Mitochondria are a quantitatively relevant source of reactive oxygen species (ROS) in the majority of cell types. Here we review the sources and metabolism of ROS in this organelle, including the conditions that regulate the production of these species, such as mild uncoupling, oxygen tension, respiratory inhibition, Ca2+ and K+ transport, and mitochondrial content and morphology. We discuss substrate-, tissue-, and organism-specific characteristics of mitochondrial oxidant generation. Several aspects of the physiological and pathological roles of mitochondrial ROS production are also addressed.

Mitochondrial permeability transition (MPT) is a non-selective inner membrane permeabilization that may precede necrotic and apoptotic cell death. Although this process has a specific inhibitor, cyclosporin A, little is known about the nature of the proteinaceous pore that results in MPT. Here, we review data indicating that MPT is not a consequence of the opening of a pre-formed pore, but the consequence of oxidative damage to pre-existing membrane proteins. ß 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

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There is an emerging consensus that pharmacological opening of the mitochondrial ATP-sensitive K(+) (K(ATP)) channel protects the heart against ischemia-reperfusion damage; however, there are widely divergent views on the effects of openers on isolated heart mitochondria. We have examined the effects of diazoxide and pinacidil on the bioenergetic properties of rat heart mitochondria. As expected of hydrophobic compounds, these drugs have toxic, as well as pharmacological, effects on mitochondria. Both drugs inhibit respiration and increase membrane proton permeability as a function of concentration, causing a decrease in mitochondrial membrane potential and a consequent decrease in Ca(2+) uptake, but these effects are not caused by opening mitochondrial K(ATP) channels. In pharmacological doses (<50 microM), both drugs open mitochondrial K(ATP) channels, and resulting changes in membrane potential and respiration are minimal. The increased K(+) influx associated with mitochondrial K(ATP) channel opening is approximately 30 nmol. min(-1). mg(-1), a very low rate that will depolarize by only 1-2 mV. However, this increase in K(+) influx causes a significant increase in matrix volume. The volume increase is sufficient to reverse matrix contraction caused by oxidative phosphorylation and can be observed even when respiration is inhibited and the membrane potential is supported by ATP hydrolysis, conditions expected during ischemia. Thus opening mitochondrial K(ATP) channels has little direct effect on respiration, membrane potential, or Ca(2+) uptake but has important effects on matrix and intermembrane space volumes.

Reactive oxygen species are a by-product of mitochondrial oxidative phosphorylation, derived from a small quantity of superoxide radicals generated during electron transport. We conducted a comprehensive and quantitative study of oxygen consumption, inner membrane potentials, and H(2)O(2) release in mitochondria isolated from rat brain, heart, kidney, liver, and skeletal muscle, using various respiratory substrates (alpha-ketoglutarate, glutamate, succinate, glycerol phosphate, and palmitoyl carnitine). The locations and properties of reactive oxygen species formation were determined using oxidative phosphorylation and the respiratory chain modulators oligomycin, rotenone, myxothiazol, and antimycin A and the uncoupler CCCP. We found that in mitochondria isolated from most tissues incubated under physiologically relevant conditions, reactive oxygen release accounts for 0.1-0.2% of O(2) consumed. Our findings support an important participation of flavoenzymes and complex III and a substantial role for reverse electron transport to complex I as reactive oxygen species sources. Our results also indicate that succinate is an important substrate for isolated mitochondrial reactive oxygen production in brain, heart, kidney, and skeletal muscle, whereas fatty acids generate significant quantities of oxidants in kidney and liver. Finally, we found that increasing respiratory rates is an effective way to prevent mitochondrial oxidant release under many, but not all, conditions. Altogether, our data uncover and quantify many tissue-, substrate-, and site-specific characteristics of mitochondrial ROS release.

