Naunyn-Schmiedeberg's Archives of Pharmacology

, Volume 369, Issue 2, pp 260–267 | Cite as

Epigallocatechin-3-gallate increases intracellular [Ca2+] in U87 cells mainly by influx of extracellular Ca2+ and partly by release of intracellular stores

  • Hee Jung Kim
  • Keun Sang Yum
  • Jong-Ho Sung
  • Duck-Joo Rhie
  • Myung-Jun Kim
  • Do Sik Min
  • Sang June Hahn
  • Myung-Suk Kim
  • Yang-Hyeok Jo
  • Shin Hee Yoon
Original Article
First Online:
Received:
Accepted:

Abstract

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+] ([Ca2+]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 [Ca2+]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+]i 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.

Keywords

Ca2+ channel EGCG Green tea Diphenylamine-2-carboxylate Phospholipase C U87 cells 
This is a preview of subscription content, log in to check access.

Notes

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.

References

  1. Ahmad N, Feyes DK, Nieminen AL, Agarwal R, Mukhtar H (1997) Green tea constituent epigallocatechin-3-gallate and induction of apoptosis and cell cycle arrest in human carcinoma cells. J Natl Cancer Inst 89:1881–1886PubMedGoogle Scholar
  2. Ahn HY, Hadizadeh KR, Seul C, Yun YP, Vetter H, Sachinidis, A (1999) Epigallocathechin-3 gallate selectively inhibits the PDGF-Rβ-induced intracellular signaling transduction pathway in vascular smooth muscle cells and inhibits transformation of sis-transfected NIH 3T3 fibroblasts and human glioblastoma cells (A172). Mol Biol Cell 10:1093–1104PubMedGoogle Scholar
  3. Bae JH, Mun KC, Park WK, Lee SR, Suh SI, Baek WK, Yim MB, Kwon TK, Song DK (2002) EGCG attenuates AMPA-induced intracellular calcium increase in hippocampal neurons. Biochem Biophys Res Commun 290:1506–1512CrossRefPubMedGoogle Scholar
  4. Berridge MJ, Bootman MD, Lipp P (1998) Calcium—a life and death signal. Nature 395:645–648CrossRefPubMedGoogle Scholar
  5. Carmignoto G, Pasti L, Pozzan T (1998) On the role of voltage-dependent calcium channels in calcium signaling of astrocytes in situ. J Neurosci 18:4637–4645PubMedGoogle Scholar
  6. Carney DH, Scott DL, Gordon EA, LaBelle EF (1985) Phosphoinositides in mitogenesis: neomycin inhibits thrombin-stimulated phosphoinositide turnover and initiation of cell proliferation. Cell 42:479–488Google Scholar
  7. Chattopadhyay N, Ye CP, Yamaguchi T, Kerner R, Vassilev PM, Brown EM (1999) Extracellular calcium-sensing receptor induces cellular proliferation and activation of a nonselective cation channel in U373 human astrocytoma cells. Brain Res 851:116–124CrossRefPubMedGoogle Scholar
  8. Chen Q, Olney JW, Lukasiewicz PD, Almli T, Romano C (1998) Fenamates protect neurons against ischemic and excitotoxic injury in chick embryo retina. Neurosci Lett 242:163–166CrossRefPubMedGoogle Scholar
  9. Choi BH, Choi JS, Min DS, Yoon SH, Rhie DJ, Jo YH, Kim MS, Hahn SJ (2001) Effects of (−)-epigallocatechin-3-gallate, the main component of green tea, on the cloned rat brain Kv1.5 potassium channels. Biochem Pharmacol 62:527–535CrossRefPubMedGoogle Scholar
  10. Dreosti IE, Wargovich MJ, Yang CS (1997) Inhibition of carcinogenesis by tea: the evidence from experimental studies. Crit Rev Food Sci Nutr 37:761–770PubMedGoogle Scholar
  11. Fujiki H, Suganuma M, Okabe S, Sueoka E, Suga K, Imai K, Nakachi K, Kimura S (1999) Mechanistic findings of green tea as cancer preventive for humans. Proc Soc Exp Biol Med 220:225–228PubMedGoogle Scholar
  12. Fukao M, Watanabe H, Takeuchi K, Tomioka H, Hattori Y (2001) Effects of SK&F 96365 and mefenamic acid on Ca2+ influx in stimulated endothelial cells and on endothelium-derived hyperpolarizing factor-mediated arterial hyperpolarization and relaxation. J Cardiovasc Pharmacol 38:130–140Google Scholar
  13. Gögelein H, Dahlem D, Englert HC, Lang HJ (1990) Flufenamic acid, mefenamic acid and niflumic acid inhibit single nonselective cation channels in the rat exocrine pancreas. FEBS Lett 268:79–82CrossRefPubMedGoogle Scholar
