Printed in Sweden Copyright ~) 1977 by Academic Press, Inc. All rights o f reproduction in any form reserved 1SSN 0014-4827
Experimental Cell Research 108 (1977) 23-30
AMMONIUM
ION INTERFERES
DEGRADATION CULTURES
WITH THE LYSOSOMAL
OF GLYCOSAMINOGLYCANS OF HUMAN
IN
GLIAL CELLS
B. GLIMELIUS,1,2 B. WESTERMARK 2 and A. WASTESON 3.
1Institute of Pathology, 2Wallenberg Laboratory and 3Institute of Medical and Physiological Chemistry, University of Uppsala, S-Uppsala 75122, Sweden
SUMMARY Supplementation of human normal glial cells with NH4C1 led to marked effects on the metabolism of sulphated glycosaminoglycans (GAG). Sulphated GAG accumulated intracellularly in the presence of NH4CI. Further, the degradation of sulphated GAG was partially inhibited. These phenomena were accompanied by an accumulation of lysosomes within the glial ceils. The characteristics of NH4Cl-treated cells were thus similar to those displayed by cultured cells from patients with mucopolysaccharidosis.
It was recently demonstrated that the administration of ammonium ion to isolated rat hepatocytes caused an inhibition of the endogenous proteolysis in the cells [1]. The inhibition was reversible and considered not to be due to non-specific toxic effects. The mechanism(s) by which this inhibitory effect was mediated has not been resolved, but in a more recent paper [2] it was correlated with lysosomal vacuolization. The effect was thought to be due either to a direct effect on the lysosomal proteases or as an indirect result of changes in the intralysosomal compartment. The present study was undertaken to see whether ammonium ion may exert a general influence on catabolic processes, not limited to the degradation of protein. Therefore * Present address: National Institute of Arthritis, Metabolism and Digestive Diseases, NIH, Bethesda, MD 20014, USA.
we examined the turnover of sulphated glycosaminoglycans (GAG) in cultures of human glial cells in the presence of ammonium chloride. These cells produce sulphated glycosaminoglycans, partly released to the surrounding medium, partly remaining associated with the cells [3]. The present experiments demonstrate that ammonium ion interferes strongly with the turnover of the latter fraction, probably by disturbing the lysosomal function. MATERIAL AND M E T H O D S Cell line and culture conditions A normal gliai cell line (U-787CG) was initiated from human brain tissue as described [4]. It was maintained in Eagle's MEM [5] supplemented with 10% calf serum and antibiotics (100 IE penicillin; 50/xg streptomycin; 1.25 /xg amphotericin B per ml) at 37°C in humidified air, containing 5 % CO2. For experimental purposes cells were pooled in routine medium and seeded sparsely in 50 mm plastic dishes (Nunclone, Roskilde, Denmark). The cells were grown with routine medium changes twice a week; after approx. 10 days they had reached a stationary growth phase [6].
Exp Cell Res 108 (1977)
24
Glimelius, Westermark and Wasteson
Incorporation experiments Dense, stationary cultures were used for all experiments. The medium was changed to 5 ml of Ham's F-10 [7], having a reduced concentration of inorganic sulphate (MgSO4 substituted with an equivalent amount of MgC12) and supplemented with 10% calf serum and 10 /~Ci/ml of inorganic carrier-free [35S]sulphate (Radiochemical Centre, Amersham, UK). The incorporation of asSO4 was allowed to proceed for various periods of time in the presence or absence of ammonium chloride added at the specified concentrations in 50/zl of aqueous solution.
Transmission electron microscopy (TEM) The cells were fixed in 2 % glutaraldehyde in 0.1 M Nacacodylate-HC1 buffer with 0.1 M sucrose (pH 7.2; total osmolality 510 mOsm, vehicle osmolality 300 mOsm) [10] and post-fixed in 2% osmium tetroxide in s-colloidine buffer. Further preparation of the cells was performed as reported in detail [11]. Sections were studied in a Jeol 100-C electron microscope run at 60 kV.
Histochemistry P u l s e - c h a s e experiments Cultures were incubated with 35SO4 for 48 h as described above in the absence of ammonium chloride. After 48 h the medium was removed and the culture dishes washed gently at 37°C three times with 2 ml of Ham's (ordinary) F-10 equilibrated with 5 % CO2. The cultures were then incubated for various periods of time with 5 ml of fresh CO2-equilibrated F-10 medium with 10 % calf serum in the presence or absence of ammonium chloride.
Termination of cultures Cultures used in incorporation or pulse-chase experiments were harvested as summarized below; details of the procedure will be described elsewhere (Glimelius et al., unpublished). The medium fraction was collected separately. After three washes the cells were subjected to a mild trypsinization (50 t~g/ml of trypsin (Difco), 60 min); the solubilized material, recovered by centrifugation, was denoted trypsin fraction. The twice washed cell pellet was denoted cell fraction.
Quantitation o f macromolecular 3~S-GAG 35S-GAG in medium, trypsin and cell fractions, respectively, was quantitated by precipitation with cetylpyridinium chloride (CPC, AB Recip, Stockholm, Sweden) on filter paper essentially as described previously [8]. However, the concentration of NaC1 in the CPC-solutions was 0.05 M instead of 0.3 M in order to increase the precipitability with CPC. Further, the cell fractions were treated for 4 h at 37°C with Protease type V (1 mg/ml) (Sigma Chemical Co., St Louis, Mo) and Triton X-100 (0.1%, v/v) prior to analysis.
