We studied whether regulated exocytosis affects the glutamate transporter density in cultured astrocytes, in which the expression of a fluorescently labeled excitatory amino acid transporter 2 (EAAT2-EGFP) predominantly labeled the plasma membrane. The addition of ionomycin that elevates cytosolic Ca(2+) strongly increased the fluorescence of FM 4-64 membrane area dye, confirming the presence of regulated exocytosis in transfected astrocytes. However, concomitant with Ca(2+)-dependent FM 4-64 fluorescence increase, ionomycin induced a significant steady-state decrease in EAAT2-EGFP fluorescence. This is likely due to a secondary inner filter effect since,(i) in the absence of FM 4-64, ionomycin stimulation was ineffective in changing the EAAT2-EGFP fluorescence, and (ii) fluorescence changes in FM 4-64 and EAAT2-EGFP were inversely correlated. To test whether subcellular EAAT2-EGFP structures are translocated from the cytoplasm to the plasma membrane during ionomycin stimulation, EAAT2-EGFP fluorescence was monitored locally at the plasma membrane and a few microns away in the adjacent cytoplasm. Measurements revealed sites with an increase in EAAT2-EGFP plasma membrane fluorescence correlated with a fluorescence decrease beneath the plasma membrane, and sites with plasma membrane fluorescence decrease correlated with fluorescence increase within the adjacent cytoplasm. The sites of rapid translocation/retrieval of EAAT2-EGFP structures to/from the plasma membrane appeared to be distributed in a punctuate pattern around the cell perimeter. The density of EAAT2-EGFP was regulated in a Ca(2+)-dependent manner, since in the absence of extracellular Ca(2+) local translocation/retrieval events were absent, revealing rapid surface density regulation of EAAT2 in astrocytes by regulated exo/endocytosis.

中文翻译：

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星形胶质细胞质膜上的EAAT2密度和Ca（2 +）调控的胞吐作用。

我们研究了调控的胞吐作用是否影响培养的星形胶质细胞中的谷氨酸转运蛋白密度，其中荧光标记的兴奋性氨基酸转运蛋白2（EAAT2-EGFP）的表达主要标记质膜。添加的离子霉素可提高胞质Ca（2+）的浓度，强烈增加了FM 4-64膜区域染料的荧光，从而证实了转染的星形胶质细胞中存在调控的胞吐作用。但是，伴随着Ca（2+）依赖的FM 4-64荧光增加，离子霉素在EAAT2-EGFP荧光上诱导了明显的稳态下降。这很可能是由于二次内部滤镜作用所致，因为（i）在不存在FM 4-64的情况下，离子霉素刺激不能有效改变EAAT2-EGFP荧光，并且（ii）FM 4-64和EAAT2- EGFP呈负相关。为了测试在离子霉素刺激期间亚细胞EAAT2-EGFP结构是否从细胞质转移到质膜，在质膜上局部监测EAAT2-EGFP荧光，在相邻细胞质中相距几微米。测量显示出具有增加的EAAT2-EGFP质膜荧光的位点与在质膜下的荧光降低相关，并且具有质膜荧光的位点减少与邻近的细胞质内的荧光增加相关。EAAT2-EGFP结构快速转移到质膜/从质膜取回的位点似乎以点状分布在细胞周围。EAAT2-EGFP的密度以Ca（2+）依赖性方式调节，