Free and bound hepatic polyribosomes after partial hepatectomy

7°

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 96941

F R E E AND BOUND HEPATIC POLYRIBOSOMES A F T E R PARTIAL HEPATECTOMY: POOL SIZES AND SEDIMENTATION PATTERNS M A R K ZWEIG AnD J O E W. GRISFIAM

Department o/ Pathology, Washington University School of Medicine, 4566 Scott Avenue, St. Louis, Mo. 63110 (U.S.A.) (Received March i6th, i97 I)

SUMMARY

To examine the response of hepatocytes to the sudden and severe demands of regeneration, we have studied free and bound ribosomal pool sizes and polyribosomal sedimentation patterns from o to 12o h after partial hepatectomy. Total ribosomal RNA increases rapidly after partial hepatectomy, reaching a peak at 24 h. Pools of both free and bound ribosomes enlarge similarly, their relative sizes remaining unchanged. Except for an early and apparently nonspecific increase of free polyribosomes of smaller size, sedimentation patterns of both free and bound polyribosomes exhibited concurrent shifts to predominance of heavier aggregates.

INTRODUCTION

Recent evidence suggests that polyribosomes attached to membranes of the endoplasmic reticulum (bound polyribosomes) are the cellular machinery for synthesizing proteins secreted from the hepatocyte and that polyribosomes lying free within the cytoplasmic matrix (free polyribosomes) synthesize proteins that remain within the cell1-e. Replacement of hepatic mass after two-thirds hepatic resection (partial hepatectomy) in the rat is accomplished by cellular proliferation, which peaks on the second day after surgery 7. Residual hepatocytes must therefore synthesize a variety of structural proteins to form new cells; normally, hepatocytes synthesize mainly serum proteins s. The total content of hepatic ribosomes increases after partial hepatectomyg; if free ribosomes are principally involved in synthesis of structural proteins one might expect a relatively greater increment in tree than in bound ribosomes at early intervals after hepatic resection. Indeed, CAMMARANO et al. 1° found that after partial hepatectomy, virtually all hepatic polyribosomes were released without use of detergent (i.e. were free). Consistent with this finding, BRAUN et al. a noted that incorporation of amino acid into structural proteins was much greater at early intervals after partial hepatectomy than was incorporation of amino acid into serum proteins. However, MAJUMDARet al. ~ and CHANDLER AND SNIDERTM have found the synthesis of serum proteins to be markedly increased early after partial hepatectomy, at the same time that formation of new cells occurs. Biochim. Biophys. Acta, 246 (1971) 70-80

FREE AND BOUND HEPATIC POLYSOMES

71

Consistent with these observations and contrary to the results of CAMMARANOet al. x°, both DELHUMEAU DE ONGAY et al. 13 and WEBB et al. ~a found no change in the distribution of free and bound ribosomes after partial hepatectomy; the content of ribosomes in both pools increased by approximately the same extent so that their relative sizes remained the same as in controls. These previous studies have been limited by inability to quantitatively recover ribosomes from liver homogenates. Recently, BLOBEL AND POTTER15 described a method of cellular fractionation which permits quantitative recovery of both free and bound ribosomes from liver. In addition, use of crude ribonuclease inhibitor allows isolation of intact polyribosomes and preparation of high quality sedimentation patterns ~e-19. We have utilized these new techniques to determine the quantity of ribosomes in free and bound pools and to study the sedimentation characteristics of polyribosomes isolated from each of these pools in livers of rats at different times after partial hepatectomy or laparotomy. In the regenerating hepatocyte, significant but similar changes occur in quantity and character of both free and bound polyribosomes.

