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• W2094704242 abstract "Oatp14/blood-brain barrier-specific anion transporter 1 (Slc21a14) is a novel member of the organic anion transporting polypeptide (Oatp/OATP) family. Northern blot analysis revealed predominant expression of Oatp14 in the brain, and Western blot analysis revealed its expression in the brain capillary and choroid plexus. Immunohistochemical staining indicated that Oatp14 is expressed in the border of the brain capillary endothelial cells. When expressed in human embryonic kidney 293 cells, Oatp14 transports thyroxine (T4; prothyroid hormone) (Km = 0.18 μm), as well as amphipathic organic anions such as 17β estradiol-d-17β-glucuronide (Km = 10 μm), cerivastatin (Km = 1.3 μm), and troglitazone sulfate (Km = 0.76 μm). The uptake of triiodothyronine (T3), an active form produced from T4, was significantly greater in Oatp14-expressed cells than in vector-transfected cells, but the transport activity for T3 was ∼6-fold lower that for T4. The efflux of T4, preloaded into the cells, from Oatp14-expressed cells was more rapid than that from vector-transfected cells (0.032 versus 0.006 min–1). Therefore, Oatp14 can mediate a bidirectional transport of T4. Sulfobromophthalein, taurocholate, and estrone sulfate were potent inhibitors for Oatp14, whereas digoxin, p-aminohippurate, or leukotriene C4, or organic cations such as tetraetheylammonium or cimetidine had no effect. The expression levels of Oatp14 mRNA and protein were up- and down-regulated under hypo- and hyperthyroid conditions, respectively. Therefore, it may be speculated that Oatp14 plays a role in maintaining the concentration of T4 and, ultimately, T3 in the brain by transporting T4 from the circulating blood to the brain. Oatp14/blood-brain barrier-specific anion transporter 1 (Slc21a14) is a novel member of the organic anion transporting polypeptide (Oatp/OATP) family. Northern blot analysis revealed predominant expression of Oatp14 in the brain, and Western blot analysis revealed its expression in the brain capillary and choroid plexus. Immunohistochemical staining indicated that Oatp14 is expressed in the border of the brain capillary endothelial cells. When expressed in human embryonic kidney 293 cells, Oatp14 transports thyroxine (T4; prothyroid hormone) (Km = 0.18 μm), as well as amphipathic organic anions such as 17β estradiol-d-17β-glucuronide (Km = 10 μm), cerivastatin (Km = 1.3 μm), and troglitazone sulfate (Km = 0.76 μm). The uptake of triiodothyronine (T3), an active form produced from T4, was significantly greater in Oatp14-expressed cells than in vector-transfected cells, but the transport activity for T3 was ∼6-fold lower that for T4. The efflux of T4, preloaded into the cells, from Oatp14-expressed cells was more rapid than that from vector-transfected cells (0.032 versus 0.006 min–1). Therefore, Oatp14 can mediate a bidirectional transport of T4. Sulfobromophthalein, taurocholate, and estrone sulfate were potent inhibitors for Oatp14, whereas digoxin, p-aminohippurate, or leukotriene C4, or organic cations such as tetraetheylammonium or cimetidine had no effect. The expression levels of Oatp14 mRNA and protein were up- and down-regulated under hypo- and hyperthyroid conditions, respectively. Therefore, it may be speculated that Oatp14 plays a role in maintaining the concentration of T4 and, ultimately, T3 in the brain by transporting T4 from the circulating blood to the brain. Brain capillary endothelial cells are characterized by tightly sealed cellular junctions (tight junctions) and the paucity of fenestra and pinocytotic vesicles, which prevent free exchange between brain and blood (1Rapoport S.I. Exp. Neurol. 