oligomycins and carboxyatractyloside

Berberine [Natural Yellow 18, 5,6-dihydro-9,10-dimethoxybenzo(g)-1,3-benzodioxolo (5,6-a) quinolizinium] is an alkaloid present in plants of the Berberidaceae family and used in traditional Chinese and North American medicine. We have previously demonstrated that berberine causes mitochondrial depolarization and fragmentation, with simultaneous increase in oxidative stress. We also demonstrated that berberine causes an inhibition of mitochondrial respiration and a decrease on calcium loading capacity through induction of the mitochondrial permeability transition (MPT). The objective of the present work is to investigate a common target for both induction of the MPT and inhibition of respiration. The hypothesis is that berberine induces the MPT through interacting with the adenine nucleotide translocator (ANT). By measuring induction of the MPT through increased mitochondrial swelling, membrane depolarization and loss of calcium retention, we observed that the effects of berberine were not inhibited by bongkrekic acid although adenosine diphosphate (ADP)/oligomycin completely prevented the MPT. Also, we observed that berberine increased the depolarization effect of oleic acid on liver mitochondria. The initial depolarization observed when berberine is added to mitochondria was not affected by ANT inhibitors. Taken together, we propose that berberine acts on the ANT, altering the binding of the protein to bongkrekic acid but not to cyclosporin A or ADP. It is also clear that the membrane potential is required for berberine effects, most likely for allowing for its mitochondrial accumulation. Mitochondrial effects of berberine can be relevant not only for its proposed antitumor activity but also for the assessment of its organ toxicity, depending on factors such as tissue accumulation or delivery.

Topics: Adenosine Diphosphate; Animals; Antineoplastic Agents, Phytogenic; Atractyloside; Berberine Alkaloids; Bongkrekic Acid; Calcium; Cell Respiration; Dose-Response Relationship, Drug; In Vitro Techniques; Male; Membrane Potential, Mitochondrial; Mitochondria, Liver; Mitochondrial ADP, ATP Translocases; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Mitochondrial Swelling; Oleic Acid; Oligomycins; Rats; Rats, Wistar; Time Factors; Uncoupling Agents

The purpose of this work was to assess the effect of oligomycin on the mitochondrial membrane permeability transition. The antibiotic was found to strengthen cyclosporin A (CSA)-induced protection of non-specific permeability, which is triggered by a matrix Ca2+ load in the absence of ADP. Oligomycin also reinforced the protective effect of CSA on carboxyatractyloside-induced pore opening in the absence of ADP, but failed to do so in mitochondria incubated under anaerobic conditions or after addition of CCCP. Analyzing the efflux of matrix Ca2+, we found that mitochondrial swelling and the collapse of the transmembrane electric gradient coincided with membrane leakage. The effects of the antibiotic were observed in phosphate-containing media but not in the presence of acetate. Furthermore, N-ethylmaleimide hindered the protective effect of oligomycin-CSA. In addition, the matrix phosphate concentration increased concurrently with a diminution in the matrix-free fraction of Ca2+. We concluded that oligomycin increases phosphate uptake by stimulating the phosphate-/OH- exchange reaction.

Topics: Acetates; Animals; Atractyloside; Calcium; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cyclosporine; Drug Synergism; Enzyme Inhibitors; Ethylmaleimide; Intracellular Membranes; Kidney; Membrane Potentials; Mitochondria; Mitochondrial Swelling; Oligomycins; Permeability; Phosphates; Rats; Uncoupling Agents

Mitochondria play a critical role in some forms of apoptosis, and the Ca(2+)-dependent permeability transition (PT) is a key initiator of this process. We quantitatively examined major control mechanisms of PT in rat brain (RBM) and liver (RLM) mitochondria. Compared with RLM, RBM were less sensitive to cyclosporin A (CsA), but the combined action of CsA+ADP was much more pronounced in RBM. Carboxyatractyloside abrogated the effects of all mPTP inhibitors in RBM but not in RLM, where the effects of CsA were not reduced. Estimated H(+)/Ca(2+) ratios were 0.81+/-0.01 for RLM and 0.84-0.93 for RBM, suggesting that Ca(2+) and Pi were sequestered in the matrix as CaHPO(4) and Ca(3)(PO(4))(2) salts, and that RBM sequester more CaPi as the least soluble salt. We conclude that: (1) RBM and RLM differ in their baseline behavior of the PT and in their responses to PT modifiers, and (2) PT modifiers can be functionally divided into those which directly affect the mitochondrial PT pore and are not energy-dependent (CsA, free Ca(2+), ADP(ex), and Mg(2+)), and those which affect the energy-dependent calcium phosphate sequestration process (ADP(mt), CATR, local anesthetics). We also conclude that ANT affects PT by changing mitochondrial capacity for energization.

Topics: Adenosine Diphosphate; Alamethicin; Animals; Atractyloside; Brain; Calcium Phosphates; Cyclosporine; Dibucaine; Hydrogen-Ion Concentration; Intracellular Membranes; Ion Channels; Light; Male; Membrane Potentials; Mitochondria; Mitochondria, Liver; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Oligomycins; Rats; Rats, Inbred Lew; Scattering, Radiation; Titrimetry

Isolated brain mitochondria were examined for their responses to calcium challenges under varying conditions. Mitochondrial membrane potential was monitored by following the distribution of tetraphenylphosphonium ions in the mitochondrial suspension, mitochondrial swelling by observing absorbance changes, calcium accumulation by an external calcium electrode, and oxygen consumption with an oxygen electrode. Both the extent and rate of calcium-induced mitochondrial swelling and depolarization varied greatly depending on the energy source provided to the mitochondria. When energized with succinate plus glutamate, after a calcium challenge, CNS mitochondria depolarized transiently, accumulated substantial calcium, and increased in volume, characteristic of a mitochondrial permeability transition. When energized with 3 mM succinate, CNS mitochondria maintained a sustained calcium-induced depolarization without appreciable swelling and were slow to accumulate calcium. Maximal oxygen consumption was also restricted under these conditions, preventing the electron transport chain from compensating for this increased proton permeability. In 3 mM succinate, cyclosporin A and ADP plus oligomycin restored potential and calcium uptake. This low conductance permeability was not effected by bongkrekic acid or carboxyatractylate, suggesting that the adenine nucleotide translocator was not directly involved. Fura-2FF measurements of [Ca(2+)](i) suggest that in cultured hippocampal neurons glutamate-induced increases reached tens of micromolar levels, approaching those used with mitochondria. We propose that in the restricted substrate environment, Ca(2+) activated a low-conductance permeability pathway responsible for the sustained mitochondrial depolarization.

