The enhancement by wortmannin of protein kinase C-dependent activation of phospholipase D in vascular endothelial cells.
Phosphatidic acid generation by phospholipase D (PLD) activation has been implicated in agonist- and oxidant-mediated endothelial cell signal transduction. We examined the effect of wortmannin on PLD activation in pulmonary artery endothelial and smooth muscle cells in culture. Pretreatment of bovine pulmonary artery endothelial cells (BPAECs) with wortmannin potentiated TPA- (100 nM), ATP- (100 microM), and bradykinin- (1 microM) induced [32P]PEt formation, an index of PLD activation. However, wortmannin by itself had no effect on PLD activity. The potentiating effect of wortmannin on TPA-induced PLD activation was dose- (1-10 microM) and time-dependent (5-30 min) and was inhibited by bisindoylmalemide, an inhibitor of protein kinase C (PKC). Furthermore, down-regulation of PKC by prolonged treatment with TPA (100 nM, 18 h) attenuated the wortmannin effect. This effect of wortmannin was specific for TPA- or agonist-induced PLD activation as no potentiation of [32P]PEt formation was observed with H2O2 (1 mM) or ionomycin (1 microM). The effect of wortmannin was not due to activation of PKC alpha as determined by western blot analysis of PKC alpha in the cytosol and membrane fractions. Also, genistein, an inhibitor of tyrosine kinases, did not attenuate the wortmannin-mediated potentiation of PLD thereby suggesting non-involvement of protein tyrosine phosphorylation. These results indicate that wortmannin potentiates PKC-dependent stimulation of PLD in vascular endothelial cells.
Natarajan
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V
Department of Medicine, Indiana University School of Medicine, Indianapolis 46202, USA.
Vepa
S
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al-Hassani
M
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Scribner
W M
WM
D000730:Androstadienes / Q000494:pharmacology*; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019833:Genistein; D020404:Glycerophospholipids; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology; D007529:Isoflavones / Q000494:pharmacology; D009025:Morpholines / Q000494:pharmacology; D016877:Oxidants / Q000494:pharmacology; D010712:Phosphatidic Acids / Q000096:biosynthesis; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology*; D010739:Phospholipase D / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D012680:Sensitivity and Specificity; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D000077191:Wortmannin
D000730:Androstadienes; D002867:Chromones; D004791:Enzyme Inhibitors; D020404:Glycerophospholipids; D007527:Isoenzymes; D007529:Isoflavones; D009025:Morpholines; D016877:Oxidants; D010712:Phosphatidic Acids; D010726:Phosphodiesterase Inhibitors; D010761:Phosphorus Radioisotopes; C051521:phosphatidylethanol; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D019833:Genistein; D011493:Protein Kinase C; D010739:Phospholipase D; D013755:Tetradecanoylphorbol Acetate; D000077191:Wortmannin