THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1985 by The American Society of Biological Chemists, Inc

Vol. 260, No. 10, Issue of May 25, pp. 6107-6114 1985 Printed in d.S.A.

5-Amino-4-imidazolecarborramide Riboside (Z-riboside) ~ e t a ~ o l i s ~ in Eukaryotic Cells*

(Received for publication, May 7, 1984)

Richard L. Sabina, David Patterson$, and Edward W. Holmes From the Howard Hughes ~ e d i c a ~ Institute ~ ~ r a t ~ r ~ e s and D e ~ r t m e n ~ of M e d ~ i ~ and E ~ c ~ ~ i s t ~ , Duke ~ n i v e r s i t ~ Medica! Center, D ~ r ~ a m , North CaroLina 27710 and the $Eleanor Roo~evelt Institute for Cancer Research and the D e ~ r t m e n ~ of Medicine and Biochemistry, Siophysics and Genetics, University of Colorado MedicaL Center, Denver, Colorado 80220

Metabolites of 5-amino-4-imidazolecarboxamide ri- boside (Z-riboside) have potential roles in the regula- tion of cellular metabolism and as pharmacological agents in several pathological situations. Before study- ing Z-riboside metabolism it was necessary to develop methods for identifying and quantitating 5(4)-amino- 4(5)-imidazolecarboxamide metabolites. These studies utilized Chinese hamster ovary fibroblast auxotrophic mutants to identify and isolate compounds relevant to Z-riboside metabolism by a combination of high per- formance liquid chromato~aphic procedures. In order to study Z-riboside metabolism wild-type and mutant cells were cultured in Z-riboside. This ribosyl precur- sor to a purine de novo intermediate does not undergo any detectable phosphorolysis but rather is phospho- rylated by adenosine kinase in an unregulated manner. This results in the intracellular accumulation of 5- amino-~- imid~l~arboxamide ribotide (ZMP), the levels of which control flow from Z-riboside to the following metabolites: 1) XMP and other purine nudeo- tides, 2) 5-amino-4-imidazole-N-succinocarboxamide ribotide (sZMP), and 3) 5-amino-4-imidazolecarbox- amide riboside 5”triphosphate (ZTP). At low ZMP concentrations, the predominant metabolic fate is IMP. Initially, IMP enters the adenylate and guanylate pools, but subsequently is hydrolyzed to inosine and this phosphorolyzed to hypoxanthine. At intermediate ZMP concentrations there is net retrograde flux through the bifunctional enzyme adenylosuccinate AMP lyase resulting in sZMP synthesis and antegrade flux leads to the accumulation of adenylosuccinate. At high ZMP concentrations, ZTP accumulates. In addi- tion to these effects on purine metabolism, pyrimidine nucleotide pools are depleted when Z M P accumulates. These results are discussed in relation to the regulation of purine nucleotide synthesis and the use of Z-riboside as a pharmacological intervention in pathophysiologi- cal situations.

The metabolism of 2-base (5(4)-amino-4(5)-imidazolecar-

* This work was supported in part by National Institutes of Health Grant AM12413, National Institute of Arthritis Grant AG00029, Grant-in-Aid 1983-1984-A-46 from the North Carolina American Heart Association, and an American Cancer Society Institutional Research Grant to Duke ~omprehensive Cancer Center. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “adver- tisement’’ in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

boxamide)’ compounds has attracted increasing attention re- cently. ZTP (5-amino-4-imidazolecarboxamide riboside 5’- triphosphate) accumulates in folate-deficient bacteria and it has been postulated that this novel riboside triphosphate might play a regulatory role in the pleiotrophic response in prokaryotes (1). In eukaryotes, ZTP accumulation can be induced in cells cultured in high concentrations of Z-riboside (5-amino-4-imidazolecarboxamide riboside), and under these conditions growth of mammalian fibroblasts is inhibited (2). This effect in eukaryotes has been attributed to inhibition of pyrimidine nucleotide synthesis (2), in part as a result of 5’- ~hosphoribosyl-1-pyrophosphate (PRPP) depletion. The re- cent demonstration that ZTP is synthesized from ZMP (5- amino-4-imidazolecarboxamide ribotide) and PRPP in a re- action catalyzed by 5‘-phosphoribosyl-l-pyrophosphate syn- thetase (EC 2.7.6.1) (3) may provide a link between these observations on the effects of Z-base metabolites on growth of eukaryotes and prokaryotes.

