[CANCER RESEARCH 35. 397-404, February 1975] Possible Interactions between the Cycle and Synthesis of Pyrimidines and Polyamines in Regenerating '

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 . 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 levels rise progressively in the liver of partially synthesisof the , 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 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 , 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 and causes liver mass caused by the operation imposes an increased a marked elevation of hepatic 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 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 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 (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

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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 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 , 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 (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. the enzyme. In the presence of high concentrations of However, the complete removal of malic acid from the ornithine (2 mM) in the assay, 5 m@i dithiothreitol was reaction mixture is easily accomplished by the following necessaryfor obtaining maximal enzyme activity (17). The procedure: the fractions containing dihydroorotate ob results obtained with assays using high substrate concentra tamed by partition chromatography are dried in a small tions and dithiothreitol agreed with those presented here. beaker by a stream of air. To the dried residue 300 @tl0.1 M was determined by the method of Lowry et a!. (24). sulfuric acid and 10 mg solid ceric sulfate are added. The Samples containing no cytosol or boiled supernatants were mixture is agitated at room temperature for 30 mm, after used as blanks. Enzyme activity is expressed in pmoles of which the mixture is filtered or centrifuged. A [email protected] “CO2produced in 30 mm per mg of liver cytosol protein. In is used for analysis on the organic acid analyzer. each assay enzyme activity was determined using 2 different Amino Acid Analysis. The free amino acids in tissuewere amounts of cytosol, generally 0.2 and 0.4 ml. A continuous run on a Technicon acid analyzer. Frozen tissues were check on the linearity of the reaction was thus assured. homogenized in a microblender using 4 ml of 3% sulfosali Ammonia and Urea Assays. Whole blood used for cylic acid for each 0.5 g of liver. A 0.4-ml aliquot of the determination of ammonia was collected from the jugular extract was analyzed using norleucine as an internal stan vein and immediately placed in a test tube containing dry dard.

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RESULTS sions showed no alterations in the 1st 6 hr following partial hepatectomy. Other investigators have reported no signifi Ammonia, Urea, and Ornithine Levels after Partial cant changes in ornithine carbamoyltransferase activity in Hepatectomy. The loss of liver cell mass caused by partial 24-hr regenerating livers (2, 38). Since urea production per hepatectomy may produce changes in the levels of sub liver 24 hr after partial hepatectomy is 60 to 70% higher strates involved in the urea cycle. We measured initially the than production in sham-operated rats (37), we conclude plasma concentrations of ammonia and urea as well as liver urea levels in sham-operated and partially hepatectomized rats. As presented in Table I, the blood ammonia nitrogen of partially hepatectomized rats does not appear to differ significantly from that of sham-operated rats in the 1st6 hr following the operations. Urea values in the plasma of partially hepatectomized rats increased slightly after the operation, but a similar increase was found in the sham operated controls. The concentrations of urea in the liver after partial hepatectomy are comparable to the concentra tions in the sham-operated rats. Although partially hepatectomized rats remove ammonia from the blood efficiently, they are unable to dispose of any excess of amino acids or ammonia. When a complete amino acid mixture or ammonium acetate is given to sham operated rats, only minimal and transient increases occur in free ammonia in the blood. In contrast, in partially hepatectomized animals ammonia levels increase by 6-fold or more after ammonium acetate injection (Chart I). Table 2 shows the levels of ornithine in the liver of sham-operated and partially hepatectomized rats. In the 1st 2 hr after partial hepatectomy, there is a rapid increase in 20 40 60 time after ammonium acetate administration (mm ornithine. The amount of ornithine doubles 2 hr after the operation and at 18 hr is 4 to 5 times higher than that of Chart I . Blood ammonia after ammonium acetate administration. Sham-operated and partially hepatectomized animals received I mmole of sham-operated rats. The progressive accumulation of orni ammonium acetate by stomach tube. Blood was withdrawn from the thine (14, 25) could have been caused by a decrease in jugular vein at the times indicated. Free ammonia nitrogen in the blood was ornithine carbamoyltransferase activity, as has been demon determined as described in Table I. A blood sample was taken during the strated in hepatomas (38, 39). Measurements ofthe activity operations, immediately preceding the administration of ammonium of this enzyme in homogenates and mitochondrial suspen acetate.

