Phillygenin Suppresses Glutamate Exocytosis in Rat Cerebrocortical Nerve Terminals (Synaptosomes) through the Inhibition of Cav2.2 Calcium Channels.

Glutamate is a major excitatory neurotransmitter that mediates neuronal damage in acute and chronic brain disorders. The effect and mechanism of phillygenin, a natural compound with neuroprotective potential, on glutamate release in isolated nerve terminals (synaptosomes) prepared from the rat cerebral cortex were examined. In this study, 4-aminopyridine (4-AP), a potassium channel blocker, was utilized to induce the release of glutamate, which was subsequently quantified via a fluorometric assay. Our findings revealed that phillygenin reduced 4-AP-induced glutamate release, and this inhibitory effect was reversed by removing extracellular Ca2+ or inhibiting vesicular transport with bafilomycin A1. However, exposure to the glutamate transporter inhibitor dl-threo-beta-benzyl-oxyaspartate (dl-TOBA) did not influence the inhibitory effect. Moreover, phillygenin did not change the synaptosomal membrane potential but lowered the 4-AP-triggered increase in intrasynaptosomal Ca2+ concentration ([Ca2+]i). Antagonizing Cav2.2 (N-type) calcium channels blocked the inhibition of glutamate release by phillygenin, whereas pretreatment with the mitochondrial Na+/Ca2+ exchanger inhibitor, CGP37157 or the ryanodine receptor inhibitor, dantrolene, both of which block intracellular Ca2+ release, had no effect. The effect of phillygenin on glutamate release triggered by 4-AP was completely abolished when MAPK/ERK inhibitors were applied. Furthermore, phillygenin attenuated the phosphorylation of ERK1/2 and its major presynaptic target, synapsin I, a protein associated with synaptic vesicles. These data collectively suggest that phillygenin mediates the inhibition of evoked glutamate release from synaptosomes primarily by reducing the influx of Ca2+ through Cav2.2 calcium channels, thereby subsequently suppressing the MAPK/ERK/synapsin I signaling cascade.

Nitron-derivative-based palladium carbene complexes: structural characterization, theoretical calculations, and catalytic applications in the Mizoroki-Heck coupling reaction.

New palladium(0) and palladium(ii) complexes with N-heterocyclic carbene (NHC) ligands derived from nitron and its derivatives were synthesized. The structures of most of these complexes were established by single-crystal X-ray diffraction studies. Among the new complexes, the palladium complex with a monodentate NHC ligand derived from nitron demonstrated the highest efficacy as a catalyst precursor in the Mizoroki-Heck coupling reaction of aryl chlorides with alkenes. Theoretical calculations provide valuable insights into the electronic parameters of both the ligands and the palladium complexes, highlighting the significance of a robust Pd-C bond and the π-accepting property of the NHC ligand in achieving enhanced catalytic activity. Notably, catalyst activation occurred much more rapidly with the preformed palladium(0) complex compared to its palladium(ii) counterpart.

Mangiferin depresses vesicular glutamate release in synaptosomes from the rat cerebral cortex by decreasing synapsin I phosphorylation.

Mangiferin is a glucosyl xanthone that has been shown to be a neuroprotective agent against brain disorders involving excess glutamate. However, the effect of mangiferin on the function of the glutamatergic system has not been investigated. In this study, we used synaptosomes from the rat cerebral cortex to investigate the effect of mangiferin on glutamate release and identify the possible underlying mechanism. We observed that mangiferin produced a concentration-dependent reduction in the release of glutamate elicited by 4-aminopyridine with an IC50 value of 25 µM. Inhibition of glutamate release was blocked by removing extracellular calcium and by treatment with the vacuolar-type H+-ATPase inhibitor bafilomycin A1, which prevents the uptake and storage of glutamate in vesicles. Moreover, we showed that mangiferin decreased the 4-aminopyridine-elicited FM1-43 release and synaptotagmin 1 luminal domain antibody (syt1-L ab) uptake from synaptosomes, which correlated with decreased synaptic vesicle exocytosis. Transmission electron microscopy in synaptosomes also showed that mangiferin attenuated the 4-aminopyridine-elicited decrease in the number of synaptic vesicles. In addition, antagonism of Ca2+/calmodulin-dependent kinase II (CaMKII) and protein kinase A (PKA) counteracted mangiferin's effect on glutamate release. Mangiferin also decreased the phosphorylation of CaMKII, PKA, and synapsin I elicited by 4-aminopyridine treatment. Our data suggest that mangiferin reduces PKA and CaMKII activation and synapsin I phosphorylation, which could decrease synaptic vesicle availability and lead to a subsequent reduction in vesicular glutamate release from synaptosomes.

Cynarin, a caffeoylquinic acid derivative in artichoke, inhibits exocytotic glutamate release from rat cortical nerve terminals (synaptosomes).

The purpose of this study was to evaluate the effect of cynarin, a caffeoylquinic acid derivative in artichoke, on glutamate release elicited by 4-aminopyridine (4-AP) in rat cortical nerve terminals (synaptosomes). We observed that cynarin decreased 4-aminopyridine-elicited glutamate release, which was prevented by the removal of external free Ca2+ with ethylene glycol bis (ß-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA) or the blockade of P/Q-type calcium channels with ω-agatoxin IVA. Molecular docking also revealed that cynarin formed a hydrogen bond with the P/Q-type Ca2+ channel, indicating a mechanism of action involving Ca2+ influx inhibition. Additionally, the inhibitory effect of cynarin on glutamate release is associated with a change in the available synaptic vesicles, as cynarin decreased 4-AP-elicited FM1-43 release or hypertonic sucrose-evoked glutamate release from synaptosomes. Furthermore, the suppression of protein kinase A (PKA) prevented the effect of cynarin on 4-AP-elicited glutamate release. 4-AP-elicited PKA and synapsin I or synaptosomal-associated protein of 25 kDa (SNAP-25) phosphorylation at PKA-specific residues were also attenuated by cynarin. Our data indicate that cynarin, through the suppression of P/Q-type Ca2+ channels, inhibits PKA activation and attenuates synapsin I and SNAP-25 phosphorylation at PKA-specific residues, thus decreasing synaptic vesicle availability and contributing to glutamate release inhibition in cerebral cortex terminals.

Plantainoside D Reduces Depolarization-Evoked Glutamate Release from Rat Cerebral Cortical Synaptosomes.

Inhibiting the excessive release of glutamate in the brain is emerging as a promising therapeutic option and is efficient for treating neurodegenerative disorders. The aim of this study is to investigate the effect and mechanism of plantainoside D (PD), a phenylenthanoid glycoside isolated from Plantago asiatica L., on glutamate release in rat cerebral cortical nerve terminals (synaptosomes). We observed that PD inhibited the potassium channel blocker 4-aminopyridine (4-AP)-evoked release of glutamate and elevated concentration of cytosolic Ca2+. Using bafilomycin A1 to block glutamate uptake into synaptic vesicles and EDTA to chelate extracellular Ca2+, the inhibitory effect of PD on 4-AP-evoked glutamate release was prevented. In contrast, the action of PD on the 4-AP-evoked release of glutamate in the presence of dl-TBOA, a potent nontransportable inhibitor of glutamate transporters, was unaffected. PD does not alter the 4-AP-mediated depolarization of the synaptosomal membrane potential, suggesting that the inhibitory effect of PD on glutamate release is associated with voltage-dependent Ca2+ channels (VDCCs) but not the modulation of plasma membrane potential. Pretreatment with the Ca2+ channel blocker (N-type) ω-conotoxin GVIA abolished the inhibitory effect of PD on the evoked glutamate release, as did pretreatment with the protein kinase C inhibitor GF109203x. However, the PD-mediated inhibition of glutamate release was eliminated by applying the mitochondrial Na+/Ca2+ exchanger inhibitor CGP37157 or dantrolene, which inhibits Ca2+ release through ryanodine receptor channels. These data suggest that PD mediates the inhibition of evoked glutamate release from synaptosomes primarily by reducing the influx of Ca2+ through N-type Ca2+ channels, subsequently reducing the protein kinase C cascade.

