Regulation of agonist binding to rat ETB receptors by cations and GTP gamma S

Endothelins exert their physiological effects through interaction with cell surface receptors that are members of the G-protein-coupled receptor famil y. The endothelin receptor subtype B (ETB receptor) is abundantly expressed in rat cerebellum. Since agonist binding to G-protein-coupled receptors ma y be modulated by cations and guanine nucleotides, we investigated the effe cts of cations and guanosine 5'-O-(2-thiotriphosphate) (GTP gammaS) on I-12 5-endothelin-1 (I-125-ET-1) binding to rat cerebellar membranes. Both Na+ a nd Mg2+-stimulated I-125-ET-1 binding causing an increase in receptor affin ity for the agonist. While the effect of the divalent cation was evident at relatively low concentrations (5-10 mM), the stimulatory activity of the m onovalent cation appeared at relatively high concentrations (50 mM). Additi ve activities of 25-50 mM NaCl and 1 mM MgCl2 suggested that monovalent and divalent cations increased receptor affinity for ET-1 by different mechani sms. In the presence of 5 mM MgCl2, 50 mM NaCl caused an additional modest reduction of the K-d value. Whereas 5 mM MgCl2 affected the displacement cu rves of both ET-3 and suc-[Glu(9), Ala(11,15)]-endothelin-1 (8-21) (IRL 162 0), the influence of 50 mM NaCl on these curves was less substantial. All t ogether, these results suggest that modulation of receptor affinity by NaCl depends on the nature of the displacing agonist. In the presence of 5 mM M gCl2 or 50 mM NaCl, a partial regulation of I-125-ET-1 binding by GTP gamma S was detectable, while in the absence of cations no GTP gammaS-dependent i nhibition was evident. (C) 2001 Elsevier Science Inc. All rights reserved.