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Biochemical and mechanical effects of adrenergic and histaminergic drugs Verma, Subhash Chander 1974

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BIOCHEMICAL AND MECHANICAL EFFECTS OF ADRENERGIC AND HISTAMINERGIC DRUGS  by  Subhash Chander Verma B.Pharm. ( 1 9 6 4 ) , M.Pharm. (Gujarat University, India, M.Sc, University of B r i t i s h Columbia, 1 9 7 2 .  A Thesis submitted i n p a r t i a l f u l f i l m e n t of the requirement f o r the degree of DOCTOR OF PHILOSOPHY  i n the  D i v i s i o n of Pharmacology and Toxicology of the Faculty of Pharmaceutical Sciences.  We accept t h i s thesis as conforming to the required standard  The University of B r i t i s h Columbia November 1 9 7 4  1967),  In p r e s e n t i n g an  this  thesis  advanced degree at  the  Library  shall  the  in p a r t i a l  fulfilment of  University  of  make i t f r e e l y  I f u r t h e r agree that permission for  s c h o l a r l y p u r p o s e s may  by  his  of  this  written  representatives.  available  granted  gain  permission.  of  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, Canada  • /QJk  Columbia  1*74  for  for extensive by  the  It i s understood  thesis for financial  Department  Date  be  British  shall  requirements  Columbia,  Head o f my  be  I agree  r e f e r e n c e and copying of  that  not  the  that  study.  this  thesis  Department  copying or  for  or  publication  allowed without  my  ABSTRACT  A time-response and dose-response study of the effects of norepinephrine and phenylephrine revealed that both agonists caused increases 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. i n the isolated perfused guinea p i g hearts»  Norepinephrine caused a nearly f i v e f o l d  increase i n c y c l i c AMP, whereas phenylephrine produced only a two-three f o l d increase i n the nucleotide.  Phenylephrine i s less potent and less  e f f e c t i v e i n elevating a l l the three  parametersaasc'comparedtto  norepinephrine. Histamine and i t s analogs, TD and betazole increased cardiac c o n t r a c t i l i t y , phosphorylase a. and l e v e l s of c y c l i c AMP i n the isolated perfused guinea p i g heart.  The order of potency f o r the three compounds  was histamine>TD>betazole.  C y c l i c AMP was found to increase p r i o r to the  increase i n c o n t r a c t i l i t y or phosphorylase _a.  In the present study, the  new ^ - r e c e p t o r blocking agent burimamide, was found to be a s p e c i f i c , competitive blocking agent of both the mechanical and biochemical effects of histamine and i t s analogs on the heart.  Burimamide d i d not a f f e c t the  norepinephrine-induced increase i n c o n t r a c t i l i t y , phosphorylase a. or c y c l i c AMP.  Promethazine did interact with cardiac histamine receptors, but the  i n t e r a c t i o n was either non-competitive or competitive non-equilibrium i n nature. Histamine, 4-methylhistamine, TD and betazole a l l stimulated cardiac -7 adenylate cyclase i n doses ranging from 10  -3 to 10 M of the agonists. The  order of potency of the compounds f o r stimulating the enzyme was histamine> 4-methylhistamine»TD>betazole.  Stimulation by the agonists was blocked i n an —6  apparently competitive manner, by burimamide, since burimamide (1x10 1x10  ~*M) produced progressive s h i f t s i n the dose-response curves of the  iii  agonists to the r i g h t . Histamine, 4-methylhistamine,  TD and betazole, i n that order increased  rat gastric adenylate cyclase a c t i v i t y of the agonists.  -in  -7 -3 doses ranging from 10 to 10 M  Burimamide an ^ - r e c e p t o r blocking agent, i n concentrations  —6 of 1-5x10  M antagonized the drug-induced enzyme stimulation.  The maximum  increase i n the enzyme a c t i v i t y was approximately three f o l d and was competitively blocked by burimamide. The r e l a t i v e order of these agonists i n stimulating cardiac or gastric adenylate cyclase was similar to that found when the effect of these agents on cardiac c y c l i c AMP, c o n t r a c t i l i t y and phosphorylase a wass measured. Histamine and histamine analogs relaxed the r a t uterus i n a dosedependent manner. betazole.  The rank order was histamine>4-methylhistamine>TD and  However, 'histamine ididjan'ot-fepro'd-uce-the' a c t i v M i d h e o f "the adenylate  cy.clase'preparedifr.pmarat^uter-iif- prepared frcrc. ~£.£ uter .. r  Histamine  (10 ~*M) , produced  the maximum response i n the guinea p i g ileum,  which wase blocked by tripelennamine and diphenhydramine (H^-receptor antagonists), but not by burimamide (^-receptor antagonist).  Histamine did  not produce any changes i n c y c l i c AMP levels i n the i s o t o n i c a l l y contracting guinea p i g ileum. The data provide some evidence that ^ - r e c e p t o r s have similar properties i n at least two tissues, gastric mucosa and heart.  The data also provide  further evidence f o r the association of the ^ - r e c e p t o r with adenylate cyclase and d i s s o c i a t i o n of adenylate cyclase from H^-receptors. The cardiac effects of the catecholamines, and histamine may be mediated through c y c l i c AMP.  The effects of the amines are potentiated or inhibited  by the drugs which a f f e c t the enzyme phosphodiesterase.  Theophylline  i n j e c t i o n (1 mg) into the perfused guinea p i g heart resulted i n an increase  iv  i n c o n t r a c t i l i t y of about 20% over the control.  Theophylline also  potentiated the inotropic and phosphorylase a. producing .norepinephrine  e f f e c t s of  and histamine but d i d not produce any changes i n cardiac  c y c l i c AMP. Imidazole,jr;a phosphodiesterase stimulator, also caused an increase i n cardiac c o n t r a c t i l i t y .  Imidazole perfusion (40 mM) decreased or abolished  the p o s i t i v e inotropic e f f e c t of norepinephrine  and histamine, and produced  a p a r a l l e l f a l l i n the amine-induced increase i n cardiac c y c l i c AMP.  However  imidazole perfusion did not affect the phosphorylase a c t i v a t i n g effect of either amine. ih'-pr.es'encea Both  s  The eff'ect:cofr.amines:.on ph6sphorylase~ractivation ofd imidazole can not be explained on the basis of c y c l i c AMP.  theophylline and imidazole produce e f f e c t s on the hearts which are  apparently unrelated to their known e f f e c t s on c y c l i c AMP phosphodiesterase. I t i s known that the methylxanthines release calcium from i n t r a c e l l u l a r storage s i t e s and that imidazole increases the i n f l u x 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 i n f l u x .  V  TABLE OF CONTENTS Page Abstract  i i  L i s t of tables  vii  L i s t of figures  viii  L i s t of abbreviations  xi  REVIEW OF LITERATURE  1  A.  Relationship between hormone and c y c l i c AMP  1  B.  Adrenergic cardiac effects  7  C.  Histaminergic effects on:  16  I 1=1 III IV D.  The The The The  heart r a t stomach r a t uterus guinea p i g ileum  16 20 22 24  Interaction of adrenergic and histaminergic drugs with agents a f f e c t i n g phosphodiesterase  S p e c i f i c goals of the present investigation MATERIALS AND METHODS  25 28 30  A.  Materials I Animals II Drugs and chemicals  30  B.  Methods  31  I II III IV V VI VII VIII IX X  C.  Phosphorylase assay C y c l i c AMP binding assay Cardiac adenylate cyclase preparation Gastric adenylate cyclase preparation Uterine*aa'e"n^ll atte cyclase preparation Adenylate cyclase assay Protein estimation Time-response study of agonists Dose-response study of agonists Dose-response study of histamine and i t s analogs on rat uterus F  S t a t i s t i c a l analysis of the data  31 32 34 34 35 35 36 36 37 38 38  vi  Page RESULTS A. B.  C.  Cardiac effects of norepinephrine and phenylephrine on c o n t r a c t i l i t y , phosphorylase a_and c y c l i c AMP  40  Cardiac effects of histamine and histamine analogs on :c) c o n t r a c t i l i t y , phosphorylase and c y c l i c AMP and their blockade by burimamide and promethazine.  43  Interaction between histamine and i t s analogs and Burimamide on: I II III IV  D.  Cardiac adenylate cyclase Gastric adenylate cyclase Myometrium adenylate cyclase Guinea pig ileum  Interaction of agents a f f e c t i n g ph@§pfe@iit^4sMie: I  II  Actions and interactions of theophylline with histamine and norepinephrine on cardiac c o n t r a c t i l i t y , phosphorylase and c y c l i c AMP  69 77 77 88 88  88  Cardiac actions and interactions of imidazole with norepinephrine and histamine  96  DISCUSSION  105  SUMMARY AND CONCLUSION  119  BIBLIOGRAPHY  122  APPENDIX  137  vii  LIST OF TABLES Table 1  2  3  4  Page Possible involvement of c y c l i c AMP i n hormone actions on various tissues.  5  Possible involvement of c y c l i c AMP i n various metabolic processes.  6  The effect of TD, betazole and TD or betazole plus burimamide on cardiac c y c l i c AMP.  58  E f f e c t of histamine (1 yg) and norepinephrine (1 yg) and the i n t e r a c t i o n of these drugs with burimamide (2xlO~^M) and propranolol (10 ^M) on cardiac c o n t r a c t i l i t y , phosphorylase and c y c l i c AMP levels i n the perfused guinea p i g heart.  59  E f f e c t of TD and betazole and TD or betazole and theophylline (10~^M) on cardiac c o n t r a c t i l i t y i n the i s o l a t e d perfused guinea p i g heart.  61  The effects of promethazine, 2 x l 0 - 16X10 6M, on the norepinephrine-induced increase i n cardiac force.  76  The effect of histamine (10~->M);; at various times on the l e v e l of c y c l i c AMP i n the guinea p i g ileum.  91  The e f f e c t of 40 mM imidazole perfusion on the p o s i t i v e inotropic effect of histamine and norepinephrine.  100  The e f f e c t of imidazole (40 mM) on the norepinephrine and histamine-induced increase i n cardiac c y c l i c AMP.  101  The e f f e c t of imidazole (40 mM) on the norepinephrine and histamine-induced a c t i v a t i o n of cardiac phosphorylase.  103  The e f f e c t of imidazole on phosphorylase a., c y c l i c AMP and c o n t r a c t i l e force i n the i s o l a t e d perfused guinea pig heart.  104  _  5  6  7  8  9  10  11  - 6  -  LIST OF FIGURES  Schematic representation of the second messenger concept Enzymes involved i n 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 i n h i b i t i o n by antagonists.  selective  Time-response effects of norepinephrine (1 yg) 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. Time-response effects 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. Dose-response e f f e c t of norepinephrine and phenylephrine on cardiac c y c l i c AMP i n the perfused guinea pig heart. The effect of histamine, t r i a z o l e derivative and betazole on cardiac c o n t r a c t i l i t y on the i s o l a t e d , perfused guinea pig heart. 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 isolated perfused guinea pig heart. The effect of time on the a b i l i t y of 1.6 yg of t r i a z o l e derivative 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 pig heart. The effect of time on the a b i l i t y of 1.6 yg of betazole 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 pig heart. The effect of histamine, t r i a z o l e derivative (TRIAZOLE) and betazole on c y c l i c AMP i n the i s o l a t e d , perfused guinea pig heart. The effect of various concentrations (2-16 x 10~^M) of burimamide on the c o n t r a c t i l e response to histamine i n the i s o l a t e d , perfused guinea pig heart.  ix  Figure 14  15  16  17  18  19  20  21  22  23  24  25  26  Page The e f f e c t o f b u r i m a m i d e ( 0 . 5 x 1 0 " % ) on t h e c o n t r a c t i l e response t o t r i a z o l e i n the i s o l a t e d , perfused guinea p i g heart.  52  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 b u r i m a m i d e 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.  53  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 b u r i m a m i d e 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.  55  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 the i s o l a t e d , perfused guinea p i g heart.  56  The e f f e c t o f b u r i m a m i d e (1 x 10~ M) on t h e c o n t r a c t i l e response t o betazole i n the i s o l a t e d , perfused guinea p i g heart.  57  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 b u r i m a m i d e 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  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 over c o n t r o l v e r s u s dose o f promethazine perfused through the heart.  64  The e f f e c t o f v a r i o u s on h e a r t r a t e .  66  6  concentrations  of promethazine  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 - 16 x 1 0 M ) o n t h e histamine-induced increase i n force of contraction.  68  T h e e f f e c t o f p r o m e t h a z i n e ( 4 x 1 0 ~ - 16 x 1 0 ~ M ) o n t h e histamine-induced increase i n heart rate.  71  T h e e f f e c t o f p r o m e t h a z i n e ( 4 x 10~^M) o n 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  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 , TD a n d b e t a z o l e o n t h e a c t i v i t y o f g u i n e a adenylate cyclase.  75  - 6  _ 6  6  6  4-methylhistamine, pig cardiac  T h e e f f e c t o f b u r i m a m i d e ( 5 t o 10 x 1 0 ~ M ) o n t h e s t i m u l a t i o n of guinea p i g c a r d i a c adenylate 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-methylhistamine. 6  79  The e f f e c t of burimamide (1 x 10 M) on the stimulation of guinea p i g cardiac adenylate cyclase by various doses of betazole and TD. -tl  The e f f e c t of various concentrations of histamine, 3- (g-aminoethyl)-l,2,4 t r i a z o l e (Triazole), 4-methylhistamine and betazole on r a t g a s t r i c adenylate cyclase a c t i v i t y . The e f f e c t of various concentrations of histamine and 4- methylhistamine and the i n t e r a c t i o n of these drugs with burimamide on r a t g a s t r i c adenylate cyclase a c t i v i t y . The e f f e c t of 3-((3-aminoethyl)-l,2,4 t r i a z o l e (Triazole) and betazole and the interaction of these drugs with burimamide on rat g a s t r i c adenylate cyclase a c t i v i t y . The e f f e c t of various concentrations of histamine, 4-methylhistamirie, 3-(B-aminoethyl)-l,2,4 t r i a z o l e (Triazole) and betazole on the r a t uterus. The e f f e c t of theophylline (1 mg) at various times following i n j e c t i o n into the perfused guinea pig heart on c o n t r a c t i l i t y , % phosphorylase a. and c y c l i c AMP. The e f f e c t of various doses of norepinephrine on cardiac phosphorylase a c t i v a t i o n i n rat hearts perfused with buffer or buffer plus theophylline (7 x lCT^M). 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 rat hearts perfused with buffer or buffer plus theophylline (7 x lCT^M or 2 x 10~3M). The e f f e c t of various doses of histamine on c y c l i c AMP, c o n t r a c t i l i t y , and phosphorylase a. i n guinea pig hearts perfused with buffer or buffer plus theophylline (10~^M). The e f f e c t of various doses of imidazole on cardiac c o n t r a c t i l i t y i n guinea pig hearts perfused with b u f f e r . The e f f e c t of perfusion of various concentrations of imidazole (0.25-40.OmM) i n the i s o l a t e d perfused guinea pig heart.  ABBREVIATIONS  ATP  adenosine 5'-triphosphate  ADP  adenosine 5'-diphosphate  AMP  adenosine monophosphate  c y c l i c AMP  adenosine 3',5'-cyclic monophosphate  db. c y c l i c AMP  d i b u t y r y l adenosine 3',5'-cyclic monophosphate  GTP  guanosine 5'-triphosphate  c y c l i c GMP  guanosine 3',5'-cyclic monophosphate  G-l-P  glucose-1-phosphate  Pi  inorganic phosphate  TD  3-(3-aminoethyl)l,2,4  Tris  tri(hydroxymethyl)aminomethane  ppo  2,5-diphenylonazole  popop  1,4-bis[2-(5-phenylonazolyl)]-benzene  triazole  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 i n pharmacology.  Special thanks are due to  Dean B.E. Reidel f o r his kind interest i n my t r a i n i n g during the course of my studies i n this faculty. I am grateful to Dr. D.M. Lyster for h i s great help with the c y c l i c nucleotide assay procedures.  I wish to thank Drs. Godin, Roufogalis,  S i n c l a i r and HajLliday'efor:•• theU'rnuseful comments.  xiii  Dedication To Asha and Subodh  1  Review of L i t e r a t u r e A.  Relationship between hormone and c y c l i c AMP Complex p h y s i o l o g i c a l and metabolic processes i n l i v i n g organisms  are p a r t i a l l y controlled by hormones.  The question as to how hormones act  was posed by Sutherland, R a i l and. their colleagues.  The study of glucagon  or epinephrine-induced mobilization of l i v e r glycogen l e d to a possible p a r t i a l answer to this question. s l i c e s caused accumulation  The addition of these hormones to l i v e r  of a heat stable, dialysable adenosine  nucleotide, which eventually was i d e n t i f i e d as adenosine 3',5' c y c l i c monophosphate, commonly referred to as c y c l i c AMP (Sutherland et a l . 1962). Extensive research revealed that c y c l i c AMP was synthesized following the action of certain hormones on adenylate cyclase, a plasma membrane bound enzyme, distributed i n many mammalian tissues (Sutherland et a l . 1962). In addition to epinephrine many polypeptide hormones increase the concentration of c y c l i c AMP or stimulate adenylate cyclase a c t i v i t y i n target tissues Fig.(_l). These findings l e d to the second messenger hypothesis of hormone action (Sutherland, 0 y e and Butcher 1965). The hormone or f i r s t messenger binds to the s p e c i f i c receptor i n the plasma membrane and activates adenylate cyclase, which i s located i n close proximity to the s p e c i f i c hormone receptor.  Adenylate cyclase  stimulation by the appropriate hormone results i n an increased conversion of ( M g P > K T P t o c y c l i c AMP, which i s referred to as a second messenger. The nucleotide influences s p e c i f i c protein kinase  to donate phosphate  moieties to those effector proteins which ultimately carry out the s p e c i f i c c e l l u l a r transactions (Walsh et a l . 1968; Garren et a l . 1971; et a l . 1973; Butcher and Sutherland 1971).  Bitensky  The a c t i v a t i o n of adenylate  Varied Stimuli  ATP  Endocrine Gland  HORMONE (first messenger)  O  Cyclic 3',5-AMP (second messenger)  >TC5  <o Inactivated Hormone  5-AMP f phosphodiesterase  -plasma membra nq of target cell  Physiological Responses  Steroids,Thyroid Hormone>etc.  Fig.l Schematic representation of the second messenger concept  N5  3  cyclase i s s p e c i f i c i n nature (Lefkowitz 1973). hormone w i l l activate adrenal adenylate adenylate  cyclase, parathormone increases  cyclase i n renal cortex and vasopressin i n renal medulla  (Chase 1968). adenylate  Adrenocorticotropic  Glucagon, catecholamines and histamine  cyclase (Murad and Vaughan 1969;  et a l . 1970; Levey 1971;  Sutherland et a l . 1968;  a l l activate cardiac  Levey and Epstein 1969;  Poch and Kukovetz 1967;  Mayer  K l e i n and  McNeill and Muschek 1972).  Glucagon and epinephrine (Birnbaumer and Rodell 1969).  stimulate l i v e r adenylate  cyclase  In l i v e r , two e n t i r e l y separate  cyclase systems e x i s t for. glucagon and epinephrine. stimulated n o n - s p e c i f i c a l l y by f l u o r i d e .  adenylate  The enzyme i s also  Fluoride stimulation occurs i n  enzyme obtained from any tissue (Sutherland et a l . 1962) and the f l u o r i d e activated enzyme cannot be further stimulated by hormones s p e c i f i c to that tissue (Sutherland et a l . 1962;  Birnbaumer et a l . 1969).  The i n t r a c e l l u l a r concentration of c y c l i c AMP i s determined by a balance i n a c t i v i t i e s of two enzymes, adenylate  cyclase and phosphodiesterase,  the enzyme responsible f o r the metabolism of c y c l i c AMP to 5' AMP.  The  a c t i v i t y of both these enzymes can be affected by drugs. In summary, the demonstration of the presence of adenylate  cyclase i n  most mammalian tissues and the fact that a given hormone can increase enzyme a c t i v i t y provides suggestive evidence that the adenylate  cyclase-  c y c l i c AMP system may serve as the mediator f o r the action of that p a r t i c u l a r hormone on i t s target tissue.  However, before a r.easonabllfe degree of  c r e d i b 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 h i s coworkers (1962), should be f u l f i l l e d : 1.  Demonstration of a response to the hormone i n a washed broken c e l l preparation.  4  2.  An appropriate increase i n c y c l i c AMP i n i n t a c t c e l l s i n response to hormone stimulation.  3.  Drugs a f f e c t i n g phosphodiesterase should potentiate or i n h i b i t the hormone action.  4.  Exogenous c y c l i c AMP or i t s derivatives, should mimic the e f f e c t of the hormone.  Tablesll"i_2, summarises the possible involvement of c y c l i c AMP as a mediator of the actions of various hormones.  TABLE Possible Involvement of on Var  r  1  c l i c AMP i n Hormone Actions  is Tissues.  Tissue  Hormone  Liver  Glucagon  Adrenohypophysis  Hypothalamic releasing hormone  Renal medulla  Vasopressin  Adrenal Cortex  Adrenocorticotropic hormone  I n t e r s t i t i a l Cells  L u t e i n i z i n g hormone  Semeniferous tubules  F o l l i c l e - S t i m u l a t i n g hormone  Melanophores  Melanocyte-Stimulating  Thyroid  Thyroid-Stimulating hormone  Bone and renal cortex  Parathyroid hormone  Brain  Histamine, Serototiinn  hormone  Catecholamines, stimulate many target tissues (Robison, G.A. 1972).  6  TABLE  2  P o s s i b l e Involvement o f C y c l i c AMP Metabolic  P r o t e i n kinase Phosphorylase  i n Various  Processes.  activation activation  Stimulation of glyconeogenesis Stimulation of  ketogenesis  S t i m u l a t i o n 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  activity  I n h i b i t i o n of l i p o g e n e s i s 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 p u r k i n j e c e l l Increased  p e r m e a b i l i t y t o water and  Increased  f o r c e of cardiac  (Robison, G.A.  1972).  firing  electrolytes  contractility  7  Of the metabolic effects i n which c y c l i c AMP i s supposed to be involved, only a few are understood.  The most studied i s the a c t i v a t i o n of the enzyme  glycogen phosphorylase i n s k e l e t a l muscle (Krebs et a l . 1966; Walsh et a l . 1968).  C y c l i c AMP increases the a c t i v i t y of a protein kinase (Walsh et a l .  1968) which catalyses the activation of phosphorylase b_ kinase.  Phosphorylase  b_ kinase i n turn catalyzes the phosphorylation of inactive phosphorylase b_ to form active phosphorylase a.. to G-l-P (Fig.  Phosphorylase a. i n turn converts glycogen  2).  