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Phorbol Ester-Evoked Ca2+ Signaling in Human Platelets Is via Autocrine Activation of P(2X1) Receptors, Not a Novel Non-Capacitative Ca2+ Entry

Phorbol Ester-Evoked Ca2+ Signaling in Human Platelets Is via Autocrine Activation of P(2X1) Receptors, Not a Novel Non-Capacitative Ca2+ Entry

M T Harper, M J Mason, S O Sage, A G S Harper

J Thromb Haemost. 2010 Jul;8(7):1604-13.

PMID: 20345709

Abstract:

Summary background:
Platelets are reported to possess a protein kinase C (PKC)-dependent non-capacitative Ca(2+)entry (NCCE) pathway. The phorbol ester, phorbol, 12-myristate, 13-acetate (PMA) has been suggested to stimulate platelet NCCE. Recently we demonstrated important roles in store-operated Ca(2+)entry in human platelets for Na(+)/Ca(2+) exchangers (NCXs) and autocrine signaling between platelets after dense granule secretion. As PMA evokes dense granule secretion, we have investigated the role of NCXs and autocrine signaling in PMA-evoked Ca(2+)entry.
Objectives:
To investigate the roles of NCXs and dense granule secretion in PMA-evoked Ca(2+)signaling in human platelets.
Methods:
Fura-2- or sodium-binding benzofuran isophthalate (SBFI)-loaded platelets were used to monitor cytosolic Ca(2+)or Na(+) concentrations. Dense granule secretion was monitored as ATP release using luciferin-luciferase.
Results:
The NCX inhibitors KB-R7943 or SN-6, and removal of extracellular Na(+), significantly reduced PMA-evoked Ca(2+)entry. PMA-evoked dense granule secretion was almost abolished by pretreatment with the PKC inhibitor Ro-31-8220 and significantly slowed by KB-R7943. The P(2X1) antagonists Ro-0437626 or MRS-2159, or desensitization of P(2X1) receptors by prior treatment with alpha,beta-Methylene-ATP or omitting apyrase from the medium, reduced PMA-evoked Ca(2+)entry. Ro-0437626 or chelation of extracellular Ca(2+) slowed but did not abolish PMA-evoked ATP release, indicating that PMA-evoked dense granule secretion does not require P(2X1) receptor activation but is accelerated by P(2X1)-mediated Ca(2+)entry. The presence of NCX3 in human platelets was demonstrated by Western blotting.
Conclusion:
PMA-evoked Ca(2+)entry results from an NCX3-dependent dense granule secretion and subsequent P(2X1) receptor activation by secreted ATP, rather than activation of a novel NCCE pathway.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
IAR4244029	MRS 2159			Price

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