Correlation of glutamate-induced apoptosis with caspase activities in cultured rat cerebral cortical neurons

In cultured rat cortical neurons lactate dehydrogenase (LDH) activity in the medium, a cell-death marker, increased gradually after exposure to glutamate (100 microM to 1 mM) for 60 min and reached a plateau at 24 to 30 h. Neuronal death was mainly apoptotic as suggested by typical electron microscopic findings, fluorescent double staining with membrane-permeating and nonpermeating chromatin dyes, nick end labeling, and assessment of DNA fragmentation by agarose gel electrophoresis. After 1 mM glutamate exposure, a rise of interleukin-1beta converting enzyme (ICE)-like protease activity in neurons was parallel to cysteine protease p32 (CPP32)-like protease activity and declined before CPP32-like protease activity reached the peak (at 6 h). LDH activity in the medium of glutamate-exposed neurons was decreased by specific ICE and/or CPP32 inhibitors, acetyl-L-tyrosyl-L-valyl-L-alanyl-L-aspart-1-al (Ac-YVAD-CHO) and acetyl-L-aspartyl-L-glutamyl-L-valyl-L-aspart-1-al (Ac-DEVD-CHO), respectively, in a dose-dependent manner. Fluorescent double staining of nuclei also demonstrated that at 100 microM each inhibitor prevented neuronal apoptosis and that this effect was additive. Among agonists corresponding to various glutamate receptor subtypes, N-methyl-D-aspartate (NMDA) and kainate induced apoptosis in cortical neuronal cultures while alpha-amino-3-hydroxy-5-methylisoxazole-4-propinate (AMPA) did not. The metabotropic glutamate receptor agonist, 1-aminocyclopentane-1S, 3R-dicarboxylate (ACPD) prevented apoptosis. Interestingly, apoptosis at 24 h after agonist or antagonist exposure correlated closely with caspase activity 6 h after exposure.