Inadequacy of high k+/nigericin for calibrating bcecf: i.
estimating steady-state intracellular ph 0.
Boyarsky, Gregory, Christopher Hanssen, Lisa A. Clyne.
Department of Physiology and Biophysics, University of Texas
Medical Branch at Galveston, Galveston, TX 77550
APStracts 3:0148C, 1996.
Intracellular pH (pHi) was measured in single vascular smooth muscle
cells (VSM), cultured from rabbit abdominal aorta, using 2',7'
-biscarboxyethyl-5(6)carboxyfluorescein (BCECF) on a microscope-based
fluorescence system. Three lines of evidence are presented that using
nigericin along with high external K+ to calibrate intracellular
BCECF produces systematic errors in pHi. First, the intrinsic
buffering power ([beta]int), measured using weak bases (e.g.
ammonium), was 2.5x smaller than that measured using weak acids (e.g.
propionic acid). This discrepancy became small if pHi had really been
0.2 lower than what was estimated using nigericin-calibrated pHi's.
Secondly, total cellular buffering power ([beta]tot) in the presence
of CO2/HCO was measured, and found to be much smaller than could
account for the [beta]int, together with the contribution of CO2/HCO
([beta]CO2: assumed to be an open system buffer). If the true pHi's
were 0.2-0.4 lower than our nigericin-calibrated values, then the sum
of [beta]int and [beta]CO2 equals [beta]tot. Thirdly, a null
technique was utilized for bracketing steady-state pHi; estimates of
steady-state pHi using this null technique were 0.2 lower than the
high K+/nigericin-calibrated estimates. Four other cell types were
examined: rat hepatocytes, rat corticotrophs, human keratinocytes,
and rabbit fibroblasts. These other cells also displayed
discrepancies between null and nigericin estimates of steady-state
pHi, as well as differences between buffering power assessed using
weak bases and acids. Finally, one potential source for these
discrepancies is described: selecting an inappropriate external K+ to
use with nigericin can produce systematic errors in pHi of 0.1.
Received 11 May 1995; accepted in final form 25 April 1996.
APS Manuscript Number C254-5.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 19 May 96