Reduction Of NADPH Oxidase Activity Research Articles

Overexpression of the dominant negative nbexo70d1 mutantionconfers tolerance to salt stress in transgenic tobacco

The vesicle trafficking process, which involves exocytotic and endocytotic pathways, has been reported to play a role in regulating plant responses to different environmental stresses. The Exo70 protein is important for the localization of the exocyst in the plasma membrane; however, its role in the physiology of stress tolerance is currently unclear. In this study, we characterized NbExo70D1, an Exo70 gene from tobacco (Nicotiana benthamiana). It was shown to have a role in the plant response to salt stress. More specifically, tolerance to salt stress is conferred by the overexpression of the dominant negative nbexo70d1 domain D mutation in transgenic tobacco. In addition, a reduced accumulation of reactive oxygen species (ROS) under salt treatment was observed in the transgenic lines compared to the wild type. Treatment with diphenylene iodonium, an NADPH oxidase inhibitor, resulted in a decrease in salt stress-triggered ROS production in the roots of both wild type tobacco and transgenic tobacco. Furthermore, there was a reduction in NADPH oxidase activity in the transgenic plants under salt treatment, which indicates NbExo70D1 is involved in NADPH oxidase-mediated ROS production. We also characterized the tissue-specific expression patterns of NbExo70D1 during salt stress response by expressing the ProNbExo70D1-β-glucuronidase reporter construct in plants. Importantly, the GFP-NbExo70D1 fusion protein was localized in both the plasma membrane and the cytoplasm; expressing the dominant negative mutation disrupted the interaction between NbExo70D1 protein and the plasma membrane. Overall, our study suggests that Exo70 plays an important role in regulating the production and transmission of ROS as part of a salt stress response in plants.

Early decrease of oxidative stress by non-invasive ventilation in patients with acute respiratory failure.

Oxidative stress plays an important role in chronic respiratory diseases where the use of non-invasive ventilation seems to reduce the oxidative damage. Data on acute respiratory failure are still lacking. The aim of the study is to investigate the interplay between oxidative stress and acute respiratory failure, and the role of non-invasive ventilation in this setting. We enrolled 60 patients suffering from acute respiratory failure (PaO2/FiO2 ratio <300): 30 consecutive patients treated with non-invasive ventilation and 30 consecutive patients treated with conventional oxygen therapy. Serum levels of soluble Nox2-derived peptide (sNOX2-dp), a marker of NADPH-oxidase activation, and 8-iso-PGF2α and H2O2, markers of oxidative stress, were evaluated at baseline and after 3h of treatment. At baseline, higher values of sNOX2-dp, 8-iso-PGF2α and H2O2 are associated with lower values of PaO2/FiO2 ratio (p<0.001). After 3h, serum levels of sNOX2-dp, H2O2, and 8-iso-PGF2α significantly decrease in patients treated with non-invasive ventilation, but not in patients treated with conventional oxygen therapy. Delta changes of oxidative stress parameters correlate inversely with the delta changes of PaO2/FiO2 (R=-0.623, p<0.001 for sNOX2-dp; R=-0.428, p<0.001 for H2O2; R=-0.548, p<0.001 for 8-iso-PGF2α). In the acute respiratory failure setting, treatment with non-invasive ventilation reduces the levels of oxidative stress in the first hours. This reduction is associated with an improvement of PaO2/FiO2 ratio as well as in a reduction of NADPH-oxidase activity.

Rac1 Pharmacological Inhibition Rescues Human Endothelial Dysfunction.

