Stimulation of the D5 dopamine receptor acidifies the lysosomal pH of retinal pigmented epithelial cells and decreases accumulation of autofluorescent photoreceptor debris

Abstract

Optimal neuronal activity requires that supporting cells provide both efficient nutrient delivery and waste disposal. The incomplete processing of engulfed waste by their lysosomes can lead to accumulation of residual material and compromise their support of neurons. As most degradative lysosomal enzymes function best at an acidic pH, lysosomal alkalinization can impede enzyme activity and increase lipofuscin accumulation. We hypothesize that treatment to reacidify compromised lysosomes can enhance degradation. Here, we demonstrate that degradation of ingested photoreceptor outer segments by retinal pigmented epithelial (RPE) cells is increased by stimulation of D5 dopamine receptors. D1/D5 receptor agonists reacidified lysosomes in cells alkalinized by chloroquine or tamoxifen, with acidification dependent on protein kinase A. Knockdown with siRNA confirmed acidification was mediated by the D5 receptor. Exposure of cells to outer segments increased lipofuscin-like autofluorescence, but SKF 81297 reduced autofluorescence. Likewise, SKF 81297 increased the activity of lysosomal protease cathepsin D in situ. D5DR stimulation also acidified lysosomes of RPE cells from elderly ABCA4−/− mice, a model of recessive Stargardt’s retinal degeneration. In conclusion, D5 receptor stimulation lowers compromised lysosomal pH, enhancing degradation. The reduced accumulation of lipofuscin-like autofluorescence implies the D5 receptor stimulation may enable cells to better support adjacent neurons.

Rescue of Compromised Lysosomes Enhances Degradation of Photoreceptor Outer Segments and Reduces Lipofuscin-Like Autofluorescence in Retinal Pigmented Epithelial Cells

“…For example, exposure of RPE cells to photoreceptor outer segments for a week increased the lipofuscin-like autofluorescence, but treatment with the D5 dopamine receptor agonist SKF81297 completely prevented this. SKF 81297 also restored access to the cathepsin D binding sites, consistent with the improved degradation [16]. Stimulation of A 2A adenosine and beta adrenergic receptors, and activation of CFTR, also enhanced the degradation of photoreceptor outer segments [6, 17].…”

“…Autofluorescence associated with photoreceptor outer segments was increased in RPE cells exposed to cholorquine [15]. Lysosomal alkalinization decreased staining of Bodipy-pepstatin–A, suggesting the lysosomal enzyme cathepsin D was less effective in RPE cells with perturbed lysosomes [15, 16]. The clearance of outer segments is also decreased when the lysosomal pH is elevated with tamoxifen [17].…”

“…The non-specific adenosine receptor agonist 5′- N -ethylcarboxamidoadenosine (NECA) and the A 2A adenosine receptor agonist CGS21680 reacidified lysosomes, while A 1 adenosine receptor agonists were not effective [6]. Dopamine agonists A68930, A77636 and SKF81297 all reacidified lysosomes in compromised RPE cells; siRNA identified the D5 dopamine receptor as mediating the response [16]. SFK81297 was particularly effective and produced a sustained reacidification of at least 12 days.…”

“…Direct activation of cAMP substantially reacidified lysosomes of RPE cells from 6 month old ABCA4 −/− mice [6], while dopamine D5 receptor agonists A68930, A77636 and SKF81297 lowered lysosomal pH in RPE cells from 11–12 month old ABCA4 −/− mice [16]. The A2A agonist CGS21680 restored lysosomal acidity in cells treated with A2E for 4 weeks [6].…”

Changes in Extra- and Intracellular pH in Hepatocytes Exposed to Gabexate Mesilate

“…The optimal activity of most pH-sensitive lysosomal enzymes ranges between 4 and 5 (23). The sharp pH dependence of these enzymes predicts that even a moderate elevation of pH will lower enzyme efficiency and reduce the clearance of material (24). A genetic disease, mucolypidosis IV, is characterized by chronic reduction of vesicular pH (25).…”