Distribution of mRNAs of fibrinogen polypeptides and albumin in free and membrane-bound polyribosomes and induction of α-fetoprotein mRNA synthesis during liver regeneration after partial hepatectomy

Biochimica et Biophysica Acta, 699 (1982) 121 - 130

121

Elsevier Biomedical Press

BBA 91135

DISTRIBUTION OF mRNAS OF FIBRINOGEN POLYPEPTIDES AND ALBUMIN IN FREE AND MEMBRANE-BOUND POLYRIBOSOMES AND INDUCTION OF ct-FETOPROTEIN mRNA SYNTHESIS DURING LIVER REGENERATION AFTER PARTIAL HEPATECTOMY H A N S M.G. P R I N C E N a, G E R A R D C.M. SELTEN a, A N N E - M A R I E E. S E L T E N - V E R S T E E G E N a G E R A P.B.M. M O L - B A C K X a, W I L L E M N I E U W E N H U I Z E N b and SING HIEM Y A P a..

~' Division of Gastrointestinal and Liver Disease, Dept. Of Medicine, St. Radboud Hospital, University of Nijmegen, 6500 HB Nijmegen and t, Gaubius Institute, Health Research Organization TNO, Leiden (The Netherlands) (Received May 5th, 1982)

Key words: Regeneration; Albumin synthesis," Fibrinogen synthesis," c~-Fetoprotein synthesis; mRNA; (Rat liver)

To study the effect of regenerative response of the liver following partial hepatectomy on the synthesis of major plasma proteins (secretory proteins), we have determined the sequence contents and the distribution of albumin and fibrinogen polypeptide mRNAs in rat liver at intervals after partial hepatectomy and sham operation. Using a quantitative technique for the isolation of polyribosomes, we demonstrated that the distribution of RNA between free and membrane-bound polyribosomal fraction was unchanged in these experiments. There was no shift in the polyribosomal population to favor free polyribosomes after partial hepatectomy. However, there was a dramatic increase (5-6-fold) of the fibrinogen polypeptide mRNA concentration during the first 24 h after resection. In contrast, the albumin mRNA concentration decreased (2-3-fold). There were no et-fetoprotein mRNA sequences detectable in any liver RNA fraction in these experimental animals. In sham-operated rats with intact livers, similar changes of fibrinogen polypeptide and albumin mRNA concentrations as described in regenerating liver after partial hepatectomy, were observed. These results suggest that albumin and fibrinogen synthesis after partial hepatectomy is reciprocally regulated at the mRNA level and represents a nonspecific acute phase response to surgical trauma.

Introduction

The regenerative response of the liver following partial hepatectomy is characterized by an increased protein synthesis and a subsequent enhanced DNA synthesis and cellular proliferation [1,2]. After partial hepatectomy, the residual hepatocytes show ultimate changes from a stage of

* To whom correspondence should be addressed. Abbreviations: Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; SDS, sodium dodecyl sulfate. Rot, the product of initial R N A concentration in mol nucleotides/l and time in s (assuming that A260 of 1.0 corresponds to 40 p g R N A / m l ) ; Rot~/2, the Rot value at 50% hybridization. 0167-4781/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press

high metabolic but low mitotic activity to an intermediate phase, in which they have to divide rapidly. Since the synthesis of a variety of structural proteins is involved in the cellular proliferation, quantitative and qualitative changes in the messenger RNA population would be expected as important determinants of the type and amount of proteins synthesized during liver regeneration. The fact that most, if not all, structural proteins are synthesized on free and secretory proteins on membrane-bound polyribosomes has suggested that there is a shift in polyribosomal population to keep the balance in protein synthesis favoring the synthesis of 'cellular' proteins over those for export in regenerating liver [3,4].

