Quantitative analysis of the cytosolic free calcium dependency of exocytosis from three subcellular compartments in intact human neutrophils
Quantitative analysis of the cytosolic free calcium dependency of exocytosis from three subcellular compartments in intact human neutrophils
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完整人中性粒细胞三个亚细胞区室胞吐作用对胞质游离钙依赖性的定量分析
作者：
P. Lew;A. Monod;F. Waldvogel;B. Dewald;M. Baggiolini;T. Pozzan
在稳态[Ca 2 +]i下，同时测量完整人中性粒细胞胞浆游离钙浓度[Ca 2 +]i和嗜天青粒细胞颗粒（β-葡萄糖醛酸酶）、特定颗粒（维生素B12结合蛋白）和分泌囊泡（明胶酶）的胞吐作用。在存在或不存在细胞外Ca 2+的情况下，用荧光钙指示剂quin 2加载细胞，并且通过添加各种浓度的细胞外钙的Ca 2+离子载体离子霉素获得范围为20至大于2，000 nM的稳态[Ca 2 +]i水平。从三个颗粒种群的胞吐的程度被认为是一个功能的[Ca 2 +]i。引起显著释放的最小[Ca 2 +]i（阈值[Ca 2 +]i）约为200-300 nM，并且所有三个隔室相似。然而，当测定半最大胞吐的[Ca 2 +]i（EC 50）时，发现了显着差异。在不存在细胞松弛素B的情况下，特异性颗粒和分泌囊泡的EC 50分别为1，100 +/- 220 nM和1，600 +/- 510 nM，嗜天青颗粒的EC 50约为6，000 nM。细胞松弛素B不影响[Ca ~（2+）]i阈值，但降低EC_（50），提高胞吐率。在细胞松弛素B存在下，分泌囊泡和特异性颗粒的EC 50均约为600 nM，嗜天青颗粒的EC 50约为2，600 nM。加入趋化肽N-甲酰-甲硫氨酰-亮氨酰-苯丙氨酸显著改变了颗粒分泌的[Ca 2 +]i依赖性：它将特异性颗粒和嗜天青颗粒的[Ca 2 +]i阈值降低至小于20和小于50 nM，EC 50分别降低至50和200 nM，并显著增加了胞吐率。因此，由受体活化提供的额外信号显著降低胞吐过程的Ca 2+需求。此外，这些结果表明，从三个不同的颗粒群在相同的细胞类型内的分泌不同的调节[Ca 2 +]i。
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.
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3.6
作者：
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--
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DOI：
--
发表时间：
1985
期刊：
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影响因子：
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影响因子：
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作者：
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DOI：
--
发表时间：
1982
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
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通讯作者：
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