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Urea production in long-term cultures of adult rat hepatocytes
Toxic. in Vitro Vol. 8, No. 2, pp. 293-299, 1994
~ ) Pergamon
0887-2333(93)E0017-A
Copyright © 1994 ElsevierScienceLtd Printed in Great Britain. All rights reserved 0887-2333/94 $7.00 + 0.00
U R E A P R O D U C T I O N IN LONG-TERM CULTURES OF ADULT RAT HEPATOCYTES A. SIERRA-SANTOYO,M. L. LrPEZ, A. HERN,ANDEZand T. MENDOZA-FIGUEROA* Pharmacology and Toxicology Department, Centro de Investigaci6n y de Estudios Avanzados del IPN, PO Box 14-740, Mexico City 07000, Mexico (Received 9 March 1993; revisions received 10 August 1993)
Abstract--To study the functionality of the urea cycle in long-term cultures of adult rat hepatocytes, urea production and the activity of two urea cycle enzymes were measured in hepatocytes cultured on 3T3 cells for 15 days. Urea production was also measured in cultures maintained with medium containing either 0.4 mM arginine or 0.4 mM ornithine and in cultures exposed to different concentrations of NH4C1, an in vivo inducer of urea production. In hepatocytes seeded on 3T3 cells, urea production decreased gradually to 50% of the initial value after 15 days. Urea production was similar in 3T3-hepatocyte cultures maintained for 11 days with medium containing ornithine or arginine. Hepatocytes exposed for 24 hr to 1, 3 and 5 mM NH4C1 showed an average increase in urea production of 25, 50 and 69%, respectively, above that of unexposed cultures over 15 days. Ornithine transcarbamylase (OTC) activity decreased by 84% after 5 days in culture and remained constant thereafter, while arginase activity remained constant over 15 days. In contrast, in hepatocytes seeded on plastic substratum, urea production decreased to 24% of the initial value after 8 days in culture. OTC and arginase activities also decreased to 13 and 10% of their initial values after 8 days in culture. These results show that 3T3-hepatocyte cultures from adult rats produce urea from ornithine and/or arginine for at least 15 days and respond to an inducer of urea production as in vivo. They also show that these cultures have decreasing and constant levels of OTC and arginase activities, respectively, owing probably to an adaptative response dependent on substrate concentrations and hormonal regulation. These findings also suggest that 3T3-hepatocyte cultures are a suitable in vitro system to study urea production, its regulation by substrates and hormones and its alteration by drugs and toxic chemicals.
INTRODUCTION A m m o n i a , a catabolite and precursor in some anabolic processes, plays a central role in nitrogen metabolism. A b o u t 90% of excess ammonia is eliminated from the blood mostly through urea synthesis by hepatocytes. Urea production involves a highly regulated cycle in which five enzymes participate: carbamylphosphate synthetase, ornithine transcarbamylase (OTC), argininosuccinate synthetase, argininosuccinate lyase and arginase. These enzymes have been studied extensively in human, rat and bovine liver, and in bacteria (Clarke, 1976; Guth6hrlein and Knappe, 1968; Harell and Sokolovsky, 1972; Kalousek et al., 1978; Lusty and Ratner, 1972; Marshall and Cohen, 1972; Ratner, 1973; Rochovansky and Ratner, 1967). Schimke (1962) reported that all urea cycle enzymes are directly associated with dietary protein consumption in rats. Administration of glucagon and glucocorticoids induces all urea cycle enzymes in vivo and in vitro (Gebhardt and Mecke, 1979; Husson et al., 1985, 1986 and 1987; Snodgrass et al., 1978), whereas *To whom reprint requests should be addressed. DMEM = Dulbecco's modified Eagle's medium; O T C = ornithine transcarbamylase; TEA = triethanolamine-HC1.
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insulin administration has an inhibitory effect on their expression (Husson et al., 1985 and 1986). To study liver functions and their alteration by hepatotoxic chemicals, several experimental systems have been used: perfused livers, liver slices, total and differential homogenates, hepatocyte suspensions, cultures of cell lines derived from liver, and primary cultures of hepatocytes. The last system is one of the most advantageous, since isolated hepatocytes can be cultured in a controlled environment maintaining several differentiated functions. However, they lose most of their characteristics and die after a few days in culture (Feliu et al., 1982; Guguen-Guillouzo et al., 1978). There are few studies on urea production by cultured hepatocytes. In adult rat hepatocytes cultured on plastic substratum, urea production decreased by 80% after 7 days, and the addition of 3 mM NH4CI on the second day increased about two-fold the amount of urea released within 24 hr (Gebhardt et al., 1978). In hepatocytes seeded on collagen membranes and maintained with serum-free medium, urea synthesis decreased by 30% after 13 days, but remained constant if the medium contained insulin, dexamethasone and high concentrations of glucagon (Dich et al., 1988). Alterations in urea production also have been used as an indicator of toxicity in the evaluation 293
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o f different classes of chemicals in hepatocyte cultures
(Acosta and Mitchell, 1981; Hems, 1983; Story et al., 1983). We have described a culture system in which adult rat hepatocytes are seeded on a feeder layer of 3T3 cells that allows their long-term (2 months) survival a n d the maintenance for at least 4 wk of their typical morphology and of several differentiated functions such as albumin secretion, lipid synthesis and secretion, and basal and inducible levels of cytochrome P-450 (Kuri-Harcuch and Mendoza-Figueroa, 1989). In the present study we investigated the functionality of the urea cycle in long-term cultures of adult rat hepatocytes by determining urea synthesis with different media, its inducibility by NH4C1, an in vivo inducer of urea production, and the activities of OTC a n d arginase, t w o urea cycle enzymes localized in the mitochondria and cytosol of parenchymal liver cells.
MATERIALS AND M E T H O D S
Materials. Collagenase type IV, insulin, d-biotin, L-arginine-HC1, L-ornithine-HCl, triethanolamineHC1 (TEA), Li2--carbamoyl phosphate, Tris(hydroxymethyl)aminomethane (Tris-HCl), antipyrine, diacetyl monoxime, citrulline, ninhydrin, hydrocortisone and trypsin type III were obtained from Sigma Chemical Co. (St Louis, MO, USA); mitomycin C w a s a gift from Bristol-Myer Co., Syracuse, NY,
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Fig. 1. Urea production by hepatocytes cultured on different substrata. Isolated hepatocytes were seeded on plastic substratum (O) and on a feeder layer of 3T3 cells (0). Culture dishes containing feeder layers (A) and Dulbecco's modified Eagle's medium (DMEM) (A) were incubated under the same conditions. Values are means + SD of duplicate determinations from five culture dishes. Similar results were obtained in two independent experiments.
