Proc. Nat. Acad. Sci. USAVol. 72, No. 4, pp. 1564-1568, 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 27710

Communicated by James B. Wyngaarden, February 6, 1975

ABSTRACT (-) PHJAlprenolol, a potent j3-adrenergicantagonist, was used to identify binding sites in a fractionof canine myocardium. Beta adrenergic agonists andantagonists compete for these binding sites in a mannerwhich directly parallels their known affinity for the cardiac_-adrenergic receptor. Thus, binding was highly stereo-

specific, with the (-) isomers of jP-aldrenergic agonists orantagonists being at least two orders of magnitude morepotent than were the (+) isomers in competing for thesesites. The order of potency for inhibition of binding by,6-adrenergic agonists was (-) isoproterenol > (-)epinephrine > (-) norepinephrine. The dissociation con-stant (KD) of (-) alprenolol for the P-adrenergic receptorswas 7-11 nM as determined independently by direct bind-ing studies or by inhibition of isoproterenol-stimulatedadenylate cyclase [ATP pyrophosphate-lyase (cyclizing),EC 4.6.1.1]. The P-adrenergic antagonist (-) propranololalso had high affinity for the binding sites (KD = 12 nM).The physiologically inactive catechol-containing com-pounds pyrocatechol and (i) dihydroxymandelic acid,as well as the metabolite (A) normetanephrine, and thea-adrenergic antagonist phentolamine did not competefor the binding sites at a concentration of 160 uM. Bind-ing was rapid (tl/, <30 sec) and was rapidly reversible (tl/,<15 sec). The binding sites were saturable and bound 0.35pmol of (-) [3Hjalprenolol per mg of membrane protein.These characteristics suggest that these binding sitesrepresent the cardiac ,-adrenergic receptors.

Binding to specific receptor sites is thought to be the initialevent leading to the modulation of cellular function by hor-mones. Recently, rapid progress has been made in the isolationand characterization of polypeptide hormone receptors (1)and nicotinic cholinergic receptors (2). In general, these re-ceptors have been studied by labeling them with high specificactivity radioactively labeled hormones or, in the case of thenicotinic cholinergic receptors, with a radioactively labeledsnake venom antagonist (1, 2).

These methods also have been applied in attempts toidentify the adrenergic ,-receptors. Tritium-labeled ,B-adrener-gic agonists such as norepinephrine (3-5), epinephrine (6-8),and isoproterenol (9, 10) have been used to identify bindingsites in various mammalian tissues and avian erythrocytes.

* "(-) [3H]Alprenolol" has been used throughout this manu-

script to identify the compound resulting from the catalyticreduction of (-) alprenolol with tritium. (-) Alprenolol con-

tains an unsaturated bond in the aliphatic chain on the 2 posi-tion of the aromatic ring. The compound, therefore, might beappropriately referred to as "(-) [3H]dihydroalprenolol." Thenature of the labeling process, however, is such that tritiumexchange might also take place, yielding (-) [3H]alprenolol.The labeled material used for these studies has biological activityand chromatographic properties identical to those of native(-) alpreidolol.

The binding characteristics of these sites have resembled, inseveral respects, those which might be expected of p-adrenergicreceptor binding sites. Several characteristics, however, havediffered from the expected properties of 3-adrenergic receptorson the basis of physiologic responses or of the activation ofadenylate cyclase which is coupled to ,-receptors (11, 12).First, these [3H]catecholamine binding sites do not exhibitstereospecificity. Physiologically, the (-) isomers of fl-adrener-gic antagonists or agonists are much more potent than are the(+) isomers (13). Second, the affinity of these binding sites forg-adrenergic antagonists such as propranolol is several ordersof magnitude lower than the affinity of the physiologic ,-receptors for these antagonists. Third, several catechol com-pounds that are devoid of physiological p-adrenergic effectsbind to the sites. Finally, whereas 3-adrenergic agonists orantagonists produce maximum physiologic effects within 1-2min (14, 15), these binding sites require many minutes toreach equilibrium binding of catecholamines (3, 16).Very recently, reports have appeared which describe bind-

ing sites in avian or amphibian erythrocytes that fulfill strictbinding criteria (affinity and stereospecificity) appropriate tothe physiologic ,B-adrenergic receptors (17-21). A commonapproach in all of these studies consisted of the labeling of thebinding sites with radioactive 3-adrenergic antagonists. Thepresent report describes the application of these techniques toa mammalian system. Thus, binding sites for (-) [8H]al-prenolol, a potent #-adrenergic antagonist, were identifiedin canine myocardium. The ability of stereo isomers of anumber of adrenergic agonists and antagonists to interactwith 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-adrenergicreceptors. The excellent correlation observed suggests that the(-) [3H]alprenolol binding sites are, in fact, equivalent to thecardiac g-adrenergic receptor binding sites.

