Simultaneous measurements of intracellular pH in the leech giant glial cell using 2',7'-bis-(2-carboxyethy])-5,6-carboxyfluorescein and ion-sensitive microelectrodes

We have employed two independent techniques to measure the intracellular pH (pH(i)) in giant glial cells of the leech Hirudo medicinalis, using the fluorescent dye 2',7'-bis-(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF) and double-barreled neutral-carrier, pH-sensitive microelectrodes, which also record the membrane potential. We have compared two procedures for calibrating the ratio of the BCECF signal, excited at 440 nm and 495 nm: 1) the cell membrane was H+-permeabilized with nigericin in high-K+ saline at different external pH (pH(o)) values, and 2) the pH(i) of intact cells was perturbed in CO2/HCO3--bufFered saline of different pH(i) and the BCECF ratio was calibrated according to a simultaneous microelectrode pH reading. As indicated by the microelectrode measurements, the pH(i) did not fully equilibrate to the pH(o) values in nigericin-containing, high-K+ saline, but deviated by -0.12 +/- 0.02 (mean +/- SEM, n = 37) pH units. In intact cells, the microelectrode readings yielded up to 0.15 pH unit lower values than the calibrated BCECF signal. In addition, larger dye injections into the cells (>100 mu M) caused an irreversible membrane potential loss indicative of some damage to the cells. The amplitude and kinetics of slow pH(i) changes were equally followed by both sensors, and the dye ratio recorded slightly higher amplitudes during faster pH(i) shifts as induced by the addition and removal of NH4+.