The relaTionship beTween The ionized Ca ConCenTraTion and miToChondrial funCTions

the aim of the study was to show the relationships between ionized ca concentration ([ca]m) in the mitochondria matrix and functional activity of this organelle. [ca]m was determined using the fluorescent probe Fluo-4, aM. total level of ca2+ accumulation in mitochondria was monitored using 45ca2+ as radioactive tracer. It was shown that incubation of myometrium mitochondria with 3 mM Mg2+ resulted in the low level of [ca]m. Subsequent addition of 100 μM ca 2+ resulted in 8 times increase of [ca]m but in low level of total calcium accumulation. Normalized fluorescence of Ca2+-sensitive probe Fluo-4 in response to the ca2+ addition was higher than 2.5. at the same time, [ca]m was considerably higher in the medium containing 3 mМ АТР and 3 mМ Mg2+. Subsequent addition of 100 μM ca2+ to the incubation medium resulted in only 2.4 times increase of [ca]m but considerably higher level of total calcium accumulation was observed. Normalized fluorescence of Fluo-4 in response to the Ca2+ addition was lower than 1.3. In liver mitochondria higher rate of oxygen consumption was detected in the presence of an oxidative substrate succinate than of pyruvate or α-ketoglutarate. At the presence of an oxidative substrate succinate normalized fluorescence of Fluo-4 in liver mitochondria in response to the ca2+ addition was lower than 1.3. It was concluded that low level of [ca]m was correlated with low functional activity of this organelle and, vise versa, high level of [ca ]m was correlated with high functional activity. It was suggested that normalized fluorescence changes in response to the Са2+ addition could be used as a test of the mitochondrial functional activity: lower normalized fluorescence values − higher functional activity.

the aim of the study was to show the relationships between ionized ca concentration ([ca 2+ ] m ) in the mitochondria matrix and functional activity of this organelle.[ca 2+ ] m was determined using the fluorescent probe Fluo-4, aM.total level of ca 2+ accumulation in mitochondria was monitored using 45 ca 2+ as radioactive tracer.It was shown that incubation of myometrium mitochondria with 3 mM Mg 2+ resulted in the low level of [ca 2+ ] m .Subsequent addition of 100 µM ca 2+ resulted in 8 times increase of [ca 2+ ] m but in low level of total calcium accumulation.Normalized fluorescence of Ca 2+ -sensitive probe Fluo-4 in response to the ca 2+ addition was higher than 2.5.at the same time, [ca 2+ ] m was considerably higher in the medium containing 3 mМ АТР and 3 mМ Mg 2+ .Subsequent addition of 100 µM ca 2+ to the incubation medium resulted in only 2.4 times increase of [ca 2+ ] m but considerably higher level of total calcium accumulation was observed.Normalized fluorescence of Fluo-4 in response to the Ca 2+ addition was lower than 1.3.In liver mitochondria higher rate of oxygen consumption was detected in the presence of an oxidative substrate succinate than of pyruvate or α-ketoglutarate.At the presence of an oxidative substrate succinate normalized fluorescence of Fluo-4 in liver mitochondria in response to the ca 2+ addition was lower than 1. 3 2+ ] m was correlated with high functional activity.It was suggested that normalized fluorescence changes in response to the Са 2+ addition could be used as a test of the mitochondrial functional activity: lower normalized fluorescence values − higher functional activity.K e y w o r d s: mitochondria, ionized Са concentration, total Са 2+ accumulation, myometrium, liver.M itochondria are known to be "power plants" of cells equally important in cell survival and death [1][2][3].Researchers are interested in studying both processes to understand how to save mitochondria and to support life of cell and, vice versa, how to kill mitochondria and, consequently, to delete undesirable cells.Intramitochondrial free calcium ([Ca 2+ ] m ) plays an important role in these processes [4][5][6][7] and it was suggested that low level of Са 2+ in the mitochondrial matrix is needed abbreviations: [Ca 2+ ] m , an intramitochondrial free calcium concentration; MCU, the mitochondrial calcium uniporter; EGTA, ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid; Hepes, 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid; Fluo-4, AM, acetoxymethyl (AM) ester derivative of fluorescent Ca 2+ indicator; Triton X-100, 4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol, non-ionic detergent; TMRM, Tetramethylrhodamine methyl ester perchlorate, sensitive probe for mitochondrial membrane potential; SEM, standard error of the mean.

