Correlation of Cyclic Amp and Cyclic Gmp Levels With

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CORRELATION OF CYCLIC AMP AND CYCLIC GMP LEVELS WITH CHANGES IN CONTRACTILE FORCE OF DOG VENTRICULAR MYOCARDIUM DURING CHOLINERGIC ANTAGONISM OF POSITIVE INOTROPIC ACTIONS OF HISTAMINE, GLUCAGON, THEOPHYLLINE AND PAPAVERINE

Masao ENDOH Department of Pharmacology, Tohoku University School of Medicine, Sendai 980, Japan

Accepted August 6, 1979

Abstract-On the dog isolated right ventricular muscle, experiments were carried out in order to elucidate the characteristics of the cholinergic antagonism against the positive isotropic action (PIA) induced via different subcellular mechanisms. The relationship between cyclic AMP and cyclic GMP levels, and contractile force during cholinergic antagonism was assessed. Carbachol (10 ,tM) by itself decreased only slightly the tension developed, but inhibited prominently the PlAs of isoprenalinc, histamine, glucagon, theophylline and papaverine. The action of dibutyryl cyclic AMP was inhibited less than PIAs of other agents mentioned above. In contrast, carbachol did not affect the PIAs of calcium and g-strophanthin. The antagonism by carbachol of PIAs of isoprenaline, histamine, glucagon, theophylline and papaverine was accompanied by a reduction of the intracellular cyclic AMP level elevated previously by these agents, and by an elevation of the intracellular cyclic GMP level. A good correlation was found between changes in the tension developed, and cyclic AMP and cyclic GMP levels during the cholinergic antagonism of PIAs induced by these agents in the dog ventricular myocardium.

In the mammalian ventricular myocardium, cholinergic stimulation via vagus nerve or by acetylcholine (ACh) affected the contractile force less than that of atrial muscle (1-4). Hollenberg and coworkers (5) found first that ACh infused into a coronary artery in dogs caused only a slight negative inotropic action, but that ACh infused in the same concen tration during sympathetic stimulation or during noradrenaline infusion induced a profound myocardial depression. These observations were confirmed in the dog (6-8), rabbit and turtle (9), and cat (10) hearts. The subcellular mechanisms underlying these adrenergic-cholinergic interactions were elucidated by the observations that cyclic AMP and cyclic GMP exerted the opposing regulatory action on the myocardial contraction (11-14). It was shown that the PIA of ,3-adrenoceptor stimulation was accompanied by an increase in cyclic AMP levels and by a concomitant decrease in cyclic GMP levels in the isolated perfused rat heart (15); ACh induced negative isotropic effect was associated with an elevation of cyclic GMP levels and with the opposite change in cyclic AI\IP levels iii the same preparation (10, 17). Based on these and their own findings in the guinea-pig heart (18), it has been proposed that the anti-adrenergic effects of cholinergic stimulation in the mammalian ventricular muscle may be mediated by the specific antagonism of the intracellular actions of cyclic AMP by cyclic GMP.

Besides ,3-adrenoceptor stimulants which stimulate adenylate cyclase , histamine (19-21) and glucagon (22, 23), as well as cyclic AMP phosphodiesterase (PDE) inhibitors, theophyl line (24) and papaverine (25, 26) increased cyclic AMP levels during induction of their positive inotropic actions. How the intracellular cyclic AMP and cyclic GMP levels cor relate with contractile force during interaction of the cholinergic stimulation with these positive inotropic stimuli in the mammalian ventricular myocardium was investigated and the findings are reported herein.

