Conventional Protein Kinase C isoforms differentially regulate ADP- and thrombin-evoked Ca²⁺ signalling in human platelets
PKC in platelet Ca²⁺ signalling
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Lever, R. A., Hussain, A., Sun, B., Sage, S., & Harper, A. G. (2015). Conventional Protein Kinase C isoforms differentially regulate ADP- and thrombin-evoked Ca²⁺ signalling in human platelets. Cell Calcium, 58 577-588. https://doi.org/10.1016/j.ceca.2015.09.005
Rises in cytosolic Ca²⁺ concentration ([Ca²⁺]cyt) are central in platelet activation, yet many aspects of the underlying mechanisms are poorly understood. Most studies examine how experimental manipulations affect agonist-evoked rises in [Ca²⁺]cyt, but these only monitor the net effect of manipulations on the processes controlling [Ca²⁺]cyt (Ca²⁺ buffering, sequestration, release, entry and removal), and cannot resolve the source of the Ca²⁺ or the transporters or channels affected. To investigate the effects of protein kinase C (PKC) on platelet Ca²⁺ signalling, we here monitor Ca²⁺ flux around the platelet by measuring net Ca²⁺ fluxes to or from the extracellular space and the intracellular Ca²⁺ stores, which act as the major sources and sinks for Ca²⁺ influx into and efflux from the cytosol, as well as monitoring the cytosolic Na⁺ concentration ([Na+]cyt), which influences platelet Ca²⁺ fluxes via Na⁺/Ca²⁺ exchange. The intracellular store Ca²⁺ concentration ([Ca²⁺]st) was monitored using Fluo-5N, the extracellular Ca²⁺ concentration ([Ca²⁺]ext) was monitored using Fluo-4 whilst [Ca²⁺]cyt and [Na⁺]cyt were monitored using Fura-2 and SFBI, respectively. PKC inhibition using Ro-31-8220 or bisindolylmaleimide I potentiated ADP- and thrombin-evoked rises in [Ca²⁺]cyt in the absence of extracellular Ca²⁺. PKC inhibition potentiated ADP-evoked but reduced thrombin-evoked intracellular Ca²⁺ release and Ca²⁺ removal into the extracellular medium. SERCA inhibition using thapsigargin and 2,5-di(tert-butyl) l,4-benzohydroquinone abolished the effect of PKC inhibitors on ADP-evoked changes in [Ca²⁺]cyt but only reduced the effect on thrombin-evoked responses. Thrombin evokes substantial rises in [Na⁺]cyt which would be expected to reduce Ca²⁺ removal via the Na⁺/Ca²⁺ exchanger (NCX). Thrombin-evoked rises in [Na⁺]cyt were potentiated by PKC inhibition, an effect which was not due to altered changes in non-selective cation permeability of the plasma membrane as assessed by Mn²⁺ quench of Fura-2 fluorescence. PKC inhibition was without effect on thrombin-evoked rises in [Ca²⁺]cyt following SERCA inhibition and either removal of extracellular Na+ or inhibition of Na⁺/K⁺-ATPase activity by removal of extracellular K⁺ or treatment with digoxin. These data suggest that PKC limits ADP-evoked rises in [Ca²⁺]cyt by acceleration of SERCA activity, whilst rises in [Ca²⁺]cyt evoked by the stronger platelet activator thrombin are limited by PKC through acceleration of both SERCA and Na⁺/K⁺-ATPase activity, with the latter limiting the effect of thrombin on rises in [Na⁺]cyt and so forward mode NCX activity. The use of selective PKC inhibitors indicated that conventional and not novel PKC isoforms are responsible for the inhibition of agonist-evoked Ca²⁺ signalling.
Calcium, Na(+)/K(+)-ATPase, Platelet, Protein kinase C, Sarco/endoplasmic reticulum Ca(2+)-ATPase
This work was funded by the British Heart Foundation (PG/07/100/23759). AGSH was supported by a Research Fellowship from St Catharine’s College, University of Cambridge. RAL was supported by a Wellcome Trust Summer Studentship and BBS by a Browning Summer Bursary from Magdalene College, Cambridge.
British Heart Foundation (PG/07/100/23759)
External DOI: https://doi.org/10.1016/j.ceca.2015.09.005
This record's URL: https://www.repository.cam.ac.uk/handle/1810/251411
Attribution-NonCommercial-NoDerivs 2.0 UK: England & Wales
Licence URL: http://creativecommons.org/licenses/by-nc-nd/2.0/uk/
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