Localization of organic anion transporting polypeptide 3 (oatp3) in mouse brain parenchymal and capillary endothelial cells

Authors

  • Sumio Ohtsuki,

    1. Department of Molecular Biopharmacy and Genetics, Graduate School of Pharmaceutical Sciences
    2. New Industry Creation Hatchery Center, Tohoku University, Aoba-ku, Sendai, Japan
    3. CREST and SORST of the Japan Science and Technology Agency (JST), Japan
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  • Takuya Takizawa,

    1. Department of Molecular Biopharmacy and Genetics, Graduate School of Pharmaceutical Sciences
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  • Hitomi Takanaga,

    1. Department of Molecular Biopharmacy and Genetics, Graduate School of Pharmaceutical Sciences
    2. New Industry Creation Hatchery Center, Tohoku University, Aoba-ku, Sendai, Japan
    3. CREST and SORST of the Japan Science and Technology Agency (JST), Japan
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  • Satoko Hori,

    1. Department of Molecular Biopharmacy and Genetics, Graduate School of Pharmaceutical Sciences
    2. New Industry Creation Hatchery Center, Tohoku University, Aoba-ku, Sendai, Japan
    3. CREST and SORST of the Japan Science and Technology Agency (JST), Japan
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  • Ken-ichi Hosoya,

    1. CREST and SORST of the Japan Science and Technology Agency (JST), Japan
    2. Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Toyama, Japan
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  • Tetsuya Terasaki

    1. Department of Molecular Biopharmacy and Genetics, Graduate School of Pharmaceutical Sciences
    2. New Industry Creation Hatchery Center, Tohoku University, Aoba-ku, Sendai, Japan
    3. CREST and SORST of the Japan Science and Technology Agency (JST), Japan
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Address correspondence and reprint requests to Tetsuya Terasaki, Ph.D, Professor, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba, Aramaki, Aoba-ku, Sendai 980–8578, Japan.
E-mail: terasaki@mail.pharm.tohoku.ac.jp

Abstract

Organic anion transporting polypeptide 3 (oatp3) transports various CNS-acting endogenous compounds, including thyroid hormones and prostaglandin E2, between extra- and intracellular spaces, suggesting a possible role in CNS function. The purpose of this study was to clarify the expression and localization of oatp3 in the mouse brain. RT-PCR analysis revealed that oatp3 mRNA is expressed in brain capillary-rich fraction, conditionally immortalized brain capillary endothelial cells, choroid plexus, brain and lung, but not in liver or kidney, where oatp1, 2 and 5 mRNAs were detected. Immunohistochemical analysis with anti-oatp3 antibody suggests that oatp3 protein is localized at the brush-border membrane of mouse choroid plexus epithelial cells. Furthermore, intense immunoreactivity was detected in neural cells in the border region between hypothalamus and thalamus, and in the olfactory bulb. Immunoreactivity was also detected in brain capillary endothelial cells in the cerebral cortex. These localizations in the mouse brain suggest that oatp3 plays roles in blood–brainand –cerebrospinal fluid barrier transport of organic anions and signal mediators, and in hormone uptake by neural cells.

Abbreviations used
BBB

blood–brain barrier

BCSFB

blood–cerebrospinal fluid barrier

CHO

Chinese hamster ovary

oatp3

organic anion transporting polypeptide 3

PGE2

prostaglandin E2

T3

triiodothyronine

T4

thyroxine

TH

thyroid hormone

TM-BBB1

conditionally immortalized mouse brain capillary endothelial cell line

TRH

thyrotropin releasing hormone

Organic anion transporting polypeptide 3 (oatp3; Slc21a7) mediates sodium-independent transport of a wide variety of amphipathic organic compounds, including opioid peptides, neurosteroid conjugates, thyroid hormones (THs), bile acids and drugs, such as fexofenadine (Abe et al. 1998; Dresser et al. 2002; Hagenbuch and Meier 2003). Since many of these substrates are active in the CNS, oatp3 may play a role in the control of CNS functions.

THs play an important role in normal CNS development and maturation. The expression of TH receptors was detected in adult brain (Puymirat et al. 1991), and hypothyroidism induces changes in the neuronal morphology of adult rat brain (Ruiz-Marcos et al. 1980) and affects memory in human patients (Burmeister et al. 2001). THs interact with nuclear TH receptors to express their effects. Although it was believed that THs enter target cells by passive diffusion, the recent reports have demonstrated that their cellular uptake by cells, including neurons and astrocytes, is carrier-mediated (Francon et al. 1989; Chantoux et al. 1995; Hennemann et al. 2001). Although roles of oatp family members in neural cells have not been considered yet, it is conceivable that oatps mediate TH uptake of the target cells in the brain.

Expression of oatp3 mRNA in rat brain has been detected by means of RNase protection assay and RT-PCR analysis (Walters et al. 2000; Ohtsuki et al. 2003). Since oatp3 mediates transport of THs, such as triiodothyronine (T3) and thyroxine (T4) (Abe et al. 1998), we hypothesize that oatp3 is expressed at the neural cells and mediates their TH uptake. To our knowledge, the brain localization of oatp3 has not been established, and clarifying the brain localization is important to understand the roles of oatp3 in the CNS.

Oatp2 and oatp14 are expressed at brain capillaries and choroid plexus, which form the blood–brain barrier (BBB) and the blood–cerebrospinal fluid barrier (BCSFB), respectively (Gao et al. 1999; Sugiyama et al. 2003). We have reported that oatp3 is localized at the rat choroid plexus (Ohtsuki et al. 2003) and this finding raises the issue of whether oatp3 is expressed at the brain capillaries. Oatp3 accepts prostaglandin E2 (PGE2) as a substrate, whereas oatp2 and oatp14 do not (Cattori et al. 2001; Sugiyama et al. 2003). PGE2 functions as a mediator of peripheral inflammation to the brain across the BBB (Engblom et al. 2002). Furthermore, a heterogeneous brain distribution of PGE2 was observed after intravenous administration (Eguchi et al. 1992). One possible explanation for this heterogeneous distribution is the existence of a heterogeneously localized BBB transport system for PGE2. Therefore, a study of the expression of oatp3 at the BBB may reveal novel CNS-related functions of the BBB transport systems.

The purpose of this study was to investigate the expression and localization of mouse oatp3 in the brain and at the BBB by means of RT-PCR and immunohistochemical analysis.

Materials and methods

Animals

Male ddY mice, weighing 25–30 g, were purchased from Japan SLC (Shizuoka, Japan). The investigations described in this report conformed to the guidelines established by the Animal Care Committee, Graduate School of Pharmaceutical Sciences, Tohoku University (approval number 1511).

Cell culture

A conditionally immortalized mouse brain capillary endothelial cell line (TM-BBB1) was established from transgenic mouse harboring the temperature-sensitive simian virus 40 (ts SV 40) large T-antigen in our laboratory (Hosoya et al. 2000). TM-BBB1 cells were routinely grown in collagen type I-coated tissue flasks (Becton Dickinson, Bedford, MA, USA) at 33°C under 5% CO2/air. The culture medium used consisted of Dulbecco's modified Eagle's medium supplemented with 20 mm sodium bicarbonate, 15 ng/mL endothelial cell growth factor, 100 U/mL benzylpenicillin potassium, 100 µg/mL streptomycin sulfate, and 10% fetal bovine serum (Moregate, Blimba, Australia).

