Ca2+ mobilization mediated by transient receptor potential canonical 3 is associated with thrombin-induced morphological changes in 1321N1 human astrocytoma cells
✍ Scribed by Kenji Nakao; Hisashi Shirakawa; Aiko Sugishita; Ikkei Matsutani; Tetsuhiro Niidome; Takayuki Nakagawa; Shuji Kaneko
- Book ID
- 102909954
- Publisher
- John Wiley and Sons
- Year
- 2008
- Tongue
- English
- Weight
- 544 KB
- Volume
- 86
- Category
- Article
- ISSN
- 0360-4012
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✦ Synopsis
Abstract
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+^]~i~) 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+^]~i~ 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. © 2008 Wiley‐Liss, Inc.