Tag Archives: mitochondria

The relationship between the ionized Ca concentration and mitochondrial functions

L. G. Babich1, S. G. Shlykov1, A. M. Kushnarova-Vakal1, N. I. Kupynyak2, V. V. Manko2, V. P. Fomin3, S. O. Kosterin1

1Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: babich@biochem.kiev.ua;
2Ivan Franko National University of Lviv, Ukraine;
3University of Delaware, Newark, USA

The aim of the study was to show the relationships between ionized Ca concentration ([Ca2+]m) in the mitochondria matrix and functional activity of this organelle. [Ca2+]m was determined using the fluorescent probe Fluo-4, AM. Total level of Ca2+ accumulation in mitochondria was monitored using 45Ca2+ as radioactive tracer. It was shown that incubation of myometrium mitochondria with 3 mM Mg2+ resulted in the low level of [Ca2+]m. Subsequent addition of 100 µM Ca2+ resulted in 8 times increase of [Ca2+]m but in low level of total calcium accumulation. Normalized fluorescence of Ca2+-sensitive probe Fluo-4 in response to the Ca2+ addition was higher than 2.5. At the same time, [Ca2+]m was considerably higher in the medium containing­ 3 mМ АТР and 3 mМ Mg2+. Subsequent addition of 100 µM Ca2+ to the incubation medium resulted in only 2.4 times increase of [Ca2+]m but considerably higher level of total calcium accumulation was observed. Normali­zed fluorescence of Fluo-4 in response to the Ca2+ addition was lower than 1.3. In liver mitochondria higher rate of oxygen consumption was detected in the presence of an oxidative substrate succinate than of pyruvate or α-ketoglutarate. At the presence of an oxidative substrate succinate normalized fluorescence of Fluo-4 in liver mitochondria in response to the Ca2+ addition was lower than 1.3. It was concluded that low level of [Ca2+]m was correlated with low functional activity of this organelle and, vise versa, high level of [Ca2+]m was correlated with high functional activity. It was suggested that normalized fluorescence changes in response to the Са2+ addition could be used as a test of the mitochondrial functional activity: lower normalized fluorescence values − higher functional activity.

Activity of the mitochondrial isoenzymes of endogenous aldehydes catabolism under the conditions of acetaminophen-induced hepatitis

O. M. Voloshchuk, G. P. Kopylchuk, Y. I. Mishyna

Yuriy Fedkovych Chernivtsi National University, Institute of Biology, Chemistry and Natural Resources, Ukraine;
e-mail: o.voloschuk@chnu.edu.ua

The research deals with the determination of the activity of aldehyde dehydrogenase (EC, aldehyde reductase (EC as well as the content of TBA reactive substances and protein carbonyl derivates in the rat liver  cytosolic fraction under the conditions of acetaminophen-induced hepatitis and protein deficiency. The most pronounced decrease in the activity of enzymes utilizing endogenous aldehydes is observed in the liver cytosolic fraction of animals with toxic liver injury maintained under the conditions of alimentary protein deficiency. Meanwhile, the accumulation of TBA reactive substances and protein carbonyl-derivates in the liver cytosolic fraction of animals of this experimental group was established. The accumulation of aldehyde products of lipid and protein oxidative damage on the background of the reduction in the activity of enzymes providing aldehyde catabolism may be considered as a possible mechanism underlying hepatocyte dysfunction under the conditions of toxic damage in protein-deficient animals.

Cаlіx[4]аrene С-956 is effective inhibitor of Н(+)-Сa(2+)-exchanger in smooth muscle mitochondria

G. V. Danylovych1, О. V. Kоlomiets1, Yu. V. Danylovych1, R. V. Rodik2, V. I. Kаlchenko2, S. О. Kоsterin1

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

It was shown that calix[4]arene C-956 exhibited a pronounced concentration-dependent (10-100 μM) inhibitory effect on the H+-Ca2+-exchanger of the inner mitochondrial membrane of rat uterine myocytes (Ki 35.1 ± 7.9 μM). The inhibitory effect of calix[4]arene C-956 was accompanied by a decrease in the initial rate (V0) and an increase in the magnitude of the characteristic time (τ1/2) of the ΔрН-induced Са2+ release. At the same time, it did not affect the potential-dependent accumulation of Ca2+ in mitochondria. Thus, the action of calix[4]arene C-956 might be directed on increasing the concentration of Ca ions in the mitochondrial matrix. The calculation of basic kinetic parameters of the Ca2+ transport from isolated organelles (in the case of its non-zero stationary level), based on changes in fluorescence of Ca2+-sensitive dye Fluo-4 AM in mitochondria was performed. The proposed approach can be used for the kinetic analysis of the exponential decrease of the fluorescence response of any probes under the same experimental conditions.

