The influence of the K+(ATP)-channel opener diazoxide on the K+ cycle and oxygen consumption has been studied in rat liver mitochondria. It was found that diazoxide activates the K+(ATP)-channel in the range of nanomolar concentrations (50-300 nM, K(1/2) ~ 140 nM), which results in activation of K+/H+ exchange in mitochondria. The latter, in turn, accelerates mitochondrial respiration in respiratory state 2. The contribution of K+(ATP)-channel to the mitochondrial potassium cycle was estimated using the selective K+(ATP)-channel blocker glibenclamide. The data show that the relative contribution of K+(ATP)-channel in the potassium cycle of mitochondria is variable and increases only with the decrease in the ATP-independent component of K+ uptake. Possible mechanisms underlying the observed phenomena are discussed. The experimental results more fully elucidate the role of K+(ATP)-channel in the regulation of mitochondrial functions, especially under pathological conditions accompanied by impairment of the mitochondrial energy state.

The opening of mitochondrial K(+) АТР-channel (mtK(+) АТР-channel) is supposed to be important in the modulation of mitochondrial functions under hypoxia, but the underlying mechanisms have not been clarified yet. The aim of this work was to study the effect of acute hypoxia on mtK(+) АТР-channel activity and to estimate the contribution of the channel in the modulation of mitochondrial functions. MtK(+) АТР-channel activity was assessed polarographically from the rate of State 4 respiration and by potentiometric monitoring of potassium efflux from deenergized mitochondria. It was shown that hypoxia reliably increased mtK(+) АТР-channel activity, which resulted in the changes of respiration rates (increase of State 4 and suppression of State 3 respiration), uncoupling (the decrease of respiratory control ratio) and suppression of phosphorylation. These effects were well mimicked by mtK(+) АТР-channel opener diazoxide (DZ) in isolated rat liver mitochondria. MtK(+) АТР-channel opening in vitro suppressed phosphorylation too, but increased phosphorylation efficiency, while mtK(+) АТР-channel blockers reduced it dramatically. The correlation was established between mtK(+) АТР-channel activity and the endurance of the rats to physical training under hypoxia. Hypoxia improved physical endurance, but treatment by mtK(+) АТР-channel blockers glibenklamide and 5-hydroxydecanoate (5-HD) prior to hypoxia strongly reduced both the channel activity and the endurance limits. This was in accord with the observation that under glibenklamide and 5-HD administration hypoxia failed to restore mtK(+) АТР-channel activity. Based on the experiments, we came to the conclusion that mtK(+) АТР-channel opening played a decisive role in the regulation of energy metabolism under acute hypoxia via the modulation of phosphorylation system in mitochondria.

The effect of potential-dependent potassium uptake on reactive oxygen species (ROS) generation in mitochondria of rat brain was studied. It was found that the effect of K+ uptake on ROS production in the brain mitochondria under steady-state conditions (state 4) was determined by potassium-dependent changes in the membrane potential of the mitochondria (ΔΨm). At K+ concentrations within the range of 0-120 mM, an increase in the initial rate of K(+)-uptake into the matrix resulted in a decrease in the steady-state rate of ROS generation due to the K(+)-induced depolarization of the mitochondrial membrane. The selective blockage of the ATP-dependent potassium channel (K(ATP)(+)-channel) by glibenclamide and 5-hydroxydecanoate resulted in an increase in ROS production due to the membrane repolarization caused by partial inhibition of the potential-dependent K+ uptake. The ATP-dependent transport of K+ was shown to be ~40% of the potential-dependent K+ uptake in the brain mitochondria. Based on the findings of the experiments, the potential-dependent transport of K+ was concluded to be a physiologically important regulator of ROS generation in the brain mitochondria and that the functional activity of the native K(ATP)(+)-channel in these organelles under physiological conditions can be an effective tool for preventing ROS overproduction in brain neurons.

