Analysis of 3-nitrotyrosine in biological fluids and protein hydrolyzates by high-performance liquid chromatography using a postseparation, on-line reduction column and electrochemical detection: results with various nitrating agents.

Nitric oxide reacts rapidly with superoxide to form the strong nitrating agent peroxynitrite, which is responsible for much of the tissue damage associated with diverse pathophysiological conditions such as inflammation. The occurrence of free or protein-bound nitrotyrosine (NTYR) has been considered as evidence for in vivo formation of peroxynitrite. However, various agents can nitrate tyrosine, and their relative significance in vivo has not been determined due to lack of a sensitive method to analyze NTYR in tissue proteins and biological fluids. We have developed a new HPLC-electrochemical detection method to analyze NTYR in protein hydrolyzates or biological fluids. The sample is injected directly into a reversed-phase HPLC column and NTYR is subsequently reduced by a platinum column to 3-aminotyrosine, which is quantified with an electrochemical detector. The method is simple, selective, and sensitive (detection limit, 0.1 pmol per 20-microl injection). We have applied this method to compare in vitro the ability of various nitrating agents to form NTYR in bovine serum albumin and human plasma. Yields of NTYR formed in human plasma proteins incubated with 1 or 10 mM nitrating agent decreased in the following order: synthetic peroxynitrite > 3-morpholinosydonimine, a generator of both NO and superoxide > Angeli's salt, which forms nitroxyl anion (NO-) > spermine-NONOate, which releases NO > sodium nitrite plus hypochlorite, which forms the nitrating agent nitryl chloride (NO2Cl). A simple purification method using a C18 Sep-Pak cartridge is also described for analysis of free NTYR in human plasma.     

Inactivation of glutathione S-transferases by nitric oxide-derived oxidants: exploring a role for tyrosine nitration

Reactive intermediates derived from nitric oxide ((*)NO) are thought to play a contributing role in disease states associated with inflammation and infection. We show here that glutathione S-transferases (GSTs), principal enzymes responsible for detoxification of endogenous and exogenous electrophiles, are susceptible to inactivation by reactive nitrogen species (RNS). Treatment of isolated GSTs or rat liver homogenates with either peroxynitrite, the myeloperoxidase/hydrogen peroxide/nitrite system, or tetranitromethane, resulted in loss of GST activity with a concomitant increase in the formation of protein-associated 3-nitrotyrosine (NO(2)Tyr). This inactivation was only partially (<25%) reversible by dithiothreitol, and exposure of GSTs to hydrogen peroxide or S-nitrosoglutathione was only partially inhibitory (<25%) and did not result in protein nitration. Thus, irreversible modifications such as tyrosine nitration may have contributed to GST inactivation by RNS. Since all GSTs contain a critical, highly conserved, active-site tyrosine residue, we postulated that this Tyr residue might present a primary target for nitration by RNS, thus leading to enzyme inactivation. To directly investigate this possibility, we analyzed purified mouse liver GST-mu, following nitration by several RNS, by trypsin digestion, HPLC separation, and matrix-assisted laser desorption/ionization-time of flight analysis, to determine the degree of tyrosine nitration of individual Tyr residues. Indeed, nitration was found to occur preferentially on several tyrosine residues located in and around the GST active site. However, RNS concentrations that resulted in near complete GST inactivation only caused up to 25% nitration of even preferentially targeted tyrosine residues. Hence, nitration of active-site tyrosine residues may contribute to GST inactivation by RNS, but is unlikely to fully account for enzyme inactivation. Overall, our studies illustrate a potential mechanism by which RNS may promote (oxidative) injury by environmental pollutants in association with inflammation.     

Inhibition of mitochondrial respiratory complex I by nitric oxide, peroxynitrite and S-nitrosothiols

NO or its derivatives (reactive nitrogen species, RNS) inhibit mitochondrial complex I by several different mechanisms that are not well characterised. There is an inactivation by NO, peroxynitrite and S-nitrosothiols that is reversible by light or reduced thiols, and therefore may be due to S-nitrosation or Fe-nitrosylation of the complex. There is also an irreversible inhibition by peroxynitrite, other oxidants and high levels of NO, which may be due to tyrosine nitration, oxidation of residues or damage of iron sulfur centres. Inactivation of complex I by NO or RNS is seen in cells or tissues expressing iNOS, and may be relevant to inflammatory pathologies, such as septic shock and Parkinson's disease.     

