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The effects of adrenergic amines and theophylline on contractile force and cyclic AMP Martinez, Terry T. 1975

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THE EFFECTS OF ADRENERGIC AMINES AND THEOPHYLLINE ON CONTRACTILE FORCE AND CYCLIC AMP by Terry T. Martinez M.S., Purdue University, U.S.A. 1970 B.S., Purdue University, U.S.A. 1969 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the D i v i s i o n of Pharmacology of the Faculty of Pharmaceutical Sciences We accept t h i s thesis as conforming to the required standard. THE UNIVERSITY OF BRITISH COLUMBIA October, 1975 In present ing th is thes is in p a r t i a l fu l f i lment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f r ee ly ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of th is thes is for scho la r ly purposes may be granted by the Head of my Department or by h is representa t ives . It is understood that copying or pub l i ca t ion of th is thes is fo r f i n a n c i a l gain sha l l not be allowed without my wri t ten permission. Department of Pharmaceutical Sciences The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date October 14, 1975 ABSTRACT Time response studies of the effects of norepinephrine and phenylephrine revealed that both agonists caused an increase i n c y c l i c AMP le v e l s p r i o r to increases i n c o n t r a c t i l e force. Norepinephrine caused a nearly s i x f o l d increase i n c y c l i c AMP, whereas phenylephrine produced only a 50% increase i n the nucleo-t i d e . Pretreatment with reserpine d i d not af f e c t the norepineph-r i n e c y c l i c AMP response; however, the phenylephrine c y c l i c AMP response was abolished. Reserpine pretreatment did not s i g n i f i c a n t -l y a f f e c t the c o n t r a c t i l e responses of either amine. In the pre-sence of propranolol, norepinephrine was found to have the a b i l i t y to produce an increase i n c o n t r a c t i l e force i n which c y c l i c AMP was apparently not involved. The time course of the c o n t r a c t i l e response induced by adrenergic amines was found to be remarkably influenced by the chronotropic response i n spontaneously beating preparations while the c y c l i c AMP response was not greatly a f f e c t -ed. This difference i n the c o n t r a c t i l e response may be due to the a b i l i t y of the chronotropic response to influence the f l u x of calcium through the c e l l membrane. At 37° C phentolamine was found to have no effect on the isoproterenol dose-response curve. Phentolamine did, however, cause the norepinephrine log dose-response (LDR) curve to s h i f t to the r i g h t and the maximum response was attenuated. Phentol-amine competitively antagonized the phenylephrine LDR curve. Propranolol caused a s h i f t to the ri g h t of the isoproterenol LDR curve. In the presence of propranolol the e f f i c a c y of i i of isoproterenol was increased, which may be related to the a b i l -i t y of propranolol to antagonize binding and sequestration of inter n a l free calcium. Propranolol competitively antagonized only the second component of phenylephrine a c t i v i t y which was probably due to catecholamine release. At 22 ° C phentolamine was found to produce an apparent non-s p e c i f i c , noncompetitive antagonism of the inotropic response to isoproterenol, norepinephrine and phenylephrine. This apparent blockade was found to be related to the a b i l i t y of phentolamine to increase the inotropic effect of low temperature so as to leave l i t t l e room within the l i m i t s of c o n t r a c t i l i t y for the agonist to produce a p o s i t i v e inotropic response. The e f f i c a c y of a l l the amines appeared to be increased i n the presence of propranolol which was found to antagonize the inotropic effects of low temperature and thus leave more room within the l i m i t s of c o n t r a c t i l i t y f o r an amine to produce an inotropic response. The c y c l i c AMP response was found to be blocked by propranolol at 37 ° C, 22° C, and 17 ° C. Phentolamine did not block the c y c l i c AMP response at any temperature tested. Exposure to phenoxy-benzamine 17 ° C f o r 45 minutes before te s t i n g at 37 ° C d i d not s i g n i f i c a n t l y a f f e c t either the c o n t r a c t i l e response or the c y c l i c AMP response from control experiments. It i s therefore concluded that there i s no interconversion of alpha and beta adrenergic receptors mediated by temperature. The interpre-t a t i o n of the effects of adrenergic antagonists at low temp-erature i s complicated by t h e i r a b i l i t y to modify the inotropic e f f e c t of temperature alone. i i i Theophylline alone produced! a 50% increase in cyclic AMP levels, however, this response was abolished in reserpine pretreated tissue. In addition, theophylline was found to exert a direct contractile effect which was unrelated to cyclic AMP. The effect of theophylline on cyclic AMP appeared to be additive with the norepinephrine and phenylephrine responses. The effect of theophylline on amine-induced cardiac cyclic AMP and contractile force showed no correlation between the contract-i l e and the cyclic AMP effects at the different times tested. It therefore seems logical that the cardiac effects of theophyl-line are not mediated through cyclic AMP. These results sup-port the view that the methylxanthines exert their effects on heart through changes in calcium metabolism. iv TABLE OF CONTENTS Chapter Page ABSTRACT i i LIST OF FIGURES v i I INTRODUCTION 1 The role of cyclic AMP in the Regulation of C e l l -ular Processes 1 The Positive Inotropic Effects of Catecholamines and Cyclic AMP 7 The Dissociation of Cardiac Inotropic and Adenylate Cyclase Activating Adrenoceptors 10 The Effect of Interaction Between Catecholamines and Theophylline on Contractility and Cardiac Cyclic AMP 13 II METHODS 16 Animals 16 Preparation of Tissues 16 Apparatus 20 Solutions 21 Cyclic AMP Assay 22 Drugs and Chemicals 25 III RESULTS 26 Time-Response Effects of Norepinephrine and Phen-ylephrine on Cardiac Cyclic AMP and Contractil-ity ' 26' The Effect of Temperature on Cardiac Inotropic and Adenylate Cyclase Activating Adrenoceptors 29 The Effect of Theophylline on Amine-Induced Cardiac Cyclic AMP and Cardiac Contractility 33 IV DISCUSSION 76 The Mechanism of Action of Norepinephrine and Phenylephrine on Cardiac Contractility 76 The Effect of Temperature on Cardiac Inotropic and Adenylate Cyclase Activating Adrenoceptors 84 The Effect of Theophylline on Amine-Induced Cardiac Cyclic AMP and Cardiac Contractility 95 V SUMMARY 102 LIST OF REFERENCES 1°5 v. LIST OF FIGURES Figure Page 1 Adenosine 3',5'-Phosphate (cyclic AMP) 2 2 Second messenger concept 4 3 The mechanism of action of cyclic AMP in regulating glycogen metabolism 6 4 Tissue bath for isolated atria 18 5 Time-response effects of norepinephrine on cardiac cyclic AMP and contractility in the driven l e f t atrium at 3 7 ° C 37 6 Time-response effects on phenylephrine on cardiac cyclic AMP and contractility in the driven l e f t atrium at 3 7 ° C 39 7 Time-response effects of norepinephrine and phenylephrine on cardiac cyclic AMP and contract-i l i t y in the spontaneously beating right atrium at 3 7 ° C 41 8 Time-response effects of, norepinephrine and phenyl-ephrine on cardiac cyclic AMP in the perfused rat heart at 3 7 ° C 43 9 Time-response effects of norepinephrine on cardiac cyclic AMP and contractility in the driven l e f t atrium in the presence of propranolol and phen-tolamine at 3 7 ° C 45 10 The effects of different voltage stimuli on cardiac cyclic AMP in the driven l e f t atrium at 3 7 ° C 47 11 The effects of different rates of stimulus on cardiac cyclic AMP in the driven l e f t atrium at 3 7 ° C 47 12 Cumulative dose-response curves of isoproterenol, norepinephrine, and phenylephrine for driven l e f t atria and spontaneously beating right atria at 3 7 ° C 49 v i . Figure Page 13 Cumulative dose-response curves of isoproterenol, norepinephrine, and phenylephrine for driven l e f t atria at 17° C 51 14 Cumulative dose-response curves of isoproterenol, norepinephrine, and phenylephrine: in the absence of antagonists, in the presence of propranolol, and in the presence of phentolamine for driven l e f t a t r ia at 37 ° C 53 15 Cumulative dose-response curves of isoproterenol, norepinephrine, and phenylephrine: in the absence of antagonists, in the presence of propranolol, and in the presence of phentolamine for driven l e f t a,tria at 22 ° C 55 16 The effect of temperature changes on the strength of contraction of driven l e f t atria 57 17 Time-response effects of norepinephrine and phenyl-ephrine on cardiac cyclic AMP in the driven l e f t atrium at 22° C 59 18 Time-response effects of norepinephrine and phenyl-ephrine on cardiac cyclic AMP in the driven l e f t atrium at 17 C 61 19 The effects of propranolol and phentolamine on cardiac cyclic AMP in response to norepinephrine at 17 ° C, 22° C, and 37 C 63 20 The effect of exposure to phenoxybenzamine for forty-five minutes at 17 ° C and 37 ° C on cardiac cyclic AMP and contractility in response to norepinephrine 65 21 Time-response effects of theophylline on cardiac contractility in the driven l e f t atrium 67 22 The effect of exposure to theophylline for three minutes, fifteen minutes and sixty minutes on cardiac contractility in response to norepineph-rine in driven l e f t atria 69 23 The effect of exposure to theophylline for fifteen minutes on cardiac contractility in response to phenylephrine in driven l e f t atria, 71 24 The effect of exposure to theophylline on cardiac cyclic AMP in the driven l e f t atrium 73 v i i Figure Page 25 The effect of exposure to theophylline f o r f i f t e e n minutes, on cardiac c y c l i c AMP i n response to nor ' epinephrine and phenylephrine i n driven l e f t a t r i a 75 v i i i CHAPTER I INTRODUCTION The Role of Cyclic AMP In the Regulation of Cellular Processes Cyclic AMP was discovered in the course of investigations on the mechanism of the glycogenolytic action of epinephrine and glucagon in li v e r (Rail et a l . . 1957-' Sutherland and Rail, 1957). It was found that the response of liver homogenates to the hormones occurred in two stages. In the f i r s t stage, a particulate fraction of homogenates produced a soluble, heat-stable factor when incubated with hormones, Mg 2 + ions, and ATP. In the second stage, this factor stimulated the formation of active phosphorylase in supernatant fractions of homogenates in which the hormones themselves were inactive. This heat-stable "factor" was isolated and eventually was determined to have the nucleotide structure depicted in figure 1 (Sutherland and Rail, 1957, 1958;fLipkin et a l . . 1959). The proposition was then advanced that cyclic AMP was an intracellular "mediator" of the glycogenolytic action of epinephrine in l i v e r and other tissues by virtue of increasing the concentration of the physiologically active species of glycogen phosphorylase (Rail and Sutherland, 1961; Sutherland and Rail, I960). Subsequent investigations have provided evidence for the involvement of cyclic AMP in the actions of 2. H ,C H NH. C HC N C-II c-•N CH •CH I OH Adenosine 3,5'-phosphate (cyclic AMP) Figure 1 3. a large number of polypeptide and amine hormones in a wide variety of tissues from a diverse array of animal species (Breckenridge, 1970; Gilman and Hall, 1971; Hardman et a l . , 1971; Liddle and Hardjaaa, 1971; Robinson et a l . . 1968; Sutherland et a l . . 1957). In considering the evidence linking cyclic AMP to hormonal regulation, Sutherland et a l . , 1965, formulated what has become known as the "second messenger concept 0 which i s illustrated in figure 2 ( i a H and. #ii»aBf» JI^Q. " , ',.). According to this concept, the f i r s t messengers, the hormones themselves, would interact with tissue-specific sites in the plasma membrane and produce an activation of the enzyme adenylate cyclase, also in the plasma membrane. The augmented level of cyclic AMP produced would then proceed to influence a variety of c e l l structures through a sequence of events that are largely unknown. Upon removal of the hormone the system would return to normal activity owing in part to the conversion of cyclic AMP to 5*AMP by one or more of a family of cyclic nucleotide phosphodiesterases, of which certain of the known members are susceptible to inhibition by the methyl-xanthines. Thus, cyclic AMP could be viewed as a kind of "trigger?, setting in motion responses determined by the programming in the individual c e l l (Ball, 1972). Subsequent investigations have served both to expand the number of hormones, tissues and cellular processes that can be linked to the regulatory function of cyclic AMP and 4. HORMONES (FIRST MESSENGERS) r ATP 5'-AMP' RECEPTORS ADENYL\ :YCLASE ^CYCLIC AMP + PPj {SECOND MESSENGER) \ rri BIOCHEMICAL RESPONSES (ENZYME ACTIVATION, ETC.) / I \ PHYSIOLOGICAL RESPONSES (Glycogenosis, Lipolysis, Steroidogenesis, Enzyme Induction, Polypeptide Secretion, Na* Extrusion, Contractile Force, Membrane Permeability, etc.) Second Messenger Concept Figure 2 5. to provide greater insight into the mechanism of action of cyclic AMP. These investigations have revealed the exist-ence of a family of protein kinases in a variety of tissues that are stimulated by cyclic AMP and that transfer phosphate groups to a variety of cellular proteins resulting in some cases in a marked change in biological properties (Greengard and Kuo, 1970; Langan, 1970; Walsh et a l . . 1970). The-mechanism of action of cyclic AMP in regulating glycogen metabolism i s the best understood. Cyclic AMP is thought to interact with a phosphoproteln kinase (phosphory-lase kinase kinase), causing an increased conversion of the inactive species of phosphorylase kinase to the active species by the transfer of phosphate from ATP to serine residues (Walsh et a l . . 1968). The phosphorylase kinase, in turn, converts the less active species of phosphorylase (e.g., phosphorylase b In muscle) to the active species (e.g., phosphorylase a in muscle) by an analogous phosphorylation reaction. Gn the other hand, cyclic AMP accelerates the conversion of the active species of glycogen synthetase to the less active species by a similar phosphorylation reaction catalyzed by an enzyme known as glycogen synthetase I kinase (Eosell-Perez and Lamer, 1964) (figure 3 ) . Other investigations have also served to provide warning that rigorous application of the second messenger concept w i l l not allow adequate explanation of certain observations. There have been a number of instances in which the application of cyclic AMP or i t s derivatives on intact c e l l preparations 6. Cyclic 3\5'-AMP in K I Protei inase A T P I ADP I Phosphorylase 6 Phosphorylase ft Kinase Kinase (PO,) 4 A T P J, 4ADP 2 Phosphorylase b Phosphorylase a ( P O « ) | 5 -AMP I p i i Glucose-1-P * < Glycogen ( n ) Glycogen ( n _ i ) r ~ t " ^ x Synthetasi ADP UDP j G-6-P | UDP Glucose Synthetase D (PO,) Synthetase/ Protein Kinase t Cyclic 3',5'-AMP The Mechanism of Action of Cyclic AMP in Regulating Glycogen Metabolism Figure 3 7. has not f a i t h f u l l y reproduced the effects of the hormone in question. One example is the failure of butyryl cyclic i' AMP to bring about release of K* ions while efficiently inducing amylase release from rat parotid slices (Batzri et a l . , 1971a). The release of amylase is mediated by stimulation of beta-adrenergic receptors (and by cyclic AMP) while the release of K + ions involves stimulation of alpha-adrenergic receptors and thus, would not be expected to be mediated by increased levels of cyclic AMP (Batzri et a l . , 197$b). These data c a l l to attention the possibility that hormones may exert effects not mediated by cyclic AMP by setting in motion parallel sequences of events that may or may not interact with those initiated by the formation of cyclic AMP. In some instances i t may be extremely d i f f i c u l t to determine whether two different populations of receptors are involved (Ball, 1972). In summary, the second messenger concept has been enormously useful in suggesting experimental approaches for the dissection of regulatory effects of a large number of hormones and in suggesting ways in which chemical agents and other environ-mental factors might influence hormone action. We realize, however, that cyclic AMP is only one of many second messengers, and that the "classical" second messenger view w i l l not allow adequate explanation of a l l biological phenomenon. The Positive Inotropic Effects of Catecholamines and Cyclic AMP Considerable evidence suggests that the positive inotropic effects of catecholamines i n the heart are mediated by cyclio AMP. Following administration of catecholamines, myocardial 8. cyclic AMP levels rise before or at least simultaneously with the positive inotropic response.(Robison et al..1965; Drummond et a l . . 1966; Wastila et a l . . 1972). Effects on phosphorylase transformation and glycogenolysis occur later after changes in contractility are evident (Williamson, 1965; Mayer, 1963). Catecholamine agonists exhibit the same general order of potency in stimulating adenylate cyclase in vitro and in increasing contractility of the intact heart (Sutherland et a l . . 1968; Mayer, 1972). Other agents with positive inotropic effects, such as glucagon, prostaglandins, and histamine also stimulate myocardial adenylate cyclase (Farah and Tuttle, I960; Murad and Vaughn, 1969; Sobel and Robinson, 1969; Klein and JLevey, 1971$. Whereas the effects of epinephrine, norepinephrine, and isoproterenol on cyclic AMP levels of cardiac muscle are well documented, previous reports on the effects of phenyl-ephrine are controversial. McNeill and Verma. 1973, have reported that phenylephrine increases cyclic AMP levels in perfused guinea pig hearts, and Drummond and Hemmings, 1973, have reported similar findings in rat heart. On the other hand previous studies by Benfey, 1971, and Benfey and Carolin, 1971» on formation of cyclic AMP by particle preparations from chicken;', and rabbit heart, as well as rabbit heart slices, did not reveal increased cyclic AMP formation on the addition of phenylephrine. McNeill et a l . . 1972, similarly did not find an increase in cyclic AMP formation with phenylephrine in a guinea pig cardiac 9. particle preparation. Lack of stimulation of cyclic AMP formation in broken c e l l preparation or tissue slices does not, however, exclude an effect in the intact heart. The response of adenylate cyclase to drugs and hormones may disappear on homogenation of the tissue (Oye and Sutherland, 1966). The adrenergic receptors of cardiac muscle have been generally classif i e d as beta-receptors on the basis of reports by some investigators that they could find no alpha-adrenergic antagonism of the positive inotropic response to adrenergic agonists (Nickerson and Chan, 1961; Moran and Perkins, 1961). Other investigators have reported evidence supporting the existence of alpha-adrenergic receptors in the myocardium. Govier, 1968, using guinea-pig atria, observed that a portion of the positive inotropic response to adrenergic agonists was blocked by alpha-antagonists and this portion was greater in response to epinephrine and norepinephrine than to isoproterenol. Similar observations were made using a second parameter of myocardial function, increased functional refractory period. Wenzel and Su, 1966, observed that phentolamine potentiated the contractile response of rat myocardium to epinephrine and norepinephrine and that complete blockade of the positive* inotropic response to adrenergic agonists could only be achieved using both alpha and-beta antagonists. Osnes and Oye, 1975f have reported that when phenylephrine was combined with the beta-blocker propranolol, a dissociation between cyclic AMP levels and contractile activity was found. 10. Cyclic AMP accumulation did not seem to be required for the inotropic response caused by alpha stimulation. Phenylephrine was shown by Trendelenburg et al„. 1962, to be a direct-acting amine, as judged by dose-response curves on the nictitating membrane of reserpine-treated cats. Daley et a l . . 1966, however, found that this compound released t r i t i a t e d norepinephrine from mouse heart and Govier, 1968, reported that phenylephrine has not only a direct effect, but also an indirect action through the release of stored catecholamines. Yoo and Lee, 1970, have reported that in isolated rabbit a t r i a in which catecholamines had been almost completely depleted by reserpine, phenylephrine exerJted a positive inotropic action which was not significantly d i f f e r -ent from that observed on normal atria. The f i r s t part of this study was undertaken to determine the time course of cyclic AMP changes in response to norepin-ephrine and phenylephrine. It was also intended to separate the direct and indirect effects through the release of stored catecholamines by phenylephrine on cyclic AMP. The Dissociation of Cardiac Inotropic and Adenylate Cyclase  Activating Adrenoceptors Observations from several laboratories have suggested that the clas s i f i c a t i o n of adrenergic receptors into alpha and beta subtypes may be susceptible to change. Several groups have reported that in isolated perfused frog hearts, stimulation of cardiac rate and contractility by catecholamines has the properties of a classical beta adrenergic response when 11. experiments are preformed at warm temperatures (25-37° C) but of an alpha adrenergic response when experiments are performed at t i l i temperatures (S55°-$S° C) ^ Kunos and Szentivanyi, 1968; Buckley and Jordan, 1970; Kunos et a l . t 1973; Benfey et a l . . 1974). At warm temperatures, the order of potency of agonists in stimulating these preparations— isoproterenol>adrenaline>noradrenaline—is classical for a beta adrenergic receptor. Similarly, effects of the catecholamines at warm temperatures are blocked by propranolol but not by the alpha adrenergic antagonist > phentolamine. When the same experiments are performed at temperatures below 25° C, the order ©f potency of agonists is reversed to that characteristic of alpha adrenergic receptors. Also at lower temperatures alpha adrenergic antagonists such as phenoxybenaamine and phentolamine block the effects of adrenaline, whereas beta adrenergic antagonists such as propranolol are ineffective. Graduations of response can be achieved by varying the temperature between 3 7 ° C and 1 0 ° C. Similar observations have been reported for the rat heart. (Kunos and Szentivany, 1968; Benfey et a l . , 1974). On the basis of such observations, Kunos et a l . , 1973, proposed that alpha and beta adrenergic receptors may represent tallosteric configurations of the same active site which could be modulated by among other factors, temperature. a Caron and Lefkowitz, 1974, however, were not able to confirm the interconversion of receptors using the adenylate cyclase system model for study of the molecular properties of 12. beta adrenergic receptors. They reasoned that i f , in fact, alpha and beta adrenergic receptors merely represent temperature sensitive transitions in the state of single macromolecule, then the a b i l i t y of catecholamines to stimulate i and of adrenergic antagonists to block stimulation of adenylate cyclase should vary with temperature in a fashion analogous to that reported above. In each case they found that stimulation of the adenylate cyclase activity by catecholamines had the characteristics of a beta adrenergic response at h&gh; (37° C.) and low (15° @) temperatures. The beta antagonists propranolol was an effective inhibitor of catecholamine-induced stimulation of the enzyme and the alpha adrenergic antagonist phentolamine was ineffective under a l l the conditions examined. Subsequently Benfey, Kunos, and Nickerson, 1974, reported the dissociation of cardiac inotropic and adenylate cyclase activating adrenoceptors. They found that when the ambient temperature was reduced, the adrenoceptors mediating cyclic AMP production changed very l i t t l e ; they were blocked as effectively as at the higher temperature by propranolol and were not blocked by phenoxybenzamine. However, the adrenoceptors mediating the inotropic response were markedly changed by the decrease in temperature; phenoxybenzamine now inhibited this response and the inhibitory activity of propranolol was reduced about tenfold. It was concluded that the adreno-ceptors that mediate cardiac inotropic responses at physiological temperatures are distinct from those that mediate the production of cyclic AMP, and that the activation of adenylate cyclase 13. and the accumulation of cyclic AMP are probably not intermediate steps in cardiac inotropic responses to catecholamines. The inotropic response in cardiac muscle mayfebe mediated through either alpha or beta receptors, However, anf inotropic response mediated by an alpha receptor would not be expected to be associated with Increased levels of cyclic AMP. A dissociation between the beta receptor, cyclic AMP, and the inotropic response would cast serious doubt on the cyclic AMP-second messenger concept. In these studies (Kunos and Szent-ivany, 1968; Kunos et a l . . 1973» Benfey et a l . , 1974), the inotropic and cyclic AMP responses have been consistently report-ed in dose ratios and not as complete dose or time response curves which would have been more revealing as to the mechan-isms involved and which might in fact change the interpretation. It was therefore decided,as the second part of this project, to throughly investigate the biochemical and physiological changes induced by lower temperature in mammalian cardiac muscle to determine i f more complete data would substantiate or fsfute the cyclic AMP-second messenger view concerning the beta-adrenergic receptor. The Effect of Interaction Between Catecholamines and Theophylline  on Contractility and Cardiac Cyclic AMP It has been suggested that one of the c r i t e r i a for involving cyclic AMP in the mechanism of action of cardiac-stimulant drugs would be to demonstrate an enhancement of the drug effect by the addition of a phosphodiesterase inhib-itor to the system. The compounds usually used to demon-14. strate this interaction are the methylxanthines, and more specifically, theophylline. Theophylline is known to have a positive inotropic effect and to inhibit phosphodiesterase,, (Butcher and Sutherland, 1962). The addition of a phospho-diesterase inhibitor should enhance the effect of drugs which increase the synthesis of cyclic AMP by decreasing the degradation of the cyclic nucleotide. It has further been suggested that the positive inotropic effect of the methy-ated xanthines, themselves, may be mediated through cyclic AMP (Sutherland et a l . . 