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The Effects of Ethanol on Growth Hormone and Prolactin Gene Expression in Male Rats Loyola University Chicago Loyola eCommons Dissertations Theses and Dissertations 1995 The Effects of Ethanol on Growth Hormone and Prolactin Gene Expression in Male Rats John James Tentler Loyola University Chicago Follow this and additional works at: https://ecommons.luc.edu/luc_diss Part of the Molecular Biology Commons Recommended Citation Tentler, John James, "The Effects of Ethanol on Growth Hormone and Prolactin Gene Expression in Male Rats" (1995). Dissertations. 3384. https://ecommons.luc.edu/luc_diss/3384 This Dissertation is brought to you for free and open access by the Theses and Dissertations at Loyola eCommons. It has been accepted for inclusion in Dissertations by an authorized administrator of Loyola eCommons. For more information, please contact [email protected]. This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License. Copyright © 1995 John James Tentler LOYOLA UNIVERSITY CHICAGO THE EFFECTS OF ETHANOL ON GROWTH HORMONE AND PROLACTIN GENE EXPRESSION IN MALE RATS A DISSERTATION SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL IN CANDIDACY FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF MOLECULAR AND CELLULAR BIOCHEMISTRY BY JOHN JAMES TENTLER CHICAGO, ILLINOIS JANUARY 1995 Copyright by John J. Tentler, 1994 All rights reserved ll ACKNOWLEDGEMENTS I would like to recognize numerous people who have contributed throughout my graduate career to help me realize my goal. First, I would especially like to thank my advisors, Drs. Mary Ann Emanuele, Mark R. Kelley, and Nick Emanuele, not only for their guidance and support, but for their close friendship and belief in me as well. Special thanks also go to Dr. Ann M. Lawrence who has served as a role model for me and was always available for support and advice over the years. I would also like to recognize my committee members, Drs. Robert Handa, John Lopes, and Mary Manteuffel for their constructive criticisms and assistance in preparing my dissertation work. I would also like to thank my fellow lab members, Jim Carney, David Wilson Ill, Peg Halloran, Dave Grabowski, Teresa Wilson, Shahabuddin, Lidia Kirsteins, Jen Jurgens, Jenny Steiner and Qin Shi, who, through their assistance and friendship, have made graduate school both an educational and enjoyable experience. Finally, I would like to extend special thanks to my parents, Robert and Helen Tentler, my entire family, and my wife Sangita, for their love, patience, and support, without which this degree would have been difficult to achieve. Ill TABLE OF CONTENTS ACKNOWLEDGMENTS .................................... m LIST OF TABLES . vu LIST OF FIGURES ........................................ viii LIST OF ABBREVIATIONS . x Chapter I. INTRODUCTION . 1 II. REVIEW OF RELATED LITERATURE . 4 A. The Growth Hormone Axis . 4 B. Regulation of GH Synthesis and Secretion .................... 7 C. Growth Hormone-Releasing Hormone (GHRH) . 7 D. Somatostatin (SRIF) . 12 E. The Prolactin (PRL) Axis . 14 F. Regulation of PRL Synthesis apd Secretion . 16 G. Similarities Between GH and PRL . 18 H. Pharmacology of Ethanol . 19 I. Effects of Ethanol on Growth Hormone . 20 J. Effects of Ethanol on Prolactin . 21 K. Alcohol, Biological Membranes, and Second Messenger Systems . 23 III. MATERIALS AND METHODS ............................. 25 A. Animals ........................................ 25 B. Acute Ethanol (EtOH) Administration . 25 C. Tissue Collection . 26 D. Blood Ethanol Determination . 26 E. GH Radioimmunoassay (RIA) . 27 F. PRL Radioimmunoassay (RIA) . 28 G. GHRH Radioimmunoassay (RIA) . 28 H. cAMP Radioimmunoassay (RIA) . 29 I. Tissue RNA Extraction . 30 J. Northern Blot Analysis . 31 K. Labelling of cDNA Probes . 33 L. Densitometric Analysis . 34 M. Reverse Transcription . 35 N. PCR Amplification . 36 0. Nuclear Run-On Transcription Assay . 38 P. Statistics . 40 IV. RESULTS . 41 A. "Binge" Ethanol Animal Model .......................... 41 B. Blood Ethanol Concentrations for Single and Double Binge Models . 41 C. Effects of Single Binge EtOH on GH Protein and mRNA levels . 42 D. Effects of Single Binge EtOH on PRL Protein and mRNA Levels ................................ 49 E. Effects of Double Binge EtOH on GH Protein and mRNA levels ................................ 53 F. Effects of Single and Double Binge EtOH on Hypothalamic Hormones ............................ 56 G. Effects of Acute EtOH on Hormone Transcription Rates ........................................ 65 H. Effects of Acute EtOH on Intrapituitary cAMP Levels ................................... 71 I. Effects of Acute EtOH on c-fos Expression . 73 V. DISCUSSION . 76 A. The Effects of Ethanol on Growth Hormone . 77 B. The Effects of Ethanol on Prolactin . 