Altered Circadian Relationship Between Serum Nitric Oxide, Carbon Dioxide, and Uric Acid in Multiple Sclerosis

The free radical nitric oxide (NO^·) is involved in a variety of diverse biological processes from acting as a vasodilator in the cardiovascular system to being the rate-limiting component in the production of peroxynitrite (ONOO^-), a contributor to neurodegenerative disorders such as multiple sclerosis (MS). Uric acid (UA), the end product of purine metabolism in humans and a selective inhibitor of toxic reactions attributed to radicals formed by the interaction of ONOO^- and CO₂, is generally low in MS patients. We investigated the relationship between serum ONOO^-, CO₂, and UA in MS patients and normal controls by comparing the circadian characteristics of the NO^· metabolites nitrite/nitrate (NO), CO₂, and UA. In this preliminary study, we found the functional relationship ascribed to the circadian timing of the peak and trough levels of NO, CO₂, and UA in healthy subjects to be clearly altered in MS patients. These findings suggest that alterations in the temporal relationship between the 24 h pattern in serum ONOO^- formation and UA may either contribute to or reflect the disease processes in MS.     

Altered Circadian Relationship Between Serum Nitric Oxide,Carbon Dioxide,and Uric Acid in Multiple Sclerosis

The free radical nitric oxide (NO^·) is involved in a variety of diverse biological processes from acting as a vasodilator in the cardiovascular system to being the rate-limiting component in the production of peroxynitrite (ONOO^?), a contributor to neurodegenerative disorders such as multiple sclerosis (MS). Uric acid (UA), the end product of purine metabolism in humans and a selective inhibitor of toxic reactions attributed to radicals formed by the interaction of ONOO^? and CO₂, is generally low in MS patients. We investigated the relationship between serum ONOO^?, CO₂, and UA in MS patients and normal controls by comparing the circadian characteristics of the NO^· metabolites nitrite/nitrate (NO), CO₂, and UA. In this preliminary study, we found the functional relationship ascribed to the circadian timing of the peak and trough levels of NO, CO₂, and UA in healthy subjects to be clearly altered in MS patients. These findings suggest that alterations in the temporal relationship between the 24 h pattern in serum ONOO^? formation and UA may either contribute to or reflect the disease processes in MS.     

The oxidative and nitrosative chemistry of the nitric oxide/superoxide reaction in the presence of bicarbonate

The primary product of the interaction between nitric oxide (NO) and superoxide () is peroxynitrite (ONOO-), which is capable of either oxidizing or nitrating various biological substrates. However, it has been shown that excess NO or can further react with ONOO- to form species which mediate nitrosation. Subsequently, the controlled equilibrium between nitrosative and oxidative chemistry is critically dependent on the flux of NO and. Since ONOO- reacts not only with NO and but also with CO2, the effects of bicarbonate () on the biphasic oxidation profile of dihydrorhodamine-123 (DHR) and on the nitrosation of both 2,3-diaminonaphthalene and reduced glutathione were examined. Nitric oxide and were formed with DEA/NO [NaEt2NN(O)NO] and xanthine oxidase, respectively. The presence of did not alter either the oxidation profile of DHR with varying radical concentrations or the affinity of DHR for the oxidative species. This suggests that the presence of CO2 does not affect the scavenging of ONOO- by either NO or. However, an increase in the rate of DHR oxidation by ONOO- in the presence of suggests that a CO2-ONOO- adduct does play a role in the interaction of NO or with a product derived from ONOO-. Further examination of the chemistry revealed that the intermediate that reacts with NO is neither ONOO- nor cis-HOONO. It was concluded that NO reacts with both trans-HOONO and a CO2 adduct of ONOO- to form nitrosating species which have similar oxidation chemistry and reactivity with and NO.     

