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Effects of cortisone and actinomycin-D upon pyrimidine nucleotide and RNA metabolism in rat liver
ARCHIVES
Effects
OF
BIOCHEMISTRY
AND
of Cortisone
BIOPHYSICS
and
and
152-157 (1969)
Actinomycin-D
RNA Metabolism
FU-LI Departments
l%,
YU
AND
PHILIP
upon
Pyrimidine
Nucleotide
in Rat Liver’ FEIGELSONZ
of Biochemistry and Medicine, and the Institute for Cancer Research, College of Physicians and Surgeons, Columbia University, New York, New York 100% Received
July
15, 1968; accepted
August
27, 1968
The effects of cortisone and actinomycin-D on the incorporation in vivo of labeled erotic acid into acid-soluble nucleotides and RNA of rat liver were studied. It was found that more than half of the stimulatory effect of cortisone on isotope incorporation into RNA was a reflection of an increased specific radioactivity of the nucleotide precursors in the acid-soluble pool. The specific activity of the acid-soluble uridine nucleotides remained a constant fraction of the elevated total acid-soluble radioactivity after cortisone treatment suggesting that the hormone may have accelerated orotate transport into the liver. Analogously, but the opposite direction, the major portion of the actinomycin-D inhibition of orotate-3H incorporation into RNA was due to an actinomycin-D-mediated diminution in the specific activity of the acid-soluble pyrimidine nucleotide pool. Pretreatment of animals with actinomycin-D resulted in complete elimination of the cortisone enhancement of radioactive precursor incorporation into RNA, whereas the hormonally enhanced incorporation of 3H-orotate into the acid-soluble fraction of liver was not inhibited suggesting that t.his latter consequence of hormone action was not dependent on transcriptional processes. Actinomycin-D, at levels that inhibit hormonal enzyme induction, did not markedly modify the hepatic uptake or subcellular distribution of 3H-cortisone.
Glucocorticoids manifest divergent effects in various target organs, among which is its specific stimulatory effect on precursor incorporation into liver RNA (l-4). Although extensive studies have been undertaken in various laboratories in recent years, the nature of the basic mechanism underlying this hormonally modified biochemical process remains controversial and obscure. Some investigators have emphasized that this hormone stimulates the synthesis of specific mRNA (5, S), whereas others have presented evidence of enhanced isotopic incorporation into all subcellular orga,nelles and into all types of RYA, 1 This study was supported by Research Grants CA 02332 and CRTY 05011 from the U.S.P.H.S. 2 Career Investigator of the Health Research Council of the City of New York (I-104).
including rRNA, tRNA, and the DNA-like RNA (l-4, 7, 8). Marked differences, however, in the magnitude of the hormonally enhanced incorporation into RNA were unexpectedly observed depending upon the nature of the labeled precursor employed (1). This may be due to either an hormonal modification in the base ratio of the hepatic RNA or to differential hormonal modulation of acid-soluble nucleotide metabolism. Indeed, accelerated acid-soluble purine nucleotide biosynthesis occurs in the livers of animals that have received glucocorticoids (9, 10). Hence, studies on isotope incorporation into RNA that neglect the effects of the hormone on the specific activit’y and size of these nucleotide pools, which are the precursors of RNA, are ambiguous. The present study is intended to evaluate the 152
CORTISONE
AND
ACTTNORIYCIN-D
effects of the glucocorticoids on pyrimidine nucleotide metabolism and to consider its relationship to hormonal regulation of RNA synthesis. It was found that more than half of the hormonally accelerated labeled orotat’e incorporation into hepatic RNA was a reflection of increased specific activit’ies of pyrimidine precursors in the acid-soluble pool. These findings prompted exploration of the effects of actinomycin-D on acid-soluble pyrimidine metabolism as well as upon RNA synthesis (11) and on the glucocort,icoidal enhancement of these processes in the presence of the anbibiotic. These studies indicate that actinomycin-D itself markedly lowers the specific activity of the hepatic acid-soluble pools that are precursors to RKA. Furthermore, actinomycin-D did not prevent hormonal effect,s upon acid-soluble pyrimidine nucleotide metabolism. MATERIALS
AND
i\lETIIODS
