Changes of Respiratory Chain Activity in Mitochondrial and Synaptosomal Fractions Isolated from the Gerbil Brain After Graded Ischaemia

Abstract: In this study we have examined (1) the integrated function of the mitochondrial respiratory chain by polarographic measurements and (2) the activities of the respiratory chain complexes I, II–III, and IV as well as the ATP synthase (complex V) in free mitochondria and synaptosomes isolated from gerbil brain, after a 30-min period of graded cerebral ischaemia. These data have been correlated with cerebral blood flow (CBF) values as measured by the hydrogen clearance technique. Integrated functioning of the mitochondrial respiratory chain, using both NAD-linked and FAD-linked substrates, was initially affected at CBF values of ∼35 ml 100 g⁻¹ min⁻¹, and declined further as the CBF was reduced. The individual mitochondrial respiratory chain complexes, however, showed differences in sensitivity to graded cerebral ischaemia. Complex I activities decreased sharply at blood flows below ∼30 ml 100 g⁻¹ min⁻¹ (mitochondria and synaptosomes) and complex II–III activities decreased at blood flows below 20 ml 100 g⁻¹ min⁻¹ (mitochondria) and 35–30 ml 100 g⁻¹ min⁻¹ (synaptosomes). Activities declined further as CBF was reduced below these levels. Complex V activity was significantly affected only when the blood flow was reduced below 15–10 ml 100 g⁻¹ min⁻¹ (mitochondria and synaptosomes). In contrast, complex IV activity was unaffected by graded cerebral ischaemia, even at very low CBF levels.     

Selective Impairment of Respiration in Mitochondria Isolated from Brain Subregions Following Transient Forebrain Ischemia in the Rat

Using Percoll density gradient centrifugation, free (nonsynaptosomal) mitochondria were isolated from the dorsal-lateral striatum and paramedian neocortex of rats during complete forebrain ischemia and reperfusion. Mitochondria prepared from either region after 30 min of ischemia showed decreased state 3 (ADP and substrate present) and uncoupled respiration rates (19-45% reductions) with pyruvate plus malate as substrates, whereas state 4 respiration (no ADP present) was preserved. At 6 h of recirculation, state 3 and uncoupled respiration rates for mitochondria from the paramedian neocortex (a region resistant to ischemic damage) were similar to or even increased compared with control values. By contrast, in mitochondria from the dorsal-lateral striatum (a region containing neurons susceptible to global ischemia), decreases in state 3 and uncoupled respiration rates (25 and 30% less than control values) were again observed after 6 h of recirculation. With succinate as respiratory substrate, however, no significant differences from control values were found in either region at this time point. By 24 h of recirculation, respiratory activity with either pyruvate plus malate or succinate was greatly reduced in samples from the dorsal-lateral striatum, probably reflecting complete loss of function in some organelles. In contrast with these marked changes in free mitochondria, the respiratory properties of synaptosomal mitochondria, assessed from measurements in unfractionated homogenates, were unchanged from controls in the dorsal-lateral striatum at each of the time points studied, but showed reductions (19-22%) during ischemia and after 24 h of recirculation in the paramedian neocortex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxidative properties of swollen rat liver mitochondria.

Rat liver mitochondria undergo extensive swelling when they are incubated in hypotonic sucrose medium containing 5 mm P_i. After 30 min of swelling at 25 °C, a three- to fourfold increase in volume has occurred, accompanied by gross disorganization of the matrix as observed by electron microscopy. Succinate-supported respiration was unchanged, but the respiration of NAD-linked substrates was reduced and there was a complete and irreversible loss of phosphorylation in both cases. β-Hydroxybutyrate-supported respiration was regained completely on addition of NAD to the swollen mitochondria. α-Ketoglutarate- and malate + pyruvate-supported respiration was only partially restored by the addition of NAD. This inhibition of respiration in swollen mitochondria may be due to a disorganization of a putative complex of Krebs cycle enzymes on the inner surface of the inner membrane.     

