Thrombin decreases expression of the glutamate transporter GLAST and inhibits glutamate uptake in primary cortical astrocytes via the Rho kinase pathway.
Thrombin decreases expression of the glutamate transporter GLAST and inhibits glutamate uptake in primary cortical astrocytes via the Rho kinase pathway.
复制标题
凝血酶可降低谷氨酸转运蛋白 GLAST 的表达，并通过 Rho 激酶途径抑制原代皮质星形胶质细胞中谷氨酸的摄取。
DOI：
10.1016/j.expneurol.2015.09.009
复制
发表时间：
2015
影响因子：
5.3
通讯作者：
Wainwright,MarkS
中科院分区：
文献类型：
--
作者：
Piao,Chunshu;RalayRanaivo,Hantamalala;Rusie,Allison;Wadhwani,Nitin;Koh,Sookyong;Wainwright,MarkS
星形胶质细胞谷氨酸转运体GLAST和GLT1在调节神经元兴奋中起关键作用，在癫痫患者和创伤性脑损伤后其水平发生改变。调节它们表达的机制还不是很清楚。我们测试了一种假设，即星形胶质细胞暴露于高水平的凝血酶，就像血脑屏障受损后可能发生的那样，会降低星形胶质细胞谷氨酸转运体的水平。在分离的大鼠皮质星形胶质细胞中，我们通过Western blotting和选择性抑制物检测凝血酶对谷氨酸转运体表达和功能的影响，以及参与这些反应的信号通路。凝血酶诱导GLAST的表达选择性降低，但不引起GLT1的表达，星形胶质细胞摄取谷氨酸的能力相应降低。用活化肽激活凝血酶受体PAR-1可引起GLAST表达的类似下降和谷氨酸摄取的损害。凝血酶诱导的GLAST下调是由丝裂原活化蛋白激酶p38MAPK、ERK和JNK介导的，但抑制这些激酶并不能阻止凝血酶诱导的谷氨酸摄取的减少。相反，使用特异性抑制剂Y27632抑制Rho激酶通路，既抑制了GLAST表达的减少，也抑制了凝血酶诱导的谷氨酸摄取的减少。在海马星形胶质细胞培养中，凝血酶引起GLAST和GLT1的降低。在顽固性癫痫儿童的脑组织中，我们发现血脑屏障的完整性降低，两种转运蛋白的免疫反应性降低，这与裂解凝血酶和反应性星形胶质细胞增多有关。体外实验结果表明，凝血酶可能导致星形胶质细胞功能受损和血脑屏障受损后突触兴奋性增强的特定机制。人类活体实验结果提供了间接的支持证据，将血脑屏障的破坏与凝血酶诱导的谷氨酸转运体表达减少和神经元兴奋增加联系起来。
Astrocyte glutamate transporters GLAST and GLT1 play a key role in regulating neuronal excitation and their levels are altered in patients with epilepsy, and after traumatic brain injury. The mechanisms which regulate their expression are not well understood. We tested the hypothesis that exposure of astrocytes to high levels of thrombin, as may occur after a compromise of the blood–brain barrier, would reduce astrocyte glutamate transporter levels. In isolated rat cortical astrocytes we examined the effects of thrombin on the expression and function of glutamate transporters, and the signaling pathways involved in these responses by using Western blotting and selective inhibitors. Thrombin induced a selective decrease in the expression of GLAST but not GLT1, with a corresponding decrease in the capacity of astrocytes to take up glutamate. Activation of the thrombin receptor PAR-1 with an activating peptide induced a similar decrease in the expression of GLAST and compromise of glutamate uptake. The downregulation of GLAST induced by thrombin was mediated by the mitogen activated protein kinases p38 MAPK, ERK and JNK, but inhibition of these kinases did not prevent the decrease in glutamate uptake induced by thrombin. In contrast, inhibition of the Rho kinase pathway using the specific inhibitor, Y27632, suppressed both the decrease in the expression of GLAST and the decrease in glutamate uptake induced by thrombin. In hippocampal astrocyte cultures, thrombin caused a decrease in both GLAST and GLT1. In tissue resected from brains of children with intractable epilepsy, we found a decrease in the integrity of the blood–brain barrier along with a reduction in immunoreactivity for both transporters which was associated with an increase in cleaved thrombin and reactive astrogliosis. Thein vitroresults suggest a specific mechanism by which thrombin may lead to a compromise of astrocyte function and enhanced synaptic excitability after the blood–brain barrier is compromised. The humanin vivoresults provide indirect support evidence linking the compromise of the blood–brain barrier to thrombin-induced reduction in glutamate transporter expression and an increase in neuronal excitation.