Kikuchi Ryota, Kusuhara Hiroyuki, Sugiyama Daisuke, Sugiyama Yuichi
Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan.
J Pharmacol Exp Ther. 2003 Jul;306(1):51-8. doi: 10.1124/jpet.103.049197. Epub 2003 Apr 8.
The role of rat organic anion transporter 3 (rOat3; Slc22a8) in the efflux transport at the blood-brain barrier (BBB) was characterized. The expression of rOat1, rOat2, and rOat3 in the brain capillary endothelial cells (BCEC) was examined using reverse transcription-polymerase chain reaction analysis, which showed that there was no expression of rOat1 or rOat2, but moderate expression of rOat3. The expression of rOat3 in the BCEC was further confirmed by Western blotting. Immunohistochemical staining showed that rOat3 is located on the abluminal and, possibly, luminal membrane of the BCEC. The contribution of rOat3 to the efflux of para-aminohippuric acid (PAH) and benzylpenicillin (PCG), substrates of rOat3, from the cerebrum into the blood circulation across the BBB was evaluated using the Brain Efflux Index method. PAH and PCG were eliminated from the cerebrum with rate constants of 0.039 and 0.043 min-1, respectively, and the elimination was saturated at high substrate concentrations. Taking account of the dilution in the brain, the Km values for the elimination of PAH and PCG were estimated to be 168 and 29 micro M, respectively. The efflux of PAH and PCG across the BBB was inhibited in a dose-dependent manner by unlabeled PCG and PAH, respectively. The Ki value of PAH for the efflux of PCG was 106 micro M and that of PCG for the efflux of PAH was 58 micro M. These values were comparable with their Km values, suggesting that they share the same efflux mechanism at the BBB. Furthermore, cimetidine and pravastatin, which are also substrates and inhibitors of rOat3, significantly inhibited the efflux of PAH and PCG from the cerebrum. These results suggest that rOat3 is responsible for the elimination of PAH and PCG from the brain across the BBB.
研究了大鼠有机阴离子转运体3（rOat3；Slc22a8）在血脑屏障（BBB）外排转运中的作用。采用逆转录-聚合酶链反应分析检测脑毛细血管内皮细胞（BCEC）中rOat1、rOat2和rOat3的表达，结果显示rOat1或rOat2无表达，但rOat3有中度表达。蛋白质印迹法进一步证实了BCEC中rOat3的表达。免疫组织化学染色显示rOat3位于BCEC的近腔面以及可能的腔面膜上。采用脑外排指数法评估rOat3对其底物对氨基马尿酸（PAH）和苄青霉素（PCG）从大脑经BBB进入血液循环的外排作用。PAH和PCG从大脑中消除的速率常数分别为0.039和0.043 min-1，且在高底物浓度下消除达到饱和。考虑到大脑中的稀释情况，PAH和PCG消除的Km值估计分别为168和29 μM。未标记的PCG和PAH分别以剂量依赖方式抑制PAH和PCG经BBB的外排。PAH对PCG外排的Ki值为106 μM，PCG对PAH外排的Ki值为58 μM。这些值与其Km值相当，表明它们在BBB处共享相同的外排机制。此外，也是rOat3底物和抑制剂的西咪替丁和普伐他汀显著抑制了PAH和PCG从大脑的外排。这些结果表明rOat3负责PAH和PCG经BBB从大脑中的消除。
