Rapid inactivation of NOS-I by lipopolysaccharide plus interferon-gamma-induced tyrosine phosphorylation.
Human astrocytoma T67 cells constitutively express a neuronal NO synthase (NOS-I) and, following administration of lipopolysaccharide (LPS) plus interferon-gamma (IFNgamma), an inducible NOS isoform (NOS-II). Previous results indicated that a treatment of T67 cells with the combination of LPS plus IFNgamma, by affecting NOS-I activity, also inhibited NO production in a very short time. Here, we report that under basal conditions, a NOS-I protein of about 150 kDa was weakly and partially tyrosine-phosphorylated, as verified by immunoprecipitation and Western blotting. Furthermore, LPS plus IFNgamma increased the tyrosine phosphorylation of NOS-I, with a concomitant inhibition of its enzyme activity. The same effect was observed in the presence of vanadate, an inhibitor of phosphotyrosine-specific phosphatases. On the contrary, genistein, an inhibitor of protein-tyrosine kinases, reduced tyrosine phosphorylation of NOS-I, enhancing its enzyme activity. Finally, using reverse transcriptase-polymerase chain reaction, we have observed that a suboptimal induction of NOS-II mRNA expression in T67 cells was enhanced by vanadate (or L-NAME) and inhibited by genistein. Because exogenous NO has been found to suppress NOS-II expression, the decrease of NO production that we have obtained from the inactivation of NOS-I by LPS/IFNgamma-induced tyrosine phosphorylation provides the best conditions for NOS-II expression in human astrocytoma T67 cells.
Colasanti
M
M
Department of Biology, University of Rome, ROMA TRE, Viale Marconi 446, I-00146 Rome, Italy.
Persichini
T
T
Cavalieri
E
E
Fabrizi
C
C
Mariotto
S
S
Menegazzi
M
M
Lauro
G M
GM
Suzuki
H
H
D001254:Astrocytoma / Q000378:metabolism; D004790:Enzyme Induction; D019833:Genistein / Q000494:pharmacology; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000096:biosynthesis* / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D052247:Nitric Oxide Synthase Type II; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000378:metabolism*; D014638:Vanadates / Q000494:pharmacology
D008070:Lipopolysaccharides; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D014638:Vanadates; D014443:Tyrosine; D007371:Interferon-gamma; D019833:Genistein; C496320:NOS1 protein, human; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; D052247:Nitric Oxide Synthase Type II