Journal of Molecular and Cellular Cardiology (1975), 7,237-248


Stimulated Effects

Myocardial of Nucleotides



ROBERT J. LEFKOWITZ Division of Cardiology, Department of Medicine and Department of Biochemistry, Duke Universify Medical Center, Durham, .North Carolina 27710, lJS.A (Received 5 March

1974, and accepted 29 March


R. J. LEPKO~ITZ, Catecholamine Stimulated Myocardial Adenylate Cyclase: Effects of Nucleotides.3ournalofMolecularandCellularCardiology (1975)7, 237-248.Theeffects of several nucleotides on canine myocardial adenylate cyclase have been investigated. Basal, fluoride and isoproterenol stimulated enzyme activities were studied. With adenosine 5’ triphosphate at - 1.5 mM guanosine 5’-triphosphate increased basal and isoproterenol stimulated cyclaseactivityseveralfold.Flouridestimulatedactivitywasinhibited.Significantstimulation occurred at 10 nM guanosine 5’-triphosphate and effects were maxima1 by I PM. In order of decreasing potency the nucleoside 5’-triphosphates were guanosine > deoxyguanosine > uridine > thymidine > q&dine. Insosine 5’-triphosphate and xanthosine 5’-triphosphatewere inactive. Although guanosine 5’-triphosphate was most potent, all other guanyl nucleotides tested, includingguanosine 5’-diphosphate, guanosine 5’-monophosphateand cyclicguanosine, 3’, 5’-monophosphate shared this effect. Guanosine was inert. Thestimulatory action of guanosine 5’-triphosphate was exhibited after a lag period of several minutes. When substrate (adenosine 5’-triphosphate) concentrations were lowered to 0.05 mM, stimulation by isoproterenol was virtually dependent on the presence of guanosine 5’-triphosphate. Nucleotides did not affect the apparent Km for enzyme stimulation by isoproterenol which was l-2 x 10-Q with or without guanosine 5’-triphosphate. Propranolol effectively blocked the augmented stimulation by isoproterenol observed in the presence of guanosine 5’triphosphate. The data indicate that guanyl nucleotides are capable of markedly altering the sensitivity of myocardial adenylate cyclase to catecholamine stimulation. KEY WORIIS: Cyclic adenosine, 3’, 5’-monophosphate; sine triphosphate; Heart; Beta adrenergic receptor.




1. Introduction In mammalian myocardium, the membrane bound enzyme adenylate cyclase is coupled to beta adrenergic receptors [17]. Thus, drugs such as isoproterenol or endogenous neurotransmitters such as norepinephrine stimulate enzyme activity and hence the formation of cyclic adenosine 3’5’-monophosphate (CAMP). Cyclic AMP can increase myocardial contractility and may be the mediator of the inotropit and chronotropic effects of catecholamines [5]. HormonalIysensitivemammalian adenylate cyclase appears to consist of at least three components [Z]: (1) the catalytic unit, (2) hormone receptors which recognize specific biologically active hormonal structures and (3) “modulators” such as phospholipids which may regulate activation of the catalytic unit by the hormone receptors.





Rodbell et al. [14] were the first to demonstrate regulatory actions of guanyl nucleotides on the glucagon sensitive adenylate cyclase of liver membranes. It was found that the nucleotides markedly enhanced the sensitivity of the liver cyclase to glucagon stimulation [14-161. Earlier experiments were interpreted as suggesting effects on the hormone receptors as responsible for the observed effects of the guanyl nucleotides. More recently, however, it has been suggested that the nucleotides might be working at some step other than hormone binding [I]. Subsequently, several reports have indicated that a variety of nucleotides can regulate hormonal control of adenylate cyclase. Reports have concerned cyclase stimulation by thryrothropin (TSH) in thyroid membrances [IS], prostaglandins in adipocyte [7] and thyroid membranes [18], epinephrine in liver membranes [II] and glucagon in pancreatic islet cell membranes [9]. To date, effects of nucleotides on myocardial adenylate cyclase have not been reported. Since the marked effects of catecholamines on the heart are likely mediated via interaction with beta adrenergic receptors and adenylate cyclase, it seemed important to determine if this process could be influenced by nucleotides. Accordingly, the studies to be reported here were undertaken to (1) determine the extent to which nucleotide regulation affects catecholamine stimulation of myocardial adenylate cyclase and (2) compare the characteristics of this regulation with that of other hormonally responsive systems such as glucagon stimulated liver adenylate cyclase. 2. Materials

