Tag Archives: diazoxide

Regulation of the mitochondrial ATP-sensitive potassium channel in rat uterus cells by ROS

O. B. Vadzyuk, Yu. Yu. Mazur, S. O. Kosterin

Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: vadzyuk@biochem.kiev.ua

In previous study we demonstrated the presence of ATP-sensitive potassium current in the inner mitochondrial membrane, which was sensitive to diazoxide and glybenclamide, in mitochondria isolated from the rat uterus. This current was supposed to be operated by mitochondrial ATP-sensitive potassium channel (mitoKATP). Regulation of the mitoKATP in uterus cells is not studied well enough yet.
It is well known that the reactive oxygen species (ROS) can play a dual role. They can dama­ge cells in high concentrations, but they can also act as messengers in cellular signaling, mediating survival of cells under stress conditions. ROS are known to activate mitoKATP during the oxidative stress in the brain and heart, conferring the protection of cells. The present study examined whether ROS mediate the mitoKATP activation in myometrium cells.
Oxidative stress was induced by rotenone. ROS generation was measured by 2′,7′-dichlorofluorescin diacetate. The massive induction of ROS production was demonstrated in the presence of rotenone. Hyperpolarization of the mitochondrial membrane was also detected with the use of the potential-sensitive dye DiOC6 (3,3′-dihexyloxacarbocyanine iodide). Diazoxide, a selective activator of mitoKATP, depolarized mitochondrial membrane either under oxidative stress or under normal conditions, while mitoKATP blocker glybenclamide effectively restored mitochondrial potential in rat myocytes. Estimated <K1/2> value for diazoxide to mitoKATP under normoxia was four times higher than under oxidative stress conditions: 5.01 ± 1.47∙10-6 М and 1.24 ± 0.21∙10-6 М respectively.
The ROS scavenger N-acetylcysteine (NAC) successfully eliminates depolarization of mitochondrial membrane by diazoxide under oxidative stress. These results suggest that elimination of ROS by NAC prevents the activation of mitoKATP under oxidative stress. Taking into account the higher affinity of diazoxide to mitoKATP under stress conditions than under normoxia, we conclude that the oxidative stress conditions are more favourable than normoxia for the activation of mitoKATP­. Thus we hypothesize that the ROS regulate the activity of the mitoKATP in myocytes.

The influence of ATP-dependent K(+)-channel diazoxide opener on the opening of mitochondrial permeability transition pore in rat liver mitochondria

O. V. Akopova

Bogomolets Institute of Physiology, National Academy of Sciences of Ukraine, Kyiv;
e-mail: luko@biph.kiev.ua

The influence of mitochondrial ATP-dependent­ K+-channel (K+АТР-channel) opener, diazoxide (DZ) on the mitochondrial permeabili­ty transition pore (MPTP) opening in rat liver mitochondria is studied. In the absence of DZ the MPTP opening leads to the increase in the rate of K+– and Ca2+-cycling supported by the simultaneous functioning of K+-channels and K+/H+-antiporter, and also Ca2+-uniporter together with MPTP as the cations influx and efflux pathways. Independent of MPTP opening, the activation of both constitutes of K+-cycle, K+-uptake as well as K+/H+-exchange, by DZ is observed. It is shown that the activation of transmembrane exchange of K+, combined with MPTP opening, results in partial inhibition of the latter. A simple methodical approach for the estimation of DZ influence on the open state of mitochondrial pore is proposed.
It is shown that MPTP closure followed by Ca2+ reentry to the matrix is accompanied by the K+/H+-exchange inhibition which takes place in the same timeframes as the increase in matrix Ca2+ content. Relevant to physiological conditions, an important physiological function of MPTP is revealed, that is the maintenance of relatively low matrix level of Ca2+ accompanied by the acceleration of transmembrane ion exchange (K+ and Ca2+) which could strongly influence the energy state and energy-dependent processes in mitochondria.

The effect of ATP-dependent K(+)-channel opener on the functional state and the opening of cyclosporine-sensitive pore in rat liver mitochondria

O. V. Akopova, V. I. Nosar, V. A. Bouryi, L. I. Kolchinskaya,
I. N. Mankovska, V. F. Sagach

Bogomoletz Institute of Physiology, National Academy of Sciences of Ukraine, Kyiv;
e-mail: a-dubensky@mail.ru

The effect of mitochondrial ATP-dependent K+-channel (K+АТР-channel) opener diazoxide (DZ) on the oxygen consumption, functional state and the opening of cyclosporine-sensitive pore in the rat liver mitochondria has been studied. It has been established that K+АТР-channel activation results in the increase of the oxygen consumption rate (V4S) and the uncoupling due to the acceleration of K+-cycling, the decrease in state 3 respiration rate (V3) and the respiratory control ratio (RCR). Under K+АТР-channel activation an inhibition of oxidative phosphorylation takes place which reduces the rate of ATP synthesis and hydrolysis as well as ATP production and consequently results in the seeming increase of P/O ratio. It has been shown that the increase in ATP-dependent K+-uptake accompanied by the opening of mitochondrial permeability transition pore (MPTP) leads to dramatic uncoupling of the respiratory chain due to simultaneous activation of K+– and Ca2+-cycling supported by MPTP and Ca2+-uniporter as well as K+-channels and K+/H+-exchange. K+АТР-channel activation leads to the partial inhibition of MPTP, but insufficient for the restoration of mitochondrial functions. Elimination of Ca2+-cycling after MPTP opening is necessary to return mitochondrial functions back to the control level which shows that MPTP could serve as the mechanism of reversible modulation of bioenergetic effects of K+АТР-channel activation.