Endothelin-1 binding to endothelin receptors in the rat anterior pituitary gland: possible formation of an ETA-ETB receptor heterodimer.

1. Interaction in the recognition of endothelin-1 (ET-1), a typical bivalent ET receptor-ligand, between ETA and ETB receptors was investigated in the rat anterior pituitary gland, using our quantitative receptor autoradiographic method with tissue sections preserving the cell-membrane structure and ET receptor-related compounds. 2. In saturation binding studies with increasing concentrations (0.77-200 pM) of 125I-ET-1 (nonselective bivalent radioligand), 125I-ET-1 binding to the rat anterior pituitary gland was saturable and single with a KD of 71 pM and a Bmax of 120 fmol mg(-1). When 1.0 microM BQ-123 (ETA antagonist) was added to the incubation buffer, binding parameters were 8.3 pM of KD and 8.0 fmol mg(-1) of Bmax, whereas 10 nM sarafotoxin S6c (ETB agonist) exerted little change in these binding parameters (KD, 72 pM; Bmax, 110 fmol mg(-1)). 3. Competition binding studies with a fixed amount (3.8 pM) of 125 I-ET-1 revealed that when 1.0 microM BQ-123 was present in the incubation buffer, ETB receptor-related compounds such as sarafotoxin S6c, ET-3, IRL1620 (ETB agonist), and BQ-788 (ETB antagonist) competitively inhibited 125I-ET-1 binding with K(i)s of 140,18,350 pM, and 14 nM, respectively, however, these compounds were not significant competitors for 125I-ET-1 binding in the case of absence of BQ-123. 4. In cold-ligand saturation studies with a fixed amount (390 pM) of 125I-IRL 1620 (ETB radioligand), IRL1620 bound to a single population of the ETB receptor, and no change was observed in binding characteristics in the presence of 1.0 microM BQ-123. 125I-IRL1620 binding was competitively inhibited by ET-1 and ET-3 in the absence of BQ-123, with K(i)s of 20 and 29 pM, respectively, the affinities being much the same as those of 29 nM, in the presence of 1.0 microM BQ-123. 5. Two nonbivalent ETA antagonists, BQ-123 and PD151242, were highly sensitive and full competitors for 125I-ET-1 binding (5.0 pM), in the presence of 10 nM sarafotoxin S6c. 6. Taken together with the present finding that mRNAs encoding the rat ETA and the ETB receptors are expressed in the anterior pituitary gland, we tentatively conclude that although there are ETA and ETB receptors with a functional binding capability for ET receptor-ligands, the ETB receptor does not independently recognize ET-1 without the aid of the ETA receptor. If this thesis is tenable, then ET-1 can bridge between the two receptors to form an ETA-ETB receptor heterodimer.