Expression of inducible nitric oxide synthase in human gastric cancer

INTRODUCTIONInduciblenitricoxidesynthase(iNOS)isanenzymethatcatalyzestheformationofnitric0xide(N0)fromL-arginine.iNOSexpressionandactivityresultsintheproduction0fhighlevelsofNO[1].ThegenerationofphysiologicallevelsofNOisimp0rtantformucosalfunctionanditalsoexertsacytoprotectiveeffectonthegastr0intestinalmucosa.However,increasediNOSexpressionhasbeenobservedinpatientswithchronicinflammatorydiseasesofthegastr0intestinaltract,suchasulcerativec0litis[2'3],andgastritis['Jandithasbeenspecul…     

Ventricular assist device therapy normalizes inducible nitric oxide synthase expression and reduces cardiomyocyte apoptosis in the failing human heart

OBJECTIVES: We examined the effect of mechanical unloading with ventricular assist device (VAD) therapy on myocardial inducible nitric oxide synthase (iNOS) expression and cardiomyocyte apoptosis in patients with end-stage heart failure (HF). BACKGROUND: Despite advances in medical therapy, HF continues to be a progressive and ultimately fatal disorder. High levels of iNOS expression are present in the myocardium of failing hearts, suggesting a potential role for iNOS in HF progression. METHODS: Inducible NOS protein expression was analyzed by Western blotting and cardiomyocyte apoptosis by terminal deoxynucleotidyltransferase dUTP nick end-labeling (TUNEL) in myocardial samples from failing hearts. Included in these analyses were tissues from 9 patients at the time of transplantation (HF-transplant group), 10 patients at the time of VAD insertion (pre-VAD group), and 11 patients undergoing transplant after VAD support (post-VAD group). Seven control samples were obtained at autopsy. RESULTS: Low or undetectable levels of iNOS were present in controls (0.005 +/- 0.002). The HF-transplant and pre-VAD myocardial specimens exhibited a marked increase in iNOS expression (1.48 +/- 0.34 and 1.29 +/- 0.26, respectively; p < 0.01 for both vs. controls). The increase in iNOS expression was reversed in the post-VAD group (0.36 +/- 0.16; p < 0.01 vs. HF-transplant and pre-VAD groups). The rate of TUNEL-positive cardiomyocytes was high in the pre-VAD group and significantly lower in the post-VAD group (0.64 +/- 0.15% in pre-VAD group and 0.16 +/- 0.07% in post-VAD group; p < 0.01). The iNOS levels correlated significantly with cardiomyocyte apoptosis (r = 0.66, p < 0.01). CONCLUSIONS: Therapy with VAD normalizes iNOS expression in association with diminished cardiomyocyte apoptosis in the failing heart. Further work is required to define whether a causal relationship exists between iNOS and cardiomyocyte apoptosis.     

Increased expression of constitutive nitric oxide synthase III,but not inducible nitric oxide synthase II,in human heart failure

Objectives. The purpose of the present study was to examine the expression of the endothelial-type nitric oxide synthase (NOS III) and the inducible-type NOS (NOS II) in human myocardium and their regulation in heart failure from patients with different etiologies.Background. In heart failure, plasma levels of nitrates were found to be elevated. However, data on myocardial NOS expression in heart failure are conflicting.Methods. Using RNase protection analysis and Western blotting, the expression of NOS III and NOS II was investigated in ventricular myocardium from nonfailing (NF) hearts (n = 5) and from failing hearts of patients with idiopathic dilated cardiomyopathy (dCMP, n = 14), ischemic cardiomyopathy (iCMP, n = 9) or postmyocarditis cardiomyopathy (mCMP, n = 7). Furthermore, immunohistochemical studies were performed to localize NOS III and NOS II within the ventricular myocardium.Results. In failing human hearts, NOS III mRNA levels were increased to 180% in dCMP, 200% in iCMP and to 210% in mCMP as compared to NF hearts. Similarly, in Western blots (using constitutively expressed beta-tubulin as a reference) NOS III protein expression was increased about twofold in failing compared to NF hearts. Immunohistochemical studies with a selective antibody to NOS III showed no obvious differences in the staining of the endothelium of cardiac blood vessels from NF and failing human hearts. However, NOS III-immunoreactivity in cardiomyocytes was significantly more intense in failing compared to NF hearts. Low expression of NOS II mRNA was detected in only 2 of 30 failing human hearts and was not found in NF hearts. Inducible-type NOS protein was undetectable in either group.Conclusions. We conclude that the increased NOS III expression in the ventricular myocardium of failing human hearts may contribute to the contractile dysfunction observed in heart failure and/or may play a role in morphologic alterations such as hypertrophy and apoptosis of cardiomyocytes.     

