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W2125402020 abstract "Two complementary DNAs for the organic anion transporter subtypes oatp2 and oatp3, which transport thyroid hormones as well as taurocholate, were isolated from a rat retina cDNA library. The sequence of oatp2 is identical to that recently reported (Noé, B., Hagenbuch, B., Stieger, B., and Meier, P. J. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 10346–10350), whereas the sequence of oatp3 is novel. oatp3 consists of 670 amino acid residues and exhibits a structural architecture common to the organic anion transporter family, possessing the 12 putative membrane-spanning segments. Oocytes injected with oatp2 and oatp3 cRNAs showed taurocholate uptake in a saturable manner. The oatp2 and oatp3 cRNA-injected oocytes also showed significant uptake of both thyroxine and triiodothyronine. Northern blot and in situ analyses showed that the oatp2 mRNA was widely expressed in neuronal cells of the central nervous system, especially in the hippocampus, cerebellum, and choroid plexus as well as in the retina and liver. The oatp3 mRNA was highly expressed in the kidney and moderately abundant in the retina. This suggests that oatp2 and oatp3 are multifunctional transporters involved in the transport of thyroid hormones in the brain, retina, liver, and kidney. Two complementary DNAs for the organic anion transporter subtypes oatp2 and oatp3, which transport thyroid hormones as well as taurocholate, were isolated from a rat retina cDNA library. The sequence of oatp2 is identical to that recently reported (Noé, B., Hagenbuch, B., Stieger, B., and Meier, P. J. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 10346–10350), whereas the sequence of oatp3 is novel. oatp3 consists of 670 amino acid residues and exhibits a structural architecture common to the organic anion transporter family, possessing the 12 putative membrane-spanning segments. Oocytes injected with oatp2 and oatp3 cRNAs showed taurocholate uptake in a saturable manner. The oatp2 and oatp3 cRNA-injected oocytes also showed significant uptake of both thyroxine and triiodothyronine. Northern blot and in situ analyses showed that the oatp2 mRNA was widely expressed in neuronal cells of the central nervous system, especially in the hippocampus, cerebellum, and choroid plexus as well as in the retina and liver. The oatp3 mRNA was highly expressed in the kidney and moderately abundant in the retina. This suggests that oatp2 and oatp3 are multifunctional transporters involved in the transport of thyroid hormones in the brain, retina, liver, and kidney. A homeostatic system controls the fluid environment in the brain and keeps its chemical composition relatively constant compared with that of plasma. One mechanism is the blood-brain barrier, which selectively transports chemical substances via capillary endothelial cells (1Brightman M.W. Reese T.S. J. Cell Biol. 1969; 40: 648-677Crossref PubMed Scopus (1813) Google Scholar). A second essential component is the choroid plexus (blood-cerebrospinal fluid barrier), which secretes or takes up specific chemical substances (2Cserr H.F. Physiol. Rev. 1971; 51: 273-311Crossref PubMed Scopus (346) Google Scholar). Although the presence of specific transporting mechanisms has long been postulated, little is known about their molecular identity. Recent molecular biological studies revealed the organic anion transporter family: the Na+-independent organic anion-transporting polypeptide oatp1 from rat liver, which transports bile acid, bromosulfophthalein (BSP), 1The abbreviations used are: BSPbromosulfophthaleinT4thyroxineT33,5,3′-triiodo-l-thyroninePCRpolymerase chain reactionbpbase pair(s).1The abbreviations used are: BSPbromosulfophthaleinT4thyroxineT33,5,3′-triiodo-l-thyroninePCRpolymerase chain reactionbpbase pair(s). and conjugated and unconjugated steroid hormones (3Jacquemin E. Hagenbuch B. Stieger B. Wolkoff A.W. Meier P.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 133-137Crossref PubMed Scopus (544) Google Scholar, 4Bossuyt X. Müller M. Hagenbuch B. Meier P.J. J. Pharmacol. Exp. Ther. 1996; 276: 891-896PubMed Google Scholar); the kidney-specific transporter OAT-K1, which transports methotrexate in the basolateral membrane of renal tubules (5Saito H. Masuda S. Inui K. J. Biol. Chem. 1996; 271: 20719-20725Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar); and the prostaglandin transporter (6Kanai N. Lu R. Satriano J.A. Bao Y. Wolkoff A.W. Schuster V.L. Science. 1995; 268: 866-869Crossref PubMed Scopus (346) Google Scholar). Moreover, physiological studies have suggested the presence of other members of the organic anion transporter family (7Hortz J.A. Honscha W. Petzinger E. Biochim. Biophys. Acta. 1996; 1300: 114-118Crossref PubMed Scopus (20) Google Scholar). Noé et al. (8Noé B. Hagenbuch B. Stieger B. Meier P.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10346-10350Crossref PubMed Scopus (389) Google Scholar) have recently reported that a new organic anion transporter subtype (oatp2) is present in rat brain and liver and that the oatp2-expressed oocytes transported cardiac glycoside as well as taurocholate. However, the endogenous substrate of oatp2 and the regional distribution in the brain have not been revealed.It has been suggested that thyroid hormones are transported into the brain via the blood-brain barrier (9Pardridge W.M. Endocrinology. 1979; 105: 605-612Crossref PubMed Scopus (103) Google Scholar) or via the choroid plexus (10Southwell B.R. Duan W. Alcorn D. Brack C. Richardson S.J. Köhrle J. Schreiber G. Endocrinology. 