alexa Na/K Pump α3-Isoform-Dependent Cell Hydration Controlling Signaling System Dysfunction as A Primary Mechanism for Carcinogenesis | OMICS International
ISSN: 0975-0851
Journal of Bioequivalence & Bioavailability
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Na/K Pump α3-Isoform-Dependent Cell Hydration Controlling Signaling System Dysfunction as A Primary Mechanism for Carcinogenesis

Sinerik Ayrapetyan1*, Liana Yeganyan1, Gagik Bazikyan2, Rafayel Muradyan3 and Flora Arsenyan3

1UNESCO Chair-Life Sciences International Postgraduate Educational Center, Yerevan, Armenia

2National Center of Oncology, Yerevan, Armenia

3NAS RA the Scientific Technological Center of Organic and Pharmaceutical Chemistry SNPO, Yerevan, Armenia

*Corresponding Author:
Sinerik Ayrapetyan
UNESCO Chair-Life Sciences International
Postgraduate Educational Center, Armenia
Tel: 374-106-241-70
Fax: 374-106-241-70
E-mail: [email protected]

Received Date: October 03, 2012; Accepted Date: October 26, 2012; Published Date: October 31, 2012

Citation: Ayrapetyan S, Yeganyan L, Bazikyan G, Muradyan R, Arsenyan F (2012) Na/K Pump α3-Isoform-Dependent Cell Hydration Controlling Signaling System Dysfunction as A Primary Mechanism for Carcinogenesis. J Bioequiv Availab 4:112-120. doi: 10.4172/jbb.1000123

Copyright: © 2012 Ayrapetyan S, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

More than 40 years cell over-hydration serves as a diagnostic marker for carcinogenesis. However, the nature of cell volume controlling mechanism dysfunction of which leads to over hydration and abnormal cell proliferation is not clear yet. The individual roles of Na+/K+ pump isoforms having different affinity to ouabain (α1-low, α2-midle and α3-high affinity) in cell hydration of different organs of healthy (H) and sarcoma-180 tumor (ST) carrying (SC) mice were studied. The tissue hydration in all organs in SC animals was higher. The pathology-induced cell hydration was accompanied by increase in α3 receptors affinity to 3H-ouabain in excitable and decrease in non-excitable cells. 10-11 M ouabain leads to dehydration while 10-8 and 10-6 M to hydration in SC mice, including ST. Tissue hydration
in H and SC mice has different sensitivity to anti cancer drug-cisplatin (cisPt): in H mice it has organo-specific effects while in SC mice it leads to dehydration in all tissues, including ST. This dehydration was accompanied by increase of receptors’ affinity to ouabain which was more pronounced in case of α3-receptors. At 10-6 M ouabain concentration cisPt has hydration effect on muscles and dehydration effect on non-excitable tissues in both H and SC mice, including ST. Cell hydration is suggested as a universal diagnostic marker for cell pathology. Na+/K+ pump α3 isoform-dependent cell hydration controlling signaling system dysfunction is supposed to be a primary mechanism for generation of carcinogenesis. Endogen ouabain circulating in mammalian blood, by its dehydration effect would
have antitumor property, and its deficit would promote carcinogenesis.

Keywords

Cancer; Cisplatin; Hydration, Na+/Ca2+ exchange; Na+/K+ pump; Ouabain

Introduction

Hydration is a fundamental parameter which determines cell’s functional state [1-3]. It is capable of shifting the pattern of cellular metabolism: cell swelling triggers proliferation, while cell shrinkage promotes the apoptosis [4]. Our early studies have shown that membrane proteins having enzymatic, receptor and channel-forming properties, are present in functionally active and inactive (reserve) states, the ratio of which changes subjected to cell volume (membrane packing) [2]. On the other side, cell hydration leads to activation of intracellular enzymes’ activity by “protein folding” mechanism [5].

It is now well established that cancerous tissue is markedly overhydrated and can contain as much as 90% water. Over-hydration of cancer cells serves as one of the essential diagnostic parameters [6]. Notwithstanding the introduction in 1971 by Raymond Damadian of nuclear resonance technique for detecting over-hydrated - cancer cells [7], the nature of the metabolic mechanism the dysfunction of which brings to cell’s over-hydration leading to carcinogenesis is not clear yet.

Among the number of mechanisms involved in cell volume regulation Na+/K+ pump has fundamental role in this process. The importance of its role is dictated by the fact that Na+ gradient serves as an energy source for a number of secondary ionic transporters, such as Na+/Ca2+, Na+/H+, Na+/sugars, amino acids & osmolytes [8].

There are two enzyme systems actively involved in metabolic regulation of cell hydration, associated with cation transport across surface membranes and ionic adsorption in cytoplasm: transport ATPases, which are the translocating structure and are fueled by the free energy derived from ATP hydrolysis, and kinases, which may regulate translocation via phosphorylation of the transporter molecules through the membrane as well as phosphorylation of associated regulatory adsorption properties of intracellular structures. The close talking between these two enzyme systems is realized through the intracellular signaling systems, the dysfunction of which leads to generation of cell pathology, accompanied by corresponding changes of cell hydration. As Na+/K+ pump is the most ATP-utilizing machine in the cell it serves as a main regulator of all other ionic pumps’ and kinases’ activity. Therefore, factors able to change the balance between ATP hydrolysis and ATP production system (mitochondria), by changing Na+/K+ pump activity, could switch on the intracellular signaling systems-induced modulation of cell katabolic and anabolic processes. Hence, the dysfunction of Na+/K+ pump can be considered as a common gate for cell pathology, including cancer. However, which of Na+/K+ pump isoforms in particular serves as the primary mechanism dysfunction of which could lead to apoptosis in excitable cells and enhance proliferation in non-excitable cells stays unclear.

The second ionic transporting mechanism in cell membrane, having a crucial role in cell volume regulation is Na+/Ca2+ exchange [9-12]. It is known that there is a close correlation between the electrogenic Na+/K+ pump and electrogenic Na+/Ca2+ exchange, which has been described since pioneering work by Baker et al. [13]. At present close correlation between these two ion transporting mechanisms on the level of different Na+/K+ pump isoforms is described by prof. Blaustein’s group who discovered and characterized different isoforms of Na+/K+ pump and indicates a key role of high affinity ouabain receptors (α32) in regulation of Ca2+ exchange [14,15]. However, the detailed mechanism of correlation between these isoforms, especially α3 one and Ca2+ exchange is not defined. Askari and coworkers suggested that α3 isoforms mainly perform signaling function and through protein-protein interactions activate the intracellular signaling cascade in cells [16]. From this point of view was explained the nanomolar ouabain induced inhibition of cell proliferation and apoptosis in breast cancer cells [17]. It increases the endocytosis and degradation of Na+/K+-ATPase in LLC-PK1, human breast (BT20), and prostate (DU145) cancer cells and the expression of Na+/K+-ATPase dictates the growth of regulatory effects of ouabain on cells [18].

The fact that ouabain in nanomolar concentrations stimulates the increase of intracellular concentration of cAMP was shown in different tissues of mammals [19]. Our earlier work also indicated that α3 isoforms could serve as a gate for activation of intracellular signaling cascades. Nanomolar concentration of ouabain elevates intracellular cAMP content, the latter, in turn, stimulates Na+/Ca2+ exchange in reversal mode without affecting Na+/K+ pump activity [20]. In is known that in mammals blood constantly circulates endogen ouabain like hormone in nanomolar concentration. It can be suggested that α3 isoforms by mentioned pathway could have a strong dehydration effect on cell by cAMP-dependent phosphorylation-induced contraction of cytoplasmatic filaments and cytoskeleton as well as by activation of electrogenic Na+/Ca2+ exchange in reverse mode, functioning in stoichiometry 3:1. Therefore, α3 isoform-dependent signaling system activation-induced cell dehydration could be responsible for nanomolar ouabain induced inhibition of proliferation. It is suggested that healthy cells by means of cGMP-dependent electroneutral Ca2+ pump [21] in membrane are able to compensate Ca2+ influx through Na+/Ca2+ exchange, but not its dehydration effect. As there is a reciprocal relation between expression of Na+/Ca2+ exchange and Na+/K+ pump proteins in development [22]. Therefore, it is predictable that in early postnatal period cAMP-dependent Na+/Ca2+ exchange – cGMP-dependent Ca2+ pump cascade could have pivotal role in regulation of cell hydration. While in maturation in norm this regulation is realized mainly through Na+/K+ pump.

It is known that in aging Na+/K+ pump dysfunction leads to weakening of Na+/Ca2+ exchange in forward mode. As a result an increase of intracellular Ca2+ content takes place, having stronger inhibitory effect on α3 isoform due to its higher affinity to Ca2+ [13,23]. Therefore, the working hypothesis in present work is that the dysfunction of α3 leads to the failure of intracellular signaling system responsible for cell adaptive function, which could be the primary reason for over hydration in cancer cells and abnormal proliferation.

With the purpose of testing this suggestion the comparative study of dose-dependent 3H-ouabain binding with membrane and cell hydration in healthy(H) and sarcoma-180 tumor carrying (SC) mice’ different tissues, including the tumor tissue were studied, as well as these parameters’ sensitivity to antitumor drug cisplatin (cisPt).

Materials and Methods

Chemicals

As a physiological solution (PS) Tyrode’s solution of following composition (in mM): 137 NaCl, 5.4 KCl, 1.8 CaCl2, 1.05 MgCl2, 5 C6H12O6, 11.9 NaHCO3, 0.42 NaH2PO4 was used adjusted to pH=7.4. All chemicals were obtained from “Medisar” Industrial Chemical Importation Company (Yerevan, Armenia).

Different doses of ouabain (10-11–10-6 M) were prepared on the basis of the same stock physiological solution. For experiments radioactive [3H]-ouabain having 12 Ci/mM specific activity, and non radioactive one (both from PerkinElmer, Boston, MA, USA) was used.

During isotopic measurements scintillation mixture (Bray’s scintillation solution) of following composition was used: 4 g PPO (2,5-diphenyloxazole), 0.2 g POPOP (1,4-bis[-2-(5-phenyloxazolyl)]-benzene), 200 ml 98% ethanol, brought to final volume of 1000 ml with p-dioxane (“Medisar” Industrial Chemical Importation Company (Yerevan, Armenia)).

Animals

Exogamic (fusion from parents that are not closely related) white male mice with the average weight of 18-20g were used for the experiments. The animals were purchased from the Animal Resources Centre at the Life Sciences International Postgraduate Educational Center (LSIPEC, Yerevan, Armenia). All procedures performed on animals were carried out following the protocols approved by Animal Care and Use Committee of Life Sciences International Postgraduate Educational Center (LSIPEC, Yerevan, Armenia).

Experimental setup

Exogamic male mice with the average weight of 18-20 g were used for the experiments. Animals were regularly examined, kept under control of the veterinary in LSIPEC and reserved in a specific pathogen free animal room under optimum conditions at temperature of 22 ± 2°C and were fed ad libitum on a standard lab chow and water.

Anticancer activity of cisPt was studied on the model of transplantable mice tumor – sarcoma-180 (Sarcoma-180, Crooker’s sarcoma).

Tumor tissues removed from sarcoma-180 carrying animal were transplanted to experimental animals. The experiments consisted of two phases: at first sarcoma transplanted animals were studied, and then experiments on normal healthy mice were performed for each ouabain concentration. Animals were divided into groups (each group consisted of 10 mice): 1. control, healthy (H); 2. cisPt, healthy (H), 3. control, sarcoma carrying (SC) 4. cisPt, SC.

During 6 days mice in the groups 1 and 3 (after 48 hours of the tumor transplantation) were intraperitoneal injected physiological solution (PS) 0.5 ml per mouse, and mice in the groups 2 and 4 (after 48 hours of the tumor transplantation) were intraperitoneal injected cisplatin (cisPt) (Cisplatin Ebeve, Austria), 0.5 ml per mouse, with concentration 0.1 mg/ml.

Tissue preparation

24 hours after the last injections (PS or cisPt) experimental animals were intraperitoneal injected PS with different concentration of ouabain (10-11–10-6 M) 0.5 ml per mouse. The mice in control groups were injected with PS, not containing ouabain molecules. 30 minutes after this injection the animals were dissected, then organs were isolated. To avoid the anesthetic effect on initial cell hydration in present experiments (Adams et al., 1977; Akbar et al., 1992) we preferred to use the sharp freezing method [Takahashi and Aprison, 1964]. The experimental animals were immobilized by dipping their heads into liquid nitrogen (for 3-4 s) which led to the freezing of the heads and death of mice (Takahashi and Aprison, 1964). At the full absence of animals’ somatic reflexes upon the extra stimuli tissue samples were removed.

Estimation of cell hydration

Determination of the water content of different tissues was performed by the traditional “tissue drying” method [Adrian, 1956]. For the estimation of cell hydration, the tissue slices with similar shape and weight, after determination of wet mass (w.m.), were dried in thermostatically controlled oven (Factory of Medical Equipment, Odessa, Ukraine) for 24 hours at 105°C. 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. and expressed as water content g/g dry mass.

Isotope measurement

Radioactive [3H]-ouabain is usually used to estimate the number of Na+/K+ pump units in the membrane. It is supposed that each binding site in membrane binds one molecule of ouabain [Baker and Willis, 1972].

After 30 min of [3H]-ouabain intraperitoneal injection (0.5 ml per mouse) the mouse (n=10 in each experimental groups) were decapitated and removed different organs. The mice in control groups were injected with PS, not containing ouabain molecules. Control and experimental tissue slices with similar shape and weight were removed. After that, the slices were washed threefold for 10, 5 and 5 min respectively to remove any radioactive ouabain absorbed by the intracellular spaces and not binded with receptors. Then, the tissue pieces were placed in special vials and each sample was homogenized in 50 μl 68% HNO3 solution. Then Bray’s scintillation fluid was added (2ml) and the radioactivity of mixture was counted by “Wallac 1450” liquid scintillation and luminescent counter (Finland production). The radioactivity of samples was calculated as counted per minute (CPM)/mg. The results were averaged on the basis of the weight of each individual tissue sample.

Statistical analysis

The mean and standard error of tissue samples hydration index and [3H]-ouabain binding 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).

Results

Cell hydration and dose-dependent 3H-ouabain binding with membrane in different tissues of H and SC mice

The data on initial cell hydration of different tissues of H and SC mice are presented in Table 1. As these data indicate, the hydration of all investigated tissues in SC mice is higher than in H mice. These data allow us to consider the increase of cell hydration as a universal marker for cells pathology. Liver being the main organ having detoxification function in organism is predicted to have a comparatively higher (59%) cell hydration in SC mice.

Organ Healthy mice Sarcoma carrying mice %
Heart muscle 2.39±0.1 3.3±0.1 ↑38.14
Skeletal muscle 2.36±0.23 3.87±0.21 ↑64,23
Lung 2.2±0.15 3.31±0.03 ↑50.47
Liver 2.05±0.12 3.27±0.15 ↑58,93
Spleen 2.23±0.13 2.8±0.14 ↑25.84
Kidney 2.05±0.13 3.36±0.19 ↑63,76

Table 1: Tissue hydration of different organs of healthy and sarcoma-180 carrying mice.

The comparative study of dose-dependent (10-11-10-6 M) 3H-ouabain binding with cell membrane in H and SC mice as well as in sarcoma tumor tissue (ST) indicate that in healthy animals only in heart muscle 3 components of ouabain binding having different dose-dependent kinetics can be distinguished. In case of non excitable cells it is difficult to distinguish these components. While in case of SC animals in heart muscle the differences of dose-dependent kinetics of binding with receptors are slightly distinguished but in non-excitable tissues they become visible. These data can probably be explained by the fact that high affinity receptors in non excitable cells are localized deeper in membrane than low affinity ones and only cell hydration-induced swelling makes these receptors accessible for 3H-ouabain binding (Figure 1, only data on heart muscle and lung are presented as examples of excitable and non-excitable tissues, respectively). The curve of dose-dependent ouabain effect on cell hydration in both types of tissue is multi-component, which indicates on heterogeneity within the three distinguished α subunit isoforms.

bioequivalence-bioavailability-dose-dependent-ouabain

Figure 1: Dose-dependent ouabain effect on cell hydration (water content) (right side) and ouabain binding with membrane (left side) in healthy (H) and sarcoma-180 tumor carrying (SC) mice.

