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 (ICa), 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). αβ-Methylene-ATP and adenosine were inactive. UTP inhibited ICa 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)-β,γ-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 Gi/o -coupled pyrimidine receptor
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