Normotonic cell shrinkage induced by Na⁺ deprivation results in apoptotic cell death in human epithelial HeLa cells

Apoptosis is a major form of cell death that occurs in response to a variety of signals in both physiological and pathological situations. A hallmark of apoptosis is normotonic cell shrinkage, called apoptotic volume decrease (AVD), the process of which involves fluxes of K⁺, Cl^-, and Na⁺. Na⁺ influx was suggested to be required in Fas-induced apoptosis in human Jurkat T cells, whereas Na⁺ efflux was found to be associated with AVD and apoptosis in human HL-60 cells. Here we examined the effects of extracellular Na⁺ deprivation on cell volume and viability in human epithelial HeLa cells. The incubation of HeLa cells in normotonic Na⁺-free Ringer solution resulted in persistent cell shrinkage after ≥30 min and reduction in cell viability after ≥1 h. After exposure to Na⁺-free solution for 5 h, a marked reduction in cell viability was found to be associated with an activation of caspase-3 without showing significant LDH release, indicating that the cells underwent apoptosis but not necrosis. Na⁺ deprivation-induced cell shrinkage and apoptotic cell death were significantly inhibited by a blocker of Na ⁺-K⁺-2Cl^? cotransporter (NKCC) or of the reverse-mode operation of Na⁺/Ca²⁺ exchanger (NCX), but not by a blocker of Na⁺/H⁺ exchanger (NHE). Therefore it is concluded that Na⁺ deprivation causes persistent cell shrinkage resulting from Na⁺ efflux mainly via NKCC and NCX and thereafter leads to apoptotic death of HeLa cells. It is also suggested that normotonic cell shrinkage per se, if persistent, provides a sufficient condition for apoptosis induction.