title: Endothelin binding and receptor down regulation in rat glomerular mesangial cells.
abstract: 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)
authors.affiliation: Department of Medicine, School of Medicine, Case Western Reserve University, Cleveland, Ohio.
authors.forename: E
authors.initials: E
authors.lastname: Baldi
authors.forename: M J
authors.initials: MJ
authors.lastname: Dunn
mesh_terms: D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism; D015536:Down-Regulation; D016232:Endothelins / Q000378:metabolism* / Q000494:pharmacology; D005920:Glomerular Mesangium / Q000737:chemistry* / Q000378:metabolism; D011493:Protein Kinase C / Q000502:physiology; D051381:Rats; D011956:Receptors, Cell Surface / Q000032:analysis*; D017466:Receptors, Endothelin; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D016232:Endothelins; D011956:Receptors, Cell Surface; D017466:Receptors, Endothelin; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium