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

Background and purpose: The G_i -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.

Experimental approach: 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.

Key results: ADP-evoked intracellular CA²⁺ responses (EC₅₀ 2.7 μM) 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 200 nM) 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 5.3 μM; PSB-0739, IC₅₀ 5.6 μM). 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.

Conclusions and implications: 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.