Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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

Cellular constituents of heart muscle contain both constitutive and inducible nitric oxide (NO) signaling pathways that modulate the contractile properties of cardiac myocytes. The identities of the inducible NO synthase (iNOS) isoform(s) expressed in cardiac muscle, and of the specific cell types expressing iNOS activity, remain poorly characterized. We amplified a 217-base pair cDNA by reverse transcriptase-polymerase chain reaction from primary cultures of inflammatory cytokine-pretreated adult rat ventricular myocytes (ARVM) that was nearly identical to other iNOS cDNA sequences. Using this 217-base pair cDNA as a probe in Northern blots, we found no evidence of iNOS mRNA in control myocytes, but both interleukin-1 beta and interferon-gamma individually increased iNOS mRNA abundance in primary cultures of ARVM, with maximal expression at 12 h. The half-life of iNOS mRNA in actinomycin C1-treated cells was 4 h. Both dexamethasone and transforming growth factor-beta attenuated the induction of iNOS mRNA abundance and enzyme activity by IL-1 beta and INF gamma. Pretreatment with dexamethasone also abolished the induction of iNOS mRNA, but not the increase in GTP cyclohydrolase mRNA in purified cardiac myocytes from lipopolysaccharide-injected rats. In order to further characterize the specific cell type producing NO, we used a NO-specific porphyrinic/Nafion-coated microsensor to record NO release from a single, isolated ARVM pretreated with IL-1 beta and IFN gamma in L-arginine-depleted medium. NO release could be detected following microinjection of L-arginine in the vicinity of the cell juxtaposed to the NO microsensor, but not following microinjection of D-arginine, and not from ARVM pretreated with L-N-monomethylarginine. Cytokine-pretreated ARVM that had been maintained in L-arginine-depleted medium also exhibited a depressed contractile response to isoproterenol after addition of L-arginine, but not D-arginine. These results indicate that altered contractile function of cardiac myocytes following exposure to specific inflammatory cytokines is due to induction of myocyte iNOS.
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PMID:Cytokine-inducible nitric oxide synthase (iNOS) expression in cardiac myocytes. Characterization and regulation of iNOS expression and detection of iNOS activity in single cardiac myocytes in vitro. 752 57

A significant induction of GTP cyclohydrolase I (GTPCH) mRNA was observed in lung, heart and kidney of rats treated with lipopolysaccharide (LPS; 10 mg/kg i.v.). GTPCH mRNA levels in liver were high even in untreated rats, and remained elevated after LPS treatment. Parallel induction of nitric oxide synthase (NOS) mRNA was observed in these tissues of LPS-treated rats. Our results demonstrate induction of GTPCH mRNA after LPS treatment in vivo and provide molecular evidence for the increased GTPCH activity which may up-regulate NOS activity in vivo.
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PMID:Lipopolysaccharide treatment in vivo induces tissue expression of GTP cyclohydrolase I mRNA. 754 62

Stimulation of nitric oxide (NO) synthase in endothelial cells by Ca2+ influx leads to increased intracellular levels of cGMP. NO synthase from various sources is known to use tetrahydrobiopterin, flavins, and NADPH as cofactors. We studied the effect of interferon-gamma, tumor necrosis factor-alpha, and lipopolysaccharide on tetrahydrobiopterin biosynthetic activities in human umbilical vein endothelial cells (HUVEC). These stimuli led to an up to 40-fold increase of GTP cyclohydrolase I (EC 3.5.4.16) activity and to increased accumulation of neopterin and tetrahydrobiopterin in HUVEC. Further enzyme activities of tetrahydrobiopterin biosynthesis, i.e. 6-pyruvoyl tetrahydropterin synthase and sepiapterin reductase (EC 1.1.1.153), remained unchanged. NO synthase activity in protein fractions from homogenates of cells treated with interferon-gamma plus tumor necrosis factor-alpha was not influenced as compared with untreated controls. However, interferon-gamma alone or in combination with tumor necrosis factor-alpha significantly increased intracellular cGMP formation in intact HUVEC by 50 and 80%, respectively. These stimuli increased intracellular tetrahydrobiopterin concentrations up to 14-fold. NO-triggered cGMP formation was similarly increased by incubation of otherwise untreated cells with sepiapterin, leading to elevated intracellular tetrahydrobiopterin levels. Thus, cytokines indirectly stimulate the activity of constitutive NO synthase in HUVEC by upregulating production of the cofactor tetrahydrobiopterin.
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PMID:Pteridine biosynthesis in human endothelial cells. Impact on nitric oxide-mediated formation of cyclic GMP. 767 11

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

The synthesis of nitric oxide by proximal tubule-inducible nitric oxide synthase requires tetrahydrobiopterin as a cofactor. To determine whether tetrahydrobiopterin synthesis is required for nitric oxide production, nitrite release by mouse proximal tubule cells treated with 2,4-diamino-6-hydroxypyrimidine, an inhibitor of the rate-limiting enzyme in the de novo synthesis of tetrahydrobiopterin from guanosine triphosphate, guanosine triphosphate cyclohydrolase I, was measured. Treatment with lipopolysaccharide (0.1 micrograms/mL) and interferon-gamma (100 U/mL) for 12 h increased nitrite production from 2.7 +/- 0.2 to 25.4 +/- 1.3 nmol/mg of protein (P < 0.001; N = 9). 2,4-Diamino-6-hydroxypyrimidine (6 mM) reduced lipopolysaccharide/interferon-gamma-induced nitrite production by 53.1 +/- 3.4%. Sepiapterin, a substrate for tetrahydrobiopterin synthesis via the dihydrofolate reductase-dependent pterin salvage pathway, prevented the inhibition by 2,4-diamino-6-hydroxypyrimidine, an effect that was blocked by methotrexate. In conclusion, guanosine triphosphate cyclohydrolase I activity is required for cytokine-induced nitric oxide production by proximal tubular epithelium. The inhibition of guanosine triphosphate cyclohydrolase I could prove useful in the treatment of nitric oxide-mediated renal disorders.
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PMID:Guanosine triphosphate cyclohydrolase I regulates nitric oxide synthesis in renal proximal tubules. 775 97

