Scavenging of peroxynitrite by oxyhemoglobin and identification of modified globin residues

Peroxynitrite is a strong oxidant involved in cell injury. In tissues, most of peroxynitrite reacts preferentially with CO(2) or hemoproteins, and these reactions affect its fate and toxicity. CO(2) promotes tyrosine nitration but reduces the lifetime of peroxynitrite, preventing, at least in part, membrane crossing. The role of hemoproteins is not easily predictable, because the heme intercepts peroxynitrite, but its oxidation to ferryl species and tyrosyl radical(s) may catalyze tyrosine nitration. The modifications induced by peroxynitrite/CO(2) on oxyhemoglobin were determined by mass spectrometry, and we found that alphaTyr42, betaTyr130, and, to a lesser extent, alphaTyr24 were nitrated. The suggested nitration mechanism is tyrosyl radical formation by long-range electron transfer to ferrylhemoglobin followed by a reaction with (*)NO(2). Dityrosine (alpha24-alpha42) and disulfides (beta93-beta93 and alpha104-alpha104) were also detected, but these cross-linkings were largely due to modifications occurring under the denaturing conditions employed for mass spectrometry. Moreover, immunoelectrophoretic techniques showed that the 3-nitrotyrosine content of oxyhemoglobin sharply increased only in molar excess of peroxynitrite, thus suggesting that this hemoprotein is not a catalyst of nitration. The noncatalytic role may be due to the formation of the nitrating species (*)NO(2) mainly in molar excess of peroxynitrite. In agreement with this hypothesis, oxyhemoglobin strongly inhibited tyrosine nitration of a target dipeptide (Ala-Tyr) and of membrane proteins from ghosts resealed with oxyhemoglobin. Erythrocytes were poor inhibitors of Ala-Tyr nitration on account of the membrane barrier. However, at the physiologic hematocrit, Ala-Tyr nitration was reduced by 65%. This "sink" function was facilitated by the huge amount of band 3 anion exchanger on the cell membrane. We conclude that in blood oxyhemoglobin is a peroxynitrite scavenger of physiologic relevance.     

Nitric oxide diffusion to red blood cells limits extracellular,but not intraphagosomal,peroxynitrite formation by macrophages

Macrophage-derived nitric oxide (^•NO) participates in cytotoxic mechanisms against diverse microorganisms and tumor cells. These effects can be mediated by ^•NO itself or ^•NO-derived species such as peroxynitrite formed by its diffusion-controlled reaction with NADPH oxidase-derived superoxide radical anion (O₂^•−). In vivo, the facile extracellular diffusion of ^•NO as well as different competing consumption routes limit its bioavailability for the reaction with O₂^•− and, hence, peroxynitrite formation. In this work, we evaluated the extent by which ^•NO diffusion to red blood cells (RBC) can compete with activated macrophages-derived O₂^•− and affect peroxynitrite formation yields. Macrophage-dependent peroxynitrite production was determined by boron-based probes that react directly with peroxynitrite, namely, coumarin-7-boronic acid (CBA) and fluorescein-boronate (Fl-B). The influence of ^•NO diffusion to RBC on peroxynitrite formation was experimentally analyzed in co-incubations of ^•NO and O₂^•−-forming macrophages with erythrocytes. Additionally, we evaluated the permeation of ^•NO to RBC by measuring the intracellular oxidation of oxyhemoglobin to methemoglobin. Our results indicate that diluted RBC suspensions dose-dependently inhibit peroxynitrite formation, outcompeting the O₂^•− reaction. Computer-assisted kinetic studies evaluating peroxynitrite formation by its precursor radicals in the presence of RBC are in accordance with experimental results. Moreover, the presence of erythrocytes in the proximity of ^•NO and O₂^•--forming macrophages prevented intracellular Fl-B oxidation pre-loaded in L1210 cells co-cultured with activated macrophages. On the other hand, Fl-B-coated latex beads incorporated in the macrophage phagocytic vacuole indicated that intraphagosomal probe oxidation by peroxynitrite was not affected by nearby RBC. Our data support that in the proximity of a blood vessel, ^•NO consumption by RBC will limit the extracellular formation (and subsequent cytotoxic effects) of peroxynitrite by activated macrophages, while the intraphagosomal yield of peroxynitrite will remain unaffected.     

