Effect of extracellular Mg(2+) on ROS and Ca(2+) accumulation during reoxygenation of rat cardiomyocytes

The effects of Mg(2+) on reactive oxygen species (ROS) and cell Ca(2+) during reoxygenation of hypoxic rat cardiomyocytes were studied. Oxidation of 2',7'-dichlorodihydrofluorescein (DCDHF) to dichlorofluorescein (DCF) and of dihydroethidium (DHE) to ethidium (ETH) within cells were used as markers for intracellular ROS levels and were determined by flow cytometry. DCDHF/DCF is sensitive to H(2)O(2) and nitric oxide (NO), and DHE/ETH is sensitive to the superoxide anion (O(2)(-).), respectively. Rapidly exchangeable cell Ca(2+) was determined by (45)Ca(2+) uptake. Cells were exposed to hypoxia for 1 h and reoxygenation for 2 h. ROS levels, determined as DCF fluorescence, were increased 100-130% during reoxygenation alone and further increased 60% by increasing extracellular Mg(2+) concentration to 5 mM at reoxygenation. ROS levels, measured as ETH fluorescence, were increased 16-24% during reoxygenation but were not affected by Mg(2+). Cell Ca(2+) increased three- to fourfold during reoxygenation. This increase was reduced 40% by 5 mM Mg(2+), 57% by 10 microM 3,4-dichlorobenzamil (DCB) (inhibitor of Na(+)/Ca(2+) exchange), and 75% by combining Mg(2+) and DCB. H(2)O(2) (25 and 500 microM) reduced Ca(2+) accumulation by 38 and 43%, respectively, whereas the NO donor S-nitroso-N-acetyl-penicillamine (1 mM) had no effect. Mg(2+) reduced hypoxia/reoxygenation-induced lactate dehydrogenase (LDH) release by 90%. In conclusion, elevation of extracellular Mg(2+) to 5 mM increased the fluorescence of the H(2)O(2)/NO-sensitive probe DCF without increasing that of the O(2)(-).-sensitive probe ETH, reduced Ca(2+) accumulation, and decreased LDH release during reoxygenation of hypoxic cardiomyocytes. The reduction in LDH release, reflecting the protective effect of Mg(2+), may be linked to the effect of Mg(2+) on Ca(2+) accumulation and/or ROS levels.     

Origin of cadmium-induced reactive oxygen species production: mitochondrial electron transfer versus plasma membrane NADPH oxidase

* Cadmium (Cd(2+)) is an environmental pollutant that causes increased reactive oxygen species (ROS) production. To determine the site of ROS production, the effect of Cd(2+) on ROS production was studied in isolated soybean (Glycine max) plasma membranes, potato (Solanum tuberosum) tuber mitochondria and roots of intact seedlings of soybean or cucumber (Cucumis sativus). * The effects of Cd(2+) on the kinetics of superoxide (O2*-), hydrogen peroxide (H(2)O(2)) and hydroxyl radical ((*OH) generation were followed using absorption, fluorescence and spin-trapping electron paramagnetic resonance spectroscopy. * In isolated plasma membranes, Cd(2+) inhibited O2*- production. This inhibition was reversed by calcium (Ca(2+)) and magnesium (Mg(2+)). In isolated mitochondria, Cd(2+) increased and H(2)O(2) production. In intact roots, Cd(2+) stimulated H(2)O(2) production whereas it inhibited O2*- and (*)OH production in a Ca(2+)-reversible manner. * Cd(2+) can be used to distinguish between ROS originating from mitochondria and from the plasma membrane. This is achieved by measuring different ROS individually. The immediate (相似文献   

Cryptogein-induced initial events in tobacco BY-2 cells: pharmacological characterization of molecular relationship among cytosolic Ca(2+) transients, anion efflux and production of reactive oxygen species

