Role Of Xanthine Oxidoreductase Research Articles

Mechanism and Physiological Relevance of Xanthine Oxidase as Nitrate Reductase in Mammalian Organism: An Overview

In living organisms nitric oxide radical is produced by the enzymes NO synthases, with consequent oxidation to nitrate by several oxidation/reduction pathways. The end step of NO oxidation is the formation of nitrite and nitrate, which after their formation enter a reduction phase of NO cycle, thereby playing a physiologically important role of NO donors. However, while in plants and microorganisms the key pathway of nitrate/nitrite reduction is the functioning of the enzymes nitrate reductases (NRs), mammalian organism lacks NRs enzymes. Instead, in mammals, the reduction of nitrates and nitrites occurs by several other, both enzymatic and non-enzymatic, pathways. Based on the present knowledge, one of the key enzymes involved in the nitrate/nitrite reduction process is xanthine oxidoreductase (XOR). XOR is capable of reducing organic and inorganic nitrates and nitrite, and the role of XOR in NO3 - → NO2 - → ·NO pathway is most studied as compared to other enzymes with nitrate reductase activity. The functions of XOR, the mechanisms of nitrate/nitrite reduction by this enzyme and their physiological relevance will be outlined in this mini review.

The XOR-IDH3α axis controls macrophage polarization in hepatocellular carcinoma

Tumor-associated macrophages (TAMs) are indispensable in the hepatocellular carcinoma (HCC) tumor microenvironment. Xanthine oxidoreductase (XOR), also known as xanthine dehydrogenase (XDH), participates in purine metabolism, uric acid production, and macrophage polarization to a pro-inflammatory phenotype. However, the role of XOR in HCC-associated TAMs is unclear. We evaluated the XOR level in macrophages isolated from HCC tissues and paired adjacent tissues. We established DEN/carbon tetrachloride (CCl4)-induced and orthotopically implanted HCC mouse models using mice with Xdh-specific depletion in the myeloid cell lineage (Xdhf/fLyz2cre) or Kupffer cells (Xdhf/fClec4fcre). We determined metabolic differences using specific methodologies, including metabolomics and metabolic flux. We found that XOR expression was downregulated in HCC TAMs and positively correlated with patient survival, which was strongly related to the characteristics of the tumor microenvironment (TME), especially hypoxia. Using HCC-inflicted mice (Xdhf/fLyz2cre and Xdhf/fClec4fcre), we revealed that XOR loss in monocyte-derived TAMs rather than Kupffer cells promoted their M2 polarization and CD8+ T-cell exhaustion, which exacerbated HCC progression. In addition, the tricarboxylic acid cycle was disturbed, and the generation of α-ketoglutarate (α-KG) was enhanced within XOR-depleted macrophages. XOR inhibited α-KG production by interacting with IDH3α catalytic sites (K142 and Q139). The increased IDH3α activity caused increased adenosine and kynurenic acid production in the TAMs, which enhanced the immunosuppressive effects of the TAMs and CD8+ T cells. The XOR-IDH3α axis mediates TAM polarization and HCC progression and may be a small-molecule therapeutic or immunotherapeutic target against suppressive HCC TAMs. HCC is one of the cancers with the highest morbidity in the world, and its 5-year survival rate is low. Currently, lots of immunotherapies have been applied to the treatment of HCC, but the curative effects are not satisfactory. TME of HCC is full of different infiltrating immune cells. TAMs are vital components in TME and involved in HCC progression. Herein, we confirm the downregulation of XOR expression in TAMs isolated from human HCC tissues. The loss of XOR in monocyte-derived macrophages increases IDH3 activity and results in an increase in α-KG production, which can promote M2-like polarization. Additionally, XOR-null TAMs derived from monocytes promote CD8+ T-cell exhaustion via the upregulation of immunosuppressive metabolites, including adenosine and KYNA. Given the prevalence and high rate of incidence of HCC and the need for improved therapeutic options for patients, our findings identify potential therapeutic targets that may be further studied to develop improved therapies.

