Cytochrome b5, not superoxide anion radical, is a major reductant of indoleamine 2,3-dioxygenase in human cells

The heme protein indoleamine 2,3-dioxygenase (IDO) initiates oxidative metabolism of tryptophan along the kynurenine pathway, and this requires reductive activation of Fe(3+)-IDO. The current dogma is that superoxide anion radical (O(2)(*-)) is responsible for this activation, based largely on previous work employing purified rabbit IDO and rabbit enterocytes. We have re-investigated this role of O(2)(*-) using purified recombinant human IDO (rhIDO), rabbit enterocytes that constitutively express IDO, human endothelial cells, and monocyte-derived macrophages treated with interferon-gamma to induce IDO expression, and two cell lines transfected with the human IDO gene. Both potassium superoxide and O(2)(*-) generated by xanthine oxidase modestly activated rhIDO, in reactions that were prevented completely by superoxide dismutase (SOD). In contrast, SOD mimetics had no effect on IDO activity in enterocytes and interferon-gamma-treated human cells, despite significantly decreasing cellular O(2)(*-) Similarly, cellular IDO activity was unaffected by increasing SOD activity via co-expression of Cu,Zn-SOD or by increasing cellular O(2)(*-) via treatment of cells with menadione. Other reductants, such as tetrahydrobiopterin, ascorbate, and cytochrome P450 reductase, were ineffective in activating cellular IDO. However, recombinant human cytochrome b(5) plus cytochrome P450 reductase and NADPH reduced Fe(3+)-IDO to Fe(2+)-IDO and activated rhIDO in a reconstituted system, a reaction inhibited marginally by SOD. Additionally, short interfering RNA-mediated knockdown of microsomal cytochrome b(5) significantly decreased IDO activity in IDO-transfected cells. Together, our data show that cytochrome b(5) rather than O(2)(*-) plays a major role in the activation of IDO in human cells.     

Early determinants of H2O2-induced endothelial dysfunction

Reactive oxygen species (ROS) can stimulate nitric oxide (NO(*)) production from the endothelium by transient activation of endothelial nitric oxide synthase (eNOS). With continued or repeated exposure, NO(*) production is reduced, however. We investigated the early determinants of this decrease in NO(*) production. Following an initial H(2)O(2) exposure, endothelial cells responded by increasing NO(*) production measured electrochemically. NO(*) concentrations peaked by 10 min with a slow reduction over 30 min. The decrease in NO(*) at 30 min was associated with a 2.7-fold increase in O(2)(*-) production (p < 0.05) and a 14-fold reduction of the eNOS cofactor, tetrahydrobiopterin (BH(4), p < 0.05). Used as a probe for endothelial dysfunction, the integrated NO(*) production over 30 min upon repeated H(2)O(2) exposure was attenuated by 2.1-fold (p = 0.03). Endothelial dysfunction could be prevented by BH(4) cofactor supplementation, by scavenging O(2)(*-) or peroxynitrite (ONOO(-)), or by inhibiting the NADPH oxidase. Hydroxyl radical (()OH) scavenging did not have an effect. In summary, early H(2)O(2)-induced endothelial dysfunction was associated with a decreased BH(4) level and increased O(2)(*-) production. Dysfunction required O(2)(*-), ONOO(-), or a functional NADPH oxidase. Repeated activation of the NADPH oxidase by ROS may act as a feed forward system to promote endothelial dysfunction.     

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.     

Autocrine/paracrine pattern of superoxide production through NAD(P)H oxidase in coronary arterial myocytes

The present study tested the hypothesis that membrane-bound NAD(P)H oxidase in coronary arterial myocytes (CAMs) is capable of producing superoxide (O(2)(*-)) toward extracellular space to exert an autocrine- or paracrine-like action in these cells. Using a high-speed wavelength-switching fluorescent microscopic imaging technique, we simultaneously monitored the binding of dihydroethidium-oxidizing product to exogenous salmon testes DNA trapped outside CAMs and to nuclear DNA as indicators of extra- and intracellular O(2)(*-) production. It was found that a muscarinic agonist oxotremorine (OXO; 80 microM) increased O(2)(*-) levels more rapidly outside than inside CAMs. In the presence of superoxide dismutase (500 U/ml) plus catalase (400 U/ml) and NAD(P)H oxidase inhibitor diphenylene iodonium (50 microM) or apocynin (100 microM), these increases in extra- and intracellular O(2)(*-) levels were substantially abolished or attenuated. The O(2)(*-) increase outside CAMs was also confirmed by detecting oxidation of nitro blue tetrazolium and confocal microscopic localization of Matrigel-trapped OxyBURST H(2)HFF Green BSA staining around these cells. By electron spin resonance spectrometry, the extracellular accumulation of O(2)(*-) was demonstrated as a superoxide dismutase-sensitive component outside CAMs. Furthermore, RNA interference of NAD(P)H oxidase subunits Nox1 or p47 markedly blocked OXO-induced increases in both extra- and intracellular O(2)(*-) levels, whereas small inhibitory RNA of Nox4 only attenuated intracellular O(2)(*-) accumulation. These results suggest that Nox1 represents a major NAD(P)H oxidase isoform responsible for extracellular O(2)(*-) production. This rapid extracellular production of O(2)(*-) seems to be unique to OXO-induced M(1)-receptor activation, since ANG II-induced intra- and extracellular O(2)(*-) increases in parallel. It is concluded that the outward production of O(2)(*-) via NAD(P)H oxidase in CAMs may represent an important producing pattern for its autocrine or paracrine actions.     

Reactive oxygen species produced up- or downstream of calcium influx regulate proinflammatory mediator release from mast cells: role of NADPH oxidase and mitochondria

Earlier studies have demonstrated that mast cells produce reactive oxygen species (ROS), which play a role in regulating Ca(2+) influx, while in other cell types ROS are produced in a Ca(2+)-dependent manner. We sought to determine whether ROS are produced downstream of the extracellular Ca(2+) entry in mast cells. Thapsigargin (TG), a receptor-independent agonist, could evoke a robust burst of intracellular ROS. However, this response was distinct from the antigen-induced burst of ROS with respect to time course and dependence on Ca(2+) and phosphatidylinositol-3-kinase (PI3K). The antigen-induced ROS generation occurred immediately, while the TG-induced ROS generation occurred with a significant lag time (~2 min). Antigen but not TG caused extracellular release of superoxide (O(2)(*-))/hydrogen peroxide (H(2)O(2)), which was blocked by diphenyleneiodonium, apocynin, and wortmannin. A capacitative Ca(2+) entry resulted in the generation of O(2)(*-) in the mitochondria in a PI3K-independent manner. Blockade of ROS generation inhibited TG-induced mediator release. Finally, when used together, antigen and TG evoked the release of leukotriene C(4), tumor necrosis factor-alpha, and interleukin-13 as well as ROS generation synergistically. These results suggest that ROS produced upstream of Ca(2+) influx by NADPH oxidase and downstream of Ca(2+) influx by the mitochondria regulate the proinflammatory response of mast cells.     

A new paradigm: manganese superoxide dismutase influences the production of H2O2 in cells and thereby their biological state