The influence of lactate,pyruvate and glucose as exogenous substrates on free radical defense mechanisms in isolated rat hearts during ischaemia and reperfusion

Previous studies have shown that exogenous lactate impairs mechanical function of reperfused ischaemic hearts, while pyruvate improves post-ischaemic recovery. The aim of this study was to investigate whether the diverging influence of exogenous lactate and pyruvate on functional recovery can be explained by an effect of the exogenous substrates on endogenous protecting mechanisms against oxygen-derived free radicals. Isolated working rat hearts were perfused by a Krebs-Henseleit bicarbonate buffer containing glucose (5 mM) as basal substrate and either lactate (5 mM) or pyruvate (5 mM) as cosubstrate. In hearts perfused with glucose as sole substrate the activity of glutathione reductase was decreased by 32% during 30 min of ischaemia (p<0.10 versus control value), while the activity of superoxide dismutase and catalase was reduced by 27 and 35%, respectively, during 5 min of reperfusion (p<0.10 versus control value). The GSH level in the glucose group was reduced by 29% following 30 min of ischaemia and 35 min of reperfusion (p<0.10). In lactate- and pyruvateperfused hearts there were no significant decreases of superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase activity during 30 min of ischaemia, 5 min of reperfusion or 35 min of reperfusion. In pyruvate-perfused hearts the glutathione peroxidase activity was even increased by 43% during 30 min of ischaemia (p<0.05). Glutathione levels (reduced and oxidized) did not markedly change in the lactate and pyruvate groups. Thus, the endogenous defense mechanism against oxygen-derived free radicals is compromised at the onset of reperfusion when glucose as sole substrate is present, while addition of lactate or pyruvate prevents reduction of the endogenous capacity to scavenge oxygen-derived free radicals. The equivocal relationship between endogenous scavenging enzyme activity and haemodynamic recovery indicates that involvement of the endogenous antioxidants, if any, in functional recovery of the post-ischaemic heart is complex. Pyruvate may exert protective effects on mechanical function after mild ischaemia by functioning as exogenous scavenger in itself, as pyruvate is able to react with hydrogen peroxide.     

l-DOPA Inhibits Complex IV of the Electron Transport Chain in Catecholamine-Rich Human Neuroblastoma NB69 Cells

Abstract: l -3,4-Dihydroxyphenylalanine ( l -DOPA) is toxic for human neuroblastoma cells NB69 and its toxicity is related to several mechanisms including quinone formation and enhanced production of free radicals related to the metabolism of dopamine via monoamine oxidase type B. We studied the effect of l -DOPA on activities of enzyme complexes in the electron transport chain (ETC) in homogenate preparations from the human neuroblastoma cell line NB69. As a preliminary step we compared the activity of ETC in cellular homogenates with that of purified mitochondria from NB69 cells and rat brain. Specific activities for complex I, complex II–III, and complex IV in NB69 cells were, respectively, 65, 96, and 32% of those in brain mitochondria. Complex I activity was inhibited in a dose-dependent way by 1-methyl-4-phenylpyridinium ion with an EC₅₀ of ∼150 µ M . Treatment with 0.25 m M l -DOPA for 5 days reduces complex IV activity to 74% of control values but does not change either complex I or citrate synthase. Ascorbic acid (1 m M ), which protects NB69 cells from l -DOPA-induced neurotoxicity, increases complex IV activity to 133% of the control and does not change other ETC complexes. Ascorbic acid also reverses l -DOPA-induced reduction of complex IV activity in NB69 cells. This observation might indicate that the protection observed with ascorbic acid is related to complex IV activation. In vitro incubation with l -DOPA (0.125–4 m M ) for 2 min produced a dose-dependent reduction of complex IV without change in complex I and II–III activities.     

Ischemic injury to rat forebrain mitochondria and cellular calcium homeostasis.

The three-vessel occlusion model of Kameyama et al. (Kameyama, M., Suzuki, J., Shirane, R. and Ogawa, A. (1985) Stroke 16, 489-493) was adapted with modifications to induce complete reversible rat forebrain ischemia. A fast and simple procedure for the isolation and purification of rat brain mitochondria, which provides high yield, is described. Mitochondria isolated from ischemic brain (12-30 min ischemia) exhibited decreases in State 3 respiratory rates of approx. 70% with NAD-linked respiratory substrates. Less effect was observed with succinate and rotenone. The State 4 respiratory activity remained near control levels except at 15 min of ischemia (25% increase) with NAD-linked substrates. Similarly, with succinate and rotenone, an approx. 30% increase in State 4 activity was observed at 20 min of ischemia. Consequently, the respiratory control indices (RCIs) were decreased. Both the respiratory rates and RCIs could be restored to near control levels upon the addition of EGTA(EDTA) or ruthenium red to the assay mixture. Analysis employing fura-2 as a Ca2+ probe, indicated a great decrease in the first order rate constant for Ca2+ uptake of ischemic mitochondria and a significant increase in Ca2+ homeostasis with an increase in the cytosolic Ca2+ concentration which results in excessive association of Ca2+ on the mitochondrial membrane and an inhibition of the respiratory chain-linked oxidative phosphorylation and Ca(2+)-transport activity of forebrain mitochondria. These deficits are proportional to the duration of ischemia.     

