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Species variations in cardiac effects of histamine Laher, Ismail 1979

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SPECIES VARIATIONS IN CARDIAC EFFECTS OF HISTAMINE by ISMAIL LAHER B.Sc, University of London, England, 197 8 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES D i v i s i o n of Pharmacology and Toxicology of the Faculty of Pharmaceutical Sciences ^ We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA, 197 9 Q I. Laher, 1979 In p r e s e n t i n g t h i s t h e s i s in 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 at the 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 that the 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 s t u d y . I f u r t h e r agree 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 the Head o f my 'Department o r by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d that 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 . Depa rtment The U n i v e r s i t y o f B r i t i s h Co lumbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 Date ABSTRACT The cardiac e f f e c t s of histamine and i t s analogs were studied i n tissues i s o l a t e d from k i t t e n s , guinea-pigs and rats. Rate changes were recorded in r i g h t a t r i a , while inotropic responses were recorded from e l e c t r i c a l l y paced (1Hz) l e f t a t r i a , r i g h t v e n t r i c l e s t r i p s and r i g h t p a p i l l a r y muscles. In the k i t t e n i s o l a t e d r i g h t atrium, both histamine and 4-methylhista-mine, a s p e c i f i c histamine l^-receptor agonist, caused dose-dependent changes in rate. These responses were not affected by a histamine H^-receptor antagonist (promethazine). Both cimetidine (a histamine l ^ -receptor antagonist) and propranolol (ayS-adrenoceptor antagonist) caused s i g n i f i c a n t reductions in the chronotropic responses produced by histamine and 4-methylhistamine. In the e l e c t r i c a l l y paced preparations from k i t t e n hearts, the inotropic responses of both histamine and 4-methylhistamine were observed at only high doses of agonist. The inotropic responses to these agonists were not affected by either promethazine or cimetidine. Propranolol caused marked i n h i b i t ion.;)of the inotropic response! - produced by histamine and 4-methyl-histamine i n the k i t t e n l e f t atrium, r i g h t v e n t r i c l e s t r i p and r i g h t p a p i l l a r y muscle. Based on these r e s u l t s , i t was concluded that the cardiac e f f e c t s of histamine in the k i t t e n were predominantly due to an i n d i r e c t /^-adrenoceptor stimulation. In addition, i t was demonstrated that part of the chronotropic e f f e c t of histamine and 4-methylhistamine was due to histamine IL^-receptor stimulation. i i i The use of 2 - (2-pyridyl) ethylamine (PEA) i n the study of cardiac histamine H-^-receptors was next investigated using guinea-pig r i g h t a t r i a , l e f t a t r i a and r i g h t v e n t r i c l e s t r i p s . The chronotropic response produced by PEA was not due to i n t e r a c t i o n with either histamine H^- or ^ - r e c e p t o r s , as indicated by experiments with the s p e c i f i c histamine receptor antagonists. In a t r i a depleted of endogenous catecholamines (using reserpine-pretreated guinea-pigs), the chronotropic response of PEA was abolished. In guinea-pig l e f t a t r i a and r i g h t v e n t r i c l e s t r i p s , inotropic re-sponses of low doses of PEA were antagonised by promethazine. However, at higher doses of PEA, promethazine was unable to antagonise the increases in force of contraction i n both the tissues used. The inotropic response due to high doses of PEA were reduced by propranolol or following reserpine pretreatment. The absence of histamine H^-receptors i n the guinea-pig r i g h t atrium was confirmed. The r e s u l t s obtained also indicate that PEA has a dual action, by causing an i n i t i a l d i r e c t histamine H^-receptor stimulation at low doses, while at higher doses causing an indirect^-adrenoceptor stimula-tion . F i n a l l y , the effect;-, of histamine i n i s o l a t e d rat a t r i a ( r i g h t and l e f t ) was studied. Large doses of histamine produced changes in rate and force that were unaffected by promethazine or cimetidine. Following pre-treatment of rats with reserpine (or by using propranolol i n untreated tissues) the inotropic response due to histamine was abolished. At the same time, a negative chronotropic response (reversed by addition of atropine) revealed a c h o l i n e r g i c component i n addi t i o n to an i n d i r e c t adrenergic component in the cardiac e f f e c t s of histamine i n the r a t . i v In summary, the cardiac e f f e c t s of histamine and i t s analogs show tremendous species v a r i a b i l i t y . The r e s u l t s indicate that some responses were due to d i r e c t histamine H^- or HL^-receptor stimulation. In addition, i t was also demonstrated that the cardiac responses of histamine were not always due to such receptor interactions. V •TABLE OF CONTENTS Page ABSTRACT 1 1 LIST OF TABLES v i LIST OF FIGURES v i i i ACKNOWLEDGMENT x INTRODUCTION 1 PURPOSE OF THE INVESTIGATION 10 METHODS AND MATERIALS 11 RESULTS 13 DISCUSSION 89 CONCLUSION AND SUMMARY 103 BIBLIOGRAPHY 104 v i LIST OF TABLES Table Page 1. E f f e c t of histamine on rate of i s o l a t e d k i t t e n r i g h t a t r i a 24 2. E f f e c t of histamine on c o n t r a c t i l e force of i s o l a t e d k i t t e n l e f t a t r i a 26 3. E f f e c t of histamine on c o n t r a c t i l e force of i s o l a t e d k i t t e n r i g h t v e n t r i c l e s t r i p s 28 4. E f f e c t of histamine on c o n t r a c t i l e force of i s o l a t e d k i t t e n r i g h t p a p i l l a r y muscles 30 5. E f f e c t of 4-methylhistamine on rate of i s o l a t e d k i t t e n r i g h t a t r i a 32 6. E f f e c t of 4-methylhistamine on c o n t r a c t i l e force of i s o l a t e d k i t t e n l e f t a t r i a 34 7. E f f e c t of 4-methylhistamine on c o n t r a c t i l e force of is o l a t e d k i t t e n r i g h t v e n t r i c l e s t r i p s 36 8. E f f e c t of 4-methylhistamine on c o n t r a c t i l e force of is o l a t e d k i t t e n r i g h t p a p i l l a r y muscles 38 9. E f f e c t of PEA on rate of i s o l a t e d r i g h t a t r i a from untreated and reserpine-pretreated guinea-pigs . . . 40 v i i Table Page 10. E f f e c t of PEA on c o n t r a c t i l e force of l e f t a t r i a i s o l a t e d from untreated guinea-pigs 42 11. E f f e c t of PEA on c o n t r a c t i l e force of l e f t a t r i a i s o l a t e d from reserpine pretreated guinea-pigs . . . 44 12. E f f e c t of PEA on c o n t r a c t i l e force of r i g h t v e n t r i c l e s t r i p s i s o l a t e d from untreated and reserpine pretreated guinea pigs 46 13. E f f e c t of 4-methylhistamine on rate of r i g h t a t r i a i s o l a t e d from untreated guinea-pigs 48 14. E f f e c t of histamine on rate of r i g h t a t r i a i s o l a t e d from untreated and reserpine pretreated rats 50 15. E f f e c t of histamine on c o n t r a c t i l e force of l e f t a t r i a i s o l a t e d from control and reserpine pretreated rats 52 v i i i LIST OF FIGURES Figure Page 1. Dose-response curve for changes in rate of k i t t e n r i g h t a t r i a produced by histamine 54 2. Histogram showing rate changes produced by 10 M histamine i n k i t t e n r i g h t a t r i a 56 3. Histogram showing maximal changes in force of contraction -3 produced by 10 M in k i t t e n l e f t a t r i a 58 4. Histogram showing maximal changes i n force of contraction _3 produced by 10 M i n k i t t e n r i g h t v e n t r i c l e s t r i p s . . 60 5. Histogram showing maximal changes i n force of contraction -3 produced by 10 M in k i t t e n r i g h t p a p i l l a r y muscles . . 62 6. Dose-response curve for changes in rate produced by 4-methylhistamine in k i t t e n r i g h t a t r i a 64 -3 7. Histograms showing maximal changes produced by 10 M 4-methylhistamine i n i s o l a t e d k i t t e n r i g h t a t r i a . . . 66 8. Histograms showing changes i n c o n t r a c t i l e force produced by 4-methylhistamine i n i s o l a t e d k i t t e n l e f t a t r i a . . 68 9. Histograms showing changes i n c o n t r a c t i l e force produced by 4-methylhistamine i n i s o l a t e d k i t t e n r i g h t v e n t r i c l e s t r i p s 70 iX) .Figure Page 10. Histograms showing changes i n c o n t r a c t i l e force produced by 4-methylhistamine i n i s o l a t e d k i t t e n r i g h t p a p i l l a r y muscles 72 11. Changes i n rate of i s o l a t e d guinea-pig r i g h t a t r i a produced by PEA 74 12. E f f e c t of PEA on rate of r i g h t a t r i a i s o l a t e d from control and reserpine pretreated guinea-pigs . . . . 76 13. Increases i n tension produced by PEA i n i s o l a t e d guinea-pig l e f t a t r i a 78 14. Changes i n force of contraction produced by PEA i n paced l e f t a t r i a i s o l a t e d from untreated and reserpine pre-treated guinea-pigs 80 15. Changes in force of contraction produced by PEA i n paced r i g h t v e n t r i c l e s t r i p s i s o l a t e d from untreated and reserpine pretreated guinea-pigs 82 16. Curve showing increases i n rate of guinea-pig r i g h t a t r i a produced by 4-methylhistamine 84 17. E f f e c t of histamine on rate of r i g h t a t r i a i s o l a t e d from untreated and reserpine pretreated rats 86 18. E f f e c t of histamine on force of contraction of paced l e f t a t r i a i s o l a t e d from untreated and reserpine pretreated rats 88 ACKNOWLEDGMENT I am indebted to Dr. J.H. McNeill for his valuable guidance and encouragement throughout t h i s study. I would l i k e to thank the committee members for t h e i r assistance, and I would also l i k e to acknowledge the f i n a n c i a l support of the Canadian Heart Foundation. 1 INTRODUCTION The p h y s i o l o g i c a l importance of histamine i n mammalian cardiovascular systems is unclear. It i s known that histamine is stored i n large amounts in cardiac t i s s u e (Vergman and Rocha e S i l v a , 1966). High le v e l s are to be found p a r t i c u l a r l y in the regions of the r i g h t atrium of the rat heart, which comprise the s i n o a t r i a l and a t r i o v e n t r i c u l a r nodes (Harvey, 1978). Even though a r o l e for cardiac histamine is not known, histamine could be of pathological importance, since i t is known that mobilisation of h i s t a -mine from storage s i t e s can be induced by s t i m u l i such as drugs (Douglas, 1975), immunological mechanisms (Mclntire, 1973) and numerous s u r g i c a l and diagnostic procedures (Lorenz, 1975). In addition, i t is possible that the cardiac effects of histamine may be potentiated by some drugs, eg. d i g i t a l i s (Levi and Capurro, 1975) and by disease states such as hyperthyroidism (McNeill and Schulze, 1972; Lee and L e v i , 1977). Histamine was synthesised over 70 years ago as an exercise in chem-i s t r y ; the r e l a t i v e l y simple organic molecule, y6-imidazolyethylamine, was prepared in the laboratory by Windaus and Vogt (1907) before i t s discovery i n nature. In the pioneering investigations published in the period 1910 to 1920, Dale and his coworkers Richards and Laidlaw revealed the diverse e f f e c t s of histamine i n various species (reviewed by Dale, 1953). The study of the actions of histamine at the receptor l e v e l has continued u t i l i s i n g mainly s p e c i f i c histamine receptor agonists and antagonists. Bovet and Straub (1937) modified an e x i s t i n g compound to provide the f i r s t synthetic histamine antagonist. I t was apparent to Ash and S c h i l d (1966) that there existed at least two histamine receptor types. They 2 designated as H^-receptors those s i t e s involved in the responses to h i s t a -mine that could be competitively blocked by mepyramine and r e l a t e d a n t i -histaminic agents, and as non-H|. receptors ( l a t e r to be termed H£ receptors) those which mediated responses to histamine that were r e f r a c t o r y to mepyramine. Trendelenburg (1960) had e a r l i e r reported that the c l a s s i c a l a n t i -histamines (mepyramine and tripelennamine) did not antagonise the cardiac e f f e c t s of histamine. Other actions of histamine such as contraction of guinea-pig ileum and bronchi (Arunlakshana and S c h i l d , 1954) could be blocked by these a n t i h i s t a m i n e drugs — and were therefore c l a s s i f i e d as tissues with H^-receptors according to the d e f i n i t i o n suggested by Ash and S c h i l d (1966). Like the cardiac histamine e f f e c t s , histamine e f f e c t s on g a s t r i c acid s ecretion and r e l a x a t i o n of the rat uterus were i n s e n s i t i v e to the c l a s s i -c a l antihistamine drugs. (These s i t e s were subsequently designated as containing I^-receptors.) The c h a r a c t e r i s t i c s of histamine H^-receptors have been defined by Black et a l . (1972).,. Careful re-evaluation of the s'tructure^-activity r e l a t i o n s i n a series of imidazole derivatives led, i n 1972, to development of the f i r s t H^-receptor antagonist, burimamide, by Black and his co-workers. Thus, c l a s s i c a l antihistamines (mepyramine, tripelennamine, and promethazine) are now accurately defined as histamine H^-receptor antagonists (Ash and S c h i l d , 1966), whereas burimamide (Black et a l . , 1972), metiamide (Black.et a l . , 1973) and cimetidine (Brimblecpme ejt a l . , 1975) are histamine I^-receptor antagonists. 