Source Of Superoxide Research Articles

Distinct oxidative stress profile in uterine fibroids versus adjacent myometrium

Oxidative stress has been shown to be a major player in common profibrotic gynecologic disorders. NAD(P)H oxidases (NOXs) are the main source of superoxide in several cell types and consists of seven isoforms: NOXs1–5, and NOX homologues, DUOX1 and DUOX2, as well binding molecules including p22phox, p40phox, p47phox/NOXO1, p67phox/NOXA1 and Rac. The objective was to investigate whether NOXs are differentially regulated in normal myometrium versus uterine fibroid lesions.

Mitochondrial-localized NADPH oxidase 4 is a source of superoxide in angiotensin II-stimulated neurons

Angiotensin II (ANG II) plays an important role in the central regulation of systemic cardiovascular function. ANG II-mediated intraneuronal signaling has been shown to be predicated by an increase in mitochondrial superoxide (O₂∙-), yet the source of this reactive oxygen species (ROS) production remains unclear. NADPH oxidase 4 (Nox4), a member of the NADPH oxidase family, has been reported to be localized in mitochondria of various cell types and has been implicated in brain angiotensinergic signaling. However, the subcellular localization and function of Nox4 in neurons has not been fully elucidated. In this study, we hypothesized that Nox4 is expressed in neuron mitochondria and is involved in ANG II-dependent O₂∙--mediated intraneuronal signaling. To query this, Nox4 immunofluorescent staining and mitochondrial enrichment were performed in a mouse catecholaminergic neuronal cell model (CATH.a). Nox4 was shown to be present in neuron mitochondria as evidenced by colocalization with both the mitochondrial-localized protein manganese superoxide dismutase (MnSOD) and dye MitoTracker Red. Moreover, Nox4 expression was significantly increased in enriched mitochondrial fractions compared with whole cell lysates. Additionally, adenoviral-encoded small interfering RNA for Nox4 (AdsiNox4) caused a robust knockdown in Nox4 mRNA and protein levels, which led to the attenuation of ANG II-induced mitochondrial O₂∙- production. Finally, in the subfornical organ (SFO) of the brain, Nox4 not only demonstrated mitochondrial localization but was induced by chronic, peripheral infusion of ANG II. Collectively, these data suggest that Nox4 is a source of O₂∙- in neuron mitochondria that contributes to ANG II intraneuronal signaling.

Abstract 72: Vascular and Cardiac Effects of Inducible Nitric Oxide Synthase in Hyperhomocysteinemic Mice

Background Diet-induced hyperhomocysteinemia (HHcy) produces endothelial and cardiac dysfunction and promotes thrombosis through a mechanism proposed to involve oxidative stress. Inducible nitric oxide synthase (iNOS) is upregulated in HHcy and can generate oxidants such as superoxide. We tested the hypothesis that iNOS mediates the adverse oxidative vascular, thrombotic, and cardiac effects of HHcy. Methods Mice deficient in iNOS ( Nos2-/ -) and their wild-type (WT) littermates were fed either a control (C) diet or a high methionine/low folate (HM/LF) diet for 3-6 months. Vasomotor function was assessed in cerebral arterioles in response to the endothelium-dependent dilator acetylcholine or the endothelium-independent dilator nitroprusside. Thrombotic occlusion of the carotid artery was measured in response to photochemical injury (rose bengal). Regional myocardial injury was measured after 30 minutes of ischemia and 2 hours of reperfusion of the left coronary artery. Results Nos2 -/- and WT mice fed the HM/LF diet developed similar levels of HHcy, with a 2-fold increase in plasma total homocysteine (P<0.001 vs. C diet). As expected, WT mice with HHcy exhibited endothelial dysfunction, with impaired dilatation to acetylcholine but not nitroprusside, and enhanced susceptibility to thrombosis, with shortened times to occlusion of the carotid artery (P<0.05 vs. C diet). Interestingly, deficiency of iNOS resulted in decreased, rather than increased dilatation responses to acetylcholine (P<0.05 vs. WT mice). Nos2 -/- mice also exhibited shortened times to thrombotic occlusion in mice fed the C diet (P<0.05 vs. WT mice) and failed to protect from endothelial dysfunction or accelerated thrombosis in mice fed the HM/LF diet. Deficiency of iNOS did not alter myocardial infarct size in mice fed the C diet but significantly increased infarct size and cardiac superoxide production in mice fed the HM/LF diet (P<0.05 vs. WT mice). Conclusions These findings suggest that endogenous iNOS protects from, rather than exacerbates, endothelial dysfunction, thrombosis, and myocardial ischemia-reperfusion injury. In the setting of HHcy, iNOS appears to function to blunt cardiac oxidative stress rather than as a source of superoxide.

