Specific receptors for endothelin on membranes from human placenta. Characterization and use in a binding assay

Abstract

High-affinity binding sites for endothelin have been found in a human placenta membrane preparation. 125I-endothelin bound to placenta membranes at 20° C with an association half-time of 30 min, whereas the binding was only slowly reversed with a dissociation half-time of 250 min. In saturation experiments, a single class of high-affinity binding sites was identified with an apparent dissociation constant (K D) of 24 pM and a maximal density of 240 fmol per mg of protein. The binding of 123I-endothelin was half-maximally inhibited by cold endothelin at a concentration (IC 50) of 140 pM. In contrast, no inhibition was found at 10 −4 M for a variety of vasoactive peptides such as angiotensin II, vasopressin, neuropeptide Y, substance P, CGRP, bradykinin, leucine enkephalin or dynorphin A. Similarly, the binding was modulated neither by the calcium channel blockers nifedipine, verapamil or diltiazem, nor by the calcium channel agonist Bay k 8644. There was also no effect with the structurally-related bee venom apamin. Using this membrane preparation, endothelin-like activity could be measured in the medium of cultured human endothelial cells by competition binding technique.