Effect of weak bases on the intralysosomal pH in mouse peritoneal macrophages.

TLDR

Fluorescent dextran probes change spectrum with pH, enabling measurement of lysosomal acidity. Macrophage lysosomes were loaded with fluorescein‑dextran and their pH was monitored under different media conditions. Weak bases raise intralysosomal pH in a concentration‑dependent manner, with the neutral form being active; the required concentration varies among bases, and although they enlarge lysosomes, volume change only slightly affects pH, supporting an energy‑dependent acidification and proton leakage model.

Abstract

The spectral characteristics of dextran, labeled with fluorescein, depend upon pH. We have loaded the lysosomes of mouse peritoneal macrophages with this fluorescence probe and used it to measure the intralysosomal pH under various conditions. The pH of the medium has no effect on the intralysosomal pH. Weakly basic substances in the medium cause a concentration-dependent increase in the intralysosomal pH. However, the concentration of base necessary to produce a significant change in the intralysosomal pH varies over a wide range for different bases. The active form of the base is the neutral, unprotonated form. Although most of these weak bases cause an increase in the volume of the lysosomes, increase in lysosomal volume itself causes only a minor perturbation of the intralysosomal pH. This was demonstrated in cells whose lysosomes were loaded with sucrose, and in cells vacuolated as a demonstrated in cells whose lysosomes were loaded with sucrose, and in cells vacuolated as a consequence of exposure to concanavalin A. The results of these studies are interpreted in terms of energy-dependent lysosomal acidification and leakage of protons out of the lysosomes in the form of protonated weak bases.