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iNOS Expression (ino + expression)
Selected AbstractsPERIPHERAL NERVOUS SYSTEM DEMYELINATION AND iNOS EXPRESSIONJOURNAL OF THE PERIPHERAL NERVOUS SYSTEM, Issue 1 2002Article first published online: 11 MAR 200 Conti G.1, Pasquale C.1, Rostami A.3, De Pol A.2, Galimberti D.1, Scarpini E.1, Baron P.L.1, Scarlato G.1 1 Milano Italy, 2 Modena, Italy, 3Philadelphia USA. Nitric oxide (NO), during CNS demyelination, is synthesised in inflammatory cells from L-arginine by the nitric oxide synthases (NOS). NO can subserve different functions, from cytotoxicity to neuroprotection and trigger either necrotic or apoptotic cell death. In this study we detected inducible form of NOS (iNOS) gene expression in experimental allergic neuritis (EAN), induced in Lewis rats by injection of "SP26," emulsified in complete Freund's adjuvant, which clinical, electrical, and pathological features resemble those of "Guillain-Barré" syndrome. Northern blot, single nerve fiber immmunostaining, and immuno-electron microscope showed that both iNOS mRNA and protein were induced in the PNS of EAN rats by day 14 after immunization, at the beginning of EAN clinical signs. However, with the same experimental procedures, we failed to find iNOS expression during Wallerian degeneration following nerve cut. These data support the hypothesis that iNOS regulation has an active role in cell-mediated demyelination. [source] Ethanol Upregulates iNOS Expression in Colon Through Activation of Nuclear Factor-kappa B in RatsALCOHOLISM, Issue 1 2010Chao Wang Background:, Alcohol inhibits colonic motility but the mechanism is unknown. The goal of this study was to test the possibility that nuclear factor-kappa B (NF-,B) is involved in the upregulation of inducible nitric oxide synthase (iNOS) expression induced by ethanol in colon. Methods:, The isometric contraction of longitudinal muscle strips of proximal colon (LP) was monitored by polygraph. Western blot analysis was used to measure the amount of iNOS and I-,B in the cytoplasm and P65 in the nucleus. Immunohistochemistry was applied to locate iNOS in colon. Results:, Ethanol (87mM) inhibited the contraction of LP. Pretreatment of S-methylisothioure (SMT) (1 mM), a specific iNOS inhibitor, Pyrrolidine dithiocarbamate (PDTC) (10 mM) and BAY11-7082(10 mM), specific inhibitors of NF-,B significantly reversed the inhibitory effect of ethanol on LP contraction. Ethanol increased the amount of iNOS and content of NO in colon, and these effects were attenuated by pretreatment of PDTC. Following ethanol administration, the amount of I-,B in the cytoplasm decreased, but that of P65, the subunit of NF-,B in the nucleus, increased. The iNOS was located in the cell body of myenteric plexus in colon. Conclusion:, Ethanol inhibited the contraction of LP in colon mainly through activation of NF-,B, the subsequent upregulation of iNOS expression and increase of NO release in myenteric plexus. [source] Imidazoline-induced amplification of glucose- and carbachol-stimulated insulin release includes a marked suppression of islet nitric oxide generation in the mouseACTA PHYSIOLOGICA, Issue 3 2009S. Meidute-Abaraviciene Abstract Aim:, The role of islet nitric oxide (NO) production in insulin-releasing mechanisms is unclear. We examined whether the beneficial effects of the imidazoline derivative RX 871024 (RX) on ,-cell function might be related to perturbations of islet NO production. Methods:, Experiments were performed with isolated islets or intact mice challenged with glucose or carbachol with or without RX treatment. Insulin was determined with radioimmunoassay, NO generation with high-performance liquid chromatography and expression of inducible NO synthase (iNOS) with confocal microscopy. Results:, RX treatment, in doses lacking effects on basal insulin, greatly amplified insulin release stimulated by the NO-generating secretagogues glucose and carbachol both in vitro and in vivo. RX also improved the glucose tolerance curve. Islets incubated at high glucose levels (20 mmol L,1) displayed increased NO production derived from both neuronal constitutive NO synthase (ncNOS) and iNOS. RX abrogated this glucose-induced NO production concomitant with amplification of insulin release. Confocal microscopy revealed abundant iNOS expression in , cells after incubation of islets at high but not low glucose levels. This was abolished after RX treatment. Similarly, islets cultured for 24 h at high glucose levels showed intense iNOS expression in , cells. This was abrogated with RX and followed by an amplified glucose-induced insulin release. Conclusion:, RX effectively counteracts the negative impact of ,-cell NO generation on insulin release stimulated by glucose and carbachol suggesting imidazoline compounds by virtue of NOS inhibitory properties being of potential therapeutic value for treatment of ,-cell dysfunction in hyperglycaemia and type 2 diabetes. [source] Nramp1 -functionality increases iNOS expression via repression of IL-10 formationEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 11 2008Gernot Fritsche Abstract In mice, resistance to certain intracellular microbes depends on the expression of a late phagosomal protein termed natural-resistance associated macrophage protein 1 (Nramp1, Slc11a1). Nramp1- functionality is associated with alterations of cellular iron homeostasis and a sustained pro-inflammatory immune response, including the formation of the antimicrobial effector molecule NO. To investigate the underlying mechanism we used RAW-264.7 murine macrophage cells stably transfected with a functional Nramp1 allele (RAW-37) or Nramp1 non-functional controls (RAW-21). We found that the production of and signalling by the anti-inflammatory cytokine IL-10 was significantly enhanced in macrophages lacking functional Nramp1. Upon infection of macrophages with Salmonella typhimurium pathogen survival was significantly better