Abstract 192: Depletion of Inflammatory Monocytes Reduces Endothelial Nitric Oxide Synthase Uncoupling and iNOS Derived Nitrosative Stress in Angiotensin II-induced Vascular Dysfunction

Background Endothelial nitric oxide synthase (eNOS) uncoupling occurs in disease states of vascular dysfunction and amplifies vascular oxidative stress and loss of NO bioactivity. Inflammatory myelomonocytic cells as a major source of superoxide are critical for angiotensin-II induced vascular dysfunction; however their role in mediating eNOS uncopuling has not been defined yet. Methods and results Angiotensin II (1mg/kg/d, 7d) increased the number of CD11b + Gr-1 low iNOS + monocytes and macrophages in mouse aorta (verified by flow cytometry) in paralell to increasing nox4 and heme oxygenase 1 expression (assessed by Western blot analysis) and inducing eNOS uncoupling, measured by differential NOS dependent superoxide formation in the endothelial layer and glutathionylation of eNOS. Toxin mediated ablation of macrophages in LysM iDTR transgenic mice restored all of these parameters. Conversely, ablation of LysM + cells reduced iNOS expression and normalized vascular levels of tetrahydrobiopterin (measured by high-performance liquid chromatography) and expression of the GTP cyclohydrolase-1, essential for tetrahydrobiopterin synthesis, all of which were increased by angiotensin II in control mice. Conclusion Depletion of LysM + monocytes reduces angiotensin II induced eNOS uncopuling in vascular dysfunction, and simultaneously protects from iNOS mediated nitrosative stress: These findings highlight the role of vascular inflammatory cells in disturbed NO-bioactivity and vascular function

Sex Differences in Aortic Endothelial Function of Streptozotocin‐induced Diabetic Rats: A Possible Role of Superoxide Production

Little is known of the interaction between diabetes and gender in the vasculature. This study was designed to investigate 1) whether there were gender differences in rat aortic endothelium‐dependent vasodilation (EDV) in streptozotocin (STZ, 60 mg/kg, iv)‐induced diabetic rats at early stage of the disease (1 wk), and 2) the potential role of superoxide in diabetes‐induced vascular dysfunction. EDV to acetylcholine (ACh; 10−8 to 10−5 M) was measured in aortic rings precontracted with phenylephrine (2 μM) before and after pretreatment with MnTMPyP (10 mM), a superoxide scavenger. In addition, the level of endothelial nitric oxide synthase (eNOS) and NADPH oxidase (Nox, a potent sources of superoxide) 1, 2 and 4 mRNA expression were determined using real‐time RT‐PCR. ACh‐induced relaxations were significantly impaired in aortic rings from both male and female diabetic rats. However, the extent of impairment was significantly greater in diabetic females than diabetic males. Preincubation with MnTMPyP increased EDV only in diabetic female group. Accordingly, our data showed that in females, the level of Nox4 mRNA expression was substantially enhanced, whereas the level of eNOS mRNA expression was decreased at 1 wk after the induction of diabetes. These data suggest that the predisposition of female rat aorta to vascular injury in diabetes is possibly due to the superoxide production (supported by NIH/NIDCR).

