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.     

Changes in Polyamine Levels in Rat Brain After Systemic Kainic Acid Administration: Relationship to Convulsant Activity and Brain Damage

We have examined the effects of systemic kainic acid (KA) administration (9 mg/kg, i.p.) on rat behavior, brain damage, and polyamine levels and the action of the specific ornithine decarboxylase inhibitor alpha-difluoromethylornithine (DFMO) on these effects. KA elicited convulsant activity in 63% of the animals. In the acute convulsant phase (1-3 h after KA), a rapid decline (-39% at 3 h) of spermidine content in frontal cortex was found. After the acute convulsant phase, levels of hippocampal spermidine and spermine were reduced (-70 and -66%, respectively, at 8 h). A dramatic increase of putrescine content (68.1, 1,382, and 336% at 8 h, 24 h, and 9 days, respectively, after KA) was found, associated with histological signs of cortical brain damage (ischemia and necrosis). There was a close relationship between the concentration of putrescine and signs of delayed toxicity (body weight losses) 24 h and 9 days after KA. DFMO partially antagonized the convulsant activity and reduced the increased putrescine levels to approximately 50% of values in KA-treated animals at 24 h but did not change the pattern of histological damage. The role of polyamines in the early and late phases of KA-induced neurotoxicity is discussed.     

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.     

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     

Development of an HPLC assay for the analysis of tetrafluoroputrescine--a putrescine analog

A simple reverse-phase liquid chromatographic system with ultraviolet detection at 254 nm for tetrafluoroputrescine (TFP), a putrescine analog, is described. The assay involves precolumn derivatization of TFP with dansyl chloride at pH 6.8-7.5 at 60 degrees C, followed by separation from putrescine (PUT) and quantitation. The derivatization procedure was adapted for the simultaneous analysis of TFP and PUT in whole blood components. Also, preliminary studies on protein binding of TFP are reported.     

GABA and its relationship to putrescine metabolism in the rat brain and pancreas

The possibility that GABA may have its origin in putrescine was investigated in the rat pancreas, relative to the brain. These studies show that radioactive putrescine is converted to GABA at a similar rate in both the pancreas and brain, but that putrescine accounts for only a small fraction of the GABA found in these organs. Inhibitors of diamine and monoamine oxidases do not significantly change the GABA level in the pancreas. In contrast to the brain, where putrescine is catabolized to GABA via monoamine oxidase, the primary catabolic pathway of putrescine to GABA in the pancreas is via diamine oxidase. In vivo studies show that AOAA inhibits GABA-T activity to the same degree in the pancreas as in the brain, elevating GABA levels more than 2-fold in 4 h. GABA is metabolized more rapidly in the brain than the pancreas. Turnover times of GABA in the pancreas and brain are 1.9 and 1.0 h, respectively. The slower turnover of GABA in the pancreas than in the brain may relate to a neuromodulatory role for GABA, similar to that for neuropeptides. Developmental studies in the postnatal pancreas suggest a role for GABA in the maturation of insulin secretion.     

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.     

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.     

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.     

Involvement of Putrescine in the Development of Kindled Seizure in Rats

During the development of kindling by daily electrical stimulations applied to the left amygdala of rats, concentrations of the polyamines putrescine, spermidine, and spermine were measured in the left amygdala and the remainder of the cerebrum. A significant increase of putrescine concentration appeared first at the left amygdala in prekindled rats and then propagated to the remainder of the cerebrum with the development of kindling. This increase in putrescine concentration in the left amygdala was higher in prekindled rats than in fully kindled rats and lasted for at least 24 h after the final kindling stimulation. The concentrations of spermidine and spermine were slightly increased in a fully kindled state. To clarify the role of putrescine in kindling, the development of amygdaloid kindling was examined in rats after microinjections of alpha-difluoromethylornithine, a specific inhibitor of polyamine synthesis, and putrescine into the ipsilateral amygdala. Pretreatment with alpha-difluoromethylornithine (50 nmol) for 10 days accelerated both the development of behavioral kindling and the propagation of the afterdischarge from the left amygdala to the frontal cortex. In contrast, pretreatment with putrescine (200 nmol) for 10 days retarded the development of kindling. These results suggest that the increase in putrescine concentration in the kindled brain has an inhibitory effect on the development of kindling.     

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 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.     

