Non-phosphorylating Mitochondria Research Articles

Succinate-mediated reactive oxygen species production in the anoxia-tolerant epaulette (Hemiscyllium ocellatum) and grey carpet (Chiloscyllium punctatum) sharks.

Anoxia/re-oxygenation (AR) results in elevated unchecked oxidative stress and mediates irreversible damage within the brain for most vertebrates. Succinate accumulation within mitochondria of the ischaemic brain appears to increase the production of reactive oxygen species (ROS) upon re-oxygenation. Two closely related elasmobranchs, the epaulette shark (Hemiscyllium ocellatum) and the grey carpet shark (Chiloscyllium punctatum) repeatedly experience near anoxia and re-oxygenation in their habitats and have adapted to survive AR at tropical temperatures without significant brain injuries. However, these anoxia-tolerant species display contrasting strategies to survive AR, with only H. ocellatum having the capacity to supress metabolism and H. ocellatum mitochondria the capacity to depress succinate oxidation post-AR. We measured oxygen consumption alongside ROS production mediated by elevated succinate in mitochondria of permeabilized cerebellum from both shark species. Although mitochondrial respiration remained similar for both species, the ROS production in H. ocellatum was half that of C. punctatum in phosphorylating and non-phosphorylating mitochondria. Maximum ROS production in H. ocellatum was mediated by succinate loads 10-fold higher than in C. punctatum mitochondria. The contrasting survival strategies of anoxia-tolerant sharks reveal the significance of mitigating ROS production under elevated succinate load during AR, shedding light on potential mechanisms to mitigate brain injury.

Membrane-modifying properties of hydrated fullerene C60 in combination with Spirulina platensis cell culture

The study is devoted to the development of new promising nanocomponent compositions with membrane-modulating properties. The effect of a solution of hydrated fullerene C60 and a cell suspension of blue-green algae Spirulina platensis on the activity of free radical lipid peroxidation and oxidative phosphorylation in mitochondria of rat liver was studied. Fullerene C60 and suspension of Spirulina platensis cells were administered to Wistar rats for 30 days, once a day, intragastrically, both jointly and singly. The rate of oxygen consumption of phosphorylating and non-phosphorylating mitochondria (in V3 and V4 states), the rate and efficiency of ADP phosphorylation (ADP/Δt and ADP/O), respiratory control coefficient (RC) were changed in rat liver mitochondria. In the same samples, the concentration of diene, triene, oxodiene, and tetraene fatty acids was measured. The results of our studies have shown that the solution of hydrated fullerene C60 serves as a structural modifier of the lipid bilayer of cell membranes, the effect of which can be corrected by using an antioxidant component such as a suspension of Spirulina platensis cells.

Endurance training increases the efficiency of rat skeletal muscle mitochondria.

Endurance training enhances mitochondrial oxidative capacity, but its effect on mitochondria functioning is poorly understood. In the present study, the influence of an 8-week endurance training on the bioenergetic functioning of rat skeletal muscle mitochondria under different assay temperatures (25, 35, and 42 °C) was investigated. The study was performed on 24 adult 4-month-old male Wistar rats, which were randomly assigned to either a treadmill training group (n = 12) or a sedentary control group (n = 12). In skeletal muscles, endurance training stimulated mitochondrial biogenesis and oxidative capacity. In isolated mitochondria, endurance training increased the phosphorylation rate and elevated levels of coenzyme Q. Moreover, a decrease in mitochondrial uncoupling, including uncoupling protein-mediated proton leak, was observed after training, which could explain the increased reactive oxygen species production (in nonphosphorylating mitochondria) and enhanced oxidative phosphorylation efficiency. At all studied temperatures, endurance training significantly augmented H2O2 production (and coenzyme Q reduction level) in nonphosphorylating mitochondria and decreased H2O2 production (and coenzyme Q reduction level) in phosphorylating mitochondria. Endurance training magnified the hyperthermia-induced increase in oxidative capacity and attenuated the hyperthermia-induced decline in oxidative phosphorylation efficiency and reactive oxygen species formation of nonphosphorylating mitochondria via proton leak enhancement. Thus, endurance training induces both quantitative and qualitative changes in muscle mitochondria that are important for cell signaling as well as for maintaining muscle energy homeostasis, especially at high temperatures.Electronic supplementary materialThe online version of this article (doi:10.1007/s00424-016-1867-9) contains supplementary material, which is available to authorized users.

C9,t11-Conjugated linoleic acid ameliorates steatosis by modulating mitochondrial uncoupling and Nrf2 pathway

Oxidative stress, hepatic steatosis, and mitochondrial dysfunction are key pathophysiological features of nonalcoholic fatty liver disease. A conjugated linoleic acid (CLA) mixture of cis9,trans11 (9,11-CLA) and trans10,cis12 (10,12-CLA) isomers enhanced the antioxidant/detoxifying mechanism via the activation of nuclear factor E2-related factor-2 (Nrf2) and improved mitochondrial function, but less is known about the actions of specific isomers. The differential ability of individual CLA isomers to modulate these pathways was explored in Wistar rats fed for 4 weeks with a lard-based high-fat diet (L) or with control diet (CD), and, within each dietary treatment, two subgroups were daily administered with 9,11-CLA or 10,12-CLA (30 mg/day). The 9,11-CLA, but not 10,12-CLA, supplementation to CD rats improves the GSH/GSSG ratio in the liver, mitochondrial functions, and Nrf2 activity. Histological examination reveals a reduction of steatosis in L-fed rats supplemented with both CLA isomers, but 9,11-CLA downregulated plasma concentrations of proinflammatory markers, mitochondrial dysfunction, and oxidative stress markers in liver more efficiently than in 10,12-CLA treatment. The present study demonstrates the higher protective effect of 9,11-CLA against diet-induced pro-oxidant and proinflammatory signs and suggests that these effects are determined, at least in part, by its ability to activate the Nrf2 pathway and to improve the mitochondrial functioning and biogenesis.

Hydroxynonenal, a lipid peroxidation end product, stimulates uncoupling protein activity in Acanthamoeba castellanii mitochondria; the sensitivity of the inducible activity to purine nucleotides depends on the membranous ubiquinone redox state

We studied the influence of exogenously generated superoxide and exogenous 4-hydroxy-2-nonenal (HNE), a lipid peroxidation end product, on the activity of the Acanthamoeba castellanii uncoupling protein (AcUCP). The superoxide-generating xanthine/xanthine oxidase system was insufficient to induce mitochondrial uncoupling. In contrast, exogenously added HNE induced GTP-sensitive AcUCP-mediated mitochondrial uncoupling. In non-phosphorylating mitochondria, AcUCP activation by HNE was demonstrated by increased oxygen consumption accompanied by a decreased membrane potential and ubiquinone (Q) reduction level. The HNE-induced GTP-sensitive proton conductance was similar to that observed with linoleic acid. In phosphorylating mitochondria, the HNE-induced AcUCP-mediated uncoupling decreased the yield of oxidative phosphorylation. We demonstrated that the efficiency of GTP to inhibit HNE-induced AcUCP-mediated uncoupling was regulated by the endogenous Q redox state. A high Q reduction level activated AcUCP by relieving the inhibition caused by GTP while a low Q reduction level favoured the inhibition. We propose that the regulation of UCP activity involves a rapid response through the endogenous Q redox state that modulates the inhibition of UCP by purine nucleotides, followed by a late response through lipid peroxidation products resulting from an increase in the formation of reactive oxygen species that modulate the UCP activation.

Molecular identification and functional characterisation of uncoupling protein 4 in larva and pupa fat body mitochondria from the beetle Zophobas atratus

Uncoupling protein 4 (UCP4) is a member of the UCP subfamily that mediates mitochondrial uncoupling, and sequence alignment predicts the existence of UCP4 in several insects. The present study demonstrates the first molecular identification of a partial Zophobas atratus UCP4-coding sequence and the functional characterisation of ZaUCP4 in the mitochondria of larval and pupal fat bodies of the beetle. ZaUCP4 shows a high similarity to predicted insect UCP4 isoforms and known mammalian UCP4s, both at the nucleotide and amino acid sequence levels. Bioenergetic studies clearly demonstrate UCP function in mitochondria from larval and pupal fat bodies. In non-phosphorylating mitochondria, ZaUCP activity was stimulated by palmitic acid and inhibited by the purine nucleotide GTP. In phosphorylating mitochondria, ZaUCP4 activity decreased the yield of oxidative phosphorylation. ZaUCP4 was immunodetected with antibodies raised against human UCP4 as a single 36-kDa band. A lower expression of ZaUCP4 at the level of mRNA and protein and a decreased ZaUCP4 activity were observed in the Z. atratus pupal fat body compared with the larval fat body. The different expression patterns and activity of ZaUCP4 during the larval-pupal transformation indicates an important physiological role for UCP4 in insect fat body development and function during insect metamorphosis.

Ion conductance pathways in potato tuber ( Solanum tuberosum) inner mitochondrial membrane

Single-ion channel activities were measured after reconstitution of potato tuber mitochondrial inner membranes into planar lipid bilayers. In addition to the recently described large-conductance Ca 2+-activated potassium channel activity (Koszela-Piotrowska et al., 2009), the following mitochondrial ion conductance pathways were recorded: (i) an ATP-regulated potassium channel (mitoK ATP channel) activity with a conductance of 164 ± 8 pS, (ii) a large-conductance Ca 2+-insensitive iberiotoxin-sensitive potassium channel activity with a conductance of 312 pS ± 23, and (iii) a chloride 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS)-inhibited channel activity with a conductance of 117 pS ± 4. In isolated non-phosphorylating potato tuber mitochondria, individual and combined potassium channel activities caused significant (up to 14 mV) but not collapsing K +-influx-induced membrane potential depolarisation. Under phosphorylating conditions, the coupling parameters were unchanged in the presence of high K + level, indicating that plant K + channels function as energy-dissipating systems that are not able to divert energy from oxidative phosphorylation. A potato tuber K + channel that is ATP-, 5-hydroxydecanonic acid-, glybenclamide-inhibited and diazoxide-stimulated caused low cation flux, modestly decreasing membrane potential (up to a few mV) and increasing respiration in non-phosphorylating mitochondria. Immunological analysis with antibodies raised against the mammalian plasma membrane ATP-regulated K + channel identified a pore-forming subunit of the Kir-like family in potato tuber mitochondrial inner membrane. These results suggest that a mitoK ATP channel similar to that of mammalian mitochondria is present in potato tuber mitochondria.

Redox state of quinone affects sensitivity of Acanthamoeba castellanii mitochondrial uncoupling protein to purine nucleotides

We studied FFA (free fatty acid)-induced uncoupling activity in Acanthamoeba castellanii mitochondria in the non-phosphorylating state. Either succinate or external NADH was used as a respiratory substrate to determine the proton conductance curves and the relationships between respiratory rate and the quinone reduction level. Our determinations of the membranous quinone reduction level in non-phosphorylating mitochondria show that activation of UCP (uncoupling protein) activity leads to a PN (purine nucleotide)-sensitive decrease in the quinone redox state. The gradual decrease in the rate of quinone-reducing pathways (using titration of dehydrogenase activities) progressively leads to a full inhibitory effect of GDP on LA (linoleic acid) induced proton conductance. This inhibition cannot be attributed to changes in the membrane potential. Indeed, the lack of GDP inhibitory effect observed when the decrease in respiratory rate is accompanied by an increase in the quinone reduction level (using titration of the quinol-oxidizing pathway) proves that the inhibition by nucleotides can be revealed only for a low quinone redox state. It must be underlined that, in A. castellanii non-phosphorylating mitochondria, the transition of the inhibitory effect of GDP on LA-induced UCP-mediated uncoupling is observed for the same range of quinone reduction levels (between 50% and 40%) as that observed previously for phosphorylating conditions. This observation, drawn from the two different metabolic states of mitochondria, indicates that quinone could affect UCP activity through sensitivity to PNs.

