Pleiotypic responses of regenerating liver

PLEIOTYPIC RESPONSES OF REGENERATING LIVER YOSHIAKI MIURA and NORIKO FUKUI Department of Biochemistry,Chiba UniversitySchool of Medicine,Chiba 280, Japan

INTRODUCTION When a quiescent culture of mouse fibroblasts is arrested in the Go phase by deficiency of serum, the cells can be stimulated to grow by adding serum and 17 hr later over 90% of the cells will be in the Gz phase and show DNA synthesis (I). The transition of cells from the Go to the GI phase of the cell cycle is accompanied by pleiotypic and mitogenic programs of the cell: that is, increased uptake of nutrients, and synthesis of RNA and DNA. Similarly, about 24 hr after partial hepatectomy of adult rats DNA synthesis is observed in approximately 30% of the hepatocytes of the remaining lobes of the liver (2). This means that at this time about 30% of the hepatocytes have entered the G~ phase from the Go phase of the cell cycle. As in the case of cultured fibroblasts, there are many reports on the activation of RNA ( 3 - 6 ) and protein synthesis ( 7 - 9 ) during the prereplicative period of liver regeneration. However, these phenomena are not always very sensitive parameters for recognition of hepatic regeneration. To examine the involvement of hormones in liver regeneration, attempts were made in our laboratory to see whether hepatic regeneration could be observed in vitro using isolated, perfused liver (I0). However, perfusion could not be continued for more than 12 hr, so no DNA synthesis was observed in this system. Therefore, the hepatic lobes remaining after partial hepatectomy were examined to see if any sensitive and specific parameters of hepatic regeneration could be detected before the beginning of DNA replication. For this purpose, we studied the in vitro inductions of several enzymes which are induced during the prereplicative period in vivo and also determined the intracellular levels of cyclic nucleotides in the same period. From the results obtained, we concluded that: 1) extrahepatic hormones are not esse'ntial for hepatic regeneration; 2)locally produced autacoids such as prostaglandins or kinins, together with serum stimulate increase in the intrahepatic level of cyclic GMP as the second messenger; 3)Cyclic GMP, in turn, induces pleiotypic responses; and 4)finally, DNA synthesis is observed 24 hr after partial hepatectomy as a late program for mitosis. 393

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Animals Usually male Donryu strain rats weighing about 100 g were used. They were obtained from Nihon Rats Co., Saitama, Japan and kept in the dark from 9.00 pm to 9.00 am and fed ad libitum. Sometimes male Sprague-Dawley strain rats were used, as indicated in the Figures. Partial hepatectomy was carried out under ether anaesthesia between 10.O0am and 12.00am. For peffusion experiments, larger rats, weighing about 250-350 g, were used and their livers were also removed between 10.00 am and 12.00 am. Perfusion Technique After brief interruption of the circulation, the liver was perfused for 10 rain to remove blood with a mixture of 80 ml of Eagle's medium or calf serum and 20ml of 02-saturated fluorocarbon suspension (perfluorotributylamine, (C4Fg)aN, and pluronic acid 2.5% w/v, Midori Juji Co., Osaka). Then it was perfused with the same medium containing glucose (200 mg), penicillin G (50,000 U), and streptomycin sulfate (100 rag). The medium was oxygenated throughout the perfusion period with a mixture of 95% oxygen and 5% carbon dioxide. After equilibration for 30 min, the flow of peffusate through the liver was maintained at approximately 30ml/min. Adequate bile production (0.6 ml/hr) and rate of flow of the perfusate were used as criteria of successful perfusion. The pressure of the peffusate entering the liver via the portal vein was maintained at 14 cm of H20. Chemicals DL- [ 1-14C] Ornithine (20 mCi/mmole), [2.14 C] deoxythymidine (50/zCi/mmole) and 3 H-deoxythymidine (5 Ci/mmole) were purchased from the Radiochemical Centre, Amersham, England. [8.14 C] Inosinic acid (33/~Ci//~xnole) and a radioimmunoassay kit for cyclic AMP and cyclic GMP were obtained from Schwarz-Mann, Orangeburg, N.Y. Prostaglandins E1 and F2a (PGE1 and PGF2a) were gifts from Upjohn Co., Kalamazoo, Mich. Crystalline indomethacin was from Merck, Sharpe & Dohrne, Rahway, NJ. Enzyme Assays Ornithine decarboxylase (EC 4.1.1.17) activity was measured in the cytosol fraction by radioassay, as described in our previous paper (10). Tyrosine aminotransferase (EC2.6.1.5) activity was determined in the supematant fraction by a modification (11) of the method of Diamondstone (12). The activity of inosinic acid dehydrogenase (EC 1.2.1.14) was determined by the radiochemical assay developed by Sarcoccia and Miech (13). Thymidine kinase (EC 2.7.1.75) was assayed using the supernatant fraction of liver

