5'1'7s i: TtlE ACTIONS AND INTERACTIONS OF NORADRENALINE, DOPAI'/IINE AND L-DOPA TTIESIS SIJBMITTED TO THE UNÏ\TERSÏTY OF ADELAÏDE FOR ÍTIE DEGREE OF DOCTOR OF PFIII.,OSOFffY. BY MARGARET ANN LAZNER DEPARTMENT OF HUMAN PHYSIOLOGY AND PHARMACOLOGY THE UNIVERSITY OF ADELAIDE SOUTH AUSTRALIA JANUARY 1975 LIST ()F CONTENTS CONTENTS PAGE i i S UMMARY -i i DECLARATION iv v AC KNOl¡,lL E DGME NTS CHAPTER 1 General IntroductÍon 1.1 Abbrevi ati ons 1.6 Tests of Significance 1.6 CHAPTER 2 GENERAL METHODS Preparat'ion of Rabbits 2.t Perfusion of the Isolated Ear Artery 2.2 Helical Strip PreParations 2"4 Pretreatment of Rabbi ts 2.6 (a) Sympathetic denervation 2.6 (b) Reserpine 2.7 (c) ¡ltalamide 2.8 Pretreatment of Isolated Arteries - Nialamide 2.8 Fl uorescence Histochemi strY 2.8 Techniques Used in Experiments with Radioisotopes 2.t0 CHAPTER 3 ACTTONS OF DA IN THE RABBIT EAR ARTERY I ntroducti on 3.1 Methods 3"3 Resul ts 3.4 1. ResPonses to doPamine 3.4 (a) Perfused segments 3"4 (b) Helicat striPs 3.5 2. Effect of chronic denervation 3.7 3. Effect of cocaine 3.8 4. Effect of reserpine pretreatment 3.10 3.10 5. Relative PotencY DA to NA 3.11 Di scussi on PAGE CONTENTS CHAPTER 4 THE EEFECT OP T^VHTBITION OF A¿'O ON RESPONSES TO DOPA](ÏNE 4.L I ntroducti on 4.3 Methods 4.3 Resul ts 4.3 1. Arteries without Pretreatment (a) ShaPes of resPonses 4.3 (b) Kinetics of the resPonses 4.5 (c) Effect of nialamide on the magnìtude 4.6 of steadY state responses 'ly'denervated 4"7 2. Chroni cal arteri es 4.8 3. ReserPine Pretreated arteries 4. Cocaine treated arteries 4.9 4"10 5. The effect of Phentolamine Di scussi on CHAPTER 5 PHARMACOLOGY OE PRECURSORS OE DOPAMTNE TN THE RABBTT ARTERY 5.1 Introducti on 5.2 Resu I ts 1. Tyrosine 5.2 5.2 2. L-dopa (a) untreated arteries 5.2 (b) Nialamide treated arteries 5"3 The effect of chronic denervation 5.3 The effect of reserPine 5.4 Restoration of monoamine fluorescence with L-dopa 5.5 5.6 Di scussi on CHAPTER 6 THE EFEECT OF TNHTBITORS OF DOPA]4TNE-\- HYDROXYLASE ON SECONDAßY SENSTTTZATION 6.1 I ntroducti on 6.1 Methods PAGE CONTENTS 6.1 Resul ts 6.2 1. The effect of DDC on the response to DA and dopa in the nialamide treated ear artery 2. Effect of cocaine 6.4 6.5 3. Effect of DDC on response to L-dopa 6.6 Di scussi on CHAPTER 7 THE EFFECT OE LOWERED TEMPERATURE ON THE RESPONSES TO NA, DA AND L-DOPA IN NIALAT4TDE TREATED AND UNTREATED ARTERTES 7.1 I ntroducti on 7.3 Methods - General IsotoPe 7.4 7.6 Resul ts 1. Effect of lowered temperature on the 7.6 response to NA 2. Effect of ìowered temperature on the 7.8 response to DA 3. Effect of lowered temperature on the 7.9 response to L-dopa after nialamide 4. Effect of lowered temperature on 7.9 uptake of NA and DA (a) Potentiation of extraluminal NA 7.10 bY cocaine at 37oC and 25oC (b) UPtake and efflux of (t) 3H t'¡R 7. 10 (c) uptate of isotoPic DA 7.t\ 7.r2 Di scussi on CHAPTER B FATE OF TNTRALUMINAL NA AND DA IN THE PERFUSED RABBTT EAß ARTERY 8.1 I ntroducti on 8.3 Methods 8.4 Resul ts 8.4 1. Fate of intraluminal NA (a) Histochemical studies with NA 8"4 PAGE CONTENTS (b) RecoverY times 8.4 (c) fne effects of some COMT inhibitors 8.5 on the sensitivitY to NA 2. Extraneuronal fate of DA 8.6 (a) HistochemistrY 8.6 (b) Effect of a COMT inhibitor on the response 8.7 to DA 8.8 Di scussi on 9.1 GENERAL DISCUSSION APPENDI X 1 Sources of drugs and chemicaìs 4.1 Preparation of drugs 4.2 APPENDIX 2 Reagent formulae 4.3 APPEND I X 3 Apparatus and techniques 4.5 BIBLIOGRAPHY i SUMMARY 1. In this thesis the vascular actions in the rabbit of dopamine and L-dopa are examined. The tissue used throughout was the isolated central artery of the rabbit ear. ear 2. Dopamine was found to exert its action in the rabbjt artery by st'imulation of the aìpha adrenergic receptors causing constriction of the artery. Djlatation due to the action of dopamine could not be demonstrated ìn this preparation. The sensitivity to dopamìne was not modified by chronìc denervatjon' by cocaine, or by reserpine. It was thus concluded that neuronal mechanisms play little part in the response to dopamine' up Fluorescence histochemistry showed that dopamine is taken into nerve terminals, but uptake reached saturation level at concentrations of dopamine whìch are threshold concentrations for the constrictor response. L-dopa was found to have no constrictor effect on the arterY' 3.Theroleplayedbythemetabol.izingenzymesinthe responsetodopamine,L.dopaandtyrosine!