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

Abstract

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-2Ј,7Ј-dichlorofluorescein (22.1-fold), and doxorubicin (1.9-fold) was significantly increased in the presence of 50, an MRP inhibitor. In addition, the intracellular accumulation of the MRP2 substrate glutathione methylfluorescein was increased by 50 M MK571 (4.3-fold), 500 M indomethacin (2.6-fold), and 50 M cyclosporin A (2.1-fold) but not by 500 M 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.

Drug Transport by the Blood–Aqueous Humor Barrier of the Eye

“…2). Thus, it is not clear based on this discrepancy The studies by Chen et al (2013), Dahlin et al (2013), Lee et al (2015), Pelis et al (2009) (Fig. 2).…”

“…Several studies have examined mRNA (using reverse-transcription polymerase chain reaction, quantitative polymerase chain reaction, or microarray) expression of ABC drug transporters in isolated human irisciliary body (Zhang et al, 2008;Chen et al, 2013) or in preparations containing only human iris or ciliary body that were isolated separately by microdissection (Pelis et al, 2009;Dahlin et al, 2013;Lee et al, 2015). Table 1 summarizes the findings from these studies.…”

“…Although Pelis et al (2009) detected BCRP in the human ciliary body from a single donor by immunoblotting, another study failed to detect BCRP protein expression in iris-ciliary body from multiple donors (Chen et al, 2013). MRP2 localizes to the apical membrane of nonpigmented epithelial cells of the human pars plana and plicata (Pelis et al, 2009), whereas MRP4 is located in the basolateral membrane of pigmented cells (Lee et al, 2015) (Fig. 2).…”

“…P-gp) showed the highest expression level in the human ciliary body. At the protein level (immunoblotting), MRP1, MRP2, MRP3, MRP4, MRP6, MRP7, and P-gp were detected in the human iris-ciliary body (Chen et al, 2013), and MRP1, MRP2, MRP4, BCRP, and P-gp were detected in the human ciliary body (Pelis et al, 2009) (Fig. 1).…”

A Renal-Like Organic Anion Transport System in the Ciliary Epithelium of the Bovine and Human Eye

“…Samples with RNA integrity number scores above 7.5 were used for expression analysis. RT-PCR was conducted using standard procedures as described previously (Pelis et al, 2009). The oligonucleotide primer sequences used are shown in Table 1.…”