BackgroundEndothelial dysfunction contributes significantly to the development of vascular diseases. However, a therapy able to reduce this derangement still needs to be identified. We evaluated the effects of pharmacological inhibition of Rac1, a small GTPase protein promoting oxidative stress, in human endothelial dysfunction.Methods and ResultsWe performed vascular reactivity studies to test the effects of NSC23766, a Rac1 inhibitor, on endothelium‐dependent vasorelaxation of saphenous vein segments collected from 85 subjects who had undergone surgery for venous insufficiency and from 11 patients who had undergone peripheral vascular surgery. The endothelium‐dependent vasorelaxation of the varicose segments of saphenous veins collected from patients with venous insufficiency was markedly impaired and was also significantly lower than that observed in control nonvaricose vein tracts from the same veins. Rac1 activity, reactive oxygen species levels, and reduced nicotine adenine dinucleotide phosphate (NADPH) oxidase activity were significantly increased in varicose veins, and NSC23766 was able to significantly improve endothelium‐dependent vasorelaxation of dysfunctional saphenous vein portions in a nitric oxide–dependent manner. These effects were paralleled by a significant reduction of NADPH oxidase activity and activation of endothelial nitric oxide synthase. Finally, we further corroborated this data by demonstrating that Rac1 inhibition significantly improves venous endothelial function and reduces NADPH oxidase activity in saphenous vein grafts harvested from patients with vascular diseases undergoing peripheral bypass surgery.ConclusionsRac1 pharmacological inhibition rescues endothelial function and reduces oxidative stress in dysfunctional veins. Rac1 inhibition may represent a potential therapeutic intervention to reduce human endothelial dysfunction and subsequently vascular diseases in various clinical settings.

Unexpected function of the phagocyte NADPH oxidase in supporting hyperglycolysis in stimulated neutrophils: key role of 6-phosphofructo-2-kinase.

The phagocyte NADPH oxidase 2 (Nox2) is an enzymatic complex that is involved in innate immunity, notably via its capacity to produce toxic reactive oxygen species. Recently, a proteomic analysis of the constitutively active Nox2 complex, isolated from neutrophil fractions, highlighted the presence of 6-phosphofructo-2-kinase (PFK-2). The purpose of this work was to study the relationship between PFK-2 and NADPH oxidase in neutrophils. Data have underlined a specific association of the active phosphorylated form of PFK-2 with Nox2 complex in stimulated neutrophils. In its active form, PFK-2 catalyzes the production of fructose-2,6-bisphosphate, which is the main allosteric activator of phosphofructo-1-kinase, the limiting enzyme in glycolysis. Pharmacologic inhibition of PFK-2 phosphorylation and cell depletion in PFK-2 by a small interfering RNA strategy led to a decrease in the glycolysis rate and a reduction in NADPH oxidase activity in stimulated cells. Surprisingly, alteration of Nox2 activity impacted the glycolysis rate, which indicated that Nox2 in neutrophils was not only required for reactive oxygen species production but was also involved in supporting the energetic metabolism increase that was induced by inflammatory conditions. PFK-2 seems to be a strategic element that links NADPH oxidase activation and glycolysis modulation, and, as such, is proposed as a potential therapeutic target in inflammatory diseases.-Baillet, A., Hograindleur, M.-A., El Benna, J., Grichine, A., Berthier, S., Morel, F., Paclet, M.-H. Unexpected function of the phagocyte NADPH oxidase in supporting hyperglycolysis in stimulated neutrophils: key role of 6-phosphofructo-2-kinase.

Calcium Channel Blocker Enhances Beneficial Effects of an Angiotensin II AT1 Receptor Blocker against Cerebrovascular-Renal Injury in type 2 Diabetic Mice

Recent clinical trials have demonstrated that combination therapy with renin-angiotensin system inhibitors plus calcium channel blockers (CCBs) elicits beneficial effects on cardiovascular and renal events in hypertensive patients with high cardiovascular risks. In the present study, we hypothesized that CCB enhances the protective effects of an angiotensin II type 1 receptor blocker (ARB) against diabetic cerebrovascular-renal injury. Saline-drinking type 2 diabetic KK-Ay mice developed hypertension and exhibited impaired cognitive function, blood-brain barrier (BBB) disruption, albuminuria, glomerular sclerosis and podocyte injury. These brain and renal injuries were associated with increased gene expression of NADPH oxidase components, NADPH oxidase activity and oxidative stress in brain and kidney tissues as well as systemic oxidative stress. Treatment with the ARB, olmesartan (10 mg/kg/day) reduced blood pressure in saline-drinking KK-Ay mice and attenuated cognitive decline, BBB disruption, glomerular injury and albuminuria, which were associated with a reduction of NADPH oxidase activity and oxidative stress in brain and kidney tissues as well as systemic oxidative stress. Furthermore, a suppressive dose of azelnidipine (3 mg/kg/day) exaggerated these beneficial effects of olmesartan. These data support the hypothesis that a CCB enhances ARB-associated cerebrovascular-renal protective effects through suppression of NADPH oxidase-dependent oxidative stress in type 2 diabetes.

Calcium dysregulation in ventricular myocytes from mice expressing constitutively active Rac1

Increased Rac1 activity and its concomitant elevation of reactive oxygen species (ROS) levels is believed to be involved in the development of cardiac diseases such as hypertrophy and arrhythmia. To study the effects of activated Rac1 on the properties of isolated ventricular myocytes we used a transgenic mouse model (RacET) expressing constitutively active Rac1. Concurrent with dilated cardiomyopathy global Ca2+ handling as well as single cell contractility was substantially decreased. Cellular ROS levels were assessed with two independent assays and unexpectedly depicted decreased ROS production in RacET that was uncoupled from hormonal stimulation. Western blot analysis illustrated a massive increase in cellular Rac1 activity concomitant with a reduction in NADPH-oxidase activity. Analysis of the Ca2+ current, the ryanodine receptor and fractional Ca2+ release uncovered defective excitation–contraction (ec) coupling and a substantial increase in sarcoplasmic reticulum Ca2+ leak together with a larger Ca2+ spark amplitude and frequency. We conclude that Rac1 activity plays an important role for cardiac diseases but can be uncoupled from NADPH-oxidase activity. Rac1-mediated partial uncoupling of the ec-coupling machinery results in a ROS-independent disarrayed cellular Ca2+ handling, contractility and impaired cardiac function.

Salicylic acid delays leaf rolling by inducing antioxidant enzymes and modulating osmoprotectant content in Ctenanthe setosa under osmotic stress

The effects of salicylic acid (SA) on leaf rolling under osmotic stress in Ctenanthe setosa (Roscoe) Eichler were evaluated. The leaves were pretreated with SA, paclobutrazol (PAC), and antioxidant enzyme inhibitors for 15 h and then kept in polyethylene glycol (PEG) for 4 h to promote osmotic stress. The degree of leaf rolling was measured every hour during the 4-h PEG treatment. Antioxidant enzymes (superoxide dismutase, catalase, ascorbate peroxidase, and guaiacol peroxidase) and NADPH oxidase (NOX) activities, endogenous SA contents, hydrogen peroxide, proline, and soluble sugar were determined immediately after PEG treatment. SA treatment retarded leaf rolling and induced antioxidant enzyme activities under osmotic stress. Antioxidant enzyme inhibitors and PAC treatments caused reductions in all antioxidant enzyme activities and accelerated leaf rolling. The PAC treatment also decreased endogenous SA, hydrogen peroxide, proline, and soluble sugar contents and NOX activity. In conclusion, the delay in leaf rolling resulting from SA application was related to the antioxidant enzyme activities and also to osmoprotectants. These were supported by the increase in leaf rolling degree due to PAC, which led to a decrease in proline and sugar contents and antioxidant enzyme activities due to a decrease in hydrogen peroxide content followed by a reduction in NOX activity.

Mechanism of Heme-Heme Oxygenase System Impairment of Endothelium Contraction in the Spontaneously Hypertensive Rat

See related article, pp 926–934 Heme oxygenase (HO) 1 and HO-2 gene expressions are known to increase cellular antioxidant and anti-inflammatory properties through activation of a battery of cytoprotective systems, including decreases in cyclooxygenase 1 and NADPH oxidase (NOX-2) and elevated levels of extracellular superoxide dismutase and NO. The heme-HO system (HO-1 and HO-2) acts as an endogenous anti-inflammatory and protective circuit critical for a self-resolving inflammatory-reparative process. A study by Li et al,1 which appears in this issue of Hypertension , shows that upregulation of heme oxygenase (HO) 1 prevents endothelium contraction in aorta of spontaneously hypertensive rats (SHRs) via a decrease in cyclooxygenase (COX) 1, not COX-2. Induction of HO-1 was observed in cultured endothelial cells and in vivo after treatment with both heme and hemoglobin. In the present article, Li et al1 showed that, in aorta isolated from either wild-type or SHR animals, levels of HO-1 are low. HO-1 under physiological conditions is expressed at low levels and does not seem to affect the basal levels of heme-dependent enzyme activity. On the other hand, HO-2, the constitutive enzyme, is the main determinant for the regulation of the basal physiological levels of various hemeproteins, including COX, NOX2, inducible NO synthase, and CYP450. COX-1/-2 are hemeproteins that catalyze the conversion of arachidonic acid (AA) to prostaglandin H2, the precursor of prostanoids that participate in the regulation of vascular function. Heme binds to the COX …

Female type 2 diabetes mellitus mice exhibit severe ischemic brain damage

Type 2 diabetes can impair the outcome of stroke as well as increase stroke risk; however, the sex difference in ischemic brain damage is not well known, and even less is known about the difference in diabetes. We therefore investigated the possible gender difference in brain damage after stroke associated with type 2 diabetes using a mouse model, KKAy. Female KKAy showed a much larger ischemic area compared with male KKAy. NADPH oxidase activity in the brain was also increased more in female than in male mice. Ovariectomy enhanced the ischemic area, and treatment with estradiol markedly attenuated the ischemic area to that in female KKAy, with a reduction of NADPH oxidase activity. Female and OVX mice showed improvement of cerebral blood flow (CBF) at 1 hour after middle cerebral artery (MCA) occlusion, but no significant difference in CBF of the ipsilateral penumbra and ipsilateral core 24 hours after MCA occlusion was observed among each group. Severe ischemic brain damage was observed in female KKAy compared with male KKAy. Estrogen showed a protective effect on the brain, at least partly from attenuation of oxidative stress in the female brain. These findings suggest that brain damage in diabetes mellitus might be more marked in women than in men.

Oxidative stress and diabetic cardiovascular disorders: roles of mitochondria and NADPH oxidaseThis review is one of a selection of papers published in a Special Issue on Oxidative Stress in Health and Disease.

Cardiovascular diseases are the predominant cause of death in patients with diabetes mellitus. Underlying mechanism for the susceptibility of diabetic patients to cardiovascular diseases remains unclear. Elevated oxidative stress was detected in diabetic patients and in animal models of diabetes. Hyperglycemia, oxidatively modified atherogenic lipoproteins, and advanced glycation end products are linked to oxidative stress in diabetes. Mitochondria are one of major sources of reactive oxygen species (ROS) in cells. Mitochondrial dysfunction increases electron leak and the generation of ROS from the mitochondrial respiratory chain (MRC). High levels of glucose and lipids impair the activities of MRC complex enzymes. NADPH oxidase (NOX) generates superoxide from NADPH in cells. Increased NOX activity was detected in diabetic patients. Hyperglycemia and hyperlipidemia increased the expression of NOX in vascular endothelial cells. Accumulated lines of evidence indicate that oxidative stress induced by excessive ROS production is linked to many processes associated with diabetic cardiovascular complications. Overproduction of ROS resulting from mitochondrial dysfunction or NOX activation is associated with uncoupling of endothelial nitric oxide synthase, which leads to reduced production of nitric oxide and endothelial-dependent vasodilation. Gene silence or inhibitor of NOX reduced oxidized or glycated LDL-induced expression of plasminogen activator inhibitor-1 in endothelial cells. Statins, hypoglycemic agents, and exercise may reduce oxidative stress in diabetic patients through the reduction of NOX activity or the improvement of mitochondrial function, which may prevent or postpone the development of cardiovascular complications.

Role of caveolin and heat shock protein 70 interaction in the antioxidative effects of an angiotensin II type 1 receptor blocker in spontaneously hypertensive rats proximal tubules

Role of caveolin and heat shock protein 70 interaction in the antioxidative effects of an angiotensin II type 1 receptor blocker in spontaneously hypertensive rats proximal tubules

High Glucose Concentration Increases Macrophage Cholesterol Biosynthesis in Diabetes Through Activation of the Sterol Regulatory Element Binding Protein 1 (SREBP1): Inhibitory Effect of Insulin

