Adipocyte-specific Nrf2 Deletion Negates Nitro-Oleic Acid Benefits on Glucose Tolerance in Diet-Induced Obesity.

Obesity is commonly linked with adipose tissue (AT) dysfunction, setting off inflammation and oxidative stress, both key contributors to the cardiometabolic complications associated with obesity. To improve metabolic and cardiovascular health, countering these inflammatory and oxidative signaling processes is crucial. Offering potential in this context, the activation of nuclear factor erythroid 2-related factor 2 (Nrf2) by nitro-fatty acids (NO2-FA) promote diverse anti-inflammatory signaling and counteract oxidative stress. Additionally, we previously highlighted that nitro-oleic acid (NO2-OA) preferentially accumulates in white adipose tissue (AT) and provides protection against already established high fat diet (HFD)-mediated impaired glucose tolerance. The precise mechanism accounting for these protective effects remained largely unexplored until now. Herein, we reveal that protective effects of improved glucose tolerance by NO2-OA is absent when Nrf2 is specifically ablated in adipocytes (ANKO mice). NO2-OA treatment did not alter body weight between ANKO and littermate controls (Nrf2fl/fl) mice on both the HFD and low-fat diet (LFD). As expected, at day 76 (before NO2-OA treatment) and notably at day 125 (daily treatment of 15 mg/kg NO2-OA for 48 days), both HFD-fed Nrf2fl/fl and ANKO mice exhibited increased fat mass and reduced lean mass compared to LFD controls. However, throughout the NO2-OA treatment, no distinction was observed between Nrf2fl/fl and ANKO in the HFD-fed mice as well as in the Nrf2fl/fl mice fed a LFD. Glucose tolerance tests revealed impaired glucose tolerance in HFD-fed Nrf2fl/fl and ANKO compared to LFD-fed Nrf2fl/fl mice. Notably, NO2-OA treatment improved glucose tolerance in HFD-fed Nrf2fl/fl but did not yield the same improvement in ANKO mice at days 15, 30, and 55 of treatment. Unraveling the pathways linked to NO2-OA's protective effects in obesity-mediated impairment in glucose tolerance is pivotal within the realm of precision medicine, crucially propelling future applications and refining novel drug-based strategies.

A FABP4-PPARγ signaling axis regulates human monocyte responses to electrophilic fatty acid nitroalkenes.

Nitro-fatty acids (NO2-FA) are electrophilic lipid mediators derived from unsaturated fatty acid nitration. These species are produced endogenously by metabolic and inflammatory reactions and mediate anti-oxidative and anti-inflammatory responses. NO2-FA have been postulated as partial agonists of the Peroxisome Proliferator-Activated Receptor gamma (PPARγ), which is predominantly expressed in adipocytes and myeloid cells. Herein, we explored molecular and cellular events associated with PPARγ activation by NO2-FA in monocytes and macrophages. NO2-FA induced the expression of two PPARγ reporter genes, Fatty Acid Binding Protein 4 (FABP4) and the scavenger receptor CD36, at early stages of monocyte differentiation into macrophages. These responses were inhibited by the specific PPARγ inhibitor GW9662. Attenuated NO2-FA effects on PPARγ signaling were observed once cells were differentiated into macrophages, with a significant but lower FABP4 upregulation, and no induction of CD36. Using in vitro and in silico approaches, we demonstrated that NO2-FA bind to FABP4. Furthermore, the inhibition of monocyte FA binding by FABP4 diminished NO2-FA-induced upregulation of reporter genes that are transcriptionally regulated by PPARγ, Keap1/Nrf2 and HSF1, indicating that FABP4 inhibition mitigates NO2-FA signaling actions. Overall, our results affirm that NO2-FA activate PPARγ in monocytes and upregulate FABP4 expression, thus promoting a positive amplification loop for the downstream signaling actions of this mediator.

Electrophilic nitro-fatty acids suppress allergic contact dermatitis in mice.

