Abstract

[125I]Tyr13-Suc-[Glu9,Ala11,15]-Endothelin-1 (8-21) ([125I]IRL-1620) displayed specific, saturable and high affinity binding to membranes prepared from rat and dog cerebellum, rat and human lung and cloned human endothelin-B (ETB) receptors. The apparent dissociation constant (Kd) for rat cerebellum, dog cerebellum, rat lung, human lung and human ETB clone were 69.1, 34.8, 128, 71.9, and 115 pM, respectively. The maximum binding (Bmax) to these membranes were 3.2, 0.71, 1.67, 1.85 and 62.3 pmol/mg of protein for rat cerebellum, dog cerebellum, rat lung, human lung and human ETB clone, respectively. Unlabeled endothelin-1 (ET-1), ET-3 and sarafotoxin 6c (S6c) displaced [125I]IRL-1620 binding to these membranes with similar affinity (IC50 = 0.1-2.5 nM), whereas cyclo(D-Trp,D-Asp,L-Pro,D-Val,L-Leu), a selective antagonist for ETA receptors, had no effect on [125I]IRL-1620 binding up to 1 microM. Time course experiments of association and dissociation indicated that [125]IRL-1620 binding to dog and human tissues and human ETB clone was rapid and reversible, whereas in rat tissues, the binding was rapid but irreversible, suggesting that this might be due to species difference. [125I]ET-1 binding was irreversible in all the tissues tested. No binding of [125I]IRL-1620 was detectable in rat vascular smooth muscle cells or cloned human ETA receptors. These data indicate that [125I]IRL-1620 is highly selective for ETB receptors and the reversible binding characteristics of this ligand appears to be species-dependent.