Glucose-6-phosphate dehydrogenase acts as a regulator of cell redox balance in rice suspension cells under salt stress

The reduced coenzyme nicotinamide-adenine dinucleotide phosphate (NADPH) is an important molecule in cellular redox balance. Glucose-6-phosphate dehydrogenase (G6PDH) is a key enzyme in the pentose phosphate pathway, the most important NADPH-generating pathway. In this study, roles of G6PDH in maintaining cell redox balance in rice suspension cells under salt stress were investigated. Results showed that the G6PDH activity decreased in the presence of 80 mM NaCl on day 2. Application of exogenous glucose stimulated the activity of G6PDH and NADPH oxidase under salt stress. Exogenous glucose also increased the ion leakage, thiobarbituric acid reactive substances and hydrogen peroxide (H₂O₂) contents in the presence of 80 mM NaCl on day 2, implying that the reduction of the G6PDH activity was necessary to avoid serious damage caused by salt stress. The NAPDH/NADP⁺ ratio increased on day 2 but decreased on day 4 under 80 mM NaCl plus glucose treatment. Diphenyleneiodonium, an NADPH oxidase inhibitor, decreased the H₂O₂ content under 80 mM NaCl treatment on day 2. These results imply that the H₂O₂ accumulation induced by glucose treatment under salt stress on day 2 was related to the NADPH oxidase. Western-blot analysis showed that the G6PDH expression was slightly induced by glucose and was obviously blocked by DPI on day 2 under salt stress. In conclusion, G6PDH plays a key role in maintaining the cell redox balance in rice suspension cells under salt stress. The coordination of G6PDH and NADPH oxidase is required in maintaining cell redox balance in salt tolerance.     

SIRT5 promotes IDH2 desuccinylation and G6PD deglutarylation to enhance cellular antioxidant defense

Excess in mitochondrial reactive oxygen species (ROS) is considered as a major cause of cellular oxidative stress. NADPH, the main intracellular reductant, has a key role in keeping glutathione in its reduced form GSH, which scavenges ROS and thus protects the cell from oxidative damage. Here, we report that SIRT5 desuccinylates and deglutarylates isocitrate dehydrogenase 2 (IDH2) and glucose‐6‐phosphate dehydrogenase (G6PD), respectively, and thus activates both NADPH‐producing enzymes. Moreover, we show that knockdown or knockout of SIRT5 leads to high levels of cellular ROS. SIRT5 inactivation leads to the inhibition of IDH2 and G6PD, thereby decreasing NADPH production, lowering GSH, impairing the ability to scavenge ROS, and increasing cellular susceptibility to oxidative stress. Our study uncovers a SIRT5‐dependent mechanism that regulates cellular NADPH homeostasis and redox potential by promoting IDH2 desuccinylation and G6PD deglutarylation.     

Nitric oxide-mediated cytosolic glucose-6-phosphate dehydrogenase is involved in aluminum toxicity of soybean under high aluminum concentration

Aims

Glucose-6-phosphate dehydrogenase (G6PDH) has been reported to be involved in resistance to various environmental stresses. However, the role of G6PDH in aluminum (Al) toxicity remains unclear.

Methods

Physiological and biochemical methods together with histochemical analysis were used to investigate the participation of G6PDH in Al-induced inhibition of root growth.

Results

Exposure to high Al concentration caused a significant increase in the activities of total and cytosolic G6PDH in roots of soybean. Al-induced inhibition of root growth and oxidative stress were alleviated by a G6PDH inhibitor. Reactive oxygen species (ROS) accumulation in Al-treated root apexes could be abolished by a NADPH oxidase inhibitor. Furthermore, treatment with a G6PDH inhibitor reduced NADPH content and NADPH oxidase activity in Al-treated root apexes. Further investigation demonstrates that nitric oxide (NO) mediates Al-induced increase in cytosolic G6PDH activity by modulating the expression of genes encoding cytosolic G6PDH. In addition, nitrate reductase pathway is mainly responsible for Al-induced NO production in root apexes.

Conclusions

These results indicate that NADPH produced by NO-modulated cytosolic G6PDH in root apexes is responsible for ROS accumulation mediated by NADPH oxidase under Al stress, subsequently suffering from oxidative stress and thus causing the inhibition of root elongation.

     

Involvement of glucose-6-phosphate dehydrogenase in reduced glutathione maintenance and hydrogen peroxide signal under salt stress

Cellular redox homeostasis is essential for plant growth, development as well as for the resistance to biotic and abiotic stresses, which is governed by the complex network of prooxidant and antioxidant systems. Recently, new evidence has been published that NADPH, produced by glucose-6-phosephate dehydrogenase enzyme (G6PDH), not only acted as the reducing potential for the output of reduced glutathione (GSH), but was involved in the activity of plasma membrane (PM) NADPH oxidase under salt stress, which resulted in hydrogen peroxide (H₂O₂) accumulation. H₂O₂ acts as a signal in regulating G6PDH activity and expression, and the activities of the enzymes in the glutathione cycle as well, through which the ability of GSH regeneration was increased under salt stress. Thus, G6PDH plays a critical role in maintaining cellular GSH levels under long-term salt stress. In this addendum, a hypothetical model for the roles of G6PDH in modulating the intracellular redox homeostasis under salt stress is presented.Key words: glucose-6-phosphate dehydrogenase, hydrogen peroxide, reduced glutathione, redox homeostasis, salt stressEnvironmental stresses inevitably induce the production of reactive oxygen species (ROS).¹ Reduced glutathione (GSH) is a key substance in the network of antioxidants that include ascorbate, glutathione, α-tocopherol and a serial of antioxidant enzymes,² which metabolizes H₂O₂ mainly via the ascorbate-glutathione cycle, the most important detoxifying system in plants.³ Thus, the regulatory ability to maintain the cellular GSH balance is crucial to confer the resistance to oxidative stress in plants. However, to our knowledge, the regulatory mechanism on the intracellular GSH-pool equilibrium under environmental stresses has been largely unknown in plants.A main source of GSH is regenerated from its oxidative form (GSSG) via glutathione cycling, which uses NADPH as the reductant.⁴ G6PDH is the key enzyme of pentose phosphate pathway that is responsible for the generation of NADPH.⁵ G6PDH has been shown to play a protective role against ROS in human and animal cells,^6,7 and the enhanced expression of G6PDH could enhance the GSH levels and the ability of resistance to oxidative stress.^5,8 In plants, it has been reported that oxidative stress induced by the elicitor stimulated G6PDH activity in tobacco cells,^9,10 and the GSH-biosynthesis inhibitor or GSH precursor could increase or suppressed G6PDH activity, respectively.¹⁰ Interestingly, after G6PDH activity was inhibited, not only GSH levels dramatically decreased, but the elicitor-induced H₂O₂ accumulation was also completely counteracted.^9,10 Thus, the functions of G6PDH under oxidative stress seem to be involved in these two contradictory courses in cells: the regeneration of GSH as well as H₂O₂ accumulation. The role of G6PDH under environmental stresses remained limited to clarify this, so we studied the G6PDH functions with a series of inhibitor or donor of GSH, H₂O₂ and G6PDH in reed calli under salt stress. Our recent studied clearly demonstrated that G6PDH activity was also simultaneously involved in intracellular GSH maintenance and H₂O₂ accumulation in salt stress. Further studies revealed that a plasma membrane (PM) NADPH oxidase, using NADPH as substrate mainly produced by G6PDH, was mainly responsible for the generation of H₂O₂. And H₂O₂, produced under salt stress, induced the increased G6PDH activity and the enzymes of glutathione cycle, which concomitantly resulted in an increased GSH contents. Foyer and Noctor (2005) suggested that the cellular “oxidative signaling” was made possibly by homeostatic regulation by antioxidant redox buffer.¹¹ Based on these, it can be speculated that G6PDH might play an important role in maintaining the cellular redox signals under salt stress in plants.Our recent work provides a new insight into G6PDH functions under environmental stresses in plants. Growing evidences suggest that PM NADPH oxidase is responsible for H₂O₂ accumulation under stresses,^12,13 and H₂O₂ is involved in various signaling pathways in plants, such as defense gene expression, stomatal closure, root growth, programmed cell death (PCD) and so on.¹¹ In addition, GSH, as a key antioxidant, also influences gene expression associated with biotic and abiotic stress responses to maximum defense.² Recent study also reported that G6PDH was involved in NR-dependent NO production, and thus played a pivotal role in establishing tolerance of red kidney bean roots to salt stress.¹⁴ Therefore, the research work is required to further clarify the regulatory mechanism underlying the roles of G6PDH in the cellular redox homeostasis as well as the related signals under environmental stresses in plants.Based on the results obtained so far, a model for G6PDH functions under salt stress is proposed (Fig. 1). In our model, the increased G6PDH activity is tightly correlated with GSH maintenance and H₂O₂ accumulation through PM NADPH oxidase under salt stress in plants. Under salt stress, H₂O₂ activities the activities of G6PDH and the enzymes in glutathione recycle, which finally result in the enhanced glutathione cycling rate and thus the increased GSH levels. This enhanced antioxidant ability can facilitate to maintain a steady-state level of H₂O₂. Eventually, the properly intracellular redox state is established under salt stress and forms a metabolic interface for signals. Thus, we suggest that G6PDH plays a crucial role in establishing this cellular redox homeostasis under salt stress.Open in a separate windowFigure 1Hypothetical model for the roles of G6PDH under salt stress. Under salt stress, G6PDH activity is involved in both GSH maintenance and H₂O₂ accumulation through PM NADPH oxidase. H₂O₂, as a signal, increases the activities of G6PDH, glutathione (GR) and glutathione peroxidase (GPX), which finally enhance glutathione cycle rate and result in the increased GSH levels. This enhanced antioxidant ability could facilitate to keep H₂O₂ in a steady state for signal in salt stress.     

