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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.

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.

The work provides a modern mathematical description of animal cell electrochemical system under a balanced state and during the transition caused by an increase in external osmolarity, considering all the main ionic pathways in the cell membrane: the sodium pump, K+, Na+, Cl- electroconductive channels and cotransporters NC, KC, and NKCC. The description is applied to experimental data obtained on U937 cells cultured in suspension, which allows the required assays to be performed, including determination of cell water content using buoyant density, cell ion content using flame photometry, and optical methods using flow cytometry. The study of these cells can serve as a useful model for understanding the general mechanisms of regulation of cellular water and ionic balance, which cannot be properly analyzed in many important practical cases, such as ischemic disturbance of cellular ionic and water balance, when cells cannot be isolated. An essential part of the results is the developed software supplied with an executable file, which allows researchers with no programming experience to calculate unidirectional fluxes of monovalent ions through separate pathways and ion-electrochemical gradients that move ions through them, which is important for studying the functional expression of channels and transporters. It is shown how the developed approach is used to reveal changes in channels and transporters underlying the RVI and AVD responses to the hyperosmolar medium in the studied living U937 cells.

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.

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.

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.We determined the intracellular sodium concentration in U937 cells by flow cytometry using the NaIt 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 NaThe sodium sensitive dye ANG-2 is a sensitive and useful probe for determination changes in Na

Monovalent ions, sodium in particular, are involved in fundamental cell functions, such as water balance and electric processes, intra- and intercellular signaling, cell movement, pH regulation and metabolite transport into and out of cells. Fluorescent probes are indispensable tools for monitoring intracellular sodium levels in single living cells in heterogeneous cell populations and tissues. 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 by changing the concentration of extracellular sodium in the presence of ionophores, making the membrane permeable to sodium and equilibrating its intra- and extracellular concentrations. The reliability of this calibration method has not been well studied. Here, we compare the determinations of the intracellular sodium concentration by flame emission photometry and flow cytometry using the Na+-sensitive probe Asante Natrium Green-2 (ANG). The intracellular Na+ concentration was altered using known ionophores or, alternatively, by blocking the sodium pump with ouabain or by causing cell apoptosis with staurosporine. The use of U937 cells cultured in suspension allowed the fluorometry of single cells by flow cytometry and flame emission analysis of samples checked for uniform cell populations. It is revealed that 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. Sodium sensitive fluorescent dyes are widely used at present, and the problem is practically significant.

A decrease in flow cytometric forward light scatter (FSC) is commonly interpreted as a sign of apoptotic cell volume decrease (AVD). However, the intensity of light scattering depends not only on the cell size but also on its other characteristics, such as hydration, which may affect the scattering in the opposite way. That makes estimation of AVD by FSC problematic. Here, we aimed to clarify the relationship between light scattering, cell hydration (assayed by buoyant density) and cell size by the Coulter technique. We used human lymphoid cells U937 exposed to staurosporine, etoposide or hypertonic stress as an apoptotic model. An initial increase in FSC was found to occur in apoptotic cells treated with staurosporine and hypertonic solutions; it is accompanied by cell dehydration and is absent in apoptosis caused by etoposide that is consistent with the lack of dehydration in this case. Thus, the effect of dehydration on the scattering signal outweighs the effect of reduction in cell size. The subsequent FSC decrease, which occurred in parallel to accumulation of annexin-positive cells, was similar in apoptosis caused by all three types of inducers. We conclude that an increase, but not a decrease in light scattering, indicates the initial cell volume decrease associated with apoptotic cell dehydration.

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.

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.

The response of fluorescent ion probes to ions is affected by intracellular environment. To properly calibrate them, intracellular and extracellular concentrations of the measured ion must be made equal. In the first, computational, part of this work, we show, using the example of potassium, that the two requirements for ion equilibration are complete dissipation of membrane potential and high membrane permeability for both potassium and sodium. In the second part, we tested the ability of various ionophores to achieve potassium equilibration in Jurkat and U937 cells and found a combination of valinomycin, nigericin, gramicidin and ouabain to be the most effective. In the third part, we applied this protocol to two potassium probes, APG-4 and APG-2. APG-4 shows good sensitivity to potassium but its fluorescence is sensitive to cell volume. Because ionophores cause cell swelling, calibration buffers had to be supplemented with 50 mM sucrose to keep cell volume constant. With these precautions taken, the average potassium concentrations in U937 and Jurkat cells were measured at 132 mM and 118 mM, respectively. The other tested probe, APG-2, is nonselective for cations; this is, however, a potentially useful property because the sum [K+] + [Na+] determines the amount of intracellular water.

