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.     

Characterization of lipid-protein interactions in acetylcholinesterase lipoprotein extracted from bovine erythrocytes.

1. The activation of human peripheral blood lymphocytes by phytohaemagglutinin in vitro was accompanied by striking increases in the concentrations of the natural polyamines putrescine, spermidine and spermine. 2. The enhanced accumulation of polyamines could be almost totally abolished by dl-alpha-difluoromethylornithine, a newly discovered irreversible inhibitor of l-ornithine decarboxylase (EC 4.1.1.17), or by methylglyoxal bis(guanylhydrazone) {1,1'-[(methylethanediylidene)dinitrilo]diguanidine}, an inhibitor of S-adenosyl-l-methionine decarboxylase (EC 4.1.1.50). The inhibition of polyamine accumulation was associated with a marked suppression of DNA synthesis, which was partially or totally reversed by low concentrations of exogenous putrescine, spermidine, spermine and cadaverine and by higher concentrations of 1,3-diaminopropane. 3. In contrast with some earlier studies, we found that methylglyoxal bis(guanylhydrazone), at concentrations that were sufficient to prevent polyamine accumulation, also caused a clear inhibition of protein synthesis in the activated lymphocytes. Similar results were obtained with difluoromethylornithine. The decrease in protein synthesis caused by both compounds preceded the impairment of DNA synthesis. The inhibition of protein synthesis by difluoromethylornithine was fully reversed by exogenous putrescine, spermidine and spermine, and that caused by methylglyoxal bis(guanylhydrazone) by spermidine and spermine. In further support of the idea that the inhibition of protein synthesis by these compounds was related to the polyamine depletion, we found that difluoromethylornithine caused a dose-dependent decrease in the incorporation of [(14)C]leucine into lymphocyte proteins which closely correlated with the decreased concentrations of cellular spermidine. 4. Difluoromethylornithine and methylglyoxal bis(guanylhydrazone) also elicited a variable depression in the incorporation of [(3)H]uridine and [(14)C]adenine into total RNA. The apparent turnover of lymphocyte RNA remained essentially unchanged in spite of severe polyamine depletion brought about by difluoromethylornithine. 5. The present results, as well as confirming the anti-proliferative action of the inhibitors of polyamine biosynthesis, suggest that polyamine depletion may interfere with reactions at different levels of gene expression.     

Polyamines in the lung: polyamine uptake and polyamine-linked pathological or toxicological conditions

The natural polyamines putrescine, cadaverine, spermidine, and spermine are found in all cells. These (poly)cations exert interactions with anions, e.g., DNA and RNA. This feature represents their best-known direct physiological role in cellular functions: cell growth, division, and differentiation. The lung and, more specifically, alveolar epithelial cells appear to be endowed with a much higher polyamine uptake system than any other major organ. In the lung, the active accumulation of natural polyamines in the epithelium has been studied in various mammalian species including rat, hamster, rabbit, and human. The kinetic parameters (Michaelis-Menten constant and maximal uptake) of the uptake system are the same order of magnitude regardless of the polyamine or species studied and the in vitro system used. Also, other pulmonary cells accumulate polyamines but never to the same extent as the epithelium. Although different uptake systems exist for putrescine, spermidine, and spermine in the lung, neither the nature of the carrier protein nor the reason for its existence is known. Some pulmonary toxicological and/or pathological conditions have been related to polyamine metabolism and/or polyamine content in the lung. Polyamines possess an important intrinsic toxicity. From in vitro studies with nonpulmonary cells, it has been shown that spermidine and spermine can be metabolized to hydrogen peroxide, ammonium, and acrolein, which can all cause cellular toxicity. In hyperoxia or after ozone exposure, the increased polyamine synthesis and polyamine content of the rat lung is correlated with survival of the animals. Pulmonary hypertension induced by monocrotaline or hypoxia has also been linked to the increased polyamine metabolism and polyamine content of the lung. In a small number of studies, it has been shown that polyamines can contribute to the suppression of immunologic reactions in the lung.     