Global profiling of reactive oxygen and nitrogen species in biological systems: high-throughput real-time analyses

Herein we describe a high-throughput fluorescence and HPLC-based methodology for global profiling of reactive oxygen and nitrogen species (ROS/RNS) in biological systems. The combined use of HPLC and fluorescence detection is key to successful implementation and validation of this methodology. Included here are methods to specifically detect and quantitate the products formed from interaction between the ROS/RNS species and the fluorogenic probes, as follows: superoxide using hydroethidine, peroxynitrite using boronate-based probes, nitric oxide-derived nitrosating species with 4,5-diaminofluorescein, and hydrogen peroxide and other oxidants using 10-acetyl-3,7-dihydroxyphenoxazine (Amplex® Red) with and without horseradish peroxidase, respectively. In this study, we demonstrate real-time monitoring of ROS/RNS in activated macrophages using high-throughput fluorescence and HPLC methods. This global profiling approach, simultaneous detection of multiple ROS/RNS products of fluorescent probes, developed in this study will be useful in unraveling the complex role of ROS/RNS in redox regulation, cell signaling, and cellular oxidative processes and in high-throughput screening of anti-inflammatory antioxidants.     

Modification of tryptophan and tryptophan residues in proteins by reactive nitrogen species.

Formation of 3-nitrotyrosine by the reaction between reactive nitrogen species (RNS) and tyrosine residues in proteins has been analyzed extensively and it is used widely as a biomarker of pathophysiological and physiological conditions mediated by RNS. In contrast, few studies on the nitration of tryptophan have been reported. This review provides an overview of the studies on tryptophan modifications by RNS and points out the possible importance of its modification in pathophysiological and physiological conditions. Free tryptophan can be modified to several nitrated products (1-, 4-, 5-, 6-, and 7-), 1-N-nitroso product, and several oxidized products by reaction with various RNS, depending on the conditions used. Among them, 1-N-nitrosotryptophan and 6-nitrotryptophan (6-NO(2)Trp) have been found as the abundant products in the reaction with peroxynitrite, and 6-NO(2)Trp has been the most abundant product in the reaction with the peroxidase/hydrogen peroxide/nitrite systems. 6-NO(2)Trp has also been observed as the most abundant nitrated product of the reactions between peroxynitrite or myeloperoxidase/hydrogen peroxide/nitrite and tryptophan residues both in human Cu,Zn-superoxide dismutase and in bovine serum albumin, as well as the reaction of peroxynitrite with myoglobin and hemoglobin. Several oxidized products have also been identified in the modified Cu,Zn-SOD. However, no 1-N-nitrosotryptophan and 1-N-nitrotryptophan has been observed in the proteins reacted with peroxynitrite or the myeloperoxidase/H(2)O(2)/nitrite system. The modification of tryptophan residues in proteins may occur at a more limited number of sites in vivo than that of tyrosine residues, since tryptophan residues are more buried inside proteins and exist less frequently in proteins, generally. However, surface-exposed tryptophan residues tend to participate in the interaction with the other molecules, therefore the modification of those tryptophans may result in modulation of the specific interaction of proteins and enzymes with other molecules.     

Recent methodological advances in the mass spectrometric analysis of free and protein-associated 3-nitrotyrosine in human plasma

