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Inhibition of δ-aminolevulinate synthetase induction by α-amanitin in avian liver cell cultures
331
Biochimica et Biophysica Acta, 361 ( 1 9 7 4 ) 3 3 1 - - 3 4 4 © Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s
BBA 98080
INHIBITION OF 5-AMINOLEVULINATE SYNTHETASE INDUCTION BY a-AMANITIN IN AVIAN LIVER CELLCULTURES
G E N E V I E V E S. I N C E F Y * , A R L E E N B. R I F K I N D * * and A T T A L L A H K A P P A S
The Rockefeller University, New York, N.Y. 10021 (U.S.A.) (Received April 1st, 1 9 7 4 )
Summary The induction of 6-aminolevulinate synthetase by the 5~-steroid etiocholanolone and the barbiturate derivative 2-allyl-2-isopropylacetamide was blocked by a-amanitin in avian h e p a t o c y t e cultures. 6-Aminolevulinate synthetase controls the initial and rate-limiting step of the porphyrin-heme pathway. ~-Amanitin was demonstrated to be a p o t e n t inhibitor of the nucleoplasmic DNA-dependent R N A polymerase in chick e m b r y o liver cells in culture. It is shown that nuclear R N A synthesis and nucleoplasmic R N A polymerase activities were inhibited by the toxin at an early time (6--6.5 h) in the induction period; whereas the nucleolar R N A polymerase was not inhibited. At this particular time of the induction period, enhancement of R N A synthesis was observed and was dependent on the effect of inducers; this enhancement was eliminated by ~-amanitin. It is concluded that the increment of new R N A synthesized by the ~-amanitin-sensitive nucleoplasmic R N A polymerase is required as one of the early steps in the induction process of 6-aminolevulinate synthetase, either as m R N A for the enzyme or for a specific protein required in the induction process.
Introduction We have shown previously that certain 5~-steroids, derived from the metabolic transformation of sex hormones or their precursors, are able to induce the de novo formation of 5-aminolevulinate synthetase, and consequently porphyrinogenesis in chick e m b r y o hepatocytes grown in primary cultures [ 1 ] . These inducing steroids, as well as a number of drugs, elicit an * Present address: Memorial Sloan-Kettering Cancer Center, 4 1 0 East 6 8 t h Street, New York, N. Y. 1 0 0 2 1 . T o w h o m requests for reprints s h o u l d be addressed.
** Present address: Cornell University Medical College, 1300 York Avenue, New York, N. Y. 10021.
332 experimental type of hepatic porphyria in vivo and in vitro [2--5], which provides an interesting system for the study of gene expression in eukaryotic cells. In this study two different types of chemicals which induce 5-aminolevulinate synthetase were utilized to study the effect of a-amanitin on the enzyme induction process; one was the 5fl-steroid, etiocholanolone (5/~-androstane-3a-ol-17-one) and the other, the barbiturate analogue, 2-allyl-2-isopropylacetamide [6]. These compounds were selected for study because they represent examples of both naturally occurring as well as foreign chemical inducers of 5-aminolevulinate synthetase; further it has been suggested that they induce the enzyme by different mechanisms [7]. In an earlier study we showed that a-amanitin, the cyclic peptide of Amanita phalloides and a potent inhibitor of RNA synthesis in mammalian systems [8,9], prevents induction of 5-aminovulinate synthetase by both etiocholanolone and 2-allyl-2-isopropylacetamide in whole chick embryo liver [10]. The findings reported here in primary cultures of avian hepatocytes indicate that a-amanitin had several effects which can be related to the de novo formation of 5-aminolevulinate synthetase. First, the toxin had a similar inhibitory effect on the induction of this enzyme in cultured liver cells as it had in vivo in the chick embryo. Second, the porphyrinogenesis which usually develops after treatment of the cultures with inducing agents, did not occur. Third, a-amanitin almost completely abolished the inhibitory effect of coproporphyrins on [~4 C] leucine incorporation into proteins [11], indicating that this potent fungal agent seems to protect, paradoxically, the induced hepatocytes from the toxic effect produced by porphyrins. Fourth, a-amanitin had marked inhibitory effects on RNA synthesis in control and induced avian liver cell cultures. In addition it prevented the specific stimulation of nuclear RNA synthesis usually observed in association with the increased activity of the nucleoplasmic DNA-dependent RNA polymerase (EC 2.7.7.6) which follows treatment with the inducing agents [12]. It is therefore concluded that the increment in new RNA synthesized by this polymerase, and which is inhibited by a-amanitin, is required for the induction of 5-aminolevulinate synthetase either as mRNA for the enzyme itself or for a specific protein required in the induction process. Materials
The inducing agents etiocholanolone and 2-allyl-2-isopropylacetamide were gifts from Mann Research and Hoffmann-La Roche respectively. a-Amanitin was a gift of Professor T. Wieland. Unlabeled ribonucleoside triphosphates were purchased from Sigma; [8 -14 C] ATP (35 Ci/mole), [3 H]uridine (5--15 Ci/mmole) and L-[14C]leucine (260 Ci/mole) were obtained from New England Nuclear. Materials for liver cell cultures and the leucine-free medium were purchased from Grand Island Biological Co. Methods
Hepatic cell cultures The technique has been described previously [6]. Briefly, liver cells from
333 15-day-old chick embryos were grown in primary cultures in an incubator at 37°C in the presence of CO2--02 (5:95, v/v), either in small vials or in Falcon plastic petri dishes {100--150 mm diameter) for 16--20 h initially to permit a cell monolayer to form. The medium was then replaced, and additions were made as required. Inducing agents, dissolved in propylene glycol, were added to cultures in volumes of 5 #l/ml of medium and a-amanitin, prepared in 0.15 M NaC1 was added in volumes o f 5--20 pl/ml of medium before or after the inducing agents. The cells were re-incubated in their presence for the various additional times indicated; additions of appropriate solvents were made to control cultures in all experiments. Porphyrins synthesized by avian hepatocytes during the induction period of 6-aminolevulinate synthetase were quantitated fluorimetrically [13] to assess the magnitude of the induction response; coproporphyrin III was used as a standard. In experiments where cells were used for determinations of 5-aminolevulinate synthetase activities, [' 4 C]leucine incorporation, nuclei isolation [14] or DNA quantitation [ 1 5 , 1 6 ] , the culture medium was removed prior to harvesting of the cells and frozen. In all other experiments, cells and medium combined were used for porphyrin determination; after lyophilization, their coproporphyrin contents were determined and compared with controls.
Enzyme assays Activities of 5-aminolevulinate synthetase were measured in cultured hepatocytes by a m e t h o d previously described [ 1 7 ] . Protein contents of cell homogenates were determined using bovine serum albumin as a standard [ 1 8 ] . DNA-dependent R N A polymerase activities of nuclei isolated from cultured hepatocytes were measured [19] in the presence of Mg :÷ or Mn :÷ and 0.3 M (NH4): SO4 with the following modification: 0.075 pmole each of GTP, CTP, UTP and 0.025 pmole of [8 -14 C] ATP, and 5--15 pg of DNA; the other components remained unchanged [14]. Incubation times were 10 and 45 min for the Mg :÷ and Mn2+--(NH4 )2 SO4 assays respectively at 36 ° C.
[3 HI uridine incorporation into RNA Hepatocytes in cultures were labeled [20] with 5 pCi of [3 H]uridine per ml of medium for 10 or 15 min (Tables V and VI respectively) in the incubator at 37°C. At the end of the incubation, medium was aspirated off and cold 0.32 M sucrose--3 mM MgC12 added, the cell monolayer was scraped off the b o t t o m of the petri dishes and homogenized. Nuclei were isolated and radioactivity determined as previously described [ 1 4 ] . Results
Inhibition of 5-aminolevulinate synthetase formation by a-amanitin Chick e m b r y o liver cells grown in primary cultures for 24 h consist almost entirely of hepatic parenchymal cells which have very low levels of 5-aminolevulinate synthetase activity. After addition of fresh medium and the inducing agents, etiocholanolone or 2-allyl-2-isopropylacetamide, there is a progressive increase in 6-aminolevulinate synthetase activity reaching 5--20 times control values after 18--20 h of incubation. When a-amanitin is added 1 h prior to the
334
TABLE I INHIBITION OF 6-AMINOLEVULINATE HEPATOCYTE CULTURES
SYNTHETASE
INDUCTION
BY a-AMANITIN
IN AVIAN
Cells f r o m c h i c k e m b r y o liver w e r e g r o w n i n i t i a l l y f o r 24 h in p e t r i d i s h e s ( 1 0 0 m m d i a m e t e r ) as desc r i b e d in M e t h o d s ; a - a m a n i t i n w a s a d d e d * to t h e m e d i u m 1 h p r i o r to t h e i n d u c i n g a g e n t s e t i o c h o l a n o l o n e a n d 2 - a U y l - 2 - i s o p r o p y l a c e t a m i d e , a n d t h e cells r e - i n c u b a t e d f u r t h e r f o r 18 h. A t t h e e n d o f t h e r e - i n c u b a t i o n p e r i o d t h e m e d i u m w a s r e m o v e d a n d its c o p r o p o r p h y r i n c o n t e n t d e t e r m i n e d [ 1 3 ] ( T a b l e II). T h e cell m o n o l a y e r s w e r e s c r a p e d f r o m t h e b o t t o m o f t h e d i s h e s , p o o l e d * * in b u f f e r a n d 5 - a m i n o l e v u l i n a t e s y n t h e t a s e a c t i v i t y d e t e r m i n e d [ 17].
Experiment and treatment
-Aminolevulinate synthetase activity (nmoles -aminolevulinate]mg p r o t e i n p e r h)
Induction (% o f control)
Inhibition of induction (%)
1. C o n t r o l E tiocho lanolone c~-Amanitin ( 0 . 4 # g ) + e t i o c h o l a n o l o n e 2. C o n t r o l 2-Allyl-2-isoprop ylacetamide ~-Amanitin (1.0 pg) + 2-allyl-2-isopropylacetamide
0.038 0.188 0.102 0.044 0.817 0.145
100 495 -100 1857 --
--57 --87
_+ 0 . 0 0 1 " * * + 0.007 + 0.002 + 0.011 + 0.160 + 0.028
* All a d d i t i o n s are p e r m l o f m e d i u m ; e t i o c h o l a n o l o n e ( 2 0 p g ) a n d 2 - a U y l - 2 - i s o p r o p y l a c e t a m i d e ( 5 0 # g ) ; c o n t r o l s w e r e t r e a t e d w i t h s o l v e n t s ( 0 . 9 % NaC1 a n d p r o p y l c n e g l y c o l ) . ** T w o p e t r i d i s h e s p o o l e d f o r e a c h c o n t r o l g r o u p a n d t h r e e f o r e a c h t r e a t e d cell cultures~ m e a n a n d S.D. are g i v e n f o r e a c h g r o u p . * * * M e a n _+ S.D.
addition of inducing agents, the stimulation of 6-aminolevulinate synthetase activity is greatly decreased and the level of enzyme induction is reduced 57 and 87% respectively {Table I). The difference observed in the degree to which the induction response is inhibited is due to the concentration of a-amanitin T A B L E II INHIBITION CULTURES
OF INDUCED
PORPHYRIN
SYNTHESIS
BY ~-AMANITIN IN AVIAN HEPATOCYTE
Cells f r o m c h i c k e m b r y o liver w e r e g r o w n a n d t r e a t e d w i t h i n d u c i n g a g e n t s as d e s c r i b e d in T a b l e I. c~-Amanitin w a s a d d e d t o t h e m e d i u m 1 h b e f o r e t h e i n d u c i n g a g e n t s a n d t h e cells r e - i n c u b a t e d f o r t h e t i m e s indicated. The m e d i u m was then r e m o v e d and its p o r p h y r i n c o n t e n t * d e t e r m i n e d [ 1 3 ] . Experiment and treatment
Time of incubation (h)
Coproporphyrin III (pmoles/ml of medium)
Induction (% o f control)
1. C o n t r o l ~-Amanitin (0.4/~g) + control Etiocholanolone ~-Amanitin (0.4 gg) + etiocholanolone 2. C o n t r o l 2-Allyl- 2 - i s o p r o p y l a c e t a m i d e ~-Amanitin (1.0 #g) + 2-aUyl-2-isopropylacetamide
18 18 18 18 24 24 24
32 32 158 23 47 609 72
-503 -100 1295 --
_+ 1 3 " * -+ 1.6 -+ 81 -+ 5.5 -+ 18 -+ 38 _+ 36
100
Inhibition of induction (%)
--
--100 --96
* C o n t e n t is p e r m l o f m e d i u m a n d d o e s n o t i n c l u d e p o r p h y r i n s in t h e cell m o n o l a y e r (see M e t h o d s ) . ** M e a n + S.D.
