Tag Archives: membrane potential

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.

Effects of thiazole derivatives on intracellular structure and functions in murine lymphoma cells

V. P. Hreniukh1, N. S. Finiuk1,2, Ya. R. Shalai1, B. O. Manko1,
B. V. Manko1, Yu. V. Ostapiuk1, O. R. Kulachkovskyy1,
M. D. Obushak1, R. S. Stoika1,2, A. M. Babsky1*

1Ivan Franko National University of Lviv, Ukraine;
2Institute of Cell Biology, Nationl Academy of Sciences of Ukraine, Lviv;
*e-mail: andriy.babsky@gmail.com

Received: 22 December 2019; Accepted: 27 March 2020

Thiazole derivatives have cytotoxic effects towards tumor cells, such as glioblastoma, melanoma, leukemia and lymphoma. However, the intracellular mechanism of this action is not clear. The aim of our study was to investigate the action of N-(5-benzyl-1,3-thiazol-2-yl)-3,5-dimethyl-1-benzofuran-2-carboxamide (BF1) and 7-benzyl-8-methyl-2-propylpyrazolo[4,3-e]thiazolo[3,2-a]pyrimidin-4(2H)-one (PP2) on cellular structure, and bioenergetic functions of mitochondria in Nemeth-Kellner lymphoma cells (NK/Ly). The structure of treated NK/Ly cells and their mitochondria was examined using electron microscopy. The rate of oxygen uptake by isolated mitochondria was recorded by a polarographic method using a Clark electrode. The mitochondrial potential relative values were registered using fluorescence dye rhodamine 123. In the short-term (15 min), incubation with BF1 and PP2 in 10 and 50 µM concentrations induced apoptotic and necrotic changes in the structure of NK/Ly cells, such as fragmentation and disintegration of the nucleus, destruction of the plasma membrane, and an increase in numbers of lysosomes and mitochondria. A polarographic method did not show significant metabolic shifts in lymphoma mitochondria, in either in vitro or ex vivo actions of the thiazole derivatives. However, fluorescent microscopy showed a significant decrease in mitochondria potential, following a 15 min incubation of cells with 50 µM of PP2. Thus, the electron and fluorescent microscopy data suggest that mitochondria are involved in the mechanism of cytotoxic action of the studied thiazole derivatives.

Transmembrane Ca(2+) exchange in depolarized rat myometrium mitochondria

L. G. Babich, S. G. Shlykov, N. V. Kandaurova, S. A. Kosterin

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

Polarization of the inner membrane is the key factor in maintenance of the physiologically significant cations accumulation, in particular Ca2+, in the mitochondria. It has been well established that mitochondria accumulate calcium through the uniporter, driven by the mitochondrial membrane potential.  Nevertheless, it has been shown that depolarized mitochondria also accumulate Ca2+. The aim of this paper is to investigate free Ca level in depolarized myometrium mitochondria. As we have shown previously Ca2+ addition to the incubation medium, that did not contain K-phosphate, ATP and Mg2+, led to inner mitochondrial membrane depolarization. Nevertheless Ca2+ addition to such medium led to the concentration-dependent accumulation of this cation in the matrix. RuR or Mg addition to the incubation medium led to the higher elevation of mitochondrial Ca2+ level in depolarized mitochondria. Mitochondrial Ca2+ level was not affected by 5 µM cyclosporine A. It was suggested that Н+/Са2+ exchanger could provide calcium accumulation in depolarized mitochondria. The elevation of mitochondrial Ca2+ level after addition of Mg2+ and RuR may be due to inhibition of Ca2+ efflux through Ca2+ uniporter.

Nicotine effects on mitochondria membrane potential: participation of nicotinic acetylcholine receptors

G. L. Gergalova, M. V. Skok

Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: gergal71@gmail.com

The effect of nicotine on the mouse liver mitochondria was studied by fluorescent flow cytometry. Mice consumed nicotine during 65 days; alternatively, nicotine was added to isolated mitochondria. Mitochondria of nicotine-treated mice had significantly lower basic levels of membrane potential and granularity as compared to those of the control group. Pre-incubation of the isolated mitochondria with nicotine prevented from dissipation of their membrane potential stimulated with 0.8 µM СаСl2 depending on the dose, and this effect was strengthened by the antagonist of α7 nicotinic receptors (α7 nAChR) methyllicaconitine. Mitochondria of mice intravenously injected with the antibodies against α7 nAChR demonstrated lower levels of membrane potential. Introduction of nicotine, choline, acetylcholine or synthetic α7 nAChR agonist PNU 282987 into the incubation medium inhibited Ca2+ accumulation in mitochondria, although the doses of agonists were too low to activate the α7 nAChR ion channel. It is concluded that nicotine consumption worsens the functional state of mitochondria by affecting their membrane potential and granularity, and this effect, at least in part, is mediated by α7 nAChR desensitization.

The effect of nitric oxide on synaptic vesicle proton gradient and mitochondrial potential of brain nerve terminals

A. S. Tarasenko

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

The effect of nitric oxide on synaptic vesicle proton gradient and membrane potential of rat brain nerve terminals was studied. It has been shown that nitric oxide in the form of S-nitrosothiols at nanomolar concentrations had no effect on the studied parameters, but caused a rapid dissipation of synaptic vesicle proton gradient and depolarization of mitochondrial membrane in the presence of a SH-reducing compound such as dithiothreitol. Both processes were reversible and the rate of H+-gradient restoration depended on the redox potential of nerve terminals, namely the molar ratio of reductant/oxidant. This facts, as well as insensitivity of the studied processes to the inhibitor of NO-sensitive guanylate cyclase such as ODQ, allow suggesting that post-translational modification of thiol residues of the mitochondrial and synaptic vesicle proteins underlies the effect of nitric oxide on the key functional parameters of presynaptic nerve terminals.

The effect of potential-dependent potassium uptake on membrane potential in rat brain mitochondria

O. V. Akopova, V. I. Nosar, L. I. Kolchinskaya,
I. N. Mankovska, M. K. Malysheva, V. F. Sagach

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

The effect of potential-dependent potassium uptake on the transmembrane potential difference (ΔΨm) in rat brain mitochondria has been studied­. It was shown that in potassium concentration range of 0-120 mM the potential-dependent K+-uptake into matrix leads to the increase in respiration rate and mitochondrial depolarization. ATP-dependent potassium channel (K+ATP-channel) blockers, gliben­clamide and 5-hydroxydecanoate, block ~35% of potential-dependent potassium uptake in the brain mitochondria. It was shown that K+ATP-channel blockage results in membrane repolarization by ~20% of control, which is consistent with experimental dependence of ΔΨm on the rate of potential-dependent potassium uptake. Obtained experimental data give the evidence that functional activity of K+ATP-channel is physiologically important in the regulation of membrane potential and energy-dependent processes in brain mitochondria.

Estimation of ATP-dependent K(+)-channel contribution to potential-dependent potassium uptake in the rat brain mitochondria

O. V. Akopova, V. I. Nosar, 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 potassium on state 4 respiration (substrate oxidation in the absence of ADP) was investigated. It was shown that potential-dependent potassium uptake in the brain mitochondria results in mitochondrial depolarization. Taking into account depolarization effect of potassium, the contribution of the endo­genous proton leak as well as K+-uptake to the re­spiration rate was calculated. It was shown that such estimation allows the share of ATP-dependent potassium channel contribution to potential-dependent potassium uptake to be determined by polarographic method.