Rates of Protein Synthesis by Hepatocytes Isolated from Rats of Various Ages GEORGE A. RICCA, DANIEL S. H. LIU, JOHN J. CONIGLIO AND ARLAN RICHARDSON Department of Chemistry, Illinois State Uniuersity, Normal, Illinois 61 761
ABSTRACT The rate of total protein synthesis in isolated hepatocytes was determined. The incorporation of L-13Hlvalineinto protein is linear for a t least two hours of incubation and is affected by the concentration of amino acids in the medium. Uptake of valine by hepatocytes from 1.5- and 18-month-old rats was identical and appears to occur by simple passive diffusion. Within five minutes, the specific activities of the intracellular and extracellular valine pools are equivalent. The specific activities of these pools are saturated by 1.6 mM valine and remain constant for 60 minutes of incubation. The rates of protein synthesis by hepatocytes from 1- to 2-month-old rats is 96.8 pmoles of valine per minute per milligram protein. This is comparable to rates of protein synthesis reported for perfused liver and liver in vivo and is approximately 64% higher than the rate of protein synthesis by hepatocytes from 18-month-old rats. Liver protein synthesis has been studied primarily in cell-free systems since the development of the first cell-free protein synthesis system by Zamecnik and Keller ('54). Cell-free systems allow an investigator to carry out routine manipulations and control experimental conditions. In addition, this type of a system allows one to study the molecular mechanism of liver protein synthesis. However, the rate of cell-free protein synthesis by liver is only 1% the rate observed in vivo (Henshaw et al., '71; Waterlow, '75). The fact that 99% of the protein synthetic activity of liver is lost in cellfree systems warrants the search for other systems which could be used to study liver protein synthesis. Although i t would be preferable to use whole animals to measure liver protein synthesis, it is difficult to manipulate and control experimental variables using whole animals. In addition, protein synthesis in whole animals is complicated by the absorption of the amino acids, compartmentation of the amino acids, and competing reactions (Neuberger and Richards, '64; Waterlow, '69; Munro, '69; Vidrich et al., '77). To allow an investigator to control and manipulate experimental variables in liver protein synthesis, the perfused liver has been used (Rannels et al., '75). However, this system is not suitable for routine J. CELL. PHYSIOL. (1978)97: 137-146.
studies and also shows compartmentation of amino acids (Mortimore et al., '72; Khairallah and Mortimore, '76). Single cell suspensions of hepatocytes offer a system for studying protein synthesis with several advantages over whole animal and perfused liver systems. It is also suited to routine studies where many homogenous samples can be studied simultaneously under a variety of experimental conditions. Since Schreiber and Schreiber ('72, '73) determined the optimum conditions for protein synthesis by hepatocytes, the use of hepatocytes to study protein synthesis has grown. In almost all studies, protein synthesis in hepatocytes has been measured by determining the amount of radioactively-labeled amino acid incorporated into protein. However, radioactive isotope incorporation data do not give a true measure of the rate of protein synthesis. The incorporation rate of a radioactively-labeled amino acid into protein is a direct function of the specific activity of the amino acid precursor pool used for protein synthesis. There have been no studies where the specific activity of the amino acid precursor pool has been measured and used to determine the rate of protein synthesis in hepatocytes. In this study, the specific activities of the intraReceived July 15, '77. Accepted Apr. 10, '78
137
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RICCA, LIU, CONIGLI 0 AND RICHARDSON
cellular and extracellular L-valine pools were measured and the actual rate of protein synthesis by hepatocytes determined. Rates of protein synthesis by hepatocytes can be easily determined. These rates are comparable to rates of protein synthesis reported for perfused liver and liver in vivo. MATERIALS AND METHODS
Isolation of hepatocytes
omitting L-valine. L-[2,33Hlvaline (0.85 mCi/ mmole) was added to the mixture at a concentration of 1.6 mM. These conditions were found to be optimal for protein synthesis. Samples (1.5 ml) were withdrawn a t various times and protein synthesis terminated by the addition of the cell suspension to an equal volume of 10% trichloroacetic acid. The suspension was heated a t 90°C for 15minutes and the precipitate collected by centrifugation. The precipitate was washed twice with 5%trichloroacetic acid and once with each of the following: NaOH neutralized ethano1:anhydrous ethyl ether (2:l), acetone, and anhydrous ethyl ether. The precipitate was air-dried and dissolved in 0.3 N NaOH. Aliquots of the NaOH solution were taken to determine t h e radioactivity by liquid scintillation counting and the protein concentration by the method of Lowry et al. ('511, using bovine serum albumin as the standard.
Female rats were purchased from ARW Sprague-Dawley and maintained as described previously (Layman et al., '76). Hepatocytes were obtained by a modification of the method of Howard et al. ('73). Animals were killed by decapitation between 9:00 and 11:OO A.M. to minimize diurnal variation. The liver was immediately removed, rinsed twice in ice-cold calcium-free Hanks' solution (Hanks and Wallace, '49), and perfused through the portal vein with 15-30ml of an enzyme solution conDetermination of the extracellular taining 0.05% collagenase in calcium-free and intracellular valine Hanks' solution supplemented with 1mM specific activity pyruvate, a vitamin mixture (Eagle, '59), twice the level of essential and non-essential Studies on valine uptake by hepatocytes amino acids described by Eagle ('59), and were performed by incubating the hepatocytes 50 mM Hepes (N-2-hydroxyethyl-piperazine- (3,000,000 cells per ml) in the protein syntheNf-2-ethanesulfonicacid) buffer pH 7.3. Using sis system described above. Inulin ([''Cleara hand-held Stadie-Riggs Tissue Slicer, slices boxylic acid, 5 mCi/mmole) was added to the 0.3-0.4 mm thick were prepared. The liver incubation medium as an extracellular slices (2 to 3 g) were incubated in 10 ml of the marker at a concentration of 1pCi/ml. After enzyme solution for 60 minutes at 37°C under incubation, the cell pellet and extracellular an atmosphere of 95: 5 0xygen:carbon dioxide. medium were obtained by low speed cenCalcium chloride (1.26 mM final concentra- trifugation. Twenty percent of the [14Clinulin tion) was added to this suspension after 30 to routinely was found associated with the cell 35 minutes of incubation. The suspension was pellet. This indicates that the cell pellet confiltered through stocking nylon and then tains a considerable portion of extracellular through a single layer of nylon mesh (pore size fluid. The acid-soluble extracts were obtained 60 pm). The cells were then obtained by low from the cell pellet and extracellular medium speed centrifugation and washed as described by treating these fractions with 5%trichloroby Howard et al. ('73). Using this method, the acetic acid. From the amount of [14Clinulin percent viable cells was routinely 90-95% as associated with the cell pellet, the extracelludetermined by trypan blue exclusion. lar valine contribution to the cell pellet could be determined. By subtracting this contribuProtein synthesis by hepatocytes tion from the total acid-soluble valine associProtein synthesis was followed by incuba- ated with the cell pellet, the intracellular (intion of approximately 3,000,000 cells per mil- ulin-impermeable) valine content of the cell liliter 37°C under an atmosphere of 95:5 pellet was determined. The specific activities of the intracellular 0xygen:carbon dioxide in a modified Eagles Minimal Essential Medium (Eagle, '59). This and extracellular acid-soluble valine were medium was composed of Hanks' solution sup- determined by passing the acid-soluble explemented with 1 m M pyruvate, a vitamin tracts over Amberlite CG-120 (H') columns mixture (Eagle, '59), 10 mM Hepes buffer pH (5.5 x 35 mm) preequilibrated with 5% tri7.3, and twice the level of essential and non- chloroacetic acid. The columns were rinsed with 2ml of distilled water and the amino essential amino acids described by Eagle ('59),
139
PROTEIN SYNTHESIS BY RAT HEPATOCYTES
Minutes Fig. 1 The time course of the appearance of acid-insolubleradioactivity in the cell pellet ( 0 )and extracelrepresent the sum of the radioactivity in the cellular lular medium (H) was determined. The open circles (0) and extracellular fractions. The initial viability of the hepatocyte preparation was 96%,and the percent of viable cells throughout the incubation period is indicated by the numbers.
