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Xerox University Microfilms 300 North Zeeb Road Ann Arbor, Michigan 48106 77-2473 O ’NEILL, Patrick Joseph, 1949- ACETALDEHYDE AND NEUROAMINE-DERIVED TETRAHYDROISOQUINOLINE ALKALOIDS: ROLE IN ALCOHOL TOXICITY AND DEPENDENCE. The Ohio State University, Ph.D., 1976 Pharmacology Xerox University Microfilms, Ann Arbor, Michigan 48106 ACETALDEHYDE AND NEUROAMINE-DERIVED TETRAHYDROISOQUINOLINE ALKALOIDS: ROLE IN ALCOHOL TOXICITY AND DEPENDENCE DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University by Patrick Joseph O'Neill, B.S ***** The Ohio State University 1976 Reading Committee: Approved By Dennis R. Feller Duane D. Miller Ralf G. Rahwan Harold H. Wolf Adviser College of Pharmacy ACKNOWLEDGMENTS Dr. Ralf G. Rahwan, for providing direction and inspiration throughout my graduate career, both in the laboratory and in the classroom. My wife, Janie, for her constant support, encouragement, and understanding. My parents, for encouraging academic achievement. Drs. Dennis R. Feller, Duane D. Miller, and Harold H. Wolf, for helpful suggestions during this work. The American Foundation for Pharmaceutical Education, for financial support during certain phases of this work. ii VITA July 12, 1949 Born-Columbus, Ohio 1972................... ...... B.S. with distinction in Pharmacy, The Ohio State University, Columbus, Ohio 1972-1974..................... Teaching Assistant, College of Pharmacy, The Ohio State University, Columbus, Ohio 1974-197 5..................... Research Assistant, College of Pharmacy, The Ohio State University, Columbus, Ohio 1975-197 6 ..................... Abe Plough Citation Fellow of The American Foundation for Pharmaceutical Education PUBLICATIONS "Pharmacological Investigations with Chlorpheniramine Isomers." B.S. Degree with Distinction, The Ohio State University, 1972. "Differential Secretion of Catecholamines and Tetrahydro- isoquinoline Alkaloids from the Bovine Adrenal Medulla." Fed. Proc. 3^:497, 1974. "Differential Secretion of Catecholamines and Tetra- hydroisoquinoline Alkaloids from the Bovine Adrenal Medulla." Life Sci. 14:1927-1938, 1974. "Stereoisomers of an Antihistamine and the Pharmacologic Receptors of Rabbit Aorta." Pharmacological Res. Comm. 7:273-279, 1975. "Experimental Evidence for Calcium-independent Catechol­ amine Secretion from the Bovine Adrenal Medulla." Fed. Proc. 3£:739' 1975. "Experimental Evidence for Calcium-independent Catechol­ amine Secretion from the Bovine Adrenal Medulla." J. Pharmacol. Exp. Ther. 193:513-522, 1975. iii "Protection Against Acute Toxicity of Acetaldehyde in Mice." Res. Conun. Chem. Path. Pharmacol. 13:125- 128, 1976. "Absence of Formation of Brain Salsolinol During Chronic Ethanol Administration to Mice." Pharmacologist. In press, 1976. "A Modified Gas Chromatographic/Electron Capture Assay Method for Salsolinol in Brain Tissue." Submitted for publication. "Absence of Formation of Brain Salsolinol in Ethanol- dependent Mice." Submitted for publication. FIELDS OF STUDY Major Field: Pharmacology Pharmacology of Ethanol and Acetaldehyde Mechanisms of Stimulus-Secretion Coupling Toxicology TABLE OF CONTENTS Page ACKNOWLEDGMENTS........................................ H VITA ................................. ii;i- LIST OF TABLES......................................... viii LIST OF FIGURES........................................ ** Chapter I. INTRODUCTION 1.1 Interaction of Ethanol with Neuro- transmitter Systems...................... 1 1.2 The Tetrahydroisoguinoline Alkaloid Hypotheses................................ 5 1.3 Biological Formation and Inhibition of Formation of Tetrahydroisoquinoline Alkaloids................................. 13 1.4 Pharmacology of the Tetrahydroiso- quinolines................................ 