Lack of effect of NH4Cl on protein synthesis in cultured fibroblasts

Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827/~0/120305-07$02.00/O

Experimental Cell Research 130 (1980) 305-3 11

LACK

OF EFFECT

OF NH&l

IN CULTURED J. S. AMENTA

ON PROTEIN

SYNTHESIS

FIBROBLASTS and S. C. BROCHER

University of Pittsburgh School of Medicine, Department of Pathology, Pittsburgh, PA 15261, USA

SUMMARY In experiments with isolated hepatocytes, Seglen [l] has shown that in the combined presence of NH&l and high concentrations of valine, incorporation of this amino acid into cell protein is inhibited. He has proposed that NH,CI, in addition to inhibiting protein degradation in lysosomes, inhibits protein synthesis in these cells as part of a general toxic effect. To determine if NH,Cl inhibits protein synthesis in cultured cells we incubated rat embryo fibroblasts, prelabeled with [*V]leucine, in the presence of 10 mM NH&I and 15 mM leucine in both growth and serum-free media. We did not detect any effect of NH: on protein synthesis or cell growth over a 3-day period. A partial inhibition of protein degradation was observed, particularly during the first 24 h of the experiment. In pulse-labeling experiments, NH,Cl had no effect on the incorporation of [3H]leucine in the media. Hieh concentrations of leucine, however, reduced re-utilization of endogenously derived leucine and-inhibited the transport of valine into the cellular acid-soluble pool. These exoeriments show that at least in cultured fibroblasts 10 mM NH,Cl shows no significant toxicity beyond an inhibition of lysosomal function. In addition these data suggest the possibility that high chase concentrations of one amino acid in the medium may be saturating a common transport mechanism, in effect reducing the transport of other amino acids utilizing this mechanism. A combined blockade by both NH&l and a high concentration of a single amino acid may in certain sensitive cells result in a significant reduction in protein synthesis.

NH:, chloroquine, and neutral red have been shown to concentrate in lysosomes and in this process inhibit protein degradation occurring in secondary lysosomes [2, 3, 41. We have utilized this phenomenon to evaluate the role of the lysosomes in protein turnover in cultured fibroblasts, in particular the quantitative contribution of autophagy under conditions of basal and augmented protein turnover [Ml. Our results indicate that under basal conditions, approx. lo-25 % of protein turnover occurs in the lysosome, while under conditions of augmented protein degradation induced by serum-free (step-down) medium, proteolysis within lysosomes is greatly increased.

These results, correlating morphologic and biochemical observations, are based on the hypotheses that the salts of these weak bases at the selected concentrations specifically inhibit lysosomal function and, most important, have no significant toxic effects upon other cellular functions. The evidence for this latter hypothesis has been mostly indirect: (1) morphologic changes in cells treated with these agents are confined to lysosomes; (2) at the selected concentrations, the agents do not significantly increase the rate of cell loss in the culture media; and (3) NH&l at least has no effect on fibroblast growth and proliferation over a 3-day period. However, Visek and coExp Cell Res 130 (1980)

306

Amenta and Brother

workers [9] observed a decreased recovery of 3T3 cells when incubated with NH&l over a 5-day period. Of particular significance is a recent report by Seglen [I], showing that NH&l inhibits the incorporation of [14C]valine into isolated hepatocytes by 60-70%, but only in the presence of high concentrations of the labeled amino acid. These observations suggested that NH: may be seriously interfering with cellular metabolism. In this study we have examined this phenomenon in cultured fibroblasts to ascertain to what extent, if any, NH: may be affecting protein synthesis and growth in this in vitro cell system. METHODS Details of the methods utilizing prelabeled cells have been previously reported [lO-121. In brief, Fisher strain rat embryo fibroblasts from frozen stock cultures were subcultured at a 1: 8 dilution and grown for 4 days in 6 cm Petri dishes containing 4 ml Eagle’s minimum essential medium (MEM) supplemented with 10% calf serum (growth medium) and 0.8 /.Ki [‘“C]leucine and 0.2 /.&Ii [sH]thymidine per 10 ml media. Cultures were grown for an additional 24 h in growth medium without radioactive isotopes. Cultures were then divided and grown for 3 days in 4 different media: (1) growth medium containing 15 mM leucine and 10 mM NaCl; (2) growth medium containing 15 mM leucine and 10 mM NH&I; (3) serum-free medium containing 15 mM leucine and 10 mM NaCI; (4) serumfree medium containing 15 mM leucine and 10 mM NH&l. The chase medium containing 15 mM [‘“Clleucine was designed to minimize re-utilization of isotope and, in addition, to reproduce the high concentration of amino acid used by Seglen [l]. We placed cells in both growth medium, containing 10% fetal calf serum (FCS), and a step-down medium, containing no serum, since cells in step-down medium more likely correspond to the condition of isolated hepatocytes in vitro, both cell systems being characterized by high rates of proteolysis within lysosomes. At 24-h intervals three dishes from each group were processed. Aliquots of media were assayed for total and acid-soluble radioactivity. Assays for media NH, [13] indicated that between 90-100% remained in the medium after 3 days’ incubation. The cell monolayer was rinsed 2 times with cold phosphate-buffered saline and fixed in situ with 5 ml cold 0.2 N perchloric acid for 30 min at 4°C. Each monolayer was scrapped, transferred to a centrifuge tube, and the dish washed once with an additional 3 ml of perchloric acid. After centrifugation at 850 g for 20 min at 4”C, an aliquot of the acid supematant was obtained to measure the intracellular radioactivity. The precipitate Exp Cell RPS 130 (1980)

