Inducible Nitric Oxide Synthase

ABSTRACT: The effects of chitosan on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) activity and gene expression in vivo or vitro were investigated in weaned piglets. In vivo, 180 weaned piglets were assigned to five dietary treatments with six replicates. The piglets were fed on a basal diet supplemented with 0 (control), 100, 500, 1000, and 2000 mg chitosan/kg feed, respectively. In vitro, the peripheral blood mononuclear cells (PBMCs) from a weaned piglet were cultured respectively with 0 (control), 40, 80, 160, and 320 µg chitosan/ml medium. Results showed that serum NO concentrations on days 14 and 28 and iNOS activity on day 28 were quadratically improved with increasing chitosan dose (P < 0.05). The iNOS mRNA expressions were linearly or quadratically enhanced in the duodenum on day 28, and were improved quadratically in the jejunum on days 14 and 28 and in the ileum on day 28 (P < 0.01). In vitro, the NO concentrations, iNOS activity, and mRNA expression in unstimulated PBMCs were quadratically enhanced by chitosan, but the improvement of NO concentrations and iNOS activity by chitosan were markedly inhibited by N-(3-[aminomethyl] benzyl) acetamidine (1400w) (P < 0.05). Moreover, the increase of NO concentrations, iNOS activity, and mRNA expression in PBMCs induced by lipopolysaccharide (LPS) were suppressed significantly by chitosan (P < 0.05). The results indicated that the NO concentrations, iNOS activity, and mRNA expression in piglets were increased by feeding chitosan in a dose- dependent manner. In addition, chitosan improved the NO production in unstimulated PBMCs but inhibited its production in LPS-induced cells, which exerted bidirectional regulatory effects on the NO production via modulated iNOS activity and mRNA expression.

Abstract: Background: The failure of intestinal mucosal barrier may induce multiple organ dysfunction and systemic inflammatory response syndrome, but little work has been done on whether hypobaric hypoxia related to the failure of intestinal mucosal barrier. Aims: To study the expression of hypoxia-inducible factor 1α (HIF-1α), inducible nitric oxide synthase (iNOS) and morphological changes of intestinal mucosa in albino rats at different altitude. Methods: 30 male Wistar rats raised in plain for one month were randomly divided into 3 groups: Plain 500 m group (n=10), High-altitude (HA) 3842 m group (n=10) and HA4767 m group (n=10). Each group was delivered to different altitude area at the same shipping time and executed after 3 days’ exposure to different altitude. Intestinal segments with the same location of all rats were removed for morphological analyses. Morphologic parameters (villous height, crypt depth, mucosal wall thickness and villous surface area) were measured by optical and scanning electron microscope. The expression of iNOS and HIF-1α were detected by immunohistochemistry. Results: Morphological indexes in higher altitude groups were exacerbated obviously compared with those of lower altitude groups. While the expression of iNOS and HIF-1α in higher altitude groups were significantly increased than those of lower altitude groups. Linear correlation analysis showed that the expression of iNOS was positively correlated with that of HIF-1α. Conclusions: Hypobaric hypoxia increases the expression of HIF-1α and iNOS in intestinal mucosa, however exac- erbates the mucous morphologic parameters with altitude increasing. HIF-1α may regulate the expression of iNOS and be involved in the damage of intestinal mucosa.

CLD = chronic lung disease; DTT = dithiothreitol; EMSA = electro- phoretic mobility shift assay; HEPES = HCO(3-)-free N-2-hydroxyethyl- piperazine-N'-2-ethanesulfonic acid; IFN-γ = gamma interferon; iNOS = inducible nitric oxide synthase; LPS = lipopolysaccaride; NF-κB = nu- clear factor-κB; NO = nitric oxide; RT-PCR = reverse transcription – polymerase chain reaction; PMSF = phenylmethysulfonyl fluoride; TAF = tracheobronchial aspirate fluid

The activated NF-κB dimer binds to the 5'-flanking region of the iNOS promoter and induces iNOS formation [12]. Inducible nitric oxide synthase (iNOS) and nitric oxide (NO) have an important role in collagen formation dur- ing wound healing [13,14]. Inhibition of NO synthase significantly decreased collagen synthesis in wound fibroblasts [13]. Dermal fibroblasts from iNOS-knock out murine fibroblasts proliferated more slowly and synthe- sized less collagen, and NO donors restored the collagen synthesis to normal level [15]. Another study implicated a role for NO in PGE 2 formation and collagen deposition in rats [16]. Therefore, endogenous iNOS and NO may link the NF- κ B pathway to PGE 2 and regulate collagen forma- tion during wound healing.

