Abstract
Vitamin E (alpha-tocopherol), one of the most important natural antioxidants, is assumed to be beneficial in the prevention of cardiovascular diseases. alpha-Tocopherol exhibits acyl-peroxyl-radical scavenger properties and exerts cell-mediated actions in the hemovascular compartment, such as inhibition of superoxide anion (O-2) production by leukocytes. The aim of this study was to examine the mechanism underlying the inhibitory effect of alpha-tocopherol on O-2 production by human monocytes. In activated monocytes O-2 is produced by the NADPH-oxidase enzyme complex. The oxidase activation elicited by phorbol myristate acetate (PMA) requires membrane translocation of several cytosolic factors. We found that in human PMA-stimulated adherent monocytes, alpha-tocopherol (but not beta-tocopherol) inhibited O-2 production in intact cells but had no effect on a membrane preparation containing activated NADPH-oxidase, suggesting that alpha-tocopherol impairs the assembly process of the enzyme complex. We showed that translocation and phosphorylation of the cytosolic factor p47(phox) were reduced in monocytes preincubated with alpha-tocopherol. We verified that the tryptic phosphopeptide map of monocyte p47(phox) was similar to that of neutrophil p47(phox), indicating that several serine residues were phosphorylated. Peptides whose phosphorylation is dependent on protein kinase C (PKC) were phosphorylated to a lesser degree when p47(phox) was immunoprecipitated from alpha-tocopherol-treated monocytes. In vitro, the activity of PKC from monocytes was inhibited by alpha-tocopherol in a specific manner compared with that of beta-tocopherol or Trolox(R). Membrane translocation of PKC was not affected. These results show that alpha-tocopherol inhibits O-2 production by human adherent monocytes by impairing the assembly of the NADPH-oxidase and suggest that the inhibition of phosphorylation and translocation of the cytosolic factor p47(phox) results from a decrease in PKC activity.
Highlights
Vitamin E (␣-tocopherol), one of the most important natural antioxidants, is assumed to be beneficial in the prevention of cardiovascular diseases. ␣-Tocopherol exhibits acyl-peroxyl-radical scavenger properties and exerts cell-mediated actions in the hemovascular compartment, such as inhibition of superoxide anion (O2.) production by leukocytes
We showed that translocation and phosphorylation of the cytosolic factor p47phox were reduced in monocytes preincubated with ␣-tocopherol
These results show that ␣-tocopherol inhibits O2. production by human adherent monocytes by impairing the assembly of the NADPH-oxidase and suggest that the inhibition of phosphorylation and translocation of the cytosolic factor p47phox results from a decrease in protein kinase C (PKC) activity
Summary
Materials—dl-␣-Tocopherol, dl--tocopherol, and PMA were from Sigma; SDS-PAGE reagents were from Bio-Rad; and [32P]orthophosphate and [␥-32P]ATP were from NEN Life Science Products. The membrane fraction containing active NADPH-oxidase was obtained by sonication of stimulated human neutrophils or monocytes (1 g/ml PMA) in 0.34 M sucrose ϩ 1 mM PMSF and ultracentrifugation (100,000 ϫ g for 30 min at 4 °C in a Beckman TL 100). Assay of PKC Activity—Resting adherent monocytes were scraped in lysis buffer (20 mM Tris-HCl, pH 7.5, 2 mM EDTA, 10 mM EGTA, 0.3 M sucrose, 2 mM dithiothreitol, 2 mM PMSF, 10 g/ml leupeptin, 10 g/ml pepstatin) at 3 ϫ 107 cells/ml. They were sonicated and ultracentrifuged to obtain the cytosolic fraction as described above. Control (100%) corresponds to 30.8 Ϯ 3.6 nmol/min/mg of protein (n ϭ 3)
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