活性氧在UV-B诱导的玉米幼苗叶片乙烯产生中的作用

 研究了活性氧在UV-B(280～320 nm)诱导的玉米(Zea mays)幼苗叶片乙烯合成中的作用。结果表明,UV-B促进了玉米幼苗活性氧和乙烯的产生;乙 烯合成抑制剂氨氧乙烯基甘氨酸 (AVG)和氨氧乙酸(AOA)能明显减弱UV-B对玉米幼苗乙烯产生的诱导作用,但对活性氧(ROS)的 产生没有明显影 响;ROS的清除剂不但能抑制UV-B诱导的 ROS的产生,而且还可以抑制UV_B诱导的乙烯的产生,但这种抑制作用可以被外源O₂^.-的供体所逆转。这 说明,乙烯的积累不能作为UV-B胁迫下ROS的诱导的因素,相反,ROS的积累则导致了乙烯的积累;因此,ROS可能参与了UV-B胁迫诱导的乙烯的产生 。质膜NADPH氧化酶的抑制剂二苯碘鎓(DPI)和H₂O₂的特异性清除剂过氧化氢酶（CAT）对UV-B胁迫诱导的乙烯积累 几乎没有影响, 这说明H₂O₂ 可能与UV-B诱导的玉米幼苗叶片乙烯的产生无关, 在UV-B诱导的玉米幼苗叶片乙烯的生物合成过程中O₂^.-起着很重要的作用,相关的O₂^.-不是由 NADPH氧化酶催化产生的。     

Purification and Properties of NADP-Dependent Sorbitol-6-Phosphate Dehydrogenase from Apple Seedlings

NADP-dependent sorbitol-6-phosphate dehydrogenase (S6PDH) waspurified from apple (Malus domestica) seedlings by a purificationprocedure that included two fractionations by affinity chromatography.The purified enzyme was a homogeneous protein that migratedas a single polypeptide chain with an apparent relative massof 36,000 during SDS-polyacrylamide gel electrophoresis andthe native enzyme was a homodimer of the polypeptide. The maximumvelocity of the reduction of glucose-6-phosphate (G6P) was muchhigher than that of the oxidation of sorbitol-6-phosphate (S6P)and the enzyme had high G6P-reducing activity over the pH rangefrom 7 to 11 even though the oxidation of S6P proceeded veryslowly at neutral pH. These results are consistent with thehypothesis that S6PDH plays a major role in the biosynthesisof sorbitol in vivo. The reduction of G6P to S6P was inhibitedby the addition of nucleotide di- or triphosphates. ATP, thestrongest inhibitor, and ADP inhibited the reduction of G6Pin a competitive manner with respect to NADPH and the K_i valueswere 0.18 mM for ATP and 0.30 mM for ADP. (Received March 24, 1992; Accepted May 25, 1993)     