Table I A mmonia and urea concentrations after partial hepatectomy Whole blood was collected from thejugular vein, immediately placed in a test tube containing dry potassium oxalate, and kept on ice. Ammonia determinations were started no later than 10 mm after blood withdrawal. Microdiffusion of ammonia was carried out for 30 mm at room temperature using a rotator apparatus. Recoveries at this step were 85 to 90%. After completion of the diffusion, ammonia was determined by the Berthelot procedure with nitroprusside as a catalyst and the combination of reagents described by Chancy and Marbach (8). Urea was determined either by urease digestion followed by colorimetric determination or with an amino acid analyzer. The values for each point were obtained from 4 to 7 rats.

after in operation ammonia N plasma in liver (zmoles/g)ShamTreatmentTime (hr)Blood (nmoles/ml)Urea (@moles/ml)Urea

operation0 3.1Partialhepa I 4.32.1 2 112(94-135) 4 123(108—141) 6l29(l00-l59)°l29(45-l94)1.1

(76-14l) tectomy0 0.5 I 3.9 1.6 2 123(100-159) 4.82.6 2.0 4 159(106-200) 6129 129(88-159)2.3

a Numbers in parentheses, range of variation.

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Table 2 for the synthesis of carbamoylphosphate from CO2 and Ornithine concentrations in normal and regenerating livers ammonia (18, 34). We gave animals injections of various Sham-operated and partially hepatectomized animals were killed dosages of N-acetyl-L-glutamate or N-carbamoyl-L-gluta immediately after or at various times following the operations. A fragment mate. Carbamoylglutamate can perform the same function of the liver was frozen in situ using a clamp previously cooled in liquid nitrogen. The frozen samples were deproteinized with sulfosalicylic acid and is more stable than the natural compound (23). and the analyses were performed using a Technicon amino acid analyzer. Injections of 50 to 350 zmoles of acetyl- or carbamoylgluta mate into adrenalectomized rats led to 20- to 60-fold ornithine elevations in liver ornithine decarboxylase activity (Table after concentration 4). As shown in Table 4 the increase in the activity of the liver)Sham0OperationTime operation (hr)Liver (@moles/g enzyme occurring 5 hr after the injection of N-acetyl-L glutamate is inhibited by actinomycin injections. Inhibition 0.75Partial 40.68 of ornithine decarboxylase activity was obtained with actinomycin given either immediately after or 1hr following hepatectomy0.5 I 1.6 the administration of N-acetyl-L-glutamate. 2 1.3 Orotate Formation and the Effect of Pyrimidines on 4 1.9 Ornithine Decarboxylase Activity. Changes in pyrimidine 180.43 3.0 metabolism, which include a 50% expansion of the endoge

