Combined regulation of ornithine and S-adenosylmethionine decarboxylases by spermine and the spermine analogue N1 N12-bis(ethyl)spermine.

Glycogenolysis was studied in glycogen-rich perfused livers in which glycogen phosphorylase was fully converted into the a form by exposure of the livers to dibutyryl cyclic AMP. We monitored intracellular Pi by 31P n.m.r. Perfusion with Pi-free medium during 30 min caused a progressive decrease of the Pi signal to 50% of its initial value. In contrast, exposure of the livers to KCN and/or 2,4-dinitrophenol resulted in a rapid doubling of the Pi signal. Alterations in the intracellular Pi coincided with proportional changes in the rate of hepatic glycogenolysis (measured as the output of glucose plus lactate). The results indicate that the rate of glycogenolysis catalysed by phosphorylase a depends linearly on the hepatic Pi concentration. Hence the Km of phosphorylase a for its substrate Pi must be considerably higher than the concentrations that occur in the cytosol, even during hypoxia.     

Hypertension in normotensive and hypertensive rats by spermine ingestion

Polyamines (putrescine, spermidine and spermine) play an important role in the development of hypertension and in the expression of atrial natriuretic peptide (ANP), a cardiac hormone involved in the regulation of blood pressure. Wistar Kyoto normotensive (WKY) and spontaneously hypertensive rats (SHR) were given spermine in drinking water (0.5%) for 15 days. The spermine intake elevated the blood pressures of both SHR and WKY rats and reduced the expression of ANP (Northern blotting) in the ventricles. ANP levels in the plasma determined by enzyme immunoassay (EIA) showed no changes in the levels of plasma ANP after spermine intake. An analysis of polyamines by high-pressure liquid chromatography showed that the levels of spermine and spermidine were elevated in SHR hearts. It was in SHR hearts alone that spermine intake was associated with increases in the levels of putrescine. The results suggest that spermine-induced increases in blood pressure may involve mechanisms other than ANP.     

Mechanism of the inhibition of cell growth by N1,N12-bis(ethyl)spermine.

The mechanism of the antiproliferation effect of N1,N12-bis(ethyl)spermine (BESPM) was studied in detail using mouse FM3A cells, since this polyamine analogue mimics the functions of spermine in several aspects [Igarashi, K., Kashiwagi, K., Fukuchi, J., Isobe, Y., Otomo, S. & Shirahata, A. (1990) Biochem. Biophys. Res. Commun. 172, 715-720]. Our results indicate that not only the decrease in sperimine and spermine caused by BESPM but also its accumulation play important roles on the inhibition of cell growth by BESPM, since BESPM accumulated in cells at a concentration fivefold that of spermidine in control cells. In comparison with the polaymine-deficient cells caused by alpha-difluoromethylornithine, an inhibitor of ornithine decarboxylase, and ethylglyoxal bis(guanylhydrazone), an inhibitor of S-adenosylmethionine decarboxylase, the behavior of polyamine-deficient cells caused by BESPM was different as follows: the inhibition of cell growth by BESPM was not abrogated by spermine or spermidine; polyamine uptake, which is stimulated during polyamine deficiency, was greatly inhibited, while spermidine/spermine N1-acetyltransferase activity, which is inhibited during polyamine deficiency, was enhanced in BESPM-treated cells; thymidine kinase activity did not decrease in BESPM-treated cells; inhibition of cell growth and macromolecule synthesis by BESPM correlated with the swelling of mitochondria and the decrease in ATP content; BESPM caused cell death when incubated together for several days. The role of BESPM accumulation on inhibition of cell growth is discussed.     

Induction of spermidine/spermine N1-acetyltransferase in rat tissues by polyamines.

Treatment of rats with spermidine, spermine or sym-norspermidine led to a substantial induction of spermidine/spermine N1-acetyltransferase activity in liver, kidney and lung. The increase in this enzyme, which was determined independently of other acetylases by using a specific antiserum, accounted for all of the increased acetylase activity in extracts from rats treated with these polyamines. Spermine was the most active inducer, and the greatest effect was seen in liver. Liver spermidine/spermine N1-acetyltransferase activity was increased about 300-fold within 6 h of treatment with 0.3 mmol/kg doses of spermine; activity in kidney increased 30-fold and activity in the lung 15-fold under these conditions. The increased spermidine/spermine N1-acetyltransferase activity led to a large increase in the liver putrescine content and a decline in spermidine. These changes are due to the oxidation by polyamine oxidase of the N1-acetylspermidine formed by the acetyltransferase. Our results indicated that spermidine was the preferred substrate in vivo of the acetylase/oxidase pathway for the conversion of the higher polyamines into putrescine. The induction of the spermidine/spermine N1-acetyltransferase by polyamines may provide a mechanism by which excess polyamines can be removed.     

Modulation of indomethacin-induced gastric injury by spermine and taurine in rats

This study investigated the involvement of neutrophil infiltration, nitric oxide (NO) generation, and oxidative stress in indomethacin-induced ulcer and the possible gastroprotective potentials of spermine and taurine, known for their tissue regenerating and antioxidant effects, respectively. Male Wistar albino rats (180-220 g) were allocated into a normal control group, ulcer control group (received a single dose of indomethacin 40 mg-kg p.o.), and two ulcer groups pretreated with spermine (150 mg-kg p.o. 1 h before ulcer induction) and taurine (250 mg-kg i.p. for three consecutive days before ulcer induction). The animals were killed 6 h after indomethacin administration, and the gastric juice, serum, and mucosal tissue were used for gastric injury evaluation. Both modulators significantly ameliorated the indomethacin-induced gastric lesions in glandular mucosa. Notably, spermine exhibited the most pronounced effect as manifested by great reduction in the gastric ulcer index, normalization of the elevated gastric acidity, and triggering of mucin production. Spermine and taurine were able to decrease the elevated levels of gastric myeloperoxidase, conjugated diene, and serum NO. However, the lowered tissue NO content was markedly elevated only by taurine. The antioxidant action of taurine was illustrated by restoration of the depressed content of glutathione, normalization of the inhibited activities of glutathione reductase, and superoxide dismutase. These results suggest that spermine and taurine confer significant gastroprotection against indomethacin-induced gastric injury with the priority of spermine.     

Enhancement of intestinal absorption of macromolecules by spermine in rats

Summary. The aim of this study was to investigate the enhancing effect of polyamines on intestinal absorption of fluorescein isothiocyanate-labeled dextran (MW 4400, FD-4) in the in situ loop study and in vivo oral absorption study. Absorption of FD-4 from the jejunum was significantly enhanced by 5 mM spermine without serious membrane damage in the jejunum. An in vivo oral absorption study was also performed, and plasma FD-4 levels increased significantly after co-administration of 30 mM spermine. In the in vitro transport studies with Caco-2 cells, prolonged incubation with spermine resulted in a gradual decrease in transepithelial electrical resistance. This finding suggests that the absorption-enhancing mechanism of spermine partly includes opening the tight junctions of the epithelium via the paracellular route. These results indicate that excess oral ingestion of polyamines may have widespread health effects via the modulation of the intestinal epithelial barrier function.     

Regulation of spermidine/spermine N1-acetyltransferase in L6 cells by polyamines and related compounds.