e x p e r i m e n T a l w o r k s e x p e r i m e n T a l w o r k s
doi: https://doi.org/10.15407/ubj90.03.032 for the normal functioning of these organelles [5,8,9].For example, Miyata et al. found that the recovery of rat cardiomyocytes from hypoxia depended on the level of [Ca 2+ ] m at the end of the hypoxic period: cells having [Ca 2+ ] m greater than about 250-300 nM invariably hypercontracted upon reperfusion [9].These results were supported by Griffiths et al. who conducted their research on individual cardiomyocytes [5,8].So, it was concluded that to decrease the excessive rise of [Ca 2+ ] m is a main task for maintenance of cell life.The Са 2+ concentration in the mitochondria matrix, undoubtedly, plays an important role in functioning of these organelles, but role of "Ca 2+ overload", which typically happens in the damaged heart during ischemia/reperfusion, has recently been challenged [10,11].It was shown that in MCU (the mitochondrial calcium uniporter) null mitochondria -where "Ca 2+ overload" does not occur during reperfusion -the extent of necrosis was the same as that observed in the hearts from wild type littermates.It was suggested that there is enough Ca 2+ in the matrix of MCU null mitochondria to allow permeability transition pore opening.
We have shown, that 1) [Ca 2+ ] m could be changed in the absence of exogenous Ca 2+ ; 2) higher [Ca 2+ ] m in the absence of exogenous Ca 2+ was the requirement for higher total Ca 2+ accumulation (myometrium) and higher rate of oxygen consumption (liver).The suggestion has been made that normalized fluorescence changes in response to the Са 2+ addition could be used as the test of the mitochondrial functional activity: lower normalized fluorescence changes -higher functional activity.

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).A chloroform anesthesia was administered before animals were sacrificed by cervical dislocation and their uteri and livers were promptly removed.Mitochondria from myometrium of non-pregnant rats were isola ted using differential centrifugation method [12].The mitochondria were suspended in a solution with the following composition (mM): sucrose -250, EGTA -1, Hepes -20, and buffered pH 7.4 at 4 °C.Fatty acid free bovine serum albumin (0.1% w/v) was also added.Protein concentration of the mitochondrial fraction was determined by Bradford assay [13].The concentration of mitochondrial protein in the sample was 25 µg/ml.Rat liver mitochondria were isolated by the method of differential centrifugation.Liver was removed rapidly and perfused with a solution of the following composition (mM): NaCl − 140, KCl -4.7, MgCl 2 − 1, glucose − 5, Hepes -10; pH 7.4 to wash out the blood.The obtained preparation was suspended in a solution with the following composition (mM): sucrose -250, EGTA -1, Hepes -10; pH 7.2 at 4 °C.Protein concentration of the mitochondria fraction was determined by Lowry assay [14].
Myometrial cells from non-pregnant rats were isolated according to Mollard et al. [15].Cell counting was performed using hemocytometer.Cell viability was determined to be higher than 95% using trypan blue method.
Rate of oxygen consumption was determined using polarographic method at 26 °C.100 µl of mitochondria suspension was added to polarographic chamber that contained the solution of appropriate oxidation substrate.The concentration of protein in the chamber was 5-7 mg/ml.Mitochondrial respiration medium contained (mM): sucrose -250, K 2 HPO 4 -2, EGTA -0.1, CaCl 2 -0.1, Hepes -10; pH 7.2.Pyruvate, α-ketoglutarate and succinate (5 mM) were used as the oxidation substrates.Respiration was stimulated by the addition of ADP (final concentration in the chamber was 200 µM).The rate of respiration was determined in state S 3 , S 4 and S 4 ATP according to Chance and Williams [16].
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 [17].
The kinetics of [Са 2+ ] m changes expressed in Fluo-4 normalized fluorescence units is shown in Fig. 2.These data show that addition of exogenous Ca 2+ to the myometrial mitochondria incubated in the Mg 2+ ,ATP-containing medium caused smaller increase of normalized fluorescence compare to the one in Mg 2+ -containing medium.It was concluded that the incubation medium composition had a great impact on the normalized fluorescence.
