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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 British Columbia, 1974 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Division of Pharmacology and Toxicology of the Faculty of Pharmaceutical Sciences We accept this thesis as conforming to the required standard THE UNIVERSITY - OF 'BRITISH COLUMBIA September, 1975 In presenting t h i s t h e s i s in p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree that permission for extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s rep r e s e n t a t i v e s . It i s understood that copying or 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 gain s h a l l not be allowed without my w r i t t e n permission. Depa rtment The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date 0 * £ / /975 i i ABSTRACT The biochemical and mechanical effects of glucagon were investigated in the isolated, perfused rat heart. Glucagon produced time and dose-dependent alteractions in myocardial force of contractions, glycogen phosphorylase activation and cyclic AMP accumulation. The positive inotropic effect was maximal following a 4.0 yg dose, after which systolic tension increased 77.1 + 7.9 % (N=6) relative to pre-injection systolic tension. This dose was also found to produce the maximal phosphorylase activation (38.8 +3.5 % in the a. form). The cyclic 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 for cyclic AMP accumulation remains undetermined. The minimum effective glucagon dose for increasing contractile force and % phosphorylase a was 0.5 yg, whereas only 0.25 yg was required to significantly elevate the ventricular cyclic AMP content over basal level. The temporal sequence of these cardiac events was determined following 2.0 yg glucagon. Cyclic AMP increased significantly at 15 seconds. The positive inotropic effect was detectable 25 seconds after injection and % phosphorylase a. elevation at 30 seconds. A l l three parameters remained sig-nificantly greater than control at least 120 seconds after glucagon admin-istration. The observed time course is consistent with the proposal that cyclic AMP mediates the glucagon-elicited alterations in force and glycogen phosphorylase activity. —8 Propranolol 10 M was found not to significantly influence glucagon-induced changes in force of contraction, % phosphorylase a or tissue cyclic AMP content, although this concentration readily, ^blocked the positive ino-i i i tropic response to norepinephrine. It is therefore unlikely that the car-diac actions of glucagon are a result of endogenous catecholamine release or an interaction with the catecholamine 3 receptor. To further elucidate the role of cyclic AMP in 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 in % phosphorylase a, con-tr a c t i l e force and cyclic AMP accumulation which were considerably greater than in buffer-perfused hearts. Systolic tension was increased 116.3 + 7.4 % over pre-injection level with 4.0 yg glucagon, and % phosphorylase a. was augmented to the maximum theoretical value of 72.2 % (N=l) with 8.0 yg glucagon. The most dramatic influence of theophylline was on ventricular cyclic 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 in buffer-perfused hearts was maintained in the presence of theophylline 1 mM. The data obtained in the present study strongly implicate an association between.myocardial cyclic AMP content and the metabolic and mechanical actions of glucagon. However, the mechanism by which theophylline potentiated the glucagon responses is not clear. One mM theophylline possessed i n t r i n s i c a b i l i t y to alter force of contraction, phosphorylase activation and cyclic AMP accumulation in a manner inconsistent with the widely-accepted theory of phosphodiesterase inhibition. Control levels of cyclic AMP were approx-imately 30 % greater than in buffer-perfused hearts yet the % active phos-phorylase was not significantly elevated. Furthermore, 1 mM theophylline was cardiodepressant in many animals. These observations•indicate that data with theophylline must be cautiously interpreted with respect to cyclic AMP involvement in the theophylline cardiac responses, and in 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 v i LIST OF FIGURES \* v i i LIST OF ABBREVIATIONS v i i i INTRODUCTION 1 1. The role of calcium in excitation-contraction coupling 1 2. The second messenger theory of catecholamine-induced actions in 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. Cyclic AMP assay 31 I. Tissue extraction • 31 II. Cyclic AMP binding reaction 32 III. Calculation of results 33 D. Sta t i s t i c a l methods 33 RESULTS 35 DISCUSSION 65 SUMMARY AND CONCLUSIONS 90 BIBLIOGRAPHY 92 APPENDIX ' 101 v i LIST OF TABLES TABLE Page 1. The effect of time on the positive inotropic response to 2 yg glucagon in the buffer-perfused and theophylline-perfused rat heart. 40 2. The effect of various doses of glucagon on contractile force in the isolated buffer-perfused and theophylline-perfused rat heart. 45 _ g 3. The influence of propranolol 10 M on the positive inotropic effect of glucagon in the isolated perfused rat heart. 47 —8 4. The influence of 10 M propranolol on the positive inotropic action of norepinephrine in the isolated per-fused rat heart. 50 5. The effect of time on glucagon-induced cardiac phosphory-lase activation in the buffer-perfused and theophylline-pe7. perfused isolated rat heart. 53 6. The effect of various doses of glucagon on cardiac glycogen phosphorylase activation in the buffer-perfused and theophylline-perfused isolated rat heart. 54 _ g 7. The influence of propranolol 10 M on glucagon-induced phosphorylase activation in the isolated perfused rat heart. 56 8. The effect of time on cardiac cyclic AMP accumulation f o l -lowing administration of 2 yg glucagon into the buffer-perfused and theophylline-perfused rat heart. 57 9. The effect of various doses of glucagon on cardiac cyclic AMP accumulation in the buffer-perfused and theophylline-perf used isolated rat heart. 59 — 8 10. The influence of propranolol 10 M on glucagon-induced cyclic AMP accumulation in the isolated perfused rat heart. 60 v i i LIST OF FIGURES FIGURE Page .11. Schematic representation of the second messenger concept. 5 2. Enzymes involved in the control of myocardial glycogenolysis. 7 3. An adaptation of the general model of c e l l activation (Rasmussen et a l . , 1972) to myocardial tissue. 13 4. Effect of glucagon (2 yg) on cyclic AMP content, con-tra c t i l e force and percentage phosphorylase a. at various times following injection into rat hearts perfused with buffer or buffer plus theophylline. 37 5. Effect of time on the absolute change in tension following injection of 2 yg glucagon into the isolated buffer-per-fused rat heart. 39 6. The effect of various doses of glucagon on cardiac cyclic AMP content, contractile force and percentage phosphorylase a. in rat hearts perfused with buffer or buffer plus theophylline (1 mM). 42 7. The effect of various doses of glucagon on the absolute change in systolic tension in buffer-perfused and theo-phylline-perfused rat hearts. 44 — 8 8. The influence of propranolol 10 M on glucagon-induced changes in cardiac cyclic AMP content, contractile force and percentage phosphorylase a.. 49 —8 9. The influence of propranolol 10 M on the positive '.inotropic effect of norepinephrine. 52 ABBREVIATIONS v i i i ATP adenosine 5' -triphosphate AMP adenosine monophosphate cyclic AMP adenosine 3',5' -cyclic monophosphate CK Chenoweth-Koelle cpm counts per minute DB-c-AMP cyclic N6-2'-0 dibutyryl-AMP EDTA ethylenediamine tetra-acetic acid G-l-P glucose-l-phosphate S.E.M. standard error of the mean TCA trichloroacetic acid Tris tri(hydroxymethyl)aminomethane i x ACKNOWLEDGEMENTS I wish to express my sincere gratitude to Dr. John McNeill for his guidance and patience throughout this project. I would also like to thank Dr. Don Lyster for his valuable assistance with the cyclic AMP assay procedure. Appreciation i s extended to Miss Elizabeth Hartley and Miss Annette Holmvang for their technical assistance in the laboratory. A special thank you is extended to Miss Barbara O'Malley and Miss Marilyn James for their help in the preparation of this manuscript. The generous financial 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 in Excitation-Contraction Coupling The intracellular concentration of free calcium is now generally ac-cepted to be a major determinant of the activation state of myocardium (Langer, 1968). However, the processes involved in the regulation of calcium are s t i l l poorly understood. Furthermore, while calcium is essential 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 potential is determined by transmembrane fluxes of sodium, calcium and potassium. A rapid inward sodium current gives rise to the spike of the action potential. This i s followed by a slower and smaller inward current responsible for the plateau phase. Voltage-clamp experiments provide evidence that this current is caused predominantly by calcium ions (Beeler and Reuter, 1970a) but sodium ions can also flow through the channel (Rougieret a l . , 1969). Sarcolemmal repolarization i s believed to be due to a sudden increase in potassium efflux. Total duration of the cardiac action potential varies from 200 to 600 milliseconds compared to 5 milliseconds in skeletal muscle (Morad and Goldman, 1973). Mechanical activity lags behind the ele c t r i c a l events. The onset of contraction follows the upstroke of the action potential by 20-40 m i l l i -seconds (Morad and Goldman, 1973). Relaxation occurs after the membrane has begun to repolarize. The duration of the active state i s approximately 200 milliseconds. According to the sliding filament theory, active tension develops when calcium binds to troponin and removes the inhibitory 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 be through sequestration by mitochondria, sarcoplasmic reticulum, or a combination of these. Katz and Repke (1967) investigated the kinetic properties of calcium binding by a cardiac microsomal preparation and proposed that the rate of binding would be sufficient for relaxation of intact muscle. Solaro and Briggs (197-4) reached similar conclusions. Mitochondrial uptake may only be of minor importance in the normal cycle (Solaro and Briggs, 197 4; Williamson et a l . , 1974).but might be necessary for relaxation of fully-activated muscle (Solaro and Briggs, 1974). To prevent the c e l l from becoming overloaded with calcium, Reuter (1974) suggests that after binding by the sarcoplasmic reticulum, the ion is transported across the sarcolemma by the sodium-calcium exchange system. By this mechanism, calcium extrusion is coupled to the passive influx of sodium. Evidence that at least part of the driving 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 efflux and extracellular 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 for release of "trigger" calcium from internal sites, possibly the lateral 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 internal membrane trans-lated from the sarcolemma. Most investigators believe the calcium ions en-tering 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 possibility (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). They suggest there are two sources of activator calcium — a superficial (sarcolemmal) source and an intracellular releasing site — which allow the calcium concentration to rise to the thres-hold for myofibrillar activation. Although the 'relaxing system' is also stimulated by the higher calcium levels, the influx rate is greater and so active state intensity increases. At a point when efflux away from the myofibrils equals influx, the contractile state is at a maximum (influx is reduced due to membrane repolarization). 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 level of membrane polarization. The action po-tential ( f i r s t 100 milliseconds) would release internal calcium. During the plateau phase inward calcium transport would be maintained by a Ca^-K exchange carrier i n the sarcolemma. (The efflux of potassium would be coupled to the influx of calcium). The degree of tension development would depend on the sum of calcium from these two components and any change i n the contri-bution of either would change the maximal force generated. Although some of the activator calcium is recycled, maintenance of releasable stores would depend on the slow inward current. Drugs may produce a positive inotropic effect by influencing one or several of the steps i n the excitation-contraction cycle. Catecholamines increase the strength of contraction in mammalian myocardium but the mech-anism of action i s s t i l l speculative. The membrane potential during the plateau phase is more positive than normal in 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 level (Reuter, 1974). These electrophysiologic experiments 4 verify earlier tracer studies (Langer, 1968) showing an enhanced influx 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 efflux (Tsien et al.,1972) or to an. effect on sarcoplasmic reticular 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 is discussed in the following section. 2. The second messenger theory of catecholamine-induced actions i n  myocardium Cyclic AMP was discovered during the course of investigations on mech-anisms of hormone-induced hepatic glycogenolysis (Sutherland and Rail, 1958). Within a few years^ cyclic AMP was implicated in a variety of hormone re-sponses. The "second messenger" theory of hormone action was proposed to account for the rapidly accumulating data (Sutherland et a l . , 1965). As originally outlined, a hormone was suggested to interact directly with adenylate cyclase located in the target c e l l membrane. The increased level of cyclic AMP then served as an intracellular messenger to modify en-zyme activity or otherwise bring about the physiological response. Tissue concentrations of the nucleotide are regulated by adenylate cyclase which catalyzes cyclic AMP formation from ATP, and specific phosphodiesterases which cause breakdown to 5'AMP. The original model has been modified to indicate the hormone receptor and adenylate cyclase are not the same entity (Figure 1). Varied Stimuli Endocrine Gland HORMONE (first messenger) Inactivated Hormone OC o O 5 - A M P ATP J d « J > < f phospho-diesterase Cyclic 3,5-AMP (second messenger) Physiological Responses Steroids,Thyroid Hormonejetc. .plasma membrane of target cell Fig-1. Schematic representation of the second messenger concept 6 The second messenger system allows hormones to be effective without permeating the c e l l membrane. It also provides a method of modifying a given hormonal stimulus. Hormone specificity 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 their cardiostimulatory effects according to the second messenger theory. The strongest evidence they cited were experiments by Robis op. Jand coworkers (1965) where epinephrine injection into isolated rat heart e l i c i t e d a marked elevation of cyclic AMP prior to the increases in contractile 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 cyclic AMP i n cardiac glycogenolysis i s well understood. A sequence of enzymic reactions i s initiated (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 is required for activity 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 intracellular cyclic AMP is increased (Namm et a l . , 1968). Als'o; anoxia w i l l stimulate phosphorylase when cyclic AMP i s not elevated (Dobson and Mayer, 1973). Therefore, the cyclic nucleotide is not independently responsible for glycogenolysis regu-lation. 