Naunyn-Schmiedeberg's Arch Pharmacol (1986) 332: 406- 409

Short communication

The ,fll-adrenoceptor antagonist CGP 20712 A unmasks p2-adrenoceptors activated by (-)-adrenaline in rat sinoatrial node Alberto J. Kaumann Department of Clinical Physiology, University of Dfisseldorf, D-4000 Diisseldorf, Federal Republic of Germany

Naunyn-Schmiedeberg's Archives of Pharmacology �9 Springer-Verlag 1986

Summary. The role of sinoatrial/31- and/32-adrenoceptors mediating positive chronotropic effects of (-)-adrenaline and (-)-noradrenaline was investigated in rat right atria. Concentration effect curves for (-)-adrenaline, but not for (-)-noradrenaline, became biphasic in the presence of the /31-adrenoceptor antagonist CGP 20712 A. The curves for (-)-adrenaline in the presence of 300 nmol/1 CGP 20712 A (equivalent to 1,000 times its KB, KB = 0.3 nmol/1 for/31- adrenoceptors) comprise a high-sensitivity component that saturates at 1/4 of maximum effect, and a low sensitivity component. The high-sensitivity component is blocked by the/32-adrenoceptor-selective antagonist ICI 118,551. These results are consistent with an involvement in the rat of both /31-adrenoceptors (to a major extent) and/3z-adrenoceptors [only at high (-)-adrenaline concentrations] in the positive chronotropic effects of (-)-adrenaline. (-)-Noradrenaline appears to activate mostly rat sinoatrial/31-adrenoceptors.

Key words: Rat sinoatrial node - (-)-adrenaline and ( - ) - noradrenaline - CGP 20712 A and ICI 118,551 - 131- and /3z-adrenoceptors - Positive chronotropic effects

Introduction

Binding inhibition experiments suggest that 2/3 of the /3- adrenoceptors of rat right atria are/31 and 1/3/32 (Juberg et al, 1985). From experiments on beating right atria Bryan et al. (1981) and Juberg et al. (1985) concluded that /3z- adrenoceptors are not involved in the generation of positive chronotropic effects of agonists in the rat. However, O'Donnell and Wanstall (1985) recently reported some blockade of the positive chronotropic effects of procaterol by/3z-selective ICI 118,551, suggesting some involvement of /32-adrenoceptors in rat fight atria. In view of these con- flicting results the question of a contribution of/32-adreno- ceptors to the positive chronotropic effects of catechol- amines is examined in the present report. The /~l-adreno- ceptor antagonist CGP 20712 A was used to saturate/31- adrenoceptors, thereby allowing the possible manifestation of/3z-adrenoceptors in the positive chronotropic effects of catecholamines. The selectivity for/31- over/321-adrenocep- tors of CGP 20712 A is approximately 10,000-fold (Dooley and Bittinger 1984; Lemoine et al. 1985 a).

Send offprint requests to A. J. Kaumann, ICI Pharmaceuticals Division, Mereside, Alderley Park, Macclesfield, Cheshire SKI0 4TG, UK

H2 H2 H20

u--N/ k=/-- I I H2~ /L ~H 3 OHH ~ "OH

~C----O NH 2

Fig. 1. Chemical structure of CGP 20712 A

lsolated atria. Male Wistar rats (250- 300 g) were pretreated i.p. with 10 mg/kg reserpine 24 h before the experiment. Spontaneously beating right atria were dissected and mounted in pairs in an apparatus with a 50 ml bath (Blinks 1965). The length of the atria was set to develop just enough force necessary to count the contractions on a polygraph. Contractions were measured with Swema (SG 4 - 4 5 ) trans- ducers. The bathing solution contained (mmol/1): Na § 140, K + 5, Ca 2+ 2.25, Mg 2+ 0.5, C1- 98.5, SOl- 0.5, HCO~ 34, HPOZ-1, fumarate 5, pyruvate 5, L-glutamate 5, glucose 10, disodium edetate (EDTA) 0.04, equilibrated with 95% Oz and 5% COz (water was deionized and redistilled). The tissues were pretreated once with 6 gmol/1 phenoxybenz- amine for 2 h. This treatment causes irreversible blockade of both tissue uptake ofcatecholamines and e-adrenoceptors (Kaumann 1972).

