Experimental Cell Research 100 (1976) 276-280







Ultrastructural Alterations Lysosomal System

in the

P. 0. SEGLEN and A. REITH Norsk Hydro’s

Institute for Cancer Research, The Norwegian Montebello, Oslo 3, Norway

Radium Hospital,

SUMMARY Ammonia (10 mM NH,CI) partially inhibited protein degradation in isolated rat hepatocytes. An electron microscopic, morphometric analysis revealed that the inhibition was accompanied by lysosomal vacuolization, i.e. a large increase in the volume fraction of more and less electronlucent vacuoles containing remnants of digested material. This observation suggests that the lysosome is the target for the feedback-inhibition of protein degradation by ammonia. Lysosomal swelling may occur because ammonia, being a weak base, selectively accumulates in the acidic interio;of the lysosome.

Isolated rat hepatocytes are in a negative nitrogen balance, degrading their endogenous protein at a high rate [ 1, 21. The isolated cells contain larger numbers of autophagosomes than liver tissue in vivo [3], which may account for the high rate of protein degradation in vitro. A similar correlation between increased protein degradation and increased numbers of autophagic vacuoles has been observed in the perfused liver [4], indicating that a high catabolic rate is a general in vitro phenomenon, most probably mediated by a high activity of the autophagic-lysosomal system. In isolated hepatocytes, protein degradation was found to be inhibited by ammonia, a product of amino acid catabolism [2]. The present investigation was undertaken to see if this feedback inhibition of protein degradation by ammonia could be correlated with morphological changes in the lysosomal system.

METHODS Isolated rat hepatocytes were prepared from the livers of 16 h fasted, male Wistar rats, 250-300 g, by the two-step method of collagenase perfusion [5, 61. The purified cells were incubated for I h at 37°C in buffered saline [7] in the presence or absence of 10 mM NH&I. The protein-catabolic activity of the hepatocytes (total loss of nitrogen from the cells during the incubation period) was calculated on the basis of the changes in amino acids, urea and ammonia [2]. Sedimented cell samples were fixed for electron microscopy either by the end of the purification procedure [S], or after the 1 h incubation with or without NHICl. Fixation was performed for I h at room temperature in 1% glutaraldehydel0.1 M cacodylate-buffer, pH 7.4, 305 mosM, followed by rinsing overnight at 4°C in 0.1 M cacodvlate/O.l M sucrose, pH 7.4, 295 mosM. Small piecesbf each sample were post-fixed for 1 h at 4°C in I % OsO,/O. I M cacodvlate. PH 7.4, 330 mosM; then dehydrated with ethanol and embedded in Epon. From each of the three experimental groups (nonFigs I, 2. Survey view of isolated liver cells incubated

for 1 h at 37°C without (fig. 1) or with (fig. 2) 10 mM NHaCl. x 1300. Fig.-3. Three autophagic vacuoles (AV) and a dense bodv (DB) in a liver cell incubated without N&Cl. x l5’odO. ’ Figs 4, 5, 6. Autophagic vacuoles (AV), dense bodies (DB) and vacuolar lysosomes (LV) in liver cells incubated with NH&I. X 15000.


Seglen and Reith

incubated, 1 h incubated controls, and 1 h incubated with ammonium chloride) 16 samples were embedded. Finally 5 blocks from each group were chosen at random, and silver-to-grey thin sections were cut and examined after lead citrate staining in an Elmiscope 1 A at 80 kV. The morphometric procedure was as described in [8, 91. Ten micrographs from each block were made at a primary magnification of x5000, and enlarged together with-a superimposed double lattice test screen. The coarse points were spaced 0.8 pm apart (for the largest vacuolar lysosomes), and the fine points were spaced 0.3 wrn apart (for the other organelles). Thecomputation andthe statistical analysis, which included mean value +S.E. and a variance analysis (Student’s t-test) was done by means of an Olivetti programmed desk computer [9].

RESULTS Three types of vacuoles belonging to the autophagic-lysosomal system could be distinguished in isolated rat hepatocytes: Autophagic vacuoles, l-2 pm in diameter, were recognizable as membranelimited cytoplasmic bodies containing other organelles such as mitochondria or endoplasmic reticulum (fig. 3). Dense bodies, up to 1 pm in diameter, were characterized by a single membrane surrounding a variety of dense granular and filamentous material dispersed in a rather dense uniform matrix (fig. 4). Vacuolar lysosomes, up to 3 pm in diameter, had a relatively electron-lucent interior, containing dispersed cytoplasmic remnants or unrecognizable debris in various forms (figs 5, 6). Freshly isolated hepatocytes have the same general ultrastructural appearance as rat liver tissue in vivo, with relatively few phagosomes and lysosomes (fig. 1, table 1). However, after 1 h of incubation at 37°C there were significant increases, relative to unincubated (freshly isolated) controls, in the volume fractions of autophagic vacuoles, dense bodies and vacuolar lysosomes (table 1). Other cytoplasmic organelles, such as the endoplasmic reticulum and the Em CdRes

100 (1976)

mitochondria, were not altered by the incubation. Incubation of the hepatocytes for 1 h in the presence of ammonium chloride (10 mM) produced a dose-dependent inhibition of protein degradation, as measured by the reduced net loss of nitrogen from the cells (fig. 7). Cycloheximide, at a dose (1 mM) which inhibits protein synthesis by 95-98 %, did not affect the basal protein degradation nor the inhibition by ammonia (fig. 7), showing that de novo synthesis of proteins (e.g. lysosomal enzymes) is not important for short-term regulation of protein degradation. Hepatocytes incubated with 10 mM NH, Cl showed great differences in vacuole content relative to controls incubated without ammonia (fig. 2). The cell volume fraction occupied by vacuolar lysosomes was more than 15 times higher in the ammoniatreated cells, whereas the content of dense bodies was only l/5 of that in the controls (table 1). The content of autophagic vacuoles was not significantly different. There were no detectable differences in mitochondrial morphology, but parts of the rough endoplasmic reticulum in many ammonia-treated cells were arranged in concentric whorls, consisting of up to 12 parallel cisternae. DISCUSSION The dramatic alterations in the lysosomal system accompanying the inhibition of protein degradation by ammonia provide suggestive evidence that this system is responsible for intracellular protein degradation, and furthermore is the target for ammonia action. It is generally recognized that cytoplasm (including organelles such as mitochondria and endoplasmic reticulum) can be degraded in bulk through a sequence

Ammonia inhibition of protein degradution


141. Both dense bodies and vacuolar lysosomes would belong to the secondary lysosome category. If the high rate of protein degradation observed in hepatocytes incubated in vitro reflects the activity of the vacuolar system described above, the autophagic activity must be particularly high. This is in good accordance with the 4-fold increase in the content of autophagic vacuoles seen in incubated versus non-incubated cells. The inI 1 1 I 1 I I creases in the volume fractions of vacuolar 2 4 6 8 10 12 lysosomes and dense bodies are probably Fig. 7. Abscissa: initial NH,CI COW. (mM); ordinate: secondary events reflecting the increased net nitrogen loss (watomslglh). Effects of ammonia and cycloheximide on protein amount of cytoplasm entering into the lysodegradation (nitrogen balance). Isolated rat hepatocytes were incubated for I h at 37°C at the initial con- somal system for digestion. centration of NH,CI indicated, with (0) or without (0) In cells incubated with ammonia, the conI mM cycloheximide. The net loss of nitrogen from the cells during this period was calculated on the basis of tent of autophagic vacuoles is not significhanges in amino acids, urea and ammonia [2]. Each cantly different from the incubated convalue is the mean of two cell samples. trols, indicating that the lowered rate of protein degradation is neither due to reduced autophagy nor to impaired fusion with secof events, starting with the sequestration ondary lysosomes. The tremendous inof parts of the cytoplasm in the form of crease in the volume fraction of vacuolar membrane-bounded autophagic vacuoles lysosomes rather suggests that ammonia (also called autophagosomes or cytosegre- acts directly on the secondary lysosomes, somes). The autophagic vacuoles transform inducing lysosomal vacuolization. Being a weak base, ammonia would be into secondary lysosomes (i.e. they acquire lysosomal enzymes) through fusion either expected to selectively accumulate in the with primary lysosomes arising from the acidic lysosomal interior in the protonated form (as NH,+), and the attendant osmotic Golgi apparatus, or-more importantlywith pre-existing secondary lysosomes [ 10- influx of water might result in swelling of Table 1. Morphometric analysis of autophagicllysosomal vacuole contents (as fractions of cell volume) in isolated rat hepatocytes Values represent means kS.E. of five cell samples

Experimental treatment

Autophagic vacuoles (mm3/cm3)

A Non-incubated (fixed directly after isolation) L3 Control, incubated 1 h without additions Significance of difference B-A C Ammonia-incubated (1 h with 10 mM NHICI) Significance of difference C-B

2.4k0.9 10.9f2.2 P

Ammonia inhibition of protein degradation in isolated rat hepatocytes. Quantitative ultrastructural alterations in the lysosomal system.

Experimental Cell Research 100 (1976) 276-280 AMMONIA INHIBITION IN ISOLATED Quantitative OF PROTEIN DEGRADATION RAT HEPATOCYTES Ultrastructura...
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