7 CYCLIC AMP Phosphorylase b Kinase (inactive) ATP Phosphorylase b Kinase Kinase \ ADP Phosphorylase b Kinase (active) Phosphorylase b (inactive) ATP +•+ ADP Phosphorylase a-(active) Glycogen + Pi Glucose-l-Phosphate FIGURE 2. Enzymes involved in the control of myocardial glycogenolysis 8 At one time phosphorylase activation was thought to be necessary for in i t i a t i o n of mechanical activity. However, low doses of epinephrine pro-ducing changes in contractility did not produce changes in 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 effect by increasing the energy supply. Most proponents of a cyclic AMP-meditated inotropism favor a more direct involvement (as opposed to an involvement through phosphorylase activation). However, the sequence of events between adenylate cyclase stimulation and contraction is not known. Early i n vitro evidence of an association was found by Murad et a l . , (1962). They noted the order of potency of different catecholamines in stimulating adenylate cyclase was the same as that for producing changes in contractility. Furthermore, dichloroisoproterenol blocked the cyclase activation. As mentioned earlier, many temporal investigations in the intact heart have established that cyclic AMP increases prior to, or at least concurrent with, the catecholamine-induced increase in force (review by Sobel and Mayer, 1973). If cyclic AMP i s an intracellular mediator then exposure to the nucleo-tide or a derivative should also increase the force of contraction. Robison and coworkers (1965) attributed their i n i t i a l failure to change the strength of contraction using cyclic AMP to the poor membrane penetrating a b i l i t y . In contrast Meinertz et a l . (1974) demonstrated that the positive inotropic action of cyclic N^-2'-O-dibutyryl-AMP (DB-c-AMP) was concentration-dependent 9 in isolated el e c t r i c a l l y driven a t r i a l and ventricular preparations.. These experiments confirmed the earlier findings of Skelton et al.(1970) and Drummond and Hemmings (1972). The literature also contains reports of dissociations between cyclic AMP and the inotropic effect of catecholamines. Shanfeld, Fraser and Hess (1969) were able to block norepinephrine-induced cyclic AMP production without a l -tering the mechanical action. Langslet and Oye (1970) noted that at low temperatures where both epinephrine and cyclic AMP increased phosphorylase activation, only epinephrine was capable of positive inotropism. Dibutyryl cyclic AMP promoted glycogenolysis in concentrations insufficient to increase contractile force (Oye and Langslet, 1972) unlike isoproterenol and suggested the cardiac response was basically different. Although contractility is augmented with dibutyryl cyclic AMP, relatively high concentrations are necessary and the effect takes longer to develop than phosphorylase activa-tion (Sobel and Mayer, 1973). Thus, while a considerable volume of evidence supports a cyclic AMP involvement in the contractile response some experi-mental observations are inconsistent with the hypothesis. The most recent experiments have focused on intracellular sites of action for cyclic AMP. Entman, Levey and Epstein (1969) demonstrated an epinephrine-sensitive cyclase in a cardiac microsomal preparation also cap-able of increasing calcium uptake. However, contamination with sarcolemma was not ruled out. Sulakhe and Dhalla (1973) and Katz and associates (1974) have obtained more purified sarcoplasmic reticulum preparations which possess an adenylate cyclase similar in properties to the sarcolemmal enzyme. Entman et a l . (1969) postulate that cyclic AMP may 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 cyclic AMP-10 stimulated calcium uptake by cardiac microsomes when protein kinase was present. The concentrations of cyclic AMP were similar to those activating 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 cyclic 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 in catecholamine-induced inotropism remains to be investigated. Cyclic AMP may induce membrane permeability changes, particularly to calcium. Scholz et al.(1975) reported that dibutyryl cyclic AMP influenced calcium-45 exchange in a manner similar to norepinephrine or theophylline. Cyclic AMP, monobutytyl cyclic AMP and dibutyryl cyclic AMP perfused into cardiac Purkinje fibres a l l increased the action potential plateau amplitude and shortened the plateau duration in an identical manner to the catechola-mines (Tsien et a l . , 1972). These results indicate a cyclic nucleotide-mediated increase in the slow inward calcium current and in the outward po-tassium current. Further evidence supporting the idea that cyclic AMP.is involved in excitation-contraction coupling .is provided i n recent combined electro-physiological-mechanical studies (Watanabe and Besch, 1974; Schneider and Sperelakis, 1975). Both isoproterenol and dibutyryl cyclic AMP restored excitability and contractions to potassium-depolarized hearts. This cardiac model assumes that excitation is. accomplished by an increase in the slow calcium current. If cyclic AMP was acting as a second messenger to increase calcium i n -11 flux then tissue levels should rise prior to this influx. Direct experimental techniques for correlative measurements are not yet available. However, Watanabe and Besch (1974) did observe isoproterenol-induced increases in myocardial cyclic AMP prior to the restoration of mechanical activity. Much of the above-mentioned experimental evidence indicates that a cyclic AMP influence on calcium homeostasis i s probable. Rasmussen and associates (1972) have reviewed the interaction of calcium and cyclic 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 third messenger (Figure 3). This model nicely accounts for 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 cyclic AMP as a mediator of i t s end-organ response. F i r s t , hormonal stimulation should produce changes in intact tissue levels of cyclic AMP, and this should either precede or. occur simultaneously with the physiological event. Second, the target tissue should possess an adenylate cyclase which is stimulatable by the hormone in broken c e l l perparations. Third, the effect of the hormone should be mimicked by the addition of exogenous cyclic AMP or one of i t s derivatives. Finally, agents which modify phosphodiesterase activity should correspondingly mod-i f y the hormonal response. These c r i t e r i a have been essentially satisfied for catecholamines in myocardium with a few exceptions mentioned earlier (Rasmussen et al . , 1 9 7 2 ; Sobel and Mayer, 1973). Experiments attempting to satisfy the last of the above-mentioned c r i -12 FIGURE 3. An adaptation of the general model of c e l l activation (Rasmussen et a l . , 1972) to myocardial tissue. When a hormone interacts with i t s receptor site, i t does two things simultaneously—(1) i t activates adenyl-ate cyclase, leading to increased intracellular levels of cyclic AMP and (2) i t increases membrane permeability to calcium, allowing an intracellular increase in con-centration of this ion. Cyclic AMP has at least two effects i n t r a c e l l u l a r l y — (a) i t initiates the enzymatic reaction sequence leading to glycogenolysis and (b), i t alters the "unavailable'1 or subcellular fraction of calcium to lead to an increase in "free? (cytosol) calcium. Then the increased cytosol calcium is responsible for several changes including (i) inhibition 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. The most important feature of this model is that each second messenger reciprocally controls the concentration of the other. This is a b u i l t - i n mechanism for stopping a signal—equally important as i n i t i a t i o n of i t . 5 A M P P h a r> G - 1 ~ P Figure 3 I 1* teria i.e. parallel alterations in phosphodiesterase activity and physio-logical response frequently employ the methylxanthines as phosphodiesterase inhibitors. Butcher and Sutherland (1962) determined the potency in this series of compounds for beef heart enzyme inhibition 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 norepinephrine-ihduced force increase when theophylline was present in the muscle bath. The influence of caffeine was less prominent. In addition, theophylline was found to augment eathecholamine-induced increases in phosphorylase a_ (Hess et a l . , 1963). Theophylline and caffeine have well-established positive inotropic effects of their 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 phos-phorylase levels which was repeated in later experiments (Hess et a l . , 1963). Vincent and E l l i s (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 their actions through a common pathway i.e. through increased intracellular levels of cyclic AMP. Further indirect evidence in support of this was obtained by Skelton et a l . (1971) who, in isolated cat papillary muscle, noted a potentiation of both the norepinephrine and dibutyryl cyclic AMP inotropic actions by theophylline I. D x 10" 4M. They assumed this was an effective concentration for phos-phodiesterase inhibition. Kukovetz and Poch (1970) also noted an augmen-tation in Langendorff preparations•of rabbit, rat and guinea pig myocardium. The sophisticated study of Watanabe and Besch (1974), using potassium-depolarized guinea pig.hearts, established a further link between phospho-15 diesterase inhibition and contractile activity. These investigators demonstrated that theophylline ( 1 - 3 mM) was capable of restoring mechanical activity and elevating intracellular cyclic AMP concentration. Unfortunately, the temporal sequence was not investigated. However, Watanabe and Besch did observe that the time required to restore contractions was longer than for catecholamines which i s consistent with an intracellular site of action. Similar observations on the ele 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 correlation in phosphodiesterase-inhibiting potency with effective concentration in inducing the slow calcium response. Imidazole was found to stimulate cardiac phosphodiesterase i n vitro (Butcher and Sutherland, 1962). Therefore an antagonism of the theophylline action and of the action of small doses of isoproterenol on contractile force and phosphorylase activation (Kukovetz and Poch, 1967) also supported the cyclic AMP hypothesis. In a more recent study, where intracellular ac-cumulation of the cyclic nucleotide was also determined, Verma and McNeill (1974) found parallel decreases i n norepinephrine-induced contractile force and in the cyclic AMP level. However phosphorylase activation was not correlated with changes in cyclic AMP caused by imidazole. Some experimental data on the cardiac actions of xanthines alone, and ^ i n combination with norepinephrine, indicate the correlation with phospho-diesterase inhibition is 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 positive inotropic response to norepinephrine. The-(preparation used was a Langendorff rat heart. McNeill and coworkers (1969) presented similar findings i n the in situ rat heart. 16 M c N e i l l , Brenner and Muschek (1973) compared, i n guinea p i g myocardium, the a b i l i t y of various methylxanthines and papaverine to p o t e n t i a t e c a t e c h o l -amine- induced i n o t r o p i s m and phosphorylase 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 . Although there was good c o r r e l a t i o n among the methylxanthines w i t h respect to the p o t e n t i a t i n g e f f e c t and the phospho-d 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 , papaverine gave anomalous r e s u l t s . The a l k a -l o i d was more potent than the n a t u r a l l y o c c u r r i n g methylxanthines i n i n -h i b i t i n g guinea p i g phosphodiesterase and enhanced the 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. However, i t d i d not augment the p o s i t i v e i n o t r o p i c a c t i o n . Furthermore the 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 not correspond to t h e i r phosphodiesterase 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 possessed the greatest i n o t r o p i c e f f e c t of the methylxanthines, w h i l e i s o b u t y l methylxanthine (SC-2964) was the most potent enzyme i n h i b i t o r . Papaverine had a negative i n o t r o p i c e f f e c t . From these data, M c N e i l l e t a l . (1973) questioned the cause and e f f e c t r e l a t i o n s h i p between phosphodiesterase i n h i b i t i o n and the c a r d i a c a c t i o n s of these drugs. Two very recent s t u d i e s have provided f u r t h e r evidence that the methyl-xanthines may not work through c y c l i c AMP. M c N e i l l et a l . (1974) found that although 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 phosphorylase-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 not change the c y c l i c AMP content of -4 the guinea p i g h e a r t . In 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 norepinephrine metabolic and i n o t r o p i c a c t i o n s but d i d not i n f l u e n c e the catecholamine-induced change i n c y c l i c AMP. A higher methylxanthine concen-t r a t i o n (2 mM) was cardiodepressant 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 to s e v e r a l doses of norepinephrine. However, i t p o t e n t i a t e d the increase i n c y c l i c AMP to the highest dose of norepinephrine (0.4 yg). These r e s u l t s suggest t h a t , although t h e o p h y l l i n e and other methylxanthines are 17 capable of inhibiting phosphodiesterase in vitro, this may not be manifest in intact preparations in concentrations producing the pharmacological responses. Henry and associates (1975) investigated the myocardial actions of papaverine. They found no positive inotropic effect after testing several concentrations. Also, the mechanical alterations following epinephrine were similarly unaffected by papaverine. However, the alkaloid increased intracellular cyclic AMP alone, and in an additive manner with epinephrine. Papaverine increased the % phosphorylase a. parallel to changes in cyclic AMP. It would appear, therefore, that the importance of phosphodiesterase inhibition as a mechanism of action for methylxanthines and papaverine may have been overestimated. If the common mechanism of inotropic action of catecholamines, methylxanthines and dibutyryl cyclic AMP is through raised intracellular levels of cyclic AMP then certain features of their mechanical effects should be identical. 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 similarity ends here. Both norepinephrine and dibutyryl cyclic 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 is 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 in rate of relaxation. Blinks et a l . (1972) could not distinguish any of these effects among the three methyl-xanthines tested, even though phosphodiesterase-inhibiting >potency; varies. 