A 2- to 3-fold increase of GTP cyclohydrolase I (E.C. 3.5.4.16), the key enzyme of tetrahydrobiopterin biosynthesis from GTP, was observed in cerebellum, remaining brain, liver, spleen, and adrenal gland of rats treated with a single dose of lipopolysaccharide (LPS). This led to increased biopterin levels in tissues but not in plasma. Parallel induction of nitric oxide (NO) synthase was indicated by a 10- to 100-fold increase of plasma nitrate levels 6 and 12 hours after injection of LPS. Furthermore, systolic blood pressure was reduced significantly by 23%. Our results demonstrate induction of tetrahydrobiopterin biosynthesis after LPS treatment in vivo.
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PMID:Induction of GTP cyclohydrolase I by bacterial lipopolysaccharide in the rat. 848 53

Nitric oxide synthesis requires the cofactor tetrahydrobiopterin. We have examined the effect on nitric oxide synthesis in experimental endotoxic shock of 2,4- diamino-6-hydroxypyrimidine (DAHP), an inhibitor of GTP cyclohydrolase I, the first and rate limiting enzyme for tetrahydrobiopterin synthesis. Rats given lipopolysaccharide (LPS, 10 mg/kg) showed a large rise in plasma nitrate at 4 and 8 hours which was significantly reduced by DAHP (1 g/kg) given at the same time as LPS. There was a 40-50% reduction in the haem-NO signal detected in kidney by electron paramagnetic resonance spectroscopy. LPS produced hypotension at 3 hours and 6 hours and this was ameliorated at 6 hours in rats given DAHP. DAHP abolished the rise in kidney tetrahydrobiopterin levels seen 4 hours after LPS but no effect was seen on induction of inducible nitric oxide synthase (iNOS) as assessed by immunohistochemistry and reverse transcriptase PCR, consistent with the effect of DAPH being by reduction of tetrahydrobiopterin levels. The results show that inhibition of tetrahydrobiopterin synthesis is an effective strategy to reduce nitric oxide synthesis by iNOS in vivo.
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PMID:Inhibition of tetrahydrobiopterin synthesis reduces in vivo nitric oxide production in experimental endotoxic shock. 860 31

GTP cyclohydrolase I is the first and rate-limiting enzyme in the synthesis of tetrahydrobiopterin, a cofactor of nitric oxide (NO) synthase. Immunostimulants increase NO and tetrahydrobiopterin synthesis in vascular smooth muscle cells by coinducing NO synthase and GTP cyclohydrolase I gene expression. Given that nuclear factor kappa(B) mediates the induction of NO synthase gene expression by lipopolysaccharide (LPS), the role of nuclear factor kappa(B) in the induction of GTP cyclohydrolase I in LPS-stimulated rat vascular smooth muscle cells was assessed by examining the effects of pyrrolidine dithiocarbamate, an inhibitor of the activation of nuclear factor kappa(B), on the abundance of GTP cyclohydrolase I mRNA and biopterin synthesis. Pyrrolidine dithiocarbamate inhibited both NO and biopterin synthesis induced by LPS in a dose-dependent manner with similar half-maximal inhibitory concentrations, 12 mu M for NO and 17 mu M for biopterin, respectively. At a concentration of 25 mu M, which inhibited NO and biopterin synthesis but caused no cytotoxicity, pyrrolidine dithiocarbamate substantially reduced the LPS-induced increase in the abundance of NO synthase and GTP cyclohydrolase I mRNAs. These results suggest that pyrrolidine dithiocarbamate inhibits LPS-induced NO and biopterin synthesis by inhibiting the expression of NO synthase and GTP cyclohydrolase I genes. Thus, the induction of both genes necessary for cellular NO synthesis in vascular smooth muscle appears to be regulated, at least in part, by a common mechanism: nuclear factor kappa(B) activation.
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PMID:Pyrrolidine dithiocarbamate inhibits immunostimulant-induced tetrahydrobiopterin synthesis in rat vascular smooth muscle. 872 Apr 83

Induction of the inducible isoform of nitric oxide synthase (iNOS) in various types of cells is implicated as the cause of septic shock. We evaluated the concentration of tetrahydrobiopterin (BH4), a cofactor of NOS, in plasma and various other tissues of rats treated with lipopolysaccharide (LPS; 10 mg/kg I.V.). The activity of GTP cyclohydrolase I (GTPCH), the first and rate-limiting enzyme in the de novo synthesis of BH4, in rat tissues was also determined. Three hours after administration of LPS, rats showed plasma levels of BH4 and NOx (NO3- and NO2-) that were elevated by 137 and 206%, respectively. GTPCH was expressed in liver and, to a lesser extent, in the lung, heart and kidney of control rats. In control rats, although a high concentration of BH4 was detected in the liver, its level was lower in lung, heart, kidney and aorta. Three hours after LPS administration, a significant increase in BH4 concentration and/or GTPCH activity was observed in all tissues examined except the liver. Our results demonstrate that the de novo synthesis of BH4 is upregulated by LPS in the rat in vivo, which may, at least in part, account for the increases in plasma level and tissue concentration of BH4 after the administration of LPS.
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PMID:Tetrahydrobiopterin and GTP cyclohydrolase I in a rat model of endotoxic shock: relation to nitric oxide synthesis. 885 74


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