Cytosolic free calcium and cell death during metabolic inhibition in a neuronal cell line

Elevated free cytosolic Ca²⁺ (Ca²⁺(i)) has been implicated as a mechanism of hypoxic neuronal death. The calcium hypothesis postulates that the basic metabolic response to hypoxic ATP depletion is a toxic increase in free cytosolic Ca²⁺(i) in all cell types. This inherent response then creates the environment in which subsequent derangements of Ca²⁺(i) may occur, for example, from glutamate excitotoxicity. Although the effect of glutamate on neuronal Ca²⁺(i) has been extensively studied, the basic neuronal response to hypoxia independent of glutamate receptor activation is not well defined. We therefore assayed both Ca²⁺(i) and plasma membrane integrity in fura-2-loaded, single SK-N-SH neuroblastoma cells, using digitized video microscopy and metabolic inhibition (2.5 mM NaCN, 10 mM 2- deoxyglucose) to model the ATP depletion of hypoxia. Median time to cell death was 90 min (n = 51 cells). Initial Ca²⁺(i) was 121 ± 67 nM. Ca²⁺(i) increased by 50 nM after 5-10 min of metabolic inhibition. Blebbing of the cell membrane was evident within 30 min. Ca²⁺(i) did not appreciably increase further until the time of cell death, when the loss of plasma membrane integrity allowed unimpeded influx of extracellular Ca²⁺. Although the increase in Ca²⁺(i) prior to cell death was statistically significant, it is unlikely to be physiologically significant, because (1) percentage change in Ca²⁺(i) accounted for only 13% of the variation in time to cell death, in a linear regression model; (2) some cells died in less than the median 90 min despite having decreases or very slight increases in Ca²⁺(i) during metabolic inhibition; and (3) the omission of Ca²⁺ from the experimental buffer prevented an increase in Ca²⁺(i) but did not prevent cell death during metabolic inhibition. In contrast, cells exposed to oxidative stress (1 mM H₂O₂) as a positive control showed a severalfold increase in Ca²⁺(i) prior to cell death, greater than the change seen in any metabolically inhibited cell. In conclusion, in the absence of glutamate receptors, Ca²⁺(i) increases minimally during metabolic inhibition in SK- N-SH cells, and this increase does not appear to contribute to the mechanisms of cell death.