122 In this study, using radioactively-labelled complementary DNAs specific for mRNAs of albumin and of fibrinogen polypeptides as markers for mRNAs of major secretory proteins synthesized by the liver and using a quantitative technique for isolation of free and membrane-bound liver polyribosomes, we have determined the sequence contents and the distribution of albumin and fibrinogen polypeptide mRNAs in rat liver at intervals following partial hepatectomy and laparotomy. Since the resynthesis of a-fetoprotein, an oncofetal protein, has been reported during liver regeneration [5-8], the sequence content of a-fetoprotein m R N A has also been determined in this study. The findings showed a dramatic increase (5-6-fold) of fibrinogen polypeptide mRNA concentration during the first 24 h after resection. In contrast, the albumin m R N A concentration decreased (2-3-fold). The distribution of RNA between free and membrane-bound polyribosomes was unchanged after partial hepatectomy. There were no a-fetoprotein mRNA sequences detectable in any R N A fraction in these experiments. In sham-operated animals, similar changes of fibrinogen polypeptide and albumin mRNA concentration as found in regenerating liver after partial hepatectomy were observed. These results indicate that there is a differential regulation at the m R N A level of fibrinogen and albumin synthesis during liver regeneration after partial hepatectomy and it represents a nonspecific acute phase response to surgical trauma. Induction of a-fetoprotein m R N A synthesis was not found in our partial hepatectomized animals. Materials and Methods

Materials. All glassware was sterilized and solutions were freshly prepared and autoclaved prior to use. Ribonuclease-free sucrose, EDTA, phenol, sodium deoxycholate, proteinase K, salts and solvents were purchased from E. Merck (Phenol was redistilled in vacuo under nitrogen prior to use). Dithiothreitol, Hepes and heparin from porcine intestinal mucosa were obtained from Sigma. Deoxyribonucleoside triphosphates from Schwarz/Mann. Glutathione was obtained from Aldrich. Triton X-100 and SDS from BDH Chemicals Ltd. [5-3H]dCTP (spec. act. 18.4 C i / m m o l )

was purchased from the Radiochemical Centre, Amersham. Oligo d(T) 12-18-cellulose (type T 2) and oligo (dT)l 0 from Collaborative Research, Inc., Waltham, MA. Avian myeloblastosis virus RNAdependent DNA polymerase was kindly supplied by Dr. J.W. Beard, National Cancer Institute, U.S.A. Nuclease S l (Aspergillus oryzae) was purchased from Miles Laboratories and stored at 2.5. 105 units/ml in 50% glycerol/100 mM NaC1/20 mM KHzPO4/5 mM Na2HPO 4 (pH 7.0) at - 2 0 ° C . E. coli strain B tRNA from Calbiochem. Animals. Male Spraque-Dawley rats weighing 250-300 g were used throughout and were maintained on standard Purina Chow and water ad libitum. Partial hepatectomy resulting in the removal of 70% of the liver was performed according to the method of Higgins and Anderson [9]. Sham-operated animals were laparotomized and the liver was palpated. Surgical procedures were performed under ether anesthesia. At different time intervals after partial hepatectomy and sham-operation, animals were killed by decapitation.

Isolation of free and membrane-bound polyribosomes. Free and membrane-bound polyribosomes were isolated according to the method of Ramsey and Steele [10] as reported previously [11,12]. Polyribosome profile analysis. Approx. 6 A260 units of free or membrane-bound polyribosomes were diluted to 300 rtl with a polyribosome buffer containing 10 mM Hepes, pH 7.4/75 mM KC1/5 m M MgCI2/3 mM glutathione and were layered over a 12 ml, 10-40% (w/v) isokinetic sucrose gradient in the same buffer, containing 0.5 mM EDTA. Centrifugation was carried out for 60 min at 280 000 × g. The gradients were withdrawn from the top of each tube and absorbance at 254 nm was monitored with a Gilford 2400-2 recording system.

