P-glycoprotein expression in human retinal pigment epithelium

Brian Kennedy, Nancy J. Mangini

Research output: Contribution to journalArticle

84 Citations (Scopus)

Abstract

Purpose: The retinal pigment epithelium (RPE) is a transporting epithelial monolayer that controls hydration and composition of the subretinal space. P-glycoprotein is an ATP-binding cassette transport protein known to transport a wide range of hydrophobic compounds. The expression of P-glycoprotein in barrier epithelial cells suggests that it could serve a normal protective function, possibly clearing potentially harmful substances from sensitive compartments, like the subretinal space. The present study is designed to determine the expression and activity of P-glycoprotein in normal human RPE. Methods: RT-PCR and direct sequencing were employed to examine the presence of mdr1 mRNA in cultured human RPE. P-glycoprotein-specific antibodies were employed in Western blotting to identify P-glycoprotein in cultured human RPE and in an established RPE cell line (D407). Anti-P-glycoprotein antibodies were also used to localize the protein in frozen, formaldehyde-fixed sections of native human RPE/choroid by immunohistochemistry. Finally, rhodamine uptake was performed in cultured human RPE monolayers to assess P-glycoprotein activity. The inhibitory antibody 4E3 and reversins 121 and 205 were used to block transport activity. Results: P-glycoprotein is expressed, and is active, in human RPE tissue not exposed to any known inducers of P-glycoprotein. RT-PCR yielded a 546 bp product that was 100% identical in sequence to published data for the mdr1 isoform of human P-glycoprotein. Western blotting demonstrated expression at the protein level, with specific bands observed at about 220 and 165 kD. In native tissue, P-glycoprotein immunoreactivity was predominantly membrane associated, with localization to both apical and basolateral cell membranes. Finally, P-glycoprotein expressed in human RPE is active. Steady-state rhodamine accumulation was increased in the presence of compounds reported to block P-glycoprotein mediated rhodamine efflux. Conclusions: Human RPE, not exposed to inducer treatment, expresses P-glycoprotein with localization to both apical and basal cell surfaces. Basolateral P-glycoprotein could serve a protective function for the neural retina helping to clear unwanted substances from subretinal space. The finding that P-glycoprotein is also on the apical surface suggests possible additional roles for P-glycoprotein in the RPE.

Original languageEnglish (US)
Pages (from-to)422-430
Number of pages9
JournalMolecular Vision
Volume8
StatePublished - 2002

Fingerprint

Retinal Pigment Epithelium
P-Glycoprotein
Rhodamines
Antibodies
Western Blotting
Polymerase Chain Reaction
Choroid

ASJC Scopus subject areas

  • Ophthalmology

Cite this

P-glycoprotein expression in human retinal pigment epithelium. / Kennedy, Brian; Mangini, Nancy J.

In: Molecular Vision, Vol. 8, 2002, p. 422-430.

Research output: Contribution to journalArticle

@article{2100fb3cbac049c393c577ff61f8a3bd,
title = "P-glycoprotein expression in human retinal pigment epithelium",
abstract = "Purpose: The retinal pigment epithelium (RPE) is a transporting epithelial monolayer that controls hydration and composition of the subretinal space. P-glycoprotein is an ATP-binding cassette transport protein known to transport a wide range of hydrophobic compounds. The expression of P-glycoprotein in barrier epithelial cells suggests that it could serve a normal protective function, possibly clearing potentially harmful substances from sensitive compartments, like the subretinal space. The present study is designed to determine the expression and activity of P-glycoprotein in normal human RPE. Methods: RT-PCR and direct sequencing were employed to examine the presence of mdr1 mRNA in cultured human RPE. P-glycoprotein-specific antibodies were employed in Western blotting to identify P-glycoprotein in cultured human RPE and in an established RPE cell line (D407). Anti-P-glycoprotein antibodies were also used to localize the protein in frozen, formaldehyde-fixed sections of native human RPE/choroid by immunohistochemistry. Finally, rhodamine uptake was performed in cultured human RPE monolayers to assess P-glycoprotein activity. The inhibitory antibody 4E3 and reversins 121 and 205 were used to block transport activity. Results: P-glycoprotein is expressed, and is active, in human RPE tissue not exposed to any known inducers of P-glycoprotein. RT-PCR yielded a 546 bp product that was 100{\%} identical in sequence to published data for the mdr1 isoform of human P-glycoprotein. Western blotting demonstrated expression at the protein level, with specific bands observed at about 220 and 165 kD. In native tissue, P-glycoprotein immunoreactivity was predominantly membrane associated, with localization to both apical and basolateral cell membranes. Finally, P-glycoprotein expressed in human RPE is active. Steady-state rhodamine accumulation was increased in the presence of compounds reported to block P-glycoprotein mediated rhodamine efflux. Conclusions: Human RPE, not exposed to inducer treatment, expresses P-glycoprotein with localization to both apical and basal cell surfaces. Basolateral P-glycoprotein could serve a protective function for the neural retina helping to clear unwanted substances from subretinal space. The finding that P-glycoprotein is also on the apical surface suggests possible additional roles for P-glycoprotein in the RPE.",
author = "Brian Kennedy and Mangini, {Nancy J.}",
year = "2002",
language = "English (US)",
volume = "8",
pages = "422--430",
journal = "Molecular Vision",
issn = "1090-0535",

}

TY - JOUR

T1 - P-glycoprotein expression in human retinal pigment epithelium

AU - Kennedy, Brian

AU - Mangini, Nancy J.

