Short- and long-chain natural polyamines play specific roles in adult cricket neuroblast proliferation and neuron differentiation in vitro

In the house cricket (Acheta domesticus) mushroom bodies, neurogenesis still occurs during adulthood. Using in vitro approaches, the respective roles of natural polyamines in neurogenesis were examined. Mushroom body neuroblast proliferation was assayed in organotypic culture using 5-bromo, 2'-deoxyuridine labeling. The number of labeled cells was significantly increased when putrescine was added to culture medium, whereas spermidine and spermine supplementation did not alter cell proliferation. Conversely, in vitro morphometric studies on mushroom body neurons cultured in a defined medium showed that putrescine addition failed to alter any morphological character of these interneurons, whereas addition of the long-chain polyamines, spermidine and spermine, stimulated neuron differentiation. These two polyamines significantly increased total neurite length; moreover, spermidine-treated cells exhibited more branches than the controls. The present data demonstrate that putrescine has a mitogenic effect on mushroom body neuronal precursors, and that spermidine and spermine, which failed to induce neuroblast proliferation, act on neuronal differentiation, inducing neurite outgrowth. Our results indicate that short- and long-chain polyamines play specific roles during neurogenesis, and provide a basis for further studies on neuronal precursor proliferation and differentiation.     

Acrolein produced from polyamines as one of the uraemic toxins

It is well known that the addition of spermine or spermidine to culture medium containing ruminant serum inhibits cellular proliferation. This effect is caused by the products of oxidation of polyamines that are generated by serum amine oxidase. Among the products, we found that acrolein is a major toxic compound produced from spermine and spermidine by amine oxidase. We then analysed the level of polyamines (putrescine, spermidine and spermine) and amine oxidase activity in plasma of patients with chronic renal failure. It was found that the levels of putrescine and the amine oxidase activity were increased, whereas spermidine and spermine were decreased in plasma of patients with chronic renal failure. The levels of free and protein-conjugated acrolein were also increased in plasma of patients with chronic renal failure. An increase in putrescine, amine oxidase and acrolein in plasma was observed in all cases such as diabetic nephropathy, chronic glomerulonephritis and nephrosclerosis. These results suggest that acrolein is produced during the early stage of nephritis through kidney damage and also during uraemia through accumulation of polyamines in blood due to the decrease in their excretion into urine.     

Short‐ and long‐chain natural polyamines play specific roles in adult cricket neuroblast proliferation and neuron differentiation in vitro

In the house cricket (Acheta domesticus) mushroom bodies, neurogenesis still occurs during adulthood. Using in vitro approaches, the respective roles of natural polyamines in neurogenesis were examined. Mushroom body neuroblast proliferation was assayed in organotypic culture using 5‐bromo, 2′‐deoxyuridine labeling. The number of labeled cells was significantly increased when putrescine was added to culture medium, whereas spermidine and spermine supplementation did not alter cell proliferation. Conversely, in vitro morphometric studies on mushroom body neurons cultured in a defined medium showed that putrescine addition failed to alter any morphological character of these interneurons, whereas addition of the long‐chain polyamines, spermidine and spermine, stimulated neuron differentiation. These two polyamines significantly increased total neurite length; moreover, spermidine‐treated cells exhibited more branches than the controls. The present data demonstrate that putrescine has a mitogenic effect on mushroom body neuronal precursors, and that spermidine and spermine, which failed to induce neuroblast proliferation, act on neuronal differentiation, inducing neurite outgrowth. Our results indicate that short‐ and long‐chain polyamines play specific roles during neurogenesis, and provide a basis for further studies on neuronal precursor proliferation and differentiation. © 2001 John Wiley & Sons, Inc. J Neurobiol 48: 315–324, 2001     

