1. Introduction

Cell hydration (water content) is a fundamental parameter determining functional activity of cell. It is realized by changing the activity of intracellular macromolecules via “folding-unfolding” mechanism [1] as well as by cell surface-dependent changes of the number of functionally active protein molecules in plasma membrane which has enzymes, receptors and ion channels forming properties [2-4]. Cell dehydration is one of the essential hallmarks for aging [5-10]. Therefore, the dysfunction of metabolic regulation of cell hydration could serve as one of the primary mechanisms for generation of cell pathology, including aging.

Although the important physiological role of water in cell functional activity is widely recognized, its messenger role in generation of various diseases [11-14] has not been clarified yet. Therefore, the elucidation of the detailed mechanism responsible for regulation of cell hydration could help understand the role of cell hydration in generation of age-related medical disorders, including heart muscle failure.

As cell membrane is highly permeable for water, cell hydration is controlled by metabolic activity of cell. The latter is realized by the following two pathways: a) transporting mechanism of membrane controlling the number of osmotically active particles in cytoplasm, b) intracellular signaling system controlling absorption properties of cytoplasm and generation of endogenous water molecules in cytoplasm during oxidative phosphorylation. However, the dysfunction of which aforementioned pathways is the primary mechanism for generation of age-dependent heart muscle dehydration is not clear yet.

As heart muscle has pacemaker contractility, it has high rate of adenosine triphosphate (ATP) utilization leading to stimulation of ATP synthesis, which is accompanied by the release of endogenous H₂O in cytoplasm. It is known that 42 water molecules are released in cytoplasm as a result of one molecule of glucose oxidation [15]. Therefore, the release of endogenous H₂O could have an essential role in metabolic control of cell hydration and its dysfunction could be one of the mechanisms responsible for heart muscle failure.

The next fundamental parameter of cell, which is determinant in heart muscle contractility is intracellular calcium ions ([Ca²⁺]_i). It is well established that aging leads to the increase of [Ca²⁺]_i [16-20]. However, the detailed mechanism of close-talking interaction between cell dehydration and the increase of [Ca²⁺]_i in aging has not been elucidated yet.

The dysfunction of Na⁺/K⁺-pump, which is a common consequence of aging, has a key role in metabolic regulation of both cardiomyocyte hydration and [Ca²⁺]_i homeostasis. Na⁺/K⁺-pump, being a high metabolic energy (ATP) utilizing mechanism and working with high intensity in cardiomyocytes, has a great intracellular signaling role in controlling Ca²⁺ sorption properties of intracellular structure as well as in generation of endogenous H₂O in cytoplasm. Therefore, Na⁺/K⁺-pump could be considered not only as an ion transporting mechanism but also as a powerful intracellular signaling system controlling cell hydration and [Ca²⁺]_i in myocytes.

At present, it is well established that Na⁺/K⁺-ATPase (working molecules of Na⁺/K⁺-pump) in myocyte membrane has three catalytic isoforms (α₁, α₂, α₃) with different affinities to ouabain (specific inhibitor for Na⁺/K⁺-ATPase) and with different functional activities [21-25]. However, the individual roles of these isoforms in regulation of myocyte hydration and [Ca²⁺]_i require further elucidation.

It is established that α₁ (low affinity) and α₂ (middle affinity) isoforms are involved in ion transporting processes, while α₃ (high affinity) has only intracellular signaling function [22, 25-28]. Although α₃ isoform is not involved in the function of transporting Na⁺ and K⁺, it has a crucial role in regulation of Na⁺/Ca²⁺ exchange [21, 25, 29-33]. By our previous study it has been shown that α₃ isoform-dependent myocyte hydration and its affinity to ouabain have more pronounced age-dependent dysfunctional character than those in case of α₁ and α₂ [34]. However, the role of α₃ isoform in determination of age-dependent myocyte dehydration and increase of [Ca²⁺]_i is not clear yet. It is suggested that the elucidation of the mechanism(s) through which α₃ isoform regulates myocyte dehydration and increase of [Ca²⁺]_i will allow us to understand the role of age-dependent dysfunction of α₃ isoform in heart muscle failure. For this purpose, in present work the following age-dependent studies have been performed both in vivo and in vitro experiments: determination of dose-dependent [³H]-ouabain binding with cell membrane and muscle hydration, measurement of ⁴⁵Ca²⁺ uptake and efflux.

2. Materials and Methods

2.1. Animals

Studies were carried out on young (6 weeks) and old (12 months) male Wistar albino rats of mass 50-60 g and 215-230 g, respectively. Animals were kept in a specific pathogen-free animal room. In present experiments the rats (N=470) were housed under optimum conditions with 12 h light/dark cycle at 22 ± 2°C and were given a sterilized diet and water ad libitum.

All procedures performed on animals were carried out following the protocols approved by the Animal Care and Use Committee of Life Sciences International Postgraduate Educational Center (Yerevan, Armenia).

2.2. Chemicals

Tyrode’s physiological solution (PS) with the following composition was used (in mM): 137 NaCl, 5.4 KCl, 1.8 CaCl₂, 1.05 MgCl₂, 5 C₆H₁₂O₆, 11.9 NaHCO₃, 0.42 NaH₂PO₄ and adjusted to pH=7.4. A radiometer PHM-22r (Radiometer, Copenhagen, Denmark) was used for pH measurements.

