Tag Archives: mitochondrial permeability transition pore
mPTP opening differently affects electron transport chain and oxidative phosphorylation at succinate and NAD-dependent substrates oxidation in permeabilized rat hepatocytes
H. M. Mazur, V. M. Merlavsky, B. O. Manko, V. V. Manko*
Ivan Franko National University of Lviv, Ukraine;
*e-mail: volodymyr.manko@lnu.edu.ua
Received: 10 October 2019; Accepted: 15 May 2020
Mitochondrial Ca2+ overload may trigger the opening of mitochondrial permeability transition pore (mPTP) and its prolonged activation leads to cell death. ATP synthase is considered as a possible molecular component of the pore. The aim of this study was to investigate the state of oxidative phosphorylation at Ca2+-induced activation of mPTP in permeabilized hepatocytes. Hepatocytes were isolated by two-stage Seglen method. Permeabilization was performed using digitonin. Oxygen consumption rate was measured with Clark electrode. Oxidative phosphorylation was determined as the ratio of the ADP-stimulated respiration and substrate-stimulated respiration rates (ADP/S). It was established that increasing of Ca2+ concentration in the medium inhibited oligomycin effects and suppressed ADP- and FCCP-stimulated respiration upon succinate or glutamate, pyruvate and malate mixture oxidation. The mPTP inhibitor cyclosporin A did not directly affect respiration and oxidative phosphorylation after elevation of Ca2+ concentration and mPTP activation. When cyclosporine A was added before increasing Ca2+ concentration, the electron transport chain function (FCCP-stimulated respiration) was not impaired while the partial disruption of oxidative phosphorylation (ADP-stimulated respiration) was observed only upon succinate oxidation. The results obtained showed that inhibition of oxidative phosphorylation was the primary event in mPTP activation, possibly due to the involvement of ATP synthase in pore opening. In the case of NAD-dependent substrates oxidation that effect was stronger and faster than at succinate oxidation, due to the lower mitochondria energization.
The effect of permeability transition pore opening on reactive oxygen species production in rat brain mitochondria
O. V. Akopova, L. I. Kolchinskaya, V. I. Nosar, A. N. Smirnov, M. K. Malysheva, I. N. Mankovska, V. F. Sagach
Bogomoletz Institute of Physiology, National Academy of Sciences of Ukraine, Kyiv;
e-mail: luko@biph.kiev.ua
The influence of mitochondrial permeability transition pore (MPTP) opening on reactive oxygen species (ROS) production in the rat brain mitochondria was studied. It was shown that ROS production is regulated differently by the rate of oxygen consumption and membrane potential, dependent on steady-state or non-equilibrium conditions. Under steady-state conditions, at constant rate of Ca2+-cycling and oxygen consumption, ROS production is potential-dependent and decreases with the inhibition of respiration and mitochondrial depolarization. The constant rate of ROS release is in accord with proportional dependence of the rate of ROS formation on that of oxygen consumption. On the contrary, transition to non-equilibrium state, due to the release of cytochrome c from mitochondria and progressive respiration inhibition, results in the loss of proportionality in the rate of ROS production on the rate of respiration and an exponential rise of ROS production with time, independent of membrane potential. Independent of steady-state or non-equilibrium conditions, the rate of ROS formation is controlled by the rate of potential-dependent uptake of Ca2+ which is the rate-limiting step in ROS production. It was shown that MPTP opening differently regulates ROS production, dependent on Ca2+ concentration. At low calcium MPTP opening results in the decrease in ROS production because of partial mitochondrial depolarization, in spite of sustained increase in oxygen consumption rate by a cyclosporine A-sensitive component due to simultaneous work of Ca2+-uniporter and MPTP as Ca2+-influx and efflux pathways. The effect of MPTP opening at low Ca2+ concentrations is similar to that of Ca2+-ionophore, A-23187. At high calcium MPTP opening results in the increase of ROS release due to the rapid transition to non-equilibrium state because of cytochrome c loss and progressive gating of electron flow in respiratory chain. Thus, under physiological conditions MPTP opening at low intracellular calcium could attenuate oxidative damage and the impairment of neuronal functions by diminishing ROS formation in mitochondria.
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.
Activation of glybenclamide-sensitive mitochondrial swelling under induction of cyclosporin of A-sensitive mitochondrial pore
O. B. Vadzyuk, S. A. Kosterin
Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: olga_vadzyuk@hotmail.com
Induction of mitochondrial swelling and increased generation of reactive oxygen forms by Ca ions have been shown in suspension of mitochondria from rat uterus. These effects were suppressed by the blocker of mitochondrial Ca2+-uniporter ruthenium red and MPTP inhibitor сyclosporin A, that evidences that the induction of mitochondrial permeability transition pore by Ca ions takes place. Ca2+-induced mitochondrial swelling was blocked by ATP-sensitive channel blocker glybenclamide but only if K+ was present in the incubation medium. We also demonstrated that Ca2+-induced mitochondrial swelling can be eliminated in the presence of ROS scavengers N-acetyl cysteine and ascorbate. This effect of scavengers was also sensitive to K+ and was not revealed in the medium that contained equimolar NaCl instead of KCl. Thus, our data gave us grounds to assume that the induction of MPTP by Ca ions evokes the activation of mitochondrial ATP-sensitive K+-channels, which are mediated by ROS.