Effects of external osmolality on polyamine metabolism in HeLa cells.

The polyamine content of Escherichia coli is inversely related to the osmolality of the growth medium. The experiments described here demonstrate that a similar phenomenon occurs in mammalian cells. When grown in media of low NaCl concentration, HeLa cells and human fibroblasts were found to contain high levels of putrescine, spermidine, and spermine. The putrescine content of HeLa cells was a function of the osmolality of the medium, as shown by growing cells in media containing mannitol or additional glucose. External osmolality per se had no effect on the contents of spermidine and spermine. For all media, the total cellular polyamine content could be correlated with the activity of ornithine decarboxylase, the first enzyme in polyamine biosynthesis. Different levels of enzyme activity appear to result solely from variations in the rate of enzyme degradation. A sudden increase in a NaCl concentration produced rapid loss of ornithine decarboxylase activity and a gradual loss of putrescine and spermidine. A sudden decrease in NaCl concentration led to rapid and substantial increases in ornithine decarboxylase activity and putrescine.     

Effects of external osmolality on polyamine metabolism in HeLa cells

The polyamine content of Escherichia coli is inversely related to the osmolality of the growth medium. The experiments described here demonstrate that a similar phenomenon occurs in mammalian cells. When grown in media of low NaCl concentration, HeLa cells and human fibroblasts were found to contain high levels of putrescine, spermidine, and spermine. The putrescine content of HeLa cells was a function of the osmolality of the medium, as shown by growing cells in media containing mannitol or additional glucose. External osmolality per se had no effect on the contents of spermidine and spermine. For all media, the total cellular polyamine content could be correlated with the activity of ornithine decarboxylase, the first enzyme in polyamine biosynthesis. Different levels of enzyme activity appear to result solely from variations in the rate of enzyme degradation.A sudden increase in NaCl concentration produced rapid loss of ornithine decarboxylase activity and a gradual loss of putrescine and spermidine. A sudden decrease in NaCl concentration led to rapid and substantial increases in ornithine decarboxylase activity and putrescine.     

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.     

Effect of starvation and refeeding on polyamine concentrations and ornithine decarboxylase antizyme in mammary gland of lactating rats.

Starvation caused a marked increase in putrescine content in mammary gland of lactating rats, together with a marked decrease in activity of ornithine decarboxylase and appearance of measurable ornithine decarboxylase antizyme. 2. Refeeding for 5 h caused disappearance of free antizyme and ornithine decarboxylase activity returned to the value in fed animals. Putrescine concentration remained elevated. 3. There was no significant change in nucleic acid content of mammary gland from starved rats, but spermidine and spermine contents increased significantly. 4. Refeeding for 5 h returned the spermidine content of mammary glands to 'fed' values, and significantly decreased the content of spermine, although it did not reach control values. Thus changes in polyamine content of mammary gland in starved rats are clearly dissociated from changes in either RNA content or activities of polyamine-synthetic decarboxylases. 5. Starvation caused a fall in the content of spermidine in liver, with no change in spermine content. Refeeding for 5 h returned the spermidine content to 'fed' values.     

Regulation of putrescine metabolism in Ehrlich ascites carcinoma cells exposed to hypotonic medium

When exposed to hypotonic growth medium, Ehrlich ascites carcinoma cells showed a rapid stimulation of ornithine decarboxylase (EC 4.1.1.17) activity in 4 h, followed by a rise in their putrescine content. This effect was totally abolished by addition of a slightly hypertonic concentration of sodium chloride or sucrose to the medium. The general protein synthesis was unaffected by the hypotonic treatment. The uptake of putrescine and, to a lesser extent, spermidine was enhanced, and the conversion of the radioactive putrescine into spermidine appeared partially inhibited during later stages of the hypotonic treatment. As a result, the half-life of putrescine increased from 2.8 h under isoosmotic conditions to 7.3 h in hypoosmotic medium. Both exogenous ([¹⁴C]-putrescine-derived) and endogenous ([¹⁴C]ornithine-derived) putrescine degraded at similar rates in control and hypotonic cells, yet the putrescine taken from the medium degraded preferably to nonpolyamine products, while the putrescine synthesized in the cell was converted evenly to spermidine and to other metabolites. Adenosylmethionine decarboxylase activity (EC 4.1.1.50), which provides the second precursor for spermidine and spermine synthesis, was distinctly inhibited in the hypotonic medium. Inhibition was likewise observed in spermidine synthase activity, while spermine synthase was marginally stimulated. It appears that the hypotonic treatment serves a special condition under which not only the formation of putrescine is enhanced dramatically but the cells also attempt to conserve the diamine by preventing its further metabolism to higher polyamines.     

