INHIBITION OF FREE-RADICAL PRODUCTION OR FREE-RADICAL SCAVENGING PROTECTS FROM THE EXCITOTOXIC CELL-DEATH MEDIATED BY GLUTAMATE IN CULTURESOF CEREBELLAR GRANULE NEURONS

Glutamate kills sensitive neurons through several steps downstream to receptor activation: increased free Ca2+ levels, activation of various enzymes and accumulation of reactive oxygen species (ROS). We have ev aluated in a well established model of neuronal cultures the neuroprot ective effects of blocking these mechanisms, either singularly or by c ombining multiple enzyme inhibition and/or ROS scavenging. In vitro cu ltures of cerebellar granule cells were exposed to a toxic concentrati on of glutamate (100 mu M for 15 min in the absence of Mg2+) combined with several pharmacological treatments. Inhibition of nitric oxide sy nthase (NOS) and phospholipase A(2) (PLA(2)) were effective in decreas ing cell death and the combined treatments showed some degree of addit ivity. By contrast, inhibition of xanthine oxidase (XO) with allopurin ol was uneffective. Antioxidants (in particular vitamin E or vitamin E analogs), protected neurons up to more than 50%. A synergistic effect was demonstrated by the combination of vitamin E and C. On the other hand, antioxidants did not increase the protection granted by enzyme i nhibitors, suggesting that they act downstream to NOS and PLA(2). In c onclusion, NOS and PLA(2) activated by Ca2+ influx give rise to reacti ve oxygen species whose deleterious action can be counteracted either by inhibiting these enzymes or by scavenging the excess of free radica ls produced by them. Finally, a moderate protection was obtained by bl ocking protein synthesis with cycloheximide, suggesting a partial cont ribution of apoptotic mechanisms to the excitotoxic cell death.