Expression of inducible nitric oxide synthase in human coronary atherosclerotic plaque.

OBJECTIVE: Macrophages in atherosclerotic plaque may express the inducible isoform of NO synthase (iNOS), which produces large amounts of NO. On one hand, the production of NO can be protective by its vasodilatory, antiaggregant and antiproliferative effects. On the other hand, the formation of peroxynitrite from NO may favour vasospasm and thrombogenesis. In this study, we investigated whether iNOS is present in human coronary atherosclerotic plaque, and we correlated these data with the clinical instability of the patients. METHODS: Fragments were retrieved by coronary atherectomy from 24 patients with unstable angina and 12 patients with stable angina. The presence of macrophages, and the production of TNF alpha, iNOS and nitrotyrosine were detected by immunocytochemistry. RESULTS: Macrophage clusters were found in 67% of stable patients and 87% of patients with unstable angina (NS). TNF alpha was expressed in about 50% of cases in both groups. iNOS was not expressed in fragments from stable patients but was found in macrophages from 58% of unstable patients (P < 0.001). The expression of iNOS was associated with the presence of nitrotyrosine residues, a marker of peroxynitrite formation. Expression of iNOS was correlated both with complaints of angina at rest (P < 0.05) and with the presence of thrombus at morphological examination (P < 0.001). CONCLUSION: The expression of iNOS may be induced in human coronary atherosclerotic plaque and is associated with different factors of instability.     

Nebivolol induces nitric oxide release in the heart through inducible nitric oxide synthase activation

Nebivolol is a beta1-adrenergic receptor antagonist that also reduces blood pressure by evoking endothelial NO production and vasodilation. We aimed at assessing whether nebivolol induces NO production also in the heart and delineating the molecular mechanisms involved. Using the fluorescent probe diaminofluorescein, we found that nebivolol induces a dose-dependent NO production in the heart, statistically significant already at 10(-7) mol/L. It is not an effect because of the blockade of beta1-adrenergic receptor, because this effect is not shared by another drug of the same class, atenolol. Because nebivolol has been reported to act as an agonist on other beta-adrenergic receptors, we tested NO production in the presence of receptor antagonists. Nebivolol was not able to induce NO production in presence of the beta3-antagonist SR59230A, indicating a fundamental role for beta3-adrenergic receptors in cardiac NO production by nebivolol. Moreover, inducible NO synthase inhibition abolishes NO release in the heart, indicating that nebivolol induces NO production by acting on the inducible isoform of the enzyme. The action of nebivolol on inducible NO synthase was confirmed by real-time PCR experiments, showing cardiac overexpression of inducible NO synthase but not neuronal NO synthase or endothelial NO synthase, after 5 hours of treatment with nebivolol. In conclusion, our study demonstrates that nebivolol also stimulates NO production in the heart. This action of nebivolol is exerted via a signaling pathway starting from the activation of beta3-adrenergic receptors and leading to overexpression of inducible NO synthase. Cardiac NO production by nebivolol could participate in the cardiovascular effects of nebivolol treatment in patients affected by hypertension and heart failure.     