2-methylhistamine (Black et a l . , 1972), 2-(2-amioethyl) pyridine (or 2-(2-pyridyl) ethyla-mine, PEA) and 2-(2-amioethyl) thiazole (Durant et a l . , 1975) are s e l e c t i v e 3 histamine H^-receptor agonists and 4-methylhistamine (Black e_t a l . , 1972) and _dimaprit (Parsons et a l . , 1977) are s e l e c t i v e histamine H^-receptor agonists. The a v a i l a b i l i t y of s e l e c t i v e receptor agonists-and antagonists has stimulated new in t e r e s t i n the nature of responses to histamine in many systems. The next section i s an attempt to discuss the cardiovascular response to histamine, with p a r t i c u l a r reference to the receptors assoc-iated with the various responses to histamine. Histamine and the Heart The e f f e c t of histamine on the heart i n vivo i s complex, and although histamine can change cardiac function i n vivo, d i s t i n c t i o n between the d i r e c t cardiac e f f e c t s of histamine and those as a consequence of i n d i r e c t mechanisms (eg. r e f l e x responses to hypotension) i s often very d i f f i c u l t . In a very extensive review on the cardiac actions of histamine, A l t u r a and Halevy (197 8) c r i t i c i z e such experiments, mainly because of i n t e r - and intra-species v a r i a b i l i t y . In addition, as pointed out by A l t u r a and Halevy, the type of response to histamine under those conditions ( i e . intact animal experiments) could also depend on experimental protocol. In 1910, Dale and Laidlaw published the f i r s t report on the cardiac ef f e c t s of histamine i n i s o l a t e d cat and rabbit hearts. The cardiac actions of the amine were then mainly ignored. In 1960, the studies of Mannaioni f i r s t suggested that histamine produced an increase i n contrac-t i l i t y by combining with s p e c i f i c histamine receptors i n the myocardium. Also i n 1960, Trendelenburg reported on the effects of histamine and 5-hydroxytryptamine on a t r i a i s o l a t e d from cats, rabbits and guinea-pigs. 4 In t h i s study, the eff e c t s of histamine and 5-hydroxytryptamine were compared to the responses produced by noradrenaline and n i c o t i n e . Based on the r e s u l t s with a v a r i e t y of pharmacological agents known to a f f e c t neurotransmitter function, Trendelenburg concluded that the eff e c t s of histamine i n the is o l a t e d k i t t e n heart were due to an i n t e r a c t i o n with s p e c i f i c histamine receptors. Since neither reserpine pretreatment (3 mg/kg) nor the presence of ay8-adrenoceptor blocking agent dichloroisoprenaline a l t e r e d the responses to histamine, Trendelenburg concluded that no i n -d i r e c t mechanisms were involved i n the chronotropic responses due to h i s t a -mine i n the k i t t e n heart. According to Trendelenburg, the reduction of the histamine response seen with dichloroisoprenaline was due to the p o s i t i v e chronotropic e f f e c t of the antagonist rather than due to an actual blockade o f - a d r e n o c e p t o r s . The author suggested that since dichloroisoprenaline i t s e l f produced a marked elevation of heart rate, the s l i g h t response due to histamine would be masked, and thus produced an apparent blockade. When histamine antagonists (H^) were used i n that study, both pyrilamine and tripelennamine f a i l e d to i n h i b i t the eff e c t s of histamine on c o n t r a c t i l i t y , except at high doses of antagonist. Using high doses of histamine (H-^ ) antagonists where s i g n i f i c a n t depression of basal rates were produced, Trendelenburg reported a pA2 value for the pyrilamine-histamine antagonism i n the cat r i g h t atrium of 5.1 and 5.3 for guinea-pig r i g h t a t r i a . Except for the study of pA^ values, the re s u l t s reported by Trendelenburg were based on si n g l e doses of agonists. In ad d i t i o n to the report by Trendelenburg (1960), numerous other investigators (Ash and Schild, 1966; Flacke et a_l., 1967; Hughes and Coret, 5 1972; L e v i and Kuye, 1974; McNeill and Mushek, 1972) have reported the ineffectiveness of the c l a s s i c a l antihistamines i n antagonising the cardiac ef f e c t s of histamine. However, with the introduction of s p e c i f i c histamine ^ - r e c e p t o r antagonists, Reinhardt et a l . , (1974) as well as Steinberg and Holland (1975) c l e a r l y demonstrated that the inotropic e f f e c t s of histamine in the guinea-pig l e f t atrium were blocked by histamine H^-receptor antagonists such as promethazine or tripelennamine, but not by H2 blocking agents. On the other hand, the chronotropic e f f e c t of histamine i n the guinea-pig r i g h t atrium was blocked by-burimamide and metiamide, but not by histamine H-^-receptor antagonists. These data contradicted the findings of McNeill and Verma (1974) i n the perfused guinea-pig heart and also those of Moroni et a l . (1973) and Ledder e_t a l . (1974) i n the guinea=pig r i g h t v e n t r i c l e s t r i p . A l l of these authors demonstrated, by using appropriate blocking agents, that the cardiac receptors mediating changes i n c o n t r a c t i l e force were"-of the h i s t a -mine H2-receptor type. In a l a t e r study by Verma and McNeill (1976), u t i l i s i n g a s p e c i f i c histamine H-^-receptor agonist, PEA, and a s p e c i f i c histamine ^ - r e c e p t o r agonist, 4-methylhistamine, demonstrated that the type of histamine receptor i n the guinea-pig heart depended on the part of the heart examined. Whereas histamine H2-receptors were found mainly i n the guinea-pig r i g h t atrium, the l e f t atrium contained H^-receptors while the r i g h t v e n t r i c l e s t r i p contained a mixture of both Hi- and ^ - r e c e p t o r s (Verma and McNeill, 1976). In the spontaneously beating Langendorff guinea-pig heart preparation used by McNeill and Verma (1974), the blockade of the 6 inotropic response of histamine reported was therefore a blockade of the increased rate produced by histamine as well as an increased force of contraction i n the r i g h t v e n t r i c l e , both histamine l^-receptor mediated ef f e c t s (Verma and McNeill, 1977). In a more recent study, N c N e i l l and Verma (1978), using i s o l a t e d rabbit hearts,ddemonstrated that the l e f t atrium, r i g h t v e n t r i c l e s t r i p and r i g h t p a p i l l a r y muscle contain histamine H-^-receptors. In the r i g h t atrium of the rabbit, however, both histamine H^- and l^-receptors were associated with chronotropic responses to histamine-like drugs. These r e s u l t s and others (for reviews see Verma and McNeill, 1976 and A l t u r a and Halevy, 197 8), c l e a r l y indicate that the type of cardiac histamine receptor depends on both the species and the part of the heart examined. A great deal of evidence has accumulated to support the hypothesis that the cardiac e f f e c t s of histamine were mediated by c y c l i c AMP through a histamine-sensitive adenylate cyclase. In 1967, Poch and Kukovetz f i r s t suggested that cardiac histamine receptors i n guinea-pig heart were assoc-iated with adenylate cyclase. Dean, i n 1968, also postulated that c y c l i c AMP mediated the cardiac e f f e c t s of histamine by stimulating adenylate cyclase in a manner analogous to that reported by Robison et al_. (1965) for the adrenergic amines. K l e i n and Levey (1971) published the f i r s t report showing an increase in cardiac c y c l i c AMP l e v e l s following histamine administration. According to K l e i n and Levey (1971), the histamine-stimulated increases of c y c l i c AMP i n the guinea-pig, cat and human heart were unaffected by the ^-adrenoceptor antagonist, DL-propranolol. In contrast, K l e i n and Levey 7 also reported that diphenhydramine, a histamine H^-receptor antagonist, could block the histamine-induced increases in c y c l i c AMP while not a f f e c t i n g increases produced by noradrenaline. McNeill and.Mushek (1972) performed a more extensive study of the e f f e c t s of histamine on c o n t r a c t i l i t y , phosphorylase and adenylate cyclase in i s o l a t e d , perfused guinea-pig hearts. These authors c l e a r l y showed that the blockade of histamine a c t i v a t i o n of adenylate cyclase by c l a s s i c a l antihistamines (tripelennamine and diphenhydramine) was nonspecific. McNeill and Mushek (1972) therefore discussed the conclusion that not only were the mechanical responses due to histamine i n the heart unaffected by histamine Hi-receptor antagonists, but that the biochemical responses (phosphorylase and adenylate cyclase activation) observed following h i s t a -mine receptor stimulation were also not competitively antagonised by the histamine antagonists a v a i l a b l e at the time. Verma and McNeill (1979) have since repeatedly demonstrated that stimulation of histamine H^-receptors (eg. guinea-pig r i g h t a t r i a , guinea-pig r i g h t v e n t r i c l e s t r i p and rabbit r i g h t a t r i a ) i s associated with i n -creases i n c y c l i c AMP. On the other hand, stimulation of histamine H^-receptors (guinea-pig and -rabbit l e f t a t r i a , rabbit v e n t r i c l e s t r i p ) did not r e s u l t i n increases of c y c l i c AMP. Therefore, increases i n c y c l i c AMP are not absolutely necessary for the inotropic action of histamine. Though the majority of l i t e r a t u r e on the cardiac e f f e c t s of histamine is based on the guinea-pig (Altura and Halevy, 1978), the presence of histamine receptors has also been reported in numerous other species. Dale and Laidlow (1910) reported on the e f f e c t s of histamine on cardiac contrac-t i l i t y i n the i s o l a t e d heart of the cat and rabbit. Trendelenburg (1960) 8 further investigated the e f f e c t s of histamine i n the cat, guinea-pig and rabbit heart. Verma and McNeill (1977) and McNeill and Verma (1979) have studied the regional d i s t r i b u t i o n of both histamine Hi- and ^ - r e c e p t o r s in the guinea-pig and rabbit myocardium. Chiba (1976) reported that the ef f e c t s of histamine on inotropic and chronotropic a c t i v i t y i n i s o l a t e d canine a t r i a l preparations were blocked by histamine Hl-receptor antago-n i s t s . Using large doses of histamine, Korasec and Erjavec (197 8) recently suggested that the e f f e c t s of histamine in the rat heart were due to stimulation of both histamine ^ - r e c e p t o r s and -adrenoceptors. These workers also claimed that the increases i n cardiac c y c l i c AMP recorded were due to stimulation of histamine ^ - r e c e p t o r s . An e a r l i e r study (Satayavivad e_t a_l., 1977) reported that the chronotropic e f f e c t of h i s t a -mine in both guinea-pig and rat a t r i a was potentiated by the phosphodie-sterase i n h i b i t o r , l-methyl-3-isobutylxanthine. However, at least i n the ra t a t r i a l preparations, very high doses of histamine had to be used again. The E D 5 0 values reported by Satayavivad et_ a_l., (1977) for the chronotropic -6 e f f e c t s of histamine i n the guinea-pig was 2.3 + 0.2 x 10 M while that for the rat was 1.7 +0.7 x 10"3M. _3 At such high doses (eg. ED^Q 1.7 +. 