Respiratory chain components involved in the glycerophosphate dehydrogenase-dependent ROS production by brown adipose tissue mitochondria

Involvement of mammalian mitochondrial glycerophosphate dehydrogenase (mGPDH, EC 1.1.99.5) in reactive oxygen species (ROS) generation was studied in brown adipose tissue mitochondria by different spectroscopic techniques. Spectrofluorometry using ROS-sensitive probes CM-H2DCFDA and Amplex Red was used to determine the glycerophosphate- or succinate-dependent ROS production in mitochondria supplemented with respiratory chain inhibitors antimycin A and myxothiazol. In case of glycerophosphate oxidation, most of the ROS originated directly from mGPDH and coenzyme Q while complex III was a typical site of ROS production in succinate oxidation. Glycerophosphate-dependent ROS production monitored by KCN-insensitive oxygen consumption was highly activated by one-electron acceptor ferricyanide, whereas succinate-dependent ROS production was unaffected. In addition, superoxide anion radical was detected as a mGPDH-related primary ROS species by fluorescent probe dihydroethidium, as well as by electron paramagnetic resonance (EPR) spectroscopy with DMPO spin trap. Altogether, the data obtained demonstrate pronounced differences in the mechanism of ROS production originating from oxidation of glycerophosphate and succinate indicating that electron transfer from mGPDH to coenzyme Q is highly prone to electron leak and superoxide generation.     

Respiratory chain components involved in the glycerophosphate dehydrogenase-dependent ROS production by brown adipose tissue mitochondria

Involvement of mammalian mitochondrial glycerophosphate dehydrogenase (mGPDH, EC 1.1.99.5) in reactive oxygen species (ROS) generation was studied in brown adipose tissue mitochondria by different spectroscopic techniques. Spectrofluorometry using ROS-sensitive probes CM-H₂DCFDA and Amplex Red was used to determine the glycerophosphate- or succinate-dependent ROS production in mitochondria supplemented with respiratory chain inhibitors antimycin A and myxothiazol. In case of glycerophosphate oxidation, most of the ROS originated directly from mGPDH and coenzyme Q while complex III was a typical site of ROS production in succinate oxidation. Glycerophosphate-dependent ROS production monitored by KCN-insensitive oxygen consumption was highly activated by one-electron acceptor ferricyanide, whereas succinate-dependent ROS production was unaffected. In addition, superoxide anion radical was detected as a mGPDH-related primary ROS species by fluorescent probe dihydroethidium, as well as by electron paramagnetic resonance (EPR) spectroscopy with DMPO spin trap. Altogether, the data obtained demonstrate pronounced differences in the mechanism of ROS production originating from oxidation of glycerophosphate and succinate indicating that electron transfer from mGPDH to coenzyme Q is highly prone to electron leak and superoxide generation.     

Tyrosine and calcium/calmodulin kinases are common signaling components in the generation of reactive oxygen species in human lymphocytes

This study examined the signaling mechanism involved in the generation of reactive oxygen species (ROS) in human lymphocytes activated by formyl-Met-Leu-Phenylalanine (fMLP; 200 nmol/L) or phorbol-myristate-acetate (PMA; 100 nmol/L). ROS were monitored spectrophotometrically using dichlorofluorescin diacetate. fMLP and PMA significantly increased ROS above the control levels (p<0.05 and 0.001, respectively). These increases were significantly inhibited by catalase, sodium azide, and dimethylsulfoxide but not by superoxide dismutase, suggesting that the ROS apparently included hydrogen peroxide, singlet oxygen and hydroxyl ion but not superoxide anion. PMA-induced responses were reduced by tyrphostin (p<0.01), ST-638 (p<0.05), KN-62 (p<0.001), bisindolylmaleimide (p<0.001), RO-31-8220 (p<0.001), and by LY-83583 (p<0.001), suggesting significant involvement of tyrosine kinase, calcium/calmodulin kinase II, protein kinase C and guanylyl cyclase. fMLP-induced responses were significantly reduced by only tyrphostin (p<0.001), ST-638 (p<0.05), and KN-62 (p<0.01). The results show that tyrosine kinase and calcium/calmodulin kinase II are common signalling components in the production of reactive oxygen species in activated lymphocytes.     

