Protective Effect Of NO Research Articles

Hemorheological changes in arterial hypertension in persons with and without COVID-19

BACKGROUND: Vascular resistance, and therefore blood pressure (BP), depends on vascular and rheological factors. Microrheological characteristics of red blood cells (RBCs) can affect blood viscosity (BV) and thus be included in the pathogenesis of increased blood pressure in arterial hypertension (AH). Signaling molecules such as gasotransmitters (NO, CO and H2S) regulate vascular tone and RBC microrheological characteristics and thus affect blood pressure and tissue perfusion. OBJECTIVE: It was evaluated the changes in the macro- and microrheological characteristics of blood and red blood cells (RBCs) under arterial hypertension in persons with and without COVID-19, as well as the protective effect of NO and H2S donors on the RBC microrheological properties. METHODS: Hemorheological profile parameters were recorded in group 1 individuals (n = 18, 9 women and 9 men) without a history of COVID-19; group 2 (n = 16; 11 females and 5 males), hypertensive patients who had COVID-19. As a control, there was a group of healthy individuals (group 3 n = 22). In experiments with red blood cells (RBCs) and their recovered ghosts (filled with an isotonic solution of known viscosity), deformability (RBCD) and aggregation (RBCA) were recorded after incubation of cells with sodium nitroprusside (SNP, 100μM) and sodium hydrosulfide (NaHS, 100μM). RESULTS: In patients with AH in both groups, the main parameters of the hemorheological profile were negatively changed, including a decrease in RBCD and an increase in BV, plasma viscosity (PV) and RBCA. SNP and NaHS significantly increased deformability and reduced their aggregation (p < 0.01). However, in healthy individuals, microrheological responses to GT donors (100μM) were more pronounced than in patients with AH, especially in the AH+COVID-19 group (p < 0.05). CONCLUSION: Both gasotransmitter donors (NO and H2S) have a positive effect on the RBC microrheological characteristics in healthy and sick individuals. However in hypertensive patients, especially those who had COVID-19, microrheological responses to GT donors were significantly lower. Therefore, on the model of red cell microrheological responses, as on a test object, it is possible to determine the decrease in the sensitivity of cells and tissues to the regulatory action of gasotransmitters.

Sulfur supplementation enhances nitric oxide efficacy in reversal of chromium-inhibited Calvin cycle enzymes, photosynthetic activity, and carbohydrate metabolism in wheat

The present study demonstrated that exogenously-sourced nitric oxide (as SNP, sodium nitroprusside; NO donor) and sulfur (S) protected photosynthesis against chromium (Cr) stress in wheat (Triticumaestivum L. cv. HD 2851). Plants grown with 100 µM Cr exhibited higher reactive oxygen species (ROS) production, resulting in photosynthetic damage. The individual application of 50 µM NO increased carbohydrate metabolism as well as photosynthetic parameters, antioxidant system with higher transcriptional gene levels that encode the key enzymes for the Calvin cycle under Cr stress. These effects were more prominent when NO was applied with 1.0 mM SO42−. An increase in the reduced glutathione (GSH) content obtained with NO was further enhanced by S and resulted in higher protection against Cr stress. The protective effect of NO with S against Cr toxicity on photosynthesis was reversed when buthionine sulfoximine (BSO; GSH biosynthetic inhibitor) was used. Application of BSO reversed the impact of NO plus S on photosynthesis under Cr stress, verifying that the ameliorating effect of NO was through S-assimilation and via GSH production. Thus, the availability of S to NO application can help reduce Cr toxicity and protect photosynthetic activity and expression of the Calvin cycle enzymes in leaves through the GSH involvement.

