Valentina E. Yurinskaya

Potassium and Sodium Balance in U937 Cells During Apoptosis With and Without Cell Shrinkage

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.

Thymocyte K + , Na + and Water Balance During Dexamethasone- and Etoposide-Induced Apoptosis

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 density of the thymocytes in a continuous Percoll gradient. The K+ and Na+ content of cells from different density fractions were assayed by flame emission analysis. Apoptosis was tested by microscopy and flow cytometry of acridine orange stained cells as well as by flow DNA cytometry. Treatment of the thymocytes with 1 µM Dex for 4-5.5 h or 50 µM Eto for 5 h resulted in the appearance of a new distinct high-density cell subpopulation. The cells from this heavy subpopulation but not those with normal buoyant density had typical features of apoptosis. Apoptotic increase of cell density was accompanied by a decrease in cellular K+ content, which exceeded the simultaneous increase in cellular Na+ content. Cellular loss of K+ contributed to most of the estimated loss of cellular osmolytes, but owing to the parallel loss of cell water, the decrease in cytosolic K+ concentration was less than one third. Due to gain of Na+ and loss of cell water the cytosolic Na+ concentration in thymocytes rose following treatment with Dex (5.5 h) or Eto (5 h) by a factor of about 3.6 and 3.1, respectively.

Differential Transcription of Ion Transporters, NHE1, ATP1B1, NKCC1 in Human Peripheral Blood Lymphocytes Activated to Proliferation

This work, using RT PCR, studied expression of mRNAs encoding ion transporters, the Na/H antiporter (NHE1), the β 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), β-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, β-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.

Balance of unidirectional monovalent ion fluxes in cells undergoing apoptosis: why does Na+/K+ pump suppression not cause cell swelling?

Non‐technical summary  Apoptosis is a crucial mechanism for tissue maintenance and deregulation of apoptosis may lead to catastrophic consequences in humans (e.g. cancer). The present work is a first attempt to quantitatively characterize rearrangement of the monovalent ion fluxes in cells during apoptosis. An established model of apoptosis induced by staurosporine in lymphoid U937 cells is used to experimentally measure cellular Cl− content and fluxes, K+, Na+ and water content as well as ouabain‐sensitive and ‐resistant Rb+ fluxes. A mathematical model is developed to account for the unidirectional ion fluxes and water balance in a cell as a whole. A decrease in the channel permeability of the plasma membrane for Na+ proved to be crucial for preventing cell swelling due to the decrease in Na+/K+ pump activity in cells undergoing apoptosis whereas opening of the K+ and Cl− channels is not required. Supplemental Table S1 is given for easy calculating flux balance under specified conditions.

Analysis of the monovalent ion fluxes in U937 cells under the balanced ion distribution : Recognition of ion transporters responsible for changes in cell ion and water balance during apoptosis

Unidirectional 22Na, 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 μM, 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.

Dual Response of Human Leukemia U937 Cells to Hypertonic Shrinkage: Initial Regulatory Volume Increase (RVI) and Delayed Apoptotic Volume Decrease (AVD)

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.

The role of potassium, potassium channels, and symporters in the apoptotic cell volume decrease: experiment and theory.

Ample evidences indicate an essential role of ion channels in apoptosis [3, 4]. The channels permeable for K + ions are of primary interest because the efflux of K + via channels is believed to be responsible for cell volume decrease, known as a hallmark of apoptosis. It has been shown that at least 14 species of K + channels are involved in apoptosis of various cells [5, 6]. The role of K + channels in the regulation of apoptosis is assumed to be related to the influence of intracellular K + concentration on apoptotic caspases and nucleases [6–8]. However, this assumption is based mostly on the study of the shift in intracellular K + concentration with use of a fluorescent probe and measurement of the forward light scattering. These methods are not adequate for the quantitative analysis of the ion and water balance of cells. We studied changes in the intracellular content and concentration of K + during apoptotic decrease in cell volume using other methods. Cell water was assayed by measuring the buoyant density of cells in a continuous Percoll gradient; cell K + and Na + , by flame emission analysis. Well-known models of apoptosis were studied, namely, the apoptosis of rat thymocytes induced by dexamethasone (1 μ M, 4–6 h) and the apoptosis of human lymphoma cell line U937 treated with staurosporine (1 μ M, 4 h). Apoptosis was examined using confocal microscopy and flow cytometry. The methods were described in details earlier [9, 10].

Regulatory volume increase (RVI) and apoptotic volume decrease (AVD) in U937 cells in hypertonic medium

Changes in intracellular water, K+ and Na+ of U937 cells incubated in hyperosmolar medium supplemented with 200 mM sucrose have been studied. Cells were stained with acrydine orange, ethydium bromide, APOPercentage dye, which marks the phosphatidyl serine distribution on the plasma membrane; and FLICA polycaspase fluorescent dye. It was found that cell shrinkage produced by direct osmotic effect induced both a regulatory volume increase and apoptotic volume decrease. The regulatory volume increase dominated at the early stage, whereas apoptotic volume decrease prevailed at the later stage. Therefore, U937 cells were capable of triggering apoptosis and apoptotic volume decrease, despite the unimpaired regulatory volume increase response, and the current opinion that the dysfunction of the regulatory volume increase is a prerequisite for apoptosis and apoptotic volume decrease (Subramanyam et al., 2010) should be revised. It is concluded that the apoptotic volume decrease plays a significant role in preventing osmotic lysis in apoptotic cells, rather than in initiating apoptosis.

Computation of Pump-Leak Flux Balance in Animal Cells

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.

Li/Na Exchange and Li Active Transport in Human Lymphoid Cells U937 Cultured in Lithium Media

Lithium transport across the cell membrane is interesting in the light of general cell physiology and because of its alteration during numerous human diseases. The mechanism of Li+ transfer has been studied mainly in erythrocytes with a slow kinetics of ion exchange and therefore under the unbalanced ion distribution. Proliferating cultured cells with a rapid ion exchange have not been used practically in study of Li+ transport. In the present paper, the kinetics of Li+ 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 is found that a balanced Li+ distribution attained at a high rate similar to that for Na+ and Cl− and that Li+/Na+ discrimination under balanced ion distribution at 1–10 mM external Li+ stays on 3 and drops to 1 following Na, K-ATPase pump blocking by ouabain. About 80% of the total Li+ flux across the plasma membrane under the balanced Li+ distribution at 5 mM external Li+ accounts for the equivalent Li+/Li+ exchange. The majority 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 understanding 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 cultured in 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.