Inducible nitric oxide synthase in inflammation

Summary Inflammation, the reaction of vascularized tissue to local injury, not only limits the effects of injury; it may also be the underlying pathological process which initiates or sustains disease. In this paper, the evidence is reviewed for a role for nitric oxide (NO) as a chemical indicator of inflammation and inflammatory diseases.     

Inducible nitric oxide synthase and Salmonella infection

Salmonella infection is associated with the increased expression of inducible nitric oxide synthase in macrophages and other cells. This review summarizes current knowledge of the molecular mechanisms involved in the induction process, and discusses the functional significance of nitric oxide production in the context of host defense against Salmonella.     

Inducible nitric oxide synthase aggresome formation is mediated by nitric oxide

Nitric oxide (NO) generated by inducible NO synthase (iNOS) contributes critically to inflammatory injury and host defense. While previously thought as a soluble protein, iNOS was recently reported to form aggresomes inside cells. But what causes iNOS aggresome formation is unknown. Here we provide evidence demonstrating that iNOS aggresome formation is mediated by its own product NO. Exposure to inflammatory stimuli (lipopolysaccharide and interferon-γ) induced robust iNOS expression in mouse macrophages. While initially existing as a soluble protein, iNOS progressively formed protein aggregates as a function of time. Aggregated iNOS was inactive. Treating the cells with the NOS inhibitor N-nitro-l-arginine methyl ester (L-NAME) blocked NO production from iNOS without affecting iNOS expression. However, iNOS aggregation in cells was prevented by L-NAME. The preventing effect of NO blockade on iNOS aggresome formation was directly observed in GFP-iNOS-transfected cells by fluorescence imaging. Moreover, iNOS aggresome formation could be recaptured by adding exogenous NO to L-NAME-treated cells. These studies demonstrate that iNOS aggresome formation is caused by NO. The finding that NO induces iNOS aggregation and inactivation suggests aggresome formation as a feedback inhibition mechanism in iNOS regulation.     

Inducible nitric oxide synthase modulates lipolysis in adipocytes

The role of inducible nitric oxide synthase (iNOS) in the modulation of adipocyte lipolysis was investigated. Treatment of white and brown adipose cell lines and mouse adipose explants with a mixture of tumor necrosis factor-alpha, interferon-gamma, and lipopolysaccharide (LPS) doubled the lipolytic rate, and this was associated with marked induction of iNOS expression and nitric oxide (NO) production. iNOS inhibition by 1400W, aminoguanidine, or L-NIL pretreatment further increased the cytokine/LPS-mediated lipolysis by 30% (P < 0.05) in cultured adipocytes and in adipose explants. However, this potentiating effect of iNOS inhibition was abolished in adipose explants isolated from iNOS knockout mice. Pharmacological inhibitors of adenylyl cyclase or protein kinase A reduced cytokine/LPS-induced lipolysis and also blunted the potentiating effect of iNOS inhibition on the lipolytic rate. Furthermore, addition of the antioxidants l-cystine and l-glutathione to cytokine/LPS-stimulated adipocytes mimicked the lipolytic effect of iNOS inhibition. In conclusion, inhibition of iNOS activity in adipocytes potentiates cytokine/LPS-induced lipolysis. This effect was fully reversed by adenylyl cyclase and protein kinase A inhibitors but was mimicked by cellular antioxidants. These data suggest that iNOS-mediated NO production counteracts cytokine/LPS-mediated lipolysis in adipocytes and that this feedback mechanism involves an oxidative process upstream of cAMP production in the signaling pathway.     

Autoinhibition of endothelial nitric oxide synthase (eNOS) in gut smooth muscle by nitric oxide

Nitric oxide in the gut is produced by nNOS in enteric neurons and by eNOS in smooth muscle cells. The eNOS in smooth muscle is activated by vasoactive intestinal peptide (VIP) released from enteric neurons. In the present study, we examined the effect of nitric oxide on VIP-induced eNOS activation in smooth muscle cells isolated from human intestine and rabbit stomach. NOS activity was measured as formation of the 1:1 co-product, l-citrulline from l-arginine. VIP caused an increase in l-citrulline production that was inhibited by NO in a concentration dependent manner (IC(50)~25 microM; maximal inhibition 72% at 100 microM NO). Basal l-citrulline production, however, was unaffected by NO. The effect was not mediated by cGMP/PKG since the PKG inhibitor KT5823 had no effect on eNOS autoinhibition. The autoinhibition was selective for NO since the co-product l-citrulline had no effect on VIP-induced NOS activation. Similar effects were obtained in rabbit gastric and human intestinal smooth muscle cells. The results suggest that NO produced in smooth muscle cells as a result of the activation of eNOS by VIP exerts an autoinhibitory restraint on eNOS thereby regulating the balance of the VIP/cAMP/PKA and NO/cGMP/PKG pathways that regulate the relaxation of gut smooth muscle.     

