Spermidine synthase is prominently expressed in the striatal patch compartment and in putative interneurones of the matrix compartment

The ubiquitous polyamines spermidine and spermine are known as modulators of glutamate receptors and inwardly rectifying potassium channels. They are synthesized by a set of specific enzymes in which spermidine synthase is the rate-limiting step catalysing the formation of the spermine precursor spermidine from putrescine. Spermidine and spermine were previously localized to astrocytes, probably reflecting storage rather than synthesis in these cells. In order to identify the cellular origin of spermidine and spermine synthesis in the brain, antibodies were raised against recombinant mouse spermidine synthase. As expected, strong spermidine synthase-like immunoreactivity was obtained in regions known to express high levels of spermidine and spermine, such as the hypothalamic paraventricular and supraoptic nuclei. In the striatum, spermidine synthase was found in neurones and the neuropil of the patch compartment (striosome) as defined by expression of the micro opiate receptor. The distinct expression pattern of spermidine synthase, however, only partially overlapped with the distribution of the products spermidine and spermine in the striatum. In addition, spermidine synthase-like immunoreactivity was seen in patch compartment-apposed putative interneurones. These spermidine synthase-positive neurones did not express any marker characteristic of the major striatal interneurone classes. The neuropil labelling in the patch compartment and in adjacent putative interneurones may indicate a role for polyamines in intercompartmental signalling in the striatum.     

Induction of angiogenesis in chick yolk-sac membrane by polyamines and its inhibition by tissue inhibitors of metalloproteinases (TIMP and TIMP-2).

Treatment of yolk-sac membranes of 4-day-old chick embryos with spermine or spermidine resulted in angiogenesis in the membranes. The angiogenic activity of spermine was stronger than that of spermidine. Putrescine, polylysine and histamine did not induce angiogenesis in the membranes. Administration of putrescine, spermidine and spermine increased their respective levels in yolk-sac membranes, but no interconversion of these amines was observed. The increases in spermidine and spermine levels in yolk-sac membranes preceded induction of angiogenesis. The angiogenesis induced by spermine was inhibited by tissue inhibitors of metalloproteinases, that is, TIMP and TIMP-2. These findings suggest that spermine and spermidine are angiogenesis factors in yolk-sac membranes of chick embryos and that matrix metalloproteinases represented by collagenase are involved in their action.     

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.     

Interconversion of putrescine,spermidine and spermine in goldfish and rat retina

Labelled putrescine is converted to spermidine and spermine in the retina of both the goldfish and of the rat, but the bulk remains as putrescine and spermidine in the goldfish retina whereas the bulk is present as spermine in the rat retina. Labelled spermidine is converted to spermine and to putrescine in the retina of both species, most remaining as spermidine in the goldfish retina whereas most is converted to spermine in the rat retina. Labelled spermine is converted to both spermidine and putrescine in the retina of both species with a greater conversion in the goldfish retina than in the rat retina. These results provide direct evidence of the interconversion of putrescine, spermidine and spermine in neural tissue from both fish and mammals and suggest that spermine should not be regarded solely as an end-product of putrescine metabolism but also as a source of spermidine and putrescine.The pattern of distribution of putrescine and the polyamines, spermidine and spermine, in goldfish retina is the reverse of that in rat retina: Putrescine is the most abundant in goldfish retina whereas spermine is most abundant in rat retina suggesting that the individual polyamines are of different importance in the two species.     

Effect of N1,N12-bis(ethyl)spermine and related compounds on growth and polyamine acetylation, content, and excretion in human colon tumor cells

Exposure of human colon tumor (HT 29 cells) to N1,N12-bis(ethyl)spermine and analogs produced a rapid loss of intracellular polyamines. This loss was brought about predominantly by an increased excretion of spermidine. N1,N11-Bis(ethyl)norspermine and N1,N12-Bis(ethyl)spermine were potent inducers of spermidine/spermine N1-acetyltransferase, and this induction facilitated the efflux of polyamines by enhancing the conversion of spermine into spermidine. N1,N14-Bis(ethyl)homospermine, which did not induce spermidine/spermine N1-acetyltransferase, also caused the loss of spermidine from the cell but was less effective in bringing about the decline in intracellular spermine. These results indicate that cellular polyamine levels can be regulated by excretion of spermidine and that the bis(ethyl)spermine derivatives deplete intracellular polyamine content by interference with this process.     

