Naunyn-Schmiedeberg's

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Naunyn-Schmiedeberg's Arch. Pharmacol. 303, 295-297 (1978)

Pharmacology

9 by Springer-Verlag 1978

Short Communication

High Affinity of Carazolol for ]3-Adrenoeeptors Coupled to the Adenylyl Cyclase in Ventricular Myocardium of Kitten and Xenopus laevis T. H. Morris t, L. Birnbaumer 1, and A. J. K a u m a n n 1,2 1 Baylor College of Medicine, Department of Cell Biology, Houston, Texas 77030, U.S.A. 2 Physiologisches Institut, Lehrstuhl fiir Klinische Physiologic der Universitfit, Universit/itsstrasse 1, D-4000 Diisseldorf, Federal Republic of Germany

Catecholamine-induced stimulation o f adenylyl cyclase in ventricular membranes o f kitten and Xenopus laevis was antagonized competitively by carazolol. A p p a r e n t equilibrium constants ranging between 1 0 0 - 1 7 0 p M were estimated for the/3-adrenoceptorcarazolol complex. Summary.

Key words: High affinity carazolol-/3-adrenoceptor C o m p l e x heart.

.Introduction

Carazolol exhibits very high and similar affinity for/3adrenoceptors mediating positive c h r o n o t r o p i c and inotropic effects o f (-)-isoprenaline in isolated heart preparations o f kitten, guinea pig and rat. The apparent equilibrium dissociation constant KB o f the /3adrenoceptor-carazolol complex in these systems was f o u n d to range f r o m 1 0 0 - 2 0 0 p M (Lemoine and K a u m a n n , 1978). We report here the nature o f the antagonism by carazolol o f catecholamine-induced stimulation o f heart adenylyl cyclase. Some differences o f heart /3-adrenoceptors o f rat and Xenopus laevis were detected by c o m p a r i n g the affinities o f certain ligands in both species ( K a u m a n n , 1977). It was f o u n d that practolol exhibited significantly lower affinity for Xenopus /3-adrenoceptors than for rat adrenoceptors; the converse was observed with the selective ligand H35/25 (Carlsson et al., 1972). We have seen that kitten and Xenopus heart fiadrenoceptors also differ in their affinity characteristics for H35/25 and practolol (unpublished experiments). To inquire whether or not carazolol is another ligand that preferentially binds more to the/3-adrenoceptors o f

Send offprint requests to A. J. Kaumann at the above address in Germany

one species than to those o f another, its affinity for cyclase-coupled/3-adrenoceptors was c o m p a r e d in ventricular membranes o f kitten and Xenopus. Methods

To avoid fl-adrenoceptor-occupancy with endogenous catecholamines (Kaumann and Birnbanmer, 1974), kittens (weighing 0.81.5 kg) and toads (weighing 90 - 150 g) were pretreated with 5 mg/kg s.c. and 50 mg/kg i.m. reserpine, respectively, 24 h before sacrificing. These doses of reserpine cause nearly complete depletion of noradrenaline in cat heart (Lee and Shideman, 1959) and of adrenaline in Xenopus heart (Strobach, personal communication). Membrane particles of kitten ventricle were prepared as before (Kaumann and Birnbaumer, 1974). Ventricular membrane particles of Xenopuswere prepared as for kitten ventricle, except that the ventricle was dissected in solution containing (mM): NaC1102.5, KC1 2.7, NaHCO 3 1.2, and CaC12 1.3 in deionized, redistilled water. Experiments were carried out on membranes which had been stored at - 70~C. In order to obtain the highest possible stimulation of the adenylyl cyclase activity by catecholamines, various incubation conditions were tried. Maximum increases of adenylyl cyclaseactivity over basal activity with catecholamines were observed with GTP in kitten membranes and 5'-guanylylimidodiphosphate in Xenopus membranes. For other incubation conditions see legend to Figure 1. In order to measure [32p]cAMPmembranes were incubated with [~32p]ATP; [3H]cAMP was added as a recovery marker. The [32p]cAMP formed and the [3H]cAMP were isolated by the method of Salomon et al. (1974) and determined by liquid scintillation counting. Protein was determined with the method of Lowry et al. (1951). A salt of( +)-carazolol (Boehringer, Mannheim) was prepared with. tartaric acid. Results and Discussion