Damage to the oxygen-evolving complex by superoxide anion, hydrogen peroxide, and hydroxyl radical in photoinhibition of photosystem II

Under strong illumination of a photosystem II (PSII) membrane, endogenous superoxide anion, hydrogen peroxide, and hydroxyl radical were successively produced. These compounds then cooperatively resulted in a release of manganese from the oxygen-evolving complex (OEC) and an inhibition of oxygen evolution activity. The OEC inactivation was initiated by an acceptor-side generated superoxide anion, and hydrogen peroxide was most probably responsible for the transportation of reactive oxygen species (ROS) across the PSII membrane from the acceptor-side to the donor-side. Besides ROS being generated in the acceptor-side induced manganese loss; there may also be a ROS-independent manganese loss in the OEC of PSII. Both superoxide anion and hydroxyl radical located inside the PSII membrane were directly identified by a spin trapping-electron spin resonance (ESR) method in combination with a lipophilic spin trap, 5-(diethoxyphosphoryl)-5-phenethyl-1-pyrroline N-oxide (DEPPEPO). The endogenous hydrogen peroxide production was examined by oxidation of thiobenzamide.     

Reactive oxygen species affect mitochondrial electron transport complex I activity through oxidative cardiolipin damage

The aim of this study was to investigate the influence of reactive oxygen species (ROS) on the activity of complex I and on the cardiolipin content in bovine heart submitochondrial particles (SMP). ROS were generated through the use of xanthine/xanthine oxidase (X/XO) system. Treatment of SMP with X/XO resulted in a large production of superoxide anion, detected by acetylated cytochrome c method, which was blocked by superoxide dismutase (SOD). Exposure of SMP to ROS generation resulted in a marked loss of complex I activity and to parallel loss of mitochondrial cardiolipin content. Both these effects were completely abolished by SOD+catalase. Exogenous added cardiolipin was able to almost completely restore the ROS-induced loss of complex I activity. No restoration was obtained with other major phospholipid components of the mitochondrial membrane such as phosphatidylcholine and phosphatidylethanolamine, nor with peroxidized cardiolipin. These results demonstrate that ROS affect the mitochondrial complex I activity via oxidative damage of cardiolipin which is required for the functioning of this multisubunit enzyme complex. These results may prove useful in probing molecular mechanisms of ROS-induced peroxidative damage to mitochondria, which have been proposed to contribute to those pathophysiological conditions characterized by an increase in the basal production of reactive oxygen species such as aging, ischemia/reperfusion and chronic degenerative diseases.     

Hyperoxia-induced NAD(P)H oxidase activation and regulation by MAP kinases in human lung endothelial cells

Hyperoxia increases reactive oxygen species (ROS) production in vascular endothelium; however, the mechanisms involved in ROS generation are not well characterized. We determined the role and regulation of NAD(P)H oxidase in hyperoxia-induced ROS formation in human pulmonary artery endothelial cells (HPAECs). Exposure of HPAECs to hyperoxia for 1, 3, and 12 h increased the generation of superoxide anion, which was blocked by diphenyleneiodonium but not by rotenone or oxypurinol. Furthermore, hyperoxia enhanced NADPH- and NADH-dependent and superoxide dismutase- or diphenyleneiodonium-inhibitable ROS production in HPAECs. Immunohistocytochemistry and Western blotting revealed the presence of gp91, p67 phox, p22 phox, and p47 phox subcomponents of NADPH oxidase in HPAECs. Transfection of HPAECs with p22 phox antisense plasmid inhibited hyperoxia-induced ROS production. Exposure of HPAECs to hyperoxia activated p38 MAPK and ERK, and inhibition of p38 MAPK and MEK1/2 attenuated the hyperoxia-induced ROS generation. These results suggest a role for MAPK in regulating hyperoxia-induced NAD(P)H oxidase activation in HPAECs.     

