O envolvimento do estresse oxidativo e nitrosativo em modelo de doença pulmonar obstrutiva crônica (DPOC) induzido por elastase / The involvement of oxidative and nitrosative stress in model of chronic obstructive pulmonary disease (COPD) induced by elastase

Este estudo buscou investigar o papel do estresse oxidativo e nitrosativo no enfisema pulmonar induzido por elastase. Foram utilizados camundongos machos C57BL/6 submetidos a dois modelos de indução do enfisema por elastase pancreática suína (PPE): intratraqueal (i.t.) e intranasal (i.n.). No modelo intratraqueal a PPE foi instilada nas doses de 0,05 U ou 0,05 U/camundongo para avaliação temporal do enfisema 7, 14 e 21 dias após instilação de PPE. Em outra etapa, o papel da iNOS foi avaliado através da sua inibição farmacológica por aminoguanidina (AMG) 1% na água de beber ou pela sua exclusão genética em camundongos deficientes em iNOS que tiveram o enfisema induzido por 0,5 U PPE i.t. após 21 dias. No modelo intranasal a dose de PPE foi 3 U/camundongo para avaliação temporal do enfisema (1, 7, 14 e 21 dias após PPE). O papel do estresse oxidativo e nitrosativo foi avaliado com diferentes tratamentos antioxidantes na água de beber: tempol, apocinina+alopurinol, n-acetilcisteína, vitamina C+E, e aminoguanidina durante os 21 dias de indução do enfisema. Os grupos controles foram submetidos à instilação de salina. Lavado broncoalveolar, imunoensaios, análises bioquímicas de estresse oxidativo e ensaios morfométricos foram realizados nos pulmões dos animais. O enfisema foi histologicamente alcançado em 21 dias após 0,5 U PPE i.t., evidenciado pelo aumento do diâmetro alveolar médio - Lm e da densidade de volume dos espaços alveolares - Vvair em comparação ao grupo controle. TNF-a foi aumentado em 7 e 14 dias após 0,05 U PPE comparados ao controle, concomitante com a redução de IL-10 nos mesmos períodos, comparados ao controle. O estresse oxidativo foi observado na fase inicial do enfisema, com aumento dos níveis de nitrito, TBARS e superóxido dismutase no grupo 7 dias após 0,5 U PPE (i.t.) quando comparados ao controle ao passo que no modelo intranasal as alterações típicas do estresse foram vistas no grupo 1 dia após 3 U de PPE. Atividade da glutationa ...

This study investigated the role of oxidative and nitrosative stress in elastase-induced pulmonary emphysema. C57BL/6 male mice were used submitted to two models of emphysema induced by porcine pancreatic elastase (PPE): intratracheal (i.t.) and intranasal (i.n.). In the intratracheal model PPE was instilled at doses of 0.05 U or 0.5 U/mouse (i.t.) to temporal evaluation of emphysema 7, 14 and 21 days post-PPE instillation. Others sets of experiments, the role of iNOS was evaluated through its pharmacology inhibition by 1% aminoguanidine (AMG) into the drinking water or bt iNOS genetic exclusion in iNOS-deficient mice which had induced emphysema by 0.5 U PPE i.t. after 21 days. In the intranasal model the PPE dose was 3 U/mouse to temporal evaluation of emphysema (1, 7, 14 and 21 days after PPE). The role of oxidative and nitrosative stress was evaluated using different antioxidant treatments into the drinking water: tempol, apocynin+allopurinol, N-acetylcysteine, vitamin C+E and aminoguanidine during the 21 days of emphysema induction. Control groups were instilled with saline. Bronchoalveolar lavage, immunoassays, biochemical analysis of oxidative stress and morphometric tests were performed in the lungs of animals. The emphysema was histologically reached 21 days after 0.5 U PPE, as evidenced by an increase in alveolar diameter - Lm and volume density of the alveolar spaces - Vvair compared to the control group. TNF-a was increased in 7 and 14 days after 0.5 U PPE compared to the control, concomitant with reduction of IL-10 at the same time-points compared to the control. Oxidative stress was observed in the early stages of emphysema, with increased levels of nitrite, TBARS and superoxide dismutase in group 7 days after 0.5 U PPE (i.t.) compared to the control, while in the intranasal model the typical stress alterations were seen in group 1 day after 3 U PPE. Glutathione peroxidase activity was increased in all PPE groups (i.t.). Exposure to 0.5 U PPE ...