1993; 133: 2116-2126Crossref PubMed Google Scholar). To reveal this mechanism, we focused on the retina. In the retina, the retinal pigment epithelium is the unique source of transthyretin synthesis, and it serves to transport thyroxine (T4) across the blood-retina barrier (11Cavallaro T. Martone R.L. Dwork A.J. Schon E.A. Herbert J. Invest. Ophthalmol. Visual Sci. 1990; 31: 497-501PubMed Google Scholar). According to this functional homology between the choroid plexus epithelium and the retinal pigment epithelium, we performed polymerase chain reaction (PCR)-based screening for an organic anion transporter subtype in the retina.In this paper, we have isolated and characterized oatp2 and a new organic anion transporter family subtype (termed oatp3) from rat retina, and this is the first report identifying the molecules involved in thyroid hormone transport.DISCUSSIONThis paper presents the characterization and tissue distribution of two cDNA clones encoding rat organic anion transporter subtypes oatp2 and oatp3 isolated from rat retina. The putative amino acid sequence of oatp3 was novel, whereas that of oatp2 was identical as recently reported (8Noé B. Hagenbuch B. Stieger B. Meier P.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10346-10350Crossref PubMed Scopus (389) Google Scholar). oatp3 is unique compared with other members of the organic anion transporter family in its tissue distribution.Both oatp2 and oatp3 appear to be responsible for transporting organic anions and thyroid hormones for the following reasons. First, the expression experiments with oatp2 and oatp3 using Xenopusoocytes showed that both oatp2 and oatp3 transport [3H]taurocholate. oatp2 transports taurocholate with aKm value similar to that reported (35.2 ± 8.9versus 34 μm) (8Noé B. Hagenbuch B. Stieger B. Meier P.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10346-10350Crossref PubMed Scopus (389) Google Scholar). The oatp3 cRNA-injected oocytes also transported [3H]taurocholate, but the apparent Km value was half of that for oatp1- and oatp2-mediated uptake (8Noé B. Hagenbuch B. Stieger B. Meier P.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10346-10350Crossref PubMed Scopus (389) Google Scholar). Second, a series ofcis-inhibition studies on the oatp2-expressing oocytes showed that the oatp2-mediated uptake of [3H]taurocholate was markedly inhibited by the addition of taurocholate, cholate, BSP, or 17β-estradiol glucuronide as well as T4 and T3. The oatp3-mediated taurocholate uptake was also inhibited by BSP. Third, the oatp2- and oatp3-expressing oocytes facilitated the uptake of thyroid hormones (T4 and T3) with simple Michaelis-Menten kinetics, and the apparentKm values are comparable to each other. Fourth, Northern blot analysis showed that the oatp2 mRNA is distributed in the brain, retina, and liver. The oatp3 mRNA is exclusively expressed in the retina and kidney. On the other hand, the oatp1 mRNA is expressed in the liver, kidney, brain, skeletal muscle, and colon (3Jacquemin E. Hagenbuch B. Stieger B. Wolkoff A.W. Meier P.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 133-137Crossref PubMed Scopus (544) Google Scholar). Finally, in situ hybridization analysis indicated that the oatp2 mRNA is widely distributed in neuronal cells of many brain regions as well as in the choroid plexus.This is the first report identifying the molecules responsive for transporting thyroid hormones across cell membrane. The transport of thyroid hormones across the plasma membrane determines the intracellular concentration of these hormones and hence the activation of the nuclear T3 receptor. The existence of mechanisms regulating the transport of thyroid hormones has been suggested in cerebrocortical neurons (17Chantoux F. Blondeau J.-P. Francon J. J. Neurochem. 1995; 65: 2549-2554Crossref PubMed Scopus (27) Google Scholar), astrocytes (18Beslin A. Chantoux F. Blondeau J.-P. Francon J. Endocrinology. 1995; 136: 5385-5390Crossref PubMed Scopus (16) Google Scholar), glial cells (19Francon J. Chantoux F. Blondeau J.-P. J. Neurochem. 1989; 53: 1456-1463Crossref PubMed Scopus (36) Google Scholar), hepatocytes (20Blondeau J.-P. Osty J. Francon J. J. Biol. Chem. 1988; 263: 2685-2692Abstract Full Text PDF PubMed Google Scholar, 21Krenning E. Docter R. Bernard B. Visser T. Hennemann G. Biochim. Biophys. Acta. 1981; 676: 314-320Crossref PubMed Scopus (103) Google Scholar), erythrocytes (22Osty J. Jego L. Francon J. Blondeau J.-P. Endocrinology. 1988; 123: 2303-2311Crossref PubMed Scopus (46) Google Scholar), and skeletal muscle (23Centanni M. Robbins J. J. Clin. Invest. 1987; 80: 1068-1072Crossref PubMed Scopus (37) Google Scholar). The following are comparisons of the pharmacological characteristics among oatp2, oatp3, and the known native thyroid hormone transporters.The Km values for T4 and T3of oatp2 and oatp3 were similar to those for neural cells (19Francon J. Chantoux F. Blondeau J.-P. J. Neurochem. 1989; 53: 1456-1463Crossref PubMed Scopus (36) Google Scholar) and hepatocytes (21Krenning E. Docter R. Bernard B. Visser T. Hennemann G. Biochim. Biophys. Acta. 1981; 676: 314-320Crossref PubMed Scopus (103) Google Scholar), but 10-fold higher than those reported by Chantouxet al. (17Chantoux F. Blondeau J.-P. Francon J. J. Neurochem. 1995; 65: 2549-2554Crossref PubMed Scopus (27) Google Scholar) and Blondeau et al. (20Blondeau J.