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From 1 Clinical Pharmacology and Toxicology, Department of Medicine, and 2 Department of Ophthalmology, University Hospital, Zürich, Switzerland; and 3 Institute of Pharmacology and Toxicology, University of Züurich, Zürich, Switzerland.
| Abstract |
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METHODS. Oatp2 was detected in rat retinal tissue by immunofluorescence confocal microscopy and by Western blot analysis, with a specific antibody. A Xenopus laevis oocyte expression system was used for functional transport studies.
RESULTS. Oatp2 immunoreactivity was abundantly present at the apical microvilli
of the rat retinal pigment epithelium and to a lesser degree in small
retinal vessels. In the oocyte expression system,
N-retinyl-N-retinylidene ethanolamine
(A2E), an unusual cationic, amphiphilic retinoid, exhibited competitive
Cis inhibition of Oatp2-mediated digoxin transport with
an estimated Ki of
37 µM.
CONCLUSIONS. In rat retina, Oatp2 is localized at the interface between the pigment epithelium and the photoreceptor outer segments. A2E is a competitive inhibitor of Oatp2-mediated substrate transport, suggesting that A2E or A2E-like compounds and some retinoids may be substrates for Oatp2 transport.
| Introduction |
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Organic anion-transporting proteins (rat, Oatps; human, OATPs) are a gene family of transmembrane transporters with a rapidly growing number of members.10 These sodium-independent transporters represent polyspecific organic solute carriers for amphiphilic compounds with mostly overlapping and partially distinct substrate specificity.11 So far, identified substrates include endogenous and exogenous compounds, such as bile salts, steroid conjugates, leukotriene C4, thyroid hormones, cardiac glycosides, peptidic drugs, and certain bulky type II organic cations.10 Members of the Oatp/OATP family (gene classification: SLC21A) are widely expressed in many organs, including the liver, kidney, intestine, and brain, where they are localized at distinct membrane domains of several polarized cells. Although the definite physiological significance(s) of Oatps/OATPs remains to be established, their broad substrate spectrum and their heterogenous distribution indicate a functional diversity of these transporters.
Oatp2 (Slc21a5), a polypeptide consisting of 661 amino acids with 12 predicted transmembrane domains, has been cloned by Noe et al.12 and by Abe et al.13 from the rat brain and retina, respectively. Besides sharing substrates with other Oatps (as just described), Oatp2 specifically mediates the high-affinity transport of the cardiovascular drug digoxin.12 It is localized at the luminal and abluminal membranes of the brain capillary endothelium, the basolateral membrane of the choroid plexus epithelium,14 and the sinusoidal membrane of hepatocytes.15 In the retina, however, its exact localization is unknown. In the present study, using immunofluorescence confocal microscopy, Oatp2 was shown to be predominantly located at the apical microvilli of the RPE. Evidence was provided that the amphiphilic molecule A2E may be an Oatp2 substrate, thus opening the possibility that A2E can be transported actively across pigment epithelial cell membranes. Because A2E may be produced within the cells of the RPE itself and therefore may not normally be a substrate for RPE import, it can be hypothesized that A2E-like molecules, especially precursors of A2E may be able to enter the pigment epithelium through Oatp2-mediated transport.
| Methods |
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Animals
Procedures concerning animals were in accordance with the
regulations of the veterinary authority of Zürich and with the
ARVO Statement for Use of Animals in Ophthalmic and Vision Research.
Male (200250 g) Sprague-Dawley rats (SUT:SDT9) were obtained from the
Institute für Labortierkunde, Universtity of Zürich
(Zürich, Switzerland). Mature Xenopus
laevis females were purchased from the African
Xenopus facility at Noor-dusek, Republic of South Africa.
Antibodies
The rabbit polyclonal Oatp2 antibody used in this study was
raised against a synthetic peptide consisting of 15 amino acids at the
C-terminal of Oatp2 and was extensively characterized by Western blot
analysis and immunofluorescence microscopy
previously.14
15
Affinity purification of this antiserum
was performed with N-hydroxy succinimide agarose gel
(Affigel 10; Bio-Rad Laboratories, Hercules, CA) coupled to synthetic
peptides according to the manufacturers instructions. The
Cy3-conjugated goat anti-rabbit and horseradish peroxidaseconjugated
goat anti-rabbit secondary antibodies were purchased from Jackson
ImmunoResearch (West Grove, PA) and Santa Cruz (Santa Cruz, CA),
respectively.
Immunoblot Analysis
For eyecup preparations, lens, vitreous, and retina were removed
through a slit in the cornea. The remaining eyecup was enucleated and
blood and opticus, conjunctiva, and muscle tissue were carefully
removed. After homogenization with an ultrasound tip in 0.1 M Tris (pH
7.5), the homogenate was centrifuged at 1000g for 5 minutes.
