Localization of multidrug resistance-associated protein 2 in the nonpigmented ciliary epithelium of the eye

Ryan M. Pelis, Mohammad - Shahidullah, Sikha Ghosh, Miguel Coca-Prados, Stephen Wright, Nicholas A Delamere

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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-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 methylfluores-cein was increased by 50 μM MK571 (4.3-fold), 500 μM in-domethacin (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.

Original languageEnglish (US)
Pages (from-to)479-485
Number of pages7
JournalJournal of Pharmacology and Experimental Therapeutics
Volume329
Issue number2
DOIs
StatePublished - May 2009

Fingerprint

Epithelium
Ciliary Body
Swine
Western Blotting
Sulfinpyrazone
Blood-Aqueous Barrier
multidrug resistance-associated protein 2
Proteins
Membranes
Aqueous Humor
Vincristine
Etoposide
Reverse Transcriptase Polymerase Chain Reaction
Human Body
Population Groups
Methotrexate
Pharmaceutical Preparations
Doxorubicin
Cyclosporine
Glutathione

ASJC Scopus subject areas

  • Pharmacology
  • Molecular Medicine
  • Medicine(all)

Cite this

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title = "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-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 methylfluores-cein was increased by 50 μM MK571 (4.3-fold), 500 μM in-domethacin (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.",
author = "Pelis, {Ryan M.} and Shahidullah, {Mohammad -} and Sikha Ghosh and Miguel Coca-Prados and Stephen Wright and Delamere, {Nicholas A}",
year = "2009",
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T1 - Localization of multidrug resistance-associated protein 2 in the nonpigmented ciliary epithelium of the eye

AU - Pelis, Ryan M.

AU - Shahidullah, Mohammad -

AU - Ghosh, Sikha

AU - Coca-Prados, Miguel

AU - Wright, Stephen

AU - Delamere, Nicholas A

PY - 2009/5

Y1 - 2009/5

N2 - 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 methylfluores-cein was increased by 50 μM MK571 (4.3-fold), 500 μM in-domethacin (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.

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