Glucocorticoids suppress and oestrogens enhance the lipopolysaccharide-induced increase in putrescine and N1-acetylspermidine in mouse liver

Previously we reported that administration of lipopolysaccharide (LPS) to mice increased the hepatic levels of putrescine (PUT) and N¹-acetylspermidine (N¹-acetyl-SPD). In the current study, we examined the in vivo effects of some steroid hormones on the LPS-induced increase in PUT and N¹-acetyl-SPD. Corticosterone, hydrocortisone and dexamethasone suppressed the LPS-induced increase in PUT and N¹-acetyl-SPD in mouse liver in a dose-dependent manner, dexamethasone being the most effective among them. On the other hand, oesterone and oestradiol-17β enhanced the LPS-induced increase in PUT and N¹-acetyl-SPD in a dose-dependent manner. Oestradiol-17 and 16β-ethyl-oestradiol, as an inactive oestradiol isomer and an antioestrogen, respectively, likewise enhanced the increase in PUT and N¹-acetyl-SPD concentrations induced by LPS. 16-hydroxy-oestradiol (oestriol), 16-hydroxyestrone, 2-hydroxyoestradiol, 2-hydroxyoestrone, progesterone, testosterone, diethylstilboestrol and nonsteroidal antioestrogen such as tamoxifen and nafoxidine had no effect on the increase. Oestradiol-17β enhanced and corticosterone had little on the carbon tetrachloride-induced increase in PUT and N¹-acetyl-SPD. These results suggest that glucocorticoids suppress the increase by preventing the immunological injury by Kupffer cells on hepatocytes and that the stimulatory effect of oestrogens may not be associated with their oestrogenic activities mediated by the oestrogen receptor system.     

Polyamines and the Cell Cycle of Catharanthus roseus Cells in Culture

Investigation was made on the effect of partial depletion of polyamines (PAs), induced by treatment with inhibitors of the biosynthesis of PAs, on the distribution of cells at each phase of the cell cycle in Catharanthus roseus (L.) G. Don. cells in suspension cultures, using flow cytometry. More cells treated with inhibitors of arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) were accumulated in the G₁ phase than those in the control, while the treatment with an inhibitor of spermidine (SPD) synthase showed no effect on the distribution of cells. The endogenous levels of the PAs, putrescine (PUT), SPD, and spermine (SPM), were determined during the cell cycle in synchronous cultures of C. roseus. Two peaks of endogenous level of PAs, in particular, of PUT and SPD, were observed during the cell cycle. Levels of PAs increased markedly prior to synthesis of DNA in the S phase and prior to cytokinesis. Activities of ADC and ODC were also assayed during the cell cycle. Activities of ADC was much higher than that of ODC throughout the cell cycle, but both activities of ODC and ADC changed in concert with changes in levels of PAs. Therefore, it is suggested that these enzymes may regulate PA levels during the cell cycle. These results indicate that inhibitors of PUT biosynthesis caused the suppression of cell proliferation by prevention of the progression of the cell cycle, probably from the G₁ to the S phase, and PUT may play more important roles in the progression of the cell cycle than other PAs.     

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.     

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.     

Changes in polyamines, c-myc and c-fos gene expression in osteoblast-like cells exposed to pulsed electromagnetic fields

Pulsed electromagnetic field (PEMF) stimulation promotes the healing of fractures in humans, though its effect is little known. The processes of tissue repair include protein synthesis and cell differentiation. The polyamines (PA) are compounds playing a relevant role in both protein synthesis processes and cell differentiation through c-myc and c-fos gene activation. Since several studies have demonstrated that PEMF acts on embryonic bone cells, human osteoblast-like cells and osteosarcoma TE-85 cell line, in this study we analyzed the effect on cell PAs, proliferation, and c-myc and c-fos gene expression of MG-63 human osteoblast-like cell cultures exposed to a clinically useful PEMF. The cells were grown in medium with 0.5 or 10% fetal calf serum (FCS). c-myc and c-fos gene expressions were determined by RT-PCR. Putrescine (PUT), spermidine (SPD), or spermine (SPM) levels were evaluated by HPLC. [(3)H]-thymidine was added to cultures for DNA analysis. The PEMF increased [(3)H]-thymidine incorporation (P < or = .01), while PUT decreased after treatment (P < or = .01); SPM and SPD were not significantly affected. c-myc was activated after 1 h and downregulated thereafter, while c-fos mRNA levels increased after 0.5 h and then decreased. PUT, SPD, SPM trends, and [(3)H]-thymidine incorporation were significantly related to PEMF treatment. These results indicate that exposure to PEMF exerts biological effects on the intracellular PUT of MG-63 cells and DNA synthesis, influencing the genes encoding c-myc and c-fos gene expression. These observations provide evidence that in vitro PEMF affects the mechanisms involved in cell proliferation and differentiation.