Role of Xanthine Oxidoreductase from Mesenchymal Cells in Retinal Inflammation after Pericyte Breakdown

Glycemic control is important for diabetic retinopathy (DR), but not sufficient for the inhibition after proliferative DR (PDR). Uric acid in the vitreous is reportedly increased in patients with DR, especially in the PDR. Here, the involvement of xanthine oxidoreductase (XO), responsible for uric acid production, in worsening DR was examined in both mouse model and co-cultured angiogenesis model. First, retinal pericytes were abolished by anti-PDGFRβ antibody injection to C57/Bl6 neonatal mouse at P1. Pericyte dropout from endothelial cells is reportedly early histological change in DR, and induces bleeding, abnormal angiogenesis, inflammation, and retinal detachment with rapid progression at P9-11. The expression of XO level in retina was increasing after pericyte breakdown, and was proportional to VEGF, and preceding the increase of TNFalpha. When 3.6mg/kg Febuxostat (Fbx), an XO inhibitor was injected at P7, widespread edema and retinal detachment/funnel-like deformation were dramatically improved by Fbx treatment at P11. ICAM-1+ cells surrounding edema and CD11b+ cells were reduced. Next, we performed a 3D culture sprouting assay using HUVEC spheroids in collagen gel co-cultured with human lung fibroblast (hLF) in fibrin gel. By Fbx treatment within the physiological concentration, the branch sprouting, cellular migration to the distal side and mitosis in branch stems were inhibited. ICAM-1 increase in HUVEC was observed with hLF co-culture but was suppressed by Fbx treatment. Finally, these effects of Fbx were not shown on HUVEC in planar culture. On the other hand, dissolution of fibrin by hLF was delayed directly due to Fbx. VEGF and PAI-1 expression from hLF and MMP-9 activity in medium were decreased by Fbx treatment. In summary, XO inhibition improved retinal inflammation and injury after pericyte loss. These results suggest the role of humoral inflammatory factors from mesenchymal cells surrounding retinal vessels in the hyperpermeable state such as DR. Disclosure A. Kushiyama: Research Support; Self; Teijin Pharma Limited, Sanwa Kagaku Kenkyusho Co., Ltd., Kowa Pharmaceutical Co., Ltd, StaGen. Consultant; Self; RIZAP. T. Kikuchi: None. H. Yamazaki: None. T. Yamamotoya: None. H. Sakoda: None. M. Fujishiro: Research Support; Self; Johnson & Johnson Services, Inc.. Y. Nakatsu: None. A. Uemura: Research Support; Self; Daiichi Sankyo Company, Limited, Boehringer Ingelheim GmbH, KAN Research Institute, Inc. T. Shirakura: Employee; Self; Teijin Pharma Limited. T. Asano: Research Support; Self; Teijin Pharma Limited, Sanwa Kagaku Kenkyusho Co., Ltd., Sanofi K.K. Y. Iwamoto: Board Member; Self; Japan Diabetes Foundation.

An effort toward molecular neuroeconomics of food deprivation induced food hoarding in mice: focus on xanthine oxidoreductase gene expression and xanthine oxidase activity.

The crucial role of xanthine oxidoreductase (XOR) gene and its active isoform, xanthine oxidase (XO), in purine metabolism and cellular oxidative status led us to investigative their fluctuations in food deprivation induced food hoarding in mice. After, 10h food deprivation, mice that hoarded lesser than 5g were considered as 'low-hoarders' while mice that hoarded higher than 20g were considered as 'high-hoarders'. Mice who hoarded between 5 to 20g of food were excluded from study. An increase (1.133-fold) in encephalic XOR expression has been found in high-hoarders compared with low-hoarders without sex consideration. An increase (~ 50-fold) in encephalic XOR in female high-hoarders vs. female low-hoarders while a decrease (0.026-fold) in encephalic XOR in male high-hoarders vs. male low-hoarders demonstrated that food deprivation is associated with sex-dependent alteration in XOR expression. The encephalic and hepatic XO activities were not different in male high-hoarders vs. male low-hoarders while encephalic XO activity has been also increased significantly in female high-hoarders (~ 4 times) compared to female low-hoarders. The plasma and hepatic XO activities tended to be increased in female high-hoarders as compared to female low-hoarders, however the uric acid levels in plasma, liver and brain tissues were not altered in female high-hoarders as compared to female low-hoarders. In sum, this study generally proposed that different gene expression space is behind of hoarding behavior in a food-deprived mouse model. Specifically, this is the first study that examined the levels of encephalic XO activity and XOR expression in hoarding behavior, although additional studies are requested.

Nitrite-Mediated Hypoxic Vasodilation Predicted from Mathematical Modeling and Quantified from in Vivo Studies in Rat Mesentery.

Nitric oxide (NO) generated from nitrite through nitrite reductase activity in red blood cells has been proposed to play a major role in hypoxic vasodilation. However, we have previously predicted from mathematical modeling that much more NO can be derived from tissue nitrite reductase activity than from red blood cell nitrite reductase activity. Evidence in the literature suggests that tissue nitrite reductase activity is associated with xanthine oxidoreductase (XOR) and/or aldehyde oxidoreductase (AOR). We investigated the role of XOR and AOR in nitrite-mediated vasodilation from computer simulations and from in vivo exteriorized rat mesentery experiments. Vasodilation responses to nitrite in the superfusion medium bathing the mesentery equilibrated with 5% O2 (normoxia) or zero O2 (hypoxia) at either normal or acidic pH were quantified. Experiments were also conducted following intraperitoneal (IP) injection of nitrite before and after inhibiting XOR with allopurinol or inhibiting AOR with raloxifene. Computer simulations for NO and O2 transport using reaction parameters reported in the literature were also conducted to predict nitrite-dependent NO production from XOR and AOR activity as a function of nitrite concentration, PO2 and pH. Experimentally, the largest arteriolar responses were found with nitrite >10 mM in the superfusate, but no statistically significant differences were found with hypoxic and acidic conditions in the superfusate. Nitrite-mediated vasodilation with IP nitrite injections was reduced or abolished after inhibiting XOR with allopurinol (p < 0.001). Responses to IP nitrite before and after inhibiting AOR with raloxifene were not as consistent. Our mathematical model predicts that under certain conditions, XOR and AOR nitrite reductase activity in tissue can significantly elevate smooth muscle cell NO and can serve as a compensatory pathway when endothelial NO production is limited by hypoxic conditions. Our theoretical and experimental results provide further evidence for a role of tissue nitrite reductases to contribute additional NO to compensate for reduced NO production by endothelial nitric oxide synthase during hypoxia. Our mathematical model demonstrates that under extreme hypoxic conditions with acidic pH, endogenous nitrite levels alone can be sufficient for a functionally significant increase in NO bioavailability. However, these conditions are difficult to achieve experimentally.

Xanthine Oxidoreductase in Drug Metabolism: Beyond a Role as a Detoxifying Enzyme.

The enzyme xanthine oxidoreductase (XOR) catalyzes the last two steps of purine catabolism in the highest uricotelic primates. XOR is an enzyme with dehydrogenase activity that, in mammals, may be converted into oxidase activity under a variety of pathophysiologic conditions. XOR activity is highly regulated at the transcriptional and post-translational levels and may generate reactive oxygen and nitrogen species, which trigger different consequences, ranging from cytotoxicity to inflammation. The low specificity for substrates allows XOR to metabolize a number of endogenous metabolites and a variety of exogenous compounds, including drugs.The present review focuses on the role of XOR as a drug-metabolizing enzyme, specifically for drugs with anticancer, antimicrobial, antiviral, immunosuppressive or vasodilator activities, as well as drugs acting on metabolism or inducing XOR expression.XOR has an activating role that is essential to the pharmacological action of quinone drugs, cyadox, antiviral nucleoside analogues, allopurinol, nitrate and nitrite. XOR activity has a degradation function toward thiopurine nucleotides, pyrazinoic acid, methylxanthines and tolbutamide, whose half-life may be prolonged by the use of XOR inhibitors.In conclusion, to avoid potential drug interaction risks, such as a toxic excess of drug bioavailability or a loss of drug efficacy, caution is suggested in the use of XOR inhibitors, as in the case of hyperuricemic patients affected by gout or tumor lysis syndrome, when it is necessary to simultaneously administer therapeutic substances that are activated or degraded by the drug-metabolizing activity of XOR.

