Intracellular H2O2 Concentration Research Articles

Control of the pericentrosomal H2O2 level by peroxiredoxin I is critical for mitotic progression.

Proteins associated with the centrosome play key roles in mitotic progression in mammalian cells. The activity of Cdk1-opposing phosphatases at the centrosome must be inhibited during early mitosis to prevent premature dephosphorylation of Cdh1-an activator of the ubiquitin ligase anaphase-promoting complex/cyclosome-and the consequent premature degradation of mitotic activators. In this paper, we show that reversible oxidative inactivation of centrosome-bound protein phosphatases such as Cdc14B by H2O2 is likely responsible for this inhibition. The intracellular concentration of H2O2 increases as the cell cycle progresses. Whereas the centrosome is shielded from H2O2 through its association with the H2O2-eliminating enzyme peroxiredoxin I (PrxI) during interphase, the centrosome-associated PrxI is selectively inactivated through phosphorylation by Cdk1 during early mitosis, thereby exposing the centrosome to H2O2 and facilitating inactivation of centrosome-bound phosphatases. Dephosphorylation of PrxI by okadaic acid-sensitive phosphatases during late mitosis again shields the centrosome from H2O2 and thereby allows the reactivation of Cdk1-opposing phosphatases at the organelle.

Involvement of an alternative oxidase in the regulation of hyphal growth and microsclerotial formation in Nomuraea rileyi CQNr01.

Mitochondria of Nomuraea rileyi contain an alternative oxidase (Aox), which reduces oxygen to water by accepting electrons directly from ubiquinol. Furthermore, through a transcriptional analysis, we found that an alternative oxidase (Nraox) was up-regulated during microsclerotial formation. To study the function of NrAox, Nraox was cloned from N. rileyi CQNr01. The full-length cDNA was 1266 bp with an open reading frame of 1068 bp encoding 355 amino acids. A phylogenetic analysis revealed that the NrAox of N. rileyi was closely related to Metarhizium acridum Aox. The relative expression level of the Nraox was up-regulated during microsclerotial (MS) initiation. A salicylhydroxamic acid, a specific alternative oxidase inhibitor, application to the culture media severely decreased MS yields, changed the hyphae morphology and slowed the H2O2 removal. Nraox silencing caused mycelial deformations, reduced the MS yields by 97.3 % and increased MS size compared with those of the control. MS virulence was decreased to 26.2 % after Nraox was silenced. However, the Nraox-silenced strain was sensitive to environmental stress, and the growth rate was reduced under stress conditions. The results obtained suggested that Nraox is required for MS differentiation by regulating the intracellular H2O2 concentration and hypha growth. Additionally, Nraox had a great impact on the virulence of N. rileyi.

α-Synuclein, a chemoattractant, directs microglial migration via H2O2-dependent Lyn phosphorylation

Malformed α-Synuclein (α-syn) aggregates in neurons are released into the extracellular space, activating microglia to induce chronic neuroinflammation that further enhances neuronal damage in α-synucleinopathies, such as Parkinson's disease. The mechanisms by which α-syn aggregates activate and recruit microglia remain unclear, however. Here we show that α-syn aggregates act as chemoattractants to direct microglia toward damaged neurons. In addition, we describe a mechanism underlying this directional migration of microglia. Specifically, chemotaxis occurs when α-syn binds to integrin CD11b, leading to H2O2 production by NADPH oxidase. H2O2 directly attracts microglia via a process in which extracellularly generated H2O2 diffuses into the cytoplasm and tyrosine protein kinase Lyn, phosphorylates the F-actin-associated protein cortactin after sensing changes in the microglial intracellular concentration of H2O2. Finally, phosphorylated cortactin mediates actin cytoskeleton rearrangement and facilitates directional cell migration. These findings have significant implications, given that α-syn-mediated microglial migration reaches beyond Parkinson's disease.

Tacrolimus increases Nox4 expression in human renal fibroblasts and induces fibrosis-related genes by aberrant TGF-beta receptor signalling.

Chronic nephrotoxicity of immunosuppressives is one of the main limiting factors in the long-term outcome of kidney transplants, leading to tissue fibrosis and ultimate organ failure. The cytokine TGF-β is considered a key factor in this process. In the human renal fibroblast cell line TK-173, the macrolide calcineurin inhibitor tacrolimus (FK-506) induced TGF-β-like effects, manifested by increased expression of NAD(P)H-oxidase 4 (Nox4), transgelin, tropomyosin 1, and procollagen α1(V) mRNA after three days. The macrolide mTOR inhibitor rapamycin had similar effects, while cyclosporine A did not induce fibrose-related genes. Concentration dependence curves were sigmoid, where mRNA expression was induced already at low nanomolar levels of tacrolimus, and reached saturation at 100–300 nM. The effects were independent of extracellular TGF-β as confirmed by the use of neutralizing antibodies, and thus most likely caused by aberrant TGF-β receptor signaling, where binding of tacrolimus to the regulatory FKBP12 protein results in a “leaky” TGF-β receptor. The myofibroblast marker α-smooth muscle actin was neither induced by tacrolimus nor by TGF-β1, indicating an incomplete activation of TK-173 fibroblasts under culture conditions. Tacrolimus- and TGF-β1-induced Nox4 protein upregulation was confirmed by Western blotting, and was accompanied by a rise in intracellular H2O2 concentration. Si-RNA mediated knock-down of Nox4 expression prevented up-regulation of procollagen α1(V) mRNA in tacrolimus-treated cells, but induced procollagen α1(V) expression in control cells. Nox4 knock-down had no significant effect on the other genes tested. TGF-β is a key molecule in fibrosis, and the constant activation of aberrant receptor signaling by tacrolimus might contribute to the long-term development of interstitial kidney fibrosis in immunosuppressed patients. Nox4 levels possibly play a regulatory role in these processes.

NADH: flavin oxidoreductase/NADH oxidase and ROS regulate microsclerotium development in Nomuraea rileyi

Based on transcriptome library, an NADH: flavinoxidore ductase/NADH oxidase gene (Nox) was cloned from Nomuraea rileyi. The 1,663-bp full-length cDNA contains an open reading frame of 1,233 bp coding 410 amino acids. The expression level of Nox was up-regulated and co-related to the intracellular H2O2 concentration during microsclerotium (MS) initiation. Rotenone inhibition showed that inhibition of Nox could cause a noticeable decrease in the MS yields. Silencing of Nox resulted in the MS yields, H2O2 and virulence decreased by 98.5, 38 and 21.5%, respectively. On the other hand, MS yields increased by 24.8-61% when induced by H2O2 or menadione. Furthermore, the reactive oxygen species (ROS) scavenger, ascorbic acid (up to 0.03 g ascorbic acid l(-1)), completely inhibited the formation of MS. In conclusion, the results obtained suggested that ROS promoted MS development, and that Nox was required for MS differentiation through regulation of intracellular H2O2 concentration. Besides, Nox had a great impact on the virulence in N. rileyi.

Reduction of hydrogen peroxide stress derived from fatty acid beta-oxidation improves fatty acid utilization in Escherichia coli.

Fatty acids are a promising raw material for substance production because of their highly reduced and anhydrous nature, which can provide higher fermentation yields than sugars. However, they are insoluble in water and are poorly utilized by microbes in industrial fermentation production. We used fatty acids as raw materials for l-lysine fermentation by emulsification and improved the limited fatty acid-utilization ability of Escherichia coli. We obtained a fatty acid-utilizing mutant strain by laboratory evolution and demonstrated that it expressed lower levels of an oxidative-stress marker than wild type. The intracellular hydrogen peroxide (H2O2) concentration of a fatty acid-utilizing wild-type E. coli strain was higher than that of a glucose-utilizing wild-type E. coli strain. The novel mutation rpsA D210Y identified in our fatty acid-utilizing mutant strain enabled us to promote cell growth, fatty-acid utilization, and l-lysine production from fatty acid. Introduction of this rpsA D210Y mutation into a wild-type strain resulted in lower H2O2 concentrations. The overexpression of superoxide dismutase (sodA) increased intracellular H2O2 concentrations and inhibited E. coli fatty-acid utilization, whereas overexpression of an oxidative-stress regulator (oxyS) decreased intracellular H2O2 concentrations and promoted E. coli fatty acid utilization and l-lysine production. Addition of the reactive oxygen species (ROS) scavenger thiourea promoted l-lysine production from fatty acids and decreased intracellular H2O2 concentrations. Among the ROS generated by fatty-acid β-oxidation, H2O2 critically affected E. coli growth and l-lysine production. This indicates that the regression of ROS stress promotes fatty acid utilization, which is beneficial for fatty acids used as raw materials in industrial production.

Exogenous melatonin improves Malus resistance to Marssonina apple blotch

We examined whether exogenously applied melatonin could improve resistance to Marssonina apple blotch (Diplocarpon mali) by apple [Malus prunifolia (Willd.) Borkh. cv. Donghongguo]. This serious disease leads to premature defoliation in the main regions of apple production. When plants were pretreated with melatonin, resistance was increased in the leaves. We investigated the potential roles for melatonin in modulating levels of hydrogen peroxide (H2O2), as well the activities of antioxidant enzymes and pathogenesis-related proteins during these plant-pathogen interactions. Pretreatment enabled plants to maintain intracellular H2O2 concentrations at steady-state levels and enhance the activities of plant defence-related enzymes, possibly improving disease resistance. Because melatonin is safe and beneficial to animals and humans, exogenous pretreatment might represent a promising cultivation strategy to protect plants against this pathogen infection.

A quantitative study of the cell-type specific modulation of c-Rel by hydrogen peroxide and TNF-α

Hydrogen peroxide (H2O2) at moderate steady-state concentrations synergizes with TNF-α, leading to increased nuclear levels of NF-κB p65 subunit and to a cell-type specific up-regulation of a limited number of NF-κB-dependent genes. Here, we address how H2O2 achieves this molecular specificity. HeLa and MCF-7 cells were exposed to steady-state H2O2 and/or TNF-α and levels of c-Rel, p65, IκB-α, IκB-β and IκB-ε were determined. For an extracellular concentration of 25µM H2O2, the intracellular H2O2 concentration is 3.7µM and 12.5µM for respectively HeLa and MCF-7 cells. The higher cytosolic H2O2 concentration present in MCF-7 cells may be a contributing factor for the higher activation of NF-κB caused by H2O2 in this cell line, when compared to HeLa cells. In both cells lines, H2O2 precludes the recovery of TNF-α-dependent IκB-α degradation, which may explain the observed synergism between H2O2 and TNF-α concerning p65 nuclear translocation. In MCF-7 cells, H2O2, in the presence of TNF-α, tripled the induction of c-Rel triggered either by TNF-α or H2O2. Conversely, in HeLa cells, H2O2 had a small antagonistic effect on TNF-α-induced c-Rel nuclear levels, concomitantly with a 50 % induction of IκB-ε, the preferential inhibitor protein of c-Rel dimers. The 6-fold higher c-Rel/IκB-ε ratio found in MCF-7 cells when compared with HeLa cells, may be a contributing factor for the cell-type dependent modulation of c-Rel by H2O2.Our results suggest that H2O2 might have an important cell-type specific role in the regulation of c-Rel-dependent processes, e.g. cancer or wound healing.

Thyroid and Hydrogen Peroxide

The thyroid gland produces high concentrations of hydrogen peroxide (H2O2) which is used for the biosynthesis of the thyroid hormones, triiodothyronine and thyroxine. The most important step in thyroid hormone synthesis is oxidation and organification of io dide, which itself is a very potent oxidant. To oxidize iodide, thyrocytes generate high intracellular concentrations of H2O2 using the NADPH-dependent dual oxidase enzymes (DUOX) and remove used H2O2 by various well-characterized antioxidant systems, such as those involving glutathione peroxidases and peroxiredoxins. Although thyrocytes have very intricate and efficient systems to regulate H2O2 generation and removal, leakage of H2O2 may be inevitable because of the high intracellular concentrations of H2O2 in thyrocytes. Leakage of the highly reactive oxidant H2O2 is likely to cause the damage to various intracellular proteins and DNA that has been demonstrated to be a causative factor in the induction of various diseases, such as thyroid cancer, chronic thyroiditis and hyperthyroidism. Therefore, ongoing investigation of the systems regulating H2O2 production and elimination in thyrocytesis essential for understanding the regulation and process of thyroid hormone synthesis and also for elucidating pathogenetic mechanisms underlying various autoimmune thyroid diseases and the initiation and promotion of thyroid cancers.

Controlled enzymatic production of astrocytic hydrogen peroxide protects neurons from oxidative stress via an Nrf2-independent pathway

Neurons rely on their metabolic coupling with astrocytes to combat oxidative stress. The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) appears important for astrocyte-dependent neuroprotection from oxidative insults. Indeed, Nrf2 activators are effective in stroke, Parkinson disease, and Huntington disease models. However, key endogenous signals that initiate adaptive neuroprotective cascades in astrocytes, including activation of Nrf2-mediated gene expression, remain unclear. Hydrogen peroxide (H(2)O(2)) plays an important role in cell signaling and is an attractive candidate mediator of adaptive responses in astrocytes. Here we determine (i) the significance of H(2)O(2) in promoting astrocyte-dependent neuroprotection from oxidative stress, and (ii) the relevance of H(2)O(2) in inducing astrocytic Nrf2 activation. To control the duration and level of cytoplasmic H(2)O(2) production in astrocytes cocultured with neurons, we heterologously expressed the H(2)O(2)-producing enzyme Rhodotorula gracilis D-amino acid oxidase (rgDAAO) selectively in astrocytes. Exposure of rgDAAO-astrocytes to D-alanine lead to the concentration-dependent generation of H(2)O(2). Seven hours of low-level H(2)O(2) production (∼3.7 nmol·min·mg protein) in astrocytes protected neurons from oxidative stress, but higher levels (∼130 nmol·min·mg protein) were neurotoxic. Neuroprotection occurred without direct neuronal exposure to astrocyte-derived H(2)O(2), suggesting a mechanism specific to astrocytic intracellular signaling. Nrf2 activation mimicked the effect of astrocytic H(2)O(2) yet H(2)O(2)-induced protection was independent of Nrf2. Astrocytic protein tyrosine phosphatase inhibition also protected neurons from oxidative death, representing a plausible mechanism for H(2)O(2)-induced neuroprotection. These findings demonstrate the utility of rgDAAO for spatially and temporally controlling intracellular H(2)O(2) concentrations to uncover unique astrocyte-dependent neuroprotective mechanisms.

Mammalian Numb-interacting Protein 1/Dual Oxidase Maturation Factor 1 Directs Neuronal Fate in Stem Cells

In this study, we describe a role for the mammalian Numb-interacting protein 1 (Nip1) in regulation of neuronal differentiation in stem cells. The expression of Nip1 was detected in the developing mouse brain, embryonic stem cells, primary neuronal stem cells, and retinoic acid-treated P19 embryonal carcinoma cells. The highest expression of Nip1 was observed in undifferentiated neuronal stem cells and was associated with Duox1-mediated reactive oxygen species ROS production. Ectopic nip1 expression in P19 embryonal carcinoma cells induced neuronal differentiation, and this phenotype was also linked to elevated ROS production. The neuronal differentiation in nip1-overexpressing P19 cells was achieved in a retinoic acid-independent manner and was corroborated by an increase in the expression of the neuronal basic helix-loop-helix transcription factors and neural-lineage cell markers. Furthermore, depletion of nip1 by short hairpin RNA led to a decrease in the expression of neuronal basic helix-loop-helix transcription factors and ROS. However, inhibition of ROS production in nip1-overexpressing P19 cells restricted but did not extinguish neuronal differentiation. Microarray and mass spectrometry analysis identified intermediate filaments as the principal cytoskeletal elements affected by up-regulation of nip1. We show here the first evidence for a functional interaction between Nip1 and a component of the nuclear lamina, lamin A/C. associated with a neuronal-specific phenotype. Taken together, our data reveal an important role for Nip1 in the guidance of neuronal differentiation through ROS generation and modulation of intermediate filaments and implicate Nip1 as a novel intrinsic regulator of neuronal cell fate.

Modulation of SCFβ-TrCP-dependent IκBα Ubiquitination by Hydrogen Peroxide

Reactive oxygen species are known to participate in the regulation of intracellular signaling pathways, including activation of NF-kappaB. Recent studies have indicated that increases in intracellular concentrations of hydrogen peroxide (H(2)O(2)) have anti-inflammatory effects in neutrophils, including inhibition of the degradation of I kappaB alpha after TLR4 engagement. In the present experiments, we found that culture of lipopolysaccharide-stimulated neutrophils and HEK 293 cells with H(2)O(2) resulted in diminished ubiquitination of I kappaB alpha and decreased SCF(beta-TrCP) ubiquitin ligase activity. Exposure of neutrophils or HEK 293 cells to H(2)O(2) was associated with reduced binding between phosphorylated I kappaB alpha and SCF(beta-TrCP) but no change in the composition of the SCF(beta-TrCP) complex. Lipopolysaccharide-induced SCF(beta-TrCP) ubiquitin ligase activity as well as binding of beta-TrCP to phosphorylated I kappaB alpha was decreased in the lungs of acatalasemic mice and mice treated with the catalase inhibitor aminotriazole, situations in which intracellular concentrations of H(2)O(2) are increased. Exposure to H(2)O(2) resulted in oxidative modification of cysteine residues in beta-TrCP. Cysteine 308 in Blade 1 of the beta-TrCP beta-propeller region was found to be required for maximal binding between beta-TrCP and phosphorylated I kappaB alpha. These findings suggest that the anti-inflammatory effects of H(2)O(2) may result from its ability to decrease ubiquitination as well as subsequent degradation of I kappaB alpha through inhibiting the association between I kappaB alpha and SCF(beta-TrCP).

Janus-faced signaling of cGMP in acute lung injury

The effect of increasing pulmonary endothelial cGMP concentration on endothelial function in acute lung injury appears to depend on 1) the presence of specific cGMP targets, 2) intracellular cGMP compartmentalization and 3) the timing of the increase in cGMP relative to the injury onset [1-4]. For example, we recently showed that pretreatment of pulmonary artery endothelial monolayers with 8pCPT-cGMP attenuated oxidant-induced barrier dysfunction by a cGMP-dependent kinase-1 (cGKI)-dependent mechanism [1,2]. More recently, however, we found that the increase in endogenous lung cGMP resulting from increased NO production in a ventilator-induced lung injury (VILI) mouse model caused lung endothelial barrier dysfunction [4]. The injurious effect of sGC-derived cGMP in VILI was mediated by the simultaneous generation of phosphodiesterase 2A (PDE2A), which was stimulated by cGMP to hydrolyze cAMP. Interestingly, in the same model, pretreatment with BAY 41-2272 (1.5 μM) to stimulate sGC before injurious tidal volume ventilation attenuated VILI. Recent evidence suggests that endothelial apoptosis may contribute to VILI [5] so we wondered if the protective effect of increasing lung endothelial cGMP before injury could be mediated by an anti-apoptotic effect of cGMP signalling. Mouse lung microvascular endothelial cells (MLMVEC) were isolated and purified by flow cytometry and shown to express cGK1 by Western blot and phosphorylation of VASP Ser235. A 6 hr pretreatment with 8pCPT-cGMP (50 μM), significantly attenuated H2O2-induced cell death assessed by flow cytometry (Annexin-, 7AAD-) and nuclear condensation. A similar protection was not observed in human pulmonary artery endothelial cells (HPAEC) which lack cGK1 expression in vitro. Restoration of cGK1 expression in HPAEC resulted in cGMP-mediated protection against oxidant cell death suggesting a cGK1-mediated effect. To determine if this protective effect was upstream of apoptotic signaling, MLMVEC from C57BL6 mice were treated with 8p-CPT-cGMP (50 uM) before exposure to increasing concentrations of H2O2. The extracellular H2O2 concentration ([H2O2]ext) was continuously measured with a H2O2 electrode. Compared with untreated cells, wildtype MLMVEC pre-treated with 8p-CPT-cGMP for 2 or 4 hrs (but not 30 min) significantly decreased the maximal Δ[H2O2]ext by 33 ± 11, 32 ± 10 and 25 ± 10% in cells exposed to 20, 50, and 100 μM H2O2, respectively (N = 8, P < 0.01). Consistent with this effect, 8pCPT-cGMP pretreatment attenuated H2O2-induced H2DCF fluorescence as well as p38MAPK and Akt phosphorylation suggesting that intracellular H2O2 concentration was also decreased. MLMVEC isolated from cGK1-/- mice failed to enhance H2O2 uptake suggesting cGK1-mediated signaling was responsible. An assessment of the major H2O2 degrading enzyme systems revealed a significant cGMP-mediated increase in catalase expression without an increase in catalase mRNA suggesting a post-translational effect. We conclude that the effects of cGMP signalling on lung endothelial function in acute lung injury are complex and include both injurious and protective mechanisms depending on the specific downstream signalling pathways that are present. Activation of lung microvascular endothelial cell cGK1 by cGMP protects against oxidant-mediated cell death possibly through an increase in endothelial antioxidant function.

S-Glutathionylation of the Rpn2 Regulatory Subunit Inhibits 26 S Proteasomal Function

Although increased intracellular concentrations of hydrogen peroxide (H2O2) are associated with inhibition of 26 S proteasomal activity, the mechanisms responsible for such effects have not been well delineated. In the present studies, we found that direct exposure of purified 26 S proteasomes to H2O2 had negligible effects on their activity, whereas incubation with glutathione and H2O2 produced >80% decrease in chymotrypsin-like and trypsin-like activities. Rpn1 and Rpn2, which are subunits of the 19 S regulatory particle, undergo S-glutathionylation after exposure of purified 26 S proteasomes to glutathione and H2O2, as well as in HEK 293 cells and neutrophils incubated with H2O2. Increased oxidation of Rpn1 and Rpn2 cysteine thiols was also found in lung extracts from mice in which catalase was inactivated, a condition associated with augmented intracellular concentrations of H2O2 and diminished 26 S proteasomal activity. Although unoxidized Rpn2 enhanced 20 S proteolytic function in vitro, such potentiation was not found when the 20 S core particle was incubated with oxidized Rpn2. The composition of 26 S proteasomes was not altered after exposure to glutathione and H2O2, with similar amounts of Rpn1 and Rpn2 in control or oxidized 26 S proteasomal complexes. These findings identify S-glutathionylation of Rpn2 as a contributory mechanism for H2O2-induced inhibition of 26 S proteasomal function.

The Effect of FK506 to Generate Reactive Oxygen Species on T Lymphocyte Death

Purpose: Tacrolimus (FK506) has been widely used as an immunosuppressant in organ transplanted recipients to suppress organ rejection phenomenon. We investigated the role of oxidative stress and heme oxygense-1 by FK506 on human Jurkat T cells. Methods: The cells viability was examined by DAPI stain, enzyme activity of caspase family proteins, and western blotting for Baks, PUMA, iNOS, HO-1. Cells were cultured in the absence or presence of CoPPIX or ZnPPIX and the fluorescence intensity was analyzed using a flow cytometry. Results: Treatment with FK506 increased the generation of reactive oxygen species (ROS), including hydrogen peroxide and superoxide anion, and NO in Jurkat cells in a dose-dependent manner. Immunohistochemistry and Western blot analysis data revealed the hemoxygenase-1 (HO-1) was induced by the addition of FK506 in Jurkat cells. Induction of CoPP, HO-1 inducer, resulted in decreased intracellular H₂O₂ and NO concentrations. Instead ZnPP, an HO-1 competitive inhibitor did it reversely. In addition, ZnPP regulates iNOS protein synthesis by inhibition of HO-1. Conclusion: Increase of HO-1 expression would induce to decrease the intracellular H₂O₂ and NO concentrations. Also, HO-1 would regulate iNOS protein synthesis. Consequently, we can expect the regulation of HO-1 expression with concomitants use of FK506 to suppress organ rejection phenomenon by enhancing apoptosis.

Hydrogen Peroxide Induces Hyphal Differentiation in Candida albicans

In this study, we demonstrate that hyphal differentiation is induced by the subtoxic concentration of exogenous H(2)O(2) in Candida albicans. This finding is confirmed by the changing intracellular concentration of H(2)O(2). In order to induce the same level of differentiation, low concentrations of exogenous H(2)O(2) are required for the null mutants of the thiol-specific antioxidant and catalase, while higher concentrations are needed for cells treated with ascorbic acid, an antioxidant chemical.

The antioxidative function of eicosapentaenoic acid in a marine bacterium, Shewanella marinintestina IK-1

When the eicosapentaenoic acid (EPA)-deficient mutant strain IK-1Δ8 of the marine EPA-producing Shewanella marinintestina IK-1 was treated with various concentrations of hydrogen peroxide (H2O2), its colony-forming ability decreased more than that of the wild type. Protein carbonylation, induced by treating cells with 0.01mM H2O2 under bacteriostatic conditions, was enhanced only in cells lacking EPA. The amount of cells recovered from the cultures was decreased more significantly by the presence of H2O2 for cells lacking EPA than for those producing EPA. Treatment of the cells with 0.1mM H2O2 resulted in much lower intracellular concentrations of H2O2 being consistently detected in cells with EPA than in those without EPA. These results suggest that cellular EPA can directly protect cells against oxidative damage by shielding the entry of exogenously added H2O2 in S. marinintestina IK-1.

Oxidative modification of M-type K+ channels as a mechanism of cytoprotective neuronal silencing

Voltage-gated K(+) channels of the Kv7 family underlie the neuronal M current that regulates action potential firing. Suppression of M current increases excitability and its enhancement can silence neurons. We here show that three of five Kv7 channels undergo strong enhancement of their activity by oxidative modification induced by physiological concentrations of hydrogen peroxide. A triple cysteine pocket in the channel S2-S3 linker is critical for this effect. Oxidation-induced enhancement of M current produced a hyperpolarization and a dramatic reduction of action potential firing frequency in rat sympathetic neurons. As hydrogen peroxide is robustly produced during hypoxia-induced oxidative stress, we used an oxygen/glucose deprivation neurodegeneration model that showed neuronal death to be severely accelerated by M current blockade. Such blockade had no effect on survival of normoxic neurons. This work describes a novel pathway of M-channel regulation and suggests a role for M channels in protective neuronal silencing during oxidative stress.

Hydrogen Peroxide: A Signaling Messenger

Hydrogen peroxide (H2O2) is a well-documented component of living cells. It plays important roles in host defense and oxidative biosynthetic reactions. In addition there is growing evidence that at low levels, H2O2 also functions as a signaling agent, particularly in higher organisms. This review evaluates the evidence that H2O2 functions as a signaling agent in higher organisms in light of the known biology and biochemistry of H2O2. All aerobic organisms studied to date from prokaryotes to humans appear to tightly regulate their intracellular H2O2 concentrations at relatively similar levels. Multiple biochemical strategies for rapidly reacting with these low endogenous levels of H2O2 have been elucidated from the study of peroxidases and catalases. Well-defined biochemical pathways involved in the response to exogenous H2O2 have been described in both prokaryotes and yeast. In animals and plants, regulated enzymatic systems for generating H2O2 have been described. In addition oxidation-dependent steps in signal transduction pathways are being uncovered, and evidence is accumulating regarding the nature of the specific reactive oxygen species involved in each of these pathways. Application of physiologic levels of H2O2 to mammalian cells has been shown to stimulate biological responses and to activate specific biochemical pathways in these cells.

Development of an in vitro model of excess intracellular reactive oxygen species.

These investigations characterize an in vitro model for generating excess intracellular reactive oxygen species (ROS). This novel model may be useful in the identification and delineation of cellular mechanisms associated with aging due to the link between age and excess oxidative events. The human cell line, MCF7, was stably transfected using the pSV3.neo plasmid housing a gene encoding the Aequorea victoria green fluorescent protein (GFP). Transfected cells were analyzed for maintenance of GFP over time, showing stability of the GFP gene. These studies demonstrate that the presence of fluorescing GFP significantly increases intracellular ROS, creating oxidative stress in these cells. Antioxidant supplementation was evaluated to determine the effectiveness of intracellular H2O2 reduction. The results demonstrate that supplementation with a potent antioxidant, such as reduced glutathione, protects cells from oxidative damage by decreasing intracellular concentrations of H2O2. This model for intracellular generation of excess ROS establishes a clear method by which the utility of antioxidant supplementation to protect against intracellularly generated reactive oxygen species may be evaluated.