The regulation of hepatic tyrosine aminotransferase

433

Biochimica et Biophysica Acta, 677 (1981) 433-444

Elsevier/North-HollandBiomedicalPress BBA 29766 THE REGULATION OF HEPATIC TYROSINE AMINOTRANSFERASE PETER ]'. EVANS * Biochemistry Laboratory, Department of Zoology, University of Oxford, South ParksRoad, Oxford OX1 3PS (U.K.}

(Received March 2nd, 1981)

Key words: Enzyme regulation; 7~yrosineaminotransferase; (Rat liver)

Tyrosine aminotransferase induction has been studied in hepatocytes from untreated, partially and fully glucocorticoid-induced rats: enzyme activities were initially 12.9 -+ 1.7 (n = 16), 41.4 -+3.2 (n = 6) and 117.9 -+ 10.5 (n =,7) munits/mg protein, respectively. Untreated or fully induced hepatocytes maintain initial levels, whereas partially induced hepatocytes increase their tyrosine aminotransferase activity even in the presence of actinomycin D. Fully induced hepatocytes possess a normal protein synthesizing machinery and the mechanisms to degrade selectively tyrosine aminotransferase. The effect of progesterone treatment is consistent with these cells retaining a high dexamethasone level. Glucagon induces tyrosine aminotransferase via its second messenger, cyclic AMP. This induction decreases dramatically with in vivo glucocorticoid treatment. Time courses and effects of inhibitors are consistent with these in vivo and in vitro treatments being alternative methods of inducing tyrosine aminotransferase by the same basic pretranslational step. Introduction Tyrosine metabolism in mammals proceeds mainly via fumaric and acetoacetic acids [1]. This pathway, besides being of intrinsic biochemical interest, is of considerable clinical importance. The enzyme-Ltyrosine: 2-oxoglutarate aminotransferase (tyrosine aminotransferase; EC 2.6.1.5) which catalyses the initial transamination reaction appears, at least in the liver, to be the rate-limiting step in the sequence [2]. Considerable attention has been paid to this enzyme because of two rather unusual properties. Firstly, it can be induced many-fold over its basal activity by a number of agents, which include hormones and nutritional factors. These effects occur in vivo [3], in perfused liver [4,5], in hepatomas [6] and in untransformed primary hepatocytes [7]. Secondly, it * Present Address: Department of Biochemistry, University of Nottingham Medical School, Queen's Medical Centre, Nottingham NG7 2UH, U.K. Abbreviation: Hepes, N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid.

has a short half-life of the order of 1.5 h [8]. This means that, in addition to its use as a model system to investigate gene expression in mammals, tyrosine aminotransferase can be used as a system in the study of intracellular protein degradation, an area of biochemistry that remains one of the least understood. The systematic investigations in established cell lines, while providing useful experimental models, may be too simplified to extrapolate directly to the situation in vivo. For example, induction of tyrosine aminotransferase is brought about by serum alone in hepatomas [9] but these often lack adenyl cyclase [10] and have a modified or uncharacteristic pattern of protein kinase activity [11 ]. The interactions between various hormones in tyrosine aminotransferase induction is incompletely understood. This is particularly true for the interplay between glucocorticoids and cyclic AMP. Wicks et al. [12] favour a translational action for cyclic AMP, while more recently Ernest and Feigelson [13] indicated that cyclic AMP can increase tyrosine aminotransferase mRNA synthesis in vivo. Primary hepato-

0304-4165/81/0000-0000/$02.50 © 1981 Elsevier/North-HollandBiomedicalPress

434 cytes appear to approximate more closely to the situation in vivo than do transformed cells. Controversy surrounds the induction of tyrosine aminotransferase in freshly prepared hepatocytes by glucocorticoids. Haung and Ebner [7] obtained a relatively high response whilst others [14,15] report at best a minimal induction. Ernest et al. [16] found that there was an absolute requirement for both glucocorticoids and cyclic AMP to obtain an increase in tyrosine aminotransferase levels. In view of these discrepancies a systematic appraisal of the inducing agent in primary rat hepatocytes was undertaken. Isolated hepatocytes still differ in many respects from their in vivo counterparts. For example, glycogen is lost during the preparative procedures [17], the cells undergo 'reversible' loss of ions and of low molecular weight metabolites [18] and, of course, cell-cell interaction and communication are eliminated. A major difficulty of in vitro work is trying to reconstruct the rather undefined highly complex medium which bathes the liver in vivo. Hepatocytes are taken from an environment in which many hormones may have been present. It is likely that the metabolic capacity of isolated hepatocytes is governed, to some extent, by the 'memory' of their previous hormonal environment. Hormonal antagonists or inhibitors may have unforeseen effects in the intact animals [19]. The removal of endocrine glands e.g. adrenalectomy, can lead to equivocal results. Complications include increased synthesis of glucocorticoid receptors [20] and unforeseen contributions from accessory adrenal glands [21]. The culture of normal hepatocytes as monolayers for a few days produces a tendency to revert to the foetal condition [22]. Lemonnier et al. [23] have suggested that the lack of response to glucocorticoids in their culture of long-term human liver cells may be due to deprivation of their in vivo environment. In addition, Weinstein et al. [24] have observed that normal cell lines of rat liver do not increase tyrosine aminotransferase levels in response to hydrocortisone while morphologically similar hepatoma cell lines do express this capacity. From the above it is clear that the direct extrapolation of in vitro hepatocytes data to the in vivo situation is not without ambiguities. In the present work, it was decided to use a combination of in vivo and in vitro affects whereby rats were treated