MATERIALS AND METHODS Mongrel dogs of either sex (7-13 kg) were anesthetized with sodium pentobarbital (30 mg/kg, i.v.) and given heparin (500 units/kg). The heart was excised, and trabeculae carneae of the right ventricular wall (smaller than 1 mm in diameter) were isolated and suspended in a 20 ml organ bath containing Krebs-Henseleit solution. The solution was equilibrated with 95% 02 and 5 % CO, at a temperature of 37'C. The muscle was electri cally driven by square wave pulses of 5 msec duration and a voltage just above the threshold at a rate of 0.5 Hz. The muscle length was adjusted to Lmax, where the tension developed was maximal, during an equilibration time of 1 hr. The tension developed was recorded isometrically on an ink-writing oscillograph (San-ei Instrument) by the use of force displace ment transducers (Grass FTO3B; Shinkoh UL). After an equilibration period, concen trations of cardiotonic agents as determined in preliminary experiments were added to the bath. When the tension developed reached a steady level, carbachol was administered. The maximal tension developed in each muscle was determined by administering calcium (10-12.5 mM) at the end of each experiment. Positive inotropic effects of drugs were expressed as the percentage of the maximal contraction or as the percentage of the basal tension developed. Experiments, except for those with isoprenaline, were carried out in the presence of 30 nM pindolol in order to avoid the ;3-adrenoceptor stimulation evoked by the noradrenaline released. All experiments for chemical assay were carried out in the paired muscle isolated from the same heart in the presence of agonists alone and agonists plus carbachol. Three minutes after the administration of carbachol in the presence of each agonist, muscles were removed from the organ bath, frozen immediately in liquid nitrogen and homogenized in 0.5 ml of 5 % trichloroacetic acid, as described in detail in the previous paper (27). After homogeni zation, 100 ,' l portions of the supernatant were extracted five times with I ml of ether after adding I N HCI. The cyclic AMP and cyclic GMP contents were determined by the radio immunoassay methods after succinylation of the nucleotides (28-31). Levels of cyclic nucleotides in the muscle were expressed as pmol/mg wet weight of the tissue. Values were given as means-S.E. Statistical significance between mean values was estimated by means of Student's t-test for paired comparison. A P value of smaller than 0.05 is con sidered to be significant. Drugs Used: (--)-Isoprenaline hydrochloride (Nikken Kagaku, Nagoya); glucagon novo (glucagon hydrochloride; Novo Industri, Copenhagen); histamine dihydrochloride, theophylline (Wako, Osaka); papaverine hydrochloride (Iwaki Seiyaku, Tokyo); dibutyryl cyclic AMP sodium (Daiichi Seiyaku, Tokyo); g-strophanthin (E. Merck, Darmstadt); carbachol chloride (K & K Laboratories, New York); (_T'_)-pindolol base (Sandoz, Basel); the anti-cyclic AMP and -cyclic GMP sera and ('23I)-succinyl cyclic AMP and cyclic GMP tyrosine methyl ester (Yamasa Shoyu Co., Choshi). The solution of isoprenaline was prepared in 1 % ascorbic acid on each experimental day and diluted with 0.9 % saline solution. Glucagon and histamine were dissolved in saline on each experimental day. These solutions were kept ice-cooled during experiments.

RESULTS Influence of carbachol on the basal tension developed and on positive inotropic actions induced by various agents: Effects of 10 /iM carbachol on the basal tension developed, and on positive inotropic actions of isoprenaline, glucagon, histamine, papaverine, theophyl line and dibutyryl cyclic AMP (dbcAMP) are shown in Fig. 1. Carbachol (10 /tM) alone decreased only slightly the basal tension developed. The same concentration of carbachol caused prominent negative inotropic actions, when administered under conditions of myocardial stimulation produced by isoprenaline, glucagon, histamine, papaverine or theophylline. The inhibitory action of carbachol on the positive inotropic response to dbcAMP was less marked. In contrast to the prominent inhibitory action of carbachol on response to the agents

FIG. 1. Effects of 10 f1M carbachol (Carb) on the basal tension developed and on positive inotropic actions of isoprenaline (Iso, I tiM), glucagon (Glu, 10 pM), histamine (Hist, 1 mM), papaverine (Papav, 30 pM), theophylline (Theoph, 1 mM) and of dbcAMP (1 mM) in the dog isolated right ventricular myocardium. Experi ments with glucagon, histamine, papaverine, theophylline and dbcAMP were performed in the presence of 30 nM pindolol. The horizontal bar indicates 10 min except for isoprenaline; for isoprenaline, 5 min. Fro. 2. Influence of 10 ;tM carbachol (Carb) on positive inotropic actions of CaCl2 (5 mM) and g-strophanthin (g-Stroph, I ' M).

F1cG.3. Effects of 10 ;tM carbachol on positive inotropic actions induced by various cardiotonic agents on the dog isolated right ventricular muscle. Iso: isoprenaline; Glu: glucagon; Hist: histamine; Papav: papaverine: Theoph: theophylline; g-Stroph: g-strophanthin. Numbers in parentheses represent number of experi ments. Vertical bars indicate S.E. of the mean. shown above, carbachol (10 ,N) failed to inhibit positive inotropic actions of calcium or g-strophanthin (Fig. 2), the actions of which occurred with no change in the intracellular cyclic AMP level (24, 32). The summarized data of these experiments are presented in Fig. 3. Changes in the tension developed caused by carbachol (10 ; tM) under myocardial stimulation produced by isoprenaline, glucagon, histamine, papaverine, theophylline or dbcAMP were highly sig nificant (P<0.001 for each agonist). Actions of calcium and g-strophanthin were not significantly affected by carbachol. Influence of carbachol on cyclic AMP and cyclic GMP levels during clrolinergic antagonism of positive inotropic actions: Intracellular cyclic AMP and cyclic GMP levels were deter mined when positive inotropic actions were caused by isoprenaline, histamine, glucagon, papaverine or theophylline, and when these actions were inhibited by 3 ,tM carbachol. Results are suntnlari,ed in 'Fable 1. Cyclic AMI' levels were increased significantly by each agonist from control levels of 0.80---0.95 pmol/ing w.w. at the time of determination described 'l- :vIr t 1. Influence of 3 ,,M carbachol on the intracellular cyclic AMP and cyclic GMP levels during inhibition of the positive inotropic action induced by various agonists in the dog isolated ventricular muscle

in Table 1. Carbachol (3 /N) administered to one of the paired muscle 3 rnin before the determination decreased significantly (P values are presented in Table IA) the cyclic AMP level elevated previously by each agonist. The cyclic AMP level in the presence of iso prenaline and carbachol was still significantly (P0.001) higher than the corresponding control value of 0.95=_0.04 pmoljmg w.w. (n--6). Cyclic AMP levels in the presence of other agonists and carbachol were not significantly different from the corresponding control value. Levels of cyclic GMP were significantly elevated by carbachol (3 tiM) compared with those in the presence of agonists alone (P values are presented in Table IB). Cyclic GMP levels of the control muscle determined simultaneously were quite variable among the groups: levels in each series of experiments were as follows: 0.0260±0.0055 (n=6), 0.0251 -0.0057 (n--7), 0.0414__:0.0027 (n=4), 0.0193-1-0.0069 (n=5) and 0.0320+0.0062 (n=6) pmol/mg w.w. for the series of isoprenaline, histamine, glucagon, theophylline and papaverine experiments, respectively. Correlation of cyclic AMP and cyclic GMP levels with changes in the tension developed: Whether or not cyclic AMP and cyclic GMP levels presented in the previous section cor related with changes in the tension developed simultaneously assessed during the interaction was then examined. The summarized data are presented in Fig. 4. There was a good FIG. 4. The correlation of cyclic AMP and cyclic GMP levels with changes in tension developed during induction of positive inotropic actions by isoprenaline (1 pM), histamine (1 mM), glucagon (3 pM), papaverine (30 aM) or by theophylline (2 mM), and during inhibition of these actions by carbachol (3 11M). *In the presence of both carbachol and each agonist . Number of experiments with each agonist and carbachol is the same as Table 1. Solid and dotted lines represent regression lines for cyclic AMP and cyclic GMP, respectively. correlation between the intracellular cyclic AMP level and changes in the tension developed: the correlation coefficient was 0.644 (n=64, P<0.001). The regression line intersected the control cyclic AMP level, when the change in tension developed was zero. This indicates that the relationship between the intracellular cyclic AMP and tension developed was not altered by agonists or by the addition of carbachol. A significant correlation was also found between the cyclic GMP level and changes in the tension developed. The correlation coefficient was lower (r= --0.402, n=60, P<0.01) than that of cyclic AMP, probably because of the variability of control levels of cyclic GMP among groups and of the considerable elevation of the cyclic G MP level induced by carbachol in the presence of papaverine.

