Nitric oxide (NO) pretreatment increases cytokine-induced NO production in cultured rat hepatocytes by suppressing GTP cyclohydrolase I feedback inhibitory protein level and promoting inducible NO synthase dimerization

Nitric oxide (NO) regulates the biological activity of many enzymes and other functional proteins as well as gene expression. In this study, we tested whether pretreatment with NO regulates NO production in response to cytokines in cultured rat hepatocytes. Hepatocytes were recovered in fresh medium for 24 h following pretreatment with the NO donor S-nitroso-N-acetyl-d,l-penicillamine (SNAP) and stimulated to express the inducible NO synthase (iNOS) with interleukin-1beta and interferon-gamma or transfected with the human iNOS gene. NO pretreatment resulted in a significant increase in NO production without changing iNOS expression for both conditions. This effect, which did not occur in macrophages and smooth muscle cells, was inhibited when NO was scavenged using red blood cells. Pretreatment with oxidized SNAP, 8-Br-cGMP, NO(2)(-), or NO(3)(-) did not increase the cytokine-induced NO production. SNAP pretreatment increased cytosolic iNOS activity measured only in the absence of exogenous tetrahydrobiopterin (BH(4)). SNAP pretreatment suppressed the level of GTP cyclohydrolase I (GTPCHI) feedback regulatory protein (GFRP) and increased GTPCHI activity without changing GTPCHI protein level. SNAP pretreatment also increased total cellular levels of biopterin and active iNOS dimer. These results suggest that SNAP pretreatment increased NO production from iNOS by elevating cellular BH(4) levels and promoting iNOS subunit dimerization through the suppression of GFRP levels and subsequent activation of GTPCHI.     

Hypoxia enhances gene expression of inducible nitric oxide synthase and matrix metalloproteinase-9 in ras/myc-transformed serum-free mouse embryo cells under simulated inflammatory and infectious conditions

Ras/myc-transformed serum-free mouse embryo (ras/myc SFME) cells were treated with interferon-gamma (IFN-gamma; 100 U/ml) and/or lipopolysaccharide (LPS; 0.5 microg/ml) for 24 h to simulate inflammatory and infectious conditions and investigate their effects on the expression of inducible nitric oxide synthase (iNOS) mRNA, nitric oxide (NO) and matrix metalloproteinase-9 (MMP-9). In addition, aminoguanidine (AG; 1 mM), a NOS inhibitor, S-nitroso-N-acetyl-DL-penicillamine (SNAP; 10-200 microM), an NO donor or (+/-)-N-[(E)-4-ethyl-2-[(Z)-hydroxyimino]-5-nitro-3-hexene-1-yl]-3-pyridine carboxamide (NOR4; 10-200 microM), an NO donor, were added to analyze possible associations of NO with MMP-9. Tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 were also measured to analyze possible relationships of NO with the MMP-9/TIMP balance. Furthermore, the cells were treated with 1% O2 under the simulated inflammatory and infectious conditions and the mRNA expressions of iNOS and MMP-9 were analyzed to investigate the possible effects of hypoxia on the expression of genes involved in tumor malignant progression and distant metastasis. Co-treatment with IFN-gamma and LPS increased the expression levels of iNOS mRNA, NO and MMP-9, but NO may not be directly associated with MMP-9 or the MMP-9/TIMP balance. Treatment with 1% O2 markedly increased the gene expression levels of iNOS and MMP-9, indicating that ras/myc SFME cells alter the expression levels of tumor-associated genes and possibly enhance their malignancy as cancer cells under inflammatory, infectious and hypoxic conditions.     

Identification of manganese superoxide dismutase as a NO-regulated gene in rat glomerular mesangial cells by 2D gel electrophoresis.