Superoxide and hydrogen peroxide stimulate renin release from juxtaglomerular cells

Renin is the rate‐limiting enzyme in angiotensin formation, playing a pivotal role in blood pressure regulation. In the kidney, superoxide and hydrogen peroxide (H202) are involved in the development of hypertension by affecting nephron transport and vascular reactivity. However, it is not known whether superoxide and H202 are endogenously produced in juxtaglomerular (JG) cells, the source of these reactive oxygen species and whether they regulate renin release. We hypothesized that superoxide and H2O2 are endogenously produced in JG cells and that they stimulate renin release. Primary cultures of mouse JG cells were treated for 60 min with different inhibitors and renin released to the supernatant measured by radioimmunoassay. Decreasing endogenous superoxide with superoxide dismutase (300U/ml) inhibited renin release by 35±10% (p<0.03). By Western Blot we found that NOX4, one of the NADPH oxidase isoforms is present in JG lysates (n=3). Apocynin (120 μM), a general inhibitor of NADPH oxidases, decreased renin release by 28±7% (p<0.04). Since dismutation of superoxide results in H202 accumulation, we tested the effect of scavenging endogenous H202. Adding catalase (100U/ml) inhibited renin release by 37±10% (p<0.02). We concluded that endogenous superoxide and H202 stimulate renin release from mouse JG cells. These are the first data identifying endogenous sources of superoxide in JG cells. HFHS Award.

Reevaluation of the Reactivity of Superoxide Radicals by Consideration of Both the Alkalinity of KO2 and the Superoxide-Scavenging Activity of Xanthine Oxidase

The rate constant, kS, for the reaction of scavengers, obtained from a competitive reaction system in which KO2 was used as a superoxide source, with superoxide has been reevaluated in electron spi...

Alternative Oxidase Expression in the Mouse Enables Bypassing Cytochrome c Oxidase Blockade and Limits Mitochondrial ROS Overproduction

Cyanide-resistant non-phosphorylating respiration is known in mitochondria from plants, fungi, and microorganisms but is absent in mammals. It results from the activity of an alternative oxidase (AOX) that conveys electrons directly from the respiratory chain (RC) ubiquinol pool to oxygen. AOX thus provides a bypath that releases constraints on the cytochrome pathway and prevents the over-reduction of the ubiquinone pool, a major source of superoxide. RC dysfunctions and deleterious superoxide overproduction are recurrent themes in human pathologies, ranging from neurodegenerative diseases to cancer, and may be instrumental in ageing. Thus, preventing RC blockade and excess superoxide production by means of AOX should be of considerable interest. However, because of its energy-dissipating properties, AOX might produce deleterious effects of its own in mammals. Here we show that AOX can be safely expressed in the mouse (MitAOX), with major physiological parameters being unaffected. It neither disrupted the activity of other RC components nor decreased oxidative phosphorylation in isolated mitochondria. It conferred cyanide-resistance to mitochondrial substrate oxidation and decreased reactive oxygen species (ROS) production upon RC blockade. Accordingly, AOX expression was able to support cyanide-resistant respiration by intact organs and to afford prolonged protection against a lethal concentration of gaseous cyanide in whole animals. Taken together, these results indicate that AOX expression in the mouse is innocuous and permits to overcome a RC blockade, while reducing associated oxidative insult. Therefore, the MitAOX mice represent a valuable tool in order to investigate the ability of AOX to counteract the panoply of mitochondrial-inherited diseases originating from oxidative phosphorylation defects.

