Journal List > Korean J Physiol Pharmacol > v.12(2) > 1025577

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

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.

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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.
kjpp-12-43f1.tif
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).
kjpp-12-43f2.tif
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).
kjpp-12-43f3.tif
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).
kjpp-12-43f4.tif
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).
kjpp-12-43f5.tif
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).
kjpp-12-43f6.tif
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).
kjpp-12-43f7.tif
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