Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P43026 (lipopolysaccharide)
 62,215 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Nitric oxide (NO) synthesis is induced in vascular smooth muscle cells by lipopolysaccharide (LPS) where it appears to mediate a variety of vascular dysfunctions. In some cell types tetrahydrobiopterin (BH4) synthesis has also been found to be induced by cytokines. Because BH4 is a cofactor for NO synthase, we investigated whether BH4 synthesis is required for LPS-induced NO production in rat aortic smooth muscle cells (RASMC). The total biopterin content (BH4 and more oxidized states) of untreated RASMC was below our limit of detection. However, treatment with LPS caused a significant rise in biopterin levels and an induction of NO synthesis; both effects of LPS were markedly potentiated by interferon-gamma. 2,4-Diamino-6-hydroxypyrimidine (DAHP), a selective inhibitor of GTP cyclohydrolase I, the rate-limiting enzyme for de novo BH4 synthesis, completely abolished the elevated biopterin levels induced by LPS. DAHP also caused a concentration-dependent inhibition of LPS-induced NO synthesis. Inhibition of NO synthesis by DAHP was reversed by sepiapterin, an agent which circumvents the inhibition of biopterin synthesis by DAHP by serving as a substrate for BH4 synthesis via the pterin salvage pathway. The reversal by sepiapterin was overcome by methotrexate, an inhibitor of the pterin salvage pathway. Sepiapterin, and to a lesser extent BH4, dose-dependently enhanced LPS-induced NO synthesis, indicating that BH4 concentration limits the rate of NO production by LPS-activated RASMC. Sepiapterin also caused LPS-induced NO synthesis to appear with an abbreviated lag period phase, suggesting that BH4 availability also limits the onset of NO synthesis. In contrast to the stimulation of LPS-induced NO synthesis, observed when sepiapterin was given alone, sepiapterin became a potent inhibitor of NO synthesis in the presence of methotrexate. This is attributable to a direct inhibitory action of sepiapterin on GTP cyclohydrolase I, an activity which is only revealed after blocking the metabolism of sepiapterin to BH4. Further studies with sepiapterin, methotrexate, and N-acetylserotonin (an inhibitor of the BH4 synthetic enzyme, sepiapterin reductase) indicated that the BH4 is synthesized in RASMC predominantly from GTP; however, a lesser amount may derive from pterin salvage. We demonstrate that BH4 synthesis is an absolute requirement for induction of NO synthesis by LPS in vascular smooth muscle. Our findings also suggest that pterin synthesis inhibitors may be useful for the therapy of endotoxin- and cytokine-induced shock.
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PMID:Tetrahydrobiopterin synthesis. An absolute requirement for cytokine-induced nitric oxide generation by vascular smooth muscle. 128 71

1. The synthesis of nitrite and citrulline from L-arginine by immune-stimulated rat alveolar macrophages and the modulation of this synthesis were studied. 2,4-Diamino-6-hydroxypyrimidine (DAHP), 6R-5,6,7,8-tetrahydro-L-biopterin (BH4) and L-sepiapterin were potent inhibitors of the recombinant interferon-gamma induced production of nitrogen oxides in intact cultured cells with I50 values for BH4 and L-sepiapterin of approximately 10 microM. They were equally effective in inhibiting the induced production of citrulline. This inhibitory effect was concentration-dependent for all three modulators investigated. 2. The inhibitory effects were not dependent on incubation times of either 24 or 48 h, on the immune-stimulus used (lipopolysaccharide, interferon-gamma), or whether these stimuli were added during or after the induction period. 3. Pterin-6-carboxylic acid (PCA), which cannot be converted into BH4, and methotrexate (MTX), which inhibits dihydrofolatereductase but not de novo biosynthesis of BH4, did not change the production of nitrite. 4. The data indicate that DAHP, an inhibitor of the de novo biosynthesis of the co-factor BH4, blocks the nitric oxide synthase activity in intact cells. Since the pterins BH4 and L-sepiapterin blocked the L-arginine dependent production of nitrite and citrulline, the activity of nitric oxide synthase in phagocytic cells may be regulated by metabolic endproducts of the de novo biosynthesis of BH4.
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PMID:Pterins inhibit nitric oxide synthase activity in rat alveolar macrophages. 128 17