Metabolic defence against oxidative stress: the road less travelled so far

Bacteria have survived, and many have thrived, since antiquity in the presence of the highly‐reactive chalcogen—oxygen (O₂). They are known to evoke intricate strategies to defend themselves from the reactive by‐products of oxygen—reactive oxygen species (ROS). Many of these detoxifying mechanisms have been extensively characterized; superoxide dismutase, catalases, alkyl hydroperoxide reductase and the glutathione (GSH)‐cycling system are responsible for neutralizing specific ROS. Meanwhile, a pool of NADPH—the reductive engine of many ROS‐combating enzymes—is maintained by metabolic enzymes including, but not exclusively, glucose‐6 phosphate dehydrogenase (G6PDH) and NADP‐dependent isocitrate dehydrogenase (ICDH‐NADP). So, it is not surprising that evidence continues to emerge demonstrating the pivotal role metabolism plays in mitigating ROS toxicity. Stemming from its ability to concurrently decrease the production of the pro‐oxidative metabolite, NADH, while augmenting the antioxidative metabolite, NADPH, metabolism is the fulcrum of cellular redox potential. In this review, we will discuss the mounting evidence positioning metabolism and metabolic shifts observed during oxidative stress, as critical strategies microbes utilize to thrive in environments that are rife with ROS. The contribution of ketoacids—moieties capable of non‐enzymatic decarboxylation in the presence of oxidants—as ROS scavengers will be elaborated alongside the metabolic pathways responsible for their homeostases. Further, the signalling role of the carboxylic acids generated following the ketoacid‐mediated detoxification of the ROS will be commented on within the context of oxidative stress.     

Increasing Glucose 6-Phosphate Dehydrogenase Activity Restores Redox Balance in Vascular Endothelial Cells Exposed to High Glucose

Previous studies have shown that high glucose increases reactive oxygen species (ROS) in endothelial cells that contributes to vascular dysfunction and atherosclerosis. Accumulation of ROS is due to dysregulated redox balance between ROS-producing systems and antioxidant systems. Previous research from our laboratory has shown that high glucose decreases the principal cellular reductant, NADPH by impairing the activity of glucose 6-phosphate dehydrogenase (G6PD). We and others also have shown that the high glucose-induced decrease in G6PD activity is mediated, at least in part, by cAMP-dependent protein kinase A (PKA). As both the major antioxidant enzymes and NADPH oxidase, a major source of ROS, use NADPH as substrate, we explored whether G6PD activity was a critical mediator of redox balance. We found that overexpression of G6PD by pAD-G6PD infection restored redox balance. Moreover inhibition of PKA decreased ROS accumulation and increased redox enzymes, while not altering the protein expression level of redox enzymes. Interestingly, high glucose stimulated an increase in NADPH oxidase (NOX) and colocalization of G6PD with NOX, which was inhibited by the PKA inhibitor. Lastly, inhibition of PKA ameliorated high glucose mediated increase in cell death and inhibition of cell growth. These studies illustrate that increasing G6PD activity restores redox balance in endothelial cells exposed to high glucose, which is a potentially important therapeutic target to protect ECs from the deleterious effects of high glucose.     

Isozymes of Glucose-6-phosphate Dehydrogenase and NAD+-malate Dehydrogenase in Shoot-forming Foliar Discs of Tobacco

Discrete pale, meristematic, shoot-forming zones (SF) and green,relatively nondividing, non-shoot-forming zones (NSF) of cellswere obtained from leaf discs of tobacco cultured for 12 dayson a shoot-forming medium. Higher chlorophyll and starch content,increased rates of O₂ evolution and CO₂ fixation in light, andincreased activities of amylases and chloroplastic enzymes suchas ribulose 1,5-bisphosphate carboxylase and NADP⁺-linked glyceraldehyde-3-phosphatedehydrogenase (G3PDH) were characteristic of the cells constitutingNSF. On the other hand, active participation of sucrose hydrolysis,dark-mediated CO₂ incorporation, an oxidative pentose phosphatepathway, glycolysis and mitochondrial complements in shoot formationwere evident from the significantly high activities of phosphoenolpyruvatecarboxylase, invertase, glucose-6-phosphate dehydrogenase (G6PDH),NAD+-G3PDH and NAD+-linked malate dehydrogenase (NAD+-MDH) respectively,in SF cells. Detection of activity of the enzymes by stainingon polyacrylamide gels disclosed synthesis of additional isoenzyme(s)of G6PDH, NAD+-MDH and peroxidase in shoot initiation sites.The much pronounced activity and isozyme groups of G6PDH andNAD+-MDH in the photosynthetically incompetent shoot-formingcells, are considered to increase the carbon budget of the differentiatingcells through non-autotrohpic CO₂ fixation and to supplementreducing power (NADPH) for the organogenetic process which requiresmuch energy. The changes in isozymes of these enzymes, as inthe isoperoxidase system, probably can serve as useful markersof the differentiation process. (Received January 23, 1981; Accepted June 17, 1981)     

