Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:1.14.13.39 (NO synthase)
 15,778 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Arginine-derived nitric oxide (NO) has been identified in some tumor cell lines and solid human tumors. The effect of tumor cell NO on tumor biology is poorly understood. The purpose of this study was to investigate the effect of NO production by EMT-6 murine breast cancer cells on tumor cell growth in vitro and subcutaneous tumor growth and experimental pulmonary metastasis in vivo. EMT-6 cells were incubated with endotoxin (LPS, 10 microgram/ml) and interferon-gamma (IFN, 50 U/ml), in the presence or absence of the NO synthase inhibitor, omega-nitro-L-arginine methyl ester (L-NAME, 2 mM), and NO production and cell number were assessed 24 hr later. EMT-6 cells were also treated overnight with LPS/IFN, in the presence or absence of L-NAME, washed and injected either subcutaneously in the dorsal flank (n = 40) or via the tail vein (n = 40) of syngeneic BALB/c mice. Two weeks following tumor cell injection, tumor size and number of pulmonary metastases were assessed. LPS/IFN stimulated NO production in EMT-6 cells and inhibited cell growth in vitro by 50%. L-NAME blocked LPS/IFN stimulation of NO production and restored cell growth to near control levels. When injected into BALB/c mice, LPS/IFN-stimulated tumor cells demonstrated a two-fold increase in subcutaneous tumor growth and experimental pulmonary metastases over control cells. L-NAME reduced tumor size and number of lung metastases to control levels, suggesting that tumor cell NO production was responsible for this effect. In summary, LPS/IFN-stimulated NO production in EMT-6 tumor cells inhibits tumor cell growth in vitro, yet paradoxically augments tumor growth and metastasis in vivo.
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PMID:Tumor cell nitric oxide inhibits cell growth in vitro, but stimulates tumorigenesis and experimental lung metastasis in vivo. 866 Nov 71

T-cell mediated immunity (CMI) is crucial for protection against genital chlamydial infection in mice. To define the underlying molecular mechanism for this protection, several T-cell clones generated against the Chlamydia trachomatis agent of mouse pneumonitis (MoPn) were analysed in an in vitro model of the mucosal epithelium, the polarized epithelial-lymphocyte co-culture (PELC) system, for immunobiological functions that correlated with chlamydial inhibition. The six clones analysed were classified as protective or non-protective on the basis of their ability to cure genital chlamydial infection in syngeneic mice. The results revealed a direct relationship between the ability of a clone to protect in vivo and to inhibit the multiplication of MoPn in vitro. Also, the protective ability of a clone correlated with its capacity to elaborate relatively high levels of interferon-gamma (IFN-gamma) and to induce nitric oxide (NO) production. Moreover, neutralizing anti-IFN-gamma antibodies used alone at 50 micrograms/ml or in combination with anti-tumour necrosis-factor (TNF-alpha), and the L-arginine analogue and NO synthase inhibitor, NG-monomethyl-L-arginine monoacetate (MLA), could significantly suppress the ability of protective clones to inhibit MoPn in epithelial cells. The results suggested that the IFN-gamma-inducible NO synthease pathway is important for chlamydial control in mice. Furthermore, IFN-gamma could stimulate infected murine epithelial cells (line TM3) to secrete NO, resulting in inhibition of MoPn growth. However, the degree of MoPn inhibition obtained with IFN-gamma alone was less than that observed when T cells were co-cultured with infected epithelial cells. T-cell-derived NO could partly explain the enhanced chlamydial inhibition when T cells were co-cultured with infected epithelial cells. These results are consistent with the hypothesis that, besides T-cell-derived IFN-gamma, other factors associated with lymphoepithelial interactions are likely to contribute an important role in chlamydial control by T cells in mice.
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PMID:The molecular mechanism of T-cell control of Chlamydia in mice: role of nitric oxide. 866 20

Infection with Mycobacterium bovis bacillus Calmette-Guerin (BCG) confers mice with strong abilities to produce nitric oxide (NO) and cytokines. Because the peritoneal macrophages taken from the mice immunized with live or heat-killed BCG can produce NO without any accessory cells and stimulants, it is difficult to clarify the immune regulation on NO production by manipulating the macrophages. Therefore, we investigated the participation of immune T cells and cytokines in NO production by using in vitro co-cultures of macrophages from non-immune mice with T cells prepared from BCG-infected mice in the presence or absence of a mycobacterial antigen, purified protein derivative (PPD). Although the non-immune thioglycollate (TGB)-elicited macrophages could not produce any detectable NO in the presence of PPD, supplementation of the macrophage cultures with CD4+ T cells prepared from BCG-infected mice enabled the macrophages to produce NO. Immunocytostaining showed that the macrophages, hut not the immune T cells, expressed inducible NO synthase (iNOS), indicating that they were NO producers. PPD could only induce NO production if there was cell-cell contact of the CD4+ T cells in the immune cells and antigen-presenting macrophages were required for the NO production in response to PPD; this interaction led to the production of soluble mediators that induced NO production by the TGB macrophages. NO production by the co-cultured cells was abrogated by adding either anti-interferon-gamma(IFN-gamma) or anti-tumor necrosis factor alpha (TNF-alpha) antibody. Furthermore, the roles of immune T cells and PPD could be replaced by adding recombinant IFN-gamma together with TNF-alpha to the macrophage cultures, but neither alone was sufficient to induce NO production by the macrophages. Our present data indicate that TNF-alpha produced by PPD-stimulated macrophages and IFN-gamma produced by cell-cell interaction of BCG-immune T cells and antigen-engulfed macrophages together activate the macrophages to produce NO.
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PMID:Nitric oxide production by peritoneal macrophages of Mycobacterium bovis BCG-infected or non-infected mice: regulatory role of T lymphocytes and cytokines. 869 Oct 77

Because the role of intracellular Ca2+ in the two-signal process for the induction of nitric oxide (NO) synthesis is controversial, this study was undertaken to examine the role of Ca2+ in the transcriptional regulation of inducible NO synthase (iNOS) in murine peritoneal macrophages. Treatment of the cells with thapsigargin (TG) or 2,5-di-(t-butyl)-1,4-benzodihydroquinone (tBuBHQ), which are the specific and potent Ca(2+)-ATPase inhibitors of endoplasmic reticulum (ER), showed modest effects on tumoricidal function, whereas TG or tBuBHQ in combination with interferon-gamma (IFN-gamma) or lipopolysaccharide (LPS) showed marked effects on tumoricidal function of the cells. The tumoricidal effects of the activated macrophages were correlated with the amount of NO synthesis, and totally abrogated by the use of NOS inhibitor, NG-monomethyl-L-arginine (NGMMA). The increases in NO synthesis was reflected as increased amounts of iNOS mRNA by Northern blotting. To confirm that iNOS induction was due to the changes in the intracellular Ca2+ level, the acetoxymethyl ester of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM), an intracellular Ca2+ chelator, was used. Blocking the increase of cytosolic free Ca2+ significantly decreased the induction of NO synthesis. To demonstrate that intracellular Ca2+ acts as a 'priming' signal rather than a 'triggering' signal on the induction of NO synthesis by murine peritoneal macrophages, we designed several experiments. When the cells were treated with TG 6 hr after the treatment with IFN-gamma, there was no increase in NO synthesis. In addition, when the cells were treated with TG or LPS 6 hr after treatment with tBuBHQ, a synergistic increase on NO synthesis was shown only in the case of LPS. When phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, was added to the cells 6 hr after the treatment with TG, there was a marked co-operative induction of NO synthesis, even though PMA alone has no effect. Based on the results obtained in this study, we suggest that cytosolic Ca2+ might be enough for the expression of iNOS gene as a priming signal and PKC might be involved in the induction of NO synthesis as a triggering signal by post-transcriptional modification of iNOS mRNA or iNOS itself in the activated murine peritoneal macrophages.
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PMID:Role of intracellular calcium as a priming signal for the induction of nitric oxide synthesis in murine peritoneal macrophages. 869 94