cAMP-dependent phosphorylation of the nuclear encoded 18-kDa (IP) subunit of respiratory complex I and activation of the complex in serum-starved mouse fibroblast cultures

A study is presented on the in vivo effect of elevated cAMP levels induced by cholera toxin on the phosphorylation of subunits of the mitochondrial respiratory complexes and their activities in Balb/c 3T3 mouse fibroblast cultures. Treatment of serum-starved fibroblasts with cholera toxin promoted serine phosphorylation in the 18-kDa subunit of complex I. Phosphorylation of the 18-kDa subunit, in response to cholera toxin treatment of fibroblasts, was accompanied by a 2-3-fold enhancement of the rotenone-sensitive endogenous respiration of fibroblasts, of the rotenone-sensitive NADH oxidase, and of the NADH:ubiquinone oxidoreductase activity of complex I. Direct exposure of fibroblasts to dibutyryl cAMP resulted in an equally potent stimulation of the NADH:ubiquinone oxidoreductase activity. Stimulation of complex I activity and respiration with NAD-linked substrates were also observed upon short incubation of isolated fibroblast mitoplasts with dibutyryl cAMP and ATP, which also promoted phosphorylation of the 18-kDa subunit. These observations document an extension of cAMP-mediated intracellular signal transduction to the regulation of cellular respiration.     

Dissociation of cytochrome c from the inner mitochondrial membrane during cardiac ischemia

Mitochondria isolated from ischemic cardiac tissue exhibit diminished rates of respiration and ATP synthesis. The present study was undertaken to determine whether cytochrome c release was responsible for ischemia-induced loss in mitochondrial function. Rat hearts were perfused in Langendorff fashion for 60 min (control) or for 30 min followed by 30 min of no flow ischemia. Mitochondria isolated from ischemic hearts in a buffer containing KCl exhibited depressed rates of maximum respiration and a lower cytochrome c content relative to control mitochondria. The addition of cytochrome c restored maximum rates of respiration, indicating that the release of cytochrome c is responsible for observed declines in function. However, mitochondria isolated in a mannitol/sucrose buffer exhibited no ischemia-induced loss in cytochrome c content, indicating that ischemia does not on its own cause the release of cytochrome c. Nevertheless, state 3 respiratory rates remained depressed, and cytochrome c release was enhanced when mitochondria from ischemic relative to perfused tissue were subsequently placed in a high ionic strength buffer, hypotonic solution, or detergent. Thus, events that occur during ischemia favor detachment of cytochrome c from the inner membrane increasing the pool of cytochrome c available for release. These results provide insight into the sequence of events that leads to release of cytochrome c and loss of mitochondrial respiratory activity during cardiac ischemia/reperfusion.     

A possible mechanism of mitochondrial dysfunction during cerebral ischemia: inhibition of mitochondrial respiration activity by arachidonic acid.

