Role Of iNOS Research Articles

Myocardial ischemia/reperfusion injury in the inducible nitric oxide synthase knockout mice

Inducible nitric oxide synthase (iNOS) plays an important role in the inflammatory process of certain major cardiac disorders including myocardial infarction and allograft rejection. However, the role of iNOS in acute myocardial ischemia has not been well defined. We determined the effects of genetically disruption of the intact iNOS system on cardiac tolerance to ischemia/reperfusion injury. Adult male wild-type(WT) and iNOS knockout (KO) B6,129 mice were subjected to 20 min global ischemia and 30 min reperfusion in a Langendorff isolated perfused heart model (37 ° C, n = 10/each group). Ventricular contractile function, heart rate, coronary flow, and leakage of intracellular enzymes (CK and LDH) were not significantly different between the groups during pre-ischemia as well as reperfusion period ( P > 0.05). Myocardial infarct size was also not significantly different between WT (20.2 ± 2.0% of risk area) and KO mice (23.5 ± 3.8%; Mean ± SEM, P > 0.05). However, the post-ischemic heart rate was significantly preserved in KO as compared to WT ( P < 0.05). We conclude that disruption of iNOS gene does not exacerbate ischemia/ reperfusion injury in the heart.

Brain iNOS: current understanding and clinical implications

Nitric oxide (NO) is a unique informational substance first identified as the endothelium-derived relaxing factor. It is generated by NO synthases and plays a prominent role in controlling a variety of organ functions in the cardiovascular, immune, reproductive and nervous systems. Inducible nitric oxide synthase (iNOS) is not normally present in the brain in youth but it can be detected in the brain after inflammatory, infectious or ischemic damage, as well as in the normal, aging brain. Brain iNOS seems to contribute to the pathophysiology of many diseases that involve the central nervous system, but the role of iNOS appears to go beyond tissue damage. Brain iNOS might be required for adequate repair following injury or damage. The effects of brain iNOS on the balance between damage and repair make this enzyme a promising therapeutic target in human disease.

Expression of inducible nitric oxide synthase in human gastric cancer.

Inducible nitric oxide synthase (iNOS) is an enzyme that catalyzes the formation of nitric oxide (NO) from L-arginine. iNOS expression and activity results in the production of high levels of NO[1]. The generation of physiological levels of NO is important for mucosal function and it also exerts a cytoprotective effect on the gastrointestinal mucosa. However, increased iNOS expression has been observed in patients with chronic inflammatory diseases of the gastrointestinal tract, such as ulcerative colitis[2,3], and gastritis[4] and it has been speculated that increased NO may induce DNA damage[5,6] and angiogenesis[7]. Nonetheless, the role of iNOS in human GI neoplasia is largely unkown. Previous studies have demonstrated increased iNOS expression in breast cancer[8,9], and increased iNOS activity and protein levels have been demonstrated in colorectal cancer[10] and adenocarcinoma of the esophagus[11]. However, to date, the role of iNOS in gastric carcinogenesis has not been elucidated.

Expression of Inducible Nitric Oxide Synthase in Human Granulomas and Histiocytic Reactions

Inducible nitric oxide synthase (iNOS) is required in immune response against infections and is involved in granuloma formation in animals; in murine macrophages, iNOS is induced by lipopolysaccharide and interferon-gamma. In contrast, the role of iNOS in human immune response against infections is still questioned, and its expression in granulomas is poorly investigated. Using Western blotting and immunohistochemistry, we investigated iNOS expression in human lymph nodes with nonspecific reactions and in tissues containing granulomas caused by mycobacteria, Toxoplasma, Cryptococcus neoformans, Leishmania, Bartonella, noninfectious granulomas (sarcoidosis, foreign body), and other hystiocitic reactions (Kikuchi's disease, Omenn syndrome). iNOS was undetectable in nonspecific reactive lymphadenitis, foreign-body granulomas, and Omenn syndrome, whereas it was strongly expressed in infectious granulomas, sarcoidosis, and Kikuchi's diseases. Immunohistochemistry demonstrated that iNOS was selectively expressed by the epithelioid and multinucleated giant cells within the granulomas. Use of an anti-nitrotyrosine antibody, recognizing nitrosilated amino acid residues derived from nitric oxide production, revealed a consistent positivity within the cells expressing iNOS, thus suggesting that iNOS is functionally active. Detection of cytokines by reverse transcriptase-polymerase chain reaction demonstrated that tissues that were positive for iNOS, also expressed the Thl-type cytokine interferon-gamma mRNA, but not the Th2-type cytokine interleukin-4. Taken together, these results indicate that iNOS is involved in different human immune reactions characterized by histiocytic/granulomatous inflammation and associated with Th1-type cytokine secretion.

