Effects of Apigenin on Glutamate-induced [Ca2+]i Increases in Cultured Rat Hippocampal Neurons

Ji-Hwa Han; Ki Jung Kim; Hyun-Jong Jang; Ju Ho Jang; Myung-Jun Kim; Ki Wug Sung; Duck-Joo Rhie; Yang-Hyeok Jo; Sang June Hahn; Mun-Yong Lee; Shin Hee Yoon

doi:10.4196/kjpp.2008.12.2.43

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Han, Kim, Jang, Jang, Kim, Sung, Rhie, Jo, Hahn, Lee, and Yoon: Effects of Apigenin on Glutamate-induced [Ca2+]i Increases in Cultured Rat Hippocampal Neurons

Original Article

Korean J Physiol Pharmacol 2008;12(2):43-49.

Published online: 17 April 2008

DOI: https://doi.org/10.4196/kjpp.2008.12.2.43

Effects of Apigenin on Glutamate-induced [Ca²⁺]_i Increases in Cultured Rat Hippocampal Neurons

Ji-Hwa Han¹, Ki Jung Kim², Hyun-Jong Jang¹, Ju Ho Jang¹, Myung-Jun Kim¹, Ki Wug Sung², Duck-Joo Rhie¹, Yang-Hyeok Jo¹, Sang June Hahn¹, Mun-Yong Lee², Shin Hee Yoon¹

¹Department of Physiology, College of Medicine, The Catholic University of Korea, Seoul 137-701, Korea

²Department of Pharmacology, College of Medicine, The Catholic University of Korea, Seoul 137-701, Korea

³Department of Anatomy, College of Medicine, The Catholic University of Korea, Seoul 137-701, Korea

Corresponding to: Shin Hee Yoon, Department of Physiology, College of Medicine, The Catholic University of Korea, 505, Banpo-dong, Seocho-gu, Seoul 137-701, Korea. (Tel) 82-2-590-1169, (Fax) 82-2-532-9575, (E-mail) s-hyoon@catholic.ac.kr

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Flavonoids have been shown to affect calcium signaling in neurons. However, there are no reports on the effect of apigenin on glutamate-induced calcium signaling in neurons. We investigated whether apigenin affects glutamate-induced increase of free intracellular Ca²⁺ concentration ([Ca²⁺]_i) in cultured rat hippocampal neurons, using fura-2-based digital calcium imaging and microfluorimetry. The hippocampal neurons were used between 10 and 13 days in culture from embryonic day 18 rats. Pretreatment of the cells with apigenin (1μM to 100μM) for 5 min inhibited glutamate (100μM, 1 min) induced [Ca²⁺]_i increase, concentration-dependently. Pretreatment with apigenin (30μM) for 5 min significantly decreased the [Ca²⁺]_i responses induced by two ionotropic glutamate receptor agonists, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA, 10μM, 1 min) and N-methyl-D-aspartate (NMDA, 100μM, 1 min), and significantly inhibited the AMPA-induced peak currents. Treatment with apigenin also significantly inhibited the [Ca²⁺]_i response induced by 50 mM KCl solution, decreased the [Ca²⁺]_i responses induced by the metabotropic glutamate receptor agonist, (S)-3,5-dihydroxy-phenylglycine (DHPG, 100μM, 90 s), and inhibited the caffeine (10 mM, 2 min)-induced [Ca²⁺]_i responses. Furthermore, treatment with apigenin (30μM) significantly inhibited the amplitude and frequency of 0.1 mM [Mg²⁺]_o-induced [Ca²⁺]_i spikes. These data together suggest that apigenin inhibits glutamate-induced calcium signaling in cultured rat hippocampal neurons.

Keywords: Apigenin, Intracellular calcium, Hippocampal neuron, Glutamate, Flavonoid

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Fig. 1.

Apigenin inhibits glutamate-induced [Ca²⁺]_i increases in cultured rat hippocampal neurons. A, Reproducible glutamate-induced [Ca²⁺]_i increases were induced by treatment with glutamate (100μM) for 1 min at 20 min intervals. B-F, Pretreatment with apigenin for 5 min inhibited the glutamate-induced responses in a concentration dependent manner. G, Plot summarizes the inhibition of the glutamate-induced [Ca²⁺]_i increases by apigenin (1μM, n=8; 3μM, n=15; 10μM, n=17; 30μM, n=14; 50μM, n=13; 100μM, n=17). Glutamate-induced response is presented as a percentage of initial glutamate-induced [Ca²⁺]_i response (peak 2/peak 1) for apigenin pretreated cells. Data are expressed as means±SEM.

