Functional aspects of oxidative phosphorylation and electron transport in cardiac mitochondria of copper-deficient rats

Although dietary copper deficiency causes physiological, morphological, and biochemical abnormalities in cardiac mitochondria, the relationship observed between abnormalities of mitochondrial structure and function have been inconsistent in previous studies. The purpose of the present study was to re-evaluate the respiration rates of cardiac mitochondria from copper-deficient rats and to use several drugs that uncouple and inhibit mitochondrial respiration in order to clarify the mechanisms of mitochondrial dysfunction found in several laboratories. Copper deficiency reduced state 4 and state 3 cardiac mitochondrial respiration rates with all substrates tested. However, neither the ratio of ADP/oxygen consumed nor the acceptor control index was affected by copper deficiency. Cardiac mitochondria of copper-deficient rats showed a resistance to respiratory blockade by oligomycin and an increased ability to hydrolyze ATP in the presence of oligomycin compared with mitochondria of copper-adequate rats. This suggests that copper deficiency affects the function of the cardiac mitochondrial ATP synthase.     

Effect of rapid freezing on the functional state of rat heart mitochondria

As evidenced by respiration, oxidative phosphorylation, ATPase and NADH-oxidase activities, mitochondria composing heart tissue slices are more damaged by freezing-thawing than isolated mitochondria. A change in the functional activity of mitochondria is manifested in an increased respiratory rate in the second metabolic state and decreased respiratory rate in the third metabolic state upon oxidation of succinate and alpha-ketoglutarate; the ability of mitochondria to synthetize ATP (inhibition of the respiratory control) varied and the ATPase and NADH-oxidase activities increased. These changes in the functional state of mitochondria appeared to be due to a rise of the proton conductivity of the inner mitochondrial membrane by freezing-thawing.     

Energy transduction in intact synaptosomes. Influence of plasma-membrane depolarization on the respiration and membrane potential of internal mitochondria determined in situ.

A method is described, based on the differential accumulation of Rb+ and methyltriphenylphosphonium, for the simultaneous estimation of the membrane potentials across the plasma membrane of isolated nerve endings (synaptosomes), and across the inner membrane of mitochondria within the synaptosomal cytoplasm. These determinations, together with measurements of respiratory rates, and ATP and phosphocreatine concentrations, are used to define the bioenergetic behaviour of isolated synaptosomes under a variety of conditions. Under control conditions, in the presence of glucose, the plasma and mitochondrial membrane potentials are respectively 45 and 148mV. Addition of a proton translocator induces a 5-fold increase in respiration, and abolishes the mitochondrial membrane potential. The addition of rotenone to inhibit respiration does not affect the plasma membrane potential, and only lowers the mitochondrial membrane potential to 128mV. Evidence is presented that ATP synthesis by anaerobic glycolysis is sufficient under these conditions to maintain ATP-dependent processes, including the reversal of the mitochondrial ATP synthetase. Addition of oligomycin under non-respiring conditions leads to a complete collapse of the mitochondrial potential. Even under control conditions the plasma membrane (Na+ + K+)-dependent ATPase is responsible for a significant proportion of the synaptosomal ATP turnover. Veratridine greatly increases respiration, and depolarizes the plasma membrane, but only slightly lowers the mitochondrial membrane potential. High K+ and ouabain also lower the plasma membrane potential without decreasing the mitochondrial membrane potential. In non-respiring synaptosomes, anaerobic glycolysis is incapable of maintaining cytosolic ATP during the increased turnover induced by veratridine, and the mitochondrial membrane potential collapses. It is concluded that the internal mitochondria must be considered in any study of synaptosomal transport.     

Simultaneous evaluation of substrate-dependent oxygen consumption rates and mitochondrial membrane potential by TMRM and safranin in cortical mitochondria

Mitochondrial membrane potential (mtMP) is critical for maintaining the physiological function of the respiratory chain to generate ATP. The present study characterized the inter-relationship between mtMP, using safranin and tetramethyl rhodamine methyl ester (TMRM), and mitochondrial respiratory activity and established a protocol for functional analysis of mitochondrial bioenergetics in a multi-sensor system. Coupled respiration was decreased by 27 and 30–35% in the presence of TMRM and safranin respectively. Maximal respiration was higher than coupled with Complex I- and II-linked substrates in the presence of both dyes. Safranin showed decreased maximal respiration at a higher concentration of carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) compared with TMRM. FCCP titration revealed that maximal respiration in the presence of glutamate and malate was not sustainable at higher FCCP concentrations as compared with pyruvate and malate. Oxygen consumption rate (OCR) and mtMP in response to mitochondrial substrates were higher in isolated mitochondria compared with tissue homogenates. Safranin exhibited higher sensitivity to changes in mtMP than TMRM. This multi-sensor system measured mitochondrial parameters in the brain of transgenic mice that model Alzheimer''s disease (AD), because mitochondrial dysfunction is believed to be a primary event in the pathogenesis of AD. The coupled and maximal respiration of electron transport chain were decreased in the cortex of AD mice along with the mtMP compared with age-matched controls. Overall, these data demonstrate that safranin and TMRM are suitable for the simultaneous evaluation of mtMP and respiratory chain activity using isolated mitochondria and tissue homogenate. However, certain care should be taken concerning the selection of appropriate substrates and dyes for specific experimental circumstances.     

Stimulation of respiration by Pb2+ in detached leaves and mitochondria of C3 and C4 plants

The exposure of detached leaves of C₃ plants (pea, barley) and C₄ plant (maize) to 5 m M Pb (NO₃)₂ for 24 h caused a reduction of their photosynthetic activity by 40–60%, whereas the respiratory rate was stimulated by 20–50%. Mitochondria isolated from Pb²⁺-treated pea leaves oxidized substrates (glycine, succinate, malate) at higher rates than mitochondria from control leaves. The respiratory control (RCR) and the ADP/O ratio were not affected. Pb²⁺ caused an increase in ATP content and the ATP/ADP ratio in pea and maize leaves. Rapid fractionation of barley protoplasts incubated at low and high CO₂ conditions, indicated that the increased ATP/ADP ratio in Pb²⁺-treated leaves resulted mainly from the production of mitochondrial ATP. The measurements of membrane potential of mitochondria with a TPP⁺-sensitive electrode further showed that mitochondria isolated from Pb²⁺-treated leaves had at least as high membrane potential as mitochondria from control leaves. The activity of NAD-malate dehydrogenase in the protoplasts from barley leaves treated with Pb²⁺ was 3-fold higher than in protoplasts from control leaves. The activities of photorespiratory enzymes NADH-hydroxypyruvate reductase and glycolate oxidase as well as of NAD-malic enzyme were not affected. The presented data indicate that stimulation of respiration in leaves treated by lead is in a close relationship with activation of malate dehydrogenase and stimulation of the mitochondrial ATP production. Thus, respiration might fulfil a protective role during heavy metal exposure.     

Control of skeletal muscle mitochondria respiration by adenine nucleotides: differential effect of ADP and ATP according to muscle contractile type in pigs

Skeletal muscle exhibits considerable variation in mitochondrial content among fiber types, but it is less clear whether mitochondria from different fiber types also present specific functional and regulatory properties. The present experiment was undertaken on ten 170-day-old pigs to compare functional properties and control of respiration by adenine nucleotides in mitochondria isolated from predominantly slow-twitch (Rhomboideus (RM)) and fast-twitch (Longissimus (LM)) muscles. Mitochondrial ATP synthesis, respiratory control ratio (RCR) and ADP-stimulated respiration with either complex I or II substrates were significantly higher (25-30%, P<0.05) in RM than in LM mitochondria, whereas no difference was observed for basal respiration. Based on mitochondrial enzyme activities (cytochrome c oxidase [COX], F0F1-ATPase, mitochondrial creatine kinase [mi-CK]), the higher ADP-stimulated respiration rate of RM mitochondria appeared mainly related to a higher maximal oxidative capacity, without any difference in the maximal phosphorylation potential. Mitochondrial K(m) for ADP was similar in RM (4.4+/-0.9 microM) and LM (5.9+/-1.2 microM) muscles (P>0.05) but the inhibitory effect of ATP was more marked in LM (P<0.01). These findings demonstrate that the regulation of mitochondrial respiration by ATP differs according to muscle contractile type and that absolute muscle oxidative capacity not only relies on mitochondrial density but also on mitochondrial functioning per se.     