0.7 x 10 M histamine) histamine would be expected to exert non-specific e f f e c t s . Hood e_t a l . (1975) have demonstrated that high doses of burimamide (34-1080yuM) produced a dose-dependent i n d i r e c t sympathomimetic e f f e c t in k i t t e n a t r i a . S i m i l a r l y , Broadley and Wilson (1978) have recently stated that high doses of PEA had both a d i r e c t e f f e c t (stimulation of histamine H^-receptors) as well as an i n d i r e c t e f f e c t (release of catecholamines). 9 In addition to the p o s i t i v e chronotropic and inotropic e f f e c t s of histamine discussed already, i t has recently been reported that histamine can also a l t e r l e f t i n t r a v e n t r i c u l a r pressure, coronary flow, a o r t i c flow, t o t a l cardiac output and external pressure-volume work in the i s o l a t e d working heart preparation of the guinea-pig (Flynn et a l . , 1979). In summary, the inotropic and chronotropic e f f e c t of histamine i n a v a r i e t y of species is well documented. Responses due to histamine H£-receptor stimulation r e s u l t in increases in c y c l i c AMP while those due to histamine H^-receptor stimulation are not associated with c y c l i c AMP. In some species (eg. r a t ) , however, mechanical and biochemical responses in the heart are produced with only high doses of histamine. 10 PURPOSE OF THE PRESENT INVESTIGATION This study set out to investigate the chronotropic and inotropic response in the i s o l a t e d k i t t e n heart. It was hoped to characterise further the nature of these responses u t i l i s i n g s p e c i f i c histamine Hi- and H2-receptor antagonists and agonists, and thereby report on the d i s t r i b u t i o n of such receptors in this species. Previous work by Trendelenburg (1960) was reported for only the e f f e c t s of s i n g l e doses of histamine, and i t was hoped to perform s i m i l a r studies using complete dose-response curves. We also wished to rein v e s t i g a t e e a r l i e r reports on the e f f e c t s of histamine on the i s o l a t e d rat heart (Satayavivad et al_., 1977) since at _3 the high doses used in that study ( E D 5 0 1.7 + 0.7 x 10 M) i t is quite l i k e l y that the e f f e c t s of histamine would be n o n - s p e c i f i c . We hoped to repeat these studies with histamine, and, i n addition, to characterise the nature of the receptors involved i n the responses to histamine in the rat heart. F i n a l l y , i t was hoped to rein v e s t i g a t e the e f f e c t s of PEA i n the i s o l a t e d guinea-pig heart, since t h i s histamine Hi-receptor agonist enjoys a widespread usage in the study of such systems ( i e . H-^-receptors) . The study was prompted by the preliminary report of Broadley and Wilson (197 8) s t a t i n g that this agonist released catecholamines in addition to i t s d i r e c t a c t i o n on histamine Hi-receptors. Since the guinea-pig l e f t atrium and r i g h t v e n t r i c l e are known to contain histamine H^-receptors (Verma and McNeill, 1977), this species was chosen to study whether the release of catecholamines was mediated by histamine H^-receptors in the heart, as i s the case in the adrenal medulla (Emmelin and Muren, 1949). 11 METHODS Cardiac preparations were obtained from three species: cat (l-2kg), guinea-pig (300-400g) and rat (150-300g). Animals of eit h e r sex were used and they received food and water ad. libitum. In studies with the i s o l a t e d cat heart, r i g h t a t r i a , l e f t a t r i a , r i g h t v e n t r i c l e s t r i p s and ri g h t p a p i l l a r y muscles were dissected free and sus-o pended i n Chenoweth-Koelle s o l u t i o n (1946) at 37 C under a 1 gram basal tension. When i s o l a t e d guinea-pig and r a t hearts were used, only the r i g h t and l e f t a t r i a were used from each animal. The buffer s o l u t i o n was aerated with a 957o oxygen, 5°L carbon dioxide mixture. Following an i n i t i a l e q u i l i b r a t i o n period of 30 minutes, a l l quiescent tissues were e l e c t r i c a l l y stimulated at 1Hz and twice the threshold voltage using a Grass model S6 stimulator. Complete dose-response curves were constructed using the cumulative method as described by Van Rossum and Van der Brink (1963). When antago-n i s t s were used, these were added to the buffer an hour p r i o r to the ad d i t i o n of agonist. In some studies, animals were pretreated with rese-pine (2.5mg/kg i.p. or 3.0mg/kg i.p.) 24 hours p r i o r to the experiment. Each t i s s u e was used for only one complete dose-response curve. Because of the gradual nature of a l l of the responses (rate and force responses), measurements were made 3 minutes a f t e r the addi t i o n of agonist. C o n t r a c t i l e force i n the l e f t atrium, r i g h t v e n t r i c l e s t r i p and ri g h t p a p i l l a r y muscle was measured i s o m e t r i c a l l y by placing a Palmer c l i p at the apex of the tiss u e and connecting t h i s to a Grass force displacement transducer. A l l recordings were made on a Grass model 79D polygraph. 12 S t a t i s t i c a l Methods S t a t i s t i c a l analysis was done by the student's t - t e s t for unpaired data. A p r o b a b i l i t y of p * 0.05 was taken as the c r i t e r i o n of s i g n i f i -cance. Materials: The following drugs were employed i n the study. Atropine Sulfate (Sigma, St. Louis, U.S.A.) Histamine Dihydrochloride (Sigma, St. Louis, U.S.A.) DL-Propranolol HCl (Sigma, St. Louis, U.S.A.) Reserpine (Serpasil ) (CIBA-GEIGY Canada) Promethazine, Cimetidine, 4 methyl-histamine and 2-pyridylethylamine were g i f t s from Smith Kline and French Laboratories, Welwyn Garden C i t y , Herts., England. 13 RESULTS Chronotropic E f f e c t of Histamine i n K i t t e n Right A t r i a The dose-response curve for the chronotropic e f f e c t of histamine i n the k i t t e n r i g h t atrium i s shown i n Figure 1 and Table 1. The threshold of the response occurs at approximately 10 histamine, and at a dose of -3 10 M, histamine was able to produce an increase i n rate of about 87 beats per minute (Table 1). Promethazine at low doses (10 \i) was unable to s i g n i f i c a n t l y reduce the changes i n heart rate produced by histamine at any dose (Table 1). When higher doses of promethazine were applied, a depression of basal heart rate was noted. In addition, the antagonism produced by higher doses of promethazine was not surmountable by increas-ing the dose of histamine. This observation i s i n agreement with the data of McNeill and Verma (1974) who demonstrated that at higher concentrations (greater than 4/uM) , promethazine was able to interact with the histamine receptor i n eit h e r a non-competitive or competitive non-equilibrium manner. - 6 - 5 Cimetidine, at doses of 1 x 10 M and 1 x 10 M, caused a displace-ment of the histamine curve to the ri g h t hand side i n a dose-dependent manner. Though the same maxima (as the control maximal histamine effect) were not reached, preliminary studies with higher doses of agonist showed that the antagonism was surmountable. With each increasing dose of cimetidine, the threshold for the histamine response was also increased (Figure 1). When propranolol (10~^M) was added to the buffer sol u t i o n , a s l i g h t elevation of the basal heart rate was observed. This dose of propranolol was s u f f i c i e n t to produce a s i g n i f i c a n t antagonism of the chronotropic 14 responses to doses of histamine greater than 1 x 10 \ (Table 1). Inotropic E f f e c t of Histamine i n Isolated K i t t e n Heart The inotropic responses of histamine i n the i s o l a t e d k i t t e n heart -4 were only demonstrable at high doses (at or greater than 10 M). For this reason, i t was decided to express the e f f e c t of histamine on the is o l a t e d k i t t e n l e f t atrium (Figure 3), r i g h t v e n t r i c l e s t r i p (Figure 4) and r i g h t p a p i l l a r y muscle (Figure 5) by means of histograms. Results for the complete dose-response curves are summarised i n Tables 2, 3 and 4. -3 At a dose of 10 M histamine, histamine caused a s i g n i f i c a n t increase i n c o n t r a c t i l e force i n a l l three tissues studied — l e f t atrium, r i g h t v e n t r i c l e s t r i p and r i g h t p a p i l l a r y muscle (Tables 2, 3 and 4). In the -5 -6 -7 presence of eit h e r cimetidine (10~ M or 10" M) or promethazine (10 M), histamine (10 M) was able to cause an increase i n force of contraction that was not s i g n i f i c a n t l y d i f f e r e n t from control conditions. However, when propranolol (10 ^ M) was added to the perfusing buffer, the inotropic e f f e c t of histamine i n the k i t t e n l e f t atrium (Figure 3), r i g h t v e n t r i c l e s t r i p (Figure 4) and r i g h t p a p i l l a r y muscle (Figure 5) was reduced by 507» or more. Chronotropic E f f e c t of 4-methyl Histamine i n Ki t t e n Right Atrium 4-methyl histamine, a s p e c i f i c histamine ^ - a g o n i s t , produced a p o s i t i v e chronotropic e f f e c t i n spontaneously beating k i t t e n r i g h t a t r i a (Figure 6, Table 5). Again, as with histamine, the onset of action was delayed. However, when compared to the e f f e c t of histamine i n the k i t t e n 15 r i g h t atrium, 4-methyl histamine had a higher threshold of response (10 M -6 4-methyl histamine compared to 10 M histamine). But i t should be noted that the mean of the maximal changes produced by either agonist is approxi-mately equal (87 beats per minute change with histamine compared to 78 beats per minute change with 4-methyl histamine). -6 -5 Both doses of cimetidine used (1 x 10 M and 1 x 10 M) produced a displacement of the control dose-response curve to the r i g h t hand side. The i n h i b i t i o n of the 4-methyl histamine e f f e c t was dependent on the dose of antagonist used, so that the i n h i b i t i o n of the maximal responses occurr-i 3 ing at 3 x 10 M and 1 x 10 M 4-methyl histamine were only s i g n i f i c a n t with the higher dose of cimetidine (10 ^ M) (Table 5). Again, as with the histamine chronotropic responses, when propranolol was added to the perfusate, a s i g n i f i c a n t i n h i b i t i o n of the chronotropic response to 4-methyl histamine occurred (Figure 6). Whereas the mean _3 maximal change produced by 10 M 4-methyl histamine under control conditions is 7 8 beats per minute, t h i s value was reduced to 37 beats per minute when propranolol was included i n the perfusate. These r e s u l t s are more c l e a r l y demonstrated when the response to a sin g l e dose of 4-methyl histamine (10"-%) under various conditions is represented by histograms, as i n Figure 7. As i s demonstrated i n Figure 7, -3 the response to 10 M 4-methyl histamine was s i g n i f i c a n t l y reduced by both cimetidine (10"^M) and propranolol (10"^M). 