Oxidative stress caused by pyocyanin impairs CFTR Cl(-) transport in human bronchial epithelial cells

Pyocyanin (N-methyl-1-hydroxyphenazine), a redox-active virulence factor produced by the human pathogen Pseudomonas aeruginosa, is known to compromise mucociliary clearance. Exposure of human bronchial epithelial cells to pyocyanin increased the rate of cellular release of H(2)O(2) threefold above the endogenous H(2)O(2) production. Real-time measurements of the redox potential of the cytosolic compartment using the redox sensor roGFP1 showed that pyocyanin (100 microM) oxidized the cytosol from a resting value of -318+/-5 mV by 48.0+/-4.6 mV within 2 h; a comparable oxidation was induced by 100 microM H(2)O(2). Whereas resting Cl(-) secretion was slightly activated by pyocyanin (to 10% of maximal currents), forskolin-stimulated Cl(-) secretion was inhibited by 86%. The decline was linearly related to the cytosolic redox potential (1.8% inhibition/mV oxidation). Cystic fibrosis bronchial epithelial cells homozygous for DeltaF508 CFTR failed to secrete Cl(-) in response to pyocyanin or H(2)O(2), indicating that these oxidants specifically target the CFTR and not other Cl(-) conductances. Treatment with pyocyanin also decreased total cellular glutathione levels to 62% and cellular ATP levels to 46% after 24 h. We conclude that pyocyanin is a key factor that redox cycles in the cytosol, generates H(2)O(2), depletes glutathione and ATP, and impairs CFTR function in Pseudomonas-infected lungs.     

Pseudomonas aeruginosa pyocyanin directly oxidizes glutathione and decreases its levels in airway epithelial cells

Production of pyocyanin enhances Pseudomonas aeruginosa virulence. Many of pyocyanin's in vitro and in vivo cytotoxic effects on human cells appear to result from its ability to redox cycle. Pyocyanin directly accepts electrons from NADH or NADPH with subsequent electron transfer to oxygen, generating reactive oxygen species. Reduced glutathione (GSH) is an important cellular antioxidant, and it contributes to the regulation of redox-sensitive signaling systems. Using the human bronchial epithelial (HBE) and the A549 human type II alveolar epithelial cell lines, we tested the hypothesis that pyocyanin can deplete airway epithelial cells of GSH. Incubation of both cell types with pyocyanin led to a concentration-dependent loss of cellular GSH (up to 50%) and an increase in oxidized GSH (GSSG) in the HBE, but not A549 cells, at 24 h. An increase in total GSH, mostly as GSSG, was detected in the culture media, suggesting export of GSH or GSSG from the pyocyanin-exposed cells. Loss of GSH could be due to pyocyanin-induced H(2)O(2) formation. However, overexpression of catalase only partially prevented the pyocyanin-mediated decline in cellular GSH. Cell-free electron paramagnetic resonance studies revealed that pyocyanin directly oxidizes GSH, forming pyocyanin free radical and O(2)(-). Pyocyanin oxidized other thiol-containing compounds, cysteine and N-acetyl-cysteine, but not methionine. Thus GSH may enhance pyocyanin-induced cytotoxicity by functioning as an alternative source of reducing equivalents for pyocyanin redox cycling. Pyocyanin-mediated alterations in cellular GSH may alter epithelial cell functions by modulating redox sensitive signaling events.     

