P2Y12-dependent modulation of ADP-evoked calcium responses in human monocytes

Micklewright, J J (2018) P2Y12-dependent modulation of ADP-evoked calcium responses in human monocytes. Other thesis, University of East Anglia.

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Abstract

The Gi-coupled, ADP-activated P2Y12 receptor is well-characterised as playing a key role in platelet activation via crosstalk with P2Y1. A crucial aspect of P2Y12-P2Y1 crosstalk in platelets involves ADP-induced intracellular calcium (Ca2+) mobilisation, however there is limited knowledge on the role of P2Y12 in ADP-evoked Ca2+ responses in other blood cells. Here, we investigate the expression of P2Y12 in human monocytes and the contribution of P2Y12 in THP-1 ADP-evoked Ca2+ responses.
RT-PCR analysis showed that all ADP-binding P2Y receptors were expressed in THP-1 monocytes at the mRNA level, with P2Y12 expressed in CD14+ primary monocytes. P2Y12 protein was found to be expressed in THP-1 cells, using immunocytochemistry. ADP-evoked Ca2+ responses in fura-2-loaded THP-1 cells were completely abolished by a sarco/endoplasmic reticulum Ca2+-ATPase inhibitor, and by a phospholipase C inhibitor, indicating that these Ca2+ responses are mediated through GPCRs. Ca2+ responses induced by ADP were significantly reduced by the P2Y12 inhibitors ticagrelor and PSB-0739 and by the P2Y6 antagonist MRS2578, but not by P2Y1 or P2Y13 inhibitors. Using P2Y6-overexpressing astrocytomas, ticagrelor was found to be selective for P2Y12 over P2Y6 at the concentrations used here, although PSB-0739 was not. ADP-induced Ca2+ responses were significantly decreased in THP-1 cells with siRNA-mediated P2Y12 knockdown. Pertussis toxin-mediated Gi inhibition caused a significant reduction in ADP-induced Ca2+ responses, as did the PI3K inhibitor LY294002. Use of the adenylate cyclase inhibitor SQ22536 had no effect on sole ADP-evoked Ca2+ responses, but caused significant recovery of Ca2+ responses inhibited by P2Y12 antagonists.
The results from this study suggest that Ca2+ responses evoked by ADP in THP-1 cells are dependent on both P2Y12 and P2Y6 activation, with P2Y12 positively modulating Gq-coupled P2Y6-mediated Ca2+ responses via adenylate cyclase inhibition and PI3K activation. Overall, this investigation reveals a new role for P2Y12 in non-platelet Ca2+ responses, and contributes to our understanding of how monocytes function in health and disease.

Item Type: Thesis (Other)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Jackie Webb
Date Deposited: 10 May 2018 11:40
Last Modified: 10 May 2018 11:40
URI: https://ueaeprints.uea.ac.uk/id/eprint/67022
DOI:

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