21 1.5 Statement of the Problem................ 27 II. METHODS AND MATERIALS M .1 Synthesis and Release of Tetrahydro­ isoquinoline Alkaloids from the Bovine Adrenal Medulla.......................... 31 M.2 Role of Intracellular Calcium in Acetaldehyde-induced Catecholamine Release from the Adrenal Medulla 36 M.3 Protection Against Acute Toxicity of Acetaldehyde in Mice..................... 3!) M.4 Chronic Alcohol Administration Experiments............................... v Page M.5 Chemical Determinations M.5.1 Measurement of Catechol­ amines and TIQs by Thin-layer Chromatography....................... 44 M.S.2 Measurement of Catechol­ amines by Colorimetry............... 46 M.S.3 Measurement of Protein in Sub- cellular Fractions of Bovine Adrenal Medulla...................... 47 45 M.S.4 Determination of Ca in Sub- cellular Fractions of Bovine Adrenal Medulla...................... 48 M.5.5 Measurement of Mitochondrial Succinic Cytochrome c Reductase in Sucrose Density Gradient Fractions of Bovine Adrenal Medulla............................... 49 M.5.6 Determination of Ethanol in Blood and Chamber Air................ 50 M.S.7 Analysis of Whole Brain Salsolinol............................ 51 M.5.8 Analysis of Salsolinol in Brain Parts...........„.............. 55 III. RESULTS R.l Synthesis and Release of Tetrahydro- isoquinoline Alkaloids from the Bovine Adrenal Medulla.......................... 56 R.2 Role of Intracellular Calcium in Acet- aldehyde-induced Catecholamine Release from the Adrenal Medulla................. 5 2 R.2.1 Concentration-Effect Relation­ ships................................. 62 R.2.2 Effect of Magnesium............. 65 R.2.3 Release of Catecholamines...... 65 R.2.4 Release of ^ C a .................. 58 vi Page R.2.5 Subcellular Distribution of Mitochondria and Chromaffin Gran­ ules . 71 R.2.6 Subcellular Distribution of 45Ca ............. 76 R.3 Protection Against Acute Toxicity of Acetaldehyde in Mice..................... 78 R.4 Absence of Formation of Brain Salsol­ inol in Ethanol-dependent Mice......... 83 R.4.1 Chamber Alcohol/Blood Alcohol Relationship......................... 83 R.4.2 Establishment of Physical Dependence............................ 90 R.4.3 Analysis of Single Whole Brains for Salsolinol....................... 93 R.4.4 Analysis of Pooled Whole Brains for Salsolinol........................ 109 R.4.5 Analysis of Pooled Brain Parts for Salsolinol........................ 109 IV. DISCUSSION D.l Synthesis and Release of Tetrahydro- isoquinoline Alkaloids from the Bovine Adrenal Medulla.................... 116 D.2 Role of Intracellular Calcium in Acetaldehyde-induced Catecholamine Release from the Adrenal Medulla......... 118 D.3 Protection Against Acute Toxicity of Acetaldehyde in Mice...................... 123 D .4 Absence of Formation of Brain Salsol­ inol in Ethanol-dependent Mice.......... 125 BIBLIOGRAPHY 128 LIST OF TABLES Table Page 1. Scoring System for Convulsions Elicited by Lifting a Mouse by theTail ................... 45 2. Enzyme Distribution in Sucrose Density Gradient Fractions of Bovine Adrenal Medulla......................................... 72 3. Effect of Acetaldehyde on Subcellular Distribution of 45ca in Isolated Bovine Adrenal Medulla................................ 77 4. Effect of Protecting Agents on Acute Toxicity of Acetaldehyde in Mice and Rats... 81 5. Retention Times and Recoveries for Tyramine, Dopamine and Salsolinol......... ............. 103 6. Summary of Chronic Alcohol Administration Studies (single brain experiments).......... 10 4 LIST OF FIGURES Figure Page 1. Structures and Routes of Formation of the Tetrahydroisoquinoline Alkaloids......... 8 2 . Schematic Representation of the Apparatus Used to Deliver Alcohol Vapors to the Test Mice.................................... 42 3. Flowsheet