Hispidulin inhibits the release of glutamate in rat cerebrocortical nerve terminals

https://doi.org/10.1016/j.taap.2012.06.015Get rights and content

Abstract

Hispidulin, a naturally occurring flavone, has been reported to have an antiepileptic profile. An excessive release of glutamate is considered to be related to neuropathology of epilepsy. We investigated whether hispidulin affected endogenous glutamate release in rat cerebral cortex nerve terminals (synaptosomes) and explored the possible mechanism. Hispidulin inhibited the release of glutamate evoked by the K+ channel blocker 4-aminopyridine (4-AP). The effects of hispidulin on the evoked glutamate release were prevented by the chelation of extracellular Ca2 + ions and the vesicular transporter inhibitor bafilomycin A1. However, the glutamate transporter inhibitor dl-threo-beta-benzyl-oxyaspartate did not have any effect on hispidulin action. Hispidulin reduced the depolarization-induced increase in cytosolic free Ca2 + concentration ([Ca2 +]C), but did not alter 4-AP-mediated depolarization. Furthermore, the effect of hispidulin on evoked glutamate release was abolished by blocking the Cav2.2 (N-type) and Cav2.1 (P/Q-type) channels, but not by blocking ryanodine receptors or mitochondrial Na+/Ca2 + exchange. Mitogen-activated protein kinase kinase (MEK) inhibition also prevented the inhibitory effect of hispidulin on evoked glutamate release. Western blot analyses showed that hispidulin decreased the 4-AP-induced phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and synaptic vesicle-associated protein synapsin I, a major presynaptic substrate for ERK; this decrease was also blocked by the MEK inhibitor. Moreover, the inhibition of glutamate release by hispidulin was strongly attenuated in mice without synapsin I. These results show that hispidulin inhibits glutamate release from cortical synaptosomes in rats through the suppression of presynaptic voltage-dependent Ca2 + entry and ERK/synapsin I signaling pathway.

Highlights

► Hispidulin inhibited glutamate release from rat cerebrocortical synaptosomes. ► This action did not involve the participation of GABAA receptors. ► A decrease in the Ca2 + influx through Cav2.2 and Cav2.1 channels was involved. ► A role for the MAPK/ERK/synapsin I pathway in the action of hispidulin was suggested. ► This study provided further understanding of the mode of hispidulin action in the brain.

Introduction

Epilepsy, which affects approximately 2% of people worldwide, is one of the most common brain disorder. Current antiepileptic drugs mainly affect transmitter receptors and ion channels. Unfortunately, because of unwanted side effects, approximately 30% of patients do not response to these drugs (Rogawski and Loscher, 2004). Therefore, seeking safe and effective antiepileptic drugs derived from natural products may enable development of novel treatments for epilepsy. Hispidulin is a naturally occurring flavone commonly found in Saussurea involucrate Kar. et Kir., a traditional Chinese medicinal herb. Several biological activities of hispidulin have emerged, for example, antioxidant, antifungal, anti-inflammatory, and antimutagenic properties (Gil et al., 1994, Tan et al., 1999). In addition to these properties, hispidulin has been confirmed to penetrate the blood–brain barrier (BBB) and possess antiepileptic activity (Kavvadias et al., 2004). However, the mechanisms involved in the antiepileptic effect of hispidulin have yet to be fully elucidated.

Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system (CNS), and evidence suggests that alterations in this neurotransmitter system may be associated with epilepsy. For instance, the experimental application of glutamate receptor agonists induces seizures in rats (Chapman, 1998, Loscher, 1998, Tizzano et al., 1995). Conversely, glutamate receptor antagonists exhibit antiepileptic-like properties and reduce seizure-induced brain damage in different animal models (Chapman et al., 2000, Clifford et al., 1990). Furthermore, a significant increase in glutamate level was observed in human epilepsy patients as well as in experimental models of epilepsy (Carlson et al., 1992, Chapman et al., 1996, During and Spencer, 1993, Millan et al., 1993, Smolders et al., 1997, Wilson et al., 1996). This evidence suggests that an overabundance of glutamatergic activity can occur in epilepsy. Thus, modulating central glutamatergic neurotransmission may provide a potential target for epilepsy treatment. Consequently, several glutamatergic modulators are being developed to treat epilepsy, including N-methyl-d-aspartic acid (NMDA) receptor antagonists, as well as metabotropic glutamate receptor agonists and antagonists. However, these drugs have been unsuccessful in clinical trials because of numerous side effects, such as ataxia, and memory impairment (Chapman, 1998, Moldrich et al., 2003).

Because excessive release of glutamate is known to be a critical factor in the neuropathology of epilepsy (Kaura et al., 1995, Meldrum, 1994), regulating its release may be an important mechanism of antiepileptic drugs. Some antiepileptic drugs have been revealed to decrease glutamate release in human and rat brain tissues (Kammerer et al., 2011, Sitges et al., 2007a, Sitges et al., 2007b). Likewise, hispidulin has an antiepileptic-like effect and whether hispidulin has an effect on endogenous glutamate release should be evaluated. Thus, the present study used isolated nerve terminals (synaptosomes) purified from the rat prefrontal cortex as a model to investigate the effects of hispidulin on glutamate release and to characterize the underlying molecular mechanisms. In contrast to brain slices, synaptosomes do not suffer from any postsynaptic interactions and are, therefore, extensively used to evaluate presynaptic phenomena. The first series of experiments investigated the effects of hispidulin on the release of endogenous glutamate, the synaptosomal plasma membrane potential, and the downstream activation of voltage dependent Ca2 + channels (VDCCs). The second series of experiments determined whether the protein kinase signaling pathway participates in the regulation of glutamate release by hispidulin. Finally, since it has been demonstrated that phosphorylation of the vesicle-associated protein synapsin I enhances vesicle mobilization and glutamate release (Jovanovic et al., 1996, Jovanovic et al., 2000, Schenk et al., 2005, Yamagata et al., 2002), we examined if the regulation of glutamate release by hispidulin is linked to a decrease in synapsin I phosphorylation.

Section snippets

Chemicals

3′, 3′, 3′-Dipropylthiadicarbocyanine iodide [DiSC3(5)] and Fura-2-acetoxy- methyl ester (Fura-2-AM) were obtained from Invitrogen (Carlsbad, CA, USA). Hispidulin, dantrolene, bafilomycin A1, 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059), N-(cyclopropylmethoxy)-3, 4, 5-trifluoro-2-[(4-iodo-2-methylphenyl)amino]-benzamide (PD198306), dl-threo-β-benzyloxyaspartate (dl-TBOA), bisindolylmaleimide I (GF109203X), 7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one

Hispidulin inhibits 4-AP-evoked glutamate release from rat cerebrocortical synaptosomes, and this phenomenon is mediated by a reduction in the Ca2 +-dependent exocytotic component of glutamate release

To examine the influence of hispidulin on glutamate release, isolated nerve terminals were depolarized with the potassium channel blocker 4-aminopyridine (4-AP) which has been shown to open voltage-dependent Ca2 + channels (VDCCs) and to induce the release of glutamate (Nicholls, 1998). In synaptosomes incubated in the presence of 1 mM CaCl2, 4-AP evoked a glutamate release of 7.2 ± 0.1 nmol/mg/5 min. Application of hispidulin (30 μM) produced an inhibition of 4-AP-evoked glutamate release to 3.4 ± 0.3 

Discussion

Epilepsy is a common neurological disorder affecting people worldwide, and obtaining antiepileptic-like medicine is highly crucial in treating this disease. Hispidulin is a naturally occurring flavonoid and has been reported to have an anticonvulsant profile (Kavvadias et al., 2004). However, the exact mechanism of its antiepileptic activity remains to be explored. To help address this, this study used isolated cerebrocortical nerve terminals to examine the effect of hispidulin on glutamate

Conflict of interest statement

There is no conflict of interest to disclose for any of the authors.

Acknowledgments

This work was supported by grants from the Far-Eastern Memorial Hospital (FEMH-2012-D-006) and the National Science Council (NSC 100-2320-B-030-006-MY3), Taiwan.

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