BACKGROUND: Reactions between nitric oxide (NO), nitrite (NO2-), and unsaturated fatty acids give rise to electrophilic nitro-fatty acids (NO2 -FAs), such as nitro oleic acid (OA-NO2 ) and nitro linoleic acid (LNO2 ). Endogenous electrophilic fatty acids (EFAs) mediate anti-inflammatory responses by modulating metabolic and inflammatory signal transduction reactions. Hence, there is considerable interest in employing NO2 -FAs and other EFAs for the prevention and treatment of inflammatory disorders. Thus, we sought to determine whether OA-NO2 , an exemplary nitro-fatty acid, has the capacity to inhibit cutaneous inflammation. METHODS: We evaluated the effect of OA-NO2 on allergic contact dermatitis (ACD) using an established model of contact hypersensitivity in C57Bl/6 mice utilizing 2,4-dinitrofluorobenzene as the hapten. RESULTS: We found that subcutaneous (SC) OA-NO2 injections administered 18 h prior to sensitization and elicitation suppresses ACD in both preventative and therapeutic models. In vivo SC OA-NO2 significantly inhibits pathways that lead to inflammatory cell infiltration and the production of inflammatory cytokines in the skin. Moreover, OA-NO2 is capable of enhancing regulatory T-cell activity. Thus, OA-NO2 treatment results in anti-inflammatory effects capable of inhibiting ACD by inducing immunosuppressive responses. CONCLUSION: Overall, these results support the development of OA-NO2 as a promising therapeutic for ACD and provides new insights into the role of electrophilic fatty acids in the control of cutaneous immune responses potentially relevant to a broad range of allergic and inflammatory skin diseases.

Quantum Hall resistance standard in graphene devices under relaxed experimental conditions.

The quantum Hall effect provides a universal standard for electrical resistance that is theoretically based on only the Planck constant h and the electron charge e. Currently, this standard is implemented in GaAs/AlGaAs, but graphene's electronic properties have given hope for a more practical device. Here, we demonstrate that the experimental conditions necessary for the operation of devices made of high-quality graphene grown by chemical vapour deposition on silicon carbide can be extended and significantly relaxed compared with those for state-of-the-art GaAs/AlGaAs devices. In particular, the Hall resistance can be accurately quantized to within 1 × 10(-9) over a 10 T wide range of magnetic flux density, down to 3.5 T, at a temperature of up to 10 K or with a current of up to 0.5 mA. This experimental simplification highlights the great potential of graphene in the development of user-friendly and versatile quantum standards that are compatible with broader industrial uses beyond those in national metrology institutes. Furthermore, the measured agreement of the quantized Hall resistance in graphene and GaAs/AlGaAs, with an ultimate uncertainty of 8.2 × 10(-11), supports the universality of the quantum Hall effect. This also provides evidence of the relation of the quantized Hall resistance with h and e, which is crucial for the new Système International d'unités to be based on fixing such fundamental constants of nature.

Quantum Hall resistance standards from graphene grown by chemical vapour deposition on silicon carbide.

Replacing GaAs by graphene to realize more practical quantum Hall resistance standards (QHRS), accurate to within 10(-9) in relative value, but operating at lower magnetic fields than 10 T, is an ongoing goal in metrology. To date, the required accuracy has been reported, only few times, in graphene grown on SiC by Si sublimation, under higher magnetic fields. Here, we report on a graphene device grown by chemical vapour deposition on SiC, which demonstrates such accuracies of the Hall resistance from 10 T up to 19 T at 1.4 K. This is explained by a quantum Hall effect with low dissipation, resulting from strongly localized bulk states at the magnetic length scale, over a wide magnetic field range. Our results show that graphene-based QHRS can replace their GaAs counterparts by operating in as-convenient cryomagnetic conditions, but over an extended magnetic field range. They rely on a promising hybrid and scalable growth method and a fabrication process achieving low-electron-density devices.

High Electron Mobility in Epitaxial Graphene on 4H-SiC(0001) via post-growth annealing under hydrogen.

We investigate the magneto-transport properties of epitaxial graphene single-layer on 4H-SiC(0001), grown by atmospheric pressure graphitization in Ar, followed by H2 intercalation. We directly demonstrate the importance of saturating the Si dangling bonds at the graphene/SiC(0001) interface to achieve high carrier mobility. Upon successful Si dangling bonds elimination, carrier mobility increases from 3 000 cm(2)V(-1)s(-1) to >11 000 cm(2)V(-1)s(-1) at 0.3 K. Additionally, graphene electron concentration tends to decrease from a few 10(12) cm(-2) to less than 10(12) cm(-2). For a typical large (30 × 280 µm(2)) Hall bar, we report the observation of the integer quantum Hall states at 0.3 K with well developed transversal resistance plateaus at Landau level filling factors of ν = 2, 6, 10, 14... 42 and Shubnikov de Haas oscillation of the longitudinal resistivity observed from about 1 T. In such a device, the Hall state quantization at ν = 2, at 19 T and 0.3 K, can be very robust: the dissipation in electronic transport can stay very low, with the longitudinal resistivity lower than 5 mΩ, for measurement currents as high as 250 µA. This is very promising in the view of an application in metrology.