Topics: Adenosine Diphosphate; Animals; Antineoplastic Agents; Atractyloside; Brain; Calcium; Electric Conductivity; Fluorescent Dyes; Fura-2; Glutamic Acid; Membrane Potentials; Mitochondria; Mitochondrial Swelling; Nerve Degeneration; Oligomycins; Onium Compounds; Organophosphorus Compounds; Oxidation-Reduction; Oxygen Consumption; Rats; Strontium; Succinic Acid; Uncoupling Agents

The effect of 3'-azido-3'-deoxythymidine on nucleoside diphosphate kinase of isolated rat liver mitochondria has been studied. This is done by monitoring the increase in the rate of oxygen uptake by nucleoside diphosphate (TDP, UDP, CDP or GDP) addition to mitochondria in state 4. It is shown that 3'-azido-3'-deoxythymidine inhibits the mitochondrial nucleoside diphosphate kinase in a competitive manner, with a Ki value of about 10 microM as measured for each tested nucleoside diphosphate. It is also shown that high concentrations of GDP prevent 3'-azido-3'-deoxythymidine inhibition of the nucleoside diphosphate kinase.

Topics: Animals; Atractyloside; Binding, Competitive; Cytidine Diphosphate; Enzyme Inhibitors; Guanosine Diphosphate; Kinetics; Mitochondria, Liver; Nucleoside-Diphosphate Kinase; Oligomycins; Oxygen Consumption; Rats; Thymine Nucleotides; Uridine Diphosphate; Zidovudine

The membrane permeability transition (MPT) induced by Ca2+ and Pi or Asi was studied in rat kidney mitochondria. Membrane potential, Ca2+ transport and swelling were used to monitor the MPT. Asi promoted a faster and more extensive collapse of membrane potential, Ca2+ release and swelling than Pi. The MPT induced by Pi was fully blocked by Mg(2+)+ADP, spermine+ADP, Mg(2+)+ cyclosporin A (CSA), and ADP+CSA. In contrast, the MPT induced by Asi was only prevented, although not completely, by CSA+Mg2+ or ADP+CSA. Asi, but not Pi, was able to cause collapse of membrane potential in the presence of Sr2+. Carboxyatractyloside (CAT) produced collapse of membrane potential at a lower concentration in the presence of Asi+Ca(2+)+ADP than with Pi+Ca(2+)+ADP. The addition of Pi+Ca2+ to [14C]-ADP loaded mitochondria brought about a greater ADP release than Asi+Ca2+. The ADP release was CAT-sensitive with Pi but it was only partially blocked by Asi. The diminution of external pH did not inhibit the MPT induced by Pi or Asi. The results of this study suggest that the adenine nucleotide translocase does not have an essential role in the MPT induced by Asi+Ca2+.

Topics: Adenosine Diphosphate; Animals; Arsenates; Arsenazo III; Atractyloside; Calcium; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cyclosporine; Female; Intracellular Membranes; Kidney; Male; Membrane Potentials; Mitochondria; Oligomycins; Permeability; Phenazines; Phosphates; Rats; Spermine; Trifluoperazine

The exogenous NADH dehydrogenase of heart mitochondria is increasingly reported to mediate cardiomyopathies following adriamycin treatment or reperfusion of ischemic hearts. A great number of studies on the biochemistry and pathobiology exists which indirectly support the existence of this dehydrogenase. Our studies exclude both the rotenone-insensitive NADH dehydrogenase of the outer membrane and the endogenous NADH dehydrogenase of damaged mitochondria as being responsible for external NADH consumption. Reducing equivalents from external NADH were demonstrated to enter complex I of the respiratory chain from the cytosolic phase. Our data support earlier reports on the physical association of the exogenous NADH dehydrogenase with the inner mitochondrial membrane excluding oxidation of external NADH via an enzyme of artefactual origin.

Topics: Animals; Artifacts; Atractyloside; Cytochrome b Group; Cytosol; Kinetics; Male; Mitochondria, Heart; Mitochondria, Liver; Myocardium; NAD; NAD(P)H Dehydrogenase (Quinone); Oligomycins; Oxidation-Reduction; Oxidative Phosphorylation; Oxygen Consumption; Rats; Rats, Sprague-Dawley

The energy state of rat liver mitochondria on day 10 of cold acclimation, when the body temperature decreases significantly concomitantly with an increase in the content of long-chain acyl-CoAs in the liver, has been studied. State 4 and uncoupling respiration rates increase in parallel; however, the integral potentials of the adenine nucleotide system in the liver diminish. In the presence of oligomycin, ADP (20 and 50 microM) decreases the II(+)-permeability and increases the Ca(2+)-capacity of mitochondria in both control and, in a lesser degree, cold-exposed rats. At 90 microM ADP has the same effect on mitochondria of both groups of rats. Carboxyatractylate abolishes the ADP effect on the mitochondria. In EGTA-containing media carboxyatractylate decreases the respiration rate in oligomycin-treated mitochondria. Palmitoyl-CoA increases the II(+)-permeability of the mitochondrial membrane and decreases the Ca(2+)-capacity of mitochondria. ADP abolished the competitive effects of this long-chain acyl-CoA. Possible involvement of the ATP/ADP antiporter in the thermoregulatory response of liver mitochondria in cold-acclimated rats and the role of fatty acids and long-chain acyl-CoAs in this process are discussed.

