Transport of proteins into mitochondria: a potassium diffusion potential is able to drive the import of ADP/ATP carrier.

The transfer of cytoplasmically synthesized precursor proteins into or across the inner mitochondrial membrane is dependent on energization of the membrane. To investigate the role of this energy requirement, a buffer system was developed in which efficient import of ADP/ATP carrier into mitochondria from the receptor-bound state occurred. This import was rapid and was dependent on divalent cations, whereas the binding of precursor proteins to the mitochondrial surface was slow and was independent of added divalent cations. Using this buffer system, the import of ADP/ATP carrier could be driven by a valinomycin-induced potassium diffusion potential. The protonophore carbonylcyanide m-chlorophenyl-hydrazone was not able to abolish this import. Imposition of a delta pH did not stimulate the import. We conclude that the membrane potential delta psi itself and not the total protonmotive force delta p is the required energy source.     

Mitochondrial energy metabolism is regulated via nuclear-coded subunits of cytochrome c oxidase

A new mechanism on regulation of mitochondrial energy metabolism is proposed on the basis of reversible control of respiration by the intramitochondrial ATP/ADP ratio and slip of proton pumping (decreased H+/e- stoichiometry) in cytochrome c oxidase (COX) at high proton motive force delta p. cAMP-dependent phosphorylation of COX switches on and Ca2+-dependent dephosphorylation switches off the allosteric ATP-inhibition of COX (nucleotides bind to subunit IV). Control of respiration via phosphorylated COX by the ATP/ADP ratio keeps delta p (mainly delta psi(m)) low. Hormone induced Ca2+-dependent dephosphorylation results in loss of ATP-inhibition, increase of respiration and delta p with consequent slip in proton pumping. Slip in COX increases the free energy of reaction, resulting in increased rates of respiration, thermogenesis and ATP-synthesis. Increased delta psi(m) stimulates production of reactive oxygen species (ROS), mutations of mitochondrial DNA and accelerates aging. Slip of proton pumping without dephosphorylation and increase of delta p is found permanently in the liver-type isozyme of COX (subunit VIaL) and at high intramitochondrial ATP/ADP ratios in the heart-type isozyme (subunit VIaH). High substrate pressure (sigmoidal v/s kinetics), palmitate and 3,5-diiodothyronine (binding to subunit Va) increase also delta p, ROS production and slip but without dephosphorylation of COX.     

Control of cellular redox potential as measured in a steady-state, cell-free system

A cell-free system consisting of rat liver mitochondria, liver cytosol, lactate, and the substrates intrinsic to the malate-aspartate shuttle was reconstituted for studies of steady-state substrate fluxes and, more specifically, to evaluate further the mechanism of control of the intra- and extramitochondrial steady states of the free NAD+/NADH ratios. Soluble (F1) ATPase or 2,4-dinitrophenol (DNP) were added in varying amounts to alter substrate fluxes and the constant energy state of this 'open' metabolizing system. The steady-state redox segregation (1.36 log NAD+/NADH ratio out vs NAD+/NADH in the mitochondrial matrix) was maximally about 3 kcal, and declined together with the membrane potential (delta psi) and log ATP/ADP, which obtain on imposing an increasing energy load on the system. It is concluded that transmembrane movement of reducing equivalents is coupled to electron transfer through delta psi, mediated by the electrogenic exchange of glutamate and aspartate. When delta psi was high (near State 4), delta G redox was approximately the same as that generated without flux of reducing equivalents [E. J. Davis, J. Bremer, and K. E. Akerman (1980) J. Biol. Chem. 255, 2277-2283], suggesting that delta Gredox is in near thermodynamic equilibrium with delta psi. If the steady-state ATP/ADP ratio was altered with an energy load (F1-ATPase), delta Gredox decreased more steeply than delta psi (tetraphenyl phosphonium-sensitive electrode used to measure delta psi). At comparable ranges of ATP/ADP, both delta Gredox and delta psi decreased more steeply with uncoupler than with an external ADP-regenerating system.     