Ion fluxes and the production of reactive oxygen species (ROS) are early events that follow elicitor treatment or microbial infection. However, molecular mechanisms for these responses as well as their relationship have been controversial and still largely unknown. We here simultaneously monitored the temporal sequence of initial events at the plasma membrane in suspension-cultured tobacco cells (cell line BY-2) in response to a purified proteinaceous elicitor, cryptogein, which induced hypersensitive cell death. The elicitor induced transient rise in cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) showing two distinct peaks, followed by biphasic (rapid/transient and slow/prolonged) Cl(-) efflux and H(+) influx. Pharmacological analyses suggested that the two phases of the [Ca(2+)](cyt) response correspond to Ca(2+) influx through the plasma membrane and an inositol 1,4,5-trisphophate-mediated release of Ca(2+) from intracellular Ca(2+) stores, respectively, and the [Ca(2+)](cyt) transients and the Cl(-) efflux were mutually dependent events regulated by protein phosphorylation. The elicitor also induced production of ROS including (*)O(2)(-) and H(2)O(2), which initiated after the [Ca(2+)](cyt) rise and required Ca(2+) influx, Cl(-) efflux and protein phosphorylation. An inhibitor of NADPH oxidase, diphenylene iodonium, completely inhibited the elicitor-induced production of (*)O(2)(-) and H(2)O(2), but did not affect the [Ca(2+)](cyt) transients. These results suggest that cryptogein-induced plasma membrane Ca(2+) influx is independent of ROS, and NADPH oxidase dependent ROS production is regulated by these series of ion fluxes.     

Impairment of endothelial nitric oxide production by acute glucose overload

We examined the effects of acute glucose overload (pretreatment for 3 h with 23 mM D-glucose) on the cellular productivity of nitric oxide (NO) in bovine aortic endothelial cells (BAEC). We had previously reported (Kimura C, Oike M, and Ito Y. Circ Res, 82: 677-685, 1998) that glucose overload impairs Ca(2+) mobilization due to an accumulation of superoxide anions (O(2)(-)) in BAEC. In control cells, ATP induced an increase in NO production, assessed by diaminofluorescein 2 (DAF-2), an NO-sensitive fluorescent dye, mainly due to Ca(2+) entry. In contrast, ATP-induced increase in DAF-2 fluorescence was impaired by glucose overload, which was restored by superoxide dismutase, but not by catalase or deferoxamine. Furthermore, pyrogallol, an O(2)(-) donor, also attenuated ATP-induced increase in DAF-2 fluorescence. In contrast, a nonspecific intracellular Ca(2+) concentration increase induced by the Ca(2+) ionophore A-23187, which depletes the intracellular store sites, elevated DAF-2 fluorescence in both control and high D-glucose-treated cells in Ca(2+)-free solution. These results indicate that glucose overload impairs NO production by the O(2)(-)-mediated attenuation of Ca(2+) entry.     

Superoxide flux in endothelial cells via the chloride channel-3 mediates intracellular signaling

Reactive oxygen species (ROS) have been implicated in both cell signaling and pathology. A major source of ROS in endothelial cells is NADPH oxidase, which generates superoxide (O(2)(.-)) on the extracellular side of the plasma membrane but can result in intracellular signaling. To study possible transmembrane flux of O(2)(.-), pulmonary microvascular endothelial cells were preloaded with the O(2)(.-)-sensitive fluorophore hydroethidine (HE). Application of an extracellular bolus of O(2)(.-) resulted in rapid and concentration-dependent transient HE oxidation that was followed by a progressive and nonreversible increase in nuclear HE fluorescence. These fluorescence changes were inhibited by superoxide dismutase (SOD), the anion channel blocker DIDS, and selective silencing of the chloride channel-3 (ClC-3) by treatment with siRNA. Extracellular O(2)(.-) triggered Ca(2+) release in turn triggered mitochondrial membrane potential alterations that were followed by mitochondrial O(2)(.-) production and cellular apoptosis. These "signaling" effects of O(2)(.-) were prevented by DIDS treatment, by depletion of intracellular Ca(2+) stores with thapsigargin and by chelation of intracellular Ca(2+). This study demonstrates that O(2)(.-) flux across the endothelial cell plasma membrane occurs through ClC-3 channels and induces intracellular Ca(2+) release, which activates mitochondrial O(2)(.-) generation.     

