Pathophysiological Role of S-Nitrosylation and Transnitrosylation Depending on S-Nitrosoglutathione Levels Regulated by S-Nitrosoglutathione Reductase

Nitric oxide (NO) mediates various physiological and pathological processes, including cell proliferation, differentiation, and inflammation. Protein S-nitrosylation (SNO), a NO-mediated reversible protein modification, leads to changes in the activity and function of target proteins. Recent findings on protein-protein transnitrosylation reactions (transfer of an NO group from one protein to another) have unveiled the mechanism of NO modulation of specific signaling pathways. The intracellular level of S-nitrosoglutathione (GSNO), a major reactive NO species, is controlled by GSNO reductase (GSNOR), a major regulator of NO/SNO signaling. Increasing number of GSNOR-related studies have shown the important role that denitrosylation plays in cellular NO/SNO homeostasis and human pathophysiology. This review introduces recent evidence of GSNO-mediated NO/SNO signaling depending on GSNOR expression or activity. In addition, the applicability of GSNOR as a target for drug therapy will be discussed in this review.

Assessment of ocular surface toxicity after topical instillation of nitric oxide donors / Avaliação da toxicidade na superfície ocular após instilação tópica de doadores de óxido nítrico

PURPOSE: To evaluate the ocular surface toxicity of two nitric oxide donors in ex vivo and in vivo animal models: S-nitrosoglutathione (GSNO) and S-nitroso-N-acetylcysteine (SNAC) in a hydroxypropyl methylcellulose (HPMC) matrix at final concentrations 1.0 and 10.0 mM. METHODS: Ex vivo GSNO and SNAC toxicities were clinically and histologically analyzed using freshly excised pig eyeballs. In vivo experiments were performed with 20 albino rabbits which were randomized into 4 groups (5 animals each): Groups 1 and 2 received instillations of 150 µL of aqueous HPMC solution containing GSNO 1.0 and 10.0 mM, respectively, in one of the eyes; Groups 3 and 4 received instillations of 150 µL of aqueous HPMC solution-containing SNAC 1.0 and 10.0 mM, respectively, in one of the eyes. The contralateral eyes in each group received aqueous HPMC as a control. All animals underwent clinical evaluation on a slit lamp and the eyes were scored according to a modified Draize eye test and were histologically analyzed. RESULTS: Pig eyeballs showed no signs of perforation, erosion, corneal opacity or other gross damage. These findings were confirmed by histological analysis. There was no difference between control and treated rabbit eyes according to the Draize eye test score in all groups (p>0.05). All formulations showed a mean score under 1 and were classified as "non-irritating". There was no evidence of tissue toxicity in the histological analysis in all animals. CONCLUSION: Aqueous HPMC solutions containing GSNO and SNAC at concentrations up to 10.0 mM do not induce ocular irritation.

OBJETIVO: Avaliar a toxidade na superfície ocular de dois compostos doadores de óxido nítrico em modelos ex vivo e in vivo: S-nitrosoglutationa (GSNO) e S-nitroso-N-acetilcisteína (SNAC), em uma matriz de hidroxipropil metilcelulose (HPMC) nas concentrações finais de 1,0 and 10,0 mM. MÉTODOS: As toxicidades de GSNO e SNAC foram avaliadas clinicamente e histologicamente em modelo ex vivo usando globos oculares porcinos recém excisados. Experimentos in vivo foram realizados com 20 coelhos albinos que foram randomizados em 4 grupos (5 animais em cada): Os grupos 1 e 2 receberam instilações de 150 µL de solução aquosa de HPMC contendo GSNO 1,0 e 10,0 mM, respectivamente, em um dos olhos; Os grupos 3 e 4 receberam instilações de 150 µL de solução aquosa de HPMC contendo SNAC 1,0 and 10,0 mM, respectivamente, em um dos olhos. Os olhos contralaterias em cada grupo receberam solução aquosa de HPMC como controle. Todos os animais foram clinicamente avaliados em lâmpada de fenda e os olhos foram pontuados de acordo com o teste de Draize modificado e analisados histologicamente. RESULTADOS: Os globos oculares porcinos não apresentaram sinais de perfuração, erosão, opacidade da córnea ou outros danos graves. Esses resultados foram confirmados pela análise histológica. Não houve diferença entre os olhos dos coelhos tratados e controles de acordo com a pontuação do teste de Draize em todos os grupos (p>0,05). Todas as formulações apresentaram um escore médio menor do que 1 e foram classificadas como "não-irritantes". Não houve evidência de toxicidade tecidual nas análises histológicas em todos os animais. CONCLUSÃO: Soluções aquosas de HPMC contendo GSNO e SNAC em concentrações até 10,0 mM não induzem irritação ocular.