Non-ohmic proton conductance of mitochondria and liposomes

Direct measurements of the proton/hydroxyl ion flux across rat liver mitochondria and liposome membranes are reported. H+/OH- fluxes driven by membrane potential (delta psi) showed nonlinear dependence on delta psi both in mitochondria and in liposomes whereas delta pH-driven H+/OH- flux shows linear dependence on delta pH in liposomes. In the presence of low concentrations of a protonophore the H+/OH- flux was linearly dependent on delta psi and showed complex dependence on delta pH. The nonlinearity of H+/OH- permeability without protonophore is described by an integrated Nernst- Plank equation with trapezoidal energy barrier. Permeability coefficients depended on the driving force but were in the range 10(-3) cm/s for mitochondria and 10(-4)-10(-6) cm/s for liposomes. The nonlinear dependence of H+/OH- flux on delta psi explains the nonlinear dependence of electrochemical proton gradient on the rate of electron transport in energy coupling systems.     

Adenosine 5''-triphosphate synthesis driven by a protonmotive force in membrane vesicles of Escherichia coli.

Adenosine 5'-triphosphate (ATP) synthesis energized by an artificially imposed protonmotive force (delta p) in adenosine 5'-diphosphate-loaded membrane vesicles of Escherichia coli was investigated. The protonmotive force is composed of an artificially imposed pH gradient (delta pH) or membrane potential (deltapsi), or both. A delta pH was established by a rapid alteration of the pH of the assay medium. A delta psi was created by the establishment of diffusion potential of K+ in the presence of valinomycin. The maximal amount of ATP synthesized was 0.4 to 0.5 nmol/mg of membrane protein when energized by a delta pH and 0.2 to 0.3 nmol/mg of membrane protein when a delta psi was imposed. Simultaneous imposition of both a delta pH and delta psi resulted in the formation of greater amounts of ATP (0.8 nmol/mg of membrane protein) than with either alone. The amount of ATP synthesized was roughly proportional to the magnitude of the artificially imposed delta p. Although p-chloromercuribenzoate, 2-heptyl-4-hydroxyquinoline-N-oxide, or NaCN each inhibits oxidation of D-lactate, and thus oxidative phosphorylation, none inhibited ATP synthesis driven by an artificially imposed delta p. Membrane vesicles prepared from uncA or uncB strains, which are defective in oxidative phosphorylation, likewise were unable to catalyze ATP synthesis when energy was supplied by an artificially imposed delta p.     

Proton pump-generated electrochemical gradients in rat liver multivesicular bodies. Quantitation and effects of chloride

Endocytic vesicles possess an electrogenic proton pump, and measurements of ATPase activity suggest that Cl- may stimulate proton pump activity. This study was undertaken to measure the steady-state pH, potential (delta psi), and total proton electrochemical gradients established by the rat liver multivesicular body (MVB) proton pump and to examine the effects of Cl- (0.5-140 mM) on these gradients. Radiolabeled [( 14C] methylamine and 36Cl-) and fluorescent (fluorescein isothiocyanate-conjugated low density lipoproteins) probes were used to assess internal pH (pHi) and delta psi. In the absence of ATP, pHi averaged 7.37 +/- 0.05 (extracellular pH 7.31 +/- 0.02), and delta psi ranged from -32 to -71 mV; but neither pHi nor delta psi varied consistently with [Cl-]. In the presence of ATP, pHi decreased progressively with increasing [Cl-] to a plateau value of about 5.89 at greater than or equal to 25 mM Cl-, and MVB exhibited an interior positive delta psi that was maximal at the lowest Cl- concentration (+65.5 mV) and decreased as medium Cl- increased. The total ATP-dependent proton electrochemical gradient (proton-motive force (delta p] averaged 118.0 +/- 4.3 mV and did not change in any consistent manner as [Cl-] varied almost 300-fold. However, initial rates of MVB acidification increased with increasing [Cl-]. These studies indicate that: (a) in the absence of ATP, isolated MVB exhibited a negative delta psi, probably a Donnan potential; (b) in the presence of ATP and at a [Cl-] similar to that in hepatocyte cytoplasm (25 mM), MVB pHi was 5.89, and delta psi was +9.6 mV; and (c) over the range of [Cl-] tested, the magnitudes of delta pH and delta psi were inversely related, apparently related to Cl- availability, but the ATP-dependent delta p did not vary. Therefore, it is concluded that Cl- increases the initial rate of vesicle acidification in MVB and also affects the relative chemical and electrical contributions of the steady-state proton pump-determined delta p. Cl-, however, does not alter steady-state delta p.     

New control of mitochondrial membrane potential and ROS formation--a hypothesis

A new control of mitochondrial membrane potential delta(psi)m and formation of reactive oxygen species (ROS) is presented, based on allosteric ATP-inhibition of cytochrome c oxidase at high intramitochondrial ATP/ADP ratios. Since the rate of ATP synthesis by the ATP synthase is already maximal at low membrane potentials (100-120 mV), the ATP/ADP ratio will also be maximal at this delta(psi)m (at constant rate of ATP consumption). Therefore the control of respiration by the ATP/ADP-ratio keeps delta(psi)m low. In contrast, the known 'respiratory control' leads to an inhibition of respiration only at high delta(psi)m values (150-200 mV) which cause ROS formation. ATP-inhibition of cytochrome c oxidase is switched on and off by reversible phosphorylation (via cAMP and calcium, respectively). We propose that 'stress hormones' which increase intracellular [Ca2+] also increase delta(psi)m and ROS formation, which promote degenerative diseases and accelerate aging.     

Coupling of carbon monoxide oxidation to CO2 and H2 with the phosphorylation of ADP in acetate-grown Methanosarcina barkeri

Cell suspensions of Methanosarcina barkeri, grown on acetate, catalyzed the conversion of carbon monoxide and H2O to CO2 and H2 in stoichiometric amounts when methane formation was inhibited by bromoethanesulfonate. The specific activity was 80-120 nmol min-1 mg protein-1 at 5% CO in the gas phase. CO oxidation was coupled with the phosphorylation of ADP as indicated by a rapid increase of the intracellular ATP level upon start of the reaction. At least 0.1 mol ATP was formed/mol CO consumed. The onset of CO oxidation was also accompanied by an increase of the proton motive force (delta p) from 100 mV to 150 mV (inside negative). Addition of the uncoupler tetrachlorosalicylanilide to CO-metabolizing cells led to a rapid decrease of the ATP level and of delta p, and to an increase of the CO oxidation rate up to 70%. In the presence of the proton-translocating ATPase inhibitor N,N'-dicyclohexylcarbodiimide the phosphorylation of ADP was inhibited and CO oxidation slowed down, whereas delta p was almost unaffected. Inhibition of CO oxidation under these conditions was relieved by the addition of the protonophore tetrachlorosalicylanilide. The results indicate that in acetate-grown M. barkeri the free-energy change associated with the formation of CO2 and H2 from CO and H2O (delta G degrees = -20 kJ/mol) can be used to drive the phosphorylation of ADP and that the coupling proceeds via a chemiosmotic mechanism. A possible role of the carbon monoxide oxidation reaction as an energy-conserving site in acetate fermentation to CH4 and CO2 is discussed.     

Control of proteoliposomal cytochrome c oxidase: the overall reaction

The control of cytochrome c oxidase incorporated into proteoliposomes has been investigated as a function of membrane potential (delta psi) and pH gradient (delta pH). The oxidase generates a pH gradient (alkaline inside) and a membrane potential (negative inside) when respiring on external cytochrome c. Low levels of valinomycin collapse delta psi and increase delta pH; the respiration rate decreases. High levels of valinomycin, however, decrease delta pH as valinomycin can also act as a protonophore. Nigericin (in the absence of valinomycin) increases delta psi and collapses delta pH; the respiration rate increases. On a millivolt equivalent basis delta pH is a more effective inhibitor of activity than is delta psi. In the absence of any ionophores the cytochrome oxidase proteoliposomes enter a steady state, in which there are both delta pH and delta psi components of control. Present and previous data suggest that the respiration rate responds in a linear way ("ohmically") to increasing delta pH but in a nonlinear way to delta psi ("non-ohmically"). High levels of both delta psi and delta pH do not completely inhibit turnover (maximal respiratory control values lie between 6 and 10). The controlled steady state involves the electrophoretic entry and electroneutral exit of K+ from the vesicles. A model is presented in which the enzyme responds to both delta pH and delta psi components of the proton-motive force, but is more sensitive to delta pH than to delta psi at an equivalent delta mu H+. The steady state of the proteoliposome system can be represented for any set of permeabilities and enzyme activity levels using the computer simulation programme Stella.     

Respiratory-driven Na+ electrical potential in the bacterium Vitreoscilla

Vitreoscilla is a Gram-negative bacterium with unique respiratory physiology in which Na+ was implicated as a coupling cation for the generation of a transmembrane electrical gradient (delta psi). Thus, cells respiring in the presence of 110 mM Na+ generated a delta psi of -142 mV compared to only -42 and -56 mV for Li+ and choline, respectively, and even the -42 and -56 mV were insensitive to the protonophore 3,5-di-tert-butyl-4-hydroxybenzaldehyde (DTHB). The kinetics of delta psi formation and collapse correlated well with the kinetics of Na+ fluxes but not with those of H+ fluxes. Cyanide inhibited respiration, Na+ extrusion, and delta psi formation 81% or more, indicating that delta psi formation and Na+ extrusion were coupled to respiration. Experiments were performed to distinguish among three possible transport systems for this coupling: (1) a Na(+)-transporting ATPase; (2) an electrogenic Na+/H+ antiport system; (3) a primary Na+ pump directly driven by the free energy of electron transport. DCCD and arsenate decreased cellular ATP up to 86% but had no effect on delta psi, evidence against a Na(+)-transporting ATPase. Low concentrations of DTHB had no effect on delta psi; high concentrations transiently collapsed delta psi, but led to a stimulation of Na+ extrusion, the opposite of that expected for a Na+/H+ antiport system. Potassium ion, which collapses delta psi, also stimulated Na+ extrusion. The experimental evidence is against Na+ extrusion by mechanisms 1 and 2 and supports the existence of a respiratory-driven primary Na+ pump for generating delta psi in Vitreoscilla.     

Depolarization of In Situ Mitochondria Due to Hydrogen Peroxide-Induced Oxidative Stress in Nerve Terminals

Mitochondrial membrane potential (delta psi(m)) was determined in intact isolated nerve terminals using the membrane potential-sensitive probe JC-1. Oxidative stress induced by H2O2 (0.1-1 mM) caused only a minor decrease in delta psi(m). When complex I of the respiratory chain was inhibited by rotenone (2 microM), delta psi(m) was unaltered, but on subsequent addition of H2O2, delta psi(m) started to decrease and collapsed during incubation with 0.5 mM H2O2 for 12 min. The ATP level and [ATP]/[ADP] ratio were greatly reduced in the simultaneous presence of rotenone and H2O2. H2O2 also induced a marked reduction in delta psi(m) when added after oligomycin (10 microM), an inhibitor of F0F1-ATPase. H2O2 (0.1 or 0.5 mM) inhibited alpha-ketoglutarate dehydrogenase and decreased the steady-state NAD(P)H level in nerve terminals. It is concluded that there are at least two factors that determine delta psi(m) in the presence of H2O2: (a) The NADH level reduced owing to inhibition of alpha-ketoglutarate dehydrogenase is insufficient to ensure an optimal rate of respiration, which is reflected in a fall of delta psi(m) when the F0F1-ATPase is not functional. (b) The greatly reduced ATP level in the presence of rotenone and H2O2 prevents maintenance of delta psi(m) by F0F1-ATPase. The results indicate that to maintain delta psi(m) in the nerve terminal during H2O2-induced oxidative stress, both complex I and F0F1-ATPase must be functional. Collapse of delta psi(m) could be a critical event in neuronal injury in ischemia or Parkinson's disease when H2O2 is generated in excess and complex I of the respiratory chain is simultaneously impaired.     