Chinese hamster ovary (CHO)-K1 cells were cultured in tissue culture flask (Corning, NY, USA) at 37°C under 5% CO2/air. The culture medium was α-Minimum Essential Medium supplemented with 2.2 g/L sodium bicarbonate, 100 U/mL penicillin, 100 µg/mL streptomycin and 10% fetal bovine serum. To establish mouse oatp3-expressing CHO-K1 cells (CHO-K1/oatp3 cells), mouse oatp3 cDNA was isolated from TM-BBB1 cells and subcloned into the XhoI and XbaI sites of pCI-neo mammalian expression vector (Promega, Madison, WI, USA). Then, the mouse oatp3 cDNA-containing expression vector (1 µg for each transfection) was transfected into CHO-K1 cells using LIPOFECTAMINETM reagent and PLUSTM reagent (Invitrogen, Carlsbad, CA, USA). The treated cells were cultured in the medium for CHO-K1 cells supplemented with 900 µg/mL G418. Fifteen lines were cloned, and the cell line which exhibited the greatest uptake activity of [3H]taurocholate was used as CHO-K1/oatp3 cells.

Reverse transcription-polymerase chain reaction (RT-PCR) analysis

Total RNA was isolated from TM-BBB1 cells at confluence using RNeasy (QIAGEN, Valencia, CA, USA), and from mouse brain capillary-rich fraction, choroid plexus, brain, lung, liver and kidney using TRIzol Reagent (Invitrogen), according to the manufacturers' instructions. The RNA was reversely transcribed using oligo(dT) primer and ReverTra Ace (Toyobo, Osaka, Japan). The specific primer sets for oatp1, 2, 3 and 5 were as follows: oatp1,5′-TGGGGAAGGTTGCTGGCCCAATTT-3′ and 5′-GGTGGTTAATCCAGCAACTGCTGC-3′; oatp2, 5′-AACAGGAATGACCATTGGCCCTTTG-3′ and 5′-ATCCGAGGCATATTGGAGGTAACATG-3′; oatp3, 5′-CAGGAAAGGTCTTTGGCCCAATAG-3′ and 5′-AGTTATAAACACCTATGAGAAGGACC-3′; oatp5, 5′-GTTGGGAAGATGATTGGCCCAATAC-3′ and 5′-ATATCCAGCAGATACTGAAGGTGTGG-3′. PCR was performed using Ex-Taq DNA polymerase with the following thermal cycle program: 40 cycles of 94°C for 30 s, 65°C for 30 s, 72°C for 1 min and an additional elongation of 72°C for 10 min. The PCR products were separated in 1% agarose gel in the presence of ethidium bromide and were visualized using an imager (EPIPRO 7000; Aisin, Aichi, Japan). The amplified products were subcloned into pBluescript SKII(+) (Stratagene, La Jolla, CA, USA) or pGEM-T Easy vector (Promega) and sequenced using a DNA Sequencer (model 4200; LI-COR, Lincoln, NE, USA). Sequences were compared using the GENETYX-WIN software package version 5 (Software Development, Tokyo, Japan).

Antibody preparation

A peptide containing 13 amino acids (ETEKRIATHGVRC, positions 2–15) near the amino-terminus of mouse oatp3 was synthesized. This peptide was linked to the maleimide-activated keyhole limpet hemocyanin (KLH; Pierce Rockford, IL, USA). The KLH-linked peptide (100 µg/injection) was emulsified by mixing with an equal volume of Freund's adjuvant and injected into male rabbits. Boosts were performed at 2, 4, 7, 10 and 13 weeks, and animals were killed at 15 weeks. The antibodies were affinity-purified using CNBr-activated Sepharose CL-4B (Amersham Pharmacia Biotech, Piscataway, CA, USA) coupled with synthetic peptides according to the standard procedure.

