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Martin Minns, Gregory Teicher, Celeste B Rich, Vickery E Trinkaus-Randall; P2X7 is Necessary for Wound-Induced Ca2+ Mobilization and Cytoskeletal Rearrangements in the Corneal Epithelium. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5829.
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© ARVO (1962-2015); The Authors (2016-present)
The purinergic receptor P2X7 is a trimeric ion channel that causes an increase in intracellular [Ca2+] in response to activation by ATP. While P2X7 has been shown to promote corneal epithelial migration, the mechanism is still relatively unknown. The purpose of this study is to determine the regulation of P2X7 after injury using a rat corneal organ culture model, and determine downstream signaling events in a cell culture model.
Experiments were performed using corneal organ culture and in vitro wound healing models. Corneal epithelial debridements were performed and the corneas were dissected, placed on agar domes, and incubated up to 48 hours. At various time points, real-time PCR, western blot analysis, and immunohistochemical analyses were performed. An established human corneal epithelial cell line was used to examine calcium waves and intracellular localization of proteins after injury via immunofluorescence.
Injury induced a translocation of P2X7 to the leading edge of the migrating corneal epithelium. The change was accompanied by an increase in p15, a marker of migratory cells. Inhibition of P2X7 with oxidized ATP (oxATP), a specific and irreversible P2X7 inhibitor, led to a loss of this translocation and delayed wound healing (p < 0.05). The in vitro scratch wound model displayed a similar delay in cell migration when P2X7 was inhibited (p < 0.01). Oxidized ATP treatment resulted in fewer focal adhesion clusters as measured by vinculin (p < 0.05). Live cell imaging confirmed immunohistochemistry results and demonstrated that P2X7 is required for membrane ruffling after scratch wounding. Finally, oxATP treatment led to a more rapid decline in the Ca2+ wave after injury as compared to control.
P2X7 activation and translocation are necessary for proper early signaling events after corneal epithelial injury.
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