Near membrane Ca 2+ changes resulting from store release in neutrophils: detection by FFP-18

Abstract

FFP-18 was incorporated into the inner face of the plasma membrane of human neutrophils by incubation with its acetoxymethyl ester. Conversion to the Ca 2+ 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 Ni 2+. Ratio fluorescence measurement of FFP-18 under these conditions permitted the detection of near membrane Ca 2+ changes resulting from the release of Ca 2+ from intracellular stores. Near membrane and cytosolic Ca 2+ changes were measured under conditions in which store release and Ca 2+ influx were triggered by FMLP, thapsigargin or immune complexes. There were significant differences in the timing and magnitude of Ca 2+ changes near the plasma membrane and bulk cytosolic Ca 2+ concentration, which were consistent with a Ca 2+ storage site deep within the neutrophil released by thapsigargin and FMLP, but Ca 2+ release sites with immune complex stimulation being close to the membrane. The use of FFP-18 to monitor Ca 2+ near the inner face of the plasma membrane thus provides evidence for the existence of two distinct Ca 2+ storage locations in neutrophils.