Medicine, Faculty of
Anesthesiology, Pharmacology and Therapeutics, Department of
DSpace
UBCV
Martinez, Terry T.
Doctor of Philosophy - PhD
University of British Columbia
The antiarrhythmic actions of prostaglandins were first investigated using arrhythmias associated with cardiac ischemia in the dog and the rat. These studies were followed by investigations of the possible mechanisms of action, using rat heart tissue in intact, isolated, and cell culture preparations. Preliminary experiments in the dog revealed that prostaglandins E₂ and F₁α markedly reduced the number of premature ventricular contractions occurring within the first 25 minutes following coronary artery ligation. Prostaglandin E₂ or F₁α did not markedly alter the cardiovascular response to occlusion, making it unlikely that modulation of autonomic reflexes is a central factor in their antiarrhythmic action. Coronary ligation in the rat was used to compare the antiarrhythmic effectiveness of prostaglandins, lidocaine, and quinidine. Prostaglandins E₂, F₂β and quinidine were found to be the most effective against arrhythmias occurring within the first 25 minutes following occlusion, reducing the number of PVCs by 40 to 50 per cent. The number of flutter episodes and the number of animals dying from arrhythmias was also markedly decreased by prostaglandins E₂ and F₂β and by quinidine. Prostaglandins F₁α and A₂, and lidocaine had lesser effects. Prostaglandins had only minor effects on blood pressure or heart rate, which were not related to their antiarrhythmic activity. No significant differences were found in the infarct size with prostaglandin treatment. The effects of prostaglandins E₂, A₂ and F₂β, quinidine, and lidocaine were tested in in situ rat heart on electrically-induced flutter threshold and maximum following frequency. Flutter threshold was not changed by any of the prostaglandins tested, although lidocaine increased and quinidine decreased it. Prostaglandins caused a dose-dependent change in maximum following frequency which was usually less than 10 per cent of control. Lidocaine produced a marked increase and quinidine a marked decrease in maximum following frequency. The slight depressive action of prostaglandins does not correlate with their antidysrhythmic actions. Prostaglandins of the E, A, and F series were found to have only minimal effects on rate and force in isolated rat hearts. However, both PGE₂ and PGF₂β, delayed the loss of contractile force with time at 10⁻⁷ M. All prostaglandins tested markedly increased coronary flow rate at 10⁻⁵ M. The effects on the beating behavior of cultured rat heart cells of fourteen prostaglandins of the A, B, D., E, and F series were investigated in cultured rat heart cells. With the exception of PGF₂α, which produced a chronotropic response, prostaglandins had limited direct action in cultured rat heart cells. The effects of ouabain, calcium, potassium, dinitrophenol, and Cyanea toxin, together with prostaglandins, lidocaine, and quinidine on cultured rat heart cells were also investigated. Ouabain and calcium increased rate and fibrillatory movements, while potassium and dinitrophenol slowed rate and decreased rhythmic beating. Cyanea toxin produced a characteristic series of arrhythmogenic changes which were also used to test for antiarrhythmic activity in cultured heart cells. Lidocaine and quinidine were effective only against cellular arrhythmias caused by high calcium concentration, and prostaglandins were effective only against dinitrophenol-induced arrhythmias, indicating that there is no over-all "protective" effect of prostaglandins in cell culture.
STUDIES ON THE ANTIARRHYTHMIC ACTIONS OP PROSTAGLANDINS by Terry Tyler Martinez M.Bc, The University of British Columbia, 1 9 7 5 fl.Sc, Purdue University, 1 9 7 0 B.S., Purdue University, 1969 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES Department of Pharmacology, School of Medicine We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA January, 1978 <g) Terry Tyler Martinez, 1978 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r a n a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l m a k e i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may b e g r a n t e d b y t h e H e a d o f my D e p a r t m e n t o r b y h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t b e a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f Pharmacology T h e U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 Wesbrook Place Vancouver, Canada V6T 1W5 March 5, 1978 D a t e ( i i ) ABSTRACT The antiarrhythmic actions of prostaglandins were f i r s t investigated using arrhythmias associated with cardiac isch-emia in the dog and the rat. These studies were followed by investigations of the possible mechanisms of action, using rat heart tissue in intact, isolated, and c e l l culture preparations. Preliminary experiments in the dog revealed that prosta-glandins Eg and F l o < markedly reduced the number of premature ventricular contractions occurring within the f i r s t 25 min-utes following coronary artery ligation. Prostaglandin Eg or did not markedly alter the cardiovascular response to occlusion, making i t unlikely that modulation of autonomic reflexes is a central factor in their antiarrhythmic action. Coronary ligation in the rat was used to compare the antiarrhythmic effectiveness of prostaglandins, lidocaine, and quinidine. Prostaglandins Eg, ^ 2 0 ' Q u i n i d i n e were found to be the most effective against arrhythmias occurring within the f i r s t 25 minutes following occlusion, reducing the number of PVCs by 40 to 50 per cent. The number of flutter episodes and the number of animals dying from arrhythmias was also markedly decreased by prostaglandins Eg and ^20' and by quinidine. Prostaglandins and Ag, and lidocaine had lesser effects. Prostaglandins had only minor effects on blood pressure or heart rate, which were not related to their antiarrhythmic activity. No significant differences were found in the infarct size with prostaglandin treatment. ( i i i ) The effects of prostaglandins Eg» A 2, Flo<, and F 2 e , quini-dine, and lidocaine were tested in in situ rat heart on elec-trically-induced flutter threshold and maximum following frequency. Flutter threshold was not changed by any of the prostaglandins tested, although lidocaine increased and quin-idine decreased it'J, Prostaglandins caused a dose-dependent change in maximum following frequency which was usually less than 10 per cent of control. Lidocaine produced a marked in-crease and quinidine a marked decrease in maximum following frequency. The slight depressive action of prostaglandins does not correlate with their antidysrhythmic actions. Prostaglandins of the E, A, and F series were found to have only minimal effects on rate and force in isolated rat hearts. However, both PGE£ and PGFgg, d e l a y e d t t l e loss of contractile force with time at 10"^ M. A l l prostaglandins tested markedly increased coronary flow rate at 10 -/ M. The effects on the beating behavior of cultured rat heart cel l s of fourteen prostaglandins of the A, B, D., E, and F series were investigated in cultured rat heart c e l l s . With the exception of PGFgpf, which produced a chronotropic response, prostaglandins had limited direct action in cultured rat heart c e l l s . The effects of ouabain, calcium, potassium, dinitrophenol, and Cyanea toxin, together with prostaglandins, lidocaine, and quinidine on cultured rat heart cells were also investigated. Ouabain and calcium increased rate and f i b r i l l a t o r y movements, while potassium and dinitrophenol slowed rate and decreased rhythmic beating. Cyanea toxin produced a characteristic (iv) series of arrhythmogenic changes which were also used to test for antiarrhythmic activity in cultured heart c e l l s . Lido-caine and quinidine were effective only against cellular arrhythmias caused by high calcium concentration, and pros-taglandins were effective only against dinitrophenol-induced arrhythmias, indicating that there is no over-all "protective" effect of prostaglandins in c e l l culture. (v) TABLE OF CONTENTS Chapter Page ABSTRACT i LIST OF TABLES v i i LIST OF FIGURES toe I INTRODUCTION 1 A. General Chemistry and Metabolism 1 B. Prostaglandins and the Heart 11 C. Antiarrhythmic Effects 21 D. Purpose of Experiments 26 II MATERIALS AND METHODS 29 Section I: Production of Arrhythmias in Dogs 29 and Rats A. Coronary Occlusion in Dogs 29 B. Acute Coronary Occlusion in Rats 31 C. Production of Arrhythmias by Electrical 38 Stimulation in the Rat Section II: Isolated Rat Hearts and Contractility 39 Losses Section III: Experiments in Cultured Heart Cells kz Section IV: General Information A. Drug Details for a l l Experiments 52 B. Calculations, S t a t i s t i c a l Methods, and 52 Experimental Design ' III RESULTS 54 Section I: Prostaglandins in Intact Dogs and Rats A. The Effects of Prostaglandins During Coro- 54 nary Occlusion in Dogs B. The Effects of Prostaglandins, Lidocaine, 55 and Quinidine on Arrhythmias Produced by Coronary Artery^Ligation in Rat C. Changes in Flutter Threshold and in Maximum 70 Following Frequency Section II: The Effect of Prostaglandin infusions on 78 Isolated Rat Heart Section III: Prostaglandins in Cultured Heart Cells 93 (vi) TABLE OF CONTENTS continued Chapter Page A. The Effect of Prostaglandins Alone on the 93 Beating Activity of Cultured Heart Cells B. The Effect of a Variety of Arrhythmogenlc 104 Agents on the Beating Activity of Cul-tured Heart Cells C. The Effects of Prostaglandins, Lidocaine, 13^ and Quinidine on Abnormal Beating In-duced in Cultured Heart Cells by various Arrhythmogenic Agents D. The Effect of Prostaglandins, Lidocaine, lk2 and Quinidine on Arrhythmias Produced by Cyanea Toxin IV DISCUSSION 151 The Effect of Prostaglandins ... 151 During Coronary Occlusion in Dogs B. The Effect of Prostaglandins, Lidocaine, and 155 Quinidine on Arrhythmias Produced by Left Coronary Artery Ligation in Bats C. The Effects of Prostaglandins, Lidocaine, 160 and Quinidine on Flutter Threshold and Maximum Following Frequency in In Situ Bat Heart D. The Effect of Prostaglandins on Isolated 163 Rat Heart E. The Effects of Prostaglandins Alone on 16? the Beating Activity of Cultured Heart Cells F. The Effects of Prostaglandins on Abnormal- 1?0 i t i e s of Beating in Cultured Heart Cells Induced by a Variety of Arrhythmogenic Agents G. The Actions of Ouabain, Ionic Manipulation, 171 Dinitrophenol, and Anoxia on Cultured Heart Cells H. The Effect of Cyanea Toxin on Cultured Heart 174 Cells I. The Effect of Prostaglandins, Lidocaine, and 175 Quinidine on Abnormal Beating. Induced in Cultured, Heart Cells j . -V SUMMARY : - •181 VI BIBLIOGRAPHY 185 VII APPENDIX 193 (vii) Table I II III IV V VI VII VIII IX X XI LIST OF TABLES Page The effects of prostaglandins, lidocaine, and 59 quinidine on arrhythmias produced by l e f t coronary artery ligation in rat. The effects of prostaglandins, lidocaine, and 75 quinidine on electrically stimulated flutter threshold and maximum following frequency in in vivo rat heart. S t a t i s t i c a l testing of the results of prostaglan- 80 din infusions on rate of beating, force of con-traction, and coronary flow in perfused rat hearts. The effect of prostaglandins on the rate of beating 9 4 of single cultured heart c e l l s . The effect of prostaglandins on the beating rate 98 range of single cultured heart c e l l s . The effect of prostaglandins on the change in 99 optical density of the c e l l image which occurs with each beat in single cultured heart c e l l s . The effect of prostaglandins on the time deriva- 101 tive, d op dens, of optical density changes in dt single cultured heart c e l l s . The effect of prostaglandins on;the percentage 102 of single':cultured heart cells spontaneously beating. The effect of Cyanea toxin-containing material on 1 2 4 the time to various arrhythmic events in single cultured heart c e l l s . The general changes in beating behavior of 1 3 6 cultured heart ce l l s , induced by different arrhythmogenic agents. The effect of prostaglandins, lidocaine, and quin- 137 idine versus control and arrhythmogenic agents-ouabain, calcium, potassium, dinitrophenol, and adrenaline- on the beating activity of cultured rat heart myoblasts. ( v i i i ) Table Page XII The a b i l i t y of prostaglandins, lidocaine, and 138 quinidine to modify the response of cultured rat heart cells to five arrhythmogenic agents-ouabain, calcium, potassium, dinitrophenol, and adrenaline. XIII The effects of prostaglandins, lidocaine, and 139 quinidine on abnormal beating induced in single cultured rat heart c e l l s . XIV The effect of quinidine, lidocaine, and prosta- 149 glandins on the time required for Cyanea toxin to produce a 50 per cent effect in the incre-ase in per cent of single rat heart cells beat-ing arrhythmically; the reduction in per cent of cells beating normally; and the increase in mean arrhythmic score. (ix) •LIST OF FIGURES Figure Page 1 The structure of prostenoic acid and several main 4 classes of prostaglandins together with pre-cursor fatty acids. 2 Several synthetic variants of naturally-occurring 7 prostaglandins. 3 Synthesis and catabolism of the two common mam- 10 malian prostaglandins: PGE2 and PGF2*;. 4 Ligation of the l e f t coronary artery in rat showing 34 the anterior aspect of the heart in a rat whose coronary arteries have been made visable by injection of a colored material. 5 Diagram of the apparatus used for measuring beating 46 activity in single cultured heart c e l l s . 6 Typical records of changes in optical density, the 46 corresponding derivative of optical density, and the tachograph measured beat-to-beat rate from five single myoblasts. 7 Quantitation of arrhythmias in single cultured heart 48 c e l l s showing the severity of arrhythmias measured using a subjective scale along with changes in the rate range of beating. 8 The effect of l e f t coronary artery occlusion on 56 the mean frequency of premature ventricular con-tractions With time for control, PGE2, and PGF1<3< treated dogs. 9 The effects of prostaglandins, lidocaine and quini- 61 dine on the average number of premature ventri-cular contractions experienced by five rats over a 25 minute period following acute l e f t coronary artery ligation. 10 The effect of prostaglandins, lidocaine, and quini- 63 dine on the average duration of flutter epi-sodes experienced by rats in a 2 5 minute period following acute l e f t coronary artery ligation. 11 The effect of prostaglandins, lidocaine, and quini- 65 dine on the average size of the infarct produced (as the percentage of the whole heart weight) following acute l e f t coronary artery ligation in rats. (X) Figure Page 12 The effect of prostaglandins, lidocaine, and quin- 67 •idine on the number of rats dying from arrhy-thmias within 25 minutes following acute l e f t coronary artery occlusion. 13 The effect of prostaglandins, lidocaine, and 69 quinidine on the average subjective score for severity of arrhythnias following acute l e f t coronary artery occlusion in rats. 14 The effect of prostaglandins, lidocaine, and quin- 71 idine on blood pressure'during l e f t coronary artery occlusion in rats. 15 The effect of prostaglandins, lidocaine, and quin- 7^ idine on heart rate during l e f t coronary artery occlusion in rats. 16 Changes in the maximum;following frequency'of in 77 vivo rat hearts determined by electrical stim-ulation with a supramaximal current during in-fusion of prostaglandins. 17 The effect of prostaglandin (lO"? M) perfusion of 82 isolated rat heart on rate. 18 The effect of prostaglandin (10"? M) perfusion of 83 isolated rat heart on maximal force of contrac-tion. 19 The effect of prostaglandin (10"^ M) perfusion of 86 isolated rat heart on coronary flow rate. 20 The effect of prostaglandin (10~5 M) perfusion of 88 isolated rat heart on heart rate. 21 The effect of prostaglandin (10"^ M) perfusion of 89 isolated rat heart on maximal force of contrac-tion. 22 The effect of prostaglandin (10"-' M) perfusion of 91 isolated rat heart on coronary flow rate. 23 Dose-response curve to prostaglandin F2oc a*14 ell- 96 adrenaline in single cultured rat heart ce l l s . 24 The effect of ouabain (5 x 10"^ M) on rate and rate range/rate, vs time in cultured heart cells. 106 25 The effect of ouabain concentration on rate and' 107 rate range/rate in cultured heart c e l l s . (xl) Figure Page 26 The effect of ouabain concentration on the per- " 107 centage of cells arrhythmic/total cells beating, and the mean subjective score of arrhythmias* 27 The effect of added calcium on rate and rate range/ 110 rate in cultured heart cell s ; 28 The effect of calcium on the percentage of cells 110 arrhythmic/total cells beating, and the mean score for arrhythmias in cultured heart c e l l s . 29 The effect of added potassium concentration on rate 114 and rate range/rate in cultured heart c e l l s . 30 The effect of dinitrophenol concentration on rate 116 and rate range/rate vs time in cultured heart c e l l s . -4 31 The effect of dinitrophenol (2 x 10 M) on rate 116 and rate range/rate vs time in cultured heart c e l l s . 32 The effect of anoxia on the percentage of cells 118 beating rhythmically/total ce l l s , and the per-centage of cells beating arrhythmically/total cells beating in culture. 33 The effect of ouabain and lidocaine concentrations 121 on the percentage of cells not beating in culture. 34 The change in beating activity over a 60 minute 122 period of a single cultured heart c e l l following exposure to Cyanea toxin-containing material. 35 The effect of Cyanea toxin-containing material on 127 the beating rate of single cultured heart c e l l s . 36 The effect of Cyanea toxin-containing material on 129 the percentage of beating cultured heart cells beating arrhythmically. 37 The effect of Cyanea toxin-containing material on 131 the percentage of total cultured heart cells beating rhythmically. 38 The effect of Cyanea toxin-containing material on 133 the mean subjective score for arrhythmias in arrhythmically beating cultured heart c e l l s . 39 The effect of prostaglandins, lidocaine, and quin- 144 idine on the percentage of single cultured heart cells arrhythmic/total beating, vs time, in the presence of Cyanea toxin, 110 ng/ml. (Xii) Figure ^ Page 40 The effect of prostaglandins, lidocaine, and quin- 146 idine on the percentage of single cultured heart cells beating normally, vs time, in the presence of Cyanea toxin, 110 ng/ml. 41 The effect of prostaglandins, lidocaine, and quin- 148 idine on the mean subjective arrhythmic score for single cultured heart cells in the presence of Cyanea toxin, 110 ng/ml. ( x i i i ) ACKNOWLEDGEMENTS I wish to thank a l l of the people who helped me throughout this study. F i r s t , Dr. Michael J.A. Walker, who guided my studies and who gave me his friendship. Mike, i t is a pleasure to thank you. To Dr. M.C. Sutter, Dr. J. 0. Runikis, Dr. V. Palaty, and Dr. A. Fessler, who gave freely of their knowledge and counsel, I also wish to offer my heartfelt gratitude. To my friends and colleagues in research: Glenn Collins, Tony Au, and Christopher Harvey who helped in so many ways, I express my appreciation. To a l l the members of the Department of Pharmacology, who taught me not only science, but through example how people of good w i l l can work together in harmony in their common search for the truth, I express my admiration. Finally, the continuing and patient support of my wife, Judith, is gratefully acknowledged. The financial support of the Canadian Heart Fund is also ;> gratefully acknowledged. - 1 -CHAPTER I INTRODUCTION A. General chemistry and Metabolism Prostaglandins are a family of unsaturated hydroxy fatty acids which have a wide spectrum of pharmacological activity. Prostaglandins are best c l a s s i f i e d as autacoids or "local hormones". They are not stored in the body but are synthe-sized in response to a particular stimulus. Due to rapid metabolic degradation their effects are mainly confined to their site of production. They have potential therapeutic uses as vasodilators, decongestants, antiarrhythmics, and as agents which prevent blood platelet aggregation. They can also be used in the treatment of asthma and stomach ulcers, as abortifacients, and to induce labor (Colbert, 1973). The experimental investigation of their possible use as ANTIARRHYTHMICS is the concern of this thesis. Although prostaglandin-like activity had been noticed in seminal f l u i d as early as 1930 (Kurzrok, 1930), i t was not u n t i l 1934 that workers in England (Goldblatt, 1935) and Sweden (von Euler, 1936) independently found that the active principle extracted from seminal plasma was an acidic, l i p i d -soluble material. The term "prostaglandins" was coined by von Euler (von Euler, 1936) in the belief that the biologically active substance was a secretion of the prostate gland. It is - 2 -now known that prostaglandins occur in widespread distribution in animal tissues, and that they are produced in response to a wide variety of stimuli. In minute amounts they produce effects on a large variety of physiological processes (Douglas, 1975). Chemistry The prostaglandins may be regarded as derivatives of a hypothetical parent structure with the name prostanoic acid^ (Figure 1, Douglas, 1975). The different prostaglandins f a l l into several main classes, known as the E , F, A, B , C, and D series of compounds. This classification is based on the , constituents of the cyclopentyl ring (Figure 2; Douglas, 1975). These classes are further subdivided with the subscript 1, 2, or 3 according to the number of double bonds in the side chains. A l l natural prostaglandins have a trans double bond at the 13, 14 position and a hydroxyl group at C^^. Thus prostaglandin E^ ( PGE^ ) has only the double bond, while K J E 2 has an additional cis double bond at and P G E ^ has a third cis double bond at C17,18. These double bonds occur in the same positions in a l l classes. The E and F series, often referred to as the 'primary prostaglandins', are the most commonly found and also the most intensively studied. A large number of stereoisomeric forms are possible, based on the several asymmetric centers. The prostaglandins obtained from natural sources have the side chains arranged trans to each other, i.e. they are oriented at opposite sides of the ring (Figure 2; Douglas, 1975). C9 and C 1 : L hydroxyl groups are - 3 -Figure 1: The structure of prostanoic acid (top) and several main classes of prostaglandins together with precursor fatty acids. In the stereochemical convention followed, the groups indicated by a hatched line l i e behind the plane of the cyclopentane ring, while those indicated by —-» l i e in front of i t . (Douglas, 1975) - 4 -- 5 -also normally in the alpha configuration, i.e. on the same side of the ring as the carboxylic acid side chain. The C-^ hydroxyl group is also normally designated as alpha. Synthetic stereochemical variants of these natural forms may arise in several different ways. (Figure 2 ) F i r s t , the side chains may be cis to one another. These are referred to as 8-iso-prostaglandins. Secondly, one or more hydroxyl groups may have the beta configuration. The C - Q and Cj^ beta hydroxyls are sometimes referred to by the term epi. The C^ J J hydroxyl group can also be named after the Chan-Ingold-Prelog Convention, i.e. S for alpha and R for beta (Butcher, 1 9 7 3 ) . Thirdly, a l l prostaglandins may exist in two optically active forms. Natural prostaglandins are levorotatory and their mirror images, which are dextrorotatory, have their side chains transposed. (The latter are also referred to as the ent forms.) A l l of these stereochemical variants may be combined with each other. Reference to the stereochemistry is often omitted in the abbreviated forms, e.g. PGE^. For the F series, however, the term alpha or beta (e.g. PGF l oc or PGF1<5) is added to denote the configuration of the C^ hydroxyl group. A method based on the structure of prostanoic acid may also be used to describe prostaglandins in terms of their f u l l systematic nomenclature when necessary (Nugteren et a l . 1966). For example, the f u l l systematic name of PGEX is (-)-ll«-, 15(5)-dihydroxy-9-oxo-13~ trans-prostanoic acid, where the (-) refers to levorotation and the trans refers to the 0 ^ x 4 double bond. Over the last decade more than two thousand prostaglandin analogs have been prepared (Schneider, 1976). It has been Figure 2: Several synthetic variants of naturally-occurring prostaglandins. The groups indicated by a dotted line l i e behind the plane of the cyclopentane ring. ( Caton, 1973) - 7 -- 8 -possible to name the great majority of these compounds after the natural group to which they are most closely related. Biosynthesis and Catabolism , Prostaglandins are not stored in the body but are syn-thesized from the essential fatty acid precursor in response to widely divergent physical, chemical, hormonal, and other influences (Figure 3; Douglas, 1975). Synthesis depends on phospholipase-catalyzed release of precursor acid from cellular phospholipid stores. Activation of phospholipase A is thought to be the rate-limiting step in prostaglandin production. The primary prostaglandins (E and F series) are synthesized in a stepwise manner by a complex of microsomal enzymes referred to as "prostaglandin synthetase". Oxygen-ation and cyclization of the precursor acid take place to form a cyclic peroxide derivative. This endoperoxide i s either isomerized or reduced to yield PGE or PGF compounds. Prosta-glandins A, B, and C arise from PGE by dehydration and isomer-ization (Samuelsson, 1972; Hamberg and Samuelsson, 1973). Prostaglandins E and F are f a i r l y stable in blood but are rapidly degraded by tissue-bound enzymes; 80 to 90$ of a dose i s destroyed during a single passage through the lungs or the l i v e r (Vane, 1969: Piper, 1973). Metabolic pathways are species dependent. In man the major metabolic step for both PGE and PGF compounds is oxidation of the secondary alcohol group at C-j^ by prostaglandin dehydrogenase, followed by reduction of the C-^ double bond. The resulting 15-dehydro-13i 14-dehydro derivatives are inactive (Samuelsson, 1972; Andersen and Ramwell, 1974). Among the most successful analogs - 9 -Figure 3: Synthesis and catabolism of the two principal mammalian prostaglandins, PGE2 and PGFgpo (Douglas, 1975) - 10 -Esential Faty Acid in Diet Esterifed Acid in Cel Lipid e.g. Phospholipids ? Also of Cel Membrane Triglyceride V a r i o u S t i m u l i : C h e m i c a l a n d M e c h a n i c a l ? A c t i v a t i o n o f P h o s p h o h p a s e A o r O t h e r A c y l h y d ' o l o s e s a. False Substrates: e.g. COOH 5,8,11,1 4-Eicosatetraynoic Acid (TYA) b. Anti-nflammatory Drugs: e.g., Aspirin and Indomethacin 5,8,1 1,14-Eicosatetraenoic / V V \/ Acid (Arachidomc Acid) \^/\^/\ / \ / Inhibiton by i COOH PGD, COOH PGA, COOH PGC, COOH PGB, i - 11 -to be prepared are the 15- and 1 6-alkyl prostaglandins, which were designed to resist enzymatic C-15- dehydrogenation. Two members of this series, 15(5)-methyl PGE2 and 16,16-dimeth-y l PGE2, have been found to possess potential gastric anti-secretory activity ln animals (Robert, 1 9 7 2 ) . PGAs are de-graded more slowly, and this series may be the only naturally occurring prostaglandins capable of functioning as circulating hormones (Lee, 1 9 7 ^ ) . New prostanoids are continually being discovered, i.e. the endoperoxides (PGG and PGH) and PGI (Schaaf, 1 9 7 6 ) . Thromboxanes, other products of endo-peroxide metabolism, have also been found to possess marked pharmacological ac t i v i t i e s (Schaaf, 1 9 7 6 ) . They are unstable compounds with extremely short half lives, but may nevertheless be responsible for physiological processess. Other analogues of these compounds are being actively investigated. B. Prostaglandins and the Heart Investigation of prostaglandins as antiarrhythmics re-quires detailed physiological, pathological and pharmacological knowledge of their importance i n cardiac tissue. Effects on Coronary Circulation Pharmacological experiments on intact animals and isolated hearts have shown that prostaglandins of the E and A series have strong vasodilating actions on coronary arteries (Berti et a l . . 