Expression of multiple P2Y receptors by MDCK-D1 cells:: P2Y1 receptor cloning and signaling

The Madin Darby canine kidney (MDCK) cell line, a well-differentiated renal epithelial cell line, is a useful model to examine P2Y receptor signaling and response. Our studies with MDCK-D1, a clonal isolate, demonstrate that these cells release ATP in response to mechanical stimulation and activation of certain G-protein-coupled receptors. Reverse transcriptase-polymerase chain reaction (RTPCR) studies document that MDCK cells express multiple F2Y receptors, including P2Y(1), P2Y(2), P2Y(6), and P2Y, I receptors. We isolated cDNAs for several of the P2Y receptor genes and expressed these in cells, such as the 1321N1 astrocytoma cell line, that lack native P2Y receptor expression. We report here the molecular cloning of the MDCK P2Y(1) receptor, heterologous expression in 1321 NI cells, and the ability of the heterologously expressed receptors to increase intracellular calcium and phosphoinositide hydrolysis. ADP, methylthioATP, and ADP S are agonists with the greatest potency, while ATP and ATPgammaS show lower potency and efficacy, and benzoylbenzoylATP, UTP, and UDP lack efficacy at the cloned P2Y(1) receptor. Several antagonists, including MRS2179, A3P5PS, suramin, and PPADS blocked response at the cloned P2Y, receptors. With their ability to respond to ADP and ATP, P2Y, receptors, along with other P2Y receptors expressed in MDCK cells, contribute to the response of these cells to ATP (or its breakdown product, ADP) released from the cells and to exogenously added nucleotides. Drug Dev. Res. 59:1-7, 2003. (C) 2003 Wiley-Liss, Inc.