Ouabain Releases Striatal Polyamines In Vivo Independently of N-Methyl-d-Aspartate Receptor Activation

Abstract: Intrastriatally infused ouabain (200 or 1,000 μ M ) markedly increased the extracellular levels of striatal spermidine and spermine in dialysis experiments in halothane-anesthetized rats. The effects of ouabain (1 m M ) on sper- midine release were rapid and unaffected by local infusion of the competitive N -methyl- d -aspartate (NMDA) antagonist 3-(2-carboxypiperazin-4-yl)propyl-1 -phosphonic acid (CPP; 100 μ M ) or by systemically administered MK-801 (0.3 mg/kg i.p.), both of which treatments markedly inhibit the effects of intrastriatally administered NMDA. The peak effects of ouabain (1 m M ) on spermine release were delayed with respect to those on spermidine release, or to the effects of NMDA, and were also insensitive to locally administered CPP (100 μ M ). However, systemically administered MK-801 (0.3 mg/kg i.p., 30 min before the striatal infusion of drugs), which totally inhibits the effects of NMDA, or CPP (10 mg/kg i.p.; 30 min before the striatal infusion of drugs) partially inhibited the effects of ouabain on spermine release, suggesting partial mediation of the delayed effects of ouabain on spermine release by indirect NMDA-receptor activation. Despite partial sensitivity of ouabain-induced spermine release to systemically administered NMDA antagonists, both spermidine and spermine can be released in vivo by sodium-pump inhibition, independently of NMDA-receptor activation.     

[3H]MK-801 Binding to N-Methyl-d-Aspartate Receptors Solubilized from Rat Brain: Effects of Glycine Site Ligands, Polyamines, Ifenprodil, and Desipramine

The N-methyl-D-aspartate (NMDA) receptor is thought to contain several distinct binding sites that can regulate channel opening. In the present experiments, the effects of ligands for these sites have been examined on [3H]MK-801 binding to a soluble receptor preparation, which had been passed down a gel filtration column to reduce the levels of endogenous small-molecular-weight substances. Glycine site agonists, partial agonists, and antagonists gave effects similar to those observed in membranes [EC50 values (in microM): glycine, 0.31; D-serine, 0.20; D-cycloserine, 1.46; (+)-HA-966, 4.06; and 7-chlorokynurenic acid, 1.81]. Spermine and spermidine enhanced [3H]MK-801 binding to the soluble receptor preparation (EC50, 4.3 and 20.1 microM, respectively), whereas putrescine and cadaverine gave small degrees of inhibitions. When spermine and spermidine were tested under conditions where [3H]MK-801 binding approached equilibrium, their ability to enhance [3H]MK-801 binding was much reduced, a result suggesting that the polyamines increase the rate to equilibrium. Putrescine antagonised the effects of spermine. Ifenprodil reduced [3H]MK-801 binding under both equilibrium and nonequilibrium conditions, although the high-affinity component of inhibition described in membranes was not observed. Ifenprodil antagonised spermine effects in an apparently noncompetitive manner. Desipramine was able to give total inhibition of specific [3H]MK-801 binding under nonequilibrium conditions with an IC50 of 4 microM, and this value was unaltered when [3H]MK-801 binding was allowed to reach equilibrium. These results suggest that the sites mediating the effects of glycine and its analogues, polyamines and desipramine are integral components of the NMDA receptor protein.     

Stimulation of the cyclic AMP-dependent protein kinase-catalyzed phosphorylation of phosphorylase kinase by micromolar concentrations of spermine

The phosphorylation of phosphorylase kinase by cyclic AMP-dependent protein kinase (A-kinase) is stimulated approximately 2-fold by spermine and spermidine. Half maximal effects were observed at 10 microM and 150 microM of spermine and spermidine, respectively. The phosphorylations of other substrates of A-kinase such as glycogen synthase, histone, and casein are not stimulated by these two polyamines. The rates, but not the final extents, of phosphorylation of both the alpha and beta subunits of phosphorylase kinase by A-kinase are stimulated by spermine. The results indicate that spermine and spermidine may play an important role in the activation of glycogenolysis in skeletal muscle.     

