ISSN:
1471-4159
Source:
Blackwell Publishing Journal Backfiles 1879-2005
Topics:
Medicine
Notes:
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 µM) 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 NG-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 O2−· andH2O2. Concurrent inhibition of O2−· and NO production nearly abolished intracellular oxidant generation. Pretreatment of cells with either chelerythrine (1 µM, protein kinase C inhibitor) or quinacrine (5 µM, phospholipase A2 inhibitor) before addition of glutamate also blocked oxidation of DCF. Generation of oxidants by glutamate was significantly reduced by incubating the cells in Ca2+-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 Ca2+-mediated process. Activation of NO synthase and phospholipaseA2 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.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1046/j.1471-4159.1995.65052016.x
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