Reactive oxygen and nitrogen species: friends or foes?

Chemical and physiological functions of molecular oxygen and reactive oxygen species (ROS) and existing equilibrium between pools of pro-oxidants and anti-oxidants providing steady state ROS level vital for normal mitochondrial and cell functioning are reviewed. The presence of intracellular oxygen and ROS sensors is postulated and few candidates for this role are suggested. Possible involvement of ROS in the process of fragmentation of mitochondrial reticulum made of long mitochondrial filaments serving in the cell as "electric cables", as well as the role of ROS in apoptosis and programmed mitochondrial destruction (mitoptosis) are reviewed. The critical role of ROS in destructive processes under ischemia/reoxygenation and ischemic preconditioning is discussed. Mitochondrial permeability transition gets special consideration as a possible component of the apoptotic cascade, resulting in excessive "ROS-induced ROS release".

Virus-induced permeability transition in mitochondria.

Isolated rat liver mitochondria undergo permeability transition after supplementation with a suspension of tobacco mosaic virus. Four mitochondrial parameters proved the opening of the permeability transition pore in the inner mitochondrial membrane: increased oxygen consumption, collapse of the membrane potential, release of calcium ions from mitochondria, and high amplitude mitochondrial swelling. All virus-induced changes in mitochondria were prevented by cyclosporin A. These effects were not observed if the virus was treated with EGTA or disrupted by heating. Protein component of the virus particle in the form of 20S aggregate A-protein, or helical polymer, as well as supernatant of the heat-disrupted virus sample, had no effect on mitochondrial functioning. Electron microscopy revealed the direct interaction of the virus particles with isolated mitochondria. The possible role of the mitochondrial permeability transition pore in virus-induced apoptosis is discussed.

The antioxidant functions of cytochrome c.

Low (C(1/2) = 1.5 x 10(-7) M) concentrations of horse cytochrome c strongly inhibit H(2)O(2) production by rat heart mitochondria under conditions of reverse electron transfer from succinate to NAD(+). The effect is abolished by binding of cytochrome c with liposomes and is not prevented by SOD. Yeast cytochrome c is much less effective than the horse protein whereas acetylated horse cytochrome c is without effect. H(2)O(2) formation stimulated by antimycin A is resistant to added cytochrome c. In inside-out submitochondrial vesicles, H(2)O(2) production is suppressed by all three cytochrome c samples tested, but at higher concentrations (C(1/2) is about 5 x 10(-7) M). In vesicles, SOD abolishes the cytochrome c inhibition. We conclude that extramitochondrial cytochrome c is competent in down-regulation of the Complex I H(2)O(2) production linked to the reverse electron transfer. Such an effect is absent in the inside-out submitochondrial vesicles where another antioxidant cytochrome c function can be observed, i.e. the oxidation of O(2-*) to O(2). A possible role of cytochrome c in the antioxidant defence is discussed.

Proteinaceous complexes from mitochondrial contact sites.

A Triton X-100 extract from rat brain mitochondria was obtained using low detergent/protein ratio. From this extract a proteinaceous complex was purified; its molecular weight was as high as 880 kD. The complex contained both hexokinase and creatine kinase activity. When incorporated into phospholipid bilayer membranes, the complex formed a channel whose activity was different than the channel activity of purified porin isolated either by adsorption chromatography or by dissociation from protein complexes. A ligand of the mitochondrial benzodiazepine receptor (Ro5-4864) in submicromolar concentrations had an apparent influence on the kinetic behavior of enzymatic coupling of hexokinase and creatine kinase. It is suggested that the 880-kD complex is formed by mitochondrial contact sites. The role of the isolated protein complex in the formation of nonspecific permeability in mitochondria is discussed.

The permeability transition pore induced under anaerobic conditions in mitochondria energized with ATP.

