Inhibition of Glutamate Release from Rat Cortical Nerve Terminals by Dehydrocorydaline, an Alkaloid from Corydalis yanhusuo

Abstract

Excessive release of glutamate induces excitotoxicity and causes neuronal damage in several neurodegenerative diseases. Natural products have emerged as potential neuroprotective agents for preventing and treating neurological disorders. Dehydrocorydaline (DHC), an active alkaloid compound isolated from Corydalis yanhusuo, possesses neuroprotective capacity. The present study investigated the effect of DHC on glutamate release using a rat brain cortical synaptosome model. Our results indicate that DHC inhibited 4-aminopyridine (4-AP)-evoked glutamate release and elevated intrasynaptosomal calcium levels. The inhibitory effect of DHC on 4-AP-evoked glutamate release was prevented in the presence of the vesicular transporter inhibitor bafilomycin A1 and the N- and P/Q-type Ca²⁺ channel blocker ω-conotoxin MVIIC but not the intracellular inhibitor of Ca²⁺ release dantrolene or the mitochondrial Na⁺/Ca²⁺ exchanger inhibitor CGP37157. Moreover, the inhibitory effect of DHC on evoked glutamate release was prevented by the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) inhibitor PD98059. Western blotting data in synaptosomes also showed that DHC significantly decreased the level of ERK1/2 phosphorylation and synaptic vesicle-associated protein synapsin I, the main presynaptic target of ERK. Together, these results suggest that DHC inhibits presynaptic glutamate release from cerebrocortical synaptosomes by suppressing presynaptic voltage-dependent Ca²⁺ entry and the MAPK/ERK/synapsin I signaling pathway.

Keywords: Ca2+ influx; MAPK/ERK/synapsin I; dehydrocorydaline; glutamate release; neuroprotection; synaptosomes.

Figure 1

Influence of DHC on 4-AP-evoked glutamate release. (A) Chemical structure of DHC. (B) DHC (20 μM) inhibited glutamate release evoked by 4-AP depolarization. Rat brain cortical synaptosomes were depolarized with 15 mM KCl, and the continuous release of glutamate followed. Representative traces of continuous glutamate release curves after treatment with DHC in the presence of 1 mM 4-AP. (C) The dose-dependent response of DHC to glutamate release. Rat synaptosomes were preincubated for 10 min in the presence of DHC, followed by the addition of 4-AP. Data were analyzed with Prism 8.4.3 using nonlinear regression. The results are expressed as the mean ± SEM (n = 5). **** p < 0.0001 versus the control group.

Figure 2

Effect of the Ca²⁺ chelator EGTA, glutamate transporter inhibitor dl-TBOA, or vacuolar ATPase inhibitor bafilomycin A1 on the action of DHC. Quantitative comparison of the extent of glutamate release by 1 mM 4-AP in the absence and presence of DHC. EGTA (300 μM), dl-TBOA (10 μM), bafilomycin A1 (0.1 μM) and DHC (20 μM) were added 10 min before depolarization. The results are the mean ± SEM values of independent experiments (n = 5). ** p < 0.01 versus the control group, **** p < 0.0001 versus the dl-TBOA-treated group.

Figure 3

Influence of DHC on synaptosomal plasma membrane potential. (A) Response of the DiSC3(5) probe in synaptosome suspension to 4-AP depolarization. The arrow indicates the moment when depolarizing stimuli (4-AP 1 mM) were added to the synaptosome. The presented curve shows the results obtained in six independent measurements. (B) Synaptosomes were depolarized with 15 mM KCl, and the continuous release of glutamate followed. DHC (20 μM) decreased KCl-evoked glutamate release from synaptosomes. **** p < 0.0001 versus the control group (n = 5).

Figure 4

DHC reduced the 4-AP-induced increase in [Ca²⁺]i in mouse cortical brain synaptosomes. Synaptosomes were incubated with Fura-2 AM, followed by treatment with DHC (20 μM) as described. Representative ([Ca²⁺]i curves in the presence of DHC and 4-AP (1 mM) are plotted versus time. **** p < 0.0001 versus the control group (n = 5).

Figure 5

DHC inhibited 4-AP-evoked glutamate release through modulation of N- and P/Q- Ca²⁺ channels. Quantitative analysis of glutamate release by depolarizing stimuli (4-AP 1 mM) when synaptosomes were incubated with or without 20 μM DHC, 2 μM ω-con-MVIIC, 50 μM dantrolene, or 100 μM CGP37157. Data are the means ± SEM. **** p < 0.0001 versus the control group, *** p < 0.001 versus the dantrolene-treated group, ** p < 0.01 versus the CGP37157-treated group (n = 5).

Figure 6

DHC-mediated inhibition of glutamate release is modulated by ERK1/2. Quantitative analysis of glutamate release by depolarizing stimuli (4-AP 1 mM) when synaptosomes were incubated with or without 20 μM DHC, 100 μM PKA inhibitor H89, 50 μM ERK kinase inhibitor PD98059, or 10 μM PKC inhibitor GF109203X. Data are the means ± SEM. *** p < 0.001 versus the control group or the H89-treated group, * p < 0.05 versus the GF109203X-treated group (n = 5).

Figure 7

Effect of DHC on the activation of ERK1/2 and synapsin I phosphorylation. The expression levels of (A) p-ERK1/2, ERK1/2, (B) p-synapsin I, and synapsin I in synaptosomes were determined by immunoblotting. Data are the means ± SEM. For p-ERK1/2 expression, *** p < 0.001 versus the 4-AP alone group (n = 3). For synapsin I expression, * p < 0.05 versus the 4-AP alone group (n = 4).