I3ISCUSSION Carbachol inhibited positive inotropic actions not only of isoprenaline, but also of histamine and theophylline in the dog ventricular muscle. Actions of calcium and g strophanthin, on the other hand, were not affected by carbachol. These observations are consistent with previous findings in the mammalian ventricular myocardium of various species (2, 3, 5-10, 18). Furthermore, it was shown in the present study that actions of glucagon and papaverine were also inhibited by carbachol. Since the positive inotropic actions, which were inhibited by cholinergic stimulation, were accompanied by a simultaneous elevation of the cyclic AMP level (15-27, 32-34, 37), while those, which were not inhibited, were not accompanied by elevations in the cyclic AMP levels (18, 24, 32), it appears that the cholinergic antagonism in the mammalian ventricular myocardium is indeed specific to actions related to an elevation of cyclic AMP levels, as previously suggested by Watanabe and Besch (18). Our recent observations that the positive inotropic action mediated via a-adrenoceptors in the rabbit papillary muscle was not inhibited by the same concentration of carbachol (35) support this hypothesis. Gardner and Allen (33) have shown that in the perfused rat heart ACh reduced cyclic AMP levels and phosphorylase activity when the levels and activity were elevated above basal values by adrenaline. In the same preparation, Keely and coworkers (34) demon strated that ACh antagonized the adrenaline-produced increases in cyclic AMP levels and in contractile force, and activations of cyclic AMP-dependent protein kinase and of phos phorylase. These changes were accompanied by increased cyclic GMP levels (33, 34). Consistent with these findings in the rat heart, carbachol, another cholinergic agent, reduced the elevation of cyclic AMP levels by isoprenaline, during the inhibition of the positive inotropic action of isoprenaline in the dog ventricular muscle. Furthermore, the elevation of cyclic AMP levels produced by histamine, glucagon, theophylline or papaverine was also abolished during the carbachol-induced inhibition of the positive inotropic actions of these agents. The findings that there was a good correlation between levels of cyclic AMP and changes in developed tension during cholinergic antagonism, and that the relationship between cyclic AMP levels and contractile force was not changed during cholinergic an tagonism suggest that the reduction of cyclic AMP levels is involved in the antagonism caused by carbachol. In adenylate cyclase preparations isolated from dog (36) and cat (37) hearts, cholinergic stimulation induced by ACh or by carbachol decreased the rate of cyclic AMP formation caused by adrenaline. These findings indicate that a reduction of the adenylate cyclase activity may be responsible for a reduction of cyclic AMP levels caused by cholinergic stimulation. However, the reduction of the adenylate cyclase activity in these experiments was not more than 300,/' (36, 37). Furthermore, the elevation of cyclic AMP levels caused by not only adenylate cyclase stimulation, but also cyclic AMP phosphodiesterase (PDE) inhibitors, theophylline and papaverine was abolished by carbachol in the present experi ments. In the PDE preparation of rabbit hearts LaRaia and Sonnenblick (37) found no significant difference between the normal activity and that with 10 1tM carbachol. Thus, it appears unlikely that carbachol stimulates directly the PDE activity. Alternatively, it is noteworthy that PDE preparations from a number of sources, including rat liver (38), rat adipose tissue (39), and human platelets (40) can be activated by a low concentration of cyclic GMP. Since the cyclic GMP level was significantly elevated by cholinergic stimulation during cholinergic antagonism of the positive inotropic actions in the ventricular myocardium in the present as well as previous studies (18, 34), stimulation of the PDE activity through the intracellular accumulation of cyclic GMP caused by carbachol is likely to be involved in reducing the intracellular cyclic AMP level. The finding that the inotropic action of dbcAMP which is resistant to hydrolysis by PDE (41) was antagonized by carbachol to a lesser extent than by those of other agonists supports this possibility. However, not all the actions of cholinergic stimulation in the mammalian ventricular myocardium can be explained by changes in cyclic AMP levels, as shown by Watanabe and Besch in the guinea-pig heart (18). Although the cyclic GMP level in the dog ventricular muscle was consistently increased during cholinergic antagonism, the observation being consistent with previous findings in various cardiac preparations (15--18, 33, 34, 37), these results cannot be simply taken as evidence for the hypothetical mediator role of cyclic GMP in producing the negative ino tropic action via cholinergic stimulation (111, 12, 15-17). Recently, detailed analyses of the dose-response relationship of carbachol in the guinea-pig atria (42) and the use of sodium nitroprusside in the cat atrial appendage (43) revealed the dissociation of the contractile force from the intracellular level of cyclic GMP. Furthermore, the derivatives of cyclic GMP, dibutyryl and 8-bromo-cyclic GMP, do not mimic the antagonistic action of carbachol regarding the positive isotropic effects of sympathomimetic amines in the dog isolated ventricular myocardium (44). In stuminary, the present results, together with. those of Keely et al. (34) strongly suggest the potential involvement of a reduction by cholinergic stimulation of cyclic AMP levels, elevated previously above basal values by 13-adrenoceptor stimulants, histamine, glucagon, theophylline or by papaverine, in the cholinergic antagonism of positive inotropic actions of these agents in both dog and rat ventricular myocardium.