Topics: Acyl Coenzyme A; Adaptation, Physiological; Animals; Atractyloside; Body Temperature Regulation; Cold Temperature; Energy Metabolism; Intracellular Membranes; Ion Transport; Male; Mitochondria, Liver; Mitochondrial ADP, ATP Translocases; Oligomycins; Oxygen; Permeability; Rats; Rats, Wistar

The effects of carboxyatractylate (CAtr) on delta psi in sunflower hypocotyl and pea stem mitochondria were compared. In sunflower mitochondria, (1) CAtr at higher concentration increased delta psi in the presence of palmitate and delta psi in metabolic state 3; (2) ]1 microM CAtr did not prevent delta psi decrease, induced by ADP addition (in contrast to pea mitochondria); (3) The ATP-generated delta psi was small and was insensitive to 40 microM CAtr. Under the same conditions, in pea mitochondria generation of delta psi by ATP was inhibited by 1 microM CAtr.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Atractyloside; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Fabaceae; Helianthus; Intracellular Membranes; Kinetics; Membrane Potentials; Mitochondria; Oligomycins; Palmitic Acid; Palmitic Acids; Plant Stems; Plants, Medicinal

Oleate (10-60 microM) stimulates oxygen consumption in hepatocytes in the presence of oligomycin. This stimulation of respiration is partially suppressed by 20 microM carboxyatractylate but is insensitive to 0.5 microM cyclosporin A. The results obtained suggest that fatty acids can uncouple oxidative phosphorylation in hepatocytes and that the uncoupling mechanism is the same as that in isolated mitochondria in the presence of EGTA.

Topics: Animals; Atractyloside; Cyclosporine; Drug Synergism; Male; Mitochondria, Liver; Oleic Acid; Oleic Acids; Oligomycins; Oxidative Phosphorylation; Oxygen; Rats; Rats, Sprague-Dawley; Uncoupling Agents

The uncoupling effect of 2-hexadecylmalonic acid (HDMA) and 2-tetradecylmalonic acid (TDMA), the dicarboxylic derivatives of stearic and palmitic acids, have been studied in heart mitochondria. The effects of these compounds and related fatty acids on the induced by ascorbate oxidation with PMS (or TMPD) in the presence of oligomycin, phosphate and rotenone have been compared. It was found that both HDMA and TDMA uncoupled mitochondria with a similar efficiency as did the corresponding fatty acids, while the uncoupling effect of low concentrations of HDMA and TDMA were almost completely suppressed by carboxyatractylate.

Topics: Animals; Atractyloside; Mitochondria, Heart; Oligomycins; Oxidation-Reduction; Palmitic Acids; Phosphates; Rats; Rotenone; Stearic Acids; Uncoupling Agents

Regulation of the mitochondrial permeability transition pore has been investigated following the release of matrix solutes which normally participate in pore regulation. Under these conditions, neither cyclosporin A nor ADP induces pore closure, as judged by restoration of delta psi, unless Mg2+ is also added. Mg2+ alone is ineffective. In liver mitochondria, the Mg2+ effect is expressed over a 0 to 0.5 mM concentration range with higher concentrations inhibiting repolarization. In heart mitochondria, the inhibitory action of high Mg2+ is not seen and it can be shown that the Mg2+ effect on repolarization increases progressively up to a concentration of 5 mM. In liver mitochondria, when the pore is closed by maximally effective concentrations of Mg2+ plus cyclosporin A or Mg2+ plus ADP, reopening occurs upon the addition of carboxyatractyloside. The latter compound, however, fails to reopen the pore when Mg2+, cyclosporin A, and ADP are present simultaneously. In heart mitochondria, where higher Mg2+ concentrations can be employed, Mg2+ plus cyclosporin A or Mg2+ plus ADP produces pore closure in a carboxyatractyloside insensitive manner. Titration experiments support the adenine nucleotide translocase as the site at which carboxyatractyloside acts to regulate the pore. However, the action of ADP appears to involve a translocase-independent site. In intact mitochondria the action of carboxyatractyloside on pore opening is counteracted by oligomycin, apparently through inhibition of the F1F0 ATP synthase, with a consequent increase in the matrix space ADP/ATP ratio. It is concluded that the permeability transition pore induced by Ca2+ plus P(i) is not formed from the adenine nucleotide translocase although the translocase conformation is one of several factors which regulate the pore. The matrix Mg2+ concentration is also one of these factors. Formation of the pore by a Ca2+ and ADP binding protein is one model which is consistent with the present data.

Topics: Adenosine Diphosphate; Animals; Atractyloside; Binding Sites; Cyclosporine; Intracellular Membranes; Kinetics; Magnesium; Mitochondria, Heart; Mitochondria, Liver; Mitochondrial ADP, ATP Translocases; Models, Biological; Oligomycins; Permeability; Proton-Translocating ATPases; Rats

The effect of TPP+ on the fatty acid or FCCP-induced uncoupling in rat heart mitochondria was studied. It was found that (a) TPP+ increases the stimulation of oxygen consumption by palmitic acid or FCCP in the presence of oligomycin, (b) TPP+ greatly enhances the palmitic acid or FCCP-induced delta psi decrease. Both effects of TPP+ were strongly suppressed by carboxyatractylate in the case of palmitate but were not in the case of FCCP. The role of ATP/ADP-antiporter in the TPP+ and palmitic acid effects is discussed.