Involvement of Nitric Oxide in Regulation of Plant Development and Resistance to Moisture Deficiency

Nitric oxide is a universal signaling molecule involved in the modulation of metabolic activity during the normal growth and development of plants, and in the formation of their resistance to environmental stressors. The review presents key information that reflects the current state of the problem of the regulatory role of NO in plants. Brief information on physicochemical properties of NO, methods of its research, ways of biosynthesis and functional activity at different stages of plant development (germination, vegetative growth, flowering, root formation, symbiosis, mineral nutrition) are given.In addition, the appearance of the protective effects of NO under conditions of moisture deficiency is described, since disturbance of the water regime and dehydration of plants is observed under the influence of various abiotic stress factors, including drought, salinity, hypo- and hyperthermia. Particular attention is paid to the molecular mechanisms of NO-dependent signaling, which are implemented in plants at the genomic, proteomic and post-proteomic levels during multiple nitration reactions. Understanding the mechanisms of regulatory action of NO in normal and under stress is of important theoretical and applied importance in connection with the need for a fundamental justification of the possibility of practical use of NO in order to increase the stability and productivity of cultivated plants.

Nitric oxide donor improves adventitious rooting in mung bean hypocotyl cuttings exposed to cadmium and osmotic stresses

Exogenous application of a nitric oxide donor, sodium nitroprusside (SNP), improved the formation and growth of adventitious roots in mung bean hypocotyl cuttings. The combined applications of SNP and CdCl2 or mannitol partially overcame the negative effects of Cd and mannitol on adventitious rooting compared with Cd or mannitol alone. SNP significantly increased the endogenous NO level and the glutathione (GSH), ascorbic acid (ASA), polyphenol, and proline (Pro) levels during the root induction and/or initiation stages. However, the malondialdehyde (MDA) levels were significantly reduced by the SNP treatment during the entire time course. To unveil the role of NO in mediating the plant response to heavy metal and osmotic stresses during adventitious rooting, SNP together with CdCl2 or mannitol was applied in the experiments. The results obtained showed that the Cd- or mannitol-induced changes in the levels of GSH, ASA, polyphenol, Pro, and MDA and the activities of ascorbate peroxidase (APX), peroxidase (POD), catalase (CAT), superoxide dismutase (SOD), and indole-3-acetic acid oxidase (IAAO) were completely or partially reversed by SNP supplementation in the SNP + Cd/mannitol treatments, suggesting that NO mediated the redox state via the regulation of antioxidative systems to prevent membrane lipid peroxidation in response to the stresses during adventitious rooting. Furthermore, the protective effects of NO on the cellular antioxidative systems under the stresses were similar to those of IBA, implying that auxin and NO shared common pathways to respond to the stresses and leading to adventitious rooting in mung bean hypocotyl cuttings under heavy metal and osmotic stresses. NO mediates the redox state and prevents membrane lipid peroxidation during adventitious rooting in plants exposed to abiotic stresses via the regulation of antioxidant levels and antioxidative enzyme activities. NO shares common signaling pathways with auxin or acts as a downstream mediator of auxin in response to abiotic stresses during adventitious rooting.

208 - Protective Role of Nitro-oleic Acid in Development of Vascular Dysfunction

It is becoming increasingly evidence that nitration products of unsaturated fatty acids represent an important class of endogenous biological mediators, generated as an adaptive response of organism to oxidative and nitrative stress. The purpose of our study was to define the role of nitro-oleic acid (NO 2 -OA) in prevention and treatment of vascular dysfunction in different models (atrial fibrosis and pulmonary hypertension). The effect of NO 2 -OA was tested in different cell types (including macrophages, neutrophils, fibroblasts, endothelial, and smooth muscle cells) both in vivo and in vitro . Our results showed that NO 2 -OA significantly improves the heart functions and overall outcome of animals with atrial fibrosis and pulmonary hypertension. These effects were associated with reduced polarization of macrophages toward pro-inflammatory and immuno-regulatory subsets, decreased activation of different signaling pathways as well as production of pro-inflammatory and pro-fibrotic mediators. NO 2 -OA prevents pathological activation of endothelial cells characterized by reduced production of pro-inflammatory cytokines and chemokines, expression of adhesive molecules as well as their transformation to the pro-fibrotic phenotype. In aggregate, our study provides unique results showing the protective effects of NO 2 -OA in progression of vascular inflammation.