REFERENCES

1) BLINKS,J.R. AND KOCH-WISER,J.: Physical factors in the analysis of the actions of drugs on myocardial contractility. Pharmacol. Rev. 15, 531-599 (1963) 2) DE, GEi:sr, H., LEVY, M.N. AND ZILSKI:, H.: Negative inotropic effect of the vagus nerves upon the canine ventricle. Science 144, 1223-1225 (1964) 3) PRIOLA, D.V. AND FULTON, R.L.: Positive and negative inotropic responses of the atria and ventricles to vagosympathetic stimulation in the isovolumic canine heart. Circu lation Res. 25, 265-275 (1969) 4) L NDOIi, M., TAMURA,K. AND HASHIMo]o, K.: Negative and positive inotropic responses of the blood-perfused canine papillary muscle to acetylcholine. J. Pharmacol. exp. Ther. 175, 377-387 (1970) 5) HOLLLNBLRG,M., CARRIERS,S. AND BARGtii, A.C.: Biphasic action of acetylcholine on ventricular myocardium. Circulation Res. 16, 527-536 (1965) 6) 1..Evy, M.N., No, M., MARTIN, P. AND ZIESKE, H.: Sympathetic and parasympathetic interactions upon the left ventricle of the dog. Circulation Res. 19, 5-10 (1966) 7) STANTON,H.C. AND VICK, R.L.: Cholinergic and adrenergic influences on right ventricular myocardial contractility in the dog. Archs int. Pharmacodvn. Ther. 176, 233-248 (1968) 8) LEvy, M.N. ANDZIESKL, H.: Effect of enhanced contractility on the left ventricular response to vagus nerve stimulation in dogs. Circulation Res. 24, 303-311 (1969) 9) MFESTER,W.D. AND HARDMANN,H.F.: Blockade of the positive inotropic actions of epin ephrine and theophylline by acetylcholine. J. Pharinacol. exp. Ther. 158, 241-247 (1967) 10) DEMPSEY,P.J. AND CooPF_R,T.: Ventricular cholinergic receptor systems: Interaction with adrenergic system. J. Pharmacol. CA-P.Tlier. 167, 282-290 (1969) 11) GOLDBERG,N.D., HADDOX, M.K., NICOL, S.L., GLASS,D.B., SANFORD,C.H., KUEHL, F.A. JR. AND ESTENSEN,R.: Biologic regulation through opposing influences of cyclic GMP and cyclic AMP: The Yin Yang hypothesis. Advances in Cyclic Nucleotide Research, Edited by DRUMMOND,G.l., GREENGARD,P. AND ROBISON,G.A., Vol. 5, p. 307-330, Raven Press, New York (1975) 12) WILKERSON,R.D., PADDOCK,R.J. ANDGEORGE, W.J.: Effects of derivative of cyclic AMP and cyclic GMP on contraction force of cat papillary muscles. Europ. J. Pharmacol. 36, 247-251 (1976) 13) NAWRATH,H.: Cyclic AMP and cyclic GMP may play opposing roles in influencing force of contraction in mammalian myocardium. Nature 262, 509-511 (1976) 14) NAWRAnI, I-I.: Evidence for opposing influences of cyclic GMP and cyclic AMP on force of' contraction in mammalian myocardium. Recent Advances in Studies on Cardiac Structure and Metabolism, Vol. 11, Heart Function and Metabolism, Edited by KOBA YASHI,T., SANO, T. AND DHALLA, N.S., p. 419-422, University Park Press, Baltimore (1978) 15) (EORGE:, W.J., POLSON,.1.13., O'TOOLE, A.G. ANDGOLDBERG, N.D.: Elevation of guanosine 3',5'-cyclic phosphate in rat heart after perfusion with acetylcholine. Proc. natn. Acad. Sci. U.S.A. 66, 398-403 (1970) 16) GEORGE, W.J., WILKERSON,R.D. AND