Homero Rubbo

Chapter 4 – The Biological Chemistry of Peroxynitrite

Publisher Summary This chapter provides a comprehensive overview of the physical and biological chemistry of peroxynitrite. A foundation is provided to rationalize the biological fate and actions of peroxynitrite and the strategies for preventing peroxynitrite-dependent biological damage and pathology. Peroxynitrite anion is formed in vivo as a result of the diffusion controlled reaction between nitric oxide (NO) and superoxide anion radicals. The anion and its conjugated acid, peroxynitrous acid, are strong oxidant species that cause molecular damage in a variety of pathophysiological conditions. Peroxynitrite reacts fast with a number of biological targets, including thiols, metalloproteins, and carbon dioxide, or more slowly decomposes to hydroxyl and nitrogen dioxide radicals by proton-catalyzed homolysis. Carbon dioxide accounts for a significant fraction of peroxynitrite consumption and leads to the secondary formation of carbonate and nitrogen dioxide radicals. At the molecular level, the predominant outcome of peroxynitrite reactions in vivo is one or two electron oxidations and nitrations. Peroxynitrite can diffuse through tissue compartments, being able to cross biomembranes by both passive diffusion and anion channels. Thus, although the biological half-life of peroxynitrite is short, it is sufficient for peroxynitrite to diffuse a couple of cell diameters and cause biological effects distant from its site of production.

Protein and lipid nitration: role in redox signaling and injury.

Protein and lipid nitration represent novel footprints of oxidative and nitrative stress processes. In this review, we first discuss the mechanisms of formation of protein 3-nitrotyrosine and nitrated fatty acids as well as their key biological and signaling actions. Elevation of protein 3-nitrotyrosine levels is associated to tissue injury, and some specific nitrated proteins play a causative role in disease progression; on the other hand, the substantiation on the role of tyrosine nitration on redox signaling is rather scarce. Herein, we also provide evidence to support that the nitration of lipids (i.e. to nitrofatty acids) results in the formation of novel endogenous modulators of redox processes, partially counteracting pro-inflammatory effects of oxidant exposure.

Nitrated fatty acids : Mechanisms of formation, chemical characterization, and biological properties

Nitrated derivatives of unsaturated fatty acids are formed under oxidative and nitrative stress conditions, and are detected and structurally characterized in cell membranes, cardiac tissue, human plasma, and urine. Nitro-fatty acids display pleiotropic activities, including modulation of macrophage activation, prevention of leukocyte and platelet activation, and promotion of blood vessel relaxation. However, mechanisms of formation and levels reached in inflammatory milieu are poorly characterized. In this review, we discuss potential mechanisms of formation of nitro-fatty acids and their key chemical and biochemical properties. A major focus is to analyze nitrated lipids as novel signaling mediators leading to secondary changes in protein function via electrophilic-based modifications as well as inhibition of inflammatory cell function, thus representing the convergence of lipid and nitric oxide signaling pathways.

Peroxynitrite-mediated lipid oxidation and nitration: mechanisms and consequences.

Lipid oxidation and nitration represents a novel area of research of relevance in the understanding of inflammatory processes. Peroxynitrite, the product of the diffusion-limited reaction between nitric oxide and superoxide anion, mediates oxidative modifications in lipid systems including cell membranes and lipoproteins. In this review, we discuss the mechanisms of lipid oxidation and nitration by peroxynitrite as well as the influence of physiological molecules and cell targets to redirect peroxynitrite reactivity. We also provide evidence to support that oxidation/nitration of lipids results in the formation of novel signaling modulators of key lipid-metabolizing enzymes.

Macrophage activation induces formation of the anti-inflammatory lipid cholesteryl-nitrolinoleate.

Nitroalkene derivatives of fatty acids act as adaptive, anti-inflammatory signalling mediators, based on their high-affinity PPARgamma (peroxisome-proliferator-activated receptor gamma) ligand activity and electrophilic reactivity with proteins, including transcription factors. Although free or esterified lipid nitroalkene derivatives have been detected in human plasma and urine, their generation by inflammatory stimuli has not been reported. In the present study, we show increased nitration of cholesteryl-linoleate by activated murine J774.1 macrophages, yielding the mononitrated nitroalkene CLNO2 (cholesteryl-nitrolinoleate). CLNO2 levels were found to increase approximately 20-fold 24 h after macrophage activation with Escherichia coli lipopolysaccharide plus interferon-gamma; this response was concurrent with an increase in the expression of NOS2 (inducible nitric oxide synthase) and was inhibited by the (*)NO (nitric oxide) inhibitor L-NAME (N(G)-nitro-L-arginine methyl ester). Macrophage (J774.1 and bone-marrow-derived cells) inflammatory responses were suppressed when activated in the presence of CLNO2 or LNO2 (nitrolinoleate). This included: (i) inhibition of NOS2 expression and cytokine secretion through PPARgamma and *NO-independent mechanisms; (ii) induction of haem oxygenase-1 expression; and (iii) inhibition of NF-kappaB (nuclear factor kappaB) activation. Overall, these results suggest that lipid nitration occurs as part of the response of macrophages to inflammatory stimuli involving NOS2 induction and that these by-products of nitro-oxidative reactions may act as novel adaptive down-regulators of inflammatory responses.

