Age-Dependent Increase in Ca2+ Exchange Magnetosensitivity in Rat Heart Muscles

Biochemistry and Biophysics (BAB) Volume 2 Issue 3, September 2014

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Age-Dependent Increase in Ca2+ Exchange Magnetosensitivity in Rat Heart Muscles Lilia Y. Narinyan1, Gayane S. Ayrapetyan2, Jaysankar De3, and Sinerik N. Ayrapetyan*4 UNESCO Chair-Life Sciences International Postgraduate Educational Center 31 Acharyan St. 0040 Yerevan, Armenia narlilia@yahoo.com; 2life@arminco.com; 3 jaysankarde@biophys.am; 4info@biophys.am

1

Abstract Previously the higher magnetosensitivity of Na+/K+ pump-α3 isoforms and its age-dependent dysfunction were shown. It was suggested that the latter could be a consequence of inhibition of Ca2+ efflux from the cell. To check this suggestion, the age-dependency of 45Ca2+ exchange, and their ouabain- and magnetosensitivities in rat heart muscles were studied. The initial rate of 45Ca2+ exchange in muscles of young rats was significantly higher than in older ones.

Intraperitoneal injections of 10-9 M ouabain led to activation of 45Ca2+ uptake as a result of its absorption by intracellular structure that had age-dependent weakening character. The static magnetic field (SMF) exposure on ouabain-poisoned rats had inhibitory effect on young and activation on older.

The rate of 45Ca2+ efflux in ouabain non-poisoned heart muscles had age-dependent weakening character but its magnetosensitivity increased. The 10-9 M ouabain had activation, while at 10-4 M concentration it had inhibition effects on 45Ca2+ efflux in young rats. Nevertheless, in older rats, both concentrations of ouabain had activation effect on 45Ca2+ efflux. The SMF exposure had age-dependent activation effect on 45Ca2+ efflux in tissues bathing in physiological (PS) and in ouabain solutions. The SMFinduced activation of 45Ca2+ efflux was more expressed in tissues of older rats poisoned by 10-4 M ouabain. We suggest that the age-dependent depression in capacity of [Ca2+]i buffer system should be the result of increase in [Ca2+]i due to dysfunction of Na+/K+ pump is responsible for agedependent increase in magnetosensitivity of 45Ca2+ exchange in heart muscles.

Key words: Na+/K+ Pump; Na+/Ca2+ Exchange; Age; Heart Muscle; Ca2+ Pump

Introduction Previously it has been shown that intracardial perfusion of isolated snail heart by magnetized physiological solution (MPS) leads to muscle relaxation which is accompanied by inhibition of 45Ca2+ uptake and increase in intracellular cyclic guanosine monophosphate (cGMP) (Ayrapetyan et al., 2005). As the similar effects were observed by NO-induced

elevation of intracellular cGMP (Azatian et al., 1998), the MPS-induced heart muscle relaxation was explained by cGMP-dependent activation of Ca2+ efflux (Ayrapetyan et al., 2005). This explanation is in harmony with well-documented facts in literature that the cGMP has activation effect on Ca2+ pump in cell membrane and Na+/Ca2+ exchanger pushes these ions from the cell (Blaustein and Lederer, 1999; Brini and Carafoli, 2009).

Thus, from these data , it was concluded that static magnetic field (SMF) –induced structural changes of cell bathing aqua medium which could activate intracellular signaling system leading to activation of Ca2+ efflux from cardiomyocites (Ayrapetyan et al., 2005).

Since the cell hydration-induced protein folding is a key mechanism for regulation of the intracellular enzymes activity (Parsegian et al., 2000), it was predicted that cell hydration could serve as a determining factor for magnetosensitivity of intracellular signaling system. By our recent study, it was shown that diet-regulation of initial water contents led to changes in heart magnetosensitivity. It was higher in initially hydrated state and lower in dehydrated state. The Na+ /K+ pump dysfunction leading to cell dehydration (Narinyan et al., 2012) explained the age-dependent decrease in magnetosensitivity of muscle hydration.

It is known that in heart muscle, the Na+/K+-ATPase, which is working molecule for Na+/K+ pump, has three catalytic isoforms having different ouabain (specific inhibitor for Na+/K+-ATPase) affinity (α1-low, α2middle and α3-high). The α1 and α2 isoforms are involved in Na+ and K+ ions transportation in membrane, while α3 is a gate for intracellular signaling system regulating intracellular Ca2+ ([Ca2+]i) homeostasis (Liu et al., 2000). The question which of these isoforms (ouabain receptors) is the most age- and magnetosensitive was the subject for our previous 39

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Biochemistry and Biophysics (BAB) Volume 2 Issue 3, September 2014

work (Narinyan et al., 2013). By the study of dosedependent [3H]-ouabain binding with cell membrane and its magnetosensitivity in heart muscle of different age rats, it was shown that among three populations of ouabain receptors, only the α3 receptors wereage- and magnetosensitive (Narinyan et al., 2013). However, the nature of mechanism(s) determining age- and magnetosensitivities of α3 receptors remains unclear.

the following composition (in mM): 137 NaCl, 5.4 KCl, 1.8 CaCl2, 1.05 MgCl2, 5 C6H12O6, 11.9 NaHCO3, and 0.42 NaH2PO4, and adjusted to pH=7.4. A radiometer PHM-22r (Radiometer, Copenhagen, Denmark) was used for pH measurements. The K-free solution consisted of 5.4 mM NaCl instead of 5.4 mM KCl. All chemicals were obtained from “Medisar” Industrial Chemical Importation Company (Yerevan, Armenia).