To find out individual role of different pump isoforms in determination cell hydration in norm and pathology effects of 10-6 M (α1), 10-8 M (α2), and 10-11 M (α3) ouabain concentration on tissue hydration in H and SC animals were studied. Data presented in Table 2A indicate that in H mice 10-11 M ouabain injection has organo-specific effect on tissue hydration while 10-8 M injection has hydration effect in all investigated tissues. The same effect was more pronounced in all tissues (except heart and lung tissues) at 10-6 M concentration.

a)

Healthy Tissue hydration (water content g/g dry mass)
Organ Ouabain concentration (M)          
0 10-11 Δ% Ouabain 10-8M Δ% Ouabain 10-6M Δ%
Heart muscle 2.389±0.1 2.422±0.02 ↑1.38 2.7±0.26 ↑13.02 2.306±0.05 ↓3.47
Skeletal muscle 2.357±0.23 2.338±0.12 ↓0.8 3.361±0.25 ↑42.6 3.421±0.3 ↑45.14
Lung 2.199±0.15 2.385±0.15 ↑8.4 2.243±0.06 ↑2.01 2.167±0.3 ↓1.46
Liver 2.054±0.12 2.337±0.22 ↑13.78 2.547±0.18 ↑24.01 2.92±0.17 ↑42.16
Spleen 2.225±0.13 1.567±0.08 ↓29.57 2.444±0.25 ↑9.84 3.147±0.3 ↑41.44
Kidney 2.05±0.13 1.722±0.08 ↓16.04 2.878±0.19 ↑40.32 3.09±0.15 ↑50.66

b)

Sarcoma carrying Tissue hydration (water content g/g dry mass)
Organ Ouabain concentration (M)          
0 10-11 Δ% Ouabain 10-8M Δ% Ouabain 10-6M Δ%
Heart muscle 3.3±0.1 3.25±0.01 ↓1.5 2.5±0.01 ↓24.24 3±0.01 ↓9.09
Skeletal muscle 3.87±0.21 2.64±0.14 ↓31.78 4.63±0.33 ↑19.64 4.41±0.23 ↑13.95
Lung 3.31±0.03 3.62±0.21 ↑9.37 5.75±0.18 ↑73.72 3.67±0.23 ↑10.88
Liver 3.265±0.15 2.95±2.27 ↓9.64 3.88±0.35 ↑18.84 3.94±0.22 ↑20.67
Spleen 2.8±0.14 2.7±0.14 ↓3.57 2.93±0.33 ↑4.64 3.14±0.23 ↑12.14
Kidney 3.357±0.19 3.52±0.16 ↑4.86 3.56±0.16 ↑6.05 3.44±0.16 ↑20.47
Sarcoma 180 4.8±0.33 4.4±0.4 ↓8.33 5.66±0.28 ↑17.92 4.97±0.32 ↑3.54

Table 2: The effect of ouabain (at 10-11, 10-8 and 10-6M concentrations) on tissue hydration of different organs: a) healthy mice; b) sarcoma-180 carrying mice.

From these data it is clear that hydration effect observed at 10-8 and 10-6 M concentrations is due to pump inactivation. The reverse effect in heart muscle and lung probably can be explained by increase of muscle contractility. But this explanation is not reliable in case of 10-11 M.

In SC mice (Table 2B), the tissues of which had higher initial hydration compared to healthy ones, 10-11 M ouabain has dehydration effect on all investigated tissues (except lung and kidney) including ST while 10-8 and 10-6 M concentrations as in case of H mice (Table 2A), in all tissues (except heart muscle) lead to increase of hydration. It is worth noting that in sarcoma tumor hydration was less sensitive to 10-6 M ouabain concentration than to 10-11 and 10-8 M concentrations. The pronounced dehydration effect of 10-11 M ouabain in SC mice and 10-8 and 10-6 M concentration hydration in both H and SC, indicate on existence of pathology sensitive α3-receptors-dependent cell hydration controlling mechanism.

Study of 3H-ouabain binding with α3(10-11 M), α2(10-8 M) and α1(10-6 M) receptors in H and SC mice’s tissues has shown that in spite that in SC mice all tissues are hydrated compared to H tissues (Table 2) 3H-ouabain binding with α3 receptors in muscle membranes was increased while in non-excitable tissues decreased (Table 3).