and Methods

d, l-propranolol hydrochloride, myoAll nucleotides, isoproterenol bitartrate, kinase, pyruvate kinase and phosphoenolpyruvate were from Sigma. [tc-32P] adenosine 5’-triphosphate (ATP) was purchased from International Chemical and Nuclear Corporation and [sH]-CAMP from Schwarz-Mann. Lubrol-PX was a gift from ICI America, Inc. Myocardial membranes were prepared by homogenization of canine ventricular myocardium in 0.25 M sucrose as previously described [IO]. Membranes sedimenting at 10 000 x g were washed twice with sucrose, then used for cyclase assays. Adenylate cyclase assay was performed using a modification of the method of Krishna et al. [S, 101 which follows the conversion of [z-33P] ATP to [s2P] CAMP in the presence of an ATP regenerating system. Incubation mixtures contained: ATP, 1.5 mM, Tris-HCl buffer, 30 mM (pH 7.5) ; MgCls, 5 mM, cyclic AMP, 0.1 mM, phosphoenolpyruvate, 5 mM; phosphoenolpyruvate kinase, 40 pg/ml and myokinase, 20 pg/ml, in a volume of 50 ~1. Incubations were performed for 10 min at 37°C. As reported previously [ZU], under these conditions CAMP formation is linear for at least 20 min and over a wide range of protein concentrations. In most assays approximately 50 pg of membrane protein were present. [ssP] CAMP was isolated by chromatography on Dowex AG-50W-X2 as described by Krishna et al.






[8] and quantitated by liquid scintilIation spectrometry. Added [sH]-CAMP was used to monitor product recovery which was generally N 50%. In certain experiments (see Figure legends) ATP concentration was lowered to 0.05 mM. In agreement with several other authors [18] we observed reasonable enzyme activities even at these low substrate ATP concentrations. Protein was determined by the method of Lowry et al. [13].











FIGURE 1. (a) The effect of GTP on basal, isoproterenol and fluoride stimulated myocardial adenylate cyclase. ATP was present at 1.5 mM. Isoproterenol concentration was 5 x 10-5~. Fluoride concentration was 0.01 M. Assay conditions are described under Methods. Each value is the mean of duplicate determinations in each of three experiments. (h) The effects of ITP on basal, isoproterenol and fluoride stimulated myocardial adenylate cyclase. (a) Fluoride stimulated; (0) isoproterenol stimulated; (0) basal. Values are the means of duplicate determinations in each of three experiments.

3. Results

Figure 1 (a) demonstrates that with substrate ATP concentration of 1.5 mM, guanosine 5’-triphosphate (GTP) stimulated both basal and isoproterenol activated adenylate cyclase. Effects were seen with GTP concentrations as low as 10 nM. Maximal effects were seen at 1 pM GTP. At higher GTP concentrations, less stimulation of the basal activity was seen. Over the same GTP concentration range, fluoride stimulated activity was inhibited. Figure 1 (b) demonstrates the effects of another purine nucleotide, inosine 5’-triphosphate (ITP) which, in thyroid membranes, causes an even greater stimulation of adenylate cyclase than GTP [18]. Although this nucleotide inhibited fluoride stimulated cyclase, it had almost no effect on basal or isoproterenol stimulated activity (slight inhibition).



GTP [M] FIGURE 2. Effects of GTP on isoproterenol stimulated myocardial adcnylate cyclasein thepresence of low substrate ATP concentration. ATP concentration was 0.05 mu. Isoproterenol was present at 5 x 10-5~. At each GTP concentration both basal and isoproterenol stimulated activity were determined and the difference has been plotted. Each value is the mean of duplicate determinations on three separate membrane preparations.

