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Effect of 5-isopropyltropolone on the adrenergic responses of the isolated guinea-pig atria Ko, Cecilia Wai Yin 1965

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THE EFFECT OF 5-ISOPROPYLTROPOLONE ON THE ADRENERGIC RESPONSES OF THE ISOLATED GUINEA-PIG ATRIA by CECILIA WAI YIN KO B.S.P. University of British Columbia 1961 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN PHARMACY in the Division of Pharmacology of the FACULTY OF PHARMACY. We accept this thesis as conforming to the required standard. THE UNIVERSITY OF BRITISH COLUMBIA August 1965. In 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 the r e q u i r e m e n t s f o r an advanced degree 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 agr e e t h a t t h e L i b r a r y s h a l l make 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 and study„ 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 be g r a n t e d by t h e Head o f my Department o r by h i s r e p r e s e n t a t i ves<, 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 not be 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 . Department o f P h a r m a c y The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada Date S e n t . X ABSTRACT 5-Isopropyltropolone(gamma-thujaplicin) i s one of the three isomeric isopropyltropolones found i n the heartwood of western red cedar(Thuja p l i c a t a D. Don). The tropolones as a class have been shown to he i n h i b i t o r s of the enzyme catechol-O-methyltransferase(COMT). The e f f e c t of this tropolone on the responses of the i s o l a t e d guinea-pig a t r i a to a* number of sympathomimetic amines has been studied. Gamma-thujaplicin was used i n the form of water soluble sodium salt(T-Na). T-Na i t s e l f was found to possess a slow but prolonged stimulating e f f e c t on the a t r i a . In the presence of T-Na greater than 0.4 meg./ml, both the posit i v e i n o t r o p i c and chronotropic e f f e c t s of a l l of the sympatho-mimetic amines studied were increased. In the case of the short acting catechol amines, prolongation of these responses was also produced. The e f f e c t of T-Na on the responses to the adrenergic amines was compared with that of cocaine and ethylenedxa-minetetraacetic acid(EDTA). Potentiation of the responses to the adrenergic s t i m u l i by T-Na and the l a s t two agents appeared to follow a sim i l a r dose-response pattern although T-Na was twice as potent. Potentiation by T-Na of responses to the d i r e c t acting catechol amines was not affected by reserpinization. 11 When used in place of EDTA for the repletion of norepinephrine stores in reserpinized atria, T-Na has been proved to be more effective than the former agent i n retard-ing the oxidation of norepinephrine. The effect of histamine on the isolated atria was also studied. In a low concentration (0.2-0. 4mcg/ml) histamine stimulated the atria to a prolonged response that can only be terminated by changing the bathing f l u i d . The response of the atria to histamine was neither affected by the presence of a high concentration of antihistamine (3mcg./ml), nor was i t blocked by the pre-addition of a beta-receptor blocking agent. Cocaine and pyrogallol had no effect on the histamine induced response. Therefore i t i s not lik e l y that norepinephrine i s involved i n the response of the atria to histamine. In the presence of T-Na, however, the response to histamine was increased i n the normal preparation, but T-Na had l i t t l e or no effect on the histamine induced res-ponse i n reserpinized atria. These results together with the fact that EDTA potentiated the action of sympathomimetic amines i n a similar manner to T-Na do not support the assumption that T-Na potentiation of adrenergic responses i s due to COMT inhibition. It would appear that T-Na produces like EDTA, a non-specific sensitization of the atria muscle. This mechanism i s not clear and no evidence has been found to indicate that i t i s due to a general depletion of ions as a result of the chelating action of these compounds. Signature of Examiners i v TABLE OF CONTENTS Page ABSTRACT 1J LIST OF TABLES v i LIST OF FIGURES v i i INTRODUCTION The Tropolones 1 Metabolism of Catechol Amines 5 Catechol-O-Methyltransferase Inhibitors 6 Sympathetic Nerve Ending as an Organized Molecular Unit 8 Gamma-Thujaplicin and Adrenergic Responses 10 METHODS AND MATERIALS Isolated Guinea-pig A t r i a 12 RESULTS E f f e c t of Gamma-Thujaplicin Sodium on Responses to Catechol Amines 15 E f f e c t of Gamma-Thujaplicin Sodium on Responses to Tyramine, Dopamine, and Amphetamine 18 E f f e c t of Cocaine on Responses to Amines 18 E f f e c t of Pyrogallol on Responses to Amines 22 E f f e c t of Ethylendiaminetetraacetic Acid on Responses to Amines 28 E f f e c t of Excess Magnesium Ions on Gamma-Thu j a p l i c i n Potentiation 28 Experiments on A t r i a from Reserpinized Guinea-pigs •••• 2 ^ V Repletion of Norepinephrine Stores.. 29 Eff e c t s s o f Drugs on Responses to Histamine Action of Histamine on the Isolated A t r i a 32 Gamma-Thu japlxcin Sodium 33 Cocaine 33 Pyrogallol 33 Ethylenediaminetetraacetic Acid 33 Diphenhydramine 35 Pr one thai o l . 35 Reserpinized A t r i a 38 DISCUSSION 39 SUMMARY 44 BIBLIOGRAPHY 46 v i LIST OF TABLES TABLES PAGE I E f f e c t of Gamma-Thujaplicin on the Responses of A t r i a to Sympathomimetic Amines 19 II E f f e c t of Cocaine on Responses to Catechol Amines 20 III E f f e c t of Cocaine on Responses to the Long-acting Sympathomimetic Amines 21 IV E f f e c t of Pyrogallol on Responses to Catechol Amines... 25 V E f f e c t of Pyrogallol on Responses to the Long-acting Sympathomimetic Amines 26 VI E f f e c t of Ethylenediaminetetraacetic Acid and Gamma-Thujaplicin on Restoration of Tyramine Responses by Incubation with Norepinephrine. Re-serpinized Guinea-Pig A t r i a 31 VII Modification of Responses of the Isolated A t r i a to Histamine by Various Drugs 36 v i i LIST OF FIGURES FIGURE PAGE 1. Structure of Alpha, Beta and Gamma-Thujaplicins 1 2. Metabolism of Epinephrine and Norepinephrine 7 3. Concept of Orgainized Molecular Unit at the Sympathetic Nerve Ending 9 4. Potentiation of Responses to Epinephrine by Gamma-Thujaplicin. Isolated Guinea-Pig A t r i a 16 5. Potentiation by Gamma-Thujaplicin of Responses to Epinephrine, Norepinephrine and Isoproterenol.... 17 6. Ef f e c t of Cocaine and Gamma-Thujaplicin on Responses To Epinephrine. Isolated Guinea-Pig A t r i a 18a 7. Potentiation of Responses to Epinephrine by Pyro-g a l l o l . Isolated Guinea-Pig A t r i a 23 8. Comparison of Effects of Gamma-Thujaplicin and Pyro-g a l l o l on Responses of Isolated Guinea-Pig A t r i a to Norepinephrine 24 9. Potentiation of Responses to Norepinephrine by Ethylenediaminetetraacetic Acid 28a 10. Comparison of Eff e c t s of Gamma-Thujaplicin and E t h y l -enediaminetetraacetic Acid on Responses of Isolated Guinea-Pig A t r i a to Norepinephrine 28b 11. Reserpinized A t r i a 29a A. Showing the Absence of Response to Tyramine B. E f f e c t of Gamma-Thujaplicin and Pyrogallol on Responses to Epinephrine. v i i i 12. E f f e c t of Gamma-Thujaplicin, Diphenhydramine and Pronethalol on Responses to Histamine 34 13. E f f e c t of Pronethalol on the Pos i t i v e Inotropic Responses of Histamine, Tyramine and Norepinephrine. 