MATERIALS AND METHODS

Animals and operations Male Wistar albino rats (Charles River) weighing 250-300 g were used in all experiments. Food and water were available ad libitum. Rats were partially hepatec: tomized under ether anesthesia by the method o~ HI6GINS AND ANDERSON20; shamoperated control animals were subjected to the same manipulation, excluding ligation and removal of hepatic lobes. Operations were performed at time intervals staggered so that animals would be killed between 8 : oo a.m. and 12 : oo noon 21. At the time of sacrifice, liver tissue was quickly removed, chilled, and weighed. All subsequent operations were performed at o to 4 °. Homogenization was carried out in a glass-teflon homogenizer using 17 strokes. Fractionation o] cellular R N A Free and bound ribosomes were quantitatively sedimented from whole cytoplasm by the method of BLOBEL AND POTTER15 or by a modification of their method. Liver was minced with scissors in two volumes of ice-cold 0.25 M sucrose in 0.05 M Tris-HC1, p H 7.5, 25 mM KC1 and 5 mM MgCI, and homogenized. The holnogenate was filtered through four layers of gauze. Nuclei were isolated b y centrifugation through 2. 3 M sucrose in 0.05 M Tris-HC1, p H 7.5, 25 mM KC1 and 5 mM MgC12 and the supernatant (postnuclear fraction) was collected and rehomogenized. Free and total ribosomes were prepared from the postnuclear homogenate and the quantity of bound ribosomes calculated b y difference is. Alternatively, 0. 5 ml of the postnuclear fraction was mixed with an equal volume of crude ribonuclease inhibitor ($3). One ml of the mixture was layered over 3.0 ml of 2.0 M sucrose in 0.05 M Tris-HC1, p H 7.5, 25 mM KC1 and 5 mM MgCI 2 in a polyallomer tube and the tube was filled to a volume of IO ml with 0.25 M sucrose in 0.o 5 M Tris-HC1, p H 7.5, 25 mM KC1 and 5 mM MgCI~. Total free cytoplasmic ribosomes were obtained in a pellet b y centrifugation in a Spinco 4 ° rotor Biochirn. Biophys. Acta, 246 (1971) 70-80

72

M. ZWEIG, J. w. GRISHAM

at 40 ooo rev./min for 25 h. The supernatant remaining after sedimentation of the free ribosomes was treated with sodium deoxycholate to solubilize membranes. Centrifugation in the Spinco 4 ° rotor at 4 ° ooo rev./min pelleted the membrane bound ribosomes released b y the detergent. The supernatant contained nonsedimentable RNA (primarily tRNA) plus t R N A added in the S3. Both methods gave comparable results. However, if bound ribosomes were isolated directly without addition of ribonuclease inhibitor a significant but variable portion of RNA was released from bound ribosomes and rendered nonsedimentable, falsely lowering the content of RNA in the bound ribosome fraction while increasing that in the supernatant.

DNA and RNA determinations DNA and RNA were quantitated in whole homogenates, in the pellets ot isolated nuclei, and in the postnuclear (cytoplasmic) fractions. RNA content was also determined in ribosomal pellets (bound, free and total) and in the final supernatants containing nonsedimentable RNA. DNA and RNA were separated by the method of FLECK AND MUNRO~2 and the RNA was quantitated b y ultraviolet absorption at 254 nm. DNA was measured colorimetrically using the diphenylamine reaction 23. Polyribosome isolation and gradient sedimentation Polyribosomes were isolated from postmitochondrial supernatants is. Throughout this procedure, crude ribonuclease inhibitor (Sa) was present in the media. For sucrose gradient sedimentation, I.o ml of the suspension of free polyribosomes or 1.2 ml of the suspension of bound polyribosomes was layered on a chilled linear gradient (IO to 4 ° %) of sucrose in o.o5 M Tris-HC1, p H 7-5, 25 mM KC1 and 5 mM MgC12 and centrifuged for lO5 rain at 25 ooo rev./min in the Spinco SW 25.1 rotor. Gradients were pumped through an ultraviolet monitor (ISCO) containing a 5 m m flow cell and a tracing of the absorption at 254 nm continuously recorded. Tissues from different rats were not pooled; a separate tracing for free and bound polyribosomes was obtained for each rat. Sodium deoxycholate was obtained from Fisher Scientific Co. Sucrose was a high purity, ribonuclease-free product obtained from Schwarz Bioresearch, Inc. Analysis of sedimentation patterns Areas under density gradient sedimentation patterns were measured directly from the tracing. Areas under curves were proportional to polyribosomal RNA content since the tracing was made at 254 nm and at constant chart speed and elution rate. To detect shifts in the proportion of heavy polyribosomes, patterns were arbitrarily divided b y passing a vertical line to the baseline from the low point of the trough separating polyribosomes containing four monomers from those containing five monomers. The area occupied b y polyribosomes containing five or more monomers was then computed as a percentage of the total area under the curve. RESULTS

Content and recovery o] RNA and DNA in homogenates and in ]ractions Unmanipulated control rats had an average of lO.44-o.51 mg RNA (mean4standard deviation) and 2.79-4-o.12 mg DNA per g wet liver (ratio of RNA to Biochim. Biopkys. Acta, 246 (1971) 70-80

73

FREE AND BOUND HEPATIC POLYSOMES

DNA -- 3.74±o.18). Temporal changes in the concentrations of hepatic RNA and DNA and in the ratio of RNA to DNA after partial hepatectomy or laparotomy are shown in Fig. I.

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F i g . I. Content of DI~/A (upper) and R N A ( m i d d l e ) and t h e ratio of R N A to D N A (lower) in livers of rats at different intervals after paxtial h e p a t e c t o m y ( O ) or l a p a r o t o m y ( O ) . Bars in this and the following figures represent 2 standard deviations about the mean.

Content of RNA in various cellular fractions from livers of unmanipulated control rats is shown in Table I, together with percentages of homogenate values in each fraction. The recovery of RNA in fractions from control rats was 97.6~2.8 %. Recoveries of RNA from laparotomized and partially hepatectomized rats was 96.4+1.3 % and 97.72LI.6 %, respectively. The relative proportions of RNA found in various fractions agrees with tile findings of BLOBEL AND POTTER15. Recovery of TABLE

I

R N A CONTENT AND PERCENT OF TOTAL R N A IN VARIOUS HEPATOCYTIC FRACTIONS

D a t a are m e a n 4 - s t a n d a r d deviation.

(rag RNA [g wet liver)

% o] h o m o g e n a t e RNA

Homogenate Nuclei Total ribosomes Free ribosomes B o u n d ribosomes Nonsedimentable RNA

l O . 3 5 -t- 0. 51 0.364-0.07 8 . 3 2 4- 0 . 5 6 2.21 + o . 12 6.13 +o.34 1.4o4-o.26

i oo 3.54-0.68 80. 4 4- 2 . 0 9 21.34-1.82 59.24-1.15 13.64-1.o9

Recovery (%)

lO.lO4-O.87

97.6±2.82

Fraction

RNA

content

Biochim. Biopkys..4eta, 2 4 6 ( 1 9 7 1 ) 7 o - 8 o

74

M. ZWEIG, J. W. GRISHAM

homogenate DNA in fractions averaged 92.2~2.3 o//o for all animals. There was no difference between unmanipulated controls and animals subjected to either laparotomy or partial hepatectomy at any time after surgery. The cytoplasmic fraction of unmanipulated controls and of rats subjected to laparotomy contained 12.3 :t: o.5 % of the homogenate DNA, Whereas for partially hepateetomized animals the comparable value was 18.5±o.8 o/,. This indicates that homogenization causes appreciable breakage of hepatocytic nuclei which appear to be more fragile after partial hepatectomy.

Content o/ RNA in pools o] /ree and bound ribosomes The content of RNA in pools of free and bound ribosomes relative to homogenate DNA (ratio of RNA in fraction to homogenate DNA) is illustrated in Fig. 2. These values fluctuated only slightly after laparotomy, but both free and bound pools clearly increased in partially hepatectomized animals as early as 9 h after surgery, reached a peak size at 24 h and then declined. Free ribosomes of unmanipulated control animals constituted 27.9 ~ i. 12 o,~ of the total ribosomal population. As shown in Table II, there was no appreciable change in this value after laparotomy and only slight increase after partial hepatectomy. Thus, the sizes of the pools of both categories of ribosomes increased proportionately after partial hepatectomy.

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Fig. 2. RNA in hepatic pools of bound (upper) or free (lower) ribosomes relative to total DNA at intervals following partial hepatectomy ( 0 ) or laparotomy ( 0 ) .