'
acids eluted from the columns with 2 ml of 5 N NH,OH. The NH,OH solutions were evaporated to dryness and the resulting residues dissolved in 0.2 M sodium phosphate buffer (pH 8.8). The amino acids in the phosphate buffer were dansylated and separated by 2-dimensional thin-layer chromatography a s described by Zanetta et al. ('70). The material migrating as dansyl-valine was identified under a n ultraviolet-light. This portion of the chromatogram was removed, and the dansylvaline was extracted with 100%methanol. The radioactivity in the methanol solution was determined by liquid scintillation counting, and t h e dansyl-valine concentration was determined with a Turner 110 fluorometer (Turner Filters: primary, 7-60; secondary, 2A-12) using dansyl-valine as a standard.
In vivo liver protein synthesis Liver protein synthesis in vivo was measured after i.p. administration of 0.33 mCi L[3,43Hlvaline ( 3 6 mCi/mmole) per 100 g body weight to female Sprague-Dawley rats. After 12 minutes the rats were killed by decapita-
tion. The liver was removed and homogenized in 3 volumes of buffer ( 0 . 0 5 ~cacodylate, 0.17~ NaCl, 0 . 3 3 M sucrose, pH 6.0) with a Potter-Elvehjem homogenizer. Samples of the homogenate were used to determine t h e amount of L- [3Hlvalineincorporated into acidprecipitable material per milligram of liver homogenate protein. This was accomplished by the procedures described for hepatocytes. Aminoacyl-tRNA was isolated from the rest of the homogenate and deacylated as described by Wallyn et al. ('74). The specific activity of the valine attached to tRNA in vivo was determined by dansylation and thin layer chromatography as described previously in the MATERIALS AND METHODS. RESULTS
Figure 1 shows the time course for the appearance of radioactive-label into intracellular and extracellular proteins. Incorporation of L-[3Hlvalineinto intracellular proteins was linear for the first 60 minutes of incubation. Acid-insoluble radioactivity appeared in the extracellular medium after 30 to 45 min-
140
RICCA, LIU, CONIGLIO AND RICHARDSON
I 2 3 4 AMINO ACID LEVELS Fig. 2 The incorporation of L-L3HHlvalineinto acid-insoluble material was determined a s described in the MATERIALS AND METHODS, using hepatocytes from 3- to 4-month-old rats. Hepatocytes were incubated for 30
minutes in the presence of the various levels of the amino acids b o t h the non-radioactively-labeledamino acids and the L-PHhaline). The levels of the amino acids are expressed in terms of the levels of essential and non-essential amino acids reported by Eagle ('591.
Utes of incubation. Several investigators (Weigand and Otto, '74; van Bezooijen et al., '76) have reported a similar lag period for the appearance of albumin in the extracellular medium following incubation of hepatocytes. When the sum of the intracellular and extracellular acid-insoluble radioactivity was plotted, protein synthesis by hepatocytes was linear for a t least two hours of incubation. During the 2-hour incubation period, the viability of the hepatocytes decreased less than 10%. Optimal conditions for protein synthesis by hepatocytes was found to be similar to that reported by Schreiber and Schreiber ('72). In determining the optimum conditions, special attention was given to the effect of the concentration of the amino acids on protein synthesis. Several laboratories have reported that protein synthesis in perfused liver or in liver in vivo is affected by the level of the amino acids (Pronczuk et al., '68; Sidransky e t al., '68; Jefferson and Korner, '69; McGown et al., '73). Extensive studies by Schreiber and Schreiber ('72, '73) with 20 different radioactively-labeled amino acids showed that the incorporation of each radioactively-labeled amino acid into protein increased when the
concentration of that amino acid in the incubation medium was increased. Using P4CCllabeled algae protein hydrolysates, Seglen ('76) showed that the incorporation of radioactivity into protein by hepatocytes exhibited saturation kinetics as the concentration of the hydrolysate was increased. However, no studies with hepatocytes have specifically determined the effect of the non-radioactively-labeled amino acids on the incorporation of the radioactively-labeled amino acid. In our studies a mixture of amino acids, as described by Eagle ('591, was used with L-VHlvaline. Valine was chosen as the radioactive tracer to measure protein synthesis because i t is widely distributed in protein, and it is neither synthesized nor degraded by liver tissue (Elwyn et al., '68; Mortimore and Mondon, '70). Figure 2 shows the effect of various concentrations of this amino acid mixture on L-L3HIvaline incorporation into acid-insoluble material. L-F3H1valine incorporation increased as the concentration of the amino acids was increased. At amino acid concentrations above 2-times Eagle's level, the hepatocyte system was saturated and L-F3HIvalineincorporation plateaued. Very high concentrations of the amino acids were found to be slightly inhib-
141
PROTEIN SYNTHESIS BY RAT HEPATOCYTES
itory. Table 1 shows the effect of the non-radioactively-labeled amino acids on protein synthesis by hepatocytes. Omission of all of t h e non-radioactively-labeled amino acids from the incubation medium decreased the incorporation of L-13Hlvalineinto acid-insoluble material over 30%.Omission of either essential or non-essential amino acids also decreased protein synthesis 30%. Addition of increasing concentrations of the non-radioactively-labeled amino acid mixture to hepatocytes containing saturating levels of L-L3Hlvaline (1.6 mM) gave a saturation curve similar to t h a t shown in figure 2 (data not shown). The stimulation of valine incorporation by amino acids could be due to an increased availability of energy (through amino acid catabolism) for the hepatocytes for protein synthesis. To test this possibility, hepatocytes were incubated with various concentrations (1-20 mM) of either pyruvate, pyruvate and lactate, or succinate in a system containing saturating levels of L-PHlvaline (1.6 mM) and low levels of the amino acid mixture (0.1times Eagle's level). In no case was protein synthesis stimulated by the addition of these compounds (results not shown). In fact, high concentrations of these compounds resulted in a slight decrease in protein synthesis. Preliminary studies in our laboratory show that amino acids do not stimulate RNA synthesis by hepatocytes and that the omission of just one amino acid from the amino acid mixture decreases protein synthesis by hepatocytes. Therefore, it appears that amino acids specifically stimulate protein synthesis in hepatocytes suspensions because of their effect on the protein synthetic apparatus. This situation appears to be analogous to the situation in perfused liver and liver in vivo (Pronczuk et al., '68; Sidransky e t al., '68; Jefferson and Korner, '69; McGown e t al., '73). I n studying protein synthesis in cell suspensions using a radioactively-labeled amino acid, attention must be given to the uptake of the radioactivity-labeled amino acid by the cells. Observations by Schreiber and Schreiber ('72) and Kletzien e t al. ('76) have shown that hepatocytes are freely permeable to methionine, leucine, and a-aminoisobutyric acid. Recently, studies have shown that the transport of a-aminoisobutyric acid (LeCam and Freychet, '77) and the branched chain amino acids, leucine, isoleucine, and valine (McGivan et al., '77) occurred by both facilitated transport and passive diffusion in hepatocytes. Initial stud-
TABLE 1
Effect o f the levels o f t he non-radioactiuely labeled amino acids on protein synthesis by hepatocytes ' Level of amino acids
dpm/pg protein
o x
1,870-t 177 2,3502 242 2,7292 194
0.5 X 2 x 2 X (minus the non-essential amino acids) 2 X (minus the essential amino acids)
1,856?216 1,910&181
z Incorporation of L-[3Hlvaline into acid~insolublematerial by hepatocytes from 3- to 4-month-old rats was determined after a 30-minUte incubation. The protein synthesis system was similar to that described in the MATERIALS AND METHODS except that the concentration of the amino acid mixture (which lacks valine) was varied as indicated above. The concentration of L-13Hlvaline was 1.6 mM in each experiment. The levels of the amino acid mixture are given in terms of the levels of essential and non~essentialamino acids reported by Eagle ('59). Each value represents the mean 2 S . E . for three experiments.
