Regulatory interrelations between GABA and polyamines. I. Brain GABA levels and polyamine metabolism

Elevation of brain GABA levels by GABA-T inhibition is accompanied by a decrease ofS-adenosylmethionine decarboxylase activity. This is followed by an increase of ornithine decarboxylase activity and a severalfold increase of brain putrescine levels. Spermidine and spermine levels are not significantly affected under these conditions. These unexpected findings support a regulatory interaction between GABA and polyamine metabolism.     

Effect of 1,25-dihydroxyvitamin D on S-adenosylmethionine decarboxylase in chick intestine

The effect of 1,25-dihydroxyvitamin D on the activity of S-adenosylmethionine decarboxylase in the duodenal mucosa of vitamin D-deficient chicks was investigated. Enzyme activity increased dose-dependently in a biphasic manner with maximal responses at 1 and 6 h, due to an increase in V_max in both cases. A second dose of 1,25-dihydroxyvitamin D, administered 6 h after the first, resulted in a significant increase in activity 1 h later, confirming the rapidity of the response. This early response was not seen with ornithine decarboxylase. The increase in S-adenosylmethionine decarboxylase activity particularly at 6 h may be due to a rise in cytosolic calcium, since hydrocortisone, an inhibitor of 1,25-dihydroxyvitamin D-stimulated calcium absorption, attenuates this enzyme's activity. Inhibitors of polyamine biosynthesis such as DL-α-difluoromethyl ornithine and methylglyoxal bis(guanylhydrazone) had no effect on calcium absorption, but the significance of this in evaluating the importance of 1,25-dihydroxyvitamin D stimulation of S-adenosylmethionine decarboxylase activity needs further investigation.     

The effect of salt stress on polyamine biosynthesis and content in mung bean plants and in halophytes

The activity of L-arginine decarboxylase (EC 4.1.1.19) and L-ornithine decarboxylase (EC 4.1.1.17), polyamine content, and incorporation of arginine and ornithine into polyamines, were determined in mung bean [Vigna radiata (L.) Wilczek] plants subjected to salt (hypertonic) stress (NaCl at 0.51–2.27 MPa). Changes in enzyme activity in response to hypotonic stress were determined as well in several halophytes [Pulicaria undulata (L.), Kostei, Salsola rosmarinus (Ehr.) Solms-Laub, Mesembryanthemum forskahlei Hochst, and Atriplex halimus L.]. NaCl stress, possibly combined with other types of stress that accompanied the experimental conditions, resulted in organ-specific changes in polyamine biosynthesis and content in mung bean plants. The activity of both enzymes was inhibited in salt-stressed leaves. In roots, however, NaCl induced a 2 to 8-fold increase in ornithine decarboxylase activity. Promotion of ornithine decarboxylase in roots could be detected already 2 h after exposure of excised roots to NaCl, and iso-osmotic concentrations of NaCl and KCl resulted in similar changes in the activity of both enzymes. Putrescine level in shoots of salt-stressed mung bean plants increased considerably, but its level in roots decreased. The effect of NaCl stress on spermidine content was similar, but generally more moderate, resulting in an increased putrescine/spermidine ratio in salt-stressed plants. Exposure of plants to NaCl resulted also in organ-specific changes in the incorporation of both arginine and ornithine into putrescine: incorporation was inhibited in leaf discs but promoted in excised roots of salt-stressed mung bean plants. In contrast to mung bean (and several other glycophytes), ornithine and arginine decarboxylase activity in roots of halophytes increased when plants were exposed to tap water or grown in a pre-washed soil—i.e. a hypotonic stress with respect to their natural habitat. NaCl, when present in the enzymatic assay mixture, inhibited arginine and ornithine decarboxylase in curde extracts of mung bean roots, but did not affect the activity of enzymes extracted from roots of the halophyte Pulicaria. Although no distinct separation between NaCl stress and osmotic stress could be made in the present study, the data suggest that changes in polyamines in response to NaCl stress in mung bean plants are coordinated at the organ level: activation of biosynthetic enzymes concomitant with increased putrescine biosynthesis from its precursors in the root system, and accumulation of putrescine in leaves of salt-stressed plants. In addition, hypertonic stress applied to glycophytes and hypotonic stress applied to halophytes both resulted in an increase in the activity of polyamine biosynthetic enzymes in roots.     

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.     

Polyamine metabolism and gene regulation during the transition of autonomous sugar beet cells in suspension culture from quiescence to division

Sugar beet cells grown in batch suspension culture have been used to study the regulation of polyamine levels during the transition from a quiescent to a proliferating state. The quiescent state was achieved by maintenance of the phytohormone autonomous cells in the stationary phase of the batch culture cycle. After subculture into fresh medium there was an increase in DNA synthesis which was accompanied by a dramatic increase in cellular polyamine levels. The levels of both free and bound cellular putrescine and spermidine within the cells reached a peak before the onset of the first synchronous division. The levels of putrescine, spermidine and to some extent spermine in the culture medium also increased dramatically shortly after subculture. The increase in polyamines was preceded by a rapid but transient increase in omithine decarboxylase (EC 4.1.1.17) and S -adenosylmethionine decarboxylase (EC 4.1.1.50). Arginine decarboxylase (EC 4.1.1.19) and S -adenosylmethionine synthetase (EC 2.5.1.6) activity did not show the same pattern of cell division-related variation. Inhibition of S -adenosylmethionine biosynthesis with methylglyoxal bis-(guanylhydra-zone) (MGBG) reduced cell division in the suspension culture. Inhibitors of ornithine decarboxylase and arginine decarboxylase individually had little effect on cell division, but in combination led to a reduction in cell division. Addition of polyamines and their precursors to cells in the stationary phase of a batch culture cycle led to the induction of expression of a mitotic cyclin sequence ( Bvcycll ).     

Polyamine metabolism and gene regulation during the transition of autonomous sugar beet cells in suspension culture from quiescence to division

Sugar beet cells grown in batch suspension culture have been used to study the regulation of polyamine levels during the transition from a quiescent to a proliferating state. The quiescent state was achieved by maintenance of the phytohormone autonomous cells in the stationary phase of the batch culture cycle. After subculture into fresh medium there was an increase in DNA synthesis which was accompanied by a dramatic increase in cellular polyamine levels. The levels of both free and bound cellular putrescine and spermidine within the cells reached a peak before the onset of the first synchronous division. The levels of putrescine, spermidine and to some extent spermine in the culture medium also increased dramatically shortly after subculture. The increase in polyamines was preceded by a rapid but transient increase in omithine decarboxylase (EC 4.1.1.17) and S -adenosylmethionine decarboxylase (EC 4.1.1.50). Arginine decarboxylase (EC 4.1.1.19) and S -adenosylmethionine synthetase (EC 2.5.1.6) activity did not show the same pattern of cell division-related variation. Inhibition of S -adenosylmethionine biosynthesis with methylglyoxal bis-(guanylhydra-zone) (MGBG) reduced cell division in the suspension culture. Inhibitors of ornithine decarboxylase and arginine decarboxylase individually had little effect on cell division, but in combination led to a reduction in cell division. Addition of polyamines and their precursors to cells in the stationary phase of a batch culture cycle led to the induction of expression of a mitotic cyclin sequence ( BvcycII ).     

The putrescine analogue 1-aminooxy-3-aminopropane perturbs polyamine metabolism in the phytopathogenic fungus<Emphasis Type="Italic"> Sclerotinia sclerotiorum</Emphasis>

