RNA Synthesizing Activities and Glucocorticoid Binding Capacities in Thymus Cells During Rat Development
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Pediat. Res. 11: 705-709 (1977) Dexamethasone RNA synthesis glucocorticoid receptor protein thymus RNA Synthesizing Activities and Glucocorticoid Binding Capacities in Thymus Cells during Rat Development K. LJpp<3 n AND N. VANDERMEULEN Universitiits-Kinderklinik, Marburg, West Germany Summary steroid-resistant ce·lls have little or no hormone binding capaci ties. In view of this functional role we studied the dexametha This investigation was designed to study specific glucocorti sone binding capacity of the cytoplasmic fraction and of nuclei of coid binding to cytoplasmic fraction and nuclei of thymus cells thymus cells during rat development. Changes in hormone bind during rat development and to find out whether these data can ing should be correlated to an altered response of the tissue. be correlated to changes of RNA synthesizing activity of nuclei Therefore, we measured nuclear RNA synthesizing activity and and cytoplasm. The dexamethasone binding capacity of cyto also the DNA-dependent RNA polymerase Illa in cytoplasm plasm rose rapidly in rats weighing up to 125 g and decreased that controls synthesis of tRNA (7), and the inhibition of RNA significantly in animals weighing more than 160 g. Hormone synthesis by dexa[llethasone in whole cells and purified nuclei binding to nuclei revealed similar but less pronounced changes. during rat development. RNA synthesizing activity of nuclei measured by [3H]uridi~e incorporation in vitro decreased from 57 ± 4.6 dpm/µg DNA m young to 23 ± 2.2 dpm/µg DNA in adult rats. RNA synthesiz MATERIALS AND METHODS ing activity of the cytoplasmic fraction fell 21.6% and that of Han-Wistar rats were obtained from the Institut fiir Versuchs purified polymerase Illa fell 27 .8% during development. Inhib tierzucht (26); animals which weighed less than 50 g were of ition of RNA synthesis by dexamethasone applied in vivo and in either sex, heavier rats were male. (5-3H]Uridine 5'-triphos vitro showed age-dependent differences. The RNA synthesizing phate ((3H]UTP), specific activity 14 Ci/mmol, and (1(2)- capacity of nuclei was inhibited up to 39% in animals weighing 3H]dexamethasone, specific activity 28 Ci/mmol, were pur 130 g and only 9% in aging rats. Similar changes were observed chased from The Biochemical Center (27). McCoy 5-A medium by incubation of intact thymocytes with and without hormone. was obtained from Gibco (28), Dextran T500 from Pharmacia The observed inhibitory effect of dexamethasone on RNA syn (29), DE-cellulose from Whatman Biochemicals Ltd. (30), calf thesis is well correlated to changes of cytoplasmic hormone re thymus DNA and TC 199 from Serva (31 ), and Scintigel from C. ceptor capacities during development. Roth (32). All other chemicals were purchased from Merck (33) and Boehringer (34). Toluene scintillation fluid contained 5 g Speculation 2,5-diphenyloxazole and 0.2 g 2,2'-p-phenylene-bis-(5-phcny If the observations on developmental changes of hormone loxazole) in 1 liter of toluene. binding capacities and on age-dependent responses of thymus The rats were killed by cervical dislocation, and the thymus cells to glucocorticoid action can be applied in general, it is not dissected out rapidly and weighed. Glands were minced with the hormone itself, but the receptor concentration which seems scissors in TSS buffer (0.25 M sucrose, 0.025 M KCI, 0.01 M MgCl in 0.067 M Tris-HCI, pH 7 .55), filtered successively to be the limiting factor for the cellular response to the hormone 2 action. Therefore, efforts should be made to find ways to induce through four and eight layers of cheese cloth, and centrifuged at synthesis of these receptor proteins. 150 x g for 1O min at 4°. All further steps were made at 4°. ISOLATION OF THYMUS CYTOPLASM Binding of steroid hormone to specific protein receptors is essential for steroid hormone action. Glucocorticoids are known For estimation of dexamethasone binding capacity thymocytes to affect a variety of different tissues. Proteins which specifically were homogenized in TSS buffer in a glass-teflon homogenizer. bind glucocorticoid hormones have been identified in these tis The crude nuclei were sedimented at 400 x g and the superna sues, e.g., in liver (4, 13), in thymocytes (20, 24), and in tant was centrifuged at 105,000 x g for 60 min in the 50 TI rotor lymphocytes (3, 23). It is generally accepted that the first step in of a Spinco ultracentrifuge. The soluble postmicrosomal super steroid hormone action is the combination of the hormone with a natant was used after removal by suction of material which specific cytoplasmic protein receptor. The cytoplasmic steroid floated. In further experiments thymocytes were allowed to swell receptor complex is then taken up by the nucleus and bound to in 1.5 mM MgCl2 for 5 min before homogenization. Isotonicity specific acceptor sites. This