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

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 to less error (only approximately 0.08 different from null estimates) than using nigericin calibrations alone (approximately 0.2 different from null estimates). Unlike high K+/nigericin calibrations, the error, pHcor, introduced by using an in vitro calibration is pHi independent. Using high K+/nigericin or in vitro calibrations, along with the respective pHcor on the same experimental days in the same cells, gave the same estimate of pHi whether in the steady state, in acid-loaded cells, or in alkali-loaded cells. Similarly, 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.-Boyarsky, G., Hanssen, C., Clyne, L. A. Superiority of in vitro over in vivo calibrations of BCECF in vascular smooth muscle cells.