Studies of the induction of spermidine/spermine N1-acetyltransferase using a specific antiserum

A specific antiserum to rat liver spermidine/spermine N1-acetyltransferase was used to study the induction of this protein. The antiserum had no effect on the spermidine acetylating capacity of crude nuclear extracts and very little effect on the activity present in crude cytosolic extracts from control rat tissues indicating that most of this activity is not due to spermidine/spermine N1-acetyltransferase. Treatment of rats with carbon tetrachloride, spermidine, thioacetamide, or methylglyoxal bis(guanylhydrazone) produced a substantial increase in the spermidine acetylating capacity of rat liver cytosolic extracts which was exclusively due to an increase in the immunoprecipitable spermidine/spermine N1-acetyltransferase protein. Exact measurement of the extent of this increase was not possible because the basal amount was too low to determine precisely but the amount of this enzyme increased about 250-fold with 6 h of treatment with carbon tetrachloride, about 25-fold at 6 h after spermidine, about 23-fold at 24 h after thioacetamide and up to 300-fold at 24 h after methylglyoxal bis(guanylhydrazone). Treatment of rats with spermidine also increased spermidine/spermine N1-acetyltransferase in other tissues including lung, kidney, and pancreas. The spermidine/spermine N1-acetyltransferase protein was found to turn over very rapidly with a half-life of about 15 min in thioacetamide-treated rats and 180 min after carbon tetrachloride.     

Kinetic studies on inhibition of aminopropyltransferases by aurintricarboxylic acid in vitro

Activities of aminopropyltransferases (spermidine synthase and spermine synthase) were inhibited by aurintricarboxylic acid (ATA). Spermidine synthase was slightly more sensitive to the inhibitor than spermine synthase. These inhibitions were not prevented by 0.15 M NaCl. Inhibition by ATA of spermidine synthase was ‘uncompetitive’ with respect to putrescine and that of spermine synthase was ‘non-competitive’ with respect to spermidine. When the amount of spermidine synthase or spermine synthase was varied, inhibition ratio hardly changed on either case implying no appreciable interaction between ATA and these enzymes.     

Tissue-specific changes in polyamine levels of neural tissue and fat body in adult crickets

The three major polyamines—putrescine, spermidine, and spermine—were studied and changes of their levels were examined in extracts of cerebral ganglia and fat body from adult Acheta domesticus. In nervous tissue, only spermidine and spermine were present and spermine was two- to three-fold more abundant than spermidine. The polyamine levels were high up to day 3, decreased on day 4, and then remained relatively unchanged up to day 10. The spermidine/spermine ratios decreased during the imaginal life. Higher spermidine titres were observed in the neural tissue of egg-laying females compared to virgin females. In the fat body, putrescine was detected together with spermidine and spermine. Spermidine and spermine levels were two-fold higher than putrescine. Fat body of virgin females contained two times more polyamines than male fat body. Low at emergence, spermidine and spermine concentrations peaked on days 2–3 only in females, and egg-laying was characterized by an increase of putrescine and spermidine titres. Starvation did not change polyamine contents, implying homeostatic regulation of the intracellular polyamine metabolism. These data showing tissue specific changes in polyamine levels during the imaginal life of Acheta domesticus point to the physiological importance of polyamines as possible intracellular regulators during adult insect development. © 1993 Wiley-Liss, Inc.     

THE DISTRIBUTION AND METABOLISM OF PUTRESCINE, SPERMIDINE AND SPERMINE INJECTED INTO THE CEREBRAL VENTRICLES OF RABBITS

Following the intracerebroventricular injection into rabbits of spermidine or spermine the highest concentrations were initially found in the caudate nucleus, hypothalamus and medulla. Subsequently there was a rapid decline in the amounts present in the caudate nucleus and hypothalamus and, particularly in the case of spermidine, an increase in the conccntration in the lower brain stem and cervical cord. This pattern of changes is consistent with the amines being redistributed by passage in CSF. Intraventricularly injected putrescine followed the same initial distribution pattern but within 2 days it had been largely converted to spermidine and spermine. Synthesized polyamines accumulated in all the regions examined. The time course of synthesis indicated that spermidine was the precursor of spermine. Spermine was also formed from injected spermidine and vice-versa. These findings concur with the pharmacological and neurotoxic actions of putrescine, spermidine and spermine.     

N1-monoacetylation abolishes the inhibitory effect of spermine and spermidine in the reticulocyte lysate translation system

At optimum magnesium, the translation of rat heart mRNA in the nuclease treated rabbit reticulocyte lysate system was inhibited by low concentrations of spermidine or spermine but not of putrescine. Spermidine and spermine cause a general reduction in the translation of all the heart mRNAs since no differential effects were observed when the translation products were examined by gel electrophoresis. Spermine was a five times more potent inhibitor than spermidine but no inhibition was obtained with N1-acetylspermidine or N1-acetylspermine. Since analyses of endogenous polyamines demonstrate that the inhibitory concentrations of spermine could be obtained by converting a small fraction of the endogenous spermidine to spermine, these results indicate that interconversions of the polyamines might be a sensitive regulatory mechanism for protein synthesis.     