In situ respiration and bioenergetic status of mitochondria in primary cerebellar granule neuronal cultures exposed continuously to glutamate

Mitochondria play a central role in neuronal death during pathological exposure to glutamate (excitotoxicity). To investigate the detailed bioenergetics of the in situ mitochondria, a method is described to monitor continuously the respiration of primary cerebellar granule neuron cultures while simultaneously imaging cytoplasmic Ca(2+) and mitochondrial membrane potential. Coverslip-attached cells were perfused in an imaging chamber with upstream and downstream flow-through oxygen electrodes. The bioenergetic consequences of chronic glutamate exposure were investigated, including ATP supply and demand, proton leak, and mitochondrial respiratory capacity during chronic glutamate exposure. In 25 mM K(+) medium supplemented with 10% dialyzed serum, cells utilized 54% of their respiratory capacity in the absence of receptor activation (37% for ATP generation, 12% to drive the mitochondrial proton leak, and the residual 5% was nonmitochondrial). glutamate initially increased mitochondrial respiration from 51 to 68% of capacity, followed by a slow decline. It was estimated that 85% of this increased respiration was because of increased ATP demand, whereas 15% was attributable to a transient mitochondrial proton leak. N-Methyl-D-aspartate receptor activation was only responsible for 62% of the increased respiration. When adjusted for cell death over 3 h of glutamate exposure, respiration of the viable cells remained near basal and protonophore stimulated respiration to the same extent as control cells. Pyruvate-supplemented media protected cells from glutamate excitotoxicity, although this was associated with mitochondrial dysfunction. We conclude that excitotoxicity under these conditions is not because of an ATP deficit or uncoupling. Furthermore, mitochondria maintain the same respiratory capacity as in control cells.     

Contribution of ATP synthase to stimulation of respiration by Ca2+ in heart mitochondria

A variety of experimental conditions were applied with the aim to estimate the correlation between the contribution of ATP synthase to the respiratory flux control and the calcium-induced activation of succinate oxidation in heart mitochondria isolated from rat, rabbit and guinea pig. The sensitivity of respiration in heart mitochondria to the decrease in temperature from 37 degrees C to 28 degrees C decreases in the order rabbit > guinea pig > rat. Ca2+ effect on succinate oxidation rate in state 3 respiration was species- and temperature-dependent and ranged from 0 (rat, 37 degrees C) to +44% (rabbit, 28 degrees C). For mitochondria from all experimental animals, the increase of Ca2+ in physiological range of concentration did not change state 2 respiration rate, and the stimulatory effect of Ca2+ on state 3 respiration was more pronounced at 28 degrees C than at 37 degrees C. The respiratory subsystem was sensitive to Ca2+ ions only in rabbit heart mitochondria. A high positive correlation between Ca2+ ability to stimulate succinate oxidation in state 3 and the control exerted by ATP synthase over the respiratory flux provides argument confirming stimulation of ATP synthase by Ca2+ ions.     

Sphingosine impairs mitochondrial function by opening permeability transition pore

Growing evidence suggest that, in the heart, sphingosine participates to contractile dysfunction by altering calcium transients and mitochondria function. However, mechanisms underlying sphingosine-induced cardiac mitochondria dysfunction are poorly understood. Here, we studied the effects of sphingosine on isolated cardiac mitochondria of either wild-type or Bcl-2 overexpressing transgenic mice. Sphingosine induced reductions in ADP-coupled respiration, membrane potential, mitochondrial cytochrome c content and ATP production, which were partially prevented by cyclosporine A and mitochondrial Bcl-2 overexpression. These data suggest that sphingosine promotes mitochondrial permeability transition pore opening, which may result in uncoupled respiration and participate in cardiac contractile dysfunction.     