Sedimentation patterns o] ]ree polyribosomes i8 profiles were obtained from unmanipulated control rats. In Fig. 3, a typical sucrose density gradient sedimentation pattern of free polyribosomes from the liver of an unmanipulated control rat is shown. At least 6 small peaks were resolved corresponding to monomeric ribosomes and aggregates of 2, 3, 4, 5, and 6 ribosomes, Biochim. Biophys. Acta, 246 (i97 I) 70-8o

FREE AND BOUND HEPATIC POLYSOMES

75

TABLE II FREE

HEPATIC

RIBOSOMES

HEPATECTOMY

AS THE

PERCENT

OF TOTAL

RIBOSOMES

AT INTERVALS

AFTER

PARTIAL

OR LAPAROTOMY

Time after surgery (h)

o 4 8 I2 18 24 36 48 72 12o

Treatment

Number o] animals

% Free ribosomes*

None Hepatectomy Laparotomy Hepatectomy Laparotomy Hepatectomy Laparotomy Hepatectomy Laparotomy Hepatectomy Lapatoromy Hepatectomy Laparotomy Hepatectomy Laparotomy Hepatectomy Hepatectomy

io 4 3 IO 4 7 3 IO 7 io 3 3 3 7 7 7 4

27.9-t- 1.12 28.34-1.2 I 28.34- 0.9 I 28.8 4- o.87 28.74- o.73 29.64-o.96 28.4 4- 0.96 3o.04-o.67 28.44- I.O7 3o.o4-o.95 28.74-o.94 3 i. 14- i.o I 28.2 4- o. 8i 30.04-o.7 I 28.64-1.37 28.3 4-1.38 28.7 4- o. 8o

* Mean+standard deviation. 1.2 "

Control

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72 h

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GRADIENT CONCENTRATION

Fig. 3. Representative sedimentation patterns of polyribosomes isolated from the pool of free ribosomes at intervals after partial hepa.e ...... ~. respectively. Most of the polyribosomes sedimented farther into the gradient a n d were n o t resolved into i n d i v i d u a l aggregate species b u t appeared as a broad m a j o r peak. F r a c t i o n s collected from gradients at various points a n d e x a m i n e d b y electron microscopy c o n t a i n e d i n t a c t polyribosomes of appropriate sizes. S e d i m e n t a t i o n p a t t e r n s were o b t a i n e d for laparotomized a n d p a r t i a l l y hepatectomized rats at 4, 6, 8, IO, 12, 18, 24, 48, 72 a n d 12o h after surgery. A total of 119 profiles were o b t a i n e d representing 3 - 7 laparotomized rats a n d 4-1o hepatectomized rats at each time point. P a t t e r n s from laparotomized rats differed from those from u n m a n i p u l a t e d controls (zero time) o n l y at 8 h after surgery, at which time there was a relative decrease in the proportion of aggregates c o n t a i n i n g 5 or more ribosomes (Fig. 5). Biockim. Biopkys. Acta, 246 (1971) 70-80

76

M. ZWEIG, J. W. GRISHAM

Sedimentation patterns from partially hepatectomized animals showed distinctive changes (Fig. 3). No change was apparent at 4 h, but at 6 and 8 h large aggregates were less prominent than in zero time controls (Fig. 5). At this time the slope of the ascending portion of the large major peak decreased, due to a broadening of the size distribution of polyribosomes contained within it (i.e. an increase in the relative proportion of smaller aggregates). By 12 h, the proportion of small aggregates had returned to normal and there was narrowing of the major peak. This narrowing (i.e. increase in proportion of very large polyribosomes with a relatively small distribution of sizes) progressed, gradually becoming maximal between 48 and 12o h. During this interval the major peak was very narrow and the proportion of small aggregates was further decreased (over 9 ° % of the free polyribosome population consisted of aggregates containing 5 or more ribosomes).

Sedimentation patterns o/bound polyribosomes 6 profiles were obtained from unmanipulated control rats. A typical sedimentation pattern of membrane-bound polyribosomes isolated from the liver of an unmanipulated control rat is shown in Fig. 4. Compared to the profiles of free polyribosomes, there is a greater proportion of smaller species of aggregates (n = I to 4) (Fig. 5) and the average size of the aggregates in the major peak is smaller (i.e. the major peak is closer to the top of the gradient in bound than in free polyribosomal patterns). Bound polyribosomal profiles were obtained from laparotomized, and partially hepatectomized rats at 4, 6, 12, 18, 24, 48, 72 and 12o h after operation. A total of

1.2

Control ~

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O.a 40%

24h

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10% 40%

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DENSITY GRADIENT CONCENTRATION

Fig. 4. Representative sedimentation patterns of polyribosomes isolated from the pool of bound ribosomes at intervals after partial hepatectomy.

