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.

5-HT1B receptor agonist CGS12066 presynaptically inhibits glutamate release in rat hippocampus.

CGS12066, a 5-hydroxytryptamine 1B (5-HT1B) receptor agonist, has been reported to exhibit antidepressant activity. Considering that glutamatergic dysfunction is implicated in depression, the effect of CGS12066 on glutamate release in rat hippocampal nerve terminals and possible underlying mechanism were investigated. We observed that CGS12066 inhibited 4-aminopyridine (4-AP)-evoked glutamate release, and that a 5-HT1B receptor antagonist blocked this inhibition. Western blot analysis and immunocytochemistry confirmed the presence of presynaptic 5-HT1B receptor proteins. CGS12066-mediated inhibition of 4-AP-evoked glutamate release was completely abolished in the synaptosomes pretreated with inhibitors of Gi/Go-protein, adenylate cyclase (AC), and protein kinase A (PKA), namely pertussis toxin, MDL12330A, and H89, respectively. CGS12066 reduced the elevation of 4-AP-evoked intrasynaptosomal Ca2+ and cyclic AMP (cAMP) levels, but did not affect the synaptosomal membrane potential. Furthermore, in the presence of ω-conotoxin MVIIC, a N- and P/Q-type channel blocker, CGS12066-mediated inhibition of 4-AP-evoked glutamate release was markedly reduced; however, the intracellular Ca2+-release inhibitors dantrolene and CGP37157 did not affect the CGS12066 effect. Furthermore, CGS12066 reduced glutamatergic miniature excitatory postsynaptic current (mEPSC) frequency but did not affect mEPSC amplitude or glutamate-activated currents in hippocampal slices. Our data are the first to suggest that CGS12066 reduces AC/cAMP/PKA activation, through the activation of Gi/Go protein-coupled 5-HT1B receptors present on hippocampal nerve terminals, subsequently reducing Ca2+ entry through voltage-dependent Ca2+ channels and reducing 4-AP-evoked glutamate release. This investigation into the role of 5-HT1B receptors in glutamate release provides crucial information regarding the potential therapeutic role of 5-HT1B receptors for treating depression.

Xanthohumol-induced presynaptic reduction of glutamate release in the rat hippocampus.

This study examined whether xanthohumol, a hop-derived prenylated flavonoid present in beer, affects glutamate release in the rat hippocampus. In the rat hippocampal nerve terminals (synaptosomes), xanthohumol inhibited the release of 4-aminopyridine (4-AP)-evoked glutamate and the elevation of cytosolic Ca(2+) concentration, whereas it had no effect on 4-AP-mediated depolarization. The inhibitory effect of xanthohumol on the evoked glutamate release was prevented by removing extracellular Ca(2+), using the Cav2.2 (N-type) and Cav2.1 (P/Q-type) channel blocker ω-CgTX MVIIC, the calmodulin antagonists W7 and calmidazolium, and the protein kinase A inhibitor H89; however, no such effect was observed when the G-protein inhibitor N-ethylmaleimide was used. In addition, immunocytochemical data demonstrated that GABAA receptors are present in the hippocampal synaptosomes and that the xanthohumol effect on evoked glutamate release was antagonized by the GABAA receptor antagonist SR95531. Furthermore, in slice preparations, xanthohumol reduced the frequency of miniature excitatory postsynaptic currents without affecting their amplitude. We conclude that xanthohumol acts at GABAA receptors present in the hippocampal nerve terminals to decrease the Ca(2+) influx through N- and P/Q-type Ca(2+) channels, which subsequently suppresses the Ca(2+)-calmodulin/PKA cascade to decrease the evoked glutamate release.

Palmitoylethanolamide inhibits glutamate release in rat cerebrocortical nerve terminals.

