Characterization of three non-peptide endothelin receptor ligands using human cloned ETA and ETB receptors.

1. A number of putative endothelin (ET) receptor ligands were synthesized with a view to assessing their relative affinity for human recombinant ET receptors. 2. Human (h) and endothelin ETA and ETB receptor open reading frames were cloned by reverse transcription-polymerase chain reaction into the mammalian expression vector pcDNA1 and stable cell lines were created by transfection of Chinese hamster ovary cells. 3. Scatchard analyses of saturation isotherms for the specific binding of [125I]-endothelin-1 ([125I]-ET-1) to membranes, prepared from Chinese hamster ovary cells transfected with hETA or hETB receptors, yielded values for equilibrium dissociation constants (Kd) of 20.5 +/- 1.8 pM and 25.5 +/- 5.5 pM, respectively. Hill coefficients did not differ significantly from unity, suggesting binding to homogeneous, non-interacting receptor populations. 4. Pharmacological characterization of the transfected hETA and hETB receptors was undertaken by measuring the relative abilities of ETA and ETB receptor-selective peptide ligands to inhibit binding of [125I]ET-1. For interaction with hETA receptors, the relative order of potency was ET-1 > ET-3 = FR139317 = BQ123 >[Ala1,3,11,15]-ET-1 = sarafotoxin S6c (S6c). In contrast, the relative order of potency, at hETB receptors, was ET-1 = ET-3 = [Ala1,3,11,15]-ET-1 = S6c >> FR139317 = BQ123. 5. The novel non-peptide ligands, Ro 46-2005, SB 209670 and BMS 182874, were found to inhibit [125I]-ET-1 binding to human recombinant ETA and ETB receptors. At hETA receptors, the calculated pIC50 values were 6.7 (Ro 46-2005), 8.7 (SB 209670) and 5.8 (BMS 182874), while at hETB receptors, the corresponding pIC50 values were 6.8, 7.5 and <5, respectively.6. In conclusion, we have characterized the pharmacology of human cloned ETA and ETB receptors and used these in membrane binding assays to determine the affinity and selectivity of three structurally diverse non-peptide ET receptor ligands. SB 209670 is, to date, the highest affinity non-peptide ligand to be described for ET receptors. As such, it may prove to be a valuable tool in further examination of the physiological and pathophysiological roles of endothelins.