When ATP concentration was lowered, the dependence of catecholamine stimulation of the enzyme on the presence of GTP was even more marked. The data shown in Figure 2 were obtained with ATP at 0.05 mM. In the absence of added GTP almost no stimulation of enzyme activity by isoproterenol was seen, Increasing concentrations of GTP from 10-E to 10-5~ progressively augmented the isoproterenol response. GTP was not unique in its ability to increase basal and isoproterenol stimulated cyclase. As noted in Figure 3, all of the guanyl nucleotides tested, including guanosine 5’-diphosphate (GDP), guanosine 5’-monophosphate (GMP), cyclic guanosine 3’5’-monophosphate (cGMP) and deoxy guanosine 5’-triphosphate (dGTP) shared this property, though to varying extents. GTP was most potent. Guanosine was inert. The effects of a number of other purine and pyrimidine nucleotides on the myocardial adenylate cyclase were investigated (Figure 4). Of those tested, only GTP stimulated basal cyclase activity. Several of the nucleotides augmented isoproterenol stimulated activity. In order of decreasing potency these were: GTP > uridine 5’-triphosphate (UTP) > thymidine 5’-triphosphate (TTP) > cytidine






c P’C 4 ‘;O %T :.c 3 mzi gg a 2 .u z u\ i7-z E ’ 5 0








FIGURE 3. Comparison of the effects of several guanyl nucleotides and guanosine on myocardial adenylate cyclase. All nucleotides were present at 0.1 mn. Isoproterenol concentration was 5 x 10-5~. All values are means of duplicate determinations for three experiments. isoproterenol. c n .g 204 522



CL; 57 .o_ , 05 55

2 I

-0 Control







FIGURE 4. Comparison of the effects of purine and pyrimidine nucleoside triphosphates on myocardial adenylate cyclase. All nucleotides were present at 0.1 mM. Isoproterenol concentration was 5 x 10-5~. Fluoride concentration was 0.01 M. All values are means & S.E.M. of duplicate determinations for three experiments. (s) Basal; (

5’-triphosphate (CTP). Inhibition of fluoride stimulated activity was seen with all of the nucleotides. 5’-AMP at 0.1 mM inhibited basal, fluoride and isoproterenol stimulated adenylate cyclase (Table 1). Figure 5 presents dose response curves for stimulation of myocardial adenylate cyclase by isoproterenol in the presence or absence of 1 PM GTP. Although enzyme activity is augmented over the entire range of isoproterenol concentrations, there was no change in the concentration of isoproterenol producing l/2 maximal stimulation of enzyme activity (l-2 X 1od6M). Propranolol is a @-adrenergic blocking compound, capable of potently and specifically inhibiting catecholamine stimulated adenylate cyclase in a variety of tissues [3, 41. The data in Figure 6 indicate that the augmented cyclase response to isoproterenol in the presence of 1 PM GTP is blocked by propranolol. The time course of cyclic AMP accumulation in this system is shown in Figure 7.




1. Effect

of adenosine



(pmol None. 5’-AMP


Adenylate cyclase activity Isoproterenol



on myocardial

832 & 30 326 5 14

CAMP/IO min/mg 1050 & 32 798 + 28

5’-AMP terenol

was added to a final concentration of 0.1 mna. Substrate and fluoride stimulated values refer to CAMP generated value is the mean & S.E.M. of triplicate determinations. 3

l/2 Maximal



protein) 4225 + 120 1798 & 55

ATP was 1.5 mM. Isoproabove basal levels. Each


z E 0

7 .c_ 2 e

g F


a E t $ b 5tii

o-0 , -





: 5 .o i3 u” 01 10-g

I 10-8



10-7 10-6 Isopro+erenol’“~~]






FIGURE 5. Stimulation of myocardial adenylate cyclase by isoproterenol in the presence and absence of GTP. Arrow indicates concentration of isoproterenol producing l/2 maximal stimulation of enzyme activity. Each value is the mean of four determinations for two separate experiments with different membrane preparations.

