Cytokines and lipopolysaccharides induce inducible nitric oxide synthase but not enzyme activity in adult rat cardiomyocytes

There is evidence that nitric oxide (NO) formation in adult cardiomyocytes stimulated with lipopolysaccharide (LPS) is not commensurate with iNOS levels. Tetrahydrobiopterin (BH(4)) is a key factor in the stabilization and NO production by iNOS homodimer. Thus we hypothesized that BH(4) is a limiting factor for NO production in adult cardiomyocytes in response to LPS and cytokines (TNF-alpha, IL-1, IFN-gamma alone, or mixed). It was verified that LPS and cytokines induced iNOS expression which did not translate into increased nitrite or [(14)C]citrulline production. This response coincided with defective BH(4) synthesis and low GTP cyclohydrolase activity. Furthermore, supplementation with BH(4) and ascorbate failed to increase iNOS activity. This effect was related to preferential accumulation of BH(2) rather than BH(4) in these cells. Uncoupled iNOS activity in stimulated cells was examined using mitochondrial aconitase activity as an endogenous marker of superoxide anion radical (O(2)(-)) formation, and found not to be significantly inhibited. 2-Hydroxyethidium also was not significantly increased. We conclude that adult cardiomyocytes are an unlikely source of NO and O(2)(-) in inflammatory conditions. This finding adds a new and unexpected layer of complexity to our understanding of the responses of the adult heart to inflammation.     

Exogenous NO suppresses flow-induced endothelium-derived NO production because of depletion of tetrahydrobiopterin

Exogenous nitric oxide (NO) suppresses endothelium-derived NO production. We were interested in determining whether this is also the case in flow-induced endothelium-derived NO production. If so, then is the mechanism because of intracellular depletion of tetrahydrobiopterin [BH4; a cofactor of NO synthase (NOS)], which results in superoxide production by uncoupled NOS? Isolated canine femoral arteries were perfused with 100 microM S-nitroso-N-acetylpenicillamine (SNAP; an NO donor) and/or 64 microM BH4. Perfusion of SNAP suppressed flow-induced NO production, which was evaluated as a change in the slope of the linear relationship between perfusion rate and NO production rate (P < 0.02 vs. control; n = 7). Subsequent BH4 perfusion returned the slope to the control level. Concomitant perfusion of SNAP and BH4 retained the control-level NO production (n = 7). Concomitant perfusion of SNAP and 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron; 1 mM; a membrane-permeable superoxide scavenger) also retained the control-level NO production (n = 7), whereas perfusion of Tiron after SNAP could not return the NO production to the control level (P < 0.02 vs. control; n = 7). We also found a significant decrease in BH4 concentration in the endothelial cells after SNAP perfusion. In conclusion, these results indicate that exogenous NO suppresses the flow-induced, endothelium-derived NO production by superoxide released from uncoupled NOS because of intracellular BH4 depletion.     

Deficient BH4 production via de novo and salvage pathways regulates NO responses to cytokines in adult cardiac myocytes

Adult rat cardiac myocytes typically display a phenotypic response to cytokines manifested by low or no increases in nitric oxide (NO) production via inducible NO synthase (iNOS) that distinguishes them from other cell types. To better characterize this response, we examined the expression of tetrahydrobiopterin (BH4)-synthesizing and arginine-utilizing genes in cytokine-stimulated adult cardiac myocytes. Intracellular BH4 and 7,8-dihydrobiopterin (BH2) and NO production were quantified. Cytokines induced GTP cyclohydrolase and its feedback regulatory protein but with deficient levels of BH4 synthesis. Despite the induction of iNOS protein, cytokine-stimulated adult cardiac myocytes produced little or no increase in NO versus unstimulated cells. Western blot analysis under nonreducing conditions revealed the presence of iNOS monomers. Supplementation with sepiapterin (a precursor of BH4) increased BH4 as well as BH2, but this did not enhance NO levels or eliminate iNOS monomers. Similar findings were confirmed in vivo after treatment of rat cardiac allograft recipients with sepiapterin. It was found that expression of dihydrofolate reductase, required for full activity of the salvage pathway, was not detected in adult cardiac myocytes. Thus, adult cardiac myocytes have a limited capacity to synthesize BH4 after cytokine stimulation. The mechanisms involve posttranslational factors impairing de novo and salvage pathways. These conditions are unable to support active iNOS protein dimers necessary for NO production. These findings raise significant new questions about the prevailing understanding of how cytokines, via iNOS, cause cardiac dysfunction and injury in vivo during cardiac inflammatory disease states since cardiac myocytes are not a major source of high NO production.     

