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Possible Interactions Between the Urea Cycle and Synthesis of Pyrimidines and Polyamines in Regenerating Liver'

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Possible Interactions Between the Urea Cycle and Synthesis of Pyrimidines and Polyamines in Regenerating Liver' [CANCER RESEARCH 35. 397-404, February 1975] Possible Interactions between the Urea Cycle and Synthesis of Pyrimidines and Polyamines in Regenerating Liver' Nelson Fausto, John T. Brandt, and Leo Kesner Division of Biological and Medical Sciences, Brown University, Providence, Rhode Island 02912 [N.F., J.T.B.]; and Department of Biochemistry. Downstate Medical Center, State University of New York, Brooklyn, New York 11203 [L.K.] SUMMARY only when injected during the 1st hr after partial hepatec tomy (1 1, 30). We suggested that, in addition to increased Ornithine levels rise progressively in the liver of partially synthesisof the enzyme, posttranscriptional control of the hepatectomized rats, probably as a consequence of the enzyme activity exists in regenerating liver (4, 11). increased flow of metabolites through the urea cycle. Although it is possible that amino acids, growth hormone Ammonia and urea concentrations in the blood and liver of (11), cyclic 3':S'-AMP (I), or a combination ofthese factors partially hepatectomized animals are not significantly dif may be the initial stimulus for the increase in ornithine ferent from those of sham-operated rats. However, in decarboxylase activity after partial hepatectomy, it seems regenerating livers, the ability to remove ammonia from the essential to consider the relationships between polyamine blood is close to its maximal limit. Ammonia overload leads synthesis and other metabolic processes (13). The loss of to the production of large amounts of orotic acid and causes liver mass caused by the operation imposes an increased a marked elevation of hepatic ornithine decarboxylase physiological demand on the liver remnant that is likely to activity. Among the pyrimidine precursors dihydroorotic alter the functioning of key biochemical pathways. acid injections increase the activity of the enzyme while Alterations of the urea cycle that may occur after partial orotic acid is without effect. A peak of labeled material that hepatectomy could be reflected in changes in the rate of corresponds to dihydroorotic acid was identified by parti polyamine biosynthesis because ornithine is a key metabo tion chromatography of acid-soluble extracts of livers of lite in the urea cycle and the 1st precursor of the polyamine partially hepatectomized rats previously given injections of pathway. In addition the injection of ammonium chloride or [14C]bicarbonate. The labeling of dihydroorotic acid from acetate into rats is associated with the excretion of large [‘4C]bicarbonate is increased in the liver of rats given amounts of orotic acid (20, 32). These observations led us to injections of ornithine. Despite the difficulties involved in explore the hypothesis that possible physiological adapta studies of ornithine decarboxylase activity in vivo, our tions in ammonia or amino acid metabolism following results suggest that mutual interactions between urea, partial hepatectomy might interact with the processes of pyrimidine, and polyamine synthesis take place during liver polyamine and pyrimidine biosynthesis. Our results suggest regeneration. that the urea cycle adapting to the metabolic load imposed on the liver remnant (36) might provide precursors for both INTRODUCTION polyamine and pyrimidine synthesisduring liver regenera tion. One of the earliest responses to partial hepatectomy in rats is the elevation of ornithine decarboxylase activity in the liver remnant (10, 16, 29, 31). This enzyme, which MATERIALS AND METHODS catalyzes the synthesis of putrescine (l,4-diaminobutane) from ornithine, is the 1st and probably the rate-limiting step Animals. The animals used in these experiments were in polyamine biosynthesis (33, 40). The elevation of putres male albino rats (The Holtzman Co., Madison, Wis.) cine synthesis during liver regeneration coincides in time weighing 130 to 170 g. They were maintained in a tempera with changes in RNA synthesis (10). ture-controlled room with 12-hr dark-light cycles. All The stimuli that are responsible for the elevation of liver animals were killed between 9 and I 1 a.m. Initially, all rats ornithine decarboxylase activity following partial hepatec used were starved for 14 hr before the experiments. We tomy have not yet been identified. While cycloheximide or found, however, that more consistent results could be puromycin injections given at any time during the 1st day obtained with rats fed ad libitum, provided that the rats following the operation block the activity of the enzyme, were maintained in a room with controlled illumination. actinomycin D inhibits ornithine decarboxylase activity Unless otherwise indicated, food was not withdrawn until the start of the experimental procedures. Hypophysecto 1 Supported by Grant AM 14706 from the NIH. mized rats were purchased from Charles River Breeding Received September 3, 1974: accepted October 25, 1974. Laboratory, Wilmington, Mass. Rats from the same sup FEBRUARY 1975 397 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1975 American Association for Cancer Research. N. Fausto et a!. plier were usedascontrols in experiments involving hypoph potassium oxalate. The tube was kept in ice and the ysectomized rats. Partial hepatectomies were performed by determination ofammonia was started not later than 10 mm the method of Higgins and Anderson (15). Animals were after blood withdrawal. The precautions indicated by Tern anesthetized with a mixture of ether and oxygen as de berg and Hershey (35) for handling of the blood were scribed by Bucher and Swaffield (6). The operation resulted followed carefully. A sample of the blood (0. 