Distinct P2Y receptor subtypes regulate calcium signaling in human retinal pigment epithelial cells.
OBJECTIVE
Nucleotide signaling plays a role in retinal pigment epithelial (RPE) function, and receptors for nucleotides are potential therapeutic targets for various ocular diseases. The purpose of this study was to investigate the expression of P2Y receptor subtypes in native and cultured human RPE cells.


METHODS
Intracellular Ca(2+) levels were monitored using real-time fluorescence imaging in cultured human RPE cells loaded with Fura-2. Expression of P2Y receptors in native and cultured RPE cells was determined by quantitative RT-PCR and Western blot analysis.


RESULTS
Adenosine triphosphate (ATP), uridine triphosphate (UTP), adenosine diphosphate (ADP), 2-methylthio ATP (2MeSATP), and uridine diphosphate (UDP) produced concentration-related increases in [Ca(2+)](i) in cultured RPE cells. However, differences between the magnitude and shape of agonist responses were observed. ATP and UTP showed similar response characteristics, including a distinct Ca(2+) influx component. ATP and UTP were equipotent (EC(50), 6 muM) and maximum responses were equivalent, suggesting activation of a P2Y(2) receptor. Maximal responses to ADP and 2MeSATP were equivalent with EC(50)s of 1 muM and 0.3 muM. The P2Y(1) antagonist MRS 2179 (10 muM) inhibited these responses, confirming functional expression of P2Y(1) receptors. The presence of a response to UDP suggested P2Y(6) expression. There was no influx component to P2Y(1)- and P2Y(6)-mediated responses. mRNA for P2Y(1), P2Y(2,) P2Y(4), and P2Y(6) receptor subtypes was found in cultured RPE cells, and for P2Y(1), P2Y(2,) P2Y(4,) P2Y(6), and P2Y(12) it was found in native RPE cells. Expression of P2Y(1), P2Y(2), and P2Y(6) protein was found in native and cultured RPE cells.


CONCLUSIONS
These data define the expression profile of P2Y receptors in human RPE and show that different P2Y subtypes control distinct calcium responses in these cells.
Tovell
Victoria E
VE
School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich, United Kingdom.
Sanderson
Julie
J
D000255:Adenosine Triphosphate / Q000494:pharmacology; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology*; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D016257:Fura-2 / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D010857:Pigment Epithelium of Eye / Q000187:drug effects / Q000378:metabolism*; D058910:Purinergic P2 Receptor Agonists; D058919:Purinergic P2 Receptor Antagonists; D012333:RNA, Messenger / Q000378:metabolism; D018048:Receptors, Purinergic P2 / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014544:Uridine Triphosphate / Q000494:pharmacology
D058910:Purinergic P2 Receptor Agonists; D058919:Purinergic P2 Receptor Antagonists; D012333:RNA, Messenger; D018048:Receptors, Purinergic P2; D000255:Adenosine Triphosphate; D002118:Calcium; D016257:Fura-2; D014544:Uridine Triphosphate