ARCHIVES OF BIOCHEMISTRY AND BIOPHYSKS Vol. 188, No. 1, May, pp. 194-205, 1978

Regulation of Pyrimidine Biosynthesis by Purine and Pyrimidine Nucleosides in Slices of Rat Tissues’

DAVID E. CRANDALL, CAROL J. LOVATT, AND GEORGE C. TREMBLAY

Department of Biochemistry and Biophysics, University of Rhode Island, Kingston, Rhode Island 02881 Received November 4, 1977; revised January 27, 1978

Evidence of the primary sites for the regulation of de novo pyrimidine biosynthesis by purine and pyrimidine nucleosides has been obtained in tissue slices through measurements of the incorporation of radiolabeled precursors into an intermediate and end product of the pathway. Both purine and pyrimidine nucleosides inhibited the incorporation of [‘“Cl- NaHCO:< into erotic acid and uridine nucleotides, and the inhibition was found to be reversible upon transferring the tissue slices to a medium lacking nucleoside. The ammonia- stimulated incorporation of [‘4C]NaHCOa into erotic acid, which is unique to liver slices, was sensitive to inhibition by pyrimidine nucleosides at physiological levels of ammonia, but this regulatory mechanism was lost at toxic levels of ammonia. Adenosine, hut not uridine, was found to have the additional effects of inhibiting the conversion of [%]orotic acid to UMP and depleting the tissue slices of PKPP. Since PKPP is required as an activator of the first enzyme of the de novo pathway, CPSase II, and a substrate of the fifth enzyme, OPRTase, these results indicate that adenosine inhibits the incorporation of [‘%]NaHC03 into erotic acid and the incorporation of [‘%]orotic acid into UMP by depriving CPSase II and ‘OPRTase, respectively, of PRPP. Uridine or its metabolites, on the other hand, appear to control the de nouo biosynthesis of pyrimidines through end product inhibition of an early enzyme, most likely CPSase II. We found no evidence of end product inhibition of the conversion of erotic acid lo UMP in tissue slices.

Studies employing cell-free extracts of genase are inhibited by erotic acid (3-5); mammalian tissues have demonstrated that and orotidine-5’-phosphate decarboxylase several of the enzymes of the orotate path- (ODCase) is inhibited by UMP and CMP way for the de nouo biosynthesis of pyrim- (6,7). Attempts to determine which of these idines are subject to regulation by end prod- enzymes has physiological importance as a ucts or intermediates of the pathway. The regulatory site include several recent stud- glutamine-dependent carbamoylphosphate ies employing intact cells. Work performed synthetase (CPSase II)” is inhibited by in our laboratory has shown that uridine UTP (1) and activated by &phosphoribo- inhibits the incorporation of [‘4C]NaHC03, syl-I-pyrophosphate (PRPP) (2); dihy- but not [‘4C]carbamoylphosphate (CP) or droorotase and dihydroorotate dehydro- [‘4C]carbamoylaspartate, into erotic acid in ’ This investigation was supported by Public Health slices of several rat tissues (8, 9), and Ito Service Research Grant No. CA17131 from the Na- and Uchino have found that uridine inhibits tional Cancer Institute. the incorporation of [‘4C]NaHC03 into car- ’ Abbreviations used: CPSase I, ATP:carbamate bamoylaspartate in human lymphocytes phosphotransferase (dephosphorylating; EC 2.7.2.5); (10). In addition, the incorporation of CPSase II, ATP:carbamate phosphotransferase (de- [14C]NaHC03, but not [14C]CP, into uridine phosphorylating, amido transferring; EC 2.7.2.9); nucleotides was stimulated in perfused liver PRPP, 5phosphoribosyl-I-pyrophosphate; CP, car- after UTP levels had been depleted by bamoylphosphate; OPRTase, orotidine-5’.phosphate: treatment with galactosamine (11). These pyrophosphate phosphoribosyltransferase (EC 2.4.2.10); ODCase, orotidine-5’-phosphate decarboxyl- results are consistent with the interpreta- ase (EC 4.1.1.23); ACTase, aspartate carbamoyltrans- tion that the regulation of pyrimidine bio- ferase (EC 2.1.3.2); uv, ultraviolet. synthesis observed in the intact cells of 194 ooO3-9861/78/1881-0194$02.00/O Copyright Q 1978 by Academic Press, Inc. All rights of reproduction in any form reserved REGULATION OF PYRIMIDINE BIOSYNTHESIS 195 mammalian tissues occurs at CPSase II. purine nucleosides on the de novo pathway We have recently demonstrated that CP for pyrimidine biosynthesis. Work in our produced from ammonia by CPSase I laboratory has demonstrated that purine within liver mitochondria constituted at nucleosides inhibit the incorporation of least 80% of the CP incorporated into erotic [‘4C]NaHC03 into erotic acid in the chick acid when liver slices were incubated with oviduct and in the rat brain and mammary ammonia and ornithine at physiological gland (9, 18), but the mechanism of inhibi- concentrations (12). Since CPSase I was tion of pyrimidine biosynthesis by purines thus revealed to be an important source of has remained a matter for conjecture. In CP for hepatic pyrimidine biosynthesis, we the present paper, we report results which sought evidence for regulation of the show that adenosine inhibits both the in- CPSase I-catalyzed production of CP or of corporation of bicarbonate into erotic acid the export of CP from liver mitochondria, and the conversion of erotic acid to uridine or both, by end products of the de novo nucleotides. Since the metabolism of aden- pathway. osine consumes PRPP and since PRPP is An additional mechanism of control, at a involved in the de novo biosynthesis of site other than CPSase II, has been sug- pyrimidine nucleotides as an activator of gested by the observation of Hoogenraad CPSase II and as a substrate for OPRTase, and Lee (13) that incubation of rat hepa- we tested the possibility that inhibition of toma cells with uridine resulted in a rapid pyrimidine biosynthesis by adenosine loss of orotate phosphoribosyltransferase might be associated with a depletion of the (OPRTase), but not any of the other en- tissue levels of PRPP. Our findings, re- zymes of the orotate pathway; this result ported herein, show that adenosine, but not led the authors to conclude that OPRTase uridine, reduces the PRPP concentrations is also a principal site of control in the de in tissue slices; thus adenosine most likely novo biosynthesis of pyrimidines. Earlier inhibits the de novo biosynthesis of pyrim- measurements showing that OPRTase may idines by removing the PRPP needed