Modulation of the NMDA receptor by polyamines.

Results of recent biochemical and electrophysiological studies have suggested that a recognition site for polyamines exists as part of the NMDA receptor complex. This site appears to be distinct from previously described binding sites for glutamate, glycine, Mg++,Zn++, and open-channel blockers such as MK-801. The endogenous polyamines spermine and spermidine increase the binding of open-channel blockers and increase NMDA-elicited currents in cultured neurons. These polyamines have been termed agonists at the polyamine recognition site. Studies of the effects of natural and synthetic polyamines on the binding of [3H]MK-801 and on NMDA-elicited currents in cultured neurons have led to the identification of compounds classified as partial agonists, antagonists, and inverse agonists at the polyamine recognition site. Polyamines have also been found to affect the binding of ligands to the recognition sites for glutamate and glycine. However, these effects may be mediated at a site distinct from that at which polyamines act to modulate the binding of open-channel blockers. Endogenous polyamines may modulate excitatory synaptic transmission by acting at the polyamine recognition site of the NMDA receptor. This site could represent a novel therapeutic target for the treatment of ischemia-induced neurotoxicity, epilepsy, and neurodegenerative diseases.     

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.     

Regional Heterogeneity of Polyamine Effects on the N-Methyl-D-Aspartate Receptor in Rat Brain

Abstract: Polyamines have pronounced effects on N-methyl-D-aspartate (NMDA) receptors in vitro and may be important modulators of NMDA receptor activity in vivo. There is considerable regional heterogeneity in the effects of polyamines on [³H]MK-801 binding in rat brain sections. For example, spermidine enhances the binding of [³H]MK-801 to a much greater extent in the striatum than in the cortex. To further explore the basis for this regional heterogeneity, the effects of polyamines on [³H]MK-801 binding were measured in well-washed membranes prepared from frontal cortex and striatum. There was no difference in the concentration-response relationship for spermidine or the K_D for [³H]MK-801 in the presence of 75 μM spermidine, suggesting that the regional difference seen in tissue sections is due to an endogenous factor that is either removed or inactivated during the preparation of membranes. Comparison of spermidine concentration-response curves in washed and unwashed tissue sections revealed that washing selectively enhanced the E_max value in the ventromedial caudate putamen without changing the EC₅₀. This is consistent with the possibility that a noncompetitive polyamine antagonist is being removed from this region during washing. There was no regional variability in the effects of the putative inverse agonist 1, 10-diaminodecane, consistent with recent suggestions that this polyamine inhibits the NMDA receptor at a site distinct from the one at which polyamines act to enhance NMDA receptor function. Agents that modulate the redox state of the NMDA receptor did not eliminate the regional heterogeneity of polyamine effects. Furthermore, the stimulatory effect of glycine in these regions did not correlate with that of spermidine. These results suggest the existence of one or more endogenous factors that noncompetitively influence the effects of polyamines in a regionspecific manner.     

Intracellular polyamine levels are involved in NMDA-evoked nitric oxide production in chick retina cells

The NMDA-sensitive glutamate receptor complex can be modulated by numerous drugs and endogenous substances such as polyamines. We studied the pathway of arginine/nitric oxide/cyclic GMP in cultured chick retina cells through NMDA receptor activation, seen as a function of both differentiation stages of culture and intracellular polyamine levels. In our experimental conditions, the nitric oxide synthase activity was stimulated by NMDA from three to four times between embryonic day (E) 8 plus 5 days in vitro (C) and E8C7. The NMDA response was blocked by MK-801 (10 microM) by >60% at stage E8C5. During culture differentiation, the NMDA-induced increase in nitric oxide synthase activity at the E8C5 stage was blocked by preliminary incubation (24 h) of the cells with alpha-difluoromethylornithine, the inhibitor of polyamine biosynthesis. This effect was assessed by a reduction of NMDA-evoked cyclic GMP formation in polyamine-depleted retina cells. Thus, intracellular polyamine levels are involved in NMDA-evoked nitric oxide production. Our results indicate that (a) the developmental pattern of polyamine levels can be associated with the modulation of NMDA-evoked events and (b) the NMDA-mediated effects have been reduced in alpha-difluoromethylornithine-treated cell cultures. These observations provide evidence for a physiological interaction between polyamines and NMDA-sensitive glutamate receptors during differentiation stages of cultured chick retina cells.     

