Intracellular K+ and water content in human blood lymphocytes during transition from quiescence to proliferation

Abstract

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.

Figure 1

Cell K⁺ and Na⁺ contents in human PBL activated to transition from quiescence to proliferation. (a) Changes in cell K⁺ (K_i) and Na⁺ (Na_i) contents in isolated human PBL stimulated by PHA (10 µg/ml), or PDBu (10 nM) with ionomycin (I, 500 nM) or anti-CD3 (3.5 µg/mL) with IL-2 (100 U/mL). K_i/g (dark symbols) and Na_i/g (light symbols) were analyzed at definite time points by flame emission photometry. (b) IL-2 induces a long-term increase in K_i and cell protein content in competent PBL. Isolated PBL were incubated with non-mitogenic PHA (0.8 µg/mL) for 20 hours, then IL-2 (100 U/mL) was introduced into cell culture. Data are means ± SEM of nine (a, PHA and PHA + I), six (a, anti-CD3 with IL-2), or five (b) experiments performed triplicate. Significant difference from the initial value at time 0 (resting PBL) was tested by one-way ANOVA with Tukey’s post hoc tests, *P < 0.01, ^**P < 0.05. (c) Costimulation with non-mitogenic PHA and IL-2 is accompanied by increased CD25 expression. The representative data of one experiment from five. Control – non-stimulated, resting PBL.

Figure 2

Long-term increase in cell K⁺ content precedes DNA synthesis and accompanies transformation of small T cells into large blasts. (A) The protein content increase in PBL stimulated by PHA (10 µg/ml) for 48 hours. Data are means ± SEM of twelve experiments performed triplicate. *Significant difference from the initial value at time 0 (resting PBL) was tested by one-way ANOVA with Tukey’s post hoc tests, P < 0.05. (B) The time course of CD25 expression on small and large PBL stimulated by PHA. Data are means ± SEM of one representative experiment of three. (C) Flow cytometric analysis of cell cycle phase distribution in PHA-stimulated PBL: the percentage of cells in S and (G₂ + M) phases. (D) The time course of CD25 expression in PBL stimulated by PHA for 48 hour. Resting or PHA-stimulated PBL were stained with PE-labeled CD25 Abs and analyzed by flow cytometry. (C) and (D) – representative data of three experiments with PBL from different donors. Control - non-stimulated, resting PBL.

Figure 3

Cell K⁺ content and inhibition of proliferation in PHA-stimulated PBL. (a) Time-course of K_i/g in PBL stimulated with PHA alone or in the presence of drugs specific for initial (CsA) or late (WHI-P131) stages of PBL activation. (b) Anti-proliferative doses of CsA and WHI-P131 inhibit growth of PHA-stimulated PBL. Cells were cultivated with 10 µg/mL PHA without or with 80 µM WHI-P131 or 1.0 µg/mL CsA for 48 and at definite time of points were analyzed for K_i by flame emission photometry and for protein content by Lowery procedure. Data are means ± SEM of one representative experiment performed triplicate. *Significant difference relative to PBL stimulated by PHA without drugs during the same time (t-test, P < 0.05).

Figure 4

Buoyant density of resting PBL compared to activated PBL and leukemia T cell Jurkat. (a) Schematic illustration of the cell distribution in discontinuous Percoll gradient. Grey areas indicate the location of T cells Jurkat, resting and PHA-activated PBL isolated for analysis. (b,c) Changes in buoyant density in PBL stimulated by PHA (b) and anti-CD3 (3.5 µg/mL) with IL-2 (100 U/mL) (c) for 24 and 48 hours, each column corresponds to a separate layer. One representative experiment of three. (d,e) Flow cytograms of resting PBL in coordinates CD25/FSC (d) and CD14/FSC (e). The representative cytograms of seven experiments on PBL from different donors. (f) The representative cell volume distributions by Scepter Counter in PBL, resting and stimulated by (anti-CD3 and IL-2) mixture for 24 and 48 hours. Control - non-stimulated, resting PBL.

Figure 5

Cell K⁺ and water content in PBL with different buoyant densities as compared to unfractionated activated PBL and cycling T cells Jurkat. (a) K_i/g in light and heavy cell populations. One representative experiment of four. (b) Changes in K_i/g in total unfractionated cell populations. Data are means ± SEM of one representative experiment performed triplicate. *Significant difference relative to control (C) (t-test, P ˂ 0.05). (c) Water content in light and heavy cell populations stimulated with anti-CD3 with IL-2 for 48 hours and in proliferating T cells Jurkat. The same experiment as in (a). (d) Water content in human PBL stimulated from quiescence to proliferation and in proliferating T cells Jurkat. The same experiments as in (b). Data are means ± SEM of one representative experiment performed triplicate. *Significant difference relative to control (C) (t- test, P < 0.05).