Next we explored the effect of different media on functional activity of mitochondria.It is known that total Са 2+ accumulation is the highest in functionally active mitochondria [20] and 45 Ca 2+ as radioactive tracer is often used to monitor these values in various cells.Therefore, we determined the total level of Са 2+ accumulation in myometrial mitochondria in both incubation media.It was shown that upon addition of 10 µM 45 Ca 2+ myometrial mitochondria accumulated 149 ± 18 and 5 ± 2 nmol Са 2+ /mg of protein/5 min in Mg 2+ ,ATP-and Mg 2+ -containing medium, respectively (Fig. 3).
These data provide evidence that incubation of mitochondria in 3 mМ АТР and 3 mМ Mg 2+ -containing medium resulted in high level of total Ca 2+ accumulation, i. e., to be functionally active, while in 3 mМ Mg 2+ -containing medium, the level of total Ca 2+ accumulation is low, meaning low activity of the organelles.
We also used another experimental model -a suspension of myometrial myocytes treated with  ,ATP-containing medium, respectively (Fig. 4).
The data suggested that: 1) the level of Са 2+ accumulation was higher in the mitochondria that were preincubated in Mg 2+ ,ATP-containing medium compared to Mg 2+ -containing medium; 2) in the case of short time myometrium mitochondria preincubation in the absence of ATP, Са 2+ accumulation did not reach the level of one at initial АТР presence in the incubation medium.So, using two experimental models (isolated mitochondria and digitonin-permeabilized myometrial cells) it was shown that the total level of Са 2+ accumulation in mitochondria was high in the case of incubation in Mg 2+ ,ATP-containing medium and low in Mg 2+ -containing medium.It was concluded that functional activity of mitochondria was high in Mg 2+ ,ATP-containing medium and low in Mg 2+containing medium.
Earlier, using isolated myometrial mitochondria loaded with potential-sensitive probe TMRM, we have shown, that addition of Ca 2+ to the Mg 2+containing medium induced mitochondrial membrane depolarization [21].This effect was observed in Mg 2+ -but not in Mg 2+ ,ATP-containing medium.It is known that Ca 2+ accumulation in the mitochondrial matrix activates Ca 2+ efflux systems, such as H + /Ca 2+ exchanger [20].It was suggested that activation of the Ca 2+ efflux through the H + /Ca 2+ exchanger led to the elevation of H + concentration in the matrix, causing the mitochondrial membrane potential dis-sipation.It is also known that mitochondria can act as ATP consumers [1].In the case of mitochondrial membrane depolarization F 1 F 0 ATP-synthase acts as an ATPase, consuming ATP and pumping protons out across the mitochondrial inner membrane.The mitochondria consume ATP 'in order' to maintain their potential [1].It was suggested that ATP addition to the incubation medium prevent Ca 2+ -induced myometrial mitochondria membrane depolarization.Perhaps these results could explain at least one of the reasons of the low level of Са 2+ accumulation in mitochondria in Mg 2+ -containing medium -Ca 2+induced depolarization resulted in inactivation of potential-sensitive Ca 2+ uniporter, that is the main Ca 2+ -transporting system in the mitochondria.
Thus, preincubation of myometrial mitochondria in Mg 2+ -containing medium resulted in low endogenous [Ca 2+ ] m , subsequent addition of 100 µM Ca 2+ caused a significant increase of free calcium concentration in the mitochondrial matrix but low level of total Ca 2+ accumulation, so -low functional activity.At the same time, preincubation of myometrial mitochondria in the Mg 2+ ,ATP-containing medium resulted in relatively high endogenous [Ca 2+ ] m , subsequent 100 µM Ca 2+ addition caused relatively low increase of free calcium concentration in the matrix but high level of total Ca 2+ accumulation meaning high functional activity.Exogenous Ca 2+ addition to the myometrial mitochondria incubated in the Mg 2+ ,ATP-containing medium caused smaller increase of normalized fluorescence compare to the one in Mg 2+ -containing medium.Thus, we concluded that (1) low endogenous [Ca 2+ ] m was not correlated with high functional activity; (2) exogenous Ca 2+ addition resulted in approximately the same level of [Ca 2+ ] m ; (3) low level of Fluo-4 normalized fluorescence correlated with high functional activity.
