| Popis: |
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. |