with glucocorticoids for varying lengths of time before the preparations of the hepatocytes. It was hoped that such a technique would lead to a better understanding of the interplay between glucocorticoids and cyclic AMP in tyrosine aminotransferase induction. Materials and Methods Animals. Male Wistar rats (200-250 g) were bred in the Zoology Department, Oxford University. They were housed under a reverse 12 h light-dark cycle (light period 00.45-12.45 h) and allowed free access to water and PMD pellets supplied by Labsure. Corticosterone and hydrocortisone are rather rapidly 'removed' by the intact animal and repeated injections of these hormones are required to maintain elevated tyrosine aminotransferase levels. Thus, dexamethasone was used in the present studies. It was injected intraperitoneally at a dose of 10 mg/ 100 g body weight, which is reported to produce a maximum response [25]. The in vivo glucocorticold response takes about 8 h to reach its peak and this level is maintained for at least another 4 h [25]. Partially induced animals were injected at appropriate times on the morning of the experiment and used for hepatocyte preparation at l l.30h. Rats which had been fully induced were generally injected at 02.00 h and the cells prepared at 11.30 h. In some cases, however, rats were injected with the synthetic hormone at 11.30 h and hepatocytes isolated 7.5 h later. The latter experiments were performed to ensure that a glucocorticoid response could be elicited at 11.30 h, since this was the time when most of the hepatocytes from untreated animals had been prepared. Chemicals. L-Tyrosine, sodium diethyldithiocarbamate, pyridoxal 5'-phosphate, dexamethasone, hydrocortisone, progesterone, actinomycin D, chloroquine, penicillin G, dibutyryl cyclic AMP, glucagon, insulin, streptomycin sulphate, bovine serum albumin (Fraction V), Norit A and cycloheximide were purchased from Sigma Chemical Co., London; 2-oxoglutarate was from BDH, Poole, Dorset; NADH and collagenase came from Boehringer Mannheim; sodium pyruvate was supplied by Koch; Joklik's modification of minimum essential medium and foetal calf serum were obtained from Flow Laboratories. Prior to use, the

435 bovine serum albumin solutions and foetal calf serum were charcoal-treated. This involved shaking the solutions with Norit A (50 mg/ml solution) for 1 h at 37°C. The resulting suspension was filtered through Whatman No. 1 paper (twice) and then through a 0.45 bun millipore filter. The filtrate was finally exhaustively dialysed against bicarbonate saline containing penicillin (75 000 units/l) and streptomycin (50 mg/ml).

Preparation and culture of primary hepatocytes Primary hepatocytes were prepared by procedures essentially similar to those of Crane and Miller [14] but with the following modifications. The perfusion medium consisted of calcium-free KrebsHenseleit bicarbonate buffer [26] supplemented with 75 000 units of penicillin and 50 mg of streptomycin per 1. A stream of water-saturated 95% 02/ 5% CO2 mixture was passed directly and constantly through the medium at a flow rate of 2 - 3 1 per min. Rats were anaesthetised by nembutal. Heparin (300 units) was injected by means of a bent syringe needle into the inferior vena cava. After approximately 25 min of recirculating perfusion in the presence of 0.02% collagenase the liver was removed from the apparatus and disrupted by the blunt scissorsspatula-plastic beaker technique of Krebs et al. [18], using a 500 /zm nylon mesh. Except where otherwise indicated the cells were pelleted by centrifuging at 5 0 × g for 2 rain. The cells were then washed (six times) at 37°C in Krebs-Henseleit bicarbonate buffer containing penicillin, streptomycin and 0.2% charcoal-treated bovine serum albumin. Each wash entailed incubating for 3 min with gentle shaking under an atmosphere of 95% 02/5% CO2. After the final wash the cells were resuspended either in the washing medium or in the cell culture medium. Cells were counted by means of an improved Neubauer haemocytometer. A 200 g rat gave ( 3 - 5 ) • 108 viable cells. Cells so prepared incorporated L- [4,5-a H] leucine into intracellular and secreted proteins in a manner essentially identical to that observed by Crane and Miller [14]. The incubation medium was Joklik's modification of minimum essential medium, pH 7.5, supplemented with 10% charcoal-treated foetal calf serum. Hormones and other chemicals were added as concentrates prior to the introduction of the cells. 20-ml

