Near membrane Ca2+ changes resulting from store release in neutrophils: detection by FFP-18.
FFP-18 was incorporated into the inner face of the plasma membrane of human neutrophils by incubation with its acetoxymethyl ester. Conversion to the Ca2+ sensitive intracellular indicator was monitored by the change in excitation spectra. The fluorescence from extracellularly facing FFP-18 was quenched by the membrane impermeant ion Ni2+. Ratio fluorescence measurement of FFP-18 under these conditions permitted the detection of near membrane Ca2+ changes resulting from the release of Ca2+ from intracellular stores. Near membrane and cytosolic Ca2+ changes were measured under conditions in which store release and Ca2+ influx were triggered by FMLP, thapsigargin or immune complexes. There were significant differences in the timing and magnitude of Ca2+ changes near the plasma membrane and bulk cytosolic Ca2+ concentration, which were consistent with a Ca2+ storage site deep within the neutrophil released by thapsigargin and FMLP, but Ca2+ release sites with immune complex stimulation being close to the membrane. The use of FFP-18 to monitor Ca2+ near the inner face of the plasma membrane thus provides evidence for the existence of two distinct Ca2+ storage locations in neutrophils.
Davies
E V
EV
Department of Surgery, University of Wales College of Medicine, Cardiff, UK.
Hallett
M B
MB
D000936:Antigen-Antibody Complex / Q000276:immunology; D002118:Calcium / Q000032:analysis*; D002462:Cell Membrane / Q000737:chemistry; D005456:Fluorescent Dyes; D016257:Fura-2 / Q000031:analogs & derivatives / Q000737:chemistry; D006801:Humans; D009504:Neutrophils / Q000166:cytology*; D019284:Thapsigargin / Q000494:pharmacology
D000936:Antigen-Antibody Complex; D005456:Fluorescent Dyes; C098105:fura-FFP18; D019284:Thapsigargin; D002118:Calcium; D016257:Fura-2