for be the rate-limiting enzyme in the orotate the reactions catalyzed by CPSase II and pathway of Ehrlich ascites cells (14) and OPRTase. the observation that a mild erotic aciduria occurs during normal human pregnancy MATERIALS AND METHODS (15) also indicate that OPRTase may be a Chemicals. All radiolabeled chemicals and Aquasol site of regulation, or at least of interruption, (liquid scintillation cocktail) were purchased from of the de novo biosynthesis of pyrimidine New England Nuclear Corp., Boston, Massachusetts. nucleotides. In order to assess the relative The mixed enzymes orotidylate phosphoribosyltrans- physiological importance of regulation at a ferase and orotidylate decarboxylase (prepared from site early in the de novo pathway (CPSase yeast) were obtained from Sigma Chemical Co., St. II) or late in the pathway (OPRTase), we Louis, Missouri. Acids, bases, and salts were purchased tested the effect of uridine on the incorpo- from Fisher Scientific Co., Boston, Massachusetts, and all other chemicals were purchased from Sigma Chem- ration of [‘4C]NaHC03 and [6-‘4C]orotic ical Co., St. Louis, Missouri. acid into uridine nucleotides in slices of rat Preparation of tissues. Tissues from adult male liver and spleen. The results of these meas- rats weighing 200-250 g were employed in these stud- urements, reported below, support the in- ies; these animals were obtained from the Charles terpretation that feedback regulation of de River Colony, Boston, Massachusetts. The animals novo pyrimidine biosynthesis occurs pri- were killed by decapitation and the tissues of interest marily at an early point in the pathway, were quickly excised and placed in ice-cold saline. most likely at the reaction catalyzed by Tissue slices were prepared using a Stadie-Riggs tissue CPSase II. slicer (Arthur H. Thomas Co.), blotted on filter paper, The observation that the addition of the weighed, and transferred to flasks containing Krebs Improved Ringer II solution (19) to which various purine nucleoside adenosine to cultures of intermediates and inhibitors of pyrimidine biosyn- mammalian cells causes pyrimidine star- thesis had been added as indicated. vation with resulting cell death (16, 17) Measurement of the incorporation of “C-labeled prompted us to examine the influence of precursors into erotic acid. Studies on the incorpo- 196 CRANDALL, LOVATT, AND TREMBLAY

ration of [‘4C]NaHC03 into erotic acid were performed tallized from 67% ethanol, as above, to constant spe- using 500 mg of tissue slices incubated at 37.5”C for 3 cific activity, which rarely required more than two h in 20 ml of Krebs Improved Ringer II solution (19) recrystallizations. The specific activity of the [‘“Cl- I which was supplemented with 6-azauridine (10 mM) to UMP obtained from the final recrystallization was inhibit the conversion of erotic acid to UMP (20). The used to calculate the incorporation of [‘4C]NaHC0:j [‘4C]orotic acid synthesized during the 3-h incubation into uridine nucleotides during the 3-h incubation was isolated from the reaction mixture by cocrystalli- period. This method, employing Krebs Improved zation with carrier monosodium orotate as described Ringer II solution made 30 mM in NaHCO:j, was also previously (12). used to measure the incorporation of [6-‘4C]orotic acid Measurement of the incorporation of “C-labeled into uridine nucleotides. precursors into uridine nucleotides. Tissue slices Preparation of [‘4CJuracil from [‘%]UMP. A sam- weighing 500 mg were incubated for 3 h at 37.5”C in ple of [‘4C]UMP was isolated by cocrystallization with 5 ml of Krebs Improved Ringer II solution made 30 carrier UMP from the acid-soluble fraction of spleen mM in [?Z]NaHCOs (1100 dpm/nmol). Each reaction slices which had been incubated with [‘?]NaHCO:, flask was sealed with a rubber cap fitted with a plastic (3667 dpm/nmol), and the [‘4C]UMP was recrystal- center well (Kontes Glassware, Vineland, New Jersey) lized to constant specific activity. An aliquot of 5 mg and a filter-paper wick. At the end of the incubation of recrystallized [“‘CIUMP was hydrolyzed in 12 N period, 0.5 ml of 6 N KOH was injected through the HCIOl at 109°C for 1 h, and the resulting [‘?]uracil rubber cap into the center well; the reaction was then was isolated by adsorption to and elution from char- terminated by the injection of 1 ml of 1.5 N HClOd into coal, followed by descending paper chromatography in the reaction medium. Following an additional 15-min isobutyric acid:0.5 N NHsOH (10:6) (21). The [‘“Cl- incubation at 37.5”C to allow ‘?02 from the acidified uracil was located by viewing the chromatogram under reaction mixture to distill into KOH, the flasks were ultraviolet light, with reference to a sample of com- opened and the center wells, containing radioactive mercial uracil developed on the same chromatogram. waste, were appropriately discarded. The contents of The uv-absorbing band was cut out, the [?Z]uracil the reaction flask were homogenized using a Kinema- was eluted with water, and its specific activity was tica Polytron tissue homogenizer (Brinkmann Instru- determined by measuring the Apal of the eluant and ments, Westbury, New York) and the acid-insoluble assaying its content of radioisotope in a liquid scintil- material was removed by centrifugation. The acid- lation spectrometer. soluble fraction so obtained was incubated at 1OO’C Measurement of the “C0, generated from [car- for 1 h to convert pyrimidine nucleotides to the mono- boxy-‘4C]orotic acid. The decarboxylation of [car- phosphate form; after cooling, the acid-soluble fraction bo.ry-14C]orotic acid was measured by trapping “‘CO2 was adjusted to pH 7.2-7.4 by the addition of KOH liberated during the conversion of erotic acid to UMP. and the precipitate of KClO, was removed by centrif- Tissue slices weighing 500 mg were incubated at 37.5”C ugation. The 14C-labeled uridine nucleotides synthe- for 3 h in 5 ml of Krebs Improved Ringer II solution sized during the incubation period were isolated as made 30 mM in NaHC03 and supplemented with 5 [‘4C]UMP by cocrystallization with carrier UMP as mM [carboxy-‘4C]orotic acid (31 dpm/nmol); the re- follows: A 2-ml aliquot of a solution containing 375 mg action flask was sealed with a rubber cap fitted with a of UMP (disodium salt)/ml was added to the neutral- plastic center well containing 0.4 ml