Typhaneoside Suppresses Glutamate Release Through Inhibition of Voltage-Dependent Calcium Entry in Rat Cerebrocortical Nerve Terminals

Glutamate is the major excitatory neurotransmitter in the brain and is involved in many brain functions. In this study, we investigated whether typhaneoside, a flavonoid from Typhae angustifolia pollen, affects endogenous glutamate release from rat cortical synaptosomes. Using a one-line enzyme-coupled fluorometric assay, glutamate release stimulated by the K⁺ channel blocker 4-aminopyridine was monitored to explore the possible underlying mechanisms. The vesicular transporter inhibitor bafilomycin A1 and chelation of extracellular CA²⁺ ions with EGTA suppressed the effect of typhaneoside on the induced glutamate release. Nevertheless, the typhaneoside activity has not been affected by the glutamate transporter inhibitor dl-threo-beta-benzyloxyaspartate. The synaptosomal plasma membrane potential was assayed using a membrane potential-sensitive dye DiSC₃(5), and cytosolic CA²⁺ concentrations ([CA²⁺]_C) was monitored by a CA²⁺ indicator Fura-2. Results showed that typhaneoside did not alter the synaptosomal membrane potential but lowered 4-aminopyridine-induced increases in [CA²⁺]_C. Furthermore, the CA_v2.2 (N-type) channel blocker ω-conotoxin GVIA blocked CA²⁺ entry and inhibited the effect of typhaneoside on 4-aminopyridine-induced glutamate release. However, the inhibitor of intracellular CA²⁺ release dantrolene and the mitochondrial Na⁺/CA²⁺ exchanger blocker 7-chloro-5-(2-chloropheny)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one have no effect on the suppression of glutamate release mediated by typhaneoside. Moreover, inhibition of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) prevented the inhibitory effect of typhaneoside on induced glutamate release. Typhaneoside reduced 4-aminopyridine-induced phosphorylation of ERK1/2 and the major presynaptic ERK target synapsin I, which is a synaptic vesicle-associated protein. In conclusion, these findings suggest a role for typhaneoside in modulating glutamate release by suppressing voltage-dependent CA²⁺ channel mediated presynaptic CA²⁺ influx and the MAPK/ERK/synapsin I signaling cascade.