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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

This report describes experiments in which a chiral alkyl methanesulfonate was used to investigate possible mechanisms by which alkylating agents cause their mutagenic, cytotoxic, and clastogenic effects. Optically active enantiomers and the racemic mixture of 2-butyl methanesulfonate (2-BMS) were cytotoxic and mutagenic in Chinese hamster V79 cells and in AS52 cells and mutagenic in Salmonella typhimurium strains TA100 and TA1535 (without the addition of exogenous metabolizing systems). Within the experimental uncertainties, the cytotoxicity and mutagenicity curves were the same for the R and S enantiomers and for the racemic mixture. The 2-BMS isomers were cytotoxic and induced sister chromatid exchanges (SCE) in CHO-K1-BH4 cells. The cytotoxicity curve was similar to that observed with V79 and AS52 cells. The induction of SCE was linear between 1 and 6 mM 2-BMS with no differences discernable between the isomers. The results can be interpreted two ways. The first interpretation is that 2-BMS reacts via a carbocation, and the second interpretation involves an SN2 reaction of 2-BMS with DNA. The latter interpretation suggests that the mechanisms of mutagenesis, cytotoxicity, or the induction of SCE cannot distinguish between small (four-carbon) optically active DNA adducts. We favor the second interpretation because of solvolysis experiments showing the complete inversion of configuration of optically active 2-octyl methanesulfonate (2-OMS, Weiner and Sneen: J American Chemical Society 87:287-291, 1965). While we assume that optically active 2-BMS will react using the same mechanism as chiral 2-OMS, we cannot exclude the possibility that 2-BMS reacts via a carbocation intermediate.
Environ Mol Mutagen 1989
PMID:Mutagenicity and induction of sister chromatid exchange by optically active enantiomers of secondary butyl methanesulfonate. 253 95

The role of endocrine organs in the regulation of tetrahydrobiopterin (BH4) levels and guanosine triphosphate cyclohydrolase (GTP-CH) activity was studied in the spleen, bone marrow and brain of rats and mice. Following hypophysectomy, BH4 levels and GTP-CH activity were significantly decreased in both spleen and bone marrow. Fourteen days after hypophysectomy GTP-CH activity and BH4 levels were approximately 25% of control levels in both tissues. In contrast, BH4 levels and GTP-CH activity in brain were not significantly different from control values. The decrease in GTP-CH activity and BH4 levels in spleen and marrow could not be reversed by high doses of ACTH or by a pituitary extract. Removal of the thyroid gland resulted in significant decreases in BH4 levels and GTP-CH activity in spleen; marrow and brain levels were not affected. BH4 levels in spleens of thyroidectomized rats returned to control values following treatment with either triiodothyronine or thyroxine. Adrenalectomy and castration had no effect on biopterin metabolism in bone marrow, spleen or brain. Tissue levels of BH4 and GTP-CH were also studied in mutant mouse strains having mutations in either pituitary or thyroid functions in order to examine further the role of these tissues in the regulation of the biosynthesis of this cofactor. The results of this study indicate that factors secreted from the pituitary are important in the regulation of BH4 levels and GTP-CH activity in spleen and bone marrow and that the thyroid gland also plays a role in regulation in the spleen. Levels of BH4 and GTP-CH in the brain, if regulated, appear to be independent of the endocrine tissues studied.
Mol Cell Endocrinol 1986 Oct
PMID:Endocrine-dependent regulation of tetrahydrobiopterin levels and guanosine triphosphate cyclohydrolase activity. 301 98

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.
Mol Pharmacol 1993 Jan
PMID:Tetrahydrobiopterin is required for cytokine-induced nitric oxide production in a murine macrophage cell line (RAW 264). 767 92

