Different behavior of agmatine in liver mitochondria: inducer of oxidative stress or scavenger of reactive oxygen species?

Agmatine, at concentrations of 10 microM or 100 microM, is able to induce oxidative stress in rat liver mitochondria (RLM), as evidenced by increased oxygen uptake, H(2)O(2) generation, and oxidation of sulfhydryl groups and glutathione. One proposal for the production of H(2)O(2) and, most probably, other reactive oxygen species (ROS), is that they are the reaction products of agmatine oxidation by an unknown mitochondrial amine oxidase. Alternatively, by interacting with an iron-sulfur center of the respiratory chain, agmatine can produce an imino radical and subsequently the superoxide anion and other ROS. The observed oxidative stress causes a drop in ATP synthesis and amplification of the mitochondrial permeability transition (MPT) induced by Ca(2+). Instead, 1 mM agmatine generates larger amounts of H(2)O(2) than the lower concentrations, but does not affect RLM respiration or redox levels of thiols and glutathione. Indeed, it maintains the normal level of ATP synthesis and prevents Ca(2+)-induced MPT in the presence of phosphate. The self-scavenging effect against ROS production by agmatine at higher concentrations is also proposed.     

Modulation of sulfide oxidation and toxicity in rat mitochondria by dehydroascorbic acid

Hydrogen sulfide is enzymatically produced in mammalian tissues and functions as a gaseous transmitter. However, H(2)S is also highly toxic as it inhibits mitochondrial respiration at the level of cytochrome c oxidase, which additionally is involved in sulfide oxidation. The accumulation of toxic sulfide levels contributes to the pathology of some diseases. This paper demonstrates that sulfide toxicity can be modified, and dehydroascorbic acid functions as an effector in this process. It significantly reduces the inhibitory effect of sulfide on cytochrome c oxidase, resulting in higher rates of respiration and sulfide oxidation in rat mitochondria. After the addition of dehydroascorbic acid mitochondria maintained more than 50% of the oxygen consumption and ATP production rates with different substrates in the presence of high concentrations of sulfide that would normally lead to complete inhibition. Dehydroascorbic acid significantly increased the sulfide concentration necessary to cause half maximal inhibition of mitochondrial respiration and thus completely prevented inhibition at low, physiological sulfide concentrations. In addition, sulfide oxidation was stimulated and led to ATP production even at high concentrations. The decrease in sulfide toxicity was more pronounced when analyzing supermolecular functional units of the respiratory chain than in isolated cytochrome c oxidase activity. Furthermore, the protective effect of dehydroascorbic acid at high sulfide concentrations was completely abolished by quantitative solubilization of mitochondrial membrane proteins with dodeclymaltoside. These results suggest that binding of cytochrome c oxidase to other proteins probably within respiratory chain supercomplexes is involved in the modulation of sulfide oxidation and toxicity by dehydroascorbic acid.     

Rapid hydrogen sulfide consumption by Tetrahymena pyriformis and its implications for the origin of mitochondria

Although sulfide is typically regarded as toxic to eukaryotic cells, it is avidly consumed by Tetrahymena pyriformis. That was observed only when the sulfide concentration was kept below 1 microM. Previously concentrations that were too high had been tested. A new device (Sulfidostat) was used to measure sulfide consumption in steady-state concentrations as low as 10(-12)M. The technique was validated non-biologically by slowly injecting AgNO(3) into buffer and using Ag(2)S precipitation to mimic sulfide consumption, confirming that rates of sulfide consumption could be measured independently of sulfide concentrations. With T. pyriformis, sulfide consumption was 0.25 micromol (gprotein)(-1)s(-1) in 0.5 microM sulfide. Sulfide consumption required O(2) and was inhibited by HCN or by too much sulfide. When cells were separated into fractions, sulfide consumption occurred in the particulate (mitochondrial) fraction. Unexpectedly, the soluble cytosolic fraction slowly produced sulfide even when aerated. The observations are consistent with the conjecture that mitochondria evolved from sulfidotrophic symbionts in a sulfidogenic host cell.     

