Sequential changes in ornithine decarboxylase thymidine kinase, and other enzyme activities in regenerating liver in rats on controlled feeding schedules.

The changes in activity of five enzymes including ornithine decarboxylase (ODC), tyrosine aminotransferase (TAT), thymidine kinase (TK), ornithine aminotransferase (OAT) and serine dehydratase (SDH) in the early stage of the regenerating rat liver have been studied under a controlled feeding and lighting schedule. The first three enzyme activities were stimulated sequentially by partial hepatectomy. The earliest response was observed in ODC activity. A significant increase in this enzyme activity was observed at 2 hrs and the maximal level was at 4 hrs after the operation. TAT began to increase at 4 hrs and the maximal level was at 8 hrs. The TK activity was induced at about 24 hrs and the highest value was at 48 hrs after partial hepatectomy.A significant decrease in OAT activity was observed at 24 hrs after the operation and subsequently. Although a decrease in SDH activity was also observed this decrease did not seem to correlate directly with the regeneration process, since a lowered level of the enzyme activity was also found in the sham operated group.     

Regulation of ornithine decarboxylase and other cell cycle-dependent genes during senescence of IMR-90 human diploid fibroblasts

Aging of IMR-90 human diploid fibroblasts in vitro is accompanied by significant changes of polyamine metabolism, most notably, a 5-fold decrease of serum-induced activity of ornithine decarboxylase, the key enzyme in the biosynthesis of polyamines (Chen, K. Y., Chang, Z. F., and Liu, A. Y.-C. (1986) J. Cell. Physiol. 129, 142-146). In this paper, we employed Northern blot hybridization and affinity radiolabeling techniques to investigate the molecular basis of this age-associated change of ornithine decarboxylase activity. Since the induction of ornithine decarboxylase by serum is a mid-G1 event, we also examined expressions of other cell cycle-dependent genes that are induced before and after the mid-G1 phase to determine if their expressions may also be age-dependent. Our results demonstrated a 3-fold decrease of the amount of active ornithine decarboxylase molecules that can be labeled by alpha-difluoromethyl[3H]ornithine in senescent IMR-90 cells (population doubling level (PDL) = 52) as compared to young cells (PDL = 22). However, the levels and kinetics of induction of ornithine decarboxylase mRNA in both young and senescent IMR-90 cells were found to be identical throughout a 24-h time period after serum stimulation. The time course and the magnitude of the expression of c-myc, an early G1 gene, were quite similar in young and senescent IMR-90 cells and appeared to be PDL-independent. In contrast, the expression of thymidine kinase, a late G1/S gene, was significantly reduced in senescent IMR-90 cells. Levels of thymidine kinase mRNA and thymidine kinase activity in senescent IMR-90 cells were 6- and 8-fold less than those in young cells, respectively. Based on these data, we proposed that impairment of cell cycling in senescent IMR-90 cells may occur at the late G1/S phase and that decreases of ornithine decarboxylase activity and putrescine accumulation during cell senescence may contribute to this impairment.     

Differential regulation of the mitogen-activated protein and stress-activated protein kinase cascades by adrenergic agonists in quiescent and regenerating adult rat hepatocytes.

To study the mechanisms by which catecholamines regulate hepatocyte proliferation after partial hepatectomy (PHX), hepatocytes were isolated from adult male rats 24 h after sham operation or two-thirds PHX and treated with catecholamines and other agonists. In freshly isolated sham cells, p42 mitogen-activated protein (MAP) kinase activity was stimulated by the alpha1-adrenergic agonist phenylephrine (PHE). Activation of p42 MAP kinase by growth factors was blunted by pretreatment of sham hepatocytes with glucagon but not by that with the beta2-adrenergic agonist isoproterenol (ISO). In PHX cells, the ability of PHE to activate p42 MAP kinase was dramatically reduced, whereas ISO became competent to inhibit p42 MAP kinase activation. PHE treatment of sham but not PHX and ISO treatment of PHX but not sham hepatocytes also activated the stress-activated protein (SAP) kinases p46/54 SAP kinase and p38 SAP kinase. These data demonstrate that an alpha1- to beta2-adrenergic receptor switch occurs upon PHX and results in an increase in SAP kinase versus MAP kinase signaling by catecholamines. In primary cultures of hepatocytes, ISO treatment of PHX but not sham cells inhibited [3H]thymidine incorporation. In contrast, PHE treatment of sham but not PHX cells stimulated [3H]thymidine incorporation, which was reduced by approximately 25 and approximately 95% with specific inhibitors of p42 MAP kinase and p38 SAP kinase function, respectively. Inhibition of the p38 SAP kinase also dramatically reduced basal [3H]thymidine incorporation. These data suggest that p38 SAP kinase plays a permissive role in liver regeneration. Alterations in the abilities of catecholamines to modulate the activities of protein kinase A and the MAP and SAP kinase pathways may represent one physiological mechanism by which these agonists can regulate hepatocyte proliferation after PHX.     

The sex difference in the regulation of liver regeneration after partial hepatectomy in the rat

The increases in the activity of hepatic thymidylate synthetase and thymidine kinase, which catalyzes the formation of thymidylate via the de novo and salvage pathways, respectively, were significantly suppressed 24 h after 70% partial hepatectomy in female rats administered either alpha- or beta-adrenoreceptor antagonists. The injection of beta-antagonist to male or ovariectomized female rats had no effect on the activities of these enzymes. Only alpha-adrenoceptor antagonist depressed these enzymatic activities of 24-h-regenerating liver in male and ovariectomized female rats. The decrease of the activities of thymidylate synthetase and thymidine kinase was accompanied by a concomitant reduction of DNA content in 24-h-regenerating liver. It is concluded that catecholamine regulates the female rat liver regeneration through both alpha- and beta-adrenergic pathways by the inductions of thymidylate synthase and thymidine kinase, while in adult male and ovariectomized female rats, only the alpha-mediated pathway is involved.     

Correlation of mitochondrial ribonucleotide reductase and thymidine kinase activities with the synthesis of mitochondrial DNA in rat liver during regeneration

3H-thymidine incorporation into DNA of heavy mitochondria from regenerating rat liver and the change of mitochondrial thymidine kinase and ribonucleotide reductase activities are studied in vivo in regenerating rat liver within 6--48 hours after hepatectomy. Synthesis of mitochondrial DNA and changes in the activity of the enzymes studied are found to be undulate. Thymidine kinase activity maxima coincide with those of 3H-thymidine incorporation. Maximal activity of ribonucleotide reductase pre-exists maxima of mitochondrial DNA synthesis.     

Involvement of cyclic GMP in the initial stage of hepatocytes proliferation.

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 [3H]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.     

Does phospholipase C stimulate thymide kinase activity of rat liver extracts prepared after partial hepatectomy.

Our purpose was to determine whether phospholipase C stimulated thymidine kinase activity of regenerating rat liver. We determined effects of phospholipase C upon TMP formation by rat liver extracts prepared at 0, 12, 24, 36 and 48 hr following partial hepatectomy. Data were obtained which supported these conclusions: (a) Commercial preparations of phospholipase C contained nucleoside phosphotransferase activity; (b) phospholipase C exerted no appreciable stimulatory influence upon thymidine kinase activity of regenerating rat liver; and (c), apparent stimulation of thymidine kinase was associated with linked activities of two enzymes, viz., liver extract-ATPase activity and nucleoside phosphotransferase activity.     

Role of endogenous TNF-alpha and sphingosine in induced DNA synthesis in regenerating rat liver after partial hepatectomy.

