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Pretreatment of tissues with potassium channel openers (KCO’s) has been observed to be cytoprotective in a broad variety of insults. This phenomenon has been proposed to be intimately linked to activation of mitochondrial potassium channels which apparently modulate the mitochondrial production of reactive oxygen species (ROS). This critical review summarizes literature findings about the mitochondrial production of ROS, the action of KCO’s on mitochondrial ROS production and the putative link to the cytoprotective action of these drugs.

The addition of 15 mM caffeine to saponin-skinned human muscle fibers from M. vastus lateralis caused in the presence of 2 mM ATP an approx. 2-fold stimulation of respiration with glutamate + malate. This effect can be abolished by either the addition of the Ca2+ chelator EGTA, the inhibitor of Ca2+ transport Ruthenium red and the inhibitor of the myosin ATPase vanadate. The caffeine concentration dependency of respiration of fibers coincided with the caffeine-caused stimulation of myosin ATPase activity. The activation of oxidative phosphorylation in saponin-skinned human muscle fibers by caffeine can be explained by a stimulation of myosin ATPase caused by Ca2+ release from sarcoplasmic reticulum.

1. 1. The energy-independent exchange of alkali metal cations against protons was investigated in intact and disintegrated mitochondria. The exchange is not specific for different alkali metal ions. 2. 2. In intact mitochondria the apparent Km for the energy-independent exchange range from 5 to 25 mM (Cs+, Rb+, K+ 5–10 mM; Na+ 15 mM; Li+ 20 mM). At all ions studied the maximum release of protons was 15–20 nval/mg protein. 3. 3. In disintegrated mitochondria there are no differences between the alkali metal ions (apparent Km 30 mM; maximum H+-release 50 nval/mg protein). 4. 4. Directly accessible binding sites seem to exist in the outer membrane. Binding sites in the inner membrane are accessible only after disintegration, but not after addition of valinomycin + rotenone.

Anaerobic potentiometric titrations of b cytochromes have been carried out in beef heart submitochondrial particles in the presence of several specific inhibitors of electron transfer through the b-c1-site of the respiratory chain. Whereas antimycin shows no significant effect on the titration curve of cytochrome b-562, NoHOQnO is found to shift the Em of b-562 by 20-30 mV to the positive. Funiculosin raises the Em of b-562 by greater than 100 mV and also appears to bring about a minor shift of b-566 midpoint potential. In the presence of myxothiazol, both b cytochromes titrate with Em values 15-30 mV more positive than in the control.

The control theory of Kacser and Burns [in: Rate Control of Biological Processes (Davies, D.D. ed) pp. 65-104, Cambridge University Press, London, 1973] and Heinrich and Rapoport [Eur. J. Biochem. (1974) 42, 97-105] has been used to quantify the amount of control exerted by different steps on mitochondrial oxidative phosphorylation in rat-liver mitochondria. Inhibitors were used to manipulate the amount of active enzyme. The control strength of the adenine nucleotide translocator was measured by carrying out titrations with carboxyatractyloside. In state 4, the control strength of the translocator was found to be zero. As the rate of respiration was increased by adding hexokinase, the control strength of the translocator increased to a maximum value of approximately 30% at approximately 80% of state 3 respiration. In state 3, control of respiration is distributed between a number of steps, including the adenine nucleotide translocator, the dicarboxylate carrier and cytochrome c oxidase. The measured values for the distribution of control agree very well with those calculated with the aid of a model for mitochondrial oxidative phosphorylation developed by Bohnensack et al. [Biochim. Biophys. Acta (1982) 680, 271-280].

A mathematical model was developed to estimate flux control coefficients (Co) from titration studies with specific non-competitive inhibitors. In contrast to the normally used graphical determination the model pays regard to the dissociation equilibrium (kD) that exists between inhibitor and its binding sites (Eo) as well as to an objective estimation of the initial slope. The model was used for the analysis of titration experiments where the respiration of rat liver mitochondria was inhibited with carboxyatractyloside and antimycin A. It is shown that the graphical estimation of Eo and Co lead to significant overestimation if the ratio Kd/Eo is larger than 10−4 which can be avoided by using our model.

Changes are described that are brought about by antimycin, NoHOQnO, funiculosin, myxothiazol and mucidin in the alpha-, beta- and gamma-absorption bands of reduced and oxidized cytochromes b in the isolated complex bc1 form beef heart mitochondria. The inhibitors can be divided into 2 groups. Antimycin, funiculosin and NoHOQnO are likely to shift the spectrum of b-562 and compete for specific binding with complex bc1, with each other but not with myxothiazol and mucidin. The spectral effects of the latter two inhibitors are more difficult to interpret and may involve contributions not only from b-562 but from b-566 as well. The existence of 2 independent inhibitor binding-sites in the complex bc1 corroborates the Q-cycle hypothesis.

