Tag Archives: mitochondria
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.
Ca ion transport in smooth muscle mitochondria
L. G. Babich, S. G. Shlykov, S. O. Kosterin
Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: babich@biochem.kiev.ua
Review focuses on the analysis of literature data and own results concerning properties of mitochondria Са2+ transporting systems and their regulation. Three mechanisms for mitochondrial Ca2+ uptake are described: the electrogenic mitochondrial Ca2+ uniporter, a rapid mode of Ca2+ uptake (RaM) and ryanodine-sensitive Ca2+ channels (RyR). Two mechanisms of Ca2+ efflux from the mitochondrial matrix have been proposed: Na+-dependent and Na+-independent Ca2+ exchange. The mitochondrial permeability transition pore can also function as a Ca2+ efflux mechanism. Ca2+ accumulation in mitochondria resulted in an increase of mitochondrial membrane potential, ATP synthesis and activation of metabolites transport. The dissipation of mitochondrial membrane potential should prevent mitochondrial Ca2+ uptake. Our data concerning depolarizing effect of calmodulin antagonists and hyperpolarizing effect of calix[4]arenes С-136 and С-137 on the myometrium mitochondria can be applied on the demand of mitochondria membrane potential correction.
Activation of glybenclamide-sensitive mitochondrial swelling under induction of cyclosporin of A-sensitive mitochondrial pore
O. B. Vadzyuk, S. A. Kosterin
Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: olga_vadzyuk@hotmail.com
Induction of mitochondrial swelling and increased generation of reactive oxygen forms by Ca ions have been shown in suspension of mitochondria from rat uterus. These effects were suppressed by the blocker of mitochondrial Ca2+-uniporter ruthenium red and MPTP inhibitor сyclosporin A, that evidences that the induction of mitochondrial permeability transition pore by Ca ions takes place. Ca2+-induced mitochondrial swelling was blocked by ATP-sensitive channel blocker glybenclamide but only if K+ was present in the incubation medium. We also demonstrated that Ca2+-induced mitochondrial swelling can be eliminated in the presence of ROS scavengers N-acetyl cysteine and ascorbate. This effect of scavengers was also sensitive to K+ and was not revealed in the medium that contained equimolar NaCl instead of KCl. Thus, our data gave us grounds to assume that the induction of MPTP by Ca ions evokes the activation of mitochondrial ATP-sensitive K+-channels, which are mediated by ROS.
Ca(2+)/H(+)-exchange in myometrium mitochondria
O. V. Kolomiets, Yu. V. Danylovych, H. V. Danylovych, S. O. Kosterin
Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: danylovych@biochem.kiev.ua
Using the fluorescent probe Fluo-4 AM the authors have identified Na+-independent Ca2+/H+-exchange in isolated mitochondria of rat myometrium and studied its individual properties. Formation of directional protons gradient in the matrix of mitochondria causes antyporte release of Ca2+, which has been previously accumulated in energetic processes (in the presence of Mg-ATP and succinate). The functioning of Ca2+/H+-exchange depends on the proton gradient and is characterized by reversibility, in case of extramitochondria environment alkalization the additional accumulation of Ca2+ by organelles is recorded. Monovalent cations gradients (Na+, K+, Li+) do not cause the release of Ca2+ from mitochondria. Rate of Ca2+/H+-exchange is growing in terms of increasing ΔpH on the mitochondria membrane and kinetics of ΔpH-induced Ca2+ release from the matrix corresponds to the laws of first order reaction. Research of Ca2+/H+-exchange some properties in the myometrium mitochondria showed that the above transport process is of electrogenic nature, perhaps it is done in a 1 : 1 stechiometry (Hill coefficient on H+ close to 1) and is able to adjust matrix Ca2+ concentration under physiological conditions (pH activation of about 6.9). Thus, in the inner membrane of the myometrium mitochondria the available system of the secondary active Ca2+-transport from the matrix of these organelles to myoplasm and the functioning of Ca2+/H+-exchanger may underlie this process.
Mitochondrial thiol-disulfide system under acute hypoxia and hypoxic-hyperoxic adaptation
O. A. Gonchar, I. N. Mankovska
Bogomoletz Institute of Physiology, National Academy of Sciences of Ukraine;
e-mail: ogonchar@yandex.ru
The authors investigated the state of mitochondrial glutathione pool (reduced and oxidized glutathione, protein-GSH mixed disulfides), content of carbonyl groups and free sulfhydryl groups of proteins, protein expression of key mitochondrial antioxidant enzymes such as glutathione peroxidase and thioredoxin reductase as well as activity of glutathione reductase, glutathione peroxidase and glutaredoxin in the liver mitochondria of rats exposed to acute hypoxia after prolonged hypoxic-hyperoxic training (HHT). It was shown that the preliminary HHT reduced the intensity of proteins oxidative modification under acute hypoxia, activated the mitochondrial antioxidant defense as well as affected the thiol-disulfide exchange, redox balance in mitochondria, modulated the S-glutathionylation/deglutathionylation process in mitochondria membranes.