Action Of Spermine Research Articles

Стрес-протекторний вплив путресцину і сперміну на рослини пшениці за ґрунтової посухи

Polyamines are considered multifunctional stress metabolites in plants. The ability of exogenous polyamines to increase plant resistance to adverse factors of various nature is well-known. At the same time, the stress-protective effects of polyamines at the level of whole plants under conditions of dehydration close to natural have not been sufficiently studied. The aim of this work was to study the effect of foliar treatment of wheat plants with solutions of putrescine and spermine on the functioning of their protective systems during drought under laboratory soil culture conditions. In the experiments, we used young wheat plants (Triticum aestivum L.), which were subjected to a 4-day drought with a gradual decrease in the water content in the soil to 25% of the total moisture capacity. Spraying plants with putrescine in a concentration range of 0,25-5 mM significantly reduced the growth-inhibiting effect of drought; the effect of spermine was less effective, but also significant at P ≤ 0,05. Putrescine significantly reduced the manifestation of water deficit caused by drought. Under the action of spermine, only a tendency towards a decrease in the water deficit of the leaves was noted. Drought caused the effect of oxidative stress, which was manifested in an increase in the content of malondialdehyde (MDA) in leaves. During the pretreatment of plants with spermine, the increase in the MDA content was partially leveled, and under the action of putrescine it was leveled almost completely. Treatment of plants with both polyamines at concentrations of 1 and 5 mM promoted preservation of the pool of chlorophylls and carotenoids in leaves under stress conditions. Moreover, when plants were treated with putrescine and spermine under drought conditions, a close to usual ratio of chlorophylls a/b was maintained. The proline content in leaves increased significantly under the influence of drought. Pretreatment with 1 and 5 mM putrescine, and 5 mM spermine reduced effect of proline content growth in leaves, caused by drought. At the same time, the treatment of plants with both polyamines caused the accumulation of sugars in the leaves. Under the influence of drought, the content of anthocyanins and flavonoids absorbing in the UV-B region significantly decreased in the leaves. Pretreatment with spermine somewhat mitigated the negative effect of drought on the anthocyanin content. Under the action of both putrescine and spermine, the content of flavonoids absorbing in UV-B stabilized in leaves. It was concluded that the protective effect of polyamines on wheat plants under drought conditions is primarily due to the regulation of water metabolism and the prevention of oxidative damage.

Structure‐Function Relationship for Spermine‐mediated Inward Rectification of Transient Receptor Potential Canonical (TRPC) 4 Channels.

Transient Receptor Potential Canonical (TRPC) channels constitute calcium‐permeable, non‐selective cation channels in mammalian cells. The expression of TRPC4 channels can be found in gastrointestinal (GI) tract, endometrium, ventricular myocytes, vascular smooth muscle cells, endothelium and many others. In such tissues, it is well‐known that TRPC4 channels exert membrane excitatory effect due to inwardly‐rectifying nature of current‐voltage (I‐V) relationship of the channels. Based on the electrophysiological similarity between inwardly‐rectifying potassium channel and TRPC4 channel, we investigated the molecular mechanism of inward‐rectification of TRPC4 channels, especially by intracellular magnesium and polyamine, which is two major cytoplasmic blocking agent of IRKs.Based on the pre‐established theory for inward‐rectification of IRKs, we have constructed a number of mutant TRPC4 channels whose acidic amino acid (glutamate or aspartate) at cytoplasmic side (C‐terminus) was changed to alanine. Such mutant channels were overexpressed in HEK293 cells and the activity and efficacy of cytoplasmic blocking action of spermine was measured using whole‐cell patch‐clamp technique. Structural analysis was based on published Cryo‐EM structure of mouse TRPC4 channels (PDB Accession Number: 5z96). The modeled 3D structures of TRPC4 mutants were visualized and analyzed using the PyMOL software. For the calculation of electrical distance, two‐barrier model with one reaction‐intermediate potential was conveyed.As a result, the electrical distance of spermine demonstrated that spermine barely penetrates into the pore but rather exerts its action on the entrance of putative inner vestibule of the channel. The voltage‐dependency of Kd value was not significant compared to other well‐known blocking agents such as proton in Na channels and polyamine in IRKs. Among various mutant channels, D629A, E648A, E728A and E729A channels showed significant recovery from spermine block, demonstrating that such residues are crucial for spermine‐mediated inward rectification. Identification of such residues in Cryo‐EM structure of TRPC4 channel showed rather consistent structural implication for residues D629, E728 and E729; they all lined inner vestibule of the channel. E648 is located at the connecting‐helix of the channel and faces the into cytoplasmic face. Hence we postulated that the residue may contribute to the inward‐rectification by reducing access resistance of spermine from channel periphery into inner vestibule. The location of residues fitted well with electrical distance.Support or Funding Information*This work was supported by the National Research Foundation of Korea, which is funded by the Ministry of Science, ICT (Information & Communication Technology), and Future Planning (MSIP) of the Korean Government (2018R1A41023822 to I. So) and by the Education and Research Encouragement Fund of Seoul National University Hospital (I. So). Christine Haewon Park was supported by the BK plus program from the MSIP.

