Abstract
Abstract: :
Purpose: ATP can stimulate P2Y receptors on the retinal pigment epithelium (RPE) to increase intracellular calcium and fluid absorption. RPE cells can release ATP, but the molecules responsible for this release have yet to be identified. The cystic fibrosis transmembrane regulator (CFTR) has been implicated in ATP release from other epithelial cells and is expressed by the RPE. We asked whether CFTR was involved in ATP release from RPE cells. Methods:ATP release into the bath was detected with luciferase reaction assay from cultured human ARPE–19 cells grown in 96 well plated and from the interior apical face of fresh bovine RPE eyecups. Intracellular calcium was monitored with fura–2. Results:Hyposmolar conditions induced a significant increase in ATP release from both ARPE–19 and fresh bovine cells. Glybenclamide (100 µM) completely blocked this release from fresh bovine cells, but only partially from ARPE–19 cells. The specific CFTR inhibitor CFTR172 blocked the hyposmotically induced ATP release from both cultured and fresh cells, with a maximum inhibition at 10 µM and an EC50∼3.5 µM. Activation of CFTR with a cAMP–stimulating mixture produced a significant increase in ATP release from both cell types. The calcium chelator BAPTA–AM inhibited the hyposmotically–induced ATP release from ARPE–19 cells, implying calcium is also necessary. The intracellular calcium concentration in ARPE–19 cells showed a biphasic increase in response to hyposmotic conditions, with a fast peak at 2–3 minutes and a slower increase with a maximum at 15 min. CFTR172 completely blocked the fast peak and most of the secondary calcium increase. Conclusions:These observations suggest that the release of ATP from RPE cells requires both CFTR and intracellular calcium. The precise roles of CFTR and calcium in the release process remain to be determined.
Keywords: retinal pigment epithelium • second messengers: pharmacology/physiology • receptors: pharmacology/physiology