MATERIALS AND METHODS

(-) [3H]Alprenolol (specific activity 17 Ci/mmol) was pre-pared by New England Nuclear by catalytic reduction of (-)alprenolol (Hassle) with tritium gas using paladium as thecatalyst (19, 20). The tritiated compound was stored inabsolute ethanol at -20°. In initial experiments labeled al-prenolol was purified daily as described previously (19, 20),although in later experiments the use of unpurified material(containing less than 5% contamination) gave identicalexperimental results. Other drugs used in this study were:

(-) and (+) propranolol hydrochloride (Ayerst); (+) iso-proterenol bitartrate, (+) epinephrine bitartrate, (+) nor-

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Cardiac fl-Adrenergic Receptors 1565

TABLE 1. Inhibition of (-) ['HJaiprenolol binding tocanine myocardial membranes by (-) propranolol

(-) [3H]Alprenolol bound Inhibi-(cpm/pellet) tion of

specificAddition Observed "Specific" binding

None 1745 4i 67 583 0(-) propranolol

1.6 X 10-9 M 1860 428 698 01.6 X 10-M 1413d 111 251 571.6 X 10-7 M 1233 4 41 71 881.6 X 10-M 1169446 0 1001.6 X 10-5 M 1214 4 86 0 1001.6 X 10-4M 1102 4 68 0 100

Values shown are mean 4 SEM of six determinations."Specific" binding is calculated by subtracting the cpm notdisplaced by high concentrations.of propranolol (>1 MM) fromthe observed cpm.

epinephrine bitartrate, (+) phenylephrine hydrochloride(Winthrop); (-) isoproterenol bitartrate, (-) epinephrinebitartrate, (-) norepinephrine bitartrate, (±) dihydroxy-mandelic acid, (d) normetanephrine, (=) dihydroxyphenyl-alanine (dopa) (Sigma); pyrocatechol (Nutritional Biochemi-cals); and phentolamine (Ciba).

Membrane Preparation. Mongrel dogs of either sex weighing40-50 pounds were used. The animals were anesthetized withintravenous pentobarbital (30 mg/kg) and the hearts wereremoved immediately and were placed in cold buffer (0.25 Msucrose, 5 mM Tris- HCl, pH 7.4, and 1 mM MgCl2). Allsubsequent procedures were carried out at 4°. After the atria,great vessels, valves, and epicardial fat were removed, theventricles (70-110 g) were weighed and placed in 4 volumes offresh buffer. The tissue was then minced with scissors, blendedin an "Osterizer" blender at medium speed for 20-30 sec,and finally homogenized in a Tekmar Tissuemizer (modelSOT-182) at- 8/4 speed for 20 sec. The homogenates werefiltered through four layers of gauze. The pellets from twopreliminary 12 min centrifugations at 700 X g and 10,000 X gin a model RC2B Sorvall centrifuge using a SS-34 rotor werediscarded. The supernatant was centrifuged a final time at29,000 X g for 15 min. The pellets from the final centrifugationof the homogenate obtained from one dog heart were resus-pended in 4-5 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 microliteraliquots (containing 0.4-1.0 mg of protein) of membrane sus-pension and 60,000 cpm (-) ['H]alprenolol (about 15 nM)were incubated with shaking with and without agonists orantagonists for 10 min (unless otherwise specified) at 370.The total incubation volume was 150 Al. At the end of theincubations duplicate 50 /J aliquots were carefully pipettedinto the upper 1/5 of horizontally positioned polyethylene 500Al centrifuge tubes which contained 300 Ml of fresh "incubationbuffer" in the lower portion of the tube. Care was taken toprevent mixing of the incubation aliquot with the fresh bufferuntil the tubes were centrifuged for 2 min in a BeckmanMicrofuge 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 containingmembranes and (-) ['H]alprenolol. Incubation conditions weredescribed in Materials and Methods. The forward and reversetime curves represent the means of seven and four experiments,respectively.

mediately. The supernatants were aspirated with a no. 20spinal needle attached to a vacuum line and the pellets werethen rinsed with 300 ,1 of incubation buffer. After the fluidwas aspirated, the tips of the centrifuge tubes were cut off intoscintillation vials and the membranes were dissolved byshaking overnight in 0.5 ml of 10% sodium dodecyl sulfate, 10mM EDTA. Fifteen milliliter of Triton X-100/toluene-basedscintillation fluid were added and "bound" (-) ['H]aIprenololwas determined by counting in a Packard Liquid ScintillationSpectrometer at an efficiency of 32%.

