"Medicine, Faculty of"@en . "Anesthesiology, Pharmacology and Therapeutics, Department of"@en . "DSpace"@en . "UBCV"@en . "Verma, Subhash Chander"@en . "2010-02-05T23:58:39Z"@en . "1974"@en . "Doctor of Philosophy - PhD"@en . "University of British Columbia"@en . "A time-response and dose-response study of the effects of norepinephrine and phenylephrine revealed that both agonists caused increases in cyclic AMP, cardiac contractility and phosphorylase a. in the isolated perfused guinea pig hearts. Norepinephrine caused a nearly five fold increase in cyclic AMP, whereas phenylephrine produced only a two-three fold increase in the nucleotide. Phenylephrine is less potent and less effective in elevating all the three parameters as compared to norepinephrine. Histamine and its analogs, TD and betazole increased cardiac contractility, phosphorylase a and levels of cyclic AMP in the isolated perfused guinea pig heart. The order of potency for the three compounds was histamine>TD>betazole. Cyclic AMP was found to increase prior to the increase in contractility or phosphorylase a. In the present study, the new H\u00E2\u0082\u0082-receptor blocking agent burimamide, was found to be a specific, competitive blocking agent of both the mechanical and biochemical effects of histamine and its analogs on the heart. Burimamide did not affect the norepinephrine-induced increase in contractility, phosphorylase a or cyclic AMP. Promethazine did interact with cardiac histamine receptors, but the interaction was either non-competitive or competitive non-equilibrium in nature. [the rest of the abstract can be found in the attached PDF file]"@en . "https://circle.library.ubc.ca/rest/handle/2429/19729?expand=metadata"@en . "BIOCHEMICAL AND MECHANICAL EFFECTS OF ADRENERGIC AND HISTAMINERGIC DRUGS by Subhash Chander Verma B.Pharm. ( 1 9 6 4 ) , M.Pharm. (Gujarat University, India, 1 9 6 7 ) , M.Sc, University of Bri t i s h Columbia, 1 9 7 2 . A Thesis submitted in pa r t i a l fulfilment of the requirement for the degree of DOCTOR OF PHILOSOPHY in the Division of Pharmacology and Toxicology of the Faculty of Pharmaceutical Sciences. We accept this thesis as conforming to the required standard The University of Bri t i s h Columbia November 1 9 7 4 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h C olumbia, I agree t h a t 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 purposes 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 . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada Date \u00E2\u0080\u00A2 /QJk 1*74 ABSTRACT A time-response and dose-response study of the effects of norepinephrine and phenylephrine revealed that both agonists caused increases in cyclic AMP, cardiac contractility and phosphorylase a. in the isolated perfused guinea pig hearts\u00C2\u00BB Norepinephrine caused a nearly five fold increase in cyclic AMP, whereas phenylephrine produced only a two-three fold increase in the nucleotide. Phenylephrine i s less potent and less effective i n elevating a l l the three parametersaasc'comparedtto norepinephrine. Histamine and i t s analogs, TD and betazole increased cardiac contractility, phosphorylase a. and levels of cyclic AMP in the isolated perfused guinea pig heart. The order of potency for the three compounds was histamine>TD>betazole. Cyclic AMP was found to increase prior to the increase in contractility or phosphorylase _a. In the present study, the new ^-receptor blocking agent burimamide, was found to be a specific, competitive blocking agent of both the mechanical and biochemical effects of histamine and i t s analogs on the heart. Burimamide did not affect the norepinephrine-induced increase in contractility, phosphorylase a. or cyclic AMP. Promethazine did interact with cardiac histamine receptors, but the interaction was either non-competitive or competitive non-equilibrium in nature. Histamine, 4-methylhistamine, TD and betazole a l l stimulated cardiac -7 -3 adenylate cyclase i n doses ranging from 10 to 10 M of the agonists. The order of potency of the compounds for stimulating the enzyme was histamine> 4-methylhistamine\u00C2\u00BBTD>betazole. Stimulation by the agonists was blocked i n an \u00E2\u0080\u00946 apparently competitive manner, by burimamide, since burimamide (1x10 1x10 ~*M) produced progressive shifts i n the dose-response curves of the i i i agonists to the right. Histamine, 4-methylhistamine, TD and betazole, in that order increased -7 -3 rat gastric adenylate cyclase activity -in doses ranging from 10 to 10 M of the agonists. Burimamide an ^-receptor blocking agent, in concentrations \u00E2\u0080\u00946 of 1-5x10 M antagonized the drug-induced enzyme stimulation. The maximum increase in the enzyme activity was approximately three fold and was competitively blocked by burimamide. The relative order of these agonists in stimulating cardiac or gastric adenylate cyclase was similar to that found when the effect of these agents on cardiac cyclic AMP, contractility and phosphorylase a wass measured. Histamine and histamine analogs relaxed the rat uterus in a dose-dependent manner. The rank order was histamine>4-methylhistamine>TD and betazole. However, 'histamine ididjan'ot-fepro'd-uce-the' activMidheof \"the adenylate cy.clase'preparedifr.pmarat^uter-iif- prepared frcrc. ~\u00C2\u00A3.\u00C2\u00A3 uterr.. Histamine (10 ~*M) , produced the maximum response in the guinea pig ileum, which wase blocked by tripelennamine and diphenhydramine (H^-receptor antagonists), but not by burimamide (^-receptor antagonist). Histamine did not produce any changes in cyclic AMP levels i n the isotonically contracting guinea pig ileum. The data provide some evidence that ^-receptors have similar properties in at least two tissues, gastric mucosa and heart. The data also provide further evidence for the association of the ^ -receptor with adenylate cyclase and dissociation of adenylate cyclase from H^-receptors. The cardiac effects of the catecholamines, and histamine may be mediated through cyclic AMP. The effects of the amines are potentiated or inhibited by the drugs which affect the enzyme phosphodiesterase. Theophylline injection (1 mg) into the perfused guinea pig heart resulted in an increase iv in contractility of about 20% over the control. Theophylline also potentiated the inotropic and phosphorylase a. producing effects of .norepinephrine and histamine but did not produce any changes in cardiac cyclic AMP. Imidazole,jr;a phosphodiesterase stimulator, also caused an increase in cardiac contractility. Imidazole perfusion (40 mM) decreased or abolished the positive inotropic effect of norepinephrine and histamine, and produced a parallel f a l l in the amine-induced increase in cardiac cyclic AMP. However imidazole perfusion did not affect the phosphorylase activating effect of either amine. The eff'ect:cofr.amines:.on ph6sphorylase~ractivation -ih'-pr.es'encea ofd imidazole can not be explained on the basis of cyclic AMP. Both s theophylline and imidazole produce effects on the hearts which are apparently unrelated to their known effects on cyclic AMP phosphodiesterase. It i s known that the methylxanthines release calcium from intracellular storage sites and that imidazole increases the influx of calcium. It i s evident from our data that both theophylline and imidazole produced positive inotropic responses which can be interpreted i n terms of the increased calcium influx. V TABLE OF CONTENTS Page Abstract i i Lis t of tables v i i Lis t of figures v i i i L i s t of abbreviations x i REVIEW OF LITERATURE 1 A. Relationship between hormone and cyclic AMP 1 B. Adrenergic cardiac effects 7 C. Histaminergic effects on: 16 I The heart 16 1=1 The rat stomach 20 III The rat uterus 22 IV The guinea pig ileum 24 D. Interaction of adrenergic and histaminergic drugs with agents affecting phosphodiesterase 25 Specific goals of the present investigation 28 MATERIALS AND METHODS 30 A. Materials 30 I Animals II Drugs and chemicals B. Methods 31 I Phosphorylase assay 31 II Cyclic AMP binding assay 32 III Cardiac adenylate cyclase preparation 34 IV Gastric adenylate cyclase preparation 34 V Uterine*aa'e\"n^llFatte cyclase preparation 35 VI Adenylate cyclase assay 35 VII Protein estimation 36 VIII Time-response study of agonists 36 IX Dose-response study of agonists 37 X Dose-response study of histamine and i t s analogs on rat uterus 38 C. S t a t i s t i c a l analysis of the data 38 v i Page RESULTS A. Cardiac effects of norepinephrine and phenylephrine on contractility, phosphorylase a_and cyclic AMP 40 B. Cardiac effects of histamine and histamine analogs on :c ) contractility, phosphorylase and cyclic AMP and their blockade by burimamide and promethazine. 43 C. Interaction between histamine and i t s analogs and Burimamide on: I Cardiac adenylate cyclase 69 II Gastric adenylate cyclase 77 III Myometrium adenylate cyclase 77 IV Guinea pig ileum 88 D. Interaction of agents affecting ph@\u00C2\u00A7pfe@iit^4sMie: 88 I Actions and interactions of theophylline with histamine and norepinephrine on cardiac contractility, phosphorylase and cyclic AMP 88 II Cardiac actions and interactions of imidazole with norepinephrine and histamine 96 DISCUSSION 105 SUMMARY AND CONCLUSION 119 BIBLIOGRAPHY 122 APPENDIX 137 v i i LIST OF TABLES Table Page 1 Possible involvement of cyclic AMP i n hormone actions on various tissues. 5 2 Possible involvement of cyclic AMP in various metabolic processes. 6 3 The effect of TD, betazole and TD or betazole plus burimamide on cardiac cyclic AMP. 58 4 Effect of histamine (1 yg) and norepinephrine (1 yg) and the interaction of these drugs with burimamide (2xlO~^M) and propranolol (10_^M) on cardiac contractility, phosphorylase and cyclic AMP levels in the perfused guinea pig heart. 59 5 Effect of TD and betazole and TD or betazole and theophylline (10~^M) on cardiac contractility in the isolated perfused guinea pig heart. 61 6 The effects of promethazine, 2xl0 - 6 - 16X10-6M, on the norepinephrine-induced increase in cardiac force. 76 7 The effect of histamine (10~->M);; at various times on the level of cyclic AMP in the guinea pig ileum. 91 8 The effect of 40 mM imidazole perfusion on the positive inotropic effect of histamine and norepinephrine. 100 9 The effect of imidazole (40 mM) on the norepinephrine and histamine-induced increase i n cardiac cyclic AMP. 101 10 The effect of imidazole (40 mM) on the norepinephrine and histamine-induced activation of cardiac phosphorylase. 103 11 The effect of imidazole on phosphorylase a., cyclic AMP and contractile force in the isolated perfused guinea pig heart. 104 LIST OF FIGURES Schematic representation of the second messenger concept Enzymes involved in the control of myocardial glycogenolysis Possible relationships between hormone-receptor interactions and calcium i n effecting an end-organ response. Responses of Hj and H2 receptors and their selective inhibition by antagonists. Time-response effects of norepinephrine (1 yg) on cardiac cyclic AMP, percent phosphorylase a. and contractility i n the perfused guinea pig heart. Time-response effects of phenylephrine (1 mg) on cardiac cyclic AMP, percent phosphorylase a. and contractility in the perfused guinea pig heart. Dose-response effect of norepinephrine and phenylephrine on cardiac cyclic AMP i n the perfused guinea pig heart. The effect of histamine, triazole derivative and betazole on cardiac contractility on the isolated, perfused guinea pig heart. The effect of time on the a b i l i t y of 1 yg of histamine to elevate (A) contractility, (B) phosphorylase and (C) cyclic AMP in the isolated perfused guinea pig heart. The effect of time on the a b i l i t y of 1.6 yg of triazole derivative to elevate (A) contractility, (B) phosphorylase and (C) cyclic AMP i n the isolated, perfused guinea pig heart. The effect of time on the a b i l i t y of 1.6 yg of betazole to elevate (A) contractility, (B) phosphorylase and (C) cyclic AMP in the isolated, perfused guinea pig heart. The effect of histamine, triazole derivative (TRIAZOLE) and betazole on cyclic AMP in the isolated, perfused guinea pig heart. The effect of various concentrations (2-16 x 10~^ M) of burimamide on the contractile response to histamine i n the isolated, perfused guinea pig heart. i x F i g u r e Page 14 The e f f e c t o f burimamide (0.5 x 1 0 \" % ) on t h e c o n t r a c t i l e r e s p o n s e t o t r i a z o l e i n t h e i s o l a t e d , p e r f u s e d g u i n e a p i g h e a r t . 52 15 The e f f e c t o f b e t a z o l e and b e t a z o l e p l u s burimamide on c a r d i a c p h o s p h o r y l a s e a c t i v a t i o n i n t h e i s o l a t e d , p e r f u s e d g u i n e a p i g h e a r t . 53 16 The e f f e c t o f h i s t a m i n e and h i s t a m i n e p l u s burimamide on c a r d i a c p h o s p h o r y l a s e a c t i v a t i o n i n t h e i s o l a t e d , p e r f u s e d g u i n e a p i g h e a r t . 55 17 The e f f e c t o f t r i a z o l e d e r i v a t i v e and t r i a z o l e d e r i v a t i v e p l u s burimamide on c a r d i a c p h o s p h o r y l a s e a c t i v a t i o n on t h e i s o l a t e d , p e r f u s e d g u i n e a p i g h e a r t . 56 18 The e f f e c t o f burimamide (1 x 10~ 6M) on t h e c o n t r a c t i l e r e s p o n s e t o b e t a z o l e i n t h e i s o l a t e d , p e r f u s e d g u i n e a p i g h e a r t . 57 19 The e f f e c t o f h i s t a m i n e and h i s t a m i n e p l u s burimamide on h i s t a m i n e - i n d u c e d i n c r e a s e s i n c y c l i c AMP. 60 20 The e f f e c t o f v a r i o u s c o n c e n t r a t i o n s o f p r o m e t h a z i n e on c a r d i a c f o r c e o f c o n t r a c t i o n . R e s u l t s a r e p r e s e n t e d as p e r c e n t a g e i n c r e a s e o v e r c o n t r o l v e r s u s dose o f p r o m e t h a z i n e p e r f u s e d t h r o u g h t h e h e a r t . 64 21 The e f f e c t o f v a r i o u s c o n c e n t r a t i o n s o f p r o m e t h a z i n e on h e a r t r a t e . 66 22 The e f f e c t o f p r o m e t h a z i n e (4 x 1 0 - 6 - 16 x 10 _ 6M) on t h e h i s t a m i n e - i n d u c e d i n c r e a s e i n f o r c e o f c o n t r a c t i o n . 68 23 The e f f e c t o f p r o m e t h a z i n e (4 x 1 0 ~ 6 - 16 x 10~ 6M) on t h e h i s t a m i n e - i n d u c e d i n c r e a s e i n h e a r t r a t e . 71 24 The e f f e c t o f p r o m e t h a z i n e (4 x 10~^M) on t h e h i s t a m i n e -i n d u c e d i n c r e a s e i n c a r d i a c c y c l i c AMP. 73 25 The e f f e c t o f v a r i o u s doses o f h i s t a m i n e , 4 - m e t h y l h i s t a m i n e , TD and b e t a z o l e on t h e a c t i v i t y o f g u i n e a p i g c a r d i a c a d e n y l a t e c y c l a s e . 75 26 The e f f e c t o f burimamide (5 t o 10 x 10~ 6M) on t h e s t i m u l a t i o n o f g u i n e a p i g c a r d i a c a d e n y l a t e c y c l a s e by v a r i o u s doses o f h i s t a m i n e and 4 - m e t h y l h i s t a m i n e . 79 The effect of burimamide (1 x 10-tlM) on the stimulation of guinea pig cardiac adenylate cyclase by various doses of betazole and TD. The effect of various concentrations of histamine, 3- (g-aminoethyl)-l,2,4 triazole (Triazole), 4-methylhistamine and betazole on rat gastric adenylate cyclase activity. The effect of various concentrations of histamine and 4- methylhistamine and the interaction of these drugs with burimamide on rat gastric adenylate cyclase activity. The effect of 3-((3-aminoethyl)-l,2,4 triazole (Triazole) and betazole and the interaction of these drugs with burimamide on rat gastric adenylate cyclase activity. The effect of various concentrations of histamine, 4-methylhistamirie, 3-(B-aminoethyl)-l,2,4 triazole (Triazole) and betazole on the rat uterus. The effect of theophylline (1 mg) at various times following injection into the perfused guinea pig heart on contractility, % phosphorylase a. and cyclic AMP. The effect of various doses of norepinephrine on cardiac phosphorylase activation in rat hearts perfused with buffer or buffer plus theophylline (7 x lCT^M). The effect of various doses of norepinephrine on cardiac contractility and cyclic AMP in rat hearts perfused with buffer or buffer plus theophylline (7 x lCT^M or 2 x 10~3M). The effect of various doses of histamine on cyclic AMP, contractility, and phosphorylase a. in guinea pig hearts perfused with buffer or buffer plus theophylline (10~^M). The effect of various doses of imidazole on cardiac contractility in guinea pig hearts perfused with buffer. The effect of perfusion of various concentrations of imidazole (0.25-40.OmM) in the isolated perfused guinea pig heart. ABBREVIATIONS ATP adenosine 5'-triphosphate ADP adenosine 5'-diphosphate AMP adenosine monophosphate cyclic AMP adenosine 3',5'-cyclic monophosphate db. cyclic AMP dibutyryl adenosine 3',5'-cyclic monophosphate GTP guanosine 5'-triphosphate cyclic GMP guanosine 3',5'-cyclic monophosphate G-l-P glucose-1-phosphate Pi inorganic phosphate TD 3-(3-aminoethyl)l,2,4 triazole Tris tri(hydroxymethyl)aminomethane ppo 2,5-diphenylonazole popop 1,4-bis[2-(5-phenylonazolyl)]-benzene ACKNOWLEDGEMENTS At the very outset, I express my deep sense of gratitude and sincere thanks to Dr. John H. McNeill, for his very valuable guidance. The unique opportunity provided by Dr. McNeill has been a major factor i n developing my understanding and interest in pharmacology. Special thanks are due to Dean B.E. Reidel for his kind interest i n my training during the course of my studies in this faculty. I am grateful to Dr. D.M. Lyster for his great help with the cyclic nucleotide assay procedures. I wish to thank Drs. Godin, Roufogalis, Sinclair and HajLliday'efor:\u00E2\u0080\u00A2\u00E2\u0080\u00A2 theU'rnuseful comments. x i i i Dedication To Asha and Subodh 1 Review of Literature A. Relationship between hormone and cyclic AMP Complex physiological and metabolic processes in livi n g organisms are partially controlled by hormones. The question as to how hormones act was posed by Sutherland, Rail and. their colleagues. The study of glucagon or epinephrine-induced mobilization of li v e r glycogen led to a possible partial answer to this question. The addition of these hormones to liver slices caused accumulation of a heat stable, dialysable adenosine nucleotide, which eventually was identified as adenosine 3',5' cyclic monophosphate, commonly referred to as cyclic AMP (Sutherland et a l . 