Inducible nitric oxide synthase immunoreactivity in healthy rat pancreas

Nitric oxide (NO) is produced by NO synthase (NOS) isoforms: neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS). It is believed that, while nNOS and eNOS are effective in regulation of normal physiological processes, iNOS is expressed at an increasing rate especially in inflammatory process. The aim of this study was to determine the presence of iNOS immunoreactivity (iNOS-IR) and, to compare the iNOS-IR in islet of Langerhans cells (LC), acinar cells (AC), centroacinar cells (CC) and ductal cells (DC) by immunohistochemical (IHC) method in healthy rat pancreata. This study revealed the presence of iNOS-IR in all cell types except AC. Statistical analysis revealed a highly significant difference (p<0.001) with respect to iNOS-IR in comparison of all cell types. However, binary comparison of cell types revealed no significant differences between LC and DC (p=0.136), significant differences LC and CC, CC and DC (p=0.001 and 0.022, respectively) and a highly significant differences LC and AC, AC and DC (P<0.001). The results of this study indicate that iNOS-IR is present in almost all LC. Thus, especially in reseach related to diabetes, it should not be disregarded that iNOS may be constitutively present in pancreatic islets.     

Inducible nitric oxide synthase promoter polymorphism in human brucellosis

Nitric oxide (NO), produced by the inducible nitric oxide synthase (NOS2) protein, is a defence mechanism against various pathogens, including bacteria of the genus Brucella. The aim of this study was to investigate the possible association between the NOS2 gene promoter polymorphism, TAAA(n) and CCTTT(n) microsatellites, and the predisposition to human brucellosis. We performed a case-control study in 85 patients with brucellosis and 100 healthy individuals, matched for age and sex, living in the same geographic area, in whom the NOS2 promoter was genotyped by using a polymerase chain reaction (PCR)-based method combined with fluorescent technology. No statistically significant differences were observed in the distribution of TAAA(n) alleles between the groups under study. When the overall NOS2 CCTTT(n) allele distribution of the brucellosis patients was compared with that of the control subjects, a significant skewing was observed (P = 0.04, by chi(2) test from 2 x 9 contingency table). Interestingly, we observed a trend towards Brucella infection protection with the 9 repeat (181 bp) allele (1.8% patients vs. 7.5% controls; P = 0.01, odds ratios = 0.22, 95% confidence interval = 0.05-0.83), which turned out to be non-significant after applying multiple testing. We concluded that the NOS2 microsatellite polymorphism might not have a major effect on brucellosis; nevertheless, the fact that a non-significant trend towards protection was detected in the CCTTT(n) alleles may be an indication for a follow-up study.     

诱导型一氧化氮合酶介导β淀粉样蛋白在体神经毒性

目的：探讨一氧化氮合酶（NOS）及一氧化氮（NO）在β淀粉样蛋白（Aβ）神经毒性和Alzheimer病（AD）发病机制中的介导作用。方法：应用行为学及病理学方法,观察海马注射Aβ1－40对大鼠Y迷宫学习记忆的影响及对局部神经元的损伤作用;观察特异性诱导型一氧化氮合酶（iNOS)抑制剂胍氢酶（AG）及特异性神经元型一氧化氮合酶(nNOS)抑制剂7－硝基吲哚（7－NI）腹腔注射对海马内注射Aβ1－40神经毒性的干预,结果：海马注射Aβ1－40后,大鼠Y迷宫学习记忆能力及海马局部神经元明显受损,特异性iNOS抑制剂AG能够阻止Aβ1－40海马注射对大鼠学习记忆和局部神经元的损伤作用,而特异性nNOS抑制剂7－NI无此干预效应。结论：iNOS/NO参与了在体条件下对Aβ神经毒性的介导,在AD发病机制中具有重要作用。     

Inducible nitric oxide synthase protection against coxsackievirus pancreatitis.

