Catabolism of polyamines

Summary. Owing to the establishment of cells and transgenic animals which either lack or over-express acetylCoA:spermidine N¹-acetyltransferase a major progress was made in our understanding of the role of polyamine acetylation. Cloning of polyamine oxidases of mammalian cell origin revealed the existence of several enzymes with different substrate and molecular properties. One appears to be identical with the polyamine oxidase that was postulated to catalyse the conversion of spermidine to putrescine within the interconversion cycle. The other oxidases are presumably spermine oxidases, because they prefer free spermine to its acetyl derivatives as substrate. Transgenic mice and cells which lack spermine synthase revealed that spermine is not of vital importance for the mammalian organism, but its transformation into spermidine is a vitally important reaction, since in the absence of active polyamine oxidase, spermine accumulates in blood and causes lethal toxic effects.Numerous metabolites of putrescine, spermidine and spermine, which are presumably the result of diamine oxidase-catalysed oxidative deaminations, are known as normal constituents of organs of vertebrates and of urine. Reasons for the apparent contradiction that spermine is in vitro a poor substrate of diamine oxidase, but is readily transformed into N⁸-(2-carboxyethyl)spermidine in vivo, will need clarification.Several attempts were made to establish diamine oxidase as a regulatory enzyme of polyamine metabolism. However, diamine oxidase has a slow turnover. This, together with the efficacy of the homeostatic regulation of the polyamines via the interconversion reactions and by transport pathways renders a role of diamine oxidase in the regulation of polyamine concentrations unlikely. 4-Aminobutyric acid, the product of putrescine catabolism has been reported to have antiproliferative properties. Since ornithine decarboxylase and diamine oxidase activities are frequently elevated in tumours, it may be hypothesised that diamine oxidase converts excessive putrescine into 4-aminobutyric acid and thus restricts tumour growth and prevents malignant transformation. This function of diamine oxidase is to be considered as part of a general defence function, of which the prevention of histamine and cadaverine accumulation from the gastrointestinal tract is a well-known aspect.     

Differential Profiles of Binding of a Radiolabeled Agonist and Antagonist at a Glycine Recognition Domain on the N- Methyl-D-Aspartate Receptor lonophore Complex in Rat Brain

Abstract: Addition of several polyamines, including spermidine and spermine, was effective in inhibiting binding of the antagonist ligand [³H] 5, 7-dichlorokynurenic acid ([³H]- DCKA) but not of the agonist ligand [³H] glycine ([³H] Gly) to a Gly recognition domain on the N -methyl-D-aspartic acid (NMDA) receptor ionophore complex in rat brain synaptic membranes. In contrast, [³H] DCKA binding was significantly potentiated by addition of proposed polyamine antagonists, such as ifenprodil and (±)-α-(4-chlorophenyl)-4- [(4-fluorophenyl)methyl]-1-piperidine ethanol, with [³H] Gly binding being unchanged. The inhibition by spermidine was significantly prevented by inclusion of ifenprodil. In addition, spermidine significantly attenuated the abilities of four different antagonists at the Gly domain to displace [³H] DCKA binding virtually without affecting those of four different agonists. Phospholipases A₂ and C and p -chloromercuribenzosulfonic acid were invariably effective in significantly inhibiting [³H] DCKA binding with [³H] Gly binding being unaltered. Moreover, the densities of [³H] DCKA binding were not significantly different from those of [³H]- Gly binding in the hippocampus and cerebral cortex, whereas the cerebellum had more than a fourfold higher density of [³H] Gly binding than of [³H] DCKA binding. These results suggest that the Gly domain may have at least two different forms based on the preference to agonists and antagonists in the rodent brain.     

Regulation by the exogenous polyamine spermidine of Na,K-ATPase activity from the gills of the euryhaline swimming crab Callinectes danae (Brachyura, Portunidae)

Euryhaline crustaceans rarely hyporegulates and employ the driving force of the Na,K-ATPase, located at the basal surface of the gill epithelium, to maintain their hemolymph osmolality within a range compatible with cell function during hyper-regulation. Since polyamine levels increase during the adaptation of crustaceans to hyperosmotic media, we investigate the effect of exogenous polyamines on Na,K-ATPase activity in the posterior gills of Callinectes danae, a euryhaline swimming crab. Polyamine inhibition was dependent on cation concentration, charge and size in the following order: spermine > spermidine > putrescine. Spermidine affected K_0.5 values for Na⁺ with minor alterations in K_0.5 values for K⁺ and NH₄⁺, causing a decrease in maximal velocities under saturating Na⁺, K⁺ and NH₄⁺ concentrations. Phosphorylation measurements in the presence of 20 µM ATP revealed that the Na,K-ATPase possesses a high affinity site for this substrate. In the presence of 10 mM Na⁺, both spermidine and spermine inhibited formation of the phosphoenzyme; however, in the presence of 100 mM Na⁺, the addition of these polyamines allowed accumulation of the phosphoenzyme. The polyamines inhibited pumping activity, both by competing with Na⁺ at the Na⁺-binding site, and by inhibiting enzyme dephosphorylation. These findings suggest that polyamine-induced inhibition of Na,K-ATPase activity may be physiologically relevant during migration to fully marine environments.     

