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IN THEIR REPORT'S-NITROSYLATION OF PARKIN regulates ubiquitination and compromises parkin's protective function' (28 May, p. 1328; Science Express 22 Apr.), K. K. K. Chung et al. raise the intriguing possibility [...]

Pseudomonas aeruginosa (PA) is a common bacterial pathogen, responsible for a high incidence of nosocomial and respiratory infections. KatA is the major catalase of PA that detoxifies hydrogen peroxide (H2O2), a reactive oxygen intermediate generated during aerobic respiration. Paradoxically, PA displays elevated KatA activity under anaerobic growth conditions where the substrate of KatA, H2O2, is not produced. The aim of the present study is to elucidate the mechanism underlying this phenomenon and define the role of KatA in PA during anaerobiosis using genetic, biochemical and biophysical approaches. We demonstrated that anaerobic wild-type PAO1 cells yielded higher levels of katA transcription and expression than aerobic cells, whereas a nitrite reductase mutant ΔnirS produced ∼50% the KatA activity of PAO1, suggesting that a basal NO level was required for the increased KatA activity. We also found that transcription of the katA gene was controlled, in part, by the master anaerobic regulator, ANR. A ΔkatA mutant and a mucoid mucA22 ΔkatA bacteria demonstrated increased sensitivity to acidified nitrite (an NO generator) in anaerobic planktonic and biofilm cultures. EPR spectra of anaerobic bacteria showed that levels of dinitrosyl iron complexes (DNIC), indicators of NO stress, were increased significantly in the ΔkatA mutant, and dramatically in a ΔnorCB mutant compared to basal levels of DNIC in PAO1 and ΔnirS mutant. Expression of KatA dramatically reduced the DNIC levels in ΔnorCB mutant. We further revealed direct NO-KatA interactions in vitro using EPR, optical spectroscopy and X-ray crystallography. KatA has a 5-coordinate high spin ferric heme that binds NO without prior reduction of the heme iron (Kd ∼6 μM). Collectively, we conclude that KatA is expressed to protect PA against NO generated during anaerobic respiration. We proposed that such protective effects of KatA may involve buffering of free NO when potentially toxic concentrations of NO are approached. [ABSTRACT FROM AUTHOR]

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) direct the activation of distinct signaling pathways that determine cell fate. In this study, the pathways activated and the mechanisms by which ROS and RNS control the viability of pancreatic β-cells were examined. Although both nitric oxide and hydrogen peroxide (H2O2) induce DNA damage, reduce cell viability, and activate AMPK, the mechanisms of AMPK activation and cell death induction differ between each reactive species. Nitric oxide activates the unfolded protein and heat shock responses and MAPK kinase signaling, whereas H2O2 stimulates p53 stabilization and poly(ADP-ribose) polymerase (PARP) activation but fails to induce the unfolded protein or heat shock responses or MAPK activation. The control of cell fate decisions is selective for the form of stress. H2O2-mediated reduction in β-cell viability is controlled by PARP, whereas cell death in response to nitric oxide is PARP independent but associated with the nuclear localization of GAPDH. These findings show that both ROS and RNS activate AMPK, induce DNA damage, and reduce cell viability; however, the pathways controlling the responses of β-cells are selective for the type of reactive species. [ABSTRACT FROM AUTHOR]

