PRODUCTION OF REACTIVE OXYGEN SPECIES AND RELEASE OF L-GLUTAMATE DURING SUPEROXIDE ANION-INDUCED CELL-DEATH OF CEREBELLAR GRANULE NEURONS

Enhanced production of superoxide anion (O-2(-)) is considered to play a pivotal role in the pathogenesis of CNS neurons. Here, we report th at O-2(-) generated by xanthine (XA) + xanthine oxidase (XO) triggered cell death associated with nuclear condensation and DNA fragmentation in cerebellar granule neuron. XA + XO induced significant increases i n amounts of intracellular reactive oxygen species (ROS) before initia ting loss of cell viability, as determined by measurement of 6-carboxy -2',7'-dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester) ( C-DCDHF-DA) for O-2(-) and other ROS and hydroethidine (HEt) specifica lly for O-2(-) by using fluorescence microscopy and flow cytometry. Ca talase, but not superoxide dismutase (SOD), significantly protected gr anule neurons from the XA + XO-induced cell death. Catalase effectivel y reduced C-DCDHF-DA but not HEt fluorescence, whereas SOD reduced HEt but not C-DCDHF-DA fluorescence, indicating that HEt and C-DCDHF-DA f luorescence correlated with O-2(-) and hydrogen peroxide, respectively . The NMDA antagonist MK-801 prevented the death. XA + XO induced an i ncrease in L-glutamate release from cerebellar granule neurons. These results indicate that elevation of O-2(-) induces cell death associate d with increasing ROS production in cerebellar granule neurons and tha t XA + XO enhanced release of L-glutamate.