Brain Polyamine Stress Response: Recurrence After Repetitive Stressor and Inhibition by Lithium

Abstract: We recently demonstrated that, unlike in peripheral tissues, the increase in activity of polyamine synthesizing enzymes observed in the brain after acute stress can be prevented by long-term, but not by short-term, treatment with lithium. In the present study we sought to examine the effects of chronic intermittent stress on two key polyamine synthesizing enzymes, ornithine decarboxylase and S-adenosylmethionine decarboxylase, and their modulation by lithium treatment. Adult male rats were subjected to 2 h of restraint stress once daily for 5 days and to an additional delayed stress episode 7 days later. Enzyme activities were assayed 6 h after the beginning of each stress episode. In contrast to the liver, where ornithine decarboxylase activity was increased (300% of the control) only after the first stress episode, the enzyme activity in the brain was increased after each stress episode (to ～170% of the control). Unlike ornithine decarboxylase activity, S-adenosylmethionine decarboxylase activity was slightly reduced after the first episode (86% of the control) but remained unchanged thereafter. After cessation of the intermittent stress period, an additional stress episode 7 days later led again to an increase in ornithine decarboxylase activity in the brain (225% of the control) but not in the liver, whereas S-adenosylmethionine decarboxylase activity remained unchanged. The latter increase in ornithine decarboxylase activity was blocked by lithium treatment during the intervening 7-day interval between stressors. The results warrant the following conclusions: (a) Repetitive application of stressors results in a recurrent increase in ornithine decarboxylase activity in the brain but to habituation of this response in the liver. (b) This brain polyamine stress response can be blocked by long-term (days) lithium treatment. (c) The study implicates an overreactive polyamine response as a component of the adaptive, or maladaptive, brain response to stressful events and as a novel molecular target for lithium action.     

The role of polyamine reutilization in depletion of cellular stores of polyamines in non-proliferating tissues

It was known from previous work that specific inhibition of neither ornithine decarboxylase activity nor polyamine oxidase activity produces spermidine depletion by more than 20% in non-growing organs, which are in a steady state with regard to polyamine metabolism. Combined treatment with inactivators of both ornithine decarboxylase and polyamine oxidase for a prolonged time caused, however, a gradual decrease of spermidine levels in liver, kidney and brain of mice by 50% and more. The method is in accordance with the previously suggested role of polyamine interconversion. Inhibition of polyamine oxidase prevents the reutilization for de novo polyamine biosynthesis of putrescine and spermidine, which are formed by oxidative splitting of N1-acetylspermine and N1-acetylspermidine, respectively, and the ornithine decarboxylase inhibitor prevents the compensatory increase of putrescine from ornithine. The findings are further evidence for the physiological significance of polyamine reutilization.     

Blood brain barrier breakdown in brain edema following cold injury is mediated by microvascular polyamines

A focal freeze injury to rat cerebral cortex induces an early (less than 5 min) increase in brain ornithine decarboxylase activity and an accumulation of polyamines involving cerebral microvessels. This polyamine synthesis correlates with the abnormal increase in microvascular permeability, monitored by uptake of Evans Blue and sod. fluorescein. The ornithine decarboxylase inhibitor alpha-difluoromethylornithine suppressed the injury-induced increment in spermidine and spermine and microvascular permeability. Putrescine nullified alpha-difluoromethylornithine inhibition and restored microvessel spermidine and spermine and the pathological increase in microvascular permeability. These results indicate that polyamine synthesis is obligatory for blood-brain barrier breakdown. alpha-Difluoromethylornithine may be useful in the treatment of vasogenic brain edema.     

Activation of polyamine biosynthetic decarboxylases during the acute phase response of rat liver

The activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase increase in the livers of rats during the acute-phase response to inflammation. The increase reaches its maximum at 2.5 hr from injection of turpentine, and is maintained at the same level for the following 2 days. Pretreatment in vivo with an inhibitor of cyclooxygenase prevents the inflammation-associated increases of both polyamine biosynthetic decarboxylases: an inhibitor of the lipoxygenase pathway seems to counteract only the increase of ornithine decarboxylase. The administration of diaminopropane, an inhibitor of ornithine decarboxylase, has only limited effects on the activation of RNA synthesis by liver nuclei, which occurs 10 hr after turpentine treatment. The results suggest that stimulation of the polyamine biosynthetic decarboxylases is surely part of the acute-phase response and depends on the previous activation of arachidonate metabolism: however its role in supporting later events of the acute-phase response will need further investigations.     