Endogenously generated or exogenously supplied nitric oxide (NO)-induced apoptotic cell death in the mouse macrophage cell line RAW 264.7. Apoptotic signaling caused an early accumulation of the tumor suppressor p53 prior to DNA fragmentation. Contrary to the notion of specific activating signals, inhibitory transduction mechanisms largely remain unknown. Therefore, RAW 264.7 macrophages were stably transfected with human Bcl-2, an anti-apoptotic protein. Bcl-2 transfectants showed substantial protection from cell death induced following the exposure to NO donors such as S-nitrosoglutathione (GSNO) and spermine-NO. In contrast, in RAW 264. 7 parent or in neomycin control-transformed cells, these NO donors induced internucleosomal DNA cleavage in a dose-dependent manner. Similarly, expression of the inducible NO synthase in response to lipopolysaccharide and interferon-gamma also caused apoptosis in RAW macrophages and neo controls within 24 h. In contrast, Bcl-2 transfectants appeared highly resistant, although inducible NO synthase levels increased along with concomitant nitrite production similar to control cells. The expression of p53 and Bax was also explored in controls and Bcl-2 transfectants after GSNO addition. GSNO induced p53 expression in Bcl-2 transfectants at levels comparable with nontransfected RAW macrophages. Moreover, GSNO induced increases in the steady-state levels of Bax protein in parental and Bcl-2-transfected cells. We conclude therefore, that Bcl-2 acts downstream of p53, presumably nullifying the NO-mediated increase in Bax protein in RAW 264.7 cells.
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PMID:Bcl-2 protects macrophages from nitric oxide-induced apoptosis. 870 45

We have elucidated the direct effects of PSK (a protein-bound polysaccharide) and OK-432 (a streptococcal preparation), both immunomodulating drugs, on the gene expression for an inducible nitric oxide synthase and on the production of nitric oxide (NO) in the RAW264.7 murine macrophage cell line. As determined by northern blot analysis, both immunomodulating drugs were potent inducers of gene expression for inducible NO synthase when cells were costimulated with interferon-gamma (IFN gamma). Expression of mRNA for the enzyme occurred in a dose-dependent manner after 3 h, when 10-50 micrograms/ml PSK or 0.001-1 KE/ml OK-432 was used. Furthermore, NO was also produced in response to these drugs, as detected by the Griess reagent reaction. The enhancement of NO synthesis was thought to be mediated, in part, through tumor necrosis factor alpha (TNF alpha) induction by these agents, since a neutralizing antibody to TNF alpha significantly suppressed NO production in RAW264.7 cells stimulated with PSK or OK432 in combination with IFN gamma. We speculate that NO production may play a role in tumoricidal and microbicidal activities of PSK or OK-432 in vivo.
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PMID:Induction of gene expression for nitric oxide synthase by immunomodulating drugs in the RAW264.7 murine macrophage cell line. 870 48

Treatment of cultured hepatocytes with a combination of cytokines, including tumour necrosis factor-alpha, interferon-gamma and interleukin-1 beta, plus lipopolysaccharide resulted in a time-dependent induction of nitric oxide (NO) synthase (as measured by NO2- (+) NO3- production) and inhibition of hepatic gluconeogenesis and glycogen breakdown. The inhibition of glucose release was comparable with the observed following treatment of rats with lipopolysaccharide or treatment of isolated hepatocytes with artificial NO donors. In addition, this effect was also evident with all substrates tested that enter the gluconeogenic pathway below the level of phosphoenolpyruvate carboxykinase, suggesting that this combination of cytokines may underlie the inhibition of gluconeogenesis observed in endotoxic shock. The maximal inhibition of glucose output required the presence of all the cytokines plus lipopolysaccharide, whereas the induction of NO synthase was independent of the lipopolysaccharide when the cytokines were employed. Inclusion of interferon-gamma was essential to obtain a maximal response for either parameter. Inclusion of 1 mM N(G)-monomethyl-L-arginine in the incubation abolished the increase in NO2- (+) NO3- observed with the complete cytokine mixture and various combinations; however, it failed to prevent the inhibition in glucose output, indicating that mechanisms other than NO underlie the cytokine-induced inhibition of glucose release.
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PMID:Effect of multiple cytokines plus bacterial endotoxin on glucose and nitric oxide production by cultured hepatocytes. 871 78

The human neuroblastoma cell line NB-39-nu expressed mRNA coding for inducible nitric oxide synthase (iNOS) following treatment with a combination of interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha). The level of iNOS mRNA peaked 24 h after stimulation and had declined by about 25% after 48 h. Trace levels of iNOS mRNA were detected after treatment with IFN-gamma alone, and its mRNA level was synergistically enhanced by simultaneous treatment with TNF-alpha. Neither bacterial lipopolysaccharide nor interleukin-1 beta (IL-1 beta) showed synergistic effects as great as that of TNF-alpha on iNOS gene expression. Dexamethasone inhibited the induction of iNOS mRNA by IFN-gamma and TNF-alpha. Induction of iNOS was confirmed by NADPH-diaphorase staining and by immunostaining with human iNOS-specific antibody.
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PMID:Nitric oxide synthase expression in human neuroblastoma cell line induced by cytokines. 872 59

We investigated the effect of nitric oxide (NO) on the induction of the stress protein heme oxygenase and its protective role in vascular endothelial cells exposed to hydrogen peroxide. Treatment of porcine aortic endothelial cells for 6 h with the NO-releasing compounds (0.1-1 mM) sodium nitroprusside (SNP), S-nitroso-N-acetylpenicillamine (SNAP), and 3-morpholinosydnonimine (SIN-1) resulted in a concentration-dependent increase in heme oxygenase activity. At 1 mM, the activity of heme oxygenase was augmented 8.5-fold with SNP, 5.8-fold with SNAP, and 5.7-fold with SIN-1 over the control value. In contrast, endothelial cells exposed to 100 microM S-bromoguanosine 3',5'-cyclic monophosphate, a tissue-permeable analogue that mimics the action of guanosine 3',5'-cyclic monophosphate, did not show any change in heme oxygenase activity. Activation of the inducible NO synthase by the synergistic action of bacterial lipopolysaccharide (250 ng/ml) and interferon-gamma (100 U/ml) also increased endothelial heme oxygenase activity by 3.2-fold (P < 0.05 vs control). Methylene blue (1 microM), an inhibitor of both NO synthase and guanylate cyclase activities, completely abolished this effect. Cells previously exposed to SNAP and SIN-1 exhibited a significant protection against the cytotoxicity mediated by hydrogen peroxide (250 microM) (P < 0.05). Conversely, SNP did not show any protective effects, possibly because of catalytic iron released during its chemical decomposition. In fact, the iron chelator deferoxamine (5 mM) completely suppressed the SNP-mediated cytotoxicity and partially attenuated the activity of heme oxygenase to a level equal to that mediated by SIN-1 and SNAP. These results indicate that NO is a determinant in the modulation of the activity of heme oxygenase leading to a major resistance of the endothelium to oxidative stress.
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PMID:NO-mediated activation of heme oxygenase: endogenous cytoprotection against oxidative stress to endothelium. 876 40

The production of nitric oxide (NO) via the inducible form of NO synthase (iNOS) is regulated by a complex network of cytokines and endogenous hormones. Among these, transforming growth factor-beta (TGF-beta 1) is known to suppress iNOS expression and NO production by many cell types. To determine the effect of TGF-beta 1 on NO production by skeletal muscle cells, we stimulated C2C12 myocytes with interferon-gamma (IFN) and interleukin-1 (IL-1) in the presence or absence of TGF-beta 1. In contrast to findings in macrophages, TGF-beta 1 markedly enhanced NO production by skeletal muscle cells. Increases in NO production reflected significant increases in iNOS immunoreactive protein and iNOS mRNA. Elevated iNOS mRNA levels associated with TGF-beta 1 treatment were not due to an alteration in mRNA stability, but rather reflected a significantly increased transcriptional rate of the iNOS gene. These findings indicate that TGF-beta 1 enhances iNOS expression in skeletal muscle cells and suggest that the regulation of NO production by TGF-beta 1 may depend on the cell type studied.
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PMID:Effects of transforming growth factor-beta 1 on nitric oxide synthesis by C2C12 skeletal myocytes. 876 96


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