In addition to the potential role which Z-base compounds play in the control of cell growth, this group of compounds may have therapeutic applicability in restoring purine nucleo- tide pools in post-ischemic myocardium (4) and in cerebral tissue of Lesch-Nyhan (hypoxanthine-guanine phosphoribo- syltransferase (EC 2.4.2.8) deficient) children (5) . Some stud- ies have shown that Z-riboside is effective in increasing the rate of purine nucleotide synthesis in myocardium (6), restor- ing purine nucleotide pools in post-ischemic myocardium (4), and improving myocardial contractility (7), while other stud- ies using different protocols for Z-riboside administration have not observed an improvement in post-ischemic myocar- dial function (8, 9). One potential explanation for these dis- parate findings is that large doses of Z-ribose lead to ZMP accumulation in amounts sufficient to inhibit adenylosuccin- ate AMP lyase (EC 4.3.2.2) (6, 10). In skeletal muscle inhi- bition of adenylosuccinate AMP lyase by ZMP leads to muscle dysfunction (11).

Before the potential effects of Z-base compounds on cell growth can be defined and before this group of compounds can be used therapeutically, a more detailed knowledge about the metabolite fates and effects of Z-riboside is needed. Pre- vious reports have demonstrated that Z-riboside is taken up and metabolized by human erythrocytes (12), mammalian

The abbreviations used are: Z-base, 5(4)-amino-4(5)-imidazole- carboxamide; Z-riboside, 5-amino-4-imidazolecarboxamide riboside; ZMP, 5-amino-4-imidazolecarboxamide ribotide; sZ-base, 5(4)- amino-4(5)-imidazole-N-succinocarboxamide; sZ-riboside, 5-amino- ~- imi~azole-N-succ in~~boxami~e riboside; sZMP, 5-amino-4-im- idazole-~-succinocar~xamide ribotide; CHO, Chinese hamster ovary; ZTP, 5-amino-4 - imi~zo lecar~xa~de riboside 5“triphos- phate; PRPP, 5’-phospboribosyl-l-pyrophosphate; ZDP, 5-amino-4- imidazolecarboxamide riboside 5“diphosphate.

6107

6108 Z-riboside Metabolism in Eukaryotic Cells

fibroblasts (2), and mammalian cardiac (6) and skeletal mus- cle (6), but limited commercial av~lability of Z-base com- pounds and metabolites has made it difficult to isolate and identify Z-riboside metabolites in cell extracts. Consequently, progress has been slow in defining the metabolism of this Z- base compound. The present study was undertaken to develop methods for isolating and identifying Z-base compounds and metabolites and to employ these methods to dissect the effect of different doses and times of exposure to Z-riboside on purine and pyrimidine metabolism in eukaryotic cells. Chinese hamster ovary cells were selected for study because of the availability of a wide range of auxotrophic mutants that facilitated identification of metabolites and delineation of pathways involved in Z-riboside metabolism.

MATERIALS AND METHODS~

RESULTS

Three sets of experimental conditions were used to study Z-riboside metabolism and to define the secondary effects this ribosyl derivative produces on purine and pyrimidine metab- olism. In the first set of experiments, Z-riboside was added to the culture medium at a fixed concentration (700 p ~ ) and samples were obtained at hourly intervals to monitor the more prolonged effects of this ribosyl derivative. After com- pleting these studies it became apparent that many interesting changes occurred within the first hour of exposure to Z- riboside and a second set of experiments was conducted at the same concentration of 2-riboside to delineate the changes which take place within the first minutes of exposure to Z- riboside. These experiments revealed that many of the changes in purine metabolism were evident by 45 min of exposure to Z-riboside, and the third set of experiments utilized the single time point of 45 min to assess the effects of variable concentrations of Z-riboside in the culture me- dium.

Effects of Prolonged (10 h) Exposure to 2-riboside During the first 10 h of exposure of CHO-K1 cells to 700

p~ Z-riboside a number of Z-riboside metabolites accumulate in the cell and medium, and intracellular pools of purine and pyrimidine nucleotides change in response to Z-riboside me- tabolism. The flow diagram depicted in Fig. 5 summarizes the changes which result from Z-riboside metabolism and pro- vides a general overview of the purine pathway indicating the relationship of the various metabolites to each other. The actual data noting the changes in metabolite content of the cell and medium are listed in Tables I and 11. For simplicity of presentation, the changes in intracellular and medium content of purine metabolites are listed in Table I, while the data on pyrimidine metabolism are listed in Table 11. The AK- mutant was used as a control for these experiments with CHO-K1 cells, and extracts of AK- were anaiyzed at each of the time points for all the metabolites listed in Tables I and XI. Since intracellular and medium content of purine and pyrimidine metabolites did not change detectably in AK- cells following 10 h of exposure to 700 p~ Z-riboside, the data