Chloride-Dependent Uncoupling of Oxidative Phosphorylation by Triethyllead and Triethyltin Increases Cytosolic Free Calcium in Guinea Pig Cerebral Cortical Synaptosomes

Metabolically competent isolated cerebral cortical nerve terminals were used to determine the effects of triethyllead (TEL) and triethyltin (TET) on cytosolic free calcium ([Ca2+]c), on plasma and mitochondrial membrane potentials, and on oxidative metabolism. In the presence of physiological concentrations of extracellular ions, 20 microM TEL and 20 microM TET increase [Ca2+]c from 185 nM to 390 and 340 nM, respectively. A simultaneous depolarization of plasma membrane potential (delta psi p) by only 3-4 mV occurs, a drop which is insufficient to open the voltage-sensitive Ca2+ channels. In contrast, an instant and substantial depolarization of mitochondrial membrane potential (delta psi m) upon addition of TEL and TET is evident, as monitored with safranine O fluorescence. At the same concentration, TEL and TET stimulate basal respiration of synaptosomes by 45%, induce oxidation of endogenous NAD(P)H, and reduce the terminal ATP/ADP ratio by 45%. Thus, TEL and TET inhibit ATP production of intrasynaptosomal mitochondria by a mechanism consistent with uncoupling of oxidative phosphorylation. This bioenergetic effect by TEL and TET can be prevented by omitting external chloride, and a concomitant reduction of the increase in [Ca2+]c by about 60% is observed. Uncoupling of mitochondrial ATP synthesis from oxidation by TEL and TET, [corrected] a process that is dependent on external chloride, is the main mechanism by which they [corrected] increase [Ca2+]c.     

Therapeutic doses of menadione reduce the rotenone-induced inhibition of respiration and membrane potential generation in mitochondria

Menadione restores the rotenone-inhibited respiration of diaphragm muscle pieces in approximately the same degree as the respiration of heart mitochondria, i.e., to 30-40%. The respiration of heart mitochondria induced by 2-5 microM menadione (after its inhibition by rotenone) is partly coupled with ATP synthesis whose rate is much lower than that of oxidation of NAD-dependent substrates. The effects of menadione and mitochondrial energetics inhibitors on lymphocyte respiration and rhodamine 123 fluorescence in individual lymphocytes and their suspensions were compared. Menadione (2--5 microM) increased the rotenone + oligomycin suppressed delta psi m in lymphocytes. At 5-40 microM menadione did not act as an uncoupler and had little effect on the uncoupled lymphocyte respiration. All these effects were observed at menadione concentrations close to therapeutic ones. Vicasol, a water-soluble analog of menadione, exerted a similar effect.     

The chloride channel blocker 5-nitro-2-(3-phenylpropyl-amino) benzoic acid (NPPB) uncouples mitochondria and increases the proton permeability of the plasma membrane in phagocytic cells.

We present evidence that the potent chloride channel blocker NPPB has protonophoric activity in the mitochondria and across the plasma membrane of phagocytic cells. The resting O2 consumption of murine peritoneal macrophages was stimulated up to 2.5-fold in the presence of NPPB, with a K0.5 of 15 microM. The stimulatory effect of NPPB on O2 consumption, like that of the classical protonophore CCCP, was prevented by the mitochondrial respiratory chain inhibitors antimycin A, rotenone or cyanide. NPPB also mediated rheogenic proton transport across the plasma membrane of human neutrophils and macrophages in the direction dictated by the electrochemical proton gradient. As a consequence of its protonophoric activity, NPPB uncoupled mitochondrial ATP synthesis, resulting in partial depletion of cellular ATP. These observations indicate that, at the concentrations frequently used for blockade of anion channels, NPPB acts as an effective protonophore, potentially disturbing cytosolic pH and mitochondrial ATP synthesis.     

Effects of 1,4-dihydropyridine derivatives (cerebrocrast, gammapyrone, glutapyrone, and diethone) on mitochondrial bioenergetics and oxidative stress: a comparative study

