JOURNAL OF CELLULAR PHYSIOLOGY 150:149-157 (1992)

Responsiveness of RNA Degradation to Amino Acids in Cultured Rat Hepatocytes: Comparison With Isolated Rat Hepatocytes SOPHIE BALAVOINE, EDITH ROGIER, GERARD FELDMANN AND BERNARD LARDEUX* Laboratoire de Biologie Ceilulaire, Unite 327 de i’lnstitut National de la Sant6 et de la Recherche Medicale, Facult6 de Medecine Xavier Bichat, Universit6 Paris 7, 75018 Paris, France The role of amino acids in the regulation of RNA degradation was investigated in cultured hepatocytes from fed rats previously labeled in vivo with [ 6 - ’ 4 C ] o r ~ t i ~ acid. Rates of RNA degradation were determined between 42 and 48 h of culture from the release of radioactive cytidine in the presence of 0.5 m M unlabeled cytidine. The fractional rate was about 4.4 t 0.4%/h in the absence of amino acids (OX).The catabolism of RNA was decreased to basal level (1.5 0.3%/h) by the addition of amino acids at 10 times normal plasma concentration (1 Ox). The inhibition of RNA degradation, expressed as percentage of maximal deprivation-induced response (OX minus l o x ) , averaged 60% at normal plasma levels of amino acids. The degree of responsiveness was greatly improved as compared to freshly isolated hepatocytes (20%) and was similar to the sensitivity previously observed with perfused livers. In cultured hepatocytes, the sensitivity of RNA degradation to amino acids was not affected by varying the volume of medium from 1 to 4 ml per dish. In freshly isolated hepatocytes, the inhibitory effect of amino acids was not modified by changing the cell density from 0.5 to 5 x 10‘ cells per ml. In the range of normal plasma concentration of amino acids, the low sensitivity of RNA degradation in isolated hepatocytes persisted with inhibition ranging from 10 to 20%. These findings suggest that the control of RNA degradation in both cultured and isolated hepatocytes is not affected by the total quantity of amino acids available in the medium, but their concentration is crucial. Electron microscopy observations and the inhibitory effect of 3-methyladenine in cultured rat hepatocytes partially confirmed the role of the lysosomal system in the increase of RNA degradation and its regulation by amino acids.

*

The RNA content of cells is affected by nutritional cytes, the sensitivity of RNA breakdown to physiologand hormonal environment. Although alterations in ical concentrations of amino acids is deeply affected the rate of RNA synthesis are important (Hirsch and (Balavoine et al., 1990) as compared to perfused rat Hiatt, 1966; Kawada et al., 1977; Conde and Franze- livers. This loss of sensitivity of RNA degradation to Fernandez, 1980; Ashford and Pain, 19861, regulation extracellular amino acids could be explained, a t least, is also achieved through modifications of the rate of by the two following reasons. First, the capacity of RNA degradation. The loss of cytoplasmic RNA during freshly isolated hepatocytes to actively transport starvation o r diabetes is associated with an increase of andlor accumulate amino acids is partially altered as a RNA breakdown in rat liver (Hirsch and Hiatt, 1966; result of collagenase dissociation (Schreiber and Enwonwu and Munro, 1970; Enwonwu et al., 1971; Schreiber, 1973; Kletzien et al., 1976). Since the culKawada et al., 1977; Conde and Franze-Fernandez, tured hepatocytes recover their capacity to transport 1980; Ashford and Pain, 1986). On the contrary, after amino acids (Kletzien et al., 1976), we used this experrefeeding or immediately after meal intake, the induc- imental model in order t o evaluate more precisely the tion of RNA accumulation is due to a moderate increase degree of responsiveness of RNA degradation to physof RNA synthesis and an almost complete suppression iological concentrations of amino acids. The contribuof RNA degradation (Conde and Franze-Fernandez, tion of the lysosomal system in RNA degradation and 1980; Lardeux et al., 1988). its regulation by amino acids in cultured rat hepatoFrom studies with perfused rat livers and isolated rat cytes was also investigated. The second reason may be related to the density of hepatocytes, it was observed that amino acids and insulin inhibit the catabolism of RNA (Lardeux and hepatocytes used in incubation flasks. Jurin and McMortimore, 1987; Balavoine et al., 1990). In addition, it was shown in these works that the lysosomal system was probably the main site of deprivation-induced RNA Received May 7, 1991; accepted July 30, 1991. degradation in rat liver. However, in isolated hepato- “To whom reprint requestdcorrespondence should be sent 0 1992 WILEY-LISS, INC.

