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.     

Synthesis of novel polyamines in Paracoccus, Rhodobacter and Micrococcus

Abstract The Gram-negative facultative chemolithotroph, Paracoccus denitrificans contains putrescine, cadaverine, agmatine, spermidine, aminopropylcadaverine, spermine, thermospermine and aminopentylnorspermidine. This bacterium has the ability to produce norspermidine from supplemented diaminopropane. The halophile, Paracoccus halodenitrificans is devoid of any polyamines. Neither decarboxylation of ornithine, lysine or arginine, nor triamine synthetic activity from diamines was detected in this halophile. Two Gram-negative facultative photoautotrophs, Rhodobacter sphaeroides and Rhodobacter capsulatus contain putrescine, cadaverine, agmatine and spermidine and can produce norspermidine from supplemented diaminopropane. A Gram-negative eubacterium, Micrococcus cryophilus , contains histamine and homospermidine in addition to putrescine, cadaverine and spermidine. Hence, polyamine distribution patterns and polyamine biosynthetic activities were very different among the four groups of Gram-negative eubacteria examined.     

Variations in endogenous polyamine content of maize calli obtained from zygotic and androgenetic embryos

A comparative study of polyamine (putrescine, spermidine and spermine) levels was conducted with maize calli originating from a) immature embryos and b) pollen embryos capable of plant regeneration. The differences observed in the studied parameters of the two kinds of calluses are related to their cellular origin and to their regeneration capacity. Moreover, only the calluses proceeding from immature embryos differentiated into preembryogenic structures, which eventually developed into plants. Although total polyamine levels in pollenderived calluses were significantly higher than those from immature embryos, spermidine and spermine were the predominant polyamines in both culture types. Furthermore, polyamine fractions of these calluses also showed differences. All these phenomena may be related with the differences observed in the callus embryogenic response. These findings may be useful in understanding the implication of polyaminesin embryogenetic processes.Abbreviations IEC immature-embryo calluses - PAs polyamines - PEC pollen-embryo calluses - PH insoluble conjugated PA fraction - Put putrescine - S free PA fraction - SH soluble conjugated PA fraction - Spd spermidine - Spm spermine 2,4d-2,4 dichlorophenoxyacetic acid     

Uptake of polyamines by the unicellular green alga Chlamydomonas reinhardtii and their effect on ornithine decarboxylase activity

Uptake of exogenous polyamines by the unicellular green alga Chlamydomonas reinhardtii and their effects on polyamine metabolism were investigated. Our data show that, in contrast to mammalian cells, Chlamydomonas reinhardtii does not contain short-living, high-affinity polyamine transporters whose cellular level is dependent on the polyamine concentration. However, exogenous polyamines affect polyamine metabolism in Chlamydomonas cells. Exogenous putrescine caused a slow increase of both putrescine and spermidine and, vice versa, exogenous spermidine also led to an increase of the intracellular levels of both spermidine and putrescine. No intracellular spermine was detected under any conditions. Exogenous spermine was taken up by the cells and caused a decrease in their putrescine and spermidine levels. As in other organisms, exogenous polyamines led to a decrease in the activity of ornithine decarboxylase, a key enzyme of polyamine synthesis. In contrast to mammalian cells, this polyamine-induced decrease in ornithine decarboxylase activity is not mediated by a polyamine-dependent degradation or inactivation, but exclusively due to a decreased synthesis of ornithine decarboxylase. Translation of ornithine decarboxylase mRNA, but not overall protein biosynthesis is slowed by increased polyamine levels.     

Polyamine auxotrophs of Saccharomyces cerevisiae.

Strains of yeast have been constructed that are unable to synthesize ornithine and are thereby deficient in polyamine biosynthesis. These strains were used to develop a protocol for isolation of mutants blocked directly in polyamine synthesis. There were seven mutants isolated that lack ornithine decarboxylase activity; these strains exhibited greatly decreased pool levels of putrescine, spermidine, and spermine when grown in the absence of polyamines. Three of the mutants lack S-adenosylmethionine decarboxylase activity; polyamine limitation of a representative mutant resulted in an accumulation of putrescine and a decrease in spermidine and spermine. When the mutants were cultured in the absence of polyamines, a continuously declining growth rate was observed.     