Peroxynitrite-mediated alpha-tocopherol oxidation in low-density lipoprotein: a mechanistic approach

Previous reports proposed that peroxynitrite (ONOO-) oxidizes alpha-tocopherol (alpha-TOH) through a two-electron concerted mechanism. In contrast, ONOO- oxidizes phenols via free radicals arising from peroxo bond homolysis. To understand the kinetics and mechanism of alpha-TOH and gamma-tocopherol (gamma-TOH) oxidation in low-density lipoprotein (LDL) (direct vs. radical), we exposed LDL to ONOO- added as a bolus or an infusion. Nitric oxide (.NO), ascorbate and CO2 were used as key biologically relevant modulators of ONOO- reactivity. Although approximately 80% alpha-TOH and gamma-TOH depletion occurred within 5 min of incubation of 0.8 microM LDL with a 60 microM bolus of ONOO-, an equimolar infusion of ONOO- over 60 min caused total consumption of both antioxidants. gamma-Tocopherol was preserved relative to alpha-TOH, probably due to gamma-tocopheroxyl radical recycling by alpha-TOH. alpha-TOH oxidation in LDL was first order in ONOO- with approximately 12% of ONOO- maximally available. Physiological concentrations of.NO and ascorbate spared both alpha-TOH and gamma-TOH through independent and additive mechanisms. High concentrations of.NO and ascorbate abolished alpha-TOH and gamma-TOH oxidation. Nitric oxide protection was more efficient for alpha-TOH in LDL than for ascorbate in solution, evidencing the kinetically highly favored reaction of lipid peroxyl radicals with.NO than with alpha-TOH as assessed by computer-assisted simulations. In addition, CO2 (1.2 mM) inhibited both alpha-TOH and lipid oxidation. These results demonstrate that ONOO- induces alpha-TOH oxidation in LDL through a one-electron free radical mechanism; thus the inhibitory actions of.NO and ascorbate may determine low alpha-tocopheryl quinone accumulation in tissues despite increased ONOO- generation.     

Reaction of peroxynitrite with carbon dioxide: intermediates and determination of the yield of CO3 •– and NO2 •

CO2 catalyses the isomerization of the biological toxin ONOO- to NO3- via an intermediate, presumably ONOOCO2-, which has an absorption maximum near 650 nm. The reflection spectrum of solid NMe4+ ONOO- exposed to CO2 shows a similar band near 650 nm; this absorption decays over minutes. Stopped-flow experiments in which CO2 solutions were mixed with alkaline ONOO- solutions indicate the formation of at least one intermediate. The initial absorption at 302 nm is less than that of ONOO-, which indicates that reactions take place within the mixing time, and this absorption is dependent (but not linearly) on the ONOO- and CO2 concentrations. We found that reaction of peroxynitrite with carbon dioxide forms some trioxocarbonate(*1-) (CO3*-) and nitrogen dioxide (NO2*) radicals via homolysis of the O-O bond in ONOOCO2-. We determined the extent of radical formation by mixing peroxynitrite, carbon dioxide and nitrogen monoxide. The later reacts with CO3*- and NO2* radicals to form, effectively, three NO2- per homolysis; ONOOCO2- that does not undergo homolysis yields NO3- and CO2. Based on the NO3- and NO2- analyses, the extent of conversion to NO3- is 96 +/- 1% and that of homolysis is 3 +/- 1%, respectively, significantly less than that reported in the literature.     

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.     

Pindolol is a potent scavenger of reactive nitrogen species

Pindolol is an indolic drug that has been shown to enhance and/or accelerate selective serotonin specific reuptake inhibitors (SSRI)-induced antidepressant (AD) effect, even though the respective mechanism is still unclear. It has been demonstrated that inhibition of nitric oxide (*NO) synthesis in CNS produces anxiolytic and AD-like behavioural effects in a variety of animal paradigms. On the other hand, sustained high levels of *NO may be deleterious to CNS, predominantly due to the formation of peroxynitrite anion (ONOO-), which is generated via reaction of *NO with superoxide radical (O2*-). Therefore, the purpose of the present study was to characterize the putative pindolol scavenging effect on *NO, ONOO-, and O2*-, using in vitro non-cellular systems. The obtained results clearly show that pindolol is a potent scavenger of *NO (IC50 of 449+/-33 microM) and ONOO- (IC50 of 131+/-24 microM). Additionally, the scavenging effect of pindolol increased almost 8 times in the presence of 25 mM NaHCO3 (IC50 of 17+/-3 microM), which indicates that pindolol efficiently scavenges reactive species that are produced from the ONOO-/CO2 reaction such as the nitrogen dioxide radical (*NO2) and the carbonate radical anion (CO3*-). These effects may contribute for the reduction of SSRI antidepressant latency that has been attributed to pindolol and may also constitute an additional value for this drug when depression is associated with pro-oxidant neurodegenerative diseases.     

Comparison of uric acid and ascorbic acid in protection against EAE

Serum levels of uric acid (UA), an inhibitor of peroxynitrite- (ONOO-) related chemical reactions, became elevated approximately 30 million years ago in hominid evolution. During a similar time frame, higher mammals lost the ability to synthesize another important radical scavenger, ascorbic acid (AA), leading to the suggestion that UA may have replaced AA as an antioxidant. However, in vivo treatment with AA does not protect against the development of experimental allergic encephalomyelitis (EAE), a disease that has been associated with the activity of ONOO- and is inhibited by UA. When compared in vitro, UA and AA were found to have similar capacities to inhibit the nitrating properties of ONOO-. However UA and AA had different capacities to prevent ONOO- -mediated oxidation, especially in the presence of iron ion (Fe3+). While UA at physiological concentrations effectively blocked dihydrorhodamine-123 oxidation in the presence of Fe3+, AA did not, regardless of whether the source of ONOO- was synthetic ONOO-, SIN-1, or RAW 264.7 cells. AA also potentiated lipid peroxidation in vivo and in vitro. In conclusion, the superior protective properties of UA in EAE may be related to its ability to neutralize the oxidative properties of ONOO- in the presence of free iron ions.     

Nitric oxide protects Cu,Zn-superoxide dismutase from hydrogen peroxide-induced inactivation

Reaction of Cu,Zn-superoxide dismutase (SOD1) and hydrogen peroxide generates a putative oxidant SOD-Cu2+-.OH that can inactivate the enzyme and oxidize 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) to DMPO-.OH. In the presence of nitric oxide (.NO), the SOD1/H2O2 system is known to produce peroxynitrite (ONOO-). In contrast to the proposed cytotoxicity of .NO conferred by ONOO-, we report here a protective role of .NO in the H2O2-induced inactivation of SODI. In a dose-dependent manner, .NO suppressed formation of DMPO-.OH and inactivation of the enzyme. Fragmentation of the enzyme was not affected by .NO. Bicarbonate retarded formation of ONOO-, suggesting that .NO competes with bicarbonate for the oxidant SOD-Cu2+-.OH. We propose that .NO protects SOD1 from H2O2-induced inactivation by reducing SOD-Cu2+.OH to the active SOD-Cu2+ with concomitant production of NO+ which reacts with H2O2 to give ONOO-.     

Reactions of peroxynitrite in the mitochondrial matrix

Superoxide radical (O2-) and nitric oxide (NO) produced at the mitochondrial inner membrane react to form peroxynitrite (ONOO-) in the mitochondrial matrix. Intramitochondrial ONOO- effectively reacts with a few biomolecules according to reaction constants and intramitochondrial concentrations. The second-order reaction constants (in M(-1) s(-1)) of ONOO- with NADH (233 +/- 27), ubiquinol-0 (485 +/- 54) and GSH (183 +/- 12) were determined fluorometrically by a simple competition assay of product formation. The oxidation of the components of the mitochondrial matrix by ONOO- was also followed in the presence of CO2, to assess the reactivity of the nitrosoperoxocarboxylate adduct (ONOOCO2-) towards the same reductants. The ratio of product formation was about similar both in the presence of 2.5 mM CO2 and in air-equilibrated conditions. Liver submitochondrial particles supplemented with 0.25-2 microM ONOO- showed a O2- production that indicated ubisemiquinone formation and autooxidation. The nitration of mitochondrial proteins produced after addition of 200 microM ONOO- was observed by Western blot analysis. Protein nitration was prevented by the addition of 50-200 microM ubiquinol-0 or GSH. An intramitochondrial steady state concentration of about 2 nM ONOO- was calculated, taking into account the rate constants and concentrations of ONOO- coreactants.     

Circadian Locomotor Rhythms in Mice with Targeted Disruption of the Gene for the Carbon Monoxide Synthesizing Enzyme,Heme Oxygenase-2

Carbon monoxide (CO), generated in neurons by the enzyme heme oxygenase-2 (HO2), is postulated to be a gaseous signaling molecule in the mammalian brain. Because of the recent evidence suggesting an important role of another endogenously produced gas, nitric oxide (NO), in entrainment of circadian rhythms in mammals, we hypothesized that CO may also be involved in regulating these rhythms. Consistent with this idea, others have found a circadian rhythm of heme turnover and CO synthesis can be induced by bright light. Furthermore, HO2 is co-localized with guanylyl cyclase, the putative target of CO, throughout the brain, with high amounts of staining in the suprachiasmatic nucleus (SCN) of the hypothalamus. The goal of the present study was to evaluate the role of CO in photic entrainment in wild-type and HO2 deficient mice. HO2–/– mice did not display any abnormalities in circadian rhythmicity. Entrainment to a light–dark cycle, the ability to phase delay locomotor activity after a four hour phase shift in photoperiod, and the period of the free running rhythm (t) were similar between HO2–/– and wild-type mice. Taken together, these data suggest that CO does not play a major role in regulating circadian activity rhythms in mice.     

Circadian Locomotor Rhythms in Mice with Targeted Disruption of the Gene for the Carbon Monoxide Synthesizing Enzyme, Heme Oxygenase-2

Carbon monoxide (CO), generated in neurons by the enzyme heme oxygenase-2 (HO2), is postulated to be a gaseous signaling molecule in the mammalian brain. Because of the recent evidence suggesting an important role of another endogenously produced gas, nitric oxide (NO), in entrainment of circadian rhythms in mammals, we hypothesized that CO may also be involved in regulating these rhythms. Consistent with this idea, others have found a circadian rhythm of heme turnover and CO synthesis can be induced by bright light. Furthermore, HO2 is co-localized with guanylyl cyclase, the putative target of CO, throughout the brain, with high amounts of staining in the suprachiasmatic nucleus (SCN) of the hypothalamus. The goal of the present study was to evaluate the role of CO in photic entrainment in wild-type and HO2 deficient mice. HO2-/- mice did not display any abnormalities in circadian rhythmicity. Entrainment to a light-dark cycle, the ability to phase delay locomotor activity after a four hour phase shift in photoperiod, and the period of the free running rhythm (t) were similar between HO2-/- and wild-type mice. Taken together, these data suggest that CO does not play a major role in regulating circadian activity rhythms in mice.     

Unique oxidative mechanisms for the reactive nitrogen oxide species, nitroxyl anion