1 9 6 5 ; Bloor and Sobel, 1 9 7 0 ; Higgins et a l . . 1 9 7 1 , 1 9 7 3 ; Hutton et a l . . 1 9 7 3 ; Nakono and McCurdy, 1 9 6 8 ; Nutter and Crumly, 1 9 7 0 ; Vergroesen, 1 9 6 7 ) . In anesthetized dogs prostaglandins E^ and E 2 have been reported to be more - 12 -potent coronary vasodilators than adenosine, exceeding i t s efficacy five to ten times (Rowe and Afonso, 1974). The duration and magnitude of the coronary vasodilator action of P G E ^ have also been shown to be greater than that of nitro-glycerin (Nakano and McCurdy, 1968). The coronary vasodilator effect of PGA-L has been reported to be much less than that caused by equivalent doses of P G E ^ , although the systemic hypotensive effect of PGA-^ exceeded that produced by P G E ^ . The increased coronary blood flow and decreased coronary vas-cular resistance produced by intracoronary administration of prostaglandins of the E and A series in the dog has been shown to occur without any change in heart rate or systemic blood pressure (Nakano and McCurdy, 1967, 1968; Nakano, 1968; Hollenberg et_al. 1 9 6 8 ; Nutter and Crumley, 1970, 1972). The increase in coronary blood flow with intravenous administration of E or A series prostaglandins appears to be independent of changes in heart rate and systemic art e r i a l pressure (Maxwell, 1967; Nakano, 1968; Higgins et a l . . 1970, 1971; Bloor and Sobol, 1970) and is not abolished by 6-adrenergic blockade or atropine. Most authors report an increase in coronary blood flow in isolated perfused hearts with prostaglandins. PGE^ has been reported to enhance coronary blood flow in rat, cat and rabbit heart without significant inotropic or chronotropic effects (Mantegazza, 1965). In the isolated perfused rat heart this effect has been reported to be concentration-dependent between 2 and 10 ng/ml PGE-p with the maximum recorded coronary flow being 2.5 to 3 times the basal level (Ten Hoor, 1975). In con-- 1 3 -trast," Berti et a l . . ( 1 9 6 5 ) f a i l e d to find any effect of PGE-^ on heart rate and coronary flow in isolated rat heart. Vergroesen et a l . ( 1 9 6 7 ) , however, reported that PGE^, PGEj> and PGA^ increased coronary flow in isolated rat heart. In the isolated guinea pig heart the increase in coronary flow produced by PGE^ was not blocked by propranolol, pro-nethalol, or reserpine pre-treatment. Most authors report that coronary blood flow in intact animals is not affected by prostaglandins of the F series, although they can cause peripheral vasoconstriction (Nakano, 1 9 6 8 ; Hollenberg et a l . . 1 9 6 8 ; Nutter and Crumly 1 9 7 0 , 1 9 7 2 ; Bloor and Sobol, 1 9 7 0 ) . In contrast, Kateri et a l . ( 1 9 7 0 ) reported that high doses of PGF^ ( 5 0 jug/ml) increased coronary flow in the isolated dog heart-lung preparation. The reports for the isolated rat heart are controversial. Vergroesen et a l . ( 1 9 7 0 ) reported that PGF-j^ or PGF i e failed to alter coronary flow, while Willebrands and Tasseman ( 1 9 6 8 ) found that PGF^ increased coronary flow. It has been suggested that prostaglandins may be endogen-ous mediators involved in coronary vasodilation, This possi-b i l i t y follows the finding of a basal release of prostaglandins when hearts are perfused by the Langendorff technique (Bader and Johnson, 1 9 7 2 ; Block et a l . . 1 9 7 4 ; Junstad and Wennmalm, 1 9 7 2 ; Neddleman et a l . . 1 9 7 5 ; Wennmalm, 1 9 7 5 ) . Both PGEX and PGE2 appear to be released at approximately 5 ng/heart/min. Prostaglandin release i n increased following hypoxia, mechan-i c a l manipulation, and acetylcholine or noradrenaline admin-istration. No increases were found with alterations in pH, - 1 4 -temperature, osmolarity, or changes in the potassium or calcium levels of perfusing solutions. Indomethacin , a prostaglandin synthetase inhibitor, has been shown to block hypoxia-induced increased coronary blood flow in intact anesthetized dogs (Afonso et a l . , 1 9 7 4 ) . Prostacyclin (PGI2) has recently been identified as the endogenous metabolite responsible for relaxation of coronary arteries induced by arachidonic acid (Gregory et a l . . 1977) • The physiological importance of such prostaglandin release has not been f u l l y elucidated; however, i t is being rapidly resolved that PGIg is probably the coronary vasodilator in hypoxic hyperemia. The hypothesis that prostaglandins E-^ and Eg are the vasodilators in hypoxic hyperemia has been rejected (Block et a l . , 1975). Nevertheless, the evidence seems to indicate that these pharmacologically active compounds are involved in the local regulation of the coronary vascular bed; they may also play a physiologic role in the cardiac response to ischemia. Inotropic and Chronotropic Effects Prostaglandins have been shown to produce cardiac ino-tropic and chronotropic effects dependent upon experimental conditions and varying qualitatively and quantitatively in different species. When given to intact animals, they increase cardiac output, partially by reflex mechanisms (Malik and McGiff, 1976). For example, in dogs the in vivo increases in myocardial contractile force and heart rate reported in response to prostaglandins of the E, A, and F series appear to be secondary to reflex sympathetic activation possibly - 1 5 -involving cardio-accelerator centers in the medulla (Malik and McGiff, 1 9 7 6 ) . Variations in inotropic and chronotropic actions of prostaglandins, and species specificity, have also been observed in vitro. Prostaglandin E± failed to alter the rate of the isolated heart of rabbit, cat, dog and chicken, but produced positive chronotropic and inotropic effects in isolated guinea pig heart which were not blocked by either pro-pranolol or reserpine pre-treatment, thus indicating a direct myocardial effect (Berti et a l . . 1 9 6 5 ; Mantegazza, 1 9 6 5 ; Sunahara and Talesnik, 1 9 7 4 ; Horton and Main, 1 9 6 7 ) . In rat myocardium the amplitude of contraction of the isolated heart was found to be only modestly increased by PGEX and PGE2 (Berti et a l . , 1 9 6 5 ; Vergroesen et a l . . 1 9 6 7 ; Vergroesen and de Boer, 1 9 6 8 ) , with a greater effect in the spontaneously beating atria of normal and genetically hypertensive rats (Levy, 1 9 7 3 ) . In the former studies no increased chronotropic action was noted, while in the latter experiments (Levy, 1 9 7 3 ) » PGE2 increased rate; i t is not known whether the inotropic and chronotropic iffects on rat myocardium are due to direct or indirect actions or combinations of both. It has been demonstrated in isolated rat and frog hearts that PGE-p which normally only modestly influences myocardial contractility, w i l l restore normal contraction i f the heart function is depressed by an increased K +/Ca 2* ratio of the perfusion f l u i d , by propranolol, by barbiturates or by excess . Mg (de Boer et a l . . 1 9 7 3 ; Vergroesen and de Boer, 1 9 6 8 ) . Such results may indicate ;a. direct membrane effect of prostaglandins. - 16 -Furthermore, some of the apparently controversial results previously discussed may not be due to differences in species, but to differences in experimental conditions. Effects of Prostaglandins on Cyclic AMP in Cardiac Tissue The mechanism by which prostaglandins produce rate and force effects is separate from the adrenergic system, although the E and A series prostaglandins increase cyclic AMP levels in homogehates of guinea pig hearts (Klein and Levey, 1971; Sobol and Robinson, 1969). PGE1 has also been shown to in-crease cyplic AMP levels in rat heart (Curnow and Nuttal, 1971) and to increase adenylate cyclase activity in guinea pig and rat heart (Sobol and Robinson, 1969). Prostaglandins of the F series have been found to be inactive in particulate membrane preparations, but active in solubilized preparations (Levey and Klein, 1973). Beta-blocking agents are not effective in preventing the activation of adenylate cyclase by prostaglandins E and A (Klein and Levey, 1971). Sen et a l . (1976) have suggested that PGE2 may modulate the cardiac cAMP level and that the latter plays an important role in the adaptive regulation of the coronary flow. Whether elevation of cyclic AMP is responsible for the rate and force effects of prostaglandins in the heart remains to be completely resolved, but there is no well-defined, obligatory association between a l l prostaglandins and cyclic AMP anymore than there is an association between a l l adrenergic agonists and cyclic AMP. Effect of Prostaglandins on Myocardial Metabolism Prostaglandins of the E series have marked effects on l i p i d and carbohydrate metabolism whereas those of the A series - 17 -are inactive and those of the F series are only minimally effective. Prostaglandin has been reported to be a potent inhibitor of lipolysis (Steinberg et a l . . 1964; Carlson, 1965) induced by catecholamines (Steinberg, 1963), sympathetic nerve stimulation (Berti and Usardi, 1 9 6 4 ) , and by a wide variety of hormones (Steinberg et a l . , 1963, 1964; Mandel and Kuehl, 1967; Fain, 1967). This action of PGE-j^ was found, however, to be dose dependent with small doses stim-ulating and large doses inhibiting l i p o l y s i s . In isolated perfused rat hearts PGE1 and PGF^ have been found to stimulate oxygen consumption, glucose uptake in hearts using glucose as a substrate, and uptake of palmitic acid in hearts given the fatty acid as a substrate (Wille-brands and Tasseron, 1 9 6 8 ) . Maxwell (1967) similarly reported a rise in oxygen uptake and coronary blood flow which was accompanied by decreased glucose and free fatty acid levels and increased lactate concentrations in response to intra-coronary administration of PGE-^ in anaesthetized dogs. In contrast to the effects of PGE^, PGFg^id not produce a sig-nificant effect upon either arterial glucose, myocardial glu-cose, or free fatty acid extraction, while i t increased both lactate and pyruvate levels in art e r i a l and in coronary sinus blood (Maxwell, 1 9 6 9 ) . Effect of Prostaglandins on Cardiac Neurotransmission Prostaglandins are thought by some to modulate autonomic ..-neurotr^ the various "types of prostaglandins synthesized in cardiovascular tissues in response to - various - 1 8 -stimuli (Barnwell and Shaw, 1 9 7 0 ; Horton, 1973; Hedqvist, 1973; Crowshaw and McGiff, 1 9 7 3 ) , those of the E series appear to be the most l i k e l y physiological modulators of adrenergic trans-mission (Hedqvist, 1 9 7 3 ) * Exogenously administered PGE^ and PGE2 ( 1 0 ~ 8 to 10"^M) inhibit the release of noradrenaline from the sympathetic nerve endings of rabbit heart (Hedqvist and Wennmalm, 1 9 7 1 ; Wennmalm, 1 9 7 6 ) . PGE compounds, however, do not alter the basal efflux of norepinephrine in isolated rabbit heart (Hedqvist et a l . . 1 9 7 0 ) which suggests that PGE compounds inhibit outflow of norepinephrine from adrenergic nerves i n -directly by interfering with some step(s) in the release mech-anism. PGE inhibition of noradrenaline release is frequency-dependent and is most marked at lower impulse frequencies (50# reduction in release at 2 to 5 Hz.). Interestingly, sym-pathetic nerve stimulation e l i c i t s release of endogenous PGE (Wennmalm and Hedqvist, 1 9 7 1 ; Wennmalm, 1976) while inhibi-tion of prostaglandin synthesis with indomethacin results in an increase of noradrenaline release with nerve stimulation. These results may indicate that the process of transmitter release i s normally modulated by endogenously formed PGE (Samuelsson and Wennmalm, 1 9 7 1 ) * Most of the available evidence indicates that PGA and PGF compounds are less l i k e l y to be physiological modulators of adrenergic transmission for they are synthesized in cardio-vascular tissues in relatively small amounts as compared to prostaglandins of the E series (Karim, 1 9 6 7 ; Karim et a l . . 1 9 6 7 , 1 9 6 8 ; Horton, 1 9 7 3 ; Crowshaw and McGiff, 1 9 7 3 ; - 19 -Papariicalaou et a l . , 1 9 7 4 ) . The effect of series A prostaglan-dins on adrenergic neurotransmission has not been studied in the heart, but in the isolated perfused rabbit kidney pGA 2 was 10 to 50 times less active than PGEi or PGE2 in inhibiting responses to sympathetic nerve stimulation. Prostaglandins of the F series have been found to either be ineffective or to exert a slight f a c i l i t o r y effect on adrenergic transmission in the cardiovascular system. pGF 2 o c ^ a i l e d ^o a l t e r ^ n e contract-i l e responses or the release of noradrenaline produced by sympathetic nerve stimulation in rabbit, cat, and dog heart (Hedqvist and Wennmalm, 1971; Brody and Kadowitz, 1 9 7 4 ) . PGE^ has also been reported to inhibit parasympathetic neurotransmission in the isolated rabbit heart. Wennmalm and Hedqvist ( 1 9 7 D reported that PGE1 inhibited the chronotropic response to exogenous acetylcholine, which would indicate a presynaptic site of action. Junstad and Wennmalm (1974) demonstrated that infusion of acetylcholine or vagal nerve stimulation at low frequency released a PGE-like substance from the isolated heart and that this release was blocked by atropine. Acetylcholine, however, is markedly less potent than noradrenaline in stimulat-ing PGE release from the heart (Wennmalm, 1 9 7 6 ) . It therefore seems doubtful that a physiological inhibition occurs with the parasympathetic nervous system. In contrast to the results in rabbit heart no presynaptic action at the cholinergic neuro-muscular junction has been found in guinea pig heart (Park et a l . . 1973. Betting and Salzman, 1974; Hadhazy et a l . . 1 9 7 3 ) . - 20 -These observations "indicate that the modulatory action of prostaglandins on cholinergic transmission may be species dependent. Cardiac Electrophysiological Actions of Prostaglandins Low concentrations of PGE-^ PGE2 (5 ng/ml) and KJF^ Cl >»g/ml) have been reported to cause an increase in the maximal rate of depolarization ( V m a x) and in some cases also of the overshoot of the transmembrane action potential in the auricles of the cat, rabbit, guinea pig and in the ventricles of the rat, with the magnitude of effect being species dependent (Kecskemeti, Keleman, and Knoll, 1973, 1974, 1976)o Higher doses of prostaglandins (20 ng/ml for PGEs; 10 /ug/ml for PGFg,^ )» however, decreased Vmax and overshoot. This dose-dependent nature of the effect of PGE^ and PGFg^ on the cardiac action potential i s ' complex for at s t i l l higher concentrations ( 0 . 1 ug/ml) PGE^ and PGE2 decreased the resting potential in cat and guinea pig auricles by a max-imum of 15 and 17$ respectively (Kecskemet!, Keleman, and Knoll, 1 9 7 6 ) . In the presence of carbaminoyl choline, which increases potassium permeability of a t r i a l fibers, a high dose of PGE2 did not decrease the resting potential. This result was interpreted to mean that the depolarizing effect of high .concentrations of PGEs ; was due to changes in the potassium permeability of the cardiac c e l l . The biphasic effect of prostaglandin E 2 and F ^ on maximum rise-rate, together with a slight increase in resting membrane potential at low concentrations has also been reported - 21 -by other workers (Porster et a l . , 1 9 7 4 ) . They found no myo-cardial depressant "quinidine-like n effects on the isolated atrium and papillary muscle of the guinea pig at antiarrhythmic concentrations. Small changes, usually not exceeding 10#, could be detected i n the e l e c t r i c a l threshold, l e f t a t r i a l conduction time, and maximum driving frequency* Prostaglandin E 2 had l i t t l e effect on the f i b r i l l a t i o n threshold of the cat heart; however, Produced a moderate increase i n f i b r i l l a t i o n threshold at high doses. Prostaglandins of the E series have been reported either to have no effect, or to . shorten the action potential duration (Kecskemeti et a l . . 1 9 7 6 ) . Prostaglandins ? 2 o c» however, has been found to significantly prolong action potential duration by 30 to 60 per cent In ventricular strips from guinea pigs and i n rat papillary muscle, respectively (Mentz et a l . . 1976; January and Schotelius, 197*0. C. ANTIARRHYTHMIC EFFECTS Almost simultaneously, and Independently, Z l j l s t r a et a l . ( 1 9 7 2 ) and Mest et a l . ( 1 9 7 2 , 1973) reported the f i r s t pre-liminary investigations of the antiarrhythmic effects of pros-taglandins. The study by Z i j l s t r a et a l . was not planned, but was suggested by the occurrence of arrhythmias In some exper-iments being conducted on the cardiovascular effects of PGE^ in dogs. It was observed that Intravenous injection ( 0 . 5 to 4 ug/Kg ) or Infusion ( 0 . 5 to 4 ng/Kg/min) of PGE^ effect-ively suppressed bigeminy occuring during the course of thio-barbitone anesthesia. In addition, ventricular tachycardia - 22 -due to acute myocardial ischemia was changed to sinus rhythm by a single intravenous injection of 2 ug/Kg PGE-L. The authors suggested that PGE^ might suppress cardiac arrhythmias through i t s moderating influence on the sympathetic-para-sympathetic balance, which would influence impulse conduction in the heart (Hedqvist and Brundin, 1969; Hedqvist, 1970; Wennmalm and Hedqvist, 1970; Wennmalm, 1 9 7 1 ) . Mest et a l . (1972, 1973) and Porster et a l . (1973) pub-lished further findings on preliminary investigations into the antiarrhythmic effects of PGE^, P G E 2 , and P G P ^ in mini-pigs, dogs, and cats. PGE-^ was found to protect the mini-pig from ventricular f l u t t e r produced by coronary artery ligation. In cats with similar ligations PGF^rf resulted in longer post-infarct survival times, together with better maintenance of cardiac output. It was suggested that such protection might be the result of maintenance of lysosomal integrity i n the ischemic tissue and prevention of the formation of a cardio-toxic peptide. Support for this suggestion has been provided by further study in dogs (Glenn et a l . . 1975) which showed that PGFg* produced higher aortic flow rates, rates of de-velopment of ventricular pressure (dp/dt) and mean ar t e r i a l blood pressures post infarct, while reducing lysosomal enzyme release. Methylprednisolone produced effects similar to that of PQF^t and both agents were presumed to act by preserving cellular integrity. Such a mechanism has also been suggested by experiments with a stable free-radical form of PGBx, PGB X , which has been reported to preserve oxidative - 23 -phosphorylation in degenerated mitochondria, and at the same time protect Rhesus monkeys from the effects of coronary ar-tery ligation, so that the mean control survival time of five minutes is prolonged to ninety minutes (Angelakos, et al». 1 9 7 5 ) . A number of comparative studies have now been carried out with PGs A l t A 2, E-p E 2, F 2 e r, F 2 P , and the precursors arachi-donic acid and l i n o l e i c acid using several other exper-imental dysrhythmias, such as those produced by aconitine, calcium chloride, barium chloride, ouabain, and electrical stimulation (Chiba et a l . . 1 9 7 2 ; Kelliher and Glenn, 1973; Mest et a l . . 1 9 7 2 , 1 9 7 3 , 1 9 7 5 , 1 9 7 6 ; Forster et a l . . 1 9 7 3 , 1 9 7 4 ; Mentz and Forster 1 9 7 4 a , 1 9 7 4 b ; Bayer et a l . . 1 9 7 6 ; Mann et a l . . 1 9 7 3 ) . The calcium chloride and aconitine ar-rhythmias were induced i n rats (Forster et a l . . 1 9 7 3 ; Mentz and Forster 1 9 7 4 a , 1 9 7 4 b ). The strongest effect against calcium induced arrhythmias was shown by PGF 2 a f with a bolus injection of 5 jug/Kg I.V. This protected a maximum of 84 per cent of animals, for 10 minutes, from lethal ventricular f i b r i l l a t i o n s . Ajmaline, the standard antiarrhythmic drug, was effective in 50 per cent of the animals. A good anti-arrhythmic effect was also shown with PGA2, followed in decreasing effectiveness by PGE-^ , E 2, A-^ , and F 2 & . Prosta-glandin Fg^. was also the most effective against aconitine-induced arrhythmias, followed by PGEg, A 2, E 1, A-p and F 2g. Barium chloride arrhythmias were induced i n unan-esthetized rabbits and a normalization of the ECG for at least one minute was taken as indication of an antiarrhyth-- 2 4 -mic effect (Forster et a l . . 1 9 7 3 ; Mentz and Forster, 1 9 7 4 ; Mest et a l . . 1 9 7 4 ) . Prostaglandins E 2 and A± at 1 n g A g/min infusion showed the strongest effect with normalization in 60 per cent and improvement i n 4 0 per cent of the animals. Sim-i l a r effects, but at higher doses, were obtained with PGA£, F 2 « a n d E l * Ajmaline was also effective i n 60 per cent of the animals, but at a dose one thousand times higher than that of the prostaglandins. In ouabain arrhythmias i n cats, PGE2 was most effective, causing 60 per cent of the animals to revert to temporary sinus rhythm (Mest et a l . . 1 9 7 6 ; Kelliher and Glenn, 1 9 7 3 ) . Similar improvement was obtained with PGE^, with lesser effects seen for PGFg^, A 2, and E ^ Ajmaline was also effective in approx-imately 5 0 per cent of the animals, but at doses 1 0 0 times higher than that of the prostaglandins. Prostaglandins have not been found to be particularly effective against arrhythmias induced by electrical stimulation (Bayer et a l . . 1 9 7 6 ; Forster et a l . . 1 9 7 ^ ) . As mentioned ear-l i e r they have most often been found to produce only small (and not s t a t i s t i c a l l y significant) changes in conduction velocity and functional refractory period. Prostaglandin F ^ produces a significant increase i n the duration of the cardiac action potential, but the PGEs have no such effect (Forster, 1 9 7 6 ; Mentz, 1 9 7 6 ) . A possibly related finding i s the a b i l i t y of PGF2, but not PGE2, to increase the f i b r i l l a t i o n threshold in isolat-ed atria from rats (Forster et a l . . 1 9 7 ^ ) . There i s , however, no correlation between this action and other antiarrhythmic act-i v i t i e s (Forster, 1 9 7 6 ) . In summary, elec t r i c a l changes - 25 -observed with prostaglandins are varied and distinctly-weaker than those seen after the application of classical antiarrhythmic agents, which may point to a mode of action different from that of quinidine-like antiarrhythmic drugs. The prostaglandin precursor fatty acids, l i n o l e i c acid and arachidonic acid, have also been tested and found to be effective against barium chloride, ouabain, and aconitine arrhythmias when infused at 0.5 to 1 mg/Kg/min (Mest and Forster, 1973; Mest et a l . . 1976; Forster et a l . . 1 9 7 6 ) . Indo-methacin, an inhibitor of prostaglandin synthetase, abolished the antiarrhythmic effect of the precursors, while prosta-glandin effects were not influenced. It therefore appears that prostaglandins biosynthesized from the precursors, rather than the precursors themselves, are responsible for the antiarrhythmic activity (Forster, 1 9 7 6 ) . C l i n i c a l Evidence Short c l i n i c a l t r i a l s with PGF 2 x have demonstrated i t s effectiveness in some dysrhythmias (Mann et a l . . 1973; Mann, 1 9 7 6 ) . Positive results were obtained with ventricular ar-rhythmias in patients suffering from myocardial infarction and possibly from the toxic administration of cardiac glyco-sides. Paroxysmal a t r i a l tachycardia did not respond. In contrast to the results obtained with other antiarrhythmic drugs, such as ajmaline, the administration of PGF2o< did not result in any essential changes in the diastolic stimulus threshold. This was interpreted as support for the conclusion that the mechanism of antiarrhythmic action of PGF2e( cannot re-present a non-specific, quinidine-like, antiarrhythmic effect. - 26 -Although prostaglandins are antiarrhythmic, i t is of interest to note that PGF 2 l < in at least one report has been cited as responsible for inducing arrhythmias (Koss and Naka-no, 1 9 7 4 ) . Transient episodes of sinus bradycardia and ven-tri c u l a r bigeminy were observed with the intravenous administra-tion of prostaglandin F 2 c < ( 5 to 15 ug/Kg) in anesthetized cats. This arrhythmogenic effect of PGF2o< was abolished following b i l a t e r a l vagotomy, which would indicate the arrhythmias were most l i k e l y due to marked stimulation of vagal tone in this species. The PGF 2 o C dose used in this experiment, however, was above the maximal antiarrhythmic dose of 1 to 2 ^ig/Kg/min, in this species (Mest et a l . , 1 9 7 4 ) . Purpose of Experiments to be Described From the literature, several conclusions may be drawn: 1 . ) Prostaglandins have a complex, and species-dependent, cardiac pharmacology affecting coronary circulation, con-t r a c t i l i t y , cyclic AMP levels, myocardial metabolism, neurotransmission, and cardiac membrane potentials. 2 . ) Prostaglandins are effective antiarrhythmic agents which are endogenously formed from precursors in the heart. 3. ) A l l prostaglandins tested have an antiarrhythmic effect, although relative potencies vary with the prostaglandin, species, and type of arrhythmia. 