ARTICLE |
Correspondence to: Peter J. Meier, Div. of Clinical Pharmacology and Toxicology, Dept. of Medicine, University Hospital, CH-8091 Zurich, Switzerland.
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Summary |
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In this study we investigated the distribution of a recently cloned polyspecific organic anion transporting polypeptide (Oatp2) in rat brain by nonradioactive in situ hybridization histochemistry and immunofluorescence microscopy. The results demonstrate that Oatp2 is expressed in brain capillary and in plexus epithelial cells. At the bloodbrain barrier (BBB), Oatp2 expression could be co-localized with the endothelial marker vWF (von Willebrand factor) but not with the astrocyte marker GFAP (glial fibrillary acidic protein). In choroid plexus epithelial cells, Oatp2 could be localized to the basolateral cell pole, whereas the first member of the Oatp gene family of membrane transporters to be cloned (Oatp1) co-localized with the 1-subunit of Na,K-ATPase at the apical plasma membrane domain. Because Oatp1 and Oatp2 have been previously shown to mediate transmembrane transport of a wide variety of amphipathic organic compounds, including many drugs and other xenobiotics, the histochemical localization of Oatp2 at the BBB and of Oatp1 and Oatp2 in the choroid plexus imply a role for these transporters in the active exchange of amphipathic solutes between the blood, brain, and cerebrospinal fluid compartments. (J Histochem Cytochem 47:12551263, 1999)
Key Words: drug transport, organic anion transporter, bloodbrain barrier, choroid plexus
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Introduction |
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The bloodbrain barrier (BBB) endothelium and the choroid plexus epithelium control the exchange of many endogenous and exogenous compounds between the blood plasma and the brain tissue and cerebrospinal fluid, respectively. Several specific and nonspecific transport systems have been described that permit access of essential nutrients and antioxidants to brain tissue and/or prevent the intracerebral accumulation of potentially neurotoxic compounds (
Recently, a novel multispecific organic anion transporting polypeptide (Oatp2) with an exceptionally high affinity for digoxin (Km ~0.24 µM) has been cloned from rat brain (
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Materials and Methods |
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Animals
Male SpragueDawley rats (SUT:SDT) weighing 200250 g were obtained from the Institute für Labortierkunde, University of Zurich (Zurich, Switzerland) and kept under standard conditions.
Riboprobes
Full-length antisense and sense Oatp2 cRNAs were labeled with digoxigenin. The 3.6-kb long probes were transcribed in vitro from an Oatp2 cDNA ligated into the Uni-ZAP XR vector (
Antibodies and Western Blotting
Antisera were raised in rabbits against a fusion protein containing the last 40 amino acids of Oatp1 (
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In Situ Hybridization Histochemistry (ISHH)
After decapitation of the animals the brains were removed and frozen immediately on dry ice. Cryostat sections (12 µm) were mounted on glass slides coated with 3-aminopropyltriethoxysilane (Sigma; St Louis, MO) and stored at -80C until use. The sections were fixed with 2% paraformaldehyde in PBS for 20 min, acetylated in 0.1 M triethanolamine hydrochloride containing 0.25% acetic anhydride, and prehybridized for 23 hr at room temperature (RT) with hybridization buffer containing 50% formamide, 5 x standard saline citrate (SSC), 5 x Denhardt's solution, 250 µg/ml yeast tRNA, and 500 µg/ml hering sperm DNA. Then the sections were hybridized overnight at 53C with the digoxigenin-labeled antisense or sense probe dissolved in the hybridization buffer at a concentration of about 1 µg/ml. Sections were then sequentially washed at 53C with SSC, using a final concentration of 0.1 x SSC/50% formamide for 20 min. Thereafter, the sections were processed for immunodetection with anti-DIGalkaline phosphatase (Boehringer) and with nitrotetrazolium blue and X-phosphate as the color substrate (Boehringer) according to the manufacturer's instructions. Sections were incubated at RT for 1 h with the anti-DIGalkaline phosphatase at a dilution of 1:5000 in 100 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 1% blocking reagent. After several washings, additional incubations were performed with nitrotetrazolium blue/X-phosphate at a dilution of 1:50 and 1 mM levamisole (Sigma) in 100 mM Tris-HCl (pH 9.5), 100 mM NaCl, and 50 mM MgCl2 at RT overnight in the dark.
Immunofluorescence Staining
To more definitively localize Oatp2 in brain capillary endothelial cells, cryostat sections subjected to ISHH analysis were also processed for immunofluorescence labeling with antibodies against von Willebrand factor (vWF; Sigma), an endothelial cell marker, and against glial fibrillary acidic protein (GFAP; Dako, Glostrup, Denmark), a marker for astrocytes. The sections were incubated overnight at 4C with the perspective antibody diluted in PBS containing 2% normal goat serum and 0.2% Triton X-100. The concentration of the rabbit anti-vWF was 1:400 and for the mouse anti-GFAP 1:5000. Finally, the sections were washed with PBS and incubated for 30 min at RT with the Cy3-labeled secondary antibody (1:300) (Jackson Immunoresearch; West Grove, PA).
