Activation of P2X receptors induces apoptosis in human retinal pigment epithelium.
OBJECTIVE
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.
Department of Ophthalmology and Visual Sciences, University of Michigan, Ann Arbor, Michigan 48105-0714, USA.
Dongli
D
Yang
Susan G
SG
Elner
Andrea J
AJ
Clark
Bret A
BA
Hughes
Howard R
HR
Petty
Victor M
VM
Elner
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling; D053148:Caspase 3 / Q000378:metabolism; D002478:Cells, Cultured; D053938:DNA Fragmentation; D004789:Enzyme Activation; D004797:Enzyme-Linked Immunosorbent Assay; D005470:Fluorometry; D006801:Humans; D018613:Microscopy, Confocal; D012333:RNA, Messenger / Q000378:metabolism; D058469:Receptors, Purinergic P2X / Q000378:metabolism*; D055213:Retinal Pigment Epithelium / Q000378:metabolism* / Q000473:pathology*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D012333:RNA, Messenger; D058469:Receptors, Purinergic P2X; C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D053148:Caspase 3; D002118:Calcium