Toxic effects of putrescine in rat brain: Polyamines can be involved in the action of excitotoxins

Summary After treatment with putrescine (PUT) 200 mg/kg, i.p., male rats displayed a behavioural pattern that included wet dog shakes and motor inco-ordination. The concentration of PUT in the brain paralleled the severity of clinical signs. Histological examination showed the presence of perivascular edema and moderate spongiosis. These biochemical and histological features were present 2 h after treatment. At 24 h PUT levels in frontal cortex decreased but the histological status of brain tissue remained. Pretreatment with hyperosmolal glycerol did not modify the effect of PUT on the brain content of polyamine or the histological condition at 2 h. These results support a neurotoxic role for putrescine. Such effects were similar to those of kainic acid at convulsant doses, suggesting a role for putrescine in the action of this excitotoxin.     

Determination of polyamines in human prostate by high-performance liquid chromatography with fluorescence detection

A high-performance liquid chromatographic method for the determination of polyamines in human prostate has been developed. This method is based on pre-column derivatization with dansyl chloride (Dns-Cl). The derivatives were separated on a μBondapak C₁₈ column (250×4.6 mm I.D.; 10 μm), and eluted with methanol and distilled water using a one-step linear gradient. The column eluate was monitored by fluorescence detection (excitation, 370 nm; emission, 506 nm). The within-assay precision of the study (C.V.) was as follows: putrescine (PUT) 2.88%, spermidine (SPD) 2.94% and spermine (SP) 1.17%. The between-assay precision (C.V.) was: PUT 2.66%, SPD 3.06%, SP 2.79%. The recovery was greater than 97%. The detection limit for PUT, SPD and SP were 0.05, 0.08 and 0.06 nmol/ml, respectively. In contrast to other studies, sample or polyamine derivatives did not require extraction with an organic solvent such as ethanol, evaporation under vacuum or other condensation procedures. This is a simple, rapid and sensitive method that can be applied to the determination of polyamines in nearly all biological tissues and body fluids, such as urine and serum.     

Polyamine metabolism in McCoy cells: III. Comparative studies of the metabolic fate of exogenous putrescine in human fibroblasts and McCoy cultures

The fate of exogenously added 14C-putrescine following incubation for 24 hours with McCoy and human skin fibroblast cultures was examined. The nature of the polyamine derivatives found were quite different indicative of a difference in the cellular metabolism of polyamines. Exogenously added putrescine (PUT) was metabolized by both McCoy and human skin fibroblast cultures to form spermidine (SPD), spermine (SPM), gamma-aminobutyric acid (GABA) and some unidentified compounds. Within the experimental period of observation, human cultured fibroblasts metabolized PUT more efficiently than McCoy cells and converted more than 50% of it into SPD, SPM, GABA and unknown compounds. Monoacetyl putrescine (MAP) was formed by human skin fibroblasts. It was mainly identified in the culture medium. No MAP was detectable either intracellularly or extracellularly in McCoy cultures. The percentage of 14C-radioactivity found as PUT in the culture medium was greater in McCoy cells (86.0%) than in human fibroblasts (53.9%). The reverse was true for the percentage distribution of 14C-radioactivity as PUT inside the cells. No low Mr conjugates of SPD or SPM were found in the medium or intracellularly with either culture type. Some low Mr putrescine conjugates were found in the culture media; these were identified by the liberation of PUT upon acid hydrolysis.     

N-caffeoyl-4-amino-n-butyric acid, a new flower-specific metabolite in cultured tobacco cells and tobacco plants

PUT cells were selected from the XD line of cultured tobacco cells (Nicotiana tabacum L. cv. Xanthi-nc) for the ability to utilize putrescine as sole nitrogen source. Previous work had indicated that hydroxycinnamoylputrescines (principally caffeoylputrescine) and 4-amino-n-butyric acid (GABA) are obligatory intermediates in the assimilation of putrescine by PUT cells. The apparent absence in these cells of diamine or polyamine oxidase and pyrroline dehydrogenase, enzymes which catalyze putrescine oxidation in some plant species, led us to propose the following pathway for putrescine oxidation in PUT cells: putrescine----hydroxycinnamoylputrescine----hydroxycinnamoyl - 4-aminobutyraldehyde----hydroxycinnamoyl-GABA----GABA. We tested the hypothesis by looking for the predicted compound, caffeoyl-GABA. A chemical synthesis was developed, and chromatographic and mass spectroscopic procedures were devised for identifying the compound in extracts of cells and plant tissues. Caffeoyl-GABA was found in extracts of PUT cells in micromolar concentrations but was not present in XD cells. Thus, its occurrence in PUT cells appears to be a direct result of selection for the ability to catabolize putrescine. Caffeoyl-GABA has the same distribution in tobacco plants as caffeoylputrescine, i.e. flower buds greater than open flowers greater than floral leaves, green fruit; absent in vegetative tissues.     