4 . ) Prostaglandins appear to be effective against ar-rhythmias induced by damage or ionic disturbance. They have l i t t l e effect against non-damaging disturbances such as electrical stimulation. - 27 -5.) The mechanism of the antiarrhythmic action of pros-taglandins i s not l i k e l y to be due to a general myocar-dial depressant (quinidine-like) effect. Although the above conclusions may be drawn from the literature, the evaluation of prostaglandins antiarrhythmic activity requires that the following question be answered: 1) which prostaglandins are antiarrhythmic, 2) against which arrhythmias and 3) by what mechanisms The answer to such a question should allow evaluation for suit-a b i l i t y and the choice of the best prostaglandin for future c l i n i c a l t r i a l . The treatment of arrhythmias i s s t i l l far from perfect. There i s need, for example, for an antiarrhy-thmic capable of being used to protect the recently infarcted patient prior to f u l l medical care. Furthermore, the extensive pharmacological spectrum of prostaglandin activity needs the f u l l e s t investigation in order to define the various acti v i t i e s with a view to the maximum u t i l i z a t i o n of each. Only the f u l l -est analysis of the pharmacological ac t i v i t i e s in adrenergic amines has allowed the development of highly selective drugs. With the above firmly i n mind the experiments described in this work were initiated to answer, at least in part, a l l the three facets of the i n i t i a l question. In view of the previously considered antiarrhythmic efficacy of prostaglandins and their a b i l i t y to reduce the deleterious effects of coronary ischemia ( Z i j l s t r a et a l . . 1972; Mest et a l . . 1972, 1973; Forster et a l . . 1973; Glenn et a l . . 1975; Angelakos, et a l . . - 28 -1975) i t was decided to test the effectiveness of prostaglandins against the various arrhythmias associated with cardiac Ischem-i a . I n i t i a l results were obtained in the dog and then recon-firmed in the rat, where a more thorough comparison of d i f f e r -ent prostaglandins was performed. Rat heart tissue in intact, isolated, and c e l l culture preparations was used to inves-tigate possible mechanisms of action. Secondary considerations of choice of experimental model, selection of specific prosta-glandins and selection of doses are more f u l l y discussed in the methods section. This work therefore I) f i r s t describes the activity of prostaglandins on arrhythmias induced in vivo in A) dogs and B) rats by coronary artery ligation together with C) the effects of prostaglandins on the electrical characteristics of in vivo rat hearts. II) This activity is then compared with an anal-ysis of the a b i l i t y of prostaglandins to prevent contractility losses in isolated rat hearts as well as their pharmacological effects in this preparation. I l l ) The possible protective actions of prostaglandins i s also described for abnormalities of beating induced in cultured neonatal rat heart ce l l s by various physiological and pharmacological interventions. Dog experiments were primarily conducted by M. J. Walker and C. Harvie with my cooperation. Experiments on isolated rat hearts were performed with the help of T. Au and G. Collins. - 29 -CHAPTER II MATERIALS AND METHODS Experimental methods are described in order of the l i s t out-lined in the Introduction. EXPERIMENTAL SECTION I Production of Arrhythmias in Dogs and Rats A. Coronary Occlusion in Dogs Acute Coronary Artery Ligation for Early and Occlusion Release Arrhythmias In order to confirm the reports of prostaglandin anti-arrhythmic activity we f i r s t tested two prostaglandins, E 2 and P^, against arrhythmias induced in anesthetized dogs by coronary artery ligation. The two prostaglandins were chosen for testing because the former is a vasodilator and the latter i s a vasoconstrictor. The dog model was chosen because i t i s a standard antiarrhythmic test preparation and because the ischemic model most closely resembles the c l i n i c a l situation following a myocardial infarction. A problem with this preparation i s the large degree of variabi l i t y in. results obtained with different animals. We f e l t , however, that i f prostaglandins were to have any potential therapeutic value, i t should be possible to demonstrate their effectiveness in this system. Prom the literature we determined the antiar-rhythmic dose of prostaglandins to f a l l between 1 and 8 jug/ Kg/min (Mest et a l . . 1974). On this basis the test dose of 1 ug/Kg/min was chosen. Studies were carried out in 18 mongrel dogs of either - 30 -sex weighing between 22.2 and 28.6 Kg. Anesthesia was induced with I.V. sodium pentobarbital (30 mg/Kg) and maintained with 1 per cent halothane, 40 per cent nitrous oxide and 59 per cent oxygen with respiratory assistance from a Bird Mark 8 respir-ator. The heart was exposed through a l e f t thoracotomy in the 5th intercostal space and suspended in a pericardial cradle. The lef t , anterior descending coronary artery was dissected free and prepared for ligation with the t i e in place, but not tightened, approximately 2 to 3 cm from the l e f t a t r i a l margin. Aortic pressures were recorded by means of a catheter introduced retrogradely from the femoral artery and connected to an appropriately calibrated P23Db Statham pressure trans-ducer. Electrocardiograms (ECG) were taken from a precordial lead and recorded on a Grass model PCPb polygraph with EKG Tachograph pre-amplif ier model 171^. Freshly prepared pros-taglandins were infused by way of a catheterized femoral vein. Experimental Protocol One hour after operation animals were monitored for 30 minutes of infusion. Five minutes after the start of the infusion the coronary artery ligature was tightened and the chest closed. The ligature was kept tightened for 25 minutes before i t s rapid release. Infusions were continued for 15 minutes after release, but i f f i b r i l l a t i o n occurred, the animal was el e c t r i c a l l y defibrillated. Determination of Arrhythmias and Analysis of Data Acute Dogs Premature ventricular contractions were counted from continuous records. Each PVC was recognized from the ECG - 31 -and corresponding anomalies in the pressure record. Pressures were measured as mean systolic or diastolic over the measure-ment period. Statistics The Student 't' test for unpaired data was used to test for differences between the drug treatments. A probability of less than 0.05 was chosen as the criterion of significance. B. Acute Coronary Occlusion in Bats In order to confirm and expand our studies on the anti-arrhythmic actions of prostaglandins we also performed cor-onary ligation experiments in rats. This allowed us to use cheaper animals and simpler surgical techniques to screen a larger number of drugs. Prostaglandins E 2, F l o 0 A 2, and F 2 P as well as lidocaine and quinidine were tested in this pre-paration. Prostaglandin E 2 and A 2 are vasodilators. Pros-taglandin A 2 is more slowly metabolized than E£ (Lee, 1 9 7 4 ) . Prostaglandin F ^ as mentioned previously is a vasoconstrictor, and K3F2(3 has l i t t l e smooth muscle activity. The standard antiarrhythmic drugs lidocaine and quinidine were additional controls. A second consideration in using rats was that i t allowed for testing of antiarrhythmic activity in a second species. A potential problem was that this was a new model for antiarrhythmic testing. Comparison of control animals and those treated with PGE2 and Fjpt which we had previously tested in dogs allowed for comparison between the rat and dog models. A prostaglandin dose of 2 jig/Kg/min was chosen as this was within the previously reported antiarrhythmic dose range. Quinidine at a bolus dose of 3 mg/Kg was also taken - 32 -from the literature (Moe and Abildskov, 1975)» and lidocaine 6 juig/Kg/min was the highest dose which could be infused throughout the test period without seriously depressing blood pressure. Male Wistar rats (300 to 4 0 0 g) were used in this exper-iment. The procedure of l e f t coronary occlusion was a mod-if i c a t i o n of techniques described by John and Olsen ( 1 9 5 4 ) and Selye et a l . . ( I 9 6 0 ) . The animal was anesthetized with pen-tabarbital sodium ( 6 0 mg/Kg I.P.), placed on a heated (37° C) pad, and immobilized with tape. A tracheotomy was performed and a blunt 15 guage needle was secured in the trachea. The animal was allowed to respire i t s e l f while cannulae were i n -serted into the carotid artery and femoral vein. Aortic pres-sures were recorded from the carotid artery using a calibrated P23Dc Stratham pressure transducer. Infusions of test drugs were made into the femoral vein. Three needle limb leads were used to monitor the ECG which was recorded on a Grass model PCPb polygraph with EKG Tachograph pre-amplifier model 7 P 4 P . An incision was then made through the skin at the base of the sternum, using large blunt scissors. The skin was loosened from the underlying muscle mass using blunt dis-section to the base of the neck. The skin was then cut from the base of the sternum to the neck.and. peeled back, revealing the chest musculature. A scalpel was used to make an incision through the chest muscles to the ribs, parallel to and approx-imately 5 mm to the l e f t of the sternum. Bleeding from the internal epigastric artery (which courses Just to the l e f t of the sternum) can thus be avoided. A r t i f i c i a l respiration was - 33 -applied at this time using room a i r with a Palmer small animal respirator having a stroke volume of 4 ml and a rate of 30 strokes per minute. Pine pointed scissors were used to cut through the ribs and remaining tissue in a line 5 mm from and parallel to the l e f t of the sternum exposing the heart. Retractors were used to hold the chest walls back, and blunt forceps were used to open the pericardium. Control measurements of ECG, rate, and blood pressure were taken and infusions of test drugs then started. Five minutes after the start of the infusion, ligation of the l e f t coronary artery was performed. Ligation of the Left Coronary Artery The l e f t coronary artery i s predominant and supplies the l e f t ventricle. There i s no true circumflex artery i n the rat. The coronary arteries l i e beneath the epicardlum and can be seen at operation i n the intact beating heart as tiny red streaks beneath the surface of the heart. In the rat the main l e f t coronary artery can be ligated at a point just beneath the l e f t auricular appendage. Occasionally, branching w i l l have already begun under the l e f t a t r i a . In this situation i t i s necessary to ligate several branches at the same time in order to obtain a good-sized area of infarct. A small curved needle (corneal needle 3/8 c i r c l e , 9 mm, atrau-matic) carrying a thread (black braided, non-slipping s i l k , size 50) held in straight hemostats was used for ligation. In the experiments, the l e f t a t r i a was l i f t e d with blunt forceps; since the coronary artery and i t s branches were v i s i -ble the needle was simply passed beneath them and the l i g a t -ture tied (Figure 4 ) . Virtually the entire anterior lateral - 3 * -Figure 4: Ligation of the l e f t coronary artery in rat , 1.,: Anterior aspect of the heart in a rat whose coronary arteries have been injected with lead oxide in latex. The superficial myocardial layers have been partially cleared in methyl benzoate to make the Intramuscular arteries more readily v i s i b l e . The l e f t coronary artery originates be-ween the pulmonary cone and the l e f t auricular appendage (right side of picture). After sending a few major branches towards the pulmonary cone, i t descends almost directly towards the apex, along the curved surface of the l e f t ven-t r i c l e . The main trunk of the right coronary artery is not visibl e , but i t s perpendicular lateral branches can be seen to advance (from the l e f t side of the picture) towards:the anterior part of the heart, within the wall of the right ventricle. 2.: Heart of a rat in which the arteries have again been made visible by injection of colored material, but the myo-cardium has been l e f t opaque to show i t s surface markings. A curved needle has been introduced between the pulmonary cone and the insertion of the l e f t auricular appendage, to demon-strate the procedure to be followed for the occlusion of the l e f t coronary artery near i t s origin. Additional ligatures (such as" are used for the production of smaller infarcts) have been placed at the lower points ?alofig the course of this vessel. Only the ligation near the origin was used in the present series of experiments to produce arrhythmias. (Selye et a l . , I960). - 35 -- 36 -wall of the l e f t ventricle then became ischemic. Arrhyth-mias usually occurred between 4 and 11 minutes after ligation and were easily detected by changes in the ECG and blood pressure. The size of the infarct produced was measured after the experiment by perfusing the hearts with Krebs solution. Blood quickly washed out of a l l areas except the infarct which was then cut out and weighed as the per cent of whole heart weight. Infusions A l l prostaglandin infusions were given as 2 jig/Kg/min in a volume of 0.1 ml saline/ min. Lidocaine was given as an infusion 6 ng/Kg/min in a volume of 0.1 ml saline/min and quinidine 3 mg/Kg in a volume of 0;3 ml was-given as a slow IV injection. Determination of Arrhythmias in Rats In i n i t i a l experiments on rats not given any type of antiarrhythmic a common series of arrhythmic events following ligation was seen. I n i t i a l l y sinus beats with occasional PVCs occurred. This was followed by bursts of ventricular flu t t e r of varying duration and then, sometimes, ventricular f i b r i l l a t i o n which did not always end in death. To quantitate these arrhythmias a number of measurements were taken. These included: 1. the number of PVCs in the 25 minute occlusion period 2. the number of PVC salvos (with one salvo being 3 or more consecutive PV€s) 3. the number of flut t e r episodes - 37 -4. the time spent in flu t t e r 5. the time for fl u t t e r to f i r s t occur 6. the time for f i b r i l l a t i o n to f i r s t occur Gn the basis of these measurements a subjective scoring system for arrhythmias was constructed as shown below. Arrhythmia Subjective Score PVC 49 or less 1 PVC 50 to 74 2 PVC 75 to 99 3 PVC 100 or more 4 Flutter after 10* 5 Flutter between 5-10' 6 Flutter total less than 1' 7 Flutter total greater than 1' 8 Flutter between 0-5' 9 Flutter before 1* 10 Flutter after 1' 11 F i b r i l l a t i o n after 10* 12 F i b r i l l a t i o n between 5-10' 13 F i b r i l l a t i o n before 5' 14 Experimental Design A l l experiments were done "blind" and a l l data were evaluated using a "blind" (drug treatment unknown to the scorer) technique. After a l l drug treatments were eval-uated the code used was broken and the scores for the treat-ments using the same drug were averaged. Five animals were included in each drug treatment group, and each animal received only one antiarrhythmic test agent. Statistics Dunnett's *t' test for unpaired data was used to test for differences between the various drug treatments and control. Chi square test was used to test for significance of the proportion of animals dying. A probability of less than 0.05 was chosen as the criterion of significance. C. Production of arrhythmias by Ele c t r i c a l Stimulation in the Bat: Flutter Threshold Current Intact and isolated rat hearts were used to investigate possible mechanisms of prostaglandin antiarrhythmic activity. We determined the effect of prostaglandins E 2 , Ag, F ^ , F 2 j 8, F2<*,..•-'lidocaine, and quinidine on flutter threshold and maximum following frequency. Flutter threshold i s defined as the current necessary to produce flu t t e r and a drop in blood pressure when the heart i s being driven at a supramaximal rate. Maximum following frequency i s the maximum rate the heart can beat when being driven by an elec t r i c a l stimulator using a supramaximal pulse current. PGF 2 o o a vasoconstrictor, was added to allow for comparison with other reports of prostaglandin effects on electrical activity. Flutter threshold measurements allowed us to determine the effective-ness of prostaglandins against a non-damaging type of ar-rhythmia, and maximum following frequency measurements allowed us to determine the quinidine-like depressive action of pros-taglandins. Male Wistar rats ( 3 0 0 to 400 g) were prepared similarly to those used in the coronary ligation experiments but with-out actual coronary ligation. Instead, two punctate silver electrodes were inserted into the heart tissue, one on the right anterior ventricular surface and the other in the apical region. The wires were attached to a Grass SD9 stimulator and an oscilloscope which had been especially calibrated to measure current, and the electrical flutter threshold was then measured by determining the threshold intensity of ser i a l shocks required to evoke flutter of the ventricles. The heart was stimulated by serial rectangular pulses of 20 cycles/sec and 1 msec duration, and the strength of the stimulus was gradually increased. Extrasystoles were seen f i r s t followed by tachycardia, flutter, and f i n a l l y by f i b r i l l a t i o n . The appear-ance of ventricular f l u t t e r was easily recognized by a sudden f a l l in blood pressure, and the current required to produce fl u t t e r with each drug treatment was measured. Periods of stimulation of approximately 4 seconds duration were required for each determination. Maximum Following Frequency The maximum following frequency was determined by a simi-l a r procedure except that a supramaximal current of 1.0 mA was used and the frequency of stimulation was rapidly increased u n t i l the heart was unable to follow. This was easily deter-mined as the blood pressure, which had been suppressed by tachycardia and fl u t t e r with increasing rate, gives a series of sharp pulses at the point where the heart begins to skip beats. The maximum following frequency i s reciprocally related to the effective refractory period (the shortest interval between two stimuli to which the heart responds with contraction). EXPERIMENTAL SECTION II Isolated Rat Hearts and Contractility Losses It was of interest to evaluate the cardiac pharmacology - 40 -of the prostaglandins used so conventional studies were performed on unpaced Langendorf f perfused hearts. As there is a decrease in contractility with time of hearts perfused by this technique i t was of interest to see whether prostaglandins would have a 'protective' effect on this contractility loss. The Langendorff rat heart model was selected mainly for convenience. We perceived a number of deficiencies of this model. A l l Langendorff hearts are in a condition of failure with edema due to the physiological solution. In addition only the ventricles are f i l l e d with solution. Measurements of coronary flow by simply collecting the outflow dripping off the hearts i s also subject to considerable error./; However, the use of five hearts for each determination tended to cancel the error and allowed sufficient accuracy for our purpose as we were looking for marked pharmacological effects on the myocardium which might explain prostaglandin antiarrhythmic activity. The same four prostaglandins^- A 2, E 2, Fi<*, and F2p-which had been used with the ln vivo rat experiments were used for comparison, and prostaglandin infusions of 10""^, 10~7, and 10*"-' M were used to allow for dose-effect comparisons. Method Male Wistar rats (250 to 350 g) were k i l l e d by a blow to the base of the skull and quickly bled from the neck. The heart was removed and immediately placed in a dissecting dish containing oxygenated Krebs solution at 2 0 ° C. Prior to mount-ing on the perfusion apparatus, which was specially constructed to allow five hearts to be perfused at the same time by the Langendorff (1895) technique, a small hook was placed in the - 41 -apex of the ventricles so that each heart could be attached at w i l l to an inverted Grass force displacement transducer by means of a second hook and piece of string. A Harvard isometric tension clamp allowed tension on the ventricles to be adjusted. No longer than 10 to 15 minutes were required to attach a l l five hearts to the cannula©. Hearts were perfused at a constant 90 mm mercury perfusion pressure at 3 3 ° C. The purpose of the reduced temperature was to allow for a more gradual 'run down' curve. A mixture of oxygen (95 per cent) and carbon dioxide (5 per cent) was bubbled through the solutions. A modified Krebs (1950) solution was used exclusively in a l l experiments. The solution was prepared by dissolving the following amounts of reagents in one l i t e r of d i s t i l l e d water ( a l l weights are expressed as grams of the anhydrous compound): NaCl, 6.92; KC1, 0.35; CaCl 2, 0.28; MgSO^ , 0.15; NaHC03, 2.1; KH2P0^, 0.16; Glucose, 2.0. For the f i r s t hour, hearts were perfused with Krebs solut-ion alone and measurements erf heart rate and force developed at 25 g, diastolic tension, and flow rates were taken every 10 minutes. For the next 20 minutes the hearts were perfused with Krebs solution containing various prostaglandins at different concentrations; measurements were taken every five minutes. During the last hour, the hearts were again perfused with only Krebs solution and measurements taken every 10 minutes. Control experiments used only Krebs solution. S t a t i s t i c a l Analysis S t a t i s t i c a l analysis were performed by assuming that - 42 -changes with time were linear. Linear regression lines were calculated for the variables over the periods 20-60*, 60-80' (infusion), and 80-140' and significant changes calculated by standard s t a t i s t i c a l tests for slopes and intercepts. EXPERIMENTAL SECTION III Experiments on Cultured Heart Cells We had three motives in choosing cultured myoblasts as a test model: 1) we wished to investigate the direct c e l l -ular cardiac pharmacology of prostaglandins; 2) we hoped to develope a screen for antiarrhythmic activity using cultur-ed heart c e l l s ; and 3) we f e l t that by choozing suitable arrhythmogenic agents i t should be possible to produce ar-rhythmias whose ionic and biochemical causes were understood, thus allowing insight into the mechanism of the antiarrhythmic activity of prostaglandins. Cultured heart cells would appear to be a particularly useful preparation in that only direct actions need be considered, the effects of metabolic poisons are readily quantifiable, and i t has been reported that anti-dysrhythmic actions can be demonstrated in such cultures (Giraidier, 1971; Hyde, 1972; Goshima, 1976). In addition the physiology of cultured heart ce l l s closely resembles that of intact tissue (Wollenberg, 1970). A potential d i f f i c u l t y with this model is related to the fact that only single cells were studied. This system lacks the factor of c e l l to c e l l com-munication and therefore the model most closely resembles ectopic f o c i . Fourteen prostaglandins of the A, B, D, E, and F series were chosen for investigation of their cellular pharm-acology. We wished to test as wide a selection as possible in - 43 -this relatively simple system. For testing against arrhythmias induced by the different arrhythmogenic agents i t was neces-sary to reduce the number of prostaglandins in order to have a manageable experiment. Prostaglandins A 2, E 2, F]^, and F20 w e r e used to allow comparison with the in vivo rat exper-iments • Heart C e l l Culture Preparation Heart c e l l cultures were prepared by a modification of a method described by Harary and Farley ( 1 9 6 3 ) . One mm cubes of ventricular tissue (one heart for each flask to be prepared) from 5-day-old Wistar rats were digested at 3 7 ° C with 0.15$ trypsin (Nutritional Biochemicals Corporation) for 20 minute periods. The resulting c e l l suspensions were collected in fe t a l bovine serum (Microbiological Associates) at 4 ° C and three such digestates were pooled for centrifugation at 220 x g for 3 minutes. Washed pellets of c e l l s were resuspended in a tissue culture medium of the following composition; 20 ml F-10 (Colo-rado Serum Co.); 4 ml fetal bovine serum (Microbiological As-sociates), and 1 ml antibiotic solution (6.2 mg p e n i c i l l i n G + 10 mg streptomycin sulfate in Puck's saline A) diluted to 100 ml with Hank's basic salt solution (without KC1 or NaHCO^). Aliquots of the suspension were incubated in Falcon Tissue culture flasks for 2 hours to remove fibroblasts (which adher-ed rapidly in this period) before being transferred to fresh flasks for the growth of a primary myoblast-enriched culture. Culture media were changed at one and three days and experi-ments were performed on single cells 4-5 days in culture. - -Measurements of Myoblast Beating Activity The beating activity of single myoblast c e l l s was moni-tored by means of a photo-optical device similar to that des-cribed by Schanne (197D. A closed circuit television system on a phase contrast microscope was employed to obtain c e l l images on a 12-inch monitor while a pair of photo-resistors placed on the monitor screen measured the variation i n op-t i c a l density accompanying each c e l l beat (see Figure 5 ) « Output was displayed on a Grass Polygraph and processed (Grass tachograph) to give beating rate, rate variation (rate range), and the f i r s t d i f f e r e n t i a l of optical density changes (Tetronix Type 0 differential amplifier). Typical records from a group of five cells are shown in Figure 6 . Although there was variation between individual c e l l s , group mean values from different cultures and flasks were very constant. The effect of arrhythmogenic agents, drugs and mixtures of such were assessed by a random *blind" observation tech-nique. Drugs (nature unknown to the observer) were added at time zero and individual c e l l s were each examined for 20 seconds after 10 and 35 minute exposure to drug. For each drug concentration six c e l l s , randomly selected, were exam-ined at each time in each of three different cultures and a l l values were pooled. Appropriate 't' tests for unpaired, pooled data were used to test for s t a t i s t i c a l significance. In contrast to the studies' performed on random samples of individual c e l l s , i t was also possible to conduct experi-ments on the same selected c e l l . A calibrated tissue flask - 4 5 -Figure 5 : Diagram of apparatus for measuring beating activity in single cultured heart c e l l s . A. Photoresistors mounted on a T.V. monitor screen detect changes in optical density with beating activity. Changes are electrically processed to give the: I., rate of beating, II., range of beating, III. optical density, and IV., the f i r s t derivative of optical density with time (d OD/dt). Figure 6: Typical records of changes in optical density, the corresponding derivative of optical density, and the tachograph measured beat-to-beat rate from five single myoblasts. The figures on the rate record give the values of rate and rate range, for that c e l l . The group means were found to be very consistent between cel l s , flasks, and cultures. - 4 6 -a 3 7 ° C H A M B E R | | F L A S K | ^ ^ M I C R O S C O P E G R A S S P O L Y G R A P H RATE b e o , V r n i n . I R A T E ••• I R A N G E T I M E —'"vf—*---*'* mllllllllll O P T I C A L D E N S H Y a r b i t r a r y u n i t s Twvm 100|— R A T E I \bsati \ n " min-5oL • A A I J U I T L H ' 23 ; G R O U P M E A N R A T E = 7 1 * 7 ( m e a n ± s . e . m . , n = 5 ) G R O U P M E A N R A N G E = 16-3 d7 Q D- ••nin,iin,tn.,.,m, fufwimwimr tmummuum m m n n n n _l 1 1_ |5SEC.