Pharmacological characterization of the rat brain P2Y1 receptor expressed in HEK293 cells:: Ca2+ signaling and receptor regulation

The increasing number of ATP- and UTP-sensitive membrane receptors identified by cloning represent either ligand-activated ion channels (P2X) or G-protein-coupled receptors (P2Y). Adenosine, ATP, and UTP have potential application in the management of pain, cancer, and some cardiovascular and pulmonary diseases and are also involved in inflammatory processes in the brain. Therefore, P2Y receptors seem to be promising therapeutic targets. Multiple P2Y receptor subtypes, classified pharmacologically, are mainly linked to activation of phospholipase C (PLC). The present study further characterizes the rat brain P2Y(1) wild-type receptor (rP2Y(1)-wt) and the eGFP-tagged receptor (rP2Y(1)-eGFP) stably expressed in HEK293 cells, thus shedding light on receptor regulation. Both receptors were analyzed by measuring Ca2+ responses in single cells. The rP2Y(1)-eGFP receptor was coupled to Ca2+ release like the rP2Y(1)-wt receptor. Experiments using the PLC inhibitor U73122 confirm the functional activation of PLC beta through rP2Y(1)-eGFP activation. The P2Y(1)-selective agonists 2-MeSADP and 2-MeSATP were most potent at the heterologously expressed receptors. We found a ligand selectivity typical for P2Y, receptors (2-MeSADP = 2-MeSATP > ADP > ATP alphaS, ATP much greater than UTP). Fluorescence microscopy and Ca2+ measurements confirm that the rP2Y1 - eGFP receptor during homologous desensitization is subjected to processes leading to agonist-induced internalization. The kinetics of receptor resensitization were also examined. Therefore, rP2Y(1)-eGFP expressing cells are suitable to determine the physiological P2Y1 receptor signaling pathway and can be a helpful tool to identify drugs acting at P2Y(1) receptors as possible therapeutic targets. (C) 2001 Wiley-Liss, Inc.