Analyzing the effects of exogeneous polyamines and growth regulators on plating efficiency of sweet potato protoplasts using a central composite test design

The effects of exogenous polyamines and growth regulators on plating efficiency of greenhouse-grown sweet potato (Ipomoea batatas Lam.) petiole protoplasts after six days were analyzed using a central composite test design. The medium components screened were 1-naphthaleneacetic acid (NAA), 6-benzylaminopurine (BAP), putrescine (PUT), spermidine (SPD), and spermine (SPM), each at five concentrations. Stepwise multiple regression analysis revealed significant interaction of NAA with BAP, PUT, and SPD as reflected in plating efficiencies. The interactions of NAA with BAP, and with SPD, were positive. The interaction of NAA and PUT appeared complex. A slight negative interaction was detected between PUT and SPM. These results indicated that plating efficiency of sweet potato protoplasts is highly sensitive to the concentrations of the medium components tested and it should be possible to further optimize the plating medium. Among the media formulations tested, the highest plating efficiency (10.8% after 6 days) was observed with NAA at 4.5 uM, BAP at 1.5 uM, PUT at 35.0 uM, SPD at 5.0 uM, and SPM at 2.5 uM.     

Superoxide dismutase and alpha-tocopherol suppress the paraquat-induced elevation of N1-acetylspermidine and putrescine in primary culture of adult rat hepatocytes

A transient increase in N1-acetylpolyamines and putrescine (PUT) was observed in hepatocytes at the early stage of primary culture of rat hepatocytes. After pre-culture for 36 hr when the polyamine content returned to constant levels, we tested the effects of superoxide dismutase (SOD) and alpha-tocopherol on the paraquat-induced increase in N1-acetylspermidine (N1-acetyl-SPD) and PUT in the culture. Paraquat increased (N1-acetyl-SPD in a dose-dependent manner. It also increased PUT at doses between 0.1 and 1.3 mM. Both SOD and alpha-tocopherol suppressed the increase in N1-acetyl-SPD and PUT induced by paraquat. These results suggested that superoxide anion is one of the factors which increase N1-acetyl-SPD and PUT in hepatocytes. Lipopolysaccharide (LPS) little affected the polyamine concentration in the cultured hepatocytes, though it increases polyamine in mouse liver when given in vivo. These findings suggested that the formation of superoxide anion after administration of LPS in vivo is mediated by Kupffer cells.     

Spermine and putrescine enhance oxidative stress tolerance in maize leaves

The protective effects of spermine (SPM) and putrescine (PUT) against paraquat (PQ), a herbicide in agriculture and oxidative stress inducer, were investigated in the leaves of maize. Maize leaves were pretreated to SPM and PUT at concentrations of 0.2 and 1 mM and treated with PQ afterwards. Pretreatment with 1 mM of SPM and PUT significantly prevented the losses in chlorophyll and carotenoid levels induced by PQ. Ascorbic acid content in the leaves pretreated with both polyamines was found to be higher than those of the leaves pretreated with water. Also, pretreatment with SPM and PUT was determined to have some effects on the activities of superoxide dismutase (SOD) and peroxidase (POD). 1 mM of SPM increased SOD activity, but PUT has no significant effect on SOD activity. On the other hand, POD activity was recorded to increase slightly in response to both concentrations of SPM and 1 mM of PUT. The results showed that such polyamine pretreated plants may become more tolerant to oxidative stress due to increases in the antioxidative enzymes and antioxidants.     

Putrescine and polyamine inhibitors in culture medium alter in vitro rooting response of <Emphasis Type="Italic">Decalepis hamiltonii</Emphasis> Wight &amp; Arn.