| i — i — i — i — i — i — i O P T I C A L D E N S I T Y ( a r b i t r a r y ! u n i t s J 11 C E L L -1 - 47 -holder allowed a particular microscope f i e l d to be selected when required and this, together with a sketch of c e l l shapes to aid identification, allowed the behavior of a number of single c e l l s to be followed in time, so that individuals in a group of ce l l s could be sampled repeatedly for any dose of drug or length of time. This method was used to obtain the complete chronotropic log dose-response curve for PG^* (Figure 2 3 ) . Quantitation of Arrhythmias in Single Cells Two methods of quantifying arrhythmias in single myoblasts were used, one subjective and one objective. Both types of arrhythmia quantitation studies were performed on random sam-ples of individual c e l l s . Subjective Measurements A qualitative account of arrhythmias in heart c e l l cul-tures has previously been described by Goshima ( 1 9 7 6 ) . In the present experiments the severity of arrhythmias was quantitated by using a scale of arrhythmias graded 0-4. A completely rhythmic c e l l scored 0. Intermediate stages were recognized; for example, a slight irregularity i n beat was given a score of 0.5; a failure to maintain rate, 1.0; fluttering (extremely rapid synchronous but weak rate), 3.0; and f i b r i l l a t i o n (rapid asynchronous contractions) were scored 4.0. The scale was based on observations of cells progressing from normal behavior to death after treatment with ouabain and Cyanea toxin (Walker, Martinez and Godin, 1 9 7 7 ) . Examples are shown i n Figure 7. The percentage of cells" beating arrhythmically was also recorded from random Figure 7; Quantitation of arrhythmias in single cultured heart c e l l s . The severity of arrhythmias was measured using a subjective scale graded from 0 to 4. The d i f f e r -ence between the highest and lowest beat-to-beat rate recorded by the tachograph is shown along with changes in rate range. - 49 -EXAMPLES OF ARRHYTHMIAS AND THEIR SUBJECTIVE SCORES DIFFERENT CELLS SUBJECTED TO VARIOUS CONCENTRATIONS OF OUABAIN NORMAL RHYTHM DISTURBANCE FLUTTER ASYNCHRONOUS FLUTTER 120-1 RATE beats mm. 120n 40-» d Q B dt TIME -^OPTICAL DENSITY^ arbitrary units 40 J 240n in in 80 J I5sed — i — , — i — 0-5 300-1 100J dQD. N O HZLiL. RECORDS I . . . . I 2-5 30 FIBRILLATION' (MEANINGFUL RATE NOT RECORDABLE) 40 SUBJECTIVE ARRHYTHMIA SCORES - 50 -samples of ten c e l l s . Objective Measurements Arrhythmogenic effects were assessed objectively i n terms of the percentage of cel l s beating, the beating rate, and rate range. The percentage of cel l s beating was deter-mined in random samples of 100 c e l l s . The rate was the mean of the tachograph readings during a 20 second sample period. Deviations from the normal rate were judged arrhythmogenic when produced by an arrhythmogenic agent. Bate range, the difference between the highest and lowest beat-to-beat rate recorded by the tachograph over a 20 second period, was also used as an objective measurement of single c e l l arrhythmias. Rate range measures the a b i l i t y of ce l l s to maintain a cons-tant rate, and may, therefore, give an index of the arrhythmic or antiarrhythmic properties of a drug. Rate range tended to increase as a function of rate. Consequently, when large changes in rate occurred the term (raterange) w a s u s e<i a s rate an index of beating behavior. This had the effect of n u l l -ifying the changes due to rate alone. Assessment of Antiarrhythmic Actions in Cultured Heart Cells To assess the antiarrhythmic and/ or "cyto-protective" effects of prostaglandins, lidocaine, and quinidine, arrhyth-mogenic agents were added for set times at appropriate concen-trations. The a b i l i t y of the various drugs to protect from or increase the effects of arrhythmogenic agents was assessed by adding the drug with the agent. Generally, i f the drug under test exaggerated the effects of the arrhythmogenic agent i t was considered to potentiate, while a lessening of the - 51 -effects of the arrhythmogenic agent was amelioration. Obvious-ly i f the drug increased the number of cells beating normally or reduced the number beating arrhythmically this would be an antiarrhythmic action. Other ameliorations were not so obviously judged antiarrhythmic. For example, dinitrophenol (DNP) slowed the rate of c e l l s , and i f this slowing was reduced by a prostaglandin (1. e. the prostaglandin increased the cell's beating rate) this was considered an antiarrhythmic action. However, with Ca which increased rate, i f the pros-taglandin further increased rate then the prostaglandin's act-ion would be arrhythmogenic. Thus the same action of a prosta-glandin alone, was not, a p r i o r i , considered arrhythmogenic or antiarrhythmic. Cyanea Toxin-containing Material Persistent and predictable changes in c e l l beating behavior were also produced by Cyanea toxin, a cardiotoxln obtained from the j e l l y f i s h Cyanea caplllata. previously described by Walker (1977a)$ This toxin was used to pro-duce the most severe types of arrhythmias encountered in cultured myoblasts. One batch of Cyanea caplllata tenacles obtained from Hong Kong coastal waters, was subjected to mac-eration and controlled sieving in order to obtain isolated nematocysts free of tentacles and mesoglial tissue (Walker, 1 9 7 7 a ) . Nematocysts were lyophilized and stored at - 1 0 ° C. Ten mg portions of the lyophilized nematocysts were suspended in d i s t i l l e d water for homogenization at 5000 revolutions per minute for 3 min at 4° C, and the volume then increased to 5 ml. The homogenate was centrifuged at 1 3 , 5 0 0 g for 30 min - 52 -to give a faintly opalescent supernatant whose protein concen-tration was obtained by the method of Lowery et a l . ( 1 9 5 D . This solution was the concentrated crude soluble toxin-contain-ing material. Lethality of the toxin to mice was assayed as previously described (Walker, 1 9 7 7 a ) . Drug Details for A l l Experiments The following prostaglandins were used A-p A g , B ^ , B g , Dl» D 2 ' El» 2-decarboxy-E-L, Eg, ( 1 5 S ) - 1 5 CH^-Eg, Flt<, F l f i , F g o < , and F g f i . A l l prostaglandins were kindly donated by The Upjohn Company, Kalamazoo, Michigan. Prostaglandins were dissolved in pure ethanol at a concentration of 3 * 5 mg/ml as a stock solution and stored at - 1 0 ° C. Final media concentrations were obtained by adding dilutions of these stock solutions to the appropriate media. Control experiments contained ethanol at the same dilution as with the prostaglandin experiments. Other drugs used in these experiments were adrenaline, lido-caine, and quinidine (Sigma Chemical Co.), and ouabain (Schwartz-Mann Co.). A l l other chemicals were of analytical grade. They were used as they were received without further purification. CALCULATIONS, STATISTICAL METHODS, AND EXPERIMENTAL DESIGN Standard s t a t i s t i c a l parametric tests such as Student's t were used where possible, together with standard linear regres-sion analysis. Dunnett's t test (Dunnett, 1955) was used for comparing several treatments with control. Multiple range and multiple F tests (Duncan, 1 9 5 5 ) were used to assess the anti-arrhythmic and or "cytoprotective" effects of prostaglandins, lidocaine, and quinidine in the presence of arrhythmogenic agents in c e l l culture. Occasionally, suitable non-parametric - 53 -tests such as c h i square had to be used. A p r o b a b i l i t y of less than 0 . 0 5 was chosen as the c r i t e r i o n of significance i n a l l cases. Where possible a l l experiments were performed double b l i n d with the drug under consideration, the arrhythmogenic agent, etc. being unknown at the time 'of the experiment or the analysis of records. Experiments were conducted accord-ing to r i g i d coded protocols. - 5 4 -CHAPTER III RESULTS The results of different experimental sections are considered in order. To recapitulate, the sections are as follows. Section I. The effect of prostaglandins on arrhythmias in A) the dog and B) the rat and C) the effect of prostaglandins on electrical vulnerability of the in vivo rat heart. Section II. The effect of prostaglandins on isolated perfused rat hearts. Section III. The effect of prostaglandins on A) the beating behavior of cultured rat heart cells and B) abnormalities of beating in cultured rat heart ce l l s induced by a variety of arrhythmogenic agents. EXPERIMENTAL SECTION I. A) The Effects of Prostaglandins During Coronary Occlusion ln Dogs In order to demonstrate possible antiarrhythmic actions of prostaglandins i n a standard experimental model an i n i t i a l experiment was performed in dogs against arrhythmias induced by coronary artery ligation. The variable measured in this experiment was the incidence of premature ventricular con-tractions (PVCs). Quantitation of this variable was d i f f i -cult, due to the non-normal distribution of PVCs occurring as the result of occlusion; even log PVCs were not normally dis-- 55 -tributed, thus making s t a t i s t i c a l analysis of these small samples d i f f i c u l t . However, by plotting the mean frequency of PVCs for each group (that is control, E 2, and F ^ — s e e Figure 8) i t can be seen that both prostaglandins reduce the incidence of arrhythmias associated with occlusion although the usual s t a t i s t i c a l tests do not show differences between individual points. The figures inserted within the table (Figure 8) are the mean log to the base e PVCs ( P V C o c c i U S i o n - P V C c o n t r o l K No significant differences were noted for blood pressure or rate within or between drug treatment groups. B) The Effect of Prostaglandins. Lldooalne. and Quinidine on Arrhythmias Produced by Coronary Artery Ligation in Bat As indicated in Methods a number of variables were measured in order to assess the possible antiarrhythmic effects of prostaglandins, lidocaine, and quinidine against arrhythmias associated with coronary artery ligation in the rat. Some of these variables were not of course independent but no standard measure of antiarrhythmic activity in this situation i s available. The effect of prostaglandins, lidocaine, and quinidine on arrhythmias produced by l e f t coronary artery ligation in rats is tabulated Table I and also shown in Figures 9 through 1 3 for c l a r i t y . The average number of premature ventricular contractions (PVCs) experienced by each rat after coronary artery ligation was reduced by a l l agents tested (Figure 9 ) . Prostaglandin E 2, A 2, and F ^ reduced the number of PVCs to approximately 40 to 5 0 per cent of control values. Lidocaine and quinidine showed approximately a 20 per cent reduction; - 56 -" Figure 8: The effect of l e f t coronary artery occlusion on the mean frequency of premature ventricular contractions with time for control, prostaglandin E 2, and prostaglandin Fw-treated dogs. Each point represents the mean of six animals. Figures inserted within the table are the Student • t 1 test of the mean log to the base e PVCs (PVCocclusion -P v c c o n t r o l ) for prostaglandin versus saline. c E in o > CL C TO a> E 150' 125-icoH 75-50-25H MEAN LOG (PVC n . . - PVC. ,) e Occlus ion Control PGE, PGF, la SALINE -30 0.87 - 1.2 p <0.05 0 . H - 0.9 p <0.05 3.38 - 1.0 # I I f f I > Oca O f f resulted 56% of animals ! ^ Fibrillating •Saline PG F l a -20 -fo To mm I occlusion on 20 30 occlusion off prrfuTIon™!"^ <_n - 58 -the reduction with F-^ex w a s 1 0 P e r cent. The number of PVC salvos (groups of 2 to 4 PVCs in rapid succession) was not taken for lidocaine and quinidine (Table I ) . The number of flutter episodes was reduced by PGE2, H^gg, and quinidine (Table I ) . Prostaglandin F ^ may have increased the number of flutter episodes. The average duration- of each flu t t e r episode was reduced by a l l drug treatments (Figure 1 0 ) . Quinidine and PGE2 were the most effective, followed in order by PGFgp, lidocaine, PGA£, and PGF]*. The time-to-flutter (Table I) did not prove to be a useful criterion as too few samples were available with the more effective antiarrhythmic agents for meaningful comparison. In general i t required approximately 5 to 6 minutes after ligation before the f i r s t f l u t t e r episode appeared. The effect of prostaglandins, lidocaine, and quinidine on the infarct size versus whole heart weight i s shown in Figure 11. No significant differences were found although there was a tendency for the size cf the infarct to be smaller with PGE2 and lidocaine. The number of animals dying from arrhythmias within the 25-minute test period following occlusion i s shown in Figure 12. Out of the five test animals for each drug, none died with PGE2; one each died with PGF^ and quinidine; two each died with PGA2, PGF^, and lidocaine; four died with control treatment. The effect of the different drug treatments on the aver-age subjective score for severity of arrhythmias i s shown i n _ Figure 13. Using this scale, PGEP reduced the severity TABLE I The effect of prostaglandins, lidocaine, and quinidine on arrhythmias produced by l e f t coronary artery ligation in rat. Standard errors of the mean are given unless too few samples were available for calculation. Statistics were calculated with Dunnettfs t test or Fishers's exact chi square test. n=5 animals for each drug treatment. Each animal received only one drug. *= significant at 0.05 level, **= significant at 0.01 level CONTROL A 2 E2 P l * F2/J Lidocaine Quinidine Number of PVC (25 min total) 131*81 61*27* 50*20* 110±49 67*15* 98±30 91*23 Number of PVC Salvos 3.6*1.8 4.6*2.4 3.0*2.2 4.0*1.2 3.2*1.0 Flutter Episodes 1.2*0.2 1.8*0.7* 0.4*0.4** 2.0*0.6* 0.6*0.4* 1.2 (n=4) 0.2 (n=l) Flutter Dur-ation Total/ heart (sec) 100*35 42*21** 2*2** 89*32 8*6** 21*6.6** 0 Time to Flutter (min) 5.5*0.4 (n-5) 4.8*1.3 (n-4) 7.5 (n=2) 5.6*1.3 (n=5) 6.8 (n=2) 6.2*0.9 (n=4) 4.0 (n=l) Infarct Size VS Whole Heart Wgt (per? cent) 27.8*1.9 28.0±2.8 22.0*3.6 24.4*3.3 27.0*4.2 22.8*2.5 24.6*5.5 Death 4/5 2/5 0/5* 2/5 1/5 2/5 1/5 Subjective Severity of Arrhythmias animals 11.8*1.2 7.0*2.3** 4 . 2 ± 1 , 2 * * 7.6*6.2** 5.0*2.3** 8.8*1.8* 5.2*2.2** - 60 -Figure 9 : The effects of prostaglandins, lidocaine, and quinidine on the average number of premature ventricular contractions (PVCs) experienced by five rats over a 25-min-ute period following acute l e f t coronary artery ligation. Each bar represents the mean of five animals - S.E.M. - 61 -140 120|-20|-Contr r 2 6 Lidocaine Quinidine -62 -Figure 10: The effect of prostaglandins, lidocaine, and quinidine on the average duration of each flutter episode experienced by rats in a 25-minute period following acute l e f t coronary artery ligation. Each bar represents the mean of five animals ± S.E.M. - 64 -Figure 11: The effect of prostaglandins, lidocaine, and quinidine on the average size of the infarct produced (as the percentage of whole heart weight) following acute l e f t coronary artery ligation i n rats. Each bar represents the mean of five hearts * S.E.M. - 65 -36 Control loo r23 Lidocaine Quin id ine - 66 -Figure 12: The effect of prostaglandins, lidocaine, and quinidine on the number of animals dying from arrhythmias within the 25-minute test period following acute l e f t coronary artery occlusion. Each group contained five rats. NUMBER OF AN IMALS DYING - 68 -Figure 13: The effect of prostaglandins, lidocaine, and quinidine on the average subjective score for severity of arrhythmias following acute l e f t coronary artery occlusion in rats. Each bar represents the mean of five rats ± S.E.M. 1 4 12 10 o u CO U LU CO to Control loc Lidocaine Quinidine - 70 -of arrhythmias to 37 per cent of control. PGF2g a n d Q u i n i-dine were nearly as effective as PGE2. PGAg, PGFle(, and lidocaine were less effective (60 - 75 per cent of control) but were s t i l l significantly better than control. Changes in blood pressure during coronary artery oc-clusion in rats are shown in Figure 14. Blood pressure was reduced by surgery alone in a l l cases. P6E2, PGA2, and quinidine further reduced blood pressure, although the pres-sure did not stay down with PGAg. PGF^, PGF2^, and li d o -caine had l i t t l e effect on blood pressure. PGF-^ m&y have caused an increase in blood pressure 10 minutes after the start of the infusion. Changes in heart rate during l e f t coronary artery occlu-sion in rats are shown in Figure 15. The heart rate tended to increase following surgery in a probable reflex response to the f a l l in blood pressure (Figure 14). None of the drug treatments produced marked effects on heart rate. P G F l c^ may have increased rate at 10 minutes after start of the infusion, however, this result i s based on only the two surviving animals and i s therefore of limited r e l i a b i l i t y . C) Changes in Flutter Threshold and. Maximum Following Frequency Flutter threshold (the current required to produce f l u t -ter) was not found to be changed by any of the prostaglandins tested (Table II). Lidocaine produced a decrease in flutter threshold at the 6 ^ ig/Kg/min dose after 10 minutes. Higher doses of lidocaine produced a f a l l in blood pressure with time and could not be used. Quinidine increased the flutter thres-r-- 71 -Figure 14: The effect of prostaglandins, lidocaine, and quinidine on blood pressure during l e f t coronary artery ligation in rats. Both systolic and diastolic pressures are shown. Each bar represents the mean of five rats * S.E.M. Values are shown at control time before surgery (C), immediately post-surgery (PS), at the start of the in -fusion of drug (I), 5 min after start of infusion (5'), and 10 min after start of infusion (10*). Standard errors are shown unless too few samples were available for calcu-.. lation. No value is shown with infusion for quinidine which was given as a bolus. (See TABLE A in appendix for tabulated data.) - 7 3 -Figure 15: The effect of prostaglandins, lidocaine, and quinidine on heart rate during l e f t coronary artery occlusion in rats. Each bar represents the mean of five rats ± S.E.M. (See Figure 16 for abbreviations) Standard errors are shown unless too few samples were available for calculation. No value is shown with infusion for quinidine which was given as a bolus. (See TABLE A in the appendix for tabulated data) -75 -TABLE II The effects of prostaglandins, lidocaine, and quinidine on electrically stimulated Flutter Threshold and Maximum Following Frequency in in vivo rat heart.. Mean Control Maximum Following Frequency = 14.42 ± 0.28 Hz (n=l6) Dose: mg/Kg/min 1 4 8 PROSTAGLANDIN Difference from i n i t i a l control value A 2 Hz -0.45 -0.8 -1.60 mA +0.02 +0.005 +0.003 E2 Hz +0.02 -0.5 -1.1 mA 0 +0.005 0 F l ~ Hz -0.03 -1.2 -0.83 mA +o.o28 -0.025 -0.007 P2/? Hz -0.07 +0.2 +0.17 mA +0.04 -0.06 0 Hz +0.50 -0.6 -1.5 mA +0.08 -0.02 -0.005 ( A l l values determined 5 min after start of infusion) Time after start of infusion (min) _ _ _ LIDOCAINE 3 ug/Kg/min Hz mA +0.1 -0.2 -0.001 +0.04 +0.1 +0.02 6 / i g A g/min Hz mA +7-3 +7.4 -0.014 -0.9 +7.2 -0.9 Time after injection (min) 5 -QUINIDINE 3 mg A g Hz mA -2.7 +0.30 6 m g A g Hz mA -4.5 +1.3 ( n « 3 to 5 for a l l determinations) r 76 -Figure 16: Maximum following frequency changes with pros-taglandin infusion. Mean control maximum following frequency was 14.42 i 0.28 Hz (n=36). Data is shown as the difference from the i n i t i a l control. Each point represents the mean of three to five animals. - 77 -hold at both 3 and 6 jug/Kg doses. Maximum following frequency was found to change in a dose-dependent manner depending on the prostaglandin; however, this change was usually less than ten per cent of the control (Figure 16, Table II). Mean control maximum following fre-quency was 14.42 - 0.28 Hz (n=36). Linear decreases in f o l -lowing frequency were found with prostaglandins Eg, Ag, and F 2 e ( (Figure 16). PGFg^ produced a small increase i n maximum following frequency, and PGF-j^ had a biphasic effect with an i n i t i a l decrease followed by an increase. Lidocaine at the 6 ;ig/Kg/min dose produced a marked increase and quinidine at 3 and 6 mg/Kg produced a marked decrease in maximum follow-ing frequency (Table II). EXPERIMENTAL SECTION II The Effect of Prostaglandins on Isolated Rat Heart Figures 1? - 22 show the effect of prostaglandin infusion of isolated rat hearts on rate, force, and coronary flow. q _7 _< Prostaglandins (10 7 , 10 f, and 10 M) were infused between minutes 60 and 80 of each experiment. Generally 1 0 " ^ M prosta-glandins did not produce a detectable change in rate, force or flow. The results of prostaglandin infusion at higher doses ( 1 0 " ? M and 1 0 " ^ M) are shown in figures 17 through 22. Table III contains the s t a t i s t i c a l testing of these results. At 10 M prostaglandins did not appear to have any marked effect on heart rate, which was subject to considerable variation (Fig-ure 1 7 ) . At the end of prostaglandin infusion there was an immediate f a l l in rate seen in a l l hearts which had been exposed to prostaglandins. At 10 ' M a l l prostaglandins kept the force - 79 -up during infusion (Figure 18). S t a t i s t i c a l analysis (see method section or Table III) showed this to be significant. It is interesting that there was a tendency for prostaglandins Eg and Fg p, but not prostaglandins Ag, F-^, or control, to delay the decline in maximum force production after the end of prostaglandin perfusion. Before exposure to prostaglandins the curves for these hearts had been lowest, but following prostaglandin treatment they became highest. Prostaglandin Eg produced a small increase while prostaglandin Fgg produced a small decrease in flow at 10"''' M perfusion (Figure 19). This effect did not persist after the end of the prostaglandin infusion period. At 10"-* M only prostaglandin Ag markedly increased rate (Figure 20). None of the other prostaglandins produced any significant effect on this parameter during i n -fusion, which was, again, subject to considerable variation. Following infusion of PGEg, F ^ , and Fg f l, there was a f a l l in heart rate. It should be noted that there was no change in rate with time for control hearts in any of these experiments. No marked change in maximum force production was seen with prostaglandins at 10~^ M (Figure 21). This was true both during and following prostaglandin infusion. A l l prostaglan-dins markedly increased flow at 10 M (Figure 22). This increase in flow may have been partially maintained for a short time following prostaglandin infusion, but the increase did not persist. TABLE III S t a t i s t i c a l testing of the results of prostaglandin , infusions on rate, force and flow in perfused hearts. Levels of significance were p<0.05 for slopes and/or intercepts with regressions. Prostaglandin concentration I'Infusion 10"7M ^Infusion 10~7M Post-?X©f. i n f u s i o n Post- ~>Jmf. RATE FORCE FLOW A 2 E 2 Fl*f*f A 2 E 2 FxJFay- A 2 E 2 • P 2# 00 o The effect of prostaglandin ^infusion on rate, force and flow was tested s t a t i s t i c a l l y by comparing drug effects against within-group controls, and against over-all controls. As heart rate did not change with time.data from the figures was grouped with control, infusion, and post- infusion groups and appropriate t-tests performed. As flow and force f e l l with time, linear regressions of response with time over the periods 20-60 min, 65-80 min (infusion), and 85-150 minutes (post-infusion) were appropriately tested. KEY: = indicates no effect + indicates an increase for rate and maintained or increased ef-fect for force and flow - indicates a decrease Figure 17: The effect of prostaglandin (10~'M) perfusion of isolated rat heart on rate. Prostaglandins were perfused for 20 minutes between minutes 60 and 80 of the perfusion. Each point represents the mean of five hearts. Figure 1 8 : The effect of prostaglandin (10"*'M) perfusion of isolated rat heart on maximal force of contraction. Each heart was tested with 25 g loading tension. Prostaglandins were perfused for 20 minutes between minutes 60 to 80 of the perfusion period. Each point represents the mean of five hearts. 100 90 80 m 70 u DC 9 60 3 50 < 40 5 30 20 101 • C O N T R O L P G A 2 P G E 2 PGF PGF I c e 2/3 Prostaglandins 1 0 ~ 7 M 00 • t 20 40 60 80 100 T IME (minutes) 120 140 160 180 - 85 -Figure 19: The effect of prostaglandin (10"'H) perfusion of isolated rat heart on coronary flow rate. Prostaglandins were perfused for 20 minutes between minutes 60 to 80 of the perfusion period. Each point represents the mean of five hearts. 10 r 1 I f i t i t i i i Prostaglandins 10 " 7 M C O N T R O L • P G A 2 • P G E 2 • P G F l c c t # 00 ON 180 20 40 60 80 100 T IME (minutes) 120 140 160 Figure 20: The effect of prostaglandin (10"->M) perfusion of isolated rat heart on rate. Prostaglandins were perfused for 20 minutes between minutes 60 to 80 of the perfusion period. Each point represents the mean of five hearts. 2 0 4 0 6 0 8 0 1 0 0 1 2 0 140 160 1 8 0 TIME (minutes) - 8 9 -Figure 21: The effect of prostaglandin (10'^M) perfusion of isolated rat heart on maximal force of contraction. Each heart was tesfced with 25 g loading tension. Prostaglandins were perfused for 20 minutes between minutes 60 to 80 of the perfusion period. Each point represents the mean of five hearts. L U U OH o 100 90 80 70 60 i 50 | ^ 40 ^° 30 20 10 0 % C O N T R O L P G A 2 P G E 2 P G F l e o PGF 2 3 -5 Prostaglandins 10 M \ 0 o 20 40 60 80 100 TIME Cminutes) 120 140 160 180 - 91 -Figure 2 2 : The effect of prostaglandin.(10"°M) perfusion of isolated rat heart on coronary flow rate. Prostaglandins were perfused for 20 minutes between minutes 60 to 80 of the perfusion period. Each point represents the mean of five hearts. i o « -8 r, 7 c E \ 6 E CONTROL P G A 2 P G E 2 P G F l 0 L P G F 2 / 3 - 5 Prostaglandins 10 M ro 20 40 60 80 100 TIME CminutesD 120 140 160 180 - 93 -EXPERIMENTAL SECTION III A) The Effects of Prostaglandins Alone on the Beating Activity of Cultured Heart Cells Table IV shows the effect of the prostaglandins as well as adrenaline, ouabain, and ethanol alone, on the beating rate of single c e l l s . Ethanol (5 or 10 JA1/5 ml of tissue culture media) produced a small increase in beating rate and, due to this small but consistent effect, ethanol alone was used as the control for prostaglandins whereas saline was the control for ouabain and adrenaline. As no changes with exposure time, to drug were detected, the results from 10 and 35 minute exposure times were combined. Only prostaglandin F 2 < < produced a marked, s t a t i s t i c a l l y significant increase i n rate, although others may have produced minimal effects (e.g. D 2 or F-j^). The reproducibility of the technique and procedures is shown with the repeated experiments using 10"""^ M prostaglandins A 2, E 2, F l c C, and Fz^* Values equal to the f i r s t experiment were ob-tained in every case. Prostaglandin F 2 0 seemed to decrease rate compared with ethanol controls although this decrease was nat s t a t i s t i c a l -ly significant. Other prostaglandins may have minimally decreased rate. Adrenaline produced a marked positive chrono-tropic effect as did ouabain when they were compared with their saline controls. Figure 23 illustrates the f u l l dose-response curve for the most active chronotropic prostaglandin, F ^ . The response curve was plotted in two ways: 1) as the mean of rate respon-ses to various concentrations of F 0 r f and 2) as the mean of TABLE IV Effect of prostaglandins on the beating rate of single cultured heart c e l l s . Prostaglandin 10' -7 M 10* -5 M Repeat 1 0 " 5 M Ethanol Alone 86 ± 7 92 ± 8 A l 76 ± 5 90 ± 4 A2 71 ± 83 ± 5 77 B l 80 ± 96 ± 6 B! 81 ± 7 83 ± 5 D l 86 ± 5 100 + 5 D| 96 ± 5 109 ± 5 EI 63 + 4. * „ 87 + 5 2-decarboxy-E^ 81 ± 6 92 ± 7 E 2 81 5 101 ± 5 J 9 ( 1 5 S)-15 G H o - E o 83 ± 7 95 ± 7 96 F l o C 86 ± 7 103 ± 7 85 ± 4 90 ± 5 F2o€ 122 ± 6 *-•-.,' 130 ± 4 * F 2/? 