Hyperleptinemia may play an important role in obesity-associated cardiovascular diseases including atherosclerosis. Leptin exerts many potentially atherogenic effects such as induction of endothelial dysfunction, stimulation of inflammatory reaction, oxidative stress, decrease in paraoxonase activity, platelet aggregation, migration, hypertrophy and proliferation of vascular smooth muscle cells. Nitric oxide (NO) is a short-lived, gaseous free radical, synthesized from L- arginine by NO synthases (NOS). The inducible nitric oxide synthase (iNOS) is a main enzyme producing nitric oxide during inflammation and thus contributes to the initiation and development of inflammatory cardiovascular diseases such as atherosclerosis. The aim of the study was to elucidate the link between leptin and inflammation by in vitro ctivation of the PBMCS with leptin and to confirm the expression of iNOS by western blotting technique in CVD. The results show that the In vitro activation of PBMC with leptin showed bands of 130 kDa which confirmed the presence of iNOS. The in vivo expression of iNOS correlates with the leptin levels in normal and overweight BMI CVD patients respectively. This shows the elevated leptin levels was associated with elevated iNOS.

Acute or chronic kidney injury results from various insults and pathological conditions, and it is accompanied by the activation of compensatory repair mechanisms. Both insults and repair mechanisms are initiated by circulating factors, whose cellular effects are mediated by the activation of selective signal transduction pathways. The inducible nitric oxide synthase (iNOS) pathway is one of the signaling pathways that are activated during these processes. iNOS is one of the three isoforms of NOS, each of which differ in their localization, regulation and catalytic properties and are encoded by different genes [7]. iNOS is responsible for the synthesis of nitric oxide (NO) and is normally undetectable under physiological conditions, but can be expressed de novo under experimental or pathological conditions in a wide range of cells and tissues. When induced by various growth factors, cytokines, signaling pathways, or agents, iNOS continuously produces NO until the enzyme is degraded [8]. Mass production of NO and NO-derived substances is mainly ascribed to excessive induction of iNOS, and these products exert various effects on the kidneys. High production of NO by iNOS can have beneficial microbicidal, antiviral, antiparasitic, and antitumor effects [3, 8, 9], but aberrant iNOS induction may have detrimental consequences and seems to be involved in the pathophysiology of conditions such as tumor development, transplant rejection, and septic shock, in diseases such as asthma, rhinitis, multiple sclerosis, psoriasis, and neurodegenerative diseases [7, 9, 10]. The regulation of iNOS expression following exposure to hypergravity, a condition that can adversely affect the kidneys, has not yet been described. The purpose of this study was to

The fact that wounds require iNOS to heal at a normal rate and that wound healing in iNOS-deficient mice can be reconstituted with iNOS cDNA Inducible Nitric Oxide Synthase: What Differe[r]

Aberrant levels of nitric oxide during inflammatory disease states have pathological consequences. This is because nitric oxide (NO), as a radical species, is capable of producing reactive intermediates which can interact with various constituents of the cell and elicit damage. The enzyme inducible nitric oxide synthase (iNOS) produces high levels of NO during the inflammatory response and thus has been implicated and investigated as a critical mediator of several pathological conditions (Iadecola et al., 1997, Koprowski et al., 1993, Merrill et al., 1993). The role of iNOS in anterior uveitis remains somewhat controversial. Initial studies in the model of EIU collectively demonstrated that iNOS activity increased during EIU and activity was reduced in response to inhibitors of nitric oxide synthases (NOS) (Goureau et al., 1995, Mandai et al., 1994). Additional studies using the selective iNOS inhibitor