Polyamine reutilization and turnover in brain

N¹, N²-bis-(2, 3-butadienyl)-1, 4-butanediamine (MDL 72527) is an irreversible, specific inhibitor of polyamine oxidase, which allows one to completely inactivate this enzyme in all organs of an experimental animal. As a result one observes a linear increase of N¹-acetylsperimidine and N¹-acetylspermine concentrations in brain. The rate of accumulation seems directly proportional to the rate of spermidine, and spermine degradation respectively, and since no compensatory changes of the polyamine synthetic enzymes, were induced by inhibition of polyamine oxidase, the rate of acetyl-polyamine accumulation is assumed to be a measure for polyamine turnover. The decrease of brain putrescine levels by 70 percent in the brains of MDL 72527-treated animals suggests the quantitative significance of putrescine reutilisation. Pretreatment of the animals with D, L--difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase reduced both, polyamine turnover rate and the extent of putrescine reutilization. Inhibition of GAPA-T produced a significant increase of polyamine turnover in brain, in agreement with the known induction of ornithine decarboxylase activity after treatment with inhibitors of GABA-T.     

Glucocorticoid Regulation of Spermidine Acetylation in the Rat Brain

The effect of glucocorticoids on polyamine metabolism has been elucidated further by measuring putrescine, spermidine, and spermine levels as well as ornithine decarboxylase, S-adenosylmethionine decarboxylase, and N1-acetylspermidine transferase activities in the hippocampus, cerebellar cortex, vermis, and deep nuclei of adrenalectomized rats. At 6 h after corticosterone or dexamethasone administration, the specific activities of ornithine decarboxylase and N1-acetylspermidine transferase showed the greatest increases in all brain tissues examined, and at 12 h, S-adenosylmethionine decarboxylase activity was not increased significantly. The hippocampus and cerebellar regions displayed different responses to corticosterone and dexamethasone, corresponding to the distribution of glucocorticoid and mineralocorticoid receptors. Corticosterone and dexamethasone increased ornithine decarboxylase and N1-acetylspermidine transferase activities in a dose-dependent manner, with dexamethasone being more active than corticosterone in all tissues. However, estradiol, progesterone, testosterone, and aldosterone were only active at doses greater than 5 mg/kg. The great increases in ornithine decarboxylase and N1-acetylspermidine transferase activities were accompanied by a marked increase in putrescine level and a small decrease in spermidine level. Our data confirm that the hippocampus and cerebellum are glucocorticoid target tissues and suggest that the increase in the content of putrescine, following acute treatment with glucocorticoids, is dependent on ornithine decarboxylase as well as N1-acetylspermidine transferase induction.     

Elevated N1-acetylspermidine levels in gerbil and rat brains after CNS injury

The polyamine system is very sensitive to different pathological states of the brain and is perturbed after CNS injury. The main modifications are significant increases in ornithine decarboxylase activity and an increase in tissue putrescine levels. Previously we have shown that the specific polyamine oxidase (PAO) inhibitor N1,N4-bis(2,3-butadienyl)-1,4-butanediamine (MDL 72527) reduced the tissue putrescine levels, edema, and infarct volume after transient focal cerebral ischemia in spontaneously hypertensive rats and traumatic brain injury of Sprague-Dawley rats. In the present study, N1-acetyl-spermidine accumulation was greater in injured brain regions compared with sham or contralateral regions following inhibition of PAO by MDL 72527. This indicates spermidine/spermine-N1-acetyltransferase (SSAT) activation after CNS injury. The observed increase in N1-acetylspermidine levels at 1 day after CNS trauma paralleled the decrease in putrescine levels after treatment with MDL 72527. This suggests that the increased putrescine formation at 1 day after CNS injury is mediated by the SSAT/PAO pathway, consistent with increased SSAT mRNA after transient ischemia.     

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.     

Developmental regulation of polyamine metabolism in growth and differentiation of carrot culture

Polyamine levels and the activities of two polyamine biosynthetic enzymes, arginine decarboxylase (EC 4.1.1.19) and S-adenosylmethionine decarboxylase (EC 4.1.1.50), were determined during somatic embryogenesis of carrot (Daucus carota L.) cell cultures. Embryogenic cultures showed severalfold increases in polyamine levels over nondifferentiating controls. A mutant cell line that failed to form embryos but grew at the same rate as the wild-type line also failed to show increases in polyamine levels, thus providing evidence that this increased polyamine content was in fact associated with the development of embryos. Furthermore, inhibition of these increases in polyamines caused by drugs inhibited embryogenesis and the effect was reversible with spermidine. The activities of arginine decarboxylase and Sadenosylmethionine decarboxylase were found to be suppressed by auxin; however, the specific effects differed between exogenous 2,4-dichlorophenoxyacetic acid and endogenous indole-3-acetic acid. The results indicate that increased polyamine levels are required for cellular differentiation and development occurring during somatic embryogenesis in carrot cell cultures.Abbreviations ADC arginine decarboxylase - 2,4-D 2,4-dichlorophenoxyacetic acid - DFMA difluoromethylarginine - DCHAS dicyclohexylammonium sulfate - SAMDC S-adenosylmethionine decarboxylase     