69 + 6 75 + 5 79 (beats /minute) Saline Control 66 + 6 Adrenaline 97 ± 11 * * 162 ± 3 * * « Ouabain 91 ± 7 * « # 103 ± 16 * * = p<0.05 * * - < 0 . 0 1 -***'m < 0 . 0 0 5 5-6 individual cells per flask from 3-4 different cultures were examined and rates calculated from records. Values for 10 and 35 min exposure to drug did not change; these time periods were therefore pooled. Each value is the mean ± S.E.M. (n=30-36). The repeat column for 10~^M shows mean results obtained in an Independent experiment performed, in exactly the same manner, several months after the main ex-periment. Statistics were calculated with Dunnett's t test. - 95 -Figure 2 3 : Dose-response curve to P G F o ^ . and dl=adrenaline. Response curves to both drugs were expressed as the mean responses (beats/min) to the various concentrations at equil-ibrium. The results for PGF2etwere individually recalculated so that the concentration necessary to produce a particular percentile response could be interpolated. Means of such con-centrations were then taken (insert). 0 & = dl-adren-aline mean responses; pi B) = PGF2oCmean responses. Each point i s the mean of 12 ce l l s * S.E.M. - 97 -concentrations required to produce a particular response in individual c e l l s . As expected, the latter plot gave a much more accurate dose-response curve, with a steeper slope, and best represents the response to this prostaglandin (Ariens, 1964). The response to FGF2<x can be compared with that of the meaned dose-response curve to dl-adrenaline (also shown in Figure 23). The catecholamine response reached much higher levels and did not reach a maximum even at 10 "^ M concentra-tion. The effects of the prostaglandins on rate range (beats/ minute) are summarized in Table V. Rate range is an index of the a b i l i t y of cel l s to maintain a constant rate as a stable c e l l can be expected to maintain a more regular rate than an unstable one. Rate range has been found to increase with a number of arrhythmogenic agents (Martinez and Walker, 1977) . No changes in this variable were detected with ethanol con-trols and therefore a l l prostaglandin values were s t a t i s t i -cally tested against the appropriate pre-drug controls. None of the prostaglandins s t a t i s t i c a l l y significantly changed rate range. Only adrenaline produced a s t a t i s t i c a l l y sig-nificant increase in this parameter, although PGFg^ d i d show a tendency to reduce rate range. Prostaglandin effects on optical density are shown in Table VI. The optical density of the c e l l image changes during a contraction cycle as areas of greater or lesser density pass before the photoresistor tubes. Changes in optical density records in cultured heart cells have been reported as corresponding to changes in the force of contrac-TABLE V Effect of prostaglandins on the beating rate range of single cultured heart c e l l s . (Prostaglandin Pre^Drug lOT? M 1Q~5 M Control (beats/min) (10 & 3 5 min comb) ?Al A 2 B l B 2 D x D 2 % 2-decarboxy-E1 E 2 ( 1 5 S ) - 1 5 CH 3-E 2 P l o G P l , < ? P2oc/ 2 8 ± 3 38 ± '5 27 ± 3 3 3 t ± 5 28 4 23 ± 3 4 0 ± 7 39 + 6 37 + 7 36 ± 6 4 0 ± 6 3 5 ± 6 3 2 + 5 30 + 4 31 + 4 3 4 + 5 36 + 5 3 5 ± 4 2 5 J t : 3 ^ 34 + 4 32 ± 3 37 ± 5 32 ± 3 4 6 ± 5 2 8 ± 5 30 ± 4 22 ± 3 3 9 + 3 3 5 + 4 31 + 4 3 5 + 7 38 + 5 4 0 + 5 25 ± 3 31 ± 3 2 8 ± 4 37 ± 2 5 34 ± 5 30 ± 6 33 ± 6 2 5 + 4 20 + 3 Ethanol Control 33 ± 6 4 1 ± 6 37 ± 6 Adrenaline 32 + 1 4 9 , + 8 * * 5P + 7 * * Ouabain ....... .. 3Q ± 5 35 ± h 31 + 4 . ** • p < 0 . 0 1 Samples were obtained as for TABLE IV and the 10 and 3 5 minute values pooled. Range i s the maximum rate minus the minimum rate occurring in a 20 sec sampling period. Each value was s t a t i s t i c a l l y compared with i t s pre-drug control using the Student t test. - 99 -TABLE VI The effect of prostaglandins on the optical density change with beats in single cultured heart c e l l s . Sampling of the optical density changes with each beat was the same as that for rate. As optical density changes (in arbritrary units) changes with prostaglandin exposure time, values for 10 and 35 minutes are not pooled and only 10 minute values given. Each value was s t a t i s t i c a l l y com-pared with i t s pre-drug control,using the Student t test. Prostaglandin Pre-Drug Control 1Q-?M IQ-5M Ethanol Alone 22 ± 3 21 ± 3 20 ± > Al 20 + 7 . 21 ± 3 20 ± 4 A 2 18 ± k 18 ± 7 13 ± 2 B l 2 6 ± 27 ± 6 15 ± 2 B 2 26 ± 5 18 ± 3 13 ± 2 D t 18 + 3 16 ± 3 16 ± 3 D 2 16 2 15 ± 2 14 ± 3 E l 14 + 2 17 • t. 3 17 ± 5 -2-decarboxy-Ei 15 2 • 11 + 3 •»8 ± 2 ** E 2 27 + 6 18 ± 5 • 13 ± 2 * ( 1 5 S ) - 1 5 C H 3 - E 2 15 ± 5 11 ± 2 *# 14 ± 2 FloC 30 + 4 21 ± 3 '* 17 ± 2 21 ± 3 20 ± 3 14 ± 3 *< F 2 o c 17 ± 3 15 ± 2 16 ± 2 12 ± 2 19 ± 3 12 ± 2 Adrenaline 16 ± 3 30 ± 6 * 14 ± 2 Ouabain 13 ± 2 18 ± 4 13 ± 3 * • p <0.05 ** =: p <o.oi Or 005 - 100 -•tion, although such a proposition has s t i l l to be f u l l y eval-uated (Butcher and Kolb, 1962; Okarma and Kalman, 1 9 7 D • Nevertheless,, density could be measured with some precision (as witnessed by the small S.E.M.s) and i t changed in the presence of prostaglandins. _7 In the presence of 10 M prostaglandins, an overall tendency for changes in optical density to decrease with ex-posure time was noted; therefore, the results from 10 minutes only are given. Values f e l l with lO"? M prostaglandins over the 35 minute drug exposure time (35 minute*value lower or equal to 10 minute value in 11 of 14 cases). Optical density changes with 10 ^ M prostaglandins did not f a l l with exposure time. Pros-taglandins A-p B-p 2-decarboxy-Ep Eg, ( 1 5 S ) - 1 5 CH^-Eg, Flo<, and F^g a l l decreased the optical density record. Only the negative chronotropic prostaglandin, Fg^, produced a s t a t i s t i c a l l y sig-nificant increase in optical density change. Adrenaline incre-_7 ased the optical density record at 10 K, and the increase re-turned to control levels at 10"^ M, possibly as a result of the marked positive chronotropic response seen at high con-centrations. No significant change was seen with ethanol con-trols, whereas possible changes with ouabain are clouded by the increasing dysrhythmias seen at higher concentrations (Martinez and Walker, 1 9 7 7 ) . The optical density changes were also electronically differentiated to give f i r s t derivative (i.e. dOD/dt) for, i f optical density gave an index of inotropism i t was expected that this second parameter would give a second index analogous to dp/dt. The derivative associated with contraction, and not - 101 -TABLE VII (Effect of prostaglandins on the time derivative d optical density o f o p t i c a l d e n s i t y changes dt Prostaglandin Pre-Drug Control 1 0 ~ 7 M 1 0 " 5 M (10 and 35 min comb) Ethanol Alone 12 + 3 9 + 2 12 ± 3 % r 13 ± 5 8 ± 2 5 ± 1 • A 2 14 ± 3 14 ± 4 14 ± 3 Bl r 7 + 3 9 2 8 + 2 B 2 r 13 ± 5 7 ± 2 4 ± 1 * D l 10 ± 3 5 ± 1 * 9 ± 2 D 2 8 ± 3 11 t 4 10 ± 3 E l 13 ± 6 6 ± 2 9 ± 3 2-dec arboxy-E^ r 8 ± 1 13 ± 4 8 ± 1 E 2 r 15 ± 5 16 ± 4 7 ± 3 « ( 1 5 S ) - CH^-Eg r 12 + 2 + 1 *# 5 + 1 p l o c r 14 ± 3 14 ± 4 11 2 P 1 0 r 11 + 4 8 ± 2 11 + 3 p 2 ^ 11 ± 3 11 ± 3 7 + 2 P 2 f 9 i 6 ± 2 17 + 5 * 10 + 2 Adrenaline Ouabain 11 8 ± 5 ± 2 17 ± 6 13 ± 3 13 ± 4 10 ± 3 * » p < 0 . 0 5 ** - p<0.01 *** = p<0.005 r * PGs reducing optical density 1 = PGs increasing optical density Since no effect of exposure time was noted r e s u l t s from 10 and 35 min combined are given for both concentrations tested. Each value was s t a t i s t i c a l l y compared with i t s pre-drug control using the Student t te s t . - 102 -TABLE VIII Effect of prostaglandins on the percentage of single cultured heart cells spontaneously beating. The percentage of cells beating was determined from five samples, of ten cells each, in three different cul-tures. The two concentrations of prostaglandins were added and the percentage of cells beating assessed after 10 minutes exposure. Figures are the mean + S.E.M. of differences from control percentages of cells beating ( r i 1 * 1 0 - 1 6 ) . Statistics were calculated with Dunnett's t test. Prostaglandin 10 M (Percentage d i f f . 1 0 " ° M from pre-drug) Ethanol Alone E 2 ( 1 5 S ) - 1 5 C H - E 2 -15153+ +4. o ± -3-0 ± -4.5 ± -3-5 ± -0.5 ± -6.5 ± +5.0 + -12.0 ± +0.8 ± -3-0 ± -4.6 ± -6.7 + -7.2 ± : 0 ± 4.0 5.0 6.5 9.0 6.4 4.9 5.0 5.1 4.5 5.0 6.5 4.0 8.8 5.6 6.5 -10.0 -7.0 -8.5 -6.5 +4.5 -5-0 -11.0 -6.3 -12.0 -17.7 -6.7 -32.0 -3.0 -33-0 -10.0 + ± 4.5 9.0 8.9 9.0 7.4 7.0 7*0 7-4 3.8 3.0 5.5 6.0 * 5.5 5.5 » 7.0 * = p< 0.05 - 103 -that associated with relaxation, was measured and the effects of the drugs are given in Table VII. No effect of exposure time was-noted; results from 10 and 35 minutes combined are therefore given. As with optical density changes, there was an overall tendency for the prostaglandins to reduce this index -7 at both concentrations. Excluding PGF_ , the 10 M values were equal to orlcwer than controls in 9 out of 13 cases, as _ 5 compared with 11 out of 13 cases with 10 M. Some of the prostaglandins producing marked decreases in optical density changes also s t a t i s t i c a l l y significantly reduced the f i r s t derivative (PGs Ap Bg, Eg, (15s)-15 CH^-Eg). As with changes in optical density, only prostaglandin Fg^ s t a t i s t i c a l l y sig-nificantly increased the derivative. Both adrenaline and oua-bain showed tendencies to increase the derivative of optical density, but this was not s t a t i s t i c a l l y significant. The ouabain results were complicated by increasing dysrhythmias. During experiments with the prostaglandins, i t was noted that some appeared to reduce the percentage of spontaneously beating c e l l s . The effect of the various prostaglandins on the per cent of cells beating spontaneously i s given i n Table VIII. At 10"^ M, most of the prostaglandins had no effect, except for prostaglandins F 1 < s < and F2o<. which reduced the per-centage of cells beating spontaneously. Reductions were dose-dependent, as is shown by a comparison of the 10 ' M with the 10"^ M results. Ethanol alone produced a small, but consistent, effect and was therefore used as the control for a l l prosta-glandins . -. 104 -B) The Effect of Prostaglandins on Abnormalities of Beating in Cultured Heart Cells Induced by a Variety of Arrhythmogenic Agents Introduction To test for a possible "cyto-protective" action of pros-taglandins, experiments were devised to determine whether or not prostaglandins protected against abnormalities of beating behavior induced in^cultured neonatal rat heart ce l l s by a variety of arrhythmogenic agents. The effect of each of the agents on beating behavior had to be determined i n i t i a l l y in order to .choose suitable times and doses for the cyto-protection studies. The Action.of Ouabain. Ionic Manipulation, Dinitrophenol. and Anoxia on Cultured Heart Cells Ouabain The effect of ouabain (5 x 10 M) on rate and rate range/rate versus time is shown in Figure 24. Rate increased to 200 per cent at 2 minutes, and to a sustained maximum of 310 per cent after 10 minutes. Rate range/rate (an object-tive measure of arrhythmic activity) f i r s t decreased to 80 per cent of control at 1 minute, then increased to 150 per cent of control at 4 minutes and 200 per cent of control at 10 minutes. Equilibrium effects were obtained within 10-14 minutes. Figures 25 and 26 show the effects of different concen-trations of ouabain on the objective (rate, rate range/rate, per cent beating) and the subjective (per cent arrhythmic, mean score for arrhythmias) indices Of arrhythmias in single c e l l s . - 105 -Figure 24: The effect of ouabain 5 x 10"- 7 M on rate and rate range/rate versus time. The i n i t i a l control measure-ment was taken as 100$. Mean values for 7 cells are shown. - 106 -4001 range I I 8 10 TIME (min) 12 14 16 18 20 - 107 -Figure 25: The effect of ouabain on rate and rate range/rate in cultured heart c e l l s . The i n i t i a l control measurement was taken as 100$. The number of cells beating out of 100 sampled is shown by the small numbers next to the data points. Mean values for 10 cells * S.E.M. are shown. Figure 26: The effect of ouabain on the percent of cells arrhythmic/total cells beating, and the mean subjective score of arrhythmias. Mean values for 10 cells * S.E.M. are shown; ^ARRHYTHMIC TOTAL BEATING CELLS O O MEAN SCORE for ARRHYTHMIAS - 109 -A l l values were determined 10 minutes after the addition of each drug dose. Increasing concentrations increased a l l in-dices, but with different dose-response relationships. Rate 7 -4 increased with doses between 10*"' and 10 M to reach 225 -h per cent of control at 10 M (Figure 25). The number of cells beating out of 100 sampled decreased rapidly above 10"^ M. The;'per cent of cells beating arrhyth-7 -k mically increased rapidly between 10 ' and 10 M, to a max-imum of 100 per cent. The mean score for severity of arrhyth-mias increased linearly throughout the dose range (Figure 26). From these data an arrhythmogenic concentration of 30 uM was chosen for studying the effects of prostaglandins on the induced beating abnormalities. Calcium Calcium chloride (2 - 12 mM) markedly increased rate (to 380$ * 30 S.E.M. of control at 12 mM); rate range/rate increased to a maximum of 200 per cent of control at 8 mM a l l at 10 min, Fig 27)- The percentage of arrhythmic cells increa-sed from 15 per cent at 2 mM to 100 per cent at 12 mM, while mean arrhythmia score increased from 0.5 - 0.2 at 2 mM to 3«0 - 0.2 at 12 mM (Figure 28). From this data a concentration of 6.0 mM Ca was chosen for the prostaglandin study. Potassium Elevation of K + decreased rate (35$ - 7 of control at 10 mM) and then increased i t (80$ of control at Zk mM) (Figure 29). Rate range/rate increased to 300 per cent of control at 10 mM and then f e l l to 160 per cent at 28 mM K +. Subjective measurements of arrhythmias on the scale used for Ca and - 110 -Figure 27; The effect of calcium on rate and rate range/rate in cultured heart c e l l s . The i n i t i a l control measurement was taken as- 100$. The number of cells beating out of 100 sampled is shown by the small numbers next to the data points. Mean values for 10 cells - S.E.M. are shown. Figure 28: The effect of calcium on the per cent of cells arrhythmic/total cells beating, and the mean subjective score for arrhythmias. Mean values for 10 cells * S.E.M. are shown. - I l l -- 112 - . ouabain.were: not appropriate for K owing to a lack of a distinct pattern of arrhythmic changes while the number of cell s beating decreased with increasing K + concentrations. A concentration of 8 mM was chosen from these data. Dinitrophenol Cells responded rapidly to dinitrophenol (DNP) 2 x lO'^M with rate f a l l i n g to 50 per cent of control after 3 minutes exposure and rising to a stable 80 per cent of control after 8 minutes; rate range/rate rose to 550 per cent of control and then f e l l to a stable 230 per cent of control (Figure 3 1 ) . The effect of different concentrations of DNP at 10 minutes i s shown in Figure 30• Maintenance of beating rate became more erratic in the presence of DNP. Subjective arrhythmias re-sembled those with K + in that they were not pronounced and did not mimic those seen with ouabain and Ca The.Effect of Anoxia on Cel l Beating Behavior Anoxia (100 per cent Ni,' atmosphere) for periods of up to 60 minutes had l i t t l e effect on the per cent of cel l s beating rhythmically (solid line) or on the per cent of cel l s beating arrhythmically out of the total number of cel l s beating (dot-ted line) (Figure 3 2 ) . Prewarmed and moistened 100 per cent nitrogen was passed through a tissue culture flask for periods of up to 60 minutes. The p 0 2 of the media f e l l to 0 mm Hg as measured by an oxygen electrode after 15 minutes. Establishment of Suitable Concentrations of Lidocaine and Quinidine For want of suitable comparison drugs for the study of -prostaglandin actions on beating behavior abnormalities indue-- 113 -Figure 29: The effect of added potassium concentration on rate and rate range/rate in cultured heart c e l l s . The i n i t i a l control measurement was taken as 100$". Mean values for 20 cells "* S.E.M. are shown. The number of cells beating out of 100 sampled is shown by the small figures beside the data points. - 115 -Figure 30: The effect of dinitrophenol concentration on rate (beats per minute) and rate range/rate in single cul-tured rat heart myoblasts. Mean values for 4 cells * S.E.M. are shown. Figure 31: The effect of dinitrophenol 2 x 10 M on rate and rate range/rate versus time in single cultured rat heart myoblasts. Mean values for 4 cells * S.E.M. are shown. R A T E ^ C O N T R O L o o O o CO o o o o • f • Cn O O O O O R A T E R A N G E R A T E % C O N T R O L "i T -r - T 1 1 — rO O ^ . Cn 0> g o o o o o ° o o o o o RATE RANGE RATE %CONTROL -117 -Figure 3 2 : The effect of anoxia on the percent of cells beating rhythmically/total cells ( • ), and the percent of cel l s beating arrhythmically/total c e l l s beating ( A -). Mean values for 10 cells are shown * S .E.M. % BEAT ING RHYTHMICALLY TOTAL CELLS - 119 -ed by the previously discussed arrhythmogenic agents, i t was decided to use two standard antiarrhythmics, namely lido-caine and quinidine. As both agents at high enough concentra-tions totally abolished c e l l beating, i n i t i a l dose-response studies were performed in order to choose a suitable concen-tration. Lidocaine and quinidine both stopped ce l l s beating f> u (Figure 3 3 ) . The ED 50*s were 10 and 10 M for quin-idine and lidocaine respectively. Threshold values were -7 -5 2 x 10 and 2 x 10 M. Parallel log-dose-response curves were observed. Exposure time for lidocaine and quinidine was 10 minutes. Cyanea Toxin In addition to examining the effects of ouabain, Ca**, K+, DNP, and anoxia on the.production of beating abnormalities in cultured heart cells the effects of a cardiotoxic substance from the j e l l y f i s h Cyanea capillata were also studied. As previously discussed, the mechanism of action of this toxin is partially understood. It was f e l t that with l i t t l e further study this toxin could be used as a tool to produce discrete, understood damage to cultured heart c e l l s . The effect of prostaglandins against such damage could then be investigated. The Effect of Cyanea Toxin on Cultured Heart Cells Cultured Heart C e l l Contractile Activity Figure 3^ shows the effect of 28 ng (Lowry protein) of Cyanea toxin per ml of culture media on a heart c e l l . An i n i -t i a l period during which the toxin produced no effect (0 to 10 min) was followed by a period of rapid increase in beating - 120 -Figure 3 3 : The effect of quinidine and lidocaine concentration on the per cent of cells not beating. Mean values for 10 cells are shown. - L 121 -L I D O C A I N E • QUINIDINE A / * / * I i I * I i * / - 7 -6 -5 -4 10 10 10 10 10 ANTIARRHYTHMIC (M) Figure 34: The response of a single cultured heart c e l l to, Cyanea toxin-containing material. A single heart c e l l was monitored before and after addition of 28 ng Cyanea toxin per ml at time zero. Optical density changes (with contractions), in a television image of the c e l l were meas-ured by photo-resistors and recordedStogether with the electronically-derived beat-to-beat rate (beats per minute) and the f i r s t derivative (dOD/dt) of the density changes. Failure to exactly reposition the photoresistors at each time sample accounts for some of the optical density changes. Records were sampled at the times shown at a chart speed indicated by the 5 and 1 sec markers. Chart speed was in-creased at the 50 min sampling period. 180 r-BEAT to BEAT RATE , (Reats/Min.) SINGLE CELL EXPOSED TO TOXIN-CONTAINING MATERIAL d OPTICAL DENSITY dt (Arbitrary Unlts/Sec.) 5 S e c CYANEA TOXIN (28 ng/ml) If 1^ (Added at 0 Mln.) OPTICAL DENSITY 'ffffffffffffj (Arbitrary Units) CONTROL 5 Min. 10 Min. 15 Min. 20 Min. d O.D. dt OPTICAL DENSITY ' ' 1 1_ CELL STOPPED 25 Mln. 40 Min. 50 Min. 60 -124 -TABLE IX;: Effect of Cyanea toxin-containing material on the time to various arrhythmic events in cultured single heart c e l l s . Cone of Cyanea toxin-containing material (ng/ml) Time to dormant period (min) Time to Time to Lonset of f i n a l - f i b r i l l a t i o n cessation (min) (min) 28 110 440 25 ± 3 8 ± 1 6 ± 1 44 ± 2 21 ± 4 12 ± 2 74 ± 7 45 ± 10 25 ± 8 A l l figures are the mean and S.E.M. of 6 to 8 determinations on single cells from different cultures of myoblasts. The time to a particular event for a single c e l l was found from inspection of records of beating activity. i - 1 2 5 -rate ( 1 5 to 2 5 min) before cellular f i b r i l l a t i o n (40 to 4 5 min) and the f i n a l cessation of a l l beating activity at 60 min. Fib-r i l l a t o r y activity is shown at a faster chart speed in the 50 minute sample period. This sequence of events was found to be the same for a l l single ce l l s and the time to each phase was dose-dependent (Table IX). A dose-dependent increase in beating rate (expressed as a percentage of control) can be seen in Figure 3 5 . From this figure i t can also be seen that there was a dose-related de-crease in the number of cells beating (due to individual cells reaching the dormant period) with time. At no time during these experiments was gross cellular disruption or vacuoliza-tion observed i n any of the toxin-treated c e l l s . The induction of arrhythmias by various doses of Cyanea toxin is illustrated in Figures 36 - 3 8 . In Figure 36 the proportion of cel l s beating arrhythmically with time can be seen to depend on Cyanea toxin concentration. A toxin con-centration of 440 ng per ml induced a l l beating cells to beat arrhythmically within 10 minutes, whereas 24 and 56 minutes exposure was required for 110 and 28 ng per ml, respectively. The percentage of total cells beating rhythmically i n i -t i a l l y increased on exposure to toxin before rapidly decreas-ing to zero at doses of 440 and 110 ng per ml (Figure 3 7 ) • No i n i t i a l increase i n those beating rhythmically was seen at the lowest toxin concentration (28 ng per ml). The time re-quired to reach 0 per cent beating rhythmically for 440, 1 1 0 , and 28 ng per ml was dose-dependent ( 1 2 , 24, and 4 4 minutes, - respectively). - 1 2 6 -Figure 3 5 : The effect.of Cyanea toxin-containing material on the beating rate of single cultured heart c e l l s . Cyanea toxin (at the concentrations of 28, 110, and 440 ng/ml) was added to cultures at time zero. Six cells were examined at 2 minute intervals for each concentration of toxin. Rate increases are expressed as percentage, increases over control rates. Each point is the mean of the number of cel l s s t i l l beating at that time. The number of cells s t i l l beating is indicated by the figure next to a point. -12? -= 440 ng/ml 6CELLS = 110 ng/ml 4 CELLS = 28 ng/ml 5CELLS ( I No. OF CELLS BEATING -4 -2 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 T IME (MIN.) Figure 36; The effect of Cyanea toxin-containing material on the percentage of beating cultured heart c e l l s beating -arrhythmically. Each point is the mean of determinations from three cultures with samples of ten c e l l s taken from each culture. Ten different c e l l s were randomly selected for each time period. Cyanea toxin ( 2 8 , 110, and 440 ng/ml) was added at time zero. An arrhythmically beating c e l l was defined subjectively as indicated in Methods and by this method approximately 10 percent of a control culture of beat! cel l s was beating arrhythmically before toxin addition. Figure 37: The effect of Cyanea toxin-containing material on the percentage of total c e l l s beating rhythmically. Each point is the mean of a sample of ten cells from three d i f f -erent cultures. Before the addition of Cyanea toxin at con-centrations of 28, 110, and kkQ ng/ml at zero time, approx-imately 50 percent of a l l (beating and non-beating) cells were beating rhythmically. - 131 -• =440ng/ml o = 1 l O ng/ml o = 28 ng/ml - 132 -Figure 38: The effect of Cyanea toxin-containing material on the degree of arrhythmia in arrhythmically beating heart c e l l s . Cyanea toxin at 440 ng/ml (•), 110 ng/ml (•), and 28 ng/ml (o) was added at zero time. The degree of arrhythmia in cells beating arrhythmically was scored subjectively on a s c a l e of 0 to 4.0 (see Methods). Prior to addition of Cyahea toxin some 10 percent of cel l s were beating arrhythm-i c a l l y with a mean score of 0.5- 1.5. This score was der-ived without consideration of the number of ^ arrhythmic c e l l s " ? and therefore is subject to the greatest error where few cel l s were^beati^carr^yt'hml'Gal-l^:..'' -'* M E A N SCORE FOR ARRHYTHMIAS O — (O w ^ i 00 o o 3 3 3 (0 (D (Q ^ ^ \ - 1 3 4 -The mean score for arrhythmias (quantitative determination) is shown in Figure 3 8 . Again, a dose-dependent relationship was seen with the severity of arrhythmias progressing rapidly from slight irregularities of rhythm, soon after addition of toxin, to cellular f i b r i l l a t i o n for a l l cells at 8, 18, and 32 minutes for 440, 1 1 0 , and 28 ng per ml toxin. Thus Cyanea toxin produced a dose-dependent progression of arrhythmic changes beginning with an i n i t i a l increase in rhythmic rate and increase in the number of ce l l s beating. These i n i t i a l changes were followed by other abnormalities in rhythm un t i l eventually a l l ce l l s " f i b r i l l a t e d " . In many respects the pattern of changes with toxin incubation time mimicked the changes seen with increasing Ca concentration. The Effects of Prostaglandins. Lidocaine. and Quinidine on Abnormal Beating Induced in Cultured Heart Cells by Various Arrhythmogenic Agents Five arrhythmogenic agents: ouabain (0) 30 nM, Ca. 6 mM, + K 8 mM, dinitrophenol (DNP) 0 . 2 mM, and adrenaline (Ad) 0 . 