Preparation of purified mRNAs for fibrinogen polypeptides, albumin and for a-fetoprotein and the preparation of 3H-labelled complementary DiVAs (cDNAs). Purification of mRNA for albumin, afetoprotein and for fibrinogen polypeptides, respectively, was performed from polyribosomes as previously reported [13-15]. The major steps in this procedure include immunoprecipitation and isolation of polyadenylated RNA from these polyribosomes by phenol extraction and oligo (dT)-cel-

123

lulose chromatography. The isolated mRNAs were then transcribed into [3H]-cDNA probes under conditions as reported previously [13]. RNA-cDNA hybridization. Analytical RNAcDNA hybridization was performed according to the method of Housman et al. [16] at 65°C in 5 ~tl sealed capillary tubes containing 0.2 M sodium phosphate buffer, pH 6.8/0.5% SDS. Determination of plasma concentration of fibrinogen and albumin. Plasma concentrations of fibrinogen and albumin were determined according to the methods of Vermijlen et al. [17] and Doumas et al. [18], respectively. Preparation of RNA fractions after sucrose gradient centrifugation. Approx. 6 A260 units of polyribosomes were centrifuged at 39 000 rev./min for 60 min in a MSE SW40 rotor at 2°C, gradients were withdrawn and RNA was prepared from each fraction by digestion with proteinase K (0.5 m g / m l ) in 0.1 M NaC1/0.5% SDS/10 mM TrisHCI (pH 7.6)/1 mM EDTA for 2 h at 37°C. The RNA was ethanol precipitated after addition of E. coli tRNA as carrier. After centrifugation, the collected RNA pellet from each fraction was resuspended in 100 ~tl of 0.5% SDS/10 mM Tris-HC1 (pH 7.4) for membrane-bound polyribosomal RNA and in 15 ~tl for free polyribosomal RNA. The samples of each fraction were analyzed for the contents of fibrinogen polypeptide mRNA and albumin mRNA sequences. Results

Preparation of fibrinogen polypeptide mRNAs and characterization of the complementary DNA probe For the immuno-precipitation of polyribosomes containing nascent chains of fibrinogen polypeptides, goat anti-rat plasma-fibrinogen antibodies were utilized followed by precipitation of immune complexes using rabbit anti-goat "y-globulins. The polyribosomes were prepared from stimulated rat livers as reported earlier [14]. Translation in a wheat germ cell-free system and hybridization kinetics showed that the isolated poly A containing RNA from immuno-precipitated polyribosomes represents the messenger RNAs for fibrinogen polypeptides [14]. As estimated by sucrose gradient centrifugation, the length of the complementary DNAs transcribed from these

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Fig. 1. Hybridization analysis of various RNA fractions prepared during the purification steps of fibrinogen polypeptide mRNAs. Fibrinogen polypeptide [3H]cDNAs were hybridized to the RNA fraction from which it was transcribed (O e); to total polyribosomal RNA prepared from livers of rats, 24 h after turpentine treatment (O O); to RNA fraction isolated from immunoprecipitated polyribosomes using antibodies specific for fibrinogen (A A); to purified albumin mRNA (× × ) and to total polyribosomal RNA of rat kidney (n U).

mRNAs was greater than 6 S (400 nucleotides) (data not shown)• When these mRNAs were hybridized in RNA excess to the cDNAs transcribed from these mRNAs, a Rotl/2 value of 3.55. 10 -3 mol × s per 1 with a 92% completion of reaction was obtained. By comparison with the R otl/2 (1.26 • 10 -3 mol x s per 1) of purified albumin mRNA (approx. 2250 nucleotides), the sequence complexity for these mRNAs can be calculated to be approx. 6400 nucleotides. From the electrophoretic mobilities on methylmercury hydroxideagarose gels, it was determined that mRNA for the Aa fibrinogen polypeptide chain contains approx. 2250 nucleotides, while the mRNAs for Bfl and 7-polypeptides contain approx. 2060 and 1780 nucleotides, respectively. Although the distribution of these three individual mRNAs has not yet been determined in this mRNA fraction isolated from the immuno-precipitated polyribosomes, the finding of the sequence complexity analysis is in good agreement with the results of the electrophoretic mobility. Fig. 1 shows the specificity of the cDNA probe for fibrinogen polypeptides mRNAs. Under the stringent reaction condition used, there was no annealing between the cDNA probe and purified

124 a l b u m i n m R N A o r the p o l y r i b o s o m a l R N A fraction obtained f r o m a d u l t rat k i d n e y . Immunoprecipitation of f i b r i n o g e n p o l y p e p t i d e s synthesizing polyribosomes from turpentinet r e a t e d rats led to an l 1-fold e n r i c h m e n t o f fibrinogen polypeptide mRNAs. Oligo(dT)-cellulose c o l u m n c h r o m a t o g r a p h y y i e l d e d ' a n o t h e r 3 0 - f o l d e n r i c h m e n t of m R N A s in the R N A fraction.

