INADEQUACY OF HIGH K+ NIGERICIN FOR CALIBRATING BCECF .1. ESTIMATING STEADY-STATE INTRACELLULAR PH/

Intracellular pH (pH(i)) was measured in single vascular smooth muscle (VSM) cells, cultured from rabbit abdominal aorta, using 2',7'-bis(ca rboxyethyl)-5(6)-carboxyfluorscein (BCECF) on a microscope-based fluor escence system. Three lines of evidence are presented that using niger icin along with high external K+ to calibrate intracellular BCECF prod uces systematic errors in pH(i). 1) The intrinsic buffering power (Pin t), measured using weak bases (e.g., ammonium), was 2.5 times smaller than that measured using weak acids (e.g., propionic acid). This discr epancy became small if pH(i) had really been similar to 0.2 lower than what was estimated using nigericin-calibrated pH(i) values. 2) Total cellular buffering power (beta(tot)) in the presence of CO2/HCO3- was measured and found to be much smaller than could account for the Pint, together with the contribution of CO2/HCO3- (beta(CO2): assumed to be an open system buffer). If the true pH(i) values were similar to 0.2- 0.4 lower than our nigericin-calibrated values, then the sum of beta(i nt) and beta(CO2) equals beta(tot.)3) A null technique was utilized fo r bracketing steady-state pH(i); estimates of steady-state pH(i) using this null technique were similar to 0.2 lower than the high K+/nigeri cin-calibrated estimates. Four other cell types were examined: rat hep atocytes, rat corticotrophs, human keratinocytes, and rabbit fibroblas ts. These other cells also displayed discrepancies between null and ni gericin estimates of steady-state pH(i), as well as differences betwee n buffering power assessed using weak bases and acids. Finally, one po tential source for these discrepancies is described: selecting an inap propriate external K+ to use with nigericin can produce systematic err ors in pH(i) of similar to 0.1.