Western blot analysis

The brain capillary-rich fraction was collected from brain homogenates as described previously (Hosoya et al. 2000). Briefly, the cerebrum was excised from mouse, dissected into 2 mm pieces, and homogenized using a Potter-Elvehjem homogenizer in extracellular fluid buffer containing 10 mm HEPES (pH 7.4), 122 mm NaCl, 25 mm NaHCO3, 3 mm KCl, 1.4 mm CaCl2, 2 mm MgSO4, 0.4 mm K2HPO4, and 10 mm d-glucose. The homogenate was added to the same volume of 32% dextran solution, resulting in a 16% dextran solution, which was centrifuged (4500 × g, 20 min, 4°C). The resulting pellets were washed three times in phosphate-buffered saline to obtain the capillary-enriched fraction. Cultured cells were suspended in ice-cold hypotonic buffer containing 10 mm Tris (pH 7.4), 10 mm NaCl, 1.5 mm MgCl2, and incubated for 15 min at 4°C. The brain capillary-rich fraction, brain, liver and kidney were homogenized in ice-cold hypotonic buffer by means of 20 passes through a Teflon homogenizer. To prepare a plasma membrane fraction, the homogenates were centrifuged at 400 × g for 10 min to remove nuclei, and then the denucleated supernatants were centrifuged at 10 000 × g for 10 min, followed by at 100 000 × g for 1 h. The choroid plexus whole lysate was prepared by sonication in cell lysis buffer (1% sodium dodecyl sulfate, 1 mm EDTA, 10% glycerin, 1 mm Tris, pH 7.4).

The samples were electrophoresed on 7.5% sodium dodecyl sulfate–polyacrylamide gels and subsequently were electrotransferred to nitrocellulose membranes (Toyo Roshi, Tokyo, Japan). Membranes were treated with blocking solution (Block Ace; Dainippon Pharmaceuticals, Osaka, Japan) for 1 h at room temperature and were incubated with anti-mouse oatp3 antibody (10 µg/mL) overnight at 4°C. Membranes were washed four times with 0.1% Tween 20/phosphate-buffered saline and then incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1 : 500; ICN, Aurora, OH, USA) for 1 h at room temperature. Immunoreactivity was visualized with an enhanced chemiluminescence kit (Supersignal west pico chemiluminescent substrate; Pierce) and exposed with X-Omat AR (Kodak, NY, USA).

Immunohistochemical analysis

A ddY mouse (6 weeks old) was anesthetized with an intramuscular injection of ketamine and xylazine, and perfused transcardially with heparin/phosphate-buffered saline (1.9 U/mL) and 4% paraformaldehyde in 0.1 m phosphate buffer. Following perfusion, the brain was dissected out and stored overnight in 4% paraformaldehyde at 4°C. Prior to sectioning, the brain was infused with 0.5 m sucrose. Then, 15-µm frozen brain sections were fixed with 2% paraformaldehyde/0.1% Triton X-100 for 30 min on ice. After incubation in Histofine (Nichirei, Tokyo, Japan) for 1 h at room temperature, the sections were reacted with 10 µg/mL anti-oatp3 antibody or 1 : 20 MRPr1 (rat monoclonal IgG2a, Kamiya Biomedical Co., Seattle, WA, USA) overnight at 4°C, and then with fluorescein-conjugated swine anti-rabbit IgG (Dako, Glostrup, Denmark) or fluorescein-conjugated anti-rat IgG (F(AB′)2 fragment (ICN) at a 1 : 40 dilution for 1 h at room temperature. The nuclei were stained with 6.6 µm propidium iodide, and sections were viewed by confocal laser microscopy (TCS SP, Leica, Heidelberg, Germany).

Results

Expression of oatp3 and OATP1A subfamily mRNAs in mouse tissues

The mRNA expression of mouse oatp3 in various mouse tissues, including brain and brain capillaries, was examined by RT-PCR (Fig. 1). Single bands of the expected size were detected in brain, lung, choroid plexus, brain capillary-rich fraction and TM-BBB1 cells (conditionally immortalized mouse brain capillary endothelial cells). The nucleotide sequence of the amplified products was identical to that of mouse oatp3. This result suggests that mouse oatp3 is expressed in mouse brain and brain capillaries.