* Portions of this paper (including “Materials and Methods” and Figs. 1-4) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Doc- ument No. 4-1347, cite the authors, and include a check or money order for $2.50 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

2-RIBOSIDE \ Y

Hx*Ino

FIG. 5. Metabolic fates of Z-riboside and effects on purine metabolism in Chinese hamster ovary fibroblasts. Abbrevia- tions are the same as described in the legends to Figs. 1-4. Symbols for the various auxotrophic cell lines are listed next to the reactions which are deficient in the respective cell lines: Ade-A = ribosylamine 5-phosphate:pyrophosphate phosphoribosyitmnsferase-deficient cell line, Ade-F = l0-formyltetrahydrofolate:5’-phosphoribosyf-5-amino- 4-imidazolecarboxamide (ZMP) formyltransferase-deficient cell line, Ade-I = adeny1osuccinate:AMP lyase-deficient cell line.

obtained from this cell line are not included in Tables I and I1 but the results are incorporated in the text.

Changes in ZMP Pools (Table I)-ZMP, the immediate product of Z-riboside phospho~lation, cannot be detected ( i e . C0.05 nmol/mg protein) in cell extracts under routine culture conditions. However, following exposure to 700 pM z- riboside this intermediate in the de mu0 pathway of purine synthesis progressively accumulates in CHO-K1 cells. In con- trast, no ZMP was detected in the AK- mutant ( i e . c0.05 nmol/mg protein) at any time during the course of this experiment.

Changes in IMP Pools (Table I)-IMP, the first complete purine nucleotide in the de mu0 pathway, cannot be detected (i.e. <0.1 nmol/mg protein) in either CHO-K1 or AK- cells under routine culture conditions. The IMP pool in CHO-K1 cells is expanded 3-fold or greater within the first hour of exposure to 700 p~ Z-riboside and remains elevated through- out the course of the experiment. In contrast, the IMP pool in AK- cells remains below the limits of detection.

Changes in A ~ n y l o s ~ ~ i n a t e Pools {Table I)-Adenylosuc- cinate, an intermediate in purine nucleotide interconversion between IMP and AMP, cannot be detected (i.e. <0.02 nmol/ mg protein) in either CHO-K1 or AK- cell extracts under routine culture conditions. Exposure to 700 p~ Z-riboside results in adenylosuccinate accumulation in CHO-K1 cell extracts within the first hour, and the concentration of this intermediate increases progressively throughout the experi- ment. No adenylosuccinate was detected in AK- cell extracts ( i e . <OB2 nmol/mg protein) at any time during exposure to 700 p~ Z-riboside.

C ~ n g e s in Purine N ~ ~ o t i d e Pools (Table I)-The time course of change in intracellular adenine and guanine ribo- nucleoside triphosphate pools in CHO-K1 cells following 10 h of exposure to 700 p~ Z-riboside is depicted in Table I. Within the first hour there is a 77% and 42% increase in the intracellular concentrations of ATP and GTP, respectively. This is followed by a leveling off of the increase in the ATP pool (1-5 h) and then a gradual decline towards basal values (5-10 h). The GTP pool follows this same pattern although the decline from 5 to 10 h is more pronounced, with this nucleotide pool falling to 6 0 % of basal values. Although not shown, the intracellular levels of the adenine and guanine ribonucleoside mono- and diphosphates parallel the changes

2-riboside Metabolism in Eukaryotic Cells 6109

TABLE I Prolonged (10 h) exposure of CHO-Kl cells to 700 p~ Z-riboside: changes in intracellular and medium content of

purine nucleotides, nucleosides, bases, and intermediates CHO-K1 cells in the log-phase of growth were cultured in nondialyzed 10% fetal calf serum for the 24 h

preceding the study. The cells were washed with phosphate-buffered saline and refed with 5 ml of F-12 medium without hypoxanthine plus 10% dialyzed serum supplemented with 700 p~ Z-riboside. Cultures and media were harvested immediately and every hour for 10 h thereafter. Extracts were prepared and analyzed.as described under "Materials and Methods." All metabolite values are expressed as nmol.mg cell protein". suc-AMP, adenylosuccin- ate.

