Localization of organic anion transporting polypeptides in the rat and human ciliary body epithelium

Experimental Eye Research 80 (2005) 61–72 www.elsevier.com/locate/yexer

Localization of organic anion transporting polypeptides in the rat and human ciliary body epithelium Bo Gaoa, Robert D. Hubera, Andreas Wenzelb, Stephan R. Vavrickaa, Manfred G. Ismaira, Charlotte Reme´b, Peter J. Meiera,* a

Division of Clinical Pharmacology and Toxicology, Department of Medicine, University Hospital Zurich, CH-8091 Zurich, Switzerland b Department of Ophthalmology, University Hospital Zurich, CH-8091 Zurich, Switzerland Received 2 June 2004; in revised form 11 August 2004; accepted in revised form 11 August 2004 Available online 7 October 2004

Abstract Purpose. To identify and localize the expression of multispecific organic anion transporting polypeptides (Oatps/OATPs) in the ciliary body epithelium and to investigate their possible involvement in the transport of the antiglaucoma agent unoprostone. Methods. Oatps/OATPs were detected by immunoblot analysis and by immunofluorescence microscopy in homogenized and fixed rat and human ciliary body samples using specific polyclonal antibodies. Transport of 3H-labelled unoprostone was measured in Oatp/OATP expressing Xenopus laevis oocytes. Results. Immunoblots of ciliary body extracts were positive for rat Oatp1a4, Oatp1a5 and Oatp1b2 and for human OATP1A2, OATP1C1, OATP2B1, OATP3A1 and OATP4A1. Confocal immunofluorecence microscopy localized Oatp1a4 and Oatp1b2 as well as all immunoblot positive human OATPs at the basolateral plasma membrane of the non-pigmented rat and human ciliary body epithelium, respectively. However, for human OATPs additional regional differences in expression were found with OATP1A2 and OATP1C1 being expressed only in the pars plana of human ciliary body epithelium. Furthermore, OATP1C1, OATP3A1 and OATP4A1 were also expressed at the basolateral plasma membrane of the pars plana pigmented epithelium. And finally, deesterified unoprostone carboxylate was found to be transported by OATP1A2, OATP2B1 and OATP4A1 with approximate Km-values of 93, 91 and 132 mM, respectively. Conclusions. Several multispecific organic anion transporting polypeptides are expressed at the basolateral plasma membrane of the nonpigmented, and to a lesser extent also of the pigmented, epithelium in rat and human ciliary body. These Oatps/OATPs can account for the previously suggested ‘liver-like’ transport functions of mammalian ciliary body epithelium. q 2004 Published by Elsevier Ltd. Keywords: organic anion transporter; ciliary body epithelium; drug transporter; unoprostone

1. Introduction The ocular ciliary epithelium is the primary site of aqueous humor secretion and absorption in all mammalian species. It is composed of two layers of polarized neuroepithelial cells with the basolateral plasma membrane domain of the outer pigmented cell layer (PE) facing the vascularized stroma and that of the inner nonpigmented epithelium (NPE) facing the aqueous humor * Corresponding author. Dr Peter J. Meier-Abt, Division of Clinical Pharmacology and Toxicology, Department of Medicine, University Hospital Zurich, CH-8091 Zurich, Switzerland. E-mail address: [email protected] (P.J. Meier). 0014-4835/$ - see front matter q 2004 Published by Elsevier Ltd. DOI:10.1016/j.exer.2004.08.013

(Macknight et al., 2000). Exchange of ions, fluid and nutrients between the ciliary blood supply and the aqueous humor is mediated by transport systems localized predominantly at the basolateral surface domains of PE and NPE (Dunn et al., 2001; Hamann et al., 1998; Takata et al., 1991). Furthermore, ‘renal-like’ and ‘liver-like’ multispecific organic anion transport systems have long been suggested to be present in the ciliary body epithelium (Barany, 1973a,b, 1974, 1975). Interestingly, the ‘liver-like‘ transport system has been suggested to be composed of several subsystems (Barany, 1972), each accepting a large variety of structurally unrelated cholephilic organic anions including the cholangiographic agent iodipamide, conjugated and unconjugated bile acids, bromosulfophthalein (BSP) and many other

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xenobiotic substances (Barany, 1973a,b). Although these ‘liver-like’ transport systems are supposed to be involved in the removal of organic anions and drugs from the aqueous humor (Barany, 1973b), their molecular identities have remained elusive. In the liver, a variety of multispecific organic anion transporting polypeptides (rodents: Oatps; humans: OATPs) have been identified in recent years (Hagenbuch and Meier, 2003; Kullak-Ublick et al., 2000; Meier and Stieger, 2002; Mizuno et al., 2003). These Oatps/OATPs form a group of 12 transmembrane domain solute carriers with a similar wide spectrum of amphipathic transport substrates as the suggested ‘liver-like‘ organic anion transporters in the ciliary body epithelium. They have recently been newly classified within the OATP/SLCO (SoLute Carrier Organic) superfamily and subdivided into families (R40% amino acid sequence identity), subfamilies (R60% amino acid sequence identity) and individual genes and gene products according to their phylogenetic relationships and chronology of identification (Hagenbuch and Meier, 2004). For clarity, the new and old nomenclature, tissue distribution and predominant substrates of selected rat and human members of the OATP/SLCO superfamily, as relevant for this study, are summarized in Table 1. A more comprehensive description of individual Oatps/OATPs can be found in recent reviews (Hagenbuch and Meier, 2004). In the present study, we investigated the hypothesis that Oatps/OATPs might represent the molecular identities of the suggested ‘liver-like’ transport system(s) in the ciliary body epithelium of rat and human eyes. Using immunoblot analysis and confocal immunofluorescence microscopy, the study demonstrates that several members of the OATP/SLCO superfamily are abundantly expressed in the ciliary body epithelium in both species. Furthermore, the data demonstrate that the antiglaucoma agent unoprostone carboxylate is a substrate of OATPs expressed in the human ciliary body epithelium.

2. Methods 2.1. Antibodies Affinity purified rabbit antibodies against Oatp1a1 and Oatp1a5 were purchased from Alpha Diagnostic (San Antonio, TX, USA). Polyclonal rabbit antibodies against rat Oatp1a4 and Oatp1b2 and against human OATP1A2, OATP1C1 and OATP2B1 were generated and characterized as previously described (Reichel et al., 1999; Cattori et al., 2001; Gao et al., 2000; Kullak-Ublick et al., 2001; Pizzagalli et al., 2002; St-Pierre et al., 2002). New polyclonal rabbit antibodies were raised against keyhole limpet hemocyanine coupled C-terminal oligopeptides of human OATP1B1 (amino acids 677–691), OATP1B3 (a.a. 688–702), OATP3A1 (a.a. 696–710) and OATP4A1 (a.a. 708–722) (Hagenbuch and Meier, 2003). Indicated antisera were affinity purified using Affigel 10 (Bio-Rad laboratorie, Hercules, CA, USA) coupled to the synthetic oligopeptide according to the manufacturer’s instructions. The secondary Cy3- and horseradish peroxidase conjugated goat anti-rabbit antibodies were purchased from Jackson Immunoresearch (West Grove, PA, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA), respectively. 2.2. Expression of human OATPs in heterologous cell lines To test for antibody specificity, individual human OATPs were expressed in appropriate cell lines and subjected to immunoblot analysis. Chinese hamster ovary (CHO) cells were transfected with the expression vector pIRESneo2 (CLONTECH, Palo Alto, CA, USA) carrying OATP1B1-, OATP1B3- or OATP2B1-cDNA (Pizzagalli, et al., 2002). Human embryonic kidney 293 (HEK 293) were transfected with the expression vector pcDNA 3$1/V5-His A (Invitrogen, Carlsbad, CA, USA) carrying OATP4A1-cDNA. Sf9

Table 1 Nomenclature/classification, tissue distribution and predominant transport substrates of Oatps/OATPs examined in the present study New protein name (new gene symbol)

Old protein name (old gene symbol)

Tissue distribution

Predominant substrates

Oatp1a1 (Slco1a1) OATP1A2 (SLCO1A2) Oatp1a4 (Slco1a4) Oatp1a5 (Slco1a5) OATP1B1 (SLCO1B1)

Oatp1, Oatp (Slc21a1) OATP1A (SLC21A3) Oatp2 (Slc21a5) Oatp3 (Slc21a7) OATP-C, LST-1, OATP2 (SLC21A6) Oatp4, Lst-1 (Slc21a10) OATP8 (SLC21A8) OATP-F, OATP-RP5 (SLC21A14) OATP-B (SLC21A9) OATP-D (SLC21A11) OATP-E (SLC21A12)

Liver, kidney, choroid plexus Brain, liver, kidney Liver, brain, choroid plexus, retina Jejunum, choroid plexus Liver

Bile salts, organic anions, organic cations Bile salts, organic anions, organic cations Bile salts, digoxin, organic anions, organic cations Bile salts, organic anions Bile salts, organic anions

Liver Liver, cancer cell lines Brain, testis

Bile salts, organic anions Bile salts, digoxin organic anions, BSP, rT3, T4

Liver, placenta Ubiquitous Ubiquitous

BSP, DHEAS, E-3-S, E-3-S, prostaglandin T3, Taurocholate, prostaglandin

Oatp1b2 (Slco1b2) OATP1B3 (SLCO1B3) OATP1C1 (SLCO1C1) OATP2B1 (SLCO2B1) OATP3A1 (SLCO3A1) OATP4A1 (SLCO4A1)

BSP, bromosulphophthalein; DHEAS, dehydroepiandrosterone sulfate; E-3-S, estrone-3-sulfate; rT3, reverse triiodo-thyronine; T4, thyroxine.

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insect cells were infected at 278C with recombinant baculovirus (Invitrogen, Carlsbad, CA, USA) carrying OATP1A2-, OATP1C1- or OATP3A1-cDNA. After 2–4 days in culture, the transfected CHO and Sf9 cells were homogenized and microsomal vesicles isolated as previously described (Gerloff, et al., 1998). Transfected HEK 293 cells were lysed with 1% Triton and centrifuged to pellet large cell debris. The CHO and Sf9 cell membrane preparations and the HEK 293 lysate were analysed for protein contents (BC assay; Interchim, Montlucon, France) and subjected to immunoblot analysis. 2.3. Animal and human tissue collection Male pigmented Brown–Norwegian rats (200–250 g) were obtained from RCC Ltd (Fu¨llinsdorf, Switzerland). They received standard care in accordance with the regulations of the veterinary authority of Zurich and with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Ciliary bodies were carefully dissected, collected in 0$1 M Tris pH 7$5 and homogenized. For immunofluorescence microscopy eyes were fixed as described below. Human ciliary bodies of both sex were collected from four deceased patients (age range: 25–88 years) during routine autopsies (range of post-mortem periods: 8–12 hr) according to a protocol approved by the local Ethics Committee. Ciliary bodies were carefully dissected from enucleated human eyes. For immunoblot analysis the dissected human ciliary bodies were quickly frozen in liquid nitrogen. For immunofluorescence microscopy ciliary bodies were fixed as described below. 2.4. Immunoblot analysis Freshly isolated rat and frozen human ciliary bodies were homogenized with an ultrasound tip. The homogenates were centrifuged at 1000!g for 5 minutes to remove larger cell debris. The supernatant was mixed with SDS sample buffer, heated to 708C for 10 minutes and stored at K208 until use. Protein content of the supernatant was assessed by the Bradford assay (BioRad, Hercules, CA) using BSA as standard. Proteins were separated on 7$5% SDS-polyacrylamide minigels and transferred to nitrocellulose membranes. Blots were blocked for 1 hr 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 48C with primary antibodies diluted in TBST containing 5% blocker. Blots were washed three times with TBST and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG diluted at 1:5000 for 1 hr at room temperature. Immunoreactivity was visualized with the Renaissance Western blot detection kit (NEN Life Science products, Boston, MA, USA).

