Adenosine Restores Lysosomal pH and Impaired Outer Segment Degradation of RPE

Abstract

Purpose:: Lysosomal enzymes of the RPE are primarily responsible for degradation of phagocytosed photoreceptor outer segments (POS). These lysosomal enzymes function maximally in a narrow range of acid pH, and the elevation of lysosomal pH (pH_L) can accompany different drug treatments. Restoration of pH_L is predicted to improve enzyme activity and reduce the accumulation of partially digested debris. Here we test the hypothesis that adenosine receptor agonists can restore a perturbed pH_L and enhance the degradative ability of compromised RPE cells.

Methods:: A high throughput screening (HTS) assay based on the pH-dependent ratiometric dye LysoSensor Yellow/ Blue was used to quantify the changes of lysosomal pH within ARPE-19 and primary-cultured bovine RPE cells. Isolated bovine POS were stained with the pH-insensitive dye calcein and added to ARPE-19 cells for 2h followed by a 2h wash, after which drugs were added for an additional 20h. Fluorescence was read immediately after the drug loading and at the end of the drug incubation, with the difference defined as the retention.

Results:: The pH_L of ARPE-19 cells was alkalinized by NH₄Cl, the vH⁺ATPase inhibitor bafilomycin-A, as predicted, and by chloroquine and tamoxifen. Tamoxifen and chloroquine both increased the retention of POS, suggesting the alkalinization led to a reduction in enzyme activity. The adenosine derivative NECA restored the acidity to lysosomes that had been compromised by tamoxifen. This restoration of pH_L was mimicked by the adenosine A_2A receptor agonist CGS21680 but not by A₁ receptor agonists ENBA or CPA. The acidification produced by CGS21680 also restored the pH of lysosomes treated with chloroquine. The rates of outer segment clearance were significantly improved by CGS21680 and NECA, consistent with an improvement of enzyme activity to accompany lysosomal acidification. The effect was not limited to ARPE-19 cells, as adenosine decreased the pH_L of fresh bovine RPE cells after elevation with tamoxifen.

Conclusions:: Tamoxifen and chloroquine elevate pH_L and reduce the degradative ability of RPE cells. Adenosine receptor agonists can restore an acidic pH_L and lysosomal activity. This restoration may have implications for the lysosomal alkalinization that occurs after treatment of cells with A2E.

Keywords: pH regulation/protons • adenosine • drusen