Lysosomal alkalinization, lipid oxidation, and reduced phagosome clearance triggered by activation of the P2X7 receptor

Abstract

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.

Purinergic signaling in retinal degeneration and regeneration

“…2F) . An upregulation of NTPDase1 was also described in the RPE in the ABCA4 À/À mouse model of Stargardt's retinal degeneration (Guha et al, 2013). Facilitated degradation of extracellular ATP/ADP by NTPDase1 (Fig.…”

“…P2X 7 activation in RPE cells may also contribute to another hallmark of AMD, the impaired digestion of peroxidized photoreceptor lipids, resulting in accumulation of lipofuscin within the cells and lipoprotein-containing drusen beneath the cells. Activation of P2X 7 in RPE cells causes lysosomal alkalinization and thus impedes lysosomal function, resulting in increased lipid oxidation of phagocytozed photoreceptor outer segments and reduction in phagosome clearance (Guha et al, 2013). Choroidal neovascularization, the hallmark of neovascular AMD, is driven by P2X 7 signaling while P2, A 2A , and A 2B receptor signaling is involved in the development of preretinal neovascularization, the hallmark of proliferative diabetic retinopathy (Taomoto et al, 2000;Mino et al, 2001;Lutty and McLeod, 2003;Sarman et al, 2008).…”

Deletion of P2X7 attenuates hyperoxia-induced acute lung injury via inflammasome suppression

“…More recently, another marker isofuran was identified, whose concentration increases under elevated O 2 levels, making combined detection of these molecules more efficient for evaluation of hyperoxia-induced oxidative stress (60). Previously, a connection has been made between P2X7 and its involvement in mediating lipid peroxidation (21,39). P2X7 was shown to increase the production of superoxide, which is known to be formed by hyperoxia-induced oxidative stress and contribute to the formation of lipid peroxidization by-products.…”

Differential oxidative stress thresholds distinguishes cellular response to vascular occlusion and chemotoxicityin vivo

“…Thus, a direct relationship exists between availability of oxygen in oxidative stress, ROS production and lipid peroxidation. In the same way, lipid peroxidation is linked with alteration of mitochondria membrane permeability and release of calcium (calcium-shift) (Guha et al, 2013). Moreover, the extent of ROS production determines the degree of permeability and threshold of the calcium-shift, in other words, an increase in ROS production will generate a more significant calcium-shift and cytotoxicity (Csordás et al, 2002;Guha et al, 2013).…”

“…ROS causes vast damage through lipid peroxidation in biomembranes such as those of the mitochondria, nucleus and lysosomes (Appelqvist et al, 2012;Guha et al, 2013). The primary site of ROS activation is the mitochondria, thus peroxidation of mitochondria membrane leads to a change in mitochondria membrane permeability and ROS leakage into the cytoplasm (Martin et al, 2011;Martin, 2012).…”