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Nitro-fatty acids (NO2-FA) are electrophilic lipid mediators derived from unsaturated fatty acid nitration. These species are produced endogenously by metabolic and inflammatory reactions and mediate anti-oxidative and anti-inflammatory responses. NO2-FA have been postulated as partial agonists of the Peroxisome Proliferator-Activated Receptor gamma (PPARγ), which is predominantly expressed in adipocytes and myeloid cells. Herein, we explored molecular and cellular events associated with PPARγ activation by NO2-FA in monocytes and macrophages. NO2-FA induced the expression of two PPARγ reporter genes, Fatty Acid Binding Protein 4 (FABP4) and the scavenger receptor CD36, at early stages of monocyte differentiation into macrophages. These responses were inhibited by the specific PPARγ inhibitor GW9662. Attenuated NO2-FA effects on PPARγ signaling were observed once cells were differentiated into macrophages, with a significant but lower FABP4 upregulation, and no induction of CD36. Using in vitro and in silico approaches, we demonstrated that NO2-FA bind to FABP4. Furthermore, the inhibition of monocyte FA binding by FABP4 diminished NO2-FA-induced upregulation of reporter genes that are transcriptionally regulated by PPARγ, Keap1/Nrf2 and HSF1, indicating that FABP4 inhibition mitigates NO2-FA signaling actions. Overall, our results affirm that NO2-FA activate PPARγ in monocytes and upregulate FABP4 expression, thus promoting a positive amplification loop for the downstream signaling actions of this mediator.

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Nitro-fatty acids are formed and detected in human plasma, cell membranes, and tissue, modulating metabolic as well as inflammatory signaling pathways. Here we discuss the mechanisms of nitro-fatty acid formation as well as their key chemical and biochemical properties. The electrophilic properties of nitro-fatty acids to activate anti-inflammatory signaling pathways are discussed in detail. A critical issue is the influence of nitroarachidonic acid on prostaglandin endoperoxide H synthases, redirecting arachidonic acid metabolism and signaling. We also analyze in vivo data supporting nitro-fatty acids as promising pharmacological tools to prevent inflammatory diseases.

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Nitro-fatty acids are formed and detected in human plasma, cell membranes, and tissue, modulating metabolic as well as inflammatory signaling pathways. Here we discuss the mechanisms of nitro-fatty acid formation as well as their key chemical and biochemical properties. The electrophilic properties of nitro-fatty acids to activate anti-inflammatory signaling pathways are discussed in detail. A critical issue is the influence of nitroarachidonic acid on prostaglandin endoperoxide H synthases, redirecting arachidonic acid metabolism and signaling. We also analyze in vivo data supporting nitro-fatty acids as promising pharmacological tools to prevent inflammatory diseases.

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Platelet activation represents a key event in normal hemostasis as well as during platelet plug formation related to thrombosis. Nitro-fatty acids are novel endogenously produced signaling mediators exerting pluripotent anti-inflammatory actions in cells and tissues. We have recently shown that nitroarachidonic acid inhibits thromboxane synthesis during platelet activation by affecting prostaglandin endoperoxide H synthase (PGHS). Herein, we investigated the regulation of human platelet activation by NO(2)AA and describe a novel mechanism involving protein kinase C (PKC) inhibition. NO(2)AA-mediated antiplatelet effects were characterized using mass spectrometry, confocal microscopy, flow cytometry, western blot and aggregometry. Incubation of NO(2)AA with human platelets caused a significant reduction in platelet sensitivity to thrombin, ADP, arachidonic acid (AA), and phorbol ester (PMA). These effects were cGMP-independent and did not involve Ca(2+) store-dependent mobilization. In contrast, signaling downstream of conventional PKC activation, such as α-granule secretion and extracellular signal regulated kinase 2 activation was strongly inhibited by NO(2)AA. Immunofluorescence confocal microscopy confirmed NO(2)AA-mediated inhibition of PKCα translocation to the membrane. In summary, we demonstrate that NO(2)AA inhibits platelet activation through modulation of PKCα activity as a potential novel mechanism for platelet regulation in vivo.