A novel high-throughput screening assay for discovery of molecules that increase cellular tetrahydrobiopterin

Tetrahydrobiopterin (BH(4)) is an essential cofactor for the nitric oxide (NO) synthases and the aromatic amino acid hydroxylases. Insufficient BH(4) has been implicated in various cardiovascular and neurological disorders. GTP cyclohydrolase 1 (GTPCH-1) is the rate-limiting enzyme for de novo biosynthesis of BH(4). The authors have recently shown that the interaction of GTPCH-1 with GTP cyclohydrolase feedback regulatory protein (GFRP) inhibits endothelial GTPCH-1 enzyme activity, BH(4) levels, and NO production. They propose that agents that disrupt the GTPCH-1/GFRP interaction can increase cellular GTPCH-1 activity, BH(4) levels, and NO production. They developed and optimized a novel time-resolved fluorescence resonance energy transfer (TR-FRET) assay to monitor the interaction of GTPCH-1 and GFRP. This assay is highly sensitive and stable and has a signal-to-background ratio (S/B) greater than 12 and a Z' factor greater than 0.8. This assay was used in an ultra-high-throughput screening (uHTS) format to screen the Library of Pharmacologically Active Compounds. Using independent protein-protein interaction and cellular activity assays, the authors identified compounds that disrupt GTPCH-1/GFRP binding and increase endothelial cell biopterin levels. Thus, this TR-FRET assay could be applied in future uHTS of additional libraries to search for molecules that increase GTPCH-1 activity and BH(4) levels.     

Insulin increases NO-stimulated guanylate cyclase activity in cultured VSMC while raising redox potential

Insulin acutely stimulates cyclic guanosine monophosphate (cGMP) production in primary confluent cultured vascular smooth muscle cells (VSMC) from canine femoral artery, but the mechanism is not known. These cells contain the inducible isoform of nitric oxide (NO) synthase (iNOS), and insulin-stimulated cGMP production in confluent cultured cells is blocked by the NOS inhibitor, N(G)-monomethyl-L-arginine (L-NMMA). In the present study, it is shown that iNOS is also present in freshly dispersed VSMC from this artery, indicating that iNOS expression in cultured VSMC is not an artifact of the culture process. Insulin did not stimulate NOS activity in primary confluent cultured cells because it did not affect citrulline or combined NO(-)(3)/NO(-)(2) production. To see whether insulin required the permissive presence of NO to stimulate cGMP production, iNOS and basal cGMP production were inhibited with L-NMMA, and the cells were incubated with or without 1 nM insulin and/or the NO donor, S-nitroso-N-acetyl-D,L-penicillamine (SNAP) at a concentration (0.1 microM) that restored cGMP production to the basal value. In the presence of L-NMMA, insulin no longer affected cGMP production but when insulin was added to L-NMMA plus SNAP, cGMP production was increased by 69% (P < 0.05 vs. L-NMMA plus SNAP). Insulin, which increases glucose uptake by these cells, increased the cell lactate content and the lactate-to-pyruvate ratio (LPR) by 81 and 97%, respectively (both P < 0.05), indicating that the hormone increased aerobic glycolysis and the redox potential. The effects of insulin on LPR and cGMP production were blocked by removing glucose or by adding 2-deoxyglucose to the incubation media and were duplicated by the reducing substrate, beta-hydroxybutyrate. We conclude that insulin does not acutely affect iNOS activity in these VSMC but it does augment cGMP production induced by the NO already present in the cell while increasing aerobic glycolysis and the cell redox potential.     

