KINETIC PARAMETERS OF RESPIRATION IN RAT PERMEABILIZED HEPATOCYTES UPON Са2+ IN VARIOUS CONCENTRATIONS..

The dependence of permeabilized rat hepatocytes respiration rate on oxidation sub­ strates concentration at presence 0.1 and 1 μM Са2+ in the medium and amino sulfonic acid – taurine – effect on this subordination was studied. Experimental animals were in­ jected with taurine (40 mg/kg of weight) for 28 days and control rats were injected with equal volume of water. Liver cells were permeabilized with digitonin (20 μg per 1 million cells). Respiration rate was determined by polarographic method using Clark’s electrode, upon either succinate or pyruvate with malate oxidation. Hill equation parameters were calculated applying {v; v/[S]h} coordinates. The kinetic dependence of respiration upon succinate and pyruvate oxidation is well described by Hill equation in the mediums with studied concentration of Са2+ in control and under taurine action. Hill coefficient h and semi activation constant K0.5 for succinate, at rotenone presence, are not changed in cont­ rol animals after increase of Са2+ сoncentration from 0.1 to 1 μM. Maximal velocity Vmax was slightly increased under such conditions. Kinetic parameters of respiration upon suc­ cinate oxidation and both Са2+ concentration, are not changed significantly under pro­ longed taurine injection. There is more essential difference between kinetic parameters of pyruvate­stimulated respiration at various Ca2+ concentrations. Thus, in control, Vmax is 1.5 times lower, and K0.5 – 10.7 times major at 0.1 μM Са2+ in comparison with 1 μM Са2+. Hill coefficient becomes less than 1 at both Са2+concentration. As a result of taurine injection, Vmax is 1.6 times less at low Са2+ and K0.5 is higher, but only 6 times. Hill coefficient of this process increases at 0.1 μM Са2+ and is 1.12, upon taurine impact. Substrate inhibition, inherent to the dependence of respiration rate on pyruvate concentration within the me­ dium with 1 μM Са2+, starts developing, under taurine effect, at higher concentration of this substrate in comparison with control (3 mM comparatively to 0.35 mM). Thus, Са2+ con­ centration rise from 0.1 to 1 μM stimulates the rat hepatocytes mitochondria respiration upon exogenous pyruvate oxidation, but not succinate. Taurine under the prolonged in vivo action does not influence succinate-stimulated rotenone-insensitive respiration and intensifies processes of pyruvate-stimulated oxygen consumption in hepatocytes.


INTRODUCTION
Hepatocytes play a vital role in providing of the entire organism functioning, be cause they are involved in the processes of endogenous and exogenous compounds detoxication, blood proteins synthesis and secretion, choleresis, glucose and tempera ture homeostasis maintenance etc.
Calcium cations have the unique properties and universal ability of diverse signals transduction on the cellular level. Increase in Са 2+ concentration in the cytosol activates various cell processes in different tissues or organs (including liver). It may cause the ATP lack in the cytosol. Living cells need the balance between producing and using of ATP for homeostasis maintenance. It requires the effective coordination of the cytosolic ATPdependent processes velocity with the mitochondrial oxidative phosphorylation in tensity [20]. It is found out that exactly Са 2+ oscillations provide the conformity between the intramitochondrial ATP synthesis and the cell energy demand in hepatocytes [6]. Properly accumulation of Са 2+ by mitochondria serves as a signal to intensify ATP pro ducing if necessary, and transduction of this signal is based on activation of the mito chondrial Са 2+ sensitive enzymes: αketoglutarate dehydrogenase, isocitrate dehydro genase and pyruvate dehydrogenase [15].
It is postulated that a change in use of substrates of oxidation by cell modulates the mitochondrial Са 2+ accumulation processes. This assumption is based greatly on the experimental data according to which the Са 2+ accumulation process in the mitochon dria is maintained during succinate oxidation mainly [22]. Conversely, the succinate oxidation limitation and switching to the predominant NADdependent substrates using prevents the excessive Са 2+ accumulation by mitochondria and overloading of their Са 2+ removal systems. It has to provide the stability of the mitochondrial energyproduc ing function, the tissues ATP and GTP pool restoring, the organs functioning mainte nance under conditions of various physiological states. However, the molecular mecha nisms and physiological direction of this process are unclear.