It is well established that ageing leads to increase in [Ca2+]i (Khachaturian, 1989), which has key role in regulation of receptors’ affinity to ligands. Therefore, this mechanism can be considered responsible for dysfunction of α3 receptors. On the other hand , it is known that [Ca2+]i exchange has a key role in realization of biological effect of electromagnetic field (EMF) (Adey, 1981; Blackman et al., 1988). Therefore, it was suggested that age-dependent increase in [Ca2+]i concentration could result in increase in its magnetosensitivity. To check this hypothesis by using 45Ca2+, we studied the age- and magnetosensitivities of Ca2+ uptake and efflux in non-poisoned and in different concentrations of ouabain-poisoned rat heart muscle tissues. It wassuggested that such study would make it possible to elucidate the individual role of Na+/K+-ATPase isoforms in determining the agedependent increase in [Ca2+]i and its magnetosensitivity. As the cytosolic gel condition determining the ratio of osmotic active and bounding water in cytoplasm highly depends on [Ca2+]i (Pollack, 2008), the cell hydration was used as a marker for [Ca2+]i buffer system. Therefore, in our present study, the muscle hydration was used as a marker to estimate the capacity of the [Ca2+]i buffer system.

Ouabain that was used for intraperitoneal injection and tissue samples incubation was obtained from Perkin Elmer (Waltham, MA, USA). The 45Ca2+ (with specific activity 40 mCi/ml) used for intraperitoneal injection and in vitro tissue samples enriched by 45Ca2+ was obtained from Perkin Elmer (Waltham, MA, USA). The volume of all injected solutions was adjusted according to the body mass of the animals (0.02 ml/g body mass).

Tissue Preparation To avoid an anesthetic effect on initial functional state (Adams et al., 1977; Akbar et al., 1992), in present experiments, we preferred to use the sharp freezing method (Takahashi and Aprison, 1964). 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).

SMF Exposure of Rats and Heart Muscle Samples

Chemicals

SMF exposure system used in the experiments was the same as used in our previous work (Narinyan et al., 2012). One day prior to the experiment, the experimental animals were selected and placed in the same cage, which was left in animal room. One hour before the experiments, this cage with animals was transferred to the experimental room without magnet and placed on a wooden table, on which the sham exposure was performed. Each sham-exposed animal after 15 min of PS, 10-9 M or 10-4 M ouabain (all injected solutions contained 45Ca2+) intraperitoneal injection was placed in a special 6×7×14 cm3 plastic cage that had free-air ventilation and kept 15 min in experimental room without SMF on the same table.

Then sham-exposed animals were immobilized by dipping their heads into liquid nitrogen and were decapitated.

Tyrode’s physiological solution (PS) was used, with

After 15 min of PS, 10-9 M or 10-4 M ouabain injection

Materials and Methods Animals Studies were carried out on young (6 weeks) and older (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=220) were housed under optimum conditions with a 12 h light/dark cycle at 22 ± 2oC and 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).

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each experimental rat was placed in the same plastic cage and at the same position as sham ones, and was exposed for 15 min to 0.2 T SMF (position of rats and magnet is shown in Figure 1 (in Narinyan et al., 2012).

For SMF and sham exposure of 45Ca2+-enriched heart muscle samples, the same procedures were used as in case of experiment with whole rats. The SMF exposure tube was placed in central part of magnet, which was used for animal exposure.

The magnetic field could be considered homogenous, because the field varied by no more than 5% within a 12 cm diameter sphere. The magnetic field intensity was measured by a Teslometer Wl-8 (Armenian Radiophysical Institute, Ashtarak, Armenia). This instrument measured magnetic fields in the range of 10-3 to 1.6 T (±5%). The magnetic induction converter was of a crystal type X511-1 (Izmeritel, Saint Petersburg, Russia), 1.5×0.2 mm2, and was fixed on non-magnetized material PX13-1 (Izmeritel, Saint Petersburg, Russia).

Definition of Heart Muscle’S Water Content Determination of the water content (hydration) of heart muscle was performed by the traditional “tissue drying” method (Adrian, 1956). For the estimation of muscle sample’s water content after determination of wet mass, the samples were dried in a thermostat (Factory of Medical Equipment, Odessa, Ukraine) for 24 h at 105 oC. 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.