Catabolism of polyamines

Summary. Owing to the establishment of cells and transgenic animals which either lack or over-express acetylCoA:spermidine N¹-acetyltransferase a major progress was made in our understanding of the role of polyamine acetylation. Cloning of polyamine oxidases of mammalian cell origin revealed the existence of several enzymes with different substrate and molecular properties. One appears to be identical with the polyamine oxidase that was postulated to catalyse the conversion of spermidine to putrescine within the interconversion cycle. The other oxidases are presumably spermine oxidases, because they prefer free spermine to its acetyl derivatives as substrate. Transgenic mice and cells which lack spermine synthase revealed that spermine is not of vital importance for the mammalian organism, but its transformation into spermidine is a vitally important reaction, since in the absence of active polyamine oxidase, spermine accumulates in blood and causes lethal toxic effects.Numerous metabolites of putrescine, spermidine and spermine, which are presumably the result of diamine oxidase-catalysed oxidative deaminations, are known as normal constituents of organs of vertebrates and of urine. Reasons for the apparent contradiction that spermine is in vitro a poor substrate of diamine oxidase, but is readily transformed into N⁸-(2-carboxyethyl)spermidine in vivo, will need clarification.Several attempts were made to establish diamine oxidase as a regulatory enzyme of polyamine metabolism. However, diamine oxidase has a slow turnover. This, together with the efficacy of the homeostatic regulation of the polyamines via the interconversion reactions and by transport pathways renders a role of diamine oxidase in the regulation of polyamine concentrations unlikely. 4-Aminobutyric acid, the product of putrescine catabolism has been reported to have antiproliferative properties. Since ornithine decarboxylase and diamine oxidase activities are frequently elevated in tumours, it may be hypothesised that diamine oxidase converts excessive putrescine into 4-aminobutyric acid and thus restricts tumour growth and prevents malignant transformation. This function of diamine oxidase is to be considered as part of a general defence function, of which the prevention of histamine and cadaverine accumulation from the gastrointestinal tract is a well-known aspect.     

Effects of polyamines on the functionality of photosynthetic membrane in vivo and in vitro

The three major polyamines are normally found in chloroplasts of higher plants and are implicated in plant growth and stress response. We have recently shown that putrescine can increase light energy utilization through stimulation of photophosphorylation [Ioannidis et al., (2006) BBA-Bioenergetics, 1757, 821-828]. We are now to compare the role of the three major polyamines in terms of chloroplast bioenergetics. There is a different mode of action between the diamine putrescine and the higher polyamines (spermidine and spermine). Putrescine is an efficient stimulator of ATP synthesis, better than spermidine and spermine in terms of maximal % stimulation. On the other hand, spermidine and spermine are efficient stimulators of non-photochemical quenching. Spermidine and spermine at high concentrations are efficient uncouplers of photophosphorylation. In addition, the higher the polycationic character of the amine being used, the higher was the effectiveness in PSII efficiency restoration, as well as stacking of low salt thylakoids. Spermine with 50 microM increase F(V) as efficiently as 100 microM of spermidine or 1000 microM of putrescine or 1000 microM of Mg(2+). It is also demonstrated that the increase in F(V) derives mainly from the contribution of PSIIalpha centers. These results underline the importance of chloroplastic polyamines in the functionality of the photosynthetic membrane.     

Polyamines are essential for the synthesis of 2-ricinoleoyl phosphatidic acid in developing seeds of castor

