NMDA RECEPTOR ACTIVATION PRODUCES CONCURRENT GENERATION OF NITRIC-OXIDE AND REACTIVE OXYGEN SPECIES - IMPLICATION FOR CELL-DEATH

The ability of glutamate to stimulate generation of intracellular oxid ant species was determined by microfluorescence in cerebellar granule cells loaded with the oxidant-sensitive fluorescent dye 2,7-dichlorofl uorescin (DCF). Exposure of cells to glutamate (10 mu M) produced a ra pid generation of oxidants that was blocked similar to 70% by MK-801 ( a noncompetitive NMDA-receptor antagonist). To determine if nitric oxi de (NO) or reactive oxygen species (ROS) contributed to the oxidation of DCF, cells were treated with compounds that altered their generatio n. NO production was inhibited with N-G-nitro-L-arginine methyl ester (L-NAME) (nitric oxide synthase inhibitor) and reduced hemoglobin (NO scavenger). Alternatively, cells were incubated with superoxide dismut ase (SOD) and catalase, which selectively metabolize O-2(-.) and H2O2. Concurrent inhibition of O-2(-.) and NO production nearly abolished i ntracellular oxidant generation. Pretreatment of cells with either che lerythrine (1 mu M, protein kinase C inhibitor) or quinacrine (5 mu M, phospholipase A(2) inhibitor) before addition of glutamate also block ed oxidation of DCF. Generation of oxidants by glutamate was significa ntly reduced by incubating the cells in Ca2+-free buffer. In cytotoxic ity studies, a positive correlation was observed between glutamate-ind uced death and oxidant generation. Glutamate-induced cytotoxicity was blocked by MK-801 and attenuated by treatment with L-NAME, chelerythri ne, SOD, or quinacrine. It is concluded that glutamate induces concurr ent generation of NO and ROS by activation of both NMDA receptors and non-NMDA receptors through a Ca2+-mediated process. Activation of NO s ynthase and phospholipase A(2) contribute significantly to this respon se. It is proposed that simultaneous generation of NO and ROS results in formation of peroxynitrite, which initiates the cellular damage.