Crosstalk of nitric oxide with calcium induced tolerance of tall fescue leaves to high irradiance

Calcium ion (Ca2+) is essential secondary messenger in plant signaling networks. In this study, the effect of Ca2+ on oxidative damage caused by a high irradiance (HI) was investigated in the leaves of two cultivars of tall fescue (Arid3 and Houndog5). Pretreatment of the tall fescue leaves with a CaCl2 solution significantly increased Ca2+ content and intrinsic HI tolerance due to a decreased ion leakage and content of malondialdehyde, hydrogen peroxide, and superoxide radicals. Moreover, the activities of superoxide dismutase, catalase, ascorbate peroxidase, and glutathione reductase increased in both the cultivars in the presence of Ca2+ under the HI stress. In contrast, treatments with a Ca2+ chelator ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) or a plasma membrane Ca2+ channel blocker LaCl3 reversed these effects. On the other hand, a pronounced increase in nitric oxide synthase-like activity and NO release by exogenous Ca2+ treatment was observed in the tolerant Arid3 plants after exposure to the HI, whereas only a small increase was observed in more sensitive Houndog5. Moreover, the inhibition of NO production by 2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide or N ω-nitro-L-arginine blocked the protective effect of exogenous Ca2+, whereas the inhibition of Ca2+ by EGTA or LaCl3 had no influence on the protective effect of NO. The results indicate that NO might be involved in the Ca2+-induced activities of antioxidant enzymes further protecting against HI-induced oxidative damage. This protective mechanism was found to be more efficient in Arid3 than in Houndog5.

Nitric oxide affects salt-induced changes in free amino acid levels in maize

It was assumed that salt-induced redox changes affect amino acid metabolism in maize (Zea mays L.), and this influence may be modified by NO. The applied NaCl treatment reduced the fresh weight of shoots and roots. This decrease was smaller after the combined application of NaCl and an NO-donor ((Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate, DETA/NO) in the shoots, while it was greater after simultaneous treatment with NaCl and nitro-l-arginine (l-NNA, inhibitor of NO synthesis) in the roots. The quantum yield efficiency of photosystem II was not influenced by the treatments. NaCl had a significant effect on the redox environment in the leaves as it was shown by the increase in the amount of glutathione disulphide and in the redox potential of the glutathione/glutathione disulphide redox pair. This influence of NaCl was modified by DETA/NO and l-NNA. Pharmacological modification of NO levels affected salt-induced changes in both the total free amino acid content and in the free amino acid composition. NaCl alone increased the concentration of almost all amino acids which effect was strengthened by DETA/NO in the case of Pro. l-NNA treatment resulted in a significant increase in the Ala, Val, Gly and Tyr contents. The Ile, Lys and Val concentrations rose considerably after the combined application of NaCl and DETA/NO compared to NaCl treatment alone in the recovery phase. NaCl also increased the expression of several genes related to the amino acid and antioxidant metabolism, and this effect was modified by DETA/NO. In conclusion, modification of NO levels affected salt-induced, glutathione-dependent redox changes and simultaneously the free amino acid composition and the level of several free amino acids. The observed much higher Pro content in plants treated with both NaCl and DETA/NO during recovery may contribute to the protective effect of NO against salt stress.

Nitric oxide alleviates adverse salt-induced effects by improving the photosynthetic performance and increasing the anti-oxidant capacity of eggplant (Solanum melongena L.)

