Binding characteristics of (+)-, (+/-)- and (-)-[125iodo] cyanopindolol to guinea-pig left ventricle membranes.

Abstract

[125Iodo]cyanopindolol [(±)-ICYP], a potent and selective ligand for β-adrenoceptors, exhibited complex biphasic dissociation kinetics. Consequently, in receptor binding studies, the pure (+)- and (-)-enantiomers of ICYP were synthesised and their equilibrium and kinetic binding characteristics were investigated on a membrane preparation of guinea pig left ventricle containing almost only β1-adrenoceptors. All three ligands, i. e. (+)-, (-)- and (±)-ICYP, bind to β-adrenoceptors as assessed by competition experiments with different β-blocking agents; irrespective of the ligand used, the same dissociation constant was found for the competitor. In a first series of saturation binding experiments performed in a low concentration range of free ligand (0–250 pM), ICYP showed the following dissociation constants: K D=93, 9 and 23 pM, and number of binding sites: B max=40,128 and 124 fmoles/mg protein for (+)-, (-)-, and (±)-ICYP, respectively. Asexpected, (±)-ICYP showed the same B max as (-)-ICYP, whereas its K D was approximately two times higher than that of (-)-ICYP. Surprisingly, the B max of (+)-ICYP represented only ∼ 30% of the B max of (-)-ICYP. All three ligands bound apparently to a single class of binding sites. In dissociation experiments, the enantiomers of ICYP showed biphasic dissociation curves as observed for the racemic ligand. (+)-, (-)- and (±)-ICYP showed a rapidly dissociating (k -1=0.488, 0.047 and 0.049 min−1) and a slowly dissociating component (k -2=0.0205, 0.0033 and 0.0025 min−1). The ratio slow dissociating/fast dissociating component represented respectively for (+)-, (-)- and (±)-ICYP 40/60, 90/10 and 90/10. For all three ligands, the association rate constants were of the same order of magnitude (ca. 109 M−1 min−1), typical for a diffusion controlled reaction. In contrast to equilibrium binding studies, the existence of multiple receptor affinity sites was evident from the biphasic dissociation behaviour observed especially with the nonracemic ligands (+)-ICYP and (-)-ICYP. Simulation of theoretical saturation curves performed with the ratios of high versus low affinity sites and the K D-values suggested by kinetic analysis, indicated that the delineation into two affinity states might be visible in saturation experiments, under certain conditions. Therefore, equilibrium binding studies were repeated with an increased number of ligand concentrations covering a large concentration range of 0–800 pM. Simultaneous analysis of saturation curves from the same experiment using three different ligands, provided more accurate estimates of the ratio of high and low affinity sites, as well as the affinity constants of the ligand for each receptor affinity state, in good agreement with the results from kinetic analysis. The contribution of the (+)enantiomer in the binding of the racemic ligand under low receptor concentrations could be neglected since dissociation characteristics of (±)- and (-)-ICYP were identical. A model that explains the biphasic dissociation of (±)-ICYP by differential binding of both enantiomers could be rejected. Kinetic and equilibrium binding characteristics of the three radioligands were not influenced by the guanylnucleotide Gpp(NH)p (10−4 M). The antagonist ICYP binds to β-adrenoceptors in a high and low affinity state which are probably interconvertible.