Purine metabolism in isolated rat hepatocytes CAMILLA M. SMITH,LIISAM. ROVAMO, MARTTIP. KEKOMAKI, A N D KARI0. RAIVIO'

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Children's Hospitcil, U n i ~ v e r s iof t ~ Helsinki, SF-00290 Helsinki 29, Finland Received March 28, 1977

Smith, C. M., Rovamo, L. M., Kekomaki, M. P. & Raivio, K . 0 . (1977) Purine metabolism in isolated rat hepatocytes. Can. J. Biochem. 55. 1134-1 139 The metabolism of adenine, hypoxanthine, guanine, and adenosine was studied in rat liver cell suspensions, prepared by collagenase perfusion. Oxygen supply was a critical variable in the preparation and subsequent incubation of the cells, a s judged on the basis of the ratio of radioactivity in ATP to that in ADP after incubation with [14C]adenine. This ratio is suggested a s an additional criterion of cell function. Adenine nucleotides synthesized from [I4C]adeninewere slowly catabolized to allantoin, with little incorporation of radioactivity into other purine compounds. [I4C]Adenineis thus suitable for prelabelling the adenine nucleotide pool. [14C]Guanine and [14C]hypoxanthinewere rapidly catabolized to allantoin, whereas nucleotide synthesis was low. [14C]Adenosinewas initially phosphorylated and deaminated at about equal rates, but with continued incubation catabolic products predominated. Isolated hepatocytes were found suitable for studies of purine metabolism, in which the liver has iniportant functions for the whole organism. Smith, C. M., Rovamo, L . M., Kekomaki, M. P. & Raivio, K . 0 . (1977) Purine metabolism in isolated rat hepatocytes. C a n . J. Biochem. 5 5 , 1134-1 139 Nous avons etudie le metabolisme de I'adenine, de I'hypoxanthine, de la guanine et de I'adenosine dans des suspensions cellulaires de foie de rat preparees par perfusion collagenasique. Si I'on se base sur le rapport entre la radioactivite dans I'ATP et celle dans I'ADP apres incubation avec la [14C]adenine, I'apport d'oxygene est une variable critique dans la preparation et l'incubation subsequente des cellules. Ce rapport est suggere comme critere additionnel de la fonction cellulaire. Les nucleotides de l'adenine synthetises a partir de la [14C]adenine sont catabolises lentement en allantoine avec peu d'incorporation de radioactivite dans les autres composes puriques. L a [I4C]adenine peut donc servir au premarquage du pool des nucleotides de I'adenine. L a [14C]guanine et la [14C]hypoxanthine sont catabolisees rapidement en allantoi'ne tandis que la synthese des nucleotides est faible. L a [14C]adenosineest d'abord phosphorylee et desaminee en quantites approximativement egales, mais si I'incubation se prolonge, les produits cataboliques predominent. Les hepatocytes isoles peuvent servir a l'etude du metabolisme des purines dans lequel le foie exerce des fonctions importantes pour l'organisme entier. [Traduit par le journal]

Introduction The importance of the liver in mammalian purine metabolism has long been recognized. Enzyme studies and isotope incorporation experiments, summarizedb~ Murray ('1, indicatea de synthesis and turnover of purine nucleotides. The liver appears to be the main Of the Purine for those tissues and Organs Of the in which de nova synthesis is inadequate. Transfer Of purines from the liver into other tissues by way of erythrocytes has been demonstrated (27 the form being Probably adenosine (4). The 3)7

ABBREVIATIONS:HEPES, N-2-hydroxyethylpiperazine-Nf-2-ethanesulfonic acid; TES, N-Tris-(hydroxymethy1)methyl-2-aminoethanesulfonic acid; Tricine, N Tris-(hydroxymethyl)methylglucine; APRT, adenine phosphoribosyltransferase; HPRT, hypoxanthine phosphoribosyltransferase; PRPP, 5-phosphoribosyl-1-pyrophosphate. 'Author to whom reprint requests should be addressed.

liver also seems to play a major role in purine catabolism and end-product formation, as suggested by the organ distribution of xanthine oxidase in man

,,,

id~solatedhepatocytes have been found useful i n the study of a number of problems of liver biochemistry (6). However, their properties with respect to purine metabolism have been studied only to a limited extent (7). In this paper we describe a method for preparing suspensions of rat hepatocytes with good viability and well-preserved energy metabolism. Using conditions that supported good energy metabolism, the metabolic pathways followed by adenine, hypoxanthine, guanine, and adenosine in these cell preparations were determined. Materials and Methods Rats Sprague-Dawley rats (200-250g) were used. They were fed an ordinary commercial chow ad libitum and used in a fed state.