C y c l i c AMP-sensitive protein kinases seem to be at l e a s t as widely distributed as c y c l i c 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.  I t seems possible, therefore,that many i f not most of the  p h y s i o l o g i c a l l y important effects of c y c l i c AMP i n mammalian c e l l s may be brought about by the a c t i v a t i o n of a protein kinase. In consideration of the vast amount of information involving c y c l i c AMP as a second messenger, this introduction w i l l be directed solely to the postulated r o l e of c y c l i c 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 i n t e r a c t i o n with cardiac adenylate cyclase.  In p a r t i c u l a r  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  c l a s s i f i e d into metabolic and mechanical e f f e c t s . e f f e c t i s the a c t i v a t i o n of phosphorylase.  The most studied metabolic  The transformation of the enzyme  8  CYCLIC  AMP  Phosphorylase b Kinase (inactive) ATP  . Phosphorylase (inactive)  P h o s p h o r y l a s e bKinase Kinase ADP Phosphorylase b Kinase (active^e)  "b"  ATP , ++  Ca  Glycogen + P i ADP  *  Phosphorylase "a" (active)  Glucose-1-Phosphate  F I G . 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 c y c l i c AMP  i n myocardial c e l l s .  Various workers have studied the r e l a t i v e potencies of adrenergic drugs on a wide range of species.  Lands and Howard (1952), reported that  epinephrine was more e f f e c t i v e than norepinephrine i n increasing the amplitude and rate of contraction i n the perfused frog heart and tortoise atria.  Epinephrine was less e f f e c t i v e than norepinephrine on isolated rabbit  a t r i a and perfused rabbit heart, but isoproterenol was much more e f f e c t i v e than epinephrine or norepinephrine i n 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 i n  cardiac c o n t r a c t i l i t y have been studied by electrophysiologists i n terms of the e l e c t r i c a l events at the c e l l membrane (Edman 1965). increased the fluxes of Na 1965).  +  and K  +  Catecholamines  across the c e l l membrane (Glitsch et a l .  An i n t e r e s t i n g approach to explain the mode of inotropic action of  catecholamines, concerns the catecholamine-induced changes i n the metabolism of cardiac muscle glycogen.  This mechanism explains how the energy i s made  available f o r increased cardiac contraction.  Catecholamine effects are  exerted upon a complex system, referred to as the c y c l i c AMP-phosphorylase system, which i s the target f o r the action of several drugs (Hess and Haugaard 1958)'. Hess and Haugaard QW:59)) reported that epinephrine caused an increase i n phosphorylase a c t i v i t y and a p o s i t i v e inotropic effect i n an isolated rat heart preparation. Kukovetz, Hess, Shanfeld and Haugaard (1959) reported that the inotropic e f f e c t and phosphorylase a c t i v a t i o n were closely associated. manner.  Both of these actions were related i n a dose-dependent  Kukovetz et a l . (1959) have presented a l i n e a r relationship between  the p o s i t i v e inotropic e f f e c t and phosphorylase a c t i v i t y .  Mayer and Moran  10  (1960) confirmed the above findings i n 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 d i s s o c i a t i o n between the inotropic responses and phosphorylase a c t i v a t i o n i n 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 i n the open-thorax dog heart preparation norepinephrine produced  s i g n i f i c a n t inotropic effects with no measurable  changes i n phosphorylase a. a c t i v i t y . . Shanfeld, Frazer and Hess (1968;  1969)  were able to dissociate the c o n t r a c t i l i t y of the heart induced by norepinephrine from the effects of the drug on myocardial c y c l i c content.  They were successful i n blocking the phosphorylase  AMP  activation  of norepinephrine by N-isopropylmethoxamine '((IMA), without the inotropic responses being s i g n i f i c a n t l y depressed.  These findings were questioned^  Wastila et a l . (1972) suggested that the r e s u l t s obtained by Shanfeld et a l . (1969) could be an a r t i f a c t introduced by their readjustment  of the  d i a s t o l i c tension after the administration of IMA to the perfused isolated rat heart.  Moreover the concentration used (15 ]iig/ml) i n their study was  probably cardiodepressent.  In a similar study, Robison et a l . (1965) using  IMA i n the i s o l a t e d perfused rat heart, reported that thenepin'ephrind-ind.uced r i s e i n c y c l i c AMP  or c o n t r a c t i l i t y was not blocked by IMA.  Murad et a l .  (1962) correlated adenylate cyclase a c t i v a t i o n by drugs to their p o s i t i v e inotropic e f f e c t .  The r e l a t i v e potencies of isoproterenol}.? norepinephrine  and epinephrine i n activating adenylate cyclase were similar to those found  11  when phosphorylase a c t i v a t i o n was obtained (Murad et a l . 1962). increased the l e v e l s o f , c y c l i c AMP  Epinephrine  i n the r a t heart and the increase preceded  the other adrenergic effects such as the inotropic, chronotropic and phosphorylase a c t i v a t i o n effects (0ye et a l . 1964;  Robison 1965).  Williamson (1966) studied the k i n e t i c changes following the i n j e c t i o n of 1 gg of epinephrine into the r a t heart i n a Langendorff preparation. 2 sec. of the epinephrine i n j e c t i o n c y c l i c AMP  Within  and c o n t r a c t i l e force both  increased two f o l d , but phosphorylase levels did not change. a. levels increased and reached a broader peak at 20-30 sec.  Phosphorylase Cyclic  AMP  levels continued to r i s e , reached a sharp peak at 10 sec. and abruptly decreased to control values within 20 sec. AMP  Both c o n t r a c t i l i t y and c y c l i c  reached their peak at about the same time.  Williamson's findings  suggested a causal relationship between the three biochemical and mechanical. parameters. I t has been shown that stimulation of beta-adrenergic receptors leads to the a c t i v a t i o n of adenylate cyclase and an increase i n the tissue levels of c y c l i c AMP 1971;  (Rail and Sutherland 1961;  Greengard and Robison 1972;  Lefkowitz, Sharp and Haber 1973;  Mayer et a l . 1963;  Robison et a l .  Lefkowitz, Roth and Pastan 11971; Lefokowitz and Levey 1972).  I t has been  suggested that adenylate cyclase i s 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 d i s t i n c t from adenylate cyclase.  Levey (1971),  Levey and Klein;(1972), Lefkowitz (1971, 1973) separated the enzyme adenylate cyclase and beta-adrenergic receptors.  A s o l u b i l i z e d 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 i n t e r a c t i o n of norepinephrine i s with cardiac beta-receptors, are coupled to adenylate cyclase by a phospholipid  which  linkage.  There i s a s p e c i f i c phospholipid linkage for each respective hormone e.g. phosphotidylserine  for glucagon and histamine and  monophosphotidylinosital  for norepinephrine (Levey 1971). P o s i t i v e support for the t h i r d c r i t e r i o n , i . e . potentiation of the amine effects by theophylline, a phosphodiesterase i n h i b i t o r was given by R a i l and West (1963) who noted that the inotropic e f f e c t of norepinephrine was greatly enhanced i n the presence of theophylline.  Antagonism of the  p o s i t i v e inotropic e f f e c t , by imidazole, a potent stimulator of phosphodiesterase (Butcher and Sutherland  1962), provided further evidence  for c y c l i c AMP mediation of the catecholamine action (Kukovetz and Poch 1967). The fourth c r i t e r i o n i s that c y c l i c AMP or i t s derivative should have the a b i l i t y to produce the end organ response d i r e c t l y .  Use of the  dibutyryl derivative of c y c l i c AMP led to the f u l f i l m e n t of the fourth c r i t e r i o n for the myb'cardium (Epstein 1970; Skelton et a l . 1969; et a l . 1970).  Skelton  Drummond and Hemmings (1972) reported that perfusion of  0.2 mM db. c y c l i c AMP into the i s o l a t e d heart caused a p o s i t i v e inotropic effect within about 2 - 4 min.  The maximum e f f e c t occured at concentrations  of 2 - 3 mM of db. c y c l i c AMP.  Skelton, Levey and Epstein (1970) reported  that db. c y c l i c AMP exhibited a p o s i t i v e inotropic e f f e c t on i s o l a t e d cat p a p i l l a r y muscle driven e l e c t r i c a l l y .  A study by Krause et a l . (1970)  demonstrated that db. c y c l i c AMP produced both p o s i t i v e inotropic and chronotropic responses i n cultures of spontaneously beating heart  cells.  Thus a l l four of Sutherland's c r i t e r i a have been s a t i s f i e d implicating  13  c y c l i c AMP  as a mediator of the p o s i t i v e inotropic action of the  catecholamines.  One of the suggestive s i t e s for c y c l i c AMP  sarcoplasmic reticulum.  Myocardial sarcoplasmic  cyclase a c t i v i t y (Katz et a l . 1970). that c y c l i c AMP  action i s the  reticulum exhibits adenylate  Entman et a l . (1969) have suggested  increases calcium accumulation by the  sarcoplasmic  reticulum and that this effect contributes to the p o s i t i v e inotropic effect of the catecholamines.  However the c y c l i c 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 i n the preparation of sarcoplasmic Preparations of sarcoplasmic  reticulum.  reticulum are extremely d i f f i c u l t to p u r i f y  and the p o s s i b i l i t y of contamination with plasma membrane fragments, a l i k e l y source of the observed enzyme a c t i v i t y , can not be excluded. E l e c t r o p h y s i o l o g i c a l and tracer studies have demonstrated that catecholamines increase the transmembrane f l u x of calcium ions into the myocardial  c e l l s (Engstfeld et a l . 1961;  Carmeliet and Vereecke 1969; It i s generally accepted  Renter 1967;  Pappano 1970). that catecholamines also increase c y c l i c  levels i n cardiac tissue (Sutherland et a l . 1968). i n t r a c e l l u l a r levels of c y c l i c AMP to be concurrent  Vassort et a l . 1969;  AMP  This increase i n the  and the membrane calcium i n f l u x appear  e f f e c t s of catecholamines on the myocardial  cells.  Rasmussen,., Goodman and Tenenhouse (1972) have cited the importance of calcium" along with c y c l i c AMP  i n adrenergic cardiac e f f e c t s .  Their hypothesis  that stimulation of beta-receptorstbjy catecholamines could cause an a c t i v a t i o n of adenylate  states  simulataneously  cyclase and an a l t e r a t i o n of sarcolemmal  2+ permeability to Ca  ;  or increased l e v e l s of c y c l i c 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.  P o s s i b l e 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 c a l c i u m i n e f f e c t i n g an end-organ response: (Rasmussen et a l . 1972).  of i n t e r a c t i o n c y c l i c AMP  and Ca^"' would both function as second messengers,  whereas i n the l a t t e r case c y c l i c AMP  would be the second messenger and  2+ Ca  would serve as third messenger (Fig. 3).  (1969) reported that norepinephrine-induced  increases i n c y c l i c AMP  caused an increase i n calcium accumulation. lend support  levels  E l e c t r o p h y s i o l o g i c a l studies  to a d i r e c t effect of c y c l i c AMP  Coraboeu (1969) showed that c y c l i c AMP  Entman, Levey and Epstein  on calcium transport.  increased the height of the plateau  of the action p o t e n t i a l i n frog a t r i a l f i b r e s , which i s i n d i c a t i v e of an increase i n calcium inward current through the'slow Na -Ca of the plasma membrane.  In-voltage clamp studies i n c a l f purkinje f i b r e s ,  Tsein (1972) concluded that db. c y c l i c AMP calcium.  channels'  increased the inward current of  Meinertz et a l . (1973) showed that i n e l e c t r i c a l l y driven l e f t  a u r i c l e s from r a t heart, suspended i n Tyrode solution with reduced calcium concentration, db. c y c l i c AMP  as w e l l as epinephrine not only increased the  force of contraction but also caused an increase i n calcium uptake.  A  recent study by Watanabe and Besch (1974), suggested that c y c l i c AMP  can  2+ activate slow Ca  channels.  In cardiac glycogeholysis, c y c l i c AMP  operates by regulating  phosphorylase b_ kinase, but calcium probably plays a r o l e i n stimulating the phosphoprotein product of the c y c l i c AMP activated form of phosphorylase lp_ kinase. calcium and c y c l i c AMP  dependent protein kinase; i . e . the In cardiac glycogenolysis, both  appear to act sequentially.  calcium metabolism, such as occur i n adrenalectomized Exton and Park 1971)  Disorders of c e l l u l a r animals ( M i l l e r ,  led to a p a r t i a l i n h i b i t i o n of the p h y s i o l o g i c a l  response without changes i n the i n i t i a l r i s e i n c e l l u l a r c y c l i c  AMP  concentration. In summary, the bulk of the evidence supports the second messenger  16  hypothesis proposed by Sutherland et a l . (1968), that c y c l i c AMP  may  be  involved i n the effects of beta adrenergic amines on the c o n t r a c t i l e 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 i n the i n t r a c e l l u l a r of c y c l i c AMP.  levels  Benfey (1971) also f a i l e d to observe any changes i n cardiac  glycogenolysis,. but he reported that phenylephrine does increase cardiac force of contraction. The lack of relationship between c y c l i c AMP and inotropic effects (atrium) may  (ventricles) .  r e f l e c t differences between v e n t r i c u l a r  and a t r i a l i(imusGfLe)'rVabbit heart rather than prove a d i s s o c i a t i o n between c y c l i c AMP  and the inotropic effects of sympathomimetic amines.  Benfey (1971) did not measure c y c l i c AMP  Moreover  i n a beating heart i n which  physiological parameters could be simultaneously monitored.  A study by  McNeill and Davis (1972) suggests that phenylephrine does activate cardiac phosphorylase as w e l l as producing a p o s i t i v e inotropic e f f e c t . Benfey (1971) claims that his r e s u l t s refute the theory that c y c l i c AMP  acts as a second messenger i n 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. present investigation, time-response and dose-response  In the  studies were carried  out and the cardiac effects of phenylephrine were compared with those obtained with norepinephrine.  C.  Histaminergic e f f e c t s : I  Cardiac effects of histamine: Dale and Laidlaw i n 1910, reported that histamine caused an increase  17  i n cardiac c o n t r a c t i l i t y i n 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 s p e c i f i c (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 i n reserpine-t.a?eatee'd animals histamine also produced  cardiac e f f e c t s .  Trendelenburg  (1960) reported that the c l a s s i c a l  antihistamines, mepyramine and tripelennamine did not antagonize cardiac histamine e f f e c t s .  But Hughes and Coret (1972) could block cardiac histamine  e f f e c t s 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 e f f e c t s , histamine  effects on gastric acid secretion and relaxation of the r a t uterus are not blocked by mepyramine and tripelennamine (Ash and Schild 1966).  From these  observations Ash and Schild (1966) c l a s s i f i e d histamine e f f e c t s , which are blocked by c l a s s i c a l antihistamines, as being due to stimulation of "H^" histamine receptors.  type  The search was continued i n order to discover a s p e c i f i c  histamine antagonist which can antagonize the effects of histamine on the heart and g a s t r i c acid secretion, which apparently are controlled by a separate or ^ - t y p e of receptor. Black et a l . (1972) i n their c l a s s i c a l publication reported that burimamide ( N - m e t h y l - N ' - (4-(4,5HinetaWp'lV^'^*7-iL thiourea) i s a compound which s p e c i f i c a l l y antagonizes the cardiac histamine e f f e c t s .  The cardiac  histamine receptors are reported to be d i f f e r e n t i n d i f f e r e n t species (El-Ackad et a l . 1974).  Histamine H^ type of receptors are reported to be  present i n guinea p i g , cat and human heart (Klein and Levey 1971).  Rat  heart however does not possess histamine receptors (Green and Erickson 1967).  18  HISTAMINERGIC RECEPTORS H  and H ) and COMPETITIVE ANTAGONISTS 2  RECEPTORS  Guinea-pig ileum  H Rat stomach  Man blood pressure  Mepyramine  +  Burimamide  +  Metiamide  +  FIG. 4.  Responses of H^ and  2  RECEPTORS  Rat uterus  Guinea-pig atrium  Rat, man gastric secretion  receptors and their selective  i n h i b i t i o n 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 i n heart rate and c o n t r a c t i l i t y i n avian heart, whereas?- burimamide (^-antagonist) f a i l e d to a l t e r the chronotropic response produced by histamine. were unaltered by reserpine treatment i n avian heart. stimulates avian heart by i t s action on 'H^'  Histamine Histamine  effects thus  type receptors.  Poch and Kukovetz (1967) suggested the receptors f o r cardiac histamine effects i n guinea p i g heart are associated with adenylate cyclase. Dean (1968) postulated that the cardiac effects of histamine are mediated by the adenylate c y c l a s e - c y c l i c AMP  system.  K l e i n and Levey (1971) indicated that  histamine has the capacity to activate adenylate cyclase i n the p a r t i c u l a t e fractions of guinea p i g , cat and human heart homogenates.  K l e i n and Levey  (1971) found that histamine i n equimolar concentrations was about as e f f e c t i v e as norepinephrine.  McNeill and Muschek (1972), reported that  histamine induced a c t i v a t i o n of adenylate cyclase was not blocked by the antihistamines diphenhydramine and tripelennamine.  Theophylline potentiated  the cardiac biochemical and mechanical e f f e c t s 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 f o r a c y c l i c AMP  mediated p o s i t i v e inotropic  e f f e c t on the heart although histamine seems to act on adenylate cyclase at a s i t e which i s d i f f e r e n t from the adrenergic beta-receptor. study, the above working hypothesis was  investigated.  In the present  Time-response and  Dose-response effects of histamine and i t s analogs, 3-(g-aminoethyl)1,2,4 t r i a z o l e (TD), and betazole, on cardiac c o n t r a c t i l i t y , a c t i v a t i o n and c y c l i c AMP were studied.  phosphorylase  The i n t e r a c t i o n of burimamide with  histamine and i t s analogs on the above parameters wase also investigated. To further characterize the cardiac histamine receptor, the i n t e r a c t i o n of  20  histamine on cardiac adenylate  cyclase was  by burimamide of the e f f e c t s of histamine, agonist), TD and betazole was  studied.  The nature of blockade  4-methylhistamine (a s p e c i f i c  also investigated.  Histamine also stimulates the secretion of the acid by the stomach (Ivy et a l . i'9Sb; ^4h^e^Va'-i.V196*6);. This e f f e c t i s also not blocked by c l a s s i c a l a  antihistamines.  Therefore i t i s probably an E^  t v  P  °f receptor.  e  The  following section i s an attempt to discuss the work supporting the concept of E^ receptors i n the stomach.  II  Gastric e f f e c t s of  histamine:  Evidence that histamine acts as a chemical mediator for g a s t r i c HC1  secretion was  provided by Ivey and F a r r e l (1925).  histamine acts d i r e c t l y upon the autotransplanted (1948) showed that histamine  They reported that  fundic pouch.  Davies  stimulated isolated rat g a s t r i c mucosa i n v i t r o .  Code (1947) reported a r e l a t i o n s h i p between the output of histamine gastric j u i c e and the volume of HC1 stimulated by a meal.  i n the  secretion, when the g a s t r i c secretion i s  Alonso and Harris (1965), reported the a c t i v a t i o n of  g a s t r i c acid secretion from g a s t r i c mucosa due to g a s t r i n and methylxanthines. Perfusion of rat g a s t r i c mucosa with c y c l i c AMP (Shaw and Ramwell 1968). or histamine  Bersinbaev et a l . (1971) observed that pentagastrin  treatment produced an increase i n adenylate  the r a t g a s t r i c mucosa. showed that histamine mucosal adenylate histamine  caused an acid secretion  cyclase a c t i v i t y i n  The studies of P e r r i e r and Laster (1969, 1970),  and the histamine  cyclase.  analog, betazole stimulated p a r i e t a l  Karppanen and Westermann (1973) reported that  caused an increase i n g a s t r i c c y c l i c AMP  i n the guinea pig.  Levine  and Washington (1973) suggested that since stimulation of acid secretion was accompanied by an increased production of c y c l i c AMP  i n gastric juice, i t  21  appeared  l i k e l y that c y c l i c AMP  plays a mediating r o l e i n the human g a s t r i c  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 c y c l i c AMP hydrochloric acid secretion. between c y c l i c AMP  and  He further reported a causal relationship  elevation and the acid output.  Karppanen and Westermann  (1973) reported that H^ receptor blocking agent, diphenhydramine even i n a -4 concentration as high as 3.3x10  M only s l i g h t l y inhibited the histamine  stimulated production,of c y c l i c AMP.  Diphenhydramine did not i n h i b i t  histamine secretion of g a s t r i c acid.  The histamine-induced gastric acid  secretion i n rats (Black et a l . 1972) and i n man  (Wyllie et a l . 1972) i s  suggested to be due to H^ receptor stimulation.  Burimamide competitively  antagonized the histamine stimulated acid secretion (Wyllie et a l . 1972). Another o r a l l y active histamine et a l . 1973;  receptor antagonist metiamide (Black  Dousa and Code, 1974) i n h i b i t e d gastric secretion apparently  by blocking the stimulation of c y c l i c AMP  formation by histamine and i t s  methyl derivative i n the gastric mucosa of guinea pig. The present study was undertaken Histamine, 4-methylhistamine  to investigate the effects of  TD and betazole on rat gastric adenylate cyclase  and to ascertain the i n t e r a c t i o n of these drugs with burimamide. Histamine relaxes the r a t uterus and the histamine-induced relaxation i s not blocked by c l a s s i c a l antihistamines (Ash and Schild 1966).  Black et a l .  (1972) suggested the presence of B.^ type of receptors i n r a t uterus. next section the nature of the histamine-receptor i n the r a t uterus i s discussed.  In the  22  III  Histamine-rat uterus i n t e r a c t i o n : The finding that histamine caused relaxation of the rat uterus  has been reported by Dale and Dudley (1921); Craver et a l . (1951).  