SummaryNitric oxide (NO) is an active molecule involved in many physiological functions in plants. To characterise the roles of NO in the tolerance of eggplant (Solanum melongena L.) to salt stress, the protective effects of exogenous sodium nitroprusside (SNP), a donor of NO, applied at different concentrations (0, 0.05, 0.1, or 0.2 mM), on plant biomass, photosynthesis, and anti-oxidant capacity were evaluated. The application of SNP alleviated the suppression of growth in eggplant under salt stress, as reflected by a higher accumulation of biomass. In parallel with growth, the application of SNP to salt-stressed plants resulted in enhanced photosynthetic parameters such as the net photosynthetic rate (Pn), stomatal conductance (gs), transpiration rate (Tr), and intercellular CO2 concentration (Ci), as well as an increased quantum efficiency of PSII (Fv/Fm), efficiency of excitation capture of open PSII centres (Fv’/Fm’), quantum yield of PSII ( psii) and photochemical quenching coefficient (qP). Furthermore, exogenous SNP also reduced significantly the rate of production of O2• – radicals and the concentrations of malondialdehyde (MDA) and H2O2. It also increased the activities of superoxide dismutase (SOD), guaiacol peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) in eggplant leaves grown under salt stress. The results indicated that the protective effects of NO against salt stress in eggplant seedlings were most likely mediated through improvements in photosynthetic performance and the stimulation of anti-oxidant capacity.

The role of induction and inhibition of nitric oxide synthesis in regulation of blood neutrophils cell death during oxidative imbalance

Modeling of oxidative stress in vitro with 5 mM H2O2 has demonstrated a protective role of nitric oxide in realization of constitutional blood neutrophil cell death. The NO synthase inductor, L-arginine, and the inhibitor of nitric oxide synthesis, L-NAME, influenced the amount of annexin-positive cells, the content of Bax protein, reactive oxygen species, cyclic nucleotides, and calcium homeostasis in neutrophils under conditions realizing programmed death during oxidative stress in vitro and under acute inflammation. During oxidative stress L-arginine normalized an increased intracellular Ca2+ level and the cAMP/cGMP ratio by increasing the cGIP level, stabilized metabolism and prolonged neutrophil lifetime. During acute inflammation NO induction was insufficient for limitation of Ca2+ release into cytosol and for onset of the apoptotic effect; blockade of NO synthesis deteriorated this situation by activating neutrophil apoptosis due to the sharp increase in the Ca2+ content and reduction of cytosolic cyclic nucleotides. The protective effect of NO on neutrophil cell death during oxidative imbalance was not associated with regulation of the proapoptotic protein Bax.

NO Inhibits Hyperoxia-Induced NF-κB Activation in Neonatal Pulmonary Microvascular Endothelial Cells

Inhaled NO (iNO) may be protective against hyperoxic injury in the premature lung, but the mechanism is unknown. We hypothesized that NO would prevent hyperoxia-induced nuclear factor kappa B (NF-κB) activation in neonatal pulmonary microvascular endothelial cells [human pulmonary microvascular endothelial cell (HPMEC)] and prevent the up-regulation of target genes. After hyperoxic exposure (O2 >95%), nuclear NF-κB consensus sequence binding increased and was associated with IκBα degradation. Both of these findings were prevented by exposure to NO. Furthermore, intracellular adhesion molecule (ICAM)-1 mRNA and protein levels increased in cells exposed to hyperoxia, an effect abrogated by NO. To evaluate the potentially toxic effect of NO plus hyperoxia, cell viability and proliferation were assessed. Cells exposed to NO plus hyperoxia demonstrated improved survival as measured by trypan blue exclusion when compared with cells exposed to hyperoxia alone. These differences in cell death could not be attributed to apoptosis measured by caspase-3 activity. Finally, cellular proliferation inhibited by hyperoxia was rescued by concurrent exposure to NO. These data demonstrate that NO prevents hyperoxia-induced NF-κB activation in HPMEC and results in decreased expression of adhesion molecules and decreased cellular toxicity. This may help to explain the protective effects of NO on hyperoxic injury in the developing lung vasculature.

The role of nitric oxide synthesis induction and inhibition in regulation of blood neutrophil cell death during oxidative disbalance

Modeling oxidative stress in vitro with 5mM H2O2 has demonstrated a protective role of nitric oxide on realization of constitutional blood neutrophil cell death. The NO-synthase inductor L-arginine and the inhibitor of nitric oxide synthesis, L-NAME, influenced on the amount of annexin-positive cells, the content of Bax protein, reactive oxygen species, cyclic nucleotides, and calcium homeostasis in neutrophils under conditions realizing programmed death during oxidative stress in vitro and under acute inflammation. During oxidative stress L-arginine normalized the increased intracellular Ca2+ level and the cAMP/cGMP ratio due to increase of cGMP level, stabilized metabolism and prolonged neutrophil life. During acute inflammation NO induction was insufficient for limitation of Ca2+ release into cytosol and for onset of the apoptotic effect; blockade of NO synthesis deteriorated this situation by activating neutrophil apoptosis due to the sharp increase in Ca2+ content and reduction of cyclic nucleotides in cytosol. The protective effect of NO on neutrophil cell death during oxidative dysbalance is not associated with regulation of apoptotic protein Bax.