Immunoperoxidase Staining
Rats were anesthetized with sodium pentobarbital (40 mg/kg IP). Brains were fixed with 2% paraformaldehyde and 15% of a saturated solution of picric acid in phosphate buffer (0.15 M, pH 7.4) through the ascending aorta. They were then removed, postfixed at 4C for 4 hr in the same fixative, and stored overnight at 4C in PBS containing 10% dimethylsulfoxide for cryoprotection. Sections of 40 µm were cut with a sliding microtome and collected in PBS. The sections were incubated overnight at 4C with the Oatp2 antiserum diluted at 1:20,000 in Trissaline (pH 7.4) containing 2% normal serum and 0.2% Triton X-100. Then, they were washed in Trissaline and stained with the ABC immunoperoxidase method using the Vectastain Elite kit (Vector Laboratories; Burlingame, CA) and diaminobenzidine hydrochloride as the chromogen. In some experiments, 12-µm cryostat sections were also used. They were fixed with 2% paraformaldehyde for 20 min and incubated with Oatp2 antiserum at a dilution of 1:5000.
Double Immunofluorescence Staining
Fresh-frozen and paraformaldehyde (2%)-fixed (20 min) cryostat sections (12 µm) were incubated overnight at 4C in PBS containing 2% normal serum, 0.2% Triton X-100, and the primary antibodies against Oatp1 (1:500), Oatp2 (1:5000), GFAP (1:5000), (1-subunit of Na,K-ATPase (2 µg/ml; Upstate Biotechnology, Lake Placid, NY), and the P-glycoprotein (antibody C219, 1:50; Signet Laboratories, Dedham, MA). After washing in PBS, the sections were incubated at RT with affinity-purified secondary goat antibodies (Jackson Immunoresearch) labeled with Cy2 (1:100) and Cy3 (1:300). The immunofluorescently stained sections were analyzed by confocal laser microscopy (MRC 600; Bio-Rad Laboratories, Hercules, CA) using dual-channel illumination with simultanous image recording for the two different fluorochromes. The pictures were processed by IMARIS software (Bitplane; Zurich, Switzerland). Control experiments for anti-Oatp2 specificity were performed by preincubating the antibody with increasing concentrations (35 µg/ml) of the peptide used for immunization. To control for crossreactivity by the secondary antibodies, one of the primary antibodies was omitted during the incubation.
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Results |
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Immunoblot Analysis of the Oatp2 Antiserum
As shown in Figure 1, the developed Oatp2 antiserum reacted with two narrow protein bands of crude rat brain membranes (Figure 1, left lane). However, whereas the 66-kD protein remained constant, the 76-kD protein band disappeared after preabsorption of the Oatp2 antibody in the presence of an excess of the oligopeptide used for immunization (Figure 1, right lane). A similar narrow 76-kD brain Oatp2 protein band has very recently also been reported by others using a different antibody (
Localization of Oatp2 in Brain Capillary Endothelial Cells
We first used ISHH to localize Oatp2 mRNA in rat brain tissue. As shown in Figure 2A2C, positive hybridization signals were associated exclusively with endothelial cells of cerebral capillaries. In contrast to the antisense probe, no positive hybridization signal was obtained with the sense probe (Figure 2A, inset), thus supporting the specific reactivity of the antisense riboprobe with Oatp2 mRNA. The ISHH-positive capillary endothelial cells were scattered diffusely over all brain regions, with the exception of the pineal gland, pituitary gland, subfornical organ, median eminance, and choroid plexus (not shown). Similar results were obtained at the protein level, where the Oatp2 antibody also selectively reacted with the capillary endothelium (Figure 2D and Figure 2E). Finally, after preabsorption of the Oatp2 antiserum with the antigenic peptide used for immunization, the immunopositive reactivity of the brain capillary endothelium was completely lost (Figure 2F), again demonstrating the specificity of the Oatp2 antiserum and supporting the idea that the preabsorption sensitive 76-kD protein band in Figure 1 corresponds to Oatp2 in brain capillary endothelium cells.
