Tag Archives: mitochondria
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.
Nicotinic acetylcholine receptors: specific antibodies and functions in humoral immunity
M. V. Skok, L. M. Koval, O. Yu. Lykhmus, O. M. Kalashnyk,
G. L. Gergalova, S. V. Komisarenko
Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: skok@biochem.kiev.ua
Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels initially discovered in muscles and neurons and further found in many non-excitable cells. The present review summarizes the results of studies performed in the Department of Molecular Immunology during the last decade and concerning the structure and functions of nAChRs in B lymphocytes and in mitochondria, as well as the role of nAChR-specific antibodies in the development of neurodegenerative disorders like Alzheimer disease.
Activity of NAD•H-generating enzymes and cytochrome content in mitochondria from rat liver and myocardium under artificial hypobiosis
S. D. Melnychuk, S. V. Khyzhnyak, V. S. Morozova, V. M. Voitsitsky
National University of Life and Environmental Sciences of Ukraine, Kyiv;
e-mail: director@quality.ua
The modification particularities of the structural and functional state of the inner mitochondrial membrane of the rat liver and myocardium were observed in conditions of artificial hypobiosis, which was created using hypoxic and hypercapnic gas medium with a body temperature reduction. Under the artificial hypobiosis the activity of NAD·H-generating enzymes of the Krebs cycle of the liver mitochondria decreases. The established changes of the enzymes activity and cytochromes content of the inner mitochondrial membrane indicate the decrease of the oxidative activity of a respiratory chain, that can be limited on a terminal (cytochrome c oxidase) site and leads to the decrease (by 49% at an average) of the H+-ATPase activity of the liver mitochondria. Under the artificial hypobiosis the detected increase of the succinate-KoQ-oxidoreductase activity (by 65% at average) causes the maintaining of the functional activity of a mitochondrial respiratory chain, considering the high (relative to control) cytochrome c oxidase and H+-ATPase activities of the mitochondria of the rats’ myocardium. The structural changes of the inner mitochondrial membrane of the liver and myocardium in experimental conditions are accompanied by the increase of hydrophobicity of tryptophan residues microenvironment and the intramolecular modifications of protein molecules.
Ca(2+) accumulation study in isolated smooth muscle mitochondria using Fluo-4 AM
O. V. Kolomiets, Yu. V. Danylovych, G. V. Danylovych, S. O. Kosterin
Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: danylovych@biochem.kiev.ua
The opportunity of Ca2+-sensitive fluorescent dye Fluo-4 AM and spectrofluorimetry method application for the study of energy-dependent Ca2+ accumulation in mitochondria from uterus smooth muscle is proved. It has been found that the presence of mitochondrial preparation increases time-dependent fluorescent response considerably and this effect depends on Ca2+ concentration in the medium. Thus, in these conditions, deesterification active probe is formed which is sensitive to Ca2+. It is shown that the accumulation of calcium ions in mitochondria in the presence of Mg-ATP and succinate depends on exogenous Ca2+ concentration and is characterized by substrate saturating. The apparent activation constant of Ca2+ accumulation is 53.9 ± 6.9 mM, which corresponds to the physiological concentration of the cation in the cell next to mitochondria. Transit addition of Ca2+-ionophore A23187 to the incubation medium caused a rapid release of ionized cation from mitochondria. When proton gradient on the inner mitochondrial membrane is dissipated by protonophore CCCP, in the case of suppressing the generation of the gradient by oligomycin and in the presence of ruthenium red that inhibits Ca2+ mitochondrial accumulation systems, Ca2+ entry is significantly reduced. The results indicate the prospects of using Fluo-4 AM to study the properties of the Ca2+ accumulation system in isolated mitochondria of the myometrium.
Electrochemical potential of the inner mitochondrial membrane and Ca(2+) homeostasis of myometrium cells
Yu. V. Danylovych, S. A. Karakhim, H. V. Danylovych, O. V. Kolomiets, S. O. Kosterin
Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: danylovych@biochem.kiev.ua
We demonstrated using Ca2+-sensitive fluorescent probe, mitochondria binding dyes, and confocal laser scanning microscopy, that elimination of electrochemical potential of uterus myocytes’ inner mitochondrial membrane by a protonophore carbonyl cyanide m-chlorophenyl hуdrazone (10 μM), and by a respiratory chain complex IV inhibitor sodium azide (1 mM) is associated with substantial increase of Ca2+ concentration in myoplasm in the case of the protonophore effect only, but not in the case of the azide effect. In particular, with the use of nonyl acridine orange, a mitochondria-specific dye, and 9-aminoacridine, an agent that binds to membrane compartments in the presence of proton gradient, we showed that both the protonophore and the respiratory chain inhibitor cause the proton gradient on mitochondrial inner membrane to dissipate when introduced into incubation medium. We also proved with the help of 3,3′-dihexyloxacarbocyanine, a potential-sensitive carbocyanine-derived fluorescent probe, that the application of these substances results in dissipation of the membrane’s electrical potential. The elimination of mitochondrial electrochemical potential by carbonyl cyanide m-chlorophenyl hуdrazone causes substantial increase in fluorescence of Ca2+-sensitive Fluo-4 AM dye in myoplasm of smooth muscle cells. The results obtained were qualitatively confirmed with flow cytometry of mitochondria isolated through differential centrifugation and loaded with Fluo-4 AM. Particularly, Ca2+ matrix influx induced by addition of the exogenous cation is totally inhibited by carbonyl cyanide m-chlorophenyl hydrazone. Therefore, using two independent fluorometric methods, namely confocal laser scanning microscopy and flow cytometry, with Ca2+-sensitive Fluo-4 AM fluorescent probe, we proved on the models of freshly isolated myocytes and uterus smooth muscle mitochondria isolated by differential centrifugation sedimentation that the electrochemical gradient of inner membrane is an important component of mechanisms that regulate Ca2+ homeostasis in myometrium cells.