L-Tyrosine and L-tyrosine residues in proteins are attacked by various reactive-nitrogen species (RNS) including peroxynitrite to form 3-nitrotyrosine (NO(2)Tyr) and protein-associated 3-nitrotyrosine (NO(2)TyrProt). Circulating NO(2)Tyr and NO(2)TyrProt have been suggested and are widely used as biomarkers of oxidative stress in humans. In this article the mass spectrometry (MS)-based analytical methods recently reported for the quantification of circulating levels of NO(2)Tyr and NO(2)TyrProt are discussed. These methodologies differ in sensitivity, selectivity, specificity and accessibility to interferences with the latter mainly arising from artifactual formation of NO(2)Tyr and NO(2)TyrProt during sample treatment such as acidification and chemical derivatization. Application of these methodologies to healthy normal humans revealed basal circulating levels for NO(2)Tyr which range between 0.7 and 64 nM, i.e. by two orders of magnitude. Application of gas chromatography-tandem mass spectrometry (GC-tandem MS) methods by two independent research groups by using two different protocols to avoid artifactual nitration of L-tyrosine revealed almost identical mean plasma levels of the order of 1.0 nM in healthy humans. The lower limits of quantitation (LOQ) of these methods were 0.125 and 0.3n M, respectively. This order of magnitude for basal NO(2)Tyr is supported by two liquid chromatography-tandem mass spectrometry (LC-tandem MS) methods with LOQ values of 4.4 and 1.4 nM. On the basis of the data provided by GC-tandem MS and LC-tandem MS the use of a range of 0.5-3 nM for NO(2)Tyr and of 0.6 pmol/mg plasma protein or a molar ratio of 3-nitrotyrosine to tyrosine in plasma proteins of the order of 1:10(6) for NO(2)TyrProt in plasma of healthy humans as reference values appear reasonably justified. Recently reported clinical studies involving 3-nitrotyrosine as a biomarker of oxidative stress are discussed in particular from the analytical point of view.     

Reactive nitrogen species induce nuclear factor-kappaB-mediated protein degradation in skeletal muscle cells

Recently, a role for NF-κB in upregulation of proteolytic systems and protein degradation has emerged. Reactive nitrogen species (RNS) have been demonstrated to induce NF-κB activation. The aim of this study was to investigate whether RNS caused increased proteolysis in skeletal muscle cells, and whether this process was mediated through the activation of NF-κB. Fully differentiated L6 myotubes were treated with NO donor SNAP, peroxynitrite donor SIN-1, and authentic peroxynitrite, in a time-dependent manner. NF-κB activation, the activation of the ubiquitin-proteasome pathway and matrix metalloproteinases, and the levels of muscle-specific proteins (myosin heavy chain and telethonin) were investigated under the conditions of nitrosative stress. RNS donors caused NF-κB activation and increased activation of proteolytic systems, as well as the degradation of muscle-specific proteins. Antioxidant treatment, tyrosine nitration inhibition, and NF-κB molecular inhibition were proven effective in downregulation of NF-κB activation and slowing down the degradation of muscle-specific proteins. Peroxynitrite, but not NO, causes proteolytic system activation and the degradation of muscle-specific proteins in cultured myotubes, mediated through NF-κB. NF-κB inhibition by antioxidants, tyrosine nitration, and molecular inhibitors may be beneficial for decreasing the extent of muscle damage induced by RNS.     

Peroxynitrite: a potent oxidizing and nitrating agent

Nitric oxide (NO*) reacts with superoxide (O2-*) forming peroxynitrite (PXN) (ONOO-), a strong oxidant which reacts with several biomolecules leading to enormous implications in biological process, holds enormous implications for the understanding of free radicals. The ONOO- formation in vivo has significant implications in free radical biology. It exerts a defensive role in large number of pathophysiological reactions and also acts as signaling molecule in activation of several protooncogenes. It decomposes rapidly to an intermediate and reacts with several biomolecules. Evidence for PXN formation in vivo has been obtained immunohistochemically through detection of a characteristic reaction product with protein tyrosine residues and 3-nitrotyrosine. This "biomarker" of PXN formation has now been identified in various pathologies such as Lou Gehrig's disease, Parkinson's disease, cancer, atherosclerosis as well as in biological aging. 3-nitrotyrosine formation has been documented in various tissues, e.g. even in non-diseased embryonic heart during normal development. Therefore, there is a great opportunity in the postgenomic period to understand the interplay of these molecular interactions with biological events such as apoptosis, gene regulation etc. This review deals with biological significance of peroxynitrite, its precursors, reactions with large range of biomolecules, including aminoacids, proteins, lipids, nucleic acids, antioxidants as well as cytotoxic aspects.     