Diabetes activates atherogenesis and macrophage foam cell formation. This study's goal was to determine whether insulin counteracts diabetes-induced macrophage foam cell formation, as well as to determine transcriptional mechanisms involved in this effect.Insulin injection to diabetic mice reduced macrophage lipid peroxides levels, Ox-LDL uptake, and CD36 mRNA levels by 40%, 29%, and by 41% respectively, compared to age-matched untreated diabetic mice. These results were further assessed using an in vitro system. Addition of insulin to glucose-enriched cells led to a significant decrease in cellular lipid peroxidation by 43% compared to cells incubated with high concentrations of glucose with no insulin. This effect was correlated with a reduction in NADPH oxidase activity.Macrophage cholesterol biosynthesis was then studied in cells from diabetic mice treated with insulin and in glucose-enriched macrophages incubated with insulin. Insulin treatment of diabetic mice significantly reduced macrophage cholesterol biosynthesis, HMG-CoA reductase mRNA expression, and protein expression by 81%, 54%, and 31% respectively, compared to macrophages isolated from nontreated diabetic mice. Similarly, insulin incubation with glucose-enriched macrophages significantly reduced macrophage cholesterol biosynthesis, HMG-CoA reductase mRNA expression, and protein expression by 84%, 42%, and 18%, respectively, compared to macrophages incubated with high glucose but without insulin. These effects were mediated by glucose and insulin ability to regulate the transcription factor SREBP-1. Whereas glucose upregulated SREBP-1 expression and maturation, insulin blocked SREBP1 cleavage, leading to reduced mature form of the transcription factor in the nucleus.In conclusion, this study presents important novel insights on the events connecting diabetes and glucose stimulation of macrophage foam cell formation leading to atherosclerosis. Most important, the inhibitory effects of insulin on diabetes-mediated (and high glucose-induced) increased cholesterol synthesis were shown to involve modulation of SREBP-1 expression and its maturation.

G allele of the −930A&gt;G polymorphism of the CYBA gene is associated with insulin resistance in obese subjects

It has been shown that NADPH oxidase plays a role in oxidative stress which has been involved in the development of metabolic syndrome. The -930A/G polymorphism of the CYBA gene (that codes p22phox, a major component of the NADPH oxidase) has been associated with human hypertension and with a reduction in NADPH oxidase activity. In this work, we have examined the influence of the -930A/G polymorphism on obesity risk and insulin resistance in a case-control study of Spanish subjects (n=313). In the obese group (n=159), there was a statistically significant association between the GG genotype of the -930A/G polymorphism of the CYBA gene and fasting insulin levels and HOMA index. This outcome agrees with previous findings concerning functional analyses of this polymorphism and reinforces the hypothesis that insulin resistance is associated with oxidative stress. In conclusion, a protective effect in carriers of the -930A/G polymorphism of the p22phox gene against insulin resistance in a population of Spanish obese adults has been found.

Microglia-Mediated Neurotoxicity Is Inhibited by Morphine through an Opioid Receptor-Independent Reduction of NADPH Oxidase Activity

Recent studies have shown that morphine modulates the function of glia cells through both opioid receptor dependent and independent mechanisms. However, the mechanism by which morphine regulates neuronal disorders through the alteration of microglia activity remains unclear. In this study, using rat primary mesencephalic neuron-glia cultures, we report that both l-morphine and its synthetic stereoenantiomer, d-morphine, an ineffective opioid receptor agonist, significantly reduced LPS- or 1-methyl-4-phenylpyridinium-induced dopaminergic neurotoxicity with similar efficacy, indicating a nonopioid receptor-mediated effect. In addition, using reconstituted neuron and glia cultures, subpicomolar concentrations of morphine were found to be neuroprotective only in the presence of microglia, and significantly inhibited the production of inflammatory mediators from LPS-stimulated microglia cells. Mechanistic studies showed that both l- and d- morphine failed to protect dopaminergic neurons in cultures from NADPH oxidase (PHOX) knockout mice and significantly reduced LPS-induced PHOX cytosolic subunit p47(phox) translocation to the cell membrane by inhibiting ERK phosphorylation. Taken together, our results demonstrate that morphine, even at subpicomolar concentrations, exerts potent anti-inflammatory and neuroprotective effects either through the inhibition of direct microglial activation by LPS or through the inhibition of reactive microgliosis elicited by 1-methyl-4-phenylpyridinium. Furthermore, our study reveals that inhibition of PHOX is a novel site of action for the mu-opioid receptor-independent effect of morphine.

