Inducible nitric oxide synthase gene promoter polymorphism is associated with increased gastric mRNA expression of inducible nitric oxide synthase and increased risk of gastric carcinoma

BACKGROUND AND AIMS: Stimulation of inducible nitric oxide synthase gene expression by Helicobacter pylori, with subsequent overproduction of nitric oxide, has been implicated in gastric carcinogenesis. We investigated whether inducible nitric oxide synthase promoter gene polymorphisms are associated with (a) inducible nitric oxide synthase mRNA expression in the gastric mucosa, and (b) the risk of gastric carcinoma. MATERIALS AND METHODS: The relationship between gastric inducible nitric oxide synthase mRNA expression and inducible nitric oxide synthase promoter polymorphisms (CCTTT repeat polymorphism and -2445 C-->G SNP) was examined in 74 H. pylori-infected patients with gastric cancer, peptic ulcer, or functional dyspepsia. In a case-control study the prevalence of the polymorphisms was examined in H. pylori-infected gastric carcinomas (n=77) and noncancerous controls (n=154). RESULTS: Inducible nitric oxide synthase mRNA levels were significantly higher in long CCTTT repeat (either allele>11) carriers than in short ones (P=0.015). Multivariate regression analysis showed that inducible nitric oxide synthase mRNA expression was significantly linked to long CCTTT repeat and gastric cancer (P=0.026), but not to -2445 C-->G SNP and other parameters. The case-control study showed that long CCTTT repeat carriers had an increased risk of gastric cancer with an odds ratio of 2.0 (P=0.021). -2445 C-->G SNP was not associated with the risk. CONCLUSIONS: Helicobacter pylori induces higher inducible nitric oxide synthase mRNA expression in carriers of long CCTTT repeats of inducible nitric oxide synthase promoter, and this polymorphism is associated with an increased risk of gastric carcinoma.     

Nitrotyrosine,inducible nitric oxide synthase (iNOS), and endothelial nitric oxide synthase (eNOS) are increased in thyroid tumors from children and adolescents

Nitric oxide (NO) is a reactive cell signal that controls vascular tone and is generated by inducible (iNOS), endothelial (eNOS) and neuronal (nNOS) NO synthase (NOS). We hypothesized that NO could be important for growth of thyroid tumors and tested this hypothesis, by staining 41 papillary thyroid carcinoma (PTC), 9 follicular thyroid carcinoma (FTC), and 15 benign thyroid lesions for iNOS, eNOS and nitrotyrosine (N-TYR). Staining intensity was determined by 2 blinded, independent examiners, and quantified from grade 1 (absent) to grade 4 (intense). Average N-TYR staining of benign adenomas (2.5+/-0.42, p=0.009), PTC (3.10+/-0.12, p=0.001), FTC (2.44+/-0.30, p=0.001), and autoimmune lesions (3.25+/-0.48, p=0.019) were greater than that of multinodular goiter (1.0 for all 3) and surrounding normal thyroid (1.1+/-0.1). Average iNOS staining of benign adenomas (2.6+/-0.37), PTC (2.7+/-0.16), FTC (2.4+/-0.26) and autoimmune lesions (3.5+/-0.29) were all greater than that of surrounding normal thyroid (1.1+/-0.1, p<0.008), but there were too few multinodular goiters to achieve a significant difference (no.=2, 3.0+/-1.0). Average eNOS staining of benign adenomas (2.9+/-0.40), multinodular goiters (3.5+/-0.5), PTC (3.24+/-0.18), FTC (3.5+/-0.50), and autoimmune lesions (2.8+/-0.6) were also greater than that of surrounding normal thyroid (mean=1.4+/-0.2, p<0.001). N-TYR staining correlated with that of vascular endothelial growth factor (VEGF, r=0.36, p=0.007) and the number of lymphocytes/high power field (r=0.39, p=0.004). Recurrent disease developed only from carcinoma with moderate-intense N-TYR staining, but there were too few recurrent tumors to achieve statistical significance (p=0.08). We conclude that NO is produced by benign adenomas, PTC and FTC suggesting that NO could be important in vascularization of thyroid tumors and autoimmune thyroid diseases.     