Regulation of the F-ATPase from mitochondria of Vigna sinensis (L.) Savi cv. Pitiuba by spermine, spermidine, putrescine, Mg2+, Na+, and K+

Mitochondria from Vigna sinensis (L.) Savi cv. Pitiuba contain the polyamines spermine, spermidine, and putrescine. The membrane-bound F1-ATPase from mitochondria of Vigna sinensis is activated by these polyamines at physiological concentrations. The effect of polyamines on the membrane-bound of F1-ATPase is dependent on the concentrations of Na+, K+, MgATP, and Mg2+. Excess Na+ or K+ prevents the activation of the membrane-bound F1-ATPase by spermine and spermidine, but not by putrescine. The most pronounced effects were observed at low MgATP concentrations in the absence of Na+ and K+. At [MgATP] = 0.08 mM, spermine activation of the membrane-bound F1-ATPase was 130%. The membrane-bound F1-ATPase is slightly activated by Mg2+ at lower concentrations and strongly inhibited by Mg2+ at higher concentrations. Activation as well as inhibition is dependent on the substrate MgATP concentration. Although there is competition between Mg2+ and MgATP, the binding sites for these two ligands are different (pseudocompetitive inhibition). The inhibition of the membrane-bound F1-ATPase can be reversed by polyamines. There is evidence that the binding sites for Mg2+ and polyamines are identical. The F1-ATPase detached from the membrane is neither activated by polyamines nor inhibited by Mg2+. Therefore, the binding sites for Mg2+ and polyamines seem to be localized on the membrane.     

Polyamine uptake in cultured cerebellar granule neurons

In the present study, we have examined the transport of polyamines in cultured cerebellar granule cells. Our results suggest the existence of two different transporters for polyamines in these neurons. Putrescine and spermidine uptake (K ap m = 2.17 and 1.39 microM, respectively), were affected when extracellular sodium was replaced with choline (about 30% inhibition over controls) or sucrose (about 2.5-fold potentiation over controls). By contrast, the substitution of sodium by choline or sucrose did not modify spermine uptake (K ap m = 13.53 microM) in cerebellar granule cells. Accordingly, alteration of membrane potential with ouabain was able to block putrescine (50% inhibition) and spermidine (60% inhibition) uptake but not spermine uptake. These results indicate that putrescine and spermidine transport in cerebellar granule cells is membrane potential dependent, whereas spermine uptake is not modulated by membrane potential.     

A role for polyamines in retinal ganglion cell excitotoxic death

Neuronal death due to excessive activation of N -methyl- d -aspartate (NMDA) receptors is a hallmark of neurodegenerative diseases. The polyamines: putrescine, spermine, and spermidine, bind to specific sites on the NMDA receptor and promote its activation, but their role in NMDA-induced neuronal death is ill defined. In this study, we characterized the role of polyamines in excitotoxic death of retinal ganglion cells (RGCs), a population of central neurons susceptible to NMDA-induced damage. Our data show that endogenous arginase I, the rate limiting enzyme for polyamine biosynthesis, is expressed in the intact, adult retina. Intraocular injection of NMDA visibly increased arginase I expression in Müller cells, the predominant glial cell-type in the mammalian retina. Inhibition of polyamine synthesis using di-fluoro-methyl-ornithine (DFMO) was markedly neuroprotective, while injection of exogenous polyamines in conjunction with NMDA exacerbated RGC death. Blockade of the polyamine binding sites on NMDA receptors using the non-competitive antagonist ifenprodil was neuroprotective, suggesting that polyamines contribute to excitotoxic death, at least partly, by binding to NMDA receptors. Importantly, we also demonstrate that NMDA leads to activation of both the Erk1/2 and PI3 K/Akt pathways, but only the PI3 K/Akt kinase was required for di-fluoro-methyl-ornithine-induced RGC survival. In summary, our study reveals that polyamines modulate neuronal death in the retina via different mechanisms that potentiate NMDA-triggered excitotoxicity.     