1976; 52: 467-479Crossref PubMed Scopus (80) Google Scholar, 2Pardridge W.M. Semin. Cell Biol. 1991; 2: 419-426PubMed Google Scholar). Therefore, the uptake of nutrients by the brain occurs through the brain capillary endothelial cells via specific transport systems (3Minn A. Ghersi-Egea J.F. Perrin R. Leininger B. Siest G. Brain Res. Brain Res. Rev. 1991; 16: 65-82Crossref PubMed Scopus (157) Google Scholar, 4Strazielle N. Ghersi-Egea J.F. J. Neurosci. 1999; 15: 6275-6289Crossref Google Scholar, 5Suzuki H. Terasaki T. Sugiyama Y. Adv. Drug. Deliv. Rev. 1997; 25: 257-285Crossref Scopus (78) Google Scholar, 6Kusuhara H. Sugiyama Y. Drug Discov. Today. 2001; 6: 150-156Crossref PubMed Scopus (155) Google Scholar, 7Lee G. Dallas M. Hong M. Bendayan R. Pharmacol. Rev. 2001; 52: 569-596Crossref Scopus (249) Google Scholar). Metabolic enzymes and efflux transporters expressed in the brain capillaries facilitate the elimination of endogenous wastes and xenobiotics from the brain, and restrict their brain accumulation (3Minn A. Ghersi-Egea J.F. Perrin R. Leininger B. Siest G. Brain Res. Brain Res. Rev. 1991; 16: 65-82Crossref PubMed Scopus (157) Google Scholar, 4Strazielle N. Ghersi-Egea J.F. J. Neurosci. 1999; 15: 6275-6289Crossref Google Scholar, 5Suzuki H. Terasaki T. Sugiyama Y. Adv. Drug. Deliv. Rev. 1997; 25: 257-285Crossref Scopus (78) Google Scholar, 6Kusuhara H. Sugiyama Y. Drug Discov. Today. 2001; 6: 150-156Crossref PubMed Scopus (155) Google Scholar, 7Lee G. Dallas M. Hong M. Bendayan R. Pharmacol. Rev. 2001; 52: 569-596Crossref Scopus (249) Google Scholar). Because of these characteristics, the brain capillaries are referred to as the blood-brain barrier (BBB). 1The abbreviations used are: BBB, blood-brain barrier; Oatp, organic anion transporting polypeptide; BSAT, BBB-specific anion transporter; HEK293, human embryonic kidney 293; CA, cholate; GCA, glycocholate; LCA, lithocholate; CDCA, chenodeoxycholate; UDCA, ursodeoxycholate; PGD2, prostaglandin D2; PGE2, prostaglandin E2; E3040, 6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridylmethyl) benzothiazole; PBS, phosphate-buffered saline; DPDPE, [d-penicillamine2,5]-enkephalin; E217βG, 17β estradiol-d-17β-glucuronide; T4, thyroxine; TLCS, taurolithocholate sulfate; 4-MUS, 4-methylumbelliferone sulfate; TRO-S, troglitazone sulfate; RT, reverse transcriptase; MMI, methimazole; T3, triiodothyronine; ES, estrone sulfate; BSP, sulfobromophthalein; LT, leukotriene; D2, type 2 iodothyronine deiodinase. The organic anion transporting polypeptides (Oatps in rodents and OATPs in human) belong to the growing gene family of organic anion/prostaglandin transporters that can mediate sodium-independent membrane transport of numerous endogenous and xenobiotic amphipathic compounds (8Kullak-Ublick G.A. Stieger B. Hagenbuch B. Meier P.J. Semin. Liver Dis. 2000; 20: 273-292Crossref PubMed Scopus (240) Google Scholar, 9Kullak-Ublick G.A. Ismair M.G. Stieger B. Landmann L. Huber R. Pizzagalli F. Fattinger K. Meier P.J. Hagenbuch B. Gastroenterology. 2001; 120: 525-533Abstract Full Text Full Text PDF PubMed Scopus (642) Google Scholar). Fourteen members of the Oatp/OATP gene family have been identified in rodents and humans, and they are classified within the gene superfamily of solute carriers as the Slc21a/SLC21A gene family (Human Gene Nomenclature Committee DataBase) (8Kullak-Ublick G.A. Stieger B. Hagenbuch B. Meier P.J. Semin. Liver Dis. 2000; 20: 273-292Crossref PubMed Scopus (240) Google Scholar, 9Kullak-Ublick G.A. Ismair M.G. Stieger B. Landmann L. Huber R. Pizzagalli F. Fattinger K. Meier P.J. Hagenbuch B. Gastroenterology. 2001; 120: 525-533Abstract Full Text Full Text PDF PubMed Scopus (642) Google Scholar). Several members of the Oatp/OATP family have been identified in the brain (Oatp1–3 and moat1 in rodents and OATP-A in human) (10Jacquemin E. Hagenbuch B. Stieger B. Wolkoff A.W. Meier P.J. Proc. Natl. Acad. Sci. U.S.A. 1994; 91: 133-137Crossref PubMed Scopus (550) Google Scholar, 11Noe B. Hagenbuch B. Stieger B. Meier P.