Structural basis of biopterin-induced inhibition of GTP cyclohydrolase I by GFRP, its feedback regulatory protein

GTP cyclohydrolase I (GTPCHI) is the rate-limiting enzyme involved in the biosynthesis of tetrahydrobiopterin, a key cofactor necessary for nitric oxide synthase and for the hydroxylases that are involved in the production of catecholamines and serotonin. In animals, the GTPCHI feedback regulatory protein (GFRP) binds GTPCHI to mediate feed-forward activation of GTPCHI activity in the presence of phenylalanine, whereas it induces feedback inhibition of enzyme activity in the presence of biopterin. Here, we have reported the crystal structure of the biopterin-induced inhibitory complex of GTPCHI and GFRP and compared it with the previously reported phenylalanine-induced stimulatory complex. The structure reveals five biopterin molecules located at each interface between GTPCHI and GFRP. Induced fitting structural changes by the biopterin binding expand large conformational changes in GTPCHI peptide segments forming the active site, resulting in inhibition of the activity. By locating 3,4-dihydroxy-phenylalanine-responsive dystonia mutations in the complex structure, we found mutations that may possibly disturb the GFRP-mediated regulation of GTPCHI.     

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

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-alpha (TNF-alpha) concomitantly potentiated the stimulation of nitric oxide (NO) and BH(4) production induced by IFN-gamma and interleukin-1beta. 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-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 BH(4) synthesis and GTPCH activity, neither BH(4) nor GTPCH expression was affected by C(2)-ceramide or sphingomyelinase in IFN-gamma- and interleukin-1beta-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-alpha or ceramide. Treatment with the antioxidant pyrrolidine dithiocarbamate, which is known to inhibit NF-kappaB and sphingomyelinase in C6 cells, or with the peptide SN-50, which blocks translocation of NF-kappaB 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 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.     

IL-15 up-regulates iNOS expression and NO production by gingival epithelial cells

To investigate the biological activity of epithelial cells in view of host defense, we analyzed the mRNA expression of inducible NOS (iNOS) as well as NO production by human gingival epithelial cells (HGEC) stimulated with IL-15. RT-PCR analysis revealed that HGEC expressed IL-15 receptor alpha-chain mRNA. In addition, stimulation with IL-15 enhanced iNOS expression by HGEC through an increase of both mRNA and protein levels. Moreover, IL-15 up-regulated the production of NO(2)(-)/NO(3)(-), a NO-derived stable end product, from HGEC. The enhanced NO production by IL-15 was inhibited by AMT, an iNOS-specific inhibitor. These results suggest that IL-15 is a potent regulator of iNOS expression by HGEC and involved in innate immunity in the mucosal epithelium.     

Ascorbate enhances iNOS activity by increasing tetrahydrobiopterin in RAW 264.7 cells

Studies on the effect of ascorbic acid on inducible nitric oxide synthase (iNOS) activity are few and diverse, likely to be dependent on the species of cells. We investigated a role of ascorbic acid in iNOS induction and nitric oxide (NO) generation in mouse macrophage cell line RAW 264.7. Although interferon- (IFN-) gamma alone produced NO end products, ascorbic acid enhanced NO production only when cells were synergistically stimulated with IFN-gamma plus Escherichia coli lipopolysaccharide (LPS). Ascorbate neither enhanced nor decreased the expression of iNOS protein in RAW 264.7 cells, in contrast to the reports that ascorbic acid augments iNOS induction in a mouse macrophage-like cell line J774.1 and that ascorbate suppresses iNOS induction in rat skeletal muscle endothelial cells. Intracellular levels of tetrahydrobiopterin (BH4), a cofactor for iNOS, were increased by ascorbate in RAW 264.7 cells. However, ascorbate did not increase GTP cyclohydrolase I mRNA, the main enzyme at the critical steps in the BH4 synthetic pathway, expression levels and activity. Sepiapterin, which supplies BH4 via salvage pathway, more efficiently enhanced NO production if ascorbate was added. These data suggest that enhanced activation of iNOS by ascorbic acid is mediated by increasing the stability of BH4 in RAW 264.7 cells.     

