Feedback inhibition of nitric oxide synthase activity by nitric oxide.

1. A murine macrophage cell line, J774, expressed nitric oxide (NO) synthase activity in response to interferon-gamma (IFN-gamma, 10 u ml-1) plus lipopolysaccharide (LPS, 10 ng ml-1). The enzyme activity was first detectable 6 h after incubation, peaked at 12 h and became undetectable after 48 h. 2. The decline in the NO synthase activity was not due to inhibition by stable substances secreted by the cells into the culture supernatant. 3. The decline in the NO synthase activity was significantly slowed down in cells cultured in a low L-arginine medium or with added haemoglobin, suggesting that NO may be involved in a feedback inhibitory mechanism. 4. The addition of NO generators, S-nitroso-acetyl-penicillamine (SNAP) or S-nitroso-glutathione (GSNO) markedly inhibited the NO synthase activity in a dose-dependent manner. The effect of NO on the enzyme was not due to the inhibition of de novo protein synthesis. 5. SNAP directly inhibited the inducible NO synthase extracted from activated J774 cells, as well as the constitutive NO synthase extracted from the rat brain. 6. The enzyme activity of J774 cells was not restored after the removal of SNAP by gel filtration, suggesting that NO inhibits NO synthase irreversibly.     

Glibenclamide-induced inhibition of the expression of inducible nitric oxide synthase in cultured macrophages and in the anaesthetized rat.

1. We have investigated whether glibenclamide, an inhibitor of ATP-sensitive potassium channels, influences the induction of the calcium-independent isoform of nitric oxide synthase (iNOS) in cultured J774.2 macrophages activated by bacterial endotoxin (E.coli lipopolysaccharide; LPS), as well as in the lung and aorta of rats with endotoxic shock. 2. Pretreatment of J774.2 macrophages with glibenclamide (10(-7) to 10(-5) M for 30 min) dose-dependently inhibited the accumulation of nitrite caused by LPS (1 microgram ml-1). In contrast, pretreatment of macrophages with tetraethylammonium (10(-4) to 10(-2) M for 30 min), a non-selective inhibitor of potassium channels, did not affect the rise in nitrite caused by LPS. At the highest concentration (10(-5) M) used, cromakalim, an opener of ATP-sensitive potassium channels, caused a small, but significant inhibition of nitrite formation in macrophages activated with LPS, while lower concentrations (10(-7) to 3 x 10(-6) M) were without effect. 3. The inhibition by glibenclamide (3 microM) of the increase in nitrite induced by LPS in J774.2 macrophages was weaker when glibenclamide was given several hours after LPS, indicating that glibenclamide inhibits the induction, but not the activity, of iNOS. In contrast, the degree of inhibition of nitrite formation caused by the nitric oxide synthase (NOS) inhibitor N omega-nitro-L-arginine methyl ester (L-NAME) was similar when this agent was given up to 10 h after LPS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chloroquine inhibits inducible nitric oxide synthase expression in murine peritoneal macrophages

Induction of inducible nitric oxide synthase in vivo or in vitro in response to stimuli is only temporary. However, chronic localized expression of inducible nitric oxide synthase in certain organs has been associated with the development of autoimmune diseases or chronic inflammatory diseases. Chloroquine is being used as an antiinflammatory drug, and its inhibitory effect on the synthesis of pro-inflammatory cytokines, such as tumour necrosis factor-alpha and interferon-gamma, has been reported. In this study, we examined whether chloroquine could inhibit nitric oxide synthesis in murine peritoneal macrophages stimulated with interferon-gamma and lipopolysaccharide. Although prolonged incubation of cells with high concentrations of chloroquine showed some cytotoxicity, the drug itself was not cytotoxic when macrophages were preincubated with chloroquine for 2 hr, washed and stimulated with interferon-gamma and lipopolysaccharide in the absence of chloroquine for another 48 hr. The nitric oxide production from stimulated macrophages was markedly reduced by chloroquine in a dose-dependent manner and induction of inducible nitric oxide synthase mRNA was also suppressed by chloroquine pretreatment. These results show that chloroquine inhibits the induction of inducible nitric oxide synthase from interferon-gamma and lipopolysaccharide-stimulated macrophages, thereby reducing nitric oxide synthesis.     

Induced RAW 264.7 macrophages express soluble and particulate nitric oxide synthase: inhibition by transforming growth factor-beta.

RAW 264.7 macrophages induced with lipopolysaccharide and interferon-gamma expressed nitric oxide (NO) synthase. Approximately two-thirds of the total induced NO synthase activity was found in the cytosolic fraction, whereas one-third was associated with the particulate fraction. Both enzymes formed L-citrulline in addition to NO-like material. NO and L-citrulline formation by both enzymes were calcium-independent and inhibited by NG-nitro-L-arginine and NG-methyl-L-arginine. Transforming growth factor-beta 1 prevented the induction of both enzymes.     