Table 3 that the ornithine levels in the liver rise as a consequence of Blood ammonia concentrations and liver ornithine decarboxylase activity the increased flow of metabolites through the urea cycle. f ollowingthe injectionof urease The ammonia load per cell in regenerating livers obviously Rats were adrenalectomized 7 days before the experiments and given 1% is elevated, but the cells of the liver remnant are able to NaCI solution. The rats were given injections of I unit of urease or 0.9% NaCl solution ( I ml volumes) and killed at the times indicated on the table. adapt to the metabolic demands. Blood for ammonia determinations werecollectedfrom the jugular vein. Urea Cycle Substrates and Ornithine Decarboxylase Ornithine decarboxylase activity was measured in liver cytosol obtained by Activity. In addition to participation in the urea cycle, centrifugation of liver homogenate for 1 hr at 105,000 x g. For each ornithine is the substrate for ornithine decarboxylase, the determination 0.2 and 0.4 ml of cytosol were used (see “Materials and 1st enzyme of the polyamine pathway. The increased levels Methods―). The enzyme activity is expressed in pmoles of 14C02 formed in 30 mm per mg of cytosol protein. Six rats were used for each determina of ornithine present in the livers of partially hepatectomized tion. rats might induce or stabilize ornithine decarboxylase activity. However, ornithine injections did not increase ornithine decarboxylase activity. Adrenalectomized rats decarboxylase were given injections of 150 @.imolesofornithine and were activity (pmoles ammonia ofCO2/mg of killed 5 hr later. Ornithine decarboxylase activity was protein)0.9%Hr after injectionBlood N (nmoles/ml)Ornithine measured in both dialyzed and undialyzed liver cytosol to avoid dilution of the labeled ornithine used as substrate in NaCl solution2 the enzyme assay. The enzyme activities of animals given 93 3 4 129 4 injections of 0.9% NaCI solution were 0.5 to 5 pmoles of 4UreaseI 6100 1082 ‘4CO2formed per 30 mm per mg of protein. The rats receiving ornithine had activities in the range of 2 to 8 8 pmoles of ‘4CO2.Injections of 100 to 150 @moles of 2 237 7 , , argininosuccinate, urea, or glucose into 3 27 adrenalectomized rats did not alter hepatic ornithine decar 4 258 47 boxylase activity. S 29 6 2003 18 Injections of urease into rats stimulate the urea cycle and 8 3 elevate blood ammonia levels and liver amino acid concen 12100 3 trations (9, 27). A single injection of 1 unit of urease produced a sustained elevation of blood ammonia and a Co2 + GLUTAMINE Co2 ÷ NH3 @ 20-fold stimulation of liver ornithine decarboxylase activity I@ETYLGLUTAMATE (Table 3). The enzyme activity is maximal between 4 and 5 CARBAMOVLPHOSPHATE CARBAM@LPH0SPHAT[ hr following urease injection and returns to normal at 8 hr. ASPARTATE Neither glucocorticoids nor growth hormone is involved in this response because the same elevations in enzyme activity I —ORNITHINE-——-—CITRULLINE CARBAMYL I were found in adrenalectomized and hypophysectomized ASR@,RTATE ______ARG rats. I UREA@ARGININEa--

Since ornithine decarboxylase activity could be markedly t@HYDR0 increased only by the urea cycle stimulation caused by OROTATE PUTRESCINE ——‘ SPERMIDINE ------‘SPERMINE urease but not by ornithine or the other intermediates, we studied the effects of N-acetyl-L-glutamate on the enzyme 0ROTATE—@OROTIDYLATE —*UMP------.UTP------.RNA activity. This compound (Chart 2) is the required Chart 2. Urea cycle, pyrimidine. and polyamine pathways.

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Table 4 Effect of urease and cofactors of the urea cycle on hepatic ornithine decarboxylase activity Rats were adrenalectomized 7 days before the experiments and given 1% sodium chloride. Animals were given injections of various compounds shown on the table and killed S hr later. Ornithine decarboxylase activity is expressed as pmoles of 14CO2 formed in 30 mm per mg of cytosol protein (see legend of Table 4). Rats received actinomycin at a dosage of 100 @g/l00 g of body weight either immediately after or 1 hr after acetylglutamate injections. Urease was inactivated by boiling for I mm. The hypophysectomized rats were not adrenalectomized. Six rats were used for each determination.

