The binding characteristics of these sites have resembled, in

Transcription

1 Proc. Nat. Acad. Sci. USA Vol. 72, No. 4, pp , April 1975 Identification of Cardiac f3-adrenergic Receptors by (-) [3H]Alprenolol Binding* (stereospecificity/binding kinetics/ft-adrenergic agonists/36-adrenergic antagonists) R. WAYNE ALEXANDER, LEWIS T. WILLIAMS, AND ROBERT J. LEFKOWITZ Department of Medicine, Division of Cardiology and the Departments of Biochemistry and Physiology, Duke University Medical Center, Durham, North Carolina 2771 Communicated by James B. Wyngaarden, February 6, 1975 ABSTRACT (-) PHJAlprenolol, a potent j3-adrenergic antagonist, was used to identify binding sites in a fraction of canine myocardium. Beta adrenergic agonists and antagonists compete for these binding sites in a manner which directly parallels their known affinity for the cardiac _-adrenergic receptor. Thus, binding was highly stereospecific, with the (-) isomers of jp-aldrenergic agonists or antagonists being at least two orders of magnitude more potent than were the (+) isomers in competing for these sites. The order of potency for inhibition of binding by,6-adrenergic agonists was (-) isoproterenol > (-) epinephrine > (-) norepinephrine. The dissociation constant (KD) of (-) alprenolol for the P-adrenergic receptors was 7-11 nm as determined independently by direct binding studies or by inhibition of isoproterenol-stimulated adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC ]. The P-adrenergic antagonist (-) propranolol also had high affinity for the binding sites (KD = 12 nm). The physiologically inactive catechol-containing compounds pyrocatechol and (i) dihydroxymandelic acid, as well as the metabolite (A) normetanephrine, and the a-adrenergic antagonist phentolamine did not compete for the binding sites at a concentration of 16 um. Binding was rapid (tl/, <3 sec) and was rapidly reversible (tl/, <15 sec). The binding sites were saturable and bound.35 pmol of (-) [3Hjalprenolol per mg of membrane protein. These characteristics suggest that these binding sites represent the cardiac,-adrenergic receptors. Binding to specific receptor sites is thought to be the initial event leading to the modulation of cellular function by hormones. Recently, rapid progress has been made in the isolation and characterization of polypeptide hormone receptors (1) and nicotinic cholinergic receptors (2). In general, these receptors have been studied by labeling them with high specific activity radioactively labeled hormones or, in the case of the nicotinic cholinergic receptors, with a radioactively labeled snake venom antagonist (1, 2). These methods also have been applied in attempts to identify the adrenergic,-receptors. Tritium-labeled,B-adrenergic agonists such as norepinephrine (3-5), epinephrine (6-8), and isoproterenol (9, 1) have been used to identify binding sites in various mammalian tissues and avian erythrocytes. * "(-) [3H]Alprenolol" has been used throughout this manuscript to identify the compound resulting from the catalytic reduction of (-) alprenolol with tritium. (-) Alprenolol contains an unsaturated bond in the aliphatic chain on the 2 position of the aromatic ring. The compound, therefore, might be appropriately referred to as "(-) [3H]dihydroalprenolol." The nature of the labeling process, however, is such that tritium exchange might also take place, yielding (-) [3H]alprenolol. The labeled material used for these studies has biological activity and chromatographic properties identical to those of native (-) alpreidolol. The binding characteristics of these sites have resembled, in several respects, those which might be expected of p-adrenergic receptor binding sites. Several characteristics, however, have differed from the expected properties of 3-adrenergic receptors on the basis of physiologic responses or of the activation of adenylate cyclase which is coupled to,-receptors (11, 12). First, these [3H]catecholamine binding sites do not exhibit stereospecificity. Physiologically, the (-) isomers of fl-adrenergic antagonists or agonists are much more potent than are the (+) isomers (13). Second, the affinity of these binding sites for g-adrenergic antagonists such as propranolol is several orders of magnitude lower than the affinity of the physiologic,- receptors for these antagonists. Third, several catechol compounds that are devoid of physiological p-adrenergic effects bind to the sites. Finally, whereas 3-adrenergic agonists or antagonists produce maximum physiologic effects within 1-2 min (14, 15), these binding sites require many minutes to reach equilibrium binding of catecholamines (3, 16). Very recently, reports have appeared which describe binding sites in avian or amphibian erythrocytes that fulfill strict binding criteria (affinity and stereospecificity) appropriate to the physiologic,b-adrenergic receptors (17-21). A common approach in all of these studies consisted of the labeling of the binding sites with radioactive 3-adrenergic antagonists. The present report describes the application of these techniques to a mammalian system. Thus, binding sites for (-) [8H]alprenolol, a potent #-adrenergic antagonist, were identified in canine myocardium. The ability of stereo isomers of a number of adrenergic agonists and antagonists to interact with these (-) [8H]alprenolol binding sites was studied. In addition the kinetics of the binding reaction were analyzed. These properties of the (-) [8H]alprenolol binding sites were related to the known characteristics of the cardiac,b-adrenergic receptors. The excellent correlation observed suggests that the (-) [3H]alprenolol binding sites are, in fact, equivalent to the cardiac g-adrenergic receptor binding sites. MATERIALS AND METHODS (-) [3H]Alprenolol (specific activity 17 Ci/mmol) was prepared by New England Nuclear by catalytic reduction of (-) alprenolol (Hassle) with tritium gas using paladium as the catalyst (19, 2). The tritiated compound was stored in absolute ethanol at -2. In initial experiments labeled alprenolol was purified daily as described previously (19, 2), although in later experiments the use of unpurified material (containing less than 5% contamination) gave identical experimental results. Other drugs used in this study were: (-) and (+) propranolol hydrochloride (Ayerst); (+) isoproterenol bitartrate, (+) epinephrine bitartrate, (+) nor- 1564

2 Proc. Nat. Acad. Sci. USA 72 (1975) Cardiac fl-adrenergic Receptors 1565 TABLE 1. Inhibition of (-) ['HJaiprenolol binding to canine myocardial membranes by (-) propranolol (-) [3H]Alprenolol bound Inhibi- (cpm/pellet) tion of specific Addition Observed "Specific" binding None i (-) propranolol 1.6 X 1-9 M X 1-M 1413d X 1-7 M X 1-M X 1-5 M X 1-4M Values shown are mean 4 SEM of six determinations. "Specific" binding is calculated by subtracting the cpm not displaced by high concentrations.of propranolol (>1 MM) from the observed cpm. epinephrine bitartrate, (+) phenylephrine hydrochloride (Winthrop); (-) isoproterenol bitartrate, (-) epinephrine bitartrate, (-) norepinephrine bitartrate, (±) dihydroxymandelic acid, (d) normetanephrine, (=) dihydroxyphenylalanine (dopa) (Sigma); pyrocatechol (Nutritional Biochemicals); and phentolamine (Ciba). Membrane Preparation. Mongrel dogs of either sex weighing 5 pounds were used. The animals were anesthetized with intravenous pentobarbital (3 mg/kg) and the hearts were removed immediately and were placed in cold buffer (.25 M sucrose, 5 mm Tris- HCl, ph 7.4, and 1 mm MgCl2). All subsequent procedures were carried out at 4. After the atria, great vessels, valves, and epicardial fat were removed, the ventricles (7-11 g) were weighed and placed in 4 volumes of fresh buffer. The tissue was then minced with scissors, blended in an "Osterizer" blender at medium speed for 2-3 sec, and finally homogenized in a Tekmar