Inhibition of Synaptic Glutamate Exocytosis and Prevention of Glutamate Neurotoxicity by Eupatilin from Artemisia argyi in the Rat Cortex.

The inhibition of synaptic glutamate release to maintain glutamate homeostasis contributes to the alleviation of neuronal cell injury, and accumulating evidence suggests that natural products can repress glutamate levels and associated excitotoxicity. In this study, we investigated whether eupatilin, a constituent of Artemisia argyi, affected glutamate release in rat cortical nerve terminals (synaptosomes). Additionally, we evaluated the effect of eupatilin in an animal model of kainic acid (KA) excitotoxicity, particularly on the levels of glutamate and N-methyl-D-aspartate (NMDA) receptor subunits (GluN2A and GluN2B). We found that eupatilin decreased depolarization-evoked glutamate release from rat cortical synaptosomes and that this effect was accompanied by a reduction in cytosolic Ca2+ elevation, inhibition of P/Q-type Ca2+ channels, decreased synapsin I Ca2+-dependent phosphorylation and no detectable effect on the membrane potential. In a KA-induced glutamate excitotoxicity rat model, the administration of eupatilin before KA administration prevented neuronal cell degeneration, glutamate elevation, glutamate-generating enzyme glutaminase increase, excitatory amino acid transporter (EAAT) decrease, GluN2A protein decrease and GluN2B protein increase in the rat cortex. Taken together, the results suggest that eupatilin depresses glutamate exocytosis from cerebrocortical synaptosomes by decreasing P/Q-type Ca2+ channels and synapsin I phosphorylation and alleviates glutamate excitotoxicity caused by KA by preventing glutamatergic alterations in the rat cortex. Thus, this study suggests that eupatilin can be considered a potential therapeutic agent in the treatment of brain impairment associated with glutamate excitotoxicity.

Antiglycation Effects of Adlay Seed and Its Active Polyphenol Compounds: An In Vitro Study.

This study aimed to evaluate the antiglycation effects of adlay on protein glycation using in vitro glycation assays. Adlay seed was divided into the following four parts: the hull (AH), testa (AT), bran (AB), and polished adlay (PA). A solvent extraction technique and column chromatography were utilized to investigate the active fractions and components of adlay. Based on a BSA-glucose assay, the ethanolic extracts of AT (ATE) and AB (ABE) revealed a greater capacity to inhibit protein glycation. ATE was further consecutively partitioned into four solvent fractions with n-hexane, ethyl acetate (ATE-Ea), 1-butanol (ATE-BuOH), and water. ATE-BuOH and -Ea show marked inhibition of glucose-mediated glycation. Medium-high polarity subfractions eluted from ATE-BuOH below 50% methanol with Diaion HP-20, ATE-BuOH-c to -f, exhibited superior antiglycation activity, with a maximum inhibitory percentage of 88%. Two phenolic compounds, chlorogenic acid and ferulic acid, identified in ATE-BuOH with HPLC, exhibited potent inhibition of the individual stage of protein glycation and its subsequent crosslinking, as evaluated by the BSA-glucose assay, BS-methylglyoxal (MGO) assay, and G.K. peptide-ribose assay. In conclusion, this study demonstrated the antiglycation properties of ATE in vitro that suggest a beneficial effect in targeting hyperglycemia-mediated protein modification.

The Effect of Isosaponarin Derived from Wasabi Leaves on Glutamate Release in Rat Synaptosomes and Its Underlying Mechanism.

Excessive glutamate release is known to be involved in the pathogenesis of neurological diseases, and suppression of glutamate release from nerve terminals is considered to be a treatment strategy. In this study, we investigated whether isosaponarin, a flavone glycoside isolated from wasabi leaves, could affect glutamate release in rat cerebral cortex nerve terminals (synaptosomes). The release of glutamate was evoked by the K+ channel blocker 4-aminopyridine (4-AP) and measured by an online enzyme-coupled fluorimetric assay. Isosaponarin produced a concentration-dependent inhibition of 4-AP-evoked glutamate release with a half-maximum inhibition of release value of 22 µM. The inhibition caused by isosaponarin was prevented by eliminating extracellular Ca2+ or by using bafilomycin A1, an inhibitor of synaptic vesicle exocytosis. Isosaponarin decreased intrasynaptosomal rises in Ca2+ levels that were induced by 4-AP, without affecting the synaptosomal membrane potential. The isosaponarin-induced inhibition of glutamate release was significantly prevented in synaptosomes that were pretreated with a combination of the calcium channel blockers ω-conotoxin GVIA (N-type) and ω-agatoxin IVA (P/Q-types). The protein kinase C (PKC) pan-inhibitor GF109203X and the Ca2+-dependent PKC inhibitor Go6976 abolished the inhibition of glutamate release by isosaponarin, while the Ca2+-independent PKC inhibitor rottlerin did not show any effect. The results from immunoblotting assays also showed that isosaponarin lowered PKC, PKCα, synaptosomal-associated protein of 25 kDa (SNAP-25), and myristoylated alanine-rich C-kinase substrate (MARCKS) phosphorylation induced by 4-AP. In addition, FM1-43-labeled synaptic vesicles in synaptosomes showed that treatment with isosaponarin resulted in an attenuation of the 4-AP-induced decrease in fluorescence intensity that is consistent with glutamate release. Transmission electron microscopy of synaptosomes also provided evidence that isosaponarin altered the number of synaptic vesicles. These results indicate that isosaponarin suppresses the Ca2+-dependent PKC/SNAP-25 and MARCKS pathways in synaptosomes, causing a decrease in the number of available synaptic vesicles, which inhibits vesicular glutamate release from synaptosomes.

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.

Anticonvulsive and Neuroprotective Effects of Eupafolin in Rats Are Associated with the Inhibition of Glutamate Overexcitation and Upregulation of the Wnt/ß-Catenin Signaling Pathway.

Several plant compounds have been found to possess neuroactive properties. The aim of this study was to investigate the anticonvulsant effect of eupafolin, a major active component extracted from Salvia plebeia, a herb used in traditional medicine for its anti-inflammatory properties. To this end, we assessed the anticonvulsant effects of eupafolin in rats intraperitoneally (i.p.) injected with kainic acid (KA) to elucidate this mechanism. Treatment with eupafolin (i.p.) for 30 min before KA administration significantly reduced behavioral and electrographic seizures induced by KA, similar to carbamazepine (i.p.), a widely used antiepileptic drug. Eupafolin treatment also significantly decreased KA seizure-induced neuronal cell death and glutamate elevation in the hippocampus. In addition, eupafolin notably reversed KA seizure-induced alterations in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunit GluR2, glutamate decarboxylase 67 (GAD67, GABAergic enzyme), and Wnt signaling-related proteins, including porcupine, Wnt1, phosphorylated-glycogen synthase kinase-3ß, ß-catenin, and Bcl-2 in the hippocampus. Furthermore, the increased level of Dickkopf-related protein 1 (Dkk-1, a Wnt signaling antagonist) and the decreased level of Disheveled1 (Dvl-1, a Wnt signaling activator) in the hippocampus of KA-treated rats were reversed by eupafolin. This study provides evidence of the anticonvulsant and neuroprotective properties of eupafolin and of the involvement of regulation of glutamate overexcitation and Wnt signaling in the mechanisms of these properties. These findings support the benefits of eupafolin in treating epilepsy.

Neferine, an Alkaloid from Lotus Seed Embryos, Exerts Antiseizure and Neuroprotective Effects in a Kainic Acid-Induced Seizure Model in Rats.

