N-methyl-D-aspartate receptor mediated toxicity in nonneuronal cell lines:characterization using fluorescent measures of cell viability and reactiveoxygen species production

Cells transfected with specific N-methyl-D-aspartate (NMDA) receptor subtyp es undergo cell death that mimics glutamate-induced excitotoxicity pharmaco logically. We have further characterized the mechanisms of cell death resul ting from NMDA receptor activation in such cells through development of cel l counting methods based on co-transfection with green fluorescent protein (GFP). When co-transfected with NMDA receptors, GFP expression was limited to live cells as indicated by the observation that GFP was only detected in cells which were positive for markers of live cells, and was found in no c ells which were trypan blue or propidium iodide positive. Using co-transfec tion with green fluorescent protein and cell counting of viable cells with a fluorescence activated cells sorter, we confirmed the subunit-specific pr ofile of NMDA receptor-mediated cell death in cells transfected with NMDA r eceptors. Toxicity was greatest in the NR1A/2A receptor, less in the NR1A/2 B receptor, and least in NR1A/2C receptors. Cell death also differed pharma cologically between subunit combinations. Cell death in cells transfected w ith NR1A/2A was blocked by amino-phosphonovaleric acid at lower concentrati ons than in cells transfected with NR1A/2B. In cells transfected with the N R1A/2A or NR1A/2B combinations but not NR1A/2C, cell death was also associa ted with production of reactive oxygen species. Tn addition, removal of the final 400 amino acids of the C-terminal region of NR2A decreased cell deat h. The use of GFP based cell counting provides a sensitive mechanism for as sessing the mechanism of excitotoxicity in transfected cell models. (C) 200 0 Elsevier Science B.V. All rights reserved.