I n h i b i t i o n of the r a t uterus by adrenergic drugs has  also been reported by M i l l e r (1967); (1970);  Huebner et a l . (1949) and  Triner et a l . (1973);  Levyy" and Tozzi (1963);  Marshall (1973);  Triner et a l .  Diamond (1973).  Adrenergic  receptors i n the r a t uterus have been c l a s s i f i e d as beta-adrenergic receptors although tevyy and Tozzi (1963) reported the presence of alpha receptors as w e l l .  Levyy and Ahlquist (1961) showed that phentolamine could  reduce adrenergic e f f e c t s i n the r a t uterus.  Various other authors have  f a i l e d to confirm the presence of alpha-receptors i n r a t uterus 1972;  Marshall 1970;  Kroeger and Marshall 1974).  Both histaminergic and  adrenergic drugs produce relaxation i n the r a t uterus. response i s blocked by beta-antagonists (Schild 1967; Szego and Davis 1969;  Kroeger and Marshall 1971).  (Leonard  The adrenergic Triner et a l . 1969;  However c l a s s i c a l  antihistaminesssuch as mepyramine do not antagonize the histamine induced relaxation i n the r a t uterus (Ash and Schild 1966).  Black et a l . (1972) were  able to block the histamine effects on r a t uterus by burimamide.  They  further reported that histamine receptors i n r a t uterus were s i m i l a r to those involved i n histamine-stimulated g a s t r i c secretion. Blyth (1973), reported that the B.^ receptor may be involved i n histamine effects on the r a t uterus. Tozzi (1973) however reported that histamine-induced i n h i b i t i o n of the r a t uterus i s similar to that of tyramine and that i t i n d i r e c t l y activates beta-adrenergic receptors by releasing catecholamines.  Propranolol, an  e f f e c t i v e beta-adrenergic blocking agent produced a s i g n i f i c a n t p a r a l l e l s h i f t of the histamine, tyramine and isoproterenol dose-response curves. Jensen and Vennerod (1961) have also previously reported that d i c h l o r i s o -  23  proterenol blocked histamine effects on the isolated r a t uterus.  The findings  of Jensen and Vennerod ( 1 9 6 1 ) and Tozzi ( 1 9 7 3 ) suggest that histamine acts either on the same structures as does epinephrine or on a subsequent stage i n an epinephrine-initiated process.  Tachyphylaxis to histamine and  "tyramine on r a t 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 i s more l i k e that of tyramine than l i k e that of isorpoterenol. The blockade of histamine by cocaine c l e a r l y indicates that the i n h i b i t o r y action of histamine on the rat uterus as the r e s u l t of i n d i r e c t a c t i v a t i o n of beta-adrenergic receptors through the release of catecholamine(Tozzi and Roth 1967;  Tozzi 1 9 7 3 ) .  Green and M i l l e r ( 1 9 6 6 ) reported that the effects  of histamine on the rat uterus were blocked i n the rats treated with reserpine.  I f histamine works through the release of catecholamines i n the  rat uterus, then: liis.taminehwou'ldhbe expeetedrto-.producebother.Acha.racteristics of betapadrenerg'ic r e c e p t o r t s t i m u ! ^  by the  catecholamines r e s u l t s i n the elevation of c y c l i c AMP Marshall 1973;  Marshall and Kroeger 1973;  (Triner et a l . 1970;  Kroeger and Marshall 1 9 7 4 ) .  The a b i l i t y of various catecholamines to relax the uterus and to increase i t s c y c l i c AMP  content i s d i r e c t l y related to the effectiveness of these  amines as activators of beta-adrenoceptors relaxation and the increase i n c y c l i c AMP (Triner et a l . 1 9 7 1 ) .  Db. c y c l i c AMP  (Kroeger and Marshall 1 9 7 4 ) .  Both  i s prevented by beta-blockers  and the phosphodiesterase  inhibitor  papaverine, relaxed the smooth muscle and potentiated the i n h i b i t o r y effects of beta-adrenoceptor  a c t i v a t i o n (McFarland et a l . 1971;  Pbch and Kukovetz 1967; Takagi et a l . 1 9 7 1 ) .  Polacek and Daniel 1971;  Mitznegg et a l . 1970;  Somlyo et a l . 1970;  Histamine activates cardiac adenylate cyclase and  24  increases c y c l i c AMP.  (McNeill and Muschek 1972, K l e i n and Levey 1971)  the cardiac e f f e c t s of histamine are s p e c i f i c and d i r e c t .  Similarly, „  histamine also stimulates the gastric mucosa"and elevates c y c l i c AMP and Ramwell 1968;  Bieck, Oates and Robison 1973).  and  (Shaw  Both cardiac and gastric  histamine receptors are c l a s s i f i e d as B.^ type receptors.I f the histamine receptor i n r a t uterus i s also of  (Black et a l . 1972).  type, then i t s  stimulatibnbby histamine may r e s u l t i n an increase i n uterine c y c l i c  AMP.  Therefore i n the present study the histamine i n t e r a c t i o n with adenylate cyclase p a r t i c l e s 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 s p e c i f i c antagonism of histamine by antihistaminicrdEugs  characterizes one type of histamine receptors f o r which Ash and Schild (1966) suggested the symbol H^.  Such receptorscbecur i n guinea p i g ileum and  bronchi (Arunlakshana and Schild 1959).  The histamine  type of receptors  such as i n the heart and i n the g a s t r i c 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 i n c y c l i c (Poch and'Kukovetz 1967;  McNeill and Muschek 1972;  Bieck, Oates and  AMP Robison  1973). receptorslhav.e not been investigated with regard to their possible involvement with adenylate cyclase.  I t was therefore of interest to  investigate the e f f e c t s of histamine on such receptors i n order to determine i f c y c l i c AMP was involved.  25  D.  Interaction of adrenergic and histaminergic drugs with agents a f f e c t i n g phosphodiesterase Adrenergic  and histaminergic drugs may  exert their inotropic and  chronotropic effect through elevation of c y c l i c AMP Sutherland Increased  et a l . 1968;  Poch and Kukovetz 1967;  l e v e l s of c y c l i c AMP  and phosphorylase a..  may  (Robison et a l .  1965;  McNeill and Muschek 1972).  cause an increase i n cardiac c o n t r a c t i l i t y  Blockade of the increase i n c y c l i c AMP  by  appropriate  blocking agentsvwaJlll r e s u l t i n the blockade of cardiac responses. c r i t e r i a to e s t a b l i s h that c y c l i c AMP  Other  i s involved as a'second messenger'  have been established by Sutherland  et a l . (1968).  For example, the e f f e c t s  of drugs working through c y c l i c AMP  should be enhanced by p r i o r administration  of a drug that i n h i b i t s the breakdown of the nucleotide by phosphodiesterase.  inhibiting  The methylxanthines are known to i n h i b i t phosphodiesterase  i n v i t r o (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 , R a i l and West (1963), reported that the inotropic e f f e c t s  of norepinephrine i n i s o l a t e d rabbit a t r i a were potentiated i n the presence of theophylline.  Subsequently, Hess and co-workers (1963) found that  theophylline pretreatment reduced the c o n t r a c t i l e response to i n i s o l a t e d perfused rat hearts.  epinephrine  The discrepancies i n their findings could  be due to the d i f f e r e n t doses of theophylline used.  McNeill, Nassar and  Brodyy (1969) established that the dose of theophylline which would potentiate the e f f e c t s of the norepinephrine on cardiac phosphorylase was  cardiodepressant.  S i m i l a r i t i e s have been reported between the cardiovascular e f f e c t s of the methylxanthines and the 3-adrenergic et a l . 1965).  e f f e c t s of adrenergic  amines (Robison  For example, both groups of drugs increase cardiac  c o n t r a c t i l i t y and relax the i n t e s t i n a l smooth muscle (Pfaffman and  26  Mcfarland  (1973).  Papaverine, another phosphodiesterase i n h i b i t o r also w i l l  produce smooth muscle relaxation (Kukovetz and Poch 1971). have also been reported for catecholamines.  Similar e f f e c t s  Since the p o s i t i v e 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 a t r i a responded with increased  concentration i n a solution containing caffeine.  In recent years, however,  a number of investigations emphasized the differences rather than s i m i l a r i t i e s betweenxanthines?and:>ica-tecfaoframfae^deGubareff and Sleator 1965; et a l . 1969; Blinks et a l . 1972).  McNeill  Since methylxanthines have been found  to release ionized calcium from intraeel'lularar; storage s i t e s , this may be the process whereby the xanthines increase myoplasmic calcium ions to cause p o s i t i v e inotropic e f f e c t s (Nayler 1963);  Nayler  (1967), demonstrated that  the enhanced i n f l u x of calcium ions, which accompanies e x c i t a t i o n i n caffeinetreated toad hearts, resulted i n a r e l a t i v e l y high myoplasmic calcium i o n concentration and hence more powerful contractions. i n producing  The action of caffeine  contractures i n s k e l e t a l 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 e f f l u x of Ca  from s k e l e t a l muscle when placed i n 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 e x c i t a t i o n , i s the probable trigger mechanism for the i n i t i a t i o n of contraction.  Chapman and Niedergerke (1970) have proposed  that the slow tension changes i n frog heart muscle could be related to accumulation and depletion of stored calcium inside the c e l l .  27  Despite the evidence (Butcher and Sutherland  that methylxanthines w i l l i n h i b i t  1962;  McNeill et a l . 1973)  phosphodiesterase  i t has not been established  that the use of these drugs results i n an increase i n cardiac c y c l i c Support for the theory that c y c l i c AMP  i s involved i n the cardiac action  of c e r t a i n drugs has been provided by Kukovetz and Poch (1967), who that, imidazole, a phosphodiesterase  AMP.  observed  stimulator, inhibited the p o s i t i v e  inotropic and phosphorylase stimulating e f f e c t s of isoproterenol. As i n the case of theophylline, however there i s not much data regarding the effect of imidazole on cardiac c y c l i c AMP.  The recent study of Knope et a l . (1973) has  raised some i n t e r e s t i n g questions regarding the cardiac actions of imidazole. These authors have shown that imidazole exerted a p o s i t i v e inotropic e f f e c t on isolated a t r i a 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 p o s i t i v e inotropic effects were not blocked by propranolol (Knope et a l . 1973).  Imidazole  the heart d i r e c t l y by a mechanism other than c y c l i c  thus appears to affect  AMP.  DeMello et a l . reported that the addition of imidazole (500 yg/ml) to depolarized heart muscle e l i c i t e d a contracture. reported that imidazole-induced  DeMello et a l . (1973) further  contractures were largely dependent on  extracel'Mlarar calcium concentration.  Contractures induced by imidazole were  quickly suppressed i n the calcium-free solution. Massingham (1969), also reported that imidazole (5x10 M) _2  s t r i p to stimulation.  increased the response of the v e n t r i c l e  The effects of imidazole on the  frequency-force  c h a r a c t e r i s t i c s of isolated l e f t rabbit a t r i a are. similar to the actions of cardiac glycosides and calcium (Tuttle and Farah 1962). suggest that the s i t e of action of imidazole may calcium  turnover.  Farah and Witt (1963)  be upon tissue calcium or  28  It i s evident that a l l the effects of theophylline and imidazole cannot be explained i n terms of their actions on phosphodiesterase.  In l i g h t  of the above findings the effects of both imidazole and.theophylline on cardiac c y c l i c AMP, studied.  phosphorylase a c t i v a t i o n and c o n t r a c t i l i t y , were  The i n t e r a c t i o n between these agents and norepinephrine or  histamine on the above parameters was also studied i n an e f f o r t to determine i f c y c l i c AMP was involved i n the action of these drugs.  S p e c i f i c 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 s e l e c t i v e histamine (I^) blocking agent, burimamide.  3.  To study the i n t e r a c t i o n between an  blocking agent promethazine,  and  the histamine cardiac e f f e c t s . 4.  To determine the i n t e r a c t i o n 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 i n t e r a c t i o n with burimamide.  6.  To study the i n t e r a c t i o n of histamine on adenylate cyclase prepared from estrogen primed r a t uterus.  7.  To investigate the effects of histamine on  receptors (guinea p i g ileum)  with respect to their association or d i s s o c i a t i o n 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 c o n t r a c t i l i t y , phosphorylase a. a c t i v a t i o n and c y c l i c  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.  C y c l i c AMP (phosphoric a c i d ) , ATP (disodium s a l t ) ,  protein kinase i n h i b i t o r , c y c l i c AMP dependent binding protein, phenylephrine hydrochloride, 1-norepinephrine HCl (l-ar.feferenolHHSl*)., Imidazole (grade I I I , c r y s t a l l i n e ) , 45<!;inj<s%$e 1-phosphate (dipotassium s a l t ) , albumin (Bovine), 5', AMP ( c r y s t a l l i n e ) , T r i s (hydroxymethyl)-Aminomethane, l-amino-2 napthol-4 s u l f o n i c acid ( p u r i f i e d grade I I I ) . The following drugs were also used i n the study.  Theiri; source of supply  3 is listed:  [ H ] - c y c l i c AMP (ammonium s a l t , 25 C i per m mole) Amersham-Searle  2- phosphoenol pyruvate (trisodium s a l t ) and pyruvate, kinase (rabbit muscle) (Calbiochem), Histamine diphosphate (Mann Research laboratories, New York), Heparin sodium ( N u t r i t i o n a l Biochemicals Corporation, Cleveland, Ohio), Propranolol (Ayerst Laboratories, Inc., New York), Betazole HCl and 3- (3-aminoethyl)-l,2,4-Traizole dihydrochloride ( E l i L i l l y and Company, Indianapolis, Ind.), Burimamide [N-methyl-N'-(4-(4,5)-imidazolyl)butyl Thiourea] (Smith Kline and French Laboratories Ltd., Welwyn Garden City, Herts., England), theophylline (Merck and Company, Inc., Rahway, N.J.),  Promethazine  31  HCl (Poulenc, Montreal, Canada). AR grade.  A l l other solvents and reagents were  They were used as they were received without further p u r i f i c a t i o n .  The chemicals used i n the study-were obtained from the following sources: Ammonium acetate ( c r y s t a l s ) , cupric sulphate, E t h y l e n e - d i n i t r i l o tetraacetic acid (EDTA) (disodium s a l t ) , Postassium chloride (granular), Sodium carbonate (granular, . anhydrous) , Sodium t^arcBaraifee (granular) , Sodium s u l f i d e (granular, anhydrous), Sodium b i s u l f i t e (granular), Sodium f l u o r i d e were a l l purchased from Mallinckrodt Chemical works.  Dowex (anion exchange r e s i n , 100-200 mesh,  Bio-Rad laboratories), DVGOJucosje- (BDH) , phenol-reagent (Folin and Ciocalteau, 5  Harleco Chemical works), POPOF (1-4,Bis[2-(5 phenyloxazolyl)]-benzene, S i n t i l l a t i o n grade), PPo, (2,5-Di-phenyloxazole, Kent Laboratories).  B. Methods I  Phosphorylase Assay: Phosphorylase was determined by the method described by McNeill  and Brodyy' (1966).  This was a modification of the o r i g i n a l method described  by Cori and Cori (1940).  The d e t a i l s of the procedure are givlnlbelow.:  From a frozen heart tissue, a 70-100 mg portion of the apex of the heart was homogenized i n 200 volumes of solution which contained 0.05M T r i s , 0.001M EDTA, 0.02M NaF and 0.3% serum albumin. centrifuged at 10,000 g for 10 minutes. added to each incubation tube.  The tissue homogenate was  0.2 ml of the clear supernatant was  There were 6 incubation tubes for each heart,  divided i ntd^tw0ig-rojips,one group of incubation tubes numbered 1, 3, 4 and :  the other group of incubation tubes numbered 2, 5, 6.  The incubation tubes  numbered 1, 3, 4 contained t r i s buffer + (glycogen + GIP) and tubes 2, 5, 6 contained (Tris buffer + 5' AMP) + (glycogen + GIP).  Tubes 1 and 2 served  32  as blanks.  The rest of the tubes 3, 4, 5, 6, were Incubated at 37°C f o r 30  minutes f o r guinea pig hearts and 15 minutes for r a t hearts.  The reaction was  terminated by adding 2.0 ml of 10% t r i c h l o r o a c e t i c acid to the blank.and incubating tubes after placing them i n i c e . The tubes were then centrifuged at 15,000 RPM f o r 10 min. at room temperature.  The clear supernatant was added to phototubes to which had been  added 6.6 ml of diluted molybdate solution and 0.4 ml of reducing reagent (Fiske and SubaRow 1925).  The tubes were allowed to stand f o r 15 minutes.  The inorganic phosphate liberated during the synthesis of glycogen from G—I-P was read i n a Coleman Junior spectrophotometer  (Colemann Instrument Inc.,  Maywood, U.S.A.) at 660mjj. Phosphorylase a. a c t i v i t y i s represented by the amount of inorganic phosphate i n the tubes which did not contain 5'-AMP. The t o t a l phosphorylase a c t i v i t y i s represented by the amount of phosphate r  liberated i n tubes containing 5'-AMP.  The r e s u l t s are expressed as percentage  phosphorylase a. which i s : Enzyme a c t i v i t y without AMP x 100. Enzyme a c t i v i t y with AMP II  C y c l i c AMP binding assay (GiMaria 1970): a.  Tissue extraction f o r c y c l i c AMP assay: A 70-100 mg piece of the frozen tissue cut from the apex  of the heart, was homogenized i n one ml. of cold 5% t r i c h l o r o a c e t i c acid. The samples were centrifuged at 15,000 RPM (room temperature) f o r 10 minutes. To the clear supernatant was added 0.1 ml of I N HC1, with (5 ml each time) ether.  and extracted 5 times  The ether was previously saturated with water.  The f i n a l traces of ether from the aqueous extract were removed by flushing with nitrogen gas.  The sample preparations were lyophylized and the lyophylized  33  samples were redissolved i n 1.5 ml of 100 mM (pH 4.0)  and 20ul of the sample was  tissue extract f o r each heart was b.  C y c l i c AMP  sodium acetate-acetic acid buffer  used i n the c y c l i c AMP  determination.  The  done i n duplicate;  standard:  Unlabelled c y c l i c AMP  (phosphoric acid) was  water to give a solution containing 2.0 p moles/ml.  One  dissolved i n  to fourteen p moles  were used routinely i n constructing the standard curve.  Iel.-' C y c l i c AMP  Binding "Assay-Mix":  The assay mix had three components:  100 mM  sodium acetate-acetic  3 acid buffer (pH 4.0), protein kinase i n h i b i t o r and  dy  AMP.  Experimental d e t a i l s : The reaction was  200 y l :  [ H]cyclic  conducted i n an ice-bath i n a f i n a l volume of  Each assay tube contained, 100 u l of assay mix, which contained:  2 p moles of l a b e l l e d c y c l i c AMP, unlabelled c y c l i c AMP,  u l , representing  and 30 y l of c y c l i c AMP  added to each assay tube. mixture was  10-70  The equilibrium was  2-14  p moles of  dependent binding protein reached i n 90 min.  d i l u t e d with 1 ml of cold 20 mM K^PO^  at 4°C.  buffer, pH 6.0.  previously rinsed with the same buffer. cold 20 mM K^PO^  buffer.  0.45  The  Four to f i v e  min l a t e r , the contents i n the incubation tubes were passed through 24 m i l l i p o r e f i l t e r s ( c e l l u l o s e ester, HAWP 02400, HA,  was  mm  y, white p l a i n ) ,  The.tubes were washed with 8.0 ml of  The m i l l i p o r e f i l t e r s were placed i n clean dry  glass v i a l s and were dried at 150°F for one hour. 10.0 ml of l i q u i d s c i n t i l l a t i o n c o c k t a i l .  To each v i a l was  The r a d i o a c t i v i t y was  added  determined,  by s c i n t i l l a t i o n counting i n a nuclear Chicago isocap/300 model f l u i d counter.  34  The e f f i c i e n c y for each sample was read from the quench curve, obtained by 3  p l o t t i n g Channels r a t i o against e f f i c i e n c y , given by the quenched [ H] standard. The CPM recorded for each sample were corrected to DPM.  The standard curve  for c y c l i c AMP was a plot of DPM against t o t a l p moles of c y c l i c  AMP  3  ([ H ] - c y c l i c AMP  and unlabelled c y c l i c AMP  standard) present i n each tube.  A straight l i n e r e l a t i o n was obtained on a logarithmic plot between 2 p moles to 14 p moles of t o t a l c y c l i c AMP unknown sample with a given DPM Ill  per tube.  Amounts of c y c l i c AMP  i n the  could thus be read from the standard curve.  Cardiac adenylate cyclase preparation: A washed p a r t i c l e preparation containing adenylate cyclase was  prepared from guinea pig heart by the method of Drummond and Duncan (1970). The d e t a i l s are as follows: The guinea p i g heart was perfused with normal saline to remove a l l blood. The heart was weighed, cut and homogenized with 10 volumes of t r i s - H C l buffer for one min. i n a S o r v a l l Omni mixer.  The homogenate was passed through a  coarse sieve, and centrifuged at 1000 g. for 15 minutes.  The  supernatant  was discarded and the p e l l e t was washed twice with t r i s buffer.  The washed  p a r t i c l e s were suspended i n 5 volumes of t r i s buffer, and s l i g h t l y homogenized. -  v st gj  IV  The prepaf ationsmwereriused within''30 minute's r met'.cf ;  (1551'*.  .'."ie p:: rparat'_m.,v .  f.b.:.: "° Hltnitfis.  Gastric adenylate cyclase preparation: The rats were starved for 24 hours before s a c r i f i c e .  The whole  stomach was removed, washed with normal saline and inverted inside out.  The  stomachs were then homogenized i n 10 mM t r i s HC1 for one min. i n a s o r v a l l omni-mixer.  The homogenate was passed through a coarse sieve and centrifuged  35  at  1000 xg f o r 15 min. at 4°C.  The p e l l e t was washed twice with t r i s buffer.  The supernatant was discarded and -pellet was resuspended and s l i g h t l y homogenized i n a hand glass homogenizer.  V  '.  Uterine adenylate cyclase preparation: Female wistar rats weighing 200-250 gms, were injected with  d i e t h y l s t i l b o e s t r a l (dissolved i n peanut o i l , 60 ug/100 gms interaperitoneally) approximately 24 hours before s a c r i f i c e .  The animals were k i l l e d by a blow  on the head, the u t e r i were removed, weighed and homogenized i n 10 mM t r i s HCl. The procedure for preparing the adenylate cyclase p a r t i c l e s was i d e n t i c a l to that described f o r cardiac or g a s t r i c adenylate cyclase.  VI  Adenylate cyclase assay: Each assay tube ( i n a t o t a l volume of 150 ul) consisted of,  t r i s HCl, 0.3M, theophylline 0.06M, MgS0 0.225M, KC1 0.083M, phosphoenol 4  pyruvate 0.3M, pyruvate kinase (1:5 d i l u t i o n ) and ATP 5 mM.  After addition of  a l l the components except enzyme, the assay tubes were preincubated f o r 4 minutes.  The reaction was started by adding 50 p i of the enzyme preparation.  Incubation was carried at 37°C f o r 10 min.  The reaction was terminated by  placing the tubes i n a b o i l i n g water bath for 3 min. removed by centrifugation at 10,000 g f o r 5 min.  