{asstudiedin nialamìde treated arteries. It was found that nialamide treat- the ment caused an increase in sensitivity and a change in recovery response to L.dopa and dopamine, the response and the after washout of the drugs becoming much prolonged' These effects were not decreased by prior treatment with cocaine, or reserpine, but were reduced by chronic denervation. cocaine appliedduringtheresponse'orduringtherecovery'phasecaused a rapid further constriction of the artery. This evidence indicated that the changes induced by nialamide treatment were in the most part medìated by neuronal mechanisms and intraneuronal stores MAO, but did not depend on the presence of intragranular of noradrenaline. nialamide 4. No response due to tyrosine was detected in treated arteries' nor was there any response to cocaine 11 after tyrosine' 5. In view of the similarity between the effects of nialamide on the response to dopa, dopamine and noradrena'lìne, a role for noradrenaline in the secondary sensitization response to dopa and dopamine is hypothesized. The role of noradrenalìne was assessed by blockade of the enzyme dopamine-ß-hydroxylase' It was shown that prior treatment with diethyldithiocarbamate (DDC) largely prevented the occurrence of secondary sensitization to dopamine in reserpine and nialamide treated arteries; however, no effect of DDC was observed in arteries treated with nialamide alone. 6. The effect of lowering the temperature from 37oC to 25oC was studied. It was found that in untreated arteries lowered temperature produced no change ìn the response to dopamine and noradrenaline; however, in nialamide treated arteries a profound effect of cooling on the parameters of aìtered response and increased sensitivity to dopamine, noradrenalìne and L-dopa !{as seen. It was also shown that cooling reduced the potentiation of noradrenaline due to cocaine and the uptake and retention of 3H noradrenaline. 7. It js concluded that there is a role for noradrenaline, and the uptakel process, in the secondary sensitization response to L-dopa and dopamine in the nialamide treated artery' However' other mechanisms may be involved, such as release of endogenous noradrenaline, or noradrenaline newly formed from the exogenous dopamine, bY the doPamine. 8. Histochemical evidence from the perfused'njalamide and reserpine pretreated ear artery segment showed that uptake by smooth muscle and metabolism by CQMT in the media were factors in the failure of intraluminal noradrenaline to penetrate across the media to the nerve endings, and to cause secondary sensitization to noradrenaline. Inhibition of C0MT alone had little effect on the sensitivity to intralum'inal noradrenaline' rtl or dopamjne. However, in nialamide treated arteries inhibition jntra- of COMT caused a two fold increase in sensit'ivity to luminal noradrenal ine. DECLARAT I ON I declare that this thesis contains no materíal which has been accepted for the award of any other degree or diploma in any University, and to the best of my know'ledge contains no material previously published by any other person' except where due reference is made in the text" Material from or related to this thesis has formed part of the following publications : cIRCULATIoN RESEARCH (tgZO) ry?7 Supp. 2" 4l'48 PROCEEDINGS AUSTRALIAN PHYSIOLOGICAL AND PHARMACOLOGICAL SOCIETY (t97L) 2, 33 ibí.d. (7e72) 1, 183 AUSTRALIAN JOURNAL OF EXPERIMENTAL BIOLOGY AND MEDTCAL SCIENCE (7974) 52, 193-200 J0URNAL 0F PHARMACY AND PHARMACOLoGY (L974) 26"62-65 J0URNAL 0F DENTAL RESEARCH (1971) 50,743-744 Results from this thesis have also been presented to meetings of the Australasian Society of Clinical and Experimental Pharmacologists, and the International Association for Dental Research. Margaret Ann Lazner, January 1975 ACKNOWLEGMENTS Professor I.S. de la Lande of the University of Adelaide who supervised my studies and experimenta'l work, and whose advice, encouragement and criticism were i nval uabl e Mrs. Maria Kay, Miss Tiina Mannik and Miss Ailsa Maxwell for their excellent technical help and Mrs. Pat Macaskill, and Mrs. Ermioni Mourtzios who typed this thesis. Mr. R.J. Head, a fellow Ph.D Student, who participated in some of the experiments described in Chapter 7. Mr. D.A.S. Parker, also a Ph.D Student, who collaborated with me in the histochemical experiments described in Chapter 8. The University of Adelaide who provided support in the form of a University Research Grant for the years 1968- 1969. The experimental work described herein u,as carried out during the years 1968 - 1973 in the Department of Human Physio'logy and Pharmacology, University of Adelaide. CHAPTER ONE GENERAL INTRODUCTION CHAPTTR 1 GENERAL INTRODUCTION It is the purpose of this thesis to examine the vascular actions in the rabbit of the precursors of noradrenaline, i'e' L-dopa and dopamine, and ìn particular to examine the part played by the sympathetic nerves and the metabolising enzymes, monoamine oxidase and catechol-o-methyltransferase, in the responses to these substances.