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2.5. Immunofluorescence staining Rat eyes were fixed in phosphate-buffered saline (PBS) containing 3% paraformaldehyde and 0$1% glutaraldehyde (pH 7$4) at 48C for 2 hrs and cryoprotected in 30% sucrose overnight at 48C. After removal of lenses and vitreous tissue, the anterior eyecups were carefully dissected, embedded in M1-embedding matrix (Lipshaw, Pittsburgh, PA, USA) and stored at K708C until used. Human ciliary bodies were fixed with 2% paraformaldehyde in PBS containing 0$2% glutaraldehyde at 48C for 1–2 hrs, cryoprotected, and stored at K708C until used. Ten micrometer cryostat sections were cut and mounted on glass slides coated with 0$5% gelatin Sections were incubated overnight at 48C with antibodies diluted in 50 mM Tris/100 mM NaCl (Tris–saline, pH 7$4) containing 2% normal goat serum and 0$05% Triton X-100. The following dilutions for primary antibodies were used: Oatp1a1 (affinity-purified), 1–50 mg mlK1; Oatp1a4 (affinity-purified), 1 mg mlK1; Oatp1a5 (affinity-purified), 1-50 mg mlK1; Oatp1b2, 1:5000; OATP1A2 (affinity-purified), 0$4 mg mlK1; OATP1B1, 1:100-1:3000; OATP1B3, 1:100-1:5000; OATP1C1, 1:1000; OATP2B1 (affinity-purified), 1$2 mg mlK1; OATP3A1, 1:3000; OATP4A1 (affinitypurified), 2 mg mlK1. 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 with Immu-mount (Shandon, Pittsburgh, PA, USA). The specificity of the immunoreactivity was verified by incubating sections with the primary antibody preabsorbed with 10–20 mg mlK1 of the corresponding antigen used for immunization or with the preimmuneserum. Stained sections were analysed by confocal laser microscopy (MRC 600 confocal imaging system, BioRad Laboratories, Richmont, CA) using a Zeiss Axioplan microscope (Oberkochen, Germany). 2.6. Transport assays in Xenopus laevis oocytes Mature X. laevis females were purchased from the African Xenopus facility, Noordhoek, Republic of South Africa and kept under standard conditions. In vitro synthesis of cRNAs for OATP1A2, OATP1C1, OATP2B1, OATP3A1, OATP4A1 was performed from the respective cDNAs as described (Kullak-Ublick et al., 1995, 2001; Pizzagalli et al., 2002). Oocytes were prepared and incubated overnight at 188C. Healthy oocytes were microinjected with either H2O (control) or 5 ng of cRNA encoding OATP1A2, OATP1C1, OATP2B1, OATP3A1 and OATP4A1. The oocytes were cultured for three days to allow expression of the transporter proteins in the plasma membrane. Tracer uptake experiments were carried out in NaC-containing

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(100 mM NaCl) and NaC-free (100 mM choline chloride) media containing in addition 2 mM KCl; 1 mM CaCl2; 1 mM MgCl2; and 10 mM HEPES/Tris, pH 7$5. Fifteen oocytes were prewashed in the uptake medium and then incubated at 258C in 100 ml of uptake medium supplemented with the indicated amounts of radiolabeled and unlabeled compounds. Subsequently, oocytes were washed with 3x6 ml of ice-cold incubation buffer and each oocyte was dissolved in 10% SDS. After addition of 5 ml of scintillation fluid (Ultima Gold; Canberra Packard, Zurich, Switzerland), the oocyte-associated radioactivity was measured in a Packard Tri-Carb 2200 CA liquid scintillation analyser (Canberra Packard). 2.7. Chemicals 3

H-labeled unoprostone isopropylester (173$5 MBq mgK1) 3H-labeled deesterified unoprostone carboxylate (also called ‘M1 metabolite’ (Kashiwagi et al., 1999), 173$5 MBq mgK1), and a stock solution of unoprostone carboxylate dissolved in dimethyl sulfoxide (DMSO) were provided by Novartis (Basel, Switzerland). All other chemicals were obtained from Fluka (Buchs, Switzerland), Merck (Whitehouse Station, NJ, USA) and Sigma Chemicals (St Louis, MO, USA).

3. Results 3.1. Specificity of Oatp/OATP antibodies Polyclonal antibodies against four rat and seven human members of the OATP/SLCO superfamily were used to detect Oatp/OATP-protein expression in the ciliary body epithelium. The specificity of the rat Oatp antibodies has been verified previously either in our laboratory (Cattori, et al., 2001; Oatp1a4 and Oatp1b2) or by the antibody provider (Alpha Diagnostic; Oatp1a1 and Oatp1a5). The specificity of the antibodies against human OATPs was tested by immunoblot analysis of OATP expressing cell lines. As illustrated in Fig. 1 the antibodies detected positive signals only in preparations expressing the corresponding OATP antigens. No cross-reactivities were observed indicating that the raised antibodies are specific and suitable to differentiate between individual OATPs in the ciliary body epithelium. 3.2. Immunochemical detection of Oatps/OATPs in rat and human ciliary body tissue samples To screen for the presence of Oatps/OATPs in the ciliary body tissue, immunoblot analysis was performed in 1000!g supernatants of rat and human ciliary body homogenates, respectively. As illustrated in Fig. 2, in rat ciliary body immunopositive protein bands of approximately 80 kDa were found for Oatp1a4, Oatp1a5 and Oatp1b2

Fig. 1. Specificity of antibodies against human OATPs shown by immunoblot analysis. Lanes were loaded with Sf9 membrane vesicles expressing OATP1A2 (50 mg/lane), OATP3A1 (2$5 mg/lane) and OATP1C1 (10 mg/lane), microsomes of Chinese hamster ovary (CHO) cells expressing OATP1B1 (10 mg/lane), OATP2B1 (10 mg/lane) and OATP1B3 (50 mg/lane), and crude lysates of human embryonic kidney (HEK) cells expressing OATP4A1 (25 mg/lane). Western blotting was performed as described in Section 2. The positions of the molecular mass markers are indicated on the right.

