RELATIONSHIP BETWEEN INTRACELLULAR PROTON BUFFERING CAPACITY AND INTRACELLULAR PH

In a recent publication the widely held view that the intracellular proton buffering power [defined as the amount of acid or base that has to be added to the cytosol to change the intracellular pH (pH(i)) by one pH unit] increases as the intracellular pH decreases, has been challenged, with the opposite relationship being proposed. In that publication, buffering was defined not in terms of pH change, but in terms of the change in proton concentration. The reason for this re-definition was the fear that the conventional analysis, using as it does a logarithmic function (pH(i)), could bias the outcome in favor of an increasing buffering power with decreasing pH(i). The new system uses a "buffering co-efficient," defined as the number of protons necessary to be added to the cytosol to change the intracellular proton concentration by 1 mM. We report the use of both of these methods to analyze the relationship of pH(i) and buffering power, using human peripheral leucocytes loaded with the pH-sensitive fluorophore BCECF examined over a very wide range of pH(i) values (pHi 6.0 to 7.5). The most common method for pH(i) perturbation for the measurement of buffering is used, the rapid diffusion of ammonia across the cell membrane. In this study, analysis for both a bicarbonate-containing "open" system and for a Hepes-buffered "closed" system was performed. Unlike the previous publication, the intracellular and extracellular conditions were such that the change in pH(i) induced by the extracellular addition of an ammonia-containing compound (NH4Cl) was the same (0.15 to 0.25 pH unit) across the pH(i) range of 6.0 to 7.5. The pH(i)-buffering relationship varied depending on the analysis used, but taking into account the known properties of Na+/H+ exchange and the desirability of defence of the cell against intracellular acidosis, the traditional relationship seems conceptually to be the more satisfactory.