figure 27. Effects of growth stimulating hormones on [ H]-thymidine incorporation in control and 10 }M R-norepinephrine (R-NE) treated primary cultured rat hepatocytes. The cells were incubated in media containing the following: 1) no insulin or epidermal growth factor (EGF), 2) insulin (0.1 juM) only, 3) EGF (10 ng/ml) only and 4) insulin (0.1 ]M) and EGF (10 ng/ml). Results are expressed as DPM/culture (x 10* ). [For example, a value of 500 on the ordinate translates to 500,000 DPM/culture.] Each bar represents the mean ± S.E.M. of determinations from 4 dishes. Asterisks indicate that the means of the cells treated with R-NE are^ significantly greater than the respective controls ( p < 0.01; * *p < 0.001, Student's unpaired t- test). 161

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Figure 28. ^ffects of Minimum Essential Medium (MEM) and Williams E (WE) medium on [ H]-thymidine incorporation in control and R—norepinephrine (R-NE) treated primary cultured rat hepatocytes. The results are expressed in three ways: 1) DPM/culture (x 10* ) [bottom panel]; 2) percent of control DPM/culture (top left panel] and 3) percent of maximal response [top left panel]. For all three manners of data expression, each point and bar represents the mean ± S.E.M. of determinations from 4 dishes. Additionally, the responses produced by 10 and 100 jjis. R-NE were significantly greater (p < 0.05) than those produced by the respective controls. Control levels of [ H]-thymidine incorporation were 187378 ± 6783 DPM/culture and 238682 ± 8658 DPM/culture for cells incubated in MEM and WE media, respectively. Asterisks (*) indicate levels of [ H]-thymidine incorporation in cells incubated with WE medium that were significantly greater (p < 0.05, Student's unpaired t-test) than those produced by the same drug treatment in cells incubated with MEM medium. Significant decreases in [ H ]-thymidine incorporation levels caused by 1 pM prazosin (PRZ) relative to the respective 100 pM R-NE responses in each incubation condition aure indicated by letters and treatments with different letters are significantly different (p < 0.05, Student-Newman-Keuls test) from each other. 162

HD CONTROL 1 2 0 0 -r Z m R-N E (100 yu-M) O CS3 R-NE (100 /iM) + rO 1000 -- PRAZOSIN (1 /iM ) O D_ Z 800 ■ • o o LÜ Z bJ =) 6 0 0 "

4 0 0 " 1 1 CL Oi 20Ô X ro 0 PRIMARIA PLATES COLLAGEN-COATED PLATES

Figure 29. Effects of dish surfaces on ( H]-thymidine incorporation in control and 100 R-norepinephrine (R-NE) treated primary cultured rat hepatocytes. The cells were incubated on either Primaria or collagen- coated plates. Results are expressed as DPM/culture (x 10" ). Each beu; represents the mean ± S.E.M. of determinations from 4 dishes. Asterisks indicate that the means of the cells treated with 100 fjii R-NE are significantly greater,^han the respective controls and 1 fM prazosin treated hepatocytes ( p < 0.001, Student's unpaired t-test). 163

O— O-NONMUUmE 0—0 -mtSKMCBE 3 5 0 - r # — • -NlUAUDE 300-■

200t R-JC + PRZ(1 lOI) CD-NawuaoE. s R-œ+PRZ(i

—7 —6 “ S - — 7 —6 —S • ifls R-tE coNŒKnanoN 00 U9C R-4E CONCEJflRXRON (U)

25% 0- O - N O N K U ^

R - œ + P ! C ( 1 )ili g 1000-■ a e a-NONMUUŒ

? a 500 '

—7 —6 —5 • Lûe COKCENTRXnOK (II)