Protein content of the supernatant was assessed using the Bradford
assay (Bio-Rad), with BSA serving as the standard. The supernatant was
mixed with SDS sample buffer, heated to 70°C for 10 minutes, and
stored at -20° until use. Basolateral rat liver plasma membrane was
isolated as described before.16
Proteins were separated on
10% SDS-polyacrylamide minigels and transferred to nitrocellulose
membranes. Blots were blocked for 1 hour at room temperature in 10 mM
Tris (pH 8.0), 150 mM NaCl, and 0.05% Tween (TBST) containing 5%
blocker (nonfat dry milk, Bio-Rad) and incubated overnight at 4°C
with the affinity-purified Oatp2 antibody (see above) diluted in TBST
containing 5% blocker. Blots were washed three times with TBST and
incubated with horseradish peroxidaseconjugated goat anti-rabbit IgG
diluted at 1:5000 for 1 hour at room temperature. Immunoreactivity was
visualized with a Western blot detection kit (Renaissance; DuPont NEN).
Immunofluorescence Staining
Rats were decapitated and the eyes were immediately enucleated,
immersed in phosphate-buffered saline (PBS) containing 3%
paraformaldehyde and 0.2% glutaraldehyde (pH 7.4) at 4°C for 2 hours
and cryoprotected in 30% sucrose overnight at 4°C. After carefully
removing the lens, 10-µm cryostat sections were cut and mounted on
glass slides coated with 3-aminopropyltriethoxysilane (Sigma). Sections
were incubated overnight at 4°C with the Oatp2 antibody diluted to
1:5000 (1:200 for the affinity-purified antiserum) in 50 mM Tris and
100 mM NaCl (Tris saline, pH 7.4) containing 2% normal goat serum and
0.05% Triton X-100. Sections were washed three times with Tris saline
and incubated for 30 minutes at room temperature with the
Cy3-conjugated goat anti-rabbit secondary antibody (1:300) diluted in
the same buffer as for the primary antibody incubation. Subsequently,
sections were washed several times with PBS and coverslipped
(Immu-mount; Shandon, Pittsburgh, PA). The specificity of the
immunoreactivity was verified by incubating sections with (1) the
primary antibody preabsorbed with 10 to 20 µg/mL of the corresponding
antigen used for immunization and (2) the secondary antibody in absence
of the primary antibody. Some sections were subjected to
double-labeling experiments with the Oatp2 antibody and
fluorescence-conjugated phalloidin (Alexa Fluor 488; Molecular Probes,
Eugene, OR). Phalloidin was used to label filamentous actin that is
abundantly present in apical microvilli of the RPE. In these
experiments, the phalloidin was added to the incubation buffer
containing secondary antibody at a concentration of 1 U/200 µL
(according to the manufacturers instructions) for 30 minutes. Stained
sections were analyzed by confocal laser microscopy (MRC 600; Bio-Rad
Laboratories).
Transport Assays in X. laevis Oocytes
Oatp2 cRNA was transcribed in vitro from
NotI-linearized cDNA12
using a kit (mMESSAGE
Mmachine T3; Ambion, Austin, TX). Oocytes were prepared and incubated
overnight at 18°C. Healthy oocytes were microinjected with either 50
nL of water (control) or 50 nL of water containing 5 ng Oatp2 cRNA.
After injection, oocytes were cultured for 3 days to allow the
expression of the transporter protein in the plasma membrane. Uptake of
radiolabeled substrates (all-trans-retinol, A2E, and
digoxin) was performed at 25°C for 30 to 40 minutes in 100 µL
Na+-free medium containing 100 mM choline
chloride, 2 mM KCl, 1 mM CaCl2, 1 mM
MgCl2, 10 mM HEPES-Tris, and 1% dimethyl
sulfoxide (DMSO, pH 7.5). Subsequently, oocytes were washed with 3 x 6 mL ice-cold incubation buffer, and each oocyte was dissolved in
10% SDS. After addition of 5 mL scintillation fluid (Ultima Gold;
Canberra Packard, Zürich, Switzerland), the oocyte-associated
radioactivity was measured in a liquid scintillation analyzer (CA
Tri-Carb 2200; Canberra Packard).
| Results |
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92 kDa, indicating that it is
considerably more glycosylated than its analogue in the brain capillary
endothelium (
76 kDa).14
15
In eyecup homogenates, the
antiserum recognized a protein band with the same molecular mass (
92
kDa, Fig. 3
) as in basolateral liver plasma membranes, thus indicating that in RPE
cells, Oatp2 undergoes an extent of glycosylation similar to that in
liver. This band disappeared when the antiserum was preabsorbed with
the peptide used for immunization (data not shown).
