Dynamic measurements of the acute and chronic effects of lysosomotropic agents on hepatocyte lysosomal pH using flow cytometry

Previous investigators measuring the pH of lysosomes have used digitized video microscopy (DVM) in freshly isolated or cultured cells. Although useful, this technique is time consuming, requires the use of an image analysis system, and is limited by the fact that measurements can be made in only a relatively small number of cells. The aim of this study was to develop and initially apply a technique using flow cytometry to make dynamic measurements of lysosomal pH in a large number of living hepatocytes. Rats were injected intraperitoneally with fluorescein isothiocyanate-dextran (FITC-Dex), a pH-sensitive fluorescent probe that is sequestered into lysosomes. Hepatocytes were isolated 16 hours after injection by collagenase perfusion. Lysosomal pH was measured in 20,000 hepatocytes per animal using flow cytometry with excitation at 488 nm and emission at 530 nm (pH sensitive) and 585 nm (pH insensitive). A standard curve of pH versus the 530/585 nm ratio was generated with FITC-Dex-loaded hepatocytes by equilibrating intralysosomal pH with extracellular pH using ionophores and metabolic inhibitors. The acute effects of chloroquine and methylamine were determined by exposing isolated hepatocytes to these lysosomotropic agents. The effect of chronic administration of chloroquine and Triton WR-1339 (Rutger Chemical, Inc., Irvington, NJ) on lysosomal pH was also measured. Intralysosomal pH was 4.67 + 0.02, nearly identical to the value 4.70 + 0.05 previously measured by us using DVM. Both chloroquine and methylamine caused both rapid (<1 minute), major (0.5 to 2.0 pH units), and dose-dependent increases in lysosomal pH as well as changes in lysosome morphology. In contrast, although chronic chloroquine and Triton WR-1339 administration caused dramatic ultrastructural changes in lysosomal morphology, lysosomal pH was not increased by either of these agents suggesting that their pharmacological mechanisms are not mediated by their effects on lysosomal pH. These results show the utility and validity of using flow cytometry in living cells to quantify dynamic changes in the pH of lysosomes and show a dissociation of morphological and pH responses of hepatocyte lysosomes to chronic treatment with lysosomotropic agents.