Produção de óxido nítrico no hipocampo: investigação do envolvimento do óxido nítrico sintase neuronal no desenvolvimento de comportamentos ansiosos em animais submetidos a estresse neonatal / Hippocampal nitric oxide production: investigation of the role of neuronal nitric oxide synthase in the development of anxiety-like behaviors in animals subjected to neonatal stress

Introdução: Estudos indicam que o trauma precoce e o sistema serotoninérgico estão relacionados ao desenvolvimento de ansiedade. Esta relação poderia ser mediada pela enzima óxido nítrico sintase neuronal (nNOS), que tem papel importante no funcionamento dos receptores de serotonina. Objetivo: Investigar, através da mensuração do óxido nítrico (NO) no hipocampo, o possível envolvimento da nNOS no desenvolvimento de ansiedade em um modelo animal de adversidade no início da vida, baseado na qualidade do cuidado materno. Métodos: Ao segundo dia de vida, genitoras Wistar e suas ninhadas foram divididas em dois grupos: intervenção, com redução do material para a confecção do ninho, ou controle. O comportamento materno foi observado do dia 1 ao dia 9 de vida. Na vida adulta, realizaram-se testes comportamentais e determinaram-se os níveis hipocampais de NO, através da mensuração de seus produtos de degradação. Resultados: Observou-se um maior comportamento do tipo ansioso no grupo intervenção, cujas genitoras apresentaram maior contato de baixa qualidade com seus filhotes. Nos machos, o cuidado materno de baixa qualidade correlacionou-se negativamente com o tempo no braço aberto e a frequência de mergulhos (r=-0,4;p=0,03) avaliados no labirinto em cruz elevado. O comportamento materno de lamber os filhotes correlacionou-se com a frequência de mergulhos em ambos os sexos (r=0,5;p<0,001). A quantidade de NO no hipocampo não diferiu entre os grupos. Conclusão: Uma maior atividade da nNOS não parece estar envolvida no comportamento ansioso observado neste modelo animal, no entanto a relação mãe-filhote, alterada por um ambiente neonatal adverso, teve impacto sobre o comportamento ansioso de forma sexo específica.

Background: Studies have shown that an adverse early life environment and the serotonergic system are related to the development of anxiety. This association could be mediated by neuronal nitric oxide synthase (nNOS), an enzyme that plays an important role in serotonin receptor functioning. Aim: To investigate the possible role of nNOS in the development of anxiety by measuring hippocampal nitric oxide (NO) in an animal model of neonatal stress, based on the quality of maternal care. Methods: On the second day of life, Wistar dams and their litters where divided in two groups: intervention, with limited access to nesting material, or control. Maternal behavior was observed from day 1 to 9 of life. In adult life, behavioral tests were performed and hippocampal NO levels were determined by measuring its degradation products. Results: There was more frequent anxiety-like behavior in the intervention group, whose dams showed low quality contact with their pups more often. In males, low quality maternal care was negatively correlated with time spent in open arms and frequency of head dips (r=-0.4; p=0.03) assessed using an elevated plus maze. Licking and grooming score was correlated with frequency of head dips in both sexes (r=0.5;p<0.001). Hippocampal NO levels were not different between groups (p=0.992). Conclusion: A higher nNOS activity does not seem to be involved in anxiety-like behavior observed in this animal model, however the relationship between dam and pup, modified by an adverse early life environment, had a different impact on anxiety behavior between sexes.

