Eur. J. Biochem. 95, 215-225 (1979)
Inhibition of the Lysosomal Pathway of Protein Degradation in Isolated Rat Hepatocytes by Ammonia, Methylamine, Chloroquine and Leupeptin Per 0. SEGLEN, Bj$m GRINDE, and Anne E. SOLHEIM Avdeling for Vevsdyrkning, Norsk Hydros Institutt foi K reftforskning, Det Norske Radiurnhospital, Oslo (Received July 20/December 8, 1978)
1. Protein degradation in isolated rat hepatocytes was measured as the release of [14C]valine from pre-labelled protein. To reduce background radioactivity, the intracellular ['4C]valine pool was depleted by serial extraction at 37 "C, effecting equilibration between the intracellular pool and the valine-free extracellular medium. After extraction, a small, non-equilibrating intracellular [14C]valine pool remained; this pool could only be labelled in the presence of ongoing protein synthesis, and might represent valine and valine-containing oligopeptides derived from protein degradation. 2. The ['4C]valine released from degraded protein was not significantly re-utilized for protein synthesis intracellularly (no effect of cycloheximide or high concentrations of unlabelled valine), reflecting the low rate of protein synthesis and the rapid transport of valine into the extracellular medium, both characteristic of isolated hepatocytes. 3. From cells pre-labelled for 24 h Ivi vivo, ['4C]valine was released linearly at a rale of 5 %/h, probably representing the true over-all protein degradation rate. The lysosomotropic inhibitor ammonia (10 mM NH4C1) inhibited 7 0 % of the degradation, presumably the contribution by the lysosomal pathway. From 1-h pre-labelled cells, [14C]valine was released at a declining rate, and ammonia inhibited degradation only by 45%, consistent with the view that the majority of short-lived proteins are degraded by the non-lysosomal pathway(s). 4. Chloroquine and methylamine, which accumulate in lysosomes by virtue of their weak base properties, inhibited hepatocytic protein degradation to the same extent as ammonia, with no additivity. These compounds therefore seem to block the lysosomal pathway of protein degradation selectively and completely. Leupeptin, which binds to and inhibits the activity of certain lysosomal proteases, also inhibited protein degradation almost to the same extent as ammonia, but with a small part of the effect ( < 20 %) being additive to the NH3 effect and thus probably reflecting a slight inhibition of non-lysosomal protein degradation as well. 5. Of the four inhibitors tested, only the effect of ammonia was rapidly reversible within the experimental period (2 h). Leupeptin, on the other hand, was the only degradation inhibitor which did not also affect protein synthesis. Chloroquine caused significant cell death at concentrations above 0.2 mmol/l in this protein-free medium, i.e. in the concentration range needed for maximal inhibition of protein degradation. 6. Incubation of hepatocytes under anoxic conditions resulted in an inhibition of protein degradation which was greater than, and partially additive to, the effect of ammonia, i.e. most of the degradation by the lysosomal pathway and more than one-half of the degradation by the nonlysosomal pathways appears to be energy-dependent. The mechanisms responsible for the general turnover of endogeneous protein in animal cells are not well known. Lysosomes (autophagy) appear to play a major role [I -41 but in addition a number of nonlysosomal proteolytic systems have been demonstrated [5-91. Ballard [lo, 1 I ] has suggested that both lyso-
somal and non-lysosomal pathways of protein degradation are operative, but with different specificities, and that e.g. short-lived proteins (abnormal proteins and others) are preferentially degraded by non-lysosomal pathways. In cultured cells, the low basal protein turnover seen under nutritionally optimal conditions
Lysosomal Pathway of Protein Degradation in Isolated Rat Hepatocytes