Nitro-oleic acid targets transient receptor potential (TRP) channels in capsaicin sensitive afferent nerves of rat urinary bladder.

Nitro-oleic acid (9- and 10-nitro-octadeca-9-enoic acid, OA-NO(2)) is an electrophilic fatty acid nitroalkene derivative that modulates gene transcription and protein function via post-translational protein modification. Nitro-fatty acids are generated from unsaturated fatty acids by oxidative inflammatory reactions and acidic conditions in the presence of nitric oxide or nitrite. Nitroalkenes react with nucleophiles such as cysteine and histidine in a variety of susceptible proteins including transient receptor potential (TRP) channels in sensory neurons of the dorsal root and nodose ganglia. The present study revealed that OA-NO(2) activates TRP channels on afferent nerve terminals in the urinary bladder and thereby increases bladder activity. The TRPV1 agonist capsaicin (CAPS, 1 µM) and the TRPA1 agonist allyl isothiocyanate (AITC, 30 µM), elicited excitatory effects in bladder strips, increasing basal tone and amplitude of phasic bladder contractions (PBC). OA-NO(2) mimicked these effects in a concentration-dependent manner (1 µM-33 µM). The TRPA1 antagonist HC3-030031 (HC3, 30 µM) and the TRPV1 antagonist diaryl piperazine analog (DPA, 1 µM), reduced the effect of OA-NO(2) on phasic contraction amplitude and baseline tone. However, the non-selective TRP channel blocker, ruthenium red (30 µM) was a more effective inhibitor, reducing the effects of OA-NO(2) on basal tone by 75% and the effects on phasic amplitude by 85%. In bladder strips from CAPS-treated rats, the effect of OA-NO(2) on phasic contraction amplitude was reduced by 65% and the effect on basal tone was reduced by 60%. Pretreatment of bladder strips with a combination of neurokinin receptor antagonists (NK1 selective antagonist, CP 96345; NK2 selective antagonist, MEN 10,376; NK3 selective antagonist, SB 234,375, 1 µM each) reduced the effect of OA-NO(2) on basal tone, but not phasic contraction amplitude. These results indicate that nitroalkene fatty acid derivatives can activate TRP channels on CAPS-sensitive afferent nerve terminals, leading to increased bladder contractile activity. Nitrated fatty acids produced endogenously by the combination of fatty acids and oxides of nitrogen released from the urothelium and/or afferent nerves may play a role in modulating bladder activity.

Nitro-oleic acid inhibits firing and activates TRPV1- and TRPA1-mediated inward currents in dorsal root ganglion neurons from adult male rats.

Nitro-oleic acid (OA-NO(2)), an electrophilic fatty acid by-product of nitric oxide and nitrite reactions, is present in normal and inflamed mammalian tissues at up to micromolar concentrations and exhibits anti-inflammatory signaling actions. The effects of OA-NO(2) on cultured dorsal root ganglion (DRG) neurons were examined using fura-2 Ca(2+) imaging and patch clamping. OA-NO(2) (3.5-35 microM) elicited Ca(2+) transients in 20 to 40% of DRG neurons, the majority (60-80%) of which also responded to allyl isothiocyanate (AITC; 1-50 microM), a TRPA1 agonist, and to capsaicin (CAPS; 0.5 microM), a TRPV1 agonist. The OA-NO(2)-evoked Ca(2+) transients were reduced by the TRPA1 antagonist 2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)-N-(4-isopropylphenyl) acetamide (HC-030031; 5-50 microM) and the TRPV1 antagonist capsazepine (10 microM). Patch-clamp recording revealed that OA-NO(2) depolarized and induced inward currents in 62% of neurons. The effects of OA-NO(2) were elicited by concentrations >or=5 nM and were blocked by 10 mM dithiothreitol. Concentrations of OA-NO(2) >or=5 nM reduced action potential (AP) overshoot, increased AP duration, inhibited firing induced by depolarizing current pulses, and inhibited Na(+) currents. The effects of OA-NO(2) were not prevented or reversed by the NO-scavenger carboxy-2-phenyl-4,4,5,5-tetramethylimidazolineoxyl-1-oxyl-3-oxide. A large percentage (46-57%) of OA-NO(2)-responsive neurons also responded to CAPS (0.5 microM) or AITC (0.5 microM). OA-NO(2) currents were reduced by TRPV1 (diarylpiperazine; 5 microM) or TRPA1 (HC-030031; 5 microM) antagonists. These data reveal that endogenous OA-NO(2) generated at sites of inflammation may initially activate transient receptor potential channels on nociceptive afferent nerves, contributing to the initiation of afferent nerve activity, and later suppresses afferent firing.