Androgenic control of polyamine concentrations in rat epididymis.

Unilateral orchidectomy resulted in a significant decrease in tissue content of putrescine and polyamines. However, no differences were detected when the results were expressed in terms of ng g-1 tissue. At 48 h after bilateral orchidectomy, a significant decrease in putrescine content was observed, but spermidine and spermine content were unaffected. The observed decrease in putrescine was prevented by treatment with testosterone propionate, but neither spermidine nor spermine were affected. Bilateral orchidectomy resulted in a significant decrease in the tissue content of putrescine, spermidine and spermine after 7 days. Treatment with testosterone propionate increased the content of putrescine, spermidine and spermine in the epididymis by about 200%, 92% and 34%, respectively. When results were expressed as nmol g-1, a significant decrease after castration in putrescine and spermidine, but not in spermine, was observed. Treatment with testosterone propionate restored putrescine concentration, but had no effect on spermidine and spermine concentrations. In castrated rats treated with testosterone propionate, the anti-androgen flutamide abolished the effect of the androgen on putrescine and spermidine content, but there was no effect on spermine. Acetylputrescine was not detected in the epididymis, while acetylpolyamines were detected at much lower concentrations than polyamines. After bilateral orchidectomy there was a decrease in the tissue content of all acetylpolyamines and an increase in their tissue concentration. The effect of castration on acetylpolyamine content was reversed by testosterone propionate treatment. We conclude that an active synthesis of polyamines occurs in the rat epididymis, and that this process depends upon the androgen environment. Regulation of ornithine decarboxylase activity appears to be the main step that is controlled by androgens.     

Treatment with phorbol esters leads to spermine depletion and inhibition of DNA synthesis in phytohemagglutinin-activated bovine lymphocytes

Phorbol 12-myristate-13-acetate (PMA) inhibited an increase in [3H]thymidine incorporation induced by phytohemagglutinin (PHA) in cultured bovine lymphocytes. Cellular levels of putrescine increased in the presence of PHA and PMA but the levels of spermidine and spermine had decreased to the control levels by 40 h. In cells treated with PHA and PMA, the activity of spermidine/spermine N1-acetyltransferase, a rate-limiting enzyme in polyamine biodegradation, was stimulated synergistically. Phorbol esters with tumor-promoting ability also stimulated the enzyme activity and a reciprocal correlation between the enzyme activity and DNA synthesis was observed. Addition of spermine reversed the PHA- and PMA-induced inhibition of DNA synthesis but putrescine and spermidine failed to restore it. These results suggest that the enhancement of spermidine/spermine N1-acetyltransferase activity results in the depletion of intracellular spermine and a concomitant decrease in DNA synthesis.     

Fluctuations of unbound whole blood polyamine levels during the menstrual cycle.

Perchloric acid-extractable whole blood spermidine and spermine concentrations were determined over a 4-week period in three men, four women, and one ovariectomized woman. Individual male spermidine/spermine ratios showed little fluctuation and similar values were obtained for each of the three males studied. Male spermidine and spermine concentrations, although stable for each male, varied from one subject to the next. Individual female spermidine/spermine ratios, as well as individual concentrations, fluctuated substantially when compared to those valued obtained for males; female ratios appear to rise and fall as a function of the menstrual cycle. The spermidine/spermine ratios obtained from a normal female receiving oral contraceptive, as well as those from an ovariectomized female, were characteristic of values obtained form men. It is suggested that a sex-related hormone(s) influences sperimidine and spermine concentrations in females.     

The biochemistry, genetics, and regulation of polyamine biosynthesis in Saccharomyces cerevisiae

We have studied the enzymes and genes involved in the biosynthesis of putrescine, spermidine, and spermine in Saccharomyces cerevisiae. Mutants have been isolated with defects in the biosynthetic pathway as follows: spe10 mutants, deficient in ornithine decarboxylase, cannot make putrescine, spermidine, or spermine; spe2 mutants, lacking S-adenosylmethionine decarboxylase, cannot make spermidine or spermine; spe3 mutants, lacking putrescine aminopropyltransferase, cannot make spermidine or spermine; and spe4 and spe40 mutants, lacking spermidine aminopropyltransferase, contain no spermine and permit growth of spe10 mutants. Studies with these mutants have shown that in yeast: 1) polyamines are absolutely required for growth; 2) putrescine is formed only by decarboxylation or ornithine; 3) two separate aminopropyltransferases are required for spermidine and spermine synthesis; 4) spermine and spermidine are important in the regulation of ornithine decarboxylase and the amines exert this control by a posttranslational modification of the enzyme; and 5) spermidine or spermine is essential for sporulation of yeast and for the maintenance of the double-stranded RNA killer plasmid. Recent studies in amine-deficient mutants of Escherichia coli have shown an important role of the polyamines in protein synthesis in vivo.     