The principal source of hydrogen peroxide in mitochondria is thought to be from the dismutation of superoxide via the enzyme manganese superoxide dismutase (MnSOD). However, the nature of the effect of SOD on the cellular production of H(2)O(2) is not widely appreciated. The current paradigm is that the presence of SOD results in a lower level of H(2)O(2) because it would prevent the non-enzymatic reactions of superoxide that form H(2)O(2). The goal of this work was to: a) demonstrate that SOD can increase the flux of H(2)O(2), and b) use kinetic modelling to determine what kinetic and thermodynamic conditions result in SOD increasing the flux of H(2)O(2). We examined two biological sources of superoxide production (xanthine oxidase and coenzyme Q semiquinone, CoQ(*-) that have different thermodynamic and kinetic properties. We found that SOD could change the rate of formation of H(2)O(2) in cases where equilibrium-specific reactions form superoxide with an equilibrium constant (K) less than 1. An example is the formation of superoxide in the electron transport chain (ETC) of the mitochondria by the reaction of ubisemiquinone radical with dioxygen. We measured the rate of release of H(2)O(2) into culture medium from cells with differing levels of MnSOD. We found that the higher the level of SOD, the greater the rate of accumulation of H(2)O(2). Results with kinetic modelling were consistent with this observation; the steady-state level of H(2)O(2) increases if K<1, for example CoQ(*-)+O(2)-->CoQ+O(2)(*-). However, when K>1, e.g. xanthine oxidase forming O(2)(*-), SOD does not affect the steady state-level of H(2)O(2). Thus, the current paradigm that SOD will lower the flux of H(2)O(2) does not hold for the ETC. These observations indicate that MnSOD contributes to the flux of H(2)O(2) in cells and thereby is involved in establishing the cellular redox environment and thus the biological state of the cell.     

Apocynin inhibits NADPH oxidase in phagocytes but stimulates ROS production in non-phagocytic cells

Apocynin is a naturally occurring methoxy-substituted catechol, experimentally used as an inhibitor of NADPH oxidase. Since it acts as a potent inhibitor in studies with neutrophils and macrophages, no inhibitory effect can often be found in non-phagocyte cells. In our experiments, apocynin even stimulated reactive oxygen species (ROS) production by vascular fibroblasts. Even when added to macrophages, apocynin initially caused an increase in ROS production. The inhibition of ROS formation followed, suggesting that in the presence of leukocyte myeloperoxidase and hydrogen peroxide, apocynin is converted to another compound. Apocynin pre-activated with H2O2 and horseradish peroxidase (HRP) inhibited ROS production immediately. In non-phagocytes, apocynin stimulated ROS production and no inhibition was observed even after 60 min. Apocynin treated with H2O2 and HRP, however, decreased ROS production in the same manner as in macrophages. The stimulatory effect on ROS production can be abolished by tiron and superoxide dismutase (SOD), suggesting that superoxide was the produced species. The effect of apocynin was inhibited by diphenylene iodinium (DPI), a non-scavenging NADPH oxidase inhibitor. It can be summarized that apocynin stimulates cell superoxide production. In the presence of peroxidase and hydrogen peroxide, however, it is converted into another compound that acts as an inhibitor of superoxide production. It strongly suggests that under conditions in vivo, apocynin can have opposite effects on phagocytes and non-phagocyte cells. It acts as an inhibitor of phagocyte NADPH oxidase but also as a ROS production stimulator in non-phagocyte cells.     

Hypoxic pulmonary hypertension: role of superoxide and NADPH oxidase (gp91phox)

Chronic exposure to low-O2 tension induces pulmonary arterial hypertension (PAH), which is characterized by vascular remodeling and enhanced vasoreactivity. Recent evidence suggests that reactive oxygen species (ROS) may be involved in both processes. In this study, we critically examine the role superoxide and NADPH oxidase plays in the development of chronic hypoxic PAH. Chronic hypoxia (CH; 10% O2 for 3 wk) caused a significant increase in superoxide production in intrapulmonary arteries (IPA) of wild-type (WT) mice as measured by lucigenin-enhanced chemiluminescence. The CH-induced increase in the generation of ROS was obliterated in NADPH oxidase (gp91phox) knockout (KO) mice, suggesting that NADPH oxidase was the major source of ROS. Importantly, pathological changes associated with CH-induced PAH (mean right ventricular pressure, medial wall thickening of small pulmonary arteries, and right heart hypertrophy) were completely abolished in NADPH oxidase (gp91phox) KO mice. CH potentiated vasoconstrictor responses of isolated IPAs to both 5-hydroxytryptamine (5-HT) and the thromboxane mimetic U-46619. Administration of CuZn superoxide dismutase to isolated IPA significantly reduced CH-enhanced superoxide levels and reduced the CH-enhanced vasoconstriction to 5-HT and U-46619. Additionally, CH-enhanced superoxide production and vasoconstrictor activity seen in WT IPAs were markedly reduced in IPAs isolated from NADPH oxidase (gp91phox) KO mice. These results demonstrate a pivotal role for gp91phox-dependent superoxide production in the pathogenesis of CH-induced PAH.     