Vitturi DA, Teng X, Toledo JC, Matalon S, Lancaster JR Jr, Patel RP. Regulation of nitrite transport in red blood cells by hemoglobin oxygen fractional saturation. Am J Physiol Heart Circ Physiol 296: H1398-H1407, 2009. First published March 13, 2009; doi: 10.11 52/ajpheart.01 303.2008.-Allosteric regulation of nitrite reduction by deoxyhemoglobin has been proposed to mediate nitric oxide (NO) formation during hypoxia. Nitrite is predominantly an anion at physiological pH, raising questions about the mechanism by which it enters the red blood cell (RBC) and whether this is regulated and coupled to deoxyhemoglobin-mediated reduction. We tested the hypothesis that nitrite transport by RBCs is regulated by fractional saturation. Using human RBCs, nitrite consumption was faster at lower fractional saturations, consistent with faster reactions with deoxyheme. A membrane-based regulation was suggested by slower nitrite consumption with intact versus lysed RBCs. Interestingly, upon nitrite addition, intracellular nitrite concentrations attained a steady state that, despite increased rates of consumption, did not change with decreasing oxygen tensions, suggesting a deoxygenation-sensitive step that either increases nitrite import or decreases the rate of nitrite export. A role for anion exchanger (AE)1 in the control of nitrite export was suggested by increased intracellular nitrite concentrations in RBCs treated with DIDS. Moreover, deoxygenation decreased steady-state levels of intracellular nitrite in AE-1-inhibited RBCs. Based on these data, we propose a model in which deoxyhemoglobin binding to AE1 inhibits nitrite export under low oxygen tensions allowing for the coupling between deoxygenation and nitrite reduction to NO along the arterial-to-venous gradient. [ABSTRACT FROM AUTHOR]

Nitrosothiols (RSNO), formed from thiols and metabolites of nitric oxide (•NO), have been implicated in a diverse set of physiological and pathophysiological processes, although the exact mechanisms by which they are formed biologically are unknown. Several candidate nitrosative pathways involve the reaction of •NO with O2, reactive oxygen species (ROS), and transition metals. We developed a strategy using extracellular ferrocyanide to determine that under our conditions intracellular protein RSNO formation occurs from reaction of •NO inside the cell, as opposed to cellular entry of nitrosative reactants from the extracellular compartment. Using this method we found that in RAW 264.7 cells RSNO formation occurs only at very low (<8 μM) O2 concentrations and exhibits zero-order dependence on •NO concentration. Indeed, RSNO formation is not inhibited even at O2 levels <1 μM. Additionally, chelation of intracellular chelatable iron pool (CIP) reduces RSNO formation by >50%. One possible metal-dependent, O2-independent nitrosative pathway is the reaction of thiols with dinitrosyliron complexes (DNIC), which are formed in cells from the reaction of •NO with the CIP. Under our conditions, DNIC formation, like RSNO formation, is inhibited by ≈50% after chelation of labile iron. Both DNIC and RSNO are also increased during overproduction of ROS by the redox cycler 5,8-dimethoxy-1,4-naphthoquinone. Taken together, these data strongly suggest that cellular RSNO are formed from free •NO via transnitrosation from DNIC derived from the CIP. We have examined in detail the kinetics and mechanism of RSNO formation inside cells. [ABSTRACT FROM AUTHOR]

Mitochondria dysfunction and hypoxic microenvironment are hallmarks of cancer cell biology. Recently, many studies have focused on isolation of brain cancer stem cells using CD133 expression. In this study, we investigated whether CD133 expression is regulated by bioenergetic stresses affecting mitochondrial functions in human glioma cells. First, we determined that hypoxia induced a reversible up-regulation of CD133 expression. Second, mitochondrial dysfunction through pharmacological inhibition of the Electron Transport Chain (ETC) produced an up-regulation of CD133 expression that was inversely correlated with changes in mitochondrial membrane potential. Third, generation of stable glioma cells depleted of mitochondrial DNA showed significant and stable increases in CD133 expression. These glioma cells, termed rho0 or ρ0, are characterized by an exaggerated, uncoupled glycolytic phenotype and by constitutive and stable upregulation of CD133 through many cell passages. Moreover, these ρ0 cells display the ability to form ''tumor spheroids'' in serumless medium and are positive for CD133 and the neural progenitor cell marker, nestin. Under differentiating conditions, ρ0 cells expressed multi-lineage properties. Reversibility of CD133 expression was demonstrated by transfering parental mitochondria to ρ0 cells resulting in stable trans-mitochondrial ''cybrid'' clones. This study provides a novel mechanistic insight about the regulation of CD133 by environmental conditions (hypoxia) and mitochondrial dysfunction (genetic and chemical). Considering these new findings, the concept that CD133 is a marker of brain tumor stem cells may need to be revised. [ABSTRACT FROM AUTHOR]