Intracellular Calcium Mediated Stiffness of Red Blood Cells Is Reversed By Hypoxic Pre-Incubation With Nitrite Ions

Normal red blood cells (RBCs) need to be highly deformable to pass through microcapillaries in order to deliver oxygen. It has been reported that an intracellular increase of calcium ions in RBCs causes them to undergo oxidative stress. Our earlier studies suggested that in hypoxia, nitrite ions react with deoxyhemoglobin (Hb(II)) to produce stable bioactive NO intermediates and Hb(II)NO that interact with the membrane and can potentially release NO and/or react with membrane protein thiols. We hypothesize that calcium-induced oxidative stress will decrease RBC deformability and thereby increase stiffness of RBCs, which will be inhibited by the production of bioactive NO in the RBCs in hypoxia. In this study we have used a newly available microfluidic ektacytometer to measure RBC-deformability in human blood expressed as the elongation index (EI, normal values 0.31-0.35) at a shear stress of 3Pa. We observed that EI of RBCs decreased to about 50% in 30minutes at 37oC when A23187 ionophore-mediated calcium ions (as low as 0.01mM) enter cells. However, when RBCs are pre-incubated with a 10:1 heme:nitrite molar ratio of nitrite ions in hypoxia prior to ionophore-mediated calcium ion entry, the decrease in EI of RBCs is inhibited. Support for NO bioactivity is provided by the observation of similar results with RBCs pre-incubated with nitroprusside, a NO-donor (at 40μM concentration). This suggests that NO released from nitrite-reacted RBCs in hypoxia blocks intracellular calcium rise-mediated decrease in RBC deformability. Experiments are underway to determine the mechanism of this protective effect of NO.(This research was supported in part by the Intramural Research Program of the NIH, National Institute on Aging and Johns Hopkins University School of Medicine)

Positive influence of Vertigoheel on signalling pathways of smooth muscle cells and vertigo

The incidence of circulatory disorders is increasing against the background of increasing life expectancy. Cardiovascular, metabolic and inflammatory disorders are directly associated with endothelial dysfunction of the small blood vessels. It is possible to influence microcirculation disorders in various ways through the use of drugs. An experimental investigation at the University of Tubingen in cooperation with the Charite Berlin has recently shown that the complex homoeopathic preparation Vertigoheel in vitro influences the contraction of smooth muscle cells. The investigation was performed on sections of rats’ arteries, which were initially contracted and then treated with various concentrations of Vertigoheel. The concentrations used corresponded to the dosages used in clinical therapy. On the one hand the complex remedy induces – through its direct effect on beta-2 receptors and a partial rise in NO – an increase in the synthesis of the messenger substances, while at the same time inhibiting their degradation. The resulting effect is a higher concentration of the molecules, leading to the development of the protective effect of NO. The study showed that under the influence of Vertigoheel there was significant, concentration-dependent relaxation in the vascular wall. In a further fundamental investigation it was possible to show the influence of Vertigoheel on the activity of human adenylate cyclase and phosphodiesterases IV and V. From the combination of these data it is now possible to describe the signalling pathways of Vertigoheel in a scientifically established manner. However, Vertigoheel not only improves the signalling pathways of smooth muscle cells but also is an effective medication for the therapy of vertigo of varying genesis. Various clinical investigations and cohort studies have shown an equivalent effect to dimenhydrinate ( n =774), betahistine ( n =117) and ginkgo biloba ( n =170). In each case the frequency, duration and intensity of the vertigo attacks were determined, as well as the tolerability of the medication. A meta-analysis of four studies also underlined the efficacy of Vertigoheel in cases of vertigo of varying genesis.

