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J Biol Chem, Vol. 275, Issue 18, 13275-13281, May 5, 2000

Divergence in Regulation of Nitric-oxide Synthase and Its Cofactor Tetrahydrobiopterin by Tumor Necrosis Factor-
CERAMIDE POTENTIATES NITRIC OXIDE SYNTHESIS WITHOUT AFFECTING GTP CYCLOHYDROLASE I ACTIVITY^*

Lewis R. Vann, Sharon Twitty, Sarah Spiegel, and Sheldon Milstien^§

From the Laboratory of Cellular and Molecular Regulation, NIMH, National Institutes of Health, Bethesda, Maryland 20892 and the  Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, Washington, D. C. 20007

Synthesis of 6(R)-5,6,7,8-tetrahydrobiopterin (BH₄), a required cofactor for inducible nitric-oxide synthase (iNOS) activity, is usually coordinately regulated with iNOS expression. In C6 glioma cells, tumor necrosis factor- (TNF-) concomitantly potentiated the stimulation of nitric oxide (NO) and BH₄ production induced by IFN- and interleukin-1. Expression of both iNOS and GTP cyclohydrolase I (GTPCH), the rate-limiting enzyme in the BH₄ biosynthetic pathway, was also markedly increased, as were their activities and protein levels. Ceramide, a sphingolipid metabolite, may mediate some of the actions of TNF-. Indeed, we found that bacterial sphingomyelinase, which hydrolyzes sphingomyelin and increases endogenous ceramide, or the cell permeable ceramide analogue, C₂-ceramide, but not C₂-dihydroceramide (N-acetylsphinganine), significantly mimicked the effects of TNF- on NO production and iNOS expression and activity in C6 cells. Surprisingly, although TNF- increased BH₄ synthesis and GTPCH activity, neither BH₄ nor GTPCH expression was affected by C₂-ceramide or sphingomyelinase in IFN-- and interleukin-1-stimulated cells. It is likely that increased BH₄ levels results from increased GTPCH protein and activity in vivo rather than from reduced turnover of BH₄, because the GTPCH inhibitor, 2,4-diamino-6-hydroxypyrimidine, blocked cytokine-stimulated BH₄ accumulation. Moreover, expression of the GTPCH feedback regulatory protein, which if decreased might increase GTPCH activity, was not affected by TNF- or ceramide. Treatment with the antioxidant pyrrolidine dithiocarbamate, which is known to inhibit NF-B and sphingomyelinase in C6 cells, or with the peptide SN-50, which blocks translocation of NF-B to the nucleus, inhibited TNF--dependent iNOS mRNA expression without affecting GTPCH mRNA levels. This is the first demonstration that cytokine-stimulated iNOS and GTPCH expression, and therefore NO and BH₄ biosynthesis, may be regulated by discrete pathways. As BH₄ is also a cofactor for the aromatic amino acid hydroxylases, discovery of distinct mechanisms for regulation of BH₄ and NO has important implications for its specific functions.

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