Polyamine Metabolism and Osmotic Stress : II. Improvement of Oat Protoplasts by an Inhibitor of Arginine Decarboxylase

We have attempted to improve the viability of cereal mesophyll protoplasts by pretreatment of leaves with dl-α-difluoromethylarginine (DFMA), a specific `suicide' inhibitor of the enzyme (arginine decarboxylase) responsible for their osmotically induced putrescine accumulation. Leaf pretreatment with DFMA before a 6 hour osmotic shock caused a 45% decrease of putrescine and a 2-fold increase of spermine titer. After 136 hours of osmotic stress, putrescine titer in DFMA-pretreated leaves increased by only 50%, but spermidine and spermine titers increased dramatically by 3.2- and 6-fold, respectively. These increases in higher polyamines could account for the reduced chlorophyll loss and enhanced ability of pretreated leaves to incorporate tritiated thymidine, uridine, and leucine into macromolecules. Pretreatment with DFMA significantly improved the overall viability of the protoplasts isolated from these leaves. The results support the view that the osmotically induced rise in putrescine and blockage of its conversion to higher polyamines may contribute to the lack of sustained cell division in cereal mesophyll protoplasts, although other undefined factors must also play a major role.     

Ornithine decarboxylase transgene in tobacco affects polyamines,in vitro-morphogenesis and response to salt stress

Transgenic tobacco plants expressing the putrescine synthesis gene ornithine decarboxylase from mouse were raised to study the effects of up-regulation of a metabolic pathway as critical as the polyamine biosynthesis on the plant growth and development, in vitro-morphogenesis and their response to salt stress. Further, the response of the alternate pathway (arginine decarboxylase) for putrescine synthesis to the modulation of the ornithine decarboxylase pathway has also been investigated. The over-expression of the odc gene and increased levels of putrescine in tobacco led to a delay in plant regeneration on selection medium which could be overcome by the exogenous application of polyamine biosynthesis inhibitors and spermidine. Further, the lines generated had a variable in vitro morphogenic potential, which could be correlated to the shifts in their polyamine metabolism. These studies have brought forward the critical role played by polyamines in the normal development of plants and also their role in plant regeneration. Since polyamines are known to accumulate in cells under abiotic stress conditions, the tolerance of the transgenics to salt stress was also investigated and the transgenics with their polyamine metabolism up-graded showed increased tolerance to salt stress.     

Adjustment of polyamine contents in Escherichia coli.

Adjustment of polyamine contents in Escherichia coli was studied with strains of Escherichia coli producing normal (DR112) and excessive amounts of ornithine decarboxylase [DR112(pODC)] or S-adenosylmethionine decarboxylase [DR112(pSAMDC)]. Although DR112(pODC) produced approximately 70 times more ornithine decarboxylase than DR112 did, the amounts of polyamines in the cells of both strains did not change significantly. The amounts of polyamines in DR112(pODC) were adjusted by excretion of excessive amounts of putrescine to the medium. When ornithine was deficient in cells, polyamine contents in DR112(pODC) were much higher than those in DR112, although polyamine contents were low in both strains. This indicates that large amounts of ornithine decarboxylase increased the utilization of ornithine for putrescine synthesis. During ornithine deficiency, strain DR112 produced 3.4 times more ornithine decarboxylase. Strain DR112(pSAMDC) produced seven times more S-adenosylmethionine decarboxylase than DR112 did. In DR112(pSAMDC) an increase (40%) in spermidine content, a decrease (35%) in putrescine content, and no significant excretion of putrescine and spermidine were observed. The amount of ornithine decarboxylase in DR112(pSAMDC) was approximately 30% less than that in DR112. In addition, S-adenosylmethionine decarboxylase activity was strongly inhibited by spermidine. A possible regulatory mechanism to maintain polyamine contents in Escherichia coli is discussed based on the results.     

Anandamide, a Brain Endogenous Compound, Interacts Specifically with Cannabinoid Receptors and Inhibits Adenylate Cyclase

Abstract: We investigated the role of polyamines and their regulatory enzyme ornithine decarboxylase in N -Methyl-D-aspartate-induced excitotoxicity in embryonic chick retina. N -Methyl-D-aspartate (200 μM) produced an early increase in ornithine decarboxylase activity, putrescine concentration, and Ca²+ entry, leading to selective neuronal death by 30 min. This response was attenuated by the ornithine decarboxylase inhibitor α-difluoromethylornithine and the N -methyl-D-aspartate receptor antagonist 5-aminophosphonovaleric acid. Exogenous putrescine increased intracellular putrescine and spermine levels and reversed neuroprotection by α-difluoromethylornithine, but not by 5-aminophosphonovaleric acid. N -Methyl-D-aspartate-receptor stimulation of putrescine/polyamine synthesis mediates abnormal Ca²+ entry and acute excitotoxic neuronal death. Postreceptor inhibition of the ornithine decar-boxylase/polyamine cascade by α-difluoromethylornithine may provide neuroprotection against N -methyl-D-aspartate-induced excitotoxicity.     

Relation of polyamine biosynthesis to the initiation of sprouting in potato tubers

The polyamines putrescine, spermidine, and spermine and their biosynthetic enzymes arginine decarboxylase, ornithine decarboxylase and S-adenosyl-l-methionine decarboxylase are present in all parts of dormant potato (Solanum tuberosum L.) tubers. They are equally distributed among the buds of apical and lateral regions and in nonbud tissues. However, the breaking of dormancy and initiation of sprouting in the apical bud region are accompanied by a rapid increase in ornithine decarboxylase and S-adenosyl-l-methionine decarboxylase activities, as well as by higher levels of putrescine, spermidine, and spermine in the apical buds. In contrast, the polyamine biosynthetic enzyme activities and titer remain practically unchanged in the dormant lateral buds and in the nonbud tissues. The rapid rise in ornithine decarboxylase, but not arginine decarboxylase activity, with initiation of sprouting suggests that ornithine decarboxylase is the rate-limiting enzyme in polyamine biosynthesis. The low level of polyamine synthesis during dormancy and its dramatic increase in buds in the apical region at break of dormancy suggest that polyamine synthesis is linked to sprouting, perhaps causally.     

Utilization of putrescine in tobacco cell lines resistant to inhibitors of polyamine synthesis

Three tobacco cell lines have been analyzed which are resistant to lethal inhibitors of either putrescine production or conversion of putrescine into polyamines. Free and conjugated putrescine pools, the enzymic activities (arginine, ornithine, and S-adenosylmethionine decarboxylases), and the growth characteristics during acidic stress were measured in suspension cultures of each cell line. One cell line, resistant to difluoromethylornithine (Dfr1) had a very low level of ornithine decarboxylase activity which was half insensitive to the inhibitor in vitro. Intracellular free putrescine in Dfr1 was elevated 10-fold which was apparently due to a 20-fold increase in the arginine decarboxylase activity. The increased free putrescine titer was not reflected in an increased level of spermidine, spermine, or putrescine conjugation. Dfr1 cultures survived acidic stress at molarities which were lethal to wild type cultures. Two other mutants, resistant to methylglyoxal bis(guanylhydrazone) (Mgr3, Mgr12), had near normal levels of the three decarboxylases and normal titers of free putrescine, spermidine, and spermine. Both mutants however had elevated levels of conjugated putrescine. Mgr12 had an increased sensitivity to acidic medium. These results suggest that increased levels of free putrescine production may enhance the ability of tobacco cells to survive acid stress. This was supported by the observation that cytotoxic effects of inhibiting arginine decarboxylase in wild type cell lines were dependent on the acidity of the medium.     

Putrescine and Acid Stress : Induction of Arginine Decarboxylase Activity and Putrescine Accumulation by Low pH

Incubation of peeled oat Avena sativa L. var Victory leaf segments on media of pH 5.0 or below leads to a rapid and massive increase in the titer of putrescine while incubation at pH values above 5.0 causes little or no change. The low pH effect is independent of the buffer system employed. Putrescine levels rise within 3 hours and reach their peak 8 to 9 hours after acidification. At this time, putrescine titer is eight times greater at pH 3.5 than at 6.0. None of the other polyamines shows a response to changes in external pH. The increase in putrescine is blocked by the addition of cycloheximide or dl-alpha-difluoromethylarginine, a specific inhibitor of the putrescine biosynthetic enzyme, arginine decarboxylase. In one experiment, arginine decarboxylase activity was 110% greater at pH 4.0 than at 6.0 after a 4-hour incubation, although the average increase over many experiments was 47%. The activity of the other possible putrescine biosynthetic enzyme, ornithine decarboxylase, falls throughout the incubation period and is virtually equal at pH 4.0 and 6.0.     

Putrescine does not support the migration and growth of IEC-6 cells

The migration of IEC-6 cells is inhibited when the cells are depleted of polyamines by inhibiting ornithine decarboxylase with alpha-difluoromethylornithine (DFMO). Exogenous putrescine, spermidine, and spermine completely restore cell migration inhibited by DFMO. Because polyamines are interconverted during their synthesis and catabolism, the specific role of individual polyamines in intestinal cell migration, as well as growth, remains unclear. In this study, we used an inhibitor of S-adenosylmethionine decarboxylase, diethylglyoxal bis(guanylhydrazone)(DEGBG), to block the synthesis of spermidine and spermine from putrescine. We found that exogenous putrescine does not restore migration and growth of IEC-6 cells treated with DFMO plus DEGBG, whereas exogenous spermine does. In addition, the normal distribution of actin filaments required for migration, which is disrupted in polyamine-deficient cells, could be achieved by adding spermine but not putrescine along with DFMO and DEGBG. These results indicate that putrescine, by itself, is not essential for migration and growth, but that it is effective because it is converted into spermidine and/or spermine.     

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.     

Control of ornithine decarboxylase in Chinese hamster ovary cells by polyamines. Translational inhibition of synthesis and acceleration of degradation of the enzyme by putrescine, spermidine, and spermine

We have recently isolated, without using any inhibitors, a mutant of Chinese hamster ovary cell line which greatly overproduces ornithine decarboxylase in serum-free culture. Addition of polyamines (putrescine, spermidine, or spermine, 10 microM) or ornithine (1 mM), the precursor of polyamines, to the culture medium of these cells caused a rapid and extensive decay of ornithine decarboxylase activity. At the same time the activity of S-adenosylmethionine decarboxylase showed a less pronounced decrease. Notably, the polyamine concentrations used were optimal for growth of the cells and caused no perturbation of general protein synthesis. Spermidine and spermine appeared to be the principal regulatory amines for both enzymes, but also putrescine, if accumulated at high levels in the cells, was capable of suppressing ornithine decarboxylase activity. The amount of ornithine decarboxylase protein (as measured by radioimmunoassay) declined somewhat more slowly than the enzyme activity, but no more than 10% of the loss of activity could be ascribed to post-translational modifications or inhibitor interaction. Some evidence for inactivation through ornithine decarboxylase-antizyme complex formation was obtained. Gel electrophoretic determinations of the [35S]methionine-labeled ornithine decarboxylase revealed a rapid reduction in the synthesis and acceleration in the degradation of the enzyme after polyamine additions. No decrease in the amounts of the two ornithine decarboxylase-mRNA species, hybridizable to a specific cDNA, was detected, suggesting that polyamines depressed ornithine decarboxylase synthesis by selectively inhibiting translation of the message.     

Polyamines and environmental challenges: recent development

In this review, we will try to summarize some recent data concerning the changes in polyamine metabolism (biosynthesis, catabolism and regulation) in higher plants subjected to a wide array of environmental stress conditions and to describe and discuss some of the new advances concerning the different proposed mechanisms of polyamine action implicated in plant response to environmental challenges. All the data support the view that putrescine and derived polyamines (spermidine, spermine, long-chained polyamides) may have several functions during environmental challenges. In several systems (except during hypoxia, and chilling tolerance of wheat and rice) an induction of polyamines (spermidine, spermine) not putrescine accumulation, may confer a stress tolerance. In several cases stress tolerance is associated with the production of conjugated and bound polyamines and stimulation of polyamine oxidation. In several environmental challenges (osmotic-stress, salinity, hypoxia, environmental pollutants) recent results indicate that both arginine decarboxylase and ornithine decarboxylase are required for the synthesis of putrescine and polyamines (spermidine and spermine). Under osmotic and salt-stresses a production of cadaverine is observed in plants. A new study demonstrates that under salt-stress putrescine catabolism (via diamine oxidase) can contribute to proline (a compatible osmolyte) accumulation.     

Polyamine Metabolism and Osmotic Stress : I. Relation to Protoplast Viability

Cereal leaves subjected to the osmotica routinely used for protoplast isolation show a rapid increase in arginine decarboxylase activity, a massive accumulation of putrescine, and slow conversion of putrescine to the higher polyamines, spermidine, and spermine (HE Flores, AW Galston 1984 Plant Physiol 75: 102). Mesophyll protoplasts from these leaves, which have a high putrescine:polyamine ratio, do not undergo sustained division. By contrast, in Nicotiana, Capsicum, Datura, Trigonella, andVigna, dicot genera that readily regenerate plants from mesophyll protoplasts, the response of leaves to osmotic stress is opposite to that in cereals. Putrescine titer as well as arginine and ornithine decarboxylase activities decline in these osmotically stressed dicot leaves, while spermidine and spermine titers increase. Thus, the putrescine:polyamine ratio in Vigna protoplasts, which divide readily, is 4-fold lower than in oat protoplasts, which divide poorly. We suggest that this differing response of polyamine metabolism to osmotic stress may account in part for the failure of cereal mesophyll protoplasts to develop readily in vitro.     

Putrescine and spermidine control degradation and synthesis of ornithine decarboxylase in Neurospora crassa

Neurospora crassa mycelia, when starved for polyamines, have 50-70-fold more ornithine decarboxylase activity and enzyme protein than unstarved mycelia. Using isotopic labeling and immunoprecipitation, we determined the half-life and the synthetic rate of the enzyme in mycelia differing in the rates of synthesis of putrescine, the product of ornithine decarboxylase, and spermidine, the main end-product of the polyamine pathway. When the pathway was blocked between putrescine and spermidine, ornithine decarboxylase synthesis rose 4-5-fold, regardless of the accumulation of putrescine. This indicates that spermidine is a specific signal for the repression of enzyme synthesis. When both putrescine and spermidine synthesis were reduced, the half-life of the enzyme rapidly increased 10-fold. The presence of either putrescine or spermidine restored the normal enzyme half-life of 55 min. Tests for an ornithine decarboxylase inhibitory protein ("antizyme") were negative. The regulatory mechanisms activated by putrescine and spermidine account for most or all of the regulatory amplitude of this enzyme in N. crassa.     

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.     

