Intracellular Transfer of Nucleic Acids II. Effect of Hormones On
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The Journal of Biochemistry, Vol. 53, No. 5, 1963 Intracellular Transfer of Nucleic Acids II. Effect of Hormones on Ribonucleic Acid Metabolism in Rat Liver Cells By TAKAO IWAMOTO, AKIKO MORTYAMA, TSUNAO TETSUKA and YOSHIAKI MIURA (From the Department of Physiological Chemistry, Chiba University, School of Medicine, Chiba) (Received for publication, November 13, 1962) Influence of hormones on the metabolism were used. With exception of the preliminary ex- of liver RNA has been studied by several periments, the litter mate rats of the same sex authors with a comprehensive review, cover- were divided into two groups and the following ing this field, being performed by Leslie hormones were administered to one of the groups. Administation of the Hormones-One mg. per 100g. (1). According to him, cortisone, insulin, body weight of parotin (a parotid gland hormone estradiol and thyroxine had a tendency of in- preparation, Teikoku-Hormone Manufacturing Co. creasing the amount of RNA in rat liver. Ltd.) was injected intramuscularly daily for 10 days However, testosterone showed practically no into female rats. ƒÀ-Androstanolone (a protein-anabolic effect on the metabolism of chick liver nucleic hormone preparation, Fujisawa Pharmaceutical & Co. acid. Ltd.) was administered intramuscularly to male rats Can tar o w et al. (2) carried out an experi- every day for 10 days with a dosage of 0.2 mg. per ments concerning the incorporation of 2-C14_ 100g. body weight. uracil into RNA with rat liver. Normal liver Prior to the injection of estradiol in female rats, did not take up C14-uracil into RNA, however, a total ovariectomy was carried out on both the regenerating liver and liver of the animal control and experimental groups. Commencing soon after the operation, the experimental group was treated with growth hormone did. given 5 ƒÊg. per 100 g. body weight of aqueous suspen- During the course of our research on the sion of estradiol benzoate intramuscularly every day metabolism of rat liver nucleic acid, we notic- for the period of 10 days.` Testectomy was performed ed that the uptake of P132 into RNA was on both the control and experimental groups. Follow- higher in female than in male rats. This fact ing this operation, the experimental group was given would suggest that estrogen stimulates the 0.2 mg. per 100g. body weight of testosterone pro- metabolic turnover of rat liver RNA. pionate (10 I.U.) once a day for 10 days. Such evidence led us to study further Half of a group of female rats who were sub- which hormone would regulate the metabolism jected total hypophysectomy, were given intramuscular injection of 3 mg. per 100 g. body weight of hypo- of RNA and what kinds of RNA are mostly physeal growth hormone (U. S. P.) every other day influenced by hormonal treatment in liver for 10 days postoperatively. cells. In this paper, the authors will describe Analytical Procedures-In order to keep radioactivity the incorporation of Pi32 into various intra- in the blood at a constant level, 50 uc of P32i for 100 cellular RNA of rat livers, under the influence g. body weight was injected intraperitoneally 5 times of hypophyseal growth hormone, estradiol, at 30 minutes intervals. Half an hour after the last testosterone, ƒÀ-androstanolone (Androstan-l7- P32i administration, the animals were sacrificed by ƒÀ-ol-3-one) and parotid gland hormone. decapitation. The blood was pooled and serum was separated, then, 10%o aqueous trichloracetic acid was added to remove protein. After wet ashing, the MATERIALS AND METHODS amount of inorganic phosphorus was determined by Animals-Wistar strain rats weighing 100-150g. F i s k e-S u b b a R o w's method. The radioactivity 408 Hormonal Effects on RNA 409 was measured by G-M counter. location of inorganic P32i was about 2-3 tubes near The livers were irrigated by intraportal injection the cathodic edge, the remaining fractions were of 30-50 ml. of cold 0.25 M sucrose solution. Livers assayed for RNA by an orcinol reaction and for the from 2-3 rats were treated in 0.25M sucrose to radioactivity by a G-M counter. The total phos- obtain 20% homogenates. Nuclei and mitochondria phorus content of RNA was estimated as 8 per cent. were separated by means of S c h n e i d e is fractiona- Specific activity and relative specific activity were tion method (3). The supernatant fraction contain- calculated according to the following equations : ing microsomes was adjusted to pH 5.2 in a pH Specific activity= c.p.m. of sample meter by adding 0.1 N HC1. The resulting precipitate amount of RNA-P (ug.) was separated by centrifugation at 700•~ g for 5 Relative specific activity =specific activity of sample minutes. specific activity of serum From each fraction, RNA was extracted by •~l00%. phenol method (4) and an alkaline hydrolysis was