Nitrite-mediated reduction of macrophage NADPH oxidase activity is dependent on xanthine oxidoreductase-derived nitric oxide but independent of S-nitrosation

BackgroundInorganic nitrite has shown beneficial effects in cardiovascular and metabolic diseases partly via attenuation of NADPH-oxidase (NOX)-mediated oxidative stress. However, the exact mechanisms are still unclear. Here we investigated the role of S-nitrosation or altered expression of NOX subunits, and the role of xanthine oxidoreductase (XOR) in nitrite-derived nitric oxide (NO) production. MethodsMouse macrophages were activated with LPS in the presence or absence of nitrite. NOX activity was measured by lucigenin-dependent chemiluminescence. Gene and protein expression of NOX2 subunits and XOR were investigated using qPCR and Western Blot. S-nitrosation of Nox2 and p22phox was studied with a Biotin Switch assay. Uric acid levels in cell culture medium were analyzed as a measure of XOR activity, and NO production was assessed by DAF-FM fluorescence. ResultsNOX activity in activated macrophages was significantly reduced by nitrite. Reduced NOX activity was not attributed to decreased NOX gene expression. However, protein levels of p47phox and p67phox subunits were reduced by nitrite in activated macrophages. Protein expression of Nox2 and p22phox was not influenced by this treatment and neither was their S-nitrosation status. Increased uric acid levels after nitrite and diminished NO production during XOR-inhibition with febuxostat suggest that XOR is more active during nitrite-treatment of activated macrophages and plays an important role in the bioactivation of nitrite. ConclusionsOur findings contribute to the mechanistic understanding about the therapeutic effects associated with nitrite supplementation in many diseases. We show that nitrite-mediated inhibition of NOX activity cannot be explained by S-nitrosation of the NOX enzyme, but that changes in NOX2 expression and XOR function may contribute.

Role of xanthine oxidoreductase in the anti-thrombotic effects of nitrite in rats in vivo

The mechanisms underlying nitrite-induced effects on thrombosis and hemostasis in vivo are not clear. The goal of the work described here was to investigate the role of xanthine oxidoreductase (XOR) in the anti-platelet and anti-thrombotic activities of nitrite in rats in vivo. Arterial thrombosis was induced electrically in rats with renovascular hypertension by partial ligation of the left renal artery. Sodium nitrite (NaNO2, 0.17 mmol/kg twice daily for 3 days, p.o) was administered with or without one of the XOR-inhibitors: allopurinol (ALLO) and febuxostat (FEB) (100 and 5 mg/kg, p.o., for 3 days). Nitrite treatment (0.17 mmol/kg), which was associated with a significant increase in NOHb, nitrite/nitrate plasma concentration, resulted in a substantial decrease in thrombus weight (TW) (0.48 ± 0.03 mg vs. vehicle [VEH] 0.88 ± 0.08 mg, p < 0.001) without a significant hypotensive effect. The anti-thrombotic effect of nitrite was partially reversed by FEB (TW = 0.63 ± 0.06 mg, p < 0.05 vs. nitrites), but not by ALLO (TW = 0.43 ± 0.02 mg). In turn, profound anti-platelet effect of nitrite measured ex vivo using collagen-induced whole-blood platelet aggregation (70.5 ± 7.1% vs. VEH 100 ± 4.5%, p < 0.05) and dynamic thromboxaneB2 generation was fully reversed by both XOR-inhibitors. In addition, nitrite decreased plasminogen activator inhibitor-1 concentration (0.47 ± 0.13 ng/ml vs. VEH 0.62 ± 0.04 ng/ml, p < 0.05) and FEB/ALLO reversed this effect. In vitro the anti-platelet effect of nitrite (1 mM) was reversed by FEB (0.1 mM) under hypoxia (0.5%O2) and normoxia (20%O2). Nitrite treatment had no effect on coagulation parameters. In conclusion, the nitrite-induced anti-platelet effect in rats in vivo is mediated by XOR, but XOR does not fully account for the anti-thrombotic effects of nitrite.

Hydralazine and Organic Nitrates Restore Impaired Excitation-Contraction Coupling by Reducing Calcium Leak Associated with Nitroso-Redox Imbalance*

