3-nitrotyrosine and peroxynitric-acid

Peroxynitrite promotes oxidative damage and is implicated in the pathophysiology of various diseases that involve accelerated rates of nitric oxide and superoxide formation. The unambiguous detection of peroxynitrite in biological systems is, however, difficult due to the combination of a short biological half-life, limited diffusion, multiple target molecule reactions, and participation of alternative oxidation/nitration pathways. In this review, we provide the conceptual framework and a comprehensive analysis of the current experimental strategies that can serve to unequivocally define the existence and quantitation of peroxynitrite in biological systems of different levels of organization and complexity.

Topics: Animals; Carbon Dioxide; Cytochrome c Group; Electron Spin Resonance Spectroscopy; Fluorescent Dyes; Free Radicals; Humans; Hydroxylation; Immunohistochemistry; Indicators and Reagents; Luminescent Measurements; Nitrates; Oxidants; Oxidative Stress; Phenols; Tyrosine

Oxidative stress, reactive oxygen (ROS), and nitrogen (RNS) species have been known to be involved in a multitude of neurodegenerative disorders such as Parkinson's disease (PD), Alzheimer's disease (AD), and amyotrophic lateral sclerosis (ALS). Both ROS and RNS have very short half-lives, thereby making their identification very difficult as a specific cause of neurodegeneration. Recently, we have developed a high performance liquid chromatography/electrochemical detection (HPLC/EC) method to identify 3-nitrotyrosine (3-NT), an in vitro and in vivo biomarker of peroxynitrite production, in cell cultures and brain to evaluate if an agent-driven neurotoxicity is produced by the generation of peroxynitrite. We show that a single or multiple injections of methamphetamine (METH) produced a significant increase in the formation of 3-NT in the striatum. This formation of 3-NT correlated with the striatal dopamine depletion caused by METH administration. We also show that PC12 cells treated with METH has significantly increased formation of 3-NT and dopamine depletion. Furthermore, we report that pretreatment with antioxidants such as selenium and melatonin can completely protect against the formation of 3-NT and depletion of striatal dopamine. We also report that pretreatment with peroxynitrite decomposition catalysts such as 5, 10,15,20-tetrakis(N-methyl-4'-pyridyl)porphyrinato iron III (FeTMPyP) and 5, 10, 15, 20-tetrakis (2,4,6-trimethyl-3,5-sulfonatophenyl) porphinato iron III (FETPPS) significantly protect against METH-induced 3-NT formation and striatal dopamine depletion. We used two different approaches, pharmacological manipulation and transgenic animal models, in order to further investigate the role of peroxynitrite. We show that a selective neuronal nitric oxide synthase (nNOS) inhibitor, 7-nitroindazole (7-NI), significantly protect against the formation of 3-NT as well as striatal dopamine depletion. Similar results were observed with nNOS knockout and copper zinc superoxide dismutase (CuZnSOD)-overexpressed transgenic mice models. Finally, using the protein data bank crystal structure of tyrosine hydroxylase, we postulate the possible nitration of specific tyrosine moiety in the enzyme that can be responsible for dopaminergic neurotoxicity. Together, these data clearly support the hypothesis that the reactive nitrogen species, peroxynitrite, plays a major role in METH-induced dopaminergic neurotoxicity and that selective antioxidants and peroxyni

Topics: Animals; Antioxidants; Biomarkers; Dopamine; Dopamine Agents; Enzyme Inhibitors; Humans; Indazoles; Methamphetamine; Nitrates; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; PC12 Cells; Rats; Tyrosine

Topics: Animals; Asthma; Bronchoalveolar Lavage Fluid; Cystic Fibrosis; Eosinophils; Humans; Nitrates; Nitric Oxide; Pneumonia; Tyrosine

Nitric oxide (NO) plays an important role as a cell-signalling molecule, anti-infective agent and, as most recently recognised, an antioxidant. The metabolic fate of NO gives rise to a further series of compounds, collectively known as the reactive nitrogen species (RNS), which possess their own unique characteristics. In this review we discuss this emerging aspect of the NO field in the context of the formation of the RNS and what is known about their effects on biological systems. While much of the insight into the RNS has been gained from the extensive chemical characterisation of these species, to reveal biological consequences this approach must be complemented by direct measures of physiological function. Although we do not know the consequences of many of the dominant chemical reactions of RNS an intriguing aspect is now emerging. This review will illustrate how, when specificity and amplification through cell signalling mechanisms are taken into account, the less significant reactions, in terms of yield or rates, can explain many of the biological responses of exposure of cells or physiological systems to RNS.

Topics: Animals; Apoptosis; Inflammation; Lipoproteins; Membranes; Nitrates; Nitric Oxide; Nitric Oxide Donors; Nitrogen; Oxidation-Reduction; Signal Transduction; Tyrosine; Vasodilation

Nitric oxide, a gaseous free radical, is poorly reactive with most biomolecules but highly reactive with other free radicals. Its ability to scavenge peroxyl and other damaging radicals may make it an important antioxidant in vivo, particularly in the cardiovascular system, although this ability has been somewhat eclipsed in the literature by a focus on the toxicity of peroxynitrite, generated by reaction of O2*- with NO* (or of NO- with O2). On balance, experimental and theoretical data support the view that ONOO- can lead to hydroxyl radical (OH*) generation at pH 7.4, but it seems unlikely that OH* contributes much to the cytotoxicity of ONOO-. The cytotoxicity of ONOO- may have been over-emphasized: its formation and rapid reaction with antioxidants may provide a mechanism of using NO* to dispose of excess O2*-, or even of using O2*- to dispose of excess NO*, in order to maintain the correct balance between these radicals in vivo. Injection or instillation of "bolus" ONOO- into animals has produced tissue injury, however, although more experiments generating ONOO- at steady rates in vivo are required. The presence of 3-nitrotyrosine in tissues is still frequently taken as evidence of ONOO- generation in vivo, but abundant evidence now exists to support the view that it is a biomarker of several "reactive nitrogen species". Another under-addressed problem is the reliability of assays used to detect and measure 3-nitrotyrosine in tissues and body fluids: immunostaining results vary between laboratories and simple HPLC methods are susceptible to artefacts. Exposure of biological material to low pH (e.g. during acidic hydrolysis to liberate nitrotyrosine from proteins) or to H2O2 might cause artefactual generation of nitrotyrosine from NO2- in the samples. This may be the origin of some of the very large values for tissue nitrotyrosine levels quoted in the literature. Nitrous acid causes not only tyrosine nitration but also DNA base deamination at low pH: these events are relevant to the human stomach since saliva and many foods are rich in nitrite. Several plant phenolics inhibit nitration and deamination in vitro, an effect that could conceivably contribute to their protective effects against gastric cancer development.

Topics: Animals; Biomarkers; Disease; Humans; Nitrates; Nitric Oxide; Superoxides; Tyrosine

Motor neuron survival is highly dependent on trophic factor supply. Deprivation of trophic factors results in induction of neuronal NOS, which is also found in pathological conditions. Growing evidence suggests that motor neuron degeneration involves peroxynitrite formation. Trophic factors modulate peroxynitrite toxicity (Estévez et al., 1995; Shin et al., 1996; Spear et al., 1997). Whether a trophic factor prevents or potentiates peroxynitrite toxicity depends upon when the cells are exposed to the trophic factor (Table 1). These results strongly suggest that a trophic factor that can protect neurons under optimal conditions, but under stressful conditions can increase cell death. In this context, it is possible that trophic factors or cytokines produced as a response to damage may potentiate rather than prevent motor neuron death. A similar argument may apply to the therapeutic administration of trophic factors to treat neurodegenerative diseases. Similarly, the contrasting actions of NO on motor neurons may have important consequences for the potential use of nitric oxide synthase inhibitors in the treatment of ALS and other related neurodegenerative diseases.

Topics: Amyotrophic Lateral Sclerosis; Animals; Apoptosis; Brain-Derived Neurotrophic Factor; Cell Death; Cell Survival; Humans; Motor Neurons; Nitrates; Nitric Oxide; Superoxide Dismutase; Tyrosine

Atrial fibrillation (AF), the most common chronic arrhythmia, increases the risk of stroke and is an independent predictor of mortality. Available pharmacological treatments have limited efficacy. Once initiated, AF tends to self-perpetuate, owing in part to electrophysiological remodeling in the atria; however, the fundamental mechanisms underlying this process are still unclear. We have recently demonstrated that chronic human AF is associated with increased atrial oxidative stress and peroxynitrite formation; we have now tested the hypothesis that these events participate in both pacing-induced atrial electrophysiological remodeling and in the occurrence of AF following cardiac surgery. In chronically instrumented dogs, we found that rapid (400 min(-1)) atrial pacing was associated with attenuation of the atrial effective refractory period (ERP). Treatment with ascorbate, an antioxidant and peroxynitrite decomposition catalyst, did not directly modify the ERP, but attenuated the pacing-induced atrial ERP shortening following 24 to 48 hours of pacing. Biochemical studies revealed that pacing was associated with decreased tissue ascorbate levels and increased protein nitration (a biomarker of peroxynitrite formation). Oral ascorbate supplementation attenuated both of these changes. To evaluate the clinical significance of these observations, supplemental ascorbate was given to 43 patients before, and for 5 days following, cardiac bypass graft surgery. Patients receiving ascorbate had a 16.3% incidence of postoperative AF, compared with 34.9% in control subjects. In combination, these studies suggest that oxidative stress underlies early atrial electrophysiological remodeling and offer novel insight into the etiology and potential treatment of an enigmatic and difficult to control arrhythmia. The full text of this article is available at http://www.circresaha.org.

Topics: Aged; Animals; Antioxidants; Ascorbic Acid; Atrial Fibrillation; Cardiac Pacing, Artificial; Coronary Artery Bypass; Dogs; Electrophysiology; Female; Heart Atria; Humans; Male; Middle Aged; Multivariate Analysis; Nitrates; Time Factors; Treatment Outcome; Tyrosine

The purpose of this study was to probe the pleiotrophic effects of Atorvastatin on intraplatelet-nitric oxide metabolism.. Hyperlipidemic subjects (n = 19) were treated for 1 month (following a 3-week washout) with either Atorvastatin or placebo in a double-blinded randomized (n = 2, crossover), placebo-controlled study. Changes in the levels of intraplatelet nitric oxide synthase, nitrotyrosine were correlated with cholesterol, LDL-C, HDL-C and triglyceride levels. These studies indicate that with atrovastatin ecNOS levels increased on average by approximately approximately 1.7-fold (paired t-test p = 0.009). Interestingly, levels of nitrotyrosylated platelet proteins, an indication of peroxynitrite damage, decreased as ecNOS levels increased in presence of the drug (paired t-test p = 0.33). Atorvastatin, at 10 mg per day, lowered cholesterol and LDL-C levels in all patients with the average lowering of approximately 21% and approximately 17% respectively. The effect on HDL was not significant whilst triglyceride levels were lowered by an average of approximately 18%.. This study adds to the volume of evidence that statins have beneficial effects other than lipid lowering. Here, Atorvastatin is shown to significantly elevate intraplatelet ecNOS levels in hyperlipidemic subjects without affecting iNOS expression. The net result of this would be the elevation of NO production which would promote platelet deaggregation and vasodilation.

Topics: Adult; Aged; Anticholesteremic Agents; Atorvastatin; Blood Platelets; Blood Proteins; Cholesterol; Cholesterol, HDL; Cholesterol, LDL; Cross-Over Studies; Double-Blind Method; Enzyme Induction; Female; Heptanoic Acids; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Hypercholesterolemia; Hypertriglyceridemia; Male; Middle Aged; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Oxidation-Reduction; Pyrroles; Treatment Outcome; Triglycerides; Tyrosine; Vasodilation

Topics: Animals; Blood Platelets; Blotting, Western; Cattle; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Humans; Nitrates; Serum Albumin, Bovine; Tyrosine

3-Nitrotyrosine (3-NT), a product of peroxynitrite reaction, is abundantly observed in hepatocytes adjacent to human metastatic colorectal carcinoma. To elucidate its biological significance, we undertook to identify nitric oxide (NO)-producing cells and apoptosis under oxidative stress. We observed strong inducible NO-synthase (iNOS) immunoreactivity in the hepatocytes adjacent to metastatic tumor, revealing an identical pattern to 3-NT immunostaining. Furthermore, intense 3-NT immunostaining of hepatocytes was associated with apoptosis whereas carcinoma cells near those hepatocytes presented high proliferating-cell nuclear antigen. Our results suggest that contact of metastatic tumor induces apoptosis in adjacent hepatocytes through peroxynitrite, thus permitting the proliferation of cancer cells.

Topics: Adenocarcinoma; Aged; Apoptosis; Cell Division; Colorectal Neoplasms; Female; Humans; Immunoenzyme Techniques; Liver Neoplasms; Male; Middle Aged; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Oxidants; Oxidative Stress; Proliferating Cell Nuclear Antigen; Reactive Oxygen Species; Tyrosine

Nitration of proteins by peroxynitrite may alter protein function. We hypothesized that reactive nitrogen species modulate fibronectin-induced fibroblast migration. To test this hypothesis, we evaluated fibroblast migration induced by fibronectin incubated with and without peroxynitrite. Peroxynitrite attenuated fibronectin-induced fibroblast migration in a dose-dependent manner but did not attenuate complement-activated serum-induced fibroblast migration. The reducing agents, deferoxamine and dithiothreitol (DTT), and L-tyrosine reversed the inhibition by peroxynitrite. PAPA-NONOate, a nitric oxide (NO) donor, and superoxide generated by the action of xanthine oxidase on lumazine or xanthine, also showed an inhibitory effect on fibroblast migration. The peroxynitrite generator, 3-morpholinosydnonimine (SIN-1), caused a concentration-dependent inhibition of fibroblast migration. Peroxynitrite reduced fibronectin binding to fibroblasts and resulted in nitrotyrosine formation. These findings are consistent with nitration of tyrosine by peroxynitrite with subsequent inhibition of fibronectin binding to fibroblasts and suggest that peroxynitrite may play a role in regulation of fibroblast migration.

Topics: Blood; Cell Line; Cell Movement; Complement Activation; Deferoxamine; Dithiothreitol; Embryo, Mammalian; Fibroblasts; Fibronectins; Humans; Lung; Molsidomine; Nitrates; Nitric Oxide Donors; Nitrogen; Reactive Oxygen Species; Reducing Agents; Superoxides; Tyrosine; Xanthine Oxidase

1. Splanchnic artery occlusion shock (SAO) causes an enhanced formation of reactive oxygen species (ROS), which contribute to the pathophysiology of shock. Here we have investigated the effects of M40401, a new S:,S:-dimethyl substituted biscyclohexylpyridine Mn-based superoxide dismutase mimetic (SODm, k(cat)=1.2x10(+9) M(-1) s(-1) at pH=7.4), in rats subjected to SAO shock. 2. Treatment of rats with M40401 (applied at 0.25, 2.5 or 25 microg kg(-1), 15 min prior to reperfusion), attenuated the mean arterial blood and the migration of polymorphonuclear cells (PMNs) caused by SAO-shock. M40401 also attenuated the ileum injury (histology) as well as the increase in the tissue levels of myeloperoxidase (MPO) and malondialdehyde (MDA) caused by SAO shock in the ileum. 3. Immunohistochemical analysis for nitrotyrosine revealed a positive staining in ileum from SAO-shocked rats. The degree of staining for nitrotyrosine was markedly reduced in tissue sections obtained from SAO-shocked rats which had received M40401. Reperfused ileum tissue sections from SAO-shocked rats showed positive staining for P-selectin and for anti-intercellular adhesion molecule (ICAM-1) in the vascular endothelial cells. M40401 treatment markedly reduced the intensity and degree of P-selectin and ICAM-1 in tissue sections from SAO-shocked rats. M40401 treatment significantly improved survival. 4. Additionally, the very high catalytic activity of this new mimetic (comparable to the native human Cu/Zn SOD enzyme and exceeding the activity of the human Mn SOD enzyme) translates into a very low dose ( approximately microg kg(-1)) required to afford protection in this SAO model of ischemia reperfusion injury. 5. Taken together, our results clearly demonstrate that M40401 treatment exerts a protective effect, and part of this effect may be due to inhibition of the expression of adhesion molecules and peroxynitrite-related pathways with subsequent reduction of neutrophil-mediated cellular injury.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Arterial Occlusive Diseases; Catalysis; Cytokines; Fluorescent Antibody Technique; Ileum; Leukocyte Count; Male; Malondialdehyde; Manganese; Nitrates; Nitrites; Organometallic Compounds; P-Selectin; Peroxidase; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Splanchnic Circulation; Superoxide Dismutase; Superoxides; Tyrosine

The use of methamphetamine (METH) leads to neurotoxic effects in mammals. These neurotoxic effects appear to be related to the production of free radicals. To assess the role of peroxynitrite in METH-induced dopaminergic, we investigated the production of 3-nitrotyrosine (3-NT) in the mouse striatum. The levels of 3-NT increased in the striatum of wild-type mice treated with multiple doses of METH (4 x 10 mg/kg, 2 h interval) as compared with the controls. However, no significant production of 3-NT was observed either in the striata of neuronal nitric oxide synthase knockout mice (nNOS -/-) or copper-zinc superoxide dismutase overexpressed transgenic mice (SOD-Tg) treated with similar doses of METH. The dopaminergic damage induced by METH treatment was also attenuated in nNOS-/- or SOD-Tg mice. These data further confirm that METH causes its neurotoxic effects via the production of peroxynitrite.

Topics: Animals; Central Nervous System Stimulants; Corpus Striatum; Dopamine; Dopamine Uptake Inhibitors; Methamphetamine; Mice; Mice, Inbred C57BL; Mice, Knockout; Neurotoxins; Nitrates; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Reference Values; Superoxide Dismutase; Tyrosine

Host inflammatory and immune responses limit viral gene expression after administration of replication-deficient adenoviruses to the lung. The current study asks whether inducible nitric oxide synthase (iNOS) expression and peroxynitrite generation accompanied the inflammatory response following intratracheal administration of adenovirus. Pulmonary iNOS mRNA and protein were increased 2, 7, and 14 days following administration of 2 x 10(9) plaque-forming units of recombinant adenovirus (Av1Luc1) to BALB/c mice. Adenovirus infection was associated with a marked increase in nitrotyrosine staining. Intense nitrotyrosine staining was observed in alveolar macrophages, respiratory epithelial cells, conducting airways, and alveolar spaces 2 days postinfection. Two weeks after exposure to adenovirus, nitrotyrosine staining was detected within alveolar macrophages, suggesting adenovirus enhanced the nitration of proteins that were subsequently taken up by alveolar macrophages. Western blot analysis using anti-nitrotyrosine antibody did not demonstrate accumulation of nitrated surfactant protein A (SP-A), although a small fraction of aggregated SP-A comigrated with a nitrotyrosine-positive protein. iNOS expression, peroxynitrite, and nitrotyrosine generation accompany and may contribute to inflammatory responses to adenovirus in the lung.

Topics: Adenoviridae Infections; Animals; Blotting, Western; Bronchoalveolar Lavage Fluid; Female; Immunohistochemistry; Lung; Male; Mice; Mice, Inbred BALB C; Nitrates; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Pulmonary Surfactants; Tyrosine

With the immersion of corn into dilute sulfur oxide during starch-manufacturing processes, corn steep liquor (CSL) remains as leftover material. CSL is often used for fermentation, but its components are not fully understood. To determine the properties of CSL, 12 p-coumaric acid-related compounds were isolated from an ethyl acetate extract of CSL with the guidance of antioxidative activity on the rabbit erythrocyte membrane ghost system. The activity of these compounds was compared against oxidative damages, and it was elucidated that the activity of p-coumaric acid derivatives was mainly affected by their functional groups at the 3-position and less by the conjugated side chain. Moreover, p-coumaric acid derivatives exhibited inhibitory activity stronger than that of tocopherols and ascorbic acid on peroxynitrite-mediated lipoprotein nitration. These findings that p-coumaric acid derivatives, which might play a beneficial role against oxidative damage, exist in CSL suggest this byproduct might be a useful resource of phenolic antioxidants.

Topics: Acetates; Animals; Antioxidants; Ascorbic Acid; Chromatography, High Pressure Liquid; Coumaric Acids; Erythrocyte Membrane; Fermentation; Lipoproteins; Magnetic Resonance Spectroscopy; Nitrates; Phenols; Rabbits; Tyrosine; Vitamin E; Zea mays

Erectile dysfunction in the aging male results in part from the loss of compliance of the corpora cavernosal smooth muscle due to the progressive replacement of smooth muscle cells by collagen fibers. We have examined the hypothesis that a spontaneous local induction of inducible nitric oxide synthase (iNOS) expression and the subsequent peroxynitrite formation occurs in the penis during aging and that this process is accompanied by a stimulation of smooth muscle apoptosis and collagen deposition. The penile shaft and crura were excised from young (3-5 mo old) and old (24-30 mo old) rats, with or without perfusion with 4% formalin. Fresh tissue was used for iNOS and proteasome 2C mRNA determinations by reverse transcription polymerase chain reaction assay, ubiquitin mRNA by Northern blot, and iNOS protein by Western blot. Penile sections from perfused animals were embedded in paraffin and immunostained with antibodies against iNOS and nitrotyrosine, submitted to the TUNEL assay for apoptosis, or stained for collagen, followed by image analysis quantitation. A 4.1-fold increase in iNOS mRNA was observed in the old versus young tissues, paralleled by a 4.9-fold increase in iNOS protein. The proteolysis marker, ubiquitin, was increased 1.9-fold, whereas a related gene, proteasome 2c, was not significantly affected. iNOS immunostaining was increased 3.6-fold in the penile smooth muscle of the old rats as compared with the young rats. The peroxynitrite indicator nitrotyrosine was increased by 1.6-fold, accompanied by a 3.6-fold increase in apoptotic cells and a 2.0-fold increase in collagen fibers in the old penis. In conclusion, aging in the penis is accompanied by an induction of iNOS and peroxynitrite formation that may lead to the observed increase in apoptosis and proteolysis and may counteract a higher rate of collagen deposition in the old penis.

Topics: Aging; Animals; Apoptosis; Blotting, Northern; Blotting, Western; Collagen; Cysteine Endopeptidases; DNA Fragmentation; Gene Expression Regulation, Enzymologic; Image Processing, Computer-Assisted; In Situ Nick-End Labeling; Male; Multienzyme Complexes; Muscle, Smooth; Nitrates; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Penis; Proteasome Endopeptidase Complex; Rats; Rats, Inbred F344; Reverse Transcriptase Polymerase Chain Reaction; RNA; RNA, Messenger; Tyrosine; Ubiquitins

Oxidative stress is implicated in the initiation and progression of congestive heart failure, but the putative reactive species and cellular targets involved remain undefined. We have previously shown that peroxynitrite (ONOO(-), an aggressive biological oxidant and nitrating agent) potently inhibits myofibrillar creatine kinase (MM-CK), a critical controller of contractility known to be impaired during heart failure. Here we hypothesized that nitration and inhibition of MM-CK participate in cardiac failure in vivo.. Heart failure was induced in rats by myocardial infarction (left coronary artery ligation) and confirmed by histological analysis at 8 weeks postinfarct (1.3+/-1.4 vs. 37.7+/-3.2% left ventricular circumference; sham control vs. CHF, n=10 each).. Immunohistochemistry demonstrated significantly increased protein nitration in failing myocardium compared to control (optical density: 0.58+/-0.06 vs. 0.93+/-0.09, sham vs. CHF, P<0.05). Significant decreases in MM-CK activity and content were observed in failing hearts (MM-CK k(cat): 6.0+/-0.4 vs. 3.0+/-0.3 micromol/nM M-CK/min, P<0.05; 6.8+/-1.3 vs. 4.7+/-1.2% myofibrillar protein, P<0.05), with no change in myosin ATPase activity. In separate experiments, isolated rat cardiac myofibrils were exposed to ONOO(-) (2-250 microM) and enzyme studies were conducted. Identical to in vivo studies, selective reductions in MM-CK were observed at ONOO(-) concentrations as low as 2 microM (IC(50)=92.5+/-6.0 microM); myosin ATPase was unaffected with ONOO(-) concentrations as high as 250 microM. Concentration dependent nitration of MM-CK occurred and extent of nitration was statistically correlated to extent of CK inhibition (P<0.001). Immunoprecipitation of MM-CK from failing left ventricle yielded significant evidence of tyrosine nitration.. These data demonstrate that cardiac ONOO(-) formation and perturbation of myofibrillar energetic controllers occur during experimental heart failure; MM-CK may be a critical cellular target in this setting.

Topics: Animals; Creatine Kinase; Creatine Kinase, MM Form; Heart Failure; Image Processing, Computer-Assisted; Immunohistochemistry; Isoenzymes; Male; Myofibrils; Myosins; Nitrates; Oxidants; Rats; Rats, Sprague-Dawley; Tyrosine

Polyamines are required during cell proliferation, whereas NO has anti-proliferative properties. Ornithine decarboxylase (ODC) is a critical enzyme for the synthesis of polyamines. We tested the hypothesis that the modification of ODC by peroxynitrite (OONO-), a short-lived free radical formed from NO and superoxide produces a fall in ODC activity, and therefore polyamine synthesis and cell proliferation. The treatment of a rat recombinant ODC (rODC) with OONO- resulted in a dose-dependent inhibition of rODC activity with an IC50 of approximately 100 microM. A Western blot employing a specific antibody to nitrotyrosine revealed a dose-dependent nitration of rODC tyrosine residues. When intact IEC-6 cells were treated with ONOO-, ODC activity decreased by 49%. These data suggest a correlation between ODC activity and nitration, and a possible mechanism by which NO synthesis may modulate polyamine synthesis.

Topics: Animals; Blotting, Western; Cell Line; Dose-Response Relationship, Drug; Enzyme Inhibitors; Epithelial Cells; Nitrates; Ornithine Decarboxylase; Ornithine Decarboxylase Inhibitors; Oxidants; Rats; Recombinant Proteins; Tyrosine

Tamoxifen (TAM), a widely used non-steroidal anti-estrogen, has recently been shown to be neuroprotective in a rat model of reversible middle cerebral artery occlusion (rMCAo). Tamoxifen has several potential mechanisms of action including inhibition of the release of excitatory amino acids (EAA) and nitric oxide synthase (NOS) activity. The question addressed in this study was whether TAM reduces ischemia-induced production of nitrotyrosine, considered as a footprint of the product of nitric oxide and superoxide, peroxynitrite. In rat brain, 2 h rMCAo produced a time-dependent increase in nitrotyrosine content in the cerebral cortex, as measured by Western blot analysis. Compared with vehicle, TAM significantly reduced nitrotyrosine levels in the ischemic cortex at 24 h. The neuronal (n)NOS inhibitor, 7-nitroindazole also tended to reduce nitrotyrosine, but this reduction was not statistically significant. Immunostaining for nitrotyrosine was seen in cortical neurons in the MCA territory and this immunostaining was reduced by TAM. In vitro, TAM and the calmodulin inhibitor trifluoperazine inhibited, with similar EC(50) values, the activity of recombinant nNOS as well as NOS activity in brain homogenates, measured by conversion of [(3)H]arginine to [(3)H]citrulline. There was marginal inhibition of recombinant inducible (i)NOS activity up to 100 microM TAM. These data suggest that TAM is an effective inhibitor of Ca(2+)/calmodulin-dependent NOS and the derived peroxynitrite production in transient focal cerebral ischemia and this may be one mechanism for its neuroprotective effect following rMCAo.

Topics: Animals; Brain; Cerebral Cortex; Disease Models, Animal; Enzyme Inhibitors; Indazoles; Ischemic Attack, Transient; Male; Middle Cerebral Artery; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Rats; Rats, Sprague-Dawley; Reference Values; Superoxides; Tamoxifen; Tyrosine

Nitric oxide (NO*) is a mediator of esophageal motility. Esophageal dysmotility accompanies esophagitis. During inflammation, superoxide and NO* form peroxynitrite (ONOO-), a reactive molecule that alters cellular function. We tested the hypotheses that ONOO- affects esophageal motility and is produced in association with esophagitis. Transverse muscle strips from the opossum esophagus were stimulated by an electrical field, and nitrotyrosine immunoblots were performed. Peroxynitrite, its decomposed form, or NaNO2 relaxed the lower esophageal sphincter (LES) and attenuated the off response. These effects were inhibited by oxyhemoglobin (Hgb). An antagonist of guanylate cyclase, 1H[1,2,4]oxadiazole[4,3]quinoxalin-1-one (ODQ), inhibited the LES relaxation produced by ONOO-. Nitrotyrosine, a footprint for ONOO- production, was detected in inflamed esophagus. These studies support the hypotheses that ONOO alters esophageal motor function and is formed in association with esophagitis. It is possible that some of the esophageal motor dysfunction seen with esophagitis may be related to the formation of ONOO-.

Topics: Animals; Enzyme Inhibitors; Esophagitis; Esophagogastric Junction; Female; Guanylate Cyclase; In Vitro Techniques; Male; Muscle Relaxation; Muscle, Smooth; Nitrates; Opossums; Oxadiazoles; Oxidants; Oxyhemoglobins; Quinoxalines; Tyrosine

To determine cytotoxic effects of activated polymorphonuclear neutrophils (PMN) and peroxynitrite on bovine mammary secretory epithelial cells before and after addition of nitric oxide synthase inhibitors, myeloperoxidase (MPO) inhibitors, and free-radical scavengers.. Polymorphonuclear neutrophils from 3 lactating cows.. Cells from the bovine mammary epithelial cell line MAC-T were cultured. Monolayers were treated with activated bovine PMN, lipopolysaccharide (LPS), phorbol 12-myristate 13-acetate (PMA), 3-morpholino-sydnonimine (SIN-1), 4-amino-benzoic acid hydrazide (ABAH), NG-monomethyl-L-arginine, histidine, and superoxide dismutase (SOD). At 24 hours, activity of lactate dehydrogenase in culture medium was used as a relative index of cell death. Tyrosine nitration of proteins in MAC-T cell lysates was determined by visual examination of immunoblots.. Lipopolysaccharide, PMA, and < or = 0.1 mM SIN-1 were not toxic to MAC-T cells. Activated PMN, > or = 6 mg of histidine/ml, and 0.5 mM SIN-1 were toxic. Together, histidine and 500,000 activated PMN/ml also were toxic. NG-monomethyl-L-arginine did not have an effect, but ABAH decreased PMN-mediated cytotoxicity. Ten and 50 U of SOD/ml protected MAC-T cells from cytotoxic effects of 0.5 mM SIN-1. Compared with control samples, nitration of MAC-T tyrosine residues decreased after addition of 500,000 PMN/ml or > or = 6 mg of histidine/ml. Superoxide dismutase increased and SIN-1 decreased tyrosine nitration of MAC-T cell proteins in a dose-responsive manner.. Peroxynitrite, MPO, and histidine are toxic to mammary secretory epithelial cells. Superoxide dismutase and inhibition of MPO activity mitigate these effects. Nitration of MAC-T cell tyrosine residues may be positively associated with viability.

Topics: Aniline Compounds; Animals; Antioxidants; Blotting, Western; Cattle; Cell Death; Enzyme Inhibitors; Epithelial Cells; Female; Free Radical Scavengers; Histidine; L-Lactate Dehydrogenase; Lipopolysaccharides; Mammary Glands, Animal; Molsidomine; Neutrophil Activation; Neutrophils; Nitrates; Nitric Oxide; Nitric Oxide Synthase; omega-N-Methylarginine; Oxidants; Peroxidase; Superoxide Dismutase; Tetradecanoylphorbol Acetate; Tyrosine

Eosinophil recruitment and enhanced production of NO are characteristic features of asthma. However, neither the ability of eosinophils to generate NO-derived oxidants nor their role in nitration of targets during asthma is established. Using gas chromatography-mass spectrometry we demonstrate a 10-fold increase in 3-nitrotyrosine (NO(2)Y) content, a global marker of protein modification by reactive nitrogen species, in proteins recovered from bronchoalveolar lavage of severe asthmatic patients (480 +/- 198 micromol/mol tyrosine; n = 11) compared with nonasthmatic subjects (52.5 +/- 40.7 micromol/mol tyrosine; n = 12). Parallel gas chromatography-mass spectrometry analyses of bronchoalveolar lavage proteins for 3-bromotyrosine (BrY) and 3-chlorotyrosine (ClY), selective markers of eosinophil peroxidase (EPO)- and myeloperoxidase-catalyzed oxidation, respectively, demonstrated a dramatic preferential formation of BrY in asthmatic (1093 +/- 457 micromol BrY/mol tyrosine; 161 +/- 88 micromol ClY/mol tyrosine; n = 11 each) compared with nonasthmatic subjects (13 +/- 14.5 micromol BrY/mol tyrosine; 65 +/- 69 micromol ClY/mol tyrosine; n = 12 each). Bronchial tissue from individuals who died of asthma demonstrated the most intense anti-NO(2)Y immunostaining in epitopes that colocalized with eosinophils. Although eosinophils from normal subjects failed to generate detectable levels of NO, NO(2-), NO(3-), or NO(2)Y, tyrosine nitration was promoted by eosinophils activated either in the presence of physiological levels of NO(2-) or an exogenous NO source. At low, but not high (e.g., >2 microM/min), rates of NO flux, EPO inhibitors and catalase markedly attenuated aromatic nitration. These results identify eosinophils as a major source of oxidants during asthma. They also demonstrate that eosinophils use distinct mechanisms for generating NO-derived oxidants and identify EPO as an enzymatic source of nitrating intermediates in eosinophils.

Topics: Eosinophil Peroxidase; Eosinophils; Free Radicals; Humans; Immunohistochemistry; Nitrates; Nitric Oxide; Nitric Oxide Donors; Nitrites; Oxidants; Oxidation-Reduction; Peroxidases; Phenylpropionates; Proteins; Reactive Oxygen Species; Status Asthmaticus; Tyrosine

Peroxynitrite, formed by nitric oxide (NO) and superoxide, can alter protein function by nitrating amino acids such as tyrosine, cysteine, trytophan, or methionine. Inducible nitric oxide synthase (Type 2 NOS or iNOS) converts arginine to citrulline, releasing NO. We hypothesized that peroxynitrite could function as a negative feedback modulator of NO production by nitration of iNOS. Confluent cultures of the murine lung epithelial cell line, LA-4 were stimulated with cytokines to express iNOS, peroxynitrite was added, and the flasks sealed. After 3 h, NO in the headspace above the culture was sampled. Peroxynitrite caused a concentration-dependent decrease in NO. Similar results were obtained when 3-morpholinosydnonimine (SIN-1), a peroxynitrite generator, was added to the flasks. PAPA-NONOate, the NO generator, did not affect the headspace NO. Nitration of the iNOS was confirmed by detection of 3-nitrotyrosine by Western blotting. These data suggest a mechanism for inhibition of NO synthesis at inflammatory sites where iNOS, NO, and superoxide would be expected.

Topics: Animals; Base Sequence; Cell Line; DNA Primers; Enzyme Inhibitors; Epithelial Cells; Gene Expression; Hydrazines; Lung; Mice; Molsidomine; Nitrates; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Oxidants; RNA, Messenger; Tyrosine

Oxidative stress has been suggested as a potential mechanism in the pathogenesis of lung inflammation. The pharmacological profile of n-acetylcysteine (NAC), a free radical scavenger, was evaluated in an experimental model of lung injury (carrageenan-induced pleurisy). Injection of carrageenan into the pleural cavity of rats elicited an acute inflammatory response characterized by fluid accumulation in the pleural cavity that contained many neutrophils (PMNs), an infiltration of PMNs in lung tissues and subsequent lipid peroxidation, and increased production of nitrite/nitrate, tumor necrosis factor alpha, and interleukin 1beta. All parameters of inflammation were attenuated by NAC treatment. Furthermore, carrageenan induced an up-regulation of the adhesion molecules ICAM-1 and P-selectin, as well as nitrotyrosine and poly (ADP-ribose) synthetase (PARS), as determined by immunohistochemical analysis of lung tissues. The degree of staining for the ICAM-1, P-selectin, nitrotyrosine, and PARS was reduced by NAC. In vivo NAC treatment significantly reduced peroxynitrite formation as measured by the oxidation of the fluorescent dihydrorhodamine-123, prevented the appearance of DNA damage, an decrease in mitochondrial respiration, and partially restored the cellular level of NAD+ in ex vivo macrophages harvested from the pleural cavity of rats subjected to carrageenan-induced pleurisy. A significant alteration in the morphology of red blood cells was observed 24 h after carrageenan administration. NAC treatment has the ability to significantly diminish the red blood cell alteration. Our results clearly demonstrate that NAC treatment exerts a protective effect and clearly indicate that NAC offers a novel therapeutic approach for the management of lung injury where radicals have been postulated to play a role.

Topics: Acetylcysteine; Animals; Blotting, Western; Carrageenan; Disease Models, Animal; DNA Damage; DNA-Binding Proteins; Erythrocytes; Free Radical Scavengers; I-kappa B Proteins; Immunohistochemistry; Intercellular Adhesion Molecule-1; Lung; Macrophages; Male; Malondialdehyde; Neutrophils; NF-KappaB Inhibitor alpha; Nitrates; Nitric Oxide; P-Selectin; Peroxidase; Pleurisy; Poly(ADP-ribose) Polymerases; Rats; Rats, Sprague-Dawley; Tyrosine

Peroxynitrite (ONOO(-)), which is generated from nitric oxide (NO) and superoxide anion (O(2)(.-)) under pathological conditions, plays an important role in pathophysiological processes. Activation of matrix metalloproteinases (MMPs) contributes to tumor angiogenesis and metastasis. NO mediates the enhanced vascular permeability and retention (EPR) effect in solid tumors, and ONOO(-)activates proMMP to MMP in vitro. In this study, we examined the role of ONOO(-)in the EPR effect in solid tumors and normal tissues as related to MMP activation. Authentic ONOO(-), at 50 nmol or higher concentrations, induced the enhanced vascular permeability in normal dorsal skin of mice. ONOO(-)scavengers ebselen and uric acid significantly suppressed the EPR effect in mouse sarcoma 180 (S-180) tumors. Indirect evidence for formation of ONOO(-)in S-180 and mouse colon adenocarcinoma (C-38) tumors included strong immunostaining for nitrotyrosine in the tumor tissue, predominantly surrounding the tumor vessels. MMP inhibitor BE16627B (66.6 mg / kg i.v., given 2 times) or SI-27 (10 mg / kg i.p., given 2 times) significantly suppressed the ONOO(-)-induced EPR effect in S-180 tumors and in normal skin. Soybean trypsin inhibitor (Kunitz type), broad-spectrum proteinase inhibitor ovomacroglobulin, and bradykinin receptor antagonist HOE 140 also significantly suppressed the ONOO(-)-induced EPR effect in normal skin tissues. These data suggest that ONOO(-)may be involved in and promote the EPR effect in tumors, which could be mediated partly through activation of MMPs and a subsequent proteinase cascade to generate potent vasoactive mediators such as bradykinin.

Topics: Adenocarcinoma; Animals; Azoles; Bradykinin; Capillary Permeability; Collagenases; Colonic Neoplasms; Dose-Response Relationship, Drug; Enzyme Precursors; Free Radical Scavengers; Gelatinases; Guinea Pigs; Isoindoles; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Metalloendopeptidases; Mice; Mice, Inbred C57BL; Nitrates; Oligopeptides; Organoselenium Compounds; Protease Inhibitors; Sarcoma 180; Skin; Tyrosine; Uric Acid

Peroxynitrite, derived from the reaction of nitric oxide (NO(.)) with superoxide (O(2)), is a potent nitrating and oxidizing agent that can induce apoptosis in a variety of different cell types. In the present study, we investigated the possible role of peroxynitrite as a mediator of colon epithelial cell death in rat colitis. Rat colon inflammation was induced by intracolonic administration of 2,4,6-trinitrobenzenesulfonic acid (TNBS) and rats were sacrificed 24 h after TNBS administration. Expression of inducible nitric-oxide synthase (iNOS) was detected by reverse transcription-polymerase chain reaction and immunohistochemistry. The enzymatic activities of Ca(2+)-independent iNOS and Ca(2+)-dependent constitutive nitric-oxide synthase were determined biochemically. Evidence of peroxynitrite-mediated cell injury was detected by immunostaining of nitrotyrosine. Apoptosis was examined by in situ terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay and DNA gel electrophoresis. To evaluate the specific contribution of peroxynitrite to the observed cell injury, a selective iNOS inhibitor, L-N(G)-[1-iminoethyl]lysine (L-NIL), was administered after TNBS induction. Morphological examination and analysis of TUNEL/cytokeratin double immunofluorescence revealed significant apoptosis in mucosal epithelial cells. Nitrotyrosine was colocalized with TUNEL, strongly demonstrating the association of peroxynitrite with the apoptotic death of colon epithelial cells. The administration of L-NIL reduced iNOS activity in 24-h lesions by 92% and also significantly attenuated both nitrotyrosine staining and apoptotic cell counts in the colon epithelium. These results strongly suggest that local elevated level of peroxynitrite produced from increased iNOS expression and activity is a major contributor to colon epithelial apoptosis during colon inflammation.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Cell Death; Colitis; Colon; Disease Models, Animal; Epithelial Cells; Immunohistochemistry; In Situ Nick-End Labeling; Isoenzymes; Lysine; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Peroxidase; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Superoxides; Trinitrobenzenesulfonic Acid; Tyrosine

Increased expression of inducible nitric oxide synthase (iNOS) has been found in inflammatory myocardial disease and increased production of nitric oxide (NO) has both an inhibitory effect on virus replication and a cytotoxic effect on host cells. To investigate the relationship between severity of enteroviral myocarditis and iNOS expression, a characterised murine model was infected with either cardiovirulent or an attenuated Coxsackievirus B3 and myocardial samples were collected on Day 7. The ability of these viruses to induce NOS expression was compared by measurement of iNOS enzyme activity and localisation of iNOS protein or peroxynitrite, a product of excessive NO production. In accordance with previous reports, high expression of iNOS was detected in mice infected with the cardiovirulent virus. The iNOS protein was located mainly in infiltrating macrophages in and around foci of necrotic myofibres where viral genomic RNA was detected. In contrast, the level of iNOS expression was significantly lower in mice infected with the attenuated virus. This correlates with fewer and smaller myocarditic lesions and less infiltrating cells in the heart. iNOS was not detected in mock-infected mice by the above assays. These findings suggest that one mechanism of attenuation may be associated with the reduced ability of the variant to induce NOS expression in the heart. This also confirms a cytotoxic role for NO in the pathogenesis of Coxsackievirus B3-induced myocarditis.

