Characterization and channel coupling of the P2Y(12) nucleotide receptor of brain capillary endothelial cells.
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 P2Y(12) ADP receptor ("P2Y(T)") of platelets. When expressed heterologously, 2-methylthio-ADP (2-MeSADP; EC(50), 2 nm), ADP, and adenosine 5'-O-(2-thio)diphosphate were agonists of cAMP decrease, and 2-propylthio-D-beta,gamma-difluoromethylene-ATP was a competitive antagonist (K(B), 28 nm), as in platelets. However, 2-methylthio-ATP (2-MeSATP) (EC(50), 0.4 nm), ATP (1.9 microm), and 2-chloro-ATP (190 nm), antagonists in the platelet, were also agonists. 2-MeSADP activated (EC(50), 0.1 nm) GIRK1/GIRK2 inward rectifier K(+) channels when co-expressed with P2Y(12) receptors in sympathetic neurons. Surprisingly, P2Y(1) receptors expressed likewise gave that response; however, a full inactivation followed, absent with P2Y(12) receptors. A new P2Y(12)-mediated transduction was found, the closing of native N-type Ca(2+) channels; again both 2-MeSATP and 2-MeSADP are agonists (EC(50), 0.04 and 0.1 nm, respectively). That action, like their cAMP response, was pertussis toxin-sensitive. The Ca(2+) channel inhibition and K(+) channel activation are mediated by beta gamma subunit release from a heterotrimeric G-protein. G alpha subunit types in B10 cells were also identified. The presence in the brain capillary endothelial cell of the P2Y(12) receptor is a significant extension of its functional range.
Simon
Joseph
J
Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, United Kingdom.
Filippov
Alexander K
AK
Göransson
Sara
S
Wong
Yung H
YH
Frelin
Christian
C
Michel
Anton D
AD
Brown
David A
DA
Barnard
Eric A
EA
D037361:Adenylate Cyclase Toxin; D000262:Adenylyl Cyclases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001254:Astrocytoma; D001667:Binding, Competitive; D001932:Brain Neoplasms; D016466:CHO Cells; D002196:Capillaries / Q000502:physiology; D002560:Cerebrovascular Circulation / Q000502:physiology*; D003001:Cloning, Molecular; D006224:Cricetinae; D004730:Endothelium, Vascular / Q000502:physiology*; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D007473:Ion Channels / Q000502:physiology*; D007700:Kinetics; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D037342:Pertussis Toxin; D010957:Plasmids; D051381:Rats; D020239:Receptor Cross-Talk / Q000502:physiology; D018048:Receptors, Purinergic P2 / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D058925:Receptors, Purinergic P2Y12; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D014407:Tumor Cells, Cultured; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D037361:Adenylate Cyclase Toxin; D007473:Ion Channels; D008565:Membrane Proteins; C548576:P2RY12 protein, human; C553058:P2ry12 protein, rat; D018048:Receptors, Purinergic P2; D058925:Receptors, Purinergic P2Y12; D011994:Recombinant Proteins; D010566:Virulence Factors, Bordetella; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases