Asiatic acid, an active substance of Centella asiatica, presynaptically depresses glutamate release in the rat hippocampus.

Inhibiting glutamate release can reduce neuronal excitability and is recognized as a key mechanism of anti-epileptic drugs. In this study, by using isolated nerve terminal (synaptosome) and slice preparations, we investigated the effect of asiatic acid, a triterpene isolated from Centella asiatica with antiepileptic activity, on glutamate release in the hippocampus of rats. In hippocampal synaptosomes, application of asiatic acid resulted in a concentration-dependent inhibition of 4-aminopyridine-evoked glutamate release. This inhibitory action was dependent on extracellular calcium, blocked by inhibiting the vesicular transporter, but was unaffected by inhibiting the glutamate transporter. In addition, asiatic acid decreased the 4-aminopyridine-induced increase in the intraterminal calcium and failed to alter the synaptosomal potential. Furthermore, the asiatic acid-mediated release inhibition was significantly suppressed by the N- and P/Q-type calcium channel inhibitor ω-conotoxin MVIIC or protein kinase C inhibitor GF109203X. Western blotting data in synaptosomes also revealed that asiatic acid reduced 4-aminopyridine-induced phosphorylation of protein kinase C. In hippocampal slices, asiatic acid decreased the frequencies of spontaneous excitatory postsynaptic currents without changing their amplitudes and glutamate-activated currents in CA3 pyramidal neurons. We also observed that asiatic acid significantly suppressed 4-aminopyridine-induced burst firing. These data suggest that, in rat hippocampal nerve terminals, asiatic acid attenuates the calcium influx via N- and P/Q-type calcium channels, subsequently suppressing protein kinase C activity and decreasing glutamate release.

Echinacoside, an Active Constituent of Cistanche Herba, Exerts a Neuroprotective Effect in a Kainic Acid Rat Model by Inhibiting Inflammatory Processes and Activating the Akt/GSK3ß Pathway.

Echinacoside is a major compound of Cistanche Herb and has glutamate release-inhibiting activity in the brain. Given the involvement of excitotoxicity caused by massive glutamate in the pathophysiology of epilepsy, we explored the antiepileptic effect of echinacoside on kainic acid-induced seizures in rats. The rats were intraperitoneally administrated echinacoside for 30 min prior to intraperitoneal injection with kainic acid. The results showed that kainic acid induced seizure-like behavioral patterns, increased glutamate concentrations, caused neuronal loss and microglial activation, and stimulated proinflammatory cytokine gene expression in the hippocampus. These kainic acid-induced alternations were found to be attenuated by echinacoside pretreatment. Furthermore, decreased Akt and glycogen synthase kinase 3ß (GSK3ß) phosphorylation as well as Bcl-2 expression in the hippocampus was reversed by the echinacoside pretreatment. These results demonstrate that echinacoside exert its antiepileptic and neuroprotective actions in a kainic acid rat model through suppressing inflammatory response and activating the Akt/GSK3ß signaling. Therefore, the present study suggests that echinacoside is the potentially useful in the prevention of epilepsy.

Baicalein, a Constituent of Scutellaria baicalensis, Reduces Glutamate Release and Protects Neuronal Cell Against Kainic Acid-Induced Excitotoxicity in Rats.

Interest in the health benefits of flavonoids, particularly their effects on neurodegenerative disease, is increasing. This study evaluated the role of baicalein, a flavonoid compound isolated from the traditional Chinese medicine Scutellaria baicalensis, in glutamate release and glutamate neurotoxicity in the rat hippocampus. In the rat hippocampal nerve terminals (synaptosomes), baicalein inhibits depolarization-induced glutamate release, and this phenomenon is prevented by chelating the extracellular Ca[Formula: see text] ions and blocking presynaptic Cav2.2 (N-type) and Cav2.1 (P/Q-type) channel activity. In slice preparations, whole cell patch-clamp experiments revealed that baicalein reduced the frequency of miniature excitatory postsynaptic currents, without affecting their amplitude. In a kainic acid rat model, intraperitoneally administering baicalein to rats before the kainic acid intraperitoneal injection substantially attenuated kainic acid-induced neuronal cell death, c-Fos expression, and the activation of the mammalian target of rapamycin in the hippocampus. This study is the first to demonstrate that the natural compound baicalein inhibits glutamate release from hippocampal nerve terminals, and executes a protective action against kainic acid-induced excitotoxicity in vivo. The findings enhance the understanding of baicalein's action in the brain, and suggest that this natural compound is valuable for treating brain disorders related to glutamate excitotoxicity.

Hesperidin inhibits glutamate release and exerts neuroprotection against excitotoxicity induced by kainic acid in the hippocampus of rats.

