Inhibition of Na+/K+-ATPase may be one mechanism contributing to potassiumefflux 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, Rb-86(+), 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 Rb- 86(+) efflux, and a decrease in cell volume compared with control cells. Pr eincubating Jurkat cells with Val-Ala-Asp-chloromethylketone (VAD-cmk), an inhibitor of caspase proteases, prevented the observed Rb-86(+) efflux and cell shrinkage induced by the anti- CD95 antibody. A wide range of inhibito rs against most types of K+ channels could not inhibit CD95-mediated efflux of Rb-86(+), however, the uptake of Rb-86(+) by Jurkat cells was severely compromised when treated with anti-CD95 antibody. Uptake of Rb-86(+) in Jur kat cells was sensitive to ouabain (a specific Na+/K+-ATPase inhibitor), de monstrating Na+/K+-ATPase dependent K+ uptake. Ouabain induced significant Rb-86(+) efflux in untreated cells, as well as it seemed to compete with Rb -86(+) efflux induced by the anti-CD95 antibody, supporting a role for Na+/ K+-ATPase in the CD95-mediated Rb-86(+) 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 ma y also facilitate apoptotic cell shrinkage.