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homogenate obtained by centrifugation at 7,000 X g for 30 rain. The method used was described in detail in a previous paper (14). For determination of the inhibitory effect of indomethacin on DNA synthesis, a method using liver slices (2) was adopted. Namely, indomethacin (0, 10 or 50 mg/kg) was given 1 hr before 70% hepatectomy and incorporation of a H-thymidine into the acidinsoluble fraction of liver slices was measured 24 hr after the operation. Estimation of Cyclic Nucleotides Soon after removal of hepatic lobes, samples were crushed with a cold metal punch and immersed in liquid nitrogen. Then cold 5% trichloroacetic acid was added to the frozen tissue and it was extracted with ether. Cyclic nucleotides were estimated using a kit for radioimmunoassay.

R E S U L T S AND D I S C U S S I O N In vivo Induction of Enzymes Ornithine decarboxylase, which catalyzes the first step of polyamine synthesis, is known to be induced by various hormones or by rapid cellular growth, such as that during liver regeneration (15). As shown in Figure 1, the enzymic activity increased rapidly for 10 hr from soon after 70% hepatectomy. Then the activity decreased for 2 hr, but remained fairly high until 32 hr after the operation. Tyrosine aminotransferase activity was reported to increase several fold after partial hepatectomy (16). To confirm this, we determined the enzymic activity in the cytosol fraction. The enzymic activity increased steadily for 6 hr after partial hepatectomy as also shown in Figure 1. However, the activity started to decrease after 8 hr. Enudeation of both adrenals did not cause any appreciable change in the induction of ornithine decarboxylase or tyrosine aminotransferase in regenerating liver (10). Inosinic acid dehydrogenase represents the unique first reaction in the de novo synthesis of guanylic acid from inosinic acid. The activity of this enzyme is known to increase in the early prereplicative period of regeneration (17). As shown in Figure 1, the increase began soon after surgical intervention and was greater 8-12 hr after the operation until it reached a ceiling. Thymidine kinase is known to be induced about 18-20 hr after partial hepatectomy (14). As also shown in Figure 1, the increase in thymidine phosphorylation began about 18hr after partial hepatectomy. As this is just before the beginning of DNA synthesis, the increase in thymidine kinase activity may be a suitable parameter to use in measuring the end of the GI phase or beginning of the S phase.

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In vitro Induction o f E n z y m e s Among the above 4 enzymes induced by partial hepatectomy, ornithine decarboxylase and tyrosine aminotransferase are suitable for use in studies on isolated, perfused liver because they are induced soon after removal of hepatic lobes. As shown in Figure 2 when synthetic medium was used, no enhancement of either enzyme activity was observed. However, using medium containing calf or horse serum instead of Eagle's MEM, the inductions of ornithine decarboxylase and tyrosine aminotransferase were observed in isolated, perfused liver after 70%

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hapatectomy. As shown in Figure 3, continuous enzymic inductions were observed for 6 hr. Thus, some factor in serum seems to be necessary for the enzymic inductions after partial hepatectomy, although this serum factor is probably not very important in liver regeneration. As seen from Figure 4, the extent o f hepatectomy plays a more important role in the inductions of both enzymes; increase in the amount o f liver removed resulted in increased inductions o f the enzyme activities. The first trigger substance for the inductions o f both enzymes must be produced in the remaining part o f the liver in the presence o f serum, and a characteristic o f this substance is that removal of a larger proportion o f the liver results in more marked inductions o f the enzymes.

