DELAYED ANTAGONISM OF CALPAIN REDUCES EXCITOTOXICITY IN CULTURED NEURONS

Background and Purpose Glutamate receptor antagonists can produce prot ection against the neurotoxicity of excessive glutamate stimulation. H owever, antagonism of the postreceptor processes that produce cell dam age may provide a longer window of opportunity for protecting neurons after the initiation of excitotoxic injury. Among various processes th at have been thought to mediate the toxic effects of glutamate are act ivation of the Ca2+-dependent proteases calpain I and 11 and the activ ation of nitric oxide synthase. We tested the potential for neuroprote ction by delayed application of calpain antagonists after excitotoxic treatment. Methods Primary cultures of cerebellar and hippocampal neur ons were exposed to the glutamate receptor agonists kainate and N-meth yl-D-aspartate (NMDA) for 20-minute periods, and survival was examined by fluorescent assay after 24 hours. Enzyme antagonists were applied at various time points during this interval. Results The neurotoxic ef fects of NMDA in cultured hippocampal neurons and of kainate in cultur ed cerebellar neurons have been previously shown to be Ca2+ dependent. Here we show that in both of these examples of glutamate receptor-med iated toxicity, activation of a calpainlike proteolytic activity occur red, which was blocked by the calpain inhibitor MDL-28170. This inhibi tor also limited the toxicity, even when applied at times up to 1 hour after the onset of the toxic exposure. Another protease inhibitor, E- 64, also blocked the proteolysis and toxicity produced by kainate in c erebellar neurons. Blocking nitric oxide synthase activity after 1 hou r with the antagonist N-G-nitro-L-arginine was also protective of cere bellar and hippocampal neurons, as was the combination of MDL-28170 an d N-G-nitro-L-arginine, Conclusions The activation of calpain is among several enzymatic processes that contribute to the toxicity of glutam ate receptor stimulation, and blocking these postreceptor mechanisms c an be effective in protecting neurons from excitotoxicity at delayed t ime points.