In order to receive PS with 50% [Na⁺], 68.5mM of NaCl was replaced with 2M of non-metabolizing substance mannitol dissolved in Tyrode’s PS for maintaining the osmolarity of solution.

All chemicals were obtained from “Medisar” Industrial Chemical Importation Company (Yerevan, Armenia).

[³H]-ouabain (specific activity 25.34 Ci/mM) and cold (non radioactive) ouabain were obtained from Perkin Elmer (Waltham, MA, USA). All doses of ouabain were prepared on the basis of the physiological solution and used for intraperitoneal injection (in vivo) or heart muscle tissue incubation (in vitro).

⁴⁵Ca²⁺ (with specific activity of 40 mCi/ml) was obtained from Perkin Elmer (Waltham, MA, USA) and was used for in vivo and in vitro experiments.

The study of ⁴⁵Ca²⁺ uptake and efflux in heart muscle tissue was performed on young and old animals. For this purpose in Tyrode’s physiological solution (PS) containing 1.8 mM CaCl₂, 0.0115 mM was substituted by labeled ⁴⁵CaCl₂. In the experiments that were aimed at studying ⁴⁵Ca²⁺ uptake and efflux, all concentrations of ouabain were prepared with this radioactive PS.

2.3. Tissue Preparation

To avoid an anesthetic effect on initial functional state [35-36] in present experiments we preferred to use sharp freezing method [37]. Animals were immobilized by dipping their heads into liquid nitrogen (for 3-4 s) and then they were decapitated. After decapitation of animals, their hearts were immediately placed in the tube with PS, and then six pieces were taken from each tested heart muscle with 50-60 mg wet mass (w. m.) per piece (time interval between these two procedures was not more than 30 sec).

In vivo experiments, 30 min before decapitation animals were intraperitoneally injected by investigated solutions. After this they were immobilized and decapitated.

In vitro experiments animals were firstly immobilized and decapitated. Their heart muscle tissues were dissected in the same manner and then incubated in investigated solutions.

For removing surface-adherent and extra-cellular tracers (³[H]-ouabain and ⁴⁵Ca²⁺), the samples were washed three times with PS in all experiments. After that the wet mass (w.m.) of samples was determined (time duration for these processes was less than 1 min for 30 samples). Similar procedures were performed with the samples of control and experimental groups.

2.4. Definition of Heart Muscle’s Water Content

Determination of the water content (hydration) of heart muscle tissues was performed by traditional “tissue drying” method [38]. After determination of wet mass, the samples were dried in thermostat (Factory of Medical Equipment, Odessa, Ukraine) during 24 h at 105°C in order to estimate water content in muscle samples. The quantity of water in 1 g of dry mass (d. m.) of tissue was derived by the following equation: (w .m. – d. m.) / d. m.

2.5. Counting of [³H]-ouabain Receptors in Membrane

Radioactive [³H]-ouabain is usually used to estimate the number of Na⁺/K⁺-pump units in membrane. It is assumed that each binding site in the membrane binds one molecule of ouabain [39]. After 30 min of [³H]-ouabain injection (in vivo experiments) the rats were decapitated and heart muscle samples were washed three times (10min-5min-5min) with normal Ringer's solution to remove surface-adherent and extra-cellular tracer. In vitro experiments after decapitation of rats the heart muscle samples were incubated in [³H]-ouabain for 30min and then washed three times (10min-5min-5min) with normal Ringer's solution. After determination of water content by the method described previously, dried tissue samples were replaced into special tubs and homogenized in 50 µl 68% HNO₃ solution. Finally, 2 ml of of Bray’s scintillation fluid was added and the radioactivity of samples was calculated as counted per minute (CPM)/mg by Wallac 1450 liquid scintillation and luminescence counter (WallacOy, Turku, Finland).

2.6. Definition of ⁴⁵Ca²⁺uptake and Efflux in Heart Muscle Tissues

⁴⁵Ca²⁺ uptake was measured in vivo as well as in vitro conditions. In vivo experiments in Tyrode’s physiological solution 0.0115 mM CaCl₂ from 1.8 mM was substituted by ⁴⁵Ca²⁺. Young and old animal groups were intraperitoneally injected with ⁴⁵Ca²⁺ (with 0.187 mCi/g radioactivity of body weight). After 30 min animals were decapitated and heart muscle samples were incubated for 30 min in PS (as a control) and PS containing different doses of ouabain. Then all samples were dried in thermostat during 24 h at 105°C.

In vitro experiments heart muscle tissue samples were incubated in 20 ml cold (7°C) K⁺-free PS containing 1.8μl ⁴⁵Ca²⁺ (as a control) or in PS containing different doses of ouabain for 30 min. Then they were dried in thermostat for 24 h at 105°C.

