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

Micklewright, J J, Layhadi, J A and Fountain, S J (2018) P2Y12 receptor modulation of ADP‐evoked intracellular Ca2+ signalling in THP‐1 human monocytic cells. British Journal of Pharmacology, 175 (12). pp. 2483-2491. ISSN 0007-1188

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    Abstract

    Background and purpose: The Gi‐coupled, ADP‐activated P2Y12 receptor is well characterised as playing a key role in platelet activation via crosstalk with P2Y1 in ADP‐evoked intracellular Ca2+ response. There is limited knowledge on the role of P2Y12 in ADP‐evoked Ca2+ responses in other blood cells. Here we investigate the role of P2Y12 receptor activation in 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 phospholipase C (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 in mediating ADP‐evoked Ca2+ responses. ADP‐evoked responses were attenuated either with pertussis toxin treatment, or P2Y12 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. Conclusion and implications: Taken together, these data suggest that P2Y12 activation positively regulates P2Y6‐mediated intracellular Ca2+ signalling through suppression of adenylate cyclase activity in human monocytic cells.

    Item Type: Article
    Faculty \ School: Faculty of Science > School of Biological Sciences
    Related URLs:
    Depositing User: Pure Connector
    Date Deposited: 05 Apr 2018 13:30
    Last Modified: 25 Jul 2018 14:44
    URI: https://ueaeprints.uea.ac.uk/id/eprint/66682
    DOI: 10.1111/bph.14218

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