ies (data not shown) indicated that when hepatocytes were incubated with L-PHlvaline, the radioactivity in the intracellular and extracellular fractions equilibrated very rapidly. One wash with 0.9% NaCl removed over 95% of the L-13HIvaline associated with the hepatocytes. Figure 3 shows that the uptake of L-PHIvaline by hepatocytes from 1.5- and 18-month-old rats was linear up to extracellular valine concentrations of 10 mM. There was no age-difference in valine uptake by hepatocytes nor was any saturation of uptake observed. The lack of saturation of valine uptake by hepatocytes is in agreement with the observations of Schreiber and Schreiber ('72) for L-methionine and L-leucine and the observations of McGivan et al. ('77) for L-leucine, L-isoleucine, and L-valine. Addition of cyanide to hepatocytes or decreasing the temperature from 37" to 4" had only a slight effect on valine uptake (table 2). McGivan et al. ('77) have reported that the transport of the TABLE 2
Effect of cyanide and temperature on d i n e uptake by hepatocytes ' L-t3Hlvaline concentration
0.4 mM 4.0 mM
dpm/lO5 cells Control
KCN
4°C
560 4,752
568 4,456
496 4,056
' Hepatocytes were incubated for five mlnutes as described in figure 3 with the two concentrations of L-[3Hlvaline.The intracellu~ lar radioactivity associated with the hepatocytes was determined for cells incubated at 37°C with and without KCN (5 x lo-' MI. and for cells incubated at 4°C.
142
RICCA, LIU, CONIGLIO AND RICHARDSON
0
10
5 Extracellular
Valine ( m M )
Fig. 3 The uptake of L-PHhaline by hepatocytes from 1.5 (0)and 18 fO)-month-old rats as a function of the extracellular L-PHlvaline concentration is shown. Cells were incubated in the protein synthesis system with various concentrations of L-13Hlvaline (0.85mCi/mmole) for five minutes. Cells were collected by centrifugation and the acid-insoluble radioactivity in the intracellular valine determined as described in the MATERIALS AND METHODS.
i
Ir)
Valine Concentration (mM)
Time (min)
Fig. 4 The specific activities of the intracellular (0) and extracellular ( 0 )valine were determined after incubating hepatocytes with L-PHlvaline in the protein synthesis system described in the MATERIALS AND METHODS. In graph A, cells were incubated with each concentration of L-PHhaline for five minutes while in graph B, cells were incubated with 1.6 m M L-VHlvaline for various times.
143
PROTEIN SYNTHESIS BY RAT HEPATOCYTES
branched chain amino acids by hepatocytes is not inhibited by ouabain or 2,4-dinitrophenol. To measure protein synthesis accurately, it is essential t h a t the specific activity of the amino acid pool used for protein synthesis be determined. Using t h e techniques described in t h e MATERIALS AND METHODS, the specific activities of the intracellular and extracellular L- [3Hlvaline were determined after hepatocytes were incubated for five minutes with 0.4 to 4.0 mM L-[3Hlvaline (fig. 4A). The specific activities of the intracellular and extracellular pools were essentially equivalent a t each concentration of L-13Hlvaline tested. At valine concentrations greater than 1.6 mM, the specific activities of both the extracellular and intracellular L-13Hlvaline pools reached steady state. The level of valine required to reach this steady state was dependent on the hepatocyte concentration. Higher hepatocyte concentrations increased the concentration of L-valine required to reach steady state. When hepatocytes were incubated with 1.6 mM L-[3Hlvaline, the specific activities of both valine pools were equivalent after five minutes of incubation and remained constant throughout 60 minutes of incubation (fig. 4B). Therefore, hepatocytes provide a system in which the rate of protein synthesis can be determined from the specific activity of either t h e extracellular or intracellular valine pool. Table 3 gives the rates of valine incorporation into acid-insoluble material by hepatocytes isolated from 1-to 18-month-oldrats. A 30-minute incubation period was chosen to determine the rates of protein synthesis by hepatocytes because very little change occurred in t h e viability of the hepatocytes during the initial 30-minute incubation (fig. 1). Furthermore, during this time period, the specific activities of the intracellular and extracellular valine were equivalent and remained constant (fig. 4B). The rates of protein synthesis by hepatocytes isolated from 1- to 18-month-old rats ranged from 96.8-34.5 pmoles of valine per minute per milligram protein. Protein synthesis by hepatocytes decreased as the age of t h e rats from which the hepatocytes were isolated increased. A 64% decrease in protein synthesis occurred from 1 to 12 months with no significant change in protein synthesis from 12 to 18 months. The decreased rate of protein synthesis by hepatocytes from older rats observed in table 3 could be a n age-related phenomenon or it could be t h a t hepatocytes from the older rats
TABLE 3
Rates ofprotein synthesis by hepatocytes from rats ofdifferent ages I Rate of valine incorporation (pmolesiminimg protein)
Age (months)
1-2 6-7 11-13 18
n
Mean
S.E.
4 5 4 5
96.8 76.4 35.2 34.5
5.6 1.5 2.5 3.3
1 Linear regression analysis of the amount of L-13Hlvalineincorporated into acid-insoluble material after 10, 20, and 30 minutes of incubation was used to determine the rate of L-PHlvaline incorporation. The pmoles of valine incorporated was determined by dividing the rate of L-PHlvaline incorporated by the specific activity of the extracellular valine pool. In all experiments, the viability of the cells at the end of the 30-minute incubation period was above 90%.The decrease in protein synthesis from 1- to S~monthto 6- to 7-month-old animals is significant at the P < 0.025 level, while the decrease in protein synthesis from 6- to 7-month to 11- to 13-month-old animals is significant at the P < 0.0005 level using the student’s t test (unpaired).
are more prone to damage during the isolation procedure than hepatocytes from younger rats. To differentiate between these two possibilities, the rate of in vivo liver protein synthesis by 3- and 15-month-old rats was determined by measuring t h e incorporation of L-[3H]~aline into acid-insoluble material and by determining the specific activity of the liver valyl-tRNA as described by Airhart et al. (‘74). Table 4 clearly shows that the in vivo rate of liver protein synthesis was lower in 15month-old rats than in 3-month-old rats. The 15-month-old rats showed a 64% decrease in the incorporation of radioactivity into acid-insoluble material compared to the 3-month-old rats; however, the specific activity of the valyl-tRNA was also lower in the 15-monthold rats. When the actual rate of valine incorporation into protein was determined from the specific activity of the valyl- tRNA, a decrease of approximately 50% was observed. TABLE 4
Rates of in uiuo protein synthesis
I
Age of animal 3 months 15 months
Rate of L-PHlvaline incorporation (dpmlminlmg protein) Specific activity of Valyl-tRNA (dpm/pmole) Rate of valine incorporation fpmole/min/mg protein)
3,070 12.0 256
1,104 8.3 133
’ The rate of valine incorporation per milligram protein in the liver homogenate was determined a8 described in the MATERIALS AND METHODS. Each value represents the mean of two animals.