The ROS Production Induced by a Reverse-Electron Flux at Respiratory-Chain Complex 1 is Hampered by Metformin

Mitochondrial reactive oxygen species (ROS) production was investigated in mitochondria extracted from liver of rats treated with or without metformin, a mild inhibitor of respiratory chain complex 1 used in type 2 diabetes. A high rate of ROS production, fully suppressed by rotenone, was evidenced in non-phosphorylating mitochondria in the presence of succinate as a single complex 2 substrate. This ROS production was substantially lowered by metformin pretreatment and by any decrease in membrane potential (Delta Phi(m)), redox potential (NADH/NAD), or phosphate potential, as induced by malonate, 2,4-dinitrophenol, or ATP synthesis, respectively. ROS production in the presence of glutamate-malate plus succinate was lower than in the presence of succinate alone, but higher than in the presence of glutamate-malate. Moreover, while rotenone both increased and decreased ROS production at complex 1 depending on forward (glutamate-malate) or reverse (succinate) electron flux, no ROS overproduction was evidenced in the forward direction with metformin. Therefore, we propose that reverse electron flux through complex 1 is an alternative pathway, which leads to a specific metformin-sensitive ROS production.

Effects of Photodynamic Action on Respiration in Nonphosphorylating Mitochondria

We have studied the effects of singlet oxygen produced by photodynamic action on respiration in nonphosphorylating mitochondria (state 4). Isolated rat liver mitochondria were incubated with 3 μM hematoporphyrin and irradiated at 365 nm with a fluence rate of 25 W/m2. After short durations of irradiation, state 4 respiration with β-hydroxybutyrate as substrate increases while respiration with succinate is negligibly affected. When mitochondria have been uncoupled with carbonylcyanide-p-trifluoromethoxyphenyl hydrazone before irradiation, no change occurs in β-hydroxybutyrate-driven respiration, while succinate-driven respiration strongly decreases. Stimulation of state 4 NADH respiration cannot be explained by slippage of the NADH ubiquinone oxidoreductase because the stoichiometry of the redox pump was found insensitive to photodynamic action. In the light of the metabolite theory for linear enzymatic chains applied to state 4 respiration (Brandet al., Biochem. J.255, 535–539, 1988), these results suggest that stimulation of NADH respiration is simply due to an increase of membrane leaks which occurs after irradiation. In the case of succinate-driven respiration, a strong inhibition of succinate dehydrogenase activity has been demonstrated after irradiation. It can be suggested that this inhibition introduces a negative control coefficient over state 4 respiration, counterbalancing the effects due to leakage.

The effect of chloroform on mitochondrial energy transduction.

The effect of chloroform on mitochondrial respiration with succinate was investigated by applying the method of Brand, Chien and Diolez [(1994) Biochem. J. 297, 27-29] to examine whether chloroform causes redox slip (fewer protons pumped per electron transferred) during mitochondrial electron transport. N,N,N',N'-Tetramethyl-p-phenylenediamine (TMPD), which lowers H+/O (the number of protons pumped to the external medium by the electron transport complexes per oxygen atom consumed) by altering the electron flow pathway, was investigated for comparison. Non-phosphorylating mitochondria that had been treated with 350 microM TMPD or 30 mM chloroform were titrated with malonate in the presence of submaximal concentrations of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP). Linear relations between CCCP-induced extra respiration and protonmotive force were obtained. These results showed that there was no measurable protonmotive force-dependent or rate-dependent slip in mitochondria treated with either TMPD or chloroform. However, both TMPD and chloroform seemed to decrease H+/O in a manner independent of protonmotive force and rate. The relationship between non-phosphorylating respiration and protonmotive force was simulated in mitochondria of which 25% of the total population were assumed to have been broken. The simulation showed that the apparent decrease in H+/O on the addition of TMPD or chloroform to mitochondria could be in principle accounted for by breakage. Assays of mitochondrial breakage (ATP hydrolysis in the presence of atractyloside and oxidation of exogenous NADH) showed that chloroform broke mitochondria but TMPD did not. We conclude that chloroform changes the measured H+/O as an artifact by causing mitochondrial breakage and does not cause measurable redox slip, whereas TMPD genuinely lowers H+/O.