Polyamine dependence of Chinese hamster ovary cells in serum-free culture is due to deficient arginase activity

We recently isolated a Chinese hamster ovary cell line which grows well without serum but requires the exogenous polyamines putrescine, spermidine or spermine for continuous replication. Here we show that these cells are defective in the arginase-catalyzed synthesis of ornithine, the precursor of polyamines, and that ornithine can replace polyamines in the medium for supporting growth of the cells. The activities of two other key enzymes of polyamine biosynthesis, ornithine decarboxylase and adenosylmethionine decarboxylase, are clearly detectable and show increase during polyamine starvation. In ornithine- and polyamine-free medium cellular putrescine and spermidine are rapidly depleted while the concentration of spermine decreases only moderately. We show further that the cells are able to grow in serum-containing medium without added ornithine or polyamines. This is explained by our finding that serum contains arginase which synthesizes ornithine from arginine in the medium. All the sera from different animal species tested contained arginase activity although in greatly varying amounts. Serum-free medium is therefore essential for expression of arginase deficiency in cells in tissue culture. The eventual importance of polyamines for serum-free cultures in general is discussed.     

Metabolism of polyamines by cultured glioma cells. Effect of asparagine on gamma-aminobutyric acid concentrations.

The activity of ornithine decarboxylase (EC 4.1.1.17) increased in confluent cultures of glioma C6BU-1 cells 3 h after adding a complete serum-containing medium, and was maximal 5 h later. The activity of S-adenoxyl-L-methionine decarboxylase (EC 4.1.1.50) increased soon after addition of the complete medium to the cells, and reached its peak after 11 h. The activity of diamine oxidase (EC 1.4.3.6) also increased soon after adding complete medium and was maximal 8h later, when the activity of ornithine decarboxylase reached its peak. The increase in the activity of S-adenosyl-L-methionine decarboxylase was accompanied by changes in cellular spermidine and spermine concentrations, whereas the increase in the activity of diamine oxidase was followed by the accumulation of gamma-aminobutyric acid, which was detected both in the cells and in the medium. Asparagine enhanced the utilization of radioactive putrescine by glioma cells suspended in buffered-salt/glucose solution and increased intracellular and extracellular gamma-aminobutyric acid concentrations. Radioactive putrescine was converted into spermidine and spermine by glioma cells after addition of a serum-containing medium, but not after adding buffered--salt/glucose solutions, in the presence or absence of asparagine. The kinetics of ornithine decarboxylase 'induction' and the half-life of the enzyme differed in cells incubated with buffered asparagine solutions and serum-containing media.     

Polyamine biogenesis in the rat mammary gland during pregnancy and lactation

Polyamines and RNA accumulate in the rat mammary gland during pregnancy, but the major increases occur after parturition. Therefore the major increases occur after the gland has obtained its maximal complement of epithelial cells. During lactation, the spermidine concentration rises above 5mm and RNA content in the lactating mammary gland reaches a value 16 times that of the unstimulated mammary gland. The ratio of spermidine/spermine, an increase of which initially signals an elevation in biosynthetic activity, is near 1 in the normal mammary gland and is greater than 10 in the lactating mammary gland. Putrescine concentration is very low during the entire course of mammary-gland development, with the exception of early pregnancy. The low putrescine concentration probably reflects the very rapid conversion of putrescine into spermidine. Both ornithine decarboxylase, the enzyme that synthesizes putrescine, and putrescine-stimulated S-adenosyl-l-methionine decarboxylase, the enzyme that synthesizes spermidine, increase in activity during middle and late pregnancy; during lactation, both enzyme activities are elevated until the 21st day of lactation, and then decline. These declines are concomitant with involution. Also, it was found that the amount of ribonuclease activity in the mammary gland was very high during lactation, almost double that in the gland during pregnancy.     

