RNA metabolism in isolated perfused normal and regenerating livers: Polyamine effects

BIOCttlMICA ET BIOPHYSICA ACTA

543

BBA 97419

RNA METABOLISM IN ISOLATED P E R F U S E D NORMAL AND R E G E N E R A T I N G LIVERS: POLYAMINE EFFECTS

NELSON FAUSTO

Brown University, Division of Biological and Medical Sciences, Providence, R. I. 02912 (U.S.A.) (Received May 26th, 1972)

SUMMARY

The labeling profiles of nuclear and cytoplasmic RNA from isolated perfused livers differ from the profiles obtained in experiments in vivo. In isolated livers rapidly sedimenting RNA species in the nucleus are not labeled and no distinct peak of radioactivity is found in 28-S cytoplasmic RNA. The addition of spermidine to the perfusate increases the specific activity of nuclear RNA of perfused livers and enhances the labeling of fast sedimenting RNA species in isolated perfused regenerating livers. These changes are not reflected in an increase in the labeling of 28-S cytoplasmic RNA. Continuous infusion or hourly addition of large amounts of amino acids are necessary to maintain the integrity of the polyribosomes of isolated perfused livers.

INTRODUCTION

Polyamine synthesis is enhanced in many.different tissues during development 1-3, compensatory growth 4-J° or following the administration of hormones u-:s and drugslF, lS. The results of these and other studies suggest that the synthesis and intracellular accumulation of polyamines may be correlated with RNA metabolism. This conclusion is supported by reports on the effects of the polyamines on ribosomes, isolated nuclei and RNA synthesizing systems in vitro which have been interpreted as resulting from a modification of the secondary structure oi RNA (see refs. 19 and 2o for references). The activity of ornithine decarboxylase, the enzyme catalyzing the synthesis of 1,4-diaminobutane (putrescine) from ornithine increases in the first hour after partial hepatectomy reaching a maximum 12-18 h alter the operation 5-9,~1. The early enhancement in polyamine synthesis during liver regeneration coincides with the increase in the synthesis and accumulation of rRNA 6,22,23. However, it is essential to determine if the relationship between RNA and polyamine synthesis during liver regeneration is the consequence ot a specific effect of the polyamines on RNA metabolism, or if it simply reflects a coincidence in the time of activation of two unrelated processes. I attempted to study the effect of polyamines on liver RNA metabolism using isolated perfused normal and regenerating livers. The goal of these experiments Biochim. Biophys. Acta, 281 (1972) 543-553

544

N. FAUSTO

was to determine if an increase in the intracellular concentration of spermidine alters the labeling profiles of nuclear and cytoplasmic RNA of isolated perfused normal and regenerating livers. To accomplish these objectives it was necessary to define some of the characteristics of RNA labeling and to examine the polyribosome profiles of isolated perfused normal and regenerating livers.

MATERIALS AND METHODS Liver per[usion

The perfusion technique for normal and regenerating livers was based on that of Miller et al. 24 and has been described in detail in a previous paper 25. Perfusions were performed in an extracorporeal perfusion unit (Ambeck) maintained at 36 °C, under an atmosphere of 02-CO 2 (95 : 5, v/v). The perfusate flow rate was approx. 2. 5 ml/g of liver per rain. The perfusing fluid (approx. 5o ml) consisted of either fresh whole rat blood diluted to one-half of its volume with Ringer's bicarbonate solution and a complete amino acid mixture at 6 times the normal blood amino acid concentration 26, or a medium containing Ringer's solution, rat red blood cells, bovine or rabbit albumin (3 g/Ioo ml of perfusate) and amino acids 26. 2ooo units of penicilin and 2 mg of streptomycin and 5oo mg of glucose were added per IOO ml of perfusate. Polyamines were dissolved in Ringer's solution and the p H adjusted to 7-4. One-half of the amount of the polyamines to be used in each experiment was added to the perfusate at the start of the perfusion. The other half was infused into the blood reservoir with a H a r v a r d P u m p at a rate calculated to deliver the entire amount of the polyamines continuously up to the end of the perfusion (3 h and 2o min to 4 h). The final concentration of spermidine in the perfusate was 2 • IO -3 M. Animals