1968). In support of this concept, i t has been shown that theophylline w i l l enhance the inotropic effect of catecholamines and histamine (Rail and West, 1963; McNeill and Muschek, 1972), and w i l l also enhance the phosphorylase-activating effect of epinephrine (Hess et a l . , 1963). In some studies, however, the dose of theophylline required to potentiate the phosphory*. lase-activating effect of epinephrine or to induce cardiac glycogenolysis wasfftomndato^prJadfoel asnegatlv© lather fchafiv y a positive, inotropic effect (Hess et a l . , 1963; Vincent and E l l i s , 1963). Kukovetz et a l . , 1973, were able to produce an elevation in cyclic AMP using a perfusion of theophylline, however, McNeill et a l . . 1974, found that when theophylline was injected into the heart, an increase in contractility and phosphorylase 'a' was noted,but cyclic AMP values were not affected. It was further reported that while theophylline did enhance the inotropic and phosphorylase-activating effect of both amines, cyclic AMP, increased by the injection of the 15. amines, was not further increased when the hearts were perfused concomitantly with theophylline. Thus, we see that the literature dealing with the inter-action between catecholamines and theophylline on contractility and cyclic AMP in the heart is controversial. It was therefore decided, as the third part of this project, to investigate this interaction, introducing as a new variable the time of exposure to theophylline before the addition of catechol-amines. We hoped by this method to be able to resolve some of the differences in the literature and at the same time to determine the importance of cyclic AMP in mediating the cardiac effects of the methylxanthines in rate controlled preparations. CHAPTER II MATERIALS AND METHODS I. Animals Wistar rats (200-300 g.) of either sex were used through-out the investigation. They received food and water ad.  libitum. II. Preparation of Tissues A l l animals were pretreated with heparin (8mg/kg, subcutaneously) one to two hours prior to sacrifice. A. Atria The animal was stunned by a heavy blow at the base of the skull and the neck was quickly broken. The chest was then opened with scissors by a parasternal incision. The heart was exposed, l i f t e d gently by grasping the apex, and removed by cutting the great vessels which suspend i t from above and behind. The heart was immediately placed in oxygenated Chenoweth-Koelle solution (Chenoweth and Koelle, 1946) at 20 C and was gently compressed a few times to express residual blood. This phase of the preparation, from sacrifice to cooling of the excised heart, consumed 30 seconds of less. At this time, the l e f t or right atrium can be recognized and removed directly from i t s position above the ventricles by cutting with small scissors. The 16. 17. two atria are easily differentiated on the basis of rhythm and shape, since the right atrium beats spontaneously and has an irregular, asymmetrical shape. The l e f t is quiescent after isolation and has a regular triangular shape with smoothly rounded corners and sides. However, speed is essential. At no time should the tissue be allowed to remain out of the dissecting dish for more than 20 seconds. Threads were then placed at one corner and the opposite side of the atrium and one was made fast to the muscle holder; then, after,tran-fer from the dissecting dish to the muscle chamber, the.other was made fast to the transducer as shown in figure 4. Using a Harvard isometric tension clamp, tension on the atrium was adjusted to one gram. Prior to the addition of any drugs, the atria were allowed to stabilize for periods of fifteen to thirty minutes. With appropriate experience, the entire procedure from sacrifice to mounting in the chamber should not take longer than three or four minutes. Experience has indicated that the longer this period, the less viable a preparation is obtained, even though the preparation is well oxygenated in the dissecting dish. This is particularly evident for the quiescent l e f t atrium, but less c r i t i c a l for the spontaneously beating right atrium. (Katzung, 1968; Levy, 1971) B. The Isolated Perfused Whole Heart For the isolated whole heart preparations, the animal was k i l l e d and the heart, with at least one cm. of aorta attached, was quickly removed and placed in dissecting dish Figure 4. (A) Typical configuration of a vertical chamber and muscle holder (MH) for in vitro a t r i a l and ventricular preparations. (B) Detail of a muscle holder incorporating punctate electrodes. The entire holder is fabricated from a single piece 6f 0.65- or 0.97-cm acrylic plastic. MC, Muscle chamber, jacketed or immersed in a constant (temperature bath; FD, f r i t t e d glass disk for dispensing O2-CO2 bubbles (a fine polyethylene capillary inserted from the top was used tn the present series of exper-iments; M, isolated myocardial tissue tied down with the thread T, and connected at i t s upper end by a second thread, or fine wire to the transducer "Trans"; S, wires to stimulator; G, groove for anchoring thread used to t i e down the muscle; £, punctate electrodes (silver or platinum); W, insulated wires to stimulator. (Ref. Katzung, 1968) 19 Figure 4 20. containing oxygenated Chenoweth-Koelle solution exactly as described for the at r i a preparation. The aorta was located and dissected free and a l l other vessels connected to the heart were trimmed away. The aorta was cut just below the point where i t divides and the heart was transferred to the perfusion apparatus where the aorta was tied onto the glass cannula. Care must be taken to see that a i r bubbles do not enter the aorta and any bubbles which have formed in the cannula should be removed. A Palmer c l i p was placed in the apex of the ventricles and an attached string was connected to the force-displacement transducer via a pair of pulleys. Using a Harvard isometric tension clamp, tension on the ventricles was adjusted to one gram. Prior to the addition of any drugs, the hearts were allowed to stabilize for periods of fifteen to thirty minutes. III. Apparatus A 100 ml. tissue bath was used for the experiments with isolated atria . Water was circulated through the outer jacket to maintain the solutions in the bath at the desired temperature. A mixture of oxygen (95%) and carbon dioxide (5%) was bubbled through the solutions by means of a fine polyethylene capillary inserted from the top. A needle and thread were used to t i e the atria to a muscle holder which incorporated punctate electrodes as shown in figure 4. A second thread attached to apex of the atria was connected to a Grass force-displacement transducer, and isometric contractions were recorded on a Grass model 79 polygraph (Grass Instruments Quincy, Mass., U.S.A.). The l e f t atria were ele c t r i c a l l y driven at one Hz. 21. with three millisecond square wave pulses at four volts by a Grass model S 6 stimulator. A mechanical beater was used |o assure rapid equilibration of a l l parts of the bath. Tension on the atria was adjusted by means of a Harvard isometric tension clamp. At various times after addition of drug, the bath was rapidly lowered and the atria were frozen with chilled Wollenberger tongs (Wollenberger et a l . , I960) which had been kept in a beaker of 2-methylbutane immersed in dry ice. A l l tissues were stored at - 8 0 ° G u n t i l assayed for.cyclic AMP. The isolated heart system was essentially that.described by Fallen, et a l . . 1967. It consists of a modified Langendorff apparatus in which the perfusion of the heart via the aorta was maintained at a constant flow rate of four millper minute rather than at a constant pressure. This was achieved by use of a Holter Microinfusion r o l l e r pump (Extracorporeal Medical Specialties, King of Prussia Pa., model HL 175). Oxygenated solutions were pumped into the heart from a reservoir. Water was circulated through the outer jacket of the reservoir and a water Jacket surrounding the f i n a l section of tubing to maintain the heart and solutions at 37° C. Contractility was monitored by means of a Palmer c l i p placed in the apex of the heart and connected to a Grass force-displacement transducer and recorded on a Grass model 7 polygraph. At various times the hearts were frozerl and stored as described for the at r i a preparation. IV. Solutions A modified Locke-Ringer solution described by Chenoweth and 22. Koelle,{1946, was used exclusively in a l l experiments. The buffer was prepared by dissolving the following amounts of reagents in one l i t e r of d i s t i l l e d water ( a l l weights are expressed as grams of the anhydrous compound): Glucose, 1.9; NaCl, 7.0; KC1, 0.42; CaCl 2, 0.24; MgCl 2, 0.20. A pH of 7-4 was obtained by adding NaHCG3 (2.0 g / l i t e r for the at r i a experiments or 1.75 g/ l i t e r for the whole heart experiments) and bubbling a mixture of 95$ oxygen and % carbon dioxide through the solution. In the isolated a t r i a experiments, concentrated solutions of drugs were pipetted into the tissue bath in amounts required to achieve the desired molar concentration. The cumulative method of determining dose response curves was used. When the maximum response for a particular dose of a drug was reached, sufficient drug was added to achieve the&next highest molar concentration of that drug. In some experiments, propranolol (1GP° M) or phentolamine (10* 6 M) were added to the bath fifteen minutes prior to drug administration. Phenoxybenzamine was added forty? five minutes before a drug treatment. Eeserpine pre-treatment consisted of a dose of 3 m g A g intraperitoneally twenty four hours prior to the experiment. In the case of isolated whole hearts, drugs dissolved in Chenoweth-Koelle solution were injected wia a side-arm cannula. A l l doses were calculated as the free base. V. Cyclic AMP Assay The method of extraction has previously been described by Gilman, 1972. Frozen tissue samples (20-35mg) were homogenized 23-in 2 ml of cold % trichloroacetic acid (TCA). The samples were centrifuged on a "bench centrifuge set at i t s maximum speed for ten minutes. TCA supernatants were then extracted five times with 5 ml of ether after the addition of 0.2 ml of 1 N H C 1 . The purpose of this extraction was to remove the TCA. Residual ether was removed byyblowing nitrogen gas over the surface of the samples for one to two minutes and the aqueous extracts were lyophylized and redissolved in 0.8 ml of Tris^EDTA buffer (pH 7.5) 0.05 M containing 4 mM EDTA. Two 50 jal portions of the sample were used in the cyclic AMP determination. B. Cyclic AMP Determination Cyclic AMP concentrations were determined using an Alersham/Searle Cyclic AMP Assay Kit code TRK 432. This kit is a commercial adaptation of a simplified competitive protein-binding assay for cyclic AMP in plasma which has been previously described by Latner and Prudhoe (1973). The method is based on the competition between unlabeled cyclic AMP and a fixed quantity of tritium labeled cyclic AMP for binding to a protein which has a high specificity for cyclic AMP (Gilman, 1970). The amount of labeled protein-cyclic AMP complex formed i s inversely related to the amount of unlabeled cyclic AMP present in the assay. The concentration of cyclic AMP in the unknown is determined by comparison with a linear standard curve. Separation of the protein bound cyclic AMP from the s unbound nucleotides is achieved by adsorption of the free nucleotide on charcoal, followed by centrifugation on aS 24. bench centrifuge set at i t s maximum speed for fifteen minutes at 4° C, as i n i t i a l l y described for this assay by Brown et a l . , 1971* The only modification we made in the use of the kit is that the entire supernatant is removed for liquid s c i n t i l l a t e ion counting by decanting instead of a 200 u l sample as is recommended. After centrifugation, even i f the tubes are maintained at 4° C, the samples must be removed for liquid s c i n t i l l a t i o n counting within ten minutes i f there is to be no significant change in the radioactivity of the supernatant. Attempts at removing the 200 u l sample required excess time and, in addition, caused disturbances to the charcoal pellet. Both of these factors contributed to a loss of accuracy. The alternative procedure for calculation of results which is presented in the booklet accompaning each cyclic AMP kit was used. By this method the standard curve in competitive protein binding assays is presented as percent radioactivity bound to the binding protein, plotted against the concentration of the standard. This was found to give a better separation of values on the calibration curve in the range of interest (0.25 to 4.0 pmole/incubation tube). The use of a high viscosity s c i n t i l l a t i o n gel i s essential (Latner and Prudhoe, 1973), and the recommended cocktail, , PCS Solubilizer (Amersham/Searle's catalog number 196097) was used. Counts per minute were used directly for calculation in a l l assays in accordance with the manufacturer's recommend-ation. Variations in the efficiency of counting betweenssamples are not normally great enough to necessitate conversion of the 25. counts per minute to disintegrations per minute. A counting time of four minutes was used for a l l samples in this assay. At no time was the count rate/sample less than at least three times the background oount. VI. Drugs and Chemicals The drugs used in these experiments were 1-phenyleporine hydrochloride (Sigma Chemical Co.), 1-norepinephrine hydro-chloride (Sigma Chemical Co.), 1-isoproterenol hydrochloride (Winthrop Pharaceutical Co.), propranolol (Ayerst Laboratories, Inc.), phentolamine hydrochloride (Ciba Pharmaceutical Co.), phenoxybenzamine hydrochloride (Ciba Pharmaceutical Co.), theophylline (Merck and Company, Inc.), reserpine (Sigma Chemical Co.), and heparin sodium (Nutritional Biochemicals Corporation). A l l other chemicals, solvents, and reagents were analytical geagent grade. They were used as they were recieved without further purification. VII. Calculations and S t a t i s t i c a l Methods The oyclic-AMP results are expressed as picomoles of cyclic AMP per milligram wet weight of tissue. Contractility i s presented as percentage increase over control or as change in contractile force (g). The results were pooled and averaged and the standard error of the mean was determined at each experi-mental point. The data were compared by means of the Students t-test for unpaired data. A probability of less than 0.05 was chosen as the criterion of significance. CHAPTER III RESULTS Time-Response Effects of Norepinephrine and Phenylephrine  on Cardiac Cyclic AMP and Contractility Preliminary experiments (figure 12) established that 10"^ M norepinephrine or 10""^ M phenylephrine produced the maximum positive inotropic effect that could be obtained with these drugs. With the use of these maximally effective dosesrtime-response studies were carried out to determine the effects of the agonists on contractility and cyclic AMP. In driven l e f t atria, norepinephrine (figure 5) significantly increased cyclic AMP from a control value of 0.26*0.03 pmole/mg wet weight to a peak of 1.16*0.13 at 10 seconds and 1.18*0.15 at 15 seconds. Cyclic AMP then decreased to 0.67*0.09 pmole/mg at 60 seconds. Contractile force was significantly elevated at 10 seconds and peaked at 60 seconds (86.9*12.2# increase over control). iBhenylephrine (figure 6) significantly increased cyclic AMP from a control value of 0.26*0.03 pmole/mg to 0.35*0.03 at 10 seconds. Cyclic AMP values remained constant at this level for 100 seconds. Contractile force was significantly elevated at 20 seconds and peaked at 100 seconds (58.3*7.3$ increase over control). 26. 27/ Reserpine pretreatment did not significantly affect the contract t i l e fespenseeto either amine or to the cyclio AMP increase produced by norepinephrine. However, the treatment abolished the phenylephrine induced cyclic AMP change. Similar results were obtained in the spontaneously beating rat atrium.(figure 7) . Norepinephrine significantly increased cyclic AMP from a control value of 0.26*0.01 . pmoles/mg to 1.02*0.06 at 10 seconds and a peak of 1.38*0.08 at 15 seconds. Cyclic AMP then decreased to 0.87*0.05 at 60 seconds. Contractile force was significantly elevated at 10 seconds and peaked at 35 seconds (0.50*0.07 g increase). Phenylephrine significantly increased cyclic AMP from a control value of 0.28*0.01 p mole/mg to 0.35*0.02 at 10 seconds. Cyclic AMP values remained constant at this level for 60 seconds. Contractile force was significantly elevated at 10 seconds and peaked at kO seconds (0.58*0.06 g increase). Again reserpine pretreatment did not significantly affect the contractile response to either amine or to the cyclic AMP produced by norepinephrine. The treatment abolished the phenylephrine induced cyclic AMP change. Attempts to measure the time-feesponse effects of nor-epinephrine and phenylephrine in driven right ventricle strips were not successful. Norepinephrine (10~^ M) increased cardiac cyclic AMP from a control value of 0.30*0.02 pmole/mg to 0.36*0.03 at 10 seconds, 0.39*0.03 at 15 seconds, and 0.31*0.03 at 20 seconds. This small cyclic AMP response may indicate that the ventricle strips had been extensively 28. damaged during dissection. Dobson et a l . , 1974, have recent^ ly reported on the problems encountered with this preparation. No attempts were made to measure the cyclic AMP response to phenylephrine in the driven ventricle strips. The time-response effects of norepinephrine and phenylephrine on cardiac cyclic AMP were next determined in the perfused rat heart (figure 8). Norepinephrine (1 ug) significantly increased?;:cyclic AMP from a control value of 0.24*0.02 pmole/mg to 0.45*0.05 at 5 seconds and a peak value of 0.71*0.08 at 10 seconds. Cyclic AMP then decreased to 0.48*0.04 at 15 seconds. Norepinephrine (2 ug) increased cyclic AMP to 1.18*0.06 pmole/mg at 10 seconds (a value near the maximum response obtained in atria) . Since a previous report from this laboratory (McNeill and Verma, 1973) had indicated that the dose of phenylephrine needed to e l i c i t an increase in cyclic AMP in the perfused heart was 1000 times greater that for norepinephrine, a 2 mg dose of phenylephrine was used. This dose of phenylephrine increased cardiac cyclic AMP from a control value of 0.24*0.02 pmole/mg to 0.38*0.04 at 10 seconds. Pretreatment with reserpine abolished the phenylephrine induced cyclic AMP response. As phenylephrine had been shown to be capable of , increasing contractile force without causing a change .in cyclic AMP levels in reserpine pretreated tissue, i t was decided to see i f norepinephrine was capable of a similar action in the presence of propranolol. Time-response ®gfmt<-effects of norepinephrine (10~^ M) on cardiac cyclic AMP and 29. contractility in the driven rat atrium in the presence of propranolol (10*^ M) and phentolamine (10"^ M) were determined (figure 9 ) . In the presence of phentolamine, norepinephrine significantly increased cyclic AMP £&?om a control value of 0.26*0.03 pmole/mg to 0.85*0.07 at 15 seconds. Contractile force was significantly elevated at 10 seconds and peaked at 70 seconds (0.70*0.08 g). In the presence of propranolol, norepinephrine did not increase cardiac cyclic AMP from control values although contractile force was significantly increased at 15 seconds and peaked at 1$0 seconds (0.53*0.06 g increase). It is well known that suprathreshold stimulation of the order of two or three times threshold intensity may produce detectable release of catecholamines. This effect is more apparent at higher stimulus frequencies (e.g. 100 to 200 beats per minute) (Levy, 1971). Since threshold voltage was 1-2 volts and the l e f t a t ria preparations were being stimulated at 4 volts, i t was dscided to test the effect of different voltages and rates of stimulus on cardiac cyclic AMP. The effects of different voltage stimuli (4,10,15, and 20 volts) can be seen in figure 10. No significant difference was detected for any of the voltages tested. Likewise, d i f -ferent rates of stimulus (2 Hz vs 1 Hz) had no effect on , cardiac cyclic AMP levels (figure 11). The Effect of Temperature on Cardiac Inotropic and Adenylate Cyclase Activating Adrenoceptors As a preliminary experiment is was necessary to establish the cumulative dose-response curves of isoproterenol, nor-30. epinephrine, and phenylephrine for driven l e f t a t r i a and spontaneously beating right a t r i a at 37° C (figure 12). As expected, the order of potency of agonists in stimulating these preparations was isoproterenol>norepinepMine^phenyl'f!^ > ephrine. The order of efficacy was norepinephrine>phenyl-ephrine > isoproterenol. The amines produced equally potent effects on the right andxleft atria respectively. However, because the spontaneously beating right atria were subject to increasing rate, the efficacy was less than in the l e f t a t r i a . The cumulative dose-response curves of isoproterenol, norepinephrine, and phenylephrine for driven l e f t a t r i a were next determined at 17° G (figure 13). The positive inotropic effect of a l l the amines was greatly reduced by lowering the temperature from 37° C to 17° C. Phenylephrine, produced a significant negative inotropic effect (-0.07*0.04 g) -4 at 10 M. The order of eificacy was now norepinephrine> isoproterenol^phenylephrine. The order of potency of agonists in stimulating these preparations at 17° C was norepinephrine=isoproterenol> phenylephrine. The effect of temperature changes on the strength of contraction of driven rat l e f t a t r ia is shown in figure 16. Contractile force f i r s t decreased from 0.78*0.08 g at 37° C to 0.61*0.07 g at 30° C, then increased to 1.36*0.13 g at 18 6 C and 1.34*0.12 g at 17° C. The effects of phentol-amine (10~^ M) and propranolol (10"^ M) on the inotropic effect of temperature change at 22° C are also shown in figure 16. 31. Phentolamine increased the inotropic effect of temperature change from 0.99*0.10 g to 1.25*0.13 g. Propranolol decreased the strength of contraction from 0.99*0.10 g to 0.74*0.08 g. Figure 14 shows the cumulative dose-response curves of isoproterenol, norepinephrine, and phenylephrine at 37° C; in the absence of antagonists, in the presence of propranolol (10"^ M), and in the presence of phentolamine (10 -^ M) for driven l e f t a t r i a at 37° C. The concentration of the agonist causing 50% of the maximum effect i s marked on each curve. Phentolamine had no effect on the isoproterenol dose-response curve. Phentolamine caused a shift of the norepineph»i rine and phenylephrine curves to the right and suppressed the maximal response of norepinephrine. Propranolol caused a ; shift to the right of both the isoproterenol and the norepin-ephrine curves. An increase in efficacy was observed with high doses of isoproterenol in the presence of propranolol. Pro-pranolol reduced the slope and diminished the maximum response of the phenylephrine log dose-response curve, but the curve was not shifted to the right. Reduction of the propranolol concentration to IO"? M resulted in a phenylephrine LDR curve intermediate between the control and the 10"^ M propranolol curve, however, increasing propranolol to 5x10*^ M did not fur-therrsuppress the inotropic effect. Figure 15 shows the cumulative dose-response curves of isoproterenol, norepinephrine, and phenylephrine; in the absence of antagonists, in the presence of propranolol (10*^ M), 32. and in the presence of phentolamine (10**^ M) for the driven l e f t a t r i a at 22° C. Phentolamine reduced the slope and diminished the maximum response for a l l the amines. The LDR curves were not, however, shifted to the right as can be seen by the concentration of the agonists causing 50% of the maximum effect. Propranolol caused the isoproterenol and norepinephrine LDR curves to shift to the right. Propranolol did not shift the phenylephrine LDR curve to the right. The apparent maximum effect of a l l the amines was increased in the presence of propranolol. The time response effects of norepinephrine (1G~^ M) and phenylephrine (10"^ M) on cardiac cyclic AMP in the driven 1 l e f t atrium at 22° C are shown in figure 17. Norepinephrine significantly increased cyclic AMP from a control value of 0.30*0.03 pmole/mg to 0.63*0.06 at 30 seconds. Cyclic AMP remained constant at this level flor 90 seconds. Phenylephrine did not increase cyclic AMP over control levels at this temperature. Figure 18 shows the time-response effects of norepinephrine (10"" 5 M) and phenylephrine (10"^ M) on cardiac cyclic AMP in the driven l e f t atrium at 1 7 ° C. Norepinephrine significantly increased cyclic AMP from a control value of 0.30*0.02 pmole/mg to 0.49*0.05 at 90 seconds. Phenylephrine did not increase cyclic AMP over control levels at this temperature. The effects of propranolol (10*^ M) and phentolamine (10~6 M) on cardiac cyclic AMP in response to norepinephrine (10-5 M) at 1 7 ° C (90 seconds), 22° C (30 seconds), and 37° C 33. (15 seconds) are seen in figure 19. Propranolol "blocked the cyclic AMP response to norepinephrine at a l l temperatures tested while phentolamine did not. The effect of exposure to phenoxybenzamine (10~^ M) for forty-five minutes at 1 7 ° C and 37° C on cardiac cyclic AMP and contractility in response to norepinephrine (10*^ M) is shown in figure 20. Driven l e f t a t r ia which had been treated with phenoxybenzamine at 1 7 ° C and 37° C were f i r s t raised to 37° C before addition of norepinephrine. Contract-i l e response and cyclic AMP were determined at their maximum response at 60 and 15 seconds respectively. If alpha and beta adrenergic receptors represent allosteric configurations of the same active site, phenoxybenzamine should irreversably block the alpha configuration at 1 7 ° C and the antagonism should remain in effect when the temperature was raised to 37° C. Phenoxybenz-amine attenuated the contractile response (P<0.02) for both temperatures of incubation. The cyclic AMP responses were not significantly changed by phenoxybenzamine. The Effect of Theophylline on Amine-Induced Cardiac Cyclic  AMP and Cardiac Contractility, Prom figure 21 i t can be seen that theophylline alone (5x10""^ M) increased the contractile response of the rat l e f t a t r i a ( 0 . 2 0 * 0 . 0 2 g) at three minutes. Reserpine pretreatment (3 mg/kg twenty-four hours before the experiment) did not significantly (P=0.10) decrease the maximum inotropic response (0.17*0.02 g). The effect of exposure to theophylline for three minutes, 34. fifteen minutes, and sixty minutes on cardiac contractility in response to norepinephrine (10""^ M) in driven l e f t atria is shown in figure 22. A l l increases in contractile force are calculated from the original base line before addition of theophylline. After fifteen minutes a concentration of 1x10-3 M theophylline did not alter the dose response curve of nor-epinephrine; 1x10*" 3 M theophylline produced f i b r i l a t i o n when combined with norepinephrine above 10"? M; and 5xl0~ i + M sig-nificantly increased the inotropic response (0.86*0.08 g vs 0.72*0.07 g control; P< 0 . 