The effects of the putrescine analogue 1-aminooxy-3-aminopropane on fungal polyamine metabolism were evaluated using Sclerotinia sclerotiorum as an experimental model. The compound inhibited ornithine decarboxylase, spermidine synthase, and S -adenosyl-methionine decarboxylase in mycelial extracts. Addition of 1-aminooxy-3-aminopropane at 1 mM to the culture medium did not reduce mycelial growth and caused a 29% decrease in free spermidine and a two-fold increase in free spermine. When added 4.5 h before the determination of ornithine decarboxylase, 1-aminooxy-3-aminopropane reduced in vivo activity of this enzyme by 40–50%. When added 48 h before the determination, 1-aminooxy-3-aminopropane at 0.01 and 0.1 mM caused a slight increase of in vivo ornithine decarboxylase activity, while it had no effect at 1 mM. Comparison of the action of 1-aminooxy-3-aminopropane with that of other inhibitors of polyamine biosynthesis suggested that its effects on in vivo ornithine decarboxylase activity resulted from a balance between direct inhibition of enzyme activity and indirect stimulation of enzyme synthesis and/or activity mediated by the decrease in spermidine levels, which in turn was due to inhibition of spermidine synthase and S -adenosyl-methionine decarboxylase. The potential of 1-aminooxy-3-aminopropane as a tool for studies on fungal polyamine metabolism and for the control of plant diseases of fungal origin is discussed.Abbreviations AdoMetDC S-Adenosyl-methionine decarboxylase - DFMO -Difluoromethylornithine - MGBG Methylglyoxal bis-[guanyl hydrazone] - ODC Ornithine decarboxylase     

Polyamines and their biosynthetic enzymes during somatic embryo development in red spruce (<Emphasis Type="Italic">Picea rubens</Emphasis> Sarg.)

Summary The major objective of this study was to determine if the observed changes in polyamines and their biosynthetic enzymes during somatic embryo development were specifically related to either the stage of the embryo development or to the duration of time spent on the maturation medium. Somatic embryos of red spruce (Picea rubens) at different developmental stages, grown in the embryo development and maturation media for various lengths of time, were separated from the associated subtending tissue (embryogenic and the suspensor cell masses) and analyzed for their polyamine content as well as for polyamine biosynthetic enzyme activities. Polyamine content was also analyzed in embryos representing different stages of developmentthat were collected from the sam culture plate at the same time and the subtending tissue surrouding them. Putrescine was the predominant polyamine in the pro-embryogenic tissue, while spermidine was predominant during embryo development. Significant changes in spermidine/putrescine and spermine/putrescine ratios were observed at all stages of embryo development as compared to the pro-embryogenic cell mass. Changes in the ratios of various polyamines were clearly correlated with the developmental stage of the embryo rather than the period of growth in the maturation medium. Whereas the activities of both ornithine decarboxylase and arginine decarboxylase increased by week 3 or 4 and stayed high during the subsequent 6 wk of growth, the activity of S-adenosylmethionine decarboxylase steadily declined during embryo development.     

NEUROCHEMICAL ASPECTS OF NEURONAL ONTOGENESIS IN THE DEVELOPING RAT CEREBELLUM: CHANGES IN NEUROTRANSMITTER AND POLYAMINE SYNTHESIZING ENZYMES

Abstract— Changes in the activities of several specific enzymes were measured in the cerebellum during development. Early transient increases were found in both ornithine decarboxylase and S -adenosylmethionine decarboxylase, enzymes involved in the initial steps of polyamine synthesis. Different patterns of changes were found in neurotransmitter synthesizing enzymes. Tyrosine hydroxylase activity achieved adult levels very early, by 3 days after birth, and remained at this level. Glutamic acid decarboxylase activity, while very low at early stages, increased rapidly before birth and then after a lag period of 10 days started to increase rapidly, directly related to the general growth of cerebellar weight and protein content. Choline acetyltransferase activity started to increase rapidly, reaching a peak of about 100% of adult levels at 3-7 days after birth; the activity then gradually declined and at 20 days, after reaching a low of about 55% of adult values, gradually started to increase, reaching adult levels later than 40 days after birth. The development of protein carboxymethylase activity was similar to that of glutamic acid decarboxylase, directly related to the general growth of the cerebellum. Several interpretations of the results are discussed.     

