Davies E V, Hallett M B
Department of Surgery, University of Wales College of Medicine, Cardiff, UK.
Cell Calcium. 1996 Apr;19(4):355-62. doi: 10.1016/s0143-4160(96)90076-7.
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.
FFP - 18通过与它的乙酰氧基甲酯孵育被整合到人中性粒细胞质膜的内表面。通过激发光谱的变化监测其向Ca2+敏感的细胞内指示剂的转化。面向细胞外的FFP - 18的荧光被膜不透性离子Ni2+淬灭。在这些条件下对FFP - 18进行比率荧光测量，可以检测到细胞内储存库释放Ca2+导致的近膜Ca2+变化。在由FMLP、毒胡萝卜素或免疫复合物触发储存库释放和Ca2+内流的条件下，测量近膜和胞质Ca2+变化。质膜附近Ca2+变化的时间和幅度与胞质溶胶中Ca2+浓度存在显著差异，这与毒胡萝卜素和FMLP从嗜中性粒细胞深处释放Ca2+储存位点一致，但免疫复合物刺激的Ca2+释放位点靠近膜。因此，使用FFP - 18监测质膜内表面附近的Ca2+为嗜中性粒细胞中存在两个不同的Ca2+储存位置提供了证据。
