The oxidation of cytochrome-c oxidase vesicles by hemoglobin.

Human hemoglobin has been used as a pro-oxidant for artificial unilamellar phospholipid vesicles, containing cytochrome-c oxidase inserted into the bilayer. This experimental system was suitable to follow directly the kinetics of lipid oxidation and the effects on both the vesicle membrane permeability and the functional state of cytochrome-c oxidase. Following mixing of vesicles with hemoglobin, an oxygen dependent, peroxyl radical mediated, rapid oxidation (taking a few minutes) of the lipid was found to occur. On a similar time scale the membrane became ion-leaky and cytochrome-c oxidase damaged. The pro-oxidant effects of hemoglobin in various oxidation and ligation states were studied and a mechanism, based on a ferric/ferryl redox cycle of the heme-iron is proposed to account for these observations.

Biological aspects of reactive nitrogen species.

Nitric oxide (NO) plays an important role as a cell-signalling molecule, anti-infective agent and, as most recently recognised, an antioxidant. The metabolic fate of NO gives rise to a further series of compounds, collectively known as the reactive nitrogen species (RNS), which possess their own unique characteristics. In this review we discuss this emerging aspect of the NO field in the context of the formation of the RNS and what is known about their effects on biological systems. While much of the insight into the RNS has been gained from the extensive chemical characterisation of these species, to reveal biological consequences this approach must be complemented by direct measures of physiological function. Although we do not know the consequences of many of the dominant chemical reactions of RNS an intriguing aspect is now emerging. This review will illustrate how, when specificity and amplification through cell signalling mechanisms are taken into account, the less significant reactions, in terms of yield or rates, can explain many of the biological responses of exposure of cells or physiological systems to RNS.

Regulation of endothelial glutathione by ICAM-1: implications for inflammation.

The role of glutathione (GSH) in inflammation is largely discussed from the context of providing reducing equivalents to detoxify reactive oxygen and nitrogen species. Inflammation is now recognized to be an underlying cause of many vascular diseases including atherosclerosis, a disease in which endothelial GSH concentrations are decreased. However, mechanisms that control GSH levels are poorly understood. Key players in the inflammatory process are endothelial adhesion molecules, including intercellular adhesion molecule-1 (ICAM-1). This adhesion molecule is present constitutively and can be induced by a variety of inflammatory stimuli. In this study, using mouse aortic endothelial cells (MAEC) deficient in ICAM-1, we demonstrate a novel interplay between constitutive ICAM-1 and cellular GSH. Deficiency of ICAM-1 was associated with an approximately twofold increase in total GSH content. Inhibiting glutamate-cysteine ligase (GCL), the enzyme that catalyses the rate-limiting step in GSH biosynthesis, prevented the increase in GSH. In addition, the catalytic subunit of GCL was increased (approximately 1.6-fold) in ICAM-1 deficient relative to wild-type cells, suggesting that constitutive ICAM-1 represses GCL expression. Furthermore, the ratio of reduced (GSH) to oxidized (GSSG) glutathione was also increased suggesting a role for ICAM-1 in modulating cellular redox status. Interestingly, increasing cytosolic GSH in wild-type mouse endothelial cells decreased constitutive ICAM-1, suggesting the presence of an inverse and reciprocal pathway. To test the effects of inducible ICAM-1 on GSH, cells were stimulated with the proinflammatory cytokine TNF-alpha. TNF-alpha stimulated production of ICAM-1, which was however not associated with induction of GSH. In contrast, supplementation of endothelial cells with GSH before TNF-alpha addition, inhibited induction of ICAM-1. These data suggest a novel regulatory pathway between constitutive ICAM-1 and GSH synthesis in the endothelium and are discussed in the context of modulating the inflammatory response.

Biphasic effects of 15-deoxy-delta(12,14)-prostaglandin J(2) on glutathione induction and apoptosis in human endothelial cells.

The lipid products derived from the cyclooxygenase pathway can have diverse and often contrasting effects on vascular cell function. Cyclopentenone prostaglandins (cyPGs), such as 15-deoxy-Delta(12,14)-prostaglandin-J(2) (15d-PGJ(2)), a peroxisome proliferator-activated receptor-gamma (PPARgamma) agonist, have been reported to cause endothelial cell apoptosis, yet in other cell types, cyPGs induce cytoprotective mediators, such as heat shock proteins, heme oxygenase-1, and glutathione (GSH). Herein, we show in human endothelial cells that low micromolar concentrations of 15d-PGJ(2) enhance GSH-dependent cytoprotection through the upregulation of glutamate-cysteine ligase, the rate-limiting enzyme of GSH synthesis, as well as GSH reductase. The effect of 15d-PGJ(2) on GSH synthesis is attributable to the cyPG structure but is independent of PPAR, inasmuch as the other cyPGs, but not PPARgamma or PPARalpha agonists, are able to increase GSH. The increase in cellular GSH is accompanied by abrogation of the proapoptotic effects of 4-hydroxynonenal, a product of lipid peroxidation present in atherosclerotic lesions. However, higher concentrations of 15d-PGJ(2) (10 micromol/L) cause endothelial cell apoptosis, which is further enhanced by depletion of cellular GSH by buthionine sulfoximine. We propose that the GSH-dependent cytoprotective pathways induced by 15d-PGJ(2) contribute to its antiatherogenic effects and that these pathways are distinct from those leading to apoptosis.

Mechanisms of the pro- and anti-oxidant actions of nitric oxide in atherosclerosis.

The association of nitric oxide (NO) with cardiovascular disease has long been recognized and the extensive research on this topic has revealed both pro- and anti-atherosclerotic effects. While these contradictory findings were initially perplexing recent studies offer molecular mechanisms for the integration of these data in the context of our current understanding of the biochemistry of NO. The essential findings are that the biochemical properties of NO allow its exploitation as both a cell signaling molecule, through its interaction with redox centers in heme proteins, and an extremely rapid reaction with other biologically relevant free radicals. The direct reaction of NO with free radicals can have either pro- or antioxidant effects. In the cell, antioxidant properties of NO can be greatly amplified by the activation of signal transduction pathways that lead to the increased synthesis of endogenous antioxidants or down regulate responses to pro-inflammatory stimuli. These findings will be discussed in the context of atherosclerosis.

Reaction of thionitrobenzoate-modified yeast cytochrome c with monomeric and dimeric forms of beef heart cytochrome c oxidase.

Thionitrobenzoate-modified yeast cytochrome c was shown to react with both monomeric and dimeric forms of beef heart cytochrome c oxidase through subunit III. This cytochrome c derivative was found to inhibit electron transfer in the dimer but not in the monomer. These results are interpreted to show that the high affinity binding site for cytochrome c is a cleft at the interface between monomers in the cytochrome c oxidase dimer.

Alpha-tocopherol mediated peroxidation in the copper (II) and met myoglobin induced oxidation of human low density lipoprotein: the influence of lipid hydroperoxides.

The principal antioxidant in human LDL, alpha-tocopherol, is converted to the alpha-tocopheroxyl radical after reaction with peroxyl radicals or Cu2+, and, if it does not terminate with peroxyl radicals, could initiate lipid peroxidation; a phenomenon called 'tocopherol mediated peroxidation'. Only in the presence of Cu2+ and low levels of lipid hydroperoxides was an alpha-tocopherol dependent decrease in the resistance of LDL to oxidation detected. This suggests that tocopherol mediated peroxidation will probably not contribute significantly as a pro-oxidant process in those individuals most at risk of developing atherosclerosis through an oxidative mechanism.

The oxidation of alpha-tocopherol in human low-density lipoprotein by the simultaneous generation of superoxide and nitric oxide.

Peroxynitrite is the product of the reaction between nitric oxide and superoxide. It is an oxidant which can also decompose to form the hydroxyl radical and nitrogen dioxide. In this report we show that a powerful oxidant with reactivity similar to that of the hydroxyl radical is formed from the generation of superoxide from xanthine oxidase and nitric oxide from S-nitroso-n-acetylpenicillamine (SNAP). Simultaneous generation of these two radicals by either xanthine oxidase/SNAP or the sydnonimine SIN-1 in the presence of low-density lipoprotein (LDL) results in the depletion of alpha-tocopherol and formation of its oxidised product alpha-tocopheroquinone. The mechanism of oxidation required both the formation of nitric oxide and superoxide. In contrast to the promotion of LDL oxidation by transition metals the oxidation of LDL by SIN-1 was not sensitive to the addition of exogenous lipid hydroperoxide.

Effects of pyrrolidine dithiocarbamate on endothelial cells: protection against oxidative stress.

