Kukoamine A activates Akt/GSK-3ß signaling pathway to inhibit oxidative stress and relieve myocardial ischemia-reperfusion injury.

PURPOSE: Myocardial ischemia/reperfusion (MI/R) injury refers to a pathological condition of treatment of myocardial infarction. Oxidative stress and inflammation are believed to be important mechanisms mediating MI/R injury. Kukoamine A (KuA), a sperm, is the main bioactive component extracted from the bark of goji berries. In this study, we wanted to investigate the possible effects of KuA on MI/R injury. METHODS: In this experiment, all rats were divided into sham operation group, MI/R group, KuA 10 mg + MI/R group, KuA 20 mg + MI/R group. After 120 min of ischemia/reperfusion treatment, left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), maximal rates of rising and fall of left ventricular pressure (±dp/dtmax), and ischemic area were detected. Serum samples of rats in each group were collected. The enzyme activities of catalase (CAT), glutathione peroxidase (GSH-PX), superoxide dismutase (SOD), levels of malondialdehyde (MDA), CK muscle/brain (CK-MB), tumor necrosis factor (TNF), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6) were detected using enzyme-linked immunosorbent assay (ELISA). The apoptosis of myocardium in each group was detected according to the instructions of the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The expressions of mammalian target of glycogen synthase kinase-3ß (GSH-3ß) and protein kinase B (Akt) mRNA level in myocardial tissues were detected via reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: MI/R rats showed a significant increase in oxidative stress and inflammation. In addition, we showed that KuA significantly improved the myocardial function such as LVSP, left ventricular ejection fraction, +dp/dt, and -dp/dt. Here, it attenuated dose-dependent histological damage in ischemia-reperfused myocardium, which is associated with the enzyme activities of SOD, GSH-PX, and levels of MDA, IL-6, TNF-α, L-1ß. CONCLUSIONS: KuA inhibited gene expression of Akt/GSK-3ß, inflammation, oxidative stress and improved MR/I injury. Taken together, our results allowed us to better understand the pharmacological activity of KuA against MR/I injury.

Role of calcium-permeable AMPA receptors in memory consolidation, retrieval and updating.

The role of the calcium-permeable AMPA receptor (CP-AMPAR) in synaptic plasticity is well established. CP-AMPAR is believed to be recruited to synapse when the memory trace is in a plastic state; however, the direct implications of its expression for memory processes is less known. Here, we investigated the contribution of CP-AMPAR expressed in the basolateral amygdala (BLA) and hippocampus (HPC) in consolidation of different types of memory, retrieval and memory update. We showed that CP-AMPAR blockade by NASPM in the BLA and HPC impaired fear memory consolidation. NASPM infusion in the HPC also impaired spatial memory consolidation in the water maze, whereas consolidation of object location memory was not affected. We found evidence of the CP-AMPAR involvement in the BLA and in the HPC upon memory retrieval. Furthermore, memory update was affected by NASPM infusion in the HPC in both immediate shock deficit and water maze reversal learning tasks. Our data indicate that the activity of CP-AMPAR in the BLA and HPC is required for the consolidation of emotional memories. Moreover, this receptor activity is required for memory retrieval in the BLA and HPC. These findings support that CP-AMPAR has a key function in memory states in which plastic changes are presumably higher, such as the beginning of memory consolidation, and retrieval-induced updating.

Thermospermine catabolism increases Arabidopsis thaliana resistance to Pseudomonas viridiflava.

This work investigated the roles of the tetraamine thermospermine (TSpm) by analysing its contribution to Arabidopsis basal defence against the biotrophic bacterium Pseudomonas viridiflava. The participation of polyamine oxidases (PAOs) in TSpm homeostasis and TSpm-mediated defence was also investigated. Exogenous supply of TSpm, as well as ectopic expression of the TSpm biosynthetic gene ACL5, increased Arabidopsis Col-0 resistance to P. viridiflava, while null acl5 mutants were less resistant than Col-0 plants. The above-mentioned increase in resistance was blocked by the PAO inhibitor SL-11061, thus demonstrating the participation of TSpm oxidation. Analysis of PAO genes expression in transgenic 35S::ACL5 and Col-0 plants supplied with TSpm suggests that PAO 1, 3, and 5 are the main PAOs involved in TSpm catabolism. In summary, TSpm exhibited the potential to perform defensive functions previously reported for its structural isomer Spm, and the relevance of these findings is discussed in the context of ACL5 expression and TSpm concentration in planta. Moreover, this work demonstrates that manipulation of TSpm metabolism modifies plant resistance to pathogens.

L-Arginine enhances glucose and lipid metabolism in rat L6 myotubes via the NO/ c-GMP pathway.

OBJECTIVE: The amino acid Arginine (Arg) is the main biological precursor of nitric oxide (NO) and has been described to improve insulin sensitivity in diabetes and obesity. We investigated the molecular mechanisms involved in the long-term effects of Arg on glucose and lipid metabolism. MATERIALS AND METHODS: L6 myotubes were treated with Arg (7 mmol/L) for 6 days. D-Mannitol (7 mmol/L) was used as control; spermine NONOate (10 µmol/L) and L-NAME (100 µmol/L) were used to evaluate the NO/c-GMP pathway role. Basal and insulin-induced (120 nmol/L) glycogen synthesis, glucose uptake and lipid oxidation, c-GMP and nitrite levels, and the intracellular signaling pathways were evaluated. RESULTS: Arg-treatment increased: 1) basal and insulin-stimulated glycogen synthesis; 2) glucose uptake; 3) palmitate oxidation; 4) p-Akt (Ser(473)), total and plasma membrane GLUT4 content, total and p-AMPK-α and p-ACC (Ser(79)), p-GSK-3α/ß (Ser(21/9)) and 5) nitrite and c-GMP levels. L-NAME treatment suppressed Arg effects on: 1) nitrite and c-GMP content; 2) glycogen synthesis and glucose uptake; 3) basal and insulin-stimulated p-Akt (Ser(473)), total and p-AMPK-α and ACC, and nNOS expression. CONCLUSION: We provide evidence that Arg improves glucose and lipid metabolism in skeletal muscle, in parallel with increased phosphorylation of Akt and AMPK-α. These effects were mediated by the NO/c-GMP pathway. Thus, arginine treatment enhances signal transduction and has a beneficial effect of metabolism in skeletal muscle through direct activation of Akt and AMPK pathways.

New insights into the role of spermine in Arabidopsis thaliana under long-term salt stress.

Polyamines (putrescine, spermidine and spermine) are traditionally implicated in the response of plants to environmental cues. Free spermine accumulation has been suggested as a particular feature of long-term salt stress, and in the model plant Arabidopsis thaliana the spermine synthase gene (AtSPMS) has been reported as inducible by abscisic acid (ABA) and acute salt stress treatments. With the aim to unravel the physiological role of free spermine during salinity, we analyzed polyamine metabolism in A. thaliana salt-hypersensitive sos mutants (salt overlay sensitive; sos1-1, sos2-1 and sos3-1), and studied the salt stress tolerance of the mutants in spermine and thermospermine synthesis (acl5-1, spms-1 and acl5-1/spms-1). Results presented here indicate that induction in polyamine metabolism is a SOS-independent response to salinity and is globally over-induced in a sensitive background. In addition, under long-term salinity, the mutants in the synthesis of spermine and thermospermine (acl5-1, spms-1 and double acl5-1/spms-1) accumulated more Na(+) and performed worst than WT in survival experiments. Therefore, support is given to a role for these higher polyamines in salt tolerance mechanisms.

Inactivation and nitration of human superoxide dismutase (SOD) by fluxes of nitric oxide and superoxide.

Human recombinant MnSOD and CuZnSOD were both inactivated when exposed to simultaneous fluxes of superoxide (JO(2)(*-)) and nitric oxide (J*NO). The inactivation was also observed with varying J*NO/JO(2)(*-) ratios. Protein-derived radicals were detected in both CuZn and MnSOD by immuno-spin trapping. The formation of protein radicals was followed by tyrosine nitration in the case of MnSOD. When MnSOD was exposed to J*NO and JO(2)(*-) in the presence of uric acid, a scavenger of peroxynitrite-derived free radicals, nitration was decreased but inactivation was not prevented. On the other hand, glutathione, known to react with both peroxynitrite and nitrogen dioxide, totally protected MnSOD from inactivation and nitration on addition of authentic peroxynitrite but, notably, it was only partially inhibitory in the presence of the more biologically relevant J*NO and JO(2)(*-). The data are consistent with the direct reaction of peroxynitrite with the Mn center and a metal-catalyzed nitration of Tyr-34 in MnSOD. In this context, we propose that inactivation is also occurring through a *NO-dependent nitration mechanism. Our results help to rationalize MnSOD tyrosine nitration observed in inflammatory conditions in vivo in the presence of low molecular weight scavengers such as glutathione that otherwise would completely consume nitrogen dioxide and prevent nitration reactions.

Nitric oxide inhibits prostanoid synthesis in the rat oviduct.

We have studied the effect of nitric oxide (NO) on the production of arachidonic acid ([14C]-AA) metabolites in the rat oviduct. The basal synthesis of eicosanoids was measured by the conversion of ([14C]-AA) to the different radiolabeled products of cyclooxygenase (COX). The oviducts incubated for 1 h with the labeled substrate of COX were able to convert 3.3 +/- 0.3% of ([14C]-AA) to 6-ceto-PGF1alpha, 10.7 +/- 1.0% to PGF2alpha, 13.5 +/- 1.2% to PGE2 and 6.3 +/- 0.5% to TXB2. The tissues were incubated with different doses of two NO donors: SIN-1 and Spermine NONOate. The results indicated that SIN-1 produces a significant decrease (50%; P < 0.05) in all prostanoids evaluated in a dose-response fashion. The inhibitory effect was completely reversed by addition of 20 microg/ml of hemoglobin (Hb), a NO scavenger. The addition of Spermine NONOate to the incubation medium diminished significantly (65%) the synthesis of COX metabolites suggesting that NO acts by inhibiting COX activity in the rat oviduct. However, NOS inhibitors, N(G)-L-arginine-methyl-ester (L-NAME) nd N(G)-L-monomethyl-arginine (L-NMMA) had no effect on basal production of the prostanoids. These results indicate that in the rat oviduct the synthesis of COX metabolites is negatively regulated by nitric oxide.

Interactions between polyamine analogs with antiproliferative effects and tRNA: a 15N NMR analysis.

N-Bisalkylpolyamine analogs have been shown to exert antiproliferative effects in many tumor models, with the bisethyl derivatives exerting the greatest activities. 15N NMR spectroscopy was used to explore the interactions between these analogs and tRNA. When tRNA was added to solutions of 15N-enriched homospermine (4-4-4), bisethylhomospermine (BE-4-4-4), bismethylhomospermine (BM-4-4-4), bisethylspermine (BE-3-4-3) and 1,19-bis(ethylamino)-5,10,15-triazanonadecane (BE-4-4-4-4), the spin-lattice relaxation times T1 of the nitrogens were strongly reduced. From the temperature dependence of these T1's we calculated the rotational activation energies (Ea) of the correlation times of the amino groups in the presence and absence of tRNA. These data indicate that: i) the N-bisethyl derivatives bind strongly to tRNA through their-NH2(+)-groups (most likely, through hydrogen bonding); ii) the binding is weakest in the N-bismethyl derivative and iii) homospermine binds very weakly and mainly through its -NH3(+)-group (most likely, through electrostatic binding). The binding of the polyamine analogs to tRNA was also estimated by the increase of the half-line widths (D1/2) of the -NH2(+)-groups, derived from the effects that tRNA has on the spin-spin relaxation time T2. The decrease of the V1/2 values of the -NH2(+)-groups in the (15N-polyamine)-tRNA complexes when the analogs were chased away by an excess of spermine confirmed the stronger binding of the bisethyl- with respect to the bismethyl derivatives, as well as the weak binding of homospermine to tRNA. A correlation was also found between the binding strengths of the analyzed polyamine analogs and their antiproliferative activities.