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 computational analysis of the whole cell flux balance. Our previous computational studies were concerned with monovalent ion fluxes in cells under the conditions of balanced ion distribution or during transition processes after stopping the Na+/K+ pump. Here we analyze a more complex case-redistribution of ions during cell apoptosis when the parameters keep changing during the process. New experimental data for staurosporine-induced apoptosis of human lymphoma cells U937 have been obtained: the time course of changes in cellular K+, Na+, Cl-, and water content, as well as Rb+ fluxes as a marker of the Na/K pump activity. Using a newly developed computational tool, we found that alteration of ion and water balance was associated with a 55% decrease in the Na+/K+-ATPase rate coefficient over a 4-h period, with a time-dependent increase in potassium channel permeability, and a decrease in sodium channel permeability. The early decrease in [Cl-]i and cell volume were associated with an ~5-fold increase in chloride channel permeability. The developed approach and the presented executable file can be used to identify the channels and transporters responsible for alterations of cell ion and water balance not only during apoptosis but in other physiological scenarios.

The results of this study of the ultrastructural changes in U-937 cells in the apoptotic state are largely consistent with analogous data available in the literature. However, we also obtained original data on the ultrastructural changes in cellular organelles and immunocytochemical localization and distribution of proteasomes. It was shown that, when apoptosis was induced by incubation of cells in a hypertonic sucrose solution (200–400 mM), the Golgi apparatus (GA) was localized close to the plasma membrane. This fact is indirect evidence that cytoskeletal elements (particularly microtubules, which hold GA in the center of the cell) are depolymerized already at the early stages of apoptosis. At the late stages of apoptosis, the distance between individual GA cisterns significantly increases, which is apparently due to the cleavage of their binding proteins, golgins. Mitochondria in apoptotic cells do not change significantly. They have regularly spaced cristae and a fairly dense matrix containing single vacuoles. At all stages of apoptosis, the nucleus and cytoplasm of U-937 cells contained proteasomes, which were represented by small rod-shaped osmiophilic particles approximately 12 × 30 nm in size. These particles formed aggregates of varying density and size, not covered with a membrane. We assumed that they are similar to the “processing bodies,” or aggresomes, which were described in the literature. The latter are detected in cells under conditions of suppressed transcription in the nucleus and, according to some researchers, are involved in the storage and degradation of various mRNAs, RNPs, and proteins. X-ray spectral analysis revealed changes in the intracellular content of Na+ and K+ at the level of single cells during apoptosis induced by osmotic shock. An increased ratio of intracellular Na+/K+ compared to the control for the majority of cells in apoptosis was shown.

Background/Aims: Many vital processes in animal cells depend on monovalent ion transport across the plasma membrane via specific pathways. Their operation is described by a set of nonlinear and transcendental equations that cannot be solved analytically. Previous computations had been optimized for certain cell types and included parameters whose experimental determination can be challenging. Methods: We have developed a simpler and a more universal computational approach by using fewer kinetic parameters derived from the data related to cell balanced state. A file is provided for calculating unidirectional Na + , K + , and Cl – fluxes via all major pathways (i.e. the Na/K pump, Na + , K + , Cl – channels, and NKCC, KC and NC cotransporters) under a balanced state and during transient processes. Results: The data on the Na + , K + , and Cl – distribution and the pump flux of K + (Rb + ) are obtained on U937 cells before and after inhibiting the pump with ouabain. There was a good match between the results of calculations and the experimentally measured dynamics of ion redistribution caused by blocking the pump. Conclusion: The presented approach can serve as an effective tool for analyzing monovalent ion transport in the whole cell, determination of the rate coefficients for ion transfer via major pathways and studying their alteration under various conditions.