The dithiocarbamates are well known for their antioxidant properties and effects on cellular transcriptional events. For example, pyrrolidine dithiocarbamate (PDTC) is widely used as an inhibitor of nuclear factor kappa B (NFkappaB) and this, or related compounds may have therapeutic potential in inhibiting atherosclerosis. However, the precise molecular mechanisms through which PDTC could elicit antioxidant or cell signaling effects in a cellular setting remain unclear. Furthermore, the mechanisms for the effects of PDTC on NFkappaB are likely to involve inhibition of binding of the transcription factor to DNA rather than an effect on the activation process as first proposed. In relation to pharmacological applications of such compounds, little is known of their interaction with endothelial cells, the anticipated site of action for inhibition of vascular related diseases. Until recently, PDTC was generally classified as an antioxidant but evidence for pro-oxidant effects have been reported. In this study, we have addressed this issue in bovine aortic endothelial cells and identified two mechanisms through which PDTC can exert antioxidant effects. At low concentrations (0-25 microM), PDTC induces a concentration dependent increase in cellular GSH levels through the increased activity of gamma-glutamylcysteine synthetase. At higher concentrations, GSH oxidation and apoptotic cell death occur. Using 2,3 dimethoxy-1,4-napthoquinone (DMNQ) as an intracellular generator of superoxide radicals, we find PDTC (10 microM) protects against the cytotoxicity of this agent through a GSH-independent mechanism.

Oxidation of human low-density lipoprotein by soybean 15-lipoxygenase in combination with copper (II) or met-myoglobin.

The enzyme 15-lipoxygenase has been implicated in the oxidation of low-density lipoprotein (LDL) in human atherosclerotic lesions. The biochemical mechanism for this oxidative process is not fully understood, and the interaction of the lipoxygenase-modified lipoprotein with metals or metalloproteins has not been explored. In the present study we have used soybean lipoxygenase to model the interaction of the enzyme with LDL and show that a direct oxygenation of fatty acids occurs, including those esterified to cholesterol, with no lag phase or change in electrophoretic mobility of the LDL particle but with some depletion of alpha-tocopherol. The enzyme-dependent oxidation may involve propagation through the release of peroxyl radicals from its active site but appears to have no requirement for free iron or copper. When lipoxygenase-treated LDL is exposed to either copper (II) or metMb, a rapid oxidation process occurs, resulting in a marked decrease in resistance to oxidation and an increase in the rate of modification to a form with increased electrophoretic mobility. This effect was not seen if lipoxygenase-treated LDL was oxidized by SIN-1, a peroxynitrite donor that oxidizes LDL with no requirement for endogenous lipid hydroperoxides. We propose that a synergistic interaction may occur between the peroxides inserted into LDL as a consequence of the enzymatic action of lipoxygenase with haem proteins or copper, which decreases the potency of the endogenous antioxidants and enhances oxidation.

Reversible inhibition of cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain, by nitric oxide. Implications for neurodegenerative diseases.

Incubation of rat skeletal muscle mitochondria with the nitric oxide generator, S-nitrosoglutathione (GSNO) reversibly inhibited oxygen utilisation with all substrates tested. The visible absorption spectra of the inhibited mitochondria showed that cytochromes c+c1, b and a+a3 were reduced, indicating a block at the distal end of the respiratory chain. Analysis of the respiratory chain enzyme activities in the presence of GSNO localised the site of inhibition of cytochrome c oxidase alone. These results indicate that nitric oxide is capable of rapidly and reversibly inhibiting the mitochondrial respiratory chain and may be implicated in the cytotoxic effects of nitric oxide in the CNS and other tissues.

Cell signaling by reactive nitrogen and oxygen species in atherosclerosis.

The production of reactive oxygen and nitrogen species has been implicated in atherosclerosis principally as means of damaging low-density lipoprotein that in turn initiates the accumulation of cholesterol in macrophages. The diversity of novel oxidative modifications to lipids and proteins recently identified in atherosclerotic lesions has revealed surprising complexity in the mechanisms of oxidative damage and their potential role in atherosclerosis. Oxidative or nitrosative stress does not completely consume intracellular antioxidants leading to cell death as previously thought. Rather, oxidative and nitrosative stress have a more subtle impact on the atherogenic process by modulating intracellular signaling pathways in vascular tissues to affect inflammatory cell adhesion, migration, proliferation, and differentiation. Furthermore, cellular responses can affect the production of nitric oxide, which in turn can strongly influence the nature of oxidative modifications occurring in atherosclerosis. The dynamic interactions between endogenous low concentrations of oxidants or reactive nitrogen species with intracellular signaling pathways may have a general role in processes affecting wound healing to apoptosis, which can provide novel insights into the pathogenesis of atherosclerosis.