in RAW-21 than in RAW-37, which inversely correlated to NO and TNF-, formation. Addition of a neutralising anti-IL-10 antibody to RAW-21 cells led to a significantly reduced survival of S. typhimurium within these cells and enhanced formation of NO and TNF-, reaching levels comparable to that observed in cells bearing functional Nramp1. Oppositely, supplementation of iron to RAW-21 cells further increased IL-10 formation. Thus, Nramp1 mediates effective host defence in part via suppression of excessive IL-10 production which may relate to Nramp1- mediated reduction of cellular iron pools, thus strengthening antimicrobial effector mechanisms. [source] The effect of ultraviolet B irradiation on nitric oxide synthase expression in murine keratinocytesEXPERIMENTAL DERMATOLOGY, Issue 6 2000M. Sasaki Abstract: Nitric oxide (NO), which has several physiological functions in skin, is generated by NO synthase (NOS). NOS has at least three isoforms; endothelial NOS (eNOS), brain NOS (bNOS), and inducible NOS (iNOS). Ultraviolet B (UVB) irradiation has been reported to stimulate NO production in skin via induction or activation of NOS, however, the exact mechanism of NOS induction by UVB irradiation remains obscure. In this study, we investigated the direct effect of UVB on the expression of NOS isoforms in murine keratinocytes, and found a significant increase in NO production within 48 h. mRNA and protein expressions of bNOS were both enhanced by UVB irradiation in murine keratinocytes, whereas iNOS mRNA expression was suppressed at 4 and 12 h after UVB irradiation. These results suggest that the enhancement of NO production observed after UVB irradiation in murine keratinocytes may be explained in part by the upregulation of bNOS expression, but not iNOS expression. [source] Smad3-dependent signaling underlies the TGF-,1-mediated enhancement in astrocytic iNOS expressionGLIA, Issue 11 2010Mary E. Hamby Abstract We previously demonstrated that transforming growth factor-,1 (TGF-,1), while having no effect alone, enhances nitric oxide (NO) production in primary, purified mouse astrocytes induced by lipopolysaccharide (LPS) plus interferon-, (IFN-,), by recruiting a latent population of astrocytes to respond, thereby enhancing the total number of cells that express Nos2. In this investigation, we evaluated the molecular signaling pathway by which this occurs. We found that purified murine primary astrocytes express mRNA for TGF,RII as well as the TGF,RI subunit activin-like kinase 5 (ALK5), but not ALK1. Immunofluorescence microscopy confirmed the expression of TGF,RII and ALK5 protein in astrocytes. Consistent with ALK5 signaling, Smad3 accumulated in the nucleus of astrocytes as early as 30 min after TGF-,1 (3 ng/mL) treatment and persisted upto 32 hr after TGF-,1 administration. Addition of ALK5 inhibitors prevented TGF-,1-mediated Smad3 nuclear accumulation and NO production when given prior to the Nos2 induction stimuli, but not after. Finally, astrocyte cultures derived from Smad3 null mutant mice did not exhibit a TGF-,1-mediated increase in iNOS expression. Overall, this data suggests that ALK5 signaling and Smad3 nuclear accumulation is required for optimal enhancement of LPS plus IFN,-induced NO production in astrocytes by TGF-,1. © 2010 Wiley-Liss, Inc. [source] Blockade of IL-15 activity inhibits microglial activation through the NF,B, p38, and ERK1/2 pathways, reducing cytokine and chemokine releaseGLIA, Issue 3 2010Diego Gomez-Nicola Abstract Reactive glia formation is one of the hallmarks of damage to the CNS, but little information exists on the signals that direct its activation. Microglial cells are the main regulators of both innate and adaptative immune responses in the CNS. The proinflammatory cytokine IL-15 is involved in regulating the response of T and B cells, playing a key role in regulating nervous system inflammatory events. We have used a microglial culture model of inflammation induced by LPS and IFN, to evaluate the role of IL-15 in the proinflammatory response. Our results indicate that IL-15 is necessary for the reactive response, its deficiency (IL-15-/-) leading to the development of a defective proinflammatory response. Blockade of IL-15, both with blocking antibodies or with the ganglioside Neurostatin, inhibited the activation of the NF,B pathway, decreasing iNOS expression and NO production. Inhibiting IL-15 signaling also blocked the activation of the mitogen-activated protein kinase (MAPK) pathways ERK1/2 and p38. The major consequence of these inhibitory effects, analyzed using cytokine antibody arrays, was a severe decrease in the production of chemokines, cytokines and growth factors, like CCL17, CCL19, IL-12, or TIMP-1, that are essential for the development of the phenotypic changes of glial activation. In conclusion, activation of the IL-15 system seems a necessarystep for the development of glial reactivity and the regulation of the physiology of glial cells. Modulating IL-15 activity opens the possibility of developing new strategies to control gliotic events upon inflammatory stimulation. © 2009 Wiley-Liss, Inc. [source] Inducible nitric oxide synthase expression in laryngeal neoplasia: Correlation with angiogenesisHEAD & NECK: JOURNAL FOR THE SCIENCES & SPECIALTIES OF THE HEAD AND NECK, Issue 1 2002Alessandro Franchi MD Abstract Background The nitric oxide (NO) pathway plays a relevant role in angiogenesis and tumor progression in squamous cell carcinoma (SCC) of the head and neck. The aim of this study was to assess whether the NO pathway may be correlated with angiogenesis in the transition from laryngeal dysplasia to invasive carcinoma. Methods We investigated the expression of the inducible NO synthase (iNOS) in 26 laryngeal precancerous lesions and 35 squamous cell carcinomas with respect to microvessel density. In addition, we determined iNOS activity and cGMP levels in specimens from SCCs. Results There was a significant increase of iNOS levels detected immunohistochemically passing from hyperplastic/mild dysplastic to moderate/severe dysplastic lesions to SCC (p = .04). Accordingly, Northern and Western analyses demonstrated higher iNOS mRNA and protein levels in SCCs than dysplastic mucosa. iNOS expression was significantly correlated with microvessel counts both in the group of preneoplastic lesions (p = .02) and in the group of SCCs (p = .01). In addition, iNOS activity was correlated with iNOS immunohistochemical expression (p = .1) and was significantly associated with increased vascularization (p = .03) in SCCs. Similarly, iNOS expression was significantly correlated with cGMP levels in SCC (p = .02) and increased tumor vascularization correlated with higher cGMP levels (rs = .4; p = .01). Conclusions Our data indicate that the NO pathway may play a relevant role in the angiogenesis associated with the progression from laryngeal dysplasia to laryngeal SCC. © 2002 John Wiley & Sons, Inc. Head Neck 24: 16,23, 2002. [source] Nuclear factor-kappaB as a molecular target for migraine therapy.HEADACHE, Issue 4 2003U Reuter Ann Neurol. 2002;51:507-516. Nitric oxide (NO) generated from inducible NO synthase (iNOS) participates in immune and inflammatory responses in many tissues. The NO donor glyceryl trinitrate (GTN) provokes delayed migraine attacks when infused into migraineurs and also causes iNOS expression and delayed inflammation within rodent dura mater. Sodium nitroprusside, an NO donor as well, also increases iNOS expression. Because inflammation and iNOS are potential therapeutic targets, we examined transcriptional regulation of iNOS following GTN infusion and the consequences of its inhibition within dura mater. We show that intravenous GTN increases NO production within macrophages. L-N(6)-(1-iminoethyl)lysine, a selective iNOS inhibitor, attenuates the NO signal, emphasizing the importance of enzymatic activity to delayed NO production. iNOS expression is preceded by significant nuclear factor kappa B (NF-kappaB) activity, as reflected by a reduction in the inhibitory protein-kappa-Balpha (IkappaBalpha) and activation of NF-kappaB after GTN infusion. IkappaBalpha degradation, NF-kappaB activation, and iNOS expression were attenuated by parthenolide (3mg/kg), the active constituent of feverfew, an anti-inflammatory drug used for migraine treatment. These findings suggest that GTN promotes NF-kappaB activity and inflammation with a time course consistent with migraine attacks in susceptible individuals. We conclude, based on results with this animal model, that blockade of NF-kappaB activity provides a novel transcriptional target for the development of anti-migraine drugs. Comment: This paper suggesting the localization of NO production in dural macrophages as part of delayed inflammation may indicate proliferation and or recruitment of these cells in migraine. Could this also be a target for drug treatment? Specifically, is the genetic transcription that leads to nitric oxide generation such a target? To amend slightly the old advertising slogan, "when Michael Moskowitz talks, we all listen." DSM and SJT [source] Terlipressin inhibits in vivo aortic iNOS expression induced by lipopolysaccharide in rats with biliary cirrhosisHEPATOLOGY, Issue 5 2002Richard Moreau In cirrhosis, lipopolysaccharide (LPS, a product of Gram-negative bacteria) in the blood may cause septic shock. LPS-elicited induction of arterial inducible nitric oxide synthase (iNOS) results in nitric oxide (NO)-induced vasodilation, which causes arterial hypotension and hyporeactivity to ,1 -adrenergic constrictors. In vitro studies have suggested that vasopressin inhibits iNOS expression in cultured vascular smooth muscle cells exposed to LPS. Thus, the aim of this study was to investigate the effects of terlipressin administration (a vasopressin analog) on in vivo LPS-induced aortic iNOS in rats with cirrhosis. LPS (1 mg/kg, intravenously) was administered followed by the intravenous administration of terlipressin (0.05 mg/kg, intravenously) or placebo 1 hour later. Arterial pressure was measured, and contractions to phenylephrine (an ,1 -adrenoceptor agonist), iNOS activity, and iNOS expressions (mRNA and protein) were investigated in isolated aortas. LPS-induced arterial hypotension and aortic hyporeactivity to phenylephrine were abolished in rats that received terlipressin. LPS-induced aortic iNOS activity and expression were suppressed in terlipressin-treated rats. In conclusion, in LPS-challenged rats with cirrhosis, terlipressin administration inhibits in vivo LPS-induced aortic iNOS expression. Terlipressin administration may be a novel approach for the treatment of arterial hypotension and hyporeactivity to ,1 -adrenergic constrictors in patients with cirrhosis and septic shock. [source] Overexpression of inducible nitric oxide synthase and accumulation of 8-OHdG in nasopharyngeal carcinomaHISTOPATHOLOGY, Issue 2 2008Y Segawa Aims:, Nitric oxide (NO), produced by inducible NO synthase (iNOS), has been suggested to cause oxidative stress, leading to 8-hydroxydeoxyguanosine (8-OHdG) accumulation and subsequent transversion mutation of DNA. The aim was to evaluate iNOS expression and the status of oxidative stress in nasopharyngeal carcinoma (NPC). Methods and results:, Seventy-three cases of NPC were investigated to examine the immunohistochemical expression of iNOS, 8-OHdG and latent membrane protein-1 (LMP-1) and Epstein,Barr virus-encoded small RNA (EBER) expression using in situ hybridization. iNOS mRNA expression and p53 gene mutations were also assessed. Overexpression of iNOS, LMP-1 and EBER was observed in 62 (84.9%), 28 (38.4%) and 53 (72.6%) cases respectively. p53 gene mutation was found in 10 of 73 (13.7%) cases. Immunohistochemical iNOS expression was associated with the 8-OHdG labelling index, iNOS mRNA expression and p53 gene alteration (P < 0.0001, P = 0.016 and 0.0082 respectively). Conclusions:, Our