Lithium transport across the cell membrane is interesting in the light of general cell physiology and becau- se of its alteration during numerous human diseases. The mechanism of Li4 transfer has been studied mainly in erythrocytes with a slow kinetics of ion exchange and therefore under the unbalanced ion distribution. Prolife- rating cultured cells with a rapid ion exchange have not been used practically in study of Li4 transport. In pre- sent paper, the kinetics of Li4 uptake and exit as well as its balanced distribution across the plasma membrane of U937 cells were studied at minimal external Li+ concentrations and after the whole replacement of external Na+ for Li+. It has been found that a steady state Li+ distribution is attained at a high rate similar to that for Na+ and Cl- and that Li+/Na+ discrimination under the balanced ion distribution at 1-10 mM external Li+ keeps on 3 and drops to 1 following blocking of the Na,K-ATPase pump by ouabain. About of 80% of the total Li+ flux across the plasma membrane under the balanced Li+ distribution at 5 mM external Li+ accounts for the equiva- lent Li+/Li+ exchange. The most part of the Li+ flux into the cell down the electrochemical gradient is a flux through channels and its small part may account for the NC and NKCC cotransport influxes. The downhill Li+ influxes are balanced by the uphill Li+ efflux involved in Li+/Na+ exchange. The Na+ flux involved in the countertransport with the Li+ accounts for about 0.5% of the total Na+ flux across the plasma membrane. The study of Li+ transport is an important approach to understand the mechanism of the equivalent Li+/Li+/Na+/Na+ exchange because no blockers of this mode of ion transfer are known and it cannot be revealed by electrophysiological methods. Cells treated with the medium where Na+ is replaced for Li+ are recommended as an object for studying cells without the Na,K-ATPase pump and with very low intracellular Na+ and K+ concentration.

Background/Aims: Osmotic cell shrinkage is a powerful trigger of suicidal cell death or apoptosis, which is paralleled and enforced by apoptotic volume decrease (AVD). Cells counteract cell shrinkage by volume regulatory increase (RVI). The present study explored the response of human U937 cells to hypertonic solution thus elucidating the relationship between RVI and AVD. Methods: Cell water, concentration of monovalent ions and the appearance of apoptotic markers were followed for 0.5-4 h after the cells were transferred to a hypertonic medium. Intracellular water, K+, Na+, and Cl– content, ouabain-sensitive and -resistant Rb+ influxes were determined by measurement of the cell buoyant density in Percoll density gradient, flame emission analysis and 36Cl– assay, respectively. Fluorescent microscopy of live cells stained by acridine orange and ethidium bromide was used to verify apoptosis. Results: After 2-4 h incubation in hypertonic media the cell population was split into light (L) and heavy (H) fractions. According to microscopy and analysis of monovalent ions the majority of cells in the L population were healthy, while the H fractions were enriched with apoptotic cells. The density of L cells was decreasing with time, while the density of H cells was increasing, thus reflecting the opposite effects of RVI and AVD. At the same time, some of the cells were shifting from L to H fractions, indicating that apoptosis was gradually extending to cells that were previously displaying normal RVI. Conclusion: The findings suggest that apoptosis can develop in cells capable of RVI.

Cells dying according to the apoptotic program, unlike cells dying via an unprogrammed mode, are able to avoid swelling and osmotic bursting with membrane disruption.There are indications that apoptosis is accompanied by suppression of the Na+/K+ pump and changes in the K+ and Cl− channels. It remains unclear how ion fluxes through individual ion pathways are integrated so as to induce loss of intracellular ions and concomitant apoptotic volume decrease. A decrease in activity of the sodium pump during apoptosis should cause cell swelling rather than shrinkage. We have made the first systemic analysis of the monovalent ion flux balance in apoptotic cells. Experimental data were obtained for human U937 cells treated with staurosporine for 4–5 h, which is known to induce apoptosis. The data include cellular Cl− content and fluxes, K+, Na+, water content and ouabain-sensitive and -resistant Rb+ fluxes.Unidirectional monovalent ion fluxeswere calculated using these data and a cell model comprising the double Donnan system with the Na+/K+ pump, Cl−, K+, Na+ channels, the Na+–K+–2Cl−cotransporter (NKCC), the Na+–Cl− cotransporter (NC), and the equivalent Cl−/Cl− exchange.Apoptotic cell shrinkage was found to be caused, depending on conditions, either by an increase in the integral channel permeability of membrane for K+ or by suppression of the pump coupledwith a decrease in the integral channel permeability of membrane for Na+. The decrease in the channel permeability of membrane for Na+ plays a crucial role in cell dehydration in apoptosis accompanied by suppression of the pump. Supplemental Table S1 is given for easy calculating flux balance under specified conditions.