In each experiment (-) ['H]alprenolol "nonspecifically"bound and/or trapped in the membrane pellet was determinedby measuring the amount of radioactivity in pellets obtainedfrom incubations performed in the presence of 100 MM (4)propranolol. This value was subtracted from the total radio-activity bound to determine (-) ['H]alprenolol "specifically"bound. In contrast to the situation in purified frog erythrocytemembranes where "specific" binding is 80-90% of the totalbinding (19-21), in these cardiac membranes "specific bind-ing" was generally about 15-30% of the total radioactivityassociated with the pellets. In all figures and tables, "(-) [1H]-alprenolol bound" refers to "specific" binding as definedabove. An example of typical experimental data from thissystem is shown in Table 1.t

Adenylate cyclase [ATP pyrophosphate-lyase (cyclizing),EC 4.6.1.11 assays in cardiac membranes were performedexactly as previously described (22) save for the presence of0.1 mM ethyleneglycol bis(5-aminoethyl ether)-N,N'-tetra-acetate (EGTA), which has been reported to increase theenzyme activity (23). ["2PIcAMP formed was isolated by themethod of Saloman et al. (24).

Proteins were determined by the method of Lowry et al.(25).

RESULTS

Binding Kinetics. Specific (-) ['H]alprenolol binding andreversal of binding were extremely rapid, as shown in Fig. 1.Specific binding was 80% complete at the earliest time pointmeasured (30 sec) and was at equilibrium at 2.5 min. The

t Note Added in Proof. We have recently found that the use ofglass fiber filters to separate bound and free (-) [3H] alprenololincreases the "specific binding" to greater than 70% of total bind-ing in cardiac membranes.

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1566 Biochemistry: Alexander et al. Proc. Nat. Acad. Sci. USA 72 (1976)

c

E"I,

0

1

Q4 - -Q--

Ql -

°! KD5llnM,

le

I,

-6

a

[(-) [3HlAlprenokI(M x I

FiG. 2. Affinity and saturability of specific (-) [(H]al-prenolol binding sites. Each point represents the mean of tripli-cate determinations from four separate experiments.

reverse rate was equally rapid and the dissociation of specifi-cally bound (-) [sH]alprenolol was essentially completewithin 15-45 see after addition of unlabeled (=) propranolol(10p&M).

Saturability and Affinity of (-) ['H]Alprenolol BindingSites. Specific (-) ['H]alprenolol binding was a saturableprocess as indicated by Fig. 2. In these experiments increasingamounts of (-) [3H]alprenolol were added to a fixed amountof membrane protein. At saturation there were 0.35 pmol of(-) ['H]alprenolol bound per mg of membrane protein (meanof four experiments). Half maximal saturation occurred atapproximately 11 nM, which provides an estimate of the dis-sociation constant of (-) alprenolol for the binding sites.

Affinity of (-) Alprenolol for Adenylate Cyclase Coupled ,-Adrenergic Receptors. In order to correlate binding of radio-labeled (-) alprenolol with its biological effect, we also studiedthe effect of (-) alprenolol on isoproterenol-stimulated ade-nylate cyclase in cardiac membranes. In agreement with pre-viously published findings in erythrocyte (19, 20) and cardiacmembranes (26), alprenolol was found to be a potent com-petitive inhibitor. This is demonstrated by the data in Fig. 3.Increasing concentrations of the antagonist caused a pro-gressive and parallel rightward shift in the isoproterenol dose-

0

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4-

0

._

C0V

4-

In

C,4)

'D

>%

KD= 1(-) Alprenol]CR-1

KD=7nM

* no blockerAi 10- M (-) Apreomlolo 107 M(-) Alprnololo 10-6 (-)Alprnolol

-log [fAdrenergic Agonist] (M)

FIG. 4. Dose-response curves for inhibition of (-) ['HI-alprenolol binding by the (+) and (-) stereoisomers of threefl-adrenergic agonists. Each point is the mean of two or threeseparate experiments, each determined in sextuplicate.