was dissolved in 1.5 ml of 0.3 N NaGH for 60 min at 37°C and ahquots obtained for estimation of protein [14] and radioactivity in the urotein and DNA. All al&rots for radioactivity were placed in vials containing 10 ml ACS (Amersham-Searle) and counted in a liquid scintillation counter. In this series of experiments, we calculated synthesis, degradation, and growth rates by measuring the specific radioactivity, the total radioactivity, and the total mass of cellular protein [ 151. Details and limitations of these calculations have been previously presented [I 1, 121.Briefly, when the values of the natural logarithm of each of the above parameters (abscissa) are plotted against time (ordinate), the slope at any time measures the fractional rates of synthesis (k,), degradation (kd). and growth (ki) of the slow-turnover protein pools. Since the calculated kd value includes losses of label due to cell loss in each experiment, we have subtracted from this value a kd calculated for the losses of labeled thymidine in DNA. This latter calculation is obtained by plotting the logarithm of the ‘“CPH ratio against time. An additional estimate of the degradation rate of [“Clprotein was obtained by measuring the rate of accumulation of acid-soluble 14Cin the medium. In a second series of experiments we evaluated protein synthesis by measuring the rate of incorporation of [r4C]leucine into cell protein; growth and stepdown media were supplemented with additional radioactive and non-radioactive leucine to provide media with different concentrations of leucine. Growth medium, prepared by adding 100 ml FCS to 900 ml of Eagle’s MEM (Gibco 410-l 100) supplemented with non-essential amino acids (Gibco 320-l 140).contained 0.38 mM/L-leucine; step-down medium without the serum contained 0.40 mM/L-leucine. To each 10 ml of media we added 4 &i of [r4C]leucine (SchwarzlMann, Orangeburg, NY; spec. act. 312 &i/mM) and 0.05 mM of [r2C]leucine. The serum-free medium contained leucine at a concentration of 5.40 mM with a spec. act. of 74.1 &i/mM, while the growth medium contained 5.38 mM leucine with a spec. act. of 74.4 @/mM. These solutions were then progressively diluted with either step-down or growth media respectively. The resulting concentrations and specific activities of each medium are presented with the experimental results. For these experiments fibroblasts were subcultured 1 : 8 into 6.0 cm diameter Petri dishes and grown in growth medium for 5 days to a subconfluent phase. At the beginning of each experiment cultures were washed with phosphate-buffered saline and placed in the labeled media with different concentrations of leutine, as previously described. NH,CI in a small volume of HZ0 was added to half of the cultures to obtain a final concentration of 10 mM; an equimolar amount of NaCl was added to the control cultures. All cultures were incubated for 2 h, washed twice with phosphate-buffered saline, and fixed in situ with 3 ml of 8 % trichloracetic acid (TCA). Cells were scrapped from each dish, quantitatively transferred to a 10 ml plastic tube, and the dish washed once with an additional 3 ml of TCA. After centrifugation at 850 g for 20 min at 4”C, an aliquot of clear supematant was transferred to a counting

Effect ofNH,Cl on protein synthesis TOO- A 600 -

loo0

I

vial containing 10 ml of ACS and counted in a liquid scintillation counter. The remaining cell pellets were washed twice with 8% TCA and dissolved in 0.5 ml of 0.5 N NaOH. Aliquots of this solution were analysed for protein and radioactivity. In these studies we calculated for each group the amount of media leucine incorporated into the cell protein. This calculation adjusted for the different specific activities of the media leucine and gave a value which reflected only the effect of media concentration on the rate of leucine incorporation into cell protein. In a third series of experiments with [YZ]valine, we reported the incorporation of label directly, since the valine concentration and specific activity were held constant in these experiments.