Denatured proteins were  A portion of the clear  supernatant (50 ul) was diluted 11-fold with 50 mM sodium acetate buffer.  A  50 u l aliquot of the sample was used f o r the determination of c y c l i c AMP by the method of Gillman (1970).  Enzyme a c t i v i t y was expressed i n p moles  c y c l i c AMP produced mg * protein min ^.  36  VII  Determination of protein: Protein content of the adenylate cyclase was determined by the  Lowry method (1951) as described by Sutherland et a l . (1949) and modified by Robson et a l . (1968). CHEMICALS AND REAGENTS A l k a l i n e copper sulfate solution was prepared by adding 1 ml of 2%  (w/v)  CuSO^ 5H 0, and 1 ml of 4% (w/v) sodium t a r t r a t e , diluted to 100 ml with 4% 2  solution.  Na^CO^  The phenol reagent was prepared by d i l u t i n g 2N f o l i n - c i o c a l t e a u  solution (1 part of reagent + 3 parts of d i s t i l l e d water).  Protein standard  solutions were made from Bovine serum albumin i n a concentration range of 10 to 200 yg/ml.  The standard protein solutions were kept frozen at -2°C.  PROCEDURE The adenylate cyclase washed p a r t i c l e s , were d i l u t e d 1:10 i n d i s t i l l e d water to place the protein concentraction within the range of the albumin standards. Five ml of the alkaline copper solution was added to 0.5 ml of the diluted suspension.  This was mixed instantaneously and was placed on a water bath kept  at 45°C f o r 12 min. added.  After exactly 12 min., 0.5 ml of the phenol reagent was  The contents of the tube were vortexed f o r 10 sec. and the tubes were  l e f t at room temperature for 15 min.  The absorbance, r e l a t i v e to a water blank,  was then determined by use of a perkin-elmer (Coleman 124) double beam spectrophotometer.  Standard albumin solutions were treated i n the same manner  i n order to generate a standard curve from which the unknown protein concentractions were estimated.  VIII  Time-response  study of the agonists:  Guinea pigs (500-700 gm) or rats (200-300 gm) of either sex were  37  injected with heparin sodium (8 mg/kg SC, i n guinea pigs and 2.8 mg/kg SC f o r r a t s ) , ninety minutes p r i o r . t o s a c r i f i c e .  The animals were stunned by a blow  to the head and the heart was rapidly removed and perfused with ChenowethKoelle solution (1946), by the Langendorff  technique.  perfusion f l u i d has been previously described.  The composition of the  The hearts were perfused at a  flow rate of 4 ml f o r both species per min. at 37°C.  The flow rate was  maintained by means of a Holter-microinfusion r o l l e r pump (Extracorporeal Medical s p e c i a l t i e s , King of prussia, PA, model RL175).  Cardiac c o n t r a c t i l i t y  was monitored by means of a palmer c l i p placed at the apex of the heart and connected to a Grass force-displacement transducer and recorded on a Grass model 7 polygraph. \ D i a s t o l i c tension was adjusted to 2.0 gm.  The hearts were  allowed to e q u i l i b r i a t e for 15 min. p r i o r to the i n j e c t i o n of the agonist. agonist was injected by means of a side arm cannula.  The  At various time-intervals  following the i n j e c t i o n the hearts were frozen by means of a pair of Wollenberger tongs (Wollenberger  et a l . 1960) previously c h i l l e d i n  2 methylbutane cooled i n an alchol-dryice mixture.  The hearts were stored  at -80°C, u n t i l assayed for c y c l i c AMP and for phosphorylase  IX  activation.  Dose-response study of the agonists: Complete dose-response curves of the agonists on cardiac force  of contraction were carried out.  From these studies the dose of the agonist  which produced the maximum effect was selected for studying the biochemical effects.  The times at which the phosphorylase  a. and c y c l i c AMP reached a  peak l e v e l , was used to study the Dose-response e f f e c t s .  The dose was  injected by the side arm cannula into the i s o l a t e d perfused guinea p i g or rat heart.  The hearts were frozen as described above at 38-40 sec. f o r  phosphorylase  measurements and at 16-18 sec. f o r the measurement of c y c l i c AMP.  38  The hearts were stored at -80° u n t i l analysed.  X  Dose-response study of histamine and i t s analogs on rat uterus: The rats were estrogen-pretreated with d i e t h y l s t i l b o e s t e r o l  dissolved i n peanut o i l (60 yg/100 gm interaperitbneally) approximately hours before s a c r i f i c e .  24  The animals were k i l l e d by a blow on the head, and  u t e r i were removed and placed i n a NaCl-tris buffered solution (Diamond at 37°C f o r 30 minutes p r i o r to suspension i n 50 ml organ baths.  1973)  A l l buffer  solutions were aerated with 100% oxygen. After 30 minutes, paired segments from one uterine horn were suspended by means pofa": small stainless s t e e l hooks i n organ baths containing the bathing media at 37°C.  Tension was measured isometrically by means of a force .  transducer (Grass FT03C) and recorded on a Grass model 79 polygraph. was slowly increased u n t i l the uterus was  contracting spontaneously with a  resting tension of approximately 0.2-0.5 gm. contractions became regular. buffered solution.  The u t e r i were l e f t u n t i l these  The bathing medium was then changed to K C l - t r i s  This caused a rapid depolarization of the muscle, and a  sustained contraction quickly developed.  After 15 minutes exposure to the  new bathing medium, histamine, 4-methylhistamine  TD and betazole i n a 1.0 ml  volume was added to the organ bath and relaxation was  C.  Tension  monitored.  S t a t i s t i c a l analyses S t a t i s t i c a l analyses were carried out using the ' t  unpaired data for c y c l i c AMP  and phosphorylase experiments  1  test f o r the  and ' t ' test f o r a  paired data f o r cardiac c o n t r a c t i l i t y , rat uterus relaxation and adenylate cyclase a c t i v a t i o n (Lewis 1966).  The results were calculated using Wange #600,  calculator using programme #9, and 10 f o r ' t ' test.  A p r o b a b i l i t y l e v e l of  39  0.05  was  chosen as indicating significance.  40 RESULTS Norepinephrine and phenylephrine cardiac effects A complete dose-response study with phenylephrine and norepinephrine on cardiac c o n t r a c t i l i t y revealed that 1.0 mg of phenylephrine or 1.0 yg of norepinephrine produced the maximum p o s i t i v e inotropic e f f e c t .  Maximum  e f f e c t i v e doses of agonists were used i n the time-response studies.  F i g . 5_  i l l u s t r a t e s the data obtained from a study of the e f f e c t of time on the norepinephrine (1 yg) response.  Norepinephrine produced a p o s i t i v e inotropic  response, an increase i n phosphorylase a and c y c l i c AMP elevation.  As a  r e s u l t of the norepinephrine i n j e c t i o n , c y c l i c AMP l e v e l s were elevated s i g n i f i c a n t l y (ELf.05) from the control value of 0.52 ± .04 nmoles/gm wet wt. at s i x seconds.  The r i s e i n c y c l i c AMP was very sharp, reaching nearly  a 5 f o l d increase over the control value i n 14 seconds.  Significant  elevations i n c o n t r a c t i l i t y and phosphorylase a i n response to norepinephrine were observed at 11 sec. C o n t r a c t i l i t y increased to a maximum of 85.0 ± 1.0% over control (20 s e c ) .  Control phosphorylase a_ values i n the experiments  were 5.6 ± 1.8% and norepinephrine increased the phosphorylase a. values to a peak of 55.0 ± 1.0% at 38 sec. S i m i l a r l y 1.0 mg of phenylephrine increased a l l three parameters. F i g . shows the e f f e c t of time on the phenylephrine responses.  C y c l i c AMP levels  were s i g n i f i c a n t l y d i f f e r e n t from control values of 0.40 ± 0.12 nmoles/gm wet wt. at 12 sec. following phenylephrine i n j e c t i o n and reached a peak at 18 sec. Cardiac c o n t r a c t i l i t y was elevated at 15 sec. and peaked at 20-25 sec. Phosphorylase a_ was s i g n i f i c a n t l y increased at 18 sec. and peaked at 30-40 sec. The maximum c y c l i c AMP value obtained with phenylephrine was 1.3 nmoles/gm wet wt. while c o n t r a c t i l i t y increased to 42.5 ± 1.5% over control and phosphorylase a increased to 35.0 ± 1.0%. A l l values were s i g n i f i c a n t l y less than those  Ui  U  CC  2  2  FIG. 5.  NOREPINEPHRINE -TIME RESPONSE  Time-response e f f e c t s of norepinephrine (1 yg) 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 ± S.E.M.  42  PHENYLEPHRINE TIME-RESPONSE  -50  o! LU  V)  -40 <1  >oc  -30  O  X  a.  Vi  o -20 CAMP FORCE  X  Ho  —PHOSPHORYLASE  30  40  TIME ( S E C . )  FIG.'6.  Time-response e f f e c t s of p h e n y l e p h r i n e (1 mg) on c a r d i a c c y c l i c AMP, p e r c e n t phosphorylase a. and c o n t r a c t i l i t y i n the p e r f u s e d guinea p i g h e a r t . Each p o i n t r e p r e s e n t s the mean o f t h r e e to f i v e h e a r t s and the v e r t i c a l b a r s r e p r e s e n t ± S.E.M.  43  obtained with norepinephrine.  In the next experiment,  a  dose-response  relationship of phenylephrine and norepinephrine i n their a b i l i t i e s to increase cardiac c y c l i c AMP was studied (Pig. § ) .  From the time-response  study, the time at which c y c l i c AMP peaked was selected f o r a dose-response study.  Different doses of the amines were injected and hearts were frozen  and analysed f o r c y c l i c AMP.  The data demonstrate that both amines produce  dose-dependent increases i n c y c l i c AMP.  Maximum values of c y c l i c AMP  obtained with norepinephrine were 2.3 ± 0.43 nmoles/gm wet wt. and with phenylephrine were 1.25 ± .101 nmoles/gm wet wt.  Phenylephrine was less  potent and less e f f e c t i v e than norepinephrine i n elevating cardiac c o n t r a c t i l i t y , phosphorylase a. and c y c l i c AMP.  B. Histamine and histamine analogs cardiac effects A dose-response contractility analogs.  study with histamine, TD and betazole on cardiac  (Fig. _8) showed that histamine was more potent than i t s two  From F i g . _8, the dose f o r histamine, TD and betazole was selected  to study their time-response relationship.  F i g . 9, l 0 , l'l ', shows: that f  1  3  histamine TD and betazole a l l increased c y c l i c AMP, phosphorylase a. and cardiac c o n t r a c t i l i t y .  S i g n i f i c a n t elevation (PL§.05) of histamine-induced  c y c l i c AMP was observed at 11 s e c , peaking at 18-19 sec. C o n t r a c t i l i t y and phosphorylase a. values were s i g n i f i c a n t l y  elevated at 15 sec. The control  values of c y c l i c AMP were 0.50 ± .08 nmoles/gm wet wt. AMP was f i v e f o l d over the control values.  The increase i n c y c l i c  Cardiac c o n t r a c t i l i t y peaked at  20-30 sec. while phosphorylase SL values peaked at a value of 41.0 ± 1.0% at a l a t e r time (30-40 s e c ) . Following an i n j e c t i o n of TD i n the i s o l a t e d guinea p i g heart; c y c l i c AMP l e v e l s increased from 0.5 ± 0 . 1 nmoles/gm wet wt. to 2.0 ± 0.lOnnmoles/gm  44  n=  © 2.5-  3-5 NOREPINEPHRINE  |jsjgj -2  E CD Vi  2.CH  O  £  1.5-  7  O.  <  1.0H  u P H E N Y L E P H R I N E (PEJ  0.2  0.4  T  0.8  T 1.6  D O S E ( u g - N E , mg-PE) FIG. 7.  Dose-response e f f e c t of norepinephrine and phenylephrine on cardiac c y c l i c AMP i n the perfused guinea pig heart. Hearts were frozen at the peak of the elevation of c y c l i c AMP (14 seconds with norepinephrine and 18 seconds with phenylephrine). 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 ± 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 a n d 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 pig heart. E a c h p o i n t r e p r e s e n t s t h e mean ± S.E.M. o f three to f i v e determinations. R e s u l t s a r e expressed 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 b e o b t a i n e d with histamine.  46  A  TIME („cj  FIG. 9.  The e f f e c t o f time on the a b i l i t y of 1 yg of h i s t a m i n 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 i n the 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 . C y c l i c AMP was s i g n i f i c a n t l y e l e v a t e d (P < .05) a t 11 seconds. C o n t r a c t i l i t y and p h o s p h o r y l a s e EL v a l u e s were s i g n i f i c a n t l y e l e v a t e d a t 15 seconds. C o n t r o l c y c l i c AMP l e v e l s were 0.50 ± 0.08 nmol/g wet wt. o f t i s s u e . C o n t r o l percent p h o s p h o r y l a s e a. l e v e l s were 5.0 ± 1.0%.  47  wet wt., a four f o l d increase over the control values.  Cardiac force of  contraction and phosphorylase a. levels were s i g n i f i c a n t l y increased (P~< 0.05) at 15 and 25 sec. respectively.  TD-induced increases i n c o n t r a c t i l i t y reached  a peak at 20-30 sec., but phosphorylase a_ elevation lagged behind, reaching a peak at 40 sec.  C o n t r a c t i l i t y increased to 48.2 ± 1.8% over control,  while phosphorylase ja values peaked at a value of 17.5 ± 2.0% at a l a t e r time. F i g . _lGf shows the time-response c o r r e l a t i o n of betazole-induced changes i n c y c l i c AMP, c o n t r a c t i l i t y and phosphorylase a., values.  C y c l i c AMP reached a  peak at 20 s e c , c o n t r a c t i l i t y and phosphorylase si reached a maximum at 20-30 sec. and 40 sec. respectively. The effect of these agonists on c y c l i c AMP formation was dose-dependent as shown i n F i g . 1-2; The order of potency i n increasing c y c l i c AMP was histamine>TD>betazole.  Peak values of c y c l i c AMP for histamine were  2.3 ± 0.05, f o r TD 2.1 ± 0.1 and for betazole 1.5 ± 0.07J/ nmoles/gm wet wt. of the tissue. In the next series of experiments, the i n t e r a c t i o n of burimamide with histamine, TD and betazole on cardiac c o n t r a c t i l i t y , phosphorylase a. a c t i v a t i o n and c y c l i c AMP was studied.  F i g . 13*shows the effect of various concentrations  (2-16x10 "*M) of burimamide on the c o n t r a c t i l e 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 e f f e c t of 0.5x10 ~*M and  —6 1x10  M burimamide on the c o n t r a c t i l e response produced by TD and betazole.  The dose-response curves of histamine, TD and betazole on cardiac c o n t r a c t i l i t y were shifted i n an apparently p a r a l l e l fashion by the doses of burimamide used. Similar effects were noted with burimamide i n s h i f t i n g the dose-response curve of histamine, TD and betazole on cardiac phosphorylase a c t i v a t i o n (Fig. _16, 17, 18).  FIG. 10.  The effect of time on the a b i l i t y of 1.6 yg of t r i a z o l e derivative 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 isolated, perfused guinea pig heart. C y c l i c AMP was s i g n i f i c a n t l y (P < .05) elevated at 11 seconds, c o n t r a c t i l i t y at 15 seconds and phosphorylase at 25 seconds. Control c y c l i c AMP levels were 0.50 ± 0.10 nmol/g wet wt. of tissue. Control percent phosphorylase a_ levels were 5.2 ± 1.0%.  75\  lu \J  n= 3-5  CONTRACTILITY  r<  O  20  15  Z  Z  * 5H  DOSE OF BETAZOLE 1.6  —r—  10  H.2fH  40  TIME30(SEC)  n=3-5  PHOSPHORYLASE  &T5H o o a:  5*  ,1 i 20  2.0-  30  40  50  ,60  TIME (SEC.}  CYCLIC AMP  E *>v. »» 1;5JU o  1.0rv.  < u >u  FIG.  11.  0:5- i rT 30 40 20 TIME (SEC.)  T h e e f f e c t o f t i m e o n 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 a n d (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 guinea p i g h e a r t .  49  50  n= 3-5  .......J  :2.OH £  --•I  Vi  £  o E  1.5H  HISTAMINE  <  y  •TRIAZOLE o.5H  BETAZOLE  U >-  u  0.2  0.4  0.8  i.«  3.2  D O S E Lug)  FIG. 12.  The effect of histamine, t r i a z o l e derivative (TRIAZOLE) and betazole on c y c l i c AMP i n the isolated, perfused guinea p i g heart. Agonists were injected and the hearts' were frozen 18 seconds l a t e r at the peak of the response. Control c y c l i c AMP levels were 0.50 ± 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 c o n t r a c t i l e response,to histamine i n the i s o l a t e d , 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  FORCE  FIG.' 14. . The effect of burimamide (0.5x10 M) on the c o n t r a c t i l e response to t r i a z o l e i n the isolated^ perfused guinea pig heart. Data are presented as percent increase,, over control.  53  % I N C R E A S E IN  FORCE O  ^-4  O O  to  m  O  FIG.  15.  3  II  CO  i  •  coH  co  The e f f e c t of 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 the i s o l a t e d , perfused guinea p i g heart. .JZ.^.'-S ••CTS .'' . P de"^:"^ad : ~ f i g -z 2 " ., . r  54  Histamine produced an increase i n c y c l i c AMP  and F i g . _19_' i l l u s t r a t e s  that 2x10 ~*M burimamide completely abolished the histamine (1 yg)-induced c y c l i c 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 c y c l i c AMP were also blocked by burimamide.(Table _3). The s p e c i f i c i t y of burimamide i n blocking the histamine effects was tested i n the next experiments.  The data presented i n Table 4- i l l u s t r a t e s  that burimamide (2x10 "*M) did not a f f e c t the norepinephrine-induced increase i n c o n t r a c t i l i t y , phosphorylase a^ or c y c l i c AMP.  Propranolol, a b e t a -  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 c o n t r a c t i l i t y i n the isolated perfused guinea pig heart.  Table _5  represents the responses due to various doses of TD.and betazole on cardiac c o n t r a c t i l i t y and their potentiation by theophylline. McNeill and Muschek _3 (1972), had previously reported that theophylline (10  M) potentiated the  histamine effects on cardiac c o n t r a c t i l i t y i n the i s o l a t e d perfused guinea pig heart. Burimamide shifted the dose-response histamine to the r i g h t .  curves f o r the cardiac effects of  The effects of promethazine (an H^ receptor  antagonist) and the nature of i t s i n t e r a c t i o n with cardiac histamine receptors —6 were investigated.  Promethazine perfusion i n a concentration of 2-4x10  M  through the guinea p i g heart caused a s l i g h t p o s i t i v e inotropic effect (Fig. 20).  r  These effects were not seen at higher promethazine concentrations g  (8-16x10 M). Promethazine (2-4x10 M) produced a p o s i t i v e chronotropic e f f e c t but a s l i g h t negative inotropic effect was noted at higher concentrations  55  %  FIG. 16.  PHOSPHORYLASE  a  The effect of histamine and histamine plus burimamide on cardiac phosphorylase a c t i v a t i o n i n the isolated, perfused guinea pig heart. Each point represents the mean percent phosphorylase ji ± S.E.M. of f i v e determinations. Burimamide was perfused through the heart for 15 to 20 minutes before histamine i n j e c t i o n . Percent phosphorylase a. values i n hearts injected with buffer solution were 5.0 ± 1.0%.  56  %  PHOSPHORYLASE Or  O  a  ho o  3 II K3  C7  O . Co oo rn  CO  Jo  FIG.  17.  T h e 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 plus b u r i m a m i d 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 ' o n t h e i s o l a t e d , perfused guinea p i g heart. Eir.t -\ ,• „ * » f  57  %  FIG.  18.  PHOSPHORYLASE  a  T h e e f f e c t o f b u r i m a m i d e ( 1 x 1 0 M) o n t h e c o n t r a c t i l e response t o b e t a z o l e i n the i s o l a t e d , perfused guinea p i g heart. 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 over c o n t r o l .  58  TABLE  3  E f f e c t of TD, betazole and TD or betazole plus burimamide on cardiac c y c l i c AMP. Hearts were frozen at the peak of the c y c l i c AMP response. Drug Treatment  Burimamide Concentration  C y c l i c AMP (nm/gm net weight)  None  0  0.48  ±  0.13  TD 1.6 yg  0  1.97  ±  0.12* 0.08  TD 1.6 yg  0.5 x 10~ M  0.51  ±  TD 6.4 yg  0.5 x 10~ M  2.10  ± 0.03*  0  1.38  ±  0.15*  0.53  ±  0.05  1.42  ± 0.08*  5  5  Betazole 1.6 yg Betazole 1.6 yg  1  x 10  M  Betazole 6.4 yg  1  x 10~ M 6  N = 3-5  * S i g n i f i c a n t l y (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 c o n t r a c t i l i t y , phosphorylase and c y c l i c AMP levels i n the perfused guinea p i g heart. Histamine and norepinephrine were injected v i a a side-arm cannula. The antagonists were perfused through the heart f o r 15-20 min. p r i o r to the i n j e c t i o n of the agonist. C y c l i c AMP was measured 18 sec. and phosphorylase a. 40 sec. after i n j e c t i o n of the agonist. Drug  C y c l i c AMP (nmole/gmwwet weight)  Buffer  solution  0.68 + 0.05  % Phosphorylase a.  5.0 + 1.0  0.00  44.7 . + 1.2  Histamine  2.50 + 0.09  Histamine & Burimamide  0.65 + 0.04  His tamine & Propranolol  2.32 + 0.12  3  38.4 + 2.2  Norepinephrine  2.30 + 0.12  a  Norepinephrine & Propranolol  0.68 + 0.03°  Norepinephrine & Burimamide  2.05 +  o.io  a  b  a  Contractility (% increase over control)  a  7.5 + 1.4  90.6 ± 1.  2  a  15 ± 1. 6  b  a  82.9 ± 2. 3  a  57.9 + 1.4  a  89.0 ± 1. 8  a  4.7 + 1.2  C  9.5 ± 0.5  C  81.1 ± 1.5  a  b  55.2 + 1.5  a  a.  S i g n i f i c a n t l y increased when compared to buffer solution injected hearts (P <0.05).  b.  S i g n i f i c a n t l y decreased when compared to histamine injected hearts.  c.  S i g n i f i c a n t l y decreased when compared to norepinephrine injected hearts.  Each value represents the mean ± 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 i n c y c l i c AMP. Each bar ^represents the mean ± S.E.M. of three to f i v e c y c l i c AMP determinations. C y c l i c AMP was determined i n hearts frozen 18 seconds after the i n j e c t i o n of histamine. ,, e  61  TABLE  5  E f f e c t of TD and betazole and TD or betazole and theophylline (10~3M) on cardiac c o n t r a c t i l i t y i n the i s o l a t e d perfused guinea pig heart. Drugs were injected or perfused as described i n the Methods. Results are expressed as a % of the maximum response obtained with histamine (1 yg).  Drug Treatment  TD TD + Theophylline Betazole Betazole & Theophylline  Dose (yg) 0.8  0.2  0.4  8.6 ± 2.9  20.0 ± 2.0  13.0 ± 1.0  27.0 ± 2.0  0.0 10.5 ± 2.3  27 a  10.0 ± 1.0 a  17.5 ± 1.9  1.6  ± 1.0  39.0 ± 2.5  40.0 ± 4 . 3 a  19.0 ± 2.0 a  25.0 ± 2.0  25.0 ± 2.5 a  N = 5  a.  (rr S i g n i f i c a n t l y (P <0.05) greater than drug treatment without theophylline.  62  (8-16x10  M) as shown i n F i g . 21.  Histamine dose-response curves of cardiac c o n t r a c t i l i t y and heart rate were obtained i n the presence and absence of promethazine (4-16x10 ^M). As seen i n F i g . 22. promethazine decreased the maximum histamine response, but did not s h i f t the histamine dose-response to the r i g h t .  Blockade by  promethazine of the histamine cardiac effects could not be overcome by increasing the concentration of the agonist, which i s an i n d i c a t i o n of a noncompetitive or competitive nonequilibrium type of antagonism.  