Isao Matsui

Effect of thioacetamide, growth hormone or partial hepatectomy on spermidine acetylase activity of rat liver cytosol.

Rat liver cytosol extracts catalyzed the formation of monoacetylspermidine when incubated with acetyl-CoA and spermidine. This activity was enhanced 15-fold by administration of thioacetamide (150 mg/kg). The peak of activity occurred 18-24 h after treatment with the drug and then declined reaching control levels by 76 h. Previous studies have shown that ornithine decarboxylase activity was also greatly increased over this time period. Putrescine content in the liver was increased 80-90-fold at 18-24 h and then declined. Spermidine levels were decreased significantly over the period 12-24 h after thioacetamide treatment and then increased substantially at later times. These results are consistent with the hypothesis that, at early times after administration of thioacetamide, the increase in putrescine content is brought about both by decarboxylation of ornithine and by degradation of monoacetylspermidine. Spermidine acetylase activity was also measured in liver extracts prepared after two other physiological stimuli known to enhance ornithine decarboxylase activity were used. Both growth hormone treatment and partial hepatectomy produced an early 2-3-fold increase in the cytosolic spermidine acetylase activity.

Increase in acetylation of spermidine in rat liver extracts brought about by treatment with carbon tetrachloride

Abstract Within three hours of the administration of hepatotoxic doses of carbon tetrachloride to rats there was a substantial increase in the ability of liver extracts to catalyze the accumulation of monoacetylspermidine when incubated with spermidine and acetyl-CoA. This increase was maximal by six hours and correlated with the period in which there was a pronounced fall in hepatic spermidine and concomitant increase in putrescine. During this time there is a large increase of the conversion of labeled spermidine into putrescine in the liver. These results therefore suggest that this conversion requires the prior acetylation of spermidine.

Formation and interconversion of putrescine and spermidine in mammalian cells

Recent findings on the control of putrescine and spermidine biosynthesis and interconversion in mammalian cells are described. 1. (a)|Ornithine decarboxylase, which is responsible for putrescine production, was also able to decarboxylase lysine but the Km for lysine was 100 times greater than the Km for ornithine rendering cadaverine production unlikely under physiological conditions. No other route to cadaverine was observed. Inactivation of ornithine decarboxylase by the suicide inhibitor, α-difluoromethylornithine, depleted SV-3T3 cells of putrescine and spermidine and prevented cell growth. Either putrescine, which was efficiently converted to spermidine, or cadaverine, which was converted to N-3-aminopropyl-cadaverine, was able to restore growth of these cells. 2. (b)|Exposure to exogeous putrescine and other diamines prevented the induction of ornithine decarboxylase activity in mouse fibroblasts. The transformed SV-3T3 cell line was less sensitive to the inhibition than the control 3T3 cells. This difference correlated with a greater rate of uptake in the serum-stimulated 3T3 cells but accumulation of the diamine from the medium could not entirely account for the difference in sensitivity of ornithine decarboxylate. Therefore, it appears that this control mechanism is in some way altered in the transformed cells permitting greater accumulation of putrescine and spermidine. 3. (c)|The rate limiting factor in the conversion of putrescine to spermidine is the supply of the tissue content of this nucleoside showed that it was present at levels of only 0.8–2.8 nmol/g. Production was regulated in liver after partial hepatectomy, in androgen stimulated ventral prostate and in cardiac hypertrophy via a change in the amount of S-adenosylmethionine decarboxylase protein and via changes in the content of putrescine which activates this enzyme. Inhibition of S-adenosylmethionine decarboxylase by 1,1′-[(methylethanediylidine)dinitrilo]-bis(3-aminoguanidine), an irreversible inactivation, prevented growth of SV-3T3 cells and led to an accumulator, or putrescine and decline in spermidine showing the essential role of this enzyme in spermidine production. Addition to spermidine reversed this growth inhibition. 4. (d)|A novel spermidine acetylase has been isolated from rat liver. This enzyme forms N1-acetylspermidine and is quite different from the previously decribed polyamine acetylase which is chromatn-bound and also acts on histones. The N1-acetylspermidine synthase was induced in rat liver by treatment with the hepatotoins, carbon tetrachloride or thioacetamide, and to a smaller extent by partial hepatectomy and treatment with growth hormone. This enzyme appears to play an essential role in the conversion of spermidine to putrescine indicating that the physiological substrate for polyamine oxidase under these conditions is N1-acetylspermidine. After treatment with carbon tetrachloride, there was a 40-fold rise in hepatic putrescine levels within six hours. The rise was not prevented when increased ornithine decarboxylase was completely inhibited by the presence of α-difluoromethylornithine. These results emphasize that putrescine accumulation can be achieved either via ornithine decarboxylase or via degradation of psermidine according to the circumstances.

