| Time course of [K + ], [Na + ] and [Cl − ] in real U937 cells treated with 1 µM STS (symbols) and calculated (lines) for different parameter datasets. Symbols-experimental data, means ± SEM from three independent experiments with duplicate determinations. Small SEM values are masked by symbols. Lines-calculated data obtained for the parameters indicated on the graphs. The initial parameters were na0 140, k0 5.8, cl0 116, B0 48.2, kv 1, na 32, k 117, cl 40, beta 0.029, gamma 1.5, pna 0.0041, pk 0.0115, pcl 0.0125, inc 0.000003, ikc = inkcc = 0, and kb 0.000068. The changed parameters are shown in the layers head. (A) Linear decrease in beta only. (B) Decrease in beta and changes in pna, pk, and pcl. (C) Additional decrease in pk. Shaded regions show significant disagreement of experimental and predicted values. The calculated data were obtained by using code BEZ01B.

| Time course of [K + ], [Na + ] and [Cl − ] in real U937 cells treated with 1 µM STS (symbols) and calculated (lines) for different parameter datasets. Symbols-experimental data, means ± SEM from three independent experiments with duplicate determinations. Small SEM values are masked by symbols. Lines-calculated data obtained for the parameters indicated on the graphs. The initial parameters were na0 140, k0 5.8, cl0 116, B0 48.2, kv 1, na 32, k 117, cl 40, beta 0.029, gamma 1.5, pna 0.0041, pk 0.0115, pcl 0.0125, inc 0.000003, ikc = inkcc = 0, and kb 0.000068. The changed parameters are shown in the layers head. (A) Linear decrease in beta only. (B) Decrease in beta and changes in pna, pk, and pcl. (C) Additional decrease in pk. Shaded regions show significant disagreement of experimental and predicted values. The calculated data were obtained by using code BEZ01B.

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Monovalent ions are involved in a vast array of cellular processes. Their movement across the cell membrane is regulated by numerous channels and transporters. Identification of the pathways responsible for redistribution of ions and cell water in living cells is hampered by their strong interdependence. This difficulty can be overcome by computati...

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... joint effect of pK, pNa and pCl shift is interesting. The cells shown in Figure 4 had initially a rather low U (−29.9 mV) and a mucl of ∼1.5 mV under the normal state ( Table 4). The variation of pCl alone at so small a mucl has no significant effect. ...

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... Our approach is based on the use of the thermodynamic classification of ion transport systems through the cell membrane and does not depend on the mechanism of ion movement. It takes into account all the main types of ion-conducting pathways through the plasma membrane: sodium pump, electrically conductive channels, and all main types of cation-chloride cotransporters [10][11][12][13][14][15]. Until now, the reliability of the proposed description has been successfully shown for transient processes such as Na/K pump blockage by ouabain or replacing extracellular Na + with Li + , as well as in staurosporine-induced apoptosis in proliferating lymphoid U937 cells [10][11][12][13][14]. ...
... It takes into account all the main types of ion-conducting pathways through the plasma membrane: sodium pump, electrically conductive channels, and all main types of cation-chloride cotransporters [10][11][12][13][14][15]. Until now, the reliability of the proposed description has been successfully shown for transient processes such as Na/K pump blockage by ouabain or replacing extracellular Na + with Li + , as well as in staurosporine-induced apoptosis in proliferating lymphoid U937 cells [10][11][12][13][14]. ...
... The TCA precipitates were dissolved in 0.1 N NaOH and analyzed for protein by the Lowry procedure, with serum bovine albumin as a standard. Cell water content was determined by measurements of the buoyant density of the cells in continuous Percoll gradient as described in our previous study [12][13][14][15]. In our experience, measurement of buoyant density of cells is the most sensitive and reliable of all currently existing method for determination of cell water. ...