AIale Sprague-Dawley rats of 250-300 g body weight were bilaterally adrenalectomized and maintained t)hereafter on O.S(i;l saline, water, and Prlrina Chow ud libitum for 557 days. Prior to the tracer experiments the animals were starved overnight. All injections were made intraperitoneally. Cortisolle-acetate snspension (Upjohn Co.) 1 or 5 mg/lOO g body weight was given 4 hr prior to sacrifice. Actinomycirl-D (kindly donated by the nlerck Sharp & I>ohme Co.) dissolved in 0.9yc N&l, was rontinely injected at 50 rgjlO0 g body weight 1 hr prior to the hormone injection. However, in the experiment in which the transport and srlbcellldar distriblltion of cortisone was studied, 100 pg actitlomycilr-I)/100 g body weight was administered 2 hr before unlabeled cortisone (5 mg/lOO g body weight,) containing 5 PCi of cort,isane-1,2-“TI (34.4 Ci/mM, New England Nuclear). The animals were sacrificed after the indicated incorpration periods (Table IV). Whelr indicated, orotir-6-14C acid (6.5 mCi/mM, New England Nllcelar Corp.) or orotic-3Tf acid (1 Ci/mM, l’olk Co.) at 12.5 PCi and 40 &i/100 g body weight respectively were itljected. III t,hese experiments the radioisotope incorporation period was 20 min. The animals were sacrificed by exanguinatioll, the livers rapidly transferred to chipped ice, homogenized, and s~~bcellrllar components and RNA were isolated as previorlsly described (12, 13). The RNA coIltent was determined according to AIllrrro and Fleck (14) asslIming that all absorbatIce of 1.0 at 260 rnp with a l-cm light pat,h corre-
EFFECTS
ON POOLS
15.3
sponds to 32.0 pg RSB/ml (14). The total acidsoluble fraction was obtained, either by mixing the liver homogenate with an equal volume of cold 0.4 M perchloric acid or by homogenizing the liver directly in 3 vol of cold 0.27 M perchloric acid. After centrifugation, the precipitate was re-extracted twice with 0.2 M perchloric acid and the combined supernates constituted the total acid-soluble fraction. The total acid-soluble uridine nucleotides were isolated as IJRIP, after acid hydrolysis, by chromatography on successive Dowex-50 and Dowex-2 columns (10). RNA-uracil was isolated on Dowex-50 by Cohn’s method (15) after hydrolysis in 70y0 perchloric acid (16). Radioisotopic scintillation counting procedures were as previously described; all values presented were corrected for quenching (10). RESULTS
Earlier studies in this laboratory revealed that cortisone stimulated both inorganic 32P and glycine-2-‘4C incorporation into the Rn’A of all subcellular components (2, 13). We now inquired as to whether this would also be true for a precursor specific for pyrimidinex and whether actinomycin-D might exert’ a selective antagonism toward the hormonally accelerated synthesis of the RNA associated with a particular subcellular entity. It’ is evident from Ta,ble I that incorporation of pyrimidine nucleotide precursors into the RNA of all subcellular components are similarly stimulated by cortisone albeit to a quantitatively lesser extent’ than previously observed using glycine-2-14C or 32P (1, 1X). As anticipated, actinomycin-D inhibited incorporation into t)he RSA of all subcellular fractions. A small, but consistent, hormonal elevation in the RNA specific activity of all fractions was observed in the presence of levels of nctinomy&-D known t,o prevent enzyme induction by cortisone (17). This apparent discrepancy is due to the complex and antagonist’ic effects of cortisone and act,inomycin-D upon acid-soluble pyrimidine nucleotide metabolism as well as upon RNA synthesis. Cortisone caused a 10% and 1‘25%increase respectively in the total and the specific radioactivitv of the acid-soluble precursor pool of the fiver and a 29 % stimulation in t)he specific activity of hepatic RNA (Table II). The hormonally
154
YU AND
FEIGELSON
TABLE
I
EFFECTS OF COILTISONE AND ACTINOMYCIN-11 ON OltoTIc-(j-W ARID INCORPORATIOX INTO THE RNA OF SUBCELLULAR FRACTIONS OF RAT LIVER” Actinomycin-D
Nuclear
Cortisane
-
-
+ +
+ +
Mitochondrial
RNA
cdw
33,400 41,800 7,600 9,800
9’
f * f f
4,100 5,500 1,000 1,700
100 125 23 29
RNA
301 405 88 100
Microsomal
%
cpmimg
f zt f zk
30 49 14 23
100 135 29 33
RXA
cpm/mg
2,080 2,310 2G4 325
f zt f +
Cell sap RNA
150 300 23 110
%
cpm/w
%
100 111 13 16
5,550 6,030 761 896
l f zk f
450 1160 40 269
100 109 14 In
Q Actinomycin-D (50 rg/lOO g body weight), cortisone (1 mg/lOO g body weight) and erotic-6-W (12.5 pCi/lOO g body weight) were respectively injected at 5 hr, 4 hr, and 20 min prior to sacrifice. Each value depicted is the mean + standard error for three animals. TABLE
II
THE EFFECTS OF COHTISONE ANI) ACTINOMYCIN-D ACID-W INTO THE HEPATIC ACID-SOLUBLE Cortisone (1 n&loo g body
Actinomycin-D (50 afmo g body wt)
wt)
-
-
To Cortisone
+ + stimulation
8,260 10,600 1,590 1,960
+ + +
7GActinomycin-D
RNA
f f f f
50 900 80 290
4,740 5,300 2,110 3,290
f f zk f
280 300 80 300
585 G43 274 398
f f f f
28.3 23.3
11.8 55.9
9.9 45.3
80.8 81.6
55.5 37.9
53.2 38.1
18 31 8 39
125 122 130 121
f zk f f
6 4 3 2
inhibition
+ a Values
Total m/mg
ON THE INCOHTORATION OF OROTIC PRECURSOR Poor. AND RNA”
are given
as means f
standard
error
for 3 animals.