The biosynthesis of nitric oxide from L-arginine. Nitric oxide formation features and its functional role in mitochondria

G. V. Danylovych, T. V. Bohach, Yu. V. Danylovych

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

Modern data on biochemical patterns of nitric oxide biosynthesis in mammal cells from L-arginine in normoxic conditions is described.  The attention of the authors is given to the results of the recent years on the structure and regulation features isoforms of NO-synthase. The emphasis is put on the latest conception of the compartmentalization of certain isoforms of these enzymes in cells and on the possibility of the directed transport of nitric oxide in the vascular wall. The central place in the review is devoted to issues on the endogenous formation of NO in mitochondria and its potential physiological significance. Our own results on the identification of NO in mitochondria of the uterine smooth muscle, biochemical characteristics of this process and NO possible role in Са2+ transport regulation by organelles are presented and discussed.

Biochemical mechanism of the o,p’-DDD effect on the adrenal cortex

A. S. Mikosha, O. I. Kovzun

V. P. Komisarenko Institute of Endocrinology and Metabolism, National Academy of Medical Sciences of Ukraine, Kyiv;
e-mail: asmikosha@gmail.com

o,p’-Dichlorodiphenyldichloroethane (o,p’-DDD, mitotane) is used in the treatment of adrenocortical cancer and Cushing’s disease. This medicine induces numerous biochemical changes in the adrenal cortex, as well as disorder in the mitochondrial structure. Therewith, the level of produced corticosteroid hormones is significantly reduced. One of the possible causes can be a decrease in the NADPH level due to inhibition of the activity of its reduction system and increased NADPH consumption during the glutathione reduction catalyzed by glutathione reductase. o,p’-DDD is partially metabolized in the adrenal glands, and   the main metabolite (in terms of quantity) is o,p’-dichlorodiphenylacetic acid. However, attempts to find a physiologically active component among metabolites were unsuccessful. The most pronounced changes caused by o,p’-DDD were found in the mitochondria of the adrenal cortex. The respiration at the level of IV and I complexes is suppressed, the protein content of these complexes decreases. The phospholipid composition of the tissue altered and the concentration of diphosphatidylglycerol, the most important component of mitochondrial membranes, decreased. In our opinion, o,p’-DDD, owing to its high lipophilicity, accumulates in the mitochondria membranes and causes conformational disorder followed by disorder in mitochondrial functions. It was shown that o,p’-DDD acts as an inhibitor of acyl-CoA-cholesterol acyltransferase (ACAT, SOAT1). Therefore, adenocorticocytes accumulate free cholesterol, causing endoplasmic reticulum stress, mitochondrial swelling and caspases activation. Increased apoptosis leads to a decline in adrenal function and to a decrease in weight of adrenal glands.

Ca(2+)-dependent regulation of the Ca(2+) concentration in the myometrium mitochondria. II. Ca(2+) 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

It is known that Ca2+ accumulation in the mitochondria undergoes complex regulation by Ca2+ itself. But the mechanisms of such regulation are still discussed. In this paper we have shown that Ca ions directly or indirectly regulate the level of myometrium mitochondria  membranes polarization.  The additions of 100 µM Ca2+ were accompanied by depolarization of the mitochondria membranes. The following experiments were designed to study the impact of Ca2+ on the myometrium mitochondria [Ca2+]m. Isolated myometrium mitochondria were preincubated without or with 10 μM Са2+ followed by 100 μM Са2+ addition. Experiments were conducted in three mediums: without ATP and Mg2+ (0-medium), in the presence of 3 mM Mg2+ (Mg-medium) and 3 mM Mg2+ + 3 mM ATP (Mg,ATP-medium). It was shown that the effects of 10 μM Са2+ addition were different in different mediums, namely in 0- and Mg-medium the [Ca2+]m values increased, whereas in Mg,ATP-medium statistically reliable changes were not registered. Preincubation of mitochondria with 10 μM Са2+ did not affect the [Ca2+]m value after the addition of 100 μM Са2+. The [Ca2+]m values after 100 μM Са2+ addition were the same in 0- and Mg,ATP-mediums and somewhat lower in Mg-medium. Preliminary incubation of mitochondria with 10 μM Са2+ in 0- and Mg-mediums reduced changes of Fluo 4 normalized fluorescence values that were induced by 100 μM Са2+ additions, but in Mg,ATP-medium such differences were not recorded. It is concluded that Са2+ exchange in myometrium mitochondria is regulated by the concentration of  Ca ions as in the external medium, so in the matrix of mitochondria. The medium composition had a significant impact on the [Са2+]m values in the absence of exogenous cation. It is suggested that light increase of [Са2+]m before the addition of 100 μM Са2+ may have a positive effect on the functional activity of the 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.