Stimulation and clustering of cytochrome b5 reductase in caveolin-rich lipid microdomains is an early event in oxidative stress-mediated apoptosis of cerebellar granule neurons

The apoptosis of cerebellar granule neurons (CGN) induced by low potassium in the extracellular medium is a model of neuronal apoptosis where an overshot of reactive oxygen species (ROS) triggers the neuronal death. In this work, using dihydroethidium and L-012 as specific dyes for superoxide anion detection we show that this ROS overshot can be accounted by an increased release of superoxide anion to the extracellular medium. The amplitude and time course of the increase of superoxide anion observed early during apoptosis correlated with the increase of the content of soluble cytochrome b(5), a substrate of the NADH-dependent oxidase activity of the cytochrome b(5) reductase associated with lipid rafts in CGN. Western blotting and immunofluorescence microscopy approaches, including fluorescence energy transfer, pointed out an enhanced clustering of cytochrome b(5) reductase within caveolins-rich lipid rafts microdomains. Protein/protein docking analysis suggests that cytochrome b(5) reductase can form complexes with caveolins 1α, 1β and 2, playing electrostatic interactions a major role in this association. In conclusion, our results indicate that overstimulation of cytochrome b(5) reductase associated with lipid rafts can account for the overshot of plasma membrane-focalized superoxide anion production that triggers the entry of CGN in the irreversible phase of apoptosis. This article is part of a Special Issue entitled: Proteomics: The clinical link.     

Mitochondrial superoxide anion radicals mediate induction of apoptosis in cardiac myoblasts exposed to chronic hypoxia

Both reactive oxygen species (ROS) and ATP depletion may be significant in hypoxia-induced damage and death, either collectively or independently, with high energy requiring, metabolically active cells being the most susceptible to damage.We investigated the kinetics and effects of ROS production in cardiac myoblasts, H9C2 cells, under 2%, 10% and 21% O₂ in the presence or absence of apocynin, rotenone and carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone.H9C2 cells showed significant loss of viability within 30 min of culture at 2% oxygen which was not due to apoptosis, but was associated with an increase in protein oxidation. However, after 4 h, apoptosis induction was observed at 2% oxygen and also to a lesser extent at 10% oxygen; this was dependent on the levels of mitochondrial superoxide anion radicals determined using dihydroethidine. Hypoxia-induced ROS production and cell death could be rescued by the mitochondrial complex I inhibitor, rotenone, despite further depletion of ATP.In conclusion, a change to superoxide anion radical steady state level was not detectable after 30 min but was evident after 4 h of mild or severe hypoxia. Superoxide anion radicals from the mitochondrion and not ATP depletion is the major cause of apoptotic cell death in cardiac myoblasts under chronic, severe hypoxia.     

Cell activating capacity of 50 Hz magnetic fields to release reactive oxygen intermediates in human umbilical cord blood-derived monocytes and in Mono Mac 6 cells

The aim of this study was to investigate the mechanism of cell activation induced by extremely low frequency magnetic fields (ELF-MF) (50 Hz) in human cells. We examined the production of free radicals in human umbilical cord blood-derived monocytes and in human Mono Mac 6 cells. The release of superoxide radical anions was analyzed using nitroblue tetrazolium chloride and the total of reactive oxygen species (ROS) was detected using dihydrorhodamine 123. Our results show a significant increase of superoxide radical anion production up-to 1.4 fold as well as an increase in ROS release up-to 1.2 fold upon exposure of monocytes to 1 mT ELF-MF (45 min). Mono Mac 6 cells exhibit higher superoxide radical anion and ROS production up-to 1.4 and 1.5 fold, respectively. These results indicate that Mono Mac 6 cells are more sensitive to ELF-MF than monocytes. Using diphenyleneiodonium chloride (DPI) a specific inhibitor for the NADPH oxidase, the MF-effect was not inhibited in Mono Mac 6 cells. Therefore, we suggest that ELF-MF exposure induces the activation of NADH oxidase in these cells. However, the MF-effect was inhibited by DPI in monocytes, indicating the activation of the NADPH oxidase after exposure to ELF-MF.     