A fundamental challenge to the study of oxidative stress responses of Mycobacterium tuberculosis (Mtb) is to understand how the protective host molecules are sensed and relayed to control bacilli gene expression. The genetic response of Mtb to hypoxia and NO is controlled by the sensor kinases DosS and DosT and the response regulator DosR through activation of the dormancy/NO (Dos) regulon. However, the regulatory ligands of DosS and DosT and the mechanism of signal sensing were unknown. Here, we show that both DosS and DosT bind heme as a prosthetic group and that DosS is rapidly autooxidized to attain the met (Fe3+ form, whereas DosT exists in the O2-bound (oxy) form. EPR and UV-visible spectroscopy analysis showed that O2, NO, and CO are ligands of DosS and DosT. Importantly, we demonstrate that the oxidation or ligation state of the heme iron modulates DosS and DosT autokinase activity and that ferrous DosS, and deoxy DosT, show significantly increased autokinase activity compared with met DosS and oxy DosT. Our data provide direct proof that DosS functions as a redox sensor, whereas DosT functions as a hypoxia sensor, and that O2, NO. and CO are modulatory ligands of DosS and DosT. Finally, we identified a third potential dormancy signal, CO. that induces the Mtb Dos regulon. We conclude that Mtb has evolved finely tuned redox and hypoxia-mediated sensing strategies for detecting O2, NO, and CO. Data presented here establish a paradigm for understanding the mechanism of bacilli persistence. [ABSTRACT FROM AUTHOR]

A fundamental challenge in the redox biology of Mycobacterium tuberculosis (Mtb) is to understand the mechanisms involved in sensing redox signals such as oxygen (O2), nitric oxide (NO), and nutrient depletion, which are thought to play a crucial role in persistence. Here we show that Mtb WhiB3 responds to the dormancy signals NO and O2 through its iron-sulfur (Fe-S) cluster. To functionally assemble the WhiB3 Fe-S cluster, we identified and characterized the Mtb cysteine desulfurase (lscS; Rv3025c) and developed a native enzymatic reconstitution system for assembling Fe-S clusters in Mtb. EPR and UV-visible spectroscopy analysis of reduced WhiB3 is consistent with a one-electron reduction of EPR silent [4Fe-4S]2+ to EPR visible [4Fe-4S]+. Atmospheric O2 gradually degrades the WhiB3 [4Fe-4S]2+ cluster to generate a [3Fe-4S]+ intermediate. Furthermore, EPR analysis demonstrates that NO forms a protein-bound dinitrosyl-iron-dithiol complex with the Fe-S cluster, indicating that NO specifically targets the WhiB3 Fe-S cluster. Our data suggest that the mechanism of WhiB3 4Fe-4S cluster degradation is similar to that of fumarate nitrate regulator. Importantly, Mtb ΔwhiB3 shows enhanced growth on acetate medium, but a growth defect on media containing glucose, pyruvate, succinate, or fumarate as the sole carbon source. Our results implicate WhiB3 in metabolic switching and in sensing the physiologically relevant host signaling molecules NO and O2 through its [4Fe-4S) cluster. Taken together, our results suggest that WhiB3 is an intracellular redox sensor that integrates environmental redox signals with core intermediary metabolism. [ABSTRACT FROM AUTHOR]

