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-related 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 Ca(2+) levels were determined by both a cell-based fluorometric Ca(2+) assay and a ratiometric Ca(2+) 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 Ca(2+) via extracellular Ca(2+) influx; both ATP- and BzATP-induced Ca(2+) 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 Ca(2+); and P2X(7) receptor mRNA and protein were expressed in RPE cells. CONCLUSIONS: These findings suggest that P2X receptors, especially P2X(7) receptors, contribute to ATP- and BzATP-induced Ca(2+) signaling and apoptosis in the RPE. Abnormal Ca(2+) homeostasis through the activation of P2X receptors could cause the dysfunction and apoptosis of RPE that underlie AMD.