Binding characterization of [3H]S-0139, an antagonist of the endothelin ET(A) receptor subtype.

S-0139 (27-O-3-[2-(3-carboxy-acryloylamino)-5-hydroxyphenyl]-acryloylo xy myricerone, sodium salt) is a highly specific nonpeptide endothelin ET(A) receptor antagonist. The binding of [3H]S-0139 was compared to that of [125I]endothelin-1 to characterize the binding of the antagonist in porcine aortic smooth muscle membranes. Scatchard analysis revealed a single class of [3H]S-0139 binding sites with a Kd value of 0.61 +/- 0.10 nM and a Bmax of 0.72 +/- 0.16 pmol/mg protein. These sites were saturable and reversible. [125I]Endothelin-1 also showed binding with high affinity (Kd = 0.12 +/- 0.02 nM) to a homogeneous population of binding sites, whose Bmax (0.71 +/- 0.20 pmol/mg protein) was almost the same as that for [3H]S-0139. In both cases, the binding could be displaced by known endothelin receptor ligands and their IC50 values in each case showed a very close correlation (r = 0.986). The potency of seven endothelin receptor antagonists to displace [3H]S-0139 binding also correlated highly to the potency for inhibiting the endothelin-1-induced increase in cytosolic Ca2+ concentration (r = 0.949). Myriceric acid A showed a more potent functional activity than expected from its binding affinity, but this seemed to result from the different assay conditions, such as incubation time. Together, the results suggest that S-0139 labels only endothelin ET(A) receptor binding sites in porcine aortic smooth muscle.