Taurine -one of the most widespread amino sulfonic acids in mammals -is also involved in the maintenance of Са 2+ homeostasis and mitochondria functioning [9]. Lobo and coauthors [11], using electron microscopy with immunohistochemical marker, revealed that the highest taurine concentration in the cell is within mitochondria. It was shown that taurine caused the increase in the rate of Са 2+ uptake by the hepatocytes mitochondria already at the minimal concentration of 1 mM [17]. However, the pecu liarities of various substrates oxidation in mitochondria upon taurine action and at the different Са 2+ concentrations are not researched. That is why a part of our work was devoted to this problem.
Thus, maintenance of cell energy supply at the physiological level is a complicated regulated process dependent on qualitative and quantitative system para me ters. The majority of papers, concerning the correlation between intracellular signaling (including Са 2+ signaling) and metabolic (mitochondria respiration) pathways, are focused on the qualitative system characteristics. The works dedicated to the quantitative characteris tics, in particular to the dependence of kinetic parameters of mitochondria respiration (during different substrates oxidation) on Са 2+ concentration are far in minority. This fact has determined the aim of our study.

MATERIALS AND METHODS
Experiments were carried out using male Wistar rats of 180-220 g weighit. All ma nipulations with animals were conducted according to "European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes" and the Law of Ukraine "On protection of animals from cruelty". Rats were kept under the statio nary vivarium conditions at constant temperature and basic allowance. Ex perimental animals were injected with taurine solution (40 mg/kg of weight) through a tube intragastrically for 28 days and control rats were injected with water. Animals were narcotized with diethyl ether after that period, then they were decapitated, abdominal dissection was made and a liver was eliminated rapidly. Decapitation procedure was executed in a laboratory from the other rats separately.
Hepatocytes were isolated by twostaged Seglen method [23]. Liver was withdrawn immediately after decapitation and perfused with noncalcium extracellular solution to wash out the blood. The next stage was recirculating perfusion of the liver with collage nase solution for 10-15 minutes. The organ was consistently rinsed with the basic ex tracellular solution to wash out collagenase after the collagen matrix destroying. All procedures listed above were carried out at 37 °С. The solutions flux velocity was sta ble. Collagenase was dissolved ex tempore because of its autoproteolysis ability.
Liver was disposed within basic extracellular solution after perfusion and hepato cytes were isolated by gentle pipetting. Suspension was filtrated through the nylon filter to exclude clots of the cells and three times centrifuged at 50 g to remove the metabo lites, the intercellular matrix residues and the damaged cells. Hepatocytes were dyed with 0.1 % tripan blue to examine their plasma membrane integrity. The portion of the cells with intact plasmalemma was 84-94 %. Cells were counted using hemocytometer.
A polarographic method was applied to estimate the intact and permeabilized he patocytes respiration. The diffuse current value was registered using a unit, constructed on a base of Clark's electrode, oxygen monitor YSI 5300, multimeter UT60E, magnetic stirrer for suspension and glass enclosed chamber with volume of 1.6 ml, connected to a thermostat.
Hepatocytes were permeabilized with digitonin for detailed researching of the respi ration and oxidative phosphorylation. This procedure was used because transport rate of exogenous substrates through intact plasma membrane is insufficient for their effective involving in the oxidative processes in real time. Isolated cells were centrifuged and ex tracellular medium was replaced with the one of solutions for permeabilized hepatocytes incubation, containing various Ca 2+ concentrations. Then digitonin was added (20 μg/ million cells within 1 ml of suspension) and the cells were incubated for 10 min at 37 °С.
Oxygen consumption rate by permeabilized liver cells was registered at 37 °С using succinate (0.1-5 mM) or pyruvate (0.01-5 mM) as substrate of oxidation. Substrates were added into the chamber in increasing concentrations. Respiration was stimulated by ADP (750 µM) before application of substrates. Rotenone (10 µM) was applied to selectively block respiratory chain complex I.