The major fatty acid component of castor (Ricinus communis L.) oil is ricinoleic acid (12-hydroxy-cis-9-octadecenoic acid), and unsaturated hydroxy acid accounts for >85% of the total fatty acids in triacylglycerol (TAG). TAG had a higher ricinoleate content at position 2 than at positions 1 and 3. Although lysophosphatidic acid (LPA) acyltransferase (EC 2.3.1.51), which catalyzes acylation of LPA at position 2, was expected to utilize ricinoleoyl-CoA preferentially over other fatty acyl-CoAs, no activity was found for ricinoleoyl-CoA in vitro at concentrations at which other unsaturated acyl-CoAs were incorporated rapidly. However, activity for ricinoleoyl-CoA appeared with addition of polyamines (putrescine, spermidine, and spermine), while polyamines decreased the rates of incorporation of other acyl-CoAs into position 2. The order of effect of polyamines on LPA acyltransferase activity was spermine > spermidine >> putrescine. At concentrations of spermine and spermidine of >0.1 mM, ricinoleoyl-CoA served as an effective substrate for LPA acyltransferase reaction. The concentrations of spermine and spermidine in the developing seeds were estimated at ∼0.09 and ∼0.63 mM, respectively. These stimulatory effects for incorporation of ricinoleate were specific to polyamines, but basic amino acids were ineffective as cations. In contrast, in microsomes from safflower seeds that do not contain ricinoleic acid, spermine and spermidine stimulated the LPA acyltransferase reaction for all acyl-CoAs tested, including ricinoleoyl-CoA. Although the fatty acid composition of TAG depends on both acyl-CoA composition in the cell and substrate specificity of acyltransferases, castor bean polyamines are crucial for incorporation of ricinoleate into position 2 of LPA. Polyamines are essential for synthesis of 2-ricinoleoyl phosphatidic acid in developing castor seeds.     

Polyamines inhibit apoptosis in porcine parthenotes developing in vitro

Polyamines, namely putrescine, spermidine, and spermine, are biogenic low-molecular-weight aliphatic amines which play essential roles in cell growth and proliferation. The aim of this study was to determine the effects of polyamines on the viability and development of porcine diploid parthenotes developing in vitro. The addition of 0.1 or 1.0 microM of putrescine, spermidine, or spermine, individually, to the culture medium did not enhance the development of 2-cell parthenotes to the blastocyst stage and did not change the total number of nuclei in the blastocysts. However, combined addition of these three compounds increased developmental rate to blastocyst and total cell numbers. Apoptosis in blastocyst stage parthenotes was decreased in the presence of exogenous polyamines. Real time PCR revealed that addition of polyamines to the culture media decreased the ratio of mRNA expression of Bak/Bcl-xL, Fas/Bcl-xL, and caspase 3, and enhanced mRNA expression of ornithine decarboxylase (ODC) and spermidine synthase, enzymes of polyamine biosynthesis. In the presence of L-alpha-difluoromethyl ornithine (an inhibitor of ODC) or cyclohexylamine (an inhibitor of spermidine synthase) development of porcine parthenotes decreased, apoptosis increased, and mRNA expression of the ratio of Bak/Bcl-xL and Fas/Bcl-xL, and caspase 3 increased. These results suggest that exogenous polyamines in the culture medium prevent apoptosis of porcine parthenotes and results in the net enhancement of porcine embryo viability.     

Transglutaminase activity is involved in polyamine-induced programmed cell death.

Natural polyamines, i.e., putrescine, spermidine, and spermine, are ubiquitous molecules essential for cell proliferation and differentiation. In the present study, the effect of polyamines on primary cultures of bovine aortic endothelial cells (BAECs), rat aortic smooth muscle cells (RASMCs), and a human melanoma cell line was examined. While in the absence of fetal calf serum (FCS) polyamines had no effect on viability, in the presence of FCS spermidine and spermine, at concentrations close to physiologic levels, induced a dose-dependent cell death, whereas putrescine was ineffective. RASMCs were significantly more sensitive than other cells. FACS analysis, oligo-nucleosome ELISA, Hoechst nuclear staining, and Annexin V-FITC quantification showed that cell death was likely due to apoptosis. Cells exposed to spermidine showed a marked increase of intracellular transglutaminase (TGase) activity ( approximately 30-fold over control). Inhibitors of polyamine oxidation or inhibitors of TGase activity prevented polyamine-induced apoptosis. Moreover, tissue TGase overexpression significantly increased cell sensitivity to polyamine, suggesting that this effect is likely related to enhanced intracellular TGase activity. These data indicate that polyamines may modulate cell viability through a novel TGase-dependent process.     

Oxidized polyamines and the growth of human vascular endothelial cells. Prevention of cytotoxic effects by selective acetylation.

The responses of human umbilical-vein vascular endothelial cells in culture to the naturally occurring polyamines spermine, spermidine and putrescine, their acetyl derivatives and oxidation products were examined. In the absence of human polyamine oxidase, exposure of cells to polyamines (up to 160 microM) had no adverse effects. In the presence of polyamine oxidase, spermine and spermidine were cytotoxic, but putrescine was not. Acetylation of the aminopropyl group of spermidine or both aminopropyl groups of spermine prevented this cytotoxicity. The amino acids corresponding to the polyamines, representing a further stage of oxidation, were also without effect. The cytotoxic effects were irreversible. Use of bovine serum amine oxidase in place of the human enzyme gave qualitatively similar results.     

Polyamines as modulators of salt tolerance in rice cultivars

The effect of NaCl on the endogenous levels of diamine, putrescine and polyamines, spermidine and spermine, was studied in the shoot system of salt-tolerant and salt-sensitive lines of rice (Oryza sativa L.) cultivars during three growth stages. Salt stress increased the levels of diamine and polyamine in varying degrees among nine rice cultivars investigated. Salt tolerant AU1, Co43, and CSC1 were effective in maintaining high concentrations of spermidine and spermine, while the content of putrescine was not significantly altered in all the growth stages when plants were exposed to salinity. The salt sensitivity in rice was associated with excessive accumulation of putrescine and with low levels of spermidine and spermine in the shoot system of salt-sensitive cultivars Co36, CSC2, GR3, IR20, TKM4, and TKM9 under saline condition. One of the possible mechanisms of saline resistance was observed to be due to the highly increased polyamines against the low increase in diamines. Alternatively, the salt sensitivity could be due to high increase of diamines and an incapacity to maintain high levels of polyamines.     

Regulation by polyamines of ornithine decarboxylase activity and cell division in the unicellular green alga Chlamydomonas reinhardtii

Polyamines are required for cell growth and cell division in eukaryotic and prokaryotic organisms. In the unicellular green alga Chlamydomonas reinhardtii, biosynthesis of the commonly occurring polyamines (putrescine, spermidine, and spermine) is dependent on the activity of ornithine decarboxylase (ODC, EC 4.1.1.17) catalyzing the formation of putrescine, which is the precursor of the other two polyamines. In synchronized C. reinhardtii cultures, transition to the cell division phase was preceded by a 4-fold increase in ODC activity and a 10- and a 20-fold increase, respectively, in the putrescine and spermidine levels. Spermine, however, could not be detected in C. reinhardtii cells. Exogenous polyamines caused a decrease in ODC activity. Addition of spermine, but not of spermidine or putrescine, abolished the transition to the cell division phase when applied 7 to 8 h after beginning of the light (growth) phase. Most of the cells had already doubled their cell mass after this growth period. The spermine-induced cell cycle arrest could be overcome by subsequent addition of spermidine or putrescine. The conclusion that spermine affects cell division via a decreased spermidine level was corroborated by the findings that spermine caused a decrease in the putrescine and spermidine levels and that cell divisions also could be prevented by inhibitors of S-adenosyl-methionine decarboxylase and spermidine synthase, respectively, added 8 h after beginning of the growth period. Because protein synthesis was not decreased by addition of spermine under our experimental conditions, we conclude that spermidine affects the transition to the cell division phase directly rather than via protein biosynthesis.     

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.     

