Direct labelling of myocardial β1-adrenoceptors
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A radioligand that selectively labels β1-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 KB (mol/l; -log KB=pKB) for a high-affinity β1-adrenoceptor of 8.8 and for a low-affinity β2-adrenoceptor of 7.0 were estimated in the three classes of tissues. In kitten sinoatrial pacemaker β1-adrenoceptors contribute 76% to the stimulus induced by (-)-adrenaline and 97% to the stimulus induced by (-)-noradrenaline. In ventricle and left atrium β1-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. pKB-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 β2-selective ICI 118,551, which did not antagonize β1 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 β2-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 β1-adrenoceptors. A koff of 1.0 min-1 was estimated. An equilibrium dissociation constant pKL*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 β1-adrenoceptors.
(-)-Bisoprolol competed with high affinity and low affinity, respectively, for β1- and β2-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 β1- and β2-adrenoceptors agrees with the selectivity for β1-adrenoceptors found in intact heart tissues.
(-)-Noradrenaline and (-)-adrenaline competed with low affinity with 3H-BIS for ventricular β1-adrenoceptors. An approximately 100-fold difference between high KL-values of these catecholamines and low inotropic EC50-values is consistent with a large β1-adrenoceptor reserve.
The binding affinity for β1-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 β1-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.
Key wordsHeart 3H-(−)-Bisoprolol Catecholamines β1-Adrenoceptors Binding and blockade
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