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10346-11035Crossref PubMed Scopus (390) Google Scholar, 12Abe T. Kakyo M. Sakagami H. Tokui T. Nishio T. Tanemoto M. Nomura H. Hebert S.C. Matsuno S. Kondo H. Yawo H. J. Biol. Chem. 1998; 273: 22395-22401Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar, 13Nishio T. Adachi H. Nakagomi R. Tokui T. Sato E. Tanamoto M. Fujiwara K. Okabe M. Onogawa T. Suzuki T. Nakai D. Shiiba K. Suzuki M. Ohtani H. Kondo Y. Unno M. Ito S. Iinuma K. Nunoki K. Matsuno S. Abe T. Biochem. Biophys. Res. Commun. 2000; 275: 831-838Crossref PubMed Scopus (70) Google Scholar, 14Kullak-Ublick G.A. Hagenbuch B. Stieger B. Schteingart C.D. Hofmann A.F. Wolkoff A.W. Meier P.J. Gastroenterology. 1995; 109: 1274-1282Abstract Full Text PDF PubMed Scopus (373) Google Scholar). Especially, in the BBB, rat Oatp2 and human OATP-A have been shown to be expressed in the plasma membrane of the brain capillary endothelial cells (15Gao B. Stieger B. Noe B. Fritschy J.M. Meier P.J. J. Histochem. Cytochem. 1999; 47: 1255-1264Crossref PubMed Scopus (269) Google Scholar, 16Gao B. Hagenbuch B. Kullak-Ublick G.A. Benke D. Aguzzi A. Meier P.J. J. Pharmacol. Exp. Ther. 2000; 294: 73-79PubMed Google Scholar). Involvement of rat Oatp2 in the uptake and efflux transport of its substrates was investigated in vivo (17Dagenais C. Ducharme J. Pollack G.M. Neurosci. Lett. 2001; 301: 155-158Crossref PubMed Scopus (64) Google Scholar, 18Sugiyama D. Kusuhara H. Shitara Y. Abe T. Meier P.J. Sekine T. Endou H. Suzuki H. Sugiyama Y. J. Pharmacol. Exp. Ther. 2001; 298: 316-322PubMed Google Scholar). The uptake of [d-penicillamine2,5]-enkephalin (DPDPE) from the blood to the brain was determined by the brain perfusion technique in the presence and absence of Oatp2 inhibitors (17Dagenais C. Ducharme J. Pollack G.M. Neurosci. Lett. 2001; 301: 155-158Crossref PubMed Scopus (64) Google Scholar). The brain uptake of DPDPE was increased in Mdr1a (P-glycoprotein) gene knockout mice, and the uptake in Mdr1a knockout mice was inhibited by the substrates and inhibitors of rat Oatp2 such as digoxin and 17β estradiol-d-17β-glucuronide (E217βG). Vice versa, when E217βG was microinjected into the cerebral cortex, the subsequent elimination of E217βG from the brain was carrier-mediated (18Sugiyama D. Kusuhara H. Shitara Y. Abe T. Meier P.J. Sekine T. Endou H. Suzuki H. Sugiyama Y. J. Pharmacol. Exp. Ther. 2001; 298: 316-322PubMed Google Scholar), and the elimination of E217βG was completely inhibited by co-administration of taurocholate and probenecid, whereas digoxin had only a partial effect (18Sugiyama D. Kusuhara H. Shitara Y. Abe T. Meier P.J. Sekine T. Endou H. Suzuki H. Sugiyama Y. J. Pharmacol. Exp. Ther. 2001; 298: 316-322PubMed Google Scholar). Partial inhibition by digoxin suggested that additional efflux transport system(s) for E217βG, which is taurocholate- and probenecid-sensitive, is involved in the brain capillary. Li et al. (19Li J.Y. Boado R. Pardridge W.M. J. Cereb. Blood Flow Metab. 2001; 21: 61-68Crossref PubMed Scopus (126) Google Scholar) recently identified BBB-specific anion transporter 1 (BSAT1) using gene microarray techniques by comparing the gene expression profile of cDNA from the brain capillary with that from the liver and kidney. BSAT1 cDNA consisted of 2148 bp that encoded a 716-amino acid residues protein with 12 putative membrane-spanning domains. BSAT1 was highly enriched in the brain capillary compared with brain homogenate, liver, and kidney. Comparison of the cDNA sequences of BSAT1 revealed that it is the 14th member of the Oatp/OATP family (Oatp14). Although the localization at the BBB and the substrates of this isoform remain unknown, BBB-specific expression prompted us to hypothesize that Oatp14 accounts for the efflux of organic anions including E217βG via the BBB, together with Oatp2. The purpose of the present study is to characterize the substrate specificity and spectrum of inhibitors of Oatp14, as well as its tissue distribution and