Troutman Matthew D, Thakker Dhiren R
Division of Drug Delivery and Disposition, School of Pharmacy, the University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
Pharm Res. 2003 Aug;20(8):1192-9. doi: 10.1023/a:1025096930604.
The purpose of this work was to elucidate transport pathways of the P-glycoprotein (P-gp) substrates rhodamine 123 (R123) and doxorubicin across Caco-2 cells.
Experiments were designed to identify saturable and nonsaturable transport processes and transport barriers for R123 and doxorubicin transport across Caco-2 cells. Confocal laser scanning microscopy (CLSM) imaged R123 transport under normal conditions and in the presence of the P-gp inhibitor, GW918 (used to abolish P-gp-mediated efflux activity).
R123 secretory P(app) (P(app,BA)) showed concentration dependence, whereas R123 absorptive P(app) (P(app,AB)) did not. Inhibition of P-gp efflux revealed that P-gp-mediated efflux had no effect on R123 or doxorubicin P(app,AB), but enhanced R123 and doxorubicin P(app,BA). In calcium-free medium, R123 P(app,AB) increased 15-fold, indicating intercellular junctions are a barrier to R123 absorption. CLSM of R123 fluorescence during absorptive transport under normal conditions and in the presence of GW918 was identical, and was limited to paracellular space, confirming that P-gp is not a barrier to R123 absorption. CLSM revealed that R123 fluorescence during secretory transport under normal conditions and in the presence of GW918 was localized intracellularly and in paracellular space. R123 and doxorubicin uptake across Caco-2 cells basolateral membrane was saturable.
R123 absorptive transport occurs primarily by paracellular route, whereas R123 secretory transport involves influx across BL membrane mediated solely by a saturable process followed by apically directed efflux via P-gp. Doxorubicin utilizes similar transport pathways to cross Caco-2 cells.
本研究旨在阐明P-糖蛋白（P-gp）底物罗丹明123（R123）和阿霉素跨Caco-2细胞的转运途径。
设计实验以确定R123和阿霉素跨Caco-2细胞转运的可饱和与不饱和转运过程及转运屏障。共聚焦激光扫描显微镜（CLSM）对正常条件下以及存在P-gp抑制剂GW918（用于消除P-gp介导的外排活性）时R123的转运进行成像。
R123的分泌表观渗透系数（P(app,BA)）呈浓度依赖性，而R123的吸收表观渗透系数（P(app,AB)）则不然。抑制P-gp外排显示，P-gp介导的外排对R123或阿霉素的P(app,AB)无影响，但增强了R123和阿霉素的P(app,BA)。在无钙培养基中，R123的P(app,AB)增加了15倍，表明细胞间连接是R123吸收的屏障。正常条件下及存在GW918时吸收转运过程中R123荧光的CLSM图像相同，且仅限于细胞旁间隙，证实P-gp不是R123吸收的屏障。CLSM显示，正常条件下及存在GW918时分泌转运过程中R123荧光定位于细胞内和细胞旁间隙。R123和阿霉素跨Caco-2细胞基底外侧膜的摄取是可饱和的。
R123的吸收转运主要通过细胞旁途径发生，而R123的分泌转运涉及仅由一个可饱和过程介导的跨基底外侧膜内流，随后通过P-gp进行顶端方向的外排。阿霉素利用类似的转运途径穿过Caco-2细胞。
