IDENTIFICATION AND CHARACTERIZATION OF P2Y(2) NUCLEOTIDE RECEPTORS INHUMAN RETINAL-PIGMENT EPITHELIAL-CELLS

P2 nucleotide receptor expression in cultured human retinal pigment ep ithelial (RPE) cells was investigated using the photoaffinity ATP anal og 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-f old increase in [Ca2+](i) followed by a prolonged elevation that was t wofold 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 t oxin, suggest that a G protein-coupled receptor mediates this response . Consistent with the expression of a P2Y(2) nucleotide receptor subty pe 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. Ad enosine had no effect on [Ca2+](i) in RPE cells, indicating that the A 2 subtype of P1 receptor described previously in human RPE is not invo lved in the response to nucleotides. Together the results indicate tha t human RPE cells express functional P2Y(2) nucleotide receptors. (C) 1997 Wiley-Liss, Inc.