The discovery that mammalian cells have the ability to synthesize the free radical nitric oxide (NO) has stimulated an extraordinary impetus for scientific research in all the fields of biology and medicine. Since its early description as an endothelial-derived relaxing factor, NO has emerged as a fundamental signaling device regulating virtually every critical cellular function, as well as a potent mediator of cellular damage in a wide range of conditions. Recent evidence indicates that most of the cytotoxicity attributed to NO is rather due to peroxynitrite, produced from the diffusion-controlled reaction between NO and another free radical, the superoxide anion. Peroxynitrite interacts with lipids, DNA, and proteins via direct oxidative reactions or via indirect, radical-mediated mechanisms. These reactions trigger cellular responses ranging from subtle modulations of cell signaling to overwhelming oxidative injury, committing cells to necrosis or apoptosis. In vivo, peroxynitrite generation represents a crucial pathogenic mechanism in conditions such as stroke, myocardial infarction, chronic heart failure, diabetes, circulatory shock, chronic inflammatory diseases, cancer, and neurodegenerative disorders. Hence, novel pharmacological strategies aimed at removing peroxynitrite might represent powerful therapeutic tools in the future. Evidence supporting these novel roles of NO and peroxynitrite is presented in detail in this review.

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Nitric Oxide and Peroxynitrite in Health and Disease

Neutrophilic polymorphonuclear leukocytes (neutrophils) are highly specialized for their primary function, the phagocytosis and destruction of microorganisms. When coated with opsonins (generally complement and/or antibody), microorganisms bind to specific receptors on the surface of the phagocyte and invagination of the cell membrane occurs with the incorporation of the microorganism into an intracellular phagosome. There follows a burst of oxygen consumption, and much, if not all, of the extra oxygen consumed is converted to highly reactive oxygen species. In addition, the cytoplasmic granules discharge their contents into the phagosome, and death of the ingested microorganism soon follows. Among the antimicrobial systems formed in the phagosome is one consisting of myeloperoxidase (MPO), released into the phagosome during the degranulation process, hydrogen peroxide (H2O2), formed by the respiratory burst and a halide, particularly chloride. The initial product of the MPO-H2O2-chloride system is hypochlorous acid, and subsequent formation of chlorine, chloramines, hydroxyl radicals, singlet oxygen, and ozone has been proposed. These same toxic agents can be released to the outside of the cell, where they may attack normal tissue and thus contribute to the pathogenesis of disease. This review will consider the potential sources of H2O2 for the MPO-H2O2-halide system; the toxic products of the MPO system; the evidence for MPO involvement in the microbicidal activity of neutrophils; the involvement of MPO-independent antimicrobial systems; and the role of the MPO system in tissue injury. It is concluded that the MPO system plays an important role in the microbicidal activity of phagocytes.

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Myeloperoxidase: friend and foe

Photo-, thermo-, solvato-, and electrochromic spiroheterocyclic compounds.

Peroxynitrite is formed by the very fast reaction of nitric oxide and superoxide radicals, a reaction that kinetically competes with other routes that chemically consume or physically sequester the reagents. It can behave either as an endogenous cytotoxin toward host tissues or a cytotoxic effector molecule against invading pathogens, depending on the cellular source and pathophysiological setting. Peroxynitrite is in itself very reactive against a few specific targets that range from efficient detoxification systems, such as peroxiredoxins, to reactions eventually leading to enhanced radical formation (e.g., nitrogen dioxide and carbonate radicals), such as the reaction with carbon dioxide. Thus, the chemical biology of peroxynitrite is dictated by the chemical kinetics of its formation and decay and by the diffusion across membranes of the species involved, including peroxynitrite itself. On the other hand, most durable traces of peroxynitrite passing (such as 3-nitrotyrosine) are derived from radicals ...

