Characterization and channel coupling of the P2Y12 nucleotide receptor of brain capillary endothelial cells

Abstract

Rat brain capillary endothelial (B10) cells express an unidentified nucleotide receptor linked to adenylyl cyclase inhibition. We show that this receptor in B10 cells is identical in sequence to the P2Y12 ADP receptor ("P2YT") of platelets. When expressed heterologously, 2-methylthio-ADP (2-MeSADP; EC50, 2 nM), ADP, and adenosine 5′-O-(2-thio)diphosphate were agonists of cAMP decrease, and 2-propylthio-D-β,γ-difluoromethylene-ATP was a competitive antagonist (KB, 28 nM), as in platelets. However, 2-methylthio-ATP (2-MeSATP) (EC50, 0.4 nM), ATP (1.9 μM), and 2-chloro-ATP (190 nM), antagonists in the platelet, were also agonists. 2-Me-SADP activated (EC50, 0.1 nM) GIRK1/GIRK2 inward rectifier K+ channels when co-expressed with P2Y12 receptors in sympathetic neurons. Surprisingly, P2Y1 receptors expressed likewise gave that response; however, a full inactivation followed, absent with P2Y12 receptors. A new P2Y12-mediated transduction was found, the closing of native N-type Ca2+ channels; again both 2-MeSATP and 2-MeSADP are agonists (EC50, 0.04 and 0.1 nM, respectively). That action, like their cAMP response, was pertussis toxin-sensitive. The Ca2+ channel inhibition and K+ channel activation are mediated by βγ subunit release from a heterotrimeric G-protein. Gα subunit types in B10 cells were also identified. The presence in the brain capillary endothelial cell of the P2Y12 receptor is a significant extension of its functional range.