Inducible Type Of Nitric Oxide Synthase Research Articles

72 - Regulation of Histone Deacetylase 6 (HDAC6) Activity via S-Nitrosylation

Nitric oxide (NO) is a gaseous molecule produced by NO synthase (NOS) and plays essential functions via S-nitrosylation. In the course of screening for the target proteins of NO, we identified histone deacetylase 6 (HDAC6). HDAC6 plays key roles in regulating cell motility and signaling through deacetylation of substrate proteins such as alpha-tubulin. In the present study, we attempted to elucidate the role of NO on HDAC6 activity. Using biotin-switch assay, we found that HDAC6 is S-nitrosylated by both NO donors and NO derive from the inducible type of NOS in A549 cells treated with inflammatory cytokines. Next, we analyzed whether NO influences on enzymatic activity of HDAC6. In vitro assays showed that treatment with S-nitrosocysteine, a physiological NO donor, significantly attenuated deacetylase activity, indicating that NO directly inhibits catalytic activity of HDAC6. Then, we examined whether NO acts on endogenous HDAC6 activity in A549 cells. The level of acetylated alpha-tubulin was significantly elevated upon treatment with cytokines, but this increment was strongly suppressed with L-NAME, a NOS inhibitor. These findings suggest that S-nitrosylation of HDAC6 plays a pivotal role in the regulation of protein acetylation.

Production and physiological role of NO in the oral cavity

SummaryNitric oxide (NO) is a free radical which is produced from a wide variety of cells and tissues in the human body. NO is involved in the regulation of many physiological processes, such as vascular relaxation, neurotransmission, immune regulation, and cell death. NO is generated by nitric oxide synthase (NOS), which has three identified isoforms: neuronal type NOS (nNOS), endothelial type NOS (eNOS), and inducible type NOS (iNOS). Different isoforms are expressed depending on the organs, tissues, and cells, and investigation of the types and functions of enzymes expressed in various tissues is underway. The oral cavity is a space in which marked changes have been detected in NO levels, and each tissue is constantly influenced by NO. NO is a component of saliva and is produced by oral bacteria in the oral cavity and released by NOS expressed in oral mucosa. NOS isoforms expressed under normal conditions differ among the oral organs. In addition, the overexpression of NOS was involved in carcinogenesis and tumor growth progression. This review summarized the expression of NOS and functions of NO in oral cavity organs, and their roles in diseases and the influences of treatments.

Increase of nitrosative stress in patients with eosinophilic pneumonia

BackgroundExhaled nitric oxide (NO) production is increased in asthma and reflects the degree of airway inflammation. The alveolar NO concentration (Calv) in interstitial pneumonia is reported to be increased. However, it remains unknown whether NO production is increased and nitrosative stress occurs in eosinophilic pneumonia (EP). We hypothesized that nitrosative stress markers including Calv, inducible type of NO synthase (iNOS), and 3-nitrotyrosine (3-NT), are upregulated in EP.MethodsExhaled NO including fractional exhaled NO (FENO) and Calv was measured in ten healthy subjects, 13 patients with idiopathic pulmonary fibrosis (IPF), and 13 patients with EP. iNOS expression and 3-NT formation were assessed by immunocytochemistory in BALf cells. The exhaled NO, lung function, and systemic inflammatory markers of the EP patients were investigated after corticosteroid treatment for 4 weeks.ResultsThe Calv levels in the EP group (14.4 ± 2.0 ppb) were significantly higher than those in the healthy subjects (5.1 ± 0.6 ppb, p < 0.01) and the IPF groups (6.3 ± 0.6 ppb, p < 0.01) as well as the FENO and the corrected Calv levels (all p < 0.01). More iNOS and 3-NT positive cells were observed in the EP group compared to the healthy subject and IPF patient. The Calv levels had significant positive correlations with both iNOS (r = 0.858, p < 0.05) and 3-NT positive cells (r = 0.924, p < 0.01). Corticosteroid treatment significantly reduced both the FENO (p < 0.05) and the Calv levels (p < 0.01). The magnitude of reduction in the Calv levels had a significant positive correlation with the peripheral blood eosinophil counts (r = 0.802, p < 0.05).ConclusionsThese results suggested that excessive nitrosative stress occurred in EP and that Calv could be a marker of the disease activity.