The role of oxygen in the induction of mitochondrial permeability transitions was studied. Oxygen consumption, swelling, membrane potential and calcium transport were recorded simultaneously in isolated rat liver mitochondria. Oxygen depletion was accomplished by saturating the medium with N2 and allowing either mitochondrial respiration or glucose/glucose oxidase to consume the residual oxygen. Upon anaerobiosis, mitochondria were supplemented with 500 microM ATP to support succinate-driven membrane potential. Under these conditions, 100 microM Ca2+ induced cyclosporin A-sensitive permeability transitions. To eliminate the possible inhibition of permeability transition by high concentrations of adenine nucleotides, anaerobic mitochondria were also energized by the combination of 20 microM ADP and phosphoenolpyruvate/pyruvate kinase. These mitochondria also underwent Ca2+-induced permeability transition. Under both of these conditions, namely the addition of ATP as a single or through actions of pyruvate kinase, the respiratory components were totally reduced. Thus, oxygen is not a necessary factor for mitochondria to undergo permeability transitions.

The Ca2+ -induced pore opening in mitochondria energized by succinate-ferricyanide electron transport.

The oxygen dependence of the mitochondria permeability transition pore was under study in non-respiring rat liver mitochondria. Oxygen in the medium was depleted by saturation of the incubation medium with N2 and spontaneously by mitochondrial respiration followed by the addition of glucose/glucose oxidase. After the anaerobic state had been reached, ferricyanide has been added to support succinate-driven energization in the absence of oxygen. In the other set of the experiments KCN was added to block operation of the respiratory chain under aerobic conditions. Again, ferricyanide was added as an electron acceptor. Superoxide dismutase was added to trap superoxide anion radicals. Under either hypoxic conditions or in the presence of cyanide, calcium ions were shown to induce the permeability transition. The concentration of Ca2+ required was lower than under conditions of active respiration. In both cases, the transition was prevented by cyclosporine A.

Mitochondria revisited. Alternative functions of mitochondria.

This review explores the alternative functions of mitochondria inside the cell. In a general picture of mitochondrial functioning, the importance and uniqueness of these intrinsic functions make them irreplaceable by other intracellular compartments. Among these are, participation in apoptosis and cellular proliferation, regulation of the cellular redox state and level of second messengers, heme and steroid syntheses, production and transmission of a transmembrane potential, detoxication and heat production. In most of the listed functions, reactive oxygen species modulate a number of non-destructive cellular activities. Some of the mitochondrial functions are reviewed in detail.

[Stimulation of mitochondrial respiration, induced by laser irradiation in the presence of rhodamine dyes]. / Stimulatsiia dykhaniia mitokhondrii, indutsiruemaia lazernym oblucheniem v prisutstvii rodaminovykh ksitelei.

The effect of micromolar concentration of rhodamine 123 (methylrhodamine) and ethyl and amyl esters of unsubstituted rhodamine on oxygen consumption by rat liver mitochondria was studied under irradiation by an argon laser (488 and 514 nm). Irradiation of mitochondria in the presence of rhodamine stimulates their respiration. Light-induced stimulation of respiration is not inhibited by free radical scavenger ionol and by inhibitor of the permeability transition pore cyclosporine A. Stimulation of respiration by moderate doses of radiation is reversed in the dark. Increase in radiation dose resulted in only partial reversal of stimulated respiration in the dark. Rhodamine efficacy in stimulation of mitochondrial respiration depends on its structure (amyl > ethyl > methylrhodamine).

Effects of amyl ester of unsubstituted rhodamine on respiration and Ca2+ transport in rat liver mitochondria.

In rat liver mitochondria the amylrhodamine is responsible for uncoupling (respiratory stimulation in state 4) by two distinct processes. Immediately after amylrhodamine addition (2-12 microM) stimulation of respiration takes place. Respiration rate for this phase is constant in time, it is independent of the potassium or inorganic phosphate content in the medium, not inhibited by oligomycin, ruthenium red, cyclosporine A, N-ethyl-maleimide and EGTA. The second phase of the respiratory stimulation is not linear in time. Respiration rate within this phase increases with rising of potassium and phosphate content in the medium. This effect is abolished by oligomycin, ruthenium red, cyclosporine A, N-ethylmaleimide and EGTA. The beginning of respiratory increment coincides with the second phase of Ca2+ release from mitochondria.