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D and haematin hydrochloride were obtained from Roth (Karlsrhue), bovine serum albumin from Sigma (St. Louis, MO.), pyridoxal-5'-phosphate from Boehringer ...
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 191, No. 1, November, 101-109, 1978

Initial Steps in the Induction Tryptophan Oxygenase JiiRGEN * Institute

VOIGT,*

THEODOR

by Glucocorticosteroids of Rat Liver and Tyrosine Aminotransferase

WIELAND,?

AND

CONSTANTIN

of Cell Research, German Cancer Research Center and t Max-Planck-lnstitut Forschung, Received February

D-6900 Heidelberg,

E. SEKERIS*’ ftir Medirinische

F.R.G.

13, 1978; revised May 25, 1978

Approximately 90 min after the application of dexamethasone, an increase in the activity of the liver enzymes tryptophan oxygenase and tyrosine aminotransferase in adrenalectomized male rats could be measured. As early as 30 set after the injection of an inducing amount of [3H]dexamethasone, significant radioactivity can be found in purified rat liver nuclei. The uptake is linear during the fast 15 min, whereas during the same time period, the increase of the radioactivity in the 10,ooOg supernatant is biphasic. No time shift could be observed in the uptake of dexamethasone by nuclei as compared to the accumulation in the 10,OOOgsupematant. Studies on the time course of sensitivity of the induction process to a-amanitin suggested that the fast induced transcripts for tryptophan oxygenase and tyrosine aminotransferase are completed about 25 min after the administration of the hormone. The ensuing processing of the transcripts to the corresponding mRNAs can be blocked by actinomycin D, the actinomycin D-sensitive phase lasting another 15-25 min. A lag phase of approximately 20 min has been calculated from the time of the entry of the first hormone molecules into the nucleus till the initiation of specific tryptophan oxygenase and tyrosine aminotransferase gene transcription.

The induction by glucocorticosteroids of TO* and TAT in the rat liver depends on a specific increase of the concentration of the TO- and TAT-coding mRNAs (l-3). Furthermore, the inhibition of the induction process by actinomycin D (4-6) and LYamanitin (7) points to a specific gene activation by these hormones (8). The time-course of the modulation of TAT and TO by hydrocortisone or dexamethasone has been described by several authors (9-13). An increase of the enzyme activities could be observed 2 h after the application of the glucocorticosteroid, although 15 to 30 min after injection of tritium-labeled hydrocortisone or dexamethasone, maximal radioactivity could be measured in the rat liver (14-16). In the case of the TAT and TO induction by insulin, glu’ Present address: National Hellenic Research Foundation, Center of Biological Research, Vassileos Konstantinou 48, Athens, Greece. * Abbreviations used: TO, tryptophan oxygenase; TAT, tyrosine aminotransferase.

cagon or dibutyryl-3’,5’-CAMP no such lagphase could be observed (17-22). There are indications that the latter compounds act via the regulation of a post-transcriptional process (19, 23, 24), whereas glucocorticosteroids regulate transcription (4-7). The question arises why the enzyme induction by glucocorticosteroids proceeds only after a lag phase of 90 min. To this end, we have investigated some of the sequential biochemical events taking place during the lag-phase of enzyme induction. The results are presented in this paper. MATERIALS

AND

METHODS

Dexamethasone was a generous gift of Schering AG (Berlin/Bergkamen). Actinomycin D and haematin hydrochloride were obtained from Roth (Karlsrhue), bovine serum albumin from Sigma (St. Louis, MO.), pyridoxal-5’-phosphate from Boehringer (Mannheim), [3H]dexamethasone (specific activity 24 Ci/mmol) and [3H]UTP (specific activity 15 Ci/mol) from Amersham-Buchler (Braunschweig), the nonlabeled nucleoside-tripbosphates from Waldhof GmbH. (Mannheim)

101 0003-9861/78/1911-0101$02.00/0 Copyright 0 1978 by Academic Press, Inc. AU rights of reproduction in any form reserved.