-P. Osty J. Francon J. J. Biol. Chem. 1988; 263: 2685-2692Abstract Full Text PDF PubMed Google Scholar). We do not know what accounts for these differences. In our study, the oatp2-mediated uptake of T4 was not dependent on extracellular Na+, whereas the transporting mechanisms of thyroid hormones are heterogeneous in Na+ dependence: Na+-dependent (21Krenning E. Docter R. Bernard B. Visser T. Hennemann G. Biochim. Biophys. Acta. 1981; 676: 314-320Crossref PubMed Scopus (103) Google Scholar), Na+-independent (19Francon J. Chantoux F. Blondeau J.-P. J. Neurochem. 1989; 53: 1456-1463Crossref PubMed Scopus (36) Google Scholar, 20Blondeau J.-P. Osty J. Francon J. J. Biol. Chem. 1988; 263: 2685-2692Abstract Full Text PDF PubMed Google Scholar, 24Topliss D.J. Kolliniatis E. Barlow J.W. Lim C.-F. Stockigt J.R. Endocrinology. 1989; 124: 980-986Crossref PubMed Scopus (30) Google Scholar) and mixed (18Beslin A. Chantoux F. Blondeau J.-P. Francon J. Endocrinology. 1995; 136: 5385-5390Crossref PubMed Scopus (16) Google Scholar, 23Centanni M. Robbins J. J. Clin. Invest. 1987; 80: 1068-1072Crossref PubMed Scopus (37) Google Scholar). In addition, it has been reported that oocytes injected with rat liver poly(A)+ RNA showed Na+-dependent uptake of T4 and T3 (25Docter R. Friesema E.C.H. van Stralen P.G.J. Krenning E.P. Everts M.E. Visser T.J. Hennemann G. Endocrinology. 1997; 138: 1841-1846Crossref PubMed Google Scholar); however, the functional fraction size is different from that of the oatp2 or oatp3 mRNA. Therefore, the molecules responsible for transporting thyroid hormones are suggested to be heterogeneous.The uptake of thyroid hormones in the native tissues appears to be inhibited by a variety of structurally unrelated drugs, including non-bile acid cholephils such as BSP, nonsteroidal anti-inflammatory drugs (20Blondeau J.-P. Osty J. Francon J. J. Biol. Chem. 1988; 263: 2685-2692Abstract Full Text PDF PubMed Google Scholar, 24Topliss D.J. Kolliniatis E. Barlow J.W. Lim C.-F. Stockigt J.R. Endocrinology. 1989; 124: 980-986Crossref PubMed Scopus (30) Google Scholar), diphenylhydantoin (26Gingrich S.A. Smith P.J. Shapiro L.E. Surks M.I. Endocrinology. 1985; 116: 2306-2313Crossref PubMed Scopus (10) Google Scholar), and propranolol (27Krenning E.P. Docter R. Bernard B. Visser T. Hennemann G. FEBS Lett. 1982; 140: 229-233Crossref PubMed Scopus (79) Google Scholar). In our study, the oatp2-mediated uptake of taurocholate was inhibited by some of these compounds, but no oatp3-mediated uptake was found for tryptophan, phenylalanine, tyrosine, and indomethacin. In the oatp1-expressing mammalian cells, T4 did not exhibit acis-inhibitory effect for BSP uptake (28Kanai N. Lu R. Bao Y. Wolkoff A.W. Schuster V.L. Am. J. Physiol. 1996; 270: F319-F325PubMed Google Scholar). Thus, further investigations will be required to understand the role and functions of the other members of the organic anion transporter family.The oatp2 mRNA is distributed exclusively in the brain, retina, and liver. In the central nervous system, most of the nuclear T3 is derived from local T4 deiodination (29Crantz F.R. Silvia J.E. Larsen P.R. Endocrinology. 1982; 110: 367-375Crossref PubMed Scopus (292) Google Scholar). However, most of the intracellular T3 in the liver and muscle is derived from plasma (30van Doorn J. van der Heide D. Roelfsema F. J. Clin. Invest. 1983; 72: 1778-1792Crossref PubMed Scopus (102) Google Scholar). Accordingly, the abundant expression of the oatp2 mRNA in the brain and liver implies that this molecule should regulate the availability of thyroid hormone in these tissues. Thyroid hormones also play an essential role in neural function of the mammalian central nervous system, particularly during a critical period of its development (31Vincent J. Legrand C. Rabié A. Legrand J. J. Physiol. ( Paris ). 1982–1983; 78: 729-738PubMed Google Scholar, 32Dussault J.H. Ruel J. Annu. Rev. Physiol. 1987; 49: 321-334Crossref PubMed Google Scholar). The absence of thyroid hormone causes serious damages to structural development and organization of the brain (especially the hippocampus and cerebellum), including biochemical maturation, and leads to irreversible mental retardation (33Porterfield S.P. Hendrich C.E. Endocr. Rev. 1993; 14: 94-106PubMed Google Scholar).In the hippocampus, the oatp2 mRNA is predominantly expressed in the pyramidal cells of CA1–4 and in the granule cells of the dentate gyrus. In the cerebellum, the oatp2 mRNA is highly expressed in Purkinje cells. The prominent expression of the oatp2 mRNA in these thyroid hormone-sensitive neurons further suggests that oatp2 may play a critical role in neuronal development and maintaining cell function.oatp2 mRNA was also moderately distributed in the choroid plexus. This is consistent with the notion that thyroid hormones are transported into the brain via the blood-brain barrier (9Pardridge W.M. Endocrinology. 1979; 105: 605-612Crossref PubMed Scopus (103) Google Scholar) or via the choroid plexus (10Southwell B.R. Duan W. Alcorn D. Brack C. Richardson S.J. Köhrle J. Schreiber G. Endocrinology. 1993; 133: 2116-2126Crossref PubMed Google Scholar). In situ hybridization of rat oatp1 revealed that rat oatp1 mRNA is also expressed in the choroid plexus (34Angeletti R.H. Novikoff P.M. Juvvadi S.R. Fritschy J.-M. Meier P.J. Wolkoff A.