Cell Activating Capacity of 50 Hz Magnetic Fields to Release Reactive Oxygen Intermediates in Human Umbilical Cord Blood-derived Monocytes and in Mono Mac 6 Cells

The aim of this study was to investigate the mechanism of cell activation induced by extremely low frequency magnetic fields (ELF-MF) (50 Hz) in human cells. We examined the production of free radicals in human umbilical cord blood-derived monocytes and in human Mono Mac 6 cells. The release of superoxide radical anions was analyzed using nitroblue tetrazolium chloride and the total of reactive oxygen species (ROS) was detected using dihydrorhodamine 123. Our results show a significant increase of superoxide radical anion production up-to 1.4 fold as well as an increase in ROS release up-to 1.2 fold upon exposure of monocytes to 1 mT ELF-MF (45 min). Mono Mac 6 cells exhibit higher superoxide radical anion and ROS production up-to 1.4 and 1.5 fold, respectively. These results indicate that Mono Mac 6 cells are more sensitive to ELF-MF than monocytes. Using diphenyleneiodonium chloride (DPI) a specific inhibitor for the NADPH oxidase, the MF-effect was not inhibited in Mono Mac 6 cells. Therefore, we suggest that ELF-MF exposure induces the activation of NADH oxidase in these cells. However, the MF-effect was inhibited by DPI in monocytes, indicating the activation of the NADPH oxidase after exposure to ELF-MF.     

SK-N-MC Cell Death Occurs by Distinct Molecular Mechanisms in Response to Hydrogen Peroxide and Superoxide Anions: Involvements of JAK2-STAT3, JNK, and p38 MAP Kinases Pathways

Oxidative stress plays a vital role in the pathogenesis of neurodegenerative diseases. Nerve cells are incessantly exposed to environmental stresses leading to overproduction of some harmful species like reactive oxygen species (ROS). ROS including hydrogen peroxide and superoxide anion are potent inducers of various signaling pathways encompassing MAPKs and JAK-STAT pathways. In the current study, we scrutinized the effects of hydrogen peroxide and/or menadione (superoxide anion generator) on JNK/p38-MAPKs and JAK2-STAT3 pathways to elucidate the mechanism(s) by which each oxidant modulated the above-mentioned pathways leading to SK-N-MC cell death. Our results delineated that hydrogen peroxide and superoxide anion radical induced distinct responses as we showed that STAT3 and p38 were activated in response to hydrogen peroxide, but not superoxide anion radicals indicating the specificity in ROS-induced signaling pathways activations and behaviors. We also observed that menadione induced JNK-dependent p53 expression and apoptotic death in SK-N-MC cells while H₂O₂-induced JNK activation was p53 independent. Thus, we declare that ROS type has a key role in selective instigation of JNK/p38-MAPKs and JAK2-STAT3 pathways in SK-N-MC cells. Identifying these differential behaviors and mechanisms of hydrogen peroxide and superoxide anion functions illuminates the possible therapeutic targets in the prevention or treatment of ROS-induced neurodegenerative diseases such as Alzheimer’s disease.     

Reactive oxygen species generated by the mitochondrial respiratory chain affect the complex III activity via cardiolipin peroxidation in beef-heart submitochondrial particles

The aim of this study was to investigate the effect of reactive oxygen species (ROS), produced by the mitochondrial respiratory chain, on the activity of complex III and on the cardiolipin content in bovine-heart submitochondrial particles (SMP). ROS were produced by treatment of nicotinamide adenine dinucleotide (NADH) respiring SMP with rotenone. This treatment resulted in a production of superoxide anion, detected by the epinephrine method, which was blocked by superoxide dismutase (SOD). Exposure of SMP to mitochondrial-mediated ROS generation resulted in a marked loss of complex III activity and in a parallel loss of mitochondrial cardiolipin content. Both these effects were completely abolished by SOD + catalase. Exogenous added cardiolipin was able to almost completely prevent the ROS-mediated loss of complex III activity. No effect was obtained with other major phospholipid components of the mitochondrial membrane such as phosphatidylcholine and phosphatidylethanolamine, or with peroxidized cardiolipin. The results demonstrate that mitochondrial-mediated ROS generation affects the activity of complex III via peroxidation of cardiolipin, which is required for the functioning of this multisubunit enzyme complex. These results may prove useful in probing molecular mechanisms of ROS-induced peroxidative damage to mitochondria, which have been proposed to contribute to those physiopathological conditions characterized by an increase in the basal production of ROS such as aging, ischemia/reperfusion and chronic degenerative diseases.     

Nonstructural 3 protein of hepatitis C virus triggers an oxidative burst in human monocytes via activation of NADPH oxidase

It has been shown that oxidative stress occurs in chronic hepatitis C. Release of reactive oxygen species (ROS) from sequestered phagocytes and activated resident macrophages represents the predominant component of oxidative stress in the liver. However, little is known about the ability of the monocyte to produce ROS in response to protein of hepatitis C virus. In this study, we investigated the ROS production in human monocytes stimulated by several viral proteins of hepatitis C virus. Human monocytes from healthy blood donors were incubated with recombinant viral protein: Core, NS3, NS4, and NS5. ROS production was measured by chemiluminescence. Only NS3 triggered ROS production in human monocytes. Generated ROS were mainly the anion superoxide. NS3 also induced a rapid and transient increase in intracellular calcium concentration measured by a video digital microscopy technique. By using different metabolic inhibitors, we showed that ROS production requires calcium influx, tyrosine kinases, and the stress-activated protein kinase, p38. The study of p47(PHOX) phosphorylation and translocation showed that NADPH oxidase was activated and involved in ROS production induced by NS3. In a second experiment, NS3 inhibited the oxidative burst induced by phorbol 12-myristate 13-acetate. These results indicate that NS3 activates NADPH oxidase and modulates ROS production, which may be involved in the natural history of hepatitis C infection.     

Superoxide anion, the main species of ROS in the development of ARDS induced by oleic acid

It is believed that reactive oxygen species (ROS) play a very important role in the pathogenesis of acute respiratory distress syndrome (ARDS), but the mechanism has not been so clear, owing to the absence of direct measurable (experimental) data. In majority of the medical studies on free radicals, the analysis of ROS has generally been done by the way of measuring their secondary and breakdown products. In our study, we used electron spin resonance (ESR), a sensitive and accurate technique to detect ROS directly and also used some other sensitive techniques including ultra-weak luminescence and chemical luminescence to identify the species and relative amount of ROS. Furthermore, superoxide dismutase (SOD) was pre-administrated in ARDS rats to verify the results from the above studies and explore the possibility of the clinical application of SOD in ARDS. The spectra of ESR showed that the concentration of ROS increased at 10 min and reached a summit at 30 min after injection of oleic acid (OA), then dropped gradually. The scavenging effects of different scavenging agents on ROS by the analysis of ultra-weak luminescence proved that superoxide anion was the main species of ROS in the development of OA-induced ARDS. Moreover, the results of quantified measure of superoxide anion by chemical luminescence also showed the accordant tendency exhibited in ESR measurement. The pre-treatment of SOD might distinctly inhibit the production of superoxide anion, obviously improve the blood gas status, lung wet/dry ratio and lung/body ratio in ARDS rats. It is suggested that ROS may play a key role in the initiation phase of ARDS, while superoxide anion may be a leading actor in this process and SOD could effectively protect rats from ARDS. These results may provide helpful information for the treatment and prevention of ARDS.     

Superoxide Anion,the Main Species of ROS in the Development of ARDS Induced by Oleic Acid

It is believed that reactive oxygen species (ROS) play a very important role in the pathogenesis of acute respiratory distress syndrome (ARDS), but the mechanism has not been so clear, owing to the absence of direct measurable (experimental) data. In majority of the medical studies on free radicals, the analysis of ROS has generally been done by the way of measuring their secondary and breakdown products. In our study, we used electron spin resonance (ESR), a sensitive and accurate technique to detect ROS directly and also used some other sensitive techniques including ultra-weak luminescence and chemical luminescence to identify the species and relative amount of ROS. Furthermore, superoxide dismutase (SOD) was pre-administrated in ARDS rats to verify the results from the above studies and explore the possibility of the clinical application of SOD in ARDS. The spectra of ESR showed that the concentration of ROS increased at 10?min and reached a summit at 30?min after injection of oleic acid (OA), then dropped gradually. The scavenging effects of different scavenging agents on ROS by the analysis of ultra-weak luminescence proved that superoxide anion was the main species of ROS in the development of OA-induced ARDS. Moreover, the results of quantified measure of superoxide anion by chemical luminescence also showed the accordant tendency exhibited in ESR measurement. The pre-treatment of SOD might distinctly inhibit the production of superoxide anion, obviously improve the blood gas status, lung wet/dry ratio and lung/body ratio in ARDS rats. It is suggested that ROS may play a key role in the initiation phase of ARDS, while superoxide anion may be a leading actor in this process and SOD could effectively protect rats from ARDS. These results may provide helpful information for the treatment and prevention of ARDS.     

In vivo reactive oxygen species production induced by ischemia in muscle arterioles of mice: involvement of xanthine oxidase and mitochondria

Reactive oxygen species (ROS) participate in tissue injury after ischemia-reperfusion. Their implication in leukocyte adherence and increase in permeability at the venular side of the microcirculation have been reported, but very little is known about ROS production in arterioles. The objective of this work was to evaluate, in the arteriole wall in vivo, the temporal changes in superoxide anion production during ischemia and reperfusion and to identify the source of this production. Mouse cremaster muscle was exposed to 1 h of ischemia followed by 30 min of reperfusion, and superoxide anion production was assessed by a fluorescent probe, i.e., intracellular dihydroethidium oxidation. During ischemia, we found a significant increase in dihydroethidium oxidation; however, we observed no additional increase in fluorescence during the subsequent reperfusion. This phenomenon was significantly inhibited by pretreatment with superoxide dismutase. Allopurinol (xanthine oxidase inhibitor) or stigmatellin [Q(o)-site (oriented toward the intermembrane space) inhibitor of mitochondrial complex III] or simultaneous administration of these two inhibitors significantly reduced superoxide production during ischemia to 80%, 88%, and 72%, respectively, of that measured in the untreated ischemia-reperfusion group. By contrast, no significant inhibition was found when NADPH oxidase was inhibited by apocynin or when mitochondrial complex I or complex II was inhibited by rotenone or thenoyltrifluoroacetone. A significant increase in ROS was found with antimycin A [Q(i)-site (located in the inner membrane and facing the mitochondrial matrix) inhibitor of mitochondrial complex III]. We conclude that a significant increase in ROS production occurs during ischemia in the arteriolar wall. This increased production involves both a cytoplasmic source (i.e., xanthine oxidase) and the mitochondrial complex III at the Q(o) site.     

Origin of reactive oxygen species in human semen: spermatozoa or leucocytes?