cultures were set up in 250 ml Erlenmeyer flasks, containing between 5 . 1 0 s and 1.75. 10 6 cells/ml, and were incubated with gentle shaking at 37°C. The contents of the flasks were gassed (95% 02/5% CO2) at a flow rate of 300 mt/min. Aliquots were taken at hourly intervals, chilled on ice and centrifuged at approximately 100 × g in a Bai}d and Tatlock bench microangular centrifuge for 3 min. The cell pellets were retained and stored in a frozen condition. Cell viability during the incubation period was monitored microscopically and the percentage of living cells determined as No. of cells excluding 0.25% trypan blue × 100. Total No. of cells

Preparation of cytosol fraction The frozen cell pellets were resuspended in 0.1 M potassium phosphate (pH 7.6) and 0.2 mM pyridoxal phosphate. The cells were then lysed by means of two 10-s bursts at an amplitude setting of 6 /am in a M.S.E. sonicator, the containing tube being cooled in ice. The preparation was finally centrifuged at 105 000 × g for 1 h and the supernatant retained for the enzyme assays.

A ssays Enzyme assays were carried out at 37°C. Tyrosine aminotransferase was assayed by two methods: A. A modified Diamondstone assay [27]. The reaction medium contained 14 btmol tyrosine in 0.2 M potassium phosphate, 30 pmol 2-oxoglutarate, 0.5 pmol pyridoxal phosphate, 12 pmol diethyldithiocarbamate in a final volume of 3 ml (pH 7.6). A minimum of 10 points were used in the measurement of the initial velocity of the reaction. B. A method based on the formation of an enolborate complex of p-hydroxyphenylpyruvatein borate buffer. Conditions were employed to obviate the need to add keto-enol tautomerase. Basically, this continuous method was performed under the same conditions as assay A but the reaction medium was supplemented with 0.322 M boric acid. The latter is a lower concentration than is usually employed because boric acid suppresses the rate of spontaneous tautomerization of the keto to the enol form of p-hydroxyphenylpyruvate [28]. The molar extinction coefficient for the enol borate p-hydroxyphenyl-

436 pyruvate complex under the condition used was 5600 M - l " c m -a at 308 nm (unpublished data). 1 munit of tyrosine aminotransferase activity catalysed the formation of 1 nmol of p-hydroxyphenylpyruvate per min at 37°C. Preliminary experiments showed that for primary hepatocytes the reaction was proportional to the number of cells sonicated; it was completely dependent upon added tyrosine but was unaffected by 35 /~rnol aspartate. In the absence of added 2-oxoglutarate only a small reaction occurred. Two systems have also been used to estimate the activity of aspartate aminotransferase (EC 2.6.1.1). A. The Karmen assay [29] obtained as a kit from the Sigma Chemical Company and carried out at 25°C. B. The assay method of Banks et al. [30] except that a standard substrate solution was prepared containing 30 ktrnol 2-oxoglutarate, 30 Bmol aspartate and 0.5 ~mol pyridoxal phosphate in 2.8 ml 0.1 triethanolamine. Reactions were started by the addition of 0.2 ml of a suitably diluted enzyme solution and run at 37°C. Lactate dehydrogenase (EC 1.1.1.27) was measured by a modification of the method of Wroblewski and La Due [31 ]. The assay mixture contained in a total volume of 2.2 ml: 0.5 M potassium phosphate, pH 7.4, 0.2 ml of a 0.1% solution of NADH, 5 mM sodium pyruvate and a suitably diluted enzyme preparation. Protein was estimated by the method of Lowry et al. [50], as modified by Houk and Ue [32] using bovine serum albumin as a standard. Absorbances were read at the appropriate wavelengths on a Zeiss PMQ 11 spectrophotometer or on a Gilford 240 spectrophotometer connected to a linear recorder. The results have been expressed in terms of a percentage of the initial tyrosine aminotransferase activity. Cell breakage was monitored by measuring the activity of lactate dehydrogenase in the same cell preparation used for tyrosine aminotransferase activity observations, since lactate dehydrogenase only appears after cell breakage.