interaction is believed to initiate the was reconstituted by addition of concentrated TSS buffer. characteristic hormone response, presumably by alteration of DNA template activity. ISOLATION OF NUCLEI The response of the tissue to glucocorticoid action is hormone Sedimented crude nuclei were purified by centrifugation and organ specific. In case of lymphatic cells glucocorticoid through a 2.2 and 2.4 M discontinuous sucrose gradient at action results in a mainly antianabolic change of intracellular 95,000 x g for 90 min. metabolism, like inhibition of synthesis of proteins (15), DNA (10), and RNA (8) and finally in cytolysis (25). In Iymphosar ASSAY OF SPECIFIC STEROID BINDING coma (11) and liver cells (6) the alteration of metabolism by steroids is proportional to the concentration of intracellular Aliquots of thymus cell cytoplasmic fraction were incubated hormone receptor. In contrast to steroid-sensitive call lines, with 5 x 1o-sM [3H]dexamethasone for 2 hr at 4°, with or 705 706 LIPP AND VAN DER MEULEN without 25 x 10-5 M nonradioactive dcxamethasone. After intact thymus cells was measured after incubation of cell suspen incubation 0.5-ml portions were mixed with 0.5 ml dextran sions in TC 199 medium with or without dexamethasone for 45 coated charcoal suspension (3 .75 g charcoal and 0.375 g dextran min at 37° followed by additional incubation for 30 min in the in 100 ml O.Ql M Tris-HCI, pH 8.0) and incubated for 10 min at presence of tritiated UTP. Cells were then transferred to filter 4°. Duplicate tubes were centrifuged for 2 min in an Eppcndorf discs and the discs treated as described for nuclei. model 3200 centrifuge. Of the supernatant 0.5 ml was counted Protein concentration was determined by the method of in scintillation fluid (Scintigel). Specific binding was measured as Lowry (14) with bovine scrum albumin as standard. DNA was the difference in radioactivity present after dextran-charcoal measured by the diphenylamine reaction according to the treatment of the cytoplasmic fraction incubated in the presence method of Burton (5) with calf thymus DNA as standard. The and absence of nonradioactive hormone. results arc expressed as the mean value ± SEM. For assay of specific steroid uptake by nuclei, thymocytes were incubated with 5 x 10-sM (3H]dexamethasone for 30 min at 37° with or without 25 x 10-5 M unlabeled dexamethasone. At the RESULTS end of the incubation period, cells were sedimented (5 min, 150 Between 14 and 70 days of age, the body weights of the x g, 2°} and washed with 500 volumes of TSS buffer. Nuclei animals rose from 20-160 g and the wet weights of the thymus were then purified by sucrose density centrifugation and lysed in glands rose steadily up to 0.33 g. During further development formic acid. DNA concentration of the solution was measured the organ weight fell significantly (P < 0.01) to 0.266 gin 4- and the radioactivity counted. month-old rats weighing 290-300 g. The ratio of thymus to body weight increased significantly up to 30-day-old animals weighing ASSAY OF RNA POLYMERASE ACTIVITY 60 g and declined steadily during further development (Fig. 1). The capacity of thymus nuclei to incorporate (3H]UMP into The protein to DNA ratio, a measure of cell size, was 2.8 ± 0.5 acid-precipitable material after. incubation with tritiated UTP and remained constant during development. was studied. The assay system contained, in a total volume of The glucocorticoid binding capacity of the cytoplasmic frac 150 µI: thymus nuclei equivalent to 500 µg DNA, 0 .2 µmol tion of thymus measured in vitro by incubation with 3 each ATP, GTP, and CTP, 0.002 µmol UTP, 1 µCi tritiatcd ( H]dexamcthasonc rose significantly (P < 0.01) in rats weigh UTP, 1 .5 µmot creatinc phosphate, 5 µg creatine phosphoki ing up to 125 g and subsequently decreased rapidly (P < 0.01) nase, 1.5 µmot {3-mercaptoethanol, 10 µmot (NH4 hSO4 , 3 in animals with a body weight more than 160-180 g. Incubation µmot MgSO4 , 10 µmot Tris-Cl, pH 7.9. The reaction was of intact thymocytes with radioactive hormone and subsequent terminated after an incubation period of 30 min at 37° by purification of the nuclei revealed maximal specific hormone 2 pipetting 100 µI aliquots on filter discs (16 cm , Schleicher und binding in 42-day-old rats weighing 90 g. The reduction of the < Schuell 2043 b) and precipitating in 5 % ice-cold trichloroacetic binding capacity in adult rats was less pronounced (P < 0.1) acid (TCA}. Discs were washed four times in TCA and two times than the decrease of glucocorticoid receptor in the cytoplasmic in 96% alcohol and then dried. The radioactivity was counted in fraction (Fig. 2). toluene scintillation fluid. In order to determine (3 H]UMP incor After incubation of cells or cell fractions with (3 H]UTP in vitro poration into RNA in the presence of cytoplasm or polymerase RNA synthesis was measured by incorporation of radioactive 111 8 containing column fractions the incubation medium was UMP into the TCA-insoluble fraction.