Casticin inhibits the release of synaptic vesicular glutamate from rat hippocampal nerve terminals

The effect of casticin, major polymethoxyflavone extracted from Vitex rotundifolia, on glutamate release and its underlying mechanisms was investigated in rat hippocampal synaptosomes. Casticin inhibited 4-aminopyridine (4-AP)-evoked glutamate release, with an IC₅₀ of approximately 7.2 μM. It reduced the 4-AP-evoked increase in intrasynaptosomal Ca²⁺ concentration without affecting the synaptosomal membrane potential. The inhibitory effect of casticin on glutamate release was markedly prevented by ω-conotoxin GVIA, an N-type Ca²⁺ channel blocker, but not by ω-agatoxin VIA, a P/Q-type Ca²⁺ channel blocker. Further analysis of FM1-43 dye release showed that casticin suppressed glutamate release by decreasing synaptic vesicle exocytosis. Consistently, casticin also reduced 4-AP-induced phosphorylation of synapsin I, a presynaptic protein that regulates synaptic vesicle mobilization. Transmission electron microscopy (TEM) revealed that casticin decreased the proportion of release-competent synaptic vesicles in 4-AP-stimulated hippocampal synaptosomes. Taken together, these findings suggest that casticin inhibits glutamate release from rat hippocampal nerve terminals, potentially by suppression of N-type Ca²⁺ channel and reducing the availability of synaptic vesicles for exocytosis.