Mechanisms of N-methyl-D-aspartate-induced apoptosis in phencyclidine-treated cultured forebrain neurons

• Published in: The Journal of pharmacology and experimental therapeutics Vol. 294; no. 1; p. 287
• Main Authors: Wang, C, Kaufmann, J A, Sanchez-Ross, M G, Johnson, K M
• Format: Journal Article
• Language: English
• Published: United States 01.07.2000
• Subjects: Animals; Apoptosis - drug effects; bcl-2-Associated X Protein; bcl-X Protein; Cells, Cultured; N-Methylaspartate - toxicity; Phencyclidine - toxicity; Prosencephalon - drug effects; Proto-Oncogene Proteins - physiology; Proto-Oncogene Proteins c-bcl-2 - physiology; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Receptors, N-Methyl-D-Aspartate - drug effects; Receptors, N-Methyl-D-Aspartate - genetics; RNA, Messenger - analysis
• ISSN: 0022-3565

Chronic administration of phencyclidine (PCP) to rats has been demonstrated to produce a sensitized locomotor response to PCP challenge that is associated with apoptotic cell death and an up-regulation of the N-methyl-D-aspartate (NMDA) receptor. To determine the underlying mechanisms, dissociated forebrain cultures were treated for 2 days with 3 microM PCP. After washout of PCP, NMDA was added (in the presence of Mg(2+)) for 20 h. The uptake of a vital dye and the release of lactate dehydrogenase measured cell viability. Apoptosis was assessed by an enzyme-linked immunosorbent assay that was specific for fragmented (histone-associated) DNA and an in situ assay for nicked DNA, terminal dUTP nick-end labeling. These assays showed that the effect of a nontoxic concentration of NMDA (30 microM) became lethal to approximately one-third of the neurons after chronic (48-h) PCP treatment. This treatment also resulted in a 47% increase in NR1 subunit mRNA, suggesting that NMDA-induced neuronal cell death after chronic PCP is due to NMDA receptor up-regulation. Furthermore, exposure of PCP-treated cultures to NMDA led to increased expression of Bax and decreased expression of Bcl-X(L). The Bcl-X(L)/Bax ratio was markedly decreased by 30 microM NMDA in the PCP-treated, but not control, cultures. Addition of superoxide dismutase and catalase prevented the decrease in Bcl-X(L)/Bax. This study suggests that NMDA-induced changes in Bax and/or Bcl-X(L) involve the formation of reactive oxygen species. By extrapolation, these data suggest that PCP-induced apoptosis in vivo may involve similar mechanisms and that cultured neurons may be a suitable model for the mechanistic study PCP toxicity in vivo.