Relationship between ATP level and respiratory rate in sea urchin embryos

In sea urchin embryos, the rate of respiration, as a result of electron transport through the mitochondrial respiratory chain, was enhanced after hatching without any change in the intrinsic capacity of electron transport in mitochondria. The increase in respiratory rate after hatching was accompanied by an evident decrease in intracellular adenosine triphosphate (ATP) concentration without any change in intracellular levels of adenosine diphosphate (ADP) and adenosine monophosphate (AMP). Adenosine triphosphate is proposed to fortify acceptor control of respiration at high concentrations and to reduce the respiratory rate even in the presence of ADP, the acceptor. The relationships between the respiratory rate and intracellular ATP concentration in embryos were the same as those in mitochondria isolated from embryos, obtained in the presence of ADP at the same concentration as in the embryos. Probably, the respiratory rate is enhanced after hatching because of the decrease in the level of ATP. In embryos kept in a medium containing adenosine, intracellular ATP concentration increased especially after hatching, without any change in the ADP level, and the respiratory rate after hatching was made as low as the rate expected, based on the relationships obtained on isolated mitochondria. The respiratory rate in embryos probably depends on intracellular ATP concentration, irrespective of the developmental stage in early development.     

Mechanism of 3-nitropropionic acid-induced membrane permeability transition of isolated mitochondria and its suppression by L-carnitine

3-Nitropropionic acid (3NP) functions as an irreversible inhibitor of succinic acid dehydrogenase (complex II) and induces neuronal disorders in rats similar to those in patients with Huntington's disease. It is well known that L-carnitine (LC), a carrier of long chain fatty acid into the mitochondrial matrix, attenuates the neuronal degeneration in 3NP-treated rats. From these findings it has been suggested that 3NP induces certain neuronal cell death through mitochondrial dysfunction and that LC preserves the neurons against the dysfunction of mitochondria caused by 3NP. However, the detailed mechanism of cell death by 3NP and the protective actions of LC against the mitochondrial dysfunction have not been fully elucidated yet. Thus, we studied the molecular mechanism of the effects of 3NP and LC on isolated rat liver mitochondria. 3NP inhibited succinate respiration and the decreased respiratory control ratio of isolated mitochondria without affecting oxidative phosphorylation. 3NP induced a membrane permeability transition (MPT), which plays an important role in the mechanism of apoptotic cell death. 3NP stimulated Ca2+ release from mitochondria, decreased membrane potential, induced mitochondrial swelling, and stimulated cytochrome c release from mitochondria. 3NP-induced swelling was suppressed by bovine serum albumin, inhibitors of phospholipase A(2) and by an inhibitor of classic MPT, cyclosporin A. Furthermore, LC suppressed the changes brought about by 3NP in mitochondrial functions in the presence of ATP. These results suggest that MPT underlies the mechanism of 3NP-induced cell death, and that LC attenuates mitochondrial MPT by decreasing long chain fatty acids generated by phospholipase A(2).     

Acetaminophen toxicity results in site-specific mitochondrial damage in isolated mouse hepatocytes

Exposure of isolated mouse hepatocytes to a toxic concentration of acetaminophen (5 mM) resulted in damage to the mitochondrial respiratory apparatus. The nature of this damage was investigated by measuring respiration stimulated by site-specific substrates in digitonin-permeabilized hepatocytes after acetaminophen exposure. Respiration stimulated by succinate at energy-coupling site 2 was most sensitive to inhibition and was decreased by 47% after 1 h. Respiration supported by NADH-linked substrates (site 1) was also decreased but to a lesser extent, while there was no decrease in the rate of ascorbate + N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD)-supported respiration (site 3). The loss of mitochondrial respiratory function was accompanied by a decrease in ATP levels and ATP/ADP ratios in the cytosolic compartment and was preceded by a loss of reduced glutathione in both the cytosol and mitochondria. All these effects occurred well before the loss of cell membrane integrity. The putative toxic metabolite of acetaminophen, N-acetyl-p-benzoquinonimine (NAPQI), produced a similar pattern of respiratory dysfunction in isolated hepatic mitochondria. Respiration stimulated by succinate- and NADH-linked substrates was very sensitive to 50 microM NAPQI, while ascorbate + TMPD-supported respiration was unaffected. The interaction between NAPQI and the respiratory chain was further investigated using submitochondrial particles. Succinate dehydrogenase (associated with respiratory complex II) was found to be very sensitive to NAPQI, while NADH dehydrogenase (respiratory complex I) was inhibited to a lesser extent. Our results indicate that a loss of the ability to utilize succinate- and NADH-linked substrates due to attack of the respiratory chain by NAPQI causes a disruption of energy homeostasis in acetaminophen hepatotoxicity.     