Rats (200-250 g) were obtained from Holtzman Co. (Madison, Wisc.), The animals were starved for approx. 14 h before each experiment. Partial hepatectomies were performed by the method of Higgins and Anderson ~7. The regenerating livers used in the perfusion experiments were obtained from rats 18 h following partial hepatectomy. Whole blood for perfusion was obtained under sterile conditions from the aorta of rats weighing 400-6o0 g. Materials

~6-14C]Orotic acid (spec. act. 8.15 mCi/mmole) was obtained from New England Nuclear Corp. (Boston, Mass.). 5 #Ci of the compound dissolved in I ml of Ringer's solution were injected into the cannula connected to the portal vein. The incorporation times are indicated in the text. Spermidine p h o s p h a t e . 6 H20 and amino acids were obtained from Schwartz Mann (Orengeburg, N. Y.). Bovine or rabbit albumin were purchased from Pentex Biochemicals. R N A determinations

At the end of the perfusion period the livers were homogenized in 0.25 M sucrose, o.oi M Tris buffer (pH 7.6). Nuclear and cytoplasmic fractions were obBiochim. Biophys. Acta, 281 (1972) 543-553

POLYAMINE EFFECTS IN PERFUSED LIVERS

545

tained after centrifugation at 6oo x g for IO min. The nuclear fraction was purified further with washings with 2 °/o citric and acetic acid ~8. The nuclei were resuspended in the homogenizing medium and extracted with phenol at 4 °C in the presence of bentonite. The cytoplasmic fraction was centrifuged at 16 ooo ×g for 9 rain (ref. 29). The pellet containing mitochondria and lysosomes was discarded. Sodium lauryl sulphate (I °/o final concn) and bentonite were added to the supernatant and the cytoplasmic RNA was extracted 3° with phenol at 4 °C. Specific activity determinations and sucrose gradient analyses were performed as previously described ~8,31. The gradients were fractionated automatically with an ISCO fractionator attached to a flow cell of an Hitachi 139 spectrophotometer. The collected fractions were precipitated with 5 % trichloroacetic acid and the radioactivity determined using Bray's solution as the scintillation fluid.

Polyribosome analysis The methods used were essentially those described by Munro et al. 8~. A 25 ~o homogenate (w/v) was prepared using the medium of Jefferson and Korner 2e. The homogenate was centrifuged at 16 o o o x g for IO rain and the supernatant of this centrifugation treated with deoxycholate (I % final concn). IO ml of deoxycholatetreated cytoplasm were layered on a discontinuous sucrose gradient made of 5 ml of 2 M sucrose and 5 ml of I M sucrose. The samples were centrifuged at 38 ooo rev./min (lO4 ooo×g) for 4 h in a B-6o International centrifuge using the rotor A-2II. The pellets were rinsed and resuspended in I ml of a solution containing 5 mM magnesium acetate, 0.02 M Tris buffer (pH 7.6), 0.04 M NaC1 and o.I M KCI. 0. 5 ml of the ribosomal solution was layered on a continuous sucrose gradient (1530 %) and centrifuged at 25 ooo rev./min (75 o o o x g ) for 3 h in an International B-6o centrifuge (SB-IIO rotor). The ultraviolet absorption was determined as in the RNA analyses.