Figure 1.

mRNA expression of mouse oatp3 and OATP1A subtypes in mouse tissues. Primer sets specific for mouse oatp1, oatp2, oatp3 or oatp5 were used for PCR amplification. Reactions without reverse transcriptase (RTase) were performed as a control for contamination [RT (–)]. The molecular weight markers are shown on the left. Lane 1, brain capillary-rich fraction; lane 2, choroid plexus; lane 3, brain; lane 4, lung; lane 5, liver; lane 6, kidney; lane 7, TM-BBB1 cells.

Oatp3 belongs to the OATP1A subfamily of the oatp superfamily (Hagenbuch and Meier 2003). Therefore, mRNA expression of oatp1, oatp2 and oatp5, which are also subtypes of the OATP1A subfamily, was compared with that of oatp3 (Fig. 1). mRNA expression of oatp2 was detected in both brain capillary-rich fraction and choroid plexus, whereas oatp1 mRNA was detected weakly in brain capillary-rich fraction and not at all in choroid plexus. Oatp1 and 2 mRNAs were detected in the liver and kidney, despite the absence of oatp3 expression. Oatp5, which has been reported to be kidney-specific (Choudhuri et al. 2001), was most abundantly expressed in kidney, but was also detected in brain capillary-rich fraction, choroid plexus, brain, lung, liver and TM-BBB1 cells.

Western blot analysis of oatp3 in mouse BBB, BCSFB and tissues

Protein expression of oatp3 was examined using an antibody prepared against an N-terminal sequence of mouse oatp3. The membrane fraction of CHO-K1/oatp3 cells gave intense immunoreactivity at 75 kDa, whereas this immunoreactivity was not detected in CHO-K1 cells (Fig. 2a). This result indicates that the prepared antibody reacts with mouse oatp3, as expected. As shown in Fig. 2(b), the band at 75 kDa was detected in isolated mouse choroid plexus, but not in the membrane fraction of mouse brain capillary-rich fraction, brain, liver or kidney.

Figure 2.

Western blot analysis of mouse oatp3. (a) Membrane proteins of mouse oatp3 expressing CHO-K1 cells (CHO-K1/oatp3 cells; lane 1) and CHO-K1 cells (lane 2) were reacted with anti-mouse oatp3 polyclonal antibodies. (b) Western blot analysis of mouse oatp3 in choroid plexus whole lysate (lane 3), membrane proteins of mouse brain capillary-rich fraction (lane 4), brain (lane 5), liver (lane 6) and kidney (lane 7). All lanes were loaded with 50 µg of protein, except in the case of choroid plexus (25 µg). The molecular weight markers are shown on the left.

Immunohistochemical analysis of oatp3 in choroid plexus

The localization of oatp3 protein in mouse choroid plexus was investigated by immunohistochemical analysis with the anti-oatp3 antibody (Fig. 3). Intense immunoreactivity was observed at one surface of the plasma membrane of choroid plexus epithelial cells (Figs 3a and b). This immunoreactivity was not detected with rabbit normal immunoglobulin (Fig. 3d). To evaluate the membrane polarity of oatp3 localization, choroid plexus was stained with MRPr1 antibody, an anti-multidrug resistance associated protein antibody that reacts with the basolateral membrane of choroid plexus epithelial cells (Rao et al. 1999). As shown in Fig. 3(c), MRPr1 immunoreactivity was detected at the basal and lateral membranes of choroid plexus epithelial cells. The staining pattern by MRPr1 is different from that by anti-oatp3 antibody. Since plasma membrane of choroid plexus epithelial cells is separated into basolateral (basal and lateral) and apical membranes by tight junctions, the one-sided pattern of oatp3 suggests that mouse oatp3 is localized at the apical (brush-border) membrane of choroid plexus epithelial cells.

Figure 3.

Localization of mouse oatp3 at choroid plexus. Choroid plexus was reacted with anti-oatp3 antibody (a, b), MRPr1 antibody (c) or normal rabbit immunoglobulin (d). Fluorescent immunoreactivity is indicated by white color in each dark field.

