Polyamine contents in the brain and liver of rats during acclimatization to the cold

The content of polyamines, spermidine and spermine in the brain and liver was studied in rats during acclimation to cold for 45 days. In the brain the amount of spermidine after one and three days does not change, on the 7th and 15th day it decreases and by the end of acclimation returns to the control value. The content of spermine lowers by 29% by the third day of acclimation and on the 7th day returns to the initial level and does not change up to it end. In the liver the amount of spermidine rises significantly on the third day of acclimation, returns to the control level on the 7th day and then its amount falls. The content of spermine is increased in all period of acclimation and only by the 45th day it returns to its initial level.     

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.     

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.     

THE REGIONAL DISTRIBUTION OF THE POLYAMINES SPERMIDINE AND SPERMINE IN BRAIN

Abstract— The distribution in brain of the polyamines spermidine and spermine is described in the rat, dog, sheep, rabbit and in man. The distribution pattern was about the same in all the species, spermidine concentration being highest in areas rich in white matter. The concentration of spermine was lower than that of spermidine and showed less variation from area to area. Rat brain polyamine content was the same in rats killed by decapitation as in those killed by rapid freezing in liquid nitrogen and was also unchanged up to 48 h after the death of the animal.     

On the subcellular localization of the polyamines

Putrescine, spermidine and spermine were determined in the nuclear fraction of rat liver which was obtained by density gradient centrifugation in non-aqueous media, i.e. under conditions which avoid migration of water-soluble compounds. Calculations of the distribution of the polyamines between nuclear and extranuclear compartments were based on the assumption that the DNA is concentrated in the nuclei. No significant losses of the polyamines occurred during fractionation. From the polyamine determination in tissue and nuclear fraction it appeared that 16-17% of the liver spermidine and spermine, and about 8% of the putrescine content was localized in the nuclei. The spermidine/spermine-ratios in nuclei and whole tissue were not significantly different. Pretreatment of the animals with inhibitors of ornithine decarboxylase caused a decrease of putrescine exclusively in the extranuclear compartments, in agreement with a higher proportion of the inhibitors in the cytoplasm. Since the nuclear volume of rat liver corresponds to about 5% of total liver volume, the concentration of spermidine and spermine is higher in the nucleus than in extranuclear compartments. Published histochemical localizations of the polyamines suggested very low polyamine concentrations in the nuclei of non-dividing liver and HeLa cells, but dramatic polyamine accumulations in metaphase and anaphase nuclei. These results are in disagreement with previously reported autoradiographic data, subcellular localizations based on density gradient centrifugations, and with our present results. Since subcellular localization is a key issue in all attempts to clarify cellular functions of the polyamines the careful revision of the techniques involved in subcellular polyamine localizations seems imperative.     

Spermine and spermidine mediate protection against oxidative damage caused by hydrogen peroxide

Summary. The polyamines spermidine and spermine have been hypothesized to possess different functions in the protection of DNA from reactive oxygen species. The growth and survival of mouse fibroblasts unable to synthesize spermine were compared to their normal counterparts in their native and polyamine-depleted states in response to oxidative stress. The results of these studies suggest that when present at normal or supraphysiological concentrations, either spermidine or spermine can protect cells from reactive oxygen species. However, when polyamine pools are pharmacologically manipulated to produce cells with low levels of predominately spermine or spermidine, spermine appears to be more effective. Importantly, when cells are depleted of both glutathione and endogenous polyamines, they exhibit increased sensitivity to hydrogen peroxide as compared to glutathione depletion alone, suggesting that polyamines not only play a role in protecting cells from oxidative stress but this role is distinct from that played by glutathione.     