Expression of endothelial nitric oxide synthase and inducible nitric oxide synthase in synovium of rheumatoid arthritis]

OBJECTIVES: To examine the localization and distribution of endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS), which participate in nitric oxide (NO) production, in synovium of rheumatoid arthritis (RA). MATERIALS AND METHODS: Immunohistochemical analysis for eNOS and iNOS in synovial tissues obtained from 10 patients with RA who were underwent total knee replacement. Synovial tissues of osteoarthritis (OA) were used as control. The percentage of cells that were positive for eNOS and iNOS was estimated in five hundred endothelial cells, synovial lining cells and interstitial cells, respectively. And mRNA expression of NOS was confirmed by in situ hybridization. In addition, to test NO production, nitration of tyrosines was assessed by immunohistochemistry. RESULTS: Not only endothelial cells but also synovial lining cells and interstitial cells exhibited immune-reactive both eNOS and iNOS. Cells which were seemed immune-reactive eNOS and iNOS expressed nitrotyrosin. By in situ hybridization, we detected mRNA expression for eNOS and iNOS. CONCLUSIONS: Endothelial cells, synovial lining cells and interstitial cells expressed both eNOS and iNOS with high frequency in RA synovium compared with OA synovium. It seemed to correlate with NO production. These results suggest that expression of iNOS may be involved in the induction of arthritis and eNOS may be participated in augmentation of inflammation in RA.     

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.     

Expression of inducible nitric oxide synthase activity in human colon epithelial cells: modulation by T lymphocyte derived cytokines

Background—Nitricoxide (NO) synthesis and inducible nitric oxide synthase (iNOS)expression are increased in colonic biopsy specimens from patients withulcerative colitis, but the cellular source of NO production is not known.
Aims—To examine thedistribution of iNOS in human colonic mucosa and to explore the abilityof T lymphocyte derived cytokines to regulate iNOS expression andactivity in human colonic epithelial cells.
Methods—iNOSexpression was examined using immunohistochemistry in colonic biopsysamples from 12 patients with ulcerative colitis and three withinfectious colitis and compared with 10normal controls. In vitro iNOSexpression and activity were determined in HT-29 cell cultures; nitritelevels were measured using a fluorescent substrate, iNOS mRNAexpression by northern blot analysis, and iNOS protein expression bywestern blot analysis.
Results—No iNOSexpression was detected (10 of 10) in non-inflamed mucosa derived fromnormal controls. In 11 of 12 cases of newly diagnosed ulcerativecolitis, iNOS protein was expressed in the epithelial cells, while noother positive cells were found in the lamina propria. Similar iNOSlabelling was found in colonic biopsy samples from patients withinfectious colitis in the acute phase, but when re-examined in samplesfrom patients in total remission, no iNOS staining was observed. Bothinterleukin (IL)-13 and IL-4, but not IL-10, are potent inhibitors ofiNOS expression and activity induced by an optimal combination ofcytokines, namely IL-1α, tumour necrosis factor α and interferonγ.
Conclusions—The datasuggest that the epithelium is the major source of iNOS activity inulcerative colitis and that IL-13 and IL-4 may act as intrinsicregulators of NO generation in intestinal inflammation.

Keywords:interleukin 13; nitric oxide; inducible nitric oxidesynthase; colonic epithelial cells; ulcerative colitis

     