Immunohistochemical analysis of oatp3 in the brain and brain capillaries

Immunohistochemical analysis with anti-oatp3 antibody was conducted to investigate the expression and localization of oatp3 in mouse brain. As shown in Fig. 4, intense immunoreactivity was detected in two brain regions. One region is the border between hypothalamus and thalamus (Fig. 4a), which is close to the 3rd ventricle (Figs 4b and c). The stained cells have neuron-like shapes (Fig. 4d). Oatp3 staining was also observed in the olfactory bulb (Figs 4e and f), and intense staining was detected in the glomerular layer. Such characteristic immunostaining was not seen following the use of rabbit normal immunoglobulin (data not shown). In cerebral cortex, positive staining was detected at the surface of capillaries (Figs 4g, h and i). Such a staining pattern was not detected with rabbit normal immunoglobulin (Fig. 4j).

Figure 4.

Localization of mouse oatp3 in the brain. Brain slices were reacted with anti-oatp3 antibody (a–i), or normal rabbit immunoglobulin (j). The immunoreactivity is indicated by green color, and red color indicates nuclei stained with propidium iodide. The images show the border regions between hypothalamus (a–d), olfactory bulb (e, f) and cerebral cortex (g–j). (c) and (f) are the overlaid images of (b) and (e) with propidium iodide staining, respectively. Asterisks in (b) and (c) indicate the 3rd ventricle. Arrows in (g) and (h) indicate staining at the brain capillary endothelial cells, and arrowheads in (j) indicate lumina of brain capillaries.

Discussion

In this study, we have examined the expression and localization of oatp3 in the brain and at the BBB of the mouse. There is controversy regarding the distribution of oatp3 in rat tissues. Northern blot analysis indicated that oatp3 is expressed in rat retina, liver and kidney, but not the brain (Abe et al. 1998), whereas the results of RNase protection assay indicated oatp3 expression in rat brain and lung (Walters et al. 2000). These apparent differences in tissue distribution may be ascribed to the similarity in nucleotide sequences among oatp family members, especially in the OATP1A subfamily. The expression in the liver and kidney, in addition to brain, is important information, since a number of oatp subtypes, including oatp1 and oatp2, function in the liver, and the kidney possesses various organic anion transport systems. We investigated the tissue distribution of mouse oatp3 by RT-PCR (Fig. 1). The detection specificity was confirmed by nucleotide sequencing of the amplified products. Mouse oatp3 is expressed in the brain and lung, but not in the liver and kidney, whereas other mouse OATP1A subtypes, oatp1, 2 and 5, are expressed in the liver and kidney.

Many oatp3 substrates and related compounds, including dehydroepiandrosterone sulfate, PGE2, T3 and T4, influence CNS functions, suggesting that oatp3 may play a role in the brain. The immunohistochemical analysis in this study showed that oatp3 is localized in limited regions of the mouse brain (Fig. 4). The prepared anti-oatp3 antibody reacted with mouse oatp3 at 75 kDa in CHO-K1/oatp3 cells and choroid plexus (Fig. 2). The band size at 75 kDa is similar to that of rat oatp3 detected in retina (Ito et al. 2002). In contrast, no band was detected in the liver or kidney (Fig. 2b). Since oatp1, 2 and 5 are expressed abundantly in liver and/or kidney (Fig. 1), the antibody appears to react selectively with mouse oatp3.