Gel chromatography Samples of medium and cell fractions were mixed with 0.5 mg of carrier chondroitin sulphate and treated with crystalline papain for 15 h at 6&C [9]. They were then subjected to gel chromatography on Sephadex G-100 (74×1.2 cm) or Sephadex G-25 (190× 1.0 cm) eluted with 1 M NaC1. Effluent fractions were analysed by liquid scintillation counting.
Exp CellRes 108 (1977)
The presence of acid phosphatase activity was demonstrated after incubation in a modified Gomori medium as described [10].
RESULTS
Effect of ammonium ion on cell morphology (fig. 1) Addition of NH4C1 to the glial cell cultures lead to a pronounced cytoplasmic vacuolization within 1-2 h. TEM of treated cultures showed an enlargement of the secondary lysosomes without any effect on the Golgi stack or on the coated vesicles forming from the Golgi apparatus (probably primary lysosomes). A Gomori reaction showed a great increase in the amount and size of acid phosphatase-containing granules. Since this enzyme is exclusively associated with lysosomes, Golgi elements and related structures [10], it was confirmed that exposure to ammonium ion caused Fig. I. Morphological effects by ammonia. Note the presence of large cytoplasmic vacuoles in (b) NH4C1treated cultures (8 mM, 3 h) not present in control cells (a). Phase contrast. The TEM pictures (c, d) are from cultures exposed to 8 mM NH4C1 for 24 h. Apart from greatly enlarged secondary lysosomes with the electron-dense content surrounded by a zone of electron lucidity, the morphology was essentially undisturbed. Note particularly the well preserved structure of the mitochondria in (c) and part of the Golgi stack in (d). The arrowed, coated vesicles forming from the Golgi apparatus probably represent primary lysosomes and do not show enlargement. If cells were incubated in a Gomori medium a sparse granular pattern of demonstrable acid phosphatase activity was obtained in controls (e). A great increase was found in NH4Cl-treated cultures, 8 mM, 3 h (f).
Ammonia inhibition of glycosaminoglycan degradation
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Fig. 2. Abscissa: NHaC1 conc. (mM); ordinate: a~S-
radioactivity (cpm × 10-3). 85S-GAG in A, medium; II, trypsin; ©, and cell fractions of glial cell cultures after incubation (24 h) with 35SO4 in the presence of various concentrations of NH4C1.
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Fig. 4. Abscissa: incubation time (hours); ordinate:
35S-radioactivity (cpm× 10-4). Turnover of 35S-GAG in ©, O, the cell fraction and r-q, II, decrease of CPC-precipitable radioactivity in whole (sum of medium, trypsin and cell fractions) glial cell cultures. The cultures were prelabelled with a~SO4 (48 h) and incubated for the indicated times in fresh medium without 35SO4, in the r~, ©, absence or m, O, presence of 8 mM NH4C1. Fig. 5. Abscissa: NH4C1 conc. (mM); ordinate: loss of CPC-precipitable radioactivity (%). Effect of NH4CI on the degradation of 35S-GAG in glial cell cultures. After prelabelling with 3sSO~ (48 h) the cultures were incubated in fresh medium, without 35SO4, for 10 h in the presence of various concentrations of NH4CI. Degradation was estimated as the loss of CPC-precipitable 35S-GAG and expressed as percentage of the loss in a non-treated control.
morphological changes in the secondary lysosomes. Maximal cellular vacuolization was found approx. 12-24 h after a single dose of NH4C1; then a slow decline took place. After 3 days of treatment an abnormal vacuolization was still visible. Substitution with fresh medium without NH4C1, led to a rapid decrease in the vacuoles, most of them disappearing within 24 h. Apart Effect of ammonium ion on the from accumulation of vacuoles, no effect on incorporation of 3~S04 into GAG cell morphology was seen after the addition of different pools of up to 16 mM NH4CI to the nutrient meAddition of NI-I4C1 to the glial cell cultures dium. The pH of the nutrient medium recaused a marked increase in ~sS-GAG of the mained at 7.4 (7.40-7.41) in the presence of cell fraction of glial cell cultures (fig. 2). At 4 mM of NH4C1. At 16 mM of NH4C1 a 4 mM NH4C1, affording maximal effect, the minimal drop in pH was observed (pH increase was 2-3-fold (range 1.7-3.3); 7.38-7.39). higher concentrations of NH4C1 failed to produce further accumulation of intracel2 3 lular 35S-GAG under the present experimental conditions. In contrast, the 3~S-GAG of the medium fraction (representing secreted material) and the trypsin fraction (representing cell surface associated material), respectively, were little affected by low doses of NH4C1 (0.5 m M - 1 . 0 mM) 6 12 24 48 (fig. 2). Since these fractions accounted for Fig. 3. Abscissa: incubation time (hours); ordinate: 35S-radioactivity (cpm× ]0-% the major part (more than 95 %) of the 3~S~sS-GAG in the cell fraction of glial cell cultures after GAG recovered after 24 h incubation, the incubation for various periods of time with 3~SO4 in the O, absence; or A, presence of NH4CI (4 mM). net production of 35S-GAG in the glial cell Exp Cell Res 108 (1977)
Ammonia inhibition of glycosaminoglycan degradation
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Fig. 6. Abscissa: effluent volume (ml); ordinate: 35S radioactivity (cpm).
