ATPase complex and oxidative phosphorylation in chloramphenicol-induced megamitochondria from mouse liver

1. 1. Functional properties of the ATPase complex are investigated in megamitochondria isolated from livers of weanling mice fed a diet containing 2% chloramphenicol, as an inhibitor of mitochondrial protein synthesis.

2. 2. Whereas the specific activity of ATPase remains unchanged in chloramphenicol-induced megamitochondria, about 40% of the enzyme activity is resistant to inhibition by oligomycin, triethyltin or venturicidin. It is concluded that the ATPase complex lacks one or more components whose synthesis or accumulation is dependent on mitochondrial translation. The inhibitor-resistant ATPase portion appears tightly bound to the mitochondrial membrane.

3. 3. Respiratory chain phosphorylation is tightly coupled in isolated megamitochondria. ATP synthesis and ATP-P_i exchange are diminished by 40%, as compared to control mitochondria, but both processes are sensitive to oligomycin, triethyltin or venturicidin.

4. 4. The decrease in ATP synthesis and ATP-P_i exchange in megamitochondria correlates quite well with the emergence of inhibitor-resistant ATPase.

5. 5. The following electron transport activities in the megamitochondria are reduced: NADH-cytochrome c reductase, by 60%, cytochrome oxidase, by 80%; the amount of antimycin required to gain complete inhibition of the bc₁-segment is diminished by more than 50%. On the other hand succinate dehydrogenase activity is increased by 50%.

6. 6. Chloramphenicol-induced megamitochondria appear to be a useful system for studying the role of mitochondrial translation in the assembly of mammalian mitochondria.

A requirement for ubiquinone in ATPase activity and oxidative phosphorylation.

The suggestion that a rapidly sedimenting rough endoplasmic reticulum fraction in close association with mitochondria, is the preferred site of cytochrome P-450 synthesis has been examined. The rate of cytochrome P-450 synthesis in the different subcellular fractions has been evaluated $\text{in}\text{vivo}\text{and}\text{in}\text{vitro}$, using the immunoprecipitation technique. The results indicate that the conventional microsomal fraction (100,000 X g sediment) is the major site of cytochrome P-450 synthesis and that the rapidly sedimenting rough endoplasmic reticulum fraction associated with mitochondria is not a preferred site for the hemoprotein synthesis.     

Effects of hydroxylated polychlorinated biphenyls on mouse liver mitochondrial oxidative phosphorylation.

The effects of three tetrachlorobiphenylols [2',3',4',5'-tetrachloro-2-biphenylol (1); 2',3',4',5'-tetrachloro-4- biphenylol (2); and 2',3',4',5'-tetrachloro-3-biphenylol (3)]; three monochlorobiphenylols [5-chloro-2-biphenylol (5), 3-chloro-2-biphenylol (6); and 2-chloro-4-biphenylol (7)] and a tetrachlorobiphenyldiol [3,3',5,5'-tetrachloro-4,4'-biphenyldiol (4) on respiration, adenosine triphosphatase (ATPase) activity, and swelling in isolated mouse liver mitochondria have been investigated. Tetrachlorobiphenylols (1-3) and the tetrachlorobiphenyldiol (4) inhibited state-3 respiration in a concentration-dependent manner with succinate as substrate (flavin adenine dinucleotide [FAD]-linked) and the tetrachlorobiphenyldiol (4) caused a more pronounced inhibitory effect on state-3 respiration than the other congeners. The monochlorobiphenylols 5-7 were less active as inhibitors of state-3 mitochondrial respiration and significant effects were observed only at higher concentration (greater than or equal to 0.4 microM). However, in the presence of the nicotinamide adenine dinucleotide (NAD)-linked substrates (glutamate plus malate), hydroxylated PCBs (1-7) significantly inhibited mitochondrial state-3 respiration in a concentration-dependent manner. Compounds 5, 6, and 7 uncoupled mitochondrial oxidative phosphorylation only in the presence of FAD-linked substrate as evidenced by increased oxygen consumption during state-4 respiratory transition, stimulating ATPase activity, releasing oligomycin-inhibited respiration, and inducing mitochondrial swelling (5, 6, and 7). Tetrachlorobiphenylols 1, 2, and 3 had no effect on mitochondrial ATPase activity while the tetrachlorobiphenyldiol, 4, decreased the enzyme activity. The possible inhibitory site of electron transport by these compounds and their toxicologic significance is discussed.     

On the mechanism of cuprizone-induced formation of megamitochondria in mouse liver

The livers of mice maintained on a diet containing 1% cuprizone for periods up to 18 days formed giant (mega) mitochondria equaling of exceeding the size of the nucleus. The megamitochondria were isolated from the liver and shown to be competent in respect to oxidative phosphorylation, although the P/O ratio and respiratory control index were somewhat reduced. When cuprizone was deleted from the diet, the size and function of the liver mitochondria returned to normal. Electron microscopic studies have made it possible to demonstrate the intermediate steps by which the membranes of paired mitochondria fuse and defuse to form megamitochondria, and from these studies a general mechanism may now be proposed for processes in which one continuous membrane splits into two separate membranes or by which two separated membranes fuse to form one continuous membrane.     