Carazolol ( 0 . 4 - 6 0 n M ) did not significantly change the basal activity o f adenylyl cyclase in kitten and Xenopus m e m b r a n e s (left abscissa o f Fig. 1 A and B). However, 0 . 4 - 60 n M carazolol caused nearly parallel and nearly surmountable shifts o f concentration-effect curves for ( )-isoprenaline in both species, suggesting competitive inhibition. Analysis for competitive be-

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296

Naunyn-Schmiedeberg's Arch. Pharmacol. 303 (1978)

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Fig.lA and B. Surmountable antagonism by carazolol of the (-)-isoprenaline-induced stimulation of adenylyl cyclase in ventricular membranes of kitten at 32.5~C (A) and toad at 24~ (B). Each symbol represents the result from a single determination. Membrane particles were incubated for 10 min in 50 gl of medium containing 0.1 mM [c~-3ap]ATP (specific activities 125 cpm/pmole and 194cpm/pmole in A and B, respectively), 2.0mM MgCIz, 1.0raM EGTA, 0.1 mM ascorbic acid, 1 mM [3H]cAMP (15000 cpm), 20 mM creatine phosphate, 0.2 mg/mI creatine kinase, 0.1 mg/ml myokinase, 25 mM Tris-HC1, pH 7.5, indicated concentrations of (-)-isoprenaline and (_+)-carazolol and either 10 gl GTP (panel A) or 10~M 5'guanylylimidodiphosphate (panel B). The reactions were stopped by addition of 100 ~tl of a solution containing 10mM cAMP, 40raM ATP and 1 sodium dodecyl sulphate, followed by immediate boiling for 3.5 rain. Protein was 55.5 gg and 26.5/ag per assay for A and B. respectively. C and D. Simple competitive antagonism of (• against the effects of catecholamines. Equieffective concentration ratios (CR) of (-)-isoprenaline (closed circles) were taken from A and B and represented doublelogarithmically as a function of (• concentration in C and D, respectively. Other symbols were from additional experiments with ( ) noradrenaline carried out at the temperature indicated on the figure

Table 1. Apparent equilibrium dissociation constants KB" for carazolol-fl-adrenoceptor complexes in ventricular membranes of kitten and

Xenopus Species

Agonist

Temperature ~

Nb

Slope of Schild plot _+ S.D.

- l o g M K B _+ 2 S.D.

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32.5 32.5 24 24 24

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1.08 _+0.07 1.06 • 0.06 1.08 _+0.06 1.05 _+0.03 1.00 • 0.03

9.87 • 0.22 9.89 _+0.18 9.98 • 0.30 9.78 _+0.10 9.79 • 0.06

Xenopus Xenopus

a KB = [Carazolol]/(CR-l); CR is ECs0 ratio of catecholamine in the presence and absence of carazolol b N = Number of concentration ratios of catecholamine

h a v i o u r was m a d e with Schild plots as s h o w n in F i g u r e l C a n d D ( A r u n l a k s h a n a a n d Schild, 1959). Slopes o f regressions o f log (ECs0 c o n c e n t r a t i o n - r a t i o o f ( - ) - i s o p r e n a l i n e - 1 ) a g a i n s t log [(_+)-carazolol] were close to 1.0 in b o t h species (Table 1) as expected f r o m simple c o m p e t i t i v e a n t a g o n i s m . C o n c e n t r a t i o n - e f f e c t curves for ( - ) - n o r a d r e n a l i n e were also a n t a g o n i z e d c o m p e t i t i v e l y by c a r a z o l o l in b o t h species (experiments n o t shown) yielding Schild plots with s l o p e s t h a t were also n o t significantly different f r o m 1.0 (Fig. 1 C a n d D, T a b l e 1). C a r a z o l o l a n t a g o n i z e d c o m p e t i t i v e l y the effect o f ( - ) - i s o p r e n a l i n e b o t h at 2 4 ~ a n d at 32.5~ ( F i g . l A a n d C a n d T a b l e 1). Because o f the simple c o m p e t i t i v e n a t u r e o f the a n t a g o n i s m o f the effects o f c a t e c h o l a m i n e s by cara-