Ethanol perturbs polyamine metabolism in the phytopathogenic fungus Pyrenophora avenae

Biomass production by the plant pathogenic fungus Pyrenophora avenae was reduced following growth in 1, 3 and 6% ethanol. Although cadaverine concentration was not affected by growth in ethanol, putrescine and spermine concentrations were increased following growth in 3% ethanol and concentrations of spermidine and spermine were substantially increased following exposure to 6% ethanol. These changes were accompanied by significant increases in the activities of the polyamine biosynthetic enzymes ornithine decarboxylase and S-adenosylmethionine decarboxylase and in the flux of label from ornithine into the polyamines. Formation of the cadaverine derivatives aminopropylcadaverine and N,N-bis(3-aminopropyl)cadaverine was greatly increased in P. avenae exposed to 6% ethanol, probably via the action of lysine decarboxylase, S-adenosylmethionine decarboxylase and the aminopropyltransferases. There was also a doubling of polyamine oxidase activity following fungal growth in 6% ethanol.     

Ornithine Decarboxylase Activity in Brain Regulated by a Specific Macromolecul, the Antizyme

Mouse brain ornithine decarboxylase activity is about 70-fold higher at the time of birth compared with that of adult mice. Enzyme activity declines rapidly after birth and reaches the adult level by 3 weeks. Immunoreactive enzyme concentration parallels very closely the decrease of enzyme activity during the first postnatal week, remaining constant thereafter. The content of brain antizyme, the macromolecular inhibitor to ornithine decarboxylase, in turn is very low during the first 7 days and starts then to increase and at the age of 3 weeks it is about six times the level of that in newborn mice. This may explain the decrease in enzyme activity during brain maturation, and suggests the regulation of polyamine biosynthesis by an antizyme-mediated mechanism in adult brain.     

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.     

Polyamine metabolism during sclerotial development of Sclerotinia sclerotiorum

A study on polyamine metabolism and the consequences of polyamine biosynthesis inhibition on the development of Sclerotinia sclerotiorum sclerotia was conducted. Concentrations of the triamine spermidine and the tetramine spermine, as well as ornithine decarboxylase and S-adenosyl-methionine decarboxylase activities, decreased during sclerotia maturation. In turn, the concentration of the diamine putrescine was reduced at early stages of sclerotial development but it increased later on. This increment was not related to de novo biosynthesis, as demonstrated by the continuous decrease in ornithine decarboxylase activity. Alternatively, it could be explained by the release of putrescine from the conjugated polyamine pool. α-Difluoro-methylornithine and cyclohexylamine, which inhibit putrescine and spermidine biosynthesis, respectively, decreased mycelial growth, but did not reduce the number of sclerotia produced in vitro even though they disrupted polyamine metabolism during sclerotial development. It can be concluded that sclerotial development is less dependent on polyamine biosynthesis than mycelial growth, and that the increase of free putrescine is a typical feature of sclerotial development. The relationship between polyamine metabolism and sclerotial development, as well as the potential of polyamine biosynthesis inhibition as a strategy for the control of plant diseases caused by sclerotial fungi are discussed.     

Age and tissue-dependent changes of ornithine decarboxylase and s-adenosylmethionine decarboxylase activities in neural tissue and fat body of adult crickets

The activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase, two key enzymes in polyamine metabolism, were determined during the first 10 days of imaginal life in the nervous tissue and the fat body of the adult cricket Acheta domesticus. The kinetic constants of the two enzymes were also determined in both tissues. Both decarboxylases presented a higher activity in fat body than in nervous tissue. In nervous tissue, the activity of the two enzymes peaked at 16 h postemergence, then slowly decreased up to day 3–4. By contrast, the enzymatic activities in fat body, low at emergence, strongly increased on day 2. Thereafter, whereas ornithine decarboxylase activity remained rather high. S-adenosyl-methionine decarboxylase activity dropped back to emergence levels by day 10. These results, examined in light of the temporal alterations of polyamine levels observed in the two tissues, demonstrate synchronous variations between polyamine contents and the enzymes involved in their biosynthesis. © 1993 Wiley-Liss, Inc.     

