This dissertation has been microfilmed exactly as received 69*22,223 WALSH, Michael Joseph, 1942- ACETALDEHYDE- INDUCED RELEASE AND ALTERS] METABOLISM OF CATECHOLAMINES: ITS ROLE IN ETHANOL ACTIONS AND THE DISULFIRAM- ALCOHOL REACTION. The Ohio State University, Ph.D., 1969 Pharmacology University Microfilms, Inc., Ann Arbor, Michigan ACETALDEHYDE-INDUCED RELEASE AND ALTERED METABOLISM OF CATECHOLAMINES: ITS ROLE IN ETHANOL ACTIONS AND THE DISULFIRAM - ALCOHOL REACTION DISSER TATION Presented in Partial Fulfil ment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohi d State University By Michael Joseph Walsh, B. S. The Ohio State University 1969 Approved by JS-QjuJBrkAx> Adviser ' Department of Pharmacology ACKNOWLEDGMENTS I want to express my gratitude first and formost, to my wife, Estelle Marilyn Walsh, for her constant encouragement, thought­ fulness, and assistance; and to dedicate this volume to Estelle in acknowledgment of her understanding and moral support, without which this research would not have been possible or purposeful. I certainly am indebted to Professor Edward B. Truitt, Jr., for his excellent advice a id training. He has remained a good friend, advisor, and teac ler throughout my graduate school years, I hope his qualities of unc erstanding and encouragement as well as his constructive critical ability have become permanently impressed on me. I am especially gra eful to Dr. Philip B. Hollander for his willingness to be my teac ler. His friendship and training and col­ laboration on certain experiments has certainly benefited my back­ ground as well as this dis sertation. Professor Bernard H. Marks has been a constant source of enlightment. As Professor,u and head of the Department of Pharmacol­ ogy, he occupies a unique position and interacts in a special capacity with all graduate students. His encouragement and sincere interest in my research and all graduate student problems is greatly appre­ ciated. Ultimately, my gratitude to the Department of Pharmacology and the graduate committee must be expressed. I will always appreciate the readiness of this department to accept me as a student transferring from another University. In retrospect, I sincerely feel that continuing my graduate studies in the Department of Pharmacology of The Ohio State University may have been the most important decision of my life. I am sure that this change has been most beneficial to my training as a pharmacologist, and hope my work has been mutually beneficial to the department. A graduate student is subject to many helping hands, there­ fore, I wish to acknowledge the frequent stimulating discussions, and constructive and helpful criticisms and comradships of the other faculty members: Drs. Daniel Couri, Rose Dagirmanjian, Saradindu Dutta, Robert Gardier, Harold Goldman and John Lindower, and especially of my fellow graduate students. The permission to perform experiments and utilize the facilities of B attelle Memorial Institute was greatly appreciated, For the effort s of Dr. Joseph Boatman, head of the Division of Physiology and Pharmacology, in my behalf, I am grateful. The efforts of Mrs. Joan Rotaru and Miss Joyce Berio for their excellent technical assistance in many phases of this research is appreciatively acknowledged. The helping hand of Daria Cverna, in both preparation of this manuscript and with any problems which arose during my graduate school days, will always be remembered. Also, the frequent sec­ retarial help of Mrs. Carol Jones and Miss Millie Flanagan of The Ohio State University, and Mrs. Nancy Handel and Miss Sharon Wells of Battelle Memorial Institute was greatly appreciated. Finally, I would like to acknowledge and express my thanks to the National Institutes of Health ( NIH) for monetary support in the form of fellow ships during my training in Pharmacology. VITA Michael Joseph Walsh Born: York, Pennsylvania April 18, 1942 Graduate Calvert Hall College High School, Baltimore, Maryland June, I960 B. S. in Pharmacy, University of Maryland, School of Pharmacy June, 1965 Ph. D. The Ohio State University June, 1969 NIH Predoctoral Trainee, Pharmacology Training Grant GM 01417 October, 1966 to June, 1969 PUBLICATIONS AND PRESENTATIONS Walsh, M. J. and Truitt, E. B. , Jr. , : Mechanism of the Effect of Disulfiram on the Sympathomimetic Action of Acetaldehyde, Fed. Proc, 26; 293, ( 1967) Abstract. Walsh, M. J. and Truitt, E. B. , Jr.; Confirmation of the Catechol­ amine Releasing Effects of Acetaldehyde and Ethanol by a .new Technique Utilizing 7-H 3 -Norepinephrine, Presented to the Ohio Valley Section of the Society for Experimental Biology and Medicine, Lexington, Kentucky, November 17, 1967. Truitt, E. B. , Jr. and Walsh, M. J. : Alcohol, Acetaldehyde and the Sympathetic