Inhibition of endothelial- and neuronal-type, but not inducible-type, nitric oxide synthase by the oxidized cholesterol metabolite secosterol aldehyde: Implications for vascular and neurodegenerative diseases

The cholesterol ozonolysis products secosterol-A and its aldolization product secosterol-B were recently detected in human atherosclerotic tissues and brain specimens, and have been postulated to play pivotal roles in the pathogenesis of atherosclerosis and neurodegenerative diseases. We examined several oxidized cholesterol metabolites including secosterol-A, secosterol-B, 25-hydroxycholesterol, 5β,6β-epoxycholesterol and 7-ketocholesterol for their effects on the activities of three nitric oxide synthases. In contrast to other oxidized metabolites, secosterol-A was found to be a potent inhibitor against the neuronal- and endothelial-type, but not the inducible-type nitric oxide synthase, with IC50 values of 22 ± 1 and 50 ± 5 µM, respectively. The calmodulin-binding regions of the neuronal- and endothelial-nitric oxide synthases contain lysine residues which are not present in the inducible-type nitric oxide synthase. Secosterol-A modifies proteins through the formation of a Schiff base with the lysine epsilon-amino group. It is possible that secosterol-A modifies lysine residues of constitutive nitric oxide synthases, leading to the inhibition of enzymatic activities. As nitric oxide is a critical signaling molecule in vascular function and in long-term potentiation, its reduced production through inhibition of constitutive nitric oxide synthases by secosterol-A may contribute to the development of atherosclerosis and memory impairment in particular neurodegenerative diseases.

Thalidomide inhibits interferon-γ-mediated nitric oxide production in mouse vascular endothelial cells

Thalidomide is known as an anti-angiogenic, anti-tumor, and anti-proliferative agent, widely used in the treatment of some immunological disorders and cancers. The effect of thalidomide on interferon (IFN)-γ induced nitric oxide (NO) production in mouse vascular endothelial cells was examined in order to elucidate the anti-angiogenic or anti-inflammatory action. Thalidomide inhibited IFN-γ-induced NO production in mouse END-D cells via reduced expression of an inducible type of NO synthase (iNOS) protein and mRNA. Since thalidomide did not alter the cell surface expression of IFN-γ receptor, the NO inhibition was suggested to be due to the impairment of IFN-γ-induced intracellular event by thalidomide. Thalidomide inhibited the phosphorylation of IRF1, which was required for the iNOS expression. Moreover, it inhibited the phosphorylation of STAT1, an upstream molecule of IRF1, in IFN-γ signaling. Thalidomide did not inhibit the JAK activation in response to IFN-γ. A phosphatase inhibitor, sodium orthovanadate, abolished the inhibitory action of thalidomide. Therefore, thalidomide was suggested to inhibit IFN-γ-induced NO production via impaired STAT1 phosphorylation.

B1 cells produce nitric oxide in response to a series of toll-like receptor ligands

The effect of a series of toll-like receptor (TLR) ligands on the production of nitric oxide (NO) in mouse B1 cells was examined by using CD5 + IgM + WEHI 231 cells. The stimulation with a series of TLR ligands, which were Pam3Csk4 for TLR1/2, poly I:C for TLR3, lipopolysaccharide (LPS) for TLR4, imiquimod for TLR7 and CpG DNA for TLR9, resulted in enhanced NO production via augmented expression of an inducible type of NO synthase (iNOS). LPS was most potent for the enhancement of NO production, followed by poly I:C and Pam3Csk4. Imiquimod and CpG DNA led to slight NO production. The LPS-induced NO production was dependent on MyD88-dependent pathway consisting of nuclear factor (NF)-κB and a series of mitogen-activated protein kinases (MAPKs). Further, it was also dependent on the MyD88-independent pathway consisting of toll-IL-1R domain-containing adaptor-inducing IFN-β (TRIF) and interferon regulatory factor (IRF)-3. Physiologic peritoneal B1 cells also produced NO via the iNOS expression in response to LPS. The immunological significance of TLR ligands-induced NO production in B1 cells is discussed.

