Effects of reactive oxygen and nitrogen species on cyclooxygenase-1 and -2 activities

The effects of reactive oxygen species (superoxide anion radical--O(2)*-, hydrogen peroxide--H(2)O(2) and hydroxyl radical--*OH; the reaction products of xanthine plus xanthine oxidase system) and reactive nitrogen species [nitric oxide--NO*; from 1-hydroxyl-2-oxo-3-(N-methyl-3-aminopropyl)-3-methyl-1-triazene--NOC7 and peroxynitrite--ONOO(-)] on the activities of purified cyclooxygenase (COX)-1 and -2 were studied. Xanthine plus xanthine oxidase suppressed the COX-1 and -2 activities in a xanthine oxidase concentration-dependent fashion. This effect was reversed by addition of catalase to the reactive oxygen species-generating system but not by superoxide dismutase or mannitol, indicating that H(2)O(2) is the responsible metabolite. NOC7 activated the COX-1 activity but inhibited the COX-2 activity at concentrations ranging from 1 to 50 microM. Experiments utilizing a NO* antidote, carboxy-2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide revealed that the observed effects of NOC7 are caused by NO*.ONOO(-), a product of NO* and O(2)*-, both activated and inhibited the COX-1 and -2 activities, depending on ONOO(-) concentration. At a low concentration of ONOO(-) (5 microM) there was enhancement of the COX-1 and -2 activities, but with higher concentrations there was suppression of these two enzyme activities (COX-1, at 200 microM; COX-2, >50 microM). These results suggest that H(2)O(2), NO* and ONOO(-) can have different modulatory effects on the COX-1 and -2 activities.     

Preconditioning with millimolar concentrations of vitamin C or N-acetylcysteine protects L6 muscle cells insulin-stimulated viability and DNA synthesis under oxidative stress

The effect of reactive oxygen/nitrogen species (ROS/RNS)(hydrogen peroxide -- H(2)O(2), superoxide anion radical O(2)*- and hydroxyl radical *OH -- the reaction products of hypoxanthine/xanthine oxidase system), nitric oxide (NO* from sodium nitroprusside -- SNP), and peroxynitrite (ONOO(-) from 3-morpholinosydnonimine -- SIN-1) on insulin mitogenic effect was studied in L6 muscle cells after one day pretreatment with/or without antioxidants. ROS/RNS inhibited insulin-induced mitogenicity (DNA synthesis). Insulin (0.1 microM), however, markedly improved mitogenicity in the muscle cells treated with increased concentrations (0.1, 0.5, 1 mM) of donors of H(2)O(2), O(2)*-, *OH, ONOO(-) and NO*. Cell viability assessed by morphological criteria was also monitored. Massive apoptosis was induced by 1 mM of donors of H(2)O(2) and ONOO(-), while NO* additionally induced necrotic cell death. Taken together, these results have shown that ROS/RNS provide a good explanation for the developing resistance to the growth promoting activity of insulin in myoblasts under conditions of oxidative or nitrosative stress. Cell viability showed that neither donor induced cell death when given below 0.5 mM. In order to confirm the deleterious effects of ROS/RNS prior to the subsequent treatment with ROS/RNS plus insulin one day pretreatment with selected antioxidants (sodium ascorbate - ASC (0.01, 0.1, 1 mM), or N-acetylcysteine - NAC (0.1, 1, 10 mM) was carried out. Surprisingly, at a low dose (micromolar) antioxidants did not abrogate and even worsened the concentration-dependent effects of ROS/RNS. In contrast, pretreatment with millimolar dose of ASC or NAC maintained an elevated mitogenicity in response to insulin irrespective of the ROS/RNS donor type used.     

Early release of arachidonic acid prevents an otherwise immediate formation of toxic levels of peroxynitrite in astrocytes stimulated with lipopolysaccharide/interferon-gamma

Addition of bacterial lipopolysaccharides (LPS) and interferon-gamma (IFN-gamma) to rat astrocytes in primary culture promotes an early release of arachidonic acid (ARA) associated with an immediate inhibition of neuronal nitric oxide synthase (nNOS). Preventing the release of constitutive nitric oxide (NO) is indeed critical for activation of the nuclear factor kappa B, and for the expression of inducible nitric oxide synthase responsible for the formation of large amounts of NO. LPS/IFN-gamma also promotes an early release of superoxide, via activation of NADPH oxidase, but the generation of peroxynitrite (ONOO-) is prevented by the different timing of superoxide (minutes) and NO (hours) formation. Upstream inhibition of the ARA-dependent nNOS inhibitory signaling, however, caused the parallel release of superoxide and constitutive NO, thereby leading to formation of ONOO- levels triggering loss of ATP and mitochondrial membrane potential followed by the mitochondrial release of cytochrome c, activation of caspase 3 and morphological evidence of apoptosis. Nanomolar levels of exogenous ARA prevented all these events via inhibition of early ONOO- formation. Thus, the ARA-dependent nNOS inhibition observed in astrocytes exposed to pro-inflammatory stimuli, as LPS/IFN-gamma, is critical for both the expression of nuclear factor kappa B-dependent genes and for survival.     

Pindolol is a potent scavenger of reactive nitrogen species

Pindolol is an indolic drug that has been shown to enhance and/or accelerate selective serotonin specific reuptake inhibitors (SSRI)-induced antidepressant (AD) effect, even though the respective mechanism is still unclear. It has been demonstrated that inhibition of nitric oxide (*NO) synthesis in CNS produces anxiolytic and AD-like behavioural effects in a variety of animal paradigms. On the other hand, sustained high levels of *NO may be deleterious to CNS, predominantly due to the formation of peroxynitrite anion (ONOO-), which is generated via reaction of *NO with superoxide radical (O2*-). Therefore, the purpose of the present study was to characterize the putative pindolol scavenging effect on *NO, ONOO-, and O2*-, using in vitro non-cellular systems. The obtained results clearly show that pindolol is a potent scavenger of *NO (IC50 of 449+/-33 microM) and ONOO- (IC50 of 131+/-24 microM). Additionally, the scavenging effect of pindolol increased almost 8 times in the presence of 25 mM NaHCO3 (IC50 of 17+/-3 microM), which indicates that pindolol efficiently scavenges reactive species that are produced from the ONOO-/CO2 reaction such as the nitrogen dioxide radical (*NO2) and the carbonate radical anion (CO3*-). These effects may contribute for the reduction of SSRI antidepressant latency that has been attributed to pindolol and may also constitute an additional value for this drug when depression is associated with pro-oxidant neurodegenerative diseases.     

3-Morpholinosyndnonimine inhibits 5-hydroxytryptamine-induced phosphorylation of nitric oxide synthase in endothelial cells.

5-Hydroxytryptamine (5-HT) is a vasoactive substance that is taken up by endothelial cells to activate endothelial nitrite oxide synthase (eNOS). The activation of eNOS results in the production of nitric oxide (NO), which is responsible for vasodilation of blood vessels. NO also interacts with superoxide anion (O2*-) to form peroxynitrite (ONOO-), a potent oxidant that has been shown to induce vascular endothelial dysfunction. We examined the ability of 3-morpholinosyndnonimine (SIN-1), an ONOO- generator, to inhibit 5-HT-induced phosphorylation of eNOS in cultured bovine aortic endothelial cells (BAECs). We observed that 5-HT phosphorylates Ser1179 eNOS in a time- and concentration-dependent manner. Maximum phosphorylation occurred at 30 sec using a concentration of 1.0 microM 5-HT. BAECs treated with SIN-1 (1-1000 microM) for 30 min showed no significant increase in eNOS phosphorylation. However, 5-HT-induced eNOS phosphorylation was inhibited in cells treated with various concentrations of SIN-1 for 30 min and stimulated with 5-HT. These data suggest that an increase in ONOO- as a result of an increase in the production of O2*-, may feedback to inhibit 5-HT-induced eNOS phosphorylation at Ser1179 and therefore, contribute to endothelial dysfunction associated with cardiovascular diseases.     

Superoxide dismutase and hydrogen peroxide cause rapid nitric oxide breakdown, peroxynitrite production and subsequent cell death.

Isolated copper/zinc superoxide dismutase (Cu/Zn-SOD) or manganese superoxide dismutase (Mn-SOD) together with hydrogen peroxide (H(2)O(2)) caused rapid breakdown of nitric oxide (NO) and production of peroxynitrite (ONOO(-)) indicated by the oxidation of dihydrorhodamine-1,2,3 (DHR) to rhodamine-1,2,3. The breakdown of NO by this reaction was inhibited by cyanide (CN(-)) or by diethyldithiocarbamate (DETC), both Cu/Zn-SOD inhibitors, and the conversion of DHR to rhodamine-1,2,3 was inhibited by incubating Cu/Zn-SOD with either CN(-) or with high levels of H(2)O(2) or by including urate, a potent scavenger of ONOO(-). In the presence of phenol, the reaction of SOD, H(2)O(2) and NO caused nitration of phenol, which is known to be a footprint of ONOO(-) formation. H(2)O(2) addition to macrophages (cell line J774) expressing the inducible form of NO synthase (i-NOS) caused rapid breakdown of the NO they produced and this was also inhibited by CN(-) and by DETC. Subsequent ONOO(-) production by the macrophages, via this reaction, was inhibited by CN(-), high levels of H(2)O(2) or by urate. H(2)O(2) addition to i-NOS macrophages also caused cell death which was, in part, prevented by DETC or urate. We also found inhibition of mitochondrial respiration with malate and pyruvate as substrates, when isolated liver mitochondria were incubated with Cu/Zn-SOD, H(2)O(2) and NO. Inhibition of mitochondrial respiration was partly prevented by urate. The production of ONOO(-) by SOD may be of significant importance pathologically under conditions of elevated H(2)O(2) and NO levels, and might contribute to cell death in inflammatory and neurodegenerative diseases, as well as in macrophage-mediated host defence.     

Synergistic depletion of astrocytic glutathione by glucose deprivation and peroxynitrite: correlation with mitochondrial dysfunction and subsequent cell death

Previously we reported that immunostimulated astrocytes were highly vulnerable to glucose deprivation. The augmented death was mimicked by the peroxynitrite (ONOO )-producing reagent 3-morpholinosydnonimine (SIN-1). Here we show that glucose deprivation and ONOO- synergistically deplete intracellular reduced glutathione (GSH) and augment the death of astrocytes via formation of cyclosporin A-sensitive mitochondrial permeability transition (MPT) pore. Astrocytic GSH levels were only slightly decreased by glucose deprivation or SIN-1 (200 microM) alone. In contrast, a rapid and large depletion of GSH was observed in glucose-deprived/ SIN-1-treated astrocytes. The depletion of GSH occurred before a significant release of lactate dehydrogenase (a marker of cell death). Superoxide dismutase and ONOO-scavengers completely blocked the augmented death, indicating that the reaction of nitric oxide with superoxide to form ONOO was implicated. Furthermore, nitrotyrosine immunoreactivity (a marker of ONOO-) was markedly enhanced in glucose-deprived/SIN-1 -treated astrocytes. Mitochondrial transmembrane potential (MTP) was synergistically decreased in glucose-deprived/SIN-1-treated astrocytes. The glutathione synthase inhibitor L-buthionine-(S,R)-sulfoximine markedly decreased the MTP and increased lactate dehydrogenase (LDH) releases in SIN-1-treated astrocytes. Cyclosporin A, an MPT pore blocker, completely prevented the MTP depolarization as well as the enhanced LDH releases in glucose-deprived/SIN-1-treated astrocytes.     

Peroxynitrite formed by mitochondrial NO synthase promotes mitochondrial Ca2+ release

Mitochondria contribute to the maintenance of the intracellular Ca2+ homeostasis by taking up and releasing the cation via separate and specific pathways. The molecular details of the release pathway are elusive but its stimulation by the cross-linking of some vicinal thiols and consequently NAD+ hydrolysis are known. Thiol cross-linking and NAD+ hydrolysis can be achieved by addition of peroxynitrite (ONOO-), the product of the reaction between superoxide (O2-) and nitric oxide (nitrogen monoxide, NO*) to mitochondria. Mitochondria contain an NO synthase (mtNOS), which is stimulated by Ca2+, and are a copious source of O2-. We show here that intramitochondrially formed ONOO- stimulates the specific, NAD+-linked Ca2+ release from mitochondria. Our findings that upon Ca2+ uptake mtNOS is stimulated, that ONOO- is formed, and that Ca2+ is subsequently released from intact mitochondria suggest the existence of a feedback loop, which prevents overloading of mitochondria with Ca2+.     

Bicarbonate-dependent superoxide release and pulmonary artery tone

Pulmonary vasoconstriction is influenced by inactivation of nitric oxide (NO) with extracellular superoxide (O2-*). Because the short-lived O2-* anion cannot diffuse across plasma membranes, its release from vascular cells requires specialized mechanisms that have not been well delineated in the pulmonary circulation. We have shown that the bicarbonate (HCO3-)-chloride anion exchange protein (AE2) expressed in the lung also exchanges O2-* for HCO3-. Thus we determined whether O2-* release involved in pulmonary vascular tone depends on extracellular HCO3-. We assessed endothelium-dependent vascular reactivity and O2-* release in the presence or absence of HCO3- in pulmonary artery (PA) rings isolated from normal rats and those exposed to hypoxia for 3 days. Lack of extracellular HCO3- in normal PA rings significantly attenuated endothelial O2-* release, opposed hypoxic vasoconstriction, and enhanced acetylcholine-mediated vasodilation. Release of O2-* was also inhibited by an AE2 inhibitor (SITS) and abolished in normoxia by an NO synthase inhibitor (NG-nitro-L-arginine methyl ester). In contrast, hypoxia increased PA AE2 protein expression and O2-* release; the latter was not affected by NG-nitro-l-arginine methyl ester or other inhibitors of enzymatic O2-* generation. Enhanced O2-* release by uncoupling NO synthase with geldanamycin was attenuated by hypoxia or by HCO3- elimination. These results indicate that O2-* produced by endothelial NOS in normoxia and unidentified sources in hypoxia regulate pulmonary vascular tone via AE2.     

Nitric oxide-induced mitochondrial dysfunction: implications for neurodegeneration

Excessive generation of nitric oxide (NO) has been implicated in the pathogenesis of several neurodegenerative disorders. Damage to the mitochondrial electron transport chain has also been implicated in these disorders. NO and its toxic metabolite peroxynitrite (ONOO(-)) can inhibit the mitochondrial respiratory chain, leading to energy failure and ultimately cell death. There appears to be a differential susceptibility of brain cell types to NO/ONOO(-), which may be influenced by factors including cellular antioxidant status and the ability to maintain energy requirements in the face of marked respiratory chain damage. Although formation of NO/ONOO(-) following cytokine exposure does not affect astrocyte survival, these molecules may diffuse out and cause mitochondrial damage to neighboring NO/ONOO(-)-sensitive cells such as neurons. Evidence suggests that NO/ONOO(-) causes release of neuronal glutamate, leading to glutamate-induced activation of neuronal NO synthase and generation of further damaging species. While neurons appear able to recover from short-term exposure to NO/ONOO(-), extending the period of exposure results in persistent damage to the respiratory chain and cell death ensues. These findings have important implications for acute infection vs. chronic neuroinflammatory disease states. The evidence for NO/ONOO(-)-mediated mitochondrial damage in neurodegenerative disorders is reviewed and potential therapeutic strategies are discussed.     

Induction of Nitric Oxide Synthase Inhibits Gap Junction Permeability in Cultured Rat Astrocytes

Abstract: Nitric oxide (^?NO) synthase (NOS) was induced in cultured rat astrocytes by incubation with lipopolysaccharide (LPS) for 18 h and gap junction permeability was assessed by the scrape-loading/Lucifer yellow transfer technique. Induction of NOS was confirmed by determining either the N^G-methyl-l -arginine (NMMA)-inhibitable production of nitrites and nitrates or the conversion of l -[³H]arginine to l -[³H]citrulline. Incubation with LPS dose-dependently inhibited gap junction permeability to 63.3% at 0.05 µg/ml LPS and no further inhibition was observed on increasing the LPS concentration up to 0.5 µg/ml. LPS-mediated gap junction inhibition was irreversible but was prevented by incubation with the NOS inhibitor NMMA and with the superoxide anion (O₂^??) scavenger superoxide dismutase. Incubation of the cells with both the ^?NO donor S-nitroso-N-acetylpenicillamine and the O₂^??-generating system xanthine/xanthine oxidase inhibited gap junction permeability. These results suggest that the in situ reaction between ^?NO and O₂^??, to form the peroxynitrite anion (ONOO^?), may be responsible for the inhibition of gap junction permeability. Scavenging the ONOO^? derivative hydroxyl radical (^?OH) with either dimethyl sulfoxide or mannitol prevented the LPS-mediated inhibition of gap junction permeability. Finally, exposure of astrocytes to authentic ONOO^? caused a dose-dependent inhibition of gap junction permeability (65.7% of inhibition at 0.5 mM ONOO^?). The pathophysiological relevance of ONOO^?-mediated inhibition of gap junctional communication in astrocytes after NOS induction by LPS is discussed, stressing the possible role played by this mechanism in some neurodegenerative diseases.     

Methylglyoxal-induced nitric oxide and peroxynitrite production in vascular smooth muscle cells

Methylglyoxal (MG) is a metabolite of glucose. Our previous study demonstrated an elevated MG level with an increased oxidative stress in vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats. Whether MG causes the generation of nitric oxide (NO) and superoxide anion (O2*-), leading to peroxynitrite (ONOO-) formation in VSMCs, was investigated in the present study. Cultured rat thoracic aortic SMCs (A-10) were treated with MG or other different agents. Oxidized DCF, reflecting H2O2 and ONOO- production, was significantly increased in a concentration- and time-dependent manner after the treatment of SMCs with MG (3-300 microM) for 45 min-18 h (n = 12). MG-increased oxidized DCF was effectively blocked by reduced glutathione or N-acetyl-l-cysteine, as well as L-NAME (p < 0.05, n = 12). Both O2*- scavenger SOD and NAD(P)H oxidase inhibitor DPI significantly decreased MG-induced oxidized DCF formation. MG significantly and concentration-dependently increased NO and O2*- generation in A-10 cells, which was significantly inhibited by L-NAME and SOD or DPI, respectively. In conclusion, MG induces significant generation of NO and O2*- in rat VSMCs, which in turn causes ONOO- formation. An elevated MG level and the consequential ROS/RNS generation would alter cellular signaling pathways, contributing to the development of different insulin resistance states such as diabetes or hypertension.     

Hydrogen peroxide induces nitric oxide and proteosome activity in endothelial cells: a bell-shaped signaling response

We investigated nitric oxide (*NO)-mediated proteosomal activation in bovine aortic endothelial cells (BAEC) treated with varying fluxes of hydrogen peroxide (H(2)O(2)) generated from glucose/glucose oxidase (Glu/GO). Results revealed a bell-shaped *NO signaling response in BAEC treated with Glu/GO (2-20 mU/ml). GO treatment (2 mU/ml) enhanced endothelial nitric oxide synthase (eNOS) phosphorylation and *NO release in BAEC. With increasing GO concentrations, phospho eNOS and *NO levels decreased. Bell-shaped responses in proteasomal function and *NO induction were observed in BAEC treated with varying levels of GO (2-10 mU/ml). Proteosomal activation induced in GO-treated BAEC was inhibited by N(omega)-nitro-L-arginine-methyl ester pretreatment, suggesting that *NO mediates proteasomal activation. Intracellular *NO induced by H(2)O(2) was detected by isolating the 4,5-diaminoflourescein (DAF-2)/*NO/O(2)-derived "green fluorescent product" using the high-performance liquid chromatography-fluorescence technique, a more rigorous and quantitative methodology for detecting the DAF-2/*NO/O(2) reaction product. Finally, the relationships between H(2)O(2) flux, proteasomal activation/inactivation, endothelial cell survival, and apoptosis are discussed.     

Interactions between nitric oxide and peroxynitrite during prostaglandin endoperoxide H synthase-1 catalysis: a free radical mechanism of inactivation

Peroxynitrite (ONOO(-)) can serve either as a peroxide substrate or as an inactivator of prostaglandin endoperoxide H synthase-1 (PGHS-1). Herein, the mechanism of PGHS-1 inactivation by ONOO(-) and the modulatory role that nitric oxide (*NO) plays in this process were studied. PGHS-1 reacted with ONOO(-) with a second-order rate constant of 1.7 x 10(7) M(-1) s(-1) at pH 7.0 and 8 degrees C. In the absence of substrates, the enzyme was dose-dependently inactivated by ONOO(-) in parallel with 3-nitrotyrosine formation. However, when PGHS-1 was incubated with ONOO(-) in the presence of substrates, the direct reaction with ONOO(-) was less relevant and ONOO(-)-derived radicals became involved in enzyme inactivation. Bicarbonate at physiologically relevant concentrations enhanced PGHS-1 inactivation and nitration by ONOO(-), further supporting a free radical mechanism. Importantly, *NO (0.4-1.5 microM min(-1)) was able to spare the peroxidase activity of PGHS-1 but it enhanced ONOO(-)-mediated inactivation of cyclooxygenase. The observed differential effects of *NO on ONOO(-)-mediated PGHS-1 inactivation emphasize a novel aspect of the complex modulatory role that *NO plays during inflammatory processes. We conclude that ONOO(-)-derived radicals inactivate both peroxidase and cyclooxygenase activities of PGHS-1 during enzyme turnover. Finally, our results reconcile the proposed alternative effects of ONOO(-) on PGHS-1 (activation versus inactivation).     

Activation of p38 MAPK induced peroxynitrite generation in LPS plus IFN-gamma-stimulated rat primary astrocytes via activation of iNOS and NADPH oxidase

We have shown that immunostimulated astrocytes produce excess nitric oxide (NO) and eventually peroxynitrite (ONOO(-)) that was closely associated with the glucose deprivation-potentiated death of astrocytes. The present study shows that activated p38 MAPK regulates ONOO(-) generation from lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma)-stimulated astrocytes. LPS+IFN-gamma-induced p38 MAPK activation and ONOO(-) generation were attenuated by SB203580 or SKF-86002, specific inhibitors of p38 MAPK. ONOO(-) generation was blocked by NADPH oxidase inhibitor, diphenyleneiodonium chloride, and nitric oxide synthase (NOS) inhibitor, N omega-nitro-L-arginine methyl ester, suggesting both enzymes are involved in ONOO(-) generation. Inhibition of p38 MAPK suppressed LPS+IFN-gamma-induced NO production through down-regulating inducible form of NOS expression. It also suppressed LPS+IFN-gamma-induced NADPH oxidase activation and eventually, the inducible form of superoxide production. Transfection with dominant negative vector of p38 alpha reduced LPS+IFN-gamma-induced ONOO(-) generation through blocking both iNOS-derived NO production and NADPH oxidase-derived O2(-) production. Our results suggest that activated p38 MAPK may serve as a potential signaling molecule in ONOO(-) generation through dual regulatory mechanisms, involving iNOS induction and NADPH oxidase activation.     

