Aged garlic extract inhibits peroxynitrite-induced hemolysis

Nitric oxide (NO), which is synthesized by constitutive NO synthase (cNOS), plays important roles in physiological functions of the cardiovascular system. However, NO, which is synthesized by inducible NOS, is detrimental when it reacts with superoxide to form peroxynitrite. Peroxynitrite is recognized as a powerful oxidant, and results in vascular or tissue damage. We have previously reported that aged garlic extract (AGE) enhances NO production through cNOS stimulation. In the present study, we determined the effect of AGE, its fractions or constituents on peroxynitrite-induced hemolysis using rat erythrocytes. Incubation of rat erythrocytes with peroxynitrite (300 microM) for 30 min at 37 degrees C caused 4-fold hemolysis. AGE (0.14-0.57 %w/v) added to an erythrocyte suspension was found to reduce peroxynitrite-induced hemolysis in a concentration-dependent manner. Of the AGE fractions, a polar fraction and a low-molecular-weight fraction both suppressed the hemolysis to the same degree as that seen with AGE. S-allylcysteine, one of the major compounds in AGE, also reduced hemolysis at 1-10 mM dose-dependently. These data indicate that AGE and its compounds protect erythrocytes from membrane damage induced by peroxynitrite, suggesting that AGE could be useful for prevention of cardiovascular diseases associated with oxidative stress or dysfunction of NO production.     

A comparative study of the effects of molsidomine and 3-morpholinosydnonimine on the redox status of rat erythrocytes and reticulocytes

After enzymic biotransformation, molsidomine (MO) acts via the metabolite 3-morpholinosydnonimine (SIN-1) through spontaneous liberation of nitric oxide (NO) and superoxide (O(2)(.-)). The aim of this study was to compare the effects of MO and its active metabolite SIN-1 on the redox status of rat erythrocytes and reticulocytes. Rat erythrocyte as well as reticulocyte-rich red blood cell (RBC) suspensions were aerobically incubated (2 h, 37 degrees C) without (control) or in the presence of different concentrations of MO or SIN-1. In rat erythrocytes, biotransformation of MO resulted in the production of NO and nitroxyl (NO(-)). Endogenous superoxide anion (O(2)(.-)) participated in peroxynitrite generation. SIN-1 simultaneously liberated NO and O(2)(.-), which formed peroxynitrite (at least in part), but the liberated NO predominantly reacted with haemoglobin, forming methaemoglobin in erythrocytes. In reticulocytes, MO and SIN-1 caused an increase in the levels of both nitrite and 3-nitrotyrosine (an indicator of peroxynitrite), whereas they decreased the level of O(2)(.-). In reticulocytes, MO was metabolized into SIN-1 which led to the generation of NO, which reacted with O(2)(.-) (endogenous or exogenous) forming reactive nitrogen species. In conclusion, there are two metabolic pathways for MO biotransformation: one causing NO and NO(-) generation predominantly in erythrocytes and the other, via SIN-1 metabolism, in reticulocytes. The main difference between the action of MO and SIN-1 was that the latter caused oxidative damage in RBCs.     

Persistent S-nitrosation of complex I and other mitochondrial membrane proteins by S-nitrosothiols but not nitric oxide or peroxynitrite: implications for the interaction of nitric oxide with mitochondria

S-nitrosation of mitochondrial proteins has been proposed to contribute to the pathophysiological interactions of nitric oxide (NO) and its derivatives with mitochondria but has not been shown directly. Furthermore, little is known about the mechanism of formation or the fate of these putative S-nitrosothiols. Here we have determined whether mitochondrial membrane protein thiols can be S-nitrosated on exposure to free NO from 3,3-bis(aminoethyl)-1-hydroxy-2-oxo-1-triazene (DETA-NONOate) by interaction with S-nitrosoglutathione or S-nitroso-N-acetylpenicillamine (SNAP) and by the NO derivative peroxynitrite. S-Nitrosation of protein thiols was measured directly by chemiluminescence detection. S-Nitrosoglutathione and S-nitroso-N-acetylpenicillamine led to extensive protein thiol oxidation, with about 30% of the modified protein thiols persistently S-nitrosated. In contrast, there was no protein thiol oxidation or S-nitrosation on exposure to 3,3-bis (aminoethyl)-1-hydroxy-2-oxo-1-triazene. Peroxynitrite extensively oxidized protein thiols but produced negligible amounts of S-nitrosothiols. Therefore, mitochondrial membrane protein thiols are S-nitrosated by preformed S-nitrosothiols but not by NO or by peroxynitrite. These S-nitrosated protein thiols were readily reduced by glutathione, so S-nitrosation will only persist when the mitochondrial glutathione pool is oxidized. Respiratory chain complex I was S-nitrosated by S-nitrosothiols, consistent with it being an important target for S-nitrosation during nitrosative stress. The S-nitrosation of complex I correlated with a significant loss of activity that was reversed by thiol reductants. S-Nitrosation was also associated with increased superoxide production from complex I. These findings point to a significant role for complex I S-nitrosation and consequent dysfunction during nitrosative stress in disorders such as Parkinson disease and sepsis.     