Synthetic triglyceride containing an arachidonic acid branch (8A8) prevents lipopolysaccharide-induced liver injury

Aims In this study, we investigated the effect of synthetic triglyceride containing an arachidonic acid branch (8A8) on lipopolysaccharide (LPS)-induced production of tumor necrosis factor (TNF)-α and nitric oxide (NO) in macrophages, and LPS-induced liver injury in the rat. Main methods RAW264.7 macrophages were co-incubated with 8A8 and LPS (100 ng/ml), and TNF-α mRNA/protein levels, nuclear factor (NF)-κB DNA binding activity, expression of inducible-type NO synthase (NOS2), and NO 2 production were measured. Male Wistar rats were given a single intraperitoneal injection of 8A8 prior to an intravenous injection of LPS (5 mg/kg), and liver histology, apoptotic cell death, serum TNF-α levels, and hepatic TNF-α mRNA were then evaluated. Key findings LPS-induced increases in TNF-α production in RAW264.7 macrophages were blunted by 8A8 in a dose-dependent manner, with 40% inhibition at 100 ppm. Further, 8A8 dose-dependently prevented LPS-induced increases in TNF-α mRNA levels, as well as NF-κB DNA binding activities, in RAW264.7 macrophages. LPS-induction of NOS2 and NO 2 release from these cells was also decreased by 8A8 in a dose-dependent manner. In vivo, LPS-induced liver injury, including hepatocyte apoptosis, was largely prevented when 8A8 (100 µl/kg) was given 30 min prior to LPS. Indeed, 8A8 blunted increases in both serum TNF-α and hepatic TNF-α mRNA levels significantly. Significance LPS-induced liver injury was prevented by 8A8 most likely through the inhibition of TNF-α and NO production from hepatic macrophages, suggesting a potential usefulness of 8A8 as an immuno-modulating nutrient for prevention/treatment of endotoxin-related organ injuries including alcoholic liver disease and non-alcoholic steatohepatitis (NASH).

Lipopolysaccharide enhances interferon-γ-induced nitric oxide (NO) production in murine vascular endothelial cells via augmentation of interferon regulatory factor-1 activation

Lipopolysaccharide (LPS) enhances the production of nitric oxide (NO) in interferon (IFN)-gamma-stimulated vascular endothelial cells. We studied the mechanism by which LPS enhances IFN-gamma-induced NO production by using the murine vascular endothelial cell line, END-D. LPS enhanced IFN-gamma-induced NO production via augmented expression of inducible type NO synthase (iNOS) mRNA. LPS significantly augmented the activation of interferon regulatory factor (IRF)-1 in IFN-gamma-stimulated END-D cells, although it did not affect the activation of either MyD88-dependent nuclear factor (NF)-kappaB or MyD88-independent IRF-3. SB203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK), prevented the nuclear translocation of IRF-1 in LPS and IFN-gamma-stimulated END-D cells, and inhibited the iNOS expression and NO production in those cells. Therefore, it is proposed that LPS enhanced NO production in IFN-gamma-stimulated END-D cells via augmenting p38 MAPKmediated IRF-1 activation.

Inhibition of extracellular signal-regulated kinase 1/2 augments nitric oxide production in lipopolysaccharide-stimulated RAW264.7 macrophage cells

The present study was conducted to determine effects of U0126, a specific inhibitor of mitogen-activated kinase kinase 1/2, on production of nitric oxide (NO) in RAW264.7 macrophage cells. U0126 significantly enhanced NO production in lipopolysaccharide (LPS) but not CpG DNA or interferon-γ-stimulated RAW264.7 cells. In contrast, U0124, a negative control for U0126, did not affect LPS-induced NO production. Further, a series of inhibitors of p38, phosphatidyl-inositol 3-kinase and Janus tyrosine kinase rather caused suppression in LPS-stimulated RAW264.7 cells. U0126 was found to definitely inhibit phosphorylation of extracellular signal-regulated kinase (Erk) 1/2 and augment the levels of inducible type of NO synthase. Antisense oligonucleotides of Erk1/2 also augmented LPS-induced NO production. Inactivation of Erk1/2 by U0126 furthermore inhibited LPS-induced activating protein-1 activation, but not nuclear factor-κB activation. The results suggest that Erk1/2 might negatively regulate NO production in LPS-stimulated RAW264.7 cells.