of 20% KOH and ized acid-soluble fraction and the total volume was a filter-paper wick. The reaction was terminated by brought to 10 ml with H20. Following thorough mix- the injection of 1.0 ml of 1.5 N HClO, into the reaction ing, an aliquot of 50 ~1 was removed and diluted to 106 mixture, and 14C02 was allowed to distill into the KOH ml in 0.01 N HCI, and the absorbance of the dilution during an additional incubation at 37.5”C for 1 h. The was measured at 260 nm. The acid-soluble fraction center well was then removed and placed in a scintil- containing carrier UMP was made 67% in ethanol by lation vial, its contents were diluted with 1.6 ml of the addition of 20 ml of ethanol and the precipitate of water and 6.5 ml of Aquasol, and the quantity of 14COZ UMP so formed was dissolved by heating the solution liberated by the decarboxylation of [carboxy-‘“C]or- to 70°C in a water bath. The [14C]UMP synthesized otic acid was measured in a Searle Isocap 300 liquid from [‘4C]NaHCOa was isolated by cocrystallization scintillation spectrometer. with carrier UMP as the ethanolic solution cooled Measurement of the activities of OPRTase and slowly to 4°C. The crystals were collected by suction ODCase in cell-free extracts of liver and spleen. The filtration, washed on the filter with ice-cold 67% combined activities of OPRTase and ODCase were ethanol, and dissolved in water. The absorbance at assayed by measuring the quantity of ?ZO, liberated 266 nm of a suitable dilution of the solution of [‘“Cl- by the decarboxylation of [carboxy-“‘Clorotic acid. UMP was measured to allow calculation of the recov- The reaction was carried out in a closed vessel sealed ery of carrier UMP, and the quantity of radioisotope with a rubber cap fitted with a plastic center well in a 2-ml aliquot of the solution of [‘?]UMP was containing 0.4 ml of 20% KOH and a filter-paper wick. measured in a Searle Isocap 300 liquid scintillation The complete reaction mixture contained, in a volume spectrometer. The remaining [“C]UMP was recrys- of 1.5 ml, the following components at the indicated REGULATION OF PYRIMIDINE BIOSYNTHESIS 197 concentrations: phosphate buffer, pH 7.4, 50 mM; vial; its contents were diluted with 1.6 ml of water MgCL, 3 mM; PRPP, 1 mM; [carboxy-‘4C]orotic acid followed by 6.5 ml of Aquasol, and the content of (142 dpm/nmol), 0.5 mM; and 0.2 ml of a 10% homog- radioisotope was assayed in a Searle Isocap 300 liquid enate of tissue prepared in isotonic saline. Following scintillation spectrometer. Each assay of PRPP was incubation at 37°C for 30 min, the reaction was ter- performed in parallel with identical samples to which minated by the injection of 0.2 ml of 25 mM NaHCO:, known quantities of PRPP (10 or 20 nmol) had been through the rubber cap, followed immediately by the added just prior to acidification; the measured loss of injection of 0.5 ml of 1.5 N HClO,; the bicarbonate was PRPP from these recovery standards was used to added to ensure complete flushing of WOs from the correct for losses of PRPP during the preparation of reaction mixture upon subsequent acidification. Dis- the tissue extracts. tillation of “‘CO2 into the center well was allowed to RESULTS proceed during an additional 30-min incubation at Effects of Pyrimidine and Purine Nucleo- 37°C. The center well was then removed and placed in a scintillation vial, and its contents were diluted sides on the Incorporation of c”C/- with 1.6 ml of Hz0 followed by 6.5 ml of Aquasol; the NaHCO3 into Orotic Acid content of radioisotope was measured in a Searle Evidence for regulation of the de novo Isocap 306 liquid scintillation spectrometer. biosynthesis of erotic acid by pyrimidine Determination of the concentration of PRPP in and purine nucleosides was obtained in ex- tissue slices. The content of PRPP in tissue slices was periments measuring the influence of added assayed by a modification of the method of May and nucleoside on the incorporation of [‘“Cl- Krooth (22), which measures the PRPP-dependent, enzyme-catalyzed decarboxylation of [car-boxy-‘%]or- NaHC03 into erotic acid by tissue slices.’ otic acid. Following a 3-h incubation in Krebs Im- In both the spleen and the liver, the incor- proved Ringer II solution (19), slices were removed poration of [‘4C]NaHCOx into erotic acid with forceps and immediately frozen by dropping them was inhibited by uridine and cytidine; the into a plastic test tube containing liquid nitrogen. A purine nucleoside, adenosine, was also an 2.5-ml aliquot of ice-cold 0.4 N HClO, was added and effective inhibitor of the incorporation of the tissue was homogenized with a Polytron tissue [‘4C]NaHC03 into erotic acid in the spleen homogenizer; the homogenizer was rinsed with 2 ml of (Table I). The liver, however, is unique ice-cold HZ0 and the rinsing was added to the acidified among the tissues we have examined in its homogenate. Acid-insoluble material was removed by centrifugation; the centrifuge was stopped rapidly and response to adenosine [(9, 18); this publi- the acid-soluble fraction was decanted into a test tube cation]; adenosine stimulated the incorpo- containing 1.0 ml of 1.0 N KOH and one drop of ration of [14C]NaHC03 into erotic acid six- phenolphthalein indicator solution. The solution was fold in the liver (Table I). We also found, in quickly adjusted to a faint-pink phenolphthalein end two separate experiments, that this stimu- point with KOH or HCl and decanted into a 25-ml lation by adenosine was reduced by an av- flask containing the other components of the assay. erage of 44% by the addition of 5 mM orni- The assay mixture, in a volume of 6.5 ml, contained thine. Since this response to adenosine and the following components; MgC12, 42 pmol; Tris-HCI, ornithine mimics the response to ammonia pH 8.0, 335 pmol; tissue extract as prepared above; and ornithine which we have previously OPRTase and ODCase (mixed enzymes, prepared from yeast), 2 U (1 U catalyzes the conversion of 1 reported to be unique to mammalian liver pmol of erotic acid to UMP per hour at 25°C); [car- (12), we suspect that adenosine is serving boxy-‘4C]orotic acid (920 dpm/nmol), 1 pmol. The as a source of ammonia, through adenosine flask was capped with a rubber seal fitted with a deaminase, and thereby stimulating the plastic center well containing 0.4 ml of 20% KOH and participation of the hepatic CPSase I in a filter-paper wick, and the reaction mixture was in- pyrimidine biosynthesis. Having estab- cubated for 2 h at 25°C. The elapsed time between lished that both purine and pyrimidine nu- acidification of the tissue slices and capping of the cleosides are effective inhibitors of an early reaction flask was 5 min. At the end of the incubation step in the de novo biosynthesis of erotic period, the reaction mixture was made 3 mM in bicar- acid [(8, 9, 18); Table I], we next sought bonate by the injection of a solution of NaHCO:r through the rubber cap, and the reaction was then evidence for regulation of the conversion of erotic acid to uridine nucleotides in tissue terminated by injection of 1.0 ml of 1.5 N HClOd through the rubber cap into the reaction mixture; slices. 