Reaction of human hemoglobin with peroxynitrite. Isomerization to nitrate and secondary formation of protein radicals

Peroxynitrite, a strong oxidant formed intravascularly in vivo, can diffuse onto erythrocytes and be largely consumed via a fast reaction (2 x 10(4) m(-1) s(-1)) with oxyhemoglobin. The reaction mechanism of peroxynitrite with oxyhemoglobin that results in the formation of methemoglobin remains to be elucidated. In this work, we studied the reaction under biologically relevant conditions using millimolar oxyhemoglobin concentrations and a stoichiometric excess of oxyhemoglobin over peroxynitrite. The results support a reaction mechanism that involves the net one-electron oxidation of the ferrous heme, isomerization of peroxynitrite to nitrate, and production of superoxide radical and hydrogen peroxide. Homolytic cleavage of peroxynitrite within the heme iron allows the formation of ferrylhemoglobin in approximately 10% yields, which can decay to methemoglobin at the expense of reducing equivalents of the globin moiety. Indeed, spin-trapping studies using 2-methyl-2-nitroso propane and 5,5 dimethyl-1-pyrroline-N-oxide (DMPO) demonstrated the formation of tyrosyl- and cysteinyl-derived radicals. DMPO also inhibited covalently linked dimerization products and led to the formation of DMPO-hemoglobin adducts. Hemoglobin nitration was not observed unless an excess of peroxynitrite over oxyhemoglobin was used, in agreement with a marginal formation of nitrogen dioxide. The results obtained support a role of oxyhemoglobin as a relevant intravascular sink of peroxynitrite.     

Reactions of desferrioxamine with peroxynitrite-derived carbonate and nitrogen dioxide radicals

The iron chelating agent desferrioxamine inhibits peroxynitrite-mediated oxidations and attenuates nitric oxide and oxygen radical-dependent oxidative damage both in vitro and in vivo. The mechanism of protection is independent of iron chelation and has remained elusive over the past decade. Herein, stopped-flow studies revealed that desferrioxamine does not react directly with peroxynitrite. However, addition of peroxynitrite to desferrioxamine in both the absence and the presence of physiological concentrations of CO2 and under excess nitrite led to the formation of a one-electron oxidation product, the desferrioxamine nitroxide radical, consistent with desferrioxamine reacting with the peroxynitrite-derived species carbonate (CO3*-) and nitrogen dioxide (*NO2) radicals. Desferrioxamine inhibited peroxynitrite-dependent free radical-mediated processes, including tyrosine dimerization and nitration, oxyhemoglobin oxidation in the presence of CO2, and peroxynitrite plus carbonate-dependent chemiluminescence. The direct two-electron oxidation of glutathione by peroxynitrite was unaffected by desferrioxamine. The reactions of desferrioxamine with CO3*- and *NO2 were unambiguously confirmed by pulse radiolysis studies, which yielded second-order rate constants of 1.7 x 10(9) and 7.6 x 10(6) M(-1) s(-1), respectively. Desferrioxamine also reacts with tyrosyl radicals with k = 6.3 x 10(6) M(-1) s(-1). However, radical/radical combination reactions between tyrosyl radicals or of tyrosyl radical with *NO2 outcompete the reaction with desferrioxamine and computer-assisted simulations indicate that the inhibition of tyrosine oxidation can be fully explained by scavenging of the peroxynitrite-derived radicals. The results shown herein provide an alternative mechanism to account for some of the biochemical and pharmacological actions of desferrioxamine via reactions with CO3*- and *NO2 radicals.     

