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Cytokine release from non-inflammatory cells is a key step in innate immunity, and agonists triggering cytokine release are central in coordinating responses. P2X7 receptor (P2X7R) stimulation by extracellular ATP is best known to active the NLRP3 inflammasome and release IL-1β, but stimulation also leads to release of other cytokines. As cytokine signaling by retinal pigmented epithelial (RPE) cells is implicated in retinal neurodegeneration, the role of P2X7R in release of cytokine IL-6 from RPE cells was investigated. P2X7R stimulation triggered IL-6 release from primary mouse RPE, human iPS-RPE and human ARPE-19 cells. IL-6 release was polarized, with predominant rise across apical membranes. IL-6 release was inhibited by P2X7R antagonists A438079, A839977, and AZ10606120, but not the NRTI lamivudine (3TC), P2X1R antagonist NF279, or P2Y1R antagonist MRS2179. P2X7R-mediated IL-6 release required extracellular Ca 2+ and was blocked by Ca 2+ chelator BAPTA. IL-6 release and Ca 2+ elevation occurred rapidly, consistent with vesicular IL-6 staining in unstimulated cells. P2X7R stimulation did not trigger IL-1β release in these unprimed cells. P2X7R-mediated IL-6 release was enhanced in RPE cells from the ABCA4 -/- mouse model of retinal degeneration. In summary, P2X7R stimulation triggers rapid Ca 2+ -dependent IL-6 release across the apical membrane of RPE cells.

Cross-reactions between innate immunity, lysosomal function, and purinergic pathways may link signaling systems in cellular pathologies. We found activation of toll-like receptor 3 (TLR3) triggers lysosomal ATP release from both astrocytes and retinal pigmented epithelial (RPE) cells. ATP efflux was accompanied by lysosomal acid phosphatase and beta hexosaminidase release. Poly(I:C) alkalinized lysosomes, and lysosomal alkalization with bafilomycin or chloroquine triggered ATP release. Lysosomal rupture with glycyl-L-phenylalanine-2-naphthylamide (GPN) eliminated both ATP and acid phosphatase release. Secretory lysosome marker LAMP3 colocalized with VNUT, while MANT-ATP colocalized with LysoTracker. Unmodified membrane-impermeant 21-nt and "non-targeting" scrambled 21-nt siRNA triggered ATP and acid phosphatase release, while smaller 16-nt RNA was ineffective. Poly(I:C)-dependent ATP release was reduced by TBK-1 block and in TRPML1 -/- cells, while TRPML activation with ML-SA1 was sufficient to release both ATP and acid phosphatase. The ability of poly(I:C) to raise cytoplasmic Ca 2+ was abolished by removing extracellular ATP with apyrase, suggesting ATP release by poly(I:C) increased cellular signaling. Starvation but not rapamycin prevented lysosomal ATP release. In summary, stimulation of TLR3 triggers lysosomal alkalization and release of lysosomal ATP through activation of TRPML1; this links innate immunity to purinergic signaling via lysosomal physiology, and suggests even scrambled siRNA can influence these pathways.

Lysosomes contribute to a multitude of cellular processes, and the pH of the lysosomal lumen plays a central mechanistic role in many of these functions. In addition to controlling the rate of enzymatic degradation for material delivered through autophagic or phagocytotic pathways, lysosomal pH regulates events such as lysosomal fusion with autophagosomes and the release of lysosomal calcium into the cytoplasm. Disruption of either the steady state lysosomal pH or of the regulated manipulations to lysosomal pH may be pathological. For example, chloroquine elevates the lysosomal pH of retinal pigmented epithelial (RPE) cells and triggers a retinopathy characterized by the accumulation of lipofuscin-like material in both humans and animals. Compensatory responses to restore lysosomal pH are observed; new data illustrate that chronic chloroquine treatment increases mRNA expression of the lysosomal/autophagy master transcription factor TcFEB and of the vesicular proton pump vHATPase in the RPE/choroid of mice. An elevated lysosomal pH with upregulation of TcFEB and vHATPase resembles the pathology in fibroblasts of patients with mutant presenilin 1 (PS1), suggesting a common link between age-related macular degeneration (AMD) and Alzheimer's disease. While the absolute rise in pH is often small in these disorders, elevations of only a few tenths of a pH unit can have a major impact on both lysosomal function and the accumulation of waste over decades. Accurate measurement of lysosomal pH can be complex, and imprecise measurements have clouded the field. Protocols to optimize pH measurement from fresh and cultured cells are discussed, and indirect measurements to confirm changes in lysosomal pH and degradative capacity are addressed. The ability of reacidifying treatments to restore degradative function confirms the central role of lysosomal pH in these disorders and identifies potential approaches to treat diseases of lysosomal accumulation like AMD and Alzheimer's disease. In summary, various approaches to determine lysosomal pH in fresh and cultured cells, as well as the potential to restore pH levels to an optimal range, can help identify and repair pathologies associated with lysosomal defects in RPE cells and perhaps also suggest new approaches to treat lysosomal storage diseases throughout the body., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

It has been previously determined that the epithelial lining of the urinary bladder, or urothelium, expresses two subtypes of nicotinic acetylcholine receptors (nAChRs) that mediate distinct physiological effects in vivo. These effects include inhibition of bladder reflexes through α7 receptors and an excitation of bladder reflexes through α3-containing (α3*) receptors. It is believed that urothelial receptors mediate their effects through modulating the release of neurotransmitters such as ATP that subsequently influence bladder afferent nerve excitability. Therefore, we examined the distribution of nAChRs in the urothelium, as well as their ability to influence the release of the neurotransmitter ATP. Immunofluorescent staining of both whole bladder tissue and primary urothelial cultures from the rat demonstrated that the urothelium contains both α3* and α7 receptors. In primary urothelial cultures, α7 stimulation with choline (10 μM to 1 mM) caused a decrease in basal ATP release while α3* stimulation with cytisine (1–100 μM) caused a concentration-dependent, biphasic response, with low concentrations (1–10 μM) inhibiting release and higher concentrations (50–100 μM) increasing release. These responses were mirrored in an in vitro, whole bladder preparation. In vivo, excitation of bladder reflexes in response to intravesical cytisine (100 μM) is blocked by systemic administration of the purinergic antagonist PPADS (1 or 3 μg kg(−1)). We also examined how each receptor subtype influenced intracellular Ca2+ levels in cultured urothelial cells. nAChR stimulation increased [Ca2+]i through distinct mechanisms: α7 through a ryanodine-sensitive intracellular mechanism and α3* through extracellular influx. In addition, our findings suggest interactions between nAChR subtypes whereby activation of α7 receptors inhibited the response to a subsequent activation of α3* receptors, preventing the increase in [Ca2+]i previously observed. This inhibitory effect appears to be mediated through protein kinase A- or protein kinase C-mediated pathways.