0 5 ) . The same dose of theophylline (5x10"*^ M) was found to produce a negative inotropic effect at three minutes (0.41*0.05 g; P^O .01), and no response at a l l could be obtained with norepinephrine after 60 minutes exposure. Figure 23 shows that no concentration of theophylline tested was able to increase the inotropic response to phenylephrine at 15 minutes. The effect of exposure to theophylline (5x10"^ M) on cardiac cyclic AMP in the driven l e f t atrium is shown in figure 24. Theophylline alone significantly increased cyclic AMP at three minutes exposure (0.38*0.04 pmole/mg vs 0.27* 0.03 control; P< 0 . 0 1 ) . Exposure to theophylline for three minutes did not significantly increase the peak cyclic AMP response to norepinephrine IO""0" M (0.73*0.09 pmole/mg vs. 0.66*0.06 control). The response of norepinephrine 10"" ^  however, was increasedsd 1.34*0.14 pmole/mg vs. 1.13*0.11 control; P< 0 . 0 5 ) . This increase was not significant when the effect of theophylline alone was subtracted. 3 5 . The effect of exposure to theophylline alone (5x10 M) for 1 5 minutes significantly increased?cyclic AMP ( 0 . 3 6 * 0 . 0 3 pmole/mg vs 0.24*0.01 control; P<0.01). Reserpine pretreatment with 3 mg/kg twenty-four hours before the experiment abolished the cyclic AMP increase seen with theophylline (0.27*0.04 pmole/mg vs 0.24*0.02 control). Theophylline significantly increased the cyclic AMP response to norepinephrine (10"^ M, 0.84*0.07 pmole/mg vs 0.66*0.06 control; P<£0.01). Again neither increase was significant when the effect of theophylline alone was subtracted. Theophylline did not significantly increase the cyclic AMP response to norepinephrine 10*"*-> M (1.24*0.09 pmole/mg vs 1.17*0.08 control; P>0.4(8; The cyclic -4 AMP response to phenylephrine 10 M (0.44*0.07 pmole/mg vs 0.37*0.04 control) was not significantly increased by theophylline. In a l l cases the effect of theophylline alone on cyclic AMP appeared to be additive with the adrenergic amine response. Figure 5. Time-response effects of norepinephrine (IO*-* M) on cardiac cyclic AMP and contractility in the driven l e f t atrium at 370 C. Each point represents the mean of five to eight a t r i a and the vertical bars represent one S1E.M. Figure 6. Time-response effects of phenylephrine (10 M) on cardiac cyclic AMP and contractility i n the driven l e f t atrium at 37° C. Reserpine pretreatment was 3 mg/kg twenty-four hours before the.experiment. Each point represents the mean of five to eight atria and the vertical bars represent one S.E.M. 2 m co C D O CAMP pmole/mg 'o> bo o O O - n O > > oz 33-1 O30 m> O 33 m (/> H m ILE RPI N m o II 00 o o o % INCREASE CONTRACTILE FORCE •6T Figure 7. Time-response effects of norepinephrine (10~^ M) and phenylephrine (10-* M) on cardiac cyclic AMP and contractility in the spontaneously beating right atrium at 37° C. Reserpine pretreatment was 3 mg/kg twenty-four hours before the experiment. Each point represents the mean of five a t r i a and the vertical bars represent one S.E.M. * 1 . CAMP NOREPINEPHRINE • PHENYLEPHRINE A PHENYLEPHRINE * RESERPENIZED * CONTRACTILE FORCE 5 10 15 20 25 30 35 4 0 45 50 55 60 TIME (sec) Figure 8. Time-response effects of norepinephrine and phenylephrine on cardiac cyclic AMP in theperfused rat heart at 37 C. Beserpine pretreatment was 3 mg/kg twenty-four hours before the experiment. Each point represents the mean of five to eight atria and the vertical bars represent one S.E.M. 12 to NOREPINEPHRINE 2 M9 O 1 M9 • PHENYLEPHRINE 2 mg A 2 mg/reserpine pretrested A f 8 O) E UJ O CL a. .4 < v .2 5 10 T I M E (sec) 15 44. Figure 9. Time-response effects of norepinephrine (10 K) on cardiac cyclic AMP and contractility in the driven l e f t atrium in the presence of propranolol (10*° M) and phentolamine (IO* 6 M) at 37 C. Each point represents the mean of five atria. CAMP PMOLE/mg ro "o> 'oo ro o o o > O 30 =0 2 > m O - o H I F 2 m z - n m o o O Z m H 30 O CHANGE IN CONTRACTILE FORCE G Figure 10. The effects of different voltage stimuli (4,10,15, and 20 V.),on cardiac cyclic AMP in the driven l e f t atrium at 37° C. The muscles were stimulated at a frequency of 1 Hz. with square-wave pulses of 3 ms duration. Each point represents thessmean of four a t r i a and the vertical bars represent one S.E.M. Figure 11. The effects of different rates of stimulus (2 Hz. vs 1 Hz.) on cardiac cyclic AMP in the driven l e f t atrium at 37° C. The muscles were stimulated with square-wave pulses of 3 ms duration and 4 volts. Each bar represents the mean of five atr i a . CD m " 0 m 3D C A M P pmole/mg ~k ro co Figure 12. Cumulative dose-response curves of isoproterenol, norepinephrine, and phenylephrine for dMvenlleft atria and spontaneously beating right atria at 37° C. Each point represents the mean of five to seven atria. 49. I S O P R O T E R E N O L R I G H T A T R I A o L E F T A T R I A * N O R E P I N E P H R I N E R I G H T A T R I A A L E F T A T R I A A P H E N Y L E P H R I N E R I G H T A T R I A • L E F T A T R I A • 10 10 -8 -7 10 - 6 10 -5 10 A G O N I S T CM) - 4 10 -3 10 Figure 13. Cumulative dose-response curves of isoproterenol, norepinephrine, and phenylephrine for driven l e f t a t ria at 1 7 ° C. Each point represents the mean of five at r i a . 51. ISOPROTERENOL • NOREPINEPHRINE * PHENYLEPHRINE • Figure 14. Cumulative dose-response curves of isoproterenol, norepinephrine, and phenylephrine; in the absence of antagonists, in the presence of propranolol (IO - 0 M), and in the presence of phentolamine (IO""6 M) for driven l e f t atria. Each point represents the mean of five atria. The concentrat-ion of the agonist causing 50% of the maximum effect is marked on each curve with a star. 53. io9 1 0 8 id7 1 0 6 id5 io4 id3 AGONIST (M) Figure 15* Cumulative dose-response curves of isoproterenol, norepinephrine, and phenylephrine:in the absence of antagonists, in the presence of propranolol (10"" M), and in the presence of phentolamine (10"° M) for driven l e f t atria at 22° G. Each point represents the mean©of five atria. The concentrat-ion of the agonist causing 50% of the maximum effect i s marked on each curve with a star? 55. AGONIST (M) Figure 1$, The effect of temperature changes on the strength of contraction of driven l e f t atria./; The effect of phentolamine (10~ b M) and propranolol (1G"*° M) on the inotropic effect of temperature change is shown at 22° C. Each point represents the mean of five a t r i a and the vertical bars represent one S.E.M. Figure 17. Time-response effects of norepinephrine (10" and phenylephrine (10- 4 M) on cardiac cyclic AMP in the driven l e f t atrium at 22° C. Each point represents the mean of five at r i a . A s * * s * / wl CAMP norepinephrine A phenylephrine • 15 30 45 60 75 90 T I M E (sec.) 60. Figure 18. Time-response effects of norepinephrine (10""5 M) and phenylephrine (10 ,M) on cardiac cyclic AMP in the driven l e f t atrium at 17° C. Each point represents the mean of 5 atria. 1 . 0 C A M P norepinephrine phenylephrine Figure 19. The effects of propranolol (10*° M) and phentolamine (10"° M) on cardiac cyclic AMP in response to norepinephrine (10*° M) at 1?° C (90 s e c ) , 22° C (30 s e c ) , and 37° C (15 s e c ) . Control values for cyclic AMP in response to norepinephrine (10~ 5 M) were as follows: 17° C (90 s e c ) , 0.49 pmole/mg; 22° C (3© s e c ) , 0.63 gmole/mg; 37 C (15 s e c ) , 1.18 pmole/mg. Each bar represents the mean of five atria. Figure 20. The effect of exposure to phenoxybenzamine ( 1 0 - 6 M) for forty-five minutes at 1 7 ° C and 37° C on cardiac cyclic AMP and contractility i n response to norepinephrine (10""-> M). Driven l e f t a t ria which had been treated with phenoxybenzamine at 1 7 ° C were f i r s t raised to 37° C before addition of norepinephrine. Contractile response and cyclic AMP were determined at their maximum response at 60 and 15 seconds respect** ively. Each bar represents the mean of fi s e atria. CHANGE IN CONTRACTILE FORCE G. ro "co bi b> x 5 . i N Oi cn m + O i cn O co O vl go O N ro C A M P P M O L E / m g *o> bo b -m + co o O i ° N OI O Figure 21. Time-response effects of theophylline (5x10-4 M) o n c a rcLiac contractility in the driven l e f t atrium. Reserpine pretreatment was 3 mg/kg twenty-four hours before the experiment. Each point represents the mean of five atr&a. THEOPHYLLINE CONTROL • RESERPINE TREATED o 30 60 90 120 150 TIME (sec) 180 210 240 68. Figure 22. The effect of exposure to theophyl l ine fo r three minutes, f i f t e e n minutes and s i x t y minutes on cardiac c o n t r a c t i l i t y i n response to norepinephrine (10-2 M) i n d r iven l e f t a t r i a . Each point represents the mean of f i v e a t r i a . 69. 60 MINUTES 15 MINUTES 3 MINUTES THEOPHYLL INE control • 10 10 10 10 10 10 NOREPINEPHRINE ( M) Figure 23. The effect of exposure to theophylline §6r fifteen minutes on. cardiac contractility in response to phenylephrine (.10--* M) in driven l e f t atria. E ach point represents the mean of five at r i a . 71. o UJ U fX ^ O 1 . 0 LU < .8 O . 6 u LU 4 O z < X O o -8 1 0 T H E O P H Y L L I N E control • — 1 X 1 0 • — - 4 1 x 1 0 M A — - 4 5 x 1 0 M * — - 7 - 6 - 5 - 4 1 0 1 0 1 0 1 0 P H E N Y L E P H R I N E (M) - 3 1 0 Figure 24. The effect of exposure to theophylline (5xl0~* M) on cardiac cyclic AMP in the driven l e f t atrium. Theophylline alone increased cyclic AMP at three minutes exposure. Expos-ure to theophylline for three minutes increased the peak cyclic AMP response to norepinephrine (10" & and 10"** M) in an additive manner; Each bar represents the mean of five to eight atria. One S.E.M. is shown. 73. 1.4 b 3 min. 3 min. Figure 25. The effect of exposure to theophylline OxlO"4* M) for fifteen minutes, on cardiac cyclic AMP in response to norepinephrine and phenylephrine in driven l e f t a t r i a . Cyclic AMP concentrations were determined 10 seconds after the addition of norepinephrine or phenylephrine. Theophyl-line alone significantly increased cyclic AMP at 1 5 minutes, exposure (P<T0.05 ). Reserpine pretreatment 3 mg/kg twenty-four hours before the experiment abolished the cyclic AMP increase seen with theophylline. Exposure to theophylline increased the cyclic AMP response to norepinephrine and phenylephrine in an additive manner. None of the increases were significant when the effect of theophylline alone was subtracted. Each bar represents the mean of five to eight atria. One S.E.M. is shown. 1.4 15 min. CHAPTER IV DISCUSSION The Mechanism of Action of Norepinephrine and Phenylephrine  on Cardiac Contractility The present series of experiments was designed to determine the time course of cyclic AMP and contractility changes in response to norepinephrine and phenylephrine. It was also intended to separate the direct and indirect effects of phenylephrine on cyclic AMP, and to determine the importance of alpha adrenergic receptors in the inotropic effects of sympathomimetic agents. The results indicate that while norepinephrine and phenylephrine do cause an increase in cyclic AMP levels prior to contractile force (figures 5 and 6), both amines have the a b i l i t y to produce an increase in cardiac contract-i l i t y in which cyclic AMP is not involved (figure 6,7, and 9). The adrenergic receptors of cardiac muscle have been gen-erally c l a s s i f i e d as beta-receptors. A number of Investigators (Benfey and Varma, 1967; Berger and Mokler; 1969; So*i©r,p}1968; NakalBhima et?al.jgi9^; WignerpEndbhf tand Reinh8i>$# 1974; Wenzel and Su, 1966), however, have reported evidence sup-porting the existence of alpha adrenergic receptors in the heart which were capable of mediating a positive inotropic 76. 