16 Inotropic E f f e c t of 4-methyl Histamine i n Isolated K i t t e n Heart -3 4-methyl histamine (10 M) produced an inotropic e f f e c t i n the k i t t e n l e f t atrium (Figure 8 ) , r i g h t v e n t r i c l e ^ s t r i p (Figure 9) and r i g h t p a p i l l a r y muscle (Figure 10). Again, as with the histamine inotropic e f f e c t , these responses were only obtained at high doses of agonist. - 6 Cimetidine (10 M) was without s i g n i f i c a n t e f f e c t on the inotropic response to 4-methyl histamine i n the k i t t e n l e f t atrium (Table 6 ) , r i g h t v e n t r i c l e s t r i p (Table 7) and r i g h t p a p i l l a r y muscle (Table 8). However, -7 -3 i n the presence of 10 M propranolol, the inotropic e f f e c t of 10 M 4-methyl histamine was reduced by more than 507o of the control response in a l l three tissues (Tables 6 , 7 and 8 ) . I t was reasoned that since promethazine had no s i g n i f i c a n t e f f e c t on e i t h e r the chronotropic (Table 1) or inotropic (Tables 2, 3 and 4) e f f e c t s of histamine i n the k i t t e n heart, i t was u n l i k e l y to a l t e r these responses when 4-methyl histamine was used as the agonist. Chronotropic E f f e c t of PEA i n Guinea-Pig Isolated Right A t r i a PEA produced a dose-dependent increase i n rate i n a t r i a obtained from untreated guinea-pigs (Figure 11). The threshold of t h i s response occurred - 6 at 10 M PEA, and the maximal change recorded under control conditions was _3 126 beats per minute with 10 M PEA (Table 9 ) . - 6 - 6 When either promethazine (3 x 10 M) or cimetidine (3 x 10""M) were added to the perfusate, the dose-response curve.; for PEA was not a l t e r e d by either treatment (Figure 1 1 ) . In other words, neither promethazine (3 x 10~^M) or cimetidine (3 x 10~^M) s i g n i f i c a n t l y a f f e c t e d the basal 17 heart rate (176 beats per minute) or the maximal change produced by 10 ~*M PEA (126 beats per minute) (Table 9). The chronotropic e f f e c t s of PEA were also studied i n a t r i a obtained from guinea-pigs pretreated with reserpine (2.5 mg/kg, 24 hours p r i o r to s a c r i f i c e ) . The res u l t s of these studies are i l l u s t r a t e d i n Figure 12. Reserpine pretreatment s i g n i f i c a n t l y reduced the maximal rate change obtained with PEA from 126 beats per minute to 44 beats per minute. The -4 -3 increases i n heart rate with 10 M and 10 M PEA i n a t r i a from reserpine pretreated guinea-pigs were s i g n i f i c a n t , and consequently propranolol -6 (10 M) was also included i n the buffer i n another series of experiments (Figure 12). When propranolol was added to the perfusate, the rate responses to PEA i n a t r i a obtained from reserpine-pretreated guinea-pigs were s i g n i f i c a n t l y depressed (Figure 12). In other words, PEA was unable to cause a s i g n i f i -cant increase i n heart rate under these conditions. Inotropic Responses to PEA in Isolated Guinea-Pig L e f t A t r i a PEA had a dose-dependent p o s i t i v e inotropic e f f e c t i n l e f t a t r i a from untreated guinea-pigs (Figure 13), increasing the force of contraction from an average basal value of 0.6g to a mean developed tension of 2.8g, an increase of 2.2g (Table 10). —ft In the presence of a non-depressant dose of promethazine (3 x 10" M) (Table 10), PEA was s t i l l able to produce an increased force of contrac-—ft t i o n at doses equal to or greater than 10" M. However, the i n h i b i t i o n of the PEA response i n the guinea-pig l e f t atrium was s i g n i f i c a n t only at f> -5 two doses, 10 M and 10 M PEA (Table 10). The responses to PEA at higher 18 doses (10 M and 10 M) were not s i g n i f i c a n t l y a l t e r e d by t h i s antagonist (promethazine). -fi Cimetidine (3 x 10 M) had l i t t l e e f f e c t on the dose-response curve for the inotropic e f f e c t of PEA i n the guinea-pig l e f t atrium (Figure 13) and aff e c t e d neither the slope nor the maximal response. As is clear from - 6 Figure 13, propranolol at a dose of 10" M i n the perfusing medium, had a s i g n i f i c a n t e f f e c t on the maximal e f f e c t of PEA i n the guinea-pig l e f t -3 atrium, reducing the inotropic e f f e c t of 10 M PEA from a developed tension of a mean 2.2g to that of 1.4g. But i t w i l l be noted that at doses of PEA less than 10 ~*M propranolol had l i t t l e e f f e c t on the slope of the dose-response curve (Figure 13). Experiments s i m i l a r to those just described above were next repeated i n l e f t a t r i a i s o l a t e d from reserpine-pretreated guinea-pigs (2.5 mg/kg, 24 hours p r i o r to s a c r i f i c e ) . The re s u l t s of these experiments are summa-r i s e d i n Figure 14 and Table 11, and for comparative purposes, a control ( i e . untreated) dose-response curve is also included. In l e f t a t r i a from reserpine-pretreated guinea-pigs, PEA was s t i l l able to produce a dose-dependent p o s i t i v e inotropic e f f e c t , but compared to the inotropic e f f e c t of PEA i n l e f t a t r i a from untreated guinea-pigs, the maximal response -3 obtained with 10 M PEA was s i g n i f i c a n t l y depressed (Table 11). In addition, the responses to lower doses of PEA were also depressed, though this was not s i g n i f i c a n t at p 4 0.05. A rightward s h i f t of the dose-response curve to PEA in l e f t a t r i a obtained from reserpine-pretreated guinea-pigs (as compared to a t r i a from untreated animals) appears to be the trend at doses greater than 10 _ 5M PEA. As with the response to PEA i n l e f t a t r i a from untreated guinea-pigs 19 (Table 10), cimetidine (3 x 10 M) was unable to a l t e r the p o s i t i v e ino-t r o p i c e f f e c t of PEA i n l e f t a t r i a from reserpine-pretreated guinea-pigs -6 (Table 11, Figure 14). When promethazine (3 x 10 M) was added to the perfusate, a further rightward s h i f t of the dose-response curve to PEA was observed (Figure 14). Under these conditions, promethazine caused a s t a t i s t i c a l l y s i g n i f i c a n t i n h i b i t i o n of the PEA-induced increases i n force of contraction at doses greater than 10 ~*M (based on a comparison with the responses obtained i n a t r i a from untreated animals (Table 11)); Inotropic Response of Guinea-Pig Right V e n t r i c l e S t r i p to PEA -6 At doses greater than 10~ M PEA, the agonist produced a p o s i t i v e i n o t r o p i c response i n right v e n t r i c l e s t r i p s from untreated guinea-pigs —6 (Figure 15). Inclusion of either promethazine (3 x 10",M) or propranolol (10 M^) s i g n i f i c a n t l y reduced the inotropic e f f e c t s of both 10~^M and 10~^M. PEA i n v e n t r i c l e s t r i p s obtained from untreated guinea-pigs (Table 12). When the response of v e n t r i c l e s t r i p s from reserpine-pretreated guinea-pigs to PEA was studied, i t was observed that under these conditions, the i n -creases i n tension produced by 10~Sl and 10~^M PEA were s i g n i f i c a n t l y reduced (Figure 15), whereas propranolol (10"%) and promethazine (3 x 10"^M) reduced the PEA-induced increases i n tension by 4270 and 2970 respec-t i v e l y (average values); following reserpine-pretreatment the reduction i n the maximal response to 10 M PEA was a mean of 527» (Table 12). Chronotropic E f f e c t of 4-methylhistamine i n Guinea-Pig Right A t r i a The response of the guinea-pig r i g h t atrium to 4-methylhistamine was 20 also studied, and the res u l t s are summarised i n Table 13 and Figure 1 6 . The maximal e f f e c t with this agonist occurred at 1 0 " ^ M 4-methylhistamine, when the spontaneous rate of the ri g h t atrium was increased from a mean of 185 beats per minute to a mean of 3 1 9 beats per minute (Table 1 3 ) . Addition of 1 0 ~ ? M propranolol a f f e c t e d neither the slope nor the maximal - 6 response of the dose-response curve (Figure 1 6 ) . In the presence of 10 M cimetidine, the rate changes seen with 4-methylhistamine were reduced, and - 6 - 5 these became s i g n i f i c a n t at 1 0 " M and 1 0 " M 4-methylhistamine (Table 1 3 ) . However, this i n h i b i t i o n due to cimetidine was overcome at higher doses of - 4 - 3 4-methylhistamine ( 1 0 M and 10 M), with the maximal chronotropic e f f e c t —6 — 3 in the presence of 10 M cimetidine now occurring at 10 M 4-methylhista-mine (Figure 1 6 ) . E f f e c t of Histamine on Spontaneously Beating Isolated Rat Right A t r i a In Figure 17 and Table 1 4 are summarised the eff e c t s of histamine on the spontaneous rate of contraction of iso l a t e d rat ri g h t a t r i a under various conditions. It i s immediately apparent that any chronotropic - 4 response due to histamine was noted only at doses greater than 10 M - 2 (Figure 1 7 ) , so that at 10 M histamine, a mean increase i n rate of 82 beats per minute was recorded. This response seen with histamine was unaffected by 10 cimetidine, _2 when a mean maximal change of 96 beats per minute was recorded with 10 M histamine (Table 1 4 ). However, in the presence of 10 promethazine, a - 2 s t a t i s t i c a l l y s i g n i f i c a n t p o t e n t i a t i o n of the response due to 10 M h i s t a -mine was observed (Table 1 4 ) , with no other changes evident. - 6 Following e i t h e r the addi t i o n of propranolol ( 1 0 M) or pretreatment 2 1 of rats with reserpine ( 3 . 0 mg/kg, 2 4 hours p r i o r to s a c r i f i c e ) , histamine - 3 - 2 (at doses of 1 0 M and 1 0 M) produced a s t a t i s t i c a l l y s i g n i f i c a n t and dose-dependent negative chronotropic response. In the presence of propra-n o l o l , the mean change with 1 0 M histamine was ( - ) 3 9 beats per minute, and the corresponding value i n a t r i a from reserpine-pretreatment rats was ( % ) 5 5 beats per minute. However, the negative chronotropic response seen with high concentra-tions of histamine when propranolol was added to the perfusate appeared to be reversed when atropine was also included i n the buffer (Figure 1 7 ) . Under these conditions, ( i e . 1 0 atropine and 1 0 propranolol) only o the highest dose of histamine used ( 1 0 M) caused a s i g n i f i c a n t increase in heart rate over basal value. E f f e c t of Histamine on Force of Contraction of E l e c t r i c a l l y Stimulated  (1Hz) Rat L e f t A t r i a Increases i n force of contraction was measured following only high doses of histamine. Consequently, the re s u l t s express only the per cent - 3 increases i n force following 1 0 M histamine (Table 1 5 , Figure 1 8 ) . A mean increase of 8 0 7 o over control tension was observed following - 3 the a d d i t i o n of 1 0 M histamine (Table 1 5 ) . The increase i n c o n t r a c t i l e force was not s i g n i f i c a n t l y a l t e r e d by the addition of either 1 0 cime-t i d i n e or 1 0 promethazine to the perfusate. - 3 When histamine ( 1 0 M) was added to rat l e f t a t r i a i n the presence - 6 of 1 0 M propranolol, a s i g n i f i c a n t reduction i n the inotropic e f f e c t of the agonist was observed, with histamine producing a mean increase i n force - 3 of only 1 5 7 o . S i m i l a r l y , histamine ( 1 0 M) produced a mean increase i n 22 force of only 107o when its inotropic response was studied in l e f t atria obtained from reserpine-pretreated rats (Table 15 ) . 23 TABLE ?1: The e f f e c t of histamine on i s o l a t e d spontaneously beating k i t t e n r i g h t a t r i a . Values are absolute heart rates (beats/min) under control conditions (basal heart rate, basal H.R.) and i n the presence of various antagonists. Results expressed as mean rate (beats/min) is.E.M. for 7-10 tiss u e s . a = sign. d i f f . (p 5- 0.05) compared to basal value, b = sign. d i f f . (p ^ 0.05) compared to control curve. .cone, of histamine (M) basal H.R. 10" 8 10" 7 10" 6 10" 5 10" 4 10 Control (10) 125±4 125±4 125±4 131±4 169±ll a 202±10 a 212±7 +10"7 Propran.(7) 133±4 133±4 133±4 133±4 136±6 b 159±6 a' b 157+-14' +10"5 Cimet.(lO) 124±5 124+5 124±5 124±5 124±5 b 133±5 a' b 175+-83' +10"6 Cimet.(8) 123+5 123+5 123+5 123+5 130±7 b 170±13 a . 186+14a +10"7 Promet .(8) 125+11 125±11 125±11 125±11 149+11 172+10a 195±6 a 25 TABLE 2: The e f f e c t of histamine on e l e c t r i c a l l y paced (1Hz) iso l a t e d k i t t e n l e f t a t r i a . Results are expressed i n absolute tension (g) both i n the absence of histamine (basal developed force, B.D.F.) and following administration of histamine under various conditions. Results expressed as mean tension (g) — S.E.M. for 7-10 tissue s . a = sign. d i f f . (p ^0.05) compared to basal value, b = sign. d i f f . (p ^0.05) compared to control curve. cone, of histamine (M) B.D. F. 10' 8 10 " 7 10" 6 IO-5. ID" 4 10" 3 Control (10) 1.6+0.2 1.6t0. 