Genetics of the mammalian oxidative phosphorylation system: characterization of a new oligomycin-resistant Chinese hamster ovary cell line.

The properties of a new type of oligomycin-resistant Chinese hamster ovary (CHO) cell line (Olir 2.2) are described in this paper. Olir 2.2 cells were approximately 50,000-fold more resistant to oligomycin than were wild-type CHO cells when tested in glucose-containing medium, but only 10- to 100-fold more resistant when tested in galactose-containing medium. Olir 2.2 cells grew with a doubling time similar to that of wild-type cells both in the presence or absence of oligomycin. Oligomycin resistance in Olir 2.2 cells was stable in the absence of drug. In vitro assays indicated that there was approximately a 25-fold increase in the resistance of the mitochondrial ATPase to inhibition by oligomycin in Olir 2.2 cells, with little change in the total ATPase activity. The electron transport chain was shown to be functional in Olir 2.2 cells. Olir 2.2 cells were cross-resistant to other inhibitors of the mitochondrial ATPase (such as rutamycin, ossamycin, peliomycin, venturicidin, leucinostatin, and efrapeptin) and to other inhibitors of mitochondrial functions (such as chloramphenicol, rotenone, and antimycin). Oligomycin resistance was expressed codominantly in hybrids between Olir 2.2 cells and wild-type cells. Cross-resistance to ossamycin, peliomycin, chloramphenicol, antimycin, venturicidin, leucinostatin, and efrapeptin was also expressed codominantly in hybrids. Fusions of enucleated Olir 2.2 cells with wild-type cells and characterization of the resulting cybrid clones indicated that resistance to oligomycin and ossamycin results from a mutation in both a nuclear gene and a cytoplasmic gene. Cross-resistance to efrapeptin, leucinostatin, venturicidin, and antimycin results from a mutation in only a nuclear gene.     

Cytoplasmically inherited mutations of a human cell line resulting in deficient mitochondrial protein synthesis.

A large number of mutants deficient in mitochondrial protein synthesis (mtPS-) have been isolated from the human cell line VA2-B by subjecting cells partially depleted of their mtDNA to mutagenic treatments thought to be specific for mtDNA. Each of these mtPS- mutants has less than 10% of the wild-type rate of mitochondrial protein synthesis, exhibits reduced cytochrome oxidase and rutamycin sensitive ATPase activities, requires high concentrations of glucose, and grows indefinitely in the presence of 100 micrograms/ml of chloramphenicol (CAP). Fusion of cytoplasts from seven mtPS- mutants to the nucleated thioguanine-resistant VA2-B derivative TG-6 has yielded numerous cybrid clones which grow in CAP plus thioguanine, whereas almost no clones have resulted from the fusion of nucleated mtPS- cells to TG-6 cells: these results suggest that the gene(s) coding for the phenotype of mtPS- cells is localized in the cytoplasm (mtDNA?).     

Water-Soluble Coenzyme Q10 Reduces Rotenone-Induced Mitochondrial Fission

Parkinson’s disease is a neurodegenerative disorder characterized by mitochondrial dysfunction and oxidative stress. It is usually accompanied by an imbalance in mitochondrial dynamics and changes in mitochondrial morphology that are associated with impaired function. The objectives of this study were to identify the effects of rotenone, a drug known to mimic the pathophysiology of Parkinson’s disease, on mitochondrial dynamics. Additionally, this study explored the protective effects of water-soluble Coenzyme Q10 (CoQ10) against rotenone-induced cytotoxicity in murine neuronal HT22 cells. Our results demonstrate that rotenone elevates protein expression of mitochondrial fission markers, Drp1 and Fis1, and causes an increase in mitochondrial fragmentation as evidenced through mitochondrial staining and morphological analysis. Water-soluble CoQ10 prevented mitochondrial dynamic imbalance by reducing Drp1 and Fis1 protein expression to pre-rotenone levels, as well as reducing rotenone treatment-associated mitochondrial fragmentation. Hence, water-soluble CoQ10 may have therapeutic potential in treating patients with Parkinson’s disease.     

Characterization of rat liver mitochondria depleted of inner membrane components by chloramphenicol treatment of regenerating rat liver.