Mitochondrial adaptations to obesity-related oxidant stress

It is not known why viable hepatocytes in fatty livers are vulnerable to necrosis, but associated mitochondrial alterations suggest that reactive oxygen species (ROS) production may be increased. Although the mechanisms for ROS-mediated lethality are not well understood, increased mitochondrial ROS generation often precedes cell death, and hence, might promote hepatocyte necrosis. The aim of this study is to determine if liver mitochondria from obese mice with fatty hepatocytes actually produce increased ROS. Secondary objectives are to identify potential mechanisms for ROS increases and to evaluate whether ROS increase uncoupling protein (UCP)-2, a mitochondrial protein that promotes ATP depletion and necrosis. Compared to mitochondria from normal livers, fatty liver mitochondria have a 50% reduction in cytochrome c content and produce superoxide anion at a greater rate. They also contain 25% more GSH and demonstrate 70% greater manganese superoxide dismutase activity and a 35% reduction in glutathione peroxidase activity. Mitochondrial generation of H(2)O(2) is increased by 200% and the activities of enzymes that detoxify H(2)O(2) in other cellular compartments are abnormal. Cytosolic glutathione peroxidase and catalase activities are 42 and 153% of control values, respectively. These changes in the production and detoxification of mitochondrial ROS are associated with a 300% increase in the mitochondrial content of UCP-2, although the content of beta-1 ATP synthase, a constitutive mitochondrial membrane protein, is unaffected. Supporting the possibility that mitochondrial ROS induce UCP-2 in fatty hepatocytes, a mitochondrial redox cycling agent that increases mitochondrial ROS production upregulates UCP-2 mRNAs in primary cultures of normal rat hepatocytes by 300%. Thus, ROS production is increased in fatty liver mitochondria. This may result from chronic apoptotic stress and provoke adaptations, including increases in UCP-2, that potentiate necrosis.     

The differential effects of superoxide anion, hydrogen peroxide and hydroxyl radical on cardiac mitochondrial oxidative phosphorylation

The involvement of reactive oxygen species (ROS) in cardiac ischemia-reperfusion injuries is well-established, but the deleterious effects of hydrogen peroxide (H(2)O(2)), hydroxyl radical (HO*) or superoxide anion (O(2)*(-) ) on mitochondrial function are poorly understood. Here, we report that incubation of rat heart mitochondria with each of these three species resulted in a decline of the ADP-stimulated respiratory rate but not substrate-dependent respiration. These three species reduced oxygen consumption induced by an uncoupler without alteration of the respiratory chain complexes, but did not modify mitochondrial membrane permeability. HO* slightly decreased F1F0-ATPase activity and HO* and O(2)*(-) partially inhibited the activity of adenine nucleotide translocase; H(2)O(2) failed to alter these targets. They inhibited NADH production by acting specifically on aconitase for O(2)*(-) and alpha-ketoglutarate dehydrogenase for H(2)O(2) and HO*. Our results show that O(2)*(-), H(2)O(2) and HO* act on different mitochondrial targets to alter ATP synthesis, mostly through inhibition of NADH production.     

Glutathione modulates the toxicity of, but is not a biologically relevant reductant for, the Pseudomonas aeruginosa redox toxin pyocyanin

Pyocyanin is an important redox toxin produced by the common human pathogen Pseudomonas aeruginosa. It generates reactive oxygen species (ROS) that alter intracellular redox status and cell function. Reducing equivalents for pyocyanin are provided by intracellular NAD(P)H and, it has been reported, glutathione (GSH). Cellular GSH levels are at least 1-2 orders of magnitude greater than NAD(P)H; therefore GSH should represent the major reductant for pyocyanin and potentiate its toxicity. Paradoxically, GSH has been found to inhibit pyocyanin toxicity in cellular models. This study was undertaken to evaluate the potential of GSH as a biologically relevant reductant for pyocyanin. As observed using spectrophotometry, under aerobic conditions pyocyanin readily oxidized NADPH, whereas oxidation of GSH could not be detected. Under anaerobic conditions pyocyanin was reduced by NADPH, but reduction by GSH could not be detected. Reduction of molecular oxygen and the formation of ROS readily proceeded in the presence of pyocyanin and NADPH, whereas GSH was without effect. Finally, exposure of normal human dermal fibroblasts to subcytotoxic concentrations of pyocyanin did not lead to depletion of endogenous GSH, but exogenous GSH provided protection against the senescence-inducing effects of the toxin. In summary, GSH does not reduce pyocyanin under physiologically relevant conditions or contribute to pyocyanin toxicity. However, GSH does provide protection against the deleterious effects of this important bacterial toxin on mammalian cells.     