Mechanisms of inhibition and uncoupling of respiration in isolated rat liver mitochondria by the general anesthetic 2,6-diisopropylphenol.

We investigated the effects of 2,6-diisopropylphenol on oxidative phosphorylation of isolated rat liver mitochondria. Diisopropylphenol strongly inhibits state-3 and uncoupled respiratory rates, when glutamate and malate are the substrates, as a direct consequence of the limitation of electron transfer at the level of complex I. In addition, diisopropylphenol acts as an uncoupler in non-phosphorylating mitochondria, which leads to an increase in respiratory rate and a large decrease in proton-motive force. However, such effects cannot be due to the classical protonophoric property of this drug, since addition of ADP plus oligomycin before diisopropylphenol avoids this increase in proton permeability, and in phosphorylating mitochondria, the ATP/O ratio is not significantly affected by diisopropylphenol addition. In the absence of added ADP, diisopropylphenol modifies some mitochondrial ATPases in such a way that they become insensitive to oligomycin and unable to couple proton movement to ATP synthesis or hydrolysis. However, these modified enzymes can catalyse passive proton permeability, which leads to uncoupling. Addition of ADP before diisopropylphenol prevents these changes. We propose that ADP induces a change in conformation of ATPase, which leads to insensitivity of this complex towards diisopropylphenol. In conclusion, we show that diisopropylphenol has two main effects on rat liver mitochondria: inhibition of the respiratory chain at the level of complex I level and modification of ATPase such that, in the absence of phosphorylation, it catalyses a H+ leak, which becomes negligible when oxidative phosphorylation is functional.

Mechanistic stoichiometry of yeast mitochondrial oxidative phosphorylation.

This study investigates the relationships between the efficiency of oxidative phosphorylation (ATP/O) and respiratory flux in yeast mitochondria. To manipulate the electron flux through the respiratory chain, different substrates leading to NAD(P)H were used. By testing the effect of ADP either on respiratory rate in the presence or absence of oligomycin or on the level of NAD(P)H, on one hand, and the effects of uncouplers on respiration, on the other, we distinguished several categories of substrates: those for which the low respiration rate was mainly controlled by dehydrogenase activities and others for which the respiration was high and controlled downstream from the dehydrogenases. By using these different substrates, we observed that the ATP/O ratio decreased irrespective of the proton-motive force when the electron flux increased, unlike the situation when the respiratory rate was modulated by addition of the respiratory inhibitor. This result suggests that the oxidative phosphorylation efficiency depends on the value of the flux crossing the proton pumps. This relationship between efficiency (ATP/O) and electron flux was linked to a main control upstream from the respiratory chain. Such changes in the ATP/O ratio at least involved changes in the stoichiometry (H+/2e-) of the respiratory chain. Indeed, in non-phosphorylating mitochondria, the ratio of stoichiometries at site 2+3 over site 3 varied according to the proton-motive force. This cannot be explained by a variation in proton leak alone but involved both (i) a variable stoichiometry (H+/2e-) in relation to the electron flux value and (ii) different relationships between the variation in stoichiometry and the flux value at each coupling site.

Swelling and contraction of heart mitochondria suspended in ammonium phosphate.

Bovine heart mitochondria which have been allowed to swell in isotonic NH4+ phosphate contract in response to initiation of oxidative phosphorylation. The contraction occurs optimally at pH 6.0 and appears from inhibition studies to result from Pi uptake being slower than removal of internal Pi via phosphorylation of external ADP. Similar results are obtained when K+ + nigericin is substituted for NH4+. Mersalyl inhibition of Pi transport in respiring, nonphosphorylating mitochondria which have been allowed to swell in NH4+ phosphate reveals a contractile process having an alkaline pH optimum. This contraction resembles closely the contraction observed in salts of strong acids and presumably occurs by electrophoretic ejection of Pi anions driven by electrogenic H+ ejection.