18 On the cardiac action potential, caffeine greatly prolongs the plateau phase (deGubareff and Sleator, 1965) while norepinephrine . has variable effects, depending on experimental protocol (deGubareff and Sleator, 1965; Spilker, 1970). In addition to their i n vitro actions on cyclic AMP phosphodies-terases, the methylxanthines have profound effects on cellular calcium homeostasis which could also account for many experimental observations. Therefore their role in establishing cyclic nucleotide involvement has been questioned (Sobel and Mayer, 1973). Caffeine increased calcium exchange-abil i t y in toad ventricle (Nayler, 1963) and mammalian atria (Guthrie and Nayler, 1967). Similarly, theophylline was found to increase calcium-45 uptake and release in guinea pig atria (Scholz, 1971). Calcium handling by intracellular organelles may be influenced by methylxanthines. For example, caffeine displaced calcium from toad ventricular mitochondria (Nayler and Hasker, 1966) arid also reduced rate of calcium uptake by rat and guinea pig microsomal preparations (Nayler et a l . , 1975). This latter observation is consistent ;with the abil 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 in skeletal muscle. Although releasing ab i l i t y by cardiac sarcoplasmic reticulum was not dir-ectly investigatedjJundt et a l . (1975) observed marked stimulation of sodium-dependent calcium-45 efflux from guinea pig atria 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 direct effect of caffeine on 19 release of bound calcium from rabbit myocardial sarcoplasmic reticulum vesicles. Methylxanthines may have an influence in the excitation phase of the cardiac cycle. Two- groups of investigators have demonstrated the a b i l i t y of these agents to restore excitability and mechanical activity to potassium-arrested hearts (Watanabe and Besch, 1974; Schneider and Sperelakis, 1975) by activating the slow calcium channels. Consistent with a postulated action during excitation, Scholz (1971) observed an increase in calcium-45 influx in beating, but not in quiescent, isolated guinea pig atria. The above-described actions of methylxanthines on calcium would explain their i n t r i n s i c effects on myocardial contractility, on glycogen phosphorylase and their a b i l i t y to potentiate the catecholamine-induced cardiac responses since calcium has a well-established role in each process. Whether phosphodiesterase inhibition 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 in myocardial function by Farah and Tuttle i n 1960. In several species- glucagon produced positive inotropic and chronotropic effects which were not altered by reserpine pretreatment or insulin administration. However, the 3-receptor blocking agent dichloroisoproterenol prevented the glucagon-induced changes. This led 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 1 tachyphylaxis' in that repeated doses gave a reduced response. 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 insensitive to the polypeptide hormone. These were the intact anaesthetized dog, the Langendorff rabbit heart and isolated rabbit atria. Glucagon was active in guinea pig atria. Thus, the preliminary experiments of Farah and Tuttle revealed fundamental properties of the glucagon cardio-tonic action. In contrast to the observations of Farah and Tuttle (1960), other investigators have demonstrated the positive inotropic effect in intact dog after intravenous glucagon administration (Glick et al.,1968; Lucchesi 1968). In si t u preparations where the drug has been directly infused into the heart (Regan et a l . , 1964; Afonso et a l . , 1972.; Hammer et a l . , 1973) have yielded qualitatively similar 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 of a cardiovascular alteration. Reserpinization does not alter 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 interfere with the inotropic action of glucagon in concentrations blocking the cat-echolamine 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 structural analog of propranolol. 'profitthalol, was also shown to be without effect on glucagon-induced inotropism (Mayer et al.,1970). The influence of glucagon on parameters of an individual contracture has been studied using isolated cat and dog papillary muscle and atria. Glucagon augments maximum developed tension in 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 like glucagon. Force of contraction may be influenced by interval between beats (Koch-Weser and Blinks, 1963). Spilker (1970) investigated the force-fre-quency relationship in isolated guinea pig atria before'and after glucagon exposure. In a control situation, there is a gradual decrease in force associated with increases in 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 at greater frequencies the curve was shifted up in a para-l l e l manner. Norepinephrine changed the shape of the curve and increased the force relative to control at a l l stimulation frequencies. The inotropic effect of glucagon is not secondary to the effect on heart rate because in preparations maintained at a constant frequency glucagon s t i l l increases contractile force (Glick et a l . , 1968; Lucchesi, 1968; Gold et a l . , 1970; Nayler et a l . , 1970; Spilker, 1970; Marcus et a l . , 1971;Henry et_al., 1975). Different ionic environments ..-may influence the action of glucagon but relatively few experiments have been done to investigate this. Manganese is believed to interfere with the influx of calcium accompanying excitation (Sabatini-Smith and Holland, 1969). Mn^? either reduced or abolished the glucagon positive inotropic effect (Nayler et a l . , 1970). Mn+^ also shifted 22 the dose-response curve for glucagon to the right (Spilker, 1970). Visscher and Lee (1972) examined the association of extra-cellular calcium concentration with the force changes induced by glucagon. The lower the extracellular calcium concentration, the greater the increase in force. In a 0.09 mM calcium medium, glucagon maintained the mechanical response. However the hormone was ineffective in a calcium-free medium. Electrophysiologic measurements in myocardial cells exposed to glucagon suggest the pancreatic hormone has l i t t l e influence on the ex-citatory phase. Spilker (1970) observed a slight prolongation of the action potential plateau but resting potential, action potential amplitude and maximum rate of depolarization were unchanged. In contrast, Prasad (1975) noticed a shortening of the action potential, and this was associated with an increase in contractility. A species variation may explain the contra-diction for Prasad (1975) used dog papillary muscle. Prasad was unable to detect any alteration of action potential features in guinea pig papillary muscle, the preparation employed by Spilker (1970) , even though similar concentrations of glucagon were investigated. It is 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 is depressed i n calf and sheep Purkinje fibres bathed in a low (0.45 mM) calcium medium (Spilker, 1970). Under conditions where both norepinephrine and calcium elevated the plateau potential glucagon had no effect. Experiments in isolated guinea pig hearts depolarized with high potassium (Watanabe and Besch, 1974; Schneider and Sperelakis, 1975) demon-strated that glucagon, unlike the catecholamines, was unable to restore 23 excitability and contractions. Similar observations were made in tetrodo-toxin-treated hearts (Watanabe and Besch, 1974). Glucagon w i l l stimulate glycogenolysis in myocardium. The spectrum of metabolic events closely resembles that of epinephrine (Kreisberg and Williamson, 1964) . In isolated rat heart (Comblath et_al., 1963; Mayer et a l . , 1970) and in situ rat heart (Williams and Mayer, 1966), glucagon initiates glycogen breakdown by activating phosphorylase apparently through the adenylate cyclase pathway. Like catecholamine-induced activity, the glucagon response depends on calcium availability (Mayer et a l . , 1970). Much experimental effort has focused on the role of cyclic AMP in the glucagon cardiac responses. I n i t i a l l y no change in cyclic AMP concentration could be detected in intact rat heart challenged with glucagon, although the inotropic response was el i c i t e d (LaRaia et a l . , 1968). However subsequent investigators (Mayer et a l . , 1970; Oye and Langslet, 1972; Henry et a l . , 1975) have been successful in demonstrating a glucagon-stimulated increase in cellular cyclic nucleotide levels. In contrast to the temporal sequence of events following catecholamines, neither Mayer's group (1970) nor Oye and Langslet (1972) detected the change prior to the increase in contractile force. Demonstration of a glucagon-sensitive adenylate cyclase in vitro was accomplished long before elevated levels were discovered in intact tissue. Rat (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 effective against catecholamines. Further evidence in support of a cyclic AMP involve-ment comes from experiments in f a i l i n g hearts where glucagon was ineffective both in increasing contractility and in stimulating adenylate cyclase in vitro (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 in this species (Henry et a l . , 1975). There have been many attempts to associate adenylate cyclase activity with calcium homeostasis. 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. The experiments of Nayler et a l . (1970) suggested that handling of calcium by sarcoplasmic reticulum or mitochondria was not influenced by glucagon, although calcium exchange across the sarcolemma was altered. Yet another study (Visscher and Lee, 1972) indicated that, while glucagon may influence calcium flux rates, there is no influx of calcium under conditions when the inotropic action is marked and hence these authors proposed some effect of glucagon on intracellular calcium stores. Cyclic AMP may alter the membrane permeability to calcium (Watanabe and Besch, 1974). Glucagon neither restored electromechanical activity (Watanabe and Besch, 1974; Schneider and Sperelakis, 1975) nor increased intracellular cyclic AMP levels (Watanabe and Besch, 1974) i n guinea pig 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 ab i l i t y of glucagon to restore excitability has not been investigated in species possessing a glucagon-stimulatable enzyme (e.g.rat)'. Another mechanism by which glucagon could alter force is through inhibition of sarcolemmal Na + - K + -ATPase. Prasad (1975) presented evidence that changes i n force accompanied glucagon inhibition of this 25 enzyme i n dog papillary muscle. Consistent with this proposal was the lack of contractile event on one hand, and lack of enzyme inhibition on the other hand in guinea pig, rabbit and pig. The a b i l i t y of phosphodiesterase inhibitors to enhance the effects of glucagon on myocardium would provide further support for an involvement of cyclic AMP. However data obtained with these agents cannot be interpreted easily because they interfere with other cellular processes. Theophylline greatly enhanced the inotropic action of glucagon on isolated cat papillary muscle (Marcus et a l . , 1971). Afonso and asso-ciates (1972) found qualitatively similar alterations in force of contrac-tion monitored following concurrent administration of glucagon and amino-phylline to in si t u dog heart. Apparently i n contradiction to these results, Lucchesi (1968) found that pre-infusion with theophylline prevented the glucagon inotropic response i n in situ canine heart. However, glucagon was administered during the period of maximum inotropic response to theophylline. Marcus et a l . (1971), in 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 papillary muscle. A higher concentration actually reduced the response to glucagon. McNeill et a l . . (1969) noted a similar influence on the norepinephrine response in i n si t u rat heart. Therefore studies on the interaction of glucagon with methylxanthines have not yet conclusively satisfied this criterion implicating cyclic AMP involvement. By virtue of the qualitative similarity of their myocardial actions, many authors have suggested that a common mechanism of action for the catecholamines and glucagon might be through the adenylate cyclase-cyclic AMP system. Although numerous studies have been undertaken to es-tablish a role for cyclic AMP i n the catecholamine responses, relatively few studies have been conducted with glucagon. The present investigation therefore undertook to characterize the myocardial actions of glucagon, by means of complete time-response and dose-response experiments, and to cor-relate the results of studies on the glucagon-induced positive inotropic effect and phosphorylase activation with' the action of glucagon on intact tissue cyclic AMP accumulation. In addition, the influence of theophylline on the dose and time dependent behaviour of glucagon in 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. MATERIALS AND METHODS 27 1. MATERIALS The cyclic 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 , deion-R ized water. PCS liquid s c i n t i l l a t i o n cocktail 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 Tris (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 in oxygenated CK solution. The injection volume was maintained at 0.25 ml throughout the study. Norepinephrine was prepared immediately prior to use in CK solution and injected i n a volume of 0.25 ml. Theophylline and propranolol were dissolved in CK solution and added to the reservoir of the perfusion medium. Fresh solutions were prepared for each experiment. 2. METHODS A. Heart perfusion 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 prior to sacrifice. Animals were stunned by a blow to the head. Hearts were rapidly 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; CaCl 2 1.8; MgCl 2 2.1; NaHCC>3 19.1; dextrose 10; 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 ro l l e r pump (Extra-Corporeal Medical Specialties, Model RL 175) or a Buchler Polystaltic Pump (Buchler Instruments, Inc., Model 2-6100). Contractile force was monitored with a Palmer c l i p placed in the apex of the ventricle and connected to a force-displacement transducer (Grass Instruments, Model FT30C) and recorder (Grass Instruments, Model 5D Polygraph). Diastolic tension was adjusted to 1 g and the heart allowed to stabilize for 10 minutes before the start of each experiment. Drugs were injected via a side arm cannula or, alternatively, dissolved in CK solution and perfused through the heart. Contractile force was determined by comparing the systolic tensions before and after drug exposure, and is expressed both as an absolute change in tension and as a percent over pre-injection level ^ e systolic tension after drug x ^OQ^ systolic tension before drug Time-Response Experiments After stabilization, glucagon ( 2yg in 0.25 ml) was injected via a side arm cannula. At selected time intervals hearts were frozen instantaneously with Wollenberger tongs (Wollenberger et a l . , I960) previously chilled in 2-methylbutane cooled in an alcohol-Dry Ice mixture. Control hearts received a 0.25 ml injection of CK solution and were frozen at 0 seconds. A l l hearts were stored at - 80 °C unti l assayed. In some experiments theophylline was added to the CK buffer in a f i n a l concentration of 1 mM and perfused through the apparatus for 15 minutes following the i n i t i a l stabilization period. Hearts were thus exposed to 29 theophylline for approximately 8 - 1 0 minutes after which 2 yg of glucagon was injected. Control hearts received 0 . 