Polyamines decrease Ca(2+) sensitivity of tension and increase rates of activation in skinned cardiac myocytes

Owing in part to their interactions with membrane proteins, polyamines (e.g., spermine, spermidine, and putrescine) have been identified as potential modulators of membrane excitability and Ca(2+) homeostasis in cardiac myocytes. To investigate whether polyamines also affect cardiac myofilament proteins, we assessed the effects of polyamines on contractility using rat myocytes and trabeculae that had been permeabilized with Triton X-100. Spermine, spermidine, and putrescine reversibly increased the [Ca(2+)] required for half-maximal tension (i.e., right-shifted tension pCa curves), with the following order of efficacy: spermine (+4) > spermidine (+3) > putrescine (+2). However, synthetic analogs that differed from spermine in charge distribution were not as effective as spermine in altering isometric tension. None of the polyamines had a significant effect on maximal tension, except at high concentrations. After flash photolysis of DM-Nitrophen (a caged Ca(2+) chelator), spermine accelerated the rate of tension development at low and intermediate but not high [Ca(2+)]. These results indicate that polyamines, especially spermine, interact with myofilament proteins to reduce apparent Ca(2+) binding affinity and speed cross-bridge cycling kinetics at submaximal [Ca(2+)].     

Polyamines and their biosynthetic enzymes in Ehrlich ascites-carcinoma cells. Modification of tumour polyamine pattern by diamines.

1. Ehrlich ascites-carcinoma cells contained relatively high concentrations of spermidine and spermine, but the putrescine content of the washed cells was less than 10% of that of higher polyamines. 2. Ascites-tumour cells likewise exhibited high activities of L-ornithine decarboxylase (EC 4.1.1.17), S-adenosyl-L-methionine decarboxylase (EC 4.1.1.50), spermidine synthase (EC 2.5.1.16) and spermine synthase. 3. During the first days after the inoculation, the polyamine pattern of the ascites cells was characterized by a high molar ratio of spermidine to spermine, which markedly decreased on aging of the cells. 4. Various diamines injected into mice bearing ascites cells rapidly and powerfully decreased ornithine decarboxylase activity in the carcinoma cells, apparently through a mechanism that was not a direct inhibition of the enzyme in vitro. Cadaverine (1,5-diaminopentane) and 1,6-diaminohexane were the most potent inhibitors of ornithine decarboxylase among the amines tested. 5. Chronic treatment of the mice with diamines resulted in a virtually complete disappearance of ornithine decarboxylase activity, and after 24h a significant decline in spermidine accumulation. 6. Cadaverine appeared to be an especially suitable compound for use as an inhibitor of the synthesis of higher polyamines, at least in Ehrlich ascites cells, since this diamine also acted as a competitive inhibitor for putrescine in the spermidine synthase reaction without being incorporated into the higher polyamines.     

Cellular content and biosynthesis of polyamines during rooster spermatogenesis.

The natural polyamines spermine and spermidine, and the diamine putrescine, were extracted from rooster testis cells separated by sedimentation at unit gravity, and from vas-deferens spermatozoa. The ratios spermine/DNA and spermidine/DNA were kept relatively constant throughout spermatogenesis, whereas the ratio putrescine/DNA rose in elongated spermatids. The cellular content of spermine, spermidine and putrescine decreased markedly in mature spermatozoa. Two rate-limiting enzymes in the biosynthetic pathway of polyamines, ornithine decarboxylase and S-adenosyl-L-methionine decarboxylase, showed their highest activities at the end of spermiogenesis and were not detectable in vas-deferens spermatozoa. A marked reduction in cell volume during spermiogenesis without a parallel decrease in the cellular content of polyamines suggests the possibility that the marked changes in chromatin composition and structure occurring in rooster late spermatids could take place in an ambience of high polyamine concentration.     

Prolactin stimulation of Nb 2 node lymphoma cell division is inhibited by polyamine biosynthesis inhibitors

The mitogenic action of prolactin in Nb 2 node lymphoma cells was inhibited by two drugs which interfere with polyamine biosynthesis. At concentrations of 0.5 mM and above alpha-difluoromethyl ornithine (DFMO), which inhibits ornithine decarboxylase and the conversion of ornithine to putrescine, significantly attenuated the mitogenic effect of prolactin. This inhibition was prevented by the addition of putrescine, spermidine, or spermine to the culture medium. At concentrations of 1 microM and above methylglyoxal bis(guanylhydrazone) (MGBG), which inhibits S-adenosylmethionine decarboxylase and hence the conversion of putrescine to spermidine and spermine, abolished the mitogenic action of prolactin. This inhibition was prevented by the addition of spermidine or spermine, but not putrescine, to the culture medium. These studies show that ongoing polyamine biosynthesis is essential for prolactin to express its mitogenic effect in this lymphoma cell line.     