Figure 30. Effects of 50 [M nialamide treatment on [ H]-thymidine incorporation in control and R-norepinephrine (R-NE) treated primary cultured rat hepatocytes. The results are expressed in three ways: 1) DPM/culture (x 10" ) [bottom panel]; 2) percent of control DPM/culture [top left panel] and 3) percent of maximal response [top left panel]. For all three manners of data expression, each point and bar represents the mean ± S.E.M. of determinations from 3 dishes. Additionally, the responses produced by 1 ;ÎM and greater concentrations of R-NE were significantly greater (p < 0.05) than those produced by the respective controls. The control level of [ H]-thymidine incorporation was 789299 ± 38857 DPM/culture and 842105 ± 9520 DPM/culture for cells not incubated and incubated with 50 ;^M nialamide, respectively. Asterisks (*) indicate levels of [ H]-thymidine incorporation in cells incubated with SO ;zM nialamide that were significantly different (p < 0.05) than those produced by the same drug treatment in cells not incubated with nialamide. Significant decreases (p < 0.05, Student-Newman-Keuls test) in [ a ]-thymidine incorporation levels caused by 1 pM prazosin (PRZ) relative to the respective 10 juM R-NE responses in each incubation condition are indicated by the letter a. 164

u CONCENTRATION-RESPONSE CURVES: []l200 DEPICTED BY UNES tIO i i iQ 6 Q' s 10 0 0 + r - n e (1 0 0 /iM) 4- PRZ (1 jM) 8 io 0_ ■ DEPICTED BY BARS ■< o 800 r*n 8ro -6 S|X m Is 600 g X - 4 m 400 X -2 § 200 \ o_ -4- II Q —8 —7 —6 —5 —4 -3 LOG R-NE CONCENTRATION (M)

Figure 31. Concentration-dependent effects of R-norepinephrine (R-NE) on [ H]-thymidine incorporation in primary cultured rat hepatocytes. For a comparison of two means of expressing the data, the results from this experiment are shown as DPM/culture (x 10" ) [left ordinate] and DPM/pg protein (x 10" ) [right ordinate]. Each point of the concentration-response curves represents the mean ± S.E.M. of determinations from 4 dishes. For concentrations of R-NE that were 3 and higher, the levels of [ H]-thymidine incorporation were significantly greater (p < 0,05, Student-Newman-Keuls test) than control values. Control levels of [ S]-thymidine incorporation were 435781 ± 27860 DPM/culture and 5253 ± 367 DPM/fig protein. Each bar, which depicts the inhibition of [ E]-thymidine incorporation by 1 fzM prazosin (PRZ), represents the mean ± S.E.M. of determinations from 4 dishes. Asterisks indicate that the mean responses of the cells exposed to 100 pM R-NE and 1 jM PRZ were significantly less (***p < 0.001, Student's unpaired t-test) than those exposed to 100 R-NE alone. 165

250 T CONCENTRATION-RESPONSE CURVES t 250 I bJ en O -----O - DPM/CULTURE s p § • ----- • - OPM/^tg PROTEIN o o_ 200 g c en 200 -- O w û. z o i c: ~n •D o _I %; u o 150-- T 150 o z o S z w 3 z o ü 100 -100 O a u. r t ; o R-NE + PRZ (1 fiM) I— g 5 0 - n n - DPM/CULTURE - 5 0 rx j - DPM//ig PROTEIN z g M LÜ Q. lio —8 —7 —6 —5 —4 - 3 en LOG R-NE CONCENTRATION (M)

Figure 32. Concentration-dependent effects of R-norepinephrine (R-NE) on [ H ]-thymidine incorporation (left ordinate) and total cellular protein (right ordinate) in primary cultured rat hepatocytes. For the [ H]-thymidine incorporation data in this graph, the results shown in Fig. 32 are now transformed to a percent of the respective control (expressed either as DPM/culture or DPM/pg protein). The total cellular protein was calculated from the same cells used to determine the effects of R-NE on DNA synthesis shown in this graph and was expressed aa a percent of the control value (84 ± 4 pg protein/dish ). Each point of the concentration-response curves represents the mean ± S.E.M. of determinations from 4 dishes. For concentrations of R-NE that were 3 pM and higher, the levels of both [ H]-thymidine incorporation and total cellular protein were significantly greater (p < 0.05, Student-Newman- Keuls test) than control values. Asterisks indicate that for a given treatment, when the data was expressed as Dg^/culture, the percent of control response is significantly greater ( p < 0.01, Student's unpaired t-test) than the percent of control response obtained when the data was originally expressed as DPM/ pg protein. Each bar, which depicts the inhibition of [ H]-thymidine incorporation by 1 pH prazosin (PRZ), represents the mean ± S.E.M. of determinations from 4 dishes. Asterisks indicate that the mean responses of tjie cells exposed to 100 pM R-NE and 1 pH PRZ were significantly less (* ’p < 0.001, Student's unpaired t-test) than those exposed to 100 R-NE alone. 166

