Clarification of xenobiotic recognition mechanism at the blood-placentabarrier by transporter array

利用大鼠合体滋养层细胞系tr-TBT 18d-1，阐明了核苷在血-胎盘屏障的摄取机制。Tr-TBT 18d-1对[~3H]尿苷和[~H]腺苷的初始摄取不依赖于钠，对硝基-苄基硫代肌苷敏感。腺苷摄取的Km值约为17μM。这些结果提示平衡的核苷转运体ENT1和ENT2参与其中。几种核苷类药物，包括齐多夫定(AZT)、阿糖胞苷、长春花碱、咪唑立宾和咖啡因，显著减少了摄取。在治疗浓度范围内，效果不明显。因此，在治疗浓度下，这些药物可能对母体到胎儿的核苷转移几乎没有影响。并对AZT的摄取机制进行了表征。AZT的初始摄取是不依赖于钠且可饱和的(Km，约.16μM)。胸腺嘧啶核苷和2‘-脱氧尿嘧啶核苷对摄取有强烈的抑制作用，而N-苄基硫代肌苷、丙磺舒则抑制…西咪替丁用量增加对摄取影响不大。环孢菌素A、Ko143和丙磺舒对AZT的稳态蓄积影响不大，提示转运体介导的AZT外排作用不明显。这些结果表明，tr-TBT 18d-1对AZT的饱和摄取是由一种未知的转运体介导的。人肠细胞模型Caco-2细胞表现出对雌酮-3-硫酸酯的摄取，但具体的转运体尚未明确。我们已经检查了OATP2B1与其同源基因在摄取中的贡献。在Caco-2细胞和人空肠组织中，OATP2B1mRNA的表达高于OATP3A1和OATP4A1。Caco-2细胞和HEK293/OATP2B1细胞对雌酮-3-硫酸酯的摄取量与OATP2B1的表达水平基本一致。OATP2B1的比活性明显高于OATP3A1和OATP4A1。这些结果表明，OATP2B1是Caco-2细胞摄取的主要原因。这种方法可以用来确定胎盘屏障中每个转运蛋白的贡献。较少

The uptake mechanisms of nucleosides at the blood-placenta barrier were clarified by using the rat syncytiotrophoblast cell line TR-TBT 18d-1. The initial uptake of [3H]uridine and [3H]adenosine by TR-TBT 18d-1 were sodium-independent and were sensitive to nitrobenzylthioinosine. The Km value of adenosine uptake was approximately 17 μM. These results suggested involvement of equilibrative nucleoside transporters ENT1 and ENT2. The uptakes were significantly reduced by several nucleoside drugs, including zidovudine (AZT), cytarabine, vidarabine, mizoribine and caffeine. The effects were small within the therapeutic concentration ranges. Therefore, these drugs at the therapeutic concentrations might have little influence on maternal-to-fetal nucleoside transfer. The uptake mechanism of AZT was characterized. Initial uptake of AZT was sodium-independent and saturable (Km, approx. 16 μM). Thymidine and 2' -deoxyuridine strongly inhibited the uptake, but nitrobenzylthioinosine, probenecid a … More nd cimetidine had little effect on the uptake. Cyclosporin A, Ko143 and probenecid had little effect on the steady state AZT accumulation, suggesting that transporter-mediated efflux of AZT is not substantial. These results indicate that the saturable AZT uptake by TR-TBT 18d-1 was mediated by a so-far unidentified transporter.A human enterocyte model Caco-2 cell exhibits estrone-3-sulfate uptake, but the responsible transporters have not been clarified, yet. We have examined the contribution of OATP2B1 to the uptake in comparison with that of its homolog genes. The OATP2B1 mRNA expression was higher than that of OATP3A1 or OATP4A1 in Caco-2 and in human jejunum biopsies. The estrone-3-sulfate uptakes normalized to OATP2B1 mRNA expression were similar in Caco-2 cells and HEK293/OATP2B1 cells. The specific activity of OATP2B1 per mRNA expression was much higher than that of OATP3A1 and OATP4A1. These results suggest that OATP2B1 is predominantly responsible for the uptake in Caco-2 cells. This method can be applied to determine the contribution of each transporter in the placenta barrier. Less