THE ROLE OF ADENOSINE 3',5'-MONOPHOSPHATE IN THE CARDIAC ACTIONS OF GLUCAGON MARGARET EDNA BRUNT. Sc. (Pharm.), University of British Columbia, 1974

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1 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

2 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Depa rtment The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date 0 * / /975

3 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 % (N=6) relative to preinjection systolic tension. This dose was also found to produce the maximal phosphorylase activation ( % in the a. form). The cyclic AMP content was 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. All three parameters remained significantly greater than control at least 120 seconds after glucagon administration. 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 glucagoninduced changes in force of contraction, % phosphorylase a or tissue cyclic AMP content, although this concentration readily, ^blocked the positive ino-

4 i i i tropic response to norepinephrine. It is therefore unlikely that the cardiac 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 methylxanthine, glucagon produced dose-dependent changes in % phosphorylase a, contractile force and cyclic AMP accumulation which were considerably greater than in buffer-perfused hearts. Systolic tension was increased % 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 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 intrinsic ability 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 approximately 30 % greater than in buffer-perfused hearts yet the % active phosphorylase 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

5 AMP involvement in the theophylline cardiac responses, and in the theophylline-glucagon interaction. Other possible mechanisms of action, such as an influence on calcium, should be given equal consideration.

6 V TABLE OF CONTENTS Page ABSTRACT LIST OF TABLES i i vi LIST OF FIGURES \* vii LIST OF ABBREVIATIONS viii 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 Cardiac actions of glucagon 19 MATERIALS AND METHODS MATERIALS 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. Statistical methods 33 RESULTS 35 DISCUSSION 65 SUMMARY AND CONCLUSIONS 90 BIBLIOGRAPHY 92 APPENDIX ' 101

7 vi LIST OF TABLES TABLE Page 1. The effect of time on the positive inotropic response to 2 yg glucagon in the buffer-perfused and theophyllineperfused rat heart The effect of various doses of glucagon on contractile force in the isolated buffer-perfused and theophyllineperfused rat heart. 45 _ g 3. The influence of propranolol 10 M on the positive inotropic effect of glucagon in the isolated perfused rat heart The influence of 10 M propranolol on the positive inotropic action of norepinephrine in the isolated perfused rat heart The effect of time on glucagon-induced cardiac phosphorylase activation in the buffer-perfused and theophyllinepe7. perfused isolated rat heart 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 The effect of time on cardiac cyclic AMP accumulation following administration of 2 yg glucagon into the bufferperfused and theophylline-perfused rat heart The effect of various doses of glucagon on cardiac cyclic AMP accumulation in the buffer-perfused and theophyllineperf used isolated rat heart The influence of propranolol 10 M on glucagon-induced cyclic AMP accumulation in the isolated perfused rat heart. 60

8 vii LIST OF FIGURES FIGURE Page.11. Schematic representation of the second messenger concept Enzymes involved in the control of myocardial glycogenolysis An adaptation of the general model of cell activation (Rasmussen et al., 1972) to myocardial tissue 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 theophylline Effect of time on the absolute change in tension following injection of 2 yg glucagon into the isolated buffer-perfused rat heart 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) The effect of various doses of glucagon on the absolute change in systolic tension in buffer-perfused and theophylline-perfused rat hearts The influence of propranolol 10 M on glucagon-induced changes in cardiac cyclic AMP content, contractile force and percentage phosphorylase a The influence of propranolol 10 M on the positive '.inotropic effect of norepinephrine. 52

9 viii ABBREVIATIONS ATP AMP cyclic AMP CK cpm DB-c-AMP adenosine 5' -triphosphate adenosine monophosphate adenosine 3',5' -cyclic monophosphate Chenoweth-Koelle counts per minute cyclic N 6-2'-0 dibutyryl-amp EDTA ethylenediamine tetra-acetic acid G-l-P S.E.M. TCA Tris glucose-l-phosphate standard error of the mean trichloroacetic acid tri(hydroxymethyl)aminomethane

10 ix 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 is 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 is gratefully acknowledged.

11 1 INTRODUCTION 1. The Role of Calcium in Excitation-Contraction Coupling The intracellular concentration of free calcium is now generally accepted to be a major determinant of the activation state of myocardium (Langer, 1968). However, the processes involved in the regulation of calcium are still 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. rise to the spike of the action potential. A rapid inward sodium current gives This is 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 al., 1969). Sarcolemmal repolarization is 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 electrical events. The onset of contraction follows the upstroke of the action potential by milliseconds (Morad and Goldman, 1973). Relaxation occurs after the membrane has begun to repolarize. The duration of the active state is 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 relaxation to occur, the calcium must be removed from the troponin (Schwartz, 1972).

12 2 Removal of calcium from the vicinity 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 al., 1974).but might be necessary for relaxation of fully-activated muscle (Solaro and Briggs, 1974). To prevent the cell 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 is supplied by the electrochemical gradient for sodium was obtained by Jundt et al. (1975) who noted an association of extent of muscle relaxation, calcium-45 efflux and extracellular sodium concentration. Theories on the initiation 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 translated from the sarcolemma. Most investigators believe the calcium ions entering the cell as the slow current do not account for all of the developed tension, although this is 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

13 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 threshold for myofibrillar activation. Although the 'relaxing system' is also stimulated by the higher calcium active state intensity increases. levels, the influx rate is greater and so 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 is that it postulates control of tension by the level of membrane polarization. The action potential (first 100 milliseconds) would release internal calcium. During the plateau phase inward calcium transport would be maintained by a Ca^-K exchange carrier in 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 contribution 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 in the excitation-contraction cycle. Catecholamines increase the strength of contraction in mammalian myocardium but the mechanism of action is still 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 increases the magnitude of the slow calcium current, which accounts for the higher plateau level (Reuter, 1974). These electrophysiologic experiments

14 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 facilitation 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 in myocardium Cyclic AMP was discovered during the course of investigations on mechanisms of hormone-induced hepatic glycogenolysis (Sutherland and Rail, 1958). Within a few years^ cyclic AMP was implicated in a variety of hormone responses. The "second messenger" theory of hormone action was proposed to account for the rapidly accumulating data (Sutherland et al., 1965). As originally outlined, a hormone was suggested to interact directly with adenylate cyclase located in the target cell membrane. The increased level of cyclic AMP then served as an intracellular messenger to modify enzyme 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).

15 Varied Stimuli.plasma Endocrine Gland ATP 5-AMP f phosphodiesterase membrane of target cell J HORMONE (first messenger) OC o d «J > Cyclic 3,5-AMP (second messenger) O < Physiological Inactivated Hormone Responses Steroids,Thyroid Hormonejetc. Fig-1. Schematic representation of the second messenger concept

16 6 The second messenger system allows hormones to be effective without permeating the cell membrane. It also provides a method of modifying a given hormonal stimulus. Hormone specificity is provided for by the fact that only those hormones which produce a physiological response in the target cell will stimulate adenylate cyclase. Sutherland and associates (1965) first proposed that catecholamines produced theory. their cardiostimulatory effects according to the second messenger The strongest evidence they cited were experiments by Robis op. Jand coworkers (1965) where epinephrine injection into isolated rat heart elicited a marked elevation of cyclic AMP prior to the increases in contractile force and phosphorylase activation. Many investigators have repeated their observations in other species and tissue preparations (review by Sutherland et al., 1968). The role of adenylate cyclase and cyclic AMP in cardiac glycogenolysis is well understood. A sequence of enzymic reactions is 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, in 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 al., 1968). Als'o; anoxia will stimulate phosphorylase when cyclic AMP is not elevated (Dobson and Mayer, 1973). Therefore, the cyclic nucleotide is not independently responsible for glycogenolysis regulation.

17 7 CYCLIC AMP Phosphorylase b Kinase Kinase Phosphorylase b Kinase (inactive) ATP 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

18 8 At one time phosphorylase activation was thought to be necessary for initiation of mechanical activity. However, low doses of epinephrine producing changes in contractility did not produce changes in phosphorylase a^ levels (Mayer et al., 1963; Drummond et al., 1964). Also, phosphorylase activation followed the initiation 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 in vitro evidence of an association was found by Murad et al., (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 is an intracellular mediator then exposure to the nucleotide or a derivative should also increase the force of contraction. Robison and coworkers (1965) attributed their initial failure to change the strength of contraction using cyclic AMP to the poor membrane penetrating ability. In contrast Meinertz et al. (1974) demonstrated that the positive inotropic action of cyclic N^-2'-O-dibutyryl-AMP (DB-c-AMP) was concentration-dependent

19 9 in isolated electrically driven atrial 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 altering 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 activation (Sobel and Mayer, 1973). Thus, while a considerable volume of evidence supports a cyclic AMP involvement in the contractile response some experimental 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 capable 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 al. (1969) postulate that cyclic AMP may facilitate 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-

20 10 stimulated present. calcium uptake by cardiac microsomes when protein kinase was 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 al., 1975: Schwartz et al., 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 al., 1974; 1975 a,b). Phosphorylase kinase can phosphorylate cardiac microsomal preparations (Schwartz et al.,1974) and also troponin (St mi et al., 1972). but the importance 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 all increased the action potential plateau amplitude and shortened the plateau duration in an identical manner to the catecholamines (Tsien et al., 1972). These results indicate a cyclic nucleotidemediated increase in the slow inward calcium current and in the outward potassium current. Further evidence supporting the idea that cyclic AMP.is involved in excitation-contraction coupling.is provided i n recent combined electrophysiological-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 If current. c y c l i c AMP was acting as a second messenger to increase calcium in-

21 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 is probable. Rasmussen and associates (1972) have reviewed the interaction of calcium and cyclic AMP on several hormonally-responsive tissues, including myocardium. They modified 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 in myocardial tissue (see figure for explanation). 3. Mechanisms of the cardiac actions of methylxanthines Sutherland and Robispn (1966) developed a set of criteria which, if fulfilled for any hormone, would strongly implicate cyclic AMP as a mediator of its end-organ response. First, 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 cell perparations. Third, the effect of the hormone should be mimicked by the addition of exogenous cyclic AMP or one of its derivatives. Finally, agents which modify phosphodiesterase activity should correspondingly modify the hormonal response. These criteria have been essentially satisfied for catecholamines in myocardium with a few exceptions mentioned earlier (Rasmussen et al.,1972; Sobel and Mayer, 1973). Experiments attempting to satisfy the last of the above-mentioned cri-

22 12 FIGURE 3. An adaptation of the general model of cell activation (Rasmussen et al., 1972) to myocardial tissue. When a hormone interacts with its receptor site, it does two things simultaneously (1) it activates adenylate cyclase, leading to increased intracellular levels of cyclic AMP and (2) it increases membrane permeability to calcium, allowing an intracellular increase in concentration of this ion. Cyclic AMP has at least two effects intracellularly (a) it initiates the enzymatic reaction sequence leading to glycogenolysis and (b), it 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, (ii) stimulation of enzymes, notably active phosphorylase b_ kinase, to produce metabolic changes and (iii) 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 built-in mechanism for stopping a signal equally important as initiation of it.

23 5 AMP Ph a r> Figure 3 G - 1 ~ P I

24 teria i.e. parallel alterations in phosphodiesterase activity and physiological response frequently employ the methylxanthines as phosphodiesterase 1* 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 electricallydriven atrial perparation, observed a potentiation of the norepinephrineihduced 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 al., 1963). Theophylline and caffeine have well-established positive inotropic effects of their own (Blinks et al., 1972). Hess and Haugaard (1958) also demonstrated the ability of aminophylline to increase active cardiac phosphorylase levels which was repeated in later experiments (Hess et al., 1963). Vincent and Ellis (1963), by measuring cardiac glycogen content, demonstrated the direct glycogenolytic action of theophylline. All 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 al. (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" 4 M. They assumed this was an effective concentration for phosphodiesterase inhibition. Kukovetz and Poch (1970) also noted an augmentation in Langendorff preparations of rabbit, rat and guinea pig myocardium. The sophisticated study of Watanabe and Besch (1974), using potassiumdepolarized guinea pig.hearts, established a further link between phospho-