144
RICCA, LIU, CONIGLIO AND RICHARDSON DISCUSSION
Although hepatocytes have been used by numerous investigators to study various aspects of protein synthesis, most of these studies have measured protein synthesis simply by following the incorporation of radioactively-labeled amino acids into protein. Generally, no attempt is made to determine t h e actual rate of total protein synthesis. Only in reports where albumin synthesis has been studied, has the rate of protein synthesis (in this case the synthesis of one specific protein) in hepatocytes been determined. Initial studies indicated that the rate of albumin synthesis by hepatocytes was only 10%of that reported for liver in vivo (East et al., '73; Crane and Miller, '74). More recently, Jeejeebhoy et al. ('75) and van Bezooijen et al. ('76) have reported rates of albumin synthesis in hepatocytes 40-80% of that reported for liver in vivo. We were interested in determining the rate of total protein synthesis in hepatocytes and comparing i t to rates of protein synthesis reported for perfused liver and liver in vivo. However, when using a radioactively-labeled amino acid to measure the rate of protein synthesis in whole cells, it is necessary to deter. mine the specific activity of the amino acid pool used for protein synthesis. Recent observations with cultured hepatoma cells, perfused liver, and liver in vivo indicate that the specific activity of the aminoacyl-tRNA is different from the specific activities of either the intracellular or extracellular amino acid pools (Airhart et al., '74; Hod and Hershko, '76; Khairallah and Mortimore, '76; Vidrich et al., '77). These studies indicate that the aminoacyl-tRNA derives its amino acids from both t h e extracellular and intracellular pools. Therefore, the specific activity of the aminoacyl-tRNA pool must be known to measure the actual rate of protein synthesis. The need for determining the specific activity of valyltRNA, however, can be circumvented by expanding the extracellular valine pool. Mortimore's group (Mortimore e t al., '72; Khairallah and Mortimore, '76) has demonstrated that when the level of valine in liver perfusates is increased to 5-10 mM, the specific activities of t h e intracellular and extracellular valine are equivalent. Furthermore, under these conditions the ratio of the specific activities of valyl-tRNA to the extracellular valine is approximately one.
Our studies indicate t h a t the transport of valine by hepatocytes occurs rapidly, is not temperature or energy dependent, and is linear up to L-valine concentrations of 10 mM. Comparison of the specific activities of t h e extracellular and intracellular valine in hepatocytes demonstrates t h a t within five minutes the specific activities of these two valine pools are equivalent. Therefore, it appears t h a t L-valine is not compartmentalized by hepatocytes; rather, the valine in the intracellular and extracellular pools rapidly exchanges. Under these conditions, the specific activity of the valyl-tRNA would be expected to be equal to the specific activity of either the intracellular or extracellular valine because tRNA derives its amino acids from these two pools (Airhart e t al., '76; Khairallah and Mortimore, '76; Vidrich et al., '77). At 1.6 mM L-valine, the specific activities of these pools remain constant for a t least 60 minutes of incubation. Because t h e incorporation of L-[3Hlvalineinto protein by hepatocytes is linear during the first 60 minutes of incubation (fig. l ) , it is possible to accurately determine the rate of valine incorporation into protein by hepatocytes from the specific activity of either the extracellular or intracellular valine pools. Using the specific activity of the extracellular valine, rates of protein synthesis by hepatocytes from 1- to 18-month-old rats were found to range from 96.8-34.5 pmoles of valine per minute per milligram protein. This is t h e first report of the rate of total protein synthesis by isolated hepatocytes. Khairallah and Mortimore ('76) have reported rates of protein synthesis in perfused rat liver of approximately 100 pmoles of valine per minute per milligram protein. Henshaw e t al. ('71) have reported in vivo rats of liver protein synthesis of 2.4-9.1 nmoles of lysine per minute per milligram RNA. We have found an RNA to protein ratio of approximately 0.04 in r a t liver. Therefore, the in vivo rate of protein synthesis reported by Henshaw e t al. ('71) would range from 100-350 pmoles of lysine per minute per milligram protein. In our studies, we observed a n in vivo rate of protein synthesis in 3month-old rats of approximately 250 pmoles of valine per minute per milligram protein (table 4). The rats used in the studies by Khairallah and Mortimore ('76) and Henshaw et al. ('71) would have been approximately one to two months old. The rate of protein synthesis by
PROTEIN SYNTHESIS BY RAT HEPATOCYTES
hepatocytes from 1- to 2-month-old rats was approximately 96.8pmoles of valine per minu t e per milligram protein (table 3). Therefore, t h e rate of protein synthesis by hepatocytes in our system appears to be similar to that reported for perfused liver and about one-half to one-third that observed in vivo. This study also shows that the protein synthetic activity of hepatocytes decreased as the age of the rats increased (table 3). This decrease was an age-related phenomenon and was representative of the liver in vivo. Cellfree protein synthesis by liver tissue from rats or mice has been reported to decrease 50-60% with age (Mainwaring, '69;Hrachovec, '71; Layman et al., '76).We observed a 64% decrease in the rate of protein synthesis by hepatocytes from 18-month-oldrats compared to hepatocytes from 1- to 2-month-old rats. The decrease in protein synthesis occurred primarily between 6 and 12 months. This emphasizes the importance of using age-matched controls when studying the effects of various manipulations on liver protein synthesis. ACKNOWLEDGMENTS
This study was supported in part by NIH grant 1 R01 AG 00344-01. LITERATURE CITED Airhart, J., A. Vidrich and E. A. Khairallah 1974 Compartmentation of free amino acids for protein synthesis in r a t liver. Biochem. J., 140: 539-548. Crane, L. J., and D. L. Miller 1974 Synthesis and secretion of fibrinogen and albumin by isolated rat hepatocytes. Biochem. Biophys. Res. Commun., 60: 1269-1277. Eagle, H. 1959 Amino acid metabolism in mammalian cell cultures. Science, 130: 432-437. East, A. G., L. N. Louis and R. Hoffenberger 1973 Albumin synthesis by isolated rat liver cells. Exptl. Cell Res., 76:
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Jeejeebhoy, K. N., J. Ho, G. R. Greenberg, M. J. Phillips, A. Bruce-Robinson and U. Sodtke 1975 Albumin, fibrinogen and transferrin synthesis in isolated rat hepatocyte suspensions. A model for the study of plasma protein synthesis. Biochem. J., 146: 141-155. Jefferson, L. S.,and A. Korner 1969 Influence of amino acid supply on ribosomes and protein synthesis of perfused rat liver. Biochem. J., 111: 703-712. Khairallah, E. A., and G. E. Mortimore 1976 Assessment of protein turnover in perfused r a t liver. J. Biol. Chem.,251:
1375-1384. Kletzien, R. F., M. W. Pariza, J. E. Becker, V. R. Potter and F. R. Butcher 1976 Induction of amino acid transport in primary cultures of adult rat liver parenchymal cells by insulin. J. Biol. Chem., 251: 3014-3020. Layman, D. K., G. A. Ricca and A. Richardson 1976 The effect of age on protein synthesis and ribosome aggregation to messenger RNA in rat liver. Arch. Biochem. Biophys., 173: 246-254. LeCam, A., and P. Freychet 1977 Neutral amino acid transport, characterization of the A and L systems in isolated rat hepatocytes. J. Biol. Chem., 252: 148-156. Lowry, 0. H., M. J. Rosebrough, A. L. Farr and F. J. Randall 1951 Protein measurement with the Folinphenol reagent. J. Biol. Chem., 193: 265-275. Mainwaring, W. I. P. 1969 The effect of age on protein synthesis in mouse liver. Biochem. J., 113: 869-878. McGivan, J. D., N. M. Bradford and J. Mendes-Mourao 1977 The transport of branched-chain amino acids into isolated rat liver cells. FEBS Lett., 80: 380-384. McGown, E., A. Richardson, L. M. Henderson and P. B. Swan 1973 Effect of amino acids on ribosome aggregation and protein synthesis in perfused rat liver. J. Nutr., 103: 109-116. Mortimore, G. E., and C. E. Mondon 1970 Inhibition by insulin of valine turnover in liver. J. Biol. Chem., 245:
2375-2383. Mortimore, G. E., K. H. Woodside and J. E. Henry 1972 Compartmentation of free valine and its relation to protein turnover in perfused rat liver. J. Biol. Chem., 247:
2776-2784. Munro, H. N. 1969 A general survey of techniques used in studying protein metabolism in whole animals and intact cells. In: Mammalian Protein Metabolism. H. N. Munro and J. B. Allison, eds. Academic Press, New York, Vol. 3, pp. 237-262. Munro, H. N., and A. Fleck 1966 Recent developments in the measurement of nucleic acids in biological materials. Analyst, 91: 78-88. Neuberger, A,, and F. F. Richards 1964 Protein biosynthesis in mammalian. In: Mammalian Protein Metabolism. H. N. Munro and J. B. Allison, eds. Academic Press, New York, Vol. 1, pp. 243-296. Pronczuk, A. W., B. S. Baliza, J. W. Triant and H. N. Munro 1968 Comparison of the effect of amino acid supply in hepatic polysome profiles in uiuo and in uitro. Biochim. Biophys. Acta, 157: 204-206. Rannels, D. E., J. B. Li, H. E. Morgan and L. S. Jefferson 1975 Evaluation of hormone effects on protein turnover in isolated perfused organs. In: Methods in Enzymology. B. W. OMalley and J. G. Hardman, eds. Academic Press, New York, Vol. 37,pp. 238-250. Schreiber, G., and M. Schreiber 1972 Protein synthesis in single cell suspensions from r a t liver. I. General properties of the system and permeability of the cells for leucine and methionine. J. Biol. Chem., 247: 6340-6346. 1973 The preparation of single cell suspensions from liver and their use for the study of protein synthesis. Sub-cell. Biochem., 2: 321-383.
