Activation of P2X Receptors Induces Apoptosis in Human Retinal Pigment Epithelium

PURPOSE. The retinal pigment epithelium (RPE) is considered a primary site of pathology in age-relatecl macular degeneration (AMD), which is the most prevalent form of irreversible blindness worldwide in the elderly population. Extracellular adenosine triphosphate (ATP) acts as a key signaling molecule in numerous cellular processes, including cell death. The purpose of this study was to determine Whether extracellular ATP induces apoptosis in cultured human RPE. METHODS. RPE apoptosis was evaluated by caspase-3 activation, Hoechst staining, and DNA fragmentation. Intracellular Ca2+ levels were determined by both a cell-based fluorometric Ca2+ assay and a ratiometric Ca2+ imaging technique. P2X(7) mRNA and protein expression were detected by reverse transcription-polymerase chain reaction (RT-PCR) and confocal microscopy, respectively. RESULTS. The authors found that both the endogenous P2X(7) agonist ATP and the synthetic, selective P2X(7) agonist 2',3'-O-(4-benzoylbenzoyl)-ATP (BzATP) induced RPE apoptosis, which was significantly inhibited by P2X(7) antagonist oxidized ATP (oATP) but not by the P2 receptor antagonist suramin; both ATP and BzATP increase intracellular Ca2+ via extracellular Ca2+ influx; both ATP- and BzATP-induced Ca2+ responses were significantly inhibited by oATP but not by suramin; ATP-induced apoptosis was significantly inhibited or blocked by BAPTA-AM or by low or no extracellular Ca2+; and P2X(7) receptor mRNA and protein were expressed in RPE cells. CONCLUSIONS. These findings suggest that P2X(7) receptors, especially P2X(7), receptors, contribute to ATP- and BzATP-induced Ca2+ signaling and apoptosis in the RPE. Abnormal Ca2+ homeostasis through the activation of P2X receptors could cause the dysfunction and apoptosis of RPE that underlie AMD. (Invest Ophthalmol Vis Sci 2011;52:1522-1530) DOI:10.1167/iovs.10-6172