SUBCELLULAR-DISTRIBUTION OF CYTOSOLIC CA2+ IN ISOLATED RAT HEPATOCYTE COUPLETS - EVALUATION USING CONFOCAL MICROSCOPY
SUBCELLULAR-DISTRIBUTION OF CYTOSOLIC CA2+ IN ISOLATED RAT HEPATOCYTE COUPLETS - EVALUATION USING CONFOCAL MICROSCOPY
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DOI：
10.1016/0143-4160(92)90002-a
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发表时间：
1992-02-01
期刊：
影响因子：
4
通讯作者：
BURGSTAHLER, AD
中科院分区：
文献类型：
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作者：
NATHANSON, MH;BURGSTAHLER, AD
Ca 2+激动剂在上皮细胞的胞质溶胶中诱导Ca 2+波和其他不均匀的Ca 2+模式。为了定义肝细胞胞质中的亚细胞Ca 2+瞬变，我们使用共聚焦显微镜检查了Fluo-3负载的离体大鼠肝细胞偶联。光学切片的厚度小于1微米，观察到的对联，和荧光细胞质Ca 2+信号很容易区分从核，线粒体，和溶酶体荧光。荧光图像的非胞质组分的性质通过线粒体染料DIOC 6（3）和溶酶体标记物吖啶橙子的双重标记来验证。使用共聚焦显微镜的线扫描模式，测量细胞质Ca 2+的频率高达250 Hz，没有显着的漂白。结果发现，苯肾上腺素诱导的Ca 2+信号一般开始于肝细胞的基底极，然后以80 μ m/s的平均速度扩散到小管。这些研究结果表明，共聚焦线扫描显微镜用于检测细胞内Ca 2+在肝细胞偶联的亚细胞分布的快速变化的效用，并建议苯肾上腺素诱导的Ca 2+波辐射在基底到顶端的方向，在这种细胞类型。
Ca2+ agonists induce Ca2+ waves and other non-uniform Ca2+ patterns in the cytosol of epithelial cells. To define subcellular Ca2+ transients in the cytosol of hepatocytes we examined Fluo-3-loaded isolated rat hepatocyte couplets using confocal microscopy. Optical sections of less than 1 micron in thickness were observed in couplets, and fluorescence from cytosolic Ca2+ signals was readily distinguished from nuclear, mitochondrial, and lysosomal fluorescence. The nature of the noncytosolic components of the fluorescent images was verified by double labelling with the mitochondrial dye DIOC6(3) and with the lysosomal marker acridine orange. Using the line scanning mode of confocal microscopy, measurements of cytosolic Ca2+ were made with a frequency of up to 250 Hz and without significant bleaching. It was found that phenylephrine-induced Ca2+ signals generally began at the basal pole of the hepatocytes, then spread to the canaliculus at average speeds of 80-mu-m/s. These findings demonstrate the utility of confocal line scanning microscopy for detecting rapid changes in the subcellular distribution of cytosolic Ca2+ in hepatocyte couplets, and suggest that phenylephrine-induced Ca2+ waves radiate in a basal-to-apical direction in this cell type.