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

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 46 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~tM,and cell death at 48 h was also markedly reduced (15.4 ± 4.5%, p < 0.001). The a-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 ±2.7%, p < 0.05) were moderately protective. Basic fibroblast growth factor (24.1 ±3.2%), the nitric oxide synthase inhibitor N°-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-mm 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 isthe first reported analysis ofthe mechanism of hypoxic cell death in cultured human neuronlike cells.

Post-hypoxic hypothermia is protective in human NT2-N neurons regardless of oxygen concentration during reoxygenation

“…Ntera2/clone D1 is a human teratocarcinoma-derived cell line which differentiates into post-mitotic neuron-like cells (NT2-N cells) upon treatment with retinoic acid [222][223][224] .…”

“…Accumulation of hypoxanthine is an indicator of energy failure 80,222 and correlates with LDH release 223 . Hypoxanthine release (ȝmol/L) to supernatant was used as a marker of cellular energy failure in the NT2-N model (paper I).…”

“…The main mechanism for hypoxic cell death in NT2-N neurons is excitotoxicity 222 where accumulated glutamate activates NMDA receptors, causing neuronal influx of Na + and Ca 2+ and activation of cytotoxic pathways. Adding the NMDA antagonist MK-801 during and after the hypoxic insult is highly protective in the NT2-N model 86 .…”

The N-methyl-D-aspartate receptor type 2A is frequently methylated in human colorectal carcinoma and suppresses cell growth

“…However, it is more possible that the antiproliferative activity of MK-801 in the study is independent of the action as a general inhibitor of NMDA receptors. The concentrations of MK-801 applied for decrease of the cell growth was much higher (200-400 mM) than those for general inhibition of NMDA receptors (10-100 mM) (Rootwelt et al, 1998). They also mentioned in discussion that 10 mM of MK-801 did not influence proliferation or levels of phosphorylated extracellular regulated kinase and CAMP responsive element binding protein when given in the absence of the growth factors.…”

Acidosis during Reoxygenation Has an Early Detrimental Effect on Neuronal Metabolic Activity

“…Several mechanisms with opposing effects may be involved. In our hypoxia model, cell death is mediated through NMDA and non-NMDA receptors, and pretreatment with MK-801 offers almost complete protection (1)(2)(3)(4). Whereas the protective effect of acidosis during hypoxia is probably to a large extent mediated through an inhibitory effect on glutaminergic NMDA receptors (1,5), the mechanisms behind the detrimental effect of acidosis during reoxygenation are less clear.…”