Effects of ethylene and auxin on polyamine levels in suspension-cultured tobacco cells

The effects of ethylene and auxin on polyamine levels were studied in suspension-cultured cells of tobacco ( Nicotiana tabacum . L). Treatment of 4-day-cultured cells with ethylene increased the levels of spermidine and spermine. The activities of arginine decarboxylase (ADC; EC 4.1.1.19), ornithine decarboxylase (ODC: EC 4.1.1.17), and S -adenosylmethionine decarboxylase (SAMDC: EC 4.1.1.50) rapidly increased between 3 and 12 h. An auxin, indole-3-acetic acid (IAA), increased polyamine levels and activities of ADC, ODC and SAMDC. The spermine level continued to increase significantly during a 24-h incubation with IAA. The increases in polyamine accumulation induced by ethylene were partially offset by an inhibitor of ethylene action, 2,5-norbornadiene. It is suggested that the auxin-induced polyamine accumulation occurred directly, without metabolic competition between ethylene and polyamine biosynthesis, and indirectly, through auxin-induced ethylene formation.     

POLYAMINE REGULATORY PROCESSES AND OXIDATIVE STRESS IN MONOCROTALINE-TREATED PULMONARY ARTERY ENDOTHELIAL CELLS

Alterations in polyamine metabolism may be a critical mechanism of monocrotaline (MCT)-induced structural remodeling of the pulmonary vasculature. In the present study, the hypothesis that MCT, through the induction of oxidative stress, modulates cellular polyamine regulatory mechanisms which in turn might be involved in the upregulation of fibronectin production in pulmonary artery endothelial cells (PAEC) was examined. A 24-h treatment with MCT significantly increased PAEC polyamine concentrations as compared to vehicle-treated cells. In addition, exposure to MCT caused an increase in abundance of ornithine decarboxylase (ODC) mRNA, upregulation of ODC activity and enhancement of spermidine import into PAEC. Inhibition ofde novopolyamine synthesis further increased spermidine uptake in MCT-treated cells. The depletion of cellular polyamine contents through the blockade of bothde novopolyamine biosynthesis and polyamine transport prevented MCT-induced increases in the medium level of fibronectin. In addition, PAEC treatment with MCT stimulated cellular oxidative stress as determined by increased levels of thiobarbituric acid reactive substances, enhanced dichlorofluorescein fluorescence and activation of NF-KB. A co-treatment with dimethylthiourea, an oxygen radical scavenger, prevented MCT-induced increases in cellular oxidation and attenuated disturbances in polyamine metabolism. These data suggest that MCT can stimulate polyamine regulatory processes in PAEC possibly through an increase in cellular oxidative stress. The present study may have significant implication in understanding mechanisms of MCT-induced pulmonary hypertension and remodeling of pulmonary vasculature.     

Regulation of polyamine transport in Chinese hamster ovary cells

Control Chinese hamster ovary (CHO) cells and mutant CHO cells lacking ornithine decarboxylase activity (CHODC-) were used to study the regulation of polyamine uptake. It was found that the transport system responsible for this uptake was regulated by intracellular polyamine levels and that this regulation was responsible for the maintenance of physiological intracellular levels under extreme conditions such as polyamine deprivation or exposure to exogenous polyamines. Polyamine transport activity was enhanced by decreases in polyamine content produced either by inhibition of ornithine decarboxylase with alpha-difluoromethylornithine in CHO cells or via polyamine starvation of CHODC- cells. The provision of exogenous polyamines resulted in rapid and large increases in intracellular polyamine content followed by decreased polyamine transport activity. Soon after this decrease in uptake activity, intracellular polyamine levels then fell to near control values. Cells grown in the presence of exogenous polyamines maintained intracellular polyamine levels at values similar to those of control cells. Protein synthesis was necessary for the increase in transport in response to polyamine depletion, but appeared to play no role in decreasing polyamine transport. Bis(ethyl) polyamine analogues mimicked polyamines in the regulation of polyamine transport but this process was relatively insensitive to regulation by methylglyoxal bis(guanylhydrazone), a spermidine analogue known to enter cells via this transport system and to accumulate to very high levels.     

