Superiority of in vitro over in vivo calibrations of BCECF in vascular smooth muscle cells

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Abstract
We measured intracellular pH (pHi) in single vascular smooth muscle cells (VSM) cultured from rabbit abdominal aorta, using 2',7'-biscarboxyethyl-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 (pHcor) to the high K+/nigericin-calibrated pHi was linearly dependent on pHi, increasing with increasing pHi (2). When the nigericin calibration data were corrected using this pHcor, the new corrected calibration was similar to the result of calibrating BCECF in vitro (higher Rmax, lower Rmin, and lower pK). Therefore, in this study the possibility is considered that in vitro calibrations might provide better estimates of pHi. Our best estimate for the absolute level of pHi derives from a null method for bracketing steady-state pHi. In VSM cells, using only in vitro calibrations to estimate steady-state pHi leads t...

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