1962). Extensive research revealed that cyclic AMP was synthesized following the action of certain hormones on adenylate cyclase, a plasma membrane bound enzyme, distributed in many mammalian tissues (Sutherland et a l . 1962). In addition to epinephrine many polypeptide hormones increase the concentration of cyclic AMP or stimulate adenylate cyclase activity in target tissues Fig.(_l). These findings led to the second messenger hypothesis of hormone action (Sutherland, 0ye and Butcher 1965). The hormone or f i r s t messenger binds to the specific receptor i n the plasma membrane and activates adenylate cyclase, which is located in close proximity to the specific hormone receptor. Adenylate cyclase stimulation by the appropriate hormone results in an increased conversion of (MgP>KTPto cyclic AMP, which i s referred to as a second messenger. The nucleotide influences specific protein kinase to donate phosphate moieties to those effector proteins which ultimately carry out the specific cellular transactions (Walsh et a l . 1968; Garren et a l . 1971; Bitensky et a l . 1973; Butcher and Sutherland 1971). The activation of adenylate Varied Stimuli Endocrine Gland HORMONE (first messenger) Inactivated Hormone O >TC5 etc. Fig.l Schematic representation of the second messenger concept N5 3 cyclase i s specific in nature (Lefkowitz 1973). Adrenocorticotropic hormone w i l l activate adrenal adenylate cyclase, parathormone increases adenylate cyclase in renal cortex and vasopressin in renal medulla (Chase 1968). Glucagon, catecholamines and histamine a l l activate cardiac adenylate cyclase (Murad and Vaughan 1969; Levey and Epstein 1969; Mayer et a l . 1970; Sutherland et a l . 1968; Poch and Kukovetz 1967; Klein and Levey 1971; McNeill and Muschek 1972). Glucagon and epinephrine stimulate l i v e r adenylate cyclase (Birnbaumer and Rodell 1969). In l i v e r , two entirely separate adenylate cyclase systems exist for. glucagon and epinephrine. The enzyme is also stimulated non-specifically by fluoride. Fluoride stimulation occurs in enzyme obtained from any tissue (Sutherland et a l . 1962) and the fluoride activated enzyme cannot be further stimulated by hormones specific to that tissue (Sutherland et a l . 1962; Birnbaumer et a l . 1969). The intracellular concentration of cyclic AMP i s determined by a balance in activities of two enzymes, adenylate cyclase and phosphodiesterase, the enzyme responsible for the metabolism of cyclic AMP to 5' AMP. The activity of both these enzymes can be affected by drugs. In summary, the demonstration of the presence of adenylate cyclase in most mammalian tissues and the fact that a given hormone can increase enzyme activity provides suggestive evidence that the adenylate cyclase-cyclic AMP system may serve as the mediator for the action of that particular hormone on i t s target tissue. However, before a r.easonabllfe degree of credib i l i t y can be attributed to such an hypothesis four c r i t e r i a , as defined by Sutherland and his coworkers (1962), should be f u l f i l l e d : 1. Demonstration of a response to the hormone in a washed broken c e l l preparation. 4 2. An appropriate increase in cyclic AMP in intact cells i n response to hormone stimulation. 3. Drugs affecting phosphodiesterase should potentiate or inhibit the hormone action. 4. Exogenous cyclic AMP or i t s derivatives, should mimic the effect of the hormone. Tablesll\"i_2, summarises the possible involvement of cyclic AMP as a mediator of the actions of various hormones. TABLE 1 Possible Involvement of on Var Tissue Liver Adrenohypophysis Renal medulla Adrenal Cortex I n t e r s t i t i a l Cells Semeniferous tubules Melanophores Thyroid Bone and renal cortex Brain r c l i c AMP in Hormone Actions is Tissues. Hormone Glucagon Hypothalamic releasing hormone Vasopressin Adrenocorticotropic hormone Luteinizing hormone Follicle-Stimulating hormone Melanocyte-Stimulating hormone Thyroid-Stimulating hormone Parathyroid hormone Histamine, Serototiinn Catecholamines, stimulate many target tissues (Robison, G.A. 1972). 6 TABLE 2 Possible Involvement of C y c l i c AMP i n Various Metabolic Processes. P r o t e i n kinase a c t i v a t i o n Phosphorylase a c t i v a t i o n Stimulation of glyconeogenesis Stimulation of ketogenesis Stimulation of l i p o l y s i s Stimulation of steriodogenesis Stimulation of exocytosis I n h i b i t i o n of glycogen synthetase a c t i v i t y I n h i b i t i o n of lipogenesis I n h i b i t i o n of c e l l growth I n h i b i t i o n of p l a t e l e t aggregation I n h i b i t i o n of c e r e b e l l a r purkinje c e l l f i r i n g Increased permeability to water and e l e c t r o l y t e s Increased force of cardiac c o n t r a c t i l i t y (Robison, G.A. 1972). 7 Of the metabolic effects i n which cyclic AMP is supposed to be involved, only a few are understood. The most studied i s the activation of the enzyme glycogen phosphorylase in skeletal muscle (Krebs et a l . 1966; Walsh et a l . 1968). Cyclic AMP increases the activity of a protein kinase (Walsh et a l . 1968) which catalyses the activation of phosphorylase b_ kinase. Phosphorylase b_ kinase in turn catalyzes the phosphorylation of inactive phosphorylase b_ to form active phosphorylase a.. Phosphorylase a. i n turn converts glycogen to G-l-P (Fig. 2 ) . Cyclic AMP-sensitive protein kinases seem to be at least as widely distributed as cyclic AMP i t s e l f (Kuo and Greengard 1969) and many proteins other than phosphorylase and glycogen synthetase can serve as substrates for them. It seems possible, therefore,that many i f not most of the physiologically important effects of cyclic AMP in mammalian cells may be brought about by the activation of a protein kinase. In consideration of the vast amount of information involving cyclic AMP as a second messenger, this introduction w i l l be directed solely to the postulated role of cyclic AMP i n the effects of adrenergic and histaminergic drugs. B. Adrenergic cardiac effects The cardiac effects of several hormones have been postulated to involve an interaction with cardiac adenylate cyclase. In particular catecholamines have been the subject of intensive investigation (Robison, Butcher, 0ye, Morgan and Sutherland 1965; Williamson 1966; Sutherland et a l . 1966; Drummond et a l . 1966; Mayer 1972). Catecholamine effects can be classified into metabolic and mechanical effects. The most studied metabolic effect i s the activation of phosphorylase. The transformation of the enzyme 8 CYCLIC AMP P h o s p h o r y l a s e b-K i n a s e K i n a s e P h o s p h o r y l a s e b K i n a s e ( i n a c t i v e ) ATP . ADP P h o s p h o r y l a s e b K i n a s e ( a c t i v e ^ e ) P h o s p h o r y l a s e \"b\" ( i n a c t i v e ) ATP , Ca ++ ADP * P h o s p h o r y l a s e \" a \" ( a c t i v e ) G l y c o g e n + P i G l u c o s e - 1 - P h o s p h a t e FIG. 2. Enzymes i n v o l v e d i n t h e c o n t r o l o f m y o c a r d i a l g l y c o g e n o l y s i s 9 phosphorylase b_ to the phosphorylated form phosphorylase a., has been used as an index of an increase i n cyclic AMP i n myocardial cel l s . Various workers have studied the relative potencies of adrenergic drugs on a wide range of species. Lands and Howard (1952), reported that epinephrine was more effective than norepinephrine i n increasing the amplitude and rate of contraction in the perfused frog heart and tortoise atria. Epinephrine was less effective than norepinephrine on isolated rabbit atria and perfused rabbit heart, but isoproterenol was much more effective than epinephrine or norepinephrine in producing increases i n rate and amplitude of contraction i n a l l the above preparations (Nickerson and Mullenberg,,1967; Robison et a l . 1970). Catecholamine-induced increases in cardiac contractility have been studied by electrophysiologists in terms of the el e c t r i c a l events at the c e l l membrane (Edman 1965). Catecholamines increased the fluxes of Na + and K + across the c e l l membrane (Glitsch et a l . 1965). An interesting approach to explain the mode of inotropic action of catecholamines, concerns the catecholamine-induced changes in the metabolism of cardiac muscle glycogen. This mechanism explains how the energy is made available for increased cardiac contraction. Catecholamine effects are exerted upon a complex system, referred to as the cyclic AMP-phosphorylase system, which is the target for the action of several drugs (Hess and Haugaard 1958)'. Hess and Haugaard QW:59)) reported that epinephrine caused an increase in phosphorylase activity and a positive inotropic effect i n an isolated rat heart preparation. Kukovetz, Hess, Shanfeld and Haugaard (1959) reported that the inotropic effect and phosphorylase activation were closely associated. Both of these actions were related in a dose-dependent manner. Kukovetz et a l . (1959) have presented a linear relationship between the positive inotropic effect and phosphorylase activity. Mayer and Moran 10 (1960) confirmed the above findings in the open-thorax dog heart preparation and reported that isoproterenol was approximately ten times as potent as norepinephrine or epinephrine. Hess et a l . (1962) found no dissociation between the inotropic responses and phosphorylase activation in the rat heart, even with small doses of epinephrine (0.005-1.0 -tag). However 0ye et a l . (1964) claimed that the inotropic response to the catecholamines could be dissociated from phosphorylase activation by the use of very small doses of epinephrine and norepinephrine. Mayer et a l . (1963) supported the findings of 0ye et a l . (1964) when they reported that in the open-thorax dog heart preparation norepinephrine produced significant inotropic effects with no measurable changes in phosphorylase a. activity. . Shanfeld, Frazer and Hess (1968; 1969) were able to dissociate the contractility of the heart induced by norepinephrine from the effects of the drug on myocardial cyclic AMP content. They were successful in blocking the phosphorylase activation of norepinephrine by N-isopropylmethoxamine '((IMA), without the inotropic responses being significantly depressed. These findings were questioned^ Wastila et a l . (1972) suggested that the results obtained by Shanfeld et a l . (1969) could be an artifact introduced by their readjustment of the diastolic tension after the administration of IMA to the perfused isolated rat heart. Moreover the concentration used (15 ]iig/ml) in their study was probably cardiodepressent. In a similar study, Robison et a l . (1965) using IMA in the isolated perfused rat heart, reported that thenepin'ephrind-ind.uced rise in cyclic AMP or contractility was not blocked by IMA. Murad et a l . (1962) correlated adenylate cyclase activation by drugs to their positive inotropic effect. The relative potencies of isoproterenol}.? norepinephrine and epinephrine in activating adenylate cyclase were similar to those found 11 when phosphorylase activation was obtained (Murad et a l . 1962). Epinephrine increased the levels of,cyclic AMP i n the rat heart and the increase preceded the other adrenergic effects such as the inotropic, chronotropic and phosphorylase activation effects (0ye et a l . 1964; Robison 1965). Williamson (1966) studied the kinetic changes following the injection of 1 gg of epinephrine into the rat heart i n a Langendorff preparation. Within 2 sec. of the epinephrine injection cyclic AMP and contractile force both increased two fold, but phosphorylase levels did not change. Phosphorylase a. levels increased and reached a broader peak at 20-30 sec. Cyclic AMP levels continued to rise, reached a sharp peak at 10 sec. and abruptly decreased to control values within 20 sec. Both contractility and cyclic AMP reached their peak at about the same time. Williamson's findings suggested a causal relationship between the three biochemical and mechanical. parameters. It has been shown that stimulation of beta-adrenergic receptors leads to the activation of adenylate cyclase and an increase in the tissue levels of cyclic AMP (Rail and Sutherland 1961; Mayer et a l . 1963; Robison et a l . 1971; Greengard and Robison 1972; Lefkowitz, Roth and Pastan 11971; Lefkowitz, Sharp and Haber 1973; Lefokowitz and Levey 1972). It has been suggested that adenylate cyclase is i n fact, the beta-adrenergic receptor (Robison et a l . 1966; Mayer 1970; Robison et a l . 1971). Evidence has been presented by Lefkowitz and Haber (1971) and Lefkowitz and Levey (1972) that beta-receptors are separate and distinct from adenylate cyclase. Levey (1971), Levey and Klein;(1972), Lefkowitz (1971, 1973) separated the enzyme adenylate cyclase and beta-adrenergic receptors. A solubilized enzyme preparation was hbrmonally unresponsive and responses could be restored upon the addition 3 of phospholipids but the binding of .[ H] epinephrine to the receptor did not 12 require phospholipid. On the basis of their data, i t appears that the i n i t i a l interaction of norepinephrine is with cardiac beta-receptors, which are coupled to adenylate cyclase by a phospholipid linkage. There i s a specific phospholipid linkage for each respective hormone e.g. phosphotidylserine for glucagon and histamine and monophosphotidylinosital for norepinephrine (Levey 1971). Positive support for the third criterion, i.e. potentiation of the amine effects by theophylline, a phosphodiesterase inhibitor was given by Rail and West (1963) who noted that the inotropic effect of norepinephrine was greatly enhanced in the presence of theophylline. Antagonism of the positive inotropic effect, by imidazole, a potent stimulator of phosphodiesterase (Butcher and Sutherland 1962), provided further evidence for cyclic AMP mediation of the catecholamine action (Kukovetz and Poch 1967). The fourth criterion i s that cyclic AMP or i t s derivative should have the a b i l i t y to produce the end organ response directly. Use of the dibutyryl derivative of cyclic AMP led to the fulfilment of the fourth criterion for the myb'cardium (Epstein 1970; Skelton et a l . 1969; Skelton et a l . 1970). Drummond and Hemmings (1972) reported that perfusion of 0.2 mM db. cyclic AMP into the isolated heart caused a positive inotropic effect within about 2 - 4 min. The maximum effect occured at concentrations of 2 - 3 mM of db. cyclic AMP. Skelton, Levey and Epstein (1970) reported that db. cyclic AMP exhibited a positive inotropic effect on isolated cat papillary muscle driven el e c t r i c a l l y . A study by Krause et a l . (1970) demonstrated that db. cyclic AMP produced both positive inotropic and chronotropic responses i n cultures of spontaneously beating heart c e l l s . Thus a l l four of Sutherland's c r i t e r i a have been satisfied implicating 13 cyclic AMP as a mediator of the positive inotropic action of the catecholamines. One of the suggestive sites for cyclic AMP action is the sarcoplasmic reticulum. Myocardial sarcoplasmic reticulum exhibits adenylate cyclase activity (Katz et a l . 