Identification of the P2Y(12) receptor in nucleotide inhibition of exocytosis from bovine chromaffin cells.
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.
Ennion
Steven J
SJ
Department of Biomedical Sciences, University of Sheffield, Alfred Denny Building, Western Bank, Sheffield, S10 2TN, United Kingdom.
Powell
Andrew D
AD
Seward
Elizabeth P
EP
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D019439:Chromaffin Cells / Q000187:drug effects* / Q000502:physiology; D003001:Cloning, Molecular; D004594:Electrophysiology; D005089:Exocytosis / Q000187:drug effects* / Q000502:physiology; D008565:Membrane Proteins / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D009865:Oocytes / Q000502:physiology; D012333:RNA, Messenger / Q000032:analysis; D018048:Receptors, Purinergic P2 / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D058469:Receptors, Purinergic P2X; D058925:Receptors, Purinergic P2Y12; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D014544:Uridine Triphosphate / Q000494:pharmacology*; D014982:Xenopus laevis
D008565:Membrane Proteins; D012333:RNA, Messenger; D018048:Receptors, Purinergic P2; D058469:Receptors, Purinergic P2X; D058925:Receptors, Purinergic P2Y12; D000255:Adenosine Triphosphate; D002118:Calcium; D014544:Uridine Triphosphate