Reactive oxygen species from NADPH oxidase contribute to altered pulmonary vascular responses in piglets with chronic hypoxia-induced pulmonary hypertension

Our main objective was to determine whether reactive oxygen species (ROS), such as superoxide (O(2)(-)) and hydrogen peroxide (H(2)O(2)), contribute to altered pulmonary vascular responses in piglets with chronic hypoxia-induced pulmonary hypertension. Piglets were raised in either room air (control) or hypoxia for 3 days. The effect of the cell-permeable superoxide dismutase mimetic (SOD; M40403) and/or PEG-catalase (PEG-CAT) on responses to acetylcholine (ACh) was measured in endothelium-intact and denuded pulmonary resistance arteries (PRAs; 90-to-300-microm diameter). To determine whether NADPH oxidase is an enzymatic source of ROS, PRA responses to ACh were measured in the presence and absence of a NADPH oxidase inhibitor, apocynin (APO). A Western blot technique was used to assess expression of the NADPH oxidase subunit, p67phox. A lucigenin-derived chemiluminescence technique was used to measure ROS production stimulated by the NADPH oxidase substrate, NADPH. ACh responses, which were dilation in intact control arteries but constriction in both intact and denuded hypoxic arteries, were diminished by M40403, PEG-CAT, the combination of M40403 plus PEG-CAT, as well as by APO. Although total amounts were not different, membrane-associated p67phox was greater in PRAs from hypoxic compared with control piglets. NADPH-stimulated lucigenin luminescence was nearly doubled in PRAs from hypoxic vs. control piglets. We conclude that ROS generated by NADPH oxidase contribute to the aberrant pulmonary arterial responses in piglets exposed to 3 days of hypoxia.     

Vanadium-induced apoptosis and pulmonary inflammation in mice: Role of reactive oxygen species

Pulmonary exposure to metals and metal-containing compounds is associated with pulmonary inflammation, cell death, and tissue injury. The present study uses a mouse model to investigate vanadium-induced apoptosis and lung inflammation, and the role of reactive oxygen species (ROS) in this process. Aspiration of the pentavalent form of vanadium, V (V), caused a rapid influx of polymorphonuclear leukocytes into the pulmonary airspace with a peak inflammatory response at 6 h post-exposure and resolution by 72 h. During this period, the number of apoptotic lung cells which were predominantly neutrophils increased considerably with a peak response at 24 h accompanied by no or minimum necrosis. After 24 h when the V (V)-induced inflammation was in the resolution phase, an increased influx of macrophages and engulfment of apoptotic bodies by these phagocytes was observed, supporting the role of macrophages in apoptotic cell clearance and resolution of V (V)-induced lung inflammation. Electron spin resonance (ESR) studies using lavaged alveolar macrophages showed the formation of ROS, including O(2)(*-), H(2)O(2), and (*)OH radicals which were confirmed by inhibition with free radical scavengers. The mechanism of ROS generation induced by V (V) involved the activation of an NADPH oxidase complex and the mitochondrial electron transport chain. The ROS scavenger, catalase (H(2)O(2) scavenger), effectively inhibited both lung cell apoptosis and the inflammatory response, whereas superoxide dismutase (SOD) (O(2)(*-) scavenger) and the metal chelator, deferoxamine (inhibitor of (*)OH generation by Fenton-like reactions) had lesser effects. These results indicate that multiple oxidative species are involved in V (V)-induced lung inflammation and apoptosis, and that H(2)O(2) plays a major role in this process.     

Antimycin A and lipopolysaccharide cause the leakage of superoxide radicals from rat liver mitochondria

Here we show that both Antimycin A, a respiratory chain inhibitor inducing apoptosis, and endotoxic shock, a syndrome accompanied by both necrosis and apoptosis, cause not only an increase but also the leakage of superoxide radicals (O(2)(*-)) from rat heart mitochondria (RHM), while O(2)(*-) generated in intact RHM do not escape from mitochondria. This was shown by a set of O(2)(*-)-sensitive spin probes with varying hydrophobicity. The levels of O(2)(*-) detected in intact RHM gradually increase as the hydrophobicity of spin probes increases and were not sensitive to superoxide dismutase (SOD) added to the incubation medium. Both Antimycin A and endotoxic shock elevated O(2)(*-) levels. Elevated O(2)(*-) levels became sensitive to SOD but in a different manner. The determination of O(2)(*-) with water-soluble PPH was fully sensitive to SOD, while the determination of O(2)(*-) with the more hydrophobic CMH and CPH was only partially sensitive to SOD, suggesting the release of a portion of O(2)(*-) into the surrounding medium.     

Aging enhances pressure-induced arterial superoxide formation

The purpose of this study was to investigate the mechanisms that regulate superoxide (O(2)(*-)) production as a function of an acute elevation of intravascular pressure and age. Mesenteric arteries isolated from young (6 mo) and aged (24 mo) male Fischer 344 rats were used. O(2)(*-) production in vessels in response to 80 (normal pressure, NP) and 180 (high pressure, HP) mmHg was determined by the superoxide dismutase-inhibitable nitroblue tetrazolium (NBT) reduction assay. In vessels exposed to NP, O(2)(*-) production was significantly higher in aged than in young vessels (32.7 +/- 7.0 vs. 15.4 +/- 2.4 nmol.mg(-1).30 min(-1)). HP enhanced O(2)(*-) production in vessels of both groups, but the enhancement was significantly greater in aged than in young vessels (63.4 +/- 6.7 vs. 32.7 +/- 4.3 nmol.mg(-1).30 min(-1)). Apocynin (100 micromol/l) attenuated HP-induced increases in O(2)(*-) production in both groups, whereas allopurinol (100 micromol/l) and N(omega)-nitro-L-arginine methyl ester (100 mumol/l) inhibited the response only in aged vessels. Confocal microscopy showed increases in O(2)(*-) in response to HP in endothelial and smooth muscle layers of both groups, with much greater fluorescent staining in aged than in young rats and in the endothelium than in smooth muscle cells. No significant changes in NAD(P)H oxidase gene and protein expressions were observed in vessels of the two groups. Upregulation of protein expression of xanthine oxidase was detected in aged vessels. We conclude that NAD(P)H oxidase contributes importantly to HP-induced enhanced O(2)(*-) production in vessels of both young and aged rats, whereas xanthine oxidase and nitric oxide synthase-dependent O(2)(*-) production also contribute to the enhancement in mesenteric arteries of aged rats.     

Detection of superoxide anion released extracellularly by endothelial cells using cytochrome c reduction, ESR, fluorescence and lucigenin-enhanced chemiluminescence techniques