Effect of inhibitors of protein synthesis on rat liver spermidine N1-acetyltransferase

The increase in spermidine N-acetyltransferase activity in rat liver produced by carbon tetrachloride was completely prevented by simultaneous treatment with inhibitors of protein and nucleic acid synthesis suggesting that the increase results from the synthesis of new protein rather than the release of the enzyme from a cryptic inactive form. Treatment with cycloheximide 2 h after carbon tetrachloride also completely blocked the rise in spermidine N-acetyltransferase seen 4 h later. Such treatment completely prevented the fall in spermidine and rise in putrescine in the liver 6 h after carbon tetrachloride confirming the importance of the induction of spermidine N-acetyltransferase in the conversion of spermidine into putrescine. When cycloheximide was administered to rats in which spermidine N-acetyltransferase activity had been stimulated by prior treatment with carbon tetrachloride or thioacetamide, the activity was lost rapidly showing that the enzyme protein has a rapid rate of turnover. The half-life for the enzyme in thioacetamide-treated rats was 40 min, whereas the half-life for ornithine decarboxylase (which is well known to turn over very rapidly) was 27 min. In carbon tetrachloride-treated rats the rate or protein degradation was reduced and the half-life of spermidine N-acetyltransferase was 155 min and that for ornithine decarboxylase was 65 min. It appears that three of the enzymes involved in the synthesis and interconversion of putrescine and spermidine namely, ornithine decarboxylase, S-adenosylmethionine decarboxylase and spermidine N-acetyltransferase have rapid rates of turnover and that polyamine levels are regulated by changes in the amount of these enzymes.

Inhibition of DNA synthesis by methylglyoxal bis(guanylhydrazone) during lymphocyte transformation

The phytohemagglutinin induced DNA synthesis in guinea pig lymph node cells was inhibited remarkably by methylglyoxal bis(guanylhydrazone). This inhibitory effect was dependent on the time of its addition to the lymph node cell culture after stimulation with phytohemagglutinin. If methylglyoxal bis(guanylhydrazone) was added 48 hr after the stimulation, no inhibition of DNA synthesis was observed. Exogenous spermidine added at an early time of cell culture reversed the inhibitory effect of methylglyoxal bis(guanylhydrazone). However, no reversion occurred when spermidine was added at a late time of the cell culture.

Induction of ornithine decarboxylase in guinea-pig lymphocytes and its relation to phospholipid metabolism

Treatment of lymphocytes with exogenous phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3.) derived from Clostridium perfringens at concentrations similar to those which induced ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) activity produced diacylglycerol and phosphatidate. A divalent cation ionophore, A23187, and phytohemagglutinin induced not only diacylglycerol formation, but also ornithine decarboxylase activity. Dibutyryl cAMP inhibited both diacylglycerol formation and ornithine decarboxylase induction to a similar extent in phytohemagglutinin-stimulated lymphocytes, but stimulated them somewhat in ionophore A23187-activated lymphocytes. This suggests that the activation of intracellular phospholipase C and the formation of diacylglycerol is involved in ornithine decarboxylase induction in lymphocytes.

Conversion of exogenous spermidine into putrescine after administration to rats

Administration of large, but non-toxic doses of spermidine (0.4-1.25 mmol/kg) led to a substantial increase in putrescine in liver, kidney and a number of other tissues including muscle. The increase in putrescine peaked at 6 h after treatment and was completely prevented by administration of cycloheximide 3 h after the spermidine suggesting that the induction of a new protein was required. This protein is likely to be spermidine N1-acetyltransferase which was induced by the treatment with spermidine and increased 3-4-fold in liver and kidney within 6 h. N1-Acetylspermidine was detected in tissues at this time after spermidine treatment and experiments in which labeled spermidine was given indicated that a substantial fraction of the administered spermidine was converted into N1-acetylspermidine and into putrescine. These results suggest that the rise in putrescine after spermidine treatment is brought about by the production of N1-acetylspermidine which is converted into putrescine by the action of polyamine oxidase. The limiting step in this conversion is the activity of the acetylase which is induced in response to the rise in spermidine content. The acetylase/oxidase pathway, therefore, provides a means by which polyamine levels can be regulated and excess polyamine disposed of.