37 ACKNOWLEDGEMENTS The author i s indebted to Dr. J.E. Halliday f o r his guidance and encouragement throughout the course of thi s work. (1) INTRODUCTION The Tropolones Tropolone i s a term used to designate the 7-uiembered carbon ring compound 2-hydroxyr- 2,4,6-cycloheptatrien-l-one, derivatives of which are r e l a t i v e l y rare i n nature. The t h u j a p l i c i n s are isopropyl derivatives of tropolones which occur i n the heartwood of western red cedar (Thuja p l i c a t a D. Don). They were the f i r s t naturally occurring compounds to be characterized conclusively as monocyclic tropolones(l). F i g . 1 alpha-thujaplicin beta-thujaplicin gamma-thujaplicin In addition to the three isomers shown i n figure 1, a fourth compound, beta-hydroxy-thujaplicin (7-hydroxy-4-isopropyltropolone) has been i s o l a t e d by Garner and Barton(2) from Thuja p l i c a t a D. Don. The chemistry of the th u j a p l i c i n s and other tropolones has been quite extensively studied(3). Antimicrobial (2) studies of the thujaplicins have been shown that a l l of the isomers possess antifungal and antibacterial properties(4). A pharmacological study was conducted by Lee(5) on beta-thujaplicin. Lee found that i t s actions were mostly depressant in nature though low concentrations may stimulate organs such as isolated heart and rabbit uterus. Katura who summarized Lee's work suggested that beta-thujaplicin i s a nerve muscle poison and related in i t s actions to camphor and thujone and other terpene compounds. Gamma-thujaplicin was studied by Halliday(6) i n the form of water soluble soduim salt. His results indicated gamma-thujaplicin exerts a mixture of stimulant and depressant action on the central nervous system of the mammals. The stimulant component of i t s action i s evident only in conscious animals and i s manifested by convulsions which have been shown to be of cerebral origin. When gamma-thujaplicin was administered in conjunction with depressant drugs to mice, there i s how-ever, sufficient evidence to indicate that gamma-thujaplicin has no value as an analeptic. In frogs, the depressant action of gamma-thujaplicin i s more predominant and no signs of stimulation could be seen when the drug was injected intravenously or intralymphatically. Its depressant effect on the isolated intestinal muscle was believed to be different from the atropine-like mechanism and probably partially involved the .myenteric ganglia. Beta-hydroxy-thujaplicin has also been studied. Sanders and Halliday (7 ) had found that beta-hydroxy-thujaplicin (3) resembled gamma-thujaplicin i n that i t produces both stimulant and depressant effects on the central nervous system of mice. Unlike gamma-thujaplicin where there i s a great difference i n the spread between the median convulsive dose (CD 50) and the l e t h a l dose (LD 50) i n mice, the CD 50 and LD 50 of beta-hydroxy-thujaplicin were found to be very close together. In doses well below those which cause convulsions, beta-hydroxy-thujaplicin causes a reduction of spontaneous a c t i v i t y without impairment of reflexes, depression of body temperature, antagonism of amphetamine, induced hyperactivity, and moderate prolongation of bar-biturate narcosis. It also interacts with chlorpromazine and reserpine to produce additive depressant e f f e c t s on a c t i v i t y and body temperature although the hypothermic e f f e c t of this t h u j a p l i c i n was not affected by a low ambient temperature. Shortly a f t e r the discovery by Axelrod (8-13), that O-methylation reaction i s an important route of i n a c t i v a t i o n of catecholamines, a number of the synthetic and naturally occurring tropolones became of i n t e r e s t to various i n v e s t i -gators. Belleau and Burba (14) f i r s t reported that 4-methyl-tropolone was a strong i n h i b i t o r of the i s o l a t e d enzyme catechol-O-methyltransferase. In t h e i r l a t e r study they had presented evidence that the catechol and tropolone rings are biochemically i s o t e r i c ( 1 5 ) . From the f a c t that excess magnesium ions produce a s l i g h t i n h i b i t o r y e f f e c t on (4 the rate of methylation, and that i n the presence of 4-methyltropolone, a five fold increase in the magnesium ion concentration had no effect on the inhibited rate, they excluded the possibility that tropolones could act by partially depriving the enzyme catechol-O-methyltransferase (COMT) of i t s magnesium ions. They suggested that tropo-lones probably form a complex with the enzyme active sites normally occupied by the substrate. COMT blockade by the tropolones therfore, has the characteristics of competitive rather than non-competitive type of inhibition(15). Ross(16) has shown that 4-methyl and 4-isopropyltropolone prolonged the tachycardia induced by isoproterenol and other sympathomimetic amines in mice. In a metabolic study, Musacchio and Goldstein(17) and Mavrides et al(l8) demon-strated that beta-thujjaplicin and 4-methyltropolone respectively decreased O-methylation of catecholamines in vivo. Potter and Axelrod(19) have noted that tropolones cause a potentiation of the pressor response to norepinephrine andri.tyramine in the rat. Murnaghan and Mazurkiewicz(20) found that an increase in toxicity of epinephrine in mice with small doses of 4-methyltropolone but a delay in the death of mice with large doses. Later they concluded that though the inhibition of the catechol-O-methyl transferase by the tropolones may have played a role in the mice experiments, the potentiation of epinephrine on isolated tissues i s due to a non-specific (5; s e n s i t i z a t i o n produced by the tropolones(21). This assump-tion i s based on observation that tropolone potentiation also occured with potassium and barium on the rabbit a o r t i c s t r i p and even with carbachol, histamine and barium on the i s o l a t e d guinea pig ileum. Metabolism of Catecholamines Although many studies have implicated cytochrome oxidase and monoamine oxidase i n the metabolism of epinephrine and other catecholamines i n vitro(22), no e v i -dence however, fo r the p a r t i c i p a t i o n of cytochrome oxidase i n the metabolism of epinephrine i n vivo has been obtained(9). Furthermore other investigators have implicated that monoamine oxidase plays only a minor role i n the i n a c t i -vation of epinephrine(24-25). The possible existence of a new but more important pathway fo r the metabolism of epinephrine was recognized by Blaschko(26) and Armstrong(27). However most of the work which has led to the elucidation of the mechanism of catecholamine degradation has been performed by Axelrod(8-13). Axelrod f i r s t demonstrated that 0-methylation of epinephrine and other catecholamines precedes deamination i n v i v o ( 8 - l l ) , then he described, i s o l a t e d and p a r t i a l l y p u r i f i e d an enzyme catechol-O-methyltransferase which i s now recognized as the enzyme primarily concerned i n the i n a c t i v a t i o n of c i r c u l a t i n g catecholamines(8-9). According to Axelrod(l3), the major pathway fo r the meta-bolism of epinephrine and norepinephrine involves (6) 0-methylation to y i e l d metanephrine and nor-metanephrine respectively. These p h y s i o l o g i c a l l y inactive metabolites are then conjugated or deaminated to 3-methoxy-4-hydroxy mandelic acid(figure 2). Monoamine oxidase i s mainly con-cerned with the deamination of the O-methylated metabolites rather than the catecholamines themselves. The enzyme COMT i s found widely d i s t r i b u t e d i n most organs and tissues including the autonomic and the central nervous system. This enzyme i s believed to be c h i e f l y concerned i n the metabolism of c i r c u l a t i n g epinephrine, but i t s role i n the i n a c t i v a t i o n of endogenously released catechol amines remains to be extablished. Catechol-O-Methy1transferase Inhibitors Besides the tropolones, other i n h i b i t o r s of the enzyme COMT include pyrogallol and other polyphenols such as catechol, adrenalone, and arterenone. Wylie(28) had studied these polyphenols and found that they augmented the t o x i c i t y of intravenous epinephrine i n the r a t . According to Wylie, the augmented t o x i c i t y being adrenergic i n nature and probably due to an increase i n the l i f e - s p a n of epinephrine. In experiments on blood pressure of the spinal cat, and on the cat n i c t i t a t i n g membrane, Wylie also found that pyrogallol prolonged the duration of pressor e f f e c t s of epinephrine and the duration of contraction of the n i c t i t a t i n g membrane i n response to supra-maximal stimulation of the preganglionic sympathetic nerve(28). Since polyphenols of the catechol type are also (7) OH H C- C.-NHCH> (1) COMT OH II OCH-Epinephrine MAO (3) 3,4 Dihydroxy Mandelic Acid MAO Metanephrine (2) MAO (10) COMT HO 0CH3 3-Methoxy-4-Hydroxy Mandelic Acid (5) MAO (6) OH H /\—C — C—NH, U H « HO I O C H 3 COMT OH Norepinephrine Normetanephrine Fig . 2 Metabolism of Epinephrine and Norepinephrine 1,6,10 catalysed by COMT; S-adenosyl methionine vserving as the methyl donor; divalent cations are required. 2,3,4,5—these reactions take place i n 2 steps. Deamination by monamine oxidase to form corresponding aldehyde followed by oxidation by aldehyde dehydrogenase and diphospho-pyridine nucleotide. (8) methylated at the 3-hydroxy position(29,30), wylie and Archer are convinced that the augmentation observed i s the re s u l t of competitive a f f i n i t y f o r the same enzyme system(28). Izquierdo and Kaumann(31) found that pyrogallol enhanced the duration of tachycardia induced by epinephrine, norepinephrine, isoprenaline and other epinephrine d e r i -vatives i n anaesthetized dogs. In the blood pressure experiments, they found that p y r o g a l l o l increased the pressor e f f e c t s of epinephrine more than norepinephrine. This has been previously explained by Wylie(28) that norepinephrine has a higher a f f i n i t y f o r COMT, thus leading to a more d i f f i c u l t displacement by a competitive i n h i b i t o r . Sympathetic Nerve Ending as an Organized Molecular Unit A l l drugs that influence the sympathetic nerve end-ings were once believed to act d i r e c t l y on the receptor s i t e s by mimicking or blocking the action of the neuro-hormone—norepinephrine . In recent years, many drugs have been shown to influence the sympathetic nervous system i n d i r e c t l y by modifying the amount of free neurohormone at the receptor s i t e s . For example, reserpine changes the quantity of a free monoamine at the receptors by impairing i t s s i t e s of storage. Drugs also a l t e r the quantity of a free monoamine by i n h i b i t i n g i t s synthesis, metabolism or physiological release(32). ( 9 ) NERVE IMPULSE ' MAO * u n u :'NE; im\ <'NEN« NE V,NE NE MAO MAO 71E COMT NE CIRCULATION NE R COMT Fi g . 3 Concept of Organized Molecular Unit at the Sympa-the t i c Nerve Ending. (32,34,35) R-Receptor NE-Norepinephrine MAO-Monoamine Oxidase COMT-Catechol-O-Methyltransferase Figure 3 shows a model of sympathetic nerve ending used to i l l u s t r a t e a concept of the storage, metabolism and re-lease of norepinephrine. S o l i d arrow indicates an active transport system or pump which serves to return the norepinephrine to i t s storage compartment within the l i p o i d membrane. The broken arrow represents the leakage of norepinephrine by passive d i f f u s i o n . Monamine Oxidase i n the mitochondria i s responsible f o r deaminating small amount of norepinephrine which constantly dif f u s e s across the membrane. The neurohormone i s stored in.two pools. The granules represent a reserve pool which i s i n (10) equilibrium with the mobile pool. Norepinephrine can:be released by nerve impulses or by the action of drugs such as tyramine. Once released, t h i s neurohormone w i l l react with the receptor and i s metabolized by COMT; or w i l l be returned to i i s s i t e s of storage. Drugs can therefore a l t e r the sympathetic responses by either stimulating or blocking d i r e c t l y at the receptor s i t e s , by releasing a small amount of amine from the mobile pool, by blocking the physiological release of norepinephrine, by impairing the active transport system, by i n h i b i t i n g the synthesis of norepinephrine or by preventing the enzymatic degradation of norepinephrine by i n a c t i v a t i o n of the enzymes monoamine oxidase or catechol-O-methyl transferase. Gamma-Thujaplicin and Adrenergic Responses Although tropolones as a class have been shown to be potent i n h i b i t o r s of the enzyme COMT i n vitro(14-16), the e f f e c t of gamma-thujaplicin on responses to adrenergic stimuli remains to be investigated. Since th i s compound was available i n adequate amounts, i t was decided to study i t s e f f e c t s i n r e l a t i o n to adrenergic responses of i s o l a t e d guinea-pig a t r i a . Experiments with i s o l a t e d organs have cer t a i n advantages over the i n vivo study. They are espe c i a l l y suitable f o r the study of basic drug properties where the interference of homeostatic mechanism, drug transport and drug metabolism are reduced to a minimum. (11) On the other hand there are certain disadvantages. This type of preparation involves the removal of the organ from the intact animal; any slight damage of the tissue during this procedure w i l l affect the experimental results. Secondly, the natural environment of the organ i s replaced by an a r t i f i c i a l medium; the results obtained from this i n vitro study may not necessarily reflect the true picture i n vivo. The purpose of this study i s to determine the extent of the influence of gamma-thujaplicin on the action of sympathomimetic agents on isolated atria, and whether or not this preparation may be a satisfactory one for the study of substances which are inhibitors of COMT. {12) METHODS AND MATERIALS Isolated Guinea-Pig Atria Guinea-pigs weighing 300-600 gm. of either sex were used throughout these experiments. The animals were k i l l e d toy a tolow at the base of the neck, bled and the heart rapidly removed. Atria were freed of ventricular muscle and connective tissue then suspended for recording spontaneous contraction in a bath containing 25 mis. of Kreb's solution maintained at 30-32 deg. C. This solution contained 6.92 gm. of NaCl, 0.35 gm. KC1, 0.28 gm. CaCl 2, 2.1 gm. of NaHC03, 0.16 gm. of KH2P04, o.29 gm. MgSO^HgO, 2.0 gm. glucose per l i t r e . A mixture of 95% 0^ and 5% CO was bubbled through the bathing f l u i d via a glass tube at the bottom of the bath. Movements of the atria were re-corded either on a smoked drum toy a spring lever or toy means of a Grass model 5D polygraph. Rate was determined either by timing with a stop-watch or toy increasing the speed of the polygraph chart paper and counting the tracings. Drugs were added to the toath toy a syringe after the preparation had attained a constant amplitude and rate of contraction. To remove drugs, the preparation was washed toy changing the toathing f l u i d three times. Control responses to the adren-ergic drugs were recorded following which responses were obtained i n the presence of gamma-thujaplicin or other test drugs. In the case of the catechol amines where the response i s short and terminates without washing of the preparation, (13) the extent of the inot r o p i c response was determined by mea-suring with a planimeter, the area of the kymograph tracing which was produced as a r e s u l t of the drug action. For those agents whose action i s long and can only be termi-nated by washing, changes i n inotropic a c t i v i t y brought about by the presence of the test drug were determined by measuring the height of maximum contraction, and comparing with control heights. Reserpinization of guinea pig was accomplished by i n j e c t i n g the animal with a single dose of reserpine 3mg/kg in t r a p e r i t o n e a l l y 24 hours p r i o r to the experiments. Reserpine was dissolved i n 10% a c e t i c acid. Gamma-thujaplicin was used i n the form of i t s sodium s a l t prepared as described by Halliday(6). Hereinafter gamma-thujaplicin sodium w i l l be referred to as T-Na. In addition to T-Na, the following drugs were used i n t h i s study. L-Epinephrine B i t a r t r a t e L-Nbrepinephrine B i t a r t r a t e Isopropyl Norepinephrine (Isoproterenol, Isoprenaline) Hydrochloride Tyramine Monohydrochloride Dopamine (3,4 dihydroxy ethyl amine) DL-Amphetamine Sulphate Cocaine Hydrochloride P y r o g a l l o l Pronethalol Hydrochloride ( A l d e r l i n R ) Histamine acid phosphate Diphenhydramine (Benadryl) hydrochloride Ethylendiamine t e t r a c e t i c acid disodium s a l t (EDTA) (15) RESULTS E f f e c t of Gamma-Thujaplicin Sodium(T-Na) on Responses to Catechol Amines. Guinea-pig a t r i a responded to epinephrine (.08-.2mcg/ml), norepinephrine (.08-s2 meg./ml) and isoprenaline (.002-.016 meg./ml) with an increase i n the rate and am-plitude of contractions. Within these ranges of concen-tra t i o n s , the responses of the a t r i a to the catechol amines were short and terminated without washing the preparation. In the presence of T-Na, both the pos i t i v e i n o t r o p i c and chronotropic e f f e c t s of epinephrine(figure 4), nor-epinephrine and isoproterenol were increased. Potentiation began at concentrations of 0.32 meg./ml - 0.40 meg./ml, depending oft the s e n s i t i v i t y of the preparation. Prolong-ation of the adrenergic response, however, did not begin u n t i l the concentration of T-Na had reached a maximum at T-Na concentration of 1.6 meg./ml - 3.2 meg./ml above which no further potentiation was observed. In most experiments, lower concentrations of T-Na (.08 meg./ml) appeared to have a s l i g h t depressive e f f e c t on the responses induced by epinephrine and norepinephrine(figure 5). T-Na also appeared to potentiate the po s i t i v e i n o t r o p i c e f f e c t of epinephrine more than that of norepinephrine while there i s l i t t l e difference i n the degree of potentiation on the pos i t i v e chronotropic e f f e c t of these amines as indicated by figure 5. (17) •H S3 15 10 300 200 100 V .05 10 15 ,20 25 F i g . 5 Potentiation by Gamma-Thujaplicin of Responses to Epinephrine, Norepinephrine and Isoproterenol * Inotropic responses determined.by planimetric measurement of area of kymograph tracing f o l -lowing addition of drugs. • Epinephrine x Norepinephrine no. of a t r i a - 5 '» - 5 o Isoproterenol it - 5 (18) E f f e c t of T-Na on Responses to Tyramine,Dopamine, Amphet-amine Unlike the catechol amines, which produced rapid hut transitor y e f f e c t s , the responses produced by tyramine, dopamine and amphetamine are slower to develop, very pro-longed and can only he terminated by washing the prepar-ation. Because of the long action of these three amines, i t was not possible to determine whether or not the presence of T-Na prolonged the duration of ac t i o n . But i n a l l cases both the height and rate of controls were increased i n the presence of T-Na. Potentiation of the response to tyramine occurred when the concentration of T-Na i n the bath was 0.4 meg./ml. Potentiation of amphetamine by T-Na was also observed at a concentration of o.4 meg./ml of T-Na pre-added to the organ bath. The action of dopamine as well was potentiated by T-Na i n a s i m i l a r manner (Table I ) . E f f e c t of Cocaine on Responses to Amines Unlike T-Na which potentiated the responses to a l l the amines used i n t h i s study, cocaine i n a concentration of 0.4 meg./ml - 0.8 meg./ml potentiated only the response to epinephrine(figure 6). norepinephrine and dopamine. In the presence of cocaine, the responses of the a t r i a to isoprenaline were unaffected or affected to a very s l i g h t extent, while the responses to tyramine and amphetamine were s l i g h t l y suppressed. (Table II, III) (18a) T B .08 F i g . 