Endothelium produces oxygen-derived free radicals (nitric oxide, NO&z.rad;; superoxide anion, O(2)(*-)) which play a major role in physiology and pathology of the vessel wall. However, little is known about endothelium-derived O(2)(*-) production, particularly due to the difficulty in assessing O(2)(*-) when its production is low and to controversies recently raised about the use of lucigenin-enhanced chemiluminescence. We compared four techniques of O(2)(*-) assessment when its production is low. In the present study, we have compared ferricytochrome c reduction, electron spin resonance (ESR) spectroscopy using DMPO as spin trap, hydroethidine fluorescence, and lucigenin-enhanced chemiluminescence to assess O(2)(*-) production in cultured bovine aortic endothelial cells (BAEC). We focused our study on extracellular O(2)(*-) production because the specificity of the signal is provided by the use of superoxide dismutase, and this control cannot be obtained intracellularly. We found that the calcium ionophore A23187 dose-dependently stimulated O(2)(*-) production, with a good correlation between all four techniques. The signals evoked by postconfluent BAEC were increased 2- to 7-fold in comparison to just-confluent BAEC, according to the technique used. Ferricytochrome c 20 microm rather than at 100 microm appears more suitable to detect O(2)(*-). However, in the presence of electron donors such as NADH or NADPH, lucigenin-enhanced chemiluminescence generated high amounts of O(2)(*-). Thus, ferricytochrome c reduction, electron spin resonance (ESR), and hydroethidine fluorescence appear as adequate tools for the detection of extracellular endothelium-derived O(2)(*-) production, whereas lucigenin may be artifactual, even when a low concentration of lucigenin is employed.     

Xanthine oxidase and mitochondria contribute to vascular superoxide anion generation in DOCA-salt hypertensive rats

Vascular superoxide anion (O(2)(*-)) levels are increased in DOCA-salt hypertensive rats. We hypothesized that the endothelin (ET)-1-induced generation of ROS in the aorta and resistance arteries of DOCA-salt rats originates partly from xanthine oxidase (XO) and mitochondria. Accordingly, we blocked XO and the mitochondrial oxidative phosphorylation chain to investigate their contribution to ROS production in mesenteric resistance arteries and the aorta from DOCA-salt rats. Systolic blood pressure rose in DOCA-salt rats and was reduced after 3 wk by apocynin [NAD(P)H oxidase inhibitor and/or radical scavenger], allopurinol (XO inhibitor), bosentan (ET(A/B) receptor antagonist), BMS-182874 (BMS; ET(A) receptor antagonist), and hydralazine. Plasma uric acid levels in DOCA-salt rats were similar to control unilaterally nephrectomized (UniNx) rats, reduced with allopurinol and bosentan, and increased with BMS. Levels of thiobarbituric acid-reacting substances were increased in DOCA-salt rats versus UniNx rats, and BMS, bosentan, and hydralazine prevented their increase. Dihydroethidium staining showed reduced O(2)(*-) production in mesenteric arteries and the aorta from BMS- and bosentan-treated DOCA-salt rats compared with untreated DOCA-salt rats. Increased O(2)(*-) derived from XO was reduced or prevented by all treatments in mesenteric arteries, whereas bosentan and BMS had no effect on aortas from DOCA-salt rats. O(2)(*-) generation decreased with in situ treatment by tenoyltrifluoroacetone and CCCP, inhibitors of mitochondrial electron transport complexes II and IV, respectively, whereas rotenone (mitochondrial complex I inhibitor) had no effect. Our findings demonstrate the involvement of ET(A) receptor-modulated O(2)(*-) derived from XO and from mitochondrial oxidative enzymes in arteries from DOCA-salt rats.     

Role of reactive oxygen species in the regulation of Arabidopsis seed dormancy

Freshly harvested seeds of Arabidopsis thaliana, Columbia (Col) accession were dormant when imbibed at 25°C in the dark. Their dormancy was alleviated by continuous light during imbibition or by 5 weeks of storage at 20°C (after-ripening). We investigated the possible role of reactive oxygen species (ROS) in the regulation of Col seed dormancy. After 24 h of imbibition at 25°C, non-dormant seeds produced more ROS than dormant seeds, and their catalase activity was lower. In situ ROS localization revealed that germination was associated with an accumulation of superoxide and hydrogen peroxide in the radicle. ROS production was temporally and spatially regulated: ROS were first localized within the cytoplasm upon imbibition of non-dormant seeds, then in the nucleus and finally in the cell wall, which suggests that ROS play different roles during germination. Imbibition of dormant and non-dormant seeds in the presence of ROS scavengers or donors, which inhibited or stimulated germination, respectively, confirmed the role of ROS in germination. Freshly harvested seeds of the mutants defective in catalase (cat2-1) and vitamin E (vte1-1) did not display dormancy; however, seeds of the NADPH oxidase mutants (rbohD) were deeply dormant. Expression of a set of genes related to dormancy upon imbibition in the cat2-1 and vet1-1 seeds revealed that their non-dormant phenotype was probably not related to ABA or gibberellin metabolism, but suggested that ROS could trigger germination through gibberellin signaling activation.     