The maximal decline of adenosine triphosphate phosphohydrolase (ATPase; EC 3.6.1.3) in mitochondria from regenerating rat liver on treatment with chloramphenicol occurs between 48 and 72 h after partial hepatectomy. The depleted mitochondria are well coupled, exhibiting a respiratory control ratio of about 7 with succinate. These mitochondria are fully sensitive to rutamycin for the inhibition of succinate state 3 respiration. In frozen-thawed mitochondria there is a 60% reduction in ATPase activity, and of this remaining ATPase activity only 50% is sensitive to rutamycin. The titers for release of succinate state 4 respiration and ATPase activity by 5-Cl, 3-tert-butyl, 2′-Cl, 4′-NO₂-salicylanilide are decreased, and the efficiency of the uncoupler is increased. The antimycin titer for inhibition of succinate state 3 respiration is decreased. In all cases where a decrease in activity or titer was observed it is about the same (50%), suggesting inhibition at a common site for these parameters.     

Mitochondria are an essential mediator of nitric oxide/cyclic guanosine 3',5'-monophosphate blocking of glucose depletion induced cytotoxicity in human HepG2 cells

It is well known that glucose is a major energy source in tumors and that mitochondria are specialized organelles required for energy metabolism. Previous studies have revealed that nitric oxide (NO) protects against glucose depletion-induced cytotoxicity in mouse liver cells and in rat hepatocytes, but the detailed mechanism is not well understood. Therefore, we investigated the involvement of mitochondria in the NO protective effect in human hepatoma HepG2 cells. In this study, we showed that glucose depletion resulted in a time-dependent decrease in intracellular NO and in the protein expression of NO synthases. This glucose depletion-induced decrease in NO was blocked by NO donors. Next, we showed that the cytoprotective effect of NO is via a cyclic guanosine 3',5'-monophosphate-dependent pathway. Additionally, SNP blocked a glucose depletion-induced decrease in mitochondrial mass, mitochondrial DNA copies, and ATP level in HepG2 cells. Moreover, glucose depletion decreased the expression of various mitochondrial proteins, including cytochrome c, complex I (NADH dehydrogenase), complex III (cytochrome c reductase), and heat shock protein 60; these glucose depletion-induced effects were blocked by SNP. Furthermore, we found that rotenone and antimycin A (mitochondria complex I and III inhibitors, respectively) blocked SNP cytoprotection against glucose depletion-induced cytotoxicity. Taken together, our results indicated that the mitochondria serve as an important cellular mediator of NO during protection against glucose deprivation-induced damage.     

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

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

An unexpectedly labile mitochondrially encoded protein is required for Mta expression

Maternally transmitted antigen (Mta) is a mouse major histocompatibility antigen recognized by cytotoxic T lymphocytes. A role for mitochondria in expression of this class I-like cell surface antigen has been previously established. We now show that a labile product of mitochondrial protein synthesis is required for Mta expression. Reexpression of Mta determinants after enzymatic removal occurred within 24 h, and the regeneration process was sensitive to chloramphenicol (CAP), a selective inhibitor of mitochondrial protein synthesis. Additionally, target cells treated with CAP for as little as 18 h showed diminished expression of Mta. The estimated half-life for Mtf products ranged from 6 to 15 h, less than the half-lives of known mitochondrial translation products. This suggests that the Mtf product is not generated by the normal turnover of stable mitochondrial respiratory proteins. Instead, these results indicate the existence of either labile unknown mitochondrially encoded peptides or a rapid turnover pathway for known mitochondrial products.     

Muscle FBPase binds to cardiomyocyte mitochondria under glycogen synthase kinase-3 inhibition or elevation of cellular Ca2+ level

A growing body of research suggests that fructose 1,6-bisphosphatase (FBPase) might be involved in regulation of cell mortality/survival. However, the precise role of FBPase in the process remains unknown. Here, we show for the first time that in HL-1 cardiomyocytes, inhibition of glycogen synthase kinase-3 results in translocation of FBPase to mitochondria. In vitro experiments demonstrate that FBPase reduces the rate of calcium-induced mitochondrial swelling, affects ATP synthesis and interacts with mitochondrial proteins involved in regulation of volume and energy homeostasis. We suggest that FBPase might be engaged in a regulation of cell survival by influencing mitochondrial function.     

Uncoupling and specific inhibition of phosphoryl transfer reactions in mitochondria by antibiotic A20668.

