29

Biochimica et Biophysica Acta, 496 (1977) 29--35 © Elsevier/North-Holland Biomedical Press

BBA 28135 AMMONIA INHIBITS PROTEIN SECRETION IN ISOLATED RAT HEPATOCYTES P.O. SEGLEN and A. REITH Norsk Hydro's Instilute for Cancer Research, The Norwegian Radium Hospital, Montebello, Oslo 3 (Norway)

(Received July 15th, 1976)

Summary The general secretion of proteins by isolated rat hepatocytes in suspension is inhibited by colchicine, anoxia, or ammonia (NH4C1). The inhibition by ammonia is accompanied by the cytoplasmic retention and swelling of protein secretory vesicles, suggesting that ammonia accumulates in the vesicles and thereby prevents their translocation to the cell periphery. General protein synthesis appears to be relatively unaffected by ammonia.

Introduction Ammonia is a natural metabolite which is generated in all cells by the deamination of amino acids, purines, pyrimidines or amino sugars. Most tissues excrete excess ammonia into the blood, from which it is taken up by the liver and converted to urea. Although ammonia is generally regarded as a metabolic waste product which is toxic to the organism at high concentrations [1,2], recent evidence indicates that it may also act as a metabolic regulator. In suspensions or cultures of isolated rat hepatocytes, ammonia may accumulate under hypoxic conditions, especially in the presence of a readily deaminated substrate like glutamine [3]. Ammonia inhibits protein degradation in isolated hepatocytes [4,5] and may also be important in the control of their gluconeogenic rate [6,7]. In liver slices, ammonia inhibits citrate cycle activity [2], and in l y m p h o c y t e cultures, ammonia formed by deamination of glutamine inhibits the lectin-induced differentiation of lymphocytes into lymphoblasts [8]. Ammonia furthermore suppresses the toxic effect of diphteria toxin in mouse macrophages [9], stimulates iodine uptake in isolated thyroid cells [10], and interferes with adrenomedullary secretion [ 11 ]. To this list of biological effects can now be added the inhibition of protein secretion in isolated hepatocytes, as will be d o c u m e n t e d in the present paper.

30 Materials and Methods Isolated hepatocytes were prepared from 16-h fasted male Wistar rats by the two-step m e t h o d of collagenase perfusion [12,13], and incubated at 37°C in buffered saline as previously described [14]. [14C]Valine (Amersham CFB.75) or a mixture of 14C-labelled amino acids (protein hydrolysate, Amersham CFB. 25) was used for the incorporation of radioactivity into protein. Separation of cells from medium was achieved by washing the cells one to three times in cold buffer. Protein (intra- or extracellular) was precipitated by the addition of cold HC10~ [14]. Sampling and processing of cells for electron microscopy and quantitative morphometric analysis [15] was performed as previously described [ 5 ]. Methylamine, phlorizin and colchicine were purchased from Sigma; cytochalasin B from Aldrich Chem. Co. Results

The isolated, hepatocytes incorporated radioactive amino acids into cellular protein at an approximately constant rate for 90 min under the experimental conditions used in Fig. 1. There was no detectable increase in protein radioactivity in the extracellular medium for the first 30 min of incubation, b u t from that time onwards, labelled protein accumulated at an increasing rate {Fig. 1B). The distinct time lag and the sensitivity to inhibitors (see below) strongly suggests that the accumulation of extracellular protein radioactivity represents secretion of plasma proteins. Ammonia (10 mM NH4C1) had little effect on over-all protein labelling (intracellular protein radioactivity, fig. 1A), b u t the secretion of labelled protein into the extracellular medium was strongly retarded (Fig. 1B). Fig. 2 shows a dose vs. response curve for the effect of ammonia on the amount of radioactive protein accumulated intra- and extracellularly in 60 min. The labelling of intracellular protein, which constituted a b o u t 95% of the total protein radioactivity, was unaffected by NH4C1 at least up to a concentration of 6 mM, whereas the secretion of labelled extracellular protein was reduced by 75% at this ammonium concentration. Fig. 2 also shows that the ammonia analogue methylamine (methyl-ammonia, ref. 16) inhibited protein secretion to the same extent as did ammonia itself. Table I compares the effects of various treatments on protein secretion. In this experiment the hepatocytes were pre-labelled with [14C]valine for 60 min; then subjected to a series of short incubations in isotope-free media (chase). The total duration of this chase (50 min) was well in excess of the minimum time required for plasma protein synthesis and secretion (30 min, Fig. 1), and subsequent release of labelled protein would therefore presumably reflect rather late stages in the protein secretory process. The inhibitory effects of ammonia and methylamine were clearly seen in this situation as well. Colchicine, which is known to inhibit the translocation of secretory vesicles from the Golgi apparatus to the liver cell surface [17], was also strongly inhibitory, whereas cytochalasin B caused only a slight inhibition. Compete anoxia (incubation of the cells in a N2 atmosphere) inhibited protein secretion dramatically,

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32 TABLE I EFFECT OF VARIOUS INHIBITORS ON PROTEIN SECRETION IN ISOLATED RAT HEPATOCYTES T h e cells wez'e p r e i n c u b a t e d f o r 6 0 r a i n a t 3 7 ° C w i t h [ 1 4 C ] v a l i n e (0.6 p C i / m l ) , w a s h e d o n c e at 0 ° C , t h e n i n c u b a t e d five t i m e s 10 m i n a t 3 7 ° C , w i t h r e n e w a l o f t h e m e d i u m ( s u s p e n s i o n b u f f e r ) e a c h t i m e . T h e cells w e r e f i n a l l y i n c u b a t e d at 3 7 ° C f o r 6 0 r a i n in t h e p r e s e n c e o f c ~ , c l o h e x i m i d e (1 r a M ) a n d t h e i n h i b i t o r i n d i c a t e d . A f t e r r e m o v a l o f t h e cells b y c e n t r i f u g a t i o n , t h e c o n t e n t o f r a d i o a c t i v e p r o t e i n in t h e m e d i u m w a s d e t e r m i n e d , a n d t a k e n as a m e a s u r e o f p r o t e i n s e c r e t i o n ( e x p r e s s e d o n a cellular w e t w e i g h t basis). E a c h v a l u e is t h e m e a n -+ S.E. o f f o u r cell s a m p l e s . T h e s i g n i f i c a n c e o f t h e d i f f e r e n c e f r o m c o n t r o l was c a l c u l a t e d b y t h e t-test. Treatment

Protein secretion ( c p m / m g p e r h)

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indicating an energy requirement for the secretory process. Phlorizin, an inhibitor of sugar transport, had no significant effect, and high concentrations of the amino acids leucine and isoleucine were also ineffective (Table I). In order to see whether the inhibition of protein secretion by ammonia was accompanied by any morphological alterations in the structural elements of the secretory apparatus, electron micrographs of untreated and ammonia-treated cells were evaluated b y quantitative morphometric methods [5,15]. In

Fig. 3. S e c r e t o r y vesicles c o n t a i n i n g d e n s l y s t a i n i n g l i p o p r o t e i n p a r t i c l e s in (a) n o n - i n c u b a t e d r a t liver cells~ ( b ) cells i n c u b a t e d f o r 1 h w i t h o u t a d d i t i o n s : a n d (c) cells i n c b u b a t e d f o r 1 h w i t h 10 m M N H 4 C I . N o t i c e vesicle s w e l l i n g in c. X 3 5 0 0 0 .

33 T A B L E II M O R P H O M E T R I C A N A L Y S I S O F T H E C O N T E N T S O F P R O T E I N S E C R E T O R Y ( V E R Y LOW D E N S I T Y L I P O P R O T E I N - C O N T A I N I N G ) V E S I C L E S IN I S O L A T E D R A T H E P A T O C Y T E S Values r e p r e s e n t m e a n s -+ S.E. of five cell s a m p l e s . Experimental treatment

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untreated cells, the number of secretory vesicles (identified by their content of densely staining lipoprotein particles) in the cytoplasm was strongly diminished after I h of incubation (Fig. 3, Table II). In ammonia-treated cells, on the other hand, there was no significant change in the number of secretory vesicles. The fraction of the cell volume occupied by secretory vesicles was significantly increased after ammonia treatment, indicating that some swelling of the vesicles had occurred (Fig. 3, Table II). A similar swelling t o o k place in the lysosomes of ammonia-treated cells, as previously reported [5].

Discussion

Isolated hepatocytes have previously been found capable of synthesizing and secreting plasma proteins in vitro [18--26]. Labelled albumin was shown to appear in the incubation medium after a distinct time lag [17], corresponding roughly to the time needed for synthesis, concentration in Golgi vesicles, and transport through the hepatic endoplasmic reticulum in vivo [27,28]. The 30 min lag observed in the present study most probably reflects the minimum transit time for passage of proteins in general through the secretory apparatus of isolated hepatocytes, The complete lack of radioactive protein release before 30 min indicates that there is no leakage of newly synthesized protein from these cells. The ability of ammonia and its analogue methylamine to inhibit general protein secretion, without affecting protein synthesis, ought to make these compounds valuable tools in the study of the secretory process. Ammonia has also been found to inhibit the secretion of an individual enzyme, lecithin-cholesterol acyltransferase (EC 2.3.1.43) in h e p a t o c y t e suspensions (Nordby, G., unpublished observations). The mechanism of action of ammonia would be expected to be different from that of colchicine, which is believed to interfere with contractile microtubuli involved in the m o v e m e n t of secretory vesicles [17]. The inhibitory effect of colchicine on protein secretion in isolated hepatocytes, incidentally, supports the contention that the secretory behaviour of these cells is similar to the in vivo state. The relative lack of effect of cytochalasin B, which inhibits protein secretion in some other cell types [29,30], can therefore probably be regarded as circumstantial evidence for the non-involvement of microfilaments in the secretory process in isolated hepatocytes.

34 Cytochalasin B has previously been shown to induce extensive zeiosis (appearance of globular surface protrusions due to interference with contractile surface microfilaments) in isolated hepatocytes [ 31], demonstrating that these cells are sensitive to the action of the drug. The electron micrographs of ammonia-treated and untreated hepatocytes suggest a possible mechanism for the inhibition of protein secretion by ammonia. In the control cells, there was a pronounced decline in the content of secretory vesicles during the course of incubation. This might indicate that secretory processes initiated in vivo are completed in vitro, b u t that the rate of formation of new secretory products is diminished. A reduced rate of plasma protein formation would be in accordance with the low rate of protein synthesis found in these isolated hepatocytes [4]. In cells prepared from fed rats, the rate of synthesis of secretory proteins was found by Weigand et al. [18,19] to be oaly 10% of the in vivo rate, and the rate of protein synthesis in fasted rats is even lower [32]. In ammonia-treated cells, the content of secretory vesicles did n o t diminish during incubation, suggesting that the inhibition of protein secretion might be due to an arrest in the movement and peripheral discharge (and disappearance) of the vesicles. The pronounced swelling of the secretory vesicles could indicate some kind of structural impairment, which might render the vesicles unable to move through the cytoplasm. It was previously shown that the inhibition of protein degradation by ammonia in isolated hepatocytes was accompanied by a swelling of secondary lysosomes [5], presumably due to the selective accumulation of this weak base in the acidic lysosomal interior, with attendant influx of water [5,33]. Since the Golgi apparatus, the endoplasmic reticulum and the lysosomes have been assumed to constitute a system of closely related membrane elements (the GERL system, ref. 34), functional similarities should perhaps n o t be unexpected. If the interior of secretory vesicles were acidic, ammonia could accumulate by the same mechanism as was proposed for lysosomal vacuolization [5], resulting both in vesicle swelling and retardation of vesicle translocation. In the adrenal gland, the chromaffin vesicles responsible for catecholamine secretion have been shown to be acidic (pH 5.5), and capable of accumulating the ammonia analogue methylamine [35]. A similar accumulation of ammonia in the neurosecretory granules of nerve cell end'~ngs could possibly interfere with neural and neuromuscular communication, and thus explain the toxic effect of ammonia observed in vivo [1,2]. Why protein secretory vesicles should be acidic is another question, b u t it could be related to the fact that most secretory proteins are formed by proteolytic cleavage of larger precursors. If the proteases involved have a low pH optimum, like the lysosomal proteases, the postulation of an acidic environment in the protein secretory apparatus could be justified. Acknowledgements The excellent technical assistance of Frances Dodman, Barbara Schiller and Anne E. Solheim is gratefully ack_~owledged. The work was supported by the Norwegiaa Cancer Society.

35

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