Topics: Animals; Atractyloside; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Fluorescent Dyes; In Vitro Techniques; Mitochondria, Heart; Oligomycins; Onium Compounds; Organophosphorus Compounds; Oxygen Consumption; Palmitic Acid; Palmitic Acids; Phenazines; Rats; Spectrometry, Fluorescence; Uncoupling Agents

Respiring mitochondria drive the electrophoretic uptake of K+ and other cations. In the presence of permeant acids this transport leads to mitochondrial swelling if it is not compensated by electroneutral K+/H+ exchange mediated by the K+/H+ antiporter. The mechanism of influx has yet to be established; however, evidence is accumulating that in addition to leak pathways a specific K+ channel or uniporter may be involved. We examine some of the properties of K+ uniport which are consistent with the existence of a specific ATP-regulated K+ channel. In contrast to the K+/H+ antiporter, K+ uniport shows little dependence on pH. K+ uniport is, however, very sensitive to inhibition by adenine nucleotides. The maximum percent inhibition is increased from 40 to 60% by treatment of mitochondria with N-ethylmaleimide (30 nmol/mg) which stimulates K+ uniport 3.6-fold. N-Ethylmaleimide, however, has no effect on the IC50 values which are 0.5, 2.3, and 8 microM for ADP, ATP, and AMP, respectively. GDP has no effect, while carboxyatractyloside is found to inhibit. The nucleotide analogs Cibacron blue 3GA and erythrosin B exhibit three effects on K+ uniport. Low doses partially inhibit K uniport (I50 = 0.13 microM Cibacron Blue), while higher doses stimulate (EC50 = 13 microM Cibacron Blue). Stimulation is especially apparent in N-ethylmaleimide-treated mitochondria. These analogs also antagonize inhibition by ATP. Since the EC50 values for this antagonism for these two drugs are similar, while the IC50 values for inhibition of ATP transport differ by a factor of five, we suggest that inhibition of K+ uniport by ATP is not mediated via the adenine nucleotide translocase. These data are consistent with the existence of an ATP-regulated K+ channel in the inner mitochondrial membrane.

Topics: Adenine Nucleotides; Adenosine Triphosphate; Animals; Atractyloside; Biological Transport; Erythrosine; Ethylmaleimide; Guanosine Diphosphate; Kinetics; Mitochondria; Mitochondrial Swelling; Oligomycins; Oxygen Consumption; Potassium; Potassium Channels; Proton-Translocating ATPases; Sulfhydryl Reagents; Tetraethylammonium; Tetraethylammonium Compounds; Triazines

Energy coupling parameters were studied in liver mitochondria of ground squirrel during arousal from hibernation. It was found that such mitochondria become uncoupled during incubation with phosphate in a salt medium. The uncoupling was revealed by respiration rate increase and membrane potential decrease in the presence of oligomycin. Both effects were reversed by addition of cyclosporin A. Under the same in vitro conditions, mitochondria from aroused (active) animals showed no uncoupling but could be uncoupled by addition of palmitate in the cyclosporin A-sensitive fashion. It is proposed that formation of cyclosporin A-sensitive pores can be involved in urgent heat production in arousing hibernators.

Topics: Animals; Atractyloside; Cyclosporine; Hibernation; Membrane Potentials; Mitochondria, Liver; Oligomycins; Oxidative Phosphorylation; Oxygen; Sciuridae

The flux control coefficients of adenine nucleotide translocase, the phosphate transporter, and H(+)-ATPase were determined in rat skeletal muscle mitochondria using glutamate plus malate as substrates and soluble F1-ATPase as load enzyme. It was observed that the flux control coefficients of adenine nucleotide translocase, H(+)-ATPase, and the load enzyme F1-ATPase, at comparable rates of respiration, strongly depend on the phosphate concentration in the incubation medium. So, the flux control exerted by adenine nucleotide translocase, in the intermediate states of mitochondrial respiration (approximately 120 nmol of O2/min/mg) at 10 mM phosphate, was found to be about 0.37. At a phosphate concentration of 1 mM and comparable rates of respiration the flux control coefficient of the translocase decreased to about 0.20. Under these conditions, a sharp increase in the controlling influence of H(+)-ATPase from 0.10 to 0.74 was detected. Furthermore, at this flux rate, the sum of flux control coefficients of adenine nucleotide translocase, H(+)-ATPase, phosphate transporter, and the load enzyme F1-ATPase was noted to be very close to unity. This indicates that under the conditions of intermediate state respiration, all of the other reactions have a negligible controlling influence on oxidative phosphorylation in skeletal muscle mitochondria.

Topics: Animals; Atractyloside; Carrier Proteins; Cattle; Kinetics; Mersalyl; Mitochondria, Heart; Mitochondria, Muscle; Mitochondrial ADP, ATP Translocases; Oligomycins; Oxidative Phosphorylation; Oxygen Consumption; Phosphate-Binding Proteins; Phosphates; Proton-Translocating ATPases; Rats; Regression Analysis

Thermoregulatory uncoupling of oxidative phosphorylation has been studied in heart and skeletal muscle mitochondria of ground squirrels. The respiratory rate of mitochondria in the presence of oligomycin was found to be much higher in winter (in hibernating, arousing, or aroused animals) than in summer. This additional respiration is strongly (arousing animals) or completely (hibernating and aroused animals) inhibited by carboxyatractylate (CAtr) and bovine serum albumin (BSA). The CAtr- and BSA-induced decreases in the rate of respiration are accompanied by membrane potential increases. The rate of the CAtr- and BSA-sensitive respiration is proportional to the content of free fatty acids which, in the heart, decreases in the order: arousing greater than aroused = hibernating greater than summer animals. Maximal respiratory rates observed in the presence of dinitrophenol (arousing greater than aroused greater than summer greater than hibernating animals) do not parallel the fatty acid level. It is assumed that some heat production in the winter animals is due to fatty acid-induced, ATP/ADP-antiporter-mediated uncoupling in heart and skeletal muscle mitochondria. The peak of heat production during arousal after hibernation also includes some other stimulatory effect on mitochondrial respiration.