Analysis of interaction between intracellular spermine and transient receptor potential canonical 4 channel: multiple candidate sites of negatively charged amino acids for the inward rectification of transient receptor potential canonical 4

Transient receptor potential canonical 4 (TRPC4) channel is a nonselective calcium-permeable cation channels. In intestinal smooth muscle cells, TRPC4 currents contribute more than 80% to muscarinic cationic current (mIcat). With its inward-rectifying current-voltage relationship and high calcium permeability, TRPC4 channels permit calcium influx once the channel is opened by muscarinic receptor stimulation. Polyamines are known to inhibit nonselective cation channels that mediate the generation of mIcat. Moreover, it is reported that TRPC4 channels are blocked by the intracellular spermine through electrostatic interaction with glutamate residues (E728, E729). Here, we investigated the correlation between the magnitude of channel inactivation by spermine and the magnitude of channel conductance. We also found additional spermine binding sites in TRPC4. We evaluated channel activity with electrophysiological recordings and revalidated structural significance based on Cryo-EM structure, which was resolved recently. We found that there is no correlation between magnitude of inhibitory action of spermine and magnitude of maximum current of the channel. In intracellular region, TRPC4 attracts spermine at channel periphery by reducing access resistance, and acidic residues contribute to blocking action of intracellular spermine; channel periphery, E649; cytosolic space, D629, D649, and E687.

Protective action of spermine and spermidine against photoinhibition of photosystem I in isolated thylakoid membranes.

The photo-stability of photosystem I (PSI) is of high importance for the photosynthetic processes. For this reason, we studied the protective action of two biogenic polyamines (PAs) spermine (Spm) and spermidine (Spd) on PSI activity in isolated thylakoid membranes subjected to photoinhibition. Our results show that pre-loading thylakoid membranes with Spm and Spd reduced considerably the inhibition of O2 uptake rates, P700 photooxidation and the accumulation of superoxide anions (O2 −) induced by light stress. Spm seems to be more effective than Spd in preserving PSI photo-stability. The correlation of the extent of PSI protection, photosystem II (PSII) inhibition and O2 − generation with increasing Spm doses revealed that PSI photo-protection is assumed by two mechanisms depending on the PAs concentration. Given their antioxidant character, PAs scavenge directly the O2 − generated in thylakoid membranes at physiological concentration (1 mM). However, for non-physiological concentration, the ability of PAs to protect PSI is due to their inhibitory effect on PSII electron transfer.

Physiological modulators of cyclosporin A-insensitive nonspecific permeability of the inner membrane of liver mitochondria induced by calcium ions and α,ω-hexadecanedioic acid

ity, inhibited mitochondrial swelling induced by Ca 2+ and HDA, despite the presence of succinate in the incubation medium. Natural uncouplers of oxidative phosphorylation, oleic and linoleic acids, produced a similar effect. These data suggest that energization of organelles, which promotes Ca 2+ transport into the matrix, is one of the basic requirements of pore opening in liver mitochondria induced by Ca 2+ and HDA. It is shown that ATP at the physiological concentration of 2 mM inhibits HDAinduced highamplitude swell� ing of mitochondria by reducing free Ca 2+ concentration in the medium. ADP at the same concentration had a similar effect. This modulating effect of nucleotides apparently is attributable to their ability to chelate cal� cium ions. Polycation spermine, which is known as an inhibitor of the classical CsAsensitive pore, at the physiological concentration of 1 mM inhibited CsAi nsensitive swelling of liver mitochondria induced by sequential addition of Ca 2+ and HDA. It is assumed that such action of spermine is due to its ability to shield the negative surface charges on the inner membrane of mitochondria. Bovine serum albumin (BSA), which is able to bind free fatty acids and thus prevent the induction of Ca 2+ �dependent pore, inhibited HDA� induced swelling of mitochondria. However, at the same BSA/fatty acid molar ratio inhibitory effect of BSA was much less pronounced if HDA was used as the pore inducer instead of palmitic acid. Apparently, this can be accounted by the fact that BSA binds α,ω�dioic acids weaker than their monocarboxylic analogues.

