Paul B. Gordon

Prelysosomal convergence of autophagic and endocytic pathways.

[14C]Lactose, electroinjected into the cytosol of isolated rat hepatocytes, was sequestered by autophagy, transferred to lysosomes and eventually hydrolysed. Asparagine prevented the fusion between prelysosomal autophagic vacuoles and lysosomes, and caused lactose to accumulate in the former. However, if the hepatocytes were simultaneously allowed to endocytose added beta-galactosidase, no lactose accumulation occurred. These results suggest that autophagically sequestered lactose and endocytosed beta-galactosidase were delivered to the same prelysosomal vacuole, where the lactose was hydrolysed by the enzyme. The name amphisome is suggested for this new functional compartment, common to the autophagic and endocytic pathways.

Amino acid inhibition of the autophagic/lysosomal pathway of protein degradation in isolated rat hepatocytes

Protein degradation in isolated rat hepatocytes, as measured by the release of [14C]valine from pre-labelled protein, is partly inhibited by a physiologically balanced mixture of amino acids. The inhibition is largely due to the seven amino acids leucine, phenylalanine, tyrosine, tryptophan, histidine, asparagine and glutamine. When the amino acids are tested individually at different concentrations, asparagine and glutamine are the strongest inhibitors. However, when various combinations are tested, a mixture of the first five amino acids as well as a combination of leucine and asparagine inhibit protein degradation particularly strongly. The inhibition brought about by asparagine plus leucine is not additive to the inhibition by propylamine, a lysosomotropic inhibitor; thus indicating that the amino acids act exclusively upon the lysosomal pathway of protein degradation. Following a lag of about 15 min the effect of asparagine plus leucine is maximal and equal to the effect of propylamine, suggesting that their inhibition of the lysosomal pathway is complete as well as specific. Degradation of endocytosed 125I-labelled asialofetuin is not affected by asparagine plus leucine, indicating that the amino acids do not affect lysosomes directly, but rather inhibit autophagy at a step prior to the fusion of autophagic vacuoles with lysosomes. The aminotransferase inhibitor, aminooxyacetate, does not prevent the inhibitory effect of any of the amino acids, i.e. amino acid metabolites are apparently not involved.

Increase in cis-dichlorodiammineplatinum(II) cytotoxicity upon reversible electropermeabilization of the plasma membrane in cultured human NHIK 3025 cells

A series of brief electrical high-voltage discharges were given to cultured NHIK 3025 cells to render the plasma membrane transiently permeable to drugs. Using [14C]sucrose as an inert marker which normally does not cross plasma membranes, increased permeability could be demonstrated for no longer than 10 min following electrical treatment, indicating that the permeabilization was entirely reversible. The reversibility of the treatment was further demonstrated by a lack of effect on cell growth and colony-forming ability. When cells were given electrical discharges immediately before or during exposure to cis-dichlorodiammineplatinum(II)(cis-DDP) the cytotoxic drug effect increased. By using electrical discharges during a 2 hr drug treatment period the cytotoxicity was enhanced to an extent corresponding to at least a 3-fold increase in drug uptake relative to unpermeabilized cells. This increase in drug uptake was confirmed by direct measurements of the amount of cell-associated Pt by atomic absorbtion spectroscopy. The results suggest that uptake across the plasma membrane may be the rate-limiting factor in the cytotoxic effect of cis-DDP. Furthermore, the methodology applied in the present study may prove useful in assessing the influence of membrane permeability on the effect of other cytotoxic drugs.

