7. Biochem., 80, 291-297 (1976) Dedicated to Prof. N. Shimazono on his 70th birthday.

Involvement of Cyclic GMP in the Initial Stage of

Yoshiaki MIURA, Hajime IWAI, ! Ryuichi SAKATA,8 Hidesato OHTSUKA, EZRA Elhanan,4 Keiko KUBOTA, and Noriko FUKUI Department of Biochemistry, Chiba University School of Medicine, Chiba, Chiba 280 Received for publication, February 14, 1976

A transient rise in cyclic guanosine 3' : 5' monophosphate (c-GMP) in the liver was observed in rats in vivo 10—20 min after partial hepatectomy. A similar increase in c-GMP in the liver was also found in rats in vivo 15 min after infusion of TGH solution (a mixture of triiodothyronine, glucagon, and heparin). In both cases, inductions of ornithine decarboxylase [EC 4.1.1.17] and tyrosine aminotransferase [EC 2.6.1.5] were found 4 hr after the beginning of the experiments. Later, 22 hr after the surgical intervention or hormone infusion, thymidine kinase [EC 2.7.1.21] was activated and liver slices were able to incorporate [!H]thymidine into DNA. These biochemical phenomena were observed commonly in regenerating liver as well as in the liver of rats infused with TGH solution. c-GMP, but not c-AMP, could induce ornithine decarboxylase and tyrosine aminotransferase in isolated, perfused liver.

Partial hepatectomy is a classical method for producing cell proliferation in the liver. This surgical intervention implicates the transfer of hepatocytes from the Go or Gi phase of the cell cycle to the S phase. In addition to partial hepatectomy, two new methods have recently been developed for the stimulation •of hepatocytes toward cell proliferation; a) Shift up to high protein content in the food of animals fed with a low protein diet ( 1 )

and b) Infusion of TAGH solution (2). TAGH solution is an infusate which contains triiodothyronine, glucagon, amino acids, and heparin; an infusate which contains only triiodothyronine, glucagon, and heparin will be referred to as TGH solution, In this report evidence will be presented that, as is the case after partial hepatectomy, there is a transient rise in intrahepatic cGMP in the livers of rats infused with TGH

1

This study was supported in part by a grant for scientific research from the Ministry of Education, Science and Culture, of Japan and by a research grant from the Naito Foundation. "* Present address: Pharmacological Research Laboratory, Tanabe Seiyaku Co., Ltd., Toda, Saitama 335. 3 Present address: Department of Brain Surgery, Tokyo Medical College, Shinjuku-ku, Tokyo 160. 4 On leave from the Department of Biological Chemistry, The Hebrew University of Jerusalem, Israel. "Vol. 80, No. 2, 1976

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Hepatocytes Proliferation1

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Y. MIURA, H. IWAI, R. SAKATA, H. OHTSUKA, EZRA E., K. KUBOTA, and N". FUKUT

MATERIALS AND METHODS Animals—Male Donryu strain rats weighing about 100 g (for partial hepatectomy) and 130 g (for TGH infusion) were obtained from Nippon Rat Co., Urawa, Saitama. They were kept in the dark from 9: 00 pm to 9 : 00 am and were fed during this period. Partial hepatectomy as well as infusion of TGH solution were carried out under ether anesthesia between 10: 00-12 : 00 am. For perfusion experiments, larger rats, weighing about 250— 300 g, were used and their livers were also removed between 10 : 00—12 : 00 am. Chemicals—DL-[l-uC]ornithine (58 mCi/ mmole), [2-uC]deoxy thymidine (59 mCi/mmole), and [8H]deoxythimidine (5 Ci/mmole) were purchased from the Radiochemical Centre, Amersham, England. Glucagon and triiodothyronine were obtained from Sigma Chemicals Co., St. Louis, Mo. Heparin was a product of Novo Co., Denmark. Authentic samples of cyclic nucleotides were from Yamasa Shoyu Co., Choshi, Chiba and fluorocarbon suspension (perfluorotributylamine, (C4F8)3N and pluronic acid, 2.5 w/v) was from Midorijuji Co., Osaka. Partial Hepatectomy—Partial hepatectomy was carried out under ether anesthesia by the method of Higgins and Anderson (3). About 70% of the hepatic lobes was usually removed, but 80% hepatectomy was sometimes carried out. As a sham operation, the abdominal wall was opened and closed. Infusion of TGH Solution—TGH solution

