Kinetic and equilibrium studies of (-)125iodocyanopindolol binding to beta-adrenoceptors on human lymphocytes: evidence for the existence of two classes of binding sites.

Abstract

Saturation experiments were performed on intact human peripheral mononuclear leucocytes (MNL) and MNL membranes with (-)125Iodocyanopindolol (125ICYP) over a large concentration range (1.5-600 pmol/l). The corresponding Scatchard plots were curvilinear suggesting two saturable classes of binding sites: A high affinity binding site (Bmax1 = 1000 +/- 400 sites/cell, Kd1 = 2.1 +/- 0.9 pmol/l for intact MNL and Bmax1 = 550 +/- 190 sites/cell, Kd1 = 4.1 +/- 0.9 pmol/l for MNL membranes) and a low affinity binding site (Bmax2 = 9150 +/- 3590 binding sites/cell, Kd2 = 440 +/- 50 pmol/l for intact MNL and Bmax2 = 11560 +/- 4690 sites/cell, Kd2 = 410 +/- 70 pmol/l for MNL membranes). Dissociation of (-)125ICYP from MNL was biphasic consisting of a slow dissociating component (dissociation rate constant k-1 = (0.5 +/- 0.2) X 10(-3) min-1 for intact MNL and k-1 = (1.0 +/- 0.1) X 10(-3) min-1 for MNL membranes) and a fast dissociating component (k-2 = (80 +/- 20) X 10(-3) min-1 for intact MNL and k-2 = (60 +/- 10) X 10(-3) min-1 for MNL membranes). In dissociation experiments started after equilibration with various (-)125ICYP concentrations k-1 and k-2 were independent of the equilibrium concentration, whereas the percentual occupancy of the slow and the fast dissociating component varied and was similar to the estimated fractional occupancy of either binding site at the same (-)125ICYP concentrations in saturation experiments. The association rate constant was in the same order of magnitude for both binding sites. These results suggest two independent classes of binding sites for (-)125ICYP on MNL.