The effect of methionine, ethylene and polyamine catabolic intermediates on polyamine accumulation in detached soybean leaves

In the present study we determined the effects of methionine, intermediates of polyamine catabolic pathways and inhibitors of either ethylene biosynthetic or polyamine catabolic pathways on polyamine accumulation in soybean leaves. Inhibitors to SAM decarboxylase and spermidine synthase, methylglyloxal-bis-(guanylhy-drazone) and cyclohexylamine, respectively, suggest that methionine may provide aminopropyl groups for the synthesis of polyamine via S-adenosylmethionine (SAM). Results from experiments that utilized a combination of compounds which altered either ethylene or polyamine biosynthesis, namely, aminoethoxyvinyl glycine, CoSO₄, 2,5-norbornadiene, and CuSO₄, suggest the two pathways compete for a common precursor. However, exogenous addition of ethylene (via ethephon treatments) had little or no effect on polyamine biosynthesis. Likewise, polyamine treatments had little or no effect on ethylene biosynthesis. These data suggest that there are few or no inhibitory effects from the end products of one pathway on the synthesis of the other. Data from leaves treated with metabolic intermediates in the catabolic pathway of polyamines and inhibitors of enzymes in the catabolic pathway, i.e. aminoguanidine, hydroxyethyldrazine and gabaculine, suggest that the observed increases in polyamine titers were not due to decreased catabolism of the polyamines. One catabolic intermediate, γ-aminobutyric acid (GABA), elevated putrescine, spermidine and spermine by 12-, 1.4-, and 2-fold, respectively, Ethylene levels decreased (25%) in GABA-treated leaves. This small decrease in ethylene could not account for such large increase in putrescine titers. Further analysis demonstrated that the GABA-mediated polyamine accumulation was inhibited by difluoromethylarginine, an inhibitor of arginine decarboxylase, but not by difluoromethylornithine, an inhibitor of ornithine decarboxylase. These data suggest that GABA directly or indirectly affects the biosynthesis of polyamines via arginine decarboxylase.     

Regulation of ornithine decarboxylase and polyamine import by hypoxia in pulmonary artery endothelial cells

In rat lung and cultured lung vascular cells, hypoxia decreases ornithine decarboxylase (ODC) activity and increases polyamine import. In this study, we used rat cultured pulmonary artery endothelial cells to explore the mechanism of hypoxia-induced reduction in ODC activity and determined whether this event was functionally related to the increase in polyamine import. Two strategies known to suppress proteasome-mediated ODC degradation, lactacystin treatment and use of cells expressing a truncated ODC incapable of interacting with the proteasome, prevented the hypoxia-induced decrease in ODC activity. Interestingly, though, cellular abundance of the 24-kDa antizyme, a known physiological accelerator of ODC degradation, was not increased by hypoxia. These observations suggest that an antizyme-independent ODC degradation pathway contributes to hypoxia-induced reductions of ODC activity. When reductions in ODC activity in hypoxia were prevented by the proteasome inhibitor strategies, hypoxia failed to increase polyamine transport. The induction of polyamine transport in hypoxic pulmonary artery endothelial cells thus seems to require decreased ODC activity as an initiating event.     

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.     

Association of increased polyamine levels with isoproterenol-stimulated mucin secretion in the rat submandibular gland

Incubation of rat submandibular gland slices with 50 microM isoproterenol for 10-40 min stimulated mucin secretion and induced a 3- to 4-fold increase in tissue concentrations of the polyamines putrescine, spermidine and spermine. alpha-Difluoromethylornithine, a specific inhibitor of ornithine decarboxylase, suppressed the isoproterenol-induced increase in submandibular polyamines and inhibited mucin secretion. Exogenous putrescine restored tissue polyamine levels and partially reversed the inhibitory effect of alpha-difluoromethylornithine on mucin secretion. Rapid increases in polyamine levels appear to mediate isoproterenol-stimulated mucin secretion in the rat submandibular gland.     

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.     

Induction of Ornithine Decarboxylase by N-Methyl-D-Aspartate Receptor Activation Is Unrelated to Potentiation of Glutamate Excitotoxicity by Polyamines in Cerebellar Granule Neurons

Abstract: Polyamines positively modulate the activity of the N -methyl- d -aspartate (NMDA)-sensitive glutamate receptors. The concentration of polyamines in the brain increases in certain pathological conditions, such as ischemia and brain trauma, and these compounds have been postulated to play a role in excitotoxic neuronal death. In primary cultures of rat cerebellar granule neurons, exogenous application of the polyamines spermidine and spermine (but not putrescine) potentiated the delayed neurotoxicity elicited by NMDA receptor stimulation with glutamate. Furthermore, both toxic and nontoxic concentrations of glutamate stimulated the activity of ornithine decarboxylase (ODC)—the key regulatory enzyme in polyamine synthesis—and increased the concentration of ODC mRNA in cerebellar granule neurons but not in glial cells. Glutamate-induced ODC activation but not neurotoxicity was blocked by the ODC inhibitor difluoromethylornithine. Thus, high extracellular polyamine concentrations potentiate glutamate-triggered neuronal death, but the glutamate-induced increase in neuronal ODC activity may not play a determinant role in the cascade of intracellular events responsible for delayed excitotoxicity.