Effect of Remifentanil on Mitochondrial Oxygen Consumption of Cultured Human Hepatocytes

During sepsis, liver dysfunction is common, and failure of mitochondria to effectively couple oxygen consumption with energy production has been described. In addition to sepsis, pharmacological agents used to treat septic patients may contribute to mitochondrial dysfunction. This study addressed the hypothesis that remifentanil interacts with hepatic mitochondrial oxygen consumption. The human hepatoma cell line HepG2 and their isolated mitochondria were exposed to remifentanil, with or without further exposure to tumor necrosis factor-α (TNF-α). Mitochondrial oxygen consumption was measured by high-resolution respirometry, Caspase-3 protein levels by Western blotting, and cytokine levels by ELISA. Inhibitory κBα (IκBα) phosphorylation, measurement of the cellular ATP content and mitochondrial membrane potential in intact cells were analysed using commercial ELISA kits. Maximal cellular respiration increased after one hour of incubation with remifentanil, and phosphorylation of IκBα occurred, denoting stimulation of nuclear factor κB (NF-κB). The effect on cellular respiration was not present at 2, 4, 8 or 16 hours of incubation. Remifentanil increased the isolated mitochondrial respiratory control ratio of complex-I-dependent respiration without interfering with maximal respiration. Preincubation with the opioid receptor antagonist naloxone prevented a remifentanil-induced increase in cellular respiration. Remifentanil at 10× higher concentrations than therapeutic reduced mitochondrial membrane potential and ATP content without uncoupling oxygen consumption and basal respiration levels. TNF-α exposure reduced respiration of complex-I, -II and -IV, an effect which was prevented by prior remifentanil incubation. Furthermore, prior remifentanil incubation prevented TNF-α-induced IL-6 release of HepG2 cells, and attenuated fragmentation of pro-caspase-3 into cleaved active caspase 3 (an early marker of apoptosis). Our data suggest that remifentanil increases cellular respiration of human hepatocytes and prevents TNF-α-induced mitochondrial dysfunction. The results were not explained by uncoupling of mitochondrial respiration.     

The interaction of flavonoids with mitochondria: effects on energetic processes

The study addressed aspects of energetics of isolated rat liver mitochondria exposed to the flavonoids quercetin, taxifolin, catechin and galangin, taking into account influences of the 2,3 double bond/3-OH group and 4-oxo function on the C-ring, and o-di-OH on the B-ring of their structures, as well as mitochondrial mechanisms potentially involved in cell necrosis and apoptosis. The major findings/hypothesis, were: The 2,3 double bond/3-OH group in conjugation with the 4-oxo function on the C-ring in the flavonoid structure seems favour the interaction of these compounds with the mitochondrial membrane, decreasing its fluidity either inhibiting the respiratory chain of mitochondria or causing uncoupling; while the o-di-OH on the B-ring seems favour the respiratory chain inhibition, the absence of this structure seems favour the uncoupling activity. The flavonoids not affecting the respiration of mitochondria, induced MPT. The ability of flavonoids to induce the release of mitochondria-accumulated Ca(2+) correlated well with their ability to affect mitochondrial respiration on the one hand, and their inability to induce MPT, on the other. The flavonoids causing substantial respiratory chain inhibition or mitochondrial uncoupling, quercetin and galangin, respectively, also decreased the mitochondrial ATP levels, thus suggesting an apparent higher potential for necrosis induction in relation to the flavonoids inducing MPT, taxifolin and cathechin, which did not decrease significantly the ATP levels, rather suggesting an apparent higher potential for apoptosis induction.     

