Early-phase occurrence of K+ and Cl− efflux in addition to Ca2+ mobilization is a prerequisite to apoptosis in HeLa cells

Abstract

Sustained rise in cytosolic Ca(2+) and cell shrinkage mainly caused by K(+) and Cl(-) efflux are known to be prerequisites to apoptotic cell death. Here, we investigated how the efflux of K(+) and Cl(-) as well as the rise in cytosolic Ca(2+) occur prior to caspase activation and are coupled to each other in apoptotic human epithelial HeLa cells. Caspase-3 activation and DNA laddering induced by staurosporine were abolished by blockers of K(+) and Cl(-) channels or cytosolic Ca(2+) chelation. Staurosporine induced decreases in the intracellular free K(+) and Cl(-) concentrations ([K(+)](i) and [Cl(-)](i)) in an early stage prior to caspase-3 activation. Staurosporine also induced a long-lasting rise in the cytosolic free Ca(2+) concentration. The early-phase decreases in [K(+)](i) and [Cl(-)](i) were completely prevented by a blocker of K(+) or Cl(-) channel, but were not affected by cytosolic Ca(2+) chelation. By contrast, the Ca(2+) response was abolished by a blocker of K(+) or Cl(-) channel. Strong hypertonic stress promptly induced a cytosolic Ca(2+) increase lasting >50 min together with sustained shrinkage and thereafter caspase-3 activation after 4 h. The hypertonic stress induced slight increases in [K(+)](i) and [Cl(-)](i) in the first 50 min, but these increases were much less than the effect of shrinkage-induced condensation, indicating that K(+) and Cl(-) efflux took place. Hypertonicity induced caspase-3 activation that was prevented not only by cytosolic Ca(2+) chelation but also by K(+) and Cl(-) channel blockers. Thus, it is concluded that not only Ca(2+) mobilization but early-phase efflux of K(+) and Cl(-) are required for caspase activation, and Ca(2+) mobilization is a downstream and resultant event of cell shrinkage in both staurosporine- and hypertonicity-induced apoptosis.