Nitric oxide (NO) synthase activity was detected in fat body and the Malpighian tubles of the silkworm, Bombyx mori. Main NO synthase activity in the fat body was Ca(2+)/calmodulin-dependent, inducible by bacterial lipopolysaccharide (LPS) and required NADPH, FAD, FMN, dithiothreitol (DTT) and tetrahydrobiopterin (BH4) as cofactors for the full expression of the activity. The Malpighian tubles contained two types of NO synthase. One was Ca(2+)-independent, calmodulin-dependent and constitutive and the other was Ca(2+)-dependent and constitutive. The former NO synthase required the same cofactors as fat body NO synthase. The activity of Malpighian tuble NO synthases increased dramatically at the end of the last instar period, just prior to spinning. These results indicate that B. mori contains new types of NO synthase, suggesting the wide distribution and different characteristics of this enzyme among vertebrates and invertebrates.
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PMID:Occurrence of novel types of nitric oxide synthase in the silkworm, Bombyx mori. 753 73

We evaluated the effects of the immunosuppressants cyclosporin A (CsA) and FK506 on the synthesis of nitric oxide (NO) induced by bacterial lipopolysaccharide (LPS) in J774 macrophages. CsA and FK506 each inhibited NO production by LPS in a concentration-dependent fashion, but the cytotoxicity was also evident at higher concentrations (100 microM). Neither CsA nor FK506 had any effect on the activity of NO synthase (NOS) that had already been induced. Findings indicated that CsA and FK506 inhibit the induction of NOS, rather than its catalytic activity. CsA and, to a lesser extent, FK506 increased the synthesis of tetrahydrobiopterin (BH4), an essential cofactor of NOS. Thus, inhibition of NO formation by CsA or FK506 is unlikely to associated with a change in BH4 synthesis caused by these agents in LPS-treated J774 macrophages.
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PMID:Effects of cyclosporin A and FK506 on nitric oxide and tetrahydrobiopterin synthesis in bacterial lipopolysaccharide-treated J774 macrophages. 754 50

1. Induction of the calcium-independent isoform of nitric oxide (NO) synthase (iNOS) in various cell types has been implicated in the circulatory failure in experimental models of septic shock. Tetrahydrobiopterin (BH4) appears to be an essential co-factor for NO formation and therefore an inhibition of its biosynthesis represents a feasible therapeutic target. We have investigated the effects of an inhibitor of BH4 synthesis, N-acetyl-5-hydroxytryptamine (N-acetylserotonin, NAS), on the expression of iNOS in cultured macrophages and smooth muscle cells in vitro, and on the hypotensive response to bacterial lipopolysaccharide (LPS) in the anaesthetized rat in vivo. 2. NAS (0.01-5 mM) caused a concentration-dependent inhibition of the accumulation of nitrite in the conditioned medium of LPS/interferon-gamma (IFN gamma)-stimulated RAW 264.7 macrophages and interleukin-1 beta (IL-1 beta)-activated vascular smooth muscle cells (VSMC). This effect was paralleled by a similar decrease in the iNOS protein content of these cells, as determined by immunoblot analysis. 3. Pretreatment of RAW 264.7 macrophages with the BH4 precursor, dihydrobiopterin (BH2, 0.1 mM) did not restore nitrite formation in the presence of NAS (1 mM). 4. Intravenous administration of NAS (1 mg kg-1 min-1 for 30 min) in anaesthetized rats significantly reduced the fall in mean arterial blood pressure, restored the pressor response to noradrenaline (1 micrograms kg-1), and ameliorated the increase in plasma nitrite following exposure to LPS (10 mg kg-1). 5. NAS pretreatment also attenuated iNOS activity in lung homogenates, as determined by the conversion of radiolabelled L-arginine to L-citrulline, and partially restored the constrictor effect of noradrenaline in aortic rings isolated from LPS-treated rats. Moreover, NAS significantly reduced the rise in the plasma concentration of tumour necrosis factor alpha (TNFalpha) in response to LPS.6. These findings suggest that NAS inhibits the expression rather than the activity of iNOS in cultured macrophages and smooth muscle cells. This effect of NAS appears to be independent of the availability of BH4, but may be related to an attenuation of the release of TNFalpha following LPS administration, as shown in the anaesthetized rat. This mechanism may also account for the beneficial haemodynamic effect of NAS in our experimental model of endotoxaemia.
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PMID:Inhibition by N-acetyl-5-hydroxytryptamine of nitric oxide synthase expression in cultured cells and in the anaesthetized rat. 758 41

