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. [125I]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 Kd, 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 GDP beta S enhances toxin-catalyzed transfer of ADP-ribose to this substrate. ET-1 potentiated GTP gamma S-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 [( Ca2+ )i]. Thus, ET-1 binds to specific receptors on rat mesangial cells and activates PLC, in part, through a pertussis toxin-sensitive G-protein.
Thomas
C P
CP
Department of Medicine, Case Western Reserve University, Cleveland, Ohio.
Baldi
E
E
Simonson
M S
MS
Kester
M
M
Dunn
M J
MJ
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D016232:Endothelins / Q000378:metabolism / Q000494:pharmacology; D004789:Enzyme Activation / Q000502:physiology; D016257:Fura-2; D019204:GTP-Binding Proteins / Q000378:metabolism*; D005920:Glomerular Mesangium / Q000201:enzymology / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D066298:In Vitro Techniques; D007700:Kinetics; D037342:Pertussis Toxin; D051381:Rats; D011919:Rats, Inbred Strains; D011956:Receptors, Cell Surface / Q000378:metabolism*; D017466:Receptors, Endothelin; D013696:Temperature; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D016232:Endothelins; D011956:Receptors, Cell Surface; D017466:Receptors, Endothelin; D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D037342:Pertussis Toxin; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D016257:Fura-2