Inadequacy of high k+/nigericin to calibrate bcecf ii.
intracellular ph-dependence of the correction in vascular smooth
muscle cells.
Boyarsky, Gregory, Chris Hanssen, Lisa A. Clyne.
Department of Physiology and Biophysics, University of Texas
Medical Branch at Galveston, Galveston, TX 77550
APStracts 3:0149C, 1996.
In the accompanying study (1), it was demonstrated that steady-state
pHi determined using high K+/nigericin calibrations was
systematically in error in vascular smooth muscle cells (VSM) by 0.2
pH unit. In this manuscript 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 were acidified either by (i) lowering external pH (pHo)
to 6.0 or (ii) by a 5 min pulse of 20 mM ammonium and monitoring the
subsequent pHi recovery in the presence of 10 [mu]M
ethylisopropylamiloride (to substantially slow recovery). Cells were
alkali-loaded by (i) raising pHo or (ii) by switching from a CO2/HCO-
to a HEPES-buffered solution. The pHcor necessary to correct pHnig
was linearly dependent on pHnig, increasing from near zero at 6.0, to
0.2 at steady-state pHi, to 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,
Rmax, increased from 16.75 to 17.28; the minimal fluorescence ratio,
Rmin, decreased from 2.15 to 1.57; and the pK of BCECF 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 non
-constancy of pHcor is shown to have profound implications for
measuring changes in pHi.
Received 11 May 1995; accepted in final form 25 April 1996.
APS Manuscript Number C256-5.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 19 May 96