A band was not detected in brain capillary-rich fraction or brain, where oatp3 mRNA expression was detected (Figs 1 and 2). As shown in Fig. 4, the immunoreactivity was detected in restricted brain regions. In addition, a previous immunohistochemical study showed that anti-oatp2 antibody gave intense immunoreactivity on endothelial cells of cerebral capillaries, whereas this antibody exhibited only a weak band of oatp2 in a similar amount (30 µg) of rat brain membrane fraction by western blotting (Gao et al. 1999). mRNA expression of oatp2 in mouse brain is greater than that of oatp3 (Fig. 1), and the immunoreactivity of oatp3 at the capillaries was weak and mainly restricted to the cerebral cortex (Fig. 4). Therefore, the lack of signal in brain and brain capillary-rich fraction is likely to be due to the lower sensitivity of western blotting as compared with RT-PCR analysis, and the small amount of oatp3 protein in the brain. It is also conceivable that the efficiency of translation from oatp3 mRNA is low. Further study is necessary to clarify this issue, and to confirm that when the protein localized at limited brain and cellular regions, immunohistochemical analysis has higher sensitivity than western blotting.

Our recent report has shown that rat oatp3, but not oatp1, is localized at the brush-border membrane of rat choroid plexus epithelial cells (Ohtsuki et al. 2003). In mouse, oatp3 mRNA was detected in choroid plexus, but oatp1 mRNA was not (Fig. 1, lane 3). Immunohistochemical analysis (Fig. 3) suggested mouse oatp3 to be localized at the brush-border membrane of choroid plexus. Furthermore, rat oatp2 is localized at the basolateral membrane of choroid plexus epithelial cells (Gao et al. 1999), and mouse oatp2 was also detected in choroid plexus (Fig. 1). These results suggest that mouse oatp2 and 3 play roles at the BCSFB similar to those in the case of rat BCSFB (Gao et al. 1999; Ohtsuki et al. 2003).

The immunohistochemical analysis in this study shows intense expression of oatp3 in two brain regions, the border region between hypothalamus and thalamus and in the olfactory bulb (Fig. 4). The expression of rat oatp3 has also been detected in a similar rat brain region close to the 3rd ventricle (T. Abe, personal communication)1. Both regions are sensitive to THs (Calza et al. 2000). The olfactory bulb expresses TH receptors and thyrotropin releasing hormone (TRH) (Kreider et al. 1985; Puymirat et al. 1991). Neural cells in the hypothalamus around the 3rd ventricle are also sensitive to THs and express TH receptors (Puymirat et al. 1991); further, the cells at the border between the hypothalamus and thalamus were reported to express TRH (Lechan and Jackson 1982). These results suggested that oatp3 expression in these regions is related to the TH response.

To interact with TH receptors, THs must enter the cells. A carrier-mediated process, rather than passive diffusion, was reported to be involved in this plasma membrane transport process (Hennemann et al. 2001). Since rat oatp3 has the ability to transport T3 and T4 (Abe et al. 1998), a likely function of oatp3 in the brain would be mediation of TH uptake into TH-sensitive neural cells. We have obtained the first in vivo evidence supporting the involvement of oatp3 in TH interaction. To date, the only known functions of the oatp family in the brain have been in transport at the BBB and BCSFB (Asaba et al. 2000; Gao et al. 2000; Sugiyama et al. 2001; Hagenbuch and Meier 2003). Therefore, our findings indicate a novel function of oatp family members in the brain.

Oatp3 may be expressed in brain capillary endothelial cells to function as a BBB transporter. The expression of oatp3 mRNA was detected in brain capillary-rich fraction and conditionally immortalized mouse brain capillary endothelial cells (TM-BBB1 cells). The possibility that neural cells contaminated the brain capillary-rich fraction cannot be ruled out, but the expression of oatp3 in the brain is limited, as shown in the present immunohistochemical study (Figs 3 and 4). Therefore, the mRNA expression of oatp3 in the capillary-rich fraction is likely to reflect its expression in brain capillaries, as in the case of oatp2 (Gao et al. 1999). Furthermore, immunohistochemical analysis revealed that oatp3 is localized in brain capillary endothelial cells in the cerebral cortex (Fig. 4).

