Hofer A M, Schlue W R, Curci S, Machen T E
Department of Molecular and Cell Biology, University of California, Berkeley 94720, USA.
FASEB J. 1995 Jun;9(9):788-98. doi: 10.1096/fasebj.9.9.7601343.
Free [Ca] within organelles of permeabilized BHK-21 cells was measured using ratio imaging of compartmentalized mag-fura-2. In BHK-21 cells, this dye monitors free [Ca] in principally one type of ATP-dependent Ca-sequestering organelle in which intrastore Ca was released uniformly and entirely by 100 nM thapsigargin or removal of ATP or Ca from the bath, and was reduced by 85% upon treatment with a supramaximal dose of InsP3 (6 microM). Examination of the spatial distribution of InsP3-sensitive Ca stores showed that InsP3 released Ca throughout all regions of the cell, although we often noted a perinuclear region (which we speculate may correspond to the Golgi apparatus) with reduced responsiveness to InsP3. InsP3-induced changes of intraluminal Mg could not be detected. Cyclic ADP-ribose, ryanodine, caffeine, mitochondrial inhibitors, and GTP, agents known to influence intraorganellar Ca sequestration in other cell types, were all without effect on the mag-fura-2 ratio. In situ calibration of the mag-fura-2 ratio with Ca ionophores revealed that the average free intraorganellar [Ca] was initially 188 +/- 21 microM in the presence of 170 nM free Ca and 3 mM ATP, and was reduced to 25 +/- 5 microM upon stimulation with 6 microM InsP3. The ionic dependence of the release and reloading process was also investigated. The presence of either K, Na, or Cl could consistently support both InsP3-induced release and the refilling of stores with Ca, but physiological concentrations of HCO3 were effective in sustaining the response in only 24% of cells examined.
使用区室化镁-呋喃-2的比率成像测量通透化BHK-21细胞细胞器内的游离[Ca]。在BHK-21细胞中，这种染料主要监测一种依赖ATP的钙螯合细胞器中的游离[Ca]，在该细胞器中，100 nM毒胡萝卜素、从浴中去除ATP或Ca可使储存的钙均匀且完全释放，用超最大剂量的InsP3（6 μM）处理后，钙含量降低85%。对InsP3敏感钙库的空间分布检查表明，InsP3在细胞的所有区域释放钙，尽管我们经常注意到一个对InsP3反应性降低的核周区域（我们推测可能对应于高尔基体）。未检测到InsP3诱导的腔内镁变化。环ADP-核糖、ryanodine、咖啡因、线粒体抑制剂和GTP，这些已知会影响其他细胞类型细胞器内钙螯合的物质，对镁-呋喃-2比率均无影响。用钙离子载体对镁-呋喃-2比率进行原位校准表明，在存在170 nM游离钙和3 mM ATP的情况下，细胞器内平均游离[Ca]最初为188±21 μM，用6 μM InsP3刺激后降至25±5 μM。还研究了释放和再填充过程的离子依赖性。K、Na或Cl的存在均可持续支持InsP3诱导的释放以及钙对储存库的再填充，但生理浓度的HCO3仅在24%的检测细胞中有效维持反应。