Intracellular solution was of such composition, mM: КCl -90.0, NaCl -15.0, MgCl 2 -1.0, KH 2 PO 4 -2, EGTA -0.5, HEPES -10.0; pH 7.2. Composition of the solution basical ly meets the ion composition of the hepatocytes intracellular medium [7]. Media with 0.1 and 1 µM Ca 2+ concentration were made on the basis of the previous one, using Са 2+ EGTA buffers. Those Ca 2+ concentrations are chosen, since our previous studies (unpub lished data) show, that the most significant differences in mitochondria functional abili ty to response the substrates, ADP and DNP influence appear upon the transition from 0.1 to 1 µM Ca 2+ within the medium. These means are close to cytosolic Ca 2+ content under con ditions of resting (0.1 µM Ca 2+ ) and activated (1 µM Ca 2+ ) states of the liver cells [1], in par ticular, physiological agonist (vasopressin) causes the rise of Ca 2+ concentration from 0.2 (approximately) to 1 µM [21]. Unbounded Са 2+ concentration was calculated by the Ca/ Mg/ATP/EGTA Calculator v1 programme (http://maxchelator.stanford.edu).
Statistical analyses were performed with computer application, via Microsoft Office Excel package. Coefficient of the difference adequacy between two statistical groups was determined using the Student's test [4]. Kinetic parameters of Hill equation were calculated within coordinates {v; v/[S] h } (modified Eadie-Hofstee coordinates) using a method of index h iteration. The best accordance of the achieved dependence to the experimental data was assessed by linear regression [14]. The approximation authen ticity was determined by applying F-statistics. The approximation coefficient was con sidered to be authentic if P ≤ 0.05.

Dependence of succinate-stimulated respiration rate on calcium concentration in the medium.
Kinetic analysis of the succinate oxidation in hepatocytes mitochon dria was carried out upon low temperatures [2], hypoxia [12], immobilization stress [22], and upon the various physiological states [3]. Brown has revealed that the substrate oxi dation kinetics is modified during hibernation and it provides the metabolism regulation by mitochondria [3]. Priority use of succinate compared to the other substrates was noted in most works [2,3,18,19]. Its oxidation rate is much higher in comparison with other sub strates of tricarboxylic acid cycle and the power of transmembrane potential generation is so large that supplies the maximal ADP phosphorylation intensity. Succinate is usually applied at saturating concentrations in the researches, but making clear of its oxidation kinetics with different substrate concentrations gives an opportunity to reveal the interest ing regularities. In particular, in experiments with hibernating animals, higher respiration rate indexes were received upon lower succinate concentration than in condition of satu rating succinate content [3]. It is considered to maintenance of provide energy processes intensity at sufficient level during hibernation, preserve cell and mitochondria against damage and give a chance to turn into active state quickly.
Although change in cytosolic Са 2+ concentration is an important instrument of mito chondria respiration processes regulation [8,24], that does not occurred succinate oxi dation. Such conclusion is a result of fact that succinate dehydrogenase is not Са 2+ dependent enzyme [18]. That is why, the first task in the approbation of a new protocolstudy in situ of mitochondria respiration kinetics, was to check a dependence of the parameters of that kinetics upon succinate oxidation and Са 2+ concentration. As a result of studying the permeabilized hepatocytes oxygen consumption in me dia containing various Са 2+ concentrations, we have revealed that: kinetics of the respi ration processes upon exogenous succinate oxidation in those cells may be well de scribed by Hill equation. Moreover, such kinetic parameters as Hill coefficient h and maximal velocity V max , are not modified with Са 2+ concentration in the medium from 0.1 to 1 µM increasing. While K 0.5 for succinate is 2.1 times less at Са 2+ concentration 1 µM (1.05 ± 0.06 mM) compared to 0.1 μM Ca 2+ (2.25 ± 0.16 mM, n = 4; Fig. 1). Such in crease of affinity to substrate shows the existence of some dependence between oxida tive processes velocity in hepatocytes mitochondria upon succinate oxidation and cyto solic Са 2+ concentration. However there is a probability of activation by Са 2+ cations (either directly or indirectly) of the other TCA cycle enzymes: αketoglutarate dehydro genase, isocitrate dehydrogenase and pyruvate dehydrogenase [15], under such experimental conditions. Elimination of effect of these enzymes is necessary for study of kinetic dependence. It can be achieved, for example, by inhibiting the respiratory chain components which are not involved in succinate oxidation. This is the reason to add rotenone -an inhibitor of the respiratory chain complex I [5] -during the later inves tigation of succinate oxidation kinetic parameters. The endogenous respiration rate of the control cells is not changed by rotenone within medium, containing 0.1 µM of Ca 2+ (0.09 ± 0.03 -before and 0.07 ± 0.02 nmol О 2 / (s × million cells) after rotenone apply ing). From other hand, rotenone lowers respiration rate in medium with higher Ca 2+ concentration 1 µM to 0.09 ± 0.02 nmol О 2 / (s × million cells), i.e. by 33 ± 9 % (n = 5, P ≤ 0.05). Thus, Ca 2+ concentration increase in nonrotenone experiments causes a reparti tion of the substrates oxidation, in fact, the limitation of succinate use in the isolated hepatocytes. Probably, it could be a mechanism of the mitochondria protection against Ca 2+ overload and it is showed in Saakyan's study [22].
