The insensitivity to uncouplers of testis mitochondrial ATPase

Albumin-free testis mitochondrial ATPase activity failed to be stimulated by either 2,4-dinitrophenol (DNP) or carbonyl cyanide rho-trifluoromethoxyphenylhydrazone (FCCP). DNP scarcely enhanced the state 4 respiration and mitochondria proved to be poorly coupled. When 1% bovine serum albumin was added to the isolation medium, DNP or FCCP stimulated ATPase nearly twofold and the dose-response curves for the uncouplers on the QO2 reached a plateau at five- to sixfold. The DNP coupling index (q) also showed a 30-40% improvement. A dose-response curve for oligomycin on the rate of [gamma-32P]ATP synthesis showed a stimulation of ATP synthase activity by 10-100 ng inhibitor/mg protein, suggesting a possible blockade of "open" F0 channels. In the albumin preparation oligomycin inhibited ATP synthesis in the range 10-100 ng/mg protein. Since testis ATPase is known to be loosely bound to the membrane, an effect of albumin, improving tightness in the interaction of the F1 and the F0 sectors of the ATPase, is suggested.     

Bax translocation to mitochondria subsequent to a rapid loss of mitochondrial membrane potential

Bax, a pro-apoptotic member of the Bcl-2 family, is a cytosolic protein that inserts into mitochondrial membranes upon induction of cell death. Using the green fluorescent protein fused to Bax (GFP-Bax) to quantitate mitochondrial binding in living cells we have investigated the cause of Bax association with mitochondria and the time course relative to endogenous and induced changes in mitochondrial membrane potential (DeltaPsi(m)). We have found that staurosporine (STS) induces a loss in DeltaPsi(m) before GFP-Bax translocation can be measured. The onset of the DeltaPsi(m) loss is followed by a rapid and complete collapse of DeltaPsi(m) which is followed by Bax association with mitochondria. The mitochondria uncoupler FCCP, in the presence of the F(1)-F(0) ATPase inhibitor oligomycin, can trigger Bax translocation to mitochondria suggesting that when ATP levels are maintained a collapse of DeltaPsi(m) induces Bax translocation. Neither FCCP nor oligomycin alone alters Bax location. Bax association with mitochondria is also triggered by inhibitors of the electron transport chain, antimycin and rotenone, compounds that collapse DeltaPsi(m) without inducing rapid ATP hydrolysis that typically occurs with uncouplers such as FCCP. Taken together, our results suggest that alterations in mitochondrial energization associated with apoptosis can initiate Bax docking to mitochondria.     

Effect of cyanide on mitochondrial membrane depolarization induced by uncouplers

In this work, it was found that the ability of common uncouplers – carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone (FCCP) and 2,4-dinitrophenol (DNP) – to reduce membrane potential of isolated rat liver mitochondria was diminished in the presence of millimolar concentrations of the known cytochrome c oxidase inhibitor – cyanide. In the experiments, mitochondria were energized by addition of ATP in the presence of rotenone, inhibiting oxidation of endogenous substrates via respiratory complex I. Cyanide also reduced the uncoupling effect of FCCP and DNP on mitochondria energized by succinate in the presence of ferricyanide. Importantly, cyanide did not alter the protonophoric activity of FCCP and DNP in artificial bilayer lipid membranes. The causes of the effect of cyanide on the efficiency of protonophoric uncouplers in mitochondria are considered in the framework of the suggestion that conformational changes of membrane proteins could affect the state of lipids in their vicinity. In particular, changes in local microviscosity and vacuum permittivity could change the efficiency of protonophore-mediated translocation.     

Inhibitors of the ATP/ADP antiporter suppress stimulation of mitochondrial respiration and H+ permeability by palmitate and anionic detergents

The action of ATP/ADP-antiporter inhibitors upon the uncoupling effect of palmitate, detergents and 'classical' uncouplers has been studied. The uncoupling effect was estimated by stimulation of succinate oxidation and of H+ permeability of rat liver mitochondria in the presence of oligomycin. It is shown that carboxyatractylate (CAtr) and pyridoxal 5-phosphate (PLP) suppress the uncoupling induced by palmitate and the anionic detergents SDS and cholate, but do not affect that induced by the cationic detergents CTAB, by the non-ionic detergent Triton X-100, as well as by the 'classical' uncouplers FCCP and DNP. The results are discussed in terms of a concept assuming that the ATP/ADP-antiporter facilitates the electrophoretic export of hydrophobic anions from mitochondria.     

Fusion of mitochondria in tobacco suspension cultured cells is dependent on the cellular ATP level but not on actin polymerization

Mitochondria in plant cells undergo fusion and fission frequently. Although the mechanisms and proteins of mitochondrial fusion are well known in yeast and mammalian cells, they remain poorly understood in plant cells. To clarify the physiological requirements for plant mitochondrial fusion, we investigated the fusion frequency of mitochondria in tobacco cultured cells using the photoconvertible fluorescent protein Kaede and some physiological inhibitors. The latter included two uncouplers, 2,4-dinitrophenol (DNP) and carbonyl cyanide m-chlorophenylhydrazone (CCCP), an inhibitor of mitochondrial ATP synthase, oligomycin, and an actin polymerization inhibitor, latrunculin B (Lat B). The frequency of mitochondrial fusion was clearly reduced by DNP, CCCP and oligomycin, but not by Lat B, although Lat B severely inhibited mitochondrial movement. Moreover, DNP, CCCP and oligomycin evidently lowered the cellular ATP levels. These results indicate that plant mitochondrial fusion depends on the cellular ATP level, but not on actin polymerization.     

Inhibition of mitochondrial bioenergetics: the effects on structure of mitochondria in the cell and on apoptosis

The effects of specific inhibitors of respiratory chain, F(o)F(1)ATP synthase and uncouplers of oxidative phosphorylation on survival of carcinoma HeLa cells and on the structure of mitochondria in the cells were studied. The inhibitors of respiration (piericidin, antimycin, myxothiazol), the F(1)-component of ATP synthase (aurovertin) and uncouplers (DNP, FCCP) did not affect viability of HeLa cells, apoptosis induced by TNF or staurosporin and the anti-apoptotic action of Bcl-2. Apoptosis was induced by combined action of respiratory inhibitors and uncouplers indicating possible pro-apoptotic action of reactive oxygen species (ROS) generated by mitochondria. Short-term incubation of HeLa cells with the mitochondrial inhibitors and 2-deoxyglucose followed by 24-48 h recovery resulted in massive apoptosis. Apoptosis correlated to transient (3-4 h) and limited (60-70%) depletion of ATP. More prolonged or more complete transient ATP depletion induced pronounced necrosis. The inhibitors of respiration and uncouplers caused fragmentation of tubular mitochondria and formation of small round bodies followed by swelling. These transitions were not accompanied with release of cytochrome c into the cytosol and were fully reversible. The combined effect of respiratory inhibitors and uncouplers developed more rapidly indicating possible involvement of ROS generated by mitochondria. More prolonged (48-72 h) incubation with this combination of inhibitors caused clustering and degradation of mitochondria.     

Selective inhibition of pyruvate and lactate metabolism in bovine epididymal spermatozoa by dinitrophenol and alpha-cyano-3-hydroxycinnamate

The disparity between the effects of the uncouplers, 2,4-dinitrophenol (DNP) and carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) on pyruvate metabolism in bovine spermatozoa has been characterized. In bovine epididymal spermatozoa metabolizing pyruvate, the uncouplers of oxidative phosphorylation, DNP (100 μm) and FCCP (0.4 or 5 μm), decreased the intracellular ATP concentration from 30 to ～10 nmol/10⁸ cells. Both uncouplers decreased, but did not abolish, sperm motility. DNP strongly inhibited pyruvate metabolism and stimulated the appearance of free carnitine from the acetylcarnitine pool. In contrast, FCCP enhanced the oxidation of pyruvate, diminished the reduction of pyruvate to lactate, and permitted the maintenance of the normal amount of acetylcarnitine. The effects of DNP and FCCP on mitochondrial pyruvate metabolism were examined in spermatozoa treated with filipin, which renders the plasma membrane permeable to small molecules. In these cells, DNP inhibited metabolism and respiration with pyruvate or lactate, but did not affect respiration supported by acetylcarnitine. Similarly, the pyruvate translocase inhibitor, α-cyano-3-hydroxycinnamate, markedly decreased the rate of metabolism of both pyruvate and lactate. With maximally inhibitory concentrations of DNP or α-cyano-3-hydroxycinnamate, the rates of pyruvate use and lactate use were the same. Metabolism of both lactate and pyruvate and production of ATP were inhibited by similar concentrations of DNP ($\text{I}{}_{50}\text{? 7}\text{μM}$). A common mitochondrial translocase for pyruvate and lactate in bovine spermatozoa is posited. This translocase is inhibited by minimally effective uncoupling concentrations of DNP.     