localization. Through this study, we found out that thyroxine (T4) is a good substrate of Oatp14, and the expression level of Oatp14 in the BBB is affected by plasma thyroid conditions. The results of the present study suggest that Oatp14 plays an important role in regulating the concentration of T4 in the central nervous system and in brain development. Chemicals—[3H]Leu-enkephalin was purchased from American Radiolabeled Chemicals (St. Louis, MO). [3H]Pravastatin was kindly donated from Sankyo (Tokyo, Japan), [14C]cerivastatin was from Bayer AG (Wuppertal, Germany), and [14C]E3040 glucuronide and [14C]E3040 sulfate were from Eisai (Tokyo, Japan). [3H]Taurolithocholate sulfate (TLCS), [35S]4-methylumbelliferone sulfate (4-MUS), and [35S]troglitazone sulfate (TRO-S) were synthesized according to a method described previously (20Izumi T. Hosiyama K. Enomoto S. Sasahara K. Sugiyama Y. J. Pharmacol. Exp. Ther. 1997; 280: 1392-40021PubMed Google Scholar, 21Akita H. Suzuki H. Ito K. Kinoshita S. Sato N. Takikawa H. Sugiyama Y. Biochim. Biophys. Acta. 2001; 1511: 7-16Crossref PubMed Scopus (173) Google Scholar). The radiochemical purity of [3H]TLCS, [35S]4-MUS, and [35S]TRO-S prepared by this method were more than 95%. Other labeled compounds were purchased from PerkinElmer Life Science. Unlabeled pravastatin, troglitazone, and its conjugated metabolites were kindly donated from Sankyo, unlabeled cerivastatin was from Bayer AG, and unlabeled E3040 glucuronide and E3040 sulfate were from Eisai. All other chemicals were commercially available, of reagent grade, and were used without any purification. Capillary Isolation—Rat brain capillaries were isolated using a modification of the procedure of Boado et al. (22Boado R.J. Pardridge W.M. J. Neurochem. 1991; 57: 2136-2139Crossref PubMed Scopus (60) Google Scholar). All steps in the isolation procedure were carried out at 4 °C in pregassed (95% O2-5% CO2) solutions. Briefly, pieces of gray matter were gently homogenized in three volumes (v/w) of an artificial extracellular fluid buffer and, after addition of dextran (final concentration 15%), the homogenate was centrifuged at low speed. The resulting pellet was resuspended in Buffer B (103 mm NaCl, 25 mm NaHCO3, 10 mm d-glucose, 4.7 mm KCl, 2.5 mm CaCl2, 1.2 mm MgSO4, 1.2 mm K2HPO4, and 15 mm HEPES, 1 mm sodium pyruvate, 0.5% (w/v) bovine serum albumin, pH 7.4) and then filtered through a 200-μm nylon mesh. The filtrate was passed over a column of glass beads, and after washing with Buffer B, the capillaries adhering to the beads were collected by gentle agitation. Northern Blot Analysis—A commercially available hybridization blot containing poly(A)+ RNA from various rat tissues (rat multi-tissue Northern blot; Clontech) was used for the Northern blot analysis. A fragment (position numbers 1–807) from Oatp14 was used as a probe, and its nucleotide sequence showed less than 60% identity with other members of the Oatp family. The master blot filter was hybridized with the 32P-labeled probe at 68 °C according to manufacturer's instructions. The filter was washed finally under high stringency conditions (0.1× SSC (1× SSC = 0.15 m NaCl and 0.015 m sodium citrate)) and 0.1% SDS at 65 °C and then exposed to Fuji imaging plates (Fuji Photo Film, Kanagawa, Japan) for 3 h at room temperature and examined using an imaging analyzer (BAS 2000; Fuji Photo Film). Western Blot Analysis—Antiserum against Oatp14 was raised in rabbits against a synthetic peptide consisting of the 17 carboxyl-terminal amino acids of Oatp14. Antiserum was purified by affinity column chromatography using the antigen and used for subsequent analyses. Choroid plexus, brain homogenate, and isolated brain capillary samples were diluted with Loading Buffer (BioLabs, Hertfordshire, United Kingdom). They were then boiled for 3 min and loaded onto an 8.5% SDS-polyacrylamide electrophoresis gel with a 3.75% stacking gel. Proteins