Bandyopadhyay A, Shin D W, Kim D H
Department of Life Science, Kwangju Institute of Science and Technology, Kwangju, Korea.
Biochem J. 2000 May 15;348 Pt 1(Pt 1):173-81.
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+)), 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.
进行了实验以研究钙调神经磷酸酶在调节哺乳动物细胞钙（Ca2+）通量中的作用。在COS-7细胞中，ATP浓度增加（1 - 10微摩尔）以剂量依赖的方式触发细胞内钙（Ca2+）释放。用钙调神经磷酸酶抑制剂如环孢菌素A（CsA）、溴氰菊酯和FK506处理细胞，导致ATP诱导的细胞内钙（Ca2+）释放增强。体外测量钙调神经磷酸酶特异性磷酸酶活性表明COS-7细胞中存在高水平的内源性钙调神经磷酸酶活性，添加溴氰菊酯或CsA可有效抑制该活性。组成型活性钙调神经磷酸酶（CnADeltaCaMAI）的表达在细胞外钙（Ca2+）存在和不存在的情况下均抑制了ATP诱导的细胞内钙（Ca2+）浓度（[Ca2+]i）升高。这些结果表明组成型活性钙调神经磷酸酶阻止了细胞内储存的钙（Ca2+）释放。在转染了钙调神经磷酸酶的细胞中，用CsA处理可恢复钙调神经磷酸酶介导的细胞内钙（Ca2+）释放抑制。蛋白激酶C介导的肌醇-1,4,5-三磷酸受体[Ins(1,4,5)P3R]磷酸化被载体转染细胞制备的提取物部分抑制，而被与CnADeltaCaMAI和钙调神经磷酸酶B共转染细胞制备的提取物完全抑制。添加10微摩尔CsA后，载体转染细胞和钙调神经磷酸酶转染细胞中Ins(1,4,5)P3R的磷酸化抑制均得以恢复。这些结果提供了直接证据表明COS-7细胞中通过Ins(1,4,5)P3R的钙（Ca2+）释放受钙调神经磷酸酶介导的去磷酸化调节。