Azidothymidine causes functional and structural destruction of mitochondria, glutathione deficiency and HIV-1 promoter sensitization.

Mitochondrial functional and structural impairment and generation of oxidative stress have been implicated in aging, various diseases and chemotherapies. This study analyzed azidothymidine (AZT)-caused failures in mitochondrial functions, in redox regulation and activation of the HIV-1 gene expression. We monitored intracellular concentrations of ATP and glutathione (GSH) as the indicators of energy production and redox conditions, respectively, during the time-course experiments with U937 and MOLT4 human lymphoid cells in the presence of AZT (0.05 mg x mL(-1)) or H(2)O(2) (0.01 mm) for 15-25 days. Mitochondrial DNA integrity and NF-kappa B-driven HIV-1 promoter activity were also assessed. ATP concentration began to decrease within several days after exposure to AZT or H(2)O(2), and the decrease continued to reach 30-40% of the normal level. However, decline of GSH was detectable after a retention period for at least 5-6 days, and progressed likewise. PCR analyses found that mitochondrial DNA destruction occurred when the ATP and GSH depletion had progressed, detecting a difference in the deletion pattern between AZT and H(2)O(2)-treated cells. The GSH decrease coincided with HIV-1 promoter sensitization detected by enhanced DNA binding ability of NF-kappa B and induction of the gene expression upon H(2)O(2)-rechallenge. Our results suggest that, in the process of AIDS myopathy development, AZT or oxidative agents directly impair the energy-producing system of mitochondria, causing dysfunction of cellular redox control, which eventually leads to loss of the mitochondrial DNA integrity. The mechanism of cellular redox condition-mediated NF-kappa B activation is discussed.     

Quinone toxicity in hepatocytes: studies on mitochondrial Ca2+ release induced by benzoquinone derivatives

Hepatocyte cytotoxicity caused by substituted benzoquinones was associated with increased cytosolic Ca2+ concentration. p-Benzoquinone-induced hepatotoxicity was enhanced when the hepatocytes were loaded with Ca2+ by preincubation with ATP. A similar order of potency of the substituted benzoquinones in releasing Ca2+ from isolated mitochondria and inducing hepatocyte cytotoxicity was found; in decreasing order, this was 2-Br-, unsubstituted-, 2-CH3-, 2,6-(CH3O)2-, 2,6-(CH3)2-, 2,5-(CH3)2-, 2,3,5-(CH3)3-, and 2,3,5,6-(CH3)4-benzoquinones (duroquinone). The cellular products of quinone metabolism, hydroquinones and glutathione conjugates, did not cause mitochondrial Ca2+ release. Benzoquinone-induced mitochondrial Ca2+ release was preceded by GSH conjugate formation and NAD(P)H oxidation but followed by mitochondrial swelling. With duroquinone, a slow GSH and NADPH oxidation preceded Ca2+ release, but GSH oxidation did not occur with Se-deficient mitochondria lacking glutathione peroxidase activity. Cyanide-insensitive respiration was also observed with duroquinone but not with benzoquinone, suggesting that duroquinone undergoes redox cycling. GSH was depleted by both arylation and oxidation with 2,6-(CH3O)2-, 2,6-(CH3)2-, 2,5(CH3)2-, and 2,3,5-(CH3)3-benzoquinones. Benzoquinone concentrations that totally depleted GSH did not cause Ca2+ release until intramitochondrial NAD(P)H was oxidized. Ca2+ release was also prevented when NAD(P)H generation was stimulated by the presence of isocitrate or 3-hydroxybutyrate. This suggests that mitochondrial Ca2+ release is associated with NAD(P)H oxidation catalyzed by NADH dehydrogenase with benzoquinone or by the glutathione peroxidase-glutathione reductase system with duroquinone.     

