Exogenous Application Of H2O2 Research Articles

Reactive Oxygen Species Are Required for 5-HT-Induced Transactivation of Neuronal Platelet-Derived Growth Factor and TrkB Receptors, but Not for ERK1/2 Activation

High concentrations of reactive oxygen species (ROS) induce cellular damage, however at lower concentrations ROS act as intracellular second messengers. In this study, we demonstrate that serotonin (5-HT) transactivates the platelet-derived growth factor (PDGF) type β receptor as well as the TrkB receptor in neuronal cultures and SH-SY5Y cells, and that the transactivation of both receptors is ROS-dependent. Exogenous application of H2O2 induced the phosphorylation of these receptors in a dose-dependent fashion, similar to that observed with 5-HT. However the same concentrations of H2O2 failed to increase ERK1/2 phosphorylation. Yet, the NADPH oxidase inhibitors diphenyleneiodonium chloride and apocynin blocked both 5-HT-induced PDGFβ receptor phosphorylation and ERK1/2 phosphorylation. The increases in PDGFβ receptor and ERK1/2 phosphorylation were also dependent on protein kinase C activity, likely acting upstream of NADPH oxidase. Additionally, although the ROS scavenger N-acetyl-l-cysteine abrogated 5-HT-induced PDGFβ and TrkB receptor transactivation, it was unable to prevent 5-HT-induced ERK1/2 phosphorylation. Thus, the divergence point for 5-HT-induced receptor tyrosine kinase (RTK) transactivation and ERK1/2 phosphorylation occurs at the level of NADPH oxidase in this system. The ability of 5-HT to induce the production of ROS resulting in transactivation of both PDGFβ and TrkB receptors may suggest that instead of a single GPCR to single RTK pathway, a less selective, more global RTK response to GPCR activation is occurring.

Hydrogen Sulfide Prevents Hydrogen Peroxide-Induced Activation of Epithelial Sodium Channel through a PTEN/PI(3,4,5)P3 Dependent Pathway

Sodium reabsorption through the epithelial sodium channel (ENaC) at the distal segment of the kidney plays an important role in salt-sensitive hypertension. We reported previously that hydrogen peroxide (H2O2) stimulates ENaC in A6 distal nephron cells via elevation of phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) in the apical membrane. Here we report that H2S can antagonize H2O2-induced activation of ENaC in A6 cells. Our cell-attached patch-clamp data show that ENaC open probability (PO) was significantly increased by exogenous H2O2, which is consistent with our previous finding. The aberrant activation of ENaC induced by exogenous H2O2 was completely abolished by H2S (0.1 mM NaHS). Pre-treatment of A6 cells with H2S slightly decreased ENaC PO; however, in these cells H2O2 failed to elevate ENaC PO. Confocal microscopy data show that application of exogenous H2O2 to A6 cells significantly increased intracellular reactive oxygen species (ROS) level and induced accumulation of PI(3,4,5)P3 in the apical compartment of the cell membrane. These effects of exogenous H2O2 on intracellular ROS levels and on apical PI(3,4,5)P3 levels were almost completely abolished by treatment of A6 cells with H2S. In addition, H2S significantly inhibited H2O2-induced oxidative inactivation of the tumor suppressor phosphatase and tensin homolog (PTEN) which is a negative regulator of PI(3,4,5)P3. Moreover, BPV(pic), a specific inhibitor of PTEN, elevated PI(3,4,5)P3 and ENaC activity in a manner similar to that of H2O2 in A6 cells. Our data show, for the first time, that H2S prevents H2O2-induced activation of ENaC through a PTEN-PI(3,4,5)P3 dependent pathway.

The role of hydrogen peroxide in chitosan-induced resistance to osmotic stress in rice (Oryza sativa L.)

Chitosan is a biopolymer with multiple agricultural applications. The objective of this research was to identify the mechanism required for the chitosan response. Chitosan clearly induced resistance to osmotic stress (a surrogate for drought stress) in the ‘Leung Pratew 123’ (‘LPT123’) rice (Oryza sativa L. ‘Leung Pratew123’) by enhancing plant growth and maintenance of the photosynthetic pigments during osmotic stress, but not in the derived mutated line, LPT123-TC171. Hydrogen peroxide (H2O2) was increased after osmotic stress in both lines, but higher levels were found in the LPT123 cultivar. Chitosan application did not affect the H2O2 or glutathione content under the osmotic stress condition in the LPT123 cultivar, but decreased H2O2 accumulation in the LPT123-TC171 line. The 20-fold lower glutathione level in the LPT123 cultivar suggested a low glutathione-ascorbate cycle activity that would lead to the higher H2O2 levels. Whereas, the chitosan-mediated reduction in glutathione levels in the LPT123-TC171 line during osmotic stress suggested a higher glutathione-ascorbate cycle activity leading to low H2O2 levels. Additionally, a higher peroxidase and catalase activity following chitosan treatment of the LPT123-TC171 line supports the lower observed H2O2 level. The lipid peroxidation after osmotic stress was decreased by chitosan treatment in LPT123, but not in LPT123-TC171. The exogenous H2O2 application with chitosan treatment in LPT123-TC171 could enhance plant growth during osmotic stress. It is concluded that the limited H2O2 level, the signal molecule for chitosan responses in the LPT123-TC171 line, resulted in no beneficial effects of chitosan application for osmotic stress. Therefore, H2O2 is proposed to be one of the key components for plant growth stimulation during osmotic (drought) stress by chitosan.

Hydrogen peroxide pretreatment of roots enhanced oxidative stress response of tomato under cold stress

In the view of physiological role of H2O2, we investigated whether exogenous H2O2 application would affect short-term cold response of tomato and induce acclimation. Pretreatments were performed by immersing roots into 1 mM H2O2 solution for 1 h when transferring seedlings from seedling substrate to soil (acclimated group). Cold stress (3 °C for 16 h) caused significant reduction in relative water content (RWC) of control and non-acclimated (distilled water treated) groups when compared with unstressed plants. H2O2 promoted maintenance of relatively higher RWC under stress. Anthocyanin level in leaves of acclimated plants under cold stress was significantly higher than that of unstressed control and non-acclimated plants. Malondialdehyde (MDA) levels demonstrated low temperature induced oxidative damage to control and non-acclimated plants. MDA remained around unstressed conditions in acclimated plants, which demonstrate that H2O2 acclimation protected tissues against cold induced lipid peroxidation. H2O2 acclimation caused proline accumulation in roots under cold stress. Ascorbate peroxidase (APX) activity in roots of cold stressed and unstressed H2O2 acclimated plants increased when compared with control and non-acclimated plants, with highest increase in roots of acclimated plants under cold stress. CAT levels in roots of acclimated plants also increased, whereas levels remained unchanged in unstressed plants. Endogenous H2O2 levels significantly increased in roots of control and non-acclimated plants under cold stress. On the other hand, H2O2 content in roots of acclimated plants was significantly lower than control and non-acclimated plants under cold stress. The results presented here demonstrated that H2O2 significantly enhanced oxidative stress response by elevating the antioxidant status of tomato.

Accelerated fat cell aging links oxidative stress and insulin resistance in adipocytes

Telomere shortening is emerging as a biological indicator of accelerated aging and aging-related diseases including type 2 diabetes. While telomere length measurements were largely done in white blood cells, there is lack of studies on telomere length in relation to oxidative stress in target tissues affected in diabetes. Therefore, the aim of this study is to induct oxidative stress in adipocytes and to test whether these adipocytes exhibit shortened telomeres, senescence and functional impairment. 3T3-L1 adipocytes were subjected to oxidative stress and senescence induction by a variety of means for 2 weeks (exogenous application of H2O2, glucose oxidase, asymmetric dimethylarginine (ADMA) and glucose oscillations). Cells were probed for reactive oxygen species generation (ROS), DNA damage, mRNA and protein expression of senescent and pro-inflammatory markers, telomere length and glucose uptake. Compared to untreated cells, both ROS generation and DNA damage were significantly higher in cells subjected to oxidative stress and senescence. Adipocytes subjected to oxidative stress also showed shortened telomeres and increased mRNA and protein expression of p53, p21, TNF alpha and IL-6. Senescent cells were also characterized by decreased levels of adiponectin and impaired glucose uptake. Briefly, adipocytes under oxidative stress exhibited increased ROS generation, DNA damage, shortened telomeres and switched to senescent/pro-inflammatory phenotype with impaired glucose uptake.

