Regulation of agonist binding to rat ETB receptors by cations and GTPγS

Abstract Endothelins exert their physiological effects through interaction with cell surface receptors that are members of the G-protein-coupled receptor family. The endothelin receptor subtype B (ET B receptor) is abundantly expressed in rat cerebellum. Since agonist binding to G-protein-coupled receptors may be modulated by cations and guanine nucleotides, we investigated the effects of cations and guanosine 5′- O -(2-thiotriphosphate) (GTPγS) on 125 I-endothelin-1 ( 125 I-ET-1) binding to rat cerebellar membranes. Both Na + and Mg 2+ -stimulated 125 I-ET-1 binding causing an increase in receptor affinity for the agonist. While the effect of the divalent cation was evident at relatively low concentrations (5–10 mM), the stimulatory activity of the monovalent cation appeared at relatively high concentrations (50 mM). Additive activities of 25–50 mM NaCl and 1 mM MgCl 2 suggested that monovalent and divalent cations increased receptor affinity for ET-1 by different mechanisms. In the presence of 5 mM MgCl 2 , 50 mM NaCl caused an additional modest reduction of the K d value. Whereas 5 mM MgCl 2 affected the displacement curves of both ET-3 and suc-[Glu 9 , Ala 11,15 ]-endothelin-1 (8–21) (IRL 1620), the influence of 50 mM NaCl on these curves was less substantial. All together, these results suggest that modulation of receptor affinity by NaCl depends on the nature of the displacing agonist. In the presence of 5 mM MgCl 2 or 50 mM NaCl, a partial regulation of 125 I-ET-1 binding by GTPγS was detectable, while in the absence of cations no GTPγS-dependent inhibition was evident.