Journal
MOLECULAR PHARMACOLOGY
Volume 64, Issue 1, Pages 104-112Publisher
AMER SOC PHARMACOLOGY EXPERIMENTAL THERAPEUTICS
DOI: 10.1124/mol.64.1.104
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The P2Y(13) receptor has recently been identified as a new P2Y receptor sharing a high sequence homology with the P2Y(12) receptor as well as similar functional properties: coupling to G(i) and responsiveness to ADP ( Communi et al., 2001). In the present study, the pharmacology of the P2Y(13) receptor and its differences with that of the P2Y(12) receptor have been further characterized in 1321N1 cells (binding of [P-33] 2-methylthio-ADP (2MeSADP) and of GTPgamma[S-35]), 1321N1 cells coexpressing Galpha(16) [AG32 cells: inositol trisphosphate (IP3) measurement, binding of GTPgamma[S-35]) and Chinese hamster ovary (CHO)-K1 cells ( cAMP assay)]. 2MeSADP was more potent than ADP in displacing [P-33] 2MeSADP bound to 1321N1 cells and increasing GTPgamma[S-35] 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 forskolin-induced 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 P2Y(13) receptor expressed in AG32 cells. The stimulatory effect of ADP on the P2Y(13) 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 N-6-methyl 2'-deoxyadenosine 3', 5'-bisphosphate (MRS-2179) (up to 100 etaM). The most potent antagonist was N-6-(2-methylthioethyl)- 2-(3,3,3-trifluoropropylthio)-5'-adenylic acid, monoanhydride with dichloromethylenebis ( phosphonic acid) ( AR-C69931MX) (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 muM.
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