The nitroxyl anion (NO-) is a highly reactive molecule that may be involved in pathophysiological actions associated with increased formation of reactive nitrogen oxide species. Angeli's salt (Na2N2O3; AS) is a NO- donor that has been shown to exert marked cytotoxicity. However, its decomposition intermediates have not been well characterized. In this study, the chemical reactivity of AS was examined and compared with that of peroxynitrite (ONOO-) and NO/N2O3. Under aerobic conditions, AS and ONOO- exhibited similar and considerably higher affinities for dihydrorhodamine (DHR) than NO/N2O3. Quenching of DHR oxidation by azide and nitrosation of diaminonaphthalene were exclusively observed with NO/N2O3. Additional comparison of ONOO- and AS chemistry demonstrated that ONOO- was a far more potent one-electron oxidant and nitrating agent of hydroxyphenylacetic acid than was AS. However, AS was more effective at hydroxylating benzoic acid than was ONOO-. Taken together, these data indicate that neither NO/N2O3 nor ONOO- is an intermediate of AS decomposition. Evaluation of the stoichiometry of AS decomposition and O2 consumption revealed a 1:1 molar ratio. Indeed, oxidation of DHR mediated by AS proved to be oxygen-dependent. Analysis of the end products of AS decomposition demonstrated formation of NO2- and NO3- in approximately stoichiometric ratios. Several mechanisms are proposed for O2 adduct formation followed by decomposition to NO3- or by oxidation of an HN2O3- molecule to form NO2-. Given that the cytotoxicity of AS is far greater than that of either NO/N2O3 or NO + O2, this study provides important new insights into the implications of the potential endogenous formation of NO- under inflammatory conditions in vivo.     

Peroxynitrite Anion Stimulates Arginine Release from Cultured Rat Astrocytes

The biosynthesis of the physiological messenger nitric oxide (*NO) in neuronal cells is thought to depend on a glial-derived supply of the *NO synthase substrate arginine. To expand our knowledge of the mechanism responsible for this glial-neuronal interaction, we studied the possible roles of peroxynitrite anion (ONOO-), superoxide anion (O2*-), *NO, and H2O2 in L-[3H]arginine release in cultured rat astrocytes. After 5 min of incubation at 37 degrees C, initial concentrations of 0.05-2 mM ONOO- stimulated the release of arginine from astrocytes in a concentration-dependent way; this effect was maximum from 1 mM ONOO- and proved to be approximately 400% as compared with control cells. ONOO(-)-mediated arginine release was prevented by arginine transport inhibitors, such as L-lysine and N(G)-monomethyl-L-arginine, suggesting an involvement of the arginine transporter in the effect of ONOO-. In situ xanthine/xanthine oxidase-generated O2*- (20 nmol/min) stimulated arginine release to a similar extent to that found with 0.1 mM ONOO-, but this effect was not prevented by arginine transport inhibitors. *NO donors, such as sodium nitroprusside, S-nitroso-N-acetylpenicillamine, or 1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium+ ++-1,2-diolate, and H2O2 did not significantly modify arginine release. As limited arginine availability for neuronal *NO synthase activity may be neurotoxic due to ONOO- formation, our results suggest that ONOO(-)-mediated arginine release from astrocytes may contribute to replenishing neuronal arginine, hence avoiding further generation of ONOO- within these cells.     

Potential of peroxynitrite to promote the conversion of oxyhemoglobin to methemoglobin

Superoxide anion and NO can react to form the highly oxidizing species peroxynitrite (ONOO-)which can react directly with hemoglobin (Hb) even in the presence of physiological concentration CO:. Thisresearch was to determine the ONOO--mediated oxidation damage to the heme of oxyhemoglobin (oxyHb)under conditions expected in blood. Results showed that 8-10 mol ONOO- was needed to quickly andcompletely convert 1 mol oxyHb to methemoglobin (metHb). ONOO- (20-140 μM) caused raoid andextensive formation of metHb from oxyHb (50 μM) mainly occurring within first 5-20 min of incubation.The conversion efficiency reached 16%, 48%, 60%, 79% and 88% output of metHb after 90 min ofincubation at 0, 20, 40, 100, and 140 μM ONOO- respectively. 1 mM CO2 caused a small decrease in theability of ONOO- to oxidize oxyHb, and ONOO--promoted conversion of oxyHb to metHb increased whenpH decreased from 8.0 to 6.0. Relatively lower temperature in blood condition will inhibit this reaction insome degree. We postulate that ONOO- can mediate oxidation damage to the heme, and cause heme lossfrom the hydrophobic cavity of Hb when its concentration exceeded 90 μM. These results indicated thatONOO- could convert oxyHb to metHb under the conditions expected in blood, and this reaction wasregulated by CO2 concentration, reaction time, temperature and pH value.     

