Endothelin-elicited stimulation of phospholipase C is mediated by guanine nucleotide binding protein(s).
In permeabilized C6 glioma cells and NIH 3T3 cells, the peptide endothelin 1 (ET-1) in combination with GTP gamma S stimulates the formation of inositol phosphates. In the presence of 10 microM GTP gamma S, ET-1 induces the formation of inositol phosphates with an EC50 value of 2.5 nM for C6 glioma cells and 1.6 nM for NIH 3T3 cells. The analogous peptide endothelin 3 (ET-3) is less potent than ET-1 in such action. In NIH 3T3 cells, ET-1+GTP gamma S-induced formation of inositol phosphates could be detected after 1 min of stimulation, and it increased for up to 30 min. ET-1-induced effects were partially reduced by pretreatment of the cells with pertussis toxin (1 microgram/ml) in C6 glioma cells, but were unaffected in NIH 3T3 cells. In binding studies in whole C6 cells and NIH 3T3 cells, specific binding for [125I]ET-1 was detected. Cross-linking of [125I]ET-1 in whole C6 cells revealed the presence of two binding proteins for ET-1 of 74 kDa and 55 kDa. ET-1 at 100 nM inhibited the labeling of both proteins by [125I]ET-1. However, ET-3 inhibited the labeling of the 55 kDa protein only. The results provide direct evidence for endothelin receptor coupling to phospholipase C through guanine nucleotide binding (G) proteins. In addition, in C6 cells, endothelin-mediated phospholipase C activation is partially inhibited by pertussis toxin pretreatment. The endothelin receptor involved in phospholipase C stimulation in C6 cells seems to correspond to a 74 kDa protein which binds ET-1 but not ET-3.
Gusovsky
F
F
Laboratory of Bioorganic Chemistry, NIDDK, NIH, Bethesda, MD 20892.
D016475:3T3 Cells; D000818:Animals; D003432:Cross-Linking Reagents; D016232:Endothelins / Q000494:pharmacology*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D005910:Glioma; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D007295:Inositol Phosphates / Q000378:metabolism*; D051379:Mice; D037342:Pertussis Toxin; D011956:Receptors, Cell Surface / Q000378:metabolism*; D017466:Receptors, Endothelin; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D003432:Cross-Linking Reagents; D016232:Endothelins; D007295:Inositol Phosphates; D011956:Receptors, Cell Surface; D017466:Receptors, Endothelin; D010566:Virulence Factors, Bordetella; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D037342:Pertussis Toxin; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins