Comparative analysis of hippocampal extracellular space uncovers widely altered peptidome upon epileptic seizure in urethane-anaesthetized rats.

BACKGROUND: The brain extracellular fluid (ECF), composed of secreted neurotransmitters, metabolites, peptides, and proteins, may reflect brain processes. Analysis of brain ECF may provide new potential markers for synaptic activity or brain damage and reveal additional information on pathological alterations. Epileptic seizure induction is an acute and harsh intervention in brain functions, and it can activate extra- and intracellular proteases, which implies an altered brain secretome. Thus, we applied a 4-aminopyridine (4-AP) epilepsy model to study the hippocampal ECF peptidome alterations upon treatment in rats. METHODS: We performed in vivo microdialysis in the hippocampus for 3-3 h of control and 4-AP treatment phase in parallel with electrophysiology measurement. Then, we analyzed the microdialysate peptidome of control and treated samples from the same subject by liquid chromatography-coupled tandem mass spectrometry. We analyzed electrophysiological and peptidomic alterations upon epileptic seizure induction by two-tailed, paired t-test. RESULTS: We detected 2540 peptides in microdialysate samples by mass spectrometry analysis; and 866 peptides-derived from 229 proteins-were found in more than half of the samples. In addition, the abundance of 322 peptides significantly altered upon epileptic seizure induction. Several proteins of significantly altered peptides are neuropeptides (Chgb) or have synapse- or brain-related functions such as the regulation of synaptic vesicle cycle (Atp6v1a, Napa), astrocyte morphology (Vim), and glutamate homeostasis (Slc3a2). CONCLUSIONS: We have detected several consequences of epileptic seizures at the peptidomic level, as altered peptide abundances of proteins that regulate epilepsy-related cellular processes. Thus, our results indicate that analyzing brain ECF by in vivo microdialysis and omics techniques is useful for monitoring brain processes, and it can be an alternative method in the discovery and analysis of CNS disease markers besides peripheral fluid analysis.

Elabela relaxes rat pulmonary artery and trachea via BKCa, KV, and KATP channels.

OBJECTIVE: Elabela is a newly discovered peptide hormone. This study aimed to determine the functional effects and mechanisms of action of elabela in rat pulmonary artery and trachea. MATERIALS AND METHODS: Vascular rings isolated from the pulmonary arteries of male Wistar Albino rats were placed in chambers in the isolated tissue bath system. The resting tension was set to 1 g. After the equilibration period, the pulmonary artery rings were contracted with 10-6 M phenylephrine. Once a stable contraction was achieved, elabela was applied cumulatively (10-10-10-6 M) to the vascular rings. To determine the vasoactive effect mechanisms of elabela, the specified experimental protocol was repeated after the incubation of signaling pathway inhibitors and potassium channel blockers. The effect and mechanisms of action of elabela on tracheal smooth muscle were also determined by a similar protocol. RESULTS: Elabela exhibited a concentration-dependent relaxation in the precontracted rat pulmonary artery rings (p < .001). Maximal relaxation level was 83% (pEC50: 7.947 CI95(7.824-8.069)). Removal of the endothelium, indomethacin incubation, and dideoxyadenosine incubation significantly decreased the vasorelaxant effect levels of elabela (p < .001). Elabela-induced vasorelaxation levels were significantly reduced after iberiotoxin, glyburide, and 4-Aminopyridine administrations (p < .001). L-NAME, methylene blue, apamin, TRAM-34, anandamide, and BaCl2 administrations did not cause a significant change in the vasorelaxant effect level of elabela (p = 1.000). Elabela showed a relaxing effect on precontracted tracheal rings (p < .001). Maximal relaxation level was 73% (pEC50: 6.978 CI95(6.791-7.153)). The relaxant effect of elabela on tracheal smooth muscle was decreased significantly after indomethacin, dideoxyadenosine, iberiotoxin, glyburide, and 4-Aminopyridine incubations (p < .001). CONCLUSIONS: Elabela exerted a prominent relaxant effect in the rat pulmonary artery and trachea. Intact endothelium, prostaglandins, cAMP signaling pathway, and potassium channels (BKCa, KV, and KATP channels) are involved in the vasorelaxant effect of elabela. Prostaglandins, cAMP signaling pathway, BKCa channels, KV channels, and KATP channels also contribute to elabela-induced tracheal smooth muscle relaxant effect.

Effects of 4-Aminopyridine on Combined Nerve and Muscle Injury and Bone Loss.

PURPOSE: Musculoskeletal injuries are common, and peripheral nerve injury (PNI) causes significant muscle and bone loss within weeks. After PNI, 4-aminopyridine (4-AP) improves functional recovery and muscle atrophy. However, it is unknown whether 4-AP has any effect on isolated traumatic muscle injury and PNI-induced bone loss. METHODS: A standardized crush injury was performed on the sciatic nerve and muscles in mice, and the mice were assigned to receive normal saline or 4-AP treatment daily for 21 days. The postinjury motor and sensory function recovery was assessed, injured muscles were processed for histomorphometry, and the tibial bone was scanned for bone density. RESULTS: 4-Aminopyridine significantly accelerated the postinjury motor and sensory function recovery, improved muscle histomorphometry, increased muscle satellite cell numbers, and shifted muscle fiber types after combined nerve and muscle injury. Importantly, the 4-AP treatment significantly reduced PNI-induced bone loss. In contrast, in the case of isolated muscle injury, 4-AP had no effect on functional recovery and bone density, but it improved muscle-specific histomorphometry to a limited extent. CONCLUSIONS: These findings demonstrate the potential beneficial effects of 4-AP on the recovery of muscle morphology and bone density after combined muscle and nerve injury. CLINICAL RELEVANCE: Nerve injuries frequently involve muscle and result in rapid muscle and bone atrophy. In this scenario, 4-AP, in addition to accelerating nerve functional recovery, might work as an adjunctive agent to improve the recovery of injured muscle and attenuate PNI-induced bone loss.

Human cerebral spheroids undergo 4-aminopyridine-induced, activity associated changes in cellular composition and microrna expression.

Activity-induced neurogenesis has been extensively studied in rodents but the lack of ante mortem accessibility to human brain at the cellular and molecular levels limits studies of the process in humans. Using cerebral spheroids derived from human induced pluripotent stem cells (iPSCs), we investigated the effects of 4-aminopyridine (4AP) on neuronal activity and associated neurogenesis. Our studies demonstrate that 4AP increases neuronal activity in 3-month-old cerebral spheroids while increasing numbers of new neurons and decreasing the population of new glial cells. We also observed a significant decrease in the expression of miR-135a, which has previously been shown to be decreased in exercise-induced neurogenesis. Predicted targets of miR-135a include key participants in the SMAD2/3 and BDNF pathways. Together, our results suggest that iPSC-derived cerebral spheroids are an attractive model to study several aspects of activity-induced neurogenesis.

Lappaconitine inhibits glutamate release from rat cerebrocortical nerve terminals by suppressing Ca2+ influx and protein kinase A cascade.

The inhibition of the excessive release of glutamate in the brain has emerged as a promising new option for developing therapeutic strategies for neurodegenerative disorders. This study investigated the effect and mechanism of lappaconitine, a diterpenoid alkaloid found in species of Aconitum, on glutamate release in rat cerebral cortex nerve terminals (synaptosomes). Here, we report that in the rat cortical synaptosomal preparation, lappaconitine reduced the K+ channel blocker 4-aminopyridine (4-AP)-evoked Ca2+-dependent release of glutamate. The inhibitory effect of lappaconitine on the evoked glutamate release was blocked by the vesicular transporter inhibitor bafilomycin A1 and calcium-chelating agent ethylene glycol tetraacetic acid (EGTA), but was unaffected by exposure to the glutamate transporter inhibitor dl-threo-beta-benzyloxyaspartate (dl-TBOA). The depolarization-induced elevation of cytosolic calcium concentration ([Ca2+]c) was inhibited by lappaconitine, while the 4-AP-mediated depolarization of the synaptosomal membrane potential was not affected. The inhibition of glutamate release by lappaconitine was markedly decreased in synaptosomes pretreated with the Cav2.3 (R-type) channel blocker SNX-482 or the protein kinase A inhibitor H89. Nevertheless, the lappaconitine-mediated inhibition of glutamate release was not abolished by the intracellular Ca2+-release inhibitors dantrolene and CGP37157. Lappaconitine also significantly decreased the 4-AP-induced phosphorylation of PKA and SNAP-25, a presynaptic substrate for PKA. Our data suggest that lappaconitine reduces Ca2+ influx through R-type Ca2+ channels, subsequently reducing the protein kinase A cascade to inhibit the evoked glutamate release from rat cerebral cortex nerve terminals.

Regional and sex differences in spontaneous striatal dopamine transmission.

Striatal dopamine release is key for learning and motivation and is composed of subregions including the dorsal striatum (DS), nucleus accumbens core, and the nucleus accumbens shell. Spontaneously occurring dopamine release was compared across these subregions. Dopamine release/uptake dynamics differ across striatal subregions, with dopamine transient release amplitude and release frequency greatest in male mice, and the largest signals observed in the DS. Surprisingly, female mice exhibited little regional differences in dopamine release for DS and nucleus accumbens core regions, but lower release in the nucleus accumbens shell. Blocking voltage-gated K+ channel (Kv channels) with 4-aminopyridine enhanced dopamine detection without affecting reuptake. The 4-aminopyridine effects were greatest in ventral regions of female mice, suggesting regional differences in Kv channel expression. The dopamine transporter blocker cocaine also enhanced detection across subregions in both sexes, with greater overall increased release in females than males. Thus, sex differences in dopamine transmission are apparent and likely include differences in the Kv channel and dopamine transporter function. The lack of regional differences in dopamine release observed in females indicates differential regulation of spontaneous and evoked dopamine release.

Structure-activity relationship studies of four novel 4-aminopyridine K+ channel blockers.

4-Aminopyridine (4AP) is a specific blocker of voltage-gated potassium channels (KV1 family) clinically approved for the symptomatic treatment of patients with multiple sclerosis (MS). It has recently been shown that [18F]3F4AP, a radiofluorinated analog of 4AP, also binds to KV1 channels and can be used as a PET tracer for the detection of demyelinated lesions in rodent models of MS. Here, we investigate four novel 4AP derivatives containing methyl (-CH3), methoxy (-OCH3) as well as trifluoromethyl (-CF3) in the 2 and 3 position as potential candidates for PET imaging and/or therapy. We characterized the physicochemical properties of these compounds (basicity and lipophilicity) and analyzed their ability to block Shaker K+ channel under different voltage and pH conditions. Our results demonstrate that three of the four derivatives are able to block voltage-gated potassium channels. Specifically, 3-methyl-4-aminopyridine (3Me4AP) was found to be approximately 7-fold more potent than 4AP and 3F4AP; 3-methoxy- and 3-trifluoromethyl-4-aminopyridine (3MeO4AP and 3CF34AP) were found to be about 3- to 4-fold less potent than 4AP; and 2-trifluoromethyl-4-AP (2CF34AP) was found to be about 60-fold less active. These results suggest that these novel derivatives are potential candidates for therapy and imaging.

Design and evolution of an enzyme with a non-canonical organocatalytic mechanism.

The combination of computational design and laboratory evolution is a powerful and potentially versatile strategy for the development of enzymes with new functions1-4. However, the limited functionality presented by the genetic code restricts the range of catalytic mechanisms that are accessible in designed active sites. Inspired by mechanistic strategies from small-molecule organocatalysis5, here we report the generation of a hydrolytic enzyme that uses Nδ-methylhistidine as a non-canonical catalytic nucleophile. Histidine methylation is essential for catalytic function because it prevents the formation of unreactive acyl-enzyme intermediates, which has been a long-standing challenge when using canonical nucleophiles in enzyme design6-10. Enzyme performance was optimized using directed evolution protocols adapted to an expanded genetic code, affording a biocatalyst capable of accelerating ester hydrolysis with greater than 9,000-fold increased efficiency over free Nδ-methylhistidine in solution. Crystallographic snapshots along the evolutionary trajectory highlight the catalytic devices that are responsible for this increase in efficiency. Nδ-methylhistidine can be considered to be a genetically encodable surrogate of the widely employed nucleophilic catalyst dimethylaminopyridine11, and its use will create opportunities to design and engineer enzymes for a wealth of valuable chemical transformations.

Piperine-mediated suppression of voltage-dependent Ca2+ influx and glutamate release in rat hippocampal nerve terminals involves 5HT1A receptors and G protein ßγ activation.

Piperine is the crucial alkaloid component of black pepper (Piper nigrum Linn.) and has neuroprotective effects. Because inhibition of glutamatergic excitatory neurotransmission is a possible mechanism involved in neuroprotection, we investigated the effect of piperine on the 4-aminopyridine (4-AP)-evoked release of glutamate from rat hippocampal synaptosomes. Piperine inhibited 4-AP-evoked glutamate release, and the inhibition was prevented by the chelation of extracellular Ca2+ ions and a vesicular transporter inhibitor. Piperine reduced the 4-AP-evoked elevation of intrasynaptosomal Ca2+ levels but did not affect the synaptosomal membrane potential. In the presence of ω-conotoxin MVIIC, an N- and P/Q-type channel blocker, the piperine-mediated inhibition of 4-AP-evoked glutamate release was markedly reduced; however, dantrolene and CGP37157, which are intracellular Ca2+-release inhibitors, did not alter the piperine effect. In addition, immunocytochemical analysis confirmed the presence of presynaptic 5-hydroxytryptamine 1A (5-HT1A) receptor proteins. The glutamate release-inhibiting effect of piperine was discovered to be prevented by the 5-HT1A receptor antagonist WAY100635 and the G protein ßγ subunit inhibitor gallein; however, it was unaffected by the adenylate cyclase inhibitor SQ22536 or the protein kinase A inhibitor PKI622. These results suggest that piperine inhibits glutamate release from rat hippocampal nerve terminals by reducing Ca2+ influx through N- and P/Q-type Ca2+ channels and that the activation of presynaptic 5-HT1A receptors and the G protein ßγ subunit is involved in this effect.

Fampridine is a Substrate and Inhibitor of Human OCT2, but not of Human MATE1, or MATE2K.

PURPOSE: The renal clearance of fampridine (Fampyra®, or Ampyra®) significantly exceeds the glomerular filtration rate, suggesting active renal secretion is likely the major elimination pathway. The goal of this study was to identify the renal transporters that are involved in the renal active secretion, and elucidate the active renal secretion mechanism of fampridine. METHODS: The uptake of fampridine to HEK-293 cells overexpressing human OCT2, MATE1 or MATE2K was determined in the absence and presence of Cimetidine, the prototypical inhibitor of the transporters. The inhibition potential of fampridine on the renal transporters was evaluated by determining the uptake of TEA and Metformin, the probe substrates of the transporters of OCT2 and MATEs, respectively, in the absence or presence of fampridine. RESULTS: Significant time- and concentration-dependent uptake of fampridine by human OCT2 was observed. The Km and Vmax were determined as 51.0 ± 17.1 µM and 1107 ± 136 pmole/min/106 cells, respectively. Fampridine also inhibited OCT2 mediated uptake of Metformin with estimated IC50 of 66.8 µM. In contrast, there was not significant uptake of fampridine by human MATE1 or MATE2K, and fampridine did not inhibit MATE1 or MATE2K mediated uptake of TEA. CONCLUSION: The studies indicated fampridine is a substrate and inhibitor of OCT2, but not MATE1 or MATE2K. Results from the study suggested the active renal secretion of fampridine is mediated by human OCT2 but not MATE1 or MATE2K. To our knowledge, fampridine is the first reported substrate specific to OCT2 but not to MATE1 or MATE2K.

Voltage-gated potassium channel blocker 4-aminopyridine induces glioma cell apoptosis by reducing expression of microRNA-10b-5p.

Accumulating evidence has demonstrated that voltage-gated potassium channels (Kv channels) were associated with regulating cell proliferation and apoptosis in tumor cells. Our previous study proved that the Kv channel blocker 4-aminopyridine (4-AP) could inhibit cell proliferation and induce apoptosis in glioma. However, the precise mechanisms were not clear yet. MicroRNAs (miRNAs) are small noncoding RNAs that act as key mediators in the progression of tumor, so the aim of this study was to investigate the role of miRNAs in the apoptosis-promoting effect of 4-AP in glioma cells. Using a microRNA array, we found that 4-AP altered the miRNA expression in glioma cells, and the down-regulation of miR-10b-5p induced by 4-AP was verified by real-time PCR. Transfection of miR-10b-5p mimic significantly inhibited 4-AP-induced caspases activation and apoptosis. Moreover, we verified that apoptosis-related molecule Apaf-1 was the direct target of miR-10b-5p. Furthermore, miR-10b-5p mimic significantly inhibited 4-AP-induced up-regulation of Apaf-1 and its downstream apoptosis-related proteins, such as cleaved caspase-3. In conclusion, Kv channel blocker 4-AP may exert its anti-tumor effect by down-regulating the expression of miR-10b-5p and then raised expression of Apaf-1 and its downstream apoptosis-related proteins. Current data provide evidence that miRNAs play important roles in Kv channels-mediated cell proliferation and apoptosis.

Resistance to protein adsorption and adhesion of fibroblasts on nanocrystalline diamond films: the role of topography and boron doping.

Boron-doped nanocrystalline diamond (BNCD) films exhibit outstanding electrochemical properties that make them very attractive for the fabrication of electrodes for novel neural interfaces and prosthetics. In these devices, the physicochemical properties of the electrode materials are critical to ensure an efficient long-term performance. The aim of this study was to investigate the relative contribution of topography and doping to the biological performance of BNCD films. For this purpose, undoped and boron-doped NCD films were deposited on low roughness (LR) and high roughness (HR) substrates, which were studied in vitro by means of protein adsorption and fibroblast growth assays. Our results show that BNCD films significantly reduce the adsorption of serum proteins, mostly on the LR substrates. As compared to fibroblasts cultured on LR BNCD films, cells grown on the HR BNCD films showed significantly reduced adhesion and lower growth rates. The mean length of fibronectin fibrils deposited by the cells was significantly increased in the BNCD coated substrates, mainly in the LR surfaces. Overall, the largest influence on protein adsorption, cell adhesion, proliferation, and fibronectin deposition was due to the underlying sub-micron topography, with little or no influence of boron doping. In perspective, BNCD films displaying surface roughness in the submicron range may be used as a strategy to reduce the fibroblast growth on the surface of neural electrodes.

Organocatalysis paradigm revisited: are metal-free catalysts really harmless?

Catalysts are commonly used in polymer synthesis. Traditionally, catalysts used to be metallic compounds but some studies have pointed out their toxicity for human health and environment, and the removal of metal impurities from synthetic polymer is quite expensive. Organocatalysts have been intensively synthesized and are now widely used in ring-opening polymerization (ROP) reactions to address these issues. However, for most of them, there is not any evidence of their safety. The present study attempts to assess whether well-established organo-based ROP catalysts used for the preparation of FDA-approved polyesters may present a certain level of cytotoxicity. In vitro toxicity is evaluated using a methyl-thiazol-tetrazolium cytotoxicity assay on two cell models (FHs74Int and HepaRG). Among the investigated organocatalysts, only functionalized thiourea shows an important cytotoxicity on both cell models. 4-Dimethylaminopyridine (DMAP), 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD), and meta-(trimethylammonio)phenolate betaine (m-BE) show cytotoxicity against HepaRG cell line only at a high concentration.

Enrichment and characterization of a bacterial culture that can degrade 4-aminopyridine.

BACKGROUND: The agrichemical 4-aminopyridine is used as a bird repellent in crop fields and has an epileptogenic action in a variety of animals, including man and mouse. 4-Aminopyridine is biodegraded in the environment through an unknown mechanism. RESULTS: A 4-aminopyridine-degrading enrichment culture utilized 4-aminopyridine as a carbon, nitrogen, and energy source, generating 4-amino-3-hydroxypyridine, 3,4-dihydroxypyridine, and formate as intermediates. 4-Amino-3-hydroxypyridine could not be further metabolized and probably accumulated as a dead-end product in the culture. Biodegradability tests and partial sequence analysis of the enrichment culture indicated that 4-aminopyridine was mainly degraded via 3,4-dihydroxypyridine and that the metabolite is probably cleaved by 3-hydroxy-4-pyridone dioxygenase. Seven culturable predominant bacterial strains (strains 4AP-A to 4AP-G) were isolated on nutrient agar plates. Changes in the bacterial populations of 4-aminopyridine, 3,4-dihydroxypyridine, or formate/ammonium chloride enrichment cultures were monitored by denaturing gradient gel electrophoresis (DGGE) profiling of PCR-amplified 16S rRNA gene fragments. Sequence analysis of the 16S rRNA gene fragments derived from predominant DGGE bands indicated that Pseudomonas nitroreducens 4AP-A and Enterobacter sp. 4AP-G were predominant in the three tested enrichment cultures and that the unculturable strains Hyphomicrobium sp. 4AP-Y and Elizabethkingia sp. 4AP-Z were predominant in 4-aminopyridine and formate/ammonium chloride enrichment cultures and in the 3,4-dihydroxypyridine enrichment culture, respectively. Among the culturable strains, strain 4AP-A could utilize 3,4-dihydroxypyridine as a growth substrate. Although we could not isolate strain 4AP-Y on several media, PCR-DGGE analysis and microscopy indicated that the unique bi-polar filamentous bacterial cells gradually became more dominant with increasing 4-aminopyridine concentration in the medium. CONCLUSIONS: Hyphomicrobium sp. 4AP-Y, P. nitroreducens 4AP-A, and Elizabethkingia sp. 4AP-Z probably play important roles in 4-aminopyridine degradation in crop fields. In the enrichment culture, 3,4-dihydroxypyridine and its metabolites including formate might be shared as growth substrates and maintain the enrichment culture, including these indispensable strains.

The spike generator in the labellar taste receptors of the blowfly is differently affected by 4-aminopyridine and 5-hydroxytryptamine.

In taste chemoreception of invertebrates the interaction of taste stimuli with specific membrane receptors and/or ion channels located in the apical membrane of taste receptor cells results in the generation of a receptor potential which, in turn, activates the 'encoder' region to produce action potentials which propagate to the CNS. This study investigates, in the labellar chemosensilla of the blowfly, Protophormia terraenovae, the voltage-gated K(+) currents involved in the action potential repolarization and repetitive firing of the neurons by way of the K(v) channel inhibitors, 4-aminopyridine and 5-hydroxytryptamine. The receptor potential and the spike activity were simultaneously recorded from the 'salt', 'sugar' and 'deterrent' cells, by means of the extracellular side-wall technique, in response to 150 mM NaCl, 100 mM sucrose and 1 mM quinine HCl, before, 0÷10 min after apical administration of 4-AP (0.01-10 mM) or 5-HT (0.1-100 mM). The results show that the receptor potential in all three cells is neither affected by 4-AP nor by 5-HT. Instead, spike activity is significantly decreased, by way of blocking different K(v) channel types: an inactivating A-type K(+) current (KA) modulating repetitive firing of the cells and responsible for the after hyperpolarization, and a sustained K(+) current that resembles the delayed rectifier (DKR) and contributes to action potential repolarization.

Engineered endothelial progenitor cells that overexpress prostacyclin protect vascular cells.

Prostacyclin (PGI2) is a potent vasodilator and important mediator of vascular homeostasis; however, its clinical use is limited because of its short (<2-min) half-life. Thus, we hypothesize that the use of engineered endothelial progenitor cells (EPCs) that constitutively secrete high levels of PGI2 may overcome this limitation of PGI2 therapy. A cDNA encoding COX-1-10aa-PGIS, which links human cyclooxygenase-1 (COX-1) to prostacyclin synthase (PGIS), was delivered via nucleofection into outgrowth EPCs derived from rat bone marrow mononuclear cells. PGI2-secreting strains (PGI2-EPCs) were established by continuous subculturing of transfected cells under G418 selection. Genomic PCR, RT-PCR, and Western blot analyses confirmed the overexpression of COX-1-10aa-PGIS in PGI2-EPCs. PGI2-EPCs secreted significantly higher levels of PGI2 in vitro than native EPCs (P < 0.05) and showed higher intrinsic angiogenic capability; conditioned medium (CM) from PGI2-EPCs promoted better tube formation than CM from native EPCs (P < 0.05). Cell- and paracrine-mediated in vitro angiogenesis was attenuated when COX-1-10aa-PGIS protein expression was knocked down. Whole-cell patch-clamp studies showed that 4-aminopyridine-sensitive K(+) current density was increased significantly in rat smooth muscle cells (rSMCs) cocultured under hypoxia with PGI2-EPCs (7.50 ± 1.59 pA/pF; P < 0.05) compared with rSMCs cocultured with native EPCs (3.99 ± 1.26 pA/pF). In conclusion, we successfully created EPC strains that overexpress an active novel enzyme resulting in consistent secretion of PGI2. PGI2-EPCs showed enhanced intrinsic proangiogenic properties and provided favorable paracrine-mediated cellular protections, including promoting in vitro angiogenesis of native EPCs and hyperpolarization of SMCs under hypoxia.

Curcumin inhibits glutamate release in nerve terminals from rat prefrontal cortex: possible relevance to its antidepressant mechanism.

There is abundant evidence suggesting the relevance of glutamate to depression and antidepressant mechanisms. Curcumin, a major active compound of Curcuma longa, has been reported to have the biological function of antidepressant. The aim of the present study was to investigate the effect of curcumin on endogenous glutamate release in nerve terminals of rat prefrontal cortex and the underlying mechanisms. The results showed that curcumin inhibited the release of glutamate that was evoked by exposing synaptosomes to the K(+) channel blocker 4-aminopyridine (4-AP). This phenomenon was blocked by the chelating the extracellular Ca(2+) ions, and by the vesicular transporter inhibitor bafilomycin A1, but was insensitive to the glutamate transporter inhibitor DL-threo-ß-benzyl-oxyaspartate (DL-TBOA). Further experiments demonstrated that curcumin decreased depolarization-induced increase in [Ca(2+)](C), whereas it did not alter the resting membrane potential or 4-AP-mediated depolarization. Furthermore, the inhibitory effect of curcumin on evoked glutamate release was prevented by blocking the Ca(v)2.2 (N-type) and Ca(v)2.1 (P/Q-type) channels, but not by blocking intracellular Ca(2+) release or Na(+)/Ca(2+) exchange. These results suggest that curcumin inhibits evoked glutamate release from rat prefrontocortical synaptosomes by the suppression of presynaptic Ca(v)2.2 and Ca(v)2.1 channels. Additionally, we also found that the inhibitory effect of curcumin on 4-AP-evoked glutamate release was completely abolished by the clinically effective antidepressant fluoxetine. This suggests that curcumin and fluoxetine use a common intracellular mechanism to inhibit glutamate release from rat prefrontal cortex nerve terminals.