Chemical Biology of Peroxynitrite: Kinetics, Diffusion, and Radicals

Uric acid is considered a major antioxidant in human blood that may protect against aging and oxidative stress. Despite its proposed protective properties, elevated levels of uric acid are commonly associated with increased risk for cardiovascular disease and mortality. Furthermore, recent experimental studies suggest that uric acid may have a causal role in hypertension and metabolic syndrome. All these conditions are thought to be mediated by oxidative stress. In this study we demonstrate that differentiation of cultured mouse adipocytes is associated with increased production of reactive oxygen species (ROS) and uptake of uric acid. Soluble uric acid stimulated an increase in NADPH oxidase activity and ROS production in mature adipocytes but not in preadipocytes. The stimulation of NADPH oxidase-dependent ROS by uric acid resulted in activation of MAP kinases p38 and ERK1/2, a decrease in nitric oxide bioavailability, and an increase in protein nitrosylation and lipid oxidation. Collectively, our results suggest that hyperuricemia induces redox-dependent signaling and oxidative stress in adipocytes. Since oxidative stress in the adipose tissue has recently been recognized as a major cause of insulin resistance and cardiovascular disease, hyperuricemia-induced alterations in oxidative homeostasis in the adipose tissue might play an important role in these derangements.

Adverse effects of the classic antioxidant uric acid in adipocytes: NADPH oxidase-mediated oxidative/nitrosative stress

The photochromic behavior of several spirooxazines (SO) containing phenanthrene or phenanthroline moieties in the oxazine part of molecules has been investigated in several solvents and phosphatidylcholine (PC) liposomes. The solvatochromic properties of the merocyanine (MC) forms of these dyes were used to probe their location within the PC membrane. Transient spectroscopic measurements revealed that, when first formed by photoexcitation, the MC forms of phenanthroline-containing spirooxazines were located at relatively nonpolar sites within the membrane, but they subsequently moved to a more polar environment typical of the aqueous−organic interface. The characteristic time for this intersite movement was τ ≈ 10-3 s, corresponding to a diffusion coefficient of D ≈ 10-11 cm2 s-1. In contrast, these spectral shifts were not observed when PC liposome-bound SO containing the phenanthrene moiety were photoexcited, suggesting that either intersite diffusion was more rapid for these compounds or the initially ...

Photochromism of Spirooxazines in Homogeneous Solution and Phospholipid Liposomes

Peroxynitrite decay in weakly alkaline media occurs by two concurrent sets of pathways which are distinguished by their reaction products. One set leads to net isomerization to NO(3)(-) and the other set to net decomposition to O(2) plus NO(2)(-). At sufficiently high peroxynitrite concentrations, the decay half-time becomes concentration-independent and approaches a limiting value predicted by a mechanism in which reaction is initiated by unimolecular homolysis of the peroxo O-O bond, i.e., the following reaction: ONOOH --> (*)OH + (*)NO(2). This dynamical behavior excludes alternative postulated mechanisms that ascribe decomposition to bond rearrangement within bimolecular adducts. Nitrate and nitrite product distributions measured at very low peroxynitrite concentrations also correspond to predictions of the homolysis model, contrary to a recent report from another laboratory. Additionally, (1) the rate constant for the reaction ONOO(-) --> (*)NO + (*)O(2)(-), which is critical to the kinetic model, has been confirmed, (2) the apparent volume of activation for ONOOH decay (DeltaV() = 9.7 +/- 1.4 cm(3)/mol) has been shown to be independent of the concentration of added nitrite and identical to most other reported values, and (3) complex patterns of inhibition of O(2) formation by radical scavengers, which are impossible to rationalize by alternative proposed reaction schemes, are shown to be quantitatively in accord with the homolysis model. These observations resolve major disputes over experimental data existing in the literature; despite extensive investigation of these reactions, no verifiable experimental evidence has been advanced that contradicts the homolysis model.

Hydroxyl radical formation by O-O bond homolysis in peroxynitrous acid.