Generation of a proton motive force by the anaerobic oxalate-degrading bacterium Oxalobacter formigenes.

The generation of transmembrane ion gradients by Oxalobacter formigenes cells metabolizing oxalate was studied. The magnitudes of both the transmembrane electrical potential (delta psi) and the pH gradient (internal alkaline) decreased with increasing external pH; quantitatively, the delta psi was the most important component of the proton motive force. As the extracellular pH of metabolizing cells was increased, intracellular pH increased and remained alkaline relative to the external pH, indicating that O. formigenes possesses a limited capacity to regulate internal pH. The generation of a delta psi by concentrated suspensions of O. formigenes cells was inhibited by the K+ ionophore valinomycin and the protonophore carbonyl cyanide-m-chlorophenylhydrazone, but not by the Na+ ionophore monensin. The H+ ATPase inhibitor N,N'-dicyclohexyl-carbodiimide inhibited oxalate catabolism but did not dissipate the delta psi. The results support the concept that energy from oxalate metabolism by O. formigenes is conserved not as a sodium ion gradient but rather, at least partially, as a transmembrane hydrogen ion gradient produced during the electrogenic exchange of substrate (oxalate) and product (formate) and from internal proton consumption during oxalate decarboxylation.     

An assessment of the role of proton leaks in the mechanistic stoichiometry of oxidative phosphorylation.

Rat liver mitochondria were incubated in the presence of varying concentrations of ATP, followed by ADP to initiate phosphorylation. Analysis of phosphorylation to oxygen ratios (P/O) was carried out with varied initial phosphorylation potentials (or ATP/ADP ratios). Rates of phosphorylation and respiration and magnitude of membrane potential (delta psi) were measured. The results are discussed in the framework of P/total O and P/"extra" O ratios in determination of the mechanistic P/O ratio. It is concluded that the former underestimates, and the latter overestimates the mechanistic P/O ratio.     

Stoichiometry of the H+-ATPase of growing and resting, aerobic Escherichia coli

The H+/ATP stoichiometry of the proton-translocating ATPase was investigated in growing and nongrowing, respiring cells of Escherichia coli. The protonmotive force, delta p, was determined by measuring the transmembrane chemical gradient of protons, delta pH, from the cellular accumulation of benzoate anions, and the electrical gradient, delta psi, from the accumulation of the lipophilic cation tetraphenylphosphonium (TPP+). The accumulation of lactose was also used to calculate the delta p in this lactose operon constitutive beta-galactosidase negative mutant. The phosphorylation potential, delta GP', was determined by measuring the cellular concentration of ATP, ADP, and inorganic phosphate. According to chemiosmotic principles, at steady state the phosphorylation potential is in thermodynamic equilibrium with the protonmotive force, and thus the ratio delta p/delta GP' can be used to determine the H+/ATP ratio. Respiring E. coli cells, in mid-exponential phase of growth or incubated in buffer, at external pHs from 6.25 to 8.25 had a constant delta GP' of about 500 mV. The H+/ATP ratio was found to be 3 when the delta p value derived from lactose accumulation levels was used. However, when the delta p values derived from delta pH and delta psi were used in the calculations, the H+/ATP ratio varied from about 2.5 at external pH 6.25 to about 4 at pH 8.25. Arguments are presented for the hypothesis that the delta psi values obtained from the TPP+ measurements are likely to be inaccurate and that a value of 3 H+/ATP, independent of the external pH, is likely to be the valid stoichiometry.     

