Noriko Fukui

Arachidonic acid metabolism in normal and regenerating liver and hepatoma

DNA synthesis in perfused rat liver after partial hepatectomy was suppressed by addition of aspirin or imidazole in the perfusion medium. Addition of PGF2α did not induce DNA synthesis without partial hepatectomy. Thus the authors postulate that thromboxane (TX) might be the trigger for DNA synthesis after partial hepatectomy. In fact, in in vivo experiments, [14C]arachidonic acid was converted to PGE2 and PGF2α in normal liver, whereas TXB2 was the major labeled product in regenerating liver. In contrast to the liver tissue, intact AH 130 ascitic hepatoma mainly incorporated [14C]arachidonic acid into phospholipids. However, the microsomal fraction of hepatoma cells synthesized PGE2, PGF2α, TXB2 and unidentified compounds from arachidonic acid. Thus the systems regulating the arachidonic acid cascade in normal and regenerating liver or hepatoma seem to be quite different.

Pleiotypic responses of regenerating liver

Abstract 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 ornithine 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 were given by a series of experiments suggesting that more extensive removal of hepatic lobes stimulates hepatic regeneration more strongly. Prostaglandin seems most likely to be the physiological stimulator of guanyl cyclase because it is produced by inflammation or traumatic intervention. The possibility is supported by the finding that indomethacin inhibited hepatic regeneration. Moreover, addition of PGE1 or PGF2α to the perfusion medium for isolated, perfused whole liver increased the intrahepatic level of cyclic GMP. This may be a strong evidence to support a hypothesis that prostaglandins are the initial triggers for hepatic regeneration after partial removal of hepatic lobes.

Cyclic AMP as a regulator of thymidine kinase.

Abstract Thymidine kinase was partially purified from Yoshida sarcoma cells by DEAE-cellulose column chromatography. The elution pattern showed two distinct peaks (I and II). The former was found both in normal and neoplastic cells and its activity was inhibited by addition of cyclic AMP, whereas the latter was only found in neoplastic cells and its enzymic activity was enhanced by addition of cyclic AMP. 3 H-Cyclic AMP did not bind with protein in peak I, but bound with that in peak II resulting in cleavage of the enzyme into several new fractions with thymidine kinase activity. The role of cyclic AMP as a regulator of cell growth is briefly discussed.

Induction of ornithine decarboxylase and DNA synthesis in isolated perfused liver after partial hepatectomy.

Characteristic biochemical events in the prereplicative period were compared in the liver of rats after partial hepatectomy and after infusion of a mixture of triiodothyronine, glucagon and heparin. The events observed were: a transient rise in cyclic GMP in the very early stage, inductions of ornithine decarboxylase and tyrosine aminotransferase during the G1 phase, induction of thymidine kinase at the end of the G1 phase and finally DNA synthesis in the S phase. Those biochemical events were observed after both the surgical and the chemical treatment. However, it was found that among these changes induction of tyrosine aminotransferase and rise in cyclic GMP were also observed without cell proliferation. Thus, the induction of ornithine decarboxylase and DNA synthesis were chosen as biochemical parameters to show that the cells had entered the proliferative process. Using isolated perfused liver it was found that regeneration occurred after partial hepatectomy: that is, ornithine decarboxylase was induced after partial hepatectomy. Addition of arachidonic acid to the perfusion fluid potentiated the enzymic induction but addition of prostaglandin antagonists suppressed it. Perfusion of PGF2μ without partial hepatectomy also induced ornithine decarboxylase. These findings suggest that prostaglandins are involved in induction of ornithine decarboxylase during the prereplicative period after partial hepatectomy. During long-term perfusion for 12 hr, partially hepatectomized liver was able to synthesize DNA. The characteristic features of DNA synthesis in isolated perfused liver were a shorter G1 phase and higher activity of thymidine incorporation into DNA than those in in vivo experiments. Addition of arachidonate to the perfusion fluid was as effective for DNA synthesis as partial hepatectomy, but previous protein-starvation was not necessary for inducing DNA synthesis in isolated perfused liver.

Uridine diphosphate reductase of Ehrlich ascites tumor is insensitive to hydroxyurea

Uridine diphosphate (UDP) reductase was isolated in the supernatant fraction obtained after the acidification of the cytosol of Ehrlich ascites tumor cells, and was found insensitive to 10 mM hydroxyurea. However, cytidine diphosphate (CDP) reductase, being separated concurrently in the precipitate fraction, was readily inhibited. In the cytosol fraction of either Ehrlich ascites tumor, Yoshida ascites sarcoma or regenerating rat liver after partial hepatectomy, UDP reduction activity, in contrast to CDP reduction activity, is not sensitive to hydroxyurea.

Regulation of the arachidonic acid cascade in normal liver and hepatoma cells

Intact liver could synthesize PGE2, PGF2α from arachidonic acid, whereas injured hepatocytes were able to synthesize TX in addition of PGE2, PGF2α. Normal liver homogenate could not produce TX from arachidonic acid because the supernatant fraction of the homogenate contained some inhibitor of TX biosynthesis. Intact hepatoma cells incorporated most of arachidonic acid into phospholipid, whereas hepatoma sonicate produced PGE2, PGF2α, TXB2, 6-keto PGF1α and 15-HPETE. The supernant fraction of hepatoma sonicate. did not contain the inhibitor of TX biosynthesis. Moreover, the supernatant fraction of hepatoma sonicate was able to synthesize PGE2, PGF2α, TXB2 6-keto PGF1α and 15-HPETE. Injured liver host temporarily the inhibitor of TX biosynthesis. TX with platelet-derived growth factor may collaborate to trigger pleiotypic responses in regenerating liver after partial hepatectomy.

Thromboxane synthesizing system in rat liver

Thromboxane B2 (TXB2), along with other primary prostaglandins, was synthesized when rat liver microsomes were incubated with radioactive arachidonic acid. TXB2 was identified directly by chemical ionization mass spectrometry and indirectly by using specific inhibitors of TX synthetase, viz., imidazole and OKY-1555 ((E)-3(4-(3-pyridyl-methyl) phenyl)-2 methyl acrylic acid HCl). The supernatant fraction obtained after centrifugation at 105,000 X g for 60 min contained a possible regulatory component that suppressed thromboxane synthesis. The regulatory influence is lost after partial hepatectomy.