Changes in Polyamine Contents Development of the Frog, Microhyla ornata

Putrescine, spermidine and spermine levels were measured during development, metamorphosis and adult life of the frog, Microhyla ornata . Development of Microhyla was accompanied by high fluctuating levels of putrescine and spermidine with low and steady levels of spermine. Putrescine was the major polyamine during development from egg to mature tadpole. During metamorphosis both putrescine and spermidine decreased significantly; but the decrease in putrescine content was more rapid than that of spermidine. Thus, in the freshly metamorphosed frog, the concentration of spermidine exceeded that of putrescine. In most of the adult tissues also spermidine concentration was higher than putrescine and spermine. While the free form of putrescine and spermidine increased during early development of the fertilized egg to tadpole, the levels of protein conjugated polyamines decreased. In the free form, putrescine was the major polyamine while in the protein conjugated form spermidine concentration was higher than putrescine and spermine. Thus polyamine pattern is different in early development, during metamorphosis and in differentiated adult tissues of this frog. ∞-Difluoromethylornithine treatment at early blastula stage did not interfere with the normal development of Microhyla embryos.     

CLEARANCE OF THE POLYAMINES FROM THE PERFUSED CEREBROVENTRICULAR SYSTEM OF THE RABBIT

Abstract— The clearance of the polyamines spermidine and spermine from cerebrospinal fluid was investigated in the rabbit by ventriculocisternal perfusion. Clearance involved both saturable and nonsaturable uptake processes. The saturable component was a high affinity system with an affinity constant of 21 μ m for spermidine and 24 μ m for spermine. The clearance of spermidine was reduced by the presence of spermine and vice-versa. Other polyamine congeners also reduced spermidine and spermine clearance and it is suggested that the two polyamines share the same carrier. Evidence for concentrative uptake of polyamines into choroid plexus is presented and it is suggested that an active system may also transport polyamines into brain tissue. At high perfusion concentrations simple diffusion may also take place.     

Polyamine regulation of ornithine decarboxylase and its antizyme in intestinal epithelial cells

Ornithine decarboxylase (ODC) is feedback regulated by polyamines. ODC antizyme mediates this process by forming a complex with ODC and enhancing its degradation. It has been reported that polyamines induce ODC antizyme and inhibit ODC activity. Since exogenous polyamines can be converted to each other after they are taken up into cells, we used an inhibitor of S-adenosylmethionine decarboxylase, diethylglyoxal bis(guanylhydrazone) (DEGBG), to block the synthesis of spermidine and spermine from putrescine and investigated the specific roles of individual polyamines in the regulation of ODC in intestinal epithelial crypt (IEC-6) cells. We found that putrescine, spermidine, and spermine inhibited ODC activity stimulated by serum to 85, 46, and 0% of control, respectively, in the presence of DEGBG. ODC activity increased in DEGBG-treated cells, despite high intracellular putrescine levels. Although exogenous spermidine and spermine reduced ODC activity of DEGBG-treated cells close to control levels, spermine was more effective than spermidine. Exogenous putrescine was much less effective in inducing antizyme than spermidine or spermine. High putrescine levels in DEGBG-treated cells did not induce ODC antizyme when intracellular spermidine and spermine levels were low. The decay of ODC activity and reduction of ODC protein levels were not accompanied by induction of antizyme in the presence of DEGBG. Our results indicate that spermine is the most, and putrescine the least, effective polyamine in regulating ODC activity, and upregulation of antizyme is not required for the degradation of ODC protein.     

