Inhibition of Na+/K+-ATPase may be one mechanism contributing to potassium efflux and cell shrinkage in CD95-induced apoptosis

To investigate the involvement of K+ efflux in apoptotic cell shrinkage, we monitored efflux of the K+ congener,86 Rb+, and cell volume during CD95-mediated apoptosis in Jurkat cells. An anti-CD95 antibody caused apoptosis associated with intracellular GSH depletion, a significant increase in 86Rb+ efflux, and a decrease in cell volume compared with control cells. Preincubating Jurkat cells with Val-Ala-Asp-chloromethylketone (VAD-cmk), an inhibitor of caspase proteases, prevented the observed 86Rb+ efflux and cell shrinkage induced by the anti- CD95 antibody. A wide range of inhibitors against most types of K+ channels could not inhibit CD95-mediated efflux of86 Rb+, however, the uptake of86 Rb+ by Jurkat cells was severely compromised when treated with anti-CD95 antibody. Uptake of86 Rb+ in Jurkat cells was sensitive to ouabain (a specific Na+/K+-ATPase inhibitor), demonstrating Na+/K+-ATPase dependent K+ uptake. Ouabain induced significant86 Rb+ efflux in untreated cells, as well as it seemed to compete with86 Rb+ efflux induced by the anti-CD95 antibody, supporting a role for Na+/K+-ATPase in the CD95-mediated86 Rb+ efflux. Ouabain treatment of Jurkat cells did not cause a reduction in cell volume, although together with the anti-CD95 antibody, ouabain potentiated CD95-mediated cell shrinkage. This suggests that the observed inhibition of Na++/K+-ATPase during apoptosis may also facilitate apoptotic cell shrinkage.