Mitochondrial ATP-sensitive K+ channels are redox-sensitive pathways that control reactive oxygen species production

Pharmacological mitochondrial ATP-sensitive K(+) channel (mitoK(ATP)) opening protects against ischemic damage and mimics ischemic preconditioning. However, physiological and pathological signaling events that open this channel are still not fully understood. We found that catalase, which removes H(2)O(2), is capable of reversing the beneficial effects of ischemic preconditioning but not of mitoK(ATP) agonist diazoxide. On the other hand, 2-mercaptopropionylglycine prevented cardioprotection in both cases, suggesting that this compound may present effects other than scavenging of reactive oxygen species. Indeed, 2-mercaptopropionylglycine and a second thiol-reducing agent, dithiothreitol, impair diazoxide-mediated activation of mitoK(ATP) in isolated heart mitochondria. This demonstrates that mitoK(ATP) activity is regulated by thiol redox status. Furthermore, stimulating the generation of endogenous mitochondrial reactive oxygen species or treating samples with H(2)O(2) strongly enhances mitoK(ATP) activity, in a manner probably dependent on redox sensors located in the channel's sulfonylurea receptor. We also demonstrate that mitoK(ATP) channel activity effectively prevents mitochondrial reactive oxygen release. Collectively, our results suggest that mitoK(ATP) acts as a reactive oxygen sensor that decreases mitochondrial free radical generation in response to enhanced local levels of oxidants. As a result, these channels regulate mitochondrial redox state under physiological conditions and prevent oxidative stress under pathological conditions such as ischemia/reperfusion.     

Oxidation-reduction and reactive oxygen species homeostasis in mutant plants with respiratory chain complex I dysfunction

Mutations in a mitochondrial or nuclear gene encoding respiratory chain complex I subunits lead to decreased or a total absence of complex I activity. Plant mutants with altered or lost complex I activity adapt their respiratory metabolism by inducing alternative pathways of the respiratory chain and changing energy metabolism. Apparently, complex I is a crucial component of the oxidation-reduction (redox) regulatory system in photosynthetic cells, and alternative NAD(P)H dehydrogenases of the mitochondrial electron transport chain (mtETC) cannot fully compensate for its impairment. In most cases, dysfunction of complex I is associated with lowered or unchanged hydrogen peroxide (H(2)O(2)) concentrations, but increased superoxide (O(2)(-)) levels. Higher production of reactive oxygen species (ROS) by mitochondria in the mosaic (MSC16) cucumber mutant may be related to retrograde signalling. Different effects of complex I dysfunction on H(2)O(2) and O(2)(-) levels in described mutants might result from diverse regulation of processes involved in H(2)O(2) and O(2)(-) production. Often, dysfunction of complex I did not lead to oxidative stress, but increased the capacity of the antioxidative system and enhanced stress tolerance. The new cellular homeostasis in mutants with dysfunction of complex I allows growth and development, reflecting the plasticity of plant metabolism.     

Down-regulation of iron regulatory protein 1 activities and expression in superoxide dismutase 1 knock-out mice is not associated with alterations in iron metabolism

Iron and oxygen (O2) are intimately associated in many well characterized patho-physiological processes. These include oxidation of the [4Fe-4S] cluster of mitochondrial aconitase and inactivation of this Krebs cycle enzyme by the superoxide anion (O2*-), a product of the one-electron of reduction O2. In contrast to the apparent toxicity of this reaction, the biological consequences of O2*- -mediated inactivation of the cytosolic counterpart of mitochondrial aconitase, commonly known as iron regulatory protein 1 (IRP1), are not clear. Apart from its ability to convert citrate to iso-citrate, IRP1 in its apo-form binds to iron-responsive elements in the untranslated regions of mRNAs coding for proteins involved in iron metabolism, to regulate their synthesis and thus control the cellular homeostasis of this metal. Here, we show that in superoxide dismutase 1 (SOD1) knock-out mice, lacking Cu,Zn-SOD, an enzyme that acts to reduce the concentration of O2*- mainly in cytosol, not only is aconitase activity of IRP1 inhibited but the level of IRP1 is also strongly decreased. Despite such an evident alteration in IRP1 status, SOD1-deficient mice display a normal iron metabolism phenotype. Our findings clearly show that under conditions of O2*- -mediated oxidative stress, IRP1 is not essential for the maintenance of iron metabolism in mammals.     