were electroblotted onto a polyvinylidene difluoride membrane (Pall Filtran, Karlstein, Germany) using a blotter (Trans-blot; Bio-Rad) at 15 V for 1 h. The membrane was blocked with TBS-T (Tris-buffered saline containing 0.05% Tween 20) and 5% skimmed milk for 1 h at room temperature. After washing with TBS-T, the membrane was incubated with the antibodies (dilution 1:1000). The membrane was allowed to bind a horseradish peroxidase-labeled anti-rabbit IgG antibody (Amersham Biosciences) diluted 1:5000 in TBS-T for 1 h at room temperature followed by washing with TBS-T. Immunohistochemical Study—Frozen sections from male Sprague-Dawley rats were prepared for the immunohistochemical study after fixing in acetone (–20 °C). The brain slices adhered to the glass cover slips were washed with PBS and fixed for 10 min on ice in acetone. After washing with PBS, the capillaries were permearized in 0.2% (v/v) Triton X-100 in PBS and incubated with peroxidase blocking reagent (DAKO, Carpinteria, CA) for 10 min at room temperature to block nonspecific peroxidase. Slices were incubated with anti-Oatp14 antibody (1:100) for 60 min at room temperature, washed three times with PBS, and subsequently incubated for 60 min at room temperature with the horseradish peroxidase-labeled anti-rabbit secondary antibody (Envision+ system; DAKO). The immune reaction was visualized using diaminobenzidine and then nuclei were stained with hematoxylin (DAKO). The specificity of the antibody reaction was verified by negative controls, which were incubated with polyclonal antibody that had been blocked with the antigenic peptide. Cloning of Rat Oatp14 cDNA—Based on the nucleotide sequence reported by Li et al. (19Li J.Y. Boado R. Pardridge W.M. J. Cereb. Blood Flow Metab. 2001; 21: 61-68Crossref PubMed Scopus (126) Google Scholar) (GenBank™ accession number NM 053441), the following primers were designed to isolate Oatp14 cDNA encoding a full open reading frame of Oatp14: forward primer, 5′-ggaattccgccaccatggacacttcatccaaaga-3′ and reverse primer, 5′-ggattccttaaagtcgggtctccttgc-3′. PCR was performed using cDNA prepared from rat brain as template according to the following protocol: 96 °C for 1 min, 55 °C for 1 min, and 72 °C for 2 min; 50 cycles. PCR products were subcloned to pGEM-T Easy Vector (Promega, Madison, WI) and sequenced. The nucleotide sequence of rat Oatp14 cDNA was identical as being that of BSAT1 except for one base change (A175G) resulting in a change of amino acid (T59A). However, it was confirmed that this change was not because of an error accumulated during PCR by sequencing the RT-PCR products directly. Stable Expression of Oatp14 cDNA in HEK293 Cells—The Oatp14-cDNA was subcloned into the pcDNA3.1(+) (Invitrogen) and introduced into HEK293 cells by lipofection with FuGENE 6 (Roche Diagnostics) according to the manufacturer's protocol and were selected by culturing them in the presence of G418 sulfate (800 μg/ml; Invitrogen). HEK293 cells were grown in minimum essential medium (Invitrogen) supplemented with 10% fetal bovine serum, penicillin (100 units/ml), streptomycin (100 μg/ml), and G418 sulfate (400 μg/ml) at 37 °C with 5% CO2 and 95% humidity. Cells were incubated for 24 h before starting the experiments with culture medium supplemented with sodium butyrate (5 mm). Transport Study—Uptake was initiated by adding the radiolabeled ligands to the incubating buffer in the presence and absence of inhibitors after cells had been washed three times and preincubated with Krebs-Henseleit buffer (142 mm NaCl, 23.8 mm NaHCO3, 4.83 mm KCl, 0.96 mm KH2PO4, 1.20 mm MgSO4, 12.5 mm HEPES, 5 mm glucose, and 1.53 mm CaCl2, adjusted to pH 7.4) at 37 °C for 15 min. For the efflux study, cells were preincubated with [125I]T4 at 37 °C for 15 min and washed three times with ice-cold Krebs-Henseleit buffer, followed by incubation in the absence of [125I]T4 with Krebs-Henseleit buffer at 37 °C. The