Anthraquinone polyamines: novel channel blockers of <Emphasis Type="Italic">N</Emphasis>-methyl-<Emphasis Type="SmallCaps">d</Emphasis>-aspartate receptors

Summary. Polyamines, in particular spermine, as well as some natural and synthetic polyamine derivatives have been found to be blockers of N-methyl-d-aspartate receptors. We developed novel, polyamine-based channel blockers to analyze the structure of NMDA receptors. Anthraquinone polyamines block NMDA receptors with some selectivity compared to other glutamate receptors. Results using mutant NR1 and NR2 subunits identified amino acid residues that influence blockade by anthraquinone polyamines. The head group (anthraquinone) may be positioned at the selectivity filter/narrowest constriction of the channel and the polyamine tail penetrates this constriction into the inner vestibule below the level of the selectivity filter. The results are consistent with other work showing that NR1 (Asn616) and NR2B (Asn616), but not NR2B (Asn615), make the narrowest constriction of NMDA channel, and that the M3 segments from the two subunits, which form the outer vestibule, are likely staggered relative to each other in the vertical axis of the channel.     

Polyamine metabolism in enterocytes isolated from newborn pigs.

In the pig, the growth of intestinal mucosa is very intense after birth. Since the polyamines are key elements affecting cell proliferation and differentiation, the present work was undertaken in order to know whether this hypertrophy is associated with an adaptation of polyamine metabolism. Villus enterocytes isolated from pig immediately after birth or 2 days later were found to contain similar amounts of putrescine, spermidine and spermine, i.e., 0.23; 0.41 and 1.24 nmol/10(6) cells, respectively. At birth, despite a relatively high ODC activity, putrescine synthesis from 1 mM L-arginine or 2 mM L-glutamine was very low in isolated enterocytes (6.4 +/- 3.8 pmol/10(6) cells per 30 min), while spermidine and spermine production were not detectable. This could be explained by a very low L-ornithine generation from both amino acids and to an inhibitory effect of polyamines on ODC activity. Two days later, polyamine synthesis from L-arginine remained undetectable despite a higher L-ornithine generation. This was concomitant with a dramatic fall in ODC activity. At both stages, enterocytes were able to take up polyamines from the extracellular medium in a temperature-dependent manner. It is concluded that de-novo synthesis of polyamines from L-arginine or L-glutamine does not play a significant role in the control of polyamine content of pig enterocytes during the postnatal period. In contrast, polyamine uptake by enterocytes would contribute to maintain a steady-state polyamine content during this period.     

Ionotropic Chemosensory Receptors Mediate the Taste and Smell of Polyamines

The ability to find and consume nutrient-rich diets for successful reproduction and survival is fundamental to animal life. Among the nutrients important for all animals are polyamines, a class of pungent smelling compounds required in numerous cellular and organismic processes. Polyamine deficiency or excess has detrimental effects on health, cognitive function, reproduction, and lifespan. Here, we show that a diet high in polyamine is beneficial and increases reproductive success of flies, and we unravel the sensory mechanisms that attract Drosophila to polyamine-rich food and egg-laying substrates. Using a combination of behavioral genetics and in vivo calcium imaging, we demonstrate that Drosophila uses multisensory detection to find and evaluate polyamines present in overripe and fermenting fruit, their favored feeding and egg-laying substrate. In the olfactory system, two coexpressed ionotropic receptors (IRs), IR76b and IR41a, mediate the long-range attraction to the odor. In the gustatory system, multimodal taste sensation by IR76b receptor and GR66a bitter receptor neurons is used to evaluate quality and valence of the polyamine providing a mechanism for the fly’s high attraction to polyamine-rich and sweet decaying fruit. Given their universal and highly conserved biological roles, we propose that the ability to evaluate food for polyamine content may impact health and reproductive success also of other animals including humans.     

Polyamine inhibition of lipoperoxidation. The influence of polyamines on iron oxidation in the presence of compounds mimicking phospholipid polar heads.

