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Renee Christine Ryals, Bryan M. Robb, Mark E Pennesi; Screening GPCR compounds for their ability to protect photoreceptors using a 661W light-induced cell death assay.. Invest. Ophthalmol. Vis. Sci. 2018;59(9):2496. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To screen compounds that target G protein coupled receptors for their ability to protect photoreceptors from light-induced degeneration.
661W cells, a transformed mouse photoreceptor cell line, were seeded in 96 well plates at a density of ~20,000 cells per well. Twenty-four hours later, in dim red light, the media was replaced with either media containing 0% FBS or media containing 0% FBS and 5 uM 9-cis retinal. Plates were then dark adapted for 4 hours. After the dark adaptation, in dim red light, the media was discarded and fresh 0% FBS media containing different concentrations of GPCR modulators were added. One plate was then exposed to ~20,000 lux of light for 4 hours, while the control plate remained in the dark. Cell death was assayed with the CellTiter 96 Aqueous One Solution Cell Proliferation Assay. Averaged absorbance readings from the light exposed group were compared to the dark control group to determine the percentage of cells that died due to the light exposure and the percentage of cells protected by GPCR modulation treatment. An unpaired t-test and an ordinary one-way ANOVA multiple comparisons test were used to determine significant differences between the groups. GPCR compounds mostly consisted of serotonergic, adrenergic, and dopaminergic modulators. All compounds were tested individually and in combination at varying concentrations in order to determine an optimal combination therapy.
Exposure to light and 5 uM 9-cis retinal resulted in ~55 % cell death. When a 10 uM and 50 uM treatment of sarpogrelate was added, ~6 % and ~13 % of cells were protected, respectively. A 100 uM concentration of sarpogrelate was toxic in both dark and light conditions. Treating light and 5 uM 9-cis retinal exposed 661W cells with 10 uM and 50 uM concentrations of 8-OH-DPAT protected ~5 % of cells. A 100 uM concentration of 8-OH-DPAT alone (no 9-cis) did not cause significant cell death in dark conditions, but caused ~13 % cell death in light conditions.
Sarpogrelate protected 661W cells from light-induced cell death in a dose dependent manner. These data are the first to show that sarpogrelate’s neuroprotective effects can occur in isolated photoreceptors suggesting the protective mechanism in the retina may, in part occur through the photoreceptors. Future studies will focus on evaluating additional GPCR compounds individually and in combination.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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