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The role of adenosine 3’, 5’-monophosphate in the cardiac actions of glucagon Brunt, Margaret Edna 1975

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THE ROLE OF ADENOSINE 3',5'-MONOPHOSPHATE IN THE CARDIAC ACTIONS OF GLUCAGON  by MARGARET EDNA BRUNT •Sc. (Pharm.), University of B r i t i s h Columbia, 1974  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the D i v i s i o n of Pharmacology and Toxicology of the Faculty of Pharmaceutical  Sciences  We accept this thesis as conforming required  to the  standard  THE UNIVERSITY - OF 'BRITISH COLUMBIA September, 1975  In p r e s e n t i n g  t h i s thesis in p a r t i a l  f u l f i l m e n t o f the requirements f o r  an advanced degree at the U n i v e r s i t y o f B r i t i s h Columbia, I agree the L i b r a r y s h a l l make i t f r e e l y  that  a v a i l a b l e f o r r e f e r e n c e and study.  I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e  copying o f t h i s  thesis  f o r s c h o l a r l y purposes may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s .  I t i s understood that c o p y i n g o r p u b l i c a t i o n  of t h i s t h e s i s f o r f i n a n c i a l written  gain  permission.  Depa rtment The U n i v e r s i t y o f B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5  Date  0 * £  /  /975  shall  not be a l l o w e d w i t h o u t my  ii ABSTRACT  The biochemical and mechanical effects of glucagon were investigated i n the isolated, perfused rat heart.  Glucagon produced time and dose-dependent  alteractions i n myocardial force of contractions, glycogen phosphorylase activation and c y c l i c AMP accumulation. The p o s i t i v e inotropic e f f e c t was maximal following a 4.0 yg dose, after which s y s t o l i c tension increased 77.1 + 7.9 % (N=6) r e l a t i v e to prei n j e c t i o n s y s t o l i c tension.  This dose was also found to produce the maximal  phosphorylase activation (38.8 +3.5 % i n the a. form).  The c y c l i c AMP content  was 0.73 + 0.01 pmol/mg wet weight following 8.0 yg glucagon.  Since higher  doses were not investigated the saturating glucagon dose f o r c y c l i c AMP accumulation remains undetermined.  The minimum e f f e c t i v e glucagon dose f o r  increasing c o n t r a c t i l e force and % phosphorylase a was 0.5 yg, whereas only 0.25 yg was required to s i g n i f i c a n t l y elevate the v e n t r i c u l a r c y c l i c AMP content over basal l e v e l . The temporal sequence of these cardiac events was determined following 2.0 yg glucagon.  C y c l i c AMP increased s i g n i f i c a n t l y at 15 seconds.  The  positive inotropic e f f e c t was detectable 25 seconds after i n j e c t i o n and % phosphorylase a. elevation at 30 seconds.  A l l three parameters remained  sig-  n i f i c a n t l y greater than control at least 120 seconds after glucagon administration.  The observed time course i s consistent with the proposal that  c y c l i c AMP mediates the glucagon-elicited alterations i n force and glycogen phosphorylase a c t i v i t y . —8 Propranolol 10  M was found not to s i g n i f i c a n t l y influence glucagon-  induced changes i n force of contraction, % phosphorylase a or tissue c y c l i c AMP content, although this concentration readily, ^blocked the p o s i t i v e ino-  iii  tropic response to norepinephrine.  I t i s therefore u n l i k e l y that the car-  diac actions of glucagon are a r e s u l t of endogenous catecholamine release or an i n t e r a c t i o n with the catecholamine  3 receptor.  To further elucidate the r o l e of c y c l i c AMP i n the cardiac mechanical and metabolic  responses to glucagon, the influence of 1 mM theophylline on  these parameters was also investigated.  In the presence of the methylxan-  thine, glucagon produced dose-dependent changes i n % phosphorylase a, cont r a c t i l e force and c y c l i c AMP accumulation which were considerably than i n buffer-perfused hearts.  greater  S y s t o l i c tension was increased 116.3 +  7.4 % over p r e - i n j e c t i o n l e v e l with 4.0 yg glucagon, and % phosphorylase a. was  augmented to the maximum t h e o r e t i c a l value of 72.2 % (N=l) with 8.0 yg  glucagon.  The most dramatic influence of theophylline was on v e n t r i c u l a r  c y c l i c AMP accumulation, for glucagon 8.0 yg elevated tissue nucleotide content to 1.64 + 0.02 pmol/mg wet weight.  The sequence of events noted  i n buffer-perfused hearts was maintained i n the presence of theophylline 1 mM. The data obtained i n the present study strongly implicate an association between.myocardial c y c l i c AMP content and the metabolic of glucagon.  and mechanical actions  However, the mechanism by which theophylline potentiated the  glucagon responses i s not c l e a r .  One mM theophylline possessed i n t r i n s i c  a b i l i t y to a l t e r force of contraction, phosphorylase a c t i v a t i o n and c y c l i c AMP accumulation i n a manner inconsistent with the widely-accepted phosphodiesterase i n h i b i t i o n .  theory of  Control levels of c y c l i c AMP were approx-  imately 30 % greater than i n buffer-perfused hearts yet the % active phosphorylase was not s i g n i f i c a n t l y elevated. was  cardiodepressant  i n many animals.  Furthermore, 1 mM theophylline  These observations•indicate that  data with theophylline must be cautiously interpreted with respect to c y c l i c  AMP  involvement i n the theophylline cardiac responses, and i n the theo-  phylline-glucagon  interaction.  Other possible mechanisms of action, such  as an influence on calcium, should be given equal consideration.  V  TABLE OF CONTENTS Page ABSTRACT  i i  LIST OF TABLES  vi  LIST OF FIGURES  \*  LIST OF ABBREVIATIONS  v i i viii  INTRODUCTION  1  1.  The role of calcium i n excitation-contraction coupling  1  2.  The second messenger theory of catecholamine-induced actions i n myocardium  4  3.  Mechanisms of the cardiac actions of methylxanthines  11  4.  Cardiac actions of glucagon  19  MATERIALS AND METHODS  27  1.  MATERIALS  27  2.  METHODS  27  A. . Heart perfusion  27  B. Phosphorylase assay  30  C. C y c l i c AMP assay  31  I. II. III.  Tissue extraction •  31  C y c l i c AMP binding reaction  32  Calculation of results  33  D. S t a t i s t i c a l methods  33  RESULTS  35  DISCUSSION  65  SUMMARY AND CONCLUSIONS  90  BIBLIOGRAPHY  92  APPENDIX  '  101  vi  LIST OF TABLES TABLE 1.  2.  3.  4.  5. pe7. 6.  7.  8.  9.  10.  Page The effect of time on the p o s i t i v e inotropic response to 2 yg glucagon i n the buffer-perfused and theophyllineperfused r a t heart. The effect of various doses of glucagon on c o n t r a c t i l e force i n the isolated buffer-perfused and theophyllineperfused rat heart. _g The influence of propranolol 10 M on the p o s i t i v e inotropic effect of glucagon i n the i s o l a t e d perfused rat heart.  40  45  47  —8  The influence of 10 M propranolol on the p o s i t i v e inotropic action of norepinephrine i n the isolated perfused rat heart.  50  The e f f e c t of time on glucagon-induced cardiac phosphorylase a c t i v a t i o n i n the buffer-perfused and theophyllineperfused i s o l a t e d rat heart.  53  The effect of various doses of glucagon on cardiac glycogen phosphorylase a c t i v a t i o n i n the buffer-perfused and theophylline-perfused i s o l a t e d rat heart. _g The influence of propranolol 10 M on glucagon-induced phosphorylase a c t i v a t i o n i n the i s o l a t e d perfused rat heart.  56  The e f f e c t of time on cardiac c y c l i c AMP accumulation f o l lowing administration of 2 yg glucagon into the bufferperfused and theophylline-perfused rat heart.  57  The e f f e c t of various doses of glucagon on cardiac c y c l i c AMP accumulation i n the buffer-perfused and theophyllineperf used isolated rat heart.  59  54  —8  The influence of propranolol 10 M on glucagon-induced c y c l i c AMP accumulation i n the isolated perfused r a t heart.  60  vii  LIST OF FIGURES FIGURE .11. 2. 3. 4.  5.  6.  7.  Page Schematic representation of the second messenger concept.  5  Enzymes involved i n the control of myocardial glycogenolysis.  7  An adaptation of the general model of c e l l a c t i v a t i o n (Rasmussen et a l . , 1972) to myocardial tissue.  13  E f f e c t of glucagon (2 yg) on c y c l i c AMP content, cont r a c t i l e force and percentage phosphorylase a. at various times following i n j e c t i o n into rat hearts perfused with buffer or buffer plus theophylline.  37  E f f e c t of time on the absolute change i n tension following i n j e c t i o n of 2 yg glucagon into the i s o l a t e d buffer-perfused rat heart.  39  The e f f e c t of various doses of glucagon on cardiac c y c l i c AMP content, c o n t r a c t i l e force and percentage phosphorylase a. i n rat hearts perfused with buffer or buffer plus theophylline (1 mM).  42  The e f f e c t of various doses of glucagon on the absolute change i n s y s t o l i c tension i n buffer-perfused and theophylline-perfused rat hearts.  44  —8 8.  9.  The influence of propranolol 10 M on glucagon-induced changes i n cardiac c y c l i c AMP content, c o n t r a c t i l e force and percentage phosphorylase a.. —8 The influence of propranolol 10 M on the p o s i t i v e '.inotropic e f f e c t of norepinephrine.  49  52  viii ABBREVIATIONS  ATP  adenosine 5' -triphosphate  AMP  adenosine monophosphate  c y c l i c AMP  adenosine 3',5' - c y c l i c monophosphate  CK  Chenoweth-Koelle  cpm  counts per minute  DB-c-AMP  c y c l i c N -2'-0 dibutyryl-AMP  EDTA  ethylenediamine tetra-acetic acid  G-l-P  glucose-l-phosphate  S.E.M.  standard error of the mean  TCA  t r i c h l o r o a c e t i c acid  Tris  tri(hydroxymethyl)aminomethane  6  ix  ACKNOWLEDGEMENTS  I wish to express my sincere gratitude to Dr. John McNeill for his guidance and patience throughout this project.  I would also l i k e to  thank Dr. Don Lyster for his valuable assistance with the c y c l i c AMP assay procedure. Appreciation i s extended to Miss Elizabeth Hartley and Miss Annette Holmvang for their technical assistance i n the laboratory.  A special  thank you i s extended to Miss Barbara O'Malley and Miss Marilyn James for t h e i r help i n the preparation of this manuscript. The generous f i n a n c i a l support from the Medical Research Council of Canada and the Geigy Pharmaceuticals Ltd. Scholarship i s gratefully acknowledged.  1 INTRODUCTION 1.  The Role of Calcium i n Excitation-Contraction Coupling The i n t r a c e l l u l a r concentration of free calcium i s now generally ac-  cepted to be a major determinant of the a c t i v a t i o n state of myocardium (Langer, 1968).  However, the processes involved i n the regulation of  calcium are s t i l l poorly understood.  Furthermore, while calcium i s e s s e n t i a l  for electromechanical coupling, the sequence of events between membrane depolarization and the development of tension remains to be elucidated. The shape of the cardiac action p o t e n t i a l i s determined by transmembrane fluxes of sodium, calcium and potassium. r i s e to the spike of the action p o t e n t i a l .  A rapid inward sodium current gives This i s followed by a slower and  smaller inward current responsible for the plateau phase.  Voltage-clamp  experiments provide evidence that this current i s caused predominantly by calcium ions (Beeler and Reuter, 1970a) but sodium ions can also flow through the channel (Rougieret a l . , 1969).  Sarcolemmal r e p o l a r i z a t i o n i s believed  to be due to a sudden increase i n potassium e f f l u x . Total duration of the cardiac action p o t e n t i a l varies from 200 to 600 milliseconds compared to 5 milliseconds i n s k e l e t a l muscle (Morad and Goldman, 1973). Mechanical a c t i v i t y lags behind the e l e c t r i c a l events.  The onset of  contraction follows the upstroke of the action p o t e n t i a l by 20-40 m i l l i seconds (Morad and Goldman, 1973).  Relaxation occurs a f t e r the membrane  has begun to repolarize. The duration of the active state i s approximately 200 milliseconds.  According to the s l i d i n g filament theory, active tension  develops when calcium binds to troponin and removes the i n h i b i t o r y effect of the troponin-tropomyosin  complex.  Calcium also activates an adenosine  triphosphatase to supply energy for the contraction.  In order for relaxa-  tion to occur, the calcium must be removed from the troponin (Schwartz, 1972).  2 Removal of calcium from the v i c i n i t y of the myofibrils may sequestration by mitochondria, these.  be through  sarcoplasmic reticulum, or a combination of  Katz and Repke (1967) investigated the k i n e t i c properties of calcium  binding by a cardiac microsomal preparation and proposed that the rate of binding would be s u f f i c i e n t for relaxation of intact muscle. Briggs (197-4) reached s i m i l a r conclusions.  Solaro and  Mitochondrial uptake may  only be  of minor importance i n the normal cycle (Solaro and Briggs, 197 4; Williamson et a l . , 1974).but might be necessary f o r relaxation (Solaro and Briggs, 1974).  of f u l l y - a c t i v a t e d muscle  To prevent the c e l l from becoming overloaded  calcium, Reuter (1974) suggests that a f t e r binding by the sarcoplasmic  with  reticulum,  the ion i s transported across the sarcolemma by the sodium-calcium exchange system.  By this mechanism, calcium extrusion i s coupled to the passive i n f l u x  of sodium.  Evidence that at least part of the d r i v i n g force i s supplied by the  electrochemical gradient for sodium was  obtained by Jundt et a l . (1975) who  noted an association of extent of muscle relaxation, calcium-45 e f f l u x and e x t r a c e l l u l a r sodium concentration. Theories on the i n i t i a t i o n of the active state are even more complex and controversial than those on relaxation.  Most include the concept of a membrane  event being responsible f o r release of " t r i g g e r " calcium from i n t e r n a l s i t e s , possibly the l a t e r a l sacs of sarcoplasmic reticulum ( Bassingthwaighte and Reuter, 1972;  Morad and Goldman, 1973).  Release might be effected through the  slow calcium current, or by a direct depolarization of i n t e r n a l membrane translated from the sarcolemma.  Most investigators believe the calcium ions en-  t e r i n g the c e l l as the slow current do not account for a l l of the developed tension, although this i s a theoretical p o s s i b i l i t y (Beeler and Reuter, 1970b) . A very comprehensive theory of excitation-contraction coupling, based on electrophysiologic evidence and correlated with anatomical structure, has been  3 presented by Morad and Goldman (1973). of activator calcium — releasing s i t e —  They suggest there are two sources  a s u p e r f i c i a l (sarcolemmal) source and an i n t r a c e l l u l a r  which allow the calcium concentration to r i s e to the thres-  hold for m y o f i b r i l l a r a c t i v a t i o n . Although the 'relaxing system' i s also stimulated by the higher calcium l e v e l s , the i n f l u x rate i s greater and so active state i n t e n s i t y increases.  At a point when e f f l u x away from the  myofibrils equals i n f l u x , the c o n t r a c t i l e state i s at a maximum ( i n f l u x i s reduced due to membrane r e p o l a r i z a t i o n ) .  The calcium sequestration then  becomes predominant and relaxation proceeds. Perhaps the most important aspect of this theory i s that i t postulates control of tension by the l e v e l of membrane p o l a r i z a t i o n . The action pot e n t i a l ( f i r s t 100 milliseconds) would release i n t e r n a l calcium.  During  the plateau phase inward calcium transport would be maintained by a Ca^-K exchange c a r r i e r i n the sarcolemma. to the i n f l u x of calcium).  (The e f f l u x of potassium would be coupled  The degree of tension development would depend  on the sum of calcium from these two components and any change i n the c o n t r i bution of either would change the maximal force generated.  Although some of the  activator calcium i s recycled, maintenance of releasable stores would depend on the slow inward current. Drugs may produce a p o s i t i v e inotropic effect by influencing one or several of the steps i n the excitation-contraction cycle.  Catecholamines  increase the strength of contraction i n mammalian myocardium but the mechanism of action i s s t i l l speculative.  The membrane p o t e n t i a l during the  plateau phase i s more p o s i t i v e than normal i n tissue exposed to norepinephrine (Reuter, 1974).  Voltage-clamp studies have shown that norepinephrine i n -  creases the magnitude of the slow calcium current, which accounts for the higher plateau l e v e l (Reuter, 1974).  These electrophysiologic experiments  4 v e r i f y e a r l i e r tracer studies (Langer, 1968)  showing an enhanced i n f l u x of  calcium after catecholamine stimulation. Norepinephrine  shortens the relaxation time of myocardium which could  be due to f a c i l i t a t i o n of potassium e f f l u x (Tsien et al.,1972) or to an. effect on sarcoplasmic r e t i c u l a r calcium sequestration.  Although Entman  and coworkers (1969) found an epinephrine-stimulated calcium uptake into canine microsomes, this observation was not repeated by others (Sulakhe  and  Dhalla, 1970). One of the-most popular theories of catecholamine-induced  positive  inotropism involves stimulation of the adenylate cyclase system.  This i s  discussed i n the following section. 2.  The second messenger theory of catecholamine-induced  actions i n  myocardium C y c l i c AMP was  discovered during the course of investigations on mech-  anisms of hormone-induced hepatic glycogenolysis (Sutherland and R a i l , 1958). Within a few years^ c y c l i c AMP sponses.  was  implicated i n a variety of hormone re-  The "second messenger" theory of hormone action was proposed to  account for the rapidly accumulating  data (Sutherland et a l . , 1965).  As o r i g i n a l l y outlined, a hormone was  suggested  to interact d i r e c t l y  with adenylate cyclase located i n the target c e l l membrane. l e v e l of c y c l i c AMP  The increased  then served as an i n t r a c e l l u l a r messenger to modify en-  zyme a c t i v i t y or otherwise bring about the p h y s i o l o g i c a l response.  Tissue  concentrations of the nucleotide are regulated by adenylate cyclase which catalyzes c y c l i c AMP  formation from ATP,  which cause breakdown to 5'AMP.  and s p e c i f i c  phosphodiesterases  The o r i g i n a l model has been modified to  indicate the hormone receptor and adenylate cyclase are not the same entity (Figure 1).  Varied Stimuli .plasma  5-AMP ATP  Endocrine Gland  f phosphodiesterase  membrane of target cell  J  HORMONE (first messenger)  d OC  o O  Inactivated Hormone  «  J  Cyclic 3,5-AMP (second messenger)  >  <  Physiological Responses  Steroids,Thyroid Hormonejetc.  Fig-1. Schematic representation of the second messenger concept  6 The second messenger system allows hormones to be e f f e c t i v e without permeating the c e l l membrane. hormonal stimulus.  I t also provides a method of modifying a given  Hormone s p e c i f i c i t y i s provided for by the fact that only  those hormones which produce a physiological response i n the target c e l l w i l l stimulate adenylate cyclase. Sutherland and associates (1965) f i r s t proposed that catecholamines produced t h e i r cardiostimulatory effects according to the second messenger theory. coworkers  The strongest evidence they c i t e d were experiments by Robis op. Jand (1965) where epinephrine i n j e c t i o n into i s o l a t e d rat heart e l i c i t e d  a marked elevation of c y c l i c AMP p r i o r to the increases i n c o n t r a c t i l e force and phosphorylase activation.  