iNOS Protein (ino + protein)

Distribution by Scientific Domains

Terms modified by iNOS Protein

  • iNO protein expression

  • Selected Abstracts

    Decreased iNOS synthesis mediates dexamethasone-induced protection of neurons from inflammatory injury in vitro

    Sabine Golde
    Abstract Brain inflammation is accompanied by transection of axons and death of neurons in the acute lesions of multiple sclerosis. We explored mechanisms of inflammatory damage to neurons in vitro using cocultures of rat embryonal cortical neurons with microglia activated by interferon-gamma (IFN,) and lipopolysaccharide (LPS). Previously, we have demonstrated that microglia are highly toxic to neurons and that nitric oxide (NO) derived from inducible nitric oxide synthase (iNOS) is necessary and sufficient to mediate this toxicity. Here, we show that addition of dexamethasone (1 M) to activated cocultures provides effective neuroprotection. We demonstrate that dexamethasone down-regulates NO production of primary microglia by ,,50% and reduces steady-state iNOS protein and mRNA expression by ,,70%. These changes were reversed by the glucocorticoid receptor blocker RU-486. Furthermore, we analysed the stability of iNOS protein and show that whilst inhibitors of the proteasome blocked iNOS degradation they did not reverse the dexamethasone effect. Our results indicate that the main mechanism of corticosteroid activity on iNOS is reduction in protein synthesis, not destabilization as previously suggested. [source]

    Inhibition of nitric oxide synthase inhibitors and lipopolysaccharide induced inducible NOS and cyclooxygenase-2 gene expressions by rutin, quercetin, and quercetin pentaacetate in RAW 264.7 macrophages

    Yen-Chou Chen
    Abstract Several natural flavonoids have been demonstrated to perform some beneficial biological activities, however, higher-effective concentrations and poor-absorptive efficacy in body of flavonoids blocked their practical applications. In the present study, we provided evidences to demonstrate that flavonoids rutin, quercetin, and its acetylated product quercetin pentaacetate were able to be used with nitric oxide synthase (NOS) inhibitors (N -nitro- L -arginine (NLA) or N -nitro- L -arginine methyl ester (L -NAME)) in treatment of lipopolysaccharide (LPS) induced nitric oxide (NO) and prostaglandin E2 (PGE2) productions, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) gene expressions in a mouse macrophage cell line (RAW 264.7). The results showed that rutin, quercetin, and quercetin pentaacetate-inhibited LPS-induced NO production in a concentration-dependent manner without obvious cytotoxic effect on cells by MTT assay using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide as an indicator. Decrease of NO production by flavonoids was consistent with the inhibition on LPS-induced iNOS gene expression by western blotting. However, these compounds were unable to block iNOS enzyme activity by direct and indirect measurement on iNOS enzyme activity. Quercetin pentaacetate showed the obvious inhibition on LPS-induced PGE2 production and COX-2 gene expression and the inhibition was not result of suppression on COX-2 enzyme activity. Previous study demonstrated that decrease of NO production by L -arginine analogs effectively stimulated LPS-induced iNOS gene expression, and proposed that stimulatory effects on iNOS protein by NOS inhibitors might be harmful in treating sepsis. In this study, NLA or L -NAME treatment stimulated significantly on LPS-induced iNOS (but not COX-2) protein in RAW 264.7 cells which was inhibited by these three compounds. Quercetin pentaacetate, but not quercetin and rutin, showed the strong inhibitory activity on PGE2 production and COX-2 protein expression in NLA/LPS or L -NAME/LPS co-treated RAW 264.7 cells. These results indicated that combinatorial treatment of L -arginine analogs and flavonoid derivates, such as quercetin pentaacetate, effectively inhibited LPS-induced NO and PGE2 productions, at the same time, inhibited enhanced expressions of iNOS and COX-2 genes. J. Cell. Biochem. 82: 537,548, 2001. 2001 Wiley-Liss, Inc. [source]

    iNOS expression requires NADPH oxidase-dependent redox signaling in microvascular endothelial cells,