Exposure of rat L6 cells in culture to exogenous polyamines led to a very large increase in the activity of spermidine/spermine N1-acetyltransferase. Spermine was more potent than spermidine in bringing about this increase, but in both cases the elevated acetyltransferase activity increased the cellular conversion of spermidine into putrescine. The N1-acetyltransferase turned over very rapidly in the L6 cells, with a half-life of 9 min after spermidine and 18 min after spermine. A wide variety of synthetic polyamine analogues also brought about a substantial induction of spermidine/spermine N1-acetyltransferase activity. These included sym-norspermidine, sym-norspermine, sym-homospermidine, N4-substituted spermidine derivatives, 1,3,6-triaminohexane, 1,4,7-triaminoheptane and deoxyspergualin, which were comparable with spermidine in their potency, and N1N8-bis(ethyl)spermidine, N1N9-bis(ethyl)homospermidine, methylglyoxal bis(guanylhydrazone), ethylglyoxal bis(guanylhydrazone) and 1,1'-[(methylethanediylidene)dinitrilo]bis(3-amino-guanidine ), which were even more active than spermidine. It is suggested that these polyamine analogues may bring about a decrease in cellular polyamines not only by inhibiting biosynthesis but by stimulating the degradation of spermidine into putrescine.     

Metabolism of N-alkylated spermine analogues by polyamine and spermine oxidases

N-alkylated polyamine analogues have potential as anticancer and antiparasitic drugs. However, their metabolism in the host has remained incompletely defined thus potentially limiting their utility. Here, we have studied the degradation of three different spermine analogues N,N′-bis-(3-ethylaminopropyl)butane-1,4-diamine (DESPM), N-(3-benzyl-aminopropyl)-N′-(3-ethylaminopropyl)butane-1,4-diamine (BnEtSPM) and N,N′-bis-(3-benzylaminopropyl)butane-1,4-diamine (DBSPM) and related mono-alkylated derivatives as substrates of recombinant human polyamine oxidase (APAO) and spermine oxidase (SMO). APAO and SMO metabolized DESPM to EtSPD [K _m(APAO) = 10 μM, k _cat(APAO) = 1.1 s⁻¹ and K _m(SMO) = 28 μM, k _cat(SMO) = 0.8 s⁻¹, respectively], metabolized BnEtSPM to EtSPD [K _m(APAO) = 0.9 μM, k _cat(APAO) = 1.1 s⁻¹ and K _m(SMO) = 51 μM, k _cat(SMO) = 0.4 s⁻¹, respectively], and metabolized DBSPM to BnSPD [K _m(APAO) = 5.4 μM, k _cat(APAO) = 2.0 s⁻¹ and K _m(SMO) = 33 μM, k _cat(SMO) = 0.3 s⁻¹, respectively]. Interestingly, mono-alkylated spermine derivatives were metabolized by APAO and SMO to SPD [EtSPM K _m(APAO) = 16 μM, k _cat(APAO) = 1.5 s⁻¹; K _m(SMO) = 25 μM, k _cat(SMO) = 8.2 s⁻¹; BnSPM K _m(APAO) = 6.0 μM, k _cat(APAO) = 2.8 s⁻¹; K _m(SMO) = 19 μM, k _cat(SMO) = 0.8 s⁻¹, respectively]. Surprisingly, EtSPD [K _m(APAO) = 37 μM, k _cat(APAO) = 0.1 s⁻¹; K _m(SMO) = 48 μM, k _cat(SMO) = 0.05 s⁻¹] and BnSPD [K _m(APAO) = 2.5 μM, k _cat(APAO) = 3.5 s⁻¹; K _m(SMO) = 60 μM, k _cat(SMO) = 0.54 s⁻¹] were metabolized to SPD by both the oxidases. Furthermore, we studied the degradation of DESPM, BnEtSPM or DBSPM in the DU145 prostate carcinoma cell line. The same major metabolites EtSPD and/or BnSPD were detected both in the culture medium and intracellularly after 48 h of culture. Moreover, EtSPM and BnSPM were detected from cell samples. Present data shows that inducible SMO parallel with APAO could play an important role in polyamine based drug action, i.e. degradation of parent drug and its metabolites, having significant impact on efficiency of these drugs, and hence for the development of novel N-alkylated polyamine analogues.     

Stimulation of RNA polymerases I, II and III from rat liver by spermine, and specific inhibition of RNA polymerase I by higher spermine concentrations.