2 1.6+0.2 1.6+0.2 1.7+0. 2 1 .8+0. 2 2.2+0.23 +10 - 7 Propran.(7) 1.5+0.2 1.6+0. 2 1.6+0.2 1.6+0.2 1.7+0. 2 1 .7+0. 2 1.8±0.2 a' b -5 +10 Cimet. (10) 1.5+0; 2 1.5+0. 2 1.5+0.2 1.5+0.2 1.5+0. 2 1 .6+0. 3 2.0+0.3a +10" Cimet. (8) 1.2+0.2 1.2+0. 2 1.2+0.2 1.2+0.2 1.2+0. 2 1 .2+0. 2 a, b 1.6±0.2 +10 Promet. (8) 1.2+0.2 1.2+0.2 1.2+0.2 1.2+0.2 1.3+0.3 1.3+0.2 1.8+0.2 27 TABLE 3: The e f f e c t of histamine on e l e c t r i c a l l y paced (1Hz) k i t t e n r i g h t v e n t r i c l e s t r i p s . Results are expressed i n absolute tension (g) both i n the absence of histamine (basal developed force, B.D.F.) and following administration of histamine under various conditions. Results are expressed as mean tension (g) — S.E.M. for 7-10 tissue s . a = sign. d i f f . (p ^.0.05) compared to basal value, b = sign. d i f f . (p ^0.05) compared to^control curve. cone, of histamine (M) B.D.F. i o " 8 i o " 7 i o " 6 i o " 5 i o " 4 i o " 3 Control (10) 1.6+0. 3 1.6+0. .3 1 .6to. .3 1 . 6 t o . 3 1.8+0. ,3 l . f i t o . , 3 3 2 . 2 t o . 3 +10-7 Prop.(7) 1.5+0. 2 1.5^0. ,2 l . s t o . .2 1 . 5 t o . 2 l . s t o . ,2 l . s t o . .2 1.7"to. b 2 +10"6 Cimet.(10) 1.6+0. 2 1 .6to. .2 1.6+0. 2 1.6t0.2 1.7+0. ,3 1.7+0. , 3 a 2 . 2 t o . 4 +10"5 Cimet.(8) 1.6+0.2 1.6to.2 1.6to.2 1 .6to.2 1.6+0.2 1.6+0.2a 2 . l t o.2 29 TABLE 4: The e f f e c t of histamine on e l e c t r i c a l l y paced (1Hz) k i t t e n r i g h t p a p i l l a r y muscles. Results are expressed i n absolute tension (g) both i n the absence of histamine (basal developed force, B.D.F.) and following administra-t i o n of histamine under various conditions. Results are expressed as mean tension (g) — S.E.M. for 7-10 tissues. a = sign. d i f f . (p $0.05) compared to basal value, b = sign. d i f f . (p ^0.05) compared to control curve. cone, of histamine (M) B.D.F. i c " 8 io- 7 io" 6 IO" 5 i o ' 4 io" 3 Control (10) l.oto.i. l . l t o . l l . l t o . l l . l t o . l l . l t o . l •+- a l . l t o . l 1.5to. 2 +10" 7 Prop.(7) 1.2+0.1 1.2+0.1 1.2+0.1 1.2+0.1 l.Zto.l 1 .2 to.l 1.4+0. b 2 -5 +10 Cimet.(10) 1.430.3 1.4+0.3 1.4to.l 1.4to.l 1.4to.l 1.4+0.1 1.8to. 3 -6 +10 Cimet. (8) 1.5+0.2 1.7+0.2 1.7+0.2 1.7+0.2 1.7to.2 1.7to.2a 2.1+0. 3 +10 - 7 Promet.(8) 1.0+0.2 1.0+0.2 1.0+0. 2 1.0+0.2 l.oto .2 + a 1.0t0.2 1.5+0. 4 3 1 TABLE 5: The e f f e c t of 4-methylhistamine (4MH) on rate of spontaneously beating i s o l a t e d k i t t e n r i g h t a t r i a . Results express the rates (beats/min) under control conditions (basal heart rate) and following administration of 4MH under various conditions. Results represent the mean rate (beats/min) — S.E.M. for SV12 tissues. a = sign. d i f f . (p ^ -0.05) compared to basal value, b = sign. d i f f . (p ^0.05) compared to control curve. 32 4-methylhistamine (M) -5 -4 -4 -3 basal H.R. 10 3 3x10 10 3x10 10 Control (10) 112+6 114t3 124*6 157t4a + a 18139 + a 1923ll +10 Cimet.(10) 111+4 115+3 121+3 14lt5 a 16l34 a 17 8 t 6 a +10"5 Cimet. ( 8 ) 122+4 122t4 124t5 139t8 15036 a' b 158t 7 a' b +10" Propran.(12) l i s t s uste- 11836 126t4a 143t 6 a' b 1523 9 a' b 33 TABLE 6: The e f f e c t of 4-methylhistamine (4MH) on e l e c t r i c a l l y paced (1Hz) k i t t e n l e f t a t r i a . Results express the tension (g) in the absence of 4MH (basal developed force, _3 B.D.F.) and in the presence of 10 M 4MH. Also shown i s the percent increase over B.D.F. following administra-t i o n of 10"3M 4MH. Results represent the mean tension (g) + S.E.M. of 10-12 tissues. a = sign. d i f f . (p ^-0.05) compared to basal value, b = sign. d i f f . (p ^0.05) compared to control curve. 34 B.D.F. ,+10"3M 4MH % Aforce Control (10) 1.8*0.3 3.3*0. 3 a 86*12 +10"7 Prop.(12) 1.7*0.3 2.1*0. 4 b 23*6 b +10" 6 Cimet.(10) 2.0*5 3.6*0.6a 80*11 35 TABLE 7: The,, e f f e c t of 4-methylhistamine (4MH) on e l e c t r i c a l l y paced (1Hz) k i t t e n r i g h t v e n t r i c l e s t r i p s . Results express the tension (g) i n the absence of 4MH (basal -3 developed force, B.D.F.) and i n the presence of 10 M 4MH. Also shown i s the percent increase over B.D.F. -3 following administration of 10 M. 4MH. Results represent the mean tension (g) + S.E.M. of 10-12 t i s s u e s . a = sign. d i f f . (p ^-0.05) compared to basal value, b = sign. d i f f . (p ^0.05) compared to control curve. 36 B.D..E. 10"3M 4MH % A force Control (10) 1.6+0.3 3.1*0. 6 a 94+11 +10"7M Propr.(12) 1.4+0.4 1.7+0. .4 21+6b +10" M Cimet.(10) 1.8+0.3 3.2+0.4a 79+12 37 TABLE 8: The e f f e c t of 4-methylhistamine (4MH) on e l e c t r i c a l l y paced (1Hz) k i t t e n r i g h t p a p i l l a r y muscles. Results express the tension (g) i n the absence of 4MH (basal -3 developed force, B.D.F.) and in the presence of 10 M 4MH. Also shown i s the percent increase over B.D.F. -3 following administration of 10 M 4MH. Results represent the mean tension (g) + S.E.M. of 10-12 tissues. a = sign. d i f f . (p $ 0.05) compared to basal value, b = sign. d i f f . (p ^0.05) compared to control curve. 38 B.D.F. 10"3M 4MH % ^ f o r c e Control (10) l . l t o . 2 1 . 6 t o . 6 46t8 +10 _ 7M Propr. (12) 1.0t0.3 l . l t o . 5 12 + 2 a +10"6M Cimet.(10) 1 2 . t o . 3 1 . 7 t o . 5 42t9 39 TABLE 9: The e f f e c t of PEA on rate of spontaneously beating guinea-pig r i g h t a t r i a . Results express the rates (beats/min) i n the absence (basal heart rate) and presence of PEA under various conditions.. Values express the mean rates (beats/min) + S.E.M. for 12-28 preparations. AtriayVere obtained from either control or reserpine-pretreated (2.5 mg/kg, 24 hours) guinea-pigs. a = sign. d i f f . (p $0.05) compared to basal value, sign. d i f f . (p ^0.05) compared to control curve. PEA (M) basal H.R. IO" 8 IO" 7 10 " 6 IO" 5 IO" 4 IO" 3 Control (28) 176+6 176+6 176+6 180+11 220+12a 276+10a 302+133 +3x10" M Promet. (18) 170+11 170+1 170+1 170+11 198+9 257+10* 295+113 +3xl0" 6M Cimet. (18) 182+4 182+4 182+4 182+4 219+8* 270+4* 292+ll E +10 Propran.(20) 166+6 167+9 167+9 168+11 179+5 3 219+8 a' b 252+12 a' b Reserpine-pretreated (20) 179+8 179+8 181+5 181+5 193+6 207+3 a' b 223+6 a' b -6 +Propran.(10 M) (12) 168+9 168+9 168+9 168+6 172+6b 176+7b 181+6b 41 TABLE 10: The e f f e c t of PEA on c o n t r a c t i l e force of e l e c t r i c a l l y paced (1Hz) guinea-pig l e f t a t r i a . Results express the absolute tension (g) for a t r i a i n the absence of PEA (basal developed force, B.D.F.) or following administration of PEA under various conditions. Results express the mean tension (g)<+ S.E.M. for 18-28 tissues. a = sign. d i f f . (p ^ 0.05) compared to basal value, b = sign. d i f f . (p ^-0.05) compared to control curve. B.D.F. I O - 8 IO" 7 IO" 6 IO" 5 - 10 " 4 10" 3 Control (28) 0.6*0.1 0.6*0. 1 0.7'to.l 1.1*0. ,2 a 1.8*0.l a 2.5+0. 3 a 2.8*0. 2 a +3x10 M Cimet. (18) 0.5*0.1 0.5*0. 1 0.6*0.1 1.1+0. a .2 -t- a 1.7*0.2 2.6*0. a 3 3.0+0. ,4a +3x10" M Promet.(24) 0.4+0.2 0.4+0. 2 0.4+0.2 0.6*0. ,2b 1.4+0.I a' b 2.2+0. 4 a 2.7+0. ,4a +10 - 6M Propran.(20) 0.5+0.1 0.5*0.1 0.6*0.1 0.8*0.2 1 . 4 * 0 . l a ' b 1.8*0.I 3' b 1.9*0.2 a , b 43 TABLE 11: The e f f e c t of PEA on c o n t r a c t i l e force of e l e c t r i c a l l y paced (1Hz) guinea-pig l e f t a t r i a . Results express the absolute tension (g) for a t r i a i n the absence of PEA (basal developed force, B.D.F.) or following administration of PEA under various conditions. Results express the mean tension (g) + S.E.M. for 12-28 tissues obtained from e i t h e r control or reserpine-pretreated animals (2.5 mg/kg, 24 hours), a = sign. d i f f . (p ^0.05) compared to basal value, b = sign. d i f f . (p ^0.05) compared to control curve. PEA (M) B.D.F. i o " 8 io" 7 i o " 6 io" 5 i o " 4 i o " 3 Control (28) 0.6 +.l 0.6+0. 1 0.7+0. 1 i.ito. 2 a l.Sto. l a 2.5to. 3 a 2. 8to. 2 a Reserpine (20) 0.7T.1 0.7+0. 1 0.8+0. 1 1.2t0. ,2 a 1.6+0. 2 a 2 a 2.lto. 3 a' b Reserpine +3x10 Cimet.(12) 0.8T.1 0.8+0. .1 0.8+0. .1 i.oto. ,2 1.sto. l a 2.oto. l a 2.2+0. ,2 a -6 Reserpine +3x10 M Prometh. (20) 0.5+0.2 0.5+0. 2 o.sto. ,2 0..9to. 2 1.3+0. 2a,b 1.7+0. 2 a' b 1.9to. 2a,b 45 TABLE 12: The e f f e c t of PEA on c o n t r a c t i l e force of e l e c t r i -c a l l y stimulated (1Hz) guinea-pig r i g h t v e n t r i c l e s t r i p s . Results express the per cent increase i n force (over basal developed force) produced by PEA under various conditions, and represent the mean + S.E.M. of 16-20 tissues, obtained from control or reserpine-pretreated animals (2.5 mg/kg, 24 hours). a = sign. d i f f . (p < 0.05) from control values. 46 PEA (M) i o " 8 i o " 7 IO" 6 IO" 5 IO" 4 IO" 3 Control (20) 0 0 11+3 25+4 90+5 122+20 -6 +10 M Propran.(16) 0 0 5+2 18+5 63±2 a 80+5 +3x10"6M Promet.(16) 0 0 4+2 10+5 66+4a 93+4 Reserpine-pre-treated (20) 0 0 12+8 23+4 58+6a 70+6* 47 TABLE 13: The e f f e c t of 4-methylhistamine on rate of spontaneously beating guinea-pig r i g h t a t r i a under various conditions. Results express the mean rate (beats/min) + S.E.M. of 11-14 tissues. a = sign. d i f f . (p ^ .0.05) compared to basal value, b = sign. d i f f . (p $0.05) compared to control curve. 4-methylhistamine (M) basal H.R. io"8 io"7 io"6 ' io"5 io"4 io"3 Control (14) 177-9 185-10 + a 219-14 + a 255-12 + a 309-13 3 1 9 t 5 a 31 s V +10"7M Propran.(11) 170-6 '• 179-6 + a 204-6 + a 248-4 + a 297-6 + a 315-5 •+ a 321-8 +10 _ 6M Cimet.(12) 183-9 . 185-13 189-6 205-7 a' b 235-8 a' b 289-6 a 316-8 a 49 TABLE 14: The e f f e c t of histamine on rate of spontaneously beating i s o l a t e d rat r i g h t a t r i a under various conditions. Rsults express the mean rate (beats/min) + S.E.M. of 18-24 tissues obtained from control or reserpine pretreated animals (3.0 mg/kg, 24 hours). a = sign. d i f f . (p ^ .0.05) compared to basal value, b = sign. d i f f . (p ^ 0.05) compared to control curve. 50 basal H.R. IO" 4 10" 3 .IO" 2 Control (20) 260 +6 260-6 286±8 a 342-1i a +10 _ 7M Prometh -(20) 2 5 835 2 5 838 289±9 a 38439 a' b +10 _ 7M Cimet.(20) 26234 26234 + a 28033 358t8 a +10_ M Propran .(24) 24036 24036 235+6b 202+ 8 a' b Reserpine pretreat-(18) 24533 24036 + b 22538 1 9 0 + l i a ' b 10"7M Propran.+ 10"7M Atropine (18) 23836 238±6 b 2483lO b 2 8 2 t l 5 a ' b 51 TABLE 15: The e f f e c t of histamine (10 M) on c o n t r a c t i l e force of e l e c t r i c a l l y paced (1Hz) i s o l a t e d rat l e f t a t r i a under various conditions. Results express the per cent.increase i n force of over basal values + S.E.M. of 18-24 tissues obtained from e i t h e r control or reserpine pretreated (3.0 mg/kg, 24 hours) animals. a •= sign. d i f f . (p ^ 0.05) from control values. 52 Treatment -3 10 Histamine Control (20) 80 T12 +10"7M Cimetidine (20) 72*18 +10"7M Promethazine (20) 80 +20 -6 +10~ M Promethazine (24) i s V Reserpine pretreated (18) 10 T12 a 53 FIGURE 1: The e f f e c t of histamine on the rate of spontaneously beating k i t t e n r i g h t a t r i a . Antagonists were added to the buffer one hour p r i o r to addi t i o n of agonist. y axis: absolute rate (beats/min). x axis: log concentration histamine (M). Results represent the mean +.S.E.M. of 7-10 tissues. 54 55 FIGURE 2: Maximal changes in rate produced by 10 M histamine i n i s o l a t e d k i t t e n r i g h t a t r i a . Bars represent the means + S.E.M. of basal rate (con t r o l ) , and maximal _3 responses to 10 M histamine and the absence and presence of various antagonists (obtained from r e s u l t s in Fig. 1). Asterisks indicate a s i g n i f i c a n t decrease in response _3 compared to the e f f e c t of 10 M i n the absence of antagonist (p^0.05). ATRIAL RATE (beats/min) CONTROL +10-7 M l -A o Propranolol I w z +10-5 M X Cimetidine </> +10 6M Cimetidine rn • +10-7 M Promethazine L } ]-3r 5 7 FIGURE 3 : Maximal changes i n force of contraction (expressed as per cent increase over basal developed force) produced by 1 0 M histamine i n the presence of various antagonists i n i s o l a t e d k i t t e n l e f t a t r i a . A sterisks represent a s i g n i f i c a n t decrease i n - 3 response compared to the e f f e c t of 1 0 M histamine i n the absence of antagonist ( p < i 0 . 