Arginine contains the guanidinium group and thus has structural similarity to ligands of imidazoline and α-2 adrenoceptors (α-2 AR). Therefore, we investigated the possibility that exogenous arginine may act as a ligand for these receptors in human umbilical vein endothelial cells and activate intracellular nitric oxide (NO) synthesis. Idazoxan, a mixed antagonist of imidazoline and α-2 adrenoceptors, partly inhibited ʟ-arginine-initiated NO formation as measured by a Griess reaction. Rauwolscine, a highly specific antagonist of α-2 AR, at very low concentrations completely inhibited NO formation. Like ʟ-arginine, agmatine (decarboxylated arginine) also activated NO synthesis, however, at much lower concentrations. We found that dexmedetomidine, a specific agonist of α-2 AR was very potent in activating cellular NO, thus indicating a possible role for α-2 AR in ʟ-arginine-mediated NO synthesis, o-arginine also activated NO production and could be inhibited by imidazoline and α-2 AR antagonists, thus indicating nonsubstrate actions of arginine. Pertussis toxin, an inhibitor of G proteins, attenuated ʟ-arginine-mediated NO synthesis, thus indicating mediation via G proteins. ʟ-type Ca2+ channel blocker nifedipine and phospholipase C inhibitor U73122 inhibited NO formation and thus implicated participation of a second messenger pathway. Finally, in isolated rat gracilis vessels, rauwolscine completely inhibited the ʟ-arginine-initiated vessel relaxation. Taken together, these data provide evidence for binding of arginine to membrane receptor(s), leading to the activation of endothelial NO synthase (eNOS) NO production through a second messenger pathway. These findings provide a previously unrecognized mechanistic explanation for the beneficial effects of ʟ-arginine in the cardiovascular system and thus provide new potential avenues for therapeutic development. [ABSTRACT FROM AUTHOR]

The reaction between nitric oxide (•NO) and oxygen yields reactive species capable of oxidizing and nitrosating proteins, as well as deaminating DNA bases. Although this reaction is considered too slow to be biologically relevant, it has been shown that membranes, lipoproteins, mitochondria and possibly proteins can accelerate this reaction. This effect stems from the higher solubility of both •NO and O2in the hydrophobic phase of these biological particles, leading to a concentration of both reagents and so a higher rate of reaction. It has been determined that this reaction occurs from 30 to 300 times more rapidly within the membrane, while even higher values have been suggested for proteins. The autoxidation of •NO in membranes is not the main route for cellular •NO consumption but an important consequence of this phenomenon is to focus the generation of significant amounts of oxidizing and nitrosating molecules (nitrogen dioxide and dinitrogen trioxide) in the small volume comprised by cellular membranes. Even so, these reactive species are diffusible and their ultimate fate will depend on the reactivity towards available substrates rather than on physical barriers. The acceleration of •NO autoxidation by biological hydrophobic phases may thus be a general phenomenon that increases in importance in cases of •NO overproduction. IUBMB Life, 59: 243-248, 2007 [ABSTRACT FROM AUTHOR]

Hydrogen sulfide (H2S) has recently been shown to have a signaling role in vascular cells. Similar to nitric oxide (NO), H25 is enzymatically produced by amino acid metabolism and can cause posttranslational modification of proteins, particularly at thiol residues. Molecular targets for H25 include ATP-sensitive K~ channels, and H2S may interact with NO and heme proteins such as cyclooxygenase. It is well known that the reactions of NO in the vasculature are O2 dependent, but this has not been addressed in most studies designed to elucidate the role of H2S in vascular function. This is important, since H2S reactions can be dramatically altered by the high concentrations of O2 used in cell culture and organ bath experiments. To test the hypothesis that the effects of H2S on the vasculature are O2 dependent, we have measured real-time levels of H2S and O2 in respirometry and vessel tension experiments, as well as the associated vascular responses. A novel polarographic H25 sensor developed in our laboratory was used to measure H25 levels. Here we report that, in rat aorta, H2S concentrations that mediate rapid contraction at high O2 levels cause rapid relaxation at lower physiological O2 levels. At high O2, the vasoconstrictive effect of H2S suggests that it may not be H2S per se but, rather, a putative vasoactive oxidation product that mediates constriction. These data are interpreted in terms of the potential for H2S to modulate vascular tone in vivo. [ABSTRACT FROM AUTHOR]