Nitrate tolerance does not increase production of peroxynitrite in the heart

Clinical studies have suggested that long-term nitrate treatment does not improve and may even worsen cardiovascular mortality, and the possible role of nitrate tolerance has been suspected. Nitrate tolerance has been recently shown to increase vascular superoxide and peroxynitrite production leading to vascular dysfunction. Nevertheless, nitrates exert direct cardiac effects independent from their vascular actions. Therefore, we investigated whether in vivo nitroglycerin treatment leading to vascular nitrate tolerance increases cardiac formation of nitric oxide (NO), reactive oxygen species, and peroxynitrite, thereby leading to cardiac dysfunction. Nitrate tolerance increased bioavailability of NO in the heart without increasing formation of reactive oxygen species. Despite elevated myocardial NO, neither cardiac markers of peroxynitrite formation nor cardiac mechanical function were affected by nitroglycerin treatment. However, serum free nitrotyrosine, a marker for systemic peroxynitrite formation, was significantly elevated in nitroglycerin-treated animals. This is the first demonstration that, although the systemic effects of nitroglycerin may be deleterious due to enhancement of extracardiac peroxynitrite formation, nitroglycerin does not result in oxidative damage in the heart.     

RBC NOS: regulatory mechanisms and therapeutic aspects

Nitric oxide (NO), one of the most important vascular signaling molecules, is primarily produced by endothelial NO synthase (eNOS). eNOS is tightly regulated by its substrate l-arginine, cofactors and diverse interacting proteins. Interestingly, an NO synthase (NOS) was described within red blood cells (RBC NOS), and it was recently shown to significantly contribute to the intravascular NO pool and to regulate physiologically relevant mechanisms. However, the regulatory mechanisms and clinical implications of RBC NOS are unknown. The aim of this review is to highlight intracellular RBC NOS interactions and the role of RBC NOS in RBC homeostasis. Furthermore, macro- and microvascular diseases affected by RBC-derived NO are discussed.     

Kinetic and mechanistic studies of the NO*-mediated oxidation of oxymyoglobin and oxyhemoglobin

The second-order rate constants for the reactions between nitrogen monoxide and oxymyoglobin or oxyhemoglobin, determined by stopped-flow spectroscopy, increase with increasing pH. At pH 7.0 the rates are (43.6 +/- 0.5) x 10(6) M(-1) x s(-1) for oxymyoglobin and (89 +/- 3) x 10(6) M(-1) x s(-1) for oxyhemoglobin (per heme), whereas at pH 9.5 they are (97 +/- 3) x 10(6) M(-1) x s(-1) and (144 +/- 3) x 10(6) M(-1) x s(-1), respectively. The rate constants for the reaction between oxyhemoglobin and NO* depend neither on the association grade of the protein (dimer/tetramer) nor on the concentration of the phosphate buffer (100-1 mM). The nitrogen monoxide-mediated oxidations of oxymyoglobin and oxyhemoglobin proceed via intermediate peroxynitrito complexes which were characterized by rapid scan UV/vis spectroscopy. The two complexes MbFe(III)OONO and HbFe(III)OONO display very similar spectra with absorption maxima around 500 and 635 nm. These species can be observed at alkaline pH but rapidly decay to the met-form of the proteins under neutral or acidic conditions. The rate of decay of MbFe(III)OONO increases with decreasing pH and is significantly larger than those of the analogous complexes of the two subunits of hemoglobin. No free peroxynitrite is formed during these reactions, and nitrate is formed quantitatively, at both pH 7.0 and 9.0. This result indicates that, as confirmed from protein analysis after reacting the proteins with NO* for 10 times, when peroxynitrite is coordinated to the heme of myoglobin or hemoglobin it rapidly isomerizes to nitrate without nitrating the globins in physiologically significant amounts.     

Blood oxygen transport in rats under hypothermia combined with modification of the L-Arginine-NO pathway.

