Abstract

The agonist selectivity for adenosine di- and triphosphates was determined for the human P2Y₁ receptor stably expressed in human 1321N1 astrocytoma cells and was studied under conditions in which nucleotide metabolism was both minimized and assessed. Cells were grown at low density on glass coverslips, encased in a flow-through chamber, and continuously superfused with medium, and Ca²⁺responses to nucleotides were quantified. Superfusion with high performance liquid chromatographically purified ADP, ATP, 2-methylthio-ADP, and 2-methylthio-ATP resulted in rapid Ca²⁺ responses, with EC₅₀ values of 10 ± 5, 304 ± 51, 2 ± 1, and 116 ± 50 nm, respectively. Similar peak responses were observed with maximal concentrations of these four agonists and with the hydrolysis-resistant adenine nucleoside triphosphate adenosine-5′-O-(3-thiotriphosphate). No conversion of [³H]ATP to [³H]ADP occurred under these conditions. Similar full agonist activities of ATP, 2-methylthio-ATP, and ADP were observed in human embryonic kidney 293 cells, which natively express the P2Y₁ receptor. In contrast to these results, Leon et al. [FEBS Lett 403:26–30 (1997)] and Hechler et al.[Mol Pharmacol 53:727–733 (1998)] recently reported that, whereas ADP and 2-methylthio-ADP were agonists, ATP and 2-methylthio-ATP were weak antagonists in studies of the human P2Y₁ receptor expressed in human Jurkat cells. To assess whether differences in the degree of receptor reserve might explain this discrepancy of results, P2Y₁ receptor-expressing 1321N1 cells were incubated for 24 hr with adenosine-5′-O-(2-thiodiphosphate), with the goal of down-regulating the level of functional receptors. Pretreatment with adenosine-5′-O-(2-thiodiphosphate) resulted in a 10-fold rightward shift in the concentration-effect curve for ADP; in contrast, the agonist activity of ATP was completely abolished. Taken together, our results indicate that adenosine di- and triphosphates are agonists at the human P2Y₁ receptor. However, the intrinsic efficacy of ATP is less than that of ADP, and the capacity of ATP to activate second messenger responses through this receptor apparently depends on the degree of P2Y₁ receptor reserve.