the effects of likely inducers. The basic culture medium was supplemented with insulin (10 munits/ ml), glucagon (10 -7 M) and dexamethasone (10 -6 M). Fig. 1 shows that rat tyrosine aminotransferase is induced approximately 3.5-fold when corrected for cell breakage under the assay conditions. 25 mM Hepes (pH 7.5), which was used as a precaution against pH changes, had no effect on the induction process and was, therefore, omitted from further experiments unless otherwise indicated. The addition of hormones, singly or in combinations, showed that the primary inducing agent is glucagon rather than insulin, glucocorticoids or a combination of glucagon and glucocorticoid (Table I). Several criteria showed that the inducation was not the result of bacterial contamination.: (i) bacteria were not observed by microscopic examination during the incubation period; (ii) in cultures using

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Fig. I. Induction of tyrosine aminotransferase in primary rat hepatocytes. Basic incubation medium supplemented with insulin (10 munits/ml), glucagon (10 -7 M), dexamethasone (10 -6 M) and Hepes (25 mM) *, Tyrosine aminotransferase activity; m,lactate dehydrogenase activity.

437 TABLE I THE EFFECTS OF HORMONES ON TYROSINE AMINOTRANSFERASE ACTIVITY IN RAT HEPATOCYTES The tyrosine aminotransferase activity at 6.5 h is expressed as a percentage of its initial activity (100%) corrected for cell breakage. The latter value was 12.9 ± 1.7 munits/mg protein (n = 16). Additions

No. of separate cell preparations

Tyrosine aminotransferase

activity (%) None Insulin (10 munits/ml) + glucagon (10 -7 M) + dexamethasone (10 -6 M) Glucagon (10-7 M)

Insulin (10 munits/ml) Dexamethasone (10- 6 M) Glucagon (10 -7 M) + dexamethasone (10- 6 M) Dibutyryl cAMP (5 10-6 M) + dexamethasone (10 -6 M) Dibutyryl cAMP (5 10-6 M) + progesterone (10 -s M) Dibutyryl cAMP (5 10- 6 M) Dibutyryl cAMP (5 10- 6 M) + actinomycin D (0.3 #g/ml) Dibutyryl cAMP (5 10- 6 M) + cycloheximide (50 #g/ml) Dibutyryl cAMP (5 10-6 M) + glucagon (10-7 M)

cells which stained with trypan blue (i.e. dead cells) an increase in tyrosine aminotransferase activity was never observed; (iii) in the occasional experiment in which the cells died after a few h culture, the decrease in enzyme activity corresponded with cell mortality; (iv) the induction was inhibited by the eukaryotic translational inhibitor cycloheximide but not by the prokaryotic translational inhibitor chloramphenicol; (v) the induced enzyme coincided on gels with the normal partially purified rat enzyme. 10 -7 M glucagon caused an increase in tyrosine aminotransferase activity of between 3- and 4-fold over a 7 h period. Neither dexamethasone nor hydrocortisone alone produced an increase in tyrosine aminotransferase activity. Glucocorticoid plus glucagon caused a minimal, if any, increase over glucagon alone. Alcoholic solutions of the glucocorticoids were added to some cultures (to make a final concentration of 0.005% alcohol) instead of aqueous suspensions. No differences in effect were observed. An increase from 10 -6 to 10 -s glucocorticoid had no effect. Since glucocorticoids have often been suggested as the primary inducing agent o f tyrosine aminotransferase messenger RNA a detailed survey of all the reagents used in the present investigation was made to ascertain whether any could act as a potential

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glucocorticoid source. The charcoal-treated 0.2% bovine serum albumin used in washing procedures in the hepatocyte preparation was replaced by Joklik's minimal essential medium, pH 7.5, supplemented with 10% treated foetal calf serum. Heparin was omitted from the procedure. Animals were killed by cervical dislocation or by decapitation instead of by nembutal injection. Omission of heparin and nembutal reduced the number of cells available for cell culture. However, the cells when cultured showed in all cases an induction pattern identical to those in Table I. Since tyrosine aminotransferase fluctuates in its diurnal activity, and the level of endogenous glucocorticoids also varies during the day, cell preparations were started at times ranging from 9.00 to 17.00 h. Great care was taken to avoid exciting the animals prior to commencing the experiments. Cervical dislocation or decapitation, in addition to avoiding any anaesthetic effects nembutal may have on tyrosine aminotransferase, eliminates the necessity of a potentially stressful intraperitoneal injection and the possible attendant release of glucocorticoids. The modifications did not alter the induction profiles. Calcium (1.8 mM) was added to the cell culture medium since this metal ion had been omitted from all the experimental solutions. Once again there was

