Korean J Physiol Pharmacol.  2008 Apr;12(2):43-49. 10.4196/kjpp.2008.12.2.43.

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

• Han JH
• Kim KJ
• Jang HJ
• Jang JH
• Kim MJ
• Sung KW
• Rhie DJ
• Jo YH
• Hahn SJ
• Lee MY
• Yoon SH

• KMID: 1486142
• DOI: http://doi.org/10.4196/kjpp.2008.12.2.43

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 (1micrometerto 100micrometer for 5 min inhibited glutamate (100 micrometer 1 min) induced [Ca2+]i increase, concentration-dependently. Pretreatment with apigenin (30micrometer 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, 10 micrometer 1 min) and N-methyl-D-aspartate (NMDA, 100 micrometer 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-dihydroxyphenylglycine (DHPG, 100micrometer 90 s), and inhibited the caffeine (10 mM, 2 min)-induced [Ca2+]i responses. Furthermore, treatment with apigenin (30micrometer 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.

Keyword

Apigenin; Intracellular calcium; Hippocampal neuron; Glutamate; Flavonoid

MeSH Terms

Animals
Apigenin
Caffeine
Calcium
Calcium Signaling
Glutamic Acid
N-Methylaspartate
Neurons
Rats
Receptors, Glutamate
Receptors, Metabotropic Glutamate
Apigenin
Caffeine
Calcium
Glutamic Acid
N-Methylaspartate
Receptors, Glutamate
Receptors, Metabotropic Glutamate

Figure

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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.

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  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 (Ipeak) (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).

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  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).

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  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).

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  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).

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  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).

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  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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