Body weight, liver weight and concentrations of serum albumin and plasma fibrinogen To examine the effect of regeneration after partial h e p a t e c t o m y o n the r e g u l a t i o n o f p r o t e i n a n d m R N A s y n t h e s i s in the liver, e x p e r i m e n t s w e r e performed simultaneously with material from control, s h a m - o p e r a t e d a n d p a r t i a l h e p a t e c t o m i z e d a n i m a l s , at i n t e r v a l s f o l l o w i n g the surgical p r o c e d u r e . T h e p r e s e n t d a t a are t h e a v e r a g e s o f the

results o f at least t h r e e e x p e r i m e n t s . A l t h o u g h t h e r e was s o m e v a r i a t i o n , these results w e r e h i g h l y r e p r o d u c a b l e . A s i l l u s t r a t e d in T a b l e I, there is a d e c r e a s e in the b o d y w e i g h t of a n i m a l s f o l l o w i n g partial hepatectomy. Although a weight reduction w a s f o u n d in s h a m - o p e r a t e d a n i m a l s on the first d a y a f t e r surgery, the w e i g h t loss was m u c h m o r e s e v e r e a n d m a i n t a i n e d for a l o n g e r p e r i o d in animals following partial hepatectomy. T h e loss of the liver tissue a l o n e a f t e r r e s e c t i o n c a n n o t a c c o u n t f o r the r e d u c t i o n o f the w h o l e b o d y weight. A s also i l l u s t r a t e d in T a b l e I, the r e p l a c e m e n t of h e p a t i c m a s s a f t e r r e s e c t i o n occ u r r e d steadily. T h e liver weight, 72 h a f t e r resection, r e a c h e d m o r e t h a n t w i c e the o r i g i n a l r e s i d u a l l i v e r mass. C o n c o m i t a n t l y w i t h the b o d y w e i g h t r e d u c t i o n , the liver o f s h a m - o p e r a t e d a n i m a l s was r e d u c e d as a c o n s e q u e n c e o f d i m i n i s h e d f o o d int a k e at the first d a y a f t e r surgery.

TABLE I YIELDS OF FREE AND MEMBRANE-BOUND POLYRIBOSOMES FROM CONTROL RATS AND ANIMALS AT DIFFERENT TIME INTERVALS AFTER PARTIAL HEPATECTOMY OR LAPAROTOMY .

Data are expressed as the average of at least three experiments with 3-5 rats per experiment _+S.D. Significance of P values (Student's t-test): Body weight: partial hepatectomy I-II: for all intervals after operation P value < 0.005; laparotomy III-IV: for 12 and 24 h after operation P value < 0.005, for 48 and 72 h N.S. Body weight (g)

Control Partial hepatectomy Interval after operation (h) 12 24 48 72

Liver weight (g)

261 _+ 10

9.9 _+0.7

Total RNA (mg/g liver)

Polyribosomal RNA free (mg/g liver)

membranebound

8.33 _+0.52

1.32 _+0.21

4.74+0.38

(1) 230 + 13 259-+ 14 240 _+12 247_+ 7

Before operation (II) 245 + 13 283_+ 15 269 _+ I 0 276_+ 9

Residual liver weight 2.2 -+0.5 3.0_+0.3 4.3 + 0.2 5.5_+0.3

10.53 + 0.95 a 9.38_+0.52 a 10.85 + 0.80 a 10.73_+0.98a

1.70 -+0.35 1.36_+0.17 1.85 _+0.38 1.42-+0.19

5.36_+0.51 4.96 _+0.27 6.40_+0.38 a 6.84_+0.63 a

(III) 259-+ 14 263 _+ 11 274--+ 16 278 _+21

(IV) 272-+ 14 275 _+ 12 273_+ 14 276 _+23

7.9-+0.3 8.5 _+0.6 9.4_+ 1.0 9.5 _+1.2

10.13_+ 1.10 a 9.42 _+0.78 10.44_+ 1.02 a 8.95 _+0.67

1.29_+0.12 1.24 _+0.18 1.29_+0.20 1.37 _+0.25

5.53_+0.31 a 4.57_+0.27 5.49_+0.38 a 5.62 _+0.42 a

Laparotomy Interval after operation (h) 12 24 48 72

a As compared to the control value, the increased RNA content is statistical significant (P < 0.05).