Ornithine decarbox Amount injected ylase activity

0.9% NaCI solution I ml 3(1-7)― Glucose 200mg 5(2-6) Urease 0.5 unit 17 (13-24) Urease(inactivated) 0.5unit 2(0-2) Urease I unit 47 (40-48) 0.9% NaCI solution (hypophysectomized) I ml 3(0-6) Urease (hypophysectomized) I unit 43 (38-39) N-Carbamoyl-L-glutamate 50 @imoles 85 (46-122) N-Carbamoyl-i-glutamate 100 @moIes 137 (73-181) N-Carbamoyl-L-glutamate 200 @moles 160(123-198) N-Carbamoyl-i-glutamate 350zmoles 209(140-268) N-Acetyl-L-glutamate 100 @moles 120(III-I36) N-Acetyl-L-glutamate (actinomycin at 100 @imoles 82 (69-100) time0) !@-Acetyl-i-glutamate(+ 100 @moles 40 (2 1-67) I hr after)

a Numbers in parentheses, range of activity.

nous UTP and CTP pools, occur in rat livers following Table 5 partial hepatectomy (5-7). As shown in Table 5, injections Conversion of metabolites to orotic acid of ammonium chloride, glutamine, or carbamoylaspartate Rats fasted for 18 hr were given injections of the metabolites indicated into fasted intact rats lead to the excretion of large amounts above. was collected and pooled. Orotic acid content was determined by silica gel column chromatography (20, 21). of orotic acid. Thus, ammonia overload appears to repro duce 3 of the early events of liver regeneration: (a) elevation excreted of ornithine concentration in the liver (27); (b) stimulation (zmoles)0.9%MetaboliteAmount injectedOrotate in 24 hr of pyrimidine biosynthesis (20, 32); and (c) the “induction― mlNoneAmmoniumNaCI solution3 of ornithine decarboxylase activity. The next logical step chloridel000Mmoles7.5Glutamine1000 was to examine the effects of various pyrimidine precursors @moles3.0DL-Carbamoylaspartate50 and derivatives on the activity of this enzyme (Table 6). .tmoles12.0 Orotic acid, methylorotic acid, and uracil did not alter the activity of the enzyme. However, further surveysof pyrimi dines showed that carbamoylaspartate injections to some were analyzed by partition chromatography in silicic acid extent and dihydroorotic acid injections in particular mark columns, and the peak corresponding to dihydroorotic acid edly increase liver ornithine decarboxylase activity. A linear was further treated by cerium sulfate to eliminate contami relationship exists between the dosageofdihydroorotic acid nation by malic acid (see “Materialsand Methods―). At 0.5 and ornithine decarboxylase activity (Chart 3). The effect of hr to 1 hr after partial hepatectomy we detected a distinct dihydroorotic acid is not altered by adrenalectomy or peak of radioactive material corresponding to dihydroorotic hypophysectomy, and the D form is 50 to 70% less active acid (Chart 4) that was approximately 5 times higher than than the L compound (Table 6). the peak found in the sham-operated controls. Further Labeling of Dihydroorotate in Regenerating Livers. The analyses of dihydroorotic acid metabolism both in regener effect ofdihydroorotic acid on putrescine synthesis suggests ating livers and in Ll210 tumor cells (L. Kesner, unpub that in rat liver a relationship between ornithine decarboxyl lished observation) revealed an effect of ornithine on ase activity and pyrimidine synthesis may exist. This could dihydroorotic acid labeling (Chart 5). Rats were given in explain some of the events occurring at the early stages of jections of 75 @Ciof[ ‘4C]bicarbonate1hr prior to killing. A liver regeneration, provided that dihydroorotic acid synthe group of rats received 3 mmoles of ornithine 0.5 hr after the sis increases after partial hepatectomy. We investigated the injection of the label. Partition chromatography of the labeling ofdihydroorotic acid in the 1st hr following partial acid-soluble extracts indicated that the labeling of dihy hepatectomy. Partially hepatectomized and sham-operated droorotate is almost 10 times higher in the animals given rats were given injections of [‘4Cjbicarbonate.The acid injections -of ornithine, with apparently little change in soluble extracts obtained from the liver of these animals orotic acid synthesis.