Differential effects of polyamine on the cytosolic and mitochondrial NADP‐isocitrate dehydrogenases

Two isozymes of NADP-dependent isocitrate dehydrogenases (EC 1.1.1.42) exist in mammalian tissues: mitochondrial (ICD1) and cytosolic (ICD2). Effects of polyamines such as spermine, spermidine, and putrescine on the cytosolic and mitochondrial NADP-isocitrate dehydrogenases were analyzed kinetically. Spermine activated ICD2, the cytosolic NADP-isocitrate dehydrogenase from rat liver with the increase in the maximal velocity and the decrease in the affinity for the substrates isocitrate and NADP. The activating action of spermine can be explained by "the uncompetitive effect," and the dissociation constant of spermine for the enzyme-substrate complex was determined to be 1.68 mM. Spermidine and putrescine showed little or no effect. ICD1, the mitochondrial form of NADP-isocitrate dehydrogenase from rat and porcine heart was inhibited by spermine effectively, and by spermidine and putrescine to a lesser extent. Spermine inhibited the enzyme competitively with respect to NADP, and noncompetitively with respect to isocitrate. K(i) value of the enzyme for spermine was 1.3 mM. These results suggest that activation by spermine of cytosolic NADP-isocitrate dehydrogenase can enhance the antioxidant activity by regeneration of GSH, and further is responsible for the stimulation of lipid biosynthesis in cytosol. Spermine may contribute to NADPH supply by enhancing transhydrogenase (EC1.6.1.2) activity through the spermine-dependent activation of Ca(2+) -incorporation to mitochondria.

Generation of a multi-layer muscle fiber sheet from mouse ES cells by the spermine action at specific timing and concentration

Here, we show that spermine can induce the generation of a multi-layer muscle fiber sheet (MMFS) in mouse embryonic stem (ES) cells. ES cells were cultured by the hanging drop method and embryoid bodies (EBs) that formed after 2 days of culture were transferred to a 24-well dish (1 EB/well) containing differentiation medium. EBs cultured in the absence of spermine showed no evidence of differentiation of contractile muscle fibers. In contrast, the addition of spermine (0.5–1.0 mM) for 24 hr on day 12 of culture was found to result in the formation of contractile muscle fibers around the EBs by day 17, with further differentiation into MMFS by day 32. We found that spermine could only induce muscle cell differentiation in EBs during a limited period of culture. Moreover, high concentrations of spermine inhibited muscle fiber generation. Histochemical analysis showed that the MMFS induced by spermine had a heterogeneous architecture. Heart muscle cells appeared to be predominant in some regions, as evidenced by the expression of the markers atrial natriuretic peptide (ANP) and connexin 40 (Cx40), while skeletal muscle appeared to predominate in other regions, as indicated by the expression of MyoD. DNA array analysis showed specific enhancement of expression of muscle cell genes, supporting our conclusion that spermine induces differentiation of muscle cells in vitro.

Effect of <i>Panax ginseng</i> Components on the Differentiation of Mouse Embryonic Stem Cells into Cardiac-like Cells

Bioactive compounds that may control the specific differentiation from mouse embryonic stem (ES) cells into cardiac-like cells have been screened from herbal medicines. Among seven preparations, Panax ginseng was found to promote the differentiation into beating cells and to sustain their beating for longer than the control. Active compounds were found in its water-soluble fraction. Although they were not isolated, their candidates were surveyed in 42 compounds selected from the database of P. ginseng. Finally we found that vitamin B12 (VB12) and methionine were active. VB12 accelerated the differentiation into beating cells and made the beating rate constantly 100%. Moreover, VB12 was effective in the recovery of beating that was inhibited by spermine action. The mechanism of action of VB12 is discussed in termo of the relevance of intercellular electrical signal transduction.