appears to occur largely by way of non-lysosomal degradation, whereas the lysosomal pathway is activated upon starvation for serum or amino acids [12151. A similar activation can be induced in the liver by glucagon treatment in vivo [ l ] or by amino acid starvation of the perfused liver [2,16,17]. Isolated rat hepatocytes in suspension have a high rate of protein turnover (4- 5 %/h), as estimated from the total nitrogen balance , and we have previously shown that most of this turnover can be prevented by ammonia, a lysosomotropic inhibitor [19-211. In the present paper we have measured protein degradation more dii-cctly as the release of ['4C]valine from pre-labelled protein, and compared the effect of ammonia with other lysosomotropic inhibitors in an attempt to elucidate the relative contributions of lysosomal and non-lysosomal degradation pathways to the total hepatocytic protein turnover. MATERIALS AND METHODS Isolated hepatocytes were prepared from the livers of 16-h fasted male Wistar rats (250-300 g) by the method of collagenase perfusion [22,23], and suspended in buffered saline (suspension buffer ). For labelling in vitro, 4-ml aliquots of concentrated cell suspension (120 150 mg cells/ml) .were incubated in 10-cm plastic petri dishes for 1 h at 37 "C with a tracer dose of ['4C]valine (2.4 pmol/l; 630 pCi/l). During the incubation, the cells were gently agitated by having the dishes standing on a tilting platform (Cenco Instruments, Breda, The Netherlands) with an axial rotation frequency of 10 rev./min. After the 1-h incubation, non-incorporated ['4C]valine was removed both from the intracellular and extracellular valine pools by re-incubating the cells 5 x 10 min at 37 "C in fresh (isotope-free) medium. The cells were recovered between extractions by centrifugation for 2 min at 400 rev./min (Sorvall RC-3). In some initial experiments the cells were first washed several times at 0 "C (to remove extracellular ['4C]valine) before being extracted at 37"C, but this was later found to be unnecessary. The [14C]valine-depleted cells were incubated as 0.4-ml aliquots (in suspension buffer) at a standard cells concentration of 75 mg/ml (range 60-90 mg/ml) in rapidly shaking(gyr0-rotating at 21 5 rev./min) 15-ml glass centrifuge tubes at 37 "C. When anoxic incubation conditions were desired, the tubes were filled with nitrogen and stoppered. Incubations were terminated by the addition of 0.1 ml perchloric acid (1 0 w/v), and after 15 min at 0 "C and centrifugation for 5 min at 5000 rev./min, duplicate 0.1-ml samples of the supernatant were assayed for acid-soluble radioactivity by liquid scintillation counting . The precipitate was washed three times in 4 ml ice-cold perchloric acid (2'4 w/v), dissolved in 0.5 ml of 0.3 M ~
NaOH (at 37"C), and assayed for total proteinincorporated radioactivity . The net release of [14C]valineduring the incubation period was expressed as a percentage of the total initial protein radioactivity of the cell sample. For labelling of the hepatocytes in vivo, 1 ml of [14C]valine (63 pCi) was injected into the tail vein of a rat 24 h before the preparation of cells. The hepatocytes were used directly for experimental incubation, as preliminary depletion of the [14C]valine pool was unnecessary in this case. For the measurement of intracellular [14C]valine, the cells were washed several times in ice-cold buffer before acid precipitation . Protein synthesis was measured as the incorporation of [14C]valine at constant specific radioactivity ( 5 mmol/l and 156 pCi/l [24,26]), or as the incorporation of a mixture of I4C-labelled amino acids at tracer concentrations . [14C]Valinewas purchased from The Radiochemical Centre (Amersham, Bucks, England, code no. CFB. 75 ; 260 Ci/mol ; 63 mCi/l ; 0.24 mmol/l). Chloroquine, methylamine and other biochemicals were obtained from Sigma (St Louis, Mo., U.S.A.); leupeptin was a kind gift from Dr H. Umezawa (Institute of Microbial Chemistry, Shinagawa-ku, Tokyo, Japan). All chemicals were added to the cell suspensions in isotonic (300 mosmol/kg HzO), neutral (pH 7.0) solutions, adjusted with NaCl, NaOH or HCl if necessary.