The induction of GSH synthesis by nanomolar concentrations of NO in endothelial cells: a role for gamma-glutamylcysteine synthetase and gamma-glutamyl transpeptidase.

Nitric oxide protects cells from oxidative stress through a number of direct scavenging reactions with free radicals but the effects of nitric oxide on the regulation of antioxidant enzymes are only now emerging. Using bovine aortic endothelial cells as a model, we show that nitric oxide, at physiological rates of production (1-3 nM/s), is capable of inducing the synthesis of glutathione through a mechanism involving gamma-glutamylcysteine synthetase and gamma-glutamyl transpeptidase. This novel nitric oxide signalling pathway is cGMP-independent and we hypothesize that it makes an important contribution to the anti-atherosclerotic and antioxidant properties of nitric oxide.

Mechanisms of cell signaling by nitric oxide and peroxynitrite: from mitochondria to MAP kinases.

Many of the biological and pathological effects of nitric oxide (NO) are mediated through cell signaling pathways that are initiated by NO reacting with metalloproteins. More recently, it has been recognized that the reaction of NO with free radicals such as superoxide and the lipid peroxyl radical also has the potential to modulate redox signaling. Although it is clear that NO can exert both cytotoxic and cytoprotective actions, the focus of this overview are those reactions that could lead to protection of the cell against oxidative stress in the vasculature. This will include the induction of antioxidant defenses such as glutathione, activation of mitogen-activated protein kinases in response to blood flow, and modulation of mitochondrial function and its impact on apoptosis. Models are presented that show the increased synthesis of glutathione in response to shear stress and inhibition of cytochrome c release from mitochondria. It appears that in the vasculature NO-dependent signaling pathways are of three types: (i) those involving NO itself, leading to modulation of mitochondrial respiration and soluble guanylate cyclase; (ii) those that involve S-nitrosation, including inhibition of caspases; and (iii) autocrine signaling that involves the intracellular formation of peroxynitrite and the activation of the mitogen-activated protein kinases. Taken together, NO plays a major role in the modulation of redox cell signaling through a number of distinct pathways in a cellular setting.

Nitration of unsaturated fatty acids by nitric oxide-derived reactive species.

Reactions of linoleate (and presumably other unsaturated fatty acids) with reactive nitrogen species that form in biological systems from secondary reactions of .NO yield two main nitration product groups, LNO2 (formed by ONOO-, .NO2, or NO2+ reaction with linoleate), and LONO2 (formed by HONO reaction with 13(S)-HPODE, or .NO termination with LOO.). Comparison of HPLC retention times and m/z for lipid nitration products indicate that the mechanisms of nitrated product formation converge at several points: (i) The initial product of HONO attack on LOOH will be LOONO, which is identical to the initial termination product of LOO. reaction with .NO. (ii) Dissociation of LOONO to give LO. and .NO2 via caged radicals, which recombine to give LONO2 (m/z 340) will occur, regardless of how LOONO is formed (Fig. 7). (iii) In some experiments, the reaction of O2- (where oxidation is initiated by xanthine oxidase-derived O2- production and metal-dependent decomposition of H2O2) with .NO will result in generation of ONOO-. Nitration of unsaturated lipid by this species will yield a species demonstrated herein to be LNO2. Lipid oxidation leads to formation of bioactive products, including hydroxides, hydroperoxides, and isoprostanes. In vivo, nitrated lipids (LNO2, LONO2) may also possess bioactivity, for example through eicosanoid receptor binding activity, or by acting as antagonists/competitive inhibitors of eicosanoid receptor-ligand interactions. In addition, nitrated lipids could mediate signal transduction via direct .NO donation, transnitrosation, or following reductive metabolism. Similar bioactive products are formed following ONOO- reaction with glucose, glycerol, and other biomolecules.

The formation of nitric oxide donors from peroxynitrite.