Cytokine-stimulated metabolism of sphingomyelin results in the accumulation of ceramide and sphingosine which play a part in the regulation of cell proliferation, differentiation, and reception, as well as in oncogenesis. Formation of TNF-alpha (a member of the cytokine family), accumulation of sphingosine, and DNA synthesis (measured by immunoblotting, HPLC, and [3H]thymidine incorporation, respectively) were studied in rat liver after partial hepatectomy. The content of TNF-alpha was found to increase during 12 h following hepatectomy. The maximum of sphingomyelinase activity and accumulation of sphingosine precede the maximum of DNA synthesis. Sphingosine is known to inhibit protein kinase C. On the other hand, it stimulates the metabolism of phosphatidylinositol, thus causing accumulation of diacylglycerol and inositol-1,4,5-triphosphate, which in turn activate protein kinase C. Hence, the release of TNF-alpha in regenerating liver may modulate DNA synthesis through the accumulation of sphingosine which is involved in regulation of protein kinase C activity and of phosphatidylinositol turnover.     

Effect of autonomic denervation on DNA synthesis during liver regeneration after partial hepatectomy

The regulatory role of autonomic nerves in liver regeneration after partial hepatectomy was studied in rats by bilateral subdiaphragmatic vagotomy or splanchnicectomy. 1. In control rats the wet weight of the regenerating liver was restored to approximately 80% of the preoperative weight 72 h after partial hepatectomy. Restoration of the liver weight was significantly impaired in vagotomy rats, but not in splanchnicectomy. Increases in the DNA and protein contents of the regenerating liver were also suppressed by vagotomy. 2. Hepatic DNA synthesis, measured as the incorporation of [methyl-3H]thymidine into DNA at various times after partial hepatectomy, was significantly less in vagotomized rats, and slightly more in splanchnicectomized rats than in control rats. The onset of DNA synthesis triggered by partial hepatectomy was also delayed by vagotomy. 3. The increases in activities of hepatic aspartate transcarbamoylase and thymidine kinase, the key enzymes in synthesis of pyrimidine nucleotides via the de novo and salvage pathways respectively, during liver regeneration, were significantly suppressed and retarded in vagotomized rats. Conversely, splanchnicectomy tended to stimulate these enzyme inductions after partial hepatectomy. 4. During starvation the plasma insulin level decreased after partial hapatectomy in control and vagotomized rats, as in sham-operated rats, but showed a transient increase 6 h after partial hepatectomy in splanchnicectomized rats. It is concluded that vagotomy inhibits and delays DNA synthesis and proliferation of liver cells after partial hepatectomy, whereas splanchnicectomy tends to stimulate these processes. The data also suggest that parasympathetic innervation of the liver may play an important regulatory role in liver regeneration.     

Ornithine decarboxylase and thymidine kinase activity in tissues of prolactin-treated rats: effect of hypophysectomy

The activities of ornithine decarboxylase and thymidine kinase were determined in tissues of young intact and hypophysectomized rats at various times after treatment with prolactin. In both types of animals, ornithine decarboxylase activity increased in liver, kidney, spleen and adrenal of prolactin treated rats. Thymidine kinase activity increased only in liver and spleen of intact rats. Increase in the kinase activity was smaller, and occurred later than the change in ornithine decarboxylase. In hypophysectomized animals, thymidine kinase activity increased in spleen, but not in liver, following prolactin treatment.     

Prereplicative enzymic changes in regenerating rat liver.

After partial hepatectomy in rats, the following changes in enzymic activities were observed in the remnant liver during the prereplicative period. In the initial period of the prereplicative process, soon after removal of part of the liver, ornithine decarboxylase [EC 4.1.1.17] and IMP dehydrogenase [EC 1.2.1.14] increase. Subsequently, for entry into the S period, thymidine kinase [EC 2.7.1.75] increases simultaneously with increase in the intracellular cyclic AMP level and decrease in its phosphodiesterase [EC 3.1.4.17].     