125 TABLE II CONCENTRATIONS OF PLASMA FIBRINOGEN A N D SERUM ALBUMIN IN CONTROL RATS AND ANIMALS AT D I F F E R E N T TIME INTERVALS AFTER PARTIAL HEPATECTOMY OR LAPAROTOMY Data are expressed as the average of at least three experiments with 3-5 rats per experiment ± S.D. As compared to the control value, the increased plasma fibrinogen concentration and the decreased serum albumin level after operation are statistically significant ( P < 0.001), except * has a P value < 0.04 (Student's t-test). Control

Intervalafteroperation(h) 12 24 48 72

Fibrinogen (mg/ml) 2.8 _+0.3

Albumin (mg/ml) 29.1 ± 2.0

Partial hepatectomy

Laparotomy

Partial hepatectomy

Laparotomy

3.6±0.3* 4.3±0.5 4.4±0.5 4.2±0.6

4.6±0.5 5.8±0.8 4.8±0.6 4.6±0.5

25.0±1.0" 23.0±0.9 22.0±1.2 21.7±0.6

26.7±0.6* 26.8±1.1 26.2±1.0 26.2±0.5

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Fig. 2. Sucrose gradient centrifugation analysis of liver free (A) and membrane-bound (B) polyribosomes and the distribution of albumin m R N A and of fibrinogen polypeptide mRNAs in these RNA fractions from control rats and animals, 48 and 72 h (left) and from animals 12 and 24 h (right) after partial hepatectomy or laparotomy. Approx. 6 A260 units of polyribosomes were analysed by sucrose gradient centrifugation (see Materials and Methods). After prote~nase K digestion and ethanol precipitation of RNA in each collected fraction, various amounts of RNA were hybridized to fibrinogen polypeptide [3H]cDNA (700 cpm) (O O) or to albumin [3H]cDNA (800 cpm) (@ O). Dilutions were made, for detection of fibrinogen polypeptide mRNA: free polyribosomes, 1 / 1 - 1 / 5 , membrane-bound: 1/20-1/100; for detection of albumin mRNA: free polyribosomes 1 / 2 - 1 / 4 , membrane-bound: 1 / 5 0 - 1 / 1 0 0 . Hybridization reactions were carried out at 65°C for 48 h.

126

The concentrations of serum albumin and plasma fibrinogen at intervals after the surgical procedure are demonstrated in Table II. In animals following partial hepatectomy as well as in shamoperated rats, the plasma fibrinogen concentration was increased as compared to the value for the control animals. Despite the loss of hepatic mass after resection, the plasma level of fibrinogen is comparable to the value obtained from stimulated animals with an intact liver (sham-operated). This finding indicates that there is an enhanced synthesis of fibrinogen in regenerating liver after resection. In contrast, the serum albumin concentration was reduced in the experimental animals. However, the reduction of serum albumin concentration was also found in sham-operated rats. Since the loss of plasma proteins due to the surgical procedure has not been ruled out, the pathogenesis of hypoalbuminaemia in these experiments was still uncertain.

given in Fig. 2. As shown in this figure, there were changes in size of free and membrane-bound liver polyribosomes from partial hepatectomized and sham-operated animals at 12 and 24 h after surgery as compared to that of control group and animals at 48 and 72 h after operation. These changes were probably the result of a temporally reduced food intake in these animals after surgery.