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Lipid-protein adducts are formed during oxidative and nitrative stress conditions associated with increasing lipid and protein oxidation and nitration. The focus of this review is the analysis of interactions between oxidative-modified lipids and proteins and how lipid nitration can modulate lipid-protein adducts formation. For this, two biologically-relevant models will be analysed: a) human low density lipoprotein, whose oxidation is involved in the early steps of atherogenesis, and b) alpha-synuclein/lipid membranes system, where lipid-protein adducts are being associated with the develop of Parkinson disease and other synucleinopathies.

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The objective of the present study was to identify disturbances of nitric oxide radical (.NO) metabolism and the formation of cholesterol oxidation products in human essential hypertension. The concentrations of.NO derivatives (nitrite, nitrate, S-nitrosothiols and nitrotyrosine), water and lipid-soluble antioxidants and cholesterol oxides were measured in plasma of 11 patients with mild essential hypertension (H: 57.8 +/- 9.7 years; blood pressure, 148.3 +/- 24.8/90.8 +/- 10.2 mmHg) and in 11 healthy subjects (N: 48.4 +/- 7.0 years; blood pressure, 119.4 +/- 9.4/75.0 +/- 8.0 mmHg). Nitrite, nitrate and S-nitrosothiols were measured by chemiluminescence and nitrotyrosine was determined by ELISA. Antioxidants were determined by reverse-phase HPLC and cholesterol oxides by gas chromatography. Hypertensive patients had reduced endothelium-dependent vasodilation in response to reactive hyperemia (H: 9.3 and N: 15.1% increase of diameter 90 s after hyperemia), and lower levels of ascorbate (H: 29.2 +/- 26.0, N: 54.2 +/- 24.9 micro M), urate (H: 108.5 +/- 18.9, N: 156.4 +/- 26.3 micro M), beta-carotene (H: 1.1 +/- 0.8, N: 2.5 +/- 1.2 nmol/mg cholesterol), and lycopene (H: 0.4 +/- 0.2, N: 0.7 +/- 0.2 nmol/mg cholesterol), in plasma, compared to normotensive subjects. The content of 7-ketocholesterol, 5alpha-cholestane-3beta,5,6beta-triol and 5,6alpha-epoxy-5alpha-cholestan-3alpha-ol in LDL, and the concentration of endothelin-1 (H: 0.9 +/- 0.2, N: 0.7 +/- 0.1 ng/ml) in plasma were increased in hypertensive patients. No differences were found for.NO derivatives between groups. These data suggest that an increase in cholesterol oxidation is associated with endothelium dysfunction in essential hypertension and oxidative stress, although.NO metabolite levels in plasma are not modified in the presence of elevated cholesterol oxides.

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The objective of the present study was to identify disturbances of nitric oxide radical (ANO) metabolism and the formation of cholesterol oxidation products in human essential hypertension. The concentrations ofANO derivatives (nitrite, nitrate, S-nitrosothiols and nitrotyrosine), water and lipid-soluble antioxidants and cholesterol oxides were measured in plasma of 11 patients with mild essential hypertension (H: 57.8 ± 9.7 years; blood pressure, 148.3 ± 24.8/90.8 ± 10.2 mmHg) and in 11 healthy subjects (N: 48.4 ± 7.0 years; blood pressure, 119.4 ± 9.4/75.0 ± 8.0 mmHg).Nitrite, nitrate and S-nitrosothiols were measured by chemiluminescence and nitrotyrosine was determined by ELISA. Antioxidants were determined by reverse-phase HPLC and cholesterol oxides by gas chromatography. Hypertensive patients had reduced endothelium-dependent vasodilation in response to reactive hyperemia (H: 9.3 and N: 15.1 percent increase of diameter 90 s after hyperemia), and lower levels of ascorbate (H: 29.2 ± 26.0, N: 54.2 ± 24.9 æM), urate (H: 108.5 ± 18.9, N: 156.4 ± 26.3 æM), ß-carotene (H: 1.1 ± 0.8, N: 2.5 ± 1.2 nmol/mg cholesterol), and lycopene (H: 0.4 ± 0.2, N: 0.7 ± 0.2 nmol/mg cholesterol), in plasma, compared to normotensive subjects. The content of 7-ketocholesterol, 5alpha-cholestane-3ß,5,6ß-triol and 5,6alpha-epoxy-5alpha-cholestan-3alpha-ol in LDL, and the concentration of endothelin-1 (H: 0.9 ± 0.2, N: 0.7 ± 0.1 ng/ml) in plasma were increased in hypertensive patients. No differences were found for ANO derivatives between groups. These data suggest that an increase in cholesterol oxidation is associated with endothelium dysfunction in essential hypertension and oxidative stress, although ANO metabolite levels in plasma are not modified in the presence of elevated cholesterol oxides

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Peroxynitrite (PN), the product of the diffusion-limited reaction between nitric oxide (*NO) and superoxide (O*-(2)), represents a relevant mediator of oxidative modifications in low-density lipoprotein (LDL). This work shows for the first time the simultaneous action of low-controlled fluxes of PN and *NO on LDL oxidation in terms of lipid and protein modifications as well as oxidized lipid-protein adduct formation. Fluxes of PN (e.g., 1 microM min(-1)) initiated lipid oxidation in LDL as measured by conjugated dienes and cholesteryl ester hydroperoxides formation. Oxidized-LDL exhibited a characteristic fluorescent emission spectra (lambda(exc) = 365 nm, lambda(max) = 417 nm) in parallel with changes in both the free amino groups content and the relative electrophoretic mobility of the particle. Physiologically relevant fluxes of *NO (80-300 nM min(-1)) potently inhibited these PN-dependent oxidative processes. These results are consistent with PN-induced adduct formation between lipid oxidation products and free amino groups of LDL in a process prevented by the simultaneous presence of *NO. The balance between rates of PN and *NO production in the vascular wall will critically determine the final extent of LDL oxidative modifications leading or not to scavenger receptor-mediated LDL uptake and foam cell formation.

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The reactions of nitric oxide ((.)NO) and alpha-tocopherol (alpha-TH) during membrane lipid oxidation were examined and compared with the pair alpha-TH/ascorbate. Nitric oxide serves as a more potent inhibitor of lipid peroxidation propagation reactions than alpha-TH and protects alpha-TH from oxidation. Mass spectrometry, oxygen and (.)NO consumption, conjugated diene analyses, and alpha-TH fluorescence determinations all demonstrated that (.)NO preferentially reacts with lipid radical species, with alpha-TH consumption not occurring until (.)NO concentrations fell below a critical level. In addition, alpha-TH and (.)NO cooperatively inhibit lipid peroxidation, exhibiting greater antioxidant capacity than the pair alpha-TH/ascorbate. Pulse radiolysis analysis showed no direct reaction between (.)NO and alpha-tocopheroxyl radical (alpha-T(.)), inferring that peroxyl radical termination reactions are the principal lipid-protective mechanism mediated by (.)NO. These observations support the concept that (.)NO is a potent chain breaking antioxidant toward peroxidizing lipids, due to facile radical-radical termination reactions with lipid radical species, thus preventing alpha-TH loss. The reduction of alpha-T(.) by ascorbate was a comparatively less efficient mechanism for preserving alpha-TH than (.)NO-mediated termination of peroxyl radicals, due to slower reaction kinetics and limited transfer of reducing equivalents from the aqueous phase. Thus, the high lipid/water partition coefficient of (.)NO, its capacity to diffuse and concentrate in lipophilic milieu, and a potent reactivity toward lipid radical species reveal how (.)NO can play a critical role in regulating membrane and lipoprotein lipid oxidation reactions.

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Atherosclerosis is one of the most common diseases and the principal cause of death in western civilization. The pathogenesis of this disease can be explained on the basis of the 'oxidative-modification hypothesis,' which proposes that low-density lipoprotein (LDL) oxidation represents a key early event. Nitric oxide (*NO) regulates critical lipid membrane and lipoprotein oxidation events by a) contributing to the formation of more potent secondary oxidants from superoxide (i.e.: peroxynitrite), and b) its antioxidant properties through termination reactions with lipid radicals to possibly less reactive secondary nitrogen-containing products (LONO, LOONO). Relative rates of production and steady state concentrations of superoxide and *NO and cellular sites of production will profoundly influence the expression of differential oxidant injury-enhancing and protective effects of *NO. Full understanding of the physiological roles of *NO, coupled with detailed insight into *NO regulation of oxygen radical-dependent reactions, will yield a more rational basis for intervention strategies directed toward oxidant-dependent atherogenic processes.

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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.

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RESUMO

Atherosclerosis is one of the most common diseases and the principal cause of death in western civilization. The pathogenesis of this disease can be explained on the basis of the 'oxidative-modification hypothesis,' which proposes that low-density lipoprotein (LDL) oxidation represents a key early event. Nitric oxide (*NO) regulates critical lipid membrane and lipoprotein oxidation events by a) contributing to the formation of more potent secondary oxidants from superoxide (i.e.: peroxynitrite), and b) its antioxidant properties through termination reactions with lipid radicals to possibly less reactive secondary nitrogen-containing products (LONO, LOONO). Relative rates of production and steady state concentrations of superoxide and *NO and cellular sites of production will profoundly influence the expression of differential oxidant injury-enhancing and protective effects of *NO. Full understanding of the physiological roles of *NO, coupled with detailed insight into *NO regulation of oxygen radical-dependent reactions, will yield a more rational basis for intervention strategies directed toward oxidant-dependent atherogenic processes.

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Nitric oxide (.NO) can mediate tissue protective reactions during oxidant stress, as well as toxic and tissue prooxidant effects. Nitric oxide regulates critical lipid membrane and lipoprotein oxidation events, by 1) contributing to the formation of more potent secondary oxidants from superoxide (i.e. peroxynitrite) and 2) termination of lipid radicals to possibly less reactive secondary nitrogen-containing products (LONO, LOONO) which are in part organic peroxynitrites and are expected to be produced in vivo. Relative rates of production and steady state concentrations of superoxide and .NO and cellular sites of production will profoundly influence expression of the differential oxidant injury-enhancing and protective effects of .NO. Full understanding of the physiological roles of .NO, coupled with detailed insight into .NO regulation of oxygen radical-dependent reactions, will yield a more rational basis for the use of .NO donors for therapeutic purposes.

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Xanthine oxidoreductase (XDH + XO, EC 1.2.3.2) is released into the circulation from organs rich in XO activity. Herein we report the specific high affinity binding of XO to glycosaminoglycans (GAGs) and the preferential association of XO with heparin, compared with heparan sulfate, chondroitin sulfate, and dematan sulfate. The binding of XO to Sepharose 6B-conjugated heparin (HS6B) occurs at physiological ionic strength and increased with pH, with Scatchard analysis revealing a nonlinear binding pattern at pH 7.4. The dissociation constant (Kd) for XO binding was 0.4 to 1.8 x 10(-7) M, similar to the heparin-reversible binding of lipoprotein lipase to vascular endothelium. The binding energy of 9-13 kcal/mol was concordant with noncovalent electrostatic interactions. Xanthine oxidase immobilization to HS6B rendered a catalytically active enzyme from that had kinetic characteristics distinct from XO in free solution. While the Km and Ki for xanthine in phosphate buffer at pH 7.4 were 3 microM and 1.6 mM, respectively, for free XO, they were 15 microM and 2.8 mM for immobilized XO. Inhibition constants for guanine and uric acid were also increased upon XO binding to HS6B. Changes in kinetic parameters were related to a real and not apparent decrease in binding affinity for substrate and inhibitors and were not due to diffusion-controlled processes within the gel matrix. Changes in Km and Ki for xanthine also had a significant influence on the relative quantities of O2.- and H2O2 generated by a given substrate concentration. Superoxide formed by HS6B-bound XO was partially consumed within the gel microenvironment which electrostatically excluded CuZn SOD. Immobilization of XO increased the half-life of enzyme activity in buffer and in the absence of substrate from 67 to 120 h at 4 degrees C. These data indicate that binding to cell surfaces will strongly influence the catalytic properties, oxidant producing capacity, and stability of XO.