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 283-286Crossref PubMed Scopus (128) Google Scholar). Although the probe for rat oatp1 (positions 2031–2090) is 80∼95% identical among organic anion transporter family members, the probe only detected signals in the choroid plexus. In our experiments, to avoid the nonspecific signals, we used the 900-bp 3′-noncoding region that has <48% identity to any organic anion transporter family. Thus, it seems likely that at least two organic anion transporter family subtypes are coexpressed in the choroid plexus: oatp1, which mediates BSP uptake that is not inhibited by T4 (28Kanai N. Lu R. Bao Y. Wolkoff A.W. Schuster V.L. Am. J. Physiol. 1996; 270: F319-F325PubMed Google Scholar); and oatp2, which transports thyroid hormones.Noé et al. (8Noé B. Hagenbuch B. Stieger B. Meier P.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10346-10350Crossref PubMed Scopus (389) Google Scholar) also showed that the oatp2 mRNA is highly expressed in the brain, liver, and kidney. However, in our experiment, no oatp2 expression was detected in the kidney. This discrepancy in the Northern blot analysis may be also attributed to the different nucleotide regions of the probe. We used the 3′-noncoding region of oatp2 that has <48% identity to oatp1, OAT-K1, and oatp3. On the other hand, Noé et al. (8Noé B. Hagenbuch B. Stieger B. Meier P.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10346-10350Crossref PubMed Scopus (389) Google Scholar) used nucleotides 1–360, which has >80% identity to oatp1 (82%) and OAT-K1 (87%). In their report, the size of the major band in the kidney was smaller than that in the brain. It is possible that their probe cross-hybridized to the other member of the organic anion transporter family,e.g. oatp3 or OAT-K1. In addition, since the probes for oatp3 clearly hybridized to the kidney mRNA, the mRNAs used were not degenerate in our study. The same result was obtained using the rat multiple-tissue Northern filter. In both cases, mRNA qualities were confirmed by β-actin (data not shown). Based on our results, we suggest that the oatp2 mRNA is not expressed in the kidney as much as shown for oatp2 (8Noé B. Hagenbuch B. Stieger B. Meier P.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10346-10350Crossref PubMed Scopus (389) Google Scholar).The oatp3 mRNA is exclusively distributed in the kidney. Most of the T4 secreted from the thyroid is deiodinated in peripheral tissues. The liver and kidney are the major peripheral organs producing T3 from T4 (35Berry M.J. Banu L. Larsen P.R. Nature. 1991; 349: 438-440Crossref PubMed Scopus (750) Google Scholar). Thus, the specific expression of oatp3 mRNA in the kidney suggests an essential role for oatp3 in transporting thyroid hormones from the circulation to the deiodination sites in the kidney.Individuals with Refetoff's syndrome, characterized by resistance to thyroid hormone, exhibit reduced clinical and biochemical activities of thyroid hormone action relative to the circulating hormone level (36Refetoff S. Weiss R.E. Usala S.J. Endocr. Rev. 1993; 14: 348-399Crossref PubMed Scopus (610) Google Scholar). Several pathophysiological mechanisms have been suggested to account for the thyroid hormone resistance seen in Refetoff's syndrome patients. One possible mechanism is reduced hormone availability to tissues due to impaired thyroid hormone entry into cells (36Refetoff S. Weiss R.E. Usala S.J. Endocr. Rev. 1993; 14: 348-399Crossref PubMed Scopus (610) Google Scholar). Accordingly, functional and genetic investigations of oatp2 and oatp3 may also help us to understand the etiology of such disorders and to aid in clinical diagnosis. A homeostatic system controls the fluid environment in the brain and keeps its chemical composition relatively constant compared with that of plasma. One mechanism is the blood-brain barrier, which selectively transports chemical substances via capillary endothelial cells (1Brightman M.W. Reese T.S. J. Cell Biol. 1969; 40: 648-677Crossref PubMed Scopus (1813) Google Scholar). A second essential component is the choroid plexus (blood-cerebrospinal fluid barrier), which secretes or takes up specific chemical substances (2Cserr H.F. Physiol. Rev. 1971; 51: 273-311Crossref PubMed Scopus (346) Google Scholar). Although the presence of specific transporting mechanisms has long been postulated, little is known about their molecular identity. Recent molecular biological studies revealed the organic anion transporter family: the Na+-independent organic anion-transporting polypeptide oatp1 from rat liver, which transports bile acid, bromosulfophthalein (BSP), 1The abbreviations used are: BSPbromosulfophthaleinT4thyroxineT33,5,3′-triiodo-l-thyroninePCRpolymerase chain reactionbpbase pair(s).1The abbreviations used are: BSPbromosulfophthaleinT4thyroxineT33,5,3′-triiodo-l-thyroninePCRpolymerase chain reactionbpbase pair(s). and conjugated and unconjugated steroid hormones (3Jacquemin E. Hagenbuch B. Stieger B. Wolkoff A.W. Meier P.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 133-137Crossref PubMed Scopus (544) Google Scholar, 4Bossuyt X. Müller M. Hagenbuch B. Meier P.J. J. Pharmacol. Exp. Ther. 1996; 276: 891-896PubMed Google Scholar); the kidney-specific transporter OAT-K1, which transports methotrexate in the basolateral membrane of renal tubules (5Saito H. Masuda S. Inui K. J. Biol. Chem. 1996; 271: 20719-20725Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar); and the prostaglandin transporter (6Kanai N. Lu R. Satriano J.A. Bao Y. Wolkoff A.W. Schuster V.L. Science. 1995; 268: 866-869Crossref PubMed Scopus (346) Google Scholar). Moreover, physiological studies have suggested the presence of other members of the organic anion transporter family (7Hortz J.A. Honscha W. Petzinger E. Biochim. Biophys. Acta. 1996; 1300: 114-118Crossref PubMed Scopus (20) Google Scholar). Noé et al. (8Noé B. Hagenbuch B. Stieger B. Meier P.