SMITH ET A L .

TABLE1. Effect of incubation conditions on nucleotide synthesis from [I4C]adenine (100 ,wM) and on the [14C]ATP: [I4C]ADP ratio in rat hepatocytes

Oxygenation

Phosphate, mM

Glucose, mM

Time of incubation, min

'4C-labelled nucleotide synthesis, nmol/h per lo6 cells

1

No Yes

10 10

5.5 5.5

60 60

8.6 8.8

3

No Yes

10 10

0 0

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Cell preparation

[14C]ATP: [14C]ADP

1 .O 4.8

NOTE: Cells (2 r lo6 per 0.1 ml) were incubated with shaking at 37°C in capped tubes. Oxygenated = 1009, O2 blown into tubes at the start and at 30 min of incubation; not oxygenated = atmospheric 02.Results are averages of duplicate experiments. For details see Materials and Methods.

Reagents Collagenase type I1 (150 Ulmg), HEPES, T E S , and Tricine were obtained from Sigma Chemical Co. (St. Louis, MO). [8-14C]Adenine (specific activity 60 mCi/mrnol(l Ci = 37 GBq)), [8-14C]hypoxanthine(specific activity 60 mCi/mmol), [8-14C]guanine (specific activity 55 mCi/mmol), and [8-14C]adenosine (specific activity 49 mCi/mmol) were products of the Radiochemical Centre (Amersham, Bucks., U.K .). The isotopes were checked by two-dimensional thin-layer chromatography (8) and found to be suitable for use without purification. Other chemicals were analytical grade. Liver Cell Prepurution Rat liver cells were prepared using the method of Seglen (9) with slight modifications. Briefly, laparotomy was performed under thiopentone anaesthesia and 1500 I U of heparin injected into the inferior vena cava. An oxygenator was not used during the perfusion, but both perfusion media, the first without and the second with collagenase and Ca2+, were bubbled with 0, plus CO, (.95:5) prior to and while being pumped through the portal vein cannula. The initial batch of cells was purified by filtration through Nylon mesh, incubated with shaking at 37OC for 30 min, followed by three washes with buffer. The final cell preparation was suspended at a density of 1-2 x lo7 cells per millilitre in a buffer pH 7.6 containing Na+ 130 mM, K + 6.5 mM, Mg2+0.6 mM, Ca2+ 1.2 mM, C 1 75 m M , inorganic phosphate 10 mM, and HEPES, TES, and Tricine each at 3 0 m M (9). The cells were counted using a Biirker haemocytometer after mixing 100 p1 of cell suspension with 300 p1 of trypan blue (0.6% solution in 0.9% NaCI). Cell viability was defined a s the percentage of cells excluding trypan blue.

Isotope Zncorporution Studies For purine incorporation studies, 100-p1 aliquots of cell suspensions were incubated at 37OC in I0 x 75 mm test tubes with shaking at 80 to 90 oscillations per minute, and 14C-labelled precursors were added. At the end of the incubation period, 10 p l of cold 4.2 N perchloric acid was added and the tubes were cooled on ice. The cell extracts were neutralized with 10 p l of 4.42 N K O H and centrifuged. Purine nucleotides were separated o n polyethyleneimine cellulose thin-layer plates by stepwise development with formate, and purine nucleosides and

