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

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Background and Purpose The G i ‐coupled, ADP‐activated P2Y 12 receptor is well characterized as playing a key role in platelet activation via crosstalk with the P2Y 1 receptor in ADP‐evoked intracellular Ca 2+ responses. However, there is limited knowledge on the role of P2Y 12 receptors in ADP‐evoked Ca 2+ responses in other blood cells. Here, we investigated the role of P2Y 12 receptor activation in the modulation of ADP‐evoked Ca 2+ responses in human THP‐1 monocytic cells. Experimental Approach A combination of intracellular Ca 2+ measurements, RT‐PCR, immunocytochemistry, leukocyte isolation and siRNA‐mediated gene knockdown were used to identify the role of P2Y 12 receptor activation. Key Results ADP‐evoked intracellular Ca 2+ responses (EC 50 2.7 μM) in THP‐1 cells were abolished by inhibition of PLC (U73122) or sarco/endoplasmic reticulum Ca 2+ ‐ATPase (thapsigargin). Loss of ADP‐evoked Ca 2+ responses following treatment with MRS2578 (IC 50 200 nM) revealed a major role for P2Y 6 receptors in mediating ADP‐evoked Ca 2+ responses. ADP‐evoked responses were attenuated either with pertussis toxin treatment, or P2Y 12 receptor inhibition with two chemically distinct antagonists (ticagrelor, IC 50 5.3 μM; PSB‐0739, IC 50 5.6 μM). ADP‐evoked responses were suppressed following siRNA‐mediated P2Y 12 gene knockdown. The inhibitory effects of P2Y 12 antagonists were fully reversed following adenylate cyclase inhibition (SQ22536). P2Y 12 receptor expression was confirmed in freshly isolated human CD14 + monocytes. Conclusions and Implications Taken together, these data suggest that P2Y 12 receptor activation positively regulates P2Y 6 receptor‐mediated intracellular Ca 2+ signalling through suppression of adenylate cyclase activity in human monocytic cells.

P2Y12 positively modulates ADP-evoked Ca2+ in monocytic cells in vitro; hypothesis-generating for non-platelet roles, clinical relevance remains open.