Colon tumor: enzymology of the neoplastic program

The biochemical strategy of colon tumor was investigated by comparing the enzymic programs of glycolysis, pentose phosphate production and purine and pyrimidine biosynthesis and degradation in liver, normal colon mucosa and transplantable colon adenocarcinoma in the mouse. In normal colon mucosa the carbohydrate and pentose phosphate enzymes were 2- to 9-fold higher in specific activity than those in liver. Among the enzymes of CTP synthesis, CTP synthetase was the rate-limiting one in both liver and colon. In colon tumor CTP synthetase, OMP decarboxylase, uracil phosphoribosyltransferase and thymidine kinase activities increased to 927, 863, 597 and 514% of activities of normal colon. In contrast, the activity of the catabolic enzymes, dihydrothymine dehydrogenase and uridine phosphorylase, decreased to 51 and 25%. The ratios of activities of uridine kinase/uridine phosphorylase and thymidine kinase/dihydrothymine dehydrogenase were elevated 6- and 10-fold. The activity of the key purine synthetic enzyme, glutamine PRPP amidotransferase, increased 7-fold and the opposing rate-limiting enzyme of purine catabolism, xanthine oxidase, decreased to 7%. The ratio of amidotransferase/xanthine oxidase was elevated to 8, 150%. Activities of glucose-6-phosphate dehydrogenase and transaldolase did not increase, but that of pyruvate kinase was elevated to 154%. Similar enzymic programs were observed in a transplantable adenocarcinoma of the colon in the rat. The alterations in gene expression in colon tumor manifested in an integrated pattern of enzymic imbalance indicate the display of a program, a segment of which is shared with rat and human liver and kidney tumors. These alterations in gene expression should confer selective advantages to colon tumor cells. The striking increases in the activities of CTP synthetase, OMP decarboxylase, glutamine PRPP amidotransferase and thymidine kinase mark out these enzymes as potentially sensitive targets for combination chemotherapy by specific inhibitors of these enzyme activities.     

Mitochondrial thymidine kinase and ribonucleotide reductase from rat liver and rat hepatoma 27]

Fractions of heavy and light mitochondria are isolated from homogenates of homologous rat tissues (intact liver, regenerating liver within 24 hours after hepatectomy and 27 hepatoma) by means of differential centrifugation. It is found that tumour mitochondria have higher heterogeneity and lower buyoant density than mitochondria from normal hepatocytes. The activity of two enzymes of DNA precursors synthesis (ribonucleotide reductase and thymidine kinase) in subcellular fractions is demonstrated to correlate with the tissue growth rate. A single injection of cyclic AMP into hepatectomised rats resulted in the retardation of the regeneration process, and the activity of both enzymes reached its normal level in all the fractions studied after 24 hours after the operation. Thymidine kinase and ribonucleotide reductase are located mainly in the mitochondrial matrix, however, pronounced enzyme activity is observed also in membrane fractions. The activity of the enzymes in the fraction of external mitochondria membranes in rapidly growing tissues is 2--3 times as high as in the same fraction from normal rat liver.     

Studies on the hyperplasia (''regeneration'') of the rat liver following partial hepatectomy. Changes in lipid peroxidation and general biochemical aspects.

Using the experimental model of partial hepatectomy in the rat, we have examined the relationship between cell division and lipid peroxidation activity. In rats entrained to a regime of 12 h light/12 h dark and with a fixed 8 h feeding period in the dark phase, partial hepatectomy is followed by a rapid regeneration of liver mass with cycles of synchronized cell division at 24 h intervals. The latter phenomenon is indicated in this study by pulses of thymidine kinase activity having maxima at 24 h, 48 h and 72 h after partial hepatectomy. Microsomes prepared from regenerating livers show changes in lipid peroxidation activity (induced by NADPH/ADP/iron or by ascorbate/iron), which is significantly decreased relative to that in microsomes from sham-operated controls, again at 24 h, 48 h and 72 h after the operation. This phenomenon has been investigated with regard to possible underlying changes in the content of microsomal fatty acids, the microsomal enzymes NADPH:cytochrome c reductase and cytochrome P-450, and the physiological microsomal antioxidant alpha-tocopherol. The cycles of decreased lipid peroxidation activity are apparently due, at least in part, to changes in microsomal alpha-tocopherol content that are closely associated in time with thymidine kinase activity.     

Hepatic pyruvate metabolism during liver regeneration after partial hepatectomy in the rat

Hepatic pyruvate kinase (PK) and pyruvate dehydrogenase (PDHa) specific activities were decreased after partial hepatectomy or sham operation. The decreases were more marked and sustained after partial hepatectomy. These activity changes ensure that hepatic carbon flux after partial hepatectomy is predominantly in the direction of gluconeogenesis. The decrease in PK specific activity observed after partial hepatectomy was associated with a decreased PK activation ratio (activity measured at 0.15 mM PEP: activity measured at 5.0 mM PEP), and hepatic concentrations of PEP were increased. The low hepatic PDHa activity observed at the first day after partial hepatectomy occurred concomitantly with an increased fatty acid concentration. PDHa activity increased after inhibition of lipolysis. The results indicate that carbohydrate utilization is unimportant for hepatic energy supply during liver regeneration. There was no evidence that the control of PK or PDH in the regenerative liver after partial hepatectomy differed from that observed in the liver of the unoperated rat.     

Lipid peroxidation in regenerating rat liver

Rats entrained to a strictly regulated lighting and feeding schedule have been subjected to partial hepatectomy or a sham operation. In the partially hepatectomised animals the period of liver regeneration is characterised by regular bursts of thymidine kinase activity. Liver microsomes from rats, at times corresponding to maximum thymidine kinase activity, have much reduced rates of lipid peroxidation compared to control preparations: this is due in part to increased levels of lipid-soluble antioxidant at times of maximal DNA synthesis. This temporal relationship between thymidine kinase and lipid peroxidation is consistent with the view that lipid peroxidation is decreased prior to cell division.     

Biopterin metabolism and phenylalanine hydroxylase activity during early liver regeneration

Rat liver biopterin content and the activities of two enzymes involved in biopterin metabolism, sepiapterin reductase and dihydropteridine reductase, were not altered twenty-four hours after partial hepatectomy. This surgical procedure did, however, produce a vigorous regenerative response as verified by an increase in ornithine decarboxylase activity. The tetrahydrobiopterin-dependent activity of phenylalanine hydroxylase was increased in homogenates of regenerating liver. The pteridine requirements for the expression of this activation, and the behavior of the enzyme on calcium-phosphate cellulose columns suggest that elevated levels of cyclic adenosine monophosphate in regenerating liver induce phosphorylation and activation of phenylalanine hydroxylase. This increase in the activity of the primary enzyme of phenylalanine catabolism was interpreted as a compensatory response designed to maintain homeostasis prior to liver regeneration.     

Effect of urethan on polyamine and DNA synthesis in the regenerating rat liver

When a single dose of urethan was injected into the peritoneal cavity of rats immediately after partial hepatectomy, DNA synthesis was delayed by 12 h. The induction of ornithine decarboxylase which was induced biphasically following partial hepatectomy was also reduced and delayed by 14–15 h by the administration of urethan. S-Adenosylmethionine decarboxylase activity in urethan-treated rat liver at 20 h and 29 h after operation was significantly lower than that of untreated animals. This enzyme activity was shown to increase thereafter, reaching a higher level than in untreated rats at 37–42 h. Hepatic spermidine content changed biphasically in a manner similar to DNA synthesis. These results suggest that the activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase may correlate with DNA synthesis and that an increase of spermidine concentration is necessary to DNA synthesis.     

Effect of portal branch ligation on polyamine metabolism in rat liver

The activity of ornithine decarboxylase and the intracellular concentrations of putrescine and spermidine in the non-ligated lobes of the liver increased after portal branch ligation. These changes were followed by increased [3H]thymidine uptake into the acid-insoluble fraction of the liver. The induction of ornithine decarboxylase and changes in intracellular polyamines are important biochemical events in liver regeneration, so our results suggest that portal branch ligation causes formation of some stimuli that trigger liver regeneration. Changes were less with ligation than with partial hepatectomy.