Gel chromatography on Sephadex G-100, of asS-labelled products, released from 35S-labelled glial cells. After pre-labelling with 35S04 (48 h) the cultures were incubated for 10 h in fresh medium without
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35S04, in (left) the absence o r (right) the presence of NH4CI (8 mM). Aliquots of the medium were chromatographed after digestion with papain. 30
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cultures was not significantly disturbed at these low concentrations of NH4C1. At increasing corkcentrations of NH4C1, however, a successive drop in the 35S-GAG of both the medium and the trypsin fractions was observed; at 16 mM of NH4C1 the net production was less than half of that of control cultures. The increased rate of intracellular accumulation of 35S-GAG after exposure to NH4C1, was demonstrable only after 1224 h of incubation; after 6 h NH4Cl-supplemented and control cultures did usually not differ (fig. 3). Thus, the intracellular accumulation of 35S-GAG in response to NH4C1 was somewhat delayed in comparison with the appearance of cytoplasmic vacuolization. After 24-48 h both incorporation curves tended to level off; at this stage the NH4Cl-treated cultures showed 2-3 times more cellular 35S-GAG than did the non-treated cultures. Gel chromatography on Sephadex G-100 revealed that a major part of the increase in cellular 35S-GAG was in a low-molecular fraction, included in the gel; however, a significant increase was also demonstrable in high-molecular (excluded) fraction of 35SGAG (not shown).
90
Effect of ammonium ion on the turnover of 35S-GAG Previous pulse-chase experiments have shown that the 35S-GAG associated with the cell surface of the glial cells (trypsin fraction) is metabolized both by degradation (end product inorganic 3ss04) and by direct release in macromolecular form to the medium (unpublished observations). As can be seen from fig. 4 the degradative route leads to a decrease, with time, in the total amount of CPC-precipitable 35S-GAG of the culture. The presence of NH4C1, however, decreased the rate of degradation (fig. 4). This effect was dependent on the dose of NH4C1; at 8-10 mM the loss of CPC-precipitable 3%-GAG was only 50 % of that found under control conditions (fig. 5). It should be pointed out that under either conditions the recovery of 35S-radioactivity was constant, i.e. the loss of CPC-precipitable radioactivity was accompanied by an equal gain in non-precipitable 35S. Gel chromatography on Sephadex G-100 of the products recovered from the growth medium (after 10 h of chase) confirmed the results; the amount of retarded, lowmolecular components was considerably smaller in NH4Cl-supplemented than in Exp Cell Res 108 (1977)
28
Glimelius, Westermark and Wasteson
700
Fig. 7, Abscissa: effluent volume (ml); ordinate: asS radioactivity (cpm). Gel chromatography on Sephadex G-100 of asS products recovered from the cell fractions of asS-labelled glial cell cultures. After prelabelling with a~SO4 (48 h) the cultures were incugated for 10 h in fresh medium, without a5SO4, in (left) the absence or (right) the presence of NH4CI (8 raM).
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control cultures (fig. 6). The asS-labelled products were of similar size in both cases, migrating with the void volume or the total volume of the column (fig. 6). The latter component migrated as inorganic sulphate on Sephadex G-25 (not shown). The absence of intermediate size products (oligosaccharides) suggests that inorganic sulphate was the only labelled degradation product released to the medium under either of the experimental conditions. Since approximately equal amounts of radioactivity were eluted with the void volume of the column in both cases (fig. 6), it was concluded that the release of macromolecular asS-GAG from the surface-associated fraction to the medium was essentially unaffected by the presence of NH4Cl. The low rate of degradation in NH4C1treated cultures was accompanied by a delay in the turnover of asS-GAG in the cell fraction (fig. 4). Thus, whereas control cultures showed a decrease in CPC-precipitable radioactivity starting promptly at the beginning of the chase periods, NH4C1 supplemented cultures showed, in contrast, an initial accumulation of asS-GAG. This finding was interpreted as an inability of the cell to degrade asS-GAG transferred from the membrane-associated compartment to the cell interior: it was estimated Exp Cell Res 108 (1977)
9'0
that during a 10 h chase period under control conditions approx. 40 % of the trypsin fraction (which is considerably larger than the cellular fraction) is metabolized via intracellular degradation (unpublished observations). Only after about 10 h a decline in radioactivity was demonstrable in the cell fraction of NH4Cl-treated (8 mM) cultures (fig. 4). An analysis of the size distribution of the cellular material (after 10 h of chase) revealed a relative increase in 35S-labelled products of intermediate size in NH4Cl-treated as compared to nontreated cultures (fig. 7). Thus, the gel chromatography patterns of cellular asS-GAG were similar to those afforded by the incorporation experiments described above.