increased specific activity of hepatic RNA is therefore the result of two factors: that derived from t,he increased specific activit,y of the precursor pool and that derived from hormonal acceleration in nucleotide polymerization to form RNA. Actinomycin-D diminished the specific activity of the acidsoluble pool by 56% and that of the RNA by 81% indicating a net actinomycin-D inhibition of RNA synthesis of but 25 %. Thus, this dose of actinomycin-D apparently exerts a greaker inhibitory effect upon acid-soluble precursor transport and/or metabolism than upon RNA synthesis. Table II also indicates that neither cortisone, nor actinomycin-D under these conditions, led to detectable alterations in the tissue concentration of nucleotides present. Actinomycin-D exerted differential eff ect>s upon the cortisone-modified acid-soluble and
RNA metabolism. The hormonal increment in acid-soluble radioactivity is not impaired by pretreatment of the animals with act.inomycin-D. However, upon correction for acid-soluble pool effects, it is evident that actinomycin-D completely prevented hormonal enhancement of nucleotide condensation into RKA. Thus, cortisone enhancement of precursor incorporation into hepatic acid-soluble pyrimidine nucleotides does not seem to be dependent upon transcriptional processes. To permit, more precise evaluation of the hormonal effects, the specific activities of RNA-uracil and of its uridine nucleotide precursors in the acid-soluble pool were directly determined (Table III). The results indicate this dosage of cortisone elicits a 54% increase in specific activity of total RNA, and a 39 % increase in specific activity
CORTISO?r’E
AND
ACTINO~IYCIN-D
EFFECTS
TABLE THE
III
EFFECTS OF CORTISONE UPON THE INCORPORATION OF OROXC ACIU-“II ACID-SOLUBLE URI~INE NUCLEOTII)ES AND INTO RNA-URKIL Specific radioactivity
-
are expressed
1,XiO 1,116 1,490 GO,800
OF THE
as means + standard
EFFECT
error
* The values
120 74 120 7,200
2,250 *
150
54
1,517
20
39 87
*
2,790 + l-20 89,200 It 5,300
47
9.0 f 0.1 120 z!z 1 1.0 + 0.1
for 4 animals. I\’
OF ACTINOMYCIN-D ON HEPATIC DISTRIBUTION OF C~RWSONE-1,
Homogenate
Actinomycin-D 1Iinutes incorporation 15 30 45 GO
‘,q; G&on
+
+ zt f I!C
9.5 f 0.3 123 zt 1 1.1 + 0.1
liver)
TABLE STIJDY
INTO
Cortisone acetatea (5 mg/lOOg body wt)
RNA (cpm/mg) Total acid-solltble fraction (cpm/ODnso,+) RNA-llracil (Cpm/~mole) Acid-soluble UMI’ (Cpm/pmole) Amount BKA (mg/g liver) Total acid-solnble fraction (ODZW,,,~units/g .4cid-soluble U1IP &mole/g liver) a Yalltes
155
ON POOLS
UPTAKE
AND
SCBCELLGL~R
2-311e
Soluble
Suclei
hlicrosomes
Mitochondria
-
+
-
+
-
+
-
+
-
+
129,000 102,000 78,000 67,000
105,000 105,000 81,000 70,000
105,000 87,000 69,000 56,000
89,000 90,000 73,000
1990 1570 1180
7000 5320 4050
57,000
B90
1950 1390 1330 750
6200 5800 5100 4300
1900 2400 2300 2600
3100 2900 2200 2400
depicted
are averages
for duplicate
of the total acid-soluble pool. When uracil was isolated from each component the hormonal effect was an S7 9% and a 47 % elevation in the specific activit.ies of t’he RNA-uracil, and acid-soluble uridine nucleotides respectively. Thus, approximately half of the hormonally elevated specific activity of RNA represented hormonal enhancement of RNA synthesis; the remainder may be attributed to hormonal elevation in t,he specific activity of the uridine nucleotide pool t’hat is the precursor of the R?;A-uracil. The size of the acid-soluble pool, expressed either as OD,G, or as acidsoluble UMI concentration remained urlchanged, nor was there an increase in the absolute amount of hepat’ic RiNA 4 hr after cortisone treatment. An experiment was undertaken to explore the possibility that actinomycirl-D might inhibit hormonal action by an inhibition of hepat,ic hormonal uptake and/or prevention
3700
animals.
of its binding to an appropriate receptor site in the target cell. The results of such a study are shown in Table IV. In the control animals, the maximal incorporation of radioactive cort’isone occurred at 15 min, 82% of the hepatic radioactivit#y was recovered in the cell sap, 5% in the microsomes, and 2% in each of the nuclear and mitochondrial fractious. After the incorporation peak at 15 min, the isotope disappeared from the liver with a Tl/z of approximately 1 hr. There was a parallel loss of radioactivity from the cell sap, the microsomal, and the nuclear fract’ions, but 110 loss of radioactivity from the mitochondrial fraction during the period studied. Animals pretreated w&h 100 pg actinomycin-D/100 g body weight 2 hr prior to labeled cortisone administration manifested a similar patt’ern for the uptake, distribution, and loss of the co&one-l, %-“H; the sole differrnce being t.hnt the maximal period of incorporation \vas somc-
156
YU AND
what broader, plateauing during the l&30min interval. Thus, actinomycin-D did not markedly hinder the transport of radioactive steroid into the liver, nor did it significantly influence its gross subcellular distribution thereafter. The possibility that actinomytin-D might act to prevent hormonal action by virtue of impaired hormonal transport’ int’o the liver therefore is seemingly obviated.
FEIGELSON
indeed involves newly made RNA. Furthermore, the specific activity of other types of RNA, including rRNA and tRNA, were elevated simultaneously with that of the mRNA. One confronts an apparently nonspecific stimulation of RNA synthesis yet select’ive induction of certain liver enzymes by the glucocorticoid hormones. Furthermore, although isotopic incorporation rates int,o various subcellular RNA fractions were similarly enhanced in response to cortisone, L)IHCUSSION greater differences in response were observed depending upon the nature of the The use of isotopically labeled organic tracers employed : glycineJ*C, which meascompounds provides unambiguous evidence ures purine nucleotide synthesis, showed a for t,he existence of metabolic routes by which one compound is the precursor of 255 % increased incorporation into hepatic another. However, the l.n vivo evaluation of RNA 4 hr after hormone treatment, whereas under similar conditions, inorganic-32P inthe rate at which these metabolic interconcorporation augmented 124 % and orotic-3H, versions occur is fraught with interpretive which