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Seglen, P. 0. 1976 Incorporation of radioactive amino acids into protein in isolated rat hepatocytes. Biochim. Biophys. Acta, 442: 391-404. Sidransky, H., D. S. R. Sarma, M. Bongiorno and E. Verney 1968 Effect of dietary tryptophan on hepatic polyribosomes and protein synthesis in fasted mice. J. Biol. Chem., 243: 1123-1132. van Bezooijen, C. F. A,, T. Grell and D. L. Knook 1976 Albumin synthesis by liver parenchymal cells isolated from young, adult and old rats. Biochem. Biophys. Res. Commun., 71: 513-519. Vidrich, A,, J. Airhart, M. K. Bruno and E. A. Khairallah 1977 Compartmentation of free amino acids for protein biosynthesis. Biochem. J., 162: 257-266. Wallyn, C. S.,A. Vidrich, J. Airhart and E. A. Khairallah 1974 Analysis of the specific radioactivity of valine isolated from aminoacyl-transfer ribonucleic acid of rat liver. Biochem. J.,140: 545-548.
Waterlow, J. C. 1969 The assessment of protein nutrition and metabolism in t h e whole animal, with special reference to man. In: Mammalian Protein Metabolism. H. N. Munro and J. B. Allison, eds. Academic Press, New York, Vol. 3,pp. 325-390. 1975 Protein turnover in t h e whole body. Nature, 253: 157. Weigand, K., and I. Otto 1974 Secretion of serum albumin by enzymatically isolated rat liver cells. FEBS Lett., 46: 127-129. Zamecnik, P.C., and E. B. Keller 1954 Relation between phosphate energy donors and incorporation of labeled amino acids into proteins. J. Biol. Chem., 209: 337-354. Zanetta, J. P., G. Vincendon, P. Mandel and G. Gombos 1970 The utilization of l-dimethylaminonaphthalene-5sulphonyl chloride for quantitative determination of free amino acids and partial analysis of primary structure of proteins. J. Chromatog, 51: 441-458.
ABSTRACT The rate of total protein synthesis in isolated hepatocytes was determined. The incorporation of L-13Hlvalineinto protein is linear for a t least two hours of incubation and is affected by the concentration of amino acids in the medium. Uptake of valine by hepatocytes from 1.5- and 18-month-old rats was identical and appears to occur by simple passive diffusion. Within five minutes, the specific activities of the intracellular and extracellular valine pools are equivalent. The specific activities of these pools are saturated by 1.6 mM valine and remain constant for 60 minutes of incubation. The rates of protein synthesis by hepatocytes from 1- to 2-month-old rats is 96.8 pmoles of valine per minute per milligram protein. This is comparable to rates of protein synthesis reported for perfused liver and liver in vivo and is approximately 64% higher than the rate of protein synthesis by hepatocytes from 18-month-old rats. Liver protein synthesis has been studied primarily in cell-free systems since the development of the first cell-free protein synthesis system by Zamecnik and Keller ('54). Cell-free systems allow an investigator to carry out routine manipulations and control experimental conditions. In addition, this type of a system allows one to study the molecular mechanism of liver protein synthesis. However, the rate of cell-free protein synthesis by liver is only 1% the rate observed in vivo (Henshaw et al., '71; Waterlow, '75). The fact that 99% of the protein synthetic activity of liver is lost in cellfree systems warrants the search for other systems which could be used to study liver protein synthesis. Although i t would be preferable to use whole animals to measure liver protein synthesis, it is difficult to manipulate and control experimental variables using whole animals. In addition, protein synthesis in whole animals is complicated by the absorption of the amino acids, compartmentation of the amino acids, and competing reactions (Neuberger and Richards, '64; Waterlow, '69; Munro, '69; Vidrich et al., '77). To allow an investigator to control and manipulate experimental variables in liver protein synthesis, the perfused liver has been used (Rannels et al., '75). However, this system is not suitable for routine J. CELL. PHYSIOL. (1978)97: 137-146.
studies and also shows compartmentation of amino acids (Mortimore et al., '72; Khairallah and Mortimore, '76). Single cell suspensions of hepatocytes offer a system for studying protein synthesis with several advantages over whole animal and perfused liver systems. It is also suited to routine studies where many homogenous samples can be studied simultaneously under a variety of experimental conditions. Since Schreiber and Schreiber ('72, '73) determined the optimum conditions for protein synthesis by hepatocytes, the use of hepatocytes to study protein synthesis has grown. In almost all studies, protein synthesis in hepatocytes has been measured by determining the amount of radioactively-labeled amino acid incorporated into protein. However, radioactive isotope incorporation data do not give a true measure of the rate of protein synthesis. The incorporation rate of a radioactively-labeled amino acid into protein is a direct function of the specific activity of the amino acid precursor pool used for protein synthesis. There have been no studies where the specific activity of the amino acid precursor pool has been measured and used to determine the rate of protein synthesis in hepatocytes. In this study, the specific activities of the intraReceived July 15, '77. Accepted Apr. 10, '78
137
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RICCA, LIU, CONIGLI 0 AND RICHARDSON
cellular and extracellular L-valine pools were measured and the actual rate of protein synthesis by hepatocytes determined. Rates of protein synthesis by hepatocytes can be easily determined. These rates are comparable to rates of protein synthesis reported for perfused liver and liver in vivo. MATERIALS AND METHODS
Isolation of hepatocytes
omitting L-valine. L-[2,33Hlvaline (0.85 mCi/ mmole) was added to the mixture at a concentration of 1.6 mM. These conditions were found to be optimal for protein synthesis. Samples (1.5 ml) were withdrawn a t various times and protein synthesis terminated by the addition of the cell suspension to an equal volume of 10% trichloroacetic acid. The suspension was heated a t 90°C for 15minutes and the precipitate collected by centrifugation. The precipitate was washed twice with 5%trichloroacetic acid and once with each of the following: NaOH neutralized ethano1:anhydrous ethyl ether (2:l), acetone, and anhydrous ethyl ether. The precipitate was air-dried and dissolved in 0.3 N NaOH. Aliquots of the NaOH solution were taken to determine t h e radioactivity by liquid scintillation counting and the protein concentration by the method of Lowry et al. ('511, using bovine serum albumin as the standard.
