Purchase this article with an account.
C. H. Mitchell, A. M. Laties, W. Lu, J. Liu; Defective Lysosomal pH in RPE Cells of ABCA4-/- Mice Restored by CFTR. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4545. doi: https://doi.org/.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
We have recently reported that the pH is elevated in lysosomes of RPE cells from ABCA4-/- mice and in cultured in ARPE-19 cells exposed to A2E for extended periods. As the degradative activity of lysosomal enzymes is optimal at acid levels, this pH elevation may reduce the ability of degradative enzymes in RPE cells to clear phagocytosed material. We hypothesize that reacidifying compromised lysosomes will thus improve enzyme activity and decrease the accumulation of partially digested material in RPE cells. We have previously identified cAMP as a key second messenger in lysosomal reacidification. Current models predict that an influx of anions through chloride channels balances the accumulation of protons as lysosomes acidify. Here we probe the role of the cAMP-activated chloride channel CFTR in the reacidification of lysosomes in compromised RPE cells.
Lysosomal pH was measured in ARPE-19 cells and RPE cells from ABCA4-/- mice using the ratiometric dye Lysosensor Yellow/Blue. Retention was determined from the decay of fluorescence after feeding cells with isolated bovine outer segments stained with the pH-insensitive dye calcein.
The lysosomal pH of ARPE-19 cells was elevated by the CFTR inhibitors CFTR-172 and glybenclamide. In cells treated to elevate lysosomal pH, the reacidification induced by cAMP was prevented by specific inhibitor CFTR-172. Activation of CFTR with CFTRact16, CFTRact11 and genestein also reacidified lysosomes. CFTR-172 slowed the degradation of rod outer segments, implying a role for CFTR in lysosomal function. Preliminary data suggest that activation of CFTR can also lower lysosomal pH of RPE cells from ABCA4-/- mice.
CFTR helps maintain an acidic pH in RPE lysosomes and improves activity of the degradative enzymes involved in outer segment clearance. The CFTR-associated pathway seems to be functional in ABCA4-/- mice, suggesting treatment to activate CFTR may be of benefit. The lack of a major RPE disorder in cystic fibrosis suggests that cells can sidestep this contribution when necessary. However, the growing number of new approaches available to activate CFTR make it a key target to prevent defective degradation by RPE cells in ocular disease.
This PDF is available to Subscribers Only