Developmental Programming of eNOS Uncoupling and Enhanced Vascular Oxidative Stress in Adult Rats After Transient Neonatal Oxygen Exposure

The authors have previously shown that neonatal hyperoxic stress leads to high blood pressure, impaired endothelium-mediated vasodilatation, and increased vascular production of superoxide anion by NAD(P)H oxidase in adulthood. However, it is unknown whether changes in nitric oxide (NO) production and/or bioinactivation prevail and whether NO synthase (NOS) is also a source of superoxide. The purpose of this study was to evaluate whether adult animals exposed to neonatal hyperoxic stress have impaired vascular NO production associated with NOS uncoupling participating to vascular superoxide production and vascular dysfunction. In adult male rats exposed to 80% oxygen from day 3 to 10 of life (H, n = 6) versus room air controls (CTRL, n = 6), vascular (aorta) NO production is decreased at baseline (CTRL: 21 ± 1 vs. H: 16 ± 2 4,5-diaminofluorescein diacetate fluorescence intensity arbitrary units; P < 0.05) and after carbachol stimulation (acetylcholine analog; CTRL: 26 ± 2 vs. H: 18±2; P < 0.05). Pretreatment with L-arginine (CTRL: 32 ± 4 vs. H: 31 ± 5) and L-sepiapterine [analog of key NOS cofactor tetrahydro-L-biopterin (BH4)] (CTRL: 30 ± 3 vs. H: 29 ± 3) normalizes NO production after carbachol. L-Sepiapterine also normalizes impaired vasodilatation to carbachol. Vascular endothelial NO synthase (eNOS) immunostaining is reduced, whereas total eNOS protein expression is increased in H (CTRL: 0.76 ± 0.08 vs. H: 1.76± 0.21; P < 0.01). The significantly higher superoxide generation (CTRL: 20 ± 2 vs. H: 28 ± 3 hydroethidine fluorescence intensity arbitrary units; P < 0.05) is prevented by pretreatment with the eNOS inhibitor N-nitro-L-arginine methyl ester (CTRL: 21 ± 4 vs. H: 22 ± 4). Taken together, the current data indicate a role for eNOS uncoupling in enhanced vascular superoxide, impaired endothelium-mediated vasodilatation, and decreased NO production in adult animals with programmed elevated blood pressure after a brief neonatal oxygen exposure.

Studies of Mitochondrial and Nonmitochondrial Sources Implicate Nicotinamide Adenine Dinucleotide Phosphate Oxidase(s) in the Increased Skeletal Muscle Superoxide Generation That Occurs During Contractile Activity

The sources of cytosolic superoxide in skeletal muscle have not been defined. This study examined the subcellular sites that contribute to cytosolic superoxide in mature single muscle fibers at rest and during contractile activity. Isolated fibers from mouse flexor digitorum brevis loaded with superoxide and nitric-oxide-sensitive fluorescent probes, specific pathway inhibitors and immunolocalization techniques were used to identify subcellular sites contributing to cytosolic superoxide. Treatment with the electron transport chain complex III inhibitor, antimycin A, but not the complex I inhibitor, rotenone, caused increased cytosolic superoxide through release from the mitochondrial intermembrane space via voltage-dependent anion or Bax channels, but inhibition of these channels did not affect contraction-induced increases in cytosolic superoxide. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitors decreased cytosolic superoxide at rest and following contractions. Protein and mRNA expression of NADPH oxidase subunits was demonstrated in single fibers. NOX2, NOX4, and p22(phox) subunits localized to the sarcolemma and transverse tubules; NOX4 was additionally expressed in mitochondria. Regulatory p40(phox) and p67(phox) proteins were found in the cytoplasm of resting fibers, but following contractions, p40(phox) appeared to translocate to the sarcolemma. Superoxide and other reactive oxygen species generated by skeletal muscle are important regulators of muscle force production and adaptations to contractions. This study has defined the relative contribution of mitochondrial and cytosolic sources of superoxide within the cytosol of single muscle fibers at rest and during contractions. Muscle mitochondria do not modulate cytosolic superoxide in skeletal muscle but NADPH oxidase is a major contributor both at rest and during contractions.

