Nitrosative Stress Conditions Research Articles

Reactive nitrogen species induce nuclear factor-κB-mediated protein degradation in skeletal muscle cells

Recently, a role for NF-κB in upregulation of proteolytic systems and protein degradation has emerged. Reactive nitrogen species (RNS) have been demonstrated to induce NF-κB activation. The aim of this study was to investigate whether RNS caused increased proteolysis in skeletal muscle cells, and whether this process was mediated through the activation of NF-κB. Fully differentiated L6 myotubes were treated with NO donor SNAP, peroxynitrite donor SIN-1, and authentic peroxynitrite, in a time-dependent manner. NF-κB activation, the activation of the ubiquitin-proteasome pathway and matrix metalloproteinases, and the levels of muscle-specific proteins (myosin heavy chain and telethonin) were investigated under the conditions of nitrosative stress. RNS donors caused NF-κB activation and increased activation of proteolytic systems, as well as the degradation of muscle-specific proteins. Antioxidant treatment, tyrosine nitration inhibition, and NF-κB molecular inhibition were proven effective in downregulation of NF-κB activation and slowing down the degradation of muscle-specific proteins. Peroxynitrite, but not NO, causes proteolytic system activation and the degradation of muscle-specific proteins in cultured myotubes, mediated through NF-κB. NF-κB inhibition by antioxidants, tyrosine nitration, and molecular inhibitors may be beneficial for decreasing the extent of muscle damage induced by RNS.

Antioxidant Properties of Nitric Oxide in Cellular Physiological and Pathophysiological Mechanisms. The Implications of Biological Balance between NO and Oxidative Stress

The function of nitric oxide ( NO) in pathophysiology remains confounding as both protective and cytotoxic effects of NO have been demonstrated in many disease processes. Nitric oxide chemistry culminating in the generation of oxidative as well as nitrosative intermediates have generally been proposed as mediators of pathophysiology and have overshadowed the antioxidant capabilities of NO. However, the counteracting role of NO in providing a balance under conditions of oxidative and nitrosative stress has been underappreciated. The purpose of this review is the discussion of the role of NO as an antioxidant and interceptor of more potent reactive intermediates in normal physiology and disease.

Vitreoscilla hemoglobin promoter is not responsive to nitrosative and oxidative stress in Escherichia coli

The Vitreoscilla hemoglobin gene ( vhb) is expressed under oxygen-limited conditions via an FNR-dependent mechanism. Furthermore, cAMP-CRP has been implicated in its regulation. Recently, VHb protein has been reported to protect a heterologous host from nitrosative stress. In this study we analyzed the regulation of the Vitreoscilla hemoglobin promoter (P vhb) in Escherichia coli under nitrosative and oxidative stress conditions. Our results show unambiguously that expression of neither VHb nor chloramphenicol acetyltransferase under the control of P vhb is induced under the experimental conditions used. Thus, a clear discrepancy between in vivo function, i.e. protection against nitrosative stress, and regulation of gene expression is obvious. The regulation of P vhb reported here is in clear contrast to the expression pattern of flavohemoglobins from various microorganisms, which are generally induced by nitrosative stress. However, the length of P vhb is only 146 bp and therefore, we cannot rule out that additional regulatory sequences may be located in the upstream region of P vhb.

Preconditioning with millimolar concentrations of Vitamin C or N-acetylcysteine protects L6 muscle cells insulin-stimulated viability and DNA synthesis under oxidative stress

The effect of reactive oxygen/nitrogen species (ROS/RNS)(hydrogen peroxide — H 2O 2, superoxide anion radical O 2 − and hydroxyl radical OH — the reaction products of hypoxanthine/xanthine oxidase system), nitric oxide (NO from sodium nitroprusside — SNP), and peroxynitrite (ONOO − from 3-morpholinosydnonimine — SIN-1) on insulin mitogenic effect was studied in L6 muscle cells after one day pretreatment with/or without antioxidants. ROS/RNS inhibited insulin-induced mitogenicity (DNA synthesis). Insulin (0.1 μM), however, markedly improved mitogenicity in the muscle cells treated with increased concentrations (0.1, 0.5, 1 mM) of donors of H 2O 2, O 2 − , OH, ONOO − and NO . Cell viability assessed by morphological criteria was also monitored. Massive apoptosis was induced by 1 mM of donors of H 2O 2 and ONOO −, while NO additionally induced necrotic cell death. Taken together, these results have shown that ROS/RNS provide a good explanation for the developing resistance to the growth promoting activity of insulin in myoblasts under conditions of oxidative or nitrosative stress. Cell viability showed that neither donor induced cell death when given below 0.5 mM. In order to confirm the deleterious effects of ROS/RNS prior to the subsequent treatment with ROS/RNS plus insulin one day pretreatment with selected antioxidants (sodium ascorbate — ASC (0.01, 0.1, 1 mM), or N-acetylcysteine — NAC (0.1, 1, 10 mM) was carried out. Surprisingly, at a low dose (micromolar) antioxidants did not abrogate and even worsened the concentration-dependent effects of ROS/RNS. In contrast, pretreatment with millimolar dose of ASC or NAC maintained an elevated mitogenicity in response to insulin irrespective of the ROS/RNS donor type used.

Regulation of microsomal and cytosolic glutathione S-transferase activities by S-nitrosylation

There is increasing evidence that S-nitrosylation is a mechanism for the regulation of protein function via the modification of critical sulfhydryl groups. The activity of rat liver microsomal glutathione S-transferase (GST) is increased after treatment with N-ethylmaleimide (NEM), a sulfhydryl alkylating reagent, and is also increased under conditions of oxidative stress. In the present study, preincubation of purified rat liver microsomal GST with S-nitrosoglutathione (GSNO) or the nitric oxide (NO) donor, 1,1-diethyl-2-hydroxy-2-nitrosohydrazine (DEA/NO), resulted in a 2-fold increase in enzyme activity. This increase in activity was reversed by dithiothreitol. The initial treatment of microsomal GST with either GSNO or DEA/NO was associated with an 85% loss of free sulfhydryl groups. After removal of the nitrosylating agents over a 6-hr period, approximately 50% of the enzyme was still nitrosylated, as determined by redox chemiluminescence. Furthermore, preincubation of either purified enzyme or hepatic microsomes with GSNO or DEA/NO prevented further enzyme activation by NEM, suggesting that NEM and the NO donors interact with a common population of sulfhydryl groups in the enzyme. In contrast, both NEM and NO donors partially inhibited the activity of cytosolic GST isoforms. The inhibitory activity of NEM and NO donors was much more evident when the GST pi isoform was used instead of a mixture of GST isoforms. These data suggest that there may be differential regulation of microsomal and cytosolic GST activities under conditions of nitrosative stress.

Cross-talk between nitric oxide and superoxide determines ceramide formation and apoptosis in glomerular cells

The modulation of cell signaling by nitric oxide (NO) and superoxide (O(-)(2)) is associated with apoptotic cell death in inflammatory kidney diseases. Recently, we have shown that NO induces ceramide production in glomerular mesangial and endothelial cells and the ratio of NO and O(-)(2) determines whether cells live or die. Glomerular endothelial and mesangial cells were labeled with [(14)C]serine, the precursor of all sphingolipids, then stimulated with reactive oxygen species- or reactive nitrogen species-generating substances and subjected to lipid extraction. Radioactive lipids were separated and analyzed by thin-layer chromatography. DNA fragmentation, as a characteristic feature of apoptosis, was measured by a nucleosome/DNA-ELISA, which quantitatively recorded the histone-associated DNA fragments. Exposure of glomerular endothelial and mesangial cells to either NO donors or superoxide-generating substances led to a delayed and sustained ceramide formation that paralleled the induction of apoptosis in both cell types. Coincubation of endothelial cells with NO and superoxide, which led to the generation of peroxynitrite, caused a synergistic enhancement of ceramide generation and apoptosis when compared to either stimulus alone. By contrast, in glomerular mesangial cells costimulation with superoxide neutralized not only NO-induced apoptosis but also NO-induced ceramide formation, although O(-)(2) alone triggered ceramide formation in mesangial cells and caused cell death. Moreover, SIN-1, a substance that simultaneously releases NO and O(-)(2) and thereby generates peroxynitrite, also stimulated a delayed ceramide formation in endothelial cells but not in mesangial cells. Furthermore, exposure of endothelial cells to glucose oxidase, which generates hydrogen peroxide, or to exogenous hydrogen peroxide, also showed a dose-dependent increase in ceramide formation and apoptosis, although to a lesser extent than did superoxide. These data suggest that ceramide represents an important mediator of reactive oxygen and nitrogen species-triggered cell responses, like apoptosis. There seem to be cell type-specific protective mechanisms that critically depend on a fine-tuned redox balance between reactive nitrogen and oxygen species to determine whether a cell undergoes apoptosis or survives when exposed to oxidative and/or nitrosative stress conditions.