120j CONCENTRATION-RESPONSE CURVES Z LU g (/) O---- O - DPM/CULTURE Z 100 -- !< O • ---- • - DPM//ig PROTEIN CL Q. o 80 CL [3 R-NE -i- PRZ (1 iM) X 0: 60-- n j - DPM/CULTURE K j - DPM//ig PROTEIN ii 40-- <

ü_ 2 0 -- o *** > X f— 0 ------r ------# ------1 LU X X I ^ -204- " , 2 ¥ -4 0 —8 —7 —6 —5 —4 - 3 LOG R-NE CONCENTRATION (M)

Figuje 33. Concentration-dependent effects of R-norepinephrine (R-NE) on [ H]-thymidine incorporation in primary cultured rat hepatocytes. For the [ H]-thymidine incorporation data in this graph, the results shown in Fig. 32 are now transformed to a percent of the maximal response (expressed either as DPM/culture or DPM/pg protein). Each point of the concentration-response curves represents the mean ± S.E.M. of determinations from 4 dishes. For concentrations of R-NE that were 3 f/M and higher, the levels of [ H]-thymidine incorporation were significantly greater (p < 0.05) than control values. When the data expressed as DPM/culture and DPM/pg protein is transformed to percent of maximal response, none of the levels of [ H]-thymidine incorporation were significantly different (p < 0.05, Student-Newmcui-Keuls test) for each concentration of R-NE. Each bar, which depicts the inhibition of [ H]-thymidine incoirporation by 1 f(M prazosin (PRZ), represents the mean ± S.E.M. of determinations from 4 dishes. Asterisks indicate that the mean responses of the,cells exposed to 100 fjM R-NE and 1 ^a^ PRZ were significantly less ( p < 0.001, Student's unpaired t-test) than those exposed to 100 R-NE alone. 167

ANTAGONIST CONC. 120 T C 3 - 0.01 /«U I E S - 0.1 /111 100 ” m - iJjjuM I m - 10/iM Ë 80 O ■■ i 60" O o 40" I 2 0 " PRAZOSIN ^ H IU B IN E

w 1 4 0 t ANTAGONBT CONC. C J -0,01/M £I 120 -0.1 jM “i 100 m -1.0 ^ X z m - 10 Adi 80" §\ \ / o 60" \ / Ü. \ o 40" \ X

20 " \ / \ I / \ i£s- mwzosiN YOHIUBINE PRWRANOLOLI

Figure 34. Effects of tk? adrenergic receptor antagonists prazosin, yohimbine and propranolol on [ H]-thymidine incorporation stimulated by 10 pM R-norepinephrine [bottom panel] and 100 ptK 2-(3,4-dihydroxy- benzyl)imidazoline (DHT) [top panel] in primary cultured rat hepatocytes. Results are expressed as a percentage of the response produced by either 10 pM R-NE or 100 pM DHT and each bar represents the inpan ± S.E.If;^ of determinations from 3-4 dishes. Asterisks ( p < 0.05; **p < 0.01; p < 0.001, Student's unpaired t-test) indicate that the mean of the stimulating drug plus inhibitor treatments is significantly less than the mean of the treatments with stimulating drug alone. The control level of [ H]-thymidine incorporation was 186809 ± 19361 DPM/culture and cells treated with 10 pM R-NE and 100 pM DHT incorporated [ H]-thymidine at a level of 250.3 ± 13.9% and 242.1 ± 12.2% (mean ± S.E.M.) of control. 168