Of note is the fact that the augmentation of activity by GTP has a lag period of several minutes. Inasmuch as Wolff and Cook have recently demonstrated that soluble adenylate cyclase from a bacterial source (Bordetella pertusis) does not respond to nucleotides [18], we were interested to see if nucleotide effects persisted when the myocardial adenylate cyclase was solubilized. The data shown in Table 2 demonstrate that the myocardial cyclase solubilized with the detergent Lubrol-PX is stimulated by GTP. Since the solubilized preparations did not respond to isoproterenol, effects of nucleotides on catecholamine stimulated cyclase activity could not be determined.






+ Propronolol

FIGURE 6. Effect of d,Lpropranolol on isoproterenol stimulated myocardial adenylate cyclase in the presence of GTP. Isoproterenol and propranolol were both present at 10 FM. GTP was present at 1 pnr. Each value is the mean + S.E.M. of three experiments.









Time (min)

FIGURE 7. Time course of GTP stimulation of myocardial mean of duplicate determinations for three experiments.


cyclase. Each value is the

4. Discussion A number of recent reports have demonstrated effects of nucleotides on a variety of different mammalian adenylate cyclase systems (Table 3) [I, 9, II, 14-16, 181. The experiments reported here are the first to extend these observations to myocardial adenylate cyclase. Taken together with findings in other systems, these data indicate several com-




2. Effect of GTP on solubilized myocardial adenylate cyclase



Control GTP 1 x IO-% GTP 1 x 10-4

(pm01 cAMP/mg/l 0 min) 416 & 25 672 f 32 971 $144

cyclase activity

Five grams of canine ventricular myocardium was solubilized as described by Levey [Z7]. The tissue was homogenized in 50 ml of 0.25 M sucrose containing 0.05 M Tris-HCI, pH 7.4, 0.001 M EDTA-MgCl s, and 0.25% Lubrol-PX. The homogenate was centrifuged at 10 000 x g for 10 min at 4°C and then the supernatant was centrifuged for an additional 90 min at 105 000 x p in a Beckman L-265B ultracentrifuge. This supernatant was used as the source of the adenylate cyclase in these experiments. Each value is the mean f S.E.M. of 4 determirmtions from 2 experiments. mon features, as well as certain variations in the patterns of nucleotide effects. In all of the systems studied (Table 3) basal cyclase is slightly to markedly stimulated by the nucleotides. Fluoride stimulated activity is either unchanged or inhibited. Hormonal stimulation is markedly augmented in all cases. At low substrate ATP or 5’ adenylyl-imidodiphosphate (AMP-PNP) concentrations, nucleotides may be obligatory for hormonal response. The augmentation of hormonal stimulation of cyclase is even more impressive, when the diversity of hormones involved is considered: glucagon [9, II, 141 TSH [18], prostaglandins [7, 181 and catecholamines [11]. In the two cases where it was specifically examined, the concentrations of hormone causing l/2 maximal stimulation of cyclase were not altered by the nucleotides [ 141. The observed effects do not appear to be confined to the guanyl nucleotides or to the nucleoside triphosphates. However, the potency of various nucleotides in producing these effects varies from tissue to tissue and may even vary from hormone to hormone within the same tissue. Thus, Wolff and Cook reported that in thyroid membranes, ITP produced greater augmentation of TSH response than GTP [18]. For prostaglandin EI (PGEi), h owever, the two nucleotides were equipotent. ITP, however, has no stimulatory effect on catecholamine stimulated myocardial adenylate cyclase. In general, pyrimidine nucleotides have been less potent than GTP [II, 181. Within the series of guanyl nucleotides, GTP has generally been equipotent with GDP, or somewhat more potent. In the studies reported here, GMP was quite potent as well. The mechanism by which the nucleotides are acting to augment hormonal stimulation of adenylate cyclase is not clear at present. Augmentation of several different hormonal responses within a single tissue by the nucleotides has been taken by some to indicate that they must be acting beyond the receptor binding sites, perhaps at some “transducer” [II] or modulator. Rodbell et al. [16] have reported that GTP enhances the dissociation of issI-


Islets of Langerhans (9) Plateets (7)

Myocardium (canine ventricular-this study) *NR = not reported

Thyroid (18)

Thyroid (18)

Only GTP tested

Rat liver (11)





