GTP gamma S increases thrombin-mediated inositol trisphosphate accumulation in permeabilized human endothelial cells.
Ca2+-mobilizing agonists stimulate phospholipase C-mediated phosphatidylinositol 4,5-bisphosphate hydrolysis and inositol trisphosphate (IP3) formation in pulmonary as well as in peripheral vascular endothelial cells (EC). In general, it is believed that receptor-phospholipase C interactions involve a guanine nucleotide regulatory (G) protein. This interaction can be inhibited by Bordetella pertussis toxin in certain cells. Here we report that pertussis toxin catalyzes the [32P]ADP ribosylation of a Mr = 41,000 protein in human umbilical vein EC. However, prior EC treatment with pertussis toxin (250 ng/ml for 20 h) does not inhibit thrombin-induced Ca2+ flux or IP3 formation, despite markedly attenuating the radiolabeling of the Mr = 41,000 protein (less than 5% control). Treatment of digitonin-permeabilized human umbilical vein EC with GTP gamma S, a stable GTP analog, or AIF4-, but not with GDP beta S, stimulates IP3 accumulation. However, GDP beta S inhibits GTP gamma S-induced IP3 accumulation. Although thrombin alone is not very effective in elevating IP3 levels in permeabilized EC, thrombin and GTP gamma S act in a synergistic fashion to increase IP3 accumulation. Overall, these observations are interpreted to indicate that a pertussis toxin-insensitive G protein is a key intermediate in the signaling pathway linking thrombin receptors to phospholipase C in human umbilical vein EC.
Brock
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TA
Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts.
Capasso
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D002118:Calcium / Q000378:metabolism; D002199:Capillary Permeability; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D004789:Enzyme Activation; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000494:pharmacology; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D037342:Pertussis Toxin; D011956:Receptors, Cell Surface / Q000378:metabolism; D013873:Thionucleotides / Q000494:pharmacology*; D013917:Thrombin / Q000494:pharmacology*; D010738:Type C Phospholipases / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D011956:Receptors, Cell Surface; D013873:Thionucleotides; D010566:Virulence Factors, Bordetella; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D015544:Inositol 1,4,5-Trisphosphate; D006160:Guanosine Triphosphate; D037342:Pertussis Toxin; D010738:Type C Phospholipases; D013917:Thrombin; D002118:Calcium