The course of inflammatory glomerular diseases is accompanied by changes in the expression of matrix-associated proteins, growth factors, and mediators in renal mesangial cells. Furthermore, the production of nitric oxide (NO) by the inducible isoform of nitric oxide synthase (iNOS) is enhanced after stimulation with pro-inflammatory cytokines. NO has been demonstrated to be a potent modulator of gene expression. To identify NO-regulated genes, we compared the expression patterns of mesangial cells treated for 24h with 500 microM (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO) with those of un-stimulated controls by applying a proteomics approach. One protein found to be NO-modulated by 2D gel electrophoresis is the manganese superoxide dismutase (Mn-SOD). Immunoblot and Northern blot analysis demonstrated a dose- and time-dependent induction of Mn-SOD expression by S-nitroso-N-acetyl-D, L-penicillamine (SNAP) and DETA-NO on both the protein and the mRNA levels. An upregulation of Mn-SOD expression by NO was accompanied by an increased Mn-SOD activity. Immunoblots of extracts of IL-1beta-treated cells cultivated with or without the iNOS inhibitor N(G)-monomethyl-L-arginine and the inhibitor of soluble guanylyl cyclase (sGC) 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ) demonstrated that the upregulation of the Mn-SOD by NO is due to a NO-dependent activation of sGC. The upregulation of Mn-SOD mRNA expression by NO was confirmed in vivo by Northern blot analysis in kidneys from rats treated with lipopolysaccharide (LPS) either in presence or absence of the iNOS inhibitor N(6)-(1-iminoethyl)-L-lysine (l-NIL).     

Age-related decline of inducible nitric oxide synthase gene expression in primary cultured rat hepatocytes

Hepatocytes exhibit a non-specific immune response by expressing the enzyme inducible nitric oxide (NO) synthase (iNOS, NOS2) through the stimulation of a mixture of cytokines, or a single cytokine such as interleukin-1beta. We examined the age-dependent inducibility of the iNOS gene expression and the capacity of NO production in response to lipopolysaccharide (LPS) or interleukin-1beta (IL-1beta) in primary cultured rat hepatocytes that were isolated from the livers of rats, 3 (young) and 24 (aging) months of age. NO production (NO2-), indicating iNOS activity, was much higher in the young rat hepatocytes following stimulation with LPS or IL-1beta. Likewise, in the young hepatocytes, Western blot analyses showed much higher protein levels in the iNOS expression; it was also a little higher in mRNA levels that were analyzed by RT-PCR. Furthermore, after stimulation with IL-1beta, the levels of transactivation of nuclear factor-KB (NF-kappaB) that were involved in the induction of the iNOS gene were reduced without a significant difference in the aged cells. Therefore, the decrease of NO formation in the aged hepatocytes was due to the belated and incomplete inducibility of the iNOS protein expression, together with a minor contribution of the reduced-transactivation of NF-kappaB. These results suggest that the age-related decline of the iNOS gene expression in primary rat hepatocytes may be associated with the increased incidence of many infective diseases with aging.     

The expression analysis of inflammatory and antimicrobial genes in the goldfish (Carassius auratus L.) infected with Trypanosoma carassii

We report results of a comprehensive analysis of inflammatory gene expression during the course of infection of Trypanosoma carassii in the goldfish. We observed significant increases in mRNA levels of genes encoding pro-inflammatory cytokines IFN-γ, TNFα1 and TNFα2; IL-1β-1 and IL-1β-2; IL-12-p35 and IL-12-p40; CCL1; CXCL8, anti-inflammatory cytokines IL-10 and TGFβ and iNOS A and iNOS B, using quantitative PCR. Expression levels and profiles of these cytokines and iNOS isoforms varied in the different tissues (kidney, spleen, liver) of goldfish during the course of T.?carassii infection. The expression of majority of genes that encode pro- and anti-inflammatory cytokines were up-regulated during the acute phase of infection (days 7-21 post-infection). The mRNA levels of these cytokines returned to normal levels or were down-regulated during the elimination phase of infection (days 28-56), with exception of IL-10 in the spleen and liver of infected fish. A parallel up-regulation of IFN-γ and IL-10 mRNA levels were observed in all tissues of infected fish during the acute phase of the infection. The expression of iNOS genes (iNOS A and B) was significantly delayed (day 14?pi) in the kidney, liver and spleen of infected fish. These results provide insights into the interaction between T.?carassii and goldfish, and suggest that Th1/Th2-like responses may be important for controlling T.?carassii infection in the goldfish.     