2 5 ml CK solution. Hearts were frozen as described above. The exposure time to theophylline was estimated by perfusing normal saline through the apparatus and measuring the time re-quired for a precipitate to form in a beaker containing AgNO^ solution. Dose-Response Experiments After stabilizing, 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 activation of phosphorylase and peak accumulation of cyclic 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 per-fused through the apparatus for 1 5 minutes (following the stabilization period) and then appropriate doses of glucagon injected. Hearts were frozen at 50 sec. Again only one injection 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 for 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 stabilize on CK solution for 1 0 minutes. They were then perfused with propranolol in a f i n a l —8 ' concentration of 1 0 M for 2 0 minutes (exposure time to propranolol approx-imately 1 3 - 15 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 efficacy of this concentration of propranolol in blocking the catecholamine response. The increase in contractile force was measured f i r s t in CK-perfused hearts f o l -lowing injections of norepinephrine through the side arm cannula. Systolic 30 tension was allowed to return to pre-injection level before the next dose was administered. After the last dose, the experiment was repeated in the —8 presence of 10 M propranolol which had been exposed to the heart for approximately 15 minutes prior to norepinephrine challenge. B. Phosphorylase assay Phosphorylase activity was determined in the direction of glycogen synthesis by a modification of the method of Cori and Cori (1940), described previously by McNeill and Brody (1966). Details are given below. From each heart a 70-100 mg frozen sample from the apex was homogen-ized in 200 volumes of a solution containing 0.05 M Tris (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) at a rheostat setting of 6 for 5 seconds and performed at 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-izing medium plus 0.4 % glycogen and 0.01 M G-l-P in 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 in 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 Tris buffer-AMP mixture after which 0.2 ml of enzyme was added. Pre-cipitated protein was pelleted by centrifugation for 10 minutes (room temp-erature) at 1500 R.P.M. in an International Centrifuge (Model EXD). The supernatant was decanted off and assayed for 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 liberation of inorganic phosphate was linear over the time studied and was proportional to the enzyme concentration. Total phos-phorylase 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 in the absence of 5' AMP. Since total enzyme activity did not change with drug treatment, the results are expressed as percent phosphorylase a_ which i s : enzyme activity without AMP ^ j enzyme activity with AMP C. Cyclic AMP assay I. Tissue extraction The method of extraction was modified from that of Gilman (1970) as follows: A 70-100 mg frozen sample from the apex of the ventricle was rapidly homogenized in 5 ml of 5 % w/v TCA (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 settle 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 five times with water'-rsaturated ether (10 ml each time). Following the f i n a l extraction step, residual ether was removed in a stream of-nitrogen. The aqueous extract was lyophilized overnight in 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. Cyclic AMP binding reaction An Amersham-Searle Cyclic AMP Assay Kit was used to determine the tissue concentration of cyclic AMP by a modification of the Gilman method (Gilman, 1970) as described below. The binding reaction was carried out at 0-4 °C i n a total volume of 200 y l consisting of: 50 y l of sample extract or known amount (0.25-8 pmol) of unlabelled cyclic AMP 3 50 y l of H-cyclic AMP (9 pmol; specific activity 20.8 c/mmol) 100 y l of binding protein (sufficient to bind 30% - 50% of the labelled 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 in the reagent vials by the manufacturer. Each unknown sample or known standard was run in duplicate. The reaction was started by the addition of the binding protein and reached equilibrium after 30 minutes at 2-4 °C. After a minimum incubation period of two hours the binding reaction was 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 in an International Centrifuge (Benchtop, Model HN) at f u l l speed for 15 minutes. The super-natant was decanted into 10 ml of Amersham-Searle PCS liquid s c i n t i l l a t i o n cocktail. Radioactivity was counted in a Nuclear-Chicago Isocap 300 counter for 10 minutes or for a time sufficient to accumulate 200,000 counts (count-ing error 0.5 %) whichever was less. With each assay of unknowns, a standard curve was run simultaneously. A" charcoal blank was run in duplicate to determine the amount of unbound radioactivity not pulled by the charcoal during centrifugation. This blank 3 contained 150 y l of 0.05 M Tris-0.004 M EDTA buffer plus 50 y l of H-33 cyclic AMP and was handled in the same manner as the other incubation tubes. To determine the total amount of radioactivity added, two tubes containing 3 150 ul of Tris buffer plus 50 ul 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 results The counting efficiency was determined to vary minimally (29.9% to 32.2%) by means of an external standard channels ratio calculation. Since the efficiency was reasonably consistent for a l l v i a l s , correction for quench was unnecessary (see Appendix). The counts per minute in each set of duplicates were averaged and then the average cpm in the charcoal blank was subtracted. The % bound activity was then determined by the following formula: average sample cpm - average blank counts ^ ^ average total radioactivity added A standard curve was obtained by plotting % bound radioactivity against the log of pmol of unlabelled cyclic AMP added per tube. The curve was linear in the range of 0.25 pmol to 8 pmol. A straight line was computed from the experimental data by the method of least squares on a Wang 600 Programmable calculator. The correlation coefficient was always between -.990 and -1.000. The amount of cyclic AMP in each unknown was determined by computer from the standard curve. This figure was corrected for dilution and original tissue weight. The two samples extracted from each heart were pooled after correction for original tissue weight. D. STATISTICAL METHODS Stat i s t i c a l analysis was performed using the Student * t 1 test for paired and unpaired data. A difference of p < 0.05 was considered to be 34. s t a t i s t i c a l l y significant. Data in tables, figures and text are presented as the mean + one S.E.M. 35 RESULTS 1. Experiments in buffer-perfused hearts Injection of glucagon into the isolated perfused rat heart produced an increase in contractile force which was time and dose dependent. Figure 4 shows the time course of the positive inotropic effect after 2.0 yg of glucagon. With this dose, systolic tension f i r s t changed s i g n i f i -cantly (p < .05) 25 seconds following drug administration (Table 1). At this time, systolic tension was 27.6 +_ 2.7 % greater than the pre-injection level of 3.7 + 0.1 g. Contractile force was maximally increased at 35 seconds when the % increase was 60.1 + 4.7 (Table 1), corresponding to an absolute tension change of 2.3+0.2 g. However there was individual variation in the time when peak tension occurred, ranging between 30 and 50 seconds. Glucagon had a long duration of action, for systolic tension had not returned to pre-injection level after 120 seconds (Figures 4, 5; Table 1). The glucagon-induced positive inotropism was dose-dependent, as illustrated in Figure 6. The maximum increase i n force of 77.1+7.9 % was obtained after 4.0 yg of glucagon (Table 2). At this concentration, the absolute increase in systolic tension was 3.1 +0.4 g (Table 2). Glucagon 8.0 yg caused an 82.7 +18.9 % increase over pre-injection level but this did not differ significantly (p > .05) from the response due to 4.0 yg. The minimum dose capable of producing a significant positive inotropic effect was 0.5 yg (Table 2). In order to eliminate the possibility that endogenous catecholamine release was responsible for the observed glucagon cardiotonic action, a study was carried, out with propranolol in which 2 dose levels 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 cyclic AMP content, contractile force and percentage phosphorylase a at various times following injection into rat, hearts perfused with buffer or buffer plus theo-phylline. 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 for points where the S.E.M. was less than the symbol size. Cyclic AMP was significantly elevated at 15 sec., contract-i l e force at 25 sec. , and % phosphorylase a at 30 sec. i n buffer-perfused hearts. In hearts perfused with 1 mM theophylline for 15 minutes, cyclic AMP and % active phosphorylase were significantly elevated at 20 sec., and contractile force at 25 sec. FIGURE 4 38 FIGURE 5. Effect of time on the absolute change in tension following injection of 2 yg glucagon into the isolated buffer-perfused and theophylline-perfused rat heart. Diastolic tension was adjusted to 1 g. Each point represents the mean + 1 S.E.M. of at least 8 deter-minations with the exception of the value at 120 sec. This i s the average of 2 hearts. The change in sys-t o l i c tension was significantly 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 positive inotropic response to 2 yg glucagon in the buffer-perfused and theophylline- perfused rat heart. Time after Percent Change Absolute Change injection in Systolic Tension in Systolic Tension (g) (sec)  Buffer- Theophylline-perfused perfused Buffer-perfused Theophylline-perfused 0 3.7 + 0.1a 3.6 + 0.2b 25 27.6 + 2.7* (35) 32.8 + 2.7 (32) (35) 1.2 + 0.1+ (35) (32) 1.1 + 0.1 (32) 30 47.4 + 3.5 (35) 57.7 + 3.4'C (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 +5.0 (24) 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 C (15) 1.6 + 0.1 (16) 2.4 + 0.2° (15) 60 39.1 +4.8 (9) 58.7 + 9.3 (8) 1.4 + 0.2 (9) 1.9 + 0.3 (8) 120 22.5 + 3.4 (2) 1.1 + 0.3 (2) •'* Mean % increase in systolic tension over pre-injection level + one S.E.M. t Mean absolute increase in systolic tension over pre-injection level +S.E.M. a Mean systolic tension (g) + one S.E.M. after a 10 minute CK perfusion b Mean systolic tension (g) + one S.E.M. after a 10 minute CK perfusion followed by a 15 minute theophylline 1 mM perfusion c Significantly enhanced over corresponding buffer perfused value (p< .05) Diastolic tension was adjusted to 1 g. Numbers in parentheses indicate the number of animals. In the theophylline experiments, mean systolic 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 cyclic AMP content, contractile force and percentage phosphorylase a_ in rat hearts perfused with buffer or buffer plus theophylline (1 mM). Cyclic AMP and % phosphorylase a_ were measured 50 sec. after glucagon injection. The % increase in systolic tension was determined at the time of maximum change relative to pre-injection level. Data points repre-senting 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 for points where the S.E. is smaller than the symbol size. In buffer-perfused hearts, cyclic AMP was significantly increased with 0.25 yg glucagon whereas the contractile force and phosphorylase a_ were increased after 0.5 yg. In 1 mM theophylline-perfused hearts, cyclic AMP and contractile 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 in systolic tension in buffer-perfused and theophylline-perfused rat hear6s. Diastolic 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 effective dose of glucagon from 0.5 to 0.25 yg. Only the 0.25 and 2.0 yg points were significantly enhanced over buffer-perfused values. FIGURE 7 45 TABLE 2 The effect of various doses of glucagon on contractile force in the isolated buffer-perfused and theophylline-perfused rat heart. Dose of Glucagon (yg) 0.25 Percent Change in Systolic Tension Buffer- Theophylline-perfused perfused 0 -6.4 + 3.2* C33) 2.6 + 1.4 28.4 + 5.5 a' b (6) (4) Absolute Change in Systolic Tension (g) Buffer- Theophylline-perfused perfused 0 -0.2 + 0.09* (33) 0.1 + 0.1 1.2 + 0.2a'b (6) (4) 0.5 1.0 2.0 4.0 8.0 17.0 + 3.9a 36.3 + 5.1 (12) (5) 52.6 + 4.6a 76.3 + 8.2a>b (8) (6) 57.2 + 4.4a 84.4 + 7.7a,b (20) (11) 77.1 + 7Z9a 116.3 + 7.4 a» b (6) (5) 82.7 + 18.9a 103.9 + 50.6a (4) (2) 0.9 + 0.2a 1.3 + 0.3a (10) (5) 2.4 + 0.2a (8) 2.1 + 0.2a (20) 3.1 + 0.4a (6) 3.0 + 1.0a (4) 2.5 + 0.3a (6) 3.1 + 0.3 a» b (11) 3.5 + 0.4a (5) 3.4 +• 1.7a (2) * Change in systolic tension relative to the pre-theophylline level. a Significantly greater (p < .05; paired "t") than pre-glucagon systolic tension b Significantly enhanced (p < .05; paired "t") over corresponding buffer-perfused value Diastolic tension was adjusted to 1 g. Numbers in parentheses indicate the number of animals. Hearts were perfused with CK for 10 min., or alternatively perfused an additional 15 minutes with 1 mM theophylline after which glucagon was injected via a side-arm cannula. Systolic tension was measured when the change was maximal relative to pre-injection level. Systolic tension was 4.1+0.1 g (N=6) after a 10 minute CK perfusion. In theophylline experi-ments, systolic tension was 3.5 + 0.1 g (N=33) before theophylline exposure and 3.3+0.1 g (N=33) after 15 minutes. 46 produced peak systolic 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 after 1.0 and 2.0 yg doses. These changes in force did not diffe r significantly from those in the buffer-perfused hearts (Figure 8; Table 3). Perfusion with propranolol lowered the mean systolic tension before glucagon injection from 5.5 + 0.5 g (CK hearts) to 4.8 + 0.3 g but this difference was not^statistically signif-icant. Figure 9 and Table 4 present data that verifies that the concentration —8 of propranolol employed (10 M) was sufficient to reduce the contractile response to norepinephrine. In addition to the contractile effect, glucagon also increased glycogen phosphorylase activity. The time course of activation was investigated with 2.0 yg glucagon, a dose producing submaximal changes in force (see Figure 6). Phosphorylase a_ levels were significantly greater than control 30 seconds following polypeptide injection (Figure 4; Table 5) and reached a maximum mean value at 60 seconds. However, during the interval between 40 and 60 seconds there was no significant difference in activity. The % phosphorylase a_ was s t i l l significantly elevated over control at 120 seconds (Figure 4; Table 5). Prior to glucagon administration (time — 0 seconds) , the % phos-phorylase a_ was 6.9 + 0.8 (N = 11). ]The dependence of phosphorylase activation on dose was examined by measuring phosphorylase a_ levels at 50 seconds, a time when activity was maximal (see Figure 4). The control level of 5.3+1.0 % was obtained by injecting 0.25 ml of CK buffer and measuring phosphorylase at 50 seconds. The minimum amount of glucagon capable of significantly elevating the % phos-phorylase a_ above control was 0.5 yg (Figure 6; Table 6) which increased activity to 12.3 + 2.2 %. The maximum activating dose was 4.0 yg glucagon, 47 TABLE 3 _ g The influence of propranolol 10 M on the positive inotropic effect of glucagon in the isolated perfused rat heart. Dose of Percent Change Absolute Change Glucagon in Systolic Tension in Systolic Tension (g) (yg) Buffer- Propranolol Buffer- Propranolol perfused 10 °M perfused 10 °M 0 5.5+0.5* 4.8+0.3- , N S 1.0 34.0 + 11.7 32.7 +-10.1NS 2.2+1.1 1.8 + 0.6 N S 2.0 61.0 + 7.4 59.2 + 9.8 ,NS 3.2 + 0.7 2.6 + 0.5N S * Mean systolic tension + one S.E.M. after 30 min. CK perfusion in 7 animals + Mean systolic tension + one S.E.M. after 10 min. CK perfusion plus 20 min. propranolol perfusion in 7 hearts NS Not significantly decreased from the corresponding buffer-perfused value (p > .05; unpaired 't' test) Diastolic tension was adjusted to 1 g. Hearts were perfused for 30 minutes with CK solutioiigOr alternatively for 10 minutes with CK followed by 20 minutes with 10 M propranolol. Glucagon was injected via a side-arm cannula. Values in table represent the mean + one S.E.M. of three hearts. 