Topics: Animals; Disease Models, Animal; Enterovirus; Guanosine Monophosphate; Heart; Immunohistochemistry; Male; Mice; Myocarditis; Myocardium; Nitrates; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nucleic Acid Hybridization; RNA, Viral; Tyrosine

The developing cortical neurons have been well documented to be extremely vulnerable to the toxic effect of methylmercury (MeHg). In the present study, a possible involvement of N-methyl-D-aspartate (NMDA) receptors in MeHg neurotoxicity was examined because the sensitivity of cortical neurons to NMDA neurotoxicity has a similar developmental profile. Rats on postnatal day 2 (P2), P16, and P60 were orally administered MeHg (10 mg/kg) for 7 consecutive days. The most severe neuronal damage was observed in the occipital cortex of P16 rats. When MK-801 (0.1 mg/kg), a non-competitive antagonist of NMDA, was administered intraperitoneally with MeHg, MeHg-induced neurodegeneration was markedly ameliorated. Furthermore, there was a marked accumulation of nitrotyrosine, a reaction product of peroxynitrite and L-tyrosine, after chronic treatment of MeHg in the occipital cortex of P16 rats. The accumulation of nitrotyrosine was also significantly suppressed by MK-801. In the present electrophysiological study, the amplitude of synaptic responses mediated by NMDA receptors recorded in cortical neurons of P16 rats was significantly larger than those from P2 and P60 rats. These observations strongly suggest that a generation of peroxynitrite through activation of NMDA receptors is a major causal factor for MeHg neurotoxicity in the developing cortical neurons. Furthermore, enhanced sensitivity of NMDA receptors may make the cortical neurons of P16 rats most susceptible to MeHg neurotoxicity.

Topics: Age Factors; Animals; Animals, Newborn; Antigens, CD; Antigens, Neoplasm; Antigens, Surface; Avian Proteins; Basigin; Blood Proteins; Cerebral Cortex; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Glutamic Acid; Membrane Glycoproteins; Mercury Poisoning, Nervous System; Methylmercury Compounds; Nerve Degeneration; Neurons; Nitrates; Quinoxalines; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Tyrosine

Most thymocytes are deleted by thymic selection. The mechanisms of cell death are far from being clear. Peroxynitrite is a powerful oxidant produced in vivo by the reaction of superoxide (O2*-) with nitric oxide (NO*) and is able to mediate apoptosis. The aim of this study was to analyze whether NO and peroxynitrite could play a role in human thymocyte apoptosis. The results indicate that 3-(4-morpholinyl)-sydnonimine (SIN-1, an O2*- and NO* donor) and chemically synthesized peroxynitrite, but not S-nitroso-N-acetyl-D,L-penicillamine (SNAP, an NO* donor), have a strong apoptotic effect on human thymocytes (annexin V staining and TUNEL reaction). This effect was inhibited by exogenous superoxide dismutase (SOD), which interacts with O2*- and inhibits the formation of peroxynitrite. Because peroxynitrite formation requires NO*, thymic stromal cells were investigated to determine if they produced NO*. Inducible NOS was synthesized in cultured thymic epithelial cells in certain conditions of cytokine stimulation, as shown by messenger RNA levels, protein analysis, and nitrite production in the supernatants. SIN-1-treated thymocytes had high levels of tyrosine nitration, abolished by the addition of exogenous SOD. Tyrosine nitration was also detected in thymus extracts and sections, suggesting the presence of peroxynitrite in situ. In thymus sections, clusters of nitrotyrosine-positive cells were found in the cortex and corticomedullary areas colocalized with cells positive in the TUNEL reaction. These data indicate an association between human thymocyte apoptosis and nitrotyrosine formation. Thus, the results support the notion of a physiologic role for peroxynitrite in human thymocyte apoptosis. (Blood. 2001;97:3521-3530)

Topics: Annexin A5; Apoptosis; Cells, Cultured; Child, Preschool; Enzyme Inhibitors; Epithelial Cells; Gene Expression; Humans; In Situ Nick-End Labeling; Molsidomine; Nitrates; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Stromal Cells; Superoxide Dismutase; Superoxides; T-Lymphocytes; Thymus Gland; Tyrosine

Peroxynitrite, formed in a rapid reaction of nitric oxide (NO) and superoxide anion radical (O(2)), is thought to mediate protein tyrosine nitration in various inflammatory and infectious diseases. However, a recent in vitro study indicated that peroxynitrite exhibits poor nitrating efficiency at biologically relevant steady-state concentrations (Pfeiffer, S., Schmidt, K., and Mayer, B. (2000) J. Biol. Chem. 275, 6346-6352). To investigate the molecular mechanism of protein tyrosine nitration in intact cells, murine RAW 264.7 macrophages were activated with immunological stimuli, causing inducible NO synthase expression (interferon-gamma in combination with either lipopolysaccharide or zymosan A), followed by the determination of protein-bound 3-nitrotyrosine levels and release of potential triggers of nitration (NO, O(2)*, H(2)O(2), peroxynitrite, and nitrite). Levels of 3-nitrotyrosine started to increase at 16-18 h and exhibited a maximum at 20-24 h post-stimulation. Formation of O(2) was maximal at 1-5 h and decreased to base line 5 h after stimulation. Release of NO peaked at approximately 6 and approximately 9 h after stimulation with interferon-gamma/lipopolysaccharide and interferon-gamma/zymosan A, respectively, followed by a rapid decline to base line within the next 4 h. NO formation resulted in accumulation of nitrite, which leveled off at about 50 microm 15 h post-stimulation. Significant release of peroxynitrite was detectable only upon treatment of cytokine-activated cells with phorbol 12-myristate-13-acetate, which led to a 2.2-fold increase in dihydrorhodamine oxidation without significantly increasing the levels of 3-nitrotyrosine. Tyrosine nitration was inhibited by azide and catalase and mimicked by incubation of unstimulated cells with nitrite. Together with the striking discrepancy in the time course of NO/O(2) release versus 3-nitrotyrosine formation, these results suggest that protein tyrosine nitration in activated macrophages is caused by a nitrite-dependent peroxidase reaction rather than peroxynitrite.

Topics: Animals; Cell Line; Cytokines; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Free Radical Scavengers; Hydrogen Peroxide; Immunoblotting; Interferon-gamma; Lipopolysaccharides; Macrophages; Mice; Nitrates; Nitric Oxide; Nitrites; Nitrogen; Oxygen; Peroxidase; Recombinant Proteins; Tetradecanoylphorbol Acetate; Time Factors; Tyrosine; Zymosan

The nitration of tyrosine residues in protein to yield 3-nitrotyrosine derivatives has been suggested to represent a specific footprint for peroxynitrite formation in vivo. However, recent studies suggest that certain hemoproteins such as peroxidases catalyze the H(2)O(2)-dependent nitration of tyrosine to yield 3-nitrotyrosine in a peroxynitrite-independent reaction. Because 3-nitrotyrosine has been shown to be present in the postischemic myocardium, we wished to assess the ability of myoglobin to catalyze the nitration of tyrosine in vitro. We found that myoglobin catalyzed the oxidation of nitrite and promoted the nitration of tyrosine. Both nitrite oxidation and tyrosine nitration were H(2)O(2)-dependent and required the formation of ferryl (Fe(+4)) myoglobin. In addition, nitrite oxidation and tyrosine nitration were pH-dependent with a pH optimum of approximately 6.0. Taken together, these data suggest that the acidic pH and low oxygen tension produced during myocardial ischemia will facilitate myoglobin-catalyzed, peroxyntrite-independent formation of 3-nitrotyrosine.

Topics: Animals; Horses; Hydrogen Peroxide; Hydrogen-Ion Concentration; Kinetics; Metmyoglobin; Myocardium; Myoglobin; Nitrates; Nitrites; Oxidation-Reduction; Tyrosine

The role for peroxynitrite (ONOO(-)) in the mechanism of preconditioning is not known. Therefore, we studied effects of preconditioning and subsequent ischemia/reperfusion on myocardial ONOO(-) formation in isolated rat hearts. Hearts were subjected to a preconditioning protocol (three intermittent periods of global ischemia/reperfusion of 5 min duration each) followed by a test ischemia/reperfusion (30 min global ischemia and 15 min reperfusion). When compared to nonpreconditioned controls, preceding preconditioning improved postischemic cardiac performance and significantly decreased test ischemia/reperfusion-induced formation of free nitrotyrosine measured in the perfusate as a marker for cardiac endogenous ONOO(-) formation. During preconditioning, however, the first period of ischemia/reperfusion increased nitrotyrosine formation, which was attenuated after the third period of ischemia/reperfusion. We conclude that classic preconditioning inhibits ischemia/reperfusion-induced cardiac formation of ONOO(-) and that subsequent periods of ischemia/reperfusion result in a gradual attenuation of ischemia/reperfusion-induced ONOO(-) generation. This mechanism might be involved in ischemic adaptation of the heart.

Topics: Animals; Aorta; Blood Flow Velocity; In Vitro Techniques; Ischemic Preconditioning, Myocardial; L-Lactate Dehydrogenase; Male; Myocardial Ischemia; Myocardial Reperfusion; Myocardial Reperfusion Injury; Myocardium; Nitrates; Rats; Rats, Wistar; Time Factors; Tyrosine; Ventricular Function, Left

Nitric oxide (.NO) attenuates hydrogen peroxide (H(2)O(2))-mediated barrier dysfunction in cultured porcine pulmonary artery endothelial cells (PAEC) (Gupta MP, Ober MD, Patterson C, Al-Hassani M, Natarajan V, and Hart, CM. Am J Physiol Lung Cell Mol Physiol 280: L116-L126, 2001). However,.NO rapidly combines with superoxide (O) to form the powerful oxidant peroxynitrite (ONOO(-)), which we hypothesized would cause PAEC monolayer barrier dysfunction. To test this hypothesis, we treated PAEC with ONOO(-) (500 microM) or 3-morpholinosydnonimine hydrochloride (SIN-1; 1-500 microM). SIN-1-mediated ONOO(-) formation was confirmed by monitoring the oxidation of dihydrorhodamine 123 to rhodamine. Both ONOO(-) and SIN-1 increased albumin clearance (P < 0.05) in the absence of cytotoxicity and altered the architecture of the cytoskeletal proteins actin and beta-catenin as detected by immunofluorescent confocal imaging. ONOO(-)-induced barrier dysfunction was partially reversible and was attenuated by cysteine. Both ONOO(-) and SIN-1 nitrated tyrosine residues, including those on beta-catenin and actin, and oxidized proteins in PAEC. The introduction of actin treated with ONOO(-) into PAEC monolayers via liposomes also resulted in barrier dysfunction. These results indicate that ONOO(-) directly alters endothelial cytoskeletal proteins, leading to barrier dysfunction.

Topics: Actins; Animals; beta Catenin; Cell Survival; Cells, Cultured; Cytoskeletal Proteins; Endothelium, Vascular; Kinetics; Molsidomine; Nitrates; Nitric Oxide; Nitric Oxide Donors; Oxidants; Pulmonary Artery; Reactive Oxygen Species; Superoxide Dismutase; Swine; Trans-Activators; Tyrosine

Topics: Administration, Inhalation; Animals; Endothelium, Vascular; Epoprostenol; Hemoglobins; Humans; Hydroxyl Radical; Hypertension, Pulmonary; Nitrates; Nitric Oxide; Nitrogen Dioxide; Oxidation-Reduction; Pneumonia; Prostaglandin H2; Prostaglandins H; Signal Transduction; Superoxides; Tyrosine

Our previous studies have demonstrated that inhaled nitric oxide (NO) decreases nitric oxide synthase (NOS) activity in vivo and that this inhibition is associated with rebound pulmonary hypertension upon acute withdrawal of inhaled NO. We have also demonstrated that inhaled NO elevates plasma endothelin-1 (ET-1) levels and that pretreatment with PD156707, an ETA receptor antagonist, blocks the rebound hypertension. The objectives of this study were to further elucidate the role of ET-1 in the rebound pulmonary hypertension upon acute withdrawal of inhaled NO. Inhaled NO (40 ppm) delivered to thirteen 4-week-old lambs decreased NOS activity by 36.2% in control lambs (P<0.05), whereas NOS activity was preserved in PD156707-treated lambs. When primary cultures of pulmonary artery smooth muscle cells were exposed to ET-1, superoxide production increased by 33% (P<0.05). This increase was blocked by a preincubation with PD156707. Furthermore, cotreatment of cells with ET-1 and NO increased peroxynitrite levels by 26% (P<0.05), whereas preincubation of purified human endothelial nitric oxide synthase (eNOS) protein with peroxynitrite generated a nitrated enzyme with 50% activity relative to control (P<0.05). Western blot analysis of peripheral lung extracts obtained after 24 hours of inhaled NO revealed a 90% reduction in 3-nitrotyrosine residues (P<0.05) in PD156707-treated lambs. The nitration of eNOS was also reduced by 40% in PD156707-treated lambs (P<0.05). These data suggest that the reduction of NOS activity associated with inhaled NO therapy may involve ETA receptor-mediated superoxide production. ETA receptor antagonists may prevent rebound pulmonary hypertension by protecting endogenous eNOS activity during inhaled NO therapy.

Topics: Administration, Inhalation; Animals; Blotting, Western; Cells, Cultured; Dioxoles; Disease Models, Animal; Endothelin Receptor Antagonists; Endothelin-1; Enzyme Activation; Humans; Hypertension, Pulmonary; Lung; Microscopy, Fluorescence; Muscle, Smooth, Vascular; Nitrates; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Pulmonary Artery; Receptor, Endothelin A; Secondary Prevention; Sheep; Superoxides; Tyrosine

Previous evidence suggests that both oxygen radicals and nitric oxide (NO) are important mediators of injury during renal ischemia-reperfusion (I-R) injury. However, the generation of reactive nitrogen species (RNS) has not been evaluated in this model at early time points. The purpose of these studies was to examine the development of oxidant stress and the formation of RNS during I-R injury. Male Sprague-Dawley rats were anesthetized and subjected to 40 min of bilateral renal ischemia followed by 0, 3, or 6 h of reperfusion. Control animals received a sham operation. Plasma urea nitrogen and creatinine levels were monitored as markers of renal injury. Glutathione (GSH) oxidation and 4-hydroxynonenal (4-HNE)-protein adducts were used as markers of oxidant stress. 3-Nitrotyrosine (3-NT) was used as a biomarker of RNS formation. Significant increases in plasma creatinine concentrations and urea nitrogen levels were found following both 3 and 6 h of reperfusion. Increases in GSH oxidation, 4-HNE-protein adduct levels, and 3-NT levels were observed following 40 min of ischemia with no reperfusion. Since these results suggested RNS generation during the 40 min of ischemia, a time course of RNS generation following 0, 5, 10, 20, and 40 min of ischemia was evaluated. Significant increases in 3-NT generation was detected as early as 10 min of ischemia and rose to values nearly 10-fold higher than Control at 40 min of ischemia. No additional increase was observed following reperfusion. The data clearly demonstrate that oxidative stress and RNS generation occur in the kidney during ischemia.

Topics: Aldehydes; Animals; Blood Urea Nitrogen; Creatinine; Free Radicals; Glutathione; Kidney; Male; Nitrates; Oxidation-Reduction; Oxidative Stress; Proteins; Random Allocation; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Time Factors; Tyrosine

Current experimental studies have demonstrated that peroxynitrite (ONOO-) has both cytotoxic and cytoprotective effects on myocardial ischemia-reperfusion injury. However, even myocardial ONOO- formation has not yet been investigated in humans undergoing open heart operation. We measured plasma nitrotyrosine as an indicator of ONOO- formation during open heart operation and examined its association with myocardial ischemia-reperfusion injury.. Twenty adult patients undergoing mitral valve replacement under cardiopulmonary bypass between 1997 and 1998 were enrolled in this study (6 men and 14 women). Arterial blood (Ao) and coronary sinus effluent (CS) were obtained: (1) before the initiation of cardiopulmonary bypass, (2) just after aortic unclamping, (3) at 5 minutes, (4) at 10 minutes, (5) at 15 minutes, and (6) at 20 minutes after aortic unclamping.. At every sampling point after reperfusion, plasma nitrate and nitrite was significantly lower in CS than in Ao, and the percentage ratio of nitrotyrosine to tyrosine (%NO2-Tyr; an index of ONOO- formation) was significantly higher in CS than in Ao. The CS-Ao difference in %NO2-Tyr, myocardium-derived ONOO-, reached its peak at 5 minutes after reperfusion (2.17+/-0.74%), which was significantly correlated with the peak CS-Ao difference in plasma malondialdehyde, and with postoperative maximum creatine kinase-MB.. These results first demonstrate that ONOO- is produced from human myocardium after ischemia-reperfusion during open heart operation, and myocardium-derived ONOO- can be determined by the CS-Ao difference in %NO2-Tyr.

Topics: Adult; Aged; Cardiopulmonary Bypass; Creatine Kinase; Creatine Kinase, MB Form; Female; Heart Valve Prosthesis Implantation; Humans; Isoenzymes; Male; Malondialdehyde; Middle Aged; Myocardial Reperfusion Injury; Myocardium; Nitrates; Tyrosine

Topics: Adult; Biomarkers; Confounding Factors, Epidemiologic; Diabetic Angiopathies; Female; Glucose Clamp Technique; Hemodynamics; Humans; Hydrogen-Ion Concentration; Hyperglycemia; Male; Nitrates; Oxidative Stress; Tyrosine; Vasoconstriction

Protein kinase C (PKC) is an important intracellular signaling molecule whose activity is essential for a number of aspects of neuronal function including synaptic plasticity. We investigated the regulation of PKC activity by reactive nitrogen species in order to examine whether such species regulate PKC in neurons. Neither autonomous nor cofactor-dependent PKC activity was altered when either hippocampal homogenates or rat brain purified PKC were incubated briefly with three different nitric oxide donor compounds. However, brief incubation of either hippocampal homogenates or purified PKC with peroxynitrite (ONOO(-)) inhibited cofactor-dependent PKC activity in a manner that correlated with the nitration of tyrosine residues on PKC, suggesting that this modification was responsible for the inhibition of PKC. Consistent with this idea, reducing agents had no effect on the inhibition of PKC activity caused by ONOO(-). Because there are numerous PKC isoforms that differ in the composition of the regulatory domain, we studied the effect of ONOO(-) on various PKC isoforms. ONOO(-) inhibited the cofactor-dependent activity of the alpha, betaII, epsilon, and zeta isoforms, indicating that inhibition of enzymatic activity by ONOO(-) was not PKC isoform-specific. We also were able to isolate nitrated PKCalpha and PKCbetaII from ONOO(-)-treated hippocampal homogenates via immunoprecipitation. Collectively, our findings support the hypothesis that ONOO(-) inhibits PKC activity via tyrosine nitration in neurons.

Topics: Animals; Brain; Cysteine; Isoenzymes; Male; Neurodegenerative Diseases; Nitrates; Nitric Oxide Donors; Nitroprusside; Nitroso Compounds; Oxidants; Oxidation-Reduction; Penicillamine; Protein Kinase C; Protein Kinase C beta; Protein Kinase C-alpha; Rats; Rats, Sprague-Dawley; S-Nitrosothiols; Tissue Extracts; Tyrosine

Although excitotoxic and oxidative stress play important roles in spinal neuron death, the exact mechanism is not fully understood. We examined cell damage of primary culture of 11-day-old rat spinal cord by addition of glutamate, nitric oxide (NO) or peroxynitrite (PN) with detection of nitrotyrosine (NT) or terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL). With addition of glutamate, NOC18 (a slow NO releaser) or PN, immunoreactivity for NT became stronger in the cytoplasm of large motor neurons in the ventral horn at 6 to 48 hr and positive in the axons of the ventral horn at 24 to 48 hr. TUNEL positive nuclei were found in spinal large motor neurons from 24 hr, and the positive cell number greatly increased at 48 hr in contrast to the vehicle. Pretreatment of cultures with alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate receptor antagonist, NO-suppressing agent, and antioxidant protected the immunoreactivity for NT or TUNEL. The present results suggest that both excitotoxic and oxidative stress play an important role in the upregulation of NT nitration and the apoptotic pathway in cultured rat spinal neurons.

Topics: Animals; Antioxidants; Apoptosis; Cells, Cultured; Central Nervous System Diseases; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Free Radicals; Glutamic Acid; Immunohistochemistry; In Situ Nick-End Labeling; Interneurons; Motor Neurons; Nerve Degeneration; Neurons; Neurotoxins; Nitrates; Nitric Oxide Donors; Organ Culture Techniques; Oxidative Stress; Rats; Rats, Sprague-Dawley; Spinal Cord; Tyrosine

Methamphetamine (METH) produces dopaminergic neurotoxicity by the production of reactive oxygen (ROS) and nitrogen (RNS) species. The role of free radicals has also been implicated in the process of aging. The present study was designed to evaluate whether METH-induced dopaminergic neurotoxicity and hyperthermia is a result of peroxynitrite production and if these effects correlate with age. One-, six- and 12-month-old male rats (n = 8) were administered a single dose of METH (0, 5, 10, 20, and 40 mg/kg, intraperitoneally). The formation of 3-nitrotyrosine (3-NT) as a marker of peroxynitrite production as well as dopamine and its metabolites DOPAC and HVA were measured in the striatum 4-h after METH-administration. Rectal temperature was monitored every 30 min after METH administration until 4 h. At 40 mg/kg METH, a 100% mortality in 12-month-old animals was observed, whereas no deaths occurred in 1- or 6-month-old rats. An age-dependent increase in hyperthermia was observed after METH-administration. A similar pattern of dose-dependent increase in the formation of 3-NT and in the depletion of dopamine and its metabolites with age was observed in the striatum. Furthermore, no effect was observed at 5 mg/kg METH in 1-month-old animals, whereas the effect was significant in 6- and 12-month-old animals. These data suggest that aging increases the susceptibility of the animals toward METH-induced peroxynitrite generation and striatal dopaminergic neurotoxicity.

Topics: 3,4-Dihydroxyphenylacetic Acid; Aging; Animals; Brain Chemistry; Central Nervous System Stimulants; Corpus Striatum; Dopamine; Dose-Response Relationship, Drug; Fever; Homovanillic Acid; Male; Methamphetamine; Nitrates; Oxidative Stress; Rats; Rats, Sprague-Dawley; Tyrosine

In a model of idiopathic pneumonia syndrome after bone marrow transplantation (BMT), injection of allogeneic T cells induces nitric oxide (.NO), and the addition of cyclophosphamide (Cy) generates superoxide (O.) and a tissue-damaging nitrating oxidant. We hypothesized that.NO and O. balance are major determinants of post-BMT survival and inflammation. Inducible nitric oxide synthase (iNOS) deletional mutant mice (-/-) given donor bone marrow and spleen T cells (BMS) exhibited improved survival compared with matched BMS controls. Bronchoalveolar lavage fluids obtained on day 7 post-BMT from iNOS(-/-) BMS mice contained less tumor necrosis factor-alpha and interferon-gamma, indicating that.NO stimulated the production of proinflammatory cytokines. However, despite suppressed inflammation and decreased nitrotyrosine staining, iNOS(-/-) mice given both donor T cells and Cy (BMS + Cy) died earlier than iNOS-sufficient BMS + Cy mice. Alveolar macrophages from iNOS(-/-) BMS + Cy mice did not produce.NO but persisted to generate strong oxidants as assessed by the oxidation of the intracellular fluorescent probe 2',7'-dichlorofluorescin. We concluded that.NO amplifies T cell-dependent inflammation and addition of Cy exacerbates.NO-dependent mortality. However, the lack of.NO during Cy-induced oxidant stress decreases survival of T cell-recipient mice, most likely by generation of.NO-independent toxic oxidants.

Topics: Animals; Bone Marrow Transplantation; Bronchoalveolar Lavage Fluid; Cyclophosphamide; Female; Immunosuppressive Agents; Interferon-gamma; Macrophages; Mice; Mice, Congenic; Mice, Inbred C57BL; Mice, Knockout; Monocytes; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitrites; Oxidative Stress; Pneumonia; Survival Rate; T-Lymphocytes; Tumor Necrosis Factor-alpha; Tyrosine

We have previously shown that the ability of overnight pretreatment with lipopolysaccharide (LPS) to suppress alveolar macrophage (AM) leukotrienes (LT) synthesis is explained by induction of nitric oxide (NO), and reactive oxygen intermediates (ROI). More recently we have demonstrated that the generation of peroxynitrite (ONOO-) from the combination of NO and ROI directly nitrotyrosinates the 5-lipoxygenase (5-LO) enzyme and reduces cell-free and intact AM 5-LO metabolism. This effect of ONOO- was associated with nitrotyrosination of the 5-LO enzyme in intact cells and after treatment of recombinant enzyme. We postulated that LPS treatment of cells resulted in activation of 5-LO with the generation of ROI, which in turn led to autoinactivation of the enzyme. In an effort to suppress ROI generated from activation of 5-LO we examined the effect of a direct 5-LO inhibitor on LPS-induced suppression of LT synthesis. Coincubation with the reversible 5-LO inhibitor zileuton during the LPS pretreatment of intact cells dose dependently blocked the inhibition of 5-LO metabolism by LPS. The effect of zileuton on LPS-induced suppression of LT synthesis was similar to that of N-monomethyl-L-arginine. Zileuton had no effect on inducible nitric-oxide synthase induction. Interestingly, zileuton blocked ONOO--induced nitrotyrosination of recombinant 5-LO in a cell-free system as well as of native enzyme in intact cells. Moreover, zileuton blocked the nitrotyrosination of other proteins. We conclude that the suppression of 5-LO activity occurring with LPS treatment can be blocked by zileuton. The mechanism by which zileuton is effective is in part explained by blocking nitrotyrosination of 5-LO.

Topics: Animals; Blotting, Western; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Female; Hydroxyurea; Leukotriene B4; Lipoxygenase Inhibitors; Nitrates; omega-N-Methylarginine; Precipitin Tests; Proteins; Rats; Rats, Wistar; Tyrosine

Evidence of an overload of reactive oxygen species and peroxynitrite, a derivative of nitric oxide, in sporadic amyotrophic lateral sclerosis suggests that peroxynitrite could impair cholinergic functions. Because of the impossibility of obtaining synaptosomes from vertebrate neuromuscular junctions, we used cholinergic synaptosomes purified from Torpedo marmorata electroneurons to characterize the defects triggered by peroxynitrite in more detail. Addition of peroxynitrite or its donor 3-morpholinosydnonimine abolished high-affinity choline uptake and synthesis of acetylcholine from acetate. T. marmorata choline acetyltransferase (ChAT) was impaired to the same extent as bovine brain ChAT. A hallmark of peroxynitrite action is the nitration of tyrosine residues in proteins. Peroxynitrite induced a concentration-dependent appearance of nitrotyrosines in several neuronal proteins from synaptosomes and, more readily, from synaptic vesicles. Peroxynitrite also triggered tyrosine nitrations in purified ChAT. Peroxynitrite-dependent nitrations were impaired when synaptosomes were pretreated with thioreductants (glutathione, N-acetyl cysteine, dithiothreitol) or antioxidants (uric acid, melatonin, bovine serum albumin, desferrioxamine). Deleterious effects of peroxynitrite on choline transport and ChAT activity were prevented by the thioreductants but only partially by the antioxidants, suggesting a mechanism other than tyrosine nitration, which may involve cysteine oxidation. Further development of protective agents acting on choline transport and on ChAT activity may offer interesting therapeutic possibilities with respect to cholinergic dysfunction occurring in neurodegenerative diseases.

Topics: Acetates; Acetylcholine; Animals; Antioxidants; Biological Transport; Carbon Radioisotopes; Choline; Choline O-Acetyltransferase; Drug Interactions; Molsidomine; Nitrates; Oxidants; Reducing Agents; Torpedo; Tyrosine; Uric Acid

Angiotensin II (ANG II) is a well-established participant in many cardiovascular disorders, but the mechanisms involved are not clear. Vascular cell experiments suggest that ANG II is a potent stimulator of free radicals such as superoxide anion, an agent known to inactivate nitric oxide and promote the formation of peroxynitrite. Here we hypothesized that ANG II reduces the efficacy of NO-mediated vascular relaxation and promotes vascular peroxynitrite formation in vivo. ANG II was infused in rats at sub-pressor doses for 3 days. Systolic blood pressure and heart rate were unchanged on day 3 despite significant reductions in plasma renin activity. Thoracic aorta was isolated for functional and immunohistochemical evaluations. No difference in isolated vascular contractile responses to KCI (125 mM), phenylephrine, or ANG II was observed between groups. In contrast, relaxant response to acetylcholine (ACh) was decreased sixfold without a change in relaxant response to sodium nitroprusside. Extensive prevalence of 3-nitrotyrosine (3-NT, a stable biomarker of tissue peroxynitrite formation) immunoreactivity was observed in ANG II-treated vascular tissues and was specifically confined to the endothelium. Digital image analysis demonstrated a significant inverse correlation between ACh relaxant response and 3-NT immunoreactivity. These data demonstrate that ANG II selectively modifies vascular NO control at sub-pressor exposures in vivo. Thus, endothelial dysfunction apparently precedes other established ANG II-induced vascular pathologies, and this may be mediated by peroxynitrite formation in vivo. Wattanapitayakul, S., Weinstein, D. M., Holycross, B. J., Bauer, J. A. Endothelial dysfunction and peroxynitrite formation are early events in angiotensin-induced cardiovascular disorders.

Topics: Acetylcholine; Angiotensin II; Animals; Aorta, Thoracic; Cardiovascular Diseases; Endothelium, Vascular; Hemodynamics; Image Processing, Computer-Assisted; Immunohistochemistry; In Vitro Techniques; Male; Nitrates; Nitroprusside; Phenylephrine; Potassium Chloride; Rats; Rats, Sprague-Dawley; Renin; Tyrosine; Vasoconstriction

Nitrotyrosine produced by NO-mediated reaction is a possible marker for cytotoxicity in brain ischemia. In this study, we aimed to determine whether iNOS is responsible for the nitrotyrosine formation and which type of cell is predominantly nitrated. Fifty-eight wild-type and 28 iNOS knockout male mice were used. Under halothane anesthesia the left middle cerebral artery was occluded for 2 h and reperfused for 0.5 or 15 h. The ratio of nitrotyrosine to total tyrosine (%NO2-Tyr) was measured by means of a hydrolysis/HPLC. After 0.5-h reperfusion, %NO2-Tyr in the ischemic cortex of wild-type and knockout mice amounted to 0.037 +/- 0.040% (n = 8) and 0.064 +/- 0.035% (n = 6), respectively, being significantly higher than that in the sham operation group (n = 7) (P < 0.05). After 15-h reperfusion, nitrotyrosine was detected only in wild-type mice (0.039 +/- 0.025%, n = 7), not in knockout or sham-operated mice (P < 0.05). Immunohistochemical reaction for nitrotyrosine was seen predominantly in the vascular wall in the peri-infarct region of the cerebral cortex in wild-type mice after 15-h reperfusion, but not in corresponding knockout mice. Our data suggest that iNOS is responsible for nitrotyrosine formation in the later phase of reperfusion, and that vascular endothelium is the major site of this reaction, at least in the case of 15-h reperfusion.

Topics: Acidosis; Animals; Blood Glucose; Blood Pressure; Carbon Dioxide; Cerebral Cortex; Cerebrovascular Circulation; Chromatography, High Pressure Liquid; Endothelium, Vascular; Infarction, Middle Cerebral Artery; Ischemic Attack, Transient; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Oxygen; Reperfusion Injury; Tyrosine

Formation of peroxynitrite from NO and O-(*2) is considered an important trigger for cellular tyrosine nitration under pathophysiological conditions. However, this view has been questioned by a recent report indicating that NO and O-(*2) generated simultaneously from (Z)-1-(N-[3-aminopropyl]-N-[4-(3-aminopropylammonio)butyl]-amino) diazen-1-ium-1,2-diolate] (SPER/NO) and hypoxanthine/xanthine oxidase, respectively, exhibit much lower nitrating efficiency than authentic peroxynitrite (Pfeiffer, S. and Mayer, B. (1998) J. Biol. Chem. 273, 27280-27285). The present study extends those earlier findings to several alternative NO/O-(*2)-generating systems and provides evidence that the apparent lack of tyrosine nitration by NO/O-(*2) is due to a pronounced decrease of nitration efficiency at low steady-state concentrations of authentic peroxynitrite. The decrease in the yields of 3-nitrotyrosine was accompanied by an increase in the recovery of dityrosine, showing that dimerization of tyrosine radicals outcompetes the nitration reaction at low peroxynitrite concentrations. The observed inverse dependence on peroxynitrite concentration of dityrosine formation and tyrosine nitration is predicted by a kinetic model assuming that radical formation by peroxynitrous acid homolysis results in the generation of tyrosyl radicals that either dimerize to yield dityrosine or combine with (*)NO(2) radical to form 3-nitrotyrosine. The present results demonstrate that very high fluxes (>2 microM/s) of NO/O-(*2) are required to render peroxynitrite an efficient trigger of tyrosine nitration and that dityrosine is a major product of tyrosine modification caused by low steady-state concentrations of peroxynitrite.

Topics: Dimerization; Flavin Mononucleotide; Free Radicals; Hydrazines; Hypoxanthine; Kinetics; Nitrates; Nitric Oxide; Nitrogen Oxides; Spermine; Tyrosine; Xanthine Oxidase

The hepatic centrilobular necrosis produced by the analgesic/antipyretic acetaminophen correlates with metabolic activation of the drug leading to its covalent binding to protein. However, the molecular mechanism of the toxicity is not known. Recent immunohistochemical analyses using an antinitrotyrosine antiserum indicated that nitrotyrosine protein adducts co-localized with the acetaminophen-protein adducts in the centrilobular cells of the liver. Nitration of proteins is believed to occur by peroxynitrite, a substance formed by the rapid reaction of superoxide with nitric oxide. Nitric oxide and superoxide may be formed by activated Kupffer cells or by other cells. Because we were unable to successfully utilize the commercial antiserum in Western blot analyses of liver fractions, we developed a new antiserum. With our antiserum, liver fractions from saline-treated control and acetaminophen-treated mice were successfully analyzed for nitrated proteins. The immunogen for this new antiserum was synthesized by coupling 3-nitro-4-hydroxybenzoic acid to keyhole limpet hemocyanin. A rabbit immunized with this adduct yielded a high titer of an antiserum that recognized BSA nitrated with peroxynitrite. Immunoblot analysis of nitrated BSA indicated that nitrotyrosine present in a protein sample could be easily detected at levels of 20 pmoles. Immunohistochemical analyses indicated that nitrotyrosine protein adducts were detectable in the centrilobular areas of the liver. Immunoblot analysis of liver homogenates from both saline-treated and acetaminophen-treated mice (300 mg/kg) indicate that the major nitrotyrosine protein adducts produced have molecular weights of 36 kDa, 44 kDa, and 85 kDa. The 85-kDa protein stained with the most intensity. The hepatic homogenates of the acetaminophen- treated mice showed significantly increased levels of all protein adducts.

Topics: Acetaminophen; Adjuvants, Immunologic; Analgesics, Non-Narcotic; Animals; Blotting, Western; Cattle; Chemical and Drug Induced Liver Injury; Enzyme-Linked Immunosorbent Assay; Hemocyanins; Immunoenzyme Techniques; Liver; Male; Mice; Mice, Inbred C57BL; Necrosis; Nitrates; Protein Binding; Proteins; Rabbits; Serum Albumin, Bovine; Sodium Chloride; Tyrosine

The inhibitory effects of various endogenous and synthetic compounds on the nitration and oxidation of L-tyrosine by peroxynitrite were examined. Nitrating and oxidizing activities were monitored by the formation of 3-nitrotyrosine and dityrosine with a HPLC-UV-fluorescence detector system, respectively. Glutathione, serotonin and synthetic sulfur- and selenium-containing compounds inhibited both the nitration and oxidation reaction of L-tyrosine effectively. However, 5-methoxytryptamine, melatonin and alpha-lipoic acid only inhibited the nitration reaction, and enhanced the formation of an oxidation product. This is important evidence that there are different intermediates in the nitrating and oxidizing reactions of L-tyrosine by peroxynitrite. It was suggested that 5-methoxytryptamine, melatonin and alpha-lipoic acid reacted only with the nitrating intermediate of peroxynitrite and inhibited nitration of L-tyrosine. Actually, the DNA strand breakage, which is believed to be a typical reaction of hydroxyl radical-like species, caused by peroxynitrite was not effectively inhibited by 5-methoxytryptamine. 5-Methoxytryptamine, melatonin and alpha-lipoic acid were viewed as useful reagents for investigating the mechanisms of damage by peroxynitrite in vitro.

Topics: Antioxidants; Chromatography, High Pressure Liquid; DNA Damage; Free Radical Scavengers; Indicators and Reagents; Nitrates; Oxidation-Reduction; Plasmids; Spectrophotometry, Ultraviolet; Spin Trapping; Thioctic Acid; Tryptamines; Tyrosine

Carrageenan causes enhanced formation of reactive oxygen species, which contribute to the pathophysiology of inflammation. We have investigated the effects of tempol, a membrane-permeable radical scavenger, in rats subjected to carrageenan-induced pleurisy. Treatment of rats with tempol (10, 30, or 100 mg/kg 15 min prior to carrageenan) attenuated the pleural exudation and the migration of polymorphonuclear cells caused by carrageenan dose dependently. Tempol also attenuated the lung injury (histology) as well as the increase in the tissue levels of myeloperoxidase and malondialdehyde caused by carrageenan in the lung. However, tempol did not inhibit the activity of inducible nitric oxide synthase in the lungs. Immunohistochemical analysis for nitrotyrosine revealed positive staining in lungs from carrageenan-treated rats. Lung tissue sections from carrageenan-treated rats also showed positive staining for poly-(ADP-ribose) synthetase (PARS). The degree of staining for nitrotyrosine and PARS was markedly reduced in tissue sections obtained from carrageenan-treated rats, which had received tempol (100 mg/kg). Furthermore, treatment of rats with tempol significantly reduced (i) the formation of peroxynitrite, (ii) the DNA damage, (iii) the impairment in mitochondrial respiration, and (iv) the fall in the cellular level of NAD(+) observed in macrophages harvested from the pleural cavity of rats treated with carrageenan. Tempol also attenuated the cell injury caused by hydrogen peroxide (1 mM) in cultured human endothelial cells. This study provides the first evidence that tempol, a small molecule which permeates biological membranes and scavenges ROS, attenuates the degree of inflammation and tissue damage associated with carageenan-induced pleurisy in the rat. The mechanisms of the anti-inflammatory effect of tempol are discussed.

Topics: Animals; Carrageenan; Cells, Cultured; Cyclic N-Oxides; DNA Damage; Free Radical Scavengers; Hydrogen Peroxide; Lung; Macrophages; Male; Mitochondria; Nitrates; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Oxidants; Oxygen Consumption; Permeability; Pleurisy; Poly(ADP-ribose) Polymerases; Rats; Rats, Sprague-Dawley; Spin Labels; Tyrosine

Peroxynitrite (ONOO(-)), a reactive oxidant produced by the reaction between nitric oxide and superoxide, was found to diffuse into the platelet cytosol and inhibit arachidonic acid-induced platelet aggregations with IC(50) value of 5.8 +/- 1.2 microM. A fluorescence assay established that ONOO(-) diffused into the platelet cytosol in a manner that was inhibited (50-70%) by 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid, an inhibitor of HCO(3)(-)/Cl(-) anion exchanger. Treatment of platelets with (-)-epigallocatechin gallate (2 microM), a tea polyphenol and inhibitor of tyrosine nitration, abolished the inhibitory effect of ONOO(-) on arachidonate-induced aggregations by 88%. ONOO(-) (50-300 microM), added to platelets 1 min before arachidonic acid, inhibited (20-100%) formation of platelet cyclooxygenase (COX) products thromboxane A(2) and 12-hydroxyheptadecatrienoic acid. Interestingly, simultaneous addition of ONOO(-) and arachidonic acid stimulated eicosanoid production by 20 to 60%. The inhibition of thromboxane A(2) generation correlated with the 5- to 10-fold increase in the 3-nitrotyrosine levels of the platelet COX. Experiments with purified COX-1 and COX-2 also showed 9-fold increase of 3-nitrotyrosine levels, which correlated with decreased (93-98%) production of prostaglandin H(2) when ONOO(-) (50 microM) was added 1 min before arachidonic acid. However, the addition of ONOO(-) (50-100 microM) simultaneously with arachidonic acid increased prostaglandin H(2) formation by 30 to 60%. Thus, the inhibitory effect of ONOO(-) involved nitration of COX tyrosine residues, whereas the stimulatory effect was likely to be a result of ONOO(-) functioning as a peroxide activator of eicosanoid signaling. Increasing doses of ONOO(-) not only inhibited platelet COX but also induced formation of unique eicosanoids: iso-prostaglandin F(2alpha), epoxyhydroxyeicosatrienoic acid, and trans-arachidonic acids, suggesting that OH and NO(2) radicals were generated from ONOO(-) in platelets. Formation of ONOO(-) from NO and superoxide may function as a platelet hormone-like COX regulatory mechanism in inflammatory processes in which large amounts of these molecules are produced.