High temperature triggers the metabolism of S-nitrosothiols in sunflower mediating a process of nitrosative stress which provokes the inhibition of ferredoxin-NADP reductase by tyrosine nitration

High temperature (HT) is considered a major abiotic stress that negatively affects both vegetative and reproductive growth. Whereas the metabolism of reactive oxygen species (ROS) is well established under HT, less is known about the metabolism of reactive nitrogen species (RNS). In sunflower (Helianthus annuus L.) seedlings exposed to HT, NO content as well as S-nitrosoglutathione reductase (GSNOR) activity and expression were down-regulated with the simultaneous accumulation of total S-nitrosothiols (SNOs) including S-nitrosoglutathione (GSNO). However, the content of tyrosine nitration (NO(2) -Tyr) studied by high-performance liquid chromatography with tandem mass spectrometry (LC-MS/MS) and by confocal laser scanning microscope was induced. Nitroproteome analysis under HT showed that this stress induced the protein expression of 13 tyrosine-nitrated proteins. Among the induced proteins, ferredoxin-NADP reductase (FNR) was selected to evaluate the effect of nitration on its activity after heat stress and in vitro conditions using 3-morpholinosydnonimine (SIN-1) (peroxynitrite donor) as the nitrating agent, the FNR activity being inhibited. Taken together, these results suggest that HT augments SNOs, which appear to mediate protein tyrosine nitration, inhibiting FNR, which is involved in the photosynthesis process.     

Tyrosine phosphorylation/dephosphorylation regulates peroxynitrite-mediated peptide nitration

Proteins are targets of reactive nitrogen species such as peroxynitrite and nitrogen dioxide. Among the various amino acids in proteins, tyrosine and tryptophan residues are especially susceptible to attack by reactive nitrogen species. On the other hand, protein tyrosine phosphorylation has gained much attention in respect to cellular regulatory events and signal transduction. Peroxynitrite-mediated nitration of peptide YPPPPPW and phosphopeptide pYPPPPPW were studied at pH 7.4. The predominant nitrated products were separated and identified by reverse phase high performance liquid chromatography coupled with electrospray ionization mass spectrometry (LC-MS). The nitration sites were established by tandem electrospray ionization-mass spectrometry (LC-MS/MS). A regulatory effect of tyrosine phosphorylation/dephosphorylation on peptide nitration was observed. YPPPPPW was predominantly nitrated at tyrosine residue while pYPPPPPW was nitrated at tryptophan one. Our results can help in understanding the biochemical significance of the relationship of tyrosine phosphorylation and nitration in proteins.     

Dopamine prevents nitration of tyrosine hydroxylase by peroxynitrite and nitrogen dioxide: is nitrotyrosine formation an early step in dopamine neuronal damage?

Peroxynitrite and nitrogen dioxide (NO2) are reactive nitrogen species that have been implicated as causal factors in neurodegenerative conditions. Peroxynitrite-induced nitration of tyrosine residues in tyrosine hydroxylase (TH) may even be one of the earliest biochemical events associated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced damage to dopamine neurons. Exposure of TH to peroxynitrite or NO2 results in nitration of tyrosine residues and modification of cysteines in the enzyme as well as inactivation of catalytic activity. Dopamine (DA), its precursor 3,4-dihydroxyphenylalanine, and metabolite 3,4-dihydroxyphenylacetic acid completely block the nitrating effects of peroxynitrite and NO2 on TH but do not relieve the enzyme from inhibition. o-Quinones formed in the reaction of catechols with either peroxynitrite or NO2 react with cysteine residues in TH and inhibit catalytic function. Using direct, real-time evaluation of tyrosine nitration with a green fluorescent protein-TH fusion protein stably expressed in intact cells (also stably expressing the human DA transporter), DA was also found to prevent NO2-induced nitration while leaving TH activity inhibited. These results show that peroxynitrite and NO2 react with DA to form quinones at the expense of tyrosine nitration. Endogenous DA may therefore play an important role in determining how DA neurons are affected by reactive nitrogen species by shifting the balance of their effects away from tyrosine nitration and toward o-quinone formation.     

Proteomic identification of nitrated proteins in Alzheimer's disease brain

Nitration of tyrosine in biological conditions represents a pathological event that is associated with several neurodegenerative diseases, such as amyotrophic lateral sclerosis, Parkinson's disease and Alzheimer's disease (AD). Increased levels of nitrated proteins have been reported in AD brain and CSF, demonstrating the potential involvement of reactive nitrogen species (RNS) in neurodegeneration associated with this disease. Reaction of NO with O2- leads to formation of peroxynitrite ONOO-, which following protonation, generates cytotoxic species that oxidize and nitrate proteins. Several findings suggest an important role of protein nitration in modulating the activity of key enzymes in neurodegenerative disorders, although extensive studies on specific targets of protein nitration in disease are still missing. The present investigation represents a further step in understanding the relationship between oxidative modification of protein and neuronal death in AD. We previously applied a proteomics approach to determine specific targets of protein oxidation in AD brain, by successfully coupling immunochemical detection of protein carbonyls with two-dimensional polyacrylamide gel electrophoresis and mass spectrometry analysis. In the present study, we extend our investigation of protein oxidative modification in AD brain to targets of protein nitration. The identification of six targets of protein nitration in AD brain provides evidence to the importance of oxidative stress in the progression of this dementing disease and potentially establishes a link between RNS-related protein modification and neurodegeneration.     

Use of a novel double-sandwich enzyme-linked immunosorbent assay method for assaying chondroitin sulfate proteoglycans that bear 3-nitrotyrosine core protein modifications, a previously unrecognized proteoglycan modification in hydrocephalus

3-Nitrotyrosine is a useful marker for nitric oxide-mediated tissue injury. However, which proteins are preferred peroxynitrite modification targets is unclear. Chondroitin sulfate proteoglycans (CSPGs) abnormally accumulate in cerebrospinal fluid of human neonates with hydrocephalus and may be a target for peroxynitrite modification. We examined (1). whether CSPG core protein can be modified by peroxynitrite in vitro; (2). to what degree in comparison to bovine serum albumin (BSA), the most commonly used nitrated protein standard; (3). whether nitrated CSPGs can be measured directly in biological samples; and (4). whether nitrated proteoglycan concentrations in cerebrospinal fluid correlate with disease. In vitro nitration of bovine aggrecan was performed by exposure to different peroxynitrite concentrations, and 3-nitrotyrosine products were measured. Bovine serum albumin (BSA) nitration was also performed in comparison. A larger percentage of tyrosine residues were nitrated in aggrecan than in BSA under all conditions tested. An enzyme-linked immunosorbent assay (ELISA) for 3-nitrotyrosine consistently overestimated aggrecan nitration when nitrated BSA was used as the standard. This is important as most current assays of nitration in biological samples use nitrated BSA as the standard. Therefore, if nitrated CPSGs were a substantial portion of the nitrated proteins in a sample, total nitrated protein content would be overestimated. Aggrecan retained its function of binding hyaluronic acid despite substantial nitration. A double-sandwich ELISA was developed for nitrated CSPGs in biological samples, using nitrated aggrecan as standard. [Nitrated CSPG] was found to be significantly elevated in preterm hydrocephalus cerebrospinal fluid (P<0.02), but correlated poorly with cerebrospinal fluid [nitric oxide] (P>0.069), suggesting that nitrated CSPG and NO levels may be independant markers of tissue injury. Peroxynitrite-mediated protein tyrosine nitration is a previously unrecognized modification of CSPGs, and may reflect level of brain injury in hydrocephalus.     

Peroxynitrite reactivity with amino acids and proteins

Summary. Peroxynitrite, the product of the fast reaction between nitric oxide (NO) and superoxide O₂ ^– radicals, is an oxidizing and nitrating agent which is able to traverse biological membranes. The reaction of peroxynitrite with proteins occurs through three possible pathways. First, peroxynitrite reacts directly with cysteine, methionine and tryptophan residues. Second, peroxynitrite reacts fast with transition metal centers and selenium-containing amino acids. Third, secondary free radicals arising from peroxynitrite homolysis such as hydroxyl and nitrogen dioxide, and the carbonate radical formed in the presence of carbon dioxide, react with protein moieties too. Nitration of tyrosine residues is being recognized as a marker of the contribution of nitric oxide to oxidative damage. Peroxynitrite-dependent tyrosine nitration is likely to occur through the initial reaction of peroxynitrite with carbon dioxide or metal centers leading to secondary nitrating species. The preferential protein targets of peroxynitrite and the role of proteins in peroxynitrite detoxifying pathways are discussed.     

