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Identification of the P2Y₁₂ Receptor in Nucleotide Inhibition of Exocytosis from Bovine Chromaffin Cells

Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, United Kingdom (S.J.E.); Department of Pharmacology, University of Bristol, Bristol, United Kingdom (A.D.P.); and Department of Biomedical Sciences, University of Sheffield, Sheffield, United Kingdom (E.P.S.)

Nucleotides are released from bovine chromaffin cells and take part in a feedback loop to inhibit further exocytosis. To identify the nucleotide receptors involved, we measured the effects of a range of exogenous nucleotides and related antagonists on voltage-operated calcium currents (I_Ca), intracellular calcium concentration ([Ca²⁺]_i), and membrane capacitance changes. In comparative parallel studies, we also cloned the bovine P2Y₁₂ receptor from chromaffin cells and determined its properties by coexpression in Xenopus laevis oocytes with inward-rectifier potassium channels made up of Kir3.1 and Kir3.4. In both systems, the agonist order of potency was essentially identical (2-methylthio-ATP 2-methylthio-ADP >> ATP ADP > UDP). -Methylene-ATP and adenosine were inactive. UTP inhibited I_Ca in chromaffin cells (pEC₅₀ = 4.89 ± 0.11) but was essentially inactive at the cloned P2Y₁₂ receptor. The relatively nonselective P2 antagonist pyridoxal-phosphate-6-azophenyl-2',4' disulfonic acid blocked nucleotide responses in both chromaffin cells and X. laevis oocytes, whereas the P2Y₁₂- and P2Y₁₃-selective antagonist N⁶-(2-methylthioethyl)-2-(3,3,3-trifluoropropylthio)-,-dichloromethylene ATP (ARC69931MX) blocked responses to ATP in both chromaffin cells and X. laevis oocytes but not to UTP in chromaffin cells. These results identify the P2Y₁₂ purine receptor as a key component of the nucleotide inhibitory pathway and also demonstrate the involvement of a UTP-sensitive G_i/o -coupled pyrimidine receptor.

Address correspondence to: Dr. Elizabeth P. Seward, Department of Biomedical Sciences, University of Sheffield, Alfred Denny Building, Western Bank, Sheffield, S10 2TN, United Kingdom. E-mail: e.p.seward{at}sheffield.ac.uk

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