A Refined Analysis of Superoxide Production by Mitochondrial sn-Glycerol 3-Phosphate Dehydrogenase

The oxidation of sn-glycerol 3-phosphate by mitochondrial sn-glycerol 3-phosphate dehydrogenase (mGPDH) is a major pathway for transfer of cytosolic reducing equivalents to the mitochondrial electron transport chain. It is known to generate H(2)O(2) at a range of rates and from multiple sites within the chain. The rates and sites depend upon tissue source, concentrations of glycerol 3-phosphate and calcium, and the presence of different electron transport chain inhibitors. We report a detailed examination of H(2)O(2) production during glycerol 3-phosphate oxidation by skeletal muscle, brown fat, brain, and heart mitochondria with an emphasis on conditions under which mGPDH itself is the source of superoxide and H(2)O(2). Importantly, we demonstrate that a substantial portion of H(2)O(2) production commonly attributed to mGPDH originates instead from electron flow through the ubiquinone pool into complex II. When complex II is inhibited and mGPDH is the sole superoxide producer, the rate of superoxide production depends on the concentrations of glycerol 3-phosphate and calcium and correlates positively with the predicted reduction state of the ubiquinone pool. mGPDH-specific superoxide production plateaus at a rate comparable with the other major sites of superoxide production in mitochondria, the superoxide-producing center shows no sign of being overreducible, and the maximum superoxide production rate correlates with mGPDH activity in four different tissues. mGPDH produces superoxide approximately equally toward each side of the mitochondrial inner membrane, suggesting that the Q-binding pocket of mGPDH is the major site of superoxide generation. These results clarify the maximum rate and mechanism of superoxide production by mGPDH.

Abstract 18877: Novel Mouse Model for Testing NADPH Oxidase (Nox2) Role in Insulin Resistance Mediated Endothelial Dysfunction

Introduction: Insulin resistance (IR) plays a pivotal role in type 2 diabetes, obesity and hypercholesterolemia. Reactive oxygen species (ROS) play a key role in IR related endothelial dysfunction. We have reported that, superoxide (SO), an important ROS is produced at higher levels in IR and established that NADPH oxidase (Nox) is the primary source of SO in IR endothelium. Here we report our study to ascertain the role of Nox2 in IR. Methods: A unique murine model of endothelium specific IR, Endothelial Specific Mutated Insulin Receptor Over-expression (ESMIRO) and mice with universal deletion of Nox2 gene (Nox2 y/- ) were used here. Nox2 y/- mice was crossed with ESMIRO to generate double transgenics (ESMIRO/Nox2 y/- ). Endothelial function was analysed by measuring 1. aortic ring relaxation. 2. superoxide production. Expression of Nox and ROS related genes were examined by PCR and Western blotting. Results: Realtime PCR and Western blot of organs and pulmonary endothelial cells (PEC) confirmed the total loss of Nox2 in ESMIRO/Nox2 y/- . Growth rate and organ weights were unaltered in ESMIRO/Nox2 y/- . However, there was a trend towards reduced BP in double transgenics (119±1 mmHg in ESMIRO Vs 114±2 mmHg in ESMIRO/Nox2 y/- ; p=0.06). Metabolic characteristics including ITT and GTT showed no difference between two lines. Blood insulin levels before and after glucose injection were similar. Basal superoxide in PEC of ESMIRO/Nox2 y/- were 240±11% (p=0.02) reduced when compared to ESMIRO. Nitric oxide (NO) mediated vascular relaxation was improved in double transgenic animals (100±4% in response to 1000 nM Ach in ESMIRO/Nox2 y/- Vs 77±6% in ESMIRO). There was no change in endothelial independent relaxation (109±5% in response to 1000 nM SNP in ESMIRO/Nox2 y/- Vs 104±6% in ESMIRO). Realtime PCR showed that the important ROS scavenger superoxide dismutase 1 (SOD1) expression was increased above 300% in ESMIRO lungs compared to ESMIRO/Nox2 y/- . Other Nox and ROS related genes (Nox4, p22, p47, p67, SOD2, SOD3, eNOS &amp; catalase) expression were unchanged. Conclusions: These results show that Nox2 NADPH oxidase is a key source of the excess superoxide seen in a model of endothelium specific insulin resistance. Targeting Nox2 may be a useful therapeutic strategy in IR conditions.

