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METABOLISM, TRANSPORT, AND PHARMACOGENOMICS

Localization of Multidrug Resistance-Associated Protein 2 in the Nonpigmented Ciliary Epithelium of the Eye

Departments of Physiology (R.M.P., M.S., S.H.W., N.A.D.) and Ophthalmology (N.A.D.), University of Arizona, College of Medicine, Tucson, Arizona; and Department of Ophthalmology and Visual Sciences, Yale University School of Medicine, New Haven, Connecticut (S.G., M.C.-P.)

The nonpigmented epithelium (NPE) of the ciliary body represents an important component of the blood-aqueous barrier of the eye. Many therapeutic drugs penetrate poorly across the NPE into the aqueous humor of the eye interior. Several of these therapeutic drugs, such as methotrexate, vincristine, and etoposide, are substrates of the multidrug resistance-associated protein 2 (MRP2). Abundant MRP2 protein was detected by Western blot in homogenates of human ciliary body and freshly dissected porcine NPE. In cultured porcine NPE, the intracellular accumulation of the MRP2 substrates calcein (1.8-fold), 5-(and-6)-carboxy-2',7'-dichlorofluorescein (22.1-fold), and doxorubicin (1.9-fold) was significantly increased in the presence of 50 µM MK571 ((E)-3-[[[3-[2-(7-chloro-2-quinolinyl)-ethenyl]phenyl]-[[3-dimethylamino)-3-oxopropyl]thio]methyl]thio]-propanoic acid), an MRP inhibitor. In addition, the intracellular accumulation of the MRP2 substrate glutathione methylfluorescein was increased by 50 µM MK571 (4.3-fold), 500 µM indomethacin (2.6-fold), and 50 µM cyclosporin A (2.1-fold) but not by 500 µM sulfinpyrazone. These data are consistent with MRP2-mediated transport activity in cultured NPE, and MRP2 mRNA (reverse transcriptase-polymerase chain reaction) and protein (Western blot) were detected in the cultured cells. Immunolocalization studies in native human and porcine eyes showed MRP2 protein at the apical interface of the NPE and pigmented cell layers. Close examination of MRP2 immunoreactivity supported the conclusion that MRP2 is localized in the apical membrane of the NPE. MRP2 at the apical membrane of NPE cells may be involved in protecting intraocular tissues from exposure to potentially harmful toxins.

Address correspondence to: Dr. Ryan M. Pelis, Novartis Pharmaceuticals Corporation, Translational Sciences-DMPK, One Health Plaza, East Hanover, NJ 07936-1080. E-mail: ryan.pelis{at}novartis.com