48 FIGURE 8. The influence of propranolol 10~° M on glucagon-induced changes in cardiac cyclic AMP content, contractile force and percentage phosphorylase a . Cyclic AMP and phosphorylase were measured 50 sec-onds after glucagon injection. Contractile force is presented as the percent increase in systolic tension relative to pre-injection level. The con-trol value represents systolic tension after ex-posure to propranolol as a percent of systolic tension in buffer-perfused hearts (N=7). Control values for phosphorylase and cyclic AMP are one measurement. Other points represent the mean +one S.E. of 3 hearts. Propranolol 10~ 8 M did not significantly reduce any of the cardiac responses to glucagon. FIGURE 8 50 TABLE 4 _g The influence of 10 M propranolol on the positive inotropic action of norepinephrine i n the isolated, perfused rat heart. Dose of , . Percent Increase in Systolic Tension Norepinephrine _„ (us) Buffer-perfused Propranolol 0.01 13.3 0 (1) (1) 0.1 74.7 16.6 (2) (2) 1.0 94.7 30.7 (2) (2) 10.0 121.4 44.3 (1) (2) a. relative to pre-injection systolic tension Diastolic tension was adjusted to 1 g. and hearts were perfused with CK solution for a stabilization period of 10 minutes. Norepinephrine was injected in a volume of 0.25 ml. Following the f i n a l dose, propranolol —8 10 M was perfused through the heart for 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 is expressed as the maximum percent increase in systolic tension relative to the pre-injection level. Each value represents 1, or alter-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 activation in the buffer-perfused and theophylline-perfused isolated rat heart. Time after injection (sec) Buffer-perfused Theophylline-perfused 0 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 + 5.9b (4) 25 9.2 + 1.5 (4) 30.9 + 2.9b'° (4) 30 19.1 + 2.8a (10) 29.1 + 2.7 b' C (7) 40 32.1 + 4.2a (8) 45.1 + 5.1b (7) 50 31.3 + 2.9a (7) 52.4 + 4.2b'c (8) 60 33.1 + 4.9a (6X 56.6 + 4.7b'C (6) 120 16.9 + 0.3a (2) Mean % phosphorylase a + one S.E.M. significantly (p < -.05) greater than the buffer-perfused, 0 sec b. significantly greater than the theophylline-perfused, 0 sec value c. significantly enhanced over the corresponding buffer-perfused value CK (0.25 ml) or glucagon (2 yg) was injected via a side-arm cannula and hearts were frozed at the indicated times. Theophylline 1 mM was perfused through the apparatus for 15 minutes. Numbers in parentheses refer to the number of animals. TABLE 6 The effect of various doses of glucagon on cardiac glycogen phosphorylase activation in the buffer-perfused and theophylline-perfused isolated rat heart. Dose of Glucagon (yg) Buffer-perfused Theophylline-perfused Control 5.3+1.0* 8.4 + 2.5 (9) (5) 0.25 8.5 + 1.1 12.5 + 2.0 (7) (6) 0.5 12.3 + 2.2a 26.0 + 3.5 b' C (8) (7) 1.0 20.2 + 3.2a 43.4 + 5.3 b , C (8) (8) 2.0 31.3 + 2.9a 52.4 + 4.2 b' C (7) (8) 4.0 38.8 + 3.5a 58.5 + 4.7 b , C (6) (5) 8.0 34.3 + 3.5a 72.2 b' C (3) (1) * Mean % phosphorylase a_ + one S.E.M. a. Significantly (p < .05) greater than the buffer-perfused control b. Significantly greater than the theophylline-perfused control c. Significantly enhanced over the corresponding buffer-perfused value CK (0.25 ml) or glucagon was injected via a side-arm cannula and hearts were frozen at 50 seconds. Theophylline 1 mM was perfused through the apparatus for 15 minutes. Numbers in parentheses refer to the number of animals. 55 which caused an increase in activity to 38.8 + 3.5 % a_. Therefore the isinimum and maximum effective doses were identical for contractile force and glycogen phosphorylase activation. —8 Propranolol 10 M did not significantly reduce active phosphorylase levels produced by glucagon (Figure 8; Table 7).. In buffer-perfused hearts, after 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. levels 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 different from values obtained in the buffer-perfused hearts (Table 7). Propranolol infusion alone had l i t t l e effect on control phosphorylase activity (8.5 % a_; N = 1 vs. 7.0 % a_, N = 1 in heart perfused with CK for 30 minutes). A possible mechanism for the stimulatory actions of glucagon i s through the adenylate cyclase system. Therefore, the influence of glucagon on the tissue accumulation of cyclic AMP was investigated in both time and dose experiments. A 2.0 yg dose of the hormone was chosen to determine the time course of cyclic AMP changes. At 0 seconds (control), the cyclic AMP concentration was 0.25 + 0.01 pmols/mg wet weight. Glucagon increased the cyclic AMP level as early as 15 seconds after injection (Figure 4; Table 8), a time preceding the contractile and phosphorylase-activating responses. Peak levels 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. Cyclic AMP was s t i l l elevated over control as long as 120 seconds after glucagon injection (Figure 4). The dose dependence of tissue cyclic AMP accumulation was studied in hearts frozeth 50 seconds following glucagon administration. In contrast to glucagon-induced changes in force and phosphorylase _a, the minimum effective 56 TABLE 7 _g The influence of propranolol 10 M on glucagon-induced phosphorylase activation in the isolated perfused rat heart. Dose of Glucagon (yg) Buffer-perfused Propranolol 10~8 M Control 7.0 (N=l) 8.5 (N=l) 1.0 26.5 + 6.1* 25.9 + 4.9* N S 2.0 42.6 + 6.5* 34.3 + 9.6*NS * Mean % phosphorylase a_ + one S.E.M. of three hearts NS Not significantly decreased from the corresponding buffer-perfused value (p > .05; unpaired 't' test) Buffer-perfused hearts were perfused with CK solution for 30 minutes and then injected with either CK (0.25 ml; control) or glucagon. Propranolol —8 10 M was perfused through the hearts for 20 minutes following a 10 TY'1 n minute stabiliaztion period on CK solution. A l l hearts were frozen 50 seconds . . seconds after injection. 57 TABLE 8 The effect of time on cardiac cyclic AMP accumulation following admin-istration of 2 yg glucagon into the buffer-perfused and theophylline-perfused rat heart. Time after injection (sec) Buffer-perfused Theophylline-perfused 0 0.25+0.01* 0.32+0.02 c (8) (4) 15 0.37+0.04 3 0.43+0.04 (A) (3) 20 0.40 + 0.05a 0.74+0.06 b' C (5) (3) 25 0.53+0.04 a 1.13+0.04 b' c (5) (2) 30 0.52 + 0.07a 1.16+0.03 b» c (5) (3) 35 0.49 + 0.03a 1.07+0.09 b> c (5) (3) 40 0.64+0.03 a 1.27+0.07 b' c (5) (3) 60 0.60 + 0.01a 1.19 b» c (2) (1) 120 0.56 + 0.04a (2) * Mean cyclic AMP content (pmol/mg wet weight) + one S.E.M. a. Significantly (p< 0.05) greater than the buffer-perfused, 0 sec value b. Significantly greater than the theophylline-perfused, 0 sec value c. Significantly enhanced over the corresponding buffer-perfused value CK (0 sec) or glucagon was injected via a side-arm cannula and hearts were frozen at the indicated times. Theophylline 1 mM was perfused through the apparatus for 15 minutes. Numbers in parentheses refer to the number of animals. 58 dose for significantly elevating cyclic AMP levels 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 cyclic AMP but this was not s t a t i s t i c a l l y higher than control tissue levels. However the experimental " t " value ( unpaired data ) of 2.207 was only slightly lower than the c r i t i c a l " t " value of 2.228 (p < .05; 10 df). Glucagon 8.0 yg was the highest dose tested. It caused the largest accumulation of cyclic 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 fl a t t e r for cyclic AMP than for either contractile force, or % phosphorylase a_. —8 Table 10 and Figure 8 show that propranolol 10 M was unable to block glucagon-induced increases in cyclic AMP. In buffer-perfused hearts, glu-cagon 1.0 yg increased cyclic AMP from 0.31 pmols/mg (N = 1) to 0.38 + 0.01 pmols/mg wet weight. The cyclic nucleotide changes induced by a higher dose of glucagon (2.0 yg) were similarly unaltered by propranolol (Table 10; —8 Figure 8). The control level of cyclic AMP after propranolol 10 M and injection 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 cyclic AMP, i t was decided to investigate the influence of theophylline perfusion on glucagon-induced increases in contractile force, phosphorylase activation and tissue cyclic AMP accumulation. Figure 4 illustrates the time course of these responses in the presence of 1 mM theophylline, a concentration sufficient to inhibit heart phosphodiesterases i n vitro (Butcher and Sutherland, 1962). The effect of theophylline on the glucagon dose-response curves i s shown in Figure 6. 59 TABLE 9 The effect of various doses of glucagon on cardiac cyclic AMP accumulation in the buffer-perfused and theophylline-perfused isolated rat heart. Dose of Glucagon (yg) Buffer-perfused Theophyllirie-peffused Control 0.28+0.04* 0.41 + 0.03^ (4) (4) 0.25 0.5 4.0 0.38 + 0.03a 0.57 + 0.03 b» c (6) (4) 0.40 + 0.04 0.66 + 0.03 b» c (7) (5) 1.0 0.62 + 0.05a 1.04+0.07 b» c (6) (6) 2.0 0.63 + 0.04a 1.33 + 0.04 b» c (2) (2) 0.64 + 0.01a 1.47+0.04 b» c (4) (5) 8.0 0.73+0.01 a 1.64+0.02 b» c (3) (2) * Mean ventricular cyclic AMP content (pmol/mg wet weight) + one S.E.M. a. Significantly (p<0.05) greater than the buffer-perfused control b. Significantly greater than the theophylline-perfused control c. Significantly enhanced over the corresponding buffer-perfused value CK (control) or glucagon was injected via a side-arm cannula and hearts were frozen at 50 seconds. Theophylline 1 mM was perfused through the apparatus for 15 minutes. Numbers in parentheses refer to the number of animals. 60 TABLE 10 The influence of propranolol 10" _8M on glucagon-induced cyclic AMP accumu-lation in the isolated perfused rat heart. Dose of Glucagon _iE£) Control 1.0 2.0 Buffer-perfused 0.31 (N=l.) 0.38 +.0.05* 0.48 + 0.02* Propranolol 10~8M 0.21 (N=l) 0.38 + 0.01*NS 0.39 + 0.07*NS * Mean cyclic AMP content (pmol/mg wet weight + one S.E.M. of 3 hearts NS Not significantly different from the corresponding buffer-perfused value (p > .05; unpaired 't' test) Buffer-perfused hearts were perfused with CK solution for 30 minutes and then injected with either CK (control) or glucagon. Propranolol 10~^ M was per-fused through the hearts for 20 minutes following a 10 minute stabilization period on CK solution. A l l hearts were frozen 50 seconds after injection. 61 With 2.0 yg glucagon, theophylline did not alter the time when peak contractile amplitude was reached (Figure 4; Table 1). However the return to pre-injection systolic tension was delayed un t i l after 50 seconds. The % increase in force was significantly enhanced over buffer-perfused 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 significant augmentation by theophylline of the glucagon response. A complete dose-response curve for force was obtained in the presence of theophylline and is illustrated in Figures 6 and 7, and Table 2. The minimum effective concentration of glucagon causing a significant increase in force was 0.25 yg. This dose was one-half of that required in 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 in 2 animals only, and gave a variable response which, when averaged, was less than that of 4.0 yg. The peak increases in force following various doses of glucagon were compared in hearts exposed to theophylline 1 mM and i n buffer-perfused hearts (Figures 6,7; Table 2). When results were calculated as a percentage increase over pre-injection systolic tension, theophylline significantly (p < .05) enhanced the inotropic response at a l l dose levels. 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 earlier that, in 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 84 .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 in a l l hearts, and the mean value at this time would tend to be distorted by the variation. If time-response comparisons were made using only those hearts which peaked at 35 seconds after 2.0 yg glucagon then a significant enhancement by theophylline did occur. Theophylline 1 mM produced i t s own alterations in cardiac force of contraction. Following the 10 minute stabilization period on CK solution, the mean systolic tension was 3.5 + 0.1 g. When the hearts were subsequently exposed to theophylline, systolic tension transiently increased 15.4 +1.5 % (CL6 + 0.1 g; N=36) over 3 to 5 minutes and then declined to pre-exposure level or below. In the dose-response experiments the mean systolic tension after exposure to theophylline (but before glucagon injection) was 3.3 + 0.1 g but this was not significantly (p > .05) lower than before methyl-xanthine infusion (Table 2). Although the 15 minute perfusion time through the apparatus was usually sufficient to return the tension to pre-theophylline level, in some hearts the force was greater at this time. The available data were insufficient to s t a t i s t i c a l l y evaluate the influence of pre-exist-ing tension level due to theophylline on the glucagon inotropic response. Figure 6 and Table 6 demonstrate that theophylline enhanced the ability of glucagon to activate glycogen phosphorylase at a l l dose levels except 0.25 yg. As was true for buffer-perfused hearts, the minimum effective dose in the presence of 1 mM theophylline was 0.5 yg. However, the largest dose tested (8.0 yg) in methylxanthine-exposed hearts was able to produce maxi-mal phosphorylase & levels (72.2 %; N=l). This degree of activity was not obtained in CK-perfused hearts (Table 6) where the maximally effective 63 dose of 4.0 yg changed phosphorylase a. to.only 38.8 +.3.5 % of the total phosphorylase content. One mM theophylline alone elevated control levels of the active enzyme (measured 50 seconds after a 0.25 ml CK injection) to 8.4 + 2.5 % from 5.3 + 1.0 % (Table 6) but this was not a significant increase (p.> .05). The time course of glucagon-induced phosphorylase activation was slightly altered in the presence of theophylline 1 mM (Figure 4; Table 5). In methylxanthine perfused hearts the peak phosphorylase _a level was measured at 60 seconds and was not significantly higher than at 50 seconds. The plateau level was therefore delayed 10 seconds compared to the CK-perfused hearts (Figure 4). Also, the activation of glycogen phosphorylase was faster in the presence of theophylline, for % a. was significantly (p < .05) elevated over control at 20 seconds (Table 5). Theophylline significantly augmented the glucagon-induced phosphorylase activation at 25 seconds and a l l subsequent times except 40 seconds (Table 5) but not at earlier intervals. The control phosphorylase <i levels in this series of experiments were determined 0 seconds after injection of 0.25 ml of CK solution. Theophylline 1 mM did not significantly alter the activity although % phosphorylase a. was elevated from 6.9 + 0.8 to 10.4 + 1.9 (Table 5). Theophylline markedly influenced the abi l i t y of glucagon to increase tissue cyclic AMP accumulation. As in buffer-perfused hearts, peak levels were present 40 seconds after glucagon 2.0 yg (Figure 4) and were maintained at least until 60 seconds. However, theophylline-perfused hearts did not show a significant elevation above control u n t i l 20 seconds after glucagon injection (Table 8). This time was s t i l l earlier than the increase in contractile force and simultaneous with the increase In phosphorylase & (Figure 4). 64 At 0 seconds (controls) theophylline-perfused hearts.had a mean cyclic AMP content of 0. 