Direct labelling of myocardial ? 1-adrenoceptors

Abstract

A radioligand that selectively labels β ₁-adrenoceptors, ³H-(−)-bisoprolol (³H-BIS), is introduced. The binding properties of ³H-BIS to membrane particles of kitten heart are compared with the blocking properties of (−)-bisoprolol against stimulant effects of (−)-adrenaline and (−)-noradrenaline in heart preparations of kitten and guinea pig. On kitten heart tissues (-)-bisoprolol antagonized the positive chronotropic and inotropic effects of catecholamines competitively. The effects of (-)-adrenaline were antagonized considerably less by (-)-bisoprolol than the corresponding effects of (-)-noradrenaline on sinoatrial pacemakers. The antagonism was nearly the same against both (-)-adrenaline and (-)-noradrenaline in left atria and papillary muscles. The data were analyzed with a model for 2-receptor subtypes by non-linear regression. Equilibrium dissociation constants K _B (mol/l; -log K _B=pK _B) for a high-affinity β ₁-adrenoceptor of 8.8 and for a low-affinity β ₂-adrenoceptor of 7.0 were estimated in the three classes of tissues. In kitten sinoatrial pacemaker β ₁-adrenoceptors contribute 76% to the stimulus induced by (-)-adrenaline and 97% to the stimulus induced by (-)-noradrenaline. In ventricle and left atrium β ₁-adrenoceptors contribute 97∓99% and 100% to the stimulus caused by (-)-adrenaline and (-)-noradrenaline, respectively. Both ³H-BIS and unlabelled (-)-bisoprolol caused competitive blockade of the positive chronotropic effects of (-)-noradrenaline in guinea-pig right atria. pK _B-values of 8.7 were estimated for both unlabelled and tritiated (-)-bisoprolol. The positive chronotropic effects of (-)-adrenaline were antagonized considerably less by (-)-bisoprolol than those of (-)-noradrenaline in guinea-pig atria. In the presence of low concentrations of β ₂-selective ICI 118,551, which did not antagonize β ₁ adrenoceptor mediated effects, (−)bisoprolol antagonized positive chronotropic effects of (−)-adrenaline to the same extent as those of (−)-noradrenaline. The results are consistent with the concept of a significant role of sinoatrial β ₂-adrenoceptors of guinea pig for the effects of (−)-adrenaline but not for those of (−)-noradrenaline. ³H-BIS associated and dissociated quickly with and from ventricular β ₁-adrenoceptors. A k _off of 1.0 min^-1 was estimated. An equilibrium dissociation constant pK _L ^*of 8.2 for ³H-BIS was estimated from saturation binding. The binding competition of the optical isomers of bupranolol with ³H-BIS revealed a degree of stereo-selectivity expected for β ₁-adrenoceptors. (-)-Bisoprolol competed with high affinity and low affinity, respectively, for β ₁- and β ₂-adrenoceptors of both ventricular and atrial membranes labelled with ³H-(-)-bupranolol bupranolol or ³H-(-)-propranolol. The difference in affinity of (-)-bisoprolol of 1.7±0.3 log units between β ₁- and β ₂-adrenoceptors agrees with the selectivity for β ₁-adrenoceptors found in intact heart tissues. (-)-Noradrenaline and (-)-adrenaline competed with low affinity with ³H-BIS for ventricular β ₁-adrenoceptors. An approximately 100-fold difference between high K _L-values of these catecholamines and low inotropic EC₅₀-values is consistent with a large β ₁-adrenoceptor reserve. The binding affinity for β ₁-adrenoceptors of both ³H-BIS and (-)-bisoprolol is 3∓8 times lower on membrane particles than on intact tissues, as also confirmed with the blockade of catecholamine-induced stimulation of the adenylate cyclase. By contrast, the affinities of (-)-propranolol and (-)-bupranolol are similar in intact tissues and on membrane particles. The low affinity of (-)-bisoprolol in membranes may be caused by a lesion of accessory recognition sites of the β ₁-adrenoceptors due to the procedure of membrane preparation. However, the lesion of these hypothetical accessory binding sites would not lower the affinity for (-)-propranolol and (-)-bupranolol.