Involvement of TNF-alpha in glutamate-induced apoptosis in a differentiated neuronal cell line.
We examined the involvement of tumor necrosis factor (TNF)-alpha on glutamate-induced cytotoxicity in a differentiated neuronal cell line. In this study, we used nerve growth factor (NGF)-differentiated PC12h cells. Glutamate cytotoxicity was assessed using the MTS and TUNEL assays. To detect TNF-alpha levels in culture supernatants after glutamate exposure, we used ELISA methods. The involvement of caspase-8, which is downstream from TNF receptor 1 (TNF-R1) in glutamate-induced cytotoxicity, was determined by Western blot analysis. The MTS assay showed that the addition of glutamate resulted in dose-dependent cell death, while the TUNEL assay showed that glutamate induced apoptosis in differentiated PC12h cells in a dose-dependent manner. TNF-alpha levels in the supernatant of glutamate-exposed cells were significantly increased compared with those in unexposed cells. In addition, glutamate caused increases in the levels of caspase-8 protein. The increases in caspase-8 levels were ameliorated by pretreatment with soluble TNF-R1. Moreover, soluble TNF-R1 significantly ameliorated the cell death induced by glutamate. These results suggest that TNF-alpha released from neuronal cells may be associated with glutamate-induced neuronal cell death.
Kogo
Jiro
J
Department of Pharmacology, St. Marianna University School of Medicine, 2-16-1 Sugao, Miyamae-ku, Kawasaki, Kanagawa 216-8511, Japan. kogo@marianna-u.ac.jp
Takeba
Yuko
Y
Kumai
Toshio
T
Kitaoka
Yasushi
Y
Matsumoto
Naoki
N
Ueno
Satoki
S
Kobayashi
Shinichi
S
D000704:Analysis of Variance; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D053181:Caspase 8 / Q000378:metabolism; D003603:Cytotoxins / Q000008:administration & dosage* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D018698:Glutamic Acid / Q000008:administration & dosage* / Q000378:metabolism; D020287:In Situ Nick-End Labeling; D009474:Neurons / Q000378:metabolism* / Q000473:pathology; D016716:PC12 Cells; D051381:Rats; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000502:physiology; D018709:Statistics, Nonparametric; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology*
D003603:Cytotoxins; D047888:Receptors, Tumor Necrosis Factor, Type I; D014409:Tumor Necrosis Factor-alpha; D018698:Glutamic Acid; D053181:Caspase 8