32+ 0.02 pmols/mg wet weight—significantly greater than in non-exposed hearts (Table 8). In fact, the only time when nucleotide levels were not enhanced by the methylxanthihe was 15 seconds after glucagon 2.0 yg..; In the presence of 1 mM theophylline, the cyclic AMP dose-response curve was much steeper than that of the CK experiments (Figure 6). A dose of 0.25 yg glucagon was sufficient to raise cyclic AMP content above the control level of 0.41 + 0.03 pmols/mg wet weight (Table 9). This was also the min-imum effective dose for increasing contractile force, but not phosphorylase a_ levels, in methylxanthine-treated hearts (Tables 2,6). Glucagon 8.0 yg caused a substantial accumulation of cyclic AMP to 1.64 + 0.02 pmols/mg. Since higher doses were not tested i t is unknown whether this produces the maximum possible tissue concentration of the cyclic nucleotide. At a l l doses of glucagon examined, theophylline augmented the glucagon-induced cyclic AMP response (Figure 6; Table 9). With a 1 mM xanthine concentration the mean control level of nucleotide was also approximately 33 % greater than that of non-exposed tissue (0.41 + 0.03 vs. 0.28 + 0.04 pmol/mg; Table 9). The response to 0.25 and 0.5 yg of glucagon appeared to be elevated by the same amount as the response to CK injection was in the control hearts after theophylline. However, a large potentiation of tissue cyclic AMP content was observed following higher doses of glucagon admin-istered in the presence of the methylxanthine. Cyclic AMP after 8.0.yg glucagon was more than double that in 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 earlier findings of the actions of glucagon on cardiac force of contraction and glycogen metabolism. Figure 6 illustrates the dose-dependent manner of these polypeptide responses. Glucagon also increased cardiac accumulation of cyclic AMP but this was less dramatically altered by dose. The greatest increases in contractile ampli-tude and % phosphorylase ji were obtained with 4.0 yg glucagon (Tables 2,6) whereas cyclic AMP was s t i l l increasing after 8.0 yg (Table 9). On the other end of the dose-response curve, cyclic nucleotide levels were elevated after only 0.25 yg glucagon but twice this concentration (0.5 yg) was required to significantly increase the mechanical and metabolic a c t i v i t i e s . The present study also investigated the temporal sequence of glucagon-induced cardiac events. Contractile force increased significantly at 25 seconds and peaked 35 seconds after glucagon. Percent phosphorylase a_ was elevated at 30 seconds but did not peak unt i l after the maximum increase in force. Cyclic AMP was increased above control as early as 15 seconds arid reached a maximum at the same time as phosphorylase a_ (Figure 4). These results therefore satisfy one of Sutherland's c r i t e r i a for implicating cyclic AMP mediation of a hormonal response i.e. tissue levels of the nucleotide increased prior to, or simultaneous with, the physiological event. An earlier temporal study in the isolated rat heart (Mayer et a l . , 1970) was unsuccessful in demonstrating an increase in cyclic AMP prior to the positive inotropic effect of glucagon, although later increases were noted. This failure was attributed, by the authors, to a difference in the relative a b i l i t y to detect increases in cyclic AMP on the one hand, and mechanical activity on the other hand. Since the present experiments measured cyclic 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 adequate. Another explanation for the discrepancy in results between the two studies might be the different perfusion rates employed. Mayer et a l . (1970) used a rate of 7 ml/minute which caused the contractile effect, as well as the cyclic AMP response, to be maximal at only 20 seconds. Phosphorylase activation also occurred earlier 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 cyclic AMP accumulation at 40 seconds. Because the experimental protocols of this investigation and that of Mayer and associates (1970) were similar, i t is appropriate to compare the data further. The maximal increase in contractile amplitude in the study of Mayer et a l . was approximately 20% following a dose of 3.0 yg glucagon. In contrast, we observed much greater increases. For example, 2.0 yg glucagon resulted in a change of 2.1 + 0.2 g., or a 57.2 + 4.4 % increase over pre-injection systolic tension (Table 2). Also, the maximally effective dose (4.0 yg) caused a 77.1 +7.9 % increase in force. This difference was not _g due to endogenous catecholamine release since propranolol 10 M did not re-duce the inotropic effect of glucagon (Figure 8; Table 3). These data also confirm that glucagon is not acting directly on the catecholamine receptor (Lucchesi, 1968; Mayer et a l . , 1970). The results on phosphorylase activation also differ quantitatively. Although control levels of the a_ form, and levels after 1.0 yg glucagon are similar, Mayer et a l . observed a greater maximal activation (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 interfere with the glucagon metabolic response. It is more d i f f i c u l t to compare cyclic AMP alterations because of the 67 discrepancy between the two studies both in time course of cardiac action and in doses of glucagon employed. Mayer et a l . (1970) obtained a much lower maximal accumulation of the cyclic nucleotide (0.48 + 0.02 pmol/mg ventricle with 3.0 yg glucagon). This was slightly more than double the control level and was measured at the peak of the inotropic response using the maximally effective dose for mechanical activity. There are three possi-ble ways to compare the results with those obtained in the present study. In our experiments the maximum tissue content determined after glucagon alone was 0.73 + 0.01 pmol/mg ventricle (Table 9) . Since this was a supra-maximal dose for the inotropic - response, a more appropriate value (for com-parison purposes) might be that obtained with 4.0 yg glucagon. In this situation, cyclic AMP content was 0.64 + 0.01 pmol/mg wet weight. This was arrroximately 130 % greater than control levels. On a percentage basis, Mayer et a l . also obtained a value 130 % greater than control. After considering the time course of cyclic AMP changes encountered in the present study, i t i s possible the heart was frozen at an inappropriate time to detect maximal accumulation in Mayer's investigation. We observed that cyclic AMP peaked at least 5 seconds after the peak inotropic response (Figure 4) . Mayer et a l . (1970) did not measure levels at this time inter-val following the mechanical peak but rather a f u l l 20 seconds later. In our experiments cyclic AMP was declining at this point. If cyclic AMP concentrations at the time of peak tension are compared (this study 35 sec-onds'; Mayer et a l . 20 seconds) then the earlier investigators obtained lower absolute values (e.g. 2.0 yg glucagon increased cyclic AMP to 0.52 + 0.07 pmols/mg; Table 9) . However, because the doses are different (3.0 yg vs. 2.0 yg) this i s not an ideal comparison either. The control values are remarkably similar in the two laboratories. Mayer and coworkers reported 68 values of 0.21. + 0.01 and 0.32 + 0.02 pmol/mg in different t r i a l s . Both means are in 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, significantly decreased the glucagon-induced cyclic AMP changes. Thus, while quantitative differences exist, the qualitative similarity of results demonstrates the validity of data obtained in this investigation. Other reports have appeared in the literature concerning the influence of glucagon on intact • tissue cyclic AMP accumulation. The f i r s t of these (LaRaia, Craig and Reddy, 1968) suggested there was a dissociation between the positive inotropic action of glucagon and cyclic AMP content in the isolated, perfused rat heart. Unlike isoproterenol, no increases in cyclic AMP were observed. However, only two time intervals after drug injection (the same for both glucagon and isoproterenol) were selected for analysis and i t is possible that a glucagon-induced increase was missed because of poor experimental design. In support of this proposal, their data indicated a slight difference in the time course of the drug-induced inotropic actions. It i s therefore unlikely that the time course for drug-induced cyclic AMP a l -terations would be identical. Oye and Langslet (1972) also speculated on the causal relationship of cyclic 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 re-sponse. They found an identical pattern following isoproterenol. This is in contrast to the classical temporal sequence obtained by Robison et a l . (1965) where epinephrine injection caused a rapid and dramatic increase in cyclic 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 in their experiments, glucagon and isoproterenol were slowly perfused through isolated rat heart rather than given in a con-centrated bolus. The evidence for a dissociation between cyclic AMP and myocardial ino-tropism which was presented in the report of Oye and Langslet (1972) is not convincing for several reasons. There is 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 cyclic AMP versus time indicates that, contrary to the authors' interpretation, intracellular levels of cyclic nucleotide may i n -crease above control prior to, or a least simultaneous with, the mechanical effect. Oye and Langslet (1972) also reported that chlorpromazine abolished the isoproterenol-induced increase in cyclic AMP without'altering the ino-tropic response. However, these findings were not verified in a subsequent study (Osnes and Oye, 1975). A recent study of the myocardial actions of glucagon demonstrated dose-dependent increases in peak l e f t ventricular pressure and cyclic AMP in 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 activity in broken c e l l preparations was sensitive to glucagon concentrations producing changes in the intact heart; Earlier Murad and Vaughan (1969) had noted the presence of a glucagon-stimulatable enzyme in rat myocardium. Cat and human heart preparations also had glucagon-sensitive adenylate cyclases (Levey and Epstein, 1969). Thus, for the above mentioned species, another of Sutherland's c r i t e r i a has been satisfied. 70 Observations of glucagon-induced changes in guinea pig myocardium vary from one laboratory to another. Farah and Tuttle (1960) and Spilker (1970) each observed the positive inotropic response in spontaneously-beating and electrically-driven atria respectively. The maximally effective con-centration produced force increases no greater than 50% (Spilker, 1970). On the other hand, Prasad (1975) could not demonstrate increases in tension using ventricular papillary muscle. It is possible that a difference i n responsiveness exists between atria and ventricle of the same species. However, in isolated perfused hearts which were elect r i c a l l y paced, glucagon increased peak l e f t ventricular pressure by approximately 100% (Henry et all, 1975) , yet the polypeptide hormone could not re-instate e l e c t r i c a l or mech-anical activity i n potassium-arrested hearts (Watanabe and Besch, 1974; Schneider and Sperelakis, 1975). There may be a sex-linked difference i n susceptibility to glucagon. In a l l studies where a positive inotropic effect was obtained, male guinea pigs were used exclusively. However, the unsuccessful studies failed to specify which sex was used. In a preliminary experiment we could not detect any force change after glucagon was administered to a Langendorff preparation of a female guinea pig. Glucagon may alter the interval-force relationship i n myocardium. A corollary to this would be that the absolute and relative magnitude of drug-induced force increases may vary with stimulation frequency. Spilker (1970) showed that at stimulation frequencies between 60 and 240/ minute, the absolute increase in isometric tension^due to glucagon was uniform. At lower and higher rates, however, glucagon had less, i f any, positive ino-tropic effect. Unfortunately the influence of glucagon on the interval-strength relationship has not been investigated in guinea pig ventricle or 71 in rat myocardium although the shape of the curve in the absence of drugs differs in these three.tissues (guinea pig atria vs. guinea pig ventricle vs. rat atria and ventricle; Koch-Weser and Blinks, 1963). When such i n -formation becomes available, i t may explain some of the discrepancies' between laboratories. In conflict with the second messenger hypothesis glucagon was found not to increase cyclic AMP in a dose-dependent manner in isolated guinea pig ventricle (Henry et a l . , 1975) although force changes paralleled those in rat heart. Furthermore adenylate cyclase activity in the guinea pig particulate preparation did not vary with glucagon concentration. These data represent an apparent dissociation between glucagon-induced inotropism and cyclic AMP. Although several time intervals after the onset of admin-istration were tested, the possibility that small increases were missed due to inappropriate freezing time cannot be entirely excluded. Henry et a l . (1975) obtained higher basal levels of cyclic AMP in the guinea pig heart' compared to the rat heart. This alone may be sufficient to mediate the ino-tropic response. A relative lack of dose-dependency was indicated in our investigation where increasing the glucagon dose 40-fold only increased tissue accumulation of cyclic AMP by approximately 3-fold (Figure 6; TablLe:.;9). Another explanation for the failure to see changes in guinea pig cyclic AMP content might be that phosphodiesterases may be degrading the cyclic AMP as fast as i t can be formed. A weak agonist lik 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 indirect-evidence that guinea pig atria 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 cyclic AMP unless papaverine was also present. The changes then resembled those in other species. The above explanation i s not entirely 72 satisfactory; ; however, because guinea pig ventricle did not require papaver-ine to respond to the cyclic AMP derivative. The adenylate cyclase responsiveness to glucagon may have been altered by the preparation of the enzyme. Although Henry et al.