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10346-10350Crossref PubMed Scopus (389) Google Scholar) have recently reported that a new organic anion transporter subtype (oatp2) is present in rat brain and liver and that the oatp2-expressed oocytes transported cardiac glycoside as well as taurocholate. However, the endogenous substrate of oatp2 and the regional distribution in the brain have not been revealed. bromosulfophthalein thyroxine 3,5,3′-triiodo-l-thyronine polymerase chain reaction base pair(s). bromosulfophthalein thyroxine 3,5,3′-triiodo-l-thyronine polymerase chain reaction base pair(s). It has been suggested that thyroid hormones are transported into the brain via the blood-brain barrier (9Pardridge W.M. Endocrinology. 1979; 105: 605-612Crossref PubMed Scopus (103) Google Scholar) or via the choroid plexus (10Southwell B.R. Duan W. Alcorn D. Brack C. Richardson S.J. Köhrle J. Schreiber G. Endocrinology. 1993; 133: 2116-2126Crossref PubMed Google Scholar). To reveal this mechanism, we focused on the retina. In the retina, the retinal pigment epithelium is the unique source of transthyretin synthesis, and it serves to transport thyroxine (T4) across the blood-retina barrier (11Cavallaro T. Martone R.L. Dwork A.J. Schon E.A. Herbert J. Invest. Ophthalmol. Visual Sci. 1990; 31: 497-501PubMed Google Scholar). According to this functional homology between the choroid plexus epithelium and the retinal pigment epithelium, we performed polymerase chain reaction (PCR)-based screening for an organic anion transporter subtype in the retina. In this paper, we have isolated and characterized oatp2 and a new organic anion transporter family subtype (termed oatp3) from rat retina, and this is the first report identifying the molecules involved in thyroid hormone transport. DISCUSSIONThis paper presents the characterization and tissue distribution of two cDNA clones encoding rat organic anion transporter subtypes oatp2 and oatp3 isolated from rat retina. The putative amino acid sequence of oatp3 was novel, whereas that of oatp2 was identical as recently reported (8Noé B. Hagenbuch B. Stieger B. Meier P.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10346-10350Crossref PubMed Scopus (389) Google Scholar). oatp3 is unique compared with other members of the organic anion transporter family in its tissue distribution.Both oatp2 and oatp3 appear to be responsible for transporting organic anions and thyroid hormones for the following reasons. First, the expression experiments with oatp2 and oatp3 using Xenopusoocytes showed that both oatp2 and oatp3 transport [3H]taurocholate. oatp2 transports taurocholate with aKm value similar to that reported (35.2 ± 8.9versus 34 μm) (8Noé B. Hagenbuch B. Stieger B. Meier P.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10346-10350Crossref PubMed Scopus (389) Google Scholar). The oatp3 cRNA-injected oocytes also transported [3H]taurocholate, but the apparent Km value was half of that for oatp1- and oatp2-mediated uptake (8Noé B. Hagenbuch B. Stieger B. Meier P.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10346-10350Crossref PubMed Scopus (389) Google Scholar). Second, a series ofcis-inhibition studies on the oatp2-expressing oocytes showed that the oatp2-mediated uptake of [3H]taurocholate was markedly inhibited by the addition of taurocholate, cholate, BSP, or 17β-estradiol glucuronide as well as T4 and T3. The oatp3-mediated taurocholate uptake was also inhibited by BSP. Third, the oatp2- and oatp3-expressing oocytes facilitated the uptake of thyroid hormones (T4 and T3) with simple Michaelis-Menten kinetics, and the apparentKm values are comparable to each other. Fourth, Northern blot analysis showed that the oatp2 mRNA is distributed in the brain, retina, and liver. The oatp3 mRNA is exclusively expressed in the retina and kidney. On the other hand, the oatp1 mRNA is expressed in the liver, kidney, brain, skeletal muscle, and colon (3Jacquemin E. Hagenbuch B. Stieger B. Wolkoff A.W. Meier P.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 133-137Crossref PubMed Scopus (544) Google Scholar). Finally, in situ hybridization analysis indicated that the oatp2 mRNA is widely distributed in neuronal cells of many brain regions as well as in the choroid plexus.This is the first report identifying the molecules responsive for transporting thyroid hormones across cell membrane. The transport of thyroid hormones across the plasma membrane determines the intracellular concentration of these hormones and hence the activation of the nuclear T3 receptor. The existence of mechanisms regulating the transport of thyroid hormones has been suggested in cerebrocortical neurons (17Chantoux F. Blondeau J.-P. Francon J. J. Neurochem. 1995; 65: 2549-2554Crossref PubMed Scopus (27) Google Scholar), astrocytes (18Beslin A. Chantoux F. Blondeau J.-P. Francon J. Endocrinology. 1995; 136: 5385-5390Crossref PubMed Scopus (16) Google Scholar), glial cells (19Francon J. Chantoux F. Blondeau J.-P. J. Neurochem. 1989; 53: 1456-1463Crossref PubMed Scopus (36) Google Scholar), hepatocytes (20Blondeau J.-P. Osty J. Francon J. J. Biol. Chem. 1988; 263: 2685-2692Abstract Full Text PDF PubMed Google Scholar, 21Krenning E. Docter R. Bernard B. Visser T. Hennemann G. Biochim. Biophys. Acta. 1981; 676: 314-320Crossref PubMed Scopus (103) Google Scholar), erythrocytes (22Osty J. Jego L. Francon J. Blondeau J.-P. Endocrinology. 1988; 123: 2303-2311Crossref PubMed Scopus (46) Google Scholar), and skeletal muscle (23Centanni M. Robbins J. J. Clin. Invest. 1987; 80: 1068-1072Crossref PubMed Scopus (37) Google Scholar). The following are comparisons of the pharmacological characteristics among oatp2, oatp3, and the known native thyroid hormone transporters.The Km values for T4 and T3of oatp2 and oatp3 were similar to those for neural cells (19Francon J. Chantoux F. Blondeau J.-P. J. Neurochem. 1989; 53: 1456-1463Crossref PubMed Scopus (36) Google Scholar) and hepatocytes (21Krenning E. Docter R. Bernard B. Visser T. Hennemann G. Biochim. Biophys. Acta. 1981; 676: 314-320Crossref PubMed Scopus (103) Google Scholar), but 10-fold higher than those reported by Chantouxet al. (17Chantoux F. Blondeau J.-P. Francon J. J. Neurochem. 1995; 65: 2549-2554Crossref PubMed Scopus (27) Google Scholar) and Blondeau et al. (20Blondeau J.-P. Osty J. Francon J. J. Biol. Chem. 1988; 263: 2685-2692Abstract Full Text PDF PubMed Google Scholar). We do not know what accounts for these differences. In our study, the oatp2-mediated uptake of T4 was not dependent on extracellular Na+, whereas the transporting mechanisms of thyroid hormones are heterogeneous in Na+ dependence: Na+-dependent (21Krenning E. Docter R. Bernard B. Visser T. Hennemann G. Biochim. Biophys. Acta. 1981; 676: 314-320Crossref PubMed Scopus (103) Google Scholar), Na+-independent (19Francon J. Chantoux F. Blondeau J.-P. J. Neurochem. 1989; 53: 1456-1463Crossref PubMed Scopus (36) Google Scholar, 20Blondeau J.-P. Osty J. Francon J. J. Biol. Chem. 1988; 263: 2685-2692Abstract Full Text PDF PubMed Google Scholar, 24Topliss D.J. Kolliniatis E. Barlow J.W. Lim C.-F. Stockigt J.R. Endocrinology. 1989; 124: 980-986Crossref PubMed Scopus (30) Google Scholar) and mixed (18Beslin A. Chantoux F. Blondeau J.-P. Francon J. Endocrinology. 1995; 136: 5385-5390Crossref PubMed Scopus (16) Google Scholar, 23Centanni M. Robbins J. J. Clin. Invest. 1987; 80: 1068-1072Crossref PubMed Scopus (37) Google Scholar). In addition, it has been reported that oocytes injected with rat liver poly(A)+ RNA showed Na+-dependent uptake of T4 and T3 (25Docter R. Friesema E.C.H. van Stralen P.G.J. Krenning E.P. Everts M.E. Visser T.J. Hennemann G. Endocrinology. 1997; 138: 1841-1846Crossref PubMed Google Scholar); however, the functional fraction size is different from that of the oatp2 or oatp3 mRNA. Therefore, the molecules responsible for transporting thyroid hormones are suggested to be heterogeneous.The uptake of thyroid hormones in the native tissues appears to be inhibited by a variety of structurally unrelated drugs, including non-bile acid cholephils such as BSP, nonsteroidal anti-inflammatory drugs (20Blondeau J.-P. Osty J. Francon J. J. Biol. Chem. 1988; 263: 2685-2692Abstract Full Text PDF PubMed Google Scholar, 24Topliss D.J. Kolliniatis E. Barlow J.W. Lim C.-F. Stockigt J.R. Endocrinology. 1989; 124: 980-986Crossref PubMed Scopus (30) Google Scholar), diphenylhydantoin (26Gingrich S.A. Smith P.J. Shapiro L.E. Surks M.I. Endocrinology. 1985; 116: 2306-2313Crossref PubMed Scopus (10) Google Scholar), and propranolol (27Krenning E.P. Docter R. Bernard B. Visser T. Hennemann G. FEBS Lett. 1982; 140: 229-233Crossref PubMed Scopus (79) Google Scholar). In our study, the oatp2-mediated uptake of taurocholate was inhibited by some of these compounds, but no oatp3-mediated uptake was found for tryptophan, phenylalanine, tyrosine, and indomethacin. In the oatp1-expressing mammalian cells, T4 did not exhibit acis-inhibitory effect for BSP uptake (28Kanai N. Lu R. Bao Y. Wolkoff A.W. Schuster V.L. Am. J. Physiol. 1996; 270: F319-F325PubMed Google Scholar). Thus, further investigations will be required to understand the role and functions of the other members of the organic anion transporter family.The oatp2 mRNA is distributed exclusively in the brain, retina, and liver. In the central nervous system, most of the nuclear T3 is derived from local T4 deiodination (29Crantz F.R. Silvia J.E. Larsen P.R. Endocrinology. 1982; 110: 367-375Crossref PubMed Scopus (292) Google Scholar). However, most of the intracellular T3 in the liver and muscle is derived from plasma (30van Doorn J. van der Heide D. Roelfsema F. J. Clin. Invest. 1983; 72: 1778-1792Crossref PubMed Scopus (102) Google Scholar). Accordingly, the abundant expression of the oatp2 mRNA in the brain and liver implies that this molecule should regulate the availability of thyroid hormone in these tissues. Thyroid hormones also play an essential role in neural function of the mammalian central nervous system, particularly during a critical period of its development (31Vincent J. Legrand C. Rabié A. Legrand J. J. Physiol. ( Paris ). 1982–1983; 78: 729-738PubMed Google Scholar, 32Dussault J.H. Ruel J. Annu. Rev. Physiol. 1987; 49: 321-334Crossref PubMed Google Scholar). The absence of thyroid hormone causes serious damages to structural development and organization of the brain (especially the hippocampus and cerebellum), including biochemical maturation, and leads to irreversible mental retardation (33Porterfield S.P. Hendrich C.E. Endocr. Rev. 1993; 14: 94-106PubMed Google Scholar).In the hippocampus, the oatp2 mRNA is predominantly expressed in the pyramidal cells of CA1–4 and in the granule cells of the dentate gyrus. In the cerebellum, the oatp2 mRNA is highly expressed in Purkinje cells. The prominent expression of the oatp2 mRNA in these thyroid hormone-sensitive neurons further suggests that oatp2 may play a critical role in neuronal development and maintaining cell function.oatp2 mRNA was also moderately distributed in the choroid plexus. This is consistent with the notion that thyroid hormones are transported into the brain via the blood-brain barrier (9Pardridge W.M. Endocrinology. 