We have examined the localization of inducible nitric oxide synthase (iNOS) and nitrotyrosine (the product of nitration of tyrosine by peroxynitrite, a highly reactive derivative of nitric oxide [NO]) in demyelinating lesions from (i) two young adult patients with acute multiple sclerosis (MS), (ii) a child with MS (consistent with diffuse sclerosis), and (iii) five adult patients with chronic MS. Previous reports have suggested a possible correlation between iNOS, peroxynitrite, related nitrogen-derived oxidants, and the demyelinating processes in MS. We have demonstrated iNOS-immunoreactive cells in both acute-MS and diffuse-sclerosis-type lesions. In acute-MS lesions, iNOS was localized in both monocytes/macrophages and reactive astrocytes. However, foamy (myelin-laden) macrophages and the majority of reactive astrocytes were iNOS negative. In specimens from the childhood MS patient, iNOS protein was present only in a subpopulation of reactive or hypertrophic astrocytes. In contrast, no iNOS staining was detected in chronic-MS lesions. Immunohistochemical staining of acute-MS lesions with an antibody to nitrotyrosine revealed codistribution of iNOS- and nitrotyrosine-positive cells, although nitrotyrosine staining was more widespread in cells of the monocyte/macrophage lineage. In diffuse- sclerosis-type lesions, nitrotyrosine staining was present in hypertrophic astrocytes, whereas it was absent in chronic-MS lesions. These results suggest that NO and nitrogen-derived oxidants may play a role in the initiation of demyelination in acute-MS lesions but not in the later phase of the disease.

Both nNOS and eNOS are normally expressed in many types of cells, but were first discovered in the neuronal and vascular endothelial cells. The expression of inducible NOS has been described from a variety of cell types, including macrophages, hepatocytes, smooth muscle cells, keratinocytes, pancreatic islet -cells and mesangial cells [3, 4]. Unlike the constitutive forms of NOS (eNOS, nNOS), inducible nitric oxide synthase (iNOS) produces a large amount (nanomolar quantities) of NO over a prolonged period of time (from hours to several days) after its activation by cytokines or bacterial endotoxins (lipopolysaccharide; LPS) [5].

We investigated the role of inducible nitric oxide synthase (iNOS) in Theiler’s murine encephalomyelitis virus (TMEV) infection of susceptible (SJL) and resistant (C57BL/6 [B6]) strains of mice. TMEV is an excellent model of virus-induced demyelinating disease, such as multiple sclerosis (MS). Previous studies of others have suggested that NO may play a role in the pathogenesis of demyelinating disease. The presence and level of iNOS were determined in the brains and spinal cords of SJL and B6 TMEV-infected mice by the following methods: (i) PCR amplification of iNOS transcripts, followed by Southern blotting with an iNOS- specific probe, and (ii) immunohistochemical staining with an anti-iNOS-specific affinity-purified rabbit antibody. iNOS-specific transcripts were determined in the brains and spinal cord of both SJL and B6 TMEV-infected mice on days 0 (control), days 3, 6, and 10 (encephalitic stage of disease), and days 39 to 42, 66, and 180 (demyelinating phase) postinfection (p.i.). iNOS-specific transcripts were found in the brains and spinal cords of both SJL and B6 TMEV-infected mice at 6, 10, and 39 (SJL) days p.i., but they were absent in mock-infected mice and in TMEV-infected SJL and B6 mice at 0, 3, 66, and 180 days p.i. Immunohistochemical staining confirmed the presence of iNOS protein in both TMEV-infected SJL and B6 mice at days 6 and 10 p.i., but not at days 0, 3, 66, and 180 days p.i. Weak iNOS staining was also observed in TMEV-infected SJL mice at 42 days p.i. iNOS-positive staining was found in reactive astrocytes surrounding areas of necrotizing inflammation, particularly in the midbrain. Weak iNOS staining was also observed in cells of the monocyte/ macrophage lineage in areas of parenchymal inflammation and necrosis (mesencephalon) and in leptomen- ingeal and white matter perivascular infiltrates of the spinal cord. Rod-shaped microglia-like cells and foamy macrophages (myelin-laden) were iNOS negative. These results suggest that NO does not play a direct role in the late phase of demyelinating disease in TMEV-infected mice.