Succinate increases the hepatocytes oxygen consumption velocity in dose depen dent manner, under conditions of inhibition of the respiratory chain complex І. Intensifi cation of oxygen consumption by liver cells at 0.1-3 mM succinate is ascertained. This process is described by the Hill equation quite strictly. K 0.5 and h are not dependent on Са 2+ concentration in the medium, and form 0.47-0.49 mM and 0.91, respectively. V max is characterised by dependence on Са 2+ concentration: it is 0.98 and 1.15 nmol О 2 / (s × million cells) at 0.1 and 1 µM, respectively. In both cases, some respiration rate de creasing is observed at 5 mM succinate. It is a result of substrate inhibition, obviously (see Fig. 1).
Received kinetic parameters of hepatocytes mitochondria respiration during suc cinate oxidation are somewhat different from ones in the pancreas cells. In particular, Hill coefficient h of permeabilized pancreas cells succinatestimulated respiration is sig nificantly higher, than 1 (1.43 and 1.40 at 0.1 and 1 µM Са 2+ [13]), but it is also practi cally not dependent on Са 2+ content in the medium. Such a difference is caused, prob ably, by the development of the substrate inhibition of succinatestimulated respiration in hepatocytes (that is why the virtual Hill coefficient becomes quite lower, than 1), but not in pancreas cells. The existence of such "negative cooperation" during succinate oxidation, as well as the substrate inhibition, indicates the presence of a complex sys tem of the feedbacks directed at oxidative processes that decrease upon the high rate of the mitochondria membrane potential.
K 0,5 amount of hepatocytes succinatestimulated respiration though has revealed two times less than in pancreas cells [13], but is also quite high and does not depend on Са 2+ concentration in the medium. Obviously, it is caused by low affinity of either suc cinate dehydrogenase or mitochondrial transporters to the exogenous succinate in he patocytes (as well as in pancreas cells [13]). Са 2+ cations in researched concentrations do not have significant influence on the metabolic flux through succinate oxidation sys tem consisting of the units for succinate transporting, succinate dehydrogenase, and respiratory chain complexes III and IV.  Finally V max index of hepatocytes succinatestimulated respiration is two times high er than this one, registered in pancreas cells upon the same experimental conditions [13]. It is taking into consideration the increased oxidative ability of the liver tissue.
Pyruvate-stimulated respiration rate dependence on calcium concentration in medium. Cell energy supply processes have a critical significance in adaptation of various living tissues to the different loads. Oxidation substrates with Са 2+ regulated use, belong to the factors, providing maximal adaptation accordingly to the demands. Ponsot and coauthors [19] have carried out studies using various rat muscles (gastroc nemius, soleus and heart muscle) with estimation of ADPstimulated respiration upon escalating pyruvate, pyruvate with malate, glutamate with malate, palmitoylcarnitine concentrations. They made a conclusion about qualitative diffe rences of mitochondria metabolic pathways dependently on the cells functions. In particular, mitochondria of glycolytic muscle fibers are adapted to the maintenance of the necessary oxidationreduction state of these cells, and heart mitochondria have deve loped an important ability to use the fatty acids. The key factor of the mitochondrial energy supply fitting to the intact neurons demand is also cytosolic Ca 2+ [8]. Ca 2+ accumulation by mitochondria proceeds at its high concentrations (> 600 nM) and it increases the level of the intrami tochondrial dehydrogenases activity. Ca 2+ induced escalation of oxygen consumption intensity in neurons mitochondria at state S 3 dimini shes, dependently on oxidation sub strate, in a such sequence: glutamate > αketoglu tarate > αglycerophosphate > pyru vate [8].