RESULTS AND DISCUSSION Pre-labelling of Hepatocytic Protein with ['"C] Valine Protein degradation can be measured as the release of a radioactive amino acid from protein pre-labelled in vivo or in vitro. We have chosen to use [14C]valine, a largely non-metabolizable amino acid , the incorporation and transport characteristics of which have been reasonably well characterized in isolated hepatocytes [24-261. Since the purpose of the prelabelling in vitro is to incorporate as much radioactivity as possible into protein during a relatively short time, a high, rather than a constant, specific radioactivity of ['4C]valine, i.e. a tracer dose, was used. Under such conditions the rate of incorporation decreases rapidly as shown in Fig. 1, due to isotope dilution by unlabelled valine derived from protein degradation [18,24,27]. The loss of acid-soluble radioactivity (intracellular + extracellular) is quantitatively accounted for by incorporation into protein (intracellular + extracellular acid-insoluble radioactivity), i.e. free and protein-incorporated [14C]valine is measured with the same efficiency. In subsequent protein degradation measurements, [14C]valine release can therefore conveniently be expressed as a percentage of the initial protein radioactivity.
P. 0. Seglen, B. Grinde, and A. E. Solheim
2 Incubation time (h) 1
Fig. 1. Time-course .f conversion oj acid-soluble [14C]valine into acid-insoluble material (protein). A concentrated suspension of isolated hepatocytes (120 mg cells/ml) was incubated for 3 h at 37 "C with a tracer dose of ['4C]valine (2.4 pmol/l; 630 pCijl). Each hour the acid-soluble radioactivity (i.e. [I4C]valine, A); the acidinsoluble radioactivity (i.e. ['4C]valine incorporated into protein,). and the total radioactivity (measured either separately, 0;o r as the sum of acid-soluble and acid-insoluble radioactivity, 0 ) in the system was measured. Each point is the mean of five cell samples
Since two-thirds of the ['4C]valine incorporation occurs during the first hour, 1 h was chosen as the standard time period for pre-labelling in vitro. It should be noted that such a short incorporation period will result in the preferential labelling of short-lived proteins [28,29], as will be discussed later.
initial extracellular [''c ] va~ine concentration ( mmoi/l 1
Fig. 2. Equilihrution oj [L4C]vulineucro.s.s the plu.wia membrane. Isolated hepatocytes (60 mg/ml) were incubated at 37 -C for 60 min with various concentrations of ['4C]valine (constant specific activity of 63 mCi/mol) in the presence or absence of NH4Cl (10 mmol/l). At the end of incubation, the concentration of acid-soluble radiocontrol; 0, NH4CI) and activity was measured intracellularly (0, extracellularly (A, control; A, NH4CI). Each point is the mean of two cell samples
Pre-labelled in absence
Depletion of the Intrucellulur (l4C/ Vuline Pool After completion of the protein pre-labelling, both the hepatocytes and the medium are filled with acidsoluble ['4C]valine which must be removed below an acceptable background level before the net release of radioactivity from protein can be measured. Extracellular ['4C]valine can easily be removed by washing the cells in the cold, whereas the intracellular pool can only be depleted under conditions which allow carrier-mediated transport of [14C]valine out of the cells. Fortunately hepatocytic valine transport is of the facilitated diffusion-type, which works equally well in both directions, and which serves to equilibrate valine between the intracellular and extracellular compartments . This is illustrated in Fig. 2, which shows that the intracellular concentration of [14C]valine at equilibrium is directly proportional to the extracellular concentration, and only slightly below the latter. Fig. 2 also shows that NH4C1, used in subsequent experiments as an inhibitor of protein degradation, does not affect the equilibrium distribution of ['4C]valine; nor does it affect the transport rate . Since valine transport in isolated hepatocytes is rapid, a 10-min period of incubation at 37°C will effect complete equilibration between the intracellular
Pre-labelled in presence of cycloheximide 1
- Number of extractions ( x 10min at 37°C) Fig. 3. DqJ.l'ketiori of llie nzajor intracelhhr [14C]rc~~ine pool and selective labelling (u'a putative degradation pool. Isolated hepatocytes (130 mgjml) were incubated for 1 h at 37'C with ['4C]valine (0.6 pCi/ml) in the presence ( 0 )or absence (0)of cycloheximide (1 mM). After incubation. the cells were washed 6 x in ice-cold buffer: then ~-cincuhalcd at 37 C incyclolicwimide-coiit~~ining(1 mM) huffcr liir h c