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Jessica Verstraelen, Stephan Reichl; Expression Analysis of ABCC and ABCG Transporters in Different In Vitro and Ex Vivo Cornea Models. Invest. Ophthalmol. Vis. Sci. 2011;52(14):283.
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© ARVO (1962-2015); The Authors (2016-present)
The aim of most drugs for eye treatment is reaching the inner space of the eye indicating that these substances need to pass the cornea. Different pathways are possible to achieve this goal, namely passive paracellular- and transcellular diffusion as well as active transport. In this context the identification and characterization of the different active carrier systems in the cornea is of interest, because the induction or inhibition of these systems can influence drug uptake and drug interactions. A well known group of efflux transporters are multidrug resistance-associated proteins (MRP), classified as ATP-binding cassette (ABC) transporters. ABC proteins represent a large family of integral membrane transporters that utilize the energy of ATP hydrolysis to translocate specific substrates across membranes. In this study the characterization of ABCC and ABCG transporter expression at the protein and activity level of different corneal models was investigated.
A comparison between an immortalized human corneal epithelial cell line (HCE-T) cultivated submerged and at the air-liquid-interface, a previously established human cornea construct, excised human and animal cornea was performed, concerning the mRNA expression of efflux transporters using reverse transcriptase PCR. The presence of protein expression was analyzed using Western blot and immunohistochemistry. The level of activity was determined using a bidirectional permeation assay with specific substrates and inhibitors for each transporter.
The mRNA expression of five efflux transporters (MRP1-3, MRP5 and BCRP) was examined and a similar pattern was obtained between the epithelial corneal cell line, the construct, as well as human and animal cornea. The permeation studies with [H3]Erythromycin and MK571 showed an absence of MRP2 in all models except in the HCE-T model and rabbit cornea. The permeation with 5(6)-Carboxy-2’,7’-dichlorofluorescein diacetate and Probenecid on the other hand showed the expression of MRP1 and MRP5 in the positive control and HCE-T cell line. The immunohistochemical experiments and Western blot confirmed the previous results.
In conclusion, a discrepancy was found between the expression of the efflux transporters at mRNA and protein level, but also between the different cornea models.
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