PY - 2002

Y1 - 2002

N2 - Purpose: The retinal pigment epithelium (RPE) is a transporting epithelial monolayer that controls hydration and composition of the subretinal space. P-glycoprotein is an ATP-binding cassette transport protein known to transport a wide range of hydrophobic compounds. The expression of P-glycoprotein in barrier epithelial cells suggests that it could serve a normal protective function, possibly clearing potentially harmful substances from sensitive compartments, like the subretinal space. The present study is designed to determine the expression and activity of P-glycoprotein in normal human RPE. Methods: RT-PCR and direct sequencing were employed to examine the presence of mdr1 mRNA in cultured human RPE. P-glycoprotein-specific antibodies were employed in Western blotting to identify P-glycoprotein in cultured human RPE and in an established RPE cell line (D407). Anti-P-glycoprotein antibodies were also used to localize the protein in frozen, formaldehyde-fixed sections of native human RPE/choroid by immunohistochemistry. Finally, rhodamine uptake was performed in cultured human RPE monolayers to assess P-glycoprotein activity. The inhibitory antibody 4E3 and reversins 121 and 205 were used to block transport activity. Results: P-glycoprotein is expressed, and is active, in human RPE tissue not exposed to any known inducers of P-glycoprotein. RT-PCR yielded a 546 bp product that was 100% identical in sequence to published data for the mdr1 isoform of human P-glycoprotein. Western blotting demonstrated expression at the protein level, with specific bands observed at about 220 and 165 kD. In native tissue, P-glycoprotein immunoreactivity was predominantly membrane associated, with localization to both apical and basolateral cell membranes. Finally, P-glycoprotein expressed in human RPE is active. Steady-state rhodamine accumulation was increased in the presence of compounds reported to block P-glycoprotein mediated rhodamine efflux. Conclusions: Human RPE, not exposed to inducer treatment, expresses P-glycoprotein with localization to both apical and basal cell surfaces. Basolateral P-glycoprotein could serve a protective function for the neural retina helping to clear unwanted substances from subretinal space. The finding that P-glycoprotein is also on the apical surface suggests possible additional roles for P-glycoprotein in the RPE.

AB - Purpose: The retinal pigment epithelium (RPE) is a transporting epithelial monolayer that controls hydration and composition of the subretinal space. P-glycoprotein is an ATP-binding cassette transport protein known to transport a wide range of hydrophobic compounds. The expression of P-glycoprotein in barrier epithelial cells suggests that it could serve a normal protective function, possibly clearing potentially harmful substances from sensitive compartments, like the subretinal space. The present study is designed to determine the expression and activity of P-glycoprotein in normal human RPE. Methods: RT-PCR and direct sequencing were employed to examine the presence of mdr1 mRNA in cultured human RPE. P-glycoprotein-specific antibodies were employed in Western blotting to identify P-glycoprotein in cultured human RPE and in an established RPE cell line (D407). Anti-P-glycoprotein antibodies were also used to localize the protein in frozen, formaldehyde-fixed sections of native human RPE/choroid by immunohistochemistry. Finally, rhodamine uptake was performed in cultured human RPE monolayers to assess P-glycoprotein activity. The inhibitory antibody 4E3 and reversins 121 and 205 were used to block transport activity. Results: P-glycoprotein is expressed, and is active, in human RPE tissue not exposed to any known inducers of P-glycoprotein. RT-PCR yielded a 546 bp product that was 100% identical in sequence to published data for the mdr1 isoform of human P-glycoprotein. Western blotting demonstrated expression at the protein level, with specific bands observed at about 220 and 165 kD. In native tissue, P-glycoprotein immunoreactivity was predominantly membrane associated, with localization to both apical and basolateral cell membranes. Finally, P-glycoprotein expressed in human RPE is active. Steady-state rhodamine accumulation was increased in the presence of compounds reported to block P-glycoprotein mediated rhodamine efflux. Conclusions: Human RPE, not exposed to inducer treatment, expresses P-glycoprotein with localization to both apical and basal cell surfaces. Basolateral P-glycoprotein could serve a protective function for the neural retina helping to clear unwanted substances from subretinal space. The finding that P-glycoprotein is also on the apical surface suggests possible additional roles for P-glycoprotein in the RPE.

UR - http://www.scopus.com/inward/record.url?scp=0036979802&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0036979802&partnerID=8YFLogxK

M3 - Article

VL - 8

SP - 422

EP - 430

JO - Molecular Vision

JF - Molecular Vision

SN - 1090-0535

ER -