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University Microfilms 300 North Zwb Road Ann Arbor, Michigan 40106 a Xarox Education Company I I INFORMATION TO USERS This dissertation was produced from a microfilm copy of the original document. While the most advanced technological means to photograph and reproduce this document have been used, the quality is heavily dependent upon the quality of the original submitted. The following explanation of techniques is provided to help you understand markings or patterns which may appear on this reproduction. 1. The sign or "target" for pages apparently lacking from the document photographed is "Missing Page(s)''. If it was possible to obtain the missing pagels} or section, they are spliced into the film along with adjacent pages. This may have necessitated cutting thru an image and duplicating adjacent pages to insure you complete continuity. 2. When an image on the film is obliterated with a large round black mark, it is an indication that the photographer suspected that the copy may have moved during exposure and thus cause a blurred image. You will find a good image of the page in the adjacent frame. 3. When a map, drawing or chart, etc., was part of the material being photographed the photographer foflowed a definite method in "sectioning" the material. It is customary to begin photoing at the upper left hand corner of a large sheet and to continue photoing from left to right in equal sections with a small overlap. If necessary, sectioning is continued again — beginning below the first row and continuing on until complete. 4. The majority of users indicate that the textual content is of greatest value, however, a somewhat higher quality reproduction could be made from "photographs" if essential to the understanding of the dissertation. Silver prints of "photographs" may be ordered at additional charge by writing the Order Department, giving the catalog number, title, author and specific pages you wish reproduced. University Microfilms 300 North Zwb Road Ann Arbor, Michigan 40106 A Xarox Education Company I I 72-20,974 KRELL, Robert Donald, 1943- THE UPTAKE, STORAGE AND RELEASE OF THE OPTICAL ISOMERS OF N0REPINEPHRINE-14C IN TISSUES OF THE RAT. The Ohio State University, Ph.D., 1972 Pharmacology University Microfilms, A XEROX Com pany, Ann Arbor, Michigan THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED. THE UPTAKE, STORAGE AND RELEASE OF THE OPTICAL ISOMERS OF NOREPINEPHRINE-14C IN TISSUES O F THE RAT DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University by R obert D. K re ll, B. S. ****************** The Ohio State University 1972 Approved by -Jlpajf/I y/jjk' f t A dvisor College of Pharmacy PLEASE NOTE: Some pages may have indistinct print. Filmed as re c eiv e d . University Microfilms, A Xerox Education Company "Science has but one language, that of quantity, and but one argument, that of experiment." Ernest Henry Starling ti ACKNOWLEDGMENTS I wish to express my sincere appreciation to DR. POPAT N. PATIL for his careful instruction and guidance in both laboratory and classroom, for his support, and for fostering an intellectually stimulating environment in which to learn MY WIFE, BECKY for her sacrifices, encouragement, endurance and understanding, and for making this undertaking both successful and enjoyable MY PARENTS for their financial support, and for encouraging me to pursue intellectual challenges MR. O. P. SETHI for technical assistance in certain phases of this re s e a rc h iii VITA D ecem b er 2 , 1943 Born - Toledo, Ohio 1 9 6 6 . ................................. B. S. in Pharm. , University of Toledo, Toledo, Ohio 1966-196 7 ................................. Teaching Assistant, College of Pharmacy, The Ohio State University, Columbus, Ohio 1967-19G 9 ................................. Research Assistant, College of Pharmacy, The Ohio State University, Columbus, Ohio 1969-197 0 ................................. Fellow of the American Foundation for Pharmaceutical Education 1970-197 1 ................................. Fellow of The Ohio State University, Columbus, Ohio 1971-197 2 ................................. Research Associate, College of Pharmacy, The Ohio State University, Columbus, Ohio PUBLICATIONS "Combination of Alpha and Beta Adrenergic Blockers in the Isolated Guinea-Pig Atrium." Fed. Proc. 28:481, 1969. "Combination of Alpha and Beta Adrenergic Blockers in Isolated Guinea-Pig A tria." J. Pharmacol. Exp. Ther. 170: 262-271, 1969. "The Sensitivity of Rabbit Aorta, Atria and Ileum to Various Agonists after Repeated Doses of (-)-Ephedrine and Related Amines." Arch. Int. Pharmacodyn. 