Although the combined use of hydralazine and isosorbide dinitrate confers important clinical benefits in patients with heart failure, the underlying mechanism of action is still controversial. We used two models of nitroso-redox imbalance, neuronal NO synthase-deficient (NOS1(-/-)) mice and spontaneously hypertensive heart failure rats, to test the hypothesis that hydralazine (HYD) alone or in combination with nitroglycerin (NTG) or isosorbide dinitrate restores Ca(2+) cycling and contractile performance and controls superoxide production in isolated cardiomyocytes. The response to increased pacing frequency was depressed in NOS1(-/-) compared with wild type myocytes. Both sarcomere length shortening and intracellular Ca(2+) transient (Δ[Ca(2+)]i) responses in NOS1(-/-) cardiomyocytes were augmented by HYD in a dose-dependent manner. NTG alone did not affect myocyte shortening but reduced Δ[Ca(2+)]i across the range of pacing frequencies and increased myofilament Ca(2+) sensitivity thereby enhancing contractile efficiency. Similar results were seen in failing myocytes from the heart failure rat model. HYD alone or in combination with NTG reduced sarcoplasmic reticulum (SR) leak, improved SR Ca(2+) reuptake, and restored SR Ca(2+) content. HYD and NTG at low concentrations (1 μm), scavenged superoxide in isolated cardiomyocytes, whereas in cardiac homogenates, NTG inhibited xanthine oxidoreductase activity and scavenged NADPH oxidase-dependent superoxide more efficiently than HYD. Together, these results revealed that by reducing SR Ca(2+) leak, HYD improves Ca(2+) cycling and contractility impaired by nitroso-redox imbalance, and NTG enhanced contractile efficiency, restoring cardiac excitation-contraction coupling.

Nitrite induced-hypotension is enhanced in hypertension: Role for xanthine oxidoreductase

Within the healthy human circulation nitrite acts as a source of NO exerting important vasodilator and blood pressure (BP)-lowering effects. In vitro studies demonstrate enhanced nitrite-derived NO generation under pathological conditions, where conventional l -arginine-derived NO synthesis is impaired. Whether nitrite-induced hypotension and reduction to NO might be enhanced in hypertension (where conventional NO synthesis is impaired) is unknown. We compared the effects of nitrite on BP in vivo and vascular reactivity in vitro in Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR). Nitrite (1–30,000 nmol/kg) caused dose-dependent decrease in BP that was enhanced in SHR compared to WKY anaesthetised rats (max decrease of 32.0 ± 6.0 and 6.3 ± 1.9 mmHg respectively, P In sum, we show that nitrite-induced decreases in blood pressure are enhanced in hypertension and likely due to XOR-mediated reduction of nitrite to NO that does not occur at the level of the blood vessel wall.

Contribution of Xanthine Oxidoreductase to Mammary Epithelial and Breast Cancer Cell Differentiation In Part Modulates Inhibitor of Differentiation-1

Loss of xanthine oxidoreductase (XOR) has been linked to aggressive breast cancer in vivo and to breast cancer cell aggressiveness in vitro. In the present study, we hypothesized that the contribution of XOR to the development of the normal mammary gland may underlie its capacity to modulate breast cancer. We contrasted in vitro and in vivo developmental systems by differentiation marker and microarray analyses. Human breast cancer microarray was used for clinical outcome studies. The role of XOR in differentiation and proliferation was examined in human breast cancer cells and in a mouse xenograft model. Our data show that XOR was required for functional differentiation of mammary epithelial cells both in vitro and in vivo. Poor XOR expression was observed in a mouse ErbB2 breast cancer model, and pharmacologic inhibition of XOR increased breast cancer tumor burden in mouse xenograft. mRNA microarray analysis of human breast cancer revealed that low XOR expression was significantly associated with time to tumor relapse. The opposing expression of XOR and inhibitor of differentiation-1 (Id1) during HC11 differentiation and mammary gland development suggested a potential functional relationship. While overexpression of Id1 inhibited HC11 differentiation and XOR expression, XOR itself modulated expression of Id1 in differentiating HC11 cells. Overexpression of XOR both inhibited Id1-induced proliferation and -stimulated differentiation of Heregulin-β1-treated human breast cancer cells. These results show that XOR is an important functional component of differentiation whose diminished expression contributes to breast cancer aggressiveness, and they support XOR as both a breast cancer biomarker and a target for pharmacologic activation in therapeutic management of aggressive breast cancer.

