Roles of NMDARs in maintenance of the mouse cerebrovascular endothelial cell-constructed tight junction barrier
Roles of NMDARs in maintenance of the mouse cerebrovascular endothelial cell-constructed tight junction barrier
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DOI：
10.1016/j.tox.2015.11.006
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发表时间：
2016-01-02
期刊：
影响因子：
4.5
通讯作者：
Chen, Ruei-Ming
中科院分区：
文献类型：
--
作者：
Chen, Jui-Tai;Chen, Tyng-Guey;Chen, Ruei-Ming
谷氨酸可激活NMDAR，导致兴奋性毒性神经元丢失。然而，NMDAR在血脑屏障(BBB)中的作用鲜为人知。本研究采用小鼠脑血管内皮细胞(MCEC)模型，探讨NMDAR激活对血脑屏障维持的影响及其可能机制。共聚焦显微镜分析显示，MCECs表达NMDAR亚单位GluN1和GluN2B。免疫印迹分析进一步证明GluN1存在于MCECs的质膜中。在脑组织中，共聚焦显微镜分析显示GluN1和第八因子(MCECs的生物标记物)共定位。此外，在MCECs和脑组织中检测到GluN1基因的表达。功能分析显示，MCECs暴露于NMDA可增加细胞内钙内流。另外，NMDA抑制跨内皮电阻值、闭塞素水平和紧密连接中的闭塞。在机制上，NMDA促进细胞外信号调节激酶(ERK)1/2和丝裂原激活的ERK(MEK)1的顺序磷酸化。有趣的是，NMDA增加了MCECs中基质金属蛋白酶(MMP2)和MMP9的含量。预先给予MEK抑制剂PD98059和NMDAR拮抗剂MK-801可逆转NMDA诱导的ERK1/2磷酸化和阻断素水平的改变。因此，本研究表明NMDAR在MCEC中的功能存在，NMDAR的激活可以通过激活MEK1/2-ERK1/2信号通路和上调MMP2/9的表达来破坏MCEC构建的紧密连接屏障。(C)2015爱思唯尔爱尔兰有限公司。保留所有权利。
Glutamate can activate NMDA receptor (NMDAR) and subsequently induces excitotoxic neuron loss. However, roles of NMDARs in the blood brain barrier (BBB) are little known. This study used a mouse cerebrovascular endothelial cell (MCEC) model to evaluate the effects of NMDAR activation on maintenance of the BBB and its possible mechanisms. Analysis of confocal microscopy revealed expressions of NMDAR subunits, GluN1 and GLUN2B, in MCECs. An immunoblot assay further showed the existence of GluN1 in plasma membranes of MCECs. In brain tissues, a confocal microscopic analysis demonstrated co-localization of GluN1 and factor VIII, a biomarker of MCECs. In addition, GluN1 mRNA was detected in MCECs and the brain. Functional assays showed that exposure of MCECs to NMDA increased calcium influx. Separately, NMDA suppressed transendothelial electrical resistance values, levels of occludin, and occludin tight junctions. As to the mechanism, NMDA stimulated sequential phosphorylations of extracellular signal-regulated kinase (ERK)1/2 and mitogen-activated ERK (MEK)1. Interestingly, amounts of matrix metalloproteinase (MMP)2 and MMP9 in MCECs were augmented by NMDA. The NMDA-induced alterations in ERK1/2 phosphorylation and occludin levels were reversed by pretreatment with PD98059, a MEK inhibitor, and MK-801, a NMDAR antagonist, respectively. Therefore, this study shows the functional presence of NMDARs in MCECs, and NMDAR activation can disrupt the MCEC-constructed tight junction barrier via activation of the MEK1/2-ERK1/2 signaling pathway and upregulation of MMP2/9 expressions. (C) 2015 Elsevier Ireland Ltd. All rights reserved.