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Cune (1985) have shown that metabolic activities of freshly isolated rat hepatocytes change as a function of the cell density. A high cell density could affect the intracellular concentrations of amino acids depending on their utilisation for protein synthesis andior their intracellular catabolism. In that case, the responsiveness of RNA degradation to amino acids in hepatocyte suspensions could also depend on the total quantity of amino acids available in the incubation medium. One might expect to improve the responsiveness by decreasing cell density. In the present investigation, we also compared the inhibitory effect of amino acids on RNA degradation in isolated and cultured rat hepatocytes maintained a t different cell densities.

MATERIALS AND METHODS Materials Reagents. [6-14Cloroticacid (50-60 mCi/mol) was obtained from the Commissariat a 1’Energie Atomique (Gif-sur-Yvette, France). Cytidine, 3-methyladenine, and amino acids were products of Sigma Chemical (St. Louis, MO, USA). Minimum essential medium with Earle’s salts (MEM), Earle’s balanced salt solution (EBSS), penicillin, streptomycin, vitamins, insulin, trypsin-EDTA were obtained from Gibco (Grand Island NY, USA). Fetal bovine serum (FBS), collagenase, collagen type I, were purchased respectively from Serovial (Neuf-Brisach, France), Boehringer (Mannheim, Germany), and the Institut Jacques Boy (Reims, France). Dowex 50W-X4 (hydrogen form, 100-200 mesh) was acquired from Bio-Rad laboratories (Richmond, VA, USA). Sodium P-glycerophosphate was product of Merck (Darmstadt, Germany). Animals and RNA labeling. Male Sprague Dawley rats (Charles River, Saint Aubin-les-Elbeuf, France), weighing 150-160g, were maintained on commercial chow (U.A.R., Villemoisson-sur-Orge, France) and water ad lib. Liver RNA was labeled in vivo by intraperitoneal injection of [6-14Cloroticacid as described previously (Lardeux et al., 1987; Balavoine et al., 1990) except for the quantity used (50 pCi per rat). Methods Hepatocyte preparation. Liver parenchymal cells were isolated under sterile conditions, 60 h after RNA labeling, by the method of collagenase perfusion described by Seglen (1976) and slightly modified by the addition of a complete mixture of amino acids in perfusion medium in order to suppress the initiation of RNA catabolism by deprived medium (Lardeux and Mortimore, 1987; Balavoine e t al., 1990). Hepatocytes were purified using Percoll gradient centrifugation as previously described (Wang et al., 1985). Hepatocyte suspensions. Isolated hepatocytes were incubated a t varying cell densities from 0.5 x lo6 cellsiml to 5 x lo6 celldml. Incubation conditions and amino acid additions were carried out as previously described (Balavoine et al., 1990). Rates of RNA degradation were measured from radioactive cytidine release (Lardeux et al., 1987; Balavoine et al., 1990). Hepatocyte cultures. Hepatocytes for monolayer cultures were resuspended a t a density of 0.4 x lo6 cellsiml in MEM containing 20 pgiml insulin, 100 IUiml penicillin, 100 pgiml streptomycin and 10% FBS.

Aliquots of hepatocyte suspensions (4ml) were seeded onto 60 mm plastic Petri dishes precoated with type I collagen. Cells were maintained at 37°C in a humidified incubator under 5 % C 0 2 in air. The unattached cells were washed out 2 h after hepatocyte spreading and the medium was replaced with insulin-free MEM supplemented as described above until 24 h. Unless otherwise noted, the culture medium was replaced with fresh MEM without FBS a t 24 h for a n additional 18 h period. The hepatocytes were maintained in EBSS containing 100 IUiml penicillin, 100 pgiml streptomycin and 0.5 mM unlabeled cytidine from 42 to 48 h of culture. Vitamins were added in EBSS in order to achieve the same concentration a s in MEM. Different additions (amino acids, 3-methyladenine) were made as described in legends of figures or tables. The complete mixture of amino acids was added as fractionimultiples of the normal (1x plasma concentration (Woodside and Mortimore, 1972; Lardeux and Mortimore, 1987). All products added were prepared under sterile conditions in EBSS. At the end of the experimental period ( 4 2 4 8 h), culture media and cells were recovered separately. Culture media recovered at 48 h were centrifuged a t 8 0 x g for 5 min to exclude cell fragments and dead cells. Cells from Petri dishes were incubated with trypsinEDTA for 5 min at 37°C. Immediately after trypsinization, cold MEM was added to the culture dishes in order to stop proteolytic activity. Hepatocyte suspensions were collected and culture dishes were washed twice with cold MEM. Washings were added to the initial cell suspensions and cells were pelleted by centrifugation. All samples were frozen a t -20°C until analysis. Analytical procedures. RNA content and radioactive RNA were measured as described by Lardeux et al., (1987) and specific radioactivity of RNA was calculated. Radioactive cytidine accumulated in incubation medium between 30 and 120 min or in culture medium between 42 and 48 h was extracted from acid-soluble fractions by Dowex chromatography (Lardeux et al., 1987). All radioactivity eluted corresponded to cytidine as controlled by HPLC. In some experiments, liquid chromatography was used to determine the specific radioactivity of cytidine in culture medium between 42 and 48 h. HPLC was performed as previously described (Balavoine et al., 1990) except that a Beckman HPLC system was used. DNA content was measured according to Munro and Fleck (1966). The viability of attached cells was determined by trypan blue exclusion test from cells obtained after trypsinization. Transmission electron microscopy and acid phosphatase demonstration. In some experiments, cell cultures were treated for morphological study and demonstration of acid phosphatase a t the ultrastructural level, either at 42 h of culture in MEM or 6 h later in EBSS, supplemented or not with amino acids (48 h of culture). For morphological study, cultures were fixed directly in plastic dishes with 1.25% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.4, a t 4°C for 10 min and washed with the same buffer. Hepatocytes were postfixed with 1% osmium tetroxide in veronal buffer, pH 7.4, a t room temperature for 20 min. For better preservation of membranes, hepatocyte mono-

