Activation of ß-adrenergic Receptors on RPE Cells Can Restore Lysosomal pH and Increase Outer Segment Breakdown

Abstract

Purpose:: The accumulation of lipofuscin by the RPE may be a factor in various ocular pathologies. The incomplete breakdown of material derived from the photoreceptor outer segments could contribute to lipofuscin formation. The lysosomal enzymes that are largely responsible for this activity operate optimally within a narrow range of acidic pH values. As lysosomal pH (pH_L) can be elevated by any of several insults, compounds that restore an acidic pH_L may increase enzyme activity and could be beneficial. This study screened drugs currently in ophthalmic use and several catecholamines for their ability to restore an optimal pH_L and to increase the rate of outer segment clearance by RPE cells.

Methods:: pH_L was measured with high throughput screening of ARPE-19 cells grown in 96-well plates using the ratiometric dye Lysosensor Yellow/Blue. Degradation of outer segments was determined from the decay of fluorescence after feeding cells with isolated bovine outer segments stained with the pH-insensitive dye calcein.

Results:: Tamoxifen and chloroquine rapidly increased pH_L, as predicted for tertiary amines. Dorzolamide, acetezolamide, timolol, latanaprost and pilocarpine were all unable to restore pH_L in cells treated with tamoxifen. However, norepinephrine, epinephrine and isoproteranol acidified pH_L by 0.3 pH units. The acidification triggered by norepinephrine was blocked by timolol, and mimicked by cell permeant cAMP, implying involvement of a ß-adrenergic receptor. Norepinephrine also acidified of lysosomes after the pH_L was increased by chloroquine, suggesting the treatment could correct damage due to multiple causes. Increasing the pH_L with tamoxifen substantially slowed the rate of outer segment clearance, but both norepinephrine and isoproterenol reversed this effect.

Conclusions:: Stimulating the ß-adrenergic receptors on RPE cells can restore an acidic pH to lysosomes compromised by selected toxins and increase the breakdown of outer segments. The acidification is predicted to increase activity of cathespin D and lysosomal acid lipase by 15-40%, suggesting the increased clearance of outer segments follows an increased activity by acidic lysosomal enzymes. These approaches could reduce lipofuscin formation in certain circumstances.

Keywords: pH regulation/protons • receptors: pharmacology/physiology • drusen