The dramatic increase in the arachidonic acid (AA) level in the brain is a well-known molecular event during cerebral ischemia. As mitochondria are known to be one possible site of the cell damage, the effects of AA on the respiratory activity of rat brain mitochondria were investigated in vitro using an oxygen electrode. In NAD-linked respiration, respiratory control ratio was decreased significantly by AA, with an IC50 of 6.0 microM. AA had the dual effect on mitochondrial respiration, a decrease in state 3 and uncoupled state and an increase in state 4 (i.e., uncoupling) as reported by Hillered and Chan (J. Neurosci. Res. 19, 94-100, 1988). Furthermore, we found that other unsaturated long-chain free fatty acids (C18:1-C18:3, C20:1-C20:5) also showed such a dual effect. Cyclooxygenase metabolites of AA such as prostaglandins (D2, E2, F2 alpha, E1) and thromboxane B2, and lipoxygenase metabolites such as leukotrienes (D4, B4) and 5- or 12-hydroperoxyeicosatetraenoic acid had no significant effect. The inhibition of the uncoupled state by AA was more marked in NAD-linked than that in FAD-linked respiration, while the degree of uncoupling by AA were the same in both respirations. In spectrophotometrical measurement, the reduction of cytochromes and flavo-protein was markedly inhibited by AA in NAD-linked respiration, but not in the FAD-linked one. In addition, the activity of cytochrome c oxidase was scarcely inhibited by AA. These data suggest that AA itself, not its metabolites, may inhibit mitochondrial ATP production during brain ischemia and that AA may act on the site(s) closely related to NAD-linked respiration, but not the FAD-linked one, in addition to its uncoupling effect.     

Hyperglycemia Preserves Brain Mitochondrial Respiration During Anoxia

We examined brain mitochondrial function in normo- (5 mM) and hyperglycemic (50 mM) cats after 8 min of anoxia. In anoxic normoglycemic cats, mitochondrial state 3 respiration with NAD-linked substrates glutamate or pyruvate (both plus malate) was inhibited 30-50%. The uncoupler carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) maximally stimulated respiration, indicating that inhibition of phosphorylation, not impairment of electron transport, substrate transport, or oxidation was present. State 3 respiration with succinate (plus rotenone) was unaffected. Mitochondrial respiratory control ratios trended toward reductions whereas ADP/O ratios remained unchanged. In contrast, brain mitochondria from anoxic hyperglycemic cats showed no such inhibition of state 3 respiration and no differences in function from normo- and hyperglycemic control animals except for trends toward loose coupling. Significantly higher brain tissue glucose concentrations were present in hyperglycemic controls as the only metabolite difference compared to normoglycemic controls. At the end of anoxia, hyperglycemic cats exhibited significantly higher cortical lactate and glucose levels but similarly reduced high-energy phosphate concentrations compared to normoglycemic cats. These results demonstrate that increased availability of glucose to gray matter as a consequence of hyperglycemia maintains normal mitochondrial state 3 respiration during exposure to anoxia. Previous survival studies have shown that lower serum glucose concentrations during anoxia are relatively brain protective. This result indicates that the presently described alterations in mitochondrial respiration must be fully reversible.     

Methotrexate: studies on the cellular metabolism. I. Effect on mitochondrial oxygen uptake and oxidative phosphorylation

Effect of methotrexate (MTX) on mitochondrial oxygen uptake, oxidative phosphorylation and on the activity of several enzymes linked to respiratory chain was studied. MTX was able to inhibit state III respiration activated by ADP and to decrease the respiratory coefficient with the substrates alpha-ketoglutarate and glutamate; these effects became pronounced when mitochondria were pre-incubated with MTX for 10 min. No effect was observed on ATPase activity of undamaged or broken mitochondria; the same was true for NADH-oxidase, NADH-dehydrogenase, NADH-cytochrome c reductase, succinate oxidase, and cytochrome c oxidase activity. The effect on the steady-state of cytochrome b, as well as, the inhibitory effect on state III of respiration with NAD+-linked substrates, offers a reasonable possibility to suggesting that the inhibition site of MTX could be in a place anterior to cytochrome b region, and not linked to respiratory chain.     

Energy-dependent accumulation of iron by isolated rat liver mitochondria V. Effect on factors controlling respiration and oxidative phosphorylation

1. Depending on the metabolic state, the addition of iron(III)-sucrose induces an inhibition or a stimulation of the respiration rate when added to isolated rat liver mitochondria.2. Under conditions identical to those used in the accumulation studies (Romslo, I. and Flatmark, T. (1973) Biochim. Biophys. Acta 305, 29?40), the ferric complex induces a decrease in the oxygen uptake concomitant to an oxidation of cytochromes c (+c₁) and a (+a₃). These results suggest that ferric iron is reduced to ferrous iron by the respiratory chain prior to or simultaneously with its energy-dependent accumulation.3. On the other hand, the addition of iron(III)-sucrose induces a stimulation of respiration in State 4 and State 3 provided Mg²⁺ is present in the suspending medium. In contrast to Ca²⁺, iron stimulates State 4 respiration in a cyclic process only within narrow concentration limits; at concentrations of iron above 100 μM the respiration remains in the activated state until anaerobiosis. The stimulation of State 4 respiration is more pronounced with succinate than with NAD-linked substrates, a difference which partly may be attributed to a stimulation of the succinate dehydrogenase complex.4. The stimulation of respiration by iron is approx. 3 times higher in State 3 than in State 4 and this difference can be attributed to a stimulation of the adenine nucleotide exchange reaction in State 3 with a concomitant increase in the rate of oxidative phosphorylation, although the $\text{P}\text{O}$ ratio is slightly diminished.     