Detection and quantification of protein adduction by electrophilic fatty acids: mitochondrial generation of fatty acid nitroalkene derivatives.

Nitroalkene fatty acid derivatives manifest a strong electrophilic nature, are clinically detectable, and induce multiple transcriptionally regulated anti-inflammatory responses. At present, the characterization and quantification of endogenous electrophilic lipids are compromised by their Michael addition with protein and small-molecule nucleophilic targets. Herein, we report a trans-nitroalkylation reaction of nitro-fatty acids with beta-mercaptoethanol (BME) and apply this reaction to the unbiased identification and quantification of reaction with nucleophilic targets. Trans-nitroalkylation yields are maximal at pH 7 to 8 and occur with physiological concentrations of target nucleophiles. This reaction is also amenable to sensitive mass spectrometry-based quantification of electrophilic fatty acid-protein adducts upon electrophoretic resolution of proteins. In-gel trans-nitroalkylation reactions also permit the identification of protein targets without the bias and lack of sensitivity of current proteomic approaches. Using this approach, it was observed that fatty acid nitroalkenes are rapidly metabolized in vivo by a nitroalkene reductase activity and mitochondrial beta-oxidation, yielding a variety of electrophilic and nonelectrophilic products that could be structurally characterized upon BME-based trans-nitroalkylation reaction. This strategy was applied to the detection and quantification of fatty acid nitration in mitochondria in response to oxidative inflammatory conditions induced by myocardial ischemia-reoxygenation.

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.

Oxidation of ubiquinol by peroxynitrite: implications for protection of mitochondria against nitrosative damage.

A major pathway of nitric oxide utilization in mitochondria is its conversion to peroxynitrite, a species involved in biomolecule damage via oxidation, hydroxylation and nitration reactions. In the present study the potential role of mitochondrial ubiquinol in protecting against peroxynitrite-mediated damage is examined and the requirements of the mitochondrial redox status that support this function of ubiquinol are established. (1) Absorption and EPR spectroscopy studies revealed that the reactions involved in the ubiquinol/peroxynitrite interaction were first-order in peroxynitrite and zero-order in ubiquinol, in agreement with the rate-limiting formation of a reactive intermediate formed during the isomerization of peroxynitrite to nitrate. Ubiquinol oxidation occurred in one-electron transfer steps as indicated by the formation of ubisemiquinone. (2) Peroxynitrite promoted, in a concentration-dependent manner, the formation of superoxide anion by mitochondrial membranes. (3) Ubiquinol protected against peroxynitrite-mediated nitration of tyrosine residues in albumin and mitochondrial membranes, as suggested by experimental models, entailing either addition of ubiquinol or expansion of the mitochondrial ubiquinol pool caused by selective inhibitors of complexes III and IV. (4) Increase in membrane-bound ubiquinol partially prevented the loss of mitochondrial respiratory function induced by peroxynitrite. These findings are analysed in terms of the redox transitions of ubiquinone linked to both nitrogen-centred radical scavenging and oxygen-centred radical production. It may be concluded that the reaction of mitochondrial ubiquinol with peroxynitrite is part of a complex regulatory mechanism with implications for mitochondrial function and integrity.

The regulation of mitochondrial oxygen uptake by redox reactions involving nitric oxide and ubiquinol.