The study of ⁴⁵Ca²⁺efflux from preliminarily ⁴⁵Ca²⁺-enriched heart muscle tissue was performed on young and old rats. For enriching heart muscle tissues by ⁴⁵Ca²⁺ they were incubated for 1 h in 16.25 ml K⁺-free (containing 50% NaCl) physiological solution. In K⁺-free solution containing 1.8 mM CaCl_2, 0.00448 mM was replaced by labeled ⁴⁵CaCl₂. Then enriched samples were washed three times in K⁺ free solution (containing 100% NaCl and cold CaCl₂) for 10 min, 5 min and 5 min, respectively, to remove ⁴⁵Ca²⁺ from extra-cellular spaces. The samples were divided into two parts. The first 30 samples (control) were dried in thermostat for 24 h at 105°C after determination of wet mass. The second set of 30 samples was incubated for 30 min in 20 ml of PS or in solutions containing different doses of ouabain and after determination of wet mass all samples were dried in thermostat for 24 h at 105°C. After determination of dry mass, all samples homogenized in 50 µl of 68% HNO₃ solution, and the radioactivity of the samples was measured as cpm/mg dry weight. The rate of ⁴⁵Ca²⁺ efflux was calculated as the residual part of absorbed ⁴⁵Ca²⁺ for control and the experimental data by the following equation:

[control – exp.]/control.

2.7. Statistical Analysis

The mean and standard error of the heart muscle hydration index, [³H]-ouabain binding and ⁴⁵Ca²⁺ changes in different samples were calculated and the statistical probability was determined by Student's paired t-test by means of computer program Sigma Plot (Version 8.02A, San Jose, CA,USA).

The statistical probability was reflected in figures by asterisks (*). For all statistical tests P value was taken as *P < 0.05; **P < 0.01; ***P < 0.001.

3. Results

In Figure 1 dose-dependent binding of ouabain with cell membrane (A) and dose-dependent muscle tissue hydration (B) in young and old rats are presented. As can be seen on the presented curves, in heart muscle tissues of young rats α₃-(10^-11-10^-9M); α₂-(10^-9-10^-7M) and α₁- (10^-7-10^-4M) can be clearly distinguished, while, in heart muscle tissues of old rats these components were less expressed. Dose-dependent [³H]-ouabain binding with α₃ receptors had downregulation character which was more pronounced in heart muscle tissues of young animals that in old ones (Figure 1A).

Figure 1. (A)-Dose-dependent binding of [3H]-ouabain with cell membrane in heart muscle tissues of young and old rats. Error bars of all points are not detected because of being blended with points. (B)-Changes of hydration (water content) in heart muscle tissues of young and old rats poisoned by different doses of [3H]-ouabain. Data of each experiment are compared to control (ouabain-free). Each point of curves is the mean ± S.E.M of 30 samples

[³H]-ouabain binding with both α₃ and α₁ receptors had dehydration effect in young animals, while it had hydration effect in old ones (Figure 1B). From these data it can be concluded that the mechanisms responsible for the effects of ouabain on heart muscle hydration in young and old animals are different. The similarity of age-dependent effects of low and high concentrations of ouabain on muscle hydration, allows us to suggest that signaling and transporting functions of Na⁺/K⁺-ATPase can modulate muscle hydration by the same mechanism. To check this suggestion the comparative study of 10^-9M and 10^-4M ouabain effects on cell hydration in vivo and in vitro experiments was performed. In vitro experiments muscle slices were incubated in cold (7^oC) K⁺-free PS in order to depress the metabolic activity of cells, particularly Na⁺/K⁺-pump activity.

As can be seen in Figure 2, the initial levels of heart muscle tissue hydration in young animals both in vivo and in vitro conditions were significantly higher than in old animals.

Figure 2. Age-dependent change of hydration in heart muscle tissues of young and old rats. (A)-Data of in vivo experiments. Animals were injected with PS (0M ouabain) and with PS containing 10-9M or 10-4M of [3H]-ouabain. (B)-Data of in vitro experiments. Heart muscle tissues samples were incubated in cold (7oC) PS (0M ouabain) and in PS contaning10-9M or 10-4M [3H]-ouabain. Data of each pair of bars are compared with each other. Each bar indicates the mean ± S.E.M of 30 samples. (*), (**) and (***) indicate P< 0.05, P<0.01 and P<0.001, respectively

Since the dysfunction of Na⁺/K⁺-pump is a consequence of aging [10, 40-43] and the pump inactivation leads to the increase of cell hydration [44], it is predicted that 10^-4M ouabain-induced pump inhibition could cause more pronounced heart muscle tissue hydration in young animals than in old ones. However, the obtained data (Figure 2A) have shown that compared with control (ouabain-free medium), 10^-4M ouabain in young animals led to heart muscle dehydration (10%), while in old animals it had hydration (6%) effect. These data clearly indicate that age-dependent effect of 10^-4M ouabain on heart muscle tissue cannot be explained only by ouabain-induced inhibition of ion-transporting function of Na⁺/K⁺-pump in protoplasmic membrane (PM). This suggestion is confirmed by the data obtained in vitro experiment where heart muscle tissue samples were incubated in cold (7^oC) K⁺-free PS (Figure 2B).

As can be seen in Figure 2B, compared with in vivo experiments, the depression of metabolic activity of tissue in vitro experiment led to the increase of tissue hydration in young animals, while in old ones hydration was not significantly changed (Figure 2A).

As can be seen in Figure 2A, in young animals, like in case of 10^-4M ouabain (10%), intraperitoneal injection of 10^-9M ouabain led to heart muscle tissue dehydration (10%) compared with control, while in old animals 10^-9 M ouabain injection led to the increase of muscle tissue hydration (11.5%). 10^-9 M ouabain-induced hydration was more pronounced than in case of 10^-4M ouabain injection (6%).