Nervous System, Presented at a Symposium on Alcoholism - 1968, Battelle Memorial Institute, March 13, 1968. Walsh, M. J. and Truitt, E. B., Jr.; Release of 7-H3-Norepinephrine in Plasma and Urine by Acetaldehyde and Ethanol in Cats and Rabbits, Federation Proceedings, 27; 601 ( 1968) Abstract. Truitt, E, B., Jr. and Walsh, M. J . : The Biochemical Pharmacology of Alcoholism, Battelle Technical Review, 17; 8 , 3-8 August, 1968. Walsh, M. J,, Truitt, E. B., Jr., and Hollander, P. B. : Adrener­ gic Effects of Acetaldehyde on Mechanical and Electrical Properties of Cardiac Muscle, Pharmacologist, 10: 183 ( 1968) Abstract. Walsh, M. J., Truitt, E.B., J r., and Hollander, P. B. : Sympath­ omimetic Effects of Acetaldehyde on Electrical and Mechan­ ical Characteristics of the Isolated Left Atria of Guinea Pigs, J. Pharmacol. Exp. Ther. 167; 173-186, 1969. Walsh, M. J., The Biochemical Pharmacology of Alcoholism: I. Acetaldehyde and Ethanol Metabolism and Effects on Lipids and Glycogenolysis, Presented at a Special Seminar at the University of Houston, Department of Pharmacology, School of Pharmacy, Houston, Texas, October 7, 1968. Walsh, M. J .: The Biochemical Pharmacology of Alcoholism: II. Role of Acetaldehyde in the Disulfiram-Alcohol Reaction. Presented at a Spec,ial Seminar at The University of Houston, Department of Pharmacology, School of Pharmacy, Houston, Texas, October 14, 1968, Walsh, M. J . : The Effects of Acetaldehyde on Contractility, Intra­ cellular Potentials and Catecholamine Release. Special Biochemistry Seminar, Department of Biochemistry, Bay­ lor University, Medical School, Houston, Texas, October 21, 1968 . Truitt, E. B ,, Jr. and Walsh, M. J. : The Role of Acetaldehyde in the Actions of Alcohol, The Biology of Alcohol, Volume I, Biochemistry, Chapter 7, to be published in 1969. Walsh, M. J. and Truitt, E. B ., Jr. : Acetaldehyde Mediation in the Mechanism of Ethanol-Induced Changes in Catecholamine Metabolism, Federation Proceedings, 28; 543 ( 1969) Abstract, Walsh, M. J .: Catecholamine Release and Altered Metabolism of Norepinephrine by Acetaldehyde. Presented at a Special Pharmacology Seminar, Battelle Memorial Institute, Col­ umbus, Ohio, May 9, 1969. v FIELDS OF STUDY Major Field; Pharmacology Autonomic Pharmacology B. H. M arks and R. Dagirmanjian Bioelectric Potentials P. B. Hollander Cardiovascular Pharmacol. S. Dutta Neuroendocrine Pharmacol. H. Goldman Drug Metabolism D. Couri, J. Merola and R. Ober Toxicology D. Couri Minor Fields; Neuromuscular Physiology E. Bozler Reproductive Physiology C. Barraclough ( Univ. of Maryland) Neurochemistry J. Allen and R. McCluer Biophysics L. Mullins and R. Sjodin ( Univ. of M aryland) TABLE OF CONTENTS Page ACKNOWLEDGMENTS ii VITA iv LIST OF TABLES x LIST OF FIGURES xiii INTRODUCTION - - 1 General Concepts 1 Scope and Purpose of the Problem 5 Acetaldehyde Measurement and Levels 7 Interactions of Ethanol and Acetaldehyde with Amine Stores 15 Adrenergic Actions of Ethanol or Acetaldehyde 21 The Disulfiram-Alcohol Reaction 27 Effects of Ethanol on Biogenic Amine Metabolism 33 Interactions of Amines, Central Acting Drugs and Ethanol Hypnosis 38 METHODS 42 Gas Chromatographic Determination of Ethanol and Acetaldehyde 42 A. General Procedures 42 B. Analysis of Biological Fluids 45 C. Excretion in Expired Air 49 D. Tissue Concentrations 50 In Vitro Release of Norepinephrine 53 A. General Procedures for Preparation of Atria 53 B. Experimental Conditions '55 C, Potential and Tension M easurem ents 58 D, Pharmacological Agents 58 vii METHODS (Continued) Page In Vivo Release of H3-Norepinephrine 60 A. General Preparations 60 B. Animal Equilibration 62 C. Injection, Sampling and Assay Procedure 63 D. Verification of Neuronal Uptake and Release 63 Autonomic Function in Whole Animals 64 Cl4-Norepinephrine and Metabolites 67 A. Administration of C14-Norepinephrine __ and Sample Collection 67 B. Analysis Interval 6 8 C. Treatment Schedules 70 D. General Procedures 72 E. Determination of C14-Norepinephrine and Normetanephrine 73 F. Determination of Deaminated C14- NE Metabolites 75 G. Identification of C14-Norepinephrine and Metabolites. 78 Evaluation of Central Nervous System Effects 80 RESULTS 82 Ethanol and Acetaldehyde Levels 82 A. In Vitro Production in Whole Blood 82 B. Human Filtrate Levels 82 C. Blood and Tissue Concentrations in Rats 84 D. Urine and Expired Air 96 Experiments with the Isolated Guinea Pig Left Atria 105 A. Controls 105 B. Effects of Norepinephrine (N E)and Tyramine 105 C. Mechanical Effects of Acetaldehyde 108 D. Electrical Effects of Acetaldehyde 115 E. Effects of Reserpine 115 F. Propranolol Blockade 118 H3-Norepinephrine Release and Blood Pressure Measurements in Whole Animals 121 A. Plasma Tritium in Cats 121 B. Blood