1. Administration of peroxynitrite (ONOO-, 30-300 microM) caused relaxation of rabbit aortic strips superfused in series in a cascade. The compound responsible for this effect had a half-life greater than 20 s and could not therefore be either nitric oxide (NO) or ONOO- which have half-lives in the order of 1-2 s under these conditions. However the relaxation was inhibited by oxyhaemoglobin, suggesting the compound could be converted to NO in the vascular tissues or in the superfusate. 2. The products of the reactions between ONOO- and Krebs buffer containing 11 mM glucose, but not glucose-free Krebs buffer, caused relaxation of the bioassay tissues. These data suggest that stable NO donor(s) were formed from the reaction of ONOO- with glucose. We therefore prepared these NO donor(s) by the reaction of glucose solutions with ONOO- in order to characterize their ability to release NO. 3. These reaction product(s) caused relaxation in the cascade and inhibition of platelet aggregation. Both effects were dependent on the concentration of D-glucose, were equally effective if L-glucose was used as a reactant and were reversed by oxyhaemoglobin. 3. The products of the reaction between ONOO- and glucose or other biological molecules containing an alcohol functional group, such as fructose, glycerol, or glyceraldehyde, released NO in the presence of Cu2+and L-cysteine. 5. These results indicate that ONOO- reacts with sugars or other compounds containing an alcohol functional group(s) to form NO donors with the characteristics of organic nitrate/nitrites. This may represent a further detoxification pathway for ONOO- in vivo.

A method for the comparative assessment of antioxidants as peroxyl radical scavengers.

Antioxidants which are peroxyl radical scavengers have been compared in a model of lipid peroxidation based on the oxidation of a suspension of linoleic acid initiated by a thermolabile azo compound. By analysing the effect of antioxidant concentration on linoleic acid peroxidation we have defined the constant kAH which characterises the rate of the reaction of the antioxidant with the peroxyl radical. This allows quantitative comparison of the efficiency of different antioxidants as peroxyl radical scavengers. By using an initiation system which is not iron dependent we were able to show that the iron chelators desferrioxamine, BW A4C and U74500A are also peroxyl radical scavengers.

The role of alpha-tocopherol as a peroxyl radical scavenger in human low density lipoprotein.

It is thought that the oxidation of low density lipoprotein (LDL) plays a key role in the pathogenesis of atherosclerosis. It is well known that lipid peroxidation reactions are propagated by peroxyl radicals and it follows, therefore, that the capacity of an individual LDL particle to scavenge these oxidants may be an important indicator of its atherogenic potential. There are several components within LDL which scavenge peroxyl radicals including chain breaking antioxidants and amino acids on the protein. It is not clear at present which of these antioxidants is most important. In attempting to address the question we have used a simple method for the measurement of the total capacity of the LDL particle to scavenge peroxyl radicals. This assay depends upon the ability of antioxidants in LDL to inhibit the peroxyl radical dependent oxidation of luminol. We have found that approximately 80% of the antioxidant capacity of LDL, isolated from a number of donors, could be accounted for by the alpha-tocopherol present in the samples. We have compared these results with those obtained when the identical samples of LDL were oxidized with copper and found, as reported by others, a wide range in the susceptibility of the different LDL preparations to oxidation by this transition metal. We suggest that this variability is unlikely to be due to differences in the ability of an LDL particle to scavenge peroxyl radicals.

Analyses of muscle proteins in a patient with a mitochondrial myopathy.

Using the small amounts of muscle available from biopsy (approximately 100 mg), from both normal controls and a patient with a previously identified defect of the mitochondrial electron transfer protein complex III, we analyzed both structural and mitochondrial proteins. The myosin light chains were found to be unchanged with respect to charge or size between patient and control. Two prominent proteins detected after two dimensional gel electrophoresis were present in the patient's total homogenised muscle protein but were not detected in the controls. One protein was positively identified as cytochrome c oxidase subunit II and the other tentatively as a component of the ATP synthetase. We suggest that the increased amounts of these proteins represents a response of the patients muscle cells to the ATP deficiency caused by the primary lesion in complex III.

Molecular mechanisms of the copper dependent oxidation of low-density lipoprotein.

There is little doubt that oxidative modification of low-density lipoprotein (LDL) is an important process during atherogenesis. This conclusion has been derived in a relatively short period of time since the initial descriptions of LDL oxidation with a significant contribution from Professor Esterbauer and colleagues. In this short overview, we have described the mechanisms by which copper promotes LDL oxidation focussing on the importance of lipid hydroperoxides in this process. These mechanisms are discussed in the context of the ongoing debate as to relevance of metal dependent LDL oxidation in vivo and as a model reaction for assessing antioxidants.