Rat cerebellar granule cells are protected from glutamate-induced excitotoxicity by S-nitrosoglutathione but not glutathione

• Published in: American Journal of Physiology: Cell Physiology 2004-04, Vol.286 (4), p.C893-C904
• Main Authors: Li, Chung-Yu, Chin, Ting-Yu, Chueh, Sheau-Huei
• Format: Article
• Language: English
• Subjects: Animals; Carbonyl Cyanide m-Chlorophenyl Hydrazone - pharmacology; Cells, Cultured; Cerebellum - cytology; Enzyme Inhibitors - pharmacology; Excitatory Amino Acid Antagonists - pharmacology; Glutamic Acid - toxicity; Glutathione - pharmacology; Glycine - pharmacology; Ionophores - pharmacology; Magnesium - pharmacology; Neurons - cytology; Neurons - drug effects; Neurons - physiology; Neuroprotective Agents - pharmacology; Neurotoxins - pharmacology; Nitric Oxide - metabolism; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species - metabolism; Receptors, N-Methyl-D-Aspartate - metabolism; S-Nitrosoglutathione - pharmacology; Thapsigargin - pharmacology
• ISSN: 0363-6143; 1522-1563
• DOI: 10.1152/ajpcell.00127.2003

Department of Biochemistry, National Defense Medical Center, Taipei 114, Taiwan, Republic of China Submitted 3 April 2003 ; accepted in final form 20 November 2003 In cultured rat cerebellar granule cells, glutamate or N -methyl- D -aspartate (NMDA) activation of the NMDA receptor caused a sustained increase in cytosolic Ca 2+ levels ([Ca 2+ ] i ), reactive oxygen species (ROS) generation, and cell death (respective EC 50 values for glutamate were 12, 30, and 38 µM) but no increase in caspase-3 activity. Removal of extracellular Ca 2+ blocked all three glutamate-induced effects, whereas pretreatment with an ROS scavenger inhibited glutamate-induced cell death but had no effect on the [Ca 2+ ] i increase. This indicates that glutamate-induced cell death is attributable to [Ca 2+ ] i increase and ROS generation, and the [Ca 2+ ] i increase precedes ROS generation. Apoptotic cell death was not seen until 24 h after exposure of cells to glutamate. S -nitrosoglutathione abolished glutamate-induced ROS generation and cell death, and only a transient [Ca 2+ ] i increase was seen; similar results were observed with another nitric oxide (NO) donor, S -nitroso- N -acetylpenicillamine, but not with glutathione, which suggests that the effects were caused by NO. The transient [Ca 2+ ] i increase and the abolishment of ROS generation induced by glutamate and S -nitrosoglutathione were still seen in the presence of an ROS scavenger. Glial cells, which were present in the cultures used, showed no [Ca 2+ ] i increase in the presence of glutamate, and glutamate-induced granule cell death was independent of the percentage of glial cells. In conclusion, NO donors protect cultured cerebellar granule cells from glutamate-induced cell death, which is mediated by ROS generated by a sustained [Ca 2+ ] i increase, and glial cells provide negligible protection against glutamate-induced excitotoxicity. cytosolic calcium concentration; N -methyl- D -aspartate; reactive oxygen species Address for reprint requests and other correspondence: S.-H. Chueh, Dept. of Biochemistry, National Defense Medical Center, 161, Section 6, Min-Chuan East Rd., Taipei 114, Taiwan, Republic of China (E-mail: shch{at}mail.ndmctsgh.edu.tw ).