Inhibition of translation of mRNAs for ornithine decarboxylase and S-adenosylmethionine decarboxylase by polyamines

The effect of spermidine and spermine on the translation of the mRNAs for ornithine decarboxylase and S-adenosylmethionine decarboxylase was studied using a reticulocyte lysate system and specific antisera to precipitate these proteins. It was found that the synthesis of these key enzymes in the biosynthesis of polyamines was much more strongly inhibited by the addition of polyamines than was either total protein synthesis or the synthesis of albumin. Translation of the mRNA for S-adenosylmethionine decarboxylase was maximal in a lysate which had been substantially freed from polyamines by gel filtration. Addition of 80 microM spermine had no significant effect on total protein synthesis and stimulated albumin synthesis but reduced the production of S-adenosylmethionine decarboxylase by 76%. Similarly, addition of 0.8 mM spermidine reduced the synthesis of S-adenosylmethionine decarboxylase by 82% while albumin and total protein synthesis were similar to that found in the gel-filtered lysate. Translation of ornithine decarboxylase mRNA was greater in the gel-filtered lysate than in the control lysate but synthesis of ornithine decarboxylase was stimulated slightly by low concentrations of polyamines and was maximal at 0.2 mM spermidine or 20 microM spermine. Higher concentrations were strongly inhibitory with a 70% reduction occurring at 0.8 mM spermidine or 150 microM spermine. Further experiments in which both polyamines were added together confirmed that the synthesis of ornithine and S-adenosylmethionine decarboxylases were much more sensitive to inhibition by polyamines than protein synthesis as a whole. These results indicate that an important part of the regulation of polyamine biosynthesis by polyamines is due to a direct inhibitory effect of the polyamines on the translation of mRNA for these biosynthetic enzymes.     

Polyamines are potent ligands for the capsaicin receptor TRPV1

Polyamines are important endogenous regulators of ion channels and are known to modulate inflammation and nociception. Here we investigated effects of polyamines on the capsaicin receptor TRPV1, a major ion channel expressed in nociceptive sensory afferents. Extracellular spermine, spermidine, and putrescine directly activated TRPV1 in a charge-dependent manner, both in heterologous expression systems and sensory neurons. The threshold for activation by spermine was approximately 500 microm at room temperature. At lower concentrations, spermine enhanced capsaicin-evoked currents with an EC50 of approximately 5 microm. Further, polyamines freely permeated TRPV1 (estimated relative permeabilities compared with Na+ were between 3 and 16), and spermine reduced the single channel conductance from 96 to 49 pS. Experiments with TRPV1 mutants identified extracellular acidic residues critical for polyamine regulation. Neutralization of aspartate 646 (D646N) abolished direct activation by spermine, whereas neutralization of this same aspartate (D646N) or glutamate 648 (E648A) inhibited spermine-induced sensitization. These data show that polyamines, by virtue of their cationic charge, can regulate the activity of TRPV1. Extracellular polyamines are present in considerable concentrations in the gastrointestinal tract and at synapses, and these levels increase during inflammation and cancer. Therefore, polyamine regulation of TRPV1 in these tissues may be relevant to a variety of physiological and pathophysiological states.     

Oxidized polyamines and the growth of human vascular endothelial cells. Prevention of cytotoxic effects by selective acetylation.

The responses of human umbilical-vein vascular endothelial cells in culture to the naturally occurring polyamines spermine, spermidine and putrescine, their acetyl derivatives and oxidation products were examined. In the absence of human polyamine oxidase, exposure of cells to polyamines (up to 160 microM) had no adverse effects. In the presence of polyamine oxidase, spermine and spermidine were cytotoxic, but putrescine was not. Acetylation of the aminopropyl group of spermidine or both aminopropyl groups of spermine prevented this cytotoxicity. The amino acids corresponding to the polyamines, representing a further stage of oxidation, were also without effect. The cytotoxic effects were irreversible. Use of bovine serum amine oxidase in place of the human enzyme gave qualitatively similar results.     

