Departments of Pharmaceutics and Obstetrics & Gynecology, Virginia Commonwealth University, Medical College of Virginia Campus, Richmond, Virginia 23298-0533, USA.
Mol Pharm. 2009 Nov-Dec;6(6):1689-702. doi: 10.1021/mp900019z.
Upon exposure to 1-chloro-2,4-dinitrobenzene (CDNB), the human placental tissue forms its glutathione conjugate 2,4-dinitrophenyl-S-glutathione (DNP-SG). The purpose of this study was to investigate the involvement of human placental ATP-binding cassette (ABC) transporters in the efflux of DNP-SG. Placental tissue samples were obtained from pregnant patients undergoing C-section deliveries following normal pregnancies; villous tissue was cultured in suspension, and DNP-SG formation and efflux upon exposure to 100 microM CDNB were measured by HPLC. DNP-SG efflux decreased by 69.1 (+/-11.3)%, 51.1 (+/-5.4)%, 56.7 (+/-8.3)% and 53.6 (+/-10.8)% (p < 0.05) in the presence of 5 mM sodium orthovanadate (ATPase inhibitor), 100 microM MK571 (MRP-inhibitor), 1 mM dipyridamole (BCRP/P-gp/MRP1-inhibitor) and 100 microM verapamil (P-gp/MRP1 inhibitor) respectively, without any change in DNP-SG formation, total tissue glutathione, GSH/GSSG ratio, tissue integrity or tissue viability. These data clearly established the role of ABC transporters in the human placental efflux of DNP-SG. To investigate the contribution of various ABC transporters toward DNP-SG transport, ATP-dependent transport of 3H-DNP-SG was determined in Sf9 membrane vesicles overexpressing P-gp, BCRP and the MRP proteins. MRP1-mediated DNP-SG transport was inhibited in the presence of sodium orthovanadate, MK571, dipyridamole and verapamil in the presence of glutathione. Furthermore, MRP1-mediated transport [K(t) = 11.3 +/- 1.3 microM and v(max) = 86.7 +/- 1.9 pmol/mg/min] was a high-affinity process compared to MRP2-mediated transport [K(t) = 168 +/- 7 microM and v(max) = 1367 +/- 18 pmol/mg/min]. The inhibition pattern and the kinetics of DNP-SG efflux in the placental villous tissue were consistent with MRP1-mediated DNP-SG efflux, suggesting a functional role and an apical localization for an MRP1-like transporter in the human placental syncytiotrophoblast.
当人体组织接触到 1-氯-2,4-二硝基苯（CDNB）时，会形成其谷胱甘肽结合物 2,4-二硝基苯基-S-谷胱甘肽（DNP-SG）。本研究的目的是探讨人胎盘 ATP 结合盒（ABC）转运蛋白在 DNP-SG 外排中的作用。胎盘组织样本取自正常妊娠剖宫产孕妇；绒毛组织在悬浮液中培养，通过 HPLC 测量暴露于 100μM CDNB 时 DNP-SG 的形成和外排。在存在 5mM 正钒酸钠（ATP 酶抑制剂）、100μM MK571（MRP 抑制剂）、1mM 双嘧达莫（BCRP/P-gp/MRP1 抑制剂）和 100μM 维拉帕米（P-gp/MRP1 抑制剂）时，DNP-SG 外排分别减少了 69.1(+/-11.3)%、51.1(+/-5.4)%、56.7(+/-8.3)%和 53.6(+/-10.8)%（p<0.05），而 DNP-SG 的形成、总组织谷胱甘肽、GSH/GSSG 比值、组织完整性或组织活力均无变化。这些数据清楚地确立了 ABC 转运蛋白在人胎盘 DNP-SG 外排中的作用。为了研究各种 ABC 转运蛋白对 DNP-SG 转运的贡献，在过表达 P-gp、BCRP 和 MRP 蛋白的 Sf9 膜囊泡中测定了 3H-DNP-SG 的 ATP 依赖性转运。在存在谷胱甘肽的情况下，MRP1 介导的 DNP-SG 转运被正钒酸钠、MK571、双嘧达莫和维拉帕米抑制。此外，MRP1 介导的转运[K(t)=11.3(+/-1.3)μM 和 v(max)=86.7(+/-1.9)pmol/mg/min]是一种高亲和力过程，与 MRP2 介导的转运[K(t)=168(+/-7)μM 和 v(max)=1367(+/-18)pmol/mg/min]相比。绒毛组织中 DNP-SG 外排的抑制模式和动力学与 MRP1 介导的 DNP-SG 外排一致，这表明在人胎盘合体滋养层中存在一种功能性的、顶端定位的 MRP1 样转运蛋白。
