Green tea has been receiving considerable attention as a possible preventive agent against cancer and cardiovascular disease. Epigallocatechin-3- gallate (EGCG) is a major polyphenol component of green tea. Using digital calcium imaging and an assay for [3H]-inositol phosphates, we determined whether EGCG increases intracellular [Ca2+] ([Ca 2+]i) in non-excitable human astrocytoma U87 cells. EGCG induced concentration-dependent increases in [Ca2+]i. The EGCG-induced [Ca2+]i increases were reduced to 20.9% of control by removal of extracellular Ca2+. The increases were also inhibited markedly by treatment with the non-specific Ca2+ channel inhibitors cobalt (3 mM) for 3 min and lanthanum (1 mM) for 5 min. The increases were not significantly inhibited by treatment for 10 min with the L-type Ca2+ channel blocker nifedipine (100 nM). Treatment with the inhibitor of endoplasmic reticulum Ca2+-ATPase thapsigargin (1 μM) also significantly inhibited the EGCG-induced [Ca2+]i increases. Treatment for 15 min with the phospholipase C (PLC) inhibitor neomycin (300 μM) attenuated the increases significantly, while the tyrosine kinase inhibitor genistein (30 μM) had no effect. EGCG increased [ 3H]-inositol phosphates formation via PLC activation. Treatment for 10 min with mefenamic acid (100 μM) and flufenamic acid (100 μM), derivatives of diphenylamine-2-carboxylate, blocked the EGCG-induced [Ca 2+]i increase in non-treated and thapsigargin-treated cells but indomethacin (100 μM) did not affect the increases. Collectively, these data suggest that EGCG increases [Ca2+]; in non-excitable U87 cells mainly by eliciting influx of extracellular Ca2+ and partly by mobilizing intracellular Ca2+ stores by PLC activation. The EGCG-induced [Ca2+]i influx is mediated mainly through channels sensitive to diphenylamine-2-carboxylate derivatives.
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Acknowledgements This work was supported by grant No. R01-1999-000-00107-0 from the Basic Research Program of the Korea Science and Engineering Foundation and the Roche Foundation made in the program year 2000.
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