Identification of the P2Y(12) receptor in nucleotide inhibition of exocytosis from bovine chromaffin cells

Mol Pharmacol. 2004 Sep;66(3):601-11. doi: 10.1124/mol.104.000224.

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 (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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / pharmacology*
  • Amino Acid Sequence
  • Animals
  • Calcium / metabolism
  • Cattle
  • Chromaffin Cells / drug effects*
  • Chromaffin Cells / physiology
  • Cloning, Molecular
  • Electrophysiology
  • Exocytosis / drug effects*
  • Exocytosis / physiology
  • Membrane Proteins / genetics
  • Membrane Proteins / physiology*
  • Molecular Sequence Data
  • Oocytes / physiology
  • RNA, Messenger / analysis
  • Receptors, Purinergic P2 / genetics
  • Receptors, Purinergic P2 / metabolism
  • Receptors, Purinergic P2 / physiology*
  • Receptors, Purinergic P2X
  • Receptors, Purinergic P2Y12
  • Reverse Transcriptase Polymerase Chain Reaction
  • Sequence Homology, Amino Acid
  • Uridine Triphosphate / pharmacology*
  • Xenopus laevis

Substances

  • Membrane Proteins
  • RNA, Messenger
  • Receptors, Purinergic P2
  • Receptors, Purinergic P2X
  • Receptors, Purinergic P2Y12
  • Adenosine Triphosphate
  • Calcium
  • Uridine Triphosphate