Epidermal keratinocytes actively maintain their intracellular polyamine levels

Increased cellular polyamine levels are thought to be essential for epidermal keratinocyte proliferation. However, a number of studies report that the induction of keratinocyte proliferation and of ornithine decarboxylase, the rate-limiting enzyme of putrescine, spermidine and spermine biosynthesis, is not concordantly expressed. The relationship between epidermal keratinocyte polyamine synthesis and proliferation was studied in neonatal mouse keratinocyte cultures using specific inhibitors of ODC activity to decrease the intracellular polyamine levels. The ODC inhibitors alpha-methyl ornithine (alpha-Me-Orn), alpha-hydrazino ornithine (alpha-HO) and difluoro-alpha-methylornithine (alpha-DFMO) did not significantly inhibit epidermal keratinocyte proliferation at 5 X 10(-3) to 10(-4) M concentrations. At these doses, only alpha-DFMO was seen to decrease (by 70%) the cellular levels of putrescine, but not of spermidine or spermine. Epidermal keratinocyte growth in the higher dose of 20 mM alpha-DFMO, however, did not decrease the cellular levels of putrescine. Polyamine analyses of the spent medium showed that growth in 10 mM alpha-DFMO decreased the normal epidermal cell transport of putrescine and spermidine into the medium. At 20 mM alpha-DFMO concentration, the keratinocytes actually transported, intracellularly, the putrescine and spermidine that are naturally found in the foetal bovine component of the growth medium. We conclude from these studies that epidermal keratinocyte polyamine levels are determined by both the rate of synthesis, and of the transport of these amines into the extracellular medium. Since epidermal keratinocytes actively maintain specific polyamine levels, it appears that these molecules are essential for epidermal keratinocyte function.     

Polyamine biosynthesis and DNA synthesis in cultured mammary gland explants from virgin mice

The mammary cells in virgin mice are essentially non-proliferative, but they can be induced to undergo DNA synthesis in vitro in the presence of insulin. Time course studies on polyamine biosynthesis and DNA synthesis showed that insulin elicits sequential stimulation of the activity of the polyamine biosynthetic enzymes, ornithine decarboxylase, S-adenosyl-L-methionine decarboxylase (SAMDC) and spermidine synthase, and an increase in the concentration of spermidine prior to the augmentation of DNA synthesis. At 48 to 72 hours of culture when DNA synthesis is maximal, the concentration of spermidine increased 2? to 3-fold, whereas the level of spermine remained unchanged. Addition of methyl glyoxal bis(guanylhydrazone) (5—10 μM), a potent inhibitor of SAMDC, to the medium at the onset of culture resulted in inhibition of spermidine formation and DNA synthesis, but when added at 24 hours or 48 hours of culture, the inhibitory effect on DNA synthesis was greatly reduced. The drug, however, produced little inhibition of RNA and protein synthesis. Inhibition of DNA synthesis by the drug can be reversed by addition of spermidine or other polyamines such as putrescine, cadaverine and spermine to the culture. Spermidine is, however, the only polyamine that is effective at physiological concentrations (100～150 pmoles/mg tissue). These results suggest a possibility that spermidine may play a key role in the regulation of mammary cell proliferation.     

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.     

Regulatory mutations affecting ornithine decarboxylase activity in Saccharomyces cerevisiae.

We isolated several strains of Saccharomyces cerevisiae containing mutations mapping at a single chromosomal gene (spe10); these strains are defective in the decarboxylation of L-ornithine to form putrescine and consequently do not synthesize spermidine and spermine. The growth of one of these mutants was completely eliminated in a polyamine-deficient medium; the growth rate was restored to normal if putrescine, spermidine, or spermine was added. spe10 is not linked to spe2 (adenosylmethionine decarboxylase) or spe3 (putrescine aminopropyltransferase [spermidine synthease]). spe 10 is probably a regulatory gene rather than the structural gene for ornithine decarboxylase, since we isolated two different mutations which bypassed spe10 mutants; these were spe4, an unliked recessive mutation, and spe40, a dominant mutation linked to spe10. Both spe4 and spe40 mutants exhibited a deficiency of spermidine aminopropyltransferase (spermine synthase), but not of putrescine aminopropyltransferase. This suggests that ornithine decarboxylase activity is negatively controlled by the presence of spermidine aminopropyltransferase.     