Effect of alpha-tocopherol and its derivatives on ATPase activity and oxidative phosphorylation in rat liver mitochondria

The effects of alpha-tocopherol and its derivatives on the energy metabolism in mitochondria were studied. It was shown that alpha-tocopherol derivatives with short hydrocarbon chain produce uncoupling effects, while alpha-tocopherol itself as well as its long-chain derivatives (tocopheryl acetate and tocopheryl stearate) exert no effect. All the derivatives studied produced an inhibitory effect on DNP-stimulated mitochondrial ATPase. As far as these compounds did not influence the activity of mitochondrial F1 factor, it was concluded that the effects of the compounds were due to membrane modification.     

The ATPase inhibitor protein in oxidative phosphorylation. The rate-limiting factor to phosphorylation in submitochondrial particles.

1. Purified luciferase and luciferin were used to study the time course of phosphorylation in submitochondrial particles. The light emitted was detected by a single-photon counter, using a multichannel analyser, and the results were analysed by an 'on-line' digital computer. 2. Using NADH as substrate, phosphorylation showed, in general, four phases. These were (i) a period of increasing rate ('lag'); (ii) a period of constant (positive) rate; (iii) a period of zero net rate (plateau), when the phosphorylation potential was maintained at its equilibrium value, and (iv) a period of negative rate (atp hydrolysis) after all the oxygen has been consumed. 3. The lag phase, several seconds in length, was a function of the inhibitor protein content of the particles. It was decreased in particles treated to remove the inhibitor protein, either by prior energisation of the particles with NADH, or by addition of aurovertin, which competes with the inhibitor protein for the ATPase. It was concluded that the ATPase inhibitor inhibits both ATP synthesis and hydrolysis by the ATPase. 4. The rate constant for the release of the inhibitor protein from the energised membrane was determined from the time course of ATP production during the lag phase. The activation energy of this process was measured from the temperature dependence of the lag, and was shown to be 13.3 kcal/mol, lower than the activation energy of ATP synthesis or NADH oxidation. 5. The rate constant for inhibitor release was dependent on 'energisation' of the membrane, being lower in the presence of uncouplers. However, it was possible to decrease the rate constant considerably with agents that collapsed the membrane potential without uncoupling the membrane. It was concluded that the inhibitor protein responded to the membrane potential component of the energisation. 6. A kinetic model for energy-dependent dissociation of the ATPase-inhibitor complex is proposed.     

Effects of hydroxylated polychlorinated biphenyls on mouse liver mitochondrial oxidative phosphorylation

The effects of three tetrachlorobiphenylols [2′,3′,4′,5′-tetrachloro-2-biphenylol (1); 2′,3′,4′,5′-tetrachloro-4-biphenylol (2); and 2′,3′,4′,5′-tetrachloro-3-biphenylol (3)]; three monochlorobiphenylols [5-chloro-2-biphenylol (5), 3-chloro-2-biphenylol (6); and 2-chloro-4-biphenylol (7)] and a tetrachlorobiphenyldiol [3,3′,5,5′-tetrachloro-4,4′-biphenyldiol (4) on respiration, adenosine triphosphatase (ATPase)] activity, and swelling in isolated mouse liver mitochondria have been investigated. Tetrachlorobiphenylols (1–3) and the tetrachlorobiphenyldiol (4) inhibited state-3 respiration in a concentration-dependent manner with succinate as substrate (flavin adenine dinucleotide [FAD]-linked) and the tetrachlorobiphenyldiol (4) caused a more pronounced inhibitory effect on state-3 respiration than the other congeners. The monochlorobiphenylols 5–7 were less active as inhibitors of state-3 mitochondrial respiration and significant effects were observed only at higher concentration (≥0.4 μM). However, in the presence of the nicotinamide adenine dinucleotide (NAD)-linked substrates (glutamate plus malate), hydroxylated PCBs (1–7) significantly inhibited mitochondrial state-3 respiration in a concentration-dependent manner. Compounds 5, 6, and 7 uncoupled mitochondrial oxidative phosphorylation only in the presence of FAD-linked substrate as evidenced by increased oxygen consumption during state-4 respiratory transition, stimulating ATPase activity, releasing oligomycin-inhibited respiration, and inducing mitochondrial swelling (5, 6, and 7). Tetrachlorobiphenylols 1, 2, and 3 had no effect on mitochondrial ATPase activity while the tetrachlorobiphenyldiol, 4, decreased the enzyme activity. The possible inhibitory site of electron transport by these compounds and their toxicologic significance is discussed.     