Nitric oxide synthase activities in white blood cells of septic patients

The bulk of data that links inducible nitric oxide synthase (iNOS) activity to the pathophysiology of sepsis originates in animal studies. However, the role of iNOS in human sepsis is controversial. Therefore, we measured in this pilot study iNOS activity in inflammatory cells from septic ICU patients compared to normal controls.

Role of iNOS in the vasodilator responses induced by L-arginine in the middle cerebral artery from normotensive and hypertensive rats.

1. The substrate of nitric oxide synthase (NOS), L-arginine (L-Arg, 0.01 microM - 1 mM), induced endothelium-independent relaxations in segments of middle cerebral arteries (MCAs) from normotensive Wistar-Kyoto (WKY) and hypertensive rats (SHR) precontracted with prostaglandin F2alpha (PGF2alpha). These relaxations were higher in SHR than WKY arteries. 2. L-N(G)-nitroarginine methyl ester (L-NAME) and 2-amine-5,6-dihydro-6-methyl-4H-1,3-tiazine (AMT), unspecific and inducible NOS (iNOS) inhibitors, respectively, reduced those relaxations, specially in SHR. 3. Four- and seven-hours incubation with dexamethasone reduced the relaxations in MCAs from WKY and SHR, respectively. 4. Polymyxin B and calphostin C, protein kinase C (PKC) inhibitors, reduced the L-Arg-induced relaxation. 5. Lipopolysaccharide (LPS, 7 h incubation) unaltered and inhibited these relaxations in WKY and SHR segments, respectively. LPS antagonized the effect polymyxin B in WKY and potentiated L-Arg-induced relaxations in SHR in the presence of polymyxin B. 6. The contraction induced by PGF2alpha was greater in SHR than WKY arteries. This contraction was potentiated by dexamethasone and polymyxin B although the effect of polymyxin B was higher in SHR segments. LPS reduced that contraction and antagonized dexamethasone- and polymyxin B-induced potentiation, these effects being greater in arteries from SHR. 7. These results suggest that in MCAs: (1) the induction of iNOS participates in the L-Arg relaxation and modulates the contraction to PGF2alpha; (2) that induction is partially mediated by a PKC-dependent mechanism; and (3) the involvement of iNOS in such responses is greater in the hypertensive strain.

Role of iNOS in regulating VCAM-1 expression in a murine model of inflammation in vivo: [formula omitted], S. Kawachi, S. Laroux, L. Gray, and H. Van der Hyde, Immunology/Vascular Biology Research Group, LSU Medical Center, Shreveport, LA. 71130

Role of iNOS in regulating VCAM-1 expression in a murine model of inflammation in vivo: [formula omitted], S. Kawachi, S. Laroux, L. Gray, and H. Van der Hyde, Immunology/Vascular Biology Research Group, LSU Medical Center, Shreveport, LA. 71130

Role of iNOS in gut inflammation and injury

Role of iNOS in gut inflammation and injury

Inducible nitric oxide synthase expression after traumatic brain injury and neuroprotection with aminoguanidine treatment in rats.