Inducible nitric oxide (NO) synthase (NOS) II has been implicated in macrophage-mediated antitumor activity. However, use of the NOS II gene in cancer therapy is problematic because of the double-edged nature of NO action. Herein we show that adeno-viral vectors mediated effective NOS II gene transfer into various human tumors. Production of NO significantly up-regulated multiple angiogenic molecules. However, the NO-producing tumor cells did not form tumors or metastases in ectopic or orthotopic xenograft nude mouse models. The dramatic loss of malignancy was due to NO-mediated apoptosis. We also generated a series of adenoviral vectors harboring mutant NOS II genes that expressed mutant NOS II proteins with defined levels of enzymatic activity. Tumor cells transduced with these NOS II genes produced NO at different levels, which directly correlated with the antitumor activity in vitro and in viva. This demonstration using a relevant biological system shows that NO produces dose-dependent anti- tumor activity in vitro and in vivo, regardless of its up-regulation of protumor factors. [ABSTRACT FROM AUTHOR]

Examines the irreversible reaction of nitric oxide with oxyhemoglobin. Formation of multiple hemoglobin species; Influence of hemoglobin concentration in preventing endothelium-derived relaxing factor-dependent relaxation; Consumption of nitric oxide by hemoglobin.

H2S is a potent gasotransmitter in eukaryotes and bacteria. Host-derived H2S has been shown to profoundly alter M. tuberculosis (Mtb) energy metabolism and growth. However, compelling evidence for endogenous production of H2S and its role in Mtb physiology is lacking. We show that multidrug-resistant and drug-susceptible clinical Mtb strains produce H2S, whereas H2S production in non-pathogenic M. smegmatis is barely detectable. We identified Rv3684 (Cds1) as an H2S-producing enzyme in Mtb and show that cds1 disruption reduces, but does not eliminate, H2S production, suggesting the involvement of multiple genes in H2S production. We identified endogenous H2S to be an effector molecule that maintains bioenergetic homeostasis by stimulating respiration primarily via cytochrome bd. Importantly, H2S plays a key role in central metabolism by modulating the balance between oxidative phosphorylation and glycolysis, and it functions as a sink to recycle sulfur atoms back to cysteine to maintain sulfur homeostasis. Lastly, Mtb-generated H2S regulates redox homeostasis and susceptibility to anti-TB drugs clofazimine and rifampicin. These findings reveal previously unknown facets of Mtb physiology and have implications for routine laboratory culturing, understanding drug susceptibility, and improved diagnostics. [ABSTRACT FROM AUTHOR]

Presents a study which looked at the impact of nitric oxide on O2 within the hydrophobic region of phospholipid or biological membranes. Role of biological membranes and tissue hydrophobic compartments; Methodology used to conduct the study; Results of the study.

Sickle cell disease (SCD) is characterized by malformed erythrocytes and results in many vascular complications, including the lysis of a minor proportion of these cells, liberating free hemoglobin, which is a potent scavenger of nitric oxide (NO). SCD involves inflammatory activation, including the upregulation of vascular coagulation. Because NO possesses important anti-coagulant and anti-adhesion properties, the increased scavenging of NO in SCD undoubtedly is a major contributor to the pathology of this disease. [Copyright &y& Elsevier]

Proposes criteria to help guide research regarding whether a bioactivity results from the S-nitrosation (or denitrosation) of a specific protein. Report from Zhang and Hogg confirming that one cellular target of oxidized forms of nitric oxide (NO) is thiol groups; Formation of protein nitrosothils (RSNOs) in macrophage cell line cystolic extracts following lipopolysaccharide (LPS) stimulation.

Provides information about sickle cell disease. Prevalence of the disease among Afro-American population; Features of the disease; Importance of the multiple biological actions of nitrogen monoxide in the cardiovascular system.