The potential protective action of 1,4-dihydropyridine derivatives (cerebrocrast, gammapyrone, glutapyrone, and diethone) against oxidative stress was assessed on mitochondrial bioenergetics, inner membrane anion channel (IMAC), Ca2+-induced opening of the permeability transition pore (PTP), and oxidative damage induced by the oxidant pair adenosine diphosphate (ADP)/Fe2+ (lipid peroxidation) of mitochondria isolated from rat liver. By using succinate as the respiratory substrate, respiratory control ratio (RCR), ADP to oxygen ratio (ADP/O), state 3, state 4, and uncoupled respiration rates were not significantly affected by gammapyrone, glutapyrone, and diethone concentrations up to 100 microM. Cerebrocrast at concentrations higher than 25 microM depressed RCR, ADP/O, state 3, and uncoupled respiration rates, but increased three times state 4 respiration rate. The transmembrane potential (deltapsi) and the phosphate carrier rate were also decreased. At concentrations lower than 25 microM, cerebrocrast inhibited the mitochondrial IMAC and partially prevented Ca2+-induced opening of the mitochondrial PTP, whereas gammapyrone, glutapyrone, and diethone were without effect. Cerebrocrast, gammapyrone, and glutapyrone concentrations up to 100 microM did not affect ADP/Fe2+-induced lipid peroxidation of rat liver mitochondria, while very low diethone concentrations (up to 5 microM) inhibited it in a dose-dependent manner, as measured by oxygen consumption and thiobarbituric acid reactive substances formation. Diethone also prevented deltapsi dissipation due to lipid peroxidation initiated by ADP/Fe2+. It can be concluded that: none of the compounds interfere with mitochondrial bioenergetics at concentrations lower than 25 microM; cerebrocrast was the only compound that affected mitochondrial bioenergetics, but only for concentrations higher than 25 microM; at concentrations that did not affect mitochondrial bioenergetics (< or = 25 microM), only cerebrocrast inhibited the IMAC and partially prevented Ca2+-induced opening of the PTP; diethone was the only compound that expressed antioxidant activity at very low concentrations (< or = 5 microM). Cerebrocrast acting as an inhibitor of the IMAC and diethone acting as an antioxidant could provide effective protective roles in preventing mitochondria from oxidative damage, favoring their therapeutic interest in the treatment of several pathological situations known to be associated with cellular oxidative stress.     

Mitochondrial adaptations to steatohepatitis induced by a methionine- and choline-deficient diet

Nonalcoholic fatty liver disease (NAFLD) has become common liver disease in Western countries. There is accumulating evidence that mitochondria play a key role in NAFLD. Nevertheless, the mitochondrial consequences of steatohepatitis are still unknown. The bioenergetic changes induced in a methionine- and choline-deficient diet (MCDD) model of steatohepatitis were studied in rats. Liver mitochondria from MCDD rats exhibited a higher rate of oxidative phosphorylation with various substrates, a rise in cytochrome oxidase (COX) activity, and an increased content in cytochrome aa3. This higher oxidative activity was associated with a low efficiency of the oxidative phosphorylation (ATP/O, i.e., number of ATP synthesized/natom O consumed). Addition of a low concentration of cyanide, a specific COX inhibitor, restored the efficiency of mitochondria from MCDD rats back to the control level. Furthermore, the relation between respiratory rate and protonmotive force (in the nonphosphorylating state) was shifted to the left in mitochondria from MCDD rats, with or without cyanide. These results indicated that, in MCDD rats, mitochondrial ATP synthesis efficiency was decreased in relation to both proton pump slipping at the COX level and increased proton leak although the relative contribution of each phenomenon could not be discriminated. MCDD mitochondria also showed a low reactive oxygen species production and a high lipid oxidation potential. We conclude that, in MCDD-fed rats, liver mitochondria exhibit an energy wastage that may contribute to limit steatosis and oxidative stress in this model of steatohepatitis.     

UCP2 regulates energy metabolism and differentiation potential of human pluripotent stem cells

It has been assumed, based largely on morphologic evidence, that human pluripotent stem cells (hPSCs) contain underdeveloped, bioenergetically inactive mitochondria. In contrast, differentiated cells harbour a branched mitochondrial network with oxidative phosphorylation as the main energy source. A role for mitochondria in hPSC bioenergetics and in cell differentiation therefore remains uncertain. Here, we show that hPSCs have functional respiratory complexes that are able to consume O(2) at maximal capacity. Despite this, ATP generation in hPSCs is mainly by glycolysis and ATP is consumed by the F(1)F(0) ATP synthase to partially maintain hPSC mitochondrial membrane potential and cell viability. Uncoupling protein 2 (UCP2) plays a regulating role in hPSC energy metabolism by preventing mitochondrial glucose oxidation and facilitating glycolysis via a substrate shunting mechanism. With early differentiation, hPSC proliferation slows, energy metabolism decreases, and UCP2 is repressed, resulting in decreased glycolysis and maintained or increased mitochondrial glucose oxidation. Ectopic UCP2 expression perturbs this metabolic transition and impairs hPSC differentiation. Overall, hPSCs contain active mitochondria and require UCP2 repression for full differentiation potential.     

