Nakao Kenji, Shirakawa Hisashi, Sugishita Aiko, Matsutani Ikkei, Niidome Tetsuhiro, Nakagawa Takayuki, Kaneko Shuji
Department of Molecular Pharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan.
J Neurosci Res. 2008 Sep;86(12):2722-32. doi: 10.1002/jnr.21711.
Activated astrocytes show various patterns of Ca(2+) mobilization under pathological conditions. In the present study we revealed a novel function of astrocytic Ca(2+) dynamics through investigation of thrombin-induced unique Ca(2+) 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 Ca(2+) concentration (Ca(2+)) showed that thrombin induced heterogeneous Ca(2+) responses with asynchronous repetitive peaks. These oscillations were found to be a result of repetitive Ca(2+) release from intracellular stores, followed by refilling of Ca(2+) from the extracellular region without a direct Ca(2+) increase. Pharmacological manipulation with BAPTA-AM, cyclopiazonic acid, and 2-aminoethoxydiphenyl borate indicated that Ca(2+) mobilization was involved in thrombin-induced morphological changes. We further addressed the role of Ca(2+) entry using small interfering RNA (siRNA) for transient receptor potential canonical 3 (TRPC3). As a result, both thrombin-induced morphological changes and oscillatory Ca(2+) responses were significantly attenuated in siRNA-transfected cells. Inhibition of TRPC3 with pyrazole-3 also provided support for the contribution of Ca(2+) influx. Moreover, TRPC3-mediated Ca(2+) dynamics regulated thrombin-induced phosphorylation of myosin light chain 2. These results suggest a novel function of astrocytic Ca(2+) dynamics, including Ca(2+) entry, in the pathophysiological effects of PAR-1-mediated astrocytic activation. TRPC3 forms a functional Ca(2+) channel and might modulate astrocytic activation in response to brain hemorrhaging.
在病理条件下，活化的星形胶质细胞表现出多种模式的钙离子动员。在本研究中，我们通过研究凝血酶诱导的独特钙离子内流，揭示了星形胶质细胞钙离子动态变化的一种新功能。使用1321N1人星形细胞瘤细胞，该细胞已被证明是检测形态动力学的良好模型，我们观察到双极突起的快速回缩，这是由0.03 - 3 U/mL凝血酶可逆性诱发的。形态学变化主要依赖于特定的凝血酶受体亚型，即蛋白酶激活受体1（PAR - 1）。同时，Fura - 2成像检测细胞内钙离子浓度（[Ca²⁺]i）显示，凝血酶诱导了具有异步重复峰值的异质性钙离子反应。这些振荡被发现是细胞内储存库中钙离子重复释放的结果，随后细胞外区域的钙离子重新填充，而细胞内[Ca²⁺]i没有直接增加。用BAPTA - AM、环匹阿尼酸和2 - 氨基乙氧基二苯硼酸进行药理操作表明，钙离子动员参与了凝血酶诱导的形态学变化。我们进一步使用针对瞬时受体电位阳离子通道亚家族C成员3（TRPC3）的小干扰RNA（siRNA）来研究钙离子内流的作用。结果，在转染siRNA的细胞中，凝血酶诱导的形态学变化和振荡性钙离子反应均显著减弱。用吡唑 - 3抑制TRPC3也为钙离子内流的作用提供了支持。此外，TRPC3介导的钙离子动态变化调节了凝血酶诱导的肌球蛋白轻链2的磷酸化。这些结果表明，星形胶质细胞钙离子动态变化，包括钙离子内流，在PAR - 1介导的星形胶质细胞激活的病理生理效应中具有新功能。TRPC3形成功能性钙离子通道，并可能在脑内出血时调节星形胶质细胞的激活。