present findings suggest that the expression of iNOS induces oxidative stress in NPC. Although the presence of p53 mutation was associated with iNOS overexpression, the type of acid,base change of p53 was transition, but not transversion, which suggests that the p53 gene is not the direct target of DNA damage by 8-OHdG accumulation. [source] Bone morphogenetic protein-6 induces the expression of inducible nitric oxide synthase in macrophagesIMMUNOLOGY, Issue 1pt2 2009Seok J. Kwon Summary Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-, (TGF-,) superfamily. In the present study, we investigated the effect of BMPs on the production of inducible nitric oxide synthase (iNOS) in the murine macrophage cell line, RAW 264.7, and in mouse peritoneal macrophages. Among the BMPs, only BMP-6 induced iNOS expression in a time-dependent and dose-dependent manner in both cell types. Induction of iNOS was inhibited by both cycloheximide and actinomycin D, indicating that the induction of iNOS expression by BMP-6 requires new protein synthesis. Mechanistic studies revealed that the BMP-6-induced iNOS expression requires both Smads and nuclear factor-kappa B (NF-,B) signalling pathways. Furthermore, induction of interleukin-1, (IL-1,) was necessary for iNOS induction by BMP-6. These observations suggest that BMP-6 stimulates macrophages to produce iNOS through IL-1, via Smad and NF-,B signalling pathways and that BMP-6 may be an important regulator of macrophages. [source] Signalling events involved in interferon-,-inducible macrophage nitric oxide generationIMMUNOLOGY, Issue 4 2003Julie Blanchette Summary Nitric oxide (NO) produced by macrophages (M,) in response to interferon-, (IFN-,) plays a pivotal role in the control of intracellular pathogens. Current knowledge of the specific biochemical cascades involved in this IFN-,-inducible M, function is still limited. In the present study, we evaluated the participation of various second messengers , Janus kinase 2 (JAK2), signal transducer and activator of transcription (STAT) 1,, MAP kinase kinase (MEK1/2), extracellular signal-regulated kinases 1 and 2 (Erk1/Erk2) and nuclear factor kappa B (NF-,B) , in the regulation of NO production by IFN-,-stimulated J774 murine M,. The use of specific signalling inhibitors permitted us to establish that JAK2/STAT1,- and Erk1/Erk2-dependent pathways are the main players in IFN-,-inducible M, NO generation. To determine whether the inhibitory effect was taking place at the pre- and/or post-transcriptional level, we evaluated the effect of each antagonist on inducible nitric oxide synthase (iNOS) gene and protein expression, and on the capacity of IFN-, to induce JAK2, Erk1/Erk2 and STAT1, phosphorylation. All downregulatory effects occurred at the pretranscriptional level, except for NF-,B, which seems to exert its role in NO production through an iNOS-independent event. In addition, electrophoretic mobility shift assay (EMSA) analysis revealed that STAT1, is essential for IFN-,-inducible iNOS expression and NO production, whereas the contribution of NF-,B to this cellular regulation seems to be minimal. Moreover, our data suggest that Erk1/Erk2 are responsible for STAT1, Ser727 residue phosphorylation in IFN-,-stimulated M,, thus contributing to the full activation of STAT1,. Taken together, our results indicate that JAK2, MEK1/2, Erk1/Erk2 and STAT1, are key players in the IFN-,-inducible generation of NO by M,. [source] Early bacterial dependent induction of inducible nitric oxide synthase (iNOS) in epithelial cells upon transfer of CD45RBhigh CD4+ T cells in a model for experimental colitisINFLAMMATORY BOWEL DISEASES, Issue 12 2007Gerard Dijkstra MD Abstract Background: Both the role of inducible nitric oxide synthase (iNOS) in the development of inflammatory bowel disease (IBD) as well as the molecular details governing its mucosal induction remain unclear. Methods: In the present study we evaluated the role of the residing intestinal microflora in the induction of epithelial iNOS upon transfer of CD45RBhigh CD4+ T cells to SCID mice. CB-17 SCID mice were reared with conventional flora (CNV) or germfree CB-17 SCID mice were monoassociated with Helicobacter muridarum, act A(,) mutant Listeria monocytogenes, segmented filamentous bacteria (SFB), or Ochrobactrum anthropi. Results: Within 2 weeks CNV SCID mice injected with CD45RBhigh CD4+ T cells showed a focal, epithelial iNOS expression on the apical site of villi that preceded the infiltration of CD4+ T cells and cytokine production followed by extension of this expression to the entire surface along the whole crypt axis as the colitis progressed. SCID mice monoassociated with H. muridarum developed a severe colitis and showed high epithelial iNOS expression. CNV-SCID mice without T cells and SCID mice monoassociated with SFB did not show any iNOS expression, whereas SCID mice monoassociated with act A(,) mutant L. monocytogenes and O. anthropi showed some scattered epithelial iNOS staining on the apical site of a few villi, but none of these mice developed colitis. Conclusions: These findings demonstrate that the expression of epithelial iNOS is highly bacterium-specific and correlates with the severity of disease, suggesting an important role for this enzyme in the development of IBD. (Inflamm Bowel Dis 2007) [source] A comparative study of the preventative effects exerted by three probiotics, Bifidobacterium lactis, Lactobacillus casei and Lactobacillus acidophilus, in the TNBS model of rat colitisJOURNAL OF APPLIED MICROBIOLOGY, Issue 4 2007L. Peran Abstract Aims:, The intestinal anti-inflammatory effects of three probiotics with immunomodulatory properties, Lactobacillus casei, Lactobacillus acidophilus and Bifidobacterium lactis, were evaluated and compared in the trinitrobenzenesulphonic acid (TNBS) model of rat colitis. Methods and Results:, Colitis was induced in rats by intracolonic administration of 10 mg of TNBS dissolved in 0·25 