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To more definitely discriminate between Oatp2 expression in capillary endothelial cells and in astrocytes, double labeling experiments were performed that combined in situ hybridization for Oatp2 mRNA with immunofluorescence staining for the endothelial marker vWF or the astrocyte marker GFAP. These experiments revealed that most, if not all, cells expressing Oatp2 mRNA were immunopositive for vWF and vice versa (Figure 3A) and that positivity for Oatp2 mRNA did not correlate with immunopositivity for GFAP, indicating that astrocytes do not express Oatp2 (Figure 3B). In addition, double immunofluorescence staining with Oatp2 and GFAP antibodies combined with confocal microscopic analysis indicated that Oatp2 is localized on both the abluminal and the luminal domain of brain capillary endothelial cells (Figure 3C3F). Furthermore, Figure 3E shows the intimate contacts between the brain capillaries and the surrounding GFAP-positive astrocyte endings that serve as ideal markers for the brain side of the blood vessels. In contrast, the blood-oriented luminal side of brain capillary endothelium is characterized by its expression of mdr1a or P-glycoprotein (
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Localization of Oatp2 Compared to Oatp1 in Choroid Plexus Epithelial Cells
Oatp2 mRNA was also detected in the choroid plexus. However, in contrast to the BBB capillary endothelium, Oatp2 mRNA (Figure 4A) and Oatp2 protein (Figure 4B) were detected only in choroid plexus epithelial cells. Moreover, the immunofluorescence staining indicated that Oatp2 is selectively expressed at the basolateral pole of choroid plexus epithelial cells (Figure 4B). This basolateral Oatp2 expression is further shown in Figure 5, in which the apical membrane of the choroid plexus epithelial cells was selectively labeled with an antibody against the 1-subunit of the Na,K-ATPase (
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Discussion |
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This study, for the first time, localizes the multispecific organic anion transporter Oatp2 at the BBB and demonstrates a polar distribution of Oatp1 and Oatp2 in the choroid plexus epithelium.
The BBB barrier is formed by a tight endothelium lining the cerebral microvessels. Effective tight junctions restrict solute uptake across the paracellular pathway to small water-soluble compounds and make transcellular routes necessary for uptake of larger organic solutes into brain tissue. These transcellular routes have been divided into a diffusional pathway for lipid-soluble agents, specific transport systems for essential nutrients such as glucose, amino acids, and purines, and receptor-mediated and adsorptive endocytosis for hormones and plasma proteins, respectively (1-opioid receptor agonist [D-Pen2,D-Pen2] enkephalin across the BBB (
Interestingly, Oatp2 is also localized at the basolateral cell pole of choroid plexus epithelial cells (Figure 4 and Figure 5). The polar localization of Oatp1 and Oatp2 at the apical and basolateral surface domains, respectively, indicates that these two transporters may serve complementary functions in the transport of amphipathic organic anions into and/or out of the cerebrospinal fluid. Previous in vitro studies have provided evidence for the presence of at least one "liver-like" organic anion transporter in the choroid plexus of mammalian brain (
In contrast to a recent study by Abe and co-workers (1998), we found no evidence for expression of Oatp2 mRNA in hippocampal neuronal cells (Figure 2). A similar discrepancy between their study and ours also exists with respect to Oatp2 mRNA expression in the cerebellar cortex (our negative data; not shown). The most probable cause of these discrepancies lies in the different ISHH methodology used. Whereas Abe and co-workers used a radiolabeled antisense cRNA probe, we used a digoxigenin-labeled antisense probe and a different ISH protocol. It is noteworthy, however, that in preliminary experiments with a radiolabeled probe we also found strong positivity in hippocampal and cerebellar neuronal cells. This neuronal labeling was judged as unspecific because similar positive labeling was also found with the sense probe. Furthermore, the neuronal cell labeling collapsed with the digoxigenin-labeled antisense probe, whereas the strong labeling of the brain capillary and choroid plexus epithelial cells remained constant independently of the labeling methods used. Finally, no immunopositivity of hippocampal and/or cerebellar neuronal cells was found with the Oatp2 antiserum used. Hence, although this study presents strong evidence for Oatp2 expression in the brain capillary endothelium and in the choroid plexus epithelium at both the mRNA and the protein level, definitive demonstration of additional expression of Oatp2 in neuronal cells requires further investigation.
In conclusion, we have localized Oatp2 at the BBB endothelium and demonstrate a polar distribution of Oatp1 and Oatp2 at the apical and basolateral cell poles of choroid plexus epithelial cells. At both sites, the expression of members of the Oatp gene family of membrane transporters can explain some phenotypic transport activities previously identified in functional in vivo and in vitro studies. The results demonstrate that more carriers than hitherto assumed (
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Acknowledgments |
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Supported by the Swiss National Science Foundation (grant 31-045536.95) and the Olga Mayenfisch Foundation, Zurich, Switzerland.
We thank Dr H.U. Luder for allowing us to use the technical facilities of the Division of Oral Structural Biology of the University Centre for Dental Medicine, Zurich, Switzerland.
Received for publication December 4, 1998; accepted April 20, 1999.
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