measures the pyrimidine nucleotide difficulties. Experiment#ally, one administers a labeled compound and evaluates the pathway, registered only 38 % above the extent of labeling in the metabolite of in- control levels (1, 13). The recognition that glucocorticoids stimulated the incorporaterest aft’er a given incorporation period. tion of labeled glycine into acid-soluble However, a precursor must be transported adenine provided a measure of clarification into the cell, converted to various interindicating that much of the increased radiomediates, and subjected to isotopic dilution activity in the RNA was a reflection of the in various pools en route to t,he formation hormonally enhanced specific activity of the of the metabolite ultimately isolated. purine nucleotide pool (10). The present experimental and physiological Clearly, manipulations may influence any of the study indicates that cortisone treatment leads to parallel elevations of radioactivity processes occurring between the labeled of both the total hepatic acid-soluble fraccompound administered and the product tion and of the acid-soluble uridine nucleoultimately measured. The first reports that tides. This observation is compatible with glucocorticoids markedly stimulate the incorthe view that cortisone enhances the transporation of various precursors into hepatic port of labeled erotic acid into the liver RNA (12, 13) apparently related hormonal action to transcription of the genome and cell, without altering its rate of conversion has been reconfirmed and extended in to UMP. The increased specific activity of this precursor pool is likewise ultimately numerous laboratories (6, 8, 15-20). Howreflectfed in the RNA. Using erotic acid, ever, t,he hormonally stimulated synthesis this pool effect was responsible for approxiof several enzymes, e.g., tryptophan oxygenase (21) and tyrosine transaminase (22), mately one half of the hormonally elevated hepatlc RNA specific activity; one half of with its relat,ively minor accompanying the stimulation was apparently due to an stimulation in over-all protein synthesis, hormonally stimulated rate of RNA syntheseemed incompatible with the several-fold hormonal increase in the specific activity of sis. It is well established that actinomycin-D hepat,ic RNA, which was unlikely to be inhibits RNA synthesis by virtue of its bindmerely the reflection of t)he enhanced synthesis of a few species of mRNAs. Yet the ing with the deoxyguanosine moieties of the thereby preventing transcription process of hormonal enzyme induction has DNA, (11). However, effects of actinomycin-D on been demonstrated to be actinomycin-Dother than transcriptional processes have sensitive (17), suggest.ing that the induction
CORTISONE
AND
ACTINOMYCIN-D
been reported (23). The present study suggests that actinomycin-D may inhibit certain transport processes within the liver. It is of furt’her interest t)hat the apparent 81 ‘Y inhibit,ion of RXA synthesis resulting from act,inomycin-D treatment is in reality merely a 25 % inhibition when corrected for the elect’ of actinomycin-D upon the specific activity of the acid-soluble pool. Thus, in an acute experiment, 500 pg actinomycin-D per kilogram permits 75 % of t,he hepatic Iir\TA synthesis to proceed. The observed sensitivity of hormonal enzyme induction to act,inomycin-D inhibition may indicate that the species of RNA involved are uniquely sensitive to the action of actinomy&l-D. It is also evident that proper eva,luation of the effects of actinomycin-D upon RNA synthesis can be made only with concomitant exploration of the effect of this agent up011 acid-soluble nucleotide metabolism. The differential effect of actiuomycin-D 011 the hormonal stimulation of RXA synthesis and on the transport and/or metabolism of orot’ic acid into the liver imply the latter hormonal effect is independent of transcriptional processes.
EFFECTS
5. 6. 7.
8. 9. 10. 11.
12. 13. 14. 15.
16. ACKNOWLEDGMENT The Marilyn
excellent Stevens
technical is gratefully
assistance of acknowledged.
Miss
17.
18. REFERENCES 1. FEIGELSON, III., AND FEIGELSON, P., in “Advances in Enzyme Regulation” (G. Weber ed.), 1.01. III, p. 11. Macmillan (Pergamon) New York (1965). 2. FEIGELSON, P., FEIGELSON, IN, AND GREENGARD, O., Recent Prog. Hormone Res. 18, 491 (1962). 3. FEIGELSON, P., AND FEIGELSON, M., in (‘ACtions of Hormones on Molecular Processes” (G. Litwack and D. Kritchevsky, eds.), p. 218. Wiley, New York (1964). 4. FEIGEISON, P., ANI)FEIGEI,SON,M., in “Rlech-
19. 20.
21. 22. 23.
Oh’ POOLS
157
anisms of Hormone Action” (P. Karlson ed.), NATO Advanced Study Institute, p. 24G. Academic Press, Xew York (1965). KIDSON, C., AND KIRBI-, K. S., Nalzcre 203, 599 (1964). SCHMID, W., GALLIVITZ, D., AED SEKERIS, C. C., Biochim. Biophys. 11&a 134, 80 (19G7). KENNEY, F. T., WICKS, W. I)., AND GREENMAN, D. L., Cellular Comp. Physiol. 66, Suppl. I., 125 (1965). DRETVS, J., AND BRA~VERMAN, G., J. Hiol. Chem. 242, 801 (1967). FEIGELSON, P., AND FEIGELSOX, II., J. Biol. Chem. 238, 1073 (1963). FEIGELSON, M., AND FEIGELSOX, P., J. Biol. Chem. 241, 5819 (1966). REICH, E., AND GOLDBERG,I. II.,in “Progress in Nucleic Acid Research and Molecular Biology” (J. !V. Davidson, and W. E. Cohn eds.), Vol. III, p. 184. Academic Press, New York (1964). FEIGELSON, P., FEIGELSON,&~.,AND FANCHER, C., Biochim. Biophys. Ada 32, 133 (1959). FEIGELSON,&I., GROSS,P.R., ANDFEIGELSON, P., Biochim. Biophys. Scta 55, 495 (1962). MUNRO, H. Xi., AND FLECK, A., Analyst 91, 78 (1966). COHN, W. E., in “The Nucleic Acids” (E. Chargaff, and J. N. I>avidson eds.), Vol. I, p. 211. Academic Press, New York (1955). MARSHAK, A., AND I-OGEL, H. J., J. Biol. Chem. 189, 597 (1951). GREENGARD, O., AND Acs, G., Biochim. Biophya. Ada 61, 652 (1962). BARNABEI, O., AND SEKENI, F., Biochim. Biophys. Acta 91, 239 (1964). GSRREN, L. D.,Ho\vELL, R.R., ANDTOMKINS, G. M., J. Mol. Biol. 9, 100 (1964). KENNEY, F.T., GREENMAN, D.L., WICKS, W. D., AND ALBKITTON, W. L., in “Advances in Enzyme Regulation” (G. Weber, ed.), 1’01. III, p. 1. Macmillan (Pergamon), New York (19G5). KNOX, W. E., Brit. J. Exptl. Palhol. 32, 462 (1951). LIN, E. C. C., AND KNOX, W. E., Biochim. Biophys. Beta 26, 85 (1957). SINGEH, M. F., AND LEDER, P., ilnn. Rev. Biochem. 36, 195 (1966).
Effects
OF
BIOCHEMISTRY
AND
of Cortisone
BIOPHYSICS
and
and
152-157 (1969)
Actinomycin-D
RNA Metabolism
FU-LI Departments
l%,
YU
AND
PHILIP
upon
Pyrimidine
Nucleotide
in Rat Liver’ FEIGELSONZ
of Biochemistry and Medicine, and the Institute for Cancer Research, College of Physicians and Surgeons, Columbia University, New York, New York 100% Received
July
15, 1968; accepted
August
27, 1968
The effects of cortisone and actinomycin-D on the incorporation in vivo of labeled erotic acid into acid-soluble nucleotides and RNA of rat liver were studied. It was found that more than half of the stimulatory effect of cortisone on isotope incorporation into RNA was a reflection of an increased specific radioactivity of the nucleotide precursors in the acid-soluble pool. The specific activity of the acid-soluble uridine nucleotides remained a constant fraction of the elevated total acid-soluble radioactivity after cortisone treatment suggesting that the hormone may have accelerated orotate transport into the liver. Analogously, but the opposite direction, the major portion of the actinomycin-D inhibition of orotate-3H incorporation into RNA was due to an actinomycin-D-mediated diminution in the specific activity of the acid-soluble pyrimidine nucleotide pool. Pretreatment of animals with actinomycin-D resulted in complete elimination of the cortisone enhancement of radioactive precursor incorporation into RNA, whereas the hormonally enhanced incorporation of 3H-orotate into the acid-soluble fraction of liver was not inhibited suggesting that t.his latter consequence of hormone action was not dependent on transcriptional processes. Actinomycin-D, at levels that inhibit hormonal enzyme induction, did not markedly modify the hepatic uptake or subcellular distribution of 3H-cortisone.
Glucocorticoids manifest divergent effects in various target organs, among which is its specific stimulatory effect on precursor incorporation into liver RNA (l-4). Although extensive studies have been undertaken in various laboratories in recent years, the nature of the basic mechanism underlying this hormonally modified biochemical process remains controversial and obscure. Some investigators have emphasized that this hormone stimulates the synthesis of specific mRNA (5, S), whereas others have presented evidence of enhanced isotopic incorporation into all subcellular orga,nelles and into all types of RYA, 1 This study was supported by Research Grants CA 02332 and CRTY 05011 from the U.S.P.H.S. 2 Career Investigator of the Health Research Council of the City of New York (I-104).
including rRNA, tRNA, and the DNA-like RNA (l-4, 7, 8). Marked differences, however, in the magnitude of the hormonally enhanced incorporation into RNA were unexpectedly observed depending upon the nature of the labeled precursor employed (1). This may be due to either an hormonal modification in the base ratio of the hepatic RNA or to differential hormonal modulation of acid-soluble nucleotide metabolism. Indeed, accelerated acid-soluble purine nucleotide biosynthesis occurs in the livers of animals that have received glucocorticoids (9, 10). Hence, studies on isotope incorporation into RNA that neglect the effects of the hormone on the specific activit’y and size of these nucleotide pools, which are the precursors of RNA, are ambiguous. The present study is intended to evaluate the 152
CORTISONE
AND
ACTTNORIYCIN-D
effects of the glucocorticoids on pyrimidine nucleotide metabolism and to consider its relationship to hormonal regulation of RNA synthesis. It was found that more than half of the hormonally accelerated labeled orotat’e incorporation into hepatic RNA was a reflection of increased specific activit’ies of pyrimidine precursors in the acid-soluble pool. These findings prompted exploration of the effects of actinomycin-D on acid-soluble pyrimidine metabolism as well as upon RNA synthesis (11) and on the glucocort,icoidal enhancement of these processes in the presence of the anbibiotic. These studies indicate that actinomycin-D itself markedly lowers the specific activity of the hepatic acid-soluble pools that are precursors to RKA. Furthermore, actinomycin-D did not prevent hormonal effect,s upon acid-soluble pyrimidine nucleotide metabolism. MATERIALS
AND
i\lETIIODS