Topics: Animals; Atractyloside; Body Temperature Regulation; Fatty Acids, Nonesterified; Kinetics; Mitochondria, Heart; Mitochondria, Liver; Mitochondria, Muscle; Oligomycins; Organ Specificity; Oxidative Phosphorylation; Sciuridae; Serum Albumin, Bovine

The respiratory capacities of hepatocytes, derived from hypothyroid, euthyroid and hyperthyroid rats, have been compared by measuring rates of oxygen uptake and by titrating components of the respiratory chain with specific inhibitors. Thyroid hormone increased the maximal rate of substrate-stimulated respiration and also increased the degree of ionophore-stimulated oxygen uptake. In titration experiments, similar concentrations of oligomycin or antimycin were required for maximal inhibition of respiration regardless of thyroid state, suggesting that the changes in respiratory capacity were not the result of variation in the amounts of ATP synthase or cytochrome b. However, less rotenone was required for maximal inhibition of respiration in the hypothyroid state than in cells from euthyroid or hyperthyroid rats, implying that hepatocytes from hypothyroid animals contain less NADH dehydrogenase. The concentration of carboxyatractyloside necessary for maximal inhibition of respiration was 100 microM in hepatocytes from hypothyroid rats, but 200 microM and 300 microM in hepatocytes from euthyroid and hyperthyroid rats, respectively, indicating a possible correlation between levels of thyroid hormone and the amount or activity of adenine nucleotide translocase. The increased capacity for coupled respiration in response to thyroid hormone is not associated with an increase in the components of the electron transport chain or ATP synthase, but correlates with an increased activity of adenine nucleotide translocase.

Topics: Animals; Antimycin A; Atractyloside; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cells, Cultured; Hyperthyroidism; Hypothyroidism; Kinetics; Liver; Male; Oligomycins; Oxygen Consumption; Rats; Rats, Inbred Strains; Reference Values; Rotenone; Thyroid Gland; Triiodothyronine

The frequency of contacts between the mitochondrial envelope membranes was determined in freeze-fractured samples of isolated mitochondria by means of quantifying the frequency of fracture plane deflections between the two membranes. It was observed that the formation of contacts correlated with the concentration of free ADP despite of inhibition of electron transport by antimycin A. The activity of ATPase partially inhibited by oligomycin or depletion of membrane potential by K+ and valinomycin had no effect on the induction of the contacts by ADP. ATP was ineffective in creating contacts irrespective of the presence or absence of a membrane potential, whereas carboxyatractyloside induced the contacts under all conditions in a manner similar to ADP. These results suggest the involvement of the ATP/ADP translocator in regulation of contact sites. As a consequence, we analyzed its distribution in the inner membrane of kidney and liver mitochondria by binding of [3H]atractyloside to subfractions of this membrane. The experiments demonstrated that the translocator was located in the peripheral part of the inner membrane as well as in the portion which formed the cristae.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Antimycin A; Atractyloside; Electron Transport; Freeze Fracturing; Intracellular Membranes; Membrane Potentials; Mitochondria, Liver; Mitochondrial ADP, ATP Translocases; Mitochondrial Swelling; Oligomycins; Oxidative Phosphorylation; Proton-Translocating ATPases; Rats

Investigations with dihydrolipoic acid in rat heart mitochondria and mitoplasts reveal an activation of ATP-synthase up to 45%, whereas ATPase activities decrease by 36%. In parallel with an increase in ATP synthesis oligomycin-sensitive mitochondrial -SH groups are activated at 2-4 nmol dihydrolipoic acid/mg protein. ATPase activation by the uncouplers carbonylcyanide-p-trifluoromethoxyphenylhydrazone and oleate is diminished by dihydrolipoic acid, and ATP synthesis depressed by oleate is partially restored. No such efficiency of dihydrolipoic acid is seen with palmitate-induced ATPase activation or decrease of ATP synthesis. This indicates different interference of oleate and palmitate with mitochondria. In addition to its known coenzymatic properties dihydrolipoic acid may act as a substitute for coenzyme A, thereby diminishing the uncoupling efficiency of oleate. Furthermore, dihydrolipoic acid is a very potent antioxidant, shifting the -SH-S-S- equilibrium in mitochondria to the reduced state and improving the energetic state of cells.

Topics: Adenosine Triphosphatases; Adenosine Triphosphate; Animals; Atractyloside; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Female; Kinetics; Mitochondria, Heart; Oleic Acid; Oleic Acids; Oligomycins; Oxidative Phosphorylation; Palmitic Acid; Palmitic Acids; Rats; Rats, Inbred Strains; Sulfhydryl Compounds; Thioctic Acid

The addition of oligomycin in the presence of Ca2+ increased the ADP pool in mitochondrial suspension. It is suggested that oligomycin inhibition of Ca(2+)-induced mitochondrial respiratory activation is the function of the increased endogenous ADP pool. Low ADP concentrations (5-20 microM) produce the same inhibitory effect as oligomycin. The increase of ADP levels in the presence of glucose plus hexokinase resulted in the inhibition of Ca(2+)-induced respiration, while the addition of phosphoenol pyruvate plus pyruvate kinase followed by a reduction in ADP levels, reversed the oligomycin inhibitory effect. One of the essential stages of ADP accumulation in mitochondrial suspensions in the presence of oligomycin and Ca2+ is proposed to be the formation of ADP from AMP and ATP, effected by adenylate kinase.

Topics: Adenine Nucleotides; Adenylate Kinase; Animals; Atractyloside; Calcium; In Vitro Techniques; Mitochondria, Liver; Mitochondrial ADP, ATP Translocases; Oligomycins; Oxygen Consumption; Rats

The ATP synthase capacity of rat heart myocytes can be measured in sonicated cell suspensions and in sonicated preparations of cultured cardiomyocytes. This procedure allows the rapid measurement of mitochondrial function in response to changes in the metabolic status of the cell. In cultured myocytes, transitions in ATP synthase capacity (with no detectable change in cellular ATP concentration) accompany a change to anoxia or electrically stimulated contraction (rise of 70%). These changes are reversed on returning to the original conditions. Exposure of myocytes to low pH has little effect on basal ATP synthase capacity (down to values less than pH 6), but markedly affects cellular ATP levels and the response of the cells to anoxia and reoxygenation, possibly mimicking changes seen in ischaemic heart. Similar effects are seen in suspensions of freshly prepared myocytes, but these preparations are less stable and more pH-sensitive than are cells in culture. It is proposed that mitochondria in vivo are directly regulated at the level of the ATP synthase, and that a regulator protein, the naturally occurring inhibitor protein from mitochondria, may be responsible for this regulation.