Female rats were purchased from ARW Sprague-Dawley and maintained as described previously (Layman et al., '76). Hepatocytes were obtained by a modification of the method of Howard et al. ('73). Animals were killed by decapitation between 9:00 and 11:OO A.M. to minimize diurnal variation. The liver was immediately removed, rinsed twice in ice-cold calcium-free Hanks' solution (Hanks and Wallace, '49), and perfused through the portal vein with 15-30ml of an enzyme solution conDetermination of the extracellular taining 0.05% collagenase in calcium-free and intracellular valine Hanks' solution supplemented with 1mM specific activity pyruvate, a vitamin mixture (Eagle, '59), twice the level of essential and non-essential Studies on valine uptake by hepatocytes amino acids described by Eagle ('59), and were performed by incubating the hepatocytes 50 mM Hepes (N-2-hydroxyethyl-piperazine- (3,000,000 cells per ml) in the protein syntheNf-2-ethanesulfonicacid) buffer pH 7.3. Using sis system described above. Inulin ([''Cleara hand-held Stadie-Riggs Tissue Slicer, slices boxylic acid, 5 mCi/mmole) was added to the 0.3-0.4 mm thick were prepared. The liver incubation medium as an extracellular slices (2 to 3 g) were incubated in 10 ml of the marker at a concentration of 1pCi/ml. After enzyme solution for 60 minutes at 37°C under incubation, the cell pellet and extracellular an atmosphere of 95: 5 0xygen:carbon dioxide. medium were obtained by low speed cenCalcium chloride (1.26 mM final concentra- trifugation. Twenty percent of the [14Clinulin tion) was added to this suspension after 30 to routinely was found associated with the cell 35 minutes of incubation. The suspension was pellet. This indicates that the cell pellet confiltered through stocking nylon and then tains a considerable portion of extracellular through a single layer of nylon mesh (pore size fluid. The acid-soluble extracts were obtained 60 pm). The cells were then obtained by low from the cell pellet and extracellular medium speed centrifugation and washed as described by treating these fractions with 5%trichloroby Howard et al. ('73). Using this method, the acetic acid. From the amount of [14Clinulin percent viable cells was routinely 90-95% as associated with the cell pellet, the extracelludetermined by trypan blue exclusion. lar valine contribution to the cell pellet could be determined. By subtracting this contribuProtein synthesis by hepatocytes tion from the total acid-soluble valine associProtein synthesis was followed by incuba- ated with the cell pellet, the intracellular (intion of approximately 3,000,000 cells per mil- ulin-impermeable) valine content of the cell liliter 37°C under an atmosphere of 95:5 pellet was determined. The specific activities of the intracellular 0xygen:carbon dioxide in a modified Eagles Minimal Essential Medium (Eagle, '59). This and extracellular acid-soluble valine were medium was composed of Hanks' solution sup- determined by passing the acid-soluble explemented with 1 m M pyruvate, a vitamin tracts over Amberlite CG-120 (H') columns mixture (Eagle, '59), 10 mM Hepes buffer pH (5.5 x 35 mm) preequilibrated with 5% tri7.3, and twice the level of essential and non- chloroacetic acid. The columns were rinsed with 2ml of distilled water and the amino essential amino acids described by Eagle ('59),
139
PROTEIN SYNTHESIS BY RAT HEPATOCYTES
Minutes Fig. 1 The time course of the appearance of acid-insolubleradioactivity in the cell pellet ( 0 )and extracelrepresent the sum of the radioactivity in the cellular lular medium (H) was determined. The open circles (0) and extracellular fractions. The initial viability of the hepatocyte preparation was 96%,and the percent of viable cells throughout the incubation period is indicated by the numbers.
'
acids eluted from the columns with 2 ml of 5 N NH,OH. The NH,OH solutions were evaporated to dryness and the resulting residues dissolved in 0.2 M sodium phosphate buffer (pH 8.8). The amino acids in the phosphate buffer were dansylated and separated by 2-dimensional thin-layer chromatography a s described by Zanetta et al. ('70). The material migrating as dansyl-valine was identified under a n ultraviolet-light. This portion of the chromatogram was removed, and the dansylvaline was extracted with 100%methanol. The radioactivity in the methanol solution was determined by liquid scintillation counting, and t h e dansyl-valine concentration was determined with a Turner 110 fluorometer (Turner Filters: primary, 7-60; secondary, 2A-12) using dansyl-valine as a standard.
In vivo liver protein synthesis Liver protein synthesis in vivo was measured after i.p. administration of 0.33 mCi L[3,43Hlvaline ( 3 6 mCi/mmole) per 100 g body weight to female Sprague-Dawley rats. After 12 minutes the rats were killed by decapita-
tion. The liver was removed and homogenized in 3 volumes of buffer ( 0 . 0 5 ~cacodylate, 0.17~ NaCl, 0 . 3 3 M sucrose, pH 6.0) with a Potter-Elvehjem homogenizer. Samples of the homogenate were used to determine t h e amount of L- [3Hlvalineincorporated into acidprecipitable material per milligram of liver homogenate protein. This was accomplished by the procedures described for hepatocytes. Aminoacyl-tRNA was isolated from the rest of the homogenate and deacylated as described by Wallyn et al. ('74). The specific activity of the valine attached to tRNA in vivo was determined by dansylation and thin layer chromatography as described previously in the MATERIALS AND METHODS. RESULTS
Figure 1 shows the time course for the appearance of radioactive-label into intracellular and extracellular proteins. Incorporation of L-[3Hlvalineinto intracellular proteins was linear for the first 60 minutes of incubation. Acid-insoluble radioactivity appeared in the extracellular medium after 30 to 45 min-
140
RICCA, LIU, CONIGLIO AND RICHARDSON
I 2 3 4 AMINO ACID LEVELS Fig. 2 The incorporation of L-L3HHlvalineinto acid-insoluble material was determined a s described in the MATERIALS AND METHODS, using hepatocytes from 3- to 4-month-old rats. Hepatocytes were incubated for 30
minutes in the presence of the various levels of the amino acids b o t h the non-radioactively-labeledamino acids and the L-PHhaline). The levels of the amino acids are expressed in terms of the levels of essential and non-essential amino acids reported by Eagle ('591.