A20668 A, B, and C are polypeptide antibiotics that inhibit phosphorylation of ADP, Mg2t-ATPase, and the ATP-driven transhydrogenase of rat liver submitochondrial particles, but not the purified F1 ATPase. In intact mitochondria, 120668 inhibits uncoupler-induced ATPase, State 3 respiration, and phosphorylation; the A and B forms are approximately equipotent with rutamycin, whereas A20668 C is less effective. Concentrations of A20668 slightly greater than required for complete inhibition of phosphoryl transfer stimulate rapid, uncoupled respiration by mitochondria under State 3 of 4 conditions. A20668 A and B are more effective uncouplers than A20668 C. In the presence of venturicidin or ossamycin, concentrations of A20668, which alone do not uncouple, stimulate oxygen consumption of mitochondria incubated under either State 3 of 4 conditions. A20668 uncoupling is not potentiated by prior inhibition of phosphoryl transfer by venturicidin X, rutamycin, aurovertin, or efrapeptin. A20668 increases mitochondrial permeability to protons in passive swelling experiments where facilitation of proton conductance correlates well with potency to uncouple. A20668 apparently binds initially at a unique locus to inhibit mitochondrial phosphoryl transfer reactions. When this site is saturated, additional antibiotic may uncouple by increasing proton conductance of mitochondria. Binding of venturicidin or ossamycin appears to interfere with the binding of A20668 to its adjacent inhibitory site, thus effectively increasing the concentration of A20668 available to uncouple.     

Functional characterization of mitochondria in neutrophils: a role restricted to apoptosis

Mitochondria are known to combine life-supporting functions with participation in apoptosis by controlling caspase activity. Here, we report that in human blood neutrophils the mitochondria are different, because they preserve mainly death-mediating abilities. Neutrophil mitochondria hardly participate in ATP synthesis, and have a very low activity of the tested marker enzymes. The presence of mitochondria in neutrophils was confirmed by quantification of mitochondrial DNA copy number, by detection of mitochondrial porin, and by JC-1 measurement of Deltapsi(m). During neutrophilic differentiation, HL-60 cells demonstrated a profound cytochrome c depletion and mitochondrial shape change reminiscent of neutrophils. However, blood neutrophils containing extremely low amounts of cytochrome c displayed strong caspase-9 activation during apoptosis, which was also observed in apoptotic neutrophil-derived cytoplasts lacking any detectable cytochrome c. We suggest that other proapoptotic factors such as Smac/DIABLO and HtrA2/Omi, which are massively released from the mitochondria, have an important role in neutrophil apoptosis.     

Cytoplasmic inheritance of oligomycin resistance in Chinese hamster ovary cells

Oligomycin-resistant clones were isolated from Chinese hamster ovary cells by treatment of cells with ethidium bromide, followed by mutagenesis with ethylmethane sulfonate and selection in oligomycin. One clone (Olir 8.1) was chosen for further study. Olir 8.1 cells grow with doubling time similar to that of wild-type cells, whether grown in the presence or absence of drug (doubling time of 13-14 h). In plating efficiency experiments, Olir 8.1 cells are approximately 100-fold more resistant to oligomycin than are wild-type cells. There is approximately a 32-fold increase in the resistance to inhibition by oligomycin of the mitochondrial ATPase from Olir 8.1 cells. The electron transport chain is functional in Olir 8.1 cells. Oligomycin resistance is stable in the absence of selective pressure. There is little or no cross-resistance of Olir 8.1 cells to venturicidin and dicyclohexylcarbodiimide, other inhibitors of the mitochondrial ATPase, or to chloramphenicol, an inhibitor of mitochondrial protein synthesis. Oligomycin resistance is dominant in hybrids between Olir 8.1 cells and wild-type cells. Fusions of enucleated Olir 8.1 cells with sensitive cells and characterization of the resulting "cybrid" clones indicates that oligomycin resistance in Olir 8.1 cells is cytoplasmically inherited.     

Similarity of lysosomal H+-ATPase to mitochondrial F0F1-ATPase in sensitivity to anions and drugs as revealed by solubilization and reconstitution

