Regulation of ATP-induced calcium release in COS-7 cells by calcineurin.
Experiments were conducted to examine the role of calcineurin in regulating Ca(2+) fluxes in mammalian cells. In COS-7 cells, increasing concentrations (1-10 microM) of ATP triggered intracellular Ca(2+) 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 Ca(2+) 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 (CnADeltaCaMAI) inhibited the ATP-induced increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), in both the presence and the absence of extracellular Ca(2+). These results suggest that the constitutively active calcineurin prevented Ca(2+) release from the intracellular stores. In the calcineurin-transfected cells, treatment with CsA restored the calcineurin-mediated inhibition of intracellular Ca(2+) release. Protein kinase C-mediated phosphorylation of Ins(1,4,5)P(3) receptor [Ins(1,4,5)P(3)R] was partly inhibited by the extracts prepared from the vector-transfected cells and completely inhibited by those from cells co-transfected with CnADeltaCaMAI and calcineurin B. On the addition of 10 microM CsA, the inhibited phosphorylation of Ins(1,4,5)P(3)R was restored in both the vector-transfected cells and the calcineurin-transfected cells. These results show direct evidence that Ca(2+) release through Ins(1, 4,5)P(3)R in COS-7 cells is regulated by calcineurin-mediated dephosphorylation.
Bandyopadhyay
A
A
Department of Life Science, Kwangju Institute of Science and Technology, Kwangju, Korea.
Shin
D W
DW
Kim
D H
DH
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D019556:COS Cells; D019703:Calcineurin / Q000235:genetics / Q000378:metabolism*; D065095:Calcineurin Inhibitors; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000096:biosynthesis / Q000378:metabolism; D002417:Cattle; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006388:Hemagglutinins / Q000378:metabolism; D053496:Inositol 1,4,5-Trisphosphate Receptors; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D018160:Receptors, Cytoplasmic and Nuclear / Q000096:biosynthesis / Q000378:metabolism; D014162:Transfection
D065095:Calcineurin Inhibitors; D015220:Calcium Channels; D006388:Hemagglutinins; D053496:Inositol 1,4,5-Trisphosphate Receptors; D018160:Receptors, Cytoplasmic and Nuclear; D000255:Adenosine Triphosphate; D019703:Calcineurin; D010744:Phosphoric Monoester Hydrolases; D002118:Calcium