Purchase this article with an account.
S. Guha, J. Liu, W. Lu, A. M. Laties, C. H. Mitchell; Stimulation of the D1-Like Dopamine Receptor Reacidifies the Lysosomal pH of Compromised Retinal Pigmented Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1856.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
The shed tips of photoreceptor outer segments are degraded by the lysosomal enzymes of retinal pigmented epithelial (RPE) cells. As the activity of these lysosomal enzymes is sharply dependent upon pH, optimal degradation of outer segments requires that lysosomal pH (pHL) is maintained within a narrow acidic range. The lysosomal pH is elevated beyond this range in fresh RPE cells from mice missing the ABCA4-/- gene mutated in a form of Stargardt’s disease, and in cultured ARPE-19 cells exposed to A2E. This suggests a pH-dependent decline in degradative enzyme activity may lead to the accumulation of partially degraded outer segments in disorders such as Stargardt’s disease. It follows that approaches to restore pHL are of interest. We have previously determined that elevation of cytoplasmic cAMP restores an acidic pHL of compromised RPE cells, suggesting receptors coupled to the Gs protein may lower pHL. As such, the ability of the dopamine D1-like receptor to restore an acidic pHL was examined in this study.
pHL was measured with dye Lysosensor Yellow/Blue from lysosomes in ARPE-19 and freshly isolated RPE cells from ABCA4-/- mice as reported. Lysosomal pH was determined from the ratio of light excited at 340nm vs 380nm (>520 nM em) using a Fluroskan Plate Reader. The pHL of ARPE-19 cells was elevated with the tertiary amine tamoxifen to enable rapid screening of acidifying agents.
The dopamine D1-like receptor agonists A68930 and A77636 reacidified pHL in compromised RPE cells, with a maximum response found at 1 microM and 100 nM respectively. The specific D1-like agonist SKF 81297 also restored lysosomal acidity, with an EC50 of 1 microM. Myristoylated protein kinase inhibitor PKI (14-22) amide, the cell-permeant inhibitor of the free catalytic subunit protein kinase A (PKA), blocked the restorative effects of SKF 81297, implying a role for PKA. Stimulation of D1- like receptors also acidified the lysosomes in RPE cells from ABCA4-/- mice. Acidification was seen even in mice over a year old.
These observations suggest that stimulation of the dopamine D1- like receptor on RPE cells restores an acidic lysosomal pH in compromised RPE cells through the activation of PKA. This receptor and its PKA -dependent mechanism can reacidify lysosomes in cells exposed to A2E for extended periods, suggesting this pathway may remain effective in RPE cells subjected to chronic damage.
This PDF is available to Subscribers Only