Control of ornithine decarboxylase activity by cyclic nucleotides in the phytohemagglutinin induced lymphocyte transformation

Summary N2,O2′-Dibutyryl guanosine 3′:5′-cyclic monophosphate enhanced the ornithine decarboxylase activity which was induced during lymphocyte transformation by phytohemagglutinin, while N6,O2′-dibutyryl adenosine 3′:5′-cyclic monophosphate tended to inhibit it. The increased ornithine decarboxylase activity was not observed when N2,O2′-dibutyryl guanosine 3′:5′-cyclic monophosphate alone was added to the non-stimulated lymphocytes. In contrast to the dibutyryl derivative, guanosine 3′:5′-cyclic monophosphate did not show any effect on enzyme induction following phytohemagglutinin stimulation.

Induction of spermidine N1-acetyltransferase in rat kidney by treatment with folic acid

The polyamine biosynthetic pathway in mammalian cells is now firmly established (reviews [l-3]). Pntrescine is formed by the action of ornithine decarboxylase and is converted into spermidine by the addition of a propylamine group from decarboxylated S-adenosylmethionine in a reaction catalyzed by spermidine synthase. Spermme synthase then catalyzes an analogous reaction in which a second propylamine group is transferred from another molecule of decarboxylated S-adenosylmethionine to spermidine forming spermine. The sperm&dine synthase and spermine synthase reactions are essentially irreversible; however, when tracer amounts of labeled spermidine or spermine are administered there is conversion of the higher polyamines back into putrescine [4,5]. This reversal involves the sequential actions of two enzymes; spermidine/spermine N’acetyltransferase and polyamine oxidase [6-S]. The first of these enzymes produces the monoacetyl derivative from the polyamine and acetyl-CoA. With spermidine as substrate it forms exclusively the N’-acetylspermidine isomer [6]. The N’-acetylspermidine is then oxidized by polyamine oxidase to form putrescine and N-acetyl3.aminopropionaldehyde [ 81. Spermine is symmetrical and only one isomer can be formed by acetylation of the terminal amino group [6]. The Ni-acetylspermine is also an excellent substrate for polyamine oxidase and is converted to spermidine and N-acetyl3aminopropionaldehyde [ 81. lated in the rat liver by the amount of the acetylase present since polyamine oxidase is present in much greater amounts and does not change substantially in response to stimuli which affect the interconversion [3,6,7,9]. We have detected large and rapid changes when acetylase has been measured in liver extracts prepared from rats pretreated with carbon tetrachloride [lo], thioacetamide [ 111, dialkylnitrosamines [ 121 and spermidine [ 121. Smaller but significant changes were observed in response to growth hormone and partial hepatectomy [ 111. In all of these cases, the enhanced acetylase activity correlated with increased conversion of spermidine into putrescine and it appears that the acetylase plays a critical role in regulation of intracellular polyamine levels. However, the induction of increased acetylase activity had only been observed in the liver and it was not known whether a similarly inducible enzyme occurs in other tissues or whether the acetylase/oxidase pathway for interconversion of polyamines was limited to liver. Administration of pharmacological amounts of folic acid to rats results in renal cell injury and subsequent hypertrophy and hyperplasia [ 13 ,141. This provides a useful model system in which to investigate biochemical changes in the kidney occurring at early stages in this process. Here, we show that the spermidine N’-acetyltransferase is induced in the kidney after treatment with folic acid and that this induction is responsible for an increase in putrescine content in this organ.

Effect of sodium butyrate on induction of ornithine decarboxylase activity in phytohemagglutinin-stimulated lymphocytes.

Effect of sodium butyrate on DNA synthesis and the induction of ornithine decarboxylase (EC 4.1.1.17), a rate-limiting enzyme of polyamine biosynthesis, was studied in phytohemagglutinin (PHA)-stimulated bovine lymphocytes. Millimolar concentrations of butyrate completely inhibited the incorporation of [3H]thymidine into the acid-insoluble fraction and reversibly suppressed the induction of ornithine decarboxylase. Other short-chain fatty acids were much less active than butyrate. These results suggest that the suppression of ornithine decarboxylase activity may be one of the reasons for the inhibition of DNA synthesis with butyrate in bovine lymphocytes, because our previous experimental results have shown that the induction of ornithine decarboxylase closely correlates with the DNA synthesis in growth-stimulated cells.