    Feng Wu
    Redox regulation of inducible nitric oxide synthase (iNOS) expression was investigated in lipopolysaccharide and interferon-, (LPS,+,IFN,)-stimulated microvascular endothelial cells from mouse skeletal muscle. Unstimulated endothelial cells produced reactive oxygen species (ROS) sensitive to inhibition of NADPH oxidase (apocynin and DPI), mitochondrial respiration (rotenone) and NOS (L-NAME). LPS,+,IFN, caused a marked increase in ROS production; this increase was abolished by inhibition of NADPH oxidase (apocynin, DPI and p47phox deficiency). LPS,+,IFN, induced substantial expression of iNOS protein. iNOS expression was prevented by the antioxidant ascorbate and by NADPH oxidase inhibition (apocynin, DPI and p47phox deficiency), but not by inhibition of mitochondrial respiration (rotenone) and xanthine oxidase (allopurinol). iNOS expression also was prevented by selective antagonists of ERK, JNK, Jak2, and NF,B activation. LPS,+,IFN, stimulated activation/phosphorylation of ERK, JNK, and Jak2 and activation/degradation of I,B, but only the activation of JNK and Jak2 was sensitive to ascorbate, apocynin and p47phox deficiency. Ascorbate, apocynin and p47phox deficiency also inhibited the LPS,+,IFN,-induced DNA binding activity of transcription factors IRF1 and AP1 but not NF,B. In conclusion, LPS,+,IFN,-induced NF,B activation is necessary for iNOS induction but is not dependent on ROS signaling. LPS,+,IFN,-stimulated NADPH oxidase activity produces ROS that activate the JNK-AP1 and Jak2-IRF1 signaling pathways required for iNOS induction. Since blocking either NF,B activation or NADPH oxidase activity is sufficient to prevent iNOS expression, they are separate targets for therapeutic interventions that aim to modulate iNOS expression in sepsis. J. Cell. Physiol. 217: 207,214, 2008. 2008 Wiley-Liss, Inc. [source]

    Molecular analysis of the vagal motoneuronal degeneration after right vagotomy

    Junfeng Ji
    Abstract The aim of this study was to investigate the vagal motoneuronal degeneration after right vagotomy using in situ hybridization, RT-PCR, and immunohistochemistry methods. The morphology of the vagal motoneurons in dorsal motor nucleus of the vagus nerve (DMV) and nucleus of ambiguus (NA) after right vagotomy was examined by using Nissl staing and TUNEL. The expression of inducible nitric oxide synthase (iNOS), bcl-2, bax, and caspase-3 in DMV and NA of rats after right vagotomy was studied. Additionally, the involvement of the N-methyl-D-aspartate (NMDA) receptor-calcium-neuronal nitric oxide synthase (nNOS) pathway in the vagal motoneuronal degeneration was addressed by double-immunolabeling analysis of nNOS with NMDAR1 and calbindin D28K in right-vagotomized rats. The neurons in right DMV and NA displayed a darkly stained, shrunken morphology at 1 day and 5 days following right vagotomy as shown by Nissl staining. Quantitative analysis revealed that, at 1 day and 5 days following right vagotomy, the number of neurons in right DMV, but not NA, was significantly reduced in comparison with that of control rats. Occasional TUNEL-positive neurons were detected in right DMV of rat at 1 day after right vagotomy. The expression of iNOS protein and mRNA was absent in DMV and NA of control rats. However, the iNOS mRNA expression was induced bilaterally in DMV and NA at 1 day postoperation and continued to be up-regulated until 5 days after vagotomy as shown by in situ hybridization. Immunohistochemistry analysis also showed the increased expression of iNOS in bilateral DMV and NA of vagotomized rats. RT-PCR analysis revealed the enhanced bcl-2 and reduced bax mRNA levels and subsequent up-regulation of both bcl-2 and bax mRNA in right sides of the vagotomized brainstems at 1 day and 5 days postoperation, respectively. In situ hybridization analysis confirmed the up-regulation of bcl-2 and bax mRNA in right DMV and NA of the rats at 5 days following operation. Immunohistochemistry analysis showed up-regulated Bcl-2 immunoreactivity and undetectable changes in Bax immunoreactivity in DMV and NA of rats at 1 day after vagotomy, whereas enhancement of both Bcl-2 and Bax immunoreactivity was observed at 5 days postoperation. In addition, the caspase-3 mRNA level was elevated ipsilaterally in DMV and NA at 1 day and 5 days following right vagotomy. Double-immunofluorescence analysis showed complete colocalization of nNOS with NMDAR1 and with calbindin in ipsilateral DMV and NA at 10 days following right vagotomy. This study suggests that the signal pathway for NMDAR1-calcium-nNOS and the up-regulation of iNOS in DMV and NA may be involved in the vagal motor neurodgeneration after right vagotomy. Furthermore, our results imply that the apoptosis pathway mediated by Bcl-2, Bax, and caspase-3 may be activated in vagal motoneurons after right vagotomy. 2002 Wiley-Liss, Inc. [source]

