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 [14C]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 [14C]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 in vivo, [14C]valine was released linearly at a rate of 5%/h, probably representing the true over-all protein degradation rate. The lysosomotropic inhibitor ammonia (10 mM NH4Cl) 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 non-lysosomal pathways appears to be energy-dependent.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1979.tb12956.x

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

Low temperature selectively inhibits fusion between pinocytic vesicles and lysosomes during heterophagy of 125I-asialofetuin by the perfused rat liver.

Publisher Summary A number of inhibitors are found to be useful in the study of lysosomal function. This chapter describes the inhibitors, which affect lysosomal function. The chapter also discusses weak-base (lysosomotropic) amines and the proteinase inhibitors, which are the most lysosome-specific of these, and the autophagy-inhibitory purines and some inhibitors that affect lysosomal function indirectly (microtubule poisons and protein synthesis inhibitors). Ammonia and the weakly basic alkylamines are lysosomotropic in the sense that, at low concentrations, they selectively accumulate in the lysosomes. The chapter also describes the biological effects of weak-base amines: interference with receptor-ligand dissociation and the recycling of receptors and membranes, inhibition of vacuolar membrane fusion and related processes, inhibition of lysosomal degradation, ionophores, proteinase-inhibitory peptides, vanadate, microtubule poisons, lectins and polymers, and protein synthesis inhibitors. Inhibitors of protein synthesis suppress the endogenous protein degradation in several cell types apparently by an action upon the lysosomal pathway. Despite their very general effects on the cell, the microtubule poisons may be useful as inhibitors of lysosomal function, acting differently from the other lysosome drugs.

Inhibitors of lysosomal function.

Changes in free cytosolic Ca2+ in hepatocytes following alpha 1-adrenergic stimulation. Studies on Quin-2-loaded hepatocytes.

AUTOPHAGOCYTOSIS is generally regarded as an important mechanism for ridding the cell of injured or unwanted cytoplasmic constituents and for degrading normal components in response to energy needs1,2. Autophagy has been noted in HeLa cells deprived of foetal calf serum and amino acids3. It has also been seen in rat liver after glucagon administration4, 5, during nutritional deprivation7, 8 and in diabetes9, 10. Thus autophagy could have a physiological role in the maintenance of body amino acid pools, although there is no direct experimental evidence for this. We reported previously that rates of proteolysis, and lysosomal sizes are seen to increase when rat livers are cyclically perfused with an unsupplemented medium and decrease in response to added amino acids11–13. We strongly suspected that autophagy was the underlying event, but the nature of the process has been difficult to define12. The possibility was considered that its expression was self-limited by the accumulation of amino acids from endogenous proteolysis. We now show that when this accumulation is prevented by perfusing livers in the single-pass mode, autophagic changes appear that are morphologically indistinguishable from those occurring after maximal doses of glucagon4–6.

Induction of autophagy by amino-acid deprivation in perfused rat liver

1. Isolated parenchymal cells were prepared by collagenase perfusion of livers from fed rats that had been previously injected with [3H]leucine to label liver proteins. When these cells were incubated in a salts medium containing glucose, gelatin and EDTA, cellular integrity was maintained over a period of 6h. 2. Cells incubated in the presence of 2mm-leucine to minimize radioactive isotope reincorporation released [3H]leucine into the medium at a rate accounting for the degradation of 4.5% of the labelled cell protein per h. 3. Degradation of [3H]protein in these cells was inhibited by insulin and by certain amino acids, of which tryptophan and phenylalanine were the most effective. 4. Protein degradation was decreased by several proteinase inhibitors, particularly those that are known to inhibit lysosomal cathepsin B, and by inhibitors of cell-energy production. 5. Ammonia inhibited degradation, but only at concentrations above 1.8mm. Aliphatic analogues of ammonia were effective at lower concentrations than was ammonia. 6. High concentrations of ammonia inhibited degradation by 50%. The extent of this inhibition could not be increased further by the addition of the cathepsin B inhibitor leupeptin, which by itself inhibited degradation by approx. 30%. 7. The sensitivity of proteolysis in isolated hepatocytes to these various inhibitory agents is discussed in relation to their possible modes of action.

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Melvyn F. Hopgood

Papers

Inhibition of protein degradation in isolated rat hepatocytes