Intracellular Time

Medium

ZMP IMP SUC-AMP ATP GTP ZTP sZMP Inosine Hypoxanthine h nmol. mg protein" nmol. mg protein" 0 c0.05 co. 1 c0.02 28.4 5.2 co.1 co.1 <2.0 <5.0 1 1 .o 0.29 0.17 50.3 7.4 co.1 co.1 15.3 66.7 2 3.2 0.24 0.19 49.2 6.4 co.1 0.33 37.5 133 3 2.8 0.27 0.17 43.8 5.7 0.3 0.41 67.1 243 4 5.3 - 0.22 47.0 5.7 0.6 0.42 76.1 304 5 7.0 - 0.26 51.5 6.1 0.9 0.60 120 6

463 10 - 0.30 44.1 5.0 1.4 0.90 137 567

7 11 - 0.29 41.2 4.4 1.3 1.1 116 8

466 21 - 0.38 43.5 4.4 3.1 1.6 NDb ND

9 22 - 0.39 37.9 3.6 3.7 1.9 129 613 10 24 - 0.47 32.9 2.8 4.1 2.3 150 754

IMP was not quantitated in CHO-K1 cells after 3 h due to interference from accumulating sZ-riboside and sZ-

ND, not determined. base.

TABLE I1 Prolonged (10 h) exposure of CHO-Kl cells to 700 p~ Z-riboside:

changes in intracellular and medium content of pyrimidine nucleotides and orotate

Culture conditions were the same as those described in the legend to Table I.

Intracellular Time

Medium

UTP CTP Orotate Orotate h 0 1 2 3 4 5 6 7 8 9

10

nmol. mg protein" 6.8 3.1 cO.1 6.5 2.5 cO.1 3.8 1.7 0.49 2.0 0.8 1.7 1.5 0.6 3.5 1.3 0.6 5.8 1.4 0.6 5.3 1.2 0.5 5.9 2.2 0.9 5.4 1.9 0.7 4.5 2.3 0.8 2.8

nml . mg protein" 4 . 0 4 . 0

14.6 29.0 57.6 116 139 N.D. 206 226

1.80

in their respective triphosphate pools in CHO-K1 cells. In contrast, the AK- mutant maintains relatively constant ATP (27.0 & 0.8, S.E.) and GTP (5.0 f 0.2, S.E.) pools throughout the course of the experiment.

Changes in ZTP Pook (Table I)-ZTP, the triphosphory- lated derivative of Z-riboside, cannot be detected in CHO-K1 or AK- cell extracts (i.e. cO.1 nmol/mg protein) under routine culture conditions. However, following a 2-h lag period during which ZMP progressively accumulates, ZTP reaches a detect- able concentration and increases progressively thereafter in CHO-K1 cells exposed to 700 ~ L M Z-riboside. (The methods used to identify ZTP in cell extracts are detailed in the Miniprint Section of this paper.) No ZTP was detected in the AK- mutant at any time during the course of this experiment. Although we were able to increase Z-ribotide levels sufficiently in the Ade-F mutant treated with Z-riboside to be able to detect and identify ZDP, we did not detect ( q O . 1 nmol/mg protein) ZDP in CHO-K1 or any mutant other than Ade-F treated with Z-riboside even under experimental conditions where Z-ribotide pools were expanded to levels that were 70%

of the purine nucleotide pool in the cell and that accounted for one-fourth of the total phosphate bound to ribotides.

Changes in sZ-ribotide Pook (Table I)-sZMP, the imme- diate precursor to ZMP in the purine de novo pathway, is present in too low a concentration to be detected (i.e. cO.10 nmol/mg protein) in cell extracts under routine culture con- ditions. However, sZMP is detected in CHO-K1 cell extracts as early as 2 h after exposure to Z-riboside, and the concen- tration of this metabolite increases progressively thereafter. sZ-base and sZ-riboside were detected after 4 h of exposure to 700 p~ Z-riboside, but we were not able to quantitate these metabolites because insufficient amounts were produced to isolate and prepare reference standards. (The methods used to isolate and identify sZMP, sZ-riboside, and sZ-base are detailed in the Miniprint Section of this paper.) sZMP and its catabolites were not detected in the AK- mutant incubated with Z-riboside.

sZMP and its catabolites could increase in CHO-K1 cells treated with Z-riboside either as a result of reversal of the sZMP lyase activity of adenylosuccinate AMP lyase or by the accumulation of sZMP produced via the first seven reactions of the de novo pathway, as the result of lyase inhibition by ZMP or one of its metabolites. The latter possibility was excluded by incubating the Ade-A mutant (deficient in ribo- sylamine 5-phosphate:pyrophosphate phosphoribosyltrans- ferase (EC 2.4.2.14) activity) with 700 p~ Z-riboside for 24 h under the condltions described in Table I. sZMP and its catabolites accumulated to the same extent in Ade-A as in CHO-K1 leading us to conclude that these metabolites of 2- riboside are produced by reversal of the lyase reaction in the cell (data not shown).