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

(6R)-5,6,7,8-Tetrahydrobiopterin (BH4), which is synthesized intracellularly from GTP, caused a concentration-dependent increase in rat pheochromocytoma (PC12) cell proliferation when added exogenously. Incubation with sepiapterin, which is converted enzymatically to BH4 within cells, also increased PC12 cell proliferation and BH4 levels concomitantly. These sepiapterin effects were mediated by BH4 as inhibition of sepiapterin conversion to BH4 by a sepiapterin reductase inhibitor, N-acetyl-serotonin, blocked the increase in proliferation and the elevation of BH4 levels. 7,8-Dihydrobiopterin (BH2) also increased BH4 levels and PC12 cell proliferation, both of which were reversed by methotrexate, which blocks the conversion of BH2 to BH4 by dihydrofolate reductase. The BH4-induced increase in PC12 cell proliferation was not related to elevated catecholamine or nitric oxide synthesis as inhibitors of tyrosine hydroxylase or nitric oxide synthase did not reduce the BH4 effect. BH4 and its precursors did not alter intracellular cAMP levels, suggesting that this second messenger is not involved in the enhancement of PC12 cell proliferation by BH4. Sepiapterin and BH4 also enhanced the proliferation of SV40-transformed human fibroblasts and rat C6 glioma cells, indicating that the stimulatory effect of BH4 on cell proliferation is not restricted to PC12 cells.
Mol Pharmacol 1996 Jan
PMID:Mitogenic effects of tetrahydrobiopterin in PC12 cells. 856

The possibility that 5,6,7,8-tetrahydrobiopterin (BH4) biosynthesis is stimulated in glial cells by treatment with lipopolysaccharide (LPS) and tumor necrosis factor (TNF-alpha) was examined in the astrocyte-derived C6 glioma cell line. Under basal culture conditions BH4 levels were found to be at the limit of detection. Concurrent treatment with 10 micrograms/ml LPS and 50 ng/ml TNF-alpha caused a time-dependent 13-fold increase in the levels of BH4. This treatment paradigm also induced nitric oxide synthase activity, as evidenced by increased levels of nitrite, an oxidized metabolite of NO, in the culture medium. LPS and TNF-alpha treatment led to a 25-fold increase in GTPCH enzyme activity, the first and rate-limiting enzyme in BH4 synthesis, and a corresponding 23-fold increase in GTPCH protein levels. Northern blot analysis showed that increased levels of GTPCH mRNA preceded changes in GTPCH protein, GTPCH enzyme activity and BH4 levels and reached a maximal of 44-fold that was sustained for at least 48 h. These results demonstrate that LPS and TNF-alpha stimulate de-novo BH4 biosynthesis and suggest that C6 cells offer a model system for studying the molecular events that control the induction of GTPCH gene expression and BH4 synthesis in glial cells.
Brain Res Mol Brain Res 1996 Sep 05
PMID:Tetrahydrobiopterin biosynthesis in C6 glioma cells: induction of GTP cyclohydrolase I gene expression by lipopolysaccharide and cytokine treatment. 888 40

Induction of the inducible isoform of nitric oxide (NO) synthase (iNOS) in the myocardium is implicated as a mechanism in the development of cardiac depression in immune activated states associated with an enhanced release of cytokines, such as septic shock. We evaluated the in vivo synthesis of NO and tetrahydrobiopterin (BH4), a cofactor of NOS, in the heart tissue using a model of LPS injection in rats (LPS: 10 mg/kg, i.v.). In control rats, iNOS activity or iNOS mRNA in the heart was negligible. Three hours after LPS administration, a marked induction of iNOS mRNA and activity was observed in the heart. A significant increase in BH4 content and GTP cyclohydrolase mRNA abundance was also observed in the heart from LPS-treated rats. Our results demonstrate induction of NO synthesis and parallel increase in BH4 concentration in the heart of rats after LPS treatment in vivo and may provide molecular evidence responsible for the increased production of BH4 which may up-regulate iNOS activity in the heart in vivo.
Mol Cell Biochem 1997 Jan
PMID:Co-induction of nitric oxide and tetrahydrobiopterin synthesis in the myocardium in vivo. 904 35