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| Discussion |
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The expression of Oatp2 at the apical microvilli of the RPE (Figs. 1 2) was somewhat unexpected, as in other types of polarized cells, such as hepatocytes and choroid plexus epithelial cells, Oatp2 is present at the basolateral domain of the plasma membrane.14 15 The specific membrane localization in different cell types suggests that Oatp2 targeting depends on cell type properties, such as anchoring and/or cytoskeleton proteins, whereas targeting signals within the Oatp2 primary sequence may be of secondary importance.
A2E is a major component of the RPE lipofuscin.1 2 Its progressive accumulation in the RPE cells appears to contribute to the pathogenesis of AMD,3 one of the leading causes of severe visual impairment, which affects 10% and 20% of people of age 65 and older in industrialized nations.18 A2E impairs lysosomal function and may finally lead to rupture of lysosomal membranes.3 Furthermore, by specifically targeting cytochrome oxidase, A2E inhibits mitochondrial function and induces apoptosis in RPE and other cells.5 Precursors of A2E form within the rod OS from the sequential condensation of one phosphatidylethanolamine with two all-trans-retinal molecules followed by proton uptake.7 8 19 20 21 A2E is thought to arise within phagolysosomes of the RPE as the degradation of ROS membranes proceeds.1 3
The evidence for the ability of Oatp2 to transport A2E comes from the Cis inhibition experiments, which is an indirect approach to identify new substrates of Oatps/OATPs.22 23 24 That A2E, but not all-trans-retinal, competitively inhibited Oatp2-mediated digoxin transport (Figs. 4 5) indicates that A2E may also be a substrate of Oatp2. Although A2E is toxic to cells and membranes at high concentrations, it is unlikely that the A2E inhibitory effect was due to oocyte damage for the following reasons: First, oocytes were exposed to A2E for only 30 minutes at the same concentration range as that used in a previous study, in which cultured rat RPE and neuronal cells showed signs of significant mitochondrial and cellular toxicity only after exposure to A2E for more than 18 hours.5 Second, we did not observe any signs of morphologic damage after oocytes had been exposed to A2E concentrations of up to 200 µM for 1 hour (data not shown). And third, A2E inhibition of Oatp2-mediated digoxin uptake could be overcome by increasing the concentration of digoxin (Fig. 5) . Thus, the data are compatible with A2Es potentially being a specific substrate of Oatp2 at the apical microvilli of the RPE.
The direction of Oatp2-mediated transport varies in different systems. For example, in the oocyte expression system, it was demonstrated that Oatp2 can mediate bidirectional substrate transport.25 In hepatocytes, Oatp2 mediates uptake of bile salts and other amphiphilic compounds from blood plasma under physiological conditions,10 whereas in the choroid plexus Oatp2 may also mediate efflux of organic solutes.25 26 The directionality of Oatp2-mediated substrate transport in RPE cells remains to be determined. The potential substrate A2E matures from precursors within the cells of the RPE. Thus, Oatp2 may not be normally involved in the import of A2E into the pigment epithelium. However, Oatp2 may instead have the capacity to transport A2E across the RPE cell membrane into the extracellular matrix under some conditions. Release of A2E from lysosomal compartments may lead to mitochondrial damage and therefore be highly toxic to the cell.5 Potential export of A2E by Oatp2 may thus lower intracellular levels of free A2E and may therefore constitute a measure to counteract the toxicity of A2E in the RPE. Alternatively, A2E may be formed under some (unknown) pathologic conditions outside the RPE and, under these conditions, Oatp2-mediated transport may contribute to the accumulation of A2E in the pigment epithelium.
Furthermore, considering the multispecificity of Oatp2, the A2E precursors NRPE and A2-PEH219 and fatty acids, particularly docosahexaenoic acid (DHA), which is known to be shuttled between photoreceptors and RPE,27 are also potential candidates for Oatp2. Finally, Oatp2 may exert synergistic effects with other transporter(s) identified recently in the RPE,28 in transporting various drugs and other xenobiotics into and/or out of the RPE.
In conclusion, this study provides evidence that A2E can be actively transported by Oatp2 across cell membranes. This may suggest a role for the Oatp2 transporter in the (patho-) physiology of A2E in the RPE. Further studies are needed to elucidate the role of a potential A2E transport into or out of the pigment epithelium. Also, whether Oatp2 is involved in the transport of other retinoids in the RPE cells in vivo should be investigated. Given the clinical relevance of the retinoid metabolism, the identification of a human orthologue of Oatp2 in human RPE is essential.
| Addendum |
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| Acknowledgements |
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| Footnotes |
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Submitted for publication July 16, 2001; revised September 4, 2001; accepted September 17, 2001.
Commercial relationships policy: N.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be marked
"advertisement" in accordance with 18 U.S.C.
1734
solely to indicate this fact.
Corresponding author: Peter J. Meier, Division Clinical Pharmacology and Toxicology, Department of Internal Medicine, University Hospital, CH-8091 Zürich, Switzerland; meierabt{at}kpt.unizh.ch
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