Changes in Purine Excretion into the Culture Medium (Ta- ble I)-Inosine and hypoxanthine concentrations in the me- dium progressively increase during the 10 h of exposure of CHO-K1 cells to 700 p~ Z-riboside. In contrast, there was no detectable accumulation of inosine or hypoxanthine in the medium of the AK- cell line (e7 nmol/mg protein) throughout the time course of the experiment. Z-base was not detected in the medium of either cell line at any time during the course of these studies ( i e . <1 p ~ ) indicating that Z-riboside is a

6110 2-riboside Metabolism in Eukaryotic Cells

poor substrate for the nucleoside phosphorylase activities in these cells.

Comparison of the changes in intracellular purine nucleo- tide pools to the amount of inosine and hypoxanthine excreted in the medium by CHO-K1 cells indicates that the latter exceeds the former by severalfold. Even during the first hour when intracellular purine nucleotide pools are expanding, the increase in the ATP and GTP pools is only 30% of the inosine and hypoxanthine excreted into the medium (i.e. 24 versus 82 nmol/mg protein). Between the second and tenth hours, the purine nucleotide pools remain constant or decrease while purine accumulation in the medium increases progressively.

Changes in Pyrimidine Nucleotide Pools (Table II)-Both CTP and UTP pools begin to fall within the first hour of incubation with 700 p~ Z-riboside, and both pyrimidine ri- bonucleoside triphosphate pools have fallen to 20% of basal levels in 4 h. Concurrently there is a transient intracellular accumulation of orotate in CHO-K1 cells and a progressive accumulation of orotate in the medium (Table 11). In addition, there is a concomitant depletion of uridine nucleotide sugars (5.2-0.9 nmol/mg protein in 4 h). There is no depletion of pyrimidine ribonucleoside triphosphate pools, orotate accu- mulation, or uridine nucleotide sugar depletion in the AK- cell line during exposure to 700 p~ Z-riboside. (The protocol used in this study leads to a transient increase in UTP and CTP content of AK- cells treated with Z-riboside. This re- sponse is not an effect of Z-riboside or its metabolites, but rather a consequence of the change from a culture medium containing purine to one that is purine-free since similar increases are observed in CHO-K1 and AK- cells following transfer to purine-free medium in the absence of Z-riboside.)

Effects of Exposure to Z-riboside for 2 h The data presented in Table I11 demonstrate that the

expansion of the ATP, GTP, and ZMP pools begins within 5

TABLE I11 Short-term effects of 700 phf Z-riboside (2-rib) on purine metabolism

in CHO-KI cells CHO-K1 cells were cultured and extracts prepared as described in

the legend of Table I, except one set of cultures received no Z-riboside and the other received 700 PM Z-riboside. Results are expressed in nmol. me of cell protein-'. suc-AMP. adenvlosuccinate.

Changes in intracellular pools

ATP GTP ZMP IMP SUC-AMP

rnin 0 5

15 30 45 60

120

nmul. mg cell protein" 29.5 28.7 5.1 4.9 <0.05 <0.05 C0.05 C0.05 0.12 0.10 33.0 29.7 6.1 5.7 0.34 <0.05 0.34 C0.05 0.13 0.10 37.9 28.8 7.3 5.1 0.94 C0.05 0.21 c0.05 0.15 0.15 41.4 29.2 7.5 4.9 2.7 <0.05 0.17 C0.05 0.24 0.11 43.2 29.3 7.2 4.7 6.0 <0.05 0.22 C0.05 0.45 0.08 47.9 28.2 7.5 4.4 6.5 <0.05 0.16 C0.05 0.39 0.11 47.4 27.8 6.3 4.8 9.8 c0.05 0.29 <0.05 0.44 0.08

Changes in medium ~

Inosine Time

min nmol. mg cell protein"

Hypoxanthine

2-rib Saline 2-rib Saline

0 5.0 4.2 0.3 0.5 5 1.4 4.0 2.3 0.5

15 4.2 3.7 10 0.7 30 9.2 4.7 20 0.6 45 13 4.8 28 0.5 60 24 4.2 49 0.5

120 47 4.3 112 0.5

TABLE IV Effect of varying concentrations of Z-riboside on purine metabolism

CHO-K1 cells were cultured and extracts prepared as described in the legend of Table I, except cells and media were harvested after 45 min of culture in Z-riboside at the concentration indicated. Results are expressed in nmol. mg cell protein". suc-AMP, adenslosuccinate.

