Materials and methods: Brush border membrane (BBM) was isolated from healthy tissues from six patients (3 male, 3 female; age range 55-81) undergoing intestinal surgery using a well-established method. Expression of OATP2B1 and other intestinal transporters were quantified in BBM and basolateral membrane by mass spectrometry-based targeted proteomics. In parallel, localization of OATP2B1 and the apically expressed P-glycoprotein (P-gp) in Caco-2 cells were characterized by immunofluorescence staining using a confocal laser scanning microscope.
Functionality of OATP2B1 was investigated in freshly exercised human intestine from other patients mounted into an Ussing chamber. The transport of the two OATP2B1 substrates sulfasalazine and pravastatin from the apical to basolateral compartment (a>b) and vice versa (b>a) were investigated in presence or absence of the OATP2B1 inhibitors rifampicin and naringin. Sulfasalazine and pravastatin were measured by a validated LC-MS/MS assay. Moreover, vectorial transport of sulfasalazine and pravastatin were measured in Transwell™ assays using Caco-2 cells.
Results: OATP2B1 and also the multidrug resistance-associated protein 3 (MRP3) and the Na+/K+-ATPase showed a significantly higher enrichment in the basolateral membrane than in the BBM. In contrast, the apically localized transporters PEPT1 and MRP2 were accumulated in the BBM. In line with these findings, basolateral localization of OATP2B1 and apical localization of P-gp could be confirmed in Caco-2 cells. In Ussing chamber experiments, sulfasalazine and pravastatin showed a predominantly b>a transport with efflux ratios (ER = Papp (b>a)/Papp (a>b)) of 7.4 and 8.4, respectively. In presence of the OATP2B1 inhibitors naringin and rifampicin, ERs were considerably reduced to 3.5 and 0.8, respectively. In Caco-2 cells, sulfasalazine and pravastatin showed a similar b>a transport with ERs of 4.7 ± 1.2 and 2.1 ± 0.6. ERs were reduced significantly to 1.3 ± 0.6 and 1.1 ± 0.1 in presence of rifampicin and naringin.
Conclusion: Our data on localization and function of OATP2B1 indicate a basolateral localization of the transporter in both, human jejunum and Caco-2 cells. Thus, intestinal uptake of OATP2B1 substrates might be dependent on other mechanisms like passive diffusion or involvement of so far unknown transporters.