Abstract
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 μM PCP. After washout of PCP, NMDA was added (in the presence of Mg2+) 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 μM) 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-XL. The Bcl-XL/Bax ratio was markedly decreased by 30 μM NMDA in the PCP-treated, but not control, cultures. Addition of superoxide dismutase and catalase prevented the decrease in Bcl-XL/Bax. This study suggests that NMDA-induced changes in Bax and/or Bcl-XL 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.
Footnotes
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Send reprint requests to: Kenneth M. Johnson, Ph.D., Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, TX 77555-1031. E-mail:kmjohnso{at}utmb.edu
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↵1 This study was supported by National Institutes of Health Grant DA 02073 and the John Sealy Memorial Endowment Fund for Biomedical Research.
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↵2 Current address: Neuroscience Graduate Program, University of Texas Medical Branch, Galveston, TX 77555-1031.
- Abbreviations:
- PCP
- phencyclidine
- NMDA
- N-methyl-d-aspartate
- DMEM
- Dulbecco's modified Eagle's medium
- MTT
- 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
- LDH
- lactate dehydrogenase
- ELISA
- enzyme-linked immunosorbent assay
- TUNEL
- terminal dUTP nick-end labeling
- PSA-NCAM
- polysialic acid-neuronal cell adhesion molecule
- ROS
- reactive oxygen species
- SOD
- superoxide dismutase
- NF-κB
- nuclear factor-κB
- Received November 16, 1999.
- Accepted March 15, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
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