    The protective action of scutellarin against immunological liver injury induced by concanavalin A and its effect on pro-inflammatory cytokines in mice

    Zheng Huai Tan
    Scutellarin is a natural compound from a Chinese herb. The purpose of this paper was to study the protective effect of scutellarin on concanavalin A (Con A)-induced immunological liver injury and its effect on liver nuclear factor ,B (NF-,B), tumor necrosis factor , (TNF-,), interferon , (IFN-,), and inducible nitric oxide synthase (iNOS) expression in mice. Mouse liver injury was produced by injection of Con A 25 mg kg,1 via the tail vein. Scutellarin 50 or 100 mg kg,1 was peritoneally administered to mice 9 or 1 h before injection of Con A. The levels of serum alanine aminotransferase (ALT) and asparatate aminotransferase (AST), NO2,/NO3, and TNF - , were determined with biochemical kits, and ELISA using Quantikine Mouse TNF-, kit according the manufacturer's instructions. Liver lesions were examined by light microscope. The expression of TNF-,, IFN-,, iNOS and Fas mRNA in the livers was detected by RT-PCR; and the expression of c-Fos, c-Jun, iNOS and I,B proteins was measured by Western Blotting. As a result, pretreatment with scutellarin 100 mg kg,1 significantly decreased the serum ALT, AST, NO2,/NO3,and TNF-, levels, and also reduced liver lesions induced by Con A. Scutellarin 100 mg kg,1 down-regulated expression of TNF-, and iNOS mRNA, and c-Fos, c-Jun and iNOS protein, while scutellarin enhanced the degradation of I,B, in the livers of mice injected with Con A. The results suggest that scutellarin has a protective action against Con A-induced liver injury in mice, and its active mechanism may be related to the inhibition of the NF-,B-TNF-,-iNOS transduction pathway. [source]

    Allicin, the active component of garlic, prevents immune-mediated, concanavalin A-induced hepatic injury in mice

