Uemura Takuji, Green Marty, Warsh Jerry J
Laboratory of Cellular and Molecular Pathophysiology, Campbell Family Mental Health Research Institute, Centre for Addiction and Mental Health, Toronto, ON, Canada.
Department of Psychiatry, University of Toronto, Toronto, ON, Canada.
Bipolar Disord. 2016 Nov;18(7):549-562. doi: 10.1111/bdi.12447.
Transient receptor potential canonical type 3 (TRPC3) channels are activated in B lymphoblast cell lines from patients with bipolar disorder (BD), and its expression is reduced by chronic lithium treatment, implicating TRPC3 in the intracellular calcium (Ca ) dyshomeostasis of BD. Thrombin, via a protease-activated receptor, moderates Ca signaling and TRPC3 in astrocytes, and also cell proliferation. We examined whether lithium pretreatment attenuates thrombin-stimulated TRPC3 expression and function in astrocytes, and levels of the calcium-binding peptide, S100B, which is expressed mainly in these cells.
Human astroglioma, U-87MG, cells were pretreated with 1 mmol L LiCl for 1 day (acute), 3 days (subacute), and 7 days (chronic). To examine the role of TRPC3, genetically stable knockdown TRPC3 cells (TRPC3 cells) were constructed using U-87MG cells. Thrombin (2.0 U/mL)-stimulated Ca mobilization was measured by ratiometric fluorimetry. Changes in TRPC3 and S100B expression levels were determined by quantitative reverse transcription-polymerase chain reaction and immunoblotting, respectively. Cell proliferation was also measured using the WST-8 assay.
In this cell model, thrombin-stimulated Ca mobilization, and both TRPC3 and S100B expression were suppressed by chronic LiCl pretreatment and the knockdown of TRPC3. Additionally, cell proliferation was attenuated in TRPC3 cells, compared with the negative control vector-transfected cell.
The reduced Ca mobilization and S100B expression levels following chronic LiCl pretreatment and in TRPC3 cells support the notion that TRPC3 modulates S100B expression and is the target of the LiCl effect. Downregulation of TRPC3 may be an important mechanism by which lithium ameliorates pathophysiological intracellular Ca disturbances as observed in BD, accounting, in part, for its mood-stabilizing effects.
瞬时受体电位香草酸亚家族3型（TRPC3）通道在双相情感障碍（BD）患者的B淋巴母细胞系中被激活，慢性锂治疗可降低其表达，提示TRPC3与BD患者细胞内钙（Ca）稳态失衡有关。凝血酶通过蛋白酶激活受体调节星形胶质细胞中的Ca信号传导和TRPC3，以及细胞增殖。我们研究了锂预处理是否能减弱凝血酶刺激的星形胶质细胞中TRPC3的表达和功能，以及主要在这些细胞中表达的钙结合肽S100B的水平。
人星形胶质瘤U-87MG细胞分别用1 mmol/L LiCl预处理1天（急性）、3天（亚急性）和7天（慢性）。为了研究TRPC3的作用，使用U-87MG细胞构建了基因稳定敲低TRPC3的细胞（TRPC3细胞）。通过比率荧光法测量凝血酶（2.0 U/mL）刺激的Ca动员。分别通过定量逆转录-聚合酶链反应和免疫印迹法测定TRPC3和S100B表达水平的变化。还使用WST-8测定法测量细胞增殖。
在该细胞模型中，慢性LiCl预处理和TRPC3的敲低抑制了凝血酶刺激的Ca动员以及TRPC3和S100B的表达。此外，与阴性对照载体转染细胞相比，TRPC3细胞中的细胞增殖减弱。
慢性LiCl预处理后以及在TRPC3细胞中Ca动员和S100B表达水平降低，支持了TRPC3调节S100B表达且是LiCl作用靶点的观点。TRPC3的下调可能是锂改善BD中观察到的病理生理细胞内Ca紊乱的重要机制，部分解释了其情绪稳定作用。