Serum-dependent potentiation of lipopolysaccharide-induced nitric oxide production is mediated by the events after the transcription of inducible type of nitric oxide synthase

The mechanism of serum-dependent potentiation of lipopolysaccharide (LPS)-induced nitric oxide (NO) production was studied by incubating mouse macrophage cell line, RAW 264.7, in the presence of fetal bovine serum (FBS). The addition of FBS definitely enhanced LPS-induced NO production through augmented expression of inducible type NO synthase (iNOS) mRNA and protein. However, nuclear run-on analysis demonstrated only marginal enhancement in the rate of LPS-induced iNOS gene transcription in the presence of FBS. Further, there was no significant difference in the luciferase reporter gene activity linked to the iNOS promoter–enhancer gene in response to LPS between the presence and absence of FBS. FBS-dependent enhancement did not appear to involve the initial step for triggering iNOS transcription in LPS-induced NO production. Rather, FBS was suggested to affect the accumulation and stabilization of iNOS mRNA leading to iNOS protein and NO production by some post-transcriptional regulatory mechanism.

Inhibitory Action of Daunorubicin on Lipopolysaccharide-Stimulated Inducible-Type Nitric Oxide Synthase Induction in Alveolar Macrophages

The purpose of this study was to investigate the effects of daunorubicin on lipopolysaccharide (LPS)-stimulated inducible-type nitric oxide synthase (iNOS) expression in macrophages. LPS-stimulated iNOS expression and NO production were significantly inhibited in alveolar macrophages from rats administrated daunorubicin (4 mg/kg body weight per day) for 5 consecutive days. Incubation of macrophages with daunorubicin at 1 µM but not at 0.1 and 0.5 µM significantly inhibited LPS-stimulated NO production and iNOS induction. Activation of extracellular regulated kinase (ERK) by LPS was markedly attenuated in both macrophages isolated from in vivo daunorubicin-treated rats and those incubated in vitro with daunorubicin at 1 µM. ERK activation, iNOS induction, and NO production following LPS stimulation were all markedly inhibited in the presence of U0126, an ERK inhibitor. The viability of macrophages was decreased by incubation with daunorubicin at 0.5 and 1 µM, while treatment of rats with daunorubicin did not affect viability of macrophages isolated from the rats. These results suggest that in vivo treatment of rats with daunorubicin attenuates LPS-induced iNOS expression of macrophages through inhibition of ERK activation, while inhibition of iNOS induction by in vitro incubation with daunorubicin may be mainly due to its cytotoxicity.

2‐Aminopurine Inhibits Lipopolysaccharide‐Induced Nitric Oxide Production by Preventing IFN‐β Production

2-aminopurine (2-AP) is widely used as a specific inhibitor for double stranded-RNA dependent protein kinase (PKR). Here we report that 2-AP can inhibit lipopolysaccharide (LPS)-stimulated nitric oxide (NO) production through the prevention of interferon (IFN)-beta production. 2-AP significantly inhibited NO production in LPS-stimulated RAW 264 murine macrophage cells. 2-AP also reduced the expression of IFN-beta and IFN-inducible genes, such as IFN-gamma-inducible protein (IP)-10 and immune-responsive gene (IRG)-1, and the inducible type of NO synthase (iNOS) mRNA in response to LPS. The addition of exogenous IFN-beta restored 2-AP-inhibited NO production in response to LPS. On the other hand, there was only partial inhibition by 2-AP of nuclear factor (NF)-kappaB activation, IL-6 mRNA expression and tumor necrosis factor (TNF)-alpha production. These results suggested that 2-AP inhibited LPS-induced IFN-beta production by preventing Toll/IL-1 receptor domain-containing adaptor-inducing IFN-beta (TRIF)-dependent signaling rather than myeloid differentiation factor (MyD) 88-dependent signaling, resulting in the inhibition of NO production.

Role of nitric oxide increase on induced programmed cell death during early stages of rat liver regeneration.