(1975) claimed that the enzyme was responsive because i t was activated by epinephrine and fluor-ide, this does not indicate that sensitivity to glucagon remained. As re-viewed by Levey (1975), glucagon and epinephrine responsiveness in sol-ubilized myocardial adenylate cyclase i s differentially restored by phos-pholipid addition. It is therefore possible that, in the experiments of Henry et al.a c r i t i c a l factor necessary only for glucagon activation was inadvertently removed. Evidence supporting a role for cyclic AMP in the mediation of glucagon-induced inotropism and phosphorylase activation was obtained in our exper-iments with theophylline. A 1 mM concentration of the methylxanthine sig-nificantly elevated both basal and glucagon-stimulated cyclic AMP accumu-lation (Figures 4,6), presumably by inhibiting i t s degradation. The time course of 2.0 yg glucagon on tissue cyclic AMP content was essentially unaltered in the presence of theophylline. It was noted, however, that theo-phylline caused a delay compared to CK-perfused hearts in the time when glucagon significantly elevated the nucleotide concentration over basal levels (20 seconds vs. 15 seconds; Table 8). Consonant with i t s actions on tissue cyclic AMP content, 1 mM theo-phylline enhanced both the phosphorylase-activating and mechanical effects of glucagon. Apart from prolonging the glucagon-induced increase in systolic 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 after glucagon 2.0 yg i.e. increases in cyclic AMP followed by in-creases in force and f i n a l l y % phosphorylase a was maintained in the pre-sence of theophylline. The dose-dependent behaviour of the glucagon-elicited changes in cyclic AMP, force of contraction and % phosphorylase a. was also examined during concurrent administration of theophylline. Phosphorylase a. levels were augmented after glucagon doses of 0.5 yg and higher, which might be expected from the greater cyclic AMP content (Figure 6). However, low doses of glucagon did not produce a greater metabolic response in the presence of theophylline 1 mM, even though cyclic nucleotide levels were enhanced. Theophylline also f a c i l i t a t e d maximal activation of glycogen phosphorylase by glucagon, as demonstrated in Figure 6. Similarly, much higher tissue con-centrations of cyclic AMP were made possible by the methylxanthine. The results on the positive inotropic effect of glucagon in combin-ation with theophylline are not easily interpreted. If data are analyzed in terms of percent increase in 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 effect only to 0.25 and 2.0 yg of glucagon (Table 2). The reason for the discrepancy i s not clear but could be due to a slightly depressed systolic tension after exposure to theophylline for 10 minutes. Because of this, the same absolute increase would result in a higher percentage in-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 relative to the pre-exposure level. This would hardly account for the magnitude of discrepancy observed. Experiments with theophylline were usually undertaken on different days from those in buffer-perfused hearts. In 74 CK-only experiments, the mean systolic tension before glucagon injection was 3.8+0.1 g while the corresponding value in the theophylline experi-ments was 3.3+0.1 g. This i s a significant (p <.05) depression and could explain the contradictory data. It is tempting to state from these results that the theophylline aug-mentation of glucagon-induced mechanical effects is more apparent than real. However, the fact that weaker hearts were able to respond to the same degree after glucagon challenge indicates some theophylline influence on myocardial contractility, although the efficacy of glucagon was not improved. This i s in contrast to the altered efficacies of glucagon on cyclic AMP accumulation and phosphorylase activation with the methylxanthine. The d i f f i c u l t y in interpreting force data reflects, to some extent, the fact that the Langendorff preparation is not ideal for determining drug-induced changes in myocardial contractility. Only .^vertical changes are recorded yet the developed tension presumably also increases i n fibres oriented in other directions. Ventricular volume i s not controlled and therefore some of the recorded change may be due to the Frank-Starling ef-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 in systolic tension. Glucagon also had a chron-otropic action which tended to reach a maximum after the peak inotropic response. The present experimental design did not include an investigation of the positive chronotropic effect and quantitative data are not obtain-able from the records. Because of the inverse frequency-force relation-ship in rat heart the inotropic response to glucagon would, at most, be 75 underestimated. In a more appropriate preparation for examining the mechanical actions of glucagon, Marcus et a l . (1971) observed a potentiation by theophylline, but with a lower concentration (2.5 x 10 - 4 M). This concentration also augmented the inotropic responses of norepinephrine and dibutyryl cyclic AMP but not of calcium (Skelton et a l . , 1971). Unfortunately, intact tissue levels of cyclic AMP were not measured and so the efficacy of this methylxanthine concentration as a phosphodiesterase inhibitor in intact preparations i s unknown. Lucchesi (1968) could not show any inotropic response to glucagon in intact dog heart simultaneously receiving 10 mg/kg theophylline which, alone, substantially increased the force of contraction. In Lucchesi's experiments, other drugs with a positive inotropic effect similarly prevented the typical glucagon increase, including dichloroisoproterenol. Lucchesi supplied evidence that glucagon did not act on the cardiac -(preceptor by showing a lack of effect of propranolol plus dichloroisoproterenol on the glucagon mechanical response. Earlier investigators (Farah and Tuttle, 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 interaction of glucagon with theophylline i n dog papillary muscle but saw only an additive tension change with a methylxanthine concentration of 3 x IO - 4 M. At a higher concentration (1 mM) they observed a depression of the glucagon response but because this concentration also impaired the tissue response to elec-t r i c a l stimulation, they suggested the effect was non-specific. For glucagon-induced phosphorylase activation, a l l of Sutherland's c r i t e r i a suggesting a role for cyclic AMP have now been met. In summary, 76 intact tissue levels of nucleotide were shown to increase prior to, or simultaneous with, the metabolic response. (Figure 4; also Mayer et a l . , 1970) Glucagon activated adenylate cyclase in 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 in phosphorylase a. (Figure 6;Table 6) in a concentration which markedly elevated amounts of intact tissue cyclic AMP (Table 9), pre-sumably due to phosphodiesterase inhibition. Finally, dibutyryl cyclic AMP (Kjekshus et a l . , 1971; Oye and Langslet, .1972) and cyclic AMP (Kjekshus et a l . , 1971) administered exogenously also increased the percent of active phosphorylase. While the metabolic actions of glucagon in the isolated 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 is a less effective agonist. This parallels the a b i l i t i e s of the two drugs to increase tissue levels of cyclic AMP. Although saturating doses were not determined for cyclic 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 in phosphorylase a.. - Glucagon presumably activates phosphorylase via cyclic AMP but consistent activation of phosphorylase kinase was not obtained (Mayer et a l . , 1970). In spite of the apparent similarity, there i s no reason to assume an identi-cal mechanism of action for phosphorylase activation by glucagon and the catecholamines. Namm et al.(1968) unquestionably established there are at least two regulators of cardiac glycogen phosphorylase activity. Calcium was essential for the catalytic activity of active phosphorylase kinase even though the amount of active enzyme was elevated apparently via cyclic AMP. As was 77 observed for epinephrine (Namm et a l . , 1968) the glucagon-induced increase in % 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 activation, the relative contribution of cyclic AMP and calcium toward the f i n a l phosphorylase a_. level is unknown; No investigator has conclusively shown that either the catecholamine or glucagon metabolic action can proceed without some increase in cyclic AMP. Mayer and coworkers (1970) reported an apparent dissociation in that 1.0 yg glucagon elevated the % phosphorylase a_ without changing the basal cyclic AMP content. However, as previously discussed, the ina b i l i t y to detect small cyclic nucleotide increases would account for the results. In the present study cyclic AMP increased after 0.25 yg glucagon but the miniinum effective dose for phosphorylase activation was 0.5 yg (Figure 6). This may be due to differences in assay sensitivities, but could also reflect the existence of other factors in the control of glycogen phos-phorylase activity. This is further indicated by the fact that glucagon 4.0 yg produced maximal activation (Table 6) i n CK-perfused hearts, yet cyclic AMP accumulation was s t i l l increasing after 8.0 yg glucagon (Table 9) . The difference, in slopes between the cyclic AMP dose-response curve and the phosphorylase dose-response curve indicates that an adenylate cyclase-cyclic AMP pathway might provide an efficient system for regulation of cardiac metabolism because very small changes in cyclic AMP were associated with large differences i n phosphorylase activity. It is conceivable that catecholamines and glucagon may increase glycogen phosphorylase activity through a simultaneous and direct influence on calcium and cyclic AMP. Alternatively, these drugs may act on adenylate cyclase and the resultant increase in cyclic AMP might, in turn, alter cal-78 cium homeostasis as well as permitting activation of phosphorylase kinase. A third possibility might be that glucagon and the catecholamines produce their metabolic response only through stimulation of adenylate cyclase, the normal cellular calcium control processes being sufficient to allow phosphorylase activation. At the present time, there are no data to rule out any of these proposals. Also there i s no indication, as yet, of the source of activator calcium—it might be intracellular, extracellular, or both. There are experimental data that would theoretically implicate calcium as the sole mediator in certain situations where glycogen phosphorylase i s activated. For example, i n anoxia the % phosphorylase a i s increased in the in situ rat heart even when cyclic AMP elevation i s blocked by practolol (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 cyclic AMP.(Namm et a l . , 1968; Hartley and McNeill, unpublished observations). In fact, Namm and associates (1968) noted a significant decrease in cyclic nucleotide concentration when rat hearts were perfused with a high calcium medium. Some drugs appear to alter myocardial phosphorylase a independently of cyclic AMP, Imidazole (1.6 mg) significantly elevated the % of the ji form without changing tissue cyclic AMP content (Verma and McNeill, 1974). How-ever only one time interval was selected for analysis and an increase might have been missed. Theophylline also may produce i t s glycogenolytic action independent of the adenylate cyclase pathway (McNeill et a l . , 1974) because increases in % phosphorylase a. were noted in the absence of significant changes in cyclic AMP. In that study, however, there was a large variation in the control nucleotide levels which may have obscurred the results. 79 In summary, cardiac glycogenolysis induced by any of the above-mentioned drugs may theoretically occur independently of the adenylate cyclase-cyclic AMP system (because of results with calcium) but most of the evidence, albeit circumstantial, militates against this, particularly i n the case of glucagon and the catecholamines. The data obtained with theophylline i n the present study do not exclude a cyclic AMP involvement in the cardiac phosphorylase activation e l i c i t e d by this methylxanthine. Experimental evidence supporting the involvement of cyclic AMP i n the positive inotropic effect of glucagon was also obtained i n the present i n -vestigation by satisfying 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 significant increase i n the intracellular cyclic nucleotide concentration preceded the onset of the positive ino-tropic response (Figure 4). Second, theophylline 1 mM potentiated the per-cent increase i n contractile force at several doses and the absolute increase at 0.25 andZOyg doses of glucagon (Table 2). Theophylline also lowered the minimum effective glucagon dose from 0.5 to .0.25 yg while concurrently raising the cyclic AMP accumulation resulting from the 0.25 yg dose (Table 2). As was true for glycogen phosphorylase activation, the catecholamines seem to be more effective than glucagon i n enhancing myocardial force of contraction. A maximum increase of 80% over pre-injection systolic 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 efficacy between glucagon and epinephrine. In our laboratory a difference between the two drugs was also noted in the time course of their mechanical action. Norepinephrine had a faster 80 onset and produced i t s peak change in tension earlier than glucagon, although the latter agent maintained the increase in force of contraction much longer. These variations in onset and duration of the inotropic effect between gluca-gon and epinephrine are reflected in the patterns of tissue cyclic AMP accu-mulation following drug administration. The time course of the norepin^-. ephrine-induced cyclic AMP response was not examined in the present study, but others have indicated that the cellular nucleotide concentration rises rapidly, reached a sharp peak and then declines to an intermediate level 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 in cyclic AMP. Also, a submaximal tissue concentration was maintained at least 120 seconds after injection of a 2.0 yg dose (Figure 4) Although the majority of experimental data strongly supports the second messenger.hypothesis, the literature does contain reports of dissociations between the positive inotropic effect of glucagon and the catecholamines and their influence on intracellular cyclic AMP. The papers on glucagon have already been discussed. Those on the catecholamines appear to be equally unconvincing. Shanf eld e_t_al. (1969) were able to block the norepinephrine-induced increase in cyclic AMP but not the positive inotropic response using a meth-oxamine congener and weak (3 adrenergic blocking agent N-isopropyl-meth-oxamine. However Wastjla et al.