Purine nucleotides reduce superoxide production by nitric oxide synthase in a murine sepsis model

Sepsis involves a systemic inflammatory response of multiple endogenous mediators, resulting in many of the injurious and sometimes fatal physiological symptoms of the disease. This systemic activation leads to a compromised vascular response and endothelial dysfunction. Purine nucleotides interact with purinoceptors and initiate a variety of physiological processes that play an important role in maintaining cardiovascular function. The purpose of the present study was to investigate the effects of ATP on vascular function in a lipopolysaccharide (LPS) model of sepsis. LPS induced a significant increase in aortic superoxide production 16 h after injection. Addition of ATP to the organ bath incubation solution reduced superoxide production by the aortas of endotoxemic animals. Reactive Blue, an antagonist of the P2Y receptor, blocked the effect of ATP on superoxide production, and the nonselective P2Y agonist MeSATP inhibited superoxide production. Nitric oxide synthase (NOS) inhibition by L-NAME blocked vascular relaxation and reduced superoxide production in LPS-treated animals. In the presence of L-NAME there was no ATP effect on superoxide production. A vascular reactivity study showed that ATP increased maximal relaxation in LPS-treated animals compared to controls. The presence of ATP induced increases in Akt and endothelial NOS phosphorylated proteins in the aorta of septic animals. ATP reduces superoxide release resulting in an improved vasorelaxant response. Sepsis may uncouple NOS to produce superoxide. We showed that ATP through Akt pathway phosphorylated endothelial NOS and “re-couples” NOS function.

Individual susceptibility to vernal keratoconjunctivitis and ameliorative effects of antioxidants on its symptoms

An allergic response is associated by an increase in the expression and activity of the inducible nitric oxide synthase [iNOS] an inducible enzyme present endothelial cells and polymorphonuclear leucocytes [PMN] and moncytes. Vernal keratoconjunctivitis [VKC], is a recurrent or chronic ocular allergic eye disease affecting young individual living in a hot climate. This study was designed to test a hypothesis that the immune changes seen in Vernal keratoconjunctivitis during the summer or spring season is a function of the effect of hot weather on the of polymorphonuclear leucocytes [PMN] and monocytes in susceptible individuals, by evaluating the role of nitric oxide [NO] and NO-synthase [iNOS] in the pathogenesis of allergic conjunctivitis and the effect of supplementing those patients with antioxidants in order to ameliorate ocular allergic effects. 40 patients with typical VKC were divided into two groups. Antioxidants were given as a supplement to one group [n=20], while the second group did not [n=20]. Both groups were followed up periodically for clinical picture and status and were compared with a third healthy age and socio-economic matching control group. Blood samples were obtained before treatment, after one month and at the end of the whole follow up period. Blood markers of oxidative stress including: protein carbonyls, nitrite. DNA fragmentation and apoptosis in circulating lymphocytes of VKC before and after antioxidant therapy during the severity of the disease were evaluated. Also neutrophil's individual ability to express iNOS was assessed. Vernal Keratoconjunctivitis patients exhibited increased individual ability in the production and expression of nitric oxide by their stimulated neutrophils [4.5 +/- 0.05 nmol/ 10[5] cell vs. 2.5 +/- 0.09 nmol/ 10[5] cell] compared to controls. Both biochemical and clinical picture proved that a supplement of antioxidants to the traditional topical treatment would significantly improve the patients ocular allergic symptoms even after seizure of topical treatment, while recurrence of symptoms and signs occurred in group two after stoppage of the topical steroidal treatment. The antioxidant supplemented group exhibited decreased markers of oxidative stress at the end of the first month follow-up period compared to those patients that were not supplemented with antioxidants: Protein carbonyls [0.57 +/- 0.34 vs. 0.93 +/- 0.05]; Percentage of DNA fragmentation per total DNA in combined plasma and leucocytes after one month [0.7% +/- 0.09 vs. 0.93 +/- 0.05] and plasma nitrite after one month [2.4 +/- 0.5 vs. 3 +/- 0.19 nMol/ml] in VKC patients compared to controls respectively. The associated immunological and inflammatory systemic condition of VKC recommends the use of antioxidant mixture in order to emeliorate the ocular allergic effects associated with VKC. Antioxidant use proved its efficiency and prevent recurrence of VKC even after stoppage of the steroid. Also individuals with increased neutrophil's activity have higher individual susceptibility to develop VKC

O bloqueio da síntese do óxido nítrico promove aumento da hipertrofia e da fibrose cardíaca em ratos submetidos a treinamento aeróbio / Nitric oxide synthesis blockade increases hypertrophy and cardiac fibrosis in rats submitted to aerobic training