Tissuemizer (model SOT-182) at- 8/4 speed for 2 sec. The homogenates were filtered through four layers of gauze. The pellets from two preliminary 12 min centrifugations at 7 X g and 1, X g in a model RC2B Sorvall centrifuge using a SS-34 rotor were discarded. The supernatant was centrifuged a final time at 29, X g for 15 min. The pellets from the final centrifugation of the homogenate obtained from one dog heart were resuspended in 5 ml of "incubation buffer" (75 mm Tris- HCl, ph 7.4, 25 mm MgCl2) by homogenization in a Potter- Elvehjem homogenizer. (-) ['H]Alprenolol Binding Assay. One hundred microliter aliquots (containing.1. mg of protein) of membrane suspension and 6, cpm (-) ['H]alprenolol (about 15 nm) were incubated with shaking with and without agonists or antagonists for 1 min (unless otherwise specified) at 37. The total incubation volume was 15 Al. At the end of the incubations duplicate 5 /J aliquots were carefully pipetted into the upper 1/5 of horizontally positioned polyethylene 5 Al centrifuge tubes which contained 3 Ml of fresh "incubation buffer" in the lower portion of the tube. Care was taken to prevent mixing of the incubation aliquot with the fresh buffer until the tubes were centrifuged for 2 min in a Beckman Microfuge 152. The membranes were pelleted almost im- Tkme (mfutes) FIG. 1. Forward and reverse rates of specific (-) ['H]- alprenolol binding to cardiac membranes. At the arrow (d) propranolol was added to the incubation mixture containing membranes and (-) ['H]alprenolol. Incubation conditions were described in Materials and Methods. The forward and reverse time curves represent the means of seven and four experiments, respectively. mediately. The supernatants were aspirated with a no. 2 spinal needle attached to a vacuum line and the pellets were then rinsed with 3,1 of incubation buffer. After the fluid was aspirated, the tips of the centrifuge tubes were cut off into scintillation vials and the membranes were dissolved by shaking overnight in.5 ml of 1% sodium dodecyl sulfate, 1 mm EDTA. Fifteen milliliter of Triton X-1/toluene-based scintillation fluid were added and "bound" (-) ['H]aIprenolol was determined by counting in a Packard Liquid Scintillation Spectrometer at an efficiency of 32%. In each experiment (-) ['H]alprenolol "nonspecifically" bound and/or trapped in the membrane pellet was determined by measuring the amount of radioactivity in pellets obtained from incubations performed in the presence of 1 MM (4) propranolol. This value was subtracted from the total radioactivity bound to determine (-) ['H]alprenolol "specifically" bound. In contrast to the situation in purified frog erythrocyte membranes where "specific" binding is 8-9% of the total binding (19-21), in these cardiac membranes "specific binding" was generally about 15-3% of the total radioactivity associated with the pellets. In all figures and tables, "(-) [1H]- alprenolol bound" refers to "specific" binding as defined above. An example of typical experimental data from this system is shown in Table 1.t Adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC assays in cardiac membranes were performed exactly as previously described (22) save for the presence of.1 mm ethyleneglycol bis(5-aminoethyl ether)-n,n'-tetraacetate (EGTA), which has been reported to increase the enzyme activity (23). ["2PIcAMP formed was isolated by the method of Saloman et al. (24). Proteins were determined by the method of Lowry et al. (25). RESULTS Binding Kinetics. Specific (-) ['H]alprenolol binding and reversal of binding were extremely rapid, as shown in Fig. 1. Specific binding was 8% complete at the earliest time point measured (3 sec) and was at equilibrium at 2.5 min. The t Note Added in Proof. We have recently found that the use of glass fiber filters to separate bound and free (-) [3H] alprenolol increases the "specific binding" to greater than 7% of total binding in cardiac membranes.

