Tag Archives: ATP
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.
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.
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.
IP(3)-sensitive Ca(2+)-channels of endoplasmic reticulum in secretory cells of the rat exorbital lacrimal gland
A. B. Kotliarova, V. V. Manko
Ivan Franko National University of Lviv, Ukraine;
e-mail: annkotliarova@gmail.com
The role of inositol-1,4,5-trisphosphate of (IP3)-sensitive Ca2+ channels in Ca2+ homeostasis maintenance under activation of M-cholinergic receptors and P2Y receptors in the secretory cells of the rat lacrimal gland was investigated. The study was carried out on intact and permeabilized secretory cells of exorbital lacrimal glands of rats. The cells were isolated using the modified Herzog, Sides, Miller method (1976) and permeabilized with digitonin (50 mg per 0.5 million cells). The functioning of the Ca2+-transport systems was estimated by changes of Ca2+ content in the studied cells, which was determined by the spectrophotometric method using arsenazo III. It was shown that IP3-sensitive Ca2+ channels (IP3Rs) of investigated cells are directly inhibited by 2-APB (10 µM/l). On the other hand, the channels are activated by IP3, cholinomimetic (carbacholine) and purine receptor agonist (ATP). When both M-cholinergic receptors and P2Y receptors were activated Ca2+ was released from the same IP3-sensitive store because the effects of ATP and carbacholine at high concentrations (1 mM/l and 10 µM/l, respectively) on the Ca2+ content were non-additive. The presence of the store-operated Ca2+-channels in secretory cells of the lacrimal gland is confirmed by the observed increase of cellular Ca2+ content as a result of Ca2+ mobilization from the store by carbacholine or thapsigargin and following restoration of Ca2+ concentration in the extracellular solution.