Regulation of ionized calcium concentRation in mitochondRia matRix in the absence of exogenous Са 2+

Functional activity of mitochondria depends on ionized calcium content in its matrix. thus, investigation of the ways to influence Ca 2+ transport and accumulation in these organelles is an urgent issue. the aim of this work was to evaluate ionized calcium concentration in the mitochondria matrix in the absence of exoge nous ca 2+ and in the presence of Mg 2+ and AtP in incubation media. the concentration of ionized calcium in mitochondria isolated from the myometrium of non-pregnant rats was measured with the fluorescent probe Fluo-4 AM, mitochondria hydrodynamic diameter was assessed using dynamic light scattering. the AtP concentration-dependent increase in ionized calcium concentration in mitochondria matrix in the absence of exogenous Са 2+ and the high level of the total Са 2+ accumulation after further addition of exogenous Са 2+ were registered. The effect of ATP on Са 2+ concentration did not depend on the blockage of mitochondrial PtP with cyclosporine A or on h + -AtPase/AtP-synthase inhibition with oligomycin, instead the addition of 10mM theophylline and 30 mM Nahco 3 to the Mg 2+ -containing medium led to Са 2+ concentration elevation in mitochondria matrix. It was shown that low ca 2+ concentration in the matrix correlated with the increase in mitochondria size, whereas high ca 2+ concentration correlated with relatively smaller size of the organelles. Activation of the soluble adenylyl cyclase by Nahco 3 with simultaneous inhibition of phosphodiesterase by theophylline was accompanied by the decline in Са 2+ probe normalized fluorescence in mitochondria matrix under conditions of exogenous ca 2+ introduction. It was suggested that the soluble adenylyl cyclase may be involved in regulation of Са 2+ concentration in mitochondria matrix.

M itochondria are high-energy organelles, which conduct a range of important functions in the cell [1]. They take part in calcium signaling, mechanisms of apoptosis, stress adaptation, steroidogenesis and ageing [2]. Dysfunction of mitochondria is one of the reasons of several neurodegenerative diseases and different forms of oncology [3,4]. Ion channels and ion transporters regulate mitochondria state and functionality directly [5]. Thus, muscles can be functionally active just on condition of corresponding calcium concentra-tion. On the one hand, activation of metabolic pathways in mitochondria by calcium ions through the Krebs cycle and electron transport chain leads to an increase in ATP synthesis [6]. On the other hand, an excessive increase of Ca 2+ concentration in mitochondria leads to apoptosis. Therefore, functional activity of mitochondria depends on the ionized calcium content in these organelles. That is why investigation of the ways to influence the Ca 2+ transport and accumulation in mitochondria is an urgent issue.
In the previous works, it has been shown that ionized calcium concentration in mitochondria matrix on condition of absence of exogenous Ca 2+ depends on incubation medium: in Mg 2+ ,ATP-containing medium this number is a couple of times higher than in Mg 2+ -containing one. The addition of exogenous Ca 2+ to Mg 2+ and Mg 2+ ,ATP-containing mediums resulted in an increase of ionized Ca 2+ concentration to approximately equal numbers. Nevertheless, the numbers of total calcium accumulation are 30 times higher in the presence of ATP than in its absence [7].
Ca 2+ accumulation in mitochondria is provided by Ca 2+ -uniporter, the activity of which is dependent on the membrane potential of these organelles [8,9]. Thus, in Mg 2+ ,ATP-containing medium, the mitochondria membrane is polarized, whereas in Mg 2+containing medium, it is depolarized. Therefore, the question arises as to the mechanisms involved in an increase of ionized calcium concentration in mitochondria matrix in the absence of exogenous Ca 2+ and in the presence of Mg 2+ and ATP, which leads to high rates of total calcium accumulation on condition of further addition of exogenous Ca 2+ .
Among different signaling pathways, which influence mitochondria function, much attention has recently been paid to cAMP signaling [10,11]. Adenylyl cyclase system affects a variety of indicators, including the level of ATP and Ca 2+ [12].
Thus, the aim of this work was to study the mechanisms of ionized calcium concentration change in mitochondria matrix in the absence of exogenous Ca 2+ and in the presence of Mg 2+ and ATP.