5 ^ '

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Figure 35. Autoradiographs of primary cultured hepatocytes incubated with [ H]-thymidine (5 pCi/ml) for 48 hr. Top: Control cells; Middle: cells treated with 10 R-NE; and Bottom: Cells treated with 10 pM R-NE + 1 prazosin. Magnification is lOOX. Labelled nuclei are seen as those which cause the precipitation of silver grains from the photographic emulsion resulting in the presence of a black deposit over the nucleus. 169

Figure 36. Autoradiographs of primary cultured hepatocytes incubated with [ H]-thymidine (5 pCi/ml) for 48 hr. Top: Control cells; Middle: Cells treated with 10 pM R-NE; and Bottom: Cells treated with 10 pM R-NE + 1 pM prazosin. Magnification is 200X. Labelled nuclei are seen as those which cause the precipitation of silver grains from the photographic emulsion resulting in the presence of a black deposit over the nucleus. REFERENCES Aggerbeck M, Ferry N, Zafrani E.-S., Billon MC, Barouki R and Hanoune J. Adrenergic regulation of glycogenolysis in rat liver after cholestasis; modulation of the balance between a.- and B„-receptors• J. Clin. Invest. 71: 476-486, 1983. Aggerbeck M, Guellaen 6 and Hanoune J. The a-adrenergic mediated effect in rat liver. Biochem. Pharmacol. 29: 1653-1662, 1980. Ahlguist RP. A study of the adrenotropic receptors. Am. J. Physiol. 153: 586-600, 1948. Ahn C-H, Hamada A, Miller DD and Feller DR. o-Adrenergic-mediated actions of optical isomers and desoxy analogs of catecholimidazoline and norepinephrine in human platelets: in vitro. Biochem. Pharmacol. 35: 4095-4102, 1986. Alexander RW, Saydjari R, MacLellan DG, Townsend CM and Thompson JC. Calmodulin antagonist trifluoperazine inhibits polyamine synthesis and liver regeneration. Br. J. Surq. 75; 1160-1162, 1988. Ashrif S, Gillespie JS and Pollack D. The effects of drugs or denervation on thymidine uptake into regenerating liver. Eur. J. Pharmacol. 29; 324-327, 1974. Assoian RK, Roberts AB, Wakefield LM, Anzano MA and Sporn MB. Transforming growth factors in nonneoplastic tissues and their role in controlling cell growth. Cancer Cells, Vol. 3: Growth Factors and Transformation, ed. by J. Feramisco, B. Oxanne and C. Stiles, pp. 15-26, Cold Spring Harbor Press, Cold Spring Harbor, NY, 1985. Banning JW, Rice PJ, Miller DD, Ruffolo RR, Hamada A and Patil PN. Differences in the adrenoreceptor activation by stereoisomerio catecholimidazolines and catecholamines. Neuronal and Extraneuronal Events in Autonomic Pharmacology, ed. by W.W. Fleming, S.Z. Langer, K.H. Grafe and N. Weiner, pp. 167-180, Raven Press, New York, 1984. Beguinot L, Hanover JA, Ito S, Richert ND, Willingham MC and Pastan I. Phorbol esters induce transient internalization without degradation of unoccupied epidermal growth factor receptors. Proc. Natl. Acad. Sci. U.S.A., 82: 2774-2778, 1985. Berthelsen S and Pettinger WA. A functional basis for the classification of a-adrenergic receptors. Life Sci. 21; 595-606, 1977. Besse JC and Furchgott RF. Dissociation constants and relative efficacies of agonists acting on a-adrenergic receptors in rabbit aorta. J. Pharmacol. Exp. Ther. 197: 66-78, 1976. Bevan JA. A comparison of the contractile responses of the rabbit basilar and pulmonary arteries to sympathomimetic agonists: Further evidence for variation in vascular adrenoceptor characteristics. J. Pharmacol. Exp. Ther. 216: 83-89, 1981.

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