Divergence in regulation of nitric-oxide synthase and its cofactor tetrahydrobiopterin by tumor necrosis factor-α -: Ceramide potentiates nitric oxide synthesis without affecting GTP cyclohydrolase I activity

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-cu (TNF-cu) concomitantly potentiated the stimulation of nitric oxide (NO) and BH4 production induced by IFN-gamma and interleukin-1 beta. 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-alpha. Indeed, we found that bacterial sphingomyelinase, which hydrolyzes sphingomyelin and increases endogenous ceramide, or the cell permeable ceramide analogue, C-2-ceramide, but not C-2-dihydroceramide (N-acetylsphinganine), significantly mimicked the effects of TNF-alpha on NO production and iNOS expression and activity in C6 cells. Surprisingly, although TNF-alpha increased BH4 synthesis and GTPCH activity, neither BH4 nor GTPCH expression was affected by C-2-ceramide or sphingomyelinase in IFN-gamma- and interleukin-1 beta-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-alpha or ceramide. Treatment with the antioxidant pyrrolidine dithiocarbamate, which is known to inhibit NF-kappa B and sphingomyelinase in C6 cells, or with the peptide SN-50, which blocks translocation of NF-kappa B to the nucleus, inhibited TNF-alpha-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.