RNA DEGRADATION IN CULTURED RAT HEPATOCYTES

151

TABLE 1. Distribution of radioactivity in culture media a s a function of medium composition (W of total radioactivity released)' Culture media

+

MEM FBS MEM - FBS EBSS FBS EBSS - FBS

+

Acidinsoluble

Acid-soluble (non-cytidine)

Acid-soluble (cytidine)

7.2 i 0.9 17.3 0.8 5.8 f 1.1 19.5 iz 0.2

59.2 f 2.4 51.5 f 0.9 57.2 f 0.7 48.0 i 0.6

33.1 k 1.5 30.6 0.9 37.0 f 1.7 32.5 k 0.4

*

+

'Labeled rat hepatocytes were maintained in cultureas described in Figure 1. Total radioactivity released in the culture medium wasmeasured directly after centrifugation a t 8Og for 5 min. Radioactive acid-soluble fraction was obtained after precipitation with perchloric acid. Acid-insoluble radioactivity was calculated hy difference between total radioactivity and acid-soluble radioactivity. Cationic radioactivity (cytidine) was eluted by Dowex 50 chromatography from acid-soluble fraction. Unbound product corresponded to non-cytidine fraction. Results are expressed a s percentage of total radioactivity released from data of three separate experiments in Figure 1.

erophosphate, and 2.4 mM lead nitrate, then finally rinsed in the same sucrose, Tris-maleate buffer. Control cell cultures were incubated without substrate. Osmium tetroxide postfixation and embedding were made as described above. Ultrathin sections were not counter-stained.