Polyamine homeostasis in arginase knockout mice

The role of ornithine decarboxylase (ODC) in polyamine metabolism has long been established, but the exact source of ornithine has always been unclear. The arginase enzymes are capable of producing ornithine for the production of polyamines and may hold important regulatory functions in the maintenance of this pathway. Utilizing our unique set of arginase single and double knockout mice, we analyzed polyamine levels in the livers, brains, kidneys, and small intestines of the mice at 2 wk of age, the latest timepoint at which all of them are still alive, to determine whether tissue polyamine levels were altered in response to a disruption of arginase I (AI) and II (AII) enzymatic activity. Whereas putrescine was minimally increased in the liver and kidneys from the AII knockout mice, spermidine and spermine were maintained. ODC activity was not greatly altered in the knockout animals and did not correlate with the fluctuations in putrescine. mRNA levels of ornithine aminotransferase (OAT), antizyme 1 (AZ1), and spermidine/spermine-N¹-acetyltransferase (SSAT) were also measured and only minor alterations were seen, most notably an increase in OAT expression seen in the liver of AI knockout and double knockout mice. It appears that putrescine catabolism may be affected in the liver when AI is disrupted and ornithine levels are highly reduced. These results suggest that endogenous arginase-derived ornithine may not directly contribute to polyamine homeostasis in mice. Alternate sources such as diet may provide sufficient polyamines for maintenance in mammalian tissues. ornithine; putrescine; spermidine; spermine; decarboxylase     

Inhibition of L-arginine iminohydrolase (EC 3.5.3.6) from Tetrahymena thermophila by putrescine and spermidine: feedback control of polyamine biosynthesis

L-Arginine iminohydrolase (arginine deiminase, ADI) from Tetrahymena thermophila was purified approx. 75-fold by means of gel permeation chromatography. The Km of the purified enzyme for L-arginine was 412 +/- 25 microM and L-ornithine inhibited the reaction competitively with a Ki of 985 +/- 105 microM. D-Ornithine was a weak inhibitor with a Ki of greater than 10mM. The polyamines putrescine and spermidine inhibited ADI incompetitively with a Kii of 2.8mM for putrescine and 4.3mM for spermidine. Since the concentrations required for inhibition were within the range of the normal intracellular polyamine concentrations in Tetrahymena (maximally 14mM putrescine and 4mM spermidine), it is suggested that the polyamine effects on ADI are of regulatory nature. Thus, polyamine biosynthesis in Tetrahymena thermophila is regulated not only on the level of ornithine decarboxylase activity, but also on an earlier step, the supply of ODC with substrates.     

Translational regulation of mammalian ornithine decarboxylase by polyamines

Ornithine decarboxylase, which catalyses the formation of putrescine, is the first and rate-limiting enzyme in the biosynthesis of polyamines in mammalian cells. The enzyme is highly regulated, as indicated by rapid changes in its mRNA and protein during cell growth. Here we report that ornithine decarboxylase is regulated at the translational level by polyamines in difluoromethylornithine-resistant mouse myeloma cells that overproduce the enzyme due to amplification of an ornithine decarboxylase gene. When such cells are exposed to putrescine or other polyamines, there is a rapid and specific decrease in the rate of synthesis of ornithine decarboxylase, assayed by pulse-labeling. Neither the cellular content of ornithine decarboxylase mRNA nor the half-life of ornithine decarboxylase protein is affected. Our results indicate that polyamines negatively regulate the translation of ornithine decarboxylase mRNA, thereby controlling their own synthesis.     

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 and Anaerobic Elongation of Rice Coleoptile

The role of polyamines in the anaerobic elongation of rice (Oryzasativa L.) coleoptiles was studied. The reduced growth of ricecoleoptiles under anoxic conditions was accompanied by a massiveaccumulation of free putrescine. Putrescine was synthesizedfrom arginine in a reaction catalyzed by arginine decarboxylase(ADC). The anoxic titer of putrescine was closely correlatedwith elongation of coleoptiles. In experiments in which putrescineand inhibitors [-difluoromethylarginine (DFMA) and -difluoromethylornithine(DFMO)] of the synthesis of polyamines were exogenously supplied,we demonstrated an absolute requirement for putrescine, synthesizedby ADC, for anaerobic elongation of coleoptiles. The presenceof exogenous putrescine (alone or in combination with DFMA)increased the rate of anaerobic elongation of coleoptile by30–40%. (Received December 1, 1988; Accepted June 19, 1989)     

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.