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Recently, we have developed software that allows, using a minimum of required experimental data, to find the characteristics of ion homeostasis and a list of all unidirectional fluxes of monovalent ions through the main pathways in the cell membrane both in a balanced state and during the transient processes. Our approach has been successfully validated in human proliferating lymphoid U937 cells during transient processes after stopping the Na/K pump by ouabain and for staurosporine-induced apoptosis. In present study, we used this approach to find the characteristics of ion homeostasis and the monovalent ion fluxes through the cell membrane of human erythrocytes in a resting state and during the transient processes after stopping the Na/K pump with ouabain and in response to osmotic challenge. Due to their physiological significance, erythrocytes remain the object of numerous studies, both experimental and computational methods. Calculations showed that, under physiological conditions, the K+ fluxes through electrodiffusion channels in the entire erythrocyte ion balance is small compared to the fluxes through the Na/K pump and cation-chloride cotransporters. The proposed computer program well predicts the dynamics of the erythrocyte ion balance disorders after stopping the Na/K pump with ouabain. In full accordance with predictions, transient processes in human erythrocytes are much slower than in proliferating cells such as lymphoid U937 cells. Comparison of real changes in the distribution of monovalent ions under osmotic challenge with the calculated ones indicates a change in the parameters of the ion transport pathways through the plasma membrane of erythrocytes in this case. The proposed approach may be useful in studying the mechanisms of various erythrocyte dysfunctions.
... Details of the experimental methods used were described in our previous study (Yurinskaya et al., 2019;Yurinskaya et al., 2020;Yurinskaya and Vereninov, 2021a). Briefly, cell water content was estimated by the buoyant density of the cells in continuous Percoll gradient, intracellular K + , Na + and Rb + content was determined by flame emission on a Perkin-Elmer AA 306 spectrophotometer, the intracellular Cl‾ was measured using a radiotracer 36 Cl. ...
... The mathematical model of the movement of monovalent ions across the cell membrane was like that used by Jakobsson (1980), and Lew with colleagues (Lew and Bookchin, 1986;Lew et al., 1991;Lew, 2000), as well as in our previous works (Vereninov et al., 2014;Vereninov et al., 2016;Yurinskaya et al., 2019;Yurinskaya et al., 2020;Yurinskaya and Vereninov, 2021a). It accounts for the Na/K pump, electroconductive channels, cotransporters NC, KC, and NKCC. ...
... Parameter characterizing a linear decrease of the pump rate coefficient β with time, min −1 respectively. The algorithm of the numerical solution of the system of these equations is considered in detail in (Vereninov et al., 2014), the using of the executable file is illustrated more in (Yurinskaya et al., 2019). The problems in determination of the multiple parameters in a system with multiple variables like cell ionic homeostasis are discussed in more detail in (Yurinskaya et al., 2019(Yurinskaya et al., , 2020. ...
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Studying the transport of monovalent ions across the cell membrane in living cells is complicated by the strong interdependence of fluxes through parallel pathways and requires therefore computational analysis of the entire electrochemical system of the cell. Current paper shows how to calculate changes in the cell water balance and ion fluxes caused by changes in the membrane channels and transporters during a normal regulatory increase in cell volume in response to osmotic cell shrinkage (RVI) followed by a decrease in cell volume associated with apoptosis (AVD). Our recently developed software is used as a computational analysis tool and the established human lymphoid cells U937 are taken as an example of proliferating animal cells. It is found that, in contrast to countless statements in the literature that cell volume restoration requires the activation of certain ion channels and transporters, the cellular responses such as RVI and AVD can occur in an electrochemical system like U937 cells without any changes in the state of membrane channels or transporters. These responses depend on the types of chloride cotransporters in the membrane and differ in a hyperosmolar medium with additional sucrose and in a medium with additional NaCl. This finding is essential for the identification of the true changes in membrane channels and transporters responsible for RVI and AVD in living cells. It is determined which changes in membrane parameters predicted by computational analysis are consistent with experimental data obtained on living human lymphoid cells U937, Jurkat, and K562 and which are not. An essential part of the results is the developed software that allows researchers without programming experience to calculate the fluxes of monovalent ions via the main transmembrane pathways and electrochemical gradients that move ions across the membrane. The software is available for download. It is useful for studying the functional expression of the channels and transporters in living cells and understanding how the cell electrochemical system works.
