Ca2+ mobilization mediated by transient receptor potential canonical 3 is associated with thrombin-induced morphological changes in 1321N1 human astrocytoma cells.
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.
Nakao
Kenji
K
Department of Molecular Pharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan.
Shirakawa
Hisashi
H
Sugishita
Aiko
A
Matsutani
Ikkei
I
Niidome
Tetsuhiro
T
Nakagawa
Takayuki
T
Kaneko
Shuji
S
D001254:Astrocytoma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D002118:Calcium / Q000378:metabolism* / Q000502:physiology; D020013:Calcium Signaling / Q000502:physiology; D006801:Humans; D044463:Receptor, PAR-1 / Q000235:genetics / Q000378:metabolism; D050052:TRPC Cation Channels / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D013917:Thrombin / Q000502:physiology*; D014407:Tumor Cells, Cultured
D044463:Receptor, PAR-1; D050052:TRPC Cation Channels; C104603:TRPC3 cation channel; D013917:Thrombin; D002118:Calcium