Research article

Protection of cortical cells by equine estrogens against glutamate-induced excitotoxicity is mediated through a calcium independent mechanism

Joel Perrella^1,3 and Bhagu R Bhavnani^1,2,3

¹Department of Obstetrics and Gynecology, University of Toronto, Toronto, Canada

²Institute of Medical Sciences, University of Toronto, Toronto, Canada

³Department of Obstetrics and Gynecology, St. Michael's Hospital, Toronto, Canada

author email corresponding author email

BMC Neuroscience 2005, 6:34doi:10.1186/1471-2202-6-34

Published:	10 May 2005

Abstract

Background

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^-). 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.

Results

Primary 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.

Conclusion

Glutamate-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.