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Yield and size of free and membrane-bound polyribosomes To study the effect of regeneration after partial hepatectomy on the distribution of free and membrane-bound polyribosomes and on the sequence content of a specific m R N A , a quantitative isolation of undegraded polyribosomes according to the method of Ramsey and Steele [10] was used in these studies. As shown in Table I, the yield of total liver R N A / g tissue as determined by the method of Fleck and Munro [19], increased after the surgical procedure. Despite the diminished food intake on the first day after operation the liver R N A content was not reduced as compared to the control value. However, the R N A concentration in the liver, 24 h after resection as well as 24 h after sham operation showed a lower value in comparison with the findings in livers at 12 and 48 h after the surgical procedure. The R N A concentration at 72 h after sham operation returned and was approaching the control value. In all experiments membrane-bound polyribosomes comprised 70-80% of the total liver polyribosomes. No shift of polyribosomal population favoring the free polyribosomes, was observed in these experiments. To examine the size of isolated polyribosomes, 10-40% ( w / v ) isokinetic sucrose gradient centrifugations were performed. A representative result is

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log Rot Fig. 3. Hybridization kinetics of R N A fractions isolated from free (open symbols) and from membrane-bound (closed symbols) liver polyribosomes of control rats and animals at different time intervals after partial hepatectomy. [ 3H]cDNA specific for fibrinogen polypeptide m R N A s (A), for albumin m R N A and for a-fetoprotein m R N A (B) were hybridized to purified m R N A s of fibrinogen polypeptides (A, × ×); to purified albumin m R N A (B, × × ); to purified a-fetoprotein m R N A (B, × . . . . . . ×); "to R N A isolated from control rat livers (O o, © ©); to liver R N A of rats, 12 h (,~ ~), ~ ~ ) , 24 h (ll I , [] []); 48 h (ll A, A r,) and 72 h (v v, v v) after partial hepatectomy and to yolk sac polysomal R N A (B, + . . . . . . . + ). The results of hybridization reactions with ~t-fetoprotein [aH]cDNA showed identical curves for free ( © - . . . . O) and for membrane-bound (O . . . . . . O) polyribosomal R N A of control and experimental animals.

127

Quantitation of mRNAs of fibrinogen polypeptides and of albumin mRNA Using molecular hybridization and a specific c D N A probe, the concentrations of mRNAs of albumin and fibrinogen polypeptides were measured in RNA fractions isolated from free and membrane-bound polyribosomes of the livers at intervals after the surgical procedure. As shown in Fig. 3A, the RNA fraction obtained from membrane-bound polyribosomes of control animals contains fibrinogen polypeptide m R N A sequences, approx. 100-times more abundant than that of free polyribosomes. Since the free polyribosomes comprise 20-25% of the total polyribosomes in the liver, it can be calculated that only 0.3% of the total fibrinogen polypeptide mRNA sequences in the liver are associated with the free polyribosomal fraction. From the Rot~/2 value of purified fibrinogen polypeptide mRNAs (3.55. 10 - 3 mol x s per 1), and the Rot~/2 value of RNA fraction prepared from membrane-bound polyribosomes of control animals (2.52 mol × s per 1) (Fig. 3A), it was determined that fibrinogen polypeptides synthesizing polyribosomes in control rat liver represent approx. 3.1% of the membranebound polyribosomes. Since the membrane-bound polyribosomal RNA prepared by the method of Ramsey and Steele comprised approx. 50% of the total cellular RNA (loss of ribosomal and monosomal materials in heavy sucrose gradient and the contribution of free polyribosomal fraction) the relative fibrinogen polypeptide mRNA content ex-

pressed as % of total cellular poly A-containing R N A is 1.5 (Table III). The concentration of fibrinogen polypeptide mRNA sequences was dramatically increased after partial hepatectomy. The maximal effect with a 5-6-fold increment of control value was found at 12-24 h after resection. Thereafter, the fibrinogen polypeptide mRNA concentration showed a gradual decrease (2-fold, 72 h after resection). Since the hepatic mass including the RNA content was increased in this period (more than 200% of the original residual liver weight, Table I) the sequence content of fibrinogen polypeptide mRNAs remained at approximately the same level in the total residual liver during the period of 72 h after partial hepatectomy. In contrast to the findings of fibrinogen polypeptide mRNAs, the concentration of albumin mRNA sequences was decreased in the residual liver after partial hepatectomy. 12-48 h after the surgical procedure, the concentration was approaching one-third of the control value, however, at 72 h after resection, the concentration was normalized (Fig. 3B, Table III). Similar patterns of changes for fibrinogen polypeptide and albumin m R N A concentration as found in the membranebound fractions were also observed in the free polyribosomal RNA fractions. Fig. 4 demonstrates the findings of hybridization kinetics in sham-operated animals. Although the maximal concentration of fibrinogen polypeptide mRNAs 12-24 h after operation was not