Inducible nitric oxide synthase (iNOS, encoded by the gene Nos2 in mice) plays a key role in the IFN- ␥ -dependent response to M. tuberculosis and several other pathogens (19–21). iNOS facilitates the production of nitric oxide (NO), a toxic metabolite with direct antimicrobial activity. NO also acts as a regulator of host responses and coordi- nates metabolic changes in IFN- ␥ -stimulated macrophages (22–24). While Nos2 ⫺/⫺ mice display increased susceptibility to infection by M. tuberculosis, evidence suggests this is not simply due to direct cell-intrinsic antimicrobial effects of NO (25). In addition, the activity of iNOS is not absolutely required to control infection by many pathogens, suggesting that there are redundant iNOS-independent mechanisms that underlie the potency of IFN- ␥ (26). Strikingly, while L. pneumophila does not display resistance to the effects of NO when cultured in broth, Nos2 ⫺/⫺ macrophages are not impaired in IFN- ␥ -dependent restriction of L. pneumophila (27–29). This indicates either that L. pneumophila is resistant to the effects of iNOS/NO during infection or, more likely, that there are redundant factors induced by IFN- ␥ that can restrict L. pneumophila in the absence of iNOS.

In this study, we have identified the source of nitric oxide (NO) produced in the human inflammatory joints by analyzing expression of inducible NO synthase. In ex vivo organ cultures, both inflammatory synovium and cartilage from patients with rheumatoid arthritis produced NO. The NO production was suppressed by NG-monomethyl-L-arginine, an inhibitor of NO synthase. The amount of NO produced by the synovium correlated with the proportion of CD14+ cells in the corresponding tissue (r = 0.8, P < 0.05).

noteworthy that similar controversial roles have been ascribed to iNOS derived NO' in a variety of pathophysiological conditions. While a number of studies have reported that genetic ablation of iNOS may exert beneficial effects in endotoxaemic shock, infection by Toxoplasma gondii and autoimmune vasculitis(i48,i6i,i64,i65), other studies have reported that genetic ablation of iNOS may exacerbate the inflammatory process in endotoxaemia, encephalomyelitis, and tuberculosis(i66-i68). Obviously further experiments are needed in order to explain these differences, which have important implications for clinical therapeutic strategies based on NOS enzyme inhibition. It can therefore be seen that the lack of understanding of the role of inducible NOS and resultant increased NO* production is not peculiar to IBD but is apparent for a lot o f other conditions in man.

To determine if SUMOylation was involved in inflamma- tory activation, rat C6 glioma cells stably transfected with luciferase reporter constructs pNF κ B (4 × NF κ B binding- element) or[r]

In liver injury, perisinusoidal cells known as lipocytes (Ito cells) undergo "activation," acquiring smooth muscle-like features and a contractile phenotype. To assess whether contraction of these cells is regulated by nitric oxide (NO), we examined the production of NO by lipocytes and the effect of NO on lipocyte contractility. Cultured lipocytes were exposed to cytokines and/or LPS. Single agents had little or no effect on the level of inducible NO synthase (iNOS) mRNA. However, interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), or LPS in combination with interferon-gamma (IFN- gamma) stimulated iNOS mRNA, which was present within 4 h after exposure. iNOS mRNA levels were paralleled by changes in nitrite (a metabolic product of NO). Intraperitoneal administration of IFN-gamma, TNF-alpha, and LPS led to rapid induction of iNOS mRNA in lipocytes, confirming in vivo the culture findings. Ligation of the common hepatic bile duct, which induces periportal-based liver injury, stimulated iNOS mRNA in lipocytes.

In cardiac transplantation, chronic rejection takes the form of an occlusive vasculopathy. The mechanism underlying this disorder remains unclear. The purpose of this study was to investigate the role nitric oxide (NO) may play in the de- velopment of allograft arteriosclerosis. Rat aortic allografts from ACI donors to Wistar Furth recipients with a strong genetic disparity in both major and minor histocompatibil- ity antigens were used for transplantation. Allografts col- lected at 28 d were found to have significant increases in both inducible NO synthase (iNOS) mRNA and protein as well as in intimal thickness when compared with isografts. Inhibiting NO production with an iNOS inhibitor increased the intimal thickening by 57.2%, indicating that NO sup- presses the development of allograft arteriosclerosis. Next, we evaluated the effect of cyclosporine (CsA) on iNOS ex- pression and allograft arteriosclerosis. CsA (10 mg/kg/d) suppressed the expression of iNOS in response to balloon- induced aortic injury. Similarly, CsA inhibited iNOS expres- sion in the aortic allografts, associated with a 65% increase in intimal thickening. Finally, we investigated the effect of adenoviral-mediated iNOS gene transfer on allograft arte- riosclerosis. Transduction with iNOS using an adenoviral vector suppressed completely the development of allograft arteriosclerosis in both untreated recipients and recipients treated with CsA. These results suggest that the early im- mune-mediated upregulation in iNOS expression partially protects aortic allografts from the development of allograft arteriosclerosis, and that iNOS gene transfer strategies may prove useful in preventing the development of this other- wise untreatable disease process. ( J. Clin. Invest. 1997. 100: 2035–2042.) Key words: gene therapy • transplantation • in- timal hyperplasia • chronic rejection • cyclosporine

