Tag Archives: mitochondria
Biochemical and molecular-physiological aspects of the nitric oxide action in the utera
H. V. Danylovych, Yu. V. Danylovych
Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: danylovych@biochem.kiev.ua
Received: 18 May 2021; Accepted: 12 November 2021
The sources of the nitric oxide (NO) formation in the uterus and the dynamics of changes in its content in different periods of organ functioning in human and animals are analyzed. The biochemical mechanisms of NO action on the myometrium contractile activity, the significance of NO in the physiological processes during pregnancy and labor, the importance of mitochondria as a reliable NO source in the smooth muscle and the possible ways of NO influence on Ca2+ transport and bioenergetic processes in mitochondria are considered. The authors’ data concerning ionic and membrane mechanisms of NO action on Ca2+-homeostasis of uterine myocytes, identification of nitric oxide in uterine smooth muscle mitochondria, biochemical characteristics of the NO-synthase reaction and the possible role of NO in the regulation of Ca2+ transport in these subcellular structures and in the electron transport chain functioning are presented and discussed.
Application of petri nets methodology to determine biophysicochemical parameters of mitochondria functioning
H. V. Danylovych*, A. Yu. Chunikhin, Yu. V. Danylovych, S. O. Kosterin
Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
*e-mail: danylovych@biochem.kiev.ua
Received: 01 Nobember 2020; Accepted: 17 May 2021
With the use of Petri net methodology a mathematical simulation model able to predict simultaneous changes in biophysicochemical parameters of mitochondria functioning was developed. The model allowed to interconnect in time the changes in mitochondria hydrodynamic diameter, electronic transport chain functioning, endogenous fluorescence of adenine nucleotides, DCF fluorescence signal of ROS production and NaN3 effects. It was shown that the calculated values of the studied biophysicalchemical parameters correspond to those obtained experimentally. The model permit to link mitochondrial functional changes and their structural representation and to optimize significantly experimental procedures.
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.
Sources and regulation of nitric oxide synthesis in uterus smooth muscle cells
H. V. Danylovych, Yu. V. Danylovych, T. V. Bohach,
V. T. Hurska, S. O. Kosterin
Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: danylovych@biochem.kiev.ua
Received: 28 February 2019; Accepted: 17 May 2019
It was proved that NO synthesis in isolated mitochondria of rat uterus smooth muscle depended on the entry of exogenous Ca2+ to mitochondria (inhibited by 1-10 mM Mg2+ in the absence of ATP and by 10 μM ruthenium red) and was suppressed by calmodulin antagonists (0.1-10 μM calmidazolium and 1-100 μM trifluoperazine). It was blocked by NG-nitro-L-arginine, a known antagonist of the constitutive NO-synthase, with a half-maximal inhibition effect at about 25 μM. Moderate deholesterinization of the plasma membrane of myocytes after processing with 0.01% digitonin was followed by increased nitric oxide biosynthesis by cells. The data obtained suggested that mitochondria and plasmalemma is a possible source of NO synthesis in uterine myocytes.
Сalix[4]arene chalcone amides effects on myometrium mitochondria
S. G. Shlykov1, A. M. Kushnarova-Vakal1, A. V. Sylenko1,
L. G. Babich1, О. Yu. Chunikhin1, O. A. Yesypenko2,
V. I. Kalchenko2, S. O. Kosterin1
1Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: sshlykov@biochem.kiev.ua;
2Institute of Organic Chemistry, National Academy of Sciences of Ukraine, Kyiv
Received: 19 November 2018; Accepted: 14 March 2019
Mitochondria are a key player in a wide range of the most important functions of the cell. Calixarenes are supramolecular compounds that have been widely used in bioorganic chemistry and biochemistry. The aim of this work was to study the effects of calix[4]arenes with two (С-1012, С-1021), three (С-1023, С-1024) and four (С-1011) chalcone amide groups on the myometrial mitochondria membranes polarization, Ca2+ concentration in the matrix of these organelles ([Ca2+]m ) and on the average hydrodynamic diameter of mitochondria. It was shown that permeabilized myometrium cells incubation with calix[4]arenes containing two or more chalcone amide groups, was accompanied by an increased level of myometrial mitochondria membranes polarization. All studied calix[4]arenes increased [Ca2+]m values in the absence and in the presence of exogenous Ca2+. The values of [Ca2+]m in the absence of exogenous Ca2+ were higher at mitochondria incubation in Mg2+-containing, than in Mg2+,ATP-containing medium. Incubation of isolated mitochondria with the studied calix[4]arenes resulted in changes of mitochondria volume: at incubation with С-1012, С-1021, C-1023 the average hydrodynamic diameter was decreased, while with С-1011 it was increased. Thus, we have shown that a short-term (5 min) incubation of mitochondria in the presence of 10 µM calix[4]arenes, which contain from two to four chalcone amide groups, increased the level of mitochondria membranes polarization, ionized Ca concentration in the matrix and had different effects on the mitochondrial volume.