77. effect in response to sympathomimetic agents. It seems reasonable, therefore, to attribute the increases in contractile force in response to norepinephrine and phenyl-ephrine,which do not involve cyclic AMP, to alpha,receptors. Ahlquist, 1948, f i r s t formulated the concept of alpha and beta adrenergic receptors based primarily on the d i f -ferent quantitative responses of end organs to catecholamines. The receptors most sensitive to isoproterenol were called beta-adrenergic receptors and those most sensitive to norepinephrine were considered alpha-adrenergic receptors. The order of effectiveness for myocardial contractility in rabbit, cat, and dog was reported by Ahlquist, 1948, as isoproterenol >epinephrineynorepinephrine. The relative order reported by Oovier, 1968, on guinea-pig atria, and by Wenzel and Su, 1966, on rat ventricle was isoproterenol > norepinephrine> phenylephrine. Phenylephrine has been con-sidered to exert a direct effect on alpha-adrenergic recep£* tors only (Ahlquist and Levy, 1959)* although i t has also been,shown to have an indirect effect on releasing nor-epinephrine stores (Govier, 1968). Wenzel and Su, 1966, working on rat ventricles, reported that phentolamine potentiated the positive inotropic response to norepinephrine and epinephrine, blocked the phenylephrine response, and exert-ed no blockade of the isoproterenol-induced response. Govier, 1968, working on guinea-pig atria, reported that phentolamine antagonized the f i r s t component of a-two-component phenylephrine-induced positive!inotropic response, but exerted no effect on the 78. responses to norepinephrine, epinephrine, or isoproterenol, i t was also reported that pronethalol did not block the phenyl-ephrine positive inotropic response. Govier, 1968, suggested that norepinephrine and epinephrine possess alpha-adrenergic receptor stimulating activity but that i t is not possible to demonstrate blockade of epinephrine and norepinephrine by alpha-adrenergic antagonists alone because the response to beta-adrenergic receptor stimulation is so great that the alpha-receptor component is s t a t i s t i c a l l y insignificant in comparison. In their experiments reduction of the beta-adrenergic receptor response with pronethalol made the alpha-adrenergic response a greater proportion of the totaib ino-tropic response and consequently alpha-receptor blockade resulted in a significant reduction in the positive inotropic response. On the basis of these experiments i t was postulated that guinea-pig a t r i a and rat ventricle myocardium contain alpha-adrenergic receptors (Wenzel and Su, 1966; Govier, 1968). The failure of pronethalol to block the positive inotcopic effect of low concentrations of phenylephrine suggested to Govier, 1968, that this effect is not mediated through the cyclic AMP system. He was "tied to the conclusion, therefore, that the positive inotropic effects of sympathomimetic agents resulting from alpha and beta adrenergic receptorsstimulation are produced through different basic mechanisms. The data in the present series of experiments support this conclusion. The f i r s t evidence that two different basic mechanisms might be involved in theiinotropic response came from the obser-79. vation that the magnitude and time course of cyclic AMP changes induced by norepinephrine and phenylephrine were remarkably different while the magnitude of the contractile responses were not (figures5,6, and 7). Pretreatment with reserpine established that the cyclic AMP response of phenyl-ephrine i s an indirect effect associated with the release of stored catecholamines. The;contractile effect of phenylephrine could,thus be dissociated from an increase in cyclic AMP, whereas with norepinephrine the two effects were s t i l l associated. It is confirmed that phenylephrine exerts a direct effect on alpha receptors only. Norepinephrine isKknown to possess bofeMibeta- and alpha-adrenergic receptor stimulating activity (Wenzel and Su, 1966). Govier, 1968, was able to separate the alpha- and beta^reeeptor components of phenylephrine by f i r s t diminishing the beta receptor response with pronethalol so that the alpha-adrenergic response became apparent. In the present series of experiments concentrations of norepinephrine (10~^ M) and propranolol (10~^ M) were chosen such that the blockade of the inotropic response produced by propranolol had just been overcome by the higher concentration of norepinephrine. It was reasoned that at this point the greater proportion of the total inotropic response would be mediated by alpha-adrenergic receptors. Under these,conditions the magnitude and time course of the contractile response resembled the phenylephrine induced inotropic effect, while there was no increase in the level of cyclic AMP (figure 9). The alpha-adrenergic antagonist phentolamine was inef-80. fective in altering either the time-course of magnitude of the contractile response induced "by norepinephrine. The cyclic AMP increase was slightly less in the presence of phentolamine than after norepinephrine alone. This could possibly be relat-ed to a nonspecific interference of phentolamine with the beta receptor. Rabinowitz, Parmley, and Katz, 1972, however, have reported that neither alpha-stimulation nor blockade affected the adenylate cyclase from cat heart muscle. Shanfeld, Prazer, and Hess, 1969, have shown that the effect of a small dose (30 ng) of norepinephrine on cyclic AMP format-ion was somewhat more inhibited in the presence of isopropyl-methoxamine (IMA) than was the positive inotropic effect. The evidence for a dissociation between the two effects did not appear to be conclusive, especially in view of the small total changes produced by this small dose of norepinephrine, since the inotropic response to norepinephrine was also reduced in the presence Hf IMA by about 50$ in these experiments, whereas cyclic AMP was elevated by about 25% (which was not sig-nificant). In contrast, the present experiment clearly separates the beta- and alpha-adrenergic receptor stimulating activity of norepinephrine on contractility and cyclic AMP. Several investigatlors (Lee and Yoo, 1964, 1970; in isolated rabbit atria; Kabela, et, a l . , 1969, in isolated dog heart) have reported that beta adrenergic antagonists inhibit the inotropic effect of phenylephrine in spontaneously beating cardiac tissue and that alpha adrenergic antagonists were ineffective. Other investigators, however, (Starke, 19724 in 81. perfused rabbit heart, and Osnes and Oye, 1975* in perfused rat heart) have found that the positive inotropic effects of phenylephrine were blocked by phentolamine but not by propranolol. A possible explanation for the divergent and contradictory experimental results may be found in a recent publication by Endoh and Schumann, 1975b. They have reported that the positive inotropic effects of the alpha-agonists methoxamlne, maphazol-ine, and oxymetazoline decreased with increasing frequency of stimuifeion (0.5-1-1.5 Hz). They considered on the basis of previous experiments (Endoh, Wagner, and Schumann, 1975) that the stimulation of alpha-adrenergic receptors is capable of increasing the intracellular calcium to a higher level, either by increasing the influx of caicium per contraction of by decreasing the efflux. Winegrad and Shanes, 1962, had shown that the frequency of stimulation strongly effects the flux of calcium through myocardial ©ell membrane. Endoh and Schuman, therefore reasoned that when intracellular calcium has already been raised by an increase of the frequency of stimulation, alpha-adrenergic receptor stimulation i s not able to, induce a further increase of intracellular calcium. In the present series of experiments the cyclic AMP response to phenylephrine (10"* M) (which has been shown to be due to the release of stored catecholamines) and norepinephrine (10~5 M) were similar in the rate controlled (1 Hz.) and the spontaneously beating preparations (figures5,6, and 7). In addition increasing the rate of stimulation from one to two Hz. had no effect on the cyclic AMP response to norepinephrine 82. (figure 11). The time course of the phenylephrine contractile response, however, was remarkably different betweentthe rate controlled and the spontaneously beating preparation (figures 6 and 7). The time required to reach peak contractile response was changed from 100 seconds in the driven preparation to 40 seconds intthe spontaneously©beating tissue. The difference in,the time course of the phenylephrine-induced contractile response in the slow-paced verses the spontaneously beating preparation is most l i k e l y due to the a b i l i t y of the chrono-tropic response to affect the flux of calcium through the c e l l membrane (Winegrad and Shanes, 1962). The chronotropic response of phenylephrine has beensahown by a number of invest-igators to be blocked by beta-adrenergic antagonists (Leong and Benfey, 1968; K r e l l and Pat11, 1969; Starke, 1972; Wagner, Endoh, and Reinhardt, 1974). The use of spontaneously beating preparations may, thus, introduce other variables such as rate which can greatly affect the contractile response. Such variables may at least partially account for the divergent experimental results reported in the liteeature. , The rate controlled preparation may offer a better system for attempting to correlate the physiological and biochemical effects. For example, the time courseof the norepinephrine-induced contractile response in slow paced atria is greatly different in the presence and absence of propranolol (lfO seoonds to reach peak tension in the presence of propranolol verses 60 seconds to reach peak tension in the absence).^figure 9). This difference may be related to the lack of cyclic AMP 83. elevation in the former situation.- This observation would have been obscured by the presence of a chronotropic response. It should always be kept in mind when pacing a tissue el e c t r i c a l l y that excessive depolarization per se may elevate cyclic AMP contents (Wollenberger, Babskii, Krause, Genz, Blohm, and Bogdanova, 1973) and may also liberate endogenous catecholamines. In the present series of experiments different voltage stimuli from four to twenty volts had no effect on cardiac cyclic AMP levels indicating that neither of the above possibilities had taken place (figure 10). In conclusion, norepinephrine and phenylephrine do cause an increase in cyclic AMP levels prior to contractile force; h however, both amines have? the a b i l i t y to produce an increase in cardiac contractility in which cyclic AMP is not involved. The phenylephrine-induced cyclic AMP response is an indirect effect associated with the release of stored catecholamines. It seems reasonable to attribute the increases in contractile force in response to norepinephrine and phenylephrine which do not involve cyclic AMP to alpha receptors. This does not, however, imply a dissociation between beta receptor function, cyclic AMP, and the inotropic response. The time-course of the contractile response induced by adrenergic amines may be remarkably influenced by the chrono-tropic response in spontaneously beating preparations while the cyclic AMP response is not greatly affected. This indicates that events beyond the receptor level, possibly involving the effect of rate changes on calcium fluxes, are taking place. It is 84 therefore suggested that rate controlled preparations may offer a better system for attempting to correlate physiological and biochemical events. The Effect of Temperature on Cardiac Inotropic and Adenylate  Cyclase Activating Adrenoceptors The second series of experiments was designed to be a through investigation of the biochemical and physiological changes induced by lower temperature in mammalian cardiac muscle. The purpose was to determine whefcnerralpha- and beta-adrenergic receptors represent allosteric configurations of the same receptor macromolecule and whether there was indeed a dissociation of cardiac inotropic and adenylate cyclase activating adrenoceptors at low temperature. The results indicate that there is no interconver&ion of alpha- and beta-adrenergic receptors mediated by temperature. I t has*' been confirmed repeatedly that the strength of contraction of isolated heart muscle from homeothermic animals increased when i t s temperature is lowered below normal (Clark, 1920; Peigen, et, a l . . 1952; Fus&chgott, and Gubareff,1958; Garb, and Chenoweth, 1949; Hollander, and Webb, 1955; Kelly, and Hoffman, I960; Saunders, and Sanyal, 1958). The inotropic effects of temperature changes and of Pharmacol logical agents are not necessarily additive. Lowering the temperature may so increase the strength of contraction as to leave l i t t l e room within the limits of contractility for a drug to exert a positive inotropic effect. The positive inotropic effects of cardiac glycosides (Brown and Cotten, 85. 1954; Meyer and Kukovetz, 1952; Saunders and Sanyal, 1958) and of epinephrine and norepinephrine (Barlow, and Sollman, 1926; Booker, I960; Brown, and Gotten, 1954; Cotten, and Cooper, 1962) have been found to be decreased by cooling, and this has beenaattributed at least in part to the influence of limits on contractility (Blinks and Koch-Weser, 1963). In the present series of experiments the Inotropic effect of temperature was again confirmed (see figure 16). The increase in contractile force mediated by cooling to 1 7 ° C was so great that the positive inotropic effects of a l l the amines were greatly reduced (figure 13). This decrease in responsiveness appeared to be due to an increase in the baseline and not to a decrease in efficacysy The fact that the order of potency i s altered by the decrease in temperature (figure 13 verses figure 12) has been interpreted by some investigators to indicate that an interconversion of receptors from beta to alpha has taken place (Amer and Byne, 1975). However phenylephrine, the most specific alpha agonist, was the least potent. In fact phenylephrine produced a negative inotropic effect which would tend to rule out a receptor change. The decrease in potency of isoproterenol i s most li k e l y due to the inotropic effects of temperature changes masking a portion of i t s dose-response curve. The Inotropic effects of the adrenergic amines at 1 7 ° C were too small to be useful in evaluating the activity of alpha and beta adrenergic antagonists. It was therefore decided 86. to compare the activity of these agents at 37° C and 22° C. It was found, however, that at 22° C phentolamine caused an increase in the positive inotropic effect of temperature change, while propranolol caused a decrease in the strength of contraction (figure 16). In order to understand how the adrenergic blocking agents may influence the inotropic effect of temperature on myocardial tissue i t is necessary to review the effects of temperature changes on the duration of the active state. Over a wide range of temperature, the direction of temperature-dependent changes i n the itrengthecBntfeactidn^seemstto bewdetermined by changes in the duration of the active state (Blinks and Koch-Weser, 1963). It is well known that the duration of the con-tractions of heart muscle increases withddecreasing temperature. This has been observed in muscle from cold-blooded (Hajdu, and Szent-Gyorgyi, 1952; Wiegmann, et a l . , 1957; Heintzen et  a l . . 