食管鳞癌组织诱导型一氧化氮合酶和增殖细胞核抗原的表达及意义

目的探讨食管鳞癌组织诱导型一氧化氮合酶（iNOS）和增殖细胞核抗原（PCNA）的表达与食管癌临床病理因素的关系。方法用免疫组化法检测2009年1至4月山东省泰安市中心医院52例食管鳞癌组织和20例癌旁正常组织iNOS与PCNA的表达,计数PCNA标记指数（PCNA LI）。食管鳞癌组织及癌旁正常组织iNOS蛋白阳性表达率、PCNA LI比较分别采用χ2检验、t检验;食管癌组织iNOS阳性表达者与iNOS阴性表达者PCNA LI比较采用t检验;不同临床病理因素食管鳞癌组织iNOS蛋白表达、PCNA LI比较分别采用χ^2检验、t检验、方差分析,进一步两两比较采用SNK-q检验;食管鳞癌组织iNOS蛋白与PCNA蛋白表达的关联性分析采用χ^2检验。结果食管鳞癌组织iNOS阳性表达率、PCNA LI均高于癌旁正常组织,且差异均有统计学意义[63.5%（33/52）与10.0%（2/20）比较,χ^2=14.455,P〈0.01;（53.29±14.55）与（2.65±1.82）比较,t=24.593,P〈0.05]。食管癌组织中,iNOS阳性表达者的PCNA LI高于iNOS阴性表达者,且差异有统计学意义[（60.89±9.98）与（40.08±11.53）比较,t=6.842,P=0.000]。不同临床分期、分化程度、有无淋巴结转移患者的食管鳞癌组织iNOS蛋白表达差异有统计学意义（χ^2=9.372,P=0.025;χ^2=12.784,P=0.002;χ2=6.361,P=0.012）。随着食管鳞癌组织癌细胞分化程度的降低PCNA LI升高,中分化和低分化的食管鳞癌组织PCNA LI明显高于高分化的食管鳞癌（q=6.000、7.378,P均〈0.05）。有淋巴结转移的食管鳞癌组织PCNA LI高于无淋巴结转移的食管鳞癌组织,且差异有统计学意义[（56.26±13.14）与（45.21±15.62）比较,t=2.556,P=0.014]。iNOS的表达与PCNA的表达有关联（χ^2=20.022,P=0.000）。结论 iNOS与PCNA蛋白在食管鳞癌组织中均有较高的阳性表达,且表达有关联。两者可能存在共同的激活机制,iNOS和PCNA蛋白表达与食管癌的恶性进程有关。     

原发性肝癌一氧化氮及一氧化氮合酶的研究

探讨一氧化氮 (Nitricoxide,NO)及诱导型一氧化氮合酶 (induciblenitricoxidesynthase ,iNOS)与原发性肝癌 (HCC)间的关系 ,用Griess反应测定 16 2例患者的血浆亚硝酸盐 /硝酸盐 (NO-2 /NO-3 )水平 ,其中HCC82例 ,非HCC80例 ,健康对照 36名。用免疫组化法检查组织中iNOS的含量 ,取正常肝脏组织 2 0例作对照 ,慢性肝炎 (CH)和肝硬化 (LC)的肝脏组织各 40例 ,HCC组织 48例。结果显示 ,正常人血浆NO-2 /NO-3 含量为 16 .8±4.9μmol/L,有HCC的CH(6 3 .4± 18.2 μmol)和LC(42 .2± 11.5 μmol/L)明显高于非HCC的患者 (CH :2 8.5±8.7μmol/L;LC :2 4.7± 6 .2 μmol/L .P <0 .0 1) ,患CH的HCC患者血浆NO-2 /NO-3 水平明显高于LC基础上的HCC患者 (P <0 .0 5 )。正常肝组织iNOS阴性 ,LC有 2 5例 (6 2 .5 % )阳性 ,CH 36例 (90 % )阳性 ,HCC 46例 (95 8% )阳性 ;且CH (P <0 .0 2 5 )及HCC(P <0 .0 0 1)的表达水平明显高于LC。提示HCC患者有NO分泌的增加 ,NO可能参与HCC的发生发展的病理过程     

N-Acetyl-cysteine modulates inducible nitric oxide synthase gene expression in human hepatocytes

BACKGROUND/AIMS: A major role has been described for inducible nitric oxide (NO) synthase in several chronic inflammatory liver diseases. N-Acetyl-cysteine (NAC) is a sulfhydryl donor molecule with antioxidant and antiinflammatory effects. It attenuates NO generation following lipopolysaccharide injection in rats. Our goal was to study the effect of NAC on NO synthase induction in hepatocytes in response to proinflammatory cytokines. METHODS: The effect of NAC on NO synthase induction was studied in the human hepatocyte cell lines HepG2 and 2.2.15 treated with a mixture of proinflammatory cytokines. Interactions between NAC and cytokines on nuclear factor-kappaB (NF-kappaB) activation and NO synthase promoter transactivation were investigated. RESULTS: NAC dose-dependently modulated the induction of NO synthase mRNA expression, the release of nitrites and the formation of NF-kappaB binding complexes in cytokine-treated hepatocytes. NAC also reduced the transactivation of the NO synthase promoter. CONCLUSIONS: Our data show that exposure of hepatocytes to NAC modulated NO synthase expression and NF-kappaB activity, the key responses of the hepatocyte to inflammatory mediators. These data constitute preliminary evidence that NAC might have hepatoprotective actions of potential relevance in chronic inflammatory liver diseases, mediated partially through the modulation of NO production.     