Only one  —6 concentration of promethazine (4x10 M) d i d not a f f e c t the chronotropic e f f e c t of histamine i n the guinea p i g heart (Fig. 23) whereas higher —6 M) of promethazine reduced the chronotropic response —6 The increase i n promethazine concentration (16x10 M) further  concentrations(8-16x10 of histamine.  reduced the histamine e f f e c t on heart rate. of "his taminenare'.reduced-by  The chronotropic-effects.  ^pr^mStHazfnS'-fe-a'm.dncbmpet±^tdve'^dr-';  ndnequlMbrium manner, s r . . : . r  - J S E typs of : . ~ : . S 3 -  Histamine (1 yg) caused abcut a fdur f d l d increase i n the tissue c y c l i c AMP content.  —6 Promethazine (4x10 M) reduced the histamine-induced cardiac  c y c l i c AMP levels (Fig. 24). Increasing the dose of histamine to 3.2 yg —6 i n the presence of promethazine (4x10 M) d i d not further increase the levels of c y c l i c AMP.  Again the type of blockade noted with promethazine was  noncompetitives or competitive nonequilibrium. The i n t e r a c t i o n between promethazine and norepinephrine was studied on guinea p i g heart.  The-data are summarized i n Table 6^. Promethazine (2-4x  —6 10  M) d i d ndt affect the p d s i t i v e indtropic respdnse td ndrepinephrine.  At 8x10 ^M, prdmethazine ldwered the maximum respdnse td ndrepinephrine. Prdmethazine (16x10 ^M) decreased the effects c f a l l ddses df ndrepinephrine. In summary, the results of the experiments described t h i s f a r suggest  63  FIG. 20.  The e f f e c t 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 ± S.E. of four hearts. Force was s i g n i f i c a n t l y elevated at 2 x l 0 ^ and 4xlO~^M. -  65  FIG. 21.  The effect of various concentrations of promethazine on heart rate. Heart rate was s i g n i f i c a n t l y elevated at 4xlO~^M promethazine and s i g n i f i c a n t l y lowered at 8 x l 0 ~ and 16xlO M promethazine. 6  -6  20  2  4  8  Promethazine  16  CulW)  67  FIG. 22.  The effect of promethazine (4x10 - 16x10 M) on the histamine-induced increase i n force of contraction.  Histamine Promethazine  4JJM J Q  '•  Promethazine 8 j j i  Promethazine 16JJM  J  2  J  —  .4  Dose Histamine (yg)  L  .8  _  _  I  1.6  69  that burimamide, competitively blocked the histamine cardiac e f f e c t s , whereas promethazine.interacted with the cardiac histamine receptors, but i n a non-competitive or non-equilibrium manner.  Histamine-cardiac adenylate cyclase i n t e r a c t i o n In order to further characterise the  receptor i n heart; the effects  of histamine on cardiac adenylate cyclasewere studied.  F i g . 25 i l l u s t r a t e s  the data obtained with several doses of histamine, 4-methylhistamine specific  (a  receptor agonist; (Black et a l . 1972), TD and betazole. A l l  the agonists, stimulated cardiac adenylate cyclase. as agonist concentration vs c y c l i c AMP  The data were plotted  formed (P Mol./mg. protein/min.).  The  order of potency of the compounds f o r stimulating cardiac adenylate cyclase was histamine>4-methylhistamine^TD>betazole.  The a c t i v i t i e s of  4-methylhistamine  and TD were not s i g n i f i c a n t l y d i f f e r e n t from each other, but were s i g n i f i c a n t l y greater than the a c t i v i t y of betazole.  Burimamide (1x10 ~*M and 5x10  ^M) —6  shifted the histamine dose-response  curve to the r i g h t .  also produced a s h i f t i n the dose-response  curve to the  Burimamide (10  M)  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 i n t e r a c t i o n with burimamide i s presented i n F i g . 27. —6 Burimamide (10  M) moved the TD and betazole-induced stimulation of the cardiac  adenylate cyclase to the r i g h t .  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 histamine-induced increase i n heart rate.  M) on the  71  72  FIG. 24.  The effect of promethazine (4x10 M) on the histamineinduced increase i n cardiac c y c l i c AMP.  ro  !—-J Control EES  Histamine 1jjg Promethazine 4pM Promethazine 4uM ~r Histamine 1jjg  s-r^ Promethazine  4JJM  + Histamine 3.2jjg  74  F I G . 25.  The e f f e c t o f v a r i o u s d o s e s 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 adenylate cyclase. Histamine,--^ 4-Methylhistamine, .... TD, Betazole.  10 0  c  £ c  80  A -  •^Histamine  V-  •v 4-IVfe thy! histamine  0-  -oTriazole • Betazole  .+-»  o &~  a cn E  60  £ a. 20i  o  10  7  -4 10  10 10 Drug concentration(M) 5  76  TABLE The effects of promethazine, 2xl0~ induced increase i n cardiac force.  6  6 - 16xlO~ M, on the norepinephrine 6  Concentration of promethazine (M) Dose of norepinephrine  0  0.2  50.2 ± 2.31  0.4 0.8 1.6  -fi ' 2 x 10  4 x 10  fi  . 8.x 10  fi  fi 16 x 10  48.6 + 1.21  50.7 ± 3.61  43.8 ± 1.43  '?38.3 ± 1.15  75.7 ± 1.52  .73.8 ± 0.89  74.2 ± 2.12  71.2 ± 0.93  62.0 ± 1.1.5  90.5 ± 0.98  89.7 ± 1.21  89.8 ± 1.80  83.2 ± 0.87  78.3 ± 1.93  98.3 ± 1.31  98.4 ± 1.51  90.0 ± 0.46  100  3  b  b  b  b  86.0 ± 0.86  a. Means ± S.E.M. of three hearts. b. S i g n i f i c a n t l y less than control P <0.01. NOTE: .The r e s u l t s are given as percentage of the maximum response that could be obtained with norepinephrine.  b  77  Histamine-gastric adenylate cyclase i n t e r a c t i o n 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 i n other tissue as well.  I t has been suggested  that histamine-induced gastric acid secretion i n frog g a s t r i c mucosa i s not blocked by H^ type of histamine antagonists. histamine g a s t r i c receptorswer.e of the  Black et a l . (1972) suggested  type.  Therefore the i n t e r a c t i o n  between histamine and gastric adenylate cyclase was that histamine, 4-methylhistamine, adenylate cyclase.  studied.  F i g . 28 shows  TD and betazole a l l stimulated gastric  Histamine-induced  stimulation of adenylate cyclase was  s i g n i f i c a n t l y greater than 4-methylhistamine,  TD and betazole.  was no s i g n i f i c a n t difference between 4Smethylhistamine  Again there  and the TD.  The rank  order of the compounds with regard to t h e i r respective a c t i v i t i e s was same as observed with cardiac adenylate cyclase. —6  the  F i g . 19. shows that  —6  burimamide (5x10  M); and 1x10  M) shifted the curves of histamine and  4-methylhistamine  respectively to the r i g h t .  The burimamide blockade  overcome by increasing the concentration of the agonists.  F i g . _3_0 i l l u s t r a t e s ,  TD and betazole stimulated g a s t r i c adenylate cyclase dose^response —6 shifted to the right by burimamide (10  M).  was  The data from these  curves were experiments  suggests, that burimamide competitively antagonized the histamine-induced a c t i v a t i o n of g a s t r i c adenylate cyclase.  Our r e s u l t s suggest that histamine  receptors i n the heart and i n the stomach are associated with adenylate cyclase. Histamine-myometrium adenylate cyclase interaction Histamine effects on the r a t uterus are not blocked by the Hj-tantihistaminic drugs (Ash and Schild 1966). v )  Black et a l . (1972) reported that burimamide,  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~ M), Histamine + burimamide (lxlO~ M). 4-Methylhistamine, - •4-Methylhistamine + burimamide (lxlO" M). 6  5  c  6  79  c A M P formed(PM/mg Protein/min) to o  4^ o  CD O  CO O  4>  o o  ©  IS  o f •  <>  X  1 I  6  oT 0)  |IU  Oi 0)  2. <& IT  + c im.  Oi  3 CP  +  CD  c  3  •  . ^ Oi  X OI  Oi  Oi  Oi  2 3  O)  80  FIG. 27.  The effect of burimamide (1x10 M) on the stimulation of guinea p i g cardiac adenylate cyclase by various doses of betazole and TD. Each point represents the mean ± S.E. of 4 determinations. TD, Betazole, -TD + burimamide (1x10"%), „Betazole + burimamide (lxlO~ M). 6  00  ©.—Q  Triazole  o—-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 t r i a z o l e ' ( T r i a z o l e ) , 4- methylhistamine and betazole on rat g a s t r i c adenylate cyclase a c t i v i t y . Each point represents the mean ± 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 g a s t r i c adenylate cyclase a c t i v i t y . Each point represents the mean ± S.E.M. of 4-6 determinations.  oo  Drug Concentration (M)  86  FIG. 30. The e f f e c t of 3-(B-aminoethyl)-l,2,4 t r i a z o l e (Triazole) and betazole and the i n t e r a c t i o n of these drugs with burimamide on rat g a s t r i c adenylate cyclase a c t i v i t y . Each point represents the mean ± S.E.M. of 4 determinations.  oo  111  C5  •  %  40  Triazole  o  •CXO  £  a /••^.  Betazole  30  -° Triazole +Burim.(1*1o|> 20  • Betazole+Burimflx10»)  e 5-.  10  a  -4  10 10 Drug Concentration (M)  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 i n F i g . 31_.  I t demonstrates that histamine,  4-methylhistamine,  TD and betazole a l l relaxed the r a t uterus i n a dose-  dependent manner.  The order of potency was histamine>4-methylhistamine>TD>  betazole.  A study of the i n t e r a c t i o n of histamine on the adenylate cyclase  p a r t i c l e s prepared from rat uterus, revealed that histamine did not stimulate the adenylate cyclase.  Histamine-guinea pig ileum i n t e r a c t i o n Preliminary experiments  established that 10 "*M histamine produced  maximum contractioricnof£ the guinea pig ileum.  Burimamide did not block the  histamine-induced contractions i n the guinea p i g ileum, diphenhydramine, a  a  whereas  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 r a t stomach resulted i n the a c t i v a t i o n of adenylate cyclase and subsequent formation of c y c l i c  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 i n t e r a c t i o n of theophylline with norepinephrine and histamine The inotropic and phosphorylase a c t i v a t i n g e f f e c t s of adrenergic and histaminergic drugs have been suggested to be mediated through c y c l i c  AMP.  Theophylline, a phosphodiesterase i n h i b i t o r potentiates the amine effects on  89  FIG. 31. The e f f e c t of various concentrations of histamine, 4-methylhistamine, 3-(3-aminoethyl)-l,2,4 t r i a z o l e (Triazole) and betazole on the r a t uterus. Each point represents the mean ± S.E.M. of 3 determinations.  o  100-  •—-^Histamine 80  -UU"B  ©4-Methylhistamine o—-oTriazolc  0  A  — Bctazole  /  A  A A  A  20-  L8  '-7  10  cr  10"  11 00  10  1  Drug Concentration CM)  o  91  TABLE  7  The effect of histamine (10 ~*M) at various times on the l e v e l of c y c l i c AMP i n the guinea p i g ileum. Each r e s u l t i s the mean of 5-6 determinations ± S.E. Time (Sec)  C y c l i c AMP (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. time were significant differences in cyclic AMP levels observed.  At no  Control  phosphorylase a. levels were 2.9 ± 0.3%, which increased to 6.5 ± 1.2% at about 20 sec. and remained elevated over the 50 sec. experimental period. Control levels of cyclic AMP were 0.54 ± 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 rat heart.  M, was perfused for 15 min. through the isolated  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. • The effect of theophylline (1 mg) at'various times following i n j e c t i o n into the perfused guinea pig heart on c o n t r a c t i l i t y , %"phosphorylase a. and'cyclic AMP.  94  60-  Buffer Perfused  Theophylline (7xlO* M) 4  ©  "x o -JC  a  *» O  u: CL  0.2 Dose of Norepinephrine  FIG.  0.4 i>g)  3 3 . The e f f e c t o f v a r i o u s d o s e s o f n o r e p i n e p h r i n e on c a r d i a c phosphorylase a c t i v a t i o n i nr a t hearts perfused with buffer or buffer plus theophylline ( 7 X 1 0 ~ 4 M ) .  0  0.1  0.2  Dose of N o r e p i n e p h r i n e  FIG.  34  0.4  (ug)  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 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 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 (7xlO~^M o r 2x10" M). 3  96  with buffer or buffer plus 10 M theophylline and injected with either buffer or with d i f f e r e n t doses of histamine.  In these experiments, theophylline  _3 (10  M) potentiated the histamine  c o n t r a c t i l i t y and phosphorylase  (0.2 and 0.4 yg) cardiac effects on  £i (Fig. 35).  Histamine (0.2 and 0.4 yg) induced increases i n c y c l i c AMP were not further increased by the presence of theophylline (Fig. 35). Cardiac actions and i n t e r a c t i o n of imidazole with norepinephrine and histamine The cardiac actions of imidazole, a phosphodiesterase  stimulator, and  the ieffg§tsio;5f^.f±hii!ss"' drug ,wonh norepinephrine and .histamine were ;  investigated.  Imidazole perfusion i n the concentration range of 0.25 to  40mM resulted i n a concentration related increase i n cardiac c o n t r a c t i l i t y (Fig. 36).  In a similar set of experiments d i f f e r e n t doses of imidazole  (0.05-1.6 mg) were injected by a side arm cannula into the perfused pig heart.  guinea  Imidazole produced a dose-dependent increase i n cardiac  contractility  (Fig. 37). The maximum increase i n force with 40mM imidazole  perfusion was 31.3 ± 6.3%. A dose of 1.6 mg of imidazole produced a 23.7 ± 3.3% increase i n c o n t r a c t i l i t y over the control l e v e l s . Further experiments were carried out to test the i n t e r a c t i o n between imidazole and histamine or norepinephrine, i n the guinea p i g hearts perfused with 40mM imidazole.  Data i n table 8 show that imidazole (40mM) reduced the  histamine and norepinephrine dose-response effects on cardiac c o n t r a c t i l i t y . Results i n Table 8, are expressed as a percentage of maximum response obtained with the agonists.  Similar effects with imidazole on amine-induced  c o n t r a c t i l i t y were reported by Poch and Kukovetz (1967).  As c y c l i c AMP i s  believed to be important f o r the amine-induced increases i n c o n t r a c t i l e force,  97  0  0.20  0.40  D o t e o f H i s t a m i n e (tig)  FIG. 35. The e f f e c t of various doses of histamine on c y c l i c AMP, * c o n t r a c t i l i t y , and phosphorylase a. i n guinea pig hearts perfused with buffer or buffer plus theophylline (lCT^M) .  98  '/ Increase in force over control cn  FIG.  36.  _A  -A  is?  o  cn  o  ro  cn  The e f f e c t o f v a r i o u s d o s e s o f i m i d a z o l e o n c a r d i a c c o n t r a c t i l i t y i n guinea p i g hearts perfused w i t h b u f f e r . E a c h p o i n t r e p r e s e n t s t h e mean ± S.E.M. o f t h r e e determinations.  99  Z Increase in force over control cn  cn  o  cn  O  IN)  cn cn O  »9  O  eo  cn  b  ro o o FIG. 37. The e f f e c t of perfusion of various concentrations of imidazole (0.25-40mM) i n the i s o l a t e d perfused guinea pig heart. Each point represents the mean ± S.E.M. of three determinations.  100  TABLE  8  . The effect of 40mM imidazole perfusion on the p o s i t i v e inotropic of histamine and norepinephrine. Histamine  effect  Norepinephrine  Dose of amine (yg)  Buffer perfused  Imidazole perfused  0.2  50.0±4.0  0.010.o  0.4  75.013.8  37.6±2.0  a  0.8  88.013.2  50.012.2  1.6  100.010.0  65.0il.4  Buffer perfused  Imidazole perfused  61.812. 4  7.-2l0.6  75.212. 5  55.6i6.0  a  a  90.012. 5  74.313.7  a  a  100.010. 0  77.3ll.4  a  a  a  n = 3-4 Results are expressed as % of the maximum response that could be obtained with the agonist. a.  S i g n i f i c a n t l y less than buffer perfused (P <0.05).  TABLE  9  The effect of imidazole (40mM) on the norepinephrine and histamine induced increase i n cardiac c y c l i c AMP.  Norepinephrine Dose of amine (yg);  Histamine  Buffer perfused  Imidazole perfused  Buffer perfused  Imidazole perfused  Control  0.45±0.017  0.4410.018  0.45±0.017  0.44±0.018  0.25  0.75+0.045  0.52±0.031  a  0,70±0.05  0.48±0.018  a  0.5  1.12±0.050  0.91±0.037  a  1.05±0.075  0.67±0.036  a  1.0  2.25±0.100  1.22±0.040  a  2.30±0.022  0.78±0.058  a  Mean c y c l i c AMP (nmoles/g wet weight) of 5-7 hearts ± S.E. a.  S i g n i f i c a n t l y less than no treatment (P <0.05).  102  the i n t e r a c t i o n of imidazole and histamine or norepinephrine ori cardiac c y c l i c AMP was investigated.  Guinea pig hearts were perfused with buffer or  imidazole (40mM) f o r 15 min. and injected with various doses of histamine or norepinephrine. i n c y c l i c AMP AMP  Imidazole perfusion decreased the amine-induced increases  at a l l the doses tested (Table 9).  Thus the decrease i n c y c l i c  correlates w e l l with the amine effects on cardiac force.  In order to  test the i n t e r a c t i o n of the histamine or norepinephrine on phosphorylase a_ activation with imidazole (40mM) , the hearts were perfused with buffer or imid.azole f o r 15 min. and injected with various doses of histamine or norepinephrine.  Table 10 shows the lack of i n t e r a c t i o n between imidazole  and the two amine agonists on cardiac phosphorylase a^.  Imidazole did not  affect phosphorylase a c t i v a t i o n of the amines at any dose tested. An investigation of the imidazole effects on cardiac c y c l i c AMP phosphorylase a. revealed that a 1.6 mg,  or  imidazole injected into the perfused  guinea pig heart, did produce an increase i n c o n t r a c t i l i t y , about 23.7 ± 3.3% over control and 11.7 ± 1.6% increase i n phosphorylase  (Table 11).  The data  suggest that imidazole can produce s i g n i f i c a n t changes i n c o n t r a c t i l i t y phosphorylase a c t i v a t i o n without measurable changes i n c y c l i c  AMP.  and  103  TABLE 10 The e f f e c t of imidazole (40mM) on the norepinephrine arid histamineinduced a c t i v a t i o n of cardiac phosphorylase.  Norepinephrine Dose of amine. (yg)  a.  Buffer perfused  Imidazole perfused  Histamine Buffer perfused  Imidazole perfused  0.0  4.810.87  6.6+1.00  4.8±0.87  5.5±1.00  0.25  20.6±1.87  17.0+0.57  .10.3±0.63  9.0±0.76  0.5  42.0±0.57  40.010.57  26.9ll.10-' 24.3±0.33  1.0  57.410.70  54.611.70  42.410.69  a  40.211.47  Mean % of phosphorylase a_ of 5-7 hearts ± S.E. measured at the peak of the phosphorylase response.  104  TABLE 11 The effect of imidazole on phosphorylase a., c y c l i c AMP and c o n t r a c t i l e force i n the isolated perfused guinea p i g heart.  Dose of Imidazole  Control 1.6 mg  %pphosphorylase a.  4.5 ± 1.5 11.7 ± 1.6  c y c l i c AMP (nmoles/gm wet weight)  % increase i n force over control  0.45 ± .017 a  0.46 ± 0.04  23.'7 ± 3.3  a  n = 3-4 C y c l i c AMP was measured i n hearts frozen at 18 sec. and phosphorylase'in hearts frozen at 38-40 sec. following the i n j e c t i o n of imidazole. a.  S i g n i f i c a n t l y 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 o f t h e c a t e c h o l a m i n e s has been p o s t u l a t e d to r e s u l t from an i n c r e a s e i n the i n t r a c e l l u l a r l e v e l o f c y c l i c AMP by t h e a c t i v a t i o n o f a d e n y l a t e c y c l a s e ( S u t h e r l a n d e t a l . 1966). h y p o t h e s i s was q u e s t i o n e d by Benfey an a d r e n e r g i c amine produced any m e t a b o l i c e f f e c t s .  produced  This  (1971) who found t h a t p h e n y l e p h r i n e ,  increased c o n t r a c t i l e f o r c e without producing  I n the p r e s e n t study dose-response  d a t a r e v e a l e d t h a t p h e n y l e p h r i n e produced  and time-response  an i n c r e a s e i n c y c l i c AMP,  c o n t r a c t i l i t y and p h o s p h o r y l a s e a c t i v a t i o n .  cardiac  These d a t a a r e i n agreement w i t h the  f i n d i n g s ofPMcNeill^et"alt^-(-1972-)-lwh6- measured phosphorylasePand. c o n t r a c t i l i t y f o l l o w i n g ' p h e n y l e p h r i n e i n j e c t i p h r l a n d a n d suggest t h a t p h e n y l e p h r i n e i s a weak b e t a - a d r e n e r g i c a g o n i s t .  P h e n y l e p h r i n e i n comparison  to norepinephrine  has l e s s potency and e f f i c a c y i n e l e v a t i n g c y c l i c AMP, p r o d u c i n g a p o s i t i v e i n o t r o p i c e f f e c t o r r a i s i n g the l e v e l s o f p h o s p h o r y l a s e _a. produce  these e f f e c t s i n a dose-dependent manner.  n o r e p i n e p h r i n e produced  P h e n y l e p h r i n e can  I n t h e time-response  an i n c r e a s e i n c y c l i c AMP which preceded  i n c o n t r a c t i l i t y and p h o s p h o r y l a s e a c t i v a t i o n .  study,  the i n c r e a s e  P h o s p h o r y l a s e a_ and  c o n t r a c t i l i t y began t o i n c r e a s e a t about the same time.  C y c l i c AMP  a peak b e f o r e the peak i n c a r d i a c c o n t r a c t i l i t y o r phosphorylase _a.  reached Similar  r e s u l t s were o b t a i n e d by Robison e t a l . (1965) i n a time-response study w i t h e p i n e p h r i n e i n the i s o l a t e d p e r f u s e d r a t h e a r t .  W i l l i a m s o n (1966) s t a t e d  t h a t c y c l i c AMP and c o n t r a c t i l i t y reached a peak a t about  the same time  (12 sec.) f o l l o w i n g t h e i n j e c t i o n o f e p i n e p h r i n e i n t o t h e p e r f u s e d r a t heart. In the time-response study w i t h p h e n y l e p h r i n e , phosphorylase _a a c t i v a t i o n appeared  t o l a g behind the i n c r e a s e i n c a r d i a c c o n t r a c t i l i t y .  Similar  results  were o b t a i n e d when t h e h i s t a m i n e a n a l o g s , TD and b e t a z o l e were used i n the  106  present study.  The apparent lag i n phosphorylase may merely r e f l e c t a  technical i n a b i l i t y to detect the smaller changes i n phosphorylase a c t i v a t i o n that occurred with less potent agonists. increase i n c y c l i c AMP with phenylephrine.  Benfey  (1971) did not find an  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 c y c l i c AMP l e v e l s .  The possible reasons f o r the  discrepancies between the r e s u l t s of Benfey and those found i n the present investigation are many. rabbit heart s l i c e s .  1.) Benfey estimated the c y c l i c AMP levels i n  2.) I t i s very l i k e l y that the f r a g i l e nature of the  enzyme (adenylate cyclase) which i s e a s i l y denatured by usual laboratory physical forces such as washing, freeze thawing, homogenization and sonication, presents serious p i t f a l l s i n the interpretation of data. 3.) Benfey (1971) did not use a beating heart i n which physiological parameters  could be simultaneously monitored.  McNeill et a l . (1972) stated  that phenylephrine did not possess s u f f i c i e n t i n t r i n s i c a c t i v i t y to activate the enzyme.  Our data suggests that phenylephrine, l i k e other adrenergic  amines, stimulates adenylate cyclase and increases the i n t r a c e l l u l a r concentration of c y c l i c AMP.  Increases i n c y c l i c AMP i n turn.may elevate  phosphorylase a. values and produce the inotropic e f f e c t (Robison et a l . 1965). I t has also previously been suggested that the cardiac biochemical and mechanical effects of histamine are mediated through the elevation of c y c l i c AMP (Poch and Kukovetz 1967; K l e i n and Levey 1971; McNeill and Muschek 1972).  