The possibility exist that our conclusions are tissue-specific and not working on others.Therefore, the next series of experiments were conducted on the liver mitochondria.We have tested kinetic of Са 2+ accumulation in liver mitochondria that were incubated in medium containing respiratory substrates such as succinate, α-ketoglutarate and pyruvate (5 mM).As shown in Fig. 5 addition of Са 2+ caused an increase of Fluo-4 normalized fluorescence with the lowest effect in the presence of succinate and the highest with pyruvate.
Next we studied the effect of the pyruvate, α-ketoglutarate and succinate oxidation on the liver mitochondria respiration rates (Fig. 6).
It was shown that in S 4 state at succinate oxidation rate of oxygen consumption in the liver mitochondria was 17.8 (ng-at.О 2 /(mg protein•min)), it is at 36.3% (p < 0.01, n = 4) higher compared with rate of oxygen consumption in mitochondria at the case of pyruvate oxidation (11.3 ng-at.О 2 /(mg protein•min) and 33.0%(p < 0.01) above regarding the indicators obtained at α-ketoglutarate oxidation (11.9 ng-at.О 2 / (mg protein•min)).
In S 3 state rates of oxygen consumption in the liver mitochondria with succinate, α-ketoglutarate and pyruvate oxidation were 20.4,14.7 and 16.8 ng-at.О 2 /(mg protein•min), respectively.That is, markers of mitochondria respiration in the presence of succinate in the medium were higher on 27.9% (p < 0.01) and 17.7% (p < 0.01) for the oxidation of pyruvate and α-ketoglutarate, respectively.
After depletion of exogenous ADP equilibrium state S 4 ATP was mounted.In this condition the rates of mitochondrial respiration were also higher at the presence of succinate compared to the NADdependent substrates -pyruvate and α-ketoglutarate, to 37.5% (p < 0.01) and 25.3% (p < 0.05) (succinate -14.9, pyruvate -9.34, and α-ketoglutarate -11.1).2+ -and Mg 2+ ,atp-containing medium.preincubation was followed by 3 mM atp addition to the Mg 2+ -containing medium.ca 2+ accumulation was started by 10 µM Са 2+ addition to both tubes.time of incubation -5 min (mean ± SeM, n = 5, P < 0.0001).Experimental model − a suspension of myometrium myocytes treated with digitonin (0.01%).total level of ca 2+ accumulation in mitochondria was monitored using 45 ca 2+ as radioactive tracer We also observed different time of ADP phosphorylation depending on oxidation substrate: succinate -155 s, pyruvate -266 s and α-ketoglutarate -183 s.

Fig. 4. the total level of ca 2+ accumulation in myometrium mitochondria after 5 minute preincubation of cells in Mg
Analysis of the data obtained on isolated liver mitochondria suggests that higher rate of oxygen consumption (took place at oxidation of succinate) is accompanied by the lower changes of Fluo-4 norfluorescence in response to exogenous Са 2+ addition.
It is well documented that a key signaling messenger that is able to transduce life or death signals to mitochondria is intracellular Ca 2+ [22][23][24].It was shown that low level of Са 2+ in the mitochondrial matrix provides the normal functioning of these organelles [5,8,9].In this study we have examined the relationship between [Са 2+ ] m and mitochondrial function (myometrium and liver).We showed that concentration of ionized Са in the myometrium and liver mitochondrial matrix depends on incubation medium composition.Specifically the presence or absence of ATP in the incubation medium had a profound effect on rat myometrium [Са 2+ ] m .For instance, incubation of myometrial mitochondria in Mg 2+ -containing medium resulted in lower [Са 2+ ] m than in Mg 2+ ,ATP-containing medium.Ca 2+ addition to the incubation medium yielded in the same [Са 2+ ] m but the different total Ca 2+ accumulation levels.We also found that oxidation of different substrates in rat liver mitochondria cause changes of [Са 2+ ] m .
[Са 2+ ] m is an important parameter of mitochondria but is not always correlated with the functional activity of this organelle.At the same time it was shown that the lower value of Fluo-4 normalized fluo rescence changes in response to the exogenous Са 2+ addition was correlated with the higher functional activity of these organelles.Our findings suggest that normalized fluorescence changes in response to the exogenous Ca 2+  К л ю ч е в ы е с л о в а: митохондрия, концентрация ионизированного Са, общая аккумуляция Ca 2+ , миометрий, печень.