Nitric oxide evolution and perception

Various experimental data indicate signalling roles for nitric oxide (NO) in processes such as xylogenesis, programmed cell death, pathogen defence, flowering, stomatal closure, and gravitropism. However, it still remains unclear how NO is synthesized. Nitric oxide synthase-like activity has been measured in various plant extracts, NO can be generated from nitrite via nitrate reductase and other mechanisms of NO generation are also likely to exist. NO removal mechanisms, for example, by reaction with haemoglobins, have also been identified. NO is a gas emitted by plants, with the rate of evolution increasing under conditions such as pathogen challenge or hypoxia. However, exactly how NO evolution relates to its bioactivity in planta remains to be established. NO has both aqueous and lipid solubility, but is relatively reactive and easily oxidized to other nitrogen oxides. It reacts with superoxide to form peroxynitrite, with other cellular components such as transition metals and haem-containing proteins and with thiol groups to form S-nitrosothiols. Thus, diffusion of NO within the plant may be relatively restricted and there might exist 'NO hot-spots' depending on the sites of NO generation and the local biochemical micro-environment. Alternatively, it is possible that NO is transported as chemical precursors such as nitrite or as nitrosothiols that might function as NO reservoirs. Cellular perception of NO may occur through its reaction with biologically active molecules that could function as 'NO-sensors'. These might include either haem-containing proteins such as guanylyl cyclase which generates the second messenger cGMP or other proteins containing exposed reactive thiol groups. Protein S-nitrosylation alters protein conformation, is reversible and thus, is likely to be of biological significance.     

S-Nitrosohemoglobin is unstable in the reductive erythrocyte environment and lacks O2/NO-linked allosteric function

Our previous results run counter to the hypothesis that S-nitrosohemoglobin (SNO-Hb) serves as an in vivo reservoir for NO from which NO release is allosterically linked to oxygen release. We show here that SNO-Hb undergoes reductive decomposition in erythrocytes, whereas it is stable in purified solutions and in erythrocyte lysates treated with an oxidant such as ferricyanide. Using an extensively validated methodology that eliminates background nitrite and stabilizes erythrocyte S-nitrosothiols, we find the levels of SNO-Hb in the basal human circulation, including red cell membrane fractions, were 46 +/- 17 nm in human arterial erythrocytes and 69 +/- 11 nm in venous erythrocytes, incompatible with the postulated reservoir function of SNO-Hb. Moreover, we performed experiments on human red blood cells in which we elevated the levels of SNO-Hb to 10,000 times the normal in vivo levels. The elevated levels of intra-erythrocytic SNO-Hb fell rapidly, independent of oxygen tension and hemoglobin saturation. Most of the NO released during this process was oxidized to nitrate. A fraction (25%) was exported as S-nitrosothiol, but this fraction was not increased at low oxygen tensions that favor the deoxy (T-state) conformation of Hb. Results of these studies show that, within the redox-active erythrocyte environment, the beta-globin cysteine 93 is maintained in a reduced state, necessary for normal oxygen affinity, and incapable of oxygen-linked NO storage and delivery.     

