(1) The removal of the nuclear envelope from isolated rat-liver nuclei by washing with Triton X-100 (TX-100) was assessed by electron microscopy. All the envelope was removed by 0.04 % (w\v) TX-100. (2) After this removal, phosphorylation of inositol lipids and diacylglycerol (DAG) from [γ-$#P]ATP still occurs, despite the near complete absence of detectable (by mass assay) DAG and PtdIns. This suggests that the majority of these two lipids in nuclei are present in the nuclear membrane, but the small amounts remaining after extraction, defined as intranuclear, are available for phosphorylation by lipid kinases (36 % for DAG and 24 % for PtdIns respectively, when expressed as a percentage of incorporation of intact nuclei). (3) PtdIns(4,5)P # did not follow the same pattern as PtdIns and DAG ; after removal of the nuclear membrane, 40 % of the mass of this lipid was left in the nucleus. Moreover, a similar amount of PtdIns(4,5)P # was also resistant to extraction with even higher concentrations of detergent, suggesting that PtdIns(4,5)P # has a discrete intranuclear location, probably bound to nuclear proteins. (4) Addition of exogenous substrates, PtdIns, PtdIns(4)P and DAG, to membrane-depleted nuclei resulted in reconstitution of the majority of lipid phosphorylations from [γ-$#P]ATP (70 %, 90% and 94 % of intact nuclei respectively),

In C6 glioma cells, the sphingolipid second messenger ceramide potentiates expression of inducible nitric-oxide synthase (iNOS) induced by tumor necrosis factor ␣ (TNF-␣) without affecting GTP cyclohydrolase I (GT-PCH), the rate-limiting enzyme in the biosynthesis of 6(R)-5,6,7,8-tetrahydrobiopterin (BH 4 ), a cofactor required for iNOS activity. TNF-␣ also stimulates sphingosine kinase, the enzyme that phosphorylates sphingosine to form sphingosine-1-phosphate (SPP), a further metabolite of ceramide. Several clones of C6 cells, expressing widely varying levels of sphingosine kinase, were used to examine the role of SPP in regulation of GTPCH and BH 4 biosynthesis. Overexpression of sphingosine kinase, with concomitant increased endogenous SPP levels, potentiated the effect of TNF-␣ on GTPCH expression and activity and BH 4 biosynthesis. In contrast, enforced expression of sphingosine kinase had no effect on iNOS expression or NO formation. Furthermore, N,N-dimethylsphingosine, a potent sphingosine kinase inhibitor, completely eliminated the increased GTPCH activity and expression induced by TNF-␣. Surprisingly, we found that, although C6 cells can secrete SPP, which is enhanced by TNF-␣, treatment of C6 cells with exogenous SPP or dihydro-SPP had no affect on BH 4 biosynthesis. However, both SPP and dihydro-SPP markedly stimulated ERK 1/2 in C6 cells, which express cell surface SPP receptors. Interestingly, although this ERK activation was blocked by PD98059, which also reduced cellular proliferation induced by enforced expression of sphingosine kinase, PD98059 had no effect on GTPCH activity. Collectively, these results suggest that only intracellularly generated SPP plays a role in regulation of GTPCH and BH 4 levels. 5,6,7, 1 is the obligate cofactor for the aromatic L-amino hydroxylases and is also required for activity of all nitric-oxide synthase isoforms (reviewed in Ref. 1). It has been proposed that BH 4 may also have cofactorindependent roles (2), including inhibition of cytokine-induced apoptosis (3) and stimulation of dopamine release (4). Cellular levels of BH 4 are regulated by the activity of GTP cyclohydrolase I (GTPCH), the first and rate-limiting enzyme in the BH 4 biosynthetic pathway (5). Its expression is increased by proinflammatory cytokines, such as IFN-␥ and TNF-␣, and is coordinately regulated with cytokine-inducible nitric-oxide synthase (iNOS) (6). BH 4 binds to NOS monomers, promoting their dimerization and subsequent activation (7), and recent crystallographic analysis suggests that it may play a direct role in the NOS reaction in a radical form (8). 6(R)-GTPCH, a homodecamer of 30-kDa subunits arranged as two pentamers facing one another (9), catalyzes the rearrangement of GTP to dihydroneopterin triphosphate. This intermediate is then converted to BH 4 in two subsequent reactions catalyzed by 6-pyruvoyltetrahydropterin synthase and sepiapterin reductase, respectively, neither of which are rate-limiting. The signal transduction pathways that regulate induction of GTPCH are not wel...

No abstract

Synthesis of 6(R)-5,6,7,8-tetrahydrobiopterin (BH 4 ), 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 BH 4 production induced by IFN-␥ and interleukin-1␤. Expression of both iNOS and GTP cyclohydrolase I (GTPCH), the rate-limiting enzyme in the BH 4 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, C 2 -ceramide, but not C 2 -dihydroceramide (N-acetylsphinganine), significantly mimicked the effects of TNF-␣ on NO production and iNOS expression and activity in C6 cells. Surprisingly, although TNF-␣ increased BH 4 synthesis and GTPCH activity, neither BH 4 nor GTPCH expression was affected by C 2 -ceramide or sphingomyelinase in IFN-␥-and interleukin-1␤-stimulated cells. It is likely that increased BH 4 levels results from increased GTPCH protein and activity in vivo rather than from reduced turnover of BH 4 , because the GTPCH inhibitor, 2,4-diamino-6-hydroxypyrimidine, blocked cytokine-stimulated BH 4 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 BH 4 biosynthesis, may be regulated by discrete pathways. As BH 4 is also a cofactor for the aromatic amino acid hydroxylases, discovery of distinct mechanisms for regulation of BH 4 and NO has important implications for its specific functions.