The K+, Na+, and Cl− balance and K+ (Rb+) and 36Cl− fluxes in U937 cells induced to apoptosis by 0.2 or 1 μM staurosporine were studied using flame emission and radioisotope techniques. It is found that two-thirds of the total decrease in the amount of intracellular osmolytes in apoptotic cells is accounted for by monovalent ions and one-third consists of other intracellular osmolytes. A decrease in the amount of monovalent ions results from a decrease in the amount of K+ and Cl− and an increase in the Na+ content. The rate of 36Cl−, Rb+ (K+), and 22Na+ equilibration between cells and the medium was found to significantly exceed the rate of apoptotic change in the cellular ion content, which indicates that unidirectional influxes and effluxes during apoptosis may be considered as being in near balance. The drift of the ion flux balance in apoptosis caused by 0.2 μM staurosporine was found to be associated with the increased ouabain-resistant Rb+ (K+) channel influx and insignificantly altered the ouabain-sensitive pump influx. Severe apoptosis induced by 1 μM staurosporine is associated with reduced pump fluxes and slightly changed channel Rb+ (K+) fluxes. In apoptotic cells, the 1.4–1.8-fold decreased Cl− level is accompanied by a 1.2–1.6-fold decreased flux.

The balance of K+, Na+, and Cl− fluxes across the cell membrane with the Na+/K+ pump, ion channels, and Na+K+2Cl− (NKCC) and Na+-Cl− (NC) cotransport was calculated to determine the mechanism of cell shrinkage in apoptosis. It is shown that all unidirectional K+, Na+, and Cl− fluxes; the ion channel permeability; and the membrane potential can be found using the principle of the flux balance if the following experimental data are known: K+, Na+, and Cl− concentrations in cell water; total Cl− flux; total K+ influx; and the ouabain-inhibited pump component of the Rb+(K+) influx. The change in different ionic pathways during apoptosis was estimated by calculations based on the data reported in the preceded paper (Yurinskaya et al., 2010). It is found that cell shrinkage and the shift in ion balance in U937 cells induced to apoptosis with 1 μM staurosporine occur due to the coupling of reduced pump activity with a decrease in the integral permeability of Na+ channels, whereas K+ and Cl− channel permeability remains almost unchanged. Calculations show that only a small part of the total fluxes of K+, Na+, and Cl− account for the fluxes mediated by NKCC and NC cotransporters. Despite the importance of cotransport fluxes for maintaining the nonequilibrium steady-state distribution of Cl−, they cannot play a significant role in apoptotic cell shrinkage because of their minority and cannot be revealed by inhibitors.

Ouabain-sensitive (OS) and -resistant (OR) Rb(+) influx was examined in three sublines of U937 cells to compare alterations of K(+) channel permeability and the Na(+),K(+)-ATPase pump leading to the shift in ion and water balance during apoptosis induced by 0.2 and 1microM staurosporine (STS) for 4-5 h. Cell K(+), Rb(+), Na(+) and Cl(-) content was determined by flame photometry and (36)Cl distribution. Changes in cell water content were monitored by measurement of buoyant cell density and distribution of [(3)H]-glycerol or 3-O-methyl-D-[(3)H]glucose. Apoptosis was detected by DNA flow cytometry and light microscopy of the native cells stained with acridine orange. Treatment with 0.2 microM STS for 5 hours led to mild apoptosis with 10-13 % cell dehydration and either moderate increase of channel mediated Rb(+) influx without significant changes in the pump activity or moderate decrease of pump Rb(+) influx without significant change of channel influx, depending on the cell line used. Treatment with 1 microM STS was followed by 18-23 % cell dehydration, a decrease of the pump activity and a small or insignificant increase in the OR Rb(+) influx in all studied sublines. It is concluded that moderate apoptotic cell shrinkage may be associated with both an increase in K(+) channel permeability and inhibition of the pump whereas more remarkable shrinkage occurs presumably due to inhibition of the pump.

Unidirectional (22)Na, Li(+) and Rb(+) fluxes and net fluxes of Na(+) and K(+) were measured in U937 human leukemic cells before and after induction of apoptosis by staurosporine (1 microM, 4 h) to answer the question which ion transporter(s) are responsible for changes in cell ion and water balance at apoptosis. The original version of the mathematical model of cell ion and water balance was used for analysis of the unidirectional ion fluxes under the balanced distribution of major monovalent ions across the cell membrane. The values of all major components of the Na(+) and K(+) efflux and influx, i.e. fluxes via the Na(+),K(+)-ATPase pump, Na(+) channels, K(+) channels, Na/Na exchanger and Na-Cl symport were determined. It is concluded that apoptotic cell shrinkage and changes in Na(+) and K(+) fluxes typical of apoptosis in U937 cells induced by staurosporine are caused by a complex decrease in the pump activity, Na-Cl symport and integral Na(+) channel permeability.