Organ Ouabain concentration (M)
10-11 10-8 10-6
H SC Δ% H SC Δ% H SC Δ%
Heart muscle 18.11 ±0.4 18.67 ±0.1 ↑3.09 21.46 ±0.64 20.72 ±0.1 ↓3.46 25.14 ±0.49 25.87 ±0.1 ↑2.9
Skeletal muscle 16.65 ±0.38 17.28 ±0.39 ↑3.79 18.81 ±0.5 20.03 ±0.87 ↑6.52 23.75 ±0.5 24.67 ±0.26 ↑3.87
Lung 18.45 ±0.75 18.23 ±0.59 ↓1.21 21.44 ±1.06 21.92 ±0.29 ↑2.24 26.04 ±0.77 25.53 ±0.29 ↓1.99
Liver 17.39 ±0.38 15.79 ±0.48 ↓9.16 18.77 ±0.55 18.97 ±0.35 ↑1.08 24.25 ±0.45 23.52 ±0.08 ↓3.02
Spleen 18.49 ±0.63 17.32 ±1.14 ↓6.31 21.56 ±0.46 20.64 ±1.22 ↓4.3 24.99 ±0.48 25.19 ±1.81 ↑0.77
Kidney 17.06 ±0.61 16.95 ±0.43 ↓0.66 19.94 ±0.6 19.64 ±0.09 ↓1.5 24.51 ±0.52 24.61 ±0.66 ↑0.42
Sarcom-180   16.64 ±0.68     19.88 ±0.75     24.4 ±0.22  

Table 3: 10-11M, 10-8M and 10-6M ouabain binding with α3, α2 and α1 receptors in tissues of different organs of healthy (H) and sarcoma-180 carrying (SC) mice.

Such differences between excitable and non-excitable tissues were not observed at level of α2 and α1 receptors. It is worth to note that in liver tissue, where cell pathology-induced increase of hydration was more pronounced, ouabain binding with α3 receptors was significantly depressed. Observed opposite effects of cell pathology on ouabain binding with α3 receptors in excitable and non-excitable tissues are new and extremely interesting from the point of view of understanding the pump dysfunction-induced activation of proliferation in non-excitable and activation of apoptosis in excitable cells. Presented data indicated that α3 receptors’ affinity, as well as cell hydration dependent on these receptors are more sensitive to pathology.

To check this statement in the next series of experiments the effect of antitumor drug cisplatin (cisPt) on cell hydration and dose-dependent ouabain binding in H and SC mice’s tissues was studied.

CisPt effect on cell hydration and ouabain binding with membrane in H and SC mice’s tissues

The data on the effect of cisPt (0.002 mg/g w.w animals) on tissue hydration in H mice which are presented in Table 4 show that it has dehydration or hydration effect on cells depending on the type of tissue. But only in case of 10-6 M ouabain injected animals (Na+/K+-pump inactivated) cisPt has specific hydration effect on excitable and dehydration effect on non-excitable tissues. These data indicate that cisPt has pump-independent specific hydration effect on excitable and dehydration effect on non-excitable cells.

a)

Organ Ouabain concentration (M)
0 10-11 10-8 10-6
control cisPt Δ% control cisPt Δ% control cisPt Δ% control cisPt Δ%
Heart muscle 2.39 ±0.1 1.93 ±0.15 ↓19.07 2.42 ±0.02 1.38 ±0.12 ↓42.89 2.7 ±0.26 1.99 ±0.13 ↓26.1 2.3 ±0.05 2.47 ±0.19 ↑7.3
Skeletal muscle 2.36 ±0.23 3.08 ±0.21 ↑30.47 2.34 ±0.06 2.76 ±0.12 ↑18.23 3.36 ±0.25 3.25 ±0.29 ↓3.2 3.42 ±0.3 3.76 ±0.27 ↑9.95
Lung 2.2 ±0.15 1.21 ±0.11 ↓45.06 2.39 ±0.15 0.88 ±0.11 ↓63.17 2.24 ±0.06 1.11 ±0.12 ↓50.5 2.17 ±0.17 1.69 ±0.16 ↓22.15
Liver 2.05 ±0.12 2.27 ±0.12 ↑10.24 2.34 ±0.22 2.32 ±0.19 ↓0.74 2.55 ±0.18 2.98 ±0.2 ↑17.2 2.92 ±0.17 2.43 ±0.17 ↓16.94
Spleen 2.23 ±0.13 2.13 ±0.12 ↓4.12 1.57 ±0.17 1.6 ±0.15 ↑2.13 2.44 ±0.13 1.55 ±0.07 ↓36.6 3.15 ±0.26 1.75 ±0.14 ↓44.4
Kidney 2.05 ±0.13 2.05 ±0.12 ↓0.12 1.72 ±0.08 2.41 ±0.13 ↑39.85 2.88 ±0.19 2.46 ±0.18 ↓14.6 3.09 ±0.15 2.67 ±0.19 ↓13.73

b)

Organ Ouabain concentration (M)
0 10-11 10-8 10-6
control cisPt Δ% control cisPt Δ% control cisPt Δ% control cisPt Δ%
Heart muscle 3.3 ±0.1 3.34 ±0.08 ↑1.3 3.25 ±0.1 2.55 ±0.07 ↓21.54 2.5 ±0.4 2.88 ±0.15 ↑15 3 ±0.12 3.05 ±0.18 ↑1.67
Skeletal muscle 3.87 ±0.21 4.07 ±0.18 ↓30.91 3.53 ±0.14 4.78 ±0.16 ↑15.37 4.62 ±0.33 4.74 ±0.16 ↓6.72 4.41 ±0.23 4.81 ±0.31 ↑1.46
Lung 3.31 ±0.03 2,83 ±0.13 ↓14.39 2.64 ±0.21 3.1 ±0.14 ↑17.54 5.75 ±0.18 3.25 ±0.15 ↓43.48 3.67 ±0.15 2.5 ±0.19 ↓31.81
Liver 3.27 ±0.15 2.4 ±0.17 ↓26.43 3.62 ±0.27 2.98 ±0.1 ↓17.58 3.88 ±0.35 3.58 ±0.2 ↓7.76 3.94 ±0.15 3.92 ±0.12 ↓0.55
Spleen 2.8 ±0.14 2.42 ±0.12 ↓13.69 2.95 ±0.06 1.88 ±0.19 ↓36.54 2.93 ±0.13 2.33 ±0.01 ↓20.33 3.14 ±0.19 2.86 ±0.2 ↓8.98
Kidney 3.36 ±0.19 2.25 ±0.2 ↓32.98 2.69 ±0.16 2.85 ±0.25 ↑5.81 3.56 ±0.16 3.17 ±0.24 ↓10.94 3.44 ±0.15 2.88 ±0.18 ↓16.36
Sarcoma-180 4.8 ±0.33 4.08 ±0.35 ↓15.16 4.41 ±0.4 4.78 ±0.15 ↓8.37 5.66 ±0.23 4.74 ±0.23 ↓16.25 4.98 ±0.32 4.81 ±0.36 ↓3.25