We investigated the time course of inducible nitric oxide synthase (iNOS) enzymatic activity and immunocytochemical localization of iNOS expression after traumatic brain injury (TBI), as well as the possible role of iNOS in the pathogenesis of TBI. Male Sprague-Dawley rats were anesthetized and underwent moderate parasagittal fluid-percussion brain injury. Rats were decapitated 5 minutes, 6 hours, 1 day, 3 days, 7 days, or 14 days later, and iNOS enzymatic activities were measured (n = 6-8). To determine whether nitric oxide produced by iNOS contributed to the histopathological consequences of TBI, inhibition of iNOS activity using aminoguanidine (intraperitoneal injections of 100 mg/kg aminoguanidine [n = 9] or vehicle [n = 8], twice each day) was conducted for 3 days. Significantly elevated iNOS activity was detected at 3 days (276.8+/-72.3% of contralateral value, means +/- standard errors; P < 0.05), and the most robust increase occurred 7 days after TBI (608.0+/-127.0%, P < 0.01) in the injured parietal cerebral cortex. Immunostaining for iNOS and glial fibrillary acidic protein, at 3 and 7 days after TBI, revealed that the major cellular sources of iNOS expression were cortical Layer 1 astrocytes and macrophages within the subarachnoid space. Administration of aminoguanidine did not reduce contusion volume significantly; however, treatment reduced total cortical necrotic neuron counts (1367.6+/-210.3; P < 0.01, compared with vehicle, 2808.5+/-325.1). These data indicate that iNOS is expressed after moderate parasagittal fluid-percussion brain injury, in a time-dependent manner, and that inhibition of iNOS synthesis improves histopathological outcomes. Thus, inhibition of iNOS activation may represent a potential therapeutic strategy for the treatment of TBI.

NADPH diaphorase-positive dendritic profiles in rat thymus are discrete from autofluorescent cells, immunoreactive for inducible nitric oxide synthase, and show strain-specific abundance differences.

Predisposition to autoimmune disorder in Lewis rats has been associated with abnormal hypothalamic regulation of circulating steroids, leading to inadequate suppression of T helper 1 (Th1) cell-mediated inflammatory reactions. In addition, autoimmune syndromes can be triggered within formerly resistant animals, following damage to the negative selection process of the thymus. A contribution to the autoimmune-susceptible phenotype may therefore derive from the status of thymic tolerance. One mechanism of intrathymic negative selection may involve nitric oxide. Because inducible nitric oxide synthase (iNOS) is known to be inhibitable by steroids, its expression might be different within strains having neuroendocrine disturbance. We report on a study to compare intrathymic iNOS expression in autoimmune-prone Lewis rats with other resistant strains. Interdigitating cells (IDC), darkly stained for diaphorase, were confirmed as immunoreactive for iNOS. They were located towards the medullary side of an accumulation of unstained, but autofluorescent cells (presumed to be macrophages) that circumscribes the corticomedullary zone. The role of iNOS+ IDC in the apoptotic deletion of T cells has been suggested by other studies. Despite the blunted steroidal condition reported for Lewis, nitrergic cell abundance was shown, by quantitative analysis of histochemical stain, to be on average approximately twofold lower compared with resistant strains (Fischer and Sprague-Dawley). This trend was evident in males and females, and confirmed by independent observers. We hypothesize that an intrathymic, iNOS-dependent mechanism may be important for the suppression of potentially autoreactive T-cell clones.

Age-Related Changes in the Immunohistochemical Localization of Inducible Nitric Oxide Synthase (iNOS) and Nuclear Factor-kappa B (NF-.KAPPA.B) in the Mouse Cochlea.

Localization of iNOS and NF-κB in the cochlear tissues of ddy-strain mice was studied immunohistochemically with reference to ageing. Severe structural disorders with a decrease in the number of cells were found, especially in the organ of Corti and in the spiral ganglion of older mice. Throughout all age-spans studied, iNOS-like immunoreactivity was found in the cells of Hensen, Claudius and the outer spiral sulcus epithelium, and it increased dramatically with age. A very small number of cells in the stroma of the spiral ligament and spiral limbus of younger mice up to 12 months of age showed weak iNOS immunoreactivity, while the spiral ganglion cells of the younger mice were almost negative. In mice after 18 months of age, however, strong iNOS immunoreactive cells remarkably increased in the spiral ligament and spiral limbus. Moreover, spiral ganglion neurons that survived from age-related cell death also showed strong reactivity. The immunohistochemical localization of NF-κB and its change with age were nearly the same as those of iNOS. The possible roles of iNOS and NF-κB in the changes of mouse cochlear tissues with advancing age were discussed.

Skin allograft rejection in mice lacking inducible nitric oxide synthase.