The impact of metal catalysis on protein tyrosine nitration by peroxynitrite

In a series of heme and non-heme proteins the nitration of tyrosine residues was assessed by complete pronase digestion and subsequent HPLC-based separation of 3-nitrotyrosine. Bolus addition of peroxynitrite caused comparable nitration levels in all tested proteins. Nitration mainly depended on the total amount of tyrosine residues as well as on surface exposition. In contrast, when superoxide and nitrogen monoxide (NO) were generated at equal rates to yield low steady-state concentrations of peroxynitrite, metal catalysis seemed to play a dominant role in determining the sensitivity and selectivity of peroxynitrite-mediated tyrosine nitration in proteins. Especially, the heme-thiolate containing proteins cytochrome P450(BM-3) (wild type and F87Y variant) and prostacyclin synthase were nitrated with high efficacy. Nitration by co-generated NO/O(2)(-) was inhibited in the presence of superoxide dismutase. The NO source alone only yielded background nitration levels. Upon changing the NO/O(2)(-) ratio to an excess of NO, a decrease in nitration in agreement with trapping of peroxynitrite and derived radicals by NO was observed. These results clearly identify peroxynitrite as the nitrating species even at low steady-state concentrations and demonstrate that metal catalysis plays an important role in nitration of protein-bound tyrosine.     

Intervention by nitric oxide, NO, and its oxide derivatives particularly in mammals

Nitric oxide (NO) is a natural and stable free radical produced in soil and water by the bacteriological reduction of nitrites and nitrates and in animals by the enzyme oxidation of L-arginine. NO is biosynthesised by finely regulated enzymatic systems called NO-synthases and readily diffuses through tissues. It reacts rapidly with hemoproteins and iron-sulphur centers to form nitrosylated compounds. It oxidises more slowly to form nitrogen oxides that nitrosate thiols into thionitrite. NO is transported in these various forms and released spontaneously or through yet unclear mechanisms into most cells; it also regulates oxygen consumption at the mitochondrial respiratory chain level through interaction with cytochrome oxidase. In the cardiovascular system, NO lowers blood pressure by activating a hemoprotein, the guanylate cyclase present in muscle cells; through such interaction it acts also as a neuromediator and neuromodulator in the nervous system. However, many of NO's roles result from rapid coupling to other radicals; for example, it reacts with the superoxide anion (O2-) to form oxoperoxinitrate (ONOO-, also known as peroxynitrite). This strong oxidant of metallic centers, thiols, and antioxidants is also able to convert tyrosine to 3-nitrotyrosine and to act upon tyrosine residues contained in proteins. The biological aspects of the roles of NO are presented with particular respect to the rapid interactions of NO with hemoproteins' iron and other radicals. Concurrently, NO oxidation enables nitrosation reactions primarily of thiols but ultimately of nucleic bases. The thionitrite function (R-S-NO) thus formed and the dimerisation and nitration of tyrosine residues are protein post-translational modifications that are being investigated in animals.     

Reactive nitrogen species control apoptosis and autophagy in K562 cells: implication of TAp73α induction in controlling autophagy

The biological outcome of nitric oxide (NO) and reactive nitrogen species (RNS) in regulating pro survival and pro death autophagic pathways still demand further investigation. In the present study, we investigated the effect of nitrosative stress in K562 cells using NO donor compound DETA-NONOate, peroxynitrite, and SIN-1. Exposure to NO, peroxynitrite, and SIN-1 caused decrease in K562 cell survival. NO induced autophagy but not apoptosis or necrosis in K562 cells. In contrast, peroxynitrite and SIN-1 treatment induced apoptosis in K562 cells. Surprisingly, inhibition of autophagic response using 3-methyladenine led to the induction of apoptosis in K562 cells. Increase in 5’adenosine monophosphate-activated protein kinase (AMPK) phosphorylation was only observed in the presence of NO donor indicated that AMPK was crucial to induce autophagy in K562 cells. We for the first time discovered a novel role of p73 in autophagy induction under nitrosative stress in K562 cells. TAp73α was only induced upon exposure to NO but not in the presence of peroxynitrite. Reduced glutathione (GSH)/oxidised glutathione (GSSG) ratio remained unaltered upon NO exposure. Our data suggest a complex network of interaction and cross regulations between NO and p73. These data open a new path for therapies based on the abilities of RNS to induce autophagy-mediated cell death.     