Dynamic determination of Ox-LDL-induced oxidative/nitrosative stress in single macrophage by using fluorescent probes

Increased oxidative/nitrosative stress, resulting from generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) appears to play an important role in the inflammatory responses to atherosclerosis. By using MitoTracker Orange CM-H(2)TMRos, CM-H(2)DCFDA (DCF-DA), Dihydrorhodamine 123 (DHR123), DAF-FM, Dihydroethidium (DHE) and JC-1 alone or in all combinations of red and green probes, the present study was designed to monitor the ROS and RNS generation in acute exposure of single monocyte U937-derived macrophage to oxidized low density lipoprotein (Ox-LDL). Acute Ox-LDL (100 microg/ml) treatment increased time-dependently production of intracellular nitric oxide (NO), superoxide (O2*-), hydrogen peroxide (H(2)O(2)) and peroxynitrite (ONOO(-)), and decreased mitochondrial membrane potential (Deltapsi) in single cell. Pretreatment of aminoguanidine (an inhibitor of inducible nitric oxide synthase (iNOS), 10 microM) and vitamin C (an antioxidant agent, 100 microM) for 2h, reduced significantly the Ox-LDL-induced increase of NO and O2*-, and vitamin C completely inhibited increase of intracellular NO and O2*-. In contrast to aminoguanidine, Vitamin C pretreatment significantly prevented Ox-LDL-induced overproduction of NO and O2*- (P<0.01), indicating that antioxidant may be more effective in therapeutic application than iNOS inhibitor in dysfunction of ROS/RNS. By demonstrating a complex imbalance of ROS/RNS via fluorescent probes in acute exposure of single cell to Ox-LDL, oxidative/nitrosative stress might be more detected in the early atherosclerotic lesions.     

Tyrosine nitration by peroxynitrite formed from nitric oxide and superoxide generated by xanthine oxidase

Peroxynitrite (ONOO(-)) is a potent nitrating and oxidizing agent that is formed by a rapid reaction of nitric oxide (NO) with superoxide anion (O(2)). It appears to be involved in the pathophysiology of many inflammatory and neurodegenerative diseases. It has recently been reported (Pfeiffer, S., and Mayer, B. (1998) J. Biol. Chem. 273, 27280-27285) that ONOO(-) generated at neutral pH from NO and O(2) (NO/O(2)) was substantially less efficient than preformed ONOO(-) at nitrating tyrosine. Here we re-evaluated tyrosine nitration by NO/O(2) with a shorter incubation period and a more sensitive electrochemical detection system. Appreciable amounts of nitrotyrosine were produced by ONOO(-) formed in situ (2.9 micrometer for 5 min; 10 nm/s) by NO/O(2) flux obtained from propylamine NONOate (CH(3)N[N(O)NO](-) (CH(2))(3)NH(2)(+)CH(3)) and xanthine oxidase using pterin as a substrate in phosphate buffer (pH 7.0) containing 0.1 mm l-tyrosine. The yield of nitrotyrosine by this NO/O(2) flux was approximately 70% of that produced by the same flux of preformed ONOO(-) (2.9 micrometer/5 min). When hypoxanthine was used as a substrate, tyrosine nitration by NO/O(2) was largely eliminated because of the inhibitory effect of uric acid produced during the oxidation of hypoxanthine. Tyrosine nitration caused by NO/O(2) was inhibited by the ONOO(-) scavenger ebselen and was enhanced 2-fold by NaHCO(3), as would be expected, because CO(2) promotes tyrosine nitration. The profile of nitrotyrosine and dityrosine formation produced by NO/O(2) flux (2.9 micrometer/5 min) was consistent with that produced by preformed ONOO(-). Tyrosine nitration predominated compared with dityrosine formation caused by a low nanomolar flux of ONOO(-) at physiological concentrations of free tyrosine (<0.5 mm). In conclusion, our results show that NO generated with O(2) nitrates tyrosine with a reactivity and efficacy similar to those of chemically synthesized ONOO(-), indicating that ONOO(-) can be a significant source of tyrosine nitration in physiological and pathological events in vivo.