6 Effe c t of Cocaine and Gamma-Thujaplicin on Responses to Epinephrine. Isolated Guinea-Pig A t r i a . E - Epinephrine i n cone, of .016 meg./ml x 10 CE - Epinephrine i n the presence of two d i f f e r e n t (C) cones, of Cocaine meg./ml x 10 TE - Epinephrine i n the presence of Gamma-Thu-j a p l i c i n ( T ) .08 meg./ml x 10 W - Washed lime Signal - 0.5 minate TABLE I Ef f e c t of Gamma-Thujaplicin on the Responses of A t r i a to Sympathomimetic Amines Amines Treatment Drug Cone. Potentiation by T-Na Maximum of Maximum Contraction Rate as Multiples of Control no. of a t r i a Tyramine T-Na 10 meg. " 20 meg. 1 1.92 2.38 135 245 275 6 n it Amphetamine T-Na 10 meg. " 20 meg, 1 1.41 1.68 193 224 268 5 II Dopamine T-Na 10 meg, " 20 meg, 1 1.17 1.20 193 226 247 6 •i it M CO TABLE II E f f e c t of Cocaine on Responses to Catechol Amines Amines Treatment Drug Cone. POTENTIATION BY COCAINE no. Inotropic E f f e c t Chronotropic E f f e c t Multiples of Maximum Rate Control * based on area of Response of Contraction of a t r i a Epinephrine Cocaine 10 meg. " 20 meg. 1 2.5 2.6 160 217 256 4 II it Norepinephrine Cocaine 10 meg. " 20 meg. 1 3.2 3.3 155 260 275 4 n Isoprenaline — 1 170 3 Cocaine 10 meg. .96 156 11 it 20 meg. .99 186 * Area of response measured toy A r i s t o Planimeter o TABLE III E f f e c t of Cocaine on Responses to the long-acting Sympathomimetic Amines Amines Treatment Drug Cone. POTENTIATION BY COCAINE no. of a t r i a Inotropic Chronotropic E f f e c t E f f e c t (Multiples Maximum Rate of Control of based on Max. Contrac-Height of tion Response) Tyramine Cocaine 10 meg. " 20 meg. .76 .72 136 102 94 5 it Amphetamine Cocaine 10 meg. " 20 meg. .83 .70 238 210 172 5 it Dopamine Cocaine 10 meg. " 20 meg. 1 1.36 1.55 173 230 263 5 n (22) Although cocaine p o t e n t i a t e d the responses of the a t r i a to e pinephrine and nor e p i n e p h r i n e , i t d i d not p r o l o n g these responses when the c o n c e n t r a t i o n exceeded 0.8 meg./ml(figure 6 ) . While the p o t e n t i a t i n g e f f e c t of T-Na i n c r e a s e s with i n c r e a s i n g c o n c e n t r a t i o n s w i t h i n the l i m i t of 0.8 meg./ml -3.2 meg./ml, the p o t e n t i a t i n g a c t i o n o f cocaine on the responses to epi n e p h r i n e , norepinephrine and dopamine was r e a d i l y changed i n t o a d e p r e s s i o n o f the a t r i a at a c o n c e n t r a t i o n g r e a t e r than 0.8 meg./ml. E f f e c t o f P y r o g a l l o l on Responses to Amines In the presence of p y r o g a l l o l , the responses to the s i x amines t e s t e d were i n c r e a s e d (Table IV, Table V ) . P o t e n t i a t i o n began a t a c o n c e n t r a t i o n of 0.4 meg./ml, but p r o l o n g a t i o n o f the responses to the c a t e c h o l amines d i d not occur u n t i l the c o n c e n t r a t i o n o f p y r o g a l l o l had reached 1.6 meg./ml(figure 7 ) . P o t e n t i a t i o n o f these amines by p y r o g a l l o l and T-Na appeared to f o l l o w a s i m i l a r dose-response p a t t e r n although T-Na was more potent. The p r o l o n g a t i o n o f the responses to the catecholamines equal to t h a t produced by T-Na a t a c o n c e n t r a t i o n of 0.8 meg./ml was not produced by p y r o g a l l o l u n t i l a concen-t r a t i o n of 1.6 meg./ml was r e a c h e d ( f i g u r e 4 , f i g u r e 7 ) . At h i g h e r c o n c e n t r a t i o n s , however, p o t e n t i a t i o n o f these amines by T-Na and p y r o g a l l o l appeared to have a p a r a l -l e l e f f e c t ( f i g u r e 8 ) . F i g . 7 Potentiation of Responses to Epinephrine by Pyr o g a l l o l . Isolated Guinea-Pig A t r i a E - Epinephrine i n cone, of .016 x 10 meg./ml PE - Epinephrine i n the presence of various cones, of Pyrogallol(P) x 10 meg./ml W - Washed to CO (24) -Sf —N CD CD CQ CO c c o o &i Oh in in CD cu o .-H •rf O a, ^ o -H> Sh C -P o o o c O c •rl 0) CD CD rH » Q, « -H o 3 S S O CD *> c8 OS c •H C O CD H 05 -rJ CQ O CD C8 S. t, c c H O o •H CO 3 0 0 2 U 0 1 0 0 10 1 5 - 3 C o n e , o f D r u g s i n x 1 0 n i i l l i m o l e s . F i g . 8 C o m p a r i s o n o f E f f e c t s o f T-Na a n d P y r o g a l l o l o n R e s p o n s e s o f I s o l a t e d G u i n e a - P i g A t r i a t o N o r e p i n -e p h r i n e . I n o t r o p i c R e s p o n s e s d e t e r m i n e d by p l a n i m e t r i c mea-s u r e m e n t o f k y m o g r a p h t r a c i n g f o l l o w i n g a d d i t i o n o f d r u g s . • - T - N a ( g a m m a - t h u j a p l i c i n s o d i u m ) n o . o f a t r i a - 6 X - P y r o g a l l o l " - 6 TABLE IV Effec t of Pyrogallol on Responses to Catechol Amines Amines Treatment Drug Cone. POTENTIATION BY PYROGALLOL no. of a t r i a Inotropic E f f e c t (Multiples of Control based on * area of Response Chronotropic E f f e c t Maximum Rate of Contraction Epinephrine Pyrogallol 10 meg. 11 20 meg. 1 1.63 3.04 143 200 216 6 it it Norepinephrine Pyrogallol 10 meg. " 20 meg. 1 2.01 2.39 155 182 205 4 •t Isoprenaline — 1 183 4 Pyrogallol 10 meg. 1.38 205 " " 20 meg. 2.33 230 " to * area of response measured by an Aristo Planimeter TABLE V Ef f e c t of Pyrogallol on Responses to the long-acting Sympathomimetic Amines Amines Treatment Drug Cone. POTENTIATION BY PYROGALLOL Inotropic Chronotropic E f f e c t E f f e c t (Multiples of Maximum Rate Control based on of Maximum Height Contraction of Contraction) no of a t r i a Tyramine Pyrogallol 10 meg. " 20 meg. 1 1.37 1.70 140 176 223 3 II it Amphetamine Pyrogallol 10 meg. n 20 meg. 1 1.45 1.75 150 220 255 2 II Dopamine — 1 130 3 ^ Pyrogallol 10 meg. 1.62 160 11 g II 20 meg. 1.90 190 (28) E f f e c t of Ethylenediaminetetraacetic Acid (EDTA) on Responses to Amines EDTA i s a chelating agent possessing a f f i n i t y for a great number of metal ions. Since i t has been postulated by Belleau(15) that chelation i s probably the primary mechanism of substrate binding to COMT, chelating agents therefore may af f e c t the rate of COMT catalysed degradation of catechol amines. EDTA has been used to retard oxidation of norepinephrine i n the bathing f l u i d of i s o l a t e d a t r i a ( 3 7 ) . Since T-Na also i s a chelating agent and has the a b i l i t y of forming stable chelates with divalent metal ions(15), there i s a p o s s i b i l i t y that i t s e f f e c t s on responses to adrenergic agents may be through a non-specific chelating mechanism rather than by i n h i b i t i o n of COMT. The response of the a t r i a to epinephrine and norepi-nephrine were increased i n the presence of EDTA. Potentia-t i o n of these responses begin at a concentration of .64 meg./ml and prolongation was also observed at higher concentrations(figure 9). Determination of the dose-response rel a t i o n s h i p indicated that EDTA was approximately one-half as potent as T-Na(figure 10). E f f e c t of Excess Magnesium Ions on T-Na Potentiation A high concentration of magnesium ions(1.2 rag./ml) i n the form of magnesium sulphate when added to the organ bath had neither e f f e c t on the normal c o n t r a c t i l i t y of the a t r i a nor on the responses stimulated by norepinephrine. F i g . 9 Potentiation of Responses to Norepinephrine by Ethylenediaminetetraacetic Acid(EDTA) NE - Norepinephrine i n cone, of .016 x 1U meg./ml E - EDTA i n various cones, x 10 meg./ml W - Washed lime Signal - 0.5 minute te oo S3 (28b) 0) CD in in fl c o o a, a, m m CD CD O rH •w o a, t, o -H> >> o s M c •rl CD CO a) Sh u c c o c_> CM O 01 CD rH a, •H 5M O CD C8 C •H CD m as CD o C u rH fl O S O s •H £0 o •H O 300 200 100 Cone, of Drugs i n x 10 millimoles F i g . 