The increase of [Ca²⁺]_i also serves as one of the essential hallmarks for aging [16, 19] and has a central role in intracellular signaling system in cells, particularly, in myocytes [45,46]. Therefore, in order to elucidate the role of [Ca²⁺]_i in determination of age-dependent muscle hydration as well as in ouabain effect on [Ca²⁺]_i, the content of ⁴⁵Ca²⁺ uptake and efflux in heart muscle tissue was the subject of our study in the next series of experiments.

The data presented in Figure 3A have shown that the initial level of ⁴⁵Ca²⁺uptake by heart muscle of young rats was much higher ( 35%) than by heart muscle tissues of old ones.

The facts that Na⁺/K⁺-pump has an age-dependent dysfunctional character and pump inactivation leads to stimulation of Na⁺/Ca²⁺ exchange in reverse mode (R Na⁺/Ca²⁺ exchange) allow us to predict that pump inhibition in young animals can lead to more pronounced activation of ⁴⁵Ca²⁺ uptake than in old ones [47, 25]. However, as it is indicated in Figure 3A, 10^-4M ouabain-induced pump inhibition led to slight increase of ⁴⁵Ca²⁺ uptake by muscle tissues of young animals, while it had inhibitory effect on ⁴⁵Ca²⁺ uptake (32.3%) by muscle tissues of old ones.

The 10^-9M ouabain in vivo experiments had strong activation effect on ⁴⁵Ca²⁺ uptake by muscle of young (20%) and old (13%) rats compared with control (ouabain-free) and 10^-4M ouabain-injected animals. However, the activation effect of 10^-9M ouabain injection on ⁴⁵Ca²⁺ uptake had age-dependent weakening character (Figure 3A). In vitro experiments10^-9M ouabain had depressing effect on⁴⁵Ca²⁺uptake by heart muscle tissue in both young (57% ) and old (15.5%) animals compared with control (Figure 3B). 10^-9M ouabain had activation effect on ⁴⁵Ca²⁺ uptake in vivo experiment, while it had inactivation effect on ⁴⁵Ca²⁺ uptake in vitro experiments in both ages of rats.

Figure 3. 45Ca2+ uptake (cpm/mg dry weight) in heart muscle tissues of young and old animals. (A)- Data of in vivo experiments after intraperitoneal injections with PS (0M ouabain) and with PS contaning 10-9M or 10-4M ouabain. (B)-Data of in vitro experiments after incubation in cold (7oC) PS (0M ouabain) and in cold PS containing 10-9M or 10-4M ouabain. In all experiments, in PS containing1.8 mM CaCl2, 0.00448 mM was replaced by labeled 45CaCl2. Each bar indicates the mean ± S.E.M. of 30 samples. Data of each pair of bars are compared with each other. (***) indicates P<0.001

It is known that Ca²⁺ uptake by cells is realized through agonist-potential-activated ionic channels and R Na⁺/Ca²⁺ exchange. To check the contribution of R Na⁺/Ca²⁺ exchange in age-dependent changes of ⁴⁵Ca²⁺ uptake and hydration of heart muscle tissue, the effects of decrease of [Na⁺] PS on ⁴⁵Ca²⁺uptake and tissue hydration were studied in vitro experiments. The fact that the decrease of extracellular Na⁺ ([Na⁺]_o) leads to stimulation of R Na⁺/Ca²⁺ exchange was shown in the experiments performed on internally dialyzed squid axon [47]. However, as it is shown in Figure 4, in vitro experiments the decrease of 50% [Na⁺] in PS led to activation of ⁴⁵Ca²⁺ uptake by heart muscle only in old rats (6.4%), while in young rats it had inactivation effect on ⁴⁵Ca²⁺uptake (27.7%) (Figure 4A). In both ages of animals PS with 50% [Na⁺] led to muscle dehydration. This effect was more pronounced in young (28.3%) animals than in old (13.2%) ones (Figure 4B).

Figure 4. Age-dependent 45Ca2+ uptake and hydration in heart muscle tissues incubated in PS containing 100% [Na+] and 50% [Na+]. (A) - 45Ca2+ uptake in heart muscle tissue incubated in PS containing 100% [Na+] and in PS containing 50% [Na+]. (B) - Mean values of hydration in heart muscle tissue incubated in PS containing 100% [Na+] and in PS containing 50% [Na+]. In all experiments, in PS containing1.8 mM CaCl2, 0.00448 mM was replaced by labeled 45CaCl2. Each bar indicates the mean ± S.E.M. of 30 samples. (***) indicates P<0.001

As can be seen in Figure 5, 50% [Na+] PS–induced muscle dehydration both in young and old animals was not changed upon the effects of 10^-4M and 10^-9M ouabain concentrations (Figure 5A, C), although they had inhibitory effect on ⁴⁵Ca²⁺ uptake (Figure 5B,D). Furthermore, 10^-9M ouabain in young animals had less (50.1%) inactivation effect on ⁴⁵Ca²⁺ uptake than 10^-4M ouabain (63.7%), while in old animals it had more depressing effect (36.6%) on ⁴⁵Ca²⁺ uptake than 10^-4M ouabain (29.3%).

