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.     

Inhibition of polyamine accumulation and deoxyribonucleic acid synthesis in regenerating rat liver.

Repeated injections of 1,3-diaminopropane into rats after partial hepatectomy caused a repression-type inhibiton of liver ornithine decarboxylase (EC 4.1.1.17) and totally prevented the marked increases in liver putrescine and spermidine concentrations that normally occur in response to partial hepatectomy. The inhibition of polyamine synthesis by diaminopropane was accompanied by a profound decrease (about 80%) in the synthesis of DNA in the regenerating rat liver without any changes in the synthesis of RNA and total liver protein.     

Uptake of amino acids and nucleic acid precursors by regenerating rat liver

1. At 0.5-1.0h after partial hepatectomy the intracellular acid-soluble fraction of rat liver took up twice as much radioactivity from [(3)H]orotic acid, [(3)H]uridine and [(3)H]thymidine as did similar fractions from sham-operated animals. This increase in penetration was not prevented by adrenalectomy or actinomycin, both of which decreased precursor uptake into nuclear RNA at this time. The increase in entry was still shown by thymidine at 22h after operation, at the height of the S period. Adenine penetration was not increased 1h after partial hepatectomy. 2. Plasma concentrations of ornithine and possibly methionine, tyrosine and lysine were raised 1.5h after partial hepatectomy. [(3)H]Lysine entry into regenerating liver at this time was increased by 60%; [(3)H]valine uptake was unaffected. Intracellular amounts of tyrosine, phenylalanine and ornithine in the liver were also increased. 3. The relation of these events to the start of liver regeneration is discussed.     

Selenomethionine induces polyamine biosynthesis in regenerating rat liver tissue

Summary. Our study was undertaken to elucidate the effects of selenomethionine (SeMet) on polyamine metabolism in regenerating rat liver tissue, as useful model of rapidly growing normal tissue. We have examined the levels of spermine, spermidine and putrescine in liver tissue. At the same time we have evaluated the activities of polyamine oxidase (PAO) and diamine oxidase (DAO), the catabolic enzymes of polyamine metabolism. The obtained results suggest that polyamine levels in regenerating liver tissue, at 7^th day after two-thirds partial hepatectomy, were higher in comparison with control group. The administration of selenomethionine to hepatectomized animals during seven days, in a single daily dose of 2.5 μg/100 g body weight, increases the amount of spermine and spermidine; the level of putrescine does not change under the influence of SeMet in regenerating rat liver tissue. PAO activity is lower in regenerating hepatic tissue than in control group. Supplementation of hepatectomized animals with SeMet significantly decreases the activity of this enzyme. DAO activity was significantly higher in hepatectomized and in operated animals treated with SeMet compared to the sham-operated and control ones. The differential sensitivity observed in our model of highly proliferating normal tissue to SeMet, compared with the reported anticancer activity of this molecule is discussed.     

The control of nucleic acid and nicotinamide nucleotide synthesis in regenerating rat liver

1. The effect of injecting nicotinamide on the incorporation of [(14)C]orotate into the hepatic nucleic acids of rats after partial hepatectomy was investigated. 2. At 3h after partial hepatectomy the rapid incorporation of [(14)C]orotate into RNA, and at 20h after partial hepatectomy the incorporation of [(14)C]orotate into both RNA and DNA, were inhibited in a dose-dependent fashion by the previous injection of nicotinamide. 3. The injection of nicotinamide at various times before the injection of [(14)C]orotate at 20h after partial hepatectomy revealed an inhibition of the incorporation of orotate into RNA and DNA which was non-linear with respect to the duration of nicotinamide pretreatment. 4. The induction of a hepatic ATP depletion by ethionine demonstrated that the synthesis of hepatic NAD and NADP in partially hepatectomized rats was more susceptible to an ATP deficiency than in control rats. 5. The total hepatic activity of ribose phosphate pyrophosphokinase (EC 2.7.6.1) was assayed at various times after partial hepatectomy and found to be only marginally greater than the maximum rate of hepatic NAD synthesis induced in vivo by nicotinamide injection between 12 and 24h after partial hepatectomy. 6. It is suggested that a competition exists between NAD synthesis and purine and pyrimidine nucleotide synthesis for available ATP and particularly 5-phosphoribosyl 1-pyrophosphate. In regenerating liver the competition is normally in favour of the synthesis of nucleic acid precursors, at the expense of NAD synthesis. This situation may be reversed by the injection of nicotinamide with a subsequent inhibition of nucleic acid synthesis.     

Nicotinamide nucleotide synthesis in regenerating rat liver

1. The concentrations and total content of the nicotinamide nucleotides were measured in the livers of rats at various times after partial hepatectomy and laparotomy (sham hepatectomy) and correlated with other events in the regeneration process. 2. The NAD content and concentration in rat liver were relatively unaffected by laparotomy, but fell to a minimum, 25 and 33% below control values respectively, 24h after partial hepatectomy. NADP content and concentration were affected similarly by both laparotomy and partial hepatectomy, falling rapidly and remaining depressed for up to 48h. 3. The effect of injecting various doses of nicotinamide on the liver DNA and NAD 18h after partial hepatectomy was studied and revealed an inverse correlation between NAD content and DNA content. 4. Injections of nicotinamide at various times after partial hepatectomy revealed that the ability to synthesize NAD from nicotinamide was impaired during the first 12h, rose to a peak at 26h and fell again by 48h after partial hepatectomy. 5. The total liver activity of NAD pyrophosphorylase (EC 2.7.7.1) remained at or slightly above the initial value for 12h after partial hepatectomy and then rose continuously until 48h after operation. The activity of NMN pyrophosphorylase (EC 2.4.2.12) showed a similar pattern of change after partial hepatectomy, but was at no time greater than 5% of the activity of NAD pyrophosphorylase. 6. The results are discussed with reference to the control of NAD synthesis in rapidly dividing tissue. It is suggested that the availability of cofactors and substrates for NAD synthesis is more important as a controlling factor than the maximum enzyme activities. It is concluded that the low concentrations of nicotinamide nucleotides in rapidly dividing tissues are the result of competition between NAD synthesis and nucleic acid synthesis for common precursor and cofactors.     

Protein,nucleic acids and cell structure in the regenerating mouse liver

Summary Prior to the onset of mitotic activity in the regenerating mouse liver, the concentrations of total protein and DNA, but not of RNA, decreased to 93 per cent of their levels in normal liver. During maximum mitosis (48–72 hours), the DNA and RNA concentrations were 110 per cent of their normal levels. The concentrations of liver nucleic acids 24 hours after a sham-operation were also about 110 per cent of normal values.Increased concentrations of insoluble protein were found at 12, 24 and 144 hours after partial hepatectomy. This may reflect an increase in the stability of the liver cell membranes during the regenerative period. Increased amounts of various cytoplasmic membranes were indicated by electronmicroscopy and may also have contributed to the increase in insoluble protein.A temporary increase in the measured solubility of the liver protein occurred during maximum mitosis. It is suggested that this was due to the association of protein with lipids during the depletion of accumulated fat from the regenerating liver.This investigation was facilitated by grants from the Royal. Physiographical Society.I wish to thank Professor I. Agrell and Docent B. Karlsson for helpful advice and criticism and Miss E. K. Persson for skilled technical assistance.     

Regulation of heme synthesis in the regenerating rat liver

This investigation shows that the regulation of heme synthesis in the regenerating rat liver does not differ from the regulation in the normal liver. The heme saturation of tryptophan pyrrolase was found to be low, indicating a reduced concentration of heme in the regulatory heme pool of the regenerating rat liver. As expected, ALAS in the mitochondrial fraction was found to be elevated. It was also shown that ALAS in the regenerating rat liver can be induced by the porphyrinogenic drugs AIA and DDC and that heme reduces its activity. The decrease observed in the activity of cytosolic ALAS might be due to impaired synthesis of the enzyme but does not affect the regulation of the heme biosynthetic pathway.     

Effect of an exogenous energy source and amino acids on DNA synthesis in regenerating rat liver

Rats on a protein-free diet synthesized less DNA after partial hepatectomy than rats on a normal diet. In regenerating livers of animals on the protein-free diet, induction of several enzymes involved in the DNA precursor synthetic pathway, and especially ribonucleotide reductase, were depressed. When young rats were maintained solely by parenteral nutrition after partial hepatectomy, exogenous amino acids were more important than the exogenous energy source for induction of enzymes involved in synthesis of DNA and its pyrimidine nucleotide precursors. In particular, induction of ribonucleotide reductase appeared to be controlled by exogenous amino acids. Tryptophan, methionine, phenylalanine, leucine, valine, isoleucine and threonine seemed to stimulate the induction of this enzyme most after partial hepatectomy.     

Stimulation of ornithine decarboxylase synthesis and its control by polyamines in regenerating rat liver and cultured rat hepatoma cells.

Ornithine decarboxylase has been induced in log phase hepatoma cells grown in suspension culture. Induction with N6, O2'-dibutyryl cyclic adenosine 3':5'-monophosphate produced a 4-fold increase in enzyme activity by 3 hours which was followed by a return to base levels by 6 hours. Induction with dexamethasone, a potent synthetic glucocorticoid, exhibited a slow steady rate of increase in enzyme activity, reaching a plateau level of approximately 5- to 6-fold stimulation by about 12 hours. Induced cell and regenerating rat liver ornithine decarboxylase were shown to be indistinguishable by titration with antibody monospecific to the latter and by heat stability. L-[14C]Leucine incorporation into immunoprecipitable enzyme protein after induction in vitro or partial hepatectomy showed an increase which, when coupled with the increase in enzymatic activity, indicated de novo synthesis of enzyme protein. Physiological concentrations of the naturally occurring polyamines, spermidine and spermine, abolish cyclic AMP induction whereas they have no effect on dexamethasone induction. Both inductions were abolished by cycloheximide; in contrast, inhibition by actinomycin D was complete for dexamethasone induction and only partial with respect to cyclic AMP induction. The different time pattern of induction seen with cyclic AMP and dexamethasone, the partial inhibition of the cyclic AMP induction seen with actinomycin D, as well as the absence of inhibition of the dexamethasone induction by polyamines, indicate that these inducers might affect different aspects of the control of the same enzyme.