1956; Eckstein, 1920) and warm-blooded animals (Trautwein and Dudel, 1954; Schmidt and Chang, 1961; Garb and Chenoweth, 1949; Hegnauer et a l . , 1950; Hirvonen, and Lybeck, 1956). The increased duration of the active state with cooling is associated with an increase in the duration of the action potential (Brooks et a l . . 1955; Cranefield, and Hoffman, 1958; Heintzen et aL, 1956; Hollander and Webb, 1955; Kelly and Hoffman, I960). During contraction the calcium ion concentration remains elevated as a result of the slow inward current carried by calcium during the plateau (sustained depolarization) phase of the action potential. Thus the action potential, by regulating the 87. calcium ion concentration, not only triggers the contraction hut influences i t s magnitude and duration as well. The positive inotropic effect of temperatue change may be a direct result of this increase in the slow calcium inward current during.the action potential. .. Phentolamine has been found to prolong the action potential duration in guinea-pig papillary muscle (Quadbeck and Belter, 19,75): guinea-pig a t r i a (Pappano, 197D* Purkinje fibers of sheep (Giotti et a l . , 1973); and dog (Bosen et a l , 1 9 7 D . Phentolamine could conceiveably mediate this effect without Interacting with any adrenoceptors. It is also possible that the effect of phentolamine on the action potential may be related to i t s direct myocardial stimulant component (Goodman and Gilman, 1970; Ahlquist et a l . . 1947; Lum and Nickerson, 1946) or to i t s a b i l i t y to cause a secondary release of endogenous catecholamines (Goodman and Gilman, 1970). Propranolol, on the other hand, has been found to shorten the action potential of guinea-pig papillary muscle (Quadbeck and Beiter, 1975). The actions of propranolol on the heart could result from at least two different mechanisms of action. The f i r s t is attributable to the beta-blocking action of propranolol which inhibits both positive inotropic and arrhythmic actions of catecholamines (Lucchesi et a l . , 1966). The second is a nonspecific 'local anaesthetic* action which appears to result from a direct action of propranolol that i s not related to i t s beta-blocking activity. The direct effect on myocardial excit-a b i l i t y predominates in the prevention of digitalis-induced 88. arrhythmias arising from myocardial ischemia (Barrett and Cullum, 1968). In the present experiment i t is unlikely that beta-adrenergic blockade is involved in the antagonism of the inotropic effect of temperature by propranolol as the control levels of cyclic AMP were not elevated by decreased temperature alone (figure 5 , 17, and 18).^ This would indicate that the beta receptor was not active in mediating the positive inotropic efSect of temperature.tjftptherefiOKelseemsliiogical that the effee* of propranolol on the inotropic effect of temperature may be due to blockade of increased calcium fluxes through the c e l l membrane. It is therefore possible that propranolol may -m antagonize the inotropic effect of temperature by acting as a local anesthetic. ,Before considering the effects of the alpha- and beta-adrenergic antagonists on the log dose-respones (LDR) curves of the adrenergic amines, i t may be appropriate to review a few important points concerning the analysis of antagonism in the framework of the LDR curve. The position of a LDR curve on the x-axis reflects the a f f i n i t y of the drug for i t s receptor. A typical LDR curve is symmetrical about the point at which 5 0 per cent of the maximum response i s obtained, and i t s maximum slope and point of inflection occur at this midpoint. The lower the ED50O(the dose for half-maximal response) the more potent the drug. In the presence of a i competitiveaantagonist, the curve for an agonist w i l l be shifted to the right, but neither the slope nor the maximum response would be expected to change. The antagonist simply 89. alters the effective a f f i n i t y of agonist drug for receptor. The effect of a noncompetitive antagonsit upon the LDR curve wll w i l l be quite different. The agonist curve may or may not be shifted to the right, however, the slope w i l l be reduced and the maximum response w i l l diminish, inrelation to the degree of noncompetitive blockade established. A through coverage of the different categories of these two basic types of antagonism is covered by Webb, 1963. Since the studies which have indicated there is an interconversion of cardiac inotropic receptors from beta to alpha mediated by temperature have consistently reported their results in dose ratios (Kunos and Szentifanyi, 1968; Kunos et a l . , 1973; Benfey et a l . . 1974) instead of complete dose or time response curves, i t is important to understand the advantages and limitations of the dose ratio method of expressing antagonism. The dose ratio i s the ratio of the two agonist concentrations producing thessame response, usually a half-maximal response, in'the presence and absence of antagonist. When the LDR curves are parallel, this ratio is constant. However, when the curves are n«*^|^iaielifehm^6se^8a*4in®*aisfes.EoMwta,not the best method of expressing an antagonist effect under these conditions. Furthermore, i t is impossible to t e l l anything about the nature of the antagonism from a simple presentation of the dose ratios. It is possible to ill u s t r a t e mathmatically the difference between competitive and noncompetitive antagonism as they pertain to dose ratios. The equation for competitive inhibition of a receptor may be reduced to the form 90. (A)j (I) . S 0 = II wHlie (A)g is the concentration of agonist to produce an arbitrarily chosen response, (A)j is the concentrations needed to produce the same response in the presence of concentration (I) of the antagonist, 1/Kj is the af f i n i t y of the antagonist for the receptor, and (A) J/(A)Q is the dose ratio (Webb, 1963; Goldstein, 1974). The equation for noncompetitive antagonism w i l l not reduce to the above form. Thus the chief advantage of the dose ratio in competitive inhibition, that i t is a convenient measure of the a f f i n i t y of the antagonist for the receptor, is lost in noncompetitive inhibition. The effects of phentolamine and propranolol on the LDR curves of the adrenergic amines at 37° C is shown in figure 14. As would be expected, phentolamine had no effect on the isoproterenol dose-response curve. Phentolamine did, however, cause a small noncompetitive antagonism of the norepinephrine curve and a small competitive antagonism of the phenylephrine curve. A similar attenuation of the posi-tive inotropic effects of norepinephrine by phentolamine has been reported by Rabinowitz et a l . , 1974,in the isolated cat papillary muscle. The shift of norepinephrine and phenylephrine curves to the right is probably mediated by a competitive antagonism of alpha adrenergic receptors. The mechanism of the phentolamine mediated supression of the nor-epinephrine maximal response remains obscure. It may be 91. related to a nonspecific interaction, possibly involving the sympathomimetic activity of phentolamine or a supression of beta receptor function. It may also reflect a specific antagonism of alpha receptors. Propranolol caused the isoproter-enol LDR curve to shift to the right. In addition, the efficacy of isoproterenol was increased in the presence of propranolol which may be due to separation of the beta-blocking and local anesthetic effects. If isoproterenol were able to overcome the beta-blockade of propranolol while cardiac microsomal and sar-coplasmic reticulum uptake and binding of calcium were s t i l l inhibited, the resultant increase in free intracellular calcium could then account for the elevation of maximal contractile force. It would be possible to test this hypothesis by repeating this experiment using sotalol or practolol which are almost devoid of local anesthetic potency instead of propranolol. Propranolol competitively antagonized the norepinephrine LDR curve. Propranolol did not cause a true noncompetitive antagonism of the phenylephrine LDR curve, although the slope was reduced and the maximum response was diminished. The fact that i t was not possible to further suppress the phenylephrine-induced positive inotropic response by increasing the concentration of antagonist suggests that propranolol was able to antagonize only the second component of phenylephrine activity which is due to catecholamine release (Govier, 1 9 6 8 ) . These data support the view that phenylephrine exerts i t s major positive inotropic effect through alpha receptors. The effects of phentolamine and propranolol on the LDR curves of the adrenergic amines at 22° C is shown in figure 15. Phentol-92. amine reduced the slope and diminished the maximum response for a l l the amines. The a f f i n i t i e s of the drugs for the receptors, however, were not altered by phentolamine as can be seen by the concentrations of the agonists causing 50% of the maximum effect. The nonspecific, noncompetitive antagonism observed in the pres-ence of phentolamine is probably due to the a b i l i t y of phentolamine to increase the strength of contraction (raise the baseline) so as to leave l i t t l e room within the limits of contractility for a drug to exert a positive inotropic effect (figure 16). Propran-olol competitively antagonized the isoproterenol and the norepin^ 1 ephrine curves, causing a shift of the LDR curves to the right in both cases. Propranolol did not antagonize the inotropic effect of phenylephrine. The apparent increase in efficacy of a l l the amines in the presence of propranolol was probably due to the a b i l i t y of propranolol at 22° C (figure 16) to decrease the inotropic effect of temperature changes and thus leave more room within the limits of contractility for the drugs to exert their positive inotropic effects. It should be noted that the use of dose ratios to characterize the changes in the LDR curves at 22° C is inappropriate due to the appearance of nonspecific, non-competitive effects which may complicate the interpretation. Lowering the temperature from 37° C to 22° C and 17° C decreased both the rate and maximum levels of cyclic AMP production in response to norepinephrine (figures 5, 17 tand 18). It was not possible to detect an increase in cyclic AMP med-iated by phenylephrine at 22° C or 17° C in contrast to the results obtained at 37° C (figures 6, 17, and 18). This probably 93. reflects the overall decrease in adenylate cyclase activity at lower, temperature. In agreement with Benf ey„et5>i$.. . the stimulation of cyclic AMP production in response to norepins ephrine remained an entirely beta-adrenoceptor response at all,temperatures tested (figure 19). Kunos and Szentivanyi, 1968, used phentolamine and propranolol as adrenergic antagonists in the experiments where they f i r s t suggested that alpha and beta adrenergic receptors may represent allosteric configuration of the same receptor macromolecule. The present series.of experiments have shown that the results upon which this conclusion was based were due to nonspecific effects. Subsequently, Kunos et  a l . . 1973* reported similar results using phenoxybenzamine and p propranolol as the antagonists. Phenoxybenzamine may, act in a manner similar to phentolamine in the present experiment to increase the inotropic effect of temperature.. An alternate explanation has also been suggested. Benfey, 1975, found the blocking effects observed with phenoxybenzamine at low temp-erature to be nonspecific in frog ventricles. He suggested that the,changes in phenoxybenzamine effects with temperature are related to differences in the rate of formation and s t a b i l -i t y of the intermediate aziridium ion and i t s rate of alkylation of nucleophilic centers. It is d i f f i c u l t , however, to see how such an explanation could account for the observed non-specific antagonism. Whatever the true explanation for the phenoxybenzamine activity at low temperature, i t is clear that i t is a nonspecific effect. 