Tag Archives: mitochondria

The use of the Petri net method in the simulation modeling of mitochondrial swelling

Yu. V. Danylovych, A. Y. Chunikhin,  G. V. Danylovych, O. V. Kolomiets

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

Using photon correlation spectroscopy, which allows investigating changes in the hydrodynamic dia­meter of the particles in suspension, it was shown that ultrahigh concentrations of Ca2+ (over 10 mM) induce swelling of isolated mitochondria. An increase in hydrodynamic diameter was caused by an increase of non-specific mitochondrial membrane permeability to Ca ions, matrix Ca2+ overload, activation of ATP- and Ca2+-sensitive K+-channels, as well as activation of cyclosporin-sensitive permeability transition pore. To formalize the experimental data and to assess conformity of experimental results with theoretical predictions we developed a simulation model using the hybrid functional Petri net method.

Ca(2+)-dependent regulation of the Ca(2+) concentration in the myometrium mitochondria. I. Trifluoperazine effects on mitochondria membranes polarization and [Ca(2+)](m)

L. G. Babich, S. G. Shlykov, A. M. Kushnarova, S. O. Kosterin

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

Са2+-dependent regulation of Ca2+ exchange in mitochondria is carried out with participation of calmodulin. We have shown previously that calmodulin antagonists reduced the level of mitochondrial membrane polarization and induced increase of the ionized Са concentration in both the mitochondrial matrix and cell cytoplasm. The concentration-dependent  influence of trifluoperazine on the level of polarization of mitochondrial membranes has been shown in this work. The coordinates of the Hill graphs were used to calculate the constant K0.5 and the Hill coefficient. K0.5 was 24.4 ± 5 μM (n = 10). The Hill coefficient was 2.0 ± 0.2, indicating the presence of two centers of the trifluoperazine binding. We have also studied [Ca2+]m changes, when incubating mitochondria in mediums of different composition: without ATP and ions of Mg (0-medium), in the presence of 3 mM Mg (Mg-medium) and 3 mM Mg + 3 mM ATP (Mg,ATP-medium). It was shown that the composition of the incubation medium affected the [Ca2+]m values in the absence of exogenous Ca2+ and did not affect them in the presence of the latter. Preincubation of mitochondria in mediums of different composition with 25 μM trifluoperazine did not affect the [Ca2+]m values both before and after the addition of  100 µМ Са2+ to the  incubation medium. It was concluded, that trifluoperazine depolarized myometrial mitochondria membranes in concentration-dependent manner. However, mitochondria preincubation with 25 μM trifluope­razine accompanied by 50% decrease in membrane polarization did not affect the [Ca2+]m values.

Effects of α-tocopherol and its anologues on rat thymocytes programmed death induced by protein kinase inhibitors

G. V. Petrova, N. V. Delemenchuk, G. V. Donchenko

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

It is established that α-tocopherol (α-ТPh) shows cytoprotective effect at the induction of rats’ thymocytes apoptosis by endocellular protein kinase inhibitors – staurosporine and phorbol ether in high concentration, and also on necrosis of the cells caused by sphyngosine. The effect of α-ТPh on thymocytes death caused by protein phosphatase type 2А inhibitor ocadaic acid is much less expressed. The obtained data testify that the known ability of α-ТPh to the inhibition of PKC and to the activation of protein phosphatase type 2А is not the main mechanism of its cytoprotective action. Partial reproduction of α-ТPh effects by its analogue α-tocopheryl acetate which is not capable to enter in redox reactions, and the absence of influence on the studied processes of an antioxidant of N-acetyl-L-cysteine do not confirm the antioxidant mechanism of α-ТPh action in this case. The inhibition by α-ТPh of the release of cytochrome c in the cytosol of cells testifies to the implementation of its cytoprotective effect at the level of mitochondrial membranes. We assume the existence of the universal mechanism of α-ТPh cytoprotective action that does not depend on the nature of apoptogenes and realized on the general for the majority of them stage of the cells death induction. The prevention by α-ТPh of mitochondria dysfunction by stabilizing mitochondrial membranes and reduction of their permeabilization is supposed as that.