uptake and efflux were terminated at designed times by adding ice-cold Krebs-Henseleit buffer. The radioactivity associated with the cells and medium specimens was determined in a liquid scintillation counter. The remaining aliquots of cell lysates were used to determine protein concentrations by the method of Lowry (23Lowry O. J. Biol. Chem. 1951; 193: 265-273Abstract Full Text PDF PubMed Google Scholar) with bovine serum albumin as a standard. Ligand uptake is given as the cell-to-medium concentration ratio determined as the amount of ligand associated with the cells divided by the medium concentration. Specific uptake was obtained by subtracting the uptake by vector-transfected cells from that by Oatp14-expressed cells. Kinetic Analyses—Kinetic parameters were obtained from the following Michaelis-Menten equation, v = V max S/(Km + S), where v is the uptake rate of the substrate (pmol/min/mg protein), S is the substrate concentration in the medium (μm), Km is the Michaelis-Menten constant (μm), and V max is the maximum uptake rate (pmol/min/mg protein). To obtain the kinetic parameters, the equation was fitted to the initial uptake velocity. The experimental data were fitted to the equation by nonlinear regression analysis with weighting as the reciprocal of the observed values, and the Damping Gauss Newton Method algorithm was used for fitting. Inhibition constants (Ki) for Oatp14-mediated transport were calculated assuming competitive inhibition. Production of Hyperthyroid and Hypothyroid Conditions—Male Sprague-Dawley rats, weighing 200–220 g, were purchased from Japan SLC (Shizuoka, Japan). Rats had free access to food and water at all times during the study. Production of hyperthyroid and hypothyroid conditions involved a modification of the procedure of Burmeister et al. (24Burmeister L.A. Pachucki J. Germain D.L.S. Endocrinology. 1997; 138: 5231-5237Crossref PubMed Google Scholar). Hypothyroidism was induced by the addition of 0.05% methimazole (MMI), an inhibitor for thyroid hormone synthesis in the thyroid gland, to the drinking water or thyroidectomy. Hypothyroidism was assessed clinically by failure to gain weight at the expected rate and could be observed within 2 weeks of the beginning MMI treatment and within 1 week after thyroidectomy. Hyperthyrodism was produced by giving L-T3 (50 μg/100 g body weight, subcutaneously, daily) 4 days before capillary isolation. Tissue Distribution of Oatp14 —The expression of Oatp14 mRNA in rat tissues was investigated by Northern blot analysis (Fig. 1A). A band was detected at 2.6 kbp, predominantly in the brain. No hybridization signals were detected in mRNA isolated from other tissues, including the heart, spleen, lung, liver, skeletal muscle, kidney, and testis. Immunoblot and Immunohistochemical Staining of Oatp14— The expression of Oatp14 in the choroid plexus, brain homogenate, and brain capillary was examined by Western blot analysis (Fig. 1B). Immunoreactive protein was detected at ∼90 kDa in the choroid plexus, brain homogenate, and brain capillary. These bands were abolished when preabsorbed polyclonal antibody for Oatp14 was used, suggesting that the positive bands were specific for the antigen peptide. To investigate the localization of Oatp14 in brain capillary endothelial cells, immunohistochemical staining was carried out using anti-Oatp14 polyclonal antibody (Fig. 2). Positive signals for anti-Oatp14 polyclonal antibody were detected in brain capillary endothelial cells. The signals were detected along the plasma membrane of brain capillary endothelial cells. The signal was abolished by preincubating the polyclonal antibody of Oatp14 with antigen (data not shown). Transport Properties of Oatp14 — Fig. 3 shows the time profiles of the uptake of [3H]E217βG (A), [14C]cerivastatin (B), [35S]TRO-S (C), and [125I]T4 (D) by Oatp14-expressed HEK293 cells and vector-transfected HEK293 cells. Their uptake by