Quantification of intraluminal free [Ca] in the agonist-sensitive internal calcium store using compartmentalized fluorescent indicators: Some considerations
Quantification of intraluminal free [Ca] in the agonist-sensitive internal calcium store using compartmentalized fluorescent indicators: Some considerations
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DOI：
10.1016/s0143-4160(96)90029-9
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发表时间：
1996-09-01
期刊：
影响因子：
4
通讯作者：
Schulz, I
中科院分区：
文献类型：
--
作者：
Hofer, AM;Schulz, I
许多荧光钙指示剂，特别是那些作为乙酰氧甲基(AM)-酯衍生物负载的指示剂，被认为是被分隔成细胞器的。这一特性可以被用来测量亚细胞隔室中游离[Ca]的变化，包括对肌醇(1，4，5)-三磷酸敏感的储存区。然而，由于染料可能在各种细胞器中积累，而且在许多情况下，指示剂对镁的敏感性，使对特定隔室内游离[Ca]的定量测量变得复杂。在这里，使用低亲和力钙指示剂Mag-Fura-2对BHK-21成纤维细胞中thapsigargin敏感存储内游离[Ca]的定量问题进行了重新研究。至少88+/-1.3%的隔间染料(扫描电子显微镜)被确定仅限于thapsigargin敏感的储存室，其余部分由[Ca]低于染料的检测下限(<；5µM)的其他隔室所占。用离子载体进行原位标定表明，膜内[Ca]的表观静息[Ca]为260+/-43muM(扫描电子显微镜)。然而，我们的分析表明，来自低[Ca]区域的染料报告对整个细胞测量的Mag-Fura-2比率的贡献不成比例，这可能导致激动剂敏感的存储中腔内[Ca]的严重低估。假设12%的Mag-Fura-2在[Ca]低于5µM的隔室中，计算出激动剂敏感商店中的游离[Ca]高达539+/-92µM。相比之下，预计商店中存在镁的干扰相对较小。
Many fluorescent Ca indicators, particularly those loaded as acetoxymethyl (AM)-ester derivatives, are known to become compartmentalized into organelles. This property can be exploited to measure changes in free [Ca] in subcellular compartments, including the inositol (1,4,5)-trisphosphate-sensitive store. However, quantitative measurement of free [Ca] within a particular compartment is complicated by the fact that dye may accumulate in a variety of organelles and, in many cases, by the Mg sensitivity of the indicator. Here the issue of the quantification of free [Ca] within the thapsigargin-sensitive store in BHK-21 fibroblasts using the low affinity Ca indicator, Mag-Fura-2, has been re-examined. At least 88 +/- 1.3% (SEM) of the compartmentalized dye was determined to be confined to the thapsigargin-sensitive store, with the remaining fraction accounted for by other compartments where [Ca] was below the detection limit for the dye (< 5 mu M). In situ calibrations with ionophores indicated that the apparent free resting intraluminal [Ca] was 260 +/- 43 mu M (SEM). Our analysis shows, however, that dye reporting from regions of low [Ca] contributes disproportionately to the Mag-Fura-2 ratio measured over the whole cell, potentially resulting in large underestimations of intraluminal [Ca] in agonist-sensitive stores. Free [Ca] in the agonist-sensitive store was calculated to be as high as 539 +/- 92 mu M, assuming 12% of the Mag-Fura-2 to be in compartments where [Ca] was below 5 mu M. In comparison, perturbations arising from the presence of Mg in stores are predicted to be relatively minor.