Single eubacterial origin of eukaryotic sulfide:quinone oxidoreductase,a mitochondrial enzyme conserved from the early evolution of eukaryotes during anoxic and sulfidic times

Mitochondria occur as aerobic, facultatively anaerobic, and, in the case of hydrogenosomes, strictly anaerobic forms. This physiological diversity of mitochondrial oxygen requirement is paralleled by that of free-living alpha-proteobacteria, the group of eubacteria from which mitochondria arose, many of which are facultative anaerobes. Although ATP synthesis in mitochondria usually involves the oxidation of reduced carbon compounds, many alpha-proteobacteria and some mitochondria are known to use sulfide (H2S) as an electron donor for the respiratory chain and its associated ATP synthesis. In many eubacteria, the oxidation of sulfide involves the enzyme sulfide:quinone oxidoreductase (SQR). Nuclear-encoded homologs of SQR are found in several eukaryotic genomes. Here we show that eukaryotic SQR genes characterized to date can be traced to a single acquisition from a eubacterial donor in the common ancestor of animals and fungi. Yet, SQR is not a well-conserved protein, and our analyses suggest that the SQR gene has furthermore undergone some lateral transfer among prokaryotes during evolution, leaving the precise eubacterial lineage from which eukaryotes obtained their SQR difficult to discern with phylogenetic methods. Newer geochemical data and microfossil evidence indicate that major phases of early eukaryotic diversification occurred during a period of the Earth's history from 1 to 2 billion years before present in which the subsurface ocean waters contained almost no oxygen but contained high concentrations of sulfide, suggesting that the ability to deal with sulfide was essential for prokaryotes and eukaryotes during that time. Notwithstanding poor resolution in deep SQR phylogeny and lack of a specifically alpha-protebacterial branch for the eukaryotic enzyme on the basis of current lineage sampling, a single eubacterial origin of eukaryotic SQR and the evident need of ancient eukaryotes to deal with sulfide, a process today germane to mitochondrial quinone reduction, are compatible with the view that eukaryotic SQR was an acquisition from the mitochondrial endosymbiont.     

Reversible effects of fatty acids on respiration, oxidative phosphorylation, and heat production of rat liver mitochondria.

1. Oleic acid at low concentrations (0--70 nmol/mg protein) stimulated mitochondrial state 4 respiration 4-fold, increased the apparent enthalpy change of the respiration per gram atom of oxygen consumed from -112 to -208 kJ/O and completely inhibited ATP synthesis without significant effect on the Mg-ATPase activity of mitochondria. 2. Similar effects on mitochondrial respiratory activities were observed with other fatty acids. 3. Bovine serum albumin (BSA) protected mitochondria from the effects of oleic acid irrespective of the order of addition of oleic acid and BSA to mitochondria. The capacity of BSA to bind oleic acid was calculated to be 3.6--7.1 (mean, 4.9) mol of oleic acid/mol of BSA. 4. The response time of mitochondrial respiration to added oleic acid or BSA was 20--25 s.     

Nitric oxide and differential effects of ATP on mitochondrial permeability transition.

The mitochondrial permeability transition pore (PTP) undergoes a calcium-dependent transition (MPT) that disrupts membrane potential and releases apoptogenic proteins. Because PTP opening is enhanced by oxidation of thiols at the so-called "S-site," we hypothesized that nitrogen monoxide (NO*) could enhance the open probability of the PTP, e.g., by S-nitrosylation or S-thiolation. At low NO donor concentrations (1 to 20 microM), PTP opening in succinate-energized liver mitochondria at nonlimiting calcium was delayed or unaffected, while it was accelerated by NO donors at 20 to 100 microM. At low donor concentrations, PTP opening was facilitated twofold by adenosine triphosphate (ATP), which normally delays PTP opening. Among NO donors, the oxatriazole GEA 3162, with an activation constant (Ka) of 1.9 microM at 500 microM ATP was more effective at enhancing pore transition than SIN-1 or SNAP. NO donor effects were superseded by diamide, which induces disulfide formation, but independent of SH-adduct formation by alkylation. NO-related changes in PTP function were accompanied by protein mixed disulfide formation, inhibited by dithiothreitol (DTT), and reversed by DTT after donor addition. PTP opening was stimulated in the presence of ATP by L-arginine-dependent NO production, i.e., mitochondrial NOS activity. ATP-facilitated pore opening was sensitive to atractyloside and depended on nucleotide interactions but not on hydrolysis, because specific nonhydrolyzable ATP analogs accelerated pore opening. These data indicate NO can influence pore transition by oxidation of thiols that produce conformational changes governing the ATP interaction at the adenine nucleotide transporter.     

