Reduction of organic nitrates catalysed by xanthine oxidoreductase under anaerobic conditions

Doel, Justin J.; Godber, Ben; Eisenthal, Robert; Harrison, Roger

doi:10.1016/s0304-4165(01)00148-9

Cited by 47 publications

(42 citation statements)

References 40 publications

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“…Besides microsomal CPR, cytosolic xanthine oxidoreductase (46,47) and mitochondrial aldehyde dehydrogenase (48) have also been reported to be able to catalyze GTN or ISDN reduction to produce nitrite. Data obtained in whole tissue, isolated mitochondria, cytosolic fractions, and microsomes have shown that clinically used GTN levels cannot produce sufficient NO 2 Ϫ as an intermediate of NO to exhibit vasodilatory effects (49 -53).…”

Section: Discussionmentioning

confidence: 99%

Characterization of the Mechanism of Cytochrome P450 Reductase-Cytochrome P450-mediated Nitric Oxide and Nitrosothiol Generation from Organic Nitrates

Cui

et al. 2006

Journal of Biological Chemistry

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Mammalian cytochrome P450 reductase (CPR) and cytochrome P450 (CP) play important roles in organic nitrate bioactivation; however, the mechanism by which they convert organic nitrate to NO remains unknown. Questions remain regarding the initial precursor of NO that serves to link organic nitrate to the activation of soluble guanylyl cyclase (sGC). To characterize the mechanism of CPR-CP-mediated organic nitrate bioactivation, EPR, chemiluminescence NO analyzer, NO electrode, and immunoassay studies were performed. With rat hepatic microsomes or purified CPR, the presence of NADPH triggered organic nitrate reduction to NO 2 ؊ .The CPR flavin site inhibitor diphenyleneiodonium inhibited this NO 2 ؊ generation, whereas the CP inhibitor clotrimazole did not. Further reaction of organic nitrite with free or microsome-associated thiols led to NO or nitrosothiol generation and thus stimulated the activation of sGC. Thus, organic nitrite is the initial product in the process of CRP-CP-mediated organic nitrate activation and is the precursor of NO and nitrosothiols, serving as the link between organic nitrate and sGC activation.Organic nitrates (R-O-NO 2 ) such as glyceryl trinitrate (GTN) 3 and isosorbide dinitrate (ISDN) have an important role in cardiovascular therapy. Over the last decade, this role has been attributed to their enzymatic metabolism to produce nitric oxide (NO) (1-4). However, the mechanism of organic nitrate bioactivation to produce NO has not been elucidated. Numerous reports have shown that the mammalian cytochrome P450 reductase (CPR)-cytochrome P450 (CP) enzyme system plays an important role in the process of organic nitrate biotransformation (5-10). However, questions remain regarding the enzymatic mechanism by which CPR and CP transform organic nitrate to NO. The chemical process of organic nitrate conversion to NO and nitrosothiols that links organic nitrates to the activation of soluble guanylyl cyclase (sGC) remains unknown.Mammalian CPR was first identified in 1950 (11). CPR is a flavoprotein containing both FAD and FMN (11,12) and is situated on the endoplasmic reticulum (microsomes) (13, 14). Previous studies have shown that CPR plays important roles in organic nitrate biotransformation. The flavoprotein inhibitor diphenyleneiodonium (DPI) greatly inhibits the metabolic activation of GTN or ISDN through inhibition of CPR (8, 9, 15). However, the enzymatic mechanism of CPR-mediated organic nitrate reduction has not been elucidated. To date, no research has been done to assess NO formation in the process of CPR-mediated organic nitrate biotransformation. It is not certain whether CPR can directly catalyze the reduction of organic nitrate to produce NO or to produce nitrite anion (NO 2 Ϫ ) or other species as intermediates that may be further transformed to NO or S-nitrosothiols. Therefore, the enzymatic mechanism of NO and S-nitrosothiol generation remains unclear in the process of organic nitrate bioactivation. CPR is the electron donor for several oxygenase enzymes, including CP, a family...

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Section: Discussionmentioning

confidence: 99%

Characterization of the Mechanism of Cytochrome P450 Reductase-Cytochrome P450-mediated Nitric Oxide and Nitrosothiol Generation from Organic Nitrates

Cui

et al. 2006

Journal of Biological Chemistry

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“…Alternative physiological sources of NO synthase-independent NO include endogenous nitrite and nitrate (39). It has been reported that xanthine oxidase can catalyze the reduction of nitrite and/or nitrate to NO under hypoxic (35,41,60) and anoxic (9,33,34) conditions. We have found that nitrite and nitrate are present in ovine fetal pulmonary veins.…”

Section: Effect Of 30 Minutes Of Hypoxia On Downregulation Of Pkg Kinmentioning

confidence: 99%

Regulation of cGMP-dependent protein kinase-mediated vasodilation by hypoxia-induced reactive species in ovine fetal pulmonary veins

Negash

Gao

Zhou

et al. 2007

American Journal of Physiology-Lung Cellular and Molecular Phys

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Regulation of cGMP-dependent protein kinase-mediated vasodilation by hypoxia-induced reactive species in ovine fetal pulmonary veins. Am J Physiol Lung Cell Mol Physiol 293: L1012-L1020, 2007. First published July 6, 2007 doi:10.1152/ajplung.00061.2007.-We previously reported that hypoxia attenuates cGMP-dependent protein kinase (PKG)-mediated relaxation in pulmonary vessels (Am J Physiol Lung Cell Mol Physiol 279: L611-L618, 2003). To determine whether hypoxia-induced reactive oxygen and nitrogen species (ROS and RNS, respectively) may be involved in the downregulation of PKGmediated relaxation, ovine fetal intrapulmonary veins were exposed to 4 h of normoxia or hypoxia, with or without scavengers of ROS [N-acetylcysteine (NAC)] or peroxynitrite (quercetin and Trolox) and preconstricted with endothelin-1. Hypoxia decreased the relaxation response to 8-bromo-cGMP, PKG protein expression, and kinase activity and increased tyrosine nitration in PKG. However, ROS and RNS scavengers prevented these changes. To determine whether increased PKG nitration diminishes PKG activity, pulmonary vein smooth muscle cells (PVSMC) were exposed to shorter-term (30 min) hypoxia, which increased PKG nitration and decreased PKG activity but did not alter PKG protein expression. Increased dihydro-2,7-dichlorofluorescein diacetate (DCFH 2-DA) fluorescence in PVSMC after 4 h or 30 min of hypoxia was not observed in the presence of NAC, quercetin, or Trolox, suggesting increased ROS and RNS production. Increased PKG nitration and the associated decrease in PKG activity in PVSMC after 30 min of hypoxia were also reversed on reoxygenation. The consequences of PKG nitration were assessed by exposure of purified PKG-I␣ to peroxynitrite, which caused increased 3-nitrotyrosine immunoreactivity and inhibition of kinase activity. Our data suggest that, after 30 min of hypoxia, reversible covalent modification of PKG by hypoxia-induced reactive species may be an important mechanism by which the relaxation response to cGMP is regulated. However, after 4 h of hypoxia, PKG nitration and decreased PKG expression are involved. hypoxic vasoconstriction; vascular smooth muscle; protein kinase G GUANOSINE 3Ј,5Ј-cyclic monophosphate (cGMP)-dependent protein kinase (PKG) is a serine/threonine kinase that plays an important role in the regulation of vascular smooth muscle (SMC) contractility (6,22,36). Activation of soluble guanylyl cyclase by nitric oxide (NO) leads to increased synthesis of the second messenger cGMP and stimulation of PKG (2, 27). PKG, in turn, mediates SMC relaxation by a number of mechanisms, including phosphorylation of myosin light chain phosphatase, lowering of intracellular free Ca 2ϩ levels, and desensitization of the contractile apparatus to Ca 2ϩ (6,22,36). Mammalian PKG exists in two major forms: PKG-I, a soluble enzyme consisting of ␣-and ␤-isoforms derived from alternative splicing from one gene, and PKG-II, a myristoylated, membrane-associated form derived from a second gene (23,36,46). PKG-I␣ is the primary isoform involv...

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“…Considering the cytosolic enzymes, XO has recently been reported to catalyze the anaerobic reduction of GTN to NO 2 Ϫ and then to NO (Millar et al, 1998;Doel et al, 2001). However, incubations of NOBA or GTN in liver cytosol in the presence of an excess of a selective inhibitor of XO did not change the metabolism of these compounds (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Although nonenzymatic pathways involving endogenous sulfhydryl groups (Ignarro et al, 1980) or hemoglobin (Bennett et al, 1986;Cosby et al, 2003) have been suggested to mediate the biotransformation to NO, the attention has also shifted to an enzyme-catalyzed mechanism (Needleman, 1976). Several enzymes have been proposed to be directly or indirectly involved in the generation of NO from organic nitrates, as the cytosolic glutathione S-transferase (GST) (Keen et al, 1976;Taylor et al, 1989;Kurz et al, 1993) and xanthine oxidoreductase (XO) (Millar et al, 1998;Doel et al, 2001), the mitochondrial aldehyde dehydrogenase (Chen et al, 2002;DiFabio et al, 2003;Kollau et al, 2005) and cytochrome bc 1 (Nohl et al, 2000), or the microsomal cytochrome P-450 (P450) (Servent et al, 1989;McDonald and Bennett, 1990;Schroder, 1992). Most likely this mechanism is not identical in all tissues or biological mediums, and a cooper-ation between different enzymes and intracellular compartments is likely to occur (Kozlov et al, 2003).…”

mentioning

confidence: 99%

In Vitro Metabolism of (Nitrooxy)butyl Ester Nitric Oxide-Releasing Compounds: Comparison with Glyceryl Trinitrate

Govoni

Casagrande²,

Maucci³

et al. 2006

J Pharmacol Exp Ther

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We investigated the in vitro metabolism of two (nitrooxy)butyl ester nitric oxide (NO) donor derivatives of flurbiprofen and ferulic acid, [1,1Ј-biphenyl]-4-acetic acid-2-fluoro-␣-methyl-4-(nitrooxy)butyl ester (HCT 1026) and 3-(4-hydroxy-3-methoxyphenyl)-2-propenoic acid 4-(nitrooxy)butyl ester (NCX 2057), respectively, in rat blood plasma and liver subcellular fractions compared with (nitrooxy)butyl alcohol (NOBA) and glyceryl trinitrate (GTN). HCT 1026 and NCX 2057 undergo rapid ubiquitous carboxyl ester hydrolysis to their respective parent compounds and NOBA. The nitrate moiety of this latter is subsequently metabolized to inorganic nitrogen oxides (NOx), predominantly in liver cytosol by glutathione S-transferase (GST) and to a lesser extent in liver mitochondria. If, however, in liver cytosol, the carboxyl ester hydrolysis is prevented by an esterase inhibitor, the metabolism at the nitrate moiety level does not occur.In blood plasma, HCT 1026 and NCX 2057 are not metabolized to NOx, whereas a slow but sustained NO generation in deoxygenated whole blood as detected by electron paramagnetic resonance indicates the involvement of erythrocytes in the bioactivation of these compounds. Differently from NOBA, GTN is also metabolized in blood plasma and more quickly metabolized by different GST isoforms in liver cytosol. The cytosolic GST-mediated denitration of these organic nitrates in liver limits their interaction with other intracellular compartments to possible generation of NO and/or their subsequent availability and bioactivation in the systemic circulation and extrahepatic tissues. We show the possibility of modulating the activity of hepatic cytosolic enzymes involved in the metabolism of (nitrooxy)butyl ester compounds, thus increasing the therapeutic potential of this class of compounds.The therapeutic potential of organic nitrates has been known for more than 120 years since the use of glyceryl trinitrate (GTN) in the treatment of angina pectoris. Moreover, the pharmaceutical development of organic nitrates containing adjunct pharmacophores was reported over 40 years ago, and these were observed to manifest biological properties beyond those of the parent compound (Hodosan et al., 1969). However, there has been an explosion of activity in the area of hybrid nitrates over the past decade, stimulated by a growing realization that nitrates may represent new therapeutic agents in different areas (Keeble and Moore, 2002). Despite this, the metabolism of several organic nitrates is still to be elucidated. The exact mechanism whereby nitric oxide (NO) is generated from organic nitrates is still unknown, and several mechanisms have been proposed and rejected. Although nonenzymatic pathways involving endogenous sulfhydryl groups (Ignarro et al., 1980) or hemoglobin (Bennett et al., 1986;Cosby et al., 2003) have been suggested to mediate the biotransformation to NO, the attention has also shifted to an enzyme-catalyzed mechanism (Needleman, 1976). Several enzymes have been proposed to be directly or i...

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Reduction of organic nitrates catalysed by xanthine oxidoreductase under anaerobic conditions

Cited by 47 publications

References 40 publications

Characterization of the Mechanism of Cytochrome P450 Reductase-Cytochrome P450-mediated Nitric Oxide and Nitrosothiol Generation from Organic Nitrates

Characterization of the Mechanism of Cytochrome P450 Reductase-Cytochrome P450-mediated Nitric Oxide and Nitrosothiol Generation from Organic Nitrates

Regulation of cGMP-dependent protein kinase-mediated vasodilation by hypoxia-induced reactive species in ovine fetal pulmonary veins

In Vitro Metabolism of (Nitrooxy)butyl Ester Nitric Oxide-Releasing Compounds: Comparison with Glyceryl Trinitrate

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