■内皮细胞中细胞质Ca2+的严格控制对于内皮屏障功能的调节是必不可少的。这里,我们研究了电压门控Ca2+(Cav)通道亚基Cavβ3的作用,在调节脑微血管内皮细胞(BMECs)中的Ca2信号传导以及这如何有助于血脑屏障的完整性。
■我们通过Ca2+成像和蛋白质印迹研究了Cavβ3在BMEC中的功能,检查了体外内皮屏障功能和体内血脑屏障的完整性,并使用Cavβ3-/-(Cavβ3缺陷型)小鼠作为对照,评估了小鼠诱导实验性自身免疫性脑脊髓炎后的病程。
■我们鉴定了BMEC中的Cavβ3蛋白,但是电生理记录没有显示出明显的Cav通道活性。在体内,在不存在Cavβ3的情况下,血脑屏障完整性降低.诱导实验性自身免疫性脑脊髓炎后,Cavβ3-/-小鼠表现出更早的疾病发作,加剧了临床残疾和增加的T细胞浸润。体外,Cavβ3-/-BMEC单层的跨内皮阻力低于野生型BMEC单层,并且连接蛋白ZO-1(闭塞带-1)的组织受损。凝血酶刺激肌醇1,4,5-三磷酸依赖性Ca2+释放,其通过MLC(肌球蛋白轻链)的Ca2+依赖性磷酸化促进细胞收缩并增强内皮屏障通透性。这些影响在Cavβ3-/-中比在野生型BMECs中更明显,而在MLCK(MLC激酶)抑制剂ML-7的存在下,差异被消除。Cacnb3cDNA在Cavβ3-/-BMECs中的表达恢复了野生型表型。共免疫沉淀和质谱表明Cavβ3与肌醇1,4,5-三磷酸受体蛋白的关联。
■独立于其作为Cav通道亚基的功能,Cavβ3与肌醇1,4,5-三磷酸受体相互作用,并参与BMEC中细胞质Ca2和Ca2依赖性MLC磷酸化的严格控制,Cavβ3在BMEC中的这种作用有助于血脑屏障的完整性,并减轻实验性自身免疫性脑脊髓炎疾病的严重程度。
UNASSIGNED: Tight control of cytoplasmic Ca2+ concentration in endothelial cells is essential for the regulation of endothelial barrier function. Here, we investigated the role of Cavβ3, a subunit of voltage-gated Ca2+ (Cav) channels, in modulating Ca2+ signaling in brain microvascular endothelial cells (BMECs) and how this contributes to the integrity of the blood-brain barrier.
UNASSIGNED: We investigated the function of Cavβ3 in BMECs by Ca2+ imaging and Western blot, examined the endothelial barrier function in vitro and the integrity of the blood-brain barrier in vivo, and evaluated disease course after induction of experimental autoimmune encephalomyelitis in mice using Cavβ3-/- (Cavβ3-deficient) mice as controls.
UNASSIGNED: We identified Cavβ3 protein in BMECs, but electrophysiological recordings did not reveal significant Cav channel activity. In vivo, blood-brain barrier integrity was reduced in the absence of Cavβ3. After induction of experimental autoimmune encephalomyelitis, Cavβ3-/- mice showed earlier disease onset with exacerbated clinical disability and increased T-cell infiltration. In vitro, the transendothelial resistance of Cavβ3-/- BMEC monolayers was lower than that of wild-type BMEC monolayers, and the organization of the junctional protein ZO-1 (zona occludens-1) was impaired. Thrombin stimulates inositol 1,4,5-trisphosphate-dependent Ca2+ release, which facilitates cell contraction and enhances endothelial barrier permeability via Ca2+-dependent phosphorylation of MLC (myosin light chain). These effects were more pronounced in Cavβ3-/- than in wild-type BMECs, whereas the differences were abolished in the presence of the MLCK (MLC kinase) inhibitor ML-7. Expression of Cacnb3 cDNA in Cavβ3-/- BMECs restored the wild-type phenotype. Coimmunoprecipitation and mass spectrometry demonstrated the association of Cavβ3 with inositol 1,4,5-trisphosphate receptor proteins.
UNASSIGNED: Independent of its function as a subunit of Cav channels, Cavβ3 interacts with the inositol 1,4,5-trisphosphate receptor and is involved in the tight control of cytoplasmic Ca2+ concentration and Ca2+-dependent MLC phosphorylation in BMECs, and this role of Cavβ3 in BMECs contributes to blood-brain barrier integrity and attenuates the severity of experimental autoimmune encephalomyelitis disease.