RESULTS

Polyribosome patterns o/ isolated per/used livers I examined the polyribosome profiles of isolated perfused livers after I or 2 h of perfusion with either rat blood diluted to one-half of its volume with Ringer's solution or with a mixture containing Ringer's solution, red blood cells and albumin. A comparison between liver polyribosome profiles obtained directly from the animals and from perfused livers is presented in Fig. IA, 1t3. With both perfusates there is a marked disaggregation of polysomes during the first hour of perfusion resulting in almost complete destruction of the large polysomal aggregates. The addition of a complete amino acid mixture at 6-1o times the normal concentration in rat blood ~s preserves the large aggregates during perfusions (Fig. IC), but the proportion of monomers and dimers in these profiles is larger than that found in experiments in vivo. When a complete amino acid mixture (at 6 times the normal amino acid concentration) is added at the start of the perfusion polyribosome profiles showing the presence of heavy aggregates (Fig. IC) are obtained up to I h after the start of the experiment. In the second hour there is an almost complete disappearance of heavy polysomal aggregates (Fig. IB). The maintenance of profiles similar Biochim. Biophys. Aaa, 281 (1972) 543-553

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Fig. I. P o l y s o m a l profiles in p e r f u s e d livers. (A) P o l y s o m a l profile f r o m t h e liver of i n t a c t r a t s ; (13) p o l y s o m a l profile of p e r f u s e d n o r m a l liver, no a m i n o acids in p e r f u s a t e ; (C) p o l y s o m a l profile of p e r f u s e d liver, c o m p l e t e a m i n o acid m i x t u r e a t 6 t i m e s t h e n o r m a l a m i n o acid c o n c e n t r a tion in r a t blood added. P o l y s o m e s were o b t a i n e d f r o m liver p o s t - m i t o c h o n d r i a l s u p e r n a t a n t s as described u n d e r Materials a n d M e t h o d s . P o l y s o m e s f r o m p e r f u s e d livers were o b t a i n e d a t t h e e n d of I h of perfusion. Blood d i l u t e d w i t h R i n g e r ' s solution w a s used as perfusate. T h e a r r o w s s h o w t h e direction of s e d i m e n t a t i o n . T h e c e n t r i f u g a t i o n w a s for 3 h a t 23 ooo r e v . / m i n in a n I n t e r n a t i o n a l B-6o c e n t r i f u g e (rotor $13-IIO). 0.5 ml of t h e p o l y s o m a l s u s p e n s i o n w a s layered on a c o n t i n u o u s 15-3o °/o sucrose gradient. T h e o r d i n a t e s h o w s t h e a b s o r b a n c e at 260 n m recorded a u t o m a t i c a l l y w i t h a flow cell.

to that of Fig. IC for 3 or 4 h of perfusion requires hourly additions (or continuous infusion) of amino acids at 6 times the normal concentrations found in rat blood.

E/]ect o/ amino acids and spermidine on R N A metabolism o/ isolated livers per/used with a defined medium In preliminary experiments designed to determine the optimum composition of the medium for studies of RNA metabolism in perfused liver, the relationship between amino acid concentration of the medium and RNA labeling was determined. Table I indicates that the specific activity of nuclear and cytoplasmic RNA of isolated perfused livers varies considerably with the amount of anaino acids added to the basic medium. The addition of a complete amino acid mixture at 2-3 times the normal rat blood amino acid concentration caused almost a io-fold increase in the specific activity of liver nuclear RNA. These amino acid concentrations are well below those necessary to maintain polyribosome aggregation in isolated livers ~6. Moreover, while a mixture of eleven amino acids appears to be sufficient to maintain the integrity of polyribosomes in isolated livers ~ such mixtures when added to the basic medium did not enhance nuclear RNA specific activity (Table I). The addition of spermidine to the basic medium containing a complete amino acid mixture at 3 times the normal blood amino acid concentration invariably produced a marked inhibition of the incorporation of labeled orotic acid into nuclear and cytoplasmic RNA of isolated perfused livers. This inhibition was detected with perfusate spermidine concentrations ranging from 2 • IO-~ to 2" lO -3 M. This inhibitory effect of spermidine was not obtained when the polyamine was added to whole rat blood containing amino acids. With this perfusing medium spermidine generally stimulated RNA labeling with maximum effects obtained with a spermidine concentration of Biochim. Biophys. Acta, 281 (1972) 543-553