Dinitrosyliron complexes (DNIC) have been found in a variety of pathological settings associated with *NO. However, the iron source of cellular DNIC is unknown. Previous studies on this question using prolonged *NO exposure could be misleading due to the movement of intracellular iron among different sources. We here report that brief *NO exposure results in only barely detectable DNIC, but levels increase dramatically after 1-2 h of anoxia. This increase is similar quantitatively and temporally with increases in the chelatable iron, and brief *NO treatment prevents detection of this anoxia-induced increased chelatable iron by deferoxamine. DNIC formation is so rapid that it is limited by the availability of *NO and chelatable iron. We utilize this ability to selectively manipulate cellular chelatable iron levels and provide evidence for two cellular functions of endogenous DNIC formation, protection against anoxia-induced reactive oxygen chemistry from the Fenton reaction and formation by transnitrosation of protein nitrosothiols (RSNO). The levels of RSNO under these high chelatable iron levels are comparable with DNIC levels and suggest that under these conditions, both DNIC and RSNO are the most abundant cellular adducts of NO. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa (PA) is a common bacterial pathogen, responsible for a high incidence of nosocomial and respiratory infections. KatA is the major catalase of PA that detoxifies hydrogen peroxide (H2O2), a reactive oxygen intermediate generated during aerobic respiration. Paradoxically, PA displays elevated KatA activity under anaerobic growth conditions where the substrate of KatA, H2O2, is not produced. The aim of the present study is to elucidate the mechanism underlying this phenomenon and define the role of KatA in PA during anaerobiosis using genetic, biochemical and biophysical approaches. We demonstrated that anaerobic wild-type PAO1 cells yielded higher levels of katA transcription and expression than aerobic cells, whereas a nitrite reductase mutant ΔnirS produced ∼50% the KatA activity of PAO1, suggesting that a basal NO level was required for the increased KatA activity. We also found that transcription of the katA gene was controlled, in part, by the master anaerobic regulator, ANR. A ΔkatA mutant and a mucoid mucA22 ΔkatA bacteria demonstrated increased sensitivity to acidified nitrite (an NO generator) in anaerobic planktonic and biofilm cultures. EPR spectra of anaerobic bacteria showed that levels of dinitrosyl iron complexes (DNIC), indicators of NO stress, were increased significantly in the ΔkatA mutant, and dramatically in a ΔnorCB mutant compared to basal levels of DNIC in PAO1 and ΔnirS mutant. Expression of KatA dramatically reduced the DNIC levels in ΔnorCB mutant. We further revealed direct NO-KatA interactions in vitro using EPR, optical spectroscopy and X-ray crystallography. KatA has a 5-coordinate high spin ferric heme that binds NO without prior reduction of the heme iron (Kd ∼6 μM). Collectively, we conclude that KatA is expressed to protect PA against NO generated during anaerobic respiration. We proposed that such protective effects of KatA may involve buffering of free NO when potentially toxic concentrations of NO are approached. [ABSTRACT FROM AUTHOR]

Xanthine oxidoreductase (XOR) generates proinflammatory oxidants and secondary nitrating species, with inhibition of XOR proving beneficial in a variety of disorders. Electrophilic nitrated fatty acid derivatives, such as nitro-oleic acid (OANO[sub2]), display anti-inflammatory effects with pleiotropic properties. Nitro-oleic acid inhibits XOR activity in a concentrationdependent manner with an IC[sub50] of 0.6 μM, limiting both purine oxidation and formation of superoxide (O[sub2]). Enzyme inhibition by OA-NO[sub2] is not reversed by thiol reagents, including glutathione, β-mercaptoethanol, and dithiothreitol. Structure-function studies indicate that the carboxylic acid moiety, nitration at the 9 or 10 olefinic carbon, and unsaturation is required for XOR inhibition. Enzyme turnover and competitive reactivation studies reveal inhibition of electron transfer reactions at the molybdenum cofactor accounts for OA-NO[sub2]-induced inhibition. Importantly, OA-NO[sub2] more potently inhibits cell-associated XOR-dependent O[sub2] production than does allopurinol. Combined, these data establish a novel role for OA-NO[sub2] in the inhibition of XOR-derived oxidant formation. [ABSTRACT FROM AUTHOR]