A novel Caenorhabditis elegans allele, smn-1(cb131), mimicking a mild form of spinal muscular atrophy, provides a convenient drug screening platform highlighting new and pre-approved compounds.

Spinal muscular atrophy (SMA), an autosomal recessive genetic disorder, is characterized by the selective degeneration of lower motor neurons, leading to muscle atrophy and, in the most severe cases, paralysis and death. Deletions and point mutations cause reduced levels of the widely expressed survival motor neuron (SMN) protein, which has been implicated in a range of cellular processes. The mechanisms underlying disease pathogenesis are unclear, and there is no effective treatment. Several animal models have been developed to study SMN function including the nematode, Caenorhabditis elegans, in which a large deletion in the gene homologous to SMN, smn-1, results in neuromuscular dysfunction and larval lethality. Although useful, this null mutant, smn-1(ok355), is not well suited to drug screening. We report the isolation and characterization of smn-1(cb131), a novel allele encoding a substitution in a highly conserved residue of exon 2, resembling a point mutation found in a patient with type IIIb SMA. The smn-1(cb131) animals display milder yet similar defects when compared with the smn-1 null mutant. Using an automated phenotyping system, mutants were shown to swim slower than wild-type animals. This phenotype was used to screen a library of 1040 chemical compounds for drugs that ameliorate the defect, highlighting six for subsequent testing. 4-aminopyridine, gaboxadol hydrochloride and N-acetylneuraminic acid all rescued at least one aspect of smn-1 phenotypic dysfunction. These findings may assist in accelerating the development of drugs for the treatment of SMA.

Ocular surface wetness is regulated by TRPM8-dependent cold thermoreceptors of the cornea.

Basal tearing is crucial to maintaining ocular surface wetness. Corneal cold thermoreceptors sense small oscillations in ambient temperature and change their discharge accordingly. Deletion of the cold-transducing ion channel Transient receptor potential cation channel subfamily M member 8 (TRPM8) in mice abrogates cold responsiveness and reduces basal tearing without affecting nociceptor-mediated irritative tearing. Warming of the cornea in humans also decreases tearing rate. These findings indicate that TRPM8-dependent impulse activity in corneal cold receptors contributes to regulating basal tear flow.

An excitatory loop with astrocytes contributes to drive neurons to seizure threshold.

Seizures in focal epilepsies are sustained by a highly synchronous neuronal discharge that arises at restricted brain sites and subsequently spreads to large portions of the brain. Despite intense experimental research in this field, the earlier cellular events that initiate and sustain a focal seizure are still not well defined. Their identification is central to understand the pathophysiology of focal epilepsies and to develop new pharmacological therapies for drug-resistant forms of epilepsy. The prominent involvement of astrocytes in ictogenesis was recently proposed. We test here whether a cooperation between astrocytes and neurons is a prerequisite to support ictal (seizure-like) and interictal epileptiform events. Simultaneous patch-clamp recording and Ca2+ imaging techniques were performed in a new in vitro model of focal seizures induced by local applications of N-methyl-D-aspartic acid (NMDA) in rat entorhinal cortex slices. We found that a Ca2+ elevation in astrocytes correlates with both the initial development and the maintenance of a focal, seizure-like discharge. A delayed astrocyte activation during ictal discharges was also observed in other models (including the whole in vitro isolated guinea pig brain) in which the site of generation of seizure activity cannot be precisely monitored. In contrast, interictal discharges were not associated with Ca2+ changes in astrocytes. Selective inhibition or stimulation of astrocyte Ca2+ signalling blocked or enhanced, respectively, ictal discharges, but did not affect interictal discharge generation. Our data reveal that neurons engage astrocytes in a recurrent excitatory loop (possibly involving gliotransmission) that promotes seizure ignition and sustains the ictal discharge. This neuron-astrocyte interaction may represent a novel target to develop effective therapeutic strategies to control seizures.