Lysosomal enzymes function optimally at low pH; as accumulation of waste material contributes to cell aging and disease, dysregulation of lysosomal pH may represent an early step in several pathologies. Here, we demonstrate that stimulation of the P2X7 receptor (P2X7R) for ATP alkalinizes lysosomes in cultured human retinal pigmented epithelial (RPE) cells and impairs lysosomal function. P2X7R stimulation did not kill RPE cells but alkalinized lysosomes by 0.3 U. Receptor stimulation also elevated cytoplasmic Ca(2+); Ca(2+) influx was necessary but not sufficient for lysosomal alkalinization. P2X7R stimulation decreased access to the active site of cathepsin D. Interestingly, lysosomal alkalinization was accompanied by a rise in lipid oxidation that was prevented by P2X7R antagonism. Likewise, the autofluorescence of phagocytosed photoreceptor outer segments increased by lysosomal alkalinization was restored 73% by a P2X7R antagonist. Together, this suggests that endogenous autostimulation of the P2X7R may oxidize lipids and impede clearance. The P2X7R was expressed on apical and basolateral membranes of mouse RPE; mRNA expression of P2X7R and extracellular ATP marker NTPDase1 was raised in RPE tissue from the ABCA4(-/-) mouse model of Stargardt's retinal degeneration. In summary, P2X7R stimulation raises lysosomal pH and impedes lysosomal function, suggesting a possible role for overstimulation in diseases of accumulation.
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.
Accumulation of lipofuscin in retinal pigmented epithelial (RPE) cell, results from incomplete degradation of phagocytic and autophagic material by lysosomes. Both increased lipofuscin and enhanced cytokine release are associated with macular degeneration in these cells, although the link between the two is unclear. Since stimulation of the P2X7 receptor (P2X7R) activates IL‐1β release in some cell types, and triggers lysosomal exocytosis in others, we investigated the effect of P2X7R activation on lysosomal pH in, and cytokine release from, RPE cells. Surprisingly, stimulation of the P2X7R with agonist BzATP did not lead to a release of IL‐1β but did trigger release of both IL‐6 and MCP‐1. BzATP led to a rise in intracellular Ca2+, and extracellular Ca2+ was necessary for IL‐6 release. Lysosomal pH was elevated by BzATP and this alkalinization was inhibited by P2X7R antagoinsts A438079 and Brilliant Blue G. BzATP on its own did not significantly increase expression of IL‐6, MCP‐1 or the ectoATPase NTPDase1, but did increase expression of all three genes in cells concurrently exposed to oxidized photoreceptor outer segments. In conclusion, stimulation of the P2X7R on RPE cells elevated lysosomal pH and enhanced cytokine release. P2X7R stimulation also upregulated cytokine gene expression in cells subjected to an oxidative challenge.Grant: EY013434
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