Identification and characterization of P2Y(2) 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 ATP gamma S 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 P2Y(2) nucleotide receptor subtype in RPE cells, [alpha-P-32]BzATP labeled a 53-kDa protein in plasma membranes, and RT-PCR revealed the presence of P2Y(2) 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 P2Y(2) nucleotide receptors. (C) 1997 Wiley-Liss, Inc.