iNOS/NO signaling regulates apoptosis induced by glycochenodeoxycholate in hepatocytes

Inducible nitric oxide synthase (iNOS) and nitric oxide (NO) can ameliorate apoptosis induced by toxic glycochenodeoxycholate (GCDC) in hepatocytes. However, the underlying molecular mechanisms are not yet understood in detail. This study is to clarify the function of iNOS/NO and its mechanisms during the apoptotic process. The apoptosis was brought about by GCDC in rat primary hepatocytes. iNOS/NO signaling was then investigated. iNOS inhibitor 1400 W enhanced the GCDC-induced apoptosis as reflected by caspase-3 activity and TUNEL assay. Exogenous NO regulated the apoptosis subsequent to NO donor S-nitroso-N-acetyl-penicillamine (SNAP) or sodium nitroprusside (SNP). The GCDC-induced apoptosis was decreased with 0.1 mM SNAP or 0.15 mM SNP, while it was increased with 0.8 mM SNAP or 1.2 mM SNP. The endogenous iNOS inhibited apoptosis, but the exogenous NO played a dual role during the GCDC-induced apoptosis. There was a potential iNOS/Akt/survivin axis that inhibited the hepatocyte apoptosis in low doses of NO donors. In contrast, high doses of NO donors activated CHOP through p38MAP-kinase (p38MAPK), upregulated TRAIL receptor DR5, and suppressed survivin. Consequently the high doses of NO donors promoted the apoptosis in hepatocytes. Our data suggest that the iNOS/NO signaling can modulate Akt/survivin and p38MAPK/CHOP pathways to mediate the GCDC-induced the apoptosis in hepatocytes. These signaling pathways may serve as targets for therapeutic intervention in cholestatic liver disease.     

Induction of glutathione peroxidase in response to inactivation by nitric oxide.

To determine effect of nitric oxide (NO) on cellular glutathione peroxidase (GPX) level in living cells, we measured the activity, protein and mRNA of GPX in rat kidney (KNRK) cells under a high NO condition. Combined treatment of lipopolysaccharide (LPS, 1 microgram/ml) and tumor necrosis factor-alpha (TNF-alpha, 50 ng/ml) synergistically enhanced (23-folds) nitrite production from KNRK cells. This was suppressed by an inducible NO synthase (iNOS) inhibitor (aminoguanidine, N-nitro-L-arginine methylester hydrochloride) and arginase. iNOS expression was detected by RT-PCR in the treated cells. GPX was inactivated irreversibly when the cells had been homogenized before exposure to a NO donor, S-nitroso-N-acetylpenicillamine (SNAP). In living KNRK cells, SNAP and LPS + TNF-alpha exerted a transient effect on the GPX activity. The treatment with SNAP (200 microM) or sodium nitroprusside (200 microM) enhanced GPX gene expression, which was blocked by a NO scavenger, 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide. GPX mRNA was markedly increased by the treatment with LPS + TNF-alpha, and aminoguanidine blocked the effect. In cells metabolically labeled with 75Se, LPS + TNF-alpha accelerated the incorporation of radioactivity into GPX molecule by 2.1-fold. These results suggest that inactivation of GPX by NO triggers a signal for inducing GPX gene expression in KNRK cells, thereby restoring the intracellular level of this indispensable enzyme.     

Vascular gene transfer of the human inducible nitric oxide synthase: characterization of activity and effects on myointimal hyperplasia.

