Systemic administration of polyaminergic agents modulate fear conditioning in rats

Rationale The polyamines putrescine, spermine, and spermidine are a group of aliphatic amines that physiologically modulate the N-methyl-d-aspartate (NMDA) receptor, a glutamate receptor implicated in memory formation. Objectives Given the potential application of these drugs in the treatment of memory disorders, we investigated whether agonists and/or antagonists of the NMDA receptor polyamine binding site alters the memory of fear conditioning and determined the time window in which fear conditioning is modulated by polyaminergic agents given by the systemic route. Results Post-training intraperitoneal administration of spermidine (10–100 mg/kg) immediately after training increased, whereas arcaine (10 mg/kg) and MK-801 (0.01–0.1 mg/kg) decreased contextual and auditory fear conditioning. Arcaine and MK-801, at doses that had no effect per se, reversed the facilitatory effect of spermidine. Memory of fear conditioning was impaired by polyaminergic blockade up to 180 min but not at 360 min after training. Conclusion These results provide evidence that systemic administration of polyamine binding site ligands modulate early consolidation of fear conditioning.     

Effects of polyamine depletion by α-difluoromethylornithine on in vitro and in vivo biological properties of 4T1 murine mammary cancer cells

Increased polyamine synthesis has been associated with proliferation and progression of breast cancer, and thus, is a potential target for anti-cancer therapy. Polyamine depletion by DFMO has been shown to decrease pulmonary and bone metastasis from human breast cancer cell xenografts. Following these observations, this study was designed to test the effects of DFMO on in vitro and in vivo features of the highly invasive and metastatic 4T1 murine mammary cancer cells. DFMO inhibited proliferation, caused G1-S arrest, and suppressed in vitro invasiveness of 4T1 cells. In contrast to our previous findings with MDA-MB-435 cells, DFMO did not affect the activation of STAT3, JNK, and ERK, but decreased phosphorylation of p38. DFMO did not alter the expression of Twist. DFMO delayed the orthotopic growth of 4T1 xenografts in association with suppressed putrescine and spermidine levels but increased levels of spermine. DFMO did not affect pulmonary metastasis when primary tumors of control and DFMO-treated mice were matched for size. Interestingly, DFMO reduced Ki-67 expression only in the primary tumors but did not affect its expression in the metastatic tumors in the lung. Cleaved caspase-3 expression was not affected by DFMO in either the primary tumors or pulmonary metastasis. In summary, DFMO treatment markedly inhibited in vitro proliferation and invasiveness of 4T1 cells and retarded the growth of orthotopic xenografts in mice. The failure of DFMO to inhibit pulmonary metastasis in this system appears to be due, at least in part, to its lack of anti-proliferative effect at the metastatic sites.     

Platinum drug effects on the expression of genes in the polyamine pathway: time-course and concentration-effect analysis based on Affymetrix gene expression profiling of A2780 ovarian carcinoma cells

Purpose As a follow-up to our previous findings that platinum drugs induce a key enzyme in polyamine catabolism, gene expression profiling and mathematical modeling were used to define the effects of cisplatin and oxaliplatin on the expression of polyamine metabolic pathway genes in A2780 human ovarian carcinoma cells. Methods Time-course and concentration–effect experiments were each carried out with cisplatin or oxaliplatin in two separate experiments and cells subjected to gene expression profiling using Affymetrix array technology. Time-course data were modeled using exponential increase and decrease models. Concentration–effect data were modeled using a four parameter Hill model. Results Gene expression profiling of human ovarian carcinoma A2780 cells after exposure to either cisplatin or oxaliplatin indicates that the expression of several genes involved in polyamine pathway is affected by the platinum drugs. Mathematical/Statistical modeling of the data from time-course and concentration–effect experiments of gene expression from nine polyamine pathway genes represented on the HGU95Av2 chip, indicates that three biosynthetic pathway genes (SAMDC, ODC1 and SRM) are down-regulated and one catabolic pathway gene (SSAT) is up-regulated. Expression changes were similar for different probesets for a given gene on the array. Studies on the induction of SSAT by platinum drugs suggested by the Affymetrix data have been previously validated from this laboratory (Hector et al. in Mol Cancer Ther 3:813–822, 2004). Here, the effects of oxaliplatin exposure on SAMDC and ODC observed by Affymetix are validated with real time QRT-PCR. Conclusion The data indicate a concerted effect of platinum drugs on the polyamine metabolic pathway with down-regulation in the expression of several enzyme genes involved in biosynthesis and many-fold up-regulation in expression of SSAT, an acetylating enzyme gene that is critically involved in polyamine catabolism and export.     

