NMDA Receptor Activation Produces Concurrent Generation of Nitric Oxide and Reactive Oxygen Species: Implications for Cell Death

Abstract: The ability of glutamate to stimulate generation of intracellular oxidant species was determined by microfluorescence in cerebellar granule cells loaded with the oxidant‐sensitive fluorescent dye 2,7‐dichlorofluorescin (DCF). Exposure of cells to glutamate (10 µ M ) produced a rapid generation of oxidants that was blocked ∼70% by MK‐801 (a noncompetitive NMDA‐receptor antagonist). To determine if nitric oxide (NO) or reactive oxygen species (ROS) contributed to the oxidation of DCF, cells were treated with compounds that altered their generation. 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 dismutase (SOD) and catalase, which selectively metabolize O 2 −· andH 2 O 2 . Concurrent inhibition of O 2 −· and NO production nearly abolished intracellular oxidant generation. Pretreatment of cells with either chelerythrine (1 µ M , protein kinase C inhibitor) or quinacrine (5 µ M , phospholipase A 2 inhibitor) before addition of glutamate also blocked oxidation of DCF. Generation of oxidants by glutamate was significantly reduced by incubating the cells in Ca 2+ ‐free buffer. In cytotoxicity studies, a positive correlation was observed between glutamate‐induced death and oxidant generation. Glutamate‐induced cytotoxicity was blocked by MK‐801 and attenuated by treatment with l ‐NAME, chelerythrine, SOD, or quinacrine. It is concluded that glutamate induces concurrent generation of NO and ROS by activation of both NMDA receptors and non‐NMDA receptors through a Ca 2+ ‐mediated process. Activation of NO synthase and phospholipaseA 2 contribute significantly to this response. It is proposed that simultaneous generation of NO and ROS results in formation of peroxynitrite, which initiates the cellular damage.