ERYTHROPOIETIN ENHANCES OXYGENATION IN CRITICALLY PERFUSED TISSUE THROUGH MODULATION OF NITRIC OXIDE SYNTHASE

The aim of this study was to investigate the effect of human recombinant erythropoietin (EPO) on the microcirculation and oxygenation of critically ischemic tissue and to elucidate the role of endothelial NO synthase in EPO-mediated tissue protection. Island flaps were dissected from the back skin of anesthetized male Syrian golden hamsters including a critically ischemic, hypoxic area that was perfused via a collateralized vasculature. Before ischemia, animals received an injection of epoetin beta at a dose of 5,000 U/kg body weight with (n = 7) or without (n = 7) blocking NO synthase by 30 mg/kg body weight L-NAME (Nomega-nitro-L-arginine methyl ester hydrochloride). Saline-treated animals served as control (n = 7). Ischemic tissue damage was characterized by severe hypoperfusion and inflammation, hypoxia, and accumulation of apoptotic cell nuclei after 5 h of collateralization. Erythropoietin pretreatment increased arteriolar and venular blood flow by 33% and 37%, respectively (P < 0.05), and attenuated leukocytic inflammation by approximately 75% (P < 0.05). Furthermore, partial tissue oxygen tension in the ischemic tissue increased from 8.2 to 15.8 mmHg (P < 0.05), which was paralleled by a 21% increased density of patent capillaries (P < 0.05) and a 50% reduced apoptotic cell count (P < 0.05). The improved microcirculation and oxygenation were associated with a 2.2-fold (P < 0.05) increase of endothelial NO synthase protein expression. Of interest, L-NAME completely abolished all the beneficial effects of EPO pretreatment. Our study demonstrates that, in critically ischemic and hypoxic collateralized tissue, EPO pretreatment improves tissue perfusion and oxygenation in vivo. This effect may be attributed to NO-dependent vasodilative effects and anti-inflammatory actions on the altered vascular endothelium.

Effect of Sodium Nitroprusside on Lipid Peroxidation and Antioxidant Enzyme Activity of Nitraria tangutorum Bobr. Callus

The experiments were conducted with the callus of Nitraria tangutorum Bobr.for studying the changes of lipid peroxidation and antioxidase activity under treatment with SNP(Sodium Nitroprusside),as a NO donor.Investigation of NO content using laser scanning confocal microscope(LSCM) technology showed that there was a significant increase of NO level in the callus of N.tangutorum Bobr.in response to SNP treatment,but a decrease induced by L-NNA.Remarkable reduce of MDA content was observed in N.tangutorum Bobr.callus treated with low SNP concentration,but 100 μmol·L-1 SNP or 0.5 mmol·L-1 L-NNA induced enhanced MDA content in comparison with the control.Compared to the control,SNP treatment induced increase in the amount of soluble protein,whereas decrease in proline content the callus of N.tangutorum Bobr..SNP treatment resulted in increasing CAT and APX activities in a concentration-dependent manner.In addition,treatment with low SNP concentration reduced POD activity,but POD activity increased to 119% of contrast in callus treated with 50 μmol·L-1 SNP.In conclusion,the results testified that exogenous NO induced synthesis of soluble protein,enhanced antioxidant capacity and antioxidase activity in the callus of N.tangutorum Bobr.,suggesting that the protective effect of NO to N.tangutorum Bobr.cell was proved.

