Involvement of tyrosine phosphorylation in inhibition of fMLP-induced PLD activation by N-acetyl-L-cysteine in differentiated HL60 cells

J Leukoc Biol. 1998 Jun;63(6):781-9. doi: 10.1002/jlb.63.6.781.

Abstract

N-acetyl-L-cysteine (NAC), which is known as a multipotential agent; an antioxidant, a thiol reagent, or a tyrosine kinase inhibitor, inhibited N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced phospholipase D (PLD) activation in HL60 cells in a concentration-dependent manner (IC50 = 2 mM). Its inhibitory mechanism was examined in this study to gain insight into the regulation of PLD activity. NAC had no direct effect on membrane PLD activity in an in vitro assay system. fMLP-induced formation of inositol phosphates via phospholipase C (PLC) was not affected by the drug, suggesting that the receptor-G protein coupling was not inhibited. H2O2, which is known to induce PLD activation in several types of cells, failed to activate PLD in HL60 cells. Pretreatment of 3-amino-1,2,4-triazole (ATZ), a catalase inhibitor, did not enhance fMLP-induced PLD activation. NAC inhibited fMLP-induced tyrosine phosphorylation of several protein bands (42, 44, 64, and 138 kDa) in a concentration-dependent manner. The temporal and concentration-dependent inhibitory profiles for tyrosine phosphorylation of 64- and 138-kDa proteins were well correlated with PLD activation. However, thiol reagents, 1 mM 2,3-dimercapto-l-propanol (2,3-DMP), 1 mM dithiothreitol (DTT), and 2 mM cysteine also did not suppress protein tyrosine phosphorylation or PBut formation by fMLP. Wortmannin, a selective phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor, inhibited these two tyrosine phosphorylation bands. These results suggest that NAC inhibits fMLP-induced PLD activation through blockage of protein tyrosine phosphorylation, which is located at the downstream of PI-3 kinase.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylcysteine / pharmacology*
  • Androstadienes / pharmacology
  • Cell Differentiation / drug effects
  • Chelating Agents / pharmacology
  • Cysteine / pharmacology
  • Dithiothreitol / pharmacology
  • Enzyme Activation / drug effects
  • Free Radical Scavengers / pharmacology*
  • HL-60 Cells / cytology
  • HL-60 Cells / drug effects
  • HL-60 Cells / enzymology
  • Humans
  • Inositol Phosphates / metabolism
  • N-Formylmethionine Leucyl-Phenylalanine / antagonists & inhibitors*
  • N-Formylmethionine Leucyl-Phenylalanine / pharmacology*
  • Oxidants / pharmacology
  • Phosphodiesterase Inhibitors / pharmacology
  • Phospholipase D / drug effects
  • Phospholipase D / metabolism*
  • Phosphorylation
  • Sulfhydryl Reagents / pharmacology
  • Tyrosine / metabolism*
  • Unithiol / pharmacology
  • Wortmannin

Substances

  • Androstadienes
  • Chelating Agents
  • Free Radical Scavengers
  • Inositol Phosphates
  • Oxidants
  • Phosphodiesterase Inhibitors
  • Sulfhydryl Reagents
  • Unithiol
  • Tyrosine
  • N-Formylmethionine Leucyl-Phenylalanine
  • Phospholipase D
  • Cysteine
  • Dithiothreitol
  • Acetylcysteine
  • Wortmannin