Topics: Blood Platelets; Cell Membrane; Cell Membrane Permeability; Chromatography, High Pressure Liquid; Cyclooxygenase 1; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Cyclooxygenase Inhibitors; Diffusion; Eicosanoids; Gas Chromatography-Mass Spectrometry; Humans; Immunoblotting; In Vitro Techniques; Indicators and Reagents; Isoenzymes; Membrane Proteins; Nitrates; Oxidants; Platelet Activation; Platelet Aggregation; Prostaglandin-Endoperoxide Synthases; Thromboxane A2; Tyrosine; Vasoconstriction

Peroxynitrite (ONOO(-)), a toxic product of the free radicals nitric oxide and superoxide, has been implicated in the pathogenesis of CNS inflammatory diseases, including multiple sclerosis and its animal correlate experimental autoimmune encephalomyelitis (EAE). In this study we have assessed the mode of action of uric acid (UA), a purine metabolite and ONOO(-) scavenger, in the treatment of EAE. We show that if administered to mice before the onset of clinical EAE, UA interferes with the invasion of inflammatory cells into the CNS and prevents development of the disease. In mice with active EAE, exogenously administered UA penetrates the already compromised blood-CNS barrier, blocks ONOO(-)-mediated tyrosine nitration and apoptotic cell death in areas of inflammation in spinal cord tissues and promotes recovery of the animals. Moreover, UA treatment suppresses the enhanced blood-CNS barrier permeability characteristic of EAE. We postulate that UA acts at two levels in EAE: 1) by protecting the integrity of the blood-CNS barrier from ONOO(-)-induced permeability changes such that cell invasion and the resulting pathology is minimized; and 2) through a compromised blood-CNS barrier, by scavenging the ONOO(-) directly responsible for CNS tissue damage and death.

Topics: Animals; Apoptosis; Blood-Brain Barrier; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Free Radical Scavengers; Mice; Multiple Sclerosis; Nitrates; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Spinal Cord; Tyrosine; Uric Acid

Peroxynitrite (ONOO-), a potent oxidizing and nitrating species, has been linked to covalent modifications of biomolecules in a number of pathological conditions. In S. cerevisiae, a model eukaryotic cell system, ONOO- was found to be more potent than hydrogen peroxide in oxidizing thiols, inducing heat shock proteins (Hsp70) and enhancing the ubiquitination of proteins. As identified by microsequence analysis following immunoprecipitation with anti-nitrotyrosine antibodies, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was especially susceptible to nitration by ONOO- in yeast cells. The activity of this enzyme was strongly inhibited upon steady-state exposure of the cells to low doses of ONOO- in yeast and in cultured rat astrocytes. Thus, ONOO- is a potent stressor in yeast capable of inducing oxidative damage and protein nitration, with GAPDH being a preferential target protein that is efficiently inactivated.

Topics: Animals; Astrocytes; Cell Culture Techniques; Cell Survival; Dithionitrobenzoic Acid; Dose-Response Relationship, Drug; Fungal Proteins; Glyceraldehyde-3-Phosphate Dehydrogenases; HSP70 Heat-Shock Proteins; Hydrogen Peroxide; Molecular Chaperones; Nitrates; Oxidants; Rats; Rats, Wistar; Saccharomyces cerevisiae; Sulfhydryl Compounds; Sulfhydryl Reagents; Tyrosine; Ubiquitins; Up-Regulation

Oxidative stress may increase production of superoxide and nitric oxide, leading to formation of prooxidant peroxynitrite to cause vascular dysfunction. Having found nitrotyrosine residues, a marker of peroxynitrite action, in placental vessels of preeclamptic and diabetic pregnancies, we determined whether vasoreactivity is altered in these placentas and treatment with peroxynitrite produces vascular dysfunction. The responses of diabetic, preeclamptic, and normal placentas to increasing concentrations of the vasoconstrictors U-46619 (10(-9)-10(-7) M) and ANG II (10(-9)-10(-7) M) and the vasodilators glyceryl trinitrate (10(-9)-10(-7) M) and prostacyclin (PGI(2); 10(-8)-10(-6) M) were compared as were responses to these agents in normal placentas before and after treatment with 3.16 x 10(-4) M peroxynitrite for 30 min. Responses to both vasoconstrictors and vasodilators were significantly attenuated in diabetic and preeclamptic placentas compared with controls. Similarly, responses to U-46619, nitroglycerin, and PGI(2), but not ANG II, were significantly attenuated following peroxynitrite treatment. The presence of nitrotyrosine residues confirmed peroxynitrite interaction with placental vessels. Overall, our data suggest that peroxynitrite formation is capable of attenuating vascular responses in the human placenta.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adult; Angiotensin II; Antihypertensive Agents; Diabetes Mellitus, Type 1; Epoprostenol; Female; Fetus; Humans; In Vitro Techniques; Muscle, Smooth, Vascular; Nitrates; Nitric Oxide; Nitroglycerin; Oxidative Stress; Placenta; Pre-Eclampsia; Pregnancy; Reactive Oxygen Species; Tyrosine; Vasoconstriction; Vasoconstrictor Agents; Vasodilator Agents

Peroxynitrite, formed by the reaction between nitric oxide and superoxide, has been shown to induce protein nitration, which compromises protein function. We hypothesized that peroxynitrite may regulate cytokine function during inflammation. To test this hypothesis, the neutrophil chemotactic activity (NCA) of interleukin-8 (IL-8) incubated with peroxynitrite was evaluated. Peroxynitrite attenuated IL-8 NCA in a dose-dependent manner (p < 0.01) but did not significantly reduce NCA induced by leukotriene B(4) or complement-activated serum. The reducing agents, dithionite, deferoxamine, and dithiothreitol, reversed and exogenous L-tyrosine abrogated the peroxynitrite-induced NCA inhibition. Papa-NONOate [N-(3-ammoniopropyl)-N-(n-propyl)amino]diazen-1-ium-1, 2-dialase or sodium nitroprusside, NO donors, or a combination of xanthine and xanthine oxidase to generate superoxide did not show an inhibitory effect on NCA induced by IL-8. In contrast, small amounts of SIN-1, a peroxynitrite generator, caused a concentration-dependent inhibition of NCA by IL-8. Consistent with its capacity to reduce NCA, peroxynitrite treatment reduced IL-8 binding to neutrophils. Nitrotyrosine was detected in the IL-8 incubated with peroxynitrite by enzyme-linked immunosorbent assay. These findings are consistent with nitration of tyrosine by peroxynitrite with subsequent inhibition of IL-8 binding to neutrophils and a reduction in NCA and suggest that oxidants may play an important role in regulation of IL-8-induced neutrophil chemotaxis.

Topics: Chemotaxis, Leukocyte; Humans; Interleukin-8; Leukotriene B4; Molsidomine; Neutrophils; Nitrates; Nitric Oxide Donors; Tyrosine

Peroxynitrite can nitrate tyrosine residues of proteins. We examined nitrotyrosine-containing proteins in cerebrospinal fluid of 66 patients with neurogenic disease by immunoblot analysis. Nitrated tyrosine residue-containing protein was observed in the cerebrospinal fluid and was concluded to be manganese superoxide dismutase (Mn-SOD). The nitrated Mn-SOD level was strikingly elevated in amyotrophic lateral sclerosis patients and was slightly increased in Alzheimer's and Parkinson's disease patients, whereas an elevated Mn-SOD level was observed only in progressive supranuclear palsy group.

Topics: Adult; Aged; Alzheimer Disease; Antibodies; Biomarkers; Female; Humans; Male; Middle Aged; Nitrates; Nitrogen; Oxidative Stress; Parkinson Disease; Precipitin Tests; Superoxide Dismutase; Tyrosine

Ribonucleotide reductase activity is rate-limiting for DNA synthesis, and inhibition of this enzyme supports cytostatic antitumor effects of inducible NO synthase. The small R2 subunit of class I ribonucleotide reductases contains a stable free radical tyrosine residue required for activity. This radical is destroyed by peroxynitrite, which also inactivates the protein and induces nitration of tyrosine residues. In this report, nitrated residues in the E. coli R2 protein were identified by UV-visible spectroscopy, mass spectrometry (ESI-MS), and tryptic peptide sequencing. Mass analysis allowed the detection of protein R2 as a native dimer with two iron clusters per subunit. The measured mass was 87 032 Da, compared to a calculated value of 87 028 Da. Peroxynitrite treatment preserved the non-heme iron center and the dimeric form of the protein. A mean of two nitrotyrosines per E. coli protein R2 dimer were obtained at 400 microM peroxynitrite. Only 3 out of the 16 tyrosines were nitrated, including the free radical Tyr122. Despite its radical state, that should favor nitration, the buried Tyr122 was not nitrated with a high yield, probably owing to its restricted accessibility. Dose-response curves for Tyr122 nitration and loss of the free radical were superimposed. However, protein R2 inactivation was higher than nitration of Tyr122, suggesting that nitration of the nonconserved Tyr62 and Tyr289 might be also of importance for peroxynitrite-mediated inhibition of E. coli protein R2.

Topics: Amino Acid Sequence; Catalysis; Chromatography, High Pressure Liquid; Conserved Sequence; Dimerization; Dose-Response Relationship, Drug; Escherichia coli; Free Radicals; Hydrogen-Ion Concentration; Mass Spectrometry; Models, Molecular; Molecular Weight; Nitrates; Nonheme Iron Proteins; Protein Structure, Quaternary; Ribonucleotide Reductases; Sequence Analysis, Protein; Spectrophotometry; Tyrosine

We carried out a partial ligation of the sciatic nerve in rats to induce nerve injury and neuropathic hyperalgesia. We showed that nitrotyrosine, a marker of peroxynitrite activity, was formed after partial nerve injury. Double-labelling immunohistochemistry showed that nitrotyrosine-immunoreactive cells were mainly macrophages and Schwann cells. Daily treatment with uric acid, a scavenger of peroxynitrite, decreased nitrotyrosine formation in the injured sciatic nerve, and produced concomitant alleviation of thermal hyperalgesia and Wallerian degeneration. These results provide the first evidence that peroxynitrite is formed after partial nerve injury, and contributes to the initiation of thermal hyperalgesia and Wallerian degeneration. We hypothesize that uric acid alleviates hyperalgesia and Wallerian degeneration by inhibiting oxidative damage caused by peroxynitrite and possibly also by decreasing the production of other inflammatory mediators such as prostaglandins.

Topics: Animals; Hyperalgesia; Hyperthermia, Induced; Immunohistochemistry; Male; Nerve Compression Syndromes; Nerve Crush; Nerve Degeneration; Neuralgia; Nitrates; Peripheral Nerve Injuries; Peripheral Nerves; Rats; Rats, Wistar; Tyrosine; Uric Acid

Reactive nitrogen species, including nitrogen oxides (N(2)O(3) and N(2)O(4)), peroxynitrite (ONOO(-)), and nitryl chloride (NO(2)Cl), have been implicated as causes of inflammation and cancer. We studied reactions of secondary amines with peroxynitrite and found that both N-nitrosamines and N-nitramines were formed. Morpholine was more easily nitrosated by peroxynitrite at alkaline pH than at neutral pH, whereas its nitration by peroxynitrite was optimal at pH 8.5. The yield of nitrosomorpholine in this reaction was 3 times higher than that of nitromorpholine at alkaline pH, whereas 2 times more nitromorpholine than nitrosomorpholine was formed at pH <7.5. For the morpholine-peroxynitrite reaction, nitration was enhanced by low concentrations of bicarbonate, but was inhibited by excess bicarbonate. Nitrosation was inhibited by excess bicarbonate. On this basis, we propose a free radical mechanism, involving one-electron oxidation by peroxynitrite of secondary amines to form amino radicals (R(2)N(*)), which react with nitric oxide ((*)NO) or nitrogen dioxide ((*)NO(2)) to yield nitroso and nitro secondary amines, respectively. Reaction of morpholine with NO(*) and superoxide anion (O(2)(*)(-)), which were concomitantly produced from spermine NONOate and by the xanthine oxidase systems, respectively, also yielded nitromorpholine, but its yield was <1% of that of nitrosomorpholine. NO(*) alone increased the extent of nitrosomorpholine formation in a dose-dependent manner, and concomitant production of O(2)(*)(-) inhibited its formation. Reactions of morpholine with nitrite plus HOCl or nitrite plus H(2)O(2), with or without addition of myeloperoxidase or horseradish peroxidase, also yielded nitration and nitrosation products, in yields that depended on the reactants. Tyrosine was nitrated easily by synthetic peroxynitrite, by NaNO(2) plus H(2)O(2) with myeloperoxidase, and by NaNO(2) plus H(2)O(2) under acidic conditions. Nitrated secondary amines, e.g., N-nitroproline, could be identified as specific markers for endogenous nitration mediated by reactive nitrogen species.

Topics: Aniline Compounds; Hydrogen Peroxide; Hypochlorous Acid; Morpholines; Nitrates; Nitrobenzenes; Nitrosamines; Sodium Nitrite; Superoxides; Tyrosine

Peroxynitrite has been receiving increasing attention as the pathogenic mediator of nitric oxide cytotoxicity. In most cases, the contribution of peroxynitrite to diseases has been inferred from detection of 3-nitrotyrosine in injured tissues. However, presently it is known that other nitric oxide-derived species can also promote protein nitration. Mechanistic details of protein nitration remain under discussion even in the case of peroxynitrite, although recent literature data strongly suggest a free radical mechanism. Here, we confirm the free radical mechanism of tyrosine modification by peroxynitrite in the presence and in the absence of the bicarbonate-carbon dioxide pair by analyzing the stable tyrosine products and the formation of the tyrosyl radical at pH 5.4 and 7.4. Stable products, 3-nitrotyrosine, 3-hydroxytyrosine, and 3, 3-dityrosine, were identified by high performance liquid chromatography and UV spectroscopy. The tyrosyl radical was detected by continuous-flow and spin-trapping electron paramagnetic resonance (EPR). 3-Hydroxytyrosine was detected at pH 5.4 and its yield decreased in the presence of the bicarbonate-carbon dioxide pair. In contrast, the yields of the tyrosyl radical increased in the presence of the bicarbonate-carbon dioxide pair and correlated with the yields of 3-nitrotyrosine under all tested experimental conditions. Taken together, the results demonstrate that the promoting effects of carbon dioxide on peroxynitrite-mediated tyrosine nitration is due to the selective reactivity of the carbonate radical anion as compared with that of the hydroxyl radical. Colocalization of 3-hydroxytyrosine and 3-nitrotyrosine residues in proteins may be useful to discriminate between peroxynitrite and other nitrating species.

Topics: Anions; Bicarbonates; Carbon Dioxide; Carbonates; Chromatography, High Pressure Liquid; Electron Spin Resonance Spectroscopy; Free Radicals; Hydrogen-Ion Concentration; Hydroxyl Radical; Hydroxylation; Kinetics; Nitrates; Nitric Oxide; Spectrophotometry, Ultraviolet; Tyrosine

The present study identifies proteins modified by nitration in the plasma of patients with ongoing acute respiratory distress syndrome (ARDS). The proteins modified by nitration in ARDS were revealed by microsequencing and specific antibody detection to be ceruloplasmin, transferrin, alpha(1)-protease inhibitor, alpha(1)-antichymotrypsin, and beta-chain fibrinogen. Exposure to nitrating agents did not deter the chymotrypsin-inhibiting activity of alpha(1)-antichymotrypsin. However, the ferroxidase activity of ceruloplasmin and the elastase-inhibiting activity of alpha(1)-protease inhibitor were reduced to 50.3 +/- 1.6 and 60.3 +/- 5.3% of control after exposure to the nitrating agent. In contrast, the rate of interaction of fibrinogen with thrombin was increased to 193.4 +/- 8.5% of the control value after exposure of fibrinogen to nitration. Ferroxidase activity of ceruloplasmin and elastase-inhibiting activity of the alpha(1)-protease inhibitor in the ARDS patients were significantly reduced (by 81 and 44%, respectively), whereas alpha(1)-antichymotrypsin activity was not significantly altered. Posttranslational modifications of plasma proteins mediated by nitrating agents may offer a biochemical explanation for the reported diminished ferroxidase activity, elevated levels of elastase, and fibrin deposits detected in patients with ongoing ARDS.

Topics: Acute Disease; Adult; alpha 1-Antichymotrypsin; Blood Proteins; Blotting, Western; Carbon Dioxide; Ceruloplasmin; Enzyme Activation; Fibrinogen; Humans; In Vitro Techniques; Nitrates; Nitric Oxide; Oxidative Stress; Precipitin Tests; Respiratory Distress Syndrome; Superoxides; Tyrosine

We assessed whether reactive oxygen-nitrogen intermediates generated by alveolar macrophages (AMs) oxidized and nitrated human surfactant protein (SP) A. SP-A was exposed to lipopolysaccharide (100 ng/ml)-activated AMs in 15 mM HEPES (pH 7.4) for 30 min in the presence and absence of 1.2 mM CO(2). In the presence of CO(2), lipopolysaccharide-stimulated AMs had significantly higher nitric oxide synthase (NOS) activity (as quantified by the conversion of L-[U-(14)C]arginine to L-[U-(14)C]citrulline) and secreted threefold higher levels of nitrate plus nitrite in the medium [28 +/- 3 vs. 6 +/- 1 (SE) nmol. 6.5 h(-1). 10(6) AMs(-1)]. Western blotting studies of immunoprecipitated SP-A indicated that CO(2) enhanced SP-A nitration by AMs and decreased carbonyl formation. CO(2) (0-1.2 mM) also augmented peroxynitrite (0.5 mM)-induced SP-A nitration in a dose-dependent fashion. Peroxynitrite decreased the ability of SP-A to aggregate lipids, and this inhibition was augmented by 1.2 mM CO(2). Mass spectrometry analysis of chymotryptic fragments of peroxynitrite-exposed SP-A showed nitration of two tyrosines (Tyr(164) and Tyr(166)) in the absence of CO(2) and three tyrosines (Tyr(164), Tyr(166), and Tyr(161)) in the presence of 1.2 mM CO(2). These findings indicate that physiological levels of peroxynitrite, produced by activated AMs, nitrate SP-A and that CO(2) increased nitration, at least partially, by enhancing enzymatic nitric oxide production.

Topics: Carbon Dioxide; Cells, Cultured; Enzyme Activation; Epithelial Cells; Humans; Hypercapnia; Lipid Metabolism; Lipopolysaccharides; Macrophages, Alveolar; Mass Spectrometry; Molsidomine; Nitrates; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitrites; Oxidation-Reduction; Proteolipids; Pulmonary Surfactant-Associated Protein A; Pulmonary Surfactant-Associated Proteins; Pulmonary Surfactants; Respiratory Distress Syndrome; Tyrosine

Proximal tubular cells produce nitric oxide (NO.). We have shown that under hyperglycemic conditions, cultured proximal tubular cells express cytochrome P450 2E1, which is capable of producing superoxide (O2.). NO. and O2. react to form peroxynitrite (ONOO.), a powerful oxidant. ONOO. nitrosylates tyrosine moieties on proteins causing tissue damage. Our hypothesis is that ONOO. plays a role in early diabetic tubular damage and perhaps disease progression.. Renal biopsies from patients with diabetic nephropathy (DM), acute allograft rejection (AAR), acute allograft tubular necrosis (ATN), and glomerulonephritis (GN) were obtained. Normal kidney specimens were taken from nephrectomy samples (N = 10 for each group). The tissues were examined for the presence of nitrotyrosine using an immunoperoxidase technique with a polyclonal antibody. Samples were then arbitrarily scored, and the results analyzed (analysis of variance and Student's t-test for unpaired data). The number of apoptotic cells in a sample of tubules in each biopsy was also assessed.. The DM biopsies showed increased staining for nitrotyrosine in proximal tubules (P = 0.0001) and in the thin limb of the loop of Henle (P = 0.0006) compared with all other groups. There was increased staining in the ascending and distal tubules in GN as compared to DM and ATN (P = 0.01). Nitrotyrosine was also found in all distal tubules and collecting ducts, including normals. There was no difference in the number of apoptotic tubular cells in diabetics compared with controls.. To our knowledge, these data provide the first evidence for the presence of nitrotyrosine in both normal and diseased kidneys. The significance of the findings in normals is unclear, but could be due to activation of constitutive NOS. However, the study clearly demonstrates increased production of ONOO. in proximal tubules of patients with DM, and suggests that oxidant injury of the proximal tubules plays an important part in the pathogenesis of DM.

Topics: Adolescent; Adult; Aged; Child; Diabetic Nephropathies; Female; Humans; Immunohistochemistry; Kidney; Male; Middle Aged; Nitrates; Tyrosine

The nitration of protein tyrosine residues by peroxynitrous acid has been associated with pathological conditions. Here it is shown, using a sensitive competitive enzyme-linked immunosorbent assay and immunoblotting for nitrotyrosine, that spontaneous nitration of specific proteins occurs during a physiological process, the activation of platelets by collagen. One of the main proteins nitrated is vasodilator-stimulated phosphoprotein. Endogenous synthesis of nitric oxide and activity of cyclo-oxygenase were required for the nitration of tyrosine. The nitration was mimicked by addition of peroxynitrite to unstimulated platelets, although the level of nitrotyrosine formation was greater and its distribution among the proteins was less specific.

Topics: Aspirin; Blood Platelets; Blood Proteins; Blotting, Western; Cell Adhesion Molecules; Collagen; Cyclooxygenase Inhibitors; Cytosol; Enzyme-Linked Immunosorbent Assay; Humans; Membrane Proteins; Microfilament Proteins; Molecular Weight; NG-Nitroarginine Methyl Ester; Nitrates; Nitric Oxide Synthase; Nitrous Acid; Oxidants; Peroxynitrous Acid; Phosphoproteins; Platelet Activation; Thrombin; Tyrosine

Peroxynitrite (PN), the product of nitric oxide (NO) reacted with superoxide, is generated at sites of inflammation. Nitrotyrosine (NT), a marker of PN formation, is abundant in lesions of acute experimental autoimmune encephalomyelitis (EAE), and in active multiple sclerosis (MS) plaques. To determine whether PN plays a role in EAE pathogenesis, mice induced to develop EAE were treated with a catalyst specific for the decomposition of PN. Because this catalyst has no effect upon NO, using it allowed differentiation of PN-mediated effects from NO-mediated effects. Mice receiving the PN decomposition catalyst displayed less severe clinical disease, and less inflammation and demyelination than control mice. Encephalitogenic T cells could be recovered from catalyst-treated mice, indicating that the PN decomposition catalyst blocked the pathogenic action of PN at the effector stage of EAE, but was not directly toxic to encephalitogenic T cells. PN plays an important role distinct from that of NO in the pathogenesis of EAE, a major model for MS.

Topics: Animals; Catalysis; Central Nervous System; Encephalomyelitis, Autoimmune, Experimental; Female; Ferric Compounds; Immunohistochemistry; Lymphocyte Transfusion; Lymphocytes; Metalloporphyrins; Mice; Mice, Inbred Strains; Nitrates; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Tyrosine

Lipopolysaccharide (LPS)-induced renal oxidant injury and the role of nitric oxide (NO) were evaluated using the inducible nitric oxide synthase (iNOS) inhibitor L-iminoethyl-lysine (L-NIL). One group of male rats received LPS (Salmonella minnesota; 2 mg/kg, i.v.). A second group received LPS plus L-NIL (3 mg/kg, i.p.). A third group received saline i.v. At 6 hr, iNOS protein was induced in the kidney cortex, and plasma nitrate/nitrite levels were increased from 4 +/- 2 nmol/mL in the Saline group to 431 +/- 23 nmol/mL in the LPS group. The value for the LPS + L-NIL group was reduced significantly to 42 +/- 9 nmol/mL. LPS increased blood urea nitrogen levels from 13 +/- 1 to 47 +/- 3 mg/dL. LPS + L-NIL reduced these levels significantly to 29 +/- 2 mg/dL. Plasma creatinine levels were unchanged in all groups. Tissue lipid peroxidation products in the kidney were increased from 0.16 +/- 0.01 nmol/mg in the Saline group to 0.30 +/- 0.03 nmol/mg in the LPS group. LPS + L-NIL reduced the values significantly to 0.22 +/- 0.02 nmol/mg. Intracellular glutathione levels were decreased in the kidneys from 1.32 +/- 0.1 nmol/mg in the Saline group to 0.66 +/- 0.08 nmol/mg in the LPS group. LPS + L-NIL increased the levels significantly to 0.99 +/- 0.13 nmol/mg. LPS increased the 3-nitrotyrosine-protein adducts in renal tubules as detected by immunohistochemistry, indicating the generation of peroxynitrite. L-NIL decreased adduct formation. These data indicated that LPS-induced NO generation resulted in peroxynitrite formation and oxidant stress in the kidney and that inhibitors of iNOS may offer protection against LPS-induced renal toxicity.

Topics: Animals; Blood Urea Nitrogen; Blotting, Western; Creatine; Enzyme Inhibitors; Glutathione; Immunohistochemistry; Kidney; Lipid Peroxidation; Lipopolysaccharides; Lysine; Male; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitrites; Oxidative Stress; Rats; Rats, Sprague-Dawley; Tyrosine

Poly(ADP-ribose) synthase (PARS), an abundant nuclear protein, has been described as an important candidate for mediation of neurotoxicity by nitric oxide. However, in cerebral ischemia, excessive PARS activation may lead to energy depletion and exacerbation of neuronal damage. We examined the effect of inhibiting PARS on the (a) degree of cerebral injury, (b) process of inflammatory responses, and (c) functional outcomes in a neonatal rat model of focal ischemia. We demonstrate that administration of 3-aminobenzamide, a PARS inhibitor, leads to a significant reduction of infarct volume: 63 +/- 2 (untreated) versus 28 +/- 4 mm(3) (treated). The neuroprotective effects currently observed 48 h postischemia hold up at 7 and 17 days of survival time and attenuate neurological dysfunction. Inhibition of PARS activity, demonstrated by a reduction in poly(ADP-ribose) polymer formation, also reduces neutrophil recruitment and levels of nitrotyrosine, an indicator of peroxynitrite generation. Taken together, our results demonstrate that PARS inhibition reduces ischemic damage and local inflammation associated with reperfusion and may be of interest for the treatment of neonatal stroke.

Topics: Animals; Animals, Newborn; Benzamides; Brain Ischemia; Cell Death; Cerebral Infarction; Encephalitis; Female; Male; Motor Activity; Neurologic Examination; Neuroprotective Agents; Neutrophils; Nitrates; Poly(ADP-ribose) Polymerase Inhibitors; Polymers; Rats; Rats, Wistar; Reperfusion Injury; Signal Transduction; Stroke; Time Factors; Tyrosine

Evidence supporting increased nitric oxide (NO) in asthma is substantial, although the cellular and molecular mechanisms leading to increased NO are not known. Here, we provide a clear picture of the events regulating NO synthesis in the human asthmatic airway in vivo. We show that human airway epithelium has abundant expression of NO synthase II (NOSII) due to continuous transcriptional activation of the gene in vivo. Individuals with asthma have higher than normal NO concentrations and increased NOSII mRNA and protein due to transcriptional regulation through activation of Stat1. NOSII mRNA expression decreases in asthmatics receiving inhaled corticosteroid, treatment effective in reducing inflammation in asthmatic airways. In addition to transcriptional mechanisms, post-translational events contribute to increased NO synthesis. Specifically, high output production of NO is fueled by a previously unsuspected increase in the NOS substrate, l -arginine, in airway epithelial cells of asthmatic individuals. Finally, nitration of proteins in airway epithelium provide evidence of functional consequences of increased NO. In conclusion, these studies define multiple mechanisms that function coordinately to support high level NO synthesis in the asthmatic airway. These findings represent a crucial cornerstone for future therapeutic strategies aimed at regulating NO synthesis in asthma.

Topics: Adult; Alternative Splicing; Arginine; Asthma; Bronchi; Bronchoalveolar Lavage Fluid; DNA-Binding Proteins; Epithelial Cells; Female; Fluocinolone Acetonide; Humans; Interferon-gamma; Janus Kinase 1; Male; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Protein Processing, Post-Translational; Protein-Tyrosine Kinases; Reactive Oxygen Species; RNA, Messenger; STAT1 Transcription Factor; Trans-Activators; Transcription, Genetic; Tyrosine

Peroxynitrite is a strong oxidant involved in cell injury. In tissues, most of peroxynitrite reacts preferentially with CO(2) or hemoproteins, and these reactions affect its fate and toxicity. CO(2) promotes tyrosine nitration but reduces the lifetime of peroxynitrite, preventing, at least in part, membrane crossing. The role of hemoproteins is not easily predictable, because the heme intercepts peroxynitrite, but its oxidation to ferryl species and tyrosyl radical(s) may catalyze tyrosine nitration. The modifications induced by peroxynitrite/CO(2) on oxyhemoglobin were determined by mass spectrometry, and we found that alphaTyr42, betaTyr130, and, to a lesser extent, alphaTyr24 were nitrated. The suggested nitration mechanism is tyrosyl radical formation by long-range electron transfer to ferrylhemoglobin followed by a reaction with (*)NO(2). Dityrosine (alpha24-alpha42) and disulfides (beta93-beta93 and alpha104-alpha104) were also detected, but these cross-linkings were largely due to modifications occurring under the denaturing conditions employed for mass spectrometry. Moreover, immunoelectrophoretic techniques showed that the 3-nitrotyrosine content of oxyhemoglobin sharply increased only in molar excess of peroxynitrite, thus suggesting that this hemoprotein is not a catalyst of nitration. The noncatalytic role may be due to the formation of the nitrating species (*)NO(2) mainly in molar excess of peroxynitrite. In agreement with this hypothesis, oxyhemoglobin strongly inhibited tyrosine nitration of a target dipeptide (Ala-Tyr) and of membrane proteins from ghosts resealed with oxyhemoglobin. Erythrocytes were poor inhibitors of Ala-Tyr nitration on account of the membrane barrier. However, at the physiologic hematocrit, Ala-Tyr nitration was reduced by 65%. This "sink" function was facilitated by the huge amount of band 3 anion exchanger on the cell membrane. We conclude that in blood oxyhemoglobin is a peroxynitrite scavenger of physiologic relevance.

Topics: Ascorbic Acid; Carbon Dioxide; Dipeptides; Erythrocytes; Free Radical Scavengers; Globins; Humans; Immunoelectrophoresis; Mass Spectrometry; Nitrates; Oxyhemoglobins; Tyrosine

The aim of the present study was to investigate the protective effect of the peroxynitrite decomposition catalyst 5,10,15, 20-tetrakis(2,4,6-trimethyl-3,5-disulfonatophenyl)-porphyrinato iron (III) (FeTMPS) in a model of splanchnic artery occlusion shock (SAO). SAO shock was induced in rats by clamping both the superior mesenteric artery and the celiac trunk for 45 min, followed by release of the clamp (reperfusion). At 60 min after reperfusion, animals were killed for histological examination and biochemical studies. There was a marked increase in the oxidation of dihydrorhodamine 123 to rhodamine (a marker of peroxynitrite-induced oxidative processes) in the plasma of the SAO-shocked rats after reperfusion, but not during ischemia alone. Immunohistochemical examination demonstrated a marked increase in the immunoreactivity to nitrotyrosine, an index of nitrogen species such as peroxynitrite, in the necrotic ileum in shocked rats. SAO-shocked rats developed a significant increase of tissue myeloperoxidase and malonaldehyde activity, and marked histological injury to the distal ileum. SAO shock was also associated with a significant mortality (0% survival at 2 h after reperfusion). Reperfused ileum tissue sections from SAO-shocked rats showed positive staining for P-selectin localized mainly in the vascular endothelial cells. Ileum tissue sections obtained from SAO-shocked rats and stained with antibody to ICAM-1 showed a diffuse staining. Administration of FeTMPS significantly reduced ischemia/reperfusion injury in the bowel, and reduced lipid and the production of peroxynitrite during reperfusion. Treatment with PN catalyst also markedly reduced the intensity and degree of P-selectin and ICAM-1 staining in tissue sections from SAO-shocked rats and improved survival. Our results clearly demonstrate that peroxynitrite decomposition catalysts exert a protective effect in SAO and that this effect may be due to inhibition of the expression of adhesion molecules and the tissue damage associated with peroxynitrite-related pathways.

Topics: Animals; Blood Pressure; Catalysis; Celiac Artery; Disease Models, Animal; Endothelium, Vascular; Ferric Compounds; Ileum; Intercellular Adhesion Molecule-1; Leukocyte Count; Lipid Peroxidation; Male; Malondialdehyde; Metalloporphyrins; Nitrates; Nitric Oxide; Nitrites; Oxidative Stress; P-Selectin; Peroxidase; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Rhodamines; Splanchnic Circulation; Tyrosine

During pancreatic tumorigenesis, the equilibrium between cell survival and cell death is altered, allowing aggressive neoplasia and resistance to radiation and chemotherapy. Local oxidative stress is one mechanism regulating programmed cell death and growth and may contribute to both tumor progression and suppression. Our recent in situ immunohistochemical studies demonstrated that levels of total nitrotyrosine, a footprint of the reactive nitrogen species peroxynitrite, are elevated in human pancreatic ductal adenocarcinomas. In this study, quantitative HPLC-EC techniques demonstrated a 21- to 97-fold increase in the overall levels of nitrotyrosine of human pancreatic tumor extracts compared to normal pancreatic extracts. Western blot analysis of human pancreatic tumor extracts showed that tyrosine nitration was restricted to a few specific proteins. Immunoprecipitation coupled with Western analysis identified c-Src tyrosine kinase as a target of both tyrosine nitration and tyrosine phosphorylation. Peroxynitrite treatment of human pancreatic carcinoma cells in vitro resulted in increased tyrosine nitration and tyrosine phosphorylation of c-Src kinase, increased (>2-fold) c-Src kinase activity, and increased association between c-Src kinase and its downstream substrate cortactin. Collectively, these observations suggest that peroxynitrite-mediated tyrosine nitration and tyrosine phosphorylation of c-Src kinase may lead to enhanced tyrosine kinase signaling observed during pancreatic ductal adenocarcinoma growth and metastasis.

Topics: Adenocarcinoma; Blotting, Western; Chromatography, High Pressure Liquid; Cortactin; Humans; Microfilament Proteins; Nitrates; Pancreatic Neoplasms; Phosphorylation; Phosphotyrosine; Precipitin Tests; Protein Conformation; Protein Folding; Proto-Oncogene Proteins pp60(c-src); Tumor Cells, Cultured; Tyrosine

Selective cardiotoxicity of doxorubicin remains a significant and dose-limiting clinical problem. The mechanisms involved have not been fully defined but may involve the production of reactive oxygen species and/or alteration of cardiac energetics. Here, we tested the hypotheses that doxorubicin causes left ventricular dysfunction in mice and is associated with dysregulation of nitric oxide in cardiac tissue, leading to the accumulation of 3-nitrotyrosine (a biomarker of peroxynitrite formation). Animals were dosed with doxorubicin (20 mg/kg i.p.), and left ventricular performance was assessed in vivo using M-mode and Doppler echocardiography. Five days after doxorubicin administration, left ventricular fractional shortening, cardiac output, and stroke volume parameters were significantly reduced relative to control values (30.0 +/- 3.6 versus 46.1 +/- 1. 6%, 8.9 +/- 0.9 versus 11.5 +/- 0.6 ml/min, and 21.2 +/- 0.1 versus 29.5 +/- 0.1 microl for doxorubicin versus control, P <.05). Statistically significant (P <.05) increases in the immunoprevalence of myocardial inducible nitric oxide synthase (33 +/- 18 versus 9 +/- 2%, via quantitative image analysis) and 3-nitrotyrosine formation (56 +/- 24 versus 0.3 +/- 0.4%) were also observed after doxorubicin. Correlation analyses revealed a highly significant inverse relationship between left ventricular fractional shortening and cardiac 3-nitrotyrosine immunoprevalence (P <.01). No such relationship was observed for inducible nitric oxide synthase. Western blot analyses of cardiac myofibrillar fractions revealed extensive nitration of an abundant 40-kDa protein, shown to be the myofibrillar isoform of creatine kinase. These data demonstrate that alteration of cardiac nitric oxide control and attendant peroxynitrite formation may be an important contributor to doxorubicin-induced cardiac dysfunction. Furthermore, nitration of key myofibrillar proteins and alteration of myocyte energetics are implicated.

Topics: Animals; Antibiotics, Antineoplastic; Creatine Kinase; Doxorubicin; Immunohistochemistry; Male; Mice; Myocardium; Nitrates; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Tyrosine; Ventricular Dysfunction, Left

In previous studies of transgenic sickle cell mice, increased renal expression of inducible nitric oxide synthase (iNOS) and endothelial cell isoform of NOS (EcNOS) was found by Western blot and immunohistochemistry. In addition, putative evidence of peroxynitrite (ONOO-) formation was found in the form of positive immunostaining and immunoblot for nitrotyrosine. Apoptosis was also detected by DNA strand breakage and TUNEL assay. The present study was carried out to examine the role of NO/ONOO- in mediating renal tubular cell apoptosis in sickle cell mouse kidneys.. Mercaptoethylguanidine (MEG), a compound that selectively inhibits iNOS and also is a scavenger of ONOO-, was administered intraperitoneally over a five-day period to control and betas mice. Immunohistochemistry of iNOS and nitrotyrosine, DNA electrophoresis, ApoTACS assay for apoptosis, and Western blot of poly(ADP-ribose) polymerase (PARP) were carried out.. MEG administration virtually eliminated renal immunostaining of iNOS and nitrotyrosine and prevented DNA strand breakage. In addition, Western blot analysis of PARP, a nuclear DNA-reparative enzyme activated in response to DNA strand breakage, was found to be cleavaged in hypoxic betas mice, but was partially protected in MEG-treated betas hypoxic mice. Finally, apoptosis was markedly reduced by MEG in betas hypoxic mice.. These observations provide evidence that NO and/or ONOO- are responsible for initiating cell damage, which leads to apoptosis in sickle cell mouse kidneys.

Topics: Alanine Transaminase; Anemia, Sickle Cell; Animals; Apoptosis; Aspartate Aminotransferases; Blotting, Western; Cell Hypoxia; DNA Fragmentation; Electrophoresis, Agar Gel; Enzyme Inhibitors; Guanidines; Immunohistochemistry; In Situ Nick-End Labeling; Kidney Diseases; Kidney Tubules; Mice; Mice, Inbred C57BL; Mice, Transgenic; Nitrates; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Poly(ADP-ribose) Polymerases; Tyrosine

Helicobacter pylori can produce a persistent infection in the human stomach, where chronic and active inflammation, including the infiltration of phagocytes such as neutrophils and monocytes, is induced. H. pylori may have a defense system against the antimicrobial actions of phagocytes. We studied the defense mechanism of H. pylori against host-derived peroxynitrite (ONOO(-)), a bactericidal metabolite of nitric oxide, focusing on the role of H. pylori urease, which produces CO(2) and NH(3) from urea and is known to be an essential factor for colonization. The viability of H. pylori decreased in a time-dependent manner with continuous exposure to 1 microM ONOO(-), i.e., 0.2% of the initial bacteria remained after a 5-min treatment without urea. The bactericidal action of ONOO(-) against H. pylori was significantly attenuated by the addition of 10 mM urea, the substrate for urease, whereas ONOO(-)-induced killing of a urease-deficient mutant of H. pylori or Campylobacter jejuni, another microaerophilic bacterium lacking urease, was not affected by the addition of urea. Such a protective effect of urea was potentiated by supplementation with exogenous urease, and it was almost completely nullified by 10 microM flurofamide, a specific inhibitor of urease. The bactericidal action of ONOO(-) was also suppressed by the addition of 20 mM NaHCO(3) but not by the addition of 20 mM NH(3). In addition, the nitration of L-tyrosine of H. pylori after treatment with ONOO(-) was significantly reduced by the addition of urea or NaHCO(3), as assessed by high-performance liquid chromatography with electrochemical detection. These results suggest that H. pylori-associated urease functions to produce a potent ONOO(-) scavenger, CO(2)/HCO(3)(-), that defends the bacteria from ONOO(-) cytotoxicity. The protective effect of urease may thus facilitate sustained bacterial colonization in the infected gastric mucosa.

Topics: Ammonia; Anti-Bacterial Agents; Carbon Dioxide; Drug Interactions; Helicobacter pylori; Microbial Sensitivity Tests; Nitrates; Oxidants; Sodium Bicarbonate; Tyrosine; Urea; Urease

Peroxynitrite is a mediator molecule in inflammation, and its biological properties are being studied extensively. Flavonoids, which are natural plant constituents, protect against peroxynitrite and thereby could play an anti-inflammatory role. Procyanidin oligomers of different sizes (monomer through nonamer), isolated from the seeds of Theobroma cacao, were recently examined for their ability to protect against peroxynitrite-dependent oxidation of dihydrorhodamine 123 and nitration of tyrosine and were found to be effective in attenuating these reactions. The tetramer was particularly efficient at protecting against oxidation and nitration reactions. Epicatechin oligomers found in cocoa powder and chocolate may be a potent dietary source for defense against peroxynitrite.

Topics: Biflavonoids; Cacao; Catechin; Chromatography, High Pressure Liquid; Drug Interactions; Nitrates; Oxidants; Oxidation-Reduction; Proanthocyanidins; Rhodamines; Structure-Activity Relationship; Tyrosine

Since nitric oxide (NO) can be involved in multiple physiological and pathological functions, we evaluated its possible involvement and that of peroxynitrite in the pathogenesis of rhinitis. Inferior nasal turbinates were obtained from allergic rhinitis and nonallergic rhinitis patients during corrective nasal surgery. The expressions of the inducible form of nitric oxide synthase (iNOS) and the production of peroxynitrite and its metabolite 3-nitrotyrosine were examined by immunohistochemistry in consecutive tissue sections. Each section (or tissue compartment) was given a score of 0-4 according to the labeling intensity seen, with the highest number representing the highest labeling intensity. The results showed that iNOS expression was present mainly in the mucosal epithelium, vascular endothelium, and submucosal glands. A significant difference was only observed in the labeling scores of glandular tissues of the allergic group, which had a higher iNOS labeling score. We also found that sections with a higher iNOS level did not necessarily exhibit a higher 3-nitrotyrosine labeling intensity. These data suggest that iNOS-derived NO may have a role in the pathophysiology of rhinitis, especially the glandular function of allergic nasal mucosa. Moreover, our findings suggest that the production of peroxynitrite in rhinitis patients is not dependent on the level of iNOS alone.

Topics: Adolescent; Adult; Enzyme Induction; Female; Humans; Immunoenzyme Techniques; Male; Middle Aged; Nasal Mucosa; Nitrates; Nitric Oxide Synthase; Rhinitis; Rhinitis, Allergic, Perennial; Turbinates; Tyrosine

Topics: Animals; Blood Physiological Phenomena; Cats; Crystalloid Solutions; Cytokines; Glutathione; Heart; Heart Diseases; Heart Ventricles; Humans; Isotonic Solutions; Myocardial Infarction; Nitrates; Nitric Oxide; Perfusion; Plasma Substitutes; Superoxides; Tyrosine

Reperfusion injury is one of the factors that unfavorably affects stroke outcome and shortens the window of opportunity for thrombolysis. Surges of nitric oxide (NO) and superoxide generation on reperfusion have been demonstrated. Concomitant generation of these radicals can lead to formation of the strong oxidant peroxynitrite during reperfusion.. We have examined the role of NO generation and peroxynitrite formation on reperfusion injury in a mouse model of middle cerebral artery occlusion (2 hours) and reperfusion (22 hours). The infarct volume was assessed by 2,3,5-triphenyl tetrazolium chloride staining; blood-brain barrier permeability was evaluated by Evans blue extravasation. Nitrotyrosine formation and matrix metalloproteinase-9 expression were detected by immunohistochemistry.. Infarct volume was significantly decreased (47%) in animals treated with the nonselective nitric oxide synthase (NOS) inhibitor N(omega)-nitro-L-arginine (L-NA) at reperfusion. The specific inhibitor of neuronal NOS, 7-nitroindazole (7-NI), given at reperfusion, showed no protection, although preischemic treatment with 7-NI decreased infarct volume by 40%. Interestingly, prereperfusion administration of both NOS inhibitors decreased tyrosine nitration (a marker of peroxynitrite toxicity) in the ischemic area. L-NA treatment also significantly reduced vascular damage, as indicated by decreased Evans blue extravasation and matrix metalloproteinase-9 expression.. These data support the hypothesis that in addition to the detrimental action of NO formed by neuronal NOS during ischemia, NO generation at reperfusion plays a significant role in reperfusion injury, possibly through peroxynitrite formation. Contrary to L-NA, failure of 7-NI to protect against reperfusion injury suggests that the source of NO is the cerebrovascular compartment.

Topics: Animals; Biomarkers; Blood-Brain Barrier; Coloring Agents; Endothelium, Vascular; Enzyme Inhibitors; Evans Blue; Indazoles; Infarction, Middle Cerebral Artery; Matrix Metalloproteinase 9; Mice; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Oxidants; Permeability; Reperfusion Injury; Tyrosine

To determine whether, and if so how, iNOS expresses and affects brain injury induced by hypoxia-ischemia in an immature brain.. Seven-day-old Wistar rat pups were exposed to right common carotid artery ligation followed by 1.5 hours of hypoxia. The time course of iNOS mRNA expression, enzymatic activity, and protein production in the cerebral cortex were determined. The extent of the infarct area in the cerebral cortex and the production of 3-nitrotyrosine (a biomarker of peroxynitrite) were compared between the control pups and pups treated with S-methyl-isothiourea (a selective iNOS inhibitor).. In the cortex ipsilateral to carotid ligation, iNOS mRNA appeared from 6 hours to 24 hours after hypoxia-ischemia and disappeared at 48 hours. The iNOS protein and its activity also increased at 12 hours and reached a maximum level at 48 hours after the insult. The percentage of damage in the cerebral cortex was significantly higher in the control pups than in treated pups (31.9 vs 10.6%). Tri-nitrotyrosine following iNOS expression-positive cells were located predominantly at the infarct and peri-infarct regions.. iNOS expression might be an important determinant of ischemic immature brain injury.

Topics: Animals; Animals, Newborn; Brain Diseases; Carotid Arteries; Hypoxia-Ischemia, Brain; Kinetics; Ligation; Nitrates; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Rats; Rats, Wistar; RNA, Messenger; Tyrosine

Topics: Alprostadil; Animals; Arginine; Burns; Enzyme Inhibitors; Humans; Ischemia; Luminescent Measurements; Models, Biological; Nitrates; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Oxidative Stress; Reperfusion Injury; Sepsis; Shock; Superoxides; Tyrosine

P-selectin participates in the development of inflammatory disorders. Cardiopulmonary bypass is thought to induce inflammatory response and increase nitric oxide production. To evaluate the role of P-selectin in bypass-induced inflammatory response and its association with nitric oxide production, we examined the effect of P-selectin monoclonal antibody in a rat model of cardiopulmonary bypass.. Twenty adult Sprague-Dawley rats undergoing cardiopulmonary bypass for 60 minutes were divided into 2 groups. A 3-mg/kg dose of anti-rat specific P-selectin monoclonal antibody (ARP2-4; Sumitomo Pharmaceuticals, Osaka, Japan) was administered into the priming solution before bypass in group P (n = 10) and a 3-mg/kg dose of PNB1.6 (nonblocking monoclonal antibody) was added in group C for control (n = 10).. At the termination of bypass and 3 hours after the termination of bypass, plasma levels of interleukins 6 and 8, nitrate/nitrite, the percentage ratio of nitrotyrosine to tyrosine (an indicator of peroxynitrite formation), and the respiratory index were significantly higher than before bypass in both groups, and they were significantly lower in group P than in group C. Plasma P-selectin level in group C and exhaled nitric oxide concentration in both groups at termination of bypass were significantly lower than those before bypass, and they were significantly higher 3 hours after termination of bypass than before bypass in both groups. Plasma P-selectin level and exhaled nitric oxide concentration in group P were significantly higher than those in group C at the end of bypass, but they were significantly lower 3 hours after the termination of bypass.. These results demonstrate that P-selectin may participate in the augmentation of bypass-induced inflammatory response in association with nitric oxide and peroxynitrite production.

Topics: Animals; Antibodies, Monoclonal; Cardiopulmonary Bypass; Inflammation; Interleukin-6; Male; Nitrates; Nitric Oxide; Nitrites; P-Selectin; Rats; Rats, Sprague-Dawley; Tyrosine

Splanchnic artery occlusion shock (SAO) causes an enhanced formation of reactive oxygen species (ROS), which contribute to the pathophysiology of shock. Here we have investigated the effects of n-acetylcysteine (NAC), a free radical scavenger, in rats subjected to SAO shock.. Treatment of rats with NAC (applied at 20 mg/kg, 5 min prior to reperfusion, followed by an infusion of 20 mg/kg/h) attenuated the mean arterial blood and the migration of polymorphonuclear cells (PMNs) caused by SAO-shock. NAC also attenuated the ileum injury (histology) as well as the increase in the tissue levels of myeloperoxidase (MPO) and malondialdehyde (MDA) caused by SAO shock in the ileum. There was a marked increase in the oxidation of dihydrorhodamine 123 to rhodamine in the plasma of the SAO-shocked rats after reperfusion. Immunohistochemical analysis for nitrotyrosine and for poly(ADP-ribose) synthetase (PARS) revealed a positive staining in ileum from SAO-shocked rats. The degree of staining for nitrotyrosine and PARS were markedly reduced in tissue sections obtained from SAO-shocked rats which had received NAC. Reperfused ileum tissue sections from SAO-shocked rats showed positive staining for P-selectin, which was mainly localised in the vascular endothelial cells. Ileum tissue section obtained from SAO-shocked rats with anti-intercellular adhesion molecule (ICAM-1) antibody showed a diffuse staining. NAC treatment markedly reduced the intensity and degree of P-selectin and ICAM-1 in tissue section from SAO-shocked rats. In addition, in ex vivo studies in aortic rings from shocked rats, we found reduced contractions to noradrenaline and reduced responsiveness to a relaxant effect to acetylcholine (vascular hyporeactivity and endothelial dysfunction, respectively). NAC treatment improved contractile responsiveness to noradrenaline, enhanced the endothelium-dependent relaxations and significantly improved survival.. Taken together, our results clearly demonstrate that NAC treatment exert a protective effect and part of this effect may be due to inhibition of the expression of adhesion molecule and peroxynitrite-related pathways and subsequent reduction of neutrophil-mediated cellular injury.

Topics: Acetylcholine; Acetylcysteine; Analysis of Variance; Animals; Aorta; Fluorescent Antibody Technique, Indirect; Free Radical Scavengers; Ileum; In Vitro Techniques; Intercellular Adhesion Molecule-1; Leukocyte Count; Lipid Peroxidation; Male; Malondialdehyde; Nitrates; Nitrites; P-Selectin; Peroxidase; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Splenic Artery; Tyrosine; Vasodilation

Peroxyacetyl nitrate (PAN) is a common gaseous photochemical compound in polluted air and cigarette smog. The toxicity of PAN has been found to depend on three pathways: (1) its oxidizing property that mimics peroxide or peroxynitrite; (2) its nitrating and hydroxylating properties similar to peroxynitrite; and (3) its acetylating property like acetic anhydride. The present investigations were intended to focus on the reactions of PAN with aromatic amino acids and guanine. When PAN interacted with tyrosine and guanine the major products were 3-nitrotyrosine, 3, 5-dinitrotyrosine, 8-hydroxyguanine and 8-nitroguanine. These compounds have been used as indicators for the presence of peroxynitrite in previous studies. When PAN interacted with phenylalanine, the products were 3-nitrotyrosine, 4-nitrophenylalanine, p-tyrosine, o-tyrosine and m-tyrosine. 5-Hydroxytryptophan is produced from the reaction of PAN with tryptophan. Furthermore, the formation of nitrated tyrosines was also found in the PAN-treated HL-60 cells. A high yield of dityrosine was formed when PAN and peroxynitrite were reacted with tyrosine, probably through free radical oxidation. We also found that peroxynitrite and PAN are similar in their oxidizing activity. From these findings, we suggest that peroxynitrite may be considered as the reactive intermediate of PAN.

Topics: Air Pollutants; Amino Acids, Cyclic; Chromatography, High Pressure Liquid; Guanine; HL-60 Cells; Humans; Hydroxylation; Mass Spectrometry; Nitrates; Nitrogen; Oxidants; Peracetic Acid; Phenylalanine; Tryptophan; Tyrosine

Occupational, airborne pollutants, such as heavy metals, are recognized for inducing injury and cytotoxicity. Chromium(VI) is a redox cycling heavy metal that has been strongly implicated in the initiation of cancer. Its proinflammatory effects, however, have not been systematically examined. In our study, we found that potassium dichromate [Cr(VI)] treatment of human umbilical vein endothelial cells (HUVEC) increased intracellular adhesion molecule (ICAM) expression at the message level. ICAM message levels remained elevated for 12-24 hours after exposure and increased with time and concentration. Cr(VI) increased the release of superoxide anion without affecting the ability of endothelial cultures to produce nitric oxide. However, Cr(VI) decreased cGMP in HUVEC, suggesting that the nitric oxide produced was scavenged intracellularly. Cr(VI) also increased nitrotyrosine in HUVEC cultures. These data are consistent with the idea that exposure to Cr(VI) increases the production of superoxide anion, which scavenges nitric oxide to increase the formation of peroxynitrite. The loss in nitric oxide activity and increased formation of peroxynitrite likely enhance endothelial cell expression of ICAM-1. Cr(VI)-induced increases in the adhesive properties of the endothelium may play a critical role in the initiation and progression of tissue injury through increased recruitment of proinflammatory white blood cells.

Topics: Cells, Cultured; Endothelium, Vascular; Free Radicals; Humans; Intercellular Adhesion Molecule-1; Nitrates; Nitric Oxide; Nitrites; Potassium Dichromate; RNA, Messenger; Superoxides; Tyrosine

Hyperhomocysteinemia (HHCy) is an independent and graded cardiovascular risk factor. HHCy is prevalent in patients with chronic renal failure, contributing to the increased mortality rate. Controversy exists as to the effects of HHCy on nitric oxide (NO) production: it has been shown that HHCy both increases and suppresses it. We addressed this problem by using amperometric electrochemical NO detection with a porphyrinic microelectrode to study responses of endothelial cells incubated with homocysteine (Hcy) to the stimulation with bradykinin, calcium ionophore, or L-arginine. Twenty-four-hour preincubation with Hcy (10, 20, and 50 microM) resulted in a gradual decline in responsiveness of endothelial cells to the above stimuli. Hcy did not affect the expression of endothelial nitric oxide synthase (eNOS), but it stimulated formation of superoxide anions, as judged by fluorescence of dichlorofluorescein, and peroxynitrite, as detected by using immunoprecipitation and immunoblotting of proteins modified by tyrosine nitration. Hcy did not directly affect the ability of recombinant eNOS to generate NO, but oxidation of sulfhydryl groups in eNOS reduced its NO-generating activity. Addition of 5-methyltetrahydrofolate restored NO responses to all agonists tested but affected neither the expression of the enzyme nor formation of nitrotyrosine-modified proteins. In addition, a scavenger of peroxynitrite or a cell-permeant superoxide dismutase mimetic reversed the Hcy-induced suppression of NO production by endothelial cells. In conclusion, electrochemical detection of NO release from cultured endothelial cells demonstrated that concentrations of Hcy >20 microM produce a significant indirect suppression of eNOS activity without any discernible effects on its expression. Folates, superoxide ions, and peroxynitrite scavengers restore the NO-generating activity to eNOS, collectively suggesting that cellular redox state plays an important role in HCy-suppressed NO-generating function of this enzyme.

Topics: Animals; Arginine; Bradykinin; Calcimycin; Cells, Cultured; Endothelium, Vascular; Folic Acid; Free Radical Scavengers; Homocysteine; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Rats; Reactive Oxygen Species; Recombinant Proteins; Superoxides; Tyrosine

Our previous study demonstrated that the aortic inducible nitric oxide synthase (iNOS) expression and the plasma nitrite level in spontaneously hypertensive rats (SHR) were greater than that in age-matched Wistar-Kyoto rats (WKY). We subsequently hypothesized that the over-expression of iNOS might play an important role in the pathogenesis of hypertension in SHR. In the present study, pyrrolidinedithiocarbamate (PDTC, 10 mg kg(-1) day(-1), p.o., antioxidant and nuclear factor-kappa B inhibitor) and aminoguanidine (15 mg kg(-1) day(-1), p.o., selective inhibitor of iNOS) was used to treat SHR and WKY from age of 5 weeks through 16 weeks. We found that PDTC and aminoguanidine significantly suppressed the development of hypertension and improved the diminished vascular responses to acetylcholine in SHR but not in WKY. Likewise, the increase of iNOS expression, nitrotyrosine immunostaining, nitric oxide production and superoxide anion formation in adult SHR were also significantly suppressed by chronic treatment with PDTC and aminoguanidine. In conclusion, this study demonstrated that both PDTC and aminoguanidine significantly attenuated the development of hypertension in SHR. The results suggest that PDTC suppresses iNOS expression due to its anti-oxidant and/or nuclear factor-kappa B inhibitory properties. However, the effect of aminoguanidine was predominantly mediated by inhibition of iNOS activity, thereby reducing peroxynitrite formation. We propose that the development of a more specific and potent inhibitor of iNOS might be beneficial in preventing pathological conditions such as the essential hypertension.

Topics: Animals; Aorta; Blood Pressure; Disease Models, Animal; Enzyme Inhibitors; Guanidines; Hypertension; Immunoassay; Male; Nitrates; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Proline; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Superoxides; Thiocarbamates; Tyrosine; Vasodilation

By the observation of chemically induced dynamic nuclear polarization in (15)N NMR spectroscopy it has been shown that nitration of N-acetyltyrosine, even under acidic conditions, is largely a radical process. In the alkaline reaction of tyrosine with peroxynitrite the main products are nitrite and nitrate, both produced by a radical pathway, and tyrosine nitration is a minor reaction. It is suggested that tyrosine catalyzes the production of NO(*)(2) and HO(*) from peroxynitrite.

Topics: Absorption; Free Radicals; Hydrogen-Ion Concentration; Kinetics; Magnetic Resonance Spectroscopy; Molecular Structure; Nitrates; Nitric Acid; Nitrites; Nitrobenzenes; Nitrogen Isotopes; Tyrosine

Much evidence supports a role of nitric oxide (.NO) and peroxynitrite (ONOO(-)) in experimental and idiopathic Parkinson's disease (PD); moreover, an overexpression of neuronal nitric oxide synthase (nNOS) was recently reported in the basal ganglia of PD patients. In accord, we previously found a 50% increased.NO production rate during the respiratory burst of circulating neutrophils (PMN) from PD patients. As PMN express the nNOS isoform, the objective of the present study was to ascertain whether this increased.NO production is representative of nNOS gene upregulation. PMN were isolated from blood samples obtained from seven PD patients and seven age- and sex-matched healthy donors; nNOS mRNA was amplified by reverse transcriptase-polymerase chain reaction and the products were hybridized with a probe for nNOS. Nitrotyrosine-containing proteins and nNOS were detected by Western blot and NO production rate was measured spectrophotometrically by the conversion of oxymyoglobin to metmyoglobin. The results showed that both.NO production and protein tyrosine nitration were significantly increased in PMN isolated from PD patients (PD 0.09 +/- 0.01 vs 0.06 +/- 0.008 nmol min(-1) 10(6) cells(-1); P < 0.05). In addition, five of the seven PD patients showed about 10-fold nNOS mRNA overexpression; while two of the seven PD patients showed an expression level similar to that of the controls; detection of nNOS protein was more evident in the former group. In summary, it is likely that overexpression of nNOS and formation of ONOO(-) in PMN cells from PD patients emphasizes a potential causal role of.NO in the physiopathology of the illness.

Topics: Blotting, Western; Enzyme Induction; Female; Humans; Hydrogen Peroxide; Male; Middle Aged; Neutrophils; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Parkinson Disease; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Spectrophotometry; Tyrosine

To determine whether reactive nitrogen species contribute to secondary damage in CNS injury, the time courses of nitric oxide, peroxynitrite, and nitrotyrosine production were measured following impact injury to the rat spinal cord. The concentration of nitric oxide measured by a nitric oxide-selective electrode dramatically increased immediately following injury and then quickly declined. Nitro-L-arginine reduced nitric oxide production. The extracellular concentration of peroxynitrite, measured by perfusing tyrosine through a microdialysis fiber into the cord and quantifying nitrotyrosine in the microdialysates, significantly increased after injury to 3.5 times the basal level, and superoxide dismutase and nitro-L-arginine completely blocked peroxynitrite production. Tyrosine nitration examined immunohistochemically significantly increased at 12 and 24 h postinjury, but not in sham-control sections. Mn(III) tetrakis(4-benzoic acid)-porphyrin (a novel cell-permeable superoxide dismutase mimetic) and nitro-L-arginine significantly reduced the numbers of nitrotyrosine-positive cells. Protein-bound nitrotyrosine was significantly higher in the injured tissue than in the sham-operated controls. These results demonstrate that traumatic injury increases nitric oxide and peroxynitrite production, thereby nitrating tyrosine, including protein-bound tyrosine. Together with our previous report that trauma increases superoxide, our results suggest that reactive nitrogen species cause secondary damage by nitrating protein through the pathway superoxide + nitric oxide peroxynitrite protein nitration.

Topics: Amino Acids; Animals; Immunohistochemistry; Male; Models, Biological; Nitrates; Nitric Oxide; Nitroarginine; Proteins; Rats; Rats, Sprague-Dawley; Spinal Cord; Spinal Cord Injuries; Superoxide Dismutase; Tyrosine; Wounds, Nonpenetrating

The role of NO and superoxide (O(2)(-)) in tissue injury during cardiac allograft rejection was investigated by using a rat ex vivo organ perfusion system. Excessive NO production and inducible NO synthase (iNOS) expression were observed in cardiac allografts at 5 days after cardiac transplantation, but not in cardiac isografts, as identified by electron spin resonance spectroscopy and Northern blotting. Cardiac isografts or allografts obtained on Day 5 after transplantation were perfused with Krebs bicarbonate buffer with or without various antidotes for NO or O(2)-, including N(omega)-monomethyl-L-arginine (L-NMMA; 1 mM), 2-phenyl-4,4,5, 5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO; 100 microM), 4-amino-6-hydroxypyrazolo[3,4-d]pyrimidine (AHPP; a xanthine oxidase inhibitor; 100 microM), and superoxide dismutase (SOD; 100 units/ml). Treatment of the cardiac allografts with PTIO showed most remarkable improvement of the cardiac injury as revealed by significant reduction in aspartate transaminase, lactate dehydrogenase, and creatine phosphokinase concentrations in the perfusate. Similar but less potent protective effect on the allograft injury was observed by treatment with L-NMMA, AHPP, and SOD. Immunohistochemical analyses for iNOS and nitrotyrosine indicated that iNOS is mainly expressed by macrophages infiltrating the allograft tissues, and nitrotyrosine formation was demonstrated not only in macrophages but also in cardiac myocytes of the allografts, providing indirect evidence for the generation of peroxynitrite during allograft rejection. Our results suggest that tissue injury in rat cardiac allografts during acute rejection is mediated by both NO and O(2)(-), possibly through peroxynitrite formation.

Topics: Acute Disease; Animals; Electron Spin Resonance Spectroscopy; Graft Rejection; Heart Transplantation; In Vitro Techniques; Male; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; omega-N-Methylarginine; Perfusion; Rats; Rats, Inbred ACI; Rats, Inbred Lew; Superoxide Dismutase; Superoxides; Transplantation, Homologous; Tyrosine

Ventilator strategies allowing for increases in carbon dioxide (CO(2)) tensions (hypercapnia) are being emphasized to ameliorate the consequences of inflammatory-mediated lung injury. Inflammatory responses lead to the generation of reactive species including superoxide (O(2)(-)), nitric oxide (.NO), and their product peroxynitrite (ONOO(-)). The reaction of CO(2) and ONOO(-) can yield the nitrosoperoxocarbonate adduct ONOOCO(2)(-), a more potent nitrating species than ONOO(-). Based on these premises, monolayers of fetal rat alveolar epithelial cells were utilized to investigate whether hypercapnia would modify pathways of.NO production and reactivity that impact pulmonary metabolism and function. Stimulated cells exposed to 15% CO(2) (hypercapnia) revealed a significant increase in.NO production and nitric oxide synthase (NOS) activity. Cell 3-nitrotyrosine content as measured by both HPLC and immunofluorescence staining also increased when exposed to these same conditions. Hypercapnia significantly enhanced cell injury as evidenced by impairment of monolayer barrier function and increased induction of apoptosis. These results were attenuated by the NOS inhibitor N-monomethyl-L-arginine. Our studies reveal that hypercapnia modifies.NO-dependent pathways to amplify cell injury. These results affirm the underlying role of.NO in tissue inflammatory reactions and reveal the impact of hypercapnia on inflammatory reactions and its potential detrimental influences.

Topics: Animals; Apoptosis; Carbon Dioxide; Cells, Cultured; Cytokines; Fetus; Hypercapnia; Interferon-gamma; Interleukin-1; Lipopolysaccharides; Nitrates; Nitric Oxide; Nitric Oxide Synthase; omega-N-Methylarginine; Pulmonary Alveoli; Rats; Respiratory Mucosa; Tumor Necrosis Factor-alpha; Tyrosine

In the present study, we investigated the role of inducible (or type 2) nitric oxide synthase (iNOS) in the development of acute inflammation by comparing the responses in wild-type mice (WT) and mice lacking (knockout [KO]). When compared with carrageenan-treated iNOS-WT mice, iNOS-KO mice that had received carrageenan exhibited a reduced degree of pleural exudation and polymorphonuclear cell migration. Lung myeloperoxidase (MPO) activity and lipid peroxidation were significantly reduced in iNOS-KO mice in comparison with iNOSWT mice. Immunohistochemical analysis for nitrotyrosine revealed positive staining in lungs from carrageenan-treated iNOS-WT mice. Lung tissue sections from carrageenan-treated iNOS-WT mice showed positive staining for poly adenosine diphosphate (ADP)-ribose synthetase that was mainly localized in alveolar macrophages and in airway epithelial cells. The intensity and degree of staining for nitrotyrosine and poly-ADP-ribose synthetase were markedly reduced in tissue sections from carrageenan-treated iNOS-KO mice. The inflamed lungs of iNOS-KO mice also showed an improved histologic status. Furthermore, a significant reduction in the suppression of energy status, in DNA strand breakage, and in decreased cellular levels of nicotinamide adenine dinucleotide (NAD(+)) was observed ex vivo in macrophages harvested from the pleural cavity of iNOS-KO mice subjected to carrageenan-induced pleurisy. Taken together, our results clearly show that iNOS plays an important role in the acute inflammatory response.

Topics: Animals; Carrageenan; Cells, Cultured; DNA Damage; Enzyme Activation; Exudates and Transudates; Immunohistochemistry; Lipid Peroxidation; Lung; Macrophages; Male; Malondialdehyde; Mice; Mice, Knockout; Neutrophils; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Peroxidase; Pleura; Pleurisy; Poly(ADP-ribose) Polymerases; Tyrosine

We show that anthocyanins can function as potent inhibitors of the formation of nitrated tyrosine in vitro, and clarified how pelargonidin (Pel), which has a mono-hydroxyl group on the B-ring, can scavenge peroxynitrite (ONOO(-)) by detection of the reaction products. Pel was reacted with ONOO(-), then the reaction mixture was analyzed using high-performance liquid chromatography (HPLC). The HPLC analyses showed two novel peaks assumed to be the reaction products. Based on the instrumental analyses, the reaction products were identified as p-hydroxybenzoic acid and 4-hydroxy-3-nitrobenzoic acid. Pel can protect tyrosine from undergoing nitration through the formation of p-hydroxybenzoic acid and 4-hydroxy-3-nitrobenzoic acid.

Topics: Anthocyanins; Chromatography, High Pressure Liquid; Free Radical Scavengers; Kinetics; Nitrates; Nitrobenzoates; Oxidants; Parabens; Tyrosine

Peroxynitrite (PN) is likely to be generated in vivo from nitric oxide and superoxide. We have previously shown that prostacyclin synthase, a heme-thiolate enzyme essential for regulation of vascular tone, is nitrated and inactivated by submicromolar concentrations of PN [Zou, M.-H. & Ullrich, V. (1996) FEBS Lett. 382, 101-104] and we have studied the effect of heme proteins on the PN-mediated nitration of phenolic compounds in model systems [Mehl, M., Daiber, A. & Ullrich, V. (1999) Nitric Oxide: Biol. Chem. 2, 259-269]. In the present work we show that bolus additions of PN or PN-generating systems, such as SIN-1, can induce the nitration of P450BM-3 (wild-type and F87Y variant), for which we suggest an autocatalytic mechanism. HPLC and MS-analysis revealed that the wild-type protein is selectively nitrated at Y334, which was found at the entrance of a water channel connected to the active site iron center. In the F87Y variant, Y87, which is directly located at the active site, was nitrated in addition to Y334. According to Western blots stained with a nitrotyrosine antibody, this nitration started at 0.5 microM of PN and was half-maximal between 100 and 150 microM of PN. Furthermore, PN caused inactivation of the P450BM-3 monooxygenase as well as the reductase activity with an IC50 value of 2-3 microM. As two thiol residues/protein molecule were oxidized by PN and the inactivation was prevented by GSH or dithiothreitol, but not by uric acid (a powerful inhibitor of the nitration), our data strongly indicate that the inactivation is due to thiol oxidation at the reductase domain rather then to nitration of Y residues. Stopped-flow data presented here support our previous hypothesis that ferryl-species are involved as intermediates during the reactions of P450 enzymes with PN.

Topics: Animals; Bacillus; Bacterial Proteins; Binding Sites; Blotting, Western; Cattle; Chromatography, High Pressure Liquid; Cytochrome P-450 Enzyme System; Dithiothreitol; Dose-Response Relationship, Drug; Erythrocytes; Glutathione; Inhibitory Concentration 50; Iron; Mass Spectrometry; Mixed Function Oxygenases; Models, Chemical; Models, Molecular; Molsidomine; NADPH-Ferrihemoprotein Reductase; Nitrates; Nitric Oxide Donors; Nitrogen; Oxygen; Superoxide Dismutase; Temperature; Time Factors; Tyrosine; Uric Acid

In inflammatory arthritis, there is evidence indicating that the affected tissues produce large amounts of oxygen-free radicals and NO. Herein, we examine the biologic effects of exposure of IgG to hypochlorous acid (HOCl) and peroxynitrite (ONOO). The concentrations of IgG modified by chlorination and nitrosation were measured in synovial fluids from inflammatory and noninflammatory arthritis. Human IgG was exposed to increasing concentrations of HOCl and ONOO, and the resulting products were tested for complement component binding; binding to FcgammaRI; activation of polymorphonuclear neutrophils; effect on the Ab-combining site of Abs; and in vivo inflammatory activity in a rabbit model of acute arthritis. Rheumatoid synovial fluids contained significantly greater concentrations of nitrosated and chlorinated IgG compared with ostearthritic specimens. In vitro exposure of human IgG to HOCl and ONOO resulted in a concentration-dependent decrease in C3 and C1q fixation. The decrease in Fc domain-dependent biologic functions was confirmed by competitive binding studies to the FcgammaRI of U937 cells. HOCl-treated IgG monomer was 10 times less effective in competing for binding compared with native IgG, and ONOO-treated IgG was 2.5 times less effective. The modified IgGs were also ineffective in inducing synthesis of H(2)O(2) by human PMN. The Ag-binding domains of IgG also showed a concentration-dependent decrease in binding to Ag. The ability of the modified IgGs to induce acute inflammation in rabbit knees decreased 20-fold as gauged by the intensity of the inflammatory cell exudates. These studies clarify the modulating role of biological oxidants in inflammatory processes in which Ag-autoantibody reactions and immune complex pathogenesis may play an important role.

Topics: Acute Disease; Animals; Arthritis, Experimental; Arthritis, Rheumatoid; Binding Sites, Antibody; Complement C1q; Complement C3; Female; Free Radicals; Gout; Humans; Hydrogen Peroxide; Hypochlorous Acid; Immunoglobulin G; Male; Neutrophils; Nitrates; Osteoarthritis; Oxidation-Reduction; Oxygen; Rabbits; Receptors, IgG; Serum Albumin, Bovine; Synovial Fluid; Tyrosine

Because overproduction of nitric oxide (NO) and peroxynitrite is known to cause tissue injury, the expression of NO synthases (NOS) and generation of peroxynitrite were investigated in adenomyosis. Immunoreactivities to endothelial and inducible NOS demonstrated phase-dependent changes in normal endometrium, and in eutopic endometrium of adenomyosis. However, NOS were expressed throughout the menstrual cycle in ectopic endometrium from the majority of patients with adenomyosis. Nitrotyrosine, a footprint of peroxynitrite, was detected concomitantly with NOS protein. This suggested that high doses of NO and superoxide are produced in the ectopic endometrium, presumably by stimulation with bioactive molecules such as cytokines and growth factors. The expression of NOS and generation of peroxynitrite were markedly reduced by administration of gonadotrophin-releasing hormone agonists (GnRHa). The suppression of serum concentrations of nitrite/nitrate, stable metabolites of NO, by long-term administration of GnRHa was also demonstrated. The suppression of synthesis of NO and/or peroxynitrite may be part of both the therapeutic and adverse effects of GnRHa therapy.

Topics: Adult; Endometriosis; Endometrium; Epithelial Cells; Female; Follicular Phase; Gonadotropin-Releasing Hormone; Humans; Luteal Phase; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Nitrites; Stromal Cells; Tyrosine

To evaluate whether an intact respiratory burst exists within the orbit of diabetics with rhinocerebral mucormycosis.. Immunohistochemical detection of nitrotyrosine in the orbital tissue of diabetics requiring exenteration due to rhinocerebral mucormycosis. Nitrotyrosine is the stable product of the nitration of tyrosine residues by peroxynitrite. Peroxynitrite is a potent oxidant produced by the combination of superoxide and nitric oxide during the respiratory burst.. Four specimens were analyzed. All showed focal areas of specific staining against nitrotyrosine of the walls and internal structures of fungal organisms.. An intact respiratory burst is present in the orbit of diabetics during infection with rhinocerebral mucormycosis. Possible mechanisms of peroxynitrite's microbicidal effects and reasons for a deficiency in diabetics are discussed.

Topics: Adolescent; Adult; Aged; Brain Diseases; Diabetes Mellitus; Eye Infections, Fungal; Female; Humans; Immunoenzyme Techniques; Male; Middle Aged; Mucormycosis; Nitrates; Orbital Diseases; Paranasal Sinus Diseases; Respiratory Burst; Tyrosine

Vascular aging is mainly characterized by endothelial dysfunction. We found decreased free nitric oxide (NO) levels in aged rat aortas, in conjunction with a sevenfold higher expression and activity of endothelial NO synthase (eNOS). This is shown to be a consequence of age-associated enhanced superoxide (.O(2)(-)) production with concomitant quenching of NO by the formation of peroxynitrite leading to nitrotyrosilation of mitochondrial manganese superoxide dismutase (MnSOD), a molecular footprint of increased peroxynitrite levels, which also increased with age. Thus, vascular aging appears to be initiated by augmented.O(2)(-) release, trapping of vasorelaxant NO, and subsequent peroxynitrite formation, followed by the nitration and inhibition of MnSOD. Increased eNOS expression and activity is a compensatory, but eventually futile, mechanism to counter regulate the loss of NO. The ultrastructural distribution of 3-nitrotyrosyl suggests that mitochondrial dysfunction plays a major role in the vascular aging process.

Topics: Acetylcholine; Aging; Animals; Aorta; Body Weight; Calcimycin; Cellular Senescence; Endothelium, Vascular; Enzyme Induction; Hemodynamics; Male; Microscopy, Immunoelectron; Mitochondria; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitroprusside; Oxidative Stress; Rats; Rats, Inbred Strains; Superoxide Dismutase; Superoxides; Tyrosine; Vasodilation

3-Nitrotyrosine (3NT) is used as a biomarker of nitrative pathology caused by peroxynitrite (PN), myeloperoxidase (MPO)-, and/or eosinophil peroxidase (EPO)-dependent nitrite oxidation. 3NT measurements in biological materials are usually based on either antibody staining, HPLC detection, or GC detection methodologies. In this report, a procedure is described for the measurement of 3NT and tyrosine (TYR) by LC-MS/MS that is simple, direct, and sensitive. Though highly specialized in its use as an assay, LC-MS/MS technology is available in many research centers in academia and industry. The critical assay for 3NT was linear below 100 ng/ml and the limit of detection was below 100 pg/ml. Regarding protein digested samples, we found that MRM was most selective with 133.1 m/z as the daughter ion. In comparison, LC-ECD was 100 times less sensitive. Basal levels of 3NT in extracted digests of rat brain homogenate were easily detected by LC-MS/MS, but were below detection by LC-ECD. The LC-MS/MS assay was used to detect 3NT in rat brain homogenate that was filtered through a 180 micron nylon mesh. Three fractions were collected and examined by phase contrast microscopy. The mass ratio (3NT/TYR) of 3NT in fractions of large vessel enrichment, microvessel enrichment, and vessel depletion was 0.6 ng/mg, 1.2 ng/mg, and 0.2 ng/mg, respectively. Ultimately, we found that the basal 3NT/TYR mass ratio as determined by LC-MS/MS was six times greater in microvessel-enriched brain tissue vs. tissue devoid of microvessels.

Topics: Animals; Brain; Chromatography, Liquid; Mass Spectrometry; Microcirculation; Microscopy, Phase-Contrast; Nitrates; Rats; Rats, Wistar; Sensitivity and Specificity; Tyrosine

Recently, it was proposed that zymosan, a nonbacterial agent, causes cellular injury by inducing the production of peroxynitrite and consequent poly-(ADP-ribose) synthetase (PARS activation). Here we investigated whether in vivo N-acetylcysteine treatment inhibits cellular injury in macrophages collected from rats subjected to zymosan-induced shock. Macrophages harvested from the peritoneal cavity exhibited a significant production of peroxynitrite, as measured by the oxidation of the fluorescent dye dihydrorhodamine 123, and by nitrotyrosine. Furthermore, zymosan-induced shock caused a suppression of macrophage mitochondrial respiration, DNA strand breakage, and reduction of cellular levels of NAD+. In vivo treatment with N-acetylcysteine (40, 20, and 10 mg/kg, intraperitoneally, 1 and 6 h after zymosan) significantly reduced in a dose-dependent manner peroxynitrite formation and prevented the appearance of DNA damage, the decrease in mitochondrial respiration, and the loss of cellular levels of NAD+. Our study supports the view that the antioxidant and anti-inflammatory effect of N-acetylcysteine is also correlated with the inhibition of peroxynitrite production. In conclusion, N-acetylcysteine may be a novel pharmacological approach to prevent cell injury in inflammation.

Topics: Acetylcysteine; Animals; Cell Membrane Permeability; Disease Models, Animal; DNA Damage; Dose-Response Relationship, Drug; Energy Metabolism; Free Radical Scavengers; Macrophages, Peritoneal; Male; NAD; Nitrates; Nitric Oxide; Rats; Rats, Sprague-Dawley; Sepsis; Shock; Tyrosine; Zymosan

The role of nitric oxide (NO) on tissue injury of hepatic allografts during rejection remains controversial. We investigated inducible nitric oxide synthase (iNOS) expression and formation of peroxynitrite in ACI rat liver grafts implanted in recipients. Animals were divided into four experimental groups: group I, isografts; group II, untreated hepatic allografts; group III, allografts treated with FK506; and group IV, allografts pretreated with donor-specific blood transfusion (DST). Serum nitrite/nitrate, interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha) concentrations increased significantly in group II rats after transplantation but were significantly lower in groups I, III, and IV. The numbers of macrophages that reacted with an antimacrophage iNOS monoclonal antibody as well as iNOS messenger RNA (mRNA) levels in liver specimens were also much lower in groups I, III, and IV as compared with group II. Immunostaining and Western blot analysis showed prominent tissue nitrotyrosine expression in untreated hepatic allografts, but not in allografts treated with FK506 or donor-specific blood. These results suggest that one of the mechanisms by which production of NO results in injury in rat hepatic allografts may be because of its reaction with superoxide to form peroxynitrite.

Topics: Animals; Blood Transfusion; Enzyme Inhibitors; Graft Rejection; Graft Survival; Liver Transplantation; Male; Nitrates; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; omega-N-Methylarginine; Rats; Rats, Inbred ACI; Rats, Inbred Lew; RNA, Messenger; Tacrolimus; Tissue Donors; Transplantation, Homologous; Transplantation, Isogeneic; Tyrosine

Peroxynitrite (ONOO-) is a potent oxidizing and nitrating agent produced by the reaction of nitric oxide with superoxide. It readily nitrates phenolic compounds such as tyrosine residues in proteins, and it has been demonstrated that nitration of tyrosine residues in proteins inhibits their phosphorylation. During immune responses, tyrosine phosphorylation of key substrates by protein tyrosine kinases is the earliest of the intracellular signaling pathways following activation through the TCR complex. This work was aimed to evaluate the effects of ONOO- on lymphocyte tyrosine phosphorylation, proliferation, and survival. Additionally, we studied the generation of nitrating species in vivo and in vitro during immune activation. Our results demonstrate that ONOO-, through nitration of tyrosine residues, is able to inhibit activation-induced protein tyrosine phosphorylation in purified lymphocytes and prime them to undergo apoptotic cell death after PHA- or CD3-mediated activation but not upon phorbol ester-mediated stimulation. We also provide evidence indicating that peroxynitrite is produced during in vitro immune activation, mainly by cells of the monocyte/macrophage lineage. Furthermore, immunohistochemical studies demonstrate the in vivo generation of nitrating species in human lymph nodes undergoing mild to strong immune activation. Our results point to a physiological role for ONOO- as a down-modulator of immune responses and also as key mediator in cellular and tissue injury associated with chronic activation of the immune system.

Topics: Apoptosis; CD3 Complex; Cell Division; Cells, Cultured; Dose-Response Relationship, Immunologic; Humans; Immunosuppressive Agents; Lymph Nodes; Lymphocyte Activation; Metalloporphyrins; Monocytes; Nitrates; Nitric Oxide Synthase; Phosphorylation; Proteins; Superoxide Dismutase; T-Lymphocytes; Time Factors; Tyrosine

Ethanol increases free radical formation; however, it was recently demonstrated that it also causes extensive hypoxia in rat liver in vivo. To address this issue, it was hypothesized that peroxynitrite formed in normoxic periportal regions of the liver lobule has its reactivity enhanced in hypoxic pericentral regions where the pH is lower. Via this pathway, peroxynitrite could lead to free radical formation in the absence of oxygen. Livers from fed rats were perfused at low flow rates for 75 min. Under these conditions, periportal regions were well oxygenated but pericentral areas became hypoxic. Low-flow perfusion caused a significant 6-fold increase in nitrotyrosine accumulation in pericentral regions. During the last 20 min of perfusion, the spin-trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone was infused and adducts were collected for electron-spin resonance analysis. A six-line radical adduct signal was detected in perfusate. Direct infusion of peroxynitrite produced a radical adduct with identical coupling constants, and a similar pattern of nitrotyrosine accumulation was observed. Retrograde perfusion at low rates resulted in accumulation of nitrotyrosine in periportal regions. Although the magnitude of the radical in perfusate was increased by ethanol, it was not derived directly from it. Both nitrotyrosine accumulation and radical formation were reduced by inhibition of nitric oxide synthase with N-nitro-L-arginine methyl ester, but not with the inactive D-isomer. Radical formation was decreased nearly completely by superoxide dismutase and N-nitro-L-arginine methyl ester, consistent with the hypothesis that the final prooxidant is a derivative from both NO. and superoxide (i.e., peroxynitrite). These results support the hypothesis that oxidative stress occurs in hypoxic regions of the liver lobule by mechanisms involving peroxynitrite.

Topics: Animals; Ethanol; Female; Free Radicals; Glycogen; Hydrogen-Ion Concentration; In Vitro Techniques; Liver; Nitrates; Oxidants; Oxidative Stress; Oxygen; Perfusion; Rats; Rats, Sprague-Dawley; Tyrosine

Oxidative stress is suggested to be involved in several neurodegenerative diseases. One mechanism of oxidative damage is mediated by peroxynitrite, a neurotoxic reaction product of superoxide anion and nitric oxide. Expression of two cytokines and two key enzymes that are indicative of the presence of reactive oxygen intermediates and peroxynitrite was investigated in brain tissue of AIDS patients with and without AIDS dementia complex and HIV-seronegative controls. RNA expression of IL-1beta, IL-10, inducible nitric oxide synthase, and superoxide dismutase (SOD) was found to be significantly higher in demented compared with nondemented patients. Immunohistochemical analysis showed that SOD was expressed in CD68-positive microglial cells while inducible nitric oxide synthase was detected in glial fibrillary acidic protein (GFAP)-positive astrocytes and in equal amounts in microglial cells. Approximately 70% of the HIV p24-Ag-positive macrophages did express SOD, suggesting a direct HIV-induced intracellular event. HIV-1 infection of macrophages resulted in both increased superoxide anion production and elevated SOD mRNA levels, compared with uninfected macrophages. Finally, we show that nitrotyrosine, the footprint of peroxynitrite, was found more intense and frequent in brain sections of demented patients compared with nondemented patients. These results indicate that, as a result of simultaneous production of superoxide anion and nitric oxide, peroxynitrite may contribute to the neuropathogenesis of HIV-1 infection.

Topics: Acquired Immunodeficiency Syndrome; Adult; Aged; AIDS Dementia Complex; Brain Chemistry; Female; HIV Infections; HIV-1; Humans; Immunohistochemistry; Interleukin-1; Interleukin-10; Macrophages; Male; Middle Aged; Nitrates; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; RNA, Messenger; Superoxide Dismutase; Superoxides; Tyrosine

Microglial activation, oxidative stress, and dysfunctions in mitochondria, including the reduction of cytochrome oxidase activity, have been implicated in neurodegeneration. The current experiments tested the effects of reducing cytochrome oxidase activity on the ability of microglia to respond to inflammatory insults. Inhibition of cytochrome oxidase by azide reduced oxygen consumption and increased reactive oxygen species (ROS) production but did not affect cell viability. Azide also attenuated microglial activation, as measured by nitric oxide (NO.) production in response to lipopolysaccharide (LPS). It is surprising that the inhibition of cytochrome oxidase also diminished the activity of the alpha-ketoglutarate dehydrogenase complex (KGDHC), a Krebs cycle enzyme. This reduction was exaggerated when the azide-treated microglia were also treated with LPS. The combination of the azide-stimulated ROS and LPS-induced NO. would likely cause peroxynitrite formation in microglia. Thus, the possibility that KGDHC was inactivated by peroxynitrite was tested. Peroxynitrite inhibited the activity of isolated KGDHC, nitrated tyrosine residues of all three KGDHC subunits, and reduced immunoreactivity to antibodies against two KGDHC components. Thus, our data suggest that inhibition of the mitochondrial respiratory chain diminishes aerobic energy metabolism, interferes with microglial inflammatory responses, and compromises mitochondrial function, including KGDHC activity, which is vulnerable to NO. and peroxynitrite that result from microglial activation. Thus, activation of metabolically compromised microglia can further diminish their oxidative capacity, creating a deleterious spiral that may contribute to neurodegeneration.

Topics: Animals; Azides; Cell Survival; Cells, Cultured; Enzyme Activation; Glutamate Dehydrogenase; Inflammation; Ketoglutarate Dehydrogenase Complex; Lipopolysaccharides; Mice; Mice, Inbred C57BL; Microglia; Mitochondria; Nitrates; Nitric Oxide; Oxidative Stress; Oxidoreductases; Oxygen Consumption; Reactive Oxygen Species; Tyrosine

In the present study we used a bioassay to study the effects of peroxynitrite (ONOO-) on angiotensin II (A-II)-triggered tension in isolated bovine coronary arteries in order to show the consequences of the previously reported PGI2-synthase inhibition by ONOO- in this model. The following results were obtained: 1. 1 micromol L(-1) ONOO- impaired A-II-induced vasorelaxation and caused a second long lasting constriction phase. Indomethacin (10(-5)M) prevented both effects. U51605, a dual blocker of PGI2-synthase and thromboxane (TX)A2-synthase mimicked the effects of ONOO-. 2. The selective TXA2/prostaglandin endoperoxide (PGH2) receptor antagonist SQ29548 antagonized the second vasoconstriction phase after ONOO- -treatment. Since a generation of TXA2 and 8-iso-prostaglandin F2alpha could be excluded a direct action of unmetabolized PGH2 on the TXA2/PGH2 receptor was postulated. 3. ONOO- dose-dependently inhibited the conversion of 14C-PGH2 into 6-keto-PGF1alpha in isolated bovine coronary arteries with an IC50-value of 100 nM. 4. Immunoprecipitation of 3-nitrotyrosine-containing proteins with a monoclonal antibody revealed PGI2-synthase as the only nitrated protein in bovine coronary arteries treated with 1 micromol 1(-1) ONOO-. 5. Using immunohistochemistry a co-localization of PGI2-synthase and nitrotyrosine-containing proteins was clearly visible in both endothelial and vascular smooth muscle cells. We concluded that ONOO- not only eliminated the vasodilatory, growth-inhibiting, antithrombotic and antiadhesive effects of PGI2 but also allowed and promoted an action of the potent vasoconstrictor, prothrombotic agent, growth promoter, and leukocyte adherer, PGH2.

Topics: Angiotensin II; Animals; Carbon Radioisotopes; Cattle; Coronary Vasospasm; Coronary Vessels; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Dinoprostone; Epoprostenol; Immunohistochemistry; In Vitro Techniques; Intramolecular Oxidoreductases; Nitrates; Oxidants; Potassium Chloride; Prostaglandin Antagonists; Prostaglandin H2; Prostaglandins; Prostaglandins H; Proteins; Tyrosine; Vasoconstriction; Vasodilation

Peroxynitrite is a mediator of toxicity in pathological processes in vivo and causes damage by oxidation and nitration reactions. Here, we report a differential induction of mitogen-activated protein kinases (MAPKs) in WB-F344 rat liver epithelial cells by peroxynitrite. For the exposure of cultured cells with peroxynitrite, we employed a newly developed infusion method. At 6.5 microM steady-state concentration, the activation of p38 MAPK was immediate, while JNK1/2 and ERK1/2 were activated 60 min and 15 min subsequent to 3 min of exposure to peroxynitrite, respectively. Protein-bound 3-nitrotyrosine was detected. When cells were grown in a medium supplemented with sodium selenite (1 microM) for 48 h, complete protection was afforded against the activation of p38 and against nitration of tyrosine residues. These data suggest a new role for peroxynitrite in activating signal transduction pathways capable of modulating gene expression. Further, the abolition of the effects of peroxynitrite by selenite supplementation suggests a protective role of selenium-containing proteins.

Topics: Animals; Calcium-Calmodulin-Dependent Protein Kinases; Cell Line; Epithelium; Glutathione Peroxidase; Hydrogen Peroxide; Liver; Nitrates; Nitric Oxide; Phosphorylation; Rats; Sodium Selenite; Time Factors; Tyrosine

In many pathological conditions such as inflammatory and neurodegenerative diseases, the in vivo toxicity of nitric oxide has been attributed to the toxic oxidant peroxynitrite. Interaction of peroxynitrite with biological molecules can modify tyrosine residues on the proteins at the ortho position resulting in the formation of the stable end-product, 3-nitro-L-tyrosine (3-NT). Recent investigations indicate that changes in the circulating concentrations of 3-NT in pathological conditions may reflect the extent of nitric oxide-dependent oxidative damage and peroxynitrite toxicity. In the present study, we examined the in vivo disposition characteristics of 3-NT in rats after either a single i.v. bolus dose (10 mg/kg) or a loading and maintenance infusion at 10 or 30 mg/kg. Plasma concentrations of 3-NT were analyzed by a reversed-phase HPLC method. After a single bolus dose of 3-NT at 10 mg/kg, the average half-life of the elimination phase for the drug was 68.5+/-18.4 min (n = 5). Infusions of 3-NT at two different doses (10 and 30 mg/kg) indicated that the pharmacokinetic properties of 3-NT below plasma concentrations of 100 microM were both linear and stationary. Urinary excretion of unchanged 3-NT was minimal, but two distinct metabolites of 3-NT were identified in the urine collected throughout the study. These findings may be useful in the interpretation of the plasma and urine 3-NT concentrations as possible indices of systemic peroxynitrite exposure.

Topics: Animals; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Half-Life; Infusions, Intravenous; Male; Nitrates; Oxidants; Rats; Rats, Sprague-Dawley; Tissue Distribution; Tyrosine

In a murine bone-marrow transplant (BMT) model designed to determine risk factors for lung dysfunction in irradiated mice, we reported that cyclophosphamide (Cy)-induced injury and lethality depended on the infusion of donor spleen T cells. In the study reported here, we hypothesized that alveolar macrophage (AM)-derived reactive oxygen/nitrogen species are associated with lung dysfunction caused by allogeneic T cells, which stimulate nitric oxide (.NO) production, and by Cy, which stimulates superoxide production.NO reacts with superoxide to form peroxynitrite, a tissue-damaging oxidant. On Day 7 after allogeneic BMT, bronchoalveolar lavage fluid (BALF) obtained from mice injected with T cells contained increased levels of nitrite, which was associated with increased lactate dehydrogenase and protein levels, both of which are indices of lung injury. The injury was most severe in mice receiving both T cells and Cy. Messenger RNA (mRNA) for inducible nitric oxide synthase was detected only in murine lungs injected with T cells +/- Cy. AMs obtained on Day 7 after BMT from mice receiving T cells +/- Cy spontaneously generated between 20 and 40 microM nitrite in culture, versus < 2 microM generated by macrophages obtained from mice undergoing BMT but not receiving T cells. The level of 3-nitrotyrosine, the stable byproduct of the reaction of peroxynitrite with tyrosine residues, was increased in the BALF proteins of mice injected with both T cells and Cy. We conclude that allogeneic T cells stimulate macrophage-derived.NO, and that the addition of Cy favors peroxynitrite formation. Peroxynitrite generation clarifies the dependence of Cy-induced lung injury and lethality on the presence of allogeneic T cells.

Topics: Animals; Bone Marrow Transplantation; Bronchoalveolar Lavage Fluid; Cyclophosphamide; Disease Models, Animal; Female; L-Lactate Dehydrogenase; Lung; Macrophages, Alveolar; Mice; Mice, Inbred C57BL; Models, Biological; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; T-Lymphocytes; Time Factors; Tyrosine

The accumulation of covalently modified proteins is an important hallmark of biological aging, but relatively few studies have addressed the detailed molecular-chemical changes and processes responsible for the modification of specific protein targets. Recently, Narayanan et al. [Narayanan, Jones, Xu and Yu (1996) Am. J. Physiol. 271, C1032-C1040] reported that the effects of aging on skeletal-muscle function are muscle-specific, with a significant age-dependent change in ATP-supported Ca2+-uptake activity for slow-twitch but not for fast-twitch muscle. Here we have characterized in detail the age-dependent functional and chemical modifications of the rat skeletal-muscle sarcoplasmic-reticulum (SR) Ca2+-ATPase isoforms SERCA1 and SERCA2a from fast-twitch and slow-twitch muscle respectively. We find a significant age-dependent loss in the Ca2+-ATPase activity (26% relative to Ca2+-ATPase content) and Ca2+-uptake rate specifically in SR isolated from predominantly slow-twitch, but not from fast-twitch, muscles. Western immunoblotting and amino acid analysis demonstrate that, selectively, the SERCA2a isoform progressively accumulates a significant amount of nitrotyrosine with age (approximately 3.5+/-0. 7 mol/mol of SR Ca2+-ATPase). Both Ca2+-ATPase isoforms suffer an age-dependent loss of reduced cysteine which is, however, functionally insignificant. In vitro, the incubation of fast- and slow-twitch muscle SR with peroxynitrite (ONOO-) (but not NO/O2) results in the selective nitration only of the SERCA2a, suggesting that ONOO- may be the source of the nitrating agent in vivo. A correlation of the SR Ca2+-ATPase activity and covalent protein modifications in vitro and in vivo suggests that tyrosine nitration may affect the Ca2+-ATPase activity. By means of partial and complete proteolytic digestion of purified SERCA2a with trypsin or Staphylococcus aureus V8 protease, followed by Western-blot, amino acid and HPLC-electrospray-MS (ESI-MS) analysis, we localized a large part of the age-dependent tyrosine nitration to the sequence Tyr294-Tyr295 in the M4-M8 transmembrane domain of the SERCA2a, close to sites essential for Ca2+ translocation.

Topics: Aging; Amino Acid Sequence; Animals; Biological Transport; Calcium; Calcium-Transporting ATPases; Cysteine; Endopeptidases; Isoenzymes; Muscle Fibers, Fast-Twitch; Muscle Fibers, Slow-Twitch; Nitrates; Nitric Oxide; Oxygen; Peptide Fragments; Protein Processing, Post-Translational; Rats; Rats, Inbred F344; Sarcoplasmic Reticulum; Sulfhydryl Compounds; Tyrosine

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.

Topics: Chromatography, High Pressure Liquid; Electrochemistry; Humans; Hypochlorous Acid; Indicators and Reagents; Molsidomine; Nitrates; Nitrites; Nitrogen Oxides; Serum Albumin, Bovine; Sodium Nitrite; Spectrophotometry, Ultraviolet; Spermine; Tyrosine

The ability of peroxynitrite to modify amino acid residues in glutamine synthetase (GS) and BSA is greatly influenced by pH and CO2. At physiological concentrations of CO2 (1.3 mM), the generation of carbonyl groups (0.2-0.4 equivalents/subunit) is little affected by pH over the range of 7.2-9.0, but, in the absence of CO2, carbonyl formation increases (from 0.1- 1.2 equivalents/subunit) as the pH is raised from 7.2 to 10.5. This increase is attributable, in part but not entirely, to the increase in peroxynitrite (PN) stability with increasing pH. Of several amino acid polymers tested, only those containing lysine residues yielded carbonyl derivatives. In contrast, the nitration of tyrosine residues of both GS and BSA at pH 7.5 almost completely depends on the presence of CO2. However, the pH profiles of tyrosine nitration in GS and BSA are not the same. With both proteins, nitration decreases approximately 65% with increasing pH over the range of 7.2-8.4, but, then in the case of GS only, there is a 3.4-fold increase in the level of nitration over the range pH 8.4-8.8. The oxidation of methionine residues in both proteins and in the tripeptide Ala-Met-Ala was inhibited by CO2 at both high and low pH values. These results emphasize the importance of controlling the pH and CO2 concentrations in studies involving PN and indicate that PN is not likely to contribute appreciably to carbonyl formation or oxidation of methionine residues of proteins at physiological pH and CO2 concentrations.

Topics: Carbon Dioxide; Escherichia coli; Glutamate-Ammonia Ligase; Glycine max; Hydrogen-Ion Concentration; Kinetics; Lipid Peroxidation; Lipoxygenase; Methionine; Models, Chemical; Nitrates; Oxidants; Oxidation-Reduction; Serum Albumin, Bovine; Tyrosine

In the present study we investigated the protective role of endogenous glutathione, a known free radical scavenger, in rats subjected to carrageenan-induced pleurisy. In vivo depletion of endogenous glutathione pools with L-buthionine-(S,R)-sulfoximine (BSO, 1 g/kg for 24 h, intraperitoneally) enhances the carrageenan-induced degree of pleural exudation and polymorphonuclear leukocyte migration in rats subjected to carrageenan-induced pleurisy. Lung myeloperoxidase activity and lipid peroxidation were significantly increased in BSO pretreated rats. However, the inducible nitric oxide (NO) synthase in lung samples was unaffected by BSO pretreatment. Immunohistochemical analysis for nitrotyrosine revealed a positive staining in lungs from carrageenan-treated rats, which was massively enhanced by BSO pretreatment. Furthermore, in vivo BSO pretreatment significantly increased peroxynitrite formation as measured by the oxidation of the fluorescent dye dihydrorhodamine 123, enhanced the appearance of DNA damage, the decrease in mitochondrial respiration and partially decreased the cellular level of NAD+ in ex vivo macrophages harvested from the pleural cavity of rats subjected to carrageenan-induced pleurisy. In vivo treatment with exogenous glutathione (50 mg/kg i.p.) significantly reverts the effects of BSO and exerts anti-inflammatory effects. Thus, endogenous glutathione plays an important protective role against carrageenan-induced local inflammation.

Topics: Animals; Carrageenan; Cells, Cultured; Disease Models, Animal; Free Radical Scavengers; Glutathione; Macrophages; Nitrates; Nitric Oxide; Oxidants; Pleurisy; Protective Agents; Rats; Tyrosine

1. Experiments were designed to explore the effects of nitric oxide (NO) donors on generation of superoxide (O2.-) and peroxynitrite (ONOO-) in rabbit aortic rings. 2. Following inhibition of endogenous superoxide dismutase (SOD), significant basal release of O2.- was revealed (0.9 +/- 0.01 x 10(-12) mol min-1 mg-1 tissue). Generation of O2.- increased in a concentration-dependent manner in response to NADH or NADPH (EC50 = 2.34 +/- 1.18 x 10(-4) and 6.21 +/- 1.79 x 10(-3) M respectively, n = 4). NADH-stimulated O2.- chemiluminescence was reduced by approximately 85% in the presence of exogenous SOD (15 x 10(3) U ml-1). 3. Incubation of aortic rings with S-nitrosoglutathione (GSNO; 1 x 10(-5)-3 x 10(-3) M) or sodium nitroprusside (SNP; 1 x 10(-8)-1 x 10(-3) M), resulted in a concentration-dependent quenching of O2.- chemiluminescence which was proportional to NO release. 4. ONOO- formation was assessed indirectly by determining protein tyrosine nitration in rabbit aorta using a specific antibody against nitrotyrosine. Basally and in the presence of NADH, a single band was detected. Incubation of aortic rings with either GSNO (1 x 10(-3) M) alone or GSNO with NADH resulted in the appearance of additional nitrotyrosine bands. Incubation of serum albumin with GSNO alone did not cause nitrotyrosine formation. In contrast, incubation with 3-morpholinosydonomine (SIN-1; 1 x 10(-3) M, 10 min), resulted in marked nitration of albumin which was reduced by oxyhaemoglobin or SOD. Incubation of albumin with GSNO and pyrogallol, a O2.- generator, also resulted in protein nitration. 5. Addition of exogenous NO results in nitrotyrosine formation in rabbit aortic rings. Nitrotyrosine formation is likely to result from the reaction of exogenous NO and basal endogenous O2.- resulting in the formation of ONOO-. Formation of ONOO- and nitration of tyrosine residues potentially could lead to vascular damage and might represent unexpected adverse effects of long-term nitrate therapy.

Topics: Animals; Aorta, Thoracic; Cattle; In Vitro Techniques; Nitrates; Nitric Oxide; Nitric Oxide Donors; Nitrogen; Rabbits; Superoxides; Tyrosine

We found that the exposure of human neuroblastoma SH-SY5Y cells to the peroxynitrite donor 3-morpholinosydnonimine (SIN-1) induced tyrosine nitration of a 130-kDa protein, and prevented tyrosine phosphorylation of the 130-kDa protein. The focal adhesion protein p130cas was identified as a component of the 130-kDa protein using specific antibody. These results suggest that p130cas is a new target protein for nitration induced by SIN-1.

Topics: Crk-Associated Substrate Protein; Humans; Molsidomine; Neuroblastoma; Nitrates; Nitric Oxide Donors; Oxidants; Phosphoproteins; Phosphorylation; Proteins; Retinoblastoma-Like Protein p130; Tumor Cells, Cultured; Tyrosine

Nitric oxide (NO) is a new intercellular messenger that occurs naturally in the brain without causing overt toxicity. Yet, NO has been implicated as a mediator of cell death in cell death. One explanation is that ischemia causes overproduction of NO, allowing it to react with superoxide to form the potent oxidant peroxynitrite. To address this question, we used immunohistochemistry for citrulline, a marker for NO synthase activity, and 3-nitrotyrosine, a marker for peroxynitrite formation, in mice subjected to reversible middle cerebral artery occlusion. We show that ischemia triggers a marked augmentation in citrulline immunoreactivity but more so in the peri-infarct than the infarcted tissue. This increase is attributable to the activation of a large population (approximately 80%) of the neuronal isoform of NO synthase (nNOS) that is catalytically inactive during basal conditions, indicating a tight regulation of physiological NO production in the brain. In contrast, 3-nitrotyrosine immunoreactivity is restricted to the infarcted tissue and is not present in the peri-infarct tissue. In nNOS(Delta/Delta) mice, known to be protected against ischemia, no 3-nitrotyrosine immunoreactivity is detected. Our findings provide a cellular localization for nNOS activation in association with ischemic stroke and establish that NO is not likely a direct neurotoxin, whereas its conversion to peroxynitrite is associated with cell death.

Topics: Animals; Brain; Cerebral Infarction; Citrulline; Corpus Striatum; Enzyme Activation; Functional Laterality; Immunohistochemistry; Injections, Intraperitoneal; Ischemic Attack, Transient; Mice; Mice, Inbred C57BL; Mice, Inbred Strains; Mice, Knockout; Microinjections; N-Methylaspartate; Neurons; Nitrates; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Oxidants; Tyrosine

Putative "protein nitratases," which catalyze denitration of peroxynitrite (PN)-treated, proteins, were detected in the crude extract of dog prostate. Nitratase activity was monitored by the decreased intensity of nitrotyrosine immunoreactive-bands in Western blot and increased nitrate level in dialysate of incubation mixture, which contained prostate crude extract, protease inhibitors and a PN-treated substrate, such as treated histone (III-S), BSA, invertase, or polylysine. Nitratases were activated by preincubation with m-calpain/Ca2+. Furthermore, after denitration, the activity of PN/DTT-treated invertase decreased to the similar activity level of DTT-treated invertase. At least two different types of nitratases may occur: type I, reductant-dependent, and type II, reductant-independent.

Topics: Animals; beta-Fructofuranosidase; Blotting, Western; Dithiothreitol; Dogs; Enzyme Activation; Glutathione Transferase; Glycoside Hydrolases; Histones; Male; Nitrates; Oxidoreductases; Polylysine; Prostate; Serum Albumin; Tissue Extracts; Tyrosine

Peroxynitrite, the product of the radical-radical reaction between nitric oxide and superoxide anion, is a potent oxidant involved in tissue damage in neurodegenerative disorders. We investigated the modifications induced by peroxynitrite in tyrosine residues of proteins from synaptosomes. Peroxynitrite treatment (> or =50 microM) induced tyrosine nitration and increased tyrosine phosphorylation. Synaptophysin was identified as one of the major nitrated proteins and pp60src kinase as one of the major phosphorylated substrates. Further fractionation of synaptosomes revealed nitrated synaptophysin in the synaptic vesicles, whereas phosphorylated pp60src was enriched in the postsynaptic density fraction. Tyrosine phosphorylation was increased by treatment with 50-500 microM peroxynitrite and decreased by higher concentrations, suggesting a possible activation/inactivation of kinases. Immunocomplex kinase assay proved that peroxynitrite treatment of synaptosomes modulated the pp60src autophosphorylation activity. The addition of bicarbonate (CO2 1.3 mM) produced a moderate enhancing effect on some nitrated proteins but significantly protected the activity of pp60src against peroxynitrite-mediated inhibition so that at 1 mM peroxynitrite, the kinase was still more active than in untreated synaptosomes. The phosphotyrosine phosphatase activity of synaptosomes was inhibited by peroxynitrite (> or =50 microM) but significantly protected by CO2. Thus, the increase of phosphorylation cannot be attributed to peroxynitrite-mediated inhibition of phosphatases. We suggest that peroxynitrite may regulate the posttranslational modification of tyrosine residues in pre- and postsynaptic proteins. Identification of the major protein targets gives insight into the pathways possibly involved in neuronal degeneration associated with peroxynitrite overproduction.

Topics: Animals; Brain; Brain Chemistry; Cattle; Dose-Response Relationship, Drug; Nitrates; Nitric Oxide; Oxidants; Phosphorylation; Proto-Oncogene Proteins pp60(c-src); Signal Transduction; Synapses; Synaptophysin; Synaptosomes; Tyrosine

Eosinophils and increased production of nitric oxide (NO) and superoxide, components of peroxynitrite, have been implicated in the pathogenesis of a number of allergic disorders including asthma. Peroxynitrite induced protein nitration may compromise enzyme and protein function. We hypothesized that peroxynitrite may modulate eosinophil migration by modulating chemotactic cytokines. To test this hypothesis, the eosinophil chemotactic responses of regulated on activation, normal T cell expressed and secreted (RANTES) and interleukin (IL)-5 incubated with and without peroxynitrite were evaluated. Peroxynitrite-attenuated RANTES and IL-5 induced eosinophil chemotactic activity (ECA) in a dose-dependent manner (P < 0.05) but did not attenuate leukotriene B4 or complement-activated serum ECA. The reducing agents deferoxamine and dithiothreitol reversed the ECA inhibition by peroxynitrite, and exogenous L-tyrosine abrogated the inhibition by peroxynitrite. PAPA-NONOate, a NO donor, or superoxide generated by lumazine or xanthine and xanthine oxidase, did not show an inhibitory effect on ECA. The peroxynitrite generator, 3-morpholinosydnonimine, caused a concentration-dependent inhibition of ECA. Peroxynitrite reduced RANTES and IL-5 binding to eosinophils and resulted in nitrotyrosine formation. These findings are consistent with nitration of tyrosine by peroxynitrite with subsequent inhibition of RANTES and IL-5 binding to eosinophils and suggest that peroxynitrite may play a role in regulation of eosinophil chemotaxis.

Topics: Chemokine CCL5; Chemotaxis; Deferoxamine; Dithiothreitol; Dose-Response Relationship, Drug; Eosinophils; Humans; Hydrazines; Interleukin-5; Leukotriene B4; Molsidomine; Nitrates; Nitric Oxide; Nitrogen; Pteridines; Reactive Oxygen Species; Superoxides; Tyrosine; Xanthine

Methamphetamine (METH)-induced dopaminergic neurotoxicity is believed to be produced by oxidative stress and free radical generation. The present study was undertaken to investigate if METH generates peroxynitrite and produces dopaminergic neurotoxicity. We also investigated if this generation of peroxynitrite can be blocked by a selective peroxynitrite decomposition catalyst, 5, 10,15, 20-tetrakis(N-methyl-4'-pyridyl)porphyrinato iron III (FeTMPyP) and protect against METH-induced dopaminergic neurotoxicity. Administration of METH resulted in the significant formation of 3-nitrotyrosine (3-NT), an in vivo marker of peroxynitrite generation, in the striatum and also caused a significant increase in the body temperature. METH injection also caused a significant decrease in the concentration of dopamine (DA), 3, 4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) by 76%, 53% and 40%, respectively, in the striatum compared with the control group. Treatment with FeTMPyP blocked the formation of 3-NT by 66% when compared with the METH group. FeTMPyP treatment also provided significant protection against the METH-induced hyperthermia and depletion of DA, DOPAC and HVA. Administration of FeTMPyP alone neither resulted in 3-NT formation nor had any significant effect on DA or its metabolite concentrations. These findings indicate that peroxynitrite plays a role in METH-induced dopaminergic neurotoxicity and also suggests that peroxynitrite decomposition catalysts may be beneficial for the management of psychostimulant abuse.

Topics: 3,4-Dihydroxyphenylacetic Acid; Animals; Body Temperature; Corpus Striatum; Dopamine; Homovanillic Acid; Male; Methamphetamine; Mice; Mice, Inbred C57BL; Neuroprotective Agents; Neurotoxins; Nitrates; Oxidants; Porphyrins; Reference Values; Tyrosine

The aim of the present study was to investigate the role of poly (ADP-ribose) synthetase (PARS) in a model of acute local inflammation (zymosan-activated plasma (ZAP)-induced paw edema), in which the oxyradicals, nitric oxide and peroxynitrite, are known to play a crucial role. Injection of zymosan-activated plasma (ZAP) into the rat paw induced edema formation. The maximal increase in paw volume was observed at three hours after administration (maximal in paw volume: 1.29+/-0.09 ml). At this time point, there was a marked increase in neutrophil infiltration in the paw, as measured by an increase in myeloperoxidase (MPO) activity in the paw tissue (260+/-25 mU/100 mg wet tissue). However, ZAP-induced paw edema was significantly reduced in a dose-dependent manner by treatment with 3-aminobenzamide (3-AB) or nicotinamide (NIC), two inhibitors of PARS, at 1, 2, 3, 4 hours after ZAP injection. PARS inhibition also caused a significant reduction of MPO activity. The paw tissues were also examined immunohistochemically for the presence of nitrotyrosine (a footprint for peroxynitrite formation). At 3 h following ZAP injection, staining for nitrotyrosine were also found to be localised within discrete cells in the inflamed paw tissue. Treatment with PARS inhibitor prevented the appearance of nitrotyrosine in the tissues. Our results suggest that in paw edema induced by ZAP, inhibition of PARS exert potent anti-inflammatory effects.

Topics: Animals; Benzamides; Complement Activation; Dose-Response Relationship, Drug; Edema; Enzyme Inhibitors; Free Radicals; Immunohistochemistry; Inflammation; Male; Niacinamide; Nitrates; Peroxidase; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Rats; Rats, Wistar; Tyrosine; Zymosan

Effective lung repair after acute injury requires elimination of proliferating mesenchymal and inflammatory cells without inducing an acute inflammatory response or disturbing concomitant repair of lung microvasculature. Previous studies have shown that endogenous NO regulates programmed cell death in fibroblasts and can modulate wound fibroblast synthetic function. We hypothesized that exposure of human lung fibroblasts to NO gas would decrease viability and induce apoptotic cell death. Primary cultures of normal human lung fibroblasts were exposed for 4 h to room air (RA), 80% oxygen, NO (at either 20 or 50 ppm) blended with RA, or NO blended with 80% O(2), then incubated for 24 to 72 h. Cell viability was determined by fluorescence viability/cytotoxicity assay and DNA fragmentation by TUNEL assay. Peroxynitrite formation was assessed using immunoblotting for S-nitrosotyrosine. NO plus O(2) induced significant cell death at 20 and 50 ppm NO when compared to either RA or O(2) alone at both 24 and 72 h (p < 0.05). Incubation with superoxide dismutase (SOD), catalase (CAT) or SOD + CAT significantly decreased cell death in fibroblasts treated with NO(20)/O(2) and NO(50)/O(2) compared with controls (p < 0.05). NO(20)/O(2) and NO(50)/O(2) exposure significantly increased TUNEL mean fluorescence intensity (MFI), consistent with increased DNA fragmentation, compared to RA at 24 and 72 h (p < 0.05). Antioxidants decreased MFI in cells exposed to NO(20)/O(2) (CAT and SOD + CAT) compared to controls at 24 h (p < 0.05). Western blot analysis for S-nitrosotyrosine showed increased signal intensity in fibroblasts exposed to NO at 20 and 50 ppm plus O(2) compared to RA or O(2) alone. Incubation with SOD + CAT reduced signal intensity for peroxynitrite in cells exposed to NO(20)/O(2). We conclude that NO in hyperoxic conditions induces fibroblast cell death and DNA fragmentation, which could be partially mediated by peroxynitrite synthesis.

Topics: Antioxidants; Catalase; Cell Survival; Cells, Cultured; DNA Fragmentation; Fibroblasts; Humans; Hyperoxia; In Situ Nick-End Labeling; Lung; Nitrates; Nitric Oxide; Oxygen; Superoxides; Tyrosine

Peroxynitrite is a reactive cytotoxic species, capable of nitrating tyrosine residues to form 3-nitrotyrosine. Little is known about the formation and loss of nitrated proteins in vivo. We have measured nitrated proteins, by enzyme-linked immunosorbent assay, in rat skin after exposure to peroxynitrite. Peroxynitrite (100-200 nmol site(-1)) was injected into the skin of anesthetized rats. At the highest dose 78.6 +/- 9.5 pmol mg(-1) protein of nitrated BSA equivalents were measured at 4 h and a significant increase was observed for 24 h after administration in skin samples. The loss of nitrated proteins from skin appeared biphasic with an initial (t(1/2) = 2 h) and slower loss (t(1/2) = 22 h). A major nitrated protein was identified as albumin by Western blot analysis. The data demonstrate that a single exposure to peroxynitrite can lead to the presence of nitrated proteins in skin for at least 24 h. The sustained presence of nitrated proteins may influence the inflammatory process in skin disease.

Topics: Animals; Kinetics; Male; Nitrates; Oxidants; Proteins; Rats; Rats, Wistar; Serum Albumin, Bovine; Skin; Time Factors; Tyrosine

This study investigated the role of peroxynitrite in an adult rat model of pneumococcal meningitis. Immunohistochemically, nitrotyrosine residues, as a marker for peroxynitrite formation, were detected perivascularly and in proximity to inflammatory cells in the subarachnoid space. Nitrotyrosine immunoreactivity was colocalized with blood-brain barrier breaching, which was visualized by fluorescence microscopy after intravenous application of Evans blue. Treatment of infected rats with uric acid (300 mg/kg intraperitoneally), a scavenger of peroxynitrite, significantly attenuated intracranial pressure, cerebrospinal fluid white blood cell count, and blood-brain barrier leakage, as indicated by Evans blue concentration in the cerebrospinal fluid (21.6+/-9.3 mm Hg, 5776+/-1790 cells/microL, 9.7+/-6.4 microgram/mL in infected, untreated rats vs. 7.2+/-1.6 mm Hg, 2004+/-904 cells/microL, 1.1+/-1.0 microgram/mL infected, uric acid-treated rats, mean+/-SD, P<.05). These data suggest that peroxynitrite plays a central role in mediating pathophysiological alterations during bacterial meningitis.

Topics: Animals; Blood-Brain Barrier; Brain; Cerebrospinal Fluid; Free Radical Scavengers; Inflammation; Intracranial Pressure; Leukocytes; Male; Meningitis, Pneumococcal; Nitrates; Oxidants; Rats; Rats, Wistar; Subarachnoid Space; Tyrosine; Uric Acid

Peroxynitrite, an oxidant generated by the interaction between superoxide and nitric oxide (NO), can nitrate tyrosine residues, resulting in compromised protein function. Monocyte chemoattractant protein-1 (MCP-1) is a chemokine that attracts monocytes and has a tyrosine residue critical for function. We hypothesized that peroxynitrite would alter MCP-1 activity. Peroxynitrite attenuated MCP-1-induced monocyte chemotactic activity (MCA) in a dose-dependent manner (P < 0.05) but did not attenuate leukotriene B4 or complement-activated serum MCA. The reducing agents dithionite, deferoxamine, and dithiothreitol reversed the MCA inhibition by peroxynitrite, and exogenous L-tyrosine abrogated the inhibition by peroxynitrite. PAPA-NONOate, an NO donor, or superoxide generated by xanthine and xanthine oxidase did not show an inhibitory effect on MCA induced by MCP-1. The peroxynitrite generator 3-morpholinosydnonimine caused a concentration-dependent inhibition of MCA by MCP-1. Peroxynitrite reduced MCP-1 binding to monocytes and resulted in nitrotyrosine formation. These findings are consistent with nitration of tyrosine by peroxynitrite, with subsequent inhibition of MCP-1 binding to monocytes, and suggest that peroxynitrite may play a role in regulation of MCP-1-induced monocyte chemotaxis.

Topics: Blood Physiological Phenomena; Cells, Cultured; Chemokine CCL2; Chemotaxis, Leukocyte; Dose-Response Relationship, Drug; Humans; Leukotriene B4; Monocytes; Nitrates; Nitrogen; Oxidants; Reactive Oxygen Species; Reducing Agents; Superoxides; Tyrosine

Nitroglycerin (NTG) is an important cardiovascular agent, but tolerance during continuous administration limits its clinical utility. Increased vascular superoxide production may mediate nitrate tolerance via a reduction in nitric oxide availability. Because superoxide anion and nitric oxide react avidly to form peroxynitrite, an aggressive cellular toxicant that nitrates protein tyrosine residues, we tested the hypotheses that protein nitration, indicative of peroxynitrite formation, occurs during vascular tolerance, and that protein nitration participates in tolerance development. Preincubation of rat thoracic aorta segments with NTG (22 microM, EC(95) for 30 min) caused a significant shift in NTG relaxation response (EC(50); 6.7 +/- 1.7 versus 0.50 +/- 0.13 microM, NTG versus vehicle, p <.05). After functional evaluations, tissues were fixed in formalin for immunohistochemistry and digital image analysis. NTG-induced vascular tolerance was associated with increased immunoprevalence of 3-nitrotyrosine (3NT, stable biomarker of protein nitration; 11.41 +/- 2.48 versus 0.04 +/- 0.02% positive pixels, NTG versus vehicle, p < .05). Staining was observed throughout vascular smooth muscle layers. Addition of 500 microM free tyrosine to the preincubation medium did not alter tolerance development (NTG EC(50) 6.5 +/- 3.0 microM) but abolished 3NT immunoprevalence (0.16 +/- 0.10%). No significant relationship between NTG potency and 3NT immunoprevalence was observed. These data support the hypothesis that protein nitration occurs during nitrate vascular tolerance, however, it apparently does not mediate this phenomenon.