Insulin-induced peroxynitrite production in human platelet-rich plasma

Recent data support the possible role of nitric oxide (NO*) in the development of insulin signalling. The aim of this study was to examine the effect of insulin on NO* production by platelets. The chemiluminescence of platelet-rich plasma prepared from the blood of healthy volunteers was measured in the presence of luminol. Indirect detection of NO* by luminol is possible in the form of peroxynitrite produced in the reaction of NO* with a superoxide free radical. Luminol oxidation induced by hydroxyl free radical and lipid peroxidation was prevented by 150 micromol/l of desferrioxamine mesylate. Insulin, in the range of 0.084-840 nmol/l, induced a concentration-dependent increase in chemiluminescence, which was inhibited both by the competitive antagonist of the NO* synthase enzyme. N(omega)-nitro-L-arginine methyl ester (at concentrations of 2.0-4.0 mmol/l, P<0.001), and by the elimination of superoxide free radicals using superoxide dismutase (72-144 IU/ml, P<0.001). In conclusion, we assume that the insulin-induced increase in chemiluminescence of platelet-rich plasma was due to increased production of NO* and superoxide free radicals forming peroxynitrite. The data are consistent with production of peroxynitrite from human platelets under insulin stimulation.     

Nitric Oxide-Induced [3H]GABA Release from Cerebral Cortical Neurons Is Mediated by Peroxynitrite

Abstract: The functional significance of peroxynitrite in the release of [³H]GABA induced by nitric oxide (NO) liberated from NO generators was investigated using cerebral cortical neurons in primary culture. NO generators such as sodium nitroprusside (SNP) and S -nitroso- N -acetylpenicillamine (SNAP) increased [³H]GABA release in a dose-dependent manner. These increases in [³H]GABA release were significantly inhibited by hemoglobin, indicating that those NO generators evoke the release of [³H]GABA by the formation of NO. Two types of superoxide scavengers, Cu²⁺/Zn²⁺ superoxide dismutase and ceruloplasmin, significantly reduced the increase in [³H]GABA release induced by both SNP and SNAP, which assumes that NO requires superoxide to induce [³H]GABA release from the neurons. In addition, synthesized peroxynitrite induced a dose-dependent increase in [³H]GABA release from the neurons. These results indicate that NO-induced [³H]GABA release is mediated by peroxynitrite formed by the reaction of NO with superoxide.     

Nitric oxide generation by hemocytes of the mussel Mytilus galloprovincialis.

The phagocytic activity of Mytilus galloprovincialis hemocytes is thought to be associated with NADPH-oxidase activity of the plasma membrane, thus producing superoxide anions. Few studies, however, have been devoted to nitric oxide release by these haemocytes. We investigated NO generation in M. galloprovincialis in order to understand its role in the defensive mechanisms of these organisms. The presence of NO-synthase-like enzymatic activity in protein homogenates from M. galloprovincialis hemocytes was revealed by the conversion of radiolabelled L-arginine to L-citrulline. We observed partial inhibition of the luminol-dependent chemiluminescence of stimulated M. galloprovincialis hemocytes by both NO-synthase inhibitors and superoxide dismutase, indicating that peroxynitrite (which results from the reaction between nitric oxide and superoxide anions) partially mediated this chemiluminescence. Furthermore, we confirmed the production of nitric oxide by M. galloprovincialis by highlighting the nitric oxide-synthase-dependence of the nitrate and nitrite production of stimulated hemocytes.     

Nitric oxide and mitochondrial respiration.

Nitric oxide (NO) and its derivative peroxynitrite (ONOO-) inhibit mitochondrial respiration by distinct mechanisms. Low (nanomolar) concentrations of NO specifically inhibit cytochrome oxidase in competition with oxygen, and this inhibition is fully reversible when NO is removed. Higher concentrations of NO can inhibit the other respiratory chain complexes, probably by nitrosylating or oxidising protein thiols and removing iron from the iron-sulphur centres. Peroxynitrite causes irreversible inhibition of mitochondrial respiration and damage to a variety of mitochondrial components via oxidising reactions. Thus peroxynitrite inhibits or damages mitochondrial complexes I, II, IV and V, aconitase, creatine kinase, the mitochondrial membrane, mitochondrial DNA, superoxide dismutase, and induces mitochondrial swelling, depolarisation, calcium release and permeability transition. The NO inhibition of cytochrome oxidase may be involved in the physiological regulation of respiration rate, as indicated by the finding that isolated cells producing NO can regulate cellular respiration by this means, and the finding that inhibition of NO synthase in vivo causes a stimulation of tissue and whole body oxygen consumption. The recent finding that mitochondria may contain a NO synthase and can produce significant amounts of NO to regulate their own respiration also suggests this regulation may be important for physiological regulation of energy metabolism. However, definitive evidence that NO regulation of mitochondrial respiration occurs in vivo is still missing, and interpretation is complicated by the fact that NO appears to affect tissue respiration by cGMP-dependent mechanisms. The NO inhibition of cytochrome oxidase may also be involved in the cytotoxicity of NO, and may cause increased oxygen radical production by mitochondria, which may in turn lead to the generation of peroxynitrite. Mitochondrial damage by peroxynitrite may mediate the cytotoxicity of NO, and may be involved in a variety of pathologies.     