for infusion (1.0 ml) was freshly prepared according to the original report of Lieberman et al. (2), except that amino acids mixture, was omitted, since it did not appear to play a major role and there were difficulties in dissolving it in 1 ml of solution. This solution is referred to as TGH solution. As we wished to check the intrahepatic c-GMP level within 30 min of the start of the experiments, we infused the TGH solution within 10 mini instead of the slow infusion procedure (3 hr period) in the original report (2). The solution was injected into the tail vein from hypodermic syringes fixed in a horizontal! position with a moving carriage assemble: acting on the plungers to deliver a constant flow of 0.1 ml per min. The TGH solution. (pH 7.2) contained 100 ug of triiodothyronine, 1 mg of glucagon, and 100 U.S.P. units of heparin in 0.15 M NaCl. Perfusion Technique— After a brief interuption of circulation, the liver was perfused, for 10 min with a mixture of 80 ml of calf serum and 20 ml of fluorocarbon suspension.. Then, it was perfused with the same medium, containing glucose (200 mg), penicillin G(50,000 U), and streptomycin sulfate (100 mg). 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 rate of the perfusatethrough the liver was maintained at approximately 30 ml/min. Adequate bile production (0.6ml/hr) and rate of flow of the perfusate: were used as criteria of successful perfusion. The pressure of the perfusate entering the liver via the portal vein was maintained a t 14 cm H,O. Estimation of Cyclic Nucleotides—Samplesof hepatic lobes were removed as described, by MacManus et al. (4) after immersion of the crushed lobes in liquid nitrogen. Cold 5% trichloroacetic acid was added to the frozen, tissue and it was extracted with ether. Cyclic, nucleotides were estimated using kits for radioimmunoassay (Schwarz Mann, N.Y., and. later Mitsui Seiyaku, Mobara, Chiba). Enzyme Assays—Ornithine decarboxylase activity was measured in the cytosol fraction, by radioassay, as described previously (5)_ / . Biochem^

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solution. Then, in the Gi phase, ornithine decarboxylase [EC 4.1.1.17] and tyrosine aminotransferase [EC 2.6.1. 5] are successively induced in regenerating liver and livers of rats infused with TGH solution. Finally, in the beginning of the S phase, thymidine kinase [EC 2.7.1.21] is activated and then liver slices could incorporate labeled thymidine into DNA. The fact that c-GMP, but not cAMP, is able to induce ornithine decarboxylase and tyrosine aminotransferase in isolated, perfused liver suggests that c-GMP may be one of the mediators producing some of the enzymes in the prereplicative period.

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CYCLIC GMP AND HEPATOCYTES REPLICATION

DNA Synthesis—Incorporation of [3H]thymidine was determined by the method described by Verly (9) using liver slices. The liver was cut into slices of approximately •0.5 mm thickness with a motor-driven microtome. The slices were placed in 25 ml Erlenmeyer flasks with 20 ftCi of [3H]deoxythymidine and Hank's solution was added to a volume of 5 ml at 0°. The flasks were shaken .gently under 02(95%)-C02(5%) in a water bath at 37° for 2 hr. After incubation, the specific activity of DNA in the acid-insoluble fraction "was measured. RESULTS Intracellular Cyclic Nucleotide Levels—The intracellular c-AMP and c-GMP levels after 80% partial hepatectomy are shown in Fig. 1. The c-AMP level was low for the first 4hr

0

1

1

8

and then showed a biphasic increase after 12 and 22 hr. On the other hand, the intracellular c-GMP level showed a small but significant sharp increase at the very beginning of hepatic regeneration, reaching a maximum after 10—20 min and returning to about the initial concentration after 60 min. Then, about 16 hr after partial hepatectomy, when the c-AMP level was low, the c-GMP level again increased to a fairly high level. Some decrease in the c-AMP: c-GMP ratio was observed 20 min and 16 hr after partial hepatectomy at times corresponding approximately to the beginning and end of the G t phase. In the case of infusion of TGH solution, a similar transient rise in the c-GMP level was also found soon after the infusion: the change was monophasic with a significant peak (3-fold increase) at 15 min, as shown in Fig. 2. In contrast to the case of partial hepatectomy, intrahepatic c-AMP was extraordinarily high : (about 200 pmole/mg protein at the very beginning of the experiment), because glucagon stimulated hepatic adenylate cyclase [EC 4.6.1.1]. Enzytnic Inductions and DNA Synthesis— Inductions of ornithine decarboxylase, tyrosine aminotransferase, and thymidine kinase in

12

16

20

TIKE AFTER PARTIAL HEPATECTOHY (

Fig. 1. Intracellular cyclic nucleotide levels after partial hepatectomy. After 80% hepatectomy, samples of the remaining liver were taken at 10, 20, and 60 min and 3, 6, 12, 16, 22, and 24 hr. Points and bars are means and standard deviations of values in 2-5 rats. The single asterisk denotes a significant difference from the zero-time value (£