Histamine, TD and betazole were found to produce a p o s i t i v e  107  inotropic effect and to increase cardiac phosphorylase i n a dose-dependent manner.  In the present study a l l of these compounds were found to elevate  cardiac c y c l i c AMP.  The order of potency and the order of effectiveness  of the compounds i n 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 c y c l i c AMP p r i o r to increasing c o n t r a c t i l i t y or phosphorylase a.. The r e l a t i v e effectiveness of these drugs i n increasing cardiac c y c l i c AMP was the same as f o r other parameters measured. A l o g i c a l sequence of events for histamine and i t s analogs would appear to be:  1.) a c t i v a t i o n of  adenylate cyclase, 2.) formation of c y c l i c AMP and 3.) enhancement of c o n t r a c t i l i t y and a c t i v a t i o n of phosphorylase a..  I t i s d i f f i c u l t to  separate the onset of 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 with histamine. However when either TD or betazole was used, the a c t i v a t i o n of phosphorylase appeared  to lag behind the positive inotropic e f f e c t .  The s p e c i f i c i t y of the  cardiac histamine receptor has been the subject of controversy.  Several  l i n e s of evidence indicate that effects of histamine are independent adrenergic nervous system.  of the  Reserpine pretreatment did not a l t e r 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 i n cardiac c o n t r a c t i l i t y and heart rate (Trendelenburg 1960; Poch and Kukovetz 1967; McNeill and Muschek 1972).  Dean (1968), reported that beta.blocking agents  l i k e propranolol and prenethanol produced dose-response  curve.  a s l i g h t s h i f t i n the histamine  This could be a non-specific antagonism.  Hearts  108  i s o l a t e d from animals pretreated with reserpine or 6-hydroxydopamine s t i l l show t y p i c a l responses to the application of exogenous histamine (Levi and G i o t t i 1967).  Such experiments  demonstrate that histamine has d i r e c t cardiac  effects. C l a s s i c a l antihistaminesssuch as diphenhydramine or tripelennamine do not antagonize the responses of isolated cat and guinea p i g a t r i a and isolated perfused guinea pig hearts. and Muschek 1972).  (Trendelenburg 1960;  B a r t l e t 1963;  McNeill  These antihistamines were also r e l a t i v e l y non-specific  antagonists of the histamine stimulation of adenylate cyclase (McNeill and Muschek 1972).  The p r i n c i p a l reports claiming the demonstration of s p e c i f i c  effects of antihistamine drugs i n blocking the cardiac actions of low concentrations of histamine are those of Mannaioni (1960) and Flacke et a l . (1967), K l e i n and Levey (1971), Hughes and Coret (1972) have reported that promethazine can antagonize the chronotropic effects of histamine on rabbit a t r i a .  Since complete dose-response  curves were not obtained i n that  study, the s p e c i f i c i t y of the blockade i s questionable.  Black et a l . (1972)  have recently c l a s s i f i e d histamine receptors into H^ and ^  receptors.  H^  receptors mediate most effects of histamine and are blocked by c l a s s i c a l antihistaminic compounds.  Histamine stimulated secretion of acid from  g a s t r i c mucosa and the inotropic and chronotropic effects of histamine can not be antagonized by mepyramine or related drugs. c l a s s i f i e d as B.^ type receptors.  These receptors are  Burimamide, however does block the g a s t r i c  secretory and p o s i t i v e chronotropic effectscof histamine (Black et a l . 1972; Wylie et a l . 1972).  In the present study the s p e c i f i c i t y of the cardiac  histamine receptor has been demonstrated. Burimamide proved to be a competitive antagonist of the p o s i t i v e inotropic, phosphorylase activating and c y c l i c AMP  increasing e f f e c t s of histamine, TD  109  and b e t a z o l e .  Burimamide d i d not a n t a g o n i z e  the e f f e c t s of n o r e p i n e p h r i n e  on these parameters and p r o p r a n o l o l d i d not a n t a g o n i z e h i s t a m i n e or h i s t a m i n e analog c a r d i a c e f f e c t s .  S t i m u l a t i o n of the a d e n y l a t e c y c l a s e then*  r e s u l t s i n an i n c r e a s e i n c y c l i c AMP, and p h o s p h o r y l a s e  a positive  a c t i v a t i n g e f f e c t s i m i l a r t o t h a t suggested  amines (Robison e t a l . 1965). Poch and Kukovetz (1973), AMP  and subsequently  inotropic  for catechol-  Our d a t a support the r e c e n t f i n d i n g s of  i n d i c a t i n g t h a t the i n o t r o p i c as w e l l as the  i n c r e a s i n g a c t i o n s of h i s t a m i n e can be i n h i b i t e d by the  cyclic  antagonist  burimamide. The  f i n d i n g s of Hughes and C o r e t  (1972) suggest  t h a t the c a r d i a c e f f e c t s  of h i s t a m i n e a r e s u s c e p t i b l e to b l o c k a d e by promethazine a H^ antagonist.  A c a r e f u l and  receptor  complete study of the i n t e r a c t i o n of promethazine  w i t h s e v e r a l doses of h i s t a m i n e on h e a r t r a t e , f o r c e of c o n t r a c t i o n and AMP  f o r m a t i o n was  C o r e t was  c a r r i e d out t o determine  correct.  cyclic  i f the p r o p o s a l of Hughes and  The d a t a p r e s e n t e d r e v e a l t h a t promethazine i s not a  c o m p e t i t i v e e q u i l i b r i u m a n t a g o n i s t o f the c a r d i a c e f f e c t s o f h i s t a m i n e but does i n t e r a c t w i t h the h i s t a m i n e r e c e p t o r i n e i t h e r a n o n - c o m p e t i t i v e c o m p e t i t i v e n o n - e q u i l i b r i u m manner.  Promethazine (4-16x10 ^M)  the h i s t a m i n e e f f e c t s on c a r d i a c c o n t r a c t i l i t y .  or  lowered  An i n c r e a s e i n the dose of  h i s t a m i n e c o u l d not overcome the promethazine b l o c k a d e pahdul/increasing the promethazine c o n c e n t r a t i o n r e s u l t e d i n a f u r t h e r d e p r e s s i o n of the maximum h i s t a m i n e response. histamine-induced  Promethazine a l s o p a r t i a l l y b l o c k e d  i n c r e a s e s i n c a r d i a c c y c l i c AMP.  I n c r e a s e s i n the dose  of h i s t a m i n e d i d not overcome the promethazine b l o c k a d e . h i s t a m i n e response produced  by promethazine was  the  The degrease  approximately  i n the  the same f o r —6  b o t h the m e c h a n i c a l  and b i o c h e m i c a l events.  not a n t a g o n i s e the i n o t r o p i c response  Promethazine (4 x 10  to norepinephrine.  M)  did  110  Higher doses of promethazine (8-16 x 10 responses.  M) did depress the norepinephrine  The r e s u l t s with promethazine stand i n contrast to those obtained  with burimamide.  Burimamide appears to be a competitive equilibrium  antagonist of histamine whereas promethazine exhibited c h a r a c t e r i s t i c s of a non-competitive antagonist of histamine.  One concentration of promethazine  (4 x 10 ^M) produced an increase i n both cardiac c o n t r a c t i l i t y 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  —6 (2-16 x 10  M) and their i n t e r a c t i o n with histamine wase investigated.  The  present study again demonstrated the importance of doing complete doseresponse curves when investigating drug interactions. The effect of histamine on adenylate cyclase was not blocked by propranolol and was poorly blocked by c l a s s i c a l antihistamines such as tripelennaminecarid diphenyhydramine (McNeill and Muschek 1972).  Our data  indicate that the stimulatory e f f e c t of histamine and histamine analogs on guinea pig cardiac adenylate cyclase i s competitively antagonised by burimamide.  The i n t e r a c t i o n between burimamide and agonists was similar to  that noted when the effects of these agents on cardiac c y c l i c c o n t r a c t i l i t y and phosphorylase were studied. from those of K l e i n and Levey (1971).  AMP,  The data presented d i f f e r )  They were able to demonstrate a  complete blockade of one concentration of histamine with diphenhydramine  Ill  (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. I t has also been proposed that histamine produces i t s secretory effect i n the gastric mucosa by interacting with 1972).  receptors (Black et a l .  C y c l i c AMP appears to act as an i n t r a c e l l u l a r 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 s p e c i f i c , competitive  blockade by burimamide provide the basis f o r suggesting that histamine and i t s analogs stimulate c y c l i c AMP formation v i a an action on receptors i n the membranes of the gastric mucosal c e l l s . The r e l a t i v e order of potency of histamine and i t s analogs on rat g a s t r i c adenylate cyclase i s similar to that found i n investigations studying the actions of these drugs on cardiac adenylate cyclase^ c y c l i c AMP formation, phosphorylase a c t i v a t i o n and cardiac c o n t r a c t i l i t y (McNeill and Muschek 1972; present study).  The rank order for the compounds i s also similar to that  noted when g a s t r i c acid secretion was measured (Lin et a l . 1963).  Karppanen  and Westermann (1973) suggested that histamine stimulated g a s t r i c secretion of acid i s mediated by c y c l i c AMP which i s formed i n response to stimulation of H£ 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; P e r r i e r and Laster 1970; who have provided evidence the receptor i n g a s t r i c mucosa i s associated with adenylate cyclase.  Thus  a l o g i c a l sequence of events for histamine on g a s t r i c adenylate cyclase would  112  appear to be; 1.) a c t i v a t i o n of adenylate cyclase; 2.) formation of c y l i c AMP;  3.) gastric acid secretion.  Histamine, 4-methylhistamine,  betazole, a l l relaxed the r a t uterus i n a dose dependent manner. of potency of the compounds for relaxing the r a t uterus was 4-methylhistamine>TD and betazole (Fig. .31).  as reported by Black et a l . (1972).  The order  histamine>  The present findings were i n  agreement with those of Black et a l . (1972) who 4-methylhistamine relaxed the rat uterus.  TD and  reported that histamine and  Rat uterus possesses  receptors  Stimulation of adenylate cyclase,  prepared from guinea pig heart or r a t stomach by histamine resulted i n an increase i n i t s a c t i v i t y and subsequently formed c y c l i c AMP. suggests that  This data  receptorsJin both the tissues were associated with adenylate  cyclase. However, we could not detect any changes i n the adenylate cyclase a c t i v i t y by histamine.  There could be two p o s s i b i l i t i e s :  1.)  receptors  i n the r a t uterus a?r.e ino.t associated with adenylate cyclase or 2.) our c y c l i c 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.  I f histamine effects on  the r a t uterus are not d i r e c t , then i t would not stimulate adenylate cyclase prepared from rat uterus. At least i n two tissues, the heart and the rat stomach, B.^ receptors are associated with adenylate cyclase, stimulation of which r e s u l t s i n an increase i n c y c l i c  AMP.  Histamine stimulates the guinea ileum by acting on  receptors (Ash  and Schild 1966).  Burimamide did not block the histamine response on the  guinea pig ileum.  In the time-response study histamine (10  -5 M) did not  113  increase c y c l i c AMP  levels at any time tested.  effects of histamine on  I t thus appears that the  receptors are not mediated through stimulation of  adenylate cyclase with a subsequent  increase i n c y c l i c AMP.  These findings  support the data of Black et a l . (1972) that burimamide does not block the H^-receptor effects of histamine.  C y c l i c AMP  a c t i v i t y of the enzyme phosphodiesterase.  l e v e l s are dependent on the  Theophylline i n h i b i t s  phosphodiesterase while imidazole activates the enzyme i n broken preparations (Butcher and Sutherland 1962).  cell  Both drugs have been used to  provide i n d i r e c t evidence for the involvement of the nucleotide i n the p o s i t i v e 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 i n cardiac c o n t r a c t i l i t y and phosphorylase a a c t i v a t i o n without elevating the levels of c y c l i c AMP.  Theophylline also enhanced the cardiac  effects of both histamine and norepinephrine without any apparent e f f e c t on the levels of c y c l i c AMP  already elevated by norepinephrine and histamine.  I t has  been assumed that because theophylline i s a phosphodiesterase i n h i b i t o r i t w i l l elevate c y c l i c AMP effects.  levels i n the i n t a c t tissue and thus produce i t s  The results of the present investigation indicate that this i s not  true for the heart.  E a r l i e r work with theophylline (Levey and Wilkenfeld  (1968) using the rat uterus showed that theophylline potentiated the i n h i b i t o r y response to n i t r o g l y c e r i n .  Polacek et a l . (1971) were able to dissociate the  relaxant e f f e c t of theophylline on the rat uterus from any e f f e c t on c y c l i c AMP.  A l l e n et a l . (1973) have shown that there was no c o r r e l a t i o n within a  group of phosphodiesterases i n h i b i t o r s , including theophylline, between the phosphodiesterase i n h i b i t i o n and l i p o l y s i s i n an isolated f a t c e l l preparation.  Some correlations have been found between various xanthine  114  derivatives with regard to t h e i r a b i l i t y to i n h i b i t phosphodiesterase and t h e i r i n t e r a c t i o n with norepinephrine  on the heart (McNeill et a l . 1973).  The p o s i t i v e inotropic response to theophylline was propranolol and i t was  not blocked  by  therefore not mediated by catecholamine release  (Massingham and Nasmyth 1972).  Skelton et a l . (1971) showed that theophylline  could enhance the inotropic e f f e c t of norepinephrine not of calcium on cat p a p i l l a r y muscle. supported a r o l e f o r c y c l i c AMP  and db. c y c l i c AMP  but  They concluded that their data  i n the theophylline i n t e r a c t i o n .  But  Massingham and Nasmyth (1972) demonstrated a p o s i t i v e i n t e r a c t i o n between the e l e c t r i c a l stimulation and theophylline i n the frog v e n t r i c l e . effect would not be mediated through c y c l i c AMP  Such an  and i s more l i k e l y  explained by an increase i n the i n t r a c e l l u l a r calcium.  Methylxanthines have  been found to release ionized calcium from the i n t r a c e l l u l a r storage Bianchi 1968;  sites  (Nayler 1963;  deGubareff and Sleator 1965;  Langer 1971).  This may be the process whereby xanthines increase myoplasmic  calcium ions to cause a p o s i t i v e inotropic e f f e c t .  Shine and  (Nayler 1967).  prolonged the active state of cardiac muscle (deGubareff and Sleator Gibbs 1967).  Caffeine, 1965;  Caffeine has been shown to markedly increase the duration of the  action p o t e n t i a l and to increase c o n t r a c t i l i t y about 45% i n isolated a t r i a . Epinephrine,  on the other hand, increased c o n t r a c t i l i t y more than 100% and  l i t t l e e f f e c t on action p o t e n t i a l duration (deGubareff and Sleator 1965).  had The  work of Blinks et a l . (1972) has pointed out that the catecholamines decrease the time to peak tension and accelerate relaxation i n cardiac muscle while  the  xanthines prolong time to peak tension and increase the t o t a l duration of contraction.  Blinks et a l . (1972) have reviewed the evidence suggesting  xanthines affect the heart by a f f e c t i n g calcium metabolism.  that  Xanthines are  known to increase the i n f l u x of calcium and to decrease the rate of calcium  115  sequestration i n mammalian a t r i a (Scholtz  1971;  Shine and Langer 1971).  E f f l u x of calcium i s also reduced by caffeine (Shine and Langer 1971). calcium i s thus available for excitation contraction increase i n positive inotropic e f f e c t . may  The  More  coupling and hence an  effects of xanthines on calcium  also explain the p o s i t i v e inotropic i n t e r a c t i o n of these drugs with  biogenic amines on both c o n t r a c t i l i t y and phosphorylase activation,i' Namm T  et a l . (1968), S t u l l and Mayer (1971) reported that calcium i s essential for cardiac c o n t r a c t i l i t y and phosphorylase a. a c t i v a t i o n . Catecholamines and xanthines could produce t h e i r synergistic effect by elevating i n t r a c e l l u l a r calcium by d i f f e r e n t mechanisms thus leading an enhanced e f f e c t . interpreted  The p o s i t i v e inotropic responses to theophylline  to  can  i n terms of the increased calcium i n f l u x which i t produces.  interpretation of the effects of theophylline  i n terms of i t s action  phosphodiesterase should be treated with reservation.  be The  on  Methylxanthines thus  appear to have d i r e c t effects on the heart, which are independent of t h e i r phosphodiesterase i n h i b i t i n g properties. Imidazole, a phosphodiesterase stimulator, appeared to produce p o s i t i v e inotropic ef f ectscon the heart which were not mediated through c y c l i c  AMP.  Poltetaeu (1970), reported similar effects of imidazole on s k e l e t a l muscle. P o s i t i v e inotropic effects were seen when guinea pig hearts were perfused with imidazole (Knope et a l . 1973).  In t h e i r study they c l e a r l y showed  that the imidazole-induced increases i n myocardial c o n t r a c t i l i t y were not blocked by propranolol or antihistamines. potentiated cyclase  by aminophylline.  Imidazole effects were not  Imidazole did not stimulate cardiac  (McNeill and Muschek 1972).  adenylate  I t thus seems u n l i k e l y that imidazole  produces i t s inotropic e f f e c t by elevating c y c l i c AMP. supported i n the present investigations  This i s further  i n that perfusion  or i n j e c t i o n of  116  imidazole did not produce any changes i n c y c l i c AMP  i n the guinea pig heart.  Knope et a l . (1973) suggested that the effect of imidazole was tissue calcium or calcium turnover.  on  DeMello et a l . (1973) suggested that  imidazole acted by increasing permeability to e x t r a c e l l u l a r calcium i n frog heart since responses to imidazole were t o t a l l y suppressed i n calcium-free media while those of caffeine were not. Imidazole reduced the amine-induced AMP  as shown i n Table 9  increases i n the levels of c y c l i c  presumably by stimulating phosphodiesterase and  thus increasing the metabolism of c y c l i c AMP.  Imidazole stimulates the  enzyme phosphodiesterase i n v i t r o (Butcher and Sutherland 1962; et a l . 1973).  McNeill  The decrease i n cardiac c y l i c AMP was p a r a l l e l e d by a  corresponding decline i n c o n t r a c t i l i t y .  Poch and Kukovetz  (1967), reported  that imidazole, i n the Langendorff jguanraa pig heart preparation, caused a depression of cardiac c o n t r a c t i l i t y and an i n h i b i t i o n of the p o s i t i v e inotropic action of catecholamines.  They attributed this effect to the  stimulation of the phosphodiesterase^e-afcalyzSdbreakdown lack of increase of c y l i c AMP  of c y c l i c AMP.  The  did not however, a f f e c t 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 c y c l i c AMP which are as important or more important, i n activating cardiac phosphorylase.  Friesen et a l . (1967),  reported an increase i n cardiac phosphorylase by increasing external calcium i n the perfusate.  In hearts perfused with no calcium, norepinephrine injections  did increase cardiac c y c l i c AMP, a. (Namm et a l . 1968).  without any detectable changes i n phosphorylase  Their study suggested that calcium was s t i l l required  for phosphorylase a c t i v a t i o n even when c y c l i c AMP was elevated.  In smooth  muscle calcium i s of primary importance and c y c l i c AMP may not be required at a l l for phosphorylase a c t i v a t i o n (Diamond and Brody 1966; Rasmussen et a l . 1972).  Namm 1971;  In a recent study, Diamond (1973) has shown increases  117  i n phosphorylase a c t i v a t i o n during spontaneous uterine contractions at various times after increasing calcium concentration from 1.8 to 7.2mM.  This further  suggests the importance of calcium i n phosphorylase a c t i v a t i o n without elevation of c y c l i c AMP.  Similar conclusions can be drawn from  experiments  i n e l e c t r i c a l l y stimulated s k e l e t a l muscles i n which phosphorylase  activity  but not c y c l i c 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 i n s k e l e t a l muscle phosphorylase a. without an effect on c y c l i c nucleotide l e v e l s i s a further i l l u s t r a t i o n of t h i s phenomenon.  In a l l the references c i t e d above, calcium has been invoked  as the probable factor i n activating the enzyme. would f i t the data of the present study.  A similar explanation  In Table 11 a single i n j e c t i o n  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 mechanisms other than c y c l i c AMP.  through  Hearts from hyperthyroid rats  responded  to norepinephrine to the same extent as controls when c o n t r a c t i l i t y and c y c l i c AMP were measured. norepinephrine was  However, phosphorylase a c t i v a t i o n by  enhanced i n the hyperthyroid hearts.  Hyperthyroidism i s  known to increase the accumulation and release of calcium i n 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 f o r the i n t e r a c t i o n of hormone, c y c l i c AMP  and calcium.  that hormone increases c y c l i c AMP,  which i n turn a l t e r s the permeability of  the membrane to calcium.  One of the suggestions i s  Our data support this suggestion.  Both histamine  and norepinephrine did not produce an inotropic effect i n the imidazole  118  perfused hearts unless c y c l i c AMP  was  elevated.  The  phosphorylase  a c t i v a t i o n , without any measurable changes i n c y c l i c AMP, for an increase i n i n t r a c e l l u l a r calcium. pharmacological agent has only one action.  could be accounted  I t i s naive to think that a Theophylline or imidazole  undoubtedly have many actions i n addition to i n h i b i t i o n or stimulation of phosphodiesterase.  119  SUMMARY AND CONCLUSIONS  1.  Phenylephrine increased cardiac c y c l i c AMP,  c o n t r a c t i l i t y and  phosphorylase a_ values i n that order i n the isolated perfused guinea pig heart.  The effects of phenylephrine on the above biochemical and  mechanical events were dose-dependent.  Phenylephrine ( i n the doses  used) i s a weak beta-adrenergic agonist, being less potent and less e f f e c t i v e than norepinephrine.  