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 (RT‐PCR) studies document that MDCK cells express multiple P2Y receptors, including P2Y1, P2Y2, P2Y6, and P2Y11 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 P2Y1 receptor, heterologous expression in 1321N1 cells, and the ability of the heterologously expressed receptors to increase intracellular calcium and phosphoinositide hydrolysis. ADP, methylthioATP, and ADPβS are agonists with the greatest potency, while ATP and ATPγS show lower potency and efficacy, and benzoylbenzoylATP, UTP, and UDP lack efficacy at the cloned P2Y1 receptor. Several antagonists, including MRS2179, A3P5PS, suramin, and PPADS blocked response at the cloned P2Y1 receptors. With their ability to respond to ADP and ATP, P2Y1 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.