title: Monitoring the intracellular store Ca2+ concentration in agonist-stimulated, intact human platelets by using Fluo-5N.
abstract: 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.
  
  
  OBJECTIVE
  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.
authors.affiliation: Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, UK.
authors.forename: S O
authors.initials: SO
authors.lastname: Sage
authors.forename: N
authors.initials: N
authors.lastname: Pugh
authors.forename: M J
authors.initials: MJ
authors.lastname: Mason
authors.forename: A G S
authors.initials: AG
authors.lastname: Harper
mesh_terms: D001792:Blood Platelets / Q000187:drug effects* / Q000378:metabolism*; D002118:Calcium / Q000097:blood*; D020013:Calcium Signaling / Q000187:drug effects; D005456:Fluorescent Dyes; D016257:Fura-2; D006801:Humans; D006873:Hydroquinones / Q000494:pharmacology; D066298:In Vitro Techniques; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000097:blood; D007424:Intracellular Fluid / Q000378:metabolism; D015759:Ionomycin / Q000494:pharmacology; D018613:Microscopy, Confocal; D009249:NADP / Q000031:analogs & derivatives / Q000097:blood; D009550:Nigericin / Q000494:pharmacology; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D019284:Thapsigargin / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology
chemical_list: D005456:Fluorescent Dyes; D006873:Hydroquinones; D053496:Inositol 1,4,5-Trisphosphate Receptors; D009249:NADP; C024376:NAADP; D015759:Ionomycin; D019284:Thapsigargin; C018855:2-tert-butylhydroquinone; D013917:Thrombin; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D009550:Nigericin; D002118:Calcium; D016257:Fura-2