Erythrocytes of humans with cystic fibrosis fail to stimulate nitric oxide synthesis in isolated rabbit lungs

Erythrocytes (red blood cells) of either rabbits or healthy humans are required to demonstrate the participation of nitric oxide (NO) in the regulation of pulmonary vascular resistance in the isolated rabbit lung. The property of the erythrocyte that is responsible for the stimulation of NO synthesis was reported to be the ability to release ATP in response to physiological stimuli, including deformation. Moreover, a signal transduction pathway that relates mechanical deformation of erythrocytes to ATP release has been described, and the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) is a component, i.e., erythrocytes of individuals with CF do not release ATP in response to deformation. Here, we investigated the hypothesis that, in contrast to those of healthy humans, erythrocytes of humans with CF fail to stimulate endogenous NO synthesis in the isolated rabbit lung. We report that CFTR is a component of the membranes of both rabbit and human erythrocytes. The addition of the NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME, 100 muM) produced increases in vascular resistance in isolated rabbit lungs perfused with physiological salt solution (PSS) containing erythrocytes of healthy humans, but L-NAME was without effect when the lungs were perfused with PSS alone or PSS containing erythrocytes of CF patients. These results provide support for the hypothesis that, in CF, a defect in ATP release from erythrocytes could lead to decreased endogenous pulmonary NO synthesis and contribute to pulmonary hypertension.     

Human erythrocyte membranes contain a cytochrome b561 that may be involved in extracellular ascorbate recycling

Human erythrocytes contain an unidentified plasma membrane redox system that can reduce extracellular monodehydroascorbate by using intracellular ascorbate (Asc) as an electron donor. Here we show that human erythrocyte membranes contain a cytochrome b(561) (Cyt b(561)) and hypothesize that it may be responsible for this activity. Of three evolutionarily closely related Cyts b(561), immunoblots of human erythrocyte membranes showed only the duodenal cytochrome b(561) (DCytb) isoform. DCytb was also found in guinea pig erythrocyte membranes but not in erythrocyte membranes from the mouse or rat. Mouse erythrocytes lost a majority of the DCytb in the late erythroblast stage during erythropoiesis. Absorption spectroscopy showed that human erythrocyte membranes contain an Asc-reducible b-type Cyt having the same spectral characteristics as recombinant DCytb and biphasic reduction kinetics, similar to those of the chromaffin granule Cyt b(561). In contrast, mouse erythrocytes did not exhibit Asc-reducible b-type Cyt activity. Furthermore, in contrast to mouse erythrocytes, human erythrocytes much more effectively preserved extracellular Asc and transferred electrons from intracellular Asc to extracellular ferricyanide. These results suggest that the DCytb present in human erythrocytes may contribute to their ability to reduce extracellular monodehydroascorbate.     

Peroxynitrite affects exocytosis and SNARE complex formation and induces tyrosine nitration of synaptic proteins

