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 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.

Searching for Mechanisms of N-Methyl-d-Aspartate-Induced Glutathione Efflux in Organotypic Hippocampal Cultures

“…The PLA 2 inhibitor quinacrine attenuates the generation of ROS, arachidonic acid release, glutamate cytotoxicity, and stroke injury (6,(60)(61)(62). In our experiments, quinacrine (500 μM) completely abolished the increased NMDA-mediated efflux rates of glutathione, PEA, and taurine.…”

Relationships between superoxide levels and delayed calcium deregulation in cultured cerebellar granule cells exposed continuously to glutamate

“…Each pair of figures traces from the same cells. ineffective against DCD, are able to decrease lactate dehydrogenase release or propidium iodide nuclear staining (Gunasekar et al 1995;Cazevieille et al 1997;Vergun et al 2001). Thus, the post-DCD increase in superoxide levels may contribute to the processes culminating in plasma membrane lysis.…”

“…Phospholipase A2 inhibitors prevent the increase in superoxide following DCD Ionomycin, metabolic inhibition or glutamate have each been reported to activate phospholipase A2 in CGNs (Lazarewicz et al 1990;Gunasekar et al 1995;Chen et al 1999) and cortical neurons (Tapia-Arancibia et al 1992;Stella et al 1995;Taylor and Hewett 2002) while PLA2 inhibitors decrease cell death measured by lactic dehydrogenase release (Ciani et al 1996). In addition Lafon-Cazal et al (1993) reported that superoxide production from glutamate-exposed CGNs was due in part to arachidonic acid release.…”

Oxidative Stress Induces Dephosphorylation of τ in Rat Brain Primary Neuronal Cultures

“…The dephosphorylation of~-is just one example of cytoskeletal changes that occur in excitotoxicity; phosphorylation changes occur in other cytoskeletal proteins such as MAPIb, MAP2, and neurofIlaments (Bigot and Hunt, 1991;Yang et a!., 1995;Pang et a!., 1996), possibly due to the compromise of the energy supply. Glutamate excitotoxicity has been shown to result in generation of free radicals, and these may make an essential contribution to the degeneration that follows exposure to toxic levels of glutamate (Cheng and Sun, 1994;Beal, 1995;Dugan et al, 1995;Gunasekar et al, 1995;Newell et al, 1995;Reynolds and Hastings, 1995;Schulz et a!., 1995). It is, therefore, significant that free radical production in the cultured neurones resulted in 'r dephosphorylation, indicating that the glutamate-induced dephosphorylation is mediated via free radicals.…”