CHARACTERIZATION OF 3 NONPEPTIDE ENDOTHELIN RECEPTOR LIGANDS USING HUMAN CLONED ET(A)-RECEPTOR AND ET(B)-RECEPTOR

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 ET(A) and ET(B) 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 [I-125]-endothelin-1 ([I-125]-ET-1) to membranes, prepared from Chinese hamster ovary cells transfected with hET(A) or hET(B) receptors, yielded values for equilibrium dissociation constants (K-d) 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 hET(A) and hET(B) receptors was undertaken by measuring the relative abilities of ET(A) and ET(B) receptor-selective peptide ligands to inhibit binding of [I-125]-ET-1. For interaction with hET(A) receptors, the relative order of potency was ET-1 > ET-3 = FR139317 = BQ123 >[Ala(1,3,11,15)]-ET-1 = sarafotoxin S6c (S6c). In contrast, the relative order of potency, at hET(B) receptors, was ET-1 = ET-3 = [Ala(1,3,11,15)]-ET-1 = S6c >> FR139317 = BQ123. 5 The novel non-peptide ligands, Ro 46-2005, SB 209670 and BMS 182874, were found to inhibit [I-125]-ET-1 binding to human recombinant ET(A) and ET(B) receptors. At hET(A) receptors, the calculated pIC(50) values were 6.7 (Ro 46-2005), 8.7 (SB 209670) and 5.8 (BMS 182874), while at hET(B) receptors, the corresponding pIC(50), values were 6.8, 7.5 and < 5, respectively. 6 In conclusion, we have characterized the pharmacology of human cloned ET(A) and ET(B) 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.