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

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 4.74.8, 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 (-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. There have been conflicting reports and theories about the pH inside lysosomes. This controversy has been the subject of a review by Tager and Reijngoud (1). Isolated lysosomes have an internal pH about 1 unit lower than that of the medium, apparently as a consequence of a Donnan equilibrium (2-4). However, there is some evidence of an energy-dependent mechanism that is capable of lowering the pH (5, 6). In living cells, various weakly basic substances are concentrated in lysosomes as a consequence of lysosomal acidity (7). When the intralysosomal concentration of these substances becomes sufficiently high, the lysosomes swell osmotically to form large vacuoles (8, 9). Vacuoles of similar appearance have been observed in cells exposed to the acidic ionophore X537A (10).Attempts have been made to estimate the pH inside lysosomes by visual inspection of color changes in pH indicator dyes (see ref. 1). Here we describe a quantitative method for the measurement of intralysosomal pH based on the pH-dependent fluorescence signals from fluorescein isothiocyanate-labeled dextran (FD) in the lysosomes of living cells. Control experiments indicate that these signals should provide an accurate measure of pH. We report the results of some measurements of pH in the lysosomes of living cells under various conditions that support the existence of an active process of pH maintenance and provide some confirmation of the theory of pHdependent concentration of certain substances in lysosomes (7). MATERIALS AND METHODSMouse peritoneal macrophages isolated by the method of Cohn and Benson (11) were cultured in modified Eagle's medium (12) The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fa...

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