Role of polyamines in the proliferation and steroidogenic activities of bovine adrenocortical cells in culture

Difluoromethylornithine (DFMO), a selective inhibitor of ornithine decarboxylase, was used to probe the possible role of polyamines in the regulation of proliferation and steroidogenic activities of bovine adrenocortical cells in primary culture. The presence of DFMO in the culture medium not only suppressed the polyamine increase observed in proliferating control cells but resulted in a rapid depletion of the putrescine and spermidine cellular content, while spermine remained at a basal level. The proliferation of DFMO-treated cells was rapidly blocked and resumed at a normal rate upon addition of putrescine to the medium. DFMO-treated cells showed an impaired steroidogenic response to ACTH while adenylate cyclase stimulation was not altered. Thus, while ornithine decarboxylase and polyamines may be required for adrenocortical cell replication, deprivation of these compounds did not facilitate the expression of differentiated cell functions, as observed with granulosa cells.     

The influence of nerve section on the metabolism of polyamines in rat diaphragm muscle.

Concentrations of spermidine, spermine and putrescine have been measured in rat diaphragm muscle after unilateral nerve section. The concentration of putrescine increased approx. 10-fold 2 days after nerve section, that of spermidine about 3-fold by day 3, whereas an increase in the concentration of spermine was only observed after 7-10 days. It was not possible to show enhanced uptake of either exogenous putrescine or spermidine by the isolated tissue during the hypertrophy. Consistent with the accumulation of putrescine, activity of ornithine decarboxylase increased within 1 day of nerve section, was maximally elevated by the second day and then declined. Synthesis of spermidine from [14C]putrescine and either methionine or S-adenosylmethionine bt diaphragm cytosol rose within 1 day of nerve section, but by day 3 had returned to normal or below normal values. Activity of adenosylmethionine decarboxylase similarly increased within 1 day of nerve section, but by day 3 had declined to below normal values. Activity of methionine adenosyltransferase was elevated throughout the period studied. The concentration of S-adenosylmethionine was likewise enhanced during hypertrophy. Administration of methylglyoxal bis(guanylhydrazone) produced a marked increase in adenosylmethionine decarboxylase activity and a large increase in putrescine concentration, but did not prevent the rise in spermidine concentration produced by denervation. Possible regulatory mechanisms of polyamine metabolism consistent with the observations are discussed.     

Regulation of growth and polyamine biosynthesis of the ciliated protozoan Tetrahymena thermophila. Effects of L-arginine metabolites and polyamines

Growth of Tetrahymena thermophila in a synthetic nutrient medium with or without the essential amino acid L-arginine was studied in the presence or absence of the arginine metabolites L-citrulline and L-ornithine and the polyamines putrescine, spermidine, and spermine. The effects of the growth conditions on the stimulations of the enzymes of the arginine metabolic and polyamine biosynthetic pathway, arginine deiminase (ADI), citrulline hydrolase (CH), ornithine decarboxylase (ODC), and ornithine-oxo-acid aminotransferase were determined. Tetrahymena cells were unable to grow in the absence of L-arginine and the amino-acid utilization was greatly impaired. None of the metabolites or polyamines was able to substitute for arginine. In the presence of arginine, Tetrahymena cultures grew well and citrulline and ornithine did not alter the growth behaviour in any way. In the presence of putrescine, the lag period was decreased from 3 h to 2 h. Spermidine and spermine acted similar to putrescine but less pronounced. The stimulation of the activity of ADI, the key enzyme of arginine degradation, was absolutely dependent upon the presence of arginine in the medium: in the absence of arginine, the low ADI activity which was present in the cells before inoculation was decreased to zero levels within 30 min. In the presence of arginine, the stimulation of ADI was not altered by citrulline and ornithine but putrescine, spermidine, and spermine decreased ADI-stimulation to half of the control values. The stimulation of CH activity in the presence of arginine was not altered by any added metabolite or polyamine. In the media without arginine, stimulation of CH was greatly reduced, in the presence of ornithine more than in its absence, and even more in the presence of putrescine and spermidine. Stimulation of ODC activity in the presence of arginine was not affected by citrulline and ornithine but in the presence of polyamines it was rapidly decreased to unstimulated levels after an initial ca. 10-fold increase. The "hyperstimulation" of ODC in the absence of free arginine was reduced to normal in the presence of citrulline, the stimulation was decreased even below normal levels in the presence of ornithine and polyamines decreased ODC activity to zero levels. O delta T activity was stimulated more in the presence of arginine than in its absence. In both cases the stimulation was enhanced in the presence of polyamines and only in the absence of arginine--by ornithine.(ABSTRACT TRUNCATED AT 400 WORDS)     

