Identification and characterization of P2Y2 nucleotide receptors in human retinal pigment epithelial cells

Abstract

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.

Agonists and antagonists acting at P2X receptors: selectivity profiles and functional implications

“…It is a very weak partial agonist at P2Y 4 and P2Y 6 receptors (Chang et al 1995;Bogdanov et al 1998). Studies at likely endogenous P2X (P2X 2 : PC-12 cells, rat pelvic ganglion neurons; Michel et al 1996a;Zhong et al 1998) and P2Y receptors (P2Y 2 : retinal pigment epithelial cells, umbilical vein endothelial cells; Sullivan et al 1997;Conant et al 1998) conform with the pharmacology of ATPγS at recombinant receptors. Using human whole blood cells or bovine pulmonary artery endothelial cells, it has been demonstrated that ATPγS reversibly inhibits ecto-nucleotidases (pIC 50 =5.2; Beukers et al 1995;Chen and Lin 1997).…”

Expression of Orai genes and ICRAC activation in the human retinal pigment epithelium

“…Various stimuli have been shown to induce the release of intracellular Ca 2+ through the activation of inositol-1,4,5-trisphosphate receptors. These include growth factors [15], carbachol [34], histamine [35], and ATP which is speculated to be the "light-peak substance" [36,37]. Accordingly, Orai channels in RPE cells provide a way to refill intracellular Ca 2+ stores after activation of ryanodine or inositol-1,4,5-trisphosphate receptors.…”

“…Some stimuli like mechanical stimulation, ATP, and bFGF have been shown to induce sustained Ca 2+ signals in RPE cells [16,36,38]. Other signals, including carbachol, lipophosphatidic acid, and stimulation with photoreceptor outer segments, lead to repetitive raises of the [Ca 2+ ] i [16,39,40].…”

Functional Expression of P2Y Receptors in WERI-Rb1 Retinoblastoma Cells

“…P2Y2 receptors have been identified in a wide variety of ocular cell types, particularly, in retina [26], retinal pigment epithelium [27][28][29], cornea [30,31], conjunctiva [32], lens [33], and ciliary epithelia [9,34]. P2Y2 receptor signaling and its function are apparent in the eye [35], and regulates multiple cellular functions in ocular physiology by calcium mobilization [9] including ion transport and fluid absorption [27,29], and mucin discharge [36].…”