Aharonovitz O, Fridman H, Livne A A, Granot Y
Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
Biochim Biophys Acta. 1996 Oct 23;1284(2):227-32. doi: 10.1016/s0005-2736(96)00135-6.
2',7'-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) is frequently used for fluorometric determination of intracellular pH (pHi) and its metabolic changes. Studies of BCECF-loaded platelets have reported different pHi values in the range of 6.98 to 7.35, despite the use of the same probe. It is now shown that intracellular BCECF (BCECFi) content affects pHi, and that its over-loading, leads to significantly lower pHi. Different pHi values can be reproduced by changing BCECFi, as reflected by fluorescence intensity. The major loading factors are: the concentration of the probe parent compound, BCECF acetoxymethyl ester (AM), and whether this ester is partly hydrolyzed externally when applied in plasma. When least affected by BCECF, platelet pHi is 7.34. High BCECFi does not affect ATP content, buffer capacity, activation of Na+/H+ exchange by protein kinase C (PKC) and basal PKC activity. On the other hand high BCECFi does inhibit the Na+/H+ exchange rate by over 50%. Since the Na+/H+ exchange strongly affects platelets pHi, it is proposed that this inhibition accounts, at least partly, for the lowered pHi in BCECF over-loaded platelets.
2',7'-双-(2-羧乙基)-5-(及-6)-羧基荧光素（BCECF）常用于荧光法测定细胞内pH值（pHi）及其代谢变化。尽管使用的是相同的探针，但对负载BCECF的血小板的研究报告的pHi值在6.98至7.35范围内有所不同。现已表明，细胞内BCECF（BCECFi）含量会影响pHi，其过载会导致pHi显著降低。通过改变BCECFi（如荧光强度所示）可以重现不同的pHi值。主要的负载因素包括：探针母体化合物BCECF乙酰氧基甲酯（AM）的浓度，以及该酯在血浆中应用时是否在外部发生部分水解。当受BCECF影响最小时，血小板pHi为7.34。高BCECFi不影响ATP含量、缓冲能力、蛋白激酶C（PKC）对Na+/H+交换的激活以及基础PKC活性。另一方面，高BCECFi确实会使Na+/H+交换率抑制超过50%。由于Na+/H+交换强烈影响血小板pHi，因此有人提出这种抑制至少部分解释了BCECF过载血小板中pHi降低的原因。
