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MOLECULAR PHARMACOLOGY 52:582-589 (1997).

Identification and Characterization of a Novel Endothelin Receptor That Binds Both ET_A- and ET_B-Selective Ligands

Ponnal Nambi, Mark Pullen, Jamie Kincaid, Parvathi Nuthulaganti, Nambi Aiyar, David P. Brooks, Miklos Gellai, and Chandrika Kumar

Departments of Renal Pharmacology (P.N., M.P., J.K., D.P.B., M.G.), Cardiovascular Pharmacology (N.A.), and Molecular Genetics (C.K.), SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406-0939

This study demonstrates the presence of a novel endothelin (ET) receptor subtype that displays high affinity for both ET_A- and ET_B-selective ligands. This subtype has been identified in canine spleen membranes using ET_B-selective agonists ET-3, IRL-1620, sarafotoxin 6c (S6c) as well as ET_A-selective antagonists BQ123 and related cyclic pentapeptides. Binding of ¹²⁵I-ET-3 to canine spleen membranes was specific and saturable with an apparent dissociation constant of 130 pM and maximum binding (B_max) of 240.0 fmol/mg protein. Although the apparent affinities obtained with ¹²⁵I-ET-1 and ¹²⁵I-ET-3 were comparable (90 and 130 pM, respectively), the maximum binding obtained with ¹²⁵I-ET-3 was ~35% of that obtained with ¹²⁵I-ET-1, which indicates that canine spleen possesses both ET_A and ET_B receptors in the ratio 65:35. Competition binding experiments using ¹²⁵I-ET-3 and unlabeled ET-1, ET-3, S6c, and IRL-1620 suggested that although ET-1 and ET-3 displayed similar high affinity, S6c and IRL-1620 were 20-300-fold weaker than ET-1 and ET-3 in competing for ¹²⁵I-ET-3 binding to canine spleen membranes. In addition, BQ123, an ET_A-selective antagonist, displaced ¹²⁵I-ET-3 binding from canine spleen with an IC₅₀ value of 30 nM. Similar profiles were obtained with related cyclic pentapeptides. Electrophysiological studies performed on Xenopus laevis oocytes injected with canine spleen poly(A)⁺ RNA indicated that the ET_B receptor present in these tissues is functional and displays the same pharmacology as that observed in binding studies using these membranes. As a comparison, both binding and functional studies were performed in canine lung and the data indicate that the ET_B receptor present in this tissue is similar to that of the cloned human ET_B receptor but different from that present in canine spleen. These observations were further confirmed by performing cross-linking experiments on these membranes. Although canine lung and cloned human ET_B receptors displayed the same molecular weight bands with similar pharmacology, canine spleen ET_B receptors displayed different molecular weight bands and different pharmacology. In addition, the ET_B receptors present in canine spleen were also identified in canine bladder, monkey spleen and human spleen. Thus, the data presented in this manuscript provide evidence for the presence of a novel ET_B receptor in different tissues as well as different species including human.