1970). Entman et a l . (1969) have suggested that cyclic AMP increases calcium accumulation by the sarcoplasmic reticulum and that this effect contributes to the positive inotropic effect of the catecholamines. However the cyclic AMP-induced stimulation of calcium accumulation has not been confirmed (Sulakhe and Dhalla 1970). The differences i n the data of Entman et a l . (1969) and Sulakhe and Dhalla (1970) could be due to the difference in the preparation of sarcoplasmic reticulum. Preparations of sarcoplasmic reticulum are extremely d i f f i c u l t to purify and the possibility of contamination with plasma membrane fragments, a li k e l y source of the observed enzyme activity, can not be excluded. Electrophysiological and tracer studies have demonstrated that catecholamines increase the transmembrane flux of calcium ions into the myocardial cells (Engstfeld et a l . 1961; Renter 1967; Vassort et a l . 1969; Carmeliet and Vereecke 1969; Pappano 1970). It i s generally accepted that catecholamines also increase cyclic AMP levels in cardiac tissue (Sutherland et a l . 1968). This increase in the intracellular levels of cyclic AMP and the membrane calcium influx appear to be concurrent effects of catecholamines on the myocardial ce l l s . Rasmussen,., Goodman and Tenenhouse (1972) have cited the importance of calcium\" along with cyclic AMP in adrenergic cardiac effects. Their hypothesis states that stimulation of beta-receptorstbjy catecholamines could simulataneously cause an activation of adenylate cyclase and an alteration of sarcolemmal 2+ permeability to Ca ; or increased levels of cyclic AMP within the c e l l could e l i c i t increased sarcolemmal conductance to calcium. In the f i r s t type 14 FIG. 3. Possible r e l a t i o n s h i p s between hormone-receptor i n t e r a c t i o n s and calcium i n e f f e c t i n g an end-organ response: (Rasmussen et a l . 1972). of interaction cyclic AMP and Ca^ \"' would both function as second messengers, whereas in the latter case cyclic AMP would be the second messenger and 2+ Ca would serve as third messenger (Fig. 3). Entman, Levey and Epstein (1969) reported that norepinephrine-induced increases in cyclic AMP levels caused an increase in calcium accumulation. Electrophysiological studies lend support to a direct effect of cyclic AMP on calcium transport. Coraboeu (1969) showed that cyclic AMP increased the height of the plateau of the action potential i n frog a t r i a l fibres, which is indicative of an increase i n calcium inward current through the'slow Na -Ca channels' of the plasma membrane. In-voltage clamp studies i n calf purkinje fibres, Tsein (1972) concluded that db. cyclic AMP increased the inward current of calcium. Meinertz et a l . (1973) showed that in ele c t r i c a l l y driven l e f t auricles from rat heart, suspended in Tyrode solution with reduced calcium concentration, db. cyclic AMP as well as epinephrine not only increased the force of contraction but also caused an increase in calcium uptake. A recent study by Watanabe and Besch (1974), suggested that cyclic AMP can 2+ activate slow Ca channels. In cardiac glycogeholysis, cyclic AMP operates by regulating phosphorylase b_ kinase, but calcium probably plays a role in stimulating the phosphoprotein product of the cyclic AMP dependent protein kinase; i.e. the activated form of phosphorylase lp_ kinase. In cardiac glycogenolysis, both calcium and cyclic AMP appear to act sequentially. Disorders of cellular calcium metabolism, such as occur in adrenalectomized animals (Miller, Exton and Park 1971) led to a partial inhibition of the physiological response without changes in the i n i t i a l rise i n cellular cyclic AMP concentration. In summary, the bulk of the evidence supports the second messenger 16 hypothesis proposed by Sutherland et a l . (1968), that cyclic AMP may be involved in the effects of beta adrenergic amines on the contractile processes of the heart. Recent reports however [Benfey and Carolin (1971), Benfey (1971)] suggest that phenylephrine does not activate the cardiac enzyme adenylate cyclase, and hence does not produce any changes in the intracellular levels of cyclic AMP. Benfey (1971) also failed to observe any changes in cardiac glycogenolysis,. but he reported that phenylephrine does increase cardiac force of contraction. The lack of relationship between cyclic AMP (ventricles) . and inotropic effects (atrium) may reflect differences between ventricular and a t r i a l i(imusGfLe)'rVabbit heart rather than prove a dissociation between cyclic AMP and the inotropic effects of sympathomimetic amines. Moreover Benfey (1971) did not measure cyclic AMP in a beating heart in which physiological parameters could be simultaneously monitored. A study by McNeill and Davis (1972) suggests that phenylephrine does activate cardiac phosphorylase as well as producing a positive inotropic effect. Benfey (1971) claims that his results refute the theory that cyclic AMP acts as a second messenger in the heart and mediates the inotropic effect of the sympathomimetic amines. Since these data ddbsnott support the second messenger theory, i t was decided to reinvestigate the problem. In the present investigation, time-response and dose-response studies were carried out and the cardiac effects of phenylephrine were compared with those obtained with norepinephrine. C. Histaminergic effects: I Cardiac effects of histamine: Dale and Laidlaw i n 1910, reported that histamine caused an increase 17 in cardiac contractility in the isolated heart of the cat and rabbit. Later on Went and Lissak (1935) and Tiffeneau (1941) reported similar effects of histamine on guinea pig and frog heart. These effects of histamine were reported to be very specific (Mannaioni 1960). In an isolated guinea pig a t r i a l preparation, dichloisoprenaline, a beta-blocker did not antagonize the cardiac histamine effects and in reserpine-t.a?eatee'd animals histamine also produced cardiac effects. Trendelenburg (1960) reported that the classical antihistamines, mepyramine and tripelennamine did not antagonize cardiac histamine effects. But Hughes and Coret (1972) could block cardiac histamine effects by promethazine. Other actions of histamine such as contraction of guinea pig ileum and bronchi (Arunlakshana and Schild 1954) are blocked by the antihistaminic drugs. Like the cardiac histamine effects, histamine effects on gastric acid secretion and relaxation of the rat uterus are not blocked by mepyramine and tripelennamine (Ash and Schild 1966). From these observations Ash and Schild (1966) classified histamine effects, which are blocked by classical antihistamines, as being due to stimulation of \"H^\" type histamine receptors. The search was continued in order to discover a specific histamine antagonist which can antagonize the effects of histamine on the heart and gastric acid secretion, which apparently are controlled by a separate or ^-type of receptor. Black et a l . (1972) in their classical publication reported that burimamide (N-methyl -N ' - (4-(4,5HinetaWp'lV^'^*7-iL thiourea) is a compound which specifically antagonizes the cardiac histamine effects. The cardiac histamine receptors are reported to be different in different species (El-Ackad et a l . 1974). Histamine H^ type of receptors are reported to be present in guinea pig, cat and human heart (Klein and Levey 1971). Rat heart however does not possess histamine receptors (Green and Erickson 1967). 18 HISTAMINERGIC RECEPTORS and H2) and COMPETITIVE ANTAGONISTS H RECEPTORS Guinea-pig ileum H 2 RECEPTORS Rat Man Rat Guinea-pig Rat, man stomach blood uterus atrium gastric pressure secretion Mepyramine Burimamide Metiamide + + + FIG. 4. Responses of H^ and receptors and their selective inhibition by antagonists (according to Black et a l . 1972). 19 El-Ackad et al.(1974) reported that tripelennamine and mepyramine (H^-antagonists) abolished the histamine-induced increase in heart rate and contractility in avian heart, whereas?- burimamide (^-antagonist) failed to alter the chronotropic response produced by histamine. Histamine effects were unaltered by reserpine treatment in avian heart. Histamine thus stimulates avian heart by i t s action on 'H^ ' type receptors. Poch and Kukovetz (1967) suggested the receptors for cardiac histamine effects i n guinea pig heart are associated with adenylate cyclase. Dean (1968) postulated that the cardiac effects of histamine are mediated by the adenylate cyclase-cyclic AMP system. Klein and Levey (1971) indicated that histamine has the capacity to activate adenylate cyclase in the particulate fractions of guinea pig, cat and human heart homogenates. Klein and Levey (1971) found that histamine in equimolar concentrations was about as effective as norepinephrine. McNeill and Muschek (1972), reported that histamine induced activation of adenylate cyclase was not blocked by the antihistamines diphenhydramine and tripelennamine. Theophylline potentiated the cardiac biochemical and mechanical effects of histamine. McNeill and Muschek's (1972) data provided good evidence that histamine f u l f i l s the c r i t e r i a as previously discussed for a cyclic AMP mediated positive inotropic effect on the heart although histamine seems to act on adenylate cyclase at a site which is different from the adrenergic beta-receptor. In the present study, the above working hypothesis was investigated. Time-response and Dose-response effects of histamine and i t s analogs, 3-(g-aminoethyl)1,2,4 triazole (TD), and betazole, on cardiac contractility, phosphorylase activation and cyclic AMP were studied. The interaction of burimamide with histamine and i t s analogs on the above parameters wase also investigated. To further characterize the cardiac histamine receptor, the interaction of 20 histamine on cardiac adenylate cyclase was studied. The nature of blockade by burimamide of the effects of histamine, 4-methylhistamine (a specific agonist), TD and betazole was also investigated. Histamine also stimulates the secretion of the acid by the stomach (Ivy et a l . i'9Sb;a^4h^e^Va'-i.V196*6);. This effect i s also not blocked by classical antihistamines. Therefore i t is probably an E^ t v P e \u00C2\u00B0f receptor. The following section is an attempt to discuss the work supporting the concept of E^ receptors in the stomach. II Gastric effects of histamine: Evidence that histamine acts as a chemical mediator for gastric HC1 secretion was provided by Ivey and Farrel (1925). They reported that histamine acts directly upon the autotransplanted fundic pouch. Davies (1948) showed that histamine stimulated isolated rat gastric mucosa in vitro. Code (1947) reported a relationship between the output of histamine in the gastric juice and the volume of HC1 secretion, when the gastric secretion is stimulated by a meal. Alonso and Harris (1965), reported the activation of gastric acid secretion from gastric mucosa due to gastrin and methylxanthines. Perfusion of rat gastric mucosa with cyclic AMP caused an acid secretion (Shaw and Ramwell 1968). Bersinbaev et a l . (1971) observed that pentagastrin or histamine treatment produced an increase in adenylate cyclase activity in the rat gastric mucosa. The studies of Perrier and Laster (1969, 1970), showed that histamine and the histamine analog, betazole stimulated parietal mucosal adenylate cyclase. Karppanen and Westermann (1973) reported that histamine caused an increase in gastric cyclic AMP in the guinea pig. Levine and Washington (1973) suggested that since stimulation of acid secretion was accompanied by an increased production of cyclic AMP in gastric juice, i t 21 appeared likely that cyclic AMP plays a mediating role in the human gastric secretory response to histamine and betazole. Bieck et a l . (1973), using denervated pouches of the stomach fundus (Heidenhain pouch dogs) reported that histamine produced a dose dependent elevation of cyclic AMP and hydrochloric acid secretion. He further reported a causal relationship between cyclic AMP elevation and the acid output. Karppanen and Westermann (1973) reported that H^ receptor blocking agent, diphenhydramine even in a -4 concentration as high as 3.3x10 M only slightly inhibited the histamine stimulated production,of cyclic AMP. Diphenhydramine did not inhibit histamine secretion of gastric acid. The histamine-induced gastric acid secretion in rats (Black et a l . 1972) and in man (Wyllie et a l . 1972) is suggested to be due to H^ receptor stimulation. Burimamide competitively antagonized the histamine stimulated acid secretion (Wyllie et a l . 1972). Another orally active histamine receptor antagonist metiamide (Black et a l . 1973; Dousa and Code, 1974) inhibited gastric secretion apparently by blocking the stimulation of cyclic AMP formation by histamine and i t s methyl derivative in the gastric mucosa of guinea pig. The present study was undertaken to investigate the effects of Histamine, 4-methylhistamine TD and betazole on rat gastric adenylate cyclase and to ascertain the interaction of these drugs with burimamide. Histamine relaxes the rat uterus and the histamine-induced relaxation i s not blocked by classical antihistamines (Ash and Schild 1966). Black et a l . (1972) suggested the presence of B.^ type of receptors i n rat uterus. In the next section the nature of the histamine-receptor in the rat uterus i s discussed. 22 III Histamine-rat uterus interaction: The finding that histamine caused relaxation of the rat uterus has been reported by Dale and Dudley (1921); Huebner et a l . (1949) and Craver et a l . (1951). Inhibition of the rat uterus by adrenergic drugs has also been reported by Miller (1967); Levyy\" and Tozzi (1963); Triner et a l . (1970); Triner et a l . (1973); Marshall (1973); Diamond (1973). Adrenergic receptors in the rat uterus have been classified as beta-adrenergic receptors although tevyy and Tozzi (1963) reported the presence of alpha receptors as well. Levyy and Ahlquist (1961) showed that phentolamine could reduce adrenergic effects in the rat uterus. Various other authors have failed to confirm the presence of alpha-receptors i n rat uterus (Leonard 1972; Marshall 1970; Kroeger and Marshall 1974). Both histaminergic and adrenergic drugs produce relaxation in the rat uterus. The adrenergic response i s blocked by beta-antagonists (Schild 1967; Triner et a l . 1969; Szego and Davis 1969; Kroeger and Marshall 1971). However classical antihistaminesssuch as mepyramine do not antagonize the histamine induced relaxation i n the rat uterus (Ash and Schild 1966). Black et a l . (1972) were able to block the histamine effects on rat uterus by burimamide. They further reported that histamine receptors in rat uterus were similar to those involved in histamine-stimulated gastric secretion. Blyth (1973), reported that the B.^ receptor may be involved in histamine effects on the rat uterus. Tozzi (1973) however reported that histamine-induced inhibition of the rat uterus is similar to that of tyramine and that i t indirectly activates beta-adrenergic receptors by releasing catecholamines. Propranolol, an effective beta-adrenergic blocking agent produced a significant parallel shift of the histamine, tyramine and isoproterenol dose-response curves. Jensen and Vennerod (1961) have also previously reported that dichloriso-23 proterenol blocked histamine effects on the isolated rat uterus. The findings of Jensen and Vennerod (1961) and Tozzi (1973) suggest that histamine acts either on the same structures as does epinephrine or on a subsequent stage in an epinephrine-initiated process. Tachyphylaxis to histamine and \"tyramine on rat uterus have been previously reported by Tozzi and Roth (1967). Lack of tachyphylaxis to isoproterenol has been reported by Levyy and Tozzi (1963). A l l of the evidence suggests that the action of histamine on the rat uterus is more like that of tyramine than like that of isorpoterenol. The blockade of histamine by cocaine clearly indicates that the inhibitory action of histamine on the rat uterus as the result of indirect activation of beta-adrenergic receptors through the release of catecholamine(Tozzi and Roth 1967; Tozzi 1973). Green and Miller (1966) reported that the effects of histamine on the rat uterus were blocked in the rats treated with reserpine. If histamine works through the release of catecholamines in the rat uterus, then: liis.taminehwou'ldhbe expeetedrto-.producebother.Acha.racteristics of betapadrenerg'ic receptortstimu!^ by the catecholamines results in the elevation of cyclic AMP (Triner et a l . 