Topics: Adenosine Triphosphate; Animals; Atractyloside; Cells, Cultured; Cold Temperature; Electric Stimulation; Energy Metabolism; Hydrogen-Ion Concentration; Hypoxia; In Vitro Techniques; Intracellular Membranes; Ischemia; Male; Mitochondria, Heart; Oligomycins; Ouabain; Proton-Translocating ATPases; Rats; Rats, Inbred Strains; Vanadium

The stimulation of respiration by long-chain fatty acids and FCCP was studied with oligomycin-inhibited mitochondria from rat liver, heart and kidney tissue. By addition of equal amounts of palmitate and oleate, mitochondrial respiration was increased in the order RLM less than RKM less than RHM. Using the classical protonophore FCCP, this difference could not be observed. Inhibition of oleate-stimulated respiration by carboxyatractyloside decreased in the order RHM greater than RKM greater than RLM. As CAT sensitivity of oleate-stimulated respiration and the mitochondrial ANT content were found to be correlated, it is suggested that the weak CAT sensitivity of oleate-stimulated respiration of RLM [(1989) Biochim. Biophys. Acta 977, 266-272] is due to the low content of ANT.

Topics: Animals; Atractyloside; Chloramphenicol O-Acetyltransferase; Fatty Acids; Female; Kidney Cortex; Mitochondria; Mitochondria, Heart; Mitochondria, Liver; Mitochondrial ADP, ATP Translocases; Nucleotidyltransferases; Oleic Acids; Oligomycins; Palmitic Acids; Rats

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.

Topics: Animals; Anions; Atractyloside; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cholic Acid; Cholic Acids; Detergents; Mitochondria, Liver; Mitochondrial ADP, ATP Translocases; Oligomycins; Oxidation-Reduction; Oxygen Consumption; Palmitic Acid; Palmitic Acids; Permeability; Protons; Pyridoxal Phosphate; Rats; Rotenone; Sodium Dodecyl Sulfate; Succinates; Succinic Acid; Uncoupling Agents

Energy coupling parameters of liver mitochondria from hibernating and arousing ground squirrels have been studied. In the oligomycin-treated mitochondria, carboxyatractylate, an inhibitor of the ATP/ADP-antiporter, is shown to decrease the respiration rate, to increase the membrane potential and to lower the rate of the membrane-potential discharge after the addition of cyanide to liver mitochondria from hibernating and arousing animals. BSA effectively substitutes for carboxyactactylate so that carboxyactactylate, added after BSA, has no effect. In mitochondria from hibernating animals, the maximal respiration rate in the presence of DNP and the rate of the membrane potential discharge in its absence are much lower than in those from arousing animals. It has been concluded that upon arousal of the animals from hibernation, the uncoupling of oxidative phosphorylation, mediated by free fatty acids and ATP/ADP-antiporter, parallels the respiratory chain activation.

Topics: 2,4-Dinitrophenol; Animals; Arousal; Atractyloside; Dinitrophenols; Fatty Acids, Nonesterified; Hibernation; Membrane Potentials; Mitochondria, Liver; Mitochondrial ADP, ATP Translocases; Oligomycins; Oxidative Phosphorylation; Oxygen Consumption; Palmitic Acid; Palmitic Acids; Sciuridae; Serum Albumin, Bovine

The effect of long-chain fatty acids (LCFA) on respiration and transmembrane potential (delta psi) in the resting state, and the rate of delta psi dissipation [d delta psi/dt)i) was investigated with oligomycin-inhibited rat liver mitochondria using succinate (plus rotenone) as substrate. The results obtained were compared with those of classical protonophores such as 2,4-dinitrophenol (DNP) and 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole (TTFB). The effects of oleate or palmitate and that of DNP or TTFB on respiration and delta psi can be described by a common force-flow relationship. These facts all in all are not compatible with a decoupler-type uncoupling mechanism of LCFA; still, they indicate that the latter are protonophores. Moreover, the oleate-induced increase in the rate of delta psi dissipation closely correlates with that in respiration, suggesting that the uncoupling activity and the protonophoric activity of LCFA are interrelated. Carboxyatractyloside (CAT) exerted only a small inhibitory effect on oleate-induced respiration and delta psi dissipation, indicating that the adenine nucleotide translocase contributes to the uncoupling effect of LCFA to a minor extent only. Proton transport through the lipid region of the membrane as mediated by permeation of the protonated and deprotonated forms of LCFA is interpreted as the main process of the uncoupling of LCFA.

Topics: 2,4-Dinitrophenol; Animals; Atractyloside; Benzimidazoles; Biological Transport; Dinitrophenols; Fatty Acids; Female; Membrane Potentials; Mitochondria, Liver; Mitochondrial ADP, ATP Translocases; Oleic Acid; Oleic Acids; Oligomycins; Oxidative Phosphorylation; Oxygen Consumption; Palmitic Acid; Palmitic Acids; Protons; Rats; Uncoupling Agents

The catabolism of intramitochondrial guanine nucleotides was examined. During 30 min incubation of rat liver mitochondria at 37 degrees C in the presence of oligomycin and carboxyatractyloside, guanine and xanthine were formed and appeared in the medium. Under these conditions, the direct conversion of GMP to guanine by hypoxanthine-guanine phosphoribosyltransferase is suggested to be the main catabolic route within the organelles. Only very small amounts of guanosine were produced and detected both inside and outside the organelles. [14C]Guanosine and [14C]inosine were taken up by the mitochondria. Therefore, guanosine is suggested to be a precursor of intramitochondrial guanine nucleotides.

Topics: Animals; Atractyloside; Chromatography, High Pressure Liquid; Guanine; Guanine Nucleotides; Guanosine; Inosine; Male; Mitochondria, Liver; Oligomycins; Rats; Rats, Inbred Strains; Subcellular Fractions; Temperature; Xanthine; Xanthines

The degradation of endogenous adenine nucleotides was compared in mitochondria isolated from mouse and rat liver, rat renal cortex and solid hepatoma. Mitochondria were incubated for 30 min at 37 degrees C in the presence of carboxyatractyloside and oligomycin. In rat liver mitochondria ATP, ADP and AMP were degraded by about 25% each, whereas in kidney and hepatoma mitochondria a rapid decline of ATP and ADP but no change in the AMP contents were observed. Main products formed were adenosine, inosine and hypoxanthine in all mitochondria examined. Compartment studies revealed that the main route of intramitochondrial adenine nucleotide catabolism seems to proceed via AMP dephosphorylation and subsequent adenosine deamination.