Regulatory role of polyamine in the acid phosphatase from potato tubers

Effects of polyamine and metal ions on the new type of acid phosphatase purified from potato ( Solanum tuberosum L. Irish Cobbler) tubers were analyzed. The enzyme belongs to nonspecific acid phosphatase family (EC 3.1.3.2), which hydrolyzes various phosphorylated substrates. The enzyme hydrolyzed inorganic pyrophosphate as a preferred substrate, and exhibited the hyperbolic kinetics with respect to the substrate, inorganic pyrophosphate in the absence of metal cations. Polyamine activated the enzyme effectively by lowering the K m value without appreciable changes in the maximal velocity. The most effective polyamines as activators were spermine and spermidine. Mg 2+ ion increased the K m value without affecting the maximal velocity of the enzyme, but Ca 2+ ion decreased both the K m and V max values. Increasing concentrations of spermine also decreased the K m value irrespective of Mg 2+ ion included, but gave a constant K m and V max values in the absence and presence of Ca 2+ ion. Action of spermine and metal ions can be explained by the complex formation with the substrate pyrophosphate. The acid phosphatase from potato can utilize the pyrophosphate–spermine or pyrophosphate–Ca 2+ complex as the preferred substrates. However, the enzyme can use the pyrophosphate–Mg complex with a weak affinity for the active site. Polyamine activates acid phosphatase in the absence and presence of metal cations, and activation by polyamine of the enzyme may contribute to the stimulation of starch biosynthesis and the control of glycolysis/gluconeogenesis by regulating PPi levels in growing potato tubers.

Effect of spermine and N 1-dansyl-spermine on epileptiform activity in mouse cortical slices

N 1-dansyl-spermine is a novel polyamine analogue, which has been demonstrated to have an antagonist action at the stimulatory polyamine site on the N-methyl- d-aspartate (NMDA) receptor macrocomplex. Cortical wedges from genetically epilepsy-prone DBA/2 mice demonstrate spontaneous epileptiform activity when perfused with Mg 2+-free artificial cerebrospinal fluid (aCSF). This epileptiform activity has been demonstrated to be primarily mediated through the NMDA receptor. N 1-dansyl-spermine reduced the spontaneous epileptiform activity at a high dose (100 μM) but had no effect at a lower dose (50 μM). The polyamine, spermine (300 μM) caused an increase in the rate of the spontaneous epileptiform discharges. This effect of spermine was antagonised by administration of the low dose of N 1-dansyl-spermine (50 μM). This further demonstrates the role of the NMDA receptor in the production of spontaneous epileptiform discharges in the cortical wedge preparation and clearly illustrates both the facilitatory action of spermine and the polyamine antagonist action of N 1-dansyl-spermine at the stimulatory polyamine site on the NMDA receptor.

Effect of L-type calcium channel antagonists on spermine-induced CNS excitation in vivo

The ability of nitrendipine, nisoldipine, verapamil and gabapentin to inhibit the development of CNS excitation induced by spermine was assessed in mice. Injection of an excitotoxic dose of spermine (100 μg, i.c.v.) in mice results in worsening tremor that culminates in the development of a fatal tonic convulsion within 8 h of spermine administration. The dihydropyridines, nitrendipine and nisoldipine, which are L-type calcium channel antagonists acting at the α1 subunit, inhibited the development of spermine-induced effects. Verapamil, which also acts at the α1 subunit of the L-type calcium channel, also inhibited the development of spermine-induced CNS excitation. Gabapentin, a postulated L-type calcium channel antagonist interacting at the α2δ subunit, did not inhibit the development of spermine-induced effects. These results show that antagonists of the α1 subunit of L-type calcium channels can effectively inhibit the effects of spermine in vivo. This may highlight the importance of L-type calcium channels in spermine action.

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)α 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. TNFα secretion was inhibited by SPM or SPD added 18 h 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 TNFα 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 TNFα and MCP-1 in different ways, depending on culture conditions. In any case, SPM was more effective than SPD.

Involvement of voltage- and ligand-gated Ca2+ channels in the neuroexcitatory and synergistic effects of putative uremic neurotoxins

Renal failure has been viewed as a state of cellular calcium toxicity due to the retention of small fast-acting molecules. We have tested this hypothesis and identified potentially neuroexcitatory compounds among a number of putative uremic neurotoxins by examining the acute in vitro effects of these compounds on cultured central neurons. The in vitro neuroexcitatory and synergistic effects of guanidinosuccinate and spermine were also examined in vivo. The acute effects of 17 candidate uremic neurotoxins on murine spinal cord neurons in primary dissociated cell culture were investigated using the tight-seal whole-cell recording technique. The compounds studied comprised low-molecular-weight solutes like urea, indoles, guanidino compounds, polyamines, purines and phenoles, homocysteine, orotate, and myoinositol. Currents evoked by these compounds were further examined using various ligand- and voltage-gated ion channel blockers. The acute in vivo effects of guanidinosuccinate and spermine were behaviorally assessed following their injection in mice. It was shown that 3-indoxyl sulfate, guanidinosuccinate, spermine, and phenol evoked significant whole-cell currents. Inward whole-cell current evoked by 3-indoxyl sulfate was not blocked by any of the applied ligand- or voltage-gated ion channel blockers, and the compound appeared to influence miscellaneous membrane ionic conductances, probably involving voltage-gated Ca2+ channels as well. Phenol-evoked outward whole-cell currents were at least partly due to the activation of voltage-gated K+ channels, but may also involve a variety of other ionic conductances. On the other hand, inward whole-cell currents evoked by guanidinosuccinate and spermine were shown to be due to specific interaction with voltage- and ligand-gated Ca2+ channels. Guanidinosuccinate-evoked current was caused by activation of N-methyl-d-aspartate (NMDA) receptor-associated ion channels. Low (micromol/L) concentrations of spermine potentiated guanidinosuccinate-evoked current through the action of spermine on the polyamine binding site of the NMDA receptor complex, whereas current evoked by high (mmol/L) concentrations of spermine alone involved direct activation of voltage-gated Ca2+ channels. Finally, intracerebroventricular administration of 0.25 micromol/L spermine potentiated clonic convulsions induced by guanidinosuccinate. These neuroexcitatory and synergistic effects of guanidinosuccinate and spermine could take place at pathophysiologic concentrations. The observed in vitro and in vivo effects of uremic retention solutes suggest that the identified compounds could play a significant role in uremic pathophysiology. Some of the compounds tested displayed in vitro and in vivo neuroexcitatory effects that were mediated by ligand- and voltage-gated Ca2+ channels. The findings suggest a mechanism for the involvement of calcium toxicity in the central nervous system complications in renal failure with particular reference to guanidinosuccinate and spermine.

Intestinal maturation induced by spermine in young animals

In suckling rats, it has been established that oral administration of spermine, a dietary polyamine, at appropriate doses, induces all the modifications in the digestive tract that occur at weaning, namely, (a) in the intestine: variations in the specific activities of disaccharidases and peptidases, gene expression, the level of receptors to polymeric immunoglobulins (RPIs), tissue histology, and the intestinal permeability to macromolecules; (b) maturation of the intestinal immunological system, an observation confirmed in mice; (c) an increase in the specific activity of enzymes contained in the pancreas; (d) a change in the growth rate and biochemical properties of the liver, e.g., in the synthesis of RPIs. The mechanism of spermine action is partly understood. In the intestine, two phases of events have been recorded. The first phase consists of the desquamation of the epithelium resulting from an activation of apoptosis, probably induced by the entrance of spermine into the enterocytes. The second phase concerns a hormonal cascade in which adrenocorticotrophic hormone, cytokines, bombesin and corticosterone intervene. A general hypothesis taking into account all the results obtained until now is presented as well as other observations recorded on the effect of exogenous spermine on the intestinal maturation of vertebrates other than the rat.

