Cramer E B, Gallin J I
J Cell Biol. 1979 Aug;82(2):369-79. doi: 10.1083/jcb.82.2.369.
Potassium pyroantimonate was used to localize sites of bound cations in human neutrophils under conditions of random migration, stimulated random migration (chemokinesis), and directed migration (chemotaxis). The cells were placed in a standard chamber in which 0.45-micron micropore filters separated the cells from the stimulus (buffer, Escherichia coli endotoxin-activated serum or the synthetic chemotactic peptide N-formyl-Met-Leu-Phe). The small pore filters permitted pseudopod formation but impeded cell imgration through the filter. Cells examined under all conditions had electron-dense precipitates of antimonate salts in some granules. However, antimonate deposits were localized in the condensed chromatin of the nucleus during random migration and associated to a large extent with the uncondensed nuclear chromatin during chemokinesis and chemotaxis. Under conditions of chemokinesis deposition of antimonate procipitates appeared on the cytoplasmic side of the plasma membrane of neutrophils whereas under conditions of chemotaxis cation deposits beneath the cell membrane were localized to the pseudopods which were directed toward the chemoattractant. In addition to endotoxin-activated serum, concentrations of N-formyl-Met-Leu-Phe which caused neutrophil chemotaxis (10(-8) M) also caused cation deposition beneath the cell membrane at the leading end of the cell regardless of whether albumin was present in the incubation media. However, with higher concentrations of the synthetic peptide (10(-5) M) which caused granule release and were not chemotactic, submembranous cation deposition was not seen. EDTA (10 mM) and EGTA (10 mM) removed nuclear, granular, and submembranous cation deposits from neutrophils examined under conditions of chemotaxis. X-ray microprobe analysis of antimonate deposits revealed the possible presence of calcium but did not detect sodium or magnesium. The data indicate that chemotactic factors induce submembranous deposition of cations, most likely Ca++, which localize to the leading edge of cells exposed to a gradient of chemoattractant.
焦锑酸钾用于在随机迁移、刺激随机迁移（化学增活作用）和定向迁移（趋化性）条件下定位人中性粒细胞中结合阳离子的位点。将细胞置于标准小室中，其中0.45微米的微孔滤膜将细胞与刺激物（缓冲液、大肠杆菌内毒素激活的血清或合成趋化肽N-甲酰甲硫氨酰亮氨酰苯丙氨酸）隔开。小孔滤膜允许伪足形成，但阻碍细胞通过滤膜迁移。在所有条件下检查的细胞在一些颗粒中都有锑酸盐的电子致密沉淀物。然而，在随机迁移过程中，锑酸盐沉积物位于细胞核的浓缩染色质中，而在化学增活作用和趋化性过程中，很大程度上与未浓缩的核染色质相关。在化学增活作用条件下，锑酸盐沉淀物出现在中性粒细胞质膜的胞质侧，而在趋化性条件下，细胞膜下的阳离子沉积物位于指向趋化因子的伪足处。除了内毒素激活的血清外，引起中性粒细胞趋化性的N-甲酰甲硫氨酰亮氨酰苯丙氨酸浓度（10^(-8) M）也会导致细胞膜下阳离子在细胞前端沉积，无论孵育培养基中是否存在白蛋白。然而，对于导致颗粒释放且无趋化性的较高浓度合成肽（10^(-5) M），未观察到膜下阳离子沉积。在趋化性条件下检查的中性粒细胞中，10 mM的乙二胺四乙酸（EDTA）和10 mM的乙二醇双四乙酸（EGTA）可去除细胞核、颗粒和膜下阳离子沉积物。对焦锑酸盐沉积物的X射线微探针分析显示可能存在钙，但未检测到钠或镁。数据表明，趋化因子诱导阳离子在膜下沉积，最有可能是Ca++，其定位于暴露于趋化因子梯度的细胞前缘。