Topics: Animals; Aorta, Thoracic; Blood Proteins; Drug Tolerance; Immunohistochemistry; Male; Nitrates; Nitroglycerin; Rats; Rats, Sprague-Dawley; Tyrosine; Vasodilator Agents

We used IL-6 knock-out (KO) mice to evaluate a possible role for IL-6 in the pathogenesis of splanchnic artery occlusion shock (SAO). SAO shock was induced by clamping both the superior mesenteric artery and the celiac trunk, followed by release of the clamp. There was a marked increase in the peroxynitrite formation in the plasma of the SAO-shocked IL-6 wild-type (WT) mice after reperfusion. Immunohistochemical examination demonstrated a marked increase in the immunoreactivity to nitrotyrosine in the necrotic ileum in shocked IL-6 WT mice. SAO-shocked WT mice developed a significant increase of tissue myeloperoxidase (MPO) and malondialdehyde (MDA) activity and marked histological injury to the distal ileum. SAO shock was also associated with a significant mortality (0% survival). Reperfused ileum tissue sections from SAO-shocked WT mice showed positive staining for P-selectin. Little specific staining was observed in sham-WT mice. Staining of ileum tissue obtained from sham-operated WT mice with anti-ICAM-1 antibody showed weak but diffuse staining, demonstrating that ICAM-1 is constitutively expressed. However, after SAO shock the staining intensity increased substantially in the ileum section from WT mice. Intensity and degree of P-selectin and ICAM-1 were markedly reduced in tissue section from SAO-shocked IL-6 KO mice. SAO-shocked IL-6 KO mice also show significant reduction of neutrophil infiltration into the reperfused intestine, as evidenced by reduced MPO activity, improved histological status of the reperfused tissues, reduced peroxynitrite formation, reduced MDA levels, and improved survival. In vivo treatment with anti-IL-6 significantly prevents the inflammatory process. Our results clearly demonstrate that IL-6 plays an important role in ischemia and reperfusion injury and allows the hypothesis that inhibition of IL-6 may represent a novel and possible strategy. Part of this effect may be due to inhibition of the expression of adhesion molecules and subsequent reduction of neutrophil-mediated cellular injury.

Topics: Animals; Antibodies, Monoclonal; Arterial Occlusive Diseases; Celiac Artery; Constriction; Cytokines; Ileum; Immunity, Innate; Intercellular Adhesion Molecule-1; Interleukin-6; Ischemia; Leukocyte Count; Lipid Peroxidation; Malondialdehyde; Mesenteric Artery, Superior; Mice; Mice, Knockout; Neutrophils; Nitrates; Nitric Oxide; P-Selectin; Peroxidase; Reperfusion Injury; Shock; Splanchnic Circulation; Tyrosine

To assess the degree, source, and patterns of oxidative damage to bronchoalveolar lavage proteins as a modification of amino acid residues in patients with acute respiratory distress syndrome (ARDS).. Prospective, controlled study.. Adult intensive care unit of a postgraduate teaching hospital.. Twenty-eight patients with established ARDS were studied and compared with six ventilated patients without ARDS and 11 normal healthy controls.. Supportive techniques appropriate to ARDS.. Evidence of oxidative modification of bronchoalveolar lavage fluid protein, indicative of the production of specific reactive oxidizing species, was sought using a high-performance liquid chromatography technique. Bronchoalveolar lavage fluid samples from patients with ARDS, ventilated intensive care controls, and normal healthy controls were analyzed. Concentrations of orthotyrosine were significantly higher in the ARDS group than in either control group (7.98 + 3.78 nmol/mg for ARDS, 0.67 + 0.67 for ventilated controls, and 0.71 + 0.22 for healthy controls; p < .05). Chlorotyrosine concentrations were also significantly increased in the ARDS group over either control group (4.82 + 1.07 nmol/mg for ARDS, 1.55 + 1.34 for ventilated controls, and 0.33 + 0.12 for healthy controls; p < .05). Nitrotyrosine concentrations were similarly significantly increased in the ARDS groups compared with each control group (2.21 + 0.65 nmol/mg for ARDS, 0.29 + 0.29 for ventilated controls, and 0.06 + 0.03 for healthy controls; p < .05). Chlorotyrosine and nitrotyrosine concentrations showed significant correlations with myeloperoxidase concentrations in bronchoalveolar lavage fluid, measured using an enzyme-linked immunosorbent assay in patients with ARDS. These findings suggest a possible relationship between inflammatory cell activation, oxidant formation, and damage to proteins in the lungs of these patients. Overall, our data strongly suggest heightened concentrations of oxidative stress in the lungs of patients with ARDS that lead to significantly increased oxidative protein damage.

Topics: Adolescent; Adult; Aged; Biomarkers; Bronchoalveolar Lavage Fluid; Case-Control Studies; Chromatography, High Pressure Liquid; Female; Humans; Hydroxyl Radical; Hydroxylation; Hypochlorous Acid; Linear Models; Male; Middle Aged; Neutrophil Activation; Neutrophils; Nitrates; Oxidants; Oxidative Stress; Proteins; Respiration, Artificial; Respiratory Distress Syndrome; Statistics, Nonparametric; Tyrosine

The peroxynitrite-scavenging ability of some phenolic antioxidants, p-coumaric acid, caffeic acid and sinapinic acid, was examined and compared with ascorbic acid and tocopherol using 3-nitrotyrosine formation as a marker. Among these, caffeic acid and sinapinic acid strongly inhibited the formation of 3-nitrotyrosine in protein. The treatment of protein with peroxynitrite in the presence of sinapinic acid, but not caffeic acid, produced a novel product determined by reversed-phase high performance liquid chromatography (HPLC). The product formed was purified and then identified as a mono-lactone type dimer (ML) of sinapinic acid by nuclear magnetic resonance (NMR) and liquid chromatography-mass spectrometry (LC-MS). This ML was converted from a di-lactone type dimer, obtained from sinapinic acid with peroxidase/hydrogen peroxide, in neutral buffer. In this report, we have proposed that the ML of sinapinic acid is generated via one-electron oxidation by peroxynitrite treatment.

Topics: Chromatography, High Pressure Liquid; Coumaric Acids; Dimerization; Magnetic Resonance Spectroscopy; Mass Spectrometry; Nitrates; Oxidation-Reduction; Proteins; Tyrosine

The role of nitric oxide in ulcer formation remains unknown. Accordingly, we assessed local expression of inducible nitric oxide synthase (NOS) and nitration of tyrosine as an indicator of peroxynitrite formation in patients with Helicobacter pylori (HP)-associated gastric ulcers compared with HP-negative ulcers. Biopsy specimens were taken from the ulcer margin and from an area remote from the ulcer portion. Inducible NOS, nitrotyrosine, and macrophage immunoreactivity were assessed immunohistochemically using a labeled streptavidin-biotin method. In HP-positive gastric ulcers, inducible NOS and nitrotyrosine immunoreactivity was frequently observed at active ulcer margins, sometimes in surface epithelial cells as well as in the lamina propria. Occasionally, inducible NOS and nitrotyrosine reactivity were found in areas remote from the lesion in cases of HP-positive ulcer and HP-related gastritis. Macrophages accumulated significantly in the margin of HP-positive ulcers. In HP-negative gastric ulcers, inducible NOS and nitrotyrosine immunoreactivity also were frequent at the ulcer margin, but no significant immunoreactivity was observed at a distance. HP eradication caused significant attenuation in inducible NOS and macrophage immunoreactivity. In conclusion, nitric oxide and peroxynitrite formation is increased in HP-infected gastric mucosa, suggesting that HP promotes nitric oxide stress.

Topics: Fluorescent Antibody Technique; Gastric Mucosa; Gastritis; Helicobacter pylori; Humans; Macrophages; Nitrates; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Peptic Ulcer; Tyrosine

The regulation of free intracellular calcium [Ca2+]i is altered in neurons from the aged brain, possibly due to reductions in the activity of Ca2+ transporters. The plasma membrane Ca(2+)-ATPase (PMCA) plays a critical role in Ca2+ homeostasis, and its kinetic properties change in aged rat brain. These changes could be due to oxidative modification of PMCA as a result of age-related chronic oxidative stresses. The present studies were undertaken to determine the sensitivity of the neuronal PMCA to in vitro exposure of synaptic plasma membranes (SPMs) to reactive oxygen species (ROS). We examined the effects of three oxidants including peroxyl radicals generated by azo-initiators, 2,2'-Azobis 2-amidinopropane dihydrochloride (AAPH) and 4,4'-Azobis 14-cyanovaleric acid (ACVA), hydrogen peroxide (H2O2), and peroxynitrite (ONOO-). Synaptic plasma membranes briefly exposed to these oxidants were analyzed for functional and structural alterations in PMCA. Although all three oxidants led to significant loss of PMCA activity, the effect of ONOO- was the most potent, followed by peroxyl radicals and H2O2. Kinetic analysis of PMCA activity after oxidant treatment showed decreases in Vmax without significant changes in K(act). Immunoblots revealed oxidant-induced cross-linking of PMCA molecules that were partially reversed under reducing conditions and completely reversed with addition of urea. The PMCA appears to be very sensitive to inhibition by ROS and hence may be a target of oxidative stress in the aging brain. Reduction in its activity may contribute to age-related alterations in neuronal [Ca2+]i regulation.

Topics: Age Factors; Amidines; Animals; Azo Compounds; Brain; Calcium; Calcium-Transporting ATPases; Hydrogen Peroxide; Kinetics; Male; Nitrates; Oxidative Stress; Phenylhydrazines; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Synaptic Membranes; Tyrosine; Urea; Valerates

In the present study we used IL-6 knockout mice (IL-6KO) to evaluate the role of IL-6 in the inflammatory response caused by injection of carrageenan into the pleural space. Compared with carrageenan-treated IL-6 wild-type (IL-6WT) mice, carrageenan-treated IL-6KO mice exhibited a reduced degree of pleural exudation and polymorphonuclear cell migration. Lung myeloperoxidase activity and lipid peroxidation were significantly reduced in IL-6KO mice compared with those in IL-6WT mice treated with carrageenan. Immunohistochemical analysis for nitrotyrosine and poly(A)DP-ribose polymerase revealed a positive staining in lungs from carrageenan-treated IL-6WT mice. No positive staining for nitrotyrosine or PARS was found in the lungs of the carrageenan-treated IL-6KO mice. Staining of lung tissue sections obtained from carrageenan-treated IL-6WT mice with an anti-cyclo-oxygenase-2 Ab showed a diffuse staining of the inflamed tissue. Furthermore, expression of inducible nitric oxide synthase was found mainly in the macrophages of the inflamed lungs from carrageenan-treated IL-6WT mice. The intensity and degree of the staining for cyclo-oxygenase-2 and inducible nitric oxide synthase were markedly reduced in tissue sections obtained from carrageenan-treated IL-6KO mice. Most notably, the degree of lung injury caused by carrageenan was also reduced in IL-6KO mice. Treatment of IL-6WT mice with anti-IL-6 (5 microg/day/mouse at 24 and 1 h before carrageenan treatment) also significantly attenuated all the above indicators of lung inflammation. Taken together, our results clearly demonstrate that IL-6KO mice are more resistant to the acute inflammation of the lung caused by carrageenan injection into the pleural space than the corresponding WT mice.

Topics: Animals; Carrageenan; Cells, Cultured; Cytokines; Dinoprostone; DNA Damage; Enzyme Induction; Interleukin-6; Leukotriene B4; Lung; Macrophages; Male; Malondialdehyde; Mice; Mice, Knockout; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Peroxidase; Pleura; Pleurisy; Poly(ADP-ribose) Polymerases; Prostaglandin-Endoperoxide Synthases; Prostaglandins F; Tyrosine

Varied intensities of nitrotyrosine immunoreactivity were detected by Western blots after the reaction of proteins or enzymes with peroxynitrite (PN), a strong oxidant derived from nitric oxide. Intense immunoreactivity of cAMP-dependent protein kinase, calmodulin and most histones may depend on greater access to tyrosine residues in the reaction, whereas the absence of immunoreactivity of caspase-3, ubiquitin and S-100 proteins may reflect lack of accessibility. In addition, the changes in UV/visible absorbency were observed after PN-treatment of polynucleotides, polypeptides or proteins. Brief PN-treatment of invertase increased its enzymatic activity. Furthermore, PN-treatment of rabbit IgG decreased its recognition by anti-IgG. The results suggest that PN may chemically modify polypeptides, proteins and polynucleotides and may subsequently alter their biological activity.

Topics: Animals; Nitrates; Nitric Oxide; Oxidants; Proteins; Rabbits; Tyrosine

Although studies in vitro have implicated oxygen-derived free radicals as possible mediators of inflammatory cytokine-induced cell injury, the role of the radicals in the cytokine-induced myocardial dysfunction in vivo remains unclear. The present study was designed to address this point in our novel canine model of cytokine-induced myocardial dysfunction in vivo.. Studies were performed in mongrel dogs, in which microspheres (MS, 15 microns in diameter) with and without interleukin-1 beta (IL-1 beta) were injected into the left main coronary artery (control and IL-1 beta group). Left ventricular ejection fraction (LVEF) was evaluated by echocardiography for 1 week.. Immediately after the intracoronary injection of MS (10(6)/kg), LVEF equally decreased to approximately 30% in both the control and IL-1 beta group. While LVEF rapidly recovered within 2 days in the control group, it remained depressed in the IL-1 beta group until day 7 (p < 0.0001 vs. control group). Pretreatment with OPC-6535 (an inhibitor of superoxide production) before (2 mg/kg i.v.) and 1 and 2 days after IL-1 beta MS application (1 mg/kg i.v.) prevented the IL-1 beta-induced myocardial dysfunction. Superoxide production in the myocardium was significantly higher in the IL-1 beta group than in the control group at day 2 (p < 0.01), and OPC-6535 significantly suppressed the IL-1 beta-induced superoxide production (p < 0.01). An HPLC assay showed that nitrotyrosine, a marker of the formation of peroxynitrite by superoxide anion and nitric oxide, was present in the myocardium treated with IL-1 beta but not in that with control MS. OPC-6535 abolished the IL-1 beta-induced formation of myocardial nitrotyrosine.. These results indicate that superoxide anion and the resultant formation of peroxynitrite may substantially be involved in the pathogenesis of the cytokine-induced myocardial dysfunction in dogs in vivo.

Topics: Analysis of Variance; Animals; Antioxidants; Biomarkers; Dogs; Echocardiography; Female; Interleukin-1; Male; Myocardium; Nitrates; Random Allocation; Stroke Volume; Superoxides; Thiazoles; Tyrosine; Ventricular Dysfunction, Left

Peroxynitrite, a potent cytotoxic oxidant formed by the reaction of nitric oxide (NO) with the superoxide anion, was recently proposed to play a major pathogenic role in the inflammatory process. Here we have investigated the effects of endogenous melatonin, a known scavenger of peroxynitrite, in rats subjected to carrageenan-induced pleurisy. Endogenous melatonin was depleted in rats maintained on 24 h light cycle for 1 wk. In vivo depletion of endogenous melatonin enhanced the carrageenan-induced degree of pleural exudation and polymorphonuclear leukocyte migration in rats subjected to carrageenan-induced pleurisy. Lung myeloperoxidase activity and lipid peroxidation were significantly increased in melatonin-deprived rats. However, the inducible NO synthase in lung samples was unaffected by melatonin depletion. Immunohistochemical analysis for nitrotyrosine revealed a positive staining in lungs from carrageenan-treated rats that was markedly enhanced in melatonin-deprived rats. Furthermore, melatonin depletion significantly increased peroxynitrite formation as measured by the oxidation of the fluorescent dye dihydrorhodamine 123, enhanced DNA damage and the decrease in mitochondrial respiration and reduced the cellular levels of NAD+ in macrophages harvested from the pleural cavity of rats subjected to carrageenan-induced pleurisy. In vivo treatment with exogenous melatonin (15 mg/kg intraperitoneal) significantly reversed the effects of melatonin depletion. Thus, endogenous melatonin plays an important protective role against carrageenan-induced local inflammation.

Topics: Animals; Carrageenan; Cells, Cultured; Energy Metabolism; Lipid Peroxidation; Macrophages; Melatonin; Nitrates; Nitric Oxide; Peroxidase; Pleurisy; Rats; Tyrosine

Tyrosine nitration is a covalent posttranslational protein modification derived from the reaction of proteins with nitrating agents. Protein nitration appears to be a selective process since not all tyrosine residues in proteins or all proteins are nitrated in vivo. To investigate factors that may determine the biological selectivity of protein tyrosine nitration, we developed an in vitro model consisting of three proteins with similar size but different three-dimensional structure and tyrosine content. Exposure of ribonuclease A to putative in vivo nitrating agents revealed preferential nitration of tyrosine residue Y(115). Tyrosine residue Y(23) and to a lesser extent residue Y(20) were preferentially nitrated in lysozyme, whereas tyrosine Y(102) was the only residue modified by nitration in phospholipase A(2). Tyrosine Y(115) was the residue modified by nitration after exposure of ribonuclease A to different nitrating agents: chemically synthesized peroxynitrite, nitric oxide, and superoxide generated by SIN-1 or myeloperoxidase (MPO)/H(2)O(2) plus nitrite (NO(-2)) in the presence of bicarbonate/CO(2). The nature of the nitrating agent determined in part the protein that would be predominantly modified by nitration in a mixture of all three proteins. Ribonuclease A was preferentially nitrated upon exposure to MPO/H(2)O(2)/NO(-2), whereas phospholipase A(2) was the primary target for nitration upon exposure to peroxynitrite. The data also suggest that the exposure of the aromatic ring to the surface of the protein, the location of the tyrosine on a loop structure, and its association with a neighboring negative charge are some of the factors determining the selectivity of tyrosine nitration in proteins.

Topics: Amino Acid Sequence; Models, Molecular; Molecular Sequence Data; Muramidase; Nitrates; Nitric Oxide; Phospholipases A; Protein Processing, Post-Translational; Protein Structure, Secondary; Ribonuclease, Pancreatic; Sequence Analysis, Protein; Superoxides; Tyrosine

The peroxynitrite contributions to hypoxic damage in brain slices that arise from N-methyl-D-aspartate (NMDA) receptor activation were studied by following the temporal-spatial course of nitrotyrosine (NT) formation during six conditions: hypoxia (pO(2)<5 mmHg) with or without 10 microM MK-801 treatment; with exposure to 10, 100 and 1000 microM NMDA; and no treatment (control). In each experiment, twenty 350-micrometer thick cerebrocortical slices, obtained from the parietal lobes of ten 7-day-old Sprague-Dawley rats, were metabolically recovered and allowed to respire in a well-oxygenated perfusion system. Thirty minutes exposures to hypoxia or NMDA were followed by 2 h of oxygenated reperfusion. MK-801 administration began 15 min prior to hypoxia and was discontinued during reperfusion. Anti-NT serum immunohistochemistry stains in 20-micrometer frozen sections of slices taken during oxygenated reperfusion, after hypoxia or NMDA exposure, were positive in both neurons and endothelial cells. NT-positive neurons were detected sooner after hypoxia than after NMDA exposure, suggesting that mechanisms of superoxide generation were different in both groups. After hypoxia and even more so after NMDA exposure, more intense NT-positive staining was observed in endothelial cells than in neurons. Cell damage after hypoxia was attenuated by MK-801. MK-801 decreased post-hypoxia counts of NT-stained endothelial cells by 78.5% (p<0. 001) and NT-stained neurons by 54.1% (p<0.05). Our findings suggest that NMDA receptor activation in hypoxic brain slices is associated with increased post-hypoxic peroxynitrite production that contributes to acute neuronal death and endothelial cell injury. Peroxynitrite injury to endothelial cells, caused either by increased peroxynitrite from within or from increased vulnerability to peroxynitrite from without, might play an important role in hypoxic-ischemic brain injury and NMDA-induced brain injury.

Topics: Animals; Brain; Dizocilpine Maleate; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Hypoxia-Ischemia, Brain; N-Methylaspartate; Neurons; Nitrates; Nitric Oxide; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Superoxides; Tyrosine

Zymosan, a non-bacterial agent, causes inflammation by inducing the production of a variety of cytokines and pro-inflammatory mediators, wherein reactive oxygen species including nitric oxide and peroxynitrite are known to play a crucial role in the inflammatory process. The current study was designed to investigate the protective effect of melatonin, a radical scavenger and antioxidant, on non-septic shock induced by zymosan in the rat. Four groups of rats (controls, melatonin-injected [5 mg/kg x 6], zymosan-injected [500 mg/kg], and zymosan + melatonin) were used in this experiment. Thiobarbituric acid reactive substances (malondialdehyde [MDA] + 4-hydroxyalkenals [4-HDA]), as an index of lipid peroxidation, were measured in the liver, lung, small intestine (ileum), kidney and pancreas. Twenty-four hours after zymosan administration, MDA + 4-HDA levels were significantly increased in the liver, lung, small intestine, and kidney while the increase in the pancreas was not statistically significant compared to levels in control rats. The percentage increases in lipid peroxidation products were 34.3%, 39.2%, 48.5%, 32.5%, and 17.4% for the liver, lung, small intestine, kidney, and pancreas, respectively. In animals given melatonin 30 minutes before zymosan, and 5 more times after zymosan (i.e., every 4 hours), the increase in MDA + 4-HDA levels was reduced in all organs studied. There was also a significant increase in the volume of peritoneal exudate in zymosan-treated rats that was reduced when the zymosan-shocked rats received melatonin. After zymosan administration, immunohistochemical and histological examination demonstrated a marked increase in the immunoreactivity to nitrotyrosine, a specific "footprint" of peroxynitrite, and tissue damage in the liver, lung, and small intestine of zymosan-shocked rats. Again, melatonin treatment reduced both nitrotyrosine immunoreactivity and tissue damage associated with zymosan administration.

Topics: Animals; Antioxidants; Disease Models, Animal; Exudates and Transudates; Immunohistochemistry; Kidney; Lipid Peroxidation; Liver; Lung; Male; Malondialdehyde; Melatonin; Nitrates; Rats; Rats, Sprague-Dawley; Shock; Tyrosine; Zymosan

Oxidative stress is implicated in the death of dopaminergic neurons in Parkinson's disease and in the 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine (MPTP) model of Parkinson's disease. Oxidative species that might mediate this damage include hydroxyl radical, tyrosyl radical, or reactive nitrogen species such as peroxynitrite. In mice, we showed that MPTP markedly increased levels of o, o'-dityrosine and 3-nitrotyrosine in the striatum and midbrain but not in brain regions resistant to MPTP. These two stable compounds indicate that tyrosyl radical and reactive nitrogen species have attacked tyrosine residues. In contrast, MPTP failed to alter levels of ortho-tyrosine in any brain region we studied. This marker accumulates when hydroxyl radical oxidizes protein-bound phenylalanine residues. We also showed that treating whole-brain proteins with hydroxyl radical markedly increased levels of ortho-tyrosine in vitro. Under identical conditions, tyrosyl radical, produced by the heme protein myeloperoxidase, selectively increased levels of o,o'-dityrosine, whereas peroxynitrite increased levels of 3-nitrotyrosine and, to a lesser extent, of ortho-tyrosine. These in vivo and in vitro findings implicate reactive nitrogen species and tyrosyl radical in MPTP neurotoxicity but argue against a deleterious role for hydroxyl radical in this model. They also show that reactive nitrogen species and tyrosyl radical (and consequently protein oxidation) represent an early and previously unidentified biochemical event in MPTP-induced brain injury. This finding may be significant for understanding the pathogenesis of Parkinson's disease and developing neuroprotective therapies.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Amino Acids; Animals; Brain; Chelating Agents; Dopamine Agents; Free Radicals; Gas Chromatography-Mass Spectrometry; Male; Mice; Mice, Inbred C57BL; Nitrates; Oxidation-Reduction; Oxidative Stress; Parkinson Disease; Pentetic Acid; Tissue Distribution; Tyrosine

1 The therapeutic efficacy of Mn(III)tetrakis (4-benzoic acid) porphyrin (MnTBAP), a novel superoxide dismutase mimetic which scavenges peroxynitrite, was investigated in rats subjected to shock induced by peritoneal injection of zymosan. 2 Our data show that MnTBAP (given at 1, 3 and 10 mg kg-1 intraperitoneally, 1 and 6 h after zymosan injection) significantly reduce in dose dependent manner the development of peritonitis (peritoneal exudation, high nitrate/nitrite and peroxynitrite plasma levels, leukocyte infiltration and histological examination). 3 Furthermore, our data suggest that there is a reduction in the lung, small intestine and liver myeloperoxidase (MPO) activity and lipid peroxidation activity from MnTBAP-treated rats. 4 MnTBAP also reduced the appearance of nitrotyrosine immunoreactivity in the inflamed tissues. 5 Furthermore, a significant reduction of suppression of mitochondrial respiration, DNA strand breakage and reduction of cellular levels of NAD+ was observed in ex vivo macrophages harvested from the peritoneal cavity of zymosan-treated rat. 6 In vivo treatment with MnTBAP significantly reduced in a dose-dependent manner peroxynitrite formation and prevented the appearance of DNA damage, the decrease in mitochondrial respiration and the loss of cellular levels of NAD+. 7 In conclusion our results showed that MnTBAP was effective in preventing the development of zymosan-induced shock.

Topics: Alanine Transaminase; Alkaline Phosphatase; Animals; Bilirubin; Disease Models, Animal; DNA Damage; Dose-Response Relationship, Drug; Energy Metabolism; Free Radical Scavengers; Immunohistochemistry; L-Lactate Dehydrogenase; Lung; Male; Malondialdehyde; Metalloporphyrins; NAD; Nitrates; Peroxidase; Rats; Rats, Sprague-Dawley; Shock; Superoxide Dismutase; Tyrosine; Zymosan

Endotoxemia promotes gut barrier failure and bacterial translocation (BT) by upregulating inducible nitric oxide synthase (iNOS) in the gut. We hypothesized that administration of a dithiocarbamate derivative, NOX, which scavenges nitric oxide (NO), may reduce intestinal injury and BT after lipopolysaccharide (LPS) challenge. Sprague-Dawley rats were randomized to receive NOX or normal saline via subcutaneously placed osmotic pumps before or after LPS challenge. Mesenteric lymph nodes, liver, spleen, and blood were cultured 24 h later. Transmucosal passage of Escherichia coli C-25 or fluorescent beads were measured in an Ussing chamber. Intestinal membranes were examined morphologically for apoptosis, iNOS expression, and nitrotyrosine immunoreactivity. NOX significantly reduced the incidence of bacteremia, BT, and transmucosal passage of bacteria and beads when administered before or up to 12 h after LPS challenge. LPS induced enterocyte apoptosis at the villus tips where bacterial entry was demonstrated by confocal microscopy. NOX significantly decreased the number of apoptotic nuclei and nitrotyrosine residues. NOX prevents LPS-induced gut barrier failure by scavenging NO and its toxic derivative, peroxynitrite.

Topics: Animals; Bacteremia; Enterobacteriaceae; Enterocytes; Free Radical Scavengers; Infusion Pumps, Implantable; Intestinal Absorption; Intestines; Lipopolysaccharides; Male; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Organometallic Compounds; Rats; Rats, Sprague-Dawley; Thiocarbamates; Tyrosine

Putative 'protein nitratases,' which catalyze denitration of peroxynitrite (PN)-treated proteins, were detected in the homogenate/crude extract of rat brains and hearts. Nitratase activity was monitored by the decreased intensity of nitrotyrosine immunoreactive-bands in Western blot and increased nitrate level in dialysate of incubation mixture, which contained homogenate/crude extract, protease inhibitors and a PN-treated substrate, such as treated histone (III-S), BSA or invertase. Enhanced activity of nitratases was noted by preincubating crude extract with Ca2+. In addition, at least two types of nitratases may occur: type I, reductant-dependent, and type II, reductant- independent. Furthermore, upon denitration, the activity of PN-treated invertase increased to the same activity level of the untreated invertase. The overall reaction catalyzed by nitratases for denitration of nitrotyrosine residues in protein could be as follows: Protein-Tyr-NO2 + H2O --> Protein-Tyr-H + H+ + NO3-. The nitration/denitration of protein-tyrosine may be crucial in regulating signal transduction.

Topics: Animals; beta-Fructofuranosidase; Blotting, Western; Brain; Brain Chemistry; Calcium; Dialysis; Enzymes; Flavin-Adenine Dinucleotide; Glutathione; Glutathione Transferase; Glycoside Hydrolases; Myocardium; NAD; NADP; Nitrates; Proteins; Rats; Serum Albumin, Bovine; Tissue Extracts; Tyrosine

Much of the damaging action of nitric oxide in heart may be due to its diffusion-limited reaction with superoxide to form peroxynitrite. Direct infusion of peroxynitrite into isolated perfused hearts fails to model the effects of in situ formation because the bulk of peroxynitrite decomposes before reaching the myocytes. To examine the direct effects of peroxynitrite on the contractile apparatus of the heart, we exposed intact and skinned rat papillary muscles to a steady state concentration of 4-microM peroxynitrite for 5 min, followed by a 30-min recovery period to monitor irreversible effects. In intact muscles developed force fell immediately to 26% of initial force, recovering to 43% by 30 min. Resting tension increased by 600% immediately, and was still elevated 500% by 30 min. Nitrotyrosine immunochemistry showed that peroxynitrite can induce tyrosine nitration at low concentrations and is capable of penetrating 200-380 microm into the papillary muscle after a 5-min infusion. Decomposed peroxynitrite had no effect on either intact or skinned muscle developed force or resting tension. Our results show that peroxynitrite directly damages both developed force and resting tension of isolated heart muscle, which can be extrapolated to systolic and diastolic injury in intact hearts.

Topics: Animals; Diastole; Immunoenzyme Techniques; Male; Myocardial Contraction; Nitrates; Oxidants; Papillary Muscles; Rats; Systole; Tyrosine

Previous work from our laboratory indicated that the bile salt sodium deoxycholate (NaDOC) induced apoptosis in cultured cells and in normal goblet cells of the colonic mucosa. We also reported that the normal-appearing flat mucosa of patients with colon cancer exhibited apoptosis resistance. Using immunofluorescence in conjunction with confocal microscopy, we now report that high physiological concentrations (0.5 mM) of NaDOC result in the formation of nitrotyrosine residues, a footprint for the formation of reactive nitrogen species, including peroxynitrite, in plasma membrane-associated proteins of HT-29 cells. Because peroxynitrite is formed from the reaction between nitric oxide and superoxide anion, we specifically looked at the role of nitric oxide and superoxide anion in NaDOC-induced apoptosis. Pretreatment of cells with the inhibitor/antioxidants, N-nitro-L-arginine methyl ester, an inhibitor of nitric oxide synthase, copper (II) 3,5-diisopropyl salicylate hydrate, a superoxide dismutase mimetic compound, and Trolox, a water-soluble analog of alpha-tocopherol, alone or in combination, sensitized cells to apoptosis induced by 0.5 mM NaDOC. These results suggest that nitric oxide may be part of a signaling pathway that is responsible for apoptosis resistance. The results also indicate that nitric oxide does not appear to protect cells against NaDOC-induced apoptosis by scavenging superoxide anion.

Topics: Apoptosis; Bile Acids and Salts; Colonic Neoplasms; Deoxycholic Acid; Enzyme Inhibitors; Fluorescent Antibody Technique; Free Radical Scavengers; Humans; Microscopy, Confocal; NG-Nitroarginine Methyl Ester; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Oxidative Stress; Salicylates; Superoxides; Tumor Cells, Cultured; Tyrosine

The inhibitory effects of endogenous and synthetic compounds on the nitration and oxidation of L-tyrosine by peroxynitrite were examined. Nitration and oxidation activities of L-tyrosine by peroxynitrite were estimated by monitoring the formation of 3-nitrotyrosine and dityrosine with a high-performance liquid chromatography-ultraviolet (HPLC-UV)-fluorescence detector system. Glutathione and synthetic compounds ((2S,3R,4S)-N-ethylmercapto-3,4-dihydroxy-2-hydroxymethylpyrrolidine, L-N-dithiocarboxyproline) inhibited both the nitration and the oxidation reactions of L-tyrosine effectively. On the other hand, 5-methoxytryptamine and lipoic acid inhibited only the nitration reaction of L-tyrosine, and instead increased the oxidation reaction. It was assumed that 5-methoxytryptamine and lipoic acid reacted only with the nitrating species of peroxynitrite. This is the first report of a selective inhibitor for the nitrating reaction of peroxynitrite.

Topics: 5-Methoxytryptamine; Antioxidants; Chromatography, High Pressure Liquid; Dopamine Agents; Levodopa; Nitrates; Oxidants; Oxidation-Reduction; Proline; Pyrrolidines; Thioctic Acid; Tyrosine

Primary cultures of rat embryonic motor neurons deprived of brain-derived neurotrophic factor (BDNF) induce neuronal nitric oxide synthase (NOS) within 18 hr. Subsequently, >60% of the neurons undergo apoptosis between 18 and 24 hr after plating. Nitro-L-arginine and nitro-L-arginine methyl ester (L-NAME) prevented motor neuron death induced by trophic factor deprivation. Exogenous generation of nitric oxide at concentrations lower than 100 nM overcame the protection by L-NAME. Manganese tetrakis (4-benzoyl acid) porphyrin, a cell-permeant superoxide scavenger, also prevented nitric oxide-dependent motor neuron death. Motor neurons cultured without trophic support rapidly became immunoreactive for nitrotyrosine when compared with motor neurons incubated with BDNF, L-NAME, or manganese TBAP. Our results suggest that peroxynitrite, a strong oxidant formed by the reaction of NO and superoxide, plays an important role in the induction of apoptosis in motor neurons deprived of trophic factors and that BDNF supports motor neuron survival in part by preventing neuronal NOS expression.

Topics: Animals; Apoptosis; Brain-Derived Neurotrophic Factor; Cells, Cultured; Enzyme Inhibitors; Fetus; Free Radical Scavengers; Motor Neurons; NG-Nitroarginine Methyl Ester; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Oxidants; Rats; Spinal Cord; Superoxides; Tyrosine

Peroxynitrite (ONOO-) exhibits potent neurotoxicity and plays an important role in neuronal death, but no evidence shows that it is formed in the brain during ischemia or subsequent reperfusion. To detect the formation of ONOO-, we used a hydrolysis/HPLC procedure to measure the formation of 3-nitro-L-tyrosine (NO2-Tyr), which is considered to reflect attack of ONOO- on L-tyrosine residues of cellular components in the brain. Focal ischemia was produced by occluding the right common carotid and right middle cerebral arteries for 2 hours, and the ischemic area was reperfused by reopening the middle cerebral artery. After 2 hours of ischemia, the values of the ratio of NO2-Tyr to L-tyrosine were 0% +/- 0%, 0.42% +/- 0.13% and 0.29% +/- 0.10% in the noninfarct, periinfarct, and core-of-infarct regions, respectively. After 3 hours of reperfusion following 2 hours of ischemia, the ratio in the periinfarct region reached 0.89 +/- 0.22%, which was significantly higher than that in the core-of-infarct region (0.35 +/- 0.09%). The NO2-Tyr was not detected in 50 mg/kg of N-monomethyl-L-arginine-treated or sham-operated rats. Regional CBF in the periinfarct region decreased to 30.8 +/- 15.9 mL/100 g/min during occlusion, but recovered more rapidly than did that in the core-of-infarct region.

Topics: Animals; Chromatography, High Pressure Liquid; Ischemic Attack, Transient; Male; Nitrates; Rats; Rats, Sprague-Dawley; Reperfusion; Tyrosine

Many lines of evidence implicate oxidative damage in aging. Possible pathways include reactions that modify aromatic amino acid residues on proteins. o-Tyrosine is a stable marker for oxidation of protein-bound phenylalanine by hydroxyl radical, whereas 3-nitrotyrosine is a marker for oxidation of protein-bound tyrosine by reactive nitrogen species. To test the hypothesis that proteins damaged by hydroxyl radical and reactive nitrogen accumulate with aging, we used isotope dilution gas chromatography-mass spectrometry to measure levels of o-tyrosine and 3-nitrotyrosine in heart, skeletal muscle, and liver from young adult (9 mo) and old (24 mo) female Long-Evans/Wistar hybrid rats. We also measured these markers in young adult and old rats that received antioxidant supplements (alpha-tocopherol, beta-carotene, butylated hydroxytoluene, and ascorbic acid) from the age of 5 mo. We found that aging did not significantly increase levels of protein-bound o-tyrosine or 3-nitrotyrosine in any of the tissues. Antioxidant supplementation had no effect on the levels of protein-bound o-tyrosine and 3-nitrotyrosine in either young or old animals. These observations indicate that the o-tyrosine and 3-nitrotyrosine do not increase significantly in heart, skeletal muscle, and liver in old rats, suggesting that proteins damaged by hydroxyl radical and reactive nitrogen species do not accumulate in these tissues with advancing age.

Topics: Aging; Animals; Antioxidants; Biomarkers; Copper; Female; Free Radicals; Gas Chromatography-Mass Spectrometry; Hydrogen Peroxide; Hydroxyl Radical; Nitrates; Nitrogen; Oxidation-Reduction; Oxidative Stress; Proteins; Rats; Serum Albumin, Bovine; Tyrosine

Increased production of reactive metabolites of oxygen and nitrogen has been implicated in chronic inflammation of the gut. The object of this study was to examine the magnitude and location of nitric oxide synthase (NOS) activity and peroxynitrite formation in the colonic mucosa of patients with ulcerative colitis in relation to the degree of inflammation.. Thirty three patients with active ulcerative colitis (17 with mild or moderate inflammation, 16 with severe inflammation).. Inducible NOS activity was determined in the colonic mucosa by measuring the conversion of L-arginine to citrulline in the absence of calcium. The localisation of NOS and nitrotyrosine immunoreactivity was assessed immunohistochemically using the labelled streptavidin biotin method.. Inducible NOS activity increased in parallel with the degree of inflammation of the mucosa. Expression of inducible NOS was found not only in the lamina propria, but also in the surface of the epithelium. Peroxynitrite formation as assessed by nitrotyrosine staining was frequently observed in the lamina propria of actively inflamed mucosa.. Nitric oxide and peroxynitrite formation may play an important role in causing irreversible cellular injury to the colonic mucosa in patients with active ulcerative colitis.

Topics: Acute Disease; Adolescent; Adult; Analysis of Variance; Antibodies, Monoclonal; Blotting, Western; Colitis, Ulcerative; Colon; Enzyme Induction; Female; Humans; Immunohistochemistry; Intestinal Mucosa; Isoenzymes; Male; Middle Aged; Nitrates; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Tyrosine

Peroxynitrite is a cytotoxic species generated by the reaction between superoxide and nitric oxide. In this study the ability of hydroxycinnamate antioxidants to decrease peroxynitrite-mediated nitration of tyrosine was investigated. The results obtained show that all compounds were able to inhibit nitration of tyrosine. The potency of inhibitory activity was in the order; caffeic acid > or = chlorogenic acid > or = ferulic acid > p-coumaric acid > ocoumaric acid > m-coumaric acid. Trolox, which was included in the study for comparative purposes, had an activity between that of ferulic acid and p-coumaric acid. The data obtained suggest that hydroxycinnamates can act by one of two possible mechanisms: preferential nitration for monophenolates and electron donation by catecholates.

Topics: Antioxidants; Caffeic Acids; Chlorogenic Acid; Chromans; Chromatography, High Pressure Liquid; Coumaric Acids; Free Radical Scavengers; Free Radicals; Nitrates; Spectrophotometry; Tyrosine

Premature infants are susceptible to bronchopulmonary dysplasia (BPD), a chronic lung disease of infancy that appears to be caused in part by oxidative stress from hyperoxia. To investigate the possible role of nitric oxide-derived oxidants such as peroxynitrite in the etiology of BPD, we measured levels of plasma 3-nitrotyrosine, which is produced by the reaction of peroxynitrite with proteins.. Ten premature infants who developed BPD, defined as requiring supplemental oxygen beyond 36 weeks' postmenstrual age, were identified retrospectively from a group of subjects enrolled in a clinical trial of antenatal therapy. Serial plasma samples had been collected on these infants during the first month of life as part of the trial. Sixteen comparison premature infants were identified from the same population: 5 had no lung disease, 6 had respiratory distress syndrome that resolved, and 5 had residual lung disease at 28 days of life that resolved by 36 weeks' postmenstrual age. Plasma 3-nitrotyrosine levels were measured using a solid phase immunoradiochemical method.. All 3-nitrotyrosine values in infants without BPD were <0.25 ng/mg protein, and levels did not change with postnatal age. Plasma 3-nitrotyrosine concentrations were significantly higher in infants with BPD, increasing approximately fourfold during the first month of life. For the 20 infants who had blood samples available at 28 days of life, plasma 3-nitrotyrosine levels correlated with the fraction of inspired oxygen that the infant was receiving (r = 0.7).. Plasma 3-nitrotyrosine content is increased during the first month of life in infants who develop BPD. This suggests that peroxynitrite-mediated oxidant stress may contribute to the development of this disease in premature infants and that 3-nitrotyrosine may be useful as an early plasma indicator of infants at risk for developing BPD.

Topics: Bronchopulmonary Dysplasia; Female; Humans; Infant, Newborn; Infant, Premature; Male; Nitrates; Oxidants; Oxidative Stress; Pilot Projects; Retrospective Studies; Tyrosine

Oxygen free radicals and nitric oxide (NO.) have been proposed to be involved in acute CNS injury produced by cerebral ischemia; however, controversy remains regarding how they cause injury. Because superoxide generation is triggered during reperfusion, the cytotoxic oxidant peroxynitrite could be formed, but it is not known if this occurs. Dot blot and immunohistochemistry studies were performed on the magnitude and time course of tyrosine nitration and inducible NO synthase (NOS2) in the postischemic rat pup brain. Neonatal ischemia was induced by permanent left middle cerebral artery occlusion in association with 1-h occlusion of the left common carotid artery in 7-day-old Wistar pups. Nitrotyrosine (NT) immunoreactivity was evident in the blood vessels close to the cortical infarct at 48-72 h of recovery, and T lymphocytes were involved with this production. NOS2 immunoreactivity was seen in neutrophils in the same vessels and in the parenchyma at 72 h of recirculation. Whereas NT staining decreased with time, NOS2-positive neutrophils could be still detected in arachnoid vessels at 14 days of recirculation. We conclude that perivascular reactions mediated by peroxynitrite are important in the cascade of events that lead to brain oxidative stress in neonatal ischemia. Moreover, NO-related species may serve as a signaling function instead of directly mediating toxicity.

