Unidirectional fluxes of monovalent ions in human erythrocytes compared with lymphoid U937 cells: Transient processes after stopping the sodium pump and in response to osmotic challenge.

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

Mesenchymal Stem/Stromal Cells in Three-Dimensional Cell Culture: Ion Homeostasis and Ouabain-Induced Apoptosis

This study describes the changes in ion homeostasis of human endometrial mesenchymal stem/stromal cells (eMSCs) during the formation of three-dimensional (3D) cell structures (spheroids) and investigates the conditions for apoptosis induction in 3D eMSCs. Detached from the monolayer culture, (2D) eMSCs accumulate Na+ and have dissipated transmembrane ion gradients, while in compact spheroids, eMSCs restore the lower Na+ content and the high K/Na ratio characteristic of functionally active cells. Organized as spheroids, eMSCs are non-proliferating cells with an active Na/K pump and a lower K+ content per g cell protein, which is typical for quiescent cells and a mean lower water content (lower hydration) in 3D eMSCs. Further, eMSCs in spheroids were used to evaluate the role of K+ depletion and cellular signaling context in the induction of apoptosis. In both 2D and 3D eMSCs, treatment with ouabain (1 µM) results in inhibition of pump-mediated K+ uptake and severe K+ depletion as well as disruption of the mitochondrial membrane potential. In 3D eMSCs (but not in 2D eMSCs), ouabain initiates apoptosis via the mitochondrial pathway. It is concluded that, when blocking the Na/K pump, cardiac glycosides prime mitochondria to apoptosis, and whether a cell enters the apoptotic pathway depends on the cell-specific signaling context, which includes the type of apoptotic protein expressed

Mesenchymal Stem/Stromal Cells in Three-Dimensional Cell Culture: Ion Homeostasis and Ouabain-Induced Apoptosis

This study describes the changes in ion homeostasis of human endometrial mesenchymal stem/stromal cells (eMSCs) during the formation of three-dimensional (3D) cell structures (spheroids) and investigates the conditions for apoptosis induction in 3D eMSCs. Detached from the monolayer culture, (2D) eMSCs accumulate Na+ and have dissipated transmembrane ion gradients, while in compact spheroids, eMSCs restore the lower Na+ content and the high K/Na ratio characteristic of functionally active cells. Organized as spheroids, eMSCs are non-proliferating cells with an active Na/K pump and a lower K+ content per g cell protein, which is typical for quiescent cells and a mean lower water content (lower hydration) in 3D eMSCs. Further, eMSCs in spheroids were used to evaluate the role of K+ depletion and cellular signaling context in the induction of apoptosis. In both 2D and 3D eMSCs, treatment with ouabain (1 µM) results in inhibition of pump-mediated K+ uptake and severe K+ depletion as well as disruption of the mitochondrial membrane potential. In 3D eMSCs (but not in 2D eMSCs), ouabain initiates apoptosis via the mitochondrial pathway. It is concluded that, when blocking the Na/K pump, cardiac glycosides prime mitochondria to apoptosis, and whether a cell enters the apoptotic pathway depends on the cell-specific signaling context, which includes the type of apoptotic protein expressed

Time-Dependent Regulation of IL-2R α-Chain (CD25) Expression by TCR Signal Strength and IL-2-Induced STAT5 Signaling in Activated Human Blood T Lymphocytes.

The expression of the IL-2R α-chain (IL-2Rα) is regulated at the transcriptional level via TCR- and IL-2R-signaling. The question is how to precede in time the activation signals to induce the IL-2Rα expression in native primary T cells. By comparing the effects of selective drugs on the dynamics of CD25 expression during the mitogen stimulation of human peripheral blood lymphocytes, we identified distinct Src- and JAK-dependent stages of IL-2Rα upregulation. PP2, a selective inhibitor of TCR-associated Src kinase, prevents CD25 expression at initial stages of T cell activation, prior to the cell growth. This early IL-2Rα upregulation underlies the T cell competence and the IL-2 responsiveness. We found that the activated with "weak" mitogen, the population of blood lymphocytes has some pool of competent CD25+ cells bearing a high affinity IL-2R. A distinct pattern of IL-2R signaling in resting and competent T lymphocytes has been shown. Based on the inhibitory effect of WHI-P131, a selective drug of JAK3 kinase activity, we concluded that in quiescent primary T lymphocytes, the constitutive STAT3 and the IL-2-induced prolonged STAT5 activity (assayed by tyrosine phosphorylation) is mostly JAK3-independent. In competent T cells, in the presence of IL-2 JAK3/STAT5 pathway is switched to maintain the higher and sustained IL-2Rα expression as well as cell growth and proliferation. We believe that understanding the temporal coordination of antigen- and cytokine-evoked signals in primary T cells may be useful for improving immunotherapeutic strategies

Stimulation with non-mitogenic PHA and IL-2 leads to long-term CD25 expression and proliferation in human blood T lymphocytes.