Adding of the exogenous pyruvate on the malate background (0.01-5 mM) leads to the dose dependent increasing of oxygen consumption velocity as at 0.1, so at 1 μM Са 2+ in the medium. K 0.5 is much less, than for succinate, in both cases. It is no wonder, because not only the endogenous pyruvate, but also that one, which arrives as lactate with the blood from glycolytic tissues, proceeds oxidation in liver [10]. Simultaneously, the V max amount of pyruvate-stimulated respiration is significantly less.
There is a significant difference between the kinetic parameters of pyruvate oxida tion processes at various Са 2+ concentrations. Thus, K 0,5 is 0.16 ± 0.01 mM at 0.1 µM Са 2+ and 0.015 ± 0.001 mM at 1 µM Са 2+ , i.e. difference amounts 10.7 times. And V max is 1.5 times larger at the higher Ca 2+ content, in comparison with Са 2+ concentration of 0.1 µM (0.4 ± 0.008 and 0.27 ± 0.01 nmol О 2 / (s × million cells) respectively, n = 4). It indicates the greater system affinity to pyruvate and more intensive its oxidation at higher Са 2+ concentration in permeabilized hepatocytes. It is harmonized with a fact that, in particular, pyruvate dehydrogenase is one of Са 2+ sensitive mitochondrial en zymes, established using only isolated organelles. Similar variations of K 0,5 and V max of pyruvate-stimulated respiration, though at some different range of Са 2+ concentration (0.1 and 0.5 µM), are inherent for pancreacytes also [13].
It was cleared up that the intensity increasing of oxygen consumption by hepato cytes at Са 2+ concentration 1 µM proceeds upon only in the range from 0.01 to 0.2 mM pyruvate application. The respiration rate indices begin to diminish at the later rise of substrate content (0.35-5 mM), i.e. substrate inhibition develops. This fact can be ex plained by the existence of homeostatic mechanisms of the reverse negative impact which decrease oxygen consumption upon reaching the mitochondria maximal oxida tive activity (that proceeds at saturating substrate concentrations), and, thus, initiate the inhibition process of the later membrane potential increasing in these organelles. Hill coefficient partly evidence of the previous hypothesis, which is less is than 1 at both Са 2+ concentration in medium. It indicates the existence of the negative feedbacks with in a system. Such well expressed substrate inhibition of pyruvatestimulated respiration (and succinatestimulated as well) is not inherent for pancreacytes in the investigated sub strate concentrations diapason [13]. It is due, quite possibly, the greater oxidative ability of liver tissue and, thus, the presence of specific protection systems.

Respiration rate dependence on calcium concentration hin the medium in the permeabilized hepatocytes of rats injected with taurine.
Endogenous metabolites, such as taurine, are also involved in energy and Са 2+ metabolism regulation. Taurine in vitro is known to enhance Са 2+ accumulation by the isolated mitochondria of rat liver [17]. It is accompanied with the mitochondrial respiration rate increasing upon succinate oxidation [16].