zolol, e q u i l i b r i u m c o n s t a n t s K B were c a l c u l a t e d (Table 1). T h e KB o f the c a r a z o l o l - f l - a d r e n o c e p t o r c o m p l e x was 1 0 0 - 1 7 0 p M in b o t h kitten a n d Xenopus m e m b r a n e s . T h e affinity o f c a r a z o l o l for m y o c a r d i a l fla d r e n o c e p t o r s was the same, regardless o f species (kitten vs xenopus), t e m p e r a t u r e (24 vs. 32.5 ~ C, kitten only) o r a g o n i s t [(-)-isoprenaline vs. ( - ) - n o r a d r e naline]. The KB o f c a r a z o l o l for adenylyl-cyclasec o u p l e d f l - a d r e n o c e p t o r s agrees with its K B o f 1 0 0 200 p M e s t i m a t e d for f l - a d r e n o c e p t o r s m e d i a t i n g positive c h r o n o t r o p i c a n d i n o t r o p i c effects o f catecholamines in m a m m a l i a n h e a r t ( L e m o i n e a n d K a u m a n n , 1978). U n l i k e the selective ligands H35/25 (Carlsson et al., 1972) a n d p r a c t o l o l which exhibit preferential affinities

T. H. Morris et al.: 100-170 pM K B of Carazolol for Heart fl-Adrenoceptors

for xenopus- and mammalian-/Ladrenoceptors, respectively (Kaumann, 1977), carazolol was non-selective in this respect. Comparison of the K B of carazolol With KB's reported for other high affinity ligands reveals that the affinity of carazolol for adenylyl cyclase-coupled fladrenoceptors is about 5 and 20 times higher than that of bupranolol and propranoloi, respectively (Kaumann and Birnbaumer, 1974). In our experience carazolol is so far the ligand with the highest affinity for cardiac fladrenoceptors. Radioactive and fluorescent carazolol analogues may serve as promising ligands for the elucidation of properties of myocardial and perhaps other fl-adrenoceptors.

Acknowledgements. Part of this work was supported by grants SFB30-Cardiologie of the Deutsche Forschungsgemeinschaft and HL19423 of the U.S. Public Health Service. We thank Dr. W. Bartsch (Boehringer, Mannheim) for the carazolol and H. Strobach (University of Dtisseldorf) for the adrenaline determination in Xenopus ventricle.

297

References Arunlakshana, O., Schild, H. O.: Some ~luantitative uses of drug antagonists. Br. J. Pharmacol. 14, 4 8 - 5 8 (1959) Carlsson,~E., A.blad, B., Brfindstr6m, A., Carlsson, B. : Differentiated blockade of the chronotropic effects of various adrenergic stimuli in the cat heart. Life Sci. 11, 953-958 (1972) Kaumann, A. J. : Some differences between heart j~-adrenoceptors of reserpine-pretreated rats (Wistar II) and frogs (Xenopus laevis), Naunyn-Schmiedeberg's Arch. Pharmacol. 297, R49 (1977) Kaumann, A. J., Birnbaumer, L. : Characteristics of the adrenergic receptor coupled to myocardial adenylyl cyclase: Stereospecificity and determination of apparent affinity constants for fl-blockers. J. Biol. Chem. 249, 7874-7885 (1974) Lee, W. C., Shideman, F. E. : Mechanism of the positive inotropic response to certain ganglionic stimulants. J. Pharmacol. Exp. Ther. 126, 239-249 (1959) Lemoine, H., Kaumann, A. J. : The affinity of carazolol for myocardial and tracheal fl-adrenoceptors activated by (-)-isoprenaline. Naunyn-Schmiedeberg's Arch. Pharmacol. 302, R53 (1978) Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J. : Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275 (1951) Salomon, Y., Londos, C., Rodbell, M. : A highly sensitive adenylate cyclase assay. Anal. Biochem. 58, 5 4 1 - 548 (1974)

Received April 7/Acc~7~tedMay 17, 1978