335 used in the cultures, the cells treated with etiocholanolone having received 0.4 pg of ~-amanitin whereas the cultures treated with 2-allyl-2-isopropylacetamide received 1.0 pg of the toxin. Porphyrin accumulation within the cells and medium also occurs during the induction period [6,12,21] as a result of the enhanced production of 5-aminolevulinate synthetase evoked by the inducing agents. This porphyrinogenesis develops after a lag period of 8--10 h; following which porphyrin synthesis rapidly occurs, reaching levels 5 - 2 0 or more times control values in the ensuing 24 h. In the presence of ~-amanitin, however, only small amounts of porphyrins could be detected when their concentrations were determined at various times during the induction period; at 18 and 24 h this inhibition of porphyrinogenesis by ~-amanitin was pronounced (Table II). In the experiments reported in Table II the treated cells were also examined for 5-aminolevulinate synthetase activity, and the results are shown in Table I. These data indicate that (a) induction of this enzyme had occurred concurrently with the enhancement of porphyrin synthesis and (b) that ~-amanitin markedly inhibited the increase in enzyme activity and in porphyrin formation. ~Amanitin, however, at 0.4 pg inhibited porphyrin synthesis much more effectively than enzyme synthesis. This is probably due to the fact that the porphyrins measured represent products accumulated over the full time period (18 h), whereas 5-aminolevulinate synthetase activity was measured at the end of the time period, when enzyme activity may have had an opportunity to recover from the low dose inhibition of ~-amanitin. It is reasonable to infer that the diminished porphyrinogenesis, resulting from pretreatment with ~-amanitin, reflects the almost total inhibition of the induction response for 5-aminolevulinate synthetase. The time required for ~-amanitin to inhibit porphyrin biosynthesis was also examined with regard to the time of addition of the steroid or barbiturate inducer agent to the cultures. As shown in Fig. 1, induction of porphyrin synthesis was inhibited from 88--83% and 95--93% respectively when ~amanitin was added 120 to 1 min prior to addition of both etiocholanolone or 2-allyl-2-isopropylacetamide; b u t when the toxin was added after the inducing agents, a less though still substantial inhibition of porphyrinogenesis occurred. Because the greatest degree of inhibition with ~-amanitin was obtained when it was added to the cultures before the inducing agents the toxin was added 40--60 min prior to the inducers in all subsequent studies.
Metabolic capacity of cells after a-amanitin treatment a-Amanitin is a p o t e n t toxin [ 8 , 2 2 , 2 3 ] ; to examine the metabolic effect of this agent on the cultured liver cells the following studies were done. (a} Cultured h e p a t o c y t e s which had been in contact with 0.5 pg of a-amanitin for 25 h were tested for their ability to convert 5-aminolevulinate to porphyrins. As shown in Table III the a-amanitin-treated hepatocytes were able to synthesize as much porphyrins as did control cultures, 205 + 52 and 174 + 19 pmoles of coproporphyrins III/ml of medium respectively. These findings indicate that in ~-amanitin-treated hepatocytes, despite the p o t e n t toxicity of this agent, the integrity of a number of enzymatic steps of the porphyrin biosynthetic p a t h w a y b e y o n d that catalyzed by ~-aminolevulinate synthetase
336
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Hours otter addition of a-amonitin Fig. 1. T i m e c o u r s e for t h e i n h i b i t o r y a c t i o n of ~ - a m a n i t i n o n p o r p h y r i n b i o s y n t h e s i s b e f o r e a n d a f t e r i n d u c t i o n w i t h e t i o c b o l a n o l o n e or 2 - a l l y l - 2 - i s o p r o p y l a c e t a m i d e . L i v e r cells w e r e g r o w n in small vials as d e s c r i b e d in M e t h o d s f o r 18 h. T h e c u l t u r e m e d i u m was c h a n g e d a n d c~-amanitin, 0.5 ~ g / m l o f m e d i u m , was a d d e d 1 2 0 , 60, 3 0 a n d 1 rain b e f o r e a d d i t i o n o f t h e i n d u c i n g a g e n t s , e t i o c h o l a n o l o n e or 2-allyl-2i s o p r o p y l a c e t a m i d e ( 1 0 p g a n d 3 0 p g / m l of m e d i u m r e s p e c t i v e l y ) , or 1, 3, 6 a n d 8.5 h a f t e r t h e i r a d d i t i o n . A t t h e e n d o f t h e 24-h i n d u c t i o n p e r i o d ( w h i c h begins a t 0 h o n t h e g r a p h ) or 20 h a f t e r t h e s t a r t o f t h e c u l t u r e s , all t h e vials w e r e f r o z e n , l y o p h i l i z e d a n d t h e p o r p h y r i n c o n t e n t of cells a n d m e d i u m d e t e r m i n e d [ 1 3 ] . T h e % i n h i b i t i o n w a s c a l c u l a t e d o n t h e basis o f t h e a m o u n t o f p o r p h y r i n s y n t h e s i z e d d u r i n g this 24-h i n c u b a t i o n in c u l t u r e s n o t t r e a t e d w i t h c~-amanitin using t h e f o l l o w i n g values as 100%; e t i o c h o l a n o l o n e - i n d u c e d c u l t u r e s : 47.9 -+ 6 . 4 p m o l e s o f c o p r o p o r p h y r i n I I I / m l ; 2 - a l l y l - 2 - i s o p r o p y l a c e t a m i d e - i n d u c e d c u l t u r e s : 1 0 5 -+ 23 p m o l e s o f c o p r o p o r p h y r i n IfI p e r ml. C o n t r o l c u l t u r e s s y n t h e s i z e d 5,1 +- 0 . 2 5 p m o l e s c o p r o p o r p h y r i n I I I / m l a n d w e r e n o t u s e d in t h e c a l c u l a t i o n s , o -o ~-amanitin- + etioeholanolonetreated cultures; • •, ~-amanitin- + 2-allyl-2-isopropylacetamide-treated cultures.
TABLE
IIl
PORPHYRIN SYNTHESIS FROM 8-AMINOLEVULINATE AFTER TREATMENT WITH ~x-AMANITIN
IN C U L T U R E D
AVIAN
HEPATOCYTES
Liver cells were g r o w n in small vials for 18 b initially as described in Methods. After changing the m e d i u m , vials were sorted into groups of five and s o m e were treated with a-amanitin*; all were then incubated for 25 h. After incubation, a-amanitin w a s r e m o v e d by aspirating off the m e d i u m and the cells washed once with 1 m l of m e d i u m prior to addition of fresh m e d i u m . T o the control and previously a-amanitin-treated ceils was added 6-aminolevulinate or propylene glycol and the cells re-incubated a second time for 24 h. T h e m e d i u m was saved at the end of the last incubation, frozen and its porphyrin content** determined [131. First t r e a t m e n t
Time of incubation (h)
Second treatment
Time of incubation (h)
Copropophyrin III (pmoleslml of m e d i u m )
Control ~ - A m a n i t i n ( 0 , 5 pg) + c o n t r o l Control ~ - A m a n i t i n ( 0 . 5 pg) + c o n t r o l
25 25 25 25
Fresh Fresh Fresh Fresh
24 24 24 24
4.2 3.2 174 205
medium medium medium medium
+ + + +
propylene glycol p r o p y l e n e glycol ~-aminolevulinate 6-aminolevulinate
_+ 0.4** + 0.2 _4-19 _+ 52
* All a d d i t i o n s are p e r m l o f m e d i u m ; ~ - a m a n i t i n in 5 pl of 0.9% NaCl a n d • - a m i n o l e v u l i n a t e ( 1 0 0 / ~ g ) in 5 #l of w a t e r ; c o n t r o l r e c e i v e d 5 ~1 of 0.9% NaC1 a n d p r o p y l e n e glycol. ** Mean + S.D. o f five d u p l i c a t e vials f o r e a c h g r o u p ; 2 0 5 is n o t d i f f e r e n t f r o m 174, P v a l u e s o f t test is n o t significant.
337
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a-omonotin (Fg/ml medium) Fig. 2. E f f e c t of v a r y i n g c o n c e n t r a t i o n s of ~ - a m a n i t i n o n t h e b i o s y n t h e s i s of p o r p h y r i n s in e t i o c h o l a n o l one- a n d 2 - a l l y l - 2 - i s o p r o p y l a c e t a m i d e - t r e a t e d h e p a t o c y t e c u l t u r e s . C o n d i t i o n s f o r cell c u l t u r e s in small vials are d e s c r i b e d in M e t h o d s ; t r e a t m e n t w i t h ~ - a m a n i t i n a n d i n d u c i n g a g e n t s are t h e s a m e as in T a b l e I V . T h e d e c r e a s e d a m o u n t o f p o r p h y r i n s a c c u m u l a t e d in t h e m e d i u m of ~ - a m a n i t i n - t r e a t e d c u l t u r e s is c o m p a r e d t o Cz-amanitin-untreated c u l t u r e s a n d t h e % i n h i b i t i o n r e p o r t e d is c a l c u l a t e d t a k i n g t h e f o l l o w i n g m e a n v a l u e s as 1 0 0 % +- S.D.: c o n t r o l 1 5 . 4 + 2.4 p m o l e s p o r p h y r i n / m l m e d i u m ; e t i o c h o l a n o l o n e - i n d u c e d 4 1 . 8 + 6 . 9 ; 2 - a l l y l - 2 - i s o p r o p y l a c e t a m i d e - i n d u c e d 53.3 + 16. T h e s e v a l u e s r e p r e s e n t 0 i n h i b i t i o n in t h e g r a p h f o r c o n t r o l , steroid- or 2 - a l l y l - 2 - i s o p r o p y l a c e t a m i d e - i n d u e e d g r o u p s a n d e a c h p o i n t t h e r e a f t e r r e p r e sents t h e m e a n a n d S.D. of five r e p l i c a t e cell c u l t u r e s , o o c~-amanitin-treated c o n t r o l c u l t u r e s ; • •, a-amanitin- + etiocholanolone-treated cultures; • • , a - a m a n i t i n - + 2-allyl-2-isopropylacetamide-treated cultures.
was maintained. (b) In addition, increasing concentrations of a-amanitin were tested for their ability to prevent porphyrin formation in cultures treated with the inducing agents, etiocholanolone or 2-ailyl-2-isopropylacetamide for 24 h. As shown in Fig. 2, inhibition of coproporphyrin formation occurred at all concentrations of ~-amanitin studied. From the graph at 0.05 pg/ml of ~amanitin, the control cultures appear to be the only ones inhibited because the steroid and barbiturate-induced hepatocytes show a slight stimulation; however this stimulation is small when compared to that usually observed when ~amanitin is absent. What is occurring here, and shown by the graph, is a partial inhibition of the stimulation usually observed, this being due to the low dose of ~-amanitin employed at that point. Inhibition of coproporphyrin synthesis was greater at the higher concentrations of the toxin and reached a leveling off value near 40% for the control cultures. A much greater inhibition was observed in the induced cells; the etiocholanolone-induced cultures rapidly reached 65% inhibition at an ~-amanitin concentration of 0.2 pg/ml medium, leveling off near 80% at the higher concentration of the toxin. Comparable degrees of inhibition were observed with a-amanitin in the 2-allyl-2-isopropylacetamide induced cultures. Therefore the induction response elicited by b o t h agents was inhibited even when very small concentrations of ~-amanitin were used and this inhibition was almost maximal at 0.5 pg, the dose chosen for our studies. The fact that this inhibition is specific to the induction re-
338 TABLE IV EFFECT OF (~-AMANITIN ON THE CULTURED AVIAN HEPATOCYTES
INCORPORATION
OF
[14C]LEUCINE
INTO PROTEINS
IN
C h i c k e m b r y o liver cells w e r e g r o w n o n c o v e r s l i p s in small vials f o r 22 h p r i o r to a d d i t i o n o f f r e s h m e d i u m a n d C~-amanitin; t h e i n d u c i n g a g e n t s * e t i o c h o l a n o l o n e a n d 2 - a l l y l - 2 - i s o p r o p y l a c e t a m i d e ( 1 0 Dg a n d 30 p g r e s p e c t i v e l y ) w e r e a d d e d 40 r a i n a f t e r a d d i t i o n o f c~-amanitin a n d t h e cells w e r e r e - i n c u b a t e d f o r 24 h. A t t h e e n d o f t h e r e - i n c u b a t i o n t h e m e d i u m w a s r e m o v e d f o r d e t e r m i n a t i o n o f its c o p r o p o r p h y r i n c o n t e n t (Fig. 2). T o e a c h vial w a s i m m e d i a t e l y a d d e d 1 m l o f w a r m l e u c i n e - f r e e m e d i u m a n d t h e cell m o n o l a y e r w a s l a b e l e d w i t h [ ! 4 C ] l e u c i n e f o r 30 m i n as d e s c r i b e d p r e v i o u s l y [ 1 1 ] . Additions*
None a-Amanitin (0.5pg) a-Amanitin (0.20 #g) ~-Amanitin(0.50pg) v~-Amanitin ( 1 . 0 p g ) W-Amanitin(2.0~zg)
[ ~ 4 C ] L e u c i n e i n c o r p o r a t i o n as c p m / c o v e r a l i p * * Control
%
Etiocholanoline
%
2- A l l y l - 2 - i s o p r o p y l a c e tamide
(%)
5 9 0 9 -+ 1 1 9 6 6112 + 1261 5 2 2 4 -+ 4 1 6 3716 + 700 2282 + 344 1696-+ 134
100 103 88 63 39 29
4 6 0 0 + 869 5776-+ 899 5 0 5 3 +- 1 0 7 6 4212 + 935 3 5 7 2 -+ 4 1 7 2762 + 538
100 125 110 92 78 60
3 0 0 4 + 981 5018-+ 735 3 3 7 7 -+ 1 1 1 3 3593 + 920 2973 + 257 2079 + 413
100 167 112 120 99 69
* All a d d i t i o n s a r e p e r m l o f m e d i u m ** M e a n a n d S.D. o f five c o v e r s l i p s f o r e a c h g r o u p .