Spermine stimulates activities of higherly purified rat liver nuclear RNA olymerases I, II and III 3 to 4 fold. Inclusion of (NH4)2SO4 at concentrations required for maximal enzyme activities does not significantly enhance the degree of stimulation of polymerase activities by spermine, but maintains the stimulatory levels of enzymes over a broader range of spermine concentrations. The stimulatory effect of spermine at a concentration of 1 mM is a useful method for the elevation of activities of all RNA polymerases and thus provides a means to measure these enzymes when extracted from small quantities of tissues or cells. Based on the differential stimulation of the polymerases by spermine, a higher concentration of spermine (5 mM) can be selected to inhibit RNA polymerase I specifically.     

The crystal structure of spermidine/spermine N1-acetyltransferase in complex with spermine provides insights into substrate binding and catalysis

The enzyme spermidine/spermine N (1)-acetyltransferase (SSAT) catalyzes the transfer of acetyl groups from acetylcoenzyme A to spermidine and spermine, as part of a polyamine degradation pathway. This work describes the crystal structure of SSAT in complex with coenzyme A, with and without bound spermine. The complex with spermine provides a direct view of substrate binding by an SSAT and demonstrates structural plasticity near the active site of the enzyme. Associated water molecules bridge several of the intermolecular contacts between spermine and the enzyme and form a "proton wire" between the side chain of Glu92 and the N1 amine of spermine. A single water molecule can also be seen forming hydrogen bonds with the side chains of Glu92, Asp93, and the N4 amine of spermine. Site-directed mutation of Glu92 to glutamine had a detrimental effect on both substrate binding and catalysis and shifted the optimal pH for enzyme activity further into alkaline solution conditions, while mutation of Asp93 to asparagine affected both substrate binding and catalysis without changing the pH dependence of the enzyme. Considered together, the structural and kinetic data suggest that Glu92 functions as a catalytic base to drive an otherwise unfavorable deprotonation step at physiological pH.     

Activation of tubulinyl-tyrosine carboxypeptidase by spermine,spermidine and putrescine

Spermine, spermidine and putrescine produce dose dependent stimulation of the invitro tubulinyl-tyrosine carboxypeptidase. Maximal stimulation was obtained with spermine, spermidine or putrescine at 0.06 mM, 1 mM and 6 mM, respectively. At higher concentrations, the enzyme activity was inhibited. The enzyme was also activated by Mg⁺⁺; the concentration formaximal effect was 4–6 mM. The stimulation produced by optimal concentration of each amine was unaffected by Mg⁺⁺ up to 2 mM; higher concentration of Mg⁺⁺ showed inhibitory effect. At optimal Mg⁺⁺ concentration, the carboxypeptidase activity was inhibited by increasing amine concentration. The amines at 0.5 or 5 mM did not produce any effect on the incorporation of tyrosine catalyzed by tubulin tyrosine ligase.     

Induction of spermidine/spermine N1-acetyltransferase by methylglyoxal bis(guanylhydrazone)

The anti-tumor agent methylglyoxal bis(guanylhydrazone) was found to be a competitive inhibitor of spermidine/spermine N1-acetyltransferase with a Ki of about 8 microM. Treatment of rats with this drug lead to a very large increase in the total amount of spermidine/spermine N1-acetyltransferase in liver, kidney and spleen. The total increase as measured using a specific antiserum amounted to 700-fold in liver and 100-fold in kidney within 18 h of treatment with 80 mg/kg doses. At least part of this induction was due to a pronounced increase in the half-life of the acetyltransferase which increased from 15 min to more than 12 h. The very large increase in the amount of the enzyme is likely to overwhelm the direct inhibition, and a net increase in the acetylation of polyamines by this enzyme would be expected to occur after treatment with methylglyoxal bis(guanylhydrazone). The acetylated polyamines are known to be rapidly degraded by polyamine oxidase producing putrescine. Direct evidence that a substantial part of the increase in the content of putrescine in the liver of rats treated with methylglyoxal bis(guanylhydrazone) occurs via the induction of this acetylase/oxidase pathway was obtained. These results indicate that methylglyoxal bis(guanylhydrazone) affects cellular polyamine levels not only by means of its inhibitory effect on S-adenosylmethionine decarboxylase and diamine oxidase but also by the induction of spermidine/spermine N1-acetyltransferase. They also raise the possibility that the enormous increase in this enzyme which occurs with higher doses may contribute to the very severe toxicity of methylglyoxal bis(guanylhydrazone).     

