P2Y12 receptor modulation of ADP-evoked intracellular Ca2+ signalling in THP-1 human monocytic cells.

Abstract

The Gi -coupled, ADP-activated P2Y12 receptor is well characterized as playing a key role in platelet activation via crosstalk with the P2Y1 receptor in ADP-evoked intracellular Ca2+ responses. However, there is limited knowledge on the role of P2Y12 receptors in ADP-evoked Ca2+ responses in other blood cells. Here, we investigated the role of P2Y12 receptor activation in the modulation of ADP-evoked Ca2+ responses in human THP-1 monocytic cells. A combination of intracellular Ca2+ measurements, RT-PCR, immunocytochemistry, leukocyte isolation and siRNA-mediated gene knockdown were used to identify the role of P2Y12 receptor activation. ADP-evoked intracellular Ca2+ responses (EC50 2.7μM) in THP-1 cells were abolished by inhibition of PLC (U73122) or sarco/endoplasmic reticulum Ca2+ -ATPase (thapsigargin). Loss of ADP-evoked Ca2+ responses following treatment with MRS2578 (IC50 200nM) revealed a major role for P2Y6 receptors in mediating ADP-evoked Ca2+ responses. ADP-evoked responses were attenuated either with pertussis toxin treatment, or P2Y12 receptor inhibition with two chemically distinct antagonists (ticagrelor, IC50 5.3μM; PSB-0739, IC50 5.6μM). ADP-evoked responses were suppressed following siRNA-mediated P2Y12 gene knockdown. The inhibitory effects of P2Y12 antagonists were fully reversed following adenylate cyclase inhibition (SQ22536). P2Y12 receptor expression was confirmed in freshly isolated human CD14+ monocytes. Taken together, these data suggest that P2Y12 receptor activation positively regulates P2Y6 receptor-mediated intracellular Ca2+ signalling through suppression of adenylate cyclase activity in human monocytic cells.