On the Mechanism of the Spermine-Exerted Inhibition on α-Thrombin-Induced Platelet Activation*

Previous reports have shown that various amines inhibited platelet activation, but no definitive conclusions on their action mechanism were drawn. We have further investigated the action of spermine on platelet responses evoked by α-thrombin and other agonists. Spermine inhibited in a concentration-dependent manner (1–10 mM), and more efficiently than spermidine and putrescine, the α-thrombin-induced (1.5 nM) platelet activation. Spermine added at a concentration that inhibited completely aggregation only partially affected the thrombin-induced increase in cytosolic Ca 2+ concentration, protein phosphorylation, and ATP secretion. The polyamine had little effect on the morphology of resting platelets, as measured by electron microscopy, thrombin hydrolytic activity, and fibrinogen clotting capacity but decreased the thrombin binding to platelets and isolated glycocalicin. Spermine partially inhibited the aggregation elicited by ADP, vasopressin, platelet-activating factor, thrombin receptor-activating peptide, fluoroaluminate, ionomycin, and dioctanoylglycerol but did not affect the cytosolic Ca 2+ increase induced by these agonists. The polyamine bound to both glycocalicin and platelets, and it inhibited the fibrinogen binding to stimulated platelets. The amount of 14C-spermine bound to resting cells decreased in the presence of the glycoprotein GPIb-antibody LJIB1, whereas the polyamine bound to activated platelets, which was higher than that tied to resting cells, was markedly reduced by LJCP8 or decorsin, a GPIIb/IIIa antibody and antagonist-peptide, respectively. These results indicate that spermine specifically inhibits the thrombin binding to GPIb of resting platelets and the fibrinogen binding to GPIIb/IIIa (integrin α IIbβ 3) of activated platelets.

Casein kinase-II regulates NMDA channel function in hippocampal neurons.

Several second-messenger-regulated protein kinases have been implicated in the regulation of N-methyl-D-aspartate (NMDA) channel function. Yet the role of calcium and cyclic-nucleotide-independent kinases, such as casein kinase II (CKII), has remained unexplored. Here we identify CKII as an endogenous Ser/Thr protein kinase that potently regulates NMDA channel function and mediates intracellular actions of spermine on the channel. The activity of NMDA channels in cell-attached and inside-out recordings was enhanced by CKII or spermine and was decreased by selective inhibition of CKII. In hippocampal slices, inhibitors of CKII reduced synaptic transmission mediated by NMDA but not AMPA receptors. The dependence of NMDA receptor channel activity on tonically active CKII thus permits changes in intracellular spermine levels or phosphatase activities to effectively control channel function.

The Ca2+-sensing receptor: a target for polyamines.

The Ca2+-sensing receptor (CaR) is activated at physiological levels of external Ca2+ (Ca(o)) but is expressed in a number of tissues that do not have well-established roles in the control of Ca(o), including several regions of the brain and the intestine. Polyamines are endogenous polyvalent cations that can act as agonists for the CaR, as shown by our current studies of human embryonic kidney (HEK-293) cells transfected with the human CaR. Cellular parameters altered by polyamines included cytosolic free Ca2+ (Ca(i)), inositol phosphate production, and the activity of a nonselective cation channel. Spermine stimulated Ca(i) transients in CaR-transfected HEK cells, with a concentration producing a half-maximal response (EC50) of approximately 500 microM in the presence of 0.5 mM Ca2+, whereas sustained increases in Ca(i) had an EC50 of approximately 200 microM. The order of potency was spermine > spermidine >> putrescine. Elevation of Ca(o) shifted the EC50 for spermine sharply to the left, with substantial stimulation below 100 microM. Addition of subthreshold concentrations of spermine increased the sensitivity of CaR-expressing HEK cells to Ca(o). Parathyroid hormone secretion from bovine parathyroid cells was inhibited by 50% in the presence of 200 microM spermine, a response similar to that elicited by 2.0 mM Ca(o). These data suggest that polyamines could be effective agonists for the CaR, and several tissues, including the brain, may use the CaR as a target for the actions of spermine and other endogenous polycationic agonists.

Permeation and block of rat GluR6 glutamate receptor channels by internal and external polyamines.