35cB 30.

,---.

2

3

307

50

2

/

3

RESULTS

‘5Fp-%-J IO

/* ,',-

/'M

,/" 5

.:'

r5'

,.P

~

4'

,*

0

Days

2

3

Days Fig. I. Ordinate: (A) total protein &g/dish); (B) [W]protein (dpmx IO-Ydish); (C) spec. act. of [“Clprotein (dpmlpg); (D) [3H]DNA (dpmx IO-Ydish); (E) [‘4C]protein/[3H]DNA (dpm/dpm): (F) acid-soluble [“C] in media (dpmx W3fdish. Effect of NH&l on metabolism of slow-turnover protein pool. Protein and DNA in fibroblasts were labeled as described in Methods. After 24 h in nonlabeled growth medium, cells were placed in either 0, fresh growth medium; 0, step-down medium; 0, growth medium with 10 mM NH,Cl, or U, step-down medium with 10 mM NH&l. At each time interval cells and media were processed for protein and radioactivity. Slopes of plots estimate (A) fractional growth rate; (B) uncorrected fractional degradation rate; and (C) fractional synthesis rate of celf proteins. Sloue of plot in (L)) estimates fractional ceil loss and in (E) fractional degradation rate of cell protein, corrected for cell loss. Acid-soluble 14Cin media (F) measures accumulation of label from degradation of cell proteins. Data from two experiments; each point represents average of 6 dishes. Pooled estimates of SD. given in inset for each experiment.

Effect ofNH: onfractional synthesis, degradation, and growth rates of slow-turnover proteins While fibroblast cultures placed in media with 10 mM NH&l for 3 days showed the expected partial inhibition of proteolysis, synthesis of the cellular slow-turnover protein pool under these conditions showed no significant change. Fibroblasts in growth medium with NH&l showed an increase in cellular protein which in fact slightly exceeded the rate observed in the corresponding control cultures during the first 2 days of the experiment (fig. 1A). This slight stimulation of the fractional growth rate was the result of the partial inhibition of proteolysis occurring at this time (fig. lB, E). We could not detect any inhibition by NH&I on protein synthesis in these cultures (fig. 1C). Similar results were obtained from cultures in step-down medium, though the interpretation of these data were complicated by a significant loss of cells into the serum-free medium over the 3-day period (fig, 1D). Therefore, the loss of [‘“Clprotein from the monolayers (fig. 1B) reflected both the rate of cell loss (fig. 10) and an enhanced rate of protein degradation (fig. 1E) induced by step-down medium. The addition of NH&l to step-down medium Exp Cell Res 130 (19801

308

Amenta and Brother

reduced slightly the rate of protein loss from the cell monolayer (fig. lA), secondary to a partial inhibition in protein degradation (fig. 1E). Again, we were not able to detect any effect of NH&l on synthesis of the slow-turnover protein pool (fig. 1C). That NH,Cl was inhibiting proteolysis was demonstrated by measuring the rate of accumulation of acid-soluble 14C in the media. This agent suppressed the hydrolysis of labeled protein in cells placed in both growth and step-down medium to the same reduced rate (fig. IF), extending results we have previously published in short-term experiments [6, 71. Of concern to us was the observation that loss of labeled protein from cells in both growth and step-down media appeared to be equal (fig. lE), while media analysis indicated that proteolysis, as expected, was stimulated by the step-down medium (fig. 1F). A likely explanation for this may be that the cell subpopulation responding to step-down medium by the induction of autophagy comprised much of the subpopulation of cells lost to the medium during the experiment (fig. 1D). The residual cells in the monolayer apparently were degrading protein at the same rate as fibroblasts in growth medium (fig. 1E). Apparently for both of these cell populations, NH4C1 slightly inhibits basal proteolysis, consistent with previous studies [6, 7, 81.