Relation between glutamate and adenine nucleotide levels of heart mitochondria during hypoxia

The effect of acute respiratory hypoxia in rats on mitochondrial respiration, adenine nucleotides and some amino acids of the heart was studied. The decrease in the total (ATP + ADP + AMP) and exchangeable (ATP + ADP) adenine nucleotide pool of the mitochondria was accompanied by a pronounced loss of state 3 respiration with glutamate plus malate and a slight decrease with succinate plus rothenone. The uncoupled respiration of mitochondria with glutamate and malate was decreased in the same degree as in the absence of 2,4-dinitrophenol. State 4 respiration with substrates of both types was unaffected by hypoxia. These data point to a hypoxia-induced impairment of complex I of the respiratory chain. The decrease of tissue and mitochondrial glutamate was accompanied by the elevation of alanine content in the heart and an increase in intramitochondrial aspartate. The ADP-stimulated respiration of mitochondria was correlated with mitochondrial glutamate and ATP as well as with exchangeable adenine nucleotide pools during hypoxia. The experimental results suggest that mitochondrial dysfunction induced by hypoxia may also be attributed to the low level of mitochondrial glutamate.     

Control of respiration and oxidative phosphorylation in isolated rat liver cells

NAD(P)H fluorescence, mitochondrial membrane potential and respiration rate were measured and manipulated in isolated liver cells from fed and starved rats in order to characterize control of mitochondrial respiration and phosphorylation. Increased mitochondrial NADH supply stimulated respiration and this accounted for most of the stimulation of respiration by vasopressin and extracellular ATP. From the response of respiration to NADH it was estimated that the control coefficient over respiration of the processes that supply mitochondrial NADH was about 0.15-0.3 in cells from fed rats. Inhibition of the ATP synthase with oligomycin increased the mitochondrial membrane potential and decreased respiration in cells from fed rats, while the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone had the opposite effect. There was a unique relationship between respiration and membrane potential irrespective of the ATP content of the cells indicating that phosphorylation potential controls respiration solely via phosphorylation (rather than by controlling NADH supply). From the response of respiration to the mitochondrial membrane potential (delta psi M) it was estimated that the control coefficients over respiration rate in cells from fed rats were: 0.29 by the processes that generate delta psi M, 0.49 by the process of ATP synthesis, transport and consumption, and 0.22 by the processes that cycle protons across the inner mitochondrial membrane other than via ATP synthesis (e.g. the passive proton leak). Control coefficients over the rate of mitochondrial ATP synthesis were 0.23, 0.84 and -0.07, respectively, by the same processes. The control distribution in cells from starved rats was similar.     

Mitochondrial alterations related to programmed cell death in tobacco cells under aluminium stress

The present investigation was undertaken to verify whether mitochondria play a significant role in aluminium (Al) toxicity, using the mitochondria isolated from tobacco cells (Nicotiana tabacum, non-chlorophyllic cell line SL) under Al stress. An inhibition of respiration was observed in terms of state-III, state-IV, succinate-dependent, alternative oxidase (AOX)-pathway capacity and cytochrome (CYT)-pathway capacity, respectively, in the mitochondria isolated from tobacco cells subjected to Al stress for 18 h. In accordance with the respiratory inhibition, the mitochondrial ATP content showed a significant decrease under Al treatment. An enhancement of reactive oxygen species (ROS) production under state-III respiration was observed in the mitochondria isolated from Al-treated cells, which would create an oxidative stress situation. The opening of mitochondrial permeability transition pore (MPTP) was seen more extensively in mitochondria isolated from Al-treated cells than in those isolated from control cells. This was Ca(2+) dependent and well modulated by dithioerythritol (DTE) and Pi, but insensitive to cyclosporine A (CsA). The collapse of inner mitochondrial membrane potential (DeltaPsi(m)) was also observed with a release of cytochrome c from mitochondria. A great decrease in the ATP content was also seen under Al stress. Transmission electron microscopy analysis of Al-treated cells also corroborated our biochemical data with distortion in membrane architecture in mitochondria. TUNEL-positive nuclei in Al-treated cells strongly indicated the occurrence of nuclear fragmentation. From the above study, it was concluded that Al toxicity affects severely the mitochondrial respiratory functions and alters the redox status studied in vitro and also the internal structure, which seems to cause finally cell death in tobacco cells.     