Characterization of the respiratory activity of mitochondria isolated from an insect cell line CP-1268 Laspeyresia pomonella

Mitochondria have been isolated from the codling moth Laspeyresia pomonella, CP-1268 cell line. The mitochondrial fraction was isolated from pooled 4 d, exponential growth phase, cultures. The mitochondria were determined to be intact based on the demonstration of respiratory control, the effects of 2,4 dinitrophenol and oligomycin on respiration, the inability to oxidize NADH, and the inability of cytochrome c to enhance respiration. The isolated mitochondria were able to oxidize succinate, pyruvate, malate, alpha-ketoglutarate, and alpha-glycerophosphate efficiently. Of the substrates tested, the CP-1268 mitochondria oxidized succinate most efficiently. The respiratory control ratios ranged from a high of 4.6 for pyruvate to a low of 1.7 with alpha-glycerophosphate. These findings confirm that the mitochondria were tightly coupled. The data also confirm the presence of three sites of oxidative phosphorylation because NAD-linked substrates had ADP-to-O ratios approaching 3 and flavoprotein linked substrates had values approaching 2.     

Thioredoxin-1 is required for the cardioprotecive effect of sildenafil against ischaemia/reperfusion injury and mitochondrial dysfunction in mice

Abstract

Sildenafil is a phosphodiesterase type 5 inhibitor which confers cardioprotection against myocardial ischaemia/reperfusion (I/R) injury. The aim of this study was to determine if Trx1 participates in cardioprotection exerted by sildenafil in an acute model of I/R, and to evaluate mitochondrial bioenergetics and cellular redox status. Langendorff-perfused hearts from wild type (WT) mice and a dominant negative (DN-Trx1) mutant of Trx1 were assigned to placebo or sildenafil (0.7?mg/kg i.p.) and subjected to 30?min of ischaemia followed by 120?min of reperfusion. WT?+?S showed a significant reduction of infarct size (51.2?±?3.0% vs. 30?±?3.0%, p < .001), an effect not observed in DN-Trx. After I/R, sildenafil preserved state 3 oxygen consumption from WT, but had a milder effect in DN-Trx1 only partially protecting state 3 values. Treatment restored respiratory control (RC) after I/R, which resulted 8% (WT) and 24% (DN-Trx1) lower than in basal conditions. After I/R, a significant increase in H₂O₂ production was observed both for WT and DN-Trx (WT: 1.17?±?0.13?nmol/mg protein and DN-Trx: 1.38?±?0.12?nmol/min mg protein). With sildenafil, values were 21% lower only in WT I/R. Treatment decreased GSSG levels both in WT and DN-Trx1. In addition, GSSG/GSH² ratio was partially restored by sildenafil. Also, an increase in p-eNOS/eNOS even before the myocardial ischaemia was observed with sildenafil, both in WT (14%, p > .05) and in DN-Trx (35%, p < .05). Active Trx1 is required for the onset of the cardioprotective effects of sildenafil on I/R injury, together with the preservation of cellular redox balance and mitochondrial function.     

Lipid peroxidation and alterations to oxidative metabolism in mitochondria isolated from rat heart subjected to ischemia and reperfusion.