1970; Marshall 1973; Marshall and Kroeger 1973; Kroeger and Marshall 1974). The a b i l i t y of various catecholamines to relax the uterus and to increase i t s cyclic AMP content is directly related to the effectiveness of these amines as activators of beta-adrenoceptors (Kroeger and Marshall 1974). Both relaxation and the increase in cyclic AMP is prevented by beta-blockers (Triner et a l . 1971). Db. cyclic AMP and the phosphodiesterase inhibitor papaverine, relaxed the smooth muscle and potentiated the inhibitory effects of beta-adrenoceptor activation (McFarland et a l . 1971; Mitznegg et a l . 1970; Pbch and Kukovetz 1967; Polacek and Daniel 1971; Somlyo et a l . 1970; Takagi et a l . 1971). Histamine activates cardiac adenylate cyclase and 24 increases cyclic AMP. (McNeill and Muschek 1972, Klein and Levey 1971) and the cardiac effects of histamine are specific and direct. Similarly, \u00E2\u0080\u009E histamine also stimulates the gastric mucosa\"and elevates cyclic AMP (Shaw and Ramwell 1968; Bieck, Oates and Robison 1973). Both cardiac and gastric histamine receptors are classified as B.^ type receptors.- (Black et a l . 1972). If the histamine receptor in rat uterus is also of type, then i t s stimulatibnbby histamine may result i n an increase i n uterine cyclic AMP. Therefore in the present study the histamine interaction with adenylate cyclase particles prepared from myometrium was investigated as well as the relaxing effect of histamine and i t s analogs. IV Histamine-guinea pig ileum interaction:. The specific antagonism of histamine by antihistaminicrdEugs characterizes one type of histamine receptors for which Ash and Schild (1966) suggested the symbol H^. Such receptorscbecur i n guinea pig ileum and bronchi (Arunlakshana and Schild 1959). The histamine type of receptors such as in the heart and in the gastric mucosa are associated with the enzyme adenylate cyclase (Pbch and Kukovetz 1967; McNeill and Mus\"chek'cl972) . Stimulation of H^-receptors causes the subsequent increase in cyclic AMP (Poch and'Kukovetz 1967; McNeill and Muschek 1972; Bieck, Oates and Robison 1973). receptorslhav.e not been investigated with regard to their possible involvement with adenylate cyclase. It was therefore of interest to investigate the effects of histamine on such receptors i n order to determine i f cyclic AMP was involved. 25 D. Interaction of adrenergic and histaminergic drugs with agents affecting phosphodiesterase Adrenergic and histaminergic drugs may exert their inotropic and chronotropic effect through elevation of cyclic AMP (Robison et a l . 1965; Sutherland et a l . 1968; Poch and Kukovetz 1967; McNeill and Muschek 1972). Increased levels of cyclic AMP may cause an increase in cardiac contractility and phosphorylase a.. Blockade of the increase in cyclic AMP by appropriate blocking agentsvwaJlll result in the blockade of cardiac responses. Other c r i t e r i a to establish that cyclic AMP is involved as a'second messenger' have been established by Sutherland et a l . (1968). For example, the effects of drugs working through cyclic AMP should be enhanced by prior administration of a drug that inhibits the breakdown of the nucleotide by inhibiting phosphodiesterase. The methylxanthines are known to inhibit phosphodiesterase in vitro (Butcher and Sutherland 1962). The influence of theophylline on the cardiac response to catecholamines hhas been the subject of several studies. I n i t i a l l y , Rail and West (1963), reported that the inotropic effects of norepinephrine in isolated rabbit atria were potentiated in the presence of theophylline. Subsequently, Hess and co-workers (1963) found that theophylline pretreatment reduced the contractile response to epinephrine in isolated perfused rat hearts. The discrepancies in their findings could be due to the different doses of theophylline used. McNeill, Nassar and Brodyy (1969) established that the dose of theophylline which would potentiate the effects of the norepinephrine on cardiac phosphorylase was cardiodepressant. Similarities have been reported between the cardiovascular effects of the methylxanthines and the 3-adrenergic effects of adrenergic amines (Robison et a l . 1965). For example, both groups of drugs increase cardiac contractility and relax the intestinal smooth muscle (Pfaffman and 26 Mcfarland (1973). Papaverine, another phosphodiesterase inhibitor also w i l l produce smooth muscle relaxation (Kukovetz and Poch 1971). Similar effects have also been reported for catecholamines. Since the positive inotropic response to theophylline was not blocked by propranolol or phentolamine, i t was therefore not mediated by catecholamine release (Massingham and Nasmyth 1972). Both normal and reserpinized atria responded with increased concentration in a solution containing caffeine. In recent years, however, a number of investigations emphasized the differences rather than similarities betweenxanthines?and:>ica-tecfaoframfae^deGubareff and Sleator 1965; McNeill et a l . 1969; Blinks et a l . 1972). Since methylxanthines have been found to release ionized calcium from intraeel'lularar; storage sites, this may be the process whereby the xanthines increase myoplasmic calcium ions to cause positive inotropic effects (Nayler 1963); Nayler (1967), demonstrated that the enhanced influx of calcium ions, which accompanies excitation in caffeine-treated toad hearts, resulted in a relatively high myoplasmic calcium ion concentration and hence more powerful contractions. The action of caffeine in producing contractures in skeletal muscle has been studied by Axelsson and Thesleff (1958) and Frank (1962), and i t has been shown that this process i s I [ intimately associated with the intraceMul'arx Ca concentration. Caffeine I | causes an increased efflux of Ca from skeletal muscle when placed in a calcium free solution (Bianchi 1961). In heart muscle, calcium flux measurements (Niedergerke 1959; Winegrade and Shanes 1962) showed that the inward movement of calcium, which occurs with membrane excitation, i s the probable trigger mechanism for the in i t i a t i o n of contraction. Chapman and Niedergerke (1970) have proposed that the slow tension changes in frog heart muscle could be related to accumulation and depletion of stored calcium inside the c e l l . 27 Despite the evidence that methylxanthines w i l l inhibit phosphodiesterase (Butcher and Sutherland 1962; McNeill et a l . 1973) i t has not been established that the use of these drugs results in an increase in cardiac cyclic AMP. Support for the theory that cyclic AMP is involved in the cardiac action of certain drugs has been provided by Kukovetz and Poch (1967), who observed that, imidazole, a phosphodiesterase stimulator, inhibited the positive inotropic and phosphorylase stimulating effects of isoproterenol. As in the case of theophylline, however there is not much data regarding the effect of imidazole on cardiac cyclic AMP. The recent study of Knope et a l . (1973) has raised some interesting questions regarding the cardiac actions of imidazole. These authors have shown that imidazole exerted a positive inotropic effect on isolated atria ahdidhbfeheVintactlh:e\"ar-t-^ Imidazole does not stimulate adenylate cyclase (McNeill and Muschek 1972), i t s cardiac effects are not enhanced by aminophylline, and i t s positive inotropic effects were not blocked by propranolol (Knope et a l . 1973). Imidazole thus appears to affect the heart directly by a mechanism other than cyclic AMP. DeMello et a l . reported that the addition of imidazole (500 yg/ml) to depolarized heart muscle elicit e d a contracture. DeMello et a l . (1973) further reported that imidazole-induced contractures were largely dependent on extracel'Mlarar calcium concentration. Contractures induced by imidazole were quickly suppressed in the calcium-free solution. Massingham (1969), also reported that imidazole (5x10 _2M) increased the response of the ventricle strip to stimulation. The effects of imidazole on the frequency-force characteristics of isolated l e f t rabbit atria are. similar to the actions of cardiac glycosides and calcium (Tuttle and Farah 1962). Farah and Witt (1963) suggest that the site of action of imidazole may be upon tissue calcium or calcium turnover. 28 It i s evident that a l l the effects of theophylline and imidazole cannot be explained in terms of their actions on phosphodiesterase. In light of the above findings the effects of both imidazole and.theophylline on cardiac cyclic AMP, phosphorylase activation and contractility, were studied. The interaction between these agents and norepinephrine or histamine on the above parameters was also studied in an effort to determine i f cyclic AMP was involved in the action of these drugs. Specific goals of the present investigation: 1. To study the cardiac effects of phenylephrine and to compare these with the cardiac effects of norepinephrine. 2. To characterize cardiac histamine receptors by means of a selective histamine (I^) blocking agent, burimamide. 3. To study the interaction between an blocking agent promethazine, and the histamine cardiac effects. 4. To determine the interaction between histamine and histamine analogs and burimamide on cardiac, adenylate cyclase. 5. To investigate the effects of histamine and histamine analogs, on rat gastric adenylate cyclase and i t s interaction with burimamide. 6. To study the interaction of histamine on adenylate cyclase prepared from estrogen primed rat uterus. 7. To investigate the effects of histamine on receptors (guinea pig ileum) with respect to their association or dissociation from adenylate cyclase. 8. To investigate the cardiac effects of theophylline and the possible interactions of theophylline with histamine and norepinephrine on their cardiac biochemical and mechanical effects.. To study the effects of the cardiac phosphodiesterase stimulator, imidazole and i t s interaction with histamine and norepinephrine on cardiac contractility, phosphorylase a. activation and cyclic AMP. 30 MATERIALS AND METHODS Materials I Animals Guinea pigs (500-700 gms) and rats (200-250 gms) of either sex were used throughout the investigation. They received food and water ad. libitum. II Drugs and Chemicals The following chemicals and drugs were purchased from Sigma Chemical Co. St. Louis, Mo. Cyclic AMP (phosphoric acid), ATP (disodium sa l t ) , protein kinase inhibitor, cyclic AMP dependent binding protein, phenylephrine hydrochloride, 1-norepinephrine HCl (l-ar.feferenolHHSl*)., Imidazole (grade III, crystalline), 45-oc O -30 X a. Vi o X -20 Ho 30 40 TIME ( S E C . ) FIG.'6. Time-response e f f e c t s of phenylephrine (1 mg) on cardiac c y c l i c AMP, percent phosphorylase a. and c o n t r a c t i l i t y i n the perfused guinea pig heart. Each point represents the mean of three to f i v e hearts and the v e r t i c a l bars represent \u00C2\u00B1 S.E.M. 43 obtained with norepinephrine. In the next experiment, a dose-response relationship of phenylephrine and norepinephrine in their a b i l i t i e s to increase cardiac cyclic AMP was studied (Pig. \u00C2\u00A7 ) . From the time-response study, the time at which cyclic AMP peaked was selected for a dose-response study. Different doses of the amines were injected and hearts were frozen and analysed for cyclic AMP. The data demonstrate that both amines produce dose-dependent increases in cyclic AMP. Maximum values of cyclic AMP obtained with norepinephrine were 2.3 \u00C2\u00B1 0.43 nmoles/gm wet wt. and with phenylephrine were 1.25 \u00C2\u00B1 .101 nmoles/gm wet wt. Phenylephrine was less potent and less effective than norepinephrine in elevating cardiac contractility, phosphorylase a. and cyclic AMP. B. Histamine and histamine analogs cardiac effects A dose-response study with histamine, TD and betazole on cardiac contractility (Fig. _8) showed that histamine was more potent than i t s two analogs. From Fig. _8, the dose for histamine, TD and betazole was selected to study their time-response relationship. Fig. 9, lf03, l'l1', shows: that histamine TD and betazole a l l increased cyclic AMP, phosphorylase a. and cardiac contractility. Significant elevation (PL\u00C2\u00A7.05) of histamine-induced cyclic AMP was observed at 11 s e c , peaking at 18-19 sec. Contractility and phosphorylase a. values were significantly elevated at 15 sec. The control values of cyclic AMP were 0.50 \u00C2\u00B1 .08 nmoles/gm wet wt. The increase in cyclic AMP was five fold over the control values. Cardiac contractility peaked at 20-30 sec. while phosphorylase SL values peaked at a value of 41.0 \u00C2\u00B1 1.0% at a later time (30-40 s e c ) . Following an injection of TD in the isolated guinea pig heart; cyclic AMP levels increased from 0.5 \u00C2\u00B10.1 nmoles/gm wet wt. to 2.0 \u00C2\u00B1 0.lOnnmoles/gm 44 \u00C2\u00A9 2.5-E CD Vi O \u00C2\u00A3 7 O . < n= 3-5 NOREPINEPHRINE |jsjgj -2 2.CH 1.5-1.0H u PHENYLEPHRINE (PEJ 0.2 0.4 T 0 .8 D O S E (ug-NE, mg-PE) T 1.6 FIG. 7. Dose-response effect of norepinephrine and phenylephrine on cardiac cyclic AMP i n the perfused guinea pig heart. Hearts were frozen at the peak of the elevation of cyclic AMP (14 seconds with norepinephrine and 18 seconds with phenylephrine). Each point represents the mean of three to five hearts and the v e r t i c a l bars represent \u00C2\u00B1 S.E.M. 45 UJ CO D O S E [>i$} PIG. 8. The e f f e c t o f h i s t a m i n e , t r i a z o l e d e r i v a t i v e and b e t a z o l e on c a r d i a c c o n t r a c t i l i t y on t h e i s o l a t e d , p e r f u s e d g u i n e a p i g h e a r t . Each p o i n t r e p r e s e n t s t h e mean \u00C2\u00B1 S.E.M. o f t h r e e t o f i v e d e t e r m i n a t i o n s . R e s u l t s a r e e x p r e s s e d as a p e r c e n t a g e o f t h e maximum r e s p o n s e t h a t c o u l d be o b t a i n e d w i t h h i s t a m i n e . 46 A TIME (\u00E2\u0080\u009Ecj FIG. 9. The e f f e c t of time on the a b i l i t y of 1 yg of histamine to elevate (A) c o n t r a c t i l i t y , (B) phosphorylase and (C) c y c l i c AMP i n the i s o l a t e d , perfused guinea p i g heart. C y c l i c AMP was s i g n i f i c a n t l y elevated (P < .05) at 11 seconds. C o n t r a c t i l i t y and phosphorylase EL values were s i g n i f i c a n t l y elevated at 15 seconds. Control c y c l i c AMP lev e l s were 0.50 \u00C2\u00B1 0.08 nmol/g wet wt. of ti s s u e . Control percent phosphorylase a. l e v e l s were 5.0 \u00C2\u00B1 1.0%. 47 wet wt., a four fold increase over the control values. Cardiac force of contraction and phosphorylase a. levels were significantly increased (P~< 0.05) at 15 and 25 sec. respectively. TD-induced increases i n contractility reached a peak at 20-30 sec., but phosphorylase a_ elevation lagged behind, reaching a peak at 40 sec. Contractility increased to 48.2 \u00C2\u00B1 1.8% over control, while phosphorylase ja values peaked at a value of 17.5 \u00C2\u00B1 2.0% at a later time. Fig. _lGf shows the time-response correlation of betazole-induced changes i n cyclic AMP, contractility and phosphorylase a., values. Cyclic AMP reached a peak at 20 s e c , contractility and phosphorylase si reached a maximum at 20-30 sec. and 40 sec. respectively. The effect of these agonists on cyclic AMP formation was dose-dependent as shown i n Fig. 1-2; The order of potency in increasing cyclic AMP was histamine>TD>betazole. Peak values of cyclic AMP for histamine were 2.3 \u00C2\u00B1 0.05, for TD 2.1 \u00C2\u00B1 0.1 and for betazole 1.5 \u00C2\u00B1 0.07J/ nmoles/gm wet wt. of the tissue. In the next series of experiments, the interaction of burimamide with histamine, TD and betazole on cardiac contractility, phosphorylase a. activation and cyclic AMP was studied. Fig. 13*shows the effect of various concentrations (2-16x10 \"*M) of burimamide on the contractile response to histamine. The data were plotted as a percentage of the maximum response that could be attained with histamine. The next two Figs. 14, 15,, show the effect of 0.5x10 ~*M and \u00E2\u0080\u00946 1x10 M burimamide on the contractile response produced by TD and betazole. The dose-response curves of histamine, TD and betazole on cardiac contractility were shifted in an apparently parallel fashion by the doses of burimamide used. Similar effects were noted with burimamide in shifting the dose-response curve of histamine, TD and betazole on cardiac phosphorylase activation (Fig. _16, 17, 18). FIG. 10. The effect of time on the a b i l i t y of 1.6 yg of triazole derivative to elevate (A) contractility, (B) phosphorylase and (C) cyclic AMP in the isolated, perfused guinea pig heart. Cyclic AMP was significantly (P < .05) elevated at 11 seconds, contractility at 15 seconds and phosphorylase at 25 seconds. Control cyclic AMP levels were 0.50 \u00C2\u00B1 0.10 nmol/g wet wt. of tissue. Control percent phosphorylase a_ levels were 5.2 \u00C2\u00B1 1.0%. 