Identification and characterization of P2Y2 nucleotide receptors in human retinal pigment epithelial cells.
P2 nucleotide receptor expression in cultured human retinal pigment epithelial (RPE) cells was investigated using the photoaffinity ATP analog BzATP, polymerase chain reaction of reverse-transcribed RNA (RT-PCR) and fura-2 fluorescence measurement of changes in intracellular free calcium concentration ([Ca2+]i). In experiments carried out in RPE cells at passage 10-15, addition of micromolar concentrations of ATP, UTP, and ATPgammaS to RPE cells resulted in a rapid, transient 3.5-fold increase in [Ca2+]i followed by a prolonged elevation that was twofold above the original baseline. Similar results were obtained from cells at passage 2. Characteristics of nucleotide-stimulated calcium mobilization in RPE cells, including partial inhibition by pertussis toxin, suggest that a G protein-coupled receptor mediates this response. Consistent with the expression of a P2Y2 nucleotide receptor subtype in RPE cells, [alpha-32P]BzATP labeled a 53-kDa protein in plasma membranes, and RT-PCR revealed the presence of P2Y2 receptor RNA. Adenosine had no effect on [Ca2+]i in RPE cells, indicating that the A2 subtype of P1 receptor described previously in human RPE is not involved in the response to nucleotides. Together the results indicate that human RPE cells express functional P2Y2 nucleotide receptors.
Sullivan
D M
DM
Laboratory of Immunology, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892, USA. dsul@helix.nih.gov
Erb
L
L
Anglade
E
E
Weisman
G A
GA
Turner
J T
JT
Csaky
K G
KG
D000241:Adenosine / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology*; D000345:Affinity Labels; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D016257:Fura-2 / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data; D037342:Pertussis Toxin; D010857:Pigment Epithelium of Eye / Q000737:chemistry* / Q000166:cytology / Q000187:drug effects; D016133:Polymerase Chain Reaction; D018048:Receptors, Purinergic P2 / Q000032:analysis* / Q000187:drug effects / Q000378:metabolism; D058473:Receptors, Purinergic P2Y2; D015398:Signal Transduction / Q000187:drug effects; D014544:Uridine Triphosphate / Q000494:pharmacology*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D000345:Affinity Labels; C546385:P2RY2 protein, human; D018048:Receptors, Purinergic P2; D058473:Receptors, Purinergic P2Y2; D010566:Virulence Factors, Bordetella; C022571:adenosine 5'-O-(3-thiotriphosphate); C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D000241:Adenosine; D002118:Calcium; D016257:Fura-2; D014544:Uridine Triphosphate