The murine macrophage cell line RAW 264 constitutively synthesizes tetrahydrobiopterin (BH4), the cofactor required for the hydroxylation of the aromatic amino acids and for the production of nitric oxide. Stimulation of the cells with interferon-gamma and lipopolysaccharide induced the production of nitric oxide and increased BH4 levels further. When the cells were stimulated in the presence of 2,4-diamino-6-hydroxypyrimidine (DAHP), an inhibitor of BH4 biosynthesis, biopterin levels decreased by 90% within 6 hr, whereas nitrite production was essentially unaffected. Pretreatment of the cells for 12 hr with DAHP decreased intracellular BH4 concentrations by > 95% yet inhibited the cytokine-stimulated production of nitric oxide by only 50%. However, pretreatment with DAHP plus N-acetylserotonin, an inhibitor of sepiapterin reductase, the terminal enzyme of the BH4 biosynthetic pathway, decreased biopterin levels by > 99% and inhibited nitric oxide synthesis by 90%. This inhibition could be reversed by loading the cells with dihydrobiopterin, a precursor of BH4 via the dihydrofolate reductase salvage pathway. In addition, these studies revealed that N-acetylserotonin has a direct inhibitory effect on nitric oxide synthesis, acting in a BH4-independent manner. The results presented here support previous suggestions, based on experiments with isolated enzymes, that BH4 is absolutely required for cytokine-stimulated nitric oxide production in macrophages and they suggest that only a small fraction of the total intracellular BH4 pool in macrophages is utilized in the production of fully active nitric oxide synthase.
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PMID:Tetrahydrobiopterin is required for cytokine-induced nitric oxide production in a murine macrophage cell line (RAW 264). 767 92

GTP cyclohydrolase I (GTPCH) is the first and rate-limiting enzyme for the synthesis of tetrahydrobiopterin (BH4), a cofactor of nitric oxide synthase (NOS). As the induction of NO synthesis by immunostimulants in vascular smooth muscle (VSM) cells requires de novo synthesis of BH4, we investigated whether immunostimulants enhance the expression of GTPCH mRNA. GTPCH mRNA and BH4 were measured in rat VSM cells after exposure to bacterial lipopolysaccharide (LPS) in combination with interferon-gamma (IFN). Reverse transcription polymerase chain reaction (RT-PCR) was used to amplify a predicted 372 bp fragment of GTPCH mRNA, deduced from the known nucleotide sequence of rat liver GTPCH cDNA. Dideoxynucleotide sequencing of the PCR fragment revealed 100% identity between LPS/IFN-induced GTPCH mRNA of VSM and the constitutive GTPCH mRNA of liver. Although BH4 was below our limit of detection in untreated VSM, low levels of GTPCH mRNA were detectable. LPS/IFN treatment triggered the appearance of BH4 and markedly increased GTPCH mRNA. Induction of GTPCH mRNA was apparent by 2 h, peaked at 4 h, and was sustained at high levels for at least 24 h. Induction of GTPCH mRNA by LPS/IFN was substantially enhanced by cycloheximide, suggesting that mRNA levels are depressed by a labile protein. Measurement of LPS/IFN-induced NOS mRNA by RT-PCR, demonstrated a timecourse of induction which mirrors that of GTPCH. Similarly, the timecourse of appearance of cytosolic NOS activity following exposure of VSM to LPS/IFN paralleled that of the increase in BH4 content. Our studies demonstrate that immunostimulants co-induce NOS and GTPCH gene expression: both events are necessary for induction of NO synthesis by VSM.
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PMID:GTP cyclohydrolase I mRNA is induced by LPS in vascular smooth muscle: characterization, sequence and relationship to nitric oxide synthase. 768 40