5 uM, induced abnormal beating in cultured rat heart myoblasts. Table X illustrates the general changes in beating behavior induced by the different agents. As indicated previously the dosages of the arrhythmogenic agents were chosen on the basis of the preceding experiments which quantitated their effect in cultured heart c e l l s . The effects of prostaglandins, lidocaine, and quinidine on abnormal beating induced in cultured heart c e l l s are shown in Tables XI, XII, and XIII. In a random double-blind fashion, arrhythmogenic agent and drug under test - were added simultaneously and the flask 'read' 10 minutes later. - 135 -TABLES X, XI, XII, and XIII: ' Five arrhythmogenle^agents (ouabain "0" 30 /uM, 6 a + + 6 mM, K + 8 mM, dinitrophenol "DNPn 0 . 2 mM, and adrenaline "Ad" 0 . 5 Aim) induced abnormal beating in cultured rat heart myoblasts. Ab-normalities were measured as: 1. ) % beating normally (N) 2 . ) % stopped (S) 2.) % beating arrhythmically (A) .) mean subjective score of arrhythmias (xA) 5 . ) rate (!) 6. ) rate range (BR) -Prostaglandins A 2, E 2, F l o C , F 2 / ? ( 1 0 " ° M), lidocaine (L) 5 x 1Q"-3 M 'and quinidine (Q) 5 x 1 0 - 7 M were teste&v for their a b i l i t y to modify responses to arrhythmogenic agents. Significant potentia-tion (p<0.05) was judged arrhythmogenic and amelioriation was considered antiarrhythmic. - 136 -TABLE X. The general changes in beating behavior of cultured heart cells induced by different arrhythmogenic agents. See Methods p 47 for definition of terms. AGENT $N %S xA R RR 0 + + • + + + ca + + + + + K + + + -DNP + + - + Ad + + + + indicates an increase - indicates a decrease = indicates no change -'137 -TABLE XI s The effect of prostaglandins, lidocaine, and quinidine versus „ control (C) and arrhythmogenic agents-ouabain ( 0 ) , calcium,(Ca ), potassium (K*):;: dinitrophenol (DNP)and adrenaline (4d)-.on beat-ing activity of cultured rat heart 'myoblasts.- Results are;ex--pressed as the^additional changf" after-the effect of the^arrhyth-mogenic agent alone has been subtracted. & NORMAL (N) % STOPPED ( S ) . G 0 Ca K DNP Ad C 0. Ca K DNP Ad A 2 +11 -7 +10 -25 +4 -4 -1 -5 +5 +19 -3 -9 E 2 -8 +11 -5 -10 +21 - 3 +25 «6 -14 +8 -24 -5 mm -5 +12 -3 -28 +10 -3 +12 -9 -4 +24 -10 o-3 F 2& +7 +17 -13 -2 +7 -3 0 -24 - 4 +2 -15 +1 L -9 -16 +20 -27 +13 *4 +20 +9 ig&t- • +29' -12 V. Q ft -17 -17 +21 -43 -3 +33 -6 . 4 +45 -10 -2 % ARRHYTHMIC U ) MEAN ARRHYTHMIC SCORE (XA) C / 0, Ca K DNP Ad C 0 Ca K DNP Ad A 2 -8 +12 - 5 +9 +7 +16 +.87 -.04 E 2 -10 -£.'5 -6 +2 +3 +8 +.65 0 +0.6 FUc -8 -4 -1 +2 0 +6 +.88 0 +0.5 -5 +7 0 0 +8 +8 +.75 +0.6 L b9 +7 -18 -2 -1 +20 +.75 - 0 . 6 0 Q -12 +22 -22 -2 +15 +20 +1.15 - 0 . 4 5 : +0.5 RATE (Beats/min) (R) BATE RANGE (Beats/min) (RR) G 0. Ca K DNP Ad C 0 Ca K DNP Ad A 2 +7 +160 -68 +12 +19 +16 +114 +73 +113 -6 -41 +46 E 2 +8 +76 -6 +34 +45 +13 -11 -66 -47 -172 -209 +48 F l * 6 +40+111 -.101 +33 +52 +49 +5 -70 -120 -132 -251 +36 -7 +1^5 -27 +5 +31 +13 -31 -45 -54 -70 +112 +108 L * -25 +68 -65 -29 +8 +15 +30 +91 -97 +226 -187 +33 Q +6 +167 ^89 -14 +6 +23 +16 +52 -19 -42 +472 +29' Where no values are shown for Mean Arrhythmic Score, too few cells were arrhythmic to provide meaningful conclusions. TABLE XII . The a b i l i t y of prostaglandins, lidocaine,, and quinidine to. modify the response of cultured rat heart myoblasts to five arrhythmogenic agents - ouabain, calcium,, potassium, dinitro-phenol, and adrenaline.. Significant potentiation-(arrhythmogenic effect) i s indicated by a Y-; amelioration (antiarrhythmic effect) is indicated by ah X. Mncan's multiple range and multiply.Ftest (Duncan, 1955) were used. See p. 50 for definition of terms. OUABAIN; CALCIUM POTASSIUM N S A xA B RR N S A xA R RR N S A xA R RR A 2 Y Y A 2 X A 2 Y E 2 E 2 X E 2 X X Y Floe Floe Y Y X X F20 X Y Y Y F2/? Y L Y L X X X X L Y Y Y Y Q Y Y Y Y Q X X X X Q Y Y Y DINITROPHENOL A 2 & nn X E 2 X X X X F r loc X X X F2fi X Y L X X X X Q Y Y Y ADRENALINE • N S A xA R RR A 2 Y Y Y Y E 2 Y Y Y F l « Y Y Y F20 Y Y Y L Y Y Y Q Y Y Y Y X = amelioration Y = potentiation - 1 3 9 -TABLE XIII . The effects of prostaglandins, lidocaine, and quinidine on abnormal beating induced in cultured heart c e l l s . The f i r s t figure of each pair in the table gives the number of variables ameliorated; the second figure is the number of variables potentiated. Table XIII is a shortened form of table XII. PROSTAGLANDIN 0 Ca"1"1" K + DNP Ad A 2 0 / 2 1 / 0 0/1 I/O O A E 2 0 / 0 1 / 0 2 / 0 4 / 0 0 / 3 F 1 K 0/1 0 / 0 2/2 3 / 0 0 / 3 P 2 £ " 1/3 0/1 0 / 0 I/O 0 / 3 L 0/1 V o O A V o 0 / 3 Q O A V o 0 / 3 0 / 3 OA - 140 -Prior to additions, overall control readings were obtained on each flask. Controls consisted of water for arrhythmo-genic agents and appropriate ethanol for prostaglandins.* The effects of prostaglandins (10~^ M), lidocaine (5 x 1 0 " ^ M ) , and quinidine (5 x 10"7 M) on 1) per cent of cells beating normally (N), 2) per cent stopped (S), 3) per cent of beating ce l l s beating arrhythmically (A), 4) mean subjects ive score for arrhythmias of arrhythmically beating cells (xA), 5) rate (R), and 6) rate range (RR) of beating cells can be seen in Table XI. Results are expressed as additional changes in the presence of drug under test (PGs L or Q) after the effect of the arrhythmogenic agent alone in the presence of appropriate controls was subtracted. Thus with ouabain and PGFgg the following results were obtained. In the presence of ouabain alone the percentage of cells beating normally f e l l from 56$ - 6.4 (S.E.M.) in the overall control situation to 37$ t 8.5 (S.E.M.) (difference 19$) in the presence of ouabain plus drug control (i.e. ethanol). With F 2 f i in the presence of ouabain the number of cel l s beating normally only f e l l from 5 0 $ to 48$ (difference 2$). Thus F 2 g treatment increased the per cent of cel l s beating normally in the presence of ouabain by 17$. Duncan's multiple .rangevahd multiple F test (Duncan,-1955) showed this change ta-be 'significant~at*-the 5$ level. Similar testingrfor*the values in Table xi allowed the summar-ies in Tables XII and.XIII to b"e "constructed. Significance at the 5$ level:was chosen-to differentiate between amelioration, potentiation and no change. * Typical data sheets for this type of experiment are given in the appendix. - 141 -A l l drugs potentiated the effects of adrenaline. Tables XI, XII, and XIII) Prostaglandins and lidocaine ameliorated the effects of dinitrophenol; however, quinidine caused potentiation, decreasing the number stopped and increasing the number arrhythmic and the rate range. Prostaglandins had mixed effects against the other agents. With high potassium, prostaglandins decreased the number beating normally. The rate was increased and the rate range was decreased by prosta-^ glandins in high potassium. With high calcium prostaglandins produced few significant changes. The mean score for arrhy-thmias was decreased with pGA2, and the number stopped was decreased with PGE2. Prostaglandin F 2 ^ reduced the number of beating normally in the presence of high Ca . In the pre-sence of ouabain prostaglandins generally made the arrhy-thmogenic changes worse. The rate was increased by prostaglan-dins and PGA2 increased the number of cells beating arrhy-thmically. The number of cells stopped was increased by PGF2p in the presence of ouabain, however, the number of cells beat^ ing normally was also increased (the only improvement seen with prostaglandins against ouabain arrhythmias). Lidocaine, and quinidine markedly opposed calcium arrhythmias but poten-tiated quabain and potassium arrhythmias. Some of these effects may not be due to a direct interaction between drug and arrhy-thmogenic agent but were probably due to physiological antag-onisms. Thus among the prostaglandins both A 2 and F 2 ( 3 reduce rate and rate range in control cultures. Thus by physiological antagonism A 2 and F 2 f i would be expected to reduce the effects pf Ca , ouabain and adrenaline on rate but potentiate the - 142 -effects of K + and DNP. On rate range such a physiological antagonism would antagonise a l l agents. Lidocaine and quinidine both reduced the percentage of cells beating and slowed rates, actions which should directly antagonize ouabain, calcium, and adrenaline effects but potentiate K + and DNP. Such expected results for lidocaine and quin-idine were seen with calcium and K + but not with adrenaline, ouabain and DNP. The Effect of Prostaglandins. Lidocaine. and Quinidine on Arrhythmias Produced by Cyanea Toxin Figure 39 shows the effect of prostaglandins, lidocaine, and quinidine on the per cent of cells arrhythmic/total beat-ing versus time in the presence of Cyanea toxin 110 ng/ml. Each curve is the line of best f i t obtained similarly to the results in Figure 3 6 . * Prostaglandins, quinidine, and li d o -caine a l l decreased the time for appearance of arrhythmias. The effect of prostaglandins, lidocaine, and quinidine on the per cent of cells beating normally versus time in the presence of Cyanea toxin is shown in Figure 40. The curves were d i s t r i -buted around the control. Similar results were obtained for the mean subjective arrhythmia score (Figure 41). Table XIV gives the tabulation of the above results in terms of the effect of the drugs on the time required for Cyanea toxin to produce a 50 per cent effect. It would therefore appear that neither prostaglandins, lidocaine, or quinidine significantly affect the course of Cyanea toxin cardiotoxicity except for a •*Note: the- scatter of points for which lines are eye-lines of best f i t in Figure 39-41 were the same as those seen in Figure 3 6 - 3 8 . - 143 -Figure 3 9 : The effect of prostaglandins, lidocaine, and quinidine on the percent of cells arrhythmic/total beating versus time, in the presence of Cyanea toxin 1(10 ng/ml. - 144 -• - 145 -Figure 40; The effect of prostaglandins, lidocaine, and quinidine on the percent of cells beating normally, versus time, in the presence of Cyanea toxin 1§0 ng/ml. - 146 -Figure 41: The effect of prostaglandins, lidocaine and quinidine on the mean subjective arrh^JtluBiclascore versus time, in the presence of Cyanea toxin 110 ng/ml. I - 148 --4 0 4 8 12 16 20 24 TIME(min) TABLE XIV The effect of quinidine, lidocaine, and^prpstaglandins on T; the time, required for Cyanea toxin, 110 ng/mlj to produce a 50 per cent effect in the increase in per cent of single rat" heart cells beating arrhythmically; the reduction in per cent of cells beating normally; and the increase in mean arrhythmic score. Shortest time (most toxic) Longest time (least toxic) Increase % Arrhythmic Fl<*> F 2 B > E 2 ' A 2 ' Q ' L » C Reduction Normal F l o c > F 2 & ' ° - , C, Eg, L, Ag Increase in Arrhythmic Score C = control Q = quinidine L = lidocaine a l l others are prostaglandins - 150 -possible slight effect with PGF-^, by increasing toxicity. - 151 -CHAPTER IV DISCUSSION The effect of Prostaglandins During Coronary Occlusion ,in Dogs The f i r s t series of experiments was designed to determine the actions of prostaglandins Eg and F-^ on arrhythmias occur-ring within the f i r s t 25 minutes following coronary artery occlusion. The results indicate that infusions of prostaglan-din Eg and F ^ are antiarrhythmic against ventricular arrhy-thmias which occur early following occlusion. The best predictive tests for antiarrhythmics are prob-ably those which closely resemble c l i n i c a l situations, e.g. those following myocardial infarction. Such tests involve ligation of coronary arteries in experimental animals (TenHorr and Vergroesen, 1 9 7 5 )• The f i r s t report of prostaglandin anti-arrhythmic activity by Z i j l s t r a et a l . ( 1 9 7 2 ) included the observation that ventricular tachycardia due to acute myo-cardial ischemia in dogs was changed to sinus rhythm by a single i.v. injection of prostaglandin E j . The authors sug-gested that PGE1 might act by moderating the sympathetic-parasympathetic balance. Other investigators (Mest et a l . . 1 9 7 2 , 1 9 7 3 , and Forster et a l . . 1973) reported on the anti-arrhythmic effects of PGE^, PGEg, and PGFg* against early arrhythmias produced by coronary artery ligation in mini-pigs, - 152 -dogs, and cats. They speculated that such protection might be the result of maintenance of lysosomal integrity in the ischemic tissue and prevention of the formantion of a cardio-toxic peptide. This suggestion was supported by a further study in dogs which showed that PGF2* helped to preserve cardiac function after coronary occlusion while reducing lysosomal enzyme release (Glenn et a l . . 1 9 7 5 ) . Prostaglan-din B x, a stable free, radical form of PGB-^ , has also been reported to protect Rhesus monkeys from the effects of cor-onary artery ligation, while preserving oxidative phosphor-ylation in degenerated mitochondria (Angelakos et a l . , 1 9 7 5 ) . Thus i t has been established that at least several different prostaglandins have antiarrhythmic activity again-st arrhythmias produced by coronary artery ligation i n exper-imental animals. Several different mechanisms of action have been postulated although none has yet been established. After a coronary occlusion, the reduction of blood supply to the deep ventricular muscle deprives the tissue of oxygen and metabolic substrates. As a result, the production of high energy phosphate bonds is drastically reduced (Braasch et a l . . 1 9 6 8 ) . Lack of sufficient supplies of high energy phosphate bonds may be responsible for the change in permeability of the myocardial c e l l membrane. There is a rapid loss of potassium and a gain of sodium in the ischemic musculature (Jennings et a l . , 1963; Harris et a l . . 1 9 5 4 ) . Since adenosine triphosphate is no longer available for proper functioning of the active ion pump, this imbalance cannot be corrected. As membrane potential declines the fast inward sodium current-carrying - 153 -mechanism i s inactivated, but the slow inward current is not (Cranefield et a l . , 1 9 7 3 ; Trautwein, 1 9 7 3 ) . Due to this de-polarization the ischemic area becomes e l e c t r i c a l l y negative with respect to the normally perfused muscle within one and one-half minutes of occlusion (Baley et a l . , 1 9 4 4 ). This i s the basis for the "injury current" - the flow of current from the pathologically depolarized to the normally polarized areas-which is seen in the electrocardiograms of patients with acute myocardial infarction (Guyton, 1 9 7 6 ). These boundary currents may contribute to the ectopic a c t i v i t y . The increased potas-sium concentration in the extracellular f l u i d also has an effect on the heart (Guyton, 1 9 7 6 ) . Depolarized nerve termin-als in the ischemic region release norepinephrine, and ischemic ventricular muscle fibers release other substances such as en-zymes and l a c t i c acid which may also contribute to the patho-logic changes (Carmiliet and Vereecke, 1 9 6 9 ; Cranefield et a l . , 1 9 7 2 ) . Powerful sympathetic reflexes develop following massive infarction due to the fail u r e of the heart to pump an adequate volume of blood (Guyton, 1 9 7 6 ) . This sympathetic stimulation further increases the i r r i t a b i l i t y of the heart muscle; however, i t should be noted that the increase in arrhythmias during these f i r s t few minutes also occurs while the animal is under morphine-barbital or other barbiturate anesthesia of surgical depth (Harris, 1 9 5 0 ; Martinez, Harvie and Walker, unpublished observations). It is therefore unlikely that reflex a c t i v i t y is a primary cause, but a local sympathetic effect cannot be entirely ruled out. - 154 -Prostaglandins E 2 and F l e < both markedly reduced the mean number of premature ventricular contractions in the 25 minute period following occlusion (Figure 1 0 ) . These results are in agreement with the reports of previous workers ( Z i j l s t r a et a l . . 1972; Mest et a l . . 1972, 1973; Forster et a l . . 1973; Glenn et a l . . 1975; and Angelakos et a l . . 1 9 7 5 ) . In agree-ment with Harris (1950). we found that massive arrhythmias were always associated with the rapid release of the occlusion after 25 minutes and that this quickly produced ventricular f i b r i l l a t i o n in many cases (Figure 1 0 ) . Prostaglandins did not seem to be effective in preventing arrhythmias associated with occlusion release. One may presume that these arrhythmias are related to the rapid release of potassium, norepinephrine, enzymes, la c t i c acid and other metabolic products from the ischemic area. The mechanism of prostaglandin antiarrhythmic activity may involve several p o s s i b i l i t i e s . F i r s t , prostaglandins might act to depress automaticity in a manner similar to lidocaine or quinidine. Secondly, prostaglandins may act in some un-known manner to suppress the effects of ionic imbalance, cat-echolamine release, or the build-up of toxic metabolic products. Finally, prostaglandins may act before the onset of necrosis to prevent degenerative changes, thus enabling the tissue to continue functioning at a more nearly normal level of activity. Such a mechanism has already been suggested (Mest et a l • . 1972, 1973; Forster et a l . . 1973: Glenn et a l . . 1975; and Angelakos et a l . . 1 9 7 5 ) . Further studies are needed to distinguish be-tween these p o s s i b i l i t i e s . - 155 -The Effect of Prostaglandins, Lidocaine, and Quinidine on Ar- rhythmias Produced "by Left Coronary Artery Ligation in Rats The second series of experiments was designed to answer at least part of the question; Which prostaglandins are antiar-rhythmic? The antiarrhythmic potency of several prostaglandins possessing different cardiovascular actions, together with l i d o -caine and quinidine, were compared to each other against early arrhythmias following l e f t coronary artery occlusion in rats. The data obtained in this study indicate that antiarrhythmic activity i s a characteristic possessed by several types of prostaglandins, although with differences in potency, and i s apparently unrelated to their other cardiovascular effects. As mentioned earlier, the best predictive tests for anti-arrhythmic drugs are probably those which closely resemble the c l i n i c a l situation. Recently, increasing attention has been paid to the problem of ventricular f i b r i l l a t i o n developing in the "pre-hospital phase" of acute myocardial ischemia, with or without overt infarction. It i s these early phase arrhythmias which account for nearly two-thirds of the mortality rate f o l -lowing acute coronary attack. (The mortality rate is approx-imately 40 per cent.) (Pantridge and Geddes, 1976) Prosta-glandins have been shown to be effective against ventricular dysrhythmias produced in dogs and monkeys by myocardial ischem-i a resulting from acute coronary artery ligation ( Z i j l s t r a et a l . , 1972; Glenn et a l . . 1975; Angelakos et_al., 1 9 7 5 ) . In the f i r s t set of experiments we confirmed this action with dogs, showing prostaglandins to be effective against early -phase arrhythmias following occlusion. In the present set of - 156 -experiments we adapted coronary ligation techniques in rats (Johns and Olsen, 1 9 5 4 ; Selye et a l . . I960) to the study of antiarrhythmics. This technique allowed inexpensive exper-imental animals and relatively simple surgical techniques to be used for comparative testing with the ischemic model. In contrast to reports of other investigatiors (Johns and Olsen, 1 9 5 4 ; Selye et a l . . I960) we did not find rats to be able to survive occlusion of the l e f t main coronary artery particu-l a r l y well. Four out of five of the untreated control animals in our experiments died in ventricular f i b r i l l a t i o n . It should be noted, however, that the previous authors used ether anesthesia, while pentobarbital was used in the present experiments. We found the early sequence of events following coronary occlusion in rats to be in good agreement with reports using dogs (Harris, 1 9 5 0 ; Sommers and Jennings, 1 9 7 2 ; Wit and Friedman, 1 9 7 5 ) . Following occlusion in rats there was an i n i t i a l latent period, of approximately 4 to 5 minutes, followed by PVCs, PVC salvos, and f i b r i l l a t i o n . We did, however, note the a b i l i t y of some hearts to spontaneously recover from prolonged flutter, an occurrence which is seldom seen in dogs. If the rat survived more than 12 minutes, sinus rhythm was restored. In comparison, Harris ( 1 9 5 0 ) reported that the period of suscept-i b i l i t y to early f i b r i l l a t i o n in dogs was brief and f i b r i l l a -tion did not occur later than the tenth minute following occlu-sion. These early arrhythmias may be attributed to electro-physiological events related to ischemia of ventricular cells in the region deprived of adequate coronary flow (Wit and - 157 -Friedman, 1 9 7 5 ) - a process which would be similar in a l l species. Other investigators have carried out studies with pros-taglandins using experimental dysrhythmias such as those pro-duced by aconitine, calcium chloride, barium chloride and oua-bain (Chiba et a l . . 1972; Kelliher and Glenn, 1973; Mest et a l . , 1972, 1973, 1976; Forster et a l . . 1974b; Bayer et a l . . 1976; Mann et a l . , 1 9 7 3 ) • The experiments appear to be in agreement that the optimum in vivo antiarrhythmic dose range for prosta-glandins in between 1 and 8 ^ ig/Kg/min. On this basis our test dose of 2 ^ ig/Kg/min was whose. We found prostaglandin E 2, followed by PGF2p and quinidine, to be the most effective agents against these early phase, ischemic arrhythmias. Prostaglandins A 2, F-^, and lidocaine were less effective, although s t i l l considerably better than control. In comparison, Forster et a l . (1973) reported the strongest effect against calcium chloride-induced arrhythmias in rats was shown by PGF2e<, which protected a maximum of 84$ of the animals for 10 minutes, followed in decreasing order of effectiveness by FGElt P G E 2> PGA-^ and PGF2/a. Against acon-itine-induced arrhythmias, PGF^ was again the most effective, followed by PGE2, PGA2, PGE-p PGA1, and PGF 2p. Against barium chloride-induced arrhythmias in unanesthetized rabbits (Mentz and Forster, 1974; Mentz et a l . . 1974) PGE2 and PGA-^ showed the strongest effect, followed by PGA2, PGF2^ and PGE-^ Finally, against ouabain arrhythmias in cats (Mest et a l . . 1976; Kelliher and Glenn, 1973) PGE2 and PGAj were most effective, with lesser effects seen with PGF 2e<, PGA2, and PGE^. It appears that the - 158 -effectiveness of a prostaglandin may vary depending on the particular arrhythmia being tested against, although PGEg consistently scored: high. The fact that we found PGFgg ^° D e highly effective against ischemic arrhythmias is also of inter-est, as this prostaglandin possesses very low smooth muscle stimulating activity. This finding gives hope that prosta-glandin analogs may be found which are devoid of any other pharmacological activity. In any case our experiment demon-strated that the prostaglandin antiarrhythmic effect is not a secondary effect, resulting from effects on blood pressure and heart rate for the prostaglandins caused only very minor changes in these variables. It should be noted that there was no significant difference in the size of the infarct for any of the antiarrhythmic treat-ments. This would tend to argue against the possibility that the antiarrhythmic action of prostaglandins in the ischemic model results from an improvement in myocardial blood supply as had been suggested by Mest et a l . , ( 1 9 7 5 ) . The standard antiarrhythmic drugs, lidocaine and quin-idine, also proved effective against these early phase arrhyth-mias although prostaglandin E 2 appeared to be clearly superior to lidocaine and in some respects superior to quinidine as well. The dose of lidocaine (6 ug/Kg/min) was determined experiment-a l l y to be the largest dose which could be given for the time of infusion without producing a depression of blood pressure. The dose of quinidine ( 3 ug/Kg/min) was determined from the literature (Moe and Abildskov, 1 9 7 5 ) . The fact that lidocaine was only moderately effective - 159 -against the early phase arrhythmias is not surprising, as con-troversy exists concerning the a b i l i t y of lidocaine to prevent spontaneous ventricular f i b r i l l a t i o n following coronary occlus-ion (Stephenson et a l . , I 9 6 0 ; Weisse et a l . , 1 9 7 D * Our work is in good agreement with a recent study done in dogs which found lidocaine to be moderately effective in preventing arrhy-thmias in the early phase after coronary occlusion; in addition, lidocaine has been reported to be less effective during the acute phase following myocardial infarction than later in the attack (Pantridge and Geddes, 1 9 7 6 ) . The effectiveness of quin-idine against these early arrhythmias in somewhat surprising since this drug is not commonly used in this situation. One may presume that this effect is related to the a b i l i t y of this drug to suppress ectopic f o c i by i t s well-known depressant action on the myocardium. Prostaglandins have not been found to have a strong "quinidine-like" depressant effect (Forster, 1 9 7 6 ; Mann, 1 9 7 6 ) , a consideration which would give them a clear therapeutic advantage. In summary, a technique for comparative*testing of antiarrhythmic drugs in the acute ischemic model using rats has been described. The time course for arrhythmia development and for types of arrhythmias produced are in good agreement with previous experiments in dogs.. Prostaglandins Eg, Fg^, and quinidine were found to be most effective against these early phase ischemic arrhythmias. The prostaglandin antiarrhythmic activity was not related to effects on blood pressure or heart rate. Since no significant differences were found in the size of the infarcts with different drug treatments, i t seems unlike-- 160 -l y that the antiarrhythmic actions of prostaglandins result from any improvement in coronary circulation. The fact that prostaglandin Fgg* a Prostaglandin with very weak activity on smooth muscle, had good antiarrhythmic activity gives hope that analogs with high specificity of action may be developed. The data in this study indicate that several classes of pros-taglandins possess antiarrhythmic activity; they compare favorably with, and in some cases are superior to, the stand-ard antiarrhythmic drugs lidocaine and quinidine. The Effects of Prostaglandins. Lidocaine. and quinidine on Flutter Threshold and Maximum Following Frequency in In Situ Rat Heart: Prostaglandins appear to be effective only against arrhy-thmias induced by damage or ionic disturbance and not against nondamaging disturbances such as electrical stimulation (Forster, 1976; Metz, 1976) . This lack of activity, together with reports of only minor or controversial electrophysiological effects (Keckskemeti et a l . . 1976; Mentz et a l . . 1974; January and Scho-telius, 1974) , led us to investigate the actions of prostaglan-dins in comparison with lidocaine and quinidine on the flutter threshold and on maximum following frequency in in vivo rat hearts. The results of the present study indicate that in the anti-arrhythmic dose range (1-8 jig/Kg/min) Mentz and Forster, 1974; Mest et a l . , 1974) prostaglandins produce alterations in maxi-mum following frequency which are dose- and prostaglandin-depen-dent. These changes are small (in the order of 10 per cent or less) in relation to the control and are much less than the - 161 -changes seen with the standard antiarrhythmic drugs lidocaine and quinidine. This finding, together with the fact that there was no change in flutter threshold with prostaglandins in con-trast to the decrease seen with lidocaine and the increase seen with quinidine, would indicate that prostaglandins act, at least in part, in a manner different from most other antiar-rhythmic drugs. These results are in accordance with the observation that in patients P G F2oc d o e s n o ^ increase the diastolic stimulation threshold when used in doses required to suppress dysrhythmias encountered c l i n i c a l l y (Mann, 1976). In isolated atria and papillary muscles of guinea pigs a biphasic effect has been reported, with low concentrations of PGE2 and PGFg* producing an increase and higher concentrations a dimunution of the maximum rate of depolarization (Forster et a l . , 1974). Very high concentrations of PGF 2 e < (1 to 10 mg/ml) were required to reduce the conduction time i n those isolated a t r i a and such doses would not be tolerated in vivo due to tox-ic effects (Karim et a l . , 1971). In rat papillary muscle a low-er dose of 10 or 100 ng/ml of H}F2«< was found to cause an in-crease in the action potential duration and plateau amplitude (January and Schotelius, 1974). In the present experiment pros-taglandins Fgoc* E2* 8 1 1 4 A2 a ^ n a d n © S a t i v e linear dose relation-ships for maximum following frequency in the antiarrhythmic dose range (1-8 ug/Kg/ml); in situ, however, the maximum change pro-duced was small. Since measurements of maximum following fre-quency are,reeeprocally related to the functional refractory period and the action potential duration (Szerkes, 1970), i t would seem that our findings of a slight depressive action of - 162 -these prostaglandins on the myocardium is in good agreement with the in vitro findings. The fact that PGFg^ i n i t i a l l y increased the maximum following frequency at 1 ug/Kg/min before causing a decrease may be related to i t s reported b i -phasic action (Forster et a l . , 1 9 7 4 ) . In situ electrophysiological effects of prostaglandins have been previously reported by Bayer et a l . (1976) and Foster et a l . (197*0 in cats. Infusion of prostaglandins at 5 ug/Kg/min resulted i n dose-dependent decreases in the ve-locity of conduction in the atria and in the AV node and extension- of the functional refractory period for prosta-glandins Fg«<» E 2 » El» w i * h maximum effects of approximate-ly 10 per cent. Prostaglandins E 2 and F 2 e < had l i t t l e effect on the ventricular f i b r i l l a t i o n threshold. Thus our results with in vivo rat heart are in close agreement with-previous reports in cats. At least some prostaglandins have been found to have slight depressive actions on the in vivo heart, however, these effects are much weaker than quinidine-like agents. In contrast, prostaglandin F g 0 caused a slightly positive linear response and prostaglandin F 1 & r had a biphasic effect (causing f i r s t a decrease, then an increase in maximum follow-ing frequency with increasing doses.) The fact that PGFg^ was found to be the second most effective antiarrhythmic prosta-glandin, after PGEg, i n the rat coronary artery ligation exper-iments while having./ virt u a l l y no effect on the ele c t r i c a l excitability measurements would tend to confirm the dissociation. In conclusion, only some prostaglandins produce a slight depressive effect on the heart which is much less than that - 163 -which is seen with quinidine-like drugs. This depressive action does not correlate with the antidysrhythmic actions of prosta-glandins . The Effect of Prostaglandins on Isolated Rat Heart Any drug which produces marked pharmacologic effects on the myocardium might have, in addition, antiarrhythmic or arrhythmogenic actions. It was therefore of interest to deter-mine the pharmacology of prostaglandins used in our in vivo studies; conventional experiments measuring rate, force, and coronary flow were performed on unpaced Langendorf perfused hearts. It was also of interest to see whether prostaglan-dins would have a "protective" effect on the force of contrac-tion at 10 M, however, this effect was not seen at other concentrations. An examination of the literature shows that prostaglandins produce cardiac inotropic and chronotropic effects which are species dependent. For example, prostaglandin E^ f a i l e d to alter the rate or force of contraction in the isolated heart of cat, dog, rabbit, or chicken, but produced positive chronotropic and inotropic effects i n isolated guinea pig hearts (Berti et a l . , 1 9 7 5 ; Mantegazza, 1965; Sunahara and Talesnik, 1974; Horton and Main, 1 9 6 7 ) . Quantitatively and qualitatively d i f -ferent results have been reported by other investigators. On the isolated cat papillary muscle, PGE1 was found to markedly increase tension (Tucker et a l . . 1 9 7 1 ) , while on dog papillary muscle only a slight increase in tension was observed (Antonaccio and Lucchesi, 1 9 7 0 ) . Wennmalm and Hedqvist (1970) also obser-ved a slight increase in both rate and contractile force of the - 164 -rabbit heart in response to PGE-j^ and PGEg. For the F series of prostaglandins the effects are just as varied. Prosta-glandin F l f < did not affect the isolated guinea pig heart (Sobel and Robison, 1969) while PGF2ot produced a slight pos-it i v e inotropic effect on isolated guinea pig atria (Nutter and Grumly, 1972) and had no effect on isolated chicken heart (Horton and Main, 1967) or dog and cat atria (Su et a l . , 1 9 7 3 ) . In rat myocardium, prostaglandin E]_ and Eg only modestly in-, creased the amplitude of contraction of the isolated heart and had no significant effect on rate (Berti et a l . , 1 9 6 5 ; Vergroesen et a l . , 1 9 6 7 ; Vergroesen and de Boer, 1 9 6 8 ) . Levy ( 1 9 7 3 ) , however, found that PGEg modestly increased both the force of contraction and the rate of atria from normal and genetically hypertensive rats. Our findings that prostaglan-dins have either no effect or produce only small positive ef-fects on rate and force are therefore in good agreement with the results of previous workers. Concerning the force of contraction. Post-infusion, hearts which had been exposed to PGE2 or PGF 2 p at 10 M did not have the same loss of contractile force with time as did control. This slight stimulatory or "protective" effect continued even though prostaglandins were no longer present. Robert ( 1 9 7 6 ) has postulated a cytoprotective property for prostaglandins in the gastrointestinal system as a partial explanation for their anti-ulcer activity; a similar suggestion has been made concern-ing the antiarrhythmic effects of prostaglandins in arrhyth-mias induced by coronary ligation (Angelakos et a l . . 1 9 7 5 ) . It should be noted that we found PGE2 and PGFpfi to be the most - 165 -effective prostaglandins A 2 and F ^ demonstrated l i t t l e "protective" action in the present experiment (see Table III). There was good cross species agreement between our in vitro and previous in vivo results. With dogs we found no significant change in rate or blood pressure when prostaglan-dins were infused. With prostaglandins in in vivo rats no change in rate and only a slight decrease in blood pressure, with PGA2 and PGE2 were noted. It has been suggested (Mest et a l . . 1975) that the anti-arrhythmic action of prostaglandins may result from improve-ment of the myocardial blood supply. This view was based on the observation of Szekeres et a l . ;(1972) who found the rate of arrhythmias due to coronary occlusion to be decreased after infusion of nitroglycerin. Only ?GE 2 produced an increase and n PGF 2£ caused a decrease in coronary flow rate at 10 ' M in the _5 present experiment. At 10 M a l l prostaglandins produced a marked increase. Most authors report an increase in coronary flow in isolated perfused hearts with prostaglandins. Mantega-zza (1965) reported that PGE1 enhanced coronary blood flow in cat, rat, and rabbit heart without significant inotropic or chronotropic effects. Vergroesen et a l . (1967) also reported that PGEp pGE 2 and PGA-^ increased coronary flow in isolated rat heart; however, Berti et a l . (1965) f a i l e d to find any effect of PGE1 on heart rate and coronary flow in isolated rat heart. With the F series prostaglandins, in rat, Vergroesen et a l . (1970) reported that PGF-^ or PGF 1 P f a i l e d to alter coronary flow. In contrast, Willebrands and Tasseman (1968) found an increase with PGF 1 V. Our data indicate marked increas-- 166 -es in coronary flow with several different series of prosta-glandins, but only at high concentrations. It is possible that this dilation of the coronary vasculature may play a role in the antiarrhythmic action of prostaglandins, however, the fact that the size of the infarct produced was not sig-nificantly decreased by any prostaglandin in our in vivo rat experiments would tend to indicate that this role i s of minor significance. In summary, we have examined the cardiac pharmacology of several prostaglandins of the E, A, and F series in isolated Langendoasff hearts and found them to have either minimal or slight effects on rate and force. These results are in good agreement with our In vivo observations, and i t is unlikely that such weak effects could account for the strong antiar-rhythmic actions of prostaglandins. Both ?GE 2 and PGF2o( (the most effective prostaglandins against coronary occlusion ar-rhythmias) delayed the loss of contractile force with time at 10 M, demonstrating a slight "protective " or stimulat-ing effect analogous to the cytoprotective proposal of Robert (1976), in the gut, or of Angelakos et a l . (1976) in the heart. The significance of this protective effect cannot be deter-mined from the present data. Finally, we found a l l prostaglan-dins tested to markedly increase coronary flow at 10 ^ M. The fact that there was no significant difference in the size of the infarct produced in previous coronary ligation experiments makes i t unlikely that dilation of the coronary vasculature i s the prime mechanism for the antiarrhythmic action. - 167 -The Effects of Prostaglandins Alone on the Beating Activity of Cultured Heart Cells Prostaglandins may produce either direct or indirect effects on rate and force in cardiac tissue. In vivo increases in heart rate reported in response to a number of prostaglan-dins have been shown in dog to be the result of reflex sym-pathetic activation and stimulation of cardioaccelerator centers in the medulla (Malik and McGiff, 1 9 7 6 ) . In contrast, direct chronotropic effects of prostaglandins have been demonstrated in vitro with guinea pig and frog heart (Berti, Lentati, and Usardi, 1 9 6 5 ; Mantegazza, 1 9 6 5 ) . No in vivo studies on the effects of prostaglandins in rats are available; however, in isolated rat heart the E-series prostaglandins have been re-ported to have either no effects or the produce modest increas-es in rate (Berti, Lentati, and Usardi, 1 9 6 5 ; Vergroesen, de Boer, and Gottenbos, 1 9 6 7 ; Vergroesen and de Boer, 1 9 6 8 ) . The lack of marked direct effects observed in the present experiment are, therefore, in agreement with the results of previous workers. Only one exception, JPGF2cC, Produced chronotropic effects, but even this was limited when compared with adrenaline. Other possible increases such as that noted with RGD2 and the de~ crease-with PGE^ were very limited and were'not: s t a t i s t i c a l l y significant. . ; Recent experiments (Goshima, 1976) have shown that the ar-rhythmic movements of both single isolated cells and c e l l clus-ters challenged with arrhythmogenic agents were improved by the addition of antiarrhythmic drugs such as quinidine or pro-caine amide. Since prostaglandins have been reported to have - 168 -antidysrhythmic properties (Forster, Mest and Mentz, 1 9 7 3 ; Kelliher, Reynolds and Roberts, 1 9 7 5 ; Mann, Meyer and Forster, 1 9 7 3 ; Mentz and Forster, 1 9 7 4 ) , we hoped i t would be possible to demonstrate this effect using the rate range as an index of rhythm va r i a b i l i t y . We found no significant chan-ge in the rate range for any of the prostaglandins tested, a l -though there was a marked significant increase in this vari-able with both doses of adrenaline, which can be contrasted with PGFg^ which increased rate without increasing'rate range. In the report noted above, antiarrhythmic drugs reduced the percentage of cells beating spontaneously. Although prosta-glandins are reported as being antiarrhythmic, only PGF1(< and PGFg^ reduced the number of beating cells (i.e. tended to abol-ish automaticity). Prostaglandin had this action despite i t s positive chronotropic action. Bucher and Kolb ( 1 9 6 2 ) and Okarma and Kalman (1971) have reported that i t is possible to interpret changes in optical density as corresponding to changes in the force of contraction in cultured heart c e l l s . In the present experiments, the f i r s t derivative of optical density was also used to provide a second index of density change. Generally prostaglandins either produced no change or reduced both indices of contract t i l e force with prostaglandins (Berti, Lentati and Usardi, 1 9 6 5 ; Vergroesen, de Boer and Gottenbros, 1 9 6 7 ; Vergroesen and de Boer, 1 9 6 8 ) . It i s , therefore, not clear whether changes in optical density and the associated derivative are measure-ments of force effects in cultured heart c e l l s . With adrenal-ine, however, density changes correlate well with what we - 169 -would expect as a force effect i f a negative treppe effect oc-curs at high beating frequencies or i f excess catecholamine^ stimulation reduces cultured c e l l energy levels. Further work is required to f u l l y evaluate optical density changes, but what-ever their exact meaning, i t is significant that the majority of prostaglandins reduced both changes in optical density and i t s derivative, with the marked exception of PGF2ig. It is also to be noted that high concentrations of prostaglandins were used and therefore the effects may be non-specific. Fatty acids can -have a depressant action on cardiac tissue (Szekeres, Borbola and Papp, 1 9 7 6 ) . In this investigation the effect of various prostaglandins on cultured heart c e l l beating activity was assessed in terms of effects on various parameters of rate and contraction. It is interesting to note that activity, on one parameter was not necessarily associated with activity on another. Thus, FQF^ increased beating rate and the number of stopped cells but did not affect the optical density or i t s derivative, while PGF-jo* affected the percentage beating and optioal density but not rate. If the prostaglandins were l i s t e d in rank order for their effect on the different parameters only F-j*, Fg**' E 2 ' a n d 15 MeE2 tended to be highly ranked, while B^, A 2 and F^ were low ranked which would appear to indicate that prostaglandins have more than one pharmacological action on cultured ce l l s , and that these actions are not necessarily related. For example, the mechanism responsible for the chronotropic action of PGF^ is probably independent of that responsible for reducing the number of beating c e l l s . Electrophysiological^ prostaglandins - 170 -can hyperpolarize or depolarize c e l l s , depending on concentra-tion (Kecskemeti, Keleman and Knoll, 1974). The possibility of prostaglandins acting on more than one mechanism is increased by considering the actions of pGF 2g, which does not change the percentage of cells beating spontaneously, despite reducing rate and increasing optical density. In conclusion, with the exception of PGF2<J<, which produc-ed a marked chronotropic response, prostaglandins have limited direct actions in cultured heart c e l l s . Furthermore, the pos-i t i v e chronotropic effect of PGF 2 o C is not associated with the dysrhythmlc tendencies seen with catecholamines. Prostaglandins either produced no change or reduced indices of contractile force, with the exception of PGF2^ which produced a positive force effect. The Effects of Prostaglandins on Abnormalities of Beating in Cultured Heart Cells Induced by a Variety of Arrhythmogenic Agents Our studies on the antiarrhythmic actions of prostaglandins have been directed at answering at least part of the question: 1) which prostaglandins are antiarrhythmic 2) under what circum-stances, and 3) by what mechanisms? The present set of experi-ments deals with the third part of the original question; by what mechanisms are prostaglandins antiarrhythmic. Several pos s i b i l i t i e s have been proposed; 1) They may modulate sympathe-tic-para-sympathetic balance 2) they may affect blood flow and distribution 3) their antiarrhythmic activity may be secondary to their general cardiovascular actions and 4) they may have some "-cellular protective" action which enables damaged cells - 171 -to carry on a more normal level,of activity. We used cultured heart cells (which are free of adrenergic innervation and blood flow considerations) together with a number of specific arrhythmogenic agents to test the hypothesis that prostaglandins may be antiarrhythmic by means of some cellular protective action. The Actions of Ouabain. Ionic Manipulation. Dinitrophenol. and Anoxia on Cultured Heart Cells Arrhythmic behavior has already been described in cultured heart cells (Goshima, 1 9 7 6 ; Boder and Johnson, 1 9 7 2 ; Sane and Sawanoberi, 1 9 7 0 ) , but such descriptions lack qualitative and quantitative depth. The evaluation of arrhythmias in previous reports u t i l i z e d subjective measurements (Goshima, 1976) and were limited quantitatively. The present experiment describes the action of a number of arrhythmogenic agents on the beating activity of single isolated cultured heart cells itiiboth qual-itative and quantitative terms using objective and subjective methods. The types of arrhythmic responses varied with the agent considered. Ouabain and calcium produced similar effects with marked increases in rate terminating in f i b r i l l a t o r y activity. The most widely quoted mechanism of action for cardiac glyco-sides is inhibition of sodium and potassium-dependent membrane ATPase (Goodman and Gilman, 1 9 7 5 ) . The rapid increase in rate to a plateau (see Figure 24) may therefore be due to a depolar-ization of the resting membrane potential toward threshold with an elevation of intracellular sodium and calcium levels. A con-tinued f a l l in membrane potential would eventually be arrhyth-- 172 -mogenic as the membrane potential approached threshold. Fur-ther depolarization would result in quiescence as the cel l s failed to repolarize. Figure 25 and 2,6 show good agreement with the expected results. Potassium and dinitrophenol produced very different types of arrhythmias from ouabain and calcium; they were characterized by decreases in rate accompanied by an increase In rate var-i a b i l i t y while none of the subjective arrhythmic patterns seen with the f i r s t two agents was observed. Elevation of potassium f i r s t decreased rate up to 10 mM (Figure 29). This decrease in rate is most l i k e l y related to a lengthening of the action potential due to delayed or slowed repolarization. A higher concentration of extracellular potassium would oppose the potassium efflux during, phase 3 of the cardiac action potential. The increase in rate seen with concentrations between 10 mM and 28 mM potassium is probably related to steepening of the slow depolarization (phase 4) portion of the cardiac action poten-t i a l . Very high (10-28 mM) extracellular potassium might then tend to oppose the declining outward flow of potassium during this phase which partially balances the Inward sodium leak (Trautwein, 1973). Dinitrophenol (DNP) is most widely known as an inhibitor of oxidative phosphorylation although i t also inhibits anerobic metabolism as well (Lehninger, 1976). The i n i t i a l rapid decrease in rate seen with DNP may be related to a partial depolarization of the cells due to inhibited sodium-and potassium-ATPase secondary to decreased ATP. The fact that although the cells partially recovered their i n i t i a l rate, they appeared to beat more.weaklywould support this view. - 173 -Anoxia was found not to be arrhythmogenic. Similar re-sults have been reported in the isolated heart (Mommaerts, 1 9 6 6 ) . Although cardiac tissue normally u t i l i z e s oxidative metabolism, apparently i t is quite capable of switching to anaerobic metabolism and deriving sufficient energy by this means to continue normal functioning (at least when not pump-ing under load). In both the isolated heart and in c e l l cul-ture the ava i l a b i l i t y of glucose for an energy source and the removal of products of metabolism are not limiting factors. Quinidine and lidocaine both decreased automaticity in cultured heart c e l l s . Similar findings had previously been reported by Mercer and Dower ( 1 9 6 6 ) although no complete log dose response curves were shown. Since we had previously found the prostaglandins to decrease automaticity up to 3 3 $ in cultured heart cells the curves allowed us to select doses of quinidine and lidocaine with similar effects for com-parison. In conclusion, we have investigated the dose and time responses of several arrhythmogenic responses of several ar-rhythmogenic agents In cultured heart c e l l s . It is possible to evaluate very different types of cellular arrhythmias in both qualitative and quantitative terms. The information from this study made i t possible to select appropriate doses of our arrhythmogenic agents for testing with antiarrhythmic drugs. It was hoped that such a study might give insight into the antiarrhythmic mechanisms involved. - 174 -The Effect of Cyanea Toxin on Cultured Heart Cells Walker ( 1 9 7 7 b ) has shown the cardiotoxicity of Cyanea toxin-containing material to result from profound ionic disturbances including a loss of intracellular potassium and a gain invsodium and calcium associated with the expected electrophysiological changes of depolarization and reduction ' of action potential height with ultimate loss of action poten-t i a l s . Cultured neonatal rat heart cells are believed to closely resemble intact heart tissue with regard to morphology and physiology (Halle and Wollenberger, 1 9 7 0 ) . Ion distribution (Halle and Wollenberger, 1 9 7 0 ) and ionic mechanism are also similar (Lieberman et a l . , 1 9 7 5 ) , apart from the possibility of a greater importance of calcium and slow sodium currents (Sperelakis, 1 9 7 2 ) . Prom the above, one could predict that exposure of such cultured c e l l s to Cyanea toxin should produce a characteristic progression of dysrhythmic changes. A toxin-induced f a l l in resting membrane potentials as seen in adult atria (Walker, 1 9 7 6 b ) , would account for the i n i t i a l increase in beating rate i f the maximum diastolic potentials in these cells are equivalent to a t r i a l resting membrane potentials. A continued f a l l in membrane potential would eventually be arrhythmogenic as is the case with cells exposed to ouabain (Goshima, 1 9 7 6 ) . The quiescent period between the loss of the f i r s t type of arrhythmic activity and the appearance of cellui-lar f i b r i l l a t i o n may indicate a membrane potential f a l l i n g below threshold for activation of a sodium dependent action potential. A further f a l l would then bring the potential into the area of spontaneous activation of slow calcium currents - 175 -(Cranefeild et a l . . 