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DISCUSSION metabolism of the urea cycle and has also been demon strated experimentally in rat liver slices (3). Alternatively, The flow of metabolites through the urea cycle is regenerating livers could clear ammonia from the blood augmented after partial hepatectomy, and the ability to through the synthesis of glutamate, which might stimulate remove ammonia from the blood is close to its maximal the 1st step in pyrimidine biosynthesis (18, 34). Some of the limit. Under these conditions carbamoylphosphate not pyrimidine precursors formed by either or both of these utilized in citrulline synthesis would be channeled to the processes may participate in the “induction―of ornithine pyrimidine pathway leading to the synthesis of orotic acid decarboxylase activity during liver regeneration. (20, 32). Cross-over of carbamoylphosphate from the urea The labeling of dihydroorotic acid from bicarbonate is to the pyrimidine pathway occurs in inborn errors of approximately 5 times higher in regenerating livers than in sham-operated controls. In addition, a distinct peak corre Table 6 sponding to dihydroorotic acid was detected in acid-soluble Effect of various pyrimidine precursors and derivatives on hepatic extracts of the liver of rats given injections of ornithine. We ornithine decarboxylase activity do not know at this time the nature or the significance of Rats were adrenalectomized 7 days before the experiments and this effect of ornithine on dihydroorotic acid metabolism. maintained on 1% NaCI solution. Ornithine decarboxylase activity is Dihydroorotate oxidation to orotic acid is the only step of expressed as pmoles of “'CO2formed in 30 mm per mg of cytosol protein the pyrimidine pathway that takes place in mitochondria (see legend of Table 4). Hypophysectomized rats were not adrenalecto mized. Six rats were used for each determination. (19). Ornithine carbamoyltransferase (28) and most of the steps of urea biosynthesis also take place in this organelle. OrnithinedecarboxylaseAmount Shifts in the intracellular distribution ofdihydroorotate and ornithine would have to be examined for their possible injectedactivity0.9% effects on urea, orotic acid, and polyamine synthesis. The -7)―Aspartatel50@zmoles32(10-72)CarbamoylaspartateINaCl solutionI ml3 (I detection of a distinct peak of dihydroorotic acid shortly after partial hepatectomy is also of interest because in -89)L-Dihydroorotic 50 @imoles32 (2 1 Ehrlich ascites cells most involved in de novo (50-73)D-DihydroOroticacid75 @moles65 synthesis of pyrimidines appear to form a complex, and no 2-27)i-Dihydrooroticacid75 Mmoles20 (I (20-108)(hypophysectomized)D-DihydrOoroticacid75 @moles57 intermediates between bicarbonate and UMP accumulate under normal circumstances (18). (10-36)(hypophysectomized)Oroticacid75 @moles22 Our results indicate that ornithine levels in the liver rise rapidly after partial hepatectomy. These results are in (3-6)Methylacidl50MmolesS orotic acid1 50 @moles2 ( I -5) agreement with the findings of Ferris and Clark (14) and Ord and Stocken (25), who reported changes in amino acid a Numbers in parentheses. range of activity. pools in liver and blood following partial hepatectomy.

Chart 3. Effects of dihydroorotic acid on ornith me decarboxylase activity. Adrenalectomized rats were given i.p. injec tions of the doses of dihydroorotic acid > shown on the abscissa and killed 45 hr later. Ornithine decarboxylase (ODC) activity was measured in the cytosol after centrifugation of the liver homogenates for I hr at 105.000 x g. Enzyme activity is expressed in pmoles of “'CO2released in 30 mm per mg of cytosol protein. Each point is the average value from 3 animals.

