The Switching of the Type of a ROS Signal from Mitochondria: The Role of Respiratory Substrates and Permeability Transition.

Flashes of superoxide anion (O) in mitochondria are generated spontaneously or during the opening of the permeability transition pore (mPTP) and a sudden change in the metabolic state of a cell. Under certain conditions, O can leave the mitochondrial matrix and perform signaling functions beyond mitochondria. In this work, we studied the kinetics of the release of O and HO from isolated mitochondria upon mPTP opening and the modulation of the metabolic state of mitochondria by the substrates of respiration and oxidative phosphorylation.

Warm Cells, Hot Mitochondria: Achievements and Problems of Ultralocal Thermometry.

Temperature is a crucial regulator of the rate and direction of biochemical reactions and cell processes. The recent data indicating the presence of local thermal gradients associated with the sites of high-rate thermogenesis, on the one hand, demonstrate the possibility for the existence of "thermal signaling" in a cell and, on the other, are criticized on the basis of thermodynamic calculations and models. Here, we review the main thermometric techniques and sensors developed for the determination of temperature inside living cells and diverse intracellular compartments.

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

Monomers, dimers, and individual FF-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 Ca-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.

Regulation of Mitochondrial Permeability Transition Pore Opening by Monovalent Cations in Liver Mitochondria.

The opening of the permeability transition pore (PTP) in mitochondria is a key event in the initiation of cell death in various pathologic states, including ischemia/reperfusion. The activation of K transport into mitochondria protects cells from ischemia/reperfusion. However, the role of K transport in PTP regulation is unclear.

Pioglitazone Is a Mild Carrier-Dependent Uncoupler of Oxidative Phosphorylation and a Modulator of Mitochondrial Permeability Transition.

Pioglitazone (PIO) is an insulin-sensitizing antidiabetic drug, which normalizes glucose and lipid metabolism but may provoke heart and liver failure and chronic kidney diseases. Both therapeutic and adverse effects of PIO can be accomplished through mitochondrial targets. Here, we explored the capability of PIO to modulate the mitochondrial membrane potential (ΔΨ) and the permeability transition pore (mPTP) opening in different models in vitro.

Dynamics of the permeability transition pore size in isolated mitochondria and mitoplasts.

The size of the permeability transition pore (PTP) is accepted to be ≤1.5 kDa. However, different authors reported values from 650 to 4000 Da.

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 Mg, adenine nucleotides (AN), and the inhibitors of AN translocase (ANT), voltage-dependent anion channel (VDAC), and cyclophilin D.

Pyridine nucleotides regulate the superoxide anion flash upon permeabilization of mitochondrial membranes: An MCLA-based study.

The permeabilization of mitochondrial membranes via permeability transition pore opening or by the pore-forming peptide alamethicin causes a flash of superoxide anion (SA) and hydrogen peroxide production and the inhibition of matrix aconitase. It was shown using the SA probe 3,7-dihydro-2-methyl-6-(4-methoxyphenyl)imidazol[1,2-a]pyrazine-3-one (MCLA) that the substrates of NAD-dependent dehydrogenases, inhibitors of the respiratory chain, and NAD(P)H at millimolar concentrations suppressed or delayed SA flashes. In the presence of added NADH and NADPH, SA flashes were observed only after considerable oxidation of pyridine nucleotides.

Glutamate contributes to alcohol hepatotoxicity by enhancing oxidative stress in mitochondria.

Chronic alcohol intoxication is associated with increased oxidative stress. However, the mechanisms by which ethanol triggers an increase in the production of reactive oxygen species (ROS) and the role of mitochondria in the development of oxidative stress has been insufficiently studied. The biochemical and proteomic data obtained in the present work suggest that one of the main causes of an increase in ROS generation is enhanced oxidation of glutamate in response to long-term alcohol exposure.

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.

External mitochondrial NADH-dependent reductase of redox cyclers: VDAC1 or Cyb5R3?

It was reported that VDAC1 possesses an NADH oxidoreductase activity and plays an important role in the activation of xenobiotics in the outer mitochondrial membrane. In the present work, we evaluated the participation of VDAC1 and Cyb5R3 in the NADH-dependent activation of various redox cyclers in mitochondria. We show that external NADH oxidoreductase caused the redox cycling of menadione ≫ lucigenin>nitrofurantoin.

Rapid fluorescent visualization of multiple NAD(P)H oxidoreductases in homogenate, permeabilized cells, and tissue slices.

Intracellular NAD(P)H oxidoreductases are a class of diverse enzymes that are the key players in a number of vital processes. The method we present and validate here is based on the ability of many NAD(P)H oxidoreductases to reduce the superoxide probe lucigenin, which is structurally similar to flavins, to its highly fluorescent water-insoluble derivative dimethylbiacridene. Two modifications of the method are proposed: (i) an express method for tissue homogenate and permeabilized cells in suspensions and (ii) a standard procedure for cells in culture and acute thin tissue slices.

Sulfur-containing compounds quench 3,7-dihydro-2-methyl-6-(4-methoxyphenyl)imidazol[1,2-a]pyrazine-3-one chemiluminescence: Discrimination between true antioxidants and quenchers using xanthine oxidase.

The probe 3,7-dihydro-2-methyl-6-(4-methoxyphenyl)imidazol[1,2-a]pyrazine-3-one (MCLA) is widely used for studying the superoxide anion production and the efficiency of antioxidants in biological systems. Here we report that a number of sulfur-containing compounds applied in biochemical and cytological studies are able to suppress MCLA-derived chemiluminescence (MDCL) independent of their capability to scavenge superoxide anion. The most effective MDCL quenchers appeared to be the substances with thiocarbamoyl and thiocarbonyl groups coupled to cyclic molecules and several thiol- and disulfide-containing compounds.

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.

Novel mycotoxin from Acremonium exuviarum is a powerful inhibitor of the mitochondrial respiratory chain complex III.

A novel mycotoxin named acrebol, consisting of two closely similar peptaibols (1726 and 1740 Da), was isolated from an indoor strain of the mitosporic ascomycete fungus Acremonium exuviarum. This paper describes the unique mitochondrial toxicity of acrebol, not earlier described for any peptaibol. Acrebol inhibited complex III of the respiratory chain of isolated rat liver mitochondria (1 mg of protein mL(-1)) with an IC(50) of approximately 80 ng mL(-1) (50 nM) after a short preincubation, and 350 ng mL(-1) caused immediate and complete inhibition.

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.

The effect of the lipophilic cation lucigenin on mitochondria depends on the site of its reduction.

The role of NAD(P)H-dependent oxidoreductases of the outer mitochondrial membrane (OMM) in the activation of lipophilic cationic dyes is poorly understood. In the present study we compared the rates of production of reactive oxygen species (ROS) and mitochondriotoxic effects of the redox-cycling lipophilic cationic dye lucigenin upon its activation by the respiratory chain and NAD(P)H-dependent oxidoreductases of the OMM. We found that, only in the presence of external NADH and NADPH, which are unable to penetrate the inner membrane, lucigenin stimulated a massive superoxide production and a fast permeabilization of mitochondrial membranes.

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.

Reactive oxygen species are involved in the stimulation of the mitochondrial permeability transition by dihydrolipoate.

Dihydrolipoic acid (DHLA) has been found to stimulate the Ca(2+)-induced mitochondrial permeability transition (MPT) in rat liver mitochondria (RLM) [Biochem. Mol. Biol.