Inadequacy of high K+/nigericin for calibrating BCECF. I. Estimating steady-state intracellular pH

Intracellular pH (pHi) was measured in single vascular smooth muscle (VSM) cells, cultured from rabbit abdominal aorta, using 2',7'-bis(carboxyethyl)-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. 1) The intrinsic buffering power (beta int), measured using weak bases (e.g., ammonium), was 2.5 times smaller than that measured using weak acids (e.g., propionic acid). This discrepancy became small if pHi had really been approximately 0.2 lower than what was estimated using nigericin-calibrated pHi values. 2) Total cellular buffering power (beta tot) in the presence of CO2/HCO-3 was measured and found to be much smaller than could account for the beta int, together with the contribution of CO2/HCO3 (beta CO2: assumed to be an open system buffer). If the true pHi values were approximately 0.2-0.4 lower than our nigericin-calibrated values, then the sum of beta int and beta CO2 equals beta tot. 3) A null technique was utilized for bracketing steady-state pHi; estimates of steady-state pHi using this null technique were approximately 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 approximately 0.1.