Production of superoxide anion and hydrogen peroxide by capacitating buffalo (Bubalus bubalis) spermatozoa

In the present study attempts were made to detect and quantify the generation of superoxide anion (O(2)(*-)) and hydrogen peroxide (H(2)O(2)) by capacitating buffalo spermatozoa. Ejaculated buffalo spermatozoa were suspended in sp-TALP medium at 50x10(6)mL(-1) and incubated at 38.5 degrees C with 5% CO(2) in air in the absence or presence of heparin (a capacitation inducer) or reduced nicotinamide adenine dinucleotide phosphate (NADPH) or diphenyleneiodonium (DPI, a flavoprotein inhibitor) for 6h. Production rate of O(2)(*-) and H(2)O(2) by spermatozoa at different hours of capacitation were measured by cytochrome c reduction and phenol red oxidation assays, respectively. Spermatozoa generated both O(2)(*-) and H(2)O(2) spontaneously and following stimulation with heparin and a significant increase of O(2)(*-) production was observed in the presence of NADPH. However, DPI inhibited this NADPH-induced O(2)(*-) production and suggested for existence of putative NADPH-oxidase that constitute a specific O(2)(*-) generating systems in buffalo spermatozoa. Results of our study indicated that buffalo spermatozoa generate O(2)(*-) and H(2)O(2) and production of these free radicals is induced during capacitation.     

Phosphatidylinositol 3-kinase plays a vital role in regulation of rice seed vigor via altering NADPH oxidase activity

Phosphatidylinositol 3-kinase (PI3K) has been reported to be important in normal plant growth and stress responses. In this study, it was verified that PI3K played a vital role in rice seed germination through regulating NADPH oxidase activity. Suppression of PI3K activity by inhibitors wortmannin or LY294002 could abate the reactive oxygen species (ROS) formation, which resulted in disturbance to the seed germination. And then, the signal cascades that PI3K promoted the ROS liberation was also evaluated. Diphenylene iodonium (DPI), an NADPH oxidase inhibitor, suppressed most of ROS generation in rice seed germination, which suggested that NADPH oxidase was the main source of ROS in this process. Pharmacological experiment and RT-PCR demonstrated that PI3K promoted the expression of Os rboh9. Moreover, functional analysis by native PAGE and the measurement of the 2, 3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazo-lium-5- carboxanilide (XTT) formazan concentration both showed that PI3K promoted the activity of NADPH oxidase. Furthermore, the western blot analysis of OsRac-1 demonstrated that the translocation of Rac-1 from cytoplasm to plasma membrane, which was known as a key factor in the assembly of NADPH oxidase, was suppressed by treatment with PI3K inhibitors, resulting in the decreased activity of NADPH oxidase. Taken together, these data favored the novel conclusion that PI3K regulated NADPH oxidase activity through modulating the recruitment of Rac-1 to plasma membrane and accelerated the process of rice seed germination.     