sponse and is not due to cell toxicity is indicated by the data in Table III in which it is shown that the ~-amanitin-treated cultures were able to synthesize large amounts of porphyrins from 6-aminolevulinate. The ability of cultured hepatocytes to incorporate amino acids into cellular proteins was also determined at the various concentrations of ~-amanitin shown in Fig. 2. It was found that steroid or barbiturate-induced hepatocytes which had been in contact with concentrations of ~-amanitin below 1.0 pg were able to incorporate [14 C]leucine into proteins very efficiently when compared to non-induced, ~-amanitin-treated controls (Table IV). In fact, the induced cultures seemed to be protected from the decreased [ 14 C] leucine uptake observed in control cells at the highest concentrations of the toxin. These results strongly suggest that the inhibition of 5-aminolevulinate synthetase activity and porphyrin formation in induced cell cultures, as shown in Tables I and II, was not due to cell toxicity. In these experiments, steroid or barbiturate-induced cells pretreated with ~-amanitin were not inhibited with respect to [ 14 C] leucine uptake, to the same degree as induced cells not treated with ~-amanitin prior to induction (Table IV}. Paradoxically, at the lower doses of ~-amanitin the incorporation of [14C]leucin e by induced cells was, in fact, enhanced over control, a finding which was unexpected since 5-aminolevulinate synthetase is markedly inhibited at this time. Because of these findings, the possible effects on protein synthesis of the excess accumulation of porphyrins in induced cells were examined. One of these effects is reflected in the data shown on the first line of Table IV, and is represented by the 23% and 43% smaller [ 14 C] leucine incorporation into total cellular proteins observed in induced cultures compared to controls. This difference is statistically significant (P < 0.05) and was noted consistently in other experiments. The decrease in [14 C]leucine incorporation found in these in-
339 duced cultures is not a characteristic of the inducer agent since it was also present when 5-aminolevulinate alone was added to the medium and allowed to be metabolized to porphyrins. Coproporphyrin which had accumulated in the cells and media of induced cultures were then shown to produce this decrease in [~4 C] leucine incorporation [11 ]. Also, in etiocholanolone- and 2-allyl-2-isopropylacetamide-treated cultures in which the accumulation of porphyrins had been inhibited by a-amanitin (0.05--0.5 pg/ml), a decrease in [,4 C]leucine incorporation was n o t observed, as shown in Table IV. At the highest a-amanitin concentrations, the cells were inhibited in their ability to incorporate [' 4C]leucine b u t again the effect in induced cultures was less marked than in controls. Since the possibility of cell damage could not be ruled out in these cultures, high concentrations of a-amanitin were not used in our studies.
Inhibition o f nuclear R N A synthesis by a-amanitin It was of interest to investigate the effect of a-amanitin on R N A synthesis because the toxin is a p o t e n t inhibitor of RNA synthesis in yeast and mammalian systems [ 22,24,25] and its effect had not been studied in avian tissue or in cell cultures. In addition a-amanitin had been shown to prevent the de novo formation of 5-aminolevulinate synthetase in whole chick e m b r y o liver previously [10] as well as in cultured avian hepatocytes as indicated in Table I. RNA synthesis was therefore determined 6 h after the start of the induction period in a-amanitin-treated cultures of both steroid- and barbiturate-induced hepatocytes and compared to respective controls (Table V). At this time, changes in R N A synthesis had been observed to occur previously [12] ; this interval precedes by 10--12 h the peak induction for 6-aminolevulinate synthetase in cultured hepatocytes [ 2 6 ] . Addition of a-amanitin was made 20 min prior to addition of inducers to the medium and R N A synthesis was measured as a short pulse of [3 H] uridine incorporated into nuclear RNA. It was found that a marked decrease in [3 H] uridine uptake characterized all a-amanitin-treated cultures, ranging from 69 to 76% in control and induced cells respectively. On the other hand enhancement of [3 H]uridine incorporation over controls was observed in steroid- and barbiturate-induced cultures not treated with a-amanitin (27 and 13% respectively). With smaller amounts of a-amanitin (0.2 pg per ml of medium) R N A synthesis was inhibited 29% in controls and 50--51% in induced cultures in a similar experiment as shown in Table V. Such inhibition was not observed, however, in control 2 h after the start of the induction period b u t reached 27 to 37% in steroid- and barbiturateinduced cultures respectively. These two concentrations of a-amanitin (0.5 and 0.2 pg) were chosen to determine if low doses of the toxin could inhibit R N A synthesis in the early part of the induction period, presumably when changes in the transcriptional process for induction of 5-aminolevulinate synthetase are taking place. These results indicate that the magnitude of the inhibition is dependent on the concentration of a-amanitin and the length of time the cells are exposed to this agent. When the cultures were exposed to a 15-min pulse of [3 HI uridine, the labeled R N A found in the cytoplasm was markedly inhibited by a-amanitin in the steroid- and barbiturate-induced cultures, 54 and 75% respectively, as compared to control (Table VI). In a short pulse label as utilized here, most labeled RNA should represent m R N A because rRNA takes longer to appear in the cytoplasm [ 2 7 ] .
340
TABLE V EFFECT
OF a-AMANITIN ON NUCLEAR
RNA SYNTHESIS IN AVIAN HEPATOCYTE
CULTURES
Cells f r o m c h i c k e m b r y o livers w e r e g r o w n i n i t i a l l y f o r 20 h in p e t r i d i s h e s ( 1 0 0 m m d i a m e t e r ) , as d e s c r i b e d in T a b l e 1, p r i o r to a d d i t i o n o f f r e s h m e d i u m a n d a - a m a n i t i n * ; t h e i n d u c i n g a g e n t s w e r e a d d e d 20 r a i n a f t e r a - a m a n i t i n a n d t h e cells r e - i n c u b a t e d f o r 6 h. Cells w e r e l a b e l e d f o r 1 0 r a i n w i t h [ 3 H ] u r i d i n e at t h e e n d o f t h e r e - i n c u b a t i o n p e r i o d ; t h e m e d i u m w a s r e m o v e d a n d ice-cold 0 . 3 2 M s u c r o s e - - 3 m M MgCI 2 a d d e d , a n d t h e cell m o n o l a y e r s c r a p e d f r o m t h e b o t t o m o f t h e p e t r i dishes. N u c l e i w e r e i s o l a t e d [ 1 4 ] f r o m cell h o m o g e n a t e s * * , p r e p a r e d in t h e s a m e s u c r o s e s o l u t i o n a n d [ 3 H ] u r i d i n e i n c o r p o r a t e d i n t o R N A p e r 1 0 r a i n w a s d e t e r m i n e d as d e s c r i b e d in M e t h o d s . Treatment
[ 3 H] U r i d i n e i n c o r p o r a t i o n
Control ~-Amanitin + control Etiocholanolone ~-Amanitin + etiocholanolone 2-Allyl-2-isoprop ylacetamide c~-Amanitin + 2 - a l l y l - 2 - i s o p r o p y l a c e t a m i d e * All a d d i t i o n s are p e r propylacetamide (30 ** Cells f r o m t w o p e t r i ** * N is n u c l e a r f r a c t i o n
cpm/0.5 ml of N***
pg DNA/0.5 ml of N
cpm//~g DNA
Incorporation
Inhibition
( M e a n -+ S.D.)
( M e a n +- S . D )
(Mean)
(%)
(%)
1984 + 188
3.0 -+ 0.2
661
100
--
3
4.3 + 0 . I
203
31
69
2 6 4 9 -+ 1 0 9 1 0 6 6 _+ 10
3.2 +- 0.1 4.3 _+ 0.2
836 246
100 29
-71
2522-+ II 7 5 0 -+ 31
3.4-+0.5 4.2 -+ 0.2
743 179
I00 24
-76
861 +
m l o f m e d i u m ; ~ - a m a n i t i n ( 0 . 5 # g ) ; e t i o c h o l a n o l o n e (5 p g ) a n d 2-aUyl-2-isopg). dishes were pooled for each homogenate. ( t o t a l v o l u m e = 5 m l f o r s a m p l e s ) ; m e a n a n d S.D. o f d u p l i c a t e d e t e r m i n a t i o n s .
TABLE VI EFFECT OF ~-AMANITIN ON [3H] URIDINE-LABELED
RNA IN THE CYTOPLASM
Cells f r o m c h i c k e m b r y o liver w e r e g r o w n a n d t r e a t e d as d e s c r i b e d i n T a b l e V. ~ - A m a n i t i n ( 0 . 2 5 / ~ g ) w a s a d d e d 1 h prior t o t h e i n d u c i n g a g e n t s a n d t h e cells r e - i n c u b a t e d f o r 5 . 5 h. A t t h e e n d o f t h e r e - i n c u b a t i o n p e r i o d , cells w e r e l a b e l e d w i t h [ 3 H ] u r i d i n e f o r 15 m i n a n d t h e m e d i u m r e m o v e d . T h e m o n o l a y e r w a s s c r a p e d f r o m t h e b o t t o m o f t h e p e t r i d i s h e s a n d n u c l e a r a n d c y t o p l a s m i c f r a c t i o n s i s o l a t e d f r o m cell h o m o g e n a t e s ; [ 3 H ] u r i d i n e i n c o r p o r a t e d i n t o R N A a f t e r 15 r a i n a n d p r e s e n t in t h e c y t o p l a s m w a s d e t e r m i n e d as d e s c r i b e d in T a b l e V. Treatment
Induction (h)
Protein (~ug/ml)*
[3H] Uridine-labeled R N A in c y t o p l a s m (cpm/ml)*
Control a-Amanitin + control Etiocholanolone 0eAmanitin + etiocholanolone 2-Allyl- 2 - i s o p r o p ylac etamide ~ - A m a n i t i n + 2-allyl-isopropylacetamide
Inhibition %
(cpm/~tg p r o t e i n )
5.5 5.5 5.5
2 4 5 +- 1 4 " * 1 4 2 -+ 5.3 1 8 3 -+ 11
4 6 1 -+ 1 6 " * 2 0 6 -+ 3 8 9 8 -+ 52
1.88 1.45 4.92
-23 --
5.5
178 +
3.5
4 0 4 +- 3
2.27
54
5.5
2 0 9 +- 4.9
1 2 6 9 -+ 59
6.07
--
5.5
1 3 7 -+ 1.8
211 -+ 33
1.55
75
* C y t o p l a s m i c f r a c t i o n ( t o t a l v o l u m e = 4 0 m l f o r all s a m p l e s ) . ** Mean-+ S.D.
341
Mn2+- (NH4)zSO,I ossoy
E
Mg 2+ OSSOy
Control
I00
/
Control
80 ~ - Am0nitln
~ ._> "6 o
6O
p
4O
o E 2
7 rr"
4
6
8
,
/(, /~q ONA
Fig. 3. I n h i b i t i o n o f t h e M n 2 + - - ( N H 4 ) 2 S O 4 - s t i m u l a t e d D N A - d e p e n d e n t R N A p o l y m e r a s e b y ~ - a m a n i t i n in v i t r o a n d r e q u i r e m e n t f o r a D N A t e m p l a t e b y b o t h e n z y m e s . Cells w e r e g r o w n as d e s c r i b e d in M e t h o d s a n d n u c l e i i s o l a t e d [ 1 4 ] f r o m c o n t r o l . C o n d i t i o n s f o r t h e R N A p o l y m e r a s e a s s a y s w e r e t h e s a m e as in T a b l e V I I e x c e p t t h a t c~-amanitin ( 0 . 2 ~tg) w a s a d d e d t o t h e r e a c t i o n m i x t u r e at t h e b e g i n n i n g o f t h e R N A p o l y m e r a s e assay.