ml of 50% ethanol. Each probiotic was administered orally (5 × 108 CFU suspended in 0·5 ml of skimmed milk) for 3 weeks, starting 2 weeks before the administration of TNBS. Colonic damage was evaluated histologically and biochemically 1 week after TNBS instillation. The results obtained revealed that all probiotics assayed showed intestinal anti-inflammatory effects, macroscopically evidenced by a significant reduction in the colonic weight/length ratio. Only B. lactis showed a lower incidence of diarrhoea in comparison with untreated rats. Biochemically, all probiotics restored colonic glutathione levels, depleted as a consequence of the oxidative stress of the inflammatory process. Bifidobacterium lactis treatment reduced colonic tumour necrosis factor (TNF)-, production, and inducible nitric oxide synthase (iNOS) and cyclo-oxygenase-2 (COX-2) expression; L. acidophilus administration reduced colonic leukotriene B4 production and iNOS expression and L. casei intake was associated with a decrease in colonic COX-2 expression. Conclusion:, The three probiotics assayed have shown intestinal anti-inflammatory activity in the TNBS model of rat colitis, although each probiotic shows its own anti-inflammatory profile. Significance and Impact of the Study:, These probiotics could be considered as potential adjuvants in the treatment of inflammatory bowel disease, although more studies are required in order to demonstrate their efficacy in humans. [source] Aging Increases the Interleukin-1,,Induced INOS Gene Expression and Nitric Oxide (NO) Production in Vascular Smooth Muscle CellsJOURNAL OF CARDIAC SURGERY, Issue 6 2002Gabriel HH Chan Objectives: Inducible form of nitric oxide synthase (iNOS) is induced by cytokines (e.g. interleukin-1, (IL-1,)) during pathological conditions, such as sepsis. Excessive NO synthesis in blood vessels during sepsis can result in massive vasodilation and life-threatening hypotension. In addition, chronic expression of iNOS contributes to onset of diabetes, autoimmune diseases, arthritis, renal toxicity, and neurodegenerative disorders. The purpose of the present study was to examine the effect of aging on the levels of expression of iNOS induced by a low concentration (5 ng/ml) of IL-1, in VSMCs. Methods: Gene expression of iNOS was determined by RT-PCR and analysis of the PCR products by both agarose gel electrophoresis and capillary electrophoresis with laser-induced fluorescence detector (CE-LIF). This new CE-LIF technique, just developed in our laboratory, provides greater than 1,000 fold better sensitivity compared to agarose gels. The production of nitrite, the stable metabolite of NO, was measured (by a modified Griess reaction) in the media of cultured VSMCs isolated from young and elderly rats (3-month and 20-months old, respectively) of both genders following the exposure to IL-1, (5 ng/ml). VSMCs were used in their 1st passage to avoid phenotypic changes that typically occur in cultures of VSMCs after 3-10 passages. Results: IL-1, (5 ng/ml) caused a much larger increase in iNOS mRNA in VSMCs of elderly rats as compared to young rats. Furthermore, IL-1, (5 ng/ml) had no significant effect on nitrite levels in VSMCs of young, but significantly increased nitrite levels by 7.9 fold in VSMCs from elderly male rats and by 2.6 fold in VSMCs from elderly female rats, as compared to young rats. A report had previously shown that the neuropeptide CGRP could synergistically enhance the expression of iNOS caused by IL-1, in later passages (10-15 passages) of rat aortic VSMCs (i.e. phenotypically modulated VSMCs). We found that IL-1, and CGRP together did not act synergistically to increase production of nitrite in our phenotypically normal (1st passage) VSMCs. Conclusion: IL-1,, at a low concentration (5 ng/ml), preferentially induces iNOS expression and increases production of NO in VSMCs of elderly rats as compared to young rats. The data suggest that aging enhances the responsiveness of VSMCs to the iNOS-inducing actions of the cytokine IL-1,. This may be a contributing factor in the increased risk of developing severe hypotension in elderly patients with sepsis. (Supported by a Direct Grant for Research). [source] Xanthine oxidase inhibitor allopurinol attenuates the development of diabetic cardiomyopathyJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 8b 2009Mohanraj Rajesh Abstract In this study, we investigated the effect of the xanthine oxidase (XO) inhibitor, allopurinol (ALP), on cardiac dysfunction, oxidative-nitrosative stress, apoptosis, poly(ADP-ribose) polymerase (PARP) activity and fibrosis associated with diabetic cardiomyopathy in mice. Diabetes was induced in C57/BL6 mice by injection of streptozotocin. Control and diabetic animals were treated with ALP or placebo. Left ventricular systolic and diastolic functions were measured by pressure,volume system 10 weeks after established diabetes. Myocardial XO, p22phox, p40phox, p47phox, gp91phox, iNOS, eNOS mRNA and/or protein levels, ROS and nitrotyrosine (NT) formation, caspase3/7 and PARP activity, chromatin fragmentation and various markers of fibrosis (collagen-1, TGF-,, CTGF, fibronectin) were measured using molecular biology and biochemistry methods or immunohistochemistry. Diabetes was characterized by increased myocardial, liver and serum XO activity (but not expression), increased myocardial ROS generation, p22phox, p40phox, p47phox, p91phox mRNA expression, iNOS (but not eNOS) expression, NT generation, caspase 3/7 and PARP activity/expression, chromatin fragmentation and fibrosis (enhanced accumulation of collagen, TGF-,, CTGF and fibronectin), and declined systolic and diastolic myocardial performance. ALP attenuated the diabetes-induced increased myocardial, liver and serum XO activity, myocardial ROS, NT generation, iNOS expression, apoptosis, PARP activity and fibrosis, which were accompanied by improved systolic (measured by the evaluation of both load-dependent