Lysosomal H+-translocating ATPase (H+-ATPase) was solubilized with lysophosphatidylcholine and reconstituted into liposomes (Moriyama, Y., Takano, T. and Ohkuma, S. (1984) J. Biochem. (Tokyo) 96, 927-930). In this study, the sensitivities of membrane-bound, solubilized and liposome-incorporated ATPase to various anions and drugs were measured in comparison with those of similar forms of mitochondrial H+-ATPase (mitochondrial F0F1-ATPase) with the following results. (1) Bicarbonate and sulfite activated solubilized lysosomal H+-ATPase, but not the membrane-bound ATPase or ATPase incorporated into liposomes. All three forms of mitochondrial F0F1-ATPase were activated by these anions. (2) All three forms of both lysosomal H+-ATPase and mitochondrial F0F1-ATPase were strongly inhibited by SCN-, NO3- and F-, but scarcely affected by Cl-, Br- and SO2-4. (3) The solubilized lysosomal H+-ATPase was strongly inhibited by azide, quercetin, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and oligomycin. Its sensitivity was almost the same as that of mitochondrial F0F1-ATPase. Neither membrane-bound ATPase nor ATPase incorporated into liposomes was affected appreciably by these drugs. These results indicate that the sensitivity to anions and drugs of lysosomal H+-ATPase depends on the form of the enzyme and that the sensitivity of the solubilized lysosomal H+-ATPase is very similar to that of mitochondrial F0F1-ATPase. On the other hand, the two ATPases differ in their sensitivity to N-ethylmaleimide and pyridoxal phosphate: only the mitochondrial ATPase is inhibited by pyridoxal phosphate whereas only the lysosomal ATPase is inhibited by N-ethylmaleimide.     

Increase of sodium delivery stimulates the mitochondrial respiratory chain H2O2 production in rat renal medullary thick ascending limb

The mitochondria-rich epithelial cells of the renal medullary thick ascending limb (mTAL) reabsorb nearly 25% of filtered sodium (Na(+)) and are a major source of cellular reactive oxygen species. Although we have shown that delivery of Na(+) to the mTAL of rats increases superoxide (O(2)(·-)) production in mTAL, little is known about H(2)O(2) production, given the lack of robust and selective fluorescent indicators for determining changes within the whole cell, specifically in the mitochondria. The present study determined the effect of increased tubular flow and Na(+) delivery to mTAL on the production of mitochondrial H(2)O(2) in mTAL. H(2)O(2) responses were determined in isolated, perfused mTAL of Sprague-Dawley rats using a novel mitochondrial selective fluorescent H(2)O(2) indicator, mitochondria peroxy yellow 1, and a novel, highly sensitive and stable cytosolic-localized H(2)O(2) indicator, peroxyfluor-6 acetoxymethyl ester. The results showed that mitochondrial H(2)O(2) and cellular fluorescent signals increased progressively over a period of 30 min following increased tubular perfusion (5-20 nl/min), reaching levels of statistical significance at ～10-12 min. Responses were inhibited with rotenone or antimycin A (inhibitors of the electron-transport chain), polyethylene glycol-catalase and by reducing Na(+) transport with furosemide or ouabain. Inhibition of membrane NADPH-oxidase with apocynin had no effect on mitochondrial H(2)O(2) production. Cytoplasmic H(2)O(2) (peroxyfluor-6 acetoxymethyl ester) increased in parallel with mitochondrial H(2)O(2) (mitochondria peroxy yellow 1) and was partially attenuated (～65%) by rotenone and completely inhibited by apocynin. The present data provide clear evidence that H(2)O(2) is produced in the mitochondria in response to increased flow and delivery of Na(+) to the mTAL, and that whole cell H(2)O(2) levels are triggered by the mitochondrial reactive oxygen species production. The mitochondrial production of H(2)O(2) may represent an important target for development of more effective antioxidant therapies.     

Syntabulin-mediated anterograde transport of mitochondria along neuronal processes

In neurons, proper distribution of mitochondria in axons and at synapses is critical for neurotransmission, synaptic plasticity, and axonal outgrowth. However, mechanisms underlying mitochondrial trafficking throughout the long neuronal processes have remained elusive. Here, we report that syntabulin plays a critical role in mitochondrial trafficking in neurons. Syntabulin is a peripheral membrane-associated protein that targets to mitochondria through its carboxyl-terminal tail. Using real-time imaging in living cultured neurons, we demonstrate that a significant fraction of syntabulin colocalizes and co-migrates with mitochondria along neuronal processes. Knockdown of syntabulin expression with targeted small interfering RNA or interference with the syntabulin-kinesin-1 heavy chain interaction reduces mitochondrial density within axonal processes by impairing anterograde movement of mitochondria. These findings collectively suggest that syntabulin acts as a linker molecule that is capable of attaching mitochondrial organelles to the microtubule-based motor kinesin-1, and in turn, contributes to anterograde trafficking of mitochondria to neuronal processes.     

Cytosol-mitochondria transfer of reducing equivalents by a lactate shuttle in heterotrophic Euglena.

