INTERACTION OF ENDOTHELIN-1 WITH CLONED BOVINE ETA-RECEPTOR - BIOCHEMICAL PARAMETERS AND FUNCTIONAL CONSEQUENCES

This paper defines the properties of interaction of endothelin-1 (Et-1 ) with cloned bovine ETA receptors. The K-d value of Et-1/ETA receptor complexes was estimated in membrane preparation to 20 pM using kineti c experiments and saturation experiments performed under quasi equilib rium conditions. Competition experiments yield a wide range of apparen t K-d(Et-1) values from 20 pM to 1 nM which were in fact measures of t he receptor concentrations rather than of K-d values. This resulted fr om the fact that complex second-order rate kinetics rather than pseudo -first-order kinetics control the association of Et-1 to its receptor when the receptor concentration is larger than K-d(Et-1). Et-1 induced a production of inositol phosphates with an apparent affinity of 2.3 nM, 100 times higher than the K-d(Et-1) value determined previously. N umerical simulation suggested that under time-limited conditions, sub- nanomolar rather than picomolar concentrations of Et-1 are necessary t o occupy an important fraction of picomolar sites. It is concluded tha t bovine ETA receptors have a single affinity state for Et-1 (K-d 20 p M) and that this affinity state can account for nanomolar actions of E t-1 in intact cells. It is suggested that the sensitivity of a prepara tion to Et-1 is a cell property rather than a receptor property. It is also suggested that the main advantage of high-affinity Et-1 binding is to promote autocrine actions rather than a high potency of the pept ide.