pools. Short-term labeling of fibroblasts places approx. 30% of the incorporated label in the fast-turnover protein pool [12, 161.We therefore considered the possibility that only the synthesis rate of this protein pool was inhibited by NH,Cl in hepatocytes. Fibroblasts were labeled for 2 h in media containing 0.65-5.4 mM leucine. The specific activity of the [3H]leucine in these solutions is given in fig. 2A. We observed a rise in intracellular leucine derived from the medium pool, coordinate with the rise in the concentration of the medium leucine (fig. 2B). The small decrement in recovery of acid-soluble intracellular leucine in the cell cultures in step-down medium was proportional to the slightly lower recovery of cell protein in these cultures. NH&l had no detectable effect on the transport of leucine from the medium into the cell, in either growth medium or step-down medium. Similarly, the incorporation of medium leucine into cell protein increased with increasing concentrations of leucine in the medium (fig. 2C), and again NH,CI was without effect. These data suggested that high concentrations of leucine either (a) were stimulating protein synthesis in cultured fibroblasts [17], or (6) more likely, were progressively blocking the re-utilization of endogenously derived leucine. In either case, it appeared clear that NH,Cl was without effect on incorporation of label Effect of NH: on pulse labeling of into cell protein. cell protein To evaluate these alternatives, we inWhile these experiments indicated, at least cubated fibroblasts with trace levels of under conditions we routinely use in ex- [14C]valine in the presence of high concenperiments with fibroblast cultures, that syn- trations of leucine, extending the concenthesis of the slow-turnover protein pool was trations of leucine in this experiment to not affected by NH&l, we had not studied 15 mM. High concentrations of leucine detibroblasts under conditions used by Seglen creased both the transport of [14C]valine [l] in which a significant fraction of the into the cell (fig. 3A) and the incorporation label was entering fast-turnover protein of [14C]valine into the cell protein to an Exp Cd Res 130 (1980)

Effect of NH&l on protein synthesis *

1500

1000 \\

309

q

500 LT.2 0

5

IO

15

:z f+Y --L----~ 1000 I--El 0

5 IO LeYCineCO”C,rnM,l,

15

(A) [L4C]valine in cell acid-soluble pool (dpm/dish); (B) incorporation of [W]valine into cell protein (dpmldish). Effect of leucine on intracellular [Wlvaline and incorporation of [Wlvaline into cell protein. Fibroblast cultures incubated for 2 h in 10 ml medium containing 0.8 &i [‘*C]valine and non-radioactive leucine as indicated. Four media were used: 0, growth medium; 0, growth medium with 10 mM NH&l; 0, step-down medium; and n , step-down medium with 10 mM NH,Cl. Cultures processed as described in Methods and assayed for (A) intracellular acid-soluble [“Clvaline; (B) incorporation of [‘*C]valine into cell protein. Each value represents the mean of six values from two different experiments. Range of SD. is given in inset. Mean protein recoveries were 0, 297 pg; 0,321 pg; 0,285 pg; n , 276 /q.

Fig. 3. Ordinate:

0

I

2

3

4

5

Media leucine ImM/Il

(A) Spec. act. of media leucine (&i/mM); (B) intracellular leucine from media nM/ dish); (C) media leucine incorporated into cell protein (nM/dish). Effect of NH,Cl on incorporation of leucine into cell protein. Fibroblast cultures were incubated for 2 h in 0, growth medium or 0, step-down medium with [Wlleucine at specific activities and concentrations shown in (A). Parallel cultures were incubated in media containing 10 mM NH&l (0, W). Cultures were processed as described in Methods. [‘*C]Leucine in TCA-soluble fractions shown in (Z?) and [‘*C]leucine in acid-insoluble fraction in (C). Each value represents the mean of six values from two separate experiments. Range of S.D. is given in inset. Mean of cell protein recovered from cultures incubated in growth medium: 0, 322 pg; 0, 321 pg; from cultures incubated in step-down medium: Cl, 291 pg; W, 2% pg.

Fig. 2. Ordinnte:

equal degree (fig. 3B). Leucine in high concentrations appeared to be partially blocking the transport of valine into the cell and the decreased incorporation of label into protein appeared to be a result of the decreased accumulation of label in the intra-

cellular amino acid pool. NH&l again had no effect on any of these phenomena. Data from both pulse label experiments thus support the hypotheses that (a) high concentrations of leucine in media decrease reutilization of endogenously generated leucine and block the transport of valine into the cell; and (b) that NH:, at least in fibroblast cultures, does not inhibit protein synthesis. DISCUSSION Rat embryo fibroblasts respond quite differently from isolated hepatocytes when placed in media containing high concentrations of leucine and NH&l. In the fibroblast cultures, both in short-term labeling Exp Cell Res 130 (1980)

3 10

Amenta and Brother

experiments and in long-term isotope clear- ids is also restricted. Thus, only when a ing experiments, we could detect no effect high valine concentration and NH&I are of NH&l on protein synthesis. Morpho- blockading together would one observe an logic studies on cells exposed to NH,Cl for inhibition of protein synthesis in hepato3 days showed no toxic effect of this agent cytes. This hypothesis would predict that upon any organelle other than the lyso- the inhibition of protein synthesis observed somal vacuolar system (unpublished ob- in liver cultures under the combined efservations). fects of high valine and NH&l should be The question remains why this agent ap- largely relieved by the addition of supplepears to have such a pronounced effect menting amino acids to the medium, exactupon incorporation of labeled amino acid ly the result observed by Seglen [l] when into cellular protein of isolated hepatocytes. an amino acid mixture at 20 times normal Seglen [I] has suggested that its effect may concentration was added. While high valine be indirect, either upon energy metabo- concentrations in the medium did not inlism or intermediary nitrogen metabolism. hibit the incorporation of labeled leucine Clearly the isolated hepatocyte is a very into hepatocyte proteins [l], the effect of different cell from the cultured fibroblast, high valine on the transport of labeled particularly in respect to ammonia metabo- leucine into the cell was not reported. lism and utilization of amino acids for Whether or not a combined blockade is gluconeogenesis. If the toxicity of NH&l operative in isolated hepatocytes exposed is related to some of these specialized func- to NH&l and a high concentration of vations of the hepatocyte, as suggested by line, the possible blockading role of a single Seglen [ 11, we would not expect to see amino acid in high concentration should be any inhibition of protein synthesis in the given due consideration in experiments on cultured fibroblast. While it is unlikely that protein synthesis when combined with the difference is significant, we would also agents which inhibit the production of note in passing that in our experiments endogenous amino acids. labeled leucine was used to assay protein synthesis, while Seglen used labeled valine. While we cannot resolve this question REFERENCES with data from fibroblast cultures, our data 1. Seglen, P 0, Biochem j 174(1978) 469. 2. Poole, B, Ohkuma, S & Warburton, M J, Acta biol suggest an additional consideration. In our med ger 36 (1977) 1777. experiments high concentrations of leucine 3. Ohkuma, S & Poole, B, Proc natl acad sci US 75 (1978) 3327. inhibited the transport of valine into the 4. Wibo; M & Poole, B, J cell bio163 (1974) 430. cells, possibly reflecting a saturating effect 5. Amenta, J S, Sargus, M J, Venkatesan, S & Shinozuka, H, J cell physio194 (1978) 77. of leucine upon a common transport mech6. Amenta, J S, Hlivko, T J, McBee, A G, Shinozuanism [18, 191. Since isolated hepatocytes, ka, H &Brocher, S, Exp cell res 115 (1978) 357. in contrast to cultured fibroblasts, have an 7. Amenta, J S & Brother, S C, Exn cell res 126 (1980) 167. extremely high rate of protein degradation 8. - J cell physiol 102 (1980) 259. 9. Visek, W J, Kolodny, G M & Gross, P R, J cell [20, 211, a valine-induced restricted transphysiol80 (1973) 373. port of some other essential amino acids 10. Amenta, J S, Baccino, F M & Sargus, M J, Intracellular protein catabolism (ed V Turk & N Marks) may not in itself be rate-limiting for protein vol. 2, p. 24. Plenum Press, NewYork (1977). synthesis. However, when NH&l is add- 11. Amenta, J S, Sargus, M J & Baccino, F M, J cell physiol 97 (1978) 267. ed, this endogenous source of amino acExp Cell Res 130 (1980)

Effect of NH&l 12. Amenta, J S & Sargus, M J, Biochem j 182 (1979) 847. 13. Lubochinsky, B & Zalta, Jr, Bull sot chim biol 36 (1954) 1363. 14. Lowry, 0 H, Rosebrough, N J, Farr, A L & Randall, R J, J biol them 193 (1951) 265. 15. Koch, A L, J theor biol 3 (1962) 283. 16. Amenta, J S, Sargus, M J & Brother, S C, J cell physiol. In press (1980). 17. Chua, B H L, Siehl, D L Morgan, H E, Fed proc 39 (1980) 349. 18. Meister, A, Science 180(1973) 33.

Printed

in Sweden

on protein synthesis

311

19. Gazzola, G C, Dall’Asta, V & Guidotti, G G, J biol them 255 (1980) 929. 20. Seglen, P 0, Use of isolated liver cells and kidney tubules in metabolic studies (ed J M Tager, N D Soling & J R Williamson) p. 245. North-Holland Publishing CO, Amsterdam (1976). 21. - Biochim biophys acta 496 (1977) 182. Received April 14, 1980 Revised version received June 18, 1980 Accepted June 24, 1980

Exp Cell Res I30 (1980)