bases on cellulose plates by two-dimensional development (8). Allantoin was located on the two-dimensional chromatogram by autoradiography. It was not completely separated from xanthosine, but since the radioactivity in xanthosine never exceeded 5% of that in allantoin, the overlapping area was routinely included in the 'allantoin' spot. For radioactivity measurements, the spots corresponding to the various purine compounds were cut out of the chromatograms and counted using an LKB-Wallac liquid scintillation system. With each experiment a control, in which the perchloric acid was added before the radioactive compound, was done for each isotope. The counts in each purine compound (except the precursor) in the experimental tubes were then corrected for these blank values. Each experiment was done in duplicate, and the results are expressed a s cpm incorporated into each metabolite per lo6 cells. Enzyme Assuys HPRT (EC 2.4.2.8) and APRT (EC 2.4.2.7) were assayed a s described by Boyle et ul. (10). Adenosine deaminase (EC 3.5.4.4) activity was determined using a radiochemical assay, essentially according to Pull and McIlwain (1 1). Protein was determined with the method of Lowry et ul. (12). T o facilitate comparison of isotope incoi-poration rates with enzyme activities, the conversion factor of 1.8 mg protein per lo6 cells was used (9).

Results Incubation Conditions To assess the effect of experimental conditions on energy metabolism and cell viability, cell suspensions were prepared and incubated under a variety of conditions in the presence of 100 pM [14C]adenine. The incorporation of radioactivity into adenine nucleotides and the ratio of radioactivity in ATP to ADP were determined. As shown in Table 1 , oxygen is the most important variable affecting this ratio. Although total incorporation of adenine into nucleotides is not affected, [14C]ATP:[14C]ADPratios exceeding 4.0 were obtained only when the cells were well oxygenated. The reduction of phosphate concentration from 10

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CAN. J . BIOCHEM. VOL. 55. 1977

mM to 1 mM resulted in a somewhat lower incorporation of [14C]adenine into nucleotides and, since extra oxygen was not supplied in this experiment, a low ATP:ADP ratio. The omission of glucose did not affect adenine incorporation into nucleotides and with oxygenation the [14C]ATP:[14C]ADPratios were satisfactory after 60 min. However, when unlabelled cells were incubated for 4 h without glucose, the percentage of cells excluding trypan blue decreased to below 50, compared to about 85% in the presence of glucose. On the basis of these studies, the subsequent experiments were performed in the presence of 10 mM phosphate and 5.5 mM glucose. Every 10 min the tubes were gassed with oxygen. Cell viability was, on the average, 85% and the [14C]ATP:[14C]ADP ratios during a 60-min incubation ranged from 2.9 to 5.8. Electron micrographs of cells from our preparation showed intact mitochondria, other subcellular organelles, and microvilli on the cell surface. This also suggests that the cells are not damaged during the isolation process. To ascertain that the hepatocytes remain undamaged during a prolonged incubation, cell suspensions were incubated in the presence of 200 pM [14C]adeninefor up to 4 h. The ratio of radioactivity in ATP:ADP and the exclusion of trypan blue were determined at intervals, as shown in Table 2. There appeared to be no significant loss of viability over the period studied and energy metabolism remained good. The slightly lower trypan blue counts at 1 and 3 h do not seem to indicate permanent damage to the cells, since subsequent counts and ratios were normal. They could reflect artefacts in the process of staining and counting. A similar experiment was done incubating a larger volume of cell suspension (2 ml) under oxygen and removing 100-p1 aliquots at timed intervals. The [14C]ATP:[4C]ADPratios were low (about 1.0 after 30 min) and after 1 h the viability by trypan blue counts rapidly fell. It is apparent that the diffusion of oxygen into the medium becomes limiting for cell function, when the surface to volume ratio is too low. Metabolism of Purine Bases and Adenosine The metabolism of radioactive hypoxanthine, guanine, adenine, and adenosine was studied. The initial concentrations were 100 pM for the other compounds but 200 p M for guanine, which was anticipated to be rapidly catabolized by deamination. As shown in Fig. 1, hypoxanthine was rapidly metabolized, primarily to allantoin. Xanthine and uric acid were also significantly labelled (7.8 x lo4 and 1.5 x lo4 cpm/106 cells, respectively, after 15 rnin of incubation). After 30 rnin of incubation, the radioactivity in purine nucleotides was only 6% of the total amount of [14C]hypoxanthinemetabolized to