Definition of 45Ca2+ Uptake in Heart Muscle Study of 45Ca2+ uptake in heart muscle tissue was performed in young and older animals. For this purpose in Tyrode’s physiological solution (PS) containing 1.8 mM CaCl2, 0.0115 mM was substituted by labeled 45CaCl2. All concentrations of ouabain were prepared with this radioactive PS. After 15 min sham-exposed injected by PS, 10-9 M or 104 M ouabain animals (N=5 in each experiment) were kept in special 6×7×14 cm3 plastic cage for 15 min in experimental room without SMF field and then they were decapitated. SMF-exposed animals were kept in the same cage at the same location for 15 min after injection and were then exposed to SMF (0.2 T) for 15 min, and then decapitated. After decapitation, the heart muscle samples were washed three times (10 min - 5 min - 5 min) with normal (non-radioactive) PS solution to remove surface-adherent and extra cellular

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traces. Then after determination of water content by the previously described method, dried tissue samples were replaced into special tubes and homogenized in 50 µl 68% HNO3 solution. Finally, 2 ml of Bray’s scintillation fluid was added and the radioactivity of samples was calculated as counts per minute (cpm)/mg by Wallac 1450 liquid scintillation and luminescence counter (Wallac Oy, Turku, Finland).

Definition of 45Ca2+ Efflux in Heart Muscle The study of 45Ca2+ efflux from preliminarily 45Ca2+enriched heart muscle tissue was performed in young and older rats (N=10 in each experiment). For enriching the 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 CaCl2, 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 the 45Ca2+ from extra-cellular spaces. The samples were divided into three parts. The first 20 samples (control) were dried in a thermostat for 24 h at 105oC after determination of wet mass. The second set of 20 samples (sham) was incubated for 30 min in 20 ml of PS or in solutions containing different doses of ouabain. The remaining set s of 20 samples (SMF) after 15 min of incubation in 20 ml PS (or solutions containing different doses of ouabain) were exposed to SMF for 15 min. Then all samples were dried in thermostat for 24 h at 105 °C. After determination of dry mass of all samples, they were homogenized in 50 µl of 68% HNO3 solution, and the radioactivity of the samples was measured as cpm/mg.

The 45Ca2+ efflux was calculated by the following equation: [control-sham (or SMF)]. The rate of 45Ca2+ efflux was calculated as the residual part of absorbed 45Ca2+ for control and sham (or SMF) data by the following equation: [control-sham (or SMF)]/control.

Statistical Analysis The mean and standard error of the heart muscle hydration index and 45Ca2+ changes in different samples was 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 the P value was taken as *P < 0.05; **P < 0.01; ***P < 0.001.

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Biochemistry and Biophysics (BAB) Volume 2 Issue 3, September 2014

Results The data presented in Table 1 indicate that in heart muscle of young animals, the 45Ca2+ uptake is much higher (35%) than in older ones. Previously it was shown that the nanomolar (nM) concentration of ouabain is agonist for α3 receptors, while 10-4 M is agonist for α1 receptors (Narinyan et al., 2012b). It was observed that the 10-9 M ouabain-induced activation of α3 receptors leads to increase in 45Ca2+ uptake in both age groups (Table 1). It is interesting to note that the 10-4 M ouabain injection had no significant effect on 45Ca2+ uptake in young but showed inhibitory effect (32.3%) in older ones (Table 1). The study of muscle hydration in the same experimental conditions showed that the 10-9 M-induced activation of 45Ca2+ uptake in young animals was not accompanied by significant changes of muscle hydration, while in older ones the dehydration occurred (Figure 1). The 10-4 M ouabain, having inhibitory effect on Na+/K+ pump in both ages of animals (Juhaszova and Blaustein, 1997), had dehydration effect which was more pronounced in older animals (Figure 1B).

We have previously shown that the [3H]-ouabain binding with α3 receptors had pronounced agedependent magnetosensitivity (Narinyan et al., 2012b).

To find out whether the observed effect was due to

changes of [Ca2+]i, the age-dependent magnetosensiivty of 45Ca2+ uptake in PS-injected, 10-9 M and 10-4 M ouabain-injected rats of both ages was studied (Table 2).

The SMF exposure had a significant inhibition effect on 45Ca2+ uptake in heart muscle tissues of ouabain non-poisoned young (26. 5%) and older animals (32.

3%), compared to their sham-exposed animals (Table 2a). It was worthy to note that despite the initial intensity of 45Ca2+ uptake in muscles of young animals was higher than in older ones (Table 1), their magnetosensitivity was less than that of the older ones (Table 2a).

In order to find out whether the observed agedependent opposite effect of SMF in 10-9 M ouabaininjected rats was due to different initial states of Na+/K+-pump activity in young and older animals (Table 2b), similar experiments were performed on both ages of rats injected with 10-4 M ouabain (Table 2c). As can be seen in Table 2c, in case of pumpinhibited state (at 10-4 M ouabain) the SMF exposure had also inactivation (2%) in young and activation (9%) effects on 45Ca2+ uptake in older rats. It was worth noting that these controversial age-dependent effects on 10-4 M were less pronounced than in case of 10-9 M ouabain-injected animals (Table 2b).

