Wortmannin is a potent phosphatidylinositol 3-kinase inhibitor: the role of phosphatidylinositol 3,4,5-trisphosphate in neutrophil responses.
Phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3) is rapidly produced upon exposure of neutrophils to the chemoattractant N-formylmethionyl-leucylphenylalanine (fMLP), and has been proposed to act as a second messenger mediating actin polymerization and respiratory-burst activity. Here we present evidence that wortmannin, a known inhibitor of respiratory-burst activity, acts on PtdIns 3-kinase, the enzyme producing PtdInsP3 from PtdIns(4,5)P2. Pretreatment of 32P-labelled human neutrophils with 100 nM wortmannin totally abolished fMLP-mediated PtdInsP3 production, raised PtdInsP2 levels, and did not affect cellular PtdInsP and PtdIns contents. The inhibitory effect on PtdInsP3 formation in intact cells was dose-dependent, with an IC50 of approximately 5 nM. Similar results were obtained with PtdIns 3-kinase immunoprecipitated by antibodies against the p85 regulatory subunit: wortmannin totally inhibited PtdIns3P production in immunoprecipitates at concentrations of 10-100 nM (IC50 approximately 1 nM). These results illustrate the direct and specific inhibition of PtdIns 3-kinase by wortmannin. Since agonist-mediated respiratory-burst activation is most sensitive to wortmannin (IC50 = 12 nM), this suggests that agonist-mediated PtdInsP3 formation is indispensable for this cell response. Neutrophils pretreated with wortmannin develop oscillatory changes in F-actin content, but actin polymerization in response to fMLP is not inhibited. This, and the absence of PtdInsP3 under these conditions, are in agreement with a modulatory role for PtdInsP3 in cytoskeletal rearrangements, but imply that PtdInsP3 production is not a primary event triggering elongation of actin filaments in neutrophils.
Arcaro
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A
Institute of Biochemistry, University of Fribourg, Switzerland.
Wymann
M P
MP
D000730:Androstadienes / Q000494:pharmacology*; D001345:Autoradiography; D006801:Humans; D007700:Kinetics; D008163:Luminescent Measurements; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D010101:Oxygen Consumption / Q000187:drug effects; D010710:Phosphates / Q000097:blood; D019869:Phosphatidylinositol 3-Kinases; D018129:Phosphatidylinositol Phosphates / Q000097:blood*; D010716:Phosphatidylinositols / Q000097:blood*; D010739:Phospholipase D / Q000037:antagonists & inhibitors; D010743:Phospholipids / Q000097:blood / Q000302:isolation & purification; D010761:Phosphorus Radioisotopes; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D000077191:Wortmannin
D000730:Androstadienes; D010710:Phosphates; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols; D010743:Phospholipids; D010761:Phosphorus Radioisotopes; C060974:phosphatidylinositol 3,4,5-triphosphate; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D010739:Phospholipase D; D013755:Tetradecanoylphorbol Acetate; D000077191:Wortmannin