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

Abstract

In cultured rat cerebellar granule cells, glutamate or N-methyl-d-aspartate (NMDA) activation of the NMDA receptor caused a sustained increase in cytosolic Ca²⁺levels ([Ca²⁺]_i), reactive oxygen species (ROS) generation, and cell death (respective EC₅₀values for glutamate were 12, 30, and 38 μM) but no increase in caspase-3 activity. Removal of extracellular Ca²⁺blocked all three glutamate-induced effects, whereas pretreatment with an ROS scavenger inhibited glutamate-induced cell death but had no effect on the [Ca²⁺]_iincrease. This indicates that glutamate-induced cell death is attributable to [Ca²⁺]_iincrease and ROS generation, and the [Ca²⁺]_iincrease 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 [Ca²⁺]_iincrease 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 [Ca²⁺]_iincrease 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 [Ca²⁺]_iincrease 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 [Ca²⁺]_iincrease, and glial cells provide negligible protection against glutamate-induced excitotoxicity.