Up to 3 cumulative concentration-effect curves for (-)-noradrenaline and (-)-adrenaline were determined, the first in the absence, the subsequent curves in the presence of antagonist. Successive concentration-effect curves for a catecholamine are reproducible (Kaumann et al. 1977). The incubation times with CGP 20712 A were 3 h at 1 nmol/1 and 2 h at 100 and 300 nmol/l, after which concentration- effect curves for a catecholamine were determined in the presence of CGP 20712 A. ICI 118,551 was added to the organ bath 2 h before the administration of catecholamines. At these incubation times equilibrium blockade was ob- served.

Drugs. 2-Hydroxy-5-{2-[hydroxy-3-(4-[(1-methyl-4-trifluo- romethyl) 1H-imidazol-2-yl-]phenoxy)propyl] aminoetho- xyl}-benzamide (CGP 20712 A, prepared as a methane sul- fonate, Fig. 1) and reserpine phosphate (CIBA-Geigy, Basel, Switzerland), phenoxybenzamine hydrochloride (Smith, Kline and French, Philadelphia, PA, USA), erythro-(_+)-l- (7-methylindan-4-yloxy)3-isopropylaminobutan-2-ol hy- drochloride (ICI 118,551) (ICI, Macclesfield, UK), ( - ) - noradrenaline bitartrate monohydrate (Sterling-Winthrop, Rensselaer, NY, USA), (-)-adrenaline bitartrate (Serva,

407

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Fig. 2. Antagonism by 300 nmol/1 CGP 20712 A (CGP) of the positive chronotropic effects of catecholamines in the absence (upper panels) and presence of 50 nmol/1 ICI 118,551 (lower panels). Two successive concentration-effect curves for a catecholamine were determined, the first in the absence (O) and the second in the presence of antagonist (@) or combination of antagonists (A). Each panel represents the results from 6 atria. Symbols are 2 + SEM; SEM not shown when smaller than size of symbol

Heidelberg, FRG). The stock solutions of (-)-adrenaline and (-)-noradrenaline (0.1 mol/1) were acidified with 0.1 mol/l HC1 to a pH of 4. For each experiment appropriate dilutions of the catecholamines were used in deionized re- distilled water containing 40 ~tmol/1 EDTA.

Results

(-)-Noradrenaline was slightly more potent than ( - ) - ad - renaline as a chronotropic stimulant (Fig. 2, Table 1). 300 nmol/1 CGP 20712 A caused surmountable antagonism of the effects of both (-)-noradrenaline and (-)-adrenaline (Fig. 2). The concentration-effect curves for ( - ) -noradren- aline were nearly parallel, but the curve for (-)-adrenaline was flatter in the presence of CGP 20712 A (Fig. 2) than in its absence. Similar results were obtained in the presence of 100 nmol/1 CGP 20712 A in 4 atria with (-)-noradrenaline and (-)-adrenaline (n -- 4 each; not shown). The curve for (-)-adrenaline was biphasic in the presence of CGP 20712 A; 1/4 of the maximum effect of (-)-adrenaline corre- sponds to a saturable high-sensitivity component with a - l o g ECs0 of 6.5 (Fig. 2 upper right panel). 50 nmol/1 ICI 118,551 abolished the high-sensitivity component of the effect of (-)-adrenaline in the presence of 300 nmol/1 CGP 20712 A (Fig. 2 lower right panel).

50 nmol/1 ICI 188,551 did not change the sensitivity of the atria to (-)-noradrenaline and (-)-adrenaline (Table 1). The shape of the concentration-effect curves of ( - ) -norad- renaline and (-)-adrenaline was not changed by 50 nmol/1 ICI t18,551 (not shown). To estimate the affinity of CGP

20712 A for/31-adrenoceptors, three successive concentra- tion-effect curves were determined for each of the catechol- amines in the presence of 50 nmol/1 ICI 118,551 (to avoid interference from/3z-adrenoceptors); the second and third curves were obtained in the presence of 1 nmol/1 and 100 nmol/1 CGP 20712 A, respectively. CGP 20712 A caused surmountable antagonism and shifted the curves of both catecholamines in a parallel way (not shown), as expected from simple competitive antagonism. Also, as expected, the Schild plots (Arunlakshana and Schild 1959) of CGP 20712 A had slopes of unity, regardless of the catecholamine (Table 1).

The degree of antagonism by CGP 20712 A to the effects of (-)-noradrenaline and (-)-adrenaline was esentially the same as revealed by the virtually identical values of affinity of CGP 20712 A (Table 1).

Discussion

The equilibrium dissociation constant of 0.3 nmol/1 for the CGP 20712 A-/3-adrenoceptor complex of rat sinoatrial node agrees with similar estimates of affinity of CGP 20712 A for/31-adrenoceptors of calf trachea, a system in which the affinity of the antagonist is 25,000 times lower for /32- than for/31-adrenoceptors (Lemoine et al. 1985 a). Thus, in a system in which both/31- and/32-adrenoceptors co-exist, CGP 20712 A can virtually eliminate the /31 population, thereby allowing the remaining/32 population to manifest its function. Such a situation was observed with (-)-adrenaline which elicited a small but conspicuous positive chronotropic

408

Table 1. Characteristics of/~l-adrenoceptors of rat sinoatrial node

- log molar concentration

ECs0 ECso (IC/) b KB ~ of CGP Slope of Schild plot for CGP d

(-)-Noradrenaline 8.32 • 0.06 (24)" 8.17 + 0.05 (4) 9.44 • 0.04 0.97 • 0.02 (4) (-)-Adrenaline 7.63 ___ 0.06 (20) 7.71 • 0.09 (5) 9.47 • 0.11 1.01 • 0.07 (5)

Number of atria in parentheses b 50 nmol/1 ICI 118,551 was in the bathing solution throughout the experiment c Equilibrium dissociation constant of CGP 20712 A determined in the presence of 50 nmol/1 ICI 118,551. KB was calculated from

catecholamine concentration-ratios (CR), taken between EC5o-values, using KB = [B]/(CR-I) d 1 nmol/1 or 100 nmol/1 CGP 20712 A was present in addition to 50 nmol/1 ICI 118,551 Data respresent 2 _+ SEM

effect in the presence of 300 nmol/1 CGP 20712 A which causes 99.9% occupancy of fll-adrenoceptors but only 4% occupancy of flz-adrenoceptors. (Receptor occupancy was calculated from [B]/([B] + KB), where [B] is the concentration of the antagonist and KB the equilibrium dissociation constant). The small effect of ( - ) -adrenal ine observed in the lower part of its concentration-effect curve in the presence of 300 nmol/1 CGP 20712 A was not seen when the atria were exposed to 50 nmol/1 ICI 118,551, a concentration causing 99.5% occupancy of flz-adrenoceptors. (The equilibrium dissociation constant of ICI 118,551 for /~2- and //1- adrenoceptors is 0.25 nmol/1 and 70 nmol/1, respectively, Lemoine et al. 1985b.) This evidence is consistent with a fl~- nature of the effects of ( - )-adrenal ine observed in the lower part of its concentration-effect curve in the presence of CGP 20712 A. The/?a-component is half maximal at 300 nmol/1 (-)-adrenaline, a concentration similar to the equilibrium dissociation constant of ( - ) -adrenal ine for /?2-adreno- ceptors (Lemoine et al. 1985b).

