Effect of trace levels of nigericin on intracellular pH and acid-base transport in rat renal mesangial cells

Nigericin is an ionophore commonly used at the end of experiments to calibrate intracellularly trapped pH-sensitive dyes. In the present study, we explore the possibility that residual nigericin from dye calibration in one experiment might interfere with intracellular pH (pH(i)) changes in the next. Using the pH-sensitive fluorescent dye 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF), we measured pHi in cultured rat renal mesangial cells. Nigericin contamination caused: (i) an increase in acid loading during the pH(i) decrease elicited by removing extracellular Na+, (ii) an increase in acid extrusion during the pH(i) increase caused by elevating extracellular [K+], and (iii) an acid shift in the pH(i) dependence of the background intracellular acid loading unmasked by inhibiting Na-H exchange with ethylisopropylamiloride (EIPA). However, contamination had no effect on the pH(i) dependence of Na-H exchange, computed by adding the pH(i) dependencies of total acid extrusion and background acid loading. Nigericin contamination can be conveniently minimized by using a separate line to deliver nigericin to the cells, and by briefly washing the tubing with ethanol and water after each experiment.