Research Article Expression of Multiple P2Y Receptors by MDCK-D1 Cells: P2Y 1 Receptor Cloning and Signaling Richard J. Hughes, Brian Torres, Alexander Zambon, David Arthur, Christine Bohmann, L. Christian Rump, and Paul A. Insel n Department of Pharmacology, University of California at San Diego, La Jolla, California Strategy, Management and Health Policy Venture Capital Enabling Technology Preclinical Research Preclinical Development Toxicology, Formulation Drug Delivery, Pharmacokinetics Clinical Development Phases I-III Regulatory, Quality, Manufacturing Postmarketing Phase IV ABSTRACT 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 P2Y 1 , P2Y 2 , P2Y 6 , and P2Y 11 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 1321N1 cells, and the ability of the heterologously expressed receptors to increase intracellular calcium and phosphoinositide hydrolysis. ADP, methylthioATP, and ADPbS are agonists with the greatest potency, while ATP and ATPgS 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 1 receptors. With their ability to respond to ADP and ATP, P2Y 1 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. INTRODUCTION Nature has orchestrated an array of cell-surface receptors that elicit responses to a broad range of pharmacologically active compounds, including pur- ines, such as adenosine, ADP, and ATP, and pyrimi- dines, such as UTP and UDP. The receptors for purines and pyrimidines can be broadly categorized as belonging to one of three major classes: the metabo- tropic P2Y nucleotide receptors, adenosine receptors, and the P2X ion channel receptors. The P2Y and adenosine receptors are members of the superfamily of G-protein-coupled receptors (GPCRs), i.e., seven transmembrane-spanning receptors, based on the presence of seven hydrophobic membrane spanning domains. These major classes are each composed of multiple receptor subtypes that have been differen- tiated on the basis of their pharmacology and, in some cases, sequence similarities. One important class of cells regulated by such receptors is epithelial cells. Epithelial cells line the gastrointestinal, bronchopulmonary, and genitourinary tracts and in those locations function as the barrier DDR Abbreviations: adenosine 3 0 -phosphate 5 0 -phosphosulfate, A3P5PS; bradykinin, BK; benzoylbenzoyl adenosine triphosphate, bzbzATP; adenosine 3 0 -phosphate 5 0 -cyclic monophosphate, cAMP; fluorescence-activated cell sorting, FACS; G protein- coupled receptor, GPCR; high throughput pharmacology system, HTPS; Madin-Darby canine kidney, MDCK; polymerase chain reaction, PCR; pyridoxal-phosphate-6-azophenyl-2 0 ,4 0 -disulfonic acid, PPADS; reverse transcription, RT. Contract grant sponsors: NIH; the UCSD Academic Senate. n Correspondence to: Paul A. Insel, Department of Pharma- cology, University of California at San Diego, La Jolla, CA 92093- 0636. Published online in Wiley InterScience (www.interscience. wiley.com) DOI: 10.1002/ddr.10196 DRUG DEVELOPMENT RESEARCH 59:1–7 (2003)  c 2003 Wiley-Liss, Inc.