FIG. 1. 45CA2+ UPTAKE (A) AND HYDRATION (B) OF YOUNG AND OLDER ANIMALS’ HEART MUSCLE TISSUES IN PS, 10-9 M AND 104 M CONCENTRATIONS OF OUABAIN INTRAPERITONEALLY INJECTED RATS. THE POINTS ON THE ORDINATE INDICATE THE DATA ON PS (CONTROL). EACH POINT OF CURVE IS THE MEAN ± S.E. OF 30 SAMPLES. SYMBOLS (**) AND (***) INDICATE P<0.01 AND P<0.001, RESPECTIVELY. A DIFFERENCE WHICH IS NOT STATISTICALLY SIGNIFICANT MEANS THAT P>0.05.

TABLE 1. 45CA2+ UPTAKE (CPM/MG) IN HEART MUSCLE TISSUES IN PS, AND 10-9 M AND 10-4 M OUABAIN INTRAPERITONEALLY INJECTED RATS (EACH EXPERIMENTAL DATUM REPRESENTS MEAN ± S.E. OF 30 SAMPLES THAT WERE OBTAINED FROM 5 ANIMALS).

Rat’s age

PS mean ± S.E.

10 -9 M ouab. mean ± S.E.

Young Older

4.76±0.2 3.1±0.1

5.7±0.4 3.5±0.2

P

Effect Δ1

P<0.05 ↑20% ↑13% Age-dependent effect

10 -4 M ouab. mean ± S.E.

P

Effect Δ2

4.76±0.3 2.1±0.2

P<0.01

0 ↓32.3%

↓56% P<0.001 Δ1- changes of 45Ca2+ uptake in heart muscle tissues after 10-9 M ouabain injection Δ2 - changes of 45Ca2+ uptake in heart muscle tissues after 10-4 M ouabain injection.

↓35% P<0.001

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↓39% P<0.001

Biochemistry and Biophysics (BAB) Volume 2 Issue 3, September 2014

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TABLE 2. EFFECT OF 0.2 T SMF ON 45CA2+ UPTAKE (CPM/MG) IN HEART MUSCLE TISSUES IN PS (A); 10-9 M (B) AND 10-4 M (C) OUABAIN INTRAPERITONEALLY INJECTED RATS (EACH EXPERIMENTAL DATUM REPRESENTS MEAN ± S.E. OF 30 SAMPLES THAT WERE OBTAINED FROM 5 ANIMALS).

(a) Effect

Rat

PS (Sham) mean ± S.E.

PS (SMF) mean ± S.E.

P

Young Older

4.76±0.2 3.1±0.1

3.5±0.2 2.1±0.1

P<0.01 P<0.001

↓26.5% ↓32.3%

Rat

10 -9 M ouab.(Sham) mean ± S.E.

10-9 M ouab.(SMF) mean ± S.E.

P

Effect

Young Older

5.7±0.4 3.5±0.2

4.5±0.3 4.8±0.3

P<0.01 P<0.01

↓21% ↑37%

Rat

10-4 M ouab.(Sham) mean ± S.E.

10-4 M ouab.(SMF) mean ± S.E.

P

Young Older

4.76±0.3 2.1±0.2

4.67±0.2 2.3±0.1

-

(b)

(c)

Age-Dependency of 45Ca2+ Efflux and Its Magnetosensitivity The study of 45Ca2+ efflux was performed on heart muscle slices, which were preliminarily enriched by 45Ca2+ through their incubation in K+-free (containing 50% NaCl) physiological solution. It was predicted that the 45Ca2+-enriched processes could have poisoning effect on heart muscles that could cause increase in the initial level of muscle hydration.

Therefore, to estimate the degree of change in the functional state of muscles, the effect of 45Ca2+-enriched processes on muscle hydration was studied.

As metabolic controlling of cell hydration is an extrasensitive and universal cellular parameter to weak environmental signals (Ayrapetyan, 2012), in present work it was used as a marker for detection of cell’s functional activities.

Data presented in Table 3 show that the process of 45Ca2+-enrichment in heart muscle samples had hydration effect on muscles in both young (36.8%) and older (13%) animals in comparison with 45Ca2+-nonenriched samples. It was worth noting that the initial tissue hydration in heart of older animals was higher by 8% than in young ones, while in case of 45Ca2+enriched samples, this age-dependency had opposite trend (decrease by 10.5%). From these data, it can be concluded that functional states of muscles used for the study of 45Ca2+ uptake and 45Ca2+ efflux were different.

Data presented in Table 4 show a strong agedependency of 45Ca2+ efflux in samples of heart muscle incubated in PS, 10-9 M and 10-4 M ouabain solutions.

The 45Ca2+ efflux in heart muscle of young rats (PS) was 21.3±3.4 cpm/mg, while in older ones , it was lower

Effect ↓2% ↑9%

than the threshold of our isotope counting system.

These data clearly indicate that Ca2+ efflux was depressed in older animals which was probably the main reason for increase in [Ca2+]i in aging tissue (Khachaturian, 1989).