Cross-talk between calcium and reactive oxygen species originated from NADPH oxidase in abscisic acid-induced antioxidant defence in leaves of maize seedlings

The signal interactions between calcium (Ca2+) and reactive oxygen species (ROS) originated from plasma membrane NADPH oxidase in abscisic acid (ABA)-induced antioxidant defence were investigated in leaves of maize (Zea mays L.) seedlings. Treatment with ABA led to significant increases in the activity of plasma membrane NADPH oxidase, the production of leaf O2-, and the activities of several antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR). However, such increases were blocked by the pretreatment with Ca2+ chelator EGTA or Ca2+ channel blockers La3+ and verapamil, and NADPH oxidase inhibitors such as diphenylene iodonium (DPI), imidazole and pyridine. Treatment with Ca2+ also significantly induced the increases in NADPH oxidase activity, O2- production and the activities of antioxidant enzymes, and the increases were arrested by pretreatment with the NADPH oxidase inhibitors. Treatment with oxidative stress induced by paraquat, which generates O2-, led to the induction of antioxidant defence enzymes, and the up-regulation was suppressed by the pretreatment of Ca2+ chelator and Ca2+ channel blockers. Our data suggest that a cross-talk between Ca2+ and ROS originated from plasma membrane-bound NADPH oxidase is involved in the ABA signal transduction pathway leading to the induction of antioxidant enzyme activity, and Ca2+ functions upstream as well as downstream of ROS production in the signal transduction event in plants.     

NADPH oxidase-dependent hydrogen peroxide production, induced by salinity stress, may be involved in the regulation of total calcium in roots of wheat

Hydrogen peroxide (H(2)O(2)) is often generated by cells and tissues under environmental stress. In this work, we provide evidence that plasma membrane (PM) NADPH oxidase-dependent H(2)O(2) production might act as an intermediate step in the NaCl-induced elevation of calcium (Ca) in roots of wheat. Remarkable increases in the content of total Ca were observed not only in roots exposed to NaCl but also in roots of seedlings exposed to exogenous H(2)O(2). In roots, H(2)O(2) production increased upon exposure to salt stress. PM vesicles were isolated from roots, and NADPH oxidase activity was determined by measuring superoxide anion (O(2)(-)) production. NADPH oxidase-dependent O(2)(-) production was 11.6nmolmg(-1)proteinmin(-1) in control vesicles, but 19.6nmol after NaCl treatment (24h), indicating that salt stress resulted in the activation of the PM NADPH oxidase. Furthermore, the NaCl-induced increase in total Ca was partially abolished by the addition of 150U/mL catalase (CAT), a H(2)O(2) scavenger, and also by 10microM diphenylane iodonium (DPI), a NADPH oxidase inhibitor. This data suggest that NADPH oxidase-dependent H(2)O(2) production might be involved in the modulation of the Ca content in wheat roots. In conclusion, our results show that salinity stress increases the total Ca content of wheat roots, which is partly due to PM NADPH oxidase-dependent H(2)O(2) generation.     

Decreased reactive oxygen species concentration in the elongation zone contributes to the reduction in maize leaf growth under salinity

Reactive oxygen species (ROS) in the apoplast of cells in the growing zone of grass leaves are required for elongation growth. This work evaluates whether salinity-induced reductions in leaf elongation are related to altered ROS production. Studies were performed in actively growing segments (SEZ) obtained from leaf three of 14-d-old maize (Zea mays L.) seedlings gradually salinized to 150 mM NaCl. Salinity reduced elongation rates and the length of the leaf growth zone. When SEZ obtained from the elongation zone of salinized plants (SEZs) were incubated in 100 mM NaCl, the concentration where growth inhibition was approximately 50%, O2*- production, measured as NBT formazan staining, was lower in these than in similar segments obtained from control plants. The NaCl effect was salt-specific, and not osmotic, as incubation in 200 mM sorbitol did not reduce formazan staining intensity. SEZs elongation rates were higher in 200 mM sorbitol than in 100 mM NaCl, but the difference could be cancelled by scavenging or inhibiting O2*- production with 10 mM MgCl2 or 200 microM diphenylene iodonium, respectively. The actual ROS believed to stimulate growth is *OH, a product of O2*- metabolism in the apoplast. SEZ(s) elongation in 100 mM NaCl was stimulated by a *OH-generating medium. Fusicoccin, an ATPase stimulant, and acetate buffer pH 4, could also enhance elongation in these segments, although both failed to increase ROS activity. These results show that decreased ROS production contributes to the salinity-associated reduction in grass leaf elongation, acting through a mechanism not associated with pH changes.     

