Alpha-tocopherol modulates tyrosine phosphorylation in human neutrophils by inhibition of protein kinase C activity and activation of tyrosine phosphatases.
Alpha-tocopherol augmentation in human neutrophils was investigated for effects on neutrophil activation and tyrosine phosphorylation of proteins, through its modulation of protein kinase C (PKC) and tyrosine phosphatase activities. Incubation of neutrophils with alpha-tocopherol succinate (TS) resulted in a dose-dependent incorporation into cell membranes, up to 2.5 nmol/2x10(6) cells. A saturating dose of TS (40 micromol/l) inhibited oxidant production by neutrophils stimulated with phorbol myristate acetate (PMA) or opsonized zymosan (OZ) by 86 and 57%, as measured by luminol-amplified chemiluminescence (CL). With PMA, TS inhibited CL generation to a similar extent to staurosporine (10 nmol/l) or genistein (100 micromol/l), and much more than Trolox (40 micromol/l). With OZ, TS inhibited CL to a similar extent to Trolox. Neutrophil PKC activity was inhibited 50% or more by TS or staurosporine. The enzyme activity was unaffected by genistein or Trolox, indicating a specific interaction of alpha-tocopherol. TS or Trolox increased protein tyrosine phosphorylation in resting neutrophils, and as with staurosporine further increased tyrosine phosphorylation in PMA-stimulated neutrophils, while the tyrosine kinase (TK) inhibitor genistein diminished phosphorylation. These effects in resting or PMA-stimulated neutrophils were unrelated to protein tyrosine phosphatase (PTP) activities, which were maintained or increased by TS or Trolox. In OZ-stimulated neutrophils, on the other hand, all four compounds inhibited the increase in tyrosine-phosphorylated proteins. In this case, the effects of pre-incubation with TS or Trolox corresponded with partial inhibition of the marked (85%) decrease in PTP activity induced by OZ. These results indicate that alpha-tocopherol inhibits PMA-activation of human neutrophils by inhibition of PKC activity, and inhibits tyrosine phosphorylation and activation of OZ-stimulated neutrophils also through inhibition of phosphatase inactivation.
Chan
S S
SS
Instituto de Quimica, Universidade de São Paulo, Brazil.
Monteiro
H P
HP
Schindler
F
F
Stern
A
A
Junqueira
V B
VB
D002839:Chromans / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D015151:Immunoblotting; D009504:Neutrophils / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism*; D016877:Oxidants / Q000378:metabolism; D010126:Ozone / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D024502:alpha-Tocopherol / Q000494:pharmacology*
D002839:Chromans; D016877:Oxidants; D019000:Phosphotyrosine; D010126:Ozone; D011493:Protein Kinase C; D017027:Protein Tyrosine Phosphatases; D024502:alpha-Tocopherol; D013755:Tetradecanoylphorbol Acetate; C010643:6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid