TY - JOUR
T1 - Identification of the P2Y12 receptor in nucleotide inhibition of exocytosis from Bovine chromaffin cells
AU - Ennion, SJ
AU - Powell, Andrew
AU - Seward, EP
PY - 2004/1/1
Y1 - 2004/1/1
N2 - 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 ([Ca(2+)](i)), and membrane capacitance changes. In comparative parallel studies, we also cloned the bovine P2Y(12) 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 approximately 2-methylthio-ADP >> ATP approximately ADP > UDP). alphabeta-Methylene-ATP and adenosine were inactive. UTP inhibited I(Ca) in chromaffin cells (pEC(50) = 4.89 +/- 0.11) but was essentially inactive at the cloned P2Y(12) 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 P2Y(12)- and P2Y(13)-selective antagonist N(6)-(2-methylthioethyl)-2-(3,3,3-trifluoropropylthio)-beta,gamma-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 P2Y(12) 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.
AB - 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 ([Ca(2+)](i)), and membrane capacitance changes. In comparative parallel studies, we also cloned the bovine P2Y(12) 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 approximately 2-methylthio-ADP >> ATP approximately ADP > UDP). alphabeta-Methylene-ATP and adenosine were inactive. UTP inhibited I(Ca) in chromaffin cells (pEC(50) = 4.89 +/- 0.11) but was essentially inactive at the cloned P2Y(12) 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 P2Y(12)- and P2Y(13)-selective antagonist N(6)-(2-methylthioethyl)-2-(3,3,3-trifluoropropylthio)-beta,gamma-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 P2Y(12) 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.
UR - http://www.scopus.com/inward/record.url?scp=4944262878&partnerID=8YFLogxK
U2 - 10.1124/mol.104.000224
DO - 10.1124/mol.104.000224
M3 - Article
C2 - 15322252
SN - 1521-0111
VL - 66
SP - 601
EP - 611
JO - Molecular Pharmacology
JF - Molecular Pharmacology
IS - 3
ER -