Studies of the specificity and kinetics of rat liver spermidine/spermine N1-acetyltransferase.

The substrate specificity and kinetic mechanism of spermidine N1-acetyltransferase from rat liver was investigated using a highly purified (18 000-fold) preparation from the livers of rats in which the enzyme was induced by treatment with carbon tetrachloride (1.5 ml/kg body wt. 6h before death). The enzyme catalysed the acetylation of spermidine, spermine, sym-norspermidine, sym-norspermine, N-(3-aminopropyl)-cadaverine, N1-acetylspermine, 3,3'-diamino-N-methyldipropylamine and 1,3-diaminopropane, but was inactive with putrescine, cadaverine, sym-homospermidine and N1-acetylspermidine. These results suggest that the enzyme is highly specific for the acetylation of a primary amino group that is separated by a three-carbon aliphatic chain from another nitrogen atom (i.e. the substrates are of the type H2N[CH2]3NHR). The maximal rates of acetylation of 1,3-diaminopropane and 3,3'-diamino-N-methyldipropylamine were much lower than the maximal rates with spermidine or sym-norspermidine as substrates, suggesting a preference for a secondary amino group bearing the aminopropyl group that is acetylated. The best substrates for acetylation were sym-norspermidine and sym-norspermine, which had Km values of about 10 micrograms and Vmax. values of about 2 mumol of product/min per mg of enzyme compared with Km of 130 microM and Vmax. of 1.3 mumol/min per mg for spermidine. N1-Acetylspermidine (the product of the reaction) and N8-acetylspermidine were weak inhibitors and were competitive with spermidine, having Ki values of about 6.6 mM and 0.4 mM respectively. N1-Acetylspermidine was a non-competitive inhibitor with respect to acetyl-CoA. CoA was also inhibitory to the reaction, showing non-competitive kinetics when either [acetyl-CoA] or [spermidine] was varied. These results suggest that the reaction occurs via an ordered Bi Bi mechanism in which spermidine binds first and N1-acetyl-spermidine is the final product to be released.     

The effect of spermine on the disaccharidase activities in suckling rats of different age.

The influence of the age of the rat on the maturation of disaccharidase activities induced by spermine was studied. Three-day- and 9-day-old rats were used in the experiment. Spermine was administered orally or directly into the stomach using thin tubing daily for 3 days, and disaccharidase activities in the jejunum were measured. While spermine caused maturation of not only lactase activity but also maltase and sucrase activities in 9-day-old rats, it only caused maturation of lactase activity in 3-day-old rats. Histological studies showed no significant changes in the jejunum of 3-day-old rats treated with spermine.     

Induction of spermidine/spermine N1-acetyltransferase by parathyroid hormone in rabbit costal chondrocytes in culture

Parathyroid hormone (PTH) increased the activity of spermidine/spermine N1-acetyltransferase, a rate-limiting enzyme of polyamine biodegradation, in rabbit costal chondrocytes in culture. The enzyme activity increased in a dose-dependent manner after addition of PTH to the culture, reaching a maximum at 8 h. The increase in the enzyme activity was abolished by cycloheximide or actinomycin D. Dibutyryl cyclic AMP also induced the acetyltransferase to some extent. These results suggest that the induction of spermidine/spermine N1-acetyltransferase by PTH may play some significant role in the expression of the differentiated phenotype of chondrocytes.