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Peroxynitrite (ONOO-), the reaction product of superoxide (O2.-) and nitric oxide (.NO), nitrates tyrosine and other phenolics. We report herein that tryptophan is also nitrated by peroxynitrite in the absence of transition metals to one predominant isomer of nitrotryptophan, as determined from spectral characteristics and liquid chromatography-mass spectrometry analysis. At high peroxynitrite to tryptophan ratios, other oxidation products were detected as well. The amount of nitrotryptophan formed from peroxynitrite increased at acidic pH, with an apparent pKa of 7.8. High concentrations of Fe(3+)-EDTA were required to enhance peroxynitrite-induced nitrotryptophan formation, while addition of up to 15 microM Cu/Zn superoxide dismutase had a minimal effect on tryptophan nitration. Cysteine, ascorbate, and methionine decreased nitrotryptophan yield to an extent similar to that predicted by their reaction rates with ground-state peroxynitrite, and typical hydroxyl radical scavengers partially inhibited nitration. Plots of the observed rate constant of nitrotryptophan formation vs tryptophan concentration presented downward curvatures. Thus, the kinetics of metal-independent nitration reactions were interpreted in terms of two parallel mechanisms. In the first one, ground-state peroxynitrous acid nitrated tryptophan with a second-order rate constant of 184 +/- 11 M-1 s-1 at 37 degrees C. The activation enthalpy was 9.1 +/- 0.3 kcal mol-1, and the activation entropy was -19 +/- 1 cal mol-1 K-1. In the second mechanism, ONOOH*, an activated intermediate derived from trans-peroxynitrous acid formed in a steady state, was the nitrating agent.

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Activated macrophages release peroxynitrite anion (ONOO-), which has been recently shown to be highly cytotoxic against Trypanosoma cruzi epimastigotes. In this work, we report that two critical enzymes for the energetic metabolism of the parasite, succinate dehydrogenase and fumarate reductase, are inactivated by biologically relevant concentrations of peroxynitrite. Enzyme inactivation was accompanied by a significant inhibition of succinate-dependent respiration in intact cells as well as in the membrane-rich fraction. Peroxynitrite also inhibited NADH-dependent oxygen consumption which depends almost exclusively on fumarate reductase activity in T. cruzi epimastigotes. Direct reactions of peroxynitrite anion with critical sulfhydryl residues of the two enzymes were responsible for most of the observed inactivation as indicated by the protection afforded by peroxynitrite scavengers and the reactivation of the enzymes by dithiothreitol. We propose that peroxynitrite-mediated inactivation of succinate dehydrogenase and fumarate reductase may be a key mechanism of macrophage-mediated cytotoxicity to T. cruzi, through inhibition of the energetic metabolism of the parasite.

