Mitochondrial F-ATP Synthase Co-Migrating Proteins and Ca2+-Dependent Formation of Large Channels.

Monomers, dimers, and individual FOF1-ATP synthase subunits are, presumably, involved in the formation of the mitochondrial permeability transition pore (PTP), whose molecular structure, however, is still unknown. We hypothesized that, during the Ca2+-dependent assembly of a PTP complex, the F-ATP synthase (subunits) recruits mitochondrial proteins that do not interact or weakly interact with the F-ATP synthase under normal conditions. Therefore, we examined whether the PTP opening in mitochondria before the separation of supercomplexes via BN-PAGE will increase the channel stability and channel-forming capacity of isolated F-ATP synthase dimers and monomers in planar lipid membranes. Additionally, we studied the specific activity and the protein composition of F-ATP synthase dimers and monomers from rat liver and heart mitochondria before and after PTP opening. Against our expectations, preliminary PTP opening dramatically suppressed the high-conductance channel activity of F-ATP synthase dimers and monomers and decreased their specific "in-gel" activity. The decline in the channel-forming activity correlated with the reduced levels of as few as two proteins in the bands: methylmalonate-semialdehyde dehydrogenase and prohibitin 2. These results indicate that proteins co-migrating with the F-ATP synthase may be important players in PTP formation and stabilization.

Regulation of permeability transition pore opening in mitochondria by external NAD(H).

BACKGROUND: The opening of the permeability transition pore (PTP) in mitochondria plays a critical role in the pathogenesis of numerous diseases. Mitochondrial matrix pyridine nucleotides are potent regulators of the PTP, but the role of extramitochondrial nucleotides is unclear. METHODS: The PTP opening was explored in isolated mitochondria and mitochondria in permeabilized differentiated and undifferentiated cells in the presence of added NAD(P)(H) in combination with Mg2+, adenine nucleotides (AN), and the inhibitors of AN translocase (ANT), voltage-dependent anion channel (VDAC), and cyclophilin D. RESULTS: Added NAD(H) and AN, but not NADP(H), inhibited the PTP opening with comparable potency. PTP suppression required neither NAD(H) oxidation nor reduction. The protective effects of NAD(H) and cyclosporin A were synergistic, and the effects of NAD(H) and millimolar AN were additive. The conformation-specific ANT inhibitors were unable to cancel the protective effect of NADH even under total ANT inhibition. Besides, NAD(H) activated the efflux of mitochondrial AN via ANT. VDAC ligand (Mg2+) and blockers (G3139 and 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid) potentiated and attenuated the protective effect of NAD(H), respectively. However, in embryonic and cancer (undifferentiated) cells, in contrast to isolated differentiated hepatocytes and cardiocytes, the suppression of PTP opening by NADH was negligible though all cells tested possessed a full set of VDAC isoforms. CONCLUSIONS: The study revealed a novel mechanism of PTP regulation by external (cytosolic) NAD(H) through the allosteric site in the OM or the intermembrane space. GENERAL SIGNIFICANCE: The mechanism might contribute to the resistance of differentiated cells under different pathological conditions including ischemia/reperfusion.

Mechanism of triclosan toxicity: Mitochondrial dysfunction including complex II inhibition, superoxide release and uncoupling of oxidative phosphorylation.

Triclosan (5-chloro-2'-(2,4-dichlorophenoxy)phenol), a widely used antibacterial agent, exerts adverse effects on the organism of mammals. Recent research reviled that triclosan at low micromolar concentrations causes mitochondrial dysfunction in many cell types, but the mechanisms of its effect are not fully understood. Here we show that exposure to triclosan disrupted membrane potential, prevented the calcium uptake-driven high-amplitude mitochondrial swelling, stimulated the respiration in the presence of complex I substrates, and suppressed the ADP-stimulated respiration in the presence of complex II substrate, succinate. Triclosan directly inhibited complex II activity. Similar to the complex II inhibitor thenoyltrifluoroacetone, triclosan induced the oxidation of the cytochromes b566 and b562 and caused the release of mitochondrial superoxide. Opposite to thenoyltrifluoroacetone, triclosan increased superoxide release synergistically with myxothiazol but not with antimycin A, indicating different topology of superoxide-producing sites. We concluded that triclosan is unique by its capability of acting as both a protonophore and an unusual complex II inhibitor, which interferes with the mitochondrial respiration by blocking the electron transfer between ubiquinone at the Qd-binding site and heme b. Our data can provide an insight into the mechanisms of the carcinogenic effect of triclosan in the liver and other tissues.