Luminal nitric oxide and epithelial expression of inducible and endothelial nitric oxide synthase in collagenous and lymphocytic colitis

BACKGROUND: Colonic nitric oxide (NO) production in collagenous colitis (CC) has been studied in a small number of patients and found increased. The cellular source of NO is believed to be the colonic epithelial cells. The aim of this study was to investigate colonic NO levels in patients with CC and lymphocytic colitis (LC), to compare with the histopathological status and with the clinical activity, and to assess the epithelial expression of inducible and endothelial nitric oxide synthase (iNOS and eNOS). METHODS: We included 19 patients with CC, 8 patients with LC and 15 controls. During colonoscopy, luminal gas was sampled and NO levels were measured using the chemiluminescence technique. Mucosal biopsies were obtained for routine histopathologic examination and immunohistochemical studies of iNOS and eNOS. Clinical activity, as measured by the mean frequency of daily bowel movements during the week prior to colonoscopy, was assessed. RESULTS: Luminal NO levels, median (25-75 percentiles), in the patients with CC and LC were greatly increased compared to the controls, 1673 (145-8143) parts per billion (ppb) and 1838 (1065-2694) ppb versus 28 (20-46) ppb (P < 0.005, both). A positive association was seen between NO levels and histopathological status as well as clinical activity. Strong expression of iNOS was seen in the surface epithelium in 5 of 6 patients with CC and in 2 of 5 patients with LC. CONCLUSIONS: The fact that luminal NO levels are related to histopathological status and correlate with clinical activity indicates that NO is involved in the pathophysiology of CC and LC. The epithelial cells are the most likely source of luminal NO.     

Fractional exhaled nitric oxide concentration is increased in asbestosis and pleural plaques

OBJECTIVE AND BACKGROUND: Asbestos exposure induces generation of reactive oxygen and nitrogen species. Nitric oxide is involved in asbestos-related lung toxicity in vitro and can be measured non-invasively in humans in exhaled breath. The authors hypothesized that fractional exhaled nitric oxide concentration (FENO) would be increased in subjects with asbestos-related lung disorders. METHODS: FENO was measured in 56 subjects with asbestos-related disorders (asbestosis: 12; pleural plaques: 32; asbestos-related diffuse pleural thickening: 12) and in 35 normal subjects. The authors also measured exhaled carbon monoxide, another marker of lung inflammation. RESULTS: Median (25-75 percentile) FENO was increased in subjects with asbestosis (7.9 (6.6-15.7) p.p.b.; P=0.001) and pleural plaques (6.3 (5.3-9) p.p.b.; P=0.03) compared with normal controls (4.6 (3.5-6) p.p.b.). Subjects with DPT had a median FENO of 5.6 p.p.b., similar to controls. No significant differences in exhaled carbon monoxide were observed between controls (1.0+/-0.3 p.p.m.) and subjects with asbestosis (1.3+/-0.3 p.p.m.), pleural plaques (1.2+/-0.3 p.p.m.) or diffuse pleural thickening (1.1+/-0.3 p.p.m.). CONCLUSIONS: FENO is raised in asbestosis consistent with lung inflammation, and also in pleural plaques.     

In stable lung transplant recipients, exhaled nitric oxide levels positively correlate with airway neutrophilia and bronchial epithelial iNOS

In conditions characterized by airway inflammation, exhaled nitric oxide (eNO) levels are increased. Variable degrees of airway inflammation are present in stable lung transplant recipients (LTR), and may lead to airway remodeling and chronic graft dysfunction. The hypothesis tested is that in stable LTR, eNO concentrations would reflect the expression of inducible (iNOS) (but not constitutive [cNOS] nitric oxide synthase) in the bronchial epithelium as well as the degree of airway inflammation. We determined eNO concentrations in 20 stable LTR, free of infection, rejection, or obliterative bronchiolitis (OB). At routine bronchoscopy, we measured the differential cell count on bronchoalveolar lavage (BAL) and a quantitative assessment of iNOS and cNOS expression in endobronchial biopsies by immunohistochemistry. Mean +/- SEM eNO concentrations in stable LTR were not significantly different from control subjects (13 +/- 0.7 ppb versus 14.2 +/- 0.49; p = 0.42). Percent BAL neutrophils was 11.5 +/- 3.2 which was significantly higher than in a group of local control subjects (1.7 +/- 0.6; p < 0.001). The bronchial epithelium and lamina propria contained abundant iNOS but cNOS was present only in the lamina propria. Using regression analysis, percent BAL neutrophils (r(2) = 0.82; p < 0.0001) and iNOS expression in the bronchial epithelium (r(2) = 0.75; p < 0.0001), but not in the lamina propria (r(2) = 0.16; p = 0.08), were positively predictive of eNO. There was an inverse relationship between cNOS and eNO. We conclude that eNO concentrations although normal for the group, still reflect the degree of airway inflammation in stable LTR. Epithelial iNOS appears to be the major source of eNO and expression of cNOS may be downregulated with increasing iNOS expression.     

