Mechanisms of N-methyl-D-aspartate-induced apoptosis in phencyclidine-treated cultured forebrain neurons.
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.
Wang
C
C
Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas 77555-1031, USA.
Kaufmann
J A
JA
Sanchez-Ross
M G
MG
Johnson
K M
KM
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002478:Cells, Cultured; D016202:N-Methylaspartate / Q000633:toxicity*; D010622:Phencyclidine / Q000633:toxicity*; D016548:Prosencephalon / Q000187:drug effects*; D011518:Proto-Oncogene Proteins / Q000502:physiology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000502:physiology; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species; D016194:Receptors, N-Methyl-D-Aspartate / Q000187:drug effects / Q000235:genetics; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein
C494828:Bax protein, rat; C494813:Bcl2l1 protein, rat; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D016194:Receptors, N-Methyl-D-Aspartate; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; D016202:N-Methylaspartate; D010622:Phencyclidine