CHARACTERIZATION OF ENDOTHELIN RECEPTORS IN MESANGIAL CELLS - EVIDENCE FOR 2 FUNCTIONALLY DISTINCT ENDOTHELIN BINDING-SITES

Endothelin (ET) peptides are growth factors that bind to G protein-cou pled receptors and serve as a useful model to study mitogenic signal t ransduction by vasoactive peptides. To begin to define the molecular m echanisms underlying mitogenic signaling by ET-1, we analyzed expressi on of ET receptor subtypes in glomerular mesangial cells and antagonis m of ET-1-induced mitogenic responses by ET receptor antagonists. Comp etitive displacement analysis of I-125-ET-1 binding revealed a shallow multicomponent curve consistent with the presence of two ET-1 binding sites (K-d values of 32 pM and 1.2 nM). Nonlinear regression analysis demonstrated that a two-site model fit the data better than a one-sit e model (p = 0.0063). Analysis of I-125-ET-1 binding sites by affinity cross-linking revealed incorporated radioactivity in two distinct pro tein bands in mesangial cell membranes. Both ET(A)-specific (BQ 123) a nd nonselective (PD 142893/PD 145065) receptor antagonists displaced I -125-ET-1 from the low affinity site. The K-i values for BQ 123 and PD 145065 were similar to the IC50 values for inhibition of ET-1-induced increases in the intracellular free Ca2+ concentration ([Ca2+](i)) by these antagonists. The ET(B)-specific ligands S6c and [Ala(1,3,11,15) ]ET-1(6-21) were unable to displace I-125-ET-1 from either low affinit y or high affinity binding sites. Analysis of ET receptor mRNA by reve rse transcription-polymerase chain reaction, using primers predicted f rom DNA sequences conserved through evolution in ET(A) and ET(B) genes , demonstrated that mesangial cells express demonstrated that mesangia l cells express a canonical ET(A) receptor. Collectively, these data s uggest that the low affinity, high capacity I-125-ET-1 binding site is an ET(A) receptor and that the high affinity, low capacity site is no t accounted for by canonical ET receptors. We further demonstrated tha t BQ 123 and PD 142893/PD 145065 inhibited ET-1-stimulated [H-3]thymid ine uptake but at higher concentrations than required for inhibition o f [Ca2+](i) increases. Preincubation (as opposed to coincubation) with antagonists was required to inhibit ET(A)-mediated increases in [Ca2]i produced by ET-1. These results suggest that the unique, nearly irr eversible binding of ET-1 to ET(A) receptors explains why high concent rations of ET receptor antagonists are required to block longer term a ctions such as mitogenesis.