methods. Intracellular Ca2+ 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 [Ca2+]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 Ca2+ influx component. ATP and UTP were equipotent (EC50, 6 µM) and maximum responses were equivalent, suggesting activation of a P2Y2 receptor. Maximal responses to ADP and 2MeSATP were equivalent with EC50s of 1 µM and 0.3 µM. The P2Y1 antagonist MRS 2179 (10 µM) inhibited these responses, confirming functional expression of P2Y1 receptors. The presence of a response to UDP suggested P2Y6 expression. There was no influx component to P2Y1- and P2Y6-mediated responses. mRNA for P2Y1, P2Y2, P2Y4, and P2Y6 receptor subtypes was found in cultured RPE cells, and for P2Y1, P2Y2, P2Y4, P2Y6, and P2Y12 it was found in native RPE cells. Expression of P2Y1, P2Y2, and P2Y6 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.