Formation of Polyamines in the Rumen of Goats During Growth

Ornithine decarboxylase (E.G. 4.1.1.17) and S-adenosylmethionine decarboxylase (E.G. 4.1.1.50) and their products putrescine, spermidine and spermine were estimated in the rumen liquid from 3 groups of growing kids and 23 adult goats. Polyamines were also estimated in the feedstuff used. Marked differences in polyamine synthesis in rumen liquid were observed between the different groups of kids. Two groups of kids growing up together with adult goats had at an age of 2–4 months a peak of a few days duration in enzyme activity as well as in polyamine concentration. In these groups ornithine decarboxylase activity reached maximal values of 158±79 s (n = 4) and 100 (66–117) (n = 3) nmol[14CO2]/ml rumen liquid/h at an age of 120 and 77 days, respectively. The corresponding activity in rumen liquid from kids who were isolated from other animals was only about 1/10 of this value. By comparison ornithine decarboxylase activity in adult goats was 30.7±20 (n = 43) nmol[14CO]/ml/h. In rumen liquid from kids grown up together with adults, concentrations of the polyamines reached maximum at about the same time as ornithine decarboxylase activity. The mean maximal concentration of putrescine in the 2 groups was about 350 and 500 nmol/ml, while the corresponding value for spermidine was about 200 nmol/ml in both groups. Relatively constant and high concentration of polyamines were present in the feedstuff used. However, in growing kids the ruminai putrescine and spermidine concentration at times far exceeded those that could be accounted for by the estimated intake of polyamines by the food. The results therefore strongly indicate that polyamines are formed in considerable amounts in rumen content of kids during the phase of rapid growth. Results from a few experiments with calves also indicate that this may be true for cattle. polyamines; putrescine; spermidine; spermine; ornithine-decarboxylase; rumen liquid.     

Active transport and metabolic characteristics of polyamines in the rat lens

Putrescine, spermidine and spermine were transported into the rat lens against a concentration gradient. This process appeared to be energy-dependent and involved a carrier system different from those for amino acids. Competition experiments suggested that the three polyamines were transported by the same system or very similar systems. Incorporated spermine was converted to spermidine and putrescine, and spermidine was converted to putrescine. In contrast, the conversion of putrescine to spermidine and spermine, or the conversion of spermidine to spermine was not observed. Furthermore, ornithine was not utilized for the synthesis of putrescine. These metabolic characteristics of the polyamines in the rat lens were correlated with the extremely low activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase. Other enzymes of polyamine metabolisms, however, were relatively active. In conclusion, the lens has a very low ability for the de novo synthesis of polyamines. The polyamines in the lens are considered to be supplied form the surrounding intraocular fluid by an active transport system specific for polyamines.     

Role of polyamines in the stimulation of synthesis and secretion of plasminogen activator from bovine aortic endothelial cells

The effects of the polyamines putrescine (PUT), spermidine (SPD), and spermidine (SPM) on the secretion of plasminogen activator (PA) and plasminogen activator inhibitor (PAI) were evaluated using cultured bovine aortic endothelial cells. All three polyamines enhanced PA secretion in a time- and dose-dependent manner, with a potency rank order of SPM greater than SPD greater than PUT. The PA stimulation required both RNA and protein synthesis, as evidenced by inhibition of polyamine-induced PA secretion by actinomycin D and cycloheximide. The inhibitors of polyamine biosynthesis methylglyoxal bis-(guanylhydrazone) (MGBG) and dl-(difluoromethyl) ornithine (DFMO) alone did not affect basal or polyamine-induced PA secretion, with the exception that MGBG reduced the effect of PUT. Polyamine-treated cells enhanced secretions of both tissue-type and urokinase-type PA. The results of the present study suggest that polyamines may play a role in the regulation of PA synthesis and secretion and that this function can be modified under pathophysiological conditions affecting cellular and tissue levels of polyamines.     