10 Comparison of E f f e c t s of Gamma-Thujaplicin(l-Na) and Ethylenediaminetetraacetic acid(EDTA) on Responses of I s o l a t e d Guinea-Pig A t r i a to Norepinephrine * Inotropic Responses determined by planimetric measurement of area of kymograph tracing following addition of drugs. • - I-Na No. of a t r i a - 6 o - ED1A " - 4 (29) In the presence of excess magnesium ions, the potentiating e f f e c t of T-Na on responses to norepinephrine was unaltered. Experiments on A t r i a from Reserpinized Guinea-pigs. The responses to tyramine, amphetamine and even i n some cases to dopamine were either greatly reduced or completely abolished i n a t r i a from reserpinized guinea-pigs. The presence of T-Na did not restore any of these responses. The reserpinized a t r i a , however, responded to epinephrine, norepinephrine and isoprenaline. Most reserpinized a t r i a were more sensitive to these catechol amines than were a t r i a from non-reserpinized animals. A very small concen-t r a t i o n of epinephrine(.008 meg./ml) was s u f f i c i e n t to produce a response corresponding to that produced by ten or twenty times t h i s concentration i n normal preparation. The e f f e c t of pyr o g a l l o l and T-Na on the responses to the amines i n these preparations were unaltered from those i n normal a t r i a ( f i g u r e 11). Potentiation s t i l l began at a concentration of .4 meg./ml f o r pyrogallol,;,and T-Na while prolongation s t i l l occurred at a concentration of .8 meg./ml f o r T-Na and 1.60 meg./ml f o r pyr o g a l l o l as i n the case of normal preparations. Repletion of Norepinephrine Stores Trendelenburg and Crout(37) found that restoration of tyramine responses can be accomplished by exposing the reserpinized a t r i a to 15 meg./ml EDTA and 3 meg./ml of norepinephrine f o r ten minutes. In the present study, i t was found that t h i s concentration of EDTA had a depressant —. ~ — • A . Showing absence of response to tyramine. B. E f f e c t of Gamma-Thujaplicin and Py r o g a l l o l on Responses to Epinepnrine. E - Epinephrine i n cone, of .008 x 10 meg./ml TY- Tyramine W - Washed /-TE- Epinephrine i n the presence of d i f f e r e n t cone, of T-Na(T). g PE- •» " " • " " Pyrogallol(P) . » (30) action on the a t r i a l muscle. However, when a lower con-centration of EDTA (7 meg./ml) was used, repletion occurred without undue depression of the atria. It was further found that when T-Na was used in place of EDTA, a more rapid and more complete restoration of tyramine response was observed. The time interval between incubation with norepinephrine and testing with tyramine was reduced by one-third when T-Na was used. The response to tyramine following incubation with norepinephrine (3 meg./ml) and T-Na (4 meg./ml) was about 65% greater than in atria i n -cubated with norepinephrine and EDTA (Table VI). TABLE VI Effect of Ethylenediaminetetraacetic(EDTA) and Gamma-Thujaplicin(T-Na) on Restoration of Tyramine Responses by Incubation with Norepinephrine Reserpinized Guinea-Pig Atria I n i t i a l Res- Treatment Drug ponse. Max. Cone. Height of Contraction 2.4 cm. 1.6 cm. EDTA 7 meg./ml NE 3 meg./ml Time of Incubation with NE 10 mins. Time required Final Response % before f i n a l Max. Height of increase response was Contraction observed 45 mins. 4.1 cm. 2.6 cm. 70.8 % 62,5 % 1,7 cm, T-Na 4 meg./ml NE 3 meg./ml 3.7 cm. 118.0 % .7 cm, 1,7 cm. 143.0 % EDTA - Ethylenediaminetetraacetic Acid NE - Norepinephrine T-Na - Gamma-Thujaplicin Sodium Tyramine cone, used - 5 meg./ml U 2 ; EFFECTS OF DRUGS ON RESPONSES TO HISTAMINE Action of Histamine on the Isolated A t r i a It i s well known that histamine stimulates i s o l a t e d a t r i a of the hearts of various species but i t s mode of ac-t i o n has not yet been c l a r i f i e d . As early as 1910, Dale and Laidlaw(38) showed that histamine increased the c o n t r a c t i l i t y of the i s o l a t e d heart of the cat and rab b i t . Later s i m i l a r e f f e c t was shown i n the heart of the guinea-pig, frog and rat(39). Trendelburg(42) summarized the work of Went who i n 1952 and 1954 reported that histamine released an e p i -nephrine-like substance from the heart of the r a t , rabbit, the cat and guinea-pig, which they considered to be sym-pathin. Since nicotine i s a substance which has t h i s l a t e r mode of action, i t i s believed that histamine w i l l a l -so stimulate the structures i n the heart which respond to n i c o t i n e . Burn and Rand(40) however, observed that a t r i a i s o l a t e d from rabbits pretreated with reserpine responded to histamine although they f a i l e d to respond to n i c o t i n e . This observation suggests that at least i n rabbits, the stimu-lant action of histamine i s not mediated through the re-lease of norepinephrine. In the present study, attempts have been made to c l a r i f y the mode of action of histamine. Histamine i n a concentration of 0.2 meg./ml when added to the organ bath increased both the rate and amplitude of contraction of the i s o l a t e d a t r i a . The response that i s produced i s very prolonged and resembles that to the i n -d i r e c t acting amines such as tyramine and amphetamine. (33) Gamma-Thujaplicin Sodium In the presence of T-Na at a concentration of p.4mcg./ml, the response to histamine was greater than control response (figure 12). In most cases, the potentiating e f f e c t was such that at least 20 - 30$ increase i n the amplitude of contraction over the preceeding control response(Table VII). Since the response to histamine i s very prolonged, the e f f e c t of T-Na on the duration of the response was un-determined. Modification of the histamine induced response by T-Na tends to resemble the T-Na potentiation of the i n d i r e c t l y acting amines such as tyramine and amphetamine. Cocaine Although cocaine potentiates the responses of nor-epinephrine and epinephrine, i t did not a l t e r the res-ponse to histamine except i n concentrations which depressed the normal a c t i v i t y of the a t r i a l muscle. Pyr o g a l l o l P y r o g a l l o l which has been shown to potentiate the responses of the a t r i a to a l l the amines previously studied, possessed very l i t t l e or no enhancing e f f e c t on the res-ponse of the a t r i a to histamine. In some cases, pyrogallol even caused a s l i g h t depression on the histamine induced response. Ethylenediaminetetraacetic Acid (EDTA) o EDTA was found to have a s l i g h t enhancing e f f e c t on (34) H W T H V 0 O 4 0^8 H D ? V ,30 ,160 F i g . 