The influence of polyamine putrescine (PUT), and polyamine inhibitors were tested for in vitro rooting response from micro shoots that initially established on Murashige and Skoog (MS) medium comprising 2.7 µM α-Naphthaleneacetic acid (NAA) and 8.9 µM 6-Benzylaminopurine (BA) by using nodal explants of Decalepis hamiltonii. Incorporation of putrescine alone in rooting medium devoid of auxins supported the best response for in vitro rooting qualitatively and quantitatively. Incorporation of putrescine at 50 µM able to induce 8.62?±?1.93 roots with a maximum root length of 9.10?±?1.65 cm wherein, the root fresh weight was also found to be high compared to all other treatments (5.248?±?1.71 g). Addition of putrescine inhibitor cyclohexylamine (CHA) in medium curtailed rooting response from microshoots. Among the three polyamine inhibitors, CHA in presence of 9.8 µM Indole-3-butyric acid (IBA) outperformed α-DL-difluromethylarginine (DFMA) and α-DL-difluoromethylornithine (DFMO) combination with 9.8?µM IBA. The least response for root number (1.55?±?0.72), root length (1.96?±?0.45 cm), and root weight (1.94?±?0.35 g) was found for IBA?+?PUT?+?DFMA and the best response was noted for IBA?+?PUT?+?CHA (2.6?±?1.1, 2.92?±?0.73 cm, 3.03?±?0.75 g) respectively. Endogenous content of putrescine, spermidine and spermine supported the rooting response from in vitro shoots. These results have clearly demonstrated that putrescine plays a crucial role in rooting of D. hamiltonii. Plantlets were transferred to micro-pots for a short acclimatization stage in greenhouse where they survived at 90?%. This highly reproducible procedure can be adopted for large scale swallow root propagation. Overall, supplementing putrescine in the rooting medium enhances the quantity and quality of roots in D. hamiltonii, thus confirming its role.     

The Response of Nitrate Reductase Activity and Nitrate Assimilation in Maize Roots to Growth Regulators at Acidic pH

Nitrate and total nitrogen contents, and nitrate reductase (NR) activity of the excised maize roots in buffered or unbuffered nitrate solution (at pH 6.5 or 4.5) as affected by putrescine (PUT), abscisic acid (ABA) and salicylic acid (SA) were investigated. In unbufferred solution, the NR activity was lower at pH 4.5 as compared to that at pH 6.5, but in bufferred solution the activity was higher at lower pH. Supply of 100 µM PUT or 500 µM SA, promoted NR activity and 50 µM ABA inhibited the activity at pH 6.5. However, at pH 4.5, PUT and SA inhibited NR activity and ABA had no effect. In most cases, the increase in NR activity was positively correlated with total organic nitrogen and a negatively with nitrate content. A reverse situation was found when NR activity was inhibited by the growth regulators.     

GC–MS measurement of spermidine and putrescine in serum of elderly subjects: intriguing association between spermidine and homoarginine

Amino Acids - Gas chromatography–mass spectrometry (GC–MS) methods were developed, validated and used to measure serum spermidine (SPD) and putrescine (PUT) in 9 seropositive...     

Putrescine alleviation of growth in salt stressed Brassica juncea by inducing antioxidative defense system

Seedlings of Indian mustard (Brassica juncea L.cv. RH-30) grown in controlled condition (irradiance 75 Wm(-2), RH 60-70% and temp. 25 +/- 2 degrees C) for 7d and watered with Hoagland's solution containing different level of NaCL (50-250 mmol/L NaCl) with or without putrescine (PUT, 0.1 mmol/L) were examined for PUT amelioration of NaCl induced inhibition in seedling growth by altering activity of antioxygenic enzymes and level of free radicals in the leaves. Salinity caused reduction in seedling growth and biomass accumulation was parallel to increased superoxide (*O2-), hydrogen peroxide (H2O2) levels, lipid peroxidation (MDA content) and electrolyte leakage in leaf tissues which were reversed significantly by PUT. The antioxygenic enzymes viz superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX) and glutathione reductase (GR) were differentially altered, depending on salt level. PUT induction of enzyme was in the following order APX>GR>CAT>SOD>POD in leaf tissues of salt stressed seedlings. PUT increased the level of glutathione and carotenoids in leaf tissues. This finding suggests that PUT might be activating antioxygenic enzymes and elevating antioxidants there by controlling free radical generation, hence preventing membrane peroxidation and denaturation of biomolecules resulting into improved seedling growth under salinity.     

Levels of endogenous polyamines and NaCl-inhibited growth of rice seedlings

The role of endogenous polyamines in the control of NaCl-inhibited growth of rice seedlings was investigated. Putrescine, spermidine and spermine were all present in shoots and roots of rice seedlings. NaCl treatment did not affect spermine levels in shoots and roots. Spermidine levels in shoots and roots were increased with increasing concentrations of applied NaCl. NaCl at a concentration of 50 mM, which caused only slight growth inhibition, drastically lowered the level of putrescine in shoots and roots. Addition of precursors of putrescine biosynthesis (L-arginine and L-ornithine) resulted in an increase in putrescine levels in NaCl-treated shoots and roots, but did not allow recovery of the growth inhibition of rice seedlings induced by NaCl. Pretreatment of rice seeds with putrescine caused an increase in putrescine level in shoots, but could not alleviate the inhibition effect of NaCl on seedling growth. The current results suggest that endogenous polyamines may not play a significant role in the control of NaCl-inhibited growth of rice seedlings.Abbreviations PUT putrescine - SPD spermidine - SPM spermine     

Tuber formation and growth of <Emphasis Type="Italic">Dioscorea cayenensis-D. rotundata</Emphasis> complex: interactions between exogenous and endogenous jasmonic acid and polyamines

Tubers can be initiated and developed in vitro from nodal cuttings of yam (Dioscorea cayenensis-D. rotundata complex). The effect of exogenous jasmonic acid, alone or in combination with putrescine, on these processes was investigated in relationship to endogenous jasmonic acid and polyamine levels. Application of exogenous jasmonic acid at various concentrations positively affected microtuber formation and growth from yam nodal cuttings. In control conditions, 3 weeks were needed to obtain 100% of tuberisation. Jasmonic acid at low level (0.1 μM) accelerated tuber formation (46% after 1 week) as did putrescine (10 μM). But endogenous levels of jasmonic acid were not significantly affected by its exogenous presence in the medium. Jasmonic acid also interacted with other growth regulators as polyamines, but the decrease in time necessary to observe tuber formation could not be correlated with endogenous modifications of PUT content. The presence of jasmonic acid (0.1–1 μM) as PUT (1 μM) induced also an increase of tuber length and weight. The combination of jasmonic acid (0.1 μM) and putrescine (1 μM) had no positive effect on tuber formation (precocity) but had an additive effect on further growth (length and weight). In the future, these results could help the optimising in vitro conditions for mass production of larger yam microtubers.     

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.     

Temporal Regulation of Somatic Embryogenesis by Adjusting Cellular Polyamine Content in Eggplant

Four critical stages of embryogenesis, including callus induction, cellular acquisition of morphogenetic competence, expression of embryogenic program, and development and maturation of somatic embryos during somatic embryogenesis from leaf discs of eggplant (Solanum melongena L.), were identified by scanning electron microscopy. Temporal changes in arginine decarboxylase (ADC) activity and polyamines (PAs) during critical stages of embryogenesis revealed that high levels of PAs (especially putrescine [PUT]), due to higher ADC activity in discs from the apical region (with high embryogenic capacity) than from the basal region of the leaf (with poor embryogenic capacity), were correlated with differential embryogenesis response. Kinetic studies of the up- and down-regulation of embryogenesis revealed that PUT and difluoromethylarginine pretreatments were most effective before the onset of embryogenesis. Basal discs pretreated with PUT for 4 to 7 d showed improved embryogenesis that was comparable to apical discs. PA content at various critical steps in embryogenesis from basal discs were found to be comparable to that of apical discs following adjustments of cellular PA content by PUT. In contrast, pretreatment of apical discs with difluoromethylarginine for 3 d significantly reduced ADC activity, cellular PA content, and embryogenesis to levels that were comparable to basal discs. Discs from the basal region of leaves treated with PUT for 3 d during the identified stages of embryogenesis improved their embryogenic potential.     