extensively in this review. The enzyme primarily responsible for the roles of NO in in- flammatory processes is the inducible NOS (iNOS; NOS2; or type II NOS), which is not typically expressed in resting cells and must first be induced by certain cytokines or micro- bial products. The molecular biology and regu- lation of NO synthases have been reviewed extensively (6,7). We would like to highlight that iNOS remains very stable at both the mRNA and protein levels, and generates large amounts of NO over a period of days (6,8,9). At least two general conclusions can be gleaned from these studies and numerous others. First, sustained production of NO at high levels will lead to the production of numerous reactive ni- trogen oxide species (RNOS), which can medi- ate a broad spectrum of physiological and pathological effects (10). Second, due to the possibility of deleterious effects to the host as a consequence of prolonged exposure to such RNOS, iNOS must be regulated carefully (11). Finally, one can hypothesize that microorgan- isms may have developed means for suppress- ing the expression and/or activity of iNOS, per- haps by co-opting the host’s own regulatory machinery. Viewed from this perspective, the balance between induction and suppression of iNOS may underlie much of the physiology and pathology of inflammation.

Abstract: Nitric oxide (NO) is a potential bioactive gas produced continuously and con- stantly in the airways of healthy subjects. In allergic airway in fl ammation, the level of exhaled NO is usually increased and mediated by inducible nitric oxide synthase (iNOS) enzyme presenting in the epithelium and different in fl ammatory cells. The measurement of NO concentration in the airway is possible with portable devices which use an electrolumi- nescence technique. In subjects with upper airway with allergic in fl ammation such as in allergic rhinitis, the measurement of nasal NO (nNO) may help to diagnose and manage the disease. In the lower airway, increased fractional exhaled NO (FENO) re fl ects directly the in fl ammatory process that occurs in the airways that are typically seen in asthma. It has been shown that there is a strong correlation between FENO levels and increased activity of airway in fl ammation mediated by immuno-allergic cells and mediators. Thus, FENO has higher speci fi city and sensitivity than other methods in diagnosing the severity of in fl amma- tion in asthmatic patients. Moreover, the correlation between increased FENO levels and a high risk of bronchial hyperresponsiveness has also been demonstrated. FENO is also a relevant biomarker to evaluate asthma status due to the change of its values occurring earlier than clinical manifestations and spirometry parameters. In addition, the measurement of FENO with portable devices helps to support the diagnosis of asthma, to follow-up the control of asthma and to personalize asthmatic patients for target treatment with biologic therapy. Therefore, measuring FENO with portable devices in the diagnosis and treatment of allergic airway in fl ammation, especially in asthma, is one of the most essential applications of NO biomarkers in exhaled breath.

Abstract—Objective: To investigate the cytokine-related molecular cascade leading to neural cell death in periventricular leukomalacia (PVL). Methods: The authors explored potential tumor necrosis factor ␣ (TNF ␣ ) signaling pathways in human brains with PVL and conducted in situ immunohistochemical investigations to search for possible expression of cytokine receptors in these brains. They also investigated likely links to molecules potentially involved in neurocytotoxic- ity, particularly pathways involving nitrosative-induced apoptosis. Results: TNF ␣ overexpression was associated with immune reactivity for p75TNF ␣ R2 and p55TNF ␣ R1 receptors in affected PVL areas. p75TNF ␣ R2 labeling was intense on cerebrovascular endothelial cells in PVL areas, whereas no vascular p55TNF ␣ R1 immunoreactivity was detected therein. Immune labeling for both receptors was detected on many white matter parenchymal cells. In contrast, there was no immune reactivity for either receptor in tissues taken from non-PVL areas. Additionally, in situ overexpression of inducible nitric oxide synthase was found in PVL brain regions where apoptotic cell death was detected. Conclusions: Both p75TNF ␣ R2 and p55TNF ␣ R1 receptors and nitric oxide may be implicated in the pathogenesis of periventricular leukomalacia.