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thesis submitted to the University of St. Andrews for the degree of Doctor of Philosophy by ROSAMONDE ELIZABETH BANKS Department of Physiology and Pharmacology, December, 1986 University of St. Andrews. QUESTIONS ANSWERED AND ANSWERS WITHOUT QUESTIONS If the human brain was so simple that we could understand it we would be so simple that we couldn't. EMERSON PUGH (Cited by Von Scheele and Nordgren, 1986, in "The mind-body problem in medicine", Lancet, February 1st., 258-261.) DECLARATION a). I, Rosamonde Elizabeth Banks, hereby certify that this thesis has been composed by myself, that it is a record of my own work, and that it has not been accepted in partial or complete fulfilment of any other degree or professional qualification. Signed b). I was admitted to the Faculty of Science of the University of St. Andrews under Ordinance General No. 12 in October 1983, and as a candidate for the degree of Ph.D. in October 1983. Signed Date 2J{./u Iff' c). We hereby certify that the candidate has fulfilled the conditions of the Resolution and Regulations appropriate to the Degree of Ph.D. Signatures of Supervisors Date COPYRIGHT UNRESTRICTED In submitting this thesis to the University of St. Andrews I understand that I am giving permission for it to be made available for use in accordance with the regulations of the University Library for the time being in force, subject to any copyright vested in the work not being affected thereby. I also understand that the title and abstract will be published, and that a copy of the work may be made and supplied to any bona fide library or research worker. ACADEMIC RECORD I graduated from the University of St. Andrews in October 1982, with a B.Sc. (Hons.) in Physiology, 1st class. In October 1983 I was awarded an M.Sc. (by instruction) in Toxicology, by the University of Surrey. ACKNOWLEDGMENTS 1 would firstly like to thank my supervisors, Dr. Jim Aiton (University of St Andrews) for his constant support and encouragement, and Dr. Graham Naylor (Ninewells Hospital, Dundee) for his enthusiasm and helpful discussions. 1 would also like to thank Dr. Gordon Cramb, Pat Ogden and Liz Rugg for their advice and friendship over the last three years, lain Laurie, Carol Voy and Mary Falls for their technical assistance, and the remaining members of the Physiology Department for their assistance at various times. 1 am also grateful to the Science and Engineering Research Council and Tayside Health Authority for financial support via a CASE studentship, which enabled this project to be carried out. Thanks are extended to Professor T. Reich (Washington University, USA), Dr. Alan Wright and Helen Ackford (MRC Clinical and Population Cytogenetics Unit, Edinburgh) and Ann Brown (Ninewells Hospital, Dundee) for supplying the lymphoblastoid cell lines used in this study. 1 would also like to thank Dr. John Walton (Chemistry Department, St. Andrews University) for performing the ESR measurements. Lastly, but by no means least, 1 would like to thank my parents for their support over the years, and my husband David for giving me the support and encouragement needed to finish this thesis • AIMS OF THE THESIS This thesis is aimed at examining the intracellular effects of chronic vanadate treatment in a cultured human cervical epithelial cell line (HeLa), with particular regard to cation transport. Following this the possible involvement of vanadate, cation transport, and phosphatidylinositol in the aetiology of bipolar manic-depressive psychosis is investigated using cultured lymphoblastoid cell lines from bipolar manic-depressive patients and control subjects. LIST OF CONTENTS PAGE NUMBER ABSTRACT i LIST OF ABBREVIATIONS iii CHAPTER 1. GENERAL INTRODUCTION 1 CHAPTER 2. THE EFFECTS OF CHRONIC VANADATE TREATMENT ON HELA CELLS; CELL GROWTH, MORPHOLOGY, AND INTRACELLULAR REDUCTION OF VANADATE I. Introduction 50 II. Materials and Methods 61 III. Results 71 IV. Discussion 78 CHAPTER 3. THE EFFECTS OF CHRONIC VANADATE TREATMENT ON THE VOLUME, ION CONTENTS, SODIUM PUMP NUMBERS, AND K+ INFLUX PATHWAYS OF HELA CELLS I. Introduction 84 II. Materials and Methods 99 III. Results 109 IV. Discussion 114 CHAPTER 4. MEASUREMENT OF INTRACELLULAR NUCLEOTIDES IN HELA CELLS BEFORE AND AFTER CHRONIC VANADATE TREATMENT, USING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) I. Introduction 123 II. Materials and Methods 136 