Influence of S-nitrosoglutathione on the membrane glycoprotein of frozen platelets / 中国实验血液学杂志

This study was purposed to explore the influence of S-nitrosoglutathione (GSNO) on membrane glycoprotein of frozen platelet. The levels of membrane glycoprotein on fresh liquid platelets, frozen platelets and frozen platelets with GSNO were measured by flow cytometry. The results showed that the GSNO obviously decreased platelet aggregation, the PAC-1 change in the three groups was not significant. The changes of CD42b and CD62P in fresh liquid platelet group, frozen platelet group and frozen platelets with GSNO were significant different. The change of membrane glycoprotein in above-mentioned three group was not significant. It is concluded that the GSNO inhibits platelet aggregation, maintains the function of platelets and may be used as a cryoprotectant. When frozen platelets were added with GSNO, the molecular rearrangement, structure change and other mechanism may occur in platelets.

Especies reactivas de oxigeno y respuesta antioxidante en pacientes VIH positivos y donantes voluntarios de sangre, Pereira, Colombia, 2007-2009 / Oxygen reactive species and anti-oxidant response in HIV positive patients and volunteer blood donors, Pereira, Colombia, 2007-2009

Introducción: El estrés oxidativo se origina por desequilibrio entre la producción de Especies Reactivas del Oxígeno (ERO) y Capacidad Antioxidante Celular (CAC). La producción de ERO mitocondrial es constante. Entre 2% y 5 % del oxígeno para la cadena respiratoria se reduce para generar el anión superóxido, •O2-, a partir de éste se producen otras moléculas y radicales libres potencialmente dañinos para la célula. El Síndrome de Inmunodeficiencia Adquirida (SIDA) se caracteriza por un estrés oxidativo persistente. El objetivo del estudio fue medir el estado redox en pacientes VIH+ del programa de SIDA de Pereira y estado redox de personas donantes voluntarios de sangre del programa de medicina de la Universidad Tecnológica de Pereira años 2007-2009 para verificar la condición de estrés oxidativo, mediante su cuantificación espectrofotométrica de marcadores asociados al sistema oxidante-antioxidante celular. Metodología: Estudio transversal realizado con un grupo de pacientes VIH-SIDA y otro de donantes voluntarios de sangre, se cuantificó el estado redox y se correlaciona con otros parámetros: edad, género, estado clínico, nivel socioeconómico, tipo de dieta, estado nutricional, consumo de estresantes inmunológicos, antecedentes personales y epidemiológicos. Resultados: La Respuesta Antioxidante Total (RAT), Peróxido Plasmático (PP), Índice de estrés oxidativo (IEO), Índice de carbonilo (IC), Malondialdehido (MDA); resultaron significativamente diferentes en los grupos estudiados. Discusión: En VIH+ no se presentaron diferencias significativas en las pruebas de estrés oxidativo entre los que consumen y no consumen antiretrovirales. Se requieren estudios poblacionales para obtener valores de referencia de las pruebas de estrés oxidativo aquí ensayadas.

