EVALUATION OF 2',7'-DICHLOROFLUORESCIN AND DIHYDRORHODAMINE 123 AS FLUORESCENT-PROBES FOR INTRACELLULAR H2O2 IN CULTURED ENDOTHELIAL-CELLS
EVALUATION OF 2',7'-DICHLOROFLUORESCIN AND DIHYDRORHODAMINE 123 AS FLUORESCENT-PROBES FOR INTRACELLULAR H2O2 IN CULTURED ENDOTHELIAL-CELLS
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DOI：
10.1006/abbi.1993.1222
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发表时间：
1993-05-01
影响因子：
3.9
通讯作者：
ISCHIROPOULOS, H
中科院分区：
文献类型：
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作者：
ROYALL, JA;ISCHIROPOULOS, H
2‘，7’-二氯荧光素和二氢罗丹明123作为检测培养内皮细胞内H_2O_2变化的探针。这些探针在细胞内的稳定水平在暴露于培养基中的探针的15分钟内建立。随着探针的持续存在，细胞内浓度在1h内保持不变，但如果在细胞内负载后使用不带探针的培养基，细胞内二氯荧光素、二氯荧光素和二氢罗丹明123的损失超过90%，而细胞内罗丹明123的损失仅为15%。外源性100μM H_2O_2作用内皮细胞1h，细胞内罗丹明123含量增加83%，而细胞内二氯荧光素含量可重复减少53%。0.05 mM BCNU+10 mM氨基三氮唑作用2 h可使细胞内罗丹明123增加111%。在二氢罗丹明123氧化的体外研究中，与以前报道的二氯荧光素氧化相似。二氢罗丹明123的氧化不是单独在H_2O_2作用下发生的，而是由H_2O_2-细胞色素c和H_2O_2-Fe~(2+)等多种依赖于H_2O_2的二级胞内反应所介导。我们的结果表明，在内皮细胞中检测到这些探针的氧化增加是最有用的，作为一般细胞氧化剂产生变化的标志。
2′,7′-Dichlorofluorescin and dihydrorhodamine 123 were evaluated as probes for detecting changes in intracellular H2O2in cultured endothelial cells. Stable intracellular levels of these probes were established within 15 min of exposure to the probe in culture medium. With continued presence of the probe in the medium, intracellular levels were unchanged for 1 h. However, if medium without the probes was used after intracellular loading had occurred, there was a greater than 90% loss of intracellular dichlorofluorescin, dichlorofluorescein, and dihydrorhodamine 123 while intracellular rhodamine 123 decreased by only 15%. Exposure of endothelial cells to exogenous 100 μM H2O2for 1 h increased intracellular rhodamine 123 by 83%, but there was a reproducible decrease of 53% in intracellular dichlorofluorescein. Exposure to 0.05 mM BCNU plus 10 mM aminotriazole for 2 h increased intracellular rhodamine 123 by 111%.In vitrostudies of dihydrorhodamine 123 oxidation were similar to previous reports of dichlorofluorescin oxidation. Oxidation of dihydrorhodamine 123 does not occur with H2O2alone, but is mediated by a variety of secondary H2O2-dependent intracellular reactions including H2O2-cytochrome c and H2O2-Fe2+. Our results suggest that detection of increased oxidation of these probes in endothelial cells is most useful as a marker of a change in general cellular oxidant production.