Abstract
Various effects of acorn extract have been reported including antioxidant activity, cytotoxicity against cancer cells, and the levels of acetylcholine and its related enzyme activities in the dementia mouse models. However, it is unclear whether acorn extract inhibits glutamate-induced calcium signaling in hippocampal neurons. This study was an investigation into the effect of acorn extract on intracellular free Ca2+ concentrations ([Ca2+]i) in cultured rat hippocampal neurons using fura-2-based digital calcium imaging and photometry. Hippocampal neurons were used between 10 and 14 d in culture from embryonic day-18 rats. Treatment with acorn extract (1 µg/mL to 1 mg/mL) for 30 min inhibited glutamate (100 µM)-induced [Ca2+]i increases in a dose-dependent manner (IC50=46.9 µg/mL). After depletion of intracellular Ca2+ stores by treatment with the inhibitor endoplasmic reticulum Ca2+-ATPase, thapsigargin (1 µM), treatment with acorn extract (50 µg/mL) for 30 min decreased the subsequent glutamate-induced [Ca2+]i increases. Acorn extract significantly inhibited (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) (30 µM)-induced [Ca2+]i increases. In addition, acorn extract inhibited the AMPA-induced [Ca2+]i responses in the presence of 1 µM nimodipine. Acorn extract also significantly inhibited N-methyl-D-aspartate (100 µM)-induced [Ca2+]i increases. Acorn extract significantly inhibited 50 mM KCl -induced [Ca2+]i increases. Acorn extract significantly inhibited (S)-3,5-dihydroxyphenylglycine-induced [Ca2+]i responses. Moreover, acorn extract almost completely blocked synaptically mediated [Ca2+]i spikes induced by decreasing extracellular Mg2+ concentration to 0.1 mM. These results suggest that acorn extract inhibits synaptically induced frequent [Ca2+]i spikes through multiple pathways such as ionotropic glutamate receptors, voltage-gated Ca2+ channels and metabotropic glutamate receptors in cultured rat hippocampal neurons.
