P-glycoprotein expression in human retinal pigment epithelium.
OBJECTIVE
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.
Kennedy
Brian G
BG
Northwest Center for Medical Education, Indiana University School of Medicine, Gary, IN 46408, USA. brkenned@iun.edu
Mangini
Nancy J
NJ
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis* / Q000235:genetics; D000328:Adult; D015153:Blotting, Western; D002478:Cells, Cultured; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D008856:Microscopy, Fluorescence; D010857:Pigment Epithelium of Eye / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012235:Rhodamines / Q000378:metabolism
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D012333:RNA, Messenger; D012235:Rhodamines