Marteau Frederic, Le Poul Emmanuel, Communi David, Communi Didier, Labouret Catherine, Savi Pierre, Boeynaems Jean-Marie, Gonzalez Nathalie Suarez
Institute for Interdisciplinary Research, School of Medicine, and Department of Medical Chemistry, Erasme Hospital, Brussels, Belgium.
Mol Pharmacol. 2003 Jul;64(1):104-12. doi: 10.1124/mol.64.1.104.
The P2Y13 receptor has recently been identified as a new P2Y receptor sharing a high sequence homology with the P2Y12 receptor as well as similar functional properties: coupling to Gi and responsiveness to ADP (Communi et al., 2001). In the present study, the pharmacology of the P2Y13 receptor and its differences with that of the P2Y12 receptor have been further characterized in 1321N1 cells (binding of [33P]2-methylthio-ADP (2MeSADP) and of GTPgamma[35S]), 1321N1 cells coexpressing Galpha16 [AG32 cells: inositol trisphosphate (IP3) measurement, binding of GTPgamma[35S]) and Chinese hamster ovary (CHO)-K1 cells (cAMP assay)]. 2MeSADP was more potent than ADP in displacing [33P]2MeSADP bound to 1321N1 cells and increasing GTPgamma[35S] binding to membranes prepared from the same cells. Similarly, 2MeSADP was more potent than ADP in stimulating IP3 accumulation after 10 min in AG32 cells and increasing cAMP in pertussis toxin-treated CHO-K1 cells stimulated by forskolin. On the other hand, ADP and 2MeSADP were equipotent at stimulating IP3 formation in AG32 cells after 30 s and inhibiting forskolininduced cAMP accumulation in CHO-K1 cells. These differences in potency cannot be explained by differences in degradation rate, which in AG32 cells was similar for the two nucleotides. When contaminating diphosphates were enzymatically removed and assay of IP3 was performed after 30 s, ATP and 2MeSATP seemed to be weak partial agonists of the P2Y13 receptor expressed in AG32 cells. The stimulatory effect of ADP on the P2Y13 receptor in AG32 cells was antagonized by reactive blue 2, suramin, pyridoxal-phosphate-6-azophenyl-2',4'disulfonic acid, diadenosine tetraphosphate, and 2-(propylthio)-5'-adenylic acid, monoanhydride with dichloromethylenebis (phosphonic acid) (AR-C67085MX), but not by N6-methyl 2'-deoxyadenosine 3',5'-bisphosphate (MRS-2179) (up to 100 microM). The most potent antagonist was N6-(2-methylthioethyl)-2-(3,3,3-trifluoropropylthio)-5'-adenylic acid, monoanhydride with dichloromethylenebis (phosphonic acid) (ARC69931MX) (IC50 = 4 nM), which behaved in a noncompetitive way. The active metabolite of clopidogrel was unable to displace bound 2MeSADP at concentrations up to 2 microM.
P2Y13受体最近被确定为一种新的P2Y受体，它与P2Y12受体具有高度的序列同源性以及相似的功能特性：与Gi偶联并对ADP有反应（Communi等人，2001年）。在本研究中，在1321N1细胞（[33P]2-甲基硫代-ADP（2MeSADP）和GTPγ[35S]的结合）、共表达Gα16的1321N1细胞[AG32细胞：肌醇三磷酸（IP3）测量、GTPγ[35S]的结合]和中国仓鼠卵巢（CHO）-K1细胞（cAMP测定）中进一步表征了P2Y13受体的药理学及其与P2Y12受体的差异。在1321N1细胞中，2MeSADP在取代与[33P]2MeSADP结合的能力以及增加与相同细胞制备的膜上的GTPγ[35S]结合方面比ADP更有效。同样，在AG32细胞中10分钟后，2MeSADP在刺激IP3积累方面比ADP更有效，在百日咳毒素处理的由福司可林刺激的CHO-K1细胞中增加cAMP方面也比ADP更有效。另一方面，ADP和2MeSADP在刺激AG32细胞30秒后IP3形成以及抑制CHO-K1细胞中福司可林诱导的cAMP积累方面效力相当。这些效力差异不能用降解速率的差异来解释，在AG32细胞中这两种核苷酸的降解速率相似。当通过酶法去除污染的二磷酸并在30秒后进行IP3测定时，ATP和2MeSATP似乎是AG32细胞中表达的P2Y13受体的弱部分激动剂。AG32细胞中ADP对P2Y13受体的刺激作用被反应性蓝2、苏拉明、磷酸吡哆醛-6-偶氮苯基-2',4'-二磺酸、四磷酸二腺苷和2-（丙硫基）-5'-腺苷酸，二氯亚甲基双（膦酸）单酐（AR-C67085MX）拮抗，但不被N6-甲基2'-脱氧腺苷3',5'-双磷酸（MRS-2179）（高达100 microM）拮抗。最有效的拮抗剂是N6-（2-甲基硫代乙基）-2-（3,3,3-三氟丙硫基）-5'-腺苷酸，二氯亚甲基双（膦酸）单酐（ARC69931MX）（IC50 = 4 nM），其表现为非竞争性。氯吡格雷的活性代谢物在浓度高达2 microM时无法取代结合的2MeSADP。
