Tzu Yu Lin

Osthole and imperatorin, the active constituents of Cnidium monnieri (L.) Cusson, facilitate glutamate release from rat hippocampal nerve terminals.

We examined the effects of osthole and imperatorin, two active compounds of Cnidium monnieri (L.) Cusson, on the release of glutamate from rat hippocampal synaptosomes and investigated the possible mechanism. The results showed that osthole or imperatorin significantly facilitated 4-aminopridine (4-AP)-evoked glutamate release in a concentration-dependent manner. The facilitatory action of osthole or imperatorin was blocked by the vesicular transporter inhibitor bafilomycin A1, not by the glutamate transporter inhibitor l-transpyrrolidine-2,4-dicarboxylic acid (l-trans-PDC), indicating that the release facilitation by osthole or imperatorin results from a enhancement of vesicular exocytosis and not from an increase of Ca(2+)-independent efflux via glutamate transporter. Examination of the effect of osthole and imperatorin on cytosolic [Ca(2+)] revealed that the facilitation of glutamate release could be attributed to an increase in voltage-dependent Ca(2+) influx. Consistent with this, omega-conotoxin MVIIC, a wide-spectrum blocker of the N- and P/Q-type Ca(2+) channels, significantly suppressed the osthole or imperatorin-mediated facilitation of glutamate release, but intracellular Ca(2+) release inhibitor dantrolene had no effect. Osthole or imperatorin did not alter the resting synaptosomal membrane potential or 4-AP-mediated depolarization; thus, the facilitation of 4-AP-evoked Ca(2+) influx and glutamate release produced by osthole or imperatorin was not due to it decreasing synaptosomal excitability. In addition, osthole or imperatorin-mediated inhibition of 4-AP-evoked release was prevented by protein kinase C (PKC) inhibitors. Furthermore, osthole or imperatorin increased 4-AP-induced phosphorylation of PKC. Together, these results suggest that osthole or imperatorin effects a facilitation of glutamate release from nerve terminals by positively modulating N-and P/Q-type Ca(2+) channel activation through a signaling cascade involving PKC.

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-β-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.

Inhibition of glutamate release by bupropion in rat cerebral cortex nerve terminals

Central glutamate neurotransmission has been postulated to play a role in pathophysiology of depression and in the mechanism of antidepressants. The present study was undertaken to elucidate the effect and the possible mechanism of bupropion, an atypical antidepressant, on endogenous glutamate release in nerve terminals of rat cerebral cortex (synaptosomes). Result showed that bupropion exhibited a dose-dependent inhibition of 4-aminopyridine (4-AP)-evoked release of glutamate. The effect of bupropion on the evoked glutamate release was prevented by the chelating the intrasynaptosomal Ca(2+) ions, and by the vesicular transporter inhibitor, but was insensitive to the glutamate transporter inhibitor. Bupropion decreased depolarization-induced increase in [Ca(2+)](C), whereas it did not alter the resting synaptosomal membrane potential or 4-AP-mediated depolarization. The effect of bupropion on evoked glutamate release was abolished by the N-, P- and Q-type Ca(2+) channel blocker, but not by the ryanodine receptor blocker, or the mitochondrial Na(+)/Ca(2+) exchanger blocker. In addition, the inhibitory effect of bupropion on evoked glutamate release was prevented by the mitogen-activated/extracellular signal-regulated kinase kinase (MEK) inhibitors. Western blot analyses showed that bupropion significantly decreased the 4-AP-induced phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2), and this effect also was blocked by MEK inhibitor. These results are the first to suggest that, in rat cerebrocortical nerve terminals, bupropion suppresses voltage-dependent Ca(2+) channel and MEK/ERK activity and in so doing inhibits evoked glutamate release. This finding may provide important information regarding the beneficial effects of bupropion in the brain.

Astaxanthin inhibits glutamate release in rat cerebral cortex nerve terminals via suppression of voltage-dependent Ca2+ entry and mitogen-activated protein kinase signaling pathway.

The purpose of this study was to examine the effect and mechanism of astaxanthin, a natural carotenoid, on endogenous glutamate release in nerve terminals of rat cerebral cortex (synaptosomes). Results showed that astaxanthin exhibited a dose-dependent inhibition of 4-aminopyridine (4-AP)-evoked release of glutamate. The effect of astaxanthin on the evoked glutamate release was prevented by chelating the intrasynaptosomal Ca(2+) ions and by the vesicular transporter inhibitor, but was insensitive to the glutamate transporter inhibitor. Astaxanthin decreased depolarization-induced increase in [Ca(2+)](C), whereas it did not alter the resting synaptosomal membrane potential or 4-AP-mediated depolarization. The effect of astaxanthin on evoked glutamate release was abolished by the N-, P- and Q-type Ca(2+) channel blockers, but not by the ryanodine receptor blocker or the mitochondrial Na(+)/Ca(2+) exchanger blocker. In addition, the inhibitory effect of astaxanthin on evoked glutamate release was prevented by the mitogen-activated protein kinase (MAPK) inhibitors PD98059 and U0126. Western blot analyses showed that astaxanthin significantly decreased the 4-AP-induced phosphorylation of MAPK, and this effect was blocked by PD98059. On the basis of these results, it was concluded that astaxanthin inhibits glutamate release from rat cortical synaptosomes through the suppression of presynaptic voltage-dependent Ca(2+) entry and MAPK signaling cascade.

Acacetin Inhibits Glutamate Release and Prevents Kainic Acid-Induced Neurotoxicity in Rats

An excessive release of glutamate is considered to be a molecular mechanism associated with several neurological diseases that causes neuronal damage. Therefore, searching for compounds that reduce glutamate neurotoxicity is necessary. In this study, the possibility that the natural flavone acacetin derived from the traditional Chinese medicine Clerodendrum inerme (L.) Gaertn is a neuroprotective agent was investigated. The effect of acacetin on endogenous glutamate release in rat hippocampal nerve terminals (synaptosomes) was also investigated. The results indicated that acacetin inhibited depolarization-evoked glutamate release and cytosolic free Ca2+ concentration ([Ca2+]C) in the hippocampal nerve terminals. However, acacetin did not alter synaptosomal membrane potential. Furthermore, the inhibitory effect of acacetin on evoked glutamate release was prevented by the Cav2.2 (N-type) and Cav2.1 (P/Q-type) channel blocker known as ω-conotoxin MVIIC. In a kainic acid (KA) rat model, an animal model used for excitotoxic neurodegeneration experiments, acacetin (10 or 50 mg/kg) was administrated intraperitoneally to the rats 30 min before the KA (15 mg/kg) intraperitoneal injection, and subsequently induced the attenuation of KA-induced neuronal cell death and microglia activation in the CA3 region of the hippocampus. The present study demonstrates that the natural compound, acacetin, inhibits glutamate release from hippocampal synaptosomes by attenuating voltage-dependent Ca2+ entry and effectively prevents KA-induced in vivo excitotoxicity. Collectively, these data suggest that acacetin has the therapeutic potential for treating neurological diseases associated with 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.

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 Ca²⁺ 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 Ca²⁺ 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⁺/Ca²⁺ exchange. These results show that ferulic acid inhibits glutamate release from cortical synaptosomes in rats through the suppression of presynaptic voltage-dependent Ca²⁺ entry.

Osthole or imperatorin-mediated facilitation of glutamate release is associated with a synaptic vesicle mobilization in rat hippocampal glutamatergic nerve endings

Osthole and imperatorin, two active compounds of Cnidium monnieri (L.) Cusson, have previously been shown to facilitate depolarization‐evoked glutamate release from rat hippocampal nerve terminals by increasing voltage‐dependent Ca2+ entry. In this study, we further investigated whether osthole and imperatorin possess an action at the exocytotic machinery itself, downstream of a Ca2+ influx. Our data showed that ionomycin‐induced glutamate release and KCl‐evoked FM1‐43 release were facilitated by osthole and imperatorin, suggesting that some steps after Ca2+ entry are regulated by these two compounds. Consistent with this, osthole or imperatorin‐mediated facilitation of ionomycin‐induced glutamate release was occluded by cytochalasin D that inhibits actin polymerization, implying that the disassembly of cytoskeleton is involved. In addition, the facilitatory action of osthole or imperatorin on ionomycin‐induced glutamate release was attenuated by the Ca2+/calmodulin‐dependent kinase II (CaMKII) inhibitor KN62. Furthermore, Western blotting analysis further showed that osthole or imperatorin significantly increased ionomycin‐induced phosphorylation of CaMKII and synapsin I, the main presynaptic target of CaMKII. These results suggest, therefore, that osthole or imperatorin‐mediated facilitation of glutamate release involves modulation of downstream events controlling synaptic vesicle recruitment and exocytosis, possibly through an increase of CaMKII activation and synapsin I phosphorylation, thereby increasing synaptic vesicle availability for exocytosis. Synapse 64:390–396, 2010.

Apigenin, a natural flavonoid, inhibits glutamate release in the rat hippocampus

The purpose of this study was to examine the effect and mechanism of apigenin, a natural flavonoid, on glutamate release in the rat hippocampus. In rat hippocampal nerve terminals (synaptosomes), apigenin inhibited glutamate release and the elevation of the cytosolic free Ca(2+) concentration evoked by 4-aminopyridine, whereas it had no effect on 4-aminopyridine-mediated depolarization and Na(+) influx. The apigenin-mediated inhibition of evoked glutamate release was prevented by chelating the extracellular Ca(2+) ions and blocking Cav2.2 (N-type) and Cav2.1 (P/Q-type) channel activity. Furthermore, we determined that gamma-aminobutyric acid type A (GABAA) receptors are present in the hippocampal nerve terminals because they are colocalized with the presynaptic marker synaptophysin. However, the effect of apigenin on 4-aminopyridine-evoked glutamate release from synaptosomes was unaffected by the GABAA receptor antagonists SR95531 and bicuculline. Furthermore, in slice preparations, whole-cell patch-clamp experiments showed that apigenin reduced the frequency of spontaneous excitatory postsynaptic currents without affecting their amplitude, suggesting a presynaptic mechanism. On the basis of these results, we suggested that apigenin exerts its presynaptic inhibition probably by reducing Ca(2+) entry mediated by the Cav2.2 (N-type) and Cav2.1 (P/Q-type) channels, thereby inhibiting glutamate release from the rat hippocampal nerve terminals.

Luteolin protects the hippocampus against neuron impairments induced by kainic acid in rats

Glutamatergic excitotoxicity is crucial in the pathogenesis of numerous brain disorders. Luteolin, a flavonoid compound, inhibits glutamate release, however, its ability to affect glutamate-induced brain injury is unknown. Therefore, this study evaluated the protective effect of luteolin against brain damage induced by kainic acid (KA), a glutamate analog. Rats were treated with luteolin (10 or 50mg/kg, intraperitoneally) 30min before an intraperitoneal injection of KA (15mg/kg). Luteolin treatment reduced the KA-induced seizure score and elevations of glutamate levels in the hippocampus. A histopathological analysis showed that luteolin attenuated KA-induced neuronal death and microglial activation in the hippocampus. An immunoblotting analysis showed that luteolin restored the KA-induced reduction in Akt phosphorylation in the hippocampus. Furthermore, a Morris water maze test revealed that luteolin effectively prevented KA-induced learning and memory impairments. The results suggest that luteolin protected rat brains from KA-induced excitotoxic damage by reducing glutamate levels, mitigating inflammation, and enhancing Akt activation in the hippocampus. Therefore, luteolin may be beneficial for preventing or treating brain disorders associated with excitotoxic neuronal damage.