The effect of palmitoylethanolamide (PEA), an endogenous fatty acid amide displaying neuroprotective actions, on glutamate release from rat cerebrocortical nerve terminals (synaptosomes) was investigated. PEA inhibited the Ca²âº-dependent release of glutamate, which was triggered by exposing synaptosomes to the potassium channel blocker 4-aminopyridine. This release inhibition was concentration dependent, associated with a reduction in cytosolic Ca²âº concentration, and not due to a change in synaptosomal membrane potential. The glutamate release-inhibiting effect of PEA was prevented by the Ca(v)2.1 (P/Q-type) channel blocker ω-agatoxin IVA or the protein kinase A inhibitor H89, not affected by the intracellular Ca²âº release inhibitors dantrolene and CGP37157, and partially antagonized by the cannabinoid CB1 receptor antagonist AM281. Based on these results, we suggest that PEA exerts its presynaptic inhibition, likely through a reduction in the Ca²âº influx mediated by Ca(v)2.1 (P/Q-type) channels, thereby inhibiting the release of glutamate from rat cortical nerve terminals. This release inhibition might be linked to the activation of presynaptic cannabinoid CB1 receptors and the suppression of the protein kinase A pathway.

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.

Cyclooxygenase 2 inhibitor celecoxib inhibits glutamate release by attenuating the PGE2/EP2 pathway in rat cerebral cortex endings.

The excitotoxicity caused by excessive glutamate is a critical element in the neuropathology of acute and chronic brain disorders. Therefore, inhibition of glutamate release is a potentially valuable therapeutic strategy for treating these diseases. In this study, we investigated the effect of celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor that reduces the level of prostaglandin E2 (PGE2), on endogenous glutamate release in rat cerebral cortex nerve terminals (synaptosomes). Celecoxib substantially inhibited the release of glutamate induced by the K(+) channel blocker 4-aminopyridine (4-AP), and this phenomenon was prevented by chelating the extracellular Ca(2+) ions and by the vesicular transporter inhibitor bafilomycin A1. Celecoxib inhibited a 4-AP-induced increase in cytosolic-free Ca(2+) concentration, and the celecoxib-mediated inhibition of glutamate release was prevented by the Cav2.2 (N-type) and Cav2.1 (P/Q-type) channel blocker ω-conotoxin MVIIC. However, celecoxib did not alter 4-AP-mediated depolarization and Na(+) influx. In addition, this glutamate release-inhibiting effect of celecoxib was mediated through the PGE2 subtype 2 receptor (EP2) because it was not observed in the presence of butaprost (an EP2 agonist) or PF04418948 [1-(4-fluorobenzoyl)-3-[[6-methoxy-2-naphthalenyl)methyl]-3-azetidinecarboxylic acid; an EP2 antagonist]. The celecoxib effect on 4-AP-induced glutamate release was prevented by the inhibition or activation of protein kinase A (PKA), and celecoxib decreased the 4-AP-induced phosphorylation of PKA. We also determined that COX-2 and the EP2 receptor are present in presynaptic terminals because they are colocalized with synaptophysin, a presynaptic marker. These results collectively indicate that celecoxib inhibits glutamate release from nerve terminals by reducing voltage-dependent Ca(2+) entry through a signaling cascade involving EP2 and PKA.

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.

Curcumin inhibits glutamate release from rat prefrontal nerve endings by affecting vesicle mobilization.

Curcumin, one of the major constituents of Curcuma longa, has been shown to inhibit depolarization-evoked glutamate release from rat prefrontocortical nerve terminals by reducing voltage-dependent Ca(2+) entry. This study showed that curcumin inhibited ionomycin-induced glutamate release and KCl-evoked FM1-43 release, suggesting that some steps after Ca(2+) entry are regulated by curcumin. Furthermore, disrupting the cytoskeleton organization using cytochalasin D abolished the inhibitory action of curcumin on ionomycin-induced glutamate release. Mitogen-activated protein kinase kinase (MEK) inhibition also prevented the inhibitory effect of curcumin on ionomycin-induced glutamate release. Western blot analyses showed that curcumin decreased the ionomycin-induced phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and synaptic vesicle-associated protein synapsin I, the main presynaptic target of ERK. These results show that curcumin-mediated inhibition of glutamate release involves modulating downstream events by controlling synaptic vesicle recruitment and exocytosis, possibly through a decrease of MAPK/ERK activation and synapsin I phosphorylation, thereby decreasing synaptic vesicle availability for exocytosis.

