Abstract
BackgroundHigh concentrations of glutamate can accumulate in the brain and may be involved in the pathogenesis of neurodegenerative disorders such as Alzheimer's disease. This form of neurotoxicity involves changes in the regulation of cellular calcium (Ca2+) and generation of free radicals such as peroxynitrite (ONOO-). Estrogen may protect against glutamate-induced cell death by reducing the excitotoxic Ca2+ influx associated with glutamate excitotoxicity. In this study, the inhibition of N-methyl-D-aspartate (NMDA) receptor and nitric oxide synthase (NOS) along with the effect of 17β-estradiol (17β-E2) and a more potent antioxidant Δ8, 17β-estradiol (Δ8, 17β-E2) on cell viability and intracellular Ca2+ ([Ca2+]i), following treatment of rat cortical cells with glutamate, was investigated.ResultsPrimary rat cortical cells were cultured for 7–12 days in Neurobasal medium containing B27 supplements. Addition of glutamate (200 μM) decreased cell viability to 51.3 ± 0.7% compared to control. Treatment with the noncompetitive NMDAR antagonist, MK-801, and the NOS inhibitor, L-NAME, completely prevented cell death. Pretreatment (24 hrs) with 17β-E2 and Δ8, 17β-E2 (0.01 to 10 μM) significantly reduced cell death. 17β-E2 was more potent than Δ8, 17β-E2. Glutamate caused a rapid 2.5 fold increase in [Ca2+]i. Treatment with 0.001 to 10 μM MK-801 reduced the initial Ca2+ influx by 14–41% and increased cell viability significantly. Pretreatment with 17β-E2 and Δ8, 17β-E2 had no effect on Ca2+ influx but protected the cortical cells against glutamate-induced cell death.ConclusionGlutamate-induced cell death in cortical cultures can occur through NMDAR and NOS-linked mechanisms by increasing nitric oxide and ONOO-. Equine estrogens: 17β-E2 and Δ8, 17β-E2, significantly protected cortical cells against glutamate-induced excitotoxicity by a mechanism that appears to be independent of Ca2+ influx. To our knowledge, this is a first such observation. Whether the decrease in NOS related products such as ONOO-, is a mechanism by which estrogens protect against glutamate toxicity, remains to be investigated. Estrogen replacement therapy in healthy and young postmenopausal women may protect against neurodegenerative diseases by these mechanisms.
Highlights
High concentrations of glutamate can accumulate in the brain and may be involved in the pathogenesis of neurodegenerative disorders such as Alzheimer's disease
The rapid effects involve the activation of the N-Methyl-D-Aspartate receptor (NMDAR) that lead to a large Ca2+ influx that may be detrimental to cell viability [6]
Effect of glutamate on cell viability Cortical neuronal cells (4 × 104) cultured for 7 days in 96well plates were treated with increasing concentrations (0.1 to1000 μM) of glutamate for 20 min and cell viability was measured after 24 hrs and compared with vehicletreated samples
Summary
High concentrations of glutamate can accumulate in the brain and may be involved in the pathogenesis of neurodegenerative disorders such as Alzheimer's disease. This form of neurotoxicity involves changes in the regulation of cellular calcium (Ca2+) and generation of free radicals such as peroxynitrite (ONOO-). Two distinct pathways for glutamate-induced cell death have been described: the excitotoxic pathway and the oxidative pathway. The excitotoxic pathway involves the overactivation of glutamate receptors that leads to both rapid and slowly triggered cytotoxic events. The rapid effects involve the activation of the N-Methyl-D-Aspartate receptor (NMDAR) that lead to a large Ca2+ influx that may be detrimental to cell viability [6]. The oxidative pathway involves the breakdown of the glutamate-cystine antiporter and a drop in glutathione levels that allows for aberrant formation of reactive oxygen species (ROS) that are neurotoxic [7,8]
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