Simon Joseph, Filippov Alexander K, Göransson Sara, Wong Yung H, Frelin Christian, Michel Anton D, Brown David A, Barnard Eric A
Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, United Kingdom.
J Biol Chem. 2002 Aug 30;277(35):31390-400. doi: 10.1074/jbc.M110714200. Epub 2002 Jun 21.
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.
大鼠脑毛细血管内皮（B10）细胞表达一种与腺苷酸环化酶抑制相关的未鉴定核苷酸受体。我们发现B10细胞中的这种受体在序列上与血小板的P2Y(12) ADP受体（“P2Y(T)”）相同。当在异源系统中表达时，2-甲硫基-ADP（2-MeSADP；EC(50)，2 nM）、ADP和腺苷5'-O-(2-硫代)二磷酸是cAMP降低的激动剂，而2-丙硫基-D-β,γ-二氟亚甲基-ATP是竞争性拮抗剂（K(B)，28 nM），与血小板中的情况相同。然而，血小板中的拮抗剂2-甲硫基-ATP（2-MeSATP）（EC(50)，0.4 nM）、ATP（1.9 μM）和2-氯-ATP（190 nM）也是激动剂。当与交感神经元中的P2Y(12)受体共表达时，2-MeSADP激活（EC(50)，0.1 nM）GIRK1/GIRK2内向整流钾通道。令人惊讶的是，同样表达的P2Y(1)受体也产生了这种反应；然而，随后会完全失活，而P2Y(12)受体则不会。发现了一种新的由P2Y(12)介导的转导过程，即天然N型钙通道的关闭；同样，2-MeSATP和2-MeSADP都是激动剂（EC(50)，分别为0.04和0.1 nM）。该作用与其cAMP反应一样，对百日咳毒素敏感。钙通道抑制和钾通道激活是由异源三聚体G蛋白释放的βγ亚基介导的。还鉴定了B10细胞中的Gα亚基类型。P2Y(12)受体在脑毛细血管内皮细胞中的存在显著扩展了其功能范围。