AIale Sprague-Dawley rats of 250-300 g body weight were bilaterally adrenalectomized and maintained t)hereafter on O.S(i;l saline, water, and Prlrina Chow ud libitum for 557 days. Prior to the tracer experiments the animals were starved overnight. All injections were made intraperitoneally. Cortisolle-acetate snspension (Upjohn Co.) 1 or 5 mg/lOO g body weight was given 4 hr prior to sacrifice. Actinomycirl-D (kindly donated by the nlerck Sharp & I>ohme Co.) dissolved in 0.9yc N&l, was rontinely injected at 50 rgjlO0 g body weight 1 hr prior to the hormone injection. However, in the experiment in which the transport and srlbcellldar distriblltion of cortisone was studied, 100 pg actitlomycilr-I)/100 g body weight was administered 2 hr before unlabeled cortisone (5 mg/lOO g body weight,) containing 5 PCi of cort,isane-1,2-“TI (34.4 Ci/mM, New England Nuclear). The animals were sacrificed after the indicated incorpration periods (Table IV). Whelr indicated, orotir-6-14C acid (6.5 mCi/mM, New England Nllcelar Corp.) or orotic-3Tf acid (1 Ci/mM, l’olk Co.) at 12.5 PCi and 40 &i/100 g body weight respectively were itljected. III t,hese experiments the radioisotope incorporation period was 20 min. The animals were sacrificed by exanguinatioll, the livers rapidly transferred to chipped ice, homogenized, and s~~bcellrllar components and RNA were isolated as previorlsly described (12, 13). The RNA coIltent was determined according to AIllrrro and Fleck (14) asslIming that all absorbatIce of 1.0 at 260 rnp with a l-cm light pat,h corre-
EFFECTS
ON POOLS
15.3
sponds to 32.0 pg RSB/ml (14). The total acidsoluble fraction was obtained, either by mixing the liver homogenate with an equal volume of cold 0.4 M perchloric acid or by homogenizing the liver directly in 3 vol of cold 0.27 M perchloric acid. After centrifugation, the precipitate was re-extracted twice with 0.2 M perchloric acid and the combined supernates constituted the total acid-soluble fraction. The total acid-soluble uridine nucleotides were isolated as IJRIP, after acid hydrolysis, by chromatography on successive Dowex-50 and Dowex-2 columns (10). RNA-uracil was isolated on Dowex-50 by Cohn’s method (15) after hydrolysis in 70y0 perchloric acid (16). Radioisotopic scintillation counting procedures were as previously described; all values presented were corrected for quenching (10). RESULTS
Earlier studies in this laboratory revealed that cortisone stimulated both inorganic 32P and glycine-2-‘4C incorporation into the Rn’A of all subcellular components (2, 13). We now inquired as to whether this would also be true for a precursor specific for pyrimidinex and whether actinomycin-D might exert’ a selective antagonism toward the hormonally accelerated synthesis of the RNA associated with a particular subcellular entity. It’ is evident from Ta,ble I that incorporation of pyrimidine nucleotide precursors into the RNA of all subcellular components are similarly stimulated by cortisone albeit to a quantitatively lesser extent’ than previously observed using glycine-2-14C or 32P (1, 1X). As anticipated, actinomycin-D inhibited incorporation into t)he RSA of all subcellular fractions. A small, but consistent, hormonal elevation in the RNA specific activity of all fractions was observed in the presence of levels of nctinomy&-D known t,o prevent enzyme induction by cortisone (17). This apparent discrepancy is due to the complex and antagonist’ic effects of cortisone and act,inomycin-D upon acid-soluble pyrimidine nucleotide metabolism as well as upon RNA synthesis. Cortisone caused a 10% and 1‘25%increase respectively in the total and the specific radioactivitv of the acid-soluble precursor pool of the fiver and a 29 % stimulation in t)he specific activity of hepatic RNA (Table II). The hormonally
154
YU AND
FEIGELSON
TABLE
I
EFFECTS OF COILTISONE AND ACTINOMYCIN-11 ON OltoTIc-(j-W ARID INCORPORATIOX INTO THE RNA OF SUBCELLULAR FRACTIONS OF RAT LIVER” Actinomycin-D
Nuclear
Cortisane
-
-
+ +
+ +
Mitochondrial
RNA
cdw
33,400 41,800 7,600 9,800
9’
f * f f
4,100 5,500 1,000 1,700
100 125 23 29
RNA
301 405 88 100
Microsomal
%
cpmimg
f zt f zk
30 49 14 23
100 135 29 33
RXA
cpm/mg
2,080 2,310 2G4 325
f zt f +
Cell sap RNA
150 300 23 110
%
cpm/w
%
100 111 13 16
5,550 6,030 761 896
l f zk f
450 1160 40 269
100 109 14 In
Q Actinomycin-D (50 rg/lOO g body weight), cortisone (1 mg/lOO g body weight) and erotic-6-W (12.5 pCi/lOO g body weight) were respectively injected at 5 hr, 4 hr, and 20 min prior to sacrifice. Each value depicted is the mean + standard error for three animals. TABLE
II
THE EFFECTS OF COHTISONE ANI) ACTINOMYCIN-D ACID-W INTO THE HEPATIC ACID-SOLUBLE Cortisone (1 n&loo g body
Actinomycin-D (50 afmo g body wt)
wt)
-
-
To Cortisone
+ + stimulation
8,260 10,600 1,590 1,960
+ + +
7GActinomycin-D
RNA
f f f f
50 900 80 290
4,740 5,300 2,110 3,290
f f zk f
280 300 80 300
585 G43 274 398
f f f f
28.3 23.3
11.8 55.9
9.9 45.3
80.8 81.6
55.5 37.9
53.2 38.1
18 31 8 39
125 122 130 121
f zk f f
6 4 3 2
inhibition
+ a Values
Total m/mg
ON THE INCOHTORATION OF OROTIC PRECURSOR Poor. AND RNA”
are given
as means f
standard
error
for 3 animals.
