title: Early-phase occurrence of K+ and Cl- efflux in addition to Ca 2+ 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.
authors.affiliation: Department of Cell Physiology, National Institute for Physiological Sciences, Okazaki, 444-8585, Japan.
authors.forename: Katsuya
authors.initials: K
authors.lastname: Dezaki
authors.forename: Emi
authors.initials: E
authors.lastname: Maeno
authors.forename: Kaori
authors.initials: K
authors.lastname: Sato
authors.forename: Tenpei
authors.initials: T
authors.lastname: Akita
authors.forename: Yasunobu
authors.initials: Y
authors.lastname: Okada
mesh_terms: D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects*; D053148:Caspase 3 / Q000378:metabolism; D061945:Caspase Inhibitors; D002712:Chlorides / Q000378:metabolism*; D003620:Dantrolene / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D011188:Potassium / Q000378:metabolism*; D026902:Potassium Channel Blockers / Q000494:pharmacology; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D012462:Saline Solution, Hypertonic; D019311:Staurosporine / Q000494:pharmacology; D013312:Stress, Physiological
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D002121:Calcium Channel Blockers; D061945:Caspase Inhibitors; D002712:Chlorides; D026902:Potassium Channel Blockers; D000644:Quaternary Ammonium Compounds; D012462:Saline Solution, Hypertonic; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D004533:Egtazic Acid; C027435:tetrapentylammonium; C505407:CASP3 protein, human; D053148:Caspase 3; D003620:Dantrolene; D019311:Staurosporine; D011188:Potassium; D002118:Calcium