Open AccessThe Role of the Intralysosomal pH in the Control of Autophagic Proteolytic Flux in Rat Hepatocytes
Author(s) -
Luiken Joost J. F. P.,
Aerts Johannes M. F. G.,
Meijer Alfred J.
Publication year - 1996
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1996.00564.x-i2
Subject(s) - intracellular , proteolysis , biochemistry , lysosome , chemistry , amino acid , substrate (aquarium) , intracellular ph , endocytosis , enzyme , biophysics , biology , cell , ecology
Current methods to estimate changes in intralysosomal pH in hepatocytes do not discriminate between lysosomes and other intracellular acidic compartments. To obtain selective information on the change in lysosomal function in response to a change in lysosomal pH we have used the liberation of fluorescent 4‐methoxy‐2‐naphthylamide from low concentrations of lysyl‐alanyl‐4‐methoxy‐2‐naphthylamide, B substrate of lysosomal dipeptidylpeptidase II. Using permeabilized and intact hepatocytes, the activity of this enzyme in response to manipulations meant to increase the intralysosomal pH was compared with intralysosomal protein degradation and with the accumulation of [ 14 C]chloroquine as a pH indicator of intracellular acidic compartments. The data show that changes in intralysosomal pH are indicated by changes in dipeptidylpeptidase II activity and that these are mainly due to a pH‐dependent change in substrate accumulation in the lysosomes. Subsequently, the method was applied to establishing the extent to which an increase in intralysosomal pH can contribute to the inhibition of autophagic proteolysis in intact hepatocytes caused by a decrease in intracellular ATP, by an increase in amino acid concentration and by hypo‐osmotic cell swelling. The following observations were made. (a) Moderate changes in intracellular ATP do not affect the lysosomal pH. (b) Hypo‐osmotic cell swelling, which promotes inhibition of proteolysis by amino acids in freshly isolated hepatocytes, does not affect the lysosomal pH. (c) In addition to their known inhibitory effect on autophagic sequestration, amino acids (leucine in particular) can increase the lysosomal pH and thus inhibit intralysosomal protein degradation directly. (d) Low concentrations of the acidotropic agent methylamine increase the lysosomal pH without having an effect on autophagic proteolytic flux. It is concluded that autophagic proteolysis is not controlled by changes in the lysosomal pH.