The 10-9 M ouabain in tissue bathing medium had strong activation effect on 45Ca2+ efflux in heart muscle of young (21 to 59 cpm/mg) animals, while in older ones it had activation effect from “0” to 15 cpm/mg. In 10-4 M ouabain medium the 45Ca2+ efflux in heart muscle of young animals decreased from 21 to 18 cpm/mg, while in older ones it increased from 0 to 9 cpm/mg, which was statistically less in degree compared to the 10-9 M ouabain.

The data presented in Figure 2 indicate that 10-9 Minduced activation of 45Ca2+ efflux (Figure 2A) was accompanied by strong dehydration (Figure 2B) effect on muscle in young, while it had no significant effect in older ones. It is interesting to note that these data were in contrast to non-enriched muscles where the 109 M ouabain had no effect on muscle hydration in young, while in older ones it had dehydration effect (Figure 1B).

To find out the individual role of different Na+/K+ATPase isoforms (α3, α2 and α1) in regulation of 45Ca2+ efflux and its age-dependency, the dose-dependent ouabain effect on 45Ca2+ efflux and muscle hydration in two age groups of animals was studied.

Data presented in Figure 3 indicate that there were pronounced age differences between curves of dosedependent (10-11-10-4 M) ouabain effect on rate of 45Ca2+ efflux in young and older rats (Figure 3A). In heart samples of young animals, the curve of dosedependent effect of ouabain on rate of 45Ca2+ efflux had 43

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Biochemistry and Biophysics (BAB) Volume 2 Issue 3, September 2014

a big variability and consisted of seven components. In older animals, all doses of ouabain had activation effect on it and its curve consisted of only three components. It was worth noting that in older animals, the ouabain-induced activation effect on rate of 45Ca2+

efflux had dose-dependent saturated character in zones of α3 and α2 isoforms, while in young animals within the population of these receptors, heterogenic ouabain-sensitive character of 45Ca2+ efflux was observed.

TABLE 3. EFFECT OF 45CA2+ ENRICHMENT ON HEART MUSCLE TISSUES HYDRATION IN YOUNG AND OLDER ANIMALS. EACH DATUM REPRESENTS THE MEAN ± S.E. OF NON-ENRICHED SAMPLES (N=30) AND 45CA2+ -ENRICHED (N=20) SAMPLES.

Rat Young Older Age-dependent effect

45

Ca2+ non-enriched muscle hydration

45

3.83±0.08 4.15±0.07 ↑8.3%

Ca2+- enriched muscle hydration 5.24±0.2 4.69±0.1 ↓10.5%

P

Effect

P<0.001 P<0.01

↑36.8% ↑13%

TABLE 4. 45CA2+ EFFLUX (CPM/MG) IN HEART MUSCLE TISSUES INCUBATED IN PS, 10-9 M AND 10-4 M OF OUABAIN.

EACH DATUM REPRESENTS MEAN ± S.E. OF 20 SAMPLES. Rat

PS mean ± S.E.

10-9 M ouab. mean ± S.E.

10-4 M ouab. mean ± S.E.

Young Older P Age-dependent effect

21.3±3.4 ~0 P<0.001 ↓ >>100%

59±8.2 15±2.8 P<0.001 ↓74.6%

18.3±2.7 9.6±1.2 P<0.05 ↓47.6%

FIG. 2. 45CA2+ EFFLUX (A) AND HYDRATION (B) OF YOUNG AND OLDER ANIMALS’ HEART MUSCLE INCUBATED IN PS, 10-9 M AND 10-4 M CONCENTRATIONS OF OUABAIN. THE POINTS ON THE ORDINATE INDICATE THE DATA ON OUABAIN-FREE PS (CONTROL). EACH POINT OF CURVE IS THE MEAN ± S.E. OF 20 SAMPLES. SYMBOLS (*) AND (***) INDICATE P< 0.05 AND P<0.001, RESPECTIVELY. A DIFFERENCE WHICH IS NOT STATISTICALLY SIGNIFICANT MEANS THAT P>0.05.

As can be seen from Figure 3B, the dose-dependent ouabain–induced variation of water contents in muscle of young animals was significantly higher than in older ones. It was interesting to note that in spite of dose-dependent ouabain effect on rate of 45Ca2+ efflux in older animals, this mechanism consisted just of three components (Figure 3A), in hydration curve (Figure 3B) in both ages seven components were seen.

In α3 receptor zones (10-11 to 10-9 M) three components can be seen, which were repeated in young and older animals, while at higher concentration the agedependent direction of muscle had opposite characters in young and older animals.

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To elucidate the role of Ca2+ efflux in determination of age-dependent magnetosensitivity of ouabain receptors observed in our previous work (Narinyan et al., 2013), in the next series of experiments , the effect of SMF exposure on 45Ca2+efflux in heart muscles of both ages animals incubated in PS and different concentrations of ouabain solutions was studied.

Data presented in Table 5 indicate that 15 min SMF exposure led to increase in rate of 45Ca2+ efflux in heart muscles of young rats by 42%, while in older animals, this increment was not statistically significant (Table 5a).