Simultaneous determination of membrane potential and pH gradient by photodiode array spectroscopy

Membrane potential (delta psi) and pH difference (delta pH) were simultaneously determined in liposomes using a photodiode array spectrophotometer. By the use of a cyanine dye (DiS-C3(5)) and 9-aminoacridine for delta psi and delta pH probes, respectively, both changes of delta psi and delta pH could be successfully determined by photodiode array spectrometry. Each dye did not disturb the fluorescence spectrum of the other probe when its concentration was lower than 5 microM. The K+-diffusion potential-driven, FCCP(protonophore)-mediated H+-influx process in the K+-loaded liposomes was analyzed by this method. Results indicate that the kinetic behavior of H+ influx changes at a FCCP concentration of approx. 30 nM. The rate of delta pH formation increased quantitatively with increasing concentrations of FCCP up to 30 nM, but was markedly enhanced at higher concentrations, although the maximal delta pH attained was about 3 pH units in any case when a K+-diffusion potential of -180 mV was applied.     

Interference of parathion with mitochondrial bioenergetics

The organophosphorus insecticide parathion depresses the phosphorylation efficiency of mitochondria as inferred from the decrease of RCR and ADP/O ratios. The transmembrane potential (delta psi) developed by energized mitochondria, and depolarization upon ADP addition are also decreased. Furthermore, repolarization is delayed and resumes at a slower rate. The inhibitory action of parathion on phosphorylation efficiency could be related with the following findings: (1) a direct effect on the succinate dehydrogenase-ubiquinone segment of the redox chain; (2) a direct action on the ATP synthetase complex; (3) partial inhibition of the phosphate transporter.     

Mechanism of citrinin-induced dysfunction of mitochondria. II. Effect on respiration, enzyme activities, and membrane potential of liver mitochondria.

The mycotoxin citrinin, depressed the phosphorylation efficiency of liver mitochondria as deduced from a decrease of respiratory coefficient and of the ADP/O ratio. Citrinin (1.0 mM) inhibited some enzymes linked to the respiratory chain, namely NADH oxidase and NADH cytochrome c reductase involved with complex I. The activities of enzymes related with other enzymatic complexes of the respiratory chain were either unaffected or enhanced. ATPase activity was inhibited by the mycotoxin. Malate, glutamate, and 2-oxoglutarate dehydrogenases were also inhibited. The transmembrane potential (delta psi), developed by energized mitochondria and depolarization on the addition of ADP, was decreased. The results suggest that citrinin promotes a partial dissipation of the transmembrane potential, different from that resulting from a classical uncoupler such as 2,4-dinitrophenol.     

The permeability transition in heart mitochondria is regulated synergistically by ADP and cyclosporin A.

Heart mitochondria respiring in a sucrose medium containing P(i) show a permeability transition when challenged with Ca2+ and an oxidant such as cumene hydroperoxide. The transition results from the opening of a Ca(2+)-dependent pore and is evidenced by loss of membrane potential (delta psi) and osmotic swelling due to uptake of sucrose and other solutes. In the absence of oxidant, high concentrations of Ca2+ (100-150 microM) are necessary to induce loss of delta psi and initiate swelling. Cyclosporin A delays the loss of delta psi but enhances swelling under these conditions, apparently by promoting better retention of accumulated Ca2+. Cyclosporin A and ADP together restore delta psi in respiring mitochondria that have undergone the permeability transition at levels that are not effective when either is added alone. When the state of the Ca(2+)-dependent pore is assessed using passive osmotic contraction in response to polyethylene glycol (Haworth, R. A., and Hunter, D. R. (1979) Arch. Biochem. Biophys. 195, 460-467), cyclosporin A is found to be a partial inhibitor of solute flow through the open pore. Cyclosporin A decreases the Vmax of passive contraction and increases the Km for Ca2+ without affecting the Hill slope. ADP in the presence of carboxyatractyloside closes the pore almost completely even in the presence of 40 microM Ca2+. ADP shows mixed type inhibition of the Ca(2+)-dependent pore, and cyclosporin A increases the affinity of the pore for ADP. It is concluded that cyclosporin A and ADP act synergistically to close the Ca(2+)-dependent pore of the mitochondrion and that the pore is probably not formed directly from the adenine nucleotide transporter.     

