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Monitoring the intracellular store Ca 2+ concentration in agonist‐stimulated, intact human platelets by using Fluo‐5N
Author(s) -
SAGE S. O.,
PUGH N.,
MASON M. J.,
HARPER A. G. S.
Publication year - 2011
Publication title -
journal of thrombosis and haemostasis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.947
H-Index - 178
eISSN - 1538-7836
pISSN - 1538-7933
DOI - 10.1111/j.1538-7836.2010.04159.x
Subject(s) - serca , intracellular , ionomycin , endoplasmic reticulum , chemistry , cytosol , agonist , platelet , plasma membrane ca2+ atpase , biophysics , fura 2 , reuptake , calcium , biochemistry , medicine , atpase , receptor , biology , serotonin , organic chemistry , enzyme
Summary. Background: Most Ca 2+ signaling research in platelets has relied solely on monitoring the cytosolic Ca 2+ concentration ([Ca 2+ ] cyt ). Changes in [Ca 2+ ] cyt constitute the net effect of Ca 2+ fluxes into the cytosol across the plasma membrane (PM) and from intracellular stores, and Ca 2+ sequestration into the stores and Ca 2+ removal across the PM. This makes interpretation of the effects of pharmacologic or genetic interventions on Ca 2+ signaling difficult and subject to error. Objectives: To validate the use of the low‐affinity Ca 2+ indicator Fluo‐5N to monitor the concentration of Ca 2+ in the intracellular stores ([Ca 2+ ] st ) of human platelets as a first step in developing assays for a systems‐level analysis of platelet Ca 2+ signaling. Methods: Fluo‐5N‐loaded and Fura‐2‐loaded human platelets were used to observe the effects of agonist stimulation and other manipulations on [Ca 2+ ] cyt and [Ca 2+ ] st . Results: Fluo‐5N fluorescence changed appropriately in response to compounds that induce passive depletion of intracellular Ca 2+ stores and to physiologic agonists. Ca 2+ reuptake inhibitors and blockers of Ca 2+ release channels had the expected effects on Fura‐2 and Fluo‐5N fluorescence. Agonist‐evoked Ca 2+ release was reversed by Ca 2+ addition to the medium, and required intact Ca 2+ reuptake mechanisms. Store refilling was observed in the presence of sarcoplasmic/endoplasmic reticulum Ca 2+ ‐ATPase (SERCA) inhibitors and ionomycin, suggesting the presence of a non‐SERCA Ca 2+ reuptake mechanism. Evidence for a role for Ca 2+ ‐induced Ca 2+ release in agonist‐evoked responses was obtained. Conclusions: Our data provide a validation of the use of Fluo‐5N as a method for monitoring changes in [Ca 2+ ] st in human platelets.