There is evidence that nitric oxide (NO) may mediate some of the functional myocardial changes caused by bacterial LPS and inflammatory cytokines. The expression of the inflammatory or inducible NO synthase (iNOS) in human cardiac myocytes, however, has not been well characterized. Therefore, we treated cultured, dedifferentiated human ventricular cardiac myocytes with the combination of TNF-alpha (500 U/ml), IL-1beta (30U/ml), IFNgamma (100 U/ml), and LPS (E.coli 0111:B4, 10 microg/ml). Northern blot analysis revealed a approximately 4.5 kb transcript for inducible NOS (iNOS) in the stimulated human heart cells but not in untreated cells. RT-PCR confirmed that iNOS mRNA was only present in stimulated cells. However, treatment of the myocytes for up to 96 h with cytokines and LPS did not result in NO synthesis as measured by nitrite + nitrate accumulation in the culture medium, and no iNOS enzymatic activity could be detected in the cell lysates. Western blot analysis failed to detect iNOS protein. Thus, despite high and persistent levels of iNOS mRNA in cytokine-treated cells, iNOS protein was absent in this experimental model. GTP-cyclohydrolase I was induced both at the mRNA and protein levels and resulted in increased biopterin levels, indicating sufficient amounts of the cofactor tetrahydrobiopterin (BH4) were present, and that the failure to express an inducible protein was specific to iNOS. To determine if the absence of iNOS protein was due to a novel cardiac iNOS gene or modified iNOS transcript in human myocytes, we cloned an iNOS cDNA from cytokine-treated myocytes. Sequencing and expression of the clone revealed a functional iNOS cDNA with >99% identity to other human iNOS cDNA clones. When human cardiac cells were transduced with a retroviral vector carrying only the coding region of the human hepatocyte iNOS cDNA, both iNOS mRNA and protein could be detected. In conclusion, these cells derived from cultured human cardiac myocytes lacked the capacity to express an endogenous iNOS protein, the basis of which appears to be a cell-specific suppression or failure of iNOS translation.
J Mol Cell Cardiol 1997 Apr
PMID:Dedifferentiated human ventricular cardiac myocytes express inducible nitric oxide synthase mRNA but not protein in response to IL-1, TNF, IFNgamma, and LPS. 916 Aug 67