Brain infarction correlates more closely with acrolein than with reactive oxygen species

Although it is thought that the major factor responsible for cell damage is reactive oxygen species (ROS), our recent studies have shown that acrolein is more toxic than ROS. Thus, the relative importance of acrolein and ROS in cell damage during brain infarction was compared using photochemically induced thrombosis model mice. The levels of acrolein-conjugated albumin, and of 4-hydroxynonenal (HNE)-conjugated albumin and 8-OHdG were evaluated as indicators of damage produced by acrolein and ROS, respectively. The increase in acrolein-conjugated albumin was much greater than the increase in HNE-conjugated albumin or 8-OHdG, suggesting that acrolein is more strongly involved in cell damage than ROS during brain infarction. It was also shown that infarction led more readily to RNA damage than to DNA or phospholipid damage. As a consequence, polyamines were released from RNA, and acrolein was produced from polyamines, especially from spermine by spermine oxidase. Production of acrolein from spermine by spermine oxidase was clarified using spermine synthase-deficient Gy mice and transglutaminase 2-knockout mice, in which spermine content is negligible or spermidine/spermine N¹-acetyltransferase activity is elevated.     

A possible role of cadaverine in the biosynthesis of polyamines in the Japanese newt testis

Polyamine content in testes of various vertebrates was studied extensively. Putrescine, spermidine and spermine were detected in all the animals examined, although the distribution pattern varied greatly from animal to animal. Cadaverine was detected only in amphibian testes; sym-homospermidine was found not only in testes but also in various other tissues of amphibians and of some reptiles. In the newt testis the concentration of cadaverine was lower than that of any other polyamines in summer, but there was a great increase in cadaverine content from autumn to winter. The testicular content of cadaverine was greater than that of other polyamines in winter. There was a gradual decrease in the cadaverine content in spring. The spermidine and spermine levels, which were rather low in winter, increased in spring and reached a peak in summer when spermatogenesis was active. The testicular concentration of putrescine that was much higher than that of spermidine or spermine throughout the year, increased only a little in summer. There was a significant negative correlation between the cadaverine levels and four other polyamine levels. Exogenous cadaverine decreased the testicular levels of putrescine. Mammalian gonadotropins decreased the cadaverine levels and increased the levels of other polyamines. A partially purified LH fraction from pituitaries of bullfrog, Rana catesbeiana, was also potent in depleting cadaverine of the testes of newts kept at 8 degrees C. These results suggest that testicular cadaverine suppresses the biosynthesis of polyamines, especially spermidine and spermine which are closely associated with spermatogenesis.     

Effects of Polyamines on Proline Endopeptidase Activity in Rat Brain

The in vitro effects of polyamines on the activity of proline endopeptidase (PEPase) in rat brain cytosol, which contains an endogenous PEPase inhibitor, have been studied. Of the three amines tested (spermine, spermidine, and putrescine), spermine and spermidine markedly enhanced the enzyme activity in brain cytosol. At 6.25 mM spermine or 25 mM spermidine, a 13- or 14-fold enhancement of the enzyme activity was observed. When Mg2+ was used, an approximately fourfold enhancement of the enzyme activity was observed at 50 mM. The enhancement produced by spermine or spermidine was unaffected by Mg2+ up to 50 mM. The activity of purified PEPase was only slightly affected by each polyamine, but it was inhibited 50% by 50 mM Mg2+. On the other hand, 50% inhibition of the enzyme produced by the purified PEPase inhibitor (Mr 7,000: Ki 0.67 mM) was completely restored by addition of 0.7 mM spermine, 3.5 mM spermidine, or 28 mM putrescine. This restoration of inhibition by polyamines was reversed by increasing the inhibitor concentration. These data suggest that polyamines effectively reverse the inhibition of PEPase by its endogenous inhibitor by the reversible formation of a kinetically significant complex. The possible functions of polyamines in the regulation of PEPase in vivo are discussed.     

Polyamines: developmental alterations in regional disposition and metabolism in rat brain

The polyamines spermidine and spermine and the activity of the polyamine synthesizing enzyme, S-adenosyl-L-methionine (SAM) decarboxylase, were measured in regions of adult rat brains and during postnatal development. In the adult, although spermidine levels tended to correlate with the relative amounts of white matter in some areas, there were striking exceptions. SAM decarboxylase activity of the adult brain was higher than in most other mammalian tissues, although brain levels of polyamines were among the lowest. SAM decarboxylase activity appeared to be localized to cellular cytoplasm. Its activity increased with age in contrast to the levels of spermine, spermidine, DNA and RNA which decreased during postnatal development.     