Inhibition of oxidative phosphorylation by organotin thiocarbamates

A series of triphenyl-, tricyclohexyl- and tribenzyltin compounds have been synthesized and examined as inhibitors of mitochondrial oxidative phosphorylation. All compounds tested inhibit oxidative phosphorylation linked to succinate oxidation by potato tuber mitochondria. All of the organotin compounds inhibit ADP-stimulated O2 uptake linked to succinate oxidation with concentrations for 50% inhibition in the range 2-50 microM. This inhibition is not due to inhibition of electron transport from succinate to O2 per se: none of the organotin compounds at 50 microM substantially inhibit the rate of succinate oxidation in the presence of 2,4-dinitrophenol. Representative organotin compounds at 0.5-50 microM do not act as uncouplers of succinate oxidation. It is concluded that the organotin compounds act as energy transfer inhibitors to inhibit oxidative phosphorylation in potato tuber mitochondria. A similar mode of action of representative organotin compounds was found with rat liver mitochondria. These organotin compounds inhibit a hydrophobic Ca2+-dependent plant protein kinase in the absence but not in the presence of thiols.     

The oxidation of long-chain unsaturated fatty acids by isolated rat liver mitochondria as a function of substrate concentration.

1. The oxidation of linoleate by rat-liver mitochondria has been studied as a function of substrate concentration. The oxidation of other long-chain unsaturated fatty acids shows similar characteristics. 2. At low concentrations, linoleate is readily oxidized in the absence of carnitine. Its rate of activation by the intramitochondrial acyl-CoA synthetase (EC 6.2.1.2) and subsequent oxidation is limited by the availability of intra-mitochondrial ATP. 3. A gradual increase of the linoleate concentration leads to (i) a strong depression of the rate of linoleate oxidation, and (ii) uncoupling of respiratory-chain phosphorylation together with induction of a mitochondrial ATPase activity. At still higher linoleate concentrations this ATPase activity is lowered rather than further stimulated and, concomitantly, the rate of linoleate oxidation increases again. 4. Evidence is presented that the inhibition by linoleate of the ATPase activity occurs at the level of the ATPase complex itself. This oligomycin-like effect of linoleate allows intramitochondrial linoleate activation to take place at the expense of ATP derived from substrate-level phosphorylation. 5.At very high concentrations of linoleate, its detergent action predominates and causes a complete inhibition of respiration as well as an extensive stimulation of an oligomycin-insensitive, Mg2+-dependent ATPase activity. 6. Measurement of the binding of radioactively labelled linoleate by isolated mitochondria shows that, at a given ratio of linoleate to mitochondrial protein, the ratio of bound to added linoleate is dependent on the concentration of the mitochondria.     

The phosphorus/oxygen ratio of mitochondrial oxidative phosphorylation.

The transport of ATP out of mitochondria and uptake of ADP and Pi into the matrix are coupled to the uptake of one proton (Klingenberg, M., and Rottenberg, H. (1977) Eur. J. Biochem. 73, 125--130). According to the chemiosmotic hypothesis of oxidative phosphorylation this coupling of nucleotide and Pi transport to proton transport implies that the P/O ratio for the synthesis and transport of ATP to the external medium is less than the P/O ratio for the synthesis of ATP inside mitochondria. A survey of previous determinations of the P/O ratio of intact mitochondria showed little convincing evidence in support of the currently accepted values of 3 with NADH-linked substrates and 2 with succinate. We have measured P/O ratios in rat liver mitochondria by the ADP pulse method and by 32 Pi esterification, measuring oxygen uptake with an oxygen electrode, and find values close to 2 with beta-hydroxybutyrate as substrate and 1.3 with succinate as substrate in the presence of rotenone to inhibit NADH oxidation. These values were largely independent of pH, temperature, Mg2+ ion concentration, Pi concentration, ADP pulse size, or amount of mitochondria used. We suggest that these are the true values of the P/O ratio for ATP synthesis and transport by mitochondria, and that previously reported higher values resulted from errors in the determination of oxygen uptake and the use of substrates which lead to ATP synthesis by succinate thiokinase.     