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FIG. 3. In vitro inductions of omithine decarboxylase and tyrosine aminotxansferase in isolated, pe~fused liver after 70% partial hepatectomy. Values are means + SEM of values in 3 to 5 separate experiments. Intracellular Cyclic Nucleotide Levels in Regenerating Liver The intracellular cyclic AMP and cyclic GMP levels found in the remaining liver at intervals after 80% hepatectomy are shown in Figure 5. The cyclic AMP level was low for the first 4 hr and then showed a biphasic increase after 12 and 22 hr. On the other hand, the intracellular cyclic GMP level showed a small but significant sharp increase at the very beginning of hepatic regeneration which reached a maximum after 1 0 - 2 0 min and returned to about the initial concentration after 60 min. Then about 16 hr after 80% hepatectomy, when the cyclic AMP level was low, the cyclic GMP level again increased to a fairly high level. Relative decreases in the cyclic AMP: cyclic GMP ratio were observed 20 rain and 16 hr after partial hepatectomy at times corresponding approximately to the beginning and end of the G1 phase. In cultured cells, Weinstein et al. (18) found that exogenous cyclic GMP and two of its derivatives stimulated resting mouse spleen lymphocytes to synthesize

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FIG. 4. (a) Effect of partial hepatectomy on the induction of tyrosine transaminase in isolated liver perfused with calf serum. , not hepatectomized; . . . . . , 70% hepateetomized at 0 time; , more than 80% hepatectomized at 0 time. (b) Effect of partial hepatectomy on the induction of ornithine decarboxylase in isolated liver perfused with calf serum. , not hepatectomized; . . . . . , 7 0 % hepatectomized at 0 time; , more than 80% hepatectomized at 0 time.

RNA and DNA, and that the time course of their effects was similar to that of concanavalin A. Moreover, Seifert and Rudland (19) showed that a relatively high concentration (10 "s to 10 "a M) of cyclic GMP stimulated DNA synthesis in a significant fraction of a quiescent culture of mouse fibroblasts. They als0 found that cyclic GMP only caused a significant increase when cultures of mouse fibroblasts which were arrested in the Go phase were stimulated to grow or when synchronized growing cells passed through the GI phase (20). This suggests that cyclic GMP acts as a specific stimulant for transition of cells from the Go or GI phase of the cell cycle by activating the pleiotypic and mitogenic program of the cell. If the initial rise of cyclic GMP is the messenger of the trigger for hepatic regeneration, then administration of cyclic GMP might induce regenerative changes in whole, perfused liver. As we have mentioned, when isolated, perfused liver was partially hepatectomized in vitro, omithine decarboxylase and tyrosine aminotransferase were induced in the remaining lobes. These findings suggested that these enzymic inductions might be prereplicative changes during the early A.I.E.R.~O

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TIME AFTER PARTIAL HEPATECTOMY( HR ) FIG. 5. Intracelhlar cyclic nucleotide levels after partial hepatectomy. Samples of the remaining liver were taken 10, 20 and 60 min and 3, 6, 12, 16, 22 and 24 hr after 80% hepatectomy. The first 2 points (10 and 20 min) are means of values in 5 rats and other points are means of values in 2 - 3 rats. Intraceliular cyclic nucleotide levels were determined as described in the methods.