RESULTS Rates of RNA degradation in cultured rat hepatocytes Preliminary experiments were designed to establish conditions of RNA degradation measurement in cultured rat hepatocytes. Different media (MEM, EBSS) and the presence or the absence of FBS were tested. Behavior of hepatocytes in culture. The removal 42 44 46 48 of FBS at 24 h of culture was rapidly followed by 17% loss of DNA probably in relation to cell detachment. HOURS OF CULTURE However, between 42 and 48 h, the quantity of DNA Fig. 1. Cumulative release of total radioactivity i n the medium from per dish was not significantly affected by the nature of cultured rat hepatocytes. Hepatocytes previously labeled in vivo with the culture medium, indicating that the number of 16-'4Clorotic acid were isolated a s described under Methods. Rat attached cells remained fairly constant throughout the hepatocytes were maintained in the presence of 10% FBS either experimental period. The viability of attached cells throughout t h e 48 h period of culture ( A ,W or only during the first 24 h of culture ( A , o ) . All cultures were carried out in MEM for 42 h and during this time period (42-48 h) was unchanged a s a maintained either in MEM ( U p ) or in EBSS ( A ,A) until 48 h . At 44, function of medium composition and ranged between 75 46 and 48 h, total radioactivity released in the medium was measured and 80%. and expressed as percentage of initial label in RNA. The latter was Release of radioactivity in culture medium from calculated by adding total radioactivity released to the radioactivity in cellular RNA a t the end of each experimental period. Values a r e hepatocytes previously labeled with [6-14Cloroti~ means i SE of three separate experiments. acid in vivo. As seen in Figure 1, the release of total radioactivity in the medium between 42 and 48 h of culture was fairly linear. The higher release was observed in deprived conditions or in the absence of layers were treated by uranyl acetate incubation ac- amino acids and FBS (EBSS without FBS). The prescording to the procedure of Karnovsky (1967). Imme- ence of amino acids and FBS decreased the accumuladiately after incubation, cells were dehydrated in tion of total radioactivity. Most of the radioactivity ethanol and embedded in a thin layer of epoxy resin. released in the medium was recovered in the acidLongitudinal ultrathin sections were stained with ura- soluble fractions (Table 1).The release of acid-insoluble nyl acetate and lead citrate before examination with a radioactive material was very limited except in the Siemens Elmiskop IA electron microscope. absence of FBS. With FBS-free medium, this radioacThe staining for acid phosphatase was performed tivity represented 17-20% of total radioactivity accuaccording to the method of Barka and Anderson (1962), mulated, or 3-4% of the initial label in RNA. As slightly modified by Lewis (1977). Cell cultures were previously shown for isolated rat hepatocytes (Balafixed in situ with 1.25% glutaraldehyde in 0.1 M voine et al., 19901, this radioactive material probably sucrose, 0.1 M cacodylate buffer, pH 7.4, a t 4°C for 10 reflected the presence of radioactive RNA released a s a min. They were washed with 0.25 M sucrose, 40 mM result of cell damage. Concerning the acid-soluble Tris-maleate buffer, pH 5.2, for 1 h at room tempera- fraction, our results indicated that the relative proporture, incubated a t 37°C for 1 h in the sucrose-Tris- tion of radioactive cytidine was unchanged as a funcmaleate buffer with the substrate, 8 mM sodium p-glyc- tion of medium composition and was equal to 3040% of

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the total radioactivity released. These results were in agreement with those obtained in isolated rat hepatocytes (Balavoine et al., 1990). The nature of the noncytidine acid-soluble radioactivity released in the medium was not identified but this radioactivity was probably composed in majority of radioactive uridine as previously observed with isolated hepatocytes (Balavoine et al., 1990). Fractional rates of RNA degradationin cultured rat hepatocytes as a function of time and medium composition. As in perfused livers (Lardeux e t al., 1987) and isolated rat hepatocytes (Balavoine et al., 19901, the release of radioactive cytidine was used to determine the fractional rates of RNA degradation. In order to consider radioactive cytidine as a marker of RNA degradation, it was necessary to prevent its reutilization and exclude its production from radioactive RNA released in the culture medium. We observed that the addition of 0.5 mM unlabeled cytidine reduced the incorporation of [2-14C]cytidineby 98% in RNA of unlabeled hepatocytes (data not shown). No additional effect was observed a t higher concentrations of unlabeled cytidine. Furthermore, the effect of different concentrations of unlabeled cytidine (0.2, 0.5 and 1 mM) was tested on the release of radioactive cytidine from previously labeled hepatocytes. The release of radioactive cytidine was increased by 15% in the presence of non-radioactive cytidine, maximum effect being obtained with 0.2 mM. As in perfused rat liver (Lardeux et al., 1987) and isolated rat hepatocytes (Balavoine et al., 1990), we can consider that the presence of 0.5 mM of non-radioactive cytidine was required to prevent reutilization of radioactive cytidine. Experiments were also carried out with unlabeled ribosomal RNA or 2' and 3'CMP to exclude the possibility of radioactive cytidine production from radioactive RNA released in the culture medium. Unlabeled ribosomal RNA was used to act directly on extracellular degradation of radioactive RNA by isotopic dilution. Unlabeled nucleotides (2' + 3'CMP) were used to saturate the dephosphorylation step of radioactive CMP possibly produced from extracellular degradation of radioactive RNA in the medium. Under such conditions, the release of radioactive cytidine from previously labeled hepatocytes was not significantly reduced (data not shown), indicating that extracellular radioactive RNA did not represent a source of radioactive cytidine. For each period, net cytidine accumulation was calculated from radioactive cytidine (dpmldish) produced during 2, 4 or 6 h divided by the specific radioactivity of CMP residues in RNA (dpminmol). The latter was determined from the specific radioactivity of RNA (dpmlpg) divided by 1.737, value representing the ratio of the specific radioactivities, RNNCMP, for rat livers submitted to similar in vivo labeling procedures (Balavoine et al., 1990). In separate experiments without unlabeled cytidine, we observed that the specific radioactivity of cytidine released was constant throughout the period of measurement (42-48 h) and equal to the specific radioactivity of CMP residues in RNA. The specific radioactivity ratio: cytidineiCMP was equal to 0.975 2 0.023 (n = 4). Each absolute rate of RNA degradation (pgRNAihldish1 was determined by dividing net cytidine accumulation by the proportion of 3'CMP