    Rafael Bruck
    Abstract: Background/Aim: Allicin, the immunologically active component of garlic, has been found to affect oxidative stress and immune response in several experimental systems. In the present study, we examined the ability of allicin to prevent immune-mediated, concanavalin A (Con A)-induced liver damage in mice. Methods: Mice were pretreated with allicin for 7 days before their inoculation with Con A (15 mg/kg). The serum levels of liver enzymes and liver histology were examined 24 h after Con A administration. The effect of Con A and allicin on serum levels of tumor necrosis factor-, (TNF-,) and nuclear factor-,B (NF-,B) activation in the liver were examined 2 h after Con A administration, in a separate group of rats, and the effect of allicin on Con A-induced expression of inducible nitric oxide synthase (iNOS) was determined by western blot analysis 24 h after Con A injection. Results: The histopathologic damage in the mouse livers, and the Con A-induced increase of aminotransferases and TNF-, were markedly inhibited in the mice pretreated with allicin before Con A injection (P<0.01). NF-,B binding activity to the nucleus, which increased 2 h after Con A administration, was attenuated by allicin. The expression of iNOS protein which was induced following Con A administration was significantly attenuated by allicin. In vitro studies showed that allicin inhibited TNF-,-mediated T cell adhesion to extracellular matrix components and to endothelial cells. Allicin also inhibited TNF-,-mediated intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 expression on human vascular endothelial cells. Conclusions: This study demonstrates that immune-mediated liver damage in mice can be prevented by allicin, probably because of its immunomodulatory effects on T cells and adhesion molecules and inhibition of NF-,B activation. [source]

    Proapoptotic Nitric Oxide Production in Amyloid , Protein-Treated Cerebral Microvascular Endothelial Cells

    MICROCIRCULATION, Issue 2 2007
    ABSTRACT Objective: The objective of this study was to investigate the effects of amyloid , protein (A,) on cerebral microvascular endothelium, and their possible involvement in A,-induced apoptosis in the neighboring cells. Methods: Cultured bovine brain microvascular endothelial cells (BBECs) were incubated with A, for 24 h. Production of nitric oxide (NO) was assessed by nitric oxide-sensitive fluorescent dye, DAF-2, and the expression of NO synthase (NOS) proteins was examined by Western blotting. Effects of A,-treated microvascular endothelium on the DNA damage of the neighboring cells were assessed by single-cell gel electrophoresis. Results: A, increased the expression of iNOS protein, but did not affect eNOS and nNOS expressions in BBECs. A,-treated BBECs showed spontaneous NO production in the presence of L-arginine. The neural cell line PC12 showed marked apoptosis after being co-cultured with A,-treated BBECs for 48 h, and the apoptosis was as potent as that induced by the inflammatory stimuli lipopolysaccharide and interferon-,. The DNA damage of PC12 cells evoked by co-culture with A,-treated BBECs was prevented by L- NG -nitroarginine methyl ester, an inhibitor of NOS. Conclusions: These results indicate that A, induces the expression of iNOS in BBECs, and that microvascular endothelium-derived NO may induce apoptosis in neighboring neural cells. [source]

    Suppressive effect of inducible nitric oxide synthase (iNOS) expression by the methanol extract of Actinodaphne lancifolia

    Youngleem Kim
    Abstract Nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) has played a crucial role in various pathophysiological processes including in,ammation and carcinogenesis. Therefore, the inhibitors of NO synthesis or iNOS gene expression have been considered as potential anti-in,ammatory and cancer chemopreventive agents. In our continuous search for iNOS inhibitors from natural products we have evaluated indigenous Korean plant extracts using an assay for inhibition of nitric oxide formation on lipopolysaccharide (LPS)-activated mouse macrophage RAW 264.7 cells. As a result, the methanolic stem extract of Actinodaphne lancifolia showed an inhibitory activity of NO production in a dose-dependent manner (IC50 = 2.5 g/ml). Additional study demonstrated that the extract of Actinodaphne lancifolia signi,cantly suppressed the iNOS protein and gene expression in a dose-dependent manner. These results suggest that Actinodaphne lancifolia could be a potential candidate for developing an iNOS inhibitor from natural products. Further elucidation of active principles for development of new cancer chemopreventive and/or anti-in,ammatory agents could be warranted. Copyright 2004 John Wiley & Sons, Ltd. [source]

    Co-localization of inducible nitric oxide synthase and phosphorylated Akt in the lesional skins of patients with melasma