Flow cytometry-based assay for the activity of NAD(P)H oxidoreductases of the outer mitochondrial membrane.

NAD(P)H oxidoreductases of the outer mitochondrial membrane (OMM) are able to activate various xenobiotics and stimulate the production of reactive oxygen species and the opening of the mitochondrial permeability transition pore. However, the role of these systems in the cell damage by xenobiotics and chemotherapeutic drugs is poorly understood because the methods for the selective assessment of their activity have not been elaborated and specific inhibitors are unknown. Here we propose a method for the semiquantitative assessment of the activity of NAD(P)H oxidoreductases of the OMM in intact and permeabilized cells that is based on the flow cytometry detection of dimethylbiacridene, a fluorescent product of two-electron reduction of lucigenin. The method uses the structural feature of mitochondrial organization: the proximity of the sites of one-electron reduction of lucigenin to cation radical (NAD(P)H oxidoreductases of the OMM) to the sites of its subsequent oxidation (cytochrome c oxidase). The inhibition of cytochrome c oxidase by cyanide selectively activates the dimethylbiacridene formation by oxidoreductases of the OMM but not by other cellular oxidoreductases. The proposed protocol allows one to assess the lucigenin reductase (two-electron) activity of NAD(P)H oxidoreductases of the OMM and to compare it with the activity of other cellular systems that can be used for the analysis of the role of these systems in the cell damage by xenobiotics and antitumor drugs.

Redox-cycling compounds can cause the permeabilization of mitochondrial membranes by mechanisms other than ROS production.

The participation of reactive oxygen species (ROS) in the regulation of mitochondrial permeability transition pore (mPTP) opening by the redox-cycling compounds menadione and lucigenin was explored. The level of ROS was modulated by antioxidants, anoxia, and switching the sites of the reduction of redox cyclers, the dehydrogenases of the inner and outer mitochondrial membranes. We found that the reduction of both lucigenin and menadione in the outer mitochondrial membrane caused a strong production of ROS. However, mPTP opening was accelerated only in the presence of the cationic acceptor lucigenin. The antioxidants and scavengers of ROS that considerably decreased the level of ROS in mitochondria did not prevent or delay the mPTP opening. If the transmembrane potential under anoxia was supported by exogenous ATP or ferricyanide, the permeabilization of mitochondrial membranes by menadione or lucigenin was the same as under normoxia or even more pronounced. Under anoxia, the lucigenin-dependent permeabilization of membranes was less sensitive to mPTP antagonists than under normoxia. We conclude that the opening of the mPTP by redox cyclers may be independent of ROS and is due to the direct oxidation of mitochondrial pyridine nucleotides by menadione and the modification of critical thiols of the mPTP by the cation radical of lucigenin.

Small heat shock protein hsp27 as a possible mediator of intercellular adhesion-induced drug resistance in human larynx carcinoma HEp-2 cells.

The confluence-dependent resistance of human larynx carcinoma HEp-2 cells to hydrogen peroxide and a new antitumor drug based on the combination of vitamins C and B12b was studied. It was found that this resistance in growing cells is suppressed by the disruption of intercellular contacts by EGTA and is related neither to the activity of P-glycoprotein nor to the content of intracellular glutathione and the activities of glutathione S-transferases, glutathione peroxidase and glutathionine reductase. Here we showed that the level of expression of the small heat shock protein hsp27, which is known to protect cells from a variety of stresses associated with apoptosis, in growing confluent cells both in the presence and absence of the vitamins B12b and C is much higher (about 20-25 times) than in non-confluent cells. Taken together, the results suggest that the confluence-dependent resistance of cells to the combination of vitamins C and B12b and to hydrogen peroxide is mediated by hsp27 overexpression, which is activated via cell-cell adhesion.