A sensitive fluorometric assay for measuring xanthine dehydrogenase and oxidase in tissues

Beckman, Joseph S.; Parks, Dale A.; Pearson, J.; Marshall, Patricia A.; Freeman, Bruce Α.

doi:10.1016/0891-5849(89)90068-3

Cited by 222 publications

(127 citation statements)

References 16 publications

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“…This NO component was suppressed by the XOR inhibitor allopurinol by approximately 71%, suggesting that the conversion of NO from nitrite in the kidney homogenates was due mainly to the enzymatic activity of XOR, as also has been reported to be the case in both rat and human heart (2). In terms of absolute nitrite reductase activity, it is worth noting that the in vitro experiments were conducted at standard laboratory conditions (25°C) and the activity of XOR is approximately 2.5-fold lower at this temperature compared with 37°C, at least with respect to xanthine oxidation (38). This demonstration of renal nitrite reductase activity confirms the findings of Okamoto et al (15), who used electron paramagnetic resonance spectroscopy.…”

Section: Discussionsupporting

confidence: 75%

Nitrite-Derived Nitric Oxide Protects the Rat Kidney against Ischemia/Reperfusion Injury In Vivo

Tripatara¹,

Patel²,

Webb³

et al. 2007

Journal of the American Society of Nephrology

212

159

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In normal conditions, nitric oxide (NO) is oxidized to the anion nitrite, but in hypoxia, this nitrite may be reduced back to NO by the nitrite reductase action of deoxygenated hemoglobin, acidic disproportionation, or xanthine oxidoreductase (XOR). Herein, is investigated the effects of topical sodium nitrite administration in a rat model of renal ischemia/reperfusion (I/R) injury. Rats were subjected to 60 min of bilateral renal ischemia and 6 h of reperfusion in the absence or presence of sodium nitrite (30 nmol) administered topically 1 min before reperfusion. Serum creatinine, serum aspartate aminotransferase, creatinine clearance, fractional excretion of Na ؉ , and plasma nitrite/nitrate concentrations were measured. The nitrite-derived NO-generating capacity of renal tissue was determined under acidic and hypoxic conditions by ozone chemiluminescence in homogenates of kidneys that were subjected to sham, ischemia-only, and I/R conditions. Nitrite significantly attenuated renal dysfunction and injury, an effect that was abolished by previous treatment of rats with the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazole-1-oxyl-3-oxide (2.5 mol intravenously 5 min before ischemia and 50 nmol topically 6 min before reperfusion). Renal tissue homogenates produced significant amounts of NO from nitrite, an effect that was attenuated significantly by the xanthine oxidoreductase inhibitor allopurinol. Taken together, these findings demonstrate that topically administered sodium nitrite protects the rat kidney against I/R injury and dysfunction in vivo via the generation, in part, of xanthine oxidoreductase-catalyzed NO production. These observations suggest that nitrite therapy might prove beneficial in protecting kidney function and integrity during periods of I/R such as those encountered in renal transplantation. U ntil recently, the nitrite anion was regarded merely as an inactive metabolite of nitric oxide (NO) oxidation produced under normal physiologic conditions, accounting for approximately 70% of the endogenous nitrite pool (1). However, we (2) and others (3) recently demonstrated that, far from being inactive, nitrite has marked protective effects in ischemia/reperfusion (I/R) injury of both the heart and the liver. Moreover, these studies demonstrate that the beneficial effects of nitrite are related to its reduction to NO, under ischemic conditions. J Am Soc NephrolThe generation of NO is attributed conventionally to the enzyme NO synthase (NOS), of which there are three isoforms. Endothelial NOS (eNOS)-derived NO plays an important role in determining and maintaining aspects of normal renal function, for instance proximal tubule sodium reabsorption (4,5), but in elevated concentrations, NO also contributes to renal pathophysiology (6), such as in proximal tubule ischemic injury (4). This dual nature of NO perhaps is an oversimplification, because NO either ameliorates or exacerbates renal injury, depending on the site and the rate of NO production and the chemical fate of NO (7,8). Further...

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Section: Discussionsupporting

confidence: 75%

Nitrite-Derived Nitric Oxide Protects the Rat Kidney against Ischemia/Reperfusion Injury In Vivo

Tripatara¹,

Patel²,

Webb³

et al. 2007

Journal of the American Society of Nephrology

212

159

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“…The XDH/XO activity was measured using a fluorimetric assay as described (Kayyali et al, 2001(Kayyali et al, , 2003Beckman et al, 1989). Cells were collected and sonicated in 50 mM sodium phosphate (pH 7.4), 1.5 mg/ml DTT and 1 × protease inhibitor mixture 3 (Calbiochem).…”

Section: Measurement Of Xdh/xo Activitymentioning

confidence: 99%

Sequential activation of JAKs, STATs and xanthine dehydrogenase/oxidase by hypoxia in lung microvascular endothelial cells

Wang

Pei

Jackson

et al. 2008

The International Journal of Biochemistry & Cell Biology

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Xanthine dehydrogenase/oxidase (XDH/XO) is associated with various pathological conditions related to the endothelial injury. However, the molecular mechanism underlying the activation of XDH/XO by hypoxia remains largely unknown. In this report, we determined whether the Janus kinases (JAKs) and signal transducers and activators of transcription (STATs) signaling pathway is involved in hypoxia-induced activation of XDH/XO in primary cultures of lung microvascular endothelial cells (LMVEC). We found that hypoxia significantly increased interleukin 6 (IL6) production in a time-dependent manner in LMVEC. Hypoxia also markedly augmented phosphorylation/activation of JAKs (JAK1, JAK2 and JAK3) and the JAK downstream effectors STATs (STAT3 and STAT5). Hypoxia-induced activation of STAT3 was blocked by IL6 antibodies, the JAK inhibitor AG490 and the suppressor of cytokine signaling 3 (SOCS3), implying that hypoxiapromoted IL6 secretion activates the JAK/STAT pathway in LMVEC. Phosphorylation and DNAbinding activity of STAT3 was also inhibited by the p38 MAPK inhibitor SB203580 and the phosphatidylinositol 3-kinase inhibitor LY294002, suggesting that multiple signaling pathways involved in STAT activation by hypoxia. Importantly, hypoxia promoted XDH/XO activation in LMVEC, which was markedly reversed by inhibiting the JAK/STAT pathway using IL6 antibodies, AG490 and SOCS3. These data demonstrated that JAKs, STATs and XDH/XO were sequentially activated by hypoxia. These data also provide the first evidence indicating that the JAK-STAT pathway is involved in hypoxia-mediated XDH/XO activation in LMVEC.

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“…Ice-cold homogenization buffer (0.25 M sucrose, 10 mM DTT, 0.2 mM PMSE, 0.1 mM EDTA, and 50 mM K + -phosphate, pH 7.4) was added to the powdered tissue (200mg/ml) and homogenized in a Wheaton 903475 homogenizer. The homogenate was centrifuged at a of speed 40,000×g at 4 °C for 30 min and xanthine dehydrogenase and xanthine oxidase activities were measured in the supernatant according to the method of Beckman et al [14]. Data (means ± SE) are shown as mIUs of xanthine oxidase/ xanthine dehydrogenase per gram protein.…”

Section: Xanthine Oxidoreductase Studies/analysismentioning

confidence: 99%

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

Baker

et al. 2007

Journal of Molecular and Cellular Cardiology

120

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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 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.

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A sensitive fluorometric assay for measuring xanthine dehydrogenase and oxidase in tissues

Cited by 222 publications

References 16 publications

Nitrite-Derived Nitric Oxide Protects the Rat Kidney against Ischemia/Reperfusion Injury In Vivo

Nitrite-Derived Nitric Oxide Protects the Rat Kidney against Ischemia/Reperfusion Injury In Vivo

Sequential activation of JAKs, STATs and xanthine dehydrogenase/oxidase by hypoxia in lung microvascular endothelial cells

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

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