12 E f f e c t of Gamma-Thujaplicin, Diphenhydramine and P r o n e t h a l o l on Responses to Histamine. H - Histamine i n cone, of .04 x 10 meg./ml T - Gamma-Thujaplicin i n cone, of .08 x 10 meg./ml D - Diphenhydramine(Benadryl) i n cone, of .30 x 10 meg./ml P - P r o n e t n a l o l i n cone, of .16 x 10 meg./ml W - Washed (35) the histamine induced contraction of the atria(Table VII). The degree of potentiation produced by EDTA s t i l l i s much less than that produced by T-Na. Diphenhydramine(Benadryl) Diphenhydramine i t s e l f did not have any e f f e c t on the normal c o n t r a c t i l i t y of the i s o l a t e d a t r i a . When this drug was added to the bathing f l u i d when the a t r i a was res-ponding to histamine, the stimulating e f f e c t of histamine was not affected(figure 12). When histamine was added to the bathing f l u i d i n the presence of diphenhydramine even when the l a t t e r concentration was as high as 3 meg./ml, the response to histamine was unaltered(Table VII). Pronethalol(Nethalide) Pronethalol i s a drug which blocks adrenergic beta receptors(43, 44). Pronethalol i n a concentration of 1.2 meg./ml, which i s s u f f i c i e n t to supress the tyramine and norepinephrine induced responses, only reduced the histamine response by 14$(figure 13, Table VII). As the concentration of pronethalol was increased, there i s however, a progressive greater decrease of the histamine induced response as i n -dicated by figure 13. Unlike the ef f e c t produced on the responses to tyramine and norepinephrine, the response to histamine was never markedly suppressed even at a con-centration of pronethalol greater than 1 x 10"^Mole(3.2 meg. 1.2 x 10"^M.). According to Koch-Weser, Pronethalol TABLE VII Modifications of the Responses of the Isolated A t r i a to Histamine by Various Drugs Drugs and Cone. Inotropic E f f e c t as % change from the Control no. of a t r i a Response (based on the area of response *) T-Na .080 x l©-mcg./ml + 30 i 7 P y r o g a l l o l .160 II 0 6 EDTA .20 it +11.65% 5 Diphenhydramine .30 it 0 3 Cocaine .080 n 0 i 3 Pronethalol .080 II -2.3 3 .120 w -14 i II .160 II -18 i n .200 II -37 i II .240 II -39 it T-Na .080 II + 1 $ it (Resperpinized Prep.) * Area of response was measured for a 10 minute i n t e r v a l by an A r i s t o Planimeter EDTA - Ethylenediaminetetraacetic Acid T-Na - Gamma-thujaplicin Sodium (37) 0 .10 .20 .30 .40 Cone, of Pronethalol i n x 10 meg./ml Fi g . 13 E f f e c t of Pronethalol on the Positive Inotropic Responses of Histamine, lyramine and Norepinephrine X - Histamine no. of a t r i a - 5 lyramine " - 3 o- Norepinephrine !'. - 2 Inotropic responses determined by planinietric measurement of area of kymograph tracing follow-ing addition of drugs. possesses c e r t a i n d i r e c t depressive e f f e c t on the cardiac — 5 muscle at a concentration exceeding 1 x 10 Mole. The pro-gressive depression of the histamine response produced by higher concentrations of pronethalol could be the r e s u l t of a d i r e c t depressive e f f e c t . On the other hand, the gradual decline of the histamine induced response due to the addition of a lower concentration of pronethalol might be due to some beta receptor blockade(figure 12). Reserpinized A t r i a Reserpinization of the guinea-pig p r i o r to the experi-ments resulted i n a b o l i t i o n or a marked reduction of the responses to tyramine and amphetamine, but the response to histamine was not affected. Although there i s no d i f -ference between the histamine response recorded from normal preparation and that obtained from reserpinized a t r i a , the presence of T-Na had l i t t l e or no e f f e c t on responses to histamine i n reserpinized preparations(Table VII). In normal preparations, T-Na caused at least a 20% increase of the positive i n o t r o p i c e f f e c t of histamine. ( 3 9 ) DISCUSSION Many studies have shown that tropolones are e f f e c t i v e i n h i b i t o r s of the enzyme COMT(14-16,17,18,20). In the present study, i t was observed that gamma-thujaplicin (T-Na) i t s e l f possessed a slow but prolonged stimulating e f f e c t on the a t r i a . This stimulating e f f e c t may be ac-counted f o r by the protection of the endogenous norepineph-rine which continuously d i f f u s e s across the c e l l membrane, as a r e s u l t of enzyme i n h i b i t i n g a c t i v i t y of T-Na. It may, therefore* be reasonable to assume that the demonstrated a b i l i t y of T-Na to increase and to prolong the responses of the a t r i a to the d i r e c t acting catechol amines, and to po-tentiate the responses of a l l other sympathomimetic amines studied i s due to COMT i n h i b i t i o n . I f the potentiation by T-Na of responses to exogenous catechol amines i s the re-s u l t of protection of the amines due to the i n h i b i t i o n of the enzyme responsible for t h e i r rapid degradation; the ef f e c t of T-Na on the responses to exogenous catechol amines should not be affected by re s e r p i n i z a t i o n . From the r e s u l t s obtained, i t i s quite evident that the e f f e c t of T-Na on the di r e c t acting catechol amines was unaltered i n reserpinized a t r i a . The fa c t that T-Na has been proved to be an e f f e c t i v e agent i n protecting the rapid degrada-ti o n of norephinepurine during the r e f i l l i n g of norepi-nephrine stores also favoured the above assumption. This assumtion appeared to be confirmed when pyr o g a l l o l , another known COMT i n h i b i t o r was found to potentiate the responses (40) to a l l of the amines studied with a dose-response pattern s i m i l a r to that of T-Na although T-Na was more potent (figure7,8). While pyrogallol has been considered to act competitively due to i t s s t r u c t u r a l resemblance to catechol amines(14), i t also serves as a substrate f o r COMT which may account f o r i t s lower potency as compared with T-Na. With regard to the mechanism of COMT i n h i b i t i o n , B e l -leau and Burba(15) excluded the p o s s i b i l i t y that tropolones could i n h i b i t COMT by p a r t i a l l y depriving the enzyme of i t s magnesium ions. Their conclusion was based on the f a c t that a f i v e - f o l d increase i n the magnesium concentration had no ef f e c t on the rate of i n h i b i t i o n of COMT produced by 4-methyltropolones. The fac t that excess magnesium ions do not reverse the i n h i b i t i o n suggested that tropolones may form a complex with the enzyme active s i t e s normally occupied by the substrate. Belleau(15) had also presented evidence that the tropolone and the catechol rings are biochemically i s o s t e r i c and they were convinced that tropolones could also display a s p e c i f i c a f f i n i t y f o r the adrenergic receptors. In the present study, a high concen-t r a t i o n of magnesium ions i n the bath had l i t t l e or no ef f e c t on the normal c o n t r a c t i l i t y of the a t r i a or on the potentiation produced by T-Na of the responses to the various amines. This observation appeared to agree with Belleau's assumption that i t i s not l i k e l y that