Schisandrin B enhances the glutathione redox cycling and protects against oxidant injury in different types of cultured cells

Tert-butylhydroperoxide (tBHP) challenge caused an initial depletion of cellular reduced glutathione (GSH), which was followed by a gradual restoration of cellular GSH in AML12, H9c2, and differentiated PC12 cells. The time-dependent changes in cellular GSH induced by tBHP were monitored as a measure of GSH recovery capacity (GRC), of which glutathione reductase (GR)-mediated glutathione redox cycling and γ-glutamate cysteine ligase (GCL)-mediated GSH synthesis were found to play an essential role. While glutathione redox cycling sustained the GSH level during the initial tBHP-induced depletion, GSH synthesis restores the GSH level thereafter. The effects of (-)schisandrin B [(-)Sch B] and its analogs (Sch A and Sch C) on GRC were also examined in the cells. (-)Sch B and Sch C, but not Sch A, ameliorated the extent of tBHP-induced GSH depletion, indicative of enhanced glutathione redox cycling. However, the degree of restoration of GSH post-tBHP challenge was not affected or even decreased. Pretreatment with (-)Sch B and Sch C, but not Sch A, protected against oxidant injury in the cells. The (-)Sch B afforded cytoprotection was abolished by N,N'-bis(chloroethyl)-N-nitrosourea pretreatment suggesting the enhancement of glutathione redox cycling is crucially involved in the cytoprotection afforded by (-)Sch B against oxidative stress-induced cell injury.     

Enhancement of quinone redox cycling by ascorbate induces a caspase-3 independent cell death in human leukaemia cells. An in vitro comparative study

Since the higher redox potential of quinone molecules has been correlated with enhanced cellular deleterious effects, we studied the ability of the association of ascorbate with several quinones derivatives (having different redox potentials) to cause cell death in K562 human leukaemia cell line. The rationale is that the reduction of quinone by ascorbate should be dependent of the quinone half-redox potential thus determining if reactive oxygen species (ROS) are formed or not, leading ultimately to cell death or cell survival. Among different ROS that may be formed during redox cycling between ascorbate and the quinone, the use of different antioxidant compounds (mannitol, desferal, N-acetylcysteine, catalase and superoxide dismutase) led to support H2O2 as the main oxidizing agent. We observed that standard redox potentials, oxygen uptake, free ascorbyl radical formation and cell survival were linked. The oxidative stress induced by the mixture of ascorbate and the different quinones decreases cellular contents of ATP and GSH while caspase-3-like activity remains unchanged. Again, we observed that quinones having higher values of half-redox potential provoke a severe depletion of ATP and GSH when they were associated with ascorbate. Such a drop in ATP content may explain the lack of activation of caspase-3. In conclusion, our results indicate that the cytotoxicity of the association quinone/ascorbate on K562 cancer cells may be predicted on the basis of half-redox potentials of quinones.     