Changes in polyamine concentration during seed germination

Phaseolus mungo seeds 0 to 10 days after germination contained putrescine, spermidine, spermine, cadaverine, agmatine and tyramine. The rate of biosynthesis of total polyamines, proteins and RNA in the developing seeds follows similar profiles, reaching maxima 3 hr from germination. Putrescine, cadaverine, spermidine, spermine and agmatine were the major amines found in Pisum sativum 0–7 days after germination. RNA and proteins seem to follow the same pattern as polyamines during the first 12 hr in the developing pea seeds. RNA reaches a peak at 15 hr and polyamines and proteins peak 24 hr after germination. A rise to total polyamine concentration was also observed in seeds of Tragopogon porrifolius, Zea mays and Triticum aestivum 2–12 hr after germination.     

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.     

Polyamines in photosynthetic eubacteria and extreme-halophilic archaebacteria

Qualitative and quantitative determinations of polyamines have been done in 4 photosynthetic eubacteria and 6 extreme-halophilic archaebacteria. For comparison, 5 moderate-halophilic eubacteria were also analyzed to determine their polyamine contents. Not only putrescine and spermidine but also homospermidine were found in the photosynthetic eubacteria, especially in the N2-fixing species, Rhodospirillum and Chromatium. Norspermidine, norspermine, and spermine were not detected in the phototrophic eubacteria. No appreciable amount of any polyamine was found in extreme-halophilic archaebacteria, Halobacterium and Halococcus, while moderate-halophilic eubacteria contained quite high concentrations of putrescine and spermidine and cadaverine. When arginine was incubated with cell lysates of these two archaebacteria, appreciable amounts of agmatine were produced; neither putrescine nor cadaverine was formed in the presence of ornithine or lysine. No detectable amount of spermidine was produced by the lysates on incubation with putrescine.     

Effects of inhibitors of polyamine biosynthesis and of polyamines on strawberry microcutting growth and development

The primary free polyamines identified during growth and development of strawberry (Fragaria × ananassa Duch.) microcuttings cultivated in vitro were putrescine, spermidine and spermine. Polyamine composition differed according to tissue and stages of development; putrescine was predominant in aerial green tissues and roots. -DL-difluoromethylarginine (DFMA), a specific and irreversible inhibitor of the putrescine-synthesizing enzyme, arginine decarboxylase (ADC), strongly inhibited growth and development. Application of agmatine or putrescine to the inhibited system resulted in a reversal of inhibition, indicating that polyamines are involved in regulating the growth and development of strawberry microcuttings. -DL-difluoromethylornithine (DFMO), a specific and irreversible inhibitor of putrescine biosynthesis by ornithine decarboxylase, promoted growth and development. We propose that ADC regulates putrescine biosynthesis during microcutting development. The application of exogenous polyamines (agmatine, putrescine, spermidine) stimulated development and growth of microcuttings, suggesting that the endogenous concentrations of these polyamines can be growth limiting.Abbreviations ADC arginine decarboxylase - ODC ornithine decarboxylase - DFMA -difluoromethylarginine - DFMO -difluoromethylornithine - Put putrescine - Spd spermidine - Sp spermine - DW dry weight - PA polyamine - PPF photosynthetic photon flux     

Polyamine transport,accumulation, and release in brain

Cycling of polyamines (spermine and spermidine) in the brain was examined by measuring polyamine transport in synaptic vesicles, synaptosomes and glial cells, and the release of spermine from hippocampal slices. It was found that membrane potential-dependent polyamine transport systems exist in synaptosomes and glial cells, and a proton gradient-dependent polyamine transport system exists in synaptic vesicles. The glial cell transporter had high affinities for both spermine and spermidine, whereas the transporters in synaptosomes and synaptic vesicles had a much higher affinity for spermine than for spermidine. Polyamine transport by synaptosomes was inhibited by putrescine, agmatine, histidine, and histamine. Transport by glial cells was also inhibited by these four compounds and additionally by norepinephrine. On the other hand, polyamine transport by synaptic vesicles was inhibited only by putrescine and histamine. These results suggest that the polyamine transporters present in glial cells, neurons, and synaptic vesicles each have different properties and are, presumably, different molecular entities. Spermine was found to be accumulated in synaptic vesicles and was released from rat hippocampal slices by depolarization using a high concentration of KCl. Polyamines, in particular spermine, may function as neuromodulators in the brain.     

Cloning and physical characterization of chromosomal conjugative elements in streptococci.