Impact of Mycoplasma hyorhinis infection on L-arginine metabolism: differential regulation of the human and murine iNOS gene

Infection with mycoplasma is a common problem in cell cultures, with Mycoplasma hyorhinis being the predominant species. Here we investigate the effect of M. hyorhinis infection on L-arginine metabolism, with focus on iNOS-mediated NO synthesis in murine keratinocytes and the human colon cancer cell line DLD-1. iNOS and arginase are L-arginine-metabolizing enzymes involved in the regulation of inflammatory processes, with NO contributing to innate immunity. In murine cells, M. hyorhinis infection enhances cytokine-induced iNOS expression and augments iNOS activity, whereas in the absence of cytokines it causes de novo induction of iNOS mRNA without subsequent translation into iNOS protein. In turn, arginase-1 mRNA expression is diminished in M. hyorhinis-infected murine keratinocytes, resulting in decreased arginase activity. One of the underlying upstream mechanisms is NF-kappaB activation. In contrast, in human cells neither iNOS mRNA nor protein expression is affected by M. hyorhinis infection, but NO synthesis is enhanced, which may be caused by increased L-arginine import. This demonstrates that infection with M. hyorhinis leads to different effects on gene regulation of the murine and human iNOS gene. Our study underlines the importance of routine checking of cell cultures for mycoplasma contamination, particularly in studies on NO-mediated effects or inflammatory processes.     

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.     

Ras/myc-transformed serum-free mouse embryo cells under simulated inflammatory and infectious conditions increase levels of nitric oxide and matrix metalloproteinase-9 without a direct association between them

Inflammatory and infectious conditions were simulated in cultures of ras/myc-transformed serum-free mouse embryo (ras/myc SFME) cells, using interferon-gamma (IFN-γ, 100 units/ml) and lipopolysaccharide (LPS, 0.5 μg/ml) co-treatment for 24 h, to investigate their effects on the expression of inducible nitric oxide synthase (iNOS) mRNA and the production of NO. Aminoguanidine (AG, 1 mM; an NOS inhibitor) along with IFN-γ and LPS, S-nitroso-N-acetyl-DL-penicillamine (SNAP, 100 μM; an NO donor) and/or (±)-N-[(E)-4-Ethyl-2-[(Z)-hydroxyimino]-5-nitro-3-hexene-1-yl]-3-pyridine carboxamide (NOR4, 100 μM; an NO donor), were also added to analyze the possible association of NO with matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1). Co-treatment of cells with IFN-γ and LPS increased iNOS mRNA expression, NO production, MMP-9 mRNA expression, and 105 kDa MMP-9 production. Additional treatment with the NOS inhibitor AG inhibited NO production, but did not down-regulate the expression of MMP-9 mRNA or 105 kDa MMP-9. The NO donors SNAP and NOR4 did not affect the expression of MMP-9 mRNA, 105 kDa MMP-9 or TIMP-1 mRNA. These results suggest that ras/myc SFME cells respond to infectious and inflammatory conditions and can enhance malignancy as cancer cells due to their increased levels of NO and MMP-9 production, but that NO is not directly associated with MMP-9 in these cells. H. Yamaguchi and Y. Kidachi contributed equally to this work     

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.     