Involvement of Glyoxalase I in NO-induced Oxidative Stress

To understand the molecular dynamics in response to nitric oxide-induced oxidative stress under infectious and inflammatory conditions, we have analyzed protein expression patterns of human endothelial cells with or without treatment of nitric oxide donors. We identified glyoxalase I as a novel nitric oxide responsive protein using two-dimensional polyacrylamide gel electrophoresis. Mammalian glyoxalase I can interact with S-nitrosoglutathione at the substrate recognition site, resulting in an inactive from with a reductant reversible chemical modification. The oxidative response of glyoxalase I is specific to nitric oxide and more sensitive than that of glyceraldehyde-3-phosphate dehydrogenase which is a well-known enzyme inactivated by nitric oxide. In this review, we introduce the characteristics of glyoxalase I as a nitric oxide responsive protein and hypothesize the roles of glyoxalase I under nitrosative stress conditions.

Study of enterostatin on appetite and preference-regulation

Oxidative and nitrosative stress are major factors in neuronal cell death underlying neurodegenerative disease. Thus, supplementation of antioxidant defenses may be an effective therapeutic strategy for diseases such as amyotrophic lateral sclerosis, Parkinson's disease, and Alzheimer's disease. In this regard, treatment with nutraceutical antioxidants has garnered increasing attention; however, the differential neuroprotective effects of structurally similar nutraceuticals, which may affect their suitability as therapeutic agents, has not been directly examined. In this study we compare the ability of two anthocyanins, callistephin (pelargonidin-3-O-glucoside) and kuromanin (cyanidin-3-O-glucoside) to protect cerebellar granule neurons from damage induced by either oxidative or nitrosative stress. These anthocyanins differ by the presence of a single hydroxyl group on the B-ring of kuromanin, forming a catechol moiety. While both compounds protected neurons from oxidative stress induced by glutamate excitotoxicity, a stark contrast was observed under conditions of nitrosative stress. Only kuromanin displayed the capacity to defend neurons from nitric oxide (NO)-induced apoptosis. This protective effect was blocked by addition of Cu, Zn-superoxide dismutase, indicating that the neuroprotective mechanism is superoxide dependent. Based on these observations, we suggest a unique mechanism by which slight structural variances, specifically the absence or presence of a catechol moiety, lend kuromanin the unique ability to generate superoxide, which acts as a scavenger of NO. These findings indicate that kuromanin and compounds that share similar chemical characteristics may be more effective therapeutic agents for treating neurodegenerative diseases than callistephin and related (non-catechol) compounds.

2012 Study of enterostatin on appetite and preference-regulation

Oxidative and nitrosative stress are major factors in neuronal cell death underlying neurodegenerative disease. Thus, supplementation of antioxidant defenses may be an effective therapeutic strategy for diseases such as amyotrophic lateral sclerosis, Parkinson's disease, and Alzheimer's disease. In this regard, treatment with nutraceutical antioxidants has garnered increasing attention; however, the differential neuroprotective effects of structurally similar nutraceuticals, which may affect their suitability as therapeutic agents, has not been directly examined. In this study we compare the ability of two anthocyanins, callistephin (pelargonidin-3-O-glucoside) and kuromanin (cyanidin-3-O-glucoside) to protect cerebellar granule neurons from damage induced by either oxidative or nitrosative stress. These anthocyanins differ by the presence of a single hydroxyl group on the B-ring of kuromanin, forming a catechol moiety. While both compounds protected neurons from oxidative stress induced by glutamate excitotoxicity, a stark contrast was observed under conditions of nitrosative stress. Only kuromanin displayed the capacity to defend neurons from nitric oxide (NO)-induced apoptosis. This protective effect was blocked by addition of Cu, Zn-superoxide dismutase, indicating that the neuroprotective mechanism is superoxide dependent. Based on these observations, we suggest a unique mechanism by which slight structural variances, specifically the absence or presence of a catechol moiety, lend kuromanin the unique ability to generate superoxide, which acts as a scavenger of NO. These findings indicate that kuromanin and compounds that share similar chemical characteristics may be more effective therapeutic agents for treating neurodegenerative diseases than callistephin and related (non-catechol) compounds.