Penetrating Cations Enhance Uncoupling Activity of Anionic Protonophores in Mitochondria

Protonophorous uncouplers causing a partial decrease in mitochondrial membrane potential are promising candidates for therapeutic applications. Here we showed that hydrophobic penetrating cations specifically targeted to mitochondria in a membrane potential-driven fashion increased proton-translocating activity of the anionic uncouplers 2,4-dinitrophenol (DNP) and carbonylcyanide-p-trifluorophenylhydrazone (FCCP). In planar bilayer lipid membranes (BLM) separating two compartments with different pH values, DNP-mediated diffusion potential of H⁺ ions was enhanced in the presence of dodecyltriphenylphosphonium cation (C₁₂TPP). The mitochondria-targeted penetrating cations strongly increased DNP- and carbonylcyanide m-chlorophenylhydrazone (CCCP)-mediated steady-state current through BLM when a transmembrane electrical potential difference was applied. Carboxyfluorescein efflux from liposomes initiated by the plastoquinone-containing penetrating cation SkQ1 was inhibited by both DNP and FCCP. Formation of complexes between the cation and CCCP was observed spectophotometrically. In contrast to the less hydrophobic tetraphenylphosphonium cation (TPP), SkQ1 and C₁₂TPP promoted the uncoupling action of DNP and FCCP on isolated mitochondria. C₁₂TPP and FCCP exhibited a synergistic effect decreasing the membrane potential of mitochondria in yeast cells. The stimulating action of penetrating cations on the protonophore-mediated uncoupling is assumed to be useful for medical applications of low (non-toxic) concentrations of protonophores.     

Effect of Metabolic Inhibitors on Membrane Potential and Ion Conductance of Rat Astrocytes

1. The aim of this study was to elucidate the effect of metabolic inhibition on the membrane potential and ion conductance of rat astrocytes. The metabolic inhibitors investigated were dinitrophenol (DNP), carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP), cyanide, and oligomycin.2. Primary cultures of astroglial cells from newborn rat cerebral cortex were cultivated for 13–20 days on chamber slides. The effect of metabolic inhibitors on the cellular ATP concentration was estimated from the decrease in peak chemiluminescence from the luciferin/luciferase reaction. The membrane potential and ion conductances were measured from whole-cell recordings with the patch-clamp technique.3. After 2.0 min of incubation ATP decreased from the control level to 43%with cyanide (2 mM), 58% with DNP (1 mM), 47% with FCCP (1 M), and 69% with oligomycin (10 M).4. Under normal conditions V was –74.4±1.0 mV. DNP and FCCP both caused a rapid and reversible depolarization equivalent to a shift in the I/V curve of 8.2±1.3 and 19.7±3.8 mV, respectively. DNP decreased the slope conductance (g) by 22.1% but FCCP had no significant effect on g. In contrast, neither oligomycin nor cyanide had any significant effect on the I/V curve.5. Tetraethylammonium (TEA; 10 mM) depolarized the cells by 7.1±2.0 mV but had no significant effect on g. In the presence of TEA, DNP caused a depolarization of 52.8±3.5 mV and increased g by 45.5±9.6%. The action of FCCP was not affected by the presence of TEA.6. Perfusion of the astrocytes with a Cl^– free solution inhibited the action of DNP and FCCP. Thus the depolarization was only 4.2±1.5mV in DNP and 3.7±0.3 mV in FCCP, which were significantly smaller effects than in the presence of a high intracellular [Cl^–].7. Block of tentative K_ATP channels with tolbutamide (1 mM) or Cl^– channels with Zn²⁺ (1 mM) did not inhibit the depolarization caused by DNP or FCCP.8. In conclusion, DNP and FCCP have specific effects on the plasmalemma in rat astrocytes which may be due to opening of Cl^– channels. This effect was not seen with cyanide or oligomycin and should be considered as a possible complication when DNP and FCCP are used for metabolic inhibition.     