Immunomodulatory action of spermine and spermidine on NR8383 macrophage line in various culture conditions

We examined the effect of spermine (SPM) and spermidine (SPD) on tumor necrosis factor (TNF)alpha and monocyte chemoattractant protein-1 (MCP-1) secretion from macrophages in various culture conditions, including several protocols of polyamines addition and media supplemented with 0, 1 or 15% fetal bovine serum. TNFalpha secretion was inhibited by SPM or SPD added 18h before stimulation in a concentration-dependent manner. Their effect was directly related to the presence of FBS. When SPM or SPD was added simultaneously to the stimulus, the TNFalpha secretion inhibition was higher than that obtained after pre-treatment. In this case, the effect was inversely proportional to the presence of FBS. The addition of polyamines also inhibited the secretion of MCP-1 in NR8383 cells. We conclude that SPM and SPD inhibited the secretion of inflammatory cytokines TNFalpha and MCP-1 in different ways, depending on culture conditions. In any case, SPM was more effective than SPD.     

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.     

Amino acids,polyamines and proteins during seed germination of two species of Dipterocarpaceae

Summary Amino acid, polyamine and protein concentrations in seeds and their evolution during seed germination of two dipterocarp species, Hopea odorata and Dipterocarpus alatus, were determined with the help of a multianalytical system. Glutamic acid and glutamine were the major amino compounds present. Hopea seeds also contain high levels of aspartic acid/asparagine, serine, threonine, arginine and alanine, while those of Dipterocarpus contain high levels of alanine, arginine and threonine. These species were quite different in their germination behavior and thus in their protein and amine metabolism rates. In Hopea, polyamines increased during the first 3 days of germination and reached a maximum by the 3rd day, 1 day before maximum germination rate. In Dipterocarpus polyamines reached their maximum at the 6th day while maximum germination rate is observed by the 7th day. This suggests that polyamine compounds could play a role in the early part of the germination process in Hopea and Dipterocarpus seeds. The possibility that control of polyamine biosynthesis could be used for the establishment of biochemical methods to improve seed storage and to control germination of these recalcitrant seeds is discussed.     

Leishmania spp.: cellular levels and synthesis of polyamines during growth cycles

Polyamines were extracted from five different leishmanial strains (Leishmania sp., L. tropica major, L. mexicana, and two L. donovani isolates) and identified as pulrescine, spermidine, and spermine by thin-layer chromatography and mass spectrometry. These sensitive methods were also used to demonstrate the conversion of radioactive putrescine into spermidine and spermine. As in other types of cells, polyamine levels fluctuated during the growth cycle, maximal levels being attained during the logarithmic growth phase. In the five leishmanial strains, which were members of four different serotypes, the spermidine—putrescine ratios also varied, and in two strains of the same serotype, polyamine ratios were practically identical, suggesting that polyamine characteristics might serve as a further criterion for strain identification and classification.     

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.     

Noncompetitive Inhibition of N-Methyl-D-Aspartate by Conantokin-G: Evidence for an Allosteric Interaction at Polyamine Sites

Conantokins T and G are polypeptide toxins present in snails of the genus Conus. These substances were recently reported to act as N-methyl-D-aspartate (NMDA) antagonists. In the present study, we examined the possible mechanisms producing this antagonism. Conantokin-G inhibited spermine- and spermidine-stimulated [3H]MK-801 binding to extensively washed rat forebrain membranes in a noncompetitive manner with IC50 values of approximately 507 and approximately 946 nM, respectively. In contrast, glutamate-enhanced [3H]MK-801 binding was unaffected by conantokin-G concentrations of less than or equal to 20 microM. At concentrations greater than or equal to 5 microM, conantokin-G effected a modest, noncompetitive inhibition of glycine-stimulated [3H]MK-801 binding and also produced a small enhancement of basal [3H]MK-801 binding. Conantokin-G reduced (IC50 approximately 1.08 microM) the NMDA-stimulated accumulation of cyclic GMP in cerebellar granule cell cultures to basal values, but did not affect kainate-mediated increases in cyclic GMP. These findings indicate that conantokin-G acts as a noncompetitive NMDA antagonist through an allosteric inhibition of polyamine responses. The neurochemical profile of this polypeptide is distinct from previously described noncompetitive NMDA antagonists.     

Gender-related differences in polyamine oxidase activity in rat tissues

Summary Variations in level of polyamines and their related enzymes are frequently observed in response to some treatments which affect in a different way male and female. The possibility of a gender-related difference in the oxidation of polyamines was investigated in rats by measuring the activity of polyamine oxidase, a ubiquitous enzyme of vertebrate tissues, which transforms spermine into spermidine and spermidine into putrescine. The study was carried out on thymus, spleen, kidney and liver of young rats of both sexes, and female rats showed a lower polyamine oxidase activity than male rats in all the tissues. We also found higher values of spermidine acetylation in female than male rats in thymus and liver. Owing to these gender-related differences, a higher spermidine N-acetyltransferase/ polyamine oxidase ratio was found in female than in male rats. A second gender-related difference was a higher spermidine/spermine ratio in female than in male, the only exception being the thymus. These basal differences possibly account for the gender-related differences of polyamine metabolic enzyme activities in response to some treatments, including drugs or hormones.