Evidence that hypoxic pulmonary vascular remodeling in rats is polyamine dependent

This study tested the hypothesis that the polyamines, a family of low-molecular-weight organic cations with documented regulatory roles in cell growth and differentiation, are mediators of chronic hypoxia-induced pulmonary vascular remodeling. Relative to room air controls, chronically hypoxic animals (inspired O2 fraction = 0.1; 21 days) exhibited higher pulmonary arterial pressures (measured in room air), thicker medial layers in pulmonary arteries of 50-100 microns diam, increased hematocrits, and right ventricular hypertrophy. In addition, lung contents of the polyamines, putrescine, spermidine, and spermine were greater in hypoxic animals than in controls. alpha-Difluoromethylornithine (DFMO), an inhibitor of polyamine synthesis, attenuated the hypoxia-induced elevations in lung putrescine and spermidine content and blunted the increases in pulmonary arterial pressure and medial thickness. Neither the increased hematocrit nor right ventricular hypertrophy associated with chronic hypoxia were abrogated by DFMO. In addition, DFMO failed to influence vasoconstrictor responses provoked by acute hypoxic ventilation in isolated, buffer-perfused rat lungs. These observations suggest that depression of polyamine biosynthesis with DFMO blunts the sustained increase in pulmonary arterial pressure by attenuating hypoxia-induced medial thickening.     

Enhanced differential synthesis of proteins in a mammalian cell-free system by addition of polyamines.

Addition of the polyamines spermidine, spermine, or putrescine to a fractionated mammalian cell-free protein-synthesis system programmed by a variety of mRNAs results in a 3- to 5-fold stimulation of amino acid incorporation over that found in the absence of added polyamine. The mRNAs used as template were adenovirus mRNA, globin 9s mRNA, and RNA from the bacteriophages R17, Qbeta, and MS2. The relative amounts of 10 adenovirus polypeptides synthesized in vitro are altered by the addition of polyamines to the translation system to reflect more closely the relative amounts of these polypeptides synthesized in vivo. This qualititive improvement in translation products on addition of polyamines allow the analysis of a number of products which are at best only marginally synthesized in the absence of added polyamines. The low level of synthesis due to endogenous mRNA is stimulated by spermidine and spermine but a lesser extent by putrescine.     

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.     

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.     

Seasonal changes of polyamines in habitat adaptation of different ecotypes of reed plants

The leaves of four reed ecotypes (Phragmites communis Trinius) growing in the desert regions of northwest China were investigated for levels of polyamines and activity of arginine decarboxylase (ADC; EC 4.1.1.19) during the growing season of 5 months. The polyamines in the leaves of all reed ecotypes consisted of putrescine, spermidine and spermine. The polyamine levels of the leaves were lower in the swamp reed than in the terrestrial reed ecotypes. Leaf polyamine levels decreased in all ecotypes over the course of the season. Compared to the swamp reed, the terrestrial reed ecotypes maintained higher ADC activity and a predominance of spermine, resulting in a lower ratio of putrescine to spermidine and spermine. It seems that the adaptation of reed plants to drought and saline habitats may be correlated with putrescine synthesis via the ADC pathway, and with a successful conversion of putrescine to spermidine and spermine.     

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.     

Polyamines in brain and heart of the neonatal rat: effects of inhibitors of ornithine decarboxylase and spermidine synthase

Daily administration of dicyclohexylamine (DCHA), an inhibitor of spermidine synthase, to neonatal rats produced a dose-dependent depletion of brain spermidine, accompanied by a rise in putrescine and spermine. Despite continued DCHA treatment, levels of all three polyamines returned toward normal within two weeks. alpha-Difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, had a much more profound and persistent effect on spermidine and also depleted putrescine throughout drug administration; furthermore, DFMO prevented both the elevation of putrescine caused by DCHA and the eventual restitution of spermidine levels. Although a similar pattern of effects was seen in the heart, the time course of onset of DCHA-induced alterations in polyamine levels and the rapidity of subsequent adaptation were considerably different from those in brain. The net activity of DCHA toward polyamines in developing tissues thus involves the direct actions of the drug on spermidine synthesis in combination with compensatory metabolic adjustments made by each tissue to polyamine depletion.     

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.     

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.