and independent indices of myocardial contractility) and diastolic performance of the hearts of treated diabetic animals. Thus, XO inhibition with ALP improves type 1 diabetes-induced cardiac dysfunction by decreasing oxidative/nitrosative stress and fibrosis, which may have important clinical implications for the treatment and prevention of diabetic cardiomyopathy and vascular dysfunction. [source] iNOS expression requires NADPH oxidase-dependent redox signaling in microvascular endothelial cells,JOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2008Feng 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] Role of cyclooxygenase-2 and inducible nitric oxide synthase in pancreatic cancerJOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, Issue 8 2002Gu 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] Hypoxic damage to the periventricular white matter in neonatal brain: role of vascular endothelial growth factor, nitric oxide and excitotoxicityJOURNAL OF NEUROCHEMISTRY, Issue 4 2006Charanjit Kaur Abstract The present study examined factors that may be involved in the development of hypoxic periventricular white matter damage in the neonatal brain. Wistar rats (1-day old) were subjected to hypoxia and the periventricular white matter (corpus callosum) was examined for the mRNA and protein expression of hypoxia-inducible factor-1, (HIF-1,), endothelial, neuronal and inducible nitric oxide synthase (eNOS, nNOS and iNOS), vascular endothelial growth factor (VEGF) and N-methyl-D-aspartate receptor subunit 1 (NMDAR1) between 3 h and 14 days after hypoxic exposure by real-time RT-PCR, western blotting and immunohistochemistry. Up-regulated mRNA and protein expression of HIF-1,, VEGF, NMDAR1, eNOS, nNOS and iNOS in corpus callosum was observed in response to hypoxia. NMDAR1 and iNOS expression was found in the activated microglial cells, whereas VEGF was localized to astrocytes. An enzyme immunoassay showed that the VEGF concentration in corpus callosum was significantly higher up to 7 days after hypoxic exposure. NO levels, measured by colorimetric assay, were also significantly higher in hypoxic rats up to 14 days after hypoxic exposure as compared with the controls. A large number of axons undergoing degeneration were observed between 3 h and 7 days after the hypoxic exposure at electron-microscopic level. Our findings point towards the involvement of excitotoxicity, VEGF and NO in periventricular white matter damage in response to hypoxia. [source] Mechanisms of inflammation in spinal cord injuryJOURNAL OF NEUROCHEMISTRY, Issue 2002D. S. Rafati The inflammatory response initiated after spinal cord injury (SCI) is characterized by an increase in cytokines, notably IL-1,. Among its effects are the transcriptional control of cyclooxygenase 2 (COX-2) and the inducible form of nitric oxide synthase (iNOS). While they may ameliorate inflammation, they may also cause cellular damage via production of reactive oxygen species (O2 , ., OH·, NO·). The transcription factor NF-,B is a key intermediary in the signalling pathways leading from IL-1, expression to COX-2 and iNOS stimulation. Binding of NF-,B can be abrogated by the use of oligonucleotide ,decoys' that compete for the cognate endogenous NF-,B proteins. Using injections of fluorescent ,decoy' oligonucleotides into two-month-old-male rats after SCI into the site of injury, we found prompt, robust and transient uptake of labelled ,decoys' into both cytoplasm and nuclei of resident cells. Injection of ,decoys' containing the NF-,B binding sequences present in the COX-2 promoter region, together with a ,nonsense' sequence, showed selective effects on iNOS expression at the site of injury. These results are consistent with the hypothesis that NF-,B transcriptional regulation of COX-2 and iNOS are linked and may be an element in the pathophysiology and recovery of mammalian spinal cord after contusion injury. Acknowledgements:, Supported in part by NINDS Grant NS-39161 and Shriners Hospital Grant 8710. [source] Thrombin and PAR-1 acitvating peptide increase iNOS expression in cytokine-stimulated C6 glioma cellsJOURNAL OF NEUROCHEMISTRY, Issue 3 2001Rosaria Meli Thrombin (THR) plays a key role in the brain under physiological and pathological conditions. Several of the biological activities of thrombin have been shown to be mainly driven through activation of protease-activated receptor-1 (PAR-1)-type thrombin receptor. Here we have studied the effect of THR and PAR-1-activating peptide (PAR1-AP), SFLLRN, on cytokine-induced expression of inducible nitric oxide (iNOS), a prominent marker of astroglial activation using the rat C6 glioma cells. In this cell line, THR (1,10 U/mL) and PAR1-AP (1,100 µm) induced a significant concentration-dependent increase both of IFN-,- (250 U/mL) or TNF-,- (500 U/mL) induced NO release. The observed increase of NO production was related to an enhancement of iNOS expression as measured in cell lysates prepared from different treatments by using SDS,PAGE followed by western blot analysis. The effect of THR, but not that of PAR1-AP, was significantly inhibited by hirulogTM (60 µg/mL), a specific and stochiometric THR inhibitor or by cathepsin-G (40 mU/mL), an inhibitor of PAR-1. In conclusion our data suggest a role for THR through activation of PAR-1 in the induction of astroglial iNOS, and further support the hypothesis that THR may function as an important pathophysiological modulator of the inflammatory response. [source] Loss of dopaminergic neurons by the induction of inducible nitric oxide synthase and cyclooxygenase-2 via CD40: Relevance to Parkinson's diseaseJOURNAL OF NEUROSCIENCE RESEARCH, Issue 6 2005Tatsusada Okuno Abstract A glial reaction associated with up-regulation of inflammatory molecules has been suggested to play an important role in dopaminergic neuron loss in Parkinson's disease (PD). Among inflammatory molecules, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) have