Endothelin (ET) exerts various biological actions in mesangial cells, including stimulation of proliferation, contraction and phospholipase C activation. We investigated the presence of specific ET receptors on cultured rat mesangial cells, incubating the cells in the presence of [125I]ET-1 both at 22 and 4 degrees C. ET binding was time- and temperature-dependent and achieved equilibrium at 2 hr at 22 degrees C and at 5 hr at 4 degrees C. Scatchard analyses of equilibrium saturation curves with [125I]ET-1 and homologous competition curves revealed the presence of a single class of high-affinity binding sites (Kd = 31.4 +/- 7.08 pM). Heterologous competition experiments with ET-3 and sarafotoxin, however, indicated the presence of two binding sites for ET-related peptides in mesangial cells with a Kd for ET-3 of 41.5 +/- 19.2 and of 374 +/- 38.5 nM. Nifedipine and arginine-vasopressin failed to compete for ET binding sites. Preincubation of the cells with 1 nM ET-1 caused a dramatic decrease in ET binding capacity (from 0.5-0.02 fmol/100,000 cells) without affecting the Kd for the receptors (38 pM). ET receptor down regulation was not prevented by protein kinase C inhibition with H-7 and sangiovamycin, or after down regulation of protein kinase C induced by 24-hr preincubation with phorbol myristate acetate. ET receptor down regulation also exerts functional effects, as we found a decrease in intracellular-free calcium response to ET-1 after long-term preincubation with the same agonist. Our results are consistent with the presence of two binding sites for ET in rat mesangial cells.(ABSTRACT TRUNCATED AT 250 WORDS)