DISCUSSION The present experiments indicate that the presence of ammonium chloride in cultures of glial cells has a profound influence on the metabolism of sulphated glycosaminoglycans (GAG) in these cells. Thus, the incorporation of 35SO4 into the cellular fraction of GAG showed a dose-dependent increase after the addition of NH4C1 (fig. 2). Since the intracellular fraction may represent a degradation pool [12], the enlarged cell fraction may reflect a defective de-
Ammonia inhibition of glycosaminoglycan degradation gradation of sulphated GAG. The increase in cellular 35S-GAG was not part of a general stimulation of GAG-biosynthesis; rather, NH4C1 at concentrations above 2 mM showed an inhibitory effect on the net production of 3~S-GAG (fig. 2). Furthermore, the pulse-chase experiments demonstrated a delay in the turnover of the cell fraction in the presence of NH4C1. The NH4Cl-treated cultures showed an initial rise in the amount of cellular 3~S-GAG in contrast to the control cultures, where the cellular fraction decreased promptly at the beginning of the chase period. It was concluded that NH4C1 impaired the degradation of 35S-GAG. Further support for this conclusion was obtained by the fact that less low molecular weight degradation products (inorganic a5SO4) appeared in the chase medium of NH4Cl-treated cultures than of non-treated ones, as demonstrated by precipitation with CPC or by gel chromatography (fig. 6). Seglen has previously shown that the catabolism of protein in cultured rat hepatocytes is restrained by ammonia [1]. The degradation of epidermal growth factor by human fibroblasts may be similarly affected by the presence of ammonium ion [13]. The present experiments extend these results to include an inhibitory effect of NH4C1 on the degradation of sulphated GAG. This observation may suggest that NH4CI has a general influence on degradative processes in the cell, not restricted to one particular kind of substance. Morphological findings lend support for this view: the accumulation of secondary lysosomes within glial cells and rat liver cells [2], after supplementation with NH4C1 suggests that the latter may interfere with the lysosomal function in general. The restrained degradation of GAG in conjunction with the accumulation of lyso-
29
somes in NH4Cl-supplemented glial cells reminds of the characteristics of cultured fibroblasts from patients with mucopolysaccharidosis [12]. Previously, a similar mucopolysaccharidosis-like picture has been induced in cultured fibroblasts by raising the pH of the growth medium [14, 15]. The elevated pH of the medium may have hampered the concentration of protons within the lysosomes [16], thereby impairing the degradation of GAG. The mechanism by which NH4C1 exerted an effect on the catabolism of GAG in glial cells is not known; however, its effect on the pH of the nutrient medium was negligible. Weak bases such as basic dyes and chloroquine may be rapidly taken up and concentrated within the lysosomes [17] causing vacuolization and possibly an increase in lysosomal pH. The accumulation of chloroquine and similar derivatives within the lysosomes of human fibroblasts is accompanied by a restrained degradation of GAG and an abnormal morphology, simulating lysosomal storage diseases [18]. Therefore it seems possible that NH4C1 and chloroquine interferes with the lysosomal function by a common mechanism, as has been suggested [2, 13]. This work was supported by the Swedish Medical Research Council (13X-4486 and 13X-2309), the Swedish Cancer Society (689-B75-01X) and Konung Gustav V:s 80-~rsfond. The skilful technical assistance of Mrs F. Carlsson, Miss Y. 0hgren, Mrs G. Fjellstrrm and Miss M. Lindstrrm is gratefully acknowledged.
REFERENCES 1. Seglen, P O, Biochem biophys res commun 66 (1975) 44. 2. Seglen, P O & Reith, A, Exp cell res 100 (1976) 276. 3. Norling, B, Glimelius, B, Westermark, B & Wasteson, ~, Abstr Vth meeting Eur fed conn tiss clubs (1976) 25. 4. Pontrn, J & Macintyre, E, Acta pathol microbiol Scand 74 (1968) 465. Exp CelI Res 108 (1977)
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5. Eagle, H, Science 130 (1959) 432. 6. Pont6n, J, Westermark, B & Hugosson, R, Exp cell res 58 (1969) 313. 7. Ham, R G, Proc natl acad sci US 53 (1965) 288. 8. Wasteson, A, Uthne, K & Westermark, B, Biochemj 136 (1973) 1069. 9. Lindahl, U, Biickstr6m, G, Jansson, L & Hall6n., A, J biol chem 248 (1973) 7234. 10. Brunk, U & Ericsson, J L E, Histochem j 4 (1972) 349. I1. Brunk, U, Ericsson, J L E, Pont6n, J & Westermark, B, Exp cell res 67 (1971) 407. 12. Fratantoni, J C, Hall, C W & Neufeld, E F, Proc natl acad sci US 60 (1968) 699. 13. Carpenter, G & Cohen, S, J cell biol 71 (1976) 159.
Exp CellRes 108 (1977)
14. Lie, S O, McKusick, V A & Neufeld, E F, Proc natl acad sci US 69 (1972) 2361. 15. Lie, S O, Schofield, B H, Taylor, H A & Doty, S B, Pediat res 7 (1973) 13. 16. Mego, J L, Farb, R M & Barkes, J, Biochem j 128 (1972) 763. 17. de Duve, C, de Barsy, T, Poole, B, Trouet, A, Tulkens, P & Hoof, F, Biochem pharmacol 23 (1974) 2495. 18. Lie, S O & Schofield, B, Biochem pharmacol 22 (1973) 3109. Received December 23, 1976 Accepted February 22, 1977
Experimental Cell Research 108 (1977) 23-30
AMMONIUM
ION INTERFERES
DEGRADATION CULTURES
WITH THE LYSOSOMAL
OF GLYCOSAMINOGLYCANS OF HUMAN
IN
GLIAL CELLS
B. GLIMELIUS,1,2 B. WESTERMARK 2 and A. WASTESON 3.
1Institute of Pathology, 2Wallenberg Laboratory and 3Institute of Medical and Physiological Chemistry, University of Uppsala, S-Uppsala 75122, Sweden
SUMMARY Supplementation of human normal glial cells with NH4C1 led to marked effects on the metabolism of sulphated glycosaminoglycans (GAG). Sulphated GAG accumulated intracellularly in the presence of NH4CI. Further, the degradation of sulphated GAG was partially inhibited. These phenomena were accompanied by an accumulation of lysosomes within the glial ceils. The characteristics of NH4Cl-treated cells were thus similar to those displayed by cultured cells from patients with mucopolysaccharidosis.
It was recently demonstrated that the administration of ammonium ion to isolated rat hepatocytes caused an inhibition of the endogenous proteolysis in the cells [1]. The inhibition was reversible and considered not to be due to non-specific toxic effects. The mechanism(s) by which this inhibitory effect was mediated has not been resolved, but in a more recent paper [2] it was correlated with lysosomal vacuolization. The effect was thought to be due either to a direct effect on the lysosomal proteases or as an indirect result of changes in the intralysosomal compartment. The present study was undertaken to see whether ammonium ion may exert a general influence on catabolic processes, not limited to the degradation of protein. Therefore * Present address: National Institute of Arthritis, Metabolism and Digestive Diseases, NIH, Bethesda, MD 20014, USA.
we examined the turnover of sulphated glycosaminoglycans (GAG) in cultures of human glial cells in the presence of ammonium chloride. These cells produce sulphated glycosaminoglycans, partly released to the surrounding medium, partly remaining associated with the cells [3]. The present experiments demonstrate that ammonium ion interferes strongly with the turnover of the latter fraction, probably by disturbing the lysosomal function. MATERIAL AND M E T H O D S Cell line and culture conditions A normal gliai cell line (U-787CG) was initiated from human brain tissue as described [4]. It was maintained in Eagle's MEM [5] supplemented with 10% calf serum and antibiotics (100 IE penicillin; 50/xg streptomycin; 1.25 /xg amphotericin B per ml) at 37°C in humidified air, containing 5 % CO2. For experimental purposes cells were pooled in routine medium and seeded sparsely in 50 mm plastic dishes (Nunclone, Roskilde, Denmark). The cells were grown with routine medium changes twice a week; after approx. 10 days they had reached a stationary growth phase [6].
Exp Cell Res 108 (1977)
24
Glimelius, Westermark and Wasteson
Incorporation experiments Dense, stationary cultures were used for all experiments. The medium was changed to 5 ml of Ham's F-10 [7], having a reduced concentration of inorganic sulphate (MgSO4 substituted with an equivalent amount of MgC12) and supplemented with 10% calf serum and 10 /~Ci/ml of inorganic carrier-free [35S]sulphate (Radiochemical Centre, Amersham, UK). The incorporation of asSO4 was allowed to proceed for various periods of time in the presence or absence of ammonium chloride added at the specified concentrations in 50/zl of aqueous solution.
Transmission electron microscopy (TEM) The cells were fixed in 2 % glutaraldehyde in 0.1 M Nacacodylate-HC1 buffer with 0.1 M sucrose (pH 7.2; total osmolality 510 mOsm, vehicle osmolality 300 mOsm) [10] and post-fixed in 2% osmium tetroxide in s-colloidine buffer. Further preparation of the cells was performed as reported in detail [11]. Sections were studied in a Jeol 100-C electron microscope run at 60 kV.
Histochemistry P u l s e - c h a s e experiments Cultures were incubated with 35SO4 for 48 h as described above in the absence of ammonium chloride. After 48 h the medium was removed and the culture dishes washed gently at 37°C three times with 2 ml of Ham's (ordinary) F-10 equilibrated with 5 % CO2. The cultures were then incubated for various periods of time with 5 ml of fresh CO2-equilibrated F-10 medium with 10 % calf serum in the presence or absence of ammonium chloride.
Termination of cultures Cultures used in incorporation or pulse-chase experiments were harvested as summarized below; details of the procedure will be described elsewhere (Glimelius et al., unpublished). The medium fraction was collected separately. After three washes the cells were subjected to a mild trypsinization (50 t~g/ml of trypsin (Difco), 60 min); the solubilized material, recovered by centrifugation, was denoted trypsin fraction. The twice washed cell pellet was denoted cell fraction.
Quantitation o f macromolecular 3~S-GAG 35S-GAG in medium, trypsin and cell fractions, respectively, was quantitated by precipitation with cetylpyridinium chloride (CPC, AB Recip, Stockholm, Sweden) on filter paper essentially as described previously [8]. However, the concentration of NaC1 in the CPC-solutions was 0.05 M instead of 0.3 M in order to increase the precipitability with CPC. Further, the cell fractions were treated for 4 h at 37°C with Protease type V (1 mg/ml) (Sigma Chemical Co., St Louis, Mo) and Triton X-100 (0.1%, v/v) prior to analysis.
Gel chromatography Samples of medium and cell fractions were mixed with 0.5 mg of carrier chondroitin sulphate and treated with crystalline papain for 15 h at 6&C [9]. They were then subjected to gel chromatography on Sephadex G-100 (74×1.2 cm) or Sephadex G-25 (190× 1.0 cm) eluted with 1 M NaC1. Effluent fractions were analysed by liquid scintillation counting.
Exp CellRes 108 (1977)
The presence of acid phosphatase activity was demonstrated after incubation in a modified Gomori medium as described [10].
RESULTS
Effect of ammonium ion on cell morphology (fig. 1) Addition of NH4C1 to the glial cell cultures lead to a pronounced cytoplasmic vacuolization within 1-2 h. TEM of treated cultures showed an enlargement of the secondary lysosomes without any effect on the Golgi stack or on the coated vesicles forming from the Golgi apparatus (probably primary lysosomes). A Gomori reaction showed a great increase in the amount and size of acid phosphatase-containing granules. Since this enzyme is exclusively associated with lysosomes, Golgi elements and related structures [10], it was confirmed that exposure to ammonium ion caused Fig. I. Morphological effects by ammonia. Note the presence of large cytoplasmic vacuoles in (b) NH4C1treated cultures (8 mM, 3 h) not present in control cells (a). Phase contrast. The TEM pictures (c, d) are from cultures exposed to 8 mM NH4C1 for 24 h. Apart from greatly enlarged secondary lysosomes with the electron-dense content surrounded by a zone of electron lucidity, the morphology was essentially undisturbed. Note particularly the well preserved structure of the mitochondria in (c) and part of the Golgi stack in (d). The arrowed, coated vesicles forming from the Golgi apparatus probably represent primary lysosomes and do not show enlargement. If cells were incubated in a Gomori medium a sparse granular pattern of demonstrable acid phosphatase activity was obtained in controls (e). A great increase was found in NH4Cl-treated cultures, 8 mM, 3 h (f).
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Fig. 2. Abscissa: NHaC1 conc. (mM); ordinate: a~S-
radioactivity (cpm × 10-3). 85S-GAG in A, medium; II, trypsin; ©, and cell fractions of glial cell cultures after incubation (24 h) with 35SO4 in the presence of various concentrations of NH4C1.
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Fig. 4. Abscissa: incubation time (hours); ordinate:
35S-radioactivity (cpm× 10-4). Turnover of 35S-GAG in ©, O, the cell fraction and r-q, II, decrease of CPC-precipitable radioactivity in whole (sum of medium, trypsin and cell fractions) glial cell cultures. The cultures were prelabelled with a~SO4 (48 h) and incubated for the indicated times in fresh medium without 35SO4, in the r~, ©, absence or m, O, presence of 8 mM NH4C1. Fig. 5. Abscissa: NH4C1 conc. (mM); ordinate: loss of CPC-precipitable radioactivity (%). Effect of NH4CI on the degradation of 35S-GAG in glial cell cultures. After prelabelling with 3sSO~ (48 h) the cultures were incubated in fresh medium, without 35SO4, for 10 h in the presence of various concentrations of NH4CI. Degradation was estimated as the loss of CPC-precipitable 35S-GAG and expressed as percentage of the loss in a non-treated control.
morphological changes in the secondary lysosomes. Maximal cellular vacuolization was found approx. 12-24 h after a single dose of NH4C1; then a slow decline took place. After 3 days of treatment an abnormal vacuolization was still visible. Substitution with fresh medium without NH4C1, led to a rapid decrease in the vacuoles, most of them disappearing within 24 h. Apart Effect of ammonium ion on the from accumulation of vacuoles, no effect on incorporation of 3~S04 into GAG cell morphology was seen after the addition of different pools of up to 16 mM NH4CI to the nutrient meAddition of NI-I4C1 to the glial cell cultures dium. The pH of the nutrient medium recaused a marked increase in ~sS-GAG of the mained at 7.4 (7.40-7.41) in the presence of cell fraction of glial cell cultures (fig. 2). At 4 mM of NH4C1. At 16 mM of NH4C1 a 4 mM NH4C1, affording maximal effect, the minimal drop in pH was observed (pH increase was 2-3-fold (range 1.7-3.3); 7.38-7.39). higher concentrations of NH4C1 failed to produce further accumulation of intracel2 3 lular 35S-GAG under the present experimental conditions. In contrast, the 3~S-GAG of the medium fraction (representing secreted material) and the trypsin fraction (representing cell surface associated material), respectively, were little affected by low doses of NH4C1 (0.5 m M - 1 . 0 mM) 6 12 24 48 (fig. 2). Since these fractions accounted for Fig. 3. Abscissa: incubation time (hours); ordinate: 35S-radioactivity (cpm× ]0-% the major part (more than 95 %) of the 3~S~sS-GAG in the cell fraction of glial cell cultures after GAG recovered after 24 h incubation, the incubation for various periods of time with 3~SO4 in the O, absence; or A, presence of NH4CI (4 mM). net production of 35S-GAG in the glial cell Exp Cell Res 108 (1977)
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Fig. 6. Abscissa: effluent volume (ml); ordinate: 35S radioactivity (cpm).
Gel chromatography on Sephadex G-100, of asS-labelled products, released from 35S-labelled glial cells. After pre-labelling with 35S04 (48 h) the cultures were incubated for 10 h in fresh medium without
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35S04, in (left) the absence o r (right) the presence of NH4CI (8 mM). Aliquots of the medium were chromatographed after digestion with papain. 30
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cultures was not significantly disturbed at these low concentrations of NH4C1. At increasing corkcentrations of NH4C1, however, a successive drop in the 35S-GAG of both the medium and the trypsin fractions was observed; at 16 mM of NH4C1 the net production was less than half of that of control cultures. The increased rate of intracellular accumulation of 35S-GAG after exposure to NH4C1, was demonstrable only after 1224 h of incubation; after 6 h NH4Cl-supplemented and control cultures did usually not differ (fig. 3). Thus, the intracellular accumulation of 35S-GAG in response to NH4C1 was somewhat delayed in comparison with the appearance of cytoplasmic vacuolization. After 24-48 h both incorporation curves tended to level off; at this stage the NH4Cl-treated cultures showed 2-3 times more cellular 35S-GAG than did the non-treated cultures. Gel chromatography on Sephadex G-100 revealed that a major part of the increase in cellular 35S-GAG was in a low-molecular fraction, included in the gel; however, a significant increase was also demonstrable in high-molecular (excluded) fraction of 35SGAG (not shown).
90
Effect of ammonium ion on the turnover of 35S-GAG Previous pulse-chase experiments have shown that the 35S-GAG associated with the cell surface of the glial cells (trypsin fraction) is metabolized both by degradation (end product inorganic 3ss04) and by direct release in macromolecular form to the medium (unpublished observations). As can be seen from fig. 4 the degradative route leads to a decrease, with time, in the total amount of CPC-precipitable 35S-GAG of the culture. The presence of NH4C1, however, decreased the rate of degradation (fig. 4). This effect was dependent on the dose of NH4C1; at 8-10 mM the loss of CPC-precipitable 3%-GAG was only 50 % of that found under control conditions (fig. 5). It should be pointed out that under either conditions the recovery of 35S-radioactivity was constant, i.e. the loss of CPC-precipitable radioactivity was accompanied by an equal gain in non-precipitable 35S. Gel chromatography on Sephadex G-100 of the products recovered from the growth medium (after 10 h of chase) confirmed the results; the amount of retarded, lowmolecular components was considerably smaller in NH4Cl-supplemented than in Exp Cell Res 108 (1977)
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Glimelius, Westermark and Wasteson
700
Fig. 7, Abscissa: effluent volume (ml); ordinate: asS radioactivity (cpm). Gel chromatography on Sephadex G-100 of asS products recovered from the cell fractions of asS-labelled glial cell cultures. After prelabelling with a~SO4 (48 h) the cultures were incugated for 10 h in fresh medium, without a5SO4, in (left) the absence or (right) the presence of NH4CI (8 raM).
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control cultures (fig. 6). The asS-labelled products were of similar size in both cases, migrating with the void volume or the total volume of the column (fig. 6). The latter component migrated as inorganic sulphate on Sephadex G-25 (not shown). The absence of intermediate size products (oligosaccharides) suggests that inorganic sulphate was the only labelled degradation product released to the medium under either of the experimental conditions. Since approximately equal amounts of radioactivity were eluted with the void volume of the column in both cases (fig. 6), it was concluded that the release of macromolecular asS-GAG from the surface-associated fraction to the medium was essentially unaffected by the presence of NH4Cl. The low rate of degradation in NH4C1treated cultures was accompanied by a delay in the turnover of asS-GAG in the cell fraction (fig. 4). Thus, whereas control cultures showed a decrease in CPC-precipitable radioactivity starting promptly at the beginning of the chase periods, NH4C1 supplemented cultures showed, in contrast, an initial accumulation of asS-GAG. This finding was interpreted as an inability of the cell to degrade asS-GAG transferred from the membrane-associated compartment to the cell interior: it was estimated Exp Cell Res 108 (1977)
9'0
that during a 10 h chase period under control conditions approx. 40 % of the trypsin fraction (which is considerably larger than the cellular fraction) is metabolized via intracellular degradation (unpublished observations). Only after about 10 h a decline in radioactivity was demonstrable in the cell fraction of NH4Cl-treated (8 mM) cultures (fig. 4). An analysis of the size distribution of the cellular material (after 10 h of chase) revealed a relative increase in 35S-labelled products of intermediate size in NH4Cl-treated as compared to nontreated cultures (fig. 7). Thus, the gel chromatography patterns of cellular asS-GAG were similar to those afforded by the incorporation experiments described above.
DISCUSSION The present experiments indicate that the presence of ammonium chloride in cultures of glial cells has a profound influence on the metabolism of sulphated glycosaminoglycans (GAG) in these cells. Thus, the incorporation of 35SO4 into the cellular fraction of GAG showed a dose-dependent increase after the addition of NH4C1 (fig. 2). Since the intracellular fraction may represent a degradation pool [12], the enlarged cell fraction may reflect a defective de-
Ammonia inhibition of glycosaminoglycan degradation gradation of sulphated GAG. The increase in cellular 35S-GAG was not part of a general stimulation of GAG-biosynthesis; rather, NH4C1 at concentrations above 2 mM showed an inhibitory effect on the net production of 3~S-GAG (fig. 2). Furthermore, the pulse-chase experiments demonstrated a delay in the turnover of the cell fraction in the presence of NH4C1. The NH4Cl-treated cultures showed an initial rise in the amount of cellular 3~S-GAG in contrast to the control cultures, where the cellular fraction decreased promptly at the beginning of the chase period. It was concluded that NH4C1 impaired the degradation of 35S-GAG. Further support for this conclusion was obtained by the fact that less low molecular weight degradation products (inorganic a5SO4) appeared in the chase medium of NH4Cl-treated cultures than of non-treated ones, as demonstrated by precipitation with CPC or by gel chromatography (fig. 6). Seglen has previously shown that the catabolism of protein in cultured rat hepatocytes is restrained by ammonia [1]. The degradation of epidermal growth factor by human fibroblasts may be similarly affected by the presence of ammonium ion [13]. The present experiments extend these results to include an inhibitory effect of NH4C1 on the degradation of sulphated GAG. This observation may suggest that NH4CI has a general influence on degradative processes in the cell, not restricted to one particular kind of substance. Morphological findings lend support for this view: the accumulation of secondary lysosomes within glial cells and rat liver cells [2], after supplementation with NH4C1 suggests that the latter may interfere with the lysosomal function in general. The restrained degradation of GAG in conjunction with the accumulation of lyso-
29
somes in NH4Cl-supplemented glial cells reminds of the characteristics of cultured fibroblasts from patients with mucopolysaccharidosis [12]. Previously, a similar mucopolysaccharidosis-like picture has been induced in cultured fibroblasts by raising the pH of the growth medium [14, 15]. The elevated pH of the medium may have hampered the concentration of protons within the lysosomes [16], thereby impairing the degradation of GAG. The mechanism by which NH4C1 exerted an effect on the catabolism of GAG in glial cells is not known; however, its effect on the pH of the nutrient medium was negligible. Weak bases such as basic dyes and chloroquine may be rapidly taken up and concentrated within the lysosomes [17] causing vacuolization and possibly an increase in lysosomal pH. The accumulation of chloroquine and similar derivatives within the lysosomes of human fibroblasts is accompanied by a restrained degradation of GAG and an abnormal morphology, simulating lysosomal storage diseases [18]. Therefore it seems possible that NH4C1 and chloroquine interferes with the lysosomal function by a common mechanism, as has been suggested [2, 13]. This work was supported by the Swedish Medical Research Council (13X-4486 and 13X-2309), the Swedish Cancer Society (689-B75-01X) and Konung Gustav V:s 80-~rsfond. The skilful technical assistance of Mrs F. Carlsson, Miss Y. 0hgren, Mrs G. Fjellstrrm and Miss M. Lindstrrm is gratefully acknowledged.
REFERENCES 1. Seglen, P O, Biochem biophys res commun 66 (1975) 44. 2. Seglen, P O & Reith, A, Exp cell res 100 (1976) 276. 3. Norling, B, Glimelius, B, Westermark, B & Wasteson, ~, Abstr Vth meeting Eur fed conn tiss clubs (1976) 25. 4. Pontrn, J & Macintyre, E, Acta pathol microbiol Scand 74 (1968) 465. Exp CelI Res 108 (1977)
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5. Eagle, H, Science 130 (1959) 432. 6. Pont6n, J, Westermark, B & Hugosson, R, Exp cell res 58 (1969) 313. 7. Ham, R G, Proc natl acad sci US 53 (1965) 288. 8. Wasteson, A, Uthne, K & Westermark, B, Biochemj 136 (1973) 1069. 9. Lindahl, U, Biickstr6m, G, Jansson, L & Hall6n., A, J biol chem 248 (1973) 7234. 10. Brunk, U & Ericsson, J L E, Histochem j 4 (1972) 349. I1. Brunk, U, Ericsson, J L E, Pont6n, J & Westermark, B, Exp cell res 67 (1971) 407. 12. Fratantoni, J C, Hall, C W & Neufeld, E F, Proc natl acad sci US 60 (1968) 699. 13. Carpenter, G & Cohen, S, J cell biol 71 (1976) 159.
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14. Lie, S O, McKusick, V A & Neufeld, E F, Proc natl acad sci US 69 (1972) 2361. 15. Lie, S O, Schofield, B H, Taylor, H A & Doty, S B, Pediat res 7 (1973) 13. 16. Mego, J L, Farb, R M & Barkes, J, Biochem j 128 (1972) 763. 17. de Duve, C, de Barsy, T, Poole, B, Trouet, A, Tulkens, P & Hoof, F, Biochem pharmacol 23 (1974) 2495. 18. Lie, S O & Schofield, B, Biochem pharmacol 22 (1973) 3109. Received December 23, 1976 Accepted February 22, 1977
