ENDOTHELIN RECEPTOR AGONISTS AND ANTAGONISTS EXHIBIT DIFFERENT DISSOCIATION CHARACTERISTICS

Endothelins (ETs) are vasoconstricting peptides that bind to membrane receptors to initiate their physiological effects. This report compare s the dissociation characteristics of selected ET agonists and antagon ists, and studies the effects of any difference in dissociation charac teristics on the potency of antagonists. Competition studies using var ious ET receptor ligands against [I-125]ET-1 or [I-125]ET-3 binding de monstrated that porcine cerebellum membranes contain predominantly ET( B) receptor. [I-125]IRL1620 associated with the receptors in a time-de pendent manner. Although bound [I-125]IRL1620 was easier to dissociate than bound [I-125]ET-3, both agonists exhibited a dissociation half l ife > 20 h. For non-radiolabeled ligands, bind-and-wash studies were e mployed in which membranes were pre-incubated with unlabeled ligand fo llowed by extensive washing before assaying for [I-125]ET-1 binding. R esults from bind-and-wash studies confirmed that bound non-radiolabele d IRL1620 and ET were as difficult to dissociate as [I-125]ligands. In contrast, bound PD142893 and Ro46-2005 were easily dissociated from E T(B) receptors. Consequently, the inhibitory effects of PD142893 and R o46-2005 on [I-125]agonist binding diminished following incubation tim e. In cloned human ET(A) and ET(B) receptors, bound ET-1 was also more difficult to dissociate than bound antagonists. These results suggest that the differences in the dissociation characteristics of ET recept or agonists vs, antagonists may account for the diminished potency of Ro46-2005 and PD142893 as a function of incubation time.