A transport system for polyamines was studied with both intact cells and membrane vesicles of an Escherichia coli polyamine-deficient mutant. Polyamine uptake by intact cells and membrane vesicles was inhibited by various protonophores, and polyamines accumulated in membrane vesicles when D-lactate was added as an energy source or when a membrane potential was imposed artificially by the addition of valinomycin to K+-loaded vesicles. These results show that the uptake was dependent on proton motive force. Transported [14C]putrescine and [14C]spermidine were not excreted by intact cells upon the addition either of carbonyl cyanide m-chlorophenylhydrazone, A23187, and Ca2+ or of an excess amount of nonlabeled polyamine. However, they were excreted by membrane vesicles, although the degree of spermidine efflux was much lower than that of putrescine efflux. These results suggest that the apparent unidirectionality in intact cells has arisen from polyamine binding to nucleic acids, thus giving rise to a negligible free intracellular concentration of polyamines. Polyamine uptake, especially putrescine uptake, was inhibited strongly by monovalent cations. The Mg2+ ion inhibited spermidine and spermine uptake but not putrescine uptake.     

Effects of dietary polyamines and clofibrate on metabolism of polyamines in the rat

The activities of catalase, polyamine oxidase, diamine oxidase, ornithine decarboxylase, and peroxisomal β-oxidation were assayed in homogenates from liver and small intestinal mucosa of rats which had been fed either a diet very low in polyamines or a diet containing five times the levels of dietary polyamines (putrescine, spermine, and spermidine) found in a standard rat diet. In rats fed the high polyamine diet, hepatic activities of catalase and polyamine oxidase were significantly decreased. Levels of the other activities were unchanged, except that intestinal ornithine decarboxylase was decreased. In rats treated simultaneously with clofibrate, the high polyamine diet restored activities of catalase, ornithine decarboxylase, and polyamine oxidase back to levels found in rats fed the low polyamine diet. The expected increase in activity of peroxisomal β-oxidation was observed, although this was somewhat diminished in rats fed the high polyamine diet. Intestinal diamine oxidase activity was stimulated by clofibrate, particularly in rats fed the high polyamine diet. For the duration of the experiment (20 days), levels of putrescine, spermine, and spermidine in blood remained remarkably constant irrespective of treatment, suggesting that polyamine homeostasis is essentially independent of dietary supply of polyamines. It is suggested that intestinal absorption/metabolism of polyamines is of significance in this respect. Treatment with clofibrate appeared to alter polyamine homeostasis.     

Polyamine metabolism in an obligately alkalophilic Bacillus alcalophilus that grows at pH 11.0

Bacillus alcalophilus, an obligately alkalophilic bacterium that grows at pH 11.0, has an intracellular pH of 9.5 or less. Unlike all other living organisms, polyamines (putrescine, spermidine and spermine) in B. alcalophilus, if present, will be largely unprotonated. HPLC analysis indicated that spermidine is the major polyamine in B. alcalophilus, accounting for more than 90% of total polyamines, and the level of spermidine varies during growth. Ornithine decarboxylase activity was not detectable in B. alcalophilus under all conditions examined. When [3H]arginine was added to the culture medium, the radioactivity can be recovered from polyamine pool; the distribution is 3% for putrescine, 94% for spermidine, and 3% for spermine, suggesting the presence of arginine pathway for polyamine biosynthesis. The polyamine transport system in B. alcalphilus appears to be Na+-dependent and is highly sensitive to the inhibition of gramicidin S and valinomycin.     

Spermidine level and protein synthesis are coregulated in nonproliferating hepatocytes

The relationship between polyamines and the rate of protein synthesis was investigated in non-proliferating cells: primary cultures of adult rat hepatocytes maintained in serum-free media, and treated with dexamethasone or dexamethasone + insulin. During the second day of culture, polyamine biosynthesis became induced along with the rate of protein synthesis. While the activity of ornithine decarboxylase and the intracellular concentration of putrescine increased only transiently and that of spermine declined, the rise of the protein synthetic rate was paralleled by that of the intracellular spermidine concentration. The polyamine analogue diamino-propanol specifically decreased spermidine content and the protein synthetic rate. The intracellular concentration of spermidine was found subject to tight homeostatic regulation, e.g. not being altered by the addition of up to 1 mM of this polyamine to the culture medium. In contrast, addition of putrescine or spermine led to an increase in their respective intracellular concentrations. These findings indicate that spermidine specifically of the polyamines is involved in protein synthesis in the intact hepatocyte. Moreover, spermidine may mediate part of the trophic action of dexamethasone and insulin upon cultured hepatocytes.     

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.     