We recently reported (Am. J. Respir. Cell Mol. Biol. 7: 471-476, 1992) that a mixture of lipopolysaccharide (LPS) and cytokines produced a time-dependent increase in mRNA and protein expression of inducible nitric oxide synthase (iNOS) in cultured rat pulmonary artery smooth muscle cells (RPASM). In the current study we extend observations on regulation of iNOS in RPASM by showing that de novo synthesis of tetrahydrobiopterin (BH4) is critical for LPS and cytokine-induced NO production. A mixture of LPS and the cytokines gamma-interferon, interleukin-1 beta, and tumor necrosis factor-alpha increased steady-state levels of mRNA of GTP-cyclohydrolase-I (GTP-CH), the rate-limiting enzyme in BH4 biosynthesis. Levels of mRNA to GTP-CH became detectable by 4 h, with further increases at 24 h by Northern blot analysis and reverse-transcriptase polymerase chain reaction. Total intracellular biopterin levels, undetectable under basal conditions, increased after 24 h exposure to LPS and cytokines (to 32.3 +/- 0.8 pmol/mg protein). LPS and cytokine-induced NO production, determined by nitrite concentrations in the medium, was decreased in a concentration-dependent manner by the GTP-CH inhibitor, 2,4-diamino-6-hydroxypyrimidine (DAHP) at 24 h. DAHP also inhibited completely the LPS- and cytokine-induced accumulation of intracellular biopterins. Sepiapterin, which supplies BH4 through a salvage pathway independent of GTP-CH, reversed the effect of DAHP on LPS and cytokine-induced NO production.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Tetrahydrobiopterin synthesis and inducible nitric oxide production in pulmonary artery smooth muscle. 780 62

Murine fibroblasts treated with interferon-gamma plus tumor necrosis factor-alpha plus lipopolysaccharide produce nitrite and EPR-observable intracellular nonheme iron-thiol-dinitrosyl species. Inhibition of .NO synthesis or de novo tetrahydrobiopterin (BH4) synthesis decreases nitrite and the EPR signal. The effects of BH4 synthesis inhibition are prevented by sepapterin, which increases BH4 through the salvage pathway.
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PMID:Coinduction of nitric oxide synthesis and intracellular nonheme iron-nitrosyl complexes in murine cytokine-treated fibroblasts. 807 Jun 94

We examined the release of tetrahydrobiopterin ((6R)-L-erythro-dihydroxypropyl-2-amino-4-hydroxy-5,6,7, 8-tetrahydropteridine; BH4) and nitric oxide induced by lipopolysaccharide from mouse neuroblastoma N1E-115 cells by measuring BH4 and nitric oxide derivatives, nitrites and nitrates, harbored in the conditioned media. The stimulation of the cells by 1 microgram/ml of lipopolysaccharide for 24 h induced 2-fold increase in the release of BH4 from the cells, but did not induce the nitric oxide release from the cells. Although such increase in BH4 release from the cells was blocked by the inhibitors of nuclear factor-kappa B or protein tyrosine kinases, the release of nitric oxide was not affected by such inhibitors. Our results may suggest that the inductions of BH4 and nitric oxide in this neuroblastoma cell line are processed in different ways and that this cell line is also different from the immune cells in the central nervous system such as microglia in this respect.
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PMID:Dissociated release of tetrahydrobiopterin and nitric oxide by lipopolysaccharide from mouse neuroblastoma cells. 883 57


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