Yes, with substrate



5 t Yes, with ATP < isoproterenol 0.1 mM I

*,,T” PGEl

AMP - PNP = i I 0.25rnM no change glucagon Yes, with ATP < epinephrine 1 0.5 rnM NR glucagon Not studied at low T ATP concentrations Yes, with substrate PGEr 1 f AMP - PNP = 0.1 RIM


Nucleotide required for hormonal effect ___

Not altered





Not altered

K, for hormone stimulated activity -.- _ __-.--.-~ __~

responsive adenylate cyclase from various tissues

Adenylate cyclase activity Hormone Basal Fluoride stimulated

ITP > dGTP > ?‘ GTP > XTP > slight CTP > UTP I GTP = dGTP = ITP > XTP 1 GTP > GDP > LJTP > TTP > CTP t

Only GTP tested



Relative potency of nucleotides

Rat liver (14)

Sources of membranes

Table 3. Effects of nucleotides on hormonally





glucagon from its binding sites in rat liver plasma membranes. It was suggested that the observed effects on binding might explain the effects on glucagon stimulated cyclase [14]. However, more recently, Birnbaumer and Pohl have pointed out that “GTP stimulates dissociation of bound labelled glucagon from liver plasma membranes under conditions which do not selectively affect the glucagon stimulated adenylate cyclase” [I]. They concluded that “previously discovered effects of purine nucleotides on hormone binding and hormonal stimulation may be unrelated phenomena” [I]. In other experiments (data not shown) we have found that nucleotides had no effect on the binding of [sH] isoproterenol to the myocardial membranes. However, until all of the catecholamine binding sites can be rigorously shown to be coupled to the cyclase, it seems hazardous to speculate on whether the adrenergic receptors or the so-called “modulator” or the catalytic sites are the sites of action of the nucleotides. It was interesting to note that the augmented cyclase response to isoproterenol in the presence of GTP could be effectively blocked by propranoIo1 in a fashion quite typical of a “p-adrenergic” receptor mediated response. In agreement with Wolff and Cook [IS] a lag of several minutes in the stimulatory effects of nucleotides was noted. The significance of this finding is not known. The stimulation of the soluble myocardial adenylate cyclase by GTP indicates that nucleotides are capable of interacting with soluble as well as particulate cyclase systems. The lack of effect of nucleotides on soluble bacterial adenylate cyclase reported by others [18] is therefore more likely due to intrinsic differences in the bacterial cyclase, than to any factors necessarily associated with the soluble state of the enzyme. The physiological significance of these nucleotide effects on the sensitivity of myocardial adenylate cyclase to catecholamines is not known at present. However, these findings suggest that guanyl nucleotides might play an important regulatory role in controlling myocardial sensitivity to catecholamines. Thus, especially at low substrate ATP levels, sensitivity of the myocardial cyclase to catecholamines was virtually dependent on the presence of GTP. These effects of GTP on myocardial adenylate cyclase might also offer an explanation for the well-known antagonistic effects of cholinergic and adrenergic agents on myocardial contractility as has been suggested by Goldberg et al. [6J. Thus, cholinergic agents such as acetylcholine are thought to influence the myocardium by stimulating increases in tissue levels of cyclic GMP, presumably by activation of the enzyme guanyl cyclase. To the extent that guanylate cyclase were activated and GTP consumed via conversion to cGMP, cellular levels of GTP might be transiently reduced. This reduction in GTP levels could then reduce sensitivity of the myocardial adenylate cyclase to catecholamines, thus blunting the effects of adrenergic agents on the myocardium.







The author wishes to thank Dr Edgar Haber in whose laboratory (Mass General Hospital) much of this work was completed and also Miss Fritzie Erlenmeyer for excellent technical assistance. This work was supported by United States Public Health Service Grant HE-5196, SCOR HE-14150, H. E. W. Grant #l ROI HL16037-01 and by a grant-in-aid from the American Heart Association with funds contributed in part by the North Carolina Heart Association. Dr Lefkowitz is an Established Investigator of the American Heart Association.


2. ‘> 5. 4. .5. 6. 7. 8. 9. 10. 11.

