Endothelin receptor antagonists exhibit diminishing potency following incubation with agonist.

Endothelins (ET) are 21-amino acid peptides that bind to membrane receptors to initiate a wide range of pathophysiological effects. ET binding to receptors has been shown to be almost irreversible because bound ET is difficult to dissociate. This report studies the dissociation characteristics of receptor antagonists and further examines the effects of ET's difficult-to-dissociate binding on the potency of antagonists. In membranes prepared from porcine cerebellum, [125I]ET-1 binding was effectively blocked by ET-1 and ET-3 with similar IC50 values (0.08 nM vs. 0.17 nM), suggesting that porcine cerebellum contains predominantly the ETB receptor subtype. [125I]ET-3 binding was inhibited by Ro46-2005 and PD142893, two non-selective antagonists, with IC50 values of 570 +/- 50 nM and 410 +/- 100 nM, respectively. Consistent with previous observations, bound [125I]ET-1 in porcine cerebellum membranes was also difficult to dissociate. In contrast, bound Ro46-2005 or PD142893, but not bound ET-1, could be readily washed away from membranes, suggesting that antagonist binding was more reversible than ET-1 binding. Although Ro46-2005 or PD142893 at 0.5 microM inhibited 0.1 nM [125I]ET-1 binding by > 80% after 15 min of incubation, the inhibitory effect decreased to approximately 50% after 3 h of incubation, and further decreased to < 10% at 24 h. This decrease in antagonizing potency was further confirmed by the results that the IC50 values of the two antagonists against [125I]ET-3 binding increased with increasing incubation time. Control experiments indicate that the observed decrease in the potency of Ro46-2005 and PD142893 was not the result of ligand degradation. These results suggest that the potency of antagonists is critically dependent on the incubation time because antagonist binding is more reversible than ET binding.