ATP-sensitive potassium channel in mitochondria of the eukaryotic microorganism Acanthamoeba castellanii

We describe the existence of a potassium ion transport mechanism in the mitochondrial inner membrane of a lower eukaryotic organism, Acanthamoeba castellanii. We found that substances known to modulate potassium channel activity influenced the bioenergetics of A. castellanii mitochondria. In isolated mitochondria, the rate of resting respiration is increased by about 10% in response to potassium channel openers, i.e. diazoxide and BMS-191095, during succinate-, malate-, or NADH-sustained respiration. This effect is strictly dependent on the presence of potassium ions in an incubation medium and is reversed by glibenclamide (a potassium channel blocker). Diazoxide and BMS-191095 also caused a slight but statistically significant depolarization of mitochondrial membrane potential (measured with a TPP(+)-specific electrode), regardless of the respiratory substrate used. The resulting steady state value of membrane potential was restored after treatment with glibenclamide or 1 mM ATP. Additionally, the electrophysiological properties of potassium channels present in the A. castellanii inner mitochondrial membrane are described in the reconstituted system, using black lipid membranes. Conductance from 90 +/- 7 to 166 +/- 10 picosiemens, inhibition by 1 mM ATP/Mg(2+) or glibenclamide, and activation by diazoxide were observed. These results suggest that an ATP-sensitive potassium channel similar to that of mammalian mitochondria is present in A. castellanii mitochondria.     

Respiratory uncoupling by UCP1 and UCP2 and superoxide generation in endothelial cell mitochondria

Mitochondria represent a major source of reactive oxygen species (ROS), particularly during resting or state 4 respiration wherein ATP is not generated. One proposed role for respiratory mitochondrial uncoupling proteins (UCPs) is to decrease mitochondrial membrane potential and thereby protect cells from damage due to ROS. This work was designed to examine superoxide production during state 4 (no ATP production) and state 3 (active ATP synthesis) respiration and to determine whether uncoupling reduced the specific production of this radical species, whether this occurred in endothelial mitochondria per se, and whether this could be modulated by UCPs. Superoxide formation by isolated bovine aortic endothelial cell (BAE) mitochondria, determined using electron paramagnetic resonance spectroscopy, was approximately fourfold greater during state 4 compared with state 3 respiration. UCP1 and UCP2 overexpression both increased the proton conductance of endothelial cell mitochondria, as rigorously determined by the kinetic relationship of respiration to inner membrane potential. However, despite uncoupling, neither UCP1 nor UCP2 altered superoxide formation. Antimycin, known to increase mitochondrial superoxide, was studied as a positive control and markedly enhanced the superoxide spin adduct in our mitochondrial preparations, whereas the signal was markedly impaired by the powerful chemical uncoupler p-(trifluoromethoxyl)-phenyl-hydrazone. In summary, we show that UCPs do have uncoupling properties when expressed in BAE mitochondria but that uncoupling by UCP1 or UCP2 does not prevent acute substrate-driven endothelial cell superoxide as effluxed from mitochondria respiring in vitro.     

Mechanical ventilation induces diaphragmatic mitochondrial dysfunction and increased oxidant production

Mechanical ventilation (MV) is a life-saving intervention used in patients with acute respiratory failure. Unfortunately, prolonged MV results in diaphragmatic weakness, which is an important contributor to the failure to wean patients from MV. Our laboratory has previously shown that reactive oxygen species (ROS) play a critical role in mediating diaphragmatic weakness after MV. However, the pathways responsible for MV-induced diaphragmatic ROS production remain unknown. These experiments tested the hypothesis that prolonged MV results in an increase in mitochondrial ROS release, mitochondrial oxidative damage, and mitochondrial dysfunction. To test this hypothesis, adult (3–4 months of age) female Sprague–Dawley rats were assigned to either a control or a 12-h MV group. After treatment, diaphragms were removed and mitochondria were isolated for subsequent respiratory and biochemical measurements. Compared to control, prolonged MV resulted in a lower respiratory control ratio in diaphragmatic mitochondria. Furthermore, diaphragmatic mitochondria from MV animals released higher rates of ROS in both State 3 and State 4 respiration. Prolonged MV was also associated with diaphragmatic mitochondrial oxidative damage as indicated by increased lipid peroxidation and protein oxidation. Finally, our data also reveal that the activities of the electron transport chain complexes II, III, and IV are depressed in mitochondria isolated from diaphragms of MV animals. In conclusion, these results are consistent with the concept that diaphragmatic inactivity promotes an increase in mitochondrial ROS emission, mitochondrial oxidative damage, and mitochondrial respiratory dysfunction.