Contribution of organic anion transporter 3 (Slc22a8) to the elimination of p-aminohippuric acid and benzylpenicillin across the blood-brain barrier
Contribution of organic anion transporter 3 (Slc22a8) to the elimination of p-aminohippuric acid and benzylpenicillin across the blood-brain barrier
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DOI：
10.1124/jpet.103.049197
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发表时间：
2003-07-01
影响因子：
3.5
通讯作者：
Sugiyama, Y
中科院分区：
文献类型：
--
作者：
Kikuchi, R;Kusuhara, H;Sugiyama, Y
研究大鼠有机阴离子转运蛋白3（rOat 3; Slc 22 a8）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（RT-PCR）检测脑毛细血管内皮细胞（BCEC）中rOat 1、rOat 2和rOat 3的表达，结果显示rOat 1和rOat 2均不表达，而rOat 3有中等程度的表达。Western blotting进一步证实了rOat 3在BCEC中的表达。免疫组织化学染色显示rOat 3位于BCEC的近腔膜上，可能也位于腔膜上。使用脑外排指数方法评价rOat 3对rOat 3底物对氨基马尿酸（PAH）和苄青霉素（PCG）从大脑穿过BBB进入血液循环的外排的贡献。PAH和PCG从大脑中消除的速率常数分别为0.039和0.043 min（-1），在高底物浓度下消除饱和。考虑到脑中的稀释，消除PAH和PCG的Km值估计分别为168和29 μ M。未标记的PCG和PAH分别以剂量依赖的方式抑制PAH和PCG穿过BBB的外排。PAH对PCG外排的Ki值为106 μ M，PCG对PAH外排的Ki值为58 μ M。这些值与它们的K-m值相当，表明它们在BBB具有相同的外排机制。此外，西咪替丁和普伐他汀，这也是底物和抑制剂的rOat 3，显着抑制PAH和PCG从大脑的流出。这些结果表明，rOat 3负责消除PAH和PCG从大脑通过BBB。
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 K-m values for the elimination of PAH and PCG were estimated to be 168 and 29 muM, respectively. The efflux of PAH and PCG across the BBB was inhibited in a dose-dependent manner by unlabeled PCG and PAH, respectively. The K-i value of PAH for the efflux of PCG was 106 muM and that of PCG for the efflux of PAH was 58 muM. These values were comparable with their K-m 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.