Hypoxia-induced alterations in Ca(2+) mobilization in brain microvascular endothelial cells

To investigate the possible cellular mechanisms of the ischemia-induced impairments of cerebral microcirculation, we investigated the effects of hypoxia/reoxygenation on the intracellular Ca(2+) concentration ([Ca(2+)](i)) in bovine brain microvascular endothelial cells (BBEC). In the cells kept in normal air, ATP elicited Ca(2+) oscillations in a concentration-dependent manner. When the cells were exposed to hypoxia for 6 h and subsequent reoxygenation for 45 min, the basal level of [Ca(2+)](i) was increased from 32.4 to 63.3 nM, and ATP did not induce Ca(2+) oscillations. Hypoxia/reoxygenation also inhibited capacitative Ca(2+) entry (CCE), which was evoked by thapsigargin (Delta[Ca(2+)](i-CCE): control, 62.3 +/- 3.1 nM; hypoxia/reoxygenation, 17.0 +/- 1.8 nM). The impairments of Ca(2+) oscillations and CCE, but not basal [Ca(2+)](i), were restored by superoxide dismutase and the inhibitors of mitochondrial electron transport, rotenone and thenoyltrifluoroacetone (TTFA). By using a superoxide anion (O(2)(-))-sensitive luciferin derivative MCLA, we confirmed that the production of O(2)(-) was induced by hypoxia/reoxygenation and was prevented by rotenone and TTFA. These results indicate that hypoxia/reoxygenation generates O(2)(-) at mitochondria and impairs some Ca(2+) mobilizing properties in BBEC.     

Activation of Rho/Rho kinase signaling pathway by reactive oxygen species in rat aorta

Evidence indicates that both the Rho/Rho kinase signaling pathway and reactive oxygen species (ROS) such as superoxide and H(2)O(2) are involved in the pathogenesis of hypertension. This study aimed to determine whether ROS-induced vascular contraction is mediated through activation of Rho/Rho kinase. Rat aortic rings (endothelium denuded) were isolated and placed in organ chambers for measurement of isometric force development. ROS were generated by a xanthine (X)-xanthine oxidase (XO) mixture. The antioxidants tempol (3 mM) and catalase (1,200 U/ml) or the XO inhibitor allopurinol (400 microM) significantly reduced X/XO-induced contraction. A Rho kinase inhibitor, (+)-(R)-trans-4-(1-aminoethyl-N-4-pyridil)cyclohexanecarboxamide dihydrochloride (Y-27632), decreased the contraction in a concentration-dependent manner; however, the Ca(2+)-independent protein kinase C inhibitor rottlerin did not have an effect on X/XO-induced contraction. Phosphorylation of the myosin light chain phosphatase target subunit (MYPT1) was increased by ROS, and preincubation with Y-27632 blocked this increased phosphorylation. Western blotting for cytosolic and membrane-bound fractions of Rho showed that Rho was increased in the membrane fraction by ROS, suggesting activation of Rho. These observations demonstrate that ROS-induced Ca(2+) sensitization is through activation of Rho and a subsequent increase in Rho kinase activity but not Ca(2+)-independent PKC.     

Cellular mechanisms underlying nitric oxide-induced vasodilation of descending vasa recta

It has been observed that vasoactivity of explanted descending vasa recta (DVR) is modulated by intrinsic nitric oxide (NO) and superoxide (O(2)(-)) production (Cao C, Edwards A, Sendeski M, Lee-Kwon W, Cui L, Cai CY, Patzak A, Pallone TL. Am J Physiol Renal Physiol 299: F1056-F1064, 2010). To elucidate the cellular mechanisms by which NO, O(2)(-) and hydrogen peroxide (H(2)O(2)) modulate DVR pericyte cytosolic Ca(2+) concentration ([Ca](cyt)) and vasoactivity, we expanded our mathematical model of Ca(2+) signaling in pericytes. We incorporated simulations of the pathways that translate an increase in [Ca](cyt) to the activation of myosin light chain (MLC) kinase and cell contraction, as well as the kinetics of NO and reactive oxygen species formation and their effects on [Ca](cyt) and MLC phosphorylation. The model reproduced experimentally observed trends of DVR vasoactivity that accompany exposure to N(ω)-nitro-L-arginine methyl ester, 8-Br-cGMP, Tempol, and H(2)O(2). Our results suggest that under resting conditions, NO-induced activation of cGMP maintains low levels of [Ca](cyt) and MLC phosphorylation to minimize basal tone. This results from stimulation of Ca(2+) uptake from the cytosol into the SR via SERCA pumps, Ca(2+) efflux into the extracellular space via plasma membrane Ca(2+) pumps, and MLC phosphatase (MLCP) activity. We predict that basal concentrations of O(2)(-) and H(2)O(2) have negligible effects on Ca(2+) signaling and MLC phosphorylation. At concentrations above 1 nM, O(2)(-) is predicted to modulate [Ca(cyt)] and MCLP activity mostly by reducing NO bioavailability. The DVR vasoconstriction that is induced by high concentrations of H(2)O(2) can be explained by H(2)O(2)-mediated downregulation of MLCP and SERCA activity. We conclude that intrinsic generation of NO by the DVR wall may be sufficient to inhibit vasoconstriction by maintaining suppression of MLC phosphorylation.     