1979; 105: 605-612Crossref PubMed Scopus (103) Google Scholar) or via the choroid plexus (10Southwell B.R. Duan W. Alcorn D. Brack C. Richardson S.J. Köhrle J. Schreiber G. Endocrinology. 1993; 133: 2116-2126Crossref PubMed Google Scholar). In situ hybridization of rat oatp1 revealed that rat oatp1 mRNA is also expressed in the choroid plexus (34Angeletti R.H. Novikoff P.M. Juvvadi S.R. Fritschy J.-M. Meier P.J. Wolkoff A.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 283-286Crossref PubMed Scopus (128) Google Scholar). Although the probe for rat oatp1 (positions 2031–2090) is 80∼95% identical among organic anion transporter family members, the probe only detected signals in the choroid plexus. In our experiments, to avoid the nonspecific signals, we used the 900-bp 3′-noncoding region that has <48% identity to any organic anion transporter family. Thus, it seems likely that at least two organic anion transporter family subtypes are coexpressed in the choroid plexus: oatp1, which mediates BSP uptake that is not inhibited by T4 (28Kanai N. Lu R. Bao Y. Wolkoff A.W. Schuster V.L. Am. J. Physiol. 1996; 270: F319-F325PubMed Google Scholar); and oatp2, which transports thyroid hormones.Noé et al. (8Noé B. Hagenbuch B. Stieger B. Meier P.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10346-10350Crossref PubMed Scopus (389) Google Scholar) also showed that the oatp2 mRNA is highly expressed in the brain, liver, and kidney. However, in our experiment, no oatp2 expression was detected in the kidney. This discrepancy in the Northern blot analysis may be also attributed to the different nucleotide regions of the probe. We used the 3′-noncoding region of oatp2 that has <48% identity to oatp1, OAT-K1, and oatp3. On the other hand, Noé et al. (8Noé B. Hagenbuch B. Stieger B. Meier P.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10346-10350Crossref PubMed Scopus (389) Google Scholar) used nucleotides 1–360, which has >80% identity to oatp1 (82%) and OAT-K1 (87%). In their report, the size of the major band in the kidney was smaller than that in the brain. It is possible that their probe cross-hybridized to the other member of the organic anion transporter family,e.g. oatp3 or OAT-K1. In addition, since the probes for oatp3 clearly hybridized to the kidney mRNA, the mRNAs used were not degenerate in our study. The same result was obtained using the rat multiple-tissue Northern filter. In both cases, mRNA qualities were confirmed by β-actin (data not shown). Based on our results, we suggest that the oatp2 mRNA is not expressed in the kidney as much as shown for oatp2 (8Noé B. Hagenbuch B. Stieger B. Meier P.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10346-10350Crossref PubMed Scopus (389) Google Scholar).The oatp3 mRNA is exclusively distributed in the kidney. Most of the T4 secreted from the thyroid is deiodinated in peripheral tissues. The liver and kidney are the major peripheral organs producing T3 from T4 (35Berry M.J. Banu L. Larsen P.R. Nature. 1991; 349: 438-440Crossref PubMed Scopus (750) Google Scholar). Thus, the specific expression of oatp3 mRNA in the kidney suggests an essential role for oatp3 in transporting thyroid hormones from the circulation to the deiodination sites in the kidney.Individuals with Refetoff's syndrome, characterized by resistance to thyroid hormone, exhibit reduced clinical and biochemical activities of thyroid hormone action relative to the circulating hormone level (36Refetoff S. Weiss R.E. Usala S.J. Endocr. Rev. 1993; 14: 348-399Crossref PubMed Scopus (610) Google Scholar). Several pathophysiological mechanisms have been suggested to account for the thyroid hormone resistance seen in Refetoff's syndrome patients. One possible mechanism is reduced hormone availability to tissues due to impaired thyroid hormone entry into cells (36Refetoff S. Weiss R.E. Usala S.J. Endocr. Rev. 1993; 14: 348-399Crossref PubMed Scopus (610) Google Scholar). Accordingly, functional and genetic investigations of oatp2 and oatp3 may also help us to understand the etiology of such disorders and to aid in clinical diagnosis. This paper presents the characterization and tissue distribution of two cDNA clones encoding rat organic anion transporter subtypes oatp2 and oatp3 isolated from rat retina. The putative amino acid sequence of oatp3 was novel, whereas that of oatp2 was identical as recently reported (8Noé B. Hagenbuch B. Stieger B. Meier P.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10346-10350Crossref PubMed Scopus (389) Google Scholar). oatp3 is unique compared with other members of the organic anion transporter family in its tissue distribution. Both oatp2 and oatp3 appear to be responsible for transporting organic anions and thyroid hormones for the following reasons. First, the expression experiments with oatp2 and oatp3 using Xenopusoocytes showed that both oatp2 and oatp3 transport [3H]taurocholate. oatp2 transports taurocholate with aKm value similar to that reported (35.2 ± 8.9versus 34 μm) (8Noé B. Hagenbuch B. Stieger B. Meier P.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10346-10350Crossref PubMed Scopus (389) Google Scholar). The oatp3 cRNA-injected oocytes also transported [3H]taurocholate, but the apparent Km value was half of that for oatp1- and oatp2-mediated uptake (8Noé B. Hagenbuch B. Stieger B. Meier P.