Extracellular ATP Induces Ca2+ Signaling and Apoptosis in Human Retinal Pigment Epithelium

Abstract

Purpose: : Studies have indicated that a variety of cell systems express P2 purinoceptors and undergo functional changes in response to extracellular adenosine triphosphate (ATP). The purpose of this study was to determine whether extracellular ATP induces Ca²⁺ signaling and apoptosis in primary cultures of human retinal pigment epithelial (RPE) cells.

Methods: : Intracellular calcium levels were determined using calcium probe, indo-1-AM. P2X₇ mRNA and protein expression were detected by reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence microscopy, respectively. RPE apoptosis was evaluated by caspase-3 detection, Hoechst staining and cell death detection ELISA.

Results: : Both ATP and the P2X₇ agonist benzoylbenzoyl ATP (BzATP) increased intracellular Ca²⁺ in RPE cells. The Ca²⁺ induced by ATP was delayed and significantly inhibited by oxidized ATP (oATP), a P2X₇ antagonist. Suramin only slightly inhibited the Ca²⁺ increase. ATP treatment increased the number of caspase-3 positive and apoptotic RPE cells, and DNA fragmentation. All these increases were significantly reduced by oATP. RT-PCR and immunofluorescence microscopy revealed the presence of P2X₇ receptor mRNA and protein in RPE cells.

Conclusions: : Extracellular ATP induces RPE Ca²⁺ signaling and apoptosis. Activation of P2 purinoceptors including P2X₇ might contribute to ATP-induced Ca²⁺ signaling and apoptosis in the RPE.

Keywords: retinal pigment epithelium • apoptosis/cell death • calcium