Topics: Animals; Animals, Newborn; Brain; Cell Death; Female; Immunoenzyme Techniques; Ischemic Attack, Transient; Male; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Oxidants; Rats; Rats, Wistar; Reperfusion Injury; Tyrosine

Peroxynitrite has recently been implicated in the inactivation of many enzymes. However, little has been reported on the structural basis of the inactivation reaction. This study proposes that nitration of a specific tyrosine residue is responsible for inactivation of recombinant human mitochondrial manganese-superoxide dismutase (Mn-SOD) by peroxynitrite. Mass spectroscopic analysis of the peroxynitrite-inactivated Mn-SOD showed an increased molecular mass because of a single nitro group substituted onto a tyrosine residue. Single peptides that had different elution positions between samples from the native and peroxynitrite-inactivated Mn-SOD on reverse-phase high performance liquid chromatography were isolated after successive digestion of the samples by staphylococcal serine protease and lysylendopeptidase and subjected to amino acid sequence and molecular mass analyses. We found that tyrosine 34 of the enzyme was exclusively nitrated to 3-nitrotyrosine by peroxynitrite. This residue is located near manganese and in a substrate O-2 gateway in Mn-SOD.

Topics: Amino Acid Sequence; Enzyme Inhibitors; Humans; Mass Spectrometry; Mitochondria; Molecular Sequence Data; Nitrates; Peptide Fragments; Recombinant Proteins; Sequence Analysis; Serine Endopeptidases; Superoxide Dismutase; Tyrosine

Treatment of mice with a toxic dose of acetaminophen (300 mg/kg, ip) significantly increased hepatotoxicity at 4 h, as evidenced by histological necrosis in the centrilobular areas of the liver, and increased serum levels of alanine aminotransferase (ALT) (from 8 +/- 1 IU/L in saline-treated mice to 3226 +/- 892 IU/L in the acetaminophen-treated mice). Serum levels of nitrate plus nitrite (a marker of nitric oxide synthesis) were also increased from 62 +/- 8 microM in saline-treated mice to 110 +/- 14 microM in acetaminophen-treated mice (P < 0.05). Regression analysis of serum ALT levels to serum nitrate plus nitrite levels in individual mice revealed a positive, linear relationship between serum ALT levels and serum nitrate plus nitrite levels with a correlation coefficient of 0.9 (P < 0.05). The y intercept value (nitrate plus nitrite level) was 63 +/- 15 microM. Immunohistochemical analysis of liver sections from acetaminophen-intoxicated mice using an anti-3-nitrotyrosine antibody indicated tyrosine nitration in the proteins of the centrilobular cells. Tyrosine nitration has been shown to occur by peroxynitrite, a reactive intermediate formed by an extremely rapid reaction of nitric oxide and superoxide and a species which also has hydroxyl radical-like activity. Analysis of liver sections using an anti-acetaminophen antiserum indicated the centrilobular cells also contained acetaminophen-protein adducts, a reaction of the metabolite N-acetyl-p-benzoquinone imine with cysteine residues on proteins. These data are consistent with acetaminophen metabolic activation leading to increased synthesis of nitric oxide and superoxide and to peroxynitrite as an important intermediate in the toxicity.

Topics: Acetaminophen; Alanine Transaminase; Analgesics, Non-Narcotic; Animals; Biotransformation; Liver; Male; Mice; Nitrates; Nitric Oxide; Proteins; Tyrosine

During inflammation nitric oxide reacts at near diffusion limited rates with superoxide to form the strong oxidant peroxynitrite. Nitration on the ortho position is a major product of peroxynitrite attack on proteins. In the present study we investigated whether immunohistochemical detection of nitrotyrosine (footprint of peroxynitrite) can be associated with human hepatitis. Paraffin-embedded liver tissue biopsies from patients with chronic active hepatitis, chronic active hepatitis plus cirrhosis and chronic persistent hepatitis exhibit significant specific immunostaining with the antibody to nitrotyrosine. Positive staining was found in 57% and 72% of tissue specimens from patients with chronic hepatitis and cirrhosis, respectively. Immunohistochemical staining of nitrotyrosine residues was found in the hepatocytes and Kuppffer cells of the necrotic area. The presence of nitrotyrosine indicates that oxidants derived from nitric oxide such as peroxynitrite are generated in human hepatitis and may be involved in its pathogenesis.

Topics: Adult; Antibodies; Antibody Specificity; Female; Hepatitis B, Chronic; Hepatitis C, Chronic; Humans; Immunohistochemistry; Liver Cirrhosis; Male; Middle Aged; Nitrates; Tyrosine

To determine the extent to which enhanced nitration of the low molecular weight neurofilament subunit protein (NFL) is of pathogenic significance in sporadic ALS, we isolated the neurofilament (NF) from the cervical spinal cord of 15 cases of sporadic ALS and 11 age-matched control cases. Of the three NF subunits, only NFL demonstrated consistent nitrotyrosine immunoreactivity on immunoblots against mouse monoclonal anti-nitrotyrosine antibodies. Regardless of whether the NFL was isolated from the Triton X-100 soluble or insoluble cytoskeletal fractions, the extent of NFL nitration did not differ between ALS and control tissue. Similarly, no differences were observed on either two dimensional isoelectric focusing or NFL peptide maps. These findings suggest that NFL is particularly susceptible to peroxynitrite-mediated nitration in vivo, but reveal no significant qualitative or quantitative modifications in the nitration of NFL isolated from sporadic ALS cervical spinal cord tissue as compared to non-ALS controls.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Amyotrophic Lateral Sclerosis; Blotting, Western; Humans; Isoelectric Focusing; Middle Aged; Neurofilament Proteins; Nitrates; Precipitin Tests; Spinal Cord; Tyrosine

The impact of neopterin and 7,8-dihydroneopterin on peroxynitrite-induced nitration of l-tyrosine was studied. Neopterin derivatives and peroxynitrite are formed during immune response. Tyrosine nitration represents one major effect of nitric oxide-mediated cytotoxicity. Peroxynitrite formed in situ was co-incubated with tyrosine and neopterin or 7,8-dihydroneopterin or other pteridine derivatives, respectively. The nitration product, 3-nitro-l-tyrosine, was measured by HPLC via UV absorption at 360 nm. Neopterin (200 microM) increased the nitration rate between pH 4.0 and 5.5 up to +60%. 7,8-Dihydroneopterin inhibited tyrosine nitration over the whole pH range examined. In a series of various pteridine derivatives, neopterin and 7,8-dihydroneopterin achieved the strongest modulating effects on tyrosine nitration. Interactions of peroxynitrite with hydroxypropyl side chains of fully aromatic pterin derivatives may increase nitration, while partially hydrated pyrazino ring structures abate the reactivity of peroxynitrite. The results of this study suggest a potential impact of neopterin derivatives on peroxynitrite-mediated cytotoxicity.

Topics: Hydrogen-Ion Concentration; Neopterin; Nitrates; Nitro Compounds; Pteridines; Pterins; Spectrophotometry; Tyrosine

Peroxynitrite, generated by the reaction of nitric oxide (NO) with superoxide at sites of inflammation, is a strong oxidant capable of damaging tissues and cells. Detection of nitrotyrosine (NT) at inflammatory sites serves as a biochemical marker for peroxynitrite-mediated damage. In this study, NT was detected immunohistochemically within autopsied CNS tissues from six of nine multiple sclerosis (MS) patients, and in most of the MS sections displaying inflammation. Nitrite and nitrate, the stable oxidation products of NO and peroxynitrite, respectively, were measured in cerebrospinal fluid samples obtained from MS patients and controls. Levels of nitrate were elevated significantly during clinical relapses of MS. These data suggest that peroxynitrite formation is a major consequence of NO produced in MS-affected CNS and implicate a role for this powerful oxidant in the pathogenesis of MS.

Topics: Central Nervous System; Humans; Immunohistochemistry; Inflammation; Multiple Sclerosis; Nervous System Diseases; Nitrates; Nitrites; Recurrence; Tyrosine

Inhaled nitric oxide is a selective pulmonary vasodilator used for the treatment of pulmonary hypertension. The potential adverse effects of inhaled nitric oxide are unknown and represent the focus of the present studies. Whereas inhalation of nitric oxide (10 to 100 ppm, 5 h) by Balb/c mice had no effect on the number or type of cells recovered from the lung, a dose-related increase in bronchoalveolar lavage protein was observed, suggesting that nitric oxide induces alveolar epithelial injury. To determine if this was associated with altered alveolar macrophage activity, we quantified production of reactive oxygen and nitrogen intermediates by these cells. Interferon-gamma, alone or in combination with lipopolysaccharide (LPS), induced expression of inducible nitric oxide synthase (iNOS) protein and nitric oxide production by alveolar macrophages. Cells from mice exposed to 20 to 100 ppm nitric oxide produced significantly more nitric oxide and expressed greater quantities of iNOS than cells from control animals. Superoxide anion production and peroxynitrite generation by alveolar macrophages were also increased after exposure of mice to nitric oxide. This was correlated with increased antinitrotyrosine antibody binding to macrophages in histologic sections. Taken together, these data demonstrate that inhaled nitric oxide primes lung macrophages to release reactive oxygen and nitrogen intermediates. Increased production of these mediators by macrophages following inhalation of nitric oxide may contribute to tissue injury.

Topics: Administration, Inhalation; Animals; Antibodies; Bronchoalveolar Lavage Fluid; Dose-Response Relationship, Drug; Epithelial Cells; Interferon-gamma; Lipopolysaccharides; Lung; Macrophages, Alveolar; Male; Mice; Mice, Inbred BALB C; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitrogen; Oxidants; Proteins; Pulmonary Alveoli; Reactive Oxygen Species; Superoxides; Tyrosine; Vasodilator Agents

Peritoneal administration of zymosan in the rat induced a severe inflammatory process characterised by an increase in the plasma levels of nitrite and nitrate, stable metabolites of nitric oxide (NO) and in the levels of peroxynitrite, as measured by the oxidation of the fluorescent dye dihydrorhodamine 123, at 18 hours zymosan challenge. Immunohistochemical examination demonstrated a marked increase in the immunoreactivity to nitrotyrosine, a specific "footprint" of peroxynitrite, in the aorta of zymosan-shocked rats. In ex vivo experiments, thoracic aorta rings of zymosan-treated rats showed a reduced contraction to noradrenaline and reduced responsiveness to the relaxant effect to acetylcholine (vascular hyporeactivity and endothelial dysfunction, respectively). Treatment of zymosan-shocked rats with 3-aminobenzamide or Nicotinamide, inhibitors of poly ADP-ribosil synthetase (PARS) activity reduced the production of peroxynitrite and significantly prevented the cardiovascular dysfunction. Our data suggest that peroxynitrite and PARS activation play a role in the zymosan-induced cardiovascular derangements in the rat.

Topics: Animals; Aortic Diseases; Benzamides; Enzyme Activation; Enzyme Inhibitors; Immunohistochemistry; Injections, Intraperitoneal; Male; Niacinamide; Nitrates; Oxidants; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Rats; Rats, Sprague-Dawley; Shock, Cardiogenic; Tyrosine; Zymosan

The mechanisms by which immature thymocyte apoptosis is induced during negative selection are poorly defined. Reports demonstrated that cross-linking of T-cell receptor leads to stromal cell activation, expression of inducible nitric oxide synthase (iNOS) and, subsequently, to thymocyte apoptosis. Therefore we examined, whether NO directly or indirectly, through peroxynitrite formation, causes thymocyte apoptosis. Immuno-histochemical detection of nitrotyrosine revealed in vivo peroxynitrite formation in the thymi of naive mice. Nitrotyrosine, the footprint of peroxynitrite, was predominantly found in the corticomedullary junction and the medulla of naive mice. In the thymi of mice deficient in the inducible isoform of nitric oxide synthase, considerably less nitrotyrosine was found. Exposure of thymocytes in vitro to low concentrations (10 microM) of peroxynitrite led to apoptosis, whereas higher concentrations (50 microM) resulted in intense cell death with the characteristics of necrosis. We also investigated the effect of poly (ADP-ribose) synthetase (PARS) inhibition on thymocyte apoptosis. Using the PARS inhibitor 3-aminobenzamide (3-AB), or thymocytes from PARS-deficient animals, we established that PARS determines the fate of thymocyte death. Suppression of cellular ATP levels, and the cellular necrosis in response to peroxynitrite were prevented by PARS inhibition. Therefore, in the absence of PARS, cells are diverted towards the pathway of apoptotic cell death. Similar results were obtained with H2O2 treatment, while apoptosis induced by non-oxidative stimuli such as dexamethasone or anti-FAS antibody was unaffected by PARS inhibition. In conclusion, we propose that peroxynitrite-induced apoptosis may play a role in the process of thymocyte negative selection. Furthermore, we propose that the physiological role of PARS cleavage by apopain during apoptosis may serve as an energy-conserving step, enabling the cell to complete the process of apoptosis.

Topics: Animals; Apoptosis; Benzamides; Caspase 1; Caspase 3; Caspases; Cell Death; Cells, Cultured; Cysteine Endopeptidases; DNA Fragmentation; Enzyme Activation; Enzyme Inhibitors; Flow Cytometry; Hydrogen Peroxide; Immunohistochemistry; Mice; Nitrates; Nitric Oxide; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Thymus Gland; Tyrosine

Human polymorphonuclear leukocytes (PMN) produce nitric oxide (NO), superoxide (O2.-) and peroxynitrite (ONOO-) upon stimulation. We investigated the role of ONOO- in PMN-induced injury to cultured bovine aortic endothelial cells (BAEC).. BAEC were cocultured with phorbol 12-myristate 13-acetate (PMA)-activated human PMN (effector-to-target ratio, 10:1) and injury to BAEC was evaluated at intervals by 51Cr release assay. The levels of NO, O2.-, ONOO- and nitrotyrosine, a reaction product of ONOO-, were also measured, and the influence of NO synthase inhibitors, O2.- and hydroxyl radical scavengers and other effectors was examined.. In BAEC cocultured with PMA-activated PMN, 51Cr release was significantly increased [14.6 +/- 2.2% at 2 h (p < 0.05) and 42.6 +/- 2.7% at 4 h (p < 0.01); control (nonactivated PMN), < 4%]. Superoxide dismutase (100 U/ml) reduced 51Cr release to 4.6 +/- 2.2% at 2 h (p < 0.05). N-Iminoethyl-L-ornithine (L-NIO, 0.1 mM) potentiated 51Cr release (30.6 +/- 3.8% at 2 h, p < 0.01), and the potentiation was eliminated by anti-CD18 monoclonal antibody. The 51Cr release was completely prevented by dimethyl sulfoxide or by deferoxamine. Treatment of PMN with L-NIO inhibited NO generation and increased O2.- production. The nitrotyrosine level did not increase in BAEC cocultured with PMA-activated PMN.. NO-derived ONOO- is not a major cytotoxic mediator in BAEC injury by activated PMN. NO may have a cytoprotective effect by inhibiting PMN adherence to endothelial cells.

Topics: Animals; Antibodies, Monoclonal; Cattle; CD18 Antigens; Cells, Cultured; Coculture Techniques; Deferoxamine; Dimethyl Sulfoxide; Endothelium, Vascular; Humans; Neutrophil Activation; Neutrophils; Nitrates; Nitric Oxide Synthase; Nitrites; Ornithine; Penicillamine; Superoxide Dismutase; Tetradecanoylphorbol Acetate; Tyrosine

Sulphite is widely used as a preservative and antioxidant in foods, beverages, and pharmaceuticals. Endogenous sulphite is generated during the normal metabolism of sulphur-containing amino acids, and alterations in sulphur amino acid metabolism occur in some neurodegenerative diseases. In particular, sulphite oxidase deficiency produces severe mental retardation, seizures, spastic quadriparesis, dislocated lenses, and early death. Exposure of a neuronal cell line (rat mesencephalic cells) to high levels of sulphite induced a time-dependent decrease in viability. Peroxynitrite was also toxic to this cell line, and sulphite affected the toxicity of ONOO-. Sulphite concentrations of < or = 0.5 mM markedly potentiated cell damage induced by 200 microM ONOO-. We propose that sulphite can act as a neurotoxic agent, especially in combination with peroxynitrite. Sulphite radicals may be involved in the neurotoxic effect.

Topics: Animals; Cell Line; Cell Survival; DNA Fragmentation; Drug Combinations; Neurons; Nitrates; Oxidants; Rats; Sulfites; Tyrosine

Exhaled nitric oxide (NO) is increased in some inflammatory airway disorders but not in others such as cystic fibrosis and acute respiratory distress syndrome. NO can combine with superoxide (O-2) to form peroxynitrite, which can decompose into nitrate. Activated polymorphonuclear neutrophils (PMNs) releasing O-2 could account for a reduction in exhaled NO in disorders such as cystic fibrosis. To test this hypothesis in vitro, we stimulated confluent cultures of LA-4 cells, a murine lung epithelial cell line, to produce NO. Subsequently, human PMNs stimulated to produce O-2 were added to the LA-4 cells. A gradual increase in NO in the headspace above the cultures was observed and was markedly reduced by the addition of PMNs. An increase in nitrate in the culture supernatant fluids was measured, but no increase in nitrite was detected. Superoxide dismutase attenuated the PMN effect, and xanthine/xanthine oxidase reproduced the effect. No changes in epithelial cell inducible NO synthase protein or mRNA were observed. These data demonstrate that O-2 released from PMNs can decrease NO by conversion to nitrate and suggest a potential mechanism for modulation of NO levels in vivo.

Topics: Animals; Cell Line; Cell Survival; Epithelial Cells; Gases; Humans; Hydrogen-Ion Concentration; Lung; Mice; Neutrophils; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Oxidants; RNA, Messenger; Superoxides; Tyrosine

In a previous study, nitric oxide synthases (NOS) were found to be strongly expressed in the tubular epithelium of kidneys of a transgenic mouse model of sickle cell disease (alphaHbetaS[betaMDD]). Because NOS activity is often associated with peroxynitrite formation when superoxide radical (.O-2) is present in abundance, we examined the kidneys of sickle cell mice for nitrotyrosine, considered to be a footprint of ONOO-.. Western blot and immunohistochemistry for nitrotyrosine was carried out. Since peroxynitrite and other reactive oxygen radicals are capable of causing apoptosis, we also performed agarose gel electrophoresis of kidney DNA and TUNEL staining of nuclei, indicators of apoptosis.. Nitration of tyrosine residues of three proteins (kD 66, 57 and 22) was found on Western blot of kidney protein extracts of the sickle cell mice. The degree of tyrosine nitration of the 66 kD protein was not significantly different in the control versus transgenic mice, whereas tyrosine nitration of the 57 and 22 kD proteins was clearly increased in transgenic mice. Strong immunostaining for nitrotyrosine was seen in tubular epithelial cells of the sickle cell mice, in close proximity to positive immunostaining of iNOS. Neither iNOS nor nitrotyrosine was expressed in the control mice. DNA "laddering" was found localized to the same zones of the kidney as nitrotyrosine and iNOS immunostaining. TUNEL assay on mouse kidney tissue sections showed minimal tubular cell apoptosis in normal mouse with hypoxia, mild tubular cell apoptosis in sickle cell mouse in room air, and moderate tubular cell apoptosis in sickle cell mouse with hypoxia.. The observations suggest that ONOO- and perhaps other reactive oxygen species are being produced in the sickle cell kidney. The mechanism may be ischemia/reperfusion due to intermittent vascular occlusion by sickle cells. The resulting hypoxia could result in iNOS activation, superoxide radical and peroxynitrite formation. Two consequences of these reactions appear to be nitration of tyrosine residues of some renal proteins and enhanced apoptosis.

Topics: Anemia, Sickle Cell; Animals; Apoptosis; Blotting, Western; Immunohistochemistry; Kidney; Mice; Mice, Inbred C57BL; Mice, Transgenic; Nitrates; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Tyrosine

The effect of mercaptoethylguanidine (MEG), a selective inhibitor of the inducible nitric oxide synthase and peroxynitrite scavenger, was evaluated in a rat model of colonic injury. A single intracolonic administration of trinitrobenzene sulfonic acid (TNBS, 20 mg/kg) dissolved in ethanol induced a severe colitis in male rats. Rats experienced bloody diarrhea and a significant loss of body weight. At 4 days after TNBS administration, the colon damage was characterized by areas of mucosal necrosis. Activity of myeloperoxidase, a marker of neutrophil infiltration, and levels of the 6-keto-prostaglandin F1alpha, were also markedly increased, whereas colonic ATP levels were reduced into the damaged tissue. Immunohistochemistry for the inducible nitric oxide synthase and nitrotyrosine, an index of nitrosative stress, showed an intense staining in the inflamed colon. Treatment with MEG (10 mg/kg i.v. b. i.d.) significantly reduced the appearance of diarrhea and the loss of body weight. This was associated with a remarkable amelioration of the disruption of the colonic architecture and suppression of the energetic failure, as well as a significant reduction of colonic myeloperoxidase activity and 6-keto-prostaglandin F1alpha levels. MEG also reduced the appearance of iNOS and nitrotyrosine immunoreactivity in the colon. The results of this study suggested that administration of MEG may be beneficial for the treatment of inflammatory bowel diseases.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Body Weight; Colitis; Enzyme Inhibitors; Free Radical Scavengers; Guanidines; Immunohistochemistry; Male; Nitrates; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Rats; Rats, Wistar; Trinitrobenzenesulfonic Acid; Tyrosine

The aim of this study was to investigate how the enhanced nitric oxide (NO) production by intra-coronary infusion of L-arginine acts in myocardial stunning in dogs by focusing on the involvement of peroxynitrite, a reaction product of NO and superoxide anion.. Dogs were divided into six groups; a control non-treated group (CON, n = 9), and NG-nitro L-arginine methyl ester (L-NAME, n = 6), 1 mM L-arginine (L-ARG, n = 8), D-arginine (D-ARG, n = 6), L-arginine plus superoxide dismutase (L-ARG + SOD, n = 6), and SOD alone (SOD, n = 6) treated groups. L-NAME, or L- or D-arginine was continuously infused into the left anterior descending coronary artery (LAD) starting just prior to reperfusion, whereas SOD was intravenously injected before occlusion. During 120 min of reperfusion after 15 min occlusion of LAD, myocardial contractile function in the ischemic region gradually recovered and reached approximately 70% of the preischemic level in CON, D-ARG and SOD, but it remained dyskinetic (-46%) in L-ARG. On the other hand, it was improved in L-NAME (90%). Tissue malondialdehyde was elevated (p < 0.005) after reperfusion, and myocardial NO metabolites measured by an intratissue-microdialyzer increased (approximately 150%, p < 0.05) in the ischemic region during reperfusion in L-ARG but not in the CON, L-NAME, D-ARG or SOD groups. In the L-ARG + SOD group, L-arginine-induced contractile dysfunction and elevation of malondialdehyde were prevented, but the increase in NO metabolites remained. These results suggest that L-arginine aggravated myocardial stunning through oxidative stress and the cytotoxicity was caused by NO derivatives but not by NO itself. The formation of nitrotyrosine, a footprint of peroxynitrite, was immunohistochemically confirmed in the ischemic region of L-ARG.. Our results demonstrate for the first time in vivo that NO has a detrimental role in myocardial stunning through the production of peroxynitrite.

Topics: Analysis of Variance; Animals; Arginine; Chromatography, High Pressure Liquid; Dogs; Enzyme Inhibitors; Female; Immunohistochemistry; Isomerism; Lipid Peroxidation; Male; Malondialdehyde; Microdialysis; Myocardial Contraction; Myocardial Stunning; Myocardium; NG-Nitroarginine Methyl Ester; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Oxidative Stress; Random Allocation; Superoxide Dismutase; Tyrosine

The aim of the present study was to investigate the protective effect of the pineal hormone melatonin in a model of acute local inflammation (zymosan-activated plasma-induced paw oedema), in which oxyradicals, nitric oxide (NO) and peroxynitrite are known to play a crucial role in the inflammatory process. The intraplantar injection of zymosan-activated plasma elicited an inflammatory response that was characterized by a time-dependent increase in paw oedema, neutrophil infiltration and increased levels of nitrite/nitrate in the paw exudate. The maximal increase in paw volume was observed at 3 h after administration (maximal in paw volume: 1.34 +/- 0.09 ml). At this time point, myeloperoxidase activity and lipid peroxidation were markedly increased in the zymosan-activated plasma-treated paw (226 +/- 10.2 mU/100 mg wet tissue, 31 +/- 2.1 mM/mg wet tissue, respectively). However, zymosan-activated plasma-induced paw oedema was significantly reduced in a dose-dependent manner by treatment with melatonin (given at 62.5 and 125 microg/paw) at 1, 2, 3, 4 h after injection of zymosan-activated plasma. Melatonin treatment also caused a significant reduction of the myeloperoxidase activity and lipid peroxidation and inhibited nitrite/nitrate levels in the paw exudate. The paw tissues were also examined immunohistochemically for the presence of nitrotyrosine (a marker of peroxynitrite formation). At 3 h following injection of zymosan-activated plasma, staining for nitrotyrosine was also found to be localised in the inflamed paw tissue. Treatment with melatonin (125 microg/paw) reduced the appearance of nitrotyrosine in the tissues. Our findings support the view that melatonin exerts anti-inflammatory effects.

Topics: Animals; Anti-Inflammatory Agents; Biomarkers; Dose-Response Relationship, Drug; Edema; Foot; Free Radical Scavengers; Male; Melatonin; Nitrates; Oxidants; Plasma; Rats; Rats, Wistar; Tyrosine

Failure of GI tract mucosa to act as a barrier against bacterial translocation (BT) has been proposed as a potential source of sepsis and subsequent multiple organ failure post thermal injury. Nitric oxide (NO) is an inorganic radical produced by NO synthase (NOS) from L-arginine. Gut mucosal constitutive NOS (cNOS) provides protection for itself. In contrast to cNOS, inducible NOS (iNOS) releases far greater amounts of NO, promotes oxidative reactions and is responsible for tissue injury. Peroxynitrite formed by the rapid reaction between superoxide and NO, is a toxic substance that contributes to tissue injury in a number of biological systems. This study was designed to investigate the effect of iNOS specific inhibitor S-methylisothiourea (SMT) on the postburn intestinal mucosal barrier function and the possible mechanism of SMT's action. Female SPF Sprague Dawley rats underwent 35% total body surface area (TBSA) or sham burn. Either SMT or the same volume of saline was given (5 mg/kg, i.p. q 12 h) for 2 days to assess the effect of iNOS inhibition. On postburn day 2, the intestinal mucosal cNOS and iNOS activity were assayed by using Griess' reagent, the mesenteric lymph node (MLN), spleen and liver were collected and cultured for BT assay and the cellular localization of nitrotyrosine, a marker for peroxynitrite activity, was examined by immunostaining. After thermal injury in rats, administration of SMT for 2 days decreased the intestinal mucosal iNOS activity/ tNOS activity ratio and the BT incidence. Nitrotyrosine immunostaining of the intestinal mucosa showed a decrease in the SMT-treated group. These findings suggest that SMT, a specific inhibitor for iNOS improves the barrier function after burn by suppression of the intestinal mucosal iNOS activity. The decrease in NO production resulted in decreased formation of peroxynitrite and subsequently decreased damage of mucosal tissue.

Topics: Animals; Antioxidants; Bacterial Translocation; Body Surface Area; Burns; Coloring Agents; Enzyme Inhibitors; Female; Intestinal Mucosa; Intestine, Small; Isothiuronium; Liver; Lymph Nodes; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Oxidants; Rats; Rats, Sprague-Dawley; Spleen; Superoxides; Tyrosine

A new sensitive and specific assay was developed and applied for the quantitative determination of 3-nitrotyrosine in proteins of human platelets. 3-Nitrotyrosine was quantitatively converted into a new pentafluorobenzyl derivative in a single step and detected as an abundant carboxylate anion at m/z 595 using negative ion chemical ionization gas chromatography/mass spectrometry. The internal standard, [13C6]-3-nitrotyrosine, was prepared via a new and efficient method using nitronium borofluorate dissolved in hydrochloric acid. The assay showed excellent linearity and sensitivity. Intact human platelets contained 1.4+/-0.6 ng of 3-nitrotyrosine per milligram of protein. Peroxynitrite increased 3-nitrotyrosine levels 4- to 535-fold at the concentration range of 10 to 300 microM. Decomposed peroxynitrite was without the effect. Nitrogen dioxide (43 microM) was also a potent tyrosine nitrating molecule, increasing the levels of 3-nitrotyrosine 153-fold. HOCl (50 microM) in the presence of nitrite (50 microM) increased the 3-nitrotyrosine levels 3-fold. Exposure of platelets to nitric oxide, nitrite, thrombin, adenosine diphosphate, platelet activating factor, and arachidonic acid had no effect on platelet 3-nitrotyrosine levels.

Topics: Blood Platelets; Fluorobenzenes; Gas Chromatography-Mass Spectrometry; Humans; Nitrates; Nitrogen Dioxide; Sensitivity and Specificity; Tyrosine

Increasing evidence suggests that oxidative damage to proteins and other macromolecules is a salient feature of the pathology of Alzheimer's disease. Establishing the source of oxidants is key to understanding what role they play in the pathogenesis of Alzheimer's disease, and one way to examine this issue is to determine which oxidants are involved in damage. In this study, we examine whether peroxynitrite, a powerful oxidant produced from the reaction of superoxide with nitric oxide, is involved in Alzheimer's disease. Peroxynitrite is a source of hydroxyl radical-like reactivity, and it directly oxidizes proteins and other macromolecules with resultant carbonyl formation from side-chain and peptide-bond cleavage. Although carbonyl formation is a major oxidative modification induced by peroxynitrite, nitration of tyrosine residues is an indicator of peroxynitrite involvement. In brain tissue from cases of Alzheimer's disease, we found increased protein nitration in neurons, including but certainly not restricted to those containing neurofibrillary tangles (NFTs). Conversely, nitrotyrosine was undetectable in the cerebral cortex of age-matched control brains. This distribution is essentially identical to that of free carbonyls. These findings provide strong evidence that peroxynitrite is involved in oxidative damage of Alzheimer's disease. Moreover, the widespread occurrence of nitrotyrosine in neurons suggests that oxidative damage is not restricted to long-lived polymers such as NFTs, but instead reflects a generalized oxidative stress that is important in disease pathogenesis.

Topics: Aged; Aged, 80 and over; Alzheimer Disease; Cerebral Cortex; Entorhinal Cortex; Hippocampus; Humans; Immunohistochemistry; Middle Aged; Neurons; Nitrates; Oxidative Stress; Tyrosine

The identification of 15N-labeled 3-nitrotyrosine (NTyr) by gas chromatography/mass spectroscopy in protein hydrolyzates from activated RAW 264.7 macrophages incubated with 15N-L-arginine confirms that nitric oxide synthase (NOS) is involved in the nitration of protein-bound tyrosine (Tyr). An assay is presented for NTyr that employs HPLC with tandem electrochemical and UV detection. The assay involves enzymatic hydrolysis of protein, acetylation, solvent extraction, O-deacetylation, and dithionite reduction to produce an analyte containing N-acetyl-3-aminotyrosine, an electrochemically active derivative of NTyr. We estimate the level of protein-bound NTyr in normal rat plasma to be approximately 0-1 residues per 10(6) Tyr with a detection limit of 0.5 per 10(7) Tyr when > 100 nmol of Tyr is analyzed and when precautions are taken to limit nitration artifacts. Zymosan-treated RAW 264.7 cells were shown to have an approximately 6-fold higher level of protein-bound NTyr compared with control cells and cells treated with N(G)-monomethyl-L-arginine, an inhibitor of NOS. Intraperitoneal injection of F344 rats with zymosan led to a marked elevation in protein-bound NTyr to approximately 13 residues per 10(6) Tyr, an approximately 40-fold elevation compared with plasma protein of untreated rats; cotreatment with N(G)-monomethyl-L-arginine inhibited the formation of NTyr in plasma protein from blood and peritoneal exudate by 69% and 53%, respectively. This assay offers a highly sensitive and quantitative approach for investigating the role of reactive byproducts of nitric oxide in the many pathological conditions and disease states associated with NO(X) exposure such as inflammation and smoking.

Topics: Animals; Blood Proteins; Cell Line; Chromatography, High Pressure Liquid; Electrochemistry; Gas Chromatography-Mass Spectrometry; Inflammation; Macrophage Activation; Male; Nitrates; Nitric Oxide; Rats; Rats, Inbred F344; Tyrosine; Zymosan

Thiourea and, more recently, dimethylthiourea, have been used as hydroxyl radical (OH.) scavengers in experiments both in vitro and in vivo. We show that both compounds can inhibit nitration of the amino acid tyrosine on addition of peroxynitrite, and also the inactivation of alpha1-antiproteinase by peroxynitrite. Hence, protective effects of (dimethyl) thiourea could be due to inhibition of peroxynitrite-dependent damage as well as to OH. scavenging, and these compounds must not be regarded as specific OH. scavengers.

Topics: alpha 1-Antitrypsin; Enzyme Inhibitors; Free Radical Scavengers; Hydroxyl Radical; Nitrates; Thiourea; Tyrosine

The production of both nitric oxide (NO) and superoxide increases in septic shock. The cogeneration of these molecules is known to yield peroxynitrite, which preferentially nitrates tyrosine residues of protein and non-protein origins. We present evidence of peroxynitrite production in septic shock by measuring plasma nitrotyrosine. The nitrotyrosine was measured by an HPLC C-18 reverse-phase column and ultraviolet detector in chronic renal failure patients with or without septic shock, and in healthy volunteers. Plasma nitrite + nitrate (NOx) was also measured to evaluate NO production. Nitrotyrosine was selected as an index for production of peroxynitrite because the direct measurement of peroxynitrite in vivo is difficult. Patients with renal failure were selected in order to minimize nitrotyrosine excretion through the kidney. Plasma nitrotyrosine levels were not detectable in volunteers, 28.0 +/- 12.3 microM (1.6 +/- 1.1% of total tyrosine) in renal failure patients without septic shock, and 118.2 +/- 22.0 microM (5.5 +/- 1.2% of total tyrosine) in patients with septic shock. NOx levels were also higher in patients with septic shock than in patients without septic shock (173.9 +/- 104.7 vs. 75.6 +/- 19.1 microM). Although renal failure itself increases plasma concentrations of both molecules, the higher levels in patients with septic shock suggest that peroxynitrite is generated and the nitration of tyrosine residues is increased in this disease.

Topics: Adult; Aged; Aged, 80 and over; Case-Control Studies; Female; Free Radicals; Humans; Kidney Failure, Chronic; Male; Middle Aged; Nitrates; Nitric Oxide; Shock, Septic; Superoxides; Tyrosine

Carboxy-PTIO reacts rapidly with NO to yield NO2 and has been used as a scavenger to test the importance of nitric oxide (NO) in various physiological conditions. This study investigated the effects of carboxy-PTIO on several NO- and peroxynitrite-mediated reactions. The scavenger potently inhibited NO-induced accumulation of cGMP in endothelial cells but potentiated the effect of the putative peroxynitrite donor SIN-1, Carboxy-PTIO completely inhibited peroxynitrite-induced formation of 3-nitrotyrosine from free tyrosine (EC50 = 36 +/- 5 microM) as well as nitration of bovine serum albumin. Peroxynitrite-mediated nitrosation of GSH was stimulated by the drug with an EC50 of 0.12 +/- 0.03 mM, whereas S-nitrosation induced by the NO donor DEA/NO (0.1 mM) was inhibited by the scavenger with an IC50 of 0.11 +/- 0.03 mM. Oxidation of NO with carboxy-PTIO resulted in formation of nitrite without concomitant production of nitrate. Our results demonstrate that the effects of carboxy-PTIO are diverse and question its claimed specificity as NO scavenger.

Topics: Animals; Benzoates; Cattle; Cells, Cultured; Cyclic GMP; Endothelium, Vascular; Free Radical Scavengers; Imidazoles; Kinetics; Molsidomine; Nitrates; Nitric Oxide; Nitrites; Serum Albumin, Bovine; Swine; Tyrosine

Lipoprotein oxidation plays a key role in the initiation and progression of atherosclerosis. Peroxynitrite is a powerful oxidant and nitrating species formed by the reaction of nitric oxide with superoxide radical. Peroxynitrite can oxidize lipoproteins and generate nitrotyrosine either from free or protein-bound tyrosine. Nitrotyrosine has been used as a fingerprint for peroxynitrite reaction in vivo. In this study, the content of nitrotyrosine bound to beta-VLDL apoproteins was determined in New Zealand rabbits before and at 15, 30, 45 and 60 days of cholesterol feeding. A significant increase of nitrotyrosine bound to beta-VLDL apoproteins was observed in parallel with the hypercholesterolemia induced by 1% cholesterol enriched diet. These data indicate that apolipoprotein-bound nitrotyrosine may be used as a biomarker of peroxynitrite production during the development of atherosclerosis in this experimental model.

Topics: Animals; Arteriosclerosis; Biomarkers; Blood Proteins; Cholesterol; Hypercholesterolemia; Lipoproteins, VLDL; Male; Nitrates; Rabbits; Triglycerides; Tyrosine

Peroxynitrite (ONOO-) is a highly reactive oxidant species produced by the reaction of the free radicals superoxide (O(2).-) and nitric oxide (NO.). Here we report a marked increase in nitrotyrosine (NT), a marker of peroxynitrite, in islet cells from NOD mice developing spontaneous autoimmune diabetes. By using specific antibodies and immunohistochemical methods, we found that NT-positive cells were significantly more frequent in islets from acutely diabetic NOD mice (22 +/- 6%) than in islets from normoglycemic NOD mice (7 +/- 1%) and control BALB/c mice (2 +/- 1%). The NT+ cells in islets were identified to be macrophages and also beta-cells. Most of the beta-cells in islets from acutely diabetic NOD mice were NT+ (73 +/- 8%), whereas significantly fewer beta-cells were NT+ in islets from normoglycemic NOD mice (18 +/- 4%) and BALB/c mice (5 +/- 1%). Also, the percentage of beta-cells in islets from NOD mice (normoglycemic and diabetic) correlated inversely with the frequency of NT+ beta-cells. This study demonstrates for the first time that peroxynitrite, a reaction product of superoxide and nitric oxide, is formed in pancreatic islet beta-cells of NOD mice developing autoimmune diabetes. This suggests that both oxygen and nitrogen free radicals contribute to beta-cell destruction in IDDM via peroxynitrite formation in the islet beta-cells.

Topics: Animals; Cell Count; Diabetes Mellitus, Type 1; Female; Islets of Langerhans; Leukocytes; Macrophages; Mice; Mice, Inbred BALB C; Mice, Inbred NOD; Nitrates; Tyrosine

Idiopathic pulmonary fibrosis (IPF) is a disease of unknown etiology characterized by alveolar inflammation, progressive proliferation of septal cells, increased production of septal matrix, and loss of lung architecture. The process of cellular injury in lung fibrosis is thought to be mediated by oxygen radicals produced by infiltrating inflammatory cells. Peroxynitrite is a potent oxidant produced by the rapid reaction of nitric oxide (NO) and superoxide. We investigated the production of nitrotyrosine, a byproduct of protein nitration by peroxynitrite, and the expression of the enzymes responsible for generating NO, in lungs of patients with IPF and compared them with lungs of normal control subjects. We used immunohistochemistry, histochemistry, and in situ hybridization to study the production of nitrotyrosine and the expression of inducible (iNOS) and constitutive endothelial (eNOS) nitric oxide synthases in 48 lungs of patients with different stages of IPF and 21 normal lungs. In lungs of control subjects, there was little expression of iNOS and nitrotyrosine in the airway epithelium and alveolar macrophages, and abundant expression of eNOS in the airway epithelium and vascular endothelium. By contrast, in lungs of patients with IPF, strong expression of nitrotyrosine and NOS was seen in macrophages, neutrophils, and alveolar epithelium. A significant increase in the expression of these molecules was only seen in lungs of patients with the early to intermediate stage of the disease. The active stage of IPF is associated with increased inflammatory and alveolar expression of nitrotyrosine and NOS. Increased production of NO and peroxynitrite may be responsible for the oxidative damage seen in this disease.