3 1566 Biochemistry: Alexander et al. Proc. Nat. Acad. Sci. USA 72 (1976) c E"I, Q4 - - Q-- le 1 Ql - I,! KD5ll nm, -u 2 4 [(-) [3HlAlprenokI(M x I FiG. 2. Affinity and saturability of specific (-) [(H]alprenolol binding sites. Each point represents the mean of triplicate determinations from four separate experiments. reverse rate was equally rapid and the dissociation of specifically bound (-) [sh]alprenolol was essentially complete within see after addition of unlabeled (=) propranolol (1p&M). Saturability and Affinity of (-) ['H]Alprenolol Binding Sites. Specific (-) ['H]alprenolol binding was a saturable process as indicated by Fig. 2. In these experiments increasing amounts of (-) [3H]alprenolol were added to a fixed amount of membrane protein. At saturation there were.35 pmol of (-) ['H]alprenolol bound per mg of membrane protein (mean of four experiments). Half maximal saturation occurred at approximately 11 nm, which provides an estimate of the dissociation constant of (-) alprenolol for the binding sites. Affinity of (-) Alprenolol for Adenylate Cyclase Coupled,- Adrenergic Receptors. In order to correlate binding of radiolabeled (-) alprenolol with its biological effect, we also studied the effect of (-) alprenolol on isoproterenol-stimulated adenylate cyclase in cardiac membranes. In agreement with previously published findings in erythrocyte (19, 2) and cardiac membranes (26), alprenolol was found to be a potent competitive inhibitor. This is demonstrated by the data in Fig. 3. Increasing concentrations of the antagonist caused a progressive and parallel rightward shift in the isoproterenol dose- m at._ KD= 1(-) Alprenol] CR-1 KD=7nM * no blocker a Ai 1- M (-) Apreomlol o 17 M(-) Alprnolol o 1-6 (-)Alprnolol -6 -log [fadrenergic Agonist] (M) FIG. 4. Dose-response curves for inhibition of (-) ['HIalprenolol binding by the (+) and (-) stereoisomers of three fl-adrenergic agonists. Each point is the mean of two or three separate experiments, each determined in sextuplicate. response curves, indicating true competitive inhibition. From such data the KD of (-) alprenolol can easily be calculated from the equation KD = [antagonist]/(cr - 1) (27). CR (concentration ratio) refers to the ratio of equipotent concentrations of isoproterenol in the presence and absence of a given fixed concentration of (-) alprenolol. The mean value calculated from three experiments each with three different (-) alprenolol concentrations (1- M, 1-7 M, 1- M) was KD = 7 nm. The value is in very close agreement with the (KD = 11 nm) KD determined from direct binding studies (Fig. 2). Specicity of Binding. The (-) ['H]alprenolol binding sites exhibited strict stereospecificity in binding P-adrenergic o i 7 -R e In C, C 4) V 'D >% -log [-HlsoproterenojJ(M) 1 FIG. 3. Affinity of (-) alprenolol for adenylate-cyclasecoupled jp-adrenergic receptors. Adenylate cyclase assays were performed as described in Materials and Methods by incubating cardiac membranes with the indicated concentrations of drugs for 1 min at 37. Each value shown is the mean of duplicate determinations from three separate experiments. Basal adenylate cyclase activity was 62.5 pmol/mg of protein per min. 2 _-(-)Pmnl C+)Prapranois -9 a log fl-adrenergic Antagonist] (M) FiG. 5. Dose-response curves for inhibition of (-) ['H]- alprenolol binding by the (+) and (-) stereoisomers of the ft-adrenergic antagonist propranolol. Each point is the mean of four separate experiments, each determined in sextuplicate.

