Characterization of efflux proteins in human corneal epithelial cells

Abstract

Abstract Purpose: Corneal epithelium is the main barrier for absorption of drugs into intraocular tissues after topical administration and part of this barrier may be formed by efflux proteins which translocate molecules from the cell interior to the extracellular space. The aim of this study was to characterize the gene expression and the activity of the efflux transporters in the cell culture model of immortalized human corneal epithelial cells (HCE cells), in primary cell line (HCEpiC), and in the human corneal epithelium. Methods: The mRNA levels of MDR1, MRP1‐MRP6, and BCRP were determined by the quantitative RT‐PCR. Immunohistochemistry was used to study protein expression and localization of efflux transporters. Functionality of these proteins was assessed with calcein‐AM efflux assay and by measuring the efflux of CDCF. Furthermore, bidirectional permeability of rhodamine 123 (Rh123) was studied. Results: The mRNA of MRP1 and MRP5 were detected in the human cornea and in both cell lines. These efflux proteins were found in the cell membranes of the human corneal epithelium. At mRNA level some efflux proteins were over‐expressed in the HCE and the primary cell lines. Increased calcein retention and decreased CDCF efflux in the presence of inhibitors suggested efflux protein activity in both primary and HCE cells. Likewise, directionality in Rh123 permeability was diminished in the presence of verapamil in HCE model. Conclusions: Functionality of the efflux proteins was demonstrated in the human corneal epithelial cells. MRP1 and MRP5 proteins may have important protecting role in corneal surface by transporting molecules out from the epithelial cells. It seems that the efflux activity in the HCE model differs from that of the corneal epithelium in vivo