BACKGROUND: Nitric oxide (NO) has been shown to decrease myointimal hyperplasia in injured blood vessels. We hypothesize inducible No synthase (iNOS) gene transfer even at low efficiency will provide adequate local no production to achieve this goal. MATERIALS AND METHODS: A retroviral vector containing the human iNOS cDNA (DFGiNOS) was used to transfer the iNOS gene into vascular cells and isolated blood vessels to answer the following questions: can vascular endothelial and smooth muscle cells support iNOS activity and will low efficiency iNOS gene transfer suppress myointimal hyperplasia in injured porcine arteries? RESULTS: DFGiNOS-infected sheep pulmonary artery endothelial cells (SPAEC) expressed significant iNOS mRNA and protein, releasing nitrite levels of 155.0 +/- 10.7 nmol/mg protein/24 h vs. 5.5 +/- 1.1 by control cells. Transduced rat smooth muscle cells (RSMC) also expressed abundant iNOS mRNA and protein, but, in contrast to SPAEC, NO synthesis was dependent on exogenous tetrahydrobiopterin (BH4) (291.8 +/- 10.4 nmol nitrite/mg protein/24 hr with BH4, 37.7 +/- 2.6 without BH4). Only porcine arteries infected with DFGiNOS following balloon injury exhibited a 3-fold increase in total NO synthesis and a 15-fold increase in cGMP levels over control vessels in a BH4 dependent fashion, despite only a 1% gene transfer efficiency. Transfer of iNOS completely prevented the 53% increase in myointimal thickness induced by balloon catheter injury; the administration of a NOS inhibitor reversed this effect. CONCLUSIONS: These in vitro findings suggest that vascular iNOS gene transfer may be feasible. Furthermore, a low gene transfer efficiency may be sufficient to inhibit myointimal hyperplasia following arterial balloon injury, although a source of BH4 may be required.     

Nitric oxide donors regulate nitric oxide synthase in bovine pulmonary artery endothelium

This study examined the notion that exogenous generation of nitric oxide (NO) modulates NOS gene expression and activity. Bovine pulmonary artery endothelial cells (BPAEC) were treated with the NO donors, 1 mM SNAP (S-nitroso-N-acetylpenicillamine), 0.5 mM SNP (sodium nitroprusside) or 0.2 microM NONOate (spermine NONOate) in medium 199 containing 2% FBS. Controls included untreated cells and cells exposed to 1 mM NAP (N-acetyl-D-penicillamine). NOS activity was assessed using a fibroblast-reporter cell assay; intracellular Ca2+ concentrations were assessed by Fura-2 microfluorometry; and NO release was measured by chemiluminescence. Constitutive endothelial (e) and inducible (i) NOS gene and protein expression were examined by northern and western blot analysis, respectively. Two hours exposure to either SNAP or NONOate caused a significant elevation in NO release from the endothelial cells (SNAP = 51.4 +/- 5.9; NONOate = 23.8 +/- 4.2; control = 14.5 +/- 2.8 microM); but A23187 (3 microM)-stimulated NO release was attenuated when compared to controls. Treatment with either SNAP or NONOate for 2 h also resulted in a significant increase in NOS activity in endothelial homogenates (SNAP = 23.6 +/- 2.5; NONOate= 29.8 +/- 7.7; control = 14.5 +/- 2.5fmol cGMP/microg per 10(6) cells). Exposure to SNAP and SNP, but not NONOate, for 1 h caused an increase in intracellular calcium. Between 4 and 8 h, SNAP and NONOate caused a 2- to 3-fold increase in eNOS, but not iNOS, gene (P < 0.05) and protein expression. NAP had little effect on either eNOS gene expression, activity or NO production. Our data indicate that exogenous generation of NO leads to a biphasic response in BPAEC, an early increase in intracellular Ca2+, and increases in NOS activity and NO release followed by increased expression of the eNOS gene, but not the iNOS gene. We conclude that eNOS gene expression and activity are regulated by a positive-feedback regulatory action of exogenous NO.     