Nitric oxide (NO) has high affinity to heme and by interaction with oxyhemoglobin (HbO2) is converted into nitrate to form methemoglobin (MetHb) as a side product. In combining with deoxy-Hb NO yields a stable molecule of nitrosyl-hemoglobin (HbFe(II)NO) that can further be converted into nitrate and hemoglobin (Hb). In addition, Hb was shown to transport NO in a form of S-nitrosohemoglobin (SNO-Hb). These features of the Hb and NO interaction are important for blood oxygen transport including hemoglobin-oxygen affinity (HOA). The present investigation was aimed to study the blood oxygen transport indices (pO2, pCO2, pH, HOA, etc.) in rats under hypothermia combined with a modification of L-arginine-NO pathway. To modify the L-arginine-NO pathway, rats were administered with N(G)-nitro-L-arginine methyl ester (L-NAME), L-arginine, or sodium nitroprusside (SNP) intravenously before cooling. A substantial impairment of oxygen delivery and development of hypoxia, with an important contribution of HOA into the latter accompanied the deep hypothermia in rats. All the experimental groups developed metabolic acidosis, less pronounced in rats treated with L-arginine only. In the experiments with a modification of the L-arginine-NO pathway, an enhanced cold resistance, attenuated oxygen deficiency, and a weaker oxyhemoglobin dissociation curve (ODC) shift leftwards were observed only after the administration of L-arginine. Neither SNP nor L-NAME had not any protective effects. L-Arginine lowered the value of standard P50 (pO2, corresponding to 50% Hb saturation with oxygen at 37 degrees C, pH 7.4, and pCO2 = 40 mmHg). The actual P50 (at actual pH, pCO2 and temperature) decreased by approximately 15 mmHg and was significantly higher than that under hypothermia without the drug treatment (21.03 +/- 0.35 vs 17.45 +/- 0.60 mmHg). NO also can contribute to this system through different mechanisms (HOA modification, vascular tone regulation, peroxynitrite formation, and effects).     

Profound biopterin oxidation and protein tyrosine nitration in tissues of ApoE-null mice on an atherogenic diet: contribution of inducible nitric oxide synthase

Diminished nitric oxide (NO) bioactivity and enhanced peroxynitrite formation have been implicated as major contributors to atherosclerotic vascular dysfunctions. Hallmark reactions of peroxynitrite include the accumulation of 3-nitrotyrosine (3-NT) in proteins and oxidation of the NO synthase (NOS) cofactor, tetrahydrobiopterin (BH(4)). The present study sought to 1) quantify the extent to which 3-NT accumulates and BH(4) becomes oxidized in organs of apolipoprotein E-deficient (ApoE(-/-)) atherosclerotic mice and 2) determine the specific contribution of inducible NOS (iNOS) to these processes. Whereas protein 3-NT and oxidized BH(4) were undetected or near the detection limit in heart, lung, and kidney of 3-wk-old ApoE(-/-) mice or ApoE(-/-) mice fed a regular chow diet for 24 wk, robust accumulation was evident after 24 wk on a Western (atherogenic) diet. Since 3-NT accumulation was diminished 3- to 20-fold in heart, lung, and liver in ApoE(-/-) mice missing iNOS, iNOS-derived species are involved in this reaction. In contrast, iNOS-derived species did not contribute to elevated protein 3-NT formation in kidney or brain. iNOS deletion also afforded marked protection against BH(4) oxidation in heart, lung, and kidney of atherogenic ApoE(-/-) mice but not in brain or liver. These findings demonstrate that iNOS-derived species are increased during atherogenesis in ApoE(-/-) mice and that these species differentially contribute to protein 3-NT accumulation and BH(4) oxidation in a tissue-selective manner. Since BH(4) oxidation can switch the predominant NOS product from NO to superoxide, we predict that progressive NOS uncoupling is likely to drive atherogenic vascular dysfunctions.     