TABLE2. Effect of prolonged incubation on [14C]ATP: [14C]ADPratio and on the percentage of cells excluding trypan blue

Time, h

[14C]ATP:[14C]ADP

Trypan blue exclusion, %

NOTE:Isolated hepatocytes (2 x lo6 cells per 0.1 ml) were incubated at 37°C with shaking, the initial concentration of [14C]adenine was 200 pM. The tubes were oxygenated every 10 min. The ATP:ADP ratios are averages of duplicate determinations; the trypan blue counts were done in single samples.

J

60: (0

s P s \

40-

E 0 a 20

-

0

-0

15

30

45

A nucleotides G nucleotides 60

Time (min)

FIG. 1. Metabolism of [14C]hypoxanthine (100 p M ) in hepatocytes. Cells (2 x lo6 per tube) in standard suspension buffer were incubated with shaking at 37°C. At timed intervals reactions were stopped with perchloric acid and the samples processed for the separation and counting of purine compounds as described in Materials and Methods. The points represent the averages of duplicate determinations, with less than 10%difference in each case.

acid-soluble products. The ratio of radioactivity in adenine nucleotides to that in guanine nucleotides was 5.8. Labelled IMP did not accumulate (0.8 x lo4 cpm/106 cells after 15 min). Guanine was also rapidly catabolized to allantoin (Fig. 2). After 30 min there was appreciable radioactivity in xanthine and uric acid (20 x lo4 and 4 x lo4 cpm/106 cells, respectively) in addition to allantoin (158 x lo4 cpm/106 cells). Only 3% of the guanine was metabolized to nucleotides after 30 min. The [14C]GTP:[14C]GDPratio at this time was 2.2. Adenine is metabolized to the adenine nucleotides via APRT. Because adenine itself is not broken down by mammalian cells, allantoin is derived solely through catabolism of nucleotides. Thus the production of allantoin is slower than with the other purine precursors, and [14C]adenineis suitable for prelabelling the adenine nucleotide pool (Fig. 3). After 30

SMITH ET A L

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Allantoin

A nucleotides

Adenosine Time (min)

FIG. 4. Metabolism of [14C]adenosine (100 p M ) in hepatocytes. Experimental conditions as in Fig. 1, for details see Materials and Methods.

FIG.2. Metabolism of [14C]guanine (200 p M ) in hepatocytes. Experimental conditions as in Fig. 1, for details see Materials and Methods.

Adenine

A nucleot~des

uric acid (1.8 x lo4, 3.4 x lo4,and 1.9 x lo4 cpm/106 cells, respectively). After 15 min, the radioactivity in the adenine nucleotides started to decrease. The rates of metabolism of the purine compounds studied were calculated on the basis of disappearance of precursor over the first 15 min of incubation; they are expressed on a protein basis. The fastest rate of disappearance was that of guanine, 0.75 nmol/min per milligram of protein, but it should be pointed out that the initial concentration of guanine was 200 pM. For adenine, hypoxanthine, and adenosine the initial concentration was 100 pM, and the disappearance rates were 0.10, 0.25, and 0.28 nmol/min per milligram of protein, respectively. Enzyme Assuys In sonicated extracts of hepatocytes the activities of adenine and hypoxanthine phosphoribosyltransferases were 9.2 and 1.9 nmol/min per milligram of protein, respectively, and that of adenosine deaminase was 3.5 nmol/min per milligram of protein.

