Abstract

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 G_i 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 GTPγ[³⁵S]), 1321N1 cells coexpressing Gα₁₆ [AG32 cells: inositol trisphosphate (IP₃) measurement, binding of GTPγ[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 GTPγ[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 μM). 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 μM.