61

T

xa

c 6

2 1 0 MEM+FUS MEM-FBS E B S S + F B S E B S S - F B S

Fig. 2. Effects of medium composition and FBS on fractional rates of RNA degradation. Rat hepatocytes from livers labeled in vivo with [6-'4Cloroticacid were cultured as described in Figure 1. As explained in the text, fractional rates of RNA degradation were calculated from radioactive cytidine release between 4 2 4 4 h (2 h), 4 2 4 6 h (4h), and 4 2 4 8 h (6 h). Results are means i SE of 3-4 separate experiments with duplicate dishes.

residues in RNA (0.87 nmolipg RNA; Lardeux et al., 1987). Fractional rates (%/h)were calculated for each time period (2, 4 and 6 h) by dividing absolute rate by initial RNA content (Fgidish). The latter was equal to RNA content a t 2, 4 or 6 h corrected for the RNA degraded during the same time period. As depicted in Figure 2, whatever the experimental conditions, fractional rates of RNA degradation measured over different periods of time (2, 4, or 6 h) between 42 and 48 h of culture, were similar with less variability a t the longest period (6 h). Thereafter, we have considered only the time period of 6 h between 42 h and 48 h of culture for the measurement of RNA degradation rates. In EBSS, fractional rates of 4.5%ih were observed, and agreed with those obtained during suspensions of freshly isolated hepatocytes incubated without amino acids (Balavoine et al., 1990).Fractional rates of RNA degradation varied with the medium composition. Addition of amino acids or FBS in the culture medium decreased RNA degradation. Our aim being to study the inhibitory effect of amino acids, we chose conditions of culture with maximum deprivation i.e., EBSS without FBS.

Effects of varying concentrations of complete mixture of amino acids on RNA degradation in cultured hepatocytes Results from experiments with graded levels of amino acids (Fig. 3) showed that the addition of a complete mixture of amino acids in the culture medium reduced RNA degradation considerably. Fractional rates obtained in the absence of amino acids ( O X ) were very high and averaged 4.4%lh. Maximal inhibition was obtained with l o x plasma amino acid concentration and in that case, rate of RNA degradation was

RNA DEGRADATION IN CULTURED RAT HEPATOCYTES

153

TABLE 2. Effect of cell density on amino acid inhibitory responses of RNA degradation in isolated rat hepatocytesl Amino acids (fraction or multiples of normal plasma concentration)

Cell densities ( 106 hepatocytes/ml)

0.5~

1

2.5

5

(n = 4)

(n = 5)

(n = 4)

(n = 3)

8.2 k 1.7 21.5 2.3 44.7 6.4 77.0 i 5.0

10.4 f 6.3 18.9 f 2.9 47.5 f 1.9 65.4 f 4.3

13.9 k 6.2 22.4 2.1 39.5 k 3.9 64.9 f 5.3

8.4 23.4 52.3 75.5

lx 2x 4x

I\\\\

0.5

f 3.7 f 6.4 k 4.7 5 3.0

**

‘Isolated hepatocytes from rats previously labeled in vivo with [6-”C]orotic acid were incubated for 2 h a s described (Balavoine et al., 1990). Fractional rates of RNA degradation weredetermined from the releaseofradioactivecytidine between 30 and 120 min of incubation. The inhibitory effect of amino acids determined from the difference with maximum rates (Ox) was expressed a s percentage of maximal deprivation responses (Ox 20~)Maximum . (Ox) rates were 5.51 0.52,4.78 0.39, 4.75 C 0.49, and 5.42 k 0.41 W h respectively for0.5,1,2.5 and 5.106hepatocytes/ml. Corresponding basal (20x1 rates were 1.29 C 0.10,1.39 i 0.16,1.51 0.24 and 1.53f 0.14 W h . Values are means f SE of three to five experiments (n).