Oxygen Radical-Nitric Oxide Reactions in Vascular Diseases

Publisher Summary The principal challenge in the research of radical biology lies in developing a solid causal relationship between the tissue productions of various reactive species, long recognized to have potent and toxic target molecule reactions, and their contribution to cell or tissue dysfunction. Not until this is accomplished can a rational therapeutic strategy for oxidant tissue injury be devised. These dilemmas amplify the immense challenge that is faced upon in development of a clear understanding of the multifaceted role that nitric oxide (NO) plays in vascular disease. The high rate of production and broad distribution of sites of production of NO, combined with its facile direct and indirect reactions with metalloproteins, thiols, and various oxygen radical species ensure that NO plays a central role in regulating vascular physiological and cellular homeostasis as well as critical intravascular free radical and oxidant reactions. This concept is emphasized in this chapter, using atherosclerosis as a prime example of the central role that reactive species play in vascular diseases. The chapter describes how superoxide anion (O 2− ) “inactivates” the vasorelaxant actions of NO in atherosclerotic vessels, leading to impaired endothelial cell (EC)-dependent relaxation and a propensity for vasospasm. The alterations in vascular reactivity associated with atherosclerosis are related to changes in EC-dependent mechanisms of relaxation. Acetylcholine and other EC agonists normally promote the relaxation of isolated vascular ring segments by stimulating the production of NO. Nitric oxide diffuses to underlying vascular smooth muscle cells, where it activates soluble guanylate cyclase and induces vessel relaxation via cGMP-dependent mechanisms.

Septic diaphragmatic dysfunction is prevented by Mn(III)porphyrin therapy and inducible nitric oxide synthase inhibition

ObjectiveDecreased diaphragmatic contractility and organ failure observed during sepsis is mediated by an overproduction of nitric oxide (.NO)-derived species, mitochondria being a major target of oxidative and nitrative stress. We tested the potential protective effects of (a) a novel synthetic antioxidant, the manganese(III) 5,10,15,20-tetrakis(N-ethylpyridinium-2-yl) porphyrin (MnTE-2-PyP5+) and (b) the inducible .NO synthase inhibitor aminoguanidine (AG) on a rat model of sepsis.SettingUniversity research laboratories.Subjects and interventionsSepsis was induced by cecal ligation and perforation in rats.Measurements and resultsSystemic hemodynamics, pulmonary gas exchange, in vitro diaphragmatic function and mitochondrial respiration were evaluated. Moreover, plasma and mitochondrial oxidative and nitrative stress parameters were investigated. Sepsis determined diaphragmatic dysfunction and a significant decrease in mitochondrial coupling and respiration. Oxidative stress was evidenced by decreased plasma antioxidants and increased lipid oxidation. Tyrosine nitration was increased in the plasma and mitochondria of the septic animals. These alterations were ameliorated or prevented by either MnTE-2-PyP5+ or AG.ConclusionsOur results demonstrate that overproduction of .NO and .NO-derived reactive species play a critical role in mitochondrial impairment and diaphragmatic function during sepsis. More importantly, AG but mainly the novel metalloporphyrin MnTE-2-PyP5+ were able to ameliorate diaphragmatic and mitochondrial dysfunction and could contribute to preventing organ failure during severe sepsis.

Interactions of nitric oxide and peroxynitrite with low-density lipoprotein.

Abstract Nitric oxide (NO) is a free radical species that diffuses and concentrates in the hydrophobic core of lowdensity lipoprotein (LDL) to serve as a potent inhibitor of lipid oxidation processes. Peroxynitrite (PN), the product of the diffusionlimited reaction between NO and superoxide (O2), represents a relevant mediator of oxidative modifications in LDL. The focus of this review is the analysis of interactions between NO and PN and its secondary reactions with oxygen radicals on LDL oxidation, which are relevant in the development of the early steps as well as progression of atherosclerosis. We propose that the balance between rates of PN and NO production, which greatly depends on oxidative stress processes within the vascular wall, will critically determine the final extent of oxidative LDL modifications leading or not to scavenger receptormediated LDL uptake and foam cell formation.

Succinate-dependent metabolism in Trypanosoma cruzi epimastigotes.

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.

Olives and Olive Oil Are Sources of Electrophilic Fatty Acid Nitroalkenes

Extra virgin olive oil (EVOO) and olives, key sources of unsaturated fatty acids in the Mediterranean diet, provide health benefits to humans. Nitric oxide (•NO) and nitrite (NO2 −)-dependent reactions of unsaturated fatty acids yield electrophilic nitroalkene derivatives (NO2-FA) that manifest salutary pleiotropic cell signaling responses in mammals. Herein, the endogenous presence of NO2-FA in both EVOO and fresh olives was demonstrated by mass spectrometry. The electrophilic nature of these species was affirmed by the detection of significant levels of protein cysteine adducts of nitro-oleic acid (NO2-OA-cysteine) in fresh olives, especially in the peel. Further nitration of EVOO by NO2 − under acidic gastric digestive conditions revealed that human consumption of olive lipids will produce additional nitro-conjugated linoleic acid (NO2-cLA) and nitro-oleic acid (NO2-OA). The presence of free and protein-adducted NO2-FA in both mammalian and plant lipids further affirm a role for these species as signaling mediators. Since NO2-FA instigate adaptive anti-inflammatory gene expression and metabolic responses, these redox-derived metabolites may contribute to the cardiovascular benefits associated with the Mediterranean diet.