Experiments were conducted to examine the role of calcineurin in regulating Ca2+ fluxes in mammalian cells. In COS-7 cells, increasing concentrations (1-10 μM) of ATP triggered intracellular Ca2+ release in a dose-dependent manner. Treatment of the cells with calcineurin inhibitors such as cyclosporin A (CsA), deltamethrin and FK506 resulted in an enhancement of ATP-induced intracellular Ca2+ release. Measurement of calcineurin-specific phosphatase activity in vitro demonstrated a high level of endogenous calcineurin activities in COS-7 cells, which was effectively inhibited by the addition of deltamethrin or CsA. The expression of constitutively active calcineurin (CnA∆CaMAI) inhibited the ATP-induced increase in intracellular Ca2+ concentration ([Ca2+]i), in both the presence and the absence of extracellular Ca2+. These results suggest that the constitutively active calcineurin prevented Ca2+ release from the intracellular stores. In the calcineurin-transfected cells, treatment with CsA restored the calcineurin-mediated inhibition of intracellular Ca2+ release. Protein kinase C-mediated phosphorylation of Ins(1,4,5)P3 receptor [Ins(1,4,5)P3R] was partly inhibited by the extracts prepared from the vector-transfected cells and completely inhibited by those from cells co-transfected with CnA∆CaMAI and calcineurin B. On the addition of 10 μM CsA, the inhibited phosphorylation of Ins(1,4,5)P3R was restored in both the vector-transfected cells and the calcineurin-transfected cells. These results show direct evidence that Ca2+ release through Ins(1,4,5)P3R in COS-7 cells is regulated by calcineurin-mediated dephosphorylation.
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Research Article|
May 09 2000
Regulation of ATP-induced calcium release in COS-7 cells by calcineurin
Arun BANDYOPADHYAY;
Arun BANDYOPADHYAY
1Department of Life Science, Kwangju Institute of Science and Technology, 1 Oryong-dong, Puk-gu, Kwangju, 500-712, Korea
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Dong-Wook SHIN;
Dong-Wook SHIN
1Department of Life Science, Kwangju Institute of Science and Technology, 1 Oryong-dong, Puk-gu, Kwangju, 500-712, Korea
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Do Han KIM
Do Han KIM
1
1To whom correspondence should be addressed (e-mail dhkim@;eunhasu.kjist.ac.kr).
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Publisher: Portland Press Ltd
Received:
December 07 1999
Revision Received:
February 18 2000
Accepted:
March 17 2000
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 2000
2000
Biochem J (2000) 348 (1): 173–181.
Article history
Received:
December 07 1999
Revision Received:
February 18 2000
Accepted:
March 17 2000
Citation
Arun BANDYOPADHYAY, Dong-Wook SHIN, Do Han KIM; Regulation of ATP-induced calcium release in COS-7 cells by calcineurin. Biochem J 15 May 2000; 348 (1): 173–181. doi: https://doi.org/10.1042/bj3480173
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