Coxsackievirus infection causes myocarditis and pancreatitis in humans. In certain strains of mice, Coxsackievirus causes a severe pancreatitis. We explored the role of NO in the host immune response to viral pancreatitis. Coxsackievirus replicates to higher titers in mice lacking NO synthase 2 (NOS2) than in wild-type mice, with particularly high viral titers and viral RNA levels in the pancreas. Mice lacking NOS have a severe, necrotizing pancreatitis, with elevated pancreatic enzymes in the blood and necrotic acinar cells. Lack of NOS2 leads to a rapid increase in the mortality of infected mice. Thus, NOS2 is a critical component in the immune response to Coxsackievirus infection.     

Induction of nitric oxide synthase by cytokines in vascular smooth muscle cells.

We investigated the mechanisms by which cytokines lead to a diminished responsiveness of vascular smooth muscle to vasoconstrictors. The attenuation of noradrenaline-induced contraction by 6 to 24 h incubations with the cytokines, tumor necrosis factor and interleukin-1, in endothelium-denuded rabbit aorta was associated with an increase in intracellular cyclic GMP level. This increase was abolished by the stereoselective inhibitor of nitric oxide-synthase, NG-nitro-L-arginine and by cycloheximide. Formation of nitric oxide was detected in the cytosol of cytokine-treated native and cultured smooth muscle cells by activation of purified soluble guanylate cyclase, and depended on tetrahydrobiopterin, but not on Ca2(+)-calmodulin. The results indicate that cytokines induce a nitric oxide-synthase of the macrophage-type in vascular smooth muscle.     

Inducible nitric oxide synthase haplotype associated with migraine and aura

Molecular and Cellular Biochemistry - Migraine is a complex neurological disorder with a clear neurogenic inflammatory component apparently including enhanced nitric oxide (NO) formation. Excessive...     

Inducible nitric oxide synthase expression is inhibited by myeloperoxidase.

Nitric oxide (NO) plays key roles in vasodilation and host defense, yet the overproduction of NO by inducible nitric oxide synthase (iNOS) at inflammatory sites can also be pathogenic. Here, we investigate the role of MPO in modulating the induction of iNOS by IFNgamma/LPS (IL). In monocyte-macrophages (Mvarphi) treated with IL, MPO gene expression was found to be downregulated as iNOS was upregulated. In Mvarphi from MPO-knockout (KO) mice, the induction of iNOS by IL was earlier and higher than in MPO-positive cells, suggesting MPO is inhibitory. Consistent with that interpretation, the addition of purified MPO enzyme to cultured macrophages inhibited iNOS induction by IL. In addition, an inhibitor of MPO enzyme, 4-aminobenzohydrazide, enhanced iNOS induction in MPO-positive cells, but not in MPO-KO cells. Similarly, taurine, a scavenger of MPO-generated HOCl, enhanced iNOS induction in MPO-positive cells, but not in MPO-KO cells. MPO affects an early event, suppressing iNOS induction when added within 2h of IL, but not when added several hours after IL. The suppression by MPO was alleviated by NO donor, sodium nitroprusside, suggesting the suppression results from scavenging of NO by MPO. This interpretation is consistent with earlier reports that MPO consumes NO, and that low levels of NO donor augment induction of iNOS by IFNgamma/LPS. The implication of these findings is that MPO acts as gatekeeper, suppressing the deleterious induction of iNOS at inflammatory sites by illegitimate signals. The combined signaling of IFNgamma/LPS overrides the gatekeeper function by suppressing MPO gene expression.     