Ischemia-reperfusion injury to cardiac myocytes involves membrane damage mediated by oxygen free radicals. Lipid peroxidation is considered a major mechanism of oxygen free radical toxicity in reperfused heart. Mitochondrial respiration is an important source of these reactive oxygen species and hence a potential contributor to reperfusion injury. We have examined the effects of ischemia (30 min) and ischemia followed by reperfusion (15 min) of rat hearts, on the kinetic parameters of cytochrome c oxidase, on the respiratory activities and on the phospholipid composition in isolated mitochondria. Mitochondrial content of malonyldialdheyde (MDA), an index of lipid peroxidation, was also measured. Reperfusion was accompanied by a significant increase in MDA production. Mitochondrial preparations from control, ischemic and reperfused rat heart had equivalent Km values for cytochrome c, although the maximal activity of the oxidase was 25 and 51% less in ischemic and reperfused mitochondria than that of controls. These changes in the cytochrome c oxidase activity were associated to parallel changes in state 3 mitochondrial respiration. The cytochrome aa3 content was practically the same in these three types of mitochondria. Alterations were found in the mitochondrial content of the major phospholipid classes, the most pronounced change occurring in the cardiolipin, the level that decreased by 28 and by 50% as function of ischemia and reperfusion, respectively. The lower cytochrome c oxidase activity in mitochondria from reperfused rat hearts could be almost completely restored to the level of control hearts by exogenously added cardiolipin, but not by other phospholipids nor by peroxidized cardiolipin. It is proposed that the reperfusion-induced decline in the mitochondrial cytochrome c oxidase activity can be ascribed, at least in part, to a loss of cardiolipin content, due to peroxidative attack of its unsaturated fatty acids by oxygen free radicals. These findings may provide an explanation for some of the factors that lead to myocardial reperfusion injury.     

Regulation of mitochondrial uncoupling respiration during exercise in rat heart: role of reactive oxygen species (ROS) and uncoupling protein 2

The physiological significance of cardiac mitochondrial uncoupling protein 2 (UCP2)-mediated uncoupling respiration in exercise is unknown. In the current study, mitochondrial respiratory function, UCP2 mRNA level, UCP2-mediated respiration (UCR), and reactive oxygen species (ROS) generation, as well as manganese superoxide dismutase (MnSOD) activity were determined in rat heart with or without endurance training after an acute bout of exercise of different duration. In the untrained rats, state 4 respiration and UCR-independent respiration rates were progressively increased with exercise time and were 64 and 70% higher, respectively, than resting rate at 150 min, whereas UCR was elevated by 86% with no significant change in state 3 respiration. UCP2 mRNA level showed a 5- and 4-fold increase, respectively, after 45 and 90 min of exercise, but returned to resting level at 120 and 150 min. Mitochondrial ROS production and membrane potential (Deltapsi) increased progressively until 120 min, followed by a decrease to the resting level at 150 min. MnSOD mRNA abundance showed a 2-fold increase at 120 min but MnSOD activity did not change with exercise. Training significantly increased mitochondrial ATP synthetase activity, ADP to oxygen consumption (P/O) ratio, respiratory control ratio, and MnSOD activity, whereas exercise-induced state 4 respiration, UCR, ROS production, and Deltapsi were attenuated in the trained rats. We conclude that (1) UCP2 mRNA expression and activity in rat heart can be upregulated during prolonged exercise, which may reduce cross-membrane Deltapsi and thus ROS production; and (2) endurance training can blunt exercise-induced UCP2 and UCR, and improve mitochondrial efficiency of oxidative phosphorylation due to increased removal of ROS.     

Hypoxia-adaptation involves mitochondrial metabolic depression and decreased ROS leakage

Through long-term laboratory selection, we have generated a Drosophila melanogaster population that tolerates severe, normally lethal, level of hypoxia. This strain lives perpetually under severe hypoxic conditions (4% O(2)). In order to shed light on the mechanisms involved in this adaptation, we studied the respiratory function of isolated mitochondria from the thorax of hypoxia-adapted flies (AF) using polarographic oxygen consumption while monitoring superoxide generation by electron paramagnetic resonance (EPR) techniques. AF mitochondria exhibited a significant 30% decrease in respiratory rate during state 3, while enhancing the resting respiratory rate during State 4-oligo by 220%. The activity of individual electron transport complexes I, II and III were 107%, 65%, and 120% in AF mitochondria as compared to those isolated from control flies. The sharp decrease in complex II activity and modest increase in complexes I and III resulted in >60% reduction in superoxide leakage from AF mitochondria during both NAD(+)-linked state 3 and State 4-oligo respirations. These results provide evidence that flies with mitochondria exhibiting decreased succinate dehydrogenase activity and reduced superoxide leakage give flies an advantage for survival in long-term hypoxia.     

