CYTOSOLIC-FREE CALCIUM AND CELL-DEATH DURING METABOLIC INHIBITION IN A NEURONAL CELL-LINE

Elevated free cytosolic Ca2+ (Ca-i(2+)) has been implicated as a mecha nism of hypoxic neuronal death. The calcium hypothesis postulates that the basic metabolic response to hypoxic ATP depletion is a toxic incr ease in free cytosolic Ca-i(2+) in all cell types. This inherent respo nse then creates the environment in which subsequent derangements of C a-i(2+) may occur, for example, from glutamate excitotoxicity. Althoug h the effect of glutamate on neuronal Ca-i(2+) has been extensively st udied, the basic neuronal response to hypoxia independent of glutamate receptor activation is not well defined. We therefore assayed both Ca -i(2+) and plasma membrane integrity in fura-a-loaded, single SK-N-SH neuroblastoma cells, using digitized video microscopy and metabolic in hibition (2.5 mM NaCN, 10 mM 2-deoxyglucose) to model the ATP depletio n of hypoxia. Median time to cell death was 90 min (n = 51 cells). Ini tial Ca-i(2+) was 121 +/- 67 nM. Ca-i(2+) increased by 50 nM after 5-1 0 min of metabolic inhibition. Blebbing of the cell membrane was evide nt within 30 min. Ca-i(2+) did not appreciably increase further until the time of cell death, when the loss of plasma membrane integrity all owed unimpeded influx of extracellular Ca2+. Although the increase in Ca-i(2+) prior to cell death was statistically significant, it is unli kely to be physiologically significant, because (1) percentage change in Ca-i(2+) accounted for only 13% of the variation in time to cell de ath, in a linear regression model; (2) some cells died in less than th e median 90 min despite having decreases or very slight increases in C a-i(2+) during metabolic inhibition; and (3) the omission of Ca2+ from the experimental buffer prevented an increase in Ca-i(2+) but did not prevent cell death during metabolic inhibition. In contrast, cells ex posed to oxidative stress (1 mM H2O2) as a positive control showed a s everalfold increase in Ca-i(2+) prior to cell death, greater than the change seen in any metabolically inhibited cell. In conclusion, in the absence of glutamate receptors, Ca-i(2+) increases minimally during m etabolic inhibition in SK-N-SH cells, and this increase does not appea r to contribute to the mechanisms of cell death.