... We believe that this is partly due to the lack of a suitable computational modeling tool for a rather complex system. The software for calculating the balance of unidirectional fluxes of monovalent ions via the main ion pathways in the cell membrane has been developed by the authors in recent years (Vereninov et al., 2014(Vereninov et al., , 2016Yurinskaya et al., 2019). The software was supplied by a simple executable file that allowed, based on the minimum necessary experimental data, to find all the characteristics of ion homeostasis and a list of all unidirectional fluxes of monovalent ions through the main pathways in the cell membrane. ...
... The software was supplied by a simple executable file that allowed, based on the minimum necessary experimental data, to find all the characteristics of ion homeostasis and a list of all unidirectional fluxes of monovalent ions through the main pathways in the cell membrane. Until now, we tested our tool in prediction of rearrangement of ion homeostasis in U937 cells caused by stopping the Na/K pump using the incomplete model with only NC cotransporter (Vereninov et al., 2014;Yurinskaya et al., 2019). The model with all major types of cotransporters for apoptotic U937 cells was considered in our recent study (Yurinskaya et al., 2020), which showed that the effects of KC and NKCC (well-known cotransporters K-Cl and Na-K-2Cl) are small. ...
... The mathematical model of the movement of monovalent ions across the cell membrane was like that used by Jakobsson (1980), Lew and Bookchin (1986), and Lew et al. (1991), as well as in our previous works (Vereninov et al., 2014(Vereninov et al., , 2016Yurinskaya et al., 2019Yurinskaya et al., , 2020. It accounts for the Na/K pump; electroconductive channels; and cotransporters NC, KC, and NKCC. ...
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Cation-coupled chloride cotransporters play a key role in generating the Cl– electrochemical gradient on the cell membrane, which is important for regulation of many cellular processes. However, a quantitative analysis of the interplay between numerous membrane transporters and channels in maintaining cell ionic homeostasis is still undeveloped. Here, we demonstrate a recently developed approach on how to predict cell ionic homeostasis dynamics when stopping the sodium pump in human lymphoid cells U937. The results demonstrate the reliability of the approach and provide the first quantitative description of unidirectional monovalent ion fluxes through the plasma membrane of an animal cell, considering all the main types of cation-coupled chloride cotransporters operating in a system with the sodium pump and electroconductive K+, Na+, and Cl– channels. The same approach was used to study ionic and water balance changes associated with regulatory volume decrease (RVD), a well-known cellular response underlying the adaptation of animal cells to a hypoosmolar environment. A computational analysis of cell as an electrochemical system demonstrates that RVD may happen without any changes in the properties of membrane transporters and channels due to time-dependent changes in electrochemical ion gradients. The proposed approach is applicable when studying truly active regulatory processes mediated by the intracellular signaling network. The developed software can be useful for calculation of the balance of the unidirectional fluxes of monovalent ions across the cell membrane of various cells under various conditions.
... We believe that this is partly due to the lack of a suitable computational modeling tool for a rather complex system. The software for calculating the balance of unidirectional fluxes of monovalent ions via main ion pathways in the cell membrane has been developed by us in recent years (Vereninov et al., 2014;2016;Yurinskaya et al., 2019). The software was supplied by a simple executable file that allowed, based on the minimum necessary experimental data, to find all the characteristics of ion homeostasis and a list of all unidirectional fluxes of monovalent ions through the main pathways in the cell membrane. ...
... The software was supplied by a simple executable file that allowed, based on the minimum necessary experimental data, to find all the characteristics of ion homeostasis and a list of all unidirectional fluxes of monovalent ions through the main pathways in the cell membrane. Until now we tested our tool in prediction of rearrangement of ion homeostasis in U937 cells caused by stopping the Na/K pump using the incomplete model with only NC cotransporter Vereninov et al., 2014;Yurinskaya et al., 2019). The model with all major types of cotransporters for apoptotic U937 cells was considered in our recent study (Yurinskaya et al., 2020) which showed that the effects of KC and NKCC are small. ...
... The mathematical model of the movement of monovalent ions across the cell membrane was similar to that used by Jakobsson (1980), and Lew with colleagues (Lew, Bookchin, 1986;Lew et al. 1991), as well as in our previous works (Vereninov et al., 2014(Vereninov et al., , 2016Yurinskaya et al., 2019Yurinskaya et al., , 2020. It accounts for the Na/K pump, electroconductive channels, cotransporters NC, KC, and NKCC. ...
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Cation-coupled chloride cotransporters play a key role in generating the Cl ⁻ electrochemical gradient on the cell membrane which is important for regulation of many cellular processes. However, the cooperation of transporters and channels of the plasma membrane in holding the ionic homeostasis of the whole cell remains poorly characterized because of the lack of a suitable tool for its computation. Our software successfully predicted in real-time changes in the ion homeostasis of U937 cells after stopping the Na/K pump, but so far considered the model with only NC cotransporter. Here the model with all main types of cotransporters is used in computation of the rearrangements of ionic homeostasis due to stopping the pump and associated with the regulatory volume decrease (RVD) of cells swollen in hypoosmolar medium. The parameters obtained for the real U937 cells are used. Successful prediction of changes in ion homeostasis in real-time after stopping the pump using the model with all major cotransporters indicates that the model is reliable. Using this model for analysis RVD showed that there is a "physical" RVD, associated with the time-dependent changes in electrochemical ion gradients, but not with alteration of channels and transporters of the plasma membrane that should be considered in studies of truly active regulatory processes mediated by the intracellular signaling network. The developed software can be useful for calculation of the balance of the partial unidirectional fluxes of monovalent ions across the cell membrane of various cells under various conditions.
... Chloride channels are key players in regulating ionic and water balance in animal cells, as chloride is the main external anion for most cells and chloride channels are the main electroconductive pathway for this ion through the cell membrane [1][2][3]. Volume regulated anion channel (VRAC) is a ubiquitously expressed chloride channel that has attracted much attention since the molecular structure of VRAC has been identified [4][5][6]. ...
... Recently, we identified changes in major pathways of monovalent ion transfer across the plasma membrane of U937 cells during apoptosis caused by staurosporine (STS). Specifically, a 5-fold increase in chloride channel permeability was found at the early stage of apoptosis along with a decrease in Na/K pump activity and changes in potassium and sodium channel permeability [3]. These findings are consistent with many electrophysiological studies which also show an increase in chloride current at the early stage of apoptosis, as well as during hypotonic stress, accompanied by the regulatory volume decrease (RVD) response [19]. ...
... Cell fluorometry using microscope did not appear to be sensitive enough for quantification of the LRRC8A subunit under the same Ab concentration. Quantification of the LRRC8A subunits via flow fluorometry revealed that membrane-bound VRAC expression did not change following STSinduced apoptosis or hypotonicity to the extent that would correspond to changes in the membrane chloride permeability determined by ion content analysis [3]. ...
Article
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Assessing the expression of channels on the cell membrane is a necessary step in studying the functioning of ion channels in living cells. We explore, first, if endogenous VRAC can be assayed using flow cytometry and a commercially available antibody against an extracellular loop of the LRRC8A, also known as SWELL1, subunit of the VRAC channel. The second goal is to determine if an increase in the number of VRAC channels at the cell membrane is responsible for an increase in chloride permeability of the membrane in two well-known cases: during staurosporine (STS)-induced apoptosis and after water balance disturbance caused by hypotonic medium. Human suspension lymphoid cells U937 were used as they are suitable for flow fluorometry and because we have recently studied their membrane chloride permeability during apoptosis. We found that surface expression of endogenous LRRC8A subunits can be quantified in living U937 cells using flow fluorometry with the Alomone Lab antibody. Further, we revealed that treatment of cells for 1 hour using STS or a hypotonic solution did not change the number of LRRC8A subunits to the extent that would correspond to changes in the membrane chloride permeability determined by ion content analysis. This indicates that prolonged increase in chloride permeability of the cell membrane during apoptotic cell shrinkage or cell volume regulation under hypotonicity in U937 cells occurs without altering cell surface expression of VRAC.
... What was clear from these early studies on AVD was while no single ionic channel or pathway could account for the loss of cell volume during apoptosis, the movement of ions was a critical part of the cell death process. More recent studies have solidified this relationship between AVD and ion flux, as a computational study on the redistribution of ions and water underlying AVD in staurosporine treated human lymphoma cells (U937) concluded that along with a significant increase in chloride and potassium permeability coupled with a decreased permeability of sodium, there was also the progressive decrease in the Na + /K + activity (Yurinskaya et al., 2019). ...
Article
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The movement of water across the cell membrane is a natural biological process that occurs during growth, cell division, and cell death. Many cells are known to regulate changes in their cell volume through inherent compensatory regulatory mechanisms. Cells can sense an increase or decrease in their cell volume, and compensate through mechanisms known as a regulatory volume increase (RVI) or decrease (RVD) response, respectively. The transport of sodium, potassium along with other ions and osmolytes allows the movement of water in and out of the cell. These compensatory volume regulatory mechanisms maintain a cell at near constant volume. A hallmark of the physiological cell death process known as apoptosis is the loss of cell volume or cell shrinkage. This loss of cell volume is in stark contrast to what occurs during the accidental cell death process known as necrosis. During necrosis, cells swell or gain water, eventually resulting in cell lysis. Thus, whether a cell gains or loses water after injury is a defining feature of the specific mode of cell death. Cell shrinkage or the loss of cell volume during apoptosis has been termed apoptotic volume decrease or AVD. Over the years, this distinguishing feature of apoptosis has been largely ignored and thought to be a passive occurrence or simply a consequence of the cell death process. However, studies on AVD have defined an underlying movement of ions that result in not only the loss of cell volume, but also the activation and execution of the apoptotic process. This review explores the role ions play in controlling not only the movement of water, but the regulation of apoptosis. We will focus on what is known about specific ion channels and transporters identified to be involved in AVD, and how the movement of ions and water change the intracellular environment leading to stages of cell shrinkage and associated apoptotic characteristics. Finally, we will discuss these concepts as they apply to different cell types such as neurons, cardiomyocytes, and corneal epithelial cells.
... This is due to specific apoptotic changes in monovalent ion homeostasis, which is closely related to cell water balance regulation (Maeno et al., 2000(Maeno et al., , 2012Okada et al., 2001;Lang et al., 2005;Hoffmann, 2012, 2013). Until now, the quantitative study of changes in channels and transporters responsible for specific apoptotic changes in the balance of Na + , K + , and Cl − has been based on the use of staurosporine-treated U937 cells and computer modeling without considering KCC and NKCC cotransporters (Yurinskaya et al., 2019). However, these cation-coupled Cl − cotransporters of the gene family SLC12 attract much attention in the recent decade (Gagnon and Delpire, 2013;Jentsch, 2016;Delpire and Gagnon, 2018). ...
... In our previous study, the cells with the sodium pump, electroconductive Na + , K + , and Cl − channels and only one cotransporter NC were considered (Yurinskaya et al., 2019). It was found that the permeability of Cl − channels significantly changes at the early stage of apoptosis. ...
... Details of the experimental methods used were described in our previous study (Yurinskaya et al., 2019). Intracellular K + , Na + , and Rb + contents were determined by flame emission on a Perkin-Elmer AA 306 spectrophotometer, and the intracellular Cl − was measured using a radiotracer 36 Cl − . ...