Constitutive NADPH oxidase and increased mitochondrial respiratory chain activity regulate chemokine gene expression

Alveolar macrophages, which generate high levels of reactive oxygen species, especially O(2)(*-), are involved in the recruitment of neutrophils to sites of inflammation and injury in the lung, and the generation of chemotactic proteins triggers this cellular recruitment. In this study, we asked whether O(2)(*-) generation in alveolar macrophages had a role in the expression of chemokines. Specifically, we hypothesized that O(2)(*-) generation is necessary for chemokine expression in alveolar macrophages after TNF-alpha stimulation. We found that alveolar macrophages have high constitutive NADPH oxidase activity that was not increased by TNF-alpha, but TNF-alpha increased the activity of the mitochondrial respiratory chain. In addition, the mitochondrial respiratory chain increased O(2)(*-) generation if the NADPH oxidase was inhibited. O(2)(*-) generation was necessary for macrophage inflammatory protein-2 (MIP-2) gene expression, because inhibition of NADPH oxidase or the mitochondrial respiratory chain or overexpression of Cu,Zn-superoxide dismutase significantly inhibited expression of MIP-2. TNF-alpha activated the ERK MAP kinase, and ERK activity was essential for chemokine gene expression. In addition, overexpression of the MEK1-->ERK pathway significantly increased IL-8 expression, and a small interfering RNA to the NADPH oxidase inhibited ERK- and TNF-alpha-induced chemokine expression. Collectively, these results suggest that in alveolar macrophages, O(2)(*-) generation mediates chemokine expression after TNF-alpha stimulation in an ERK-dependent manner.     

Role of reactive oxygen species in LPS-induced production of prostaglandin E2 in microglia

We determined the roles of reactive oxygen species (ROS) in the expression of cyclooxygenase-2 (COX-2) and the production of prostaglandin E2 (PGE2) in lipopolysaccharide (LPS)-activated microglia. LPS treatment increased intracellular ROS in rat microglia dose-dependently. Pre-treatment with superoxide dismutase (SOD)/catalase, or SOD/catalase mimetics that can scavenge intracellular ROS, significantly attenuated LPS-induced release in PGE2. Diphenylene iodonium (DPI), a non-specific NADPH oxidase inhibitor, decreased LPS-induced PGE2 production. In addition, microglia from NADPH oxidase-deficient mice produced less PGE2 than those from wild-type mice following LPS treatment. Furthermore, LPS-stimulated expression of COX-2 (determined by RT-PCR analysis of COX-2 mRNA and western blot for its protein) was significantly reduced by pre-treatment with SOD/catalase or SOD/catalase mimetics. SOD/catalase mimetics were more potent than SOD/catalase in reducing COX-2 expression and PGE2 production. As a comparison, scavenging ROS had no effect on LPS-induced nitric oxide production in microglia. These results suggest that ROS play a regulatory role in the expression of COX-2 and the subsequent production of PGE2 during the activation process of microglia. Thus, inhibiting NADPH oxidase activity and subsequent ROS generation in microglia can reduce COX-2 expression and PGE2 production. These findings suggest a potential therapeutic intervention strategy for the treatment of inflammation-mediated neurodegenerative diseases.     

Intracellular GSH levels rather than ROS levels are tightly related to AMA-induced HeLa cell death

Antimycin A (AMA) inhibits succinate oxidase and NADH oxidase, and also inhibits mitochondrial electron transport between cytochromes b and c. We investigated the involvement of ROS and GSH in AMA-induced HeLa cell death. AMA increased the intracellular H(2)O(2) and O(2)(*-) levels and reduced the intracellular GSH content. ROS scavengers (Tempol, Tiron, Trimetazidine and NAC) did not down-regulate the production of ROS and inhibit apoptosis in AMA-treated cells. Treatment with NAC and N-propylgallate showing the enhancement of GSH depletion in AMA-treated cells significantly intensified the levels of apoptosis. Calpain inhibitors I and II (calpain inhibitor III) and Ca(2+)-chelating agent (EGTA/AM) significantly reduced H(2)O(2) levels in AMA-treated HeLa cells. However, treatment with calpain inhibitor III intensified the levels of O(2)(*-) in AMA-treated cells. In addition, calpain inhibitor III strongly depleted GSH content with an enhancement of apoptosis in AMA-treated cells. Conclusively, the changes of ROS by AMA were not tightly correlated with apoptosis in HeLa cells. However, intracellular GSH levels are tightly related to AMA-induced cell death.