Control of state 3 respiration in liver mitochondria from rats subjected to chronic ethanol consumption

Male Sprague-Dawley rats were pair-fed a liquid diet containing 36% of calories as ethanol for at least 31 days. Mitochondria were isolated from the livers and assayed for state 3, state 4 and uncoupled respiration at all three coupling sites. Assay conditions were established that maximized state 3 respiration with each substrate while maintaining a high respiratory control ratio. In mitochondria from ethanol-fed animals, state 3 respiratory rates were decreased at all three coupling sites. The decreased state 3 rate observed at site III was still significantly higher than the state 3 rates observed at site II in mitochondria from either ethanol-fed or control animals. Moreover, the maximal (FCCP-uncoupled) rates with succinate and alpha-ketoglutarate were the same in mitochondria from ethanol-fed and control animals, whereas with glutamate-malate as substrate it was lowered 23% by chronic ethanol consumption. To investigate the role of cytochrome oxidase in modulating the respiratory rate with site I and site II substrates, the effects of cyanide on state 3 and FCCP-uncoupled respiration were determined. When the mitochondria were uncoupled there was no decrease in the rate of succinate oxidation until the rates of ascorbate and succinate oxidation became equivalent. Conversely, parallel inhibition of ascorbate, succinate and glutamate-malate state 3 respiratory rates were observed at all concentrations (1-50 microM) of cyanide utilized. These observations suggest strongly that in coupled mitochondria ethanol-elicited decreases in cytochrome oxidase activity depress the state 3 respiratory rates with site I and II substrates.     

Protection of Respiratory Chain Enzymes from Ischaemic Damage in Adult Rat Brain Slices

The effect of aglycaemic hypoxia (AH) on the activity of the mitochondrial respiratory chain complexes was measured in superfused adult cortical brain slices. After 15 min of AH the activity of complex II–III was significantly reduced (by 45%) with no change in complex I or IV. Following 30 min of reperfusion the activities of complex II–III and IV were significantly reduced (by 45% and 20% respectively). These reductions in enzyme activity were abolished by removing the external calcium or by the addition of Nω-nitro-l-arginine (LNNA) or an analogue of superoxide dismutase (SOD) manganese [III] tetrakis 4-benzoic acid porphyrin (Mn-TBAP). These data suggest that a reactive oxygen species (ROS) such as peroxynitrite is involved in the reduction of mitochondrial complex activities following AH.     

Inhaled Methane Limits the Mitochondrial Electron Transport Chain Dysfunction during Experimental Liver Ischemia-Reperfusion Injury

Background

Methanogenesis can indicate the fermentation activity of the gastrointestinal anaerobic flora. Methane also has a demonstrated anti-inflammatory potential. We hypothesized that enriched methane inhalation can influence the respiratory activity of the liver mitochondria after an ischemia-reperfusion (IR) challenge.

Methods

The activity of oxidative phosphorylation system complexes was determined after in vitro methane treatment of intact liver mitochondria. Anesthetized Sprague-Dawley rats subjected to standardized 60-min warm hepatic ischemia inhaled normoxic air (n = 6) or normoxic air containing 2.2% methane, from 50 min of ischemia and throughout the 60-min reperfusion period (n = 6). Measurement data were compared with those on sham-operated animals (n = 6 each). Liver biopsy samples were subjected to high-resolution respirometry; whole-blood superoxide and hydrogen peroxide production was measured; hepatocyte apoptosis was detected with TUNEL staining and in vivo fluorescence laser scanning microscopy.

Results

Significantly decreased complex II-linked basal respiration was found in the normoxic IR group at 55 min of ischemia and a lower respiratory capacity (~60%) and after 5 min of reperfusion. Methane inhalation preserved the maximal respiratory capacity at 55 min of ischemia and significantly improved the basal respiration during the first 30 min of reperfusion. The IR-induced cytochrome c activity, reactive oxygen species (ROS) production and hepatocyte apoptosis were also significantly reduced.

Conclusions

The normoxic IR injury was accompanied by significant functional damage of the inner mitochondrial membrane, increased cytochrome c activity, enhanced ROS production and apoptosis. An elevated methane intake confers significant protection against mitochondrial dysfunction and reduces the oxidative damage of the hepatocytes.