8-hydroxydeoxyguanosine suppresses NO production and COX-2 activity via Rac1/STATs signaling in LPS-induced brain microglia

Free 8-hydroxydeoxyguanosine (oh 8dG), a nucleoside of 8-hydroxyguanine (oh 8Gua), present in cytosol is not incorporated into DNA. However, nothing is known about its biological function when it presents in cytosol as a free form. We demonstrate here for the first time that oh 8dG inhibits lipopolysaccharide (LPS)-induced nitric oxide (NO) production and cyclooxygenase-2 (COX-2) activity, and both gene transcriptions in microglia. Furthermore, oh 8dG reduced mRNA levels of pro-inflammatory cytokine, such as IL-1β, IL-6, and TNF-α, in activated BV2 cells. We also found that oh 8dG suppressed reactive oxygen species (ROS) production through reduction of NADPH oxidase activity and blocked Rac1/STATs signal cascade. Finally, oh 8dG suppressed recruitment of STATs and p300 to the iNOS and COX-2 promoters, and inhibited H3 histone acetylation. Taken together, these results provide new aspects of oh 8dG as an anti-inflammatory agent.

Pulmonary surfactant protein a inhibits macrophage reactive oxygen intermediate production in response to stimuli by reducing NADPH oxidase activity.

Alveolar macrophages are important host defense cells in the human lung that continuously phagocytose environmental and infectious particles that invade the alveolar space. Alveolar macrophages are prototypical alternatively activated macrophages, with up-regulated innate immune receptor expression, down-regulated costimulatory molecule expression, and limited production of reactive oxygen intermediates (ROI) in response to stimuli. Surfactant protein A (SP-A) is an abundant protein in pulmonary surfactant that has been shown to alter several macrophage (Mphi) immune functions. Data regarding SP-A effects on ROI production are contradictory, and lacking with regard to human Mphi. In this study, we examined the effects of SP-A on the oxidative response of human Mphi to particulate and soluble stimuli using fluorescent and biochemical assays, as well as electron paramagnetic resonance spectroscopy. SP-A significantly reduced Mphi superoxide production in response to the phorbol ester PMA and to serum-opsonized zymosan (OpZy), independent of any effect by SP-A on zymosan phagocytosis. SP-A was not found to scavenge superoxide. We measured Mphi oxygen consumption in response to stimuli using a new oxygen-sensitive electron paramagnetic resonance probe to determine the effects of SP-A on NADPH oxidase activity. SP-A significantly decreased Mphi oxygen consumption in response to PMA and OpZy. Additionally, SP-A reduced the association of NADPH oxidase component p47(phox) with OpZy phagosomes as determined by confocal microscopy, suggesting that SP-A inhibits NADPH oxidase activity by altering oxidase assembly on phagosomal membranes. These data support an anti-inflammatory role for SP-A in pulmonary homeostasis by inhibiting Mphi production of ROI through a reduction in NADPH oxidase activity.

Evidence for the interaction between protein disulfide isomerase and NADPH oxidase as a regulatory mechanism for superoxide generation in phagocytic and vascular smooth muscle cells