The reactive species peroxynitrite, formed via the near diffusion-limited reaction of nitric oxide and superoxide anion, is a potent oxidant that contributes to tissue damage in neurodegenerative disorders. Peroxynitrite readily nitrates tyrosine residues in proteins, producing a permanent modification that can be immunologically detected. We have previously demonstrated that in the nerve terminal, nitrotyrosine immunoreactivity is primarily associated with synaptophysin. Here we identify two other presynaptic proteins nitrated by peroxynitrite, Munc-18 and SNAP25, both of which are involved in sequential steps leading to vesicle exocytosis. To investigate whether peroxynitrite affects vesicle exocytosis, we used the fluorescent dye FM1-43 to label a recycling population of secretory vesicles within the synaptosomes. Bolus addition of peroxynitrite stimulated exocytosis and glutamate release. Notably, these effects were strongly reduced in the presence of NaHCO(3), indicating that peroxynitrite acts mainly intracellularly. Furthermore, peroxynitrite enhanced the formation of the sodium dodecyl sulfate-resistant SNARE complex in a dose-dependent manner (100-1000 microm) and induced the formation of 3-nitrotyrosine in proteins of SNARE complex. These data suggest that modification(s) of synaptic vesicle proteins induced by peroxynitrite may affect protein-protein interactions in the docking/fusion steps, thus promoting exocytosis, and that, under excessive production of superoxide and nitric oxide, neurons may up-regulate neuronal signaling.     

Androgen deprivation increases neuronal nitric oxide metabolism and its vasodilator effect in rat mesenteric arteries.

This study examines the effects of male sex hormones on the vasoconstrictor response to electrical field stimulation (EFS), as well as neuronal NO modulation of this response. For this purpose, denuded superior mesenteric artery from orchidectomized and control male Sprague-Dawley rats was used. EFS induced similar frequency-dependent contractions in segments from both groups. The NO synthase (NOS) inhibitor N(omega)-nitro-L-arginine methyl ester strengthened EFS-elicited contractions more in arteries from orchidectomized than from control male rats. The expression of nNOS was more pronounced in segments from control than from orchidectomized animals. Basal and EFS-induced NO release was similar in segments from both groups. In noradrenaline (NA)-precontracted segments, sodium nitroprusside (SNP) induced a concentration-dependent relaxation, that was greater in segments from orchidectomized than control male rats. 8-Bromo-cGMP induced a similar concentration-dependent relaxation in NA-precontracted segments from either group, and the cGMP levels induced by SNP were also similar in the two groups. Superoxide dismutase (SOD), a superoxide anion scavenger, did not modify the relaxation in segments from control male rats. In contrast, SOD enhanced the relaxation induced by SNP in segments from orchidectomized rats, and the effect was reversed by preincubation with SOD plus catalase. The generation of superoxide anion and of peroxynitrite was greater in segments from orchidectomized than control rats. In NA-precontracted segments from control or orchidectomized rats, exogenous peroxynitrite and H(2)O(2) induced a concentration-dependent relaxation. These results suggest that EFS induces a similar nNOS-derived NO release in segments from orchidectomized and control male rats, despite the decrease in nNOS expression in orchidectomized rats. The NO metabolism is higher in segments from orchidectomized male rats due to the increases in anion superoxide generation and peroxynitrite formation. The vasodilator effects of the peroxynitrite and H(2)O(2)0 generated from the NO metabolism are what enhance the functional role of the nNOS-derived NO release in the orchidectomized rats.     

Can erythrocytes release biologically active NO?

Under physiological conditions, endothelial cells and the endothelial nitric oxide (NO) synthase (eNOS) are the main source of NO in the cardiovascular system. However, several other cell types have also been implicated in the NO-dependent regulation of cell function, including erythrocytes. NO derived from red blood cells has been proposed to regulate erythrocyte membrane fluidity, inhibit platelet activation and induce vasodilation in hypoxic areas, but these proposals are highly controversial. In the current issue of Cell Communication and Signaling, an elegant study by Gambaryan et al., assayed NO production by erythrocytes by monitoring the activation of the platelet intracellular NO receptor, soluble guanylyl cyclase, and its downstream kinase protein kinase G. After systematically testing different combinations of erythrocyte/platelet suspensions, the authors found no evidence for platelet soluble guanylyl cyclase/protein kinase G activation by erythrocytes and conclude that erythrocytes do not release biologically active NO to inhibit platelet activation.     

Protection of primary glial cells by grape seed proanthocyanidin extract against nitrosative/oxidative stress.

