Human NTera2
teratocarcinoma cells were differentiated into postmitotic NT2-N neurons and exposed to
hypoxia for 6 h. The cultures were evaluated microscopically, and percent
lactate dehydrogenase (LDH) release after 24 and 48 h was used as an assay for cell death. After 48 h LDH release was 24.3 +/- 5.6% versus 13.8 +/- 3.7% in controls (p < 0.001). Cell death was greatly diminished by
MK-801 pretreatment (15.4 +/- 5.1%, p < 0.001). If
glutamine was omitted from the medium,
glutamate levels after 6 h of
hypoxia were reduced from 101 +/- 63 to 2.3 +/- 0.3 microM, and cell death at 48 h was also markedly reduced (15.4 +/- 4.5%, p < 0.001). The alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate antagonist
6-cyano-7-nitroquinoxaline-2,3-dione (18.7 +/- 5.1%, p < 0.001) and mild
hypothermia (33.5-34 degrees C) during
hypoxia (19.5 +/- 2.7%, p < 0.05) were moderately protective.
Basic fibroblast growth factor (24.1 +/- 3.2%), the
nitric oxide synthase inhibitor
N(G)-nitro-L-arginine methyl ester (22.8 +/- 8.1%), the
antioxidant N-tert-butyl-o-phenyinitrone (18.9 +/- 5.9%), and the 21-aminosteroid U74389G (24.0 +/- 3.4%) did not protect the cells.
N-Acetyl-L-cysteine even tended to increase cell death (30.1 +/- 2.5%, p = 0.06). Treatment with
MK-801 at the end of
hypoxia did not reduce cell death (23.3 +/- 2.3%). In separate experiments, a 15-min exposure to 1 mM
glutamate without
hypoxia did not result in significant cell death (14.7 +/- 2.4 vs. 12.2 +/- 2.1%, p = 0.07). We conclude that, although somewhat resistant to
glutamate toxicity when normoxic, NT2-N neurons die via an
ionotropic glutamate receptor-mediated mechanism when exposed to
hypoxia in the presence of
glutamate. As far as we know, this is the first reported analysis of the mechanism of hypoxic cell death in cultured human neuronlike cells.