Inhibitory effects of water-soluble cationic manganese porphyrins on peroxynitrite-induced SOS response in Salmonella typhimurium TA4107/pSK1002

We have investigated the protective effects of water-soluble cationic Mn(III) porphyrins against peroxynitrite (ONOO-)-induced DNA damage in the cells of Salmonella typhimurium TA4107/pSK1002 and lipid peroxidation of red blood cell membranes. Mn(III) tetrakis (N-methylpyridinium-4-yl) porphine (TMPyP) and the brominated form, Mn(III) octabromo-tetrakis (N-methylpyridinium-4-yl) porphine (OBTMPyP) effectively reduced the damage and peroxidation induced by N-morpholino sydnonimine (SIN-1), which gradually generates ONOO- from O2*- and *NO produced through hydrolysis. Mn(III)OBTMPyP became 10-fold more active than the non-brominated form. In the presence of authentic ONOO-, the Mn(III) porphyrins were ineffective against damage and strongly enhanced lipid peroxidation, while the coexistence of ascorbic acid inhibited peroxidation. Using a diode array spectrophotometry, the reactions of Mn(III)TMPyP with authentic ONOO- and SIN-1 were measured. Mn(III)TMPyP is known to be catalytic for ONOO- decomposition in the presence of antioxidants. OxoMn(IV)TMPyP with SIN-1 was rapidly reduced back to Mn(III) without adding any oxidants. Further, in the SIN-1 system, the concentration of NO2- and NO3- were colorimetrically determined by Griess reaction based on the two-step diazotization. NO2- increased by addition of Mn(III) porphyrin and the ratio of NO2- to NO3- was 4-7 times higher than that (1.05) of SIN-1 alone. This result suggests that O2*- from SIN-1 acts as a reductant and *NO cogenerated is oxidized to NO2-, a primarily decomposition product of *NO. Under the pathological conditions where biological antioxidants are depleted and ONOO- and O2*- are extensively generated, the Mn(III) porphyrins will effectively cycle ONOO- decomposition using O2*-.     

Reactions of Peroxynitrite and Nitrite with Organic Molecules and Hemoglobin

In this work, the reactions of nitrite (NO2-) and peroxynitrite (ONOO-) with organic molecules as well as with hemoglobin (Hb) were examined and the potential interference with the detection of hydrogen peroxide and Hb was investigated. ONOO- at low concentrations (35-140 microM) induced a concentration-dependent oxidation of o-phenylenediamine and guaiacol, and this process can be improved by the addition of Hb in a concentration-dependent manner. This enhancing effect of Hb was possibly due to the formation of such highly reactive species as ferrylHb during the reaction of ONOO- and Hb. NO2- also oxidized the aromatic amine o-phenylenediamine, but its efficiency was much lower than that of ONOO-. A 300-fold excess of NO2- over hydrogen peroxide inhibited the oxidation of Pyrogallol Red mediated by hydrogen peroxide and Hb, which was due in part to the reaction of NO2- with Hb ferryl species compound I and compound II and the phenoxyl radical. These data suggest that ONOO- and NO2- can interfere with the detection of hydrogen peroxide. The overestimation or underestimation of the hydrogen peroxide detected is dependent upon the organic molecule utilized for detection and the relative rate of NO2-, superoxide, and ONOO- generation.     

