Hypoxic Cell Death in Human NT2-N Neurons: Involvement of NMDA and Non-NMDA Glutamate Receptors

Authors


Address correspondence and reprint requests to Dr. T. Rootwelt at Department of Pediatric Research, National Hospital, University of Oslo, N-0027 Oslo, Norway.

Abstract

Abstract: 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 glutamate was omitted from the medium, glutamate levels after 6 h of hypoxia were reduced from 101 ± 63 to 2.3 ± 0.3 µM, and cell death at 48 h was also markedly reduced (15.4 ± 4.5%, p < 0.001). The α-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°C) during hypoxia (19.5 ± 2.75, p < 0.05) were moderately protective. Basic fibroblast growth factor (24.1 ± 3.2%), the nitric oxide synthase inhibitor NG-nitro-l-arginine methyl ester (22.8 ± 8.1%), the antioxidant N-tert-butyl-o-phenylnitrone (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.

Abbreviations used: AMPA, α-amino-3-hydroxy-5-methylisoxazole-4-propionate; b-FGF, basic fibroblast growth factor; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; DIC, differential interference contrast; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; HG, high glucose; LDH, lactate dehydrogenase; l-NAME, NG-nitro-l-arginine methyl ester HCl; NF-L, neurofilament light chain; NMDA, N-methyl-d-aspartate; NO, nitric oxide; NOS, nitric oxide synthase; NT2-N neurons, neurons derived from the human teratocarcinoma cell line NTera2; PBN, N-tert-butyl-o-phenylnitrone.

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