We have investigated the taurine in vivo influence on oxygen consumption by iso lated hepatocytes dependently on Ca 2+ and oxidation substrates concentration. Pro longed taurine injection to the rats does not modify the endogenous respiration intensity in permeabilized hepatocytes of these animals. Oxygen consumption velocity in liver cells decreases as consequence of rotenone adding to the polarographic chamber upon the endogenous substrates oxidation. Such reduction occurs in the medium containing either 0.1 or 1 µM Са 2+ -for 47 ± 8 % (n = 4, P ≤ 0.05) and 26 ± 7 % (n = 4, P ≤ 0.01) respectively. Since rotenone does not modify the endogenous respiration rate at 0.1 µM  Са 2+ in control group, taurine, nevertheless, enhances inflow of NAD-dependent endog enous substrates. Taurine effect is smoothed over at higher Са 2+ concentration. It cor roborates the assumption that its impact is realized due to the intensification of Са 2+ accumulation within mitochondria. At rotenone presence V max index of succcinatestimulated respiration increases upon taurine influence, as well as in control animals, with Ca 2+ concentration rise (from 0.83 ± 0.03 (at 0.1 µM Ca 2+ ) to 0.91 ± 0.06 (at 1 µM Ca 2+ ) nmol О 2 / (s × million cells). Whereas K 0,5 conversely diminishes upon these conditions (from 0.52 to 0.41 mM), un like the control. This reduction is nonessential and can be neglected. At the same time, the Hill coefficient h stays unchangeable in control rats also. The V max amounts them selves after prolonged taurine injection are lower, in comparison with control, at both Ca 2+ concentrations. Development of "substrate inhibition" is also observed at the high est succinate concentration (5 mM) in animals receiving taurine (Fig. 3). No significant changes in hepatocytes respiration kinetics upon succinate oxidation after prolonged taurine injection were registered, as well as a dependence of this kinetics on Ca 2+ con centration. V max index of pyruvatestimulated respiration in rats, receiving taurine for a long time, 1.6 times increases with Са 2+ concentration rise in the medium from 0.1 to 1 µM (Fig. 4), i.e. practically the same as in control animals (see Fig. 2). K 0.5 , conversely, dimin ishes 6 times -essentially less than in control -first of all due to its reduction already at 0.1 µM Са 2+ in the medium under taurine effect. Virtual Hill coefficient is significantly  higher at 0.1 µM Са 2+ in experimental animals in comparison with control ones, and has a value of 1.12. That is negative cooperativity, inherent to the respiration rate depen dence on pyruvate concentration in control, disappears or even is replaced with the positive cooperativity upon a prolonged taurine effect. Са 2+ concentration increase with in mitochondria matrix underlies such transformation, because this transformation is eliminated at 1 µM Са 2+ in the medium. Generally, the appearance of the respiration rate positive cooperativity with pyruvate concentration and some decrease of K 0.5 at 0.1 µM Са 2+ in the medium have to be interpreted as an evidence of pyruvate oxidation intensi fication upon prolonged taurine effect. It occurs despite the calculated V max amounts are lower in experimental animals at both Са 2+ concentrations in the medium. Besides, substrate inhibition inherent to the respiration rate dependence on pyru vate concentration at 1 µM Са 2+ in the medium, starts to develop at one fold higher than in control, this substrate concentration (3 mM in comparison with 0.35 mM), under a prolonged taurine effect. Such elimination of substrate inhibition by taurine is an evi dence of pyruvate oxidation intensification.
It has to be noted, that dispersion (deviations range) of pyruvatestimulated respira tion rate (not succinate-stimulated) is larger upon taurine influence, in comparison with control rats. Two experimental points, characterizing the respiration rate upon the lowest pyruvate concentration (0.01 and 0.05 mM) at Са 2+ concentration 0.1 µM, are not situ ated on a curve computed with use of Hill equation. There is only one such point in con trol -0.01 mM pyruvate. This "basic" pyruvate concentration dislocation at 0.1 µM Са 2+ 0. to the right caused by prolonged injection of taurine, is similar to substrate inhibition displacement at 1 μM Са 2+ . The curves reflecting pyruvate oxidation kinetics upon tau rine effect come closer to each other at both Са 2+ concentrations (it is well observed within semilogarithmic coordinates). It also indicates indirectly a change of Са 2+ depen dent component of pyruvatestimulated oxygen consumption in hepatocytes.

CONCLUSION
A prolonged in vivo effect of taurine, does not influence the succinate-stimulated rotenone-insensitive respiration and intensifies the processes of pyruvate-stimulated oxygen consumption in rat hepatocytes. A change in the parameters of Са 2+ regulation of mitochondrial respiration underlies such intensification. It causes a reduction in K 0.5 and appearance of positive cooperativity in pyruvate oxidation at 0.1 µM Са 2+ in the medium and substrate inhibition decrease at 1 µM Са 2+ .