Carboxyatractylate inhibits the uncoupling effect of free fatty acids

The ATP/ADP-antiporter inhibitors and ADP decrease the palmitate-induced stimulation of the mitochondrial respiration in the controlled state. The degree of inhibition decreases in the order: carboxyatractylate greater than bongkrekic acid, palmitoyl-CoA, ADP greater than atractylate. GDP is ineffective. The inhibiting concentration of carboxyatractylate coincides with this arresting the state 3 respiration. Carboxyatractylate inhibition decreases when the palmitate concentration increases. Stimulation of controlled respiration by FCCP or gramicidin D at any concentration of these uncouplers is carboxyatractylate-resistant, whereas that by low concentrations of DNP is partially suppressed by carboxyatractylate. These data together with observations that palmitate does not increase H+ conductance in bilayer phospholipid membranes and in cytochrome oxidase-asolectin proteoliposomes indicate that the ATP/ADP-antiporter is somehow involved in the uncoupling by low concentrations of fatty acids (or DNP), whereas that by FCCP and gramicidin D is due to their effect on the phospholipid bilayer. It is suggested that the antiporter facilitates translocation of palmitate anion across the mitochondrial membrane.     

Role of the ADP/ATP and aspartate/glutamate antiporters in the uncoupling effect of fatty acids, lauryl sulfate, and 2, 4-dinitrophenol in liver mitochondria.

Study of the uncoupling effect of various saturated fatty acids (from caprylic to palmitic) revealed that the glutamate recoupling effect was more pronounced in the case of short chain fatty acids, whereas recoupling of mitochondria by carboxyatractylate was more effective in the case of long chain fatty acids. The overall recoupling effect, however, did not depend on the fatty acid chain length. Besides carboxyatractylate, glutamate and aspartate also exhibited a recoupling effect under uncoupling by lauryl sulfate. The uncoupling effect of lauryl sulfate was markedly weaker in the presence of DNP or laurate (but not FCCP) which were added in concentrations causing twofold increase in mitochondrial respiration. In the presence of lauryl sulfate the uncoupling action of laurate and DNP was insensitive to carboxyatractylate and glutamate. With laurate and DNP as uncouplers increasing the pH from 7.0 to 7.8 potentiated the recoupling effect of carboxyatractylate and attenuated the recoupling effect of glutamate. In the case of uncoupling by lauryl sulfate similar changes in the recoupling effect of carboxyatractylate and glutamate were observed only in the presence of 10 microM tetraphenylphosphonium. Thus, when uncoupling is induced by fatty acids, DNP, and lauryl sulfate, the ADP/ATP and aspartate/glutamate antiporters function as two parallel and independent pathways for mitochondrial membrane potential dissipation. We suggest that the role of the ADP/ATP antiporter in uncoupling includes proton capture from the intermembrane space with subsequent protonation of uncoupler anions, their transport as neutral molecules on the internal side, and deprotonation followed by proton release into the matrix and transfer of the uncoupler anion in the reverse direction. During uncoupling the aspartate/glutamate antiporter cyclically carries the uncoupler anion with simultaneous proton transfer from the intermembrane space into the matrix.     

Mitochondria, Calcium Regulation, and Acute Glutamate Excitotoxicity in Cultured Cerebellar Granule Cells

Abstract: Exposure of cultured cerebellar granule cells to 100 µ M glutamate plus glycine in the absence of Mg²⁺ causes calcium loading of the in situ mitochondria and is excitotoxic, as demonstrated by a collapse of the cellular ATP/ADP ratio, cytoplasmic Ca²⁺ deregulation (the failure of the cell to maintain a stable cytoplasmic free Ca²⁺ concentration), and extensive cell death. Glutamate-evoked Ca²⁺ deregulation is exacerbated by the mitochondrial respiratory chain inhibitor rotenone. Cells maintained by glycolytic ATP, i.e., in the presence of the mitochondrial ATP synthase inhibitor oligomycin, remain viable for several hours but are still susceptible to glutamate; thus, disruption of mitochondrial ATP synthesis is not a necessary step in glutamate excitotoxicity. In contrast, the combination of rotenone (or antimycin A) plus oligomycin, which collapses the mitochondrial membrane potential, therefore preventing mitochondrial Ca²⁺ transport, allows glutamate-exposed cells to maintain a high ATP/ADP ratio while accumulating little ⁴⁵Ca²⁺ and maintaining a low bulk cytoplasmic free Ca²⁺ concentration determined by fura-2. It is concluded that mitochondrial Ca²⁺ accumulation is a necessary intermediate in glutamate excitotoxicity, whereas the decreased Ca²⁺ flux into cells with depolarized mitochondria may reflect a feedback inhibition of the NMDA receptor mediated by localized Ca²⁺ accumulation in a microdomain accessible to the mitochondria.     

Mitochondrial membrane potential in lymphocytes as monitored by fluorescent cation diS-C3-(5)

A lipophilic fluorescent cation diS-C3-(5) and rotenone suppress the oxygen consumption rate of thymocytes in similar concentrations. Seventy percent inhibition corresponds to an inhibitor:cytochrome a molar ratio of about 1:1. Addition of uncouplers decreases the inhibition of respiration by diS-C3-(5) (but not rotenone). FCCP in similar concentrations increases O2 consumption in the absence of diS-C3-(5) and the diS-C3-(5) fluorescence intensity in the presence of TMPD in thymocyte suspensions. In most thymocyte preparations, oligomycin (0.05-0.1 microgram/mL) increases the fluorescence of diS-C3-(5) and further addition of TMPD (50-100 microM) decreases the fluorescence. Addition of NaCN (400 microM) after oligomycin leads to a fluorescence increase that is hardly affected by subsequent addition of 0.2 microM FCCP. Nigericin (10-50 nM) decreases the diS-C3-(5) fluorescence. The data indicate that the diS-C3-(5) fluorescence associated with mitochondrial transmembrane potential (delta psi m) may be an essential part of the diS-C3-(5) fluorescence in lymphocyte suspensions. The changes of the diS-C3-(5) fluorescence intensity in the presence of TMPD after FCCP addition reflect delta psi m.     

Ca2+ Homeostasis in the Agonist-sensitive Internal Store: Functional Interactions Between Mitochondria and the ER Measured In Situ in Intact Cells

Mitochondria have a well-established capacity to detect cytoplasmic Ca²⁺ signals resulting from the discharge of ER Ca²⁺ stores. Conversely, both the buffering of released Ca²⁺ and ATP production by mitochondria are predicted to influence ER Ca²⁺ handling, but this complex exchange has been difficult to assess in situ using conventional measurement techniques. Here we have examined this interaction in single intact BHK-21 cells by monitoring intraluminal ER [Ca²⁺] directly using trapped fluorescent low-affinity Ca²⁺ indicators. Treatment with mitochondrial inhibitors (FCCP, antimycin A, oligomycin, and rotenone) dramatically prolonged the refilling of stores after release with bradykinin. This effect was largely due to inhibition of Ca²⁺ entry pathways at the plasma membrane, but a significant component appears to arise from reduction of SERCA-mediated Ca²⁺ uptake, possibly as a consequence of ATP depletions in a localized subcellular domain. The rate of bradykinin-induced Ca²⁺ release was reduced to 51% of control by FCCP. This effect was largely overcome by loading cells with BAPTA-AM, highlighting the importance of mitochondrial Ca²⁺ buffering in shaping the release kinetics. However, mitochondria-specific ATP production was also a significant determinant of the release dynamic. Our data emphasize the localized nature of the interaction between these organelles, and show that competent mitochondria are essential for generating explosive Ca²⁺ signals.     

The effects of mitochondrial energetics inhibitors on the fluorescence of potential-sensitive dyes rhodamine 123 and DiS-C3-(5) in lymphocyte suspensions