Reduced efficiency, but increased fat oxidation, in mitochondria from human skeletal muscle after 24-h ultraendurance exercise.

The hypothesis that ultraendurance exercise influences muscle mitochondrial function has been investigated. Athletes in ultraendurance performance performed running, kayaking, and cycling at 60% of their peak O(2) consumption for 24 h. Muscle biopsies were taken preexercise (Pre-Ex), postexercise (Post-Ex), and after 28 h of recovery (Rec). Respiration was analyzed in isolated mitochondria during state 3 (coupled to ATP synthesis) and state 4 (noncoupled respiration), with fatty acids alone [palmitoyl carnitine (PC)] or together with pyruvate (Pyr). Electron transport chain activity was measured with NADH in permeabilized mitochondria. State 3 respiration with PC increased Post-Ex by 39 and 41% (P < 0.05) when related to mitochondrial protein and to electron transport chain activity, respectively. State 3 respiration with Pyr was not changed (P > 0.05). State 4 respiration with PC increased Post-Ex but was lower than Pre-Ex at Rec (P < 0.05 vs. Pre-Ex). Mitochondrial efficiency [amount of added ADP divided by oxygen consumed during state 3 (P/O ratio)] decreased Post-Ex by 9 and 6% (P < 0.05) with PC and PC + Pyr, respectively. P/O ratio remained reduced at Rec. Muscle uncoupling protein 3, measured with Western blotting, was not changed Post-Ex but tended to decrease at Rec (P = 0.07 vs. Pre-Ex). In conclusion, extreme endurance exercise decreases mitochondrial efficiency. This will increase oxygen demand and may partly explain the observed elevation in whole body oxygen consumption during standardized exercise (+13%). The increased mitochondrial capacity for PC oxidation indicates plasticity in substrate oxidation at the mitochondrial level, which may be of advantage during prolonged exercise.     

The decrease of mitochondrial substrate uptake caused by trialkyltin and trialkyl-lead compounds in chloride media and its relevance to inhibition of oxidative phosphorylation.

1. In a 100 mM-KCl medium (pH 6.8) containing ATP, triethyltin (1 muM) causes a decrease in the uptake of pyruvate, malate, citrate or beta-hydroxybutyrate by rat liver mitochondria, but no decrease is observed in a 100 mM-KNO3 medium. This response is not modified by the presence of rotenone in the incubation medium. 2. In the KCl medium at least 1 muM-triethyltin is required to cause maximum inhibition of pyruvate uptake. 3. Trimethyltin, tributyltin and the trialkyl-lead analogues at 1 muM, to varying degrees, also cause a decrease in pyruvate uptake by mitochondria only in the KCl medium. 4. Triethyltin stimulates resting respiration of mitochondria with all the substrates tested in the KCl medium but not in the KNO3 medium, yet this stimulation of O2 uptake occurs under conditions when substrate uptake is decreased. 5. In contrast, both O2 uptake during state 3 respiration and ATP synthesis when linked to the oxidation of pyruvate, malate or citrate are strongly inhibited by 1 muM-triethyltin in a KCl medium, but O2 uptake and ATP synthesis during the oxidation of beta-hydroxybutyrate are only slightly affected. In a KNO3 medium O2 uptake and ATP synthesis linked to the oxidation of all substrates are only slightly affected. 6. The relevance of the decrease in substrate uptake by mitochondria caused by triethyltin in a KCl medium to the greater sensitivity of various mitochondrial functions observed in vitro is discussed. It is concluded that decrease of matrix substrate content is probably not the major cause of the greater sensitivity of oxidative phosphorylation to triethyltin in a KCl medium observed previously.     

Oxyconformity in the intertidal worm Sipunculus nudus: the mitochondrial background and energetic consequences

