Inhibition of the Lysosomal Pathway of Protein Degradation in Isolated Rat Hepatocytes by Ammonia, Methylamine, Chloroquine and Leupeptin

Abstract

1. Protein degradation in isolated rat hepatocytes was measured as the release of [14C]valine from pre-labelled protein. To reduce background radioactivity, the intracellular ['4C]valine pool was depleted by serial extraction at 37 "C, effecting equilibration between the intracellular pool and the valine-free extracellular medium. After extraction, a small, non-equilibrating intracellular [14C]valine pool remained; this pool could only be labelled in the presence of ongoing protein synthesis, and might represent valine and valine-containing oligopeptides derived from protein degradation.2. The ['4C]valine released from degraded protein was not significantly re-utilized for protein synthesis intracellularly (no effect of cycloheximide or high concentrations of unlabelled valine), reflecting the low rate of protein synthesis and the rapid transport of valine into the extracellular medium, both characteristic of isolated hepatocytes.3. From cells pre-labelled for 24 h Ivi vivo, ['4C]valine was released linearly at a rale of 5 %/h, probably representing the true over-all protein degradation rate. The lysosomotropic inhibitor ammonia (10 mM NH4C1) inhibited 70% of the degradation, presumably the contribution by the lysosomal pathway.From 1-h pre-labelled cells, [14C]valine was released at a declining rate, and ammonia inhibited degradation only by 45%, consistent with the view that the majority of short-lived proteins are degraded by the non-lysosomal pathway(s).4. Chloroquine and methylamine, which accumulate in lysosomes by virtue of their weak base properties, inhibited hepatocytic protein degradation to the same extent as ammonia, with no additivity. These compounds therefore seem to block the lysosomal pathway of protein degradation selectively and completely. Leupeptin, which binds to and inhibits the activity of certain lysosomal proteases, also inhibited protein degradation almost to the same extent as ammonia, but with a small part of the effect ( < 20 %) being additive to the NH3 effect and thus probably reflecting a slight inhibition of non-lysosomal protein degradation as well.5. Of the four inhibitors tested, only the effect of ammonia was rapidly reversible within the experimental period (2 h). Leupeptin, on the other hand, was the only degradation inhibitor which did not also affect protein synthesis. Chloroquine caused significant cell death at concentrations above 0.2 mmol/l in this protein-free medium, i.e. in the concentration range needed for maximal inhibition of protein degradation.6. Incubation of hepatocytes under anoxic conditions resulted in an inhibition of protein degradation which was greater than, and partially additive to, the effect of ammonia, i.e. most of the degradation by the lysosomal pathway and more than one-half of the degradation by the nonlysosomal pathways appears to be energy-dependent.The mechanisms responsible for the general turnover of endogeneous protein in animal cells are not well known. Lysosomes (autophagy) appear to play a major role [I -41 but in addition a numbe...

Cleavage of Focal Adhesion Kinase by Different Proteases during Src-regulated Transformation and Apoptosis

“…Calpain inhibitor I-ALLN has also been reported to inhibit the lysosomal proteases cathepsin B, cathepsin L, and the ubiquitin-dependent proteasome complex (27,30,31). Treatment of ts v-Src-expressing CEF undergoing transformation with ammonium chloride, a reputed inhibitor of lysosomal cathepsins (32,33), or the specific proteasome inhibitor, lactacystin (34), had no effect on the characteristics of v-Src transformation (results not shown), indicating that the effects of ALLN were most likely due to calpain inhibition. In addition, treatment of ts v-Src-expressing CEF with ALLM (100 M), which is inhibitory against calpains, but not the …”

Inhibition of autophagic sequestration and endogenous protein degradation in isolated rat hepatocytes by methylated adenosine derivatives

“…Degradation rates (%) were calculated as percentage of the total protein radioactivity at the start of the experiment. Sample preparation and analysis was performed as in [9,10].…”

Characterization of ligand binding and processing by gastrin-releasing peptide receptors in a small-cell lung cancer cell line

“…The present study indicates that in COR-L42 cells, 1251I-GRP is degraded by endo/exopeptidases. Chloroquine is a weak base which acts by increasing intralysosomal pH, thus inhibiting further processing of ligand-receptor complexes (Wibo & Poole, 1974;Seglen et al, 1979;Marshall & Olefsky, 1979;Poole & Ohkuma, 1981;Maxfield, 1982). Binding studies carried out in the presence of chloroquine showed that after 1251-GRP binding and internalization, cell-associated radioactivity did not decline, indicating that the subsequent degradation of I251-GRP was inhibited by this agent.…”