To correlate with the inhibition of RNA synthesis by ~-amanitin shown in Table V, endogenous activities of RNA polymerase were also measured in nuclei from control and induced hepatocytes grown in the presence of ~amanitin and induced for 6.5 h. When ~-amanitin (0.2 pg/ml) was added 1 h prior to the inducing agents, the Mn2+--(NH4 )2 SO4 -stimulated RNA TABLE VII IN VIVO EFFECT OF (~-AMANITIN ON DNA-DEPENDENT AVIAN HEPATOCYTES
RNA POLYMERASE
IN NUCLEI FROM
Cells f r o m c h i c k e m b r y o livers w e r e g r o w n in p e t r i d i s h e s ( 1 5 0 m m d i a m e t e r ) f o r 24 h i n i t i a l l y . ~A m a n i t i n w a s a d d e d * 2 h a f t e r c h a n g i n g t h e m e d i u m a n d 1 h p r i o r t o a d d i t i o n o f i n d u c i n g a g e n t s . Cells w e r e r e - i n c u b a t e d f o r 6 . 5 h; a t t h i s t i m e t h e m e d i u m w a s r e m o v e d a n d t h e cells s c r a p e d f r o m t h e b o t t o m o f t h e p e t r i d i s h e s . N u c l e i w e r e i s o l a t e d [ 1 4 ] f r o m cell h o m o g e n a t e s p r e p a r e d in 0 . 3 2 M s u c r o s e - - 3 m M MgCI 2 f r o m t w o pctri dishes. D N A - d e p e n d e n t R N A p o l y m e r a s e activities of nuclei were d e t e r m i n e d and a r e e x p r e s s e d as c p m o f [ 8 - 1 4 C ] A M P i n c o r p o r a t e d i n t o R N A p e r /~g o f D N A ; i n c u b a t i o n s w e r e 10 a n d 4 5 r a i n a t 36 ° C f o r t h e Mg 2 ÷ a n d M n 2 + - - ( N H 4 ) 2 S O 4 a s s a y s r e s p e c t i v e l y . R a d i o a c t i v i t y i n c o r p o r a t e d i n t o n e w l y s y n t h e s i z e d R N A w a s d e t e r m i n e d as p r e v i o u s l y d e s c r i b e d [ 1 4 ] . T r e a t m e n t o f cells
DNA-dependent RNA polymerase activities of nuclei Mn2÷--(NH4)~SO4 assay
M g 2+ a s s a y
(cpm#tg DNA)
cpm/ktg D N A
Inhibition
(%) Control ~-Amanitin + control Etiocholanolone ~-Amanitin + etiocholanolone 2-Allyl-2-isopropylacetamide ~-Amanitin + 2-allyl-2-isopropylaeetamide
19.0
4.5 29.4 5.3 20.6
+ 0.08**
+ 0.15 -+ 6 . 4 + 0.28 -+ 0 . 6 4 ~5.1 + 1.24
Inhibition
(%)
--
16.9
+ 3.5**
--
76 -82 -75
20.4 15.3 16.0 14.8 22.8
+ 0.23 + 0.56 + 1.1 -+ 2.1 + 0.74
None -None --
None
* All a d d i t i o n s a r e p e r m l o f m e d i u m , ~ - a m a n i t i n ( 0 . 2 / ~ g ) ; e t i o c h o l a n o l o n e ( 1 0 ~tg) a n d 2-allyl-2-isoprop y l a c e t a m i d e (30/~g). ** M e a n s a n d S . D . o f d u p l i c a t e d e t e r m i n a t i o n s .
342 polymerase activities of control and induced cell nuclei were inhibited 76, 82 and 68% respectively, whereas the Mg 2+ activities were not inhibited, and even showed some enhancement (Table VII). This very significant depression of Mn2÷--(NH4 )2 SO4-stimulated R N A polymerase by a-amanitin in avian tissue confirms prior reports that the nucleoplasmic polymerases are markedly inhibited by the toxin in vitro (as shown in Fig. 3) and in vivo [28]. The results obtained in Table VII with the Mg2+-stimulated activity, corresponding to the nucleolar polymerase, do not show any inhibition and these data differ from those of others who show inhibition of rRNA in vivo by a-amanitin [28,29]. To further substantiate the increase in R N A synthesis observed in induced cultures (i.e. for the steroid treated 27% above control, Table V) enhancement of the Mn2+--(NH4)2SO4-stimulated activity of the nucleoplasmic R N A polymerase was also found in nuclei isolated from etiocholanolone-treated hepatocytes (Table VII); the increase from 19.0 to 29.4 cpm/pg DNA represents a change of 55% over control value in this particular experiment, a very significant enhancement which did not occur in the R N A polymerase activity of the 2-allyl-2-isopropylacetamide-treated nuclei. The Mg2÷-stimulated RNA polymerase of both control and induced nuclei, isolated from hepatocytes not treated with a-amanitin, showed no significant changes in activities b u t the a-amanitin-treated samples showed a certain pattern of increase, most pronounced in the nuclei isolated from the barbiturate-treated hepatocytes. Discussion
a-Amanitin is a p o t e n t inhibitor of the DNA-dependent polymerase in mammalian liver and other tissues [8,9,30--32] ; and as shown in these studies the toxin markedly inhibits the enzyme in cultured avian hepatocytes as well. The induction of 5-aminolevulinate synthetase by a 5~-steroid or 2-allyl2-isopropylacetamide was strongly inhibited by a-aminitin in avian liver cells grown in primary cultures. This inhibition was most pronounced at a toxin concentration of 1 pg/ml of medium reaching a level of inhibition of 87%, confirming our earlier observations on a-amanitin inhibition of the induction of this enzyme in vivo [ 1 0 ] . Its inhibitory action with respect to chemicalinduced porphyrinogenesis is presumably a secondary effect, resulting from inhibition of the induction of 5-aminolevulinate synthetase. The pronounced ability of a-amanitin to block induction of 6-aminolevulinate synthetase suggests that the synthesis of new R N A is a necessary step in the de novo formation of this enzyme. The toxin inhibited 69 to 76% the incorporation of [3 H] uridine into RNA at the low concentration of 0.5 pg/ml of medium, eliminating the increased uptake of this precursor observed in both steroid- and drug-induced nuclei isolated from hepatocytes cultured in the absence of the toxin. These findings indicate that DNA transcription by RNA polymerase is required for the induction of d-aminolevulinate synthetase, and that certain area(s) of the genome are transcribed into new species of R N A as a result of the action of these inducer agents. Such a process is an initial step in the induction mechanism for this enzyme as shown here and occurred 14--16 h prior to full induction of the protein. The induction was eliminated in the absence of R N A synthesis, or its magnitude greatly reduced depending on the
343
concentration of the toxin used. The inhibition produced by low doses of a-amanitin, as used in our studies, is quite significant since protein synthesis was not inhibited concomitantly; on the contrary it was slightly enhanced and more significantly so in the induced cultures even though 5-aminolevulinate synthetase formation was markedly decrease at 18--24 h after addition of the inducers. In keeping with these observations, a-amanitin inhibited in vivo the Mn2÷--(NH4 )2 SO4 -stimulated nucleoplasmic DNA-dependent RNA polymerase in nuclei isolated from control and induced hepatocytes b u t to different degrees. This enzyme is responsible for the synthesis of heterogeneous nuclear RNA~ a precursor molecule from which m R N A is cleaved upon transfer to the cytoplasm [ 3 3 ] . This R N A polymerase is very sensitive to the toxic effect of ~-amanitin in vitro [ 3 4 ] , suggesting therefore that ~-amanitin inhibited the synthesis of m R N A in the toxin-treated cells. As shown in the [3 H]uridine incorporation studies of Table V, the increased activity of this R N A polymerase in the steroid-treated nuclei was not present in the drug-induced nuclei of Table VII; however in the cytoplasm of both steroid- and barbiturate-treated hepatocytes (Table VI), an increase in labeled R N A species was present and inhibited when the toxin was added to the culture medium. This inhibitory effect on the enhancement of R N A polymerase activity coincides in time with the inhibition observed in the enhancement of R N A synthesis found after induction. The inhibition was only manifest in the R N A polymerase present in the nucleoplasm and n o t in the nucleolar enzyme. The latter is stimulated by Mg 2÷ and was n o t inhibited by a-amanitin when examined in our studies at 6 or 6.5 h after the start of the induction period; however in a related experiment, this polymerase was inhibited slightly {20--30%} at 1.5 h after addition of the inducers b u t not at 5 h. In fact a slight tendency for an increase in its activity was evident in the control and barbiturate-induced nuclei. This could suggest that in the a-amanitin-treated nuclei in vivo a controlling factor, translated from an RNA species synthesized by the nucleoplasmic RNA polymerase, regulates the activity of the nucleolar polymerase in synthesizing pre-rRNA, this protein factor being eliminated by the inhibitory effect of a-amanitin on the nucleoplasmic enzyme [ 2 8 ] . The synthesis of another t y p e of RNA, possibly rRNA, not sensitive at the time studied (6.5 h} to a-amanitin and n o t involved with the triggering of the induction process for 5-aminolevulinate synthetase could be stimulated by the barbiturate inducer, particularly if the Mg2+-stimu lated R N A polymerase had a short half life. This would correlate with the slight stimulation of protein synthesis observed in the a-amanitin-treated induced cultures, an effect which is present only at the latter part of the induction period. Coproporphyrins which have accumulated in the induced cells and their culture medium have also been shown to inhibit protein synthesis [ 1 1 ] , and the prevention of their formation by low doses of a-amanitin restores protein synthesis to control values or values even slightly higher than control, as shown in Table IV. There is therefore only a slight difference evident between the actions of the inducers examined in this study; the difference being manifested for the steroid in the magnitude of the response for stimulation of the nucleoplasmic RNA polymerase and for the barbiturate in the nucleolar enzyme. 2-allyl-2-
344
isopropylacetamide may act post-transcriptionally in addition to transcriptionally since there appear to be several sites of control for enzyme induction in mammalian tissues. Our findings indicate, however, that continued m R N A synthesis is necessary for enzyme induction b y both inducers, even though both may not directly stimulate m R N A synthesis. It is possible also that induction proceeds via stabilization of, or faster translation of m R N A s synthesized at the same rate as in the absence of inducers. The data available here do not permit a choice between these alternatives. Acknowledgements We wish to thank Mrs Maureen Morgan and Miss Mary Horan for skilled technical assistance and Miss Ann Marie Quatela for typing the manuscript. This work was supported by Grant No. HD-04313 from the National Institutes of Health. References 1 Granick, S. and Kappas, A. (1967) J. Biol. Chem. 242, 4587 2 Schmid, R. and Schwartz, S. (1952) Proc. Soc. Exp. Biol. Med, 8 1 , 6 8 5 3 Goldberg, A. and R i m i n g t o n , C. (1962) Diseases of Porphyrin Metabolism, Charles C. Thomas, Springfield 4 Granick, S. and Urata, G. (1963) J. Biol. Chem. 238, 821 5 Kappas, A., Song, C.S., Levere, R.D., Saehson, R,A. and Granick, S. (1968) Proc. Natl. Acad. Sci. U.S. 61, 509 6 Graniek, S. (1966) J. Biol. Chem. 241, 1359 7 Sassa, S. and Granick, S. (1970) Proc. Natl. Acad. Sci. U.S. 67, 517 8 Wieland, T. (1969) Science 159, 946 9 Jacob, S.T., Sajdel, E.M. and Munro, J.N. (1970) Nature, 225, 60 10 Incefy, G.S. and Kappas, A. (1971) FEBS Lett. 15, 153 11 Incefy, G.S. and Kappas, A. (1972) FEBS Lett. 23, 37 12 Incefy, G.S. and Kappas, A. (1971) Fed. Proc. 30, 1302 13 Kappas, A., Song, C.S., Sassa, S., Levere, R.D. and Graniek, S. (1969) Proe. Natl. Aead. Sci. U.S. 64, 557 14 Incefy, G.S. and Kappas, A. (1971) J. Cell Biol. 50, 385 15 Kissane, M.J. and Robins, E. (1958) J. Biol. Chem. 2 3 3 , 1 8 4 16 Hinegardner, R.T. (1971) Anal, Biochem. 3 9 , 1 9 7 17 Poland, A. and Glover, E. (1973) Science 179, 476 18 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randal, R.J. (1951) J. Biol. Chem., 193, 265 19 Widnell, C.C. and Tata, J.R. (1966) Biochim. Biophys. Aeta 123, 478 20 Incefy, G.S. and Kappas, A. (1974) Proc. Natl. Acad. Sci. U.S., in press 21 Doss, M. and Kaltepolh, B. (1971) in Proceedings of the I n t e r n a t i o n a l Conference on P orphyri n Metabolism and the Porphyrias (South African Medical J.) 25 September, p. 73, Special issue S.A.J. Lab. and Clin. Med. 22 Stirpe, F. and Fiume, L. (1967) Bioehem. J. 105, 779 23 Buku, A., Campadelli-Fiume, G., Flume, L. and Wieland, Th. (1971) FEBS Lett. 14, 42 24 Ponta, H., Ponta, U. and Winterberger, E. (1972) Eur. J. Biochem. 29, l l O 25 Seifart, K.H. and Sekeris, C.E. (1969) Z. Naturforsch. 24b, 1538 26 Strand, L.J., Manning, J. and Marver, H.S. (1972) J. Biol. Chem. 247, 2820 27 Willems, M., Penman, M. and Penman, S. (1969) J. Cell Biol. 4 1 , 1 7 7 28 Tata, J.R., Hamilton, M.J. and Shields, D. (1972) Nat. New Biol. 238, 161 29 Bucci, S., Nardi, I., Maneino, G. and Flume, L. (1971) Exp. Cell. Res. 69, 462 30 Lindell, T.J., Weinberg, F., Morris, P.W., Roeder, R.G. and Rutter, W.J. (1970) Science 170, 447 31 Kedinger, C., Nuret, P. and Chambon, P. (1971) FEBS Lett. 15, 169 32 Dezelee, S., Sentenae, A. and Fromageot, P. (1970) FEBS Lett. 7 , 2 2 0 33 Jelinek, W., Adesnik, M., Salditt, M., Sheiness, D., Wall, R., Molloy, G., Phillipson, L. and Darnell, J.E. (1973) J. Mol. Biol. 75, 515 34 Kedinger, C., Gissinger, F., Gniazdowski, M., Mandel, J.L. and Chambon, P. (1972) Eur. J. Biochem. 28, 269
Biochimica et Biophysica Acta, 361 ( 1 9 7 4 ) 3 3 1 - - 3 4 4 © Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s
BBA 98080
INHIBITION OF 5-AMINOLEVULINATE SYNTHETASE INDUCTION BY a-AMANITIN IN AVIAN LIVER CELLCULTURES
G E N E V I E V E S. I N C E F Y * , A R L E E N B. R I F K I N D * * and A T T A L L A H K A P P A S
The Rockefeller University, New York, N.Y. 10021 (U.S.A.) (Received April 1st, 1 9 7 4 )
Summary The induction of 6-aminolevulinate synthetase by the 5~-steroid etiocholanolone and the barbiturate derivative 2-allyl-2-isopropylacetamide was blocked by a-amanitin in avian h e p a t o c y t e cultures. 6-Aminolevulinate synthetase controls the initial and rate-limiting step of the porphyrin-heme pathway. ~-Amanitin was demonstrated to be a p o t e n t inhibitor of the nucleoplasmic DNA-dependent R N A polymerase in chick e m b r y o liver cells in culture. It is shown that nuclear R N A synthesis and nucleoplasmic R N A polymerase activities were inhibited by the toxin at an early time (6--6.5 h) in the induction period; whereas the nucleolar R N A polymerase was not inhibited. At this particular time of the induction period, enhancement of R N A synthesis was observed and was dependent on the effect of inducers; this enhancement was eliminated by ~-amanitin. It is concluded that the increment of new R N A synthesized by the ~-amanitin-sensitive nucleoplasmic R N A polymerase is required as one of the early steps in the induction process of 6-aminolevulinate synthetase, either as m R N A for the enzyme or for a specific protein required in the induction process.
Introduction We have shown previously that certain 5~-steroids, derived from the metabolic transformation of sex hormones or their precursors, are able to induce the de novo formation of 5-aminolevulinate synthetase, and consequently porphyrinogenesis in chick e m b r y o hepatocytes grown in primary cultures [ 1 ] . These inducing steroids, as well as a number of drugs, elicit an * Present address: Memorial Sloan-Kettering Cancer Center, 4 1 0 East 6 8 t h Street, New York, N. Y. 1 0 0 2 1 . T o w h o m requests for reprints s h o u l d be addressed.
** Present address: Cornell University Medical College, 1300 York Avenue, New York, N. Y. 10021.
332 experimental type of hepatic porphyria in vivo and in vitro [2--5], which provides an interesting system for the study of gene expression in eukaryotic cells. In this study two different types of chemicals which induce 5-aminolevulinate synthetase were utilized to study the effect of a-amanitin on the enzyme induction process; one was the 5fl-steroid, etiocholanolone (5/~-androstane-3a-ol-17-one) and the other, the barbiturate analogue, 2-allyl-2-isopropylacetamide [6]. These compounds were selected for study because they represent examples of both naturally occurring as well as foreign chemical inducers of 5-aminolevulinate synthetase; further it has been suggested that they induce the enzyme by different mechanisms [7]. In an earlier study we showed that a-amanitin, the cyclic peptide of Amanita phalloides and a potent inhibitor of RNA synthesis in mammalian systems [8,9], prevents induction of 5-aminovulinate synthetase by both etiocholanolone and 2-allyl-2-isopropylacetamide in whole chick embryo liver [10]. The findings reported here in primary cultures of avian hepatocytes indicate that a-amanitin had several effects which can be related to the de novo formation of 5-aminolevulinate synthetase. First, the toxin had a similar inhibitory effect on the induction of this enzyme in cultured liver cells as it had in vivo in the chick embryo. Second, the porphyrinogenesis which usually develops after treatment of the cultures with inducing agents, did not occur. Third, a-amanitin almost completely abolished the inhibitory effect of coproporphyrins on [~4 C] leucine incorporation into proteins [11], indicating that this potent fungal agent seems to protect, paradoxically, the induced hepatocytes from the toxic effect produced by porphyrins. Fourth, a-amanitin had marked inhibitory effects on RNA synthesis in control and induced avian liver cell cultures. In addition it prevented the specific stimulation of nuclear RNA synthesis usually observed in association with the increased activity of the nucleoplasmic DNA-dependent RNA polymerase (EC 2.7.7.6) which follows treatment with the inducing agents [12]. It is therefore concluded that the increment in new RNA synthesized by this polymerase, and which is inhibited by a-amanitin, is required for the induction of 5-aminolevulinate synthetase either as mRNA for the enzyme itself or for a specific protein required in the induction process. Materials
The inducing agents etiocholanolone and 2-allyl-2-isopropylacetamide were gifts from Mann Research and Hoffmann-La Roche respectively. a-Amanitin was a gift of Professor T. Wieland. Unlabeled ribonucleoside triphosphates were purchased from Sigma; [8 -14 C] ATP (35 Ci/mole), [3 H]uridine (5--15 Ci/mmole) and L-[14C]leucine (260 Ci/mole) were obtained from New England Nuclear. Materials for liver cell cultures and the leucine-free medium were purchased from Grand Island Biological Co. Methods
Hepatic cell cultures The technique has been described previously [6]. Briefly, liver cells from
333 15-day-old chick embryos were grown in primary cultures in an incubator at 37°C in the presence of CO2--02 (5:95, v/v), either in small vials or in Falcon plastic petri dishes {100--150 mm diameter) for 16--20 h initially to permit a cell monolayer to form. The medium was then replaced, and additions were made as required. Inducing agents, dissolved in propylene glycol, were added to cultures in volumes of 5 #l/ml of medium and a-amanitin, prepared in 0.15 M NaC1 was added in volumes o f 5--20 pl/ml of medium before or after the inducing agents. The cells were re-incubated in their presence for the various additional times indicated; additions of appropriate solvents were made to control cultures in all experiments. Porphyrins synthesized by avian hepatocytes during the induction period of 6-aminolevulinate synthetase were quantitated fluorimetrically [13] to assess the magnitude of the induction response; coproporphyrin III was used as a standard. In experiments where cells were used for determinations of 5-aminolevulinate synthetase activities, [' 4 C]leucine incorporation, nuclei isolation [14] or DNA quantitation [ 1 5 , 1 6 ] , the culture medium was removed prior to harvesting of the cells and frozen. In all other experiments, cells and medium combined were used for porphyrin determination; after lyophilization, their coproporphyrin contents were determined and compared with controls.
Enzyme assays Activities of 5-aminolevulinate synthetase were measured in cultured hepatocytes by a m e t h o d previously described [ 1 7 ] . Protein contents of cell homogenates were determined using bovine serum albumin as a standard [ 1 8 ] . DNA-dependent R N A polymerase activities of nuclei isolated from cultured hepatocytes were measured [19] in the presence of Mg :÷ or Mn :÷ and 0.3 M (NH4): SO4 with the following modification: 0.075 pmole each of GTP, CTP, UTP and 0.025 pmole of [8 -14 C] ATP, and 5--15 pg of DNA; the other components remained unchanged [14]. Incubation times were 10 and 45 min for the Mg :÷ and Mn2+--(NH4 )2 SO4 assays respectively at 36 ° C.
[3 HI uridine incorporation into RNA Hepatocytes in cultures were labeled [20] with 5 pCi of [3 H]uridine per ml of medium for 10 or 15 min (Tables V and VI respectively) in the incubator at 37°C. At the end of the incubation, medium was aspirated off and cold 0.32 M sucrose--3 mM MgC12 added, the cell monolayer was scraped off the b o t t o m of the petri dishes and homogenized. Nuclei were isolated and radioactivity determined as previously described [ 1 4 ] . Results
Inhibition of 5-aminolevulinate synthetase formation by a-amanitin Chick e m b r y o liver cells grown in primary cultures for 24 h consist almost entirely of hepatic parenchymal cells which have very low levels of 5-aminolevulinate synthetase activity. After addition of fresh medium and the inducing agents, etiocholanolone or 2-allyl-2-isopropylacetamide, there is a progressive increase in 6-aminolevulinate synthetase activity reaching 5--20 times control values after 18--20 h of incubation. When a-amanitin is added 1 h prior to the
334
TABLE I INHIBITION OF 6-AMINOLEVULINATE HEPATOCYTE CULTURES
SYNTHETASE
INDUCTION
BY a-AMANITIN
IN AVIAN
Cells f r o m c h i c k e m b r y o liver w e r e g r o w n i n i t i a l l y f o r 24 h in p e t r i d i s h e s ( 1 0 0 m m d i a m e t e r ) as desc r i b e d in M e t h o d s ; a - a m a n i t i n w a s a d d e d * to t h e m e d i u m 1 h p r i o r to t h e i n d u c i n g a g e n t s e t i o c h o l a n o l o n e a n d 2 - a U y l - 2 - i s o p r o p y l a c e t a m i d e , a n d t h e cells r e - i n c u b a t e d f u r t h e r f o r 18 h. A t t h e e n d o f t h e r e - i n c u b a t i o n p e r i o d t h e m e d i u m w a s r e m o v e d a n d its c o p r o p o r p h y r i n c o n t e n t d e t e r m i n e d [ 1 3 ] ( T a b l e II). T h e cell m o n o l a y e r s w e r e s c r a p e d f r o m t h e b o t t o m o f t h e d i s h e s , p o o l e d * * in b u f f e r a n d 5 - a m i n o l e v u l i n a t e s y n t h e t a s e a c t i v i t y d e t e r m i n e d [ 17].
Experiment and treatment
-Aminolevulinate synthetase activity (nmoles -aminolevulinate]mg p r o t e i n p e r h)
Induction (% o f control)
Inhibition of induction (%)
1. C o n t r o l E tiocho lanolone c~-Amanitin ( 0 . 4 # g ) + e t i o c h o l a n o l o n e 2. C o n t r o l 2-Allyl-2-isoprop ylacetamide ~-Amanitin (1.0 pg) + 2-allyl-2-isopropylacetamide
0.038 0.188 0.102 0.044 0.817 0.145
100 495 -100 1857 --
--57 --87
_+ 0 . 0 0 1 " * * + 0.007 + 0.002 + 0.011 + 0.160 + 0.028
* All a d d i t i o n s are p e r m l o f m e d i u m ; e t i o c h o l a n o l o n e ( 2 0 p g ) a n d 2 - a U y l - 2 - i s o p r o p y l a c e t a m i d e ( 5 0 # g ) ; c o n t r o l s w e r e t r e a t e d w i t h s o l v e n t s ( 0 . 9 % NaC1 a n d p r o p y l c n e g l y c o l ) . ** T w o p e t r i d i s h e s p o o l e d f o r e a c h c o n t r o l g r o u p a n d t h r e e f o r e a c h t r e a t e d cell cultures~ m e a n a n d S.D. are g i v e n f o r e a c h g r o u p . * * * M e a n _+ S.D.
addition of inducing agents, the stimulation of 6-aminolevulinate synthetase activity is greatly decreased and the level of enzyme induction is reduced 57 and 87% respectively {Table I). The difference observed in the degree to which the induction response is inhibited is due to the concentration of a-amanitin T A B L E II INHIBITION CULTURES
OF INDUCED
PORPHYRIN
SYNTHESIS
BY ~-AMANITIN IN AVIAN HEPATOCYTE
Cells f r o m c h i c k e m b r y o liver w e r e g r o w n a n d t r e a t e d w i t h i n d u c i n g a g e n t s as d e s c r i b e d in T a b l e I. c~-Amanitin w a s a d d e d t o t h e m e d i u m 1 h b e f o r e t h e i n d u c i n g a g e n t s a n d t h e cells r e - i n c u b a t e d f o r t h e t i m e s indicated. The m e d i u m was then r e m o v e d and its p o r p h y r i n c o n t e n t * d e t e r m i n e d [ 1 3 ] . Experiment and treatment
Time of incubation (h)
Coproporphyrin III (pmoles/ml of medium)
Induction (% o f control)
1. C o n t r o l ~-Amanitin (0.4/~g) + control Etiocholanolone ~-Amanitin (0.4 gg) + etiocholanolone 2. C o n t r o l 2-Allyl- 2 - i s o p r o p y l a c e t a m i d e ~-Amanitin (1.0 #g) + 2-aUyl-2-isopropylacetamide
18 18 18 18 24 24 24
32 32 158 23 47 609 72
-503 -100 1295 --
_+ 1 3 " * -+ 1.6 -+ 81 -+ 5.5 -+ 18 -+ 38 _+ 36
100
Inhibition of induction (%)
--
--100 --96
* C o n t e n t is p e r m l o f m e d i u m a n d d o e s n o t i n c l u d e p o r p h y r i n s in t h e cell m o n o l a y e r (see M e t h o d s ) . ** M e a n + S.D.
335 used in the cultures, the cells treated with etiocholanolone having received 0.4 pg of ~-amanitin whereas the cultures treated with 2-allyl-2-isopropylacetamide received 1.0 pg of the toxin. Porphyrin accumulation within the cells and medium also occurs during the induction period [6,12,21] as a result of the enhanced production of 5-aminolevulinate synthetase evoked by the inducing agents. This porphyrinogenesis develops after a lag period of 8--10 h; following which porphyrin synthesis rapidly occurs, reaching levels 5 - 2 0 or more times control values in the ensuing 24 h. In the presence of ~-amanitin, however, only small amounts of porphyrins could be detected when their concentrations were determined at various times during the induction period; at 18 and 24 h this inhibition of porphyrinogenesis by ~-amanitin was pronounced (Table II). In the experiments reported in Table II the treated cells were also examined for 5-aminolevulinate synthetase activity, and the results are shown in Table I. These data indicate that (a) induction of this enzyme had occurred concurrently with the enhancement of porphyrin synthesis and (b) that ~-amanitin markedly inhibited the increase in enzyme activity and in porphyrin formation. ~Amanitin, however, at 0.4 pg inhibited porphyrin synthesis much more effectively than enzyme synthesis. This is probably due to the fact that the porphyrins measured represent products accumulated over the full time period (18 h), whereas 5-aminolevulinate synthetase activity was measured at the end of the time period, when enzyme activity may have had an opportunity to recover from the low dose inhibition of ~-amanitin. It is reasonable to infer that the diminished porphyrinogenesis, resulting from pretreatment with ~-amanitin, reflects the almost total inhibition of the induction response for 5-aminolevulinate synthetase. The time required for ~-amanitin to inhibit porphyrin biosynthesis was also examined with regard to the time of addition of the steroid or barbiturate inducer agent to the cultures. As shown in Fig. 1, induction of porphyrin synthesis was inhibited from 88--83% and 95--93% respectively when ~amanitin was added 120 to 1 min prior to addition of both etiocholanolone or 2-allyl-2-isopropylacetamide; b u t when the toxin was added after the inducing agents, a less though still substantial inhibition of porphyrinogenesis occurred. Because the greatest degree of inhibition with ~-amanitin was obtained when it was added to the cultures before the inducing agents the toxin was added 40--60 min prior to the inducers in all subsequent studies.
Metabolic capacity of cells after a-amanitin treatment a-Amanitin is a p o t e n t toxin [ 8 , 2 2 , 2 3 ] ; to examine the metabolic effect of this agent on the cultured liver cells the following studies were done. (a} Cultured h e p a t o c y t e s which had been in contact with 0.5 pg of a-amanitin for 25 h were tested for their ability to convert 5-aminolevulinate to porphyrins. As shown in Table III the a-amanitin-treated hepatocytes were able to synthesize as much porphyrins as did control cultures, 205 + 52 and 174 + 19 pmoles of coproporphyrins III/ml of medium respectively. These findings indicate that in ~-amanitin-treated hepatocytes, despite the p o t e n t toxicity of this agent, the integrity of a number of enzymatic steps of the porphyrin biosynthetic p a t h w a y b e y o n d that catalyzed by ~-aminolevulinate synthetase
336
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Hours otter addition of a-amonitin Fig. 1. T i m e c o u r s e for t h e i n h i b i t o r y a c t i o n of ~ - a m a n i t i n o n p o r p h y r i n b i o s y n t h e s i s b e f o r e a n d a f t e r i n d u c t i o n w i t h e t i o c b o l a n o l o n e or 2 - a l l y l - 2 - i s o p r o p y l a c e t a m i d e . L i v e r cells w e r e g r o w n in small vials as d e s c r i b e d in M e t h o d s f o r 18 h. T h e c u l t u r e m e d i u m was c h a n g e d a n d c~-amanitin, 0.5 ~ g / m l o f m e d i u m , was a d d e d 1 2 0 , 60, 3 0 a n d 1 rain b e f o r e a d d i t i o n o f t h e i n d u c i n g a g e n t s , e t i o c h o l a n o l o n e or 2-allyl-2i s o p r o p y l a c e t a m i d e ( 1 0 p g a n d 3 0 p g / m l of m e d i u m r e s p e c t i v e l y ) , or 1, 3, 6 a n d 8.5 h a f t e r t h e i r a d d i t i o n . A t t h e e n d o f t h e 24-h i n d u c t i o n p e r i o d ( w h i c h begins a t 0 h o n t h e g r a p h ) or 20 h a f t e r t h e s t a r t o f t h e c u l t u r e s , all t h e vials w e r e f r o z e n , l y o p h i l i z e d a n d t h e p o r p h y r i n c o n t e n t of cells a n d m e d i u m d e t e r m i n e d [ 1 3 ] . T h e % i n h i b i t i o n w a s c a l c u l a t e d o n t h e basis o f t h e a m o u n t o f p o r p h y r i n s y n t h e s i z e d d u r i n g this 24-h i n c u b a t i o n in c u l t u r e s n o t t r e a t e d w i t h c~-amanitin using t h e f o l l o w i n g values as 100%; e t i o c h o l a n o l o n e - i n d u c e d c u l t u r e s : 47.9 -+ 6 . 4 p m o l e s o f c o p r o p o r p h y r i n I I I / m l ; 2 - a l l y l - 2 - i s o p r o p y l a c e t a m i d e - i n d u c e d c u l t u r e s : 1 0 5 -+ 23 p m o l e s o f c o p r o p o r p h y r i n IfI p e r ml. C o n t r o l c u l t u r e s s y n t h e s i z e d 5,1 +- 0 . 2 5 p m o l e s c o p r o p o r p h y r i n I I I / m l a n d w e r e n o t u s e d in t h e c a l c u l a t i o n s , o -o ~-amanitin- + etioeholanolonetreated cultures; • •, ~-amanitin- + 2-allyl-2-isopropylacetamide-treated cultures.
TABLE
IIl
PORPHYRIN SYNTHESIS FROM 8-AMINOLEVULINATE AFTER TREATMENT WITH ~x-AMANITIN
IN C U L T U R E D
AVIAN
HEPATOCYTES
Liver cells were g r o w n in small vials for 18 b initially as described in Methods. After changing the m e d i u m , vials were sorted into groups of five and s o m e were treated with a-amanitin*; all were then incubated for 25 h. After incubation, a-amanitin w a s r e m o v e d by aspirating off the m e d i u m and the cells washed once with 1 m l of m e d i u m prior to addition of fresh m e d i u m . T o the control and previously a-amanitin-treated ceils was added 6-aminolevulinate or propylene glycol and the cells re-incubated a second time for 24 h. T h e m e d i u m was saved at the end of the last incubation, frozen and its porphyrin content** determined [131. First t r e a t m e n t
Time of incubation (h)
Second treatment
Time of incubation (h)
Copropophyrin III (pmoleslml of m e d i u m )
Control ~ - A m a n i t i n ( 0 , 5 pg) + c o n t r o l Control ~ - A m a n i t i n ( 0 . 5 pg) + c o n t r o l
25 25 25 25
Fresh Fresh Fresh Fresh
24 24 24 24
4.2 3.2 174 205
medium medium medium medium
+ + + +
propylene glycol p r o p y l e n e glycol ~-aminolevulinate 6-aminolevulinate
_+ 0.4** + 0.2 _4-19 _+ 52
* All a d d i t i o n s are p e r m l o f m e d i u m ; ~ - a m a n i t i n in 5 pl of 0.9% NaCl a n d • - a m i n o l e v u l i n a t e ( 1 0 0 / ~ g ) in 5 #l of w a t e r ; c o n t r o l r e c e i v e d 5 ~1 of 0.9% NaC1 a n d p r o p y l e n e glycol. ** Mean + S.D. o f five d u p l i c a t e vials f o r e a c h g r o u p ; 2 0 5 is n o t d i f f e r e n t f r o m 174, P v a l u e s o f t test is n o t significant.
337
I00
801"-
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6O
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40
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a-omonotin (Fg/ml medium) Fig. 2. E f f e c t of v a r y i n g c o n c e n t r a t i o n s of ~ - a m a n i t i n o n t h e b i o s y n t h e s i s of p o r p h y r i n s in e t i o c h o l a n o l one- a n d 2 - a l l y l - 2 - i s o p r o p y l a c e t a m i d e - t r e a t e d h e p a t o c y t e c u l t u r e s . C o n d i t i o n s f o r cell c u l t u r e s in small vials are d e s c r i b e d in M e t h o d s ; t r e a t m e n t w i t h ~ - a m a n i t i n a n d i n d u c i n g a g e n t s are t h e s a m e as in T a b l e I V . T h e d e c r e a s e d a m o u n t o f p o r p h y r i n s a c c u m u l a t e d in t h e m e d i u m of ~ - a m a n i t i n - t r e a t e d c u l t u r e s is c o m p a r e d t o Cz-amanitin-untreated c u l t u r e s a n d t h e % i n h i b i t i o n r e p o r t e d is c a l c u l a t e d t a k i n g t h e f o l l o w i n g m e a n v a l u e s as 1 0 0 % +- S.D.: c o n t r o l 1 5 . 4 + 2.4 p m o l e s p o r p h y r i n / m l m e d i u m ; e t i o c h o l a n o l o n e - i n d u c e d 4 1 . 8 + 6 . 9 ; 2 - a l l y l - 2 - i s o p r o p y l a c e t a m i d e - i n d u c e d 53.3 + 16. T h e s e v a l u e s r e p r e s e n t 0 i n h i b i t i o n in t h e g r a p h f o r c o n t r o l , steroid- or 2 - a l l y l - 2 - i s o p r o p y l a c e t a m i d e - i n d u e e d g r o u p s a n d e a c h p o i n t t h e r e a f t e r r e p r e sents t h e m e a n a n d S.D. of five r e p l i c a t e cell c u l t u r e s , o o c~-amanitin-treated c o n t r o l c u l t u r e s ; • •, a-amanitin- + etiocholanolone-treated cultures; • • , a - a m a n i t i n - + 2-allyl-2-isopropylacetamide-treated cultures.
was maintained. (b) In addition, increasing concentrations of a-amanitin were tested for their ability to prevent porphyrin formation in cultures treated with the inducing agents, etiocholanolone or 2-ailyl-2-isopropylacetamide for 24 h. As shown in Fig. 2, inhibition of coproporphyrin formation occurred at all concentrations of ~-amanitin studied. From the graph at 0.05 pg/ml of ~amanitin, the control cultures appear to be the only ones inhibited because the steroid and barbiturate-induced hepatocytes show a slight stimulation; however this stimulation is small when compared to that usually observed when ~amanitin is absent. What is occurring here, and shown by the graph, is a partial inhibition of the stimulation usually observed, this being due to the low dose of ~-amanitin employed at that point. Inhibition of coproporphyrin synthesis was greater at the higher concentrations of the toxin and reached a leveling off value near 40% for the control cultures. A much greater inhibition was observed in the induced cells; the etiocholanolone-induced cultures rapidly reached 65% inhibition at an ~-amanitin concentration of 0.2 pg/ml medium, leveling off near 80% at the higher concentration of the toxin. Comparable degrees of inhibition were observed with a-amanitin in the 2-allyl-2-isopropylacetamide induced cultures. Therefore the induction response elicited by b o t h agents was inhibited even when very small concentrations of ~-amanitin were used and this inhibition was almost maximal at 0.5 pg, the dose chosen for our studies. The fact that this inhibition is specific to the induction re-
338 TABLE IV EFFECT OF (~-AMANITIN ON THE CULTURED AVIAN HEPATOCYTES
INCORPORATION
OF
[14C]LEUCINE
INTO PROTEINS
IN
C h i c k e m b r y o liver cells w e r e g r o w n o n c o v e r s l i p s in small vials f o r 22 h p r i o r to a d d i t i o n o f f r e s h m e d i u m a n d C~-amanitin; t h e i n d u c i n g a g e n t s * e t i o c h o l a n o l o n e a n d 2 - a l l y l - 2 - i s o p r o p y l a c e t a m i d e ( 1 0 Dg a n d 30 p g r e s p e c t i v e l y ) w e r e a d d e d 40 r a i n a f t e r a d d i t i o n o f c~-amanitin a n d t h e cells w e r e r e - i n c u b a t e d f o r 24 h. A t t h e e n d o f t h e r e - i n c u b a t i o n t h e m e d i u m w a s r e m o v e d f o r d e t e r m i n a t i o n o f its c o p r o p o r p h y r i n c o n t e n t (Fig. 2). T o e a c h vial w a s i m m e d i a t e l y a d d e d 1 m l o f w a r m l e u c i n e - f r e e m e d i u m a n d t h e cell m o n o l a y e r w a s l a b e l e d w i t h [ ! 4 C ] l e u c i n e f o r 30 m i n as d e s c r i b e d p r e v i o u s l y [ 1 1 ] . Additions*
None a-Amanitin (0.5pg) a-Amanitin (0.20 #g) ~-Amanitin(0.50pg) v~-Amanitin ( 1 . 0 p g ) W-Amanitin(2.0~zg)
[ ~ 4 C ] L e u c i n e i n c o r p o r a t i o n as c p m / c o v e r a l i p * * Control
%
Etiocholanoline
%
2- A l l y l - 2 - i s o p r o p y l a c e tamide
(%)
5 9 0 9 -+ 1 1 9 6 6112 + 1261 5 2 2 4 -+ 4 1 6 3716 + 700 2282 + 344 1696-+ 134
100 103 88 63 39 29
4 6 0 0 + 869 5776-+ 899 5 0 5 3 +- 1 0 7 6 4212 + 935 3 5 7 2 -+ 4 1 7 2762 + 538
100 125 110 92 78 60
3 0 0 4 + 981 5018-+ 735 3 3 7 7 -+ 1 1 1 3 3593 + 920 2973 + 257 2079 + 413
100 167 112 120 99 69
* All a d d i t i o n s a r e p e r m l o f m e d i u m ** M e a n a n d S.D. o f five c o v e r s l i p s f o r e a c h g r o u p .