  14. Grynkiewicz G, Poenie M, Tsien RY (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260:3440–3450PubMedGoogle Scholar
  15. Haslam E (1996) Natural polyphenols (vegetable tannins) as drugs: possible modes of action. J Nat Prod 59:205–215CrossRefPubMedGoogle Scholar
  16. Hildebrandt J P, Plant TD, Meves H (1997) The effects of bradykinin on K+ currents in NG108-15 cells treated with U73122, a phospholipase C inhibitor, or neomycin. Br J Pharmacol 120:841–850PubMedGoogle Scholar
  17. Hofmann CS, Sonenshein GE (2003) Green tea polyphenol epigallocatechin-3 gallate induces apoptosis of proliferating vascular smooth muscle cells via activation of p53. FASEB J 17:702–704PubMedGoogle Scholar
  18. Kagaya N, Tagawa Y, Nagashima H, Saijo R, Kawase M, Yagi K (2002) Suppression of cytotoxin-induced cell death in isolated hepatocytes by tea catechins. Eur J Pharmacol 450:231–236CrossRefPubMedGoogle Scholar
  19. Kitano K, Nam KY, Kimura S, Fujiki H, Imanishi Y (1997) Sealing effects of (−)-epigallocatechin gallate on protein kinase C and protein phosphatase 2A. Biophys Chem 65:157–164CrossRefPubMedGoogle Scholar
  20. Lee YS, Sayeed MM, Wurster RD (1994) Inhibition of cell growth and intracellular Ca2+ mobilization in human brain tumor cells by Ca2+ channel antagonists. Mol Chem Neuropathol 22:81–95PubMedGoogle Scholar
  21. Lee S, Suh S, Kim S (2000) Protective effects of the green tea polyphenol (−)-epigallocatechin gallate against hippocampal neuronal damage after transient global ischemia in gerbils. Neurosci Lett 287:191–194CrossRefPubMedGoogle Scholar
  22. Lerma J, Martin del Rio R (1992) Chloride transport blockers prevent N-methyl-d-aspartate receptor-channel complex activation. Mol Pharmacol 41:217–222PubMedGoogle Scholar
  23. Levites Y, Weinreb O, Maor G, Youdim MB, Mandel S (2001) Green tea polyphenol (−)-epigallocatechin-3-gallate prevents N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced dopaminergic neurodegeneration. J Neurochem 78:1073–1082CrossRefPubMedGoogle Scholar
  24. Levites Y, Amit T, Mandel S, Youdim MB (2003) Neuroprotection and neurorescue against Abeta toxicity and PKC-dependent release of nonamyloidogenic soluble precursor protein by green tea polyphenol (−)-epigallocatechin-3-gallate. FASEB J 17:952–954PubMedGoogle Scholar
  25. Lin YL, Lin JK (1997) (−)-Epigallocatechin-3-gallate blocks the induction of nitric oxide synthase by down-regulating lipopolysaccharide-induced activity of transcription factor nuclear factor-kappa B. Mol Pharmacol 52:465–472PubMedGoogle Scholar
  26. Lo TM, Thayer SA (1993) Refilling the inositol 1,4,5-trisphosphate-sensitive Ca2+ store in neuroblastoma×glioma hybrid NG108-15 cells. Am J Physiol 264:C641–C653PubMedGoogle Scholar
  27. Lu YP, Lou YR, Xie JG, Peng QY, Liao J, Yang CS, Huang MT, Conney AH (2002) Topical applications of caffeine or (−)-epigallocatechin gallate (EGCG) inhibit carcinogenesis and selectively increase apoptosis in UVB-induced skin tumors in mice. Proc Natl Acad Sci USA 99:12455–12460CrossRefPubMedGoogle Scholar
  28. Mukhtar H, Ahmad N (1999) Mechanism of cancer chemopreventive activity of green tea. Proc Soc Exp Biol Med 220:234–238CrossRefPubMedGoogle Scholar
  29. Munaron L, Distasi C, Carabelli V, Baccino FM, Bonelli G, Lovisolo D (1995) Sustained calcium influx activated by basic fibroblast growth factor in Balb-c 3T3 fibroblasts. J Physiol (Lond) 484:557–566Google Scholar
  30. Nie G, Cao Y, Zhao B (2002) Protective effects of green tea polyphenols and their major component, (−)-epigallocatechin-3-gallate (EGCG), on 6-hydroxydopamine-induced apoptosis in PC12 cells. Redox Rep 7:171–177CrossRefPubMedGoogle Scholar
  31. Nilius B (1994) Ion channels in nonexcitable cells. In: Sperelakis N (ed) Cell physiology Source book 2nd edn. Academic Press, San Diego, pp 436–455Google Scholar
  32. Pan CY, Kao YH, Fox AP (2002) Enhancement of inward Ca2+ currents in bovine chromaffin cells by green tea polyphenol extracts. Neurochem Int 40:131–137CrossRefPubMedGoogle Scholar
  33. Poronnik P, Ward MC, Cook DI (1992) Intracellular Ca2+ release by flufenamic acid and other blockers of the non-selective cation channel. FEBS Lett 296:245–248CrossRefPubMedGoogle Scholar