POLYAMINE EFFECTS IN PERFUSED LIVERS TABLE I RNA S P E C I F I C

ACTIVITY

OF ISOLATED

LIVERS

547

PERFUSED

XVITH M I X T U R E S

OF DIFFERENT

COMPO-

SITION

Normal livers were perfused for 4 h. 5/~Ci of [6-14CJorotic acid were added to the perfusate 80 rain prior to the termination of the experiment. Nuclear and cytoplasmic RNA specific activities are expressed in Cpm/mg of RNA. The basic medium contains Ringer's solution, bovine or rabbit albumin, glucose and rat red blood cells. The eleven essential amino acids are those used by Jefferson and Korner2e. The concentration of these amino acids as well as that of the complete amino acid mixture was approx. 6 times the normal concentration of each amino acid in rate blood 8°. The spermidine concentration in the perfusate at the end of the experiment was 2 • I O - a M . The results presented are averages and the range of variation obtained from three different experiments with each perfusion mixturex.

Medium used for pevfusion

Specific activity Nuclear RNA

Basic medium Basic medium+ i i essential amino acids Basic medium + complete amino acid mixture Basic medium + spermidine Basic medium+complete amino acid mixture+spermidine

Cytoplasmic RNA

6 777 ( 6 378- 7 160)

3267 (1989-3719)

7968 ( 6432- 93 lo )

4254 (3312-46o3)

66 419 (52 145-74 994) 2229 ( 2o63- 24o6)

5619 (4329-6261) 3719 (153o-4472 )

38 044 (34 044-48 037)

4148 (4oo3-4412)

2 • 10 -3 M. F o r this reason all e x p e r i m e n t s described below were p e r f o r m e d using whole blood c o n t a i n i n g a m i n o acids a n d glucose as t h e perfusing m e d i u m .

Incorporation o/orotic acid into R N A o[ isolated per[used livers The i n c o r p o r a t i o n of [6-14C]orotic acid into R N A of isolated perfused livers was m e a s u r e d at various times after t h e a d d i t i o n of the labeled precursor to the perfusate. The specific a c t i v i t y of t o t a l liver R N A , expressed as c p m / m g of R N A increases l i n e a r l y for at least 40 min (Fig. 2). The labeling of c y t o p l a s m i c R N A increases l i n e a r l y for at least I h (Fig. 2) while i n c o r p o r a t i o n of orotic a c i d into nuclear R N A increases l i n e a r l y u p to 4 ° min after orotic acid a d m i n i s t r a t i o n b u t reaches a p l a t e a u thereafter.

Ornithine decarboxylase activity in per[used livers There are two possible w a y s to increase t h e i n t r a c e l l u l a r c o n c e n t r a t i o n of sperm i d i n e of isolated perfused livers: (a) s t i m u l a t i o n of t h e endogenous p o l y a m i n e m e t a b o l i s m a n d (b) the a d d i t i o n of s p e r m i d i n e to t h e perfusate. Since it a p p e a r s t h a t p u t r e s c i n e synthesis is t h e r a t e - l i m i t i n g s t e p of p o l y a m i n e m e t a b o l i s m in t h e l i v e r it is necessary to s t i m u l a t e ornithine d e c a r b o x y l a s e a c t i v i t y of t h e isolated perfused livers to p r o d u c e increases in t h e s p e r m i d i n e pool. Moreover, because t h e a c t i v i t y of this e n z y m e is e l e v a t e d i m m e d i a t e l y following p a r t i a l h e p a t e c t o m y , if it were possible to s t i m u l a t e p o l y a m i n e m e t a b o l i s m in the isolated organ it is conc e i v a b l e t h a t some of t h e biochemical changes occurring d u r i n g liver regeneration in vivo would be r e p r o d u c e d in t h e isolated liver. U n f o r t u n a t e l y some of t h e inducers t h a t are effective in increasing t h e a c t i v i t y of this e n z y m e e,7 in the liver of i n t a c t a n i m a l s (amino acids, casein h y d r o l y z a t e , t h i o a c e t a m i d e ) p r o v e d to be w i t h o u t effect on ornithine d e c a r b o x y l a s e a c t i v i t y of isolated livers. The a d d i t i o n of g r o w t h h o r m o n e to the perfusate occasionally p r o d u c e d a m o d e r a t e elevation in o r n i t h i n e d e c a r b o x y l a s e a c t i v i t y . Studies with isolated perfused r e g e n e r a t i n g livers or with

