TY - JOUR
T1 - Cytosolic free calcium and cell death during metabolic inhibition in a neuronal cell line
AU - Johnson, Michael E.
AU - Gores, Gregory J.
AU - Uhl, Cindy B.
AU - Christopher Sill, J.
PY - 1994/7
Y1 - 1994/7
N2 - 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.
AB - 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.
KW - 2-deoxyglucose
KW - calcium
KW - cyanide
KW - fura-2
KW - hypoxia
KW - metabolic inhibition
KW - neuroblastoma
KW - neuronal ischemia
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UR - http://www.scopus.com/inward/citedby.url?scp=0028301620&partnerID=8YFLogxK
U2 - 10.1523/jneurosci.14-07-04040.1994
DO - 10.1523/jneurosci.14-07-04040.1994
M3 - Article
C2 - 8027761
AN - SCOPUS:0028301620
SN - 0270-6474
VL - 14
SP - 4040
EP - 4049
JO - Journal of Neuroscience
JF - Journal of Neuroscience
IS - 7
ER -