Tag Archives: calcium

The effect of permeability transition pore opening on reactive oxygen species production in rat brain mitochondria

O. V. Akopova, L. I. Kolchinskaya, V. I. Nosar, A. N. Smirnov, M. K. Malysheva, I. N. Mankovska, V. F. Sagach

Bogomoletz Institute of Physiology, National Academy of Sciences of Ukraine, Kyiv;
e-mail: luko@biph.kiev.ua

The influence of mitochondrial permeability transition pore (MPTP) opening on reactive oxygen species (ROS) production in the rat brain mitochondria was studied. It was shown that ROS production is regulated differently by the rate of oxygen consumption and membrane potential, dependent on steady-state or non-equilibrium conditions. Under steady-state conditions, at constant rate of Ca2+-cycling and oxygen consumption, ROS production is potential-dependent and decreases with the inhibition of respiration and mitochondrial depolarization. The constant rate of ROS release is in accord with proportional dependence of the rate of ROS formation on that of oxygen consumption. On the contrary, transition to non-equilibrium state, due to the release of cytochrome c from mitochondria and progressive respiration inhibition, results in the loss of proportionality in the rate of ROS production on the rate of respiration and an exponential rise of ROS production with time, independent of membrane potential. Independent of steady-state or non-equilibrium conditions, the rate of ROS formation is controlled by the rate of potential-dependent uptake of Ca2+ which is the rate-limiting step in ROS production. It was shown that MPTP opening differently regulates ROS production, dependent on Ca2+ concentration. At low calcium MPTP opening results in the decrease in ROS production because of partial mitochondrial depolarization, in spite of sustained increase in oxygen consumption rate by a cyclosporine A-sensitive component due to simultaneous work of Ca2+-uniporter and MPTP as Ca2+-influx and efflux pathways. The effect of MPTP opening at low Ca2+ concentrations is similar to that of Ca2+-ionophore, A-23187. At high calcium MPTP opening results in the increase of ROS release due to the rapid transition to non-equilibrium state because of cytochrome c loss and progressive gating of electron flow in respiratory chain. Thus, under physiological conditions MPTP opening at low intracellular calcium could attenuate oxidative damage and the impairment of neuronal functions by diminishing ROS formation in mitochondria.

Responsiveness to progesterone and potassium channel blockers 4-aminopyridine, tetraethylammonium and free Ca(2+) contentration in spermatozoa of patients with oligozoospermia/leucocytospermia

R. V. Fafula1, G. V. Danylovych2, A. S. Besedina1, O. V. Melnyk1, Z. D. Vorobets1

1Danylo Halytsky Lviv National Medical University, Ukraine;
2Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: roman_fafula@ukr.net

The present study was undertaken to evaluate [Ca2+]i signals that occur in human sperm cells exposed in vitro to three diverse compounds; progesterone, 4-aminopyridine (a highly effective inducer of hyperactivation in human sperm) and tetraethylammonium. The [Ca2+]i reached after the extracellular calcium treatment was always higher in normozoospermic samples pretreated with progesterone than in pathozoospermic samples pretreated with progesterone. There were no changes in calcium signal in spermatozoa pretreated with progesterone from patients with oligozoospermia and leucocytospermia compared to control samples (without progesterone). [Ca2+]i was always higher in pathozoospermic samples without 4-aminopyridine and always lower in pathozoospermic samples with 4-aminopyridine compared to these values in normozoospermic men. The 2 mM extracellular calcium administration to spermatozoa pretreated with tetraethylammonium did not result in a detectable increase in [Ca2+]i in normo- and pathozoospermic samples. The mechanisms of progesterone-dependent activation of the Ca2+-entry and the functioning of the voltage gated Ca2+-channels of plasmalemma are disturbed in pathologies – there was no increase in the Ca2+ level in the conditions of K+-depolarization (in the presence of inhibitors of K+-channels).

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.

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.

Nitric oxide as the regulator of intracellular homeostasis in the uterus myocytes

Yu. V. Danylovych

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

The published data on the mechanisms and regulation of active and passive Ca2+ transport in the myometrium have been analyzed. Particular attention is paid to the cGMP-dependent and independent pathways of action of nitric oxide or its derivatives on intracellular Ca2+ homeostasis of uterine smooth muscle and its contractile activi­ty. Information on the effect of nitric oxide on Ca2+-transport systems of other types of smooth muscles is provided in a comparative aspect. Based on own experimental results and literature data a scheme of NO action in the myometrium is suggested in which nitric oxide or its derivatives cause­ Ca2+-dependent polarization of the sarcolemma. In accordance with our results, this effect may be based on the increase of sarcolemma Ca2+ permeability under the influence of NO or its derivatives and the stimulation of at least the initial passive transport of the cation in the myocytes mediated by dihydropyridine-sensitive channels. Additional factors that contribute to the polarization of the membrane are the increase of protons transport from the muscle cells and stimulation of Na+, K+-ATPase. Acting on the sarcoplasmic reticulum, nitrosactive compounds activate the inclusion of calcium in this compartment and inhibit Ca2+-induced release of the cation. The latter effects are able to provide compensation for NO-induced Ca2+ increase in myocytes and supress the electro-mechanical coupling at Ca2+ release from the reticulum. NO-derivates also inhibit a key link in the smooth muscle contractile act – the formation of the Ca2+-calmodulin complex.