KADOWITZ, P.J.: Influence of acetylcholine on contractile force and cyclic nucleotide levels in the isolated perfused rat heart. J. Pharmacol. exp. Ther. 184, 228-235 (1973) 17) GOLDBERG,N.D., HADDOX,M.K., HARTLE, D.K. AND HADDEN,J.W.: The biological role of cyclic 3',5'-guanosine monophosphate. Pharmacology and the Future of Man, Fifth International Congress on Pharmacology, San Francisco, Vol. 5, p. 146-169, Karger, Basel (1973) 18) WATANABE,A.M. AND BESCH,H.R. JR.: Interaction between cyclic adenosine monophos phate and cyclic guanosine monophosphate in guinea pig ventricular myocardium. Circulation Res. 37, 309-317 (1975) 19) McNEILL, J.H. AND MUSCIIEK, L.D.: Histamine effects on cardiac contractility, phos phorylase and adenyl cyclase. J. Mol. Cell. Cardiol. 4, 611-624 (1972) 20) KUKOVETZ,W.R., POCH, G. AND WURM, A.: Effect of catecholamines, histamine and oxyfedrine on isotonic contraction and cyclic AMP in the guinea-pig heart. Arch. Pharmacol. 278, 403-424 (1973) 21) VERMA,S.C. AND McNEILL, J.H.: Cardiac histamine receptors: Differences between left and right atria and right ventricle. J. Pharmacol. exp. Ther. 200, 352-362 (1977) 22) ENTMAN,M.L., LEVEY,G.S. AND EPSTEIN, S.E.: Mechanism of action of epinephrine and glucagon on the canine heart: Evidence for increase in sarcotubular calcium stores mediated by cyclic 3',5'-AMP. Circulation Res. 25, 429-438 (1969) 23) EPSTEIN, S.E., LEVEY, G.S. AND SKELTON,C.L.: Adenyl cyclase and cyclic AMP: Bio chemical links in the regulation of myocardial contractility. Circulation 43, 437-450 (1971) 24) DZNGES, C., HEITMANN,M., JUNGBLUTH,H., MEINERTZ,T., SCHMELZLE,B. AND SCHOLZ, H.: Effectiveness of theophylline to increase cyclic AMP levels and force of contraction in electrically paced guinea-pig auricles. Comparison with isoprenaline, calcium and ouabain. Arch. Pharmacol. 301, 87-97 (1977) 25) REINHARDT,D., ROGGENBACH,W., BRODDE, O.-E. AND SCHUMANN,H.J.: Influence of papaverine and isoprenaline on contractility and cyclic AMP level of left guinea-pig atria at different rates of beat. Arch. Pharmacol. 299, 9-15 (1977) 26) ENDOH, M. AND HONMA, M.: Effects of papaverine and its interaction with isoprenaline and carbachol on the contractile force and cyclic nucleotide levels of the canine ven tricular myocardium. Arch. Pharmacol. 306, 241-248 (1979) 27) SCHUMANN,H.J., ENDOH, M. AND BRODDE,O.-E.: The time course of the effects of S and a-adrenoceptor stimulation by isoprenaline and methoxamine on the contractile force and cAMP level of the isolated rabbit papillary muscle. Arch . Pharmacol. 289, 291-302 (1975) 28) STEINER,A.L., PARKER,C.W. ANDKIPNIS, D.M.: Radioimmunoassay for cyclic nucleotides: 1. Preparation of antibodies and iodinated cyclic nucleotides. J. biol. Chem . 247, 1106-1113 (1972) 29) CAILLA,H.L., RACINE-WEISBUCH,M.S. AND DELAAGE,M.A.: Adenosine 3',5'-cyclic mono phosphate assay at 10-15 mole level. Analyt. Biochem. 