Ubiquitination of inducible nitric oxide synthase is required for its degradation

Inducible nitric oxide synthase (iNOS) is responsible for nitric oxide (NO) synthesis from l-arginine in response to inflammatory mediators. We have previously shown that iNOS is degraded through the 26S proteasome. Targeting of proteins for proteasomal degradation may or may not require their covalent linkage to multiubiquitin chains (ubiquitination). In addition, ubiquitination of a protein can serve functions other than signaling proteolysis. In this context, it is not known whether iNOS is subject to ubiquitination or whether ubiquitination is required for its degradation. In this study, we show that iNOS, expressed in HEK293 cells or induced in primary bronchial epithelial cells, A549 cells, or murine macrophages, is subject to ubiquitination. To investigate whether iNOS ubiquitination is required for its degradation, HEK293T cells were cotransfected with plasmids containing cDNAs of human iNOS and of the dominant negative ubiquitin mutant K48R. Disruption of ubiquitination by K48R ubiquitin resulted in inhibition of iNOS degradation. ts20 is a mutant cell line that contains a thermolabile ubiquitin-activating enzyme (E1) that is inactivated at elevated temperature, preventing ubiquitination. Incubation of ts20 cells, stably expressing human iNOS, at the nonpermissive temperature (40 degrees C) resulted in inhibition of iNOS degradation and marked accumulation of iNOS. These studies indicate that iNOS is subject to ubiquitination and that ubiquitination is required for its degradation.     

Role of neuronal nitric oxide synthase and inducible nitric oxide synthase in intestinal injury in neonatal rats

AIM: To investigate the dynamic change and role of neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) in neonatal rat with intestinal injury and to define whether necrotizing enterocolitis (NEC) is associated with the levels of nitric oxide synthase (NOS) in the mucosa of the affected intestine tissue. METHODS: Wistar rats less than 24 h in age received an intraperitoneal injection with 5 mg/kg lipopolysaccharide (IPS). Ileum tissues were collected at 1, 3, 6, 12 and 24 h following LPS challenge for histological evaluation of NEC and for measurements of nNOS and iNOS. The correlation between the degree of intestinal injury and levels of NOS was determined. RESULTS: The LPS-injected pups showed a significant increase in injury scores versus the control. The expression of nNOS protein and mRNA was diminished after LPS injection. There was a negative significant correlation between the nNOS protein and the grade of median intestinal injury within 24 h. The expression of iNOS protein and mRNA was significantly increased in the peak of intestinal injury. CONCLUSION: nNOS and iNOS play different roles in LPS-induced intestinal injury. Caution should be exerted concerning potential therapeutic uses of NOS inhibitors in NEC.     

Course of FEV(1) after onset of bronchiolitis obliterans syndrome in lung transplant recipients

RATIONALE: Bronchiolitis obliterans syndrome (BOS), defined by loss of lung function, develops in the majority of lung transplant recipients. However, there is a paucity of information on the subsequent course of lung function in these patients. OBJECTIVES: To characterize the course of FEV(1) over time after development of BOS and to determine the predictors that influence the rate of functional decline of FEV(1). METHODS: FEV(1)% predicted (FEV(1)%pred) trajectories were studied in 111 lung transplant recipients with BOS by multivariate, linear, mixed-effects statistical models. MEASUREMENTS AND MAIN RESULTS: FEV(1)%pred varied over time after BOS onset, with the steepest decline typically seen in the first 6 months (12% decline; p < 0.0001). Bilateral lung transplant recipients had significantly higher FEV(1)%pred at BOS diagnosis (71 vs. 47%; p < 0.0001) and at 24 months after BOS onset (58 vs. 41%; p = 0.0001). Female gender and pretransplant diagnosis of idiopathic pulmonary fibrosis were associated with a steeper decline in FEV(1)%pred in the first 6 months after BOS diagnosis (p = 0.02 and 0.04, respectively). A fall in FEV(1) greater than 20% in the 6 months preceding BOS (termed "rapid onset") was associated with shorter time to BOS onset (p = 0.01), lower FEV(1)%pred at BOS onset (p < 0.0001), steeper decline in the first 6 months (p = 0.03), and lower FEV(1)%pred at 2 years after onset (p = 0.0002). CONCLUSIONS: Rapid onset of BOS, female gender, pretransplant diagnosis of idiopathic pulmonary fibrosis, and single-lung transplantation are associated with worse pulmonary function after BOS onset.     

