Different capacities of various NMDA receptor antagonists to prevent ischemia-induced neurodegeneration in human cultured NT2 neurons.

Abstract 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 μM glycine)-induced inward currents (EC 50  = 43.7 μM) 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 3 h the loss of cell viability measured directly after 3 h 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 μM) and CGS19755 (a competitive NMDA receptor antagonist; 10 μM) reduced ischemia-induced cell death, in contrast to DCKA (10 μM). 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.