94 Kunos et a l , , 1973» have also reported that the heart binds twice as much phenoxybenzamine at 14° C than at 24° C. This was interpreted as proof of additional binding to receptors which had been converted from their beta to their alpha configuration. There is no reason to suppose, however, that the a b i l i t y to bind phenoxybenzamine should be the same at 14° C and 24° C. The great majority of the binding in any case w i l l be nonspecific, and differences encountered between the&two temperatures may be related to such a phenomenon as phase changes in membrane lipids which have been shown to alter the permeability of membranes (DeGier et a l . , 1968). It is also possible that the different binding characteristics may be related to the rate of formation and s t a b i l i t y of the intermediate aziridium ion as has been suggested by Benfey, 1975. The fact that the temperature of incubation did not influence the effectioflphenoxybenzam tpietojp m^'m^^iuil^'meBW^^^^^o^pinepArine (figure 20) would Indicate that there is no specific binding of phenoxy-benzamine to the beta receptor at either temperature. In conclusion, the results indicate that there is no interconversion of alpha- and beta-adrenergic receptors mediated by temperature. The apparent blockade of the adren-ergic amine-induced inotropic response by phentolamine and the apparent lack of blockade by propranolol has been found to be related to the a b i l i t y of the blocking agents to modify the inotropic effect of low temperatures. Although adrenergic amines have the a b i l i t y to cause increases in contractile force 9£. Which are mediated by alpha receptors and which do not involve cyclic AMP (Benfey and Carolin, 1971; Osnes and Oye, 1975; Martinez and McNeill, 1975), no dissociation has yet been found between the beta receptor, cyclic AMP, and the inotropic response, It therefore seems logical, in view of the close association of these parameters, that cyclic AMP may be one of the second messengers mediating the inotropic response in cardiac tissue. These data support the role of cyclic AMP as a mediator of beta-adrenergic receptor function. The Effect of Theophylline on Amine-Induced Cardiac Cyclic  AMP and Cardiac Contractility The third series of experiments was designed to invest-igate the effect of theophylline on adrenergic amine-induced cardiac cyclic AMP and cardiac contractility. The positive inotropic effects of the methylated xanthine derivatives caffeine, theophylline, theobromine, and papaverine are well known, however, the mechanism responsible for this action remains unclear. T wo recent developments: 1.) the demonstrat-ion that caffeine causes the release of calcium from and decreases the net uptake of calcium in microsomes (Weber, 1968; Johnson and Inesi,,1969; Fuchs, 1969; Ogawa, 1970), and 2.) the discovery that the methylxanthines are inhibitors of phospho-diesterase (Sutherland and Rail, 1958; Butcher and Sutherland, 1962), the enzyme responsible for the breakdown of cyclic AMP in muscle, have stimulated interest in these compounds. The fact that catecholamines stimulate the production of cardiac cyclic AMP has led to the suggestion that catechol-96. amines and the methylxanthines may both exert their inotropic effects through increased concentrations of cyclic AMP. The present results inddate the inotropic effects of the methylxan-thines are not mediated through cyclic AMP but are more readily explained by the effects of these agents on calcium metabolism. The methylxanthine phosphodiesterase inhibitors are known to release catecholamines from heart tissue (Westfall and Fleming, 1968). Several investigators have, therefore, proposed that theophylline may influence cardiac muscle through the secondary release of endogenous catecholamines (Nuzher et a l . , 1967: Westfall and Fleming, 1968). In the present series of experiments, however, theophylline was found to exert a direct contractile effect in reserpine pretreated tissue which was not significantly different from the control response (figure 21). In contrast, reserpine pretreatment abolished the cyclic AMP increase seen with theophylline (figure 25). Thus, the theophylline-induced cyclic AMP response is an indirect effect associated with the release of endogenous amines while the greater part of the contractile reasponse is not. A further dissociation between the contractile and the cyclic AMP responses can be seen in figure 22. Theophylline (5xl0~^ M) significantly (P<0.05) enhanced the contractile response to norepinephrine after 15 minutes. The contractile response to norepinephrine after Q minutes,exposure, however, was attenuated (P< 0 . 0 1 ) . There was no difference in the cyclic AMP concentrations at either of these times (figure 24 97. and 25). After 60 minutes exposure, the tissue was complete-ly unresponsive to norepinephrine. It is d i f f i c u l t to see how these results can be resolved on the basis of phospho-diesterase inhibition and cyclic AMP elevation, however, they may be readily explained on the basis of calcium met-abolism. The methylxanthines are known to alter i n t r a c e l l -ular calcium concentrations by decreasing the rate of cal -cium sequestration by the sarcoplasmic reticulum (Weber, 1968), and mitochondrial accumulation of calcium (Nayler, 1967). Transport of calcium by the c e l l membrane either to increase influx (Scholz, 1971) or to decrease efflux (Shine and Langer, 1971) or both, may also be affected, more calcium is thus available for excitation-contraction coupling and a positive inotropic effect results. In the ,&?-•*••. present series of experiments, the attenuation of the nor-epinephrine contractile response observed after 3 minutes exposure to theophylline may result from excessive release of calcium ions. De Gubareff and Sleator, 1965, have demon-strated that guinea-pig atria respond in a positive fashion to concentrations of caffeine up to 1.5amM in the presence of a normal amount of calcium in the bath. Higher concentra-tions of caffeine produced a negateve inotropic effect n unless the ©calcium concentration in the bath was lower-ed. In the presence of calcium , caffeine produced only a negative inotropic effect. 98. These authors suggested that increasing intracellular calcium beyond this optimal level (by increasing the dose of caf-feine or raising the concentration of calcium in the bath) would result in a decrease in contractility. It seems reason-able that the rate of theophylline-induced calcium release would be greatest when the contractile response induced by theophylline f i r s t reached i t s peak. At this time release would be maximally stimulated and the calcium reserves would not yet be seriously depMed. Norepinephrine might then increase the intracellular level of ionized calcium beyond the optimal level for ©contraction and produce a negative inotropic effect ( f i g -ure 22). After 15 minutes exposure to the same dose of theo-phylline, the calcium reserves might be sufficiently deplet-ed that the combined effect of theophylline and norepineph-rine on calcium mobilization would then be in the optimal range for contraction, and an enhancement of the inotropic response would result. After 60 minutes exposure, the intracel-lular calcium reserves may be so much further depleted that no inotropic response to norepinephrine would be possible f f i g -ure 22). It has previously been shown that the methylxan-thines can both enhance and depress the contractile effect of norepinephrine depending on the dose of both drugs and on the amount of calcium present in the physiological.solution ^McNeill et a l . , 1969, 1973b). In the present study the time of exposure to theophylline has also been shown to affect the results. A number of Investigators (Hamakawa et a l . , 1972; Endoh 99. and Schumann, 1974; Kalsner, 1971) have reported that theophylline in a sufficient concentration to enhance the contractile effect of isoproterenol, epinephrine, or nor-epinephrine, did not alter the positive inotropic effect of phenylephrine. This observation is again confirmed in the present series 6f experiments (figure 23). Since a l l increases in cardiac contractility were plotted against the original baseline before the addition of theophylline the increase in contractile force seen with low doses of phenylephrine are due to the inotropic response of theophylline alone. It can clearly be seen that there was no increase in the maximum contractile effect of phenylephrine in contrast to the results obtained with norepinephrine (figure 22). Endoch aM Schumann, 19,71,yhaveplateftpffited thesetsesults to be inconsistent with the a b i l i t y of theophylline to act through a calcium-dependent mechanism as proposed by Blinks et a l . , 1972, and by McNeill et a l . , 1969. However, one would not expect the maximum response mediated mostly by alpha-adrenergic receptors (phenylephrine stimulated) to be enhanced by the methylxanthines i f the same sources of calcium were u t i l i z e d by both groups of compounds. Only i f an additional factor (such as a high intracellular level of cyclic AMP) which was able to mobilize additional resources of calcium, was introduced would an increase in efficacy be apparent® Endoch et a l . , 1975, have recently:, demonstrated differences in the calcium fluxes mobilized by alpha and beta-adrenergic receptors. Similarly, one would not expect the methylxanthines to affect calcium dose-response 100. curves (Endoch and Schumann, 1975a; Skelton et a l . , 1971). The amount of calcium released fieom internal stores by theophylline might be too small to significantly affect calcium dose-response curves which occur between 10 and 10 molar (Endoch and Shumann, 1975a) • In any case, i t should be noted that merely elevating extracellular calcium may not be sufficient to demonstrate an intracellular calcium effect. Several investigators (MassIngham and Nasmyth, 1972; Endoch and Schumann, 1975a)have demonstrated a positive interaction between el e c t r i c a l stimulation and theophylline in cardiac muscle. Such an effect would not li k e l y be mediated through cyclic AMP and is more l i k e l y explained by an increase in intracelluai? calcium. Alternate explanations are also possible. Kalsner, 1971» has demonstrated that the methylxanthines inhibit the extraneuronal inactivat-ion of catecholamines by inhibiting catechol-o-meibhyl-trans-ferase in vascular smooth muscle. A similar mechanism might operate in cardiac muscle and would thus specifically enhance the norepinephrine but not the phenylephrine nesponse. Further studies are needed before any definite conclusions can be reached on the interpretation of this data. Theophylline alone caused a small increase in cyclic AMP which was found to be due to the secondary release of endogenous catecholamines (figures 24 and 25). In a l l cases the effect of theophylline@nn cyclic AMP appeared to be additive and not potentiative with the adrenergic amine response. This would further tendto rule out phosphodiesterase inhibition as a factor 101. In the effect of theophylline on the amine-induced cardiac contractility. Many other discrepancies between phosphodies-terase inhibition and pharmacological effects have been noted in other tissues and have been reviewed by Blinks et a l . . 1972;. and McNeill et a l . , 1969, 1974. In conclusion our data support the work of McNeill et a l . , 1974, who reported a lack of interaction between norepinephrine and theophylline on cardiac cyclic AMP. The theophylline-induced cyclic AMP response is an indirect effect associat-ed with the release of stored catecholamines. . Futhermoxej, theophylline exerts a direct contractile effect which is un-related to cyclic AMP. The a b i l i t y of theophylline to enhance the norepinephrine and not the phenylephrine response may be due to mobilization of different sources of calcium by the alpha and beta receptors. The fact that the effect of theo-phylline on cyclic AMP appeared to be additive and not potentiative would tend to rule out phosphodiesterase inhibition as a"factor in the effect of theophylline on the amine-induced cardiac contractility. In addition, there was no correlation between the contractile and the cyclic AMP effects at the d i f f e r -ent times tested. It therefore seems logical, in yiew of the lack of correlation observed, that the cardiac effects of the methylxanthines are not mediated through cyclic AMP. These results support the view that the methylxanthines exert their effects on cardiac tissue through calcium metabolism. CHAPTER V SUMMARY 1. Norepinephrine and phenylephrine cause an increase in cyclic AMP levels prior to contractile force, however, both amines have the a b i l i t y to produce an increase in cardiac contractile force in which cyclic AMP is not involved. 2. The phenylephrine-induced cyclic AMP response is an indirect effect associated with the release of stored cate-cholamines. 3. It seems reasonable to attribute the increase in contract-i l e force in response to norepinephrine and phenylephrine which do not involve cyclic AMP to alpha receptors. 4. The time course of the contractile response induced by adrenergic amines may be remarkably influenced by the chron-otropic response in spontaniously beating preparations while the cyclic AMP response is not greatly affected. 5. It is therefore suggested that rate controlled preparations may offer a better system for attempting to correlate physiolog i c a l and biochemical events. 6. There is no interconversion of alpha- and beta adrenergic receptors mediated by temperature. 102. 103. 7. The apparent blockade of the adrenergic amine-induced inotropic response by phentolamine and the apparent lack of blockade by propranolol have been found to be related to the a b i l i t y of the blocking agents to modify the inotropic effect of low temperatures. 8. Although adrenergic amines have the a b i l i t y to cause increases in contractile force which are mediated by alpha receptors and which do not involve cyclic AMP, no dissociation has yet been found between the beta receptor, cyclic AMP, and the inotropic response. 9 . It therefore seems logical, in view of the close association of these parameters, that cyclic AMP may be one of the second messengers mediating the inotropic response in cardiac tissue. 10. The theophylline-induced cyclic AMP response is an indirect effect associated with the release of stored catecholamines. 11. Furthermore, theophylline exerts a direct contractile effect which is unrelated to cyclic AMP. 12. The a b i l i t y of theophylline to enhance the norepinephrine and not phenylephrine response may be due to mobilization of different sources of calcium by the alpha and beta receptors. 13. 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