Biochim. Biophys. Acta, 281 (i972) 543-553

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Incorporation time (rnln) Fig. 2. R N A specific activity of isolated perfused n o r m a l livers. 5/zCi of [6-1~C]orotic acid were added 2 h after the s t a r t of the perfusion. Liver biopsies and a lobe obtained from the liver at t h e end of the perfusion were used for extraction of whole cell RNA. To obtain the specific activity of nuclear and cytoplasmic RNA, perfusion e x p e r i m e n t s were t e r m i n a t e d at IO, 20 or 4 ° rain after orotic acid addition. After homogenization, nuclear and p o s t - m i t o c h o n d r i a l s u p e r n a t a n t s were obtained as described u n d e r Materials and Methods. R N A was extracted w i t h phenol at 4 °C. U1---[N, spec. act. of whole cell liver R N A expressed in c p m / m g of R N A × io-a; • - & , nuclear R N A spec. act. expressed in c p m / m g of nuclear R N A x io 4; O O , spec. act. of cytoplasmic R N A expressed in c p m / m g of cytoplasmic R N A x IO-a. The abscissa indicates the t i m e after addition of labeled orotic acid to the perfusate. Averages and s t a n d a r d errors o b t a i n e d from three different e x p e r i m e n t s are presented.

perfused livers obtained from rats injected with small doses of thioacetamide indicate that ornithine decarboxylase activity is unstable in isolated livers and progressively disappears in the course of a 4-h perfusion as shown in Table II. T A B L E ]I ORNITHINE DECARBOXYLASE ACTIVITY IN ISOLATED PERFUSED REGENERATING LIVERS I6-h regenerating livers were perfused w i t h blood for 4 h. At the s t a r t of the perfusion a s a m p l e of the liver was obtained (o time) and biopsies were t a k e n at one, 2 and 3 h after. Ornithine decarboxylase activity of the isolated perfused liver is expressed in c p m in CO, per mg of liver supern a t a n t fraction used in the assay 5,~.

Time after start of perfusion (h )

Ornithine decarboxylase activity

o

489 376

I

2 3 4

200

223 134 6o 63 89 44 47

Bioehim. Biophys. Acta, 281 (1972) 543-553

POLYAMINE EFFECTS IN PERFUSED LIVERS

549

Alteration o/the intracellular concentration o/ spermidine in isolated per/used liver Since ornithine decarboxylase activity could not be stimulated in a consistent way in the isolated organ, I attempted to increase the intracellular concentration of polyamines in perfused livers by adding spermidine to the perfusate. Preliminary experiments with radioactive putrescine and spermidine showed that both labeled compounds were rapidly picked up by the liver. Table III indicates that the intracellular concentration of spermidine in the perfused liver is doubled after 3 h of perfusion, when the concentration of spermidine in the perfusate is 2 • 10 -3 M. Special care was taken in these experiments to eliminate the possibility that the spermidine contained in the blood vessels of the isolated liver was interfering with the determination of the true intracellular concentration of the polyamine. The liver samples used for spermidine analysis were approximately of the same size and were carefully perfused and washed with saline before homogenization. It is clear (Table III) that any significant interference by blood spermidine has been eliminated since almost the same spermidine concentration per g of liver was obtained from determinations in a small fragment of the liver (500 rag) or in the whole organ (4.7 g) at the end of the perfusion period.