III. Results 143 IV. Discussion 148 CHAPTER 5. LONG-TERM CULTURE OF LYMPHOBLASTOID CELL LINES FROM BIPOLAR MANIC-DEPRESSIVE PATIENTS AND CONTROLS; TRANSFORMATION, GROWTH AND MORPHOLOGY I. Introduction 157 II. Materials and Methods 161 III. Results 166 IV. Discussion 168 CHAPTER 6. K+ FLUXES AND SODIUM PUMP NUMBERS IN LYMPHOBLASTOID CELL LINES FROM BIPOLAR MANIC- DEPRESSIVE PATIENTS AND CONTROL SUBJECTS; EFFECTS OF CHRONIC INCUBATION WITH OUABAIN, LITHIUM OR VANADATE I. Introduction 170 II. Materials and Methods 176 III. Results 188 IV. Discussion 198 CHAPTER 7. PHOSPHATIDLYINOSITOL TURNOVER IN LYMPHOBLASTOID CELL LINES FROM BIPOLAR MANIC- DEPRESSIVE PATIENTS AND CONTROL SUBJECTS I. Introduction 211 II. Materials and Methods 225 III. Results 239 IV. Discussion 246 CHAPTER 8. CONCLUDING REMARKS 257 APPENDICES 262 REFERENCES 268 ABSTRACT The symptoms, classification, occurrence and possible aetiologies of bipolar manic-depressive psychosis have been reviewed, with particular emphasis on the possible role of the vanadate ion (VS+) and cation transport in the illness. The effect of vanadate on cation transport in intact cells has been determined using the well-characterised HeLa cell line. Cation transport in virally transformed lymphob1astoid cell lines from 13 bipolar manic-depressive patients and 13 control subjects has been examined, under normal conditions and after treatment (24 hours) with lithium, ouabain or vanadate. The phosphatidylinositol system has also been examined in these cell lines, in view of the therapeutic effect of lithium, and its known inhibitory actions on inositol I-phosphatase. In HeLa cells, no effects of vanadate on cation transport were seen until concentra tions greater than 3.2 x 10-6M. This was attributed to the intracellular reduction of VS+ to V4+ shown to occur using ESR. Similar decreases were seen in all the K+ influx pathways, with maximum decreases of approximately 30% at 10-4M vanadate extracel1ularly. Significant toxicity was also seen at these concentrations, with a maximum decrease in cell number of 40% at lO-4M vanadate. No change in the energy charge was seen and changes in ATP levels occurred subsequently to the changes in cell number, with a decrease of 40% at 10-4M vanadate. Using the lymphoblastoid cell lines, no significant differences were seen in any of the cation transport parameters examined, with the exception of mean sodium pump number which was i 30% greater in the bipolar group compared with the control group. Lithium or vanadate treatment produced either no effect or inconsistent changes in cation transport. Ouabain treatment produced similar decreases in sodium pump number in both groups. Inositol uptake was similar in both groups, but the percentage incorporation into phosphoinositides was reducerl in bipolar cell lines compared with controls. ii LIST OF ABBREVIATIONS Abbreviations are as illustrated in the text. In addition, the more extensively-used abbreviations are listed below. Abbreviations of measurements follow the S.I. system of nomenclature. ADP Adenosine 5'-diphosphate AMP Adenosine 5'-monophosphate ATP Adenosine 5'-triphosphate AUFS Absorbance units full scale Maximum binding CDP Cytidine 5'-diphosphate CMP Cytidine 5'-monophosphate cpm Counts per minute CSF Cerebrospinal fluid CTP Cytidine 5'-triphosphate DAG Diacylglycerol DIOS 4,4'-Oiisothiocyanatostilbene-2,2'-disulfonic acid DOPAC Dehydroxyphenylacetic acid DST Dexamethasone suppression test Cl- equilibrium potential K+ equilibrium potential Equilibrium membrane potential Na+ equilibrium potential EGF Epidermal growth factor Endoplasmic reticulum ESR Electron spin resonance GOP Guanosine 5'-diphosphate GFR Glomerular filtration rate Hi Guanosine 5'-monophosphate Gpp(NH)p Guanyl imidodiphosphate GSH Glutathione (reduced) G~ Guanosine 5'-triphosphate HEPES N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid HPLC High performance liquid chromatography . HPTLC High performance thin layer chromatography HVA Homovanillic acid IBMX Isobutylmethylxanthine Internal diameter IP Inositol I-phosphate Inositol 1,4-bisphosphate Inositol l,4,5-trisphosphate or the 1,3,4 isomer Inositol 1,3,4,5-tetrakisphosphate Inositol pentakisphosphate Inositol hexakisphosphate Affinity constant Dissociation constant Inhibitory constant Michaelis-Menten constant lew Litre of cell water MAO I Monoamine oxidase inhibitor MHPG 3-methoxy-4-hydroxyphenylglycol N~ Nicotinamide