Topics: Adenine Nucleotides; Animals; Atractyloside; Kidney Cortex; Kinetics; Liver Neoplasms, Experimental; Mice; Mitochondria; Mitochondria, Liver; Oligomycins; Organ Specificity; Rats

The lipophilic triphenylmethylphosphonium cation (TPMP+) has been employed to measure delta psi m, the electrical potential across the inner membrane of the mitochondria of intact hepatocytes. The present studies have examined the validity of this technique in hepatocytes exposed to graded concentrations of inhibitors of mitochondrial energy transduction. Under these conditions, TPMP+ uptake allows a reliable measure of delta psi m in intracellular mitochondria, provided that the ratio [TPMP+]i/[TPMP+]e is greater than 50:1 and that at the end of the incubation more than 80% of the hepatocytes exclude Trypan blue. Hepatocytes, staining with Trypan blue, incubated in the presence of Ca2+, do not concentrate TPMP+. The relationships between delta psi m and two other indicators of cellular energy state, delta GPc and Eh, or between delta psi m and J0, were examined in hepatocytes from fasted rats by titration with graded concentrations of inhibitors of mitochondrial energy transduction. Linear relationships were generally observed between delta psi m and delta GPc, Eh or J0 over the delta psi m range of 120-160 mV, except in the presence of carboxyatractyloside or oligomycin, where delta psi m remained constant. Both the magnitude and the direction of the slope of the observed relationships depended upon the nature of the inhibitor. Hepatocytes from fasted rats synthesized glucose from lactate or fructose, and urea from ammonia, at rates which were generally linear functions of the magnitude of delta psi m, except in the presence of oligomycin or carboxyatractyloside. Linear relationships were also observed between delta psi m and the rate of formation of lactate in cells incubated with fructose and in hepatocytes from fed rats. The linear property of these force-flow relationships is taken as evidence for the operation of thermodynamic regulatory mechanisms within hepatocytes.

Topics: Adenine Nucleotides; Animals; Atractyloside; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Energy Metabolism; Gluconeogenesis; Indicators and Reagents; Intracellular Membranes; Liver; Male; Membrane Potentials; Mitochondria, Liver; Oligomycins; Onium Compounds; Rats; Rats, Inbred Strains; Staining and Labeling; Thermodynamics; Trityl Compounds; Trypan Blue; Urea; Valinomycin

The activating anions are found to induce an unexpectedly high (up to 8-fold for sulphite) increase of ATPase activity in intact rat liver mitochondria. This effect is not determined by the observed changes in Km and Ki (ADP) values. The stimulation seems to be caused by dissociation of the inactive complex of ATPase with Mg.ADP. The quantity of this complex formed in the course of ATP hydrolysis is approx. 90% of the total ATPase content in intact mitochondria. The data on toluene-permeabilized mitochondria suggest that the high content of the complex is a result of the stabilizing effect of some matrix macromolecules.

Topics: Adenosine Triphosphate; Animals; Anions; Atractyloside; Azides; Enzyme Activation; Kinetics; Magnesium; Mitochondria, Liver; Oligomycins; Proton-Translocating ATPases; Rats; Sodium Azide; Sulfites

The effects of Reye's plasma, allantoin, and salicylates on mitochondrial structure and Ca2+ transport have been investigated. Measurements of Ca2+ transport showed that when 20-30 microM Ca2+ was added to isolated rat liver mitochondria preincubated with one of these agents, Ca2+ uptake was followed by its spontaneous release into the medium. This was accompanied by large-amplitude swelling; the onset preceded the Ca2+ release. No further Ca2+ release was induced by uncoupler or the Ca2+ ionophore, A23187. The mitochondria continued to swell even after all of the Ca2+ had been released. The time between the addition of Ca2+ and the onset of swelling (or Ca2+ release) depended on the concentration of the agent added and the preincubation time; the extent of swelling did not. These effects were prevented, but not reversed, by ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid, ruthenium red, rotenone, or adenine nucleotides. The massive swelling and membrane disruption were confirmed by electron microscopy of the treated vs untreated mitochondria. Similar results concerning swelling and Ca2+ release were also seen with Ca2+ alone, but the time scale was much longer (i.e., greater than 3-4 min), indicating that these agents act by potentiating Ca2+-induced alterations in mitochondrial structure, as suggested by our earlier work (T.Y. Segalman and C.P. Lee (1982) Arch. Biochem. Biophys. 214, 522-530; M.E. Martens and C.P. Lee (1984) Biochem. Pharmacol. 33, 2869-2876). Our data show, therefore, that allantoin, salicylates, and the "toxic" agent in Reye's plasma severely limit the ability of isolated rat liver mitochondria to maintain their structural integrity under conditions of limited Ca2+ loading.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Allantoin; Animals; Atractyloside; Biological Transport; Calcimycin; Calcium; Humans; In Vitro Techniques; Male; Microscopy, Electron; Mitochondria, Liver; Mitochondrial Swelling; Oligomycins; Oxygen Consumption; Rats; Rats, Inbred Strains; Reye Syndrome; Rotenone; Salicylates; Salicylic Acid

The regulation of the rate of mitochondrial oxidative phosphorylation and arsenylation was studied at two external free Ca2+ concentrations. The rate of arsenate-stimulated respiration in absence of added ADP was not affected by external 10(-9) and 10(-6) M Ca2+ levels or carboxyatractyloside, while state 3 respiration was profoundly modified. In addition, the kinetic analysis showed that the rate of arsenylation in the presence of ADP was more efficient (Vm/Km ratio 3.5 times higher) in the catalytic process than phosphorylation. Therefore, this suggests that the activity of the ATP/ADP carrier is importantly controlled by Ca2+. The evaluation of the control in phosphorylation showed that the flux-control coefficients (Ci) exerted by the ATP/ADP carrier (ranged between 0.23 and 0.48) and the ATP synthase (0.05-0.57) were modified in a reciprocal way by Ca2+ and Pi concentrations. This suggests that these two enzymes are coupling sequentially through a common intermediate, the intramitochondrial ATP/ADP ratio. Other important steps controlling phosphorylation were the b-c1 complex (Ci = 0.30) and the cytochrome oxidase (Ci = 0.23) but they were not modified by Ca2+. It was also found that the main step controlling arsenylation was the ATP synthase (Ci = 0.74). The increment in the inorganic arsenate concentration induced a diminution in the control exerted by the ATP synthase (from 0.73 to 0.56). The results suggest that Ca2+ and Pi (or inorganic arsenate) could be regulated by ATP synthesis through an activating effect on ATP/ADP carrier and/or ATP synthase.

