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+.(ABSTRACT TRUNCATED AT 250 WORDS)
Johnson
M E
ME
Department of Anesthesiology, Mayo Foundation, Rochester, Minnesota 55905.
Gores
G J
GJ
Uhl
C B
CB
Sill
J C
JC
D002118:Calcium / Q000378:metabolism*; D016923:Cell Death; D003486:Cyanides / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism*; D003847:Deoxyglucose / Q000494:pharmacology; D016257:Fura-2; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D017470:Receptors, Glutamate / Q000378:metabolism; D014407:Tumor Cells, Cultured
D003486:Cyanides; D017470:Receptors, Glutamate; D003847:Deoxyglucose; D006861:Hydrogen Peroxide; D002118:Calcium; D016257:Fura-2