In previous work it was shown that the immune cytokine interferon-gamma (IFN-gamma) inhibits hormone secretion in anterior pituitary (AP) cell cultures, an action most likely mediated by folliculostellate (FS) cells. In the present study, we wanted to investigate whether nitric oxide (NO) is involved in this inhibitory action of IFN-gamma. NO synthase (NOS) inhibitors with affinity for the inducible (iNOS) and the constitutive (cNOS) isoform such as N(G)-monomethyl-L-arginine (L-NMMA) and S-methyl-L-thiocitrulline (SMLT) dose-dependently blocked the inhibitory action of IFN-gamma on GHRH-stimulated GH secretion, and partially reversed the inhibitory effect on basal prolactin (PRL) release. In the absence of IFN-gamma these inhibitors significantly augmented basal PRL release and slightly enhanced GHRH-stimulated GH release. L-N6-(1-iminoethyl)lysine (L-NIL), a NOS inhibitor with preferential affinity for iNOS, abrogated the IFN-gamma effect on GHRH-stimulated GH secretion and partially reversed IFN-gamma inhibition of PRL release. However, L-NIL did not exert a stimulatory effect on basal PRL and GHRH-stimulated GH release by its own. 2,4-diamino-6-hydroxypyrimidine (DAHP), a NOS inhibitor by interfering with tetrahydrobiopterin (BH4) cofactor availability, showed the same activity profile as L-NIL. NOS inhibitors blocked or reduced the production of NO as detected by measuring nitrite (NO2-) levels in AP cell cultures and cGMP levels in the NO-reporter cell line RFL-6. The NOS inhibiting action of L-NMMA was confirmed by competition experiments with the natural NOS substrate L-arginine. Thus, in culture medium with lower amounts of L-arginine, L-NMMA blocked the IFN-gamma-induced inhibition of GHRH-stimulated GH release at a lower dose. The inhibition of PRL and GH release by IFN-gamma was markedly reduced in L-arginine-depleted medium. The NO donor sodium nitroprusside (SNP) mimicked the inhibitory action of IFN-gamma on GHRH-stimulated GH and basal PRL release. Similarly to IFN-gamma, SNP did not affect basal GH release. As previously reported, inhibition by IFN-gamma occurred only in AP cell populations containing a minimal proportion of FS cells. As studied in different cell populations obtained by unit gravity sedimentation in a serum albumin gradient, L-NMMA reversed the IFN-gamma effect in the same populations enriched in FS cells. Interestingly, in the absence of IFN-gamma L-NMMA strongly stimulated basal PRL release in the population most enriched in FS cells. It is concluded that IFN-gamma through activation of the iNOS pathway probably in FS cells enhances the production of NO and that this effect is responsible for the inhibitory action of IFN-gamma on GHRH-stimulated GH release and partially for the IFN-gamma-induced decrease in basal PRL release. On the other hand, NO, likely produced by cNOS, appears to exert a tonic inhibitory effect on GHRH-stimulated GH and basal PRL release. It seems therefore that low amounts of NO produced constitutively may take charge of subtle physiological adaptations, and higher levels of NO produced by iNOS under the influence of IFN-gamma may attenuate PRL and GH release during emergency conditions of immune and inflammatory reactions.
Mol Cell Endocrinol 1997 May 16
PMID:Involvement of nitric oxide in the interferon-gamma-induced inhibition of growth hormone and prolactin secretion in anterior pituitary cell cultures. 920 99

The nitric oxide (NO) signalling pathway is thought to play a direct role in regulating the contractile properties of cardiac muscle both in vitro and in vivo. The inducible isoform of NO synthase (iNOS) mediates a sustained increase in NO production in response to cytokines in the cardiac myocytes; however, the regulation of NO synthesis in these cells remains poorly understood. Tetrahydrobiopterin (BH4) is an essential cofactor for NO formation. Cytokines induce the de novo synthesis of BH4 in cardiac myocytes, an event that is essential for the induction of NO synthesis. Activation of NO formation by cytokines in cardiac myocytes requires transcriptional induction of the genes that encode iNOS and guanosine triphosphate cyclohydrolase I (GTPCH), the first and rate-limiting enzyme in de novo BH4 synthesis. Given that nuclear factor kappa B (NF-kappa B) mediates the induction of iNOS gene expression in various cell types, the role of NF-kappa B in the induction of iNOS in cytokine-stimulated rat neonatal cardiac myocytes was assessed by examining the effects of pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-kappa B activation, on the abundance of iNOS mRNA and NO synthesis. The effects of PDTC on GTPCH mRNA abundance and biopterin synthesis were also investigated. PDTC inhibited in a dose-dependent manner both NO and BH4 synthesis induced by a combination of interleukin-1 alpha (IL-1 alpha) and interferon-gamma (IFN gamma), with a half-maximal inhibitory concentration of 22 muM. PDTC also prevented the accumulation of iNOS and GTPCH mRNAs induced by IL-1 alpha and IFN gamma. Cytokine-induced NO and BH4 synthesis was also inhibited by tosyl-lysine-chloromethyl ketone. another inhibitor of NF-kappa B activation. Results suggest that PDTC inhibits cytokine-induced NO and BH4 synthesis by inhibiting the expression of iNOS and GTPCH genes. Thus, the induction of both genes necessary for NO synthesis in cardiac myocytes appears to be regulated, at least in part, by a common mechanism: NF-kappa B activation.
J Mol Cell Cardiol 1997 Jun
PMID:Role of nuclear factor kappa B in cytokine-induced nitric oxide and tetrahydrobiopterin synthesis in rat neonatal cardiac myocytes. 922 Mar 44


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