Degradation of lambda-carrageenan by Pseudoalteromonas carrageenovora lambda-carrageenase: a new family of glycoside hydrolases unrelated to kappa- and iota-carrageenases

NOX4 is an enigmatic member of the NOX (NADPH oxidase) family of ROS (reactive oxygen species)-generating NADPH oxidases. NOX4 has a wide tissue distribution, but the physiological function and activation mechanisms are largely unknown, and its pharmacology is poorly understood. We have generated cell lines expressing NOX4 upon tetracycline induction. Tetracycline induced a rapid increase in NOX4 mRNA (1 h) followed closely (2 h) by a release of ROS. Upon tetracycline withdrawal, NOX4 mRNA levels and ROS release decreased rapidly (<24 h). In membrane preparations, NOX4 activity was selective for NADPH over NADH and did not require the addition of cytosol. The pharmacological profile of NOX4 was distinct from other NOX isoforms: DPI (diphenyleneiodonium chloride) and thioridazine inhibited the enzyme efficiently, whereas apocynin and gliotoxin did not (IC(50)>100 muM). The pattern of NOX4-dependent ROS generation was unique: (i) ROS release upon NOX4 induction was spontaneous without need for a stimulus, and (ii) the type of ROS released from NOX4-expressing cells was H(2)O(2), whereas superoxide (O(2)(-)) was almost undetectable. Probes that allow detection of intracellular O(2)(-) generation yielded differential results: DHE (dihydroethidium) fluorescence and ACP (1-acetoxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine) ESR measurements did not detect any NOX4 signal, whereas a robust signal was observed with NBT. Thus NOX4 probably generates O(2)(-) within an intracellular compartment that is accessible to NBT (Nitro Blue Tetrazolium), but not to DHE or ACP. In conclusion, NOX4 has a distinct pharmacology and pattern of ROS generation. The close correlation between NOX4 mRNA and ROS generation might hint towards a function as an inducible NOX isoform.     

Local production of O2- by NAD(P)H oxidase in the sarcoplasmic reticulum of coronary arterial myocytes: cADPR-mediated Ca2+ regulation

The present study was designed to determine whether the sarcoplasmic reticulum (SR) could locally produce superoxide (O2-) via NAD(P)H oxidase (NOX) in coronary arterial myocytes (CAMs) and to address whether cADPR-RyR/Ca2+ signaling pathway regulates this local O2- production from the SR. Using confocal microscopic imaging analysis in intact single CAMs, a cell-permeable indicator CM-H2DCFDA for dynamic changes in intracellular ROS (in green color) and a highly selective ER-Tracker Red dye for tracking of the SR were found co-localized. A quantitative analysis based on the intensity of different spectra demonstrated a local O2- production derived from the SR. M(1)-receptor agonist, oxotremorine (Oxo) and a Ca2+ ionophore, A23187, time-dependently increased this O2- production colocalized with the SR. NOX inhibitors, diphenylene iodonium (DPI) and apocynin (Apo), or superoxide dismutase (SOD) and catalase, and Nox4 (a major intracellular NOX subunit) siRNA all substantially blocked this local production of O2-, demonstrating an involvement of NOX. This SR-derived O2- production was also abolished by the inhibitors of cyclic ADP-ribose (cADPR)-mediated Ca2+ signaling, such as nicotinamide (Nicot, 6 mM), ryanodine (Rya, 50 muM) or 8-Br-cADPR (30 microM). However, IP3 antagonist, 2-APB (50 microM) had no effect. In CAMs transfected with siRNA of ADP-ribosyl cyclase or RyR, this SR O2- production was attenuated. Electron spin resonance (ESR) spectromic assay in purified SR also demonstrated the production of O2- that was dependent on NOX activity and Ca2+ concentrations. These results provide direct evidence that O2- could be locally produced via NOX on the SR and that this local O2- producing system is controlled by cADPR-RyR/Ca2+ signaling pathway.     