Quantitative kinetic models have been developed for the reaction between peroxynitrite and membrane lipids in vesicles and for transmembrane oxidation of reactants located within their inner aqueous cores. The models were used to analyze TBARS formation and oxidation of entrapped Fe(CN)64- ion in egg lecithin liposomes and several artificial vesicles. The analyses indicate that permeation of the bilayers by ONOOH and NO2•, a radical formed by homolysis of the ONOOH bond, is unusually rapid but that permeation by ONOO- and CO3•-, a radical formed when CO2 is present, is negligible. Bicarbonate protects the vesicles against both membrane and Fe(CN)64- oxidation by rapid competitive CO2-catalyzed isomerization of ONOOH to NO3-; this effect is partially reversed by addition of nitrite ion, which reacts with CO3•- to generate additional NO2•. Under medium conditions mimicking the physiological milieu, a significant fraction of the oxidants escape to inflict damage upon the vesicular assemblies. Rate constants ...

Permeation of phospholipid membranes by peroxynitrite.

Solar energy is being used for power generation, but also attracts increasing interest as a renewable energy source for the photocatalytic production of useful chemicals. Simple systems based on vesicles with transmembrane redox mediators have been used to transform photon energy into long-lived, membrane-separated photoredox products1,2,3,4,5,6. However, these systems are not suitable for high-throughput applications because the transmembrane electron carriers are oxidized inside the vesicle into charged species that are no longer able to readily traverse the membrane bilayer. This leads to continuous trapping of these carriers during photolysis and, ultimately, to the termination of the redox reaction due to accumulation of the available carriers within the vesicle interior. Living cells circumvent this problem by using quinones to simultaneously transport electrons and protons, thus allowing the carrier to remain neutral in its reduced and oxidized states and so retain the ability to undergo transmembrane diffusion throughout the redox cycle. But the incorporation of quinones into artificial systems is not practical because of their susceptibility to oxidative degradation and slow transmembrane diffusion7. Here we describe an alternative mechanism for rapid electroneutral charge transport across vesicle membranes: we use pyrylium cations as the electron carrier, which undergo reversible ring-opening hydrolysis to form neutral diketones after deposition of the electron inside the vesicle. As the pyrylium cations are also the primary acceptors for the photoproduced electrons, our approach greatly simplifies the design of vesicle-based photocatalytic devices.

Cyclic transmembrane charge transport by pyrylium ions in a vesicle-based photocatalytic system

Amide-linked spiropyran−anthraquinone (SP−AQ) conjugates were shown to mediate ZnTPPS4--photosensitized transmembrane reduction of occluded Co(bpy)33+ within unilamellar phosphatidylcholine vesicles by external EDTA. Overall quantum yields for these reactions were dependent upon the isomeric state of the dye; specifically, 30−35% photoconversion of the closed-ring spiropyran (SP) moiety to the open-ring merocyanine (MC) form caused the quantum yield to decrease by 6-fold in the simple conjugate and 3-fold for an analogue containing a lipophilic 4-dodecylphenoxy substituent on the anthraquinone moiety. Transient spectroscopic and fluorescence quenching measurements revealed that two factors contributed to these photoisomerization-induced changes in quantum yields:  increased efficiencies of fluorescence quenching of 1ZnTPPS4- by the merocyanine group and lowered transmembrane diffusion rates of the merocyanine-containing redox carriers. Transient spectrophotometry also revealed the sequential formation and...

Rafail F. Khairutdinov

Papers

Photochromism of Spirooxazines in Homogeneous Solution and Phospholipid Liposomes

Hydroxyl radical formation by O-O bond homolysis in peroxynitrous acid.

Permeation of phospholipid membranes by peroxynitrite.

Cyclic transmembrane charge transport by pyrylium ions in a vesicle-based photocatalytic system

Photoregulated Transmembrane Charge Separation by Linked Spiropyran−Anthraquinone Molecules