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K. Russell Randall, A. K. Lindsay, J. H. Boatright; RGC-5 Cells Express Opioid Receptors: Role in Neuroprotection. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4056.
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Opioid receptor agonists are neuroprotective in various models of brain injury. We hypothesize that they will similarly protect retinal neurons. This study was designed to characterize the expression of kappa and delta opioid receptors (KOR and DOR) in the rat ganglion cell line (RGC-5) and test whether activation of kappa receptors on these cells is neuroprotective.
RGC-5 cells were cultured in 75 cm2 flasks with Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 µg/ml streptomycin and maintained at 37 °C in a humidified chamber of 5% CO2. Differentiation of RGC-5 cells was promoted by media supplementation with succinyl concanavalin A (sConA; 50 µg/ml) for 7 days. Cells were analyzed by western blot analysis and immunocytochemistry for the expression of kappa and delta opioid receptors. Confocal microscopy was used for localization. To test the possible protective effects of KOR ligands, differentiated RGC-5 cells were pretreated with the KOR agonist BRL 52537 followed by 24 hr exposure to culture medium containing glutamate in the presence of the agonist. In other experiments, cells were maintained in serum-free medium for 24 hr followed by exposure to the agonist for 24 hr in the serum-free medium. The presence of DNA fragmentation in differentiated RGC-5 cells was evaluated in glutamate-treated and serum-free cultures via terminal dUTP nick end labeling (TUNEL) analysis. The effect of acute exposure to glutamate or growth factor withdrawal on cell viability was tested using the Live/Dead fluorescent assay.
Kappa and delta opioid receptors are expressed in RGC-5 cells, localizing to cell membranes. Glutamate treatment and serum-deprivation resulted in RGC-5 cell death. Pre-treatment with the KOR-specific ligand BRL 52537 reduced cell death induced by glutamate toxicity and serum-deprivation.
The results indicate that activation of kappa opioid receptors on RGC-5 cells protect against glutamate cytotoxicity and growth factor withdrawal.
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