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Identification of the P2Y12 receptor in nucleotide inhibition of exocytosis from bovine chromaffin cells

By Steven J. Ennion, A. Powell and E. Seward

Abstract

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

Year: 2004
DOI identifier: 10.1124/mol.104.000224
OAI identifier: oai:lra.le.ac.uk:2381/1727
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