Suppression of TNF-alpha production by S-adenosylmethionine in human mononuclear leukocytes is not mediated by polyamines

Endotoxin-induced cytokine production is an important mechanism in the development of several types of liver damage. Methionine, some of its precursors and metabolites were reported to have protective effects against such injury. The aim of this study was to investigate whether methionine, its precursors or metabolites [phosphatidylcholine, choline, betaine, S-adenosylmethionine (SAM)] have a modulating effect on tumor necrosis factor alpha (TNF-alpha) production by endotoxin-stimulated human mononuclear leukocytes and whether SAM-dependent polyamines (spermidine, spermine) are mediators of SAM-induced inhibition of TNF-alpha synthesis. Methionine and betaine had a moderate stimulatory effect on TNF-alpha production, whereas phosphatidylcholine (ID(50) 5.4 mM), SAM (ID(50) 131 microM), spermidine (ID(50) 4.5 microM) and spermine (ID(50) 3.9 microM) had a predominantly inhibitory effect. Putrescine did not alter TNF-alpha release. Inhibitors of polyamine synthesis that blocked either putrescine (difluoromethylornithine) or spermine (CGP48664A) production did not affect TNF-alpha synthesis. Endotoxin stimulation of leukocytes did not alter the intracellular levels of polyamines. In addition, supplementation with SAM did not change the intracellular concentration of either polyamine measured. We conclude that phosphatidylcholine-induced immunosuppression is not caused by methionine and polyamines are not involved in SAM-induced inhibition of TNF-alpha production. The limitation of TNF-alpha release by spermidine is specific and is not due to its conversion into spermine.     

Effects of polyamines on the functionality of photosynthetic membrane in vivo and in vitro

The three major polyamines are normally found in chloroplasts of higher plants and are implicated in plant growth and stress response. We have recently shown that putrescine can increase light energy utilization through stimulation of photophosphorylation [Ioannidis et al., (2006) BBA-Bioenergetics, 1757, 821-828]. We are now to compare the role of the three major polyamines in terms of chloroplast bioenergetics. There is a different mode of action between the diamine putrescine and the higher polyamines (spermidine and spermine). Putrescine is an efficient stimulator of ATP synthesis, better than spermidine and spermine in terms of maximal % stimulation. On the other hand, spermidine and spermine are efficient stimulators of non-photochemical quenching. Spermidine and spermine at high concentrations are efficient uncouplers of photophosphorylation. In addition, the higher the polycationic character of the amine being used, the higher was the effectiveness in PSII efficiency restoration, as well as stacking of low salt thylakoids. Spermine with 50 microM increase F(V) as efficiently as 100 microM of spermidine or 1000 microM of putrescine or 1000 microM of Mg(2+). It is also demonstrated that the increase in F(V) derives mainly from the contribution of PSIIalpha centers. These results underline the importance of chloroplastic polyamines in the functionality of the photosynthetic membrane.     

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.     

Effects of S-adenosyl-1,8-diamino-3-thio-octane and S-methyl-5''-methylthioadenosine on polyamine synthesis in Ehrlich ascites-tumour cells.