During allograft rejection, up-regulation of cytokine-inducible nitric oxide synthase (iNOS) leads to the production of large amounts of nitric oxide (NO). The net effect of NO on the alloimmune response is, however, difficult to predict because of its diverse biological effects, which include potentially opposing roles as an effector and immunoregulatory molecule. In this study, the role of iNOS on the in vitro and in vivo alloimmune response was defined using mutant mice that lack a functional iNOS gene. The ability of spleen cells obtained from iNOS-deficient mutants to proliferate and to produce cytokines in response to irradiated BALB/c stimulator cells was determined, and the rate at which iNOS-deficient mice were able to reject BALB/c skin allografts was observed. Spleen cells from homozygous iNOS-deficient (129xMF1)F1 mice, when compared with cells from heterozygous control mice, showed an increased in vitro proliferative response and produced substantially higher levels of interferon-gamma, and also more interleukin-2 and interleukin-12, in response to allogeneic stimulation. The kinetics of BALB/c skin graft rejection were comparable in heterozygous control animals and iNOS-deficient mice. Moreover, no net effect of iNOS on skin allograft rejection was apparent in mice treated with depleting monoclonal antibodies (mAb) to CD4 or CD8 T cells, either alone or in combination, or in mice treated with both anti-CD8 mAb and a neutralizing anti-tumor necrosis factor mAb. These results show that iNOS has an immunomodulatory effect on the in vitro alloimmune response but lack of iNOS has no net influence on the kinetics of murine skin allograft rejection in either unmodified recipients or recipients in which the early contribution of T-cell subsets and tumor necrosis factor-alpha to graft rejection has been abrogated.

NO as an Indicator of Portal Hemodynamics and the Role of iNOS in Increased NO Production in CCl4-Induced Liver Cirrhosis

It is well known that nitric oxide, a vasodilator, is overproduced in liver cirrhosis. This study was designed to elucidate the role of nitric oxide (NO) in portal hemodynamics and to determine the mechanism underlying the increased serum NO levels in rats with liver cirrhosis induced by the oral intake of CCl4. Using rats, liver cirrhosis was induced by oral administration of CCl4. The serum levels of NO2−/NO3−(NOx) were measured, and portal hemodynamic parameters were evaluated with and without the administration of the NO synthase inhibitorNω-nitro-l-arginine (NNA). Furthermore, Northern blot analysis was used to detect iNOS and cNOS mRNA, and immunohistochemical methods were used to detect iNOS-like immunoreactivity. In cirrhotic rats, the portal flow had increased significantly and the portal resistance had decreased significantly when compared with normal control rats. Hepatic capillary flow in the cirrhotic rats was similar to the control rats. NNA decreased portal flow and increased portal resistance in both groups, but the change was greater in the cirrhotic rats than in controls. The serum levels of NOxwere significantly higher in cirrhotic rats than in normal control rats and were positively correlated with portal flow and negatively correlated with portal resistance. The expression of iNOS mRNA, which was barely detectable in control rats, had increased in all organs of the cirrhotic rats, whereas no significant increase in cNOS mRNA was found in any of the organs from cirrhotic rats. The immunohistochemical analysis was generally consistent with the results of the Northern blot analysis. In the control rats, only the bronchial epithelial cells were stained with the anti-iNOS antibody, but in cirrhotic rats, the bronchial cells in the lungs as well as the histiocytic mesenchymal cells in all organs, and the alveolar epithelial cells of the lungs, were stained. This study demonstrated that NO plays a significant role in portal hypertensive hemodynamics in CCl4-induced liver cirrhosis, and that NO is a useful indicator for the evaluation of portal hypertension. Furthermore, the increased serum levels of NO were found to be derived at least in part from the increased expression of iNOS mRNA in the liver, spleen, and lung.

A proinflammatory activity of interleukin 8 in human skin: expression of the inducible nitric oxide synthase in psoriatic lesions and cultured keratinocytes.