*

~

0

+

*

+

AMINO ACIDS

1

TABLE 3. Effect of volume of culture medium on amino acid inhibitory responses of RNA degradation in cultured rat hepatocytes’ Amino acids Volume of medium (ml) (fraction or multiples of normal plasma 1 2 4 concentration) (0.8 x 106/ml) (0.4 x 106/ml) (0.2 x 106/ml)

0.5~

38.5 58.4 76.7 88.9

1X

2x 4x

f 4.2 i 5.2 i 5.1 f 2.8

42.8 f 7.8 60.9 f 9.5 79.6 f 6.6 87.5 f 8.6

42.2 f 3.3 60.7 k 2.4 79.1 f 2.3 93.3 k 1.5

‘Hepatocytes from rats labeled in vivo with [B-“C]orotic acid were maintained in culture for 42 h a s mentioned under Methods. From 42 to 48 h, hepatocyte cultures were carried out a s described in Figure 3 except that varying volumes of medium from 1 to 4 ml per dish were used. Values in parentheses indicate the estimate of hepatocytenumberperml ofmediumadded. Thenumberofhepatocytesperdish was calculated from the quantity of DNA per dish (15.3 k 2.3 pg, n = 7) and the concentration of DNAper lohhepatocytes (18.4 j, 1.1 pg, n = 5 ) . Fractional rates of RNA degradation were calculated from the release of radioactive cytidine during 6 h. The inhibitory effect of amino acids determined from the difference with maximum rates (Ox) was expressed a s percentage of maximal deprivation responses (Ox minus lox). Maximum (Ox) rates were 4.79 0.30, 5.58 k 0.73, and 4.37 k 0.37 %/h respectively for 1, 2, and 4 ml of medium. Corresponding basal ( 1 0 ~ )rates were 2.02 i- 0.07, 2.34 0.25, and 1.58 f 0.27 W/h.Values are means SE of four to six exueriments.

*

Fig. 3. Effect of varying concentrations of amino acids on RNA degradation in cultured rat hepatocytes. Hepatocytes from rat previously labeled in vivo with [6-14Cloroti~acid were cultured for 42 h as described under Methods. Between 42 and 48 h of culture, hepatocytes were maintained in EBSS ( O X ) or EBSS with fraction ( 0 . 5 ~ or ) multiples (1X, 2 X , 4 x , 10 x , 2 0 1 ~of a complete plasma amino acid mixture (Woodside and Mortimore, 1972; Lardeux and Mortimore, 1987). RNA degradation rates were determined from the release of radioactive cytidine accumulated during 6 h between 42 and 48 h. Each symbol represents the rates of RNA degradation in cultured rat hepatocytes from a different rat. Inset: all values were corrected for basal rates (10 x ) and expressed as percentage of maximal deprivation responses (Ox minus 1 0 ~ )Results . are means t SE of six separate experiments.

equal to 1.5%/h. No further inhibition was seen with 2 0 plasma ~ amino acid concentration. RNA degradation in cultured hepatocytes was very sensitive to physiological concentration of amino acids (Fig. 3 inset): for 0.5 x and 1x plasma amino acid concentration, RNA degradation under amino acid control (OX minus l o x ) was inhibited by 40 and 60% respectively.

Effect of cell density on RNA degradation responses to amino acids in isolated hepatocytes and cultured hepatocytes When isolated hepatocytes were incubated a t varying cell densities from 0.5 to 5 x lo6 cells/ml, the inhibition of RNA degradation to a given concentration

+

*

of amino acids was not modified (Table 2). At 0.5 x and 1X normal plasma concentrations of amino acids, RNA degradation was decreased by 10 and 20% respectively. In cultured hepatocytes maintained with different volumes of culture media from 1 to 4 ml per dish during the experimental period ( 4 2 4 8 h), the sensitivity of RNA degradation to amino acids was not affected (Table 3). However, the inhibitory effect of physiological concentration of amino acids was 4 to 3 times higher in cultured than isolated hepatocytes, even if the total quantity of amino acids available was approximately identical (compare first column Table 2 with second column Table 3 and second column Table 2 with first column Table 3).

Inhibitory effect of 3-methyladenine on RNA degradation in cultured hepatocytes Addition of 10 mM of 3-methyladenine, a n inhibitor of autophagic vacuole formation (Seglen and Gordon, 1982), decreased RNA degradation rates when hepatocytes were maintained in culture without amino acids or a t their physiological concentration (0.5 to 2 x ; Fig. 4).However, no effect was observed when hepato-

154

6

BALAVOINE ET AL

I

1

4L\

1

32-

revealed by a dark staining of the lysosomal structures as observed by Lake et al., (1987) and Dunn (1990). The vacuoles positive for acid phosphatase were more abundant with an increased size in cells incubated in EBSS without amino acids (Figs. 5C, D), than in cells cultured with media containing amino acids (MEM and EBSS with 1 0 amino ~ acids) as illustrated in Figs. 5A and B. Moreover, autophagosomes usually negative for acid phosphatase (Schworer et al., 1981; Pfeifer, 1987; Dunn, 1990) were easily seen and appeared more numerous in cells cultured without amino acids (Figs. 5C, D).