TABLE III SPERMIDINE CONCENTRATION IN ISOLATED PERFUSED LIVERS Livers were perfused w i t h blood containing spermidine (2 • lO -8 M). Half of the a m o u n t of the spermidine was added at the s t a r t of the perfusion and the rest was infused continuously u p to the end of the perfusion period. Liver biopsies were t a k e n at 5 min, i h and 3 h. The whole liver was perfused w i t h saline at the end of the e x p e r i m e n t and homogenized. An aliquot of the h o m o genate was used for spermidine determination 17. Liver spermine concentration was also determined in all samples. The ratio between spermidine and spermine concentration 17 in the perfused livers is shown.

Time after start of perfusion

Spermidine conch (nmoles/g of liver)

Spermidine/spermine ratio

5 min I h 3h

684 1183 1931 (biopsy) 1774 (whole liver)

1.14 1.51 1.69

E//ect o/ spermidine on the incorporation o~ [14C]orotic acid into RNA o/ isolated livers per/used with blood Livers were perfused with heparinized whole rat blood diluted with Ringer's solution, containing a complete mixture of amino acids at 6 times the amino acid concentration of rat blood. Spermidine was added to the perfusate 5 min after the beginning of the perfusion. After 2 h, 5 pCi of E14C]orotic acid were added to the perfusate and the experiment terminated I h and 20 min following the addition of the labeled compound. In the experiments with regenerating livers, the organ was removed from partially hepatectomized rats 18 h following the operation and placed in the perfusion apparatus. The same experimental conditions were used for the perfusion of normal and regenerating livers. In the absence of spermidine the specific activity of perfused regenerating liver nuclear RNA was approx. 3--5 times higher Biochim. Biophys. Aeta, 281 (1972) 543-553

55 °

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than that of perfused normal livers while cytoplasmic RNA specific activities were approx. 4 times higher in regenerating perfused livers (Table IV). The addition of T A B L E IV N U CL E A R AND CYTOPLASMIC R N A

OF ISOLATED P E R F U S E D NORMAL AND R E G E N E R A T I N G L1VERS

N o r n m l and 18-h regenerating livers were perfused for 3 h and 2o min with blood diluted with one-half of its volume with Ringer's solution containing a complete a m i n o acid m i x t u r e at 6 times the n o r m a l blood amino acid concentration 8°. 5/zCi of [6-~4C]orotic acid were added -, h after the s t a r t of the perfusion experiment. The final concentration of spermidine in the perfusate was approx. 2 • IO -3 M. Nuclear and cytoplasmic R N A specific activities are expressed in c p m / mg of RNA. The figures presented are averages and the range of variation obtained from three different e x p e r i m e n t s .

Spec~[ic activity

N o r m a l liver Regenerating liver N o r m a l liver + spermidine Regenerating liver + s p e r m i d i n e

Nuclear R N A

Cyloplasmic RN.4

31 I26 45 157

3 ° 4 9 ( 2444 !o3o) It 287 (lO7O 9 I 2 3 3 7 ) 4 226 ( 3 227 5 344)

020 256 5oo 897

( 26 627- 35 220) (115o88-I37424) ( 39 116- 49 6o4) (116 114 177 681)

It 906

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spermidine to the perfusate produced an increase of approx. 50 % in the specific activity of nuclear RNA of perfused normal livers and of approx. 25 % in the specific activity of nuclear RNA of perfused regenerating livers. Spermidine did not significantly change the specific activity of cytoplasmic RNA of isolated perfused normal or regenerating livers, or the amount of radioactivity present in the acid-soluble fraction of perfused liver homogenates, but I did not measure the effect of spermidine on the labeling of the nucleotide pool. Despite the increase in the absolute nmnber of counts found in nuclear RNA of normal livers perfused in the presence of spermidine, the polyamine did not produce any change in the distribution of label or in the ultraviolet profiles of nuclear and cytoplasmic RNA of the perfused normal liver. Fig. 3 shows the effect of spermidine on nuclear (Fig. 3C) and cytoplasmic RNA (Fig. 3D) profiles from isolated perfused regenerating livers. Spermidine caused an increase in the absolute numbeI of counts and a shift in the distribution of tile label in nuclear RNA from regenerating livers. In perfusions performed in the presence of spermidine, labeled material is found in the area of the gradient corresponding to fast sedimenting RNA species while in the absence of the polyamine no radioactivity is detected in RNA species larger than 28 S (Fig. 3C), The nuclear RNA profiles obtained from isolated regenerating livers perfused with whole blood containing 2 • io-* M spermidine is similar to the RNA profiles obtained from 18 h regenerating livers in experiments In viuo.