(1972), using butoxamine, instead observed a simultaneous reduction.in contractile force with the decrease in cyclic AMP. These later investigators also noted the direct depressant effect of the blocking agent on myocardial force, as well as basal tissue cyclic 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 diastolic tension (fibre length) after perfusion with isopropylmethoxamine. Another indication of a dissociation was presented by Oye and Langslet (1972) who found that isoproterenol perfused through the rat heart did not increase cyclic AMP prior to the onset of the positive inotropic event. The shortcomings of this report were discussed previously with respect to the glucagon dissociation. Exogenous cyclic AMP perfusion, in the presence of DMSO to f a c i l i t a t e membrane transport, was found to increase glycogen phosphorylase activation in the isolated guinea pig heart without elevating the force of contraction (Kjekshus et a l . , 1971). The authors believed this represented a dissocia-tion of cyclic AMP from positive inotropism because the % phosphorylase a was enhanced and that this was a consequence of intracellular cyclic nucleo-tide accumulation. However others have shown that active phosphorylase levels can be elevated independently of cyclic AMP (Namm et a l . , 1968; Dobson and Mayer, 1973; Hartley and McNeill, unpublished observations) and so the interpretation of Kjekshus and coworkers (1971) must be questioned. Furthermore, the data on phosphorylase activation are also suspect because the control levels are very high, and DMSO by i t s e l f had a considerable stimulatory effect (which the authors claim i s unimportant). The dose dependency of the cyclic AMP-produced phosphorylase activation was also not investigated and the possibility of a nonspecific activation remains open. Cyclic AMP is believed to be a common mediator of the inotropic effects of the catecholamines and glucagon, and also of the methylxanthines. On this basis, the influence of these drugs on properties of a single contraction would be expected to be similar, and should be mimicked by dibutyryl cyclic 82 AMP. To summarize what was mentioned earlier, a l l four agents increase maximal developed tension and rate of rise of tension. The catecholamines and dibutyryl cyclic AMP decrease time to peak tension and shorten the dur-ation of the active state (Skelton et a l . , 1970; Spilker, 1970; Blinks et a l . , 1972; Meinertz et a l . , 1974; 1975a) but glucagon has no influence on these parameters (Glick et a l . , 1968; Spilker, 1970; Marcus et a l . , 1971). Theo-phylline and other methylxanthines increase time to peak tension and prolong relaxation time (Skelton et a l . , 1971; Blinks et a l . , 1972). These d i f f e r -ences can only be explained i f the drugs are assumed to have multiple mech-anisms of action, one of which might be elevation of the intracellular cyclic AMP concentration. The f i n a l drug response would thus represent the sum total of various effects on cellular processes leading to tension devel-opment. Since the catecholamines, methylxanthines and glucagon a l l alter myo-cardial calcium exchangeability, this may be a possible consequence of their actions on cyclic AMP. Meinertz et al.. (1973 a,b) have shown similar ef-fects of dibutyryl cyclic AMP and norepinephrine on calcium-45 exchange and contractile force. Glucagon w i l l also promote calcium-45 influx (Nayler et a l . , 1970). In guinea pig atria, theophylline increased calcium-45 uptake and release (Scholz, 1971). The extent to which a drug interferes with normal calcium control processes may determine the magnitude of i t s inotropic effect. One of several sites where cyclic AMP may act to influence calcium exchangeability is 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 potential (review by Reuter, 83 1974). Tsien et a l . (1972) have demonstrated that, like the catecholamines, dibutyryl cyclic AMP and cyclic AMP increase action potential 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 directly determined at present but Watanabe and Besch (1974) performed sophisticated experiments to show that cyclic AMP did increase prior to isoproterenol-restored mechanical activity in arrested hearts. Dibutyryl cyclic AMP also restored contractions but the onset of action was slower (Watanabe and Resch, 1974; Schneider and Sperelakis, 1975). If cyclic AMP mediates the catecholamine effects on the cardiac action potential, other agents which elevate cellular accumulation of the nucleo-tide would be expected to display similar properties. Glucagon has not been widely investigated from the electrophysiological standpoint, but those studies which have been conducted indicate glucagon does not raise the plat-eau amplitude (Spilker, 1970; Prasad, 1975). No consistent influence on the duration of the action potential has been shown, for Prasad (1975) noted a dose-dependent shortening, whereas Spilker (1970) observed a slight pro-longation. Spilker also de-emphasized the importance of action potential duration because variable 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 in electrophysio-logic measurements. Therefore these data are of questionable value. Manganese ion can antagonize the positive inotropic effect of glucagon (Spilker, 1970; Nayler et a l . , 1970) i n a manner similar to what was observed with catecholamines (Sabatini-Smith and Holland, 1969). This suggests some influence of glucagon on calcium entry. However because glucagon could not 84 elevate the depressed action potential amplitude to a normal level (Spilker, 1970) while norepinephrine could restore the plateau potential indicates there are differences in their effects on calcium influx. Part of the di f -ference may be due to the type of tissue investigated since, in this case, Spilker (1970) used Purkinje fibres which are specialized 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 reflect their respective chronotropic rather than inotropic actions. Watanabe and Besch (1974) and Schneider and Sperelakis (1975) could not restore excitability and contractions to potassium-depolarized guinea pig hearts with glucagon. This was evidently considered together with the failure of glucagon to elevate cyclic AMP in this preparation (Watanabe and Besch,1974) as support for the idea of a cyclic AMP-mediated increase in the slow calcium current which is apparently essential for electro^ mechanical activity. The intriguing results of Henry et al.(1975X who demonstrated a pos-i t i v e inotropic effect of glucagon i n guinea pig independent of adenylate cyclase stimulation, together with the data of Watanabe and Besch, must be interpreted cautiously. Superficially the results indicate that glucagon (in guinea pig) does not activate the slow calcium channels to increase contractile force but must instead influence some other cellular process.. It i s implied that slow calcium channels are not activated because glucagon was incapable of restoring tension development. It i s also implied that the reason glucagon failed to increase membrane calcium transport was be-cause i t did not stimulate adenylate cyclase. Because the positive ino-tropic action of glucagon is not consistently observed in guinea pig i t is 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 really 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 cyclic AMP) may s t i l l be a possibility. The study of Henry et a l . (1975) indicated that glucagon does not require elevated levels of intracellular cyclic AMP to produce i t s positive inotropic action but does not suggest the absence of a causal relationship in situations where adeny-late cyclase is stimulated. The above-described investigations also do not permit speculation of the role of cyclic AMP in the glucagon response in other species. Measurements of the influence of glucagon on calcium influx are contradictory. Nayler et a l . (1970) did observe that glucagon increased calcium-45 influx in dog papillary muscle. In contrast, Visscher and Lee (1972) obtained negative results with the isolated cat heart, even though the doses of glucagon used had a considerable inotropic effect. Indirectly, the competitive antagonism of glucagon-induced force changes by manganese (Nayler et a l . , 1970; Spilker, 1970) is consistent with a hormonal action on membrane calcium transport, yet this does not explain why glucagon was incapable of restoring excitation-contraction coupling in the experiments of Watanabe and Besch (1974) or Schneider and Sperelakis (1975). Visscher and Lee (1972) reported that while glucagon could prevent cardiac arrest in the isolated, perfused cat heart exposed to 0.09 M calcium, i t could not restore contractions in a zero-calcium medium. This suggests that a major action through intracellular calcium release is unlikely. In agreement with the observation by Visscher and Lee, Nayler et al.(1970) noticed no action of glucagon on either calcium binding or release by intra-86 cellular organelles. Katz and associates (1975) proposed that cyclic AMP mediates the i n -creased rate of relaxation after catecholamine administration. They have demonstrated in vitro that cyclic AMP stimulates sarcoplasmic reticular membrane phosphorylation in parallel with calcium transport and suggest a causal relationship between cyclic AMP and the accelerated relaxation rate. Meinertz et al.(1975 b) provided further evidence by showing that dibutyryl cyclic AMP mimicked the behaviour of norepinephrine on duration of the active state following twitch stimulation. Both agents were alsonnoted to depress high-potassium induced contracture. On the basis of the hypothesis of Katz et a l . (1975), other agents which increase cyclic AMP might be expected to also produce abbreviation of systole, yet glucagon does not (Glick et a l . , 1968; Spilker, 1970; Marcus et a l . , 1971). 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. This is d i f f i c u l t to explain i f each drug is assumed to. work via cyclic 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 is appropriate to consider other mechanisms by which such potentiations may occur. A blockade of catechol-amine uptake by theophylline appears unlikely because Rail 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 shift to the l e f t of the dose-response curve of tyramine and isoproterenol on phosphorylase activation and a further shift of the nor-87 epinephrine response in tripelennamine-treated rats. Methylxanthines are capable of releasing endogenous catecholamines (Westfall and Fleming, 1968). McNeill et al.(1969) demonstrated that the slightly elevated basal phosphorylase activity as a result of theophylline was blocked by propranolol or reserpine. Therefore, i n the present study, the possibility that 1 mM theophylline is releasing endogenous catecholamines cannot be disregarded. Control cyclic AMP levels were significantly greater after theophylline perfusion for 15 minutes (Tables 8,9). Also, % phos-phorylase a was greater in the theophylline-exposed hearts but the d i f f e r -ence was not significant (Tables 5,6) and from dose-response studies i t was evident that cyclic AMP increases could be detected to 0.25 yg glucagon when phosphorylase elevation could not (Figure 6). Therefore small amounts of catecholamines may be liberated sufficient to affect basal cyclic AMP accumulation without detectably raising phosphorylase activity. The trans-ient positive inotropic effect of 1 mM theophylline perfusion might also be explained by catecholamine release. The data from the present investigation strongly implicates cyclic AMP as the mediator of the myocardial actions of glucagon. It is tempting to suggest that the potentiation of the glucagon responses in the presence of theophylline was due to greater tissue levels of cyclic AMP. The disso-ciation at low doses of glucagon between cyclic AMP on the one hand, and contractile force and phosphorylase activation on the other hand, may be attributed to differencesi-in the limits of measurement sensitivity. In fact, theophylline 1 mM abolished the apparent dissociation between con-tra c t i l e force and cyclic AMP following 0.25 yg glucagon (Figure 6). The theophylline perfusion slightly altered the time-course of glucagon-induced phosphorylase activation and contractile force increases, but the time course of cyclic AMP accumulation was altered in a parallel fashion (Figure 4). 88 Even though theophylline appeared to act by phosphodiesterase inhibition, the data must be interpreted cautiously. The results obtained with theo-phylline alone illustrate this point. If theophylline exhibited i t s positive inotropic effect by raising intracellular cyclic AMP then pre-injection sys-t o l i c tension should be greater after methylxanthine exposure to correspond to increased control cyclic AMP concentration (Table 9 ) . However, a mean depression i n systolic tension of approximately 6 % was instead observed (Table 2 ) . Active phosphorylase was also not increased significantly (Table 6 ) . A l l of the observed potentiations could be explained on the basis of a methylxanthine effect on calcium. This may be a direct effect or mediated through cyclic AMP, or both. Watanabe and Besch (1974) and Schneider and Sperelakis (1975) each determined the ab i l i t y of theophylline to restore mechanical activity presumably via the slow calcium channels when theo-phylline also raised intracellular tissue cyclic AMP levels (Watanabe and Besch, 1974). If, as Katz et a l . (1975) propose, the catecholamine-induced acceleration of relaxation is mediated by cyclic AMP, then methylxanthines must not act via phosphodiesterase inhibition entirely ( i f at al l ) because they prolong systole and antagonize the isoproterenol shortening (Blinks et a l . , 1972). Other investigators have noted an imperfect correlation of phosphodiesterase inhibition with cardiac actions of methylxanthines and papaverine (McNeill et a l . , 1973; McNeill et a l . , 1974; Henry et al. ,1975) . A direct effect on intracellular calcium homeostasis by methylxanthines would explain these differences as well as account for the augmentation of the glucagon responses. However such an influence on intracellular calcium is not an entirely satisfactory explanation either, because theophylline failed to potentiate the inotropic effect of calcium, in concentrations which 89 potentiated the inotropic effects of norepinephrine and dibutyryl cyclic 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 is ubiquitous among drugs. To expect that glucagon also works entirely through the adenylate cyclase system is also naive and while the present investigation strongly implicates cyclic 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 cyclic AMP, elevated % phosphorylase _a, and enhanced the force of contraction in a time and dose dependent manner. Time experiments in the present study established that following glucagon challenge, tissue cyclic AMP concentration increased prior to significant increases in contractile force or phosphorylase a.. Cyclic AMP remained elevated for prolonged periods as did systolic tension and the % active phophorylase. Dose experiments revealed that cyclic AMP could increase after doses of glucagon which would not e l i c i t a detectable positive inotropic effect or glycogen phosphorylase activation. These results suggest that cyclic AMP is involved in 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 in sensitivity of the various measuring systems. Further evidence supporting the second messenger hypothesis was supplied in the experiments with theophylline . A 1 mM concentration of the xanthine was found to be effective in inhibiting phosphodiesterases in the intact rat hearti as indicated by the dramatic enhancement of glucagon-induced cyclic AMP accumulation. Theophylline perfusion also augmented the positive inotropic effect of glucagon and the % of active phosphorylase. The efficacy of glucagon in activating phosphorylase was improved in the presence of the methylxanthine. The data strongly implicate an association between myocardial cyclic 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 systolic tension, and also produced slight changes in phosphorylase activity. Cyclic nucleotide accumulation was elevated relative to buffer-perfused hearts after theophylline exposure. .Therefore, the methylxanthine.' appeared to be working through phosphodiesterase inhibition. However, theophylline can cause release of endogenous catecholamines and has pro-found effects on calcium homeostasis in vitro. The results of the present investigation do not rule out these alternate mechanisms of methylxanthine action. 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Recent Advances in studies on Cardiac Structure and Metabolism 4^: 375-392, 1974. Wollenberger, A., Ristau, 0. and Schoffa, G. : Eine einfache technik der extremen schnellen abkulung grosserer gewebstucke. Arch. Ges. Physiol. 270: 399-412, 1960. APPENDIX Comparison of t i s s u e c y c l i c AMP concentration c a l c u l a t e d assuming a u n i f o counting e f f i c i e n c y i n a l l v i a l s w i t h t i s s u e c y c l i c n u c l e o t i d e l e v e l s i n v i a l s c o r r e c t e d f o r quench. % C y c l i c AMP (pmol/mg C y c l i c AMP (pmol/mg uncorrected , ' uncorrected c o r r e c t e d -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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