The nature of cell volume sensor in shrinkage‐induced apoptosis

Potassium loss and persistent shrinkage have been both implicated in apoptosis, but their relationship and respective roles remain obscure. We approached this problem by clamping intracellular sodium and potassium in HeLa, MDBK or MDCK cells by a combination of ionophores and ouabain. Although ionophores cause significant cell swelling, the initial volume can be restored and further reduced by applying external sucrose. We showed that swollen cells treated with ionophores remain viable for at least 4–8 hours without developing any signs of apoptosis. Application of sucrose and the resulting shrinkage caused volume‐dependent intrinsic apoptosis with all its classical features: inversion of phosphatidylserine, caspase activation, cytochrome c release from mitochondria. Furthermore, overexpression of Bcl‐2 prevents mitochondrial loss of cytochrome c in shrunken cells. All these responses occurred to approximately the same degree regardless of whether the cells were kept in a high potassium/zero sodium or in a high sodium/zero potassium buffer, with only a slight enhancement under potassium‐free conditions. However, apoptosis induced by protein kinase inhibitor staurosporine in ionophore‐treated and swollen cells was strongly inhibited by as little as 5 mM potassium. Addition of 50 mM sucrose to restore the initial cell volume eliminated potassium dependence of staurosporine‐induced caspase activation. We have eliminated potassium as a stimulus in shrinkage induced apoptosis and are left with the choice between macromolecular crowding and cytoskeleton, which according to our unconfirmed preliminary data may be cytoskeleton. Thus, our results show that: (1) persistent shrinkage (presumably mediated through macromolecular crowding) acts as a direct proapoptotic signal independently of potassium; (2) strong potassium dependence of caspase activation is only observed in swollen cells with reduced cytoplasmic density.