  34. Putney JW Jr (1993) Excitement about calcium signaling in inexcitable cells. Science 262:676–678PubMedGoogle Scholar
  35. Rhee SG, Bae YS (1997) Regulation of phosphoinositide-specific phospholipase C isozymes. J Biol Chem 272:15045–15048PubMedGoogle Scholar
  36. Rhie D-J, Sung J-H, Ha U-S, Kim HJ, Min DS, Hahn SJ, Kim M-S, Jo Y-H, Yoon SH (2003) Endogenous somatostatin receptors mobilize calcium from inositol 1,4,5-trisphosphate-sensitive stores in NG108-15 cells. Brain Res 975:120–128CrossRefPubMedGoogle Scholar
  37. Sachinidis A, Seul C, Seewald S, Ahn H, Ko Y, Vetter H (2000) Green tea compounds inhibit tyrosine phosphorylation of PDGF beta-receptor and transformation of A172 human glioblastoma. FEBS Lett 471:51–55CrossRefPubMedGoogle Scholar
  38. Sachinidis A, Skach RA, Seul C, Ko Y, Hescheler J, Ahn HY, Fingerle J (2002) Inhibition of the PDGF beta-receptor tyrosine phosphorylation and its downstream intracellular signal transduction pathway in rat and human vascular smooth muscle cells by different catechins. FASEB J 16:893–895PubMedGoogle Scholar
  39. Semenchuk LA, Di Salvo J (1995) Receptor-activated increases in intracellular calcium and protein tyrosine phosphorylation in vascular smooth muscle cells. FEBS Lett 370:127–130PubMedGoogle Scholar
  40. Thastrup O, Cullen PJ, Drobak BK, Hanley MR, Dawson AP (1990) Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2+-ATPase. Proc Natl Acad Sci USA 87:2466–2470Google Scholar
  41. Thayer SA, Sturek M, Miller RJ (1988) Measurement of neuronal Ca2+ transients using simultaneous microfluorimetry and electrophysiology. Pflugers Arch 412:216–223PubMedGoogle Scholar
  42. Tijburg LB, Mattern T, Folts JD, Weisgerber UM, Katan MB (1997) Tea flavonoids and cardiovascular disease: a review. Crit Rev Food Sci Nutr 37:771–785PubMedGoogle Scholar
  43. Weinreb O, Mandel S, Youdim MB (2003) cDna gene expression profile homology of antioxidants and their antiapoptotic and proapoptotic activities in human neuroblastoma cells. FASEB J 17:935–937PubMedGoogle Scholar
  44. White MM, Aylwin M (1990) Niflumic and flufenamic acids are potent reversible blockers of Ca2+-activated Cl− channels in Xenopus oocytes. Mol Pharmacol 37:720–724PubMedGoogle Scholar
  45. Yamane T, Takahashi T, Kuwata K, Oya K, Inagake M, Kitao Y, Suganuma M, Fujiki H (1995) Inhibition of N-methyl-N′-nitro-N-nitrosoguanidine-induced carcinogenesis by (−)-epigallocatechin gallate in the rat glandular stomach. Cancer Res 55:2081–2084PubMedGoogle Scholar
  46. Yamashita K, Suzuki Y, Matsui T, Yoshimaru T, Yamaki M, Suzuki-Karasaki, M, Hayakawa S, Shimizu K (2000) Epigallocatechin gallate inhibits histamine release from rat basophilic leukemia (RBL-2H3) cells: role of tyrosine phosphorylation pathway. Biochem Biophys Res Commun 274:603–608CrossRefPubMedGoogle Scholar
  47. Yang CS, Chen L, Lee M J, Balentine D, Kuo MC, Schantz SP (1998) Blood and urine levels of tea catechins after ingestion of different amounts of green tea by human volunteers. Cancer Epidemiol Biomarkers Prev 7:351–354PubMedGoogle Scholar
  48. Yang CS, Maliakal P, Meng X (2002) Inhibition of carcinogenesis by tea. Annu Rev Pharmacol Toxicol 42:25–54Google Scholar
  49. Yoon SH, Lo T-M, Loh HH, Thayer SA (1999) δ-Opioid-induced liberation of Gβγ mobilizes Ca2+ stores in NG108-15 cells. Mol Pharmacol 56:902–908PubMedGoogle Scholar
  50. Zheng L, Stojilkovic SS, Hunyady L, Krsmanovic LZ, Catt KJ (1994) Sequential activation of phospholipase-C and -D in agonist-stimulated gonadotrophs. Endocrinology 134:1446–1454PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • Hee Jung Kim
    • 1
  • Keun Sang Yum
    • 1
  • Jong-Ho Sung
    • 1
  • Duck-Joo Rhie
    • 1
  • Myung-Jun Kim
    • 1
  • Do Sik Min
    • 1
  • Sang June Hahn
    • 1
  • Myung-Suk Kim
    • 1
  • Yang-Hyeok Jo
    • 1
  • Shin Hee Yoon
    • 1
  1. 1.Department of Physiology, College of MedicineThe Catholic University of KoreaSeoulKorea

Personalised recommendations