The citrus flavonoid hesperidin exerts neuroprotective effects and could cross the blood-brain barrier. Given the involvement of glutamate neurotoxicity in the pathogenesis of neurodegenerative disorders, this study was conducted to evaluate the potential role of hesperidin in glutamate release and glutamate neurotoxicity in the hippocampus of rats. In rat hippocampal nerve terminals (synaptosomes), hesperidin inhibited the release of glutamate and elevation of cytosolic free Ca(2+) concentration evoked by 4-aminopyridine (4-AP), but did not alter 4-AP-mediated depolarization. The inhibitory effect of hesperidin on evoked glutamate release was prevented by chelating the extracellular Ca(2+) ions and blocking the activity of Cav2.2 (N-type) and Cav2.1 (P/Q-type) channels or protein kinase C. In hippocampal slice preparations, whole-cell patch clamp experiments showed that hesperidin reduced the frequency of spontaneous excitatory postsynaptic currents without affecting their amplitude, indicating the involvement of a presynaptic mechanism. In addition, intraperitoneal (i.p.) injection of kainic acid (KA, 15 mg/kg) elevated the extracellular glutamate levels and caused considerable neuronal loss in the hippocampal CA3 area. These KA-induced alterations were attenuated by pretreatment with hesperidin (10 or 50 mg/kg, i.p.) before administering the KA. These results demonstrate that hesperidin inhibits evoked glutamate release in vitro and attenuates in vivo KA-induced neuronal death in the hippocampus. Our findings indicate that hesperidin may be a promising candidate for preventing or treating glutamate excitotoxicity related brain disorders such as neurodegenerative diseases.

Protective effect of hispidulin on kainic acid-induced seizures and neurotoxicity in rats.

Hispidulin is a flavonoid compound which is an active ingredient in a number of traditional Chinese medicinal herbs, and it has been reported to inhibit glutamate release. The purpose of this study was to investigate whether hispidulin protects against seizures induced by kainic acid, a glutamate analog with excitotoxic properties. The results indicated that intraperitoneally administering hispidulin (10 or 50mg/kg) to rats 30 min before intraperitoneally injecting kainic acid (15 mg/kg) increased seizure latency and decreased seizure score. In addition, hispidulin substantially attenuated kainic acid-induced hippocampal neuronal cell death, and this protective effect was accompanied by the suppression of microglial activation and the production of proinflammatory cytokines such as interleukin-1ß, interleukin-6, and tumor necrosis factor-α in the hippocampus. Moreover, hispidulin reduced kainic acid-induced c-Fos expression and the activation of mitogen-activated protein kinases in the hippocampus. These data suggest that hispidulin has considerable antiepileptic, neuroprotective, and antiinflammatory effects on kainic acid-induced seizures in rats.

Myricetin inhibits the release of glutamate in rat cerebrocortical nerve terminals.

The excessive release of glutamate is a critical element in the neuropathology of acute and chronic brain disorders. The purpose of the present study was to investigate the effect and possible mechanism of myricetin, a naturally occurring flavonoid with a neuroprotective profile, on endogenous glutamate release in the nerve terminals (synaptosomes) of the rat cerebral cortex. The release of glutamate was evoked by the K(+) channel blocker 4-aminopyridine (4-AP) and measured by one-line enzyme-coupled fluorometric assay. We also used a membrane potential-sensitive dye to assay the synaptosomal plasma membrane potential, and a Ca(2+) indicator Fura-2 to monitor cytosolic Ca(2+) concentrations ([Ca(2+)]C). Results show that myricetin inhibited 4-AP-evoked glutamate release, and this effect was prevented by chelating extracellular Ca(2+) ions and the vesicular transporter inhibitor bafilomycin A1. However, the glutamate transporter inhibitor dl-threo-beta-benzyl-oxyaspartate had no effect on myricetin action. Myricetin did not alter the synaptosomal membrane potential, but decreased 4-AP-induced increases in the cytosolic free Ca(2+) concentration. Furthermore, the myricetin effect on 4-AP-evoked glutamate release was prevented by blocking the Cav2.2 (N-type) and Cav2.1 (P/Q-type) channels, but not by blocking intracellular Ca(2+) release. These results suggest that myricetin inhibits glutamate release from cerebrocortical synaptosomes by attenuating voltage-dependent Ca(2+) entry. This implies that the inhibition of glutamate release is an important pharmacological activity of myricetin that may play a critical role in the apparent clinical efficacy of this compound.

Tanshinone IIA, a constituent of Danshen, inhibits the release of glutamate in rat cerebrocortical nerve terminals.