Palmitate-induced NO production has a dual action to reduce cell death through NO and accentuate cell death through peroxynitrite formation

The objective of this study was to determine the role of palmitate-induced stimulation of nitric oxide synthase (NOS) on palmitate-induced cell death, specifically distinguishing the effects of the subtype NOS2 from NOS3, defining the effect of NO on mitochondria death pathways, and determining whether palmitate induces peroxynitrite formation which may impact cardiomyocyte cell survival. Cardiomyocytes from embryonic chick hearts were treated with palmitate 300–500 μM. Cell death was assessed by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. The ability of palmitate to induce NO production and its consequences were tested by using the NOS inhibitor 7-nitroindazole (7-N) and the peroxynitrite scavenger (5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron (III) chloride) (FeTPPS). The effect of palmitate on the mitochondria was assessed by Western blotting for cytochrome c release into the cytosol, and assessment of mitochondrial transmembrane potential (ΔΨ m) by 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazolyl-carbocyanine iodide staining and immunocytochemistry. The NOS inhibitor 7-N, which is selective for NOS2 and not for NOS3, significantly ( p<0.05) increased palmitate-induced cell death. In contrast, 7-N did not alter cell death produced by the combination of potassium cyanide and deoxyglucose, which, respectively, inhibit glycolysis and oxidative phosphorylation. The mitochondrial actions of palmitate, specifically palmitate-induced translocation of mitochondrial cytochrome c to cytosol and loss of mitochondrial transmembrane potential, were not altered by pretreatment with 7-N. FeTPPS, which isomerizes peroxynitrite to nitrate and thereby reduces the toxic effects of peroxynitrite, produced a significant reduction in palmitate-induced cell death. In summary, these data suggest that palmitate stimulates NO production, which has a dual action to protect against cell death or to induce cell death. Palmitate-induced cell death is mediated, in part, through NO generation, which leads to peroxynitrite formation. The protective effect of NO is operative through stimulation of NOS2 but not NOS3. The actions of NO on palmitate-induced cell death are independent of mitochondrial cell death pathways.

Endothelial iNOS versus platelet iNOS: Responsibility for the platelet/leukocyte endothelial cell interaction in murine antigen induced arthritis in vivo

Since an increase of platelet-endothelial cell interactions has been observed in mice with Antigen- induced-Arthritis (AiA) as well as an increase of NO expression, the aim of our study was to investigate in vivo the influence of NO, especially the platelet and endothelial inducible NO Synthase, on the platelet- and leukocyte endothelial cell interaction. C57/Bl6 mice and iNOS deficient mice were disposed in 6 groups (each=7). After induction of AiA, rolling and adherent fluorescence labelled platelets and leukocytes were investigated by intravital microscopy (IVM) on day 8 after AiA. Rank SUM Test and ANOVA on ranks have been performed regarding the data. All arthritic mice presented an increase in platelet and leukocyte interaction with the endothelium compared to control groups. The arthritic iNOS deficient mice showed a more intense interaction of platelets and leukocytes with the endothelium in comparison with the wild-type arthritic mice. The group using arthritic wild-type recipient and iNOS deficient donor mice showed an increase in cell-interactions, leading to an endothelial effect, compared to the group using iNOS deficient arthritic recipient and wild-type donor mice. The IVM data lead to an anti-inflammatory effect of NO, since NO followed an increase in platelet- and leukocyte- endothelial cell interaction in iNOS deficient mice with AiA. In addition, we have shown for the first time in vivo that platelet NO produced by iNOS seems to have a minor influence on the leukocyte induced tissue damage in contrast to endothelial iNOS. Therefore, selective platelet inhibition would not interfere with the protective effect of NO.

NO mobilizes intracellular Zn2+ via cGMP/PKG signaling pathway and prevents mitochondrial oxidant damage in cardiomyocytes.

