In the present study, human NT2 neurons obtained from embryonic
teratocarcinoma (NT2) cells were established as human in-vitro model to investigate the mechanisms associated with
hypoxia/
ischemia-induced neuronal injury. NT2 neurons express functional
NMDA receptors that are of particular significance for
hypoxia/
ischemia-related neuronal damage. In patch-clamp recordings under normoxic conditions,
NMDA (plus 10 microM
glycine)-induced inward currents (EC(50)=43.7 microM) were distinctly antagonized by
memantine, a blocker of the receptor channel, but only slightly by
5,7-dichlorokynurenic acid (DCKA), a
glycine(B) binding site antagonist. Immunohistochemistry demonstrated that the NT2 neurons are mostly GABAergic; they predominantly express the
NMDA receptor subunits NR2B and NR2C, and lower levels of NR1 and, particularly, of NR2A. Upon
glucose and
oxygen deprivation for 3h the loss of cell viability measured directly after 3h was higher than after application of either
hypoxia or aglycemia as assessed by
propidium iodide flow cytometry. Ischemic conditions significantly reduced the
NMDA responses associated with a decrease in EC(50) and decreased mitochondrial membrane potential as detected by
JC-1 flow cytometry.
Memantine (50 microM) and CGS19755 (a competitive
NMDA receptor antagonist; 10 microM) reduced
ischemia-induced cell death, in contrast to DCKA (10 microM). In conclusion, in the present human in-vitro model for studying the molecular mechanisms associated with ischemic injury, neuroprotection could be achieved with
NMDA receptor antagonists but not with a
glycine(B) binding site antagonist. Accordingly,
glycine antagonists might not represent an optimal therapeutic strategy for preventing ischemic neuronal damage in contrast to
NMDA receptor antagonists like
memantine.