81 profiles were obtained with from 2-3 laparotomized rats and from 4-1o hepatectomized rats at each time point. The sedimentation patterns from laparotomized rats exhibited an early shift toward heavier aggregates, but at 18 h and later after surgery there was no difference from the control patterns (Fig. 5). Sedimentation patterns of bound polyribosomes obtained from partially hepatectomized rats appear in Fig. 4. Compared to controls, there was no change until 18 h when a relative increase in the proportion of larger aggregates was clearly evident (Fig. 5). This trend progressed and persisted through 12o h (at this time almost 80 % of the bound polyribosome population consisted of aggregates containing 5 or more ribosomes). From 72 to 12o h the large major peak was clearlylocated farther Biochim. Biophys. Acta, 246 (1971) 7o-8o

FREE AND BOUND HEPATIC POLYSOMES

77

,~ 78

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70

62

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HOURS AFTER OPERATION

Fig. 5. P r o p o r t i o n of b o u n d and free polyribosomes containing 5 or more m o n o s o m e s at intervals after partial h e p a t e c t o m y ( 0 ) or l a p a r o t o m y ( 0 ) .

down in the gradient compared to unmanipulated controls, reflecting an increase in the average size of the aggregates comprising this peak. There is, then, an increase in the size of aggregates as well as an increase in the proportion of these larger species.

DISCUSSION

Relative " h y p e r t r o p h y " of the free ribosomal pool does not occur after partial hepatectomy; both ribosomal pools enlarge to the same extent. These results agree with those of DELHUMEAU DE ONGAYet al. 13. However, CAMMARANOel al. z°, although they did not attempt to quantitate free and bound ribosomes, found that at 48 h after partial hepatectomy essentially all of the ribosomes in postmitochondrial supernatants were released without use of detergent (i.e. were free). As BLOBEL AND POTTER15 have pointed out, free ribosomes may be recovered quantitatively from postmitochondrial supernatants, but only a variable fraction (depending on homogenization technique and sedimentation times) of bound ribosomes are recoverable from such a preparation. Recovery of ribosomes from postnuclear homogenates, as used in the study reported here, obviates this problem 15. Sedimentation patterns of polyribosomes isolated from free and bound pools are distinctly different ~8. Free polyribosomes contain a larger proportion of heavy aggregates than do bound polyribosomes. This disparity in size distribution may reflect in vitro breakdown of bound polyribosomes. Such breakdown could occur because of the release of ribonuclease activity by the detergent action of deoxycholate. However, there is reason to doubt this explanation. HOWELLet al. ~4 reported no measurable increase in ribonuclease activity after deoxycholate treatment of postmitochondrial supernatants. We have prepared profiles of total polyribosomes by treating the postBiochim. Biophys. Acta, 246 (I97 x) 70-80

