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Mariel Piechowicz, Anna Figueroa, Nicole R. Congrove, Sara Ann Sillik, Brian S McKay; L-DOPA activation of GPR143 decreases lysosomal pH in RPE. Invest. Ophthalmol. Vis. Sci. 2018;59(9):2467.
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© ARVO (1962-2015); The Authors (2016-present)
GPR143 is a G-protein coupled receptor expressed in pigmented cells including RPE. The ligand of GPR143 is L-DOPA, which acts as an agonist, but the related molecule, dopamine, functions as an antagonist. Dopamine receptors in RPE participate in the circadian digestion of photoreceptor outer segments. In this study, we tested the hypothesis that GPR143 signaling impacts short-term RPE degradative capacity, akin to dopamine receptor signaling, by decreasing lysosomal pH.
The lysosomes in primary bovine RPE were visualized with LysoSensor Green DND-189, a pH-dependent dye which increases fluorescent emission upon lysosome acidification. Cells were treated with L-DOPA or dopamine and images were captured by confocal microscopy, with control cells not treated with either drug. To terminate the assay and control for cell loading, a low pH standard was used to achieve maximum signal intensity of each cell. Each group had three replicates in 9 separate experiments. Four 5x5 micron regions spanning lysosomes were selected for each cell and IOD was measured for time 0, 90sec, 5min, and 8min after drug treatment. Unpaired T-tests between the groups were completed at each time point to test for significant changes in pH response to the treatments.
Variability in IOD of the lysosomes at t = 0s was observed both between and within cells in all experiments. However, there was no significant difference between the groups at time 0. As early as 90 seconds after treatment, a trend emerged; the mean IOD of the L-DOPA and dopamine groups exceeded that of the negative control, though neither reached statistical significance at 90sec (p = 0.057 and p = 0.07, respectively). By 5 minutes after treatment, the mean fluorescence intensity of the L-DOPA treated cells was significantly greater compared to untreated cells (p = 0.02) and this persisted through 8 minutes (p = 0.03). The mean fluorescence intensity of the dopamine treated cells was not significantly greater than the untreated cells at 5 and 8 minutes (p=0.12).
Our results suggest L-DOPA activation of GPR143 significantly increases lysosomal activity, judged by lowering pH in the compartment. Our results varied from cell to cell and this variability may have impacted our ability to reach statistical significance in cells treated with dopamine. These results suggest GPR143 activation may relate to RPE activity in circadian degradation of photoreceptor outer segments.
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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