Fig. 3 (B, D) shows the cytoplasmic RNA profiles obtained from isolated perfused regenerating livers in presence or absence of spermidine. Spermidine did not affect the labeling of regenerating liver cytoplasmic RNA. Labeled material was detected in the areas of the gradient corresponding to 4-S and I8-S RNA forming two sharp peaks. However, no defined radioactivity peak was present in the 28-S cytoplasmic RNA in normal or regenerating perfused livers even in the presence of spermidine. Biochim. Biophys. Acta, 281 (1972) 543-553

POLYAMINE EFFECTS IN PERFUSED LIVERS

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Fig. 3- Effect of s p e r m i d i n e on t h e i n c o r p o r a t i o n of I6-14C]orotic acid into n u c l e a r a n d cytop l a s m i c R N A of p e r f u s e d r e g e n e r a t i n g livers. R e g e n e r a t i n g livers for t h e p e r f u s i o n s were o b t a i n e d f r o m p a r t i a l l y h e p a t e c t o m i z e d r a t s 18 h following t h e operation. (A) N u c l e a r 1RNA of p e r f u s e d r e g e n e r a t i n g livers; (B) c y t o p l a s m i c R N A of perfused r e g e n e r a t i n g livers; (C) n u c l e a r R N A of p e r f u s e d r e g e n e r a t i n g liver, s p e r m i d i n e added; (D) c y t o p l a s m i c R N A of p e r f u s e d r e g e n e r a t i n g liver, s p e r m i d i n e added. F i n a l s p e r m i d i n e c o n c e n t r a t i o n w a s 2 • lO -8 M. H a l f of t h e t o t a l a m o u n t of s p e r m i d i n e w a s a d d e d at t h e s t a r t of t h e p e r f u s i o n a n d t h e rest w a s c o n t i n u o u s l y i n f u s e d into t h e blood reservoir. 5/~Ci of E6-1*C]orotic acid were a d d e d 2 h a f t e r t h e s t a r t of t h e p e r f u s i o n a n d a n d t h e e x p e r i m e n t t e r m i n a t e d 6o rain after t h e a d d i t i o n of t h e labeled precursor. N u c l e a r a n d p o s t - m i t o c h o n d r i a l s u p e r n a t a n t were o b t a i n e d f r o m t h e liver h o m o g e n a t e . R N A w a s e x t r a c t e d w i t h p h e n o l as described u n d e r Materials a n d Methods. A p p r o x . 0.8 m g of n u c l e a r or c y t o p l a s m i c R N A w a s l a y e r e d on t o p of a c o n t i n u o u s lO-35 % sucrose g r a d i e n t (for nuclei) or 5 - 3 0 % (for c y t o p l a s m ) . T h e t u b e s were c e n t r i f u g e d in t h e r o t o r SB-IIO of a n I n t e r n a t i o n a l B-6o c e n t r i f u g e for 16 h a t 22 ooo rev./min. A b s o r b a n c e w a s d e t e r m i n e d a u t o m a t i c a l l y w i t h a flow cell. T e n drop fractions were u s e d for r a d i o a c t i v i t y d e t e r m i n a t i o n . - - - , a b s o r b a n c e a t 260 n m ; × - × , radioactivity.