Characterization of a carrot callus line resistant to high concentrations of putrescine

A mutant carrot callus line resistant to a high concentration (1 mM) of putrescine has been isolated. A high level of endogenous putrescine, about 13-fold higher than in the controls grown in the absence of putrescine, characterized this resistant mutant. Ornithine-, arginine- and S-adenosylmethionine decarboxylase activities were similar in the resistant line and in the controls by the fifth subculture. The uptake of putrescine when supplied to the medium at high concentration (1 mM), was similar in both the putrescine-treated calli and in the untreated controls. At low concentrations (0.64 M) however the putrescine absorbed by the resistant calli was less than that absorbed by the controls. Putrescine uptake took place almost always against a concentration gradient and might be due to an active mechanism.Abbreviations ADC arginine decarboxylase - 2,4D 2,4-dichlorophenoxyacetic acid - ODC ornithine decarboxylase - SAMDC S-adenosylmethionine decarboxylase - TCA trichloroacetic acid - TLC thin layer chromatography     

Ornithine Decarboxylase in Human Brain: Influence of Aging, Regional Distribution, and Alzheimer's Disease

Abstract: Although experimental animal data have implicated ornithine decarboxylase, a key regulatory enzyme of polyamine biosynthesis, in brain development and function, little information is available on this enzyme in normal or abnormal human brain. We examined the influence, in autopsied human brain, of postnatal development and aging, regional distribution, and Alzheimer's disease on the activity of ornithine decarboxylase. Consistent with animal data, human brain ornithine decarboxylase activity was highest in the perinatal period, declining sharply (by ∼60%) during the first year of life to values that remained generally unchanged up to senescence. In adult brain, a moderately heterogeneous regional distribution of enzyme activity was observed, with high levels in the thalamus and occipital cortex and low levels in cerebellar cortex and putamen. In the Alzheimer's disease group, mean ornithine decarboxylase activity was significantly increased in the temporal cortex (+76%), reduced in occipital cortex (−70%), and unchanged in hippocampus and putamen. In contrast, brain enzyme activity was normal in patients with the neurodegenerative disorder spinocerebellar ataxia type I. Our demonstration of ornithine decarboxylase activity in neonatal and adult human brain suggests roles for ornithine decarboxylase in both developing and mature brain function, and we provide further evidence for the involvement of abnormal polyamine system activity in Alzheimer's disease.     

Chronic lithium treatment prevents the dexamethasone-induced increase of brain polyamine metabolizing enzymes.

The paper describes the effects of various regimens of lithium chloride treatment on dexamethasone-induced increases in brain polyamine metabolizing enzymes. In contrast to peripheral tissues where acute lithium treatment suppresses the increase in ornithine decarboxylase activity, in the brain only chronic treatment was effective in preventing this increase and also the increases in the activities of S-adenosylmethionine decarboxylase and spermidine/spermine N1-acetyltransferase. This findings indicate a novel brain target for lithium's action and in turn provide new avenues for exploring polyamine function in the brain.     

Purification,properties and regulation of the level of bovine S-adenosylmethionine decarboxylase during lymphocyte mitogenesis

S-Adenosylmethionine decarboxylase was purified from the livers of calves treated with methylglyoxal bis (guanylhydrazone) to elevate the level of the enzyme. Purified bovine S-adenosylmethionine decarboxylase was similar in specific activity and subunit molecular weight (32 000) to the enzymes previously isolated from rat and mouse. The bovine liver enzyme immunologically crossreacted with S-adenosylmethionine decarboxylase from resting and mitogenically activated bovine lymphocytes. The rate of enzyme synthesis in activated lymphocytes was determined by labeling the cells with [³H]leucine and isolating the radioactive decarboxylase by affinity chromatography and sodium dodecyl sulfate gel electrophoresis. The rate of enzyme syntheis was increased 10-fold by 9 h after mitogen treatment, which accounts for the initial increase in cellular enzymatic. There was no further incraese in the rate of S-adenosylmethionine decarboxylase synthesis that correlated with a second elevation of activity occuring at approx. 24 h after mitogenic activation. It was concluded that the second increase in enzyme activity was due to lengthening the intracellular half-life of the enzyme by 2-fold.     

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.