Oatp14-expressed cells is markedly greater than that by vector-transfected cells. This Oatp14-mediated uptake showed saturation kinetics and followed the Michaelis-Menten equation (Fig. 3, E-H). The kinetic parameters for the uptake by Oatp14 were determined by nonlinear regression analysis and summarized in Table I. The uptake of various organic anions by Oatp14 was investigated, and the results are summarized in Table II. The uptake of [14C]E3040 glucuronide, [14C]E3040 sulfate, [14C]4-MUS, and [125I]reverse T3 by Oatp14-expressed cells was significantly greater compared with that by vector-transfected (Table II). Although the triiodothyronine (T3) uptake by Oatp14-expressed cells was significantly greater than that by vector-transfected cells, the Oatp14-mediated uptake for T3 was ∼6-fold smaller than that of T4 and reverse T3 by Oatp14 (Table II). The difference in the uptake of [3H]taurocholate, [3H]TLCS, [3H]testosterone, [3H]dihydrotestosterone, [3H]estrone, [3H]estrone sulfate (ES), [3H]dehydroepiandrosterone sulfate, [3H]leukotriene E4 (LTE4), [3H]Leu-enkephalin, [3H]cholecystokinin-octapeptide (CCK-8), [125I]T3, [3H]pravastatin, [3H]ketoprofen, and [3H]ochratoxin A was statistically significant between Oatp14-expressed and vector-transfected cells, although the rates of uptake were very low (Table II).Table ISubstrate specificity of Oatp14SubstratesOatp14pcDNARatio, Oatp14/pcDNAμl/mg protein/15 minCA5.66 ± 0.305.24 ± 0.431.1 ± 0.1GCA18.7 ± 2.315.7 ± 1.61.2 ± 0.2TCA8.09 ± 0.526.06 ± 0.101.3 ± 0.1aStatistically significant uptake is indicated. p < 0.05.LCA576 ± 6535 ± 181.1 ± 0.0CDCA65.7 ± 6.784.1 ± 11.50.8 ± 0.1UDCA10.5 ± 0.19.71 ± 0.351.1 ± 0.0TLCS61.5 ± 1.943.7 ± 1.71.4 ± 0.1Estradiol204 ± 4186 ± 131.1 ± 0.1Testosterone52.3 ± 1.539.2 ± 1.41.3 ± 0.1bStatistically significant uptake is indicated. p < 0.01.Dihydrotestosterone147 ± 15106 ± 51.4 ± 0.2bStatistically significant uptake is indicated. p < 0.01.Corticosterone24.7 ± 1.520.9 ± 0.51.2 ± 0.1Estrone303 ± 15248 ± 61.2 ± 0.1aStatistically significant uptake is indicated. p < 0.05.DHEAS9.68 ± 0.247.72 ± 0.481.3 ± 0.1aStatistically significant uptake is indicated. p < 0.05.Estrone-sulfate11.1 ± 0.86.3 ± 0.11.7 ± 0.1aStatistically significant uptake is indicated. p < 0.05.E217βG50.1 ± 4.72.4 ± 0.221.2 ± 2.9bStatistically significant uptake is indicated. p < 0.01.LTC414.5 ± 0.613.4 ± 0.11.1 ± 0.0bStatistically significant uptake is indicated. p < 0.01.LTD419.6 ± 0.818.0 ± 2.01.1 ± 0.1LTE430.5 ± 1.326.0 ± 0.91.2 ± 0.1aStatistically significant uptake is indicated. p < 0.05.PGD23.50 ± 0.153.61 ± 0.121.0 ± 0.1PGE26.99 ± 0.465.80 ± 0.241.2 ± 0.1Leu-Enkephalin54.2 ± 2.943.3 ± 2.51.3 ± 0.1aStatistically significant uptake is indicated. p < 0.05.CCK-82.58 ± 0.211.81 ± 0.131.4 ± 0.2aStatistically significant uptake is indicated. p < 0.05.T3951 ± 16733 ± 41.3 ± 0.0bStatistically significant uptake is indicated. p < 0.01.Reverse T31397 ± 7971 ± 519.7 ± 1.7bStatistically significant uptake is indicated. p < 0.01.T41456 ± 10124 ± 311.8 ± 0.3bStatistically significant uptake is indicated. p < 0.01.Ketoprofen9.53 ± 0.426.91 ± 0.261.4 ± 0.1bStatistically significant uptake is indicated. p < 0.01.Ibuprofen3.18 ± 0.113.99 ± 1.180.8 ± 0.2Indomethacin31.3 ± 0.934.3 ± 2.00.9 ± 0.1Benzylpenicillin5.76 ± 0.475.45 ± 0.121.1 ± 0.1OchratoxinA8.58 ± 1.295.81 ± 0.141.5 ± 0.2bStatistically significant uptake is indicated. p < 0.01.Qunidine1390 ± 341274 ± 1501.1 ± 0.1Cerivastatin105 ± 233 ± 13.1 ± 0.1bStatistically significant uptake is indicated. p < 0.01.Pravastatin5.62 ± 0.383.50 ± 0.751.6 ± 0.4aStatistically significant uptake is indicated. p < 0.05.Digoxin8.70 ± 0.139.96 ± 0.220.9 ± 0.0E3040106 ± 393 ± 51.1 ± 0.1E3040G10.7 ± 1.02.12 ± 0.225.1 ± 0.7aStatistically significant uptake is indicated. p < 0.05.E3040S4.78 ± 0.351.69 ± 0.262.8 ± 0.5bStatistically significant uptake is indicated. p < 0.01.4MUS1.82 ± 0.350.90 ± 0.032.0 ± 