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S. Guha, L.-A. Tu, G. Baltazar, A. Argalla, A. M. Laties, C. H. Mitchell; Reduction of Lipofuscin-Like Autofluorescence in RPE Cells by Sustained D1/D5 Receptor Stimulation. Invest. Ophthalmol. Vis. Sci. 2010;51(13):476.
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© ARVO (1962-2015); The Authors (2016-present)
Spent photoreceptor outer segments are engulfed and degraded by RPE cells. Incomplete or faulty degradation may lead to the accumulation of amorphous material including autofluorescent lipofuscin. Degradative lysosomal enzymes are pH dependent and function optimally in an acidic environment. We have previously demonstrated that elevation of lysosomal pH can reduce the ability of RPE cells to clear labeled outer segments, that elevation of cAMP can restore an acidic pH, and that receptors coupled to the Gs protein can restore lysosomal pH in compromised cells. In this regard, the joint D1/D5 dopamine receptor agonist SKF81297 reacidifies lysosomes of RPE cells. In this study, we asked whether SKF81297 can induce a prolonged reacidification of RPE cells, whether the agonist can actually reduce outer segment autofluorescence, and which receptor subtype mediates the reacidification.
Lysosomal pH was determined from ARPE-19 cells grown in 96 well plates using Lysosensor Yellow/Blue dye. Cells were treated with chloroquine to raise the lysosomal pH and the ability of SKF81297 to restore this pH was tracked over time. Cells were transfected with RNAi against the D1 or D5 dopamine receptors and lysosomal pH measured 2-3 days later to determine which receptor was mediating the effects of SKF81297. Cells were exposed to bovine photoreceptor outer segments, then SKF8197, 3x over a week before autofluorescence at 488 nm was determined using a flow cytometer.
A single exposure to 10 µM SKF81297 lowered lysosomal pH in chloroquine-treated RPE cells for 7 days. The restoration of acidity was cumulative, with the pH fully recovered after 7 days. SKF81297 acidified lysosomal pH in control cells, but had no effect in cells transfected with RNAi for the D5 receptor. Transfection with RNAi against the D1 receptor did not interfere with the acidifying actions of SKF81297. The autofluorescence excited at 488 nM was substantially increased in cells fed outer segments, but treatment with SKF81297 decreased this autofluorescence by 54 ±4%.
SKF81297 induced a sustained restoration of lysosomal pH in compromised RPE cells and a reduction of lipofuscin-like autofluorescence. These actions are likely mediated by stimulation of the D5 receptor. It remains to be determined whether stimulation of the D5 receptor can reduce lipofuscin accumulation in vivo.
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