One of the most important biological reactions of nitric oxide (nitrogen monoxide, •NO) is its reaction with transition metals, of which iron is the major target. This is confirmed by the ubiquitous formation of EPR-detectable g = 2.04 signals in cells, tissues, and animals upon exposure to both exogenous and endogenous •NO. The source of the iron for these dinitrosyliron complexes (DNIC), and its relationship to cellular iron homeostasis, is not clear. Evidence has shown that the chelatable iron pool (CIP) may be at least partially responsible for this iron, but quantitation and kinetic characterization have not been reported. In the murine cell line RAW 264.7, •NO reacts with the CIP similarly to the strong chelator salicylaldehyde isonicotinoyl hydrazone (SIH) in rapidly releasing iron from the iron-calcein complex. SIH pretreatment prevents DNIC formation from •NO, and SIH added during the •NO treatment "freezes" DNIC levels, showing that the complexes are formed from the CIP, and they are stable (resistant to SIH). DNIC formation requires free •NO, because addition of oxyhemoglobin prevents formation from either •NO donor or S-nitrosocysteine, the latter treatment resulting in 100-fold higher intracellular nitrosothiol levels. EPR measurement of the CIP using desferroxamine shows quantitative conversion of CIP into DNIC by •NO. In conclusion, the CIP is rapidly and quantitatively converted to paramagnetic large molecular mass DNIC from exposure to free •NO but not from cellular nitrosothiol. These results have important implications for the antioxidative actions of •NO and its effects on cellular iron homeostasis. [ABSTRACT FROM AUTHOR]

The aqueous decay and concomitant release of nitric oxide (˙NO) by nitrolinoleic acid (10-nitro-9,12-octadecadienoic acid and 12-nitro-9,12-octadecadienoic acid; LNO2) are reported. Mass spectrometric analysis of reaction products supports a modified Nef reaction as the mechanism accounting for the generation of ˙NO by the aqueous reactions of fatty acid nitroalkene derivatives. Nitrolinoleic acid is stabilized by an aprotic milieu, with LNO2 decay and ˙NO release strongly inhibited by phosphatidylcholine/cholesterol liposome membranes and detergents when present at levels above their critical micellar concentrations. The release of ˙NO from LNO2 was induced by UV photolysis and triiodide-based ozone chemiluminescence reactions currently used to quantify putative protein nitrosothiol and N-nitrosamine derivatives. This reactivity of LNO2 complicates the qualitative and quantitative analysis of biological oxides of nitrogen when applying UV photolysis and triiodide-based analytical systems to biological preparations typically abundant in nitrated fatty acids. The results reveal that nitroalkene derivatives of linoleic acid are pluripotent signaling mediators that act not only via receptor-dependent mechanisms, but also by transducing the signaling actions of ˙NO via pathways subject to regulation by the relative distribution of LNO2 to hydrophobic versus aqueous microenvironments. [ABSTRACT FROM AUTHOR]

Nitric oxide (NO[sup •]) inhibits mitochondrial respiration by binding to the binuclear heme a[sub 3]/Cu[sub B] center in cytochrome c oxidase. However, the significance of this reaction at physiological O[sub 2] levels (5-10 µM) and the effects of respiratory state are unknown. In this study mitochondrial respiration, absorption spectra, [O[sub 2]], and [NO[sup •]] were measured simultaneously at physiological O[sub 2] levels with constant O[sub 2] delivery, to model in vivo respiratory dynamics. Under these conditions NO[sup •] inhibited mitochondrial respiration with an IC[sub 50] of 0.14 ± 0.01 µM in state 3 versus 0.31 ± 0.04 µM in state 4. Spectral data indicate that the higher sensitivity of state 3 respiration to NO[sup •] is due to greater control over respiration by an NO[sup •]-dependent spectral species in the respiratory chain in this state. These results are discussed in the context of regulation of respiration by NO[sup •] in vivo and its implications for the control of vessel-parenchymal O[sub 2] gradients. [ABSTRACT FROM AUTHOR]