Many investigators have repeated their obser-  vations i n other species and tissue preparations (review by Sutherland et a l . , 1968). The role of adenylate cyclase and c y c l i c AMP i n cardiac glycogenolysis i s w e l l understood.  A sequence of enzymic reactions i s i n i t i a t e d (Figure 2) which  ultimately leads to glycogen phosphorylase activation as described below. Epinephrine (and other catecholamines) interacts with the cardiac g receptor to stimulate adenylate cyclase.  Cyclic AMP i s required for a c t i v i t y of a  protein kinase which phosphorylates phosphorylase kinase to the active form. Active phosphorylase kinase, i n the presence of calcium, catalyzes the conversion of inactive glycogen phosphorylase (phosphorylase b) to active phosphorylase (phosphorylase a.) which causes glycogen breakdown.  In the absence of calcium,  phosphorylase activation does not proceed even though i n t r a c e l l u l a r c y c l i c AMP i s increased (Namm et a l . , 1968).  Als'o; anoxia w i l l stimulate phosphorylase  when c y c l i c AMP i s not elevated (Dobson and Mayer, 1973).  Therefore, the  c y c l i c nucleotide i s not independently responsible for glycogenolysis regulation.  7  CYCLIC  AMP  Phosphorylase b Kinase (inactive) ATP \  Phosphorylase b Kinase Kinase ADP  Phosphorylase b Kinase (active)  Phosphorylase b (inactive) ATP +•+  Glycogen + P i  ADP Phosphorylase a(active)  Glucose-l-Phosphate  FIGURE 2.  Enzymes involved i n the control of myocardial  glycogenolysis  8 At one time phosphorylase activation was thought to be necessary f o r i n i t i a t i o n of mechanical a c t i v i t y .  However, low doses of epinephrine pro-  ducing changes i n c o n t r a c t i l i t y did not produce changes i n phosphorylase a^ levels (Mayer et a l . , 1963; Drummond et a l . , 1964).  Also, phosphorylase  activation followed the i n i t i a t i o n of contraction (Cheung and Williamson, 1965; Williamson and Jamieson, 1965). ?  More recently Drummond and Hemmings  (1973) could not separate the two events either on the basis of dose or time :  and hence suggest that catecholamine-induced glycogenolysis secondary to adenylate cyclase stimulation may support the inotropic e f f e c t by increasing the energy supply. Most proponents of a c y c l i c AMP-meditated inotropism favor a more direct involvement (as opposed to an involvement through phosphorylase a c t i v a t i o n ) . However, the sequence of events between adenylate cyclase stimulation and contraction i s not known.  Early i n v i t r o evidence of an association was  found by Murad et a l . , (1962).  They noted the order of potency of d i f f e r e n t  catecholamines i n stimulating adenylate cyclase was the same as that f o r producing changes i n c o n t r a c t i l i t y . the cyclase activation.  As mentioned  Furthermore, dichloroisoproterenol blocked e a r l i e r , many temporal investigations  i n the i n t a c t heart have established that c y c l i c AMP increases p r i o r to, or at least concurrent with, the catecholamine-induced increase i n force (review by Sobel and Mayer, 1973). I f c y c l i c AMP i s an i n t r a c e l l u l a r mediator then exposure to the nucleotide or a derivative should also increase the force of contraction. and coworkers  Robison  (1965) attributed t h e i r i n i t i a l f a i l u r e to change the strength  of contraction using c y c l i c AMP to the poor membrane penetrating a b i l i t y . In contrast Meinertz et a l .  (1974) demonstrated  that the p o s i t i v e inotropic  action of c y c l i c N^-2'-O-dibutyryl-AMP (DB-c-AMP) was concentration-dependent  9  i n i s o l a t e d e l e c t r i c a l l y driven a t r i a l and v e n t r i c u l a r preparations.. These experiments confirmed the e a r l i e r findings of Skelton et al.(1970)  and  Drummond and Hemmings (1972). The l i t e r a t u r e also contains reports of d i s s o c i a t i o n s between c y c l i c  Shanfeld, Fraser and Hess (1969)  and the inotropic e f f e c t of catecholamines. were able to block norepinephrine-induced t e r i n g the mechanical action.  c y c l i c AMP  Langslet and Oye  c y c l i c AMP  production without a l -  (1970) noted that at low  temperatures where both epinephrine and c y c l i c AMP activation, only epinephrine was  AMP  increased  phosphorylase  capable of p o s i t i v e inotropism.  Dibutyryl  promoted glycogenolysis i n concentrations i n s u f f i c i e n t to increase  c o n t r a c t i l e force (Oye and Langslet, 1972)  unlike isoproterenol and  the cardiac response was b a s i c a l l y d i f f e r e n t . augmented with dibutyryl c y c l i c AMP,  Although  contractility is  r e l a t i v e l y high concentrations are  necessary and the e f f e c t takes longer to develop than phosphorylase tion (Sobel and Mayer, 1973). supports a c y c l i c AMP  suggested  activa-  Thus, while a considerable volume of evidence  involvement  i n the c o n t r a c t i l e response some experi-  mental observations are inconsistent with the hypothesis. The most recent experiments have focused on i n t r a c e l l u l a r s i t e s of action for c y c l i c AMP.  Entman, Levey and Epstein (1969) demonstrated an  epinephrine-sensitive cyclase i n a cardiac microsomal preparation also capable of increasing calcium uptake. was not ruled out.  However, contamination with sarcolemma  Sulakhe and Dhalla (1973) and Katz and associates (1974)  have obtained more p u r i f i e d sarcoplasmic reticulum preparations which possess an adenylate cyclase s i m i l a r i n properties to the sarcolemmal enzyme. et a l . (1969) postulate that c y c l i c AMP  may  Entman  f a c i l i t a t e more rapid calcium  binding and greater accumulation  of calcium so that on subsequent stimulation  more calcium might be released.  Kirchberger et al.(1972) noted a c y c l i c  AMP-  10 stimulated calcium uptake by cardiac microsomes when protein kinase was present.  The concentrations of c y c l i c AMP were s i m i l a r to those a c t i v a t i n g  phosphorylase kinase.  Similar experiments have been performed by other  workers (LaRaia and Morkin, 1974; Kirchberger et a l . , 1975: Schwartz et a l . , 1975).  This action on sarcoplasmic  reticulum may explain the increased rate  of relaxation following catecholamine administration for dibutyryl c y c l i c AMP can mimic the relaxing effects (Meinertz et a l . , 1974; 1975 a,b). Phosphorylase kinase can phosphorylate cardiac microsomal preparations (Schwartz et al.,1974) and also troponin (St m i  et a l . , 1972). but the im-  portance i n catecholamine-induced inotropism remains to be investigated. C y c l i c AMP may induce membrane permeability changes, p a r t i c u l a r l y to calcium.  Scholz et al.(1975) reported that dibutyryl c y c l i c AMP influenced  calcium-45 exchange i n a manner s i m i l a r to norepinephrine  or theophylline.  C y c l i c AMP, monobutytyl c y c l i c AMP and dibutyryl c y c l i c AMP perfused into cardiac Purkinje fibres a l l increased the action p o t e n t i a l plateau amplitude and shortened the plateau duration i n an i d e n t i c a l manner to the catecholamines (Tsien et a l . , 1972).  These results indicate a c y c l i c nucleotide-  mediated increase i n the slow inward calcium current and i n the outward potassium  current.  Further evidence supporting the idea that c y c l i c AMP.is involved i n excitation-contraction coupling .is provided i n recent combined e l e c t r o physiological-mechanical studies (Watanabe and Besch, 1974; Schneider and Sperelakis, 1975).  Both isoproterenol and dibutyryl c y c l i c AMP restored  e x c i t a b i l i t y and contractions to potassium-depolarized  hearts.  This cardiac  model assumes that excitation is. accomplished by an increase i n the slow calcium current. I f c y c l i c AMP was acting as a second messenger to increase calcium i n -  11  f l u x then tissue levels should r i s e p r i o r to this i n f l u x .  Direct experimental  techniques f o r c o r r e l a t i v e measurements are not yet available.  However,  Watanabe and Besch (1974) d i d observe isoproterenol-induced increases i n myocardial c y c l i c AMP p r i o r to the restoration of mechanical a c t i v i t y . Much of the above-mentioned experimental evidence indicates that a c y c l i c AMP influence on calcium homeostasis i s probable.  Rasmussen and  associates (1972) have reviewed the i n t e r a c t i o n of calcium and c y c l i c AMP on several hormonally-responsive tissues, including myocardium.  They mod-  i f i e d the second messenger theory to include calcium as either a dual second messenger or a t h i r d messenger (Figure 3).  This model n i c e l y accounts f o r  presently accumulated data on the inotropic and glycogenolytic actions of catecholamines i n myocardial tissue (see figure for explanation).  3.  Mechanisms of the cardiac actions of methylxanthines Sutherland and Robispn  (1966) developed a set of c r i t e r i a which, i f  f u l f i l l e d for any hormone, would strongly implicate c y c l i c AMP as a mediator of i t s end-organ response.  F i r s t , hormonal stimulation should produce changes  i n i n t a c t tissue l e v e l s of c y c l i c AMP, and this should either precede or. occur simultaneously with the p h y s i o l o g i c a l event.  Second, the target tissue  should possess an adenylate cyclase which i s stimulatable by the hormone i n broken c e l l perparations. Third, the e f f e c t of the hormone should be mimicked by the addition of exogenous c y c l i c AMP or one of i t s derivatives.  Finally,  agents which modify phosphodiesterase a c t i v i t y should correspondingly modi f y the hormonal response.  These c r i t e r i a have been e s s e n t i a l l y s a t i s f i e d  f o r catecholamines i n myocardium with a few exceptions mentioned  earlier  (Rasmussen et a l . , 1 9 7 2 ; Sobel and Mayer, 1973). Experiments attempting to s a t i s f y the l a s t of the above-mentioned c r i -  12  FIGURE  3.  An adaptation of the general model of c e l l a c t i v a t i o n (Rasmussen et a l . , 1972) to myocardial t i s s u e .  When a hormone interacts with i t s receptor s i t e , i t does two things simultaneously—(1) i t activates adenylate cyclase, leading to increased i n t r a c e l l u l a r levels of c y c l i c AMP and (2) i t increases membrane permeability to calcium, allowing an i n t r a c e l l u l a r increase i n concentration of this ion. Cyclic AMP has at least two e f f e c t s i n t r a c e l l u l a r l y — (a) i t i n i t i a t e s the enzymatic reaction sequence leading to glycogenolysis and (b), i t alters the "unavailable' or subcellular f r a c t i o n of calcium to lead to an increase i n "free? (cytosol) calcium. Then the increased cytosol calcium i s responsible for several changes including ( i ) i n h i b i t i o n of adenylate cyclase and/or stimulation of phosphodiesterase, ( i i ) stimulation of enzymes, notably active phosphorylase b_ kinase, to produce metabolic changes and ( i i i ) mediation of excitation-contraction coupling. 1  The most important feature of this model i s that each second messenger r e c i p r o c a l l y controls the concentration of the other. This i s a b u i l t - i n mechanism for stopping a s i g n a l — e q u a l l y important as i n i t i a t i o n of i t .  5 AMP  Ph  a  r> G -  Figure I  3  1  ~ P  1* t e r i a i . e . p a r a l l e l alterations i n phosphodiesterase a c t i v i t y and physiol o g i c a l response frequently employ the methylxanthines as phosphodiesterase inhibitors.  Butcher and Sutherland (1962) determined the potency i n t h i s  series of compounds f o r beef heart enzyme i n h i b i t i o n to be theophylline > caffeine > theobromine.  Later,Rail and West (1963), using an e l e c t r i c a l l y -  driven a t r i a l perparation, observed a potentiation of the norepinephrineihduced force increase when theophylline was present i n the muscle bath. The influence of caffeine was less prominent.  In addition, theophylline  was found to augment eathecholamine-induced increases i n phosphorylase a_ (Hess et a l . ,  1963).  Theophylline and caffeine have well-established p o s i t i v e inotropic e f f e c t s of t h e i r own  (Blinks et a l . , 1972).  Hess and Haugaard (1958) also  demonstrated the a b i l i t y of aminophylline to increase active cardiac phosphorylase levels which was repeated i n l a t e r experiments (Hess et a l . , Vincent and E l l i s  1963).  (1963), by measuring cardiac glycogen content, demonstrated  the direct glycogenolytic action of theophylline. A l l of the above observations led Sutherland and associates (1968) to propose that methylxanthines and catecholamines exerted t h e i r actions through a common pathway i . e . through increased i n t r a c e l l u l a r levels of c y c l i c  AMP.  Further i n d i r e c t evidence i n support of this was obtained by Skelton et a l . (1971) who,  i n i s o l a t e d cat p a p i l l a r y muscle, noted a potentiation of both  the norepinephrine and dibutyryl c y c l i c AMP I. D x 10"M. 4  inotropic actions by theophylline  They assumed this was an e f f e c t i v e concentration f o r phos-  phodiesterase i n h i b i t i o n .  Kukovetz and Poch (1970) also noted an augmen-  tation i n Langendorff preparations•of rabbit, rat and guinea p i g myocardium. The sophisticated study of Watanabe and Besch (1974), using potassiumdepolarized guinea pig.hearts, established a further l i n k between phospho-  15  diesterase i n h i b i t i o n and c o n t r a c t i l e a c t i v i t y . demonstrated  that theophylline ( 1 - 3  These investigators  mM) was capable of restoring mechanical  a c t i v i t y and elevating i n t r a c e l l u l a r c y c l i c AMP concentration. Unfortunately, the temporal sequence was not investigated.  However, Watanabe and Besch d i d  observe that the time required to restore contractions was longer than f o r catecholamines which i s consistent with an i n t r a c e l l u l a r s i t e of action. Similar observations on the e l e c t r i c a l and mechanical restorative a b i l i t y of methylxanthines were obtained by Schneider and Sperelakis (1975).  They  noted a c o r r e l a t i o n i n phosphodiesterase-inhibiting potency with e f f e c t i v e concentration i n inducing the slow calcium response. Imidazole was found to stimulate cardiac phosphodiesterase i n v i t r o (Butcher and Sutherland, 1962).  Therefore an antagonism of the theophylline  action and of the action of small doses of isoproterenol on c o n t r a c t i l e force and phosphorylase activation (Kukovetz and Poch, 1967) also supported the c y c l i c AMP hypothesis.  In a more recent study, where i n t r a c e l l u l a r ac-  cumulation of the c y c l i c nucleotide was also determined, Verma and McNeill (1974) found p a r a l l e l decreases i n norepinephrine-induced c o n t r a c t i l e force and i n the c y c l i c AMP l e v e l .  However phosphorylase activation  was not  correlated with changes i n c y c l i c AMP caused by imidazole. Some experimental data on the cardiac actions of xanthines alone, and ^ i n combination with norepinephrine, indicate the c o r r e l a t i o n with phosphodiesterase i n h i b i t i o n i s less than perfect.  Hess et a l .  (1963) found the  dose of theophylline required to potentiate the catecholamine phosphorylase activation was cardiodepressant on basal tension and decreased the p o s i t i v e inotropic response to norepinephrine. rat heart.  The-(preparation used was a Langendorff  McNeill and coworkers (1969) presented s i m i l a r findings i n the  i n s i t u r a t heart.  16 M c N e i l l , Brenner and Muschek (1973) compared, i n g u i n e a p i g myocardium, the a b i l i t y o f v a r i o u s m e t h y l x a n t h i n e s and p a p a v e r i n e t o p o t e n t i a t e c a t e c h o l amine- i n d u c e d i n o t r o p i s m and p h o s p h o r y l a s e a c t i v a t i o n w i t h t h e i r potency as phosphodiesterase i n h i b i t o r s .  A l t h o u g h t h e r e was good c o r r e l a t i o n among t h e  m e t h y l x a n t h i n e s w i t h r e s p e c t t o the p o t e n t i a t i n g e f f e c t and t h e phosphod i e s t e r a s e i n h i b i t i n g a c t i o n , p a p a v e r i n e gave anomalous r e s u l t s .  The a l k a -  l o i d was more p o t e n t t h a n the n a t u r a l l y o c c u r r i n g m e t h y l x a n t h i n e s i n i n h i b i t i n g g u i n e a p i g p h o s p h o d i e s t e r a s e and enhanced t h e 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 of norepinephrine. inotropic action.  However, i t d i d n o t augment t h e p o s i t i v e  Furthermore t h e d i r e c t i n o t r o p i c a c t i o n s ' o f t h i s s e r i e s  d i d n o t c o r r e s p o n d t o t h e i r p h o s p h o d i e s t e r a s e i n h i b i t i n g a b i l i t y because t h e o p h y l l i n e p o s s e s s e d the g r e a t e s t i n o t r o p i c e f f e c t o f t h e m e t h y l x a n t h i n e s , w h i l e i s o b u t y l m e t h y l x a n t h i n e (SC-2964) was t h e most p o t e n t enzyme i n h i b i t o r . P a p a v e r i n e had a n e g a t i v e i n o t r o p i c e f f e c t .  From t h e s e d a t a , M c N e i l l e t a l .  (1973) q u e s t i o n e d the cause and e f f e c t r e l a t i o n s h i p between p h o s p h o d i e s t e r a s e i n h i b i t i o n and t h e c a r d i a c a c t i o n s of t h e s e drugs. Two v e r y r e c e n t s t u d i e s have p r o v i d e d f u r t h e r e v i d e n c e t h a t t h e m e t h y l x a n t h i n e s may not work through c y c l i c AMP.  M c N e i l l e t a l . (1974) found t h a t  a l t h o u g h t h e o p h y l l i n e (1 mg) had a weak p o s i t i v e i n o t r o p i c and p h o s p h o r y l a s e a c t i v a t i n g e f f e c t on i t s own, i t d i d n o t change t h e c y c l i c AMP c o n t e n t o f -4 the g u i n e a p i g h e a r t .  I n a d d i t i o n , t h e o p h y l l i n e 7 x 10  M p o t e n t i a t e d the  n o r e p i n e p h r i n e m e t a b o l i c and i n o t r o p i c a c t i o n s b u t d i d n o t i n f l u e n c e the c a t e c h o l a m i n e - i n d u c e d change i n c y c l i c AMP. tration  A h i g h e r m e t h y l x a n t h i n e concen-  (2 mM) was c a r d i o d e p r e s s a n t by i t s e l f and reduced the p o s i t i v e i n o -  t r o p i c response t o s e v e r a l doses o f n o r e p i n e p h r i n e .  However, i t p o t e n t i a t e d  the i n c r e a s e i n c y c l i c AMP t o the h i g h e s t dose o f n o r e p i n e p h r i n e (0.4 y g ) . These r e s u l t s suggest t h a t , a l t h o u g h t h e o p h y l l i n e and o t h e r m e t h y l x a n t h i n e s a r e  17 capable of i n h i b i t i n g phosphodiesterase i n v i t r o , this may not be manifest in intact preparations i n concentrations producing the pharmacological responses. Henry and associates (1975) investigated the myocardial actions of papaverine.  They found no p o s i t i v e inotropic effect after testing several  concentrations.  Also, the mechanical alterations following epinephrine  were s i m i l a r l y unaffected by papaverine.  However, the a l k a l o i d increased  i n t r a c e l l u l a r c y c l i c AMP alone, and i n an additive manner with epinephrine. Papaverine increased the % phosphorylase a. p a r a l l e l to changes i n c y c l i c AMP. It would appear, therefore, that the importance of phosphodiesterase i n h i b i t i o n as a mechanism of action for methylxanthines and papaverine may have been overestimated. I f the common mechanism of inotropic action of catecholamines, methylxanthines and dibutyryl c y c l i c AMP i s through raised i n t r a c e l l u l a r levels of c y c l i c AMP then certain features of their mechanical effects should be i d e n t i c a l .  A l l three agents increase maximum developed isometric tension  and rate of tension development (Skelton et a l . , 1970; Skelton et a l . , 1971; Blinks et a l . , 1972).  However the s i m i l a r i t y ends here.  Both norepinephrine  and dibutyryl c y c l i c AMP decreased time to peak tension (Skelton et a l . , 1970) whereas the methylxanthines increased time to peak tension (Blinks et a l . , 1972).  In contrast to isoproterenol, the active state of myocardium i s  prolonged by methylxanthines  (Blinks et a l . , 1972).  Gibbs (1967) and Blinks  and coworkers (1972) have both observed the antagonistic action of caffeine toward catecholamine-induced increases i n rate of relaxation.  Blinks et a l .  (1972) could not distinguish any of these effects among the three methylxanthines tested, even though phosphodiesterase-inhibiting >potency; varies.  18 On the cardiac action p o t e n t i a l , caffeine greatly prolongs the plateau phase (deGubareff and Sleator, 1965) while norepinephrine . has variable e f f e c t s , depending on experimental protocol (deGubareff and Sleator, 1965; Spilker, 1970). In addition to t h e i r i n v i t r o actions on c y c l i c AMP  phosphodies-  terases, the methylxanthines have profound effects on c e l l u l a r calcium homeostasis which could also account for many experimental observations. Therefore t h e i r role i n establishing c y c l i c nucleotide involvement has been questioned (Sobel and Mayer, 1973).  Caffeine increased calcium exchange-  a b i l i t y i n toad v e n t r i c l e (Nayler, 1963) and mammalian a t r i a (Guthrie and Nayler, 1967). S i m i l a r l y , theophylline was found to increase calcium-45 uptake and release i n guinea p i g a t r i a (Scholz, 1971). Calcium handling by i n t r a c e l l u l a r organelles may be influenced by methylxanthines.  For example, caffeine displaced calcium from toad  v e n t r i c u l a r mitochondria (Nayler and Hasker, 1966) arid also reduced rate of calcium uptake by r a t and guinea p i g microsomal preparations (Nayler et a l . , 1975).  This l a t t e r observation i s consistent ;with the a b i l i t y of  caffeine to prolong the active state (deGubareff and Sleator, 1965).  Earlier  Weber and Herz (1968) demonstrated that caffeine both released and prevented reaccumulation of calcium by sarcoplasmic reticulum i n s k e l e t a l muscle. Although releasing a b i l i t y by cardiac sarcoplasmic reticulum was not d i r ectly investigatedjJundt et a l . (1975) observed marked stimulation of sodiumdependent calcium-45 e f f l u x from guinea p i g a t r i a by caffeine (and to a lesser extent by theophylline).  Since they could not observe a caffeine effect on  calcium release from incubated mitochondria (contrary to the results of Nayler and Hasker, 1966) they speculated the source of the calcium was sarcoplasmic reticulum.  Thorpe (1973) found a d i r e c t e f f e c t of caffeine on  19 release of bound calcium from rabbit myocardial sarcoplasmic reticulum vesicles. Methylxanthines may have an influence i n the excitation phase of the cardiac cycle. of  Two- groups of investigators have demonstrated the a b i l i t y  these agents to restore e x c i t a b i l i t y and mechanical a c t i v i t y to potassium-  arrested hearts (Watanabe and Besch, 1974; Schneider and Sperelakis, by activating the slow calcium channels.  1975)  Consistent with a postulated  action during e x c i t a t i o n , Scholz (1971) observed an increase i n calcium-45 i n f l u x i n beating, but not i n quiescent, i s o l a t e d guinea p i g a t r i a . The above-described actions of methylxanthines on calcium would explain their i n t r i n s i c effects on myocardial c o n t r a c t i l i t y , on glycogen phosphorylase and t h e i r a b i l i t y to potentiate the catecholamine-induced cardiac responses since calcium has a well-established role i n each process. Whether phosphodiesterase i n h i b i t i o n i s causally related to the calcium effects remains to be determined.  4.  Cardiac actions of glucagon Glucagon was  f i r s t observed to e l i c i t changes i n myocardial function  by Farah and Tuttle i n 1960.  In several species-  glucagon produced p o s i t i v e  inotropic and chronotropic effects which were not altered by reserpine pretreatment or i n s u l i n administration.  However, the 3-receptor blocking  agent dichloroisoproterenol prevented the glucagon-induced changes. led  This  to the conclusion that glucagon was acting on the catecholamine receptor. Farah and Tuttle (1960) noted some differences between the re-  sponses of glucagon and epinephrine.  The glucagon effect took longer to  develop and had a longer duration. Also, glucagon showed the phenomenon of tachyphylaxis' i n that repeated doses gave a reduced response. 1  In  contrast, the f i f t h dose of epinephrine increased the force of contraction as much as the f i r s t dose. Of the many species and tissue myocardial preparations studies only three were i n s e n s i t i v e to the polypeptide hormone.  These were the intact  anaesthetized dog, the Langendorff rabbit heart and isolated rabbit a t r i a . Glucagon was active i n guinea p i g a t r i a .  Thus, the preliminary experiments  of Farah and Tuttle revealed fundamental properties of the glucagon cardiotonic action. In contrast to the observations of Farah and Tuttle (1960), other investigators have demonstrated the p o s i t i v e inotropic e f f e c t i n intact dog after intravenous glucagon administration (Glick et al.,1968; Lucchesi 1968). the  In s i t u preparations where the drug has been d i r e c t l y infused into  heart (Regan et a l . , 1964; Afonso et a l . , 1972.; Hammer et a l . , 1973)  have yielded q u a l i t a t i v e l y s i m i l a r results.  A l l . i n s i t u experiments have  produced data to suggest a direct inotropic action rather than a secondary result o f a cardiovascular a l t e r a t i o n .  Reserpinization does not a l t e r the  responses to glucagon (Glick et a l . , 1968; Lucchesi, 1968; Hammer et a l . , 1973) and therefore an action through endogenous catecholamine release may be ruled out. The Vg-^adrenergic receptor antagonist propranolol does not i n t e r f e r e with the inotropic action of glucagon i n concentrations blocking the catecholamine response (Glick et al.,1968; LaRaia et a l , . 1968; Lucchesi,.1968; Spilker, 1970; Hammer et a l . , 1973).  Consequently, the theory that glucagon  acts at the .'Yg -receptor has now been, abandoned (Glick et a l . , 1968; Lucchesi, 1968).  A s t r u c t u r a l analog of propranolol. 'profitthalol, was also shown to  be without e f f e c t on glucagon-induced inotropism (Mayer et al.,1970).  The influence of glucagon on parameters  of an i n d i v i d u a l contracture  has been studied using i s o l a t e d cat and dog p a p i l l a r y muscle and a t r i a . Glucagon augments maximum developed tension i n a dose-dependent manner (Glick et a l . ,  1968; Gold et a l . ,  1970; Nayler et a l . ,  1970; Spilker, 1970).  The drug also increases rate of force development although time to peak tension i s not altered (Glick et a l . ,  1968; Gold et a l . ,  1970;Spilker, 1970).  The catecholamines decrease time to peak, tension (Skelton et a l . , 1970; Spilker, 1970) but otherwise behave l i k e glucagon. Force of contraction may be influenced by i n t e r v a l between beats (Koch-Weser and Blinks, 1963).  Spilker (1970) investigated the f o r c e - f r e -  quency relationship i n i s o l a t e d guinea p i g a t r i a before'and a f t e r glucagon exposure.  In a control s i t u a t i o n , there i s a gradual decrease i n force  associated with increases i n frequency up to 20/minute.  At greater stim-  ulation rates up to approximately 180/minute, force increases.  In the  presence of glucagon, frequency-force behaviour was not altered up to 20/ minute.  However a t greater frequencies the curve was s h i f t e d up i n a para-  l l e l manner.  Norepinephrine changed the shape of the curve and increased  the force r e l a t i v e to control at a l l stimulation frequencies.  The inotropic  effect of glucagon i s not secondary to the e f f e c t on heart rate because i n preparations maintained at a constant frequency glucagon s t i l l increases contractile force (Glick et a l . , Nayler et a l . ,  1968; Lucchesi, 1968; Gold et a l . , 1970;  1970; Spilker, 1970; Marcus et a l . ,  1971;Henry e t _ a l . , 1975).  Different i o n i c environments ..-may influence the action of glucagon but r e l a t i v e l y few experiments have been done to investigate t h i s .  Manganese  is believed to i n t e r f e r e with the i n f l u x of calcium accompanying excitation (Sabatini-Smith and Holland, 1969).  Mn^? e i t h e r reduced or abolished the  glucagon positive inotropic effect (Nayler et a l . ,  1970). Mn ^ also s h i f t e d +  22 the dose-response curve for glucagon to the right (Spilker, 1970). Visscher and Lee (1972) examined the association of extrac e l l u l a r calcium concentration with the force changes induced by glucagon. The lower the e x t r a c e l l u l a r calcium concentration, the greater the increase i n force.  In a 0.09  mM  calcium medium, glucagon maintained  response.  However the hormone was  the mechanical  i n e f f e c t i v e i n a calcium-free medium.  Electrophysiologic measurements i n myocardial  c e l l s exposed to  glucagon suggest the pancreatic hormone has l i t t l e influence on the excitatory phase.  Spilker (1970) observed a s l i g h t prolongation of the action  potential plateau but resting p o t e n t i a l , action p o t e n t i a l amplitude and maximum rate of depolarization were unchanged.  In contrast, Prasad  noticed a shortening of the action p o t e n t i a l , and this was an increase i n c o n t r a c t i l i t y . d i c t i o n for Prasad  A species v a r i a t i o n may  (1975) used dog p a p i l l a r y muscle.  (1975)  associated with  explain the contraPrasad was  unable to  detect any a l t e r a t i o n of action p o t e n t i a l features i n guinea pig p a p i l l a r y muscle, the preparation employed by Spilker (1970) , even though s i m i l a r concentrations of glucagon were investigated.  It i s noteworthy that Spilker  considered the.slight prolongation of the plateau unimportant because action potential durations with norepinephrine  or ouabain could vary with exper-  imental conditions. The amplitude of the plateau phase i s depressed i n c a l f and sheep Purkinje fibres bathed i n a low (0.45 mM) Under conditions where both norepinephrine  calcium medium (Spilker, 1970). and calcium elevated the  plateau p o t e n t i a l glucagon had no e f f e c t . Experiments i n isolated guinea pig hearts depolarized with high potassium (Watanabe and Besch, 1974;  Schneider and Sperelakis, 1975)  strated that glucagon, unlike the catecholamines,  was  demon-  unable to restore  23  e x c i t a b i l i t y and contractions.  Similar observations were made i n tetrodo-  toxin-treated hearts (Watanabe and Besch, 1974). Glucagon w i l l stimulate glycogenolysis i n myocardium.  The spectrum  of metabolic events closely resembles that of epinephrine (Kreisberg and Williamson, 1964) .  In isolated rat heart (Comblath e t _ a l . , 1963; Mayer  et a l . , 1970) and i n s i t u rat heart (Williams and Mayer, 1966), i n i t i a t e s glycogen breakdown by activating phosphorylase the adenylate cyclase pathway.  Like catecholamine-induced  glucagon  apparently  through  a c t i v i t y , the  glucagon response depends on calcium a v a i l a b i l i t y (Mayer et a l . , 1970). Much experimental e f f o r t has focused on the role of c y c l i c AMP i n the glucagon cardiac responses.  I n i t i a l l y no change i n c y c l i c AMP  concentration could be detected i n intact rat heart challenged with although the inotropic response was e l i c i t e d  (LaRaia et a l . , 1968).  glucagon, However  subsequent investigators (Mayer et a l . , 1970; Oye and Langslet, 1972; Henry et a l . , 1975) have been successful i n demonstrating increase i n c e l l u l a r c y c l i c nucleotide l e v e l s .  a glucagon-stimulated  In contrast to the temporal  sequence of events following catecholamines, neither Mayer's group (1970) nor Oye and Langslet (1972) detected the change p r i o r to the increase i n c o n t r a c t i l e force. Demonstration of a glucagon-sensitive adenylate cyclase i n v i t r o was accomplished Rat  long before elevated levels were discovered i n i n t a c t tissue.  (Murad and Vaughan, 1969; Henry et a l . , 1975), cat and human heart  (Levey and Epstein, 1969) preparations possess  glucagon-stimulatable  cyclases which are not blocked by concentrations of propranolol e f f e c t i v e against catecholamines.  Further evidence i n support of a c y c l i c AMP involve-  ment comes from experiments i n f a i l i n g hearts where glucagon was i n e f f e c t i v e both i n increasing c o n t r a c t i l i t y and i n stimulating adenylate cyclase i n  v i t r o (Gold et a l . , 1970).  Apparently  guinea pigs do not have a glucagon-  responsive cyclase system, although glucagon can e l i c i t changes i n force i n this species (Henry et a l . , 1975). There have been many attempts to associate adenylate a c t i v i t y with calcium homeostasis.  cyclase  Entman and coworkers (1969) found a  glucagon-sensitive cyclase i n a microsomal preparation' which also increased calcium uptake i n the presence of glucagon.  However this paper does not  present data on the purity of the preparation. et a l .  The experiments of Nayler  (1970) suggested that handling of calcium by sarcoplasmic  reticulum  or mitochondria was not influenced by glucagon, although calcium exchange across the sarcolemma was altered. 1972)  Yet another study (Visscher and Lee,  indicated that, while glucagon may influence calcium f l u x rates,  there i s no i n f l u x of calcium under conditions when the inotropic action i s marked and hence these authors proposed some e f f e c t of glucagon on i n t r a c e l l u l a r calcium stores. C y c l i c AMP may a l t e r the membrane permeability to calcium (Watanabe and Besch, 1974).  Glucagon neither restored electromechanical  activity  (Watanabe and Besch, 1974; Schneider and Sperelakis, 1975) nor increased i n t r a c e l l u l a r c y c l i c AMP levels (Watanabe and Besch, 1974) i n guinea p i g hearts depolarized with high potassium.  This i s consistent with the ob-  servations of Henry et a l . (1975)indicating lack of a glucagon-sensitive cyclase i n this species.. Unfortunately, the a b i l i t y of glucagon to restore e x c i t a b i l i t y has not been investigated i n species possessing a glucagonstimulatable enzyme (e.g.rat)'. Another mechanism by which glucagon could a l t e r force i s through i n h i b i t i o n of sarcolemmal N a - K +  +  -ATPase.  Prasad (1975) presented  evidence that changes i n force accompanied glucagon i n h i b i t i o n of this  25 enzyme i n dog p a p i l l a r y muscle.  Consistent with this proposal was the lack  of c o n t r a c t i l e event on one hand, and lack of enzyme i n h i b i t i o n on the other hand i n guinea p i g , rabbit and p i g . The a b i l i t y o f phosphodiesterase i n h i b i t o r s to enhance the e f f e c t s of glucagon on myocardium would provide further support for an involvement of c y c l i c AMP.  However data obtained with these agents cannot be interpreted  e a s i l y because they i n t e r f e r e with other c e l l u l a r processes. Theophylline greatly enhanced the inotropic action of glucagon on isolated cat p a p i l l a r y muscle (Marcus et a l . , 1971).  Afonso and asso-  ciates (1972) found q u a l i t a t i v e l y s i m i l a r alterations i n force of contraction monitored following concurrent administration of glucagon and aminop h y l l i n e to i n s i t u dog heart.  Apparently i n contradiction to these r e s u l t s ,  Lucchesi (1968) found that pre-infusion with theophylline prevented the glucagon inotropic response i n i n s i t u canine heart.  However, glucagon was  administered during the period of maximum inotropic response to theophylline. Marcus et a l .  (1971), i n contrast, used a methylxanthine concentration with  minimal i n t r i n s i c actions.  Antonaccio and Lucchesi (1970) found the ino-  tropic actions of glucagon with low concentrations of theophylline were additive i n isolated dog p a p i l l a r y muscle. reduced the response to glucagon.  A higher concentration actually  McNeill et a l . . (1969) noted a s i m i l a r  influence on the norepinephrine response i n i n s i t u rat heart.  Therefore  studies on the i n t e r a c t i o n of glucagon with methylxanthines have not yet conclusively s a t i s f i e d this c r i t e r i o n implicating c y c l i c AMP involvement. By v i r t u e of the q u a l i t a t i v e s i m i l a r i t y of t h e i r myocardial actions, many authors have suggested that a common mechanism of action f o r the catecholamines and glucagon might be through the adenylate cyclasec y c l i c AMP system.  Although numerous studies have been undertaken to es-  t a b l i s h a role f o r c y c l i c AMP  i n the catecholamine responses, r e l a t i v e l y  few studies have been conducted with glucagon.  The present i n v e s t i g a t i o n  therefore undertook to characterize the myocardial actions of glucagon, by means of complete time-response and dose-response experiments, and to correlate the results of studies on the glucagon-induced p o s i t i v e i n o t r o p i c e f f e c t and phosphorylase a c t i v a t i o n with' the action of glucagon on intact tissue c y c l i c AMP  accumulation.  In addition, the influence of theophylline  on the dose and time dependent behaviour of glucagon i n e l i c i t i n g i t s cardiac responses was also examined i n order to further elucidate the mechanism of action of the polypeptide hormone.  27 MATERIALS AND METHODS  1.  MATERIALS The c y c l i c AMP assay k i t was obtained from Amersham-Searle Ltd. (Oak-  v i l l e , Ont.).  A l l k i t reagents were reconstituted with d i s t i l l e d , deionR  ized water.  PCS  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 was also obtained from  Amersham-Searle. Drugs used:  Theophylline, norepinephrine, DL-propranolol HC1, and  heparin sodium (Sigma Chem. Corp.); and glucagon ( E l i L i l l y & Co.). Glucagon was reconstituted from the commercially available preparation with 1 mM T r i s (Sigma Chem. Corp.), pH 8.5 - 9.0, and stored as a 0.5 mg/ml stock solution, at 15 °C. Just prior to use, the stock solution was diluted i n oxygenated CK solution.  The i n j e c t i o n volume was maintained at 0.25 ml  throughout the study. Norepinephrine was prepared immediately prior to use i n CK solution and injected i n a volume of 0.25 ml. Theophylline and propranolol were dissolved i n CK solution and added to the reservoir of the perfusion medium. for  each  2.  METHODS  A.  Heart perfusion  Fresh solutions were prepared  experiment.  Female Wistar rats weighing 200 to 250 g- were used throughout the study.  They received food and water ad libitum.  Heparin sodium (500 U.S.P.  units/kg) was administered subcutaneously sixty minutes p r i o r to s a c r i f i c e . Animals were stunned by a blow to the head.  Hearts were r a p i d l y excised  and perfused by the Langendorff technique with Chenoweth-Koelle solution (CK) (Chenoweth and Koelle, 1946) at 37 °C. The perfusion medium contained  28 (in mM) NaCl 119; KC1 5.6; C a C l  2  1.8; MgCl  2  2.1; NaHCC> 19.1; dextrose 10; 3  and was aerated with 95% 0^ - 5% CO2 to f i r s t adjust and then maintain the pH at 7.4. A perfusion rate of 2.8 ml/min was maintained using either a Holter microinfusion r o l l e r pump (Extra-Corporeal Medical S p e c i a l t i e s , Model RL 175) or a Buchler P o l y s t a l t i c Pump (Buchler Instruments, Model 2-6100).  C o n t r a c t i l e force was monitored with a Palmer c l i p placed i n  the apex of the v e n t r i c l e and connected to a force-displacement (Grass Instruments, Polygraph).  Inc.,  transducer  Model FT30C) and recorder (Grass Instruments, Model 5D  D i a s t o l i c tension was adjusted to 1 g  and the heart allowed to  s t a b i l i z e for 10 minutes before the s t a r t of each experiment.  Drugs were  injected v i a a side arm cannula or, a l t e r n a t i v e l y , dissolved i n CK solution and perfused through the heart. Contractile force was determined by comparing the s y s t o l i c tensions before and a f t e r drug exposure, and i s expressed both as an absolute change i n tension and as a percent over p r e - i n j e c t i o n l e v e l ^  e  s y s t o l i c tension a f t e r drug s y s t o l i c tension before drug  x  ^OQ^  Time-Response Experiments After s t a b i l i z a t i o n , glucagon ( 2yg i n 0.25 ml) was injected v i a a side arm cannula.  At selected time intervals hearts were frozen instantaneously  with Wollenberger tongs (Wollenberger  et a l . , I960) previously c h i l l e d i n  2-methylbutane cooled i n an alcohol-Dry Ice mixture.  Control hearts received  a 0.25 ml i n j e c t i o n of CK solution and were frozen at 0 seconds. were stored at - 80 °C u n t i l  A l l hearts  assayed.  In some experiments theophylline was added to the CK buffer i n a f i n a l concentration of 1 mM and perfused through the apparatus f o r 15 minutes following the i n i t i a l s t a b i l i z a t i o n period.  Hearts were thus exposed to  29  theophylline f o r approximately 8 - 1 0 minutes a f t e r which 2 yg was injected.  Control hearts received 0 . 2 5 ml CK solution.  frozen as described above.  of glucagon  Hearts were  The exposure time to theophylline was estimated  by perfusing normal saline through the apparatus and measuring the time r e quired f o r a p r e c i p i t a t e to form i n a beaker containing AgNO^ solution. Dose-Response Experiments After s t a b i l i z i n g , hearts were injected with appropriate doses of glucagon and frozen at 5 0 sec. This time, established from the time-response experiments, was when peak a c t i v a t i o n of phosphorylase and peak accumulation of c y c l i c AMP occurred.  Each heart received only one dose of glucagon.  Control hearts were injected with 0 . 2 5 ml of CK solution and frozen at 5 0 sec. In some experiments theophylline ( 1 mM f i n a l concentration ) was perfused through the apparatus f o r 1 5 minutes  (following the s t a b i l i z a t i o n  period) and then appropriate doses of glucagon injected.  Hearts were frozen  at 5 0 sec. Again only one i n j e c t i o n of glucagon was given per heart. Other experiments were conducted to investigate the interaction..of glucagon with propranolol.  Some hearts were perfused with CK buffer f o r 3 0  minutes after which they were injected with either glucagon or 0 . 2 5 ml of CK. Hearts were frozen at 5 0 sec. Some hearts were allowed to s t a b i l i z e on CK solution f o r 1 0 minutes.  They were then perfused with propranolol i n a f i n a l '  —8  concentration of 1 0  M f o r 2 0 minutes  (exposure time to propranolol approx-  imately 1 3 - 1 5 minutes) and. then injected with glucagon (or 0 . 2 5 ml CK) and frozen at 5 0 sec. An experiment was also conducted to establish the e f f i c a c y of t h i s concentration of propranolol i n blocking the catecholamine response. The increase i n c o n t r a c t i l e force was measured f i r s t i n CK-perfused hearts f o l lowing injections of norepinephrine through the side arm cannula.  Systolic  30 tension was allowed to return to p r e - i n j e c t i o n l e v e l before the next dose was administered.  After the l a s t dose, the experiment was repeated i n the  —8 presence of 10  M propranolol which had been exposed to the heart f o r  approximately 15 minutes prior to norepinephrine challenge. B.  Phosphorylase assay Phosphorylase a c t i v i t y was determined i n the d i r e c t i o n of glycogen  synthesis by a modification of the method of Cori and C o r i (1940), described previously by McNeill and Brody  (1966).  D e t a i l s are given below.  From each heart a 70-100 mg frozen sample from the apex was homogenized i n 200 volumes of a solution containing 0.05 M T r i s (pH 6.8-6.9), 0.001 M EDTA, 0.02 M NaF, and 0.3 % bovine serum albumin.  Homogenization  was accomplished using a Polytron (Brinkman Instruments, Ltd., Model PT 10203500) a t a rheostat setting of 6 for 5 seconds and performed a t 0-4 °C. Following centrifugation of the tissue homogenate at 7,500 x g (0 °C) for 10 minutes, a 0.2 ml aliquot of the supernatant was incubated for 15 minutes at 37 °C.  The reaction mixture consisted of the above-described homogen-  i z i n g medium plus 0.4 % glycogen and 0.01 M G-l-P i n a f i n a l volume of 1.0 ml. A duplicate supernatant sample was incubated with the same reaction mixture containing, i n addition, 5'-AMP i n a f i n a l concentration of 0.001 M. The :  reaction was terminated by adding 2.0 ml of 10% w/v TCA.  To serve as zero  time controls 2.0 ml of 10 % TCA were also added to one tube containing the T r i s buffer-AMP mixture a f t e r which 0.2 ml of enzyme was added. c i p i t a t e d protein was pelleted by centrifugation for 10 minutes  Pre-  (room temp-  erature) at 1500 R.P.M. i n an International Centrifuge (Model EXD).  The  supernatant was decanted o f f and assayed f o r inorganic phosphate by the method of Fiske and SubbaRow (1925).  Spectrophotometric measurements were  performed on a Coleman Junior Spectrophotometer  (Model 6C) at 660  my.  The rate of l i b e r a t i o n of inorganic phosphate was l i n e a r over the time studied and was proportional to the enzyme concentration. Total phosphorylase was represented by the amount of inorganic phosphate released i n the presence of 5' AMP,  while active phosphorylase (phosphorylase a) was  represented by the amount released i n the absence of 5' AMP.  Since t o t a l  enzyme a c t i v i t y did not change with drug treatment, the r e s u l t s are expressed as percent phosphorylase a_ which i s : enzyme a c t i v i t y without AMP enzyme a c t i v i t y with AMP  C.  C y c l i c AMP  I.  Tissue extraction  ^  j  assay  The method of extraction was modified from that of Gilman (1970) as follows: A 70-100 mg frozen sample from the apex of the v e n t r i c l e was homogenized i n 5 ml of 5 % w/v TCA  rapidly  (4 °C) using a Kontes Dupour ground glass  tube and pestle driven at maximum speed by a Fisher Dyna-Mix motor (Model 143).  After 20 passes the homogenate was allowed to s e t t l e for 30-60 sec-  onds and subjected to 5 more passes.  The homogenate was then centrifuged  at 8,000 x g (0 °C) for 10 minutes and the supernatant decanted into a test tube containing 0.5 ml of 1 N HC1.  TCA was removed from the supernatant  by extracting f i v e times with water'-rsaturated ether (10 ml each time). Following the f i n a l extraction step, r e s i d u a l ether was removed i n a stream of-nitrogen.  The aqueous extract was l y o p h i l i z e d overnight i n a V i r t i s  Freeze-Dryer (Model 10-100 Unitrap).  The dried samplecwas reconstituted  with 1.0 ml of cold 0.05 M Tris-0.004 M EDTA buffer (pH 7.5) and assayed within 2 hours.  32 II.  C y c l i c AMP  binding reaction  An Amersham-Searle C y c l i c AMP tissue concentration (Gilman, 1970)  of c y c l i c AMP  Assay K i t was  used to determine the  by a modification of the Gilman method  as described below.  The binding reaction was  c a r r i e d out at 0-4  °C i n a t o t a l volume of  200 y l consisting of: 50 y l of sample extract or known amount (0.25-8 pmol) of unlabelled c y c l i c AMP 3 50 y l of  H-cyclic AMP  (9 pmol; s p e c i f i c a c t i v i t y 20.8  c/mmol)  100 y l of binding protein ( s u f f i c i e n t to bind 30% - 50% of the l a b e l l e d nucleotide) A l l reagents were reconstituted with d i s t i l l e d water and buffered at pH 7.5 by 0.05  M Tris-0.004 M EDTA incorporated  manufacturer.  i n the reagent v i a l s by  Each unknown sample or known standard was  The reaction was  the  run i n duplicate.  started by the addition of the binding protein and  reached equilibrium a f t e r 30 minutes at 2-4 period of two hours the binding reaction was  °C.  After a minimum incubation  terminated by the addition of  100 y l of charcoal suspension supplied by the manufacturer.  Within 3  minutes of addition, the tubes were centrifuged at 4 °C i n an International Centrifuge  (Benchtop, Model HN)  at f u l l speed for 15 minutes.  natant was  decanted into 10 ml of Amersham-Searle PCS  cocktail.  Radioactivity was  liquid  The  super-  scintillation  counted i n a Nuclear-Chicago Isocap 300 counter  for 10 minutes or for a time s u f f i c i e n t to accumulate 200,000 counts (counting error 0.5  %) whichever was  less.  With each assay of unknowns, a standard curve was A" charcoal blank was  run simultaneously.  run i n duplicate to determine the amount of unbound  r a d i o a c t i v i t y not pulled by the charcoal during centrifugation. contained  150 y l of 0.05  This blank 3 M Tris-0.004 M EDTA buffer plus 50 y l of H-  33 c y c l i c AMP and was handled i n the same manner as the other incubation tubes. To determine the t o t a l amount of r a d i o a c t i v i t y added, two tubes containing 3  150 u l of T r i s buffer plus 50 u l of H-cyclic AMP were also incubated. Instead of adding charcoal, 100 \il of k i t buffer was added and the tubes centrifuged with the other samples. III.  Calculation of r e s u l t s The counting e f f i c i e n c y was determined to vary minimally (29.9% to  32.2%) by means of an external standard channels r a t i o c a l c u l a t i o n .  Since  the e f f i c i e n c y was reasonably consistent for a l l v i a l s , correction f o r quench was unnecessary (see Appendix).  The counts per minute i n each set  of duplicates were averaged and then the average cpm i n the charcoal blank was subtracted. The % bound a c t i v i t y was then determined by the following formula: average sample cpm - average blank counts average t o t a l r a d i o a c t i v i t y added  ^  ^  A standard curve was obtained by p l o t t i n g % bound r a d i o a c t i v i t y against the log of pmol of unlabelled c y c l i c AMP added per tube. i n the range of 0.25 pmol to 8 pmol.  The curve was l i n e a r  A straight l i n e was computed from the  experimental data by the method of least squares on a Wang 600 Programmable calculator.  The c o r r e l a t i o n c o e f f i c i e n t was always between -.990 and -1.000.  The amount of c y c l i c AMP i n each unknown was determined by computer from the standard curve. tissue weight.  This figure was corrected for d i l u t i o n and o r i g i n a l  The two samples extracted from each heart were pooled after  correction for o r i g i n a l tissue weight.  D.  STATISTICAL METHODS S t a t i s t i c a l analysis was performed using the Student * t test for 1  paired and unpaired data.  A difference of p < 0.05 was considered to be  34.  statistically significant. as the mean + one S.E.M.  Data i n tables, figures and text are presented  35 RESULTS 1.  Experiments i n buffer-perfused hearts Injection of glucagon into the isolated perfused r a t heart produced  an increase i n c o n t r a c t i l e force which was  time and dose dependent.  Figure 4 shows the time course of the p o s i t i v e inotropic effect a f t e r yg of glucagon.  2.0  With this dose, s y s t o l i c tension f i r s t changed s i g n i f i -  cantly (p < .05) 25 seconds following drug administration (Table 1). t h i s time, s y s t o l i c tension was l e v e l of 3.7 + 0.1 g.  At  27.6 +_ 2.7 % greater than the p r e - i n j e c t i o n  C o n t r a c t i l e force was maximally increased at 35  seconds when the % increase was  60.1 + 4.7  absolute tension change of 2.3+0.2 g.  (Table 1), corresponding  However there was  to an  individual  v a r i a t i o n i n the time when peak tension occurred, ranging between 30 and 50 seconds.  Glucagon had a long duration of action, for s y s t o l i c tension  had not returned to p r e - i n j e c t i o n l e v e l a f t e r 120 seconds (Figures 4, 5; Table 1). The glucagon-induced p o s i t i v e inotropism was i l l u s t r a t e d i n Figure 6.  The maximum increase i n force of 77.1+7.9 %  was obtained a f t e r 4.0 yg of glucagon (Table 2). the absolute increase i n s y s t o l i c tension was Glucagon 8.0  dose-dependent, as  yg caused an 82.7  +18.9  At this concentration,  3.1 +0.4  g (Table 2).  % increase over p r e - i n j e c t i o n l e v e l  but this did not d i f f e r s i g n i f i c a n t l y (p > .05) from the response due to 4.0 yg.  The minimum dose capable of producing a s i g n i f i c a n t p o s i t i v e  inotropic effect was  0.5 yg (Table 2).  In order to eliminate the p o s s i b i l i t y that endogenous catecholamine release was study was  responsible for the observed glucagon cardiotonic action, a  carried, out with propranolol i n which 2 dose l e v e l s of glucagon  were tested.  In buffer-perfused hearts, 1.0 "'and 2j0 yg doses of glucagon  36 •  FIGURE 4.  Effect of glucagon (2 yg) on c y c l i c AMP content, c o n t r a c t i l e force and percentage phosphorylase a at various times following i n j e c t i o n into r a t , hearts perfused with buffer or buffer plus theophylline. Points plotted at 0 sec. represent the mean + S.E. of hearts injected with 0.25 ml CK. Data points representing only one determination are indicated by (1). Error bars are not shown f o r points where the S.E.M. was less than the symbol s i z e . Cyclic AMP was s i g n i f i c a n t l y elevated at 15 sec., contracti l e force at 25 sec. , and % phosphorylase a at 30 sec. i n buffer-perfused hearts. In hearts perfused with 1 mM theophylline f o r 15 minutes, c y c l i c AMP and % active phosphorylase were s i g n i f i c a n t l y elevated at 20 sec., and c o n t r a c t i l e force at 25 sec.  FIGURE  4  38  FIGURE 5.  E f f e c t of time on the absolute change i n tension following i n j e c t i o n of 2 yg glucagon into the i s o l a t e d buffer-perfused and theophylline-perfused rat heart. D i a s t o l i c tension was adjusted to 1 g. Each point represents the mean + 1 S.E.M. of at least 8 determinations with the exception of the value at 120 sec. This i s the average of 2 hearts. The change i n syst o l i c tension was s i g n i f i c a n t l y elevated at 25 sec. 1 mM theophylline enhanced the change at 50 sec. only.  39  FIGURE  5  TIME  (sec)  40 TABLE 1 The effect of time on the p o s i t i v e i n o t r o p i c response to 2 yg glucagon i n the buffer-perfused and theophylline- perfused rat heart. Time a f t e r injection (sec)  Percent Change i n S y s t o l i c Tension Bufferperfused  Absolute Change i n S y s t o l i c Tension  Theophyllineperfused  Theophyllineperfused  Bufferperfused  0  3.7 +  0.1  a  3.6 +  0.2  b  (32)  (35) 1.2 + 0.1 (35)  (g)  1.1 + 0.1 (32)  25  27.6 + 2.7* (35)  32.8 + 2.7 (32)  30  47.4 + 3.5 (35)  57.7 + 3.4' (32)  1.8 + 0.2 (35)  1.9 +0.1 (32)  35  60.1 + 4.7 (27)  70.6 ± 5.2 «26)  2.3 + 0.2 (27)  2.3+0.2 (26)  40  58.4  67.1 + 5.2 (25)  2.2 + 0.2 (24)  2.2 + 0.2 (25)  50  46.1 +4.2 (16)  69.9 + 5.9 (15)  1.6 + 0.1 (16)  2.4 + 0.2° (15)  60  39.1 +4.8 (9)  58.7 + (8)  1.4 + 0.2 (9)  1.9 + 0.3 (8)  120  22.5 + 3.4 (2)  +5.0 (24)  C  C  9.3  +  1.1 + 0.3 (2)  •'* Mean % increase i n s y s t o l i c tension over p r e - i n j e c t i o n l e v e l + one S.E.M. t Mean absolute increase i n s y s t o l i c tension over p r e - i n j e c t i o n l e v e l +S.E.M. a Mean s y s t o l i c tension (g) + one S.E.M. a f t e r a 10 minute CK perfusion b Mean s y s t o l i c tension (g) + one S.E.M. a f t e r a 10 minute CK perfusion followed by a 15 minute theophylline 1 mM perfusion c S i g n i f i c a n t l y enhanced over corresponding  buffer perfused value  (p<  .05)  D i a s t o l i c tension was adjusted to 1 g. Numbers i n parentheses indicate the number of animals. In the theophylline experiments, mean s y s t o l i c tension after CK perfusion but before theophylline exposure was 3.6 + .0.2 g. (N=32).  41  FIGURE  6.  The effect of various doses of glucagon on cardiac c y c l i c AMP content, c o n t r a c t i l e force and percentage phosphorylase a_ i n r a t hearts perfused with buffer or buffer plus theophylline (1 mM). C y c l i c AMP and % phosphorylase a_ were measured 50 sec. a f t e r glucagon i n j e c t i o n . The % increase i n s y s t o l i c tension was determined at the time of maximum change r e l a t i v e to p r e - i n j e c t i o n l e v e l . Data points representing only one measurement are indicated by (1). Other data points are the mean + one S.E. M. of N determinations. No error bars are shown f o r points where the S.E. i s smaller than the symbol s i z e . In buffer-perfused hearts, c y c l i c AMP was s i g n i f i c a n t l y increased with 0.25 yg glucagon whereas the c o n t r a c t i l e force and phosphorylase a_ were increased a f t e r 0.5 yg. In 1 mM theophylline-perfused hearts, c y c l i c AMP and c o n t r a c t i l e force were increased at 0.25 yg and % phosphorylase a_ was increased at 0.5 yg glucagon.  FIGURE  6  43  FIGURE  7.  The effect of various doses of glucagon on the absolute change i n s y s t o l i c tension i n bufferperfused and theophylline-perfused rat hear6s. D i a s t o l i c tension was adjusted to 1 g. Each point represents the mean + one S.E.M. of at least 4 determinations, unless otherwise indicated. Theophylline 1 mM lowered the minimum e f f e c t i v e dose of glucagon from 0.5 to 0.25 yg. Only the 0.25 and 2.0 yg points were s i g n i f i c a n t l y enhanced over buffer-perfused values.  FIGURE  7  45 TABLE 2  The effect of various doses of glucagon on c o n t r a c t i l e force i n the i s o l a t e d buffer-perfused and theophylline-perfused rat heart. Dose of Glucagon (yg)  Percent Change i n S y s t o l i c Tension  Absolute Change i n S y s t o l i c Tension  Bufferperfused  Theophyllineperfused  Bufferperfused  0  -6.4 + 3.2* C33)  0  0.25  2.6 + 1.4 (6)  28.4 + 5.5 ' (4)  0.5  17.0 + 3.9 (12)  a  36.3 + 5.1 (5)  1.0  52.6 + 4.6 (8)  a  76.3 + 8.2a> (6)  2.0  57.2 + 4.4 (20)  a  4.0  77.1 + 7Z9 (6)  8.0  82.7 + 18.9 (4)  a  Theophyllineperfused -0.2 + 0.09* (33)  0.1 + 0.1 (6)  b  0.9 + 0.2 (10)  (g)  1.2 + 0.2 ' (4) a  1.3 + 0.3 (5)  a  b  a  b  84.4 + 7.7a,b (11)  a  a  116.3  + 7.4 » (5)  103.9  + 50.6 (2)  a  a  b  2.4 + 0.2a (8)  2.5 + 0.3 (6)  2.1 + 0.2 (20)  a  3.1 + 0 . 3 » (11)  3.1 + 0.4 (6)  a  3.5 + 0.4 (5)  a  3.4 +• 1.7 (2)  a  3.0 + 1.0 (4)  a  a  a  *  Change i n s y s t o l i c tension r e l a t i v e to the pre-theophylline l e v e l .  a  S i g n i f i c a n t l y greater (p < .05; paired "t") than pre-glucagon s y s t o l i c tension  b  S i g n i f i c a n t l y enhanced (p < .05; paired "t") over corresponding b u f f e r perfused value  b  D i a s t o l i c tension was adjusted to 1 g. Numbers i n parentheses indicate the number of animals. Hearts were perfused with CK for 10 min., or a l t e r n a t i v e l y perfused an additional 15 minutes with 1 mM theophylline a f t e r which glucagon was injected v i a a side-arm cannula. S y s t o l i c tension was measured when the change was maximal r e l a t i v e to p r e - i n j e c t i o n l e v e l . S y s t o l i c tension was 4.1+0.1 g (N=6) after a 10 minute CK perfusion. In theophylline experiments, s y s t o l i c tension was 3.5 + 0.1 g (N=33) before theophylline exposure and 3.3+0.1 g (N=33) a f t e r 15 minutes.  46  produced peak s y s t o l i c tension elevations of 34.0+11.7 and 61.0 + 7.4 % —8 respectively (Table 3).  In hearts exposed to 10  M propranolol, mean  increases of 32.7 + 10.1 and 59.2 + 9.8 % were observed a f t e r 1.0 and 2.0 yg doses.  These changes i n force did not d i f f e r s i g n i f i c a n t l y from those i n  the buffer-perfused hearts (Figure 8; Table 3).  Perfusion with propranolol  lowered the mean s y s t o l i c tension before glucagon i n j e c t i o n from 5.5 + 0.5 g (CK hearts) to 4.8 + 0.3 g but t h i s difference was n o t ^ s t a t i s t i c a l l y s i g n i f icant.  Figure 9 and Table 4 present data that v e r i f i e s that the concentration —8  of propranolol employed (10  M) was s u f f i c i e n t to reduce the c o n t r a c t i l e  response to norepinephrine. In addition to the c o n t r a c t i l e e f f e c t , glucagon also increased glycogen phosphorylase a c t i v i t y .  The time course of a c t i v a t i o n was investigated with  2.0 yg glucagon, a dose producing submaximal changes i n force (see Figure 6). Phosphorylase a_ l e v e l s were s i g n i f i c a n t l y greater than control 30 seconds following polypeptide i n j e c t i o n (Figure 4; Table 5) and reached a maximum mean value at 60 seconds.  However, during the i n t e r v a l between 40 and 60  seconds there was no s i g n i f i c a n t difference i n a c t i v i t y .  The % phosphorylase  a_ was s t i l l s i g n i f i c a n t l y elevated over control at 120 seconds Table 5).  (Figure 4;  Prior to glucagon administration (time — 0 seconds) , the % phos-  phorylase a_ was 6.9 + 0.8 (N = 11). ]The dependence of phosphorylase a c t i v a t i o n on dose was examined by measuring phosphorylase a_ l e v e l s at 50 seconds, a time when a c t i v i t y was maximal (see Figure 4).  The control l e v e l of 5.3+1.0 % was obtained by  i n j e c t i n g 0.25 ml of CK buffer and measuring phosphorylase at 50 seconds. The minimum amount of glucagon capable of s i g n i f i c a n t l y elevating the % phosphorylase a_ above control was 0.5 yg (Figure 6; Table 6) which increased a c t i v i t y to 12.3 + 2.2 %.  The maximum a c t i v a t i n g dose was 4.0 yg glucagon,  47  TABLE  3  _g The influence of propranolol 10 M on the positive inotropic e f f e c t of glucagon i n the i s o l a t e d perfused rat heart. Dose of Glucagon  Percent Change i n S y s t o l i c Tension  Absolute Change i n S y s t o l i c Tension (g)  (yg)  Bufferperfused  Propranolol 10 °M  Bufferperfused  0  5.5+0.5*  1.0  34.0 + 11.7  32.7 +-10.1  2.0  61.0 +  59.2 +  7.4  NS  ,NS 9.8  Propranolol 10 °M  4.8+0.3-  2.2+1.1  1.8 + 0.6  NS  3.2 + 0.7  2.6 + 0.5  NS  ,NS  *  Mean s y s t o l i c tension + one S.E.M. after 30 min. CK perfusion i n 7 animals  +  Mean s y s t o l i c tension + one S.E.M. after 10 min. CK perfusion plus 20 min. propranolol perfusion i n 7 hearts  NS  Not s i g n i f i c a n t l y decreased from the corresponding value (p > .05; unpaired ' t ' test)  buffer-perfused  D i a s t o l i c tension was adjusted to 1 g. Hearts were perfused f o r 30 minutes with CK solutioiigOr a l t e r n a t i v e l y f o r 10 minutes with CK followed by 20 minutes with 10 M propranolol. Glucagon was injected v i a a side-arm cannula. Values i n table represent the mean + one S.E.M. of three hearts.  48  FIGURE 8.  The influence of propranolol 10~° M on glucagoninduced changes i n cardiac c y c l i c AMP content, contractile force and percentage phosphorylase a . Cyclic AMP and phosphorylase were measured 50 seconds after glucagon i n j e c t i o n . Contractile force i s presented as the percent increase i n s y s t o l i c tension r e l a t i v e to p r e - i n j e c t i o n l e v e l . The cont r o l value represents s y s t o l i c tension after exposure to propranolol as a percent of s y s t o l i c tension i n buffer-perfused hearts (N=7). Control values f o r phosphorylase and c y c l i c AMP are one measurement. Other points represent the mean +one S.E. of 3 hearts. Propranolol 10~ M did not s i g n i f i c a n t l y reduce any of the cardiac responses to glucagon. 8  FIGURE  8  50  TABLE  4  _g The influence of 10 M propranolol on the p o s i t i v e inotropic action of norepinephrine i n the isolated, perfused r a t heart.  Dose of , . Norepinephrine (us) 0.01  0.1  1.0  10.0  a.  Percent Increase i n S y s t o l i c Tension _„ Propranolol Buffer-perfused 13.3  0  (1)  (1)  74.7  16.6  (2)  (2)  94.7  30.7  (2)  (2)  121.4  44.3  (1)  (2)  r e l a t i v e to p r e - i n j e c t i o n s y s t o l i c tension  D i a s t o l i c tension was adjusted to 1 g. and hearts were perfused with CK solution f o r a s t a b i l i z a t i o n period of 10 minutes. injected i n a volume of 0.25 ml. —8 10  Norepinephrine was  Following the f i n a l dose, propranolol  M was perfused through the heart f o r 10 minutes and the dose-response  curve repeated.  The numbers i n parentheses indicate the number of animals.  51  FIGURE  9.  The influence of propranolol 10 M on the positive inotropic effect of norepinephrine. Contractile force i s expressed as the maximum percent increase i n s y s t o l i c tension r e l a t i v e to the prei n j e c t i o n l e v e l . Each value represents 1, or a l t e r natively, the average of 2 determinations, as indicated by the numbers oh each bar.  52  FIGURE  9  I 140J  DOSE  OF NOREPINEPHRINE (ug)  53 TABLE  5  The effect of time on glucagon-induced cardiac phosphorylase a c t i v a t i o n i n the buffer-perfused and theophylline-perfused isolated r a t heart. Time after injection (sec)  Buffer-perfused  0  Theophylline-perfused  6.9 + 0.8* (11)  10.4 + 1.9 (9)  15  6.0 + 1.4 (3)  12.9 + 3.7 (4)  20  10.8 + 2.8 (4)  25.8 + 5.9 (4)  25  9.2 + 1.5 (4)  30  19.1 + 2.8 (10)  a  29.1 + 2.7 ' (7)  40  32.1 + 4.2 (8)  a  45.1 + 5.1 (7)  50  31.3 + 2.9 (7)  a  52.4 + 4.2 ' (8)  c  60  33.1 + 4.9  a  56.6 + 4.7 ' (6)  C  120  16.9 + 0.3 (2)  a  significantly  30.9 + 2.9 '° (4) b  (6X  Mean % phosphorylase a  +  b  b  C  b  b  b  one S.E.M.  (p < -.05) greater than the buffer-perfused, 0 sec  b.  s i g n i f i c a n t l y greater than the theophylline-perfused, 0 sec value  c.  s i g n i f i c a n t l y enhanced over the corresponding  buffer-perfused value  CK (0.25 ml) or glucagon (2 yg) was injected v i a a side-arm cannula and hearts were frozed at the indicated times. Theophylline 1 mM was perfused through the apparatus f o r 15 minutes. Numbers i n parentheses refer to the number of animals.  TABLE  6  The effect of various doses of glucagon on cardiac glycogen phosphorylase a c t i v a t i o n i n the buffer-perfused and theophylline-perfused isolated r a t heart. Dose of Glucagon (yg)  Control  0.25  Buffer-perfused  Theophylline-perfused  5.3+1.0*  8.4 + 2.5  (9)  (5) 12.5 + 2.0  8.5 + 1.1  (6)  (7) 0.5  12.3 + 2.2  a  26.0 + 3.5 ' b  C  43.4 + 5 . 3  b , C  (7)  (8) 1.0  20.2 + 3.2  a  (8)  (8) 2.0  31.3 + 2.9  a  52.4 + 4.2 ' b  C  58.5 + 4 . 7  b , C  (8)  (7) 4.0  38.8 + 3.5  a  (5)  (6) 8.0  34.3 + 3.5  a  72.2 '  (3)  *  Mean % phosphorylase a_ +  b  C  (1)  one S.E.M.  a.  S i g n i f i c a n t l y (p < .05) greater than the buffer-perfused  control  b.  S i g n i f i c a n t l y greater than the theophylline-perfused  c.  S i g n i f i c a n t l y enhanced over the corresponding buffer-perfused value  control  CK (0.25 ml) or glucagon was injected v i a a side-arm cannula and hearts were frozen at 50 seconds. Theophylline 1 mM was perfused through the apparatus for 15 minutes. Numbers i n parentheses refer to the number of animals.  55  which caused an increase i n a c t i v i t y to 38.8 + 3.5 % a_. Therefore the isinimum and maximum e f f e c t i v e doses were i d e n t i c a l f o r c o n t r a c t i l e force and glycogen phosphorylase a c t i v a t i o n . —8 Propranolol 10  M did not s i g n i f i c a n t l y reduce active phosphorylase  l e v e l s produced by glucagon (Figure 8; Table 7).. In buffer-perfused hearts, a f t e r 1.0 and 2.0 yg of the polypeptide, phosphorylase a_ increased from 8.0 % (control, N = 1) to 26.5 + 6.1 and 42.6 + 6.5 % respectively.  When hearts  were exposed to propranolol and then challenged with the same doses of glucagon, phosphorylase a. l e v e l s were 25.9 +_ 4.9 and 34.3 + 9.6 % respectively. These are not s t a t i s t i c a l l y d i f f e r e n t from values obtained i n the b u f f e r perfused hearts (Table 7). Propranolol infusion alone had l i t t l e e f f e c t on control phosphorylase a c t i v i t y (8.5 % a_; N = 1 vs. 7.0 % a_, N = 1 i n heart perfused with CK f o r 30 minutes). A possible mechanism f o r the stimulatory actions of glucagon i s through the adenylate cyclase system.  Therefore, the influence of glucagon on the  tissue accumulation of c y c l i c AMP was investigated i n both time and dose experiments.  A 2.0 yg dose of the hormone was chosen to determine the time  course of c y c l i c AMP changes.  At 0 seconds (control), the c y c l i c AMP  concentration was 0.25 + 0.01 pmols/mg wet weight.  Glucagon increased the  c y c l i c AMP l e v e l as early as 15 seconds a f t e r i n j e c t i o n (Figure 4; Table 8), a time preceding the c o n t r a c t i l e and phosphorylase-activating responses. Peak l e v e l s of the nucleotide were not obtained u n t i l 40 seconds but were maintained at least u n t i l 60 seconds.  C y c l i c AMP was s t i l l elevated over  control as long as 120 seconds a f t e r glucagon i n j e c t i o n (Figure 4). The dose dependence of tissue c y c l i c AMP accumulation was studied i n hearts frozeth 50 seconds following glucagon administration.  In contrast to  glucagon-induced changes i n force and phosphorylase _a, the minimum e f f e c t i v e  56  TABLE  7  _g The influence of propranolol 10 M on glucagon-induced a c t i v a t i o n i n the isolated perfused r a t heart.  Dose of Glucagon (yg)  Control  *  Propranolol 10~ M 8  7.0 (N=l)  8.5 (N=l)  1.0  26.5 + 6.1*  25.9 + 4.9*  NS  2.0  42.6 + 6.5*  34.3 + 9.6*  NS  Mean % phosphorylase  NS  Buffer-perfused  phosphorylase  a_ + one S.E.M. of three hearts  Not s i g n i f i c a n t l y decreased from the corresponding buffer-perfused value (p > .05; unpaired ' t ' test)  Buffer-perfused hearts were perfused with CK solution f o r 30 minutes and then injected with either CK (0.25 ml; control) or glucagon. Propranolol —8 10 M was perfused through the hearts f o r 20 minutes following a 10 TY' n minute s t a b i l i a z t i o n period on CK solution. A l l hearts were frozen 50 seconds .. seconds after i n j e c t i o n . 1  57  TABLE 8 The effect of time on cardiac c y c l i c AMP accumulation following admini s t r a t i o n of 2 yg glucagon into the buffer-perfused and theophyllineperfused rat heart.  Time a f t e r injection (sec) 0  Buffer-perfused 0.25+0.01* (8)  0.32+0.02 (4)  15  0.37+0.04 (A)  0.43+0.04 (3)  20  0.40 + 0.05 (5)  25  0.53+0.04 (5)  3  c  0.74+0.06 ' (3)  C  a  1.13+0.04 ' (2)  c  30  0.52 + 0.07 (5)  1.16+0.03 » (3)  c  35  0.49 + 0.03a  1.07+0.09 >  c  a  a  b  b  (3)  40  0.64+0.03 (5)  60  0.60 + 0.01 (2)  a  0.56 + 0.04 (2)  a  120  b  b  (5)  *  Theophylline-perfused  a  1.27+0.07 ' (3) b  1.19 » (1) b  c  c  Mean c y c l i c AMP content (pmol/mg wet weight) + one S.E.M.  a. S i g n i f i c a n t l y (p< 0.05) greater than the buffer-perfused, 0 sec value b. S i g n i f i c a n t l y greater than the theophylline-perfused, 0 sec value c. S i g n i f i c a n t l y enhanced over the corresponding buffer-perfused value CK (0 sec) or glucagon was injected v i a a side-arm cannula and hearts were frozen at the indicated times. Theophylline 1 mM was perfused through the apparatus for 15 minutes. Numbers i n parentheses refer to the number of animals.  58  dose f o r s i g n i f i c a n t l y elevating c y c l i c AMP l e v e l s was 0.25 yg (Figure 6) which increased nucleotide concentration to 0.38 + 0.03 pmols/mg wet weight (Table 9).  Doubling  the glucagon dose also elevated tissue c y c l i c AMP but  this was not s t a t i s t i c a l l y higher than control tissue l e v e l s . the experimental  However  " t " value ( unpaired data ) of 2.207 was only s l i g h t l y  lower than the c r i t i c a l " t " value of 2.228 (p < .05; 10 d f ) . Glucagon 8.0 yg was the highest dose tested. accumulation  I t caused the largest  of c y c l i c AMP (0.73 +0.01 pmols/mg wet weight, Table 9) i n  buffer-perfused hearts.  As can be seen from Figure 6, the dose-response  curve i s much f l a t t e r f o r c y c l i c AMP than for either c o n t r a c t i l e force, or % phosphorylase a_. —8 Table 10 and Figure 8 show that propranolol 10 glucagon-induced increases i n c y c l i c AMP.  M was unable to block  In buffer-perfused hearts, g l u -  cagon 1.0 yg increased c y c l i c AMP from 0.31 pmols/mg (N = 1) to 0.38 + 0.01 pmols/mg wet weight.  The c y c l i c nucleotide changes induced by a higher dose  of glucagon (2.0 yg) were s i m i l a r l y unaltered by propranolol (Table 10; —8 Figure 8).  The control l e v e l of c y c l i c AMP a f t e r propranolol 10  M and  i n j e c t i o n of 0.25 ml of CK buffer was 0.21 pmol/mg wet weight (N = 1). 2. Experiments with theophylline In order to gain further information on the role of c y c l i c AMP, i t was decided to investigate the influence of theophylline perfusion on glucagoninduced increases i n c o n t r a c t i l e force, phosphorylase a c t i v a t i o n and tissue c y c l i c AMP accumulation.  Figure 4 i l l u s t r a t e s the time course of these  responses i n the presence of 1 mM theophylline, a concentration s u f f i c i e n t to i n h i b i t heart phosphodiesterases  i n v i t r o (Butcher and Sutherland, 1962).  The e f f e c t of theophylline on the glucagon dose-response curves i s shown i n Figure 6.  59 TABLE 9 The e f f e c t of various doses of glucagon on cardiac c y c l i c AMP accumulation i n the buffer-perfused and theophylline-perfused i s o l a t e d rat heart. Dose of Glucagon (yg)  Buffer-perfused  Theophyllirie-peffused  Control  0.28+0.04* (4)  0.41 + 0.03^ (4)  0.25  0.38 + 0.03 (6)  0.57 + 0.03 » (4)  c  0.5  0.40 + 0.04 (7)  0.66 + 0.03 » (5)  c  1.0  0.62 + 0.05 (6)  1.04+0.07 » (6)  c  2.0  0.63 + 0.04 (2)  1.33 + 0.04 » (2)  c  4.0  0.64 + 0.01 (4)  1.47+0.04 » (5)  c  8.0  0.73+0.01 (3)  1.64+0.02 » (2)  c  *  a  b  b  a  a  a  a  Mean v e n t r i c u l a r c y c l i c AMP content  b  b  b  b  (pmol/mg wet weight) + one S.E.M.  a. S i g n i f i c a n t l y (p<0.05) greater than the buffer-perfused control b. S i g n i f i c a n t l y greater than the theophylline-perfused control c. S i g n i f i c a n t l y enhanced over the corresponding buffer-perfused value CK (control) or glucagon was injected v i a a side-arm cannula and hearts were frozen at 50 seconds. Theophylline 1 mM was perfused through the apparatus f o r 15 minutes. Numbers i n parentheses refer to the number of animals.  60  TABLE 10 The influence of propranolol 10" M on glucagon-induced c y c l i c AMP accumul a t i o n i n the isolated perfused rat heart. _8  Dose of Glucagon  _iE£)  Buffer-perfused  Control  0.31  (N=l.)  Propranolol  0.21  10~M 8  (N=l)  1.0  0.38 +.0.05*  0.38 + 0.01*NS  2.0  0.48 + 0.02*  0.39 + 0.07*NS  * NS  Mean c y c l i c AMP content  (pmol/mg wet weight + one S.E.M. of 3 hearts  Not s i g n i f i c a n t l y d i f f e r e n t from the corresponding buffer-perfused value (p > .05; unpaired ' t ' test)  Buffer-perfused hearts were perfused with CK s o l u t i o n f o r 30 minutes and then injected with either CK (control) or glucagon. Propranolol 10~^M was perfused through the hearts f o r 20 minutes following a 10 minute s t a b i l i z a t i o n period on CK solution. A l l hearts were frozen 50 seconds a f t e r i n j e c t i o n .  61 With 2.0 yg glucagon, theophylline did not a l t e r the time when peak c o n t r a c t i l e amplitude was reached (Figure 4; Table 1).  However the return  to pre-injection s y s t o l i c tension was delayed u n t i l after 50 seconds. % increase i n force was s i g n i f i c a n t l y enhanced over buffer-perfused  The  hearts  only at 30 and 50 seconds, while the absolute tension change due to glucagon was greater only at 50 seconds (Table 1).  At 35 seconds, the mean time of  peak increase, there was no s i g n i f i c a n t augmentation by theophylline of the glucagon response. A complete dose-response curve for force was obtained i n the presence of theophylline and i s i l l u s t r a t e d i n Figures 6 and 7, and Table 2. The minimum e f f e c t i v e concentration of glucagon causing a s i g n i f i c a n t increase i n force was 0.25 yg. This dose was one-half of that required i n the absence of theophylline.  The maximum force increase (116.3 + 7.4 %) was  achieved with glucagon 4.0 yg. The 8.0 yg dose was tested i n 2 animals only, and gave a variable response which, when averaged, was less than that of 4.0 yg. The peak increases i n force following various doses of glucagon were compared i n hearts exposed to theophylline 1 mM and i n buffer-perfused (Figures 6,7; Table 2).  hearts  When r e s u l t s were calculated as a percentage increase  over pre-injection s y s t o l i c tension, theophylline s i g n i f i c a n t l y (p < .05) enhanced the inotropic response at a l l dose l e v e l s .  However, i f results  were compared on the basis of absolute tension changes, then only the r e sponses to 0.25 and 2.0 yg were augmented by the methylxanthine. It was mentioned e a r l i e r that, i n the time-response experiments, 1 mM theophylline did not ".enhance the force obtained with 2. Oyg glucagon when determined at 35 seconds.  This apparent contradiction to the dose-response  experiments, where theophylline altered the 2.0 yg glucagon force change  62 from 57.2 + 4.4% to 8 4 . 4 + 7 . 7 % (Table 2), may be explained by the fact that the time of maximum force varied from 30 to 50 seconds.  Hence the  tension change at 35 seconds was not representative of peak increase i n a l l hearts, and the mean value at t h i s time would tend to be d i s t o r t e d by the v a r i a t i o n .  I f time-response comparisons were made using only those  hearts which peaked at 35 seconds after 2.0 yg glucagon then a s i g n i f i c a n t enhancement by theophylline did occur. Theophylline 1 mM produced i t s own alterations i n cardiac force of contraction.  Following the 10 minute s t a b i l i z a t i o n period on CK solution,  the mean s y s t o l i c tension was 3.5 + 0.1 g.  When the hearts were subsequently  exposed to theophylline, s y s t o l i c tension t r a n s i e n t l y increased 15.4 +1.5 % (CL6 + 0.1 g; N=36) over 3 to 5 minutes and then declined to pre-exposure l e v e l or below.  In the dose-response experiments the mean s y s t o l i c tension  after exposure to theophylline (but before glucagon injection) was 3.3 + 0.1 g but this was not s i g n i f i c a n t l y (p > .05) lower than before methylxanthine infusion (Table 2).  Although the 15 minute perfusion time through  the apparatus was usually s u f f i c i e n t to return the tension to pre-theophylline l e v e l , i n some hearts the force was greater at this time.  The available  data were i n s u f f i c i e n t to s t a t i s t i c a l l y evaluate the influence of pre-existing tension l e v e l due to theophylline on the glucagon i n o t r o p i c response. Figure 6 and Table 6 demonstrate that theophylline enhanced the a b i l i t y of glucagon to activate glycogen phosphorylase at a l l dose l e v e l s except 0.25 yg. As was true for buffer-perfused hearts, the minimum e f f e c t i v e dose i n the presence of 1 mM theophylline was 0.5 yg. However, the largest dose tested (8.0 yg) i n methylxanthine-exposed hearts was able to produce maximal phosphorylase & l e v e l s (72.2 %; N=l).  This degree of a c t i v i t y was not  obtained i n CK-perfused hearts (Table 6) where the maximally e f f e c t i v e  63  dose of 4.0 yg changed phosphorylase a. to.only 38.8 +.3.5 phosphorylase content.  % of the t o t a l  One mM theophylline alone elevated control l e v e l s  of the active enzyme (measured 50 seconds after a 0.25 ml CK i n j e c t i o n ) to 8.4 + 2.5 % from 5.3 + 1.0 % (Table 6) but this was not a s i g n i f i c a n t increase (p.> .05). The time course of glucagon-induced phosphorylase activation was altered i n the presence of theophylline 1 mM  (Figure 4; Table 5).  slightly  In  methylxanthine perfused hearts the peak phosphorylase _a l e v e l was measured at 60 seconds and was not s i g n i f i c a n t l y higher than at 50 seconds.  The  plateau l e v e l was therefore delayed 10 seconds compared to the CK-perfused hearts (Figure 4).  Also, the activation of glycogen phosphorylase was  faster i n the presence of theophylline, for % a. was s i g n i f i c a n t l y (p < .05) elevated over control at 20 seconds (Table 5). Theophylline s i g n i f i c a n t l y augmented the glucagon-induced phosphorylase activation at 25 seconds and a l l subsequent times except 40 seconds (Table 5) but not at e a r l i e r i n t e r v a l s .  The control phosphorylase <i l e v e l s i n this  series of experiments were determined 0 seconds after i n j e c t i o n of 0.25 of CK solution.  ml  Theophylline 1 mM did not s i g n i f i c a n t l y a l t e r the a c t i v i t y  although % phosphorylase a. was elevated from 6.9 + 0.8 to 10.4 + 1.9 (Table 5). Theophylline markedly influenced the a b i l i t y of glucagon to increase tissue c y c l i c AMP  accumulation.  As i n buffer-perfused hearts, peak levels  were present 40 seconds a f t e r glucagon 2.0 yg (Figure 4) and were maintained at least u n t i l 60 seconds.  However, theophylline-perfused hearts did not  show a s i g n i f i c a n t elevation above control u n t i l 20 seconds a f t e r glucagon i n j e c t i o n (Table 8).  This time was s t i l l e a r l i e r than the increase i n  contractile force and simultaneous with the increase In phosphorylase & (Figure 4).  64  At 0 seconds (controls) theophylline-perfused hearts.had a mean c y c l i c AMP  content of 0. 32+  0.02 pmols/mg wet w e i g h t — s i g n i f i c a n t l y greater than  i n non-exposed hearts (Table 8).  In f a c t , the only time when nucleotide  l e v e l s were not enhanced by the methylxanthihe was 15 seconds after glucagon 2.0 yg..  ;  In the presence of 1 mM theophylline, the c y c l i c AMP  dose-response curve  was much steeper than that of the CK experiments (Figure 6). yg glucagon was s u f f i c i e n t to raise c y c l i c AMP  A dose of 0.25  content above the control  l e v e l of 0.41 + 0.03 pmols/mg wet weight (Table 9).  This was also the min-  imum e f f e c t i v e dose for increasing c o n t r a c t i l e force, but not phosphorylase a_ l e v e l s , i n methylxanthine-treated hearts (Tables 2,6). caused a substantial accumulation of c y c l i c AMP  Glucagon 8.0 yg  to 1.64 + 0.02 pmols/mg.  Since higher doses were not tested i t i s unknown whether this produces the maximum possible tissue concentration of the c y c l i c nucleotide. At a l l doses of glucagon examined, theophylline augmented the glucagoninduced c y c l i c AMP  response (Figure 6; Table 9).  With a 1 mM xanthine  concentration the mean control l e v e l of nucleotide was also approximately 33 % greater than that of non-exposed tissue (0.41 + 0.03 vs. 0.28 + pmol/mg; Table 9).  0.04  The response to 0.25 and 0.5 yg of glucagon appeared to  be elevated by the same amount as the response to CK i n j e c t i o n was i n the control hearts after theophylline. c y c l i c AMP  However, a large potentiation of tissue  content was observed following higher doses of glucagon admin-  i s t e r e d i n the presence of the methylxanthine.  C y c l i c AMP  a f t e r 8.0.yg  glucagon was more than double that i n buffer-perfused hearts (1.64 +0.02 vs. 0.73 + 0.01 pmol/mg; Table 9).  Furthermore, the slope of the curve, from  1.0 to 8.0 yg glucagon, was increased considerably (Figure 6).  65 DISCUSSION The present study has confirmed e a r l i e r findings of the actions of glucagon on cardiac force of contraction and glycogen metabolism.  Figure 6  i l l u s t r a t e s the dose-dependent manner of these polypeptide responses. Glucagon also increased cardiac accumulation of c y c l i c AMP but this was less dramatically altered by dose.  The greatest increases i n c o n t r a c t i l e ampli-  tude and % phosphorylase ji were obtained with 4.0 yg glucagon (Tables 2,6) whereas c y c l i c AMP was s t i l l increasing a f t e r 8.0 yg (Table 9).  On the other  end of the dose-response curve, c y c l i c nucleotide l e v e l s were elevated a f t e r only 0.25 yg glucagon but twice t h i s concentration (0.5 yg) was required to s i g n i f i c a n t l y increase the mechanical and metabolic a c t i v i t i e s . The present study also investigated the temporal sequence of glucagoninduced cardiac events.  Contractile force increased s i g n i f i c a n t l y at 25  seconds and peaked 35 seconds after glucagon.  Percent phosphorylase a_ was  elevated at 30 seconds but did not peak u n t i l a f t e r the maximum increase i n force.  C y c l i c AMP was increased above control as early as 15 seconds arid  reached a maximum at the same time as phosphorylase a_ (Figure 4).  These  r e s u l t s therefore s a t i s f y one of Sutherland's c r i t e r i a for implicating c y c l i c AMP mediation of a hormonal response i . e . tissue levels of the nucleotide increased prior to, or simultaneous with, the physiological event. An e a r l i e r temporal study i n the isolated rat heart (Mayer et a l . , 1970) was  unsuccessful i n demonstrating an increase i n c y c l i c AMP p r i o r to the  p o s i t i v e inotropic effect of glucagon, although l a t e r increases were noted. This f a i l u r e was attributed, by the authors, to a difference i n the r e l a t i v e a b i l i t y to detect increases i n c y c l i c AMP on the one hand, and mechanical a c t i v i t y on the other hand.  Since the present experiments measured c y c l i c  AMP by the protein binding method, which w i l l detect increases i n the test tube as low as 0.05 pmol (Gilman, 1970), the explanation given by Mayer and  66 coworkers  (1970) appears  Another explanation  adequate. for the discrepancy i n results between the two  studies might be the d i f f e r e n t perfusion rates employed.  Mayer et a l . (1970)  used a rate of 7 ml/minute which caused the c o n t r a c t i l e e f f e c t , as well as the c y c l i c AMP  response, to be maximal a t only 20 seconds.  Phosphorylase  activation also occurred e a r l i e r with the plateau range occurring from 20 to 40 seconds.  In contrast, the slower perfusion rate used here (2.8 ml/  minute) allowed a greater separation of these events because the peak force occurred at 35 seconds and peak c y c l i c AMP  accumulation at 40 seconds.  Because the experimental protocols of this investigation and that of Mayer and associates data further.  (1970) were s i m i l a r , i t i s appropriate to compare the  The maximal increase i n c o n t r a c t i l e amplitude i n the study of  Mayer et a l . was approximately 20% following a dose of 3.0 yg glucagon. contrast, we observed much greater increases.  In  For example, 2.0 yg glucagon  resulted i n a change of 2.1 + 0.2 g., or a 57.2 + 4.4 % increase over prei n j e c t i o n s y s t o l i c tension (Table 2). (4.0 yg) caused a 77.1 +7.9  Also, the maximally e f f e c t i v e dose  % increase i n force.  This difference was not _g  due to endogenous catecholamine release since propranolol 10 duce the inotropic e f f e c t of glucagon (Figure 8; Table 3).  M did not reThese data also  confirm that glucagon i s not acting d i r e c t l y on the catecholamine receptor (Lucchesi, 1968; Mayer et a l . , 1970). The results on phosphorylase a c t i v a t i o n also d i f f e r quantitatively. Although control levels of the a_ form, and levels after 1.0 yg glucagon are s i m i l a r , Mayer et a l . observed a greater maximal a c t i v a t i o n (55.0 +_ 4.0 % a. vs. 38.8 + 3.5 % a.;Table 6)  In both studies cardiac  P r e c e p t o r blockade did  not i n t e r f e r e with the glucagon metabolic response. It i s more d i f f i c u l t to compare c y c l i c AMP  alterations because of the  67  discrepancy between the two studies both i n time course of cardiac action and i n doses of glucagon employed.  Mayer et a l . (1970) obtained a much  lower maximal accumulation of the c y c l i c nucleotide (0.48 v e n t r i c l e with 3.0  yg glucagon).  + 0.02  pmol/mg  This was s l i g h t l y more than double the  control l e v e l and was measured at the peak of the inotropic response using the maximally e f f e c t i v e dose f o r mechanical a c t i v i t y .  There are three possi-  ble ways to compare the results with those obtained i n the present study. In our experiments the maximum tissue content determined a f t e r glucagon alone was 0.73  + 0.01  pmol/mg v e n t r i c l e (Table 9).  Since this was a supra-  maximal dose f o r the i n o t r o p i c - response, a more appropriate value (for comparison purposes) might be that obtained with 4.0 s i t u a t i o n , c y c l i c AMP  content was 0.64  + 0.01  yg glucagon.  In this  pmol/mg wet weight.  arrroximately 130 % greater than control levels.  This was  On a percentage basis,  Mayer et a l . also obtained a value 130 % greater than control. After considering the time course of c y c l i c AMP  changes encountered i n  the present study, i t i s possible the heart was frozen at an inappropriate time to detect maximal accumulation i n Mayer's investigation.  We observed  that c y c l i c AMP peaked at least 5 seconds a f t e r the peak inotropic response (Figure 4).  Mayer et a l . (1970) did not measure levels at this time i n t e r -  v a l following the mechanical peak but rather a f u l l 20 seconds l a t e r . our experiments c y c l i c AMP was declining at this point.  If cyclic  In  AMP  concentrations at the time of peak tension are compared (this study 35 seconds'; Mayer et a l . 20 seconds) then the e a r l i e r investigators obtained lower absolute values (e.g. 2.0 pmols/mg; Table 9). 2.0  yg glucagon increased c y c l i c AMP  to 0.52  However, because the doses are different  yg) t h i s i s not an i d e a l comparison either.  remarkably similar i n the two laboratories.  (3.0  +  0.07  yg vs.  The control values are  Mayer and coworkers reported  68  values of 0.21. + 0.01 and 0.32 + 0.02 pmol/mg i n d i f f e r e n t t r i a l s .  Both  means are i n the range of nucleotide levels observed'here (Time experiments: 0.25 + 0.01, Table 8; Dose experiments: 0.28 + 0.04, Table 9).  Another common  finding was that neither pronethalol (Mayer et a l . , 1970) nor propranolol (Table 10), both  3 blocking agents, s i g n i f i c a n t l y decreased the glucagon-  induced c y c l i c AMP changes.  Thus, while quantitative differences e x i s t , the  q u a l i t a t i v e s i m i l a r i t y of results demonstrates the v a l i d i t y of data obtained in this investigation. Other reports have appeared i n the l i t e r a t u r e concerning the influence of glucagon on i n t a c t • tissue c y c l i c AMP accumulation.  The f i r s t of these  (LaRaia, Craig and Reddy, 1968) suggested there was a d i s s o c i a t i o n between the positive inotropic action of glucagon and c y c l i c AMP content i n the isolated, perfused rat heart. AMP were observed.  Unlike isoproterenol, no increases i n c y c l i c  However, only two time intervals after drug i n j e c t i o n  (the same for both glucagon and isoproterenol) were selected f o r analysis and i t i s possible that a glucagon-induced increase was missed because of poor experimental design.  In support of this proposal, t h e i r data indicated  a s l i g h t difference i n the time course of the drug-induced inotropic actions. It i s therefore unlikely that the time course f o r drug-induced c y c l i c AMP a l terations would be i d e n t i c a l . Oye and Langslet (1972) also speculated on the causal relationship of c y c l i c AMP and the glucagon inotropic response.  These investigators deter-  mined that a concentration capable of increasing force would also increase tissue accumulation of the nucleotide, but not preceding the mechanical response.  They found an i d e n t i c a l pattern following isoproterenol.  This i s i n  contrast to the c l a s s i c a l temporal sequence obtained by Robison et a l . (1965) where epinephrine i n j e c t i o n caused a rapid and dramatic increase i n c y c l i c AMP followed by the positive inotropic response.  Oye and Langlet (1972)  69 concluded that such a temporal sequence was a function of the method of drug administration fbr i n their experiments, glucagon and isoproterenol were slowly perfused through i s o l a t e d rat heart rather than given i n a concentrated bolus. The evidence f o r a d i s s o c i a t i o n between c y c l i c AMP and myocardial inotropism which was presented i n the report of Oye and Langslet (1972) i s not convincing for several reasons.  There i s no s t a t i s t i c a l analysis of the data.  In fact, many of the conclusions were apparently based on single observations. Also, only p i c t o r i a l representations of the results, were published.  Further-  more, the graph of c y c l i c AMP versus time indicates that, contrary to the authors' interpretation, i n t r a c e l l u l a r levels of c y c l i c nucleotide may i n crease above control p r i o r to, or a least simultaneous with, the mechanical effect.  Oye and Langslet (1972) also reported that chlorpromazine  abolished  the isoproterenol-induced increase i n c y c l i c AMP without'altering the inotropic response. study  However, these findings were not v e r i f i e d i n a subsequent  (Osnes and Oye, 1975). A recent study of the myocardial actions of glucagon demonstrated dose-  dependent increases i n peak l e f t v e n t r i c u l a r pressure and c y c l i c AMP i n the isolated, perfused rat heart (Henry et a l . , 1975).  The present investigation  has confirmed the influence of dose on these parameters (Figure 6).  Henry  and associates (1975) also observed that adenylate cyclase a c t i v i t y i n broken c e l l preparations was sensitive to glucagon concentrations changes i n the intact heart;  producing  E a r l i e r Murad and Vaughan (1969) had noted  the presence of a glucagon-stimulatable enzyme i n rat myocardium. Cat and human heart preparations also had glucagon-sensitive adenylate cyclases (Levey and Epstein, 1969). of Sutherland's  Thus, for the above mentioned species, another  c r i t e r i a has been s a t i s f i e d .  70 Observations of glucagon-induced changes i n guinea pig myocardium vary from one laboratory  to another.  Farah and Tuttle (1960) and  Spilker  (1970) each observed the p o s i t i v e inotropic response i n spontaneously-beating and e l e c t r i c a l l y - d r i v e n a t r i a respectively. centration produced force increases  The maximally e f f e c t i v e con-  no greater than 50%  (Spilker, 1970).  On the other hand, Prasad (1975) could not demonstrate increases using v e n t r i c u l a r p a p i l l a r y muscle.  i n tension  I t i s possible that a difference i n  responsiveness exists between a t r i a and v e n t r i c l e of the same species. However, i n i s o l a t e d perfused hearts which were e l e c t r i c a l l y paced, glucagon increased  peak l e f t v e n t r i c u l a r pressure by approximately 100%  all,  (Henry et  1975) , yet the polypeptide hormone could not re-instate e l e c t r i c a l or mechanical a c t i v i t y i n potassium-arrested hearts (Watanabe and Besch,  1974;  Schneider and Sperelakis, 1975). There may  be a sex-linked  difference i n s u s c e p t i b i l i t y to glucagon.  In a l l studies where a positive inotropic e f f e c t was  obtained, male guinea  pigs were used exclusively.  However, the unsuccessful studies f a i l e d to  specify which sex was  In a preliminary  any  used.  force change after glucagon was  experiment we  could not  detect  administered to a Langendorff preparation  of a female guinea p i g . Glucagon may  a l t e r the i n t e r v a l - f o r c e relationship i n myocardium.  A  corollary to this would be that the absolute and r e l a t i v e magnitude of druginduced force increases may  vary with stimulation  frequency.  Spilker (1970)  showed that at stimulation frequencies between 60 and 240/  minute, the  absolute increase i n isometric tension^due to glucagon was  uniform.  lower and higher rates, however, glucagon had tropic effect. strength  At  l e s s , i f any, p o s i t i v e ino-  Unfortunately the influence of glucagon  on the i n t e r v a l -  relationship has not been investigated i n guinea pig v e n t r i c l e or  71 i n rat myocardium although the shape of the curve i n the absence of drugs d i f f e r s i n these three.tissues (guinea p i g a t r i a vs. guinea pig v e n t r i c l e vs. rat a t r i a and v e n t r i c l e ; Koch-Weser and Blinks, 1963).  When such i n -  formation becomes available, i t may explain some of the discrepancies' between laboratories. In c o n f l i c t with the second messenger hypothesis glucagon was found not to increase c y c l i c AMP i n a dose-dependent manner i n i s o l a t e d guinea pig v e n t r i c l e (Henry et a l . , 1975) although force changes p a r a l l e l e d those in rat heart.  Furthermore adenylate cyclase a c t i v i t y i n the guinea p i g  p a r t i c u l a t e preparation did not vary with glucagon concentration. These data represent an apparent d i s s o c i a t i o n between glucagon-induced inotropism and c y c l i c AMP.  Although several time i n t e r v a l s after the onset of admin-  i s t r a t i o n were tested, the p o s s i b i l i t y that small increases were missed due to inappropriate freezing time cannot be e n t i r e l y excluded.  Henry et a l .  (1975) obtained higher basal levels of c y c l i c AMP i n the guinea p i g heart' compared to the rat heart. t r o p i c response.  This alone may be s u f f i c i e n t to mediate the ino-  A r e l a t i v e lack of dose-dependency was indicated i n our  investigation where increasing the glucagon dose 40-fold only increased tissue accumulation of c y c l i c AMP by approximately 3-fold (Figure 6; TablLe:. 9). ;  Another explanation for the f a i l u r e to see changes i n guinea p i g c y c l i c AMP content might be that phosphodiesterases may be degrading the c y c l i c AMP as fast as i t can be formed.  A weak agonist l i k e glucagon might not be able  to overcome this whereas a stronger agonist (e.g.epinephrine, as i n Henry et a l . , 1975) could.  Meinertz and associates (1974) presented some i n d i r e c t -  evidence that guinea p i g a t r i a may have a high i n t r i n s i c phosphodiesterase, activity.  They were unable to observe the positive inotropic response to  dibutyryl c y c l i c AMP unless papaverine was also present. resembled those i n other species.  The changes then  The above explanation i s not e n t i r e l y  72  s a t i s f a c t o r y ; however, because guinea p i g v e n t r i c l e did not require papaver;  ine  to respond to the c y c l i c AMP  derivative.  The adenylate cyclase responsiveness to glucagon may have been altered by the preparation of the enzyme. the ide,  Although Henry et al.(1975) claimed that  enzyme was responsive because i t was activated by epinephrine and f l u o r this does not indicate that s e n s i t i v i t y to glucagon remained.  As re-  viewed by Levey (1975), glucagon and epinephrine responsiveness i n s o l u b i l i z e d myocardial adenylate cyclase i s d i f f e r e n t i a l l y restored by phospholipid addition.  It i s therefore possible that, i n the experiments of  Henry et a l . a c r i t i c a l factor necessary only for glucagon activation was inadvertently removed. Evidence supporting a role f o r c y c l i c AMP  i n the mediation of glucagon-  induced inotropism and phosphorylase a c t i v a t i o n was obtained i n our experiments with theophylline.  A 1 mM concentration of the methylxanthine s i g -  n i f i c a n t l y elevated both basal and glucagon-stimulated c y c l i c AMP l a t i o n (Figures 4,6), presumably by i n h i b i t i n g i t s degradation. course of 2.0 yg glucagon on tissue c y c l i c AMP unaltered i n the presence of theophylline.  content was  accumuThe time  essentially  I t was noted, however, that theo-  phylline caused a delay compared to CK-perfused hearts i n the time when glucagon s i g n i f i c a n t l y elevated the nucleotide concentration over basal levels (20 seconds vs. 15 seconds; Table 8). Consonant with i t s actions on tissue c y c l i c AMP  content, 1 mM theo-  phylline enhanced both the phosphorylase-activating and mechanical e f f e c t s of glucagon.  Apart from prolonging the glucagon-induced increase i n s y s t o l i c  tension, theophylline did not otherwise influence the time course of the inotropic response.  In contrast, the methylxanthine shortened the time re-  quired to i n i t i a l l y increase % phosphorylase ji but lengthened the attainment  73  of plateau levels (Figure 4).  The temporal sequence of cardiac events ob~  served a f t e r glucagon 2.0 yg i . e . increases i n c y c l i c AMP followed by i n creases i n force and f i n a l l y % phosphorylase a was maintained i n the presence of theophylline. The dose-dependent behaviour of the glucagon-elicited changes i n c y c l i c AMP, force of contraction and % phosphorylase a. was also examined during concurrent  administration of theophylline.  Phosphorylase a. l e v e l s  were augmented after glucagon doses of 0.5 yg and higher, which might be expected from the greater c y c l i c AMP content (Figure 6).  However, low doses  of glucagon did not produce a greater metabolic response i n the presence of theophylline 1 mM, even though c y c l i c nucleotide l e v e l s were enhanced. Theophylline  also f a c i l i t a t e d maximal a c t i v a t i o n of glycogen phosphorylase by  glucagon, as demonstrated i n Figure 6.  S i m i l a r l y , much higher tissue con-  centrations of c y c l i c AMP were made possible by the methylxanthine. The results on the p o s i t i v e inotropic e f f e c t of glucagon i n combination with theophylline are not e a s i l y interpreted. i n terms of percent  I f data are analyzed  increase i n force then theophylline potentiated the  inotropic response to a l l but the largest glucagon dose (8.0 yg).  However,  when absolute tension changes are calculated, 1 mM theophylline enhanced the inotropic e f f e c t only to 0.25 and 2.0 yg of glucagon (Table 2). for the discrepancy  The reason  i s not clear but could be due to a s l i g h t l y depressed  s y s t o l i c tension after exposure to theophylline f o r 10 minutes.  Because  of t h i s , the same absolute increase would result i n a higher percentage i n crease.  Table 2 shows that a 15 minute theophylline perfusion  (exposure  time approximately 8 to 10 minutes) caused a mean tension depression of 0. '2+0.09 g r e l a t i v e to the pre-exposure l e v e l . for the magnitude of discrepancy  observed.  This would hardly account  Experiments with theophylline were  usually undertaken on d i f f e r e n t days from those i n buffer-perfused hearts. In  74 CK-only experiments, the mean s y s t o l i c tension before glucagon i n j e c t i o n was 3.8+0.1 g while the corresponding value i n the theophylline experiments was 3.3+0.1 g.  This i s a s i g n i f i c a n t (p <.05) depression and could  explain the contradictory data. It i s tempting to state from these results that the theophylline augmentation of glucagon-induced mechanical effects i s more apparent than r e a l . However, the fact that weaker hearts were able to respond to the same degree after glucagon challenge indicates some theophylline influence on myocardial c o n t r a c t i l i t y , although the e f f i c a c y of glucagon was not improved. This i s i n contrast to the altered e f f i c a c i e s of glucagon on c y c l i c AMP accumulation and phosphorylase activation with the methylxanthine. The d i f f i c u l t y i n interpreting force data r e f l e c t s , to some extent, the fact that the Langendorff preparation i s not i d e a l f o r determining druginduced changes i n myocardial c o n t r a c t i l i t y .  Only .^vertical changes are  recorded yet the developed tension presumably also increases i n fibres oriented i n other directions.  Ventricular volume i s not controlled and  therefore some of the recorded change may be due to the Frank-Starling e f fect (Blinks and Koch-Weser, 1963). Other uncontrolled parameters , which may have influenced the force results were coronary flow and contraction frequency.  Theophylline per-  fusion increased heart rate by approximately 12% and this might contribute to the observed depression i n s y s t o l i c tension.  Glucagon also had a chron-  otropic action which tended to reach a maximum a f t e r the peak inotropic response.  The present experimental design d i d not include an investigation  of the p o s i t i v e chronotropic e f f e c t and quantitative data are not obtainable from the records.  Because of the inverse frequency-force r e l a t i o n -  ship i n rat heart the inotropic response to glucagon would, at most, be  75  underestimated. In a more appropriate preparation f o r examining the mechanical actions of glucagon, Marcus et a l .  (1971) observed  but with a lower concentration (2.5 x 1 0  -4  a potentiation by theophylline, M).  This concentration also  augmented the inotropic responses of norepinephrine and dibutyryl c y c l i c AMP  but not of calcium (Skelton et a l . , 1971).  Unfortunately, i n t a c t  tissue levels of c y c l i c AMP were not measured and so the e f f i c a c y of this methylxanthine concentration as a phosphodiesterase  i n h i b i t o r i n intact  preparations i s unknown. Lucchesi (1968) could not show any inotropic response to glucagon i n i n t a c t dog heart simultaneously receiving 10 mg/kg theophylline which, alone, substantially increased the force of contraction.  In Lucchesi's experiments,  other drugs with a p o s i t i v e inotropic e f f e c t s i m i l a r l y prevented the t y p i c a l glucagon increase, including dichloroisoproterenol.  Lucchesi supplied  evidence that glucagon did not act on the cardiac -(preceptor by showing a lack of e f f e c t of propranolol plus dichloroisoproterenol on the glucagon mechanical response.  E a r l i e r investigators (Farah and T u t t l e , 1960; Regan  et a l . , 1964) had interpreted the blockade by dichloroisoproterenol as evidencing a common receptor for catecholamines  and  glucagon.  Antonaccio; and Lucchesi (1970) examined the i n t e r a c t i o n of glucagon with theophylline i n dog p a p i l l a r y muscle but saw only an additive tension change with a methylxanthine concentration of 3 x I O concentration (1 mM)  - 4  M.  At a higher  they observed a depression of the glucagon response  but because this concentration also impaired the tissue response to elect r i c a l stimulation, they suggested For glucagon-induced  the e f f e c t was non-specific.  phosphorylase  activation, a l l of Sutherland's  c r i t e r i a suggesting a role f o r c y c l i c AMP  have now been met.  In summary,  76  i n t a c t tissue l e v e l s of nucleotide were shown to increase p r i o r to, or simultaneous with, the metabolic response. (Figure 4; 1970)  also Mayer et a l . ,  Glucagon activated adenylate cyclase i n broken c e l l preparations  of myocardium from several species (Levey and Epstein, 1969; Murad and Vaughan, 1969; Henry et a l . , 1975).  Theophylline potentiated glucagon-  induced increases i n phosphorylase a. (Figure 6;Table 6) i n a concentration which markedly elevated amounts of i n t a c t tissue c y c l i c AMP sumably due to phosphodiesterase i n h i b i t i o n .  (Table 9), pre-  F i n a l l y , dibutyryl c y c l i c  (Kjekshus et a l . , 1971; Oye and Langslet, .1972) and c y c l i c AMP  AMP  (Kjekshus  et a l . , 1971) administered exogenously also increased the percent of active phosphorylase. While the metabolic actions of glucagon i n the i s o l a t e d perfused rat heart resemble those of epinephrine, data from the present study (Table 6) and from the report of Mayer et a l . (1970) suggest that glucagon i s a less e f f e c t i v e agonist.  This p a r a l l e l s the a b i l i t i e s of the two drugs to increase  tissue levels of c y c l i c AMP.  Although saturating doses were not determined  for c y c l i c nucleotide accumulation, Mayer and associates (1970) obtained a 5-fold increase after epinephrine and a 2.5-fold increase after glucagon, using drug doses producing the respective maximal increases i n phosphorylase a.. - Glucagon presumably  activates phosphorylase v i a c y c l i c AMP  but consistent  activation of phosphorylase kinase was not obtained (Mayer et a l . ,  1970).  In spite of the apparent s i m i l a r i t y , there i s no reason to assume an i d e n t i c a l mechanism of action f o r phosphorylase activation by glucagon and the catecholamines. Namm et al.(1968) unquestionably established there are at least regulators of cardiac glycogen phosphorylase a c t i v i t y .  Calcium was  two  essential  for the c a t a l y t i c a c t i v i t y of active phosphorylase kinase even though the amount of active enzyme was elevated apparently v i a c y c l i c AMP.  As was  77  observed for epinephrine (Namm et a l . , 1968)  the glucagon-induced increase  i n % phosphorylase a. was markedly diminished, but not abolished, when hearts were perfused with a calcium-free medium (Mayer et a l . , 1970). For  drug-induced glycogen phosphorylase a c t i v a t i o n , the r e l a t i v e  contribution of c y c l i c AMP l e v e l i s unknown;  and calcium toward the f i n a l phosphorylase a_.  No investigator has conclusively shown that either the  catecholamine or glucagon metabolic action can proceed without some increase i n c y c l i c AMP. i n that 1.0  Mayer and coworkers  (1970) reported an apparent d i s s o c i a t i o n  yg glucagon elevated the % phosphorylase a_ without changing the  basal c y c l i c AMP  content.  However, as previously discussed, the i n a b i l i t y  to detect small c y c l i c nucleotide increases would account for the results. In the present study c y c l i c AMP  increased after 0.25  yg glucagon but  the miniinum e f f e c t i v e dose f o r phosphorylase activation was 0.5 6).  yg (Figure  This may be due to differences i n assay s e n s i t i v i t i e s , but could also  r e f l e c t the existence of other factors i n the control of glycogen phosphorylase a c t i v i t y .  This i s further indicated by the fact that glucagon  4.0  yg produced maximal a c t i v a t i o n (Table 6) i n CK-perfused hearts, yet c y c l i c AMP  accumulation was s t i l l increasing after 8.0  difference, i n slopes between the c y c l i c AMP  yg glucagon (Table 9).  The  dose-response curve and the  phosphorylase dose-response curve indicates that an adenylate cyclasec y c l i c AMP  pathway might provide an e f f i c i e n t system f o r regulation of  cardiac metabolism because very small changes i n c y c l i c AMP were associated with large differences i n phosphorylase  activity.  It i s conceivable that catecholamines and glucagon may increase glycogen phosphorylase a c t i v i t y through a simultaneous and d i r e c t influence on calcium and c y c l i c AMP.  A l t e r n a t i v e l y , these drugs may  act on adenylate  cyclase and the resultant increase i n c y c l i c AMP might, i n turn, a l t e r c a l -  78  cium homeostasis  as w e l l as permitting activation of phosphorylase kinase.  A t h i r d p o s s i b i l i t y might be that glucagon and the catecholamines produce t h e i r metabolic response only through stimulation of adenylate cyclase, the normal c e l l u l a r calcium control processes being s u f f i c i e n t to allow phosphorylase activation.  At the present time, there are no data to rule  out any of these proposals.  Also there i s no i n d i c a t i o n , as yet, of the  source of activator c a l c i u m — i t might be i n t r a c e l l u l a r , e x t r a c e l l u l a r , or both. There are experimental data that would t h e o r e t i c a l l y implicate calcium as the sole mediator i n certain situations where glycogen phosphorylase i s activated.  For example, i n anoxia the % phosphorylase a i s increased i n  the i n s i t u rat heart even when c y c l i c AMP elevation i s blocked by p r a c t o l o l (Dobson and Mayer, 1973).  High concentrations of calcium can activate  phosphorylase i n the isolated rat heart (Friesen et a l . , 1967: Namm et a l . , 1968) without increasing c y c l i c AMP.(Namm et a l . , 1968; Hartley and McNeill, unpublished observations).  In f a c t , Namm and associates (1968) noted a  s i g n i f i c a n t decrease i n c y c l i c nucleotide concentration when r a t hearts were perfused with a high calcium medium. Some drugs appear to a l t e r myocardial phosphorylase a independently of c y c l i c AMP,  Imidazole (1.6 mg) s i g n i f i c a n t l y elevated the % of the ji form  without changing tissue c y c l i c AMP content (Verma and McNeill, 1974).  How-  ever only one time i n t e r v a l was selected f o r analysis and an increase might have been missed. independent  Theophylline also may produce i t s glycogenolytic action  of the adenylate cyclase pathway (McNeill et a l . , 1974) because  increases i n % phosphorylase a. were noted i n the absence of s i g n i f i c a n t changes i n c y c l i c AMP.  In that study, however, there was a large v a r i a t i o n  i n the control nucleotide levels which may have obscurred the r e s u l t s .  79  In summary, cardiac glycogenolysis induced by any of the above-mentioned drugs may t h e o r e t i c a l l y occur independently of the adenylate c y c l a s e - c y c l i c AMP  system (because of results with calcium) but most of the evidence, a l b e i t  circumstantial, m i l i t a t e s against t h i s , p a r t i c u l a r l y i n the case of glucagon and the catecholamines.  The data obtained with theophylline i n the present  study do not exclude a c y c l i c AMP  involvement i n the cardiac phosphorylase  activation e l i c i t e d by t h i s methylxanthine. Experimental evidence supporting the involvement of c y c l i c AMP i n the positive inotropic e f f e c t of glucagon was also obtained i n the present i n vestigation by s a t i s f y i n g two of Sutherland's c r i t e r i a .  F i r s t , a temporal  sequence consistent with the second messenger hypothesis was observed following glucagon2 ..0yg> when a s i g n i f i c a n t increase i n the i n t r a c e l l u l a r ;  c y c l i c nucleotide concentration preceded the onset of the p o s i t i v e inotropic response (Figure 4).  Second, theophylline 1 mM potentiated the per-  cent increase i n c o n t r a c t i l e force at several doses and the absolute increase at 0.25 andZOyg doses of glucagon (Table 2).  Theophylline also lowered  the minimum e f f e c t i v e glucagon dose from 0.5 to .0.25 yg while concurrently r a i s i n g the c y c l i c AMP  accumulation r e s u l t i n g from the 0.25 yg dose (Table 2).  As was true for glycogen phosphorylase a c t i v a t i o n , the catecholamines seem to be more e f f e c t i v e than glucagon i n enhancing myocardial force of contraction.  A maximum increase of 80% over p r e - i n j e c t i o n s y s t o l i c tension  was e l i c i t e d by 4.0,yg glucagon (Table 2) compared with a 120% increase e l i c i t e d by lO.CLyg norepinephrine (Table 4).  Mayer and associates (1970)  also demonstrated a difference i n maximal e f f i c a c y between glucagon and epinephrine. In our laboratory a difference between the two drugs was also noted i n the time course of t h e i r mechanical action.  Norepinephrine had a faster  80 onset and produced i t s peak change i n tension e a r l i e r than glucagon, although the l a t t e r agent maintained the increase i n force of contraction much longer. These variations i n onset and duration of the inotropic e f f e c t between glucagon and epinephrine are r e f l e c t e d i n the patterns of tissue c y c l i c AMP mulation following drug administration. ephrine-induced c y c l i c AMP  accu-  The time course of the norepin^-.  response was not examined i n the present study,  but others have indicated that the c e l l u l a r nucleotide concentration r i s e s rapidly, reached a sharp peak and then declines to an intermediate l e v e l within 30 seconds of bolus administration of catecholamine (Cheung and Williamson, 1965; Robinson et a l . , 1965; McNeill and Verma, 1973).  In  contrast, glucagon produced a more gradual increase i n c y c l i c AMP.  Also,  a submaximal tissue concentration was maintained at least 120  seconds  after i n j e c t i o n of a 2.0 yg dose (Figure 4) Although the majority of experimental data strongly supports the second messenger.hypothesis, the l i t e r a t u r e does contain reports of dissociations between the positive inotropic e f f e c t of glucagon and the catecholamines and their influence on i n t r a c e l l u l a r c y c l i c AMP. have already been discussed.  The papers on glucagon  Those on the catecholamines appear to be  equally unconvincing. Shanf eld e_t_al. (1969) were able to block the norepinephrine-induced increase i n c y c l i c AMP but not the positive inotropic response using a methoxamine congener and weak (3 adrenergic blocking agent N-isopropyl-methoxamine.  However Wastjla et al.(1972), using butoxamine, instead observed  a simultaneous reduction.in c o n t r a c t i l e force with the decrease i n c y c l i c AMP.  These l a t e r investigators also noted the direct depressant e f f e c t of  the blocking agent on myocardial force, as w e l l as basal tissue c y c l i c  AMP  content and therefore proposed that the force changes seen by Shanfeld and  81  associates (1969) may only have been a consequence of their readjustment of d i a s t o l i c tension ( f i b r e length) a f t e r perfusion with  isopropylmethoxamine.  Another i n d i c a t i o n of a d i s s o c i a t i o n was presented by Oye and Langslet (1972) who found that isoproterenol perfused through the rat heart did not increase c y c l i c AMP p r i o r to the onset of the p o s i t i v e inotropic event. The shortcomings of this report were discussed previously with respect to the glucagon d i s s o c i a t i o n . Exogenous c y c l i c AMP perfusion, i n the presence of DMSO to f a c i l i t a t e membrane transport, was found to increase glycogen phosphorylase a c t i v a t i o n i n the i s o l a t e d guinea pig heart without elevating the force of contraction (Kjekshus et a l . , 1971).  The authors believed this represented a d i s s o c i a -  t i o n of c y c l i c AMP from p o s i t i v e inotropism because the % phosphorylase a was enhanced and that this was a consequence of i n t r a c e l l u l a r c y c l i c nucleotide accumulation.  