sponse and is not due to cell toxicity is indicated by the data in Table III in which it is shown that the ~-amanitin-treated cultures were able to synthesize large amounts of porphyrins from 6-aminolevulinate. The ability of cultured hepatocytes to incorporate amino acids into cellular proteins was also determined at the various concentrations of ~-amanitin shown in Fig. 2. It was found that steroid or barbiturate-induced hepatocytes which had been in contact with concentrations of ~-amanitin below 1.0 pg were able to incorporate [14 C]leucine into proteins very efficiently when compared to non-induced, ~-amanitin-treated controls (Table IV). In fact, the induced cultures seemed to be protected from the decreased [ 14 C] leucine uptake observed in control cells at the highest concentrations of the toxin. These results strongly suggest that the inhibition of 5-aminolevulinate synthetase activity and porphyrin formation in induced cell cultures, as shown in Tables I and II, was not due to cell toxicity. In these experiments, steroid or barbiturate-induced cells pretreated with ~-amanitin were not inhibited with respect to [ 14 C] leucine uptake, to the same degree as induced cells not treated with ~-amanitin prior to induction (Table IV}. Paradoxically, at the lower doses of ~-amanitin the incorporation of [14C]leucin e by induced cells was, in fact, enhanced over control, a finding which was unexpected since 5-aminolevulinate synthetase is markedly inhibited at this time. Because of these findings, the possible effects on protein synthesis of the excess accumulation of porphyrins in induced cells were examined. One of these effects is reflected in the data shown on the first line of Table IV, and is represented by the 23% and 43% smaller [ 14 C] leucine incorporation into total cellular proteins observed in induced cultures compared to controls. This difference is statistically significant (P < 0.05) and was noted consistently in other experiments. The decrease in [14 C]leucine incorporation found in these in-
339 duced cultures is not a characteristic of the inducer agent since it was also present when 5-aminolevulinate alone was added to the medium and allowed to be metabolized to porphyrins. Coproporphyrin which had accumulated in the cells and media of induced cultures were then shown to produce this decrease in [~4 C] leucine incorporation [11 ]. Also, in etiocholanolone- and 2-allyl-2-isopropylacetamide-treated cultures in which the accumulation of porphyrins had been inhibited by a-amanitin (0.05--0.5 pg/ml), a decrease in [,4 C]leucine incorporation was n o t observed, as shown in Table IV. At the highest a-amanitin concentrations, the cells were inhibited in their ability to incorporate [' 4C]leucine b u t again the effect in induced cultures was less marked than in controls. Since the possibility of cell damage could not be ruled out in these cultures, high concentrations of a-amanitin were not used in our studies.
Inhibition o f nuclear R N A synthesis by a-amanitin It was of interest to investigate the effect of a-amanitin on R N A synthesis because the toxin is a p o t e n t inhibitor of RNA synthesis in yeast and mammalian systems [ 22,24,25] and its effect had not been studied in avian tissue or in cell cultures. In addition a-amanitin had been shown to prevent the de novo formation of 5-aminolevulinate synthetase in whole chick e m b r y o liver previously [10] as well as in cultured avian hepatocytes as indicated in Table I. RNA synthesis was therefore determined 6 h after the start of the induction period in a-amanitin-treated cultures of both steroid- and barbiturate-induced hepatocytes and compared to respective controls (Table V). At this time, changes in R N A synthesis had been observed to occur previously [12] ; this interval precedes by 10--12 h the peak induction for 6-aminolevulinate synthetase in cultured hepatocytes [ 2 6 ] . Addition of a-amanitin was made 20 min prior to addition of inducers to the medium and R N A synthesis was measured as a short pulse of [3 H] uridine incorporated into nuclear RNA. It was found that a marked decrease in [3 H] uridine uptake characterized all a-amanitin-treated cultures, ranging from 69 to 76% in control and induced cells respectively. On the other hand enhancement of [3 H]uridine incorporation over controls was observed in steroid- and barbiturate-induced cultures not treated with a-amanitin (27 and 13% respectively). With smaller amounts of a-amanitin (0.2 pg per ml of medium) R N A synthesis was inhibited 29% in controls and 50--51% in induced cultures in a similar experiment as shown in Table V. Such inhibition was not observed, however, in control 2 h after the start of the induction period b u t reached 27 to 37% in steroid- and barbiturateinduced cultures respectively. These two concentrations of a-amanitin (0.5 and 0.2 pg) were chosen to determine if low doses of the toxin could inhibit R N A synthesis in the early part of the induction period, presumably when changes in the transcriptional process for induction of 5-aminolevulinate synthetase are taking place. These results indicate that the magnitude of the inhibition is dependent on the concentration of a-amanitin and the length of time the cells are exposed to this agent. When the cultures were exposed to a 15-min pulse of [3 HI uridine, the labeled R N A found in the cytoplasm was markedly inhibited by a-amanitin in the steroid- and barbiturate-induced cultures, 54 and 75% respectively, as compared to control (Table VI). In a short pulse label as utilized here, most labeled RNA should represent m R N A because rRNA takes longer to appear in the cytoplasm [ 2 7 ] .
340
TABLE V EFFECT
OF a-AMANITIN ON NUCLEAR
RNA SYNTHESIS IN AVIAN HEPATOCYTE
CULTURES
Cells f r o m c h i c k e m b r y o livers w e r e g r o w n i n i t i a l l y f o r 20 h in p e t r i d i s h e s ( 1 0 0 m m d i a m e t e r ) , as d e s c r i b e d in T a b l e 1, p r i o r to a d d i t i o n o f f r e s h m e d i u m a n d a - a m a n i t i n * ; t h e i n d u c i n g a g e n t s w e r e a d d e d 20 r a i n a f t e r a - a m a n i t i n a n d t h e cells r e - i n c u b a t e d f o r 6 h. Cells w e r e l a b e l e d f o r 1 0 r a i n w i t h [ 3 H ] u r i d i n e at t h e e n d o f t h e r e - i n c u b a t i o n p e r i o d ; t h e m e d i u m w a s r e m o v e d a n d ice-cold 0 . 3 2 M s u c r o s e - - 3 m M MgCI 2 a d d e d , a n d t h e cell m o n o l a y e r s c r a p e d f r o m t h e b o t t o m o f t h e p e t r i dishes. N u c l e i w e r e i s o l a t e d [ 1 4 ] f r o m cell h o m o g e n a t e s * * , p r e p a r e d in t h e s a m e s u c r o s e s o l u t i o n a n d [ 3 H ] u r i d i n e i n c o r p o r a t e d i n t o R N A p e r 1 0 r a i n w a s d e t e r m i n e d as d e s c r i b e d in M e t h o d s . Treatment
[ 3 H] U r i d i n e i n c o r p o r a t i o n
Control ~-Amanitin + control Etiocholanolone ~-Amanitin + etiocholanolone 2-Allyl-2-isoprop ylacetamide c~-Amanitin + 2 - a l l y l - 2 - i s o p r o p y l a c e t a m i d e * All a d d i t i o n s are p e r propylacetamide (30 ** Cells f r o m t w o p e t r i ** * N is n u c l e a r f r a c t i o n
cpm/0.5 ml of N***
pg DNA/0.5 ml of N
cpm//~g DNA
Incorporation
Inhibition
( M e a n -+ S.D.)
( M e a n +- S . D )
(Mean)
(%)
(%)
1984 + 188
3.0 -+ 0.2
661
100
--
3
4.3 + 0 . I
203
31
69
2 6 4 9 -+ 1 0 9 1 0 6 6 _+ 10
3.2 +- 0.1 4.3 _+ 0.2
836 246
100 29
-71
2522-+ II 7 5 0 -+ 31
3.4-+0.5 4.2 -+ 0.2
743 179
I00 24
-76
861 +
m l o f m e d i u m ; ~ - a m a n i t i n ( 0 . 5 # g ) ; e t i o c h o l a n o l o n e (5 p g ) a n d 2-aUyl-2-isopg). dishes were pooled for each homogenate. ( t o t a l v o l u m e = 5 m l f o r s a m p l e s ) ; m e a n a n d S.D. o f d u p l i c a t e d e t e r m i n a t i o n s .
TABLE VI EFFECT OF ~-AMANITIN ON [3H] URIDINE-LABELED
RNA IN THE CYTOPLASM
Cells f r o m c h i c k e m b r y o liver w e r e g r o w n a n d t r e a t e d as d e s c r i b e d i n T a b l e V. ~ - A m a n i t i n ( 0 . 2 5 / ~ g ) w a s a d d e d 1 h prior t o t h e i n d u c i n g a g e n t s a n d t h e cells r e - i n c u b a t e d f o r 5 . 5 h. A t t h e e n d o f t h e r e - i n c u b a t i o n p e r i o d , cells w e r e l a b e l e d w i t h [ 3 H ] u r i d i n e f o r 15 m i n a n d t h e m e d i u m r e m o v e d . T h e m o n o l a y e r w a s s c r a p e d f r o m t h e b o t t o m o f t h e p e t r i d i s h e s a n d n u c l e a r a n d c y t o p l a s m i c f r a c t i o n s i s o l a t e d f r o m cell h o m o g e n a t e s ; [ 3 H ] u r i d i n e i n c o r p o r a t e d i n t o R N A a f t e r 15 r a i n a n d p r e s e n t in t h e c y t o p l a s m w a s d e t e r m i n e d as d e s c r i b e d in T a b l e V. Treatment
Induction (h)
Protein (~ug/ml)*
[3H] Uridine-labeled R N A in c y t o p l a s m (cpm/ml)*
Control a-Amanitin + control Etiocholanolone 0eAmanitin + etiocholanolone 2-Allyl- 2 - i s o p r o p ylac etamide ~ - A m a n i t i n + 2-allyl-isopropylacetamide
Inhibition %
(cpm/~tg p r o t e i n )
5.5 5.5 5.5
2 4 5 +- 1 4 " * 1 4 2 -+ 5.3 1 8 3 -+ 11
4 6 1 -+ 1 6 " * 2 0 6 -+ 3 8 9 8 -+ 52
1.88 1.45 4.92
-23 --
5.5
178 +
3.5
4 0 4 +- 3
2.27
54
5.5
2 0 9 +- 4.9
1 2 6 9 -+ 59
6.07
--
5.5
1 3 7 -+ 1.8
211 -+ 33
1.55
75
* C y t o p l a s m i c f r a c t i o n ( t o t a l v o l u m e = 4 0 m l f o r all s a m p l e s ) . ** Mean-+ S.D.
341
Mn2+- (NH4)zSO,I ossoy
E
Mg 2+ OSSOy
Control
I00
/
Control
80 ~ - Am0nitln
~ ._> "6 o
6O
p
4O
o E 2
7 rr"
4
6
8
,
/(, /~q ONA
Fig. 3. I n h i b i t i o n o f t h e M n 2 + - - ( N H 4 ) 2 S O 4 - s t i m u l a t e d D N A - d e p e n d e n t R N A p o l y m e r a s e b y ~ - a m a n i t i n in v i t r o a n d r e q u i r e m e n t f o r a D N A t e m p l a t e b y b o t h e n z y m e s . Cells w e r e g r o w n as d e s c r i b e d in M e t h o d s a n d n u c l e i i s o l a t e d [ 1 4 ] f r o m c o n t r o l . C o n d i t i o n s f o r t h e R N A p o l y m e r a s e a s s a y s w e r e t h e s a m e as in T a b l e V I I e x c e p t t h a t c~-amanitin ( 0 . 2 ~tg) w a s a d d e d t o t h e r e a c t i o n m i x t u r e at t h e b e g i n n i n g o f t h e R N A p o l y m e r a s e assay.
