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

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 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.In a previous paper (6) we reported the results of experiments showing that exposure of macrophages to medium containing any one of a large number of weak bases results in the uptake of the bases into lysosomes, and, when the concentration in lysosomes becomes sufficiently high, osmotic swelling of these particles to form large phase-lucent vacuoles occurs. We showed that the concentration of the free base in the medium is the determining factor for uptake and vacuolation, and that the protonated base in the medium has no effect. Widely different concentrations are required to elicit vacuolation by different bases.We have described previously a fluorescence probe technique for the measurement of intralysosomal pH (5) . In this study we use this technique to explore, in some detail, the pH changes caused in the lysosomes by some of these weak bases, and we discuss the results in terms of the leakage of protons out of lysosomes in the form of protonated base. We conclude that the high acidity in lysosomes is the consequence of an active process of proton secretion into lysosomes . MATERIALS AND METHODS Cell CultureOur culture procedures for mouse peritoneal macrophages, adapted from This paper is appearing without fmal revision due to the untimely death of Dr . Brian Poole.THE JOURNAL OF CELL BIOLOGY " VOLUME 90 SEPTEMBER 1981 665-669 © The Rockefeller University Press " 0021-9525/81/09/0665/05 $1 .00Cohn and Benson (2), have been described fully in the previous paper in this series (6). Lysosomal pH MeasurementThis technique has been described in more detail elsewhere (5) . Briefly, cells were exposed to medium containing 1 mg/ml fluorescein-labeled dextran for 24 h. They were then washed free of medium and the cover slips were mounted in a special holder in a regular fluorescence cell that was perfused with various media . The fluorescence of the cells was measured with a Hitachi Perkin-Elmer MPF...

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