When Does Low Oxygen Become Hypoxia? Implications for Nitrite Reduction

To the Editor: Webb and Milsom et al1 present the intriguing finding that endothelial nitric oxide synthase (eNOS) in human umbilical vein endothelial cells (HUVECs) and potentially RBCs is responsible under hypoxic conditions for nitrite (NO2•) reduction to nitric oxide (NO). Based on several practical and theoretical points of view, the conclusions that the authors draw regarding their data are flawed. It is important that these are considered by the wider Circulation Research readership. The authors conclude that lack of oxygen (O2) availability to eNOS promotes NO2• reduction to NO. Whereas the RBC experiments involve measurement of NO produced by RBC under nitrogen and acidic conditions, the HUVEC experiments were carried out at 5% O2 and 5% CO2 (refer to online data supplement that accompanies the article for details). Although there is no question 5% …

Role of Xanthine Oxidoreductase in the Reversal of Diastolic Heart Failure by Candesartan in the Salt-Sensitive Hypertensive Rat

The role of angiotensin II and reactive oxygen species in the exacerbation of diastolic heart failure is unknown. We examined the therapeutic effect of angiotensin blockade on hypertensive diastolic heart failure, focusing on the role of xanthine oxidoreductase and reduced nicotinamide-adenine dinucleotide phosphate oxidase, major enzymes producing reactive oxygen species. Dahl salt-sensitive hypertensive rats (DS rats) with established diastolic heart failure were given vehicle, candesartan (an angiotensin II receptor subtype 1 receptor blocker), oxypurinol (a xanthine oxidoreductase inhibitor), apocynin (a reduced nicotinamide-adenine dinucleotide phosphate oxidase inhibitor), or hydralazine (a vasodilator), and their therapeutic effects on diastolic heart failure were compared. Candesartan treatment of DS rats with established diastolic heart failure reversed cardiac remodeling, improved cardiac relaxation abnormality, and prolonged survival, being accompanied by the attenuation of the increase in cardiac superoxide, reduced nicotinamide-adenine dinucleotide phosphate oxidase, and xanthine oxidoreductase activities. Thus, the beneficial effect of candesartan in DS rats appears to be mediated by the inhibition of cardiac reactive oxygen species. Cardiac xanthine oxidoreductase inhibition with oxypurinol significantly reduced cardiac superoxide, prevented the progression of cardiac remodeling, and delayed the mortality in DS rats. Apocynin, which significantly inhibited cardiac reduced nicotinamide-adenine dinucleotide phosphate oxidase activity, prevented the exacerbation of diastolic heart failure more than hydralazine. However, compared with candesartan or oxypurinol, apocynin did not improve cardiac reactive oxygen species, remodeling, and function in DS rats. In conclusion, candesartan slowed the exacerbation of hypertensive diastolic heart failure in DS rats by causing reverse cardiac remodeling. Cardiac xanthine oxidoreductase contributed to these beneficial effects of candesartan.

Nitrite confers protection against myocardial infarction: Role of xanthine oxidoreductase, NADPH oxidase and K ATP channels

Reduction of nitrite to nitric oxide during ischemia protects the heart against injury from ischemia/reperfusion. However the optimal dose of nitrite and the mechanisms underlying nitrite-induced cardioprotection are not known. We determined the ability of nitrite and nitrate to confer protection against myocardial infarction in two rat models of ischemia/reperfusion injury and the role of xanthine oxidoreductase, NADPH oxidase, nitric oxide synthase and K ATP channels in mediating nitrite-induced cardioprotection. In vivo and in vitro rat models of myocardial ischemia/reperfusion injury were used to cause infarction. Hearts ( n = 6/group) were treated with nitrite or nitrate for 15 min prior to 30 min regional ischemia and 180 min reperfusion. Xanthine oxidoreductase activity was measured after 15 min aerobic perfusion and 30 min ischemia. Nitrite reduced myocardial necrosis and decline in ventricular function following ischemia/reperfusion in the intact and isolated rat heart in a dose- or concentration-dependent manner with an optimal dose of 4 mg/kg in vivo and concentration of 10 μM in vitro. Nitrate had no effect on protection. Reduction in infarction by nitrite was abolished by the inhibition of flavoprotein reductases and the molybdenum site of xanthine oxidoreductase and was associated with an increase in activity of xanthine dehydrogenase and xanthine oxidase during ischemia. Inhibition of nitric oxide synthase had no effect on nitrite-induced cardioprotection. Inhibition of NADPH oxidase and K ATP channels abolished nitrite-induced cardioprotection. Nitrite but not nitrate protects against infarction by a mechanism involving xanthine oxidoreductase, NADPH oxidase and K ATP channels.

Role of xanthine oxidoreductase as an antimicrobial agent.