Signal mediators at induction of heat resistance of wheat plantlets by short-term heating

Yu. V. Karpets, Yu. E. Kolupaev, T. O. Yastreb

V. V. Dokuchaev Kharkiv National Agrarian University, Ukraine;
e-mail: plant_biology@mail.ru

The effects of functional interplay of calcium ions, reactive oxygen species (ROS) and nitric oxide (NO) in the cells of wheat plantlets roots (Triticum aestivum L.) at the induction of their heat resistance by a short-term influence of hyperthermia (heating at the temperature of 42 °С during 1 minute) have been investigated. The transitional increase of NO and H2O2 content, invoked by heating, was suppressed by the treatment of plantlets with the antagonists of calcium EGTA (chelator of exocellular calcium), lanthanum chloride (blocker of calcium channels of various types) and neomycin (inhibitor of phosphatidylinositol-dependent phospholipase C). The rise of hydrogen peroxide content, caused by hardening, was partially suppressed by the action of inhibitors of nitrate reductase (sodium wolframate) and NO-synthase (NG-nitro-L-arginine methyl ester – L-NAME), and the increasing of nitric oxide content was suppressed by the treatment of plants with the antioxidant ionol and with the scavenger of hydrogen peroxide (dimethylthiourea). These compounds and antagonists of calcium also partially removed the effect of the rise of plantlets’ heat resistance, invoked by hardening heating. The conclusion on calcium’s role in the activation of enzymatic systems, generating reactive oxygen species and nitric oxide, and on the functional interplay of these signal mediators at the induction of heat resistance of plantlets by hardening heating is made.

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.

Reactive oxygen forms and Ca ions as possible intermediaries under the induction of heat resistance of plant cells by jasmonic acid

Yu. V. Karpets, Yu. E. Kolupaev, T. O. Yastreb, O. I. Oboznyi,
M. V. Shvydenko, G. A. Lugova, A. O. Vayner

Dokuchayev Kharkiv National Agrarian University, Ukraine;
e-mail: plant_biology@mail.ru

The participation of reactive oxygen species (ROS) and calcium ions in realization of influen­ce of exogenous jasmonic acid (JA) on the heat resistance of wheat coleoptiles has been investigated­. Influence of 1 µM JA caused the transitional intensifying of generation of superoxide anion-radi­cal (O2•–) and hydrogen peroxide in coleoptiles with the maximum within 15-30 minutes after the treatment beginning. Within the first hour after the beginning of coleoptiles treatment with JA the increase of superoxide dismutase (SOD) activi­ty was noted. Later on (within 5-24 hours after the treatment beginning) there was the lowering of ROS generation by coleoptiles of experimental variant, and the SOD activity approached the control value. Intensifying of generation of superoxi­de radical induced by JA was suppressed by the antioxidant ionol and was partially levelled by imidazole (inhibitor of NADPH-oxidase), EGTA (chelator of extracellular calcium) and lanthanum chloride (calcium channels blocker). Pretreatment of coleoptiles with the ionol, imidazole, EGTA and LaCl3 also partially removed the effect of increase of their resistance to the damaging heating caused by exogenous JA. It is supposed that the ROS gene­rated with participation NADPH-oxidase, which activity depends on the receipt of calcium ions from extracellular space in the cytosol, are involved in realization of physio­logical effects of JA.

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.

NAADP-sensitive Сa(2+) stores in permeabilized rat hepatocytes

S. V. Bychkova1, T. I. Chorna2

1Ivan Franko National University of Lviv, Ukraine;
2National Centre for Biological Sciences, Tata Institute
of Fundamental Research, Bangalore, India;
e-mail: s.bychkova@gmail.com

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a nucleotide that is potent to release calcium from intracellular stores in different cell types. NAADP was shown to target specific type of intracellular store namely endolysosomal system or acidic store. Despite intense studies, its effect on endoplasmatic reticulum (ER) still remains to be elucidated. The main aim of our work was to investigate NAADP-sensitive store in permeabilized rat hepatocytes monitoring the level of Ca2+ inside intracellular organelles using chlorotetracycline (CTC). We have shown that NAADP triggered changes of stored Ca2+ in rat hepatocytes are dependent on concentration of EGTA-Ca2+-buffer in cell incubation medium, i.e. the higher is the EGTA concentration in incubation medium the smaller or absent is the effect of NAADP. Besides, the effect of NAADP was more pronounced upon cells pretreatment with the inhibitory concentration of ryanodine (100 µM). This might suggest that the effect of NAADP is dependent on ER luminal calcium. We have also found that NAADP-evoked Ca2+ release in permeabilized hepatocytes is sensitive to nigericin, bafilomycin A and thapsigargin. Additionally, NAADP triggered changes in stored Ca2+ were completely abolished by NED-19 as antagonist of NAADP.