Peroxidative damage induced by reactive oxygen species (ROS) has been proposed as one of the major causes of defective sperm function. In previous studies of the production of ROS in semen, the contribution of contaminating leucocytes was not assessed. We determined the levels of ROS in 60 semen samples from men attending our infertility clinic and demonstrated by performing extraction experiments with antibody-coated magnetic beads that, within this unselected population of patients, leucocytes were the major source of ROS in the low-density Percoll fraction. Of the sperm motion parameters examined using computerized semen analysis, beat-cross frequency was the only one significantly affected by the ROS in semen.     

Mass Spectrometry Reveals New Insights into the Production of Superoxide Anions and 4‐Hydroxynonenal Adducted Proteins in Human Sperm

The free‐radical theory of male infertility suggests that reactive oxygen species produced by the spermatozoa themselves are a leading cause of sperm dysfunction, including loss of sperm motility. However, the field is overshadowed on several fronts, primarily because: i) the probes used to measure reactive oxygen species (ROS) are imprecise; and ii) many reports suggesting that oxygen radicals are detrimental to sperm function add an exogenous source of ROS. Herein, a more reliable approach to measure superoxide anion production by human spermatozoa based on MS analysis is used. Furthermore, the formation of the lipid‐peroxidation product 4‐hydroxynonenal (4‐HNE) during in vitro incubation using proteomics is also investigated. The data demonstrate that neither superoxide anion nor other free radicals that cause 4‐HNE production are related to the loss of sperm motility during incubation. Interestingly, it appears that many of the 4‐HNE adducted proteins, found within spermatozoa, originate from the prostate. A quantitative SWATH analysis demonstrate that these proteins transiently bind to sperm and are then shed during in vitro incubation. These proteomics‐based findings propose a revised understanding of oxidative stress within the male reproductive tract.     

Reactive oxygen species in the killing of Pseudomonas aeruginosa by human leukocytes

Pseudomonas aeruginosa (PA) infects hosts with compromised host defenses. An important defense mechanism is the generation of reactive oxygen species (ROS) by white blood cells (WBCs). What roles do ROS play in host defense against PA? Human WBCs killed PA in vitro, and they generated a respiratory burst as measured by the production of H₂O₂. ROS efficiently killed PA; in acellular assays, less than 10mm of H₂O₂ or OCl^- eliminated all bacteria in 90 min. However, WBCs with suppressed production of ROS (caused by hypoxia) killed PA normally. In addition, none of the antioxidants vitamin C, N-acetylcysteine, superoxide dismutase, or catalase affected PA killing by WBCs. Thus, PA stimulates WBCs to produce ROS, which can kill the bacteria, but disturbances of WBC ROS production do not interfere with the killing of PA. WBCs have robust, redundant mechanisms for PA elimination.     

Increased reactive oxygen species production with antisense oligonucleotides directed against uncoupling protein 2 in murine endothelial cells.

Uncoupling protein 2 (UCP-2) belongs to the mitochondrial anion carrier family. It is ubiquitously expressed but is most abdundant in the reticuloendothelial system. In addition to uncoupling function, UCP-2 modulates the production of reactive oxygen species (ROS) by isolated mitochondria. Using an antisense oligonucleotide strategy, we investigated whether a defect in UCP-2 expression modulates ROS in intact endothelial cells. Murine endothelial cells (CRL 2181) pretreated by antisense oligonucleotides directed against UCP-2 mRNA exhibited a significant and specific increase in membrane potential and intracellular ROS level compared with control scrambled or anti-UCP-1 and -UCP-3 antisense oligonucleotides. These specific changes induced by UCP-2 antisense oligonucleotides were correlated with a rise in extracellular superoxide anion production and oxidative stress assessed by thiobarbituric acid reactive substance values. Taken together, these data suggest a role for UCP-2 in control of ROS production and subsequent oxidation of surrounding compounds mediating oxidative stress of endothelial cells. These data also support the notion that manipulations of UCP-2 at the genetic level could control ROS metabolism at the cellular level.