25 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 is consistent with an intracellular site of action. Similar observations on the electrical and mechanical restorative ability 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 in 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 accumulation of the cyclic nucleotide was also determined, Verma and McNeill (1974) found parallel decreases in 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 ^ in combination with norepinephrine, indicate the correlation with phosphodiesterase inhibition is less than perfect. Hess et al. (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 in the in situ rat heart.

26 16 McNeill, Brenner and Muschek (1973) compared, in guinea pig myocardium, the ability of various methylxanthines and papaverine to potentiate catecholamine- induced inotropism and phosphorylase activation with their potency as phosphodiesterase inhibitors. Although there was good correlation among the methylxanthines with respect to the potentiating effect and the phosphodiesterase inhibiting action, papaverine gave anomalous results. The alkaloid was more potent than the naturally occurring methylxanthines in in hibiting guinea pig phosphodiesterase and enhanced the phosphorylase-activating effect of norepinephrine. However, i t did not augment the positive inotropic action. Furthermore the direct inotropic actions'of this series did not correspond to their phosphodiesterase inhibiting ability because theophylline possessed the greatest inotropic effect of the methylxanthines, while isobutyl methylxanthine (SC-2964) was the most potent enzyme inhibitor. Papaverine had a negative inotropic effect. From these data, McNeill et al. (1973) questioned the cause and effect relationship between phosphodiesterase inhibition and the cardiac actions of these drugs. Two very recent studies have provided further evidence that the methylxanthines may not work through cyclic AMP. McNeill et al. (1974) found that although theophylline (1 mg) had a weak positive inotropic and phosphorylaseactivating effect on its own, it did not change the cyclic AMP content of -4 the guinea pig heart. In addition, theophylline 7 x 10 M potentiated the norepinephrine metabolic and inotropic actions but did not influence the catecholamine-induced change in cyclic AMP. A higher methylxanthine concentration (2 mm) was cardiodepressant by itself and reduced the positive inotropic response to several doses of norepinephrine. However, i t potentiated the increase in cyclic AMP to the highest dose of norepinephrine (0.4 yg). These results suggest that, although theophylline and other methylxanthines are

27 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. All three agents increase maximum developed isometric tension and rate of tension development (Skelton et al., 1970; Skelton et al., 1971; Blinks et al., 1972). However the similarity ends here. Both norepinephrine and dibutyryl cyclic AMP decreased time to peak tension (Skelton et al., 1970) whereas the methylxanthines increased time to peak tension (Blinks et al., 1972). In contrast to isoproterenol, the active state of myocardium is prolonged by methylxanthines (Blinks et al., 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 al. (1972) could not distinguish any of these effects among the three methylxanthines tested, even though phosphodiesterase-inhibiting >potency; varies.

28 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 in vitro actions on cyclic AMP phosphodiesterases, the methylxanthines have profound effects on cellular calcium homeostasis which could also account Therefore their role in establishing questioned (Sobel and Mayer, 1973). for many experimental observations. cyclic nucleotide involvement has been Caffeine increased calcium exchangeability 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 al., 1975). This latter observation is consistent ;with the ability 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 ability by cardiac sarcoplasmic reticulum was not directly investigatedjjundt et al. (1975) observed marked stimulation of sodiumdependent 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

29 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 ability of these agents to restore excitability and mechanical activity to potassiumarrested 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 intrinsic effects on myocardial contractility, on glycogen phosphorylase and their ability to potentiate the catecholamine-induced cardiac responses since calcium has a well-established role in each process. Whether phosphodiesterase inhibition is causally related to the calcium effects remains to be determined. 4. Cardiac actions of glucagon Glucagon was first observed to elicit changes in myocardial function by Farah and Tuttle in 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 responses 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