The rate-limiting enzymes in polyamine biosynthesis, ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC), are negatively regulated by the polyamines spermidine and spermine. In the present work the spermidine synthase inhibitor S-adenosyl-1,8-diamino-3-thio-octane (AdoDATO) and the spermine synthase inhibitor S-methyl-5'-methylthioadenosine (MMTA) were used to evaluate the regulatory role of the individual polyamines. Treatment of Ehrlich ascites-tumour cells with AdoDATO caused a marked decrease in spermidine content together with an accumulation of putrescine and spermine. Treatment with MMTA, on the other hand, gave rise to a marked decrease in spermine, with a simultaneous accumulation of spermidine. A dramatic increase in the activity of AdoMetDC, but not of ODC, was observed in MMTA-treated cells. This increase appears to be unrelated to the decrease in spermine content, because a similar rise in AdoMetDC activity was obtained when AdoDATO was given in addition to MMTA, in which case the spermine content remained largely unchanged. Instead, we show that the increase in AdoMetDC activity is mainly due to stabilization of the enzyme, probably by binding of MMTA. Treatment with AdoDATO had no effects on the activities of ODC and AdoMetDC, even though it caused a precipitous decrease in spermidine content. The expected decrease in spermidine-mediated suppression of ODC and AdoMetDC was most probably counteracted by the simultaneous increase in spermine. The combination of AdoDATO and MMTA caused a transient rise in ODC activity. Concomitant with this rise, the putrescine and spermidine contents increased, whereas that of spermine remained virtually unchanged. The increase in ODC activity was due to increased synthesis of the enzyme. There were no major effects on the amount of AdoMetDC mRNA by treatment with the inhibitors, alone or in combination. However, the synthesis of AdoMetDC was slightly stimulated in cells treated with MMTA or AdoDATO plus MMTA. The present study demonstrates that regulation of neither ODC nor AdoMetDC is a direct function of the polyamine structure. Instead, it appears that the biosynthesis of the polyamines is feedback-regulated by the various polyamines at many different levels.     

N1-monoacetylation abolishes the inhibitory effect of spermine and spermidine in the reticulocyte lysate translation system

At optimum magnesium, the translation of rat heart mRNA in the nuclease treated rabbit reticulocyte lysate system was inhibited by low concentrations of spermidine or spermine but not of putrescine. Spermidine and spermine cause a general reduction in the translation of all the heart mRNAs since no differential effects were observed when the translation products were examined by gel electrophoresis. Spermine was a five times more potent inhibitor than spermidine but no inhibition was obtained with N1-acetylspermidine or N1-acetylspermine. Since analyses of endogenous polyamines demonstrate that the inhibitory concentrations of spermine could be obtained by converting a small fraction of the endogenous spermidine to spermine, these results indicate that interconversions of the polyamines might be a sensitive regulatory mechanism for protein synthesis.     

Polyamines are unevenly distributed within the rat and rabbit kidney

Summary. Aliphatic polyamines have generally been measured on the whole kidney. Since the kidney is composed of a variety of cells, whole organ data are of limited value for the interpretation of the functions of the polyamines. The aim of this study was to establish the distribution pattern of putrescine, spermidine and spermine within the kidneys of male and female rats and rabbits. It is shown that the polyamines are unevenly distributed along the cortico-papillary axis. Each amine exhibited its own distinct distribution pattern. The polyamines are predominantly located in the cortex. Putrescine levels increased gradually from the cortex to the papillary tip in rabbits, whereas, in rats, fluctuations in putrescine level were marked. In the six zones of the rabbit kidney studied, spermidine and spermine concentrations were markedly higher in females than in males. This difference was less marked in rats. Received April 1, 1999, Accepted May 17, 1999     

Inhibition of mammalian spermine synthase by N-alkylated-1,3-diaminopropane derivatives in vitro and in cultured rat hepatoma cells

A number of N-alkylated-1,3-diaminopropane derivatives [H2N-(CH2)3-NH-(CH2)nH, where n = 1-9] have been tested as potential inhibitors of partially purified rat hepatoma (HTC) cell or pure bovine spleen spermine synthase. Among the compounds described in this paper, the most potent competitive inhibitor of spermine synthase, with respect to spermidine, is N-butyl-1,3-diaminopropane with Ki values of 11.9 nM and 10.4 nM for the HTC cell and bovine spleen enzymes respectively. Inhibition of spermine synthase by this alkylated amine is selective since spermidine synthase activity is not affected up to 100 microM N-butyl-1,3-diaminopropane at a range of 5-200 microM putrescine. Added to the culture medium of growing HTC cells, N-butyl-1,3-diaminopropane causes the expected changes in the polyamine levels with a marked decrease of spermine and an increase of spermidine. Under these conditions cell growth continues unabated. Such N-alkylated-1,3-diaminopropane derivatives may have considerable potential as tools for studying the role of polyamines and in particular the functions of spermine in cell multiplication and differentiation.     