Utes of incubation. Several investigators (Weigand and Otto, '74; van Bezooijen et al., '76) have reported a similar lag period for the appearance of albumin in the extracellular medium following incubation of hepatocytes. When the sum of the intracellular and extracellular acid-insoluble radioactivity was plotted, protein synthesis by hepatocytes was linear for a t least two hours of incubation. During the 2-hour incubation period, the viability of the hepatocytes decreased less than 10%. Optimal conditions for protein synthesis by hepatocytes was found to be similar to that reported by Schreiber and Schreiber ('72). In determining the optimum conditions, special attention was given to the effect of the concentration of the amino acids on protein synthesis. Several laboratories have reported that protein synthesis in perfused liver or in liver in vivo is affected by the level of the amino acids (Pronczuk et al., '68; Sidransky e t al., '68; Jefferson and Korner, '69; McGown et al., '73). Extensive studies by Schreiber and Schreiber ('72, '73) with 20 different radioactively-labeled amino acids showed that the incorporation of each radioactively-labeled amino acid into protein increased when the
concentration of that amino acid in the incubation medium was increased. Using P4CCllabeled algae protein hydrolysates, Seglen ('76) showed that the incorporation of radioactivity into protein by hepatocytes exhibited saturation kinetics as the concentration of the hydrolysate was increased. However, no studies with hepatocytes have specifically determined the effect of the non-radioactively-labeled amino acids on the incorporation of the radioactively-labeled amino acid. In our studies a mixture of amino acids, as described by Eagle ('591, was used with L-VHlvaline. Valine was chosen as the radioactive tracer to measure protein synthesis because i t is widely distributed in protein, and it is neither synthesized nor degraded by liver tissue (Elwyn et al., '68; Mortimore and Mondon, '70). Figure 2 shows the effect of various concentrations of this amino acid mixture on L-L3HIvaline incorporation into acid-insoluble material. L-F3H1valine incorporation increased as the concentration of the amino acids was increased. At amino acid concentrations above 2-times Eagle's level, the hepatocyte system was saturated and L-F3HIvalineincorporation plateaued. Very high concentrations of the amino acids were found to be slightly inhib-
141
PROTEIN SYNTHESIS BY RAT HEPATOCYTES
itory. Table 1 shows the effect of the non-radioactively-labeled amino acids on protein synthesis by hepatocytes. Omission of all of t h e non-radioactively-labeled amino acids from the incubation medium decreased the incorporation of L-13Hlvalineinto acid-insoluble material over 30%.Omission of either essential or non-essential amino acids also decreased protein synthesis 30%. Addition of increasing concentrations of the non-radioactively-labeled amino acid mixture to hepatocytes containing saturating levels of L-L3Hlvaline (1.6 mM) gave a saturation curve similar to t h a t shown in figure 2 (data not shown). The stimulation of valine incorporation by amino acids could be due to an increased availability of energy (through amino acid catabolism) for the hepatocytes for protein synthesis. To test this possibility, hepatocytes were incubated with various concentrations (1-20 mM) of either pyruvate, pyruvate and lactate, or succinate in a system containing saturating levels of L-PHlvaline (1.6 mM) and low levels of the amino acid mixture (0.1times Eagle's level). In no case was protein synthesis stimulated by the addition of these compounds (results not shown). In fact, high concentrations of these compounds resulted in a slight decrease in protein synthesis. Preliminary studies in our laboratory show that amino acids do not stimulate RNA synthesis by hepatocytes and that the omission of just one amino acid from the amino acid mixture decreases protein synthesis by hepatocytes. Therefore, it appears that amino acids specifically stimulate protein synthesis in hepatocytes suspensions because of their effect on the protein synthetic apparatus. This situation appears to be analogous to the situation in perfused liver and liver in vivo (Pronczuk et al., '68; Sidransky e t al., '68; Jefferson and Korner, '69; McGown e t al., '73). I n studying protein synthesis in cell suspensions using a radioactively-labeled amino acid, attention must be given to the uptake of the radioactivity-labeled amino acid by the cells. Observations by Schreiber and Schreiber ('72) and Kletzien e t al. ('76) have shown that hepatocytes are freely permeable to methionine, leucine, and a-aminoisobutyric acid. Recently, studies have shown that the transport of a-aminoisobutyric acid (LeCam and Freychet, '77) and the branched chain amino acids, leucine, isoleucine, and valine (McGivan et al., '77) occurred by both facilitated transport and passive diffusion in hepatocytes. Initial stud-
TABLE 1
Effect o f the levels o f t he non-radioactiuely labeled amino acids on protein synthesis by hepatocytes ' Level of amino acids
dpm/pg protein
o x
1,870-t 177 2,3502 242 2,7292 194
0.5 X 2 x 2 X (minus the non-essential amino acids) 2 X (minus the essential amino acids)
1,856?216 1,910&181
z Incorporation of L-[3Hlvaline into acid~insolublematerial by hepatocytes from 3- to 4-month-old rats was determined after a 30-minUte incubation. The protein synthesis system was similar to that described in the MATERIALS AND METHODS except that the concentration of the amino acid mixture (which lacks valine) was varied as indicated above. The concentration of L-13Hlvaline was 1.6 mM in each experiment. The levels of the amino acid mixture are given in terms of the levels of essential and non~essentialamino acids reported by Eagle ('59). Each value represents the mean 2 S . E . for three experiments.
ies (data not shown) indicated that when hepatocytes were incubated with L-PHlvaline, the radioactivity in the intracellular and extracellular fractions equilibrated very rapidly. One wash with 0.9% NaCl removed over 95% of the L-13HIvaline associated with the hepatocytes. Figure 3 shows that the uptake of L-PHIvaline by hepatocytes from 1.5- and 18-month-old rats was linear up to extracellular valine concentrations of 10 mM. There was no age-difference in valine uptake by hepatocytes nor was any saturation of uptake observed. The lack of saturation of valine uptake by hepatocytes is in agreement with the observations of Schreiber and Schreiber ('72) for L-methionine and L-leucine and the observations of McGivan et al. ('77) for L-leucine, L-isoleucine, and L-valine. Addition of cyanide to hepatocytes or decreasing the temperature from 37" to 4" had only a slight effect on valine uptake (table 2). McGivan et al. ('77) have reported that the transport of the TABLE 2
Effect of cyanide and temperature on d i n e uptake by hepatocytes ' L-t3Hlvaline concentration
0.4 mM 4.0 mM
dpm/lO5 cells Control
KCN
4°C
560 4,752
568 4,456
496 4,056
' Hepatocytes were incubated for five mlnutes as described in figure 3 with the two concentrations of L-[3Hlvaline.The intracellu~ lar radioactivity associated with the hepatocytes was determined for cells incubated at 37°C with and without KCN (5 x lo-' MI. and for cells incubated at 4°C.
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RICCA, LIU, CONIGLIO AND RICHARDSON
0
10
5 Extracellular
Valine ( m M )
Fig. 3 The uptake of L-PHhaline by hepatocytes from 1.5 (0)and 18 fO)-month-old rats as a function of the extracellular L-PHlvaline concentration is shown. Cells were incubated in the protein synthesis system with various concentrations of L-13Hlvaline (0.85mCi/mmole) for five minutes. Cells were collected by centrifugation and the acid-insoluble radioactivity in the intracellular valine determined as described in the MATERIALS AND METHODS.
i
Ir)
Valine Concentration (mM)
Time (min)
Fig. 4 The specific activities of the intracellular (0) and extracellular ( 0 )valine were determined after incubating hepatocytes with L-PHlvaline in the protein synthesis system described in the MATERIALS AND METHODS. In graph A, cells were incubated with each concentration of L-PHhaline for five minutes while in graph B, cells were incubated with 1.6 m M L-VHlvaline for various times.
143
PROTEIN SYNTHESIS BY RAT HEPATOCYTES
branched chain amino acids by hepatocytes is not inhibited by ouabain or 2,4-dinitrophenol. To measure protein synthesis accurately, it is essential t h a t the specific activity of the amino acid pool used for protein synthesis be determined. Using t h e techniques described in t h e MATERIALS AND METHODS, the specific activities of the intracellular and extracellular L- [3Hlvaline were determined after hepatocytes were incubated for five minutes with 0.4 to 4.0 mM L-[3Hlvaline (fig. 4A). The specific activities of the intracellular and extracellular pools were essentially equivalent a t each concentration of L-13Hlvaline tested. At valine concentrations greater than 1.6 mM, the specific activities of both the extracellular and intracellular L-13Hlvaline pools reached steady state. The level of valine required to reach this steady state was dependent on the hepatocyte concentration. Higher hepatocyte concentrations increased the concentration of L-valine required to reach steady state. When hepatocytes were incubated with 1.6 mM L-[3Hlvaline, the specific activities of both valine pools were equivalent after five minutes of incubation and remained constant throughout 60 minutes of incubation (fig. 4B). Therefore, hepatocytes provide a system in which the rate of protein synthesis can be determined from the specific activity of either t h e extracellular or intracellular valine pool. Table 3 gives the rates of valine incorporation into acid-insoluble material by hepatocytes isolated from 1-to 18-month-oldrats. A 30-minute incubation period was chosen to determine the rates of protein synthesis by hepatocytes because very little change occurred in t h e viability of the hepatocytes during the initial 30-minute incubation (fig. 1). Furthermore, during this time period, the specific activities of the intracellular and extracellular valine were equivalent and remained constant (fig. 4B). The rates of protein synthesis by hepatocytes isolated from 1- to 18-month-old rats ranged from 96.8-34.5 pmoles of valine per minute per milligram protein. Protein synthesis by hepatocytes decreased as the age of t h e rats from which the hepatocytes were isolated increased. A 64% decrease in protein synthesis occurred from 1 to 12 months with no significant change in protein synthesis from 12 to 18 months. The decreased rate of protein synthesis by hepatocytes from older rats observed in table 3 could be a n age-related phenomenon or it could be t h a t hepatocytes from the older rats
TABLE 3
Rates ofprotein synthesis by hepatocytes from rats ofdifferent ages I Rate of valine incorporation (pmolesiminimg protein)
Age (months)
1-2 6-7 11-13 18
n
Mean
S.E.