The energetic consequences of strict oxyconformity in the intertidal worm S. nudus were studied by characterizing the Po2 dependence of respiration in mitochondria isolated from the body wall tissue. Mitochondrial respiration rose in a Po2 range between 2.8 and 31.3 kPa from a mean of 56.5 to 223.9 nmol O mg protein(-1) h(-1). Respiration was sensitive to both salicylhydroxamic acid (SHAM) and KCN. Po2 dependence remained unchanged with saturating and non-saturating substrate levels (malate, glutamate and ADP). A concomitant decrease of the ATP/O ratio revealed a lower ATP yield of aerobic metabolism at elevated Po2. Obviously, oxyconforming respiration implies progressive uncoupling of mitochondria. The decrease in ATP/O ratios at higher Po2 was completely reversible. Addition of 90.9 micromol H2O2 l(-1) did not inhibit ATP synthesis. Both observations suggest that oxidative injury did not contribute to oxyconformity. The contribution of the rates of mitochondrial ROS production and proton leakiness to mitochondrial oxygen consumption and uncoupling was investigated by using oligomycin as a specific inhibitor of the ATP synthase. The maximum contribution of oligomycin independent respiration to state 3 respiration remained below 6% and showed a minor, insignificant increase at elevated Po2, at a slope significantly lower than the increment of state 3 respiration. Therefore, Po2 dependent mitochondrial proton leakage or ROS production cannot explain oxyconformity. In conclusion Po2 dependent state 3 respiration likely relates to the progressive contribution of an alternative oxidase (cytochrome o), which is characterized by a low affinity to oxygen and an ATP/O ratio similar to the branched respiratory system of bacteria. The molecular nature of the alternative oxidase in lower invertebrates is still obscure.     

Involvement of the energy-dissipating systems in modulating the energetic efficiency of respiration in mitochondria from etiolated winter wheat seedlings

Effects of cyanide-resistant alternative oxidase (AOX) and modulators of plant uncoupling mitochondrial proteins (PUMP) on respiration rate and generation of transmembrane electric potential (ΔΨ) were investigated during oxidation of various substrates by isolated mitochondria from etiolated coleoptiles of winter wheat (Triticum aestivum L.). Oxidative phosphorylation in wheat mitochondria during malate and succinate oxidation was quite effective (it was characterized by high respiratory control ratio as defined by Chance, high ADP/O ratio, and rapid ATP synthesis). Nevertheless, the effectiveness of oxidative phosphorylation was substantially modulated by operation of energy-dissipating systems. The application of safranin dye revealed the partial dissipation of ΔΨ during inhibition of cytochrome-mediated malate oxidation by cyanide and antimycin A and demonstrated the operation of AOX-dependent compensatory mechanism for ΔΨ generation. The complex I of mitochondrial electron transport chain was shown to play the dominant role in ΔΨ generation and ATP synthesis during AOX functioning upon inhibition of electron transport through the cytochrome pathway. Effects of linoleic acid (PUMP activator) at physiologically low concentrations (4–10 μM) on respiration and ΔΨ generation in mitochondria were examined. The uncoupling effect of linoleic acid was shown in activation of the State 4 respiration, as well as in ΔΨ dissipation; this effect was eliminated in the presence of BSA but was insensitive to purine nucleotides. The uncoupling effect of linoleic acid was accompanied by reversible inhibition of AOX activity. The results are discussed with regard to possible physiological role of mitochondrial energy-dissipating systems in regulation of energy transduction in plant cells under stress conditions.     

Deleterious action of FA metabolites on ATP synthesis: possible link between lipotoxicity, mitochondrial dysfunction, and insulin resistance

Insulin resistance is a characteristic feature of type 2 diabetes and obesity. Insulin-resistant individuals manifest multiple disturbances in free fatty acid (FFA) metabolism and have excessive lipid accumulation in insulin target tissues. Although much evidence supports a causal role for altered FFA metabolism in the development of insulin resistance, i.e., "lipotoxicity", the intracellular mechanisms by which elevated plasma FFA levels cause insulin resistance have yet to be completely elucidated. Recent studies have implicated a possible role for mitochondrial dysfunction in the pathogenesis of insulin resistance in skeletal muscle. We examined the effect of FFA metabolites [palmitoyl carnitine (PC), palmitoyl-coenzyme A (CoA), and oleoyl-CoA] on ATP synthesis in mitochondria isolated from mouse and human skeletal muscle. At concentrations ranging from 0.5 to 2 microM, these FFA metabolites stimulated ATP synthesis; however, above 5 microM, there was a dose-response inhibition of ATP synthesis. Furthermore, 10 microM PC inhibits ATP synthesis from pyruvate. Elevated PC concentrations (> or =10 microM) inhibit electron transport chain activity and decrease the mitochondrial inner membrane potential. These acquired mitochondrial defects, caused by a physiological increase in the concentration of FFA metabolites, provide a mechanistic link between lipotoxicity, mitochondrial dysfunction, and muscle insulin resistance.