THE EFFECT OF BCECF ON INTRACELLULAR PH OF HUMAN PLATELETS

-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) is frequent ly used for fluorometric determination of intracellular pH (pH(i)) and its metabolic changes. Studies of BCECF-loaded platelets have reporte d 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(i) ) content affects pH(i), and that its over-loading, leads to significa ntly lower pH(i). Different pH(i) values can be reproduced by changing BCECF(i), as reflected by fluorescence intensity. The major loading f actors are: the concentration of the probe parent compound, BCECF acet oxymethyl ester (AM), and whether this ester is partly hydrolyzed exte rnally when applied in plasma. When least affected by BCECF, platelet pH(i) is 7.34. High BCECF(i) does not affect ATP content, buffer capac ity, 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 lea st paltry, for the lowered pH(i) in BCECF over-loaded platelets.