Nuñez-Lumbreras María de Los Ángeles, Castañeda-Cabral José Luis, Valle-Dorado María Guadalupe, Sánchez-Valle Vicente, Orozco-Suárez Sandra, Guevara-Guzmán Rosalinda, Martínez-Juárez Iris, Alonso-Vanegas Mario, Walter Fruzsina, Deli Maria A, Carmona-Cruz Francia, Rocha Luisa
Departamento de Farmacobiología, Centro de Investigación y de Estudios Avanzados, Mexico City, Mexico.
Departamento de Farmacología, Centro de Investigación y de Estudios Avanzados, Mexico City, Mexico.
Front Behav Neurosci. 2021 Jan 20;14:611780. doi: 10.3389/fnbeh.2020.611780. eCollection 2020.
Cannabinoid receptors 1 and 2 (CB1 and CB2, respectively) play an important role in maintaining the integrity of the blood-brain barrier (BBB). On the other hand, BBB dysfunction is a common feature in drug-resistant epilepsy. The focus of the present study was to characterize protein expression levels and Gαi/o protein-induced activation by CB1 and CB2 receptors in the microvascular endothelial cells (MECs) isolated from the brain of patients with drug-resistant mesial temporal lobe epilepsy (DR-MTLE). MECs were isolated from the hippocampus and temporal neocortex of 12 patients with DR-MTLE and 12 non-epileptic autopsies. Immunofluorescence experiments were carried out to determine the localization of CB1 and CB2 receptors in the different cell elements of MECs. Protein expression levels of CB1 and CB2 receptors were determined by Western blot experiments. [S]-GTPγS binding assay was used to evaluate the Gαi/o protein activation induced by specific agonists. Immunofluorescent double-labeling showed that CB1 and CB2 receptors colocalize with tight junction proteins (claudin-5, occludin, and zonula occludens-1), glial fibrillary acidic protein and platelet-derived growth factor receptor-β. These results support that CB1 and CB2 receptors are expressed in the human isolated microvessels fragments consisting of MECs, astrocyte end feet, and pericytes. The hippocampal microvasculature of patients with DR-MTLE presented lower protein expression of CB1 and CB2 receptors (66 and 43%, respectively; < 0.001). However, its Gαi/o protein activation was with high efficiency (CB1, 251%, < 0.0008; CB2, 255%, < 0.0001). Microvasculature of temporal neocortex presented protein overexpression of CB1 and CB2 receptors (35 and 41%, respectively; < 0.01). Their coupled Gαi/o protein activation was with higher efficiency for CB1 receptors (103%, < 0.006), but lower potency ( < 0.004) for CB2 receptors. The present study revealed opposite changes in the protein expression of CB1 and CB2 receptors when hippocampus (diminished expression of CB1 and CB2) and temporal neocortex (increased expression of CB1 and CB2) were compared. However, the exposure to specific CB1 and CB2 agonists results in high efficiency for activation of coupled Gαi/o proteins in the brain microvasculature of patients with DR-MTLE. CB1 and CB2 receptors with high efficiency could represent a therapeutic target to maintain the integrity of the BBB in patients with DR-MTLE.
大麻素受体1和2（分别为CB1和CB2）在维持血脑屏障（BBB）的完整性方面发挥着重要作用。另一方面，BBB功能障碍是耐药性癫痫的一个常见特征。本研究的重点是表征从耐药性内侧颞叶癫痫（DR-MTLE）患者大脑中分离出的微血管内皮细胞（MEC）中CB1和CB2受体的蛋白表达水平以及Gαi/o蛋白诱导的激活情况。从12例DR-MTLE患者和12例非癫痫尸检的海马体和颞叶新皮质中分离出MEC。进行免疫荧光实验以确定CB1和CB2受体在MEC不同细胞成分中的定位。通过蛋白质印迹实验确定CB1和CB2受体的蛋白表达水平。使用[S]-GTPγS结合测定法评估特异性激动剂诱导的Gαi/o蛋白激活。免疫荧光双标记显示CB1和CB2受体与紧密连接蛋白（claudin-5、occludin和小带闭合蛋白-1）、胶质纤维酸性蛋白和血小板衍生生长因子受体-β共定位。这些结果支持CB1和CB2受体在由MEC、星形胶质细胞终足和周细胞组成的人分离微血管片段中表达。DR-MTLE患者的海马微血管呈现出CB1和CB2受体蛋白表达降低（分别为66%和43%；<0.001）。然而，其Gαi/o蛋白激活效率很高（CB1为251%，<0.0008；CB2为255%，<0.0001）。颞叶新皮质微血管呈现出CB1和CB2受体蛋白过表达（分别为35%和41%；<0.01）。它们偶联的Gαi/o蛋白激活对CB1受体效率更高（103%，<0.006），但对CB2受体效力较低（<0.004）。本研究揭示，当比较海马体（CB1和CB2表达减少）和颞叶新皮质（CB1和CB2表达增加）时，CB1和CB2受体的蛋白表达存在相反变化。然而，暴露于特异性CB1和CB2激动剂会导致DR-MTLE患者脑微血管中偶联的Gαi/o蛋白高效激活。高效的CB1和CB2受体可能代表维持DR-MTLE患者BBB完整性的治疗靶点。