Polyamine metabolism in BHK21/C13 cells : Loss of spermidine from cells following transfer to serum-depleted medium

The growth rate of BHK21/C13 cells in culture was slowed down by transferring growing cells to serum-depleted medium Following deprivation of serum, the intracellular concentration of polyamines decreased. The amount of spermidine relative to spermine decreased, and this change was the result of the spermidine content per cell decreasing more than the spermine content. The decrease in cell content of polyamines was accompanied by release of polyamines from the cells into the culture medium. The polyamines released were examined using cells whose polyamines had been labelled by prior incubation of the cells with radioactive putrescine. Almost all of the radioactivity released into the medium was found in spermidine, even though the cells contained most of their radioactivity in spermine. It is suggested that specific release of spermidine may be an important mechanism by which these cells can regulate their intracellular content of polyamines.     

Polyamines in liver and their influence on chromatin condensation after 17-β estradiol treatment of Atlantic salmon

Atlantic salmon (Salmo salar) were treated with 17- estradiol to induce vitellogenin synthesis in liver. This led to an increase in liver wet weight and total DNA. After incubation with micrococcal nuclease (EC 3.1.31.1) less soluble chromatin was obtained from nuclei of the estradiol treated than the control fish, but active gene regions were solubilized by the nuclease. Thus, in the estradiol treated fish soluble mononucleosomes contained hybridizable vitellogenin gene sequences. As a result of estradiol treatment the content in total liver of putrescine rose 3-fold, that of spermidine 2-fold, while spermine was unchanged. In muscle no significant changes were observed. The regulatory functions of polyamines during gene expression were investigated by binding (¹⁴C)spermine to isolated liver nuclei depleted of endogenous polyamines. The number of binding sites was higher in nuclei of estradiol treated than control fish. (^14C)spermine associated preferentially with micrococcal nuclease insensitive chromatin. Thus, the high content of putrescine and spermidine in liver supported the view of polyamine accumulation in proliferating tissues. The preferential binding to condensed chromatin indicated a stabilizing effect of polyamines on the organization of inactive chromatin structures.Abbreviations MNase micrococcal nuclease - PMSF phenylmethylsulfonylfluoride     

The role of polyamine depletion and accumulation of decarboxylated S-adenosylmethionine in the inhibition of growth of SV-3T3 cells treated with alpha-difluoromethylornithine.

The effects of alpha-difluoromethylornithine, a specific inhibitor of ornithine decarboxylase, on cell growth rate, polyamine content and the content of decarboxylated S-adenosylmethionine in SV-3T3 transformed mouse fibroblasts were studied. DL-alpha-Difluoromethylornithine at 1 mM or higher concentrations decreased the growth rate by over 90% after 2 or more days of exposure, but the cells remained viable, although quiescent for at least 9 days. Addition of 10 microM-spermidine or -spermine or 50 microM-putrescine at any time throughout this period completely reversed the inhibition of growth. Treatment with alpha-difluoromethylornithine decreased putrescine and spermidine contents by more than 98% and that of spermine by 60%, but cells exposed to exogenous polyamines did not require complete replenishment of the polyamine pools to resume growth. In fact, a virtually normal growth rate was obtained in cells lacking putrescine, having 2% of normal spermidine content and 156% of normal spermine. These results suggest that the well-known increase in putrescine and spermidine in cells stimulated for growth is not essential for this to occur and that mammalian cells can utilize spermine as their only polyamine. A substantial reversal of the growth-inhibitory effect of alpha-difluoromethylornithine was produced by a number of polyamines not normally found in mammalian cells, including the spermidine analogues aminopropylcadaverine and sym-homospermidine, which were partially converted into their respective spermine analogues by addition of an aminopropyl group within the cell. The spermine analogue sym-norspermine was also effective, but the maximal growth rate produced by these unphysiological polyamines was only 60-70% of that produced by the normal polyamines. These results indicate that spermidine and spermine have the optimal length for activation of the cellular processes critically dependent on polyamines and should help in identifying these processes. Exposure to alpha-difluoromethylornithine leads to an enormous rise in the concentration of decarboxylated S-adenosylmethionine, which reached a peak at 530-fold after 3 days of exposure and steadily declined to 140-fold after 11 days. This increase was abolished by addition of exogenous polyamines, which rapidly decreased the activity of S-adenosylmethionine decarboxylase. The increase in decarboxylated S-adenosylmethionine is unlikely to be solely responsible for the decrease to the same extent by spermine, sym-norspermidine and sym-homospermidine, which produce 97%, 16% and 60% of the control growth rate, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Changes in Polyamine Concentrations in Amygdaloid-Kindled Rats

