Ca2+ mobilization mediated by transient receptor potential canonical 3 is associated with thrombin-induced morphological changes in 1321N1 human astrocytoma cells
Ca2+ mobilization mediated by transient receptor potential canonical 3 is associated with thrombin-induced morphological changes in 1321N1 human astrocytoma cells
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DOI：
10.1002/jnr.21711
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发表时间：
2008-09-01
影响因子：
4.2
通讯作者：
Kaneko, Shuji
中科院分区：
文献类型：
--
作者：
Nakao, Kenji;Shirakawa, Hisashi;Kaneko, Shuji
活化的星形胶质细胞在病理条件下表现出不同的Ca2+动员模式。在本研究中，我们通过研究凝血酶诱导的独特Ca 2+内流，揭示了星形胶质细胞Ca 2+动力学的新功能。使用1321N1人星形细胞瘤细胞，这已被证明是一个很好的模型，用于检测形态学动力学，我们观察到快速回缩的双极突起，可逆地诱发0.03 - 3 U/mL凝血酶。形态学变化主要依赖于一个特定的凝血酶受体亚型，蛋白酶激活受体1（PAR-1）。同时，Fura-2细胞内Ca2+浓度（[Ca2 +]（i））成像显示凝血酶诱导具有异步重复峰的异质性Ca2+反应。这些振荡被认为是重复的Ca2+释放从细胞内的商店，然后再填充的Ca2+从细胞外区域没有直接的[Ca2 +] i增加的结果。药理学操作与BAPTA-AM，cyclopiazonic酸，和2-氨基乙氧基二苯基硼酸盐表明，Ca2+动员参与凝血酶诱导的形态学变化。我们进一步解决了Ca2+进入的作用，使用小干扰RNA（siRNA）的瞬时受体电位典型3（TRPC 3）。结果，凝血酶诱导的形态学变化和振荡Ca2+反应在siRNA转染的细胞中均显著减弱。用吡唑-3抑制TRPC 3也为Ca2+内流的贡献提供了支持。此外，TRPC 3介导的Ca2+动力学调节凝血酶诱导的肌球蛋白轻链2磷酸化。这些结果表明，星形胶质细胞的钙动力学，包括钙离子进入，在PAR-1介导的星形胶质细胞激活的病理生理效应的一种新的功能。TRPC 3形成功能性Ca 2+通道，并可能调节星形胶质细胞活化以响应脑老化。(C)2008 wiley-liss，Inc.
Activated astrocytes show various patterns of Ca2+ mobilization under pathological conditions. In the present study we revealed a novel function of astrocytic Ca2+ dynamics through investigation of thrombin-induced unique Ca2+ entry. Using 1321N1 human astrocytoma cells, which have been shown to be a good model for detecting morphological dynamics, we observed rapid retraction of bipolar protrusions that were reversibly evoked by 0.03-3 U/mL thrombin. Morphological changes were predominantly dependent on a specific thrombin receptor subtype, proteinase-activated receptor 1 (PAR-1). In parallel, Fura-2 imaging of intracellular Ca2+ concentration ([Ca2+](i)) showed that thrombin induced heterogeneous Ca2+ responses with asynchronous repetitive peaks. These oscillations were found to be a result of repetitive Ca2+ release from intracellular stores, followed by refilling of Ca2+ from the extracellular region without a direct [Ca2+]i increase. Pharmacological manipulation with BAPTA-AM, cyclopiazonic acid, and 2-aminoethoxydiphenyl borate indicated that Ca2+ mobilization was involved in thrombin-induced morphological changes. We further addressed the role of Ca2+ entry using small interfering RNA (siRNA) for transient receptor potential canonical 3 (TRPC3). As a result, both thrombin-induced morphological changes and oscillatory Ca2+ responses were significantly attenuated in siRNA-transfected cells. Inhibition of TRPC3 with pyrazole-3 also provided support for the contribution of Ca2+ influx. Moreover, TRPC3-mediated Ca2+ dynamics regulated thrombin-induced phosphorylation of myosin light chain 2. These results suggest a novel function of astrocytic Ca2+ dynamics, including Ca2+ entry, in the pathophysiological effects of PAR-1-mediated astrocytic activation. TRPC3 forms a functional Ca2+ channel and might modulate astrocytic activation in response to brain hemorrhaging. (C) 2008 wiley-Liss, Inc.