Intracellular Ca²⁺ thresholds that determine survival or death of energy-deprived cells

Increase of intracellular ionized or free Ca²⁺ is thought to play a central role in cell death due to ATP depletion. However, concurrently operative mechanisms of injury that do not require intracellular Ca²⁺ increases have made it difficult to test this hypothesis or to determine the concentrations at which intracellular Ca²⁺ becomes lethal. The predominant Ca²⁺-independent mechanism of injury during ATP depletion involves the loss of cellular glycine. This type of damage can be fully inhibited by adding the amino acid exogenously. Using glycine to suppress Ca²⁺-independent plasma membrane damage, we have examined the effect of intracellular Ca²⁺ elevations on cell viability during ATP depletion. Madin-Darby canine kidney (MDCK) cells were depleted of ATP by incubation with a mitochondrial uncoupler in glucose-free medium. Free Ca²⁺ concentration in the medium was varied between 26 nmol/L and 1.25 mmol/L in the presence of a Ca²⁺ ionophore. Measurements with the Ca²⁺ probes fura-2, furaptra, and fura- 2FF showed that intracellular Ca²⁺ was clamped at extracellular levels under these conditions. Cell survival during ATP depletion was indicated by viable cells recovered 24 hours later. The results show that ATP depleted cells can sustain high levels of intracellular Ca²⁺ (100 μmol/L) for prolonged periods and remain viable if plasma membrane damage is prevented by glycine. Cell death was observed only when intracellular free Ca²⁺ was allowed to increase beyond 100 μmol/L, and this was associated with dramatic nuclear alterations: chromatin condensation, loss of nuclear lamins, and breakdown of DNA into large 50- to 150-kb fragments. Our studies demonstrate unexpectedly high resistance of cells to calcium cytotoxicity if glycine that is lost during ATP depletion is restored. In addition, they provide insights into novel mechanisms of nuclear disintegration and DNA damage that are triggered when the high thresholds of intracellular Ca²⁺ required for cell death are exceeded.