Glucose-6-phosphate dehydrogenase plays a pivotal role in tolerance to drought stress in soybean roots

Key message

Two soybean cultivars showed markedly different drought tolerance. G6PDH plays a central role in the process of H ₂ O ₂ regulated GR, DHAR, and MDHAR activities to maintain GSH and Asc levels.

Abstract

Glucose-6-phosphate dehydrogenase (G6PDH) plays a pivotal role in plant resistance to environmental stresses. In this study, we investigated the role of G6PDH in modulating redox homeostasis under drought stress induced by polyethylene glycol 6000 (PEG6000) in two soybean cultivars JINDOU21 (JD-21) and WDD00172 (WDD-172). The G6PDH activity markedly increased and reached a maximum at 96 h in JD-21 and 72 h in WDD-172 during PEG6000 treatments, respectively. Glucosamine (Glucm, a G6PDH inhibitor) obviously inhibited G6PDH activity in both soybeans under PEG6000 treatments. After PEG6000 treatment, JD-21 showed higher tolerance than WDD-172 not only in higher activities of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), glutathione reductase (GR), dehydroascorbate reductase (DHAR), and monodehydroascorbate reductase (MDHAR), but also in higher content of glutathione (GSH) and ascorbate (Asc). And we found that hydrogen peroxide (H₂O₂) regulated the cell length in root elongation zone. Diphenylene iodonium (DPI, a plasma membrane NADPH oxidase inhibitor) counteracted the PEG6000-induced H₂O₂ accumulation and decreased the activities of GR, DHAR, and MDHAR as well as GSH and Asc content. Furthermore, exogenous application of H₂O₂ increased the GR, DHAR, and MDHAR activities that were decreased by Glucm under drought stress. Western blot analysis showed that the G6PDH expression was stimulated by PEG6000 and buthionine sulfoximine (BSO, glutathione biosynthesis inhibitor), and blocked by Glucm, DPI and N-acetyl-l-cysteine (NAC, GSH precursor) in both cultivars. Taken together, our evidence indicates that G6PDH plays a central role in the process of H₂O₂ regulated GR, DHAR, and MDHAR activities to maintain GSH and Asc levels.     

Involvement of G6PDH in heat stress tolerance in the calli from Przewalskia tangutica and Nicotiana tabacum

Glucose-6-phosphate dehydrogenase (G6PDH) has been implicated in supplying reduced nicotine amide cofactors for biochemical reactions and in modulating the redox state of cells. In this study, the role of G6PDH in thermotolerance of the calli from Przewalskia tangutica and tobacco (Nicotiana tabacum L.) was investigated. Results showed that Przewalskia tangutica callus was more sensitive to heat stress than tobacco callus. The activity of G6PDH and antioxidant enzymes (ascorbate peroxidase, catalase, peroxidase and superoxide dismutase) in calli from Przewalskia tangutica and tobacco increased after 40 °C treatment, although two calli exhibited a difference in the degree and timing of response to heat stress. When G6PDH was partially inhibited by glucosamine pretreatment, the antioxidant enzyme activities and thermotolerance in both calli significantly decreased. Simultaneously, the heat-induced H₂O₂ content and the plasma membrane NADPH oxidase activity were also reduced. Application of H₂O₂ increased the activity of G6PDH and antioxidant enzymes in both calli. Diphenylene iodonium, a NADPH oxidase inhibitor, counteracted heatinduced H₂O₂ accumulation and reduced the heat-induced activity of G6PDH and antioxidant enzymes. Moreover, exogenous H₂O₂ was effective in restoring the activity of G6PDH and antioxidant enzymes after glucosamine pretreatment. Western blot analysis showed that G6PDH gene expression in both calli was also stimulated by heat and H₂O₂, and blocked by DPI and glucosamine under heat stress. Taken together, under heat stress G6PDH promoted H₂O₂ accumulation via NADPH oxidase and the elevated H₂O₂ was involved in regulating the activity of antioxidant enzymes, which in turn facilitate to maintain the steady-state H₂O₂ level and protect plants from the oxidative damage.     