(Fig. 2A), whereas no immunoreactivity was found for Oatp1a1 (data not shown). Furthermore, human ciliary body tissue samples were immunopositive for the 90-97 kDa proteins OATP1A2, OATP1C1, OATP2B1, OATP3A1 and OATP4A1 (Fig. 2B), whereas no or only weak

Fig. 2. Immunochemical identification of Oatps/OATPs in rat and human ciliary body. 1000!g supernatants of ciliary body homogenates (40 mg protein/lane) were subjected to immunoblot analysis as described in Section 2. (A) Rat ciliary body: Western blotting was performed with affinity-purified antibodies against Oatp1a4 (6 mg ml K1 ), Oatp1a5 (10 mg mlK1) and with an antiserum against Oatp1b2 (1:1000), (B) Human ciliary body: Western blotting was performed with affinity-purified antibodies against OATP1A2 (0$33 mg mlK1), OATP2B1 (0$33 mg mlK1) and OATP4A1 (5 mg mlK1) and with antisera against OATP1C1 (1:1000) and OATP3A1 (1:1000). The positions of the molecular mass markers are indicated on the right.

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immunoreactivity was found for OATP1B1 and OATP1B3, respectively, (data not shown). No immunopositive reactions were observed in parallel control experiments with preimmunsera and/or after preabsorption of the antibody preparations with the corresponding antigenic peptides (data not shown). 3.3. Cellular expression of Oatps in rat ciliary body epithelium Immunofluorescence analysis of fixed rat ciliary body sections revealed strong positive staining of the inner ciliary body border facing the aqueous humor for Oatp1a4 (Fig. 3A1) and Oatp1b2 (Fig. 3B1), which is consistent with the immunopositivity observed in the Western blots (Fig. 2). In contrast, no immunostaining was obtained for Oatp1a5 (data not shown) indicating that Oatp1a5 is not expressed in rat ciliary body epithelium. Immunolocalization of rat Oatp1a4 and Oatp1b2 was confined to the pars plicata as well as the pars plana (data not shown) of the ciliary body epithelium. No specific immunoreactivities were observed in the iris or cornea. In addition, no specific immunostainings were observed after preabsorption of antibodies with the corresponding antigenic peptides (inserts of Fig. 3A1, B1). At higher magnification, specific immunolocalization of Oatp1a4 (Fig. 3A2) and Oatp1b2 (Fig. 3B2) was observed exclusively at the basolateral plasma membrane of the NPE. These results demonstrate the occurrence of multispecific ‘liver-like’ Oatps in the rat ciliary body epithelium. 3.4. Cellular expression of OATPs in human ciliary body epithelium Fig. 4 shows expression of OATP2B1, OATP3A1 and OATP4A1 in the pars plicata of human ciliary body. Similar to the rat, the immunopositivities were exclusively confined to the inner layer of the ciliary body epithelium facing the aqueous humor, which at higher magnification, corresponded also to the basolateral plasma membrane of the NPE (Fig. 4B). In the pars plicata, no positive immunofluorescence signals were obtained for OATP1A2 and OATP1C1 (data not shown). However, all immunoblot positive OATPs, i.e. OATP1A2, OATP1C1, OATP2B1, OATP3A1 and OATP4A1 (Fig. 1) were found to be expressed in the pars plana of human ciliary body epithelium (Fig. 5). Interestingly, only OATP1A2 and OATP2B1 exhibited predominant or even selective basolateral NPE expression, while OATP1C1 and to a lesser extent OATP3A1 and OATP4A1 could also be localized at the basolateral plasma membrane of the PE (Fig. 6). Hence, various human OATPs are strongly expressed along the ciliary body epithelium with some of them exhibiting clearcut regional differences in their cellular expression pattern.