Superoxide Reaction with Tyrosyl Radicals Generates para-Hydroperoxy and para-Hydroxy Derivatives of Tyrosine

Tyrosine-derived hydroperoxides are formed in peptides and proteins exposed to enzymatic or cellular sources of superoxide and oxidizing species as a result of the nearly diffusion-limited reaction between tyrosyl radical and superoxide. However, the structure of these products, which informs their reactivity in biology, has not been unequivocally established. We report here the complete characterization of the products formed in the addition of superoxide, generated from xanthine oxidase, to several peptide-derived tyrosyl radicals, formed from horseradish peroxidase. RP-HPLC, LC-MS, and NMR experiments indicate that the primary stable products of superoxide addition to tyrosyl radical are para-hydroperoxide derivatives (para relative to the position of the OH in tyrosine) that can be reduced to the corresponding para-alcohol. In the case of glycyl-tyrosine, a stable 3-(1-hydroperoxy-4-oxocyclohexa-2,5-dien-1-yl)-L-alanine was formed. In tyrosyl-glycine and Leu-enkephalin, which have N-terminal tyrosines, bicyclic indolic para-hydroperoxide derivatives were formed ((2S,3aR,7aR)-3a-hydroperoxy-6-oxo-2,3,3a,6,7,7a-hexahydro-1H-indole-2-carboxylic acid) by the conjugate addition of the free amine to the cyclohexadienone. It was also found that significant amounts of the para-OH derivative were generated from the hydroxyl radical, formed on exposure of tyrosine-containing peptides to Fenton conditions. The para-OOH and para-OH derivatives are much more reactive than other tyrosine oxidation products and may play important roles in physiology and disease.

In vitro study of poly(ethylene carbonate) as a drug-eluting stent coating

An in vitro feasibility study of the use of poly(ethylene carbonate) (PEC) as a biodegradable coating material for drug-eluting stents is reported, and the performance of PEC is compared with that of poly(lactic-co-glycolic acid) (PLGA). Scanning electron microscopy (SEM) images of PEC and PLGA discs after treatment with an alkaline KO2 solution as a superoxide source showed that the PEC maintained its integrity whereas holes and small particles appeared during the treatment of PLGA. Sirolimus and paclitaxel were loaded into PEC and PLGA in order to study drug release performance. Attenuated total reflectance–infrared (ATR–FTIR) spectroscopy of sirolimus, PEC and the sirolimus-loaded PEC coating showed that no chemical reaction occurred between sirolimus and PEC. The results of atomic force microscopy (AFM) revealed that the mean roughness (Ra) values of the bare metal stent (BMS) and the drug-eluting stent (DES) were 2.3nm and 1.0nm, respectively. After balloon expansion experiments, no delamination or destruction of the PEC coating was observed. The drug release profile of sirolimus was different from that of paclitaxel when PEC was employed as the drug carrier, and the release curves of sirolimus were different when PEC and PLGA were used as drug carriers. All the experimental results demonstrated that PEC was one of the best potential stent-coating materials.

Expression of NADPH oxidase in human pancreatic islets

AimsNADPH oxidase (NOX) is a known source of superoxide anions in phagocytic and non-phagocytic cells. In this study, the presence of this enzyme in human pancreatic islets and the importance of NADPH oxidase in human β-cell function were investigated. Main methods and key findingsIn isolated human pancreatic islets, the expression of NADPH oxidase components was evidenced by real-time PCR (p22PHOX, p47PHOX and p67PHOX), Western blotting (p47PHOX and p67PHOX) and immunohistochemistry (p47PHOX, p67PHOX and gp91PHOX). Immunohistochemistry experiments showed co-localization of p47PHOX, p67PHOX and gp91PHOX (isoform 2 of NADPH oxidase—NOX2) with insulin secreting cells. Inhibition of NADPH oxidase activity impaired glucose metabolism and glucose-stimulated insulin secretion. SignificanceThese findings demonstrate the presence of the main intrinsic components of NADPH oxidase comprising the NOX2 isoform in human pancreatic islets, whose activity also contributes to human β-cell function.