Table 4: The effect of cisPt on tissue hydration of different organs: a) healthy mice; b) sarcoma-180 carrying mice.

The study cisPt effect on different tissues’ hydration in SC animals shows that (except heart muscle) it has dehydration effect on all tissues, including ST (Table 4B). The strongest dehydration effect was observed in kidney (33%), skeletal muscle (31%) and liver (24%). On ST cisPt has 15% dehydration effect.

As can be seen from presented data, 10-11 M ouabain in SC animals depressed the dehydration effect of cisPt, including ST. For example in kidney dehydration effect even changed to hydration (6%). From these data can be concluded that in SC animals 10-11 M ouabain and cisPt have dehydration effect on cell by different pathways.

At 10-8 M ouabain concentration cisPt in SC mice also has dehydration effect, except heart muscle. It is extremely interesting that at 10-6 M ouabain, when pump is inactive, cisPt has dehydration effect on both healthy and SC excitable tissues and dehydration effect on non-excitable ones. It is worth noting that cisPt dehydration effect on ST at 10-6 M was just 3%. The differences between cisPt effects in two types of cells can be explained by different nature of mechanism involved in cell volume regulation.

To find out which of Na+/K+ pump isoforms is responsible for cisPt-induced modulation of cell hydration in different tissues of H and SC animals the comparative study of cisPt effect on dose-dependent ouabain binding with cell membrane in tissues of H and SC animals was performed.

From the curves of dose-dependent ouabain binding with membrane of H tissues in absence and presence of cisPt (Figure 2 left side) and data obtained in SC animals (Figure 2 right side) can be seen that the dose-dependent ouabain binding with membrane in tissues of SC have significantly higher sensitivity to cisPt than H tissues. The differences of this sensitivity were pronounced in ouabain binding with α3 receptors. In all studied excitable and non excitable tissues of SC animals, including ST (Figure 3), cisPt increased ouabain binding with membrane, but the most pronounced activation effect was expressed on ouabain binding with α3 receptors (except heart muscle tissue where the effect is pronounced on α2 and α1 receptors).

bioequivalence-bioavailability-effect-ouabain-healthy

Figure 2: Effect of cisPt on ouabain binding with cell membrane in healthy (left side) and sarcoma-180 tumor carrying (right side) mice.

bioequivalence-bioavailability-effect-ouabain-cancer

Figure 3: The effect of cisPt on ouabain binding with cell membrane of cancer tissue (number of ouabain molecules/mg dry mass (Ln)) (A) and the effect of cisPt on cell hydration of sarcoma-180 tissue (B).

As can be seen from data presented in Table 5 cisPt basically has elevation effect on ouabain binding at 10-11 M and 10-6 M ouabain concentrations in H (Table 5A) and SC (Table 5B) mice. But this effect was more pronounced in SC, including ST at 10-11 M ouabain injection. At 10-8 cisPt has organ specific effect on ouabain binding and has no effect on ST.

a)

Organ Ouabain concentration (M)
10-11 10-8 10-6
control cisPt Δ% control cisPt Δ% control cisPt Δ%
Heart muscle 18.11 ±0.41 18.33 ±0.55 ↑1.2 21.46 ±0.64 20.72 ±0.52 ↓3.45 25.14 ±0.49 25.11 ±0.28 ↓0.13
Skeletal muscle 16.65 ±0.38 16.58 ±0.83 ↓0.4 18.8 ±0.51 19.8 ±0.28 ↑5.3 23.75 ±0.51 24.05 ±0.68 ↑1.25
Lung 18.45 ±0.75 18.67 ±0.33 ↑1.21 21.44 ±1.06 21.37 ±0.53 ↓0.33 26.04 ±0.77 25.91 ±0.22 ↓0.5
Liver 17.39 ±0.38 17.66 ±0.68 ↑1.54 18.77 ±0.55 19.97 ±0.38 ↑6.38 24.25 ±0.45 24.65 ±0.55 ↑1.64
Spleen 18.49 ±0.63 18.69 ±0.67 ↑1.15 21.56 ±0.46 21.04 ±0.55 ↓2.43 24.99 ±0.48 25.43 ±0.29 ↑1.75
Kidney 17.06 ±0.61 17.52 ±0.18 ↑2.69 19.94 ±0.61 19.92 ±0.15 ↓0.1 23.75 ±0.52 24.55 ±0.42 ↑0.26

b)

