Increase In Nitrotyrosine Research Articles

Protection against myocardial ischemia and reperfusion injury by 3-aminobenzamide, an inhibitor of poly (ADP-ribose) synthetase.

Peroxynitrite and hydroxyl radical, reactive oxidants produced during reperfusion, are potent triggers of DNA single strand breakage. DNA injury triggers the activation of the nuclear enzyme poly (ADP-ribose) synthetase (PARS), which contributes to cellular energetic depletion. Using 3-aminobenzamide, an inhibitor of PARS, we investigated the role of PARS in the pathogenesis of myocardial reperfusion injury in a rat model. Occlusion of the left main coronary artery (one hour) followed by reperfusion (one hour) in the anesthetized rat caused severe cardiac necrosis, neutrophil infiltration, and increased plasma creatine phosphokinase activity. There was significant peroxynitrite production during reperfusion, as indicated by a massive increase in nitrotyrosine in the necrotic myocardium. Reperfusion was also associated with a significant loss of myocardial ATP. In vivo administration of the PARS inhibitor 3-aminobenzamide (10 mg/kg i.v.) to rats subjected to myocardial ischemia and reperfusion, reduced myocardial infarct size and blunted the increase in plasma creatine phosphokinase activity and myeloperoxidase activity in infarcted hearts. In addition, 3-aminobenzamide partially preserved the myocardial ATP levels. In vitro, pharmacological inhibition of PARS also ameliorated peroxynitrite-induced cytotoxicity in rat cardiac myocytes and human endothelial cells. 3-aminobenzamide has significant protective effects in myocardial reperfusion injury. We hypothesize that activation of PARS activation plays a role in the pathophysiology of acute myocardial infarction.

Development of autoimmune diabetes in NOD mice is associated with the formation of peroxynitrite in pancreatic islet beta-cells.

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.

Nitrotyrosine Bound to β-VLDL-Apoproteins: A Biomarker of Peroxynitrite Formation in Experimental Atherosclerosis

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 β-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 β-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.

Nitric oxide production and perivascular nitration in brain after carbon monoxide poisoning in the rat.

Nitric oxide is a short-lived free radical and physiological mediator which has the potential to cause cytotoxicity. Studies were conducted to investigate whether nitric oxide, and the potent oxidant peroxynitrite, were generated in brain during experimental carbon monoxide (CO) poisoning in the rat. Nitric oxide production was documented by electron paramagnetic resonance spectroscopy, and found to be increased by ninefold immediately after CO poisoning. Evidence that peroxynitrite was generated was sought by looking for nitrotyrosine in the brains of CO-poisoned rats. Nitrotyrosine was found deposited in vascular walls, and also diffusely throughout the parenchyma in inummocytochemical studies. The affinity and specificity of an anti-nitrotyrosine antibody was investigated and a solid phase immunoradiochemical assay was developed to quantity nitrotyrosine in brain homogenates. A 10-fold increase in nitrotyrosine was found in the brains of CO-poisoned rats. Platelets were involved with production of nitrotyrosine in the early phase of exposure to CO. However, nitrotyrosine formation and leukocyte sequestration were not decreased in thrombocytopenic rats poisoned with CO according to the standard model. When rats were pre-treated with the nitric oxide synthase inhibitor, L-nitroarginine methyl ester, formation of both nitric oxide and nitrotyrosine in response to CO poisoning were abolished, as well as leukocyte sequestration in the microvasculature, endothelial xanthine dehydrogenase conversion to xanthine oxidase, and brain lipid peroxidation. We conclude that perivascular reactions mediated by peroxynitrite are important in the cascade of events which lead to brain oxidative stress in CO poisoning.

Nitration of surfactant protein A results in decreased ability to aggregate lipids

We assessed the extent to which nitration of surfactant protein (SP) A, isolated from the bronchoalveolar lavage of patients with alveolar proteinosis, alters its ability to enhance lipid aggregation, bind lipids, and act synergistically with surfactant apoproteins B and C (SP-B, SP-C) in lowering the surface activity of surfactant lipids. SP-A was treated with various concentrations of tetranitromethane (TNM) at pH 6, 7.4, 8, or 10. Depending on the pH, TNM acts either as a nitrating (pH > or = 7.4) or an oxidizing agent (pH < or = 6). Exposure of SP-A to TNM (0.1-1 mM) at pH 7.4 or 8 for 30 min resulted in dose-and pH-dependent increases in nitrotyrosine, detected by Western blotting, enzyme-linked immunosorbent assay, and direct amino acid analysis. Treatment of SP-A with 0.5 mM TNM decreased its ability to aggregate lipids by 30% at pH 7.4, and 90% at pH 8, but had no effect on the disulfide-dependent oligomeric state of SP-A. In contrast, SP-A exposed to 1 mM TNM at pH 6 had background levels of nitrotyrosine and exhibited normal lipid aggregation properties. TNM, but not a hydroxyl radical-generating system, resulted in a pH-dependent loss of SP-A fluorescence, suggesting that tryptophan also may have been nitrated. Nitration of SP-A did not affect its ability to bind lipids. In addition, SP-A (1-3% by weight), treated with 0.25-0.5 mM TNM at pH 8, restored the surface-active properties of calf lung surfactant extract, previously damaged by exposure to peroxynitrite. We conclude that tyrosine nitration selectively inhibits the SP-A-mediated lipid aggregation without affecting its ability to bind lipids.

Structural and Functional Alterations of Surfactant Protein A by Peroxynitrite

We assessed the extent of functional and structural injury to surfactant protein-A (SP-A) after exposure to peroxynitrite or reactive oxygen species. Functional injury was quantified by measuring the extent of SP-A-induced lipid aggregation of small unilamellar vesicles of calf lung surfactant extract (CLSE 0.25 mg/ml) in the presence of 10 mM CaCl2, and by its ability to act synergistically with SP-B and SP-C in lowering the minimum surface tension (Tmin) of surfactant mixtures. Exposure of SP-A to peroxynitrite (0.1 to 1 mM) for 10 min decreased its ability to induce lipid-aggregation with a 50 percent inhibition of about 0.2 mM. When CLSE, suspended in HEPES buffer, pH 10, was compressed to 50 percent of its area in an oscillating bubble, it reached a Tmin of no less than 12 dynes/cm. Addition of control SPA to CLSE (3 percent by weight) decreased the Tmin to less than 2 dynes/cm, however, after SP-A exposure to 1 mM peroxynitrite for 10 min, the Tmin failed to drop below 12 dynes/cm. Peroxynitrite injury to SP-A was prevented by the addition of cysteine but not mannitol prior to peroxynitrite exposure. Western blotting studies of peroxynitrite exposed SP-A, separated under reducing conditions with 12 percent polyacrylamide, revealed a decreased intensity of the 29 kd band and the appearance of low molecular weight peptides, consistent with protein degradation. Amino acid analysis of SP-A incubated with 1 mM peroxynitrite revealed a significant decrease in tyrosine (from 35 to 27 residues/1,000) and a significant increase in nitrotyrosine (from 0.9 to 4.2 residues/1,000). Exposure of SP-A to 10 mU/ml xanthine oxidase and 500 µM xanthine had no effect on SP-A structure or function. These data indicate the significant cytotoxic potential of peroxynitrite towards components of the pulmonary surfactant system.