Matrix metalloproteinase-2-mediated inhibition of Na+-dependent Ca+ uptake by superoxide radicals (O2*-) in microsomes of pulmonary smooth muscle

Treatment of microsomes (preferably enriched with endoplasmic reticulum) isolated from bovine pulmonary artery smooth muscle tissue with the O2*- -generating system (hypoxanthine (HPX) plus xanthine oxidase (XO)), markedly stimulated matrix metalloproteinase-2 (MMP-2) activity and also enhanced Ca2+ ATPase activity and ATP-dependent Ca2+ uptake. Pretreatment with superoxide dismutase (SOD) and tissue inhibitor of metalloproteinase (TIMP-2) (50 microg ml(-1)), preserved the increase in MMP-2 activity, Ca2+ ATPase activity and also ATP-dependent Ca2+ uptake in the microsomes. In contrast, Na+-dependent Ca2+ uptake in the microsomes was found to be inhibited by the O2*- - generating system. Additionally, O2*- -induced inhibition of Na+-dependent Ca2+ uptake was reversed by SOD and TIMP-2 (50 microg ml(-1)). Electron microscopy revealed that treatment with the O2*- -generating system did not cause any noticeable damage to the microsomes. O2*- -induced changes in MMP-2 activity, ATP-dependent Ca2+ uptake and Na+-dependent Ca2+ uptake, were not reversed upon pretreatment of the microsomes with a low dose (5 microg ml(-1)) of TIMP-2 which, on the contrary, reversed MMP-2 (1 microg ml(-1))-mediated alteration on these parameters. The inhibition of Na+-dependent Ca2+ uptake by O2*- and MMP-2, overpowered the stimulation of ATP-dependent Ca2+ uptake in the microsomes. Treatment of TIMP-2 (5 microg ml(-1)) with the O2*- -generating system abolished the inhibitory effect of TIMP-2 (5 microg ml(-1)) on MMP-2 (1 microg ml(-1)) (measured by (14)C-gelatin degradation). Overall, the present study suggests that O2*- inactivated TIMP-2, the ambient inhibitor of MMP-2, leading to activation of the ambient proteinase, MMP-2, which subsequently stimulated Ca2+ ATPase activity and ATP-dependent Ca2+ uptake, but inhibited Na+-dependent Ca2+ uptake, resulting in a marked decrease in Ca2+ uptake in the smooth muscle microsomes.     

Novel redox-dependent regulation of NOX5 by the tyrosine kinase c-Abl

We investigated the mechanism of H(2)O(2) activation of the Ca(2+)-regulated NADPH oxidase NOX5. H(2)O(2) induced a transient, dose-dependent increase in superoxide production in K562 cells expressing NOX5. Confocal studies demonstrated that the initial calcium influx generated by H(2)O(2) is amplified by a feedback mechanism involving NOX5-dependent superoxide production and H(2)O(2). H(2)O(2) NOX5 activation was inhibited by extracellular Ca(2+) chelators, a pharmacological inhibitor of c-Abl, and overexpression of kinase-dead c-Abl. Transfected kinase-active GFP-c-Abl colocalized with vesicular sites of superoxide production in a Ca(2+)-dependent manner. In contrast to H(2)O(2), the Ca(2+) ionophore ionomycin induced NOX5 activity independent of c-Abl. Immunoprecipitation of cell lysates revealed that active GFP-c-Abl formed oligomers with endogenous c-Abl and that phosphorylation of both proteins was increased by H(2)O(2) treatment. Furthermore, H(2)O(2)-induced NOX5 activity correlated with increased localization of c-Abl to the membrane fraction, and NOX5 proteins could be coimmunoprecipitated with GFP-Abl proteins. Our data demonstrate for the first time that NOX5 is activated by c-Abl through a Ca(2+)-mediated, redox-dependent signaling pathway and suggest a functional association between NOX5 NADPH oxidase and c-Abl.     