Nitric oxide suppresses the expression of Bcl-2 binding protein BNIP3 in hepatocytes

Nitric oxide (NO) is not only an important signaling molecule, but it also regulates the expression of a number of genes in the liver. We have previously shown that apoptosis in hepatocytes exposed to tumor necrosis factor-alpha and actinomycin D is prevented by NO derived from the inducible nitric-oxide synthase (iNOS), by mechanisms that are both dependent on and independent of modulation of cyclic guanosine monophosphate (cGMP) subsequent to activation of soluble guanylyl cyclase (sGC). We hypothesize that one mechanism by which NO exerts these effects is by regulating the expression of genes involved in apoptosis. We used differential display-polymerase chain reaction to isolate NO-regulated genes in hepatocytes from iNOS knockout mice (to eliminate endogenous inducible NO production). Using this analysis, we identified a NO-suppressed gene fragment homologous with the pro-apoptotic Bcl-2 binding protein BNIP3. Northern analysis confirmed the NO-dependent suppression of BNIP3 in cultured cells. Similarly, the NO donor S-nitroso-N-acetyl-dl-penicillamine (1-1000 microm) down-regulated the expression of BNIP3 in both iNOS knockout and wild-type hepatocytes. This effect of NO was reversed by the sGC inhibitor 1H-(1,2,4)-oxadiazole[4,3-a]quinoxalon-1-one (ODQ),suggesting the involvement of the sGC/cGMP pathway in the modulation of BNIP3 by NO. We propose that suppression of BNIP3 expression is one sGC/cGMP-dependent mechanism by which NO might affect the process of hepatocyte apoptosis.     

The herbal medicine sho-saiko-to induces nitric oxide synthase in rat hepatocytes

We have examined the effects of the herbal medicine sho-saiko-to (SST) on nitric oxide (NO) biosynthesis in rat hepatocytes by measuring the stable end-product nitrite and the mRNA of inducible NO synthase (iNOS). Interferon-γ (IFN) by itself failed to induce NO synthesis (IFN: 1-1,000 u/ml). SST also did not elicit NO synthesis at concentrations up to 300 μg/ml when administered alone, but dose-dependently induced NO production in the presence of IFN. Whereas SST or IFN induced barely detectable levels of iNOS mRNA when administered alone, a combination of SST and IFN markedly induced iNOS mRNA in the cells. SST also modestly increased NO synthesis caused by interleukin-1 or bacterial lipopolysaccharide as a single agent, or in combination with IFN. On the other hand, SST had no effects on the NO synthesis produced by iNOS which were already induced. Thus, we found that SST stimulates cultured hepatocytes to produce NO by inducing iNOS gene expression under appropriate conditions. The capability of SST to induce NO biosynthesis might be related to the therapeutic efficacy of SST on the liver diseases.     

Carbon monoxide decreases the level of iNOS protein and active dimer in IL-1β-stimulated hepatocytes

There is evidence that NO can regulate CO production, however less is known about CO regulation of NO synthesis. Our studies were undertaken to define how CO regulates iNOS in cultured hepatocytes. CO (250 ppm) exposure resulted in a significant decrease in iNOS protein, nitrite production, level of active iNOS dimer and cytosolic iNOS activity in cells stimulated with cytokines (IL-1β) or transfected with the human iNOS gene. However, IL-1β-stimulated iNOS mRNA expression was unaffected by CO. These effects of CO on iNOS protein levels were inhibited when CO was scavenged using hemoglobin. HO-1 induction with an adenoviral vector carrying HO-1 showed a decrease in total iNOS protein, nitrite production, and iNOS dimer level from cells stimulated by IL-1β. iNOS protein level was significantly higher in lung endothelial cells isolated from HO-1 knockout mice compared to wild type cultures stimulated with cytokines mixture. CO was found to increase p38 phosphorylation and p38 inhibition using SB203580 increased iNOS protein levels in response to IL-1β. Interestingly, proteasome inhibitors (MG132 and Lactacystin) and an autophagy inhibitor (3-methyladenine) reversed CO influence iNOS levels. Our results imply that CO exposure decreases NO production by suppressing dimer formation and increasing iNOS degradation through a process involving p38 activation.