Biochemistry and Biophysics (BAB) Volume 2 Issue 3, September 2014

The effect of SMF exposure on rate of 45Ca2+ efflux in heart muscles after 30 min incubation in 10-9 M and 10-4 M ouabain solution also indicated the age-dependent activation effect of SMF on rate of 45Ca2+ efflux (Table 5b, c). But it was worth noting that the SMF-induced activation of rate of 45Ca2+ efflux in heart samples of young animals was significantly less pronounced than in older ones (Table 5b). This SMF-induced activation effect of rate of 45Ca2+ efflux at 10-4 M ouabain concentration (Table 5c) as well as its age-dependent increase effect were significantly higher than at 10-9 M ouabain (Table 5b,c).

The study of SMF exposure effects in presence of different concentrations of ouabain in tissue bathing solution in both ages of animals showed that the age-

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dependent magnetosensitivity of 45Ca2+ efflux appeared more pronounced in the range of high affinity ouabain receptors (α3 and α2) and at concentration of 10-4 M ouabain (Figure 4). It was interesting to note that although SMF exposure at 10-4 M ouabain concentration had activation effect on 45Ca2+ efflux in both ages, at low ouabain concentrations (1011-10-9 M) the magnetosensitivity in the samples of young animals was significantly higher than in older rats (Figures 4A,B). The fact that in the heart of older animals , SMF exposure had activation effect on 45Ca2+ efflux at 10-10 M ouabain and inactivation effect at 10-8 M (α2 receptors), while the latter in young ones was magnetosensitive, deserve to be a subject of detailed investigation.

FIG. 3. DOSE-DEPENDENT CHANGES OF 45CA2+ EFFLUX RATE (A) AND HYDRATION (B) OF HEART MUSCLE SAMPLES FROM YOUNG AND OLDER RATS INCUBATED IN PS (CONTROL) AND DIFFERENT CONCENTRATIONS (10-11-10-4 M) OF OUABIAN. THE HORIZONTAL LINES INDICATE CONTROL LEVEL OF 45CA2+ EFFLUX AND HYDRATION OF MUSCLES TISSUES FROM BOTH AGES OF ANIMALS INCUBATED IN PS. EACH POINT IS THE MEAN ± S.E. OF 20 SAMPLES. STATISTICAL ANALYSIS WAS PERFORMED WITHIN EACH GROUP AND SYMBOLS (*), (**) AND (***) INDICATE P< 0.05, P<0.01 AND P<0.001.

TABLE 5. EFFECT OF 0.2 T SMF ON RATE OF 45CA2+ EFFLUX IN HEART MUSCLE INCUBATED IN PS (A), 10-9 M (B) AND 10-4 M (C) OUABAIN. EACH DATUM REPRESENTS MEAN ± S.E. OF 20 SAMPLES. (a) Rat

PS (Sham) mean ± S.E.

PS (SMF) mean ± S.E.

P

Effect

Young Older

21.3±3.4 ~0

30.4±2.3 2.5±1.6

P<0.001 -

↑42% ↑ >>100%

Rat

10 -9 M ouab.(Sham) mean ± S.E.

10-9 M ouab.(SMF) mean ± S.E.

P

Effect

Young Older

59±8.2 15±2.8

61.2±7.8 16.4±2.7

P<0.05 -

↑3.7% ↑9.3%

Rat

10 -4 M ouab.(Sham) mean ± S.E.

10-4 M ouab.(SMF) mean ± S.E.

P

Effect

Young Older

18.3±2.7 9.6±1.2

26.5±3.8 15.6±1.9

P<0.001 P<0.05

↑44.8% ↑62.5%

(b)

(c)

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Biochemistry and Biophysics (BAB) Volume 2 Issue 3, September 2014

FIG. 4. AGE-DEPENDENT EFFECT OF 0.2 T SMF EXPOSURES ON RATE OF 45CA2+ EFFLUX OF HEART MUSCLES SAMPLES FROM YOUNG (A) AND OLDER (B) RATS INCUBATED IN PS (CONTROL) AND DIFFERENT CONCENTRATIONS (10-11-10-4 M) OF OUABAIN. THE POINTS ON THE ORDINATE INDICATE THE DATA ON PS. EACH POINT OF CURVE IS THE MEAN ± S.E. OF 20 SAMPLES. EACH EXPERIMENTAL DATUM IS COMPARED TO SHAM ONES AND SYMBOLS (*), (**) AND (***) INDICATE P< 0.05, P<0.01 AND P<0.001.

Discussion Two parallel mechanisms operate in cell membrane for Ca2+ influx: Ca2+-selective channels and Na+/Ca2+ exchange in reverse mode (R-Na+/Ca2+exchange).

It is known that in normal (with low [Na]i) cells , the Ca2+ influx which is realized by Ca2+-channels is compensated by Ca2+ pump and Na+/Ca2+ exchange in forward mode (F-Na+/Ca2+ exchange) in cell membrane, i.e 45Ca2+ influx=Ca2+ efflux (Blaustein and Lederer, 1999). Therefore, in present in vivo experiments with ouabain non-poisoned animals, the differences (35%) between 45Ca2+ uptakes in young and older animals can be explained by age-dependent decrease in channels activity in heart muscle (Table 1).

