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N-Methyl-D-aspartate receptor mediated toxicity in nonneuronal cell lines: Characterization using fluorescent measures of cell viability and reactive oxygen species production

Department of Pharmacology, University of Pennsylvania School of Medicine, Children's Seashore House, and Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA.
Molecular Brain Research (Impact Factor: 2). 06/2000; 77(2):163-75. DOI: 10.1016/S0169-328X(00)00050-4
Source: PubMed

ABSTRACT Cells transfected with specific N-methyl-D-aspartate (NMDA) receptor subtypes undergo cell death that mimics glutamate-induced excitotoxicity pharmacologically. We have further characterized the mechanisms of cell death resulting from NMDA receptor activation in such cells through development of cell counting methods based on co-transfection with green fluorescent protein. 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 cells which were trypan blue or propidium iodide positive. Using co-transfection with green fluorescent protein and cell counting of viable cells with a fluorescence activated cells sorter, we confirmed the subunit-specific profile of NMDA receptor-mediated cell death in cells transfected with NMDA receptors. Toxicity was greatest in the NR1A/2A receptor, less in the NR1A/2B receptor, and least in NR1A/2C receptors. Cell death also differed pharmacologically between subunit combinations. Cell death in cells transfected with NR 1A/2A was blocked by amino-phosphonovaleric acid at lower concentrations than in cells transfected with NR 1A/2B. In cells transfected with the NR1A/2A or NR1A/2B combinations but not NR1A/2C, cell death was also associated with production of reactive oxygen species. In addition, removal of the final 400 amino acids of the C-terminal region of NR2A decreased cell death. The use of GFP based cell counting provides a sensitive mechanism for assessing the mechanism of excitotoxicity in transfected cell models.

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    • "In addition, several authors reported that GFP, stably integrated into the genome of test cells, provides an excellent and simple tool to measure apoptosis or necrosis by flow cytometry (Strebel et al., 2001) and that GFP is an useful ''live-cell biosensor'' for many cellular activities, representing an accurate measure of viable cells (Anegawa et al., 2000). Because the transfection efficiency and GFP intensity depend upon several factors, such as the culture time of tissue biopsies after transfection, lipofectamine ratios, and capacitance for EP, we have tried to optimize the transfection conditions testing different peri- Fig. 3. "
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    • "Firstly, it is not clear whether NSC components can be disrupted without affecting pro-survival or plasticity signaling. Secondly, NMDAR-dependent cell death can be reconstituted in non-neuronal cells lacking the NSC simply by expressing NMDARs (Cik et al., 1993; Anegawa et al., 2000) which shows that certain NMDAR-induced death pathways do not require the NSC. "
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    • "Importantly , similar to young wild-type cultures, excitotoxicity can be mediated via the NR2B subunit also in older mice if NR2A receptor signalling is impaired. The importance of the C-terminal domain of the NR2AR subunit was also shown in HEK293 cells in which truncation of the last 406 amino acids (NR2A D1050 ) reduced NMDAR dependent toxicity (Anegawa et al., 2000). In contrast to this, truncation of the C-terminal 190 amino acids (NR2A D1266 ) of NR2A does not alter NMDA-mediated toxicity in HEK293 cells (Wagey et al., 2001), indicating that amino acids 1051-1266 of this subunit might be involved in mediating the toxic NMDA effect. "
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