438 no change in the response. The glucocorticoid anti-inducer progesterone (10 -s M) did not inhibit the induction process. Replacement of glucagon by 5 • 10 -6 M dibutyryl cyclic AMP, however, led to an induction of tyrosine aminotransferase essentially identical to that caused by glucagon (Table I). The glucagon and dibutyryl cyclic AMP were not additive. In all these experiments the measured tyrosine aminotransferase activity was unaffected by 35 grnol of aspartate and the activity of aspartate aminotransferase remained approximately constant during the culture period. These observations establish that the tyrosine aminotransferase activity being measured was due to cytosolic tyrosine aminotransferase and was not due to the release of mitochondrial aspartate aminotransferase [33]. The mechanism of action of dibutyryl cyclic AMP was investigated. As shown in Fig. 2, the potential for tyrosine aminotransferase induction persists for at least 4 h after the incubation commences and the addition of dibutyryl cyclic AMP at this time results in an identical time course for tyrosine aminotrans-

ferase induction. The induction is inhibited by both actinomycin D (0.3 btg/ml) and cycloheximide (50

~/ml). The presence of tyrosine (6.08 mM) in the culture medium did not modify the tyrosine aminotransferase induction caused by dibutyryl cyclic AMP or by the combined presence of dibutyryl cyclic AMP and dexamethasone (cf. Ref. 34).

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Fig. 3. Induction of tyrosine aminotransferase in rat hepatocytes pretreated in vivo with dexamethasone. Timing: preparation started 1.5 h after dexamethasone injection and the hepatocytes put into culture 1.5 h later. Tyrosine aminotransferase activity in basic culture medium supplemented with: o, Hepes (25 mM), CaC12 (1.8 mM) and dibutyryl cyclic AMP (5 • 10-6 M); A, Hepes (25 mM) and CaC12 (1.8 mM); o, Hepes (25 mM), CaC12 (1.8 mM) and actinomycin D (0.3 ~g/ml); =, lactate dehydrogenase activity in basic culture medium supplemented with Hepes (25 mM), CaC12 (1.8 mM) and actinomycin D (0.3 ~tg/ml) (representative of the other lactate dehydrogenase activities).

439

Induction of tyrosine aminotransferase in primary hepatocytes obtained from partially glucocorticoidinduced rats Cultures were started some 3 - 4 h after glucocorticoid injection. The initial tyrosine aminotransferase activity in these hepatocytes when freshly isolated was 41.4-+ 3.2 munits/mg protein (n --- 6, where n is the number o f separate cell preparations). The control flask, without hormonal additions, repeatedly showed an increase in tyrosine aminotransferase activity during culture (Figs. 3 and 4) and this

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was only slightly affected b y the addition o f actinomycin D (Fig. 3). The presence of dexamethasone in the culture medium did not enhance the in vivo dexamethasone induction, however, dibutyryl cyclic AMP did have this effect (Figs. 3 and 4). The dibutyryl cyclic AMP response could not be mimicked by the cyclic AMP phosphodiesterase (EC 3.1.4.17) inhibitor theophylline (Fig. 4).

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Fig. 4. Induction of tyrosine aminotransferase in rat liepatocytes pretreated in vivo with dexamethasone. Timing: preparation started 2.5 h after dexamethasone injection and the hepatocytes put into culture 1.5 h later. Tyrosine aminotransferase activity in basic culture medium, supplemented with: A, Hepes (25 mM) and CaCl2 (1.8 mM); e, Hepes (25 mM), CaC12 (1.8 mM) and dibutyryl cyclic AMP (5 • 10 -6 M); o, Hepes (25 mM), CaCI2 (1.8 mM) and theophylline (3.3 mM). Lactate dehydrogenase activity in basic culture medium supplemented with: % Hepes (25 mM) and CaC12 (1.8 mM); • , Hepes (25 mM), CaC12 (1.8 mM) and dibutyryl cyclic AMP (5 • 10 -6 M); D, Hepes (25 mM), CaCI2 (1.8 mM) and theophylline (3.3 mM).

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Fig. 5. The response of hepatocytes in vitro prepared from rats fully induced with glucoeortieoids in vivo. Timing: preparation started 9_5 h after injection of dexamethasone and hepatocytes put into culture 1.5 h later. Tyrosine aminotransferase activity in basic culture medium supplemented with: o, Hepes (25 mM), CaCI2 (1.8 mM) and dibutyryl cyclic AMP (5 • 10--6 M); o, Hepes (25 mM), CaCI2 (1.8 mM) and progesterone (10 --4 M). Lactate dehydrogenase activity in basic culture medium supplemented with: =, Hepes (25 mM), CaC12 (1.8 mM) and dibutyryl cyclic AMP (5 • 10-6 M); % Hepes (25 mM), CaCI2 (1.8 raM) and progesterone (10 -4 M).

440

Induction of tyrosine aminotransferase in primary hepatocytes obtained from fully glucocorticoidinduced rats Some of the results of incubation of hepatocytes from rats previously treated with glucocorticoids for 8 h and longer is shown in Figs. 5 and 6. Such hepatocytes showed a tyrosine aminotransferase activity of I 17.9 +_ 10.5 munits/mg protein (n = 7, where n is the

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Fig. 6. The effect of inhibitors of gone expression on tyrosine aminotransferase activity in hepatocytes prepared from rats fully induced with glucocorticoids in vivo. Timing: preparation started 9.5 h after injection of dexamethasone and hepatocytes put into culture 1.5 h later. Tyrosine aminotransferase activity in basic culture medium supplemented with: o, Hopes (25 mM), CaC12 (1.8 mM) and cycloheximide (50/sg/ ml);.., Hopes (25 mM), CaCI2 (1.8 rnM) and eordycepin (5 ~g/ml); A, Hopes (25 mM), CaCI2 (1.8 mM) and aetinomycin D (0.3 ,ug/ml). Lactate dehydrogenase activity in basic culture medium supplemented with: o, Hopes (25 mM), CaC12 (1.8 mM) and cycloheximide (50/ag/ml); t~, Hopes (25 raM), CaC12 (1.8 mM) and cordycepin (5 /~g/ml); % Hopes (25 mM), CaC12 (1.8 mM) and actinomycin D (0.3 ,ug/ml).

number of separate cell preparations). However, the tyrosine aminotransferase activity in the induced hepatocytes showed no tendency to fall when cultured in media devoid of added hormones. Moreover, the tyrosine aminotransferase activity in these cells was not increased by dibutyryl cyclic AMP (Fig. 5), by dibutyryl cyclic AMP and dexamethasone, nor by insulin. 10 -s M progesterone had no effect, at 10 -~ M its addition had a relatively small effect (Fig. 5) while higher concentrations lysed the hepatocytes due to the detergent action of the steroid (both tyrosine aminotransferase and lactate dehydrogenase activities rapidly fell to zero). The level of tyrosine aminotransferase activity in induced hepatocytes was assayed by both the continuous and the Diamondstone [27] methods. The induced hepatocytes incorporated L- [4,5-aH] leucine into intra- and extracellular protein in a manner identical to that of untreated hepatocytes. The addition of inhibitors of gene expression such as actinomycin D, cordycepin and cycloheximide (Fig. 6) resulted in a sharp decrease in tyrosine aminotransferase levels relative to the corresponding lactate dehydrogenase activities. When cycloheximide was added to cultures of induced hepatocytes tyrosine aminotransferase was selectively degraded over an initial 2 h period, with a half-life of 1.88 -+0.16 h (n --4 separate cell prep, arations). The corresponding tu2 of tyrosine aminotransferase in cultures of hepatocytes from animals not pretreated with dexamethasone was 2.29 -+0.33 h (n = 4 separate cell preparations). The slowing down of tyrosine aminotransferase degradation at longer time periods (Fig. 6) is due to the fact that cycloheximide is an inhibitor of intracellular protein degradation. Discussion

Under the conditions used in the present investigations, either glucagon or dibutyryl cyclic AMP alone bring about the induction of tyrosine aminotransferase in primary rat hepatocytes in suspension culture. These results are at variance with those obtained by Ernest et al. [16] but they are not really surprising as it is known that these agents increase tyrosine aminotransferase messenger RNA and tyrosine aminotransferase activity in vivo in the absence of any change in the levels of glucocorticoids [13].

