The effect of BCECF on intracellular pH of human platelets

2',7'-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) is frequently used for fluorometric determination of intracellular pH (pH(i)) and its metabolic changes. Studies of BCECF-loaded platelets have reported different pH(i) values in the range of 6.98 to 7.35, despite the use of the same probe. It is now shown that intracellular BCECF (BCECF,) content affects pH(i), and that its over-loading, leads to significantly lower pH(i). Different pH(i) values can be reproduced by changing BCECF(i) as reflected by fluorescence intensity. The major lending 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(i) platelet pH(i) is 7.34. High BCECF(i) 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 BCECF(i) does inhibit the Na⁺/H⁺ exchange rate by over 50%. Since the Na⁺/H⁺ exchange strongly affects platelets pH(i), it is proposed that this inhibition accounts, at least partly, for the lowered pH(i), in BCECF over-loaded platelets.