We analysed the possible cellular mechanism involved in the NO action in the balance between apoptosis and cell proliferation in liver regeneration process. We determined p53, proapoptotic protein Bax, antiapoptotic Bcl-xL, proliferating cell nuclear antigen (PCNA) and apoptotic index at the early stages of regenerative process after NO increase by lipopolysaccharide-induction (LPS) of inducible-type nitric oxide synthase (iNOS) and by direct NO donor (sodium nitroprusside, SNP). Male Wistar rats were randomised in four experimental groups: sham operated control (Sh), partial hepatectomised control (PH-C), partial hepatectomised pretreated with LPS (2 mg/kg body weight, i.p.) (PH-LPS), and partial hepatectomised pretreated with SNP (2.5 mg/kg body weight, i.v. at a rate of 1 ml/h) (PH-SNP). Animals were killed 5 h post-surgery. Hepatic cytosolic iNOS showed an increase of 34% in PH-C animals with respect to Sh, and LPS-treatment increased iNOS protein levels 30% compared with PH-C. Bax and p53 protein levels showed significant increases in LPS- and SNP-treated hepatectomised rats with respect to PH-C. The apoptotic indexes were increased 75% in both, PH-LPS and PH-SNP rats versus PH-C. The increase of NO did not show any change in the proliferation process. These results suggest that NO is involved in apoptosis via p53 and Bax proteins after PH, showing a tightly regulated growth process in liver regeneration.

Butyrate enhances the production of nitric oxide in mouse vascular endothelial cells in response to gamma interferon

The effect of butyrate, a natural bacterial product of colonic bacterial flora, on nitric oxide (NO) production in murine vascular endothelial cell line END-D in response to IFN-γ and/or LPS was studied. Butyrate significantly augmented NO production in END-D cells in response to IFN-γ or IFN-γ + LPS, but not LPS alone. The NO production was augmented by the addition of butyrate until 6 h after the stimulation with IFN-γ or IFN-γ + LPS. The augmentation was abolished by the removal of butyrate from the cultures. Butyrate enhanced the expression of inducible type NO synthase (iNOS) in the stimulated END-D cells. Furthermore, butyrate-enhanced NO production in the presence of various signal inhibitors down-regulating the signal pathways using nuclear factor (NF)-κB, mitogen-activated protein (MAP) kinases and Janus tyrosine kinase. The putative mechanism of butyrate-induced augmentation of NO production in response to IFN-γ or IFN-γ + LPS is discussed.

Butyrate enhances the production of nitric oxide in mouse vascular endothelial cells in response to gamma interferon.

The effect of butyrate, a natural bacterial product of colonic bacterial flora, on nitric oxide (NO) production in murine vascular endothelial cell line END-D in response to IFN-gamma and/or LPS was studied. Butyrate significantly augmented NO production in END-D cells in response to IFN-gamma or IFN-gamma + LPS, but not LPS alone. The NO production was augmented by the addition of butyrate until 6 h after the stimulation with IFN-gamma or IFN-gamma + LPS. The augmentation was abolished by the removal of butyrate from the cultures. Butyrate enhanced the expression of inducible type NO synthase (iNOS) in the stimulated END-D cells. Furthermore, butyrate-enhanced NO production in the presence of various signal inhibitors down-regulating the signal pathways using nuclear factor (NF)-kappaB, mitogen-activated protein (MAP) kinases and Janus tyrosine kinase. The putative mechanism of butyrate-induced augmentation of NO production in response to IFN-gamma or IFN-gamma + LPS is discussed.

Gamma Interferon‐Induced Nitric Oxide Production in Mouse CD5+ B1‐Like Cell Line and Its Association with Apoptotic Cell Death

The in vitro effect of gamma interferon (IFN-gamma) on nitric oxide (NO) production in a mouse CD5+ B1-like cell line, TH2.52, was studied. The TH2.52 cell line is the hybridoma line between mouse B lymphoma line and mouse splenic B cells and expresses a series of B1 markers. IFN-gamma induced a marked NO production in TH2.52 cells through the expression of an inducible type of NO synthase (iNOS). IFN-gamma-induced NO production was triggered by the Janus tyrosine kinase (JAK)/signal transducer and activator of transcription (STAT) pathway since it was inhibited by AG490, a JAK2 inhibitor. The growth of TH2.52 cells significantly was inhibited in the presence of IFN-gamma. A significant number of cells underwent apoptotic cell death, accompanied by the DNA fragmentation, annexin V binding, and caspase 3 activation. N(G)-monomethyl-L-arginine, an iNOS inhibitor, prevented IFN-gamma-induced cell death. Therefore, IFN-gamma-induced NO production was possible in causing cell death in TH2.52 cells. Further, IFN-gamma-induced NO production and cell death significantly were prevented by interleukin-4, a representative Th2 cytokine. The immunological significance of IFN-gamma-induced NO production in a mouse B1-like cell line is discussed.

C 2-ceramide inhibits LPS-induced nitric oxide production in RAW 264.7 macrophage cells through down-regulating the activation of Akt

The effect of C2-ceramide, a membrane-permeable ceramide analogue, on nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells was studied. The non-toxic concentration of C2-ceramide inhibited LPS-induced NO production. It was due to the attenuated expression of the inducible type of NO synthase (iNOS). C2-ceramide did not influence the phosphorylation of a series of mitogen-activated protein (MAP) kinases in response to LPS. On the other hand, C2-ceramide down-regulated the phosphorylation of Akt in LPS-stimulated RAW 264.7 cells, followed by the impairment of nuclear factor (NF)-κB activation. Moreover, the Akt dominant-negative mutant inhibited LPS-induced NO production. C2-ceramide was suggested to inhibit LPS-induced NO production through down-regulating the activation of Akt.

C2-ceramide inhibits LPS-induced nitric oxide production in RAW 264.7 macrophage cells through down-regulating the activation of Akt.

The effect of C(2)-ceramide, a membrane-permeable ceramide analogue, on nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells was studied. The non-toxic concentration of C(2)-ceramide inhibited LPS-induced NO production. It was due to the attenuated expression of the inducible type of NO synthase (iNOS). C(2)-ceramide did not influence the phosphorylation of a series of mitogen-activated protein (MAP) kinases in response to LPS. On the other hand, C(2)-ceramide down-regulated the phosphorylation of Akt in LPS-stimulated RAW 264.7 cells, followed by the impairment of nuclear factor (NF)-kappaB activation. Moreover, the Akt dominant-negative mutant inhibited LPS-induced NO production. C(2)-ceramide was suggested to inhibit LPS-induced NO production through down-regulating the activation of Akt.

Stimulation of tetrahydrobiopterin synthesis by cyclosporin A in mouse brain microvascular endothelial cells

We examined the effect of the immunosuppressant, cyclosporin A (CsA) on the synthesis of tetrahydrobiopterin (BH4), a cofactor for nitric oxide (NO) synthase and a scavenger of reactive oxygen species (ROS), in mouse brain microvascular endothelial cells. Treatment with CsA increased the BH4 content and the expression of mRNA level of GTP cyclohydrolase I, the rate-limiting enzyme of BH4 synthesis. 2,4-Diamino-6-hydroxypyrimidine, an inhibitor of GTP cyclohydrolase I, strongly reduced the CsA-induced increase in BH4 content. Cycloheximide (CHX), a protein synthesis inhibitor, also reduced CsA-induced BH4 synthesis. These findings suggest that CsA stimulates BH4 synthesis via a de novo pathway with the induction of GTP cyclohydrolase I. Moreover, CsA-induced the mRNA level of the inducible type of NO synthase, and stimulated the l-citrulline formation from l-arginine, which is a marker for NO synthesis. The CsA-stimulated l-citrulline formation was attenuated by the co-treatment with GTP cyclohydrolase I inhibitor. The expression of the endothelial type of NO synthase was low under basal condition, and was not affected by the treatment with CsA. These findings suggest that increase in BH4 content induced by CsA is coupled with NO production by inducible type of NO synthase.

The inhibitory action of quercetin on lipopolysaccharide-induced nitric oxide production in RAW 264.7 macrophage cells

The effect of quercetin on lipopolysaccharide (LPS)-induced nitric oxide (NO) production was studied. Quercetin pretreatment significantly inhibited NO production in an LPS-stimulated RAW 264.7 murine macrophage cell line. Post-treatment with quercetin partially inhibited NO production. The inhibitory action of quercetin was due to neither the cytotoxic action nor altered LPS binding. The expression of inducible-type NO synthase (iNOS) was markedly down-regulated by quercetin. Quercetin suppressed the release of free nuclear factor (NF)-κB by preventing degradation of IκB-α and IκB-β. Moreover, quercetin blocked the phosphorylation of extracellular signal regulated kinase 1/2 (Erk1/2), p38, and c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) and, further, the activity of tyrosine kinases in LPS-stimulated RAW cells. Quercetin also inhibited interferon (IFN)-γ-induced NO production. Taken together, these results indicate that the inhibitory action of quercetin on NO production in LPS- and/or IFN-γ-stimulated macrophages might be due to abrogation of iNOS protein induction by impairment of a series of intracellular signal pathways.