0 5 ) . Results express the mean + S.E.M. of 7 - 1 0 t i s s u e s . 5 8 111 O OC o LU cn < HI oc o 50-± 10-3M HISTAMINE 59 FIGURE 4: Maximal increase i n force of contraction of k i t t e n r i g h t v e n t r i c l e s t r i p s (expressed as per cent increase over basal developed force) produced by 10 ^histamine i n the presence of various antagonists. A s t e r i s k indicates a s i g n i f i c a n t l y decreased response -3 compared to the e f f e c t of 10 M histamine i n the absence of antagonist (p ^0.05). Results express the mean + S.E.M. of 7-10 tissues. 60 501 UJ O CC O UJ < UJ cc o 0-1 10- 3M HISTAMINE o S o - £ 2 a + 2 a. 4) C s i in J3 L „ 6 1 FIGURE 5: Maximal increase i n force of contraction of k i t t e n r i g h t p a p i l l a r y muscles (expressed as per cent increase over basal developed force) produced by -3 10 Mhistamine i n the presence of various antagonists. A s t e r i s k indicates a s i g n i f i c a n t l y decreased response -3 compared to the e f f e c t of 10 M histamine i n the absence of antagonist (p $-0.05). Results express the mean + S.E.M. of 7-10 t i s s u e s . 62 LU o cc o 50 UJ < LU CC O 10- 3M HISTAMINE o Z o 2 a + 2 a. a> s i i n + o s | e l + o 0 s § O T= 0) + E o 63 FIGURE 6: The e f f e c t of 4-methylhistamine on the rate of spontaneously beating k i t t e n r i g h t a t r i a . Antagonists were added to perfusate one hour p r i o r to addi t i o n of agonist. y axis: absolute rate (beats/min) x axis: log concentration 4-methylhistamine (M). Results express the mean + S.E.M. of 8-12 tissues. 64 log 4-METHYLHISTAMINE (M) 65 FIGURE 7: Maximal changes i n rate of spontaneously beating -3 k i t t e n r i g h t a t r i a produced by 10 M 4-methylhistamine (4MH). Antagonists were added to perfusate one hour p r i o r to a d d i t i o n of agonist, and bars represent the mean + S.E.M. of basal rate (control) and the e f f e c t _3 of 10 M 4MH i n the absence or presence of antagonists. Asterisks represent a s i g n i f i c a n t decrease i n response _3 compared to the e f f e c t of 10 M 4MH i n the absence of antagonist (p ^ 0.05). A T R I A L R A T E b e a t s / m i n o o U l o IN) o o C O N T R O L H o I* •Ft Cimetidine rn 10~6M 10-5 M x < Cimetidine 55 4-10- 6M £ Propranolol z J r -0 67 FIGURE 8: Changes i n force of contraction i n i s o l a t e d k i t t e n l e f t a t r i a produced by 4-methylhistamine (4MH) (expressed as per cent increase i n force over basal developed force) i n the absence or presence of antagonists. A s t e r i s k represents a s i g n i f i c a n t decrease of -3 response compared to the e f f e c t of 10 M 4MH i n the absence of antagonist (p ^0.05). Bars represent the mean + S.E.M. of 10-12 determinat ions. % INCREASE IN FORCE (g) cn O 10-7 M Proranolol 10-6 M Cimetidine O cb I s i 3 ' m CO 69 FIGURE 9: Changes i n force of contraction i n is o l a t e d k i t t e n r i g h t v e n t r i c l e s t r i p s produced by 4-methylhistamine (4MH) (expressed as per cent increase i n force over basal developed force) i n the absence or presence of antagonists. A s t e r i s k represents a s i g n i f i c a n t decrease of response compared to the e f f e c t of 10 M 4MH i n the absence of antagonist (p ^0 . 0 5 ) . Bars represent the mean + S.E.M. of 10-12 determinat ions. % INCREASE IN FORCE (g) cn O O O 10-7 M Proranolol 4* 10-6 M 5 Cimetidine 5 ] m } o 71 FIGURE 10: Changes i n force of contraction i n i s o l a t e d k i t t e n r i g h t p a p i l l a r y muscles produced by 4-methylhistamine (4MH) (expressed as per cent increase i n force over basal developed force) i n the absence or presence of antagonists. Aster i s k represents a s i g n i f i c a n t decrease of _3 response compared to the e f f e c t of 10 M 4MH i n the absence of antagonist (p-^0.05). Bars represent the mean + S.E.M. of 10-12 determinat ions. X INCREASE IN FORCE (g) o cn o O cb. 10-7 M P r o r a n o l o l 10-6 M Cimetidine i x CO ae m 73 FIGURE 11: Changes i n rate of i s o l a t e d guinea-pig r i g h t a t r i a produced by PEA i n the absence or presence of antagonists. y axis: absolute rates (beats/min) x axis: log concentration PEA (M) Results express the mean + S.E.M. of 18-28 experiments. 320-280-ATRIAL RATE beats/min 240« 200-160" 75 FIGURE 12: E f f e c t of PEA on rate of spontaneously beating ' r i g h t a t r i a from untreated (control) and reserpine-pretreated (2.5 mg/kg, 24 hours) guinea-pigs. y axis: absolute rate (beats/min) x axis: log concentration PEA (M) Rsults express the mean + S.E.M. of 12-28 experiments. 320 280 ATRIAL RATE beats/min 240 200-160-control Reserpine Reserpine + 10"*M Propranolol -5 -4 loq PEA (M) -3 77 FIGURE 13: Increases i n tension (g) of e l e c t r i c a l l y paced (1Hz) guinea-pig l e f t a t r i a produced by PEA i n the absence (control) or presence of various antagonists. y axis: absolute developed tension (g) x axis: log concentration PEA (M) Results express the mean + S.E.M. of 18-28 exerpiments. 78 3.5n i • • i 1 1 C -7 -6 -5 -4 -3 log PEA (M) 7 9 FIGURE 14: Increases i n tension (g) produced by PEA i n e l e c t r i c a l l y paced (1Hz) l e f t a t r i a obtained from, untreated (control) and reserpine-pretreated (2.5 mg/kg, 24 hours) guinea-pigs. y axis: absolute developed tension (g) x axis: log concentration PEA (M) Results express the mean + S.E.M. of 12-28 experiments. log PEA (M) 81 FIGURE 15: Increases i n tension of guinea=pig i s o l a t e d r i g h t v e n t r i c l e s t r i p s produced by PEA i n the absence or presence of antagonists. Responses were obtained i n e l e c t r i c a l l y paced (1Hz) v e n t r i c l e s t r i p s from untreated and reserpine pretreated guinea-pigs (2.5 mg/kg, 24 hours). y axis: per cent increase i n force (over basal developed tension) x axis: log concentration PEA (M) Results express the mean + S.E.M. of 16-20 experiments. 82 83 FIGURE 16: Increases i n rate of i s o l a t e d guinea-pig r i g h t a t r i a produced by 4-methylhistamine i n the absence (control) or presence of antagonists. y axis: absolute rate (beats/min) x axis: log concentration 4-methylhistamine (M) Results express the mean + S.E.M. of 12-14 experiments. 84 85 FIGURE 17: Changes i n rate of i s o l a t e d r at a t r i a produced by histamine i n the absence (control) or presence of antagonists. Where a t r i a were obtained from reserpine-pretreated animals, rats were pretreated 24 hours p r i o r to experiment at a dose of 3.0 mg/kg. y axis: absolute rate (beats/min) x axis: log concentration histamine (M) Results express the mean + S.E.M. of 18-20 experiments. 86 87 FIGURE 18: E f f e c t of 10 M histamine on force of contraction (expressed as per cent increase over basal developed tension) of i s o l a t e d l e f t a t r i a obtained from un-treated (control) and reserpine-pretreated rats (3.0 mg/kg, 24 hours). Results express the mean + S.E.M. of 18-24 experiments. 88 1001 — 50 0-* 1 0 - 3 M HISTAMINE 89 DISCUSSION The p o s i t i v e inotropic and chronotropic e f f e c t of histamine has been demonstrated to be the r e s u l t of an i n i t i a l drug-receptor i n t e r a c t i o n (Ash and S c h i l d , 1966; Black et a l . , 1972; Verma and McNeill, 1976 and L e v i et a l . , 1976). The r e s u l t s of the present study indicate that i n some species, the cardiac responses due to histamine are due to an i n t e r -a c t i o n of the agonist with s p e c i f i c cardiac histamine receptors. In addition, the r e s u l t s obtained also demonstrate that cardiac responses due to histamine and i t s analogs are not always due to such drug-receptor int e r a c t i o n s , but are due to histamine-induced release of endogenous noradrenaline. Trendelenburg (1960) demonstrated that i n the i s o l a t e d k i t t e n heart, histamine produced a p o s i t i v e chronotropic response. Since only changes i n rate were reported by.Trendelenburg, i t was an aim of the present study to more c l e a r l y define any changes i n both force and rate of contrac-t i o n produced by histamine i n the i s o l a t e d k i t t e n heart. To do t h i s , a v a r i e t y of tissues were used -- r i g h t a t r i a to measure rate changes and l e f t a t r i a , r i g h t v e n t r i c l e s t r i p s and r i g h t p a p i l l a r y muscles to measure changes in c o n t r a c t i l e force. It is c l e a r from t h i s study that histamine produced pronounced changes i n both rate and force of contraction i n a l l preparations used from the k i t t e n heart. The slow nature of the responses observed i s i n agreement with the fi n d i n g by Trendelenburg that the f u l l e f f e c t to any dose of histamine u s u a l l y takes about 5 minutes to develop. Closer examination of the Trendelenburg study reveals that when the 90 e f f e c t of histamine was studied i n i s o l a t e d k i t t e n a t r i a , only a s i n g l e -6 dose of histamine (6.7 x 10 M) was used throughout. The inappropriatness of such an experimental protocol is demonstrated when the re s u l t s of t h i s study are considered. When histamine was added to i s o l a t e d k i t t e n r i g h t a t r i a , a p o s i t i v e chronotropic response was recorded (Figure 1). Both the histamine E^-receptor antagonist cimetidine and theJS -adrenoceptor antagonist, propranolol caused an i n h i b i t i o n of the histamine e f f e c t . However, this i n h i b i t i o n was only s i g n i f i c a n t at higher doses of histamine, and conse-quently may explain why Trendelenburg (1960) was unable to record any i n h i b i t i o n with a low test-dose of histamine in i s o l a t e d k i t t e n r i g h t a t r i a . Propranolol caused the greatest i n h i b i t i o n of the maximal chrono-t r o p i c response to histamine (Figure 1). This indicates a strong i n -volvement of p -adrenoceptors i n the response to histamine, i n addition to the a c t i v a t i o n of histamine ^ - r e c e p t o r s as suggested by the antagonism with cimetidine. However, Trendelenburg (1960) claimed that the i n h i b i -t i o n of the rate response to histamine following administration of a fS -adrenoceptor antagonist, dichloroisoprenaline, was due to an elevated basal heart rate because of the i n t r i n s i c a c t i v i t y of dichloroisoprena-l i n e , thus reducing any chronotropic changes produced by histamine. The r e s u l t s obtained i n the present study, using propranolol, a -adrenoceptor antagonist with • no i n t r i n s i c a c t i v i t y , indicate that the chronotropic ac t i o n of histamine can be blocked by^-adrenoceptor antagonists. This suggests that the blockade of the histamine response produced by both dichloroisoprenaline and propranolol was due to Q -adrenoceptor blockade 91 and that histamine produces part of i t s chronotropic response by releasing endogenous catecholamines. The presence of histamine l^-receptors i n the k i t t e n r i g h t atrium is indicated by a competitive blockade of the histamine dose-response curve by two doses of cimetidine (Figure 1). The pAg'for the antagonism of histamine at histamine E^-rec'eptors (eg. guinea-pig r i g h t a t r i a ) using burimamide as the antagonist is 5.11 (Black et a l . , 1972). Trendelenburg (1960).reported a mean pA 2 value of 5.1 i n cat r i g h t a t r i a using pyrilamine as the antagonist. The pA 2 value i n organs i n which pyrilamine acts as a s p e c i f i c antagonist of the H-^  e f f e c t i n g histamine, such as the guinea-pig ileum, i s approximately 9.3 (Arunlakshana and Schil d , 1959). Based on these reported values, and on the r e s u l t s of t h i s study i n d i c a t i n g the presence of histamine H 2-receptors i n the k i t t e n r i g h t atrium, i t would appear quite l i k e l y that the