However, in case of activation of electrogenic RNa+/Ca2+ exchange (Baker et al., 1969) , the 45 2+ contribution of channels in total Ca uptake is excluded for following two reasons. They are, a) because R-Na+/Ca2+ exchange-induced membrane hyper-polarization leads to potential-dependent ionic channels inactivation (Hodgkin, 1964), and b) the RNa+/Ca2+ exchange-induced water efflux through the membrane has inactivation effect on inward ionic channels including Ca2+ channels (Ayrapetyan et al., 1988). Therefore, activation of 45Ca2+ uptake at low and higher ouabain concentrations was due to activation of R-Na+/Ca2+ exchange (Ayrapetyan et al., 1984; Sagian et al., 1996), and contribution of Ca2+ channels in total 45Ca2+ uptakes could be fully ignored. Consequently, the obtained data in present work on age-dependent 46

decrease in 45Ca2+ uptake in 10-9 M and 10-4 M ouabainpoisoned rats (39% and 56%, respectively) (Table 1) can be explained by the age-dependent decrease in RNa+/ 45Ca2+ exchange activity.

The data that at 10-4 M ouabain, having strong inhibitory effect on Na+/K+ pump (Blaustein and Lederer, 1999) have no significant changes of the 45Ca2+ uptake in young but have inhibition effect (32.3%) on older ones (Table 1), and indicate on the existence of strong age-dependent decrease in the capacity of intracellular [Ca2+]i buffer system. This suggestion is in close agreement with literature data that in older animals, intracellular structures are Ca2+saturated because of higher [Ca2+]i (Khachaturian, 1989; Kostyuk and Lukyanetz, 2006).

It is known that besides plasma membrane mechanisms, there are two cytoplasmic close–talking mechanisms determining [Ca2+]i–buffering properties: the thermodynamic activity of intracellular water (gel state of cytoplasm) (Pollack, 2008) and [Ca2+]i sorption properties of intracellular structure (sarcoplasmic reticulum (SR), mitochondria, calmodulin and other proteins) (Brini and Carafoli, 2009). Since the [Ca2+]i has strong coagulation effect on cytoplasm and effect on thermodynamic activity of intracellular water, in present work the cell hydration was used as the extra sensitive marker for detection of capacity of [Ca2+]i buffer system.

Originally it was thought that ouabain-induced increase in [Ca2+]i was due to activation of the RNa+/Ca2+ exchange, which was the consequence of

Biochemistry and Biophysics (BAB) Volume 2 Issue 3, September 2014

Na+/K+ pump inactivation (Baker et al., 1969).

However, data in present work on the 10-9 M ouabain (which has no effect on Na+/K+ pump)-induced activation of 45Ca2+ uptake significantly being higher than 10-4 M ouabain (which has strong inhibitory effect on Na+/K+ pump) effect on it (Figure 1) clearly indicate that this effect cannot be explained only by Na+/K+ pump inactivation.

Since the difference between electrochemical gradients of Na+ and Ca2+ ions (ENa -ECa ) serves as energy source for Na+/Ca2+ exchange, the increase in [Na+]i and decrease in [Ca2+]i could bring in activation of the RNa+/Ca2+ exchange (Baker et al., 1969). Therefore, 10-9 M ouabain-induced activation of R-Na+/Ca2+ exchange can be considered as a result of [Ca2+]i decrease leading to increase in ECa. It is obvious that the latter could be done by increase in absorption of [Ca2+]i by intracellular structure. It is known that such absorption can be realized by cyclic adenosine monophosphate (cAMP)-activated Ca2+-pump localized in SR membrane, transporting Ca2+ from cytoplasm into the SR (Brini and Carafoli, 2009).

The fact that the nM ouabain could elevate the intracellular cAMP was demonstrated in different tissues including dog renal cortex, goldfish intestinal mucosa, mouse pancreatic islets, murine epithelioid and fibroblastic cell lines, rat brain, rat renal collecting tubule cells in culture and astrocytes (Siegel, 1999).

Earlier we have also shown that low ouabain activation of R-Na+/Ca2+ exchange was accompanied by increase in intracellular cAMP content in snail neurones (Sagian et al., 1996).

Thus, based on above data, the α3 receptors-induced activation of R-Na+/Ca2+ exchange can be considered as a consequence of cAMP-activated protein kinase activity leading to activation of Ca2+ pump in SR membrane. The fact that in young animals , the 10-9 M ouabain-induced activation of R-Na+/Ca2+ exchange was not accompanied by muscle dehydration as was seen in older rats (Figure 3) which can be explained by age-dependent dysfunction of cAMP-dependent Ca2+ pump in SR membrane as a result of high [Ca2+]i. Such age differences were more pronounced in vitro experiments when muscles were preliminary enriched by 45Ca2+ (Figure 3B). This fact could serve as additional evidence for this suggestion.