1. Polyamine block of rat GluR6(Q) glutamate receptor channels was studied in outside-out patches from transiently transfected HEK 293 cells. With symmetrical 150 mM Na+ and 30 microM internal spermine there was biphasic voltage dependence with 95% block at +40 mV but only 20% block at +140 mV. Dose-inhibition analysis for external spermine also revealed biphasic block; the Kd at +40 mV (54 microM) was lower than at +80 (167 microM) and -80 mV (78 microM). 2. For internal polyamines relief from block was most pronounced for spermine, weaker for N-(4-hydroxyphenylpropanoyl)-spermine (PPS), and virtually absent for philanthotoxin 343 (PhTX 343), suggesting that permeation of polyamines varies with cross-sectional width (spermine, 0.44 nm; PPS, 0.70 nm; PhTX 343, 0.75 nm). 3. With putrescine, spermidine, or spermine as sole external cations, inward currents at -120 mV confirmed permeation of polyamines. For bi-ionic conditions with 90 mM polyamine and 150 mM Na+i, reversal potentials were -12.4 mV for putrescine (permeability ratio relative to Na+, PPut/PNa = 0.42) and -32.7 mV for spermidine (PSpd/PNa = 0.07). Currents carried by spermine were too small to analyse accurately in the majority of patches. 4. Increasing [Na+]i from 44 to 330 mM had no effect on the potential for 50% block (V1/2) by 30 microM internal spermine; however, relief from block at positive membrane potentials increased with [Na+]i. In contrast, raising [Na+]o from 44 to 330 mM resulted in a depolarizing shift in V1/2, indicating a strong interaction between internal polyamines and external permeant ions. 5. The Woodhull infinite barrier model of ion channel block adequately described the action of spermine at membrane potentials insufficient to produce relief from block. For 30 microM internal spermine such analysis gave Kd(O) = 2.5 microM, z theta = 1.97; block by 30 microM external spermine was weaker and less voltage dependent (Kd(O) = 37.8 microM and z delta = 0.55); delta and theta are electrical distances measured from the outside and inside, respectively. 6. Fits of the Woodhull equation for a permeable blocker adequately described both onset and relief from block by spermine over a wide range of membrane potentials. However, the rate constants and z delta values estimated for block by internal spermine predicted much stronger external block than was measured experimentally, and vice versa. 7. An Eyring rate theory model with two energy wells and three barriers explained qualitatively many characteristic features of the action of polyamines on GluRs, including biphasic I-V relationships, weaker block by external than internal spermine and low permeability.

Dietary spermine supplementation induces intestinal maturation in sea bass (Dicentrarchus labrax) larvae

Sea bass (Dicentrarchus labrax) larvae were fed microparticulated compound diet containing 0 (FP0), 0.10 (FP10) and 0.33% (FP33) of a polyamine, spermine, from day 20 to day 38. LP group was fed live prey. This group exhibited the highest growth and survival. The addition of spermine did not lead to growth enhancement. A 33% survival improvement was obtained in FP33 group compared to FP0 group. The spermine addition affected the activity of pancreatic enzymes, trypsin, chymotrypsin and amylase, during larvae development. This non specific effect suggested that the action of spermine would be mediated by hormones. In the intestine, the FP33 group exhibited from day 31 higher activities of brush border membrane enzymes (leucine aminopeptidase and alkaline phosphatase) and lower level in a cytosolic enzyme (leucine-alanine peptidase) compared to FP10 and FP0 group. The diet containing the highest spermine level induced an enzymatic profile similar to that obtained in LP group and characteristic of a mature enterocyte. The initiation of enterocyte maturation at a proper development stage was associated to the survival improvement observed in FP33 group.

The in vitro action of polyamines on rat basilar and femoral artery contractile activity

This study was performed to assess the role of exogenously administered polyamines on rat basilar and femoral artery contractile activity in vitro. With the endothelium removed, rings of tissue were set up in organ chambers to measure isometric tension. The polyamines (0.1–3mM), putrescine, spermidine, and spermine, were added to the tissue baths; after 30 min of incubation a cumulative concentration response curve (CRC) was obtained with either KCl or serotonin (5-HT). Additional CRCs were run with Ca ++ in high K + Krebs (60mM). In both tissues, the CRCs to KCl were shifted to the right in a dose-dependent manner for spermidine and spermine (1 & 3 mM) but not putrescine. Spermine (3mM) depressed the KCl maxima by 18.6% and 10.1% in the basilar and femoral artery respectively. For 5-HT CRCs, only spermine (3mM) slightly inhibited the maximal response in both tissues. The most potent action of spermine was on inhibition of Ca ++ responses in high K +where the EC 50s were shifted 3.5 and 10 fold over control values in the basilar and femoral respectively. We conclude spermidine and spermine, but not putrescine, attenuate vascular smooth muscle contractions on the basilar and femoral arteries in vitro. The exact nature of the inhibition remains to be fully explored, but blockade of calcium entry through voltage operated Ca channels may play a role. Thus, certain polyamines may affect cerebral perfusion by inhibition of vascular contractility.