Article
Full-text available
Fluxes of monovalent ions through the multiple pathways of the plasma membrane are highly interdependent, and their assessment by direct measurement is difficult or even impossible. Computation of the entire flux balance helps to identify partial flows and study the functional expression of individual transporters. Our previous computation of unidirectional fluxes in real cells ignored the ubiquitous cotransporters NKCC and KCC. Here, we present an analysis of the entire balance of unidirectional Na+, K+, and Cl- fluxes through the plasma membrane in human lymphoid U937 cells, taking into account not only the Na/K pump and electroconductive channels but all major types of cotransporters NC, NKCC, and KCC. Our calculations use flux equations based on the fundamental principles of macroscopic electroneutrality of the system, water balance, and the generally accepted thermodynamic dependence of ion fluxes on the driving force, and they do not depend on hypotheses about the molecular structure of the channel and transporters. A complete list of the major inward and outward Na+, K+, and Cl- fluxes is obtained for human lymphoid U937 cells at rest and during changes in the ion and water balance for the first 4 h of staurosporine-induced apoptosis. It is shown how the problem of the inevitable multiplicity of solutions to the flux equations, which arises with an increase in the number of ion pathways, can be solved in real cases by analyzing the ratio of ouabain-sensitive and ouabain-resistant parts of K+ (Rb+) influx (OSOR) and using additional experimental data on the effects of specific inhibitors. It is found that dynamics of changes in the membrane channels and transporters underlying apoptotic changes in the content of ions and water in cells, calculated without taking into account the KCC and NKCC cotransporters, differs only in details from that calculated for cells with KCC and NKCC. The developed approach to the assessment of unidirectional fluxes may be useful for understanding functional expression of ion channels and transporters in other cells under various conditions. Attached software allows reproduction of all calculated data under presented conditions and to study the effects of the condition variation.
... Human suspension lymphoid cells U937 were used, as they are suitable for flow fluorometry and because changes in all major monovalent ions, Na + , K + and Cl -, while blocking the sodium pump and STS-induced apoptosis have been studied in detail [6][7][8][9][10][11][12]. We report here that the same intracellular Na + concentration in ionophore-treated and untreated cells produces different ANG fluorescence signals, indicating that Gram and AmB reduce ANG fluorescence in cells. ...
... We compared the relative changes in cellular sodium, as measured by flame emission analysis, with the relative changes in ANG-Na fluorescence detected by a flow cytometer, which reflects the total fluorescence of a single cell, averaged over 20 thousand measurements. An increase in cellular Na + was induced by stopping the pump with ouabain or by STS; the effects of these stimuli on cell ion composition have been previously characterized in detail [6][7][8][9][10][11][12][13]. The treatment of cells with Gram commonly used in ANG calibration gave another way to increase cellular Na + . ...
... It is believed that in the media where Clis partly replaced with gluconate, Donnan's rules become insignificant [1]. We calculated changes in the K + and Na + concentrations using our recently developed computational tool based on Donnan's rules [9] for model cells with parameters similar to U937 cells. Increasing cell membrane permeability for K + and Na + (pK and pNa in the calculation, respectively) by approximately 10 and 50 times were taken, imitating the gramicidin effect. ...
Article
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Background/aims: Sodium is a key player in the fundamental cell functions. Fluorescent probes are indispensable tools for monitoring intracellular sodium levels in single living cells. Since the fluorescence of sodium-sensitive dyes in cells is significantly different from that in an aqueous solution, the fluorescence signal is calibrated in situ indirectly using ionophores for equalizing external and intracellular ion concentration. Attempts to compare data obtained using fluorescent probes and by direct flame emission analysis are sparse and results are inaccurate. Methods: We determined the intracellular sodium concentration in U937 cells by flow cytometry using the Na+-sensitive probe Asante Natrium Green-2 (ANG), and by standard flame emission photometry combined with the cellular water determination by cell density in Percoll gradient. The intracellular Na+ concentrations was modified using known ionophores or, alternatively, by blocking the sodium pump with ouabain or by causing cell apoptosis with staurosporine. Results: It is revealed that both methods are comparable when intracellular sodium concentration was modified by ouabain-mediated blockage of the sodium pump or staurosporine-induced apoptosis. The ANG fluorescence of cells treated with ionophores is approximately two times lower than that in cells with the same Na+ concentration but not treated with ionophores. Although the mechanism is still unknown, this effect should be taken into account when a quantitative assessment of the concentration of intracellular sodium is required. Conclusion: The sodium sensitive dye ANG-2 is a sensitive and useful probe for determination changes in Na+ content and concentration both in single cells and subcellular microparticles. The ANG fluorescence determined in the studied cells in the absence of ionophores, cannot be used as a measure of the real intracellular concentration of Na+ if calibration was carried out in the presence of ionophores.