Tamoxifen depresses glutamate release through inhibition of voltage-dependent Ca2+ entry and protein kinase Cα in rat cerebral cortex nerve terminals.

This study was aimed at examining the effect of tamoxifen, a selective estrogen receptor modulator, on the release of endogenous glutamate in rat cerebral cortex nerve terminals (synaptosomes) and exploring the possible mechanism. Tamoxifen inhibited the release of glutamate that was evoked by the K(+) channel blocker 4-aminopyridine (4-AP), and this phenomenon was concentration-dependent and insensitive to the estrogen receptor antagonist. The effect of tamoxifen on the evoked glutamate release was prevented by the chelating extracellular Ca(2+) ions, and by the vesicular transporter inhibitor bafilomycin A1. However, the glutamate transporter inhibitor dl-threo-beta-benzyloxyaspartate did not have any effect on the action of tamoxifen. Tamoxifen did not alter the resting synaptosomal membrane potential or 4-AP-mediated depolarization whereas it decreased the 4-AP-induced increase in cytosolic [Ca(2+)]. Furthermore, the inhibitory effect of tamoxifen on the evoked glutamate release was abolished by the Ca(v)2.2 (N-type) and Ca(v)2.1 (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. In addition, the protein kinase C (PKC) inhibitors GF109203X or Ro318220 prevented tamoxifen from inhibiting glutamate release. Western blotting showed that tamoxifen significantly decreased the 4-AP-induced phosphorylation of PKC and PKCα. Together, these results suggest that tamoxifen inhibits glutamate release from rat cortical synaptosomes, through the suppression of presynaptic voltage-dependent Ca(2+) entry and PKC activity.

Short-term versus long-term intermittent hypobaric hypoxia on cardiac fibrosis and Fas death receptor dependent apoptotic pathway in rat hearts.

It is unknown if short-term and long-term intermittent hypobaric hypoxic challenges both exert pro-apoptotic effects on Fas death receptor-dependent apoptotic pathway in rat hearts. Seventy-two Sprague-Dawley rats were randomly assigned into two groups. First, short-term intermittent hypobaric hypoxia (STIHH)-normobaric normoxia (n = 12), hypobaric hypoxia (380 mmHg, 12% O2, 8 hrs/day) for 1 day (n = 12), and for 4 days (n = 12) and second, long-term intermittent hypobaric hypoxia (LTIHH)-normobaric normoxia (n = 12), hypobaric hypoxia for 1 week (n = 12) and 2 weeks (n = 12). After STIHH or LTIHH challenge, Fas receptor related pathway and histopathological analysis in the excised left ventricle was determined by Western blotting, RT-PCR, Hematoxylin-eosin staining, Masson trichrome staining and TUNEL assay. Fas death receptor and TNFalpha were significantly decreased after STIHH whereas Fas receptor, TNFalpha, FAS-associated death domain (FADD), and caspase 8 were increased after LTIHH. In addition, cardiomyocyte disarray and fibrosis were observed in 1 week LTIHH. Cardiac hypertrophy and more severe disarray, fibrosis and cardiac apoptotic activities were observed in 2 week LTIHH. STIHH exerts anti-apoptotic effects on hearts such as downregulation of TNFalpha and Fas receptor whereas LTIHH exerts pro-apoptotic effects such as upregulation of TNFalpha and Fas-mediated apoptotic pathways and lead to cardiac fibrosis and apoptosis. Our findings imply that short-term versus long-term intermittent hypobaric hypoxia exerted protective versus deleterious effects on hearts.