Tissue-specific changes in polyamine levels of neural tissue and fat body in adult crickets

The three major polyamines—putrescine, spermidine, and spermine—were studied and changes of their levels were examined in extracts of cerebral ganglia and fat body from adult Acheta domesticus. In nervous tissue, only spermidine and spermine were present and spermine was two- to three-fold more abundant than spermidine. The polyamine levels were high up to day 3, decreased on day 4, and then remained relatively unchanged up to day 10. The spermidine/spermine ratios decreased during the imaginal life. Higher spermidine titres were observed in the neural tissue of egg-laying females compared to virgin females. In the fat body, putrescine was detected together with spermidine and spermine. Spermidine and spermine levels were two-fold higher than putrescine. Fat body of virgin females contained two times more polyamines than male fat body. Low at emergence, spermidine and spermine concentrations peaked on days 2–3 only in females, and egg-laying was characterized by an increase of putrescine and spermidine titres. Starvation did not change polyamine contents, implying homeostatic regulation of the intracellular polyamine metabolism. These data showing tissue specific changes in polyamine levels during the imaginal life of Acheta domesticus point to the physiological importance of polyamines as possible intracellular regulators during adult insect development. © 1993 Wiley-Liss, Inc.     

Characterization of lipid-protein interactions in acetylcholinesterase lipoprotein extracted from bovine erythrocytes.

1. The activation of human peripheral blood lymphocytes by phytohaemagglutinin in vitro was accompanied by striking increases in the concentrations of the natural polyamines putrescine, spermidine and spermine. 2. The enhanced accumulation of polyamines could be almost totally abolished by dl-alpha-difluoromethylornithine, a newly discovered irreversible inhibitor of l-ornithine decarboxylase (EC 4.1.1.17), or by methylglyoxal bis(guanylhydrazone) {1,1'-[(methylethanediylidene)dinitrilo]diguanidine}, an inhibitor of S-adenosyl-l-methionine decarboxylase (EC 4.1.1.50). The inhibition of polyamine accumulation was associated with a marked suppression of DNA synthesis, which was partially or totally reversed by low concentrations of exogenous putrescine, spermidine, spermine and cadaverine and by higher concentrations of 1,3-diaminopropane. 3. In contrast with some earlier studies, we found that methylglyoxal bis(guanylhydrazone), at concentrations that were sufficient to prevent polyamine accumulation, also caused a clear inhibition of protein synthesis in the activated lymphocytes. Similar results were obtained with difluoromethylornithine. The decrease in protein synthesis caused by both compounds preceded the impairment of DNA synthesis. The inhibition of protein synthesis by difluoromethylornithine was fully reversed by exogenous putrescine, spermidine and spermine, and that caused by methylglyoxal bis(guanylhydrazone) by spermidine and spermine. In further support of the idea that the inhibition of protein synthesis by these compounds was related to the polyamine depletion, we found that difluoromethylornithine caused a dose-dependent decrease in the incorporation of [(14)C]leucine into lymphocyte proteins which closely correlated with the decreased concentrations of cellular spermidine. 4. Difluoromethylornithine and methylglyoxal bis(guanylhydrazone) also elicited a variable depression in the incorporation of [(3)H]uridine and [(14)C]adenine into total RNA. The apparent turnover of lymphocyte RNA remained essentially unchanged in spite of severe polyamine depletion brought about by difluoromethylornithine. 5. The present results, as well as confirming the anti-proliferative action of the inhibitors of polyamine biosynthesis, suggest that polyamine depletion may interfere with reactions at different levels of gene expression.