Introduction: Oxidative stress is caused by imbalance between the production of Reactive Oxygen Species (ROS) and cellular antioxidant capacity (CAC). Mitochondrial ROS production is constant. Between 2% and 5% of oxygen to the respiratory chain is reduced to generate superoxide anion, • O2-, from this there are other molecules and free radicals potentially harmful to the cell. Acquired Immunodeficiency Syndrome (AIDS) is characterized by persistent oxidative stress. The objective of this study was to measure the redox status in HIV + AIDS program of the Municipal Institute of Health Pereira and redox status of people volunteer blood donors medicine program of the Technological University of Pereira years 2007 to 2009 to verify the condition of oxidative stress by spectrophotometric quantification of markers associated with oxidant-antioxidant cell system. Materials and methods: Cross-sectional study with a group of HIV-AIDS and other blood donors, the redox state was measured and correlated with other parameters: age, gender, clinical status, socioeconomic status, diet, nutritional status, immunological stressor consumption, personal history and epidemiologists. Results: Total antioxidant response (TAR), peroxide plasma (PP), oxidative stress index (IEO), carbonyl index (CI), malondialdehyde (MDA) were significantly different in the groups studied. Discussion: In HIV+ there were no significant differences in oxidative stress tests among those who consume and consume no antiretrovirals. Population studies are required to obtain reference values of oxidative stress tests tested here.

Influence of S-nitrosoglutathione on agglutination and nitric oxide concentration in frozen platelets / 中国实验血液学杂志

The aim of this study was to investigate the influence of S-nitrosoglutathione (GSNO) on agglutination and nitric oxide (NO) concentration in frozen platelets. The agglutination of platelets was detected by using platelet agglutination apparatus, the level of NO in platelets was detected by the nitrate enzyme reduction method. The results showed that the rates of agglutination in freeze platelets and frozen platelets treated with GSNO were (35.47 ± 2.93) and (24.43 ± 3.07), which were significantly lower than that in fresh liquid platelets (63.44 ± 2.96). The level of NO concentration in frozen platelets was (22.16 ± 6.38), which was significantly lower than that in fresh liquid platelets (31.59 ± 16.88). The level of NO concentration in frozen platelets treated with GSNO was (45.64 ± 6.31), which was significantly higher than that in fresh liquid platelets (P < 0.01). It is concluded that GSNO increases the concentration of NO in frozen platelets, inhibits platelet activation and maintains platelet function, thus GSNO can be used as a frozen protective agent.

Effect of Nitric Oxide on the Cryopreservation of Platelets / 대한진단검사의학회지

BACKGROUND: To determine whether nitric oxide (NO) could inhibit activation of platelets stored in a cold or frozen state, we measured platelet P-selectin expression and platelet-bound fibrinogen in platelet-rich plasma (PRP) with S-nitrosoglutathione (GSNO) (Sigma, USA) by flow cytometry. METHODS: PRP was prepared by centrifuging venous blood collected in a 3.2% sodium citrate tube from 10 healthy donors. It was aliquotted into 4 groups (no cryoprotectant, GSNO, GSNO/dimethyl sulfoxide [DMSO] [Sigma], and DMSO), and stored at room, cold and freezing temperatures for 24 hrs. We performed a flow cytometric analysis of all specimens stained with FITC-fibrinogen and PE-CD62P monoclonal antibodies (Becton Dickinson, USA). The results were compared according to the storage temperature and agonist among 4 groups. RESULTS: GSNO inhibited significantly the activation of frozen platelets, but not in the presence of DMSO. GSNO was also shown to preserve the aggregability of frozen platelets because in the presence of GSNO the delta percent change of P-selectin expression and fibrinogen binding of frozen platelets increased significantly irrelevant to DMSO. CONCLUSIONS: GSNO inhibited the activation of frozen platelets and preserved the platelet aggregability; therefore, it may be used as a protectant for platelet cryopreservation.

Modulation of glucose uptake in adipose tissue by nitric oxide-generating compounds.