4 Proc. Nat. Acad. Sci. USA 72 (1975) TABLE 2. Effects of adrenergic agonists and antagonists, catecholamine metabolites and precursors, and pyrocatechol on (-) ['H]alprenolol binding to cardiac membranes Half-maximal inhibition of (-) ['H]alprenolol binding, Compound (AM) (-) Propranolol.12 (-) Isoproterenol.5 (-)Epinephrine 2. (-) Norepinephrine 1. (+) Propranolol 12. (4 ) Phenylephrine 12. (+) Isoproterenol 32. (+) Epinephrine 5. (+) Norepinephrine * (d) 3,Dihydroxymandelic acid NI, 16 I&M (A ) Dihydroxyphenylalanine (dopa) NI, 16 ym (A ) Normetanephrine NI, 16 AM Pyrocatechol NI, 16 ;M Phentolamine NI, 16juM * Indicates that even the highest concentration tested displaced less than 5% of specifically bound (-) ['H]alprenolol. NI indicates no inhibition of binding. agonists and antagonists (Figs. 4 and 5). The order of potency of agonists in inhibiting (-) ['H]alprenolol binding was (-) isoproterenol > (-) epinephrine > (-) norepinephrine > (+) isoproterenol > (+) epinephrine > (+) norepinephrine (Fig. 4). Similarly, the fl-adrenergic antagonist propranolol competed for the (-) ['HJalprenolol binding sites in a potent and stereospecific manner (Fig. 5). Thus, (-) propranolol is bound with much greater affinity than is (+) propranolol. For both agonists and antagonists the (-) isomers are at least two orders of magnitude more potent in inhibiting binding than are the corresponding (+) isomers. The inhibition of (-) ['H]alprenolol binding by all drugs tested is summarized in Table 2. Where possible the concentration of drug that inhibited binding by 5% was calculated. This concentration provided an estimate of the apparent dissociation constant of the drug for the receptor and is inversely related to its affinity. Of the drugs tested, the binding sites have the greatest affinity for (-) propranolol and progressively decreasing affinity for the (-) isomers of the #-adrenergic agonists isoproterenol, epinephrine, and norepinephrine. The physiologically inactive catechol-containing compounds pyrocatechol, (i) dihydroxymandelic acid, and (i) dihydroxyphenylalanine (dopa), as well as the metabolite (=) normetanephrine, and the a-adrenergic antagonist phentolamine, all showed no affinity for the binding sites at the concentration tested (16,uM) (Table 2). DISCUSSION The data presented here demonstrate in a cardiac membrane preparation the presence of (-) ['HJalprenolol binding sites which have the characteristics of fl-adrenergic receptor binding sites. Binding is rapid, reversible, saturable, and stereospecific and exhibits high affinity for fl-adrenergic agents. The rapid rate of binding of (-) ['H]alprenolol to cardiac membranes is consistent with the rapid activation of adenylate cyclase by,-adrenergic agents in cardiac membranes (14, 15). TABLE 3. Cardiac g3-adrenergic Receptors 1567 Concentrations of fl-adrenergic agonists causing 6%1 displacement of (-) ['HJalprenolol binding and 6% maximal adenylate cyclase activation in cardiac membrane preparations Half-maximal stimulation Half-maximal of adenylate inhibition cyclase of (-) ['H]- (23, 28, 29) alprenolol (AM) binding (MM) (-) Isoproterenol (-) Epinephrine (-) Norepinephrine The adenylate cyclase values are taken reported studies. from previously Binding was 8% complete within 3 sec and was maximal by 2.5 min, a time course much faster than the previously reported rate of ['HJnorepinephrine binding to cardiac membranes (3, 16). The reversible nature of the binding is indicated by the extremely rapid dissociation rate of the (-) ['H]- alprenolol from its binding sites (til2 < 15 sec). The specificity of the (-) ['Hjalprenolol binding sites is virtually identical to the specificity of fl-adrenergic