To correlate with the inhibition of RNA synthesis by ~-amanitin shown in Table V, endogenous activities of RNA polymerase were also measured in nuclei from control and induced hepatocytes grown in the presence of ~amanitin and induced for 6.5 h. When ~-amanitin (0.2 pg/ml) was added 1 h prior to the inducing agents, the Mn2+--(NH4 )2 SO4 -stimulated RNA TABLE VII IN VIVO EFFECT OF (~-AMANITIN ON DNA-DEPENDENT AVIAN HEPATOCYTES
RNA POLYMERASE
IN NUCLEI FROM
Cells f r o m c h i c k e m b r y o livers w e r e g r o w n in p e t r i d i s h e s ( 1 5 0 m m d i a m e t e r ) f o r 24 h i n i t i a l l y . ~A m a n i t i n w a s a d d e d * 2 h a f t e r c h a n g i n g t h e m e d i u m a n d 1 h p r i o r t o a d d i t i o n o f i n d u c i n g a g e n t s . Cells w e r e r e - i n c u b a t e d f o r 6 . 5 h; a t t h i s t i m e t h e m e d i u m w a s r e m o v e d a n d t h e cells s c r a p e d f r o m t h e b o t t o m o f t h e p e t r i d i s h e s . N u c l e i w e r e i s o l a t e d [ 1 4 ] f r o m cell h o m o g e n a t e s p r e p a r e d in 0 . 3 2 M s u c r o s e - - 3 m M MgCI 2 f r o m t w o pctri dishes. D N A - d e p e n d e n t R N A p o l y m e r a s e activities of nuclei were d e t e r m i n e d and a r e e x p r e s s e d as c p m o f [ 8 - 1 4 C ] A M P i n c o r p o r a t e d i n t o R N A p e r /~g o f D N A ; i n c u b a t i o n s w e r e 10 a n d 4 5 r a i n a t 36 ° C f o r t h e Mg 2 ÷ a n d M n 2 + - - ( N H 4 ) 2 S O 4 a s s a y s r e s p e c t i v e l y . R a d i o a c t i v i t y i n c o r p o r a t e d i n t o n e w l y s y n t h e s i z e d R N A w a s d e t e r m i n e d as p r e v i o u s l y d e s c r i b e d [ 1 4 ] . T r e a t m e n t o f cells
DNA-dependent RNA polymerase activities of nuclei Mn2÷--(NH4)~SO4 assay
M g 2+ a s s a y
(cpm#tg DNA)
cpm/ktg D N A
Inhibition
(%) Control ~-Amanitin + control Etiocholanolone ~-Amanitin + etiocholanolone 2-Allyl-2-isopropylacetamide ~-Amanitin + 2-allyl-2-isopropylaeetamide
19.0
4.5 29.4 5.3 20.6
+ 0.08**
+ 0.15 -+ 6 . 4 + 0.28 -+ 0 . 6 4 ~5.1 + 1.24
Inhibition
(%)
--
16.9
+ 3.5**
--
76 -82 -75
20.4 15.3 16.0 14.8 22.8
+ 0.23 + 0.56 + 1.1 -+ 2.1 + 0.74
None -None --
None
* All a d d i t i o n s a r e p e r m l o f m e d i u m , ~ - a m a n i t i n ( 0 . 2 / ~ g ) ; e t i o c h o l a n o l o n e ( 1 0 ~tg) a n d 2-allyl-2-isoprop y l a c e t a m i d e (30/~g). ** M e a n s a n d S . D . o f d u p l i c a t e d e t e r m i n a t i o n s .
342 polymerase activities of control and induced cell nuclei were inhibited 76, 82 and 68% respectively, whereas the Mg 2+ activities were not inhibited, and even showed some enhancement (Table VII). This very significant depression of Mn2÷--(NH4 )2 SO4-stimulated R N A polymerase by a-amanitin in avian tissue confirms prior reports that the nucleoplasmic polymerases are markedly inhibited by the toxin in vitro (as shown in Fig. 3) and in vivo [28]. The results obtained in Table VII with the Mg2+-stimulated activity, corresponding to the nucleolar polymerase, do not show any inhibition and these data differ from those of others who show inhibition of rRNA in vivo by a-amanitin [28,29]. To further substantiate the increase in R N A synthesis observed in induced cultures (i.e. for the steroid treated 27% above control, Table V) enhancement of the Mn2+--(NH4)2SO4-stimulated activity of the nucleoplasmic R N A polymerase was also found in nuclei isolated from etiocholanolone-treated hepatocytes (Table VII); the increase from 19.0 to 29.4 cpm/pg DNA represents a change of 55% over control value in this particular experiment, a very significant enhancement which did not occur in the R N A polymerase activity of the 2-allyl-2-isopropylacetamide-treated nuclei. The Mg2÷-stimulated RNA polymerase of both control and induced nuclei, isolated from hepatocytes not treated with a-amanitin, showed no significant changes in activities b u t the a-amanitin-treated samples showed a certain pattern of increase, most pronounced in the nuclei isolated from the barbiturate-treated hepatocytes. Discussion
a-Amanitin is a p o t e n t inhibitor of the DNA-dependent polymerase in mammalian liver and other tissues [8,9,30--32] ; and as shown in these studies the toxin markedly inhibits the enzyme in cultured avian hepatocytes as well. The induction of 5-aminolevulinate synthetase by a 5~-steroid or 2-allyl2-isopropylacetamide was strongly inhibited by a-aminitin in avian liver cells grown in primary cultures. This inhibition was most pronounced at a toxin concentration of 1 pg/ml of medium reaching a level of inhibition of 87%, confirming our earlier observations on a-amanitin inhibition of the induction of this enzyme in vivo [ 1 0 ] . Its inhibitory action with respect to chemicalinduced porphyrinogenesis is presumably a secondary effect, resulting from inhibition of the induction of 5-aminolevulinate synthetase. The pronounced ability of a-amanitin to block induction of 6-aminolevulinate synthetase suggests that the synthesis of new R N A is a necessary step in the de novo formation of this enzyme. The toxin inhibited 69 to 76% the incorporation of [3 H] uridine into RNA at the low concentration of 0.5 pg/ml of medium, eliminating the increased uptake of this precursor observed in both steroid- and drug-induced nuclei isolated from hepatocytes cultured in the absence of the toxin. These findings indicate that DNA transcription by RNA polymerase is required for the induction of d-aminolevulinate synthetase, and that certain area(s) of the genome are transcribed into new species of R N A as a result of the action of these inducer agents. Such a process is an initial step in the induction mechanism for this enzyme as shown here and occurred 14--16 h prior to full induction of the protein. The induction was eliminated in the absence of R N A synthesis, or its magnitude greatly reduced depending on the
343
concentration of the toxin used. The inhibition produced by low doses of a-amanitin, as used in our studies, is quite significant since protein synthesis was not inhibited concomitantly; on the contrary it was slightly enhanced and more significantly so in the induced cultures even though 5-aminolevulinate synthetase formation was markedly decrease at 18--24 h after addition of the inducers. In keeping with these observations, a-amanitin inhibited in vivo the Mn2÷--(NH4 )2 SO4 -stimulated nucleoplasmic DNA-dependent RNA polymerase in nuclei isolated from control and induced hepatocytes b u t to different degrees. This enzyme is responsible for the synthesis of heterogeneous nuclear RNA~ a precursor molecule from which m R N A is cleaved upon transfer to the cytoplasm [ 3 3 ] . This R N A polymerase is very sensitive to the toxic effect of ~-amanitin in vitro [ 3 4 ] , suggesting therefore that ~-amanitin inhibited the synthesis of m R N A in the toxin-treated cells. As shown in the [3 H]uridine incorporation studies of Table V, the increased activity of this R N A polymerase in the steroid-treated nuclei was not present in the drug-induced nuclei of Table VII; however in the cytoplasm of both steroid- and barbiturate-treated hepatocytes (Table VI), an increase in labeled R N A species was present and inhibited when the toxin was added to the culture medium. This inhibitory effect on the enhancement of R N A polymerase activity coincides in time with the inhibition observed in the enhancement of R N A synthesis found after induction. The inhibition was only manifest in the R N A polymerase present in the nucleoplasm and n o t in the nucleolar enzyme. The latter is stimulated by Mg 2÷ and was n o t inhibited by a-amanitin when examined in our studies at 6 or 6.5 h after the start of the induction period; however in a related experiment, this polymerase was inhibited slightly {20--30%} at 1.5 h after addition of the inducers b u t not at 5 h. In fact a slight tendency for an increase in its activity was evident in the control and barbiturate-induced nuclei. This could suggest that in the a-amanitin-treated nuclei in vivo a controlling factor, translated from an RNA species synthesized by the nucleoplasmic RNA polymerase, regulates the activity of the nucleolar polymerase in synthesizing pre-rRNA, this protein factor being eliminated by the inhibitory effect of a-amanitin on the nucleoplasmic enzyme [ 2 8 ] . The synthesis of another t y p e of RNA, possibly rRNA, not sensitive at the time studied (6.5 h} to a-amanitin and n o t involved with the triggering of the induction process for 5-aminolevulinate synthetase could be stimulated by the barbiturate inducer, particularly if the Mg2+-stimu lated R N A polymerase had a short half life. This would correlate with the slight stimulation of protein synthesis observed in the a-amanitin-treated induced cultures, an effect which is present only at the latter part of the induction period. Coproporphyrins which have accumulated in the induced cells and their culture medium have also been shown to inhibit protein synthesis [ 1 1 ] , and the prevention of their formation by low doses of a-amanitin restores protein synthesis to control values or values even slightly higher than control, as shown in Table IV. There is therefore only a slight difference evident between the actions of the inducers examined in this study; the difference being manifested for the steroid in the magnitude of the response for stimulation of the nucleoplasmic RNA polymerase and for the barbiturate in the nucleolar enzyme. 2-allyl-2-
344
isopropylacetamide may act post-transcriptionally in addition to transcriptionally since there appear to be several sites of control for enzyme induction in mammalian tissues. Our findings indicate, however, that continued m R N A synthesis is necessary for enzyme induction b y both inducers, even though both may not directly stimulate m R N A synthesis. It is possible also that induction proceeds via stabilization of, or faster translation of m R N A s synthesized at the same rate as in the absence of inducers. The data available here do not permit a choice between these alternatives. Acknowledgements We wish to thank Mrs Maureen Morgan and Miss Mary Horan for skilled technical assistance and Miss Ann Marie Quatela for typing the manuscript. This work was supported by Grant No. HD-04313 from the National Institutes of Health. References 1 Granick, S. and Kappas, A. (1967) J. Biol. Chem. 242, 4587 2 Schmid, R. and Schwartz, S. (1952) Proc. Soc. Exp. Biol. Med, 8 1 , 6 8 5 3 Goldberg, A. and R i m i n g t o n , C. (1962) Diseases of Porphyrin Metabolism, Charles C. Thomas, Springfield 4 Granick, S. and Urata, G. (1963) J. Biol. Chem. 238, 821 5 Kappas, A., Song, C.S., Levere, R.D., Saehson, R,A. and Granick, S. (1968) Proc. Natl. Acad. Sci. U.S. 61, 509 6 Graniek, S. (1966) J. Biol. Chem. 241, 1359 7 Sassa, S. and Granick, S. (1970) Proc. Natl. Acad. Sci. U.S. 67, 517 8 Wieland, T. (1969) Science 159, 946 9 Jacob, S.T., Sajdel, E.M. and Munro, J.N. (1970) Nature, 225, 60 10 Incefy, G.S. and Kappas, A. (1971) FEBS Lett. 15, 153 11 Incefy, G.S. and Kappas, A. (1972) FEBS Lett. 23, 37 12 Incefy, G.S. and Kappas, A. (1971) Fed. Proc. 30, 1302 13 Kappas, A., Song, C.S., Sassa, S., Levere, R.D. and Graniek, S. (1969) Proe. Natl. Aead. Sci. U.S. 64, 557 14 Incefy, G.S. and Kappas, A. (1971) J. Cell Biol. 50, 385 15 Kissane, M.J. and Robins, E. (1958) J. Biol. Chem. 2 3 3 , 1 8 4 16 Hinegardner, R.T. (1971) Anal, Biochem. 3 9 , 1 9 7 17 Poland, A. and Glover, E. (1973) Science 179, 476 18 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randal, R.J. (1951) J. Biol. Chem., 193, 265 19 Widnell, C.C. and Tata, J.R. (1966) Biochim. Biophys. Aeta 123, 478 20 Incefy, G.S. and Kappas, A. (1974) Proc. Natl. Acad. Sci. U.S., in press 21 Doss, M. and Kaltepolh, B. (1971) in Proceedings of the I n t e r n a t i o n a l Conference on P orphyri n Metabolism and the Porphyrias (South African Medical J.) 25 September, p. 73, Special issue S.A.J. Lab. and Clin. Med. 22 Stirpe, F. and Fiume, L. (1967) Bioehem. J. 105, 779 23 Buku, A., Campadelli-Fiume, G., Flume, L. and Wieland, Th. (1971) FEBS Lett. 14, 42 24 Ponta, H., Ponta, U. and Winterberger, E. (1972) Eur. J. Biochem. 29, l l O 25 Seifart, K.H. and Sekeris, C.E. (1969) Z. Naturforsch. 24b, 1538 26 Strand, L.J., Manning, J. and Marver, H.S. (1972) J. Biol. Chem. 247, 2820 27 Willems, M., Penman, M. and Penman, S. (1969) J. Cell Biol. 4 1 , 1 7 7 28 Tata, J.R., Hamilton, M.J. and Shields, D. (1972) Nat. New Biol. 238, 161 29 Bucci, S., Nardi, I., Maneino, G. and Flume, L. (1971) Exp. Cell. Res. 69, 462 30 Lindell, T.J., Weinberg, F., Morris, P.W., Roeder, R.G. and Rutter, W.J. (1970) Science 170, 447 31 Kedinger, C., Nuret, P. and Chambon, P. (1971) FEBS Lett. 15, 169 32 Dezelee, S., Sentenae, A. and Fromageot, P. (1970) FEBS Lett. 7 , 2 2 0 33 Jelinek, W., Adesnik, M., Salditt, M., Sheiness, D., Wall, R., Molloy, G., Phillipson, L. and Darnell, J.E. (1973) J. Mol. Biol. 75, 515 34 Kedinger, C., Gissinger, F., Gniazdowski, M., Mandel, J.L. and Chambon, P. (1972) Eur. J. Biochem. 28, 269