75\ l u \J r< O 20 Z 15 Z * 5H H.2fH &T5H o o a: 5 * 2.0-E *>v. \u00C2\u00BB\u00C2\u00BB 1;5-J U o 1.0-rv. 0:5-< u >-u n= 3-5 CONTRACTILITY 49 DOSE OF BETAZOLE 1.6 10 \u00E2\u0080\u0094r\u00E2\u0080\u0094 30 40 TIME (SEC) n=3-5 PHOSPHORYLASE ,1 i 20 30 40 50 ,60 TIME (SEC.} CYCLIC AMP T 20 - i r-30 40 TIME (SEC.) FIG. 11. The e f f e c t o f t i m e on t h e a b i l i t y o f 1.6.yg o f b e t a z o l e t o e l e v a t e (A) c o n t r a c t i l i t y , (B) p h o s p h o r y l a s e and (C) c y c l i c AMP.in t h e i s o l a t e d , p e r f u s e d g u i n e a p i g h e a r t . 50 n= 3-5 : 2 . O H \u00C2\u00A3 Vi \u00C2\u00A3 1 . 5 H o E < y o . 5 H U >-u .......J --\u00E2\u0080\u00A2I HISTAMINE \u00E2\u0080\u00A2TR IAZOLE B E T A Z O L E 0 . 2 0.4 0.8 i . \u00C2\u00AB D O S E Lug) 3 . 2 FIG. 12. The effect of histamine, triazole derivative (TRIAZOLE) and betazole on cyclic AMP in the isolated, perfused guinea pig heart. Agonists were injected and the hearts' were frozen 18 seconds later at the peak of the response. Control cyclic AMP levels were 0.50 \u00C2\u00B1 0.09 nmol/g wet wt. of tissue. 51 % OF MAXIMUM RESPONSE FIG. 13. The effect of various concentrations (2-16x10 M) of burimamide on the contractile response,to histamine in the isolated, perfused guinea pig heart. The data are plotted as a percentage of the maximum response that could be attained with histamine. 52 % I N C R E A S E IN F O R C E FIG.' 14. . The effect of burimamide (0.5x10 M) on the contractile response to triazole in the isolated^ perfused guinea pig heart. Data are presented as percent increase,, over control. 53 % INCREASE IN F O R C E O ^-4 O O to \u00E2\u0080\u00A2 m coH O co 3 II CO i FIG. 15. The e f f e c t of betazole and betazole plus burimamide on cardiac phosphorylase a c t i v a t i o n i n the i s o l a t e d , perfused guinea p i g heart. .JZ.^.'-S \u00E2\u0080\u00A2 \u00E2\u0080\u00A2 C T S . ' -' . P de\"^:\"^ad :r~ f i g -z 2 \" . , . 54 Histamine produced an increase in cyclic AMP and Fig. _19_' illustrates that 2x10 ~*M burimamide completely abolished the histamine (1 yg)-induced cyclic AMP elevation. The blockade was overcome by increasing the dose of histamine to 6.4 yg. The effects of TD (1,6 yg) and betazole (1.6 yg) on cardiac cyclic AMP were also blocked by burimamide.(Table _3). The specificity of burimamide in blocking the histamine effects was tested in the next experiments. The data presented i n Table 4- illustrates that burimamide (2x10 \"*M) did not affect the norepinephrine-induced increase in contractility, phosphorylase a^ or cyclic AMP. Propranolol, a beta-antagonist, did not decrease the histamine-induced biochemical and mechanical effects. In other words, propranolol blocked the effects o^rndregirfephrine and burimamide blocked the effects of histamine and i t s analogs. _3 Theophylline (10 M) potentiated the effects of TD and betazole on cardiac contractility in the isolated perfused guinea pig heart. Table _5 represents the responses due to various doses of TD.and betazole on cardiac contractility and their potentiation by theophylline. McNeill and Muschek _3 (1972), had previously reported that theophylline (10 M) potentiated the histamine effects on cardiac contractility in the isolated perfused guinea pig heart. Burimamide shifted the dose-response curves for the cardiac effects of histamine to the right. The effects of promethazine (an H^ receptor antagonist) and the nature of i t s interaction with cardiac histamine receptors \u00E2\u0080\u00946 were investigated. Promethazine perfusion in a concentration of 2-4x10 M through the guinea pig heart caused a slight positive inotropic effect (Fig. 20). These effects were not seen at higher promethazine concentrations r g (8-16x10 M). Promethazine (2-4x10 M) produced a positive chronotropic effect but a slight negative inotropic effect was noted at higher concentrations 55 % P H O S P H O R Y L A S E a FIG. 16. The effect of histamine and histamine plus burimamide on cardiac phosphorylase activation in the isolated, perfused guinea pig heart. Each point represents the mean percent phosphorylase ji \u00C2\u00B1 S.E.M. of five determinations. Burimamide was perfused through the heart for 15 to 20 minutes before histamine injection. Percent phosphorylase a. values in hearts injected with buffer solution were 5.0 \u00C2\u00B1 1.0%. 56 % P H O S P H O R Y L A S E a K3 O Or C7 O . Co oo rn C O Jo ho o 3 II FIG. 17. The e f f e c t o f t r i a z o l e d e r i v a t i v e arid t r i a z o l e d e r i v a t i v e p l u s burimamide on c a r d i a c p h o s p h o r y l a s e a c t i v a t i o n ' o n t h e i s o l a t e d , p e r f u s e d g u i n e a p i g h e a r t . Eir. t - \ f - ,\u00E2\u0080\u00A2 \u00E2\u0080\u009E * \u00C2\u00BB 57 % P H O S P H O R Y L A S E a FIG. 18. The e f f e c t o f burimamide (1x10 M) on t h e c o n t r a c t i l e r e s p o n s e t o b e t a z o l e i n t h e i s o l a t e d , p e r f u s e d g u i n e a p i g h e a r t . D a t a a r e p r e s e n t e d as p e r c e n t i n c r e a s e o v e r c o n t r o l . 58 TABLE 3 Effect of TD, betazole and TD or betazole plus burimamide on cardiac cyclic AMP. Hearts were frozen at the peak of the cyclic AMP response. Drug Treatment Burimamide Concentration Cyclic AMP (nm/gm net weight) None 0 0.48 \u00C2\u00B1 0.13 TD 1.6 yg 0 1.97 \u00C2\u00B1 0.12* TD 1.6 yg 0.5 x 10~5M 0.51 \u00C2\u00B1 0.08 TD 6.4 yg 0.5 x 10~5M 2.10 \u00C2\u00B1 0.03* Betazole 1.6 yg 0 1.38 \u00C2\u00B1 0.15* Betazole 1.6 yg 1 x 10 M 0.53 \u00C2\u00B1 0.05 Betazole 6.4 yg 1 x 10~6M 1.42 \u00C2\u00B1 0.08* N = 3-5 * Significantly (P <0.05) greater than no drug treatment. 59 TABLE - 4 The effect of histamine (1 yg) and norepinephrine (1 yg) and the interaction of these drugs with burimamide (2xlO~^M) and propranolol (10~%) on cardiac contractility, phosphorylase and cyclic AMP levels in the perfused guinea pig heart. Histamine and norepinephrine were injected via a side-arm cannula. The antagonists were perfused through the heart for 15-20 min. prior to the injection of the agonist. Cyclic AMP was measured 18 sec. and phosphorylase a. 40 sec. after injection of the agonist. Drug Cyclic AMP % Phosphorylase a. Contractility (nmole/gmwwet (% increase over weight) control) Buffer solution 0.68 + 0.05 5.0 + 1.0 0.00 Histamine 2.50 + 0.09a 44.7 . + 1.2a 90.6 \u00C2\u00B1 1. 2 a Histamine & Burimamide 0.65 + 0.04b 7.5 + 1.4b 15 \u00C2\u00B1 1. 6 b His tamine & Propranolol 2.32 + 0.123 38.4 + 2.2a 82.9 \u00C2\u00B1 2. 3 a Norepinephrine 2.30 + 0.12a 57.9 + 1.4a 89.0 \u00C2\u00B1 1. 8 a Norepinephrine & Propranolol 0.68 + 0.03\u00C2\u00B0 4.7 + 1.2C 9.5 \u00C2\u00B1 0. 5 C Norepinephrine & Burimamide 2.05 + o. ioa 55.2 + 1.5a 81.1 \u00C2\u00B1 1. 5 a a. Significantly increased when compared to buffer solution injected hearts (P <0.05). b. Significantly decreased when compared to histamine injected hearts. c. Significantly decreased when compared to norepinephrine injected hearts. Each value represents the mean \u00C2\u00B1 S.E.M. of 3-5 determinations. 60 Cyclic A M P C n moles/gm wet weight; o ro fp ch o cn o oi CO o i FIG. 19. The effect of histamine and histamine plus burimamide on histamine-induced increases in cyclic AMP. Each bar ^represents the mean \u00C2\u00B1 S.E.M. of three to five cyclic AMP determinations. Cyclic AMP was determined in hearts frozen 18 seconds after the injection of histamine. ,,e 61 TABLE 5 Effect of TD and betazole and TD or betazole and theophylline (10~3M) on cardiac contractility in the isolated perfused guinea pig heart. Drugs were injected or perfused as described in the Methods. Results are expressed as a % of the maximum response obtained with histamine (1 yg). Dose (yg) Drug Treatment 0.2 0.4 0.8 1.6 TD 8.6 \u00C2\u00B1 2.9 20.0 \u00C2\u00B1 2.0 27 \u00C2\u00B1 1.0 40.0 \u00C2\u00B14.3 TD + Theophylline 13.0 \u00C2\u00B1 1.0 27.0 \u00C2\u00B1 2.0a 39.0 \u00C2\u00B1 2.5a Betazole 0.0 10.0 \u00C2\u00B1 1.0 19.0 \u00C2\u00B1 2.0 25.0 \u00C2\u00B1 2.5 Betazole & Theophylline 10.5 \u00C2\u00B1 2.3a 17.5 \u00C2\u00B1 1.9a 25.0 \u00C2\u00B1 2.0a N = 5 a. (rr Significantly (P <0.05) greater than drug treatment without theophylline. 62 (8-16x10 M) as shown in Fig. 21. Histamine dose-response curves of cardiac contractility and heart rate were obtained in the presence and absence of promethazine (4-16x10 ^M). As seen in Fig. 22. promethazine decreased the maximum histamine response, but did not shift the histamine dose-response to the right. Blockade by promethazine of the histamine cardiac effects could not be overcome by increasing the concentration of the agonist, which i s an indication of a noncompetitive or competitive nonequilibrium type of antagonism. Only one \u00E2\u0080\u00946 concentration of promethazine (4x10 M) did not affect the chronotropic effect of histamine i n the guinea pig heart (Fig. 23) whereas higher \u00E2\u0080\u00946 concentrations(8-16x10 M) of promethazine reduced the chronotropic response \u00E2\u0080\u00946 of histamine. The increase in promethazine concentration (16x10 M) further reduced the histamine effect on heart rate. The chronotropic-effects. of \"his taminenare'.reduced-by ^pr^mStHazfnS'-fe-a'm.dncbmpet\u00C2\u00B1^tdve'^dr-'; ndnequlMbrium manner, s r . . : . r - J S E typs of : . ~ : . S 3 -Histamine (1 yg) caused abcut a fdur fdld increase i n the tissue cyclic \u00E2\u0080\u00946 AMP content. Promethazine (4x10 M) reduced the histamine-induced cardiac cyclic AMP levels (Fig. 24). Increasing the dose of histamine to 3.2 yg \u00E2\u0080\u00946 in the presence of promethazine (4x10 M) did not further increase the levels of cyclic AMP. Again the type of blockade noted with promethazine was noncompetitives or competitive nonequilibrium. The interaction between promethazine and norepinephrine was studied on guinea pig heart. The-data are summarized in Table 6^. Promethazine (2-4x \u00E2\u0080\u00946 10 M) did ndt affect the pdsitive indtropic respdnse td ndrepinephrine. At 8x10 ^ M, prdmethazine ldwered the maximum respdnse td ndrepinephrine. Prdmethazine (16x10 M^) decreased the effects cf a l l ddses df ndrepinephrine. In summary, the results of the experiments described this far suggest 63 FIG. 20. The effect of various concentrations of promethazine on cardiac force of contraction. Results are presented as percentage increase over control versus dose of promethazine perfused through the heart. Each poinf represents the mean \u00C2\u00B1 S.E. of four hearts. Force was significantly elevated at 2xl0 -^ and 4xlO~^M. 65 FIG. 21. The effect of various concentrations of promethazine on heart rate. Heart rate was significantly elevated at 4xlO~^M promethazine and significantly lowered at 8xl0~ 6 and 16xlO-6M promethazine. 20 2 4 8 16 Promethazine C u l W ) 67 FIG. 22. The effect of promethazine (4x10 - 16x10 M) on the histamine-induced increase in force of contraction. Dose Histamine Promethazine 4 J J M '\u00E2\u0080\u00A2 J Q Promethazine 8 j j i Promethazine 16JJM J J \u00E2\u0080\u0094 L _ _ I 2 .4 .8 1.6 Histamine (yg) 69 that burimamide, competitively blocked the histamine cardiac effects, whereas promethazine.interacted with the cardiac histamine receptors, but in a non-competitive or non-equilibrium manner. Histamine-cardiac adenylate cyclase interaction In order to further characterise the receptor in heart; the effects of histamine on cardiac adenylate cyclasewere studied. Fig. 25 illustrates the data obtained with several doses of histamine, 4-methylhistamine (a specific receptor agonist; (Black et a l . 1972), TD and betazole. A l l the agonists, stimulated cardiac adenylate cyclase. The data were plotted as agonist concentration vs cyclic AMP formed (P Mol./mg. protein/min.). The order of potency of the compounds for stimulating cardiac adenylate cyclase was histamine>4-methylhistamine^TD>betazole. The activities of 4-methylhistamine and TD were not significantly different from each other, but were significantly greater than the activity of betazole. Burimamide (1x10 ~*M and 5x10 M^) \u00E2\u0080\u00946 shifted the histamine dose-response curve to the right. Burimamide (10 M) also produced a shift in the dose-response curve to the 4-methylhistamine stimulation of cardiac adenylate cyclase (Fig. 26). The study of the two other histamine analogs, TD and betazole, on cardiac adenylate cyclase and their interaction with burimamide is presented i n Fig. 27. \u00E2\u0080\u00946 Burimamide (10 M) moved the TD and betazole-induced stimulation of the cardiac adenylate cyclase to the right. The burimamide blockade could be overcome by increasing the concentration of the agonists. Thus burimamide competitively blocked the histamine and histamine analog-induced stimulation of cardiac adenylate cyclase. 70 FIG. 23.' The effect of promethazine (4xl0~ - 16x10 M) on the histamine-induced increase in heart rate. 71 72 FIG. 24. The effect of promethazine (4x10 M) on the histamine-induced increase i n cardiac cyclic AMP. ro !\u00E2\u0080\u0094-J Control EES Histamine 1jjg Promethazine 4pM Promethazine 4uM ~r Histamine 1jjg s-r^ Promethazine 4 J J M + Histamine 3.2jjg 74 FIG. 25. The e f f e c t o f v a r i o u s doses of h i s t a m i n e , 4 - m e t h y l h i s t a m i n e , TD and b e t a z o l e on t h e a c t i v i t y o f g u i n e a p i g c a r d i a c a d e n y l a t e c y c l a s e . H i s t a m i n e , - - ^ 4 - M e t h y l h i s t a m i n e , .... TD, B e t a z o l e . 10 0 80 c \u00C2\u00A3 c .+-\u00C2\u00BB o &~ a cn 6 0 E \u00C2\u00A3 a. o A -V -0 -\u00E2\u0080\u00A2^Histamine \u00E2\u0080\u00A2v 4-IVfe thy! histamine -oTriazole \u00E2\u0080\u00A2 Betazole 2 0 i 10 7 10 10 5 - 4 1 0 Drug concentration(M) 76 TABLE 6 The effects of promethazine, 2xl0~ 6 - 16xlO~6M, on the norepinephrine induced increase in cardiac force. Concentration of promethazine (M) Dose of -fi ' fi fi fi norepinephrine 0 2 x 10 4 x 10 . 8.x 10 16 x 10 0.2 50.2 \u00C2\u00B1 2.313 48.6 + 1.21 50.7 \u00C2\u00B1 3.61 43.8 \u00C2\u00B1 1.43 '?38.3 \u00C2\u00B1 1.15b 0.4 75.7 \u00C2\u00B1 1.52 .73.8 \u00C2\u00B1 0.89 74.2 \u00C2\u00B1 2.12 71.2 \u00C2\u00B1 0.93 62.0 \u00C2\u00B1 1.1.5b 0.8 90.5 \u00C2\u00B1 0.98 89.7 \u00C2\u00B1 1.21 89.8 \u00C2\u00B1 1.80 83.2 \u00C2\u00B1 0.87 78.3 \u00C2\u00B1 1.93b 1.6 100 98.3 \u00C2\u00B1 1.31 98.4 \u00C2\u00B1 1.51 90.0 \u00C2\u00B1 0.46b 86.0 \u00C2\u00B1 0.86b a. Means \u00C2\u00B1 S.E.M. of three hearts. b. Significantly less than control P <0.01. NOTE: .The results are given as percentage of the maximum response that could be obtained with norepinephrine. 77 Histamine-gastric adenylate cyclase interaction At this stage i t was of interest to determine whether the association of receptorsiwith adenylate cyclase was confined to the heart or whether this association was found in other tissue as well. It has been suggested that histamine-induced gastric acid secretion in frog gastric mucosa is not blocked by H^ type of histamine antagonists. Black et a l . (1972) suggested histamine gastric receptorswer.e of the type. Therefore the interaction between histamine and gastric adenylate cyclase was studied. Fig. 28 shows that histamine, 4-methylhistamine, TD and betazole a l l stimulated gastric adenylate cyclase. Histamine-induced stimulation of adenylate cyclase was significantly greater than 4-methylhistamine, TD and betazole. Again there was no significant difference between 4Smethylhistamine and the TD. The rank order of the compounds with regard to their respective activities was the same as observed with cardiac adenylate cyclase. Fig. 19. shows that \u00E2\u0080\u00946 \u00E2\u0080\u00946 burimamide (5x10 M); and 1x10 M) shifted the curves of histamine and 4-methylhistamine respectively to the right. The burimamide blockade was overcome by increasing the concentration of the agonists. Fig. _3_0 illustrates, TD and betazole stimulated gastric adenylate cyclase dose^response curves were \u00E2\u0080\u00946 shifted to the right by burimamide (10 M). The data from these experiments suggests, that burimamide competitively antagonized the histamine-induced activation of gastric adenylate cyclase. Our results suggest that histamine receptors in the heart and in the stomach are associated with adenylate cyclase. Histamine-myometrium adenylate cyclase interaction Histamine effects on the rat uterus are not blocked by the Hj-tantihistaminic drugs (Ash and Schild 1966). Black et a l . (1972) reported that burimamide, v ) 78 FIG. 26. The effect of burimamide (5 to 10x10 M) on the stimulation of guinea pig cardiac adenylate cyclase by various doses of histamine and 4-methylhistamine. - Histamine^ Histamine + burimamide (5xlO~6M), c Histamine + burimamide (lxlO~ 5M). 