To assess the expression and physiological role of the mitochondrial NAD(+)-independent lactate dehydrogenase (iLDH) in Euglena gracilis, cells were grown with different carbon sources, and the d- and l-iLDH activities and several key metabolic intermediates were examined. iLDH activity was significant throughout the growth period, increasing by three- to fourfold from latency to the stationary phase. Intracellular levels of D- and L-lactate were high (5-40 mm) from the start of the culture and increased (20-80 mm) when the stationary phase was entered. All external carbon sources were actively consumed, reaching a minimum upon entering the stationary phase, when degradation of paramylon started. The level of ATP was essentially unchanged under all experimental conditions. Oxalate, an inhibitor of iLDH, strongly inhibited oligomycin-sensitive respiration and growth, whereas rotenone, an inhibitor of respiratory complex I, only slightly affected these parameters in lactate-grown cells. Isolated mitochondria exhibited external NADH-supported respiration, which was sensitive to rotenone and flavone, and an inability to oxidize pyruvate. Addition of cytosol, NADH and pyruvate to mitochondria incubated with rotenone and flavone prompted significant O2 uptake, which was blocked by oxalate. The data suggested that iLDH expression in Euglena is independent of substrate availability and that iLDHs play a key role in the transfer of reducing equivalents from the cytosol to the respiratory chain (lactate shuttle).     

Lack of oxidative phosphorylation and low mitochondrial membrane potential decrease susceptibility to apoptosis and do not modulate the protective effect of Bcl-x(L) in osteosarcoma cells

We explored the role of low mitochondrial membrane potential (DeltaPsim) and the lack of oxidative phosphorylation in apoptosis by assessing the susceptibility of osteosarcoma cell lines with and without mitochondrial DNA to staurosporine-induced death. Our cells without mitochondrial DNA had low DeltaPsim and no functional oxidative phosphorylation. Contrary to our expectation, these cells were more resistant to staurosporine-induced death than were the parental cells. This reduced susceptibility was associated with decreased activation of caspase 3 but not with the mitochondrial permeability transition pore or cytochrome c release from the mitochondria. Apoptosis in both cell lines was associated with an increase in DeltaPsim. Bcl-x(L) could protect both cell types against caspase 3 activation and apoptosis by a mechanism that does not appear to be mediated by mitochondrial function or modulation of DeltaPsim. Nevertheless, we found that Bcl-x(L) expression can stimulate cell respiration in cells with mitochondrial DNA. Our results showed that the lack of functional oxidative phosphorylation and/or low mitochondrial membrane potential are associated with an antiapoptotic effect, possibly contributing to the development of some types of cancer. It also reinforces a model in which Bcl-x(L) can exert an antiapoptotic effect by stimulating oxidative phosphorylation and/or inhibiting caspase activation.     

Two subcellular locations for peripheral-type benzodiazepine acceptors in rat liver

Subcellular fractionation of rat liver by differential centrifugation showed the mitochondrial fractions to have the greatest enrichment of 'peripheral-type' benzodiazepine acceptor. Two peaks of acceptor sites were found on isopycnic density-gradient centrifugation, one peak (rho = 1.19 g/ml) corresponding to the peak of mitochondria as judged by marker enzyme distribution and by transmission electron microscopy, and the other peak (rho = 1.17 g/ml) which is not mitochondrial as judged by the lack of mitochondrial enzyme markers. Whereas the density of the mitochondrial acceptor was sensitive to sonication and was shown to have an outer-membrane location, the density of the non-mitochondrial acceptor was insensitive to sonication. The non-mitochondrial acceptor was shown not to be associated with Golgi, lysosomes, rough endoplasmic reticular microsomes, peroxisomes, or some types of plasma membranes, as judged by differences in the distribution of marker activities. No enrichment of benzodiazepine acceptor was found in the purified nuclear fraction. Both acceptors were shown to be peripheral-type high-affinity acceptors as judged by ligand specificities and by photoaffinity labelling.     

Mitochondrial fragmentation in apoptosis

Mitochondrial outer membrane permeabilization (MOMP) is associated with most pro-apoptotic stimuli. MOMP results in the release of cytochrome c from the mitochondria into the cytosol, triggering caspase activation and subsequent apoptosis. Several theories explaining the mechanisms of MOMP have been proposed, one of which suggests that MOMP relies on the activation of the molecular machinery involved in fission, resulting in mitochondrial fragmentation. By contrast, several recent studies suggest that mitochondrial fragmentation occurs following MOMP. Moreover, under some conditions. MOMP occurs without mitochondrial fragmentation and, in fact, fragmentation even inhibits MOMP. Here, I discuss the apparently conflicting data and conclude that mitochondrial fragmentation is probably not a prerequisite for MOMP and cytochrome c release.