Different capacities of various NMDA receptor antagonists to prevent ischemia-induced neurodegeneration in human cultured NT2 neurons.
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.
Garcia de Arriba
Susana
S
Rudolf-Boehm-Institute of Pharmacology and Toxicology, University of Leipzig, Leipzig, Germany.
Wegner
Florian
F
Grüner
Katia
K
Verdaguer
Ester
E
Pallas
Mercé
M
Camins
Antoni
A
Wagner
Armin
A
Wohlfahrt
Kai
K
Allgaier
Clemens
C
D002470:Cell Survival; D002478:Cells, Cultured; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology*; D005434:Flow Cytometry; D006801:Humans; D007511:Ischemia / Q000503:physiopathology*; D008564:Membrane Potentials; D008928:Mitochondria / Q000502:physiology; D009474:Neurons / Q000187:drug effects*; D018408:Patch-Clamp Techniques; D016194:Receptors, N-Methyl-D-Aspartate / Q000037:antagonists & inhibitors*
D018691:Excitatory Amino Acid Antagonists; D016194:Receptors, N-Methyl-D-Aspartate