Current anti-seizure drugs fail to control approximately 30% of epilepsies. Therefore, there is a need to develop more effective anti-seizure drugs, and medicinal plants provide an attractive source for new compounds. This study aimed to evaluate the possible anti-seizure and neuroprotective effects of neferine, an alkaloid from the lotus seed embryos of Nelumbo nucifera, in a kainic acid (KA)-induced seizure rat model and its underlying mechanisms. Rats were intraperitoneally (i.p.) administrated neferine (10 and 50 mg/kg) 30 min before KA injection (15 mg/kg, i.p.). Neferine pretreatment increased seizure latency and reduced seizure scores, prevented glutamate elevation and neuronal loss, and increased presynaptic protein synaptophysin and postsynaptic density protein 95 expression in the hippocampi of rats with KA. Neferine pretreatment also decreased glial cell activation and proinflammatory cytokine (interleukin-1ß, interleukin-6, tumor necrosis factor-α) expression in the hippocampi of rats with KA. In addition, NOD-like receptor 3 (NLRP3) inflammasome, caspase-1, and interleukin-18 expression levels were decreased in the hippocampi of seizure rats pretreated with neferine. These results indicated that neferine reduced seizure severity, exerted neuroprotective effects, and ameliorated neuroinflammation in the hippocampi of KA-treated rats, possibly by inhibiting NLRP3 inflammasome activation and decreasing inflammatory cytokine secretion. Our findings highlight the potential of neferine as a therapeutic option in the treatment of epilepsy.

An Anthranilate Derivative Inhibits Glutamate Release and Glutamate Excitotoxicity in Rats.

The neurotransmitter glutamate plays an essential role in excitatory neurotransmission; however, excessive amounts of glutamate lead to excitotoxicity, which is the most common pathogenic feature of numerous brain disorders. This study aimed to investigate the role of butyl 2-[2-(2-fluorophenyl)acetamido]benzoate (HFP034), a synthesized anthranilate derivative, in the central glutamatergic system. We used rat cerebro-cortical synaptosomes to examine the effect of HFP034 on glutamate release. In addition, we used a rat model of kainic acid (KA)-induced glutamate excitotoxicity to evaluate the neuroprotective potential of HFP034. We showed that HFP034 inhibits 4-aminopyridine (4-AP)-induced glutamate release from synaptosomes, and this inhibition was absent in the absence of extracellular calcium. HFP034-mediated inhibition of glutamate release was associated with decreased 4-AP-evoked Ca2+ level elevation and had no effect on synaptosomal membrane potential. The inhibitory effect of HFP034 on evoked glutamate release was suppressed by blocking P/Q-type Ca2+ channels and protein kinase C (PKC). Furthermore, HFP034 inhibited the phosphorylation of PKC and its substrate, myristoylated alanine-rich C kinase substrate (MARCKS) in synaptosomes. We also observed that HFP034 pretreatment reduced neuronal death, glutamate concentration, glial activation, and the levels of endoplasmic reticulum stress-related proteins, calpains, glucose-regulated protein 78 (GRP 78), C/EBP homologous protein (CHOP), and caspase-12 in the hippocampus of KA-injected rats. We conclude that HFP034 is a neuroprotective agent that prevents glutamate excitotoxicity, and we suggest that this effect involves inhibition of presynaptic glutamate release through the suppression of P/Q-type Ca2+ channels and PKC/MARCKS pathways.

Inhibition of Glutamate Release from Rat Cortical Nerve Terminals by Dehydrocorydaline, an Alkaloid from Corydalis yanhusuo.

Excessive release of glutamate induces excitotoxicity and causes neuronal damage in several neurodegenerative diseases. Natural products have emerged as potential neuroprotective agents for preventing and treating neurological disorders. Dehydrocorydaline (DHC), an active alkaloid compound isolated from Corydalis yanhusuo, possesses neuroprotective capacity. The present study investigated the effect of DHC on glutamate release using a rat brain cortical synaptosome model. Our results indicate that DHC inhibited 4-aminopyridine (4-AP)-evoked glutamate release and elevated intrasynaptosomal calcium levels. The inhibitory effect of DHC on 4-AP-evoked glutamate release was prevented in the presence of the vesicular transporter inhibitor bafilomycin A1 and the N- and P/Q-type Ca2+ channel blocker ω-conotoxin MVIIC but not the intracellular inhibitor of Ca2+ release dantrolene or the mitochondrial Na+/Ca2+ exchanger inhibitor CGP37157. Moreover, the inhibitory effect of DHC on evoked glutamate release was prevented by the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) inhibitor PD98059. Western blotting data in synaptosomes also showed that DHC significantly decreased the level of ERK1/2 phosphorylation and synaptic vesicle-associated protein synapsin I, the main presynaptic target of ERK. Together, these results suggest that DHC inhibits presynaptic glutamate release from cerebrocortical synaptosomes by suppressing presynaptic voltage-dependent Ca2+ entry and the MAPK/ERK/synapsin I signaling pathway.

Enmein Decreases Synaptic Glutamate Release and Protects against Kainic Acid-Induced Brain Injury in Rats.

This study investigated the effects of enmein, an active constituent of Isodon japonicus Hara, on glutamate release in rat cerebrocortical nerve terminals (synaptosomes) and evaluated its neuroprotective potential in a rat model of kainic acid (KA)-induced glutamate excitotoxicity. Enmein inhibited depolarization-induced glutamate release, FM1-43 release, and Ca2+ elevation in cortical nerve terminals but had no effect on the membrane potential. Removing extracellular Ca2+ and blocking vesicular glutamate transporters, N- and P/Q-type Ca2+ channels, or protein kinase C (PKC) prevented the inhibition of glutamate release by enmein. Enmein also decreased the phosphorylation of PKC, PKC-α, and myristoylated alanine-rich C kinase substrates in synaptosomes. In the KA rat model, intraperitoneal administration of enmein 30 min before intraperitoneal injection of KA reduced neuronal cell death, glial cell activation, and glutamate elevation in the hippocampus. Furthermore, in the hippocampi of KA rats, enmein increased the expression of synaptic markers (synaptophysin and postsynaptic density protein 95) and excitatory amino acid transporters 2 and 3, which are responsible for glutamate clearance, whereas enmein decreased the expression of glial fibrillary acidic protein (GFAP) and CD11b. These results indicate that enmein not only inhibited glutamate release from cortical synaptosomes by suppressing Ca2+ influx and PKC but also increased KA-induced hippocampal neuronal death by suppressing gliosis and decreasing glutamate levels by increasing glutamate uptake.

Suppression on allergic airway inflammation of dehulled adlay (Coix lachryma-jobi L. var. ma-yuen Stapf) in mice and anti-degranulation phytosterols from adlay bran.

Adlay (Coix lachryma-jobi L. var. ma-yuen Stapf) seeds have been used in Asia for thousands years to treat warts, chapped skin, rheumatism, and neuralgia. The anti-allergic activity of dehulled adlay (DA) seeds was identified, and the bran (AB) is regarded as the main functional constituent in the edible part. However, no study has focused on in vivo acute anti-allergic airway inflammation. In the present report, we investigated DA methanolic extract (DAM) reversed ovalbumin (OVA)/methacholine (Mch)-induced airway hypersensitivity, decreased interleukin (IL)-4, IL-5, and IL-13 levels from splenocytes, suppressed tumor necrosis factor (TNF)-α, IL-1ß, and IL-13 levels and reduced eosinophil counts and eotaxin in bronchoalveolar lavage fluid (BALF), which imply that the modulatory effects of DA should involve allergic degranulation. Further, seven phytosterols were isolated from AB ethanolic extract (ABE); among them, 3-O-caffeoyl-5ß-sitostan-3-ol, ß-sitosterol 3-O-glucopyranoside and ß-sitosterol inhibited ß-hexosaminidase release from A23187-stimulated RBL-2H3 cells with percentages of 54.1%, 52.0% and 48.5%, respectively, at 50 µM. In addition, ß-sitosterol reduced immunoglobulin (Ig)E-stimulated degranulation on RBL-2H3 cells in a dose-dependent manner. The phytosterols were the predominant components based on gas chromatography (GC) analysis. This is the first study to demonstrate that DA suppressed OVA/Mch-induced acute airway inflammation. The phytosterols in AB showed significant anti-degranulation activities, and may be regarded as the indicative components of AB for anti-allergy effects.

Natural Product Isoliquiritigenin Activates GABAB Receptors to Decrease Voltage-Gate Ca2+ Channels and Glutamate Release in Rat Cerebrocortical Nerve Terminals.