blockade observed by Trendelenburg-' (1960) using high doses of pyrilamine was a blockade of histamine Il^-receptors by a histamine H^-receptor antagonist. The reported e f f e c t s of histamine H-^-receptor antagonists on cardiac c o n t r a c t i l i t y and adenylate cyclase a c t i v i t y is unclear. Several workers (Trendelenburg, 1960; Mannaioni, 1960; McNeill and Muschek, 1972) reported that high concentrations of histamine H-^-receptor antagonists resulted i n a blockade of histamine H^-receptor mediated c o n t r a c t i l i t y of the heart i n a non-competitive or i r r e v e r s i b l e manner. When the effects of h i s t a -mine on adenylate cyclase a c t i v i t y was measured, i t was also found that high concentrations of diphenhydramine (Klein and Levey, 1971; McNeill and Muschek, 1972) and mepyramine (Weinryb and Michel, 1975) blocked this e f f e c t . The blockade of the e f f e c t of histamine on cardiac adenylate 92 cyclase by tripelennamine and diphenhydramine was through a non-competitive mechanism according to the study of McNeill and Muschek (1972). On the other hand, Johnson and Mizoguchi (1977) l a t e r reported that the i n h i b i -t i o n of the e f f e c t of histamine on cardiac v e n t r i c u l a r adenylate cyclase by tripelennamine was competitive. Kanof and Greengard (1979) have recently studied the pharmacological properties of a histamine-sensitive adenylate cyclase preparation from guinea-pig cardiac v e n t r i c u l a r muscle. This preparation compared favour-ably with the properties of other histamine ^ - r e c e p t o r s as defined by p h y s i o l o g i c a l experiments in peripheral tissues (Black et_ a_l. , 1972; Verma and McNeill, 1977). For example, the r e l a t i v e potencies of compounds as agonists of p h y s i o l o g i c a l responses mediated by histamine ^ - r e c e p t o r s (but not H^-receptors) agree well with t h e i r r e l a t i v e potencies as a c t i v a -tors of the adenylate cyclase preparation. In addition, the i n h i b i t i o n constants for antagonists (eg. cimetidine, metiamide) for the e f f e c t s of histamine on the chronotropic e f f e c t s on guinea-pig a t r i a and on stimula-ti o n of the adenylate cyclase preparation also agreed (Kanof and Greengard, 1979). However, the adenylate cyclase preparation of Kanof and Greengard (1979) that had a l l the c h a r a c t e r i s t i c s of histamine ^ - r e c e p t o r s was also aff e c t e d by the c l a s s i c a l antihistamines (eg. mepyramine, diphenhydramine, promethazine) in a competitive manner. This fi n d i n g confirmed the e a r l i e r report by Johnson and Mizoguchi (1977) who also found that histamine H-^ -receptor antagonists acted as competitive i n h i b i t o r s of histamine-sensitive adenylate cyclase. According to Kanof and Greengard (197 9) the histamine H^-receptor antagonists were much less potent i n h i b i t o r s of histamine H2 _ receptors coupled to adenylate cyclase than as i n h i b i t o r s of the physio-93 l o g i c a l e f f e c t s of histamine mediated by H-^-receptors. The c l a s s i c a l antihistamines are known to possess l o c a l anaesthetic properties at high concentrations (Dutta, 1949). I t i s cl e a r from the re s u l t s of Kanof and Greengard (1979) that under c e r t a i n conditions (eg. in c e l l - f r e e preparations of guinea-pig cardiac v e n t r i c u l a r muscle) histamine H^-antagonists can competitively i n h i b i t the eff e c t s of histamine on H^-receptors. Kanof and Greengard (1979) also demonstrated that t h i s e f f e c t was dependent on the chemical class of the -histamine H^-antagonist. These authors speculated that the discrepancies i n the l i t e r a t u r e regard-ing the eff e c t s of histamine H^-antagonists on the myocardial e f f e c t s of histamine could i n part be due to differences i n concentrations and types of antagonists used. In view of this suggestion, i t would have been i n t e r e s t i n g to study the eff e c t s of other histamine H^-receptor antagonists i n some of the experiments discussed. It i s evident that histamine l^-receptors are involved i n the chrono-t r o p i c e f f e c t of histamine i n the k i t t e n r i g h t atrium. This r e s u l t was next v e r i f i e d using a s p e c i f i c histamine H2-recept agonist, 4-methyl-histamine. Using this agonist, a dose-dependent increase i n rate was again antagonised by both cimetidine and propranolol (Figures 6 and 7 ) . Again i t is apparent that histamine and i t s analogs produce a p o s i t i v e chronotropic, e f f e c t both v i a a d i r e c t histamine I^-receptor stimulation and through an i n d i r e c t mechanism, namely a release of endogenous cate-cholamines. As was mentioned i n an e a r l i e r discussion of the r e s u l t s , high doses of 4-methylhistamines were required to produce a chronotropic e f f e c t ; this would be expected since Black et a l . , (1972) have pointed out that this agonist possesses a maximum of only 43.07o of the maximum a c t i v i t y 94 of histamine on histamine H.2-receptor mediated responses such as in the guinea-pig r i g h t atrium. Using a test dose of histamine, Trendelenburg (1960) was able to record only small changes i n force of contraction. However, due to a large v a r i a b i l i t y i n the response, Trendelenburg f a i l e d to report any inotropic data for histamine i n the i s o l a t e d k i t t e n heart. In the present study, when the inotropic e f f e c t of histamine was studied i n three elec-t r i c a l l y paced tissues from the k i t t e n heart, neither a histamine H-^ - or ^ - r e c e p t o r antagonist a l t e r e d the responses (Figures 3, 4 and 5). Some potentiation of the histamine response was observed i n the k i t t e n l e f t atrium and r i g h t p a p i l l a r y muscle i n the presence of promethazine (Figures 3 and 5). No r e s u l t s for the e f f e c t of promethazine i n the k i t t e n r i g h t v e n t r i c l e s t r i p are shown, since under these conditions the v a r i a b i l i t y of both the basal tension and the developed tension was e x t r a o r d i n a r i l y high. No immediate explanation for this observation can be provided. Propranolol s i g n i f i c a n t l y reduced the inotropic response to both histamine and 4-methylhistamine i n the k i t t e n l e f t atrium, r i g h t v e n t r i c l e s t r i p and r i g h t p a p i l l a r y muscle (Figures 6 to 10). Since only propranolol and neither of the histamine receptor-antagonists.\ a l t e r e d the inotropic responses of histamine and 4-methylhistamine, i t was assumed that the inotropic responses to these amines i n the k i t t e n heart were due e n t i r e l y to i n d i r e c t mechanisms involving a release of endogenous catecholamines. Histamine i s known to cause a release of catecholamines from the adrenal medalla v i a histamine H^-receptor stimulation (Burn and.Dale, 1926; Emmelin and Muren,. 1949). In the r a t uterus, histamine is thought to release catecholamines through mediation by histamine Ho-receptors (Verma 95 and McNeill, 1976; McNeill and Verma, 1975). I f either histamine H^- or ^ - r e c e p t o r s were involved i n the release of catecholamines during the ino t r o p i c responses of histamine i n the k i t t e n heart, then ad d i t i o n of either promethazine or cimetidine would have reduced the responses to a greater or equal extent than the reduction observed when low doses of propranolol were used. In fact, inotropic responses to 2-methylhistamine and PEA (r e s u l t s not shown) as well as to 4-methylhistamine were also unaffected by the respective blocking agents, but were decreased by the use of propranolol. These r e s u l t s would argue against a receptor-mediated release of catecholamines during the inotropic or chronotropic responses of histamine i n the k i t t e n heart. Owen (1977), i n a preliminary study, reported that histamine-induced changes i n heart rate and force of contraction i n anaesthetised cats could be reduced or abolished by treatment with e i t h e r low doses of propranolol (1 mg/kg) or a high dose of mepyramine (5 mg/kg). In these animals which were pretreated with mecamylamine to block autonomic ganglia and so prevent r e f l e x increases i n heart rate associated with the depressor response to histamine, no d i r e c t e f f e c t of histamine on the heart was reported. Higher doses of histamine caused an increased heart rate that was anta-gonised by metiamide. Owen (1977) therefore concluded that histamine caused tachycardia both by a d i r e c t i n t e r a c t i o n with histamine ^ - r e c e p t o r s as well as by a release of catecholamines from chromaffin tissue. It was previously mentioned in the introduction to t h i s thesis that K l e i n and Levey (1971) were able to obtain an a c t i v a t i o n of myocardial adenylate cyclase by histamine i n guinea-pig, cat and human heart. These authors reported that the histamine-mediated a c t i v a t i o n of adenylate 9 6 cyclase in a l l three preparations was abolished by a dose of diphenhydra-mine (histamine H^-receptor antagonist) that caused no e f f e c t on a s i m i l a r response produced by noradrenaline i n these tissues. In contrast, DL-propranolol antagonised only the noradrenaline effects i n these tissues, and not the histamine a c t i v a t i o n of myocardial adenylate cyclase. While histamine produced a 3 0 0 % stimulation of adenylate cyclase a c t i v i t y i n the guinea-pig heart, under s i m i l a r conditions histamine produced only a 6 0 7 o stimulation of adenylate cyclase a c t i v i t y i n the cat heart (Klein and Levey, 1 9 7 1 ) . Interpretation of the r e s u l t s of the present study on the ef f e c t s of histamine i n the i s o l a t e d k i t t e n heart are at variance with the report by K l e i n and Levey ( 1 9 7 1 ) ; but confirms the lack of a histamine-s e n s i t i v e adenylate cyclase in the k i t t e n heart suggested i n recent experiments by Johnson (personal communication). While studying the nature of the p o s i t i v e inotropic response to histamine i n the guinea-pig l e f t atrium, Broadley and Wilson ( 1 9 7 8 ) reported that. PEA caused an indirecty&-^-adrenoceptor stimulation i n addi-t i o n to i t s d i r e c t histamine H^-receptor stimulation. The p o s i t i v e ino-t r o p i c response of the l e f t atrium in t h i s species is known to be mediated v i a histamine H^-receptors (Reinhard et_ al_. , 1 9 7 4 ; Steinburg and Holland, 1 9 7 5 and Verma and McNeill, 1 9 7 7 ) . Numerous studies i n our laboratory u t i l i s e t his agent as a s p e c i f i c histamine H^-agonist (Verma and McNeill, 1 9 7 7 ; McNeill and Verma, 1 9 7 9 ) i n cardiac preparations of the guinea-pig and rabbit. Histamine H-^-receptors, as well as ^ - r e c e p t o r s , are known to mediate the inotropic responses of histamine i n the guinea-pig r i g h t v e n t r i c l e s t r i p . I t was of i n t e r e s t during this study to reinvestigate the s p e c i f i c i t y of PEA as a d i r e c t receptor stimulant at the histamine H^-97 receptor in both the guinea-pig l e f t atrium and r i g h t v e n t r i c l e s t r i p ; i n addition, i t was also of i n t e r e s t to characterise any chronotropic changes produced by t h i s agonist. PEA produced a dose-dependent chronotropic e f f e c t i n the guinea-pig i s o l a t e d r i g h t atrium that was unaltered i n the presence of either prometha-zine or cimetidine (Figure 11). This r e s u l t was expected because the guinea-pig r i g h t atrium i s known not to contain histamine H^-receptors, based on the c l a s s i c a l studies of Ash and S c h i l d (1966). In addition, cimetidine would not have been expected to antagonise the e f f e c t s of PEA at histamine H-^-receptors, since Black e_t <al. (1972) have demonstrated that the histamine I^-receptor antagonists (at very high concentrations) are non-competitive antagonists at histamine H^-receptors. Based on the r e s u l t s observed using PEA i n the guinea-pig r i g h t atrium, i t can be stated that the chronotropic e f f e c t of this agent is not mediated through either histamine H^- or .