Different temperature dependences of oxidative phosphorylation in the inner and outer layers of tuna heart ventricle

1. The oxidative phosphorylation and the temperature dependence of mitochondria prepared from the outer compact layer and the inner spongy layer of adult tuna heart ventricle have been examined. 2. Mitochondria of the inner layer show higher succinoxidase and NADH-oxidase activities as compared with those of the outer layer. 3. Arrhenius plots for succinate oxidation by phosphorylating mitochondria show that the temperature dependence of the inner layer is higher than that of the outer layer. 4. Experiments performed with disrupted non-phosphorylating mitochondria demonstrate that this difference in temperature dependence of the two cardiac compartments depends on the integrity of the mitochondrial membranes. 5. These findings are discussed in relation to the physiology of the fish.

Functional relationship between the ADP/ATP-carrier and the F 1-ATPase in mitochondria

1. 1. The distribution of labeled and unlabeled adenine-nucleotides inside and outside mitochondria was followed after addition of [ 14C]ADP to rat liver mitochondria. Two types of mitochondria were used: 1, respiring mitochondria which were carrying out oxidative phosphorylation and which had been replenished in ATP by incubation in a medium supplemented with succinate and phosphate; 2, non-respiring mitochondria which had been partially depleted of ATP by incubation in a medium supplemented with rotenone and phosphate. During the first minute following addition of [ 14C]ADP to the respiring mitochondria, the pre-existing intramitochondrial (internal) [ 12C]ATP was released into the medium and replaced by newly synthesized [ 14C]ATP. No [ 14C]ADP accumulated in the mitochondria. It is suggested that extramitochondrial (external) ADP entering respiring mitochondria in exchange for internal ATP is phosphorylated to ATP before its complete release in the matrix space. In non-respiring mitochondria, the entry of [ 14C]ADP into the mitochondria was accompanied by the appearance in the external space of [ 12C]ADP and [ 12C]-ATP, with a marked predominance of [ 12C]ADP. Thus in non-respiring mitochondria, the residual internal ATP is dephosphorylated to ADP in the inner membrane before being released outside the mitochondria. 2. 2. When mitochondria were incubated with glutamate, ADP and [ 32P]phosphate, the [ 32P]ATP which accumulated in the matrix space became rapidly labeled in both the P γ and P β groups of the ATP, due to the presence of a transphosphorylation system in the mitochondrial matrix. The [ 32P]ATP which accumulated outside the mitochondria was also labeled in the P β group, although less rapidly than the internal ATP. Our data show that a large fraction (75–80%) of the ATP produced by phosphorylation of added ADP within the inner mitochondrial membrane is released into the matrix space before being transported out from the mitochondria; only a small part (20–25%) is released directly outside the mitochondria without penetrating the matrix space. 3. 3. In respiring and phosphorylating mitochondria, the value of the K m of the ADP-carrier for external ADP was 2–4 times lower than its value in non-respiring and non-phosphorylating mitochondria. 4. 4. The above experimental data are discussed with reference to the topological and functional relationships between the ADP-carrier and the oxidative phosphorylation complex in the inner mitochondrial membrane. They strongly suggest that the ADP-carrier comes to the close neighbourhood of the ATP synthetase on the matrix side of the inner membrane.

Swelling and contraction of heart mitochondria suspended in ammonium chloride.

Bovine heart mitochondria which have been allowed to swell in isotonic NH 4 + phosphate contract in response to initiation of oxidative phosphorylation. The contraction occurs optimally at pH 6.0 and appears from inhibition studies to result from Pi uptake being slower than removal of internal Pi via phosphorylation of external ADP. Similar results are obtained when K+ + nigericin is substituted for NH 4 + . Mersalyl inhibition of Pi transport in respiring, nonphosphorylating mitochondria which have been allowed to swell in NH 4 + phosphate reveals a contractile process having an alkaline pH optimum. This contraction resembles closely the contraction observed in salts of strong acids and presumably occurs by electrophoretic ejection of Pi anions driven by electrogenic H+ ejection.