Hispidulin inhibits the release of glutamate in rat cerebrocortical nerve terminals
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.
References (57)
- et al.
PD98059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo
J. Biol. Chem.
(1995) Neuronal calcium signaling
Neuron
(1998)A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding
Anal. Biochem.
(1976)- et al.
Seizure related elevations of extracellular amino acids in human focal epilepsy
Neurosci. Lett.
(1992) Glutamate receptors in epilepsy
Prog. Brain Res.
(1998)- et al.
Anticonvulsant activity of two metabotropic glutamate group I antagonists selective for the mGlu5 receptor: 2-methyl-6-(phenylethynyl)-pyridine (MPEP), and (E)-6-methyl-2-styryl-pyridine (SIB 1893)
Neuropharmacology
(2000) - et al.
Synaptic vesicle mobilization is regulated by distinct synapsin I phosphorylation pathways at different frequencies
Neuron
(2003) - et al.
Extracellular hippocampal glutamate and spontaneous seizure in the conscious human brain
Lancet
(1993) - et al.
Extracellular-signal-regulated kinase signaling in neurons
Curr. Opin. Neurobiol.
(1999) - et al.
Somatostatin inhibits glutamate release from mouse cerebrocortical nerve endings through presynaptic sst2 receptors linked to the adenylyl cyclase-protein kinase A pathway
Neuropharmacology
(2004)
A new generation of Ca2 + indicators with greatly improved fluorescence properties
J. Biol. Chem.
Cellular mechanisms of acute decrease of glutamate release induced by raloxifene in rat cerebral cortex
Neuropharmacology
Presynaptic, extrasynaptic and axonal GABAA receptors in the CNS: where and why?
Prog. Biophys. Mol. Biol.
Nerve terminal GABAA receptors activate Ca2 +/calmodulin-dependent signaling to inhibit voltage-gated Ca2 + influx and glutamate release
J. Biol. Chem.
Pharmacology of glutamate receptor antagonists in the kindling model of epilepsy
Prog. Neurobiol.
Differential coupling of N and P/Q-type calcium channels to glutamate exocytosis in the rat cerebral cortex
Neurosci. Lett.
Extracellular amino acid levels in hippocampus during pilocarpine-induced seizures
Epilepsy Res.
Glutamate metabotropic receptors as targets for drug therapy in epilepsy
Eur. J. Pharmacol.
Presynaptic modulation of glutamate release
Prog. Brain Res.
Non-specific effects of the MEK inhibitors PD98059 and U0126 on glutamate release from hippocampal synaptosomes
Neuropharmacology
Structure, pharmacology, and function of GABAA receptor subtypes
Adv. Pharmacol.
Localized Ca2 + entry preferentially effects protein dephosphorylation, phosphorylation, and glutamate release
J. Biol. Chem.
Effects of carbamazepine, phenytoin, lamotrigine, oxcarbazepine, topiramate and vinpocetine on Na+ channel-mediated release of [3H]glutamate in hippocampal nerve endings
Neuropharmacology
Effects of carbamazepine, phenytoin, valproic acid, oxcarbazepine, lamotrigine, topiramate and vinpocetine on the presynaptic Ca2 + channel-mediated release of [3H]glutamate: comparison with the Na+ channel-mediated release
Neuropharmacology
Induction or protection of limbic seizures in mice by mGluR subtype selective agonists
Neuropharmacology
Opposing facilitatory and inhibitory modulation of glutamate release elicited by cAMP production in cerebrocortical nerve terminals (synaptosomes)
Neuropharmacology
Comparison of seizure related amino acid release in human epileptic hippocampus versus a chronic, kainate rat model of hippocampal epilepsy
Epilepsy Res.
Presynaptic inhibition of elicited neurotransmitter release
Trends Neurosci.
Cited by (28)
Clerodendrum petasites S. Moore: The therapeutic potential of phytochemicals, hispidulin, vanillic acid, verbascoside, and apigenin
2019, Biomedicine and PharmacotherapyCitation Excerpt :A massive increase in glutamate is also strongly linked to epilepsy, thus one treatment target aims to moderate this glutamatergic neurotransmission. It has been reported that hispidulin decreased phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK 1/2) and vesicle-associated protein synapsin I phosphorylation, consequently contributing to a decrease in glutamate level [41]. Hispidulin also suppressed presynaptic voltage-dependent Ca2+ entry, Cav2.2, and Cav2.1 channels.
Autophagy induction by hispidulin provides protection against sevoflurane-induced neuronal apoptosis in aged rats
2018, Biomedicine and PharmacotherapyCitation Excerpt :Due to its ability to pass through blood brain barrier, the effect of hispidulin on conditions of central nervous system has been investigated since last decade [11]. Hispidulin exerts anti-epileptic activities by inhibiting glutamate release from cortical synaptosomes in rats through the suppression of presynapticvoltage-dependent Ca2+ entry and ERK/synapsin I signaling pathway [25]. The anxiolytic and pro-cognitive effects of hispidulin has also been reported [11].
Hispidulin prevents sevoflurane— Induced memory dysfunction in aged rats
2018, Biomedicine and Pharmacotherapy(+)-Dehydrofukinone modulates membrane potential and delays seizure onset by GABAa receptor-mediated mechanism in mice
2017, Toxicology and Applied PharmacologyCitation Excerpt :Nevertheless, we do not rule out that DHF interacts directly with calcium channels, since 1 μM FMZ did not fully prevent the effect of DHF on calcium influx. Moreover, other natural compounds with GABAa modulatory properties also decrease calcium influx in synaptosomes through a GABAergic-independent pathway (Lin et al., 2012). Additionally, compounds with highly similar chemical structure to DHF have been addressed as VGCC blockers/modulators (Asakura et al., 1999; Asakura et al., 2000).
Medicinal importance, pharmacological activities, and analytical aspects of hispidulin: A concise report
2017, Journal of Traditional and Complementary MedicineCiproxifan, a histamine H<inf>3</inf> receptor antagonist and inverse agonist, presynaptically inhibits glutamate release in rat hippocampus
2017, Toxicology and Applied Pharmacology