0.1bStatistically significant uptake is indicated. p < 0.01.Troglitazone-sulfate64.1 ± 14.38.4 ± 0.47.6 ± 1.7bStatistically significant uptake is indicated. p < 0.01.a Statistically significant uptake is indicated. p < 0.05.b Statistically significant uptake is indicated. p < 0.01. Open table in a new tab Table IIKm, Vmax and Vmax/Km values for Oatp14SubstrateKmVmaxV max/Kmμmpmol/min/mg proteinμl/min/mg proteinE217βG10.7 ± 1.693.4 ± 10.48.73 ± 1.63Cerivastatin1.34 ± 0.2514.5 ± 2.210.8 ± 2.6TRO-S0.76 ± 0.0969.0 ± 6.791.3 ± 14.2T40.18 ± 0.0332.1 ± 2.5147 ± 14 Open table in a new tab To investigate whether Oatp14 can mediate bidirectional transport, cells were preloaded with [125I]T4 for 15 min followed by incubation in the absence of [125I]T4. The radioactivity associated with cell specimens was rapidly reduced in Oatp14-expressed HEK293 cells compared with that in vector-transfected cells, and the elimination rate constants were 0.032 ± 0.002 and 0.006 ± 0.001 min–1, respectively (Fig. 4). cis-inhibitory effects on the Oatp14-mediated uptake of [3H]E217βG were investigated (Fig. 5). Sulfobromophthalein (BSP), pravastatin, ES, and trichloroacetic acid were potent inhibitors of Oatp14, whereas probenecid was a moderate inhibitor (Fig. 5). p-Aminohippurate and cimetidine, typical substrates of organic anion and cation transporters, had no effect on the Oatp14-mediated uptake, whereas benzylpenicillin was a weak inhibitor (Fig. 6). Leukotriene C4 (LTC4) and glutathione (GSH) had no effect, but dinitrophenyl-s-glutathione (DNP-SG) was a weak inhibitor (see Figs. 5 and 6). No inhibitory effect by folates (methotrexate, folate, and 5-methyltetrahydrofolate) or tetraethylammonium was observed (Fig. 6). The Ki values of probenecid, BSP, trichloroacetic acid, ES, DNP-SG, DPDPE, T3, and T4 for the uptake of [3H]E217βG by Oatp14-expressed HEK cells are summarized in Table III.Fig. 5Effects of unlabeled probenecid, BSP, trichloroacetic acid, ES, DNP-SG, DPDPE, T3, and T4 on the uptake of [3H]E217βGby Oatp14-transfected HEK293 cells. The effects of unlabeled probenecid (A), BSP (B), taurocholate (TCA; C), ES (D), DNP-SG (E), DPDPE (F), T3 (G), and T4 (H) on the uptake of [3H]E217βG by Oatp14-transfected HEK293 cells were examined at 37 °C. The specific uptake was obtained by subtracting the uptake by vector-transfected cells from that by gene-transfected cells. Open and closed circle" @default.
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• W2094704242 title "Functional Characterization of Rat Brain-specific Organic Anion Transporter (Oatp14) at the Blood-Brain Barrier" @default.
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• W2094704242 cites W1775749144 @default.
• W2094704242 cites W1972326404 @default.
• W2094704242 cites W1972626220 @default.
• W2094704242 cites W1973870850 @default.
• W2094704242 cites W1985259581 @default.
• W2094704242 cites W1988193927 @default.
• W2094704242 cites W1994138655 @default.
• W2094704242 cites W1994709174 @default.
• W2094704242 cites W1998275215 @default.
• W2094704242 cites W2013714261 @default.
• W2094704242 cites W2030640565 @default.
• W2094704242 cites W2031595392 @default.
• W2094704242 cites W2045401856 @default.
• W2094704242 cites W2057422423 @default.
• W2094704242 cites W2057526688 @default.
• W2094704242 cites W2064620605 @default.
• W2094704242 cites W2071101614 @default.
• W2094704242 cites W2074823355 @default.
• W2094704242 cites W2075417942 @default.
• W2094704242 cites W2088776629 @default.
• W2094704242 cites W2089487642 @default.
• W2094704242 cites W2118867675 @default.
• W2094704242 cites W2119471148 @default.
• W2094704242 cites W2121537182 @default.
• W2094704242 cites W2125402020 @default.
• W2094704242 cites W2130538167 @default.
• W2094704242 cites W2130695898 @default.
• W2094704242 cites W2135301946 @default.
• W2094704242 cites W2292239568 @default.
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