Inducible nitric oxide synthase and guinea-pig ileitis induced by adjuvant

We sought to establish a model of inflammatory bowel disease by augmenting the activity of the local immune system with Freund's complete adjuvant, and to determine if inducible nitric oxide synthase (iNOS) expression and peroxynitrite formation accompanied the inflammatory condition. In anaesthetized guinea-pigs, a loop of distal ileum received intraluminal 50% ethanol followed by Freund's complete adjuvant. Control animals were sham operated. When the animals were killed 7 or 14 days later, loop lavage fluid was examined for nitrite and PGE(2) levels; mucosal levels of granulocyte and macrophages were estimated by myeloperoxidase (MPO) and N-acetyl-D-glucosaminidase (NAG) activity, respectively. Cellular localization if iNOS and peroxynitrite formation were determined by immunohistochemistry with polyclonal antibodies directed against peptide epitopes of mouse iNOS and nitrotyrosine, respectfully. Adjuvant administration resulted in a persistent ileitis, featuring gut thickening, crypt hyperplasia, villus tip swelling and disruption, and cellular infiltration. Lavage levels of PGE(2) and nitrite were markedly elevated by adjuvant treatment. Immunoreactive iNOS and nitrotyrosine bordered on detectability in normal animals but were markedly evident with adjuvant treatment at day 7 and particularly day 14. Immunohistochemistry suggested that enteric neurons and epithelia were major sites of iNOS activity and peroxynitrite formation. We conclude that local administration of adjuvant establishes a chronic ileitis. Inducible nitric oxide synthase may contribute to the inflammatory process.     

Inducible nitric oxide synthase and control of intracellular bacterial pathogens

Inducible nitric oxide synthase (iNOS) has important functions in innate immunity and regulation of immune functions. Here, the role of iNOS in the pathogenesis of various intracellular bacterial infections is discussed. These pathogens have also evolved a broad array of strategies to repair damage by reactive nitrogen intermediates, and to suppress or inhibit functions of iNOS.     

Antithrombin III enhances inducible nitric oxide synthase gene expression in vascular smooth muscle cells

Evidence suggests that antithrombin III (ATIII) exerts anti-inflammatory properties in addition to its anti-coagulative mechanisms. In animal models of sepsis, ATIII affected cytokine plasma concentrations with a decrease of pro-inflammatory cytokines. In addition to cytokines, excessive production of nitric oxide (NO) derived from inducible nitric oxide synthase (iNOS) might represent another important mediator of the cytotoxic events during sepsis. Regarding ATIII as a potential anti-inflammatory modulator, one may speculate that ATIII inhibits the synthesis of iNOS-derived NO. However, our data demonstrate that ATIII further stimulates iNOS gene expression when applied together with either interleukin-1 beta or the combination of lipopolysaccharide plus interferon-gamma. The most prominent synergistic effects on NO synthesis were found when ATIII was given at higher concentrations (1, 5, and 10 U/ml). Although the mechanisms of ATIII signal transduction remain to be established, intensification of interleukin-1 beta or interferon-gamma/lipopolysaccharide-induced NO synthesis by ATIII does not attribute to the anti-inflammatory properties of ATIII.     

Inducible nitric oxide synthase and apoptosis in human B cell lymphomas

Nitric oxide synthases are isoenzymes that catalyse the synthesis of nitric oxide (NO). NO plays both pathological and physiological roles depending on its rate of synthesis and concentration in cellular source and microenvironment. Apoptosis is an important biological factor in lymphomas. This study evaluates expression of inducible nitric oxide synthase (iNOS) in human lymphomas and its relation with apoptosis. This study comprised 46 cases of B-cell lymphoma. The lymphomas were classified as 3 mantle cell, 5 marginal zone, 4 follicular, 2 Burkitt, 25 diffuse large cell, 2 anaplastic large cell, 3 lymphoblastic, 2 lymphoplasmacytic according to WHO classification of lymphoid neoplasms. Hematoxylin eosin slides of the cases were reviewed and immunoperoxidase technique was performed iNOS and Caspase monoclonal antibodies to selected sections of each case. Antigen staining was carried out with iNOS and Caspase proteins and Ultravision Polyvalent, HRP-AEC kit (Neomarkers-Biogen USA). For the evaluation of iNOS and Caspase, tumor areas with a high density of expression were chosen. Positive stained cells were counted in 5 different areas at a magnification ×40 by an Olympus B × 51 microscope in each case. The iNOS and Caspase expressions were independently recorded by four pathologists and the results were averaged. All of the cases were positive for the iNOS and Caspase. But there is not a statistically important relation between lymphoma grade and iNOS activity. We could not find a correlation between iNOS and patients age. This study reveals the capacity of B-cell neoplasms to express iNOS in situ. In conclusion, our study revealed that there is a positive relation between iNOS expression and apoptosis (p $=$ 0.032 spearman correlation).