4-Methylhistamine, - \u00E2\u0080\u00A2- 4-Methylhistamine + burimamide (lxlO\" 6M). c A M P formed(PM/mg Protein/min) 79 Oi 0) Oi Oi Oi Oi t o o 4 ^ o CD O CO O 4> \u00C2\u00A9 I S 2. <& IT o o o f 1 \u00E2\u0080\u00A2 I <> 6 X oT 0) |IU CP + + CD c c im. 3 \u00E2\u0080\u00A2 X . ^ OI O i O) 2 3 3 80 FIG. 27. The effect of burimamide (1x10 M) on the stimulation of guinea pig cardiac adenylate cyclase by various doses of betazole and TD. Each point represents the mean \u00C2\u00B1 S.E. of 4 determinations. TD, Betazole, -- TD + burimamide (1x10\"%), \u00E2\u0080\u009E- Betazole + burimamide (lxlO~ 6M). 0 0 \u00C2\u00A9.\u00E2\u0080\u0094Q Triazole o\u00E2\u0080\u0094-o Betazole 1 x I6H) 6 x Betazole + Burim(ixl6^j) FIG. 28. The effect of various concentrations of histamine, 3- (B-aminoethyl)-l,2,4 triazole'(Triazole), 4- methylhistamine and betazole on rat gastric adenylate cyclase activity. Each point represents the mean \u00C2\u00B1 S.E.M. of 4 determinations. ro oo 5= e 10 10' 10' Drug Concentration(M) 84 FIG. 29. The effect of various concentrations of histamine and 4-methylhistamine and the interaction of these drugs with burimamide on rat gastric adenylate cyclase activity. Each point represents the mean \u00C2\u00B1 S.E.M. of 4-6 determinations. oo Drug Concentration (M) 86 FIG. 30. The effect of 3-(B-aminoethyl)-l,2,4 triazole (Triazole) and betazole and the interaction of these drugs with burimamide on rat gastric adenylate cyclase activity. Each point represents the mean \u00C2\u00B1 S.E.M. of 4 determinations. oo % C5 \u00E2\u0080\u00A2 111 40 o \u00E2\u0080\u00A2CXO \u00C2\u00A3 30 a . /\u00E2\u0080\u00A2\u00E2\u0080\u00A2^ 20 e 5 - . 10 a -4 10 10 Drug Concentration (M) Triazole Betazole -\u00C2\u00B0 Triazole +Burim.(1*1o|> \u00E2\u0080\u00A2 Betazole+Burimflx10\u00C2\u00BB) 88 an H^-receptor antagonist blocked the histamine effects on rat uterus. A dose-response study of the effects of histamine and i t s analogs on the rat uterus i s shown in Fig. 31_. It demonstrates that histamine, 4-methylhistamine, TD and betazole a l l relaxed the rat uterus in a dose-dependent manner. The order of potency was histamine>4-methylhistamine>TD> betazole. A study of the interaction of histamine on the adenylate cyclase particles prepared from rat uterus, revealed that histamine did not stimulate the adenylate cyclase. Histamine-guinea pig ileum interaction Preliminary experiments established that 10 \"*M histamine produced a maximum contractioricnof\u00C2\u00A3 the guinea pig ileum. Burimamide did not block the histamine-induced contractions i n the guinea pig ileum, whereas diphenhydramine, a receptor antagonist competitively blocked the histamine effects on guinea pig ileum (Ash and Schild 1966). Stimulation of cardiac 0 H^-receptors i n either guinea pig heart or rat stomach resulted in the activation of adenylate cyclase and subsequent formation of cyclic AMP (present study). In a time-response study histaminet.(-l'0t^M); di'dthotgiricreasegcyclic AMP ataamy:.time 1 tested.'.(Table ,7)'.T. t a i i a 7_. Cardiac actions and interaction of theophylline with norepinephrine and histamine The inotropic and phosphorylase activating effects of adrenergic and histaminergic drugs have been suggested to be mediated through cyclic AMP. Theophylline, a phosphodiesterase inhibitor potentiates the amine effects on 89 FIG. 31. The effect of various concentrations of histamine, 4-methylhistamine, 3-(3-aminoethyl)-l,2,4 triazole (Triazole) and betazole on the rat uterus. Each point represents the mean \u00C2\u00B1 S.E.M. of 3 determinations. o 1 0 0 -80 - U U \" B A \u00E2\u0080\u00A2\u00E2\u0080\u0094-^Histamine 0 \u00C2\u00A94-Methylhistamine o\u00E2\u0080\u0094 - o T r i a z o l c \u00E2\u0080\u0094ABctazole / 2 0 -A A A cr L 8 10 '-7 10 10\" 10 10 1 Drug Concentration CM) o 91 TABLE 7 The effect of histamine (10 ~*M) at various times on the level of cyclic AMP in the guinea pig ileum. Each result i s the mean of 5-6 determinations \u00C2\u00B1 S.E. Time Cyclic AMP (Sec) (nmol/gm wet weight) 0 0.58 + 0.088 5 0.66 + 0.080 10 0.56 + 0.070 20 0.50 + 0.050 30 0.63 + 0.051 50 0.64 + 0.030 92 the heart and is also capable of directly stimulating the heart. Therefore the effects of theophylline on cardiac contractility, cyclic AMP and phosphorylase activation as well as the interaction between norepinephrine or histamine and theophylline on these parameters were investigated. Theophylline (1.0 mg) when injected into the perfused guinea pig heart caused a 20.0% increase in cardiac contractility over the control (Fig. 32). In about 20-30 seconds slight phosphorylase a. activation was noted. At no time were significant differences in cyclic AMP levels observed. Control phosphorylase a. levels were 2.9 \u00C2\u00B1 0.3%, which increased to 6.5 \u00C2\u00B1 1.2% at about 20 sec. and remained elevated over the 50 sec. experimental period. Control levels of cyclic AMP were 0.54 \u00C2\u00B1 0.07 nmoles/gm wet wt. and did not change over the experimental time interval. In the next set of experiments, the interaction between theophylline and norepinephrine or histamine were investigated. Theophylline, at a -4 concentration of 7.0x10 M, was perfused for 15 min. through the isolated rat heart. This was the minimal concentration of theophylline necessary to enhance the inotropic effects of norepinephrine. Theophylline significantly potentiated the 0.2 and 0.4 yg doses of norepinephrine effects on phosphorylase a. activation (Fig. 33). -4 As stated before, theophylline (7x10 M) produced an increase in cardiac contractility.However an increase in the dose of theophylline to -3 2x10 M, resulted in a negative inotropic effect. When the hearts were -3 perfused with theophylline (2x10 M) and injected with different doses of norepinephrine, the norepinephrine response was lowered (Fig. 34). Theophylline - 4 , . . . -3 :.(7-x-10~yM ors2xl0 A M ) did3n6tE.potehti'at\"e^the',nbrepinephrine-induced'elevation of cyclic AMP (Fig. 34). Similar experiments were carried out in the guinea pig hearts, perfused Time [sec] FIG. 32. \u00E2\u0080\u00A2 The effect of theophylline (1 mg) at'various times following injection into the perfused guinea pig heart on contractility, %\"phosphorylase a. and'cyclic AMP. 9 4 60-\u00C2\u00A9 \"x o -JC a *\u00C2\u00BB O u: C L Buffer Perfused Theophylline (7xlO* 4M) 0.2 0.4 Dose of Norepinephrine i>g) FIG. 3 3 . The e f f e c t o f v a r i o u s doses o f n o r e p i n e p h r i n e on c a r d i a c p h o s p h o r y l a s e a c t i v a t i o n i n r a t h e a r t s p e r f u s e d w i t h b u f f e r o r b u f f e r p l u s t h e o p h y l l i n e ( 7 X 1 0 ~ 4 M ) . 0 0.1 0 . 2 0 . 4 D o s e o f N o r e p i n e p h r i n e (ug) FIG. 34 The e f f e c t of various doses of norepinephrine on cardiac c o n t r a c t i l i t y and c y c l i c AMP i n r a t hearts perfused with buffer or buffer plus theophylline (7xlO~^M or 2x10\" 3M). 96 with buffer or buffer plus 10 M theophylline and injected with either buffer or with different doses of histamine. In these experiments, theophylline _3 (10 M) potentiated the histamine (0.2 and 0.4 yg) cardiac effects on contractility and phosphorylase \u00C2\u00A3i (Fig. 35). Histamine (0.2 and 0.4 yg) induced increases in cyclic AMP were not further increased by the presence of theophylline (Fig. 35). Cardiac actions and interaction of imidazole with norepinephrine and histamine The cardiac actions of imidazole, a phosphodiesterase stimulator, and the i;effg\u00C2\u00A7tsio;5f^ .f\u00C2\u00B1hii!ss\"' drug ,wonh norepinephrine and .histamine were investigated. Imidazole perfusion in the concentration range of 0.25 to 40mM resulted in a concentration related increase in cardiac contractility (Fig. 36). In a similar set of experiments different doses of imidazole (0.05-1.6 mg) were injected by a side arm cannula into the perfused guinea pig heart. Imidazole produced a dose-dependent increase in cardiac contractility (Fig. 37). The maximum increase in force with 40mM imidazole perfusion was 31.3 \u00C2\u00B1 6.3%. A dose of 1.6 mg of imidazole produced a 23.7 \u00C2\u00B1 3.3% increase in contractility over the control levels. Further experiments were carried out to test the interaction between imidazole and histamine or norepinephrine, in the guinea pig hearts perfused with 40mM imidazole. Data in table 8 show that imidazole (40mM) reduced the histamine and norepinephrine dose-response effects on cardiac contractility. Results in Table 8, are expressed as a percentage of maximum response obtained with the agonists. Similar effects with imidazole on amine-induced contractility were reported by Poch and Kukovetz (1967). As cyclic AMP i s believed to be important for the amine-induced increases in contractile force, 97 0 0.20 0.40 Dote of Histamine (tig) FIG. 35. The effect of various doses of histamine on cyclic AMP, * contractility, and phosphorylase a. in guinea pig hearts perfused with buffer or buffer plus theophylline (lCT^M) . 9 8 '/ Increase in force over control _ A - A is? r o cn o cn o cn FIG. 36. The e f f e c t o f v a r i o u s doses o f i m i d a z o l e on c a r d i a c c o n t r a c t i l i t y i n g u i n e a p i g h e a r t s p e r f u s e d w i t h b u f f e r . Each p o i n t r e p r e s e n t s t h e mean \u00C2\u00B1 S.E.M. of t h r e e d e t e r m i n a t i o n s . 99 eo Z Increase in force over control IN) cn cn O \u00C2\u00BB9 O cn b ro o o cn cn o cn O FIG. 37. The effect of perfusion of various concentrations of imidazole (0.25-40mM) in the isolated perfused guinea pig heart. Each point represents the mean \u00C2\u00B1 S.E.M. of three determinations. 100 TABLE 8 . The effect of 40mM imidazole perfusion on the positive inotropic effect of histamine and norepinephrine. Histamine Norepinephrine Dose of amine Buffer Imidazole Buffer Imidazole (yg) perfused perfused perfused perfused 0.2 5 0 . 0 \u00C2\u00B1 4 . 0 0 . 0 1 0 .o a 61 .812. 4 7 . -2l0.6 a 0 .4 75.013.8 3 7 . 6 \u00C2\u00B1 2 . 0 a 7 5 . 2 1 2 . 5 5 5.6i6 . 0 a 0 . 8 88.013.2 50 . 0 1 2 . 2 a 90 .012. 5 7 4 . 3 1 3 . 7 a 1.6 100 .010.0 6 5 . 0 i l . 4 a 100 .010. 0 7 7 . 3 l l . 4 a n = 3-4 Results are expressed as % of the maximum response that could be obtained with the agonist. a. Significantly less than buffer perfused (P <0.05). TABLE 9 The effect of imidazole (40mM) on the norepinephrine and histamine induced increase in cardiac cyclic AMP. Norepinephrine Histamine Dose of amine Buffer (yg); perfused Control 0.25 0.5 1.0 0.45\u00C2\u00B10.017 0.75+0.045 1.12\u00C2\u00B10.050 2.25\u00C2\u00B10.100 Imidazole perfused 0.4410.018 0.52\u00C2\u00B10.031a 0.91\u00C2\u00B10.037a 1.22\u00C2\u00B10.040a Buffer perfused 0.45\u00C2\u00B10.017 0,70\u00C2\u00B10.05 1.05\u00C2\u00B10.075 2.30\u00C2\u00B10.022 Imidazole perfused 0.44\u00C2\u00B10.018 0.48\u00C2\u00B10.018a 0.67\u00C2\u00B10.036a 0.78\u00C2\u00B10.058a Mean cyclic AMP (nmoles/g wet weight) of 5-7 hearts \u00C2\u00B1 S.E. a. Significantly less than no treatment (P <0.05). 102 the interaction of imidazole and histamine or norepinephrine ori cardiac cyclic AMP was investigated. Guinea pig hearts were perfused with buffer or imidazole (40mM) for 15 min. and injected with various doses of histamine or norepinephrine. Imidazole perfusion decreased the amine-induced increases in cyclic AMP at a l l the doses tested (Table 9). Thus the decrease in cyclic AMP correlates well with the amine effects on cardiac force. In order to test the interaction of the histamine or norepinephrine on phosphorylase a_ activation with imidazole (40mM) , the hearts were perfused with buffer or imid.azole for 15 min. and injected with various doses of histamine or norepinephrine. Table 10 shows the lack of interaction between imidazole and the two amine agonists on cardiac phosphorylase a^ . Imidazole did not affect phosphorylase activation of the amines at any dose tested. An investigation of the imidazole effects on cardiac cyclic AMP or phosphorylase a. revealed that a 1.6 mg, imidazole injected into the perfused guinea pig heart, did produce an increase in contractility, about 23.7 \u00C2\u00B1 3.3% over control and 11.7 \u00C2\u00B1 1.6% increase in phosphorylase (Table 11). The data suggest that imidazole can produce significant changes in contractility and phosphorylase activation without measurable changes in cyclic AMP. 103 TABLE 10 The effect of imidazole (40mM) on the norepinephrine arid histamine-induced activation of cardiac phosphorylase. Norepinephrine Histamine Dose of amine. Buffer Imidazole Buffer Imidazole (yg) perfused perfused perfused perfused 0.0 4.810.87a 6.6+1.00 4.8\u00C2\u00B10.87 5.5\u00C2\u00B11.00 0.25 20.6\u00C2\u00B11.87 17.0+0.57 .10.3\u00C2\u00B10.63 9.0\u00C2\u00B10.76 0.5 42.0\u00C2\u00B10.57 40.010.57 26.9ll.10-' 24.3\u00C2\u00B10.33 1.0 57.410.70 54.611.70 42.410.69 40.211.47 a. Mean % of phosphorylase a_ of 5-7 hearts \u00C2\u00B1 S.E. measured at the peak of the phosphorylase response. 104 TABLE 11 The effect of imidazole on phosphorylase a., cyclic AMP and contractile force in the isolated perfused guinea pig heart. Dose of Imidazole %pphosphorylase cyclic AMP % increase in force a. (nmoles/gm over control wet weight) Control 4.5 \u00C2\u00B1 1.5 0.45 \u00C2\u00B1 .017 1.6 mg 11.7 \u00C2\u00B1 1.6a 0.46 \u00C2\u00B1 0.04 23.'7 \u00C2\u00B1 3.3a n = 3-4 Cyclic AMP was measured in hearts frozen at 18 sec. and phosphorylase'in hearts frozen at 38-40 sec. following the injection of imidazole. a. Significantly greater than no treatment (P<0.05). 105 DISCUSSION The p o s i t i v e i n o t r o p i c e f f e c t of the catecholamines has been postulated to r e s u l t from an increase i n the i n t r a c e l l u l a r l e v e l of c y c l i c AMP produced by the a c t i v a t i o n of adenylate cyclase (Sutherland et a l . 1966). This hypothesis was questioned by Benfey (1971) who found that phenylephrine, an adrenergic amine produced increased c o n t r a c t i l e force without producing any metabolic e f f e c t s . In the present study dose-response and time-response data revealed that phenylephrine produced an increase i n c y c l i c AMP, cardiac c o n t r a c t i l i t y and phosphorylase a c t i v a t i o n . These data are i n agreement with the findings ofPMcNeill^et\"alt^-(-1972-)-lwh6- measured phosphorylasePand. c o n t r a c t i l i t y following'phenylephrine injectiphrlandand suggest that phenylephrine i s a weak beta-adrenergic agonist. Phenylephrine i n comparison to norepinephrine has less potency and e f f i c a c y i n elevating c y c l i c AMP, producing a p o s i t i v e i n o t r o p i c e f f e c t or r a i s i n g the l e v e l s of phosphorylase _a. Phenylephrine can produce these e f f e c t s i n a dose-dependent manner. In the time-response study, norepinephrine produced an increase i n c y c l i c AMP which preceded the increase i n c o n t r a c t i l i t y and phosphorylase a c t i v a t i o n . Phosphorylase a_ and c o n t r a c t i l i t y began to increase at about the same time. C y c l i c AMP reached a peak before the peak i n cardiac c o n t r a c t i l i t y or phosphorylase _a. Similar r e s u l t s were obtained by Robison et a l . (1965) i n a time-response study with epinephrine i n the i s o l a t e d perfused r a t heart. Williamson (1966) stated that c y c l i c AMP and c o n t r a c t i l i t y reached a peak at about the same time (12 sec.) following the i n j e c t i o n of epinephrine into the perfused r a t heart. In the time-response study with phenylephrine, phosphorylase _a a c t i v a t i o n appeared to lag behind the increase i n cardiac c o n t r a c t i l i t y . Similar r e s u l t s were obtained when the histamine analogs, TD and betazole were used i n the 106 present study. The apparent lag in phosphorylase may merely reflect a technical in a b i l i t y to detect the smaller changes in phosphorylase activation that occurred with less potent agonists. Benfey (1971) did not find an increase in cyclic AMP with phenylephrine. Benfey and Carolin (1971) have also shown that phenylephrine does not activate adenylate cyclase. Benfey and Carolin (1971) have interpreted their data to mean that phenylephrine did not stimulate adenylate cyclase and subsequently caused no elevation i n cyclic AMP levels. The possible reasons for the discrepancies between the results of Benfey and those found i n the present investigation are many. 1.) Benfey estimated the cyclic AMP levels i n rabbit heart slices. 