It has been previously shown that rats led to dehydration of different the heart muscle (Danielyan et al., age-induced decrease in character

SMF exposure in tissues, including 1999), which had (Narinyan et al.,

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2012). Based on the present data (Table 5) it can be stated that SMF has activation effect on 45Ca2+ efflux leading to cell dehydration because of reactivation of Na+/K+ pump.

However, the question which of Na+/K+-ATPase isoforms is or are responsible for age-dependent magnetosensitivity of heart muscle hydration was elucidated recently by studying the magnetosensitivity of [3H]-ouabain binding with cell membrane (Narinyan et al., 2013). It was shown that the [3H]ouabain binding only with α3 receptors was pronounced magnetosensitive.

The fact that SMF effect on Ca2+ uptake in 10-9 M ouabain was more pronounced than in 10-4 M ouabain clearly indicates that it cannot be explained by modulation of Na+/K+ pump activity. Therefore, it is suggested that both of nM ouabain and SMF modulate 45Ca2+ uptake by intracellular signaling systems but in opposite age-dependent manner. The ouabain has activation and age-dependent decreasing, while SMF has inactivation and age-dependent increasing effects on 45Ca2+ uptake. We reached the same conclusion from the data on study of age-dependent magnetosensitivity of 45Ca2+ efflux at different concentrations of ouabain in medium.

It is known that 45Ca2+ efflux from the cells is realized by the ATP-driven Ca2+ pump and F-Na+/Ca2+ exchanger. The F-Na+/Ca2+ exchanger has about a 10fold lower affinity to Ca2+ but 10- to 50-fold higher turnover rate than the ATP-driven Ca2+ pump (Blaustein and Lederer, 1999; Dipolo and Beaugé, 2006; Brini and Carafoli, 2009). Therefore, the Ca2+ pump is active only in low [Ca2+]i, while in higher [Ca2+]i such as in our experimental condition, the Ca2+ pump is in inactive state and the 45Ca2+ efflux is due to F-Na+/Ca2+ exchange. Therefore, the Ca2+ efflux can be considered because of Na+/Ca2+ exchange.

The obtained data on age-dependent decrease in 45Ca2+ efflux (Table IV) is in agreement with literature data on age-dependent dysfunction of Ca2+ efflux leading to increase in [Ca2+]i (Khachaturian, 1989).

The fact that in 45Ca2+-enriched muscle , the 10-9 M ouabain leads to strong activation of 45Ca2+ efflux, which was more pronounced in young than older animals, seems extremely interesting, from the point of protective effect of endogenic ouabain (Blaustein et al., 2009). The fact that in samples of young animals’ heart, the 10-9 M ouabain had strong er dehydration effect on muscle (Figure 3) than was observed in vivo experiments (Figure 1) which indicate d on high 47

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Biochemistry and Biophysics (BAB) Volume 2 Issue 3, September 2014

capacity intracellular buffer system. The latter is realized by activation of cAMP-dependent Ca2+ pump by pushing these ions from cytoplasm to the SR. This mechanism could have important physiological significance because activation of cAMP-dependent Ca2+ pump in SR would reactivate cGMP-dependent Ca2+ pump in membrane. However, this conclusion cannot be considered as a final since it needs further detailed investigation.

In present study on dose-dependent effect of ouabain on 45Ca2+ efflux as well as on cell hydration, the heterogeneity of ouabain sensitivity was shown (Figure 4). This heterogeneity was more pronounced and age-dependent within the family of high affinity ouabain receptors (α3 and α2). The fact that in older animals , the variation of ouabain-dependent muscle hydration (free [Ca2+]i) was rather small er than in young ones which indicates on the depression of intracellular Ca2+-buffer system capacity.

It is interesting to note that as in case of study on Ca2+ uptake, 10-9 M ouabain has more pronounced activation effect on Ca2+ efflux than 10-4 M ouabain.

This effect has age-dependent depression, while SMFinduced activation of Ca2+ efflux has age-dependent elevation character. These data that in case of Na+/K+ pump inhibited state , the SMF had more pronounced activation effect on F-Na+/Ca2+ exchange, allow us to explain the age-dependent increase in magnetosensitivity of Ca2+ efflux by age-dependent dysfunction of Na+/K+ pump.

is a consequence of dysfunction of cGMP-dependent Ca2+ efflux leading to increase in [Ca2+]i.

The nM ouabain-induced activation of α3 receptors leads to R-Na+/Ca2+ exchange as a result of activation of cAMP-dependent Ca2+ pump in SR membrane. The age-dependent weakening of cGMP-dependent Ca2+ efflux from the cells through both Ca2+ pump and FNa+/Ca2+ exchange can be considered as a consequence of dysfunction of cGMP formation process. The SMFinduced elevation of [cGMP]i leads to activation of Ca2+ efflux, which is more pronounced in older rats' heart muscles having high [Ca2+]i.

ACKNOWLEDGEMENTS

We express our gratitude to Mrs. Tatevik Arzumanyan for secretarial work.

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