The reversible inhibitory effects of nitric oxide (.NO) on mitochondrial cytochrome oxidase and O(2) uptake are dependent on intramitochondrial.NO utilization. This study was aimed at establishing the mitochondrial pathways for.NO utilization that regulate O-(2) generation via reductive and oxidative reactions involving ubiquinol oxidation and peroxynitrite (ONOO(-)) formation. For this purpose, experimental models consisting of intact mitochondria, ubiquinone-depleted/reconstituted submitochondrial particles, and ONOO(-)-supplemented mitochondrial membranes were used. The results obtained from these experimental approaches strongly suggest the occurrence of independent pathways for.NO utilization in mitochondria, which effectively compete with the binding of.NO to cytochrome oxidase, thereby releasing this inhibition and restoring O(2) uptake. The pathways for.NO utilization are discussed in terms of the steady-state levels of.NO and O-(2) and estimated as a function of O(2) tension. These calculations indicate that mitochondrial.NO decays primarily by pathways involving ONOO(-) formation and ubiquinol oxidation and, secondarily, by reversible binding to cytochrome oxidase.

Circulating plasma factors in Parkinson's disease enhance nitric oxide release of normal human neutrophils.

Nitric oxide (*NO)-mediated toxicity has been involved in neurodegenerative diseases, including Parkinson's disease (PD). We have recently reported an increase of about 50% in *NO production rate in PMA-activated polymorphonuclear leukocytes (PMN) from either newly diagnosed or chronically treated PD patients. As humoral factors in sera from PD patients could inhibit cell dopaminergic activity, the aim of this study was to determine whether a plasma circulating factor from PD patients could modify *NO metabolism in PMN from healthy control subjects. To this purpose, we determined simultaneously the maximal production rate of *NO and hydrogen peroxide (H2O2) of PMA-activated PMN isolated from healthy control subjects in the presence of aliquots of plasma of PD patients. The results showed that, after 30 min incubation, plasma from newly diagnosed (n=4) or from L-Dopa chronically treated (n=7) PD patients enhanced *NO release in neutrophils isolated from healthy controls by about 50% and 47% respectively, with respect to non-parkinsonian control plasma (n = 10); in the same condition, H2O2 production did not differ among the groups. These data suggest that an overproduction of *NO related to plasma circulating factors, already detected at initial stages of the disease, participates in the pathophysiology of Parkinson's disease.

The reaction of nitric oxide with ubiquinol: kinetic properties and biological significance.

The reaction of nitric oxide (*NO) with ubiquinol-0 and ubiquinol-2, short-chain analogs of coenzyme Q, was examined in anaerobic and aerobic conditions in terms of formation of intermediates and stable molecular products. The chemical reactivity of ubiquinol-0 and ubiquinol-2 towards *NO differed only quantitatively, the reactions of ubiquinol-2 being slightly faster than those of ubiquinol-0. The ubiquinol/*NO reaction entailed oxidation of ubiquinol to ubiquinone and reduction of *NO to NO-, the latter identified by its reaction with metmyoglobin to form nitroxylmyoglobin and indirectly by measurement of nitrous oxide (N2O) by gas chromatography. Both the rate of ubiquinone accumulation and *NO consumption were linearly dependent on ubiquinol and *NO concentrations. The stoichiometry of *NO consumed per either ubiquinone formed or ubiquinol oxidized was 1.86 A 0.34. The reaction of *NO with ubiquinols proceeded with intermediate formation of ubisemiquinones that were detected by direct EPR. The second order rate constants of the reactions of ubiquinol-0 and ubiquinol-2 with *NO were 0.49 and 1.6 x 10(4) M(-1)s(-1), respectively. Studies in aerobic conditions revealed that the reaction of *NO with ubiquinols was associated with O2 consumption. The formation of oxyradicals - identified by spin trapping EPR- during ubiquinol autoxidation was inhibited by *NO, thus indicating that the O2 consumption triggered by *NO could not be directly accounted for in terms of oxyradical formation or H2O2 accumulation. It is suggested that oxyradical formation is inhibited by the rapid removal of superoxide anion by *NO to yield peroxynitrite, which subsequently may be involved in the propagation of ubiquinol oxidation. The biological significance of the reaction of ubiquinols with *NO is discussed in terms of the cellular O2 gradients, the steady-state levels of ubiquinols and *NO, and the distribution of ubiquinone (largely in its reduced form) in biological membranes with emphasis on the inner mitochondrial membrane.

[Shock: concepts for a definition]. / Shock: conceptos para una definición.