Background/Aims: NO and reactive nitrogen species (RNS) are thought to be physiologically important effectors of mitochondrial calcium transport, but this issue was not studied in a living organism. According to literature, the modulation of Ca²⁺ uptake could influence RNS production via the action on mitochondrial NO synthase (mtNOS). The aim of this work was to study the effect of in vivo administration of NO donor nitroglycerine (NG) on matrix Ca²⁺ accumulation, RNS production and mtNOS activity. Methods: Ca²⁺ uptake was studied spectrophotometrically with arsenazo-III. The amounts of stable RNS (nitrite, nitrate and nitrosothiols) and L-citrulline, the product of enzymatic NOS activity, were determined analytically. Results: NG administration resulted in dose-dependent short-term increase in Ca²⁺-uptake accompanied by essential rise in L-citrulline and RNS content in mitochondria. In parallel, dose-dependent elevation of hydroperoxide production was detected. Ca²⁺-uniporter activity was not affected, but mitochondrial permeability transition pore (MPTP) was effectively blocked by NO. Conclusion: Our results indicate that MPTP blockage by NO was the primary cause for the increase in calcium uptake which eventually resulted in the activation of mtNOS and RNS production. Improved Ca²⁺ accumulation in mitochondria, together with MPTP blockage, may contribute to well-known cardioprotective effects of pharmacological donors of nitric oxide.

Cytochrome c as an amplifier of ROS release in mitochondria The influence of exogenous cytochrome c on reactive oxygen species (ROS) formation and its dependence on mitochondrial permeability transition pore (MPTP) opening is studied in rat liver mitochondria. Fluorescent probe dichlorofluorescein (DCF) was used. It was shown that MPTP activation by increasing concentrations of Ca 2+ in the medium results in the increase in mitochondrial ROS production and oxygen consumption, but the decrease in matrix calcium retention, dependent on the amount of added Ca 2+. Cytochrome c in the incubation medium does not much influence ROS formation when MPTP opening is blocked by cyclosporine A. However, in the presence of cytochrome c MPTP opening is accompanied by dramatic increase in ROS production. Steep rise in DCF fluorescence because of matrix ROS formation is sensitive to MPTP opening and is not resulted from the direct interaction between the probe and cytochrome c outside the mitochondria. To explain obtained data the hypothesis is put forward that MPTP could serve for ROS exchange between the matrix and the medium where heme iron of cytochrome c would act as a catalytic center to enhance ROS production. We suppose that apart of its conventional function, cytochrome c which is not involved in electron transport, could serve in such way as the amplifier of ROS production which in turn would provide a background for the development of apoptosis due to MPTP opening.

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Nitroglycerine (NG) affords cardioprotection via NO formation, but the impact of NG application on reactive nitrogen species (RNS) metabolism remains little studied yet. Mitochondrial NO synthase (mtNOS) is an important endogenous source of RNS. Our aim was to study the effect of NG application on mtNOS activity and RNS production in rat heart and liver mitochondria. Different regulation of mtNOS activity in the heart and liver under NG treatment was found. While in heart mitochondria it increased dose-dependently, in liver mitochondria only moderate elevation and bell-shaped dose dependence of mtNOS activity on NG was observed. Nitrite and nitrate, which are the end products of L-arginine transformation by NOS, showed similar dose dependence on NG. To find an explanation for the observed dependences, we studied the effects of NG administration on the activity of arginase which competes with NOS for physiological substrate, Larginine. A strong reciprocal dependence between mtNOS and arginase activities was found. As we observed, the arginase activity increased under NG application. However, while in heart mitochondria high mtNOS activity agreed with moderate arginase activation, in liver mitochondria high arginase activity coincided with suppression of mtNOS activity at high doses of NG. Low arginase and high mtNOS activities observed in heart mitochondria were consistent with high NO2 − and NO3 − production and low hydroperoxide (H2O2) formation, whereas high arginase activity in liver mitochondria was accompanied by the reduction of NO2− /NO3− formation and simultaneous elevation of H2O2 production. A linear correlation between the arginase activity and hydroperoxide formation was found. We came to the conclusion that under NG administration arginase activation resulted in reciprocal regulation of RNS and ROS production in mitochondria, dependent on the proportion of mtNOS to arginase activity. Suppression of RNS metabolism could be the cause of ROS overproduction caused by high arginase and low mtNOS activity.