12. 13. 14.

BIRNBAUMER,L. & POHL, S. L. Relation of glucagon-specific binding sites to glucagondependent stimulation of adenylyl cyclase activity in plasma membranes of rat liver. Journal of Biological Chemistry 248, 2056-2061 (1973). BIRNBAUMER,L., POHL, S. L. & RODBELL, M. The glucagon-sensitive adenyl cyclase system in plasma membranes of rat liver, II. Journal of Biological Chemistry 246, 18571860 (1971). BIRNBAUMER, L. & RODBELL, M. Adenyl cyclase in fat cells, II. Journal of Biological Chemistry 244, 3477-3482 (1969). ENTMAN, M. L., LEVEY, G. S. & EPSTEIN, S. E. Mechanism of action of epinephrine and glucagon on the canine heart. Circulation Research 25, 429-438 (1969). EPSTEIN, S. E., LEVEY, G. S. & SKELTON, C. L. Adenyl cyclase and cyclic AMP, biochemical links in the regulation of myocardial contractility. Circulation 43, 437-450 (1971). GOLDBERG, N. D., O’DEA, R. F. & HADDOX, M. K., Cyclic GMP. In Advances in Cyclic Nucleotide Research, Vol. 3, pp. 155-224, Greengard, Robison (1973). KRISHNA, G. & HARWOOD, J. P. Requirement for guanosine triphosphate in the prostaglandin activation of adenylate cyclase of platelet membranes. Journal of Biological Chemistry 247,2253-2254 (1972). KFUSHNA, G., WEISS, B. & BRODIE, B. B. Simple, sensitive method for the assay of adenyl cyclase. Journal of Pharmacology and Experimental Therapeutics 163,379-388 (1968). Kuo, W., HODGINS, D. S. & Kuo, J. F. Adenylate cyclase in islets of langerhans. Journal of Biological Chemistry 248, 2705-27 11 (1973). LEFKOWITZ, R. J., SHARP, G. W. G. & HABER, E. Specific binding of beta-adrenergic catecholamines to a subcellular fraction from cardiac muscle. Journal qf Biological Chemistry 248, 342-249 (1973). LERAY, F., CHAMBAUT, A. & HANOUNE, J. Role of GTP in epinephrine and glucagon activation of adenyl cyclase of liver plasma membrane. Biochemical and Biophysical Research Communications 48, 1385-1391 (1972). LEVEY, G. S. Solubilization of myocardial adenyl cyclase. Biochemical and Biophysical Research Communications 38, 86-92 (1970). LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L. & Randall, R. J. Protein measurement with the folin-phenol reagent. Journal of Biological Chemistry 193,265271 (1951). RODBELL, M., BIRNBAUMER, L., POHL, S. L. & KRANS, H. M. J. The glaucagonsensitive adenyl cyclase system in plasma membranes of rat liver, V. Journal of Biological Chemistry 246, 1877-1882 (1971).




15. RODBELL, M., KRANS, H. M. J., POHL, S. L. & BIRNBAUMER, L. The glucagon sensitive adenyl cyclase system in plasma membranes of rat liver, III. Journal of Biological Chemistry 246, 1861-1871 (1971). 16. RODBELL, M., KRANS, H. M. J., POHL, S. L. & BIRNBAUMER, L., The glucagonsensitive adenyl cyclase system in plasma membranes of rat liver, IV. Journal of Biological Chmistry 246, 1872-1876 (1971). 17. SUTHERLAND, E. W., ROBISON, G. A. & BUTCHER, R. W. Some aspects of the biological role of adenosine 3’, 5’-monophosphate (cyclic AMP). Circulation 37, 279-306 (1968). 18. WOLFF, J. & COOK, G. H. Activation of thyroid membrane adenylate cyclase by purine nucleotides. Journal of Biological Chemistry 248, 350-355 (1973).

Catecholamine stimulated myocardial adenylate cyclase: effects of nucleotides.

Journal of Molecular and Cellular Cardiology (1975), 7,237-248 Catecholamine Stimulated Effects Myocardial of Nucleotides Adenylate Cyclase: ROB...
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