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 pH i 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.