ZusammenfassungDisentegration oder Alterung führt bei intakten Lebermitochondrien und bei der Innenmembrant + Matrix-Fraktion (Digitonin) zu einer Zunahme der K+-bedingten Protonenfreisetzung, nicht jedoch bei Aussenmembranen. Es wird geschlossen, dass auch die Innenmembran + Matrix-Fraktion Austauschstallen für K+ enthält, die jedoch bei struktureller Integrität nicht frei zugänglich sind. Die dem intermembrane space zugewandte Seite der Innenmembran stellt möglicherweise eine Barriere für das K+-Ion dar.AbstractDisintegration or altering of intact mitochondria or digitonin inner membrane + matrix particles causes an increase in K+-linked release of protons. This effect is not obtainable in the case of outer membranes. It is assumed, that also the inner membrane + matrix possesses exchange sites for K+, which are freely accessible for the potassium ion if the structural integrity is damaged. The outer layer of inner membrane seems to be a barrier for the K+-movement.

The oxygen dependence of cytochrome c oxidase control on succinate oxidation was investigated in saponin-permeabilized muscle fibers and isolated mitochondria from mouse quadriceps muscle applying metabolic control analysis. For this cyanide titrations of the oxygen consumption in the presence of succinate+rotenone were performed at different oxygen concentrations in the medium. While with isolated mitochondria high flux control coefficients were detected only at oxygen concentrations close to the K M value of cytochrome c oxidase, with saponin-permeabilized fibers a significant increase of cytochrome c oxidase flux control was already observed below 130 μM oxygen. The result is in line with the high oxygen sensitivity of maximal respiration of saponin-permeabilized muscle fibers (P 50 =33 μM) caused most probably by oxygen diffusion gradients through the fiber lattice. The oxygen dependence of cytochrome c oxidase flux control in muscle fibers can explain the pathological phenotype of mild cytochrome c oxidase deficiencies in mitochondrial myopathies.

ZusammenfassungEs wurde die energieunabhängige Protonenfreisetzung durch intakte Mitochondrien und submitochondriale Partikel in Abhängigkeit vom K+-Gehalt des Inkubationsmediums gemessen. Die Protonenfreisetzung war korreliert mit der specifischen Aktivität der Monoaminooxidase. Es wird geschlossen, dass der energieunabhängige K+-H+-Austausch in der Aussenmembran der Mitochondrien lokalisiert ist.AbstractThe energy-independent K+-H+-exchange was measured in intact mitochondria and submitochondrial particles at different K+-concentration of the incubation medium. Correlation was found between H+-displacement and specific activity of monoamine oxidase. It is concluded, that the energy-independent K+-H+-exchange is localized in the outer membrane of mitochondria.

The different flavoproteins contributing to flavin fluorescence of isolated rat liver mitochondria have distinct excitation and emission spectra. The NAD-linked flavin component was identified as α-lipoamide dehydrogenase, while the non-NAD-linked component was found to be electron transfer flavoprotein. The differences in excitation and emission properties of the mitochondrial flavoproteins permit selective recording of their redox state changes in isolated mitochondria.

A respiratory chain inhibitor antimycin has long been known to bring about a shift of the ferrous cytochrome b-562 a-absorption band by about l-2 nm to the red [l-3]. This red shift was sometimes considered to be a specific effect of antimycin [4,5] since it had not been observed in animal mitochondria with an antimycin-type inhibitor HOQNO [3]. At the same time, rather similar perturbations of the b cytochrome a-band by antimytin, HOQNO and mucidin have been described in yeast mitochondria [6,7]. Here, we report on the spectral changes of b cytochromes brought about by HOQNO and mucidin in beef heart SMP. A spectral response very similar to that induced by HOQNO is also observed upon addition of ATP to dithionite-reduced SMP. The effects of the ligands and of the energization are tentatively ascribed to redistribution of ferrocytochrome b-566/b-562 between 2 conformational states.

When succinate oxidation by submitochondrial particles is blocked by antimycin, NoHOQnO or funiculosin, addition of ferricyanide restores oxygen uptake coupled to membrane potential generation. The effect of ferricyanide is abolished by mucidin or myxothiazol, as well as by KCN. The data strongly favor a cyclic redox loop mechanism in site 2 and show that either heme of the ferrous cytochrome b or ubisemiquinone formed in the QH2-oxidizing center of complex b−c1 is accessible to ferricyanide at the outer (M) side of the submitochondrial particle membrane.

In the presence of ascorbate, hexaamineruthenium mediates rapid reduction of cytochrome b -562 in submitochondrial particles but not in mitochondria. The reaction is obsreved in the combined presence of antimycin (or funiculosin) and myxothiazol, which implies direct interaction of Ru(NH 3 ) 2+ 6 with b cytochrome(s). We assume that contrary to previous conclusions (Case and Leigh (1976) Biochem. J., 160, 769-783) redox centre of at least one of the oxidized cytochromes b , most probably of b -562, is exposed to the M-aqueous phase.