2.) It is very li k e l y that the fragile nature of the enzyme (adenylate cyclase) which is easily denatured by usual laboratory physical forces such as washing, freeze thawing, homogenization and sonication, presents serious p i t f a l l s in the interpretation of data. 3.) Benfey (1971) did not use a beating heart in which physiological parameters could be simultaneously monitored. McNeill et a l . (1972) stated that phenylephrine did not possess sufficient intrinsic activity to activate the enzyme. Our data suggests that phenylephrine, like other adrenergic amines, stimulates adenylate cyclase and increases the intracellular concentration of cyclic AMP. Increases i n cyclic AMP in turn.may elevate phosphorylase a. values and produce the inotropic effect (Robison et a l . 1965). It has also previously been suggested that the cardiac biochemical and mechanical effects of histamine are mediated through the elevation of cyclic AMP (Poch and Kukovetz 1967; Klein and Levey 1971; McNeill and Muschek 1972). Histamine, TD and betazole were found to produce a positive 107 inotropic effect and to increase cardiac phosphorylase in a dose-dependent manner. In the present study a l l of these compounds were found to elevate cardiac cyclic AMP. The order of potency and the order of effectiveness of the compounds in producing these effects was histamine>TD>betazole. The order obtained i s the same as that previously found when the compounds were examined f qr their a b i l i t y to activate cardiac adenylate cyclase (McNeill and Muschek 1972). Time-response relationships revealed that a l l three agonists elevated cyclic AMP prior to increasing contractility or phosphorylase a.. The relative effectiveness of these drugs in increasing cardiac cyclic AMP was the same as for other parameters measured. A logical sequence of events for histamine and i t s analogs would appear to be: 1.) activation of adenylate cyclase, 2.) formation of cyclic AMP and 3.) enhancement of contractility and activation of phosphorylase a.. It is d i f f i c u l t to separate the onset of contractility and phosphorylase activation with histamine. However when either TD or betazole was used, the activation of phosphorylase appeared to lag behind the positive inotropic effect. The specificity of the cardiac histamine receptor has been the subject of controversy. Several lines of evidence indicate that effects of histamine are independent of the adrenergic nervous system. Reserpine pretreatment did not alter the responsiveness of the heart to histamine.(Trendelenburg, 1960, Mannaioni 1960). In fact reserpine-induced supersensitivity to the cardiac effects of histamine has been demonstrated (McNeill and Schulze 1972). Dichloroisoproterenol and propranolol did not abolish the histamine-induced changes in cardiac contractility and heart rate (Trendelenburg 1960; Poch and Kukovetz 1967; McNeill and Muschek 1972). Dean (1968), reported that beta.blocking agents like propranolol and prenethanol produced a slight shift in the histamine dose-response curve. This could be a non-specific antagonism. Hearts 108 isolated from animals pretreated with reserpine or 6-hydroxydopamine s t i l l show typical responses to the application of exogenous histamine (Levi and Giotti 1967). Such experiments demonstrate that histamine has direct cardiac effects. Classical antihistaminesssuch as diphenhydramine or tripelennamine do not antagonize the responses of isolated cat and guinea pig atria and isolated perfused guinea pig hearts. (Trendelenburg 1960; Bartlet 1963; McNeill and Muschek 1972). These antihistamines were also relatively non-specific antagonists of the histamine stimulation of adenylate cyclase (McNeill and Muschek 1972). The principal reports claiming the demonstration of specific effects of antihistamine drugs in blocking the cardiac actions of low concentrations of histamine are those of Mannaioni (1960) and Flacke et a l . (1967), Klein and Levey (1971), Hughes and Coret (1972) have reported that promethazine can antagonize the chronotropic effects of histamine on rabbit atria. Since complete dose-response curves were not obtained in that study, the specificity of the blockade is questionable. Black et a l . (1972) have recently classified histamine receptors into H^ and ^ receptors. H^ receptors mediate most effects of histamine and are blocked by classical antihistaminic compounds. Histamine stimulated secretion of acid from gastric mucosa and the inotropic and chronotropic effects of histamine can not be antagonized by mepyramine or related drugs. These receptors are classified as B.^ type receptors. Burimamide, however does block the gastric secretory and positive chronotropic effectscof histamine (Black et a l . 1972; Wylie et a l . 1972). In the present study the specificity of the cardiac histamine receptor has been demonstrated. Burimamide proved to be a competitive antagonist of the positive inotropic, phosphorylase activating and cyclic AMP increasing effects of histamine, TD 109 and betazole. Burimamide did not antagonize the e f f e c t s of norepinephrine on these parameters and propranolol did not antagonize histamine or histamine analog cardiac e f f e c t s . Stimulation of the adenylate cyclase then* r e s u l t s i n an increase i n c y c l i c AMP, and subsequently a p o s i t i v e i n o t r o p i c and phosphorylase a c t i v a t i n g e f f e c t s i m i l a r to that suggested f o r catechol-amines (Robison et a l . 1965). Our data support the recent findings of Poch and Kukovetz (1973), i n d i c a t i n g that the i n o t r o p i c as w e l l as the c y c l i c AMP increasing actions of histamine can be i n h i b i t e d by the antagonist burimamide. The findings of Hughes and Coret (1972) suggest that the cardiac e f f e c t s of histamine are susceptible to blockade by promethazine a H^ receptor antagonist. A c a r e f u l and complete study of the i n t e r a c t i o n of promethazine with several doses of histamine on heart rate, force of contraction and c y c l i c AMP formation was c a r r i e d out to determine i f the proposal of Hughes and Coret was correct. The data presented reveal that promethazine i s not a competitive equilibrium antagonist of the cardiac e f f e c t s of histamine but does i n t e r a c t with the histamine receptor i n either a non-competitive or competitive non-equilibrium manner. Promethazine (4-16x10 M^) lowered the histamine e f f e c t s on cardiac c o n t r a c t i l i t y . An increase i n the dose of histamine could not overcome the promethazine blockade pahdul/increasing the promethazine concentration resulted i n a further depression of the maximum histamine response. Promethazine also p a r t i a l l y blocked the histamine-induced increases i n cardiac c y c l i c AMP. Increases i n the dose of histamine did not overcome the promethazine blockade. The degrease i n the histamine response produced by promethazine was approximately the same for \u00E2\u0080\u00946 both the mechanical and biochemical events. Promethazine (4 x 10 M) did not antagonise the i n o t r o p i c response to norepinephrine. 110 Higher doses of promethazine (8-16 x 10 M) did depress the norepinephrine responses. The results with promethazine stand in contrast to those obtained with burimamide. Burimamide appears to be a competitive equilibrium antagonist of histamine whereas promethazine exhibited characteristics of a non-competitive antagonist of histamine. One concentration of promethazine (4 x 10 M^) produced an increase in both cardiac contractility and rate. The stimulatory effects were not noted by McNeill and Brodyy (1968); Hughes and Coret (1972) or Davis and McNeill (1973). The effect was blocked by propranolol and this probably involved an adrenergic mechanism. Promethazine i s known to block the uptake of adrenergic amines (McNeill and Brody, 1968; Davis and McNeill 1973). Such an affect could account for the results obtained. Hughes and Coret (1972) have used only one concentration of promethazine (4.7 x 10 ^M). In our study a complete range of promethazine concentrations \u00E2\u0080\u00946 (2-16 x 10 M) and their interaction with histamine wase investigated. The present study again demonstrated the importance of doing complete dose-response curves when investigating drug interactions. The effect of histamine on adenylate cyclase was not blocked by propranolol and was poorly blocked by classical antihistamines such as tripelennaminecarid diphenyhydramine (McNeill and Muschek 1972). Our data indicate that the stimulatory effect of histamine and histamine analogs on guinea pig cardiac adenylate cyclase is competitively antagonised by burimamide. The interaction between burimamide and agonists was similar to that noted when the effects of these agents on cardiac cyclic AMP, contractility and phosphorylase were studied. The data presented d i f f e r ) from those of Klein and Levey (1971). They were able to demonstrate a complete blockade of one concentration of histamine with diphenhydramine I l l (8 x 10 M). However (10 M) diphenhydramine appeared to lower the maximum response to histamine (McNeill and Muschek 1972) and thus may again be an example of a non-competitive antagonism. It has also been proposed that histamine produces i t s secretory effect in the gastric mucosa by interacting with receptors (Black et a l . 1972). Cyclic AMP appears to act as an intracellular mediator of histaminic action on gastric mucosa, (Levine and Wilson 1971; Mao and Jacobson 1973,; Bieck, Oates and Robison 1973). The stimulation of gastric adenylate cyclase by histamine, 4-methylhistamine, TD and betazole and their specific, competitive blockade by burimamide provide the basis for suggesting that histamine and i t s analogs stimulate cyclic AMP formation via an action on receptors in the membranes of the gastric mucosal c e l l s . The relative order of potency of histamine and i t s analogs on rat gastric adenylate cyclase is similar to that found i n investigations studying the actions of these drugs on cardiac adenylate cyclase^ cyclic AMP formation, phosphorylase activation and cardiac contractility (McNeill and Muschek 1972; present study). The rank order for the compounds i s also similar to that noted when gastric acid secretion was measured (Lin et a l . 1963). Karppanen and Westermann (1973) suggested that histamine stimulated gastric secretion of acid i s mediated by cyclic AMP which i s formed in response to stimulation of H\u00C2\u00A3 receptors. Our data agree with the findings of Dosa and Code (1974) that burimamide i s a competitive antagonist of the stimulatory effects of histamine on gastric adenylate cyclase. The results are further supported by the findings of Narumi and Maki 1973; Bersimbaeve et a l . 1971; Mao et a l . 1973; Perrier and Laster 1970; who have provided evidence the receptor in gastric mucosa i s associated with adenylate cyclase. Thus a logical sequence of events for histamine on gastric adenylate cyclase would 112 appear to be; 1.) activation of adenylate cyclase; 2.) formation of cy l i c AMP; 3.) gastric acid secretion. Histamine, 4-methylhistamine, TD and betazole, a l l relaxed the rat uterus i n a dose dependent manner. The order of potency of the compounds for relaxing the rat uterus was histamine> 4-methylhistamine>TD and betazole (Fig. .31). The present findings were in agreement with those of Black et a l . (1972) who reported that histamine and 4-methylhistamine relaxed the rat uterus. Rat uterus possesses receptors as reported by Black et a l . (1972). Stimulation of adenylate cyclase, prepared from guinea pig heart or rat stomach by histamine resulted in an increase in i t s activity and subsequently formed cyclic AMP. This data suggests that receptorsJin both the tissues were associated with adenylate cyclase. However, we could not detect any changes in the adenylate cyclase activity by histamine. There could be two p o s s i b i l i t i e s : 1.) receptors in the rat uterus a?r.e ino.t associated with adenylate cyclase or 2.) our cyclic AMP method was not sensitive enough to pick up cHangeschifgahyiriinycyclip AMP. Recently, Tozzi (1973), reported that histamine effects on the rat uterus were through the release of catecholamines. If histamine effects on the rat uterus are not direct, then i t would not stimulate adenylate cyclase prepared from rat uterus. At least in two tissues, the heart and the rat stomach, B.^ receptors are associated with adenylate cyclase, stimulation of which results i n an increase in cyclic AMP. Histamine stimulates the guinea ileum by acting on receptors (Ash and Schild 1966). Burimamide did not block the histamine response on the -5 guinea pig ileum. In the time-response study histamine (10 M) did not 113 increase cyclic AMP levels at any time tested. It thus appears that the effects of histamine on receptors are not mediated through stimulation of adenylate cyclase with a subsequent increase in cyclic AMP. These findings support the data of Black et a l . (1972) that burimamide does not block the H^-receptor effects of histamine. Cyclic AMP levels are dependent on the activity of the enzyme phosphodiesterase. Theophylline inhibits phosphodiesterase while imidazole activates the enzyme in broken c e l l preparations (Butcher and Sutherland 1962). Both drugs have been used to provide indirect evidence for the involvement of the nucleotide in the positive inotropic response to catecholamines (Rail' and West 1963; Kukovetz and Poch 1967; McNeill 1970; McNeill and Muschek 1972). The results presented here indicate that theophylline can produce an increase in cardiac contractility and phosphorylase a activation without elevating the levels of cyclic AMP. Theophylline also enhanced the cardiac effects of both histamine and norepinephrine without any apparent effect on the levels of cyclic AMP already elevated by norepinephrine and histamine. It has been assumed that because theophylline i s a phosphodiesterase inhibitor i t w i l l elevate cyclic AMP levels in the intact tissue and thus produce i t s effects. The results of the present investigation indicate that this i s not true for the heart. Earlier work with theophylline (Levey and Wilkenfeld (1968) using the rat uterus showed that theophylline potentiated the inhibitory response to nitroglycerin. Polacek et a l . (1971) were able to dissociate the relaxant effect of theophylline on the rat uterus from any effect on cyclic AMP. Allen et a l . (1973) have shown that there was no correlation within a group of phosphodiesterases inhibitors, including theophylline, between the phosphodiesterase inhibition and lipolysis in an isolated fat c e l l preparation. Some correlations have been found between various xanthine 114 derivatives with regard to their a b i l i t y to inhibit phosphodiesterase and their interaction with norepinephrine on the heart (McNeill et a l . 1973). The positive inotropic response to theophylline was not blocked by propranolol and i t was therefore not mediated by catecholamine release (Massingham and Nasmyth 1972). Skelton et a l . (1971) showed that theophylline could enhance the inotropic effect of norepinephrine and db. cyclic AMP but not of calcium on cat papillary muscle. They concluded that their data supported a role for cyclic AMP in the theophylline interaction. But Massingham and Nasmyth (1972) demonstrated a positive interaction between the el e c t r i c a l stimulation and theophylline in the frog ventricle. Such an effect would not be mediated through cyclic AMP and is more lik e l y explained by an increase in the intracellular calcium. Methylxanthines have been found to release ionized calcium from the intracellular storage sites (Nayler 1963; deGubareff and Sleator 1965; Bianchi 1968; Shine and Langer 1971). This may be the process whereby xanthines increase myoplasmic calcium ions to cause a positive inotropic effect. (Nayler 1967). Caffeine, prolonged the active state of cardiac muscle (deGubareff and Sleator 1965; Gibbs 1967). Caffeine has been shown to markedly increase the duration of the action potential and to increase contractility about 45% i n isolated atria. Epinephrine, on the other hand, increased contractility more than 100% and had l i t t l e effect on action potential duration (deGubareff and Sleator 1965). The work of Blinks et a l . (1972) has pointed out that the catecholamines decrease the time to peak tension and accelerate relaxation in cardiac muscle while the xanthines prolong time to peak tension and increase the total duration of contraction. Blinks et a l . (1972) have reviewed the evidence suggesting that xanthines affect the heart by affecting calcium metabolism. Xanthines are known to increase the influx of calcium and to decrease the rate of calcium 115 sequestration in mammalian atria (Scholtz 1971; Shine and Langer 1971). Efflux of calcium is also reduced by caffeine (Shine and Langer 1971). More calcium is thus available for excitation contraction coupling and hence an increase in positive inotropic effect. The effects of xanthines on calcium may also explain the positive inotropic interaction of these drugs with biogenic amines on both contractility and phosphorylase activation,i'TNamm et a l . (1968), St u l l and Mayer (1971) reported that calcium is essential for cardiac contractility and phosphorylase a. activation. Catecholamines and xanthines could produce their synergistic effect by elevating intracellular calcium by different mechanisms thus leading to an enhanced effect. The positive inotropic responses to theophylline can be interpreted in terms of the increased calcium influx which i t produces. The interpretation of the effects of theophylline in terms of i t s action on phosphodiesterase should be treated with reservation. Methylxanthines thus appear to have direct effects on the heart, which are independent of their phosphodiesterase inhibiting properties. Imidazole, a phosphodiesterase stimulator, appeared to produce positive inotropic ef f ectscon the heart which were not mediated through cyclic AMP. Poltetaeu (1970), reported similar effects of imidazole on skeletal muscle. Positive inotropic effects were seen when guinea pig hearts were perfused with imidazole (Knope et a l . 