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period of hepatic regeneration. Table 1 shows the effects of addition of cyclic AMP and of cyclic GMP to the perfusate on enzymic induction in whole, perfused liver. Addition of cyclic AMP did not always induce ornithine decarboxylase or tyrosine aminotransferase in isolated, perfused liver. However, addition of cyclic GMP induced both enzymes during a 3-hr period. This suggests that cyclic GMP is the intraceUular activator of the positive pleiotypic response of the cells, while cyclic AMP may mediate the negative pleiotypic response. TABLE 1. INDUCTIONSOF ORNITHINEDECARBOXYLASE AND TYROSINE AMINOTRANSFERASEIN ISOLATI~D, PERFUSED LIVER AFTER ADMINISTRATIONOF CYCLIC NUCLEOTIDES Conc. of cyclic nueleotides None (without hepatectomy) None (with 80% hepatectomy) c-AMP 10-4 M c-AMP 10az M c-AMP c-GMP c-GMP c-GMP

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Possible Involvement of Prostaglandins in the Regeneration of Liver Assuming that cyclic GMP is the messenger of the trigger for hepatic regeneration, the problem arises of what kind of substance is the actual trigger. Surgical operation may cause a local inflammatory change which produces prostaglandins (21). In addition, Kuehl (22) found that 10 "s M PGF2t~ caused a 4-fold increase in cyclic GMP levels in rat uterus within 45 seconds. Thus, prostaglandin might be the actual trigger for hepatic regeneration. Indomethacin is known to inhibit the biosynthesis of prostaglandins (23). So we examined the effect of administration of ifidomethacin 60 rain before partial hepatectomy on hepatic regeneration in vivo. Regeneration was estimated as DNA synthesis and induction of thymidine kinase after 24 hr. The results are shown in Figure 6. A dose of 50 mg/kg body weight of indomethacin decreased s H-incorporation into DNA by liver slices of the rats and the thymidine kinase activity in the cytosol of their liver. In addition, there was a slight but significant decrease, instead of increase, in the intracellular cyclic GMP level at the very beginning of hepatic regeneration, as shown in

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FIG. 6. (a) Inhibitory effect of indomethacin on induction of thymidine kinase activity by partial hepatectomy. Columns and bars show means and standard deviations of values in 3 experiments. IDM: Indomethacin; Sham: sham operated rats. (b) Inhibitory effect of indomethacin on incorporation of 3H-thymidine into DNA 24 hr after partial hepatectomy. Columns and bars show means and standard deviations of values in 3 experiments. IDM: Indomethacin; Sham: sham operated rats. Table 2. These results suggest that regeneration.

prostaglandins are involved in liver

TABLE 2. EFFECT OF INDOMETHACINON INTRACELLULAR CYCLIC GMP LEVELS AFTER 80% HEPATECTOMY Cyclic GMP (pmole/mg protein) 10 min after Before hepatectomy hepatectomy Control rats lndomethacin-treated rats

0.25 + 0.027 0.26 + 0.019

0.30 + 0.032* 0.24 + 0.006*

*Significantly different from the value before hepatectomy (P < 0.01).

Effects o f Prostaglandins on Cyclic Nucleotide Levels in Isolated Perfused Liver Based on the above results we studied the effects of PGE1 and PGF2a on perfused liver in vitro. For this, the prostagiandins were dissolved in ethyl alcohol and 0.1 ml of the solution was added to the perfusion medium to give a final

403

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concentration of 10 6 M. The cyclic nucleotide concentrations of the isolated perfused fiver were determined 30 min after perfusion with the prostaglandins. Table 3 shows the changes in the intracefiular levels of cyclic nucleotides. TABLE 3. EFFECTS OF PROSTAGLANDINSON THE CYCLIC NUCLEOTIDE LEVELS IN ISOLATED,PERFUSED WHOLELIVER Conc. of cyclic nucleotides (pmoles/mg protein) c-AMP c-GMP Level 30 min after perfusion without prostaglandins With 10"6 M PGEI With 10 "$ M PGF2ol

2.24 + 0.27* 3.61 + 0.23* 2.38 ","0.41

0.094 + O.Ollt 0.160 - 0.027 0.171 4- 0.026t

The differencesbetween * to * and I" to I" are significant(p <(0.005).

