The effect of BCECF on intracellular pH of human platelets

Abstract2′,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