Inadequacy of high K+/nigericin for calibrating BCECF. II. 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 (pHi) determined using high K+/nigericin calibrations was systematically in error in vascular smooth muscle (VSM) cells by approximately 0.2 pH units. In this paper the possibility is explored that this correction (pHcor) to the nigericin-calibrated pHi (pHnig) might not be a constant but could vary as pHi varies. The range of pHi during exposures to "null solutions" designed to bracket pHi was extended to acidic and alkaline levels relative to the starting pHi in VSM cells. The pHcor necessary to correct pHnig was linearly dependent on pHnig, increasing from near zero at approximately 6.0 to approximately 0.2 at steady-state pHi, to approximately 0.3 at alkaline pHnig. It is shown how to retrieve previously acquired (tabulated) data using the linear relationship between pHcor and pHnig. Also examined were what corrections must be made to high K+/nigericin calibration curves to correct for this pHi-dependent pHcor. 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 (carboxy-ethyl)-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 pHcor is shown to have profound implications for measuring changes in pHi.