The level of cyclic AMP was increased by PGEt but not affected by PGF2a. However, addition of PGE~ or more especially PGF2 a remarkably increased the intracellular level of cyclic GMP. In 1972, Hadden et al. (24) proposed that growth of lymphocytes was regulated by the balance between cyclic AMP and cyclic GMP in the cell, a high cyclic AMP:cyclic GMP ratio being associated with proliferation. If so, PGE1 cannot be one of the triggers for cell proliferation, because it increases the levels of both cyclic AMP and cyclic GMP, as shown in Table 3. However, Rudland et al. (25) succeeded in increasing the intracellular level of cyclic GMP in mouse fibroblasts by treatment with the fibroblast growth factor (FGF), together with hydrocortisone and bovine serum albumin. It is interesting that FGF initiates the pleiotypic program without causing a significant fall in the cyclic AMP concentration. Furthermore, studies on isolated membranes from fibroblast cells show that FGF specifically stimulates guanyl cyclase, and does not affect adenyl cyclase. These data strongly suggest that increase in the intracellular cyclic GMP concentration may alone be sufficient to initiate growth without concomitant decrease in cyclic AMP. These facts suggest that both PGE~ and PGF2ol are triggers for liver regeneration. SUMMARY A sudden rise in cyclic GMP was observed in rats in vivo 10-20 min after partial hepatectomy. Addition of cyclic GMP to the perfusate of isolated whole liver induced tyrosine aminotransferase and omithine decarboxylase. Similar phenomena were observed in isolated, perfused liver in vitro after removal of hepatic lobes. These facts suggest that cyclic GMP is involved in the initiation of hepatic regeneration. In order to know what triggered off regeneration, hints

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were given by a series o f experiments suggesting that more extensive removal o f hepatic lobes stimulates hepatic regeneration more strongly. Prostaglandin seems most likely to be the physiological stimulator o f guanyl cyclase because it is produced b y inflammation or traumatic intervention. The possibility is supported by the finding that indomethacin inhibited hepatic regeneration. Moreover, addition o f PGEI or PGF2~ to the perfusion medium for isolated, perfused whole liver increased the intrahepatic level o f cyclic GMP. This may be a strong evidence to support a hypothesis that prostaglandins are the initial triggers for hepatic regeneration after partial removal o f hepatic lobes.

ACKNOWLEDGEMENT This work was supported in part by grants from the Japanese Ministry o f Education and from the Mishima Kaiun Foundation.

REFERENCES 1. P.S. RUDLAND, M. SEELEY and W. SEIFERT, Cyclic GMP and cyclic AMP levels in normal and transformed fibroblasts, Nature 251,417-419 (1974). 2. W. G. VERLY, The hepatic chalone, Natl. Cancer Inst. Monograph 38, 175-184 (1973). 3. R. CHURCH and B. J. McCARTHY, Ribonucleic add synthesis in regenerating and embryonic liver, 1. The synthesis of new species of RNA during regeneration of mouse liver after partial hepatectomy, Z Mol. Biol. 23, 459-475 (1967). 4. K. TSUKADA and I. LIEBERMAN, Metabolism of nucleolar ribonucleic acid after partial hepatectomy, J. Biol. Chem. 239, 1564-1568 (1964). 5. M. MURAMATSU and H. BUSCH, Studies on the nuclear and nucleolar ribonucleic acid of regenerating rat liver, J. Biol. Chem. 240, 3960- 3966 (1965). 6. S. SAKIYAMA, S. USUI and Y. MIURA, Regulation of ribosomal RNA synthesis, Advances in Enzyme Regulation 7, 207-218 (1969). 7. P. N. CAMPBELL, E. LOWE and G. SERCK-HANSSEN, Protein synthesis by microsomal particles from regenerating rat liver, Biochem. J. 103, 280-288 (1967). 8. M. B. HOAGLAND, O. A. SCORNIK and L. C. PFEFFERKORN, Aspects of control of protein synthesis in normal and regenerating rat liver, II. A microsomal inhibitor of amino acid incorporation whose action is antagonized by guanosine triphosphate,Proc. Natl. Acad. Sci. U. S. 51, 1184-1191 (1964). 9. O. A. SCORNIK, M. B. HOAGLAND, L. C. PFEFFERKORN and E. A. BISHOP, Inhibitors of protein synthesis in rat liver microsome fractions, Jr. Biol. Chem. 242, 131-139 (1967). 10. N. FUKUI, A. FUJITA, H. OHTSUKA and Y. MIURA, Induction of tyrosine aminottansferase and ornithine decarboxylase in isolated perfused regenerating rat liver, J. Biochem. 75,867-873 (1974). 11. H. OHTSUKA, Hormonal regulation of tyrosine transaminase synthesis in isolated rat liver perfused with synthetic medium, J. BiocherrL 75, 53-58 (1974). 12. T. I. DIAMONDSTONE, Assay of tyrosine transaminase activity by conversion of p-hydroxybenzaldehyde,Analyt. B i o c h e ~ 16, 395-401 (1966). 13. P. A. SARCOCCIA, Jr. and R. P. MIECH, Inosinic acid dehydrogenase in mammalian tissues, Mol. Pharmacol. 5, 26-29 (1969). 14. N. FUKUI, Factors regulating thymidine kinase in regenerating liver, J. Biochem. 69, 1075-1082 (1971). 15. D. RUSSELL and S. H. SNYDER, Amine synthesis in rapidly growing tissues: ornithine decarboxylase activity in regenerating rat liver, chick embryo and various tumors, Proc. Natl. Acad. ScL U. S. 60, 1420-1427 (1968).

