Role of NMDA Receptor in Apoptosis of Retinal Pigment Epithelial Cells After Exposure To Oxidized Cholesterol

Abstract

Abstract: : Purpose: To investigate the role of NMDAR₁ receptor in apoptosis of ARPE–19 cells induced by oxidized cholesterol. Methods: ARPE–19 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum. Cells were exposed to 40µg/ml 7–ketocholesterol dissolved in ethanol (final concentration 0.4% vol./vol.), 0.4% (vol./vol.) ethanol alone for 2, 6 or 24 hours or were left untreated. Immunochemistry was performed with rabbit anti–NMDAR₁ polyclonal antibody (Chemicon, Temecula, CA) after the cells were fixed with 2% paraformaldehyde. Semi–quantitative RT–PCR was performed to analyze the expression of NMDAR₁ receptor gene. Also, the mitochondrial dehydrogenase activity of ARPE–19 cells treated with 7–ketocholesterol was studied by the WST–1 colorimetry assay (490 nm) after the addition of 100 µM of the NMDA antagonist MK–801. Results: ARPE–19 cells treated with 7–ketocholesterol 40 µg/ml for 24 hours showed bright fluorescence when stained with the NMDAR₁ antibody. An approximately 344 base pairs (bp) size band was seen in the cells treated with 40µg/ml 7–ketocholesterol for 24 hours, suggesting upregulation of the NMDAR₁ gene. Cells treated with 40 µg/ml 7–ketocholesterol for 2 or 6 hours, and the untreated ARPE–19 cells did not show upregulation of the NMDAR₁ gene. Restriction digestion of the NMDAR₁ RT–PCR product with Stu I enzyme showed two bands of approximately 163 bp and 181 bp. Cells treated with 7–ketocholesterol 40µg/ml for 24 hours had a significantly lower mitochondrial dehydrogenase activity compared to the untreated ARPE–19 cells (0.2434 ± 0.1695 vs. 0.537 ± 0.177, p=0.0008) whereas cells treated with 7–ketocholesterol 40µg/ml and MK–801 for 24 hours had a mean mitochondrial dehydrogenase activity that was not significantly lower than the untreated ARPE–19 cells (0.3853 ± 0.3127 vs. 0.537 ± 0.177, p>0.05). Conclusions: NMDAR₁ receptor expression is upregulated in ARPE–19 cells exposed to 7–ketocholesterol. NMDA antagonists can block the apoptosis induced by 7–ketocholesterol and may play a role in diseases of the RPE caused by oxidative stress such as macular degeneration.

Keywords: apoptosis/cell death • retinal pigment epithelium • oxidation/oxidative or free radical damage