Mechanism of vitamin E inhibition of cyclooxygenase activity in macrophages from old mice: role of peroxynitrite

Vitamin E inhibits cyclooxygenase activity in macrophages from old mice by reducing peroxynitrite production. PGE(2) is a proinflammatory mediator that has been linked to a variety of age-associated diseases such as cancer, arthritis, and cardiovascular disease. Furthermore in the aged, increased cyclooxygenase (COX)-2-mediated PGE(2) production contributes to decline in T-cell-mediated function. Previously we reported that increased macrophage PGE(2) production in the aged is due to higher COX-2 activity and that supplementation with vitamin E significantly reduced the age-associated increase in macrophage PGE(2) production posttranslationally without changing COX-2 expression. Peroxynitrite, a product of nitric oxide (NO) and superoxide (O(-)(2)), increases the activity of COX without affecting its expression. Thus, we investigated if vitamin E inhibits COX activity through decreasing peroxynitrite formation. Macrophages from old mice had higher PGE(2) levels, COX activity, and NO levels than those from young mice, all of which were significantly reduced by vitamin E. When added individually, inhibitors of NO and O(-)(2) did not significantly reduce COX activity; however, when the inhibitors were combined, COX activity was significantly reduced in macrophages from old mice fed 30 ppm vitamin E. Increasing NO levels alone using SNAP or O(-)(2) levels, using X/XO, had no effect; however, increasing peroxynitrite levels using Sin-1 or X/XO + SNAP significantly increased COX activity in macrophages from old mice fed 500, but not those fed 30 ppm vitamin E. These data strongly suggest that peroxynitrite plays an important role in the vitamin E-induced inhibition of COX activity. These findings have important implications for designing interventions to reverse and/or delay age-associated dysregulation of immune and inflammatory responses and diseases associated with them.     

Differential regulation of NO availability from macrophages and endothelial cells by the garlic component S-allyl cysteine

Garlic has been used as a traditional medicine for prevention and treatment of cardiovascular diseases. However, the molecular mechanism of garlic's pharmacological action has not been clearly elucidated. We examined here the effect of garlic extract and its major component, S-allyl cysteine (SAC), on nitric oxide (NO) production by macrophages and endothelial cells. The present study demonstrates that these reagents inhibited NO production through the suppression of iNOS mRNA and protein expression in the murine macrophage cell line RAW264.7, which had been stimulated with LPS and IFNgamma. The garlic extract also inhibited NO production in peritoneal macrophages, rat hepatocytes, and rat aortic smooth muscle cells stimulated with LPS plus cytokines, but it did not inhibit NO production in iNOS-transfected AKN-1 cells or iNOS enzyme activity. These reagents suppressed NF-kappaB activation and murine iNOS promoter activity in LPS and IFNgamma-stimulated RAW264.7 cells. In contrast, these reagents significantly increased cGMP production by eNOS in HUVEC without changes in activity, protein levels, and cellular distribution of eNOS. Finally, garlic extract and SAC both suppressed the production of hydroxyl radical, confirming their antioxidant activity. These data demonstrate that garlic extract and SAC, due to their antioxidant activity, differentially regulate NO production by inhibiting iNOS expression in macrophages while increasing NO in endothelial cells. Thus, this selective regulation may contribute to the anti-inflammatory effect and prevention of atherosclerosis by these reagents.     

Changes in tetrahydrobiopterin levels in endothelial cells and adult cardiomyocytes induced by LPS and hydrogen peroxide—A role for GFRP?

Alterations in tetrahydrobiopterin (BH4) levels have significant consequences in vascular pathophysiology. However, the mechanisms regulating BH4 remain poorly understood. The activity of GTP cyclohydrolase I (GTPCH-I), the first enzyme in BH4 biosynthesis, is controlled by protein levels, posttranslational modifications and interaction with GTPCH-I feedback regulatory protein (GFRP). This work examined the correlation between GTPCH-I protein levels and activity and changes in BH4 in human endothelial cells (HAECs) and adult rat cardiomyocytes (ARCM). Changes in BH4 were stimulated with LPS in HAECs and ARCM, and with hydrogen peroxide in HAECs only. Biopterin production by HAECs and ARCM were attained with concentrations of LPS >1 microg/ml and responses were nonlinear with respect to LPS concentrations. Western blot analysis demonstrated that induction of biopterin synthesis in HAECs and ARCM by LPS does not entail augmentation of constitutive GTPCH-I protein levels. However, LPS diminished GFRP mRNA, suggesting that disruption of GTPCH-I:GFRP complex enhances de novo biopterin synthesis. Conversely, treatment with hydrogen peroxide increased GTPCH-I and GFRP mRNA levels in HAECs while depleting BH4 and GSH, which was counteracted by catalase. This indicates that GFRP may override increases in GTPCH-I protein inhibiting enzyme activity. This conclusion is further supported by depletion of biopterin in cells transiently transfected with GFRP. Thus, allosteric regulation of GTPCH-I activity in the cardiovascular system maybe an important mechanism regulating BH4 levels through GFRP signaling.     