4 5 4 5
96.8 76.4 35.2 34.5
5.6 1.5 2.5 3.3
1 Linear regression analysis of the amount of L-13Hlvalineincorporated into acid-insoluble material after 10, 20, and 30 minutes of incubation was used to determine the rate of L-PHlvaline incorporation. The pmoles of valine incorporated was determined by dividing the rate of L-PHlvaline incorporated by the specific activity of the extracellular valine pool. In all experiments, the viability of the cells at the end of the 30-minute incubation period was above 90%.The decrease in protein synthesis from 1- to S~monthto 6- to 7-month-old animals is significant at the P < 0.025 level, while the decrease in protein synthesis from 6- to 7-month to 11- to 13-month-old animals is significant at the P < 0.0005 level using the student’s t test (unpaired).
are more prone to damage during the isolation procedure than hepatocytes from younger rats. To differentiate between these two possibilities, the rate of in vivo liver protein synthesis by 3- and 15-month-old rats was determined by measuring t h e incorporation of L-[3H]~aline into acid-insoluble material and by determining the specific activity of the liver valyl-tRNA as described by Airhart et al. (‘74). Table 4 clearly shows that the in vivo rate of liver protein synthesis was lower in 15month-old rats than in 3-month-old rats. The 15-month-old rats showed a 64% decrease in the incorporation of radioactivity into acid-insoluble material compared to the 3-month-old rats; however, the specific activity of the valyl-tRNA was also lower in the 15-monthold rats. When the actual rate of valine incorporation into protein was determined from the specific activity of the valyl- tRNA, a decrease of approximately 50% was observed. TABLE 4
Rates of in uiuo protein synthesis
I
Age of animal 3 months 15 months
Rate of L-PHlvaline incorporation (dpmlminlmg protein) Specific activity of Valyl-tRNA (dpm/pmole) Rate of valine incorporation fpmole/min/mg protein)
3,070 12.0 256
1,104 8.3 133
’ The rate of valine incorporation per milligram protein in the liver homogenate was determined a8 described in the MATERIALS AND METHODS. Each value represents the mean of two animals.
144
RICCA, LIU, CONIGLIO AND RICHARDSON DISCUSSION
Although hepatocytes have been used by numerous investigators to study various aspects of protein synthesis, most of these studies have measured protein synthesis simply by following the incorporation of radioactively-labeled amino acids into protein. Generally, no attempt is made to determine t h e actual rate of total protein synthesis. Only in reports where albumin synthesis has been studied, has the rate of protein synthesis (in this case the synthesis of one specific protein) in hepatocytes been determined. Initial studies indicated that the rate of albumin synthesis by hepatocytes was only 10%of that reported for liver in vivo (East et al., '73; Crane and Miller, '74). More recently, Jeejeebhoy et al. ('75) and van Bezooijen et al. ('76) have reported rates of albumin synthesis in hepatocytes 40-80% of that reported for liver in vivo. We were interested in determining the rate of total protein synthesis in hepatocytes and comparing i t to rates of protein synthesis reported for perfused liver and liver in vivo. However, when using a radioactively-labeled amino acid to measure the rate of protein synthesis in whole cells, it is necessary to deter. mine the specific activity of the amino acid pool used for protein synthesis. Recent observations with cultured hepatoma cells, perfused liver, and liver in vivo indicate that the specific activity of the aminoacyl-tRNA is different from the specific activities of either the intracellular or extracellular amino acid pools (Airhart et al., '74; Hod and Hershko, '76; Khairallah and Mortimore, '76; Vidrich et al., '77). These studies indicate that the aminoacyl-tRNA derives its amino acids from both t h e extracellular and intracellular pools. Therefore, the specific activity of the aminoacyl-tRNA pool must be known to measure the actual rate of protein synthesis. The need for determining the specific activity of valyltRNA, however, can be circumvented by expanding the extracellular valine pool. Mortimore's group (Mortimore e t al., '72; Khairallah and Mortimore, '76) has demonstrated that when the level of valine in liver perfusates is increased to 5-10 mM, the specific activities of t h e intracellular and extracellular valine are equivalent. Furthermore, under these conditions the ratio of the specific activities of valyl-tRNA to the extracellular valine is approximately one.
Our studies indicate t h a t the transport of valine by hepatocytes occurs rapidly, is not temperature or energy dependent, and is linear up to L-valine concentrations of 10 mM. Comparison of the specific activities of t h e extracellular and intracellular valine in hepatocytes demonstrates t h a t within five minutes the specific activities of these two valine pools are equivalent. Therefore, it appears t h a t L-valine is not compartmentalized by hepatocytes; rather, the valine in the intracellular and extracellular pools rapidly exchanges. Under these conditions, the specific activity of the valyl-tRNA would be expected to be equal to the specific activity of either the intracellular or extracellular valine because tRNA derives its amino acids from these two pools (Airhart e t al., '76; Khairallah and Mortimore, '76; Vidrich et al., '77). At 1.6 mM L-valine, the specific activities of these pools remain constant for a t least 60 minutes of incubation. Because t h e incorporation of L-[3Hlvalineinto protein by hepatocytes is linear during the first 60 minutes of incubation (fig. l ) , it is possible to accurately determine the rate of valine incorporation into protein by hepatocytes from the specific activity of either the extracellular or intracellular valine pools. Using the specific activity of the extracellular valine, rates of protein synthesis by hepatocytes from 1- to 18-month-old rats were found to range from 96.8-34.5 pmoles of valine per minute per milligram protein. This is t h e first report of the rate of total protein synthesis by isolated hepatocytes. Khairallah and Mortimore ('76) have reported rates of protein synthesis in perfused rat liver of approximately 100 pmoles of valine per minute per milligram protein. Henshaw e t al. ('71) have reported in vivo rats of liver protein synthesis of 2.4-9.1 nmoles of lysine per minute per milligram RNA. We have found an RNA to protein ratio of approximately 0.04 in r a t liver. Therefore, the in vivo rate of protein synthesis reported by Henshaw e t al. ('71) would range from 100-350 pmoles of lysine per minute per milligram protein. In our studies, we observed a n in vivo rate of protein synthesis in 3month-old rats of approximately 250 pmoles of valine per minute per milligram protein (table 4). The rats used in the studies by Khairallah and Mortimore ('76) and Henshaw et al. ('71) would have been approximately one to two months old. The rate of protein synthesis by
PROTEIN SYNTHESIS BY RAT HEPATOCYTES
hepatocytes from 1- to 2-month-old rats was approximately 96.8pmoles of valine per minu t e per milligram protein (table 3). Therefore, t h e rate of protein synthesis by hepatocytes in our system appears to be similar to that reported for perfused liver and about one-half to one-third that observed in vivo. This study also shows that the protein synthetic activity of hepatocytes decreased as the age of the rats increased (table 3). This decrease was an age-related phenomenon and was representative of the liver in vivo. Cellfree protein synthesis by liver tissue from rats or mice has been reported to decrease 50-60% with age (Mainwaring, '69;Hrachovec, '71; Layman et al., '76).We observed a 64% decrease in the rate of protein synthesis by hepatocytes from 18-month-oldrats compared to hepatocytes from 1- to 2-month-old rats. The decrease in protein synthesis occurred primarily between 6 and 12 months. This emphasizes the importance of using age-matched controls when studying the effects of various manipulations on liver protein synthesis. ACKNOWLEDGMENTS
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