Vitamin C improves the effect of a new nitric oxide donor on the vascular smooth muscle from renal hypertensive rats.

Impaired relaxation induced by the new nitric oxide (NO) donor [Ru(NH.NHq)(terpy)NO(+)](3+) (TERPY) has been observed in the aortic rings from renal hypertensive rats (2K-1C). An increased production of reactive oxygen species (ROS) in the aortas from 2K-1C rats are capable of reducing NO bioavailability. Therefore, this study aimed at investigating the effects of an antioxidant (vitamin C) on the relaxant effect of NO released from TERPY on the 2K-1C rat aorta. As for vascular reactivity, the potency of TERPY is greater in the control rats (2K) than in 2K-1C whereas the maximum relaxation (ME) is not significantly different between the 2K and 2K-1C rat aortas. The relaxation of TERPY is potentiated only in the 2K-1C aortic ring treated with vitamin C. TERPY has a lower effect in decreasing cytosolic Ca(2+) concentration ([Ca(2+)]c) in vascular smooth muscle cells (VSMCs) from 2K-1C rats. This effect is also potentiated in 2K-1C aortic cells treated with vitamin C, but it is not altered in 2K cells. The basal cytosolic NO concentration ([NO]c) is lower in 2K-1C than in 2K cells, and the bioavailability of the NO released from TERPY is larger in 2K than in 2K-1C VSMCs. The superoxide radical concentration ([O(2)(*-)]) is higher in the 2K-1C aorta, and vitamin C reduces the [O(2)(*-)] in the 2K-1C aorta. Taken together, these results show that in the aortas of renal hypertensive 2K-1C rats, released NO from the new NO donor is not available to produce a similar effect in 2K aorta due to increased [O(2)(*-)].

Role of peroxynitrite in macrophage microbicidal mechanisms in vivo revealed by protein nitration and hydroxylation.

The cytotoxins produced by phagocytic cells lacking peroxidases such as macrophages remain elusive. To elucidate macrophage microbicidal mechanisms in vivo, we compared the lesion tissue responses of resistant (C57Bl/6) and susceptible (BALB/c) mice to Leishmania amazonensis infection. This comparison demonstrated that parasite control relied on lesion macrophage activation with inducible nitric oxide synthase expression (iNOS), nitric oxide synthesis, and extensive nitration of parasites inside macrophage phagolysosomes at an early infection stage. Nitration and iNOS expression were monitored by confocal microscopy; nitric oxide synthesis was monitored by EPR. The main macrophage nitrating agent was shown to be peroxynitrite derived because parasite nitration occurred in the virtual absence of polymorphonuclear cells (monitored as peroxidase activity) and was accompanied by protein hydroxylation (monitored as 3-hydroxytyrosine levels). In vitro studies confirmed that peroxynitrite is cytotoxic to parasites whereas nitric oxide is cytostatic. The results indicate that peroxynitrite is likely to be produced close to the parasites and most of it reacts with carbon dioxide to produce carbonate radical anion and nitrogen dioxide whose concerted action leads to parasite nitration. In parallel, some peroxynitrite decomposition to the hydroxyl radical should occur due to the detection of hydroxylated proteins in the healing tissues. Consequently, peroxynitrite and derived radicals are likely to be important macrophage-derived cytotoxins.

Role of the carbonate radical anion in tyrosine nitration and hydroxylation by peroxynitrite.

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.

Direct evidence for apo B-100-mediated copper reduction: studies with purified apo B-100 and detection of tryptophanyl radicals.

Copper binding to apolipoprotein B-100 (apo B-100) and its reduction by endogenous components of low-density lipoprotein (LDL) represent critical steps in copper-mediated LDL oxidation, where cuprous ion (Cu(I)) generated from cupric ion (Cu(II)) reduction is the real trigger for lipid peroxidation. Although the copper-reducing capacity of the lipid components of LDL has been studied extensively, we developed a model to specifically analyze the potential copper reducing activity of its protein moiety (apo B-100). Apo B-100 was isolated after solubilization and extraction from size exclusion-HPLC purified LDL. We obtained, for the first time, direct evidence for apo B-100-mediated copper reduction in a process that involves protein-derived radical formation. Kinetics of copper reduction by isolated apo B-100 was different from that of LDL, mainly because apo B-100 showed a single phase-exponential kinetic, instead of the already described biphasic kinetics for LDL (namely alpha-tocopherol-dependent and independent phases). While at early time points, the LDL copper reducing activity was higher due to the presence of alpha-tocopherol, at longer time points kinetics of copper reduction was similar in both LDL and apo B-100 samples. Electron paramagnetic resonance studies of either LDL or apo B-100 incubated with Cu(II), in the presence of the spin trap 2-methyl-2-nitroso propane (MNP), indicated the formation of protein-tryptophanyl radicals. Our results supports that apo B-100 plays a critical role in copper-dependent LDL oxidation, due to its lipid-independent-copper reductive ability.

Uric acid oxidation by peroxynitrite: multiple reactions, free radical formation, and amplification of lipid oxidation.

Uric acid has been considered to be an efficient scavenger of peroxynitrite but the reaction between urate and peroxynitrite has been only partially characterized. Also, previous studies have indicated that urate may increase peroxynitrite-mediated oxidation of low density lipoprotein (LDL). Here, we examined the reaction between urate and peroxynitrite by combining kinetic, oxygen consumption, spin trapping, and product identification studies; in parallel, we tested the effect of urate upon peroxynitrite-mediated lipid oxidation. Our results demonstrated that urate reacts with peroxynitrite with an apparent second order rate constant of 4.8 x 10(2) M(-1). s(-1) in a complex process, which is accompanied by oxygen consumption and formation of allantoin, alloxan, and urate-derived radicals. The main radical was identified as the aminocarbonyl radical by the electrospray mass spectra of its 5, 5-dimethyl-l-pyrroline N-oxide adduct. Mechanistic studies suggested that urate reacts with peroxynitrous acid and with the radicals generated from its decomposition to form products that can further react with peroxynitrite anion. These many reactions may explain the reported efficiency of urate in inhibiting some peroxynitrite-mediated processes. Production of the aminocarbonyl radical, however, may propagate oxidative reactions. We demonstrated that this radical is likely to be the species responsible for the effects of urate in amplifying peroxynitrite-mediated oxidation of liposomes and LDL, which was monitored by the formation of lipid peroxides and thiobarbituric acid-reactive substances. The aminocarbonyl radical was not detectable during urate attack by other oxidants and consequently it is unlikely to be responsible for all previously described prooxidant effects of uric acid.

Toxic effects of alcohol intake on prostate of rats.

BACKGROUND: The present report was carried out to determine whether alcohol intake could induce prostate lesions. METHODS: We tested male rats for 300 days. Animals were divided into three groups: controls received only tap water as liquid diet; the chronic alcohol intake group received only ethanol solution in semivoluntary research; and the withdrawal group received the same treatment as chronic alcohol intake until 240 days, after which they reverted to drinking water. RESULTS: Chronic alcohol intake increased lipoperoxide concentrations and acid phosphatase activities. Cu-Zn superoxide dismutase (SOD) was decreased at 60 days, but approached controls values at 300 days following treatment. The serum increased alkaline phosphatase, and alanine transaminase activities reflected the chronic toxic effect of ethanol. CONCLUSIONS: Since SOD activity was unable to scavenge superoxide radical and lipoperoxide formation, we can conclude that superoxide is an important intermediate in prostate damage of chronic alcohol intake.