Increased neuronal nitric oxide synthase-derived NO production in the failing human heart

Experimental data suggest that nitric oxide (NO) generated from neuronal NO synthase (nNOS) modulates the myocardial inotropic state. To assess the contribution of NO, derived from endothelial and neuronal isoforms, to the pathophysiology of congestive heart failure in human beings, we compared expression, localisation, and specific activity of NOS isoforms in myocardium from patients with dilated cardiomyopathy with those in controls who had died from head trauma or intracranial bleeds. Diseased hearts had a significant increase in nNOS mRNA and protein expression, and activity associated with the translocation of nNOS to the sarcolemma through interactions with caveolin 3. Enhanced nNOS activity counteracted a decrease in eNOS expression and activity. Our results provide evidence of increased nNOS-derived NO in the failing human heart. Such altered regulation may be important in the pathophysiology of cardiac dysfunction in human congestive heart failure.     

Discrepant effects of inducible nitric oxide synthase modulation on systemic and splanchnic endothelial nitric oxide synthase activity and expression in cirrhotic rats

BACKGROUND: Arterial vasodilatation, which is a major factor in the pathogenesis of the hyperkinetic circulatory state and portal hypertension in cirrhosis, is due to arterial nitric oxide (NO) overproduction secondary to endothelial NO synthase (eNOS) and inducible NOS (iNOS) upregulation. However, in cirrhosis, the respective roles of eNOS and iNOS isoforms in NO overproduction are still unknown and the effect of iNOS modulation on eNOS activity and expression has not been evaluated in the systemic or splanchnic vessels. The aim of this study was to evaluate the effects of modulating aortic and superior mesenteric arteries (SMA) iNOS on arterial eNOS activity and expression in rats with cirrhosis. METHODS: eNOS and iNOS protein expression and eNOS activity (assessed by its phosphorylation at serine 1177) were measured in the aortas and SMA in untreated and treated cirrhotic rats with lipopolysaccharide (LPS), N-iminoethyl-L-lysine (L-NIL), a selective iNOS inhibitor, and LPS plus L-NIL. RESULTS: LPS administration significantly increased eNOS and iNOS protein expression and eNOS activity in the aortas of both sham-operated and cirrhotic rats. However, in SMA, LPS administration induced a decrease in eNOS protein expression and activity and an increase in iNOS protein expression. CONCLUSION: The results of this study may explain the worsening of the hyperdynamic state in cirrhosis during septic shock by direct LPS-induced eNOS activation in large systemic vessels, and its inhibition in concomitant small splanchnic vasculature by iNOS synthesized NO.     

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.     

人胃癌组织中一氧化氮合酶的表达

目的探讨NOS与胃癌的关系.方法用NADPH-d组织化学法测定了正常胃组织、癌旁组织和癌组织中一氧化氮合酶(NOS)表达水平.结果正常胃组织中粘膜上皮细胞、各种有分泌功能的细胞及肌层神经纤维中均有NOS表达,测一个视野NOS阳性细胞的平均灰度,正常胃组织为112、癌旁组织为120、胃癌组织为145.各组间差异有显著意义.表明正常胃组织NOS活性最高,胃癌组织NOS活性最低.结论①正常胃组织有广泛的NOS分布,提示NO对维持正常胃功能具有重要作用;②胃粘膜细胞癌变过程中,NOS活性明显降低,提示NOS活性与胃粘膜细胞癌变有高度相关性.