Effect of wortmannin and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) on N-formyl-methionyl-leucyl-phenylalanine-induced phospholipase D activation in differentiated HL60 cells: possible involvement of phosphatidylinositol 3-kinase in phospholipase D activation.
Phospholipase D (PLD) plays an important role in neutrophil activation. However, despite various proposed mechanisms, its detailed regulatory mechanism is not fully understood. The functional coupling between phosphatidylinositol 3-kinase (PI 3-kinase) and PLD was investigated in N-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated human promyelocytic leukemia HL60 cells, using wortmannin, a fungal metabolite that is known as a selective inhibitor for phosphatidylinositol 3-kinase. Treatment of cells with this drug inhibited the formation of both phosphatidylinositol 3,4,5-trisphosphate (PIP3), a product of PI 3-kinase, and phosphatidylbutanol (PBut), the specific product of transphosphatidylation due to PLD in the presence of butanol, with similar concentration dependence (IC50 = 30-70 nM). Another PI 3-kinase inhibitor, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) also inhibited PBut formation in a concentration-dependent manner. However, wortmannin failed to inhibit phorbol 12-myristate 13-acetate-induced PLD activation in whole cells and membrane PLD activity in an in vitro assay system, indicating that inhibition of fMLP-induced PLD activation by wortmannin was not due to its direct effect on PLD activity. These results suggest that a major part of inhibition of PLD activation by wortmannin might be mediated through its effect on PI 3-kinase.
Nakamura
M
M
Department of Pharmacy, Gifu University Hospital, Japan.
Nakashima
S
S
Katagiri
Y
Y
Nozawa
Y
Y
D000730:Androstadienes / Q000494:pharmacology*; D002454:Cell Differentiation; D002462:Cell Membrane / Q000187:drug effects; D002867:Chromones / Q000494:pharmacology*; D004789:Enzyme Activation; D018922:HL-60 Cells; D006801:Humans; D009025:Morpholines / Q000494:pharmacology*; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019869:Phosphatidylinositol 3-Kinases; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology*; D010739:Phospholipase D / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors; D013755:Tetradecanoylphorbol Acetate; D000077191:Wortmannin
D000730:Androstadienes; D002867:Chromones; D009025:Morpholines; D018129:Phosphatidylinositol Phosphates; D010726:Phosphodiesterase Inhibitors; C060974:phosphatidylinositol 3,4,5-triphosphate; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D010739:Phospholipase D; D013755:Tetradecanoylphorbol Acetate; D000077191:Wortmannin