Exercise training decreases rat heart mitochondria free radical generation but does not prevent Ca2+-induced dysfunction.

Exercise provides cardioprotection against ischemia-reperfusion injury, a process involving mitochondrial reactive oxygen species (ROS) generation and calcium overload. This study tested the hypotheses that isolated mitochondria from hearts of endurance-trained rats have decreased ROS production and improved tolerance against Ca(2+)-induced dysfunction. Male Fischer 344 rats were either sedentary (Sed, n = 8) or endurance exercise trained (ET, n = 11) by running on a treadmill for 16 wk (5 days/wk, 60 min/day, 25 m/min, 6 degrees grade). Mitochondrial oxidative phosphorylation measures were determined with glutamate-malate or succinate as substrates, and H(2)O(2) production and permeability transition pore (PTP) opening were determined with succinate. All assays were carried out in the absence and presence of calcium. In response to 25 and 50 microM CaCl(2), Sed and ET displayed similar decreases in state 3 respiration, respiratory control ratio, and ADP:O ratio. Ca(2+)-induced PTP opening was also similar. However, H(2)O(2) production by ET was lower than Sed (P < 0.05) in the absence of calcium (323 +/- 12 vs. 362 +/- 11 pmol.min(-1).mg protein(-1)) and the presence of 50 microM CaCl(2) (154 +/- 3 vs. 197 +/- 7 pmol.min(-1).mg protein(-1)). Rotenone, which blocks electron flow from succinate to complex 1, reduced H(2)O(2) production and eliminated differences between ET and Sed. Mitochondrial superoxide dismutase and glutathione peroxidase were not affected by exercise. Catalase activity was extremely low but increased 49% in ET (P < 0.05). In conclusion, exercise reduces ROS production in myocardial mitochondria through adaptations specific to complex 1 but does not improve mitochondrial tolerance to calcium overload.     

Activation by saturated and monounsaturated fatty acids of the O2- -generating system in a cell-free preparation from neutrophils

Saturated and monounsaturated fatty acids with appropriate chain length such as laurate and oleate activated an O2- -generating enzyme system in a cell-free preparation from porcine neutrophils. The activated preparation catalyzed a stoichiometric conversion of O2 to O2- by utilizing NADPH as the electron donor. The preparation contained both membrane and soluble fractions and, upon separation into subfractions, the O2- -generating activity resided exclusively in the membrane fraction. Polyunsaturated fatty acids including arachidonate also activated the system, but they concurrently stimulated NADPH-independent O2 consuming reactions which yield neither O2- nor H2O2. The amount of such a non-O2- -producing O2 consumption often reached twice as much as that of O2- production. For the activation of the O2- -generating system in the membrane, the presence of the soluble fraction was essential. However, the soluble fraction was no longer effective when once used for the activation, suggesting that the effective component(s) in the fraction was consumed or translocated to the membrane during the activation. When the activated membrane was incubated with delipidated albumin, the activity was lost with concomitant decreases in the amount of membrane-associated fatty acids. The lost activity was restored by the replenishment of the fatty acid in the presence of a fresh soluble fraction. We also found that Ca2+ augmented a non-O2- -producing O2 consumption in the cell-free preparation by unsaturated fatty acids and interfered with the activation of the O2- -generating system, especially that by saturated fatty acids.     

Antioxidant pyruvate inhibits cardiac formation of reactive oxygen species through changes in redox state