78

M. ZWEIG, J. w. GRISHAM

mitochondrial supernatant with deoxycholate and then sedimenting both free and deoxycholate-released bound polyribosomes together. The sedimentation pattern of total polyribosomes prepared in this way was essentially a summation of the separate patterns of free and bound polyribosomes as routinely prepared. If deoxycholatereleased ribonuclease activity produces the typical bound polyribosomal sedimentation pattern, then when total polyribosomes are prepared by deoxycholate treatment their profile should more closely resemble that of bound polyribosomes, rather than a summation of the patterns of the two types. Furthermore, bound polyribosomes were in the presence of 45 % $3 when deoxycholate was added. Following partial hepatectomy the sedimentation patterns of both free and bound polyribosomes change. Earliest changes occur in polyribosomes of the free pool. Free polyribosomes undergo a change resembling breakdown at 6 to 8 h after hepatic resectiod. This early change in sedimentation pattern did not occur in bound polyribosomes. Nutritional factors could adversely affect the integrity of free polyribosomes in partially hepatectomized rats, since these animals may not eat for I2h or longer after surgery. Prolonged fasting has been shown to cause a shift to smaller aggregates in preparations of total polyribosomes 25,2s which may occur as early as I2 h after initiation of fasting 26. Patterns with predominance of small aggregates, noted in total polyribosomes by other investigators .5,28, may reflect the changes found by us in the flee pool. Since a similar change occurred in free polyribosomes of laparotomized animals, we consider this early breakdown to be nonspecific and probably related to stress and temporary inanition. After I2 h the sedimentation patterns of both free and bound polyribosomes behaved similarly, shifting toward a greater predominance of heavy aggregates. Even though the normal sedimentation patterns of free and bound polyribosomes are distinct, the changes occurring after partial hepatectomy caused them to become more alike. A similar but less marked shift to heavier aggregates has been noted after laparotomy and was ascribed to the effect of injury ~. However, changes described by us occurred only in polyribosomes from livers of partially hepatectomized rats and were not present after laparotomy alone. These changes, thus, appear to be dependent on reduction of hepatic mass. In a study of total polyribosomes, WEBB et al. 25 found that at 12 1l after partial hepatectomy there was a decrease in monomers and a shift of mean distribution of polyribosomes to heavier species; at 21 h this shift was more pronounced. CAMMARANO gt al. a° noted a sinfilar build-up of heavy polyribosomes at 25 h after partial hepatectomy and they also described a continuing trend toward accumulation of heavy aggregates during the first 48 h after surgery. They did not utilize laparotomized rats as controls for operative trauma or stress. The changes observed in sedimentation patterns of free and bound polyribosomes are probably intimately related to the increased rates of synthesis of different types of proteins which occur after partial hepatectomy 8,n'12. However, more than just the length of the mRNA molecule is possibly involved. Factors goverlfing peptide chain initiation, growth, termination, and release; rate of reading of mRNA; spacing of ribosomes, and availability of tRNA, amino acids, enzymes, and energy supply may all play a significant role in influencing polyribosome size and configuration 2s. It is unlikely that there is a single simple mechanism underlying the shift to heavier aggregates that occurs in both free and bound polyribosome pools after Biochim. Biophys. Acta,

246 (1971) 70-80

FREE AND BOUND HEPATIC POLYSOMES

79

partial hepatectomy. Other factors that could also affect polyribosome size have been described. Polyribosomes from regenerating liver are said to be inhelently more stable than those from normal livers 29,3°. Furthermore, several investigators have described a decline in ribonuclease activity and an increase in ribonuclease inhibitor in liver after partial hepatectomy31,32. This study shows that the cellular machinery for synthesis of both intracellular and extracellular proteins (free and bound polyribosomes) expands rapidly after partial hepatectomy. Additionally, polyribosomes from each pool (free or bound) contain a larger proportion of heavy aggregates than before surgery. These results support the concept that the "average" residual hepatocyte increases its capacity to synthesize extracellular T M as well as intracellular proteins 8. In addition to the need to form new cells, the residual hepatocyte is confronted with an increased metabolic load. Since the functional capacity of the liver is reduced by partial hepatectomy, many biochemical reactions, including synthesis of proteins, must be accelerated in residual cells merely to maintain homeostasis. Unfortunately, hepatocellular events leading specifically to cell proliferation cannot be readily distinguished from events whose activity is augmented only as a result of increased metabolic load. Enlargement of the pool of free ribosomes may well occur because these organelles must synthesize protein to make new cells; however, this function is not temporally separated in residual hepatocytes from the concurrent necessity to make more extracellular proteins, a need met by similar enlargement of the pool of bound ribosomes. ACKNOWLEDGEMENTS Mhis work was supported by grants AM-o7568 and 5II-GM-897 from the National Institutes of Health. We thank Miss Mary Stenstrom for technical assistance. REFERENCES I 2 3 4 5 6 7 8 9 IO Ii 12 13 14 15 16 17 18 19 2o 21 22 23

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M. ZWEIG, J. w . GRISHAM

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Biochim. Biophys. Acta, 246 (1971) 7o-8o