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.     

Presence of the distinct systems responsible for superoxide anion and hydrogen peroxide generation in red tide phytoplankton Chattonella marina and Chattonella ovata

Raphidophycean flagellates, Chattonella marina and C. ovata,are harmful red tide phytoplankters; blooms of these phytoplanktersoften cause severe damage to fish farming. Previous studieshave demonstrated that C. marina and C. ovata continuously producereactive oxygen species (ROS) such as superoxide anion (O₂^–)hydrogen peroxide (H₂O₂) under normal growth conditions, andan ROS-mediated toxic mechanism against fish and other marineorganisms has been proposed. Although the exact mechanism ofROS generation in these phytoplankters still remains to be clarified,our previous study suggested that NADPH oxidase-like enzymelocated on the cell surface of C. marina may be involved inO₂^– generation. To investigate the localization of O₂^–and H₂O₂ generation in C. marina and C. ovata, we employed 2-methyl-6(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazin-3-oneand 5-(and-6)-carboxy-2',7'-dichlorodihydrodihydrofluoresceindictate, acetyl ester, which are specific fluorescent probefor detecting O₂^– and H₂O₂, respectively. Observationby fluorescence microscopy of live phytoplankters incubatedwith each probe revealed that O₂^– is mainly generatedon the cell surface, whereas H₂O₂ is generated in the intracellularcompartment in these phytoplankters. When the cells were rupturedby ultrasonic treatment, O₂^– levels of C. marina and C.ovata decreased significantly, whereas a few times higher levelsof H₂O₂ were detected in the ruptured cell suspensions whencompared with the levels of the live cell suspension. In immunoblottinganalysis, the protein recognized by anti-human gp91 phox wasdetected in both species. These results suggest that, in bothphytoplankters, the underlying mechanisms of O₂^– and H₂O₂generation may be distinct and such systems are independentlyoperating in the cells.     

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 H₂O₂ 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.     

ROS scavenging before 27 degrees C ischemia protects hearts and reduces mitochondrial ROS, Ca2+ overload, and changes in redox state

We have shown that cold perfusion of hearts generates reactive oxygen and nitrogen species (ROS/RNS). In this study, we determined 1) whether ROS scavenging only during cold perfusion before global ischemia improves mitochondrial and myocardial function, and 2) which ROS leads to compromised cardiac function during ischemia and reperfusion (I/R) injury. Using fluorescence spectrophotometry, we monitored redox balance (NADH and FAD), O₂^– levels and mitochondrial Ca²⁺ (m[Ca²⁺]) at the left ventricular wall in 120 guinea pig isolated hearts divided into control (Con), MnTBAP (a superoxide dismutase 2 mimetic), MnTBAP (M) + catalase (C) + glutathione (G) (MCG), C+G (CG), and N^G-nitro-L-arginine methyl ester (L-NAME; a nitric oxide synthase inhibitor) groups. After an initial period of warm perfusion, hearts were treated with drugs before and after at 27°C. Drugs were washed out before 2 h at 27°C ischemia and 2 h at 37°C reperfusion. We found that on reperfusion the MnTBAP group had the worst functional recovery and largest infarction with the highest m[Ca²⁺], most oxidized redox state and increased ROS levels. The MCG group had the best recovery, the smallest infarction, the lowest ROS level, the lowest m[Ca²⁺], and the most reduced redox state. CG and L-NAME groups gave results intermediate to those of the MnTBAP and MCG groups. Our results indicate that the scavenging of cold-induced O₂^– species to less toxic downstream products additionally protects during and after cold I/R by preserving mitochondrial function. Because MnTBAP treatment showed the worst functional return along with poor preservation of mitochondrial bioenergetics, accumulation of H₂O₂ and/or hydroxyl radicals during cold perfusion may be involved in compromised function during subsequent cold I/R injury. hypothermic ischemia; mitochondrial Ca²⁺; reactive oxygen species     