Figure 5. Age-dependent hydration and 45Ca2+ uptake in heart muscle tissues incubated in PS solutions containing 100% [Na+] and 50% [Na+]. (A, C)-Changes of hydration in heart muscle tissues of young and old rats incubated in 100%, 50% [Na+]o PS (control), and incubated in100% [Na+] PS or 50% [Na+] PS containing 10-9M and 10-4Mconcentrations of non-labeled ouabain. (B, D)- 45Ca2+ uptake in heart muscle tissues of young and old rats incubated in 100% [Na+] PS and 50% [Na+] PS (control groups), and incubated in 100% [Na+] PS or 50% [Na+] PS containing 10-9M and 10-4M concentrations of non-labeled ouabain. In all experiments, in PS containing1.8 mM CaCl2, 0.00448 mM was replaced by labeled 45CaCl2. In all experiments the osmolarity of solutions was the same. Each bar indicates the mean ± S.E.M of 30 samples. (*), (**) and (***) indicate P< 0.05, P<0.01 and P<0.001, respectively

In vitro experiments PS with 50% [Na⁺] both in young and old animals had the same dehydration effect on heart muscle compared with 100% [Na⁺] PS. The same dehydration effect was observed at 50% [Na⁺] PS upon different concentrations of ouabain. These data allow us to consider that this kind of dehydration is a result of high [Ca²⁺]_i-induced contraction of myocytes. Therefore, in order to elucidate the age-dependent effect of 50% [Na⁺] PS on muscle hydration and [³H]-binding, the effects of intraperitoneal injection of 50% [Na⁺] PS on dose-dependent ouabain-induced muscle hydration and [³H]-binding were studied in the next series of experiments.

The data presented in Figure 6 indicate that the decrease of [Na⁺]_o in vivo experiments led to muscle tissue hydration both in young and old rats. However, dose-dependent effect of ouabain on muscle hydration had age-dependent character; in young animals <10^-9M ouabain had hydration (except 10^-10M), while higher concentrations of ouabain had dehydration effects on heart muscle. In old animals all concentrations of ouabain had dehydration effect on muscle tissue (Figure 6A, C).

Figure 6. Dose-dependent [3H]-ouabain effect on heart muscle hydration and binding of [3H]-ouabain with cell membrane in heart muscle tissues of young and old rats injected with 100% [Na+]and 50% [Na+]PS. (A, C)-Changes of hydration in heart muscle tissues of young and old rats after injection with 100% [Na+]and 50% [Na+]PS. (B, D) -Dose-dependent [3H]-ouabain binding with cell membrane in heart muscle tissues of young and old rats after injection with 100% [Na+]and 50% [Na+]PS. In all experiments the osmolarity of solutions was the same. Each point of curves is the mean ± S.E.of 30 samples. (*), (**) and (***) indicate P< 0.05, P<0.01 and P<0.001, respectively

Injection with 50% [Na⁺] PS in muscle tissues of both ages of animals had depression effect on dose-dependent [³H]-ouabain binding with cell membrane (Figure 6B,D). Furthermore, injection of 50% [Na⁺] PS led to depression of downregulation of [³H]-ouabain binding with cell membrane in muscle tissue of young animals (Figure 6B). It is worth to note that the depressing effect of 50% [Na⁺] PS on [³H]-ouabain binding was more pronounced with α₃ receptors in young animals than in old ones.

As the rate of Ca²⁺ uptake also depends on the activity of Ca²⁺ efflux, the effects of 10^-9M and 10^-4M ouabain on the rate of ⁴⁵Ca²⁺ efflux from preliminary Ca²⁺-enriched heart muscle tissues of young and old animals were studied.

As can be seen in Figure 7, the rate of Ca²⁺efflux from heart muscle tissues of young rats was significantly higher than in old ones.

Figure 7. Dose-dependent changes of rate constant of 45Ca2+ efflux in heart muscle samples of young and old rats incubated in PS (control) and in 10-9and 10-4M concentrations of ouabain. Each point is the mean ± S.E. of 20 samples. Statistical analysis was performed within each group. (***) indicate P<0.001

10^-9M ouabain had activation effect on ⁴⁵Ca²⁺ efflux from heart muscle tissues of young and old animals. This effect had age-dependent weakening character. However, 10^-4M ouabain-induced activation of ⁴⁵Ca²⁺ efflux from heart muscle tissues had age-dependent elevation character.

4. Discussion

Approximately 50% of cardiomyocyte volume is made up of myofibrils, and the remainder consists of mitochondria, nucleus, sarcoplasmic reticulum (SR) and the cytosol [48].Therefore, it is obvious that myofibril contractility could have an essential role in regulation of myocyte volume and the latter could be considered as a marker for myocyte contraction.

As cell membrane is highly permeable for water, cardiomyocyte volume is controlled by cell metabolic activity, which is realized by two close-talking pathways; by regulation of cytoplasm osmolarity and myofibril contraction.

It is known that the inhibition of Na⁺/K⁺-pump on one hand leads to the activation of cAMP-Ca²⁺-dependent phosphorylation processes bringing to myofibril contraction (cell dehydration) and on the other hand to the increase of osmotically active particles in cytoplasm (cell hydration). Previously it has been shown that the ouabain affinity of α₃ receptors has more pronounced age-dependent character than α₂ and α₁ receptors [10]. The obtained data in present work indicate (Figure 1A, B) that <10^-9M ouabain-induced activation of α₃ in heart muscle of young rats had dehydration effect, while in old rats it had hydration effect. In our previous experiment performed on snail neurons it was shown that <10^-9M ouabain had activation effect on ²²Na⁺ efflux in exchange to Ca²⁺uptake (R Na⁺/Ca²⁺ exchange), which was accompanied by elevation of intracellular cAMP, without changing Na⁺/K⁺-pump activity [30]. Therefore,<10^-9M ouabain-induced dehydration effect on muscle of young rats can be considered as a result of activation of both R Na⁺/Ca²⁺ exchange and Ca²⁺-induced contraction of myofibrils. On the other hand, such factors as NO-donor (SNAP) and magnetic fields, having elevation effect on intracellular cGMP content, have relaxation effects on heart muscle tissues as a result of activation of cGMP-dependent F Na⁺/Ca²⁺ exchange. The latter has hydration effect on cells [49]. However, the question of whether nM ouabain-induced muscle dehydration in young and hydration in old rats is due to the activation of R Na⁺/Ca²⁺ exchange and F Na⁺/Ca²⁺ exchange or contraction and relaxation of myofibrils, respectively, requires further elucidation.

