Thrombin-mediated phosphoinositide hydrolysis in Chinese hamster ovary cells overexpressing phospholipase C-delta 1.
The regulatory mechanism(s) of a phosphoinositide-specific phospholipase C, PLC-delta 1, was investigated using a clone of stably overexpressed PLC-delta 1 (PLC delta 30 cells) in Chinese hamster ovary cells. Thrombin stimulation of PLC delta 30 cells exhibited 6.5-fold increase in total inositol phosphates (InsP), which was significantly higher than that in the vector-transfected (V1) cells (2.0-fold). AIF-4 increased InsP accumulation in both V1 and PLC delta 30 cells, and pertussis toxin partially blocked InsP accumulation in thrombin-stimulated PLC delta 30 cells. Guanosine thiotriphosphate (GTP gamma S) markedly potentiated thrombin-stimulated InsP generation in permeabilized PLC delta 30 cells compared with V1 cells, suggesting possible involvement of a G-protein (s) in the activation of PLC-delta 1. In PLC delta 30 cells, ionomycin-induced significant InsP generation and thrombin-stimulated InsP generation were completely inhibited by addition of EGTA. Furthermore, the stimulatory effects of thrombin plus GTP gamma S in PLC delta 30 cells were more sensitive to change in free calcium concentration than in V1 cells. Suppression by 12-O-tetradecanoylphorbol 13-acetate of thrombin-stimulated InsP accumulation was not affected by increasing Ca2+ concentration. These results indicate that thrombin-induced PLC-delta 1 activation is regulated via both G-protein(s) and calcium.
Banno
Y
Y
Department of Biochemistry, Gifu University School of Medicine, Japan.
Okano
Y
Y
Nozawa
Y
Y
D000818:Animals; D015153:Blotting, Western; D001920:Bradykinin / Q000494:pharmacology; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D003001:Cloning, Molecular; D006224:Cricetinae; D003600:Cytosol / Q000201:enzymology; D004533:Egtazic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006868:Hydrolysis; D015151:Immunoblotting; D007294:Inositol / Q000378:metabolism; D007527:Isoenzymes / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010716:Phosphatidylinositols / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D012969:Sodium Fluoride / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology*; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism*
D007527:Isoenzymes; D010716:Phosphatidylinositols; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D007294:Inositol; D004533:Egtazic Acid; D012969:Sodium Fluoride; D010727:Phosphoric Diester Hydrolases; D010738:Type C Phospholipases; D013917:Thrombin; D019204:GTP-Binding Proteins; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D013755:Tetradecanoylphorbol Acetate; D001920:Bradykinin; D002118:Calcium