USA; urea, manganous chloride, ferric chloride and other reagents of analytical grade were purchased from J. T. Baker (Mexico City, Mexico). Cell cultures. Hepatocytes were isolated from male Wistar rats weighing 180-200 g by the collagenase perfusion method of Berry and Friend (1969), as modified (Mendoza-Figueroa et al., 1983), and cultivated as described (Kuri-Harcuch and MendozaFigueroa, 1989). Briefly, the liver was perfused with TD solution (5.5 mM glucose, 137 mM NaCI, 50 mM KCI, 0.4raM Na2HPO4, 25 mM Tris-HC1, pH 7.4, 0.01% phenol red) for 3 min at 37°C and a flow rate of 15ml/min and again for 10min at a rate of 10 mi/min with 100 U collagenase type IV/ml in TD solution at 37°C. Cells were dispersed in 40 ml Dulbecco's modified Eagle's medium (DMEM), supplemented with 7% calf serum, 5 pg insulin/ml and 0.1 pM d-biotin. The cell suspension was filtered through a nylon mesh and allowed to stand for 10 min; the supernatant was discarded and the cells were resuspended in 20 ml DMEM. 3T3 cells were lethally treated with 10 #g mitomycin C/ml for 2 hr (Rheinwald, 1978), seeded at 35,000 cells/cm2 and incubated at 37°C in a humidified 90% ai~10% C02 atmosphere. Hepatocytes were cultured in 35-ram dishes at 35,000 celis/cm2 on a feeder layer of 3T3 cells seeded 1 day earlier. After 2 hr, non-attached cells were discarded, cultures were rinsed twice with serum-free medium and re-fed with D M E M supplemented as before together with 10/~g hydrocortisone/ml. Culture media (1 ml/35-mm dish) were changed daily. Urea production by hepatocytes cultured with different media. 24 hr before the indicated times, the cultures were re-fed with 1 ml fresh culture medium. Then used medium was collected, denatured with 1 ml HCIO4 (5 M) and centrifuged for 3 min in an Eppendorf microfuge. Urea was determined in the supernatant by the method of Ceriotti and Spandrio (1965) with some modifications. In a test-tube 1.6 ml sulfuric acid-antipyrine-Fe ÷3 reagent and 0.2ml 0.5% diacetylmonoxime solution were added to 50/~1 supernatant; the test-tubes were closed with marbles and incubated in a boiling water-bath for 30min. Then the tubes were cooled and the optical density measured at 460 nm. Urea production was expressed as the amount accumulated in 24 hr per culture dish (/~g/dish/day). To determine the functionality of the urea cycle, hepatocytes were incubated with DMEM containing 0.4 mu arginine, or D M E M containing either 0.4mu ornithine or 0.4mu ornithine plus 0.04 mM or 0.4mu arginine. Culture media were added 24hr after seeding. To study the effect of NH4C1 on urea production, hepatocytes were maintained with DMEM, and 24 hr before the indicated times were incubated with 1 ml DMEM containing 1, 3 or 5 mM NH4C1. Enzymatic assays. I U of enzyme activity was referred to as the amount catalysing the production of 1 ~mol product/hr at 37°C. For the assay of OTC
Urea production in rat hepatocyte cultures (a)
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Days in culture Fig, 2. Effect of ornithine and arginine on urea production. Hepatocytes were seeded on a feeder layer of 3T3 cells. The cultures were incubated with different media containing (a) 0.4 mM arginine, (b) 0.4 mM ornithine, (c) 0.4 mM ornithine + 0.04 mM arginine and (d) 0.4 mM ornithine + 0.4 mM arginine. Values are means _ SD of duplicate determinations from five culture dishes. Similar results were obtained in two independent experiments.
activity, hepatocytes cultured on 60-mm dishes were rinsed twice with 0.2 M T E A buffer, pH 7.6, scraped with a rubber policeman in 0.4 ml of the same buffer, and disrupted by sonication at 40 W for 15sec. A 0.3-ml sample of the resulting cell suspension was diluted with 1.2 ml cold water, The assay of OTC, based on the colorimetric determination of the citrulline produced, was performed with the method of Raijman (1983) with some modifications. OTC activity was measured at two protein concentrations, because previous determinations showed that the amount of citrulline produced was proportional to the amount of the diluted cell suspension used in the assay. The tested volumes of the diluted cell suspension were 0.1 and 0.2 ml per assay. The reaction was stopped by adding 0.5 ml 5M perchloric acid, and the tubes were centrifuged at 3000 rpm for 5 rain. Citrulline was determined using
diacetyl monoxime as described earlier using 0.1 ml supernatant. For the assay of arginase activity, hepatocyte cultures were rinsed twice with 0.2 ml 5 mM Tris-1 mM MnCI 2 buffer, pH 7,5, scraped with a rubber policeman in 0.2 ml of the same buffer, and disrupted by sonication at 40 W for 15 sec. 20 #1 of the cell suspension were diluted with 0.98 ml TrisMnCI 2 buffer. The assay of arginase activity, based on the colorimetric determination of the ornithine produced, was carried out as described by Colombo and Konarska (1983) using 50#1 diluted cell suspension. Protein was measured with the method of Lowry et al. (1951), using bovine serum albumin as standard. Statistical analysis. Results are expressed as the mean + SD of duplicate determinations from four culture dishes in a typical experiment. Similar results were obtained in two independent experiments.
A. SIERRA-SANTOYOet al.
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Statistical analysis of the results was made with Student's t-test at a significance level of P < 0.05. 60
RESULTS
Urea production Hepatocytes are the only cells able to produce urea. About 99% of the total urea produced by hepatocyte cultures was released into the culture medium. Urea production by hepatocytes cultured on 3T3 cells for 2 days was 186/tg/dish/day and decreased steadily to about 50% of the initial value after 15 days in culture (Fig. 1). Hepatocytes cultured on a plastic substratum for 1 day produced 179.5 pg urea/dish/day, a value similar to that seen in the 3T3-hepatocyte cultures. However, urea production decreased rapidly during the first days, representing only 24% of the initial value after 8 days in culture (Fig. 1). The urea content in the 24-hr medium of 3T3 cells represented less than 10% of the initial production by the 3T3-hepatocyte cultures, remained constant for 11 days and was similar to that measured in culture medium incubated for 24 hr in the same conditions.
Urea production by hepatocytes cultured with different media Since DMEM contains arginine, urea present in the medium may result from hydrolysis of arginine.
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Days in culture Fig. 4. Ornithine transcarbamylase (OTC) activity of cultured hepatocytes. Hepatocytes were seeded on plastic substratum (©) or on a feeder layer of 3T3 cells (0). Values are means + SD of duplicate determinations from five culture dishes. Similar results were obtained in two independent experiments.
To test the functionality of the urea cycle, hepatocytes seeded on 3T3 cells were incubated with medium containing either 0.4 m u arginine or 0.4 mM ornithine, or 0.4 mM ornithine plus 0.04 and 0.4 mM arginine. No significant changes in urea production were observed in the different media for at least 11 days in culture (Fig. 2).
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Days in culture Fig. 3. Effect of NH4CI on urea production. Hepatocytes were seeded on a feeder layer of 3T3 cells and maintained with Dulbecco's modified Eagle's medium (DMEM). 24 hr before the indicated times cultures were incubated with DMEM (O), DMEM containing NH4CI I mM (C)), 3 mM (A) or 5 mM (A). Values are means _+SD of duplicate determinations from five culture dishes. Similar results were obtained in two independent experiments. Asterisks indicate significant differences from control values (P < 0.05).
The steady-state concentration of total ammonia in the liver is about 0.7 mM. All urea cycle enzymes and urea production increase when this level is exceeded in vivo (Schimke, 1962). 24hr before the indicated times 3T3-hepatocyte cultures were exposed to 1, 3 and 5 mM NH4CI. Urea production increased significantly at all the tested concentrations during the 15 days in culture (Fig. 3). Urea content in the medium was proportional to NH4C1 concentration, showing mean increases of 25, 50 and 69%, respectively. Our results show that over 15 days, 3T3-hepatocyte cultures maintain the capacity to respond to ammonia (generated from NH4CI), as in vivo. Continuous exposure of the 3T3-hepatocyte cultures to NH4C1 altered the polygonal morphology, producing cytoplasmic vacuolizations, enlargement of intercellular spaces, degeneration and cell death after 5 days; however, the inducing effect of NH4CI on urea production was maintained over the first 3 days of continuous exposure (data not shown).
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Days in c u l t u r e Fig. 5. Arginase activity of cultured hepatocytes. Hepatocytes were seeded on plastic substratum (O) or on a feeder layer of 3T3 cells (Q). Cultures of 3T3 cells were incubated in the same conditions (A). Values are means + SD of duplicate determinations from five culture dishes. Similar results were obtained in two independent experiments. Activity o f O T C and arginase in cultured hepatocytes OTC, a mitochondrial enzyme of the parenchymal liver cells, is also present in small amounts in other organs. The initial OTC activity of hepatoeytes cultured on 3T3 cells was 61.1 U/60-mm dish, decreased to about 16% of the initial value after 5 days in culture, and remained constant thereafter (Fig. 4). OTC activity in hepatocytes cultured on plastic substratum for 24 hr was similar to that observed in 3T3-hepatocyte cultures and decreased to 13% of the initial value after 8 days in culture (Fig. 4). No OTC activity was observed in cultures of 3T3 cells. Arginase is the urea cycle enzyme with higher activity in the liver. It is also present in other cells but with lower activity. The initial arginase activity of hepatocytes seeded on 3T3 cells was 54.0 + 6.8 U/ 35-mm dish and remained constant over 15 days in culture (Fig. 5). The initial arginase activity of hepatocytes cultured on plastic substratum was similar to that measured in 3T3-hepatocyte cultures, but it decreased rapidly to only 10% of the initial value after 8 days in culture (Fig. 5). No arginase activity was observed in cultures of 3T3 cells. DISCUSSION
Hepatocytes seeded on a feeder layer of 3T3 cells maintain their typical morphology and several liver functions for at least 1 month in culture (KuriHarcuch and Mendoza-Figueroa, 1989); however, the functionality of the urea cycle had not been explored
297
in these cultures. In this study we showed that 3T3-hepatocyte cultures produce urea through the urea cycle for at least 15 days, although urea production decreases to 50% of the initial value. In contrast, urea production in hepatocytes seeded on plastic substratum decreased to 24% of the initial value after 8 days. The urea production by 3T3-hepatocyte cultures was due only to hepatocytes, since 3T3 cells do not produce urea. The urea detected in 3T3 cell cultures may be due to the serum added, or produced by spontaneous hydrolysis of arginine. The differences in urea production by hepatocytes seeded on different substrata were probably due to the presence of 3T3 cells, which maintained viability and the expression of differentiated functions for longer times as shown earlier (Kuri-Harcuch and Mendoza-Figueroa, 1989), because most of the hepatocytes seeded on plastic substratum die and detach from the cultures dishes after 1 wk (Feliu et al., 1982; Guguen et al., 1975; Guguen-Guillouzo et al., 1978). The decrease in urea production by the hepatocytes cultured on 3T3 cells could represent the cell response to the new micro-environment, which is different from the in vivo condition in the concentration of nutrients, suppression of stimuli mediated by the pancreatic hormone glucagon necessary to maintain urea production (Dich et al., 1988)and adrenaline (Hern~ndez-Sotomayor and Garcia-Sfiinz, 1984), a decrease in exchange of specific signals with other hepatic cells, or accumulation of catabolic products in the culture medium. Urea production by the 3T3-hepatocyte cultures was kept at lower levels than those observed in hepatocytes seeded on collagen membranes and maintained with serum-free medium or medium supplemented with hormones (Dich et al., 1988). 3T3-hepatocyte cultures were maintained with culture medium supplemented with 7% serum, insulin and hydrocortisone in the absence of glucagon. Glucagon and glucocorticoids are necessary for the expression of urea cycle enzymes (Dich et al., 1988; Husson et al., 1986; Lin and Snodgrass, 1975), while insulin has an inhibitory effect on their expression (Husson et al., 1985 and 1986). High concentrations of glucagon are necessary to maintain urea production by cultured hepatocytes in the presence of insulin (Dichet al., 1988). Further work is necessary to study the effect of glucagon on urea production in 3T3-hepatocyte cultures. Since arginine synthesis from ornithine through the urea cycle is a unique property of hepatocytes, it permits the culture of hepatocytes in a selective medium that does not allow the growth of other cell types (Acosta et al., 1985; Leffert and Paul, 1972). Since arginase activity in hepatocytes is very high, the urea produced may be due to hydrolysis of arginine by hepatocytes or spontaneous hydrolysis of arginine in the culture conditions. Our results show that in selective .media with 0.4mM ornithine or with
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ornithine plus different concentrations of arginine, urea production was maintained at the same level as in medium containing 0.4mM arginine (Fig. 2), indicating that the urea cycle is functional in 3T3-hepatocyte cultures since they can synthesize urea using ornithine as substrate. Moreover, it is known that ornithine improves the hydrogen supply for ATP-consuming processes of hepatocytes (Letko and Halangk, 1986). Microscopic examination of the cultures showed that hepatocytes maintained with ornithine formed the typical cords with polygonal cells and intercellular spaces, and these were better preserved than in hepatocytes maintained with arginine (data not shown). Hepatocytes in vivo respond to different stimuli, such as hyperproteosis, a prolonged fast or accelerated catabolic process, increasing enzymatic activities of the urea cycle with a consequent increase in urea production (Schimke, 1962). This response is mediated by rapid adaptation of essential metabolic functions, such as RNA and protein synthesis, and concentration of the substrates involved. The 3T3-hepatocyte cultures exposed to NH4CI for 24 hr responded by increasing urea production, as occurs in vivo when ammonia concentration rises. This increase was proportional to NH4C1 concentration for at least 15 days in culture (Fig. 3). Although hepatocytes in suspension or seeded on plastic produce urea and increase its production after exposure to NH4C1 (Drew et al., 1985; Gebhardt et al., 1978), the presence of 3T3 cells maintains these liver functions for longer periods. Of the urea cycle enzymes arginase and OTC have the higher specific activities. The levels of arginase and OTC activity observed in 3T3-hepatocyte cultures (Figs 5 and 4) could be due to the high arginine concentration in the medium, since high arginine concentrations increase arginase activity while all the other four enzyme activities decrease (Powers and Meister, 1982). The arginine concentration in the culture medium was nearly 10 times higher than the hepatic concentration (Powers and Meister, 1982). The activity of the urea cycle enzymes is also modulated by the presence of hormones and serum in the culture medium (Dich et al., 1988; Husson et al., 1986; Lin and Snodgrass, 1975). Further work is necessary to study the effect of ornithine and arginine concentrations on urea cycle enzymes, and the regulation of urea cycle enzymes by different substrate concentrations in the presence of various combinations of hormones. There are few studies on the urea cycle in long-term cultures of adult rat hepatocytes (Haggerty et al., 1983). The cell line H4-II-E-C3 has been proposed as a suitable culture system to study how the urogenetic pathway is regulated in hepatocytes in vivo, although this cell line derives from a rat hepatoma (Haggerty et al., 1983). Our results show that 3T3-hepatocyte cultures from adult rats maintain the functionality of the urea
cycle for at least 15 days in culture, since the cells produce urea from ornithine and respond to a ureagenesis inducer as they do in vivo. Our findings also show that these cultures have decreasing or constant levels of OTC and arginase activities, respectively, representing probably an adaptive response dependent on substrate concentrations and hormonal regulation. They also suggest that the 3T3-hepatocyte cultures could be a suitable in vitro system to study urea production, its regulation by substrates and hormones and its alteration by drugs and toxic chemicals. Acknowledgements--The authors wish to thank Dr Arnulfo Albores and Dr Rubrn Lrpez Revilla for their critical review of the manuscript, and Alfredo Padilla for the artwork. This work was partially supported by grant 667.87 from COSNET-SEP, Mexico. A. Sierra-Santoyo was supported by a scholarship from COSNET-SEP, Mexico.
REFERENCES
Acosta D. and Mitchell D. B. (1981) Metabolic activation and cytotoxicity of cyclophosphamidein primary cultures of postnatal rat hepatocytes. Biochemical Pharmacology 30, 3225-3230. Acosta D., Sorensen E. M. B., Anuforo D. C., Mitchell D. B., Ramos K., Santone K. S. and Smith M. A. (1985) An in vitro approach to the study of target organ toxicity of drugs and chemicals. In vitro Cellular and Developmental Biology 21, 495-504. Berry M. N. and Friend D. S. (1969) High-yield preparation of isolated rat liver parenchymal cells. Journal of Cell Biology 43, 506-520. Ceriotti G. and Spandrio L. (1965) Catalytic acceleration of the urea~liacetylmonoxime-phenazone reaction and its application to automatic analysis. Clinica Chimica Acta 11, 519-522. Clarke S. (1976) A major polypeptide component of rat liver mitochondria: carbamyl phosphate synthetase. Journal oJ Biological Chemistry 251, 950-961. Colombo J. P. and Konarska L. (1983) Arginase. In Methods in Enzymatic Analysis. Edited by J. Bergmeyer and M. Grable. pp. 285-294. Verlag-Chimie,Weinheim. Dich J., Vind C. and Grunnet N. (1988) Long-term culture of hepatocytes: effect of hormones on enzyme activities and metabolic capacity. Hepatology 8, 39-45. Drew P. J. T., Monzon J. P., Metcalfe H. K., Evans S. J. W., Iles R. A. and Cohen R. D. (1985) The effect of arginine vasopressin on ureagenesis in isolated hepatocytes. Clinical Science 69, 231-233. Feliu J. E., Coloma J., Grmez-Lechrn M. J., Garcia M. D. and Baguena J. (1982) Effect of dexamethasone on the isozyme pattern of adult rat liver parenchymal cells in primary cultures. Molecular and Cellular Biochemistry 45, 73-81. Gebhardt R., Belleman P. and Mecke D. (1978) Metabolic and enzymatic characteristics of adult rat liver parenchyreal cells in non-proliferating primary monolayers cultures. Experimental Cell Research 112, 431-441. Gebhardt R. and Mecke D. (1979) Permissive effect of dexamethasone on glucagon induction of urea-cycle enzymes in perfused primary monolayer cultures of rat hepatocytes. European Journal of Biochemistry 97, 29-35. Guguen C., Gregori C. and Schapira F. (1975) Modification of pyruvate kinase isozymes in prolonged primary cultures of adult rat hepatocytes. Biochimie 57, 1065-1071. Guguen-Guillouzo C., Tichonicky L., Szajnert M. F., Schapira F. and Kruh J. (1978) Changes of some chro-
Urea production in rat hepatocyte cultures matin and cytoplasmic enzymes in primary cultures of adult rat hepatocytes. Biology of the Cell 31, 225-233. Guth6hrlein G. and Knappe J. (1968) Structure and function of carbamoylphosphate synthase. European Journal of Biochemistry 7, 119-127. Haggerty D. F., Spector E. B., Lynch M., Kern R., Frank L. B. and Cederbaum S. D. (1983) Regulation of expression of genes for enzymes of the mammalian urea cycle in permanent cell-culture lines of hepatic and nonhepatic origin. Molecularand Cellular Biochemistry 53/54, 57-76. Harell D. and Sokolovsky M. (1972) Beef-liver arginase. European Journal of Biochemistry 25, 102-108. Hems R. (1983) The effect of dichloroacetate and hydroxypyruvate on the entry of t4C from [l-~4C]alanine into urea in rat hepatocytes. FEBS Letters 160, 255-258. Hern/mdez-Sotomayor T. and Garcia-S~inz A. (1984) Adrenergic regulation of ureogenesis in bepatocytes from adrenalectomized rats. FEBS Letters 166, 285-388. Husson A., Bouazza M., Buquet C. and Vaillant R. (1985) Role of dexamethasone and insulin on the development of the five urea-cycle enzymes in cultured rat foetal hepatocytes. Biochemical Journal 225, 271-274. Husson A., Buquet C. and Vaillant R. (1987) Induction of the five urea-cycle enzymes by glucagon in cultured foetal rat hepatocytes. Differentiation 35, 212-218. Husson A., Guechairi M., Fairand A., Bouazza M., Ktorsa A. and Vaillant R. (1986) Effects of pancreatic hormones and glucocorticosteroids on argininosuccinate synthetase and argininosuccinase activities of rat liver during perinatal period: in viva and in vitro studies. Endocrinology 119, 1171-1177. Kalousek F., Franqois B. and Rosenberg L. E, (1978) Isolation and characterization of ornithine transcarbamylase from normal human liver. Journal of Biological Chemistry 253, 3939-3944. Kuri-Harcuch W. and Mendoza-Figueroa T. (1989) Cultivation of adult rat hepatocytes on 3T3 cells: expression of various liver differentiated functions. Differentiation 41, 148-157. Leffert H. L. and Paul D. (1972) Studies on primary cultures of differentiated fetal liver ceils. Journal of Cell Biology 52, 559-568. Letko G. and Halangk W. (1986) Effect of improved hydrogen supply on energy state, ureogenesis and gluco-
299
neogenesis in isolated hepatocytes. Biomedica Biochimica
Acta 45, 265-271. Lin R. C. and Snodgrass J. (1975) Primary culture of normal adult rat liver cells which maintain stable urea cycle enzymes. Biochemical and Biophysical Research Communications 64, 725-734. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193, 265-275. Lusty C. J. and Ratner S. (1972) Biosynthesis of urea. Journal of Biological Chemistry 247, 7010-7022. Marshall M. and Cohen P. P. (1972) Ornithine transcarbamylase from Streptococcus faecalis and bovine liver. Journal of Biological Chemistry 247, 1641-1653, 1654-1668 & 1669-1682. Mendoza-Figueroa T., L6pez-Revilla R. and Villa-Trevifio S. (1983) DNA breaks induced by micromolar concentrations of dimethylnitrcsamine in liver primary cell cultures from untreated and phenobarbital treated rats. Toxicology 27, 55-69. Powers S. G. and Meister A. (1982) Urea synthesis and ammonia metabolism. In The Liver: Biology and Pathobiology. Edited by I. Arias, H. Popper, D. Schachter and D. A. Shafritz. pp. 251 263. Raven Press, New York. Raijman L. (1983) Ornithine carbamoyltransferase in liver. In Methods of Enzymatic Analysis. Edited by J. Bergmeyer and M. Grable. pp. 32(~334. Verlag Chimie, Weinheim. Ratner S. (1973) Enzymes of arginine and urea synthesis. Advances in Enzymology 39, 140. Rheinwald J. G. (1978) Serial cultivation of normal human epidermal keratinocytes. Methods of Cell Biology 21A, 229-254. Rochovansky D. and Ratner S. (1967) Biosynthesis of urea. Journal of Biological Chemistry 242, 3839-3849. Schimke R. T. (1962) Adaptive characteristics of urea cycle enzymes in the rat. Journal of Biological Chemistry 237, 459-468. Snodgrass P. J., Lin R. C., Miiller W. A. and Aoki T. T. (1978) Induction of urea cycle enzymes of rat liver by glucagon. Journal of Biological Chemistry 7, 3047 3051. Story D. L., Gee S. J., Tyson C. A. and Gould D. H. (1983) Response of isolated hepatocytes to organic and inorganic cytotoxins. Journal of Toxicology and Environmental Health 11, 483-501.
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U R E A P R O D U C T I O N IN LONG-TERM CULTURES OF ADULT RAT HEPATOCYTES A. SIERRA-SANTOYO,M. L. LrPEZ, A. HERN,ANDEZand T. MENDOZA-FIGUEROA* Pharmacology and Toxicology Department, Centro de Investigaci6n y de Estudios Avanzados del IPN, PO Box 14-740, Mexico City 07000, Mexico (Received 9 March 1993; revisions received 10 August 1993)
Abstract--To study the functionality of the urea cycle in long-term cultures of adult rat hepatocytes, urea production and the activity of two urea cycle enzymes were measured in hepatocytes cultured on 3T3 cells for 15 days. Urea production was also measured in cultures maintained with medium containing either 0.4 mM arginine or 0.4 mM ornithine and in cultures exposed to different concentrations of NH4C1, an in vivo inducer of urea production. In hepatocytes seeded on 3T3 cells, urea production decreased gradually to 50% of the initial value after 15 days. Urea production was similar in 3T3-hepatocyte cultures maintained for 11 days with medium containing ornithine or arginine. Hepatocytes exposed for 24 hr to 1, 3 and 5 mM NH4C1 showed an average increase in urea production of 25, 50 and 69%, respectively, above that of unexposed cultures over 15 days. Ornithine transcarbamylase (OTC) activity decreased by 84% after 5 days in culture and remained constant thereafter, while arginase activity remained constant over 15 days. In contrast, in hepatocytes seeded on plastic substratum, urea production decreased to 24% of the initial value after 8 days in culture. OTC and arginase activities also decreased to 13 and 10% of their initial values after 8 days in culture. These results show that 3T3-hepatocyte cultures from adult rats produce urea from ornithine and/or arginine for at least 15 days and respond to an inducer of urea production as in vivo. They also show that these cultures have decreasing and constant levels of OTC and arginase activities, respectively, owing probably to an adaptative response dependent on substrate concentrations and hormonal regulation. These findings also suggest that 3T3-hepatocyte cultures are a suitable in vitro system to study urea production, its regulation by substrates and hormones and its alteration by drugs and toxic chemicals.
INTRODUCTION A m m o n i a , a catabolite and precursor in some anabolic processes, plays a central role in nitrogen metabolism. A b o u t 90% of excess ammonia is eliminated from the blood mostly through urea synthesis by hepatocytes. Urea production involves a highly regulated cycle in which five enzymes participate: carbamylphosphate synthetase, ornithine transcarbamylase (OTC), argininosuccinate synthetase, argininosuccinate lyase and arginase. These enzymes have been studied extensively in human, rat and bovine liver, and in bacteria (Clarke, 1976; Guth6hrlein and Knappe, 1968; Harell and Sokolovsky, 1972; Kalousek et al., 1978; Lusty and Ratner, 1972; Marshall and Cohen, 1972; Ratner, 1973; Rochovansky and Ratner, 1967). Schimke (1962) reported that all urea cycle enzymes are directly associated with dietary protein consumption in rats. Administration of glucagon and glucocorticoids induces all urea cycle enzymes in vivo and in vitro (Gebhardt and Mecke, 1979; Husson et al., 1985, 1986 and 1987; Snodgrass et al., 1978), whereas *To whom reprint requests should be addressed. DMEM = Dulbecco's modified Eagle's medium; O T C = ornithine transcarbamylase; TEA = triethanolamine-HC1.
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insulin administration has an inhibitory effect on their expression (Husson et al., 1985 and 1986). To study liver functions and their alteration by hepatotoxic chemicals, several experimental systems have been used: perfused livers, liver slices, total and differential homogenates, hepatocyte suspensions, cultures of cell lines derived from liver, and primary cultures of hepatocytes. The last system is one of the most advantageous, since isolated hepatocytes can be cultured in a controlled environment maintaining several differentiated functions. However, they lose most of their characteristics and die after a few days in culture (Feliu et al., 1982; Guguen-Guillouzo et al., 1978). There are few studies on urea production by cultured hepatocytes. In adult rat hepatocytes cultured on plastic substratum, urea production decreased by 80% after 7 days, and the addition of 3 mM NH4CI on the second day increased about two-fold the amount of urea released within 24 hr (Gebhardt et al., 1978). In hepatocytes seeded on collagen membranes and maintained with serum-free medium, urea synthesis decreased by 30% after 13 days, but remained constant if the medium contained insulin, dexamethasone and high concentrations of glucagon (Dich et al., 1988). Alterations in urea production also have been used as an indicator of toxicity in the evaluation 293
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o f different classes of chemicals in hepatocyte cultures
(Acosta and Mitchell, 1981; Hems, 1983; Story et al., 1983). We have described a culture system in which adult rat hepatocytes are seeded on a feeder layer of 3T3 cells that allows their long-term (2 months) survival a n d the maintenance for at least 4 wk of their typical morphology and of several differentiated functions such as albumin secretion, lipid synthesis and secretion, and basal and inducible levels of cytochrome P-450 (Kuri-Harcuch and Mendoza-Figueroa, 1989). In the present study we investigated the functionality of the urea cycle in long-term cultures of adult rat hepatocytes by determining urea synthesis with different media, its inducibility by NH4C1, an in vivo inducer of urea production, and the activities of OTC a n d arginase, t w o urea cycle enzymes localized in the mitochondria and cytosol of parenchymal liver cells.
MATERIALS AND M E T H O D S
Materials. Collagenase type IV, insulin, d-biotin, L-arginine-HC1, L-ornithine-HCl, triethanolamineHC1 (TEA), Li2--carbamoyl phosphate, Tris(hydroxymethyl)aminomethane (Tris-HCl), antipyrine, diacetyl monoxime, citrulline, ninhydrin, hydrocortisone and trypsin type III were obtained from Sigma Chemical Co. (St Louis, MO, USA); mitomycin C w a s a gift from Bristol-Myer Co., Syracuse, NY,
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Fig. 1. Urea production by hepatocytes cultured on different substrata. Isolated hepatocytes were seeded on plastic substratum (O) and on a feeder layer of 3T3 cells (0). Culture dishes containing feeder layers (A) and Dulbecco's modified Eagle's medium (DMEM) (A) were incubated under the same conditions. Values are means + SD of duplicate determinations from five culture dishes. Similar results were obtained in two independent experiments.
USA; urea, manganous chloride, ferric chloride and other reagents of analytical grade were purchased from J. T. Baker (Mexico City, Mexico). Cell cultures. Hepatocytes were isolated from male Wistar rats weighing 180-200 g by the collagenase perfusion method of Berry and Friend (1969), as modified (Mendoza-Figueroa et al., 1983), and cultivated as described (Kuri-Harcuch and MendozaFigueroa, 1989). Briefly, the liver was perfused with TD solution (5.5 mM glucose, 137 mM NaCI, 50 mM KCI, 0.4raM Na2HPO4, 25 mM Tris-HC1, pH 7.4, 0.01% phenol red) for 3 min at 37°C and a flow rate of 15ml/min and again for 10min at a rate of 10 mi/min with 100 U collagenase type IV/ml in TD solution at 37°C. Cells were dispersed in 40 ml Dulbecco's modified Eagle's medium (DMEM), supplemented with 7% calf serum, 5 pg insulin/ml and 0.1 pM d-biotin. The cell suspension was filtered through a nylon mesh and allowed to stand for 10 min; the supernatant was discarded and the cells were resuspended in 20 ml DMEM. 3T3 cells were lethally treated with 10 #g mitomycin C/ml for 2 hr (Rheinwald, 1978), seeded at 35,000 cells/cm2 and incubated at 37°C in a humidified 90% ai~10% C02 atmosphere. Hepatocytes were cultured in 35-ram dishes at 35,000 celis/cm2 on a feeder layer of 3T3 cells seeded 1 day earlier. After 2 hr, non-attached cells were discarded, cultures were rinsed twice with serum-free medium and re-fed with D M E M supplemented as before together with 10/~g hydrocortisone/ml. Culture media (1 ml/35-mm dish) were changed daily. Urea production by hepatocytes cultured with different media. 24 hr before the indicated times, the cultures were re-fed with 1 ml fresh culture medium. Then used medium was collected, denatured with 1 ml HCIO4 (5 M) and centrifuged for 3 min in an Eppendorf microfuge. Urea was determined in the supernatant by the method of Ceriotti and Spandrio (1965) with some modifications. In a test-tube 1.6 ml sulfuric acid-antipyrine-Fe ÷3 reagent and 0.2ml 0.5% diacetylmonoxime solution were added to 50/~1 supernatant; the test-tubes were closed with marbles and incubated in a boiling water-bath for 30min. Then the tubes were cooled and the optical density measured at 460 nm. Urea production was expressed as the amount accumulated in 24 hr per culture dish (/~g/dish/day). To determine the functionality of the urea cycle, hepatocytes were incubated with DMEM containing 0.4 mu arginine, or D M E M containing either 0.4mu ornithine or 0.4mu ornithine plus 0.04 mM or 0.4mu arginine. Culture media were added 24hr after seeding. To study the effect of NH4C1 on urea production, hepatocytes were maintained with DMEM, and 24 hr before the indicated times were incubated with 1 ml DMEM containing 1, 3 or 5 mM NH4C1. Enzymatic assays. I U of enzyme activity was referred to as the amount catalysing the production of 1 ~mol product/hr at 37°C. For the assay of OTC
Urea production in rat hepatocyte cultures (a)
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Days in culture Fig, 2. Effect of ornithine and arginine on urea production. Hepatocytes were seeded on a feeder layer of 3T3 cells. The cultures were incubated with different media containing (a) 0.4 mM arginine, (b) 0.4 mM ornithine, (c) 0.4 mM ornithine + 0.04 mM arginine and (d) 0.4 mM ornithine + 0.4 mM arginine. Values are means _ SD of duplicate determinations from five culture dishes. Similar results were obtained in two independent experiments.
activity, hepatocytes cultured on 60-mm dishes were rinsed twice with 0.2 M T E A buffer, pH 7.6, scraped with a rubber policeman in 0.4 ml of the same buffer, and disrupted by sonication at 40 W for 15sec. A 0.3-ml sample of the resulting cell suspension was diluted with 1.2 ml cold water, The assay of OTC, based on the colorimetric determination of the citrulline produced, was performed with the method of Raijman (1983) with some modifications. OTC activity was measured at two protein concentrations, because previous determinations showed that the amount of citrulline produced was proportional to the amount of the diluted cell suspension used in the assay. The tested volumes of the diluted cell suspension were 0.1 and 0.2 ml per assay. The reaction was stopped by adding 0.5 ml 5M perchloric acid, and the tubes were centrifuged at 3000 rpm for 5 rain. Citrulline was determined using
diacetyl monoxime as described earlier using 0.1 ml supernatant. For the assay of arginase activity, hepatocyte cultures were rinsed twice with 0.2 ml 5 mM Tris-1 mM MnCI 2 buffer, pH 7,5, scraped with a rubber policeman in 0.2 ml of the same buffer, and disrupted by sonication at 40 W for 15 sec. 20 #1 of the cell suspension were diluted with 0.98 ml TrisMnCI 2 buffer. The assay of arginase activity, based on the colorimetric determination of the ornithine produced, was carried out as described by Colombo and Konarska (1983) using 50#1 diluted cell suspension. Protein was measured with the method of Lowry et al. (1951), using bovine serum albumin as standard. Statistical analysis. Results are expressed as the mean + SD of duplicate determinations from four culture dishes in a typical experiment. Similar results were obtained in two independent experiments.
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Statistical analysis of the results was made with Student's t-test at a significance level of P < 0.05. 60
RESULTS
Urea production Hepatocytes are the only cells able to produce urea. About 99% of the total urea produced by hepatocyte cultures was released into the culture medium. Urea production by hepatocytes cultured on 3T3 cells for 2 days was 186/tg/dish/day and decreased steadily to about 50% of the initial value after 15 days in culture (Fig. 1). Hepatocytes cultured on a plastic substratum for 1 day produced 179.5 pg urea/dish/day, a value similar to that seen in the 3T3-hepatocyte cultures. However, urea production decreased rapidly during the first days, representing only 24% of the initial value after 8 days in culture (Fig. 1). The urea content in the 24-hr medium of 3T3 cells represented less than 10% of the initial production by the 3T3-hepatocyte cultures, remained constant for 11 days and was similar to that measured in culture medium incubated for 24 hr in the same conditions.
Urea production by hepatocytes cultured with different media Since DMEM contains arginine, urea present in the medium may result from hydrolysis of arginine.
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Days in culture Fig. 4. Ornithine transcarbamylase (OTC) activity of cultured hepatocytes. Hepatocytes were seeded on plastic substratum (©) or on a feeder layer of 3T3 cells (0). Values are means + SD of duplicate determinations from five culture dishes. Similar results were obtained in two independent experiments.
To test the functionality of the urea cycle, hepatocytes seeded on 3T3 cells were incubated with medium containing either 0.4 m u arginine or 0.4 mM ornithine, or 0.4 mM ornithine plus 0.04 and 0.4 mM arginine. No significant changes in urea production were observed in the different media for at least 11 days in culture (Fig. 2).
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Days in culture Fig. 3. Effect of NH4CI on urea production. Hepatocytes were seeded on a feeder layer of 3T3 cells and maintained with Dulbecco's modified Eagle's medium (DMEM). 24 hr before the indicated times cultures were incubated with DMEM (O), DMEM containing NH4CI I mM (C)), 3 mM (A) or 5 mM (A). Values are means _+SD of duplicate determinations from five culture dishes. Similar results were obtained in two independent experiments. Asterisks indicate significant differences from control values (P < 0.05).
The steady-state concentration of total ammonia in the liver is about 0.7 mM. All urea cycle enzymes and urea production increase when this level is exceeded in vivo (Schimke, 1962). 24hr before the indicated times 3T3-hepatocyte cultures were exposed to 1, 3 and 5 mM NH4CI. Urea production increased significantly at all the tested concentrations during the 15 days in culture (Fig. 3). Urea content in the medium was proportional to NH4C1 concentration, showing mean increases of 25, 50 and 69%, respectively. Our results show that over 15 days, 3T3-hepatocyte cultures maintain the capacity to respond to ammonia (generated from NH4CI), as in vivo. Continuous exposure of the 3T3-hepatocyte cultures to NH4C1 altered the polygonal morphology, producing cytoplasmic vacuolizations, enlargement of intercellular spaces, degeneration and cell death after 5 days; however, the inducing effect of NH4CI on urea production was maintained over the first 3 days of continuous exposure (data not shown).
