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 inter feron-gamma (IFN gamma), an inducible NOS isoform (NOS-II). Previous result s indicated that a treatment of T67 cells with the combination of LPS plus IFN gamma, by affecting NOS-I activity, also inhibited NO production in a v ery short time. Here, we report that under basal conditions, a NOS-I protei n of about 150 kDa was weakly and partially tyrosine-phosphorylated, as ver ified by immunoprecipitation and Western blotting. Furthermore, LPS plus IF N gamma increased the tyrosine phosphorylation of NOS-I, with a concomitant inhibition of its enzyme activity. The same effect was observed in the pre sence of vanadate, an inhibitor of phosphotyrosine-specific phosphatases. O n the contrary, genistein, an inhibitor of protein-tyrosine kinases, reduce d tyrosine phosphorylation of NOS-I, enhancing its enzyme activity. Finally , using reverse transcriptase-polymerase chain reaction, we have observed t hat a suboptimal induction of NOS-II mRNA expression in T67 cells was enhan ced by vanadate (or L-NAME) and inhibited by genistein. Because exogenous N O has been found to suppress NOS-II expression, the decrease of NO producti on that we have obtained from the inactivation of NOS-I by LPS/IFN gamma-in duced tyrosine phosphorylation provides the best conditions for NOS-II expr ession in human astrocytoma T67 cells.