TECHNIQUE FOR INSITU MEASUREMENT OF CALCIUM IN INTRACELLULAR INOSITOL 1,4,5-TRISPHOSPHATE-SENSITIVE STORES USING THE FLUORESCENT INDICATOR MAG-FURA-2
TECHNIQUE FOR INSITU MEASUREMENT OF CALCIUM IN INTRACELLULAR INOSITOL 1,4,5-TRISPHOSPHATE-SENSITIVE STORES USING THE FLUORESCENT INDICATOR MAG-FURA-2
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DOI：
10.1073/pnas.90.7.2598
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发表时间：
1993-04-01
影响因子：
11.1
通讯作者：
MACHEN, TE
中科院分区：
文献类型：
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作者：
HOFER, AM;MACHEN, TE
钙激活激动剂对细胞的刺激常常导致1，4，5-三磷酸肌醇(InsP3)介导的细胞内钙释放。我们在这里报告了一种直接监测胃上皮细胞中这个和其他存储中的钙的技术。这项技术利用了荧光染料MAG-Fura-2的乙酰氧甲酯衍生物(对5微米以上的钙浓度敏感)在亚细胞内积累的倾向，在那里它可以报告游离钙浓度的变化。完整的染料负载细胞对胆碱能刺激的反应是350 nm/385 nm激发比降低，这与数字成像显微镜在单个细胞中测量的结果一致，与一个或多个细胞隔室中钙浓度的降低一致。当细胞被洋地黄素渗透并在“细胞内缓冲液”中孵育时，细胞质染料被释放，将mag-Fura-2留在内部存储中。InSP3以剂量依赖的方式使捕获指示剂的比例降低(即钙释放)，这一作用可被InsP3受体拮抗剂肝素阻断。钙离子进入内库是依赖于ATP的，而进入InsP3敏感池的重摄取被内库Ca-ATPase的特异性抑制剂thapsigargin所阻断。我们利用这一技术研究了氯在InsP3敏感内库释放和重新加载中的作用，发现在无氯溶液中钙摄取减少，这表明氯在这个库的再填充中起着重要的作用。
Stimulation of cells with calcium-mobilizing agonists frequently results in inositol 1,4,5-trisphosphate (InsP3)-mediated discharge of Ca from an internal store. We report here a technique for directly monitoring Ca within this and other stores in gastric epithelial cells. This technique takes advantage of the propensity of the acetoxymethyl ester derivative of the fluorescent dye mag-fura-2 (which is sensitive to Ca concentrations above 5 muM) to accumulate in subcellular compartments where it can report changes in the free Ca concentration. Intact dye-loaded cells responded to cholinergic stimulation with a decrease in the 350 nm/385 nm excitation ratio, as measured in individual cells with a digital imaging microscope, consistent with reduced Ca concentration in one or more cellular compartments. When cells were permeabilized with digitonin and incubated in an ''intracellular buffer,'' the cytoplasmic dye was released, leaving the mag-fura-2 in the internal store. InSP3 caused the ratio from the trapped indicator to decrease (i.e., Ca was released) in a dose-dependent manner, and this effect was blocked by the InsP3 receptor antagonist heparin. Ca sequestration into the internal store was ATP-dependent, and reuptake into the InsP3-sensitive pool was blocked by thapsigargin, a specific inhibitor of the Ca-ATPase of the internal store. We used this technique to investigate the role of Cl on the release and reloading of the InsP3-sensitive internal store and found that Ca uptake was reduced in Cl-free solutions, suggesting an important function for Cl in the refilling of this pool.