1 to 0.2 ml) was in the removal of approximately 70% of the liver. pipetted into a flask containing 1.0 ml of saturated sodium Adrenalectomies were performed 7 to 10 days before the carbonate. The flask was closed with a rubber stopper experiments. Adrenalectomized rats were given 1% NaC1 perforated by a glass rod. The tip of the rod was dipped into solution instead of water. I N sulfuric acid. The glass flasks were placed in a rotator Materials. DL-[ I -‘4C]Ornithine-HCI (specific activity, apparatus (Scientific Industries Inc., Springfield, Mass.) 12.8 mCi/mmole) and [‘4C]sodium bicarbonate (specific for 30 mm. After the completion of the microdiffusion, the activity, 50 mCi/mmole) were purchased from New Eng ammonia was determined by the Berthelot procedure using land Nuclear, Boston, Mass. Urease (jack bean meal, nitroprusside as a catalyst. The combination of reagents URPC, 240 units/mg, lyophilized) was obtained from described by Chaney and Marbach (8) was used for the Worthington Biochemical Corp., Freehold, N. J. A unit of color development. The absorbance of the samples was activity corresponds to the liberation of I @imoleof am measured at 625 nm and compared to that of ammonium monia per mm at 25°. All other chemicals used were sulfate standards processed in a similar manner. A linear obtained either from Schwarz/Mann, Orangeburg, N. Y., relationship between ammonia content in blood and A was or from Sigma Chemical Co., St. Louis, Mo. obtained for samples containing 1 to 8 zg of ammonia. The Ornithine Decarboxylase Activity. Rats were killed by recoveries of the microdiffusion step was 85 to 90%. Blood decapitation. The livers were rapidly removed, weighed, cut urea was determined either by urease digestion followed by into small pieces, and homogenized in a Potter-Elvehjem the colorimetric procedure described by Chaney and Mar homogenizer in 0.25 M sucrose, 10 m@iTris buffer (pH 7.5), bach (8) or with an amino acid analyzer. 0.5 m@i EDTA, and I m@imercaptoethanol. The homoge Determination of Orotic Acid and Dihydroorotic Acid. nates (20%, w/v) were centrifuged for 60 mm at I 15,000 x g Sham-operated or partially hepatectomized rats were given in an International Centrifuge (Model B-60). The cytosol injections of 75 @iCi of [‘4C]sodium bicarbonate. The was collected and used immediately for the enzyme assay. animals were killed 60 mm after the injection of the The incubation mixture contained, in a final volume of 1.0 precursor. The livers were removed and homogenized in ml: 0.2 ml of 0.05 M Tris buffer (pH 7.8), 0.2 zmole of pyri 0.25 M sucrose. A sample of the homogenate was precipi doxal phosphate, and 0.2 to 0.4 ml of the cytosol. After 10 tated with 5% perchloric acid and centrifuged. The superna mm ofpreincubation at 37°,0.4 ml (0.8,uCi)of[l-'4C]orni tant was saved and mixed with 2 more perchloric acid thine was added and the flasks were closed with rubber stop extractions of the pellet. The combined supernatants were pers fitted with a polyethylene center well and a capillary neutralized with potassium hydroxide. After centrifugation tube. The center well contained 0.25 ml of a CO2-trapping the volumes ofthe supernatants were measured and samples solution of ethanolamine in ethylene glycol (1:2, v/v). The of the extracts were stored at —20°untilused. Orotate and samples were incubated for 30 mm at 37°and the reaction dihydroorotate analyses were performed by the partition was stopped by adding 0.5 ml of 5% trichloroacetic acid chromatography procedure of Kesner and Muntwyler (20, through the capillary tube (4, 10). After 30 mm at room 21). Dihydroorotic acid appears as a peak on the organic temperature, 0. 1 ml of the CO2-trapping solution was acid analyzer in the area of malic acid (21, 22). The pipetted into a vial containing 10 ml of scintillation fluid. separation of these 2 substances is often imperfect, making The radioactivity was determined in a Nuclear Chicago quantitation difficult. It is possible to cause a sample counter and all determinations were corrected for counting containing a mixture of these 2 substances to react with efficiency. Under the conditions used for determining ceric sulfate and sulfuric acid. We have found that varia ornithine decarboxylase activity, addition of dithiothreitol tions in concentration of sulfuric acid and ceric sulfate will to the incubation mixture slightly inhibited the activity of alter the nature of the oxidation products of malic acid.
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