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Superiority of in vitro over in vivo calibrations of BCECF in vascular smooth muscle cells
Author(s) -
Boyarsky Gregory,
Hanssen Chris,
Clyne Lisa A.
Publication year - 1996
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.10.10.8751723
Subject(s) - nigericin , in vitro , biophysics , intracellular , chemistry , calibration , in vivo , vascular smooth muscle , intracellular ph , steady state (chemistry) , analytical chemistry (journal) , smooth muscle , biochemistry , biology , chromatography , mathematics , statistics , membrane , endocrinology , microbiology and biotechnology
We measured intracellular pH (pH i ) in single vascular smooth muscle cells (VSM) cultured from rabbit abdominal aorta, using 2‘, 7‘‐biscar‐boxyethyl‐5(6)carboxyfluorescein (BCECF) on a microscope‐based fluorimetric system. We previously found substantial errors introduced by using high K + /nigericin to calibrate intracellular BCECF (1). We also previously demonstrated that the necessary correction (pH cor ) to the high K + /nigericin‐calibrated pH i was linearly dependent on pH i , increasing with increasing pH i (2). When the nig‐ericin calibration data were corrected using this pH cor , the new corrected calibration was similar to the result of calibrating BCECF in vitro (higher R max , lower R min, and lower pK). Therefore, in this study the possibility is considered that in vitro calibrations might provide better estimates of pH i . Our best estimate for the absolute level of pH i derives from a null method for bracketing steady‐state pH i In VSM cells, using only in vitro calibrations to estimate steady‐state pH i leads to less error (only ~0.08 different from null estimates) than using nigericin calibrations alone (~0.2 different from null estimates). Unlike high K + /nigericin calibrations, the error, pH cor , introduced by using an in vitro calibration is pH i independent. Using high K + /nigericin or in vitro calibrations, along with the respective pH cor on the same experimental days in the same cells, gave the same estimate of pH i whether in the steady state, in acid‐loaded cells, or in alkali‐loaded cells. Simi‐larly, when appropriately corrected, both methods gave indistinguishable calibration curves. Thus, the two methods are entirely equivalent from the standpoint of accuracy. Because nigericin is toxic, expensive, and complicated in its use, we suggest that in vitro calibrations, along with simple null determinations to assess the small, constant correction factor, be used to calibrate intracellular BCECF.—Boyar‐sky, G., Hanssen, C., Clyne, L. A. Superiority of in vitro over in vivo calibrations of BCECF in vascular smooth muscle cells. FASEB J. 10, 1205‐1212 (1996)