INTRODUCTION: Hypertonic media causes cells to shrink due to water loss through aquaporin channels. After acute shrinkage, cells either regulate their volume or, alternatively, undergo a number of metabolic changes which ultimately lead to cell death. In many cell types, hypertonic shrinkage is followed by apoptosis. Due to the complex 3D morphology of skeletal muscle and the difficulty in obtaining isolated human tissue, we have begun skeletal muscle volume regulation studies using the human skeletal muscle cell line TE671RD. In this study we investigated whether hypertonic challenge of the human skeletal muscle cell line TE671RD triggered cell death or evoked a cell volume recovery response. METHODS: The cellular volume of TE671RD cells was calculated from the 2D surface area. Cell death was assessed by both the trypan blue live/dead assay and the TUNEL assay. RESULTS: Medium osmolality was increased by addition of up to 200mM sucrose. Addition of 200mM sucrose resulted in mean cell shrinkage of 44±1% after 30mins. At later time points (2 and 4 hrs) two separate cell subpopulations with differing mean cell volume became apparent. The first subpopulation (15±2% of the total cell number) continued to shrink whereas the second subpopulation had an increased cell volume. Cell death was observed in a small proportion of cells (approximately 6-8%). CONCLUSION: We have established that a substantial proportion of TE671RD cells respond to hypertonic challenge with RVI, but that these cells are resistant to hypertonicity triggered cell death.

Introduction Hypertonic media causes cells to shrink due to water loss through aquaporin channels. After acute shrinkage, cells either regulate their volume or, alternatively, undergo a number of metabolic changes which ultimately lead to cell death. In many cell types, hypertonic shrinkage is followed by apoptosis. Due to the complex 3D morphology of skeletal muscle and the difficulty in obtaining isolated human tissue, we have begun skeletal muscle volume regulation studies using the human skeletal muscle cell line TE671RD. In this study we investigated whether hypertonic challenge of the human skeletal muscle cell line TE671RD triggered cell death or evoked a cell volume recovery response. Methods The cellular volume of TE671RD cells was calculated from the 2D surface area. Cell death was assessed by both the trypan blue live/dead assay and the TUNEL assay. Results Medium osmolality was increased by addition of up to 200mM sucrose. Addition of 200mM sucrose resulted in mean cell shrinkage of 44±1% after 30mins. At later time points (2 and 4 hrs) two separate cell subpopulations with differing mean cell volume became apparent. The first subpopulation (15±2% of the total cell number) continued to shrink whereas the second subpopulation had an increased cell volume. Cell death was observed in a small proportion of cells (approximately 6-8%). Conclusion We have established that a substantial proportion of TE671RD cells respond to hypertonic challenge with RVI, but that these cells are resistant to hypertonicity triggered cell death.

Cell proliferation and apoptosis are paralleled by altered regulation of ion channels that play an active part in the signaling of those fundamental cellular mechanisms. Cell proliferation must--at some time point--increase cell volume and apoptosis is typically paralleled by cell shrinkage. Cell volume changes require the participation of ion transport across the cell membrane, including appropriate activity of Cl- and K+ channels. Besides regulating cytosolic Cl- activity, osmolyte flux and, thus, cell volume, most Cl- channels allow HCO3- exit and cytosolic acidification, which inhibits cell proliferation and favors apoptosis. K+ exit through K+ channels may decrease intracellular K+ concentration, which in turn favors apoptotic cell death. K+ channel activity further maintains the cell membrane potential, a critical determinant of Ca2+ entry through Ca2+ channels. Cytosolic Ca2+ may trigger mechanisms required for cell proliferation and stimulate enzymes executing apoptosis. The switch between cell proliferation and apoptosis apparently depends on the magnitude and temporal organization of Ca2+ entry and on the functional state of the cell. Due to complex interaction with other signaling pathways, a given ion channel may play a dual role in both cell proliferation and apoptosis. Thus, specific ion channel blockers may abrogate both fundamental cellular mechanisms, depending on cell type, regulatory environment and condition of the cell. Clearly, considerable further experimental effort is required to fully understand the complex interplay between ion channels, cell proliferation and apoptosis.