Previous studies showed that proanthocyanidins provide potent protection against oxidative stress. Here we investigate the effects of grape seed proanthocyanidin extract (GSPE) as a novel natural antioxidant on the generation and fate of nitric oxide (NO) in rat primary glial cell cultures. GSPE treatment (50 mg/L) increased NO production (measured by NO(2-) assay) by stimulation of the inducible isoform of NOS. However, GSPE failed to affect the LPS/IFN-gamma-induced NO production or iNOS expression. Similar responses were found in the murine macrophage cell line RAW264.7. GSPE did not show any effect on dihydrodichlorofluorescein fluorescence (ROS marker with high sensitivity toward peroxynitrite) either in control or in LPS/IFN-gamma-induced glial cultures even in the presence of a superoxide generator (PMA). GSPE treatment alone had no effect on the basal glutathione (GSH) status in glial cultures. Whereas the microglial GSH level declined sharply after LPS/IFN-gamma treatment, the endogenous GSH pool was protected when such cultures were treated additionally with GSPE, although NO levels did not change. Glial cultures pretreated with GSPE showed higher tolerance toward application of hydrogen peroxide (H(2)O(2)) and tert-butylhydroperoxide. Furthermore, GSPE-pretreated glial cultures showed improved viability after H(2)O(2)-induced oxidative stress demonstrated by reduction in lactate dehydrogenase release or propidium iodide staining. We showed that, in addition to its antioxidative property, GSPE enhances low-level production of intracellular NO in primary rat astroglial cultures. Furthermore, GSPE pretreatment protects the microglial GSH pool during high output NO production and results in an elevation of the H(2)O(2) tolerance in astroglial cells.     

Reversal of nitric oxide-, peroxynitrite- and S-nitrosothiol-induced inhibition of mitochondrial respiration or complex I activity by light and thiols

Nitric oxide (NO) and its derivatives peroxynitrite and S-nitrosothiols inhibit mitochondrial respiration by various means, but the mechanisms and/or the reversibility of such inhibitions are not clear. We find that the NO-induced inhibition of respiration in isolated mitochondria due to inhibition of cytochrome oxidase is acutely reversible by light. Light also acutely reversed the inhibition of respiration within iNOS-expressing macrophages, and this reversal was partly due to light-induced breakdown of NO, and partly due to reversal of the NO-induced inhibition of cytochrome oxidase. NO did not cause inhibition of complex I activity within isolated mitochondria, but 0.34 mM peroxynitrite, 1 mM S-nitroso-N-acetylpenicillamine or 1 mM S-nitrosoglutathione did cause substantial inhibition of complex I activity. Inhibition by these reagents was reversed by light, dithiothreitol or glutathione-ethyl ester, either partially or completely, depending on the reagent used. The rapid inhibition of complex I activity by S-nitroso-N-acetylpenicillamine also occurred in conditions where there was little or no release of free NO, suggesting that the inhibition was due to transnitrosylation of the complex. These findings have implications for the physiological and pathological regulation of respiration by NO and its derivatives.     

Interactions of peroxynitrite,tetrahydrobiopterin, ascorbic acid,and thiols: implications for uncoupling endothelial nitric-oxide synthase

Tetrahydrobiopterin (BH4) serves as a critical co-factor for the endothelial nitric-oxide synthase (eNOS). A deficiency of BH4 results in eNOS uncoupling, which is associated with increased superoxide and decreased NO* production. BH4 has been suggested to be a target for oxidation by peroxynitrite (ONOO-), and ascorbate has been shown to preserve BH4 levels and enhance endothelial NO* production; however, the mechanisms underlying these processes remain poorly defined. To gain further insight into these interactions, the reaction of ONOO- with BH4 was studied using electron spin resonance and the spin probe 1-hydroxy-3-carboxy-2,2,5-tetramethyl-pyrrolidine. ONOO- reacted with BH4 6-10 times faster than with ascorbate or thiols. The immediate product of the reaction between ONOO- and BH4 was the trihydrobiopterin radical (BH3.), which was reduced back to BH4 by ascorbate, whereas thiols were not efficient in recycling of BH4. Uncoupling of eNOS caused by peroxynitrite was investigated in cultured bovine aortic endothelial cells (BAECs) by measuring superoxide and NO* using spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine and the NO*-spin trap iron-diethyldithiocarbamate. Bolus ONOO-, the ONOO- donor 3-morpholinosydnonimine, and an inhibitor of BH4 synthesis (2,4-diamino-6-hydroxypyrimidine) uncoupled eNOS, increasing superoxide and decreasing NO* production. Exogenous BH4 supplementation restored endothelial NO* production. Treatment of BAECs with both BH4 and ascorbate prior to ONOO- prevented uncoupling of eNOS by ONOO-. This study demonstrates that endothelial BH4 is a crucial target for oxidation by ONOO- and that the BH4 reaction rate constant exceeds those of thiols or ascorbate. We confirmed that ONOO- uncouples eNOS by oxidation of tetrahydrobiopterin and that ascorbate does not fully protect BH4 from oxidation but recycles BH3. radical back to BH4.     