Tetrandrine concentrations not affecting oxidative phosphorylation protect rat liver mitochondria from oxidative stress

The effects of tetrandrine (6,6', 7,12-tetramethoxy-2, 2'-dimethyl-berbaman) on the mitochondrial function were assessed on oxidative stress, mitochondrial permeability transition (MPT), and bioenergetics of rat liver mitochondria. At concentrations lower than 100nmol/mg protein, tetrandrine decreased the hydrogen peroxide formation, the extent of lipid peroxidation, the susceptibility to Ca(2+)-induced opening of MPT pore, and inhibited the inner membrane anion channel activity, not significantly affecting the mitochondrial bioenergetics. High tetrandrine concentrations (100-300nmol/mg protein) stimulated succinate-dependent state 4 respiration, while some inhibition was observed for state 3 and p-trifluoromethoxyphenylhydrazone-uncoupled respirations. The respiratory control ratio and the transmembrane potential were depressed but the adenosine diphosphate to oxygen (ADP/O) ratio was less affected. A slight increase of the inner mitochondrial membrane permeability to H(+) and K(+) by tetrandrine was also observed. It was concluded that low concentrations of tetrandrine afford protection against liver mitochondria injury promoted by oxidative-stress events, such as hydrogen peroxide production, lipid peroxidation, and induction of MPT. Conversely, high tetrandrine concentrations revealed toxicological effects expressed by interference with mitochondrial bioenergetics, as a consequence of some inner membrane permeability to H(+) and K(+) and inhibition of the electron flux in the respiratory chain. The direct immediate protective role of tetrandrine against mitochondrial oxidative stress may be relevant to clarify the mechanisms responsible for its multiple pharmacological actions.     

Cycloheximide and 4-OH-TEMPO suppress chloramphenicol-induced apoptosis in RL-34 cells via the suppression of the formation of megamitochondria

Toxic effects of chloramphenicol, an antibiotic inhibitor of mitochondrial protein synthesis, on rat liver derived RL-34 cell line were completely blocked by a combined treatment with substances endowed with direct or indirect antioxidant properties. A stable, nitroxide free radical scavenger, 4-hydroxy-2,2,6, 6-tetramethylpiperidine-1-oxyl, and a protein synthesis inhibitor, cycloheximide, suppressed in a similar manner the following manifestations of the chloramphenicol cytotoxicity: (1) Oxidative stress state as evidenced by FACS analysis of cells loaded with carboxy-dichlorodihydrofluorescein diacetate and Mito Tracker CMTH2MRos; (2) megamitochondria formation detected by staining of mitochondria with MitoTracker CMXRos under a laser confocal microscopy and electron microscopy; (3) apoptotic changes of the cell detected by the phase contrast microscopy, DNA laddering analysis and cell cycle analysis. Since increases of ROS generation in chloramphenicol-treated cells were the first sign of the chloramphenicol toxicity, we assume that oxidative stress state is a mediator of above described alternations of RL-34 cells including MG formation. Pretreatment of cells with cycloheximide or 4-hydroxy-2,2, 6,6-tetramethylpiperidine-1-oxyl, which is known to be localized into mitochondria, inhibited the megamitochondria formation and succeeding apoptotic changes of the cell. Protective effects of cycloheximide, which enhances the expression of Bcl-2 protein, may further confirm our hypothesis that the megamitochondria formation is a cellular response to an increased ROS generation and raise a possibility that antiapoptotic action of the drug is exerted via the protection of the mitochondria functions.     

Oligomycin-resistant mitochondrial ATPase from mouse fibroblasts.

Fourteen oligomycin-resistant LM(TK-) clones were isolated following the mutagenesis of minicells. In the absence of oligomycin, the mutants grew with population doubling times similar to that of the wild type (1 day). In 3 or 5 microgram oligomycin/ml the doubling times of the mutants were 1.2-2.5 days. Both stable and unstable classes were represented among the oligomycin-resistant mutants. Mitochondrial ATPase activities of the mutants were 1.3-1130 times more resistant to oligomycin than the wild type. The mitochondrial ATPase of OLI 14 was found to be bound firmly to the mitochondrial membrane, showed no alteration in the pH optimum compared to wild-type, and exhibited increased resistance to DCCD and venturicidin. These results are consistent with the conclusion that oligomycin resistance in these mutants results from altered mitochondrial ATPase.     

Anandamide inhibits oxidative phosphorylation in isolated liver mitochondria

A study on the effect of anandamide (AEA) in energy coupling of rat liver mitochondria is presented. Micromolar concentrations of AEA, while almost ineffective on substrate supported oxygen consumption rate and on uncoupler stimulated respiration, strongly inhibited the respiratory state III. AEA did not change the rate and the extent of substrate generated membrane potential, but markedly delayed rebuilding by respiration of the potential collapsed by ADP addition. Overall, these data suggest that anandamide inhibits the oxidative phosphorylation process. Direct measurement of the F_oF₁ ATP synthase activity showed that the oligomycin sensitive ATP synthesis was inhibited by AEA, (IC₅₀, 2.5 μM), while the ATP hydrolase activity was unaffected. Consistently, AEA did not change the membrane potential generated by ATP hydrolysis.