physiological responses. A comparison of fl-adrenergic agonist binding affinities determined here with previously reported concentrations causing half-maximal activation of cardiac membrane adenylate cyclase (Table 3) reveals a remarkable similarity (23, 28, 29). Not only is the order of potency for activation of adenylate cyclase and for inhibition of binding the same, but the absolute values of the dissociation constants computed from binding data are in excellent agreement with the previously reported dissociation constants derived from adenylate cyclase data. The KD of (-) alprenolol for the receptors determined by direct binding studies and by competitive inhibition of isoproterenol-stimulated adenylate cyclase were virtually identical. Similarly the fl-adrenergic antagonist (-) propranolol demonstrated very high affinity for the binding sites (KD = 12 nm), which is in agreement with the reported potency of propranolol as an antagonist of isoproterenol-stimulated adenylate cyclase in cardiac membranes (KD = 3-5 nm) (14, 23, 26). The high affinity of propranolol for the binding sites reported here is in marked contrast to the previously reported low affinity of propranolol for (-) ['Hlnorepinephrine binding sites in cardiac membranes (3, 4). The conclusion that (-) ['H]alprenolol binding specificity and the well-known jb-adrenergic specificity are identical was strengthened further by the observation that catechol compounds that are devoid of fl-adrenergic effects do not interact with these (-) ['H]alprenolol binding sites. The a-blocking agent phentolamine also did not compete for the binding sites. (i) Phenylephrine, a predominantly a-adrenergic agent, caused weak inhibition of (-) ['H ]alprenolol binding at high concentrations (KD = 1 MM), which is consistent with the weak j-adrenergic positive inotropic effects of phenylephrine (3). The remarkable stereospecificity observed in binding further suggests that these (-) ['H]alprenolol binding sites are identical with the physiological cardiac #-adrenergic receptors. For the adrenergic agonists and antagonists tested the (-)

5 1568 Biochemistry: Alexander et al. isomers were from 25- to 1-fold more potent as inhibitors of binding than were the corresponding (+) isomers. Previous attempts to identify directly P-adrenergic receptors in the heart with ['H]norepinephrine (3-5) identified sites that have several inappropriate characteristics as noted above. Attempts to use (-) [3H ]propranolol in binding studies with cardiac membranes were complicated by the presence of a large number of low affinity, nonstereospecific sites which obscured identification of any high affinity j3-adrenergic receptor binding sites (31, 32). The present data report binding sites in a mammalian tissue that appear to demonstrate the kinetics, specificity, affinity, and stereospecificity that are expected of physiological P-adrenergic receptors. (-) [8H]Alprenolol was chosen for these studies of cardiac f3-adrenergic receptors for several reasons: (1) the tritiumlabeled compound is biologically active as a potent 8-adrenergic antagonist with high affinity for the,8-receptors (2); (2) the compound lacks a catechol moiety and hence does not bind to catechol-specific binding sites in the membranes; (3) (-) [3H]alprenolol recently has been used in our laboratory to demonstrate frog erythrocyte binding sites which have the characteristics of physiological fl-adrenergic receptors (19-21). The heart is a particularly relevant organ for study of,- adrenergic binding sites since there is a large body of physiological and biochemical data available characterizing interaction of a variety of adrenergic agents with intact and subcellular