Occurrence of uncommon polyamines in cultured tissues of maize

Summary The uncommon polyamines, norspermidine and norspermine, were detected in maizein vitro cultures of three different genotypes. The common polyamines, spermidine and spermine, along with the diamine, putrescine, were also observed. The total amounts of the uncommon polyamines, norspermidine and norspermine, were comparable to the total amounts of the common polyamines, spermidine and spermine, in the maize tissues. The titer for norspermidine was 6- to 15-fold greater than that of its common counterpart (spermidine) in the three genotypes. Norspermidine was the predominant polyamine among all triamines and tetramines detected in cell cultures of two of the three genotypes of maize examined and was predominant along with spermine in the third genotype. Enzyme assays performed with extracts from callus of one of the genotypes suggested a likely mechanism to account for the biosynthesis of the uncommon polyamines in cultured maize cells, through the actions of putrescine aminopropyltransferase, polyamine oxidase, and Schiff-base reductase/decarboxylase enzyme activities. This is the first report of the detection of uncommon polyamines in maize tissues, as well as the first report of these uncommon polyamines in a monocotyledonous plant.     

Effects of polyamines on the functionality of photosynthetic membrane in vivo and in vitro

The three major polyamines are normally found in chloroplasts of higher plants and are implicated in plant growth and stress response. We have recently shown that putrescine can increase light energy utilization through stimulation of photophosphorylation [Ioannidis et al., (2006) BBA-Bioenergetics, 1757, 821-828]. We are now to compare the role of the three major polyamines in terms of chloroplast bioenergetics. There is a different mode of action between the diamine putrescine and the higher polyamines (spermidine and spermine). Putrescine is an efficient stimulator of ATP synthesis, better than spermidine and spermine in terms of maximal % stimulation. On the other hand, spermidine and spermine are efficient stimulators of non-photochemical quenching. Spermidine and spermine at high concentrations are efficient uncouplers of photophosphorylation. In addition, the higher the polycationic character of the amine being used, the higher was the effectiveness in PSII efficiency restoration, as well as stacking of low salt thylakoids. Spermine with 50 μM increase F_V as efficiently as 100 μM of spermidine or 1000 μM of putrescine or 1000 μM of Mg²⁺. It is also demonstrated that the increase in F_V derives mainly from the contribution of PSIIα centers. These results underline the importance of chloroplastic polyamines in the functionality of the photosynthetic membrane.     

Fourier transform Raman study of the structural specificities on the interaction between DNA and biogenic polyamines

Biogenic polyamines putrescine, spermidine, and spermine are essential molecules for proliferation in all living organisms. Direct interaction of polyamines with nucleic acids has been proposed in the past based on a series of experimental evidences, such as precipitation, thermal denaturation, or protection. However, binding between polyamines and nucleic acids is not clearly explained. Several interaction models have also been proposed, although they do not always agree with one another. In the present work, we make use of the Raman spectroscopy to extend our knowledge about polyamine-DNA interaction. Raman spectra of highly polymerized calf-thymus DNA at different polyamine concentrations, ranging from 1 to 50 mM, have been studied for putrescine, spermidine, and spermine. Both natural and heavy water were used as solvents. Difference Raman spectra have been computed by subtracting the sum of the separated component spectra from the experimental spectra of the complexes. The analysis of the Raman data has supported the existence of structural specificities in the interactions, at least under our experimental conditions. These specificities lead to preferential bindings through the DNA minor groove for putrescine and spermidine, whereas spermine binds by the major groove. On the other hand, spermine and spermidine present interstrand interactions, whereas putrescine presents intrastrand interactions in addition to exo-groove interactions by phosphate moieties.