NaCl-induced heme oxygenase in roots of rice seedlings is mediated through hydrogen peroxide

Recent findings have suggested that H2O2 is an important signaling molecule for regulating plant responses to abiotic stress. H2O2 plays a critical role in NaCl stress. Heme oxygenase (HO) is known to play a protective role against oxidative stress. In this study, we examined the possible involvement of H2O2 in regulating NaCl-promoted HO activity in rice roots. Treatment with NaCl increased HO activity and H2O2 content in rice roots. As well, NaCl could induce OsHO1 mRNA expression. NaCl (150 mM) and NaNO3 (150 mM) were equally effective in inducing HO activity. However, mannitol at the concentration (276 mM) iso-osmotic with 150 mM NaCl had no effect on HO activity. NaCl-promoted HO activity and OsHO1 expression in rice roots was reduced by NADPH oxidase inhibitors i.e. dipehnyleneiodonium and imidazole. Moreover, exogenous application of H2O2 enhanced the activity of HO and the mRNA level of OsHO1. Our data suggest that H2O2 production plays a positive role in NaCl- induced HO activity by enhancing its mRNA level in rice roots.

Expression and functional properties of TRPM2 channels in dopaminergic neurons of the substantia nigra of the rat

Transient receptor potential melastatin 2 (TRPM2) channels are sensitive to oxidative stress, and their activation can lead to cell death. Although these channels have been extensively studied in expression systems, their role in the brain, particularly in the substantia nigra pars compacta (SNc), remains unknown. In this study, we assessed the expression and functional properties of TRPM2 channels in rat dopaminergic SNc neurons, using acute brain slices. RT-PCR analysis revealed TRPM2 mRNA expression in the SNc region. Immunohistochemistry demonstrated expression of TRPM2 protein in tyrosine hydroxylase-positive neurons. Channel function was tested with whole cell patch-clamp recordings and calcium (fura-2) imaging. Intracellular application of ADP-ribose (50-400 μM) evoked a dose-dependent, desensitizing inward current and intracellular free calcium concentration ([Ca(2+)](i)) rise. These responses were strongly inhibited by the nonselective TRPM2 channel blockers clotrimazole and flufenamic acid. Exogenous application of H(2)O(2) (1-5 mM) evoked a rise in [Ca(2+)](i) and an outward current mainly due to activation of ATP-sensitive potassium (K(ATP)) channels. Inhibition of K(+) conductance with Cs(+) and tetraethylammonium unmasked an inward current. The inward current and/or [Ca(2+)](i) rise were partially blocked by clotrimazole and N-(p-amylcinnamoyl)anthranilic acid (ACA). The H(2)O(2)-induced [Ca(2+)](i) rise was abolished in "zero" extracellular Ca(2+) concentration and was enhanced at higher baseline [Ca(2+)](i), consistent with activation of TRPM2 channels in the cell membrane. These results provide evidence for the functional expression of TRPM2 channels in dopaminergic SNc neurons. Given the involvement of oxidative stress in degeneration of SNc neurons in Parkinson's disease, further studies are needed to determine the pathophysiological role of these channels in the disease process.

Regulation of Substantia Nigra Pars Reticulata GABAergic Neuron Activity by H2O2 via Flufenamic Acid-Sensitive Channels and KATP Channels

Substantia nigra pars reticulata (SNr) GABAergic neurons are key output neurons of the basal ganglia. Given the role of these neurons in motor control, it is important to understand factors that regulate their firing rate and pattern. One potential regulator is hydrogen peroxide (H2O2), a reactive oxygen species that is increasingly recognized as a neuromodulator. We used whole-cell current clamp recordings of SNr GABAergic neurons in guinea-pig midbrain slices to determine how H2O2 affects the activity of these neurons and to explore the classes of ion channels underlying those effects. Elevation of H2O2 levels caused an increase in the spontaneous firing rate of SNr GABAergic neurons, whether by application of exogenous H2O2 or amplification of endogenous H2O2 through inhibition of glutathione peroxidase with mercaptosuccinate. This effect was reversed by flufenamic acid (FFA), implicating transient receptor potential (TRP) channels. Conversely, depletion of endogenous H2O2 by catalase, a peroxidase enzyme, decreased spontaneous firing rate and firing precision of SNr neurons, demonstrating tonic control of firing rate by H2O2. Elevation of H2O2 in the presence of FFA revealed an inhibition of tonic firing that was prevented by blockade of ATP-sensitive K+ (KATP) channels with glibenclamide. In contrast to guinea-pig SNr neurons, the dominant effect of H2O2 elevation in mouse SNr GABAergic neurons was hyperpolarization, indicating a species difference in H2O2-dependent regulation. Thus, H2O2 is an endogenous modulator of SNr GABAergic neurons, acting primarily through presumed TRP channels in guinea-pig SNr, with additional modulation via KATP channels to regulate SNr output.

Glucose‐6‐phosphate dehydrogenase‐dependent hydrogen peroxide production is involved in the regulation of plasma membrane H+‐ATPase and Na+/H+ antiporter protein in salt‐stressed callus from Carex moorcroftii

Glucose-6-phosphate dehydrogenase (G6PDH) is important for the activation of plant resistance to environmental stresses, and ion homeostasis is the physiological foundation for living cells. In this study, we investigated G6PDH roles in modulating ion homeostasis under salt stress in Carex moorcroftii callus. G6PDH activity increased to its maximum in 100 mM NaCl treatment and decreased with further increased NaCl concentrations. K+/Na+ ratio in 100 mM NaCl treatment did not exhibit significant difference compared with the control; however, in 300 mM NaCl treatment, it decreased. Low-concentration NaCl (100 mM) stimulated plasma membrane (PM) H+-ATPase and NADPH oxidase activities as well as Na+/H+ antiporter protein expression, whereas high-concentration NaCl (300 mM) decreased their activity and expression. When G6PDH activity and expression were reduced by glycerol treatments, PM H+-ATPase and NADPH oxidase activities, Na+/H+ antiporter protein level and K+/Na+ ratio dramatically decreased. Simultaneously, NaCl-induced hydrogen peroxide (H₂O₂) accumulation was abolished. Exogenous application of H₂O₂ increased G6PDH, PM H+-ATPase and NADPH oxidase activities, Na+/H+ antiporter protein expression and K+/Na+ ratio in the control and glycerol treatments. Diphenylene iodonium (DPI), the NADPH oxidase inhibitor, which counteracted NaCl-induced H₂O₂ accumulation, decreased G6PDH, PM H+-ATPase and NADPH oxidase activities, Na+/H+ antiporter protein level and K+/Na+ ratio. Western blot result showed that G6PDH expression was stimulated by NaCl and H₂O₂, and blocked by DPI. Taken together, G6PDH is involved in H₂O₂ accumulation under salt stress. H₂O₂, as a signal, upregulated PM H+-ATPase activity and Na+/H+ antiporter protein level, which subsequently resulted in the enhanced K+/Na+ ratio. G6PDH played a central role in the process.

Salt stress-induced expression of rice AOX1a is mediated through an accumulation of hydrogen peroxide

Treatment with 300 mM NaCl increased the capacity of the alternative respiratory pathway and induced the expression of AOX1a of the leaves of rice (Oryza sativa L.) seedlings. A significant increase in the content of hydrogen peroxide (H2O2) was observed in rice leaves treated with 300 mM NaCl. However, NaCl at 150 mM did not significantly affect the capacity of the alternative respiratory pathway, the content of H2O2, and the transcript level of AOX1a. Exogenous application of H2O2 enhanced the levels of the capacity of the alternative respiratory pathway and AOX1a expression. The accumulation of H2O2 in rice leaves in response to 300 mM NaCl was inhibited by the pretreatment with dimethylthiourea (DMTU, scavenger of H2O2). This treatment also suppressed the induction of AOX1a expression and the increase in the capacity of the alternative respiratory pathway under 300 mM NaCl stress. Moreover, the salt-stressed (300 mM NaCl) seedlings pretreated with 1 mM salicylhydroxamic acid (SHAM, a special inhibitor of alternative oxidase) had higher level of H2O2 production than the seedlings either subjected to 300 mM NaCl stress or SHAM treatment alone did. These observations suggest that the expression of AOX1a in response to higher salt stress is mediated through an accumulation of H2O2 and alternative oxidase could play a role in antioxidant protection under the condition of higher salt stress.

Acclimatory response to hydrogen peroxide and glutathione under salt-boron stress through their impact on mineral nutrition and antioxidant defense system in pigeonpea (Cajanus cajan L.)

Investigations were carried out on a salt tolerant (Manak, H77-216) and a comparatively salt sensitive (ICPL 88039) genotypes of pigeonpea (Cajanus cajan L. Millsp.) under NaCl, B and NaCl + B stress to examine the acclimatory response to H2O2, glutathione and H2O2 + glutathione through their effect on mineral nutrition, morpho-physiological parameters and antioxidant defense system. Both B and NaCl alone and their combinations had deleterious effect on dry biomass of plumule, enhanced relative stress injury (RSI), lipid peroxidation with concomitant increase in Na, Cl and B contents. However it did not bear any correlation with osmotic potential of plumule and K contents. Antioxidative enzymes like superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), peroxidase (POX) and glutathione reductase (GR) also decreased with salt, B and salt + B treatments. However contents of H2O2 enhanced and that of ascorbate declined under aforementioned treatments. These injurious effects are partially alleviated by exogenous application of H2O2; glutathione (GSH) and H2O2 + GSH treatments. The role of H2O2 and GSH in the present study is suggestive of triggering multifunctional signal transduction in plant defense mechanisms to prevent cellular oxidation, membrane injury, lipid peroxidation and protein enzyme inactivation.

Salicylic acid-mediated hydrogen peroxide accumulation and protection against Cd toxicity in rice leaves

The role of H2O2 in salicylic acid (SA)-induced protection of rice leaves against subsequent Cd toxicity was investigated. SA pretreatment resulted in an increase in the contents of endogenous SA, as judged by the expression of OsWRKY45 (a SA responsive gene), and H2O2 in rice leaves. Diphenyleneiodonium (DPI) and imidazole (IMD), inhibitors of NADPH oxidase, prevented SA-increased H2O2 production, suggesting that NADPH oxidase is a H2O2-generating enzyme in SA-pretreated rice leaves. DPI and IMD also inhibited SA-increased activities of superoxide dismutase (SOD), ascorbate peroixdase (APX), and glutathione reductase (GR) activities, but had no effect on SA-increased catalase (CAT) activity. Moreover, SA-induced protection against subsequent Cd toxicity could also be prevented by DPI and IMD. The inhibitory effect of DPI and IMD on SA-induced protection against subsequent Cd toxicity could be reversed by exogenous H2O2. All these results suggested that SA-induced protection against subsequent Cd toxicity is mediated through H2O2. This conclusion is supported further by the observations that exogenous H2O2 application resulted in an increase in SOD, APX, and GR activities, but not CAT activity and a protection against subsequent Cd toxicity of rice leaves.

Glycinebetaine‐induced water‐stress tolerance in codA‐expressing transgenic indica rice is associated with up‐regulation of several stress responsive genes

Rice (Oryza sativa L.), a non-accumulator of glycinebetaine (GB), is highly susceptible to abiotic stress. Transgenic rice with chloroplast-targeted choline oxidase encoded by the codA gene from Arthrobacter globiformis has been evaluated for inheritance of transgene up to R5 generation and water-stress tolerance. During seedling, vegetative and reproductive stages, transgenic plants could maintain higher activity of photosystem II and they show better physiological performance, for example, enhanced detoxification of reactive oxygen species compared to wild-type plants under water-stress. Survival rate and agronomic performance of transgenic plants is also better than wild-type following prolonged water-stress. Choline oxidase converts choline into GB and H2O2 in a single step. It is possible that H2O2/GB might activate stress response pathways and prepare transgenic plants to mitigate stress. To check this possibility, microarray-based transcriptome analysis of transgenic rice has been done. It unravelled altered expression of many genes involved in stress responses, signal transduction, gene regulation, hormone signalling and cellular metabolism. Overall, 165 genes show more than two-fold up-regulation at P-value < 0.01 in transgenic rice. Out of these, at least 50 genes are known to be involved in plant stress response. Exogenous application of H2O2 or GB to wild-type plants also induces such genes. Our data show that metabolic engineering for GB is a promising strategy for introducing stress tolerance in crop plants and which could be imparted, in part, by H2O2- and/or GB-induced stress response genes.

Hydrogen peroxide increases cerebral arterial KCa channel opening through activation of Akt signaling pathway

The oxidant hydrogen peroxide (H2O2) is not only an inducer of cellular damage, but can also play a role as a modulator of vascular function. Exogenous application of H2O2 (3‐300 µM) resulted in a concentration‐dependent increase in NPo of KCa single‐channel currents recorded from cell attached patches at a patch potential of +40 mV, whereas this oxidant did not change the NPo of KCa single‐channel current when recorded from excised inside‐out membrane patches of cerebral arterial muscle cells (CAMCs). The membrane permeant organic peroxide tert‐butyl hydroperoxide (t‐BOOH) (30 µM) also induced similar increase in NPo of KCa single‐channel currents in the cell‐attached patches but had no effect on NPo of the single‐channel KCa currents recorded from excised inside‐out patches at a the same patch potential. In an attempt to elucidate possible signaling mechanism mediating the effects of these oxidants on KCa channel currents in CAMCs, we examined the impact of inhibition of the phospoinositide 3‐kinase‐Akt (PI3K‐Akt) pathway on the stimulatory actions of both H2O2 and t‐BOOH on KCa channel current. Pretreatment of CAMCs with two structurally different PI3K‐Akt pathway inhibitors wortmannin (10 nM) and LY294002 (5 µM) significantly attenuated the H2O2‐ or t‐BOOH‐induced increase in NPo of single‐channel KCa currents in CAMCs. On the other hand, stimulation of cerebral arterial muscle with either H2O2 or t‐BOOH for 10 minutes induced activation of Akt as determined by western blot analysis. These findings suggest that activation of the Akt signaling pathway contributes to the H2O2‐or t‐BOOH‐induced enhancement of KCa channel currents in cerebral arterial muscle cells.

Chilling Tolerance of Wheat Seedlings Is Related to an Enhanced Alternative Respiratory Pathway

Pretreatment with exogenous hydrogen peroxide (H2O2) mitigated the water loss of wheat leaves exposed to 4°C for 24 h. The same treatment also partially reduced the increases of electrolyte leakage and H2O2 production of the chilled plants. These observations suggest that H2O2 pretreatment could effectively induce chilling tolerance. The capacity of the alternative respiratory pathway and the expression of the gene encoding the alternative oxidase (AOX1) were substantially elevated by H2O2 treatment under either normal or chilling conditions. Salicylhydroxamic acid (SHAM, an inhibitor of the alternative respiratory pathway) decreased the tolerance of plants to chilling conditions and reversed the chilling tolerance induced by exogenous application of H2O2. These observations indicate that the tolerance of plants to chilling might be related to an enhanced alternative respiratory pathway.

Variations of vinblastine accumulation and redox state affected by exogenous H2O2 in Catharanthus roseus (L.) G. Don

Effects of exogenous H2O2 application on vinblastine (VBL) and its precursors, vindoline (VIN), catharanthine (CAT) and α-3′,4′-anhydrovinblastine (AVBL), were measured in Catharanthus roseus seedlings in order to explore possible correlation of VBL formation with oxidative stress. VBL accumulation has previously been shown to be regulated by an in vitro H2O2-dependent peroxidase (POD)-like synthase. Experimental exposure of plants to different concentrations of H2O2 showed that endogenous H2O2 and alkaloid concentrations in leaves were positively elevated. The time-course variations of alkaloid concentrations and redox state, reflected by the concentrations of H2O2, ascorbic acid (AA), oxidative product of glutathione (GSSG) and POD activity, were significantly altered due to H2O2 application. The further correlation analysis between alkaloids and redox status indicated that VBL production was tightly correlated with redox status. These results provide a new link between VBL metabolisms and redox state in C. roseus.

Glucose-6-phosphate dehydrogenase plays a central role in modulating reduced glutathione levels in reed callus under salt stress

In the present study, we investigated the role of glucose-6-phosphate dehydrogenase (G6PDH) in regulating the levels of reduced form of glutathione (GSH) to the tolerance of calli from two reed ecotypes, Phragmites communis Trin. dune reed (DR) and swamp reed (SR), in a long-term salt stress. G6PDH activity was higher in SR callus than that of DR callus under 50-150 mM NaCl treatments. In contrast, at higher NaCl concentrations (300-600 mM), G6PDH activity was lower in SR callus. A similar profile was observed in GSH contents, glutathione reductase (GR) and glutathione peroxidase (GPX) activities in both salt-stressed calli. After G6PDH activity and expression were reduced in glycerol treatments, GSH contents and GR and GPX activity decreased strongly in both calli. Simultaneously, NaCl-induced hydrogen peroxide (H2O2) accumulation was also abolished. Exogenous application of H2O2 increased G6PDH, GR, and GPX activities and GSH contents in the control conditions and glycerol treatment. Diphenylene iodonium (DPI), a plasma membrane (PM) NADPH oxidase inhibitor, which counteracted NaCl-induced H(2)O(2) accumulation, decreased these enzymes activities and GSH contents. Furthermore, exogenous application of H2O2 abolished the N-acetyl-L: -cysteine (NAC)-induced decrease in G6PDH activity, and DPI suppressed the effect of buthionine sulfoximine (BSO) on induction of G6PDH activity. Western-blot analyses showed that G6PDH expression was stimulated by NaCl and H2O2, and blocked by DPI in DR callus. Taken together, G6PDH activity involved in GSH maintenance and H2O2 accumulation under salt stress. And H2O2 regulated G6PDH, GR, and GPX activities to maintain GSH levels. In the process, G6PDH plays a central role.

Spatial variation in H2O2 response of Arabidopsis thaliana root epidermal Ca2+ flux and plasma membrane Ca2+ channels

Hydrogen peroxide is an important regulatory agent in plants. This study demonstrates that exogenous H2O2 application to Arabidopsis thaliana root epidermis results in dose-dependent transient increases in net Ca2+ influx. The magnitude and duration of the transients were greater in the elongation zone than in the mature epidermis. In both regions, treatment with the cation channel blocker Gd3+ prevented H2O2-induced net Ca2+ influx, consistent with application of exogenous H2O2 resulting in the activation of plasma membrane Gd3+-sensitive Ca2+-influx pathways. Application of 10 mm H2O2 to the external plasma membrane face of elongation zone epidermal protoplasts resulted in the appearance of a hyperpolarization-activated Ca2+-permeable conductance. This conductance differed from that previously characterized as being responsive to extracellular hydroxyl radicals. In contrast, in mature epidermal protoplasts a plasma membrane hyperpolarization-activated Ca2+-permeable channel was activated only when H2O2 was present at the intracellular membrane face. Channel open probability increased with intracellular [H2O2] and at hyperpolarized voltages. Unitary conductance decreased thus: Ba2+ > Ca2+ (14.5 pS) > Mg2+ > Zn2+ (20 mM external cation, 1 mM H2O2). Lanthanides and Zn2+ (but not TEA+) suppressed the open probability without affecting current amplitude. The results suggest spatial heterogeneity and differential sensitivity of Ca2+ channel activation by reactive oxygen species in the root that could underpin signalling.

Depletion of intracellular Ca2+ stores enhances flow‐induced vascular dilatation in rat small mesenteric artery

The effect of depleting intracellular Ca2+ stores on flow-induced vascular dilatation and the mechanism responsible for the vasodilatation were examined in rat isolated small mesenteric arteries. The arteries were pressurized to 50 mmHg and preconstricted with phenylephrine. Intraluminal flow reversed the effect of phenylephrine, resulting in vasodilatation. Flow dilatation consisted of an initial transient peak followed by a sustained plateau phase. The magnitude of dilatation was markedly reduced by removing Ca2+ from the intraluminal flow medium. Depletion of intracellular Ca2+ stores with either cyclopiazonic acid (CPA, 2 microM) or 1,4-dihydroxy-2,5-di-tert-butylbenzene (BHQ, 10 microM) significantly augmented the magnitude of flow dilatation. Flow-induced endothelial cell Ca2+ influx was also markedly enhanced in arteries pretreated with CPA or BHQ.Flow-induced dilatation was insensitive to Nw-nitro-L-arginine methyl ester (100 microM) plus indomethacin (3 microM) or to oxyhemoglobin (3 microM), but was markedly reduced by 30 mM extracellular K+ or 2 mM tetrabutylammonium (TBA), suggesting an involvement of EDHF. Catalase at 1200 U ml-1 abolished the flow-induced dilatation, while the application of exogenous H2O2 (90-220 microM) induced relaxation in phenylephrine-preconstricted arteries. Relaxation to exogenous H2O2 was blocked in the presence of 30 mM extracellular K+, and H2O2 (90 microM) hyperpolarized the smooth muscle cells, indicating that H2O2 can act as an EDHF. In conclusion, flow-induced dilatation in rat mesenteric arteries can be markedly enhanced by prior depletion of intracellular Ca2+ stores. Furthermore, these data are consistent with a role for H2O2 as the vasodilator involved.

Endogenous Hydrogen Peroxide Regulates the Excitability of Midbrain Dopamine Neurons via ATP-Sensitive Potassium Channels

ATP-sensitive K+ (K(ATP)) channels link metabolic state to cell excitability. Here, we examined regulation of K(ATP) channels in substantia nigra dopamine neurons by hydrogen peroxide (H2O2), which is produced in all cells during aerobic metabolism. Blockade of K(ATP) channels by glibenclamide (100 nM) or depletion of intracellular H2O2 by including catalase, a peroxidase enzyme, in the patch pipette increased the spontaneous firing rate of all dopamine neurons tested in guinea pig midbrain slices. Using fluorescence imaging with dichlorofluorescein to visualize intracellular H2O2, we found that moderate increases in H2O2 during partial inhibition of glutathione (GSH) peroxidase by mercaptosuccinate (0.1-0.3 mM) had no effect on dopamine neuron firing rate. However, with greater GSH inhibition (1 mM mercaptosuccinate) or application of exogenous H2O2, 50% of recorded cells showed K(ATP) channel-dependent hyperpolarization. Responsive cells also hyperpolarized with diazoxide, a selective opener for K(ATP) channels containing sulfonylurea receptor SUR1 subunits, but not with cromakalim, a selective opener for SUR2-based channels, indicating that SUR1-based K(ATP) channels conveyed enhanced sensitivity to elevated H2O2. In contrast, when endogenous H2O2 levels were increased after inhibition of catalase, the predominant peroxidase in the substantia nigra, with 3-amino-1,2,4-triazole (1 mM), all dopamine neurons responded with glibenclamide-reversible hyperpolarization. Fluorescence imaging of H2O2 indicated that catalase inhibition rapidly amplified intracellular H2O2, whereas inhibition of GSH peroxidase, a predominantly glial enzyme, caused a slower, smaller increase, especially in nonresponsive cells. Thus, endogenous H2O2 modulates neuronal activity via K(ATP) channel opening, thereby enhancing the reciprocal relationship between metabolism and excitability.