82 C. The Effects of Ethanol on Hypothalamic Regulatory Hormones . 84 D. Effects of Acute EtOH on Second Messenger Systems in the Pituitary . 85 REFERENCES . 90 LIST OF TABLES Table Page I. Summary of Results in Pituitary . 75 II. Summary of Results in the Hypothalamus . 75 Vll LIST OF FIGURES Figure Page 1. Diagrammatic representation of the growth hormone axis, with feedback loops . 8 2. Location of oligonucleotide primers used for PCR amplification . 37 3. Blood ethanol concentrations after single binge treatment . 43 4. Blood ethanol concentrations after double binge treatment . 44 5. Serum GH levels after single binge EtOH treatment . 45 6. Pituitary GH content after single binge EtOH treatment . 47 7. Pituitary GH steady-state mRNA levels after single binge EtOH treatment . 48 8. Serum PRL levels after single binge EtOH treatment . 50 9. Pituitary PRL content after single binge EtOH treatment . 51 10. Pituitary PRL steady-state mRNA levels after single binge EtOH treatment . 52 11. Pit-1 steady-state mRNA levels after single binge EtOH treatment . 54 12. Serum GH levels after double binge EtOH treatment . 55 13. Pituitary GH steady-state mRNA levels after double binge EtOH treatment . 57 Vlll 14. Effects of EtOH on hypothalamic GHRH content ............................................ 58 15. Semi-quantitative PCR standardization . 60 16. Correlation between the amount of RNA added and Linearity of the RT-PCR assay . 61 17. Composite autoradiograph of effects of single and double binge EtOH on hypothalamic GHRH mRNA levels as measured by semi-quantitative RT-PCR . 63 18. Graphic representation of effects of single and double binge EtOH on hypothalamic GHRH mRNA levels as measured by semi-quantitative RT-PCR . 64 19. Hypothalamic SRIF mRNA levels after single binge EtOH treatment as measured by semi-quantitative RT-PCR . 66 20. Hypothalamic SRIF mRNA levels after double binge EtOH treatment as measured by semi-quantitative RT-PCR . 67 21. Representative autoradiograph of effects of EtOH treatment on transcription rates of pituitary hormones 3 hours after EtOH treatment as measured by the nuclear run-on transcription assay . 68 22. Graphic representation of the effects of EtOH on hormone transcription rates at 1.5 and 3 hours after treatment as measured by the nuclear run-on transcription assay . 69 23. Graphic representation of the effects of EtOH on hormone transcription rates at 6 and 24 hours after treatment as measured by the nuclear run-on transcription assay . 70 24. The effects of EtOH on intrapituitary cAMP levels at various time points after EtOH treatment . 72 25. Time course experiment showing the effects of EtOH on pituitary c-fos steady-state mRNA levels . 74 lX LIST OF ABBREVIATIONS ADU arbitrary densitometer units a alpha ~ beta bp base pairs BSA bovine serum albumin cAMP cyclic adenosine 3', 5'-monophosphate cc cubic centimeter cDNA complimentary deoxyribonucleic acid Ci cune cpm counts per minute Da daltons DA dopamine DEPC diethyl pyrocarbonate DNA deoxyribonucleic acid dNTP deoxynucleotide triphosphate DTT dithiothreitol EDTA ethylenediamine tetraacetic acid g gram x GH growth hormone GHRH growth hormone releasing-hormone GI gastro-intestinal GIT guanidinium isothiocyanate HEP ES N-2-h ydroxyethylp iperazine- N '-2-ethanesulfonic acid Ip intraperitoneal kb kilobase kDa kilo Dalton L liter M molar µCi microcurie µg microgram µ1 micro liter µM micromolar mg microgram MgCl2 magnesium chloride ml milliliter mM millimolar XI mm millimeter mRNA messenger ribonucleic acid MOPS 3-(N-morpholino) propanesulfonic acid ng nano gram nM nano molar O.D. optical density PBS phosphate buffered saline PCR polymerase chain reaction PEG polyethylene glycol pg picogram Prl prolactin RIA radioimmunoassay RNA ribonucleic acid rRNA ribosomal RNA RT reverse transcription SDS sodium dodecyl sulfate SSC sodium chloride/sodium citrate buffer Taq thermus aquaticus DNA polymerase TBE tris-borate-EDTA buffer Xll TE tris-EDTA TRH thyrotropin-releasing hormone tRNA transfer RNA TSH thyroid-stimulating hormone UV ultraviolet v/v percent volume per volume xg times gravity xiii CHAPTER I INTRODUCTION Alcohol (ethyl alcohol, or EtOH) is the most important drug of dependence in all industrialized countries. The social and clinical problems that arise from alcohol's widespread abuse are now well documented. In the U.S. alone, approximately 18 million adults exhibit some form of alcoholism, alcohol dependence, or alcohol abuse (Van Natta, et al 1984). Recent studies of current trends in alcohol drinking behavior indicate that bouts of high level alcohol consumption over short periods of time, termed "binge-drinking" episodes, are becoming more common, especially amongst young adults in the U.S. (Meilman et al 1990). Binge drinking is believed to be one of several behavioral characteristics
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