Adenine triphosphate nucleotides are antagonists at the P2Y₁₂ receptor

Summary

The aim of the present study was to characterize the pharmacological profile of the P2Y₁₂ receptor for several adenine triphosphate nucleotides in view of their possible roles as partial agonists or true antagonists. Two distinct cellular systems were used: P2Y₁ receptor deficient mouse platelets (

platelets) previously shown to express a native and functional P2Y₁₂ receptor and 1321 N1 astrocytoma cells stably expressing the human P2Y₁₂ receptor (1321 N1 P2Y₁₂). ADP and its structural analogues inhibited cAMP accumulation in a dose‐dependent manner in both

platelets and 1321 N1 P2Y₁₂ cells with a similar rank order of potency, 2 methylthio‐ADP (2MeSADP) >>ADP – Adenosine 5′‐(βthio) diphosphate (ΑDPβS). Commercial ATP, 2 chloro; ATP (2ClATP) and 2 methylthio‐ATP (2MeSATP) also inhibited cAMP accumulation in both cell systems. In contrast, after creatine phosphate (CP)/creatine phosphokinase (CPK) regeneration, adenine triphosphate nucleotides lost their agonistic effect on

platelets and behaved as antagonists of ADP (0.5 µm)‐induced adenylyl cyclase inhibition with IC₅₀ of 13.5 ± 4.8, 838 ± 610, 1280 ± 1246 µm for 2MeSATP, ATP and 2ClATP, respectively. In 1321 N1 P2Y₁₂ cells, CP/CPK regenerated ATP and 2ClATP lost their agonistic effect only when CP/CPK was maintained during the cAMP assay. The stable ATP analogue ATPγS antagonized ADPβS‐induced inhibition of cAMP accumulation in both

platelets and 1321 N1 P2Y₁₂ cells. Thus, ATP and its triphosphate analogues are not agonists but rather antagonists at the P2Y₁₂ receptor expressed in platelets or transfected cells, provided care is taken to remove diphosphate contaminants and to prevent the generation of diphosphate nucleotide derivatives by cell ectonucleotidases.