Adaptive respiratory response of rat pancreatic acinar cells to mitochondrial membrane depolarization
B. O. Manko, O. O. Bilonoha, V. V. Manko
Ivan Franko National University of Lviv, Ukraine;
e-mail: bohdan.manko@lnu.edu.ua
Received: 06 December 2018; Accepted: 14 March 2019
The dependence of uncoupled respiratory capacity of intact pancreatic acini on oxidative substrate supply and functional cell state has not yet been studied in detail. In this study, the respiratory responses of isolated pancreatic acini to FCCP were measured with Clark electrode and mitochondrial membrane potential was assessed with rhodamine123 fluorescence. The response of acini to FCCP was characterized with maximal uncoupled respiration rate, optimal FCCP concentration, respiration acceleration and deceleration. Maximal uncoupled respiration rate substantially increased upon the oxidation of glucose + glutamine (3.03 ± 0.54 r.u.), glucose + glutamine + pyruvate (2.82 ± 0.51 r.u.), glucose + isocitrate (2.71 ± 0.33 r.u.), glucose + malate (2.75 ± 0.38 r.u.), glucose + monomethyl-succinate (2.64 ± 0.42 r.u.) or glucose + dimethyl-α-ketoglutarate (2.36 ± 0.33 r.u.) comparing to glucose alone (1.73–2.02 r.u.) or no substrate (1.76 ± 0.33 r.u.). The optimal FCCP concentration was the highest (1.75 μM) upon glucose + glutamine + pyruvate combination and the lowest (0.5 μM) upon glutamate, combinations of glucose with isocitrate, malate, succinate or α-ketoglutarate. Respiration acceleration after FCCP application was the highest with dimethyl-α-ketoglutarate. Following the peak respiration, time-dependent deceleration was observed. It increased with FCCP concentration and depended on oxidative substrate type. Deceleration was the highest upon malate or isocitrate oxidation but was not observed in case of glutamine or dimethyl-α-ketoglutarate oxidation. Pyruvate alone or in combination with glutamine and glucose significantly decreased the depolarizing effect of FCCP on mitochondrial membrane potential and increased respiration elasticity coefficient with respect to the membrane potential change. Thus, in pancreatic acinar cells, the combination of pyruvate, glutamine and glucose enables the optimal adaptive respiratory response to membrane depolarization.
Glutathione influence on energy metabolism in rat liver mitochondria under experimental nephropathy
Ye. O. Ferenchuk, I. V. Gerush
Higher State Educational Establishment of Ukraine “Bukovinian State Medical University”, Chernivtsi;
e-mail: yelena_f@ukr.net
Received: 17 October 2018; Accepted: 14 March 2019
Mitochondrial oxidative damage and disorders of energy metabolism contribute to a wide range of pathologies and disease progression. In our work, the effect of glutathione on the activity of respiratory chain enzymes and the content of free SH-groups in rat liver mitochondria was examined with the use of folic acid-induced nephropathy model. Mitochondria were isolated by differential centrifugation, NADH-dehydrogenase, succinate dehydrogenase, cytochrome oxidase and H+-ATPase activity were determined. The activity of these enzymes and the content of the free SH-groups in the liver were shown to be decreased under conditions of nephropathy, evidently due to the intensification of the free radical processes. The introduction of glutathione increased the content of SH-groups and the activity of the Complexes II and V enzymes of mitochondrial respiratory chain but did not change the activity of cytochrome oxidase in mitochondria isolated from the liver of rats under experimental nephropathy. The results obtained demonstrate a positive effect of glutathione on mitochondrial succinate dehydrogenase and H+-ATPase activity normalization in the liver of rats with nephropathy. These findings may help to extend the understanding of mitochondrial energy metabolism under development of kidney diseases.
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 1.2.1.3), aldehyde reductase (EC 1.1.1.21) 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.