INADEQUACY OF HIGH K+ NIGERICIN FOR CALIBRATING BCECF .2. INTRACELLULAR PH-DEPENDENCE OF THE CORRECTION/

In the accompanying study [G. Boyarsky, C. Hanssen, and L. A. Clyne. A m. J. Physiol. 271 (Cell Physiol. 40): C1131-C1145, 1996], it was demo nstrated that steady-state intracellular pH (pH(i)) determined using h igh K+/nigericin calibrations was systematically in error in vascular smooth muscle (VSM) cells by similar to 0.2 pH units. In this paper th e possibility is explored that this correction (pH(cor)) to the nigeri cin-calibrated pH(i) (pH(nig)) might not be a constant but could vary as pH(i) varies. The range of pH(i) during exposures to ''null solutio ns'' designed to bracket pH(i) was extended to acidic and alkaline lev els relative to the starting pH(i) in VSM cells. The pH(cor) necessary to correct pH(nig) was linearly dependent on pH(nig), increasing from near zero at similar to 6.0 to similar to 0.2 at steady-state pH(i), to similar to 0.3 at alkaline pH(nig). It is shown how to retrieve pre viously acquired (tabulated) data using the linear relationship betwee n pH(cor) and pH(nig). Also examined were what corrections must be mad e to high K+/nigericin calibration curves to correct for this pH(i)-de pendent pH(cor). The following changes in the calibration parameters w ere found: the maximal fluorescence ratio increased from 16.75 to 17.2 8; the minimal fluorescence ratio decreased from 2.15 to 1.57; and the pK of 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein decreased from 7.13 to 6.93. Three potential explanations for these changes are discu ssed: external [K+] in the nigericin solutions could have been too low ; internal [K+] changes during the calibration because of the finite b uffering power of cells; and other acid-base transport/generation coul d have been contributing during the nigericin calibrations (i.e., nige ricin does not overwhelm to insignificance other processes generating/ consuming H+). The nonconstancy of pH(cor) is shown to have profound i mplications for measuring changes in pH(i).