Our aim was to determine if NO prevents mitochondrial oxidant damage by mobilizing intracellular free zinc (Zn(2+)). Zn(2+) levels were determined by imaging enzymatically isolated adult rat cardiomyocytes loaded with Newport Green DCF. Mitochondrial membrane potential (DeltaPsi(m)) was assessed by imaging cardiomyocytes loaded with tetramethylrhodamine ethyl ester (TMRE). S-nitroso-N-acetylpenicillamine (SNAP) dramatically increased Zn(2+), which was blocked by both ODQ and NS2028, two specific inhibitors of guanylyl cyclase. The protein kinase G (PKG) inhibitor KT5823 blocked the effect of SNAP while the PKG activator 8-Br-cGMP mimicked the action of SNAP, indicating that the cGMP/PKG pathway is responsible for the effect of SNAP. The increased Zn(2+) was prevented by 5-hydroxydecanoate (5HD) but was mimicked by diazoxide, implying that mitochondrial K(ATP) channel opening may account for this effect. Since chelation of Zn(2+) blocked the preventive effect of SNAP on H(2)O(2)-induced loss of DeltaPsi(m) and exogenous zinc (1 microM ZnCl(2)) prevented dissipation of DeltaPsi(m), Zn(2+) may play a critical role in the protective effect of NO. The MEK (mitogen-activated protein kinase or extracellular signal-regulated kinase) inhibitor PD98059 blocked the preventive effects of SNAP and zinc on DeltaPsi(m), indicating that extracellular signal-regulated kinase (ERK) mediates the protective effect of both these compounds on mitochondrial oxidant damage. A Western blot analysis further showed that ZnCl(2) significantly enhances phosphorylation of ERK, confirming the involvement of ERK in the action of Zn(2+). In isolated cardiomyocytes, NO mobilizes endogenous zinc by opening mitochondrial K(ATP) channels through the cGMP/PKG pathway. In these cells, Zn(2+) may be an important mediator of the action of NO on the mitochondrial death pathway.

SNAP, a NO donor, induces cellular protection only when cortical neurons are submitted to some aggression process

Nitric oxide is a versatile molecule, which plays important physiological and pathological roles. Its protective and toxic actions have been already evidenced in several cell types. However, the protective effect in cortical neurons remains elusive. In this work, we demonstrate that the NO-donor SNAP may induce both neuroprotection and neurotoxicity in this sort of cells. The protective effect of NO was evidenced when cortical neurons were exposed to deleterious conditions, such as serum deprivation. Serum deprivation induces apoptotic cortical neuron death through a caspase-dependent mechanism. Under these conditions, SNAP was able to oppose cell death through both caspase-3 inhibition and/or increase of antiapoptotic protein levels (Bcl-2 and Bcl-x L). On the other hand, in a normally serum-supplemented medium, high dose of SNAP behaves as a neurotoxic agent, through a mechanism which involves caspase-3 activation.

Selenium deficiency increases the expression of inducible nitric oxide synthase in RAW 264.7 macrophages: role of nuclear factor-kappaB in up-regulation.

The inducible isoform of nitric oxide synthase (iNOS) is implicated in atherosclerosis, malignancy, rheumatoid arthritis, tissue and reperfusion injuries. A key determinant of the pro-oxidant versus protective effects of NO is the underlying redox status of the tissue. Selenoproteins, such as glutathione peroxidases (GPxs) and thioredoxin reductases, are key components of cellular defence and promote optimal antioxidant/oxidant balance. In this study, we have investigated the relationship between Se status, iNOS expression and NO production in Se-deficient and Se-supplemented RAW 264.7 macrophage cell lines. The cellular GPx activity, a measure of Se status, was 17-fold lower in Se-deficient RAW 264.7 cells and the total cellular oxidative tone, as assessed by flow cytometry with 2',7'-dichlorodihydrofluorescein diacetate, was higher in the Se-deficient cells than the Se-supplemented cells. Upon lipopolysaccharide (LPS) stimulation of these cells in culture, we found significantly higher iNOS transcript and protein expression levels with an increase in NO production in Se-deficient RAW 264.7 cells than the Se-supplemented cells. Electrophoretic mobility-shift assays, nuclear factor-kappaB (NF-kappaB)-luciferase reporter assays and Western blot analyses indicate that the increased expression of iNOS in Se deficiency could be due to an increased activation and consequent nuclear localization of the redox-sensitive transcription factor NF-kappaB. These results suggest an inverse relationship between cellular Se status and iNOS expression in LPS-stimulated RAW 264.7 cells and provide evidence for the beneficial effects of dietary Se supplementation in the prevention and/or treatment of oxidative-stress-mediated inflammatory diseases.