30 contrast, the fifth dose of epinephrine increased the force of contraction as much as the first 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 cardiotonic 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 situ preparations where the drug has been directly infused into the heart (Regan et al., 1964; Afonso et al., 1972.; Hammer et al., 1973) have yielded qualitatively similar results. All.in situ 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 al., 1968; Lucchesi, 1968; Hammer et al., 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 catecholamine response (Glick et al.,1968; LaRaia et al,. 1968; Lucchesi,.1968; Spilker, 1970; Hammer et al., 1973). Consequently, the theory that glucagon acts at the.'yg -receptor has now been, abandoned (Glick et al., 1968; Lucchesi, 1968). A structural analog of propranolol. 'profitthalol, was also shown to be without effect on glucagon-induced inotropism (Mayer et al.,1970).

31 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 al., 1968; Gold et al., 1970; Nayler et al., 1970; Spilker, 1970). The drug also increases rate of force development although time to peak tension is not altered (Glick et al., 1968; Gold et al., 1970;Spilker, 1970). The catecholamines decrease time to peak, tension (Skelton et al., 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-frequency 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 stimulation 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 parallel manner. Norepinephrine changed the shape of the curve and increased the force relative to control at all 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 still increases contractile force (Glick et al., 1968; Lucchesi, 1968; Gold et al., 1970; Nayler et al., 1970; Spilker, 1970; Marcus et al., 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 al., 1970). Mn +^ also shifted

32 22 the dose-response curve for glucagon to the right (Spilker, 1970). Visscher and Lee (1972) examined the association of extracellular 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 little influence on the excitatory 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 contradiction 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 experimental conditions. The amplitude of the plateau phase is depressed in 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) demonstrated that glucagon, unlike the catecholamines, was unable to restore

33 23 e x c i t a b i l i t y and contractions. Similar observations were made in tetrodotoxin-treated hearts (Watanabe and Besch, 1974). Glucagon will 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 al., 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 al., 1970). Much experimental effort has focused on the role of cyclic AMP in the glucagon cardiac responses. Initially no change in cyclic AMP concentration could be detected in intact rat heart challenged with glucagon, although the inotropic response was elicited (LaRaia et al., 1968). However subsequent investigators (Mayer et al., 1970; Oye and Langslet, 1972; Henry et al., 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 al., 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 involvement comes from experiments in failing hearts where glucagon was ineffective both in increasing contractility and in stimulating adenylate cyclase in

34 vitro (Gold et al., 1970). Apparently guinea pigs do not have a glucagonresponsive cyclase system, although glucagon can elicit changes in force in this species (Henry et al., 1975). There have been many attempts to associate adenylate cyclase activity with calcium homeostasis. Entman and coworkers (1969) found a glucagon-sensitive cyclase in a microsomal preparation' which also increased calcium uptake in the presence of glucagon. However this paper does not present data on the purity of the preparation. The experiments of Nayler et al. (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) in guinea pig hearts depolarized with high potassium. This is consistent with the observations of Henry et al. (1975)indicating lack of a glucagon-sensitive cyclase in this species.. Unfortunately, the ability of glucagon to restore excitability has not been investigated in species possessing a glucagonstimulatable 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 in force accompanied glucagon inhibition of this

35 25 enzyme in 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 ability 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 al., 1971). Afonso and associates (1972) found qualitatively similar alterations in force of contraction monitored following concurrent administration of glucagon and aminophylline to in situ dog heart. Apparently in contradiction to these results, Lucchesi (1968) found that pre-infusion with theophylline prevented the glucagon inotropic response in in situ canine heart. However, glucagon was administered during the period of maximum inotropic response to theophylline. Marcus et al. (1971), in contrast, used a methylxanthine concentration with minimal intrinsic actions. Antonaccio and Lucchesi (1970) found the inotropic actions of glucagon with low concentrations of theophylline were additive in isolated dog papillary muscle. A higher concentration actually reduced the response to glucagon. McNeill et al.. (1969) noted a similar influence on the norepinephrine response in in situ 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 cyclasecyclic AMP system. Although numerous studies have been undertaken to es-