... It is known from the theory of monovalent ion distribution between animal cells and the medium that the amount of K + in cell essentially depends on the amount of so called "impermeant (through cell membrane) anions" sequestered in cell. It is the amount of these anions in combination with Na, K ATPase pump that determines the water balance of the cell and the accumulation of K + in the cells [45][46][47][48][49][50][51][52][53][54][55] . We can suggest that in activated PBL, an increase in dry mass (total cell protein) during blasttransformation is accompanied by an increase in the amount of impermeant anions per g dry mass, inevitably leading to an increase water influx to restore osmotic balance of cell with medium. ...
Article
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Many evidence shows that K⁺ ions are required for cell proliferation, however, changes in intracellular K⁺ concentration during transition of cells from quiescence to cycling are insufficiently studied. Here, we show using flame emission assay that a long-term increase in cell K⁺ content per g cell protein is a mandatory factor for transition of quiescent human peripheral blood lymphocytes (PBL) to proliferation induced by phytohemagglutinin, phorbol ester with ionomycin, and anti-CD3 antibodies with interleukin-2 (IL-2). The long-term increase in K⁺ content is associated with IL-2-dependent stage of PBL activation and accompanies the growth of small lymphocytes and their transformation into blasts. Inhibition of PBL proliferation with drugs specific for different steps of G0/G1/S transit prevented both blast-transformation and an increase in K⁺ content per cell protein. Determination of the water content in cells by measuring the density of cells in the Percoll gradient showed that, unlike the K⁺ content, the concentration of K⁺ in cell water remains unchanged, since water and K⁺ change in parallel. Correlation of proliferation with high cell K⁺ and water content has been confirmed by the data obtained in comparative study of PBL and permanently cycling Jurkat cells. Our data suggest that K⁺ is important for successful proliferation as the main intracellular ion that participates in regulation of cell water content during cell transition from quiescence to proliferation. We concluded that high K⁺ content in cells and the associated high water content is a characteristic feature of proliferating cells.
... ( Yurinskaya et al., 2005Yurinskaya et al., , 2010Yurinskaya et al., , 2011Yurinskaya et al., , 2017Yurinskaya et al., , 2019Vereninov et al., 2007Vereninov et al., , 2014Vereninov et al., , 2016). The 215 treatment of cells with Gram commonly used in ANG calibration gave another way to increase 216 cell Na + . ...
Preprint
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Fluorescent probes are a popular and indispensable tool for monitoring sodium concentration in living cells in situ. Calibration of fluorescent probes inside cells commonly uses ionophores to equilibrate intracellular and external ion concentrations. Here we test this calibration method using in parallel classical flame emission assay. Suspension human lymphoma cells allow both flow cytometry fluorometric study and flame emission assay. The most sensitive Na+ fluorescent probe ANG-2 and the most common ionophores were tested. Cellular Na+ was altered for calibration in three different ways: by stopping the sodium pump with ouabain, by inducing of apoptosis with staurosporine, and by gramicidin or amphotericin B treatment. We found that ANG-2 fluorescence in cells treated with gramicidin or amphotericin was about two fold lower than in the cells with the same sodium concentration but without ionophores. The equal fluorescence measured in the absence and in the presence of ionophores corresponds to different cell sodium concentrations. No effect of gramicidin on hydrolyzed ANG was observed in vitro. The mechanism, by which gramicidin decreases ANG fluorescence in cells is unlikely to be physical quenching and remains obscure. We conclude that ANG fluorescence does not display realistic cell Na+ if fluorescence in cell is measured in ionophore absence while calibrated in its presence.