441 The effects of glucagon and dibutyryl cyclic AMP are not additive (Table I), indicating that the action of the hormone was mediated through its second messenger cyclic AMP. It is far more surprising that glucocorticoids fail or have only very limited effects on tyrosine aminotransferase induction. The specific activity of tyrosine aminotransferase in the hepatocytes (Table I) is very similar to that reported by Lin and Knox [3] in uninduced animals. In addition, uninduced rats were not maximally primed with glucocorticoids prior to hepatocyte preparation since injection of dexamethasone (10 mg/ 100 g body weight) 7.5-11 h before killing the animal resulted in a 9-fold increase in tyrosine aminotransferase activity in isolated hepatocytes. The possible release of glucocorticoids during the operative procedure seems unlikely to have contributed to the induction process because the addition of dibutyryl cyclic AMP after 4 h in culture (Fig. 2) gave an identical induction profile to that added at zero time and was still inhibited by actinomycin D (cf. Ref. 19). This premise is supported by the fact that the glucocorticoid antagonist, progesterone [35], had no effect on the action of dibutyryl cyclic AMP (Table I). Explanations for the lack of a glucocorticoid response in the cells might lie in the requirement for a recovery period following collagenase treatment [36], too high a rate of culture oxygenation or that some cell contact is required for glucocorticoid action. The mechanism of the action of cyclic AMP in tyrosine aminotransferase induction is controversial. At least three independent sites of action are possible, namely enzyme activation, gene transcription and translation. The fact that the increase in activity takes 7 h to reach a maximum value does not support a simple conversion of the enzyme from an inactive to an active form. The time taken together with the inhibition of the tyrosine aminotransferase increase by cycloheximide suggests that the phosphorylation activation of tyrosine aminotransferase as expounded by Hamm and Seubert [37] may have no physiological significance. Cells incubated in the absence of any hormonal additions for 4 h are still capable of being induced by the addition of cyclic AMP. This fact could be explained on the translational model by the presence of long.lived tyrosine aminotransferase messenger RNA. However, the iden-

tical time course for the tyrosine aminotransferase induction process at 0 and 4 h plus the total inhibition of induction by actinomycin D at both these times is more consistent with cyclic AMP exerting its effect at the pretranslational level. Results obtained from hepatocytes isolated at various times after a glucocorticoid injection to the donor animal appear to shed some light on the mechanism of action and interplay between the hormones in tyrosine aminotransferase induction. A clear difference between basal and partly induced hepatocytes is that the latter show an increase in tyrosine aminotransferase activity during subsequent culture (Figs. 3 and 4). This rise in tyrosine aminotransferase activity is inhibited only slightly by actinomycin D (Fig. 3). The results are consistent with glucocorticoid treatment in vivo resulting in an increased level of tyrosine aminotransferase messenger RNA [38]. The latter is then present in a large enough quantity, initially, to allow tyrosine aminotransferase to be synthesized in the isolated hepatocytes in vitro in the presence of the transcriptional inhibitor actinomycin D. Although dexamethasone increases tyrosine aminotransferase in vivo, its addition to cells in vitro, which have been partially induced, does not potentiate the action of this hormone. Hence, in vivo priming does not enhance the glucocorticoid response in vitro. The induction in the hepatocyte preparations caused by dibutyryl cyclic AMP is of particular interest. The magnitude of its inducing power and its value relative to controls, fell off dramatically as the in vivo glucocorticoid affect was extended. The values were 3.2-, 2.5-, 2.0- and 0-fold, respectively, relative to the initial value when the cultures were started 0, 3, 4 and 8 h (or longer) after the in vivo glucocorticoid injection. These represent increases in tyrosine aminotransferase activity relative to the initial values of approximately 300, 80, 45 and 0 when compared with controls without dibutyryl cyclic AMP. The absolute increases were 30.2, 26.0, 18.6 and 0 munits/mg of protein. Examination of the time course of tyrosine aminotransferase induction in partially induced hepatocytes (Figs. 3 and 4) show that, although dibutyryl cyclic AMP increases the tyrosine aminotransferase activity, its presence does not modify the initial rate of tyrosine aminotransferase synthesis in these cells. The latter would have been expected if it were acting at a translational step.