Organ Ouabain concentration (M)
10-11 10-8 10-6
  control cisPt Δ% control cisPt Δ% control cisPt Δ%
Heart muscle 18.67 ±0.11 18.88 ±0.76 ↑1.1 20.72 ±0.11 21.37 ±0.81 ↑3.17 25.87 ±0.11 26.27 ±0.65 ↑1.56
Skeletal muscle 17.28 ±0.39 18.41 ±0.74 ↑6.49 20.03 ±0.87 20.62 ±0.25 ↑2.9 24.67 ±0.26 24.75 ±0.42 ↑0.33
Lung 18.23 ±0.59 20.45 ±0.35 ↑12.18 21.92 ±0.29 21.57 ±0.61 ↓1.62 25.52 ±0.29 26.73 ±0.81 ↑4.74
Liver 15.79 ±0.48 18.52 ±0.73 ↑17.26 18.97 ±0.35 20.39 ±0.23 ↑7.46 23.52 ±0.08 25.06 ±0.44 ↑6.54
Spleen 17.32 ±1.14 19.81 ±0.46 ↑14.4 20.64 ±1.22 21.22 ±0.37 ↑2.84 25.19 ±1.81 26.41 ±0.41 ↑4.87
Kidney 16.95 ±0.43 19.09 ±0.81 ↑12.67 19.64 ±0.09 20.62 ±0.32 ↑5.08 24.61 ±0.66 25.21 ±0.69 ↑2.45
Sarcoma-180 16.64 ±0.68 18.43 ±0.54 ↑10.76 19.88 ±0.75 19.75 ±0.34 ↓0.66 24.41 ±0.33 25.04 ±0.26 ↑2.63

Table 5: The effect of cisPt on ouabain binding with cell membrane (number of ouabain molecules/mg dry mass (Ln)) of different organs of a) healthy mice; b) sarcoma-180 carrying mice.

Discussion

As it is known cell’s over-hydration is responsible for abnormal proliferation. Data presented in this work have shown that in pathology (SC mice) hydration increases in all tissues. This indicates that cell hydration increase could serve as an extra-sensitive diagnostic parameter for cell pathology. As was noted above there are two metabolic cascades controlling osmotic gradient on membrane: ionic transport mechanism and intracellular signaling system, regulating adsorption properties of intracellular macromolecules and organs. The question is which of these two mechanisms is the one more sensitive to cell pathology the dysfunction of which leads to decontrolling of cell hydration and generating carcinogenesis.

It is known there are 3 mammalian α subunit isoforms (α1 to α3) of Na+/K+ pump which are products of different genes but have ≈90% sequence identity, different expression patterns, and different kinetics, and they are differently regulated [9-12,13]. It is documented that the low affinity isoforms (α1) have a “housekeeping” function: they control, primarily, Na+ in bulk cytosol while the real function of α2 and α3 isoforms and their functional significance, are uncertain [15,23]. It is suggested that α2 and α3 may regulate Na+ and, indirectly, Ca2+ in a restricted cytosolic space between the cell membrane and reticulum. These isoforms may thereby modulate reticulum Ca2+ content and Ca2+ signaling [14]. By α2 pump isoform-induced activation of Na+/Ca2+ exchange in reversal mode, was explained the generation of hypertension in mammals [13].

A relationship between cancer and Na+/K+ pump α3 isoforms has been repeatedly documented: it is acquiring special interest, as accumulating evidence now indicates that the altered cellular ionic homeostasis may be involved in the abnormal cell proliferation that is a hallmark of the malignant transformation. The matter has aspects that are seemingly paradoxical. On one hand, the Na+/K+ pump α3 isoforms’ dysfunction promotes cell proliferation in non- excitable cells. On the other hand, Na+/K+ pump inhibition in excitable cells triggers apoptotic death pathways. Somehow, therefore, tumor cells regulate the cell signaling machinery to promote proliferation while at the same time protecting themselves from apoptosis. A remodeling of Na+/K+ pump α3 and α2 isoforms is thus increasingly considered important in the process of malignant transformation and it is thus only to be expected that alterations of higher affinity to ouabain Na+/K+ pump isoform should have a role in the process of carcinogenesis [17]. It is known that α3 isoforms’ function closely correlates with intracellular signaling systems [16,17,21]. Our previous data showed that nanomolar concentration of ouabain could stimulate cAMP dependent Na+/Ca2+ exchange in reversed mode without changing Na+/K+ pump activity [20]. It is known that the energy for this transporting mechanism is the difference between electrochemical gradients of Na+ and Ca2+ (ΔENa/Ca=ENa-ECa). Therefore, low ouabain-induced activation of Na+/Ca2+ exchange in reverse mode can be explained by decrease of intracellular Ca2+ ([ Ca2+]i) in result of its adsorption by intracellular structure. Obtained data indicate that 10-11 M ouabain has dehydration effect on tissues of SC mice, including ST, while the concentrations, which inhibit Na+/K+ pump (10-8 and 10-6 M), have tissue hydration effect. This can be explained also by activation of electrogenic Na+/Ca2+ exchange in reverse mode, having dehydration effect on cells. This suggestion is in accordance with our resent data on nM concentration induced heart muscle and brain tissue dehydration which is induced by activation of 45 Ca2+ uptake through Na+/Ca2+ exchange, weakened [23]. It is worth mentioning that because of SC mice were in young age they have still high capacity of intracellular Ca2+ buffering system because of which the α3 receptors activation could lead to cell hydration while in higher concentration ouabain injected animals we had only hydration effect on tissues (Table 2B).