Submolecular adventures of brain tyrosine: what are we searching for now?

This overview summarizes recent findings on the role of tyrosyl radical (TyrO(*)) in the multitudinous neurochemical systems of brain, and theorizes on the putative role of TyrO(*) in neurological disorders [Parkinson's disease (PD), Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS)]. TyrO(*) and tyrosine per se can interact with reactive oxygen species (ROS) and reactive nitrogen species (RNS) via radical mechanisms and chain propagating reactions. The concentration of TyrO(*), ROS and RNS can increase dramatically under conditions of generalized stress: oxidative, nitrative or reductive as well, and this can induce damage directly (by lipid peroxidation) or indirectly (by proteins oxidation and/or nitration), potentially causing apoptotic neuronal cell death or autoschizis.Evidence of lesion-induced neuronal oxidative stress includes the presence of protein peroxides (TyrOOH), DT (o,o'-dityrosine) and 3-NT (3-nitrotyrosine). Mechanistic details of protein- and enzymatic oxidation/nitration in vivo remain unresolved, although recent in vitro data strongly implicate free radical pathways via TyrO(*). Nitration/denitration processes can be pathological, but they also may play: 1). a signal transduction role, because nitration of tyrosine residues through TyrO(*) formation can modulate, as well the phosphorylation (tyrosine kinases activity) and/or tyrosine hydroxylation (tyrosine hydroxylase inactivation), leading to consequent dopamine synthesis failure and increased degradation of target proteins, respectively; 2). a role of "blocker" for radical-radical reactions (scavenging of NO(*), NO(*)(2) and CO(3)(*-) by TyrO(*)); 3). a role of limiting factors for peroxynitrite formation, by lowering O(2)(*-) formation, which is strongly linked to the pathogenesis of neural diseases.It is still not known if tyrosine oxidation/nitration via TyrO(*) formation is 1). a footprint of generalized stress and neuronal disorders, or 2). an important part of O(2)(*-) and NO(*) metabolism, or 3). merely a part of integral processes for maintaining of neuronal homeostasis. The full answer to these questions should be of top research priority, as the problem of increased free radical formation in brain and/or imbalance of the ratios ROS/RNS/TyrO(*) may be all important in defining whether oxidative stress is the critical determinant of tissue and neural cell injury that leads to pathological end-points.     

Specificity of antioxidant enzyme inhibition in skeletal muscle to reactive nitrogen species donors

Nitric oxide (*NO) and its by-products modulate many physiological functions of skeletal muscle including blood flow, metabolism, glucose uptake, and contractile function. However, growing evidence suggests that an overproduction of nitric oxide contributes to muscle wasting in a number of pathologies including chronic heart failure, sepsis, COPD, muscular dystrophy, and extreme disuse. Limited data point to the potential of inhibition various enzymes by reactive nitrogen species (RNS), including (.)NO and its downstream products such as peroxynitrite, primarily in purified systems. We hypothesized that exposure of skeletal muscle to RNS donors would reduce or downregulate activities of the crucial antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX). Diaphragm muscle fiber bundles were extracted from 4-month-old Fischer-344 rats and, in a series of experiments, exposed to either (a) 0 (control), 1, or 5 mM diethylamine NONOate (DEANO: *NO donor); (b) 0, 100, 500 microM, or 1 mM sodium nitroprusside (SNP: *NO donor); (c) 0 or 2 mM S-nitroso-acetylpenicillamine (SNAP: *NO donor); or (d) 0 or 500 microM SIN-1 (peroxynitrite donor) for 60 min. DEANO resulted in a 50% reduction in CAT, GPX, and a dose-dependent inhibition of Cu, Zn-SOD. SNP resulted in significantly lower activities for total SOD, Mn-SOD isoform, Cu, Zn-SOD isoform, CAT, and GPX in a dose-dependent fashion. Two millimolar SNAP and 500 microM SIN-1 also resulted in a large and significant inhibition of total SOD and CAT. These data indicate that reactive nitrogen species impair antioxidant enzyme function in an RNS donor-specific and dose-dependent manner and are consistent with the hypothesis that excess RNS production contributes to skeletal muscle oxidative stress and muscle dysfunction.