materials and methods
The treatment of the lab animals was carried out according to "European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes" (Strasbourg, 1986). Animals were euthanized by chloroform followed by cervical dislocation. After that, their uteri were promptly removed. Mitochondria from myometrium of non-pregnant rats were isolated using the differential centrifugation method [13]. The mitochondria were suspended in a medium with the following composition: 250 mM sucrose, 1 mM EGTA, 20 mM Hepes, and buffered pH 7.4 at 4°C. Fatty acid free bovine serum albumin (0.1% w/v) was also added. The protein concentration of the mitochondrial fraction was determined by Bradford assay [14]. The concentration of mitochondrial protein in the sample was 25 µg/ml. The hydrodynamic diameter of mitochondria in different mediums was investigated with the method of dynamic light scattering, using Zeta Sizer-3 (Malvern Instruments, UK) with Multi8 computing correlator 7032ce type. Incubation medium contained 20 mM HEPES, 250 mM sucrose, 5 mM succinate, 2 mM K + -phosphate buffer, ±3 mM MgCl 2 and ±3 mM ATP.
The calibration of the Fluo-4 fluorescence was performed at the end of the experiments by adding 0.1% Triton X-100 and, in 1 min, 5 mM EGTA (fluorescence intensities F max and F min , respectively). The concentration of ionized Ca in the mitochondria matrix was calculated using the Grynkiewicz equation [14].

Results and discussion
Dependence of ionized calcium concentration in matrix on AtP and calculating the hill coefficient. Results of ionized calcium concentration measurements in mitochondria matrix in the absence of exogenous Ca 2+ have shown that this number is higher after an incubation in Mg 2+ ,ATP-containing medium than after incubation in Mg 2+ -containing medium (Fig. 1).
After getting these results, the questions, whether the concentration of ionized calcium in mitochondria matrix depends on ATP concentration in an incubation medium and on the absence of exogenous Ca 2+ and, if so, what mechanisms underlie this process, were raised. At constant Mg 2+ concentration (3 mM), an increase of ATP concentration in the incubation medium was accompanied by an increase of ionized calcium concentration in mito- chondria matrix with a plateau at ATP concentration over 2 mM (Fig. 2, a). In the presence of 100 µM Ca 2+ ATP concentration effect was not registered (Fig. 2, b). Linearization of the obtained results in the coordinates of Hill graph allowed calculating the Hill coefficient. Several evaluations were made, thus the Hill coefficient equals 3.18 ± 0.27 and activation constant for ATP equals 0.97 ± 0.07 mM. The rate of Hill coefficient indicates the positive cooperativity, which means that the binding rate of every subsequent ATP molecule is being increased after binding one ATP molecule. Besides, it may also indicate that the effect of ATP on ionized calcium concentration in mitochondria matrix is a multi-stage process. The activation constant for ATP is greatly comparable with physiological ATP concentrations inside of cells. Thus, an ATP-dependent increase of ionized calcium concentration in mitochondria matrix in the absence of exogenous Ca 2+ was shown and high rates of total Ca 2+ accumulation in condition of further addition of exogenous Ca 2+ arising from this was indicated.
the hydrodynamic diameter of mitochondria change after incubation in different mediums. Due to the fact that in the absence of ATP and the presence of exogenous Ca 2+ , the level of total calcium accumulation in mitochondria matrix is low, this process may be connected to low level of mitochondria membrane potential. Depolarization of mitochondria membrane is usually accompanied by an increase of their hydrodynamic diameter and death.
It was shown that the hydrodynamic diameter of mitochondria was different after incubating it in Mg 2+ -containing medium and Mg 2+ ,ATP-containing medium: in Mg 2+ -containing medium it was higher than in Mg 2+ ,ATP-containing one (Fig. 3).
Thus, in the absence of exogenous Ca 2+ , low Ca 2+ concentration in matrix correlates with relatively bigger size of the organelles, which is associated with mitochondria membrane depolarization and organelle death. The high Ca 2+ concentration in matrix correlates with relatively smaller size of the organelles, which corresponds to the functional activity of mitochondria in normal conditions. Which mechanisms underlie the mitochondria hydrodynamic diameter change, caused by presence or absence of ATP in the incubation medium? Perhaps, there may be a role of mitochondrial pore in this process. This is a known fact that adenine nucleotides are blockers of mitochondrial pore [15]. Besides, the mitochondrial pore opening is accompanied by an increase in mitochondria size. We assumed that an increase in mitochondria membrane permeability may be accompanied by an increase of ionized calcium exit from mitochondria matrix. To check this hypothesis, we investigated the ionized calcium concentration in mitochondria matrix in the presence of cyclosporine A, classical blocker of mitochondrial pore.
