Fluorescence probe measurement of the intralysosomal pH in living cells and the perturbation of pH by various agents.

TLDR

The study develops a quantitative method to measure intralysosomal pH in living cells. The method uses fluorescein‑isothiocyanate–labeled dextran that is endocytosed and accumulates in lysosomes, and live cells are monitored in a spectrofluorometer while perfused with various media. Fluorescence of the dextran changes with pH 4–7 and is insensitive to concentration, ionic strength, or protein levels; normal macrophage lysosomal pH is 4.7–4.8, dropping to 4.5 transiently; exposure to weak bases, acidic potassium ionophores, and combined glycolytic/oxidative inhibitors rapidly raises pH, supporting an active proton‑accumulation mechanism and the proton‑trapping theory.

Abstract

A quantitative method is described for the measurement of intralysosomal pH in living cells. Fluorescein isothiocyanate-labeled dextran (FD) is endocytized and accumulates in lysosomes where it remains without apparent degradation. The fluorescence spectrum of this compound changes with pH in the range 4-7 and is not seriously affected by FD concentration, ionic strength, or protein concentration. Living cells on coverslips are mounted in a spectrofluorometer cell and can be perfused with various media. The normal pH inside macrophage lysosomes seems to be 4.7-4.8, although it can drop transiently as low as 4.5. Exposure of the cells to various weak bases and to acidic potassium ionophores causes the pH to increase. The changes in pH are much more rapid than is the intralysosomal accumulation of the weak bases. Inhibitors of glycolysis (2-deoxyglucose) and of oxidative phosphorylation (cyanide or azide) added together, but not separately, cause the intralysosomal pH to increase. These results provide evidence for the existence of an active proton accumulation mechanism in the lysosomal membrane and support the theory of lysosomal accumulation of weak bases by proton trapping.