Ursodeoxycholic acid may inhibit deoxycholic acid-induced apoptosis by modulating mitochondrial transmembrane potential and reactive oxygen species production.

BACKGROUND: The hydrophilic bile salt ursodeoxycholate (UDCA) inhibits injury by hydrophobic bile acids and is used to treat cholestatic liver diseases. Interestingly, hepatocyte cell death from bile acid-induced toxicity occurs more frequently from apoptosis than from necrosis. However, both processes appear to involve the mitochondrial membrane permeability transition (MPT). In this study, we determined the inhibitory effect of UDCA on deoxycholic acid (DCA)-induced MPT in isolated mitochondria by measuring changes in transmembrane potential (delta psi m) and production of reactive oxygen species (ROS). In addition, we examined the expression of apoptosis-associated proteins in mitochondria isolated from livers of bile acid-fed animals. MATERIALS AND METHODS: Adult male rats were maintained on standard diet supplemented with DCA and/or UDCA for 10 days. Mitochondria were isolated from livers by sucrose/percoll gradient centrifugation and MPT was measured using spectrophotometric and fluorimetric assays. delta psi m and ROS generation were determined by FACScan analysis. Cytoplasmic and mitochondrial protein abundance were determined by Western blot analysis. RESULTS: DCA increased mitochondrial swelling 25-fold over controls (p < 0.001); UDCA reduced the swelling by > 40% (p < 0.001). Similarly, UDCA inhibited DCA-mediated release of calcein-loaded mitochondria by 50% (p < 0.001). delta psi m was significantly decreased in mitochondria incubated with DCA but not with UDCA. delta psi m disruption was followed closely by increased superoxide anion and peroxides production (p < 0.01). Coincubation of mitochondria with UDCA significantly inhibited the changes associated with DCA (p < 0.05). In vivo, DCA feeding was associated with a 4.5-fold increase in mitochondria-associated Bax protein levels (p < 0.001); combination feeding with UDCA almost totally inhibited this increase (p < 0.001). CONCLUSION: UDCA significantly reduces DCA-induced disruption of delta psi m, ROS production, and Bax protein abundance in mitochondria, suggesting both short- and long-term mechanisms in preventing MPT. The results suggest a possible role for UDCA as a therapeutic agent in the treatment of both hepatic and nonhepatic diseases associated with high levels of apoptosis.     

Reversible depolarization of in situ mitochondria by oxidative stress parallels a decrease in NAD(P)H level in nerve terminals

We have reported recently (Chinopoulos et al., 1999 J. Neurochem. 73, 220 228) that mitochondrial membrane potential (delta(psi)m) in isolated nerve terminals is markedly reduced by H2O2 in the absence of F0F1-ATPase working as a proton pump. Here we demonstrate that delta(psi)m reduced by H2O2 (0.5 mM) in the presence of oligomycin (10 mM), an inhibitor of the F0F1-ATPase, was able to recover by the addition of catalase (2000 U). Similarly, a decrease in the NAD(P)H level due to H2O2 can be reversed by catalase. In addition, H2O2 decreased the ATP level and the [ATP]:[ADP] ratio measured in the presence of oligomycin reflecting an inhibition of glycolysis by H2O2, but this effect was not reversible. The effect of H2O2 on delta(psi)m in the presence of the complex I inhibitor, rotenone, was also unaltered by addition of catalase. These results provide circumstantial evidence for a relationship between the decreased NAD(P)H level and the inability of mitochondria to maintain delta(psi)m during oxidative stress.