Superoxide dictates the mode of U937 cell ascorbic acid uptake and prevents the enhancing effects of the vitamin to otherwise nontoxic levels of reactive oxygen/nitrogen species

Exposure of U937 cells to low micromolar levels of ascorbic acid or dehydroascorbic acid, while resulting in identical ascorbic acid accumulation, is unexpectedly associated with remarkably different responses to exogenous oxidants. We observed that otherwise nontoxic levels of hydrogen peroxide, tert-butylhydroperoxide or peroxynitrite promote toxicity in cells preloaded with ascorbic acid, whereas hardly any effect was detected in cells pretreated with dehydroascorbic acid. Further experiments performed with peroxynitrite in cells preloaded with ascorbic acid provided evidence for a very rapid nonapoptotic death, preceded by early Bax mitochondrial translocation and by mitochondrial permeability transition. The notion that conversion of extracellular ascorbic acid to dehydroascorbic acid prevents the enhancing effects on oxidant toxicity and nevertheless preserves the net amount of vitamin C accumulated was also established using ascorbate oxidase as well as various sources of superoxide, namely, xanthine/xanthine oxidase or ATP-driven NADPH oxidase activation. These findings suggest that superoxide-dependent conversion of extracellular ascorbic acid to dehydroascorbic acid represents an important component of the overall survival strategy of some cell types to reactive oxygen/nitrogen species.

Impairment of the extrusion transporter for asymmetric dimethyl-l-arginine: A novel mechanism underlying vasospastic angina

A 37-year old male patient presented with frequent angina attacks (up to 40/day) largely resistant to classical vasodilator therapy. The patient showed severe coronary and peripheral endothelial dysfunction, increased platelet aggregation and increased platelet-derived superoxide production. The endothelial nitric oxide synthase (eNOS)-inhibitor NG-nitro-l-arginine methyl ester (L-NAME) reduced superoxide formation in platelets identifying “uncoupled” eNOS as a superoxide source. Oral l-arginine normalized coronary and peripheral endothelial dysfunction and reduced platelet aggregation and eNOS-derived superoxide production. Plasma concentrations of the endogenous NOS inhibitor asymmetric dimethyl-l-arginine (ADMA), representing an independent risk factor for cardiovascular disease, were normal in the patient. However, immediately after oral administration of cationic amino acid (CAA), plasma ADMA levels rose markedly, demonstrating increased ADMA efflux from intracellular stores. ADMA efflux from mononuclear cells of the patient was accelerated by CAA, but not neutral amino acids (NAA) demonstrating impairment of y+LAT (whose expression was found reduced in these cells). These data suggest that impairment of y+LAT may cause intracellular (endothelial) ADMA accumulation leading to systemic endothelial dysfunction. This may represent a novel mechanism underlying vasospastic angina and vascular dysfunction in general. Moreover, these new findings contribute to the understanding of the l-arginine paradox, the improvement of eNOS activity by oral l-arginine despite sufficient cellular l-arginine levels to ensure proper function of this enzyme.

Mitochondria‐targeted antioxidant therapy reverses age‐related arterial stiffening