increased specific activity of hepatic RNA is therefore the result of two factors: that derived from t,he increased specific activit,y of the precursor pool and that derived from hormonal acceleration in nucleotide polymerization to form RNA. Actinomycin-D diminished the specific activity of the acidsoluble pool by 56% and that of the RNA by 81% indicating a net actinomycin-D inhibition of RNA synthesis of but 25 %. Thus, this dose of actinomycin-D apparently exerts a greaker inhibitory effect upon acid-soluble precursor transport and/or metabolism than upon RNA synthesis. Table II also indicates that neither cortisone, nor actinomycin-D under these conditions, led to detectable alterations in the tissue concentration of nucleotides present. Actinomycin-D exerted differential eff ect>s upon the cortisone-modified acid-soluble and
RNA metabolism. The hormonal increment in acid-soluble radioactivity is not impaired by pretreatment of the animals with act.inomycin-D. However, upon correction for acid-soluble pool effects, it is evident that actinomycin-D completely prevented hormonal enhancement of nucleotide condensation into RKA. Thus, cortisone enhancement of precursor incorporation into hepatic acid-soluble pyrimidine nucleotides does not seem to be dependent upon transcriptional processes. To permit, more precise evaluation of the hormonal effects, the specific activities of RNA-uracil and of its uridine nucleotide precursors in the acid-soluble pool were directly determined (Table III). The results indicate this dosage of cortisone elicits a 54% increase in specific activity of total RNA, and a 39 % increase in specific activity
CORTISO?r’E
AND
ACTINO~IYCIN-D
EFFECTS
TABLE THE
III
EFFECTS OF CORTISONE UPON THE INCORPORATION OF OROXC ACIU-“II ACID-SOLUBLE URI~INE NUCLEOTII)ES AND INTO RNA-URKIL Specific radioactivity
-
are expressed
1,XiO 1,116 1,490 GO,800
OF THE
as means + standard
EFFECT
error
* The values
120 74 120 7,200
2,250 *
150
54
1,517
20
39 87
*
2,790 + l-20 89,200 It 5,300
47
9.0 f 0.1 120 z!z 1 1.0 + 0.1
for 4 animals. I\’
OF ACTINOMYCIN-D ON HEPATIC DISTRIBUTION OF C~RWSONE-1,
Homogenate
Actinomycin-D 1Iinutes incorporation 15 30 45 GO
‘,q; G&on
+
+ zt f I!C
9.5 f 0.3 123 zt 1 1.1 + 0.1
liver)
TABLE STIJDY
INTO
Cortisone acetatea (5 mg/lOOg body wt)
RNA (cpm/mg) Total acid-solltble fraction (cpm/ODnso,+) RNA-llracil (Cpm/~mole) Acid-soluble UMI’ (Cpm/pmole) Amount BKA (mg/g liver) Total acid-solnble fraction (ODZW,,,~units/g .4cid-soluble U1IP &mole/g liver) a Yalltes
155
ON POOLS
UPTAKE
AND
SCBCELLGL~R
2-311e
Soluble
Suclei
hlicrosomes
Mitochondria
-
+
-
+
-
+
-
+
-
+
129,000 102,000 78,000 67,000
105,000 105,000 81,000 70,000
105,000 87,000 69,000 56,000
89,000 90,000 73,000
1990 1570 1180
7000 5320 4050
57,000
B90
1950 1390 1330 750
6200 5800 5100 4300
1900 2400 2300 2600
3100 2900 2200 2400
depicted
are averages
for duplicate
of the total acid-soluble pool. When uracil was isolated from each component the hormonal effect was an S7 9% and a 47 % elevation in the specific activit.ies of t’he RNA-uracil, and acid-soluble uridine nucleotides respectively. Thus, approximately half of the hormonally elevated specific activity of RNA represented hormonal enhancement of RNA synthesis; the remainder may be attributed to hormonal elevation in t,he specific activity of the uridine nucleotide pool t’hat is the precursor of the R?;A-uracil. The size of the acid-soluble pool, expressed either as OD,G, or as acidsoluble UMI concentration remained urlchanged, nor was there an increase in the absolute amount of hepat’ic RiNA 4 hr after cortisone treatment. An experiment was undertaken to explore the possibility that actinomycirl-D might inhibit hormonal action by an inhibition of hepat,ic hormonal uptake and/or prevention
3700
animals.
of its binding to an appropriate receptor site in the target cell. The results of such a study are shown in Table IV. In the control animals, the maximal incorporation of radioactive cort’isone occurred at 15 min, 82% of the hepatic radioactivit#y was recovered in the cell sap, 5% in the microsomes, and 2% in each of the nuclear and mitochondrial fractious. After the incorporation peak at 15 min, the isotope disappeared from the liver with a Tl/z of approximately 1 hr. There was a parallel loss of radioactivity from the cell sap, the microsomal, and the nuclear fract’ions, but 110 loss of radioactivity from the mitochondrial fraction during the period studied. Animals pretreated w&h 100 pg actinomycin-D/100 g body weight 2 hr prior to labeled cortisone administration manifested a similar patt’ern for the uptake, distribution, and loss of the co&one-l, %-“H; the sole differrnce being t.hnt the maximal period of incorporation \vas somc-
156
YU AND
what broader, plateauing during the l&30min interval. Thus, actinomycin-D did not markedly hinder the transport of radioactive steroid into the liver, nor did it significantly influence its gross subcellular distribution thereafter. The possibility that actinomytin-D might act to prevent hormonal action by virtue of impaired hormonal transport’ int’o the liver therefore is seemingly obviated.
FEIGELSON
indeed involves newly made RNA. Furthermore, the specific activity of other types of RNA, including rRNA and tRNA, were elevated simultaneously with that of the mRNA. One confronts an apparently nonspecific stimulation of RNA synthesis yet select’ive induction of certain liver enzymes by the glucocorticoid hormones. Furthermore, although isotopic incorporation rates int,o various subcellular RNA fractions were similarly enhanced in response to cortisone, L)IHCUSSION greater differences in response were observed depending upon the nature of the The use of isotopically labeled organic tracers employed : glycineJ*C, which meascompounds provides unambiguous evidence ures purine nucleotide synthesis, showed a for t,he existence of metabolic routes by which one compound is the precursor of 255 % increased incorporation into hepatic another. However, the l.n vivo evaluation of RNA 4 hr after hormone treatment, whereas under similar conditions, inorganic-32P inthe rate at which these metabolic interconcorporation augmented 124 % and orotic-3H, versions occur is fraught with interpretive which