Changes in polarization of myometrial cells plasma and internal mitochondrial membranes under calixarenes action as inhibitors of plasma membrane Na(+), K(+)-ATPase

G. V. Danylovych1, Yu. V. Danylovych1, O. V. Kolomiets1,
S. O. Kosterin1, R. V. Rodik2, S. O. Cherenok2, V. I. Kalchenko2,
A. Ju. Сhunikhin1, V. F. Gorchev1, S. A. Karakhim1

1Palladine Institute of Biochemistry, National Academy of Science of Ukraine, Kyiv
2Institute of Organic Chemistry, National Academy of Science of Ukraine, Kyiv
e-mail: danylovych@biochem.kiev.ua;  vik@bpci.kiev.ua

The influence of supramolecular macrocyc­lic compounds – calix[4]arenes C-97, C-99, C-107, which are ouabainomymetic high affinity inhibitors of Na+, K+-ATPase, on the polarization level of plasmic and mitochondrial membranes of rat uterine smooth muscle cells was investigated. The influence of these compounds on the myocytes characteristic size was studied.
By using a confocal microscopy and specific for mitochondrial MitoTracker Orange CM H2TMRos­ dye it was proved that the potential-sensitive fluorescent probe DiOC6(3) interacts with mitochondria. Artificial potential collapse of plasmic membrane in this case was modeled by myocytes preincubation with ouabain (1 mM).
Further experiments performed using the method of flow cytometry with DiOC6(3) have shown that the compounds C-97, C-99 and C-107 at concentration 50-100 nM caused depolarization of the plasma membrane (at the level of 30% relative to control values) in conditions of artificial collapse of mitochondrial potential by myocytes preincubation in the presence of 5 mM of sodium azide.
Under artificial sarcolemma depolarization by ouabain, calixarenes C-97, C-99 and C-107 at 100 nM concentrations caused a transient increase of mitochondrial membrane potential, that is 40% of the control level and lasted about 5 minutes. Calixarenes C-99 and C-107 caused a significant increase in fluorescence of myocytes in these conditions, which was confirmed by confocal microscopy too.
It was proved by photon correlation spectroscopy method that the C-99 and C-107 caused an increase of characteristic size of myocytes.

Endoplasmic-mitochonrial Са(2+)-functional unit: dependence of respiration of secretory cells on activity of ryanodine- and IP(3)-sensitive Ca(2+)-channels

O. Yu. Velykopolska, B. O. Manko, V. V. Manko

Ivan Franko National University of Lviv, Ukraine;
e-mail: vvmanko@franko.lviv.ua

Using Clark oxygen electrode, dependence of mitochondrial functions on Ca2+-release channels activity of Chironomus plumosus L. larvae salivary glands suspension was investigated. Cells were ATP-permeabilized in order to enable penetration of exogenous oxidative substrates. Activation of plasmalemmal P2X-receptors (as well as P2Y- receptors) per se does not modify the endogenous respiration of salivary gland suspension. That is, Ca2+-influx from extracellular medium does not influence functional activity of mitochondria, although they are located along the basal part of the plasma membrane. Activation of RyRs intensifies endogenous respiration and pyruvate-malate-stimulated respiration, but not succinate-stimulated respiration. Neither activation of IP3Rs (via P2Y-receptors activation), nor their inhibition alters endogenous respiration. Nevertheless, IP3Rs inhibition by 2-APB intensifies succinate-stimulated respiration. All abovementioned facts testify that Са2+, released from stores via channels, alters functional activity of mitochondria, and undoubted­ly confirm the existence of endoplasmic-mitochondrial Ca2+-functional unit in Ch. plumosus larvae salivary glands secretory cells. In steady state of endoplasmic-mitochondrial Ca2+-functional unit the spontaneous activity of IP3Rs is observed; released through IP3Rs, Ca2+ is accumulated in mitochondria via uniporter and modulates oxidative processes. Activation of RyRs induces the transition of endoplasmic-mitochondrial Ca2+-functional unit to the active state, which is required to intensify cell respiration and oxidative phosphorylation. As expected, the transition of endoplasmic-mitochondrial Ca2+-functional unit to inactivated state (i. e. inhibition of Ca2+-release channels at excessive [Ca2+]i) limits the duration of signal transduction, has protective nature and prevents apoptosis.

The effect of ionizing radiation with low dose rate on the state of electron transfer chain of enterocyte mitochondria of rat small intestine

L. V. Grubska1, V. M. Voitsitskiy2, S. V. Khyzhnayk3

1Іnstitute of Bioorganic Chemistry and Petrochemistry, National Academy of Sciences of Ukraine, Kyiv;
2National University of Life and Environmental Sciences of Ukraine, Kyiv;
3Taras Shevchenko Kyiv National University, Ukraine;
е-mail: hsv@univ.kiev.ua

The influence of ionizing radiation with low absorbed dose rate (55 mGy·min-1) in 1, 12 and 24 hours after irradiation in doses of 0.1; 0.5 and 1.0 Gy on functional state of the electron transfer  chain of the rat small intestine mitochondria was investigated by assessment of the oxygen consumption rate. The uncoupling of oxidation and phosphorylation, a decrease of phosphorylation rate and inhibition of ATP hydrolysis reactions were established in mitochondria in dependence on the irradiation dose and time interval after irradiation. The functional peculiarities of the oxidation-phosphorylation coupling sites of the mitochondrial electron transfer chain were detected.