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Macrophages produce and release superoxide anion (O2.-) and nitric oxide (.NO) as part of their microbicidal effector molecules. The simultaneous production of O2.- and .NO results in the rapid formation of peroxynitrite anion (ONOO-) by macrophages. Peroxynitrite is a strong oxidant with a half-life of less than 1 s in biological systems. There is solid experimental evidence implicating .NO and O2.- in macrophage-induced cytotoxicity against bacteria, parasites, and tumor cells. However, the cytotoxic role of peroxynitrite in these processes remains to be studied. In this work we demonstrate the parasiticidal activity of ONOO- against Trypanosoma cruzi. Peroxynitrite was highly trypanocidal, killing T. cruzi in a dose-dependent manner. Addition of 500 microM ONOO- as a single bolus resulted in 50% inhibition of cell proliferation as followed by growth curves. Fifty percent inhibition of [3H]thymidine incorporation measured at 6 h postaddition of ONOO- was obtained at 150 microM. Addition of ONOO- as a continuous infusion rather than a single bolus resulted in an even stronger inhibition in cell growth. Other cytotoxic effects of ONOO- included cellular swelling and inhibition of cell motility. Classical hydroxyl radical scavengers and metal chelators afforded minimal protection against ONOO(-)-mediated cytotoxicity, indicating that peroxynitrite anion itself, rather than the .OH-like oxidant derived from its proton-catalyzed decomposition, was the main damaging species. From literature data we estimated the production of ONOO- by activated macrophages inside phagolysosomes to be around 500 microM/min. Therefore, our results demonstrate that ONOO- may operate in vivo as a critical macrophage-derived reactive intermediate against T. cruzi.

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Trypanosoma cruzi epimastigotes permeabilized with digitonin (65 micrograms (mg protein)-1) to measure mitochondrial respiration were exposed to different substrates. Although none of the NADH-dependent substrates stimulated respiration, succinate supported not only oxygen consumption but also oxidative phosphorylation (respiratory control ratio of 1.9 +/- 0.3) indicating that the mitochondria were coupled. The rate of NADH-dependent oxygen consumption by membrane fractions (9.4 +/- 0.7 nmol min-1 (mg protein)-1) was reduced by 50% upon addition of catalase indicating that the electrons from NADH oxidation reduced oxygen to H2O2. NADH-dependent H2O2 production (16 +/- 1 nmol min-1 (mg protein)-1) was confirmed using cytochrome c peroxidase. This activity was inhibited by fumarate by 70%, suggesting a competition between fumarate and oxygen for the electrons from NADH, probably at the fumarate reductase level. The respiratory chain inhibitor antimycin blocked both respiration by intact cells and succinate-dependent cytochrome c by isolated membranes. No inhibition by antimycin was observed when NADH replaced succinate as an electron donor, indicating that the electrons from NADH oxidation reduced cytochrome c through a different route. Malonate blocked not only succinate-cytochrome c reductase and fumarate reductase, but also intact cell motility. These results suggest that succinate has a central role in the intermediate metabolism of i. cruzi, as it may be used for respiration or excreted to the extracellular space under anaerobic conditions. In addition, 2 potential sources of H2O2 were tentatively identified as: (a) the enzyme fumarate reductase; and (b) a succinate-dependent site, which may be the semiquinone form of Coenzyme Q9, as in mammalian mitochondria.

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The influence of substrate inhibition on xanthine oxidase-intramolecular electron transport was studied by steady-state kinetic analysis. Experiments with hypoxanthine and xanthine up to 900 microM indicated an inhibition pattern which fitted an equation of the general form nu 0 = nu max . [S]/(Km + a[S] + b[S]2/Ki). Univalent electron flux to oxygen was favored at substrate concentrations above 50 microM. This augmentation of univalent flux percentage that appeared at a high substrate concentration was greater for hypoxanthine that xanthine and at pH 8.3 than at 9.5. Our results support a mechanism of inhibition in which a substrate-reduced enzyme, non-productive Michaelis complex was formed. It is possible that this non-productive complex favored the univalent pathway of enzyme reoxidation (superoxide production) by increasing the midpoint redox potential of the molybdenum active site.

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