The effects of uncouplers (FCCP, DNF), oligomycin, and rotenone on the fluorescence of potential-sensitive dyes, rhodamine 123 and diS-C₃-(5), in lymphocyte suspensions were compared. The fluorescence of these optical probes gradually increased at higher FCCP concentrations. The dependences of fluorescence intensities and FCCP concentrations were similar for both dyes, and only diS-C₃-(5) fluorescence started increasing at lower FCCP concentrations. Rotenone (1 µM) significantly increased rhodamine 123 fluorescence. TMPD-induced and uncoupler-induced diS-C₃-(5) fluorescence changes increased 1.5- to 2-fold if the incubation mixture was supplemented with oligomycin (0.1–0.2 µg/ml). The fluorescence responses of the dyes in the lymphocyte suspension correlate with the effects of mitochondrial energetics inhibitors on _m in isolated mitochondria. The results suggest the possibility of using these dyes for estimating the direction of the _m changes in the lymphocyte suspension.Abbreviations _m difference in electrical potential across the mitochondrial inner membrane - _p difference in electrical potentials across the plasma membrane - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - DNP 2,4-dinitrophenol - FCCP carbonyl cyanidep-trifluoromethoxyphenylhydrazone - diS-C₃-(5) 3,3-dipropylthiodicarbocyanine - MOPS morpholinopropane sulfonic acid     

Effect of inhibitors of energy metabolism and protein synthesis on the process of neutral red segregation in frog erythrocytes

Effects of inhibitors of energy metabolism and protein synthesis on Neutral red segregation in frog erythrocytes were studied. Inhibitors of both glycolysis and respiration significantly reduced formation of segregation zones. This influence was most striking with antimycin A, rotenone and cyanide. This indicates that intact respiratory pathways may play an important part in the process of Neutral red segregation. Such uncouplers as FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) and 2,4-dinitrophenol (DNP) as well as inhibitors of oxidative phosphorylation (arsenate and azide) are also very effective in inhibiting the Neutral red segregation at low concentrations. The effects of these uncouplers and of olygomycin suggest an important role of ATP as an energy source for the segregation process. An inhibitor of protein synthesis, such as cycloheximide, produces some reduction in segregation zones formation. Trapping of Neutral red by protonation could readily explain the high level of this dye accumulation in nucleated erythrocytes. The fact that low concentrations of FCCP and DNP inhibit the process of segregation brings a supporting evidence for the possibility of the ATP-driven proton pump involved in Neutral red segregation.     

Mitochondrial porin regulates the sensitivity of anion carriers to inhibitors

In mitoplasts, respiratory stimulation by ADP, palmitate, DNP and CCCP and sensitivity of respiration to carboxyatractylate are considerably less pronounced than in mitochondria. Addition of porin-containing preparations (purified outer membranes or solubilized mitochondrial porin) to mitoplasts results in partial restoration of the oxygen consumption and sensitivity to carboxyatractylate (CAT). The uncoupling effect of FCCP in mitoplasts is CAT-resistant and does not depend on added porin. It is suggested that mitochondrial porin may be a natural activator of ADP/ATP antiporter and succinate carrier in mitochondria.     

Uncoupling of oxidative phosphorylation by fatty acids and detergents suppressed by ATP/ADP antiporter inhibitors

The effects of ATP/ADP-antiporter inhibitors on the uncoupling of oxidative phosphorylation by palmitic acid, detergents and protonophore FCCP in liver mitochondria were studied. The uncoupling activity of these compounds was estimated by their stimulating effect on succinate oxidation and H+ conductivity of the inner mitochondrial membrane in the presence of oligomycin. Carboxyatractylate and pyridoxal 5-phosphate suppressed the uncoupling effects of palmitic acid and anionic detergents but had no effect on the uncoupling action of the nonionic detergent Triton X-100, the cationic detergent CTAB and FCCP. The data obtained are discussed in terms of the putative role of the ATP/ADP-antiporter in the electrophoretic transport of hydrophobic anions from the mitochondria.     

Studies on the active transfer of reducing equivalents into mitochondria via the malate-aspartate shuttle.

1. The effects of mitochondrial energy states onthe extramitochondrial NADH/NAD ratio via a reconstituted malate-aspartate shuttle have been investigated. 2. The transfer of reducing equivalents into isolated mitochondria is stimulated by ATP and by electron transport. The effect of ATP is inhibited by oligomycin. The effect of electron transport is inhibited by uncouplers. 3. Uncoupling of the mitochondria is required for rapid transfer of reducing equivalents out of the mitochondria. 4. A glutamate-stimulated entry of aspartate into energized mitochondria suggests that the malate-aspartate shuttle is to some extent reversible even in a high energy state of the mitochondria. 5. It is concluded that the malate-aspartate shuttle contributes to the formation of the skewed redox situation across the inner mitochondrial membrane, which has a more reduced inside.