Nitric oxide and peroxynitrite interactions with mitochondria

Nitric oxide (*NO) and peroxynitrite (ONOO-) avidly interact with mitochondrial components, leading to a range of biological responses spanning from the modulation of mitochondrial respiration, mitochondrial dysfunction to the signaling of apoptotic cell death. Physiological levels of *NO primarily interact with cytochrome c oxidase, leading to a competitive and reversible inhibition of mitochondrial oxygen uptake. In turn, this leads to alterations in electrochemical gradients, which affect calcium uptake and may regulate processes such as mitochondrial transition pore (MTP) opening and the release of pro-apoptotic proteins. Large or persistent levels of *NO in mitochondria promote mitochondrial oxidant formation. Peroxynitrite formed either extra- or intra-mitochondrially leads to oxidative damage, most notably at complexes I and II of the electron transport chain, ATPase, aconitase and Mn-superoxide dismutase. Mitochondrial scavenging systems for peroxynitrite and peroxynitrite-derived radicals such as carbonate (CO3*-) and nitrogen dioxide radicals (*NO2) include cytochrome c oxidase, glutathione and ubiquinol and serve to partially attenuate the reactions of these oxidants with critical mitochondrial targets. Detection of nitrated mitochondrial proteins in vivo supports the concept that mitochondria constitute central loci of the toxic effects of excess reactive nitrogen species. In this review we will provide an overview of the biochemical mechanisms by which *NO and ONOO- regulate or alter mitochondrial functions.     

Peroxynitrite and protein tyrosine nitration of prostacyclin synthase

When working on the regulation of prostacyclin synthase (PGIS), we found that PGIS was selectively inhibited by peroxynitrite (ONOO-), a potent oxidant formed by the combination of superoxide anion and nitric oxide (NO) at a rate of diffusion-controlled. None of the cellular antioxidants studied (i.e. GSH, Vitamins C and E, and others) prevented the inhibition of ONOO- on PGIS. This unexpected behavior was explained by a catalytic reaction of the iron-thiolate center of PGIS with ONOO- anion. In contrast, ONOO- activated both thromboxane A2-synthase and cyclooxygenases. In addition, we demonstrated that sub-micromolar levels of ONOO- inhibited PGI2-dependent vasorelaxation and triggered a PGH2-dependent vasospasm, indicating that ONOO- increased PGH2 formation as a consequence of PGIS nitration. We have subsequently demonstrated that endogenous ONOO- caused PGIS nitration and TxA2 activation in several diseased conditions such as atherosclerotic vessels, hypoxia-reperfusion injury, cytokines-treated cells, diabetes, as well as hypertension. Since NO is produced physiologically it seems that excessive formation of superoxide not only eliminates the vasodilatory, growth-inhibiting, anti-thrombotic and anti-adhesive effects of NO and PGI2 but also allows and promotes an action of the potent vasoconstrictor, prothrombotic agent, growth promoter, and leukocyte adherer, PGH2. We conclude that the nitration of PGIS nitration might be a new pathogenic mechanism for superoxide-induced endothelium dysfunction often observed in vascular diseases such as atherosclerosis, hypertension, ischemia, endotoxic shock, and diabetes.     

Anin VitroModel of Oligodendrocyte Destruction by Nitric Oxide and Its Relevance to Multiple Sclerosis

There is mounting evidence that nitric oxide (NO) is produced in the brains of patients with multiple sclerosis (MS) and in the experimental model of MS, experimental autoimmune encephalomyelitis, after the induction of Type II nitric oxide synthase (iNOS). Because NO can cause a variety of biological insults that compromise or even kill normal cells, we studied the effects of NO on oligodendrocytes since they are a target in MS tissue. In anin vitromodel, we have been able to demonstrate that NO causes damage to oligodendrocytes preferentially, sparing microglia almost completely and affecting some but not all astrocytic functions. This article describes the types of assays used to measure morphological changes, mitochondrial dysfunction, DNA strand breaks, and cell death brought on by NO or peroxynitrite (ONOO-) as well as a comprehensive review of the various techniques and sensitivities of NO and iNOS assays that would be applicable to similarin vitromodels.