DISCUSSION

In this study I examined the effect of spermidine on RNA labeling of isolated perfused normal and regenerating livers. Spermidine added to the perfusate stimulated the incorporation of [14C]orotic acid into nuclear RNA of perfused normal and regenerating livers. Labeling of high molecular weight nuclear RNA was only detected in isolated perfused regenerating livers when spermidine was present in the perfusing fluid. These results, by directly demonstrating an effect of the polyamines on RNA metabolism in whole livers strengthen the possibility that the enhanced polyamine synthesis found in growing tissues is directly related to RNA metabolism. It is important to stress that the RNA labeling patterns of isolated perfused regenerating livers differ from those obtained from regenerating livers in vivo ~5,2s,31. Biochim. Biophys. Acta, 28i (1972) 543-553

552

N. FAUSTO

The presence of spermidine in the perfusate corrects this situation and makes the nuclear RNA profiles of perfused regenerating livers almost identical to those obtained in vivo. However, the labeling of high molecular weight nuclear RNA (presumably ribosomal precursor RNA) in perfused regenerating livers is not reflected in an increase of rRNA labeling in the cytoplasm or the appearance of label in the 28-S RNA area of gradients of cytoplasmic RNA. There are at least two explanations for these findings: (a) the possibility that in the present experiments spernlidine prevents the processing of 45-S nuclear RNA into rRNA; (b) that while polyamine are necessary for 45-S synthesis or stabilization, the transport of rRNA to the cytoplasm is dependent on the presence of proteins that are degraded or not synthesized in perfused livers. Direct effects of polyamines in stimulating RNA labeling in vivo have been demonstrated by Caldarera and Moruzzi 3~ in chick embryos, Raina et al. a4 in polyauxotrophic strains of E. coli, and by Goldstein 35 in Walker tmnor cells. Moreover, Dion and Herbst 36 have shown that high concentrations of spermidine enhance the incorporation of uridine into salivary gland nuclei of D. melanogaster. All these results can be explained either by postulating a stimulatory effect of spermidine on RNA polymerase or a stabilizing effect of the polyamine on the helical loops of RNA. In Ehrlich ascites cells as well as in isolated rat liver nuclei and nucleoli it appears that polyamines both stimulate RNA polymerase and prevent the degradation of newly synthesized RNA following a "chase" with actinomycin or unlabeled precursor 18. The present results showing an effect of spermidine on the incorporation of orotic acid into RNA of normal and regenerating livers and a change of the labeling profile of nuclear RNA of regenerating liver, do not permit a definite conclusion as to the mechanism of spermidine action. However, it is of interest that the effect of spermidine on 45-S RNA labeling could be demonstrated only in perfused regenerating livers but not in normal livers. Since regenerating livers have a much higher rate of 45-S nuclear RNA labeling than normal livers in vivo 28'37"3s it nlight be suggested that the action of the polyamine is primarily on the stabilization of RNA species already synthesized. Extrapolation of this tentative conclusion to the situation occurring during liver regeneration in vivo makes it conceivable that RNA and polyamine synthesis are triggered in a coordinate fashion ahnost immediately after partial hepatectomy but that the amines exert their effect on the newly synthesized RNA rather than directly stinmlating RNA synthesis. A more detailed study of the mechanism of action of the polyamines and their interaction with amino acids in isolated perfused livers can only be attempted in isolated livers perfused with a defined medium. However, with this procedure spermidine causes a marked inhibition of RNA labeling. It is likely that the inhibitory effect is not physiological but derives from the production of toxic products from spermidine in the presence of bovine albumin a9'4°. ACKNOWLEDGMENTS I t h a n k Mrs Paula Kelly and Miss Ann DiPippo for their help at different stages of this study. The work was supported b y Grants NP-52P from tile American Cancer Society and R o I AM-I47O6 from the N.I.H. Biochim. Biophys. Acta, 281 (1972) 543 553

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553

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Biochim. Biophys. Acta, 281 (1972) 543-553