The shock syndrome has been classically considered as a consequence of both decreased tissue perfusion and O2 supply; however, in some types of shock like septic or traumatic ones, regional blood flows may be increased. A decade ago, mitochondrial alterations consistent with uncoupling of oxidative phosphorylation were reported in either endotoxemic or hemorrhagic experimental shock or in humans. Recently, the discovery of nitric oxide (NO) and its increase in the shock state, has opened new perspectives in the understanding of this problem. Nitric oxide produces vasodilatation and, at the same time, increases the mitochondrial production of O2 active species like superoxide anion. Both radicals react to form a strong oxidant that is able to nitrate the phenolic rings of proteins: peroxynitrite. This effect leads to the impairment of the activities of different mitochondrial enzymes like succinate dehydrogenase and ATPase and the mitochondrial function and finally, to decreased energy levels and to multiorgan failure. The increase in NO release is due to the effects of circulating peptides and of increased adhesion of neutrophils to the endothelium and to the positive effects of inflammatory mediators like TNF-alpha and cytokines on inducible NOS (iNOS) expression in endothelium and tissues. It is suggested that the shock state is the consequence of an imbalance between NO and O2 and their metabolites.

Nitric oxide regulates oxygen uptake and hydrogen peroxide release by the isolated beating rat heart.

Isolated rat heart perfused with 1.5-7.5 microM NO solutions or bradykinin, which activates endothelial NO synthase, showed a dose-dependent decrease in myocardial O2 uptake from 3.2 +/- 0.3 to 1.6 +/- 0.1 (7.5 microM NO, n = 18, P < 0.05) and to 1.2 +/- 0.1 microM O2.min-1.g tissue-1 (10 microM bradykinin, n = 10, P < 0.05). Perfused NO concentrations correlated with an induced release of hydrogen peroxide (H2O2) in the effluent (r = 0.99, P < 0.01). NO markedly decreased the O2 uptake of isolated rat heart mitochondria (50% inhibition at 0.4 microM NO, r = 0.99, P < 0.001). Cytochrome spectra in NO-treated submitochondrial particles showed a double inhibition of electron transfer at cytochrome oxidase and between cytochrome b and cytochrome c, which accounts for the effects in O2 uptake and H2O2 release. Most NO was bound to myoglobin; this fact is consistent with NO steady-state concentrations of 0.1-0.3 microM, which affect mitochondria. In the intact heart, finely adjusted NO concentrations regulate mitochondrial O2 uptake and superoxide anion production (reflected by H2O2), which in turn contributes to the physiological clearance of NO through peroxynitrite formation.

Nitric oxide inhibits electron transfer and increases superoxide radical production in rat heart mitochondria and submitochondrial particles.

Nitric oxide (.NO) released by S-nitrosoglutathione (GSNO) inhibited enzymatic activities of rat heart mitochondrial membranes. Cytochrome oxidase activity was inhibited to one-half at an effective .NO concentration of 0.1 microM, while succinate- and NADH-cytochrome-c reductase activities were half-maximally inhibited at 0.3 microM .NO. Submitochondrial particles treated with .NO (either from GSNO or from a pure solution) showed increased O(-)(2) and H202 production when supplemented with succinate alone, at rates that were comparable to those of control particles with added succinate and antimycin. Rat heart mitochondria treated with .NO also showed increased H2O2 production. Cytochrome spectra and decreased enzymatic activities in the presence of .NO are consistent with a multiple inhibition of mitochondrial electron transfer at cytochrome oxidase and at the ubiquinone-cytochrome b region of the respiratory chain, the latter leading to the increased O2- production. Electrochemical detection showed that the buildup of a .NO concentration from GSNO was interrupted by submitochondrial particles supplemented with succinate and antimycin and was restored by addition of superoxide dismutase. The inhibitory effect of .NO on cytochrome oxidase was also prevented under the same conditions. Apparently, mitochondrial O2- reacts with .NO to form peroxynitrate and, by removing .NO, reactivates the previously inhibited cytochrome oxidase. It is suggested that, at physiological concentrations of .NO, inhibition of electron transfer, .NO-induced O2- production, and ONOO- formation participate in the regulatory control of mitochondrial oxygen uptake.

Heavy ion effects on yeast cells: reappearance of the oxygen effect with very high LET.

Survival of a diploid and a haploid wild type and a radiation-sensitive rad52-mutant was investigated after exposure to accelerated ions in the presence or absence of oxygen. Ar, Kr, Xe, Sm, Pb and U ions were used with specific energies between 0.3 and 12 MeV/u. The results demonstrate that the oxygen enhancement ratios (o.e.r.) do not only depend on LET or Z*2/beta 2 but even more so on ion specific energy. The o.e.r.s are always higher with greater E/m values pointing to the importance of delta-electron action.