DISCUSSION Our knowledge concerning the mechanism of RNA degradation in liver and its regulation by nutritional and hormonal factors has been recently improved by using in vitro experimental models derived from rat liver (Lardeux and Mortimore, 1987; Balavoine et al., 1990). Both perfused livers and isolated hepatocytes 011 I I 1 I have shown advantages and limits in their application. 0 1 2 4 10 AMINO ACIDS Considering the practical aspect, the use of isolated (multiples of normal concentration) cells presents evident advantages compared to perfused tissues. Because different cell populations exist in the Fig. 4. Effect of 3-methyladenine on RNA degradation in cultured liver, the isolation and the purification of a subset of rat hepatocytes. Hepatocytes previously labeled in vivo with cells (hepatocytes) allow studies concerning these cells [6-14C]orotic acid were cultured for 42 h as described under Methods. 3-Methyladenine ( A -- - - A1 at lOmM final concentration in EBSS was directly without interaction of other cell populations. In tested in the absence or the presence of amino acid at fraction or addition, isolated cells allow comparisons between cells multiples of normal plasma concentration; @-A), cultured hepatocytes without 3-methyladenine. Fractional rates of RNA degradation from a single tissue reducing the variability that appears between rats. However, the major limitation of were determined from the release of radioactive cytidine between 42 and 45 h of culture. Each point represents the mean of duplicate freshly isolated rat hepatocytes is their reduced sensidishes. tivity to external regulators. We have recently observed such alteration for the regulation of RNA degradation by amino acids. The degree of responsiveness was largely reduced in isolated hepatocytes compared to perfused livers (Balavoine et al., 1990). In order to resolve this important question of sensitivity, we decided to study the regulation of RNA degradation in cytes were incubated in medium with l o x plasma hepatocytes by using another experimental model amino acid concentration. These results suggested that where cells have recovered from the stress due to the the macroautophagy is involved in the increase of RNA isolation by collagenase. Primary cultures of hepatocytes have been shown to recover a high degree of degradation in cultured rat hepatocytes. sensitivity for many functions to external regulators. Effects of amino acid deprivation on For example, the capacity to actively transport amino ultrastructural aspects of cultured acids was largely improved after few days of culture rat hepatocytes (Kletzien et al., 1976). General ultrastructural features of the hepatocytes As expected from previous studies with perfused maintained in MEM without FBS between 24 and 42 h, livers (Lardeux et al., 1987) and isolated rat hepatowere similar to those described by Wanson et al., (1977) cytes (Balavoine et al., 1990), the release of radioactive and Yu and Marzella (1988). We confirmed these cytidine from in vivo-labeled RNA is also a valuable observations particularly for lysosomal structures (au- marker of RNA degradation in cultured rat hepatotophagosomes, secondary lysosomes and dense bodies) cytes. Because RNA is pulse-labeled at least 60 h before which were more abundant than in livers from normal hepatocyte isolation and for the reasons mentioned fed rats (Loud, 1968; Weibel et al., 1969; Yu and earlier (Blobel and Potter, 1968; Lardeux et al., 1987; Marzella, 1988). For hepatocytes maintained in EBSS Balavoine et al., 19901, degradation rates are mainly alone between 42 h and 48 h, lysosomal structures as representative of the ribosomal RNA fraction. defined above were more numerous as compared with After 48 h of culture, hepatocytes clearly recover hepatocytes a t 42 h of culture in MEM, or those their sensitivity with high degree of responsiveness of maintained in EBSS with amino acids at 10 times RNA degradation around physiological concentrations normal plasma concentration (data not shown). of amino acids (Fig. 3 and Table 3). RNA degradation For a better morphological appreciation of lysosomal under amino acid control is reduced by 40 and 60% modifications, we located the acid phosphatase, the respectively at half ( 0 . 5 ~ )and normal ( l x ) plasma most characteristic lysosomal enzyme, by histochemi- concentration of amino acids, inhibition similar to that cal staining (Fig. 5). The presence of this enzyme was observed in perfused livers (Lardeux and Mortimore,

RNA DEGRADATION IN CULTURED RAT HEPATOCYTES

155

Fig. 5. Effects of amino acids on lysosomal structures of rat hepatocytes cultured in different media. Histochemical staining of acid phosphatase at the ultrastructural level. (A) MEM at 42 h. (B) EBSS with amino acids ( l o x normal plasma amino acids) at 48 h. (C) and (D) EBSS without amino acids at 48 h. The presence of acid phos-

phatase is demonstrated by electron dense products located only in different lysosomal structures except the autophagosomes (arrows).A non-specific reaction product is visible in the nuclei and the cytoplasm. (A-C: x 3,000; D x 6,500).

1987). The degree of inhibition in both experimental models was also identical for higher amino acid concentrations. In contrast, the results presented in Table 2 extend and confirm our previous observation from freshly isolated rat hepatocytes. At normal levels of amino acids, RNA degradation is inhibited by only 10-20%, several times less than in primary cultures of hepatocytes or perfused livers. This difference of sensitivity was also observed for intracellular protein degradation where unphysiologically high concentrations of amino acids are required in order to obtain substantial inhibition in isolated rat hepatocytes compared to perfused livers (Seglen et al., 1980; Schworer et al., 1981; Mortimore, 1987; Seglen, 1987). Two main reasons could explain these differences of sensitivity: (1)the rapid modification of amino acid

concentrations when high densities of hepatocytes are maintained in the same medium, and (2) the alteration of amino acids transport in freshly isolated hepatocytes as a result of collagenase digestion. Using the perifusion of freshly isolated hepatocytes, a method which maintains constant concentrations of amino acids, Leverve et al., (1987) showed that the control of intracellular protein breakdown by amino acids was improved. It is clear from our results (Table 2) that decreasing cell density by 10-fold from 5 x lo6 cells/ml to 0.5 x lo6 cells/ml, did not improve the responsiveness of RNA degradation to amino acids. We can consider that the total quantity of extracellular amino acids is not a limiting factor of their inhibitory effect. This observation was confirmed in cultured rat hepatocytes (Table 3 ) indicating that the concentration of amino

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acids determined the inhibitory effect of RNA degradation. We must consider alterations of amino acid transport and/or intracellular concentration as the most probable explanation for the differential response between our two experimental models. It is known that the capacity of amino acid transport is higher in cultured hepatocytes than in freshly isolated hepatocytes (Kletzien et al., 1976). However, there is no information concerning alterations of intracellular concentrations of amino acids when their extracellular concentrations were modified. In order to study the possible relationship between amino acid transport, intracellular concentrations of amino acids, and rates of RNA degradation in hepatocytes, it is important to have more details concerning the qualitative aspect of RNA degradation control by amino acids. As for intracellular protein degradation in perfused rat livers (Mortimore et al., 1989), high levels of a limited number of amino acids suppressed RNA degradation by the same extent than a complete mixture (Lardeux and Mortimore, 1987). However, at physiological concentrations, the inhibition of RNA degradation by these regulatory amino acids is not yet known. These important questions must be resolved in the future. Although the responsiveness of RNA degradation to amino acids was greatly improved in cultured hepatocytes, the fraction of RNA catabolism insensitive to amino acids or basal rate remained unexpectedly high as compared to perfused livers (1.5%/h vs 0.3%/h). One can explain such high basal rates of RNA degradation by the presence of non-viable hepatocytes in our cultures without fetal bovine serum. These hepatocytes could degrade RNA molecules without amino acid control. However, the presence of serum which improved cell viability, was not associated with a decrease of the basal rate of RNA degradation (data not shown). Another explanation could be postulated from our observations concerning the lysosomal system. It was previously shown (Lardeux et al., 1987; Balavoine et al., 1990))from morphological and biochemical data, that macroautophagy is the cellular mechanism responsible for the acceleration of intracellular RNA degradation above basal rates. Addition of amino acids strongly inhibited the macroautophagic process (Schworer et al., 1981). We have partially confirmed these observations in primary cultures of hepatocytes. However, with high concentrations of amino acids, lysosomal structures remained abundant in the cytoplasm of cultured hepatocytes as compared with perfused livers. In addition, the inhibition of autophagic vacuole formation by 3-methyladenine, was less efficient in suppressing induced macroautophagy and had no effect on the basal rates of RNA degradation (Fig. 4). It seems that in cultured hepatocytes, the mechanism of macroautophagy will proceed even with high levels of amino acids. This incomplete suppression of macroautophagy could explain, a t least in part, the high basal rates of RNA degradation in cultured hepatocytes. Furthermore, lysosomal pathways other than macroautophagy exist in rat liver. It has been shown by Mortimore et al., (1988) that microautophagy is involved in basal degradation of intracellular proteins. However, a role of microautophagy in basal rates of

RNA degradation is not documented, although recent observations (Heydrick et al., 1991) indicate that the lysosomal pathway is probably involved in the basal degradation of liver RNA.

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