Reduction in glutamate release is a key mechanism for neuroprotection and we investigated the effect of isoliquiritigenin (ISL), an active ingredient of Glycyrrhiza with neuroprotective activities, on glutamate release in rat cerebrocortical nerve terminals (synaptosomes). ISL produced a concentration-dependent inhibition of glutamate release and reduced the intraterminal [Ca2+] increase. The inhibition of glutamate release by ISL was prevented after removing extracellular Ca2+ or blocking P/Q-type Ca2+ channels. This inhibition was mediated through the γ-aminobutyric acid type B (GABAB) receptors because ISL was unable to inhibit glutamate release in the presence of baclofen (an GABAB agonist) or CGP3548 (an GABAB antagonist) and docking data revealed that ISL interacted with GABAB receptors. Furthermore, the ISL inhibition of glutamate release was abolished through the inhibition of Gi/o-mediated responses or Gßγ subunits, but not by 8-bromoadenosine 3',5'-cyclic monophosphate or adenylate cyclase inhibition. The ISL inhibition of glutamate release was also abolished through the inhibition of protein kinase C (PKC), and ISL decreased the phosphorylation of PKC. Thus, we inferred that ISL, through GABAB receptor activation and Gßγ-coupled inhibition of P/Q-type Ca2+ channels, suppressed the PKC phosphorylation to cause a decrease in evoked glutamate release at rat cerebrocortical nerve terminals.

Asiatic Acid Prevents Cognitive Deficits by Inhibiting Calpain Activation and Preserving Synaptic and Mitochondrial Function in Rats with Kainic Acid-Induced Seizure.

Cognitive impairment is not only associated with seizures but also reported as an adverse effect of antiepileptic drugs. Thus, new molecules that can ameliorate seizures and maintain satisfactory cognitive function should be developed. The antiepileptic potential of asiatic acid, a triterpene derived from the medicinal herb Centella asiatica, has already been demonstrated; however, its role in epilepsy-related cognitive deficits is yet to be determined. In this study, we evaluated the effects of asiatic acid on cognitive deficits in rats with kainic acid (KA)-induced seizure and explored the potential mechanisms underlying these effects. Our results revealed that asiatic acid administrated intraperitoneally 30 min prior to KA (15 mg/kg) injection ameliorated seizures and significantly improved KA-induced memory deficits, as demonstrated by the results of the Morris water maze test. In addition, asiatic acid ameliorated neuronal damage, inhibited calpain activation, and increased protein kinase B (AKT) activation in the hippocampus of KA-treated rats. Asiatic acid also increased the levels of synaptic proteins and the number of synaptic vesicles as well as attenuated mitochondrial morphology damage in the hippocampus of KA-treated rats. Furthermore, proteomic and Western blot analyses of hippocampal synaptosomes revealed that asiatic acid reversed KA-induced changes in mitochondria function-associated proteins, including lipoamide dehydrogenase, glutamate dehydrogenase 1 (GLUD1), ATP synthase (ATP5A), and mitochondrial deacetylase sirtuin-3 (SIRT3). Our data suggest that asiatic acid can prevent seizures and improve cognitive impairment in KA-treated rats by reducing hippocampal neuronal damage through the inhibition of calpain activation and the elevation of activated AKT, coupled with an increase in synaptic and mitochondrial function.

Typhaneoside Suppresses Glutamate Release Through Inhibition of Voltage-Dependent Calcium Entry in Rat Cerebrocortical Nerve Terminals.