Inhibition of mitochondrial respiratory complex I by nitric oxide, peroxynitrite and S-nitrosothiols

NO or its derivatives (reactive nitrogen species, RNS) inhibit mitochondrial complex I by several different mechanisms that are not well characterised. There is an inactivation by NO, peroxynitrite and S-nitrosothiols that is reversible by light or reduced thiols, and therefore may be due to S-nitrosation or Fe-nitrosylation of the complex. There is also an irreversible inhibition by peroxynitrite, other oxidants and high levels of NO, which may be due to tyrosine nitration, oxidation of residues or damage of iron sulfur centres. Inactivation of complex I by NO or RNS is seen in cells or tissues expressing iNOS, and may be relevant to inflammatory pathologies, such as septic shock and Parkinson's disease.     

Red blood cells in the metabolism of nitric oxide-derived peroxynitrite

In this review we have analyzed the reactions of nitric oxide (.NO) with superoxide radical (O(2).-) at the vascular compartment which results in limitation of the bioavailability of .NO and the formation of peroxynitrite (ONOO-), a strong oxidant species. The intravascular formation of peroxynitrite can result in oxidative modifications of plasma and vessel wall proteins including the formation of protein-3-nitrotyrosine. The role of red blood cells (RBC) and oxyhemoglobin in the metabolism of intravascular peroxynitrite will be discussed. While RBC constitute an important 'sink' of both .NO and peroxynitrite, redox reactions of these species with oxyhemoglobin may in part contribute to erythrocyte aging. The intravascular formation, reactions and detoxification of peroxynitrite are revealed as important factors controlling vascular dysfunction and degeneration in a variety of pathophysiologically-relevant conditions.     

Protein tyrosine nitration in cytokine-activated murine macrophages. Involvement of a peroxidase/nitrite pathway rather than peroxynitrite.

Peroxynitrite, formed in a rapid reaction of nitric oxide (NO) and superoxide anion radical (O(2)), is thought to mediate protein tyrosine nitration in various inflammatory and infectious diseases. However, a recent in vitro study indicated that peroxynitrite exhibits poor nitrating efficiency at biologically relevant steady-state concentrations (Pfeiffer, S., Schmidt, K., and Mayer, B. (2000) J. Biol. Chem. 275, 6346-6352). To investigate the molecular mechanism of protein tyrosine nitration in intact cells, murine RAW 264.7 macrophages were activated with immunological stimuli, causing inducible NO synthase expression (interferon-gamma in combination with either lipopolysaccharide or zymosan A), followed by the determination of protein-bound 3-nitrotyrosine levels and release of potential triggers of nitration (NO, O(2)*, H(2)O(2), peroxynitrite, and nitrite). Levels of 3-nitrotyrosine started to increase at 16-18 h and exhibited a maximum at 20-24 h post-stimulation. Formation of O(2) was maximal at 1-5 h and decreased to base line 5 h after stimulation. Release of NO peaked at approximately 6 and approximately 9 h after stimulation with interferon-gamma/lipopolysaccharide and interferon-gamma/zymosan A, respectively, followed by a rapid decline to base line within the next 4 h. NO formation resulted in accumulation of nitrite, which leveled off at about 50 microm 15 h post-stimulation. Significant release of peroxynitrite was detectable only upon treatment of cytokine-activated cells with phorbol 12-myristate-13-acetate, which led to a 2.2-fold increase in dihydrorhodamine oxidation without significantly increasing the levels of 3-nitrotyrosine. Tyrosine nitration was inhibited by azide and catalase and mimicked by incubation of unstimulated cells with nitrite. Together with the striking discrepancy in the time course of NO/O(2) release versus 3-nitrotyrosine formation, these results suggest that protein tyrosine nitration in activated macrophages is caused by a nitrite-dependent peroxidase reaction rather than peroxynitrite.