Myocardial ischemia-reperfusion is associated with bursts of reactive oxygen species (ROS) such as superoxide radicals (O(2)(-).). Membrane-associated NADH oxidase (NADHox) activity is a hypothetical source of O(2)(-)., implying the NADH concentration-to-NAD(+) concentration ratio ([NADH]/[NAD(+)]) as a determinant of ROS. To test this hypothesis, cardiac NADHox and ROS formation were measured as influenced by pyruvate or L-lactate. Pre- and postischemic Langendorff guinea pig hearts were perfused at different pyruvate/L-lactate concentrations to alter cytosolic [NADH]/[NAD(+)]. NADHox and ROS were measured with the use of lucigenin chemiluminescence and electron spin resonance, respectively. In myocardial homogenates, pyruvate (0.05, 0.5 mM) and the NADHox blocker hydralazine markedly inhibited NADHox (16 +/- 2%, 58 +/- 9%). In postischemic hearts, pyruvate (0.1-5.0 mM) dose dependently inhibited ROS up to 80%. However, L-lactate (1.0-15.0 mM) stimulated both basal and postischemic ROS severalfold. Furthermore, L-lactate-induced basal ROS was dose dependently inhibited by pyruvate (0.1-5.0 mM) and not the xanthine oxidase inhibitor oxypurinol. Pyruvate did not inhibit ROS from xanthine oxidase. The data suggest a substantial influence of cytosolic NADH on cardiac O(2)(-). formation that can be inhibited by submillimolar pyruvate. Thus cytotoxicities due to cardiac ischemia-reperfusion ROS may be alleviated by redox reactants such as pyruvate.     

Fullerene derivatives protect against oxidative stress in RAW 264.7 cells and ischemia-reperfused lungs

Fullerene derivatives have often been used as effective scavengers for reactive oxygen species (ROS). This study was designed to test whether polyhydroxylated fullerene derivatives [C(60)(OH)(7+/-2)] protect against oxidative stress in cultured RAW 264.7 cells and ischemia-reperfused (IR) lungs. In RAW 264.7 cells, sodium nitroprusside (SNP, 1 mM) and H(2)O(2) (400 microM) caused a marked (90%) decrease in cell viability, and this decrease was dose dependently reversed by pretreatment with C(60)(OH)(7+/-2) (10-50 microM). The increase in ROS production induced by SNP and H(2)O(2) was significantly suppressed by C(60)(OH)(7+/-2). Also, the decrease in mitochondrial membrane potential induced by SNP and H(2)O(2) was significantly reversed by C(60)(OH)(7+/-2). However, high concentration of C(60)(OH)(7+/-2) (1 and 1.5 mM) lead to cell death (apoptosis or necrosis). In the isolated rat lung, the increases in pulmonary artery pressure and capillary filtration pressure induced by SNP during IR were reversed significantly by C(60)(OH)(7+/-2) (10 mg/kg). These results indicate that polyhydroxylated fullerene derivatives C(60)(OH)(7+/-2) at low concentrations protect against oxidative stress in RAW 264.7 cells and IR lungs.     

Effect of nitric oxides and hydrogen peroxide on Ca2+, Mg(2+)-ATPase and Mg(2+)-ATPase activity in myometrium sarcolemma

The action of sodium nitroprusside, nitrite-anions and hydrogen peroxide on Ca2+, Mg(2+)-ATPase and Mg(2+)-ATPase (Ca(2+)-independent) enzymatic activity in myometrium sarcolemma fraction is investigated. It is established, that 0.1 mM sodium nitroprusside and 10(-8)-10(-5) M nitrite-anions essentially reduce Ca2+, Mg(2+)-ATPase activity whereas Mg(2+)-ATPase proved to be absolutely resistant to them. At rather high concentration of nitrite-anions (0.1 mM) appreciable stimulation of Ca2+, Mg(2+)-ATPase was observed. Hydrogen peroxide (10(-8)-10(-4)), depending on the concentration suppressed both enzymes activity. However, Ca2+, Mg(2+)-ATPase proved to be more sensitive to the action of H2O2 (seeming K(i) = 0.42 +/- 0.1 microM), than Mg(2+)-ATPase (seeming K(i) = 3.1 +/- 0.9 microM). At presence of 1 mM ditiothreitole (a reducer of SH groups of the membrane surface) action of investigated substances considerably decreased. Reagents on carboxic- (dicyclogexilcarbodiimid) and amino- groups of the membrane (trinitrobenzolsulfonic acid) inhibited both Ca2+, Mg(2+)-ATPase, and Mg(2+)-ATPase activity in membrane fractions. In the presence of noted reagents sodium nitroprusside and nitrite-anions action was not almost shown. Hence, nitrogen oxide, nitrite-anions and hydrogen peroxide suppress Ca2+, Mg(2+)-ATPase and Mg(2+)-ATPase (only hydrogen peroxide) activity in the plasmatic membrane of myometrium cells, and this action can be connected with direct updating of superficial chemical groups of the membrane.