Transglutaminase activity decrease during acclimation to hyposaline conditions in marine seaweed Grateloupia doryphora (Rhodophyta, Halymeniaceae)

Polyamines (PAs), such as diamine putrescine (PUT), triamine spermidine (SPD) and tetraamine spermine (SPM) have been related to environmental stress, including salt stress. A marine red macrophyte alga Grateloupia doryphora (Montagne) Howe was used to investigate the role of PAs during acclimation to moderate hyposaline conditions (incubation 24h in 18 psu seawater as compared to 36 psu of natural seawater). The results obtained showed that a moderate hyposaline shock caused an increase in the free fraction of PUT, SPD and SPM, mainly due to a decrease in TGase activity, together with an apparent increase in the l-arginine dependent PAs synthesis (ODC and arginase decreased, and ADC slightly increased). The photosynthetic rate increased in thalli when exposed to free SPD at 18 psu, but it did not increase at 36 psu.     

Polyamine Changes and Chilling Injury in Cold-stored Loquat Fruits

Free polyamine levels (spermine (SPM), spermidine (SPD), and putrescine (PUT)) were determined using thin-layer chromatography and fluorometric method in loquat (Eriobotrya japonica Lindl. cv. Dahongpao) fruits stored at 1℃ and 12℃ and in postharvest SPM treated fruits stored at 1℃ respectively to investigate the relationship between changes in polyamines and chilling injury. In the loquat fruits stored at 1℃, SPM level decreased gradually in the first two weeks, then increased sharply and reached a peak value after three weeks, thereafter it decreased rapidly. SPD level decreased steadily during the first three weeks and increased significantly afterwards. PUT level evolved in a similar way as the SPM level did except that it increased slowly in the first two weeks. The fruit showed symptom of chilling injury manifested as flesh leatheriness after three weeks. However, no significant increase and decrease of these three polyamines was detected during storage at the nonchilling temperature (12℃). The SPM-treated fruits maintained high levels of SPM and SPD and remained low level of PUT during storage at 1℃, and no symptom of chilling injury was observed. These results suggested that the increase in SPM level in response to chilling exposure might serve as a defense mechanism against chilling injury while the accumulation of PUT could be a cause of the stress-induced injury and the increase in SPD level could be a consequence of this kind of stress.     

Efficient plant regeneration from long-term callus cultures of rice by spermidine

A significant reduction in regeneration potential with increasing age (upto 12months) in rice (Oryza sativa L. cv.TN-1) embryogenic callus cultures was observed. Spermidine, while having an inhibitory effect on plant regeneration in fresh callus cultures, promoted morphogenesis in long-term callus cultures. A massive accumulation of polyamines, particularly putrescine (5-fold) was observed in 12 month old cultures resulting in a change of putrescine /spermidine ratio, which seems to be important for maintaining the morphogenetic response. Application of exogenous spermidine to 12 month old cultures showed increased levels of polyamines and restored the putrescine/spermidine ratio comparable to that found in freshly induced cultures, concomitantly, promoting the plant regeneration via somatic embryogenesis in long-term rice callus cultures.Abbreviations PA Polyamines - PCA Perchloric acid - PUT Putrescine - SPD Spermidine - SPM Spermine     

Differential response of rat brain polyamines to convulsant agents.

The polyamines putrescine (PUT), spermidine (SD) and spermine (SM) have been studied in rat brain after treatment with several convulsant agents. Kainic acid (10 mg/kg), picrotoxinin (1.5 mg/kg), pentylenetetrazol (60 mg/kg) and lindane (gamma-hexachlorocyclohexane) (60 mg/kg) were given to male Wistar rats. Twenty-four hours later, the animals were sacrificed and their brains removed. Cortical polyamines were analyzed by HPLC with fluorimetric detection of their respective dansyl derivatives, using 1,6-diaminohexane as internal standard for the measurements. Polyamine levels are not affected by short periods of time (30 min) of brain exposure to room temperature before freezing the samples, as compared to a quick procedure (less than 40 s from animal death). Kainic acid induced a 14-fold increase of cortical PUT with respect to control values, leaving unchanged the other polyamines. Lindane also increased cortical PUT (4-fold) without affecting SM or SD. Neither picrotoxinin, nor pentylenetetrazol groups were different from controls for any of the polyamines assayed. The results are discussed in relation to the possible mechanism of action of these convulsant agents and the role of the polyamines in cell injury.