Relationships between polyamine metabolism and RNA synthesis in post-ischemic liver cell repair

In liver cells recovering from reversible ischemia the increase in RNA synthesis by isolated nuclei is preceded by activation of ornithine decarboxylase, leading in turn to an increase in putrescine concentration. Treatment of the animals with 1,3-diaminopropane and putrescine prevents ornithine decarboxylase activation but does not hinder the enhancement of RNA synthesis in post-ischemic liver nuclei; therefore, ornithine decarboxylase activation does not seem to be a necessary prerequisite for the increase in RNA synthesis. Hypophysectomy does not prevent the post-ischemic increases of ornithine decarboxylase and RNA synthesis; but pre-treatment of the animals with cycloheximide—which has a dual effect on the activity of ornithine decarboxylase—abolishes the post-ischemic enhancement of RNA synthesis. In contrast with regenerating liver, changes in ornithine decarboxylase activity and putrescine concentrations in reversible ischemia are not associated to changes in S-adenosylmethionine decarboxylase activity and in spermine and spermidine concentrations that seem to be characteristic of tissues where increases in RNA synthesis are followed by DNA synthesis and cell multiplication.     

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 appear to be second messengers in mediating Ca2+ fluxes and neurotransmitter release in potassium-depolarized synaptosomes

High potassium (50 mM) depolarization induces a rapid (less than 15 sec) increase in the levels of the polyamines putrescine, spermidine and spermine and their rate-regulating synthetic enzyme ornithine decarboxylase in synaptosomes from rat cerebral cortex. The ornithine decarboxylase inhibitor alpha-difluoromethylornithine blocked the K+-stimulated increase in enzyme activity and polyamines and also suppressed the increase in 45Ca2+ influx and efflux and the Ca2+-dependent release of GABA and norepinephrine. Added putrescine attenuated or negated the effects of alpha-difluoromethylornithine. These results suggest that enhanced polyamine synthesis is required for potassium depolarized stimulation of synaptic function.     

Concomitant Changes in Polyamine Pools and DNA Methylation during Growth Inhibition of Human Colonic Cancer Cells

The effects of CGP 48664 and DFMO, selective inhibitors of the key enzymes of polyamine biosynthesis, namely, ofS-adenosylmethionine decarboxylase (AdoMetDC) and ornithine decarboxylase (ODC), were investigated on growth, polyamine metabolism, and DNA methylation in the Caco-2 cell line. Both inhibitors caused growth inhibition and affected similarly the initial expression of the differentiation marker sucrase. In the presence of the AdoMetDC inhibitor, ODC activity and the intracellular pool of putrescine were enhanced, whereas the spermidine and spermine pools were decreased. In the presence of the ODC inhibitor, the AdoMetDC activity was enhanced and the intracellular pools of putrescine and spermidine were decreased. With both compounds, the degree of global DNA methylation was increased. Spermine and spermidine (but not putrescine) selectively inhibited cytosine–DNA methyltransferase activity. Our observations suggest that spermidine (and to a lesser extent spermine) controls DNA methylation and may represent a crucial step in the regulation of Caco-2 cell growth and differentiation.