Changes in intracellular uools

2-riboside concentration ATP GTP ZMP IMP SUC-AMP

P M nmol. mg cell protein" 0 25.9 4.9 cO.10 <0.05 C0.05

50 26.8 5.1 <0.10 C0.05 C0.05 150 32.3 6.0 0.16 0.09 <0.05 450 38.5 6.9 2.6 0.24 0.26 700 37.3 6.5 8.7 0.23 1.0

Changes in medium

2-riboside concentration Inosine Hypoxanthine

P M

0 50

150 450 700

nmol. mg cell protein" 2.9 1.2 3.1 3.0 5.2 12

16 34 18 35

~~~

min of exposure to 700 p~ Z-riboside. The IMP pool is maximally expanded by the first time point sampled ( 5 min) and remains elevated throughout. Excretion of purine catab- olites is not detected until 15 min into the experiment. Ac- cumulation of adenylosuccinate is delayed until 30 min after exposure to Z-riboside, a point at which the ZMP level has increased markedly. CHO-K1 cells supplemented with me- dium not containing Z-riboside maintain constant ATP (28.8 f 0.2, S.E.) and GTP (4.9 f 0.2, S.E.) pools; they do not accumulate ZMP, IMP, or adenylosuccinate, and they do not excrete inosine or hypoxanthine into the medium.

Effects of Variable Concentrations of Z-riboside on Purine Metabolism

The data in Table IV demonstrate the changes in purine nucleotide pool sizes and catabolite excretion when Z-riboside concentration in the medium is varied from 50 to 700 p ~ . Fifty micromolar Z-riboside has no effect on pool sizes or purine excretion, at least not during the 45-min period in which the experiment was conducted. Z-riboside at 150 p~ does increase purine nucleotide synthesis as evidenced by the increase in cellular content of IMP, ATP, GTP and excretion of inosine and hypoxanthine into the medium. At 450 p~ Z- riboside, purine synthesis is maximally stimulated since in- creasing the concentration to 700 p~ has no incremental effect on IMP, ATP, or GTP pools and there is no further increase in inosine or hypoxanthine excretion. While 450 p~ 2-riboside stimulates maximal production of ATP and GTP, ZMP content is 3-fold greater in cells treated with 700 p~ compared to 450 p~ Z-riboside, suggesting that phosphoryla- tion of this riboside is not tightly controlled in the cell. Adenylosuccinate accumulation reaches detectable levels only after the ZMP concentration in the cell is markedly increased, i.e. at 450 and 700 FM Z-riboside.

DISCUSSION

The Z-base nucleus is formed as an intermediate in the de novo pathway of purine nucleotide synthesis, but little is known about the metabolism of Z-base compounds under normal conditions because of the very small amount of these intermediates found in the cell. Recent interest in Z-base

2-riboside ~ e t a b o l ~ ~ in Eukaryotic Cells 6111

compounds as metabolites that potentially control cell growth (1, 2) and which may be useful as therapeutic agents in restoring purine nucleotide pools in pathological situations (4,s) prompted us to develop methods for identifying Z-base metabolites and characterizing the effects of Z-riboside on purine and pyrimidine metabolism.

Results of this study demonstrate that Z-riboside does not undergo phosphorolysis in CHO cells, and the only known metabolic fate of this riboside is phosphorylation to ZMP. Although Schnebli et al. (29) found Z-riboside to be a poor substrate for pigeon liver adenosine kinase, the results of this study document that adenosine kinase is the predominant activity responsible for phosphorylation of Z-riboside in CHO cells. The AK- mutant, which was demonstrated to have functional uridine-cytidine and deoxycytidine kinase activi- ties, produced no ZMP or other Z-base metabolites, and no purine products were excreted in the medium when AK- was cultured in Z-riboside.

The dose-response and time course data (Tables I, 111, and IV) taken together demonstrate that ZMP has a number of metabolic fates in CHO cells which are summarized in Fig. 5. At low intracellular concentrations of ZMP and for a brief period of time after administration of Z-riboside, essentially all of the ZMP proceeds in antegrade fashion along the de novo pathway to IMP and subsequently to ATP and GTP. Within a few minutes after the ATP and GTP pools begin to expand, inosine and hypoxanthine appear in the medium. Once the ATP and GTP pools become expanded to approxi- mately 150% of basal levels, there is no further increase in purine nucleoside triphosphate pools in the cell. However, ZMP continues to be metabolized to IMP at a fairly constant rate for a prolonged period of time, as evidenced by the monotonic accumulation of inosine and h~oxanth ine in the medium for up to 10 h. These results are most easily inter- preted in light of current concepts regarding the