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

ABSTRACT:

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 ?M) 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 ?M; PSB-0739, IC₅₀ 5.6 ?M). 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.

SUBMITTER: Micklewright JJ 

PROVIDER: S-EPMC5980558 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature