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. Am. J. Physiol. 271 (Cell Physiol. 40): C1131-C1145, 1996], it was demonstrated that steady-state intracellular pH (pH(i)) determined using high K+/nigericin calibrations was systematically in error in vascular smooth muscle (VSM) cells by similar to 0.2 pH units. In this paper the possibility is explored that this correction (pH(cor)) to the nigericin-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 solutions'' designed to bracket pH(i) was extended to acidic and alkaline levels 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 previously acquired (tabulated) data using the linear relationship between pH(cor) and pH(nig). Also examined were what corrections must be made to high K+/nigericin calibration curves to correct for this pH(i)-dependent pH(cor). The following changes in the calibration parameters were found: the maximal fluorescence ratio increased from 16.75 to 17.28; 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 discussed: external [K+] in the nigericin solutions could have been too low; internal [K+] changes during the calibration because of the finite buffering power of cells; and other acid-base transport/generation could have been contributing during the nigericin calibrations (i.e., nigericin does not overwhelm to insignificance other processes generating/consuming H+). The nonconstancy of pH(cor) is shown to have profound implications for measuring changes in pH(i).