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Molecular evidence and functional expression of multidrug resistance associated protein (MRP) in rabbit corneal
Pradeep K Karla1, Dananjay Pal, Ashim K Mitra
1Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, 5005 Rockhill Road, Kansas City, MO 64110-2499, USA.
Abstract:
Multidrug resistance associated protein (MRP) is a major family of efflux transporters involved in drug efflux leading to drug resistance. The objective of this study was to explore physical barriers for ocular drug absorption and to verify if the role of efflux transporters. MRP-2 is a major homologue of MRP family and found to express on the apical side of cell membrane. Cultured Rabbit Corneal Epithelial Cells (rCEC) were selected as an in vitro model for corneal epithelium. [14C]-erythromycin which is a proven substrate for MRP-2 was selected as a model drug for functional expression studies. MK-571, a known specific and potent inhibitor for MRP-2 was added to inhibit MRP mediated efflux. Membrane fraction of rCEC was used for western blot analysis. Polarized transport of [14C]-erythromycin was observed in rCEC and transport from B-->A was significantly high than from A-->B. Permeability's increased significantly from A-->B in the presence of MK-571 and ketoconozole. Uptake of [14C]-erythromycin in the presence of MK-571 was significantly higher than control in rCEC. RT-PCR analysis indicated a unique and distinct band at approximately 498 bp corresponding to MRP-2 in rCEC and MDCK11-MRP-2 cells. Immunoprecipitation followed by Western Blot analysis indicated a specific band at approximately 190 kDa in membrane fraction of rCEC and MDCK11-MRP-2 cells. For the first time we have demonstrated high expression of MRP-2 in rabbit corneal epithelium and its functional activity causing drug efflux. RT-PCR, immunoprecipitation followed by Western blot analysis further confirms the result.
Insights
Multidrug resistance associated protein 2 (MRP-2) efflux transporters are highly expressed in rabbit corneal epithelial cells, impacting ocular drug absorption. This study confirms MRP-2
Area of Science:
- Ocular Pharmacology
- Drug Transport
- Biochemistry
Background:
- Multidrug resistance associated proteins (MRPs) are key efflux transporters contributing to drug resistance.
- Understanding ocular drug absorption barriers is crucial for effective ophthalmic therapies.
- MRP-2 is a significant MRP family member expressed on apical cell membranes.
Purpose of the Study:
- To investigate physical barriers affecting ocular drug absorption.
- To determine the role of efflux transporters, specifically MRP-2, in ocular drug disposition.
- To validate the expression and function of MRP-2 in rabbit corneal epithelial cells (rCEC).
Main Methods:
- Utilized cultured rCEC as an in vitro model for corneal epithelium.
- Employed [14C]-erythromycin as a model drug substrate for MRP-2.
- Applied MK-571, an MRP-2 inhibitor, to assess transporter-mediated efflux.
- Performed Western blot analysis on rCEC membrane fractions.
- Conducted RT-PCR and immunoprecipitation assays to confirm MRP-2 expression.
Main Results:
- Demonstrated polarized transport of [14C]-erythromycin in rCEC, with higher efflux from basal to apical (B-->A) than apical to basal (A-->B).
- Observed a significant increase in permeability from A-->B in the presence of MK-571 and ketoconazole.
- Showed significantly higher uptake of [14C]-erythromycin with MK-571 compared to control.
- RT-PCR confirmed MRP-2 expression at approximately 498 bp in rCEC.
- Western blot analysis identified MRP-2 at approximately 190 kDa in rCEC membrane fractions.
Conclusions:
- This study provides the first evidence of high MRP-2 expression in rabbit corneal epithelium.
- Functional activity of MRP-2 was confirmed, demonstrating its role in causing drug efflux across the corneal barrier.
- Molecular techniques (RT-PCR, immunoprecipitation, Western blot) robustly support the presence and activity of MRP-2 in rCEC.
