Intracellular glutathione levels determine cerebellar granule neuron sensitivity to excitotoxic injury by kainic acid

Glutathione (GSH) is a key component of the cellular defence cascade agains t injury caused by reactive oxygen species. Kainic acid (KA) is a potent ce ntral nervous system excitotoxin. KA-elicited neuronal death may result fro m the generation of ROS. The present study was undertaken to characterize t he role of GSH in KA-induced neurotoxicity. Cultures of cerebellar granule neurons were prepared from 8-day-old rats, and used at 8, 14 and 20 days in vitro (DIV). Granule neurons displayed a developmental increase in their s ensitivity to KA injury, as quantified by an ELISA-based assay with the tet razolium salt MTT. At DIV 14 and 20, a 30-min challenge with KA (500 mu M) reduced cell viability by 45% after 24 h, significantly greater (P<0.01) th an the 22% cell loss with DIV 8 cultures. Moreover acute (30 min) KA exposu re concentration-dependently reduced intracellular GSH and enhanced reactiv e oxygen species generation (evaluated by 2',7'-dichlorofluorescein diaceta te). In comparison to control, KA (500 mu M) lowered GSH levels in DIV 8 gr anule neurons by 16% (P=0.0388), and by 36% (P=0.0001) in both DIV 14 and D IV 20 neurons, after 30 min. Preincubation of granule neurons with the memb rane permeant GSH delivery agent, GSH ethyl ester (5 mM), for 30 min signif icantly increased intracellular GSH content. Importantly, GSH ethyl ester r educed the toxic effects of KA, becoming significant at 1 mM (P=0.007 vs. K A-treated group), and was maximal at greater than or equal to 2.5 mM (P<0.0 001). GSH ethyl ester displayed a similar dose-dependence in its ability to counteract KA-induced depletion of cellular GSH. The data strengthen the n otion that cellular GSH levels have a fundamental role in KA-induced neurot oxicity. (C) 2000 Elsevier Science B.V. All rights reserved.