AMINO ACID CONTROL OF PROTEIN SYNTHESIS AND DEGRADATION IN ISOLATED RAT HEPATOCYTES

Cell protein is a term that covers an extremely heterogeneous collection of enzymes and structural elements; yet in certain respects it is justified to consider cell protein as a biological entity. The growth of cells is essentially (at least in quantitative terms) an accumulation of cell protein as such, with no significant alterations in the ratios between its individual components. Conversely, during starvation, cell protein may be degraded en bloc to provide metabolic fuel and materials for synthetic processes. As is the case for many other metabolic entities, separate pathways are used for the synthesis and degradation of protein, creating a metabolic cycle. However, the pathways are extraordinarily complex in the case of protein, as is its precursor/ degradation product: the full complement of twenty amino acids. Amino acids stimulate protein synthesis and inhibit protein degradati~n,’-~ but the mechanisms of regulation are as obscure as the reasons for our ignorance are obvious: in addition to the complex nature of the precursors, products and pathways, regulation of protein metabolism involves both overall rate controls and regulation of the synthesis and degradation of each hdividual protein by a multitude of hormonal, metabolic and environmental factors. Furthermore, the simple quantitative relationship between overall protein balance and the availability of amino acids that would be expected to prevail during growth, may be perturbed in multicellular organisms by the growth restrictions imposed upon all normal cells. To lead to a better understanding of these cell growth restrictions, and their relative absence in malignant cells, protein metabolism is a particularly pertinent field of study. At the cellular level, regulatory mechanisms are best studied with isolated cells, since the systematic testing of regulatory agents may be carried out without interference from homeostatic control by the organism. Isolated hepatocytes are a most suitable material for such studies, being normal cells that can be prepared intact, in large quantities, from rat liver by the method of collagenase perfusion.6*7 In the present article, the role of amino acids in the control of protein synthesis, degradation in hepatocyte suspensions, and early cultures will be discussed.

Use of a hydrolysable probe, [14C]lactose, to distinguish between pre-lysosomal and lysosomal steps in the autophagic pathway

[14C]Lactose, introduced into the cytosol of isolated rat hepatocytes by means of electropermeabilization, is sequestered autophagically in the same way as the established sequestration probe, [14C]sucrose. However, unlike the inert sucrose molecule, lactose is rapidly hydrolysed in the lysosomes, and can therefore be used to probe the last step of the autophagic pathway (i.e. fusion with the lysosome). During autophagy lactose is present only at a low, steady-state level in pre-lysosomal vacuoles (probably autophagosomes), serving as a useful marker for these organelles. If autophagosome-lysosome fusion is blocked with vinblastine (Kovács et al., Exp cell res 137 (1982) 191), [14C]lactose will accumulate continuously as a function of the sequestration rate, and reach a high level in the pre-lysosomal vacuoles. Density gradient analysis, using chloroquine (CLQ) to alter lysosomal density, suggests that these organelles have a broad density distribution (1.08-1.13 g/ml), thus differing significantly from the distribution of lysosomes.

6-Substituted purines: A novel class of inhibitors of endogenous protein degradation in isolated rat hepatocytes

Abstract About 100 different purine derivatives and analogs were tested for their effect on protein synthesis and protein degradation in isolated rat hepatocytes. These included 6-aminopurines (adenine and adenosine analogs), 6-mercaptopurines, chloropurines, oxypurines, cytokinins, methylxanthines, methylindoles, benzimidazoles, and benzodiazepines. Most of the compounds were either inactive or inhibited protein synthesis as much as or more than they inhibited protein degradation. However, three methylated 6-aminopurines (3-methyladenine, 6-dimethylaminopurine riboside, and puromycin aminonucleoside) and four 6-mercaptopurines (6-methylmercaptopurine, 6-methylmercaptopurine riboside, 6-mercaptopurine riboside, and 2′,3′,5t - triacetyl-6-mercaptopurine riboside) had a markedly stronger effect on protein degradation than on synthesis, and might therefore be potentially useful as selective degradation inhibitors. None of the seven above-mentioned purines had any significant effect on the degradation of the exogenous protein, asialofetuin, and would therefore seem to selectively inhibit endogenous protein degradation. Since the degradation was not further affected by purines in the presence of amino acids or lysosomotropic amines, it is suggested that the purines exert their effect specifically upon the autophagic/lysosomal pathway. All the mercaptopurines significantly depressed cellular ATP levels, whereas the methylated aminopurines did not. For this reason, the latter are probably more useful as degradation inhibitors. 3-Methyladenine had no effect on protein synthesis at a concentration (5 m m ) which inhibited protein degradation by more than 60%, and may therefore be regarded as a highly specific inhibitor of autophagy.

Autophagic sequestration of [14C]sucrose introduced into isolated rat hepatocytes by electrical and non-electrical methods

Isolated rat hepatocytes were found to become permeable to [14C]sucrose at 0 degree C under three different conditions: Immediately following their liberation from the collagenase-perfused liver. Following a short incubation under hypoxic conditions. After electropermeabilisation. All three conditions were characterised by the formation of small protuberances (blebs) indicative of localised cell surface damage, and it is possible that the stretched plasma membrane of such blebs acted as a high-permeability region. Disappearance of blebs and restoration of normal plasma membrane impermeability could be achieved by a short (15 min) incubation at 37 degrees C. It could be shown that [14C]sucrose introduced into rat hepatocytes by non-electrical means was autophagically sequestered at the same rate as [14C]sucrose introduced electrically. In both cases the sequestration was inhibited by the specific autophagy inhibitor 3-methyladenine to a similar extent. The subcellular distribution of sequestered isotope in metrizamide/sucrose density gradients was found to be independent of the conditions of its introduction into cells.

Temperature dependence of protein degradation, autophagic sequestration and mitochondrial sugar uptake in rat hepatocytes

Lysosomal (propylamine-sensitive) protein degradation as well as the energy-dependent (chymostatin-sensitive) part of the non-lysosomal protein degradation was found to be strongly affected by temperature in isolated rat hepatocytes, the activation energy (Ea) being about 25 kcal/mol for both processes. In contrast, the energy-independent (chymostatin-resistant) part of the non-lysosomal degradation had an Ea of approx. 10 kcal/mol only. Sequestration of electroinjected [14C]sucrose into sedimentable organelles showed a pronounced temperature dependence. By means of digitonin extraction it was possible to distinguish between a moderately temperature-sensitive mitochondrial sugar uptake (Ea approx. 12 kcal/mol) and a strongly temperature-dependent autophagic sequestration (Ea approx. 22 kcal/mol). There was no significant autophagic sequestration below 20 degrees C. The sequestration process is more temperature-sensitive than, for example, the early steps of endocytosis, and is likely to represent the major controlling step in the overall autophagic-lysosomal pathway.

Trapping of electro-injected [14C]sucrose by hepatocyte mitochondria: A mechanism for cellular autofiltration?

[14C]Sucrose was introduced, by means of electropermeabilization, into the cytosol of isolated rat hepatocytes to serve as an inert marker of the intracellular fluid. Radioactivity accumulated in mitochondria (as well as in lysosomes) at a rate of 2-3%/h for 4 h of incubation at 37 degrees C, as shown by subcellular fractionation in isotonic density gradients. This unprecedented mitochondrial trapping of a molecule which does not cross most biological membranes points to the possibility that mitochondria participate in some kind of autofiltration of cellular fluid.

Inhibition of cell spreading by lysosomotropic amines

Ammonia and various amines with weak-base properties have been shown to accumulate in acidic cellular vacuoles such as lysosomes, and to inhibit lysosomal degradative processes in several types of cells in vitro [l-l I]. In addition these compounds may: inhibit protein secretion [ 121 as well as other vesicle-mediated secretory processes [ 131, inhibit the adsorbtive uptake of macromolecules [ 14161, suppress the activity of certain receptor-dependent toxins [ 17,181, and prevent the focal patching of occupied cell-surface receptors normally preceding internalization and degradation of receptor-ligand complexes [ 191. That all of these effects are observed only at relatively high amine concentrations (2-10 mM) may indicate that they are consequential of a generalized perturbation of cellular membrane mobility, caused by the accumulation of amines in certain compartments of the cellular membrane system. Isolated rat hepatocytes have been shown to attach to and spread readily on substrata of adsorbed serum (fibronectin) or collagen [20-221, and scanning electron microscopy has revealed that the spreading is mediated by a continuous flow of a thin, basal layer of lamellar cytoplasm [21] directed towards the cell periphery. A general interference with cellular membrane flow might therefore be expected to cause inhibition of hepatocyte spreading, and such inhibition by lysosomotropic amines is demonstrated here. 2. Materials and methods