Polyamine changes during the development of zygotic embryos in Prunus avium

A study of the polyamine profile was carried out during zygotic embryo development in Prunus avium. Zygotic embryos were collected from 2 donor trees and sorted into 3 size classes: C1 [2.5 to 3.5 mm]; C2 [3.6 to 4.5 mm] and C3 [5.5 to 7 mm]. Evolution of the various polyamines was similar for the two donor trees. Changes in the relative amount of the various free polyamines were observed during zygotic embryo development. Agmatine and spermine levels increased from C1 to C3. Spermidine, the predominant polyamine, showed a two-fold decrease in C3 compared with C1 and C2; the evolution of putrescine was opposed, showing an increase in the last developmental stage. The putrescine/spermidine ratio could be a marker for these 3 developmental stages with a higher ratio in C3 compared with C1 and C2. Polyamine changes in cotyledons from class C1 were investigated during in vitro culture. A 10-day induction on a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin caused a strong decline in free spermidine levels and a dramatic increase in free putrescine. The formation of conjugated putrescine occurred simultaneously, and twenty days after removal of growth regulators, the various polyamine contents were still at the same level.Abbreviations Agm agmatine - Dap diaminopropane - 2,4-D 2,4-dichlorophenoxyacetic acid - Put putrescine - Spd spermidine - Spm spermine     

Changes in Polyamine Titer in Etiolated Pea Seedlings Following Red Light Treatment

The polyamines agmatine, cadaverine, putrescine, spermidineand spermine were measured by means of thin layer chromatographyand high performance liquid chromatography in buds and in 5mm long subapical sections of the 3rd internode of 6-day-oldetiolated pea seedlings. The polyamine pattern of each organwas specific, relative quantities varying with age and growth.While agmatine, putrescine, spermidine and spermine were presentin buds and in tissues of the 3rd internode, cadaverine wasfound in the 3rd internode only. Concentrations of spermidineand spermine were higher in the bud than in the 3rd internode,and the highest putrescine titer was found in the internode.Short exposure of etiolated seedlings to red light (5 min) increasedbud development while inhibiting growth of the 3rd internode.In general, exposure to red light increased the titer of putrescine,agmatine and spermidine in the bud, whereas in the internodea reverse pattern was found, i.e., internodes of seedlings growingin the dark yielded higher titer of polyamines in general, andagmatine in particular. These results are particularly pronounced18 hr after exposure to red light. A link between phytochrome-controlledgrowth and polyamine titer is suggested. ² On sabbatical leave from the Hebrew University of Jerusalem,Department of Horticulture, Rehovot, Israel. ³ Supported by a grant from the Turkish Government; Permanentaddress: Department of General Botany, University of Istanbul,Suleymaniye, Istanbul, Turkey. ¹ Supported by a grant from NSF to A.W.G. (Received August 24, 1981; Accepted October 22, 1981)     

Targeted disruption of spermidine/spermine N1-acetyltransferase gene in mouse embryonic stem cells. Effects on polyamine homeostasis and sensitivity to polyamine analogues

We have generated mouse embryonic stem cells with targeted disruption of spermidine/spermine N(1)-acetyltransferase (SSAT) gene. The targeted cells did not contain any inducible SSAT activity, and the SSAT protein was not present. The SSAT-deficient cells proliferated normally and appeared to maintain otherwise similar polyamine pools as did the wild-type cells, with the possible exception of constantly elevated (about 30%) cellular spermidine. As expected, the mutated cells were significantly more resistant toward the growth-inhibitory action of polyamine analogues, such as N(1),N(11)-diethylnorspermine. However, this resistance was not directly attributable to cellular depletion of the higher polyamines spermidine and spermine, as the analogue depleted the polyamine pools almost equally effectively in both wild-type and SSAT-deficient cells. Tracer experiments with [C(14)]-labeled spermidine revealed that SSAT activity is essential for the back-conversion of spermidine to putrescine as radioactive N(1)-acetylspermidine and putrescine were readily detectable in N(1),N(11)-diethylnorspermine-exposed wild-type cells but not in SSAT-deficient cells. Similar experiments with [C(14)]spermine indicated that the latter polyamine was converted to spermidine in both cell lines and, unexpectedly, more effectively in the targeted cells than in the parental cells. This back-conversion was only partly inhibited by MDL72527, an inhibitor of polyamine oxidase. These results indicated that SSAT does not play a major role in the maintenance of polyamine homeostasis, and the toxicity exerted by polyamine analogues is largely not based on SSAT-induced depletion of the natural polyamines. Moreover, embryonic stem cells appear to operate an SSAT-independent system for the back-conversion of spermine to spermidine.