T A B L E 1II C O N T E N T S OF F I B R I N O G E N P O L Y P E P T I D E A N D A L B U M I N m R N A SEQUENCES IN T O T A L C E L L U L A R R N A AT DIFFERENT TIME INTERVALS AFTER PARTIAL HEPATECTOMY AND LAPAROTOMY Relative m R N A content expressed as % of total cellular poly A containing RNA. Control

lntervalafteroperation(h) 12 24 48 72

Fibrinogen polypeptide 1.5

Albumin 10.0

Partial hepatectomy

Laparatomy

Partial hepatectomy

Laparotomy

10.0 8.9 6.3 3.1

6.3 5.6 4.4 2.8

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Distribution of fibrinogen polypeptide mRNA sequences in sucrose gradient fractions of free and membrane-bound polyribosomcs To determine whether changes of distribution of fibrinogen polypeptides mRNA sequences in the polyribosomal population could be observed in stimulated livers, free and membrane-bound polyribosomes from control and experimental animals were sedimented in 10-40% (w/v), isokinetic sucrose gradients. An RNA sample from each fraction was assayed for sequences complementary to fibrinogen polypeptide and albumin mRNA. As shown in Fig. 2 the albumin mRNA sequences as well as the fibrinogen polypeptide mRNA sequences were found equally in all fractions of membrane-bound polyribosomes and in much less quantity in free polyribosomes. No particular peak of mRNA concentration was observed in these fractions. Discussion

4

log Rot

Fig. 4. Hybridization kinetics of R N A fractions isolated from free (open symbols) and from membrane-bound (closed symbols) liver polyribosomes of control rats and animals at different time intervals after sham operation. [3H]cDNA specific for fibrinogen polypeptide m R N A s (A), for albumin m R N A and for a-fetoprotein m R N A (B) were hybridized to various R N A fractions as indicated in Fig. 3, except that the experimental animals were sham-operated rats.

as high as the value found after partial hepatectomy, the pattern of concentration changes for fibrinogen polypeptide mRNAs was almost identical in these two conditions (sham-operation and partial hepatectomy). The measurement of albumin mRNA sequence content in the livers of sham-operated rats showed similar findings as found in regenerating liver after partial hepatectomy. In all experiments up to 7 days after partial hepatectomy, there was no a-fetoprotein mRNA detectable in the liver RNA fractions even when hybridization reactions were carried out to significantly high Rot values (Figs. 3B and 4B).

In order to study the effect of regeneration after partial hepatectomy on the distribution of free and membrane-bound polyribosomes, it is important to use an isolation procedure with high yields of undegraded polyribosomes. Methods for separation of membrane-bound and free polyribosomes from post-mitochondrial supernatant have been subjected to criticism, because the yields of membrane-bound polyribosomes may be quite low and significant degradation of membrane-bound polyribosomes may occur, particularly in physiological or pathological conditions, wherein a high ribonuclease activity could be found in the liver [10,20-22]. In the present study and in previous experiments [11,12,23] using the isolation technique described by Ramsey and Steele [10], we have obtained an excellent recovery of undegraded polyribosomes. Although a shift to favor a substantial increase in the free polyribosomal content [3] and changes in.polyribosomal size [24,25,31] have been reported in regenerating liver after resection, the results of our present study indicate that partial hepatectomy or sham operation does not affect the distribution and the average size of free and membrane-bound polyribosomes in rat liver. However, the RNA content showed no change or a slight increase after the operation,