Silencing of nicotinamide nucleotide transhydrogenase impairs cellular redox homeostasis and energy metabolism in PC12 cells

Mitochondrial NADPH generation is largely dependent on the inner-membrane nicotinamide nucleotide transhydrogenase (NNT), which catalyzes the reduction of NADP(+) to NADPH utilizing the proton gradient as the driving force and NADH as the electron donor. Small interfering RNA (siRNA) silencing of NNT in PC12 cells results in decreased cellular NADPH levels, altered redox status of the cell in terms of decreased GSH/GSSG ratios and increased H(2)O(2) levels, thus leading to an increased redox potential (a more oxidized redox state). NNT knockdown results in a decrease of oxidative phosphorylation while anaerobic glycolysis levels remain unchanged. Decreased oxidative phosphorylation was associated with a) inhibition of mitochondrial pyruvate dehydrogenase (PDH) and succinyl-CoA:3-oxoacid CoA transferase (SCOT) activity; b) reduction of NADH availability, c) decline of mitochondrial membrane potential, and d) decrease of ATP levels. Moreover, the alteration of redox status actually precedes the impairment of mitochondrial bioenergetics. A possible mechanism could be that the activation of the redox-sensitive c-Jun N-terminal kinase (JNK) and its translocation to the mitochondrion leads to the inhibition of PDH (upon phosphorylation) and induction of intrinsic apoptosis, resulting in decreased cell viability. This study supports the notion that oxidized cellular redox state and decline in cellular bioenergetics - as a consequence of NNT knockdown - cannot be viewed as independent events, but rather as an interdependent relationship coordinated by the mitochondrial energy-redox axis. Disruption of electron flux from fuel substrates to redox components due to NNT suppression induces not only mitochondrial dysfunction but also cellular disorders through redox-sensitive signaling.     

Mitochondrial superoxide radicals mediate programmed cell death in Trypanosoma cruzi: cytoprotective action of mitochondrial iron superoxide dismutase overexpression

Trypanosoma cruzi undergo PCD (programmed cell death) under appropriate stimuli, the mechanisms of which remain to be established. In the present study, we show that stimulation of PCD in T. cruzi epimastigotes by FHS (fresh human serum) results in rapid (<1 h) externalization of phosphatidylserine and depletion of the low molecular mass thiols dihydrotrypanothione and glutathione. Concomitantly, enhanced generation of oxidants was established by EPR and immuno-spin trapping of radicals using DMPO (5,5-dimethylpyrroline-N-oxide) and augmentation of the glucose flux through the pentose phosphate pathway. In the early period (<20 min), changes in mitochondrial membrane potential and inhibition of respiration, probably due to the impairment of ADP/ATP exchange with the cytosol, were observed, conditions that favour the generation of O2*-. Accelerated rates of mitochondrial O2*- production were detected by the inactivation of the redox-sensitive mitochondrial aconitase and by oxidation of a mitochondrial-targeted probe (MitoSOX). Importantly, parasites overexpressing mitochondrial FeSOD (iron superoxide dismutase) were more resistant to the PCD stimulus, unambiguously indicating the participation of mitochondrial O2*- in the signalling process. In summary, FHS-induced PCD in T. cruzi involves mitochondrial dysfunction that causes enhanced O(2)(*-) formation, which leads to cellular oxidative stress conditions that trigger the initiation of PCD cascades; moreover, overexpression of mitochondrial FeSOD, which is also observed during metacyclogenesis, resulted in cytoprotective effects.     

鱼藤酮诱导线粒体轻度损伤细胞氧化应激时硫氧还蛋白转录水平降低

观察鱼藤酮诱导的线粒体轻度损伤细胞氧化应激时硫氧还蛋白转录水平的变化,探讨细胞氧化损伤的可能机制。通过荧光素发光法检测ATP生成、细胞内活性氧(ROS)水平的变化,流式细胞术检测线粒体膜电位,了解低剂量鱼藤酮对线粒体功能的影响;继而用H2O2诱导细胞氧化损伤,MTT法检测细胞活性,观察正常及线粒体缺陷细胞氧化应激时,胞内硫氧还蛋白(Trx)mRNA水平的变化。结果表明,鱼藤酮以剂量依赖方式抑制线粒体ATP的产生、降低线粒体膜电位,而细胞内ROS水平增高;当线粒体损伤细胞氧化应激时胞内Trx mRNA水平降低,提示鱼藤酮诱导线粒体轻度损伤细胞抗氧化能力降低与Trx转录受到抑制有关。     

Redox cycling compounds generate H2O2 in HTS buffers containing strong reducing reagents--real hits or promiscuous artifacts?