Role of increased guanosine triphosphate cyclohydrolase-1 expression and tetrahydrobiopterin levels upon T cell activation

Tetrahydrobiopterin (BH(4)) is an essential co-factor for the nitric-oxide (NO) synthases, and in its absence these enzymes produce superoxide (O(2)(·-)) rather than NO. The rate-limiting enzyme for BH(4) production is guanosine triphosphate cyclohydrolase-1 (GTPCH-1). Because endogenously produced NO affects T cell function, we sought to determine whether antigen stimulation affected T cell GTPCH-1 expression and ultimately BH(4) levels. Resting T cells had minimal expression of inducible NOS (NOS2), endothelial NOS (NOS3), and GTPCH-1 protein and nearly undetectable levels of BH(4). Anti-CD3 stimulation of T cells robustly stimulated the coordinated expression of NOS2, NOS3, and GTPCH-1 and markedly increased both GTPCH-1 activity and T cell BH(4) levels. The newly expressed GTPCH-1 was phosphorylated on serine 72 and pharmacological inhibition of casein kinase II reduced GTPCH-1 phosphorylation and blunted the increase in T cell BH(4). Inhibition of GTPCH-1 with diaminohydroxypyrimidine (1 mmol/liter) prevented T cell BH(4) accumulation, reduced NO production, and increased T cell O(2)(·-) production, due to both NOS2 and NOS3 uncoupling. GTPCH-1 inhibition also promoted TH(2) polarization in memory CD4 cells. Ovalbumin immunization of mice transgenic for an ovalbumin receptor (OT-II mice) confirmed a marked increase in T cell BH(4) in vivo. These studies identify a previously unidentified consequence of T cell activation, promoting BH(4) levels, NO production, and modulating T cell cytokine production.     

Paradoxical effects of two oximes on nitric oxide production by purified NO synthases, in cell culture and in animals.

We have studied the impact of two novel compounds TO-85 (2,6-di-(alpha-aziridino-alpha-hydroxyiminomethyl)pyridine and TO-133 (bis-(diaziridinoglyoximato)copper), designed as NO donors, on nitrite production by cell cultures, NO production in rat tissues and their ability to inhibit purified NO synthases (NOS). Both substances induced considerable increase of nitrite production in cell cultures. When NO production was assayed in rat organs by means of ESR using Fe(DETC) as a spin trap the anticipated NO-increasing activity of TO-85 was observed only in kidneys; the NO level increasing almost 10-fold. Treatment of rats with TO-133, decreased the NO concentration in brain cortex, cerebellum and liver. When the drugs were administered to animals with high level of iNOS expression induced by LPS, TO-85 did not significantly modify the LPS-induced NO production; administration of TO-133 caused a significant decrease of NO production in blood, brain cortex and cerebellum. Only high concentrations of TO-85 were capable of inhibiting iNOS (IC50=7 mM), the substance inhibited eNOS at lower concentrations (IC50=250 microM). Inhibitory activities of TO-85 on nNOS were dependent on BH4 concentrations, suggesting eventual competition of TO-85 with BH4 when the substance interacts with nNOS. TO-133 reduced eNOS activity with IC50=200 microM, nNOS activity with IC50=200 microM, iNOS activity was not much affected by this substance. Thus, the two tested compounds manifest opposite effects on NO production by purified enzymes and in cell culture. The pattern of the NO synthesis modification in a living animal appears to be even more complex. Our results stress the importance of direct measurements of NO in the tissues using the ESR method.