14COswas allowed to distill into the center well during .’ Properties of the system for measuring the inror- an additional incubation for 1 h at 25°C. The center poration of [“C]NaHCO:, into ortic acid have been well was then removed and placed in a scintillation reported previously (12). 198 CRANDALL, LOVATT. AND TREMBLAY TABLE I periments as possible contaminants of EFFECTS OF URIDINE, CYTIDINE, AND ADENOSINE [14C]UMP isolated by cocrystallization, no ON THE INCORPORATION OF [‘%]NaHC03 INTO detectable radioisotope was isolated with OROTIC ACID IN RAT LIVER AND SPLEEN” the carrier UMP. The metabolite of [‘“Cl- Additions Incorporation of [‘%]NaHC03 into NaHC03 which cocrystallizes with carrier h-d erotic acid 1 (% of corresponding control) UMP was identified as [‘4C]UMP by de- scending paper chromatography on What- Spleen, Liver, man 3 MM paper in 95% ethanol:1 M am- 125.0 k 6.1 (24)* 27.0 f 2.4 (26)h monium acetate (75:30), isobutyric acid:0.5 None (control) 100 109 N NH40H (10:6), and isopropanol: concen- Uridine (10) 24.7 -c 1.1 (18) 29.4 f 2.6 (20) trated HCl:HzO (170:41:39) (21); in each of Cytidine (10) 46.5 f 2.4 (3) 61.2 +. 6.4 (7) these solvent systems, more than 96% of Adenosine (2.5) 63.3 rt_ 5.1 (4) 669.6 + 126.9 (5) the radioisotope cocrystallizing with carrier n The experimental method employed in measuring UMP was found to cochromatograph with the incorporation of [‘%]NaHCOa into erotic acid has the carrier. been described previously (12). Each value for the incorporation of [%]NaHC03 into erotic acid ob- Some Properties of the System Used for tained when a nucleoside was present in the incubation Measuring the Incorporation of [‘“C/- medium is presented as a percentage of its respective control -+ standard error, with the number of obser- NaHCO3 and [6-‘“C/Orotic Acid into vations included in each value listed in parentheses. Uridine Nucleotides b These control values are presented as averages f Mild homogenization of slices of liver or standard errors, in nanomoles of [‘%]NaHC03 incor- spleen before incubation with [‘“Cl- porated into erotic acid per gram of tissue during a 3- NaHC03 or [6-14C]orotic acid resulted in a h incubation; the number of observations for each loss of 88% or more of the amount of ra- value is listed in parentheses. dioisotope isolated by cocrystahization with carrier UMP. Thus, the observed incorpo- Characterization of the Method Employed ration of 14C-labeled precursors into uridine for the Isolation of [‘4C]Uridine Nucleo- nucleotides in tissue slices is a measure of tides as [‘“C] UMP pyrimidine biosynthesis by intact cells, and To ensure that hydrolysis of the acid- not an artifact of our procedure or incor- soluble fraction in 0.25 N HClO( at 1OO’C poration by a small number of ruptured for 1 h was sufficient for the quantitative cells. conversion of uridine nucleotides to UMP, The possibility that the observed incor- a recovery experiment was performed in poration of [‘4C]NaHC03 into uridine nu- which a sample of commercial [14C]UTP cleotides might reflect incorporation into was added to an incubation mixture just the ribose moiety rather than the pyrimi- prior to acidification. Following hydrolysis dine ring was ruled out by comparing the of the acid-soluble fraction at 100°C for 1 specific activity of the [14C]UMP isolated h, nucleotides were extracted by adsorption from the acid-soluble fraction with that of to and elution from charcoal (23) and frac- the [‘4C]uracil derived from the same sam- tionated by descending paper chromatog- ple of [14C]UMP by hydrolysis of the N- raphy in 1 M ammonium acetate:95% glycosidic bond. The specific activity of the ethanol (3:7) (21); more than 95% of the original sample of [14C]UMP was 259 radioisotope recovered from the chromat- dpm/pmol, and that of the [‘4C]uracil de- ogram was found to cochromatograph with rived from this sample of [14C]UMP by UMP. In two experiments in which a sam- hydrolysis of the N-glycosidic bond was ple of [14C]UMP was added to a typical found to be 277 dpm/pmol; this result in- incubation mixture just prior to acidifica- dicates that all of the radioisotope was lo- tion, 98.2 and 94.5% of the radioisotope cated in the pyrimidine ring, and that little were recovered upon subsequent cocrystal- or no incorporation of [‘4C]NaHC03 into lization with carrier UMP. In addition, the ribose moiety of the nucleotide had when [‘4C]orotic acid and [ 14C]orotidine occurred. On the basis of the results pre- monophosphate were tested in similar ex- sented above, our method was judged to be REGULATION OF PYRIMIDINE BIOSYNTHESIS 199 a simple and reliable means for measuring the incorporation of radiolabeled precur- sors into the pyrimidine moiety of uridine nucleotides. Effects of Uridine and Adenosine on the Incorporation of [‘4C]NaHC03 into Ur- idine Nucleotides The incubation of spleen slices with uri- dine or adenosine produced, in both cases, a significant inhibition of the incorporation of [‘4C]NaHC03 into uridine nucleotides (Fig. 1). In addition, uridine (10 mM) was also found to inhibit the incorporation of [‘4C]NaHC03 into uridine nucleotides by 52 f 7% (average f SE; N = 3) in similar experiments with liver slices.4 These results are consistent with those reported above FIG. 1. The effect of uridine and adenosine on the (Table I) showing inhibition by uridine and incorporation of [‘%]NaHC03 into uridine nucleotides in rat spleen. The experimental method employed in’ adenosine of the incorporation of precursor measuring the incorporation of [‘%]NaHCOa into ur- into erotic