Redox cycling compounds (RCCs) generate μM concentrations of hydrogen peroxide (H(2)O(2)) in the presence of strong reducing agents, common buffer components used to maintain the catalytic activity and/or folding of target proteins for high throughput screening (HTS) assays. H(2)O(2) generated by RCCs can indirectly inhibit the catalytic activity of proteins by oxidizing accessible cysteine, tryptophan, methionine, histidine, or selenocysteine residues, and indeed several important classes of protein targets are susceptible to H(2)O(2)-mediated inactivation; protein tyrosine phosphatases, cysteine proteases, and metalloenzymes. The main sources of H(2)O(2) in cells are the Nox enzyme/SOD systems, peroxisome metabolism, and the autoxidation of reactive chemicals by enzyme mediated redox cycling at both the microsomal and mitochondrial sites of electron transport. Given the role of H(2)O(2) as a second messenger involved in the regulation of many signaling pathways it is hardly surprising that compounds that can generate intracellular H(2)O(2) by enzyme mediated redox cycling would have pleiotropic effects. RCCs can therefore have serious negative consequences for the probe and/or lead generation process: primary HTS assay hit rates may be inflated by RCC false positives; crucial resources will be diverted to develop and implement follow up assays to distinguish RCCs from real hits; and screening databases will become annotated with the promiscuous activity of RCCs. In an attempt to mitigate the serious impact of RCCs on probe and lead generation, two groups have independently developed assays to indentify RCCs.     

Hydrogen peroxide inhibits alveolar macrophage 5-lipoxygenase metabolism in association with depletion of ATP

We have previously shown that the biologically important reactive oxygen metabolite hydrogen peroxide (H2O2) stimulates arachidonic acid (AA) release and thromboxane A2 synthesis in the rat alveolar macrophage. We have now investigated the effects of H2O2 on alveolar macrophage 5-lipoxygenase metabolism. H2O2 failed to stimulate detectable synthesis of leukotriene B4, leukotriene C4, or 5-hydroxyeicosatetraenoic acid (5-HETE) as determined by reverse-phase high performance liquid chromatography (RP-HPLC) and sensitive radioimmunoassays (RIAs). This was not explained by oxidative degradation of leukotrienes by H2O2 at the concentrations used. Moreover, RIA and RP-HPLC analyses demonstrated that H2O2 dose-dependently inhibited synthesis of leukotriene B4, leukotriene C4, and 5-HETE induced by the agonists A23187 (10 microM) and zymosan (100 micrograms/ml), over the same concentration range at which it augmented synthesis of the cyclooxygenase products thromboxane A2 and 12-hydroxy-5,8,10-heptadecatrienoic acid. Four lines of evidence suggested that H2O2 inhibited alveolar macrophage leukotriene and 5-HETE synthesis by depleting cellular ATP, a cofactor for 5-lipoxygenase. 1) H2O2 depleted ATP in A23187- and zymosan-stimulated alveolar macrophages with a dose dependence very similar to that for inhibition of agonist-induced leukotriene synthesis. 2) The time courses of ATP depletion and inhibition of leukotriene B4 synthesis by H2O2 were compatible with a rate-limiting effect of ATP on leukotriene synthesis in H2O2-exposed cultures. 3) Treatment of alveolar macrophages with the electron transport inhibitor antimycin A prior to A23187 stimulation depleted ATP and inhibited leukotriene B4 and C4 synthesis to equivalent degrees, while thromboxane A2 production was spared. 4) Incubation with the ATP precursors inosine plus phosphate attenuated both ATP depletion and inhibition of leukotriene B4 and C4 synthesis in alveolar macrophages stimulated with A23187 in the presence of H2O2. Our results show that H2O2 has the capacity to act both as an agonist for macrophage AA metabolism, and as a selective inhibitor of the 5-lipoxygenase pathway, probably as a result of its ability to deplete ATP. Depletion of cellular energy stores by oxidants generated during inflammation in vivo may be a means by which the inflammatory response is self-limited.     