102

VOIGT,

WIELAND,

and all other chemicals from Merck (Darmstadt). Animals and injections. Seven- to lo-week-old male Wistar rats (160-240 g), kept under standard conditions, were used throughout. Adrenalectomy was performed bilaterally under ether anesthesia through the paravertebral dorsal approach. After adrenalectomy, the animals received a 0.14 M NaCl solution instead of normal drinking water. Dexamethasone and actinomycin D were dissolved in dimethylformamide and then mixed with a 0.9% NaCl solution. a-Amanitin was directly dissolved in 0.14 M NaCl. The solutions were injected intraperitoneally between 9 and 10 a.m. The control animals obtained the same volume of 0.9% NaCl solution and all animals received the same amount of dimethylformamide, the dose of the organic solvent never exceeding 0.5 ml per kg body weight. The animals were killed by cervical dislocation at the times indicated in the legends. Preparation of homogenates for the determination of TO and TA actiuities. The livers were perfused in situ with 10 ml ice cold buffer consisting of 0.25 M sucrose, 50 mM KCl, 10 mM 2-mercaptoethanol, 10 mM r,-tryptophan, 20 mM Tris-HCl, pH 7.5, via the portal vein and removed within 30 s after the death of the animal. Homogenization was performed in 2.5 ml of ice cold buffer per g wet weight liver by use of an Ultra Turrax (IO set at 10,900 rpm). The homogenates were centrifuged for 20 min at 30,OOOg.One milliliter of the supernatant was adjusted to 0.2 mM pyridoxal-5’-phosphate, 2.5 mM a-ketoglutarate, and 8.5 mM hemin and heated for 10 min at 55°C. The samples were centrifuged for 10 min at 3O,OOOg,and the supernatant was tested for enzyme activities. Preparation of nuclei and mitochondria. Livers were perfused with 10 ml ice cold buffer consisting of 0.3 M sucrose, 70 mM KCl, 2 mM MgC12, 10 mM Tris-HCl, pH 7.5, via the potal vein and immediately removed. The tissue was minced and homogenized in 5 ml of ice cold buffer by six strokes at 1,200 rpm in a motor-driven Potter-Elvejehm homogenizer with a loose-fitting Teflon pestle. All further procedures were performed at 0-4°C. The homogenate was filtered through two layers of cheesecloth and centrifuged for 10 min at 1,200g. The crude nuclear pellet was taken up in 2.2 M sucrose containing 70 mM KCl, 2 mM MgCL, 10 mM Tris-HCl, pH 7.5. The nuclei were further purified, essentially as described by Chauveau et al. (25) as follows. The suspension was layered on top of a cushion of 2.2 M sucrose buffer and centrifuged for 90 min in the Beckman SW27 rotor at 25,000 rpm. The supernatant was carefully discarded and the centrifuge tube carefully cleaned. The pellet was taken up in 0.3 M sucrose, 70 mM KCl, 2 mM MgC12, 10 mM Tris-HCl, pH 7.8, and the suspension centrifuged for 10 min at 1,200g. The last washing procedure was repeated three times.

AND

SEKERIS

Mitochondria were prepared by the method of Johnson and Lardy (26) starting from the 1,200g supernatant. The washing procedure described by these authors was, however, modified as far as the washing buffer contained additionally 2 mM EDTA. Assays. Tyrasine aminotransferase was determined according to Diamondstone (27), tryptophan oxygenase essentially as described by Schlitz and Feigelson (28). The activities of TAT and TO are expressed in micromoles of p-hydroxybenzaldehyde and micromoles of N-formyl-kynurenine plus kynurenine, respectively, formed per min and mg protein. Protein concentrations were determined according to Lowry et al. (29) using bovine serum albumin as a standard. DNA concentrations were measured as described by Burton (30). Activities of DNA-dependent RNA polymerases were determined by measuring the incorporation of tritium-labeled UTP into insoluble material in 5% TCA (31). The reaction mixtures contained in a total volume of 150 pl: 50 mu Tris-HCl, pH 7.5, 100 mM KCl, 10 mM MgC12, 3.5 mM MnSO+ 5 m&r 2-mercaptoethanol, 1 mM ATP, 1 mM GTP, 1 mM CTP, 6.4 pM UTP, 1 pCi[3H]UTP (specific activity, 15 Ci/mol) and varying amounts of nuclei (corresponding to 4-30 pg DNA). After an incubation for 10 min at 3O”C, 100~1.11 aliquots were pipetted onto 2.5-cm discs of Whatman 3MM paper. The ftiters were successively washed with 5% TCA (4x), ethanol (2x), and ether (2x), dried and measured for radioactivity using a liquid scintillation spectrometer (Nuclear Chicago, Mark II). The scintillation cocktail contained 5 g of PPO per liter toluene. In order to estimate the contribution of RNA polymerase B to the overall incorporation of radioactivity, all measurements were performed in the presence and absence of 0.1 pg of a-amanitin, because at this concentration, the toxin inhibits selectively the activity of RNA polymerase B (32). The remaining activity is accounted for by enzymes A and C. RESULTS

Time-course of Enzyme Induction and of Hormone Uptake The time-course of the induction of TO and TAT by glucocorticoids has been described by many authors (9-13). As we were specifically interested in the duration of the lag-phase, we reinvestigated in the present studies the time-course of the modulation of TO and TAT after a single dose of dexamethasone. The results obtained with adrenalectomized male rats (see Fig. 1) revealed that the activities of both enzymes begin to increase 90 to 105 min after hormone administration. After that time period and for some hours, enzyme activities

TRYPTOPHAN

/

*-t-.-r

d

1

OXYGENASE

4

AND

TYROSINE

l’,C

:

3

4

FIG. 1. Activation of TO and TAT during the fust 5 hr after administration of dexamethasone. Adrenalectomized male rata weighing 250-300 g were injected intraperitoneahy with 10 pg dexamethasone per 100 g body weight. At the appropriate time periods, the animals were killed and the enzyme activity assayed as described in Materials and Methods. The enzyme activities represent the average of individual determinations in nine animals. The standard deviations are also depicted. -, TAT activity; ---, TO activity. Statistical analysis TAT: 60, 75, and 90 min versus zero time, nonsignificant; 105 min versus zero time, P < 0.025; ah the other values versus zero time, P < 0.0005. Statistical analysis TO: 60, 75, and 90 min versus zero time, nonsignificant; 105 min versus zero time, P < 0.01; other values versus zero time, P