Spatial mapping of pulmonary and vascular nitrotyrosine reveals the pivotal role of myeloperoxidase as a catalyst for tyrosine nitration in inflammatory diseases

Nitrotyrosine (NO(2)Tyr) formation is a hallmark of acute and chronic inflammation and has been detected in a wide variety of human pathologies. However, the mechanisms responsible for this posttranslational protein modification remain elusive. While NO(2)Tyr has been considered a marker of peroxynitrite (ONOO(-)) formation previously, there is growing evidence that heme-protein peroxidase activity, in particular neutrophil-derived myeloperoxidase (MPO), significantly contributes to NO(2)Tyr formation in vivo via the oxidation of nitrite (NO(2)(-)) to nitrogen dioxide (.NO(2)). Coronary arteries from a patient with coronary artery disease, liver and lung tissues from a sickle cell disease patient, and an open lung biopsy from a lung transplant patient undergoing rejection were analyzed immunohistochemically to map relative tissue distributions of MPO and NO(2)Tyr. MPO immunodistribution was concentrated along the subendothelium in coronary tissue and hepatic veins as well as in the alveolar epithelial compartment of lung tissue from patients with sickle cell disease or acute rejection. MPO immunoreactivity strongly colocalized with NO(2)Tyr formation, which was similarly distributed in the subendothelial and epithelial regions of these tissues. The extracellular matrix protein fibronectin (FN), previously identified as a primary site of MPO association in vascular inflammatory reactions, proved to be a major target protein for tyrosine nitration, with a strong colocalization of MPO, NO(2)Tyr, and tissue FN occurring. Finally, lung tissue from MPO(-/-) mice, having tissue inflammatory responses stimulated by intraperitoneal zymosan administration, revealed less subendothelial NO(2)Tyr immunoreactivity than tissue from wild-type mice, confirming the significant role that MPO plays in catalyzing tissue nitration reactions. These observations reveal that (i) sequestration of neutrophil-derived MPO in vascular endothelial and alveolar epithelial compartments is an important aspect of MPO distribution and action in vivo, (ii) MPO-catalyzed NO(2)Tyr formation occurs in diverse vascular and pulmonary inflammatory pathologies, and (iii) extracellular matrix FN is an important target of tyrosine nitration in these inflammatory processes.     

Interactions of nitrosylhemoglobin and carboxyhemoglobin with erythrocyte.

Nitrosylhemoglobin (HbFe(II)NO) has been detected in vivo, and its role in NO transport and preservation has been discussed. To gain insight into the potential role of HbFe(II)NO, we performed in vitro experiments to determine the effect of oxygenated red blood cells (RBCs) on the dissociation of cell-free HbFe(II)NO, using carboxyhemoglobin (HbFe(II)CO) as a comparison. Results show that the apparent half-life of the cell-free HbFe(II)CO was reduced significantly in the presence of RBCs at 1% hematocrit. In contrast, RBC did not change the apparent half-life of extracellular HbFe(II)NO, but caused a shift in the HbFe(II)NO dissociation product from methemoglobin (metHbFe(III)) to oxyhemoglobin (HbFe(II)O(2)). Extracellular hemoglobin was able to extract CO from HbFe(II)CO-containing RBC, but not NO from HbFe(II)NO-containing RBC. Although these results appear to suggest some unusual interactions between HbFe(II)NO and RBC, the data are explainable by simple HbFe(II)NO dissociation and hemoglobin oxidation with known rate constants. A kinetic model consisting of these reactions shows that (i) deoxyhemoglobin is an intermediate in the reaction of HbFe(II)NO oxidation to metHbFe(III), (ii) the rate-limiting step of HbFe(II)NO decay is the dissociation of NO from HbFe(II)NO, (iii) the magnitude of NO diffusion rate constant into RBC is estimated to be approximately 10(4)M(-1)s(-1), consistent with previous results determined from a competition assay, and (iv) no additional chemical reactions are required to explain these data.     

