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

We measured intracellular pH (pH(i)) 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 (pH(cor)) to the high K+/nigericin-calibrated pH(i) was linearly dependent on pH(i), increasing with increasing pH(i) (2), When the nigericin 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 similar to 0.08 different from null estimates) than using nigericin calibrations alone (similar to 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. Similarly, when appropriately corrected, both methods gave indistinguishable calibration curves. Thus, the two methods are entirely equivalent from the stand-point 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.