ALTERED PGP AND MRP TRANSPORTERS IN BLOOD BRAIN BARRIER

血脑屏障（BBB）内转运蛋白的表达和功能的变化已知发生在自然衰老过程中。 在本提案中，年龄对P-糖蛋白（P-gp）和多药耐药相关蛋白（MRP）在形成血脑屏障的脑微血管内皮细胞的表达和功能的影响将进行评估。 P-gp和MRP都是主动将多种药物和大分子转运出细胞的外排转运蛋白。 P-gp和MRP的过度表达是肿瘤细胞产生耐药性的重要原因。 这些相同的外排转运蛋白在正常细胞（例如形成BBB的脑微血管内皮细胞）中的表达被认为起保护作用，防止潜在毒性化合物在脑中的积累。 目前提出的假设是，P-gp和MRP在BBB中的表达和/或功能活性作为年龄的函数而变化。 本研究采用3月龄和18月龄雌性Fisher 344大鼠的脑微血管内皮细胞，具体目的是：1）检测新鲜分离的脑微血管内皮细胞和原代培养的大鼠BMEC单层中P-gp和MRP表达的年龄相关差异;和2）检查在汇合的大鼠BMEC单层中P-gp和MRP探针的细胞积累和跨细胞渗透性的年龄相关差异。 将分别使用定量免疫印迹和RT-PCR技术在蛋白质和RNA水平评价P-gp和MRP的表达。将通过检查原代大鼠BMEC单层中选定P-gp和MRP探针的细胞蓄积和渗透性的双向差异来评估P-gp和MRP在BBB中的功能活性。 考虑到这两种药物外排转运蛋白在BBB中的假定保护作用，P-gp和MRP的表达或功能的年龄相关变化可能导致对损害中枢神经系统功能的内源性因子和外源性物质的易感性增加。

Changes in the expression and function of transport proteins within the blood-brain barrier (BBB) are known to occur during the natural aging process. In the present proposal, the effects of age on the expression and function of P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP) in the brain microvessel endothelial cells that form the BBB will be evaluated. Both P-gp and MRP are efflux transport proteins that actively transport a wide variety of drugs and macromolecules out of the cell. The development of drug resistance in cancer cells has been attributed to over expression of P-gp and MRP. The expression of these same efflux transport proteins in normal cells, such as the brain microvessel endothelial cells that form the BBB, are believed to play a protective role, preventing the accumulation of potentially toxic compounds in the brain. The hypothesis of the current proposal is that the expression and/or functional activity of P-gp and MRP in the BBB changes as a function of age. Using brain microvessel endothelial cells harvested from female Fisher 344 rats at 3, and 18 months of age, the specific aims of the proposal are to: 1) examine age-related differences in the expression of P-gp and MRP in freshly isolated brain microvessel endothelial cells and primary cultured rat BMEC monolayers; and 2) examine age-related differences in the cellular accumulation and transcellular permeability of P-gp and MRP probes in confluent rat BMEC monolayers. Expression of P-gp and MRP will be evaluated at both the protein and RNA level using quantitative immunoblot and RT-PCR techniques, respectively. Functional activity of P-gp and MRP in the BBB will be assessed by examining the cellular accumulation and bi-directional differences in the permeability of selected P-gp and MRP probes in primary rat BMEC monolayers. Given the putative protective role of these two drug efflux transporters in the BBB, age- related alterations in the expression or function of P-gp and MRP could lead to increased susceptibility to endogenous factors and xenobiotics that impair central nervous system function.