Polyamines appear to inhibit peroxidation of vesicles containing acidic phospholipids. A correlation exists between polyamine binding to phospholipid vesicles and its protective effect. However, phosphatidylinositol-containing vesicles which bind spermine are not protected by the polyamine [Tadolini, Cabrini, Landi, Varani & Pasquali (1985) Biogenic Amines 3, 97-106]. In the present paper I tested the hypothesis that polyamines, in particular spermine, by forming a ternary complex with iron and the phospholipid polar head may change the susceptibility of Fe2+ to autoxidation and thus its ability to generate free oxygen radicals. Different compounds mimicking phospholipid polar heads were studied, namely AMP, mimicking phosphatidic acid, CDP-choline, mimicking phosphatidylcholine, and glycerophosphoinositol, mimicking phosphatidylinositol. The results support the proposed hypothesis. In the presence of CDP-choline or of glycerophosphoinositol, spermine poorly affects Fe2+ autoxidation, whereas a considerable inhibition is observed in the presence of AMP. The ability of other phosphorus-containing compounds (ATP, ADP, cyclic AMP, sodium phosphate) to affect Fe2+ autoxidation in the presence of polyamines was also evaluated to understand the molecular mechanism of this phenomenon. It is proposed that polyamines may be part of the passive cellular defence mechanism against the oxidative damage caused by Fe2+.     

Polyamine-mediated reduction in human airway epithelial migration in response to wounding is PGE2 dependent through decreases in COX-2 and cPLA2 protein levels.

Both ornithine decarboxylase inhibition to deplete polyamines and cyclooxygenase inhibition diminish the migration response to injury of human airway epithelial cells in tissue culture monolayers by approximately 75%. Restoration of normal migration responses is achieved in the polyamine depleted system either by exogenous reconstitution of polyamines or the addition of prostaglandin E(2) (PGE(2)). However, only PGE(2) was able to restore migration in the cyclooxygenase-inhibited systems. Western blot for cyclooxygenase-2 and cytosolic phospholipase A(2) protein levels and ELISAs for PGE(2) secretion demonstrate dramatic increases over 24-48 h after monolayer wounding. These increases are completely abolished by polyamine depletion or cyclooxygenase inhibition. We conclude that polyamine inhibition decreases cellular migration in response to injury in airway epithelial cells at least in part through inhibiting normal PGE(2) production in response to injury. This may be brought about by decreases in cytosolic phospholipase A(2) and cyclooxygenase-2 protein levels.     

Differential effects of linear and cyclic polyamines on NMDA receptor activities

The linear polyamine spermine enhances N-methyl-d-aspartate (NMDA) receptors activity at depolarized membrane potential and shows a voltage-dependent block. Spermine potentiates NMDA receptor currents in the presence of saturating concentrations of glutamate and glycine, but cyclic polyamines such as CP2323 do not. CP2323 inhibited the currents most potently amongst 10 kinds of cyclic polyamines tested. The inhibition was prominent at heteromeric NR1/NR2A and NR1/NR2B receptors but not at NR1/NR2C and NR1/NR2D receptors expressed in Xenopus oocytes. Inhibition by CP2323 was voltage-dependent, because the degree of inhibition was in the order -100mV>-70mV>-20mV. It was 10-100 times more prominent than inhibition by spermine. The inhibitory potency of both CP2323 and spermine was attenuated by the mutations around the vestibule of the channel pore at NR1 W563, N650, T807, and NR2B Y646. Inhibition by CP2323 was hardly affected by the mutations of NR1 N616 and E621, whereas inhibition by spermine was reduced by these mutations. The results suggest that CP2323 interacts with the vestibule region of the NMDA receptor and does not enter deep into the channel. Mutations of NR2B W607 greatly reduced the inhibition by CP2323 and spermine, suggesting that the mutation of this residue may cause the change of the channel structure. Neuroprotective effects of cyclic polyamines against cell damage caused by NMDA were compared with those of spermine in cultured rat hippocampal neurons. Addition of CP2323, but not spermine, into the medium attenuated the neurotoxicity induced by NMDA. These results indicate that CP2323 functions as a channel blocker of the NMDA receptor.     

