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Hormonal Regulation of Two Urea-Cycle Enzymes in Cultured

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Hormonal Regulation of Two Urea-Cycle Enzymes in Cultured Biochem. J. (1983) 216, 281-285 281 Printed in Great Britain Hormonal regulation oftwo urea-cycle enzymes in cultured foetal hepatocytes Annie HUSSON, Mimouna BOUAZZA, Catherine BUQUET and Rene VAILLANT Laboratoire d'Endocrinologie ERA 891, Faculte des Sciences et des Techniques, 76130 Mont-Saint-A ignan, France (Received 5 April 1983/Accepted 11 July 1983) Foetal-rat hepatocytes were cultured in primary monolayer culture, and activity changes of argininosuccinate synthetase (ASS, EC 6.3.4.5) and argininosuccinase (ASL, EC 4.3.2.1) were followed under defined hormone conditions. In hormone-free medium, cultured cells maintained the enzyme activities at values equal to those of freshly isolated cells for at least 3 days. Continuous addition of dexamethasone produced the development of the two enzyme activities, but only after the first 20h of culture. Under these conditions, urea production by the foetal hepatocytes was concomitantly increased in the culture medium. Pretreatment with dexamethasone for 20h was sufficient to produce the development of ASL activity within the 2 following days. Introduced alone, glucagon induced an increase of ASL activity, but did not affect the ASS activity. The most powerful stimulation of ASS and ASL could be observed in cultured hepatocytes if glucagon and dexamethasone were added simultaneously or sequentially. These results indicated that the development of the receptor complex for the induction of urea-cycle enzymes appears early before birth and established that glucocorticoids amplify the glucagon stimulation of these enzyme activities during foetal life. During development of the foetal rat liver, the To obtain more definite information, we have used appearance of a number of enzymes has been shown primary culture of foetal liver cells, which has been to result from hormone action (Jost & Picon, 1970; shown to be a very attractive model for studying Greengard, 1970). Previous studies with whole development of some specific liver functions (Plas & foetal rat livers have indicated that four urea-cycle Nunez, 1976; Leffert & Paul, 1973). A preliminary enzymes, namely carbamoyl phosphate synthetase study has shown that, in such cells, the urea-cycle (EC 6.3.4.16), ornithine transcarbamoylase (EC enzyme activities were stable without hormone 2.1.3.3), argininosuccinate synthetase (ASS, EC addition in the culture medium (Husson et al., 1982). 6.3.4.5) and arginase (EC 3.5.3.1), appeared in the In the present investigation, cultured hepatocytes liver of 17.5-day foetuses and could not develop derived from 17.5- or 18.5-day foetuses have been without glucocorticosteroids (Gautier et al., 1977). tested for their enzyme response to dexamethasone Argininosuccinate lyase (ASL, EC 4.3.2.1) was the and glucagon. Several studies on urea-cycle enzymes only enzyme of the cycle that could not be affected in cultured adult cells had been made (Schimke, by steroid addition or deprivation before term 1964; Gebhardt & Mecke, 1979; Lin et al., 1982), (Husson & Vaillant, 1979). In addition, ASL but the development of urea-cycle enzyme activities activity could be induced prematurely by administra- in cultured foetal hepatocytes has never been tion of glucagon (or dibutyryl cyclic AMP) and by investigated. thyroxine in utero (Husson & Vaillant, 1978). We also reported that, in corticosteroid-deprived foetuses, glucagon failed to promote ASL activity, Materials and methods although simultaneous administration of glucagon and cortisol increased the activity far more than the Materials increases caused by a single hormone (Husson & Animals. Adult female rats of the Wistar strain Vaillant, 1982). These results showed that a syner- were mated overnight and recognized as pregnant by gistic interaction occurred between steroid and a vaginal smear on the next morning. This was glucagon on ASL activity. designated as day 0.5 of pregnancy. Livers from Vol. 216 282 A. Husson, M. Bouazza, C. Buquet and R. Vaillant 17.5-18.5-day foetuses were used to prepare the Cell viability was tested by exclusion of the dye primary cultures. Trypan Blue, and the proportion of viable cells in Chemicals. Glucagon was from Novo Terapeut- final cell suspensions was always over 90%. isk Laboratorium (Copenhagen, Denmark). Dexa- Fresh medium with 5% foetal-calf serum was methasone, ornithine and Hepes [4-(2-hydroxy- given 5h after plating and every 24h thereafter. ethyl)-1-piperazine-ethanesulphonic acid] were ob- Additions of hormones were eventually performed tained from Sigma Chemical Co. (St. Louis, MO, 5h after plating or when otherwise indicated, and U.S.A.). Collagenase and foetal-calf serum were enzyme activities were measured at the specified purchased from Boehringer (Mannheim, Germany). times thereafter. Samples of culture medium were The culture medium was a mixture of Eagle's removed at daily intervals and assayed for urea Minimal Essential Medium and Medium 199 with concentration, as described by Husson et al. (1981). Hanks salts, deficient in arginine, obtained in powder Urea production is expressed as,umol/h per mg of form from Eurobio (Paris, France). Glucose con- total protein. centration was 5.5 mm. This medium was supple- Preparation of cell extracts. After removal of mented with 0.1 mM-ornithine, 5% or 10% (v/v) culture medium, cell monolayers were washed twice foetal-calf serum and Fungizone (0.25,ug/ml) and with ice-cold Hepes buffer and the cells were kanamycin (lOO1ug/ml), both from GIBCO (Grand harvested by scraping with a rubber policeman. The Island, NY, U.S.A.). cell suspension was collected by centrifugation at All liquids were filtered through 0.22,um-pore- 700g for 5min, and the pellet was homogenized in diameter membranes (Millipore, London, U.K.) 0.1% N-cetyl-NNN-trimethylammonium bromide by before use. freezing and thawing twice. All enzyme assays were made on fresh extracts. Methods Enzyme assay procedures. The homogenate was Culture offoetal rat hepatocytes. This was done centrifuged at 4000g for 15 min, and samples by the method of Leffert & Paul (1972), with some of the supernatant fraction were used for enzyme modifications. For a typical culture, about 30 assays and protein determination (Lowry et al., foetuses (17.5 or 18.5 days old) were delivered by 1951). The activities of ASS and ASL were caesarean section and decapitated immediately. The determined by the procedures of Ratner (1955). All livers were collected aseptically, and cut into six to enzyme activities are in units of umol/h at eight pieces, which were then pooled and washed 380 C, and specific activities are units/mg of pro- twice in lOmM-Hepes buffer, pH 7.5. The liver pieces tein. Results are expressed as means + S.E.M., the were next incubated in a dissociation medium numbers of observations being given in parentheses. (Hepes buffer) with 0.03% collagenase for 10min Significance was determined by using a paired at 370C, with gentle magnetic stirring. This enzymic Student's t test. digestion was performed twice, and the cell sus- pensions were then centrifuged for 1min at 50g Results and discussion in conical centrifuge tubes. The resulting pellets were pooled and washed three times in Hepes buffer, and Freshly isolated cells became firmly attached to packed cells were finally suspended in the growth the flasks 5 h after inoculation, and within the first medium. This selective medium, without arginine, 24 h they formed a monolayer in which the cells were prevented overgrowth of fibroblasts (Leffert & Paul, in close contact with neighbouring cells. ASS and 1972) which cannot synthesize arginine from orni- ASL activities could be assayed reproducibly in thine. Cell suspension (1 ml; approx. 2 x 106 cells/ homogenates prepared from freshly isolated cells or ml) was added to each 25cm2 tissue-culture flask from cultured cells. In such extracts (Fig. 1), the two with 4 ml of growth medium and incubated at 370C enzyme activities from 18.5-day foetal hepatocytes in a humidified incubator under 5% CO2 in air (CO2 did not show any significant increase during 3 days incubator; Forma Scientific, Marietta, OH, U.S.A.). in control medium. The rate of urea synthesis in The plating medium contained 10% foetal-calf culture medium measured each day also remained serum to promote cell attachment. unaltered. As shown for other specific functions To remove endogenous steroids, the foetal-calf (Plas & Nunez, 1976; Leffert & Paul, 1973), these serum was charcoal-treated before use. The serum data demonstrate that the capacity for urea syn- was incubated for 20h at 4°C in the presence of thesis is maintained in cultured foetal hepato- activated charcoal (10mg/ml) and centrifuged cytes and can be studied under various hormonal (lOOOOOg for 30min) at 4°C to eliminate the conditions. charcoal. This procedure removes all the endo- genous corticosteroids, as measured by using a Effect ofdexamethasone competitive protein-binding radioassay (Murphy, The requirements for the glucocorticoid inducer 1967). are shown in Fig. 1 and Table 1. When dexa- 1983 Regulation of urea-cycle enzymes in foetal hepatocytes 283 1.5 (a) 0.7 r (b) ._ 0 u s- m 1.0 t-0~ 0.5t- 0 0 A7 A m -E E .0 0tCt 0.31- 3= 0.51 _- ._ - cn CLI0.1 0 0 1 2 3 0 1 2 3 Time in culture (days) Time in culture (days) nn7 _ r E (c) 0. 0.05 1I _E - 0. 0 .° '- 0.03 c) u U o. o0 1 2 3 Time in culture (days) Fig. 1. Effects ofdexamethasone on the activities ofASS (a) and ASL (b) and on urea production (c) in 18.5-dayfoetal hepatocytes Cells were maintained in control medium (@) or in the presence of 1 4uM-dexamethasone (A). Hormone was added 5 h after seeding, or 48h thereafter. After the first 20h of exposure to hormone, the medium was removed from some flasks and the cells were washed twice before reincubation in control medium.
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