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3.5. Transport of unoprostone carboxylate (metabolite M1) by human OATPs expressed in the ciliary body epithelium Although Oatps/OATPs are in general multispecific transport systems that mediate transport of a wide variety of amphipathic substrates, some members of the OATP/SLCO superfamily have a more restricted substrate spectrum and possess rather specific transport functions in distinct organs (e.g. high affinity thyroxin transport by OATP1C1 in brain and testis (Pizzagalli et al., 2002); prostaglandin transport by OATP2A1 (previously called PGT/SLC21A9) and OATP3A1 (Adachi et al., 2003; Kanai et al., 1995). Therefore, we wondered whether Oatps/ OATPs could also mediate some ‘eye-specific’ transport functions at the ciliary body epithelium. For this purpose we tested OATP-dependent transport of the prostaglandin analog and antiglaucoma agent ‘unoprostone’ in Xenopus leavis oocytes. As shown in Fig. 7, an approximately three fold increased uptake of de-esterified unoprostone carboxylate (Zmetabolite M1) was observed in OATP1A2, OATP2B1 and OATP4A1 expressing (Fig. 7B) as compared to water injected oocytes (Fig. 7A). In contrast, OATP1C1 and OATP3A1 expressing oocytes exhibited similar unoprostone M1 uptake as water injected oocytes. Although OATP-mediated unoprostone M1 uptakes were higher in the presence of NaCl, significantly increased uptakes were still found in the absence of sodium (Fig. 7C) indicating no obligatory (or direct) coupling of OATP-mediated unoprostone uptake with sodium. Furthermore, OATP-mediated unoprostone M1 uptakes were saturable with apparent Km-values of 93, 91 and 132 mM for OATP1A2, OATP2B1 and OATP4A1, respectively (Fig. 8). Hence, unoprostone M1 represents a new transport substrate of three human OATP isoforms that are expressed in the ciliary body epithelium.

4. Discussion The present study identifies several members of the OATP/SLCOsuperfamily of organic anion transporters in the ciliary body epithelia of rat and human eyes (for summary see Table 2). In rat ciliary body, Oatp1a4 and Oatp1b2 are exclusively expressed at the basolateral plasma membrane of the NPE facing the aqueous humor (Fig. 3). In human ciliary body, a similar exclusive basolateral NPE localization was found for OATP2B1, OATP3A1 and OATP4A1 in the pars plicata (Fig. 4) and for OATP1A2 and OATP2B1 in the pars plana (Figs. 5 and 6). In addition, a bipolar expression at the basolateral membranes of NPE and PE was found for OATP1C1, OATP3A1 and OATP4A1 in the pars plana (Fig. 6) indicating regional differences in OATP-dependent transport functions in human ciliary body epithelium. Finally, unoprostone carboxylate (metabolite M1) was identified as a new substrate for OATP1A2, OATP2B1 and OATP4A1 (Figs. 7 and 8).

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Fig. 3. Immunolocalization of Oatp1a4 (A) and Oatp1b2 (B) in the pars plicata of rat ciliary body epithelium. A1, B1: Low magnification images: Strong positive immunoreactivities were found at the inner border of the ciliary body epithelium facing the aqueous humor. Immunostaining was abolished after preabsorption of the antibody with the antigenic peptides (inserts). Bars, 65 mm. A2/3, B2/3: High-power confocal microscopy analysis localized Oatp1a4 (A2) and Oatp1b2 (B2) exclusively at the basolateral surface of the non-pigmented epithelium (NPE) as evidenced by comparison with differential interference contrast (DIC) images (A3, B3). PE, pigmented epithelium. Bars, 5$5 mm.

The identification of distinct Oapts/OATPs in rat and human ciliary body epithelium provides the molecular basis for the previous functional characterization of one (or several) multispecific ‘liver-like’ organic anion transport system(s) in mammalian ciliary body epithelium

(Barany, 1972, 1973a,b, 1974). Among rodent Oatps, Oatp1a4 and Oatp1b2 are expressed in liver and ciliary body epithelium. In contrast, among human OATPs only OATP1A2 and OATP2B1 are expressed in both liver and ciliary body epithelium, whereas no clear evidence for

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Fig. 4. Immunolocalization of OATP2B1, OATP3A1 and OATP4A1 in the pars plicata of the human ciliary body. (A): Strong positive immunoreactivities were found at the inner border of the ciliary body epithelium facing the aqueous humor. Immunostaining was abolished after preabsorption of the correspoding antibodies with the antigenic peptides indicating specific immunoreactivities (data not shown). Bar, 50 mm, (B): High-power confocal microscopy analysis localized the indicated human OATPs exclusively at the basolateral surface of the non-pigmented epithelium (NPE) as evidenced by comparison with the differential interference contrast (DIC) images. PE, pigmented epithelium. Bar, 15 mm.