Transglutaminase catalyzed dissociation and association of protein-polyamine complex

Transglutaminase 2 (TG2) has been reported to be involved in cell growth through the formation of epsilon-(gamma-glutamyl) lysine (Gln-Lys) or N-(gamma-glutamyl) polyamine (Gln-polyamine). We have recently reported that the inhibition of Gln-Lys cross-linking by the formation of Gln-spermidine led to the increase of DNA synthesis in regenerating rat liver. TG2 may catalyze the replacement reaction between Lys residues in protein and polyamines. In the present study, we attempted to develop an experimental model for ascertaining this replacement reaction. We examined whether or not TG2 exhibited the association and dissociation reaction of Gln-polyamine bond in protein, using N,N-dimethylcasein (DC). The dissociated polyamines were identified by autoradiography. The dissociation of [(14)C] polyamines from DC bond [(14)C] polyamines complex by TG2 could occur in the presence of non-radioactive polyamines as second amine donor, whereas in the absence, could not almost occur. Moreover, it was indicated that this release of old [(14)C] polyamine bonded to DC was due to binding of added new [(14)C] polyamine to Gln residues in DC. These results demonstrate that TG2 catalyzes the replacement reaction between added [(14)C] polyamine and DC bond [(14)C] polyamine. The dissociation and association reaction may both occur together, the new DC-polyamine complex being formed at the same time as the dissociation of old DC-polyamine complex, since readying a second amine donor is necessary to dissociate DC-polyamine complex. These results indicate that this experimental model is successful in the study of TG2-catalyzed dissociation and association reaction of Gln-polyamine bond in protein.     

Formation of polyamine-modified peptides during protein digestion

The effect of polyamines on the digestion of proteins by serine proteases was examined. Based on the mechanism of action of serine proteases, it was anticipated that nucleophilic functionalities such as amino groups in polyamine, rather than hydroxyl ions, would react with peptide bonds during the hydrolysis process. If this were the case, polyamine might be covalently linked to the C-terminus of the product peptides during protein digestion. In order to test this hypothesis, hemoglobin was used as a model protein and was digested with trypsin in the presence of polyamine. The product peptides were separated, collected by HPLC, and analyzed by MALDI-TOF MS using post-source decay. The results showed that some peptides were indeed modified with polyamine at the C-terminus.     

Polyamine uptake by bovine adrenocortical cells

Bovine adrenocortical cells of fasciculo-reticulata origin in primary culture actively accumulate polyamines from the extracellular medium in an energy-dependent process. At low extracellular concentration (e.g., 1 microM putrescine), the transport system resulted in a several-hundred-fold concentration of polyamine in the cellular compartment within 1-2 h of incubation. Putrescine uptake appeared to be the sum of a sodium-dependent, saturable process, with an apparent Km of about 10 microM and of a non-saturable, sodium-independent component. By contrast, spermine was taken up by the cells mostly in a sodium-independent manner. Cross-competition experiments suggested that both polyamines were at least partly transported by the same system. Using specific corresponding probes, it was shown that the polyamine uptake was independent of the amino acid transport systems of the A, L and N types known in a number of cell systems. Adrenocortical cell polyamine content is known to be modulated by adrenocorticotropin through induction of ornithine decarboxylase activity. The existence of a specific uptake system in these cells opens the possibility of a more rapid pathway for the regulation of cellular polyamine levels. It remains to be examined whether this polyamine transport system is under hormonal control, and whether this can support the suggestion that polyamines may represent a form of intracellular messengers in the mechanism of hormone action.     

Biphasic effect of polyamines on chromatin-bound histone deacetylase

Calf thymus chromatin preparations contain a bound histone deacetylase. The activity of the deacetylase is increased by addition to the reaction mixture of 0.1 to 1 mm polyamine. Further increase in polyamine levels cause a progressive inhibition of enzyme activity. This biphasic action was shown to result from two opposite activities. First, low levels of polyamines are able to release several-fold greater amounts of enzyme from the bound state to free solution. Second, the free enzyme activity is progressively inhibited by polyamines from 0.1 to 20 mm. The combination of the two activities accounts for a peak of activity at around 1 mm polyamine.     

Atmospheric pressure chemical ionization-mass spectrometry method to improve the determination of dansylated polyamines

Determination of polyamine pools is still a step impossible to circumvent in studies aimed at determining the pathophysiological role of natural polyamines. In addition, polyamine measurement in biological fluids and tissues may have clinical relevance, especially in cancer patients. Among the wide panel of analytical methods developed for the quantification of polyamines, high-performance liquid chromatographic (HPLC) separation of polyamines after derivatization with dansyl chloride remains the most commonly used method. In this work, we show that atmospheric pressure chemical ionization-mass spectrometry (MS) can be used to detect and quantify biologically relevant polyamines after dansylation, without chromatographic separation. Positive-ion mass spectra for each dansylated polyamine were generated after optimization by flow injection analysis (FIA). FIA coupled with MS detection by selected ion monitoring greatly increased the sensitivity of the polyamine detection. The method is linear over a wide range of polyamine concentrations and allows detection of quantities as low as 5 fmol. The FIA/MS method is about 50-fold more sensitive than the conventional HPLC/fluorimetry procedure. A good correlation (r>0.98) between these two methods was observed. The FIA/MS method notably reduces the time of analysis per sample to 1.5 min and turns out to be rapid, efficient, cost saving, reproducible, and sufficiently simple to allow its routine application.     