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16. K. TSUKADA, H. OURA, S. NAKASHIMA and N. HAYASAKI, Adaptive response of tryptophan pyrrolase and tyrosine transaminase in rat liver after partial hepatectomy and laparotomy, Biochim. Biophys. Acta 165,218-224 (1968). 17. T. ADACHI, R. WATANABE and K. OKUDA, Guanine nucleotides synthesis in rat organs, (in Japanese) Seikagaku 4 5 , 5 9 8 - 5 9 8 (1973). 18. Y. WEINSTEIN, D. A. CHAMBERS, H. R. BOURNE and K. L. MELMON, Cyclic GMP stimulates lymphocyte nucleic acid synthesis, Nature 251, 352-353 (1974). 19. W.E. SEIFERT and P. S. RUDLAND, Possible involvement of cyclic GMP in gTowth control of cultured mouse cells, Nature 248, 138-140 (1974). 20. W. SEIFERT and P. S. RUDLAND, Cyclic nucleotides and growth control in cultured mouse cells: Correlation of changes in intracellular 3':5' c GMP concentration with a specific phase of the cell cycle, Proc. Natl. Acad. Sci. U. S. 71, 4920-4924 (1974). 21. A. L. WILLIS, Release of histamine, kinin and prostaglandins during carrageenininduced inflammation in the rat, pp. 39-49 in Prostaglandins, Peptides and Amines (P. MANTAGAZZA and E. W. HORTON, eds.), Academic Press, New York (1969). 22. F.A. KUEHL, Prostaglandins 5, 325-340 (1974). 23. R. V. TOMLINSON, H. J. RINGOLD, M. C. QURESHI and E. FORCHIELLI, Relationship between inhibition of prostaglandin synthesis and drug efficacy: Support for the current theory on mode of action of aspirin-like drugs, Biochent Biophys. Res. Communs. 46, 552-559 (1972). 24. J.W. HADDEN, E. M. HADDEN, M. K. HADDOX and N. D. GOLDBERG, Guanosine 3':5'-cyclic monophosphate: A possible intracellular mediator of mitogenic influences in lymphocytes, Proc. Natl. Acad. Sci. U. S. 69, 3024-3027 (1972). 25. P. S. RUDLAND, D. GOSPODAROWICZ and W. SEIFERT, Activation of guanyl cyclase and intraceUulax cyclic GMP by fibroblast growth factor, Nature 250, 7 4 1 - 7 4 2 , 7 7 3 - 7 7 4 (1974).