Investigation of ionized calcium concentration in mitochondria matrix under the influence of several substances. The key regulator of mitochondrial pore is cyclophilin D. Its typical blocker is cyclosporine A [16]. Thus, to study the influence of mitochondrial pore on an increase of Ca 2+ concentration in mitochondria matrix, we added 5 µM cyclosporine A to the incubation medium, which also contained Mg 2+ or Mg 2+ ,ATP. Using the spectrofluorometer, level of ionized calcium in the organelles was measured. Ionized calcium concentration was calculated with Grynkiewicz equation. As shown in Fig. 4, there were no changes in given parameters. Thus, it was concluded that mitochondrial pore was not likely to be involved in an increase of Ca 2+ concentration in mitochondria matrix after incubation in Mg 2+ ,ATP-containing medium in comparison with incubation in Mg 2+ -containing medium.
Next ATP-dependent process in mitochondria, which could possibly influence the change of ion- ized calcium concentration, is functioning of ATP synthase in the opposite direction, i.e. as H + -ATPase. It is known that the proton pump provides a sufficiently high level of polarization of mitochondria inner membrane, which is a guarantee of high values of total Ca 2+ accumulation [17]. Typical inhibitor of F 1 F 0 ATP-synthase is oligomycin. Thus, the next step was to investigate the effect of oligomycin in concentration of 0.5 µg/ml. Using the spectrofluorometer, level of ionized calcium concentration in the organelles was measured. The results are shown in Fig. 5; it can be seen that 0.5 µg/ml of oligomycin did not have an influence on given parameters. Thus, a blocker of cyclophilin D (cyclosporine A) and an inhibitor of F 1 F 0 ATP-synthase (oligomycin) do not affect an ionized calcium concentration in mitochondria matrix in the absence and the presence of exogenous Ca 2+ .
Investigation of the role of adenylyl cyclase system in regulation of calcium concentration in mitochondria matrix. Lately, much attention is paid to investigation of mitochondria adenylyl cyclase system, which includes adenylyl cyclase, cAMP, cAMP-dependent protein kinase A and phosphodiesterase [11]. Membrane form of adenylyl cyclase is activated by forskolin [18]. Although, it has recently been shown that there is a soluble adenylyl cyclase in mitochondria matrix, which is regulated by NaHCO 3 [19]. Soluble adenylyl cyclase has ATP binding constant in millimolar concentration range (substrate of adenylyl cyclase). Typical inhibitor of phosphodiesterase is theophylline. It is known that soluble form of cAMP takes part in the regulation of electron transport chain activity, ion channels activity, oxidative phosphorylation, mitochondrial DNA phosphorylation, which influences mitochondria homeostasis, cell survival and apoptosis [19,20]. Mistakes in these processes lead to a range of pathologies, including metabolism disorders, neurodegenerative disorders, heart diseases, diabetes and different forms of oncolo gy. Besides, new studies claim that dysregulation of cAMP signaling pathway is connected to the development of ageing.
We have shown that forskolin with concentrations 10 and 100 µM did not have an influence on the Ca 2+ concentration in mitochondria matrix (data is not presented). The next step was to measure the ioni zed calcium concentration in mitochondria matrix under the influence of theophylline and NaHCO 3 . Incubation was conducted in Mg 2+ -containing medium in the absence of exogenous Ca 2+ .
As it can be seen from the results presented in Fig. 6, addition of mentioned above substances led to an increase of ionized calcium concentration in mitochondria matrix after their incubation in Mg 2+containing medium. Thus, it was shown that activation of adenylyl cyclase in the presence of 30 mM NaHCO 3 with simultaneous inhibition of phosphodiesterase by 10 mM theophylline was accompanied by an increase in ionized calcium concentration in mitochondria matrix (P < 0.05).
The next step was to analyze the kinetics of changes in the normalized fluorescence of Ca 2+sensitive probe Fluo-4 in response to the addition of exogenous Ca 2+ . Normalized fluorescence is calculated with the data received from spectrofluorometer. The values of the fluorescence intensity of the probe after the addition of exogenous Ca 2+ are divided by the values of the fluorescence intensity of the probe before the addition of exogenous Ca 2+ . A graph of normalized fluorescence is constructed based on the obtained results (Fig. 7).