@mc@esofdihydrooro@cacid injected

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0 10 15 20 25 frac@,onnumber fract@,nnumber Chart 4. Dihydroorotic acid labeling in regenerating livers. PartialLy Chart 5. Effect of ornithine on dihydroorotic acid labeling in regener hepatectomized and sham-operated rats were given injections of 75 zCi of ating livers. Partially hepatectomized rats were given injections of either [‘4C]sodiumbicarbonate. Thirts' mm after the injection the liver was frozen 0.9% NaCl solution or 3 mmoles ofornithine 18 hr after the operation. All in situ using a clamp previously chilled in liquid nitrogen. The liver samples animals received 50 @Ciof [‘4C]sodium bicarbonate 30 mm after 0.9% were deproteinized with perchloric acid and neutralized with potassium NaCI solution or ornithine injection and were killed 30 mm after the hydroxide. The acid-soluble extracts were analyzed by partition chroma injection of the labeled precursor. The livers were frozen in situ and the tography in silicic acid columns as described by Kesner and Muntwyler acid-soluble extracts were analyzed by partition chromatography in silicic (20, 21). Unlabeled compounds were used as markers. The fractions acid columns. Ordinate, radioactivity present in each fraction. —, rats corresponding to the dihydroorotate peak were collected and treated with given injections of ornithine; ----, rats given injections of 0.9% NaCI cerium sulfate to eliminate contamination by malic acid. After this solution. The location of the appropriate markers is shown. treatment the samples were rechromatographed and the fractions corre sponding to dihydroorotic acid were collected and counted in a liquid scintillation counter. considerable range of variation in enzyme activity exists both under basal conditions and after stimulation. None of However, it does not appear that the elevated levels of the compounds that stimulated ornithine decarboxylase ornithine in regenerating livers cause the increase in orni activity in vivo appears to have an effect when tested in thine decarboxylase activity after partial hepatectomy. crude cell extracts in vitro. Within these limitations, our Ornithine, citrulline, argininosuccinate, arginine, and urea results point to some interrelationships between polyamine injections do not alter hepatic ornithine decarboxylase and pyrimidine synthesis and the functioning of the urea activity. This activity is markedly elevated after urease, cycle after partial hepatectomy. acetylglutamate, or carbamoylglutamate injections, all of It is not yet known whether the changes in RNA which may lead to increases in ammonia, glutamine, or metabolism and polyamine synthesis occurring in regenera carbamoylphosphate. tion are triggered by a common stimulus or by independent Orotic acid, methylorotic acid, uracil, and dihydrouracil mechanisms. Whatever the case may be, polyamine and injections have no effect on liver ornithine decarboxylase RNA metabolism in regenerating rat liver might be linked activity. In contrast, dihydroorotic acid in dosages above 20 at various levels: (a) by the effect of pyrimidines on jzmoles causes a very large increase in ornithine decarboxyl ornithine decarboxylase activity, the 1st and rate-limiting ase activity, which is proportional to the amount of the step of the polyamine pathway; (b) by an effect of ornithine compound injected. The same effect of L-dihydroorotic acid on dihydroorotate metabolism; and (c) by a possible effect on liver ornithine decarboxylase activity was obtained in of polyamines on RNA transcription (12). intact, adrenalectomized, or hypophysectomized animals. In an attempt to circumvent the difficulties involved with in vivo studies of ornithine decarboxylase activity (3 1), most REFERENCES of the metabolites used were tested for their effect on the activity of the enzyme in adrenalectomized and hypophysec I. Beck, W. T., Bellantone, R. A., and Canellakis, E. S. The In Vivo tomized rats. Although it has been possible to exclude the Stimulation of Rat Liver Ornithine Decarboxylase Activity by Dibuty ryl Cyclic Adenosine 3'-5'-Monophosphate, Theophylline and Dexa participation of adrenal and growth hormones, it is ex methasone. Biochem. Biophys. Res. Commun.. 48: 1649-1655, 1973. tremely difficult to ascertain if other hormones (glucagon, 2. Bhide, S. V. Comparative Study of Metabolic Profiles of Primary insulin) are released under these conditions (26). In our Hepatoma. Regenerating Liver, and Liver in Newborn Swiss Mice. J. experiments there was an obvious effect of various com NatI. Cancer Inst., 47: 797-800, 1971. pounds on liver ornithine decarboxylase activity, but a 3. Bourget, P. A., Natale, P. J., and Tremblay. G. C. Pyrimidine

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Nelson Fausto, John T. Brandt and Leo Kesner

Cancer Res 1975;35:397-404.

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