Allantoin

Discussion Time (min)

FIG.3. Metabolism of [14C]adenine (100 p M ) in hepatocytes. Experimental conditions as in Fig. 1, for details see Materials and Methods.

min the radioactivity in the nucleotide fraction was 70% of the adenine metabolized to acid-soluble products, and the fraction remained about the same for the rest of the 60-min incubation. As shown in Fig. 4, there was relatively rapid initial nucleotide synthesis from [14C]adenosine. At 15 min, the radioactivity in adenine nucleotides was 49% and that in catabolic products 48% of the total adenosine metabolized. In addition to allantoin, there was significant labelling of hypoxanthine (Fig. 4) and, to a smaller extent, of inosine, xanthine, and

For studies of liver biochemistry, the currently available in vitro methods include tissue slice preparations, perfusion, and the use of isolated parenchymal cells. Oxygenation problems and tissue damage in the process of slicing are the main disadvantages in liver slice experiments. In the isolated perfused liver, nucleotide concentrations are better preserved, and the metabolic capabilities of such a preparation are also otherwise well maintained as compared with the intact organ (13). However, only one set of experimental conditions can usually be applied to a single liver, and repeated tissue sampling in a time-course experiment is problematic. For these reasons, we wanted to work out suitable conditions for obtaining isolated cell preparations that could be used for studies of purine metabolism.

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CAN. J . BIOCHEM. VOL. 55. 1977

Other workers have established the viability and metabolic performance of similarly obtained hepatocyte preparations in terms of trypan blue exclusion, oxygen consumption, glucose production rate, and a number of other criteria (6). Our data corroborate these conclusions about cell viability and energy metabolism on the basis of trypan blue exclusion, electron microscopic appearance, and the ability of the cells to synthesize nucleotides and maintain normal [14C]ATP:[14C]ADPratios with radioactive adenine as the precursor. The increased nucleotide synthesis at higher phosphate concentrations is in agreement with other studies using other isolated cell systems (14), and presumably reflects increased availability of PRPP. In cells from our preparations, hypoxanthine, and guanine were predominantly and rapidly catabolized to allantoin, and only slightly used for nucleotide synthesis. Adenine was primarily converted into nucleotides followed by slow catabolism. The physiological significance of adenine metabolism in mammalian systems is unclear, since adenine has not been detected in normal serum. However, adenine is a suitable precursor for labelling the adenine nucleotide pool of hepatocytes. There was no significant labelling of adenosine, the purine moiety postulated to be produced by the liver (4, 15). This suggests that, under the conditions used, hepatocytes catabolized adenine nucleotides via the adenylate deaminase pathway, rather than dephosphorylation. The circumstances, under which adenosine is generated, remain to be studied. Since essentially all of the [14C]adenine metab~ l l z e amust have undergone the APRT reaction, the rate of disappearance of the precursor gives an estimate of the enzyme activity in the intact cell. Comparison of this rate with the maximal APRT activity, measured in cell extracts under optimal conditions, shows that the enzyme functions in vivo at about 1% of the maximal rate, even though the initial concentration of adenine was severalfold higher than the K , of the enzyme, 0.9-2.6 pM (16). Similar comparisons of observed metabolic rates with enzyme activities are less meaningful in the case of hypoxanthine and adenosine, since each may undergo more than one initial reaction. Even so the metabolic rate for total hypoxanthine utilization (catabolism and synthesis) is only about 10% of the HPRT activity measured in extracts. Adenosine utilization is about 10% of adenosine deaminase activity. Possible causes for the relative slowness of these reactions in cells include slow transport into the cell or lack of cosubstrates. PRPP is likely limiting for APRT and HGPRT (14, 17). The metabolic fate of adenosine has been postulated to depend on its concentration and to be predictable by the relative K , values and activities of adenosine kinase and adenosine deaminase (18).

Several studies have indicated that the K , for adenosine of adenosine kinase is an order of magnitude lower than that of adenosine deaminase, or 1-3 pM vs. 25-60 pM. Hence, nucleotide synthesis would be expected to predominate at low adenosine concentrations and deamination at higher concentrations. This prediction seems to be fulfilled in the case of human erythrocyte ghosts (19) and erythrocytes (20), Ehrlich ascites tumor cells (21), human fibroblasts (22), and phytohemagglutinin-stimulated human lymphocytes (23). Our results show that even at relatively high adenosine concentrations, 100pM, about equal amounts are phosphorylated and deaminated. Whether this is due to differences in the kinetics or distribution of the two enzymes is at present unknown. In any case, adenosine salvage for nucleotide synthesis seems more efficient in liver cells than in a number of other cell types studied. Acknowledgments