<p>(A) The representative histograms of one experiment on PBL from one donor are shown. PBL were not stimulated (CTR) or stimulated with 0.7 μg/ml (0.7PHA), or 10 μg/ml (10PHA) PHA, or 200 U/ml IL-2 (IL-2) for 48 h. In the same experiment, PBL were preincubated in culture medium with 0.7 μg/ml PHA and 80 μM WHI-P131 (WHI) or 1.0 μM PP2 for 24 h prior to IL-2 stimulation, or drugs were added simultaneously with 0.7 μg/ml PHA for 24 h and thereafter IL-2 was added for the next 24 h. Additional Y-axis is 4 decades logarithmic scale. (B) CD25 expression in resting PBL in response to 0.7 μg/ml PHA (2) and 10 μg/ml PHA (8), or in competent PBL in response to IL-2 in the absence (3) or presence of WHI-P131 (4) or PP2 (5), or in response to IL-2 in PBL preactivated with 0.7 μM PHA in the presence of WHI-P131 (6) or PP2 (7). Summary data of independent experiments on PBL from different donors are shown as mean + SEM (n = 4, p˂0.05). (C) Proliferation of resting PBL in response to 0.7 μg/ml PHA (2) and 10 μg/ml PHA (6, 7), or competent PBL in response to IL-2 in the absence (3) or presence of PP2 (4) or WHI-P131 (5). Bars represent the number of cells in (S+G2+M) phases of cell cycle at 48h after PBL stimulation. Summary data of independent experiments on PBL from different donors is shown as mean + SEM (n = 4, p˂0.05).</p

The inhibitory effect of WHI-P131 and PP2 on CD25 expression in PHA-stimulated human blood T lymphocytes.

<p>(A) The PP2-inhibitable expression of CD25 is timely associated with the initial stages of PBL activation. Cells were cultivated with 10 μg/ml PHA without or with 80 μM WHI-P131 (WHI) or 1.0 μM PP2 for 19 h (middle row) or cells were cultivated with 10 μg/ml PHA for 19 h and thereafter WHI-P131 or PP2 were added for the next 21 h (bottom row). Additional Y-axis is 4 decades logarithmic scale. Representative data on PBL from one donor are shown. (B) Summary data of four independent experiments are shown as mean + SEM (n = 4, p˂0.05). (C) The inhibitory effects of WHI-P131 (WHI) and PP2 on CD25 expression are additive. Cells were cultivated with 10 μg/ml PHA without or with WHI-P131 (WHI, 20 and 50 μM), or PP2 (0.4 and 0.8 μM), or WHI-P131 (20 μM) and PP2 (0.4 μM) were given simultaneously for 24 h. Summary of independent experiments are presented as mean + SEM (n = 4). ⃰, <i>p<</i>0.05. CTR—control, non-stimulated PBL.</p

PHA stimulation leads to long-term CD25 expression in human blood T lymphocytes.

<p>(A) The representative histograms of nine experiments on PBL from different donors are shown. PBL were cultivated with 10 μg/ml PHA in the absence or presence of 80 μM WHI-P131 (WHI) or 1.0 μM PP2 and after 5, 24 or 48 h were analyzed by flow cytometry. The dot plots have been obtained with Win MDI Version 2.8. In order to identify the axis labels, the additional vertical axis is shown in the upper row of histograms. Y-axis is 4 decades logarithmic scale. <b>1, 2 –</b>The gate was chosen considering the cell size increase during blasttransformation: 1- CTR—control, non-stimulated PBL, 2 –after 48 h of culture with 10 μg/ml PHA. X-axis–forward scatter (FSC), Y-axis–side scatter (SSC). (B) Time-course of CD25+ expression in PHA-induced PBL. 1 –Total number of CD25+ cells, 2—large CD25+ cells, 3—small CD25+ cells. (C) WHI-P131- and PP2-inibitable portions of CD25+ cells in PHA-stimulated PBL. Summary of independent experiments is presented as mean ±SEM (n = 9, p˂0.05). In each experiment, when analyzing the PHA-induced CD25 expression changes, the background CD25 expression in resting cells (CTR) was subtracted. Summary data are presented as mean ± SEM (n = 10, p˂0.05).</p

The growth, proliferation and viability of human blood T lymphocytes, stimulated with PHA or IL-2 in the absence or presence of WHI-P131 and PP2 for 48 h.

<p>The growth, proliferation and viability of human blood T lymphocytes, stimulated with PHA or IL-2 in the absence or presence of WHI-P131 and PP2 for 48 h.</p

PHA induces delayed and sustained STAT5 activation in human blood T lymphocytes.

<p>(A) Representative Western blots of total lysates of PBL stimulated with 100 units/ml IL-2 (IL-2) or 10 μg/ml PHA (PHA) for 2, 24 or 48 h in the absence or presence of 80 μM WHI-P131 (W). (B) and (C) Densitometric analysis of STAT5 (B) and STAT3 (C) tyrosine phosphorylation in PBL stimulated with PHA (line 1) or IL-2 (line2) in the absence or presence of 80 μM WHI-P131 (W, lines 3 and 4). At each time point P-STAT5 and P-STAT3 levels relative to total STAT5 and total STAT3 levels were calculated and these values were normalized to those from samples at 24 h on the same protein gel blot to determine fold change. Summary of independent experiments on PBL from six donors are presented as mean ± SEM (n = 6, p˂0.05). Significant changes from P-STAT/STAT ratio at 24 h are indicated with an asterisk, p˂0.05. (D) Sustained STAT5 activity is provided by JAK3 kinase in mitogen-induced PBL. PBL were stimulated with 10 μg/ml PHA (10PHA) or with 100U/ml IL-2 (IL-2) or IL-2 after pretreatment of resting PBL with 0.7 μg/ml PHA (0.7PHA) in the absence or presence of 80 μM WHI-P131 (WHI). Data are representative of three experiments on PBL from different healthy donors. Ctrl—control, unstimulated PBL.</p