Psoriasis is a common chronic skin disease mediated by cellular immune mechanisms and characterized by an intense neutrophil cell infiltrate and proliferative activation of epidermal keratinocytes. We have previously described the expression of the inducible nitric oxide synthase (iNOS) in epidermal keratinocytes of psoriatic skin lesions. In this study, the role of iNOS in psoriatic inflammation was explored ex vivo in psoriatic skin biopsies and in vitro in primary cultures of human keratinocytes. Messenger RNA for the iNOS enzyme (iNOS mRNA) was detected by reverse transcriptase polymerase chain reaction in skin biopsies from patients with psoriasis, but not in skin specimens from patients with atopic eczema or from healthy volunteers. As demonstrated by in situ hybridization and immunohistochemistry, expression of iNOS mRNA and its gene product was localized to the epidermal keratinocytes of psoriatic skin lesions. In situ hybridization further revealed a complete colocalization of mRNA expression for iNOS with interleukin (IL) 8 receptor-specific mRNA either in the basal germinative cell layer or at focal sites of ongoing neutrophil inflammation in suprabasal cell layers. Because psoriatic keratinocytes have previously been shown to express mRNA transcripts for IL-8, it seemed reasonable to hypothesize that iNOS expression could be induced in an autocrine loop by IL-8. This hypothesis was substantiated by our in vitro experiments showing that a combination of IL-8 and interferon gamma induces the expression of iNOS-specific mRNA and of the functional enzyme in cultured human keratinocytes. These results suggest an important role for iNOS in concert with IL-8 and its receptor early during the formation of psoriatic lesions.

Inducible nitric oxide synthase gene expression in vascular cells after transient focal cerebral ischemia.

We investigated whether inducible nitric oxide synthase (iNOS) is expressed after transient cerebral ischemia and, if so, we sought to define the temporal profile and cellular localization of the expression and the role of iNOS in the mechanism of ischemic brain injury. The middle cerebral artery in rats was occluded for 2 hours by an intraluminal filament. The occurrence of transient ischemia and reperfusion was confirmed by laser-Doppler flowmetry (n = 5). iNOS message in the ischemic neocortex was determined by reverse-transcription polymerase chain reaction. iNOS enzymatic activity was assessed by citrulline assay. The cellular localization of iNOS expression was determined by immunohistochemistry. iNOS mRNA was maximally expressed in postischemic brain at 12 hours and was not present at 4 days (n = 3 per time point). iNOS mRNA was not observed in the contralateral cerebral cortex. iNOS enzymatic activity developed in the postischemic brain between 12 and 24 hours (P < .05) and subsided at 4 days (n = 4 to 8 per time point). iNOS immunoreactivity in the ischemic region was restricted to the wall of capillaries and of larger blood vessels at 12 to 24 hours. In regions of early necrosis, inflammatory cells were iNOS positive. Treatment with the iNOS inhibitor aminoguanidine (n = 5; 100 mg/kg IP, BID for 4 days), starting 6 hours after ischemia, reduced infarct size in neocortex by 36 +/- 7% in comparison with vehicle-treated controls (n = 5) (P < .05). Transient focal ischemia leads to iNOS expression in postischemic brain. However, the spatial and temporal patterns of expression differ from those occurring in permanent ischemia: iNOS is induced earlier and predominantly in vascular cells rather than in neutrophils. Thus, the temporal profile and localization of postischemic iNOS expression depend on the nature of the ischemic insult. The finding that aminoguanidine reduces infarct size adds further support to the hypothesis that postischemic iNOS expression contributes to ischemic brain damage.

Light and Electron Microscopic localization of inducible nitric oxide synthase in the heart of rats with myocarditis

Nitric oxide synthase (NOS) is an enzyme involved in the synthesis of nitric oxide (NO). The inducible form of NOS (iNOS) is induced in the presence of cytokines and produces massive amounts of NO which may be harmful to living tissue. In acute myocarditis, a serious disease which can be fatal, extensive myocardial cell damage associated with massive inflammatory cell infiltration is usually observed and large amounts of cytokines are released from inflammatory cells. However, the mechanism of the heart tissue injury is still unclear. In this study, we investigated the role of iNOS in the myocardial tissue injury in acute myocarditis.The heart tissue specimens were taken from Lewis rats with experimentally induced autoimmune myocarditis. An immunohistochemical study was performed using iNOS antibody raised in New Zealand white rabbits immunized with synthetic peptides (17 amino acids) corresponding to the C-terminal of macrophage iNOS. The specificity of the antibody was confirmed by Western blot analysis. Immunoelectron microscopic studies were carried out by the gold-labeled IgG method using ultrathin sections of LR white resin-embedded material.