Delayed cardioprotection afforded by nitroglycerin is mediated by alpha-CGRP via activation of inducible nitric oxide synthase

Previous investigations have demonstrated that delayed preconditioning induced by nitroglycerin is mediated by endogenous calcitonin gene-related peptide (CGRP). In the present study, we examined whether CGRP-mediated delayed preconditioning induced by nitroglycerin is involved in activation of inducible nitric oxide synthase (iNOS). Male Wistar rats were pretreated with nitroglycerin 24 h before the experiment, and then the left main coronary artery of rat heart was subjected to 60-min occlusion followed by 3 h reperfusion. Infarct size, the plasma level of cGMP and CGRP, and expression of CGRP isoforms (alpha-CGRP and beta-CGRP) mRNA in lumbar dorsal root ganglia were measured. Pretreatment with nitroglycerin (120 microg/kg, i.v.) markedly reduced infarct size. Nitroglycerin caused a significant increase in the expression of alpha-CGRP mRNA, but not beta-CGRP mRNA, concomitant with an increase in plasma concentrations of cGMP and CGRP. These effects of nitroglycerin were completely abolished by pretreatment with aminoguanidine (300 mg/kg, i.p.), a selective inhibitor of iNOS activity, or dexamethasone (5 mg/kg, i.p.), the iNOS expression inhibitor. The present results suggest that delayed cardioprotection afforded by nitroglycerin is mediated by the alpha-CGRP isoform via generation of NO derived from iNOS.     

Myeloperoxidase up-regulates the catalytic activity of inducible nitric oxide synthase by preventing nitric oxide feedback inhibition

Kinetic and structure analysis of inducible nitric oxide synthase (iNOS) revealed that, in addition to the increase of iNOS expression in inflamed areas, the major pathway causing overproduction of NO is destabilization of the iNOS-nitrosyl complex(es) that form during steady-state catalysis. Formation of such a complex allows iNOS to operate at only a fraction (20-30%) of its maximum activity. Thus, bioavailability of NO scavengers at sites of inflammation may play an essential role in up-regulation of the catalytic activity of iNOS, by preventing the catalytic activity inhibition that is attributed to nitrosyl complex formation. Myeloperoxidase (MPO), a major NO scavenger, is a pivotal enzyme involved in leukocyte-mediated host defenses. It is thought to play a pathogenic role under circumstances such as acute inflammatory tissue injury and chronic inflammatory conditions. However, a detailed understanding of the interrelationship between iNOS and MPO at sites of inflammation is lacking. We used direct spectroscopic, HPLC, and selective NO-electrode measurements to determine the interdependent relationship that exists between iNOS and MPO and the role of the MPO/H2O2 system in up-regulating the catalytic activity of iNOS that occurs at sites of inflammation. Scavenging free NO from the iNOS milieu by the MPO/H2O2 system subsequently restores the full capacity of iNOS to convert L-arginine to product (NO), as judged by the increase in the rates of citrulline and nitrite/nitrate production. Studies of iNOS catalytic mechanisms and function are essential to a more fundamental understanding of these factors, which govern iNOS-dependent processes in human health and disease.     

Raised exhaled nitric oxide in healthy children is associated with domestic formaldehyde levels

Exposure to domestic levels of formaldehyde has been associated with adverse respiratory symptoms in both adults and children. The underlying mechanisms responsible for these findings have not been established. In order to investigate possible inflammatory effects of formaldehyde at levels typically found in the home, we measured exhaled nitric oxide (eNO) in 224 healthy children 6 to 13 yr of age (116 girls) and monitored formaldehyde levels in their homes. Formaldehyde was monitored using a passive sampling technique. Exhaled NO was measured directly into a fast response chemiluminescence nitric oxide analyzer. The children also undertook a lung function (spirometry) test. There was no effect of formaldehyde levels measured in homes on spirometric variables. However, eNO levels were significantly elevated in children living in homes with average formaldehyde levels >/= 50 ppb. Exhaled NO levels (geometric mean) were 15.5 ppb (95% CI: 10.5 to 22.9 ppb) for children from homes with formaldehyde concentrations >/= 50 ppb compared with 8.7 ppb (7.9 to 9.6) for children from homes with formaldehyde concentrations < 50 ppb (p < 0.05). These results suggest that exposure to formaldehyde in homes may invoke a subclinical inflammatory response in the airways of healthy children.     