Taurine prevents high-glucose-induced human vascular endothelial cell apoptosis

Elevated blood glucose in uncontrolled diabetes is causallycorrelated with diabetic microangiopathy. Hyperglycemia-triggered accelerated endothelial cell apoptosis is a critical event in theprocess of diabetes-associated microvascular disease. The conditionallysemiessential amino acid taurine has been previously shown to protectagainst human endothelial cell apoptosis. Therefore, this study wasdesigned to investigate the role of taurine in the prevention ofhigh-glucose-mediated cell apoptosis in human umbilical veinendothelial cells (HUVEC) and the mechanisms involved. Exposure ofHUVEC to 30 mM glucose for 48 h (short-term) and 14 days (long-term)resulted in a significant increase in apoptosis, compared with normalglucose (5.5 mM; P < 0.05).High-glucose-induced DNA fragmentation preferentially occurred in the Sphase cells. Mannitol (as osmotic control) at 30 mM failed to induceHUVEC apoptosis. Taurine prevented high-glucose-induced HUVECapoptosis, which correlates with taurine attenuation ofhigh-glucose-mediated increased intracellular reactive oxygen species(ROS) formation and elevated intracellularCa²⁺ concentration([Ca²⁺]_i)level. Antioxidants, DMSO, N-acetylcysteine, and glutathione, only partly attenuated high-glucose-inducedHUVEC apoptosis. Glucose at 30 mM did not cause HUVEC necrosis.However, both glucose and mannitol at 60 mM caused HUVEC necrosis asrepresented by increased lactate dehydrogenase release and cell lysis.Taurine failed to prevent hyperosmolarity-induced cell necrosis. Theseresults demonstrate that taurine attenuates hyperglycemia-induced HUVECapoptosis through ROS inhibition and[Ca²⁺]_istabilization and suggest that taurine may exert a beneficial effect inpreventing diabetes-associated microangiopathy.

     

Reactive oxygen species formation in the transition to hypoxia in skeletal muscle

Many tissues produce reactive oxygen species (ROS) during reoxygenation after hypoxia or ischemia; however, whether ROS are formed during hypoxia is controversial. We tested the hypothesis that ROS are generated in skeletal muscle during exposure to acute hypoxia before reoxygenation. Isolated rat diaphragm strips were loaded with dihydrofluorescein-DA (Hfluor-DA), a probe that is oxidized to fluorescein (Fluor) by intracellular ROS. Changes in fluorescence due to Fluor, NADH, and FAD were measured using a tissue fluorometer. The system had a detection limit of 1 µM H₂O₂ applied to the muscle superfusate. When the superfusion buffer was changed rapidly from 95% O₂ to 0%, 5%, 21%, or 40% O₂, transient elevations in Fluor were observed that were proportional to the rise in NADH fluorescence and inversely proportional to the level of O₂ exposure. This signal could be inhibited completely with 40 µM ebselen, a glutathione peroxidase mimic. After brief hypoxia exposure (10 min) or exposure to brief periods of H₂O₂, the fluorescence signal returned to baseline. Furthermore, tissues loaded with the oxidized form of the probe (Fluor-DA) showed a similar pattern of response that could be inhibited with ebselen. These results suggest that Fluor exists in a partially reversible redox state within the tissue. When Hfluor-loaded tissues were contracted with low-frequency twitches, Fluor emission and NADH emission were significantly elevated in a way that resembled the hypoxia-induced signal. We conclude that in the transition to low intracellular PO₂, a burst of intracellular ROS is formed that may have functional implications regarding skeletal muscle O₂-sensing systems and responses to acute metabolic stress. dihydrofluorescein; tissue fluorometer; ebselen; N-acetylcysteine; rat     

Activation and inactivation of the volume-sensitive taurine leak pathway in NIH3T3 fibroblasts and Ehrlich Lettre ascites cells

Hypotonic exposure provokes the mobilization of arachidonic acid, production of ROS, and a transient increase in taurine release in Ehrlich Lettre cells. The taurine release is potentiated by H₂O₂ and the tyrosine phosphatase inhibitor vanadate and reduced by the phospholipase A₂ (PLA₂) inhibitors bromoenol lactone (BEL) and manoalide, the 5-lipoxygenase (5-LO) inhibitor ETH-615139, the NADPH oxidase inhibitor diphenyl iodonium (DPI), and antioxidants. Thus, swelling-induced taurine efflux in Ehrlich Lettre cells involves Ca²⁺-independent (iPLA₂)/secretory PLA₂ (sPLA₂) plus 5-LO activity and modulation by ROS. Vanadate and H₂O₂ stimulate arachidonic acid mobilization and vanadate potentiates ROS production in Ehrlich Lettre cells and NIH3T3 fibroblasts under hypotonic conditions. However, vanadate-induced potentiation of the volume-sensitive taurine efflux is, in both cell types, impaired in the presence of BEL and DPI and following restoration of the cell volume. Thus, potentiation of the volume-sensitive taurine efflux pathway following inhibition of tyrosine phosphatase activity reflects increased arachidonic acid mobilization and ROS production for downstream signaling. Vanadate delays the inactivation of volume-sensitive taurine efflux in NIH3T3 cells, and this delay is impaired in the presence of DPI. Vanadate has no effect on the inactivation of swelling-induced taurine efflux in Ehrlich Lettre cells. It is suggested that increased tyrosine phosphorylation of regulatory components of NADPH oxidase leads to increased ROS production and a subsequent delay in inactivation of the volume-sensitive taurine efflux pathway and that NADPH oxidase or antioxidative capacity differ between NIH3T3 and Ehrlich Lettre cells. organic osmolytes; reactive oxygen species; vanadate; H₂O₂; tyrosine phosphatases; arachidonic acid mobilization     

Apolipoprotein A-IV attenuates oxidant-induced apoptosis in mitotic competent, undifferentiated cells by modulating intracellular glutathione redox balance

Oxidant-mediated modulation of the intracellular redox state affects the apoptotic cascade by altering the balance between cellular signals for survival and suicide. Apolipoprotein A-IV (Apo A-IV) is known to possess antioxidant-like activity. In the present study, we tested 1) whether Apo A-IV could influence redox-dependent apoptosis and, if so, 2) whether such an effect could be mediated by modulation of intracellular redox balance. Mitotic competent, undifferentiated PC-12 cells were incubated with either tert-butyl hydroperoxide (TBH) or diamide with or without preincubation with human Apo A-IV. Apo A-IV significantly decreased apoptosis produced by both TBH and diamide, and washout of A-IV before incubation with TBH and diamide did not eliminate its protective effect. Apo A-I had no such protective effect. The Apo A-IV effect was not blocked by D,L-buthionine-[S,R]-sulfoximine, but it was reversed by both dehydroisoandrosterone and transfection with an antisense oligodeoxynucleotide to glucose-6-phosphate dehydrogenase (G6PD). Apo A-IV abolished the transient, oxidant-induced rise in glutathione disulfide (GSSG) and cellular redox imbalance previously shown to initiate the apoptotic cascade. Apo A-IV had no effect on GSSG reductase activity, but it stimulated G6PD activity 10-fold. These results suggest a novel role for Apo A-IV in the regulation of intracellular glutathione redox balance and the modulation of redox-dependent apoptosis via stimulation of G6PD activity. tert-butyl hydroperoxide; diamide; dehydroisoandrosterone; glucose-6-phosphate dehydrogenase; antisense     

Cyclic strain and motion control produce opposite oxidative responses in two human endothelial cell types

The phenotype of endothelial cells (ECs) is specific to the vascular bed from which they originate. To examine how mechanical forces alter the phenotype of different ECs, we compared the effects of cyclic strain and motion control on reactive oxygen species (ROS) production and metabolism and cell adhesion molecule expression in human umbilical vein endothelial cells (HUVEC) vs. human aortic endothelial cells (HAEC). HUVEC and HAEC were subjected to cyclic strain (10% or 20%, 1 Hz), to a motion control that simulated fluid agitation over the cells without strain, or to static conditions for 24 h. We measured H₂O₂ production with dichlorodihydrofluorescein acetate and superoxide with dihydroethidium fluorescence changes; superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx) activities spectrophotometrically; and vascular cell adhesion molecule (VCAM)-1 and intercellular adhesion molecule (ICAM)-1 protein expression with Western blot analyses. HUVEC under cyclic strain showed 1) higher intracellular H₂O₂ levels, 2) increased SOD, catalase, and GPx activities, and 3) greater VCAM-1 and ICAM-1 protein expression, compared with motion control or static conditions. However, in HAEC, motion control induced higher levels of ROS, enzyme activities associated with ROS defense, and VCAM-1 and ICAM-1 expression than cyclic strain. The opposite responses obtained with these two human EC types may reflect their vessels of origin, in that HAEC are subjected to higher cyclic strain deformations in vivo than HUVEC. phenotype; reactive oxygen species; inflammation; shear stress