The Buffering Power in Human Monocytes

Intracellular pH (pHi) plays a vital role in the regulation of many cell functions. Apart from active transmembrane pHi regulators, passive intracellular buffering power acts in the first line to attenuate the impact of pHi changes. Moreover, the quantification of the total intracellular buffering power (βtot) is essential for calculating transmembrane acid-equivalent fluxes from pHi recordings. The βtot has two components: intrinsic buffering power (βi) and CO2-dependent buffering power (βCO2). By microspectrofluorimetry with a fluorescence probe BCECF (2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein), we calculated the buffering power in human monocytes.Experiments were performed under conditions free of Na^+, Cl^- and high K^+ to prevent the operation of active transmembrane pH regulators. Small stepwise reductions of external NH4Cl (from 30 to 0 mM) resulted in stepwise reductions of pHi. Similar procedures were performed either in the CO2/HCO3^--or the HEPES-buffered solution. The results showed in the pHi ranges of 6.9~7.5, under the CO2/HCO3^- -buffered condition, the values of βtot can be described as βtot=1403.1[pHi]^2-19169.7[pHi]+65538 (R^2=0.81). Under HEPES-buffered condition, the values of βi can be described as βi=-1293.2[pHi]^2-18539.6[pHi]+66519.9 (R^2=0.64). Note, the factor of βtot becomes more important while in the alkaline direction. In addition, the magnitude of intracellular βCO2, derived from βtot-βi, has been described as βCO2=745.7[pHi]^2-9832.1[pHi]+32306.3 (R^2=0.99). This demonstrated the CO2-dependent buffering power in the human monocytes was not consistent with a fully open cell-system for CO2, i.e. βCO2 is not equal to 2.3×[HCO3^-]. In other words, CO2-permeation and-hydration/dehydration reaction are not rapid enough to behave as an open system. In conclusion, our present study, for the first time, quantifies the buffering power in human monocytes.