Column-Chromatography-The procedure of DEAE undertaken with N KOH incubating at 37•Ž for 15 cellulose chromatography was identical to what des- hours. Phenol extraction was performed as follows : cribed in detail in the previous paper (6). Equal volume of 90% (v/v) phenol was added to each fraction and shaken for 30 minutes at room temperature; after centrifugation at 700•~ g for 30 minutes, the upper aqueous layer was collected; crude RNA in the aqueous layer was precipitated by add- ing two volumes of ethanol with potassium acetate (final concentration of 2%); thus precipitated RNA was redissolved in 25 ml. of cold water, and 2 ml. of 8% perchloric acid and 1 ml, of 10% phosphate was added as its carrier; the acid-soluble fraction was centrifuged off and the residual polynucleotides were digested overnight at 37°C by adding N KOH; the solution of mononucleotide was neutralized with perchloric acid and resulting precipitate of potassium FIG. 1. Elution pattern of the supernatant perchloride was centrifuged off. after removal of sediment caused by acidification To avoid contamination of highly radioactive of AH 130 cytoplasmic supernatant by 0.1 N HGI inorganic phosphorus into mononucleotides, electro- to pH 5.2. DEAE cellulose (Schleicher & Schuell, Stand- phoretic fractionation was accomplished by continuous- ard type No. 70, 0.87 Meq/g.) columns used in flow electrophoresis on Tokyo filter paper No. 52 this experiment were 30 cm. in height and 1 cm. in diameter. The pH of the sample was adjust- (40 X 40 cm.) (5). Separations were achieved in 0.02 ed at pH 7.6 before pouring in the column. M citrate buffer (pH 3.5) at a constant voltage of The elution was first carried out with Tris-buffer, 550 volt. with currents of 15-28 mA. Since the then with increasing concentration of NaC1. TABLE I Difference of Relative SpecificActivities of RNA between Male and Female Rats 410 T. IWAMOTO, A. MORIYAMA, T. TETSUKA and Y. MIURA in male and female rats are shown in Table RESULTS I. After precipitation at pH 5.2, the super- In this table, animals of groups 2 and 3, natant fraction was analyzed by DEAE cel- and of 5 and 6 were litter mates respectively. lulose chromatography. Elution pattern is Statistically fluctuations of the relative specific shown in Fig. 1. It is clear that S-RNA activities in S-RNA or ribosomal RNA ob- fraction which should appear at 0.4-0.7M tained from the litter mates in a series of NaCI eluate, was not found in the supernatant experiments, are much smaller than the ones fraction after the acidic treatment at pH 5.2. when litter mates are not used (standard error Thus, the main components of the precipitate of 4 cases of litter mates was 0.017 whereas, should be S-RNA and microsomal RNA, that of 4 cases of difference litters was 0.131). whereas those of the supernatant fraction In subsequent experiments, litter mate mainly consists of RNA secreted from nuclei rats were used throughout the study. The as previously reported (6). Therefore, the greatest difference observed between male and intracellular fractionation adopted in this female rats was found the relative specific experiment would be interpreted as nuclear activity of nuclear RNA. The value of female RNA, mitochondrial RNA, cytoplasmic S- rats was significantly higher than that of male RNA plus microsomal RNA and supernatant- rats (1% level). RNA. This fact would suggest the possibility of Results of preliminary studies on the the hormonal control of nuclear RNA P32i-incorporation into various types of RNA metabolism. To observe the influence of sex TABLE II Effect of Esiradiol-Administration on Ovariectomized Rats Hormonal Effects on RNA 411 hormone more specifically, castrated male into nuclear RNA of male rats but administ- rats were used for the experiments with ad- ration of the hormone to the testectomized ministration of estradiol or testosterone. The rats lowered its relative specific activity. enhancing effect of estradiol on the uptake This indicates that the lower metabolic rate of P32i into nuclear RNA is again demonst- of nuclear RNA in male rats shown in Table rated in Table II. I was based not only on the estrogenic hor- Since the actual counts in mitochondrial mone deficiency but also on the effect of and S-RNA-microsomal RNA in the experi- testosterone which positively decreased the ment concerning estradiol and testosterone relative specific activity of nuclear RNA. were too low to calculate, specific activities Hypophysectomy did not exert too much are shown in parentheses. influence upon the metabolic activity of nu- The relative specific activity of the super- clear RNA in female rats. However, as shown natant fraction was small in the ovariecto- Table in IV, the administration of growth mized group but was great in the estradiol- hormone to the hypophysectomized rats in- administered group as was seen in nuclear crease the uptake of P32i, into nuclear RNA fraction.