response curves, indicating true competitive inhibition. Fromsuch data the KD of (-) alprenolol can easily be calculatedfrom the equation KD = [antagonist]/(CR - 1) (27). CR(concentration ratio) refers to the ratio of equipotent con-centrations of isoproterenol in the presence and absence of agiven fixed concentration of (-) alprenolol. The mean valuecalculated from three experiments each with three different(-) alprenolol concentrations (10- M, 10-7 M, 10- M)was KD = 7 nM. The value is in very close agreement with theKD determined from direct binding studies (KD = 11 nM)(Fig. 2).Specicity of Binding. The (-) ['H]alprenolol binding sites

exhibited strict stereospecificity in binding P-adrenergic

8o

i 70

-R

e 60

3 50

040

20

-log [-HlsoproterenojJ(M) 10

FIG. 3. Affinity of (-) alprenolol for adenylate-cyclase-coupled jp-adrenergic receptors. Adenylate cyclase assays wereperformed as described in Materials and Methods by incubatingcardiac membranes with the indicated concentrations of drugsfor 10 min at 370. Each value shown is the mean of duplicatedeterminations from three separate experiments. Basal adenylatecyclase activity was 62.5 pmol/mg of protein per min.

_-0(-)PmnlC+)Prapranois

-9 a 7 5 4 3-log fl-Adrenergic Antagonist] (M)

FiG. 5. Dose-response curves for inhibition of (-) ['H]-alprenolol binding by the (+) and (-) stereoisomers of theft-adrenergic antagonist propranolol. Each point is the mean offour separate experiments, each determined in sextuplicate.

-u 2 4

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Cardiac g3-Adrenergic Receptors 1567

TABLE 2. Effects of adrenergic agonists and antagonists,catecholamine metabolites and precursors, and pyrocatechol

on (-) ['H]alprenolol binding to cardiac membranes

Half-maximalinhibition of

(-) ['H]alprenololbinding,

Compound (AM)(-) Propranolol 0.012(-) Isoproterenol 0.5(-)Epinephrine 2.0(-) Norepinephrine 10.0(+) Propranolol 12.0(4 ) Phenylephrine 120.0(+) Isoproterenol 320.0(+) Epinephrine 500.0(+) Norepinephrine *(d) 3,4-Dihydroxymandelic acid NI, 160 I&M(A ) Dihydroxyphenylalanine (dopa) NI, 160 yM(A ) Normetanephrine NI, 160 AM

Pyrocatechol NI, 160 ;MPhentolamine NI, 160juM

* Indicates that even the highest concentration tested dis-placed less than 50% of specifically bound (-) ['H]alprenolol.NI indicates no inhibition of binding.

agonists and antagonists (Figs. 4 and 5). The order of potencyof agonists in inhibiting (-) ['H]alprenolol binding was (-)isoproterenol > (-) epinephrine > (-) norepinephrine >(+) isoproterenol > (+) epinephrine > (+) norepinephrine(Fig. 4). Similarly, the fl-adrenergic antagonist propranololcompeted for the (-) ['HJalprenolol binding sites in a potentand stereospecific manner (Fig. 5). Thus, (-) propranolol isbound with much greater affinity than is (+) propranolol.For both agonists and antagonists the (-) isomers are atleast two orders of magnitude more potent in inhibiting bind-ing than are the corresponding (+) isomers.The inhibition of (-) ['H]alprenolol binding by all drugs

tested is summarized in Table 2. Where possible the concentra-tion of drug that inhibited binding by 50% was calculated.This concentration provided an estimate of the apparent dis-sociation constant of the drug for the receptor and is inverselyrelated to its affinity. Of the drugs tested, the binding siteshave the greatest affinity for (-) propranolol and progres-sively decreasing affinity for the (-) isomers of the #-adrener-gic agonists isoproterenol, epinephrine, and norepinephrine.The physiologically inactive catechol-containing compounds

pyrocatechol, (i) dihydroxymandelic acid, and (i) dihy-droxyphenylalanine (dopa), as well as the metabolite (=)normetanephrine, and the a-adrenergic antagonist phentol-amine, all showed no affinity for the binding sites at the con-centration tested (160,uM) (Table 2).

DISCUSSIONThe data presented here demonstrate in a cardiac membranepreparation the presence of (-) ['HJalprenolol binding siteswhich have the characteristics of fl-adrenergic receptor bindingsites. Binding is rapid, reversible, saturable, and stereospecificand 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 adenylatecyclase by ,-adrenergic agents in cardiac membranes (14, 15).

TABLE 3. Concentrations of fl-adrenergic agonists causing60%10 displacement of (-) ['HJalprenolol binding and60% maximal adenylate cyclase activation in cardiac

membrane preparations

Half-maximalstimulation Half-maximalof adenylate inhibition

cyclase of (-) ['H]-(23, 28, 29) alprenolol

(AM) binding (MM)

(-) Isoproterenol 0.1-0.9 0.5(-) Epinephrine 1-8 2(-) Norepinephrine 3-8 10

The adenylate cyclase values are takenreported studies.

from previously

Binding was 80% complete within 30 sec and was maximal by2.5 min, a time course much faster than the previously re-ported rate of ['HJnorepinephrine binding to cardiac mem-branes (3, 16). The reversible nature of the binding is indi-cated 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 physio-logical responses. A comparison of fl-adrenergic agonist bind-ing affinities determined here with previously reported con-centrations causing half-maximal activation of cardiac mem-brane adenylate cyclase (Table 3) reveals a remarkable simi-larity (23, 28, 29). Not only is the order of potency for activa-tion of adenylate cyclase and for inhibition of binding thesame, but the absolute values of the dissociation constantscomputed from binding data are in excellent agreement withthe previously reported dissociation constants derived fromadenylate cyclase data.The KD of (-) alprenolol for the receptors determined by

direct binding studies and by competitive inhibition of iso-proterenol-stimulated adenylate cyclase were virtually identi-cal. Similarly the fl-adrenergic antagonist (-) propranololdemonstrated very high affinity for the binding sites (KD = 12nM), which is in agreement with the reported potency ofpropranolol as an antagonist of isoproterenol-stimulatedadenylate cyclase in cardiac membranes (KD = 3-50 nM)(14, 23, 26). The high affinity of propranolol for the bindingsites reported here is in marked contrast to the previouslyreported low affinity of propranolol for (-) ['Hlnorepi-nephrine binding sites in cardiac membranes (3, 4).The conclusion that (-) ['H]alprenolol binding specificity

and the well-known jB-adrenergic specificity are identical wasstrengthened further by the observation that catechol com-pounds that are devoid of fl-adrenergic effects do not interactwith these (-) ['H]alprenolol binding sites. The a-blockingagent phentolamine also did not compete for the bindingsites. (i) Phenylephrine, a predominantly a-adrenergicagent, caused weak inhibition of (-) ['H ]alprenolol binding athigh concentrations (KD = 100 MM), which is consistent withthe weak j-adrenergic positive inotropic effects of phenyl-ephrine (30).The remarkable stereospecificity observed in binding fur-

ther suggests that these (-) ['H]alprenolol binding sites are

identical with the physiological cardiac #-adrenergic receptors.For the adrenergic agonists and antagonists tested the (-)

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1568 Biochemistry: Alexander et al.

isomers were from 250- to 1000-fold more potent as inhibitorsof binding than were the corresponding (+) isomers.

Previous attempts to identify directly P-adrenergic re-ceptors in the heart with ['H]norepinephrine (3-5) identifiedsites that have several inappropriate characteristics as notedabove. Attempts to use (-) [3H ]propranolol in binding studieswith cardiac membranes were complicated by the presence ofa large number of low affinity, nonstereospecific sites whichobscured identification of any high affinity j3-adrenergic re-ceptor binding sites (31, 32). The present data report bindingsites in a mammalian tissue that appear to demonstrate thekinetics, specificity, affinity, and stereospecificity that areexpected of physiological P-adrenergic receptors.

(-) [8H]Alprenolol was chosen for these studies of cardiacf3-adrenergic receptors for several reasons: (1) the tritium-labeled compound is biologically active as a potent 8-adren-ergic antagonist with high affinity for the ,8-receptors (20);(2) the compound lacks a catechol moiety and hence does notbind to catechol-specific binding sites in the membranes; (3)(-) [3H]alprenolol recently has been used in our laboratory todemonstrate frog erythrocyte binding sites which have thecharacteristics 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 physio-logical and biochemical data available characterizing inter-action of a variety of adrenergic agents with intact and sub-cellular cardiac preparations. Future studies will compare thebinding affinities of a wide spectrum of adrenergic agents withthe potencies of their effects on cardiac adenylate cyclase.In addition it seems likely that the use of purified cardiacmembrane preparations may decrease the amount of non-specific binding and thus facilitate the study of the cardiac j3-receptors. The use of (-) [3H ]alprenolol to identify j3-adrener-gic receptors will also make possible the study of alterations innumber and/or affinity of cardiac a-receptors in states of pos-sible altered sensitivity to catecholarnines, such as congestiveheart failure (33), hyperthyroidism (34), and denervation(35).This work was supported by National Institutes of Health

Grant HL-16037-01 and by a grant-in-aid from The AmericanHeart Association with funds contributed in part by The NorthCarolina Heart Association. L.T.W. is a student in the MedicalScientist Training Program supported by National Institutes ofHealth Grant ST 05-6MO1678. R.J.L. is an Established Inves-tigator of the American Heart Association.

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