56, 394-407 (1973) 30) CAILLA,H.L., VANNIER,C.J. AND DELAAGE,M.A.: Guanosine 3',5'-cyclic monophosphate assay at 10-'' -mole level. Analyt. Biochem. 70, 195-202 (1976) 31) HONMA,M., SATOH,T., TAKEZAWA,J. AND UI, M.: An ultrasensitive method for the simul taneous determination of cyclic AMP and cyclic GMP in small-volume samples from blood and tissue. Biochem. Med. 18, 257-273 (1977) 32) KEELY,S.L. JR. AND CORBIN,J.D.: Involvement of cAMP-dependent protein kinase in the regulation of heart contractile force. Am. J. Physiol. 233, H269-H275 (1977) 33) GARDNER, R.M. AND ALLEN, D.O.: Regulation of cyclic nucleotide levels and glycogen phosphorylase activity by acetylcholine and epinephrine in perfused rat heart. J. Pharmacol. exp. Ther. 198, 412-419 (1976) 34) KEELY,S.L. JR., LINCOLN,T.M. ANDCORBIN, J.D.: Interaction of acetylcholine and epin ephrine on heart cyclic AMP-dependent protein kinase. Am. J. Physiol. 234, H432 H438 (1978) 35) ENDOH, M. AND MOTOMURA,S.: Differentiation by cholinergic stimulation of positive inotropic actions mediated via a and ~-adrenoceptors in the rabbit heart. Life Sci. 25,759-768(1979) 36) MURAD, F., CHI, Y.-M., RALL, T.W. AND SUTHERLAND,E.W.: Adenyl cyclase. 111. The effect of catecholamines and choline esters on the formation of adenosine 3',5'-phos phate by preparations from cardiac muscle and liver. J. biol. Chem. 237, 1233-1238 (1962) 37) LARAIA,P.J. AND SONNENBLICK,E.H.: Autonomic control of cardiac c-AMP. Circulation Res. 28, 377-384 (1971) 38) RUSSELL, T.R., TERASAKI,W.L. AND APPLEMAN,M.M.: Separate phosphodiesterase for the hydrolysis of cyclic adenosine 3',5'-monophosphate and cyclic guanosine 3',5' monophosphate in rat liver. J. biol. Chem. 248, 1334-1340 (1973) 39) KLOTZ, U. AND STOCK, K.: Influence of cyclic guanosine-3',5'-monophosphate on the enzymatic hydrolysis of adenosine 3',5'-monophosphate. Arch. Pharmacol. 274, 54-62 (1972) 40) ASANO,T., OCHIAI, Y. ANDHIDAKA, H.: Selective inhibition of separated forms of human platelet cyclic nucleotide phosphodiesterase by platelet aggregation inhibitors. Mol. Pharmacol. 13, 400-406 (1977) 41) SIMON, L.N., SHUMAN,D.A. AND ROBINS, R.K.: The chemistry and biological properties of nucleotides related to nucleoside 3',5'-cyclic phosphates. Advances in Cyclic Nucleo tide Research, Edited by GREENGARD,P. ANDROBISON, G.A., Vol. 3, p. 225-353 (1973) 42) BROOKER,G.: Dissociation of cyclic GMP from the negative inotropic action of carbachol in guinea pig atria. J. Cyclic Nucleotide Res. 3, 407-413 (1977) 43) DIAMOND,J., TEN EICK, R.E. AND TRAPANI, A.J.: Are increases in cyclic GMP levels re sponsible for the negative inotropic effects of acetylcholine in the heart? Biochem. biophys. Res. Commun. 79, 912-918 (1977) 44) ENDOH, M. AND SHIMIZU,T.: Failure of dibutyryl and 8-bromo-cyclic GMP to mimic the antagonistic action of carbachol on the positive inotropic effects of sympathomimetic amines in the canine isolated ventricular myocardium. Japan. J. Pharmacol. 29, 423-433 (1979)