Dexamethasone inhibits inducible nitric-oxide synthase expression and nitric oxide production by destabilizing mRNA in lipopolysaccharide-treated macrophages

Nitric oxide (NO) production through the inducible nitric-oxide synthase (iNOS) pathway is increased in inflammatory diseases and leads to cellular injury. Anti-inflammatory steroids inhibit the expression of various inflammatory genes, including iNOS. In the present study, we investigated the mechanism how dexamethasone decreased NO production in murine J774 macrophages. Dexamethasone (0.1-10 microM) inhibited the production of NO and iNOS protein in a dose-dependent manner in cells stimulated with lipopolysaccharides (LPS). In contrast, in cells treated with a combination of LPS and interferon-gamma (IFN-gamma), dexamethasone did not reduce iNOS expression and NO formation. Dissociated glucocorticoid RU24858 inhibited iNOS expression and NO production to levels comparable with that of dexamethasone, suggesting that the reduced iNOS expression by dexamethasone is not a GRE-mediated event. In further studies, the effect of dexamethasone on iNOS mRNA levels was tested by actinomycin assay. The half-life of iNOS mRNA after LPS treatment was 5 h 40 min, and dexamethasone reduced it to 3 h. The increased degradation of iNOS mRNA was reversed by a protein synthesis inhibitor cycloheximide. iNOS mRNA was more stabile in cells treated with a combination of LPS plus IFN-gamma (half-life = 8 h 20 min), and dexamethasone had a minor effect in these conditions. In conclusion, dexamethasone decreases iNOS-dependent NO production by destabilizing iNOS mRNA in LPS-treated cells by a mechanism that requires de novo protein synthesis. Also, decreased iNOS mRNA and protein expression and NO formation by dexamethasone was not found in cells treated with a combination of LPS plus IFN-gamma, suggesting that the effect of dexamethasone is stimulus-dependent.     

Pterins inhibit nitric oxide synthase activity in rat alveolar macrophages.

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.     

8-Prenylkaempferol suppresses inducible nitric oxide synthase expression through interfering with JNK-mediated AP-1 pathway in murine macrophages

8-Prenylkaempferol is a prenylflavonoid isolated from the roots of Sophora flavescens, a Chinese herb with anti-inflammatory properties. However whether 8-prenylkaempferol itself displayed an anti-inflammatory activity remained unclear. In this study, we evaluated the effect of 8-prenylkaempferol on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW264.7 macrophages. 8-Prenylkaempferol inhibited significantly LPS-induced NO production through suppressing inducible NO synthase (iNOS) expression at both protein and mRNA levels but failed to affect sodium nitroprusside-triggered NO production, iNOS enzyme activity, and cell viability. Further investigation of the mechanisms revealed that 8-prenylkaempferol inhibited LPS-induced c-Jun phosphorylation (a major component of activator protein-1, AP-1), but did not attenuate IkB-alpha degradation nor NF-kappaB nuclear translocation. Cellular signaling analysis using mitogen-activating protein kinase (MAPK) inhibitors including 2'-amino-3'-methoxyflavone (PD98059, MEK1/2 inhibitor), 4-[5-(4-fluorophenyl)-2-[4-(methylsulfonyl)phenyl]-1H-imidazol-4-yl]pyridine (SB203580, p38 kinase inhibitor) and anthra[1-9-cd]pyrazol-6(2H)-one (SP600125, c-Jun N-terminal kinase inhibitor) demonstrated that extracellular signal-regulated kinase1/2 (ERK1/2), p38 and JNK all participated in LPS-stimulated iNOS expression and NO production, but 8-prenylkaempferol interfered selectively with JNK phosphorylation. On the other hand, LPS-induced c-Jun phosphorylation was attenuated in the presence of SP600125. We suggested that interfering with JNK-mediated c-Jun phosphorylation and thus blocking AP-1 activation might contribute to the suppression effects of 8-prenylkaempferol on iNOS. These findings provided the first molecular basis that 8-prenylkaempferol is an effective agent for attenuating pro-inflammatory NO induction.     

Dithiolane analogs of lignans inhibit interferon-gamma and lipopolysaccharide-induced nitric oxide production in macrophages

AIM: To investigate the effect of a group of novel synthetic dithiolane analogs of lignans and a well characterized platelet-activating factor (PAF) receptor antagonist, L659,989 on PAF-receptor binding, IFN-gamma- and lipopolysaccharide (LPS)-induced NO production, and steady-state inducible nitric-oxide synthase (iNOS) mRNA expression. METHODS: PAF-receptor binding study was performed by displacement of 3H-PAF from rabbit platelet membrane; NO production was quantitated by measuring the NO oxidation product, nitrite, in conditioned culture medium; expression of iNOS mRNA was assessed by Northern blot analysis. RESULTS: The dithiolane analogs inhibited the production of NO, decreased iNOS mRNA expression and antagonized PAF-receptor binding. L659,989 had no effect on NO production and iNOS mRNA expression. Among the compounds tested, there was no simple correlation between their PAF-receptor antagonistic and iNOS inhibitory activities. CONCLUSION: The dithiolane analogs are a new synthetic chemical class of iNOS expression regulators with dual biologic functions: inhibiting iNOS induction and blocking PAF-receptor.     

Activation of inducible nitric oxide synthase by Taraxacum officinale in mouse peritoneal macrophages.

The objective of the current study was to determine the effect of Taraxacum officinale (TO) on the production of nitric oxide (NO). Stimulation of mouse peritoneal macrophages with TO after the treatment of recombinant interferon-gamma (rIFN-gamma) resulted in increased NO synthesis. TO had no effect on NO synthesis by itself. When TO was used in combination with rIFN-gamma, there was a marked cooperative induction of NO synthesis in a dose-dependent manner. The optimal effect of TO on NO synthesis was shown 6 h after treatment with rIFN-gamma. This increase in NO synthesis was manifested as an increased amount of inducible NO synthase (iNOS) protein. NO production was inhibited by N(G)-monomethyl-L-arginine. The increased production of NO from rIFN-gamma plus TO-stimulated cells was decreased by treatment with a protein kinase C inhibitor such as staurosporin. In addition, synergy between rIFN-gamma and TO was mainly dependent on TO-induced tumor necrosis factor-alpha (TNF-alpha) secretion. All the preparations of TO were endotoxin free. These results suggest that the capacity of TO to increase NO production from rIFN-gamma-primed mouse peritoneal macrophages is the result of TO-induced TNF-alpha secretion.