Traditionally, ouabain effect on cells is explained by the fact that it has inactivation effect on Na⁺/K⁺-ATPase [50]. However, the fact that in vivo experiments 10^-9M ouabain had more pronounced dehydration effect on muscle tissues in young and hydration effect in old animals than 10^-4M ouabain indicates that the above mentioned explanation cannot be considered reliable.

In vitro experiments, where metabolic activity of heart muscle was inhibited (muscle slices bathing in cold K⁺-free PS), the increase of initial level of muscle hydration in young animals compared with those in vivo experiments can be explained by excitation-induced ions uptake during preparation. In vitro experiments the insensitivity of muscle hydration to depression of cell metabolic activity in old animals can be explained by high initial level of [Ca²⁺]_i-induced muscle contraction (Figure 2A, B). The fact that 10^-4M ouabain–induced Na⁺/K⁺-pump inhibition had dehydration effect on muscle tissues of young animals (having low [Ca²⁺]_i), and hydration effect on muscle tissues (having high [Ca²⁺]_i) of old animals indicates that ouabain, besides inhibition of transporting function of Na⁺/K⁺-ATPase, also has effect on intracellular signaling function leading to muscle contraction in young and hydration (release of endogenous H₂O) in old animals, which was depressed in vivo experiments. This suggestion is confirmed by the data on the effect of 10^-9M ouabain (having no effect on Na⁺/K⁺-pump) on muscle hydration in vitro experiments (Figure 2B). It is worth to note that in vivo experiments 10^-9M ouabain-induced muscle hydration in old animals was higher than 10^-4M ouabain-induced hydration, which allows us to consider that there are different mechanisms which are responsible for the effects of low and high ouabain concentrations on myocyte hydration.

There are minimum two intracellular signaling systems which could be responsible for 10^-9M ouabain-induced changes of myocyte hydration in young rats: a) cAMP and Ca²⁺-dependent myofibril contraction (dehydration) and b) release of endogenous H₂O in cytoplasm (hydration) as a result of cAMP-dependent activation of Ca²⁺-ATPase in ER membrane.

In muscle tissues of old animals, where myocytes are in contracted state because of high [Ca²⁺]_i, 10^-9M ouabain-induced hydration of myocytes can be explained by activation of Ca²⁺ efflux from cytoplasm leading to reactivation of membrane ATP-ases and release of endogenous H₂O. On the basis of our previous and literature data, we hypothesize that 10^-9M ouabain-induced stimulation of Ca²⁺-calmodulin-NO-GMP-F Na⁺/Ca²⁺ exchange cascade could be responsible for myocyte hydration in old animals.

Previously we have shown that there is a negative correlation between Na⁺/K⁺ATP-ase and cAMP formation (adenylatecyclase activity) [51]. This correlation is disturbed by the increase of phospholipase A₂ activity [52-53]. It is known that the increase of [Ca²⁺]_i leads to the activation of phospholipase activity in membrane [54]. Therefore, it is suggested that in old animals inositol triphosphate (I3P) leading to activation of phosphatidylinositol cycle producing cGMP could serve as a sensor for 10^-9M ouabain and activate Na⁺/Ca²⁺exchange in ER [46] and cell membrane [54]. This conclusion cannot be final and needs more detailed investigation.

The data that in vivo experiments ⁴⁵Ca²⁺ uptake by heart muscle tissues of young animals was significantly higher than in old ones can be explained by age-dependent increase of [Ca²⁺]_i. Traditionally, cardiac glycoside-induced stimulation of heart muscle contractility is explained by Na⁺/K⁺-pump inhibition-induced increase of [Ca²⁺]_i through the activation of R Na⁺/Ca²⁺ exchange in cell membrane [26,55]. As Na⁺/K⁺-pump has age-dependent dysfunctional character [56], it was predicted that pump inactivation-induced stimulation of R Na⁺/Ca²⁺ exchange could be more expressed in heart muscle tissues of young animals than in old ones. However, the obtained data have shown that poisoning by10^-4M ouabain led to the depression of ⁴⁵Ca²⁺ uptake by muscle tissues of old animals, while it had slight activation effect on ⁴⁵Ca²⁺ uptake in muscle tissues of young ones (Figure 3A). These data witness the absence of negative correlation between Na⁺/K⁺-pump and R Na⁺/Ca²⁺exchange in heart muscle, which was present in intracellularly perfused squid axon [55]. The obtained data speak about the existence of metabolic mechanisms controlling ⁴⁵Ca²⁺ absorption properties of intracellular structure, such as ER and mitochondria, which have age-dependent dysfunctional character. The data obtained in vitro experiments (Figure 3B), where 10^-4M ouabain had strong depressing effect on⁴⁵Ca²⁺ uptake in both young and old animals and this effect had no age-dependent character, clearly indicate that 10^-4M ouabain-induced changes of ⁴⁵Ca²⁺ uptake cannot be explained only by inhibition of ion transporting function of Na⁺/K⁺-pump.