    Ho-Youn JO
    ABSTRACT Activation of the inducible nitric oxide synthase (iNOS)/nitric oxide (NO) pathway in keratinocytes has been reported to be associated with the pathogenesis of melanogenesis. Akt activation plays an important role in the activation of the transcription factor nuclear factor (NF)-,B and subsequent elevation of iNOS expression. In the present study, we highly detected both iNOS protein and Akt phosphorylation in keratinocytes of the basal layer of the epidermis at the junction with the dermis of melasma skin biopsy specimens, but not in normal skin tissues, from nine patients using immunohistological analysis. iNOS protein and phosphorylated Akt were co-localized in the lesional skins, and their levels were highly correlated (R2 = 0.69). Furthermore, iNOS mRNA was also detected in an additional three skin biopsy specimens, but not in normal skin, by reverse transcription polymerase chain reaction. Our results describe that iNOS expression is elevated in human melasma lesions, probably via activation of the Akt/NF-,B pathway, indicating that NO production plays an important role in the mechanism of hyperpigmentation in human facial melasma. [source]

    Up-regulation of autophagy in small intestine Paneth cells in response to total-body ,-irradiation,,

    Nikolai V. Gorbunov
    Abstract Macroautophagy (mAG) is a lysosomal mechanism of degradation of cell self-constituents damaged due to variety of stress factors, including ionizing irradiation. Activation of mAG requires expression of mAG protein Atg8 (LC3) and conversion of its form I (LC3-I) to form II (LC3-II), mediated by redox-sensitive Atg4 protease. We have demonstrated upregulation of this pathway in the innate host defense Paneth cells of the small intestine (SI) due to ionizing irradiation and correlation of this effect with induction of pro-oxidant inducible nitric oxide synthase (iNOS). CD2F1 mice were exposed to 9.25 Gy ,-ionizing irradiation. Small intestinal specimens were collected during 7 days after ionizing irradiation. Assessment of ionizing irradiation-associated alterations in small intestinal crypt and villus cells and activation of the mAG pathway was conducted using microscopical and biochemical techniques. Analysis of iNOS protein and the associated formation of nitrites and lipid peroxidation products was performed using immunoblotting and biochemical analysis, and revealed increases in iNOS protein, nitrate levels and oxidative stress at day 1 following ionizing irradiation. Increase in immunoreactivity of LC3 protein in the crypt cells was observed at day 7 following ionizing irradiation. This effect predominantly occurred in the CD15-positive Paneth cells and was associated with accumulation of LC3-II isoform. The formation of autophagosomes in Paneth cells was confirmed by transmission electron microscopy (TEM). Up-regulation of LC3 pathway in the irradiated SI was accompanied by a decreased protein,protein interaction between LC3 and chaperone heat shock protein 70. A high-level of LC3-immunoreactivity in vacuole-shaped structures was spatially co-localized with immunoreactivity of 3-nitro-tyrosine. The observed effects were diminished in iNOS knockout B6.129P2- NOS2tm1Lau/J mice subjected to the same treatments. We postulate that the observed up-regulation of mAG in the irradiated small intestine is at least in part mediated by the iNOS signalling mechanism. Published in 2009 by John Wiley & Sons, Ltd. [source]

    Increased inducible nitric oxide synthase in lung carcinoma of smokers

    CANCER, Issue 2 2008
    George G. Chen MD
    Abstract BACKGROUND. Cigarette smoking is well known to play an important role in the development of lung cancer. Inducible nitric oxide synthase (iNOS) can either promote or inhibit cell proliferation and growth, which makes its role in the development of malignant tumors controversial. The relation between cigarette smoking and iNOS in human lung cancer is unknown. METHODS. The study examined the levels of iNOS/NO in nonsmall-cell lung cancer (NSCLC) tissues of smokers and nonsmokers and in NSCLC cells (NCI-H23) treated by 4-(N-Methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a potent tobacco-specific carcinogen. RESULTS. The level of iNOS/NO was significantly higher in lung cancer tissues of smokers than that of nonsmokers. Unlike iNOS/NO, the activity of caspase-3 was reduced in the former compared with the latter. The expression of the cleaved caspase-3 was deceased in NCI-H23 cells treated with S-Nitroso-N-acetylpenicillamine (SNAP), an NO donor, whereas treatment with NG-methyl-L-arginine (NMA), an NO inhibitor, caused an increase in cleaved caspase-3. Consistent with the change in caspase-3, SNAP treatment inhibited cell death induced by UCN01, a potent cell death-inducer. NMA treatment greatly enhanced the sensitivity of the cells to UCN01. Further, the cells treated by NNK showed an increase in iNOS protein, accompanied by an elevation of cell proliferation. CONCLUSIONS. The study demonstrates that cigarette smoking promotes the level of iNOS/NO but suppresses the activity of caspase-3, which may lead to the proliferation and growth of lung cancer cells. Cancer 2008. 2007 American Cancer Society. [source]

