Abstract

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 and¹²⁵I-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.