The fact that intracellular signaling system controlling ⁴⁵Ca²⁺ absorption properties of intracellular structure has a crucial role in regulation of ⁴⁵Ca²⁺ uptake by heart muscle tissues is supported by the data presented in Figure 3A, B. 10^-9M ouabain had more pronounced activation effect on ⁴⁵Ca²⁺ uptake by muscle tissues of young and old animals than 10^-4M ouabain in vivo experiments. These data witness the existence of different mechanisms responsible for low and high concentration effects of ouabain on ⁴⁵Ca²⁺ uptake by muscle tissues. Furthermore, the reverse effect (inhibition) of 10^-9M ouabain on ⁴⁵Ca²⁺ uptake in vitro experiments compared with the data obtained in vivo experiments indicates the metabolism-dependent character of this effect (Figure 3B).

This suggestion is confirmed by the data of the comparative study of dose-dependent ouabain effects on R Na⁺/Ca²⁺ exchange and hydration in muscle tissues bathing in 100% and 50% [Na⁺] PS.

It is known that the decrease of [Na⁺]_o brings to the activation of R Na⁺/Ca² exchange [55]. However, the obtained data indicate that ⁴⁵Ca²⁺ uptake was decreased in young animals upon the effect of 50% [Na⁺] PS, whereas, in old animals 50% [Na⁺] PS led to the increase of ⁴⁵Ca²⁺ uptake (Figure 4A). Although 50% [Na⁺] PS had depressing effect on ⁴⁵Ca²⁺ uptake by heart muscle tissues in young and activation effect in old animals, it had dehydration effect on heart muscle tissues in both ages of animals (Fig. 4B). Furthermore, dehydration effect of 50% [Na⁺] PS on muscle tissues of young animals was more pronounced than in old ones. Therefore, it is suggested that 50% [Na⁺] PS-induced muscle dehydration cannot be explained by R Na⁺/Ca² exchange, but by [Ca²⁺]_i-induced myosin contraction, which had age-dependent weakening character (Figure 4B). The data on the study of ouabain effects on ⁴⁵Ca²⁺ uptake at 50% [Na⁺] PS in vitro experiments bring us to the same conclusion.

The data that both low and high concentrations of ouabain (10^-4M and 10^-9Mouabain) had depressing effect on ⁴⁵Ca²⁺ uptake, but had no effect on muscle tissue hydration clearly indicate that in vitro condition, when muscle tissues were enriched by [Ca²⁺]_i, myocytes were in contracted state and were insensitive to the changes of [Ca²⁺]_i (Figure 5). Furthermore, 10^-9M ouabain in young animals had less inactivation effect on ⁴⁵Ca²⁺ uptake than 10^-4M ouabain, while in old animals it had more inhibitory effect on ⁴⁵Ca²⁺ uptake than 10^-4M ouabain. These data allow us to suggest that 10^-9M ouabain effect depends on the initial level of [Ca²⁺]_i.

The data indicating that dose-dependent ouabain binding with cell membrane in muscle tissues of both young and old animals was depressed at 50% [Na⁺] PS compared with 100% [Na⁺] PS (Figure 6B,D) can be explained by the increase of [Ca²⁺]_i..It is suggested that the increase of [Ca²⁺]_i can bring to the decrease of ouabain binding with membrane by two mechanisms; a) by myosin contraction leading to the decrease of the number of ouabain receptors on cell surface b) by the decrease of affinity of membrane receptors to ouabain. At the range of <10^-9M ouabain concentrations (α₃ receptors), 50% [Na⁺] PS-induced decrease of ouabain binding in young animals was accompanied by the increase of muscle hydration (Figure 6B) (except at 10^-10M ouabain). This allows us to exclude myosin contraction-induced decrease of the number of α₃ receptors in membrane and consider this depression as a result of the decrease of ouabain receptors affinity. Therefore, the data indicating that ouabain binding with α₃ receptors in young animals is more pronounced (Figure 6A) that in old ones (Figure 6C), allow us to explain it by high initial level of [Ca²⁺]_i in old animals. These explanations are in harmony with literature data indicating that the affinity of α₃ receptors to Ca²⁺ is higher than of α₂ and α₁ receptors [57].

The data showing that all concentrations (10^-11-10^-4M) of ouabain had dehydration effect on heart muscle tissues of old animals (Figure 6D), while <10^-9M ouabain had hydration effect on heart muscle tissues of young animals allow us to suggest the existence of an age-dependent signaling system, through which 50% [Na⁺] PS leads to formation of endogenous H₂O. Thus, on the basis of the above presented data it can be concluded that the activation of R Na⁺/Ca²⁺ exchange in young animals was accompanied by the stimulation of intracellular signaling system leading to the release of endogenous H₂O which was depressed in old animals (Figure 6B,D). This suggestion is supported by the data of in vitro experiments on age-dependent study of the effects of 10^-9 M and10^-4 M ouabain on the rate of ⁴⁵Ca²⁺ efflux from heart muscle tissues (Figure 7).