1 9 7 2 ) . Indeed, f i b r i l l a t o r y activity in Purkinje fibers may involve slow currents resistant to tetro-dotoxin (TTX) and revealed by bathing with high K solutions (Aronson and Cranefield, 1 9 7 3 ) . The f i n a l proof that the arrhythmic activity pattern results from membrane potential changes must await detailed electrophysiological investigation. Despite the profound changes in activity with Cyanea toxin-containing material, i t is of interest to note that morpholog-i c a l signs indicative of gross membrane damage (vacuolization and lysing), were not apparent. However, cells treated with Cyanea toxin did not recover activity when placed for 48 hours in control media. This lack of evidence of direct l y s i s is analogous to the finding of an unaltered intracellular space (Walker, 1977b) in toxin-treated atria. In conclusion, Cyanea toxin produced the most severe type of arrhythmia encountered in cultured heart c e l l s . We decided to use the characteristic series of changes in beating behavior produced by Cyanea toxin cultured cardiac cells as a further arrhythmogenic test for anti-arrhythmic drugs and prostaglandins. The Effects of Lidocaine, Quinidine, and Prostaglandins on Abnormal Beating Induced in Cultured Heart Cells The primary purpose of the present series of experiments was to determine the a b i l i t y of prostaglandins to protect heart cells from a variety of damaging agents. The underlying ration-ale was that the anti-dysrhythmic actions of prostaglandins may be related toan lability to protect cardiac cells against damage which results in Ionic redistribution, electrophysiological disturbances, and subsequent dysrhythmias. A similar "cyto-- 176 -protective" mechanism has been proposed by Robert ( 1 9 7 6 ) for \ the anti-ulcer effects of prostaglandins and by Angelakos ( 1 9 7 5 ) for the antiarrhythmic action in coronary occlusion. The data from the present study indicate that prostaglandins do not have any general "cytoprotective" action, although they do appear to oppose the inhibition of c e l l beating activity seen with dinitrophenol. Part of the rationale underlying the present experiments was that behavior in cultured heart cells reflects that seen in intact tissue, to the extent that disturbances of beating rhy-thm may involve similar mechanisms to those occurring in the intact tissue. This is not withstanding the fact that re-entry models have an absolute dependency on heterogeneity in a pop-ulation of c e l l s . The morphology and physiology of cultured heart cells has been found to closely resemble the intact heart (Halle and Wollenberg, 1 9 7 0 ) . Single isolated myocardial cells obtained from mammalian or chicken heart are known to beat spontaneously and indepen-dently in culture (Goshima, 1 9 7 6 ; Wollenberger, 1 9 6 4 ; Sperelakis and Lehmkuhl, 1 9 6 4 ) . The great majority of single isolated cells show regular, rhythmical beating under normal conditions and have regular arrhythmic movements analogous to f i b r i l l a t i o n upon the addition of aconitine (Boder and Johnson, 1972) or of d i g i t a l i s (Marke and Strasser, 1 9 6 6 ; Mercer and Dower, 1 9 6 6 ; Goshima, 1 9 7 6 ) or in a medium of low potassium (Goshima, 1 9 7 5 ) or of high calcium concentration (Goshima, 1 9 7 5 ) . The a b i l i t y of standard antiarrhythmic drugs to improve these arrhythmic movements is controversial. Mercer and Dower ( 1 9 6 6 ) and Klein-- 177 -feld et a l . ( 1 9 6 9 ) found that single isolated cells from embry-onic chick heart showed arrhythmic movements on addition of antiarrhythmic drugs such as quinidine or procainamide. In contrast, Boder and Johnson ( 1 9 7 2 ) reported that pre-treatment with lidocaine partially prevented aconitine-induced arrhythmias in c e l l clusters from neonatal mouse heart. Goshima ( 1 9 7 5 , 1 9 7 6 , 1977) reported that both c e l l clusters and single isolated cells from fetal mouse heart showed arrhythmic movements in . " media of low potassium, high calcium concentrations or with ouabain; these arrhythmic movements were markedly improved or completely disappeared post-treatment with quinidine, procain-amide, or lidocaine. In the present experiments we found lidocaine and quini-dine to be effective only against cellular arrhythmias caused by high calcium concentration. In agreement with Mercer and Dower ( 1 9 6 6 ) and Rleinfeld et a l • . ( 1 9 6 9 ) and in contrast to the results of Goshima ( 1 9 7 6 ) , we did not find lidocaine or quinidine to be effective against cardiac glycoside-induced arrhythmias. In fact, our standard antiarrhythmic drugs made both ouabain and high potassium concentration arrhythmias worse, although the results with potassium could have been at least partially due to the cell's response to the drug alone. With dinitrophenol, lidocaine ameliorated the arrhythmias while quinidine had mixed effects which would tend to indicate some action other than -direct depression of automaticity. A l l agents made adrenaline arrhythmias worse, indicating no direct sympathetic blocking action for quinidine, lidocaine, or prostaglandins. The prostaglandins had mixed effects except - 1?8 -for an a b i l i t y to improve cells inhibited with dinitrophenol and the previously mentioned adverse effect seen with adrenaline. Dinitrophenol is a well-known inhibitor of oxidative phosphor-ylation which is capable of inhibiting'anaerobic metabolism as well(Lehninger, 1 9 7 6 ) . Polis et a l . ( 1 9 7 2 ) have previously reported that prostaglandins are capable of stimulating ox-idative phosphorylation in depressed mitochondrial prepara-tions. Our results indicate that a similar effect i s pre-sent at the cellular level. We therefore found no over-all "protective" effect with prostaglandins in c e l l cultures. Only the effects of prosta-glandins against dinitrophenol-induced arrhythmias appeared to bear any relationship to the antiarrhythmic actions of prosta-glandins in vivo. The correlation, however, is not exact. Prostaglandin Eg was most effective against DNP-induced c e l l -ular dysrhythmias and in previous experiments against in vivo coronary occlusion arrhythmias in rats. Prostaglandin Eg had also previously been found to be most effective in maintaining contractile force in Langendbrff hearts. In in vivo and in Langendorff hearts, however, PGFg^ w a s * n e s e c o n d niost effective prostaglandin, followed by K x F l a < and PGAg. In comparison, PGF^ was more effective than PGFgfi in c e l l culture. Lido-caine was effective against DNP-induced cellular arrhythmias, while quinidine was not, which may indicate that there i s a fundamental difference in their mechanisms of action. At least with the prostaglandins, however, there is substantial agree-ment between the effects on DNP-induced cellular arrhythmias, maintenance of contractility in Langendorff hearts, and in vivo - 179 -antiarrhythmic activity in the rat coronary occlusion model. It is therefore possible that at least part of the antiarrhy-thmic action of prostaglandins may be due to their a b i l i t y to maintain metabolic processes in depressed tissue. Such a mech-anism would be compatible with the suggestions of Mest et a l . ( 1 9 7 2 , 1 9 7 3 ) , Forster et a l . ( 1 9 7 3 ) , Glenn et a l . ( 1 9 7 5 ) , and Angelakos ( 1 9 7 5 ) that the "protective" effect of prostaglan-dins following coronary artery ligation may be the result of maintenance of cellular function. The Effect of Prostaglandins, Lidocaine, and Quinidine on Arrhythmic Changes in Cultured Rat Heart Cells with Cyanea Toxin We had determined that the cardiotoxic activity of Cyanea toxin was not related to an ouabain-like inhibition of the mem-brane ATPase cation transport systems, and that Cyanea toxin produces a characteristic series of changes in beating behav-ior in cultured cardiac c e l l s . This toxin was found to produce the most severe types of arrhythmias encountered in cultured myoblasts, and we therefore decided to make use of i t as a fur-ther test for a "protective" effect with our antiarrhythmic drugs. The results indicate that neither prostaglandins or the standard antiarrhythmic drugs, lidocaine and quinidine, protect against Cyanea toxin-induced arrhythmias. None of the antiarrhythmic agents altered the maximum ef-fect produced by the toxin on arrhythmic score, per cent beat-ing, or the per cent arrhythmic. The time to produce 100 per cent arrhythmic beating was, however, decreased by the prosta-glandins. This change would be interpreted as being arrhythmo-genic, not antiarrhythmic. Such a change may be related to an - 180 -increase in membrane permeability. The arrhythmias produced ky Cyanea toxin are similar to the arrhythmias seen with high ++ + Ca or with low K media (Goshima, 1 9 7 6 ) , and the membrane depolarization seen with Cyanea toxin (Walker, 1977b) would also be consistent with increased membrane permeability. This may account for the fact that both Cyanea toxin and adrenaline arrhythmias were made worse by prostaglandins. This experiment provides further evidence that there is no over-all "cellular protective" action responsible for the antiarrhythmic actions of the prostaglandins. Only the pre-viously described effects against dinitrophenol-induced arrhy-thmias appear to bear any correlation with the in vivo results. - 181 -CHAPTER V SUMMARY 1. Prostaglandins Eg and Flct are effective against early-arrhythmias associated with coronary occlusion in dogs. 2. Prostaglandins E 2 and Flu were not effective against arrhythmias associated with release of occlusion. 3. A ..technique for comparative testing of~ ahtArrhythmic" ' ^ drugs in the acute ischemic model using rats has been described. 4. The time course for arrhythmia development and for types of arrhythmias produced in rats are in agreement with previous experiments in dogs. 5. Prostaglandins Eg, Pgg, and quinidine were found to be most effective against early phase ischemic arrhythmias in rats. 6. The antiarrhythmic act i v i t y of prostaglandins i s not related to effects on blood pressure or heart rate. 7. Since the size of infarct i s not altered by prostaglandins, i t i s unlikely that the antiarrhythmic actions of prosta-glandins result from any marked improvement in coronary c i r -culation. - 182 -8. The antiarrhythmic activity of prostaglandins does not correlate with their effects on smooth muscle, as shown "by the results obtained with PGFgg. 9. Several classes of prostaglandins possess antiarrhythmic activity; they compare favorably with, and in some cases are superior to, the standard antiarrhythmic drugs lidocaine and quinidine. 10. Results with representive prostaglandins of the E, A , and F series show that some, but not a l l , prostaglandins produce a slight depressive effect on maximum following frequency in in vivo rat heart at antiarrhythmic doses. No effect on f l u t -ter threshold;;.is detectable,. This depressive action does not correlate with the antidysrhythmic actions of prostaglandins. 11. Prostaglandins of the E, A , and F series have only minimal effects on rate and force in Langendorff rat hearts. It is unlikely that such weak effects could account for the strong antiarrhythmic actions of prostaglandins. 12. Both PGE2 and PGF-j^ delayed the loss of contractile force - 7 with time at 10 M in isolated hearts. 13. Prostaglandins E 2, A 2 , F l e <, and F 2^ a l l markedly increase coronary flow at 10"^ M in isolated rat hearts. - 183 -14. With the exception of PGF2^, which produced a marked chronotropic response, prostaglandins have limited direct action in cultured heart c e l l s . 15- In cultured heart ce l l s the positive chronotropic effect of PGF2o< is not associated with the dysrhythraic tendencies seen with catecholamines. 16. The effect of ouabain, calcium, potassium, and dinitro-phenol on cultured heart cells has been investigated. 17. It is possible to evaluate very different types of c e l l -ular arrhythmias in both qualitative and quantitative terms. 18. Quinidine and lidocaine produce similar decreases in auto-mat i c i t y in cultured heart cells; however, quinidine is approx-imately 100 times more potent. 19. Cyanea toxin produces a characteristic series of arrhyth-mogenic changes in cultured heart cells which may be useful as a test agent for antiarrhythmic drugs. 2 0 . Lidocaine and quinidine are effective only against cellu -lar arrhythmias caused by high calcium concentration. 21. Prostaglandins were effective only against dinitrophenol-induced cellular arrhythmias. - 184 -22. There is no over-all "protective" effect with prostaglan-dins in c e l l culture. 2 3 . At least part of the antiarrhythmic action of prostaglan-dins may he due to their a b i l i t y to maintain metabolic pro-cesses in depressed tissue. 24. Cyanea toxin-induced cellular arrhythmias were made worse by prostaglandins, providing further evidence that there is no over-all "cellular protective" action responsible for anti-arrhythmic action of prostaglandins. - 183' -BIBLIOGRAPHY Afonso, S., Bandow, G.T., Rowe, G.G., J. Physiol., 2 4 1 : 2 9 9 , 1 9 7 4 . Andersen, N.H., and Ramwell, P.W., Arch. Intern. Med., 133:30, 1 9 7 4 . Angelakos, E.T., Polls, D. and Riley, R., 6 t h I n t . Cong. Pharmacol, Abstract 9, 1 9 7 5 . Antonaccio, M.J. and Lucchesi, B.R., Life Sci., 9 : 1 0 8 1 , 1 9 7 0 . Ariens, E.J., Molecular Pharmacology, Academic Press, New York, 1 9 6 4 . Aronson, R.S. and Cranefield, P.P. J. gen Physiol. 6 1 : 7 8 6 , 1 9 7 3 . Bayer, B.L., Forster, W., and Sperling, J., Archs int. Pharmacodyn. Ther. 2 2 1 : 3 2 8 , 1 9 7 6 . Bayley, R.H., La Due, J.S., and York, D.J., Am. Heart J. 27 : 1 6 4 , 1 9 4 4 . Berti, F. Lentati, R. and Usardi, M.M., Med. Pharmacol. Exp. 13: 233, 1 9 6 5 . Berti, F. a n d Usardi, M.M., G. Arterioscler, 2 : 2 6 1 , 1 9 6 4 . Block, A.J., Poole, S. and Vane, J.R., Prostaglandins, 7 : 4 7 3 , 1 9 7 4 . Bloor, CM. ;and Sobel, B.E., Circulation, 4 2 Suppl. 111:123, 1 9 7 0 . Braasch, W., Gudbjamason, S. and Puri, P.S., Circ. Res. 2 3 : 4 2 9 , 1 9 6 8 . Boder, G.B., and Johnson, I.S., J. Mol. C e l l . Cardiol., 4 : 4 5 3 , 1 9 7 2 . Brody, M.J. and Kadowitz, P.J., Fed. Proc, 3 3 : 4 8 , 1 9 7 4 . Bucher, P., and Kolb, I., Med. Exp., 6 : 1 4 , 1 9 6 2 . Butcher, R.W., Baird, C.E., J. Biol., Chem., 2 4 3:1713, 1 9 6 8 . Carmeliet, E., and Vereecke, J., Pfluegerfe Arch., 313:300, 1 9 6 9 . Carlson, L.A., Ann. N.Y. Acad. Sci., 1 3 1 : 1 1 9 , 1 9 6 5 . Calton, G.J. and Burnett, J.W., Toxicon 1 1 : 3 5 7 , 1 9 7 3 . - 186 -Caton, M.P.L., Chemistry Structure and Availability. In: The Prostaglandins: Pharmacological and Therapeutic Advances, (ed. M.F. Cuthbert) London, Whitefriars Press, 1 9 7 3 . Chiba, S., Nakajima, T. and Nakano, J., Jap. J. Pharmacol. 2 2 , 7 3 ^ , 1 9 7 2 . ^ Colbert, J.C, Prostaglandins Isolation and Synthesis, London, Noyes Data Corporation, 1973* Cranefield, P.F., Wit, A.L. and Hoffman, B.F., Gen. Physiol. 5 9 : 2 2 7 , 1 9 7 2 . Cranefeld, P.F., Wit, A.L., Hoffman, B.F., Circulation, 4 7 : 1 9 0 , 1 9 7 3 -Crowshaw, K., and McGiff, J.C, Prostaglandins in the kidney: a correlative study of their biochemistry and renal function. In: Mechanisms of Hypertension (ed. M.P. Sambhi) pp 2 5 4 - 2 7 3 , Amsterdam, Excerpta Medica, 1973* Curnow, R.T. and Nuttal, F.Q., Fed. Proc, 3 0 : 6 2 5 , 1 9 7 1 -DeBoer, J., Houtsmuller, U.M.T. and Vergroesen, A.J., Prostaglandins, 3 : 8 0 5 , 1 9 7 3 -Douglas, W.W., Polypeptides-Angiotensin, Plasma Kinins, and Other Vasoactive Agents: Prostaglandins. Louis S. Goodman and Alfred Gilman (eds.) The Pharmacological Basis of Therapeutics F i f t h Edition, New York, Macmillan, 1 9 7 5 , P 640. Duncan, D.B., Biometrics, 1 1 : 1 , 1 9 5 5 « Dunnett, CW., J. Amer. statist. Ass., 5 0 : 1 0 9 6 , 1 9 5 5 . Dusting, G.J., Moncada, S. and Vane, J.R., Prostaglandins, 1 3 : 3 , 1 9 7 7 . Fain, J.N., Ann. N.Y. Acad. Sci. 1 3 9 : 8 7 9 , 1 9 6 7 . Forster, W., Acta b i o l . Med. Germ., 3 5 : 1 1 0 1 , 1 9 7 6 . Forster, W., Borbola, J., Papp, J.G., Schror, K., and Szekeres, L., Arch. int. Pharmacodyn. 2 1 1 : 1 3 3 , 197^-Forster, W., Mest, H.J., and Mentz, P., Prostaglandins, 3 : 8 9 5 , 1 9 7 3 -Freeman, S.E., Toxicon, 1 2 : 3 9 5 , 1 9 7 4 . Friedman, P.L. Stewart, J.R., and Wit, A.L., Circ. Res. 3 3 : 6 1 2 , 1 9 7 3 -Goldblatt, M.W., J. Physiol. (Lond.) 84:208, 1 9 3 5 -Glenn, T., Tauber, P.L. and Goldfarb, R.D., 6 t h Int. Cong. Phar-macol., Abstract 5 7 , 1 9 7 5 . - 187 -Girardier, L., Cardiology 5 6 : 8 8 , 1971. Glynn, I.M., Br. med. Bull, 2 4 : 1 6 5 , 1968. Godin, D.V., Ng, T.W., and Tuchek, J.M., Biochim. biophys. Acta 4 3 6 : 7 5 7 , 1976. Godin, D.V., and Schrier, S.L., Biochemistry 9 : 4 0 6 8 , 1970. Goodman, L.S., and Gilman, A., The Pharmacological Basife of Thera-peutics, F i f t h Edition, New York, Macmillan, 1975-Goshima, K., J. Mol. C e l l . Cardiol., 9 : 7 , 1 9 7 7 . Goshima, K., J. Mol. C e l l . Cardiol., 8:217, 1976. Goshima, K., Exp. C e l l . Bes. 9 2 : 3 3 9 . 1975* Gould, W.M., and Burnett, J.W., J. invest. Derm. 5 7 : 2 6 6 , 1971. Guyton, A.C., Textbook of Medical Physiology, F i f t h Edition, Philadelphia, Saunders, 1976, pp. 207-211. Hadhazy, P., I l l e s , B., and Knoll, J., Eur. J. Pharmac, 2 3 : 2 5 1 , 1973-Halle, W., and Wollenberger, A. Myocardial and Other Muscle C e l l Cultures, in Methods in Pharmacology, Vol. 1 (A. Schwartz, Ed.), Meredith, New York, 1970, Chapter 7, pv 191. Hamberg, M., and Samuelsson, B., Proc. natn. Acad. Sci. U.S.A., 7 0 : 8 9 9 , 1973-Harary, I., and Farley, B., Exp. C e l l . Bes. 2 9 : 4 5 1 , 1963. Harris, A.S., Bisteni, A., and Russel, R.A., Science 119:200, 1954. Harris, A.S., Circulation, 1:1318, 1950. Hedqvist, P., Acta Physiol. Scand. 79; Supp. 3 4 5 : 1 , 1970. Hedqvist, P., and Brundin, J., Life Sci. (part 1) 8 : 3 8 9 , 1969. Hedqvist, P., Autonomic Neurotransmission, in The Prostaglandins (P.W. Ramwell, Ed.), Plenum Press, New York, 1 9 7 3 , PP- 101-131. Hedqvist, P., and Wennmalm, A., Acta Physiol. Scand., 8 3 : 1 5 6 , 1971. Hedqvist, P., Stjarne, L., and Wennmalm, A., Acta Physiol. Scand. 7 9 : 1 3 9 , 1970. Higgins, C.B., .$»Snd Braunwald, E., Am. Heart J. 8 5 : 3 4 9 , 1973. Higgins, C.B., Vatner, S.F., Franklin, D., Patrick, T., and Braun-wald, E., Circ. Res., 2 8 : 6 3 8 , 1971. - 188 - -Higgins, C.B., Vatner, S.f Franklin, D., and Braunwald, E., Cir-culation, 42> Supp. 1 1 1 : 1 2 3 , 1 9 7 0 . Hollenberg, M., Walker, R.S., and McCormick, D.P., Arch. int. Pharmacodyn. Ther., 1 7 4 : 6 6 , 1 9 6 8 . Horton, E.W., Br. med. Bull., 29:148, 1 9 7 3 . Horton, E.W., and Main, I.H.M., Br i t . J. Pharmacol. 3 0 : 5 6 8 , 1 9 6 7 . Hutton, I., Parratt, J.B., and Lawrie, T.D.U., Cardiol. Res. 7:149, 1 9 7 3 -Hyde, A., Cheneval, J.P., Blondel, B., and Girardier, L., J. Physiol. (Paris) 64 : 2 6 9 , 1 9 7 2 . January, C.T., and Schottelius, B.A., Proc. Soc. Exp. Biol. Med., 143:403, 1 9 7 4 . Jennings, R.B., Sommers, H.M., and Kaltenbacn, J.P., Circ. Res. 14:260, 1 9 6 3 . Johns, T.N., and Olson, B.J., Ann. of Surg. 140:675, 1 9 5 4 . Junstad, M. and Wennmalm, A., Br. J. Pharmac, 5 2 : 3 7 5 , 1 9 7 4 . Junstad, M., and Wennmalm, A., Acta Physiol. Scand. 8 7 : 5 7 3 , 1 9 7 3 . Karim, S.M.M., Somers, K., and H i l l i e r , K., Cardiovasc. Res. 5 : 2 5 5 , 1 9 7 1 . Karim, S.M.M., H i l l i e r , K., and Devlin, J., J. Pharm. Pharmac. 2 0 : 7 4 9 , 1968. Karim, S.MF*!M., Sandler, M., and Williams, E.D., Br. J. Pharmac. 3 1 : 3 ^ 0 , 1 9 6 7 . Karim, S.M.M., Br. J. Pharmac. 2 9 : 2 3 0 , 1 9 6 7 -Kateri, M., Takeda, K., and Imai, S., Tohoke, J. Exp. Med. 1 0 1 : 6 7 , 1 9 7 0 . Kecskemeti, v . , Kelemen, K., and Knoll, J.,Pol. J. Pharmacol. 2 6 : 1 7 1 , 1 9 7 4 . Kecskemeti, V., Kelemen, K., and Knoll, J., Adv. Biosci. 9 : 3 7 3 , 1 9 7 3 . Keen, T.E.B., Toxicon 1 1 : 2 9 3 , 1973* Kelliher, G.J., Reynolds, R.D., and Roberts, J., 6 t h Int. Congr. Pharmac, Abs. 981, 1 9 7 5 . Kelliher, G.J., and Glenn, T.M., Eur. J. Pharmac 24:410, 1 9 7 3 . - 189 -Klein, I., and Levey, G.S., Metabolism, 2 0 : 8 9 0 , 1 9 7 1 . Kleinfeld, M., Shade, 0 . , and Gruen, F., J. Pharmac. and Exp. Ther., 170:84, 1 9 6 9 . Koss, M.C., and Nakano, J., Prostaglandins, 8 : 1 7 9 , 1 9 7 4 . Krebs, H.A., Biochem. biophys. Acta., 4:249, 1 9 5 0 . Kurzrok, B., Lieb, C , Proc. Soc. Exp. B i o l . Med. 28:268, 1 9 3 0 . Lazzara, R., El-Sherif, N., and Scherlag, B.J., Circ. Res. 3 3 : 7 2 2 , 1 9 7 3 -Lee, J.B., Archs. intern. Med. 1 3 3 : 5 6 , 1 9 7 4 . Lehninger, A.L., Biochemistry, Second Edition, New York, Worth Publishers, 1 9 7 5 , p. 5 1 9 -Levey, G.S., and Klein, I., Life Sci. 13:41, 1 9 7 3 -Levy, J.V., Prostaglandins 4 : 7 3 1 , 1 9 7 3 -Lieberman, M., Sawanobori, T., Shigeto, M., and Johnson, E., Phys-iological Implications of Heart Muscle in Tissue Culture, in De-velopmental and .'Physiological Correlates of Cardiac Muscle (M. Lieberman and T. Sano, Eds.), Raven Press, New York, 1 9 7 5 , P« 1 3 9 . Lowry, O.H., Roseborough, N.J., Farr, A.L., and Randall, R.J., J. b i o l . Chem. 1 9 3 = 2 6 5 , 1 9 5 1 . Malik, K.U., and McGiff, J.C., Cardiovascular Actions of Prosta-glandins, in Prostaglandins: Physiological Pharmacological and Pathological Aspects (S.M.M. Karim, Ed.), MTP Press, Lancaster, 1 9 7 6 , pp. 118-124. Mandel, L.R., and Kuehl, F.A. Jr., Biochem. Biophys. Res. Commun. 28:13, 1 9 6 7 . Mann, D., Acta b i o l . Med. Germ., 3 5 : 1 1 1 3 , 1 9 7 6 . Mann, D., Meyer, H.G., and Forster, W., Prostaglandins 3 : 9 0 5 , 1 9 7 3 -Mantegazza, P., A t t i . Acad. Med. 2 0 : 6 6 , 1 9 6 5 . Mark, G.E., and Strasser, F.F., Exp. Ce l l Res. 4 4 ; 2 1 7 , 1 9 6 6 . Martinez, T.T., and Walker, M.J.A., Proc. West. Pharmac. Soc. 2 0 : 2 6 9 , 1 9 7 7 . Martinez, T.T., and Walker, M.J.A., Canadian Heart Foundation Meeting, Edmonton, Alberta, Canada, October, 1 9 7 6 . Maxwell, G.M., Br. J. Pharmac, 3 1 : 1 6 2 , 1 9 6 7 -Maxwell, G.M., Austr. J. Exp. B i o l . Med. Sci. 4 7 : 7 1 3 , 1 9 6 9 -- 190 -Mentz, P., Opitz, H., and Forster, W., Acta Biol. Med. Germ 3 5 : 1 1 6 5 , 1 9 7 6 . Mentz, P., and Forster, W., Acta Biol. Med. Germ., 3 2 : 3 9 3 , 1 9 7 4 a . Mentz, P., and Forster, W., Acta Biol. Med. Germ., 3 3 : 2 4 5 , 1 9 7 4 b . Mercer, E.N., and Dower, G.E., J. Pharmac. Exp. Ther. 1 5 3 : 2 0 3 , 1 9 6 6 . Mest, H . J C , Blass, K.E., and Forster, W., Acta Biol. Med. Germ. 3 5 : 6 3 , 1 9 7 6 . Mest, H.J., and Forster, W., Archs. int. Pharmacodyn. Ther. 2 1 7 : 1 5 2 , 1 9 7 5 . Mest, H.J., Mentz, P., and Forster, W., Pol. J. Pharmac. Pharm. 2 6 : 1 5 1 , 1 9 7 4 . Mest, H.J., and Forster, W., Prostaglandins 4 : 7 5 1 , 1 9 7 3 . Mest, H.J., Schror, K., and Forster, W.*.,, Adv. B i o s c i . 9 : 3 8 5 , 1 9 7 2 . Mee, G.K., and Abildskov, J.A., Antiarrhythmic Drugs in The Pharma-cological Basis of Therapeutics, F i f t h Edition, (L.S. Goodman and A. Gilman, Eds.), New York, MacMillan, 1 9 7 5 , P. 6 8 8 . Mommaerts, W.F.M., in Cardiovascular and Respiratory Effects of Hypoxia, (J.D. Hatcher and D.B. Jennings, Eds.), Hafnerf/Co., New York, 1 9 6 6 , p.70* Nakano, J., and McCurdy, J.R., Proc. Soc. Exp. Biol. Med. 1 2 8 : 3 9 , 1 9 6 8 . Nakano, J., Proc. Soc. Exp. Biol. Med. 1 2 7 : 1 1 6 0 , 1 9 6 8 . Needleman, P., Key, S.L., Isakson, P.C, and Kelkarni, P.S., Pros-taglandins 9 : 1 2 3 , 1 9 7 5 . Nugteren, D.H., Beerthuis, R.K., and VanDorp, D.A., Biosynthesis of Prostaglandins, in Prostaglandins, Proceedings of the Second Nobel Symposium, Stockholm, Stockholm, Almquist and Wiksell, 1 9 6 7 , p . 4 5 . Nutter, D.O., and Crumly, H.R. Jr., Cardiovasc. Res. 6 : 2 1 7 , 1 9 7 2 . Nutter, D.O., and Crumley, H., Circulation Supp. I l l , 4 2 : 1 2 4 , 1 9 7 0 . Pantridge, J.F., and Geddes, J.S., Br. Med. J. 2 : 1 6 8 , 1 9 7 6 . Papanicolaou, N., Makrakis, S., Bariety, J., and Mi l l i e z , P., J. Pharm. Pharmac. 2 6 : 2 7 0 , 1 9 7 4 . Park, M.K., Dyer, D.C, and Vineenzi, F.F., Prostaglandins, 4:717, 1 9 7 3 . Piper, P.J., Distribution and Metabolism in The Prostaglandins: Pharmacological and Therapeutic Advances, (M.P. Cuthbert, Ed.), London, Whitefriars Press, 1 9 7 3 . - 191 -Polls, B.D., Grandizio, A.M., and Polls, E., Proceedings Aerospace Med. Assoc. 4 3 : 2 1 3 , 1 9 7 2 . Barnwell, P.W., and Shaw, J.E., Recent Prog. Herm. Res. 2 $ : 1 3 9 , 1 9 7 0 . Robert, A., Antisecretory, Antiulcer,Cytoprotective and Diarrheo-genic Properties of Prostaglandins in Advances in Prostaglandin and Thromboxane Research, Vol. 2 (B.Samuelsson and R. Paoletti, Eds.), New York, Raven Press, 1 9 7 6 . Rowe, G.G., and Afonso, S., Am. Heart J. 8 8 : 5 1 , 1974. Samuelsson, B., and Wennmalm, A., Acta Physiol. Scand. 8 3 : 1 6 3 , 1 9 7 1 -Sano, T., and Sawanobori, T., Circ. Res. 26:201, 1 9 7 0 . Schaaf, T.K., Annual Reports in Medicinal Chemistry, p. 80, 1 9 7 6 . Schanne, 0.*v,J. Appl. Physiol. 2 9 : 8 9 2 , 1 9 7 0 . Schatzmann, H.J., and Vineenzi, P.F., J. Physiol, Lond. 2 0 1 : 3 6 9 , 1 9 6 9 . Schneider, W.P., The Chemistry of the Prostaglandins in The Prosta-glandins (S.M.M. Karim, Ed.), New York, Wiley-Interscience, 1 9 7 2 , P. 2 9 3 . Selye, H., Bajusz, E., Grasso, S., Mendell, P., Angiology 2 : 3 9 8 , i 9 6 0 . Sen, A.K., Sunahara, F.A., and Talesnik, J., Can. J. Phys. Pharmac. 54:128, 1 9 7 6 . Sobel, B.E., and Robinson, A,K., Circulation 39/40, Supp. I l l , 1 8 9 , 1 9 6 9 . Sommers, H.M., and Jennings, R.B., Arch. Intern. Med. 1 2 9 : 7 8 0 , 1 9 7 2 . Sperelakis, N., Elec t r i c a l Phenomena in the Heart, Academic Press, New York, 1 9 7 2 , p. 1. Steinberg, D., Vaughan, M., Nestel, P.J., Strand, 0 . , and Bergstrom, S., J. Clin. Invest. 4 3 : 1 5 3 3 , 1 9 6 4 . Steinberg, D., Vaughn, M., Nestel, P.J., and Bergstrom, S., Biochem. Pharm. Pharmac. 1 2 : 7 6 4 , 196-3. Stephenson, S.E., Cole, R.K., and Parrish, T.F., Am. J. Cardiol. 5 : 8 8 , I960. Su, J.Y., Higgins, G.B., and Friedman, W.F., Proc. Soc. Exp. Biol. Med. 143:1227, 1 9 7 3 -Sunahara, F.A., and Talesnik, J., J. Pharmac. Exp. Ther. 188:135, 1 9 7 4 . Szekeres, L., Borbola, J., and Papp, J.G., Acta Biol. Med. German. 3 5 : 1 1 1 9 , 1 9 7 6 . - 192 -TenHoor, F., and Vergroesen, A.J., J. Mol. Ce l l . Cardiol 7 : 5 3 7 , 1 9 7 5 . TenHoor, F., and Vergroesen, A.J., Prostaglandins 7 : 5 3 5 , 1 9 7 5 . Toom, P.M., and Phi l l i p s , T.D., Toxicon 1 3 : 2 6 1 , 1 9 7 5 . Trautwein, W., Physiol. Eev. 5 3 : 7 9 3 , 1 9 7 3 . Turker, E.K., Kiran, B.K., and Vural, H., Arzneim. Forsch. 2 1 : 9 8 9 , 1 9 7 1 . Vane, J.B., Br. J. Pharmac, 3 5 : 2 0 9 , 1 9 6 9 . Vergroesen, A.J., and de Boer, J., Europ. J. Pharmac. 3 : 1 7 1 , 1 9 6 8 . Vergroesen, A.J., deBoer, J., and Gottenbes, J.J., Effects jof Prostaglandins on Perfused Isolated i a t Hearts, in Prostaglandins, Nobel Symposium 2 (S. Bergstrom and B. Samuelsson, Eds.), Stockholm, Almquist and Wiksell, 1 9 6 7 . von Euler, U.S., J. Physiol. (Lond.) 8 8 : 2 1 3 , 1 9 3 6 . Walker, M.J.&., Martinez, T.T., and Godin, D.V., Toxicon 1 5 : 3 3 9 , 1 9 7 7 -Walker, M.J.A., Toxicon 1 5 : 3 , 1 9 7 7 a . Walker, M.J.A., Toxicon 1 5 : 1 5 , 1 9 7 7 b . Weisse, A.B., Moschos, C.B., and Passannante, A.J., Am. Heart J. 81 : 5 0 3 , 1 9 7 1 . Wennmalm, A., Acta Biol. Med. German. 38:1127,* 1 9 7 6 . Wennmalm, A., Acta Mysio^ScaM^ ; j 9 3 : 1 5 , 1 9 7 5 . Wennmalm, A., Acta Physiol. Scand. 8 3 , Supp. 3 6 5 : 1 , 1 9 7 1 . Wennmalm, A., and Hedqvist, P., Life Sci. (part 1 ) , 1 0 : 4 6 5 , 1 9 7 1 . Wennmalm, A., and Hedqvist, P., Life Sci. (part 1 ) , 9 : 9 3 1 , 1 9 7 0 . Willebrands, A.F., and Tasseron, S.J.A., Am. J. Physiol. 2 1 5 : 1 0 8 9 , 1 9 6 8 . Wit, A.L., and Friedman, P.L., Arch. Intern. Med., 1 3 5 : 4 5 9 , 1 9 7 5 . Zjt^lstra, W.G., Brunsting, J.B., TenHoor, F., and Vergroesen, A.J. Eur. J. Pharmac 18:392, 1 9 7 2 . - 193 -APPENDIX TABLE A The effeot of prostaglandins, lidocaine, and quinidine on blood pressure and heart rate during l e f t coronary artery ligation in rat. Standard errors are given unless too few samples were available for calculation. Control A 2 E 2 F i o r F2£ Lidocaine Quinidine B.P. Control 150 125 ± 7 ± 4 130 102 ± 6 ±8 162 131 ± 1 3 ± 1 0 130 104 ±24 ± 2 2 XifUS 145 112 ± 2 3 ±21 147 117 ± 6 ± 5 162 128 ±8 ±12 Post Surg 114 95 ± 1 7 ±14 115 89 ± 1 3 ± 1 3 127 105 ±18 ±20 87 63 ±21 ± 2 2 120 92 ±14 ±18 125 93 ± 1 3 ±18 96 58 ± 1 1 ±12 Infusion 116 96 ± 1 1 ±10 87 63 ± 4 ± 5 95 70 ±12 ±14 91 67 ±17 ±17 124 99 ± 1 5 ±12 109 80 ±12 ± 1 5 5 min post occlusion 104 87 ±10 ± 1 1 104 80 ±20 ± 2 3 99 78 ± 1 5 ±16 73 51 ± 2 3 ± 2 2 112 90 ± 1 1 ± 1 1 93 70 14 17 69 35 ± 1 1 16 10 min post occlusion 129 112 ± 5 ±8 98 75 ±21 ±26 99 75 ± 1 5 ±17 135 120 112 87 ± 1 1 ± 1 1 99 78 16 19 72 40 17 18 Bate: Control 425±17 443± 14 427117- 412±11 404118 424125 399125 Post Surg 44l±30 445 493±38 448115 467±18 443124 397127 Infusion 421±13 486122 463±24 425*18 503±27 432134 — — 5 min post 446t6 occlusion i 10 Min post 455 475±20 496±17 453*20 458±17 469±45 520 483±18 4 9 0 ± 2 3 458127 447 410137 398 occlusion - 195 -PG BOOK t PAGE FLASK#_ DATE & TIME ; DRUG OPERATOR CONC. CONTROLS _ DRUG Rate Rate Range Rate X Cont. Rate Range Z Cont. Rate Rate C e l l #1 2 ' . 3 • • 4 . 5 Mean S.E.M. - 196 -PC BOOK t , PAGE DATE & TIME_ OPERATOR FLASK t_ DRUG CONC. No. 1 1 2 3 4 5 6 7 8 9 10 S S S S S S S S S S A A A A A A A A A A 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 1 2 3 4 5 6 7 8 9 10 S S S S S S S S S S A A A A A A A A A A 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 No. 2 1 2 3 4 5 6 7 8 9 10 S S S S S S S S S S A A A A A A A A A A 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 5 1 2 3 4 5 6 7 8 9 10 S S S S S S S S S S A A A A A A A A A A 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 No. No. No. 3 1 2 3 4 5 6 7 8 9 10 S S S S S S S S S S A A A A A A A A A A 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 S.E.M. Z Norn beat Z Stop Z Arrhy. x Arr. NOTES C e l l Appearance: Fibroblasts: Age (days): Good Many 1 2 3 4 Bad Fev _ Other None 5 6 7 8 9 10 Special drug d e t a i l s ( d l l n . , soln., etc.) General Notes:
Thesis/Dissertation
eng
Pharmacology
Vancouver : University of British Columbia Library
For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.
Graduate
Studies on the antiarrhythmic actions of prostaglandins
Text