cardiac preparations. Future studies will compare the binding affinities of a wide spectrum of adrenergic agents with the potencies of their effects on cardiac adenylate cyclase. In addition it seems likely that the use of purified cardiac membrane preparations may decrease the amount of nonspecific binding and thus facilitate the study of the cardiac j3- receptors. The use of (-) [3H ]alprenolol to identify j3-adrenergic receptors will also make possible the study of alterations in number and/or affinity of cardiac a-receptors in states of possible altered sensitivity to catecholarnines, such as congestive heart failure (33), hyperthyroidism (34), and denervation (35). This work was supported by National Institutes of Health Grant HL and by a grant-in-aid from The American Heart Association with funds contributed in part by The North Carolina Heart Association. L.T.W. is a student in the Medical Scientist Training Program supported by National Institutes of Health Grant ST 5-6MO1678. R.J.L. is an Established Investigator of the American Heart Association. 1. Roth, J. (1973) Metabolism 22, Hall, Z. W. (1972) Annu. Rev. Biochem. 41, Lefkowitz, R. J. & Haber, E. (1971) Proc. Nat. Acad. Sci. USA 68, Lefkowitz, R. J., Sharp, G. & Haber, E. (1973) J. Biol. Chem. 248, Proc. Nat. Acad- Sci. USA 72 (1975) 5. Lefkowitz, R. J., Haber, E. & O'Hara, D, (1972) Proc. Nat. Acad. Sci. USA 69, Marinetti, G. V., Ray, T. K. & Tomasi, V. (1969) in Biochem. Biophys. Res. Commun. 36, Tomasi, V., Koretz, S., Ray, T. K., Dunnick, J. & Marinetti, G. V. (197) Biochim. Biophys. Acta 211, Schramm, M., Feinstein, H., Naim, E,, Long, M. & Lasser, M. (1972) Proc. Nat. Acad. Sci. USA 69, Bilezikian, J. P. & Aurbach, G. D. (1973) J. Biol. Chem. 248, Bilezikian, J. P. & Aurbach, G. D. (1973) J. Biol. Chem. 248, Lefkowitz, R. J. (1974) Biochem. Pharmacol. 23, Cuatrecasas, P., Tell, G. P. E., Sica, V., Parikh, I. & Chang, K. (1974) Nature 247, Innes, I. R. & Nickerson, M. (197) in The Pharmacological Basis of Therapeutics, eds. Goodman, L. S. & Gilman, A. (The Macmillan Co., New York), p Birnbaumer, L., Pohl, S. L. & Kaumann, A. J. (1974) in Advances in Cyclic Nucleotide Research, eds. Greengard, P. & Robison, G. A. (Raven Press, New York), Vol. 4, pp Bar, H. (1974) Mol. Pharmacol. 1, Maguire, M. E., Goldman, P. H. & Gilman, A. G. (1974) Mol. Pharmacol. 1, Levitzki, A., Atlas, D. & Steer, M. L. (1974) Proc. Nat. Acad. Sci. USA 71, Aurbach, G. D., Fedak, S. A., Woodard, C. J. Palmer, J. S., Hauser, D. & Troxler, F. (1974) Science 186, Lefkowitz, R. J., Mukherjee, C., Coverstone, M. & Caron, M. G. (1974) Biochem. Biophys. Res, Commun. 6, 73-79, 2. Mukherjee, C., Caron, M. G., Coverstone, M. & Lefkowitz, R. J. (1975) J. Biol. Chem. 25, in press. 21. Lefkowitz, R. J. (1975) in Methods in Receptor Research, ed. Blecher, M. (M. Dekker, New York), in press. 22. Lefkowitz, R. J. (1974) J. Biol. Chem. 249, Mayer, S. E. (1972) J. Pharmacol. Exp. Therap. 181, Saloman, Y., Londos, C. & Rodbell, M. (1974) Anal. Biochem. 58, Lowry,. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chemn. 193, Kaumann, A. J. & Birnbaumer, L. (1974) J. Biol. Chem. 249, Furchgott, R. F. (1967) Ann. N. Y. Acad. Sci. 139, Murad, F., Chi, Y. M., Rall, T. W. & Sutherland, E. W. (1962) J. Biol. Chem. 237, Lefkowitz, R. J. (1975) Biochem. Pharmacol. 24, Benfey, B. G. & Carolin, T. (1971) Can. J. Physiol. Pharmacol. 49, Potter, L. T. (1967) J. Pharmacol. Exp. Therap. 155, Vatner, D. E. & Lefkowitz, R. J. (1974) Mol. Pharmacol. 1, Sobel, B. E., Henry, P. D., Robison, A., Bloor, C. & Ross, J. (1969) Circ. Res. 24, Wildenthal, K. (1974) J. Pharmacol. Exp. Therap. 19, Dempsey, P. J. & Cooper, T. (1968) Amer. J. Physiol. 215,