We thank Dr. Juhani Rapola for preparing the electron micrographs, Miss Ritva Metsola for skilled technical assistance, and Dr. J. F. Henderson for comments during the preparation of the manuscript. The study was supported by the Sigrid Juselius Foundation and the Foundation for Pediatric Research. 1 . Murray, A. W. (1971) A n n ~ i .Rev. Biochem. 4 0 , 81 1-826 2. Henderson, J . F. & LePage, G. A. (1959) J. Biol. Chem. 234,3219-3223 3. Pritchard, J . B., Chavez-Peon, F. & Berlin, R. D. (1970)Am.J. Physiol. 219, 1263-1267 4 . Lerner, M. H. & Lowy, B. A. (1974)J. Biol. Chem. 249,959-966 5. Watts, R. W. E., Watts, J. E. M. & Seegmiller, J. E. (1965)J. Lab. Clin. Med. 66,688-697 6 . Tager, J . M., Soling, H. D. & Williamson, J. R. (1976) U s e of Isolated Liver Cells and Kidney Tubules in Metabolic Studies, North-HollandIAmerican Else-

vier, AmsterdamINew York 7 . Lund, P., Cornell, N. W. & Krebs, H. A . (1975) Biochem. J. 152,593-599 8 . Crabtree, G. W. & Henderson, J. F. (1971) Cancer Res. 31,985-991 9 . Seglen, P. 0. (1973)Exp. Cell Res. 8 2 , 391-398 10. Boyle, J . A., Raivio, K. O., Astrin, K. H., Schulman, J. D., Graf, M. L., Seegmiller, J. E. & Jacobsen, C. B. (1970)Science 169,688-689 11. Pull, I . & Mcllwain, H. (1974)Biochem. J. 144, 37-41 12. Lowry, 0. H., Rosebrough, N. J . , Farr, A. L. & Randall, R. J . (1951)J. Biol. Chem. 193,265-275 13. Krebs, H. A., Cornell, N. W., Lund, P. & Hems, R. (1974) in Alfred Benzon Symposium V I , pp. 718-743, Munksgaard , Copenhagen 14. Henderson, J . F., Bagnara, A. S., Crabtree, G. W., Lomax, C. A., Shantz, G. D. & Snyder, F. F. (1975) Adv. Enzyme Regul. 13, 37-64

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SMITH ET A L .

15. Pritchard. J . B., O'Connor, N.. Oliver, J. M. & Berlin, R. D. (1975)A m . J. Physiol. 229,967-972 16. Murray. A. W., Elliott, D. C . & Atkinson, M. R. (1970) Progr. Nllc.leic Acid R e s . Mol. Biol. 10,87- 1 19 17. Henderson, J. F . , Brox, L . W., Fraser, J . H . , Lomax. C. A., McCoy, E. E., Snyder, F. F. & Zombor, G. (1975) Pharmacological Basis of Cancer Chemorherapy, pp. 663-680, The Williams and Wilkins Co., Baltimore, MD 18. Meyskens, F . L . & Williams, H . E . (1971) Biochim. Biophys. Acru 240, 170- 179

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19. Schrader, J . , Berne, R. M. & Rubio, R. (1972)A m . J. Pl~ysiol.223, 159- 166 20. Parks. R . E., Jr. & Brown, P. R. (1973) Biochemisrry 12.3294-3302 21. Snyder, F . F. & Henderson, J . F . (1973) J. Biol. Chem. 248,5899-5904 22. Benke, P. J . & Dittmar, D. (1976) Pcdiclrr. R r s . 10, 642-646 23. Snyder, F. F., Mendelsohn, J . & Seegmiller, J. E . (1976) J. Clin. Inl-esr. 58,654-666

Purine metabolism in isolated rat hepatocytes.

Purine metabolism in isolated rat hepatocytes CAMILLA M. SMITH,LIISAM. ROVAMO, MARTTIP. KEKOMAKI, A N D KARI0. RAIVIO' Can. J. Biochem. Downloaded fr...
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