The data are consistent with the  hypothesis that adrenergic drugs produce their cardiac effects by stimulating adenylate cyclase and producing an increase i n c y c l i c  2.  (a)  AMP.  Histamine and the histamine analogs, TD and betazole, stimulated  cardiac adenylate cyclase and increased cardiac c y c l i c AMP  levels.  The  drugs also increased c o n t r a c t i l i t y and phosphorylase a.. (b)  Histamine was most potent as compared to i t s analogs, TD and betazole  i n increasing cardiac c o n t r a c t i l i t y , phosphorylase a. and c y c l i c 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 s p e c i f i c i t y for  histamine by not a f f e c t i n g any of the norepinephrine-induced cardiac e f f e c t s , namely c o n t r a c t i l i t y , phosphorylase and c y c l i c AMP 3.  Promethazine^. H  1  elevation.  receptor antagonist also blocked the cardiac effects  of histamine, including the histamine-induced increases i n c y c l i c  AMP.  Themblbekade ofocardiacfhistami^ noncompetitive br-ncompeiiMvemri^  of a; . Promethazine  —6 (4x10  M) produced inotropic effects but at a higher concentration ^ _^ -(8-16x10 M) was cardiodepressanttc Promethazine (4x10 M) did not  120  a f f e c t the p o s i t i v e inotropic response to norepinephrine.  At 8-16x10 °M  promethazine lowered the maximum response of norepinephrine. 4.  Histamine and i t s analogs, 4-methylhistamine, cardiac adenylate cyclase.  TD and betazole stimulated  The order of potency of the compounds for  stimulating cardiac adenylate cyclase was histamine>4-methylhistamine>TD> betazole.  Stimulation by the agonists was blocked, i n an apparently  competitive manner, by burimamide.  5.  Histamine, 4-methylhistamine,  TD and betazole stimulated the gastric  —6 —2 adenylate cyclase, over a dose-range of 10 - 10 M.  The rank order  of stimulation was histamine>4-methylhistamine^TD and betazole. —6 Burimamide (1-10 M) antagonized the effect of the drugs i n an apparently competitive manner. 6.  Histamine, TD and betazole relaxed the estrogen primed r a t uterus.  The  order of relaxation was histamine>4-methylhistamine>TD and betazole. However, we f a i l e d to detect any changes i n theladehylateecyclase a c t i v i t y dueiVt6=Jiis taminee interaction. 7.  Histamine i n a dose of 10 ~*M produced a maximum effect on the i s o t o n i c a l l y contracting guinea pig ileum. histamine effect on the guinea pig ileum.  Burimamide d i d not block the Histamine at no time  increased the levels of c y c l i c AMP over control, when measured i n 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 c y c l i c AMP.  receptors appear to be associated with  adenylate cyclase, at least i n two tissues, the heart and the stomach.  121  Histamine receptors i n guinea p i g ileum are of  type.  Stimulation  o f H^. r e c e p t o r s d o e s n o t i n c r e a s e c y c l i c AMP a n d _ h e n c e H^ r e c e p t o r s a r e not a s s o c i a t e d w i t h adenylate 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 , effects  cyclase. theophylline potentiated the cardiac  of norepinephrine or histamine.  inotropic  T h e o p h y l l i n e enhanced t h e  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  T h e o p h y l l i n e , when i n j e c t e d  into  of both  t h e h e a r t , produced  amines.  an  inotropic  e f f e c t a n d p h o s p h o r y l a s e <i a c t i v a t i o n b u t d i d n o t e l e v a t e c y c l i c The  data presented suggest  are  not mediated  by  9.  the effects  that cardiac effects  of methylxanthines  t h r o u g h c y c l i c AMP, b u t a r e m o r e r e a d i l y e x p l a i n e d  o f these agents  i n c a l c i u m metabolism.  Imidazole, a phosphodiesterase stimulator, AMP i n t h e h e a r t f o l l o w i n g and  also decreased  lowered the l e v e l s o f c y c l i c  the i n j e c t i o n of histamine or norepinephrine  the inotropic  however d i d n o t a f f e c t  e f f e c t of these agents.  Imidazole  the phosphorylase a c t i v a t i n g e f f e c t  a m i n e s , s u g g e s t i n g t h a t m e c h a n i s m s o t h e r t h a n c y c l i c AMP,  of other presumably  c a l c i u m , a r e a s i m p o r t a n t f o r a c t i v a t i n g t h e enzyme i n c a r d i a c as t h e y a r e i n smooth and s k e l e t a l m u s c l e . imidazole suggest i n cardiac  AMP.  muscle  However d a t a o b t a i n e d w i t h  t h a t c y c l i c AMP i s r e q u i r e d f o r am±rie indueedpincreases  contractility.  s  122  BIBLIOGRAPHY A l l e n , D.O., Clark, J..F. and Ashmore, J . : Study of. phosphodiesterase i n h i b i t o r s on l y p o l y s i s , phosphodiesterase a c t i v i t y and c y c l i c 3', 5'-adenosine monophosphate l e v e l s i n isolated f a t c e l l s . J . Pharmacol. Exp. Ther., 185_: 379-385, 1973. Alonso, D. and. Harris, J.B..: E f f e c t s of xanthines and histamine on ion .transport and r e s p i r a t i o n by frog gastric mucosa. Am. J . Physiol., 208: 18-23, 1965. Arunlakshana, 0. and Schild, H£0.: Potentiation, of pharmacological e f f e c t s of histamine..by histamine, inhibitors.. J.. Physiol.., 123: 32-54, 1954. Ash, A.S.F. and Schild, H.O.: Receptors mediating some a c t i v i t i e s of histamine. Br. J . Phamac, 27: 427-439, 1966 Axelsson, J.. and.Theslef f.,.. S.:. A c t i v a t i o n of the. c o n t r a c t i l e mechanism i n striated.muscle. Acta Physiol. Scand., 44: 55-66, 1958. B a r t l e t t , A.L.: The action of histamine on the isolated heart. Pharmac. Chem., 21: .450-561, 1963.  Br. J .  Benfey, B.G.: Lack of relationship between myocardial c y c l i c AMP concentrations and inotropic e f f e c t s of.sympathomimetic amines. Br. J . Pharmac, 43_: 757-763, 1971. Benfey, B.G. and Carolin, T.: E f f e c t s of phenylephrine on cardiace c o n t r a c t i l i t y and adenyl cyclase a c t i v i t y . Can. J . Physiol. Pharmacol., 49: 508-512, 1971. Bersimbaev, R.I., Argutinskaya, S.V.. and .Salganik,. R.L..:. The stimulating action of gastrin pentapeptide and histamine on adenyl cyclase a c t i v i t y i n r a t stomach. Experientia, 27: 1389-1390, 1971. . Bianchi, C P . : The effect of caffeine on radiocalcium movement i n frog s a r t o r i u s . J . Gen. Physiol., 44_: 845-858, 1961. Bianchi, C P . :  C e l l calcium.  Butterworths, London. 1968, pp 85-99.  Bieck, P.R., Oates, J.A. and Robison, G.A.: C y c l i c AMP i n the regulation of g a s t r i c secretion in. dogs and humans. Am. J . Physiol., 224: 158-164, 1973. Birnbaumer, L.. and Rodbell, M.: Adenyl cyclase i n f a t c e l l s . receptors. J . . B i o l . Chem., 244: 3477-3482, 1969.  I I Hormone  Bitensky, M..W. and Gorman, R.E.: C e l l u l a r responses to c y c l i c AMP. Prog. Biophys. Molec. B i o l . , 26: 409-461, 1973.  123 Black, J.W., Duncan, W.A.M.., Durant, C.J., Ganellin, C.R., and Parsons, E.M.: Definition.and antagonism of histamine H -receptors. Nature, 236: 385-390, 1972. —"——  \  Black, J.W., Duncan, W.A.M.,, Emmet,. J.C., Ganellin., C.R.., Hesselbo, J . , Parsons, E.M. and Wyllie, J.H..: . Metiamide - an.orally active histamine H receptor antagonist. Agents, and Actions, _3: 133-137, 1973. Blinks, J.R.., Olson, C.B.., Jewell, B.R. and Braveny, P..: Influence of caffeine and other, methylxanthines. on mechanical, .properties of isolated . mammalian.heart, muscle.:-,..Evidence, for .a. dual .mechanism of action. C i r . Res., 30:. 367-392,1972. Blyth, D.I.: Some e f f e c t s of. histamine i n the depolarized rat uterus. Br. J . Pharmac.., 49: 445-456, 1973. Butcher, R.W. and Sutherland, E.W.. : Adenosine 3', 5'-phosphate i n b i o l o g i c a l . materials. . I.. P u r i f i c a t i o n and. properties .of . c y c l i c .3', 5'nucleotide, phosphodiesterase, and, use..of - this enzyme to, characterize adenosine 3', 5.'-phosphate, i n human,urine. J . B i o l . Chem., 237: .. 1244-1250, 1962. Carmeliet, E. and Vereecks, J . : Adrenaline.and the plateu.phase of the cardiac action potential., . Pf luegers-Arch.., 313: 300-315, 1969. Chapman,. R.A., .and..Niedergerke,.R..: ... Effects., of.. calcium.on, the contraction of-the hypodynamic frog's, heart....... J..-Physiol., 211: 389-4211, 1970. Chase, L.R. and Ausbach, G..D..: Renal, adenyl cyclase: Anatomically separate s i t e s for parathyroid,..hormone..and. vasopressin. Science, 159: 545-547, 1968. Chenoweth, M.B. and Koelle, E.S.: An isolated, heart perfusion system adapted to the. determination of non-gaseous metabolites. J . Lab. C l i n . Med.., 31: 600-608, 1946. Code, C.F., Hallenbeck, G.A. and ..Gregory,R.A. : . Histamine content of canine gastric j u i c e . Am. ..J. Physiol.., 151: 593-605, 1947. Coraboeu, E.: Influence.de certaines substances biologiques sur l a permeabilite. membranaire. myocardique. . In:. Drugs and metabolism of.myocardium, and. striated.muscle. Proc. of the International Symposium. .... Nancy, pp 305-316, Ed. by M. Lamarche.and R. Royer (1969). Cori, G.T. and Cori, C F . : The k i n e t i c s of the enzymatic synthesis of . glycogen_from.glucose-l-;phosphate. J . B i o l . Chem., 135: 733-756, 1940.  124 Craver, B.N., Barrett, W., Cameron,-A. and Herrold., E. : Pharmacological •actions of .35. derivatives, of :4-methyl, 5-ethyl, or 2-substituted imidazoles. Arch. Int. Pharmacodyn. ..Ther..,. 87: 33-48, 1951. Dale, H.H. and Laidlaw,.P.P.: . The p h y s i o l o g i c a l . a c t i o n of 3-iminazolethylamine. J..Physiol.., 41:.. 318-344, 1910. Dale, H.H. and Dudley,. H.W..: The. physiological action of B-iminazolethylamine. ...J.. Pharmacol.. Exp. T h e r . 1 8 : 103-110, 1921. Davies, R.E. and ..Roughton,-F...J..W. : Hydrochloric acid production by isolated gastric-mucosa... Biochem. J..,. 4_2: .609-621, 1948. Davis, R.A. and McNeill, J.H.: The cardiac effects.of cocaine and certain . . antihistamines and antidepressants. Arch.. Int. Pharmacodyn., . 201: 262-279, 1973. Dean, P.M.: . Investigation into the mode.of action of histamine on the isolated rabbit.heart. . Br. J . Pharmac.-Chem...,, ,32: 65-77, 1968. DeGubareff., T., and. Sleator., W..: E f f e c t s of caffeine on mammalian a t r i a l muscle.and.its interaction, with adenosine,and calcium. J./Pharmacol. Exp. Ther., 148: 202-214, 1965. DeMello, D.C, Mendo.za.,. T.., and Perez, B.: Caffeine contractures i n depolarized heart, muscle. Res.. Commun.. Chem. Pathol., Pharmacol., 6: 1-17, 1973. Diamond, J....:. Phosphorylase.,, calcium, and c y c l i c ..AMP-in smooth-muscle c contraction.. Am... J . Physiol. ,. 225: 930-937, 1973. Diamond, J . and.Brody, T..M..: Relationship, between smooth muscle contraction ,and ..phosphorylase .activation. J . Pharmacol. Exp. Ther., 152: .212-220, 1966. :  Dousa, T.P.. and Code C F . : Effect of histamine, and its.methyl derivatives on cyclic. AMP .metabolism. in,gastric.mucosa,. and -its. blockade by an H^ receptor, antagonist. J . C l i n . Invest., 53_: 334-337, 1974. Drummond, G.I., Duncan,.-L.. and .Her tzman, ,E.:. Effect of epinephrine on phosphorylase .b a c t i v i t y i n the perfused r a t hearts. . J . B i o l . Chem., 241: 5899-5903, 1966. Drummond, G.I. and.Duncan,. L.: Adenyl cyclase-in. cardiac tissue. J..Biol..Chem., 245: 979-983, 1970. Drummond, G„I., and ..Hemmings,.. S.J..:. Inotropic and-chronotropic e f f e c t s of d i b u t y r y l c y c l i c AMP. Ad.. C y c l i c Nuc..-Res. Wol'sdlV.eedlLted by . .Greengard:, .P..ju-Paoletti,. R.., Robison, G.A., 1972, p 307. Edman, K.A.P.:, Drugs and properties, of heart muscle. 5: 99-118, 1965.  Ann. Rev. Pharmacol.  125  E l - A c k a d , T.N., Meyer, M.J. and S t u r k l e , P.D.: I n o t r o p i c and c h r o n o t r o p i c a c t i o n s o f h i s t a m i n e on the avian, h e a r t . .Fed.. P r o c . , 3J3: 585, 1974. E n g s t f e l d , G., A n t o n i , H..and. F l e c k e n s t e i n , . .A: Die. R e s t i t u t i o n d e r . . e r r e g u n g s f o r t l e i . t u n g and. K o n t r a k t i o n s k r a t t . . d e s . K"!" gelahmten F r o s c h und s a u g e r t i e r m y o k a r d s . d u r c h - a d r e n a l i n e . - P f l u e g e r s A r c h . , 273: . . 145-163, 1961. Entman, M.L.., Levey, G.S. - ' e p i n e p h r i n e , and. . i n .sarcotubular C i r . Res.,, 25:.  and Epstein,, S.E.: . Mechansim o f a c t i o n o f glucagon on. canine..heart.. Evidence f o r i n c r e a s e c a l c i u m s t o r e s . m e d i a t e d by c y c l i c AMP. 429-438, 1969.  F a r a h , A. and W i t t , P...: C a r d i a c g l y c o s i d e s and calcium.... In. p r o c e e d i n g s of the f i r s t I n t e r n a t i o n a l pharmacological.meeting,.Stockholm, V o l . Ill,,.New. a s p e c t s of . c a r d i a c g l y c o s i d e s , ed. by.W. W i l b r a n d t , pp 137-171, Pergamon,Press,. New York, 1963. F i s k e , C .H. and . Subbarow, Y..: The c o l o r i m e t r i c d e t e r m i n a t i o n of . phosphorus. J . . B i o l . Chem... 66:. 375-400, 1925 F l a c k e , W., A t a n a c k o y i c , D., G i l l i s , R.A. and A l p e r , M.H.: The a c t i o n s o f h i s t a m i n e on the.mammalian h e a r t . J . Pharamcol. Exp. Ther., 155: 217-278, 1967. Frank, G.B.: E f f e c t s o f changes i n e x t r a c e l l u l a r c a l c i u m c o n c e n t r a t i o n on the p o t a s s i u m - i n d u c e d . c o n t r a c t u r e , of. f r o g ' s s k e l e t a l muscle. J . - P h y s i o l . , 151:. 518-538, 1960. F r i e s e n , A.J.D., A l l e n , G. and V a l a d a r e s , J.R.E.: Calcium-induced a c t i v a t i o n of p h o s p h o r y l a s e i n r a t h e a r t s . S c i e n c e , 155: 1108-1109, 1967. Garren,. L . D . . , . G i l l , G..N. and. Walton, G.M. : The i s o l a t i o n of. a r e c e p t o r f o r adenosine,. 3 ' , 5 ' - r c y c l i c monophosphate (cAMP). from the. a d r e n a l c o r t e x . The r o l l , o f . t h e r e c e p t o r i n the mechanism o f a c t i o n of cAMP. Ann. New.York Acad. Sc., 185: 210-226, 1971. Gilman, A.G.: A p r o t e i n b i n d i n g assay f o r adenosine 3 ' , 5' c y c l i c monophosphate. P r o c N a t l . Acad.. S c i . , _67_: 305-312, 1970. . Gli.tsgh^dH.G. ,aHaas9b.Hs:Gn,andATran,twein(jeW.::.n T h e l e f f e c t l o f t a d r e n a i i n e c o n . ''therKIan'dyNaGf'luxesninPthemfrogsgatriumyclNau/iMSch.RAreh.PExptl. ..Pathol:;-Pharmacol., 250: 59-71, 1965. Green, H. and E r i c k s o n , R.W.: E f f e c t . o f some drugs upon the h i s t a m i n e c o n c e n t r a t i o n , of r a t h e a r t . A r c h . I n t . Pharmacodyn., 166: 127-135, 1967. Green, R.D. and M i l l e r , J.W.: The e f f e c t o f v a r i o u s substances on the e f f l u x of . l a b e l l e d catecholamines from the u t e r u s of the r a t . .J.. Pharmacol. Exp,. Therp..,. 152: 439-444, 1966. Greengard,_P.. and Robison, G.A.: Advances i n . c y c l i c , n u c l e o t i d e r e s e a r c h , Vol.. I , P h y s i o l o g y , and .Pharmacology, o f . . c y c l i c . AMP,. Raven P r e s s . New.York, 1972.  126  Harwood,. J.P. and Drummond, G.1.: Effects_.of. e l e c t r i c a l stimulation and adrenaline on.phosphorylase, ..phosphorylase h kinase and c y c l i c 3 ' , 5 '-AMP i n skeletal muscle'. Fed. P r o c , 28: 541, 1969. Hess. M.E. and Haugaard, N.: The effect of epinephrine.and aminophylline on the phosphorylase a c t i v i t y of the perfused, contracting heart muscle. J . Pharmacol. Exp. Therp. .122: . 169-175, 1958. Hess, M.E.., Hohenstein, D., Shanfeld, J . and Haugaard,.N.:. Metabolic e f f e c t s of theophylline .in cardiac and s k e l e t a l muscle. J . Pharmacol. Exp. Ther.,.141: 274-279,. 1963. Huebner., C.F., Turner.,. R.A., and Scholz,. C.R.: Studies of imidazole compounds. IV. Derivatives of. 4-ethylimidazole.. J.. Am. Chem. S o c , 71: 3942-3944, 1949. Hughes,.M.J. and Coret, .I.A.: On s p e c i f i c i t y of histamine receptors i n the heart. Am. J . Physiol. 223: 1257-1262, 1972. Ivy, A.C. and F a r r e l , J . J . : Contribution to the physiology of gastric secretion. Am. J.. Physiol., 7_4: 639-649, 1925. Jensen, K.B. and Veenerod, A.M.: Reversal of the i n h i b i t o r y action of adrenaline and histamine, on.rat uterus. Acta Pharmacol. Exp. Ther., 18: 307-312, 1961. Karppanen,. H.O. and.Westermann, E.: Increased production o f . c y c l i c AMP i n gastric tissue by stimulation of histamine (H„) receptors. Naun. Sch..Arch. .Pharmac.., 279:. .83-87, 1973. Katz,. A.M., Repke,. D ...I.., Upshaw, . J .E. and Polascik, M.A.:, Characterization of dog cardiac, microsomes: use of ..zonal.centrifugation..to fractionate fragmented sarcoplasmic reticulum (Na~t + K^.)-activated ATPase and mitochondrial fragments.. . Biochemica et Biophysica Acta, 25: 47-3-490,. 1970. K l e i n , I. and Levey, G.S.: A c t i v a t i o n of myocardial adenyl cyclase by histamine i n guinea, p i g , cat. and human heart.. J . C l i n . Invest. 50:. 1012-1012, 1971. Knope, R., Moe, G.K., Saunder,, J.. and. Tuttle,. R.: Myocardial effects of imidazole. J . Pharmacol., Exp... Ther., 185: 29-34, 1973. Krause, E.G., Halle, W., K a l l a b i s , E. and Wollenberger, A.: Positive chronotropic .response of cultured isolated rat. c e l l s to N -2 -0-dibutyryl-r3', 5'-adenosine monophosphate. J . Mol. C e l l . Cardiol., .L: 1-^10,1970. 6  Krebs, E., Delange, R.., Kemp, R. and.Riley, W.: A c t i v a t i o n of skeletal . muscle phosphorylase. Pharmacol. Rev., J_8_: 163, 1966. Kroeger, E.A. and Marshall, J.M..: Beta-adrenergic e f f e c t s on r a t myometrium: r o l e of c y c l i c AMP. Am. J . Physiol, 226: 1298-1303, 1974.  127  'Kukovetz, W.R., Hess, M.E.., Shanfeld, J . and. Haugaard, . N. : The action of sympathomimetic amines .on the isometric contraction and phosphorylase a c t i v i t y of ..the isolated r a t heart. J.. Pharmacol. Exp. Ther., 127: 122-127, 1959. Kukovetz, W.R. and .Poch, G.: The action of imidazole on.the e f f e c t s of methylxanthines.and catecholamines.on cardiac contraction and phosphorylase a c t i v i t y . J . Pharmacol. Exp. Ther.., 156: 514-521, 1967. Kukovetz, W.R. and Poch,.. G.: I n h i b i t i o n of c y c l i c - 3 ' , 5'-nucleotidephosphodiesterase as a possible, mode.of. action.of papaverine and similar acting drugs. .Naun. Sch. Arch.. Pharmac.., 267: 189-194, 1970. Kukovetz, W.R.. and Poch,. G.:.. . The p o s i t i v e inotropic e f f e c t of c y c l i c AMP. In: Advances.in c y c l i c Nucleotide Research, Vol.. I, edited by P. Greengard.,. R... P a o l e t t i and G.A. Robison,. 1972 pp 261-290. Raven Press, New York. Kukovetz, W.R., Poch, G. and Wurm, A..: E f f e c t of catecholamines, histamine and oxyfedrine on.isotonic.contraction and c y c l i c AMP i n the guinea p i g heart. Naun. Sch. Arch. Pharmac., 278: 403-424, 1973. Kuo, J.F. and..Greengard, P..:.. C y c l i c nucleotide dependent protein kinases. VI. Isolation and p u r i f i c a t i o n of. a protein kinase, activated by Guanosine, 3',5'-monophosphate. J . B i o l . , Chem.,245: 2493-2498, 1970. Lands, A.M. and.Howard, J.W.: A comparative study of the e f f e c t s of 1-arterenol, epinephrine and isopropylarterenol on the heart. J . Pharmacol. Exp. Ther., 106: 65-76, 1952. Langendorff, 0.: Uber elektrische Reinung des Herzens. . _8: 284-298, 1885. Lefkowitz,  Arch. ges. Physiol.  R.J.: Isolated Hormone receptors: physiologic and c l i n i c a l .implications,. New. Eng.. J . Med., 288: 1061^1066, 1973.  Lefkowitz, R.J.. and Haber, E.: A f r a c t i o n of the v e n t r i c u l a r myocardium that has.the s p e c i f i c i t y of. the beta adrenergic receptor. Proc. Natl. Acad. S c i . U.S.A., 68: 1773-1777, 1971. Lefkowitz,  R.J., Roth, J . , Pastan, I..: ACTH-receptor i n t e r a c t i o n i n the adrenal: A.model .for the i n i t i a l , step.in the action of hormones that stimulate adenylate cyclase. Ann. N.Y. Acad. S c i . , 185_: 195-209, 1971.  Lefkowitz, R.J., Sharp, G.W.G. and Haber, E.: S p e c i f i c binding of .^-adrenergic catecholamines to a subcellular fraction,from cardiac milscle. J . B i o l . Chem., 248: 342-349, 1973.  128  Leonard, S.L.: Studies of theophylline induced a c t i v a t i o n of glycogen phosphorylase i n r a t u t e r i . Proc. Soc. Exp. B i o l , and Med. 140: 1350-1355, 1972. Levey, G.S. and Kleing, I.: Solublized myocardial adenylate cyclase: Restoration of.histamine responsiveness to phosphatidylserine. J . C l i n . Invest.., 51: 1578-1582, 1972. Levey, G.S...: Restoration of norepinephrine responsiveness of s o l u b i l i z e d myocardial, adenylate cyclase by phosphotidylinositol. J . B i o l . Chem...246: 7405*7407, 1971. Levey, G.S. and Epstein, S.E.: A c t i v a t i o n of.adenyl cyclase by glucagon i n cat and human heart. C i r . Res., 24:, 151-156,^.1969. L e v i , R. and G i o t t i , A.: Effect of histamine on s i n o - a r t i a l node c e l l s of rabbit heart. Experientia, 23^: 66-67, 1967. Levine, R.A. and Wilson, D.E.: The r o l e of c y c l i c AMP i n gastric secretion, Ann. N..Y. Acad. S c i . , 185: 363-375, 1971. Levine, R.A. and Washington, A.: Increased c y c l i c AMP production i n human g a s t r i c j u i c e i n response to secretagogues. Gastroenterology, 64_: A117-863, 1973. Levy, B. and Ahlquist,.R.P.: An analysis of adrenergic.blocking a c t i v i t y . J . Pharmacol. Exp. Ther., 133: 202-210, 1961. Levy, B. and Tozzi, S.: The adrenergic .receptive mechanism, of the r a t uterus. J . Pharmacol- Exp. Ther., 142: 178-184, 1963. Levy, B. and Wilkenfeld, B.E.: The potentiation of r a t uterine i n h i b i t o r y responses to .noradrenaline by theophylline and nitroglycaine. Br. J . Pharmac, 34: 604-612, 1968. Lewis, A.E.:  B i o s t a t i s t i c s , Reinhold Publishing Corp..., New York, 1966.  L i n , T.M., Henderson, E.G, Chen, K.K. and ..Benslay, D..N.: Structure-activity . and. enzyme substrate, relationships, of .histamine.,, histamine metabolites and. analogues, i n stimulation, of..gastric secretion. Proc. of 1st International Pharmacological Meeting... V o l . 7_:. . 351-358, 1963. Mannaioni, P.F..: Interaction, between, histamine., and.dichloroisoproterenol, hexamethonium, pempidine...and. diphenhydramine .in normal and reserpine-treated.heart preparations.. Br. J . Pharmacol. Chem., 15:- 500-505, 1960. MagV C?C^. .^acpbs-ph.,..E.t"D?. apd .Shanbour., L.L.: Mucosal c y c l i c AMP and secretion i n the dog stomach... Am.. J . ..Physiol,. 225: 893-896, 1973. -  129  Marshall, J.M.: Innervation of the female reproductiyeotract: Anatomy, physiology and pharmacology. Ergebn. Physiol., 62_: 6-67, 1970. Marshall, J.M.: E f f e c t s of catecholamines on smooth muscle of the female reproductive t r a c t . Ann. Rev. Pharmacol., 13_: 19-32, 1973. Marshall, J.M. and Kroeger, E.A.: Adrenergic influence on uterine smooth muscle. P h i l , Trans. R. Soc. Lond. B. 265: 135-148, 1973. Massingham, R.: The mechanism of potentiation of inotropic responses to phenylephrine by theophylline. Br. J . Pharmac, 370: 540P, 1969. Massingham, R. and Nasmyth, R.A.: The nature of the positive inotropic response of the isolated frog heart to theophylline and imidazole. Br. J . Pharmac, 45_: 229-239, 1972. Mayer, S.E.: Adenyl cyclase as a component of the adrenergic receptor. In Molecular properties of drug receptors, Ed. by J.A. C h u r c h i l l , Ciba foundation, 1970, pp 43-56. Mayer, S.E.: E f f e c t s of adrenergic agonists and antagonists on adenylate cyclase a c t i v i t y of dog heart and l i v e r . J . Pharmacol. Exp. Ther., 181: 116-125, 1972. Mayer, S.E. and Moran, N.C.: Relation between pharmacologic augmentation of cardiac c o n t r a c t i l e force and the a c t i v a t i o n of myocardial glycogen phosphorylase, J . Pharmacol. Exp. Ther., 129: 271-281, 1960. Mayer, S.E., Cotton, M. and Moran, N.C.: Dissociation of the augmentation of cardiac c o n t r a c t i l e force from the a c t i v a t i o n of myocardial phosphorylase by catecholamines. J . Pharmacol. Exp. Ther., 139: 275-282, 1963. Mayer, S.E., Namm, D.H. and Hickenbottom, J.P.: Regulation of the phosphoryla a c t i v a t i n g pathway i n intact cardiac and skeletal muscle. Adv. Enz. Reg., 8: 205-216, 1970. Mayer, S.E., Namm, D.H., Rice.L.: Effect of glucagon on c y c l i c 3',5'-AMP, phosphorylase a c t i v i t y and c o n t r a c t i l i t y of heart muscle of the r a t . C i r . Res., 2j6: 225-33, 1970. Meinertz, T., Nawrath, H., Schmitz, W. and Scholz, H.: F a i l u r e of reserpiniza t i o n to decrease the positive inotropic effect of d i b u t y r l c y c l i c AMP. Res. Comm. Chem. Path. Pharmacol., 6_: 383-390, 1973. M i l l e r , T.B., Exton, J.H. and Park, C.R.: A block i n epinephrine-induced glycogenolysis i n the hearts from adrenalectomized r a t s . J . B i o l . Chem., 246: 3672-3678, 1971. M i l l e r , J.W.: Adrenergic receptors i n the myometrium, 139: 788-798, 1967. '  Ann. N.Y. Acad. S c i .  130  Mitznegg, P., Hoch, B. and Heim, F.: The influence of c y c l i c 3', 5'-AMP on c o n t r a c t i l e responses induced by vasopressin i n the i s o l a t e d rat uterus. L i f e S c i . , 10: 169-174," 1971. Murad, F. and Vaughan, M.: E f f e c t of glucagon on r a t heart adenyl cyclase. Bioch. Pharmacol., 18: 1053-1059, 1969. Murad, F., Chi., Y.-M., R a i l , T.W. and Sutherland, E.W.: Adenyl cyclase. I I I . The e f f e c t of catecholamines and choline esters on the formation of adenosine-3', 5'-phosphate by preparations from cardiac muscle and l i v e r . J . B i o l . Chem., 237: 1233-1238, 1962. McNeill, J.H.: Potentiation of norepinephrine-induced cardiac phosphorylase by theophylline and reserpine. Can. J . Physiol. Pharmacol., 48: 149-151, 1970. McNeill, J.H. and Brody, T.M.: The e f f e c t of of antihistamines, cocaine and reserpine on amine-induced r a t cardiac phosphorylase a c t i v a t i o n . J . Pharmacol. Exp. Ther., 152: 478-487, 1966. McNeill, J.H., Nassarr,MM.anddBModyheT.;Nt:D.TheIeffect of theophylline on amine -induced cardiac phosphorylase a c t i v a t i o n and cardiac c o n t r a c t i l i t y . J . Pharmacol. Exp. Ther., 165: 234-241, 1969. McNeill, J.H., Davis, R.S. and Muschek, L.D.: Phenylephrine e f f e c t s on cardiac c o n t r a c t i l i t y , adenyl cyclase and phosphorylase. Arch. Int. Pharmacodyn. Ther., 197: 317-327, 1972. McNeill, J.H. and Muschek, L£D. : Histamine e f f e c t s on cardiac c o n t r a c t i l i t y , phosphorylase and adenyl cyclase. J . Mol. C e l l . C a r d i o l , 4_: 611-624, 1972.. McNeill, J.H. and Schulze, S.: Reserpine-induced s u p e r s e n s i t i v i t y to histamine activated cardiac phosphorylase and cardiac c o n t r a c t i l i t y . Res. Commun. Chem. Pathol. Pharmacol., 3^: 339-347, 1972. McNeill, J.H., Brenner, M. and Muschek, L.D.: Interaction of four methylxanthine compounds and norepinephrine on cardiac phosphorylase a c t i v a t i o n andiGardiacacontractilityijtrRecenttadvahcesninastudies on cardiac structure and metabolism. Ed. N.S. Dhalla, _3: 261-273, 1973. McNeill, J.H. and Young, B.A.: The e f f e c t of thyroid pretreatment on the cardiac e f f e c t s of tyramine and noradrenaline. C l i n . Res., 21: 815, 1973. Namm, D.H.: The a c t i v a t i o n of glycogen phosphorylase i n a r t e r i a l smooth muscle. J . Pharmacol. Exp. Ther., 178: 299-310, 1971  131 Namm, D.H., Mayer, S.E. and Maltbie, M.: The role of potassium and calcium ions i n the e f f e c t of epinephrine on cardiac c y c l i c adenosine 3', 5'-monophosphate, phosphorylase kinase and phosphorylase. Mol. Pharmacol., 4_: 5229530, 1968. Narumi, S. and Maki, Y.: Possible r o l e of c y c l i c AMP i n gastric acid secretion i n r a t . Biochem. Biophys. Acta, 311: 90-97, 1973. Nayler, W.G.: E f f e c t of caffeine on cardiac c o n t r a c t i l e a c t i v i t y and radiocalcium movement. Am. J . Physiol., 204: 969-974, 1963. Nayler, W.G.: Calcium exchange i n cardiac muscle: Basic mechanism of drug action. Am. Heart. J . , 73_: 379-394, 1967. Nayler, W.G., M e r r i l l e e s , N.C.R., C h i p p e r f i e l d , D. and Kurtz, U.B.: Influence of hyperthyroidism on the uptake and binding of calcium by cardiac microsomal f r a c t i o n s and on mitochondrial structure. Cardiovascular Res., 4_: 469M82, 1971. Nickerson, M. and Hollenberg, N.K.: Blockade of alpha-adrenergic receptors. In: P h y s i o l o g i c a l Pharmacology, V o l . 4 ed. by W.S. Root, F.G. Hofman, New York, Academic Press, 1967, pp 243-305. Niedergreeke, R.: Calcium and the a c t i v a t i o n of contraction. 15: ' 128-130, 1959.  Experienta,  0ye, I.: The action of adrenaline i n cardiac muscle. D i s s o c i a t i o n between phosphorylase a c t i v a t i o n and inotropic response. Acta Physiol. Scand., 65: 251-258, 1965. 0ye, I., Butcher, R.W., Morgan, M.E. and Sutherland, E.W.: Epinephrine and c y c l i c 3 , 5'-AMP l e v e l s i n working r a t heart. Fed. P r o c , 23: 262, 1964. r  P e r r i e r , C.V. and Laster, L.: Adenyl cyclase a c t i v i t y of guinea pig gastric mucosa. C l i n . Res., 1_7: 596, 1969. P e r r i e r , C.V. and Laster, L.: Adenyl cyclase a c t i v i t y of guinea p i g g a s t r i c mucosa: stimulation of histamine and prostaglandins. J. C l i n . Invest., 49: 73, 1970. Pfaffman, M.A. and Mcfarland, S.A.: The e f f e c t s of theophylline and c y c l i c AMP on i n t e s t i n a l smooth muscle contractions. European J . Pharmacol. 23: 147-152, 1973. Poch, G. and. Kukovetz, W.R. : DEug.-sinduc.ed-.j're'leas'e iand.tp'harmaGQdynamiGicef f ects Q'feehiSjfraimine' in\ t%.et'iguine^ip.i&^ .Pharanaco.l'.HExp 7 f.T-her !5j6-:r. 522-527, 1967. Poch-.;, G,,. an]ijkKuko;v£etzj, TW.R;ri:l SP.%ay^r±iie-ii3duc.64:iinKiMM o.n\cof op'ho:sphodiestera'seeaG;igivjityiiinava"riiou'Si mamma-l'ianptdss^es.yclL'ifeMS'ci>y, blO;•• 133-144, WJ:h., Sch. Arch. Pharmacol., Woy 223-228, 19/3. f  s  132 Poch, G., Kukovetz, W.R. and Scholz, N.: Specific i n h i b i t i o n of the cardiac e f f e c t s of histamine on contractionaand c y c l i c AMP by burimamide. Naun. Sch. Arch. Pharmacol., 280: 223-228, 1973. Polacek, I. and Daniel, E.E.: E f f e c t s of a- and 3-adrenergic stimulation on the uterine m o t i l i t y and adenosine 3,',5 -monophosphate l e v e l . Can. J . Physiol. Pharmacol., 49: 488-998, 1971. 1  Poltetaev, G.I.: Mechanism of imidazole effect on the function of myoneural junction i n the frog. Sechenov. Physiol. J . (U.S.S.R.), 1,: 64-69, 1970. Posner, J.P., Stern, R. and Krebs, E.G.: E f f e c t s of e l e c t r i c a l stimulation and epinephrine on phosphorylase, phosphorylase b_ kinase and adenosine 3',5'-phosphate. J . B i o l . Chem., 240: 982-985, 1965. R a i l , T.W. and Sutherland, E.W. : 27_: 347, 1961.  Cold Spring Harbor Symp. Quant. B i o l ,  R a i l , T.W. and West, T.C.: The potentiation of cardiac inotropic responses to norepinephrine by theophylline. J . Pharmacol. Exp. Ther., 139: 269-274, 1963. Rasmussen, H., Goodman, D.B.P. and Tenenhouse, A.: The role of c y c l i c AMP and calcium i n c e l l a c t i v a t i o n . C r i t i c a l Rev. Biochem., 1: 95-148, 1972. Rasmussen, H. and Tenenhouse, A.: In Biochemical Actions of Hormones, Litwack, G. (Ed.), Academic Press, New York, pp 365-413, 1970. Reuter, H.: Dependence of slow inward current i n Purkinje f i b r e s on the e x t r a c e l l u l a r calcium concentration. J . Physiol, 192: 479-492, 1967. Robison, G.A.: C y c l i c AMP and hormone action. 36: 723-733, 1972.  Am. J . Pharm. Ed.  Robison, G.A., Butcher, R.W., 0ye, I., Morgan, H.E. and Sutherland, E.W.: The effect of epinephrine on adenosine 3', 5'-phosphate levels i n the isolated perfused rat heart. Mol. Pharmacol., 1: 168-177, 1965. Robson, R.M., G o l l , D.E. and Temple, M.J.: Determination of proteins i n T r i s buffer by the Biruet reaction. Anal. Biochem., 2A_: 339-341, 1968. Schild, H.O.: The action of isoprenaline i n the depolarized rat uterus. Br. J . Pharmac, 31: 578-592, 1967. Scholtz, H.: Uberden Mechanismus der p o s i t i v inotropen wirkung von theophyllin am warmibluterherzen. II Wirkung von Theophyllin auf Aufnalime ung Abgabe von ^^Ca. Naun. Sch. Arch. Pharmack., 271: 396-409, 1971,  133 Shanfeld, J . , Frazer, A., Hess, M.E.: Effect of isopropylmethoxamine on norepinephrine-lnduced elevation of c y c l i c 3',5'-AMP, phosphorylase activation and c o n t r a c t i l i t y i n the i s o l a t e d perfused rat heart. Fed. Proc., TU 352, 1968. Shanfeld, J . , Frazer, A. and Hess, M.E.: Dissociation of the increased formation of cardiac adenosine 3',5'-monophosphate from the p o s i t i v e inotropic effect of norepinephrine. J . Pharmacol. Exp. Ther., 169: 315-320, 1969. Shaw, J.E. and Ramwell, P.W.':' I n h i b i t i o n of gastric secretion i n rats by prostaglandin E^. In: 'Prostaglandins, Symposium of the Worcester Foundation for Experimental Biology', ed. P.W. Ramwell and J.E. Shaw, New York: Interscience. 1968, p 55-56. Shine, K.I. and Langer, G.A.: Caffeine effects upon contraction and calcium exchange i n rabbit myocardium. J . Mol. C e l l . Cardiol., 3: 255-270, 1971. Skelton, C.L., Levey, G.S. and Epstein, S.E.: P o s i t i v e inotropic effects of dibutyryl c y c l i c adenosine 3',5'-monophosphate. C i r . Res., 26: 35-43, 1970. Skelton, C L . , Karch, F.E., Hougen, T.J., Marcus, M.L. and Epstein, S.E.: Potentiation of the inotropic effects of norepinephrine and dibutyryl c y c l i c AMP by theophylline. J . Mol. C e l l . C a r d i o l . , 3: 243-253, 1971. Sobel, B.E. and Mayer, S.E.: C y c l i c adenosine monophosphate and cardiac c o n t r a c t i l i t y . C i r . Res., 32: 407-414, 1973. S t u l l , J.T. and Mayer, S.E.: Regulation of phosphorylase a c t i v a t i o n i n s k e l e t a l muscle i n vivo. J . B i o l . Chem., 246: 5716-5723, 1971. Suko, J . : A l t e r a t i o n s of calcium uptake and calcium activated ATPase of cardiac sarcoplasmic reticulum i n hyper- and hypo-thyroidesim. Biochem., Biophys., Acta, 252: 324-327, 1971. Sulakhe, P.V., and Dhalla, N.S.: Excitation-contraction coupling i n heart: I I I . Evidence against the involvement of adenosine c y c l i c 3',5'-monophosphate i n calcium transport by sarcotubular v e s i c l e s of canine myocardium. Mol. Pharmacol. 6_: 659-666, 1970. Sutherland, E.W., C o r i , C.F., Haynes, R. and Olsen, N.S.: P u r i f i c a t i o n of the hyperglycemic-glycogenolytic factor from i n s u l i n and from gastric mucosa. J . B i o l . Chem., 180: 825-837, 1949. Sutherland, E.W., R a i l , T.W. and Menon, T.: Adenylate cyclase. 1. D i s t r i b u t i o n , preparation and properties. J . B i o l . Chem., 237: 1220-1227, 1962. Sutherland, E.W., Oye, I. and Butcher, R.W.: The.,action of epinephrine and the role of the adenyl cyclase system i n hormone action. Recent Prog. Hormone Res., 21: 623-646, 1965.  134  Sutherland, E.W. and Robison, G.A.: The role of c y c l i c 3',5'-AMP i n responses to catecholamines and other hormones. Pharmacol. Rev., 18: 145-161, 1966. Sutherland, E.W., Robison, G.A. and Butcher, R.W.: Some aspects of the b i o l o g i c a l role of adenosine 3',5'-monophosphate ( c y c l i c AMP). C i r c u l a t i o n , 37. 279-306, 1968. :  Szego, CM. and Davis, J.S.: Inhibition of estrogen-induced c y c l i c AMP elevation i n r a t uterus. L i f e A c i . , 8: 1109-1116, 1969. Tiffeneau, R.C.R.: Action inotropic negative De L'Histamine Sur Le Coeur De Grenouille. Effect secondaire inotrope p o s i t i f , D u A LA formation D'une. Substance antagoniste. Soc. Bio., 135: 1037, 1941. ?  Tozzi, S.: The mechanism of action of histamine on the isolated rat uterus. J. Pharmacol. Exp. Ther., 187: 511-517, 1973. Tozzi, S. and Roth, E.F.: The effect of histamine on the spontaneous m o t i l i t y of the rat uterus i n v i t r o . Fed. P r o c , J26: 785, 1967. Trendelenburg, U.: The action of histamine and 5-hydroxytryptamine on i s o l a t e d mammalian a t r i a . J . Pharmacol. Exp. Ther., 130: 450460, 1960. Triner, L., Overweg, N.I.A. and Nahas, G.G.: C y c l i c 3',5'-AMP and uterine c o n t r a c t i l i t y . Nature 225: 282-283, 1970. Triner, L., Nahas, G.G., Vulliemoz, Y., Overweg, N.I.A., Verosky, M., Habif, D.V. and Ngal, S.H.: Cyclic AMP and smooth muscle function. Ann. N.Y. Acad. S c i . , 185: 458-476, 1971. Triner, L., Nahas, G.G.,Vulliemoz, N., Overweg, N.I.A., Verosky, M., Habif, D.V. and Ngai, S.H.: Cyclic AMP and smooth muscle function. Ann. N.Y. Acad. S c i . , 185: 458-476, 1973. Triner, L., Vulliemoz, Y., Overweg, N.I.A., Verosky, M. and Nahas, G.G.: Adenyl cyclase i n rat uterine smooth muscle. Fed. P r o c , 32: 615, 1973. Tsien, R.W., G i l e s , W., Greengard, P.: Mediation of the e f f e c t s of adrenaline on cardiac Purkinje f i b r e s . Nature (New Biology), 240: 181-183, 1972. T u t t l e , R.S. and Fatah, A.: The e f f e c t of Ouabain on the frequency-force r e l a t i o n and on post-stimulation potentiation i s isolated a t r i a l and v e n t r i c u l a r muscle. J . Pharmacol. Exp. Ther., 135: 142-150, 1962. Vassort, G., Rougier, 0., Garnier, D., Sauviat, M.P., Coraboeuf, E. and Gargouil, Y.M.: Effects of adrenaline on membrane inward currents during the cardiac action p o t e n t i a l . Pfluegers Arch., 309: 70-81, 1969.  135  Walsh, D.A., Perkins, J.P. and Krebs, E.G.: An adenosine 3',5'-monophosphate dependent protein kinase from rabbit. J . B i o l . Chem., 243: 37633765, 1968. Wantanabe, A.M. and Besch, H.R. : C y c l i c adenosine monophosphate modulation of slow calcium i n f l u x channels i n guinea pig hearts. C i r . Res., 35: 316-324, 1974. Wastila, W.B., Su, J.Y., Friedman, W.F. and Mayer, S.E.: Blockade of Biochemical and Physiological responses of cardiac muscle to norepinephrine by N-Tert-butylmethoxamine (Butoxamine), J . Pharmacol. Exp. Ther. , 181: 126-138, 1972. Weber, A.:  Mechanism of the action of caffeine on sarcoplasmic reticulum. J . Gen. Physiol., 52: 760-772, 1968.  Went, S., Lissak, K.: Histamin-Und proteinwirkung am normalen und s e n s i b i l i s i e - e r t e r n Meerschweinchenherz. Naunyn-Schmiedeberg's Arch. Exp. Path. Pharmacol., 17_7: 609-615, 1935. Williamson, J.E. : Metabolic effects of epinephrine i n the perfused rat heart. I I . Control steps of glucose and glycogen metabolism. Mol. Pharmacol., 2: 206-219, 1966. Williamson, J.E.: K i n e t i c studies of epinephrine effects i n the perfused rat heart. Pharmacol. Rev., 18: 205-210, 1966. Winegrad, S. and Shanes, A.M.: Calcium f l u x and c o n t r a c t i l i t y i n guinea pig a t r i a . J . Gen. Physiol., 45: 371-394, 1962. Wollenberger, A., Ristau, 0. and Schoffa, G.: Eine einfache Tecknik der extrem schnellen Abkuhlung grosserer Gewebestucke. Pflugers Arch. Gesamte Physio. Menschen Tiere, 270: 399-412, 1960. Wollenberger, A., Babskii, E.B., Krause, E.G., Genz, S., Blohm, D., and Bogdanova, E.V.: C y c l i c changes i n levels of c y c l i c AMP and c y c l i c GMP i n frog myocardium during the cardiac cycle. Biochem. Biophys. Res. Comm., 55: 446-452, 1973. Wylie, J.H., Hesselbo, T. and Black, J.W.: receptor blockade by burimamide.  Effects i n man of histamine R^Lancet, 2: 1117-1120, 1972.  136  Young, B.A. and McNeill, J.H.: The effect of. noradrenaline and tyramine on cardiac c o n t r a c t i l i t y , c y c l i c AMP and phosphorylase a^ i n normal and hyperthyroid rats. Can. J . Physiol. Pharmacol., _5_2: 375-383, 1974.  APPENDIX REAGENTS USED FOR PHOSPHORYLASE ASSAY: I  10%  TCA  II Fiske and Subbarow reagent,  I t contains:  l-amin62-naphthol-4-sulfonic Acid  NaHSO 3  500  mg  1.0  gm  30.0  Dist. Water  to make  gm  200.0 ml  Na2S0^ and NaHSO^ were mixed i n 120 ml of d i s t i l l e d water, and to this added, with constant for several hours.  s t i r r i n g , s u l f o n i c acid. I t was  I l l TRIS. buffer (Sigma 7-9  The solution was  stirred  f i l t e r e d and stored protected from l i g h t . standard  pH, adjusted to 6.9,  and Biochem buffer)  using concentrated  HC1.  IV Shelf Molybdate Stock Solution. Working solution of shelf molybdate was made fresh from stock solution by appropriate d i l u t i o n 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 I  CYCLIC AMP  BINDING ASSAY:  Sodium acetate lOOmM.  II Potassium phosphate buffer 20mM.  III 5% Trichloroacetic Acid  IV  1N-HC1  V  C y c l i c AMP dependent protein kinase 3.2 ug/30A  VI  Protein kinase i n h i b i t o r (from beef heart) 1.1 mg/ml  VII Unlabelled c y c l i c AMP VIII §[H] c y c l i c AMP  -12 200 ,p moles (10 moles)/ml 200 p moles/ml  REAGENTS USED FOR ADENYLATE CYCLASE ASSAY: I  T r i s buffer 0.3M  II  Sodium f l u o r i d e  III Theophylline IV  KC1 0.166M  V  MgS0,.7H 0 o  4 VI  0.06M  0.06M  0.45M  2.  Salt mix:  KC1, MgSO^, i n l ' : l r a t i o  VII Phosphoenol pyruvate VIII ATP IX  0.3M  5mM  Pyruvate.kinase; 1 to 5 d i l u t i o n (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.Cl .2H 0  6.4 gm  MgCl .6H 0  8.6 gm  2  2  2  2  Dist. water to make  2000 ml  The working Chenoweth and Koelle buffer was made by d i l u t i o n 200 ml. of the stock solution to 1000 ml with d i s t i l l e d water. with sodium bicarbonate. gases, 95% 0  2  The pH was adjusted  The perfusion f l u i d was aerated with mixer of  + 5% C0 . 2  COMPOSITION OF THE BATHING SOLUTION FOR THE RAT UTERUS: (Diamond, 1973) 1.  Sodium Chloride T r i s buffer: Chemical  m mol./litre  Sodium chloride  125.0  Potassium chloride Magnesium chloride  2.4 '  0.5  Glucose  11.0  Tris*  23.8  Calcium c h l o r i d e . .  1.8  140  2.  Potassium Chloride T r i s buffer: Chemical  m mol./litre  Potassium chloride  127.4  Magnesium chloride  0.5  Glucose  11.0  Tris*  23.8 1.8  Calcium chloride  *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: ( A l l solutions are expressed i n mM). NaCl,  136.89;  NaHPO^.^O,  KC1,  0.36;  2.68; NaHC0 , 3  CaCl .6H 0, 2  11.90;  2  1.76;  Glucose,  MgCl .6H 0, 2  5.55;  2  0.98;  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 and 5% C0„. 2  2  Publications:  SUBHASH C. VERMA  Stewart, W.D., Runikis, J.O., Verma, S.C., Wallace S.: Problems i n S e l e c t i o n of t o p i c a l antiinflammatory c o r t i c o s t e r o i d s , Can. J . Med. Assoc., 108: 33, 1973. ;  Verma, S.C., Runikis, J.O., Stewart, W.D.: Fluorinated and nonfluorinated c o r t i c o s t e r o i d s : A re-evaluation. Ind. J . Hosp. Pharm. 10_. 167-1.73, 1973Verma, S.C., Chauhan, G.M.: S t a b i l i t y studies of selected combinations of thiamine, pyredoxine and hydroxocobalamine i n parental form. Bangladesh Pharm. J . 2_: 13-16, 1973. Verma, S.C., McNeill, J.H.: Action of imidazole on the cardiac i n o t r o p i c phosphorylase a c t i v a t i n g and c y c l i c AMP producing e f f e c t s of norepinephrine and histamine. Res. Commun. Chem. Pathol. Pharmacol. 305-319, 1974 McNeill, J.H. and Verma, S.C.: Phenylephrine induced increases i n cardiac c o n t r a c t i l i t y , c y c l i c AMP and phosphorylase a_. J . Pharmacol. Exp. Therap., 187: 296-299, 1973. McNeill, J.H. and Verma, S.C.: Blockade by burimamide of the e f f e c t s of histamine and histamine analogs on cardiac c o n t r a c t i l i t y , phosphorylase a c t i v a t i o n and c y c 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 . P h y s i o l , and Pharmacol. 52: 23-27, 1974. Verma, S.C. and M c N e i l l , J.H.: Blockade by burimamide of the e f f e c t s 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 r a t g a s t r i c adenylate cyclase by Histamine and Histamine analogues and blockade by Burimamide. B r i t . J . Pharmacol. In Press.  Verma, S . C : The involvement of c y c l i c AMP i n altered drug metabolism. Pharmagram (1973). McNeill, J.H. Coutinho, F.E. and Verma, S . C : Lack of i n t e r a c t i o n between norepinephrine or histamine and theophylline on cardiac c y c l i c AMP. Can. J . P h y s i o l . Pharmacol. McNeill, J.H., Verma, S . C : Blockade of the cardiac e f f e c t s of histamine, C l i n . Res., 21, 238, 1973. McNeill, J.H., Verma, S . C , Lyster, D.M. : Blockade of cardiac mechanical e f f e c t s of histamine and histamine analogs. Fed. P r o c , ^2: 808, 1973.  - 2McNeill, J.H., Young, B.A. and Verma, S . C : Adrenergic amine induced increases i n cardiac c y c l i c AMP, phosphorylase a_ and c o n t r a c t i l i t y . Pharmacologist 15_: 418, 1973. M c N e i l l , J.H. and Verma, S . C : Histamine and histamine analogue stimulated formation of cardiac c y c l i c AMP. Proc. Can. Fed. B i o l . S c i . , JL6: 228, 1973. McNeill, J.H. and Verma, S.C: Blockade by burimamide of the cardiac mechanical and biochemical e f f e c t s of histamine. Presented to the Canadian Cardiovascular Society, H a l i f a x , Oct. 1973. McNeill, J.H. and Verma, S . C : Res., 21: 951, 1973.  The cardiac e f f e c t s of imidazole.  Clin.  McNeill, J.H., Verma, S.C and Coutinho, F.E.: Cardiac actions and i n t e r actions of theophylline. C l i n . Res., 22: 9A, 1974. M c N e i l l , 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 i n t e r a c t i o n between morepinephrine or histamine and theophylline on cardiac c o n t r a c t i l i t y , phosphorylase a c t i v a t i o n and c y c l i c AMP. Fed. Proc. 33: 479, 1974 M c N e i l l , J.H., Verma, S.C.,: Caridac actions and i n t e r a c t i o n s of noradrenaline and histamine. Presented at the Internatiorf^conference on C y c l i c AMP, Vancouver, Canda, J u l y 1974 Verma, S.C, and M c N e i l l , J.H.,: Antagonism by Burimamide of histamine and histamine analog a c t i v a t i o n of cardiac and g a s t r i c adenylate cyclase. Presented a t the Internatiori^conference on C y c l i c AMP, Vancouver, Canada, J u l y 1974. Verma, S.C, and M c N e i l l , J.H.,: Histamine and Histamine agalogueinduced a c t i v a t i o n of adenylate cyclase and blockade by Burimamide. Proc. Can. Fedn. B i o l . S c i . , 17, 122. 1974  

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