Staurosporine (STS) and etoposide (Eto) induced apoptosis of the human histiocytic lymphoma cells U937 were studied to determine the role of monovalent ions in apoptotic cell shrinkage. Cell shrinkage, defined as cell dehydration, was assayed by measurement of buoyant density of cells in continuous Percoll gradient. The K + and Na + content in cells of different density fractions was estimated by flame emission analysis. Apoptosis was evaluated by confocal microscopy and flow cytometry of acridine orange stained cells, by flow DNA cytometry and by effector caspase activity. Apoptosis of U937 cells induced by 1 µM STS for 4 h was found to be paralleled by an increase in buoyant density indicating cell shrinkage. An increase in density was accompanied by a decrease in K + content (from 1.1 to 0.78 mmol/g protein), which exceeded the increase in Na + content (from 0.30 to 0.34 mmol/g) and resulted in a significant decrease of the total K + and Na + content (from 1.4 to 1.1 mmol/g). In contrast to STS, 50 µM Eto for 4 h or 0.8-8 µM Eto for 18-24 h induced apoptosis without triggering cell shrinkage. During apoptosis of U937 cells induced by Eto the intracellular K + /Na + ratio decreased like in the cells treated with STS, but the total K + and Na + content remained virtually the same due to a decrease in K + content being nearly the same as an increase in Na + content. Apoptotic cell dehydration correlated with the shift of the total cellular K + and Na + content. There was no statistically significant decrease in K + concentration per cell water during apoptosis induced by either Eto (by 13.5%) or STS (by 8%), whereas increase in Na + concentration per cell water was statistically significant (by 27% and 47%, respectively). The data show that apoptosis can occur without cell shrinkage-dehydration, that apoptosis with shrinkage is mostly due to a decrease in cellular K + content, and that this decrease is not accompanied by a significant decrease of K + concentration in cell water.

The mechanism of apoptotic cell volume decrease was studied in rat thymocytes treated with dexamethasone (Dex) or etoposide (Eto). Cell shrinkage, i.e. dehydration, was quantified by using buoyant den

Ample pharmacological evidence points to a role of kinases in the regulation of cell volume. Given the limited selectivity of most inhibitors, however, the specific molecules involved have remained largely elusive. The search for cell volume regulated genes in liver HepG2 cells led to the discovery of the human serum- and glucocorticoid-dependent serine/threonine kinase hsgk1. Transcription and expression of hsgk1 is markedly and rapidly upregulated by osmotic and isotonic cell shrinkage. The effect of osmotic cell shrinkage on hsgk1 is mediated by p38 kinase. Further stimuli of hsgk1 transcription include glucocorticoids, aldosterone, TGF-β1, serum, increase of intracellular Ca 2+ and phorbolesters, whereas cAMP downregulates hsgk1 transcription. The hsgk1 protein is expressed in several epithelial tissues including human pancreas, intestine, kidney, and shark rectal gland. Co-expression of hsgk1 with the renal epithelial Na + -channel ENaC or the Na + /K + /2Cl − -cotransporter NKCC2 (BSC1) in Xenopus oocytes , accelerates insertion of the transport proteins into the cell membrane and thus, stimulates channel or transport activity. Thus, hsgk1 participates in the regulation of transport by steroids and secretagogues increasing intracellular Ca 2+ -activity. The stimulation of hsgk1 transcription by TGF-β1 may further bear pathophysiological relevance.

This work, using RT PCR, studied expression of mRNAs encoding ion transporters, the Na/H antiporter (NHE1), the beta subunit of the Na,K-ATPase pump (ATP1B1), the NaK2Cl symporter (NKCC1), and some proteins unrelated to ion transport: the serum and glucocorticoid dependent kinase (hSGK), beta-actin, a glycolytic enzyme (GAPDH), and regulators of proliferation and apoptosis (p53, Bcl-2) during activation of human lymphocytes with phytohemagglutinin for 4-24 h. Within 24 hours the mRNA levels of NHE1, beta-actin, Bcl-2, and p53 increased by more than 100%, the mRNA levels of ATP1B1, GAPDH, and hSGK, by about 50%, while the mRNA levels of NKCC1 decreased transiently. These results indicate a differential transcriptional control of NHE1, ATP1B1, and NKCC1 following a proliferative stimulus of human lymphocytes.

During the winter pre-spawning migration, lampreys Lampetra fluviatilis stop feeding, and their liver metabolism is reduced substantially. Aerobic ATP production in hepatocytes decreased to one third and ATP content decreased by 50% as compared with the values in autumn. In spite of the decrease of endogenous and phosphorylating (oligomycin-sensitive) respiration in winter, the oxygen consumption used to drive sodium and potassium pumping through Na,K-ATPase activity (ouabain-sensitive respiration) remained virtually constant. Consequently its share in phosphorylating respiration increased from 16·3% in November to 54·2% in February. Potassium influx was similar within the range of ATP content between 2·5 and 1 nmol 10−6 cells and decreases only in hepatocytes which contained

Functional expression of Na,K-ATPase pump as determined by ouabain-sensitive Rb influxes has been investigated in human peripheral blood lymphocytes, activated by phytohemagglutinin (PHA) from resting state to proliferation. It is found that a rapid twofold elevation of ouabain-sensitive Rb influx in response to PHA is followed by a long-term increase in pump activity, which precedes the DNA synthesis and is temporally related to the growth phase of mitogenic response. Unlike the early pump activation, the late enhanced pump activity is not the result of elevated cell Na content, it is inhibited by cycloheximide and requires new protein synthesis. Actinomycin D and α-amanitin, in doses, which suppress the PHA-induced increase in the RNA synthesis, do not abolish the elevated Rb influx until 20–24h of mitogenic activation and inhibit the late, growth-associated increase in Rb influx. It is concluded that (1) in mitogen-activated cells both short- and long-term control is involved in the enhanced pump activity, and (2) translational and transcriptional mechanisms may contribute to the long-term up-regulation of Na,K-ATPase pump during blast transformation of human lymphocytes.

Monovalent ion traffic across the cell membrane occurs via various pathways. Evaluation of individual fluxes in whole cell is hampered by their strong interdependence. This difficulty can be overcome by computational analysis of the whole cell flux balance. However, the previous computational studies disregarded ion movement of the self-exchange type. We have taken this exchange into account. The developed software allows determination of unidirectional fluxes of all monovalent ions via the major pathways both under the balanced state and during transient processes. We show how the problem of finding the rate coefficients can be solved by measurement of monovalent ion concentrations and some of the fluxes. Interdependence of fluxes due to the mandatory conditions of electroneutrality and osmotic balance and due to specific effects can be discriminated, enabling one to identify specific changes in ion transfer machinery under varied conditions. To test the effectiveness of the developed approach we made use of the fact that Li/Na exchange is known to be an analogue of the coupled Na/Na exchange. Thus, we compared the predicted and experimental data obtained on U937 cells under varied Li+ concentrations and following inhibition of the sodium pump with ouabain. We found that the coupled Na/Na exchange in U937 cells comprises a significant portion of the entire Na+ turnover. The data showed that the loading of the sodium pump by Li/Na exchange involved in the secondary active Li+ transport at 1-10 mM external Li+ is small. This result may be extrapolated to similar Li+ and Na+ flux relationships in erythrocytes and other cells in patients treated with Li+ in therapeutic doses. The developed computational approach is applicable for studying various cells and can be useful in education for demonstrating the effects of individual transporters and channels on ion gradients, cell water content and membrane potential.

Increase in Na + ,K + -ATPase mRNAs was detected in activated lymphocytes by the RT-PCR method. α1 subunit mRNA gradualy increased with time and by 36 h was 2.4 times higher than at the start. Increase in the β1 mRNA was transient reaching a maximum in the 8 h probe and declining to the initial level in the 24 and 36 h probes. The elevation of Na + ,K − -ATPase mRNAs does not underlie a cycloheximide-inhibited increase in cation pumping peculiar to the prereplicative period as can be judged from the fact that Act D fails to eliminate PHA-induced enhancement of pump fluxes.

Ouabain-sensitive (OS) and -resistant (OR) Rb(+) influx was examined in three sublines of U937 cells to compare alterations of K(+) channel permeability and the Na(+),K(+)-ATPase pump leading to the shift in ion and water balance during apoptosis induced by 0.2 and 1microM staurosporine (STS) for 4-5 h. Cell K(+), Rb(+), Na(+) and Cl(-) content was determined by flame photometry and (36)Cl distribution. Changes in cell water content were monitored by measurement of buoyant cell density and distribution of [(3)H]-glycerol or 3-O-methyl-D-[(3)H]glucose. Apoptosis was detected by DNA flow cytometry and light microscopy of the native cells stained with acridine orange. Treatment with 0.2 microM STS for 5 hours led to mild apoptosis with 10-13 % cell dehydration and either moderate increase of channel mediated Rb(+) influx without significant changes in the pump activity or moderate decrease of pump Rb(+) influx without significant change of channel influx, depending on the cell line used. Treatment with 1 microM STS was followed by 18-23 % cell dehydration, a decrease of the pump activity and a small or insignificant increase in the OR Rb(+) influx in all studied sublines. It is concluded that moderate apoptotic cell shrinkage may be associated with both an increase in K(+) channel permeability and inhibition of the pump whereas more remarkable shrinkage occurs presumably due to inhibition of the pump.