control of purine nucleotide interconversion and catabolism (30). The initial expansion of the IMP pool leads to an increase in flux through the adenylosuccinate synthetase and IMP dehydro- genase reactions, since the Km for both these enzymes is greater than the estimated concentration of IMP in the basal state (31,32). Once the adenylate and guanylate pools become expanded the branch-point enzymes leading from IMP to AMP and GMP are subject to feedback inhibition (30). Con- comitantly, the expansion of the ATP pool leads to activation of cytosol 5’-nucleotidase (33), and most of the newly formed IMP undergoes hydrolysis to inosine with subsequent phos- phorolysis to hypoxanthine with excretion of the nucleoside and base into the medium.

Apparently ZMP transformylase becomes saturated with ZMP at relatively low concentrations of this ribotide or the availability of formyl donors becomes limiting. The ultimate effect regardless of the explanation is that transformylase activity becomes the rate-limiting step in purine nucleotide synthesis from Z-riboside and ZMP accumulates.

As the cell’s ability to metabolize ZMP in an antegrade direction in the purine pathway becomes limiting and with phosphorylation of Z-riboside proceeding, ZMP accumulates to progressively higher levels. In conjunction with the further i n c ~ m e n t in ZMP, adenylosuccinate begins to accumulate and thereafter this purine nucleotide increases in parallel with increases in ZMP. The accumulation of adenylosuccinate is probably the result of inhibition of adenylosuccinate AMP lyase by ZMP. ZMP has been shown to be a potent inhibitor ( K ; of 4-27 PM) of the adenylosuccinate AMP lyase activity of this enzyme from various mammalian sources (6, 10). This is not a surprising observation, since ZMP is a product of the

sZMP lyase activity of this bifunctional enzyme. Temporally, the next change noted in purine metabolism

as a result of ZMP accumulation is also attributable to this bifunctional enzyme. sZMP and its catabolites, sZ-riboside and sZ-base, accumulate as a result of ZMP metabolism in a retrograde fashion in the purine pathway. Reversal of the sZMP lyase activity of adenylosuccinate AMP lyase was es- tablished by using a CHO mutant defective in the first step of the de novo pathway of purine synthesis. The combined effects of inhibition of the adenylosuccinate lyase activity and reversal of the sZMP lyase activity of this bifunctional enzyme by high concentrations of ZMP may act in concert to decrease AMP synthesis, and this in turn may contribute to the reduc- tion in ATP pools observed as a late (5 h) response to Z- riboside admin~st~t ion.

The next change observed in purine metabolism as a con- sequence of the continued increase in ZMP concentration is the appearance of ZTP. Unlike the other metabolites of ZMP that are present in low but undetectable concentrations as normal intermediates in purine metabolism, it is not clear that ZTP is produced in the cell except in the unusual circum- stances where ZMP concentrations reach very high levels, i.e. following Z-riboside a~ in i s t r a t ion (6, ll), reduction in ZMP trans for my la^ activity secondary to inhibition of dihydrofo- late reductase (1) or genetic deficiency of this enzyme (34). It is not surprising that ZTP is not produced until the ZMP concentration in the cell becomes markedly elevated, since the only mechanism known for producing this ribosyl tri- phosphate is through pyrophosphorylation of ZMP in a re- action catalyzed by reversal of the 5-phosphoribosyl-1-pyro- phosphate synthetase reaction (3). The K,,, for ZMP is 3.2 mM (3), and this may explain the requirement for high ZMP concentrations in the cell before ZTP is produced.

The metabolic consequences of ZTP production have yet to be defined in eukaryotic cells, but in prokaryotes it has been proposed that this unique ribosyl triphosphate is an alarmone which plays a role in the plieotrophic response to folate depletion (1). If ZTP is shown to have an effect on the metabolism of eukaryotic cells, it may be that the unique mechanism by which this novel ribosyl triphosphate is pro- duced is important. Exposure of another Chinese hamster fibroblast line to Z-riboside has been shown to result in depletion of PRPP stores, as well as inhibition of PRPP synthesis (2). Depletion of PRPP almost certainly plays a role in the reduction of CTP and UTP pools, as well as orotate accumulation, observed in this and a previous study (2) in which Chinese hamster fibroblasts were cultured in high concentrations of Z-riboside for a prolonged period of