been focused upon as key factors in the pathogenesis. However, the mechanism of how these molecules are induced in PD brains is not clearly understood. We focused on CD40, which is expressed on neural cells and could be implicated in the neuroinflammation by inducing inflammatory molecules. We showed that both iNOS and COX-2 were up-regulated in microglia and astrocytes by CD40 stimulation in association with a low dose of interferon-, (IFN-,) in vitro. Selective loss of dopaminergic neurons was induced by costimulation with CD40 and IFN-, in mesencephalic cultures, which was protected by selective inhibitors of iNOS and/or COX-2. We also found in CD40-stimulated astrocytes an increase of a low-affinity IgE receptor CD23, which is known to induce iNOS expression. Together these data suggest that up-regulated iNOS and COX-2 via the CD40 pathway may lead to dopaminergic neuron loss and may participate in the neuroinflammaory pathway of PD. © 2005 Wiley-Liss, Inc. [source] An extract of Apium graveolens var. dulce leaves: structure of the major constituent, apiin, and its anti-inflammatory propertiesJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 6 2007T. Mencherini Flavonoids, natural compounds widely distributed in the plant kingdom, are reported to affect the inflammatory process and to possess anti-inflammatory as well as immunomodulatory activity in-vitro and in-vivo. Since nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) is one of the inflammatory mediators, the effects of the ethanol/water (1:1) extract of the leaves of Apium graveolens var. dulce (celery) on iNOS expression and NO production in the J774.A1 macrophage cell line stimulated for 24 h with Escherichia coli lipopolysaccharide (LPS) were evaluated. The extract of A. graveolens var. dulce contained apiin as the major constituent (1.12%, w/w, of the extract). The extract and apiin showed significant inhibitory activity on nitrite (NO) production in-vitro (IC50 0.073 and 0.08 mg mL,1 for the extract and apiin, respectively) and iNOS expression (IC50 0.095 and 0.049 mg mL,1 for the extract and apiin, respectively) in LPS-activated J774.A1 cells. The croton-oil ear test on mice showed that the extract exerted anti-inflammatory activity in-vivo (ID50 730 ,g cm,2), with a potency seven-times lower than that of indometacin (ID50 93 ,g cm,2), the non-steroidal anti-inflammatory drug used as reference. Our results clearly indicated the inhibitory activity of the extract and apiin in-vitro on iNOS expression and nitrite production when added before LPS stimulation in the medium of J774.A1 cells. The anti-inflammatory properties of the extract demonstrated in-vivo might have been due to reduction of iNOS enzyme expression. [source] Effects of phenylethanoid glycosides from Digitalis purpurea L. on the expression of inducible nitric oxide synthaseJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 7 2005Jae Wook Oh We have isolated four different phenylethanoid glycosides (purpureaside A, desrhamnosyl acteo-side, calceolarioside B and plantainoside D) from the leaves of Digitalis purpurea (foxglove). The effects of these glycosides on activator protein-1 (AP-1)-mediated inducible nitric oxide synthase (iNOS) gene expression in the Raw264.7 macrophage cell line have been studied. Of these four glycosides, purpureaside A potently inhibited iNOS induction by lipopolysaccharide (LPS). Increase in iNOS mRNA by LPS was completely suppressed by purpureaside A. Purpureaside A did not significantly affect LPS-inducible nuclear factor- kB (NF- kB) activation or the nuclear translocation of p65. Moreover, a reporter gene assay using AP-1 specific luciferase reporter revealed that the enhanced activity of AP-1 by LPS was completely abolished in cells treated with purpureaside A. These results demonstrated that purpureaside A inhibited LPS-inducible iNOS expression in macrophages through the suppression of AP-1, but not of NF- kB. [source] Nitric Oxide Plays a Crucial Role in the Development/Progression of Nonalcoholic Steatohepatitis in the Choline-Deficient, l-Amino Acid-Defined Diet-Fed Rat ModelALCOHOLISM, Issue 2010Koji Fujita Background:, The pathogenesis of nonalcoholic steatohepatitis (NASH) is still unclear. Recently, the 2-hit hypothesis was proposed, in which nitric oxide production, representing oxidative stress, was proposed as a very important candidate for the second hit. Methods:, The total study period was 10 weeks. A total of 20 rats were randomly divided into 2 groups. Group 1 was administered the Choline-Deficient, l-Amino Acid-Defined diet to produce a NASH model, and Group 2 as control received the Choline-Sufficient, l-Amino Acid-defined diet. The blood and tissue concentrations of nitrate + nitrite were measured using the Griess reagent and the expression levels of inducible nitric oxide synthase (iNOS) proteins and mRNA was determined by Western blotting. Results:, In regard to nitric oxide (NO) and NO metabolites, there were significant differences in the blood (especially portal venous blood) as well as tissue (liver and visceral fat) concentrations between the 2 animal groups; the amounts of NO metabolites in the tissues were much higher in the NASH models. The level of nitrotyrosine was much markedly higher in the NASH models than in the controls. In regard to the tissue expression of iNOS a significant difference between the 2 groups was found in the visceral fat, especially in the mesenterium. Conclusions:, Based on these results, we hypothesize that the iNOS expression and NO levels in the visceral fat increase, with increased diffusion of NO and its metabolites into the liver, resulting in increased nitrotyrosine formation in the liver; this, in turn, induces inflammation, apoptosis, and fibrosis in the liver, which are one of the characteristic features of NASH. [source] Ethanol Upregulates iNOS Expression in Colon Through Activation of Nuclear Factor-kappa B in RatsALCOHOLISM, Issue 