Oatp2 and oatp14 have been reported to be expressed in brain capillary endothelial cells (Gao et al. 1999; Sugiyama et al. 2003). Oatp3 and these transporters have similar substrate specificity, so that oatp2, 3 and 14 are likely to cooperate in BBB transport. On the other hand, the expression of oatp3 at the BBB can explain an important BBB function which is not mediated by oatp2 or 14, i.e. PGE2 is a substrate of oatp3, whereas oatp2 and 14 do not transport it (Cattori et al. 2001; Sugiyama et al. 2003). PGE2 functions as a signal to the brain of peripheral inflammation (Engblom et al. 2002). Two major pathways have been reported; first, plasma PGE2 is transferred into the brain across the BBB via a transport system (Engblom et al. 2002), and second, inflammation-induced cytokines in plasma induce PGE2 synthesis in endothelial cells via a receptor-mediated response (Davidson et al. 2001). The oatp3 expressed at the BBB is likely to be involved in the transport of PGE2. Since oatp1 and 2 are bidirectional anion exchangers (Li et al. 1998, 2000), the transport mode and expression polarity of oatp3 are important issues for further studies to improve our understanding of the biological function of oatp3 at the BBB.

The expression of oatp3 at brain capillaries was detected in the cerebral cortex (Figs 4g and h), but not in other brain regions, including hypothalamus (Figs 4a and b). Eguchi and coworkers have reported a heterogeneous regional distribution of [3H]PGE2 after intravenous administration (Eguchi et al. 1992). A significant distribution to the cerebral cortex was observed, and so this distribution appears to involve oatp3 expressed at the BBB. Their report also showed that the highest distribution of [3H]PGE2 was observed in the olfactory bulb and basal forebrain (Eguchi et al. 1992), though oatp3 was not detected at the brain capillaries in those regions. In addition, oatp3 localization in neural cells did not completely overlap with the distribution of TH-sensitive cells. Therefore, our findings indicate a novel oatp function in the brain, and, at the same time, raise the question of what subtypes of the oatp family are involved in TH uptake in neural cells and in PGE2 transport in the brain regions where oatp3 is not expressed.

Oatp5 has been identified as a kidney-specific organic anion transporter, and also belongs to the OATP1A subfamily (Choudhuri et al. 2001). Oatp5 has been detected selectively in mouse kidney by northern blotting (Choudhuri et al. 2001), whereas it was detected in mouse brain, choroid plexus, brain capillary-rich fraction and TM-BBB1 cells by RT-PCR, as shown in Fig. 1. This difference presumably reflects the difference in sensitivity between northern blot and RT-PCR analyses. This result suggests that oatp5 functions at the BBB and BCSFB, and also plays a role in the brain, although its transport properties have not yet been clarified.

In conclusion, this study has revealed the localization of oatp3 in the brain and at the BBB. Our histochemical results suggest two novel functions of oatp3. One is TH uptake into TH-sensitive neural cells, and the other is PGE2 transport at the BBB. This also throws new light on the CNS functions of oatp family members. OATP-A is the only human subtype belonging to the OATP1A subfamily that has been reported to be expressed at the BBB (Gao et al. 2000). Therefore, the brain localization of other oatp subtypes, including OATP-A, should be examined in order to understand further the CNS functions of these transporters.

Footnotes

  • 1

    Dr Takaaki Abe and his coworkers (Tohoku University, Sendai, Japan) have demonstrated the localization of rat oatp3 in rat brain parenchymal cells close to the 3rd ventricle by means of immunohistochemical analysis using anti-rat oatp3 antibody (Ito et al. 2002) in the 12th International Thyroid Congress at Kyoto, Japan (abstract SB-1).

Acknowledgements

The authors wish to thank Ms. N. Funayama for secretarial assistance. This study was supported, in part, by a Grant-in-Aid for Scientific Research, and a grant for the 21st Century Center of Excellence (COE) Program from the Japan Society for the Promotion of Science. It was also supported in part by the Industrial Technology Research Grant Program from New Energy and the Industrial Technology Development Organization (NEDO) of Japan.

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