[Ca²⁺]_i can be decreased by removing Ca²⁺ from the cytosol by various mechanisms; a) by Ca²⁺-pumps in membrane of endoplasmatic reticulum (ER) and in PM pushing Ca²⁺ into ER or outside the cell, respectively, b) by Na⁺/Ca²⁺ exchange in forward mode (F Na⁺/Ca²⁺ exchange) extruding cytosolic Ca²⁺ in exchange for extracellular Na⁺. Ca²⁺-pump in cell membrane controls the rest Ca²⁺ concentrations because of their high-affinity/low-capacity transport properties, whereas, Na⁺/Ca²⁺ exchangers display low-affinity/high-capacity transport properties [21]. Therefore, in case of high [Ca²⁺]_i, when Ca²⁺-pumps in ER and PM are in inhibited state, the net Ca²⁺ efflux is mainly determined by F Na⁺/Ca²⁺ exchange [55].

The fact that the initial rate of ⁴⁵Ca²⁺ efflux from the muscle tissues of young animals was much higher than of old ones (Figure 7) can be considered as a determining factor for age-dependent weakening character of ⁴⁵Ca²⁺ uptake intensity (Figure 3). The fact that 10^-9M ouabain had stronger stimulation effect on the rate of ⁴⁵Ca²⁺ efflux from the muscle tissues of young animals than of old ones allows us to consider age-dependent weakening of nM ouabain-induced activation effect of ⁴⁵Ca²⁺ uptake as a result of activation of ⁴⁵Ca²⁺ efflux from the myocytes (Figure 3A).

Both in young and old animals 10^-9M ouabain had stronger activation effect on ⁴⁵Ca²⁺ efflux than 10^-4M ouabain (Figure 7). Moreover, the activation effect of 10^-9M ouabain on the rate of ⁴⁵Ca²⁺ efflux had age-dependent weakening, while 10^-4M ouabain had age-dependent elevation character. These data serve as strong evidence that different metabolic mechanisms are responsible for the activation effect of 10^-9M ouabain and 10^-4M ouabain on the rate of ⁴⁵Ca²⁺ efflux.

Thus, the obtained data of the present work allow us to make the following conclusions:

1. In young animals muscle hydration is significantly higher than in old ones.

2. In heart muscle of young animals dose-dependent ouabain binding with cell membrane in the range of 10^-11-10^-9M has downregulation, while in old ones it has upregulation character. These concentrations of ouabain have hydration and dehydration effects on heart muscles of young and old rats, correspondingly.

3. In vivo experiments the intensity of ⁴⁵Ca²⁺ uptake by heart muscle of young animals is significantly higher than in old ones, while in vitro experiments such age-dependency of ⁴⁵Ca²⁺ uptake intensity is eliminated.

4. In vivo experiments 10^-9 M ouabain has activation effect on ⁴⁵Ca²⁺ uptake by muscles which has age-dependent weakening character, while in vitro experiments it has depression effect on ⁴⁵Ca²⁺ uptake which also has age-dependent weakening character.

5. In vitro experiments PS with 50% [Na⁺] has depression effect on ⁴⁵Ca²⁺ uptake by muscles in young and activation effect in old animals.Whereas, in both age groups PS with 50% [Na⁺] leads to muscle dehydration, which in young animals is more pronounced, than in old ones.

6. The intraperitonal injection of PS with 50% [Na⁺] has depression effect on [³H]-ouabain binding with membrane with α₃ receptors but has different dose-dependent ouabain effect on muscle hydration in the range of 10^-11 -10^-9 M ouabain in young and old animals. 10^-11, 10^-9 M ouabain have hydration and 10^-10 M ouabain has no effect on heart muscles of young animals. Whereas, in old animals 10^-11 -10^-9 M ouabain have dehydration effect.

7. The rate of ⁴⁵Ca²⁺ efflux from heart muscle has strong age-dependnet depressing character. 10^-9,10^-4M ouabain have stimulating effect on ⁴⁵Ca²⁺ efflux from muscle which has age-dependent weakening character. The activation effect of 10^-9 M ouabain is more pronounced on the rate of ⁴⁵Ca²⁺ efflux than the activation effect of 10^-4 M ouabain.

8. The data that ≤10^-9 M ouabain has stronger effect on both muscle hydration and ⁴⁵Ca²⁺ exchange than 10^-4 M ouabain, indicate that the effects of ≤10^-9 M ouabain on muscle hydration and ⁴⁵Ca²⁺ uptake cannot be explained by inactivation of Na⁺/K⁺ pump which takes place in case of 10^-4 M ouabain.

9. Based on the previous data that ≤10^-9 M ouabain has activation effect on cAMP-dependent R Na⁺/Ca²⁺ exchange and the present data that ≤10^-9 M ouabain has activation effects on muscle hydration and ⁴⁵Ca²⁺ uptake which has age-dependent weakening character and disappears in case of depression of metabolic activity of muscles (in vitro experiments at 7^oC) allow us to conclude that age-dependent dehydration of heart muscle is a result of dysfunction of cAMP-dependent Na⁺/Ca²⁺ exchange-induced activation of oxidation processes bringing to the release of endogenous water molecules in cytoplasm.

ACKNOWLEDGEMENTS

We express our gratitude to Ani Gyurjinyan and Anna Sargsyan for editing the article.