1 2010Chao Wang Background:, Alcohol inhibits colonic motility but the mechanism is unknown. The goal of this study was to test the possibility that nuclear factor-kappa B (NF-,B) is involved in the upregulation of inducible nitric oxide synthase (iNOS) expression induced by ethanol in colon. Methods:, The isometric contraction of longitudinal muscle strips of proximal colon (LP) was monitored by polygraph. Western blot analysis was used to measure the amount of iNOS and I-,B in the cytoplasm and P65 in the nucleus. Immunohistochemistry was applied to locate iNOS in colon. Results:, Ethanol (87mM) inhibited the contraction of LP. Pretreatment of S-methylisothioure (SMT) (1 mM), a specific iNOS inhibitor, Pyrrolidine dithiocarbamate (PDTC) (10 mM) and BAY11-7082(10 mM), specific inhibitors of NF-,B significantly reversed the inhibitory effect of ethanol on LP contraction. Ethanol increased the amount of iNOS and content of NO in colon, and these effects were attenuated by pretreatment of PDTC. Following ethanol administration, the amount of I-,B in the cytoplasm decreased, but that of P65, the subunit of NF-,B in the nucleus, increased. The iNOS was located in the cell body of myenteric plexus in colon. Conclusion:, Ethanol inhibited the contraction of LP in colon mainly through activation of NF-,B, the subsequent upregulation of iNOS expression and increase of NO release in myenteric plexus. [source] PERIPHERAL NERVOUS SYSTEM DEMYELINATION AND iNOS EXPRESSIONJOURNAL OF THE PERIPHERAL NERVOUS SYSTEM, Issue 1 2002Article first published online: 11 MAR 200 Conti G.1, Pasquale C.1, Rostami A.3, De Pol A.2, Galimberti D.1, Scarpini E.1, Baron P.L.1, Scarlato G.1 1 Milano Italy, 2 Modena, Italy, 3Philadelphia USA. Nitric oxide (NO), during CNS demyelination, is synthesised in inflammatory cells from L-arginine by the nitric oxide synthases (NOS). NO can subserve different functions, from cytotoxicity to neuroprotection and trigger either necrotic or apoptotic cell death. In this study we detected inducible form of NOS (iNOS) gene expression in experimental allergic neuritis (EAN), induced in Lewis rats by injection of "SP26," emulsified in complete Freund's adjuvant, which clinical, electrical, and pathological features resemble those of "Guillain-Barré" syndrome. Northern blot, single nerve fiber immmunostaining, and immuno-electron microscope showed that both iNOS mRNA and protein were induced in the PNS of EAN rats by day 14 after immunization, at the beginning of EAN clinical signs. However, with the same experimental procedures, we failed to find iNOS expression during Wallerian degeneration following nerve cut. These data support the hypothesis that iNOS regulation has an active role in cell-mediated demyelination. [source] Hyperbaric oxygen attenuation of lipopolysaccharide-induced acute lung injury involves heme oxygenase-1ACTA ANAESTHESIOLOGICA SCANDINAVICA, Issue 9 2005T.-Y. Huang Background:, Hyperbaric oxygen (HBO) attenuates lipopolysaccharide (LPS)-induced acute lung injury. This beneficial effect of HBO involves inhibition of inducible nitric oxide synthase (iNOS) expression and subsequent nitric oxide (NO) biosynthesis. We sought to investigate the role of heme oxygenase-1 (HO-1) on this HBO inhibition of iNOS induction and acute lung injury in septic rat lungs. Methods:, Before the experiment, 72 rats were randomly allocated to receive HBO or air treatment. With or without HBO pre-treatment, the rats were further divided into the following subgroups (n = 6): (i) LPS injection, (ii) normal saline (N/S) injection, (iii) hemin (a HO-1 inducer) plus LPS, (iv) hemin alone, (v) tin protoporphyrin (SnPP; a HO-1 inhibitor) plus LPS, and (vi) SnPP alone. All rats were maintained for 6 h and then sacrificed with a high-dose pentobarbital injection. Lung injuries and relevant enzymes expression were thus assayed. Results:, Histological analysis, PMNs/alveoli ratio, and wet/dry weight ratio measurements demonstrated that LPS caused significant lung injury and HBO and/or hemin significantly attenuated this LPS-induced lung injury. Increased pulmonary iNOS expression and NO production were associated with lung injury. Induction of HO-1, by HBO and/or hemin, significantly attenuated this LPS-induced iNOS expression and acute lung injury. SnPP, on the contrary, offset the effects of HBO and worsened the LPS-induced lung injury. Conclusions:, HBO may act through inhibiting pulmonary iNOS expression to attenuate LPS-induced acute lung injury in septic rats. Furthermore, this HBO attenuation of iNOS expression involves HO-1 induction. [source] Endothelial, but not the inducible, nitric oxide synthase is detectable in normal and portal hypertensive ratsLIVER INTERNATIONAL, Issue 6 2002Michael Martin Stumm Abstract:Background: Chronic portal hypertension is accompanied by a nitric oxide (NO) dependent vasodilation. Three isoforms of NO producing synthases (NOS) are characterized: neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS). Sources of increased NO levels in chronic hypertension is disputed. Methods: To determine eNOS and iNOS expression in different organs of portal hypertensive and control rats, we divided Sprague-Dawley rats in 6 groups: (1) Partial portal vein ligated rats, (2) Bile duct ligated rats, (3) Carbon tetrachloride treated rats, (4) Sham operated rats, (5) Untreated control rats, and (6) LPS treated rats. Immunohistochemistry (IHC) and immunoblotting (IB) using antibodies against eNOS or iNOS were carried out on samples from thymus, aorta, heart, lung, oesophagus, liver, spleen, kidney, pancreas, small and large intestine. Results: IHC revealed an even eNOS expression in all groups. Expression of iNOS was restricted to macrophages in organs of LPS treated and the thymus of rats. IB mirrored these results. Conclusion: In chronic portal hypertension, the main source for NO production depends on eNOS activity. [source] | ||||||||||||||||||||||||||||||||||