1973). In their study they clearly showed that the imidazole-induced increases in myocardial contractility were not blocked by propranolol or antihistamines. Imidazole effects were not potentiated by aminophylline. Imidazole did not stimulate cardiac adenylate cyclase (McNeill and Muschek 1972). It thus seems unlikely that imidazole produces i t s inotropic effect by elevating cyclic AMP. This is further supported in the present investigations in that perfusion or injection of 116 imidazole did not produce any changes in cyclic AMP in the guinea pig heart. Knope et a l . (1973) suggested that the effect of imidazole was on tissue calcium or calcium turnover. DeMello et a l . (1973) suggested that imidazole acted by increasing permeability to extracellular calcium in frog heart since responses to imidazole were totally suppressed i n calcium-free media while those of caffeine were not. Imidazole reduced the amine-induced increases in the levels of cyclic AMP as shown in Table 9 presumably by stimulating phosphodiesterase and thus increasing the metabolism of cyclic AMP. Imidazole stimulates the enzyme phosphodiesterase i n vitro (Butcher and Sutherland 1962; McNeill et a l . 1973). The decrease in cardiac c y l i c AMP was paralleled by a corresponding decline in contractility. Poch and Kukovetz (1967), reported that imidazole, i n the Langendorff jguanraa pig heart preparation, caused a depression of cardiac contractility and an inhibition of the positive inotropic action of catecholamines. They attributed this effect to the stimulation of the phosphodiesterase^e-afcalyzSdbreakdown of cyclic AMP. The lack of increase of cylic AMP did not however, affect the a b i l i t y of either amine to elevate cardiac phosphorylase a.. Our data suggest the presence of a factor or factors other than cyclic AMP which are as important or more important, in activating cardiac phosphorylase. Friesen et a l . (1967), reported an increase in cardiac phosphorylase by increasing external calcium in the perfusate. In hearts perfused with no calcium, norepinephrine injections did increase cardiac cyclic AMP, without any detectable changes in phosphorylase a. (Namm et a l . 1968). Their study suggested that calcium was s t i l l required for phosphorylase activation even when cyclic AMP was elevated. In smooth muscle calcium is of primary importance and cyclic AMP may not be required at a l l for phosphorylase activation (Diamond and Brody 1966; Namm 1971; Rasmussen et a l . 1972). In a recent study, Diamond (1973) has shown increases 117 in phosphorylase activation during spontaneous uterine contractions at various times after increasing calcium concentration from 1.8 to 7.2mM. This further suggests the importance of calcium in phosphorylase activation without elevation of cyclic AMP. Similar conclusions can be drawn from experiments in e l e c t r i c a l l y stimulated skeletal muscles in which phosphorylase activity but not cyclic AMP was found to be increased (Harwood and Drummond 1969; Posner et a l . 1965). The recent demonstration by S t u l l and Mayer (1971) of an isoproterenol-induced increase in skeletal muscle phosphorylase a. without an effect on cyclic nucleotide levels is a further i l l u s t r a t i o n of this phenomenon. In a l l the references cited above, calcium has been invoked as the probable factor in activating the enzyme. A similar explanation would f i t the data of the present study. In Table 11 a single injection of 1.6 mg imidazole alone was able to increase phosphorylase _a. A recent study of McNeill and Young (1973) and Young and McNeill (1974), supports the concept that drugs can activate cardiac phosphorylase through mechanisms other than cyclic AMP. Hearts from hyperthyroid rats responded to norepinephrine to the same extent as controls when contractility and cyclic AMP were measured. However, phosphorylase activation by norepinephrine was enhanced in the hyperthyroid hearts. Hyperthyroidism is known to increase the accumulation and release of calcium in the heart (Suko 1971; Nayler et a l . 1971). Again the involvement of calcium could explain the data. Rasmussen and Tenenhouse (1970) have made several suggestions for the interaction of hormone, cyclic AMP and calcium. One of the suggestions is that hormone increases cyclic AMP, which in turn alters the permeability of the membrane to calcium. Our data support this suggestion. Both histamine and norepinephrine did not produce an inotropic effect in the imidazole 118 perfused hearts unless cyclic AMP was elevated. The phosphorylase activation, without any measurable changes in cyclic AMP, could be accounted for an increase in intracellular calcium. It is naive to think that a pharmacological agent has only one action. Theophylline or imidazole undoubtedly have many actions in addition to inhibition or stimulation of phosphodiesterase. 119 SUMMARY AND CONCLUSIONS 1. Phenylephrine increased cardiac cyclic AMP, contractility and phosphorylase a_ values in that order in the isolated perfused guinea pig heart. The effects of phenylephrine on the above biochemical and mechanical events were dose-dependent. Phenylephrine (in the doses used) i s a weak beta-adrenergic agonist, being less potent and less effective than norepinephrine. The data are consistent with the hypothesis that adrenergic drugs produce their cardiac effects by stimulating adenylate cyclase and producing an increase in cyclic AMP. 2. (a) Histamine and the histamine analogs, TD and betazole, stimulated cardiac adenylate cyclase and increased cardiac cyclic AMP levels. The drugs also increased contractility and phosphorylase a.. (b) Histamine was most potent as compared to i t s analogs, TD and betazole in increasing cardiac contractility, phosphorylase a. and cyclic AMP. The order of potency observed wase histamine>TD>betazole. (c) Burimamide competitively antagonized the cardiac effects of histamine and i t s analogs. Burimamide showed i t s specificity for histamine by not affecting any of the norepinephrine-induced cardiac effects, namely contractility, phosphorylase and cyclic AMP elevation. 3. Promethazine^. H1 receptor antagonist also blocked the cardiac effects of histamine, including the histamine-induced increases in cyclic AMP. Themblbekade ofocardiacfhistami^ of a; . noncompetitive br-ncompeiiMvemri^ Promethazine \u00E2\u0080\u00946 (4x10 M) produced inotropic effects but at a higher concentration ^ _^ -(8-16x10 M) was cardiodepressanttc Promethazine (4x10 M) did not 120 affect the positive inotropic response to norepinephrine. At 8-16x10 \u00C2\u00B0M promethazine lowered the maximum response of norepinephrine. 4. Histamine and i t s analogs, 4-methylhistamine, TD and betazole stimulated cardiac adenylate cyclase. The order of potency of the compounds for stimulating cardiac adenylate cyclase was histamine>4-methylhistamine>TD> betazole. Stimulation by the agonists was blocked, in an apparently competitive manner, by burimamide. 5. Histamine, 4-methylhistamine, TD and betazole stimulated the gastric \u00E2\u0080\u00946 \u00E2\u0080\u00942 adenylate cyclase, over a dose-range of 10 - 10 M. The rank order of stimulation was histamine>4-methylhistamine^TD and betazole. \u00E2\u0080\u00946 Burimamide (1-10 M) antagonized the effect of the drugs in an apparently competitive manner. 6. Histamine, TD and betazole relaxed the estrogen primed rat uterus. The order of relaxation was histamine>4-methylhistamine>TD and betazole. However, we failed to detect any changes i n theladehylateecyclase activity dueiVt6=Jiis taminee interaction. 7. Histamine in a dose of 10 ~*M produced a maximum effect on the isotonically contracting guinea pig ileum. Burimamide did not block the histamine effect on the guinea pig ileum. Histamine at no time increased the levels of cyclic AMP over control, when measured in the frozen tissues. The present data are consistent with the hypothesis that histamine and i t s analogs produce their action by stimulating adenylate cyclase and elevating cyclic AMP. receptors appear to be associated with adenylate cyclase, at least in two tissues, the heart and the stomach. 121 H i s t a m i n e r e c e p t o r s i n g u i n e a p i g i l e u m a r e o f t y p e . S t i m u l a t i o n o f H^. r e c e p t o r s does n o t i n c r e a s e c y c l i c AMP and_hence H^ r e c e p t o r s a r e n o t a s s o c i a t e d w i t h a d e n y l a t e c y c l a s e . 8. The m e t h y l x a n t h i n e d e r i v a t i v e , t h e o p h y l l i n e p o t e n t i a t e d t h e c a r d i a c e f f e c t s o f n o r e p i n e p h r i n e o r h i s t a m i n e . T h e o p h y l l i n e enhanced t h e i n o t r o p i c and p h o s p h o r y l a s e a c t i v a t i n g e f f e c t s o f b o t h amines. 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It was f i l t e r e d and stored protected from light. I l l TRIS. buffer (Sigma 7-9 standard and Biochem buffer) solution by appropriate dilution with d i s t i l l e d water. V Glycogen 4.0% The glycogen solution was made by passing through the Dowex-column which was previously treated with hydrochloric acid and washed free of chloride. REAGENTS USED FOR CYCLIC AMP BINDING ASSAY: pH, adjusted to 6.9, using concentrated HC1. IV Shelf Molybdate Stock Solution. Working solution of shelf molybdate was made fresh from stock I Sodium acetate lOOmM. II Potassium phosphate buffer 20mM. III 5% Trichloroacetic Acid IV 1N-HC1 V Cyclic AMP dependent protein kinase 3.2 ug/30A VI Protein kinase inhibitor (from beef heart) 1.1 mg/ml -12 VII Unlabelled cyclic AMP 200 ,p moles (10 moles)/ml VIII \u00C2\u00A7[H] cyclic AMP 200 p moles/ml REAGENTS USED FOR ADENYLATE CYCLASE ASSAY: I Tris buffer 0.3M II Sodium fluoride 0.06M III Theophylline 0.06M IV KC1 0.166M V MgS0,.7Ho0 0.45M 4 2. VI Salt mix: KC1, MgSO^, i n l' : l ratio VII Phosphoenol pyruvate 0.3M VIII ATP 5mM IX Pyruvate.kinase; 1 to 5 dilution (freshly prepared). 139 LIQUID SCINTILATION COCKTAIL PPO 4.0 gm POPOP 50.0 mg Toluene to make 1000 ml COMPOSITION OF THE HEART PERFUSION FLUID: (Stock Solution) (Chenoweth and Koelle, 1946) Sodium chloride 140.0 gm Dextrose 36.0 gm Pot. chloride 8.4 gm Ca.Cl2.2H20 6.4 gm MgCl2.6H20 8.6 gm Dist. water to make 2000 ml The working Chenoweth and Koelle buffer was made by dilution 200 ml. of the stock solution to 1000 ml with d i s t i l l e d water. The pH was adjusted with sodium bicarbonate. The perfusion f l u i d was aerated with mixer of gases, 95% 0 2 + 5% C0 2. COMPOSITION OF THE BATHING SOLUTION FOR THE RAT UTERUS: (Diamond, 1973) 1. Sodium Chloride Tris buffer: Chemical m mol./litre Sodium chloride 125.0 Potassium chloride 2.4 Magnesium chloride ' 0.5 Glucose 11.0 Tris* 23.8 Calcium chloride. . 1.8 140 2. Potassium Chloride Tris buffer: Chemical m mol./litre Potassium chloride 127.4 Magnesium chloride 0.5 Glucose 11.0 Tris* 23.8 Calcium chloride 1.8 *Tris Tris(hydroxymethyl)-aminomethane A l l buffers were adjusted to pH 7.4 with concentrated HCl, and aerated with 100% oxygen. COMPOSITION OF THE TYRODE SOLUTION FOR THE GUINEA PIG ILEUM: (All solutions are expressed in mM). NaCl, 136.89; KC1, 2.68; CaCl 2.6H 20, 1.76; MgCl2.6H20, 0.98; NaHPO^.^O, 0.36; NaHC03, 11.90; Glucose, 5.55; d i s t i l l e d water to make 1000 ml of the-solution. The tyrode solution was adjusted to pH 7.4 and aerated with 95% 0 2 and 5% C0\u00E2\u0080\u009E. 2 Publications: SUBHASH C. VERMA Stewart, W.D., Runikis, J.O., Verma, S.C., Wallace ; S.: Problems in Selection of topical antiinflammatory corticosteroids, Can. J. Med. Assoc., 108: 33, 1973. Verma, S.C., Runikis, J.O., Stewart, W.D.: Fluorinated and nonfluorinated corticosteroids: A re-evaluation. Ind. J. Hosp. Pharm. 10_. 167-1.73, 1973-Verma, S.C., Chauhan, G.M.: St a b i l i t y studies of selected combinations of thiamine, pyredoxine and hydroxocobalamine in parental form. Bangladesh Pharm. J. 2_: 13-16, 1973. Verma, S.C., McNeill, J.H.: Action of imidazole on the cardiac inotropic phosphorylase activating and cyclic AMP producing effects of norepinephrine and histamine. Res. Commun. Chem. Pathol. Pharmacol. 305-319, 1974 McNeill, J.H. and Verma, S.C.: Phenylephrine induced increases in cardiac contractility, cyclic AMP and phosphorylase a_. J. Pharmacol. Exp. Therap., 187: 296-299, 1973. McNeill, J.H. and Verma, S.C.: Blockade by burimamide of the effects of histamine and histamine analogs on cardiac c o n t r a c t i l i t y , phosphorylase activation and cyc l i c AMP. J. Pharmacol. Exp. Therap. 188: 180-188, 1974. McNeill, J.H. and Verma, S.C.: Blockade of cardiac histamine receptors by promethazine. Can. J. Physiol, and Pharmacol. 52: 23-27, 1974. Verma, S.C. and McNeill, J.H.: Blockade by burimamide of the effects of histamine and histamine analogs on cardiac adenylate cyclase. J. Pharm. Pharmacol. 26_: 372-73, 1974. McNeill, J.Ho and Verma, S.C: Stimulation of rat gastric adenylate cyclase by Histamine and Histamine analogues and blockade by Burimamide. Brit. J . Pharmacol. In Press. Verma, S.C: The involvement of cy c l i c AMP in altered drug metabolism. Pharmagram (1973). McNeill, J.H. Coutinho, F.E. and Verma, S.C: Lack of interaction between norepinephrine or histamine and theophylline on cardiac c y c l i c AMP. Can. J. Physiol. Pharmacol. McNeill, J.H., Verma, S.C: Blockade of the cardiac effects of histamine, Cl i n . Res., 21, 238, 1973. McNeill, J.H., Verma, S.C, Lyster, D.M. : Blockade of cardiac mechanical effects of histamine and histamine analogs. Fed. P r o c , ^2: 808, 1973. - 2 -McNeill, J.H., Young, B.A. and Verma, S.C: Adrenergic amine induced increases in cardiac cyclic AMP, phosphorylase a_ and co n t r a c t i l i t y . Pharmacologist 15_: 418, 1973. McNeill, J.H. and Verma, S.C: Histamine and histamine analogue stimulated formation of cardiac cyclic AMP. Proc. Can. Fed. B i o l . S ci., JL6: 228, McNeill, J.H. and Verma, S.C: Blockade by burimamide of the cardiac mechanical and biochemical effects of histamine. Presented to the Canadian Cardiovascular Society, Halifax, Oct. 1973. McNeill, J.H. and Verma, S.C: The cardiac effects of imidazole. C l i n . Res., 21: 951, 1973. McNeill, J.H., Verma, S.C and Coutinho, F.E.: Cardiac actions and inter-actions of theophylline. C l i n . Res., 22: 9A, 1974. McNeill, J.H. and Verma, S.C: Blockade of cardiac histamine receptors. C l i n . Res., 22i 148a, 1974. McNeill, J.H., Verma, S.C and Coutinho, F.E.: The interaction between morepinephrine or histamine and theophylline on cardiac c o n t r a c t i l i t y , phosphorylase activation and c y c l i c AMP. Fed. Proc. 33: 479, 1974 McNeill, J.H., Verma, S.C.,: Caridac actions and interactions of noradrenaline and histamine. Presented at the Internatiorf^conference on Cyclic AMP, Vancouver, Canda, July 1974 Verma, S.C, and McNeill, J.H.,: Antagonism by Burimamide of histamine and histamine analog activation of cardiac and gastric adenylate cyclase. Presented at the Internatiori^conference on Cyclic AMP, Vancouver, Canada, July 1974. 1973. Verma, S.C, and McNeill, J.H.,: Histamine and Histamine agalogue-induced activation of adenylate cyclase and blockade by Burimamide. Proc. Can. Fedn. Bi o l . Sci., 17, 122. 1974 "@en . "Thesis/Dissertation"@en . "10.14288/1.0100090"@en . "eng"@en . "Pharmacology"@en . "Vancouver : University of British Columbia Library"@en . "University of British Columbia"@en . "For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use."@en . "Graduate"@en . "Biochemical and mechanical effects of adrenergic and histaminergic drugs"@en . "Text"@en . "http://hdl.handle.net/2429/19729"@en .