Effect of Trace Levels of Nigericin on Intracellular pH and Acid-Base Transport in Rat Renal Mesangial Cells |
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Authors: | MO Bevensee E Bashi WF Boron |
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Institution: | (1) Department of Cellular and Molecular Physiology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA, US |
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Abstract: | 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.
Received: 14 October 1998/Revised: 2 March 1999 |
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Keywords: | : Acid extrusion — Acid loading — BCECF — Dye — Ionophore — pHi |
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