NITRIC-OXIDE, SUPEROXIDE AND PEROXYNITRITE - PUTATIVE MEDIATORS OF NMDA-INDUCED CELL-DEATH IN CEREBELLAR GRANULE CELLS

In this study, we analysed the implication of superoxide (O2.-) and ni tric oxide (NO.) free radicals and their resulting product peroxynitri te (ONOO-) in the neuronal death induced by the activation of the glut amatergic receptor of the N-methyl-D-aspartate (NMDA) subtype using cu ltured cerebellar granule cells. The NO. donor SIN-1 (3-morpholinosydn onimine N-ethylcarbamide), at concentrations which produced a much hig her guanylate cyclase activation (i.e. NO. concentration) than NMDA, w as not neurotoxic and did not increase the NMDA-induced neuronal death . The absence of involvement of NO. in NMDA-induced neuronal death was confirmed by the ineffectiveness of L-N(G)-nitroarginine (L-Narg) as a neuroprotective compound. Electron paramagnetic resonance (EPR) expe riments, using 5,5-dimethyl pyrroline 1-oxide (DMPO) as a spin trap, i ndicated that NMDA receptor stimulation led to the generation of O2.- from at least 15-30 min. The generation of O2.- by xanthine (XA)-xanth ine oxidase (XO) induced a neuronal death similar to that of NMDA. XA- XO-induced neuronal death was suppressed by addition of either superox ide dismutase (SOD) plus catalase (CAT), or DMPO in the incubation med ium. In contrast, NMDA-induced neuronal death was widely blocked by DM PO and other spin trap compounds, but not by SOD +/- CAT. XA-XO-induce d neuronal death was not potentiated by SIN-1 indicating that ONOO- is not more toxic than O2.- in our neuronal model.