acid in spleen slices, and inhibi- idine nucleotides is described under Materials and tion by uridine in liver slices. Methods. Each value is presented as a percentage of In making such comparisons between the the control value f standard error, with a minimum results obtained from measurements of the of three observations represented by each point incorporation of precursor into erotic acid shown. The control value is 66.8 -t 3.7 (average f SE; and the incorporation of precursor into N = 20) nmol of [14C]NaHCOn incorporated into uri- UMP, attention is drawn to the differences dine nucleotides per gram of spleen during the 3-h in the two procedures. The incorporation of incubation period. precursor into erotic acid is measured in the presence of Gazauridine, a drug which ically labeled uridine nucleotides being syn- is quite effective in preventing the further thesized de novo, albeit at a lower rate. metabolism of erotic acid in tissue slices Since no such interference by uridine oc- and minces (9, 12). For want of a suitable curs in measurements of the incorporation drug, no such measure is taken to prevent of precursor into erotic acid, a comparison the further metabolism of uridine nucleo- of the results of both measurements would tides. Thus a comparison of the rate of suggest a difference in sensitivity to end incorporation of precursor into erotic acid product inhibition which may not exist. with the rate of incorporation of precursor Thus, while the absolute values reported into uridine nucleotides would suggest a herein can only be directly compared with wider disparity than actually exists, owing others obtained by the same experimental to differences in the rates at which the procedure, each of these procedures is ad- newly synthesized end products are being equately sensitive to detect regulation of depleted by their further metabolism. Sim- the de novo pathway in the intact cell and ilarly, a comparison of the sensitivity of to confirm the interpretation of the results each incorporation to end product inhibi- obtained by the other. tion requires additional consideration. The addition of uridine to effect end product Effects of Uridine and Adenosine on the inhibition also expands the pool size of uri- Incorporation of [6-‘4C]Orotic Acid into dine nucleotides, thus trapping the isotop- Uridine Nucleotides

4 The control value for the liver was 19.1 f 3.7 We tested the possibility that OPRTase (average f SE; N = 4) nmol of [‘%]NaHC03 incor- may have a regulatory role in the de novo porated into uridine nucleotides per gram of liver biosynthesis of pyrimidines in slices of rat during 3 h of incubation. liver and spleen by measuring the effects of 200 CHANDALL, LOVATT, AND TREMBLAY

uridine and adenosine on the incorporation were corroborated in another assay system of [6-‘4C]orotic acid into uridine nucleo- which did not depend on the isolation of tides. We found that the addition of 10 mM [14C]UMP, but which instead measured the uridine to the incubation medium was with- 14C02 liberated from [carbo3cy-‘4C]orotic out effect on the incorporation of [6-14C]- acid during its conversion to UMP. In two erotic acid into uridine nucleotides in either separate experiments, the addition of 10 tissue (Fig. 2); the results were essentially mM uridine to the incubation medium pro- the same over a loo-fold range in substrate duced no inhibition of the decarboxylation concentrations. The addition of 5 mM aden- of [carboxy-y-‘4C]orotic acid, while the addi- osine, however, inhibited the incorporation tion of 10 mM 6-azauridine inhibited the of [6-‘4C]orotic acid into uridine nucleotides decarboxylation of [carboxy-‘4C]orotic acid by 80% in the liver and 38% in the spleen. by 93%; these results are virtually identical In eight experiments employing slices of to those obtained by measuring the incor- liver and spleen, the addition of 10 mM 6- poration of [6-‘4C]orotic acid into uridine azauridine, a known inhibitor of the con- nucleotides and argue strongly that the con- version of erotic acid to UMP (20), reduqed version of erotic acid to UMP is not affected the incorporation of [6-14C]orotic acid into by uridine or its metabolites in the intact uridine nucleotides by an average of 88.5% cells of mammalian liver or spleen. (Fig. 2), thus demonstrating that our exper- In addition to the above studies employ- imental system is adequately sensitive to ing intact cells, we also tested directly for a detect inhibition of the conversion of erotic possible uridine-promoted depletion of the acid to UMP in the intact cell. These results levels of one or both of the last two enzymes in the de novo pathway by assaying the 1 combined activities of OPRTase and ODCase in cell-free extracts of slices of liver and spleen following incubation with this pyrimidine nucleoside. In an initial experi- ment using liver slices incubated without uridine, the combined activities of OPRTase and ODCase declined sharply, reaching 18 + 2% (average + SE; N = 5) of the activity of freshly prepared slices after 3 h of incubation; since this rapid loss of activity occurred in the absence of uridine, we judged liver slices to be an inappropriate system for testing for a uridine-promoted depletion of the levels of OPRTase and LT L u-- 50 1 CONCENTRATIONOF[6 ‘Y] OR”TlC 4Clb ,rnMl ODCase. In contrast to the liver, OPRTase FIG. 2. The effect of uridine, adenosine, and 6. and ODCase in spleen slices declined only azauridine on the incorporation of [6-‘%]orotic acid slightly after a 3-h incubation without uri- into uridine nucleotides in rat liver and spleen. The dine, to 85 f 6% (average f SE; N = 3) of experimental method employed in measuring the in- the value of freshly prepared slices, while corporation of [6-‘%]orotic acid into uridine nucleo- incubation with 10 mM uridine resulted in tides is described under Materials and Methods. Each a decline to 72 + 7% (average + SE; N = 3) value is presented as a percentage of its corresponding of the control level; these values are not control and represents the average of two to four significantly different, indicating that in- observations. The control values for the liver averaged cubation with uridine has no effect on the 44 + 11,73 f 13, and 127 f 16, and those for the spleen combined activity of OPRTase and averaged 13 f 4, 56 f 8, and 104 f 11, when the concentrations of [6-‘%]orotic acid were 0.05, 0.5, and ODCase in slices of spleen. 5.0 mM, respectively. These values are given in nano- We also tested the ability of spleen slices moles of [6-‘%]orotic acid incorporated into uridine to recover from inhibition by uridine in an nucleotides per gram of tissue.3 h f standard error; experiment which extended the incubation each value is an average of three to four assays. period from 3 to 6 h. We found that the REGULATION OF PYRIMIDINE BIOSYNTHESIS 201 rates of incorporation of [“‘C]NaHC03 into was linear with respect to added PRPP, at uridine nucleotides over a 3-h incubation least up to 40 nmol. No additional genera- period were the same whether the radiois- tion of 14C02 resulted from doubling either otope was added at zero time or at 3 h by the incubation time, the concentration of transfer of the slices to fresh medium; sim- [carboxy-‘4C]orotate, or the amount of the ilarly, the rates of incorporation measured yeast enzymes, OPRTase and ODCase, in in the presence of uridine also remained the reaction mixture. The concentration of constant at reduced levels over the entire PRPP in tissue slices after 3 h of incubation 6-h incubation period. In two experiments was found to be 7.2 PM in the liver and 16.5 designed to test for recovery from inhibi- PM in the spleen (Table II). The addition of tion, slices were transferred after 3 h of 10 mM uridine to the incubation medium incubation in medium containing uridine to produced either no change or a slight in- fresh medium lacking uridine, and the in- crease in the concentration of PRPP, both corporation of [‘4C]NaHC03 into uridine in liver and in spleen. The effect of adeno- nucleotides was measured during a second sine, however, was strikingly different from 3-h incubation period. The incorporation that of uridine; when tissue slices were in- measured after transfer to the uridine-free cubated for 3 h with 5 mM adenosine, the medium was 1.9 to 2.0 times the value ob- concentrations of PRPP decreased to 52 tained during the first incubation period in and 36% of the values obtained in the ab- the presence of uridine; since 2.4 times the sence of adenosine in the liver and spleen, inhibited value would have constituted full respectively. recovery, the observed reversal of inhibi- tion was almost complete. Effects of Uridine and Adenosine on the de Novo Biosynthesis of Pyrimidines in Effects of of Uridine and Adenosine on the the Ammonia-Stimulated Liver Concentration of PRPP in Tissue Slices Since we had previously determined that Adenosine was found to be a potent in- CPSase I of hepatic mitochondria is an hibitor both of the incorporation of [‘“Cl- important source of CP for pyrimidine bio- NaHCOR into erotic acid and of the incor- poration of [6-‘4C]orotic acid into uridine TABLE II nucleotides (Figs. 1 and 2). Since PRPP is EFFECTS OF UHDINE ANI) ADENOSINF.ON THE required in both of these reaction se- CONCENTKATION OF PRPP IN TISSIJE SIKES” quences, as an activator of CPSase II and Additions Concentration of PRPP bM) (R of corresponding control) as a substrate for OPRTase, we explored .- the possibility that the incubation of tissue Liver Spleen slices with adenosine resulted in a decrease in the concentration of PRPP. Since PRPP Expt 1 Expt 2 has been shown to be unstable in acid so- None (control) 100 100 100 lution (24), commercial PRPP (the purity Uridine (IO) 121.8 f 9.7 97.6 130.9 of which had been determined in separate Adenosine (5) 52.2 f 5.9 38.1 34.6 assays) was added to some of the tissue n The content of PRPP was determined by meas- preparations before acidification, and sev- uring the PRPP-dependent, enzyme-catalyzed decar- eral determinations of the recovery of the boxylation of [carbory-‘%]orotic acid, as described added PRPP were made in parallel with under Materials and Methods. The values of PRPP each assay of the concentration PRPP in concentrations in tissue slices incubated with uridine tissue slices. Each determination was then or adenosine are presented as percentages of the cor- corrected for the loss of PRPP as measured responding control. For the liver, the values are given for that individual experiment; the average as averages + SE for three determinations; the results of two experiments with spleen slices are presented recovery of PRPP (10 or 20 nmol) added to separately. The control values, given as micromolar tissue slices just prior to acidification was concentrations of PRPP in tissue slices incubated for 31.3 + 1.4% (average + SE; N = 22). Assays 3 h in the absence of nucleoside, were 7.2 + 1.9 pM (N of commercial PRPP revealed that the gen- = 3) for the liver and 18.8 and 16.2 pM for two experi- eration of 14C02 from [carboxy-‘4C]orotate ments with spleen slices. 202 CRANDALL, LOVATT, AND TREMBLAY synthesis in the liver (12), we explored the creasing the production of [‘“CICP by possibility that the synthesis of CP by CPSase I. CPSase I or the export of CP from the When liver slices were supplemented mitochondria was subject to regulation by with 5 mM NH4C1, a concentration suffi- end products of the de novo pyrimidine cient to stimulate fully the incorporation of pathway. In a test for feedback regulation [14C]NaHC03 into erotic acid, the inhibi- of CPSase I, the incorporation of [‘“Cl- tory effects of uridine and cytidine were NaHC03 into erotic acid in liver slices in- greatly diminished or lost (Table III). The cubated with the physiological concentra- pyrimidine nucleosides were thus found to tion (0.7 mM) of NH4CI was found to be be much less effective as inhibitors of the sensitive to inhibition by uridine and cyti- incorporation of [‘4C]NaHC03 into erotic dine, whether or not ornithine was added acid in liver slices exposed to toxic levels of (Table III). Since the addition of 0.7 mM ammonia than in liver slices incubated NH4Cl stimulated the incorporation of either with physiological concentrations of [‘4C]NaHCOz into erotic acid sixfold over ammonia or without ammonia. In contrast the value obtained when NH&l was omit- to the liver, the addition of 5 mM NH,Cl to ted, and since this ammonia-enhanced in- slices of the spleen, a tissue which does not corporation was completely prevented by possess CPSase I activity, had no effect on the addition of hyperphysiological (5 mM) the rate of incorporation of [‘4C]NaHC03 ornithine, we interpret these results to in- into erotic acid, nor did the presence of dicate that the ammonia-enhanced activity ammonia modify the inhibitory effects of was due to CPSase I, and that pyrimidine uridine and cytidine in this tissue (Table nucleosides or their metabolites are effec- III). tive inhibitors of the production of CP by Although we found that adenosine was CPSase I or of the export of CP from the without effect on the incorporation of mitochondria. The slight stimulation of the [‘4C]NaHC03 into erotic acid in liver slices incorporation of [‘4C]NaHC03 into erotic incubated with toxic concentrations of am- acid that was observed when liver slices monia, this purine nucleoside was a pow- incubated with 0.7 mM NH&I were supple- erful inhibitor of the incorporation of pre- mented with adenosine (Table III) can be cursors into uridine nucleotides under the attributed, as it was previously (Table I), to same conditions. In two separate experi- adenosine’s role as ammonia donor, ments employing liver slices supplemented through adenosine deaminase, thereby in- with 5 mM NH4Cl, the incorporations of

TABLE III EFFECTS OF UHIDINE AND ADENOSINE ON THE INCORPOHATION OF [14C]NaHCO:I INTO OROTIC ACID IN THF: AMMONIA-STIMIJLATEDLIVE:& Additions Incorporation of [‘Y!]NaHCOzj into erotic acid bM) (% of corresponding control) Liver slices Spleen slices

0.7 mM NH&I, 0.7 mM NRC1 + 0.5 mM 5 mM NH&I, 5 mM NRCl, 164.9 f 14.7 (14)h ornithine, 60.6 + 12.2 (8)h 481.4 f 50.1 (12)h 120.0 + 9.0 (3)h None 100 100 100 100 Uridine (IO) 57.2 -t 4.6 (11) 66.9 +- 9.8 (8) 88.7 +- 10.1 (5) 30.7 + 2.3 (3) Cytidine (10) 73.3 + 4.5 (7) 62.2 -+ 12.8 (8) 81.2 -c 9.8 (4) 41.0 f 4.8 (3) Adenosine (5) 145.0 f 10.1 (3) 89.5 + 5.1 (3) (1The experimental conditions are described under Materials and Methods. Each value for the incorporation of [‘4C]NaHCO:I into erotic acid obtained when the incubation medium was supplemented with a nucleoside is presented as a percentage of its respective control f standard error, with the number of observations included in each value listed in parentheses. h Control values are presented as averages f standard errors, in nanomoles of [“‘C]NaHCO:, incorporated into erotic acid per gram of tissue during a 3-h incubation; the number of observations for each value is listed in parentheses. REGULATION OF PYRIMIDINE BIOSYNTHESIS 203

[‘4C]NaHC03 and [6-‘4C]orotic acid into lian cells by a process which involves initial uridine nucleotides were inhibited by aver- cleavage of the sugar moiety by a mem- ages of 87 and 77%, respectively. These brane-bound nucleoside phosphorylase, observations are consistent with the inter- producing intracellular ribose-l-phosphate; pretation that adenosine prevents the con- subsequent uptake of the free base involves version of erotic acid to UMP by depleting a condensation with PRPP catalyzed by the cells of PRPP, but does not inhibit the adenine phosphoribosyltransferase (25). synthesis of erotic acid in liver slices which Since PRPP is required both as an activator have been provided with ammonia because, for CPSase II (2) and as a substrate for under these conditions, CPSase I displaces OPRTase, reduction of cellular levels of CPSase II in pyrimidine biosynthesis and PRPP to concentrations below those re- CPSase I is not influenced by PRPP. quired for saturation would result in de- creases in the rate of pyrimidine biosyn- DISCUSSION thesis. We found that the incubation of Evidence presented in this paper and in liver and spleen slices with 5 mM adenosine previous reports (8, 9, 18) demonstrates decreased PRPP concentrations by 48 and that uridine or its metabolites are effective 64%, respectively. Thus it is likely that the end product inhibitors of the de novo path- incorporation of [14C]NaHC03 into erotic way for pyrimidine biosynthesis in the in- acid and the incorporation of [‘4C]orotic tact cell. The observations reported herein, acid into uridine nucleotides are inhibited that uridine inhibits the incorporation of by adenosine through the depletion of [‘4C]NaHC0,y, but not [6-‘4C]orotic acid, PRPP; this inhibition may, however, be into uridine nucleotides, extend these ear- peculiar to the availability of an exogenous lier studies and strengthen the interpreta- supply of purines for salvage.” Since incu- tion that CPSase II is the primary site of bation with uridine did not result in a de- regulation by uridine. In addition, the re- crease in the intracellular concentrations of covery of spleen slices from inhibition by PRPP or inhibition of the conversion of uridine, as measured by the rate of incor- erotic acid to UMP, the possibility that poration of [‘4C]NaHC03 into uridine nu- uridine acts similarly to adenosine can be cleotides, was nearly complete upon trans- excluded. fer of the slices to uridine-free medium, a The failure of adenosine to inhibit the result consistent with the observation that incorporation of [‘4C]NaHCO:( into erotic levels of OPRTase and ODCase in the acid in liver slices incubated with NH4C1 spleen were not altered by incubation with (Table III) is consistent both with the in- this pyrimidine nucleoside. While our ex- terpretation that CPSase I provides much perimental method readily yielded evi- of the CP for pyrimidine biosynthesis dur- dence of end product regulation of the de ing ammonia stimulation (12) and with the novo biosynthesis of pyrimidines at an early interpretation that adenosine inhibits the reaction in the pathway, we were unable to biosynthesis of pyrimidines by reducing cel- detect any regulatory effect by uridine or lular levels of PRPP. If these interpreta- its metabolites governing the activities of tions are correct, one would expect adeno- the last enzymes in the pathway, OPRTase sine not to inhibit the incorporation of and ODCase. It is important to emphasize [‘4C]NaHC03 into erotic acid in liver slices that these results were obtained with pro- supplemented with ammonia, since CPSase cedures which were well suited to the dem- I, unlike CPSase II, does not require PRPP onstration inhibition of the combined ac- as an activator. The incorporation of tion of OPRTase and ODCase by adenosine and by 6-azauridine. Thus, we conclude ‘The suggestion that adenosine might interfere with pyrimidine biosynthesis by competition for PRPP that the regulatory mechanism demon- was offered earlier by Ishii and Green (16); Meuth and strated in rat hepatoma cells by Hoogen- Green subsequently demonstrated that the inclusion raad and Lee (13) does not operate in slices of adenosine in the growth medium resulted in a of rat liver or spleen. reduction of the PRPP levels in cultured mammalian Adenosine is thought to enter mamma- cells (personal communication). 