129 despite the fact that these animals did not eat for 12 h or for a longer period after surgery. Prolonged fasting has been shown to cause a shift in polyribosomal size and a decrease in RNA content as early as 12 h after initiation of fasting [23,24,26,27]. Previously, we have reported that practically all albumin m R N A sequences in rat liver polyribosomes are contained in the membrane-bound fraction [12]. In the present study, using a [3H]cDNA probe to determine the sequence content of fibrinogen polypeptide mRNAs, we demonstrate that more than 99% of fibrinogen polypeptide mRNA sequences are associated with the membrane-bound fraction. These findings as expected support again the hypothesis that secretory proteins are synthesized primarily on membranebound polyribosomes, whereas 'structural' proteins are synthesized primarily on free polyribosomes. Although the results of our experiments indicate that free polyribosomes do contain a low level of mRNAs sequences for albumin as well as for fibrinogen polypeptides, it was unclear whether this small amount of mRNAs was due to contamination or represented a fraction of albumin or fibrinogen polypeptides synthesizing polyribosomes prior to their attachment to endoplasmic reticulum membranes, as suggested by Blobel and Dobberstein [28]. In stimulated livers after partial hepatectomy and after sham operation the dramatic increase of m R N A sequences for fibrinogen polypeptides was found not only in the membrane-bound polyribosomal fraction but also in free polyribosomes. These findings could be consistent with the model proposed by Blobel and Dobberstein [28], however, analysis of sucrose gradient fractions for fibrinogen polypeptide mRNA sequences established that the increased level of these m R N A sequences in the free polyribosomal fraction was not particularly found in the small aggregates. Therefore, the increased level of fibrinogen polypeptide mRNAs in the free polyribosomal fraction in these experiments is due to cross contamination with the membrane-bound fraction during preparation. A rise in the serum ~x-fetoprotein level has been observed during liver regeneration after partial hepatectomy and after CC14 intoxication [5-8,29].

However, the augmentation of the production depends on the age and the species of the experimental animals used [5]. The reexpression of this oncofetal protein in the liver has been suggested to be associated with the stimulated synthesis of DNA in damaged liver [6,30], but liver cell injury per se may also play an important role for the augmented production of this protein [7]. In our present study, using c~-fetoprotein cDNA probe and molecular hybridization, no c~-fetoprotein mRNA sequences could be detected in the liver RNA fractions up to 7 days after partial hepatectomy in adult rats (weight 250-300 g). Although Sell et al. [6] using a sensitive radioimmunoassay, have demonstrated a small ~-fetoprotein elevation in serum after 70% liver resection in adult rats, our findings suggest that there is no significant synthesis of ~xfetoprotein mRNA in the residual liver after operation. Using molecular hybridization, Atryzek and Fausto [31] demonstrated that, during liver hypertrophy after partial hepatectomy, the amount of polyadenylylated RNA doubles, while Scholla et al. [32] and Wilkes et al. [33] showed that the total sequence complexity, which corresponds to approx. 10000-20000 different mRNA sequences was similar to that of sham-operated rats. However, Grady et al. [34] demonstrated that at 24 to 48h after 70% liver resection there was a 10-14% difference in RNA sequences in this liver as compared to those found in sham-operated animals. In this study a dramatic increase (5-6-fold) of fibrinogen polypeptide mRNA content was found during the first 24h after resection. In contrast, the albumin mRNA content decreased (2-fold). Similar changes in fibrinogen polypeptide and albumin mRNA concentration were found in shamoperated animals. These results indicate that albumin and fibrinogen synthesis after partial hepatectomy is reciprocally regulated at the mRNA level and represents a nonspecific acute phase response to surgical trauma. Although further studies using specific DNA probes for the mRNA of structural proteins remain to be established for the exact mechanism of regenerating response, from our findings we can conclude that the residual liver after partial hepatectomy is still able to maintain its functions as intact liver in the synthesis of plasma proteins.

130

Acknowledgements The authors would like to thank Mr. H.W. Verbruggen for performing plasma fibrinogen determinations. This research was supported in part by a grant from the Foundation for Fundamental Medical Research (FUNGO) and a grant from KWF (Koningin Wilhelmina Fonds).

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