Topics: Adenosine Diphosphate; Animals; Arsenates; Arsenic; Atractyloside; Calcium; Kinetics; Mitochondria, Liver; Mitochondrial ADP, ATP Translocases; Nucleotidyltransferases; Oligomycins; Oxidative Phosphorylation; Proton-Translocating ATPases; Rats

The mechanism of stimulation of hepatic respiration by fatty acids was studied in isolated rat hepatocytes. Stimulation of respiration by fatty acids varied from about 35% to about 105% depending on chain length. The stimulatory effect of octanoate (1 mM) or oleate (0.5 mM) was prevented by oligomycin (2 micrograms/ml). With carboxyatractyloside (100 microM) and ouabain (2 mM) the stimulation of respiration was partially inhibited (by 50-70 and 50-60%, respectively). From these results it can be concluded that the increased rate of respiration after addition of fatty acids is coupled to ATP synthesis. A large part (50-60%) of this ATP is utilized by the (Na+ + K+)-ATPase.

Topics: 2,4-Dinitrophenol; Adenine Nucleotides; Animals; Atractyloside; Caprylates; Dinitrophenols; Energy Metabolism; Fatty Acids; In Vitro Techniques; Liver; Male; Oleic Acid; Oleic Acids; Oligomycins; Ouabain; Oxygen Consumption; Rats; Rats, Inbred Strains

Adenosine diphosphatase (ADPase) activity and ATPase activity were assayed in rat liver mitochondria and outer mitochondrial membrane preparations with [beta-32P]ADP and [gamma-32P]ATP as substrates. Inhibition studies were performed with the mitochondrial ATPase inhibitor oligomycin and the adenine nucleotide transport inhibitor, carboxyatractyloside. Kinetic studies were also performed with the nucleotide thiophosphate analogs adenosine 5'-O-thiophosphate, adenosine 5'-O-(2-thiodiphosphate) and adenosine 5'-O-(3-thiotriphosphate) which can act as inhibitors of phosphohydrolases. It is concluded that part of the apparent ADPase activity of intact mitochondria is mediated via ATPase, presumably in conjunction with adenylate kinase. In addition the outer mitochondrial membrane appears to show a distinct ADPase not attributable to contamination by inner membrane ATPase.

Topics: Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphatases; Adenosine Triphosphate; Animals; Apyrase; Atractyloside; Female; In Vitro Techniques; Intracellular Membranes; Mitochondria, Liver; Oligomycins; Phosphoric Monoester Hydrolases; Rats; Rats, Inbred Strains; Thionucleotides

ATP hydrolysis catalysed by the H+-ATPase of intact mitochondria can be induced by addition of ATP in the presence of valinomycin and KCl. This leads to an increase in intramitochondrial Pi and therefore allows investigation of potential Pi efflux pathways in intact mitochondria. Combining this approach with the direct measurement of both internal and external Pi, we have attempted to determine whether Pi efflux occurs via an atractyloside-sensitive transporter, by the classical operation of the Pi/H+ and Pi/dicarboxylate carriers, and/or by other mechanisms. Initial experiments re-examined the evidence that led to the current view that one efflux pathway for Pi is an atractyloside-sensitive ATP/ADP,0.5Pi transporter. No evidence was found in support of this efflux pathway. Rather, atractyloside-sensitivity of the low rate of Pi efflux observed in previous studies (oligomycin present) was accounted for by ATP entry on the well known ATP/ADP transport system followed by hydrolysis of ATP and subsequent Pi efflux. Thus, under these conditions, where ATP hydrolysis is not completely inhibited, Pi efflux becomes atractyloside sensitive most likely because this inhibitor blocks ATP entry, not because it directly inhibits Pi efflux. Substantial efflux of Pi from rat liver mitochondria is observed on generation of high levels of matrix Pi by ATP hydrolysis induced by valinomycin and K+ (oligomycin absent). A portion of this efflux can be inhibited by thiol-specific reagents at concentrations that normally inhibit the Pi/H+ and Pi/dicarboxylate carriers. However, a significant fraction of efflux continues even in the presence of p-chloromercuribenzoate, N-ethylmaleimide plus n-butylmalonate or mersalyl. The mersalyl-insensitive Pi efflux, which is also insensitive to carboxyatractyloside, is a saturable process, thus suggesting carrier mediation. During this efflux the mitochondrial inner membrane retains considerable impermeability to other low-molecular-weight anions (i.e., malate, 2-oxoglutarate). In conclusion, results presented here rule out an atractyloside-sensitive ATP/ADP,0.5Pi transport system as a mechanism for Pi efflux in rat liver mitochondria. Rather Pi efflux appears to occur on the classical Pi/H+ transport system as well as via a mersalyl-insensitive saturable process. The inhibitor-insensitive Pi efflux may occur on a portion of the Pi/H+ carrier molecules that exist in a state different from that normally catalysing Pi influx. Alternatively, a

Topics: Adenosine Triphosphate; Animals; Atractyloside; Chloromercuribenzoates; Male; Mersalyl; Mitochondria, Liver; Oligomycins; p-Chloromercuribenzoic Acid; Phosphates; Potassium; Rats; Sucrose; Valinomycin