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首页> 外文期刊>Journal of cell biology >Quantitative analysis of the cytosolic free calcium dependency of exocytosis from three subcellular compartments in intact human neutrophils.
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Quantitative analysis of the cytosolic free calcium dependency of exocytosis from three subcellular compartments in intact human neutrophils.

机译：定量分析完整人类嗜中性粒细胞中三个亚细胞区的胞吐作用对胞质游离钙的依赖性。

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Cytosolic free calcium concentration, [Ca2+]i, and exocytosis of azurophil granules (beta-glucuronidase), specific granules (vitamin B12-binding protein), and secretory vesicles (gelatinase) were measured concomitantly in intact human neutrophils under steady state [Ca2+]i. The cells were loaded with the fluorescent calcium indicator quin2 in the presence or absence of extracellular Ca2+, and steady state [Ca2+]i levels ranging from 20 to greater than 2,000 nM were obtained by adding the Ca2+ ionophore ionomycin at various concentrations of extracellular calcium. The extent of exocytosis from the three granule populations was found to be a function of [Ca2+]i. The minimal [Ca2+]i that caused significant release (threshold [Ca2+]i) was approximately 200-300 nM and was similar for all three compartments. Marked differences, however, were found when the [Ca2+]i for half-maximal exocytosis (EC50) was determined. In the absence of cytochalasin B the EC50 was 1,100 +/- 220 nM and 1,600 +/- 510 nM for specific granules and secretory vesicles, respectively, and approximately 6,000 nM for azurophil granules. Cytochalasin B did not affect the threshold [Ca2+]i but decreased the EC50 and enhanced the rate of exocytosis. In the presence of cytochalasin B the EC50 was approximately 600 nM both for secretory vesicles and specific granules, and approximately 2,600 nM for azurophil granules. The addition of the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine dramatically changed the [Ca2+]i dependency of granule secretion: It decreased the threshold [Ca2+]i to less than 20 and less than 50 nM, and the EC50 to 50 and 200 nM for specific and azurophil granules, respectively, and it significantly increased the rate of exocytosis. Thus, the additional signal(s) provided by receptor activation markedly lower(s) the Ca2+ requirement of the exocytotic process. Furthermore, these results indicate that the secretion from three different granule populations within the same cell type are differently modulated by [Ca2+]i.
机译：完整状态下完整人嗜中性粒细胞在稳定状态[Ca2 +]的同时测定了胞液中游离钙的浓度，[Ca2 +] i和嗜酸性粒细胞颗粒（β-葡萄糖醛酸苷酶），特定颗粒（维生素B12结合蛋白）和分泌性囊泡（明胶酶）的胞吐作用。一世。在存在或不存在细胞外Ca2 +的情况下，将荧光钙指示剂quin2加载到细胞中，并通过以各种浓度的细胞外钙添加Ca2 +离子载体离子霉素来获得从20到大于2,000 nM的稳态[Ca2 +] i水平。发现来自三个颗粒群的胞吐程度是[Ca 2+] i的函数。导致显着释放的最小[Ca2 +] i（阈值[Ca2 +] i）约为200-300 nM，并且对于所有三个隔室都是相似的。但是，当确定半数最大胞吐作用的[Ca2 +] i（EC50）时，发现明显差异。在不存在细胞松弛素B的情况下，特定颗粒和分泌小泡的EC50分别为1,100 +/- 220 nM和1,600 +/- 510 nM，而嗜蓝粒颗粒的EC50约为6,000 nM。细胞松弛素B不会影响[Ca2 +] i阈值，但会降低EC50并提高胞吐速率。在细胞松弛素B的存在下，分泌性囊泡和特定颗粒的EC 50均为约600 nM，嗜蓝粒颗粒的EC 50约为2,600 nM。趋化肽N-甲酰基-甲硫酰基-亮氨酰-苯丙氨酸的添加极大地改变了颗粒分泌的[Ca2 +] i依赖性：将[Ca2 +] i阈值降低至小于20和小于50 nM，并将EC50降低至50和200 nM分别用于特异颗粒和嗜蓝粒颗粒，它显着提高了胞吐速率。因此，由受体激活提供的附加信号显着降低了胞吐过程的Ca 2+需求。此外，这些结果表明，同一细胞类型中三个不同颗粒种群的分泌受到[Ca2 +] i的不同调节。

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