Fig. 2.

Apigenin inhibits AMPA-induced [Ca²⁺]_i increases and currents. A, Reproducible AMPA-induced [Ca²⁺]_i increases were induced by treatment with 10μM (s)-AMPA for 1 min. B, Pretreatment with apigenin (30μM) for 5 min decreased the AMPA-induced [Ca²⁺]_i increases. C, Graph summarizes the effect of apigenin on the AMPA-induced [Ca²⁺]_i increases (AMPA, n=16; + apigenin, n=16). D, Inhibitory effects of apigenin on AMPA-induced inward currents. Application of AMPA (10μM, 10 s) evoked inward currents. Pretreatment with apigenin (30μM) for 5 min inhibited the AMPA-induced inward currents. E, Graph summarizes the effect of apigenin on AMPA-induced peak current (I_peak) (AMPA, n=6; + apigenin, n=6). Data are expressed as means±SEM. ∗p<0.05 relative to AMPA (unpaired Student's t-test) ∗∗p<0.05 relative to AMPA (paired Student's t-test).

Fig. 3.

Apigenin inhibits NMDA-induced [Ca²⁺]_i increases. A, Reproducible NMDA- induced [Ca²⁺]_i increases were induced by treatment with 100μM NMDA for 1 min. B, Pretreatment with apigenin (30μM) for 5 min decreased the NMDA-induced responses. C, Graph summarizes the effect of apigenin on the NMDA-induced responses (NMDA, n=17; + apigenin, n=13). Data are expressed as means±SEM. ∗p<0.05 relative to NMDA (unpaired Student's t-test).

Fig. 4.

Apigenin inhibits the high K⁺-induced [Ca²⁺]_i increases. A, Reproducible high K⁺-induced [Ca²⁺]_i increases were induced by treatment with HHSS containing 50 mM KCl for 1 min at 30 min intervals. B, Pretreatment with apigenin (30μM) for 5 min decreased the high K⁺-induced responses. C, Graph summarizes the effect of apigenin on the high K⁺-induced responses (KCl, n = 63; + apigenin, n=17). Data are expressed as means±SEM. ∗p<0.05 relative to KCl (unpaired Student's t-test).

Fig. 5.

Apigenin inhibits [Ca²⁺]_i increases induced by group I mGluR agonist DHPG. A, Reproducible DHPG-induced [Ca²⁺]_i increases were induced by treatment with 100μM DHPG for 90 s at 30 min intervals. B, Pretreatment with apigenin (30μM) for 5 min decreased the DHPG-induced responses. C, Graph summarizes the effect of apigenin on the DHPG-induced responses (DHPG, n=35; + apigenin, n=25). Data are expressed as means±SEM. ∗p<0.05 relative to DHPG (unpaired Student's t-test).

Fig. 6.

Apigenin inhibits the caffeine-induced [Ca²⁺]_i increases. A, Reproducible caffeine-induced [Ca²⁺]_i increases were induced by treatment with 10 mM caffeine for 2 min at 20 min intervals. B, Pretreatment with apigenin (30μM) for 5 min decreased the caffeine-induced responses. C, Graph summarizes the effect of apigenin on the caffeine-induced responses (10 mM caffeine, n=11; + apigenin, n=20). Data are expressed as means±SEM. ∗p<0.05 relative to caffeine (unpaired Student's t-test).

Fig. 7.

Apigenin inhibits synaptically mediated Ca²⁺ spikes induced by treatment with 0.1 mM [Mg²⁺]_o in a cultured rat hippocampal neuron. A, Reducing the extracellular Mg²⁺ concentration ([Mg²⁺]_o) to 0.1 mM induced [Ca²⁺]_i spikes. B, Treatment with apigenin (30 μM) inhibits the [Ca²⁺]_i spikes. C1 & C2, Graph summarizes the effect of apigenin on 0.1mM [Mg²⁺]_o-induced [Ca²⁺]_i spikes (control, n=4; + apigenin, n=4) Data are expressed as means±SEM. ∗p<0.05 relative to 0.1mM [Ma²⁺]_o (unpaired Student's t-test).

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