188: 257-270, 1970. "Steric Aspects of Adrenergic Drugs XV. Use of Isomeric Activity Ratio as a Criterion to Differentiate Adrenergic Receptors." J. Pharmacol. Exp. Ther. 176: 622-633, 1971. "Uptake and Subcellular Distribution of d- and i- fc 147-Norepinephrine (NE-C14)." Fed. Proc. 30: 445, 1971. iv "Steric Aspects of Adrenergic Drugs XIX. Influence of Various Agents and Procedures on the Stereoselectivity of Pre- and Post-Junctional Sites to Norepinephrine Isomers in Isolated Rat Vasa Deferentia." J. Pharmacol. Exp. Ther. In press, 1972. FIELDS OF STUDY Major Field: Pharmacology Structure-Activity Relationships of Adrenergic Drugs Adrenergic Mechanisms Biochemistry of the Sympathetic Nervous System Neonatal Autonomic Pharmacology Associate Professor Popat N. Patil v TABLE OF CONTENTS Page ACKNOWLEDGMENTS............................................................................................... ill VTTA................................................................................................................................. iv LIST OF TABLES........................................................................................................ ix LIST OF ILLUSTRATIONS......................................................................................... x C hapter I. INTRODUCTION The Discovery of "Uptake" and Subsequent Develop­ ments in "Uptake" Theory............................................................... 1 Stereoselectivity of the Adrenergic Neuronal Membrane Transport Site for the Optical Isomers of Epinephrine and Norepinephrine.............................................. 6 Stereoselectivity of Intraneuronal Mechanisms of Inactivation for the Optical Isomers of Epinephrine and Norepinephrine.................................................. 12 Statement of the Problem .................................................................... 16 H. METHODS AND MATERIALS General Considerations...................................................................... 19 Cold-Storage of Vasa Deferentia.................................................... 19 Surgical Denervation of Vasa Deferentia ..................................... 20 Determination of Radiochemical Purity of the Iso m e rs.................................................................................................. 20 Pretreatment with Various Agents .................................................. 21 Determination of the Subcellular Distribution Rittern of Endogenous Norepinephrine in Vasa Deferentia 21 vi Page Determination of the Accumulation and Subcellular Distribution Pattern of (-)- and ^-N orepin­ ephrine-1^ in Isolated Vasa Deferentia...................................... 23 Determination of the Accumulation of (-)- and (+)-Norepinephrine-i4c slices of Ventricle, Spleen and Vas Deferens............................................. * ...................... 24 hi Vivo Infusion of (-)- and (-fJ-Norepinephrine-^C.................... 25 Release of (-)- and ^-Norepinephrine-1^ from Isolated, Superfused Vasa Deferentia ...................... 26 Separation of Metabolites, Sample Preparation and Liquid Scintillation Counting ..................................................................29 Statistical Analysis........................ 30 Drugs and Solutions ........................................................................................30 ID. RESULTS Influence of Varying Homogenization and Centrifugation Parameters on the Subcellular Distribution Pattern of Endogenous Norepinephrine in Vasa Deferentia.................. 32 Influence of Incubation on Endogenous Norepinephrine Content and its Subcellular Distribution Pattern in Isolated Vasa Deferentia............... 34 Accumulation and Subcellular Distribution Pattern of (-)- and (+)-Norepinephrine-14c after Various Periods of Incubation in Isolated Vasa Deferentia .................. 37 Influence of Inhibition of Intraneuronal Mechanisms of Inactivation on the Accumulation of (-)- and (+)-Norepinephrine-1 4c by Isolated Vasa Deferentia 39 Influence of 6 -hydroxy dopamine, Cocaine, Cold-Storage and Surgical Denervation on the Uptake of (-)- and {+)-Norepinephrine-1 4c by Isolated Vasa Deferentia 47 Accumulation of (-)- and ^-norepinephrine-1^ by Slices of Ventricle, Spleen and Vas Deferens......................... 49 vii P age Determination of Kinetic Constants in Normal and Guanethidine, Iproniazid and Tropolone Pretreated Slices of Ventricle, Spleen and Vas Deferens............................ 55 Inhibition of Uptake by Isomers of Cocaine ..................................... 65 In Vivo Infusion of (-)- and (+)-Norepinephrine-14C................... 5 7 "Spontaneous" Release of {-)- and (+)-Norepinephrine-^C from Isolated, Superfused Vasa Deferentia ................................. 71 Tyramine-induced Release of (-)- and ^-N orepin­ ephrine-1^ from the Isolated, Superfused Vasa Deferentia 76 IV. DISCUSSION Inhibition of Litraneuronal Mechanisms of Inactivation
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