The effect of amixin and agmatine on cytochrome c release from isolated mitochondria

K. R. Uspenska, G. L. Gergalova, O. Yu. Lykhmus, M. V. Skok

Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;

e-mail: kate.uspenska@gmail.com

Mitochondrial nicotinic acetylcholine receptors (nAChRs) control permeability transition pore formation and cytochrome c release in the presence of apoptogenic factors. This study demonstrates that pharmacological agents amixin and agmatine affect mitochondrial nAChR functioning: they slightly suppress cytochrome c release from mouse brain and liver mitochondria stimulated with apoptogenic dose of Са2+ and prevent the effect of α7 nAChR agonist PNU282987. We conclude that mitochondria may be one of therapeutic targets of amixin and agmatine.

Experimental substantiation of permeabilized hepatocytes model for investigation of mitochondria in situ respiration

V. M. Merlavsky, B. O. Manko, O. V. Ikkert, V. V. Manko

Ivan Franko National University of Lviv, Ukraine;
e-mail: vvmanko@lnu.edu.ua

TTo verify experimentally the model of permeabilized hepatocytes, the degree of cell permeability was assessed using trypan blue and polarographycally determined cell respiration rate upon succinate (0.35 mM) and α-ketoglutarate (1 mM) oxidation. Oxidative phosphorylation was stimulated by ADP (750 μM). Hepatocyte permeabilization depends on digitonin concentraion in medium and on the number of cells in suspension. Thus, the permeabilization of 0.9-1.7 million cells/ml was completed by 25 μg/ml of digitonin, permeabilization of 2.0-3.0 million cells/ml – by 50 μg/ml of digitonin and permeabilization of 4.0-5.6 million cells/ml – by 100 μg/ml. Thus, the higher is the suspension density, the higher digitonin concentration is required. Treatment of hepatocytes with digitonin resulted in a decrease of endogenous respiration rate to a minimum upon 20-22 μg of digitonin per 1 million cells. Supplementation of permeabilized hepatocytes with α-ketoglutarate maintained stable respiration rate on the level higher than endogenous respiration at the corresponding digitonin concentration, unlike the intact cells. Respiration rate of permeabilized hepatocytes at the simultaneous addition of α-ketoglutarate and ADP increased to the level of intact cell respiration, irrespective of digitonin concentration. Addition of solely succinate and especially succinate plus ADP markedly intensified the respiration of permeabilized hepatocytes to the level higher than that of intact cells. The dependence of succinate-stimulated respiration on digitonin concentration reached maximum at 20-22 μg of digitonin per 1 million cells. Optimal ratio of digitonin amount and the cell number in suspension is expected to be different in various tissues.

Protective effects of potassium transport in mitochondria from rat myometrium under activation of mitochondrial permeability transition pore

O. B. Vadzyuk

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

We demonstrated using PBFI K+-sensitive fluorescent probe an enhancement of both components of K+-cycle – the ATP-sensitive K+-uptake and quinine-sensitive K+/H+-exchange – under the Ca2+ induced opening­ of mitochondrial permeability transition pore (MPTP) in rat myometrium mitochondria. Addition of CaCl2 (100 μM) to K+-free medium results in the enhancement of reactive oxygen species (ROS) production, which was eliminated by cyclosporine A. Addition of CaCl2 to K+-rich medium did not increase the rate of ROS production, but blocking of mitoK+ATP-channels with glybenclamide (10 μM) increased production of ROS. We conclude that K+-cycle exerts a protective influence in mitochondria from rat myometrium by regulation of matrix volume and rate of ROS production under the condition of Ca2+-induced MPTP.

Reprogramming of mitochondrial energy metabolism in malignant neoplasms

A. A. Kaplia1, L. V. Sorokina2, S. V. Khyzhnyak3

1Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: kaplya@biochem.kiev.ua;
2ESC “Institute of Biology”, Taras Shevchenko National University of Kyiv, Ukraine;
3National University of Life and Environmental Sciences of Ukraine, Kyiv

The novel ideas of fundamental role of mitochondria in the maintenance of viability of malignant cells have been reviewed. The modern state of research is considered in detail, including: mitochondrial control of the cellular redox state, sites of reactive oxygen species (ROS) production in inner mitochondrial membrane and antioxidant protection   systems. Specificities of the structural-functional mitochondrial remodelling in malignant tumors, the mechanisms of the energy metabolism reprogramming, enhancement of the ROS production and adaptation to the hypoxic conditions and metabolic stress are analyzed. The available data including our research on transplanted tumors indicate that cytotoxic action of sodium dichloroacetate (the inhibitor of pyruvate dehydrogenase kinase) depends on biological properties of tumors and intensity of structural-functional mitochondrial rearrangement. Dichloroacetate turned out to be effective for sarcoma 37, but not for Lewis lung carcinoma.