Endothelin Receptors and Coupled GTP-Binding Proteins in Glomerular Mesangial Cells

Endothelins (ETs) are a family of vasoactive peptides with profound biological actions in diverse cell systems. Among its varied actions, ET stimulates phospholipase C (PLC) in cultured mesangial cells. We investigated the presence of specific ET receptors in rat mesangial cells in culture, and studied the role of GTP-binding proteins (G proteins) in coupling PLC to the endothelin receptor. [I]ET binding was time- and temperature-dependent, and Scatchard analysis of saturation data showed a single class of high-affinity binding sites. Heterologous displacement with two related peptides, ET-3 and sarafotoxin (SFTX), revealed the presence of two binding sites for these isopeptides. Preincubation of cells with ET-1 reduced the receptor number without affecting K, and this effect was not prevented by protein kinase C inhibition or downregulation. We confirmed the presence of a 41− to 43-kDa pertussis toxin substrate in rat mesangial cell membranes in an ADP ribosylation assay. ET-1 inhibits and GDPβS enhances toxin-catalyzed transfer of ADP-ribose to this substrate. ET-1 potentiated GTPγS-induced phosphatidylinositol (PI) hydrolysis in a concentration-dependent manner. In addition, pertussis toxin partially inhibited ET-stimulated PI hydrolysis in intact mesangial cells. Pertussis toxin also reduced the magnitude of ET-stimulated intracellular free calcium [(Ca )]. Thus, ET-1 binds to specific receptors on rat mesangial cells and activates PLC, in part, through a pertussis toxin-sensitive G-protein.