However others have shown that active phosphorylase  l e v e l s can be elevated independently of c y c l i c AMP (Namm et a l . , 1968; Dobson and Mayer, 1973; Hartley and McNeill, unpublished observations) and so the i n t e r p r e t a t i o n of Kjekshus and coworkers  (1971) must be questioned.  Furthermore, the data on phosphorylase a c t i v a t i o n are also suspect because the control levels are very high, and DMSO by i t s e l f had a considerable stimulatory e f f e c t (which the authors claim i s unimportant).  The dose  dependency of the c y c l i c AMP-produced phosphorylase a c t i v a t i o n was also not investigated and the p o s s i b i l i t y of a nonspecific activation remains open. C y c l i c AMP i s believed to be a common mediator of the inotropic effects of the catecholamines and glucagon, and also of the methylxanthines.  On this  b a s i s , the influence of these drugs on properties of a single contraction would be expected to be s i m i l a r , and should be mimicked by dibutyryl c y c l i c  82 AMP.  To summarize what was mentioned e a r l i e r , a l l four agents increase  maximal developed tension and rate of r i s e of tension. and d i b u t y r y l c y c l i c AMP  decrease time to peak tension and shorten the dur-  ation of the active state (Skelton et a l . , 1970; 1972;  The catecholamines  Meinertz et a l . , 1974;  S p i l k e r , 1970;  Blinks et a l . ,  1975a) but glucagon has no influence on these  parameters (Glick et a l . , 1968;  S p i l k e r , 1970; Marcus et a l . , 1971).  p h y l l i n e and other methylxanthines increase time to peak tension and relaxation time (Skelton et a l . , 1971;  Blinks et a l . , 1972).  Theoprolong  These d i f f e r -  ences can only be explained i f the drugs are assumed to have multiple mechanisms of action, one of which might be elevation of the i n t r a c e l l u l a r c y c l i c AMP  concentration.  The f i n a l drug response would thus represent the  t o t a l of various e f f e c t s on c e l l u l a r processes  sum  leading to tension devel-  opment. Since the catecholamines, methylxanthines and glucagon a l l a l t e r myoc a r d i a l calcium exchangeability, this may actions on c y c l i c AMP.  Meinertz et al.. (1973  fects of d i b u t y r y l c y c l i c AMP c o n t r a c t i l e force. et a l . , 1970).  be a possible consequence of t h e i r a,b) have shown s i m i l a r ef-  and norepinephrine  on calcium-45 exchange and  Glucagon w i l l also promote calcium-45 i n f l u x  (Nayler  In guinea pig a t r i a , theophylline increased calcium-45  uptake and release (Scholz, 1971).  The extent to which a drug i n t e r f e r e s  with normal calcium control processes may  determine the magnitude of i t s  inotropic e f f e c t . One of several s i t e s where c y c l i c AMP  may  act to influence calcium  exchangeability i s at the sarcolemma by increasing membrane permeability to calcium (Rasmussen et a l . , 1972).  Catecholamines have been shown to  enhance the transmembrane calcium current passing through the slow calcium channels during the plateau phase of the action p o t e n t i a l (review by Reuter,  83  1974).  Tsien et a l . (1972) have demonstrated that, l i k e the catecholamines,  dibutyryl c y c l i c AMP  and c y c l i c AMP  increase action p o t e n t i a l plateau am-  plitude and shorten i t s duration. These data suggest that the catecholamines therefore increase the slow current by f i r s t acting on adenylate cyclase. The temporal sequence of events cannot be d i r e c t l y determined at present but Watanabe and Besch (1974) performed sophisticated experiments to show that c y c l i c AMP  did increase p r i o r to isoproterenol-restored mechanical a c t i v i t y  i n arrested hearts.  Dibutyryl c y c l i c AMP  also restored contractions but  the onset of action was slower (Watanabe and Resch, 1974; Schneider and Sperelakis, 1975). I f c y c l i c AMP mediates the catecholamine effects on the cardiac action p o t e n t i a l , other agents which elevate c e l l u l a r accumulation of the nucleotide would be expected to display s i m i l a r properties.  Glucagon has not been  widely investigated from the e l e c t r o p h y s i o l o g i c a l standpoint, but those studies which have been conducted indicate glucagon does not raise the p l a t eau amplitude (Spilker, 1970; Prasad, 1975).  No consistent influence on the  duration of the action potential has been shown, f o r Prasad (1975) noted a dose-dependent shortening, whereas Spilker (1970) observed a s l i g h t prolongation.  Spilker also de-emphasized the importance of action p o t e n t i a l  duration because v a r i a b l e effects have been produced by catecholamines, depending on the experimental procedure.  Morad and Goldman (1973) pointed  out numerous technical d i f f i c u l t i e s which are encountered i n electrophysiol o g i c measurements.  Therefore these data are of questionable value.  Manganese ion can antagonize the positive inotropic e f f e c t of glucagon (Spilker, 1970; Nayler et a l . , 1970) i n a manner s i m i l a r to what was with catecholamines (Sabatini-Smith and Holland, 1969). influence of glucagon on calcium entry.  observed  This suggests some  However because glucagon could not  84 elevate the depressed action p o t e n t i a l amplitude to a normal l e v e l (Spilker, 1970) while norepinephrine  could restore the plateau p o t e n t i a l indicates  there are differences i n t h e i r effects on calcium i n f l u x .  Part of the d i f -  ference may be due to the type of tissue investigated since, i n this case, Spilker (1970) used Purkinje fibres which are s p e c i a l i z e d for conduction rather than contraction.  Catecholamines have marked effects on heart rate  whereas we observed lesser effects with glucagon.  Consequently the i n f l u -  ence of these drugs on action potentials i n Purkinje fibres may r e f l e c t t h e i r respective chronotropic rather than inotropic actions. Watanabe and Besch (1974) and Schneider and Sperelakis (1975) could not restore e x c i t a b i l i t y and contractions to potassium-depolarized pig hearts with glucagon.  guinea  This was evidently considered together with the  f a i l u r e of glucagon to elevate c y c l i c AMP i n this preparation (Watanabe and Besch,1974) as support f o r the idea of a c y c l i c AMP-mediated increase i n the slow calcium current which i s apparently e s s e n t i a l f o r e l e c t r o ^ mechanical a c t i v i t y . The i n t r i g u i n g results of Henry et al.(1975X who demonstrated a posi t i v e inotropic effect of glucagon i n guinea p i g independent of adenylate cyclase stimulation, together with the data of Watanabe and Besch, must be interpreted cautiously.  S u p e r f i c i a l l y the results indicate that glucagon  (in guinea pig) does not activate the slow calcium channels to increase c o n t r a c t i l e force but must instead influence some other c e l l u l a r process.. It i s implied that slow calcium channels are not activated because glucagon was  incapable of restoring tension development.  I t i s also implied that  the reason glucagon f a i l e d to increase membrane calcium transport was because i t did not stimulate adenylate  cyclase.  Because the p o s i t i v e ino-  tropic action of glucagon i s not consistently observed i n guinea pig i t i s  85  d i f f i c u l t to determine whether the preparation of Watanabe.'.and Besch (1974) was  merely unresponsive, perhaps due to the absence of a cardiac glucagon  receptor, or whether the polypeptide hormone r e a l l y does not act on the slow calcium channels.  Because of the experimental  protocol, the hypothetical  glucagon receptor may have been inactivated and therefore an action on calcium((and  c y c l i c AMP) may  s t i l l be a p o s s i b i l i t y .  The study of Henry  et a l . (1975) indicated that glucagon does not require elevated levels of i n t r a c e l l u l a r c y c l i c AMP  to produce i t s p o s i t i v e inotropic action but does  not suggest the absence of a causal relationship i n situations where adenyl a t e cyclase i s stimulated.  The above-described investigations also do not  permit speculation of the role of c y c l i c AMP  i n the glucagon response i n  other species. Measurements of the influence of glucagon on calcium i n f l u x are contradictory.  Nayler et a l . (1970) did observe that glucagon increased  calcium-45 i n f l u x i n dog p a p i l l a r y muscle.  In contrast, Visscher and  Lee  (1972) obtained negative results with the i s o l a t e d cat heart, even though the doses of glucagon used had a considerable inotropic e f f e c t .  Indirectly,  the competitive antagonism of glucagon-induced force changes by manganese (Nayler et a l . , 1970;  S p i l k e r , 1970)  i s consistent with a hormonal action  on membrane calcium transport, yet this does not explain why  glucagon was  incapable of restoring excitation-contraction coupling i n the experiments of Watanabe and Besch (1974) or Schneider and Sperelakis (1975). Visscher and Lee  (1972) reported that while glucagon could prevent  cardiac arrest i n the i s o l a t e d , perfused cat heart exposed to 0.09 i t could not restore contractions i n a zero-calcium medium.  M calcium,  This suggests  that a major action through i n t r a c e l l u l a r calcium release i s u n l i k e l y .  In  agreement with the observation by Visscher and Lee, Nayler et al.(1970) noticed no action of glucagon on e i t h e r calcium binding or release by i n t r a -  86  c e l l u l a r organelles. Katz and associates (1975) proposed that c y c l i c AMP  mediates the i n -  creased rate of relaxation after catecholamine administration. demonstrated i n v i t r o that c y c l i c AMP membrane phosphorylation  stimulates sarcoplasmic  They have reticular  i n p a r a l l e l with calcium transport and suggest a  causal relationship between c y c l i c AMP  and the accelerated relaxation rate.  Meinertz et al.(1975 b) provided further evidence by showing that d i b u t y r y l c y c l i c AMP  mimicked the behaviour of norepinephrine  state following twitch stimulation. high-potassium induced  Both agents were alsonnoted  to depress  contracture.  On the basis of the hypothesis which increase c y c l i c AMP  of Katz et a l . (1975), other agents  might be expected to also produce abbreviation  of systole, yet glucagon does not Marcus et a l . , 1971).  on duration of the active  (Glick et a l . , 1968;  S p i l k e r , 1970;  Furthermore, methylxanthines prolong the duration  of the active state (Blinks et a l . , 1972).  Also, Gibbs (1967) and Blinks  and coworkers (1972) have demonstrated an antagonism by methylxanthines of catecholamine-induced shortening. drug i s assumed to. work v i a c y c l i c  This i s d i f f i c u l t to explain i f each AMP.  In the present study, the concurrent  administration of glucagon and  methylxanthines produced augmented responses which were consistent with the second messenger theory.  However i t i s appropriate to consider other  mechanisms by which such potentiations may  occur.  A blockade of catechol-  amine uptake by theophylline appears u n l i k e l y because R a i l and West (1963) obtained a potentiation of the inotropic response with tyramine rather than an antagonism.  McNeill and associates (1969) also noted that theophylline  produced a s h i f t to the l e f t of the dose-response curve of tyramine and isoproterenol on phosphorylase a c t i v a t i o n and a further s h i f t of the nor-  87  epinephrine  response i n tripelennamine-treated  rats.  Methylxanthines are capable of releasing endogenous catecholamines (Westfall and Fleming, 1968).  McNeill et al.(1969) demonstrated that the  s l i g h t l y elevated basal phosphorylase a c t i v i t y as a r e s u l t of theophylline was blocked by propranolol or reserpine. the p o s s i b i l i t y that 1 mM cannot be disregarded.  Therefore, i n the present study,  theophylline i s releasing endogenous catecholamines  Control c y c l i c AMP  levels were s i g n i f i c a n t l y greater  a f t e r theophylline perfusion for 15 minutes (Tables 8,9). phorylase a was ence was  greater i n the theophylline-exposed  not s i g n i f i c a n t (Tables 5,6)  evident that c y c l i c AMP  hearts but the d i f f e r -  and from dose-response studies i t was  increases could be detected to 0.25  when phosphorylase elevation could not (Figure 6). of catecholamines may  Also, % phos-  yg glucagon  Therefore small amounts  be l i b e r a t e d s u f f i c i e n t to affect basal c y c l i c  accumulation without detectably r a i s i n g phosphorylase a c t i v i t y . ient p o s i t i v e inotropic effect of 1 mM  AMP  The trans-  theophylline perfusion might also be  explained by catecholamine release. The data from the present i n v e s t i g a t i o n strongly implicates c y c l i c as the mediator of the myocardial  actions of glucagon.  AMP  I t i s tempting to  suggest that the potentiation of the glucagon responses i n the presence of theophylline was  due to greater tissue levels of c y c l i c AMP.  c i a t i o n at low doses of glucagon between c y c l i c AMP  The disso-  on the one hand, and  c o n t r a c t i l e force and phosphorylase a c t i v a t i o n on the other hand, may attributed to differencesi-in the l i m i t s of measurement s e n s i t i v i t y . fact, theophylline 1 mM  be In  abolished the apparent d i s s o c i a t i o n between con-  t r a c t i l e force and c y c l i c AMP  following 0.25  yg glucagon (Figure 6).  The  theophylline perfusion s l i g h t l y altered the time-course of glucagon-induced phosphorylase a c t i v a t i o n and c o n t r a c t i l e force increases, but the time course of c y c l i c AMP  accumulation was  altered i n a p a r a l l e l fashion (Figure 4).  88  Even though theophylline appeared to act by phosphodiesterase i n h i b i t i o n , the data must be interpreted cautiously. p h y l l i n e alone i l l u s t r a t e this point.  The results obtained with theo-  I f theophylline exhibited i t s p o s i t i v e  inotropic e f f e c t by r a i s i n g i n t r a c e l l u l a r c y c l i c AMP then p r e - i n j e c t i o n syst o l i c tension should be greater a f t e r methylxanthine exposure to correspond to increased control c y c l i c AMP concentration (Table 9 ) .  However, a mean  depression i n s y s t o l i c tension of approximately 6 % was instead observed (Table 2 ) .  Active phosphorylase was also not increased s i g n i f i c a n t l y  (Table 6 ) . A l l of the observed potentiations could be explained on the basis of a methylxanthine e f f e c t on calcium. through c y c l i c AMP, or both.  This may be a direct e f f e c t or mediated  Watanabe and Besch (1974) and Schneider and  Sperelakis (1975) each determined the a b i l i t y of theophylline to restore mechanical a c t i v i t y presumably v i a the slow calcium channels when theop h y l l i n e also raised i n t r a c e l l u l a r tissue c y c l i c AMP levels (Watanabe and Besch, 1974).  I f , as Katz et a l . (1975) propose, the catecholamine-induced  acceleration of relaxation i s mediated by c y c l i c AMP, then methylxanthines must not act v i a phosphodiesterase i n h i b i t i o n e n t i r e l y ( i f at a l l ) because they prolong systole and antagonize the isoproterenol shortening (Blinks et a l . , 1972).  Other investigators have noted an imperfect c o r r e l a t i o n of  phosphodiesterase i n h i b i t i o n with cardiac actions of methylxanthines and papaverine (McNeill et a l . , 1973; McNeill et a l . , 1974; Henry et a l . , 1 9 7 5 ) . A d i r e c t e f f e c t on i n t r a c e l l u l a r calcium homeostasis by methylxanthines would explain these differences as well as account f o r the augmentation of the glucagon responses.  However such an influence on i n t r a c e l l u l a r calcium  i s not an e n t i r e l y s a t i s f a c t o r y explanation e i t h e r , because  theophylline  f a i l e d to potentiate the inotropic e f f e c t of calcium, i n concentrations which  89  potentiated the inotropic effects of norepinephrine and dibutyryl c y c l i c AMP  (Skelton et a l . , 1971). Thus i t appears l i k e l y that methylxanthines work through more than  one mechanism, a property which i s ubiquitous among drugs.  To expect that  glucagon also works e n t i r e l y through the adenylate cyclase system i s also naive and while the present investigation strongly implicates c y c l i c  AMP,  i t does not establish a causal.relationship, nor does i t exclude other mechanisms of action.  90  SUMMARY AND CONCLUSIONS  Glucagon increased myocardial accumulation of c y c l i c AMP, elevated % phosphorylase _a, and enhanced the force of contraction i n a time and dose dependent manner. Time experiments i n the present study established that following glucagon challenge, tissue c y c l i c AMP concentration increased p r i o r to s i g n i f i c a n t increases i n c o n t r a c t i l e force or phosphorylase a.. C y c l i c AMP remained elevated for prolonged periods as did s y s t o l i c tension and the % active phophorylase. Dose experiments revealed that c y c l i c AMP could increase a f t e r doses of glucagon which would not e l i c i t a detectable p o s i t i v e inotropic e f f e c t or glycogen phosphorylase activation.  These results suggest that c y c l i c  AMP i s involved i n the metabolic and mechanical actions of glucagon but that other factors may contribute to the end-organ response.  The apparent  dissociation may only be a consequence of differences i n s e n s i t i v i t y of the various measuring  systems.  Further evidence supporting the second messenger hypothesis was supplied i n the experiments with theophylline .  A 1 mM concentration of the xanthine  was found to be e f f e c t i v e i n i n h i b i t i n g phosphodiesterases i n the i n t a c t rat hearti as indicated by the dramatic enhancement of glucagon-induced c y c l i c AMP accumulation.  Theophylline perfusion also augmented the  positive inotropic e f f e c t of glucagon and the % of active phosphorylase. The e f f i c a c y of glucagon i n a c t i v a t i n g phosphorylase was improved i n the presence of the methylxanthine.  The data strongly implicate an association  between myocardial c y c l i c AMP content and the glucagon-induced inotropic and metabolic responses.  However the results do not prove a causal  91  relationship, nor do they exclude other mechanisms of action. Theophylline 1 mM was observed to increase and then depress s y s t o l i c tension, and also produced s l i g h t changes i n phosphorylase a c t i v i t y . Cyclic nucleotide accumulation was elevated r e l a t i v e to buffer-perfused hearts after theophylline exposure.  .Therefore, the methylxanthine.'  appeared to be working through phosphodiesterase i n h i b i t i o n .  However,  theophylline can cause release of endogenous catecholamines and has profound effects on calcium homeostasis i n v i t r o . 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C y c l i c AMP (pmol/mg uncorrected  C y c l i c AMP (pmol/mg corrected  % uncorrected , ' -corrected  0.324  0.328  98.8  0.172  0.183  94.0  0.469  0.460  102.0  0.467  0.460  101.5  0.434  0.423  102.6  0.496  0.511  97.1  0.575  0.564  102.0  0.553  0.545  101.5  0.268  0.259  103.5  0.264  0.261  101.1  0.221  0.214  103.3  0.183  0.174  105.2  0.359  0.352  102.0  0.302  0.288  104.9  0.523  0.515  101.6  0.420  0.433  97.0  0.262  0.255  102.7  0.228  0.238  95.8  0. 378  0.366  103.3  0.438  0.375  116.8  

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