Aging leads to large elastic artery stiffening mediated by increases in superoxide‐dependent oxidative stress and related changes/damage to structural proteins. Mitochondria are a major source of cellular superoxide and may therefore be a therapeutic target for the treatment of arterial stiffening. We studied young (Y: 5 mo, n=5) and old (O: 27–29 mo, n=5) male C57BL6 mice, and O mice treated with the mitochondria targeted antioxidant MitoQ10 (OMQ: 28–29 mo, n=5). Aortic pulse wave velocity (aPWV), an index of arterial stiffness, was higher in O vs. Y mice (485 ± 24 vs. 405 ± 15 cm/s, p&lt;0.05) and was associated with increased aortic superoxide production (electron paramagnetic resonance, O: 7200 ± 1950 vs. Y: 2800 ± 515 AU, p&lt;0.05). O mice also had greater aortic levels of nitrotyrosine (NT), a marker of oxidative stress, and lower levels of manganese (mitochondrial) superoxide dismutase (MnSOD) (western blotting, both p&lt;0.05 vs. Y). MQ treatment (250 μM in water x 3w) normalized aortic stiffening (OMQ: 378 ± 33 cm/s), superoxide production (OMQ: 2835 ± 1131 AU), MnSOD and NT in O mice. MQ also increased expression of complex IV and the mitochondrial biogenesis marker PGC‐1α (both p&lt;0.05 vs. O). Incubation of aortas from Y mice with the mitochondrial superoxide inducer rotenone (0.5 μM, 48h) increased NT by ~300% (p&lt;0.05) and this was inhibited by coincubation with MQ, an effect associated with enhanced MnSOD expression. MQ treatment may be a novel therapy for age‐related stiffening of large elastic arteries by reducing mitochondrial oxidative stress and perhaps increasing mitochondrial biogenesis.NIH AG013038, AG039210

Reevaluation of the Reactivity of Superoxide Radicals by Consideration of Both the Alkalinity of KO2 and the Superoxide-Scavenging Activity of Xanthine Oxidase

Abstract The rate constant, kS, for the reaction of scavengers, obtained from a competitive reaction system in which KO2 was used as a superoxide source, with superoxide has been reevaluated in electron spin resonance (ESR) spin-trapping studies. In consequence, it has been found that the local pH in a reaction field of our KO2 system was 12. As a result, the rate constants for the reaction of xanthine oxidase (XOD) with superoxide at pH 7.8 and 12 were established to be 2.1 × 106 and 8.0 × 105 M−1 s−1, respectively. On the basis of these values, discrepancies between reports in the literature have been examined, and it has been found that systematic errors appearing in values of the rate constant, kT, for the reaction of spin-trapping agents with superoxide due to variations in determination method had originated from omission of the superoxide-scavenging activity of XOD and that the errors could be explained almost completely quantitatively. For this reason, the kT values latterly reported by Allouch et al. are inferior to those formerly reported by Finkelstein et al. as reference values when the kS values are determined by the kinetic competition using these kT values.

NOX Inhibitors as a Therapeutic Strategy for Stroke and Neurodegenerative Disease

NADPH oxidase was originally identified in immune cells as playing an important microbicidal role. In neurodegenerative and cerebrovascular diseases, inflammation is increasingly being recognized as contributing negatively to neurological outcome, with NADPH-oxidase as an important source of superoxide. Recently, several forms of this oxidase have been found in a variety of non-immune cells. Neuronal NADPH oxidase is thought to participate in longterm potentiation and intercellular signaling. However, excessive superoxide production is damaging and has been shown to play an important role in the progression of brain injury. NADPH oxidase is a multisubunit complex composed of membrane-associated gp91(phox) and p22(phox) subunits and cytosolic subunits, p47(phox), p67(phox), and p40(phox) and Rac. When NADPH oxidase is activated through phosphorylatoin of p47(phox), cytosolic subunits translocate to the cell membrane and fuse with the catalytic subunit, gp91(phox). The activated enzyme complex transports electrons to oxygen, thus producing the superoxide anion (O₂˙⁻), a precursor of reactive oxygen species. The advantage of a targeted NADPH oxidase inhibitor that would inhibit the production of superoxide non-phagocytic cells is clear. To date no such therapeutically viable inhibitor exists but recent research using current inhibitors has enhanced our knowledge of the role of NADPH oxidase in CNS diseases and provides impetus to develop a very specific, potent and safe NADPH oxidase inhibitor.