Concentrations of the polyamines putrescine, spermidine, and spermine were investigated in the left and right amygdala and in the remaining cerebrum, in which kindling was induced by repeated application of electrical stimulation of the left amygdala of rats. In kindled rats, the concentrations of spermidine and spermine increased slightly, but elevations did not reach significant levels in any brain regions. The most profound increase was detected in the putrescine concentration in all parts of the cerebrum 1-8 h after the final stimulation. These results suggest that the increases in concentrations of polyamines, particularly of putrescine, are involved in the pathogenesis of amygdaloid kindling.     

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.     

INTERRELATIONS BETWEEN POLYAMINES AND NUCLEIC ACIDS: CHANGES OF POLYAMINE AND NUCLEIC ACID CONCENTRATIONS IN THE DEVELOPING RAT BRAIN

The rat brain concentrations of protein, RNA, DNA, putrescine, and of the polyamines spermidine and spermine, were studied during development. Putrescine formation is apparently controlled by ornithine decarboxylase. Spermidine and spermine concentrations change in inverse directions to their anabolic enzymes. It has been presumed, therefore, that the low concentrations of the polyamine-synthesizing enzymes in immature brain are compensated for, by high putrescine and S-adenosylmethionine concentrations. In agreement with previous findings for fish brain, the changes in RNA and spermidine concentrations were most closely correlated. The functions of DNA: spermine are directly correlated only during the periods of brain maturation, after cell proliferation has nearly ceased.     

POLYAMINE METABOLISM IN THE BRAIN AND LIVER OF THE DEVELOPING MONKEY

Abstract— The concentrations of putrescine and the polyamines, spermidine and spermine, along with the specific activities of the enzymes involved in their biosynthesis, ornithine decarboxylase, S -adenosylmethionine decarboxylase and spermidine synthase have been measured in brain and liver of the developing Rhesus monkey from mid-gestation, through birth and neonatal life to maturity. The results suggest that it is an increased concentration of putrescine and an increased specific activity of ornithine decarboxylase which are associated with the rapid growth of fetal brain during the middle of gestation. By the end of two-thirds of gestation, both of these parameters have attained values similar to those found in mature brain. The concentration of spermidine in brain and the specific activities of S -adenosylmethionine decarboxylase and spermidine synthase are lower in fetal brain than adult brain and increase slowly after birth to reach values similar to those of the adult only after several months. These results provide additional evidence that in the mature brain spermidine serves some function other than one associated with rapid growth.
Fetal liver at mid-gestation was characterized by increased concentrations of both putrescine and spermidine and increased specific activities of the enzymes which synthesize them. By two-thirds of gestation, values similar to those found in adult liver had been attained. Liver has thus reached maturity with regard to polyamine metabolism by this time.     

Changes in the nuclear polyamine content of chick erythrocytes during embryonic development.

The polyamine content of the circulating erythrocyte population in the embryonic chick was studied during its development. Total cellular polyamine content fell dramatically between 5 and 7 days of development, paralleling the decrease in metabolic activity exhibited by these cells. Nuclei were isolated from the erythrocytes by a non-aqueous technique, which not only eliminated the polyamine loss that occurred with aqueous isolation, but also prevented redistribution of the polyamines from the cytoplasm. Nuclear spermidine and spermine contents decreased markedly between 5 and 6 days of development from 31 to 10 pmol/microgram of DNA and from 33 to 18 pmol/microgram of DNA respectively. Thereafter the spermine content remained constant, but the spermidine content continued to decline. Good correlations between spermidine and RNA contents were observed in both cells and nuclei, and similarly between spermine and RNA contents in cells, but no such correlation was observed between spermine and RNA in nuclei.     

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.