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Days in c u l t u r e Fig. 5. Arginase activity of cultured hepatocytes. Hepatocytes were seeded on plastic substratum (O) or on a feeder layer of 3T3 cells (Q). Cultures of 3T3 cells were incubated in the same conditions (A). Values are means + SD of duplicate determinations from five culture dishes. Similar results were obtained in two independent experiments. Activity o f O T C and arginase in cultured hepatocytes OTC, a mitochondrial enzyme of the parenchymal liver cells, is also present in small amounts in other organs. The initial OTC activity of hepatoeytes cultured on 3T3 cells was 61.1 U/60-mm dish, decreased to about 16% of the initial value after 5 days in culture, and remained constant thereafter (Fig. 4). OTC activity in hepatocytes cultured on plastic substratum for 24 hr was similar to that observed in 3T3-hepatocyte cultures and decreased to 13% of the initial value after 8 days in culture (Fig. 4). No OTC activity was observed in cultures of 3T3 cells. Arginase is the urea cycle enzyme with higher activity in the liver. It is also present in other cells but with lower activity. The initial arginase activity of hepatocytes seeded on 3T3 cells was 54.0 + 6.8 U/ 35-mm dish and remained constant over 15 days in culture (Fig. 5). The initial arginase activity of hepatocytes cultured on plastic substratum was similar to that measured in 3T3-hepatocyte cultures, but it decreased rapidly to only 10% of the initial value after 8 days in culture (Fig. 5). No arginase activity was observed in cultures of 3T3 cells. DISCUSSION
Hepatocytes seeded on a feeder layer of 3T3 cells maintain their typical morphology and several liver functions for at least 1 month in culture (KuriHarcuch and Mendoza-Figueroa, 1989); however, the functionality of the urea cycle had not been explored
297
in these cultures. In this study we showed that 3T3-hepatocyte cultures produce urea through the urea cycle for at least 15 days, although urea production decreases to 50% of the initial value. In contrast, urea production in hepatocytes seeded on plastic substratum decreased to 24% of the initial value after 8 days. The urea production by 3T3-hepatocyte cultures was due only to hepatocytes, since 3T3 cells do not produce urea. The urea detected in 3T3 cell cultures may be due to the serum added, or produced by spontaneous hydrolysis of arginine. The differences in urea production by hepatocytes seeded on different substrata were probably due to the presence of 3T3 cells, which maintained viability and the expression of differentiated functions for longer times as shown earlier (Kuri-Harcuch and Mendoza-Figueroa, 1989), because most of the hepatocytes seeded on plastic substratum die and detach from the cultures dishes after 1 wk (Feliu et al., 1982; Guguen et al., 1975; Guguen-Guillouzo et al., 1978). The decrease in urea production by the hepatocytes cultured on 3T3 cells could represent the cell response to the new micro-environment, which is different from the in vivo condition in the concentration of nutrients, suppression of stimuli mediated by the pancreatic hormone glucagon necessary to maintain urea production (Dich et al., 1988)and adrenaline (Hern~ndez-Sotomayor and Garcia-Sfiinz, 1984), a decrease in exchange of specific signals with other hepatic cells, or accumulation of catabolic products in the culture medium. Urea production by the 3T3-hepatocyte cultures was kept at lower levels than those observed in hepatocytes seeded on collagen membranes and maintained with serum-free medium or medium supplemented with hormones (Dich et al., 1988). 3T3-hepatocyte cultures were maintained with culture medium supplemented with 7% serum, insulin and hydrocortisone in the absence of glucagon. Glucagon and glucocorticoids are necessary for the expression of urea cycle enzymes (Dich et al., 1988; Husson et al., 1986; Lin and Snodgrass, 1975), while insulin has an inhibitory effect on their expression (Husson et al., 1985 and 1986). High concentrations of glucagon are necessary to maintain urea production by cultured hepatocytes in the presence of insulin (Dichet al., 1988). Further work is necessary to study the effect of glucagon on urea production in 3T3-hepatocyte cultures. Since arginine synthesis from ornithine through the urea cycle is a unique property of hepatocytes, it permits the culture of hepatocytes in a selective medium that does not allow the growth of other cell types (Acosta et al., 1985; Leffert and Paul, 1972). Since arginase activity in hepatocytes is very high, the urea produced may be due to hydrolysis of arginine by hepatocytes or spontaneous hydrolysis of arginine in the culture conditions. Our results show that in selective .media with 0.4mM ornithine or with
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ornithine plus different concentrations of arginine, urea production was maintained at the same level as in medium containing 0.4mM arginine (Fig. 2), indicating that the urea cycle is functional in 3T3-hepatocyte cultures since they can synthesize urea using ornithine as substrate. Moreover, it is known that ornithine improves the hydrogen supply for ATP-consuming processes of hepatocytes (Letko and Halangk, 1986). Microscopic examination of the cultures showed that hepatocytes maintained with ornithine formed the typical cords with polygonal cells and intercellular spaces, and these were better preserved than in hepatocytes maintained with arginine (data not shown). Hepatocytes in vivo respond to different stimuli, such as hyperproteosis, a prolonged fast or accelerated catabolic process, increasing enzymatic activities of the urea cycle with a consequent increase in urea production (Schimke, 1962). This response is mediated by rapid adaptation of essential metabolic functions, such as RNA and protein synthesis, and concentration of the substrates involved. The 3T3-hepatocyte cultures exposed to NH4CI for 24 hr responded by increasing urea production, as occurs in vivo when ammonia concentration rises. This increase was proportional to NH4C1 concentration for at least 15 days in culture (Fig. 3). Although hepatocytes in suspension or seeded on plastic produce urea and increase its production after exposure to NH4C1 (Drew et al., 1985; Gebhardt et al., 1978), the presence of 3T3 cells maintains these liver functions for longer periods. Of the urea cycle enzymes arginase and OTC have the higher specific activities. The levels of arginase and OTC activity observed in 3T3-hepatocyte cultures (Figs 5 and 4) could be due to the high arginine concentration in the medium, since high arginine concentrations increase arginase activity while all the other four enzyme activities decrease (Powers and Meister, 1982). The arginine concentration in the culture medium was nearly 10 times higher than the hepatic concentration (Powers and Meister, 1982). The activity of the urea cycle enzymes is also modulated by the presence of hormones and serum in the culture medium (Dich et al., 1988; Husson et al., 1986; Lin and Snodgrass, 1975). Further work is necessary to study the effect of ornithine and arginine concentrations on urea cycle enzymes, and the regulation of urea cycle enzymes by different substrate concentrations in the presence of various combinations of hormones. There are few studies on the urea cycle in long-term cultures of adult rat hepatocytes (Haggerty et al., 1983). The cell line H4-II-E-C3 has been proposed as a suitable culture system to study how the urogenetic pathway is regulated in hepatocytes in vivo, although this cell line derives from a rat hepatoma (Haggerty et al., 1983). Our results show that 3T3-hepatocyte cultures from adult rats maintain the functionality of the urea
cycle for at least 15 days in culture, since the cells produce urea from ornithine and respond to a ureagenesis inducer as they do in vivo. Our findings also show that these cultures have decreasing or constant levels of OTC and arginase activities, respectively, representing probably an adaptive response dependent on substrate concentrations and hormonal regulation. They also suggest that the 3T3-hepatocyte cultures could be a suitable in vitro system to study urea production, its regulation by substrates and hormones and its alteration by drugs and toxic chemicals. Acknowledgements--The authors wish to thank Dr Arnulfo Albores and Dr Rubrn Lrpez Revilla for their critical review of the manuscript, and Alfredo Padilla for the artwork. This work was partially supported by grant 667.87 from COSNET-SEP, Mexico. A. Sierra-Santoyo was supported by a scholarship from COSNET-SEP, Mexico.
REFERENCES
Acosta D. and Mitchell D. B. (1981) Metabolic activation and cytotoxicity of cyclophosphamidein primary cultures of postnatal rat hepatocytes. Biochemical Pharmacology 30, 3225-3230. Acosta D., Sorensen E. M. B., Anuforo D. C., Mitchell D. B., Ramos K., Santone K. S. and Smith M. A. (1985) An in vitro approach to the study of target organ toxicity of drugs and chemicals. In vitro Cellular and Developmental Biology 21, 495-504. Berry M. N. and Friend D. S. (1969) High-yield preparation of isolated rat liver parenchymal cells. Journal of Cell Biology 43, 506-520. Ceriotti G. and Spandrio L. (1965) Catalytic acceleration of the urea~liacetylmonoxime-phenazone reaction and its application to automatic analysis. Clinica Chimica Acta 11, 519-522. Clarke S. (1976) A major polypeptide component of rat liver mitochondria: carbamyl phosphate synthetase. Journal oJ Biological Chemistry 251, 950-961. Colombo J. P. and Konarska L. (1983) Arginase. In Methods in Enzymatic Analysis. Edited by J. Bergmeyer and M. Grable. pp. 285-294. Verlag-Chimie,Weinheim. Dich J., Vind C. and Grunnet N. (1988) Long-term culture of hepatocytes: effect of hormones on enzyme activities and metabolic capacity. Hepatology 8, 39-45. Drew P. J. T., Monzon J. P., Metcalfe H. K., Evans S. J. W., Iles R. A. and Cohen R. D. (1985) The effect of arginine vasopressin on ureagenesis in isolated hepatocytes. Clinical Science 69, 231-233. Feliu J. E., Coloma J., Grmez-Lechrn M. J., Garcia M. D. and Baguena J. (1982) Effect of dexamethasone on the isozyme pattern of adult rat liver parenchymal cells in primary cultures. Molecular and Cellular Biochemistry 45, 73-81. Gebhardt R., Belleman P. and Mecke D. (1978) Metabolic and enzymatic characteristics of adult rat liver parenchyreal cells in non-proliferating primary monolayers cultures. Experimental Cell Research 112, 431-441. Gebhardt R. and Mecke D. (1979) Permissive effect of dexamethasone on glucagon induction of urea-cycle enzymes in perfused primary monolayer cultures of rat hepatocytes. European Journal of Biochemistry 97, 29-35. Guguen C., Gregori C. and Schapira F. (1975) Modification of pyruvate kinase isozymes in prolonged primary cultures of adult rat hepatocytes. Biochimie 57, 1065-1071. Guguen-Guillouzo C., Tichonicky L., Szajnert M. F., Schapira F. and Kruh J. (1978) Changes of some chro-
Urea production in rat hepatocyte cultures matin and cytoplasmic enzymes in primary cultures of adult rat hepatocytes. Biology of the Cell 31, 225-233. Guth6hrlein G. and Knappe J. (1968) Structure and function of carbamoylphosphate synthase. European Journal of Biochemistry 7, 119-127. Haggerty D. F., Spector E. B., Lynch M., Kern R., Frank L. B. and Cederbaum S. D. (1983) Regulation of expression of genes for enzymes of the mammalian urea cycle in permanent cell-culture lines of hepatic and nonhepatic origin. Molecularand Cellular Biochemistry 53/54, 57-76. Harell D. and Sokolovsky M. (1972) Beef-liver arginase. European Journal of Biochemistry 25, 102-108. Hems R. (1983) The effect of dichloroacetate and hydroxypyruvate on the entry of t4C from [l-~4C]alanine into urea in rat hepatocytes. FEBS Letters 160, 255-258. Hern/mdez-Sotomayor T. and Garcia-S~inz A. (1984) Adrenergic regulation of ureogenesis in bepatocytes from adrenalectomized rats. FEBS Letters 166, 285-388. Husson A., Bouazza M., Buquet C. and Vaillant R. (1985) Role of dexamethasone and insulin on the development of the five urea-cycle enzymes in cultured rat foetal hepatocytes. Biochemical Journal 225, 271-274. Husson A., Buquet C. and Vaillant R. (1987) Induction of the five urea-cycle enzymes by glucagon in cultured foetal rat hepatocytes. Differentiation 35, 212-218. Husson A., Guechairi M., Fairand A., Bouazza M., Ktorsa A. and Vaillant R. (1986) Effects of pancreatic hormones and glucocorticosteroids on argininosuccinate synthetase and argininosuccinase activities of rat liver during perinatal period: in viva and in vitro studies. Endocrinology 119, 1171-1177. Kalousek F., Franqois B. and Rosenberg L. E, (1978) Isolation and characterization of ornithine transcarbamylase from normal human liver. Journal of Biological Chemistry 253, 3939-3944. Kuri-Harcuch W. and Mendoza-Figueroa T. (1989) Cultivation of adult rat hepatocytes on 3T3 cells: expression of various liver differentiated functions. Differentiation 41, 148-157. Leffert H. L. and Paul D. (1972) Studies on primary cultures of differentiated fetal liver ceils. Journal of Cell Biology 52, 559-568. Letko G. and Halangk W. (1986) Effect of improved hydrogen supply on energy state, ureogenesis and gluco-
299
neogenesis in isolated hepatocytes. Biomedica Biochimica
Acta 45, 265-271. Lin R. C. and Snodgrass J. (1975) Primary culture of normal adult rat liver cells which maintain stable urea cycle enzymes. Biochemical and Biophysical Research Communications 64, 725-734. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193, 265-275. Lusty C. J. and Ratner S. (1972) Biosynthesis of urea. Journal of Biological Chemistry 247, 7010-7022. Marshall M. and Cohen P. P. (1972) Ornithine transcarbamylase from Streptococcus faecalis and bovine liver. Journal of Biological Chemistry 247, 1641-1653, 1654-1668 & 1669-1682. Mendoza-Figueroa T., L6pez-Revilla R. and Villa-Trevifio S. (1983) DNA breaks induced by micromolar concentrations of dimethylnitrcsamine in liver primary cell cultures from untreated and phenobarbital treated rats. Toxicology 27, 55-69. Powers S. G. and Meister A. (1982) Urea synthesis and ammonia metabolism. In The Liver: Biology and Pathobiology. Edited by I. Arias, H. Popper, D. Schachter and D. A. Shafritz. pp. 251 263. Raven Press, New York. Raijman L. (1983) Ornithine carbamoyltransferase in liver. In Methods of Enzymatic Analysis. Edited by J. Bergmeyer and M. Grable. pp. 32(~334. Verlag Chimie, Weinheim. Ratner S. (1973) Enzymes of arginine and urea synthesis. Advances in Enzymology 39, 140. Rheinwald J. G. (1978) Serial cultivation of normal human epidermal keratinocytes. Methods of Cell Biology 21A, 229-254. Rochovansky D. and Ratner S. (1967) Biosynthesis of urea. Journal of Biological Chemistry 242, 3839-3849. Schimke R. T. (1962) Adaptive characteristics of urea cycle enzymes in the rat. Journal of Biological Chemistry 237, 459-468. Snodgrass P. J., Lin R. C., Miiller W. A. and Aoki T. T. (1978) Induction of urea cycle enzymes of rat liver by glucagon. Journal of Biological Chemistry 7, 3047 3051. Story D. L., Gee S. J., Tyson C. A. and Gould D. H. (1983) Response of isolated hepatocytes to organic and inorganic cytotoxins. Journal of Toxicology and Environmental Health 11, 483-501.