Data obtained in present work indicate that it is ouabain binding with α3 receptors that suffers most in cell pathology. Appearing of high affinity receptors in pathology in non-excitable tissues when the cells are over-hydrated correspond to our earlier data that the number of these receptors is more sensitive to hypotonic solution [2]. Such direct correlation between hydration and number of these receptors can be explained by Blaustein’s data that α3 isoforms are localized deeper in membrane [12]. From this point of view probably in non-excitable tissues the localizing of these receptors is deeper than in excitable cells. It is obvious that for final explanation of these differences a more detailed investigation is needed.

In spite pathology leads to increase of cell hydration both in excitable and non-excitable tissues the correlation between cell hydration and number of binding 3H-ouabin with α3 receptors is opposite: i.e. in excitable tissues it increases, while in non-excitable ones it decreases. As the number of ouabain receptors increases by cell swelling in hypotonic medium [2], it is obvious that its decrease can be explained by decrease of receptors affinity to agonist, which can be increase of [Ca2+]i. It is suggestible that the elucidation of differences in signaling system of excitable and non-excitable cells would make clear the reason of α3 dysfunction-induced activation of apoptosis in excitable and proliferation in non-excitable cells (carcinogenesis).

As is widely known that with aging increases the risk of different medical disorders including cancer. Our previous data have shown that aging leads to inhibition of dose-dependent ouabain binding with α3 isoforms which is accompanied by inhibition of Ca2+ efflux from the cell leading to accumulation of [Ca2+]i [23-25]. Probably age-dependent dysfunction of α3 pump isoforms could be responsible for age-dependent increase of cancer risk. Moreover, because of α3 isoform has much higher affinity to intracellular Ca2+ than α2 and α1 isoforms [13], it is predictable that any pathology-induced intracellular accumulation of Ca2+ ions could inhibit α3 function first of all.

The experimental results on antitumor drug cisPt effect on cell hydration and ouabain binding in H and SC animals demonstrated the higher cisPt-sensitivity of α3 receptors in cell pathology. Although cisPt is widely used in clinics, the detailed mechanism of its antitumor effect is not fully understood. It is widely recognized that antitumor effect of cisPt is realized by modification of DNA structure. However, the question of its effects on membrane transporting function as well on intracellular signaling systems controlling cell hydration and cell proliferation is not sufficiently elucidated. Obtained data demonstrate that cisPt has dehydration effect on all tissues (except that of heart muscle) in SC animals, including sarcoma tumor (Table 4B). According to literature data on the close correlation between cell hydration and cell proliferation [3] cisPt-induced cell dehydration can be considered as one of the essential pathways through which its inhibition effect on cell proliferation in tumor tissues is realized. However, the nature of membrane mechanism as well as intracellular signaling system responsible for cisPt-induced cell dehydration remains unclear. Comparing cisPt effect on tissue hydration in H and SC animals can be concluded that the target for cisPt should be a pump independent cell volume regulating mechanism leading to hydration of excitable tissues (muscles) and dehydration of non-excitable tissues (lung, liver, spleen, kidney, sarcoma). A potential candidate for such a mechanism could be also Na+/Ca2+ exchange in forward and reverse mode respectively. However, to prove this suggestion the study of Na+/Ca2+ exchange in these tissues is necessary and is the subject for our current study. The disappearance of differences of cisPt effect on tissue hydration at 10-6 M ouabain concentration in H and SC animals could serve as strong evidence that these differences are due to different initial activity of Na+/K+ pump i.e. because in H mice’s tissues pump is active while in SC and tumor tissue pump is inactivated.

The fact that cisPt, having dehydration effect on cells has an elevation effect on ouabain binding with cells membrane in all tissues of SC animals indicates that it has increasing effect on receptors affinity to ouabain (Figure 2 and Table 5). From the data on cisPt effect on dose-dependent binding of 3H-ouabain with cell membrane of H and SC animals (Figure 2) can be concluded that the effect of cisPt on receptors affinity depends also on initial functional activity of cell, probably on cell hydration. For example in H animals in muscles it has depressing effect on receptors affinity while in SC animals it has elevation effect on these receptors’ affinity. It is worth to note that cisPt effect on α3 receptors in SC animals, especially in sarcoma tumor, was more pronounced. The fact that the elevation effect of cisPt on the affinity of ouabain receptors appeared at the level of α1 also, (Figure 2) when the Na+/K+ pump was in inactive state (at 10-6 M ouabain), indicates on pump independency of this effect.

Thus all the above presented data bring us to the following conclusions:

• Cell hydration, extra sensitive to pathological changes of cells, could serve as a universal diagnostic parameter for cell pathology;

• α3-dependent intracellular signaling system controlling cell hydration is a primary mechanism dysfunction of which causes decontrolling of cell hydration and activation of abnormal proliferation in non-excitable cells leading to carcinogenesis;

• α3-dependent intracellular signaling system controlling cell hydration in excitable and non-excitable tissues has different nature.

• α3 isoform serves as a target of antitumor drag - cisPt, which leads to increase of α3 isoform’s affinity to ouabain;

• Taking together the fact that in mammalian blood constantly circulate endogen ouabain-like species in nM concentration, and the data presented on low ouabain dehydration effect on tissues, including ST (Table 2B), can be concluded that the deficit of the endogen ouabain could be a promotional factor for cell over-hydration and carcinogenesis.

Thus, presented results give new insight into the role of α3-dependent cell hydration controlling signaling system in the generation of cell pathologies, including carcinogenesis. Further detailed elucidation of the mechanism of this intracellular signaling system could provide us new avenues for cancer diagnosis and treatment.

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