Tyrosine nitration in prostaglandin H(2) synthase

In this study, we investigated the effects of various nitrogen oxide (NO(x)) species on the extent of prostaglandin H(2) synthase-1 (PGHS-1) nitration in purified protein and in vascular smooth muscle cells. We also examined PGHS-1 activity under these conditions and found the degree of nitration to correlate inversely with enzyme activity. In addition, since NO(x) species are thought to invoke damage during the pathogenesis of atherosclerosis, we examined human atheromatous tissue for PGHS-1 nitration. Both peroxynitrite and tetranitromethane induced Tyr nitration of purified PGHS-1, whereas 1-hydroxy-2-oxo-3-(N-methyl-aminopropyl)-3-methyl-1-triazene (NOC-7; a nitric oxide-releasing compound) did not. Smooth muscle cells treated with peroxynitrite showed PGHS-1 nitration. The extent of nitration by specific NO(x) species was determined by electrospray ionization mass spectrometry. Tetranitromethane was more effective than peroxynitrite, NOC-7, and nitrogen dioxide at nitrating a tyrosine-containing peptide (12%, 5%, 1%, and <1% nitration, respectively). Nitrogen dioxide and, to a lesser extent, peroxynitrite, induced dityrosine formation. Using UV/Vis spectroscopy, it was estimated that the reaction of PGHS-1 with excess peroxynitrite yielded two nitrated tyrosines/PGHS-1 subunit. Finally, atherosclerotic tissue obtained from endarterectomy patients was shown to contain nitrated PGHS-1. Thus, prolonged exposure to elevated levels of peroxynitrite may cause oxidative damage through tyrosine nitration.     

Nitrite reduction to nitric oxide by deoxyhemoglobin vasodilates the human circulation

Nitrite anions comprise the largest vascular storage pool of nitric oxide (NO), provided that physiological mechanisms exist to reduce nitrite to NO. We evaluated the vasodilator properties and mechanisms for bioactivation of nitrite in the human forearm. Nitrite infusions of 36 and 0.36 micromol/min into the forearm brachial artery resulted in supra- and near-physiologic intravascular nitrite concentrations, respectively, and increased forearm blood flow before and during exercise, with or without NO synthase inhibition. Nitrite infusions were associated with rapid formation of erythrocyte iron-nitrosylated hemoglobin and, to a lesser extent, S-nitroso-hemoglobin. NO-modified hemoglobin formation was inversely proportional to oxyhemoglobin saturation. Vasodilation of rat aortic rings and formation of both NO gas and NO-modified hemoglobin resulted from the nitrite reductase activity of deoxyhemoglobin and deoxygenated erythrocytes. This finding links tissue hypoxia, hemoglobin allostery and nitrite bioactivation. These results suggest that nitrite represents a major bioavailable pool of NO, and describe a new physiological function for hemoglobin as a nitrite reductase, potentially contributing to hypoxic vasodilation.     

Peroxynitrite reactions and formation in mitochondria

Mitochondria constitute a primary locus for the intracellular formation and reactions of peroxynitrite, and these interactions are recognized to contribute to the biological and pathological effects of both nitric oxide ((*)NO) and peroxynitrite. Extra- or intramitochondrially formed peroxynitrite can diffuse through mitochondrial compartments and undergo fast direct and free radical-dependent target molecule reactions. These processes result in oxidation, nitration, and nitrosation of critical components in the matrix, inner and outer membrane, and intermembrane space. Mitochondrial scavenging and repair systems for peroxynitrite-dependent oxidative modifications operate but they can be overwhelmed under enhanced cellular (*)NO formation as well as under conditions that lead to augmented superoxide formation by the electron transport chain. Peroxynitrite can lead to alterations in mitochondrial energy and calcium homeostasis and promote the opening of the permeability transition pore. The effects of peroxynitrite in mitochondrial physiology can be largely rationalized based on the reactivities of peroxynitrite and peroxynitrite-derived carbonate, nitrogen dioxide, and hydroxyl radicals with critical protein amino acids and transition metal centers of key mitochondrial proteins. In this review we analyze (i) the existing evidence for the intramitochondrial formation and reactions of peroxynitrite, (ii) the key reactions and fate of peroxynitrite in mitochondria, and (iii) their impact in mitochondrial physiology and signaling of cell death.     

