Tag Archives: rat liver mitochondria
Functional activity of permeability transition pore in energized and deenergized rat liver mitochondria
O. V. Akopova*, L. I. Kolchinskaya, V. I. Nosar
Bogomoletz Institute of Physiology, National Academy of Sciences of Ukraine, Kyiv;
*e-mail: ov_akopova@ukr.net
Received: 15 June 2020; Accepted: 13 November 2020
Permeability transition pore (mPTP) opening was studied under energized and deenergized conditions in rat liver mitochondria, and the effect of membrane depolarization on mPTP activity was evaluated. To assess mPTP activity, cyclosporine-sensitive swelling and cyclosporine sensitive Ca2+ efflux from mitochondria was studied using light absorbance techniques. In energized mitochondria, mPTP opening in sub-conductance states, at [Ca2+] ≤ Ka, contributed positively to the rate of respiration, without affecting ΔΨm. Threshold Ca2+ concentrations were found, which excess resulted in fast mitochondrial depolarization upon mPTP opening. An estimate of mPTP activity by cyclosporine-sensitive Ca2+ transport under energized and deenergized conditions have shown that membrane depolarization by protonophore CCCP essentially increased initial rate (V0), at simultaneous decrease of the half-time (t1/2) of Ca2+ efflux, which indicated mPTP activation, as compared to energized mitochondria. However, only partial release of Ca2+ via mPTP upon membrane depolarization was observed. With the use of selective blockers of Ca2+ uniporter and mPTP, ruthenium red (RR) and cyclosporine A (CsA), partial contribution of Ca2+ uniporter and mPTP in Ca2+ transport was found. “Titration” of Ca2+ transport by adding RR at different times from the onset of depolarization showed that depolarization dramatically reduced “life span” of mPTP as compared to energized mitochondria, which agreed with the kinetic characteristics of CsA-sensitive Ca2+ transport after the abolition of ΔΨm. Ca2+ added from the outer side of mitochondrial membrane produced dual effect on mPTP activity: activation at the onset of depolarization, but consequent promotion of mPTP closure. Based on the experiments, it was concluded that mitochondrial energization was required for prolonged mPTP functioning in sub-conductance states, whereas membrane depolarization promoted the transition of mPTP to inactive state during calcium release from mitochondria.
The effect of Са(2+)-induced opening of cyclosporine-sensitive pore on the oxygen consumption and functional state of rat liver mitochondria
O. V. Akopova, V. I. Nosar, I. N. Mankovska, V. F. Sagach
A. A. Bogomoletz Institute of Physiology, National Academy of Sciences of Ukraine, Kyiv;
e-mail: a-dubensky@mail.ru
The effect of Ca2+-induced opening of cyclosporine-sensitive pore (mitochondrial permeability transition pore, MPTP) on the oxygen consumption and mitochondrial functional state was studied in the rat liver mitochondria. It was shown that, with the use of glutamate as oxidation substrate, in the absence of depolarization MPTP opening results in the increase of steady state respiration rate because of the activation of cyclosporine-sensitive Ca2+/H+-exchange and Ca2+ cycling, which was supported by the simultaneous work of MPTP and Ca2+-uniporter. With the aid of selective blockers, cyclosporine A and ruthenium red, it was shown that MPTP and Ca2+-uniporter contribute equally to the Ca2+-cycling and mitochondrial respiration. It was shown that bioenergetic effects of MPTP opening under steady state conditions (increase in the oxygen consumption rate under substrate oxidation without ADP, decrease in respiratory control ratio as well as the effectiveness of ATP synthesis, P/O) are close to the functional alterations, which result from the increase of endogenous proton conductance of mitochondrial membrane. Uncoupling effect of MPTP opening, by itself, had no effect on phosphorylation rate, which remains relatively stable because the fall of P/O is compensated by the activation of respiratory chain and the increase in the rate of state 3 respiration. It was concluded that under physiologically normal conditions MPTP might function as the endogenous mechanism of mild uncoupling of respiratory chain.
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.