Topics: Endothelium, Vascular; Epithelium; Female; Humans; Immunohistochemistry; In Situ Hybridization; Lung; Macrophages, Alveolar; Male; Middle Aged; Nitrates; Oxidants; Pulmonary Fibrosis; Tyrosine

Nitrotyrosine in human and animal tissues has been associated with pathologic conditions such as atherosclerosis, renal failure, and acute lung disease. In this study, free and protein-associated nitrotyrosine were determined in plasma and tissue samples using a dual-channel electrochemical detection method. Free nitrotyrosine was quantified in acetonitrile-extracted samples while protein-associated nitrotyrosine was determined in proteinase K-digested samples. In human plasma, total nitrotyrosine increased from 2.3 to 4.3 and 13.2 mumol/mol Tyr following addition of 0, 0.5, and 1 mM ONOO-. To determine if nitrotyrosine was produced during ex vivo hypothermic preservation, rat livers were stored in University of Wisconsin solution (UW) for 0, 6, or 8 h and reperfused for 3 h. Total nitro-tyrosine increased 359 and 908% after 6 and 8 h preservation compared to 0 h. To determine if nitrotyrosine was produced in vivo following hepatic ischemia, a rat preservation-transplantation model was utilized in which livers were flushed with cold UW (0-h group) or transplanted following 6 h hypothermic preservation in UW. Free nitrotyrosine increased from 15.7 +/- 0.3 in the 0-h group to 23.6 +/- 2.5 mumol/mol Tyr, 24 h posttransplant of 6-h preserved livers. Protein-associated nitrotyrosine increased from 9.5 +/- 1.1 in the 0-h group to 27.5 +/- 0.7 mumol/mol Tyr in the 6-h preservation-transplantation group. Protein-associated nitrotyrosine provides an integrative determination of nitration. Detection of free and protein-associated nitrotyrosine in biologic samples may allow insight into the role of .NO-derived oxidants in tissue injury associated with various pathologic conditions.

Topics: Animals; Chromatography, High Pressure Liquid; Electrochemistry; Free Radicals; Humans; Liver; Liver Transplantation; Nitrates; Organ Preservation; Protein Binding; Rats; Rats, Sprague-Dawley; Time Factors; Tyrosine

Production of nitric oxide via the cytokine-mediated activation of myocardial inducible nitric oxide synthase decreases myocardial contractility. Whether myocardial dysfunction is mediated directly by nitric oxide or indirectly through the formation of secondary reaction products, such as peroxynitrite, has not been established. Peroxynitrite, but not nitric oxide, reacts with the phenolic ring of tyrosine to form the stable product 3-nitro-L-tyrosine. Demonstration of tissue nitrotyrosine residues, therefore, infers the presence of peroxynitrite or related nitrogen-centered oxidants.. Retrospective analysis of human autopsy specimens.. University pathology and basic science laboratories.. Formalin-fixed, paraffin-embedded myocardial tissue samples were obtained from 11 patients with a diagnosis of sepsis, seven patients with a diagnosis of viral myocarditis, and five control patients without clinical or pathologic cardiac disease.. None.. Specific antibodies to nitrotyrosine were utilized to detect nitrotyrosine residues in human autopsy specimens. Cardiac tissue obtained from patients with myocarditis or sepsis demonstrated intense nitrotyrosine immunoreactivity in the endocardium, myocardium, and coronary vascular endothelium and smooth muscle. In contrast, connective tissue elements were without appreciable immunohistochemical staining. Nitrotyrosine antibody binding was blocked by coincubation with nitrotyrosine or nitrated bovine serum albumin, but not by aminotyrosine, phosphotyrosine, or bovine serum albumin. In situ reduction of tissue nitrotyrosine to aminotyrosine by sodium hydrosulfite also blocked antibody binding. Densitometric analysis of nitrotyrosine immunoreactivity demonstrated significantly higher values for specimens from myocarditis and sepsis patients when compared with control tissue specimens.. These results demonstrate the formation of peroxynitrite within the myocardium during inflammatory disease states, suggesting a role for peroxynitrite in inflammation-associated myocardial dysfunction.

Topics: Adolescent; Autopsy; Child; Child, Preschool; Densitometry; Female; Humans; Infant; Infant, Newborn; Male; Myocarditis; Nitrates; Nitrosation; Retrospective Studies; Sepsis; Tyrosine

Nitric oxide (NO) production has been associated with disease activity in multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). This free radical can be transformed by superoxide to peroxynitrite, an extremely toxic oxidant which causes lipid peroxidation. In addition, peroxynitrite nitrates tyrosine residues, resulting in nitrotyrosine, which can be identified immunohistochemically. The results of this study indicate that peroxynitrite is formed very early during EAE development, correlating with clinical disease activity. Nitrotyrosine-positive cells display a widespread distribution in brain and spinal cord during severe disease and are associated with both perivascular infiltrates and parenchymal sites. Double-staining procedures demonstrated that a subpopulation of CD11b-positive cells (macrophages/microglia) reacted with nitrotyrosine antibodies. Immunostaining for inducible NO synthase demonstrated a similar distribution as nitrotyrosine staining. These experiments indicate that peroxynitrite is formed during progressive stages of disease activity.

Topics: Animals; Antibody Specificity; Brain; Disease Progression; Encephalomyelitis, Autoimmune, Experimental; Free Radicals; Immunohistochemistry; Lipid Peroxidation; Mice; Mice, Inbred Strains; Neuritis, Autoimmune, Experimental; Neurons; Nitrates; Nitric Oxide; Spinal Cord; Staining and Labeling; Tyrosine

Peroxynitrite (ONOO-) can react with a wide range of biomolecules resulting in peroxidation, oxidation, and/or nitration and as a consequence cause their inactivation. In this study mass spectrometry (MS) combined with both liquid (LC) and gas chromatography (GC) has been employed to identify the products formed following ONOO- treatment of three peptides at physiological pH: leucine-enkephalin (YGGFL), V3 loop (GPGRAF), and LVV-hemorphin7 (LVVYPWTQRF). LC-MS analysis of leucine-enkephalin following ONOO treatment indicated the formation of products corresponding in mass to mono- and dinitrated forms of the starting material. LC-MS-MS and GC-MS analyses revealed no evidence for the formation of nitrophenylalanine; however, both 3-nitrotyrosine and 3,5-dinitrotyrosine were observed and characterized. GC-MS analysis of hydrolyzed peptides following ONOO- treatment confirmed the presence of nitrated and dinitrated tyrosine. However, when a 20-fold molar excess of ONOO- was reacted with leucine-enkephalin, only about half of the tyrosine originally present in the peptide could be accounted for in the acid hydrolysate. The main product was 3-nitrotyrosine which represented ca. 50% of the original tyrosine; traces of 3,5-dinitrotyrosine (ca. 3% of the original tyrosine) were also present.

Topics: Chromatography, Liquid; Enkephalin, Leucine; Gas Chromatography-Mass Spectrometry; Hemoglobins; HIV Envelope Protein gp120; Mass Spectrometry; Nitrates; Peptide Fragments; Trimethylsilyl Compounds; Tyrosine

Peroxynitrite (ONOO-) is a 'reactive nitrogen species' that can be formed (among other reactions) by combination of superoxide (O2.-) and nitric oxide (NO.) radicals. It is being increasingly proposed as a contributor to tissue injury in several human diseases. The evidence presented for peroxynitrite participation usually includes the demonstration of increased nitrotyrosine levels in the injured tissue. Indeed, this is often the only evidence presented: the assumption is that formation of nitrotyrosine is a biomarker specifically diagnostic of ONOO- production. The present article examines this assumption and concludes that nitrotyrosine is a biomarker for 'nitrating species' rather than being specific for ONOO-.

Topics: Biomarkers; Free Radicals; Humans; Molecular Structure; Nitrates; Nitric Oxide; Nitrogen Dioxide; Nitrous Acid; Peroxidase; Superoxides; Tyrosine

Vascular tone critically depends on the endothelial release of nitric oxide and prostacyclin. Superoxide anions counteract these relaxations by trapping nitric oxide under formation of peroxynitrite. As we have recently reported, peroxynitrite is able to inhibit prostacyclin formation in aortic microsomes (Zou et al., 1996). Here we show that peroxynitrite also blocks purified prostacyclin synthase with an IC50 value of about 50 nM and with a similar sensitivity also inhibits the enzyme activity in the EaHy 926 endothelial cell line. Thromboxane synthase, having the same heme-thiolate (P450) structure and a closely-related mechanism was unaffected by peroxynitrite. Anti-nitrotyrosine antibodies reacted positive by a Western blot after treatment of the purified enzyme with 1 microM peroxynitrite. Tetranitromethane also inhibited the enzyme activity which, like the inhibition by peroxynitrite, could be partially prevented in the presence of the substrate analog U46619. The simultaneous generation of superoxide and nitric oxide proved to be as efficient as a bolus of peroxynitrite which supports a possible inactivation of prostacyclin synthase under in vivo conditions. This substantiates an often suggested crucial role of superoxide in the pathophysiology of the cardiovascular system.

Topics: Animals; Cattle; Cell Line; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Endothelium, Vascular; Humans; Intramolecular Oxidoreductases; Nitrates; Nitric Oxide; Superoxides; Tyrosine

Carbon monoxide causes a perivascular oxidative injury in animals, and we tested the hypothesis that endothelial cells could be a source of the injurious oxidants. Studies were undertaken to assess whether exposure to carbon monoxide would cause cultured bovine pulmonary artery endothelial cells to liberate reactive species. Concentrations of carbon monoxide between 11 and 110 nM caused progressively higher concentrations of nitric oxide to be released by endothelial cells based on measurements of nitrite and nitrate. Intracellular production of peroxynitrite was indicated by elevated concentrations of nitrotyrosine, and extracellular liberation of peroxynitrite was indicated by oxidation of p-hydroxyphenylacetic acid and dihydrorhodamine-123. Carbon monoxide did not disturb mitochondrial function based on the rate of oxygen consumption, intracellular production of hydrogen peroxide, and the ability of cells to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Carbon monoxide also did not alter arginine transport by cells or nitric oxide synthase activity, but it was found to increase steady state levels of nitric oxide by competing for intracellular binding sites. Acute cytotoxicity from carbon monoxide, assessed as radioactive chromium leakage, was due to nitric oxide-derived oxidants. A delayed cell death, whose mechanism is not entirely clear, was also demonstrated by chromium leakage and uptake of vital stain. These findings offer a possible mechanism for adverse health effects caused by carbon monoxide at concentrations ranging from the relatively low levels in polluted environments to levels typically encountered with life-threatening poisoning. Carbon monoxide causes oxidative stress by a novel mechanism involving a competition for intracellular binding sites which increases steady state levels of nitric oxide and allows for generation of peroxynitrite by endothelium.

Topics: Animals; Arginine; Carbon Monoxide; Cattle; Cell Survival; Cells, Cultured; Chromium; Endothelium, Vascular; Nitrates; Nitric Oxide Synthase; Nitrites; Oxidants; Oxidation-Reduction; Oxygen Consumption; Sulfhydryl Compounds; Tetrazolium Salts; Thiazoles; Tyrosine

Topics: Humans; Lipoproteins, LDL; Nitrates; Oxidants; Serum Albumin; Tyrosine

In vitro studies have demonstrated that melatonin is a scavenger of oxyradicals and peroxynitrite and an inhibitor of nitric oxide (NO) production. In the present study, we evaluated the effect of melatonin treatment in two models of acute inflammation (carrageenan-induced paw edema and pleurisy), where oxyradicals, NO, and peroxynitrite play a crucial role in the inflammatory process. Our data show that melatonin (given at 62.5 and 125 microg/paw in the paw edema model or 25 and 50 mg/kg in the pleurisy model) inhibits the inflammatory response (paw swelling, pleural exudate formation, mononuclear cell infiltration, and histological injury) in dose-dependent manner in both models. Furthermore, our data suggest that melatonin exerts an inhibitory effect on the expression of the inducible isoform of NO synthase. Melatonin also prevented the formation of nitrotyrosine, an indicator of peroxynitrite, in both models of inflammation. Taken together, the present results demonstrate that melatonin exerts potent antiinflammatory effects. Part of these antiinflammatory effects may be related to an inhibition of the expression of the inducible NO synthase, while another part may be related to oxyradical and peroxynitrite scavenging.

Topics: Animals; Antioxidants; Carrageenan; Disease Models, Animal; Dose-Response Relationship, Drug; Edema; Free Radical Scavengers; Immunoenzyme Techniques; Inflammation; Male; Melatonin; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Peroxidase; Pleurisy; Rats; Rats, Sprague-Dawley; Tyrosine

Peroxynitrite, which is generated by the reaction of nitric oxide (NO) with superoxide, is a strong oxidant that can damage subcellular organelles, membranes and enzymes through its actions on proteins, lipids, and DNA, including the nitration of tyrosine residues of proteins. Detection of nitrotyrosine (NT) serves as a biochemical marker of peroxynitrite-induced damage. In the present studies, NT was detected by immunohistochemistry in CNS tissues from mice with acute experimental autoimmune encephalomyelitis (EAE). NT immunoreactivity was displayed by many mononuclear inflammatory cells, including CD4+ cells. It was also observed in astrocytes near EAE lesions. Immunostaining for the inducible isoform of NO synthase (iNOS) was also observed, particularly during acute EAE. These data strongly suggest that peroxynitrite formation is a major consequence of NO produced via iNOS, and implicate this powerful oxidant in the pathogenesis of EAE.

Topics: Acute Disease; Animals; Chronic Disease; Encephalomyelitis, Autoimmune, Experimental; Female; Immunohistochemistry; Mice; Mice, Inbred Strains; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Recurrence; Remission Induction; Serum Albumin, Bovine; Spinal Cord; Tyrosine

Since the production of peroxynitrite may contribute to the pathophysiology of endotoxemia or sepsis, the quantities of the produced peroxynitrite were evaluated in rats after lipopolysaccharide (LPS) treatment by measuring plasma nitrotyrosine concentrations with a new method. The intraperitoneal administration of LPS caused a persistent increase in plasma nitrotyrosine concentrations, which reached a maximum with 6-fold level of the base line (105 pmol ml-1) at 24 h and gradually declined to 3-fold level of the base line at 7 days. However, plasma concentrations of nitrite and nitrate peaked at 18 h, returning to base line within 48 h. The effect of LPS on the increase in plasma concentration of nitrotyrosine was dose-dependent and consistent with that of nitrite and nitrate concentrations. On the other hand, intravenous injection of nitrotyrosine revealed a rapid clearance with a plasma half-life of 1.67 h. These results indicate that the elevation of plasma nitrotyrosine concentrations may persist for more than a week after LPS treatment, and that the determination of plasma nitrotyrosine concentrations may be useful to detect the previous peroxynitrite-dependent oxidative damages.

Topics: 4-Chloro-7-nitrobenzofurazan; Animals; Lipopolysaccharides; Male; Nitrates; Nitrites; Rats; Rats, Wistar; Tyrosine

The SR Ca-ATPase in skeletal muscle SR vesicles isolated from young adult (5 months) and aged (28 months) rats was analyzed for nitrotyrosine. Only the SERCA2a isoform contained significant amounts with approximately one and four nitrotyrosine residues per young and old Ca-ATPase, respectively. The in vitro exposure of SR vesicles of young rats to peroxynitrite yielded selective nitration of the SERCA2a Ca-ATPase even in the presence of excess SERCA1a. No nitration was observed during the exposure of SR vesicles to nitric oxide in the presence of O2. These data suggest the vivo presence of peroxynitrite in skeletal muscle. The greater nitrotyrosine content of SERCA2a from aged tissue implies an age-associated increase in susceptibility to oxidation by this species.

Topics: Aging; Animals; Calcium; Calcium-Transporting ATPases; Male; Muscle, Skeletal; Nitrates; Rats; Rats, Inbred F344; Sarcoplasmic Reticulum; Tyrosine

Peroxynitrite ion (ONO2-) reacted rapidly with CO2 to form a short-lived intermediate provisionally identified as the ONO2CO2- adduct. This adduct was more reactive in tyrosine oxidation than ONO2- itself and produced 3-nitrotyrosine and 3,3'-dityrosine as the major oxidation products. With tyrosine in excess, the rate of 3-nitrotyrosine formation was independent of the tyrosine concentration and was determined by the rate of formation of the ONO2CO2- adduct. The overall yield of oxidation products was also independent of the concentration of tyrosine and medium acidity; approximately 19% of the added ONO2- was converted to products under all reaction conditions. However, the 3-nitrotyrosine/3,3'-dityrosine product ratio depended upon the pH, tyrosine concentration, and absolute reaction rate. These data are in quantitative agreement with a reaction mechanism in which the one-electron oxidation of tyrosine by ONO2CO2- generates tyrosyl and NO2 radicals as intermediary species, but are inconsistent with mechanisms that invoke direct electrophilic attack on the tyrosine aromatic ring by the adduct. Based upon its reactivity characteristics, ONO2CO2- has a lifetime shorter than 3 ms and a redox potential in excess of 1 V, and oxidizes tyrosine with a bimolecular rate constant greater than 2 x 10(5) M-1 s-1. In comparison, in CO2-free solutions, oxidation of tyrosine by peroxynitrite was much slower and gave significantly lower yields (approximately 8%) of the same products. When tyrosine was the limiting reactant, 3,5-dinitrotyrosine was found among the reaction products of the CO2-catalyzed reaction, but this compound was not detected in the uncatalyzed reaction.

Topics: Carbon Dioxide; Chromatography, High Pressure Liquid; Kinetics; Nitrates; Oxidation-Reduction; Time Factors; Tyrosine

To examine in vitro the ability of several drugs to protect against deleterious effects of peroxynitrite, a cytotoxic agent formed by reaction of nitric oxide with superoxide radical, that may be generated in the rheumatoid joint and could cause joint damage.. The ability of several drugs to protect against such possible toxic actions of peroxynitrite as inactivation of alpha 1-antiproteinase and nitration of tyrosine was evaluated.. Most non-steroidal anti-inflammatory drugs were moderately (indomethacin, diclofenac, naproxen, tolmetin) or only weakly (sulindac, ibuprofen, aurothioglucose, flurbiprofen, sulphasalazine, salicylate, penicillamine disulphide) effective in preventing tyrosine nitration and alpha 1-antiproteinase inactivation by peroxynitrite, but 5-aminosalicylate and penicillamine were much more effective, as was the antibiotic tetracycline (but not ampicillin). Phenylbutazone and flufenamic acid protected effectively against tyrosine nitration, but could not be tested in the alpha 1-antiproteinase system. The analgesic paracetamol was highly protective in both assay systems.. Many drugs used in the treatment of rheumatoid arthritis are unlikely to act by scavenging peroxynitrite. The feasibility of peroxynitrite scavenging as a mechanism of penicillamine, 5-aminosalicylate, and paracetamol action in vivo is discussed.

Topics: Acetaminophen; Aminosalicylic Acids; Anti-Inflammatory Agents, Non-Steroidal; Antirheumatic Agents; Chromatography, High Pressure Liquid; Enzyme Inhibitors; Flufenamic Acid; Free Radical Scavengers; Humans; Mesalamine; Nitrates; Pancreatic Elastase; Penicillamine; Phenylbutazone; Tetracycline; Tyrosine

The interaction of nitric oxide and superoxide produces peroxynitrite anion, a strong, long-lived oxidant with pronounced deleterious effects that may cause vascular damage. The formation and action of peroxynitrite can be detected by immunohistochemical localization of nitrotyrosine residues. We compared the presence and localization of nitrotyrosine and of the endothelial isoform of nitric oxide synthase in placental villous tissue from normotensive pregnancies (n = 5) with pregnancies complicated by preeclampsia (n = 5), intrauterine growth restriction (n = 5), and preeclampsia plus intrauterine growth restriction (n = 4), conditions characterized by increases in fetoplacental vascular resistance, fetal platelet consumption, and fetal morbidity and mortality. In all tissues, absent or faint nitrotyrosine immunostaining but prominent nitric oxide synthase immunostaining were found in syncytiotrophoblast. In tissues from normotensive pregnancies, faint nitrotyrosine immunostaining was found in vascular endothelium, and nitric oxide synthase was present in stem villous endothelium but not in the terminal villous capillary endothelium. In contrast, in preeclampsia and/or intrauterine growth restriction, moderate to intense nitrotyrosine immunostaining was seen in villous vascular endothelium, and immunostaining was also seen in surrounding vascular smooth muscle and villous stroma. The intensity of nitrotyrosine immunostaining in preeclampsia (with or without intrauterine growth restriction) was significantly greater than that of controls. Intense nitric oxide synthase staining was seen in endothelium of stem villous vessels and the small muscular arteries of the terminal villous region in these tissues and may be an adaptive response to the increased resistance. The presence of nitrotyrosine residues, particularly in the endothelium, may indicate the formation and action of peroxynitrite, resulting in vascular damage that contributes to the increased placental vascular resistance.

Topics: Adult; Drug Residues; Female; Fetal Growth Retardation; Humans; Immunohistochemistry; Nitrates; Placenta; Pre-Eclampsia; Pregnancy; Tyrosine

Following CNS trauma or ischemia, peroxynitrite may be a toxic intermediate which forms in vivo when nitric oxide condenses with superoxide. Alone, peroxynitrite appears to directly react with aromatic and sulfhydryl nucleophiles. However, at physiological pH, peroxynitrite is protonated and, in that form, will rapidly (within seconds) decompose to species with hydroxyl radical and nitrogen dioxide characteristics. These reactive species are shown to initiate lipid peroxidation, hydroxylate aromatic residues, and nitrate aromatic residues. This reactivity may contribute to differential toxicity in vivo and in vitro. Tirilazad mesylate (TZ) is a lipid-soluble antioxidant shown to inhibit iron-dependent lipid peroxidation. It is an effective therapy in a variety of CNS injury and ischemia models and is currently undergoing human clinical evaluation in stroke, head injury, and spinal injury. This study was designed to investigate the cytoprotective properties of TZ in a cerebellar granule cell model of peroxynitrite toxicity. Cytoprotective efficacy of TZ was based on viability measurements, blockade of lipid hydroperoxide generation, and blockade of nitrotyrosine formation. Cell viability was determined by [3H]-aminoisobutyric acid (3H-AIB) uptake, and lipid hydroperoxide and nitrotyrosine content were determined by HPLC assays. Tirilazad mesylate was found to have similar cytoprotective effects (approximately 50% protection at 100 microM) when applied before or after exposure of cells to peroxynitrite. In contrast, post-treatment with superoxide dismutase (50 units/ml) or allopurinol (100 microM) failed to produce any cytoprotection. Furthermore, we discovered that TZ inhibited the peroxynitrite-induced increase of phosphatidylethanolamine hydroperoxide (PEOOH), but did not affect the peroxynitrite-induced formation of nitrotyrosine formation. This suggests that the ability of TZ to afford cytoprotection in this peroxynitrite toxicity model is due to the inhibition of membrane-localized lipid peroxidation, and not to the inhibition of nitration of tyrosine residues.

Topics: Aminoisobutyric Acids; Animals; Biomarkers; Cells, Cultured; Cerebellum; Emulsions; Free Radical Scavengers; Lipid Peroxides; Nitrates; Pregnatrienes; Rats; Rats, Sprague-Dawley; Tyrosine

Reaction with peroxynitrite at pH 7.4 and 37 degrees C was found to increase the 8-oxodeoxyguanosine levels in calf thymus DNA 35- 38-fold. This oxidation of deoxyguanosine, as well as the peroxynitrite-mediated nitration of tyrosine to 3-nitrotyrosine, was significantly inhibited by ascorbic acid, glutathione and (-)-epigallocatechin gallate, a polyphenolic antioxidant present in tea. For 50% inhibition of the oxidation of deoxyguanosine to 8-oxodeoxyguanosine, 1.1, 7.6 or 0.25 mM ascorbate, glutathione or (-)-epigallocatechin gallate, respectively, was required. For 50% inhibition of tyrosine nitration, the respective concentrations were 1.4, 4.6 or 0.11 mM. Thus, (-)-epigallocatechin gallate is a significantly better inhibitor of both reactions than either ascorbate or glutathione. Reaction of (-)-epigallocatechin gallate with peroxynitrite alone resulted in the formation of a number of products. Ultraviolet spectra of two of these suggest that the tea polyphenol and/or its oxidation products are nitrated by peroxynitrite.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Antioxidants; Catechin; Cattle; Deoxyguanosine; DNA; Free Radical Scavengers; Nitrates; Oxidation-Reduction; Tea; Tyrosine

Inflammatory processes in chronic rejection remain a serious clinical problem in organ transplantation. Activated cellular infiltrate produces high levels of both superoxide and nitric oxide. These reactive oxygen species interact to form peroxynitrite, a potent oxidant that can modify proteins to form 3-nitrotyrosine. We identified enhanced immunostaining for nitrotyrosine localized to tubular epithelium of chronically rejected human renal allografts. Western blot analysis of rejected tissue demonstrated that tyrosine nitration was restricted to a few specific polypeptides. Immunoprecipitation and amino acid sequencing techniques identified manganese superoxide dismutase, the major antioxidant enzyme in mitochondria, as one of the targets of tyrosine nitration. Total manganese superoxide dismutase protein was increased in rejected kidney, particularly in the tubular epithelium; however, enzymatic activity was significantly decreased. Exposure of recombinant human manganese superoxide dismutase to peroxynitrite resulted in a dose-dependent (IC50 = 10 microM) decrease in enzymatic activity and concomitant increase in tyrosine nitration. Collectively, these observations suggest a role for peroxynitrite during development and progression of chronic rejection in human renal allografts. In addition, inactivation of manganese superoxide dismutase by peroxynitrite may represent a general mechanism that progressively increases the production of peroxynitrite, leading to irreversible oxidative injury to mitochondria.

Topics: Amino Acid Sequence; Analysis of Variance; Blotting, Western; Graft Rejection; Humans; Immunohistochemistry; Kidney; Kidney Transplantation; Models, Biological; Nitrates; Peptide Fragments; Superoxide Dismutase; Transplantation, Homologous; Tyrosine

Peroxynitrite, which attenuates catecholamine-mediated hemodynamic responses in vivo, nitrates free tyrosine residues to form the specific product, 3-nitro-L-tyrosine. The chemical structure of 3-nitro-L-tyrosine is similar to that of the endogenous catecholamines. Therefore, 3-nitro-L-tyrosine may interfere with catecholamine hemodynamic function in vivo. The hemodynamic responses produced by norepinephrine (1-4 micrograms/kg, i.v., n = 6), epinephrine (0.5-4 micrograms/kg, i.v., n = 7), phenylephrine (1-8 micrograms/kg, i.v., n = 5), and isoproterenol (100-400 ng/kg, i.v., n = 5) were attenuated, while the hemodynamic responses produced by arginine vasopressin (50-250 ng/kg; i.v., n = 5) were unaffected following the administration of 3-nitro-L-tyrosine (2.5 mumol/kg, i.v.) in pentobarbital-anesthetized rats. These results demonstrate substantial and selective attenuation of the hemodynamic effects produced by alpha- and beta-adrenoceptor agonists, raising the possibility that 3-nitro-L-tyrosine may play a role in the hemodynamic dysfunction associated with inflammatory conditions in which the formation of peroxynitrite is favored.

Topics: Adrenergic Agonists; Animals; Arginine Vasopressin; Hemodynamics; Male; Nitrates; Rats; Rats, Sprague-Dawley; Tyrosine

Peroxynitrite, formed by reaction of superoxide and nitric oxide, appears to be an important tissue-damaging species generated at sites of inflammation. In this paper, we compare the abilities of several biological antioxidants to protect against peroxynitrite-dependent inactivation of alpha 1-antiproteinase, and to inhibit tyrosine nitration upon addition of peroxynitrite. GSH and ascorbate protected efficiently in both systems. Uric acid inhibited tyrosine nitration but not alpha 1-antiproteinase inactivation. The possibility that ascorbic acid is an important scavenger of reactive nitrogen species in vivo is discussed.

Topics: alpha 1-Antitrypsin; Antioxidants; Arthritis, Rheumatoid; Ascorbic Acid; Humans; Lipid Peroxidation; Nitrates; Oxidation-Reduction; Oxidative Stress; Pancreatic Elastase; Synovial Fluid; Tyrosine

Peroxynitrite is a potent oxidant formed endogenously by the near diffusion-limited reaction of nitric oxide with superoxide anion. Peroxynitrite specifically adds a nitro group to the ortho position of the phenolic ring of free and protein-associated tyrosines to form the stable product 3-nitro-L-tyrosine. Systemic administration of 3-nitro-L-tyrosine markedly inhibits the subsequent hemodynamic responses to alpha 1- and beta-adrenoceptor agonists in anesthetized rats. Angiotensin II is an important modulator of vascular tone. The vasoconstrictor effects of this hormone are known to involve the release of catecholamines from sympathetic tissues. In the present study, we examined whether 3-nitro-L-tyrosine (2.5 mumol/kg i.v.) would attenuate the hemodynamic responses produced by angiotensin II (0.1-1.0 microgram/kg i.v.). Angiotensin II produced increases in mean arterial pressure, and renal and mesenteric vascular resistances, but no changes in hindquarter vascular resistance. The pressor and renal and mesenteric vasoconstrictor responses produced by angiotensin II were significantly attenuated 30-60 min following the administration of 3-nitro-L-tyrosine. Further attenuation of these responses was evident 120-180 min following the administration of 3-nitro-L-tyrosine. The alpha 1-adrenoceptor antagonist prazosin also diminished the pressor and renal and mesenteric vasoconstrictor responses produced by angiotensin II. These results demonstrate that 3-nitro-L-tyrosine inhibits the hemodynamic responses to angiotensin II, possibly through the inhibition of alpha 1-adrenoceptor-mediated events. The effect of 3-nitro-L-tyrosine on the hemodynamic action of angiotensin II raises the possibility that 3-nitro-L-tyrosine may be involved in the pathogenesis of the hemodynamic disturbances associated with inflammatory conditions, such as atherosclerosis, ischemia-reperfusion, and sepsis, where formation of peroxynitrite is favored.

Topics: Anesthesia; Angiotensin II; Animals; Hemodynamics; Male; Nitrates; Pentobarbital; Rats; Rats, Sprague-Dawley; Tyrosine; Vasoconstrictor Agents

Peroxynitrite, the potent oxidant formed by the fast reaction between nitric oxide and superoxide anion, has been suggested to be the reactive intermediate responsible for some of the pathologies associated with an over-production of nitric oxide. In this report, we demonstrate that both nitric oxide and peroxynitrite are formed during infection of the susceptible mouse strain, BALB/c, with Leishmania amazonensis. Nitric oxide was detected as the nitrosyl hemoglobin complex by EPR analysis of blood drawn from mice at 35, 64 and 148 days of infection. The levels of nitrosyl hemoglobin complex increased with disease evolution, which in the murine model used is characterized by skin lesions, ulceration and visceralization of the parasites. Peroxynitrite formation was inferred from immunoreaction of homogenates obtained from footpad lesions in the late stages of the infection with anti-nitrotyrosine antibody; homogenates from parasites drawn from the lesions were also immunoreactive, although to a lesser extent. Analysis of protein homogenates by gel electrophoresis and western blots suggests that peroxynitrite may degrade proteins in vivo, in addition to nitrating them. The results demonstrate that peroxynitrite is formed during murine leishmaniasis and may play a role in the aggravation of the disease.

Topics: Animals; Disease Models, Animal; Female; Hemoglobins; Leishmania mexicana; Leishmaniasis, Cutaneous; Mice; Mice, Inbred BALB C; Nitrates; Nitric Oxide; Photobiology; Tyrosine

Lung ischemia-reperfusion represents a potentially important mechanism for diverse forms of tissue injury associated with decreased pulmonary flow. Previous studies demonstrated oxidative injury in ischemic-reperfused lungs. The present study was designed to evaluate the contribution of nitric oxide and peroxynitrite in tissue injury. The levels of the stable decomposition products of nitric oxide and peroxynitrite, nitrite plus nitrate, were twofold greater than control during reperfusion after 60 min of ischemia. Inhibition of nitric oxide synthesis by endotracheal insufflation of 5 mM NG-nitro-L-arginine methyl ester, 30 min before the induction of ischemia, decreased the production of lung thiobarbituric acid reactive substances (TBARS) by 67% (P < 0.05, n = 5), TBARS released into the lung perfusate by 55% (P < 0.05, n = 5), lung-conjugated dienes by 61% (P < 0.05, n = 5), and dinitrophenylhydrazine-reactive protein carbonyl levels by 86% (P < 0.05, n = 5). Amino acid analysis of tissue homogenates from lungs exposed to 60 min of ischemia and 60 min of reperfusion revealed a 1.8-fold (P < 0.05, n = 5) increase in nitrotyrosine concentration compared with 2 h continuously perfused lungs. Inhibition of nitric oxide synthesis abolished the increase in nitrotyrosine levels. Furthermore, lungs exposed to 60 min of reperfusion after 60 min of ischemia showed specific binding of an anti-nitrotyrosine antibody. In reperfused tissues, antibody binding was observed throughout the lung. The binding was blocked with excess of nitrotyrosine, and minimal binding was observed in nonperfused blood-free control lungs.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Arginine; Immunohistochemistry; Ischemia; Lung; Male; NG-Nitroarginine Methyl Ester; Nitrates; Nitric Oxide; Nitrites; Perfusion; Pressure; Pulmonary Circulation; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Staining and Labeling; Tyrosine

Experimental evidence has implicated oxidative stress in the development of Parkinson's disease, amyotrophic lateral sclerosis, and other degenerative neuronal disorders. Recently, peroxynitrite, which is formed by the nearly diffusion-limited reaction of nitric oxide with superoxide, has been suggested to be a mediator of oxidant-induced cellular injury. The potential role of peroxynitrite in the pathology associated with Parkinson's disease was evaluated by examining its effect on DOPA synthesis in PC12 pheochromocytoma cells. Peroxynitrite was generated from the compound 3-morpholinosydnonimine (SIN-1), which releases superoxide and nitric oxide simultaneously. Exposure of PC12 cells to peroxynitrite for 60 min greatly diminished their ability to synthesize DOPA without apparent cell death. The inhibition was due neither to the formation of free nitrotyrosine nor the oxidation of DOPA by peroxynitrite. The inhibition in DOPA synthesis by SIN-1 was abolished when superoxide was scavenged by the addition of superoxide dismutase. These data indicated that neither nitric oxide nor hydrogen peroxide generated by the dismutation of superoxide is responsible for the SIN-1-mediated inhibition of DOPA production. The inhibition of DOPA synthesis at high concentration of SIN-1 persisted even after removal of SIN-1. The inactivation of the tyrosine hydroxylase may be responsible for the significant decline in DOPA formation by peroxynitrite. Inactivation of tyrosine hydroxylase may be part of the initial insult in oxidative damage that eventually leads to cell death.

Topics: Animals; Dihydroxyphenylalanine; Molsidomine; Nitrates; Oxidation-Reduction; PC12 Cells; Rats; Tyrosine

Proteins are targets of reactive species and detection of oxidatively modified proteins is often used as an index of oxidative stress. Peroxynitrite is a strong oxidant formed by reaction of nitric oxide with superoxide. Using fatty acid-free bovine serum albumin as a model we examined peroxynitrite-mediated protein modifications. The reaction of protein with peroxynitrite resulted in the oxidation of tryptophan and cysteine, in the nitration of tyrosine, in the formation of dityrosine, in the production of 2,4 dinitrophenylhydrazine-reactive carbonyls and in protein fragmentation. The formation of 3-nitrotyrosine represents a specific peroxynitrite-mediated protein modification that is different from modifications mediated by reactive oxygen species.

Topics: Animals; Cysteine; Electrophoresis, Polyacrylamide Gel; Fluorescence; Hydrogen-Ion Concentration; Male; Nitrates; Oxidation-Reduction; Oxidative Stress; Phenylhydrazines; Rats; Rats, Sprague-Dawley; Serum Albumin, Bovine; Tryptophan; Tyrosine

We measured the amounts of tyrosine and 3-nitrotyrosine (NO2-tyrosine) in proteins of plasma and polymorphonuclear leukocytes (PMN) from human whole blood before and after activation with phorbol ester (PMA) or calcium ionophore (A 23187). In unstimulated blood, no significant nitration of tyrosine was detected into PMN proteins, but a NO2-tyrosine/tyrosine ratio of 0.7% was detected in plasma proteins. When blood was activated with PMA, the NO2-tyrosine/tyrosine ratio stayed at 0.7% in plasma proteins, but it increased to 1.4% in PMN proteins, indicating a peroxynitrite production within the cells. In blood activated with calcium ionophore, the NO2-tyrosine/tyrosine ratio was 1.2% in plasma proteins and 2.1% in PMN proteins. Incubation of blood with a NO-synthase inhibitor before stimulation inhibited such a protein tyrosine nitration. To ensure that NO2-tyrosine detected in intracellular proteins did not result from the enzymatic posttranslational tyrosylation of PMN proteins, the incorporation of 14C labeled tyrosine into PMN proteins after activation with PMA or A23187 was studied. The addition of a 10 fold excess of NO2-tyrosine did not modify the course of protein tyrosylation. Because tyrosine nitration is an irreversible reaction, NO2-tyrosine could be accumulated into proteins and could act as a cumulative index of peroxynitrite production.

Topics: Blood Proteins; Calcimycin; Humans; Ionophores; Kinetics; Neutrophils; Nitrates; Tetradecanoylphorbol Acetate; Time Factors; Tyrosine

Peroxynitrite anion is a powerful oxidant which can initiate nitration and hydroxylation of aromatic rings. Peroxynitrite can be formed in several ways, e.g. from the reaction of nitric oxide with superoxide or from hydrogen peroxide and nitrite at acidic pH. We investigated pH dependent nitration and hydroxylation resulting from the reaction of hydrogen peroxide and nitrite to determine if this reaction proceeds at pH values which are known to occur in vivo. Nitration and hydroxylation products of tyrosine and salicyclic acid were separated with an HPLC column and measured using ultraviolet and electrochemical detectors. These studies revealed that this reaction favored hydroxylation between pH 2 and pH 4, while nitration was predominant between pH 5 and pH 6. Peroxynitrite is presumed to be an intermediate in this reaction as the hydroxylation and nitration profiles of authentic peroxynitrite showed similar pH dependence. These findings indicate that hydrogen peroxide and nitrite interact at hydrogen ion concentrations present under some physiologic conditions. This interaction can initiate nitration and hydroxylation of aromatic molecules such as tyrosine residues and may thereby contribute to the biochemical and toxic effects of the molecules.

Topics: Hydrogen Peroxide; Hydrogen-Ion Concentration; Hydroxylation; Inflammation; Iron; Nitrates; Oxidative Stress; Salicylates; Salicylic Acid; Sodium Nitrite; Tyrosine

Activated alveolar macrophages and epithelial type II cells release both nitric oxide and superoxide which react at near diffusion-limited rate (6.7 x 10(9) M-1s-1) to form peroxynitrite, a potent oxidant capable of damaging the alveolar epithelium and pulmonary surfactant. Peroxynitrite, but not nitric oxide or superoxide, readily nitrates phenolic rings including tyrosine. We quantified the presence of nitrotyrosine in the lungs of patients with the adult respiratory distress syndrome (ARDS) and in the lungs of rats exposed to hyperoxia (100% O2 for 60 h) using quantitative immunofluorescence. Fresh frozen or paraffin-embedded lung sections were incubated with a polyclonal antibody to nitrotyrosine, followed by goat anti-rabbit IgG coupled to rhodamine. Sections from patients with ARDS (n = 5), or from rats exposed to hyperoxia (n = 4), exhibited a twofold increase of specific binding over controls. This binding was blocked by the addition of an excess amount of nitrotyrosine and was absent when the nitrotyrosine antibody was replaced with nonimmune IgG. In additional experiments we demonstrated nitrotyrosine formation in rat lung sections incubated in vitro with peroxynitrite, but not nitric oxide or reactive oxygen species. These data suggest that toxic levels of peroxynitrite may be formed in the lungs of patients with acute lung injury.

Topics: Adolescent; Animals; Child; Child, Preschool; Female; Fluorescent Antibody Technique; Humans; Infant; Infant, Newborn; Lung; Male; Microtomy; Nitrates; Rats; Rats, Sprague-Dawley; Respiratory Distress Syndrome, Newborn; Tyrosine

Peroxynitrite (ONOO-), the reaction product of superoxide (O2-) and nitric oxide (NO), may be a major cytotoxic agent produced during inflammation, sepsis, and ischemia/reperfusion. Bovine Cu,Zn superoxide dismutase reacted with peroxynitrite to form a stable yellow protein-bound adduct identified as nitrotyrosine. The uv-visible spectrum of the peroxynitrite-modified superoxide dismutase was highly pH dependent, exhibiting a peak at 438 nm at alkaline pH that shifts to 356 nm at acidic pH. An equivalent uv-visible spectrum was obtained by Cu,Zn superoxide dismutase treated with tetranitromethane. The Raman spectrum of authentic nitrotyrosine was contained in the spectrum of peroxynitrite-modified Cu,Zn superoxide dismutase. The reaction was specific for peroxynitrite because no significant amounts of nitrotyrosine were formed with nitric oxide (NO), nitrogen dioxide (NO2), nitrite (NO2-), or nitrate (NO3-). Removal of the copper from the Cu,Zn superoxide dismutase prevented formation of nitrotyrosine by peroxynitrite. The mechanism appears to involve peroxynitrite initially reacting with the active site copper to form an intermediate with the reactivity of nitronium ion (NO2+), which then nitrates tyrosine on a second molecule of superoxide dismutase. In the absence of exogenous phenolics, the rate of nitration of tyrosine followed second-order kinetics with respect to Cu,Zn superoxide dismutase concentration, proceeding at a rate of 1.0 +/- 0.1 M-1.s-1. Peroxynitrite-mediated nitration of tyrosine was also observed with the Mn and Fe superoxide dismutases as well as other copper-containing proteins.

Topics: Animals; Binding Sites; Cattle; Erythrocytes; Hydrogen-Ion Concentration; Kinetics; Lasers; Nitrates; Nitric Oxide; Protein Conformation; Spectrum Analysis, Raman; Superoxide Dismutase; Tyrosine