tropolones act by p a r t i a l l y depriving COMT of i t s magnesium ions. Since the tropolones are powerful chelating agents, i t i s possible that the potentiation of reponses to (41) adrenergic agents could be due to a non-specific chelating mechanism such as observed by Shida(45) i n h i s studies of the potentiating e f f e c t s of thyroxine on responses of aor-t i c s t r i p s to epinephrine. According to Shida's experiment, the presence of copper as a contaminant i n the bathing f l u i d often had an i n h i b i t o r y e f f e c t on the c o n t r a c t i l i t y of the muscle. The removal of t h i s metal by chelating agents often improved the condition of the muscle thus r e s u l t i n g i n a potentiation of epinephrine responses by thyroxine. In the present study, when EDTA was added to the bathing f l u i d , the response to epinephrine was potentiated. When T-Na was added i n addition to EDTA, further potentiation was observed. This would suggest that the potentiating e f f e c t of T-Na cannot be explained by the removal of copper ions or other heavy metals by chelation, since these would have been removed by EDTA. A s i m i l a r conclusion with regard to 4-methyltropolone and beta-thujaplicin was made by Murnaghan(21). The study of the e f f e c t of histamine on the i s o l a t e d a t r i a gave r e s u l t s which suggested that the potentiation produced by T-Na of the adrenergic responses i s due to a non-specific mechanism. Prom the r e s u l t s obtained i n t h i s study, i t i s quite evident that histamine did not act through a tyramine-like mechanism. While pronethalol greatly supressed and py r o g a l l o l potentiated the res-ponses to tyramine, these two substances had l i t t l e or no e f f e c t on the histamine response(figure 13, Table V, Table VII). Reserpinization did not a l t e r the response of the a t r i a to histamine, although the responses to tyramine were suppressed by th i s procedure. These r e s u l t s suggest that histamine stimulation of the a t r i a does not involve adrenergic mechanism. This conclusion has also been reached by others(39-42). However, the response to histamine was greatly increased i n the presence of a low concentration of T-Na(Table VII). This potentiation of the histamine res-ponse by T-Na could best be explained by postulating a non-specific s e n s i t i z i n g e f f e c t of T-Na. When EDTA, a chelating agent, was used i n place of T-Na, i t was also found to potentiate the responses of the a t r i a to the various adrenergic agents(figure 0 ) . Poten-t i a t i o n produced by EDTA appeared to follow a very si m i l a r dose-response pattern to that of T-Na(figure 10). The resemblance of the e f f e c t s of T-Na to those of EDTA on both histamine and adrenergic responses casts doubt on the assumption that COMT i n h i b i t i o n played a predominant role i n the T-Na induced potentiation of sympathomimetic res-ponses. It appears then that the potentiating e f f e c t shown here by T-Na to the various drugs studied may be the r e s u l t of a non-specific s e n s i t i z a t i o n of the a t r i a . It i s of i n t e r e s t that Murnaghan(21) had reached a si m i l a r con-clusion i n his studies of 4-methyltropolone and beta-t h u j a p l i c i n on i s o l a t e d tissues. He concluded that potentiation of epinephrine stimulated responses to be (43) unspecific as i t also occurred with potassium and barium induced responses on the rabbit a o r t i c s t r i p and with cabachol, histamine and barium stimulated contractions on the guinea-pig ileum. From the study of Murninghan and McConail(46) related substituted phenols had been shown to sens i t i z e the frog's s k e l e t a l muscle to potassium; Murnaghan was convinced that the potentiation of epineph-rine induced responses on is o l a t e d tissues i s due to a non-specific s e n s i t i z a t i o n e f f e c t produced by the tropo-lones . On the other hand, i n normal a t r i a T-Na alone produced an increase i n a c t i v i t y which may have been due to protection of passively release norepinephrine. The enhanced action of histamine i n the presence of T-Na i n normal but not i n reserpinized a t r i a was l i k e l y the resul t of the additive e f f e c t s of the two drugs. However the fac t that these effects occurred only i n a t r i a which had not been depleted of catecholamines suggests that COMT i n h i b i t i o n may be i n -volved to some extent i n the action of T-Na. It i s concluded that the i s o l a t e d guinea-pig a t r i a i s not a sa t i s f a c t o r y preparation f o r the testing of drugs pharmacologically f o r COMT i n h i b i t o r y a c t i v i t i e s . (44) SUMMARY The action of gamma-thujaplicin(T-Na) on the res-ponses of the i s o l a t e d a t r i a to the various sympatho-mimetic amines has been studied and compared with the modifications of the adrenergic responses produced by co-caine, p y r o g a l l o l , ethylenediametetraacetic acid(EDTA). 1. Gamma-thujaplicin i t s e l f possessed a slow but very prolonged stimulating e f f e c t on the i s o l a t e d a t r i a . 2. In the presence of T-Na, the responses of the a t r i a to the sympathomimetic amines studied were increased. In the case of the short acting catechol amines, the duration of responses was prolonged. 3. Cocaine potentiated the responses to epinephrine, norepinephrine, and dopamine while p y r o g a l l o l and EDTA potentiated the responses to a l l of the amines studied. The potentiating e f f e c t of EDTA and pyrogallol appeared to resemble that produced by T-Na. 4. The potentiating e f f e c t of T-Na on the catechol amines was not affected by reserpinization. 5. T-Na was also found to be e f f e c t i v e i n delaying the oxidation of norepinephrine during the repletion of norepinephrine stores i n reserpinized a t r i a . 6. Histamine was found to increase the normal con-t r a c t i l i t y of the a t r i a . The response to histamine was not affected by the presence of cocaine, pyro-(45) g a l l o l , EDTA, diphenhydramine and pronethalol, but increased i n the presence of T-Na. 7. Reserpinization did not a l t e r the response to h i s r tamine, but the potentiating e f f e c t normally produced by T-Na was absent i n reserpinized a t r i a . 8. I t has been concluded that potentiation of sym-pathomimetic amines on i s o l a t e d guinea-pig a t r i a cannot be completely attributed to COMT i n h i b i t i o n . (46) BIBLIOGRAPHY 1. Cook, J.W., Raphael, R.A., Scott, A.I., Tropolones: The Synthesis of alpha-, beta-?, and gamma-Thujaplicins. J . Chem. S o c , 695(1951). 2. Gardner, J.A.F., Barton, G.M., Maclean H., Occurrence of 2,7 Dihydroxy-4-isopropyl-2, 4, 6 cycloheptatrian-1-one (7-hydroxy-4-isopropyltrbpolone) i n Western Red Cedar. Can. J . Chem., 55:1059(1957). 3. Pauson, L.P., Tropones and Tropolones. Chem. Rev., 55: 10(1955) 4. 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