Hypoxic cell death in human NT2-N neurons: involvement of NMDA and non-NMDA glutamate receptors.
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.
Rootwelt
T
T
Neurology Research, Children's Hospital of Philadelphia, Pennsylvania, USA.
Dunn
M
M
Yudkoff
M
M
Itoh
T
T
Almaas
R
R
Pleasure
D
D
D016923:Cell Death / Q000502:physiology; D015687:Cell Hypoxia; D002470:Cell Survival; D018698:Glutamic Acid / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity; D019271:Hypoxanthine / Q000378:metabolism; D007501:Iron / Q000633:toxicity; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D009474:Neurons / Q000378:metabolism* / Q000473:pathology; D017470:Receptors, Glutamate / Q000502:physiology*; D016194:Receptors, N-Methyl-D-Aspartate / Q000502:physiology*; D013194:Staining and Labeling; D018243:Teratocarcinoma; D014407:Tumor Cells, Cultured
D017470:Receptors, Glutamate; D016194:Receptors, N-Methyl-D-Aspartate; D019271:Hypoxanthine; D018698:Glutamic Acid; D006861:Hydrogen Peroxide; D007501:Iron; D007770:L-Lactate Dehydrogenase