OBJETIVO: O presente estudo avaliou as adaptações teciduais cardíacas em ratos submetidos a treinamento aeróbio, após o bloqueio da síntese de óxido nítrico (NO). MÉTODOS: Os animais (n = 48) foram divididos em quatro grupos: sedentários (grupo CONTROLE), hipertensos após administração de Ng-nitro-L-arginina metil éster durante sete dias (grupo L-NAME), treinados por meio de natação durante oito semanas (grupo TREINADO) e treinados e tratados com L-NAME na última semana (grupo TREINADO L-NAME). Em todos os animais foi registrada a pressão arterial (PA) e realizada a avaliação morfométrica cardíaca. RESULTADOS: Os grupos L-NAME e TREINADO L-NAME apresentaram-se hipertensos em relação aos demais (p < 0,05), porém a elevação da PA no grupo TREINADO L-NAME foi significativamente menor em relação ao L-NAME (p < 0,05). Os grupos TREINADO e TREINADO L-NAME apresentaram índice de peso cardíaco maior que os grupos CONTROLE e L-NAME (p < 0,05). Também apresentaram maiores índices de área cardíaca macroscópica e de fibrose cardíaca em relação aos demais (p < 0,05) e, quando comparados, o grupo TREINADO L-NAME mostrou-se significativamente superior (p < 0,05). CONCLUSÃO: O bloqueio a curto prazo da síntese de NO, em animais sedentários, induziu hipertensão, sem no entanto causar hipertrofia cardíaca. Nos animais treinados, a inibição da síntese de NO atenuou a hipertensão e promoveu hipertrofia cardíaca com aumento expressivo da fibrose miocárdica, sugerindo importante papel do NO nas adaptações teciduais cardíacas induzidas pelo treinamento físico aeróbio.

OBJECTIVE: The objective of the present study was to evaluate cardiac tissue adaptations in rats submitted to aerobic training after nitric oxide (NO) synthesis blockade. METHODS: The animals (n=48) were divided into four groups: sedentary (CONTROL group); hypertensive after administration of NG-nitro-L-arginine methyl ester for 7 days (L-NAME Group); trained for 8 weeks through swimming exercises (TRAINED Group);trained and treated with L-NAME during the last week (L-NAME TRAINED Group). All the animals were submitted to the experiment procedures for blood pressure (BP) readings and cardiac morphometric evaluation. RESULTS: In comparison to the other groups, the L-NAME and L-NAME TRAINED groups were hypertensive (p<0.05); however, BP elevation in the L-NAME TRAINED group was significantly lower than the L-NAME group (p<0.05). The heart weight indexes for the TRAINED and L-NAME TRAINED groups were higher than the CONTROL and L-NAME groups (p<0.05). Also they had presented higher rates of macroscopic cardiac area and cardiac fibrosis in relation to the rest (p<0.05); comparisons revealed that the values for the L-NAME TRAINED group were significantly higher (p<0.05) than the others. CONCLUSION: Short term NO synthesis blockade in sedentary animals induced hypertension but did not cause cardiac hypertrophy. In the trained animals, the inhibition of NO synthesis attenuated hypertension, induced cardiac hypertrophy and significantly increased myocardial fibrosis, indicating that NO plays an important role in cardiac tissue adaptations caused by aerobic exercise.

El estado hipovolémico modula la actividad y la expresión de la óxido nítrico sintetasa en el sistema cardiovascular / Hypovolemic state modulates nitric oxide synthase activity and expression in the cardiovascular system