measures the pyrimidine nucleotide difficulties. Experiment#ally, one administers a labeled compound and evaluates the pathway, registered only 38 % above the extent of labeling in the metabolite of in- control levels (1, 13). The recognition that glucocorticoids stimulated the incorporaterest aft’er a given incorporation period. tion of labeled glycine into acid-soluble However, a precursor must be transported adenine provided a measure of clarification into the cell, converted to various interindicating that much of the increased radiomediates, and subjected to isotopic dilution activity in the RNA was a reflection of the in various pools en route to t,he formation hormonally enhanced specific activity of the of the metabolite ultimately isolated. purine nucleotide pool (10). The present experimental and physiological Clearly, manipulations may influence any of the study indicates that cortisone treatment leads to parallel elevations of radioactivity processes occurring between the labeled of both the total hepatic acid-soluble fraccompound administered and the product tion and of the acid-soluble uridine nucleoultimately measured. The first reports that tides. This observation is compatible with glucocorticoids markedly stimulate the incorthe view that cortisone enhances the transporation of various precursors into hepatic port of labeled erotic acid into the liver RNA (12, 13) apparently related hormonal action to transcription of the genome and cell, without altering its rate of conversion has been reconfirmed and extended in to UMP. The increased specific activity of this precursor pool is likewise ultimately numerous laboratories (6, 8, 15-20). Howreflectfed in the RNA. Using erotic acid, ever, t,he hormonally stimulated synthesis this pool effect was responsible for approxiof several enzymes, e.g., tryptophan oxygenase (21) and tyrosine transaminase (22), mately one half of the hormonally elevated hepatlc RNA specific activity; one half of with its relat,ively minor accompanying the stimulation was apparently due to an stimulation in over-all protein synthesis, hormonally stimulated rate of RNA syntheseemed incompatible with the several-fold hormonal increase in the specific activity of sis. It is well established that actinomycin-D hepat,ic RNA, which was unlikely to be inhibits RNA synthesis by virtue of its bindmerely the reflection of t)he enhanced synthesis of a few species of mRNAs. Yet the ing with the deoxyguanosine moieties of the thereby preventing transcription process of hormonal enzyme induction has DNA, (11). However, effects of actinomycin-D on been demonstrated to be actinomycin-Dother than transcriptional processes have sensitive (17), suggest.ing that the induction
CORTISONE
AND
ACTINOMYCIN-D
been reported (23). The present study suggests that actinomycin-D may inhibit certain transport processes within the liver. It is of furt’her interest t)hat the apparent 81 ‘Y inhibit,ion of RXA synthesis resulting from act,inomycin-D treatment is in reality merely a 25 % inhibition when corrected for the elect’ of actinomycin-D upon the specific activity of the acid-soluble pool. Thus, in an acute experiment, 500 pg actinomycin-D per kilogram permits 75 % of t,he hepatic Iir\TA synthesis to proceed. The observed sensitivity of hormonal enzyme induction to act,inomycin-D inhibition may indicate that the species of RNA involved are uniquely sensitive to the action of actinomy&l-D. It is also evident that proper eva,luation of the effects of actinomycin-D upon RNA synthesis can be made only with concomitant exploration of the effect of this agent up011 acid-soluble nucleotide metabolism. The differential effect of actiuomycin-D 011 the hormonal stimulation of RXA synthesis and on the transport and/or metabolism of orot’ic acid into the liver imply the latter hormonal effect is independent of transcriptional processes.
EFFECTS
5. 6. 7.
8. 9. 10. 11.
12. 13. 14. 15.
16. ACKNOWLEDGMENT The Marilyn
excellent Stevens
technical is gratefully
assistance of acknowledged.
Miss
17.
18. REFERENCES 1. FEIGELSON, III., AND FEIGELSON, P., in “Advances in Enzyme Regulation” (G. Weber ed.), 1.01. III, p. 11. Macmillan (Pergamon) New York (1965). 2. FEIGELSON, P., FEIGELSON, IN, AND GREENGARD, O., Recent Prog. Hormone Res. 18, 491 (1962). 3. FEIGELSON, P., AND FEIGELSON, M., in (‘ACtions of Hormones on Molecular Processes” (G. Litwack and D. Kritchevsky, eds.), p. 218. Wiley, New York (1964). 4. FEIGEISON, P., ANI)FEIGEI,SON,M., in “Rlech-
19. 20.
21. 22. 23.
Oh’ POOLS
157
anisms of Hormone Action” (P. Karlson ed.), NATO Advanced Study Institute, p. 24G. Academic Press, Xew York (1965). KIDSON, C., AND KIRBI-, K. S., Nalzcre 203, 599 (1964). SCHMID, W., GALLIVITZ, D., AED SEKERIS, C. C., Biochim. Biophys. 11&a 134, 80 (19G7). KENNEY, F. T., WICKS, W. I)., AND GREENMAN, D. L., Cellular Comp. Physiol. 66, Suppl. I., 125 (1965). DRETVS, J., AND BRA~VERMAN, G., J. Hiol. Chem. 242, 801 (1967). FEIGELSON, P., AND FEIGELSOX, II., J. Biol. Chem. 238, 1073 (1963). FEIGELSON, M., AND FEIGELSOX, P., J. Biol. Chem. 241, 5819 (1966). REICH, E., AND GOLDBERG,I. II.,in “Progress in Nucleic Acid Research and Molecular Biology” (J. !V. Davidson, and W. E. Cohn eds.), Vol. III, p. 184. Academic Press, New York (1964). FEIGELSON, P., FEIGELSON,&~.,AND FANCHER, C., Biochim. Biophys. Ada 32, 133 (1959). FEIGELSON,&I., GROSS,P.R., ANDFEIGELSON, P., Biochim. Biophys. Scta 55, 495 (1962). MUNRO, H. Xi., AND FLECK, A., Analyst 91, 78 (1966). COHN, W. E., in “The Nucleic Acids” (E. Chargaff, and J. N. I>avidson eds.), Vol. I, p. 211. Academic Press, New York (1955). MARSHAK, A., AND I-OGEL, H. J., J. Biol. Chem. 189, 597 (1951). GREENGARD, O., AND Acs, G., Biochim. Biophya. Ada 61, 652 (1962). BARNABEI, O., AND SEKENI, F., Biochim. Biophys. Acta 91, 239 (1964). GSRREN, L. D.,Ho\vELL, R.R., ANDTOMKINS, G. M., J. Mol. Biol. 9, 100 (1964). KENNEY, F.T., GREENMAN, D.L., WICKS, W. D., AND ALBKITTON, W. L., in “Advances in Enzyme Regulation” (G. Weber, ed.), 1’01. III, p. 1. Macmillan (Pergamon), New York (19G5). KNOX, W. E., Brit. J. Exptl. Palhol. 32, 462 (1951). LIN, E. C. C., AND KNOX, W. E., Biochim. Biophys. Beta 26, 85 (1957). SINGEH, M. F., AND LEDER, P., ilnn. Rev. Biochem. 36, 195 (1966).