Redox signaling: bioinorganic chemistry at its best

Oxidative modifications of amino acids in proteins can serve to regulate enzyme activity. This emerging field of redox regulation is related to other cellular signaling pathways, however, neither the chemical mechanisms in the cellular environment nor the affected metabolic and physiological changes are well understood. From data on endotoxin action in vascular tissue and reports on thiol modifications and tyrosine nitrations a unified scheme with five key components is proposed, governed solely by variations in the fluxes of nitrogen monoxide (NO) and superoxide (O(2)(-)). Crucial to the interactions is the formation of peroxynitrite which at concentrations of 10(-9)-10(-6)M elicits events like activation of prostanoid formation, metal catalyzed nitrations and two electron oxidations at cysteines and methionines. As a new concept we postulate that peroxynitrite formed in situ from NO and O(2)(-) is in rapid equilibrium with excess NO to form a nitrosating species that transfers NO(+). The resulting S-nitrosations occur prior to oxidative peroxynitrite action and seem to be involved in the down-regulation of reductive pathways. As the flux of O(2)(-) exceeds the one of NO, cellular damage develops induced by one-electron oxidations caused by nitrogen dioxide and by the Fenton reaction.     

Local oxidative and nitrosative stress increases in the microcirculation during leukocytes-endothelial cell interactions

Leukocyte-endothelial cell interactions and leukocyte activation are important factors for vascular diseases including nephropathy, retinopathy and angiopathy. In addition, endothelial cell dysfunction is reported in vascular disease condition. Endothelial dysfunction is characterized by increased superoxide (O(2) (?-)) production from endothelium and reduction in NO bioavailability. Experimental studies have suggested a possible role for leukocyte-endothelial cell interaction in the vessel NO and peroxynitrite levels and their role in vascular disorders in the arterial side of microcirculation. However, anti-adhesion therapies for preventing leukocyte-endothelial cell interaction related vascular disorders showed limited success. The endothelial dysfunction related changes in vessel NO and peroxynitrite levels, leukocyte-endothelial cell interaction and leukocyte activation are not completely understood in vascular disorders. The objective of this study was to investigate the role of endothelial dysfunction extent, leukocyte-endothelial interaction, leukocyte activation and superoxide dismutase therapy on the transport and interactions of NO, O(2)(?-) and peroxynitrite in the microcirculation. We developed a biotransport model of NO, O(2)(?-) and peroxynitrite in the arteriolar microcirculation and incorporated leukocytes-endothelial cell interactions. The concentration profiles of NO, O(2)(?-) and peroxynitrite within blood vessel and leukocytes are presented at multiple levels of endothelial oxidative stress with leukocyte activation and increased superoxide dismutase accounted for in certain cases. The results showed that the maximum concentrations of NO decreased ~0.6 fold, O(2)(?-) increased ~27 fold and peroxynitrite increased ~30 fold in the endothelial and smooth muscle region in severe oxidative stress condition as compared to that of normal physiologic conditions. The results show that the onset of endothelial oxidative stress can cause an increase in O(2)(?-) and peroxynitrite concentration in the lumen. The increased O(2) (?-) and peroxynitrite can cause leukocytes priming through peroxynitrite and leukocytes activation through secondary stimuli of O(2)(?-) in bloodstream without endothelial interaction. This finding supports that leukocyte rolling/adhesion and activation are independent events.     

Biochemical characterization of S-nitrosohemoglobin. Mechanisms underlying synthesis, no release, and biological activity.