There is increasing evidence that endogenous nitric oxide (NO) influences adipogenesis, lipolysis and insulin-stimulated glucose uptake. We investigated the effect of NO released from S-nitrosoglutathione (GSNO) and S-nitroso-N-acetylpenicillamine (SNAP) on basal and insulin-stimulated glucose uptake in adipocytes of normoglycaemic and streptozotocin (STZ)-induced diabetic rats. GSNO and SNAP at 0.2,0.5, and 1 mM brought about a concentration-dependent increase in basal and insulin-stimulated 2-deoxyglucose uptake in adipocytes of normoglycaemic and STZ-induced diabetic rats. SNAP at 1.0 mM significantly elevated basal 2-deoxyglucose uptake (115.8+/-10.4% compared with GSNO at the same concentration (116.1+/-9.4%; P less than 0.05) in STZ-induced diabetic rats. Conversely, SNAP at concentrations of 10 mM and 20 mM significantly decreased basal 2-deoxyglucose uptake by 50.0+/-4.5% and 61.5+/-7.2% respectively in adipocytes of STZ-induced diabetic rats (P less than 0.05). GSNO at concentrations of 10 mM and 20 mM also significantly decreased basal 2-deoxyglucose uptake by 50.8+/-6.4% and 55.2+/-7.8% respectively in adipocytes of STZ-induced diabetic rats (P less than 0.05). These observations indicate that NO released from GSNO and SNAP at 1 mM or less stimulates basal and insulin-stimulated glucose uptake,and at concentrations of 10 mM and 20 mM inhibits basal glucose uptake. The additive effect of GSNO or SNAP, and insulin observed in this study could be due to different mechanisms and warrants further investigation.

Formation of platelets from cord blood CD34+ cells-derived megakaryocytes induced by S-nitrosoglutathione / 生理学报

To investigate the effect of S-nitrosoglutathione (GSNO), a nitric oxide donor, on platelet production from megakaryocytes differentiated from cord blood CD34(+) cells in vitro, the CD34 (+) cells from eight fresh umbilical cord blood samples by a high-gradient magnetic cell sorting (MACS) system were cultured in serum-free medium for 14 d with thrombopoietin (TPO) 50 ng/ml, IL-3 10 ng/ml, stem cell factor (SCF) 50 ng/ml and rHuGM-CSF 20 ng/ml. Then, CD61 (+) cells were purified by MACS system from these CD34 (+) cells, and were cultured in serum-free medium supplemented with TPO 50 ng/ml, IL-3 10 ng/ml and SCF 50 ng/ml in the presence (treatment group) and absence (control group) of GSNO for 30 min or 2 h. Platelet-sized particles were counted by flow cytometry; megakaryocyte structure was detected by scanning electron microscope. Aggregation of the thrombin-induced platelet particle was observed under inversion microscope. cGMP was assessed by commercial ELISA kit. The results showed that, compared with the control group, the number of platelet-sized particles significantly increased (P<0.05) in the treatment group, in which megakaryocytes presented significant pseudopod formation and extensive membrane blebbing. The platelet particle aggregation could be observed under microscope after thrombin induction. cGMP activity was significantly increased after treatment with GSNO (P<0.05). These results propose that GSNO can facilitate platelet production from megakaryocyte, and it may be partly through cGMP pathway.

Implicações do estresse oxidativo sobre o metabolismo eritrocitário de pessoas com Síndrome de Down / Implications of the oxidative stress on the erythrocyte metabolism of Down Syndrome individuals

Portadores da Síndrome de Down estão sob estresse oxidativo endógeno e crônico que pode ser resultado do excesso de atividade da SOD-1. Este trabalho descreve alguns indicadores e adaptações das defesas frente aos danos oxidativos. Observamos que, nas pessoas com Síndrome de Down, a presença de estresse oxidativo (aumento de 48 por cento na atividade da SOD-1) induziu várias adaptações no metabolismo eritrocitário, sendo que a redução da meta-hemoglobina, via aumento da atividade da meta-hemoglobina redutase (29 por cento), garantiria a eficiência do transporte de oxigênio, enquanto o aumento da GSH (61 por cento) propiciaria a integridade funcional do eritrócito. A diminuição dos valores de concentração de hemoglobina e hematócrito, possivelmente, resulta do aumento de atividade da enzima Glicose-6-fosfato desidrogenase e redução da meia-vida eritrocítica. Os efeitos destas adaptações sob a oxigenação do sangue merecem maiores investigações.

Down’s Syndrome carriers are under endogenous andchronic oxidative stress that can result from anexcessive SOD-1enzyme activity. This work describessome indicators and adaptations of the defense systemfaced with oxidative damage. We observed that, inDown’s Syndrome individuals, the presence of oxidativestress (with an increase of 48% in the SOD-1 activity)induced several adaptations of the erythrocytemetabolism, with a reduction of methemoglobin,through an increase of methemoglobin reductaseactivity (29%) which guaranteed the efficiency ofoxygen transportation. The increase of GSH (61%)would propitiate the maintenance of the integrity ofthe membrane, essential to the functional integrity oferythrocytes. The decrease in the hemoglobin andhematocrit concentrations, possibly, results from theincrease in the activity of the glucose-6-phosphatedehydrogenase enzyme, and a reduction in the halflifeof erythrocytes. The effects of these adaptations onblood oxygenation require further investigations.

Redox regulation of calcium release in skeletal and cardiac muscle

In skeletal and cardiac muscle cells, specific isoforms of the Ryanodine receptor channels mediate Ca2+ release from the sarcoplasmic reticulum. These channels are highly susceptible to redox modifications, which regulate channel activity. In this work, we studied the effects of Ca2+ (endogenous agonist) and Mg2+ (endogenous inhibitor) on the kinetics of Ca2+ release from sarcoplasmic reticulum vesicles isolated from skeletal or cardiac mammalian muscle. Native skeletal vesicles exhibited maximal stimulation of release kinetics by 10-20 microM [Ca2+], whereas in native cardiac vesicles, maximal stimulation of release required only 1 microM [Ca2+]. In 10 microM [Ca2+], free [Mg2+] < 0.1 mM produced marked inhibition of release from skeletal vesicles but free [Mg2+] < or = 0.8 mM did not affect release from cardiac vesicles. Incubation of skeletal or cardiac vesicles with the oxidant thimerosal increased their susceptibility to stimulation by Ca2+ and decreased the inhibitory effect of Mg2+ in skeletal vesicles. Sulfhydryl-reducing agents fully reversed the effects of thimerosal. The endogenous redox species, glutathione disulfide and S-nitrosoglutathione, also stimulated release from skeletal sarcoplasmic reticulum vesicles. In 10 microM [Ca2+], 35S-nitrosoglutathione labeled a protein fraction enriched in release channels through S-glutathiolation. Free [Mg2+] 1 mM or decreasing free [Ca2+] to the nM range prevented this reaction. Possible physiological and pathological consequences of redox modification of release channels on Ca2+ signaling in heart and muscle cells are discusse

Neuroprotective Effect of Estrogen on Motor Neuron Cells Expressing Wild Type or Mutant Cu/Zn Superoxide Dismutase

BACKGROUND: Recently, the mechanism involved in nitric oxide (NO)-mediated motor neuron death is under extensive investigation. The role of Cu/Zn superoxide dismutase (SOD) mutation, which is found in about 2% of all ALS patients, has been implicated in selective motor neuron death and it is said to play an important role in NO-mediated motor neuron death. Estrogen is reported to have neuroprotective effect in various neurological diseases. However, neuroprotective effect on estradiol on spinal motor neuron exposed to NO has rarely been studied. METHODS: Motor neuron-neuroblastoma hybrid cell expressing wild-type or mutant (G93A or A4V) SOD gene was treated with 200 micro M Snitrosoglutathione. After 24 hours, cell viability was measured by MTT assay. To see the neuroprotective effect of estradiol, pretreatment with 5 nM or 50 nM 17 beta-estradiols was done 24 hours before S-nitrosoglutathione treatment. RESULTS: S-nitrosoglutathione showed significant neurotoxic effect in all three cell lines. Percentage of cell death was significantly different in each cell line. Both 5 nM and 50 nM estradiols showed neuroprotective effect in G93A cell line. In wild-type cell line, 50 nM estradiol showed neuroprotective effect, but 5 nM estradiol did not. In A4V cell line, estradiol did not showed neuroprotective effect. CONCLUSIONS: This study showed that NO-mediated motor neuron death could be influenced by presence or absence of mutation and type of mutation in SOD gene. Neuroprotective effect of estradiol is also influenced by SOD gene mutation. This study implies that estrogen might be beneficial to some ALS patients.