UPREGULATION OF NMDA–GLUTAMATE RECEPTORS BY 7–KETOCHOLESTEROL IN CULTURED ARPE–19 CELLS

Abstract

Abstract: : Purpose: To evaluate the role of N–methyl–D–aspartic acid (NMDA)–glutamate receptor 1 subunit (NMDAR1) in apoptosis induced by 7–ketocholesterol in human retinal pigment epithelial (ARPE–19) cells. Methods: ARPE–19 cells were cultured in 8–well chamber slides with Dulbeccos modified Eagles medium (DMEM) containing 10% fetal bovine serum. Cells were incubated for 48 hours at 37^oC and 5% carbon dioxide either in 20ug/ml of 7–ketocholesterol dissolved in ethanol (final concentration 0.2%), 0.2% ethanol alone or untreated. For immunostaining, the cells were fixed with 2% paraformaldehyde and then incubated with primary antibody (rabbit NMDAR1 polyclonal antibody, 1:50, Chemicon) for 1 hour. Rhodamine conjugated antibody (donkey anti–rabbit, 1:50, Chemicon) was used as secondary antibody. Chamber slides were examined with a Nikon fluorescent microscope with a digital camera. Results: There was high immunoreactivity to NMDAR1 receptor in the cells treated with 7–ketocholesterol. ARPE–19 cells treated with 0.2% ethanol alone and the untreated negative control cells had very low immunoreactivity. Conclusion: Our previous studies showed that 7–ketocholesterol induced apoptosis and decreased cell viability in ARPE–19 cells. This study showed that 7–ketocholesterol upregulated the expression of NMDAR1–glutamate receptor in cultured ARPE–19 cells. Glutamate mediated excitotoxicity may play a role in oxidized cholesterol induced apoptosis of retinal pigment epithelial cells.

Keywords: apoptosis/cell death • retinal pigment epithelium • receptors