S-Nitrosohemoglobin (SNO-Hb) has been suggested to act as an endogenous NO donor and physiological regulator of blood pressure. However, the mechanisms responsible for the formation of SNO-Hb and those underlying the release of NO and subsequent biological activity have yet to be elucidated. In the present study, a number of nitrosated oxyhemoglobin (HbO(2)) derivatives have been synthesized and characterized. HbO(2) can be nitrosated at up to three distinct residues, one in the alpha-globin chain and two in the beta-chain. A beta-chain mononitrosated species (designated "SNO-Hb"), generated by the reaction of HbO(2) and S-nitrosoglutathione, released NO via a thiol-dependent mechanism involving nucleophilic attack at the nitrosated thiol functionality of SNO-Hb; in the case of glutathione, this process was associated with the formation of a mixed disulfide. In contrast, multinitrosated hemoglobin species released NO and relaxed vascular smooth muscle by a thiol-independent mechanism. HbO(2) scavenged potently NO released from SNO-Hb and inhibited its vasorelaxant properties. These data show that the predominant vasoactive species released from SNO-Hb is NO, with HNO a putative intermediate; the presence of a low molecular weight thiol is a prerequisite for this process. Such observations have important implications for the generation, metabolic fate, and biological activity of S-nitrosothiols.     

Nitric oxide in dengue and dengue haemorrhagic fever: necessity or nuisance?

Advances in free radical research show that reactive oxygen and nitrogen oxide species, for example superoxide, nitric oxide (NO) and peroxynitrite, play an important role in the pathogenesis of different viral infections, including dengue virus. The pathogenic mechanism of dengue haemorrhagic fever (DHF) is complicated and is not clearly understood. The hallmarks of the dengue disease, the antibody-dependent enhancement, the shift from T-helper type 1 (Th1) to Th2 cytokine response and the cytokine tsunami resulting in vascular leakage can now be explained much better with the knowledge gained about NO and peroxynitrite. This paper makes an effort to present a synthesis of the current opinions to explain the pathogenesis of DHF/shock syndrome with NO on centre stage.     

Reactions of peroxynitrite with globin proteins and their possible physiological role

It is now widely accepted that, besides their well-established function in O(2) transport, hemoglobin and myoglobin also undergo several redox reactions aimed to scavenge toxic free radicals and reactive oxygen and nitrogen species. At least some of these reactions are believed to play an important physiological role in the defense against oxidative stress. This aspect is exemplified by the recently discovered neuroglobin, a globin expressed in the brain. Rather than being considerably involved in reversible O(2) binding, neuroglobin is likely to undergo redox reactions to protect neurons against oxidative and potentially pathogenic pathways, as those operating after episodes of tissue hypoxia or ischemia. A major part of the cellular damage occurring under such conditions has been ascribed to formation of peroxynitrite, that originates from the reaction between two biologically important free radicals, nitric oxide (NO ) and superoxide. Here we review the current knowledge of the reactions of different forms of hemoglobin, myoglobin, and neuroglobin with peroxynitrite and discuss their physiological role on the basis of measured rate constants and on the probability of occurrence of these reactions in vivo.     

Mechanistic studies of the oxidation of oxyhemoglobin by peroxynitrite

The strong oxidizing and nitrating agent peroxynitrite has been shown to diffuse into erythrocytes and oxidize oxyhemoglobin (oxyHb) to metHb. Because the value of the second-order rate constant for this reaction is on the order of 10(4) M(-)(1) s(-)(1) and the oxyHb concentration is about 20 mM (expressed per heme), this process is rather fast and oxyHb is considered a sink for peroxynitrite. In this work, we showed that the reaction of oxyHb with peroxynitrite, both in the presence and absence of CO(2), proceeds via the formation of oxoiron(iv)hemoglobin (ferrylHb), which in a second step is reduced to metHb and nitrate by its reaction with NO(2)(*). In the presence of physiological relevant amounts of CO(2), ferrylHb is generated by the reaction of NO(2)(*) with the coordinated superoxide of oxyHb (HbFe(III)O(2)(*)(-)). This reaction proceeds via formation of a peroxynitrato-metHb complex (HbFe(III)OONO(2)), which decomposes to generate the one-electron oxidized form of ferrylHb, the oxoiron(iv) form of hemoglobin with a radical localized on the globin. CO(3)(*)(-), the second radical formed from the reaction of peroxynitrite with CO(2), is also scavenged efficiently by oxyHb, in a reaction that finally leads to metHb production. Taken together, our results indicate that oxyHb not only scavenges peroxynitrite but also the radicals produced by its decomposition.