204 CRANDALL, LOVATT, AND TREMBLAY [‘4C]NaHCO:1 and [6-‘4C]orotic acid into prevented by hyperphysiological ornithine, uridine nucleotides, however, does require the data support the interpretation that the PRPP as a substrate for OPRTase, and the uridine-sensitive incorporation in the pres- incorporation of both of these radioactive ence of ammonia is predominantly a reflec- precursors into uridine nucleotides in liver tion of regulation of the contribution of CP slices supplemented with 5 IIIM NH,Cl was by CPSase I. These results indicate that, strongly inhibited by adenosine; these ob- under normal physiological conditons, the servations strengthen the interpretation intramitochondrial production of CP, or the that the effect of adenosine on the de nouo export of CP from the mitochondria, or biosynthesis of pyrimidines is due to the both, are inhibited by uridine or its metab- reduction in PRPP concentration following olites. the administration of this purine nucleo- When the concentration of ammonia was side. increased to toxic levels, where the incor- The incorporation of [‘4C]NaHCO:3 into poration of [‘4C]NaHC03 into erotic acid in erotic acid in liver slices incubated with liver slices is increased 20-fold, the sensitiv- physiological concentrations of ammonia ity to pyrimidine nucleosides was lost (Ta- and ornithine was found to be sensitive to ble III). Our observations are consistent inhibition by pyrimidine nucleosides or with the interpretation that, when ammo- their metabolites (Table III); this observa- nia levels are high, uridine or its metabo- tion confirms the earlier report of Kerson lites no longer inhibit the production of CP and Appel (26), who demonstrated that the by CPSase I or the export of CP from the ammonia-dependent CPSase I of rat liver mitochondria, and the detoxification of am- was inhibited by pyrimidine nucleotides in monia via the synthesis of erotic acid a cell-free assay system. Since uridine was supersedes the regulation of pyrimidine bio- found to be a potent inhibitor of the incor- synthesis. Support for this interpretation is poration of [‘4C]NaHC0:3 into erotic acid in provided by Pausch et al. (27), who found liver slices incubated without NH&l (Table that the perfusion of liver with 15 mM I), a basal activity which may be attributed NH,Cl resulted in a 20-fold increase in the to CPSase II, it may be tempting to specu- incorporation of [‘4C]bicarbonate into uri- late that the uridine-sensitive incorporation dine nucleotides and that this ammonia- of [‘4C]NaHCO:3 into erotic acid represents stimulated incorporation could not be fur- the contribution of CPSase II, while the ther increased by the administration of ga- uridine-insensitive fraction represents that lactosamine. Although Pausch et al. sug- of CPSase I; the evidence, however, does gested that the effect of ammonia was to not support this interpretation. Of the sev- abolish the regulatory effect of uridine nu- eral tissues we have examined, only in cleotides on CPSase II, we found no evi- mammalian liver is the de nouo synthesis dence for this mechanism. The addition of of erotic acid stimulated by ammonia, and 5 mM NH4Cl to slices of spleen, a tissue this accelerated synthesis is progressively containing only CPSase II, did not alter the dampened by increasing concentrations of potent inhibition by uridine of the incor- ornithine (12); we attribute this ammonia- poration of [‘4C]NaHC03 into erotic acid in stimulated, ornithine-sensitive incorpora- this tissue (Table III). Thus, lack of feed- tion of [‘4C]NaHC0:3 into erotic acid, which back regulation of CPSase I during ammo- is unique to mammalian liver, to the partic- nia toxicity is a more likely explanation of ipation of CPSase I in the de nouo biosyn- their observation. In a more recent inves- thesis of pyrimidines. Since the uridine-sen- tigation employing whole animals, Pausch sitive fraction of the incorporation of et al. (28) found that repeated hourly injec- [‘4C]NaHCO:3 into erotic acid in the pres- tions of ammonium acetate resulted in large ence of physiological ammonia is 2.6 times increases in the concentrations of hepatic greater than the total uninhibited incorpo- uridine nucleotides, an observation which ration observed in the absence of ammonia is consistent with both an increased contri- (Tables I and III), and since the ammonia- bution by CPSase I to the orotate pathway stimulated incorporation can be completely and a lack of regulation of this contribution REGULATION OF PYRIMIDINE BIOSYNTHESIS 205 by uridine nucleotides at high concentra- 248,4782-4785. tions of ammonia. The additional observa- 11. PA~JSCF~, J., WILKF,NING, J., NOWACK, J., AND tion that hepatic ammonia concentrations DECKF,K, K. (1975) Eur. J. Biochem. 53, 349-356. had returned to the normal range 30-60 12. THEMBLAY, G. C., CIIANDALI,, D. E., KNOTT, C. min after the injection of ammonium ace- E., AND ALFANT, M. (1977) Arch. Biochem. Bio- tate does not alter our interpretation that phys. 178,264-277. the increased synthesis of uridine nucleo- 13. HOOGENRAAD, N. J., AND LEE, D. C. (1974) J. tides reflects mitochondrial production and Biol. Chem. 249, 2763-2768. export of CP in response to transient in- 14. SHOAF, W. T., AND JONES, M. E. (1973) Biochem- creases in ammonia concentrations. Thus, istry 12,4039-4051. since their study was based on responses to 15. WOOD, M. H., AND O’SULLIVAN, W. J. (1973) high doses of ammonia, the experimental Amer. J. Obstet. Gynecol. 116, 57-61. system employed does not permit either an 16. ISHII, K., AND GREEN, H. (1973) J. Cell Sci. 13, 429-439. estimate of the relative contributions of CP 17. GIIEEN, H., AND CHAN, T. (1973) Science 182, from CPSase I and CPSase II to the de 836-837. nouo pathway or a statement about feed- 18. GULEN, S., SMITH, P. C., AND TREMBLAY, G. C. back regulation of CPSase I under normal (1974) Biochem. Biophys. Res. 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