Reactive oxygen species generation has been implicated with diverse vascular and inflammatory diseases. NADPH oxidase is the major source of reactive oxygen species within phagocytes. It is activated by the phosphorylation and recruitment of cytosolic subunits p47phox, p67phox and rac2 to the membrane-bound cytochromeb558 formed by the p22phox and gp91phox subunits. In vascular cells, an analogous enzymatic complex is found within the membrane or cytosol but it has primarily signaling purposes. Both oxidases are inhibitable by thiol oxidants, not affected by the global redox cell state. Thiol oxidoreductases are effective modulators of the thiol redox state on cell membranes, in the ER and cytosol. Considering that spontaneous thiol–disulfide exchange reactions occur at rates too slow to be involved with regulatory pathways, we postulate that thiol oxidoreductases may control the redox state of important thiol sites on the NADPH oxidase, affecting its superoxide generating activity. Protein disulfide isomerase (PDI) is a ubiquitous multifunctional enzyme of the thiol oxidoreductase family, involved in regulation of diverse cellular mechanisms. Our objective is to investigate a possible interaction between PDI and the oxidases in vascular smooth muscle cells and neutrophils. Western blot analysis disclosed PDI in neutrophils within granules and the membrane, the same place where cytochrome b558 is found. In smooth muscle cells PDI was also found on the membrane fraction, and immunofluorescence disclosed a spatial colocalization between PDI and the oxidase. Superoxide generation was evaluated by superoxide dismutase-inhibitable cytochrome c reduction spectrophotometric assay using the cell free system (isolated membrane and cytosol from human neutrophils). Inhibition of PDI activity caused a 60% reduction of NADPH oxidase activity. With vascular smooth muscle cell homogenates, PDI inhibition caused similar oxidase activity. Angiotensin II, a known vascular NADPH oxidase agonist, induced in vascular cells a parallel increase in PDI activity. PDI modulation of thiol redox state on the vascular and phagocytic oxidases may thus represent a new regulatory mechanism of reactive oxygen species generation.

In vitro knockout of human p47phox blocks superoxide anion production and LDL oxidation by activated human monocytes

AbstractWe previously reported that superoxide dismutase (SOD) blocked human monocyte oxidation of LDL and therefore concluded that superoxide anion ( O2.‒ ) was required for oxidation. Others, however, have suggested that SOD may inhibit by mechanisms alternative to the dismutation of O2.‒ . This study definitively addresses the involvement of O2.‒ in monocyte oxidation of LDL. Using an antisense ODN designed to target p47phox mRNA, we found that treatment of monocytes with antisense ODN caused a substantial and selective decrease in expression of p47phox protein, whereas sense ODN was without effect. Corresponding functional assays demonstrated that antisense ODN inhibited production of O2.‒ . As sense ODN caused no inhibition of O2.‒ production, these results suggested that inhibition of p47phox expression caused reduction in O2.‒ production. Evaluation of the contribution of O2.‒ production to monocyte-mediated oxidation of LDL lipids confirmed that O2.‒ production is required for LDL lipid oxidation as antisense ODN treatment significantly inhibited LDL oxidation whereas sense ODN treatment caused no inhibition. This is the first report of the reduction of NADPH oxidase activity in intact human monocytes by directly targeting the mRNA of a significant member of this enzyme complex. Our results provide convincing data that O2.‒ is indeed required for monocyte-mediated LDL oxidation. —Bey, E. A., and M. K. Cathcart. In vitro knockout of human p47phox blocks superoxide anion production and LDL oxidation by activated human monocytes. J. Lipid Res. 2000. 41: 489–495.

Biosynthesis of the Phagocyte NADPH Oxidase Cytochromeb558: ROLE OF HEME INCORPORATION AND HETERODIMER FORMATION IN MATURATION AND STABILITY OF gp91 phox and p22 phox SUBUNITS

The NADPH oxidase cytochrome b558 is a membrane heterodimer comprised of a glycosylated 91-kDa subunit, gp91(phox), and a nonglycosylated 22-kDa subunit, p22(phox). The role of heme in cytochrome b558 biosynthesis was studied using succinyl acetone, an inhibitor of heme synthesis, in PLB-985 myeloid cells undergoing granulocytic differentiation. Succinyl acetone markedly reduced expression of p22(phox) and the mature 91-kDa form of gp91(phox) but not its 65-kDa high mannose precursor, in association with a profound reduction in NADPH oxidase activity. Expression of non-heme-containing cytosolic oxidase components was unaffected. The reduction in cytochrome b558 expression and NADPH oxidase activity was prevented by adding exogenous heme and was reversible upon removal of succinyl acetone. Transgenic expression of gp91(phox) in monkey COS-7 and murine 3T3 cells, both of which lacked endogenous p22(phox) mRNA, demonstrated that p22(phox) was not required for maturation of gp91(phox) carbohydrate to complex oligosaccharides. However, coexpression of transgenic p22(phox) increased the abundance of the mature gp91(phox) glycoprotein. These results suggest that heme incorporation plays an important role in cytochrome b558 assembly and provide further support for the concept that stability of p22(phox) and the mature gp91(phox) subunit is increased by heterodimer formation.