ETHNOPHARMACOLOGICAL RELEVANCE: Danshen is a commonly used traditional Chinese medicine and has received considerable attention due to their beneficial effects on the health, including prevention of cardiovascular disease, and cancer. Tanshinone IIA, a major active constituent of Danshen, has been reported to have a neuroprotective profile. AIM OF THE STUDY: An excessive release of glutamate is considered to be related to neuropathology of several neurological diseases. In this study, we investigated whether tanshinone IIA could affect endogenous glutamate release and explored the possible mechanism. MATERIALS AND METHODS: The experimental model was the isolated nerve terminals (synaptosomes) purified from the rat cerebral cortex. The release of glutamate was evoked by the K(+) channel blocker 4-aminopyridine (4-AP) and measured by one-line enzyme-coupled fluorometric assay. We also used a membrane potential-sensitive dye to assay nerve terminal excitability and depolarization, and a Ca(2+) indicator, Fura-2-acetoxymethyl ester, to monitor cytosolic Ca(2+) concentrations ([Ca(2+)]C). RESULTS: Tanshinone IIA inhibited the release of glutamate evoked by 4-AP in a concentration-dependent manner. Inhibition of glutamate release by tanshinone IIA was prevented by the chelating the extracellular Ca(2+) ions, and by the vesicular transporter inhibitor bafilomycin A1. However, the glutamate transporter inhibitor DL-threo-beta-benzyl-oxyaspartate did not have any effect on the action of tanshinone IIA. Tanshinone IIA decreased the depolarization-induced increase in [Ca(2+)]C, whereas it did not alter the resting synaptosomal membrane potential or 4-AP-mediated depolarization. Furthermore, the effect of tanshinone IIA on evoked glutamate release was prevented by the Cav2.2 (N-type) and Cav2.1 (P/Q-type) channel blocker ω-conotoxin MVIIC, but not by the ryanodine receptor blocker dantrolene or the mitochondrial Na(+)/Ca(2+) exchanger blocker CGP37157. Mitogen-activated protein kinase (MEK) inhibition also prevented the inhibitory effect of tanshinone IIA on evoked glutamate release. CONCLUSION: These results show that tanshinone IIA inhibits glutamate release from cortical synaptosomes in rats through the suppression of presynaptic voltage-dependent Ca(2+) entry and MEK signaling cascade.

Ferulic acid suppresses glutamate release through inhibition of voltage-dependent calcium entry in rat cerebrocortical nerve terminals.

This study investigated the effects and possible mechanism of ferulic acid, a naturally occurring phenolic compound, on endogenous glutamate release in the nerve terminals of the cerebral cortex in rats. Results show that ferulic acid inhibited the release of glutamate evoked by the K⁺ channel blocker 4-aminopyridine (4-AP). The effect of ferulic acid on the evoked glutamate release was prevented by chelating the extracellular Ca²âº ions, but was insensitive to the glutamate transporter inhibitor DL-threo-beta-benzyl-oxyaspartate. Ferulic acid suppressed the depolarization-induced increase in a cytosolic-free Ca²âº concentration, but did not alter 4-AP-mediated depolarization. Furthermore, the effect of ferulic acid on evoked glutamate release was abolished by blocking the Ca(v)2.2 (N-type) and Ca(v)2.1 (P/Q-type) channels, but not by blocking ryanodine receptors or mitochondrial Na⁺/Ca²âº exchange. These results show that ferulic acid inhibits glutamate release from cortical synaptosomes in rats through the suppression of presynaptic voltage-dependent Ca²âº entry.

HTDP-2, a new synthetic compound, inhibits glutamate release through reduction of voltage-dependent Ca²âº influx in rat cerebral cortex nerve terminals.

AIM: The present study was aimed at investigating the effect of trans-6-(4-chlorobutyl)-5-hydroxy-4-(phenylthio)-1-tosyl-5,6-dihydropyridine-2(1H)-one (HTDP-2), a novel synthetic compound, on the release of endogenous glutamate in rat cerebrocortical nerve terminals (synaptosomes) and exploring the possible mechanism. METHODS: The release of glutamate was evoked by the K⁺ channel blocker 4-aminopyridine (4-AP) and measured by an on-line enzyme-coupled fluorimetric assay. We also used a membrane potential-sensitive dye to assay nerve terminal excitability and depolarization, and a Ca²âº indicator, Fura-2-acetoxymethyl ester, to monitor cytosolic Ca²âº concentrations ([Ca²âº](c)). RESULTS: HTDP-2 inhibited the release of glutamate evoked by 4-AP in a concentration-dependent manner. Inhibition of glutamate release by HTDP-2 was prevented by the chelating intraterminal Ca²âº ions, and by the vesicular transporter inhibitor bafilomycin A1, but was insensitive to the glutamate transporter inhibitor DL-threo-ß-benzyloxyaspartate. HTDP-2 did not alter the resting synaptosomal membrane potential or 4-AP-mediated depolarization whereas it decreased the 4-AP-induced increase in [Ca²âº](c). Furthermore, the inhibitory effect of HTDP-2 on the evoked glutamate release was abolished by the N-, and P/Q-type Ca²âº channel blocker ω-conotoxin MVIIC, but not by the ryanodine receptor blocker dantrolene, or the mitochondrial Na⁺/Ca²âº exchanger blocker CGP37157. CONCLUSION: Based on these results, we suggest that, in rat cerebrocortical nerve terminals, HTDP-2 decreases voltage-dependent Ca²âº channel activity and, in so doing, inhibits the evoked glutamate release.