Alterations of cell volume are key events during both cell proliferation and apoptotic cell death. Cell proliferation eventually requires an increase of cell volume, and apoptosis is typically paralleled by cell shrinkage. Alterations of cell volume require the participation of ion transport across the cell membrane, including appropriate activity of Cl(-) and K(+) channels. Cl(-) channels modify cytosolic Cl(-) activity and mediate osmolyte flux, and thus influence cell volume. Most Cl(-) channels allow exit of HCO(3)(-), leading to cytosolic acidification, which in turn inhibits cell proliferation and favors apoptosis. K(+) exit through K(+) channels decreases cytosolic K(+) concentration, which may sensitize the cell for apoptotic cell death. K(+) channel activity further maintains the cell membrane potential, a critical determinant of Ca(2+) entry through Ca(2+) channels. Ca(2+) may, in addition, enter through Ca(2+)-permeable cation channels, which, in some cells, are activated by hyperosmotic shock. Increases of cytosolic Ca(2+) activity may trigger both mechanisms required for cell proliferation and mechanisms, leading to apoptosis. Thereby cell proliferation and apoptosis depend on magnitude and temporal organization of Ca(2+) entry, as well as activity of other signaling pathways. Accordingly, the same ion channels may participate in the stimulation of both cell proliferation and apoptosis. Specific ion channel blockers may thus abrogate both cellular mechanisms, depending on cell type and condition.

Cell proliferation must be accompanied by increase of cell volume and apoptosis is typically paralleled by cell shrinkage. Moreover, profound osmotic cell shrinkage may trigger apoptosis. In isotonic environment cell volume changes require the respective alterations of transport across the cell membrane. Cell proliferation is typically paralleled by activation of K(+) channels, which is required for the maintenance of the cell membrane potential, a critical determinant of Ca(2+) entry through Ca(2+) channels. The Ca(2+) entry leads to oscillations of cytosolic Ca(2+) activity which is followed by activation of Ca(2+) dependent transcription factors and by depolymerization of the actin filament network. The latter disinhibits the Na(+) H(+) exchanger and Na(+) , K(+) , 2Cl(-)cotransport thus leading to cell swelling. At some point transient activation of Cl(-) channels is required leading to transient decrease of cell volume. Apoptosis is typically paralleled by sustained activation of Cl(-) channels leading to Cl(-) , HCO-(3) and osmolyte exit. The subsequent cell shrinkage and cytosolic acidification are not counter-regulated by activation of the Na(+) /H(+) exchanger, which is inhibited and eventually degraded during apoptosis. At a later stage K(+) exit through K(+) channels decreases intracellular K(+) concentration and facilitates cell shrinkage. Sustained or excessive increase of Ca(+) triggers apoptotic cell death, typically paralleled by cell shrinkage due to activation of Ca(2+) sensitive K(+) channels. Cellular K(+) loss and cell shrinkage are supportive but not required for the induction of apoptosis. On the other hand, several studies point to a critical role of K(+) -channel inhibition in the initiation of apoptosis. Thus, alterations of K(+) channel and Ca(2+) channel activities may participate in the triggering of both, cell proliferation and apoptosis. The impact of those channels depends on magnitude and temporal organization of channel activation and on the activity of further signaling mechanisms. Accordingly, the same ion channel blockers may interfere with both, cell proliferation and apoptosis depending on cell type, regulatory environment and condition of the cell.

The transition of phytohemagglutinin-activated human lymphocytes from resting state to proliferation is accompanied by a long-term increase in ouabain-sensitive Rb(K) influx which is closely related to a cyclosporin A-sensitive step of G0/G1/S progression. At least two distinct phases of the up-regulation of cation pump has been revealed: the initial stage (5–20 h) which is cycloheximide-inhibitable and actinomycin D (α-amanitin)-unaffected, and the later stage (after 20 h) which is cycloheximide- and actinomycin D (α-amanitin)-inhibitable. Thus, the enhanced Na,K-ATPase pump during the cell progression from quiescence to proliferation is controlled both at translational and transcriptional levels.