Xanthine oxidoreductase (XOR) is widely distributed in mammalian tissues and has long been known to be a major constituent of the milk fat globule membrane (MFGM), which surrounds fat globules in cow's milk ([36][1], [50][2]). This source has been exploited for isolation and characterization of the

Xanthine oxidoreductase and cardiovascular disease: molecular mechanisms and pathophysiological implications.

There is substantial evidence that oxidative stress participates in the pathophysiology of cardiovascular disease. Biochemical, molecular and pharmacological studies further implicate xanthine oxidoreductase (XOR) as a source of reactive oxygen species in the cardiovascular system. XOR is a member of the molybdoenzyme family and is best known for its catalytic role in purine degradation, metabolizing hypoxanthine and xanthine to uric acid with concomitant generation of superoxide. Gene expression of XOR is regulated by oxygen tension, cytokines and glucocorticoids. XOR requires molybdopterin, iron-sulphur centres, and FAD as cofactors and has two interconvertible forms, xanthine oxidase and xanthine dehydrogenase, which transfer electrons from xanthine to oxygen and NAD(+), respectively, yielding superoxide, hydrogen peroxide and NADH. Additionally, XOR can generate superoxide via NADH oxidase activity and can produce nitric oxide via nitrate and nitrite reductase activities. While a role for XOR beyond purine metabolism was first suggested in ischaemia-reperfusion injury, there is growing awareness that it also participates in endothelial dysfunction, hypertension and heart failure. Importantly, the XOR inhibitors allopurinol and oxypurinol attenuate dysfunction caused by XOR in these disease states. Attention to the broader range of XOR bioactivity in the cardiovascular system has prompted initiation of several randomised clinical outcome trials, particularly for congestive heart failure. Here we review XOR gene structure and regulation, protein structure, enzymology, tissue distribution and pathophysiological role in cardiovascular disease with an emphasis on heart failure.

Role of xanthine oxidoreductase and NAD(P)H oxidase in endothelial superoxide production in response to oscillatory shear stress.

Oscillatory shear stress occurs at sites of the circulation that are vulnerable to atherosclerosis. Because oxidative stress contributes to atherosclerosis, we sought to determine whether oscillatory shear stress increases endothelial production of reactive oxygen species and to define the enzymes responsible for this phenomenon. Bovine aortic endothelial cells were exposed to static, laminar (15 dyn/cm2), and oscillatory shear stress (+/-15 dyn/cm2). Oscillatory shear increased superoxide (O2.-) production by more than threefold over static and laminar conditions as detected using electron spin resonance (ESR). This increase in O2*- was inhibited by oxypurinol and culture of endothelial cells with tungsten but not by inhibitors of other enzymatic sources. Oxypurinol also prevented H2O2 production in response to oscillatory shear stress as measured by dichlorofluorescin diacetate and Amplex Red fluorescence. Xanthine-dependent O2*- production was increased in homogenates of endothelial cells exposed to oscillatory shear stress. This was associated with decreased xanthine dehydrogenase (XDH) protein levels and enzymatic activity resulting in an elevated ratio of xanthine oxidase (XO) to XDH. We also studied endothelial cells lacking the p47phox subunit of the NAD(P)H oxidase. These cells exhibited dramatically depressed O2*- production and had minimal XO protein and activity. Transfection of these cells with p47phox restored XO protein levels. Finally, in bovine aortic endothelial cells, prolonged inhibition of the NAD(P)H oxidase with apocynin decreased XO protein levels and prevented endothelial cell stimulation of O2*- production in response to oscillatory shear stress. These data suggest that the NAD(P)H oxidase maintains endothelial cell XO levels and that XO is responsible for increased reactive oxygen species production in response to oscillatory shear stress.

A new route to peroxynitrite: a role for xanthine oxidoreductase

Peroxynitrite, a potent oxidising, nitrating and hydroxylating agent, results from the reaction of nitric oxide with superoxide. We show that peroxynitrite can be produced by the action of a single enzyme, xanthine oxidoreductase (XOR), in the presence of inorganic nitrite, molecular oxygen and a reducing agent, such as pterin. The effects of oxygen concentration on peroxynitrite production have been examined. The physiologically predominant dehydrogenase form of the enzyme is more effective than the oxidase form under aerobic conditions. It is proposed that XOR-derived peroxynitrite fulfils a bactericidal role in milk and in the digestive tract.