442 However, this observation, together with a progressive dimunition in the dibutyryl cyclic AMP induction as the tyrosine aminotransferase activity is increased by glucocorticoids, suggest that, in this system, both hormones are acting on the same process. The results can be interpreted in another way. The 'permissive' action of steroids including glucocorticoids, could be mediated through increased levels of cyclic AMP caused by the ability of these substances to inhibit cyclic AMP phosphodiesterase [39]. In the present work this theory was tested by adding theophylline, the known cyclic AMP phosphodiesterase inhibitor, to partially induced hepatocytes in culture. The results (Fig. 4) revealed that theophylline was unable to mimic the action of dibutyryl cyclic AMP and suggests that in this system dexamethasone does not have its effect by inhibiting the degradation of cyclic AMP. Recent in vivo studies [13,40], together with the work reported in this investigation, indicate that dibutyryl cyclic AMP exerts at least part of its action at the pretranslational level by increasing the tyrosine aminotransferase messenger RNA. This seems to rule out mechanisms of cyclic AMP which only affect translational machinery because the transcription of tyrosine aminotransferase is not tightly coupled to its translation [41]. It is interesting that recently a cyclic AMP receptor protein has been reported which on binding to cyclic AMP is translocated to the nucleus in a manner identical to the glucocorticoid receptor complex [42]. Hepatocytes fully induced by dexamethasone in vivo maintain high tyrosine aminotransferase activities when subsequently incubated in vitro in the absence of added hormones. Attempts at removing the glucocorticoids from hepatocytes which have been exposed to these hormones in vivo have not been reported. However, steroids appear to be washed out of hepatomas very easily (e.g. Ref. 43). The washing procedures used in the present work were extensive. This may indicate a fundamental difference between the permeability of normal hepatocytes and hepatoma ceils, or possibly that the hepatocytes used in this work had so high an intracellular concentration of dexarnethasone that 'wash-out' was impracticable. Glucocorticoids are known to affect the uptake of amino acids, sugars, purine and pyrimidine bases and

nucleosides, and metal ions in a variety of tissues [44-46]. Hepatocytes obtained from animals pretreated with glucocorticoids, however, preserve a normal protein synthesizing machinery as shown by the fact that they incorporate L-[4,5PH]leucine into their intracellular and secreted proteins in a manner indistuinguishable from that of hepatocytes obtained from untreated rats. At least one report has suggested that, during the early phase of tyrosine aminotransferase induction by glucocorticoids (first 4 h), the degradation of tyrosine aminotransferase is inhibited [47]. It was possible, in the present work, in vivo treatment with dexamethasone in some way prevented the normal degradation of tyrosine aminotransferase. This theory was tested by adding cycloheximide, the protein synthesis inhibitor, to cultures of induced hepatocytes. The results are shown in Fig. 6. Over the initial period of culture the tyrosine aminotransferase disappears with a half-life of 1.88-+ 0.16 h (n = 4 separate cell preparations), which is the in vivo half-life of the enzyme. Actinomycin D is a transcriptional inhibitor which exerts its effect by intercalating between guanine and cytosine residues in the DNA [48]. Fig. 6 shows that, in the presence of actinomycin D, induced tyrosine aminotransferase levels fall while the activity of lactate dehydrogenase remains constant. This suggest that continued transcription is required to maintain the higher tyrosine aminotransferase activity. An alternative transcriptional inhibitor which does not bind to DNA was also used in this investigation. Cordycepin (3'-deoxyadenosine) is phosphorylated by the hepatocytes to produce 3'-deoxy ATP which then serves as a potential substrate for RNA polymerase (EC 2.7.7.6) [49]. As shown in Fig. 6, cordycepin causes a time-dependent decrease in tyrosine aminotransferase activity, relative to lactate dehydrogenase, in induced hepatocytes. The effects of the three inhibitors, actinomycin D, cordycepin and cycloheximide suggest that both continuing transcription and translation are required for the maintenance of induced tyrosine aminotransferase activity in hepatocytes taken from rats which have been exposed in vivo to glucocorticoids for 8 h or longer. These observations, together with the normal protein synthesis in these cells, confirm that failure of the in vitro occurrence of deinduction is

443

due to the continued presence of inducer rather than to a metabolic defect. Progesterone is recognised as possessing anti-glucocorticoid activity in vitro [35]. Addition of progesterone to hepatomas, the tyrosine aminotransferase activity of which has been induced by glucocorticoids, is known to result in a rapid deinduction of tyrosine aminotransferase [43]. Progesterone was used in the present work to attempt a similar operation in dexamethasone-induced hepatocytes. Progesterone at 10 -s M had no effect on tyrosine aminotransferase levels, while at a concentration of 10 -3 M it rapidly lysed the hepatocytes due to its detergent properties. The result of adding 10 -4 M progesterone to induced hepatocytes is shown in Fig. 5. Relative to lactate dehydrogenase, tyrosine aminotransferase activity fell and its level later stabilized at a somewhat lower value. This is consistent with the induced ceils having a high dexamethasone concentration. The initial fall in tyrosine aminotransferase activity is attributable to the antiglucocorticoid effect of 10-4M progesterone and the stabilization is due to the establishment of an equilibrium state between dexamethasone and its antagonist.

Acknowledgement This work was supported by a grant from the Medical Research Council.

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