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ABSTRACT: Background and purpose
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.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
British journal of pharmacology 20180429 12
<h4>Background and purpose</h4>The G<sub>i</sub> -coupled, ADP-activated P2Y<sub>12</sub> receptor is well characterized as playing a key role in platelet activation via crosstalk with the P2Y<sub>1</sub> receptor in ADP-evoked intracellular Ca<sup>2+</sup> responses. However, there is limited knowledge on the role of P2Y<sub>12</sub> receptors in ADP-evoked Ca<sup>2+</sup> responses in other blood cells. Here, we investigated the role of P2Y<sub>12</sub> receptor activation in the modulation of ...[more]