    Lycopene Inhibits LPS-Induced Proinflammatory Mediator Inducible Nitric Oxide Synthase in Mouse Macrophage Cells

    Mohamed M. Rafi
    ABSTRACT:, Lycopene is a fat-soluble red-orange carotenoid found primarily in tomatoes and tomato-derived products, including tomato sauce, tomato paste, and ketchup, and other dietary sources, including dried apricots, guava, watermelon, papaya, and pink grapefruit. In this study, we have demonstrated the molecular mechanism underlying the anti-inflammatory properties of lycopene using a mouse macrophage cell line (RAW 264.7). Treatment with lycopene (10 ,M) inhibited lipopolysaccharide (LPS)-stimulated nitric oxide (NO) production (40% compared with the control). Western blotting and reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that lycopene treatment decreased LPS-induced inducible nitric oxide synthase (iNOS) protein and mRNA expression in RAW 264.7 cells, respectively. These results suggest that lycopene has anti-inflammatory activity by inhibiting iNOS proteins and mRNA expressions in mouse macrophage cell lines. Furthermore, cyclooxygenase-2 (COX-2) protein and mRNA expression were not affected by treatment with lycopene. [source]

    Role of cyclooxygenase-2 and inducible nitric oxide synthase in pancreatic cancer

    Gu Kong
    Abstract Background and Aim: Recently, it has been recognized that both cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) produce important endogenous factors of human tumor progression. However, the clinicopathological and biological significance of the expression of COX-2 and iNOS in pancreatic cancer remains unclear. The objective of this study is to find the possible roles and clinical significance of COX-2 and iNOS expression in pancreatic cancer. Methods: Seventy-two pancreatic adenocarcinoma tissue specimens were obtained through surgical resection. We investigated the immunohistochemical expression of COX-2 and iNOS in respect to variable clinicopathological characteristics, proliferation activity (by Ki-67 expression), apoptosis (by terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling stain), and microvessel density (by CD34 expression; angiogenesis). Results: Immunohistochemical investigations demonstrated immunolabeling of tumor cells with the primary antibodies, bovine anti-iNOS and anti-COX-2 antibodies. The COX-2 and iNOS positive rates were 41.7 and 66.7%, respectively. There was significant correlation between positive COX-2 and positive iNOS expression (P = 0.043). The proliferation index (Ki-67 labeling index) was higher in COX-2 positive specimens compared to COX-2 negative specimen (P = 0.015). The apoptotic index of positive iNOS expressions was significantly higher than negative expressions (P < 0.001). The expression of COX-2 and iNOS proteins did not correlate with age, sex, serum bilirubin, CA-19,9, location, size, American Joint Committee on Cancer stage, differentiation, distant metastasis, patient survival, or microvessel density. Conclusions: Although the pattern of positive expression was similar in both enzymes, the effect on tumor progression differed; iNOS expression may play a role in apoptosis of tumor cell, while COX-2 expression may contribute to tumor proliferation. However, COX-2 and iNOS expression is not related to prognosis in patients with pancreatic cancer. 2002 Blackwell Publishing Asia Pty Ltd [source]