Interleukin-1beta signals through a c-Jun N-terminal kinase-dependent inducible nitric oxide synthase and nitric oxide production pathway in Sertoli epithelial cells

Our recent Sertoli cell (SC) studies showed that the c-Jun N-terminal kinase (JNK) and inducible cyclooxygenase-2 (COX-2) pathways are key regulatory components of IL (IL-1alpha, IL-1beta, and IL-6) expression and START-domain containing StARD1 and StARD5 proteins. IL-1beta regulates SC autocrine/paracrine activities and subsequently influences developing germ cells and spermatogenesis. This study was designed to evaluate whether IL-1beta mediates high-output inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production in these specialized epithelial cells and characterize gonadotropin and cytokine-regulation of NO. Purified SCs were maintained in serum-free cultures and treated with FSH (100 ng-1 microg/ml) or IL-1beta (10 ng/ml) in time-course studies. To determine obligatory intracellular pathways, treatments were conducted with or without activity inhibitors: COX-2 selective (NS-398, 10 microM) or JNK (SP600125, 10 microM) for 1, 3, 6, and 24 h. NOS mRNAs and proteins were evaluated by RT-PCR and Western analysis, respectively. NO and reactive oxygen species were measured by flow cytometry and ELISA. IL-1beta transiently induces intracellular NO (30 min) but not reactive oxygen species. Subsequently, iNOS mRNA and protein expression (3-6 h) significantly increased after IL-1beta but not FSH stimulation, and in time-dependent manner, markedly increased extracellular NO (24 h, 8-fold). No change in the constitutive endothelial NOS isoform was observed. Inhibition of JNK, but not COX-2, activity inhibits IL-1beta-induced iNOS expression and NO production. Such findings suggest that intra- and extracellular NO within the tubule may alert SCs monitoring the microenvironment to an aberrant cytokine, triggering antioxidant and antiinflammatory activities to avoid disruption of spermatogenesis.     

Reduction of myocardial infarct size by inhibition of inducible nitric oxide synthase

The inducible nitric oxide synthase isoform (iNOS) is upregulated by cytokines and endotoxins in many types of cells, including cardiac myocytes. Nitric oxide (NO) induced by cytokines can be cytotoxic, and has been implicated in the pathophysiology of myocardial infarction, cardiomyopathy, and septic shock. To examine the role of iNOS in the ischemic myocardium, we studied: 1) the time course of expression of iNOS mRNA after myocardial infarction (MI) in male Sprague-Dawley rat hearts and expression of iNOS protein in the infarcted region; 2) whether hypoxia in vitro is a potential mediator of the induction of iNOS mRNA; and 3) whether inhibition of iNOS by two different selective inhibitors (aminoguanidine and S-methylisothiourea sulfate) in vivo influences infarct size. Myocardial infarction was induced by ligation of the left anterior descending coronary artery (LAD), and tissue was collected at selected times thereafter from both ligated and sham-operated rats. iNOS mRNA was induced in the infarcted region of the left ventricle for 7 days; iNOS protein was also detected in the infarcted area. We next tested whether hypoxia would induce iNOS in vitro. In cultured neonatal ventricular myocytes, iNOS mRNA was slightly induced by 6 to 24 h of hypoxia; however, iNOS protein was only detected when the cytokine interleukin-1β was present. To study whether iNOS activity contributed to myocardial damage (eg, infarct size), we administered the first dose of the NOS inhibitors 24 h before LAD occlusion and then a second dose after surgery. Inhibition of iNOS activity with aminoguanidine reduced infarct size by 20% but had no effect on infiltration by neutrophils, whereas the more selective inhibitor S-methylisothiourea sulfate reduced infarct size by 41%. These data suggest that NO derived from the iNOS isoform contributes to some of the myocardial injury following MI, possibly by causing myocardial cell death in areas bordering the ischemic region of the heart.