Hoffmann Katrin, Löscher Wolfgang
Department of Pharmacology, Toxicology, and Pharmacy, University of Veterinary Medicine, Hannover, Germany.
Epilepsia. 2007 Apr;48(4):631-45. doi: 10.1111/j.1528-1167.2006.00939.x.
The multidrug resistance protein 2 (MRP2) is a drug efflux transporter that is expressed predominantly at the apical domain of hepatocytes but seems also to be expressed at the apical membrane of brain capillary endothelial cells that form the blood-brain barrier (BBB). MRP2 is absent in the transport-deficient (TR(-)) Wistar rat mutant, so that this rat strain was very helpful in defining substrates of MRP2 by comparing tissue concentrations or functional activities of compounds in MRP2-deficient rats with those in transport-competent Wistar rats. By using this strategy to study the involvement of MRP2 in brain access of antiepileptic drugs (AEDs), we recently reported that phenytoin is a substrate for MRP2 in the BBB. However, one drawback of such studies in genetically deficient rats is the fact that compensatory changes with upregulation of other transporters can occur. This prompted us to study the brain expression of P-glycoprotein (Pgp), a major drug efflux transporter in many tissues, including the BBB, in TR(-) rats compared with nonmutant (wild-type) Wistar rats.
The expression of MRP2 and Pgp in brain and liver sections of TR(-) rats and normal Wistar rats was determined with immunohistochemistry, by using a novel, highly selective monoclonal MRP2 antibody and the monoclonal Pgp antibody C219, respectively.
Immunofluorescence staining with the MRP2 antibody was found to label a high number of microvessels throughout the brain in normal Wistar rats, whereas such labeling was absent in TR(-) rats. TR(-) rats exhibited a significant up-regulation of Pgp in brain capillary endothelial cells compared with wild-type controls. No such obvious upregulation of Pgp was observed in liver sections. A comparable overexpression of Pgp in the BBB was obtained after pilocarpine-induced seizures in wild-type Wistar rats. Experiments with systemic administration of the Pgp substrate phenobarbital and the selective Pgp inhibitor tariquidar in TR(-) rats substantiated that Pgp is functional and compensates for the lack of MRP2 in the BBB.
The data on TR(-) rats indicate that Pgp plays an important role in the compensation of MRP2 deficiency in the BBB. Because such a compensatory mechanism most likely occurs to reduce injury to the brain from cytotoxic compounds, the present data substantiate the concept that MRP2 performs a protective role in the BBB. Furthermore, our data suggest that TR(-) rats are an interesting tool to study consequences of overexpression of Pgp in the BBB on access of drugs in the brain, without the need of inducing seizures or other Pgp-enhancing events for this purpose.
多药耐药蛋白2（MRP2）是一种药物外排转运体，主要表达于肝细胞的顶端结构域，但似乎也表达于构成血脑屏障（BBB）的脑毛细血管内皮细胞的顶端膜。在转运缺陷型（TR(-)）Wistar大鼠突变体中不存在MRP2，因此通过比较MRP2缺陷型大鼠与具有转运功能的Wistar大鼠中化合物的组织浓度或功能活性，该大鼠品系对于确定MRP2的底物非常有帮助。通过使用这种策略来研究MRP2在抗癫痫药物（AEDs）脑内摄取中的作用，我们最近报道苯妥英是血脑屏障中MRP2的一种底物。然而，在基因缺陷型大鼠中进行此类研究的一个缺点是可能会发生其他转运体上调的代偿性变化。这促使我们研究TR(-)大鼠与非突变（野生型）Wistar大鼠相比，P-糖蛋白（Pgp）在脑内的表达情况，Pgp是包括血脑屏障在内的许多组织中的一种主要药物外排转运体。
分别使用一种新型、高度选择性的单克隆MRP2抗体和单克隆Pgp抗体C219，通过免疫组织化学法测定TR(-)大鼠和正常Wistar大鼠脑和肝切片中MRP2和Pgp的表达。
发现用MRP2抗体进行免疫荧光染色可标记正常Wistar大鼠全脑的大量微血管，而TR(-)大鼠中则没有这种标记。与野生型对照相比，TR(-)大鼠脑毛细血管内皮细胞中Pgp显著上调。在肝切片中未观察到Pgp有如此明显的上调。在野生型Wistar大鼠中，毛果芸香碱诱发癫痫后，血脑屏障中也出现了类似的Pgp过表达。在TR(-)大鼠中全身给予Pgp底物苯巴比妥和选择性Pgp抑制剂他林洛尔的实验证实，Pgp具有功能并可代偿血脑屏障中MRP2的缺失。
关于TR(-)大鼠的数据表明，Pgp在血脑屏障中对MRP2缺陷的代偿中起重要作用。由于这种代偿机制很可能是为了减少细胞毒性化合物对脑的损伤，目前的数据证实了MRP2在血脑屏障中发挥保护作用的概念。此外，我们的数据表明，TR(-)大鼠是一种有趣的工具，可用于研究血脑屏障中Pgp过表达对脑内药物摄取的影响，而无需为此目的诱发癫痫或其他增强Pgp的事件。