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Fig. 5. Immunolocalization of OATP1A2, OATP1C1, OATP2B1, OATP3A1 and OATP4A1 in the pars plana of human ciliary body. Strong positive immunoreactivities were found for all indicated human OATPs. Immunostaining was abolished after preabsorption of the corresponding antibodies with the antigenic peptides (data not shown) indicating specific immunoreactivities. Bar, 50 mm.

expression of the prominent human liver OATP1B1 and OATP1B3 in the ciliary body epithelium was found. Nevertheless, because of overlapping substrate specificities OATPs not expressed in human liver can also mirror ‘liverlike’ organic anion transport in the human ciliary body epithelium. For example, similar to OATP1A2 and OATP2B1, OATP1C1, OATP3A1 and OATP4A1 have also been shown to transport BSP, bile salts, steroidconjugates, thyroid hormones and/or prostaglandin E2 (Hagenbuch and Meier, 2003; Ito et al., 2003). Hence, our findings confirm the previous suggestion that the ‘liver-like’ organic anion transport system of the mammalian ciliary body epithelium is actually composed of several overlapping ‘subsystems’ (Barany, 1972). Furthermore, Oatp4a1 has

been suggested to play an additional special role in the ciliary transport of the thyroid hormone T3 in the rat eye (Ito et al., 2003). The most prominent cellular expression of rat and human Oatps/OATPs was found to be at the basolateral plasma membrane of NPE. Furthermore, regional differences in expression were found for OATP1A2 and OATP1C1, which are only expressed in the pars plana of human ciliary body (Fig. 5). In the same region, OATP1C1, OATP3A1 and OATP4A1 are expressed at the basolateral plasma membranes of PE and NPE cells (Fig. 6). These regional differences in expression support the concept of functional heterogeneity along the major axis of the ciliary body epithelium (McLaughlin et al., 2001). Since Oatps/OATPs function as anion exchanger and, thus, can mediate bidirectional transmembrane transport, the ultimate directionality of Oatp/OATP-mediated organic solute transport is dependent on the prevailing local anion gradients such as for example bicarbonate and/or reduced glutathione gradients (Li et al., 1998, 2000; Satlin et al., 1997; Shi et al., 1995). However, whether intracellular bicarbonate and/or reduced glutathione represent indeed important counter-anions for Oatp/OATP-mediated amphipathic organic solute transport at the ciliary body epithelium remains to be determined in isolated and cultured ciliary epithelial cells. These studies should also elucidate the relationship between the regional and cellular differences of Oatp/OATP-mediated transport functions and the heterogeneity of inorganic ion transporters along the ciliary body epithelium (Dunn et al., 2001; Gosh et al., 1990; McLaughlin et al., 2001; Wetzel and Sweadner, 2001). The identification of the antiglaucoma agent unoprostone as an additional transport substrate of OATP1A2, OATP2B1 and OATP4A1 (Figs. 7 and 8) supports the important role of Oatps/OATPs in the cellular bioavailability and elimination of prostaglandins and prostaglandin analogs (Hagenbuch and Meier, 2003). The exact reason for the partial NaC-dependency of unoprostone uptake (Fig. 7) is not known, but has been seen also with other Oatp substrates (e.g. phalloidin; Meier-Abt et al., 2004) and might reflect indirect effects of NaC on for example out to in proton gradients (Meier-Abt et al., 2004). While most Oatps/OATPs transport leukotriene C4 and prostaglandin E2 with rather low affinities, high affinity prostaglandin transport has been shown for OATP2A1 (previously called PGT/SLC21A9) (Kanai et al., 1995; Lu et al., 1996), Oatp2b1 (Nishio et al., 2000) and OATP3A1 (Adachi et al., 2003). Although OATP2A1 is widely expressed in ocular tissues including the ciliary body (Schuster et al., 1997), its cellular and subcellular expression has not been investigated on the protein level. Also, the affinity of unoprostone and its major metabolites as inhibitors of OATP2A1-mediated prostaglandin E2 transport have been reported to be higher than 10 mM (Kashiwagi et al., 2002). These Ki-values are similar to the Km-values for OATP1A2-, OATP2B1- and OATP4A1-mediated unoprostone carboxylate (metabolite

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Fig. 6. Immunolocalization of OATP1A2, OATP1C1, OATP2B1, OATP3A1 and OATP4A1 at the basolateral plasma membrane of non-pigmented (NPE) and/or pigmented (PE) epithelial layers in the pars plana of the human ciliary body. All indicated human OATPs were localized at the basolateral plasma membrane of NPE as evidenced by comparison with the differential interference contrast (DIC) images (right panel). In addition, for OATP1C1, OATP3A1 and OATP4A1 immunopositive reactivities were also found at the basolateral surface of the PE. Bar, 15 mm.