Previamente mostramos que el pretratamiento con el inhibidor de la óxido nítrico sintetasa, NG-nitro-L-arginina metil éster (L-NAME), revierte la hipotensión y anula los cambios en la frecuencia cardíaca inducidos por la hemorragia. Objetivo: Evaluar la actividad histoquímica (técnica NADPH-diaforasa) y la expresión (Western blot) de la óxido nítrico sintetasa en la aurícula derecha y en el ventrículo izquierdo de animales sometidos a una hemorragia del 20 por ciento de la volemia. Material y métodos: Se conformaron cuatro grupos de animales (n = 14, por grupo): S ( sham ), H (hemorragia), SL-NAME ( sham + L-NAME) (0,5 mg/kg/h IV = 100 µl/h) y HL-NAME (hemorragia + LNAME). El sacrificio de los animales se realizó por decapitación a los 60 y a los 120 min posteriores al sangrado y se extrajo el corazón para su estudio. Resultados: La pérdida de sangre aumentó la actividad de la NOS en la aurícula y en el ventrículo a los 60 y 120 min (aurícula: 8 por ciento y 21 por ciento respectivamente; ventrículo: 21 por ciento y 45 por ciento, respectivamente) del sangrado. El análisis de Western blot con empleo de un anticuerpo antióxido nítrico sintetasa inducible reveló la presencia de dicha proteína en la aurícula y en el ventrículo a los 120 min del sangrado (654 ± 13 y 465 ± 9 unidades arbitrarias, respectivamente). La expresión de la óxido nítrico sintetasa endotelial aumentó en la aurícula y en el ventrículo a los 60 min (18 por ciento y 147 por ciento respectivamente) en comparación con S, normalizándose a los 120 min de la hemorragia. Conclusiones: El estado hipovolémico inducido por una hemorragia del 20 por ciento de la volemia está asociado con un patrón heterogéneo y dinámico de regulación de la actividad y de la expresión de la óxido nítrico sintetasa en el tejido cardíaco.

Effect of berberine on the mRNA expression of nitric oxide synthase (NOS) in rat corpus cavernosum / 华中科技大学学报（医学）（英德文版）

In order to further investigate the mechanisms of action of berberine (Ber), we assessed the effects of Ber on the mRNA expression of nitric oxide synthases (NOS) in rat corpus cavernosum. After incubation with Ber for 1 or 3 h respectively, the levels of NOS mRNA were examined by reverse transcription polymerase chain reaction (RT-PCR). Our results showed that there were iNOS and eNOS mRNA expressions in rat corpus cavernosum. Ber enhanced eNOS mRNA expression in rat penis, but exhibited no effect on the expression of iNOS mRNA (P > 0.05). The present study indicated that the relaxation of Ber involved the NO-cGMP signal transduction pathway. The enhancing effect of Ber on eNOS mRNA expression might associated with its relaxation of corpus cavernosum.

Expression of nitric oxide synthase isoforms in the porcine ovary during follicular development

The expression of nitric oxide synthase (NOS) isoforms in the ovaries of pigs was examined to study the involvement of nitric oxide, a product of NOS activity, in the function of the ovary. Western blot analysis detected three types of NOS in the ovary, including constitutive neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS); eNOS immunoreactivity was more intense compared with that of iNOS or nNOS. Immunohistochemical studies demonstrated the presence of nNOS and eNOS in the surface epithelium, stroma, oocytes, thecal cells, and endothelial cells of blood vessels. Positive immunoreactions for nNOS and iNOS were detected in the granulosa cells from multilaminar and antral follicles, but not in those of unilaminar follicles. iNOS was detected in the surface epithelium, oocytes, and theca of multilaminar and antral follicles. Taking all of the findings into consideration, the observed differential expression of the three NOS isoforms in the ovary suggests a role for nitric oxide in modulating reproduction in pigs.

Expression of Inducible Nitric Oxide Synthase Is Increased in Rat Barrett's Esophagus Induced by Duodenal Contents Reflux

Barrett's esophagus is a premalignant condition of esophageal adenocarcinoma. Inducible nitric oxide synthase (iNOS) is induced by cytokines and can generate locally high concentrations of nitric oxide (NO), whose metabolites can mediate genotoxicity and influence multistage carcinogenesis by causing DNA damage. Therefore, we evaluated the immunolocalization and expression of iNOS in surgically induced rat Barrett's esophagus. Esophagoduodenal anastomosis was performed in rats for inducing reflux of duodenal contents. Rats were killed at postoperative 10, 20, 30 and 40 weeks. We examined histologic changes and iNOS expression in esophagus by immunohistochemistry and reverse transcription-poly-merase chain reaction. Eighty six percent of experimental rats showed Barrett's esophagus above esophagoduodenal junction. iNOS immunoreactivity was clearly observed in the epithelial cells of Barrett's esophagus, predominantly at the apical surface of epithelial cells. Cytoplasmic staining was also seen only in atypical Barrett's esophagus. iNOS mRNA was detected only in the lower esophagus of experimental group. In conclusion, this study suggests that iNOS has some roles on Barrett's esophagus formation.

Nitric oxide suppresses inducible nitric oxide synthase expression by inhibiting post-translational modification of I kappa B

The expression of inducible nitric oxide synthase (iNOS) is a critical factor in both normal physiological functions and the pathogenesis of disease. This study was undertaken to determine the molecular mechanism by which nitric oxide (NO) exerts negative feedback regulation on iNOS gene expression. Isolated rat hepatocytes stimulated with cytokines exhibited a marked increase in NO production as well as iNOS mRNA and protein levels, which were significantly reduced by pretreatment of the NO donors S-nitroso-N-acetyl-D, L-penicillamine (SNAP) and V-PYRRO/NO. This effect of SNAP was inhibited when NO was scavenged using red blood cells. Pretreatment with oxidized SNAP, 8-Br-cGMP, NO2-, or NO3- did not suppress the cytokine-induced NO production. Moreover, LPS/ IFN-gamma-stimulated RAW264.7 cells, which produce endogenous NO, expressed lower levels of iNOS, IL-1beta, IL-6 and TNF-alpha mRNAs, without changes in their mRNA half-lives, than those in the presence of the iNOS inhibitor NG-monomethyl- L-arginine. The iNOS gene transcription rate exhibited an 18-fold increase after cytokine stimulation, which was significantly inhibited by SNAP pretreatment. SNAP also blocked cytokine- induced increase in NF-kappa B activation, iNOS promoter activity, nuclear translocation of cytosolic NF-kappa B p65 subunit, and I kappa B alpha degradation, which correlated with its inhibitory effect on phosphorylation and ubiquitination of I kappa B. These data indicate that NO down-regulates iNOS gene expression and NO production by inhibiting the post-translational processes of I kappa B alpha thereby preventing NF-kappa B activation. These results identify a novel negative feedback mechanism whereby NO down-regulates iNOS gene expression.

Induction of nitric oxid esynthase(NOS) by soluble glucocorticoid induced tumor necrosis factor receptor(sGITR) is modulated by IFN-g in murine macrophage

Earlier study showed that glucocorticoid induced tumor necrosis factor receptor (GITR), a new TNFR family, activated murine macrophages to express inducible nitric oxide synthase (iNOS) and to generate nitric oxide (NO). A possible involvement of pro-inflammatory cytokines on NO production by GITR was investigated in vitro systems and signaling molecules contributing to sGITR-induced iNOS production are determined in Raw 264.7 cells, a murine macrophage cell line. The result showed that the synergy was afforded by the combination of GITR with IFN-gamma in a dose-dependent manner but IFN-gamma alone was not able to induce NOS. No effects were observed with TNF-alpha, IL-1beta, or IL-6 co-treated with GITR. To determine signaling molecules contributing to sGITR-induced iNOS production, a specific inhibitor for signal pathway proteins tested showed that PDTC (NF- kB) and genistein (tyrosine kinase) inhibited NOS induction significantly, while sodium orthovanadate (tyrosine phosphatase) potentiated NOS expression. These results suggest that activations of NF-kB were involved in induction of iNOS by GITR and IFN-gamma priming caused earlier and stronger NF-kB activation.

Lack of association between CSF nitrate and sera nitrate in falciparum malaria infection.

Nitrate levels in CSF and sera from 16 coma and 19 noncoma falciparum malaria patients were determined using nitric oxide colorometric assay. The medians (range lower, upper limits) of nitrate in sera of comatose and noncomatose patients were 0.28 (0.11, 1.24) and 0.23 (0.05, 0.87) microM, respectively. The medians of nitrate level in CSF of coma and noncoma cases were 0.09 (0.01, 0.28) and 0.15 (0, 1.18) microM, respectively. There was no difference of nitrate level in sera and CSF from comatose or noncomatose patients compared to that in normal sera and CSF. The amount of nitrate in sera and CSF of both groups was not significantly correlated with coma depth, parasitemia, parasite clearance time and time to recovery. Contrast to our in vitro study using immunoperoxidase staining, we found inducible nitric oside synthase production by brain endothelial cells during 4-24 hours of coculturing with late stage of P. falciparum infected red blood cells. These results suggests that malaria severity can not be differentiated by nitrate level in body fluid.