The/~2-component comprises 1/4 of the maximum effects of (-)-adrenaline, while the remainder corresponds to a /?~-component because the middle and upper part of the concentration-effect curve of ( - )-adrenal ine is shifted by 300 nmol/1 CGP 20712 as expected from its affinity for/?l- adrenoceptors. These results are in line with 1/3 /?2- adrenoceptors and 2/3 /~a-adrenoceptors encountered in binding experiments in rat right atria by Juberg et al. (1985).

Based on the observation that ICI 118,551 antagonized with low potency the positive chronotropic effects of the/?z- selective agonist procaterol, Juberg et al. (1985) concluded that/~2-adrenoceptors may not exist in rat sinoatrial node but instead have some metabolic function in right atrium. O'Donnell and Wanstall (1985) performed experiments in rat atria similar to those of Juberg et al. (1985), but using more procaterol concentrations per log unit and found that the slope of the concentration-effect curve as flatter than that encountered by Juberg et al. (1985). Furthermore, O'Donnell and Wanstall (1985) were able to antagonize with ICI 118,551 the high-sensitivity component of the effects of procaterol, a result consistent with the existence and functional involvement of rat sinoatrial/?2-adrenoceptors. Remarkably, the flat concentration-effect curve for pro- caterol resembles the curve for ( - ) -adrenal ine in the pres- ence of CGP 20712 A. However, this is not surprising, be- cause with 99.9% blockade of fll-adrenoceptors, ( - ) - adrenaline becomes relatively/~2-selective.

The importance of rat sinoatrial fl2-adrenoceptors should not be overestimated because they only come into play at relatively high (-)-adrenal ine concentrations as shown by the unchanged effects and ECs0 for ( - ) - adrenaline in the presence of 50 nmol ICI 117,551. In addi- tion, a considerable reserve of/~l-adrenoceptors could exist. Thus, fll-adrenoceptors mediate predominantly the effects of (-)-adrenal ine and mostly those of (-)-noradrenaline in rat sinoatrial node.

To estimate from blockade the affinity of an antagonist or partial agonist for both/71- and/~2-adrenoceptors in the same system and to estimate the contribution of each re- ceptor subtype to the effects of the agonist, Kaumann and Marano (1982) and Lemoine and Kaumann (1983) had to assume spare receptors for the activation of each subtype by the agonist. The present method of unmasking the function of the smaller/~-subtype population renders the spare re- ceptor assumption unnecessary. The present data in rat sinoatrial node show that there are no spare /~z-adreno- ceptors for (-)-adrenaline. On the other hand, there appears to exist a considerable reserve of/?l-adrenoceptors in rat sinoatrial node. The equilibrium dissociation constant of both (-)-noradrenal ine and (-)-adrenal ine for heart/~l- adrenoceptors is approximately 1 gmol/1 (Kaumann and Lemoine 1985; Lemoine et al. 1985b). The EC5o-values of 0.005 gmol/1 for (-)-noradrenaline and 0.03 gmol/1 for ( - ) - adrenaline (Table 1) are 200 and 30 times lower than the corresponding affinity values, supporting the existence of a considerable reserve of/~l-adrenoceptors.

Acknowledgement. The author is grateful to Dr. Maitre (CIBA- Geigy, Basel, Switzerland) for a gift of CGP 20712 A, to Dr. Fitzgerald (ICI, Macclesfield, UK) for a gift of ICI 118,551, and to Julie Edwards for typing the manuscript.

R e f e r e n c e s

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Lemoine H, Ehle B, Kaumann AJ (1985b) Direct labelling of fl2- adrenoceptors. Comparison of binding potency of 3H-ICI 118,551 and blocking potency of ICI 118,551. Naunyn- Schmiedeberg's Arch Pharmacol 331:40- 51

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Received November 11, 1985/Accepted January 31, 1986