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B. Gao et al. / Experimental Eye Research 80 (2005) 61–72 Table 2 Summary of expression of Oatps/OATPs in rat and human ciliary body epithelium Transporters

Fig. 7. Uptake of unoprostone into OATP expressing Xenopus laevis oocytes. Oocytes were injected with water or 5 ng of OATP1A2-, OATP1C1-, OATP2B1-, OATP3A1-, and OATP4A1-cRNAs. Uptake studies were performed for 15 min at 258C with (A) 3H-labelled unoprostone isopropyl ester (4 mM) in NaCl (100 mM) containing uptake medium (see Section 2), (B) 3H-labelled unoprostone carboxylate (M1 metabolite; 4 mM) in NaCl (100 mM) containing medium, and C) 3Hlabelled unoprostone carboxylate (M1 metabolite; 4 mM) in cholineCl (100 mM) containing medium. Uptake values are presented as meanGS.D. of 12–15 oocyte uptake measurements. *p!0$0001 (paired TTEST, Microsoft Excel, Cambridge, MA).

Detection by Western blotting

Localization by Immunofluorescence

Rat Oatp1a1 Oatp1a4

n.d. Strong

n.d. Pars plicata: BLM of NPE

Oatp1a5 Oatp1b2

Weak Strong

pars plana: BLM of NPE n.d. Pars plicata: BLM of NPE pars plana: BLM of NPE

Human OATP1A2

Strong

OATP1B1 OATP1B3 OATP1C1

n.d. n.d. Strong

OATP2B1

Strong

OATP3A1

Strong

OATP4A1

Strong

Pars plicata: n.d. pars plana: BLM of NPE n.d. n.d. Pars plicata: n.d. pars plana: BLM of NPE and PE Pars plicata: BLM of NPE pars plana: BLM of NPE Pars plicata: BLM of NPE pars plana: BLM of NPE and PE Pars plicata: BLM of NPE pars plana: BLMof NPE and PE

n.d., not detected; BLM, basolateral membrane; NPE, non-pigmented epithelium; PE, pigmented epithelium.

M1) transport (Fig. 8) indicating that multiple members of the OATP/SLCO superfamily are involved in the clearance of unoprostone carboxylate from the aqueous humor. Furthermore, since unoprostone and its metabolites induce the intraocular synthesis of prostaglandin E2 (Kashiwagi et al., 2002), and since prostaglandin E2 (Adachi et al., 2003), but not unoprostone carboxylate (Fig. 7), represents a high affinity substrate of OATP3A1, complementary prostaglandin transport functions may be mediated by different Oatps/OATPs. In conclusion, we have identified several members of the OATP/SLCO superfamily of organic anion transporting polypeptides in rat and human ciliary body epithelia. Given their prominent expression at the basolateral plasma membrane of NPE, Oatps/OATPs are most probably involved in the removal of amphipathic organic anions such as prostaglandins, prostaglandin analogs (e.g. unoprostone carboxylate) and numerous drugs from aqueous humor as well as in organic anion exchange between ciliary body epithelium and vitreous tissue. These assumptions, 3 Fig. 8. Kinetics of OATP1A2, OATP2B1 and OATP4A1-mediated uptake of unoprostone M1 metabolite (unoprostone carboxylate) in cRNA (5 ng) injected Xenopus laevis oocytes. Uptake measurements were performed during the initial linear uptake phase (10 min) in NaCl (100 mM) containing uptake medium. Non-specific uptake into water-injected oocytes was subtracted from all uptake measurements. Values are presented as meanG S.E. of 12–15 uptake measurements. The curves were fitted by computerized nonlinear regression analysis (Systat, Spss Inc., Chicago, IL, USA).

B. Gao et al. / Experimental Eye Research 80 (2005) 61–72

however, as well as the functional significance of the basolateral expression of selected human OATPs at the NPE and PE require further investigations in a cultured in vitro system of ciliary epithelial cells.

Acknowledgements This study was supported by the NCCR on Neural Plasticity and Repair, Neuroscience Center Zurich, and the Swiss National Science Foundation (Grant 31-64140$00).

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