time. PRPP is a central metabolite in purine and pyrimidine nu- cleotide synthesis, and some authors have suggested the in- tracellular conc~ntration of PRPP may play a role in coordi- nating the synthesis of these two classes of nucleotides (2, 35). It is also important to note that the state of pyrimidine starvation produced by Z-riboside appears to be limited to cultured cells since pyrimidine nucleotide and uridine sugar depletion is not observed in vivo in situations where Z-riboside is administered in large doses (6, 11). This disparity between changes produced in cultured cells and intact animals parallels the results observed previously with adenosine (for a review see Ref. 35).

Several hours following incubation with Z-riboside in high concentrations, expansion of the ATP pool ceases and intra- cellular concentrations of this nucleoside triphosphate fall toward basal levels over the next 5 h. One explanation for this effect is the documented inhibition of adenylosuccinate AMP lyase by ZMP. GTP pools which expand to approxi-

6112 2-riboside ~ e ~ a b o ~ ~ ~ in Eukuryotic Cells

mately the same extent as ATP within the first hour of exposure to Z-riboside begin to fall before ATP levels and ultimately decline to or below basal levels of this nucleoside triphosphate. These changes in guanine nucleotide pools are not explained at present. One hypothesis that could account for these results is that ZMP or ZTP inhibits one or more of the reactions leading from IMP to GTP as a result of the ability of Z-nucleotides to assume a configuration which re- sembles that of a guanine nucleotide (3).

While there are a number of questions which remain to be answered about the effects of Z-base compounds on cellular metabolism in general and purine and pyrimi~ne metabolism in particufar, the studies cited in this report provide definite guidelines for using Z-riboside to replete purine nucleotide pools in pathological situations. Clearly the administration of too high a concentration of Z-riboside over too short a period of time can lead to a striking elevation of ZMP concentration in the cell with a resultant inhibition of adenine, and possibly, guanine nucleotide synthesis. It would appear that the optimal dose and rate of administration of ‘2-riboside for increasing ATP and GTP synthesis is that amount which leads to an increase in IMP pools without concomitant increases in ad- enylosuccinate and sZMP. When the latter two metabolites increase it is probable that the rate of ZMP formation from Z-riboside has eclipsed ZMP t r~sformy~ase activity, the lat- ter being the rate-limiting step in converting ZMP into purine nucleotides. Further increases in ZMP concentration are likely to have counterproductive effects on adenine and guan- ine nucleotide synthesis.

Acknowledgment-We would like to acknowledge the technical assistance of Bill Laas, Diane Vannais, Jean C. Meade, and Margaret Evans in performing these studies and the secretarial support of Carolyn Mills in preparing the manuscript.

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2-riboside Metabolism in

MATERIALS AM METHODS

Cell Culture ?he parentar ce l l l i n e o f Chinese hamster ovary f l b m b l a s t s (CHO-Kl) and the purine

duxotroohs derlred frm It 1Ade-A. Me-1. and M e - F l have been described oreviourlr (13-16).

;&30 minutes through a C e n t r l f l o tF25 cone;llrlcon Corp.. Lexington. PA) anto a Uaterr Nucleosides and bases were separated by i n j e c t l o g 50ul o f u l t r a f i l t r a t e d medium (1000xg

NHqH2PO4 i n 2% methanol Cvlv l ) and Buffer 0 (5W IIH4H2W4 i n 5X r t h a n o l Crlvl) a t a fln Y Bondapak reverse phase c o l u n (3Ocm x 3.9nn) using a gradlent of Buffer C ( W I

r a t e of 1 d h i n . The c o l m n was i n i t i a l l y developed over 15 mlnuter using a nonllnear

Ulcers ClR-u Bondapat reverse-phase i o l u n u s I n g ' 1 0 W potassium phosphate. pH 6.0 (Buffer E ) 25 minutes a t 100% Buffer D. Z-base 2-rlboside and ZMP were separated lsocrat lcal ly on a

a t a fln r a t e of 1.0 allmin. 51-base, r2-riboside. sur-adenine. ruc-adenosine, and opotate I n tlrruc cul ture d i u " were separated using the gradient syrtea dcscrlbed above for nucleotlder. Since t he l a t te r compounds are a l l negatively charged a t the pH o f the buffem

ch rwa tognph ic f ron t . used. they a r e retained on the c o l u m w h i l e t h e u j o r l t y of mdim c q m e n t s e l u t e In the

gradlent of 0% Buffer 0 t o 100% Buffer 0 ( X p l r p Buffer 0 - 100 (t iE(mln)40)3) fol lowed by

Eukuryotic Cells 6113

a t

r ibot idcr .

1 2 A 2 8 336