Mitochondrially generated reactive oxygen species are involved in a myriad of signaling and damaging pathways in different tissues. In addition, mitochondria are an important target of reactive oxygen and nitrogen species. Here, we discuss basic mechanisms of mitochondrial oxidant generation and removal and the main factors affecting mitochondrial redox balance. We also discuss the interaction between mitochondrial reactive oxygen and nitrogen species, and the involvement of these oxidants in mitochondrial diseases, cancer, neurological, and cardiovascular disorders.

Increased replicative longevity in Saccharomyces cerevisiae because of calorie restriction has been linked to enhanced mitochondrial respiratory activity. Here we have further investigated how mitochondrial respiration affects yeast life span. We found that calorie restriction by growth in low glucose increased respiration but decreased mitochondrial reactive oxygen species production relative to oxygen consumption. Calorie restriction also enhanced chronological life span. The beneficial effects of calorie restriction on mitochondrial respiration, reactive oxygen species release, and replicative and chronological life span could be mimicked by uncoupling agents such as dinitrophenol. Conversely, chronological life span decreased in cells treated with antimycin (which strongly increases mitochondrial reactive oxygen species generation) or in yeast mutants null for mitochondrial superoxide dismutase (which removes superoxide radicals) and for RTG2 (which participates in retrograde feedback signaling between mitochondria and the nucleus). These results suggest that yeast aging is linked to changes in mitochondrial metabolism and oxidative stress and that mild mitochondrial uncoupling can increase both chronological and replicative life span.The only intervention known to increase average and maximum life span in mammals is caloric restriction (CR), 1 a reduction of 25-60% in calorie intake without essential nutrient deficiency. This diet not only extends life span but also delays many unwanted effects of aging and age-related pathologies. CR is highly effective in a wide range of organisms, increasing life span by up to 50% in some species (reviewed in Refs. 1-3). Unfortunately, the mechanisms through which it results in increased life span are still controversial (see Ref. 4 for a critical review).A leading hypothesis on the mechanism through which CR prevents aging is that this process decreases reactive oxygen species (ROS) generation and, hence, the oxidation of cellular components (5-8). Indeed, aging is usually accompanied by oxidative damage of DNA, proteins, and lipids (9, 10). CR promotes a metabolic shift resulting in more efficient electron transport in the mitochondrial respiratory chain (1, 5). Faster and more efficient electron transport may lead to lower production of ROS by mitochondria, one of the major intracellular ROS sources. This occurs because of reduced leakage of electrons from the respiratory chain and/or lower oxygen concentrations in the mitochondrial microenvironment (11,12). Indeed, artificially increasing mitochondrial respiration using uncouplers such as 2,4-dinitrophenol (DNP) strongly prevents mitochondrial ROS release (11). Furthermore, CR decreases ROS release/O 2 consumed in isolated mammalian mitochondria (13), possibly because of enhanced expression of mitochondrial uncoupling proteins (14,15). Despite this evidence supporting a correlation between ROS-induced damage and aging, a clear cause-effect relationship has been hard to establish, and conflicting results are often p...

Protection of heart against ischemia-reperfusion injury by ischemic preconditioning and K ATP channel openers is known to involve the mitochondrial ATPsensitive K ؉ channel (mitoK ATP ). Brain is also protected by ischemic preconditioning and K ATP channel openers, and it has been suggested that mitoK ATP may also play a key role in brain protection. However, it is not known whether mitoK ATP exists in brain mitochondria, and, if so, whether its properties are similar to or different from those of heart mitoK ATP . We report partial purification and reconstitution of a new mitoK ATP from rat brain mitochondria. We measured K ؉ flux in proteoliposomes and found that brain mitoK ATP is regulated by the same ligands as those that regulate mitoK ATP from heart and liver. We also examined the effects of opening and closing mitoK ATP on brain mitochondrial respiration, and we estimated the amount of mitoK ATP by means of green fluorescence probe BODIPY-FL-glyburide labeling of the sulfonylurea receptor of mitoK ATP from brain and liver. Three independent methods indicate that brain mitochondria contain six to seven times more mitoK ATP per milligram of mitochondrial protein than liver or heart.