Polyamines and ”scavenging system”: influence of exogenous spermidine on catalase and guaiacol peroxidase activities,and free polyamine level in barley leaves under water deficit

Barley (Hordeum vulgare) seedlings were treated with spermidine prior to water deficit treatment to determine whether this polyamine is able to influence the activity of catalase (EC 1.11.1.6) and guaiacol peroxidase (EC 1.11.1.7). The content of endogenous polyamines was determined as well. This stress caused a visible increase of catalase activity and a much higher increase of guaiacol peroxidase activity. Treatment with exogenous spermidine induced a significant decrease in the activity of the both enzymes, whereas the polyamine level was enhanced, suggesting that polyamines are able to influence the activity of hydrogen peroxide scavenging enzymes and to moderate in that way this signal molecule level.     

Regulation of plant cell-wall pectin methyl esterase by polyamines--interactions with the effects of metal ions.

The kinetic study of the de-esterification of natural pectin by soya bean or orange pectin methyl esterase shows that the rate of the reaction is highly controlled by the presence of polyamines. The reaction rate versus the polyamine concentration is a bell-shaped curve similar to that which is obtained when the concentration of salts is varied in the reaction mixture. However polyamines, in particular the largest ones, are more efficient than salts. The results may be interpreted by assuming that polyamines mainly interact with the negative charges of the pectic substrate which condition the binding of the pectin methyl esterase. Activating effects were observed at polyamine concentrations that have been shown to exist in the plant cell wall in vivo. Thus, polyamines may act as efficient regulators of the cell-wall pH via the control of the electrostatic cell-wall potential. If such is the case, they might have a role in all regulatory mechanisms in which cell-wall enzymes are involved.     

Reproducibility of erythrocyte polyamine measurements and correlation with plasma micronutrients in an antioxidant vitamin intervention study

Erythrocyte polyamine measurements have been previously investigated as candidate biomarkers for hyperproliferation and recently as a potential intermediate endpoint in clinical chemoprevention trials with difluoromethylornithine, an inhibitor of polyamine biosynthesis. This study was performed to determine the reproducibility of erythrocyte polyamine measurements and their possible correlation with plasma micronutrients in seven healthy adults in an antioxidant vitamin intervention study. As part of this cross-over intervention study, three subjects took β-carotene (31.4 mg/day) plus D-α-tocopherol acetate (720 IU/day) supplements during the first 3 months and four subjects took the supplements during the second 3 months. Heparinized blood samples were collected at baseline and every month over total 6 months for simultaneous determination of erythrocyte polyamines and plasma micronutrients by the high-performance liquid chromatographic method. For all the measures of erythrocyte polyamines the intraindividual variation was smaller than that between subjects, and three or four measurements required to accurately characterize long-term erythrocyte polyamines for an individual. The intra-class correlations were moderately high for all erythrocyte polyamine measurements, indicating a good reproducibility for intra-individual erythrocyte polyamine measurements. Based on monthly values, significant inverse correlations were found between erythrocyte spermidine and the plasma levels of retinol (r = -0.50) and lutein (r = -0.52). There were also significant inverse associations between erythrocyte spermine and plasma levels of α-tocopherol (r = -0.29), lutein (r = -0.44), lycopene (r = -0.29), β-cryptoxanthin (r = -0.30), and total carotenoids (r = -0.29). The effects of supplementation upon the associations between erythrocyte polyamines and plasma nutrient levels were additionally addressed. The results indicate an acceptable longitudinal reproducibility of erythrocyte polyamine measurements, support the hypothesis that erythrocyte polyamine measurements may be correlated with plasma levels of certain nutrients, and suggest a further biomarker application in cancer prevention trials involving dietary modifications or specific relevant micronutrients. © 1996 Wiley-Liss, Inc.