^-receptors . Following t h i s r e s u l t , r i g h t a t r i a were next obtained from animals depleted of endogenous catecholamines by pretreatment with reserpine (some tissues were challenged with tyramine to check the pretreatment schedule). In a t r i a that were depleted of catecholamines, PEA produced a modest i n -crease i n rate that became s i g n i f i c a n t only at the higher doses (Figure 12). The r e s i d u a l increase in rate (due to a release of catecholamines) was abolished by using t h e ^ -adrenoceptor antagonist, propranolol. These r e s u l t s indicate that the rate changes produced by PEA i n the guinea-pig r i g h t atrium r e s u l t s from a release of endogenous catecholamines, thus causing a stimulation of | S-adrenoceptors. A part of the inotropic response to PEA i n the guinea-pig l e f t atrium 98 and r i g h t v e n t r i c l e s t r i p also appeared to r e s u l t from i n d i r e c t ^ - a d r e n o -ceptor stimulation, as suggested by the res u l t s obtained i n this study (Figures 13-15). The increases i n c o n t r a c t i l e force produced by PEA i n both the guinea-pig l e f t atrium and r i g h t v e n t r i c l e s t r i p was unaffected by cimetidine and reduced i n the presence of promethazine or propranolol, or following reserpine pretreatment. Since the antagonism by promethazine occurred only at the lower doses of PEA, while the antagonism due to propranolol only at higher doses of PEA, i t would appear that i n the guinea-pi g l e f t atrium (Figure 13) PEA i n i t i a l l y stimulates histamine H^-receptors d i r e c t l y , while at higher doses the agonist causes a non-specific release of catecholamines. Reserpine pretreatment s i g n i f i c a n t l y reduced the maximal response observed with PEA, while at the same time producing no change in s e n s i t i v i t y to the d i r e c t actions of histamine at H^-receptors i n either the l e f t atrium (Figure 14) or the ri g h t v e n t r i c l e s t r i p (Figure 15) of the guinea-pig heart. Verma and McNeill (1977) have previously demonstrated that inotropic responses due to 4-methylhistamine i n the guinea-pig r i g h t v e n t r i c l e s t r i p were associated with increases i n c y c l i c AMP, while changes i n force due.to PEA were not preceded by changes i n c y c l i c AMP in the same tissue. In addition, these workers showed that while changes i n the maximal increase i n force due to 4-methylhistamine took under 30 seconds to develop i n the r i g h t v e n t r i c l e , an equal increase i n force produced by PEA i n the same tissue required over 60 seconds to develop. The slow nature of the response due to i n d i r e c t l y acting doses of PEA is again apparent. However, i t is evident that i n the guinea-pig r i g h t v e n t r i c l e s t r i p , a very narrow dose-range exists for the inotropic response of PEA (Figure 15). 99 This i s i n agreement with Verma and McNeill (1977), and to be expected i n view of the report by Black et al_. (1972) s t a t i n g that the a c t i v i t i e s of histamine H-^-receptor s p e c i f i c agonists r e l a t i v e to that of histamine are poor. Flynn et a l . (1979) in t h e i r studies on the roles of histamine H-p and ILp-receptors i n the working heart preparation of the guinea-pig also reported that PEA had l i t t l e e f f e c t on cardiac function (sinus rate, l e f t i n t r a v e n t r i c u l a r pressure, t o t a l cardiac output and external pressure-volume work) unless administered i n large doses. Even at these doses, the increases were modest. Based on a comparison of potency values, Flynn e_t a l . (1979) concluded that some of the actions of PEA i n t h e i r preparation was due to a possible involvement of histamine l^-receptors. In addition, Flynn e_t a l . (1979) reported that mepyramine did not antagonise the stimulant e f f e c t s of PEA on any parameter, while cimetidine antagonised the ef f e c t s of PEA on a l l parameters with the exception of sinus rate and coronary flow. The d i f f e r e n c e between the re s u l t s obtained i n the present study, and by Verma and McNeill (1977), and those of Flynn e_t a_l. (1979) with regard to the involvement of histamine l^-receptors during the inotropic responses due to PEA i s not immediately apparent. A possible explanation could be that while this study and that of Verma and McNeill (1977) u t i l i s e d the e l e c t r i c a l l y driven guinea-pig r i g h t v e n t r i c l e s t r i p , Flynn et al_. (1979) based t h e i r r e s u l t s on the l e f t v e n t r i c l e of an i s o l a t e d working heart preparation. Therefore, the p o s s i b i l i t y of a difference between l e f t and r i g h t v e n t r i c l e e x i s t s . It has already been mentioned that the chronotropic response due to PEA was unaffected by either promethazine or cimetidine. This f i n d i n g is 1 0 0 in agreement with those of Flynn e_t al_., who were also able to abolish the chronotropic effects of PEA only by using propranolol and not by using either cimetidine alone or i n combination with mepyramine. Since the lack of s p e c i f i c i t y of PEA i s apparent, i t seemed l o g i c a l to next investigate whether a s i m i l a r problem existed with histamine H^-receptor agonists, eg. 4-methylhistamine. The chronotropic response due to this agonist i n the guinea-pig r i g h t atrium was unaffected by the ^ -adrenoceptor antagonist, propranolol, while cimetidine caused a p a r a l l e l displacement, of the dose-response curve c h a r a c t e r i s t i c of competitive i n h i b i t i o n at the histamine H^-receptor. Black et: a l . ( 1 9 7 2 ) reported that t h i s agonist pos s e s s e d ' 0 . 2 7 o of the a c t i v i t y r e l a t i v e to histamine at the guinea-pig ileum ^n v i t r o . Preliminary data (results not shown) indicated that a s i m i l a r s i t u a t i o n existed in the guinea-pig l e f t atrium, where 4 -methylhistamine caused no s i g n i f i c a n t increase i n c o n t r a c t i l i t y . Similar r e s u l t s were reported by Verma and McNeill ( 1 9 7 7 ) . The r e s u l t s of this study using PEA indicate that compared to histamine ^ - r e c e p t o r s , H-^-receptors play a lesser r o l e i n the mediation of the cardiac e f f e c t s of histamine i n the guinea-pig. Whether or not s i m i l a r conclusions are v a l i d i n other cardiac systems that predominantly contain histamine H^-receptors, eg. rabbit heart (McNeill and Verma, 1 9 7 9 ) i s un-c l e a r . However, i n the dog heart, which i s known to respond to histamine predominantly v i a histamine H^-receptors (Powell and Brady, 1 9 7 6 ) histamine has been shown to cause i t s inotropic and chronotropic responses by both a promethazine and a propranolol s e n s i t i v e mechanism (Flacke et_ a l . , 1 9 6 7 ) . Whether the problem of the histamine H^-receptor mediated responses out-l i n e d is a r e f l e c t i o n of a poor a f f i n i t y or a poor e f f i c a c y of the 101 agonists is s t i l l unclear. The inotropic response of PEA i n guinea-pig l e f t a t r i a was also studied i n our laboratory by Tenner and McNeill (1978). These authors attempted to characterise the response by a l t e r i n g the environmental conditions of the preparation. It-was found that conditions enhancing basal developed force (eg. hypothermia, increased frequency of stimulation and increased e x t r a c e l l u l a r calcium concentration) enhanced the s e n s i t i v i t y of the l e f t atrium to isoprenaline, while at the same time producing an opposite e f f e c t on the s e n s i t i v i t y to histamine. In some instances, eg. O o | when the tissue was paced at 4Hz (37 C, 2.2 mM Ca . ) the inotropic response of histamine was not c l e a r l y evident. These authors concluded that the p o s i t i v e inotropic e f f e c t of histamine could be demonstrated only w i t h i n a very narrow range of experimental conditions. Based on the study by Tenner and McNeill (1978), i t is apparent that under normal p h y s i o l o g i c a l conditions, the inotropic response due to h i s t a -mine H^-receptor stimulation i s of l i t t l e importance during the o v e r a l l cardiac response to histamine (that i s , at least i n the guinea-pig). The presence of histamine ^ - r e c e p t o r s i n the rat heart was suggested by Korosec and Erjavec (197 8). Using both cimetidine and promethazine, the involvement of histamine H-^ - and/or ^ - r e c e p t o r s i n the cardiac responses of histamine i n the rat was excluded i n the present study. However, in the presence of propranolol or following reserpine pretreatment, large doses of histamine produced a negative chronotropic e f f e c t (Figure 17). At the same time, the i n o t r o p i c - e f f e c t of histamine i n the rat l e f t atrium was markedly reduced (Figure 18) i n d i c a t i n g that the inotropic responses to large doses of histamine i n the rat was due e n t i r e l y to a release of catecholamines. 102 The negative chronotropic response due to large doses of histamine ( i n the presence of propranolol or i n reserpine pretreated a t r i a ) was reversed by using the muscarinic antagonist, atropine (Figure 17). I t is cl e a r that i n the rat r i g h t atrium, large doses of histamine can release both ac e t y l c h o l i n e and endogenous catecholamines. I t is also abundantly c l e a r that the rat heart possesses no histamine receptors, as claimed by Korosec and Erjavec (197 8) and by Satayavivad et al_. (1977). The rat heart i s known to be i n s e n s i t i v e to histamine (McNeill, un-published; B a r t l e t , 1963). Went et a l . (1954) noted that histamine f i r s t decreases and subsequently increases the force of contraction of the rat heart, while B a r t l e t (1963) reported that the response of histamine i n a perfused rat heart was vari a b l e , producing either a negative inotropic response or a weak p o s i t i v e change. I t i s quite l i k e l y that the responses recorded by Went et a l . (1954) and those of B a r t l e t (1963) were due. to the simultaneous release of both catecholamines and acetyl c h o l i n e by large doses of histamine i n the rat heart. 103 CONCLUSION AND SUMMARY It i s clear from the re s u l t s of this study that a tremendous species difference exists with regard to the d i r e c t actions of histamine i n the heart. In agreement with other workers (Steinberg and Holland, 1975; Verma and McNeill, 1977) i t was also found that the type of receptor i n -volved in the cardiac responses of histamine also depends on the part of the heart examined. The majority of the cardiac responses of histamine i n the is o l a t e d k i t t e n heart were found to be due to a release of endogenous catecholamines-,, with some contribution of histamine ^ - r e c e p t o r s i n the chronotropic responses i n the right atrium. On the other hand, a l l the cardiac responses due to histamine i n the rat heart were through i n d i r e c t mechanisms, i n -volving both catecholamines and acetylcholine. Therefore, the cardiac e f f e c t s of histamine and i t s analogs are not always due to d i r e c t receptor stimulation, since such agents are also able to produce changes due to i n d i r e c t receptor stimulation through release of catecholamines. F i n a l l y , the p h y s i o l o g i c a l s i g n i f i c a n c e of histamine H^-receptors i n the guinea-pig heart is questioned with the find i n g that d i r e c t stimulation of these receptors produced only weak changes i n c o n t r a c t i l i t y , under conditions which normally e x i s t i n vivo. 103a APPENDIX Summary of the cardiac e f f e c t s of histamine i n d i f f e r e n t species. ( A l l - e f f e c t s and muscarinic e f f e c t s (M) are due to i n d i r e c t mechanisms.) 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