Graduate Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada.
J Pharmacol Exp Ther. 2012 Jan;340(1):96-108. doi: 10.1124/jpet.111.186916. Epub 2011 Oct 11.
The blood-testis barrier (BTB), composed primarily of Sertoli cells, is responsible for protecting developing germ cells from xenobiotic exposure. ATP-binding cassette (ABC) membrane-associated drug efflux transporters, P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and the multidrug resistance-associated proteins (Mrps), have been shown to restrict antiretroviral drug permeability at blood-tissue barriers such as the blood-brain barrier. However, it remains unclear whether these transporters are functional at the level of Sertoli cells and can regulate anti-HIV drug permeability at the BTB. This study investigated the functional expression of ABC transporters in a mouse Sertoli cell line system (TM4) and in primary cultures of human Sertoli cells (HSECs). Expression of multidrug resistance Mdr1a/1b/MDR1/P-gp, Mrp1/MRP1, and Mrp4/MRP4 is confirmed by quantitative polymerase chain reaction and immunoblotting analysis in TM4 cells and HSECs. Immunofluorescence studies revealed plasma membrane localization of P-gp, Mrp1/MRP1, and Mrp4/MRP4 in both cell systems. However, Bcrp expression and localization was only detected in rodent cells. Accumulation of 1) rhodamine-6G (R-6G), a fluorescent P-gp substrate, 2) [³H]atazanavir, a HIV protease inhibitor and known P-gp substrate, 3) 2'7'-bis-(2-carboxyethyl)-5-(and-6)carboxyfluorescein (BCECF), a fluorescent Mrp substrate, and 4) [³H]mitoxantrone, a BCRP substrate, by TM4 monolayer cells in the presence of established inhibitors demonstrates that these transporters are functional. In addition, several anti-HIV drugs significantly enhance the accumulation of R-6G, [³H]atazanavir, BCECF, and [³H]mitoxantrone by TM4 cells. This study provides the first evidence of ABC transporter expression and activity in Sertoli cells and suggests that these transporters could play an important role in restricting antiretroviral drug permeability at the BTB.
血睾屏障（BTB）主要由支持细胞组成，负责保护发育中的生殖细胞免受外源性物质的暴露。已证实，三磷酸腺苷结合盒（ABC）膜相关药物外排转运体，如 P 糖蛋白（P-gp）、乳腺癌耐药蛋白（BCRP）和多药耐药相关蛋白（Mrps），可限制如血脑屏障等血组织屏障中抗逆转录病毒药物的通透性。然而，这些转运体在支持细胞水平上是否具有功能，以及能否调节 BTB 处抗 HIV 药物的通透性尚不清楚。本研究在小鼠支持细胞系（TM4）和人支持细胞原代培养物（HSECs）中研究了 ABC 转运体的功能表达。通过定量聚合酶链反应和免疫印迹分析，在 TM4 细胞和 HSECs 中证实了多药耐药 Mdr1a/1b/MDR1/P-gp、Mrp1/MRP1 和 Mrp4/MRP4 的表达。免疫荧光研究显示，P-gp、Mrp1/MRP1 和 Mrp4/MRP4 均定位于两种细胞系的质膜上。然而，BCRP 的表达和定位仅在啮齿动物细胞中检测到。在 TM4 单层细胞中，荧光 P-gp 底物 1）罗丹明-6G（R-6G）、2）HIV 蛋白酶抑制剂[³H]阿扎那韦和已知的 P-gp 底物、3）荧光 Mrp 底物 2'7'-双（2-羧乙基）-5-（和-6）羧基荧光素（BCECF）和 4）BCRP 底物[³H]米托蒽醌的积累，在建立的抑制剂存在下，证明了这些转运体具有功能。此外，几种抗 HIV 药物显著增加了 TM4 细胞中 R-6G、[³H]阿扎那韦、BCECF 和[³H]米托蒽醌的积累。本研究首次提供了支持细胞中 ABC 转运体表达和活性的证据，并表明这些转运体可能在限制 BTB 处抗逆转录病毒药物的通透性方面发挥重要作用。