Inhibition of NMDA-induced currents by conantokin-G and conantokin-T in cultured embryonic murine hippocampal neurons

Conantokin-G (con-G) and conantokin-T (con-T) are small (17 and 21 amino acids, respectively) gamma-carboxyglutamate (Gla) containing peptides derived from the venoms of marine cone snails that are potent and selective inhibitors of N-methyl-D-aspartate (NMDA) receptors. In this study, the effects of con-G and con-T on NMDA-evoked responses were evaluated in mouse primary hippocampal neuronal cultures using the whole-cell patch-clamp technique. Under equilibrium conditions, NMDA-induced currents were inhibited by con-G and con-T (10 nM-100 microM) in a dose-dependent manner while maintaining a holding potential of -70 mV. In the presence of saturating amounts of NMDA (100 microM) and glycine (1 microM), the IC50 values obtained were 487 +/- 85 nM for con-G and 1030 +/- 130 nM for con-T. NMDA (10 microM-1 mM) dose-response curves produced in the presence of con-G or con-T (1 or 3 microM) resulted in a downward shift of the current response at saturation with NMDA, without affecting the EC50. The maximum response obtainable in the absence of peptide could not be achieved by increasing concentrations of NMDA. The same effect was also observed for conantokin inhibition of spermine-potentiated responses. Association rate constants (k(on)) for the peptides were determined in the presence of NMDA and glycine, with and without the addition of spermine. Using a single binding site bimolecular model, k(on) values were 3.1 +/- 0.2 x 10(3) M(-1) s(-1) for con-G and 3.2 +/- 0.1 x 10(3) M(-1) s(-1) for con-T in the absence of spermine. The added presence of a saturating amount of spermine (300 microM) resulted in an approximate 60% increase in the k(on) values for both con-G and con-T. These results demonstrate that con-T and con-G inhibit NMDA-evoked currents, as well as the potentiation by spermine, in what appears to be a noncompetitive manner, and that spermine increases the rate of conantokin inhibition.     

Inhibition of platelet aggregation by putrescine, spermidine, and spermine in hypercholesterolemic rabbits

BACKGROUND: Hypercholesterolemia causes alterations in platelet function. Platelet hyperaggregation is considered a predisposing factor for atherosclerosis. In this paper, the antiaggregating effect of the polyamines putrescine, spermidine, and spermine was studied on platelets of normal and hypercholesterolemic rabbits. METHODS: New Zealand rabbits were fed with a cholesterol-enriched diet for 10 weeks. Lipids and glucose were determined in serum. The assays of platelet aggregation were carried out using platelet-rich plasma (PRP) obtained from both control and cholesterol-fed rabbits. We used 2.5 micromol /mL ADP and 2 microg/mL collagen as inductors of platelet aggregation. In addition, arginase activity and L-arginine content were determined in PRP. RESULTS: Serum total cholesterol and LDL-cholesterol concentrations were increased from 26.3 +/- 8.1 to 1,485.0 +/- 26.8 mg/dL and from 15.9 +/- 5.9 to 1,383.8 +/- 58.9 mg/dL, respectively, whereas triglyceride concentration increased from 88.3 +/- 35.6 to 411.0 +/- 154.5 mg/dL upon cholesterol feeding. Seventy-five percent of platelet aggregation inhibition was observed with 10 microM of polyamines in PRP of normal rabbits. Spermine inhibited platelet aggregation by 54% in PRP of hypercholesterolemic rabbits when ADP was used as agonist. The order of polyamine action was spermine > spermidine > putrescine. In addition, we found that platelet arginase activity and L-arginine content were unaltered upon hypercholesterolemia. CONCLUSIONS: These results show that the polyamines putrescine, spermidine, and spermine have antagonist action in platelet aggregation and suggest a key role of polyamines in platelet aggregation under normal and hypercholesterolemic conditions.     

Characterization of Phycomyces blakesleeanus mutants temperature-sensitive for heat-shock induced germination

Summary Sixty-nine mutants of the fungus Phycomyces blakesleeanus, temperature-sensitive for heat-shock induced germination, have been characterized. All of them show glow viability at 26 °C and normal viability at 16 °C. Eleven mutants recover the wild type phenotype if yeast extract is added to the minimal medium; the mutant phenotype of eight of these mutants is also suppressed by the addition of putrescine or other polyamines. The majority of the mutants are affected very early in germination. Spontaneous, heat-shock and acetate induced germination are not equally impaired by some of the mutations, so specific and independent steps seem to be involved in part of the activation mechanism of germination.     

Characterization of spermidine synthase from Trypanosoma brucei brucei

Spermidine synthase from Trypanosoma brucei brucei was characterized and found to be similar to spermidine synthase from other sources. The Km for putrescine was found to be 0.2 mM and the Km for decarboxylated S-adenosylmethionine 0.1 microM. The approximate molecular weight of the enzyme was 74 000 as determined by a combination of molecular sieve chromatography and sucrose density gradient centrifugation. Spermidine synthase activity was markedly inhibited in vitro by dicyclohexylamine (50% inhibition at 3 microM) and cyclohexylamine (50% inhibition at 15 microM); both being competitive inhibitors with respect to putrescine. S-Adenosyl-1,8-diamino-3-thiooctane, a nucleoside bisubstrate analog, was also a potent inhibitor of enzyme activity (50% inhibition at 25 microM). Administration of dicyclohexylamine to mice with trypanosomiasis resulted in no increase in survival time probably due to the lack of effect on trypanosome spermidine concentrations. Other possible inhibitors remain to be tested in vivo.     

脑梗死患者血清腐胺、精脒、精胺水平变化

目的 动态观察脑梗死患者血清府胺、精脒、精胺水平变化。方法 选择１６例脑梗死患者及２６例正常人,应用高效液相色谱法测定血清腐胺、精脒、精胺水平。结果 脑梗患者发病７天内府胺水平显著升高并达峰值,７天后开始下降,１４天后仍明显高于对照组,精脒、精胺水平７天内显著低于对照组,７天后恢复并逐渐达对照组水平。脑梗死急性期腐胺水平随病情另重显著升高,精胺水平随病情加重而下降。结论聚胺代谢紊乱参与了脑梗死的病     

Effects of gestational or neonatal treatment with alpha-difluoromethylornithine on ornithine decarboxylase and polyamines in developing rat brain and on adult rat neurochemistry

Pregnant rats were treated for five consecutive days during gestation with s.c. injections of the ornithine decarboxylase (ODC) inhibitor alpha-difluoromethylornithine (DFMO). Treatment beginning at gestational days 13 or 14 was effective in inhibiting ODC and altering polyamine levels, and resulted in relatively small decreases in body and forebrain weight, but not in significant differences in adult neurochemistry. Neonatal rats were treated with DFMO from postnatal day 0 (PD 0) to PD 24. In addition to some somatic effects (decreased body weight, delayed eyelid opening and delayed fur growth) the postnatal treatment resulted in a permanent decrease in brain weight, which was mainly due to a dramatic decrease in cerebellar size. During treatment, and 3 days after the end of it, the levels of putrescine and spermidine, but not those of spermine, were consistently lower in the cerebellum and forebrain of DFMO-treated rats than in controls. On the other hand, ODC appeared strongly inhibited only during the first phase of the treatment and showed recovery, and also rebound of the activity, during the second part of the treatment. A screening of neurochemical markers related to cholinergic, GABAergic and glutamatergic neurons, as well to astrocytes and oligodendrocytes was performed in several brain regions (cerebellum, olfactory bulbs, cortex, striaturn, hippocampus) of some of these rats once they became adults. Significant alterations for all the parameters tested, with the exception of the marker for the glutamatergic transmission, were measured in the undersized cerebellum of the neonatally DFMO-treated rats. A shorter neonatal treatment with DFMO (from PD 1 to 6) resulted, in the adult, in decreased cerebellar size and in neurochemical alterations, both very similar to those occurring after the prolonged treatment. In the other brain regions a few minor differences were noticed. The present results show that: (1) the brain polyamine system is differently regulated in foetuses with respect to newborns; (2) the effects of chronic ODC blockade are different on prenatally or postnatally proliferating neurons, due either to a lower sensitivity of gestation ally proliferating neurons or to a subsequent recovery; and (3) chronic postnatal ODC inhibition has a strong effect on proliferating neurons, but little effect on further maturation of postmitotic neurons.     

Characterisation of a functional polyamine site on rat mast cells: association with a NMDA receptor macrocomplex

Polyamines can modulate activation of N-methyl-d-aspartate (NMDA) receptors by binding to a specific polyamine site associated with a NMDA receptor macrocomplex. Polyamines induce histamine release from mast cells, although the mechanism had not been defined. We have examined whether spermine, a natural polyamine, and compound , regarded as a synthetic polyamine, activate mast cells by a polyamine site associated with a NMDA receptor macrocomplex. Spermine induced secretion of histamine from rat peritoneal mast cells and rat brain mast cells in a concentration-dependent manner. Rat peritoneal mast cells were used as a model system to explore the effects of NMDA antagonists on polyamine-induced histamine release. Ifenprodil, MK801 and arcaine inhibited histamine secretion from mast cells exposed to polyamines; the percentage inhibition was greater against spermine than compound . These data support the proposal that spermine (and possibly compound ) induce histamine release from mast cells by interacting with a specific polyamine site on a NMDA receptor complex.