Nitric oxide, complex I, and the modulation of mitochondrial reactive species in biology and disease

Mitochondria are the specialized organelles for energy metabolism but also participate in the production of O(2) active species, cell cycle regulation, apoptosis and thermogenesis. Classically, regulation of mitochondrial energy functions was based on the ADP/ATP ratio, which dynamically stimulates the transition between resting and maximal O(2) uptake. However, in the last years, NO was identified as a physiologic regulator of electron transfer and ATP synthesis by inhibiting cytochrome oxidase. Additionally, NO stimulates the mitochondrial production of O(2) active species, primarily O(2)(-) and H(2)O(2), and, depending on NO matrix concentration, of ONOO(-), which is responsible for the nitrosylation and nitration of mitochondrial components. By this means, alteration in mitochondrial complexes restricts energy output, further increases O(2) active species and changes cell signaling for proliferation and apoptosis through redox effects on specific pathways. These mechanisms are prototypically operating in prevalent generalized diseases like sepsis with multiorgan failure or limited neurodegenerative disorders like Parkinson's disease. Complex I appears to be highly susceptible to ONOO(-) effects and nitration, which defines an acquired group of mitochondrial disorders, in addition to the genetically induced syndromes. Increase of mitochondrial NO may follow over-expression of nNOS, induction and translocation of iNOS, and activation and/or increased content of the newly described mtNOS. Likewise, mtNOS is important in the modulation of O(2) uptake and cell signaling, and in mitochondrial pathology, including the effects of aging, dystrophin deficiency, hypoxia, inflammation and cancer.     

Metabolic control of mitochondrial properties by adenine nucleotide translocator determines palmitoyl-CoA effects. Implications for a mechanism linking obesity and type 2 diabetes

Inhibition of the mitochondrial adenine nucleotide translocator (ANT) by long-chain acyl-CoA esters has been proposed to contribute to cellular dysfunction in obesity and type 2 diabetes by increasing formation of reactive oxygen species and adenosine via effects on the coenzyme Q redox state, mitochondrial membrane potential (Deltapsi) and cytosolic ATP concentrations. We here show that 5 microm palmitoyl-CoA increases the ratio of reduced to oxidized coenzyme Q (QH(2)/Q) by 42 +/- 9%, Deltapsi by 13 +/- 1 mV (9%), and the intramitochondrial ATP/ADP ratio by 352 +/- 34%, and decreases the extramitochondrial ATP/ADP ratio by 63 +/- 4% in actively phosphorylating mitochondria. The latter reduction is expected to translate into a 24% higher extramitochondrial AMP concentration. Furthermore, palmitoyl-CoA induced concentration-dependent H(2)O(2) formation, which can only partly be explained by its effect on Deltapsi. Although all measured fluxes and intermediate concentrations were affected by palmitoyl-CoA, modular kinetic analysis revealed that this resulted mainly from inhibition of the ANT. Through Metabolic Control Analysis, we then determined to what extent the ANT controls the investigated mitochondrial properties. Under steady-state conditions, the ANT moderately controlled oxygen uptake (control coefficient C = 0.13) and phosphorylation (C = 0.14) flux. It controlled intramitochondrial (C = -0.70) and extramitochondrial ATP/ADP ratios (C = 0.23) more strongly, whereas the control exerted over the QH(2)/Q ratio (C = -0.04) and Deltapsi (C = -0.01) was small. Quantitative assessment of the effects of palmitoyl-CoA showed that the mitochondrial properties that were most strongly controlled by the ANT were affected the most. Our observations suggest that long-chain acyl-CoA esters may contribute to cellular dysfunction in obesity and type 2 diabetes through effects on cellular energy metabolism and production of reactive oxygen species.