Feedback regulation of polyamine synthesis in Ehrlich ascites tumor cells. Analysis using nonmetabolizable derivatives of putrescine and spormine

Ornithine decarboxylase (ODC) is subject to feedback regulation by the polyamines. Thus, addition of putrescine, spermidine or spermine to cells causes inhibition of ODC mRNA translation. Putrescine and spermine are readily converted into spermidine. Therefore, it is conceivable that the inhibition of ODC synthesis observed in putrescine- and spermine-supplemented cells is instead an effect of spermidine. To examine this possibility we have used two analogs of putrescine and spermine, namely 1,4-dimethylputrescine and 5,8-dimethylspermine, which cannot be converted into spermidine. Both analogs were found to inhibit the incorporation of [³⁵S]methionine into ODC protein to approximately the same extent, suggesting that putrescine as well as spermine exert a negative feedback control of ODC mRNA translation in the cell. In addition to suppressing ODC synthesis, both analogs were found to increase the turnover rate of the enzyme. 5,8-Dimethylspermine caused a marked decrease in the activity of S-adenosylmethionine decarboxylase (AdoMetDC). This effect was not obtained with 1,4-dimethylputrescine, indicating that spermine, but not putrescien, exerts a negative control of AdoMetDC. Treatment with 1,4-dimethylputrescine caused extensive depletion of the cellular putrescine and spermidine content, but accumulation of spermine. 5,8-Dimethylspermine treatment, on the other hand, effectively depleted the spermine content and had less effect on the putrescine and spermidine content, at least initially. Nevertheless, the total polyamine content was more extensively reduced by treatment with 5,8-dimethylspermine than with 1,4-dimethylputrescine. Accordingly, only 5,8-dimethylspermine treatment exerted a significant inhibitory effect on Ehrlich ascites tumor cell growth.     

Characteristics of the polyamine transporter TPO1 and regulation of its activity and cellular localization by phosphorylation

The subcellular localization of the polyamine transporter TPO1 of Saccharomyces cerevisiae was determined by sucrose gradient centrifugation and indirect immunofluorescence microscopy. When expressed from a multi-copy vector, TPO1 was located mainly on the plasma membrane, but with some localization on the vacuolar membrane. Polyamine transport by TPO1 was dependent on pH. Uptake of spermidine and spermine occurred at alkaline pH (pH 8.0), whereas inhibition of spermidine uptake, but not spermine uptake, was observed at acidic pH (pH 5.0). This suggests that TPO1 catalyzes polyamine excretion at acidic pH, similar to the PotE transporter in Escherichia coli. Paraquat, a polyamine analogue, was excreted by TPO1 at a rate comparable with the excretion of spermidine (deduced from the inhibition of spermidine uptake) at pH 5.0. However, excretion of preloaded radiolabeled spermidine and spermine was not observed in intact cells, suggesting that preloaded spermidine (or spermine) exists mainly as spermidine (or spermine)-ribosome complex in cells. The transport activity of TPO1 was enhanced through phosphorylation at Ser19 by protein kinase C and at Thr52 by casein kinase 1. Sorting of TPO1 from the endoplasmic reticulum to the plasma membrane was enhanced through phosphorylation at Ser342 by cAMP-dependent protein kinases 1 and 2.     

Feedback regulation of polyamine synthesis in Ehrlich ascites tumor cells. Analysis using nonmetabolizable derivatives of putrescine and spermine

Ornithine decarboxylase (ODC) is subject to feedback regulation by the polyamines. Thus, addition of putrescine, spermidine or spermine to cells causes inhibition of ODC mRNA translation. Putrescine and spermine are readily converted into spermidine. Therefore, it is conceivable that the inhibition of ODC synthesis observed in putrescine- and spermine-supplemented cells is instead an effect of spermidine. To examine this possibility we have used two analogs of putrescine and spermine, namely 1,4-dimethylputrescine and 5,8-dimethylspermine, which cannot be converted into spermidine. Both analogs were found to inhibit the incorporation of [35S]methionine into ODC protein to approximately the same extent, suggesting that putrescine as well as spermine exert a negative feedback control of ODC mRNA translation in the cell. In addition to suppressing ODC synthesis, both analogs were found to increase the turnover rate of the enzyme. 5,8-Dimethylspermine caused a marked decrease in the activity of S-adenosylmethionine decarboxylase (AdoMetDC). This effect was not obtained with 1,4-dimethylputrescine, indicating that spermine, but not putrescine, exerts a negative control of AdoMetDC. Treatment with 1,4-dimethylputrescine caused extensive depletion of the cellular putrescine and spermidine content, but accumulation of spermine. 5,8-Dimethylspermine treatment, on the other hand, effectively depleted the spermine content and had less effect on the putrescine and spermidine content, at least initially. Nevertheless, the total polyamine content was more extensively reduced by treatment with 5,8-dimethylspermine than with 1,4-dimethylputrescine. Accordingly, only 5,8-dimethylspermine treatment exerted a significant inhibitory effect on Ehrlich ascites tumor cell growth.