Glutamate is the major excitatory neurotransmitter in the brain and is involved in many brain functions. In this study, we investigated whether typhaneoside, a flavonoid from Typhae angustifolia pollen, affects endogenous glutamate release from rat cortical synaptosomes. Using a one-line enzyme-coupled fluorometric assay, glutamate release stimulated by the K+ channel blocker 4-aminopyridine was monitored to explore the possible underlying mechanisms. The vesicular transporter inhibitor bafilomycin A1 and chelation of extracellular Ca2+ ions with EGTA suppressed the effect of typhaneoside on the induced glutamate release. Nevertheless, the typhaneoside activity has not been affected by the glutamate transporter inhibitor dl-threo-beta-benzyloxyaspartate. The synaptosomal plasma membrane potential was assayed using a membrane potential-sensitive dye DiSC3(5), and cytosolic Ca2+ concentrations ([Ca2+]C) was monitored by a Ca2+ indicator Fura-2. Results showed that typhaneoside did not alter the synaptosomal membrane potential but lowered 4-aminopyridine-induced increases in [Ca2+]C. Furthermore, the Cav2.2 (N-type) channel blocker ω-conotoxin GVIA blocked Ca2+ entry and inhibited the effect of typhaneoside on 4-aminopyridine-induced glutamate release. However, the inhibitor of intracellular Ca2+ release dantrolene and the mitochondrial Na+/Ca2+ exchanger blocker 7-chloro-5-(2-chloropheny)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one have no effect on the suppression of glutamate release mediated by typhaneoside. Moreover, inhibition of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) prevented the inhibitory effect of typhaneoside on induced glutamate release. Typhaneoside reduced 4-aminopyridine-induced phosphorylation of ERK1/2 and the major presynaptic ERK target synapsin I, which is a synaptic vesicle-associated protein. In conclusion, these findings suggest a role for typhaneoside in modulating glutamate release by suppressing voltage-dependent Ca2+ channel mediated presynaptic Ca2+ influx and the MAPK/ERK/synapsin I signaling cascade.

Inhibition of glutamatergic transmission and neuronal excitability by oxycodone in the rat hippocampal CA3 neurons.

Oxycodone, a semisynthetic opioid analgesic with actions similar to morphine, is extensively prescribed for treatment of moderate to severe acute pain. Given that glutamate plays a crucial role in mediating pain transmission, the purpose of this study was to investigate the effect of oxycodone on glutamatergic synaptic transmission in rat hippocampal CA3 area, which is associated with the modulation of nociceptive perception. Whole-cell patch-clamp recordings revealed that oxycodone effectively reduced presynaptic glutamate release, as detected by decreased frequencies of spontaneous excitatory postsynaptic currents (sEPSCs) and miniature EPSCs (mEPSCs), without eliciting significant changes in the amplitudes of sEPSCs and mEPSCs and glutamate-evoked inward currents. The inhibitory effect of oxycodone on the frequency of sEPSCs was blocked by the nonselective opioid receptor antagonist naloxone. In addition, oxycodone suppressed burst firing induced by 4-aminopyridine and tonic repetitive firing evoked by the applied depolarizing current. These results suggest that oxycodone inhibits spontaneous presynaptic glutamate release possibly by activating opioid receptors and consequently suppressing the neuronal excitability of hippocampal CA3 neurons.

Silymarin Inhibits Glutamate Release and Prevents against Kainic Acid-Induced Excitotoxic Injury in Rats.

Silymarin, a polyphenoic flavonoid derived from the seeds of milk thistle (Silybum marianum), exhibits neuroprotective effects. In this study, we used a model of rat cerebrocortical synaptosomes to investigate whether silymarin affects the release of glutamate, an essential neurotransmitter involved in excitotoxicity. Its possible neuroprotective effect on a rat model of kainic acid (KA)-induced excitotoxicity was also investigated. In rat cortical synaptosomes, silymarin reduced glutamate release and calcium elevation evoked by the K+ channel blocker 4-aminopyridine but did not affect glutamate release caused by the Na+ channel activator veratridine or the synaptosomal membrane potential. Decreased glutamate release by silymarin was prevented by removal of extracellular calcium and blocking of N- and P/Q-type Ca2+ channel or extracellular signal-regulated kinase 1/2 (ERK1/2) but not by blocking of intracellular Ca2+ release. Immunoblotting assay results revealed that silymarin reduced 4-aminopyridine-induced phosphorylation of ERK1/2. Moreover, systemic treatment of rats with silymarin (50 or 100 mg/kg) 30 min before systemic KA (15 mg/kg) administration attenuated KA-induced seizures, glutamate concentration elevation, neuronal damage, glial activation, and heat shock protein 70 expression as well as upregulated KA-induced decrease in Akt phosphorylation in the rat hippocampus. Taken together, the present study demonstrated that silymarin depressed synaptosomal glutamate release by suppressing voltage-dependent Ca2+ entry and ERK1/2 activity and effectively prevented KA-induced in vivo excitotoxicity.