Pump and channel K (Rb+) fluxes in apoptosis of human lymphoid cell line U937.

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.

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 muM 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 microM Eto for 4 h or 0.8-8 microM 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.