[I-125] BQ3020, A POTENT ET(B)-SELECTIVE AGONIST, DISPLAYS SPECIES-DIFFERENCES IN ITS BINDING CHARACTARISTICS

Binding of [I-125]-BQ3020 to membranes prepared from rat cerebellum, dog lung and CHO cells stably transfected with the human endothelin ET(B) receptor clone was specific, saturable and of high affinity. The apparent dissociation constants (K(d)s) were 63, 53 and 112 pM for rat cerebellum, dog lung and human ET(B), respectively. The maximum bindings (B-max) were 3.8, 0.430 and 20 pmol/mg protein for rat cerebellum, dog lung and human clone, respectively. Competition binding experiments using [I-125]-BQ3020 and unlabelled ET-1, ET-3, S6c and BQ123 indicated that ET-1, ET-3 and S6c competed for [I-125]-B03020 binding with similar high affinity (IC50 similar to 0.3 nM), whereas BQ123 was ineffective in competing for [I-125]-BQ3020 binding. Time course experiments suggested that the binding of [I-125]-BQ3020 to all three preparations was rapid, and reached steady-state by similar to 40 min at 30 degrees C. Addition of excess unlabelled ET-1 resulted in partial dissociation (25-40%) from human ET(B) and dog lung receptors, whereas from rat cerebellum it was essentially irreversible. These data suggest that [I-125]-BQ3020 is a potent and selective ET(B) agonist and its reversible binding property is species-dependent.