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10346-10350Crossref PubMed Scopus (389) Google Scholar). Second, a series ofcis-inhibition studies on the oatp2-expressing oocytes showed that the oatp2-mediated uptake of [3H]taurocholate was markedly inhibited by the addition of taurocholate, cholate, BSP, or 17β-estradiol glucuronide as well as T4 and T3. The oatp3-mediated taurocholate uptake was also inhibited by BSP. Third, the oatp2- and oatp3-expressing oocytes facilitated the uptake of thyroid hormones (T4 and T3) with simple Michaelis-Menten kinetics, and the apparentKm values are comparable to each other. Fourth, Northern blot analysis showed that the oatp2 mRNA is distributed in the brain, retina, and liver. The oatp3 mRNA is exclusively expressed in the retina and kidney. On the other hand, the oatp1 mRNA is expressed in the liver, kidney, brain, skeletal muscle, and colon (3Jacquemin E. Hagenbuch B. Stieger B. Wolkoff A.W. Meier P.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 133-137Crossref PubMed Scopus (544) Google Scholar). Finally, in situ hybridization analysis indicated that the oatp2 mRNA is widely distributed in neuronal cells of many brain regions as well as in the choroid plexus. This is the first report identifying the molecules responsive for transporting thyroid hormones across cell membrane. The transport of thyroid hormones across the plasma membrane determines the intracellular concentration of these hormones and hence the activation of the nuclear T3 receptor. The existence of mechanisms regulating the transport of thyroid hormones has been suggested in cerebrocortical neurons (17Chantoux F. Blondeau J.-P. Francon J. J. Neurochem. 1995; 65: 2549-2554Crossref PubMed Scopus (27) Google Scholar), astrocytes (18Beslin A. Chantoux F. Blondeau J.-P. Francon J. Endocrinology. 1995; 136: 5385-5390Crossref PubMed Scopus (16) Google Scholar), glial cells (19Francon J. Chantoux F. Blondeau J.-P. J. Neurochem. 1989; 53: 1456-1463Crossref PubMed Scopus (36) Google Scholar), hepatocytes (20Blondeau J.-P. Osty J. Francon J. J. Biol. Chem. 1988; 263: 2685-2692Abstract Full Text PDF PubMed Google Scholar, 21Krenning E. Docter R. Bernard B. Visser T. Hennemann G. Biochim. Biophys. Acta. 1981; 676: 314-320Crossref PubMed Scopus (103) Google Scholar), erythrocytes (22Osty J. Jego L. Francon J. Blondeau J.-P. Endocrinology. 1988; 123: 2303-2311Crossref PubMed Scopus (46) Google Scholar), and skeletal muscle (23Centanni M. Robbins J. J. Clin. Invest. 1987; 80: 1068-1072Crossref PubMed Scopus (37) Google Scholar). The following are comparisons of the pharmacological characteristics among oatp2, oatp3, and the known native thyroid hormone transporters. The Km values for T4 and T3of oatp2 and oatp3 were similar to those for neural cells (19Francon J. Chantoux F. Blondeau J.-P. J. Neurochem. 1989; 53: 1456-1463Crossref PubMed Scopus (36) Google Scholar) and hepatocytes (21Krenning E. Docter R. Bernard B. Visser T. Hennemann G. Biochim. Biophys. Acta. 1981; 676: 314-320Crossref PubMed Scopus (103) Google Scholar), but 10-fold higher than those reported by Chantouxet al. (17Chantoux F. Blondeau J.-P. Francon J. J. Neurochem. 1995; 65: 2549-2554Crossref PubMed Scopus (27) Google Scholar) and Blondeau et al. (20Blondeau J.-P. Osty J. Francon J. J. Biol. Chem. 1988; 263: 2685-2692Abstract Full Text PDF PubMed Google Scholar). We do not know what accounts for these differences. In our study, the oatp2-mediated uptake of T4 was not dependent on extracellular Na+, whereas the transporting mechanisms of thyroid hormones are heterogeneous in Na+ dependence: Na+-dependent (21Krenning E. Docter R. Bernard B. Visser T. Hennemann G. Biochim. Biophys. Acta. 1981; 676: 314-320Crossref PubMed Scopus (103) Google Scholar), Na+-independent (19Francon J. Chantoux F. Blondeau J.-P. J. Neurochem. 1989; 53: 1456-1463Crossref PubMed Scopus (36) Google Scholar, 20Blondeau J.-P. Osty J. Francon J. J. Biol. Chem. 1988; 263: 2685-2692Abstract Full Text PDF PubMed Google Scholar, 24Topliss D.J. Kolliniatis E. Barlow J.W. Lim C.-F. Stockigt J.R. Endocrinology. 1989; 124: 980-986Crossref PubMed Scopus (30) Google Scholar) and mixed (18Beslin A. Chantoux F. Blondeau J.-P. Francon J. Endocrinology. 1995; 136: 5385-5390Crossref PubMed Scopus (16) Google Scholar, 23Centanni M. Robbins J. J. Clin. Invest. 1987; 80: 1068-1072Crossref PubMed Scopus (37) Google Scholar). In addition, it has been reported that oocytes injected with rat liver poly(A)+ RNA showed Na+-dependent uptake of T4 and T3 (25Docter R. Friesema E.C.H. van Stralen P.G.J. Krenning E.P. Everts M.E. Visser T.J. Hennemann G. Endocrinology. 1997; 138: 1841-1846Crossref PubMed Google Scholar); however, the functional fraction size is different from that of the oatp2 or oatp3 mRNA. Therefore, the molecules responsible for transporting thyroid hormones are suggested to be heterogeneous. The uptake of thyroid hormones in the native tissues appears to be inhibited by a variety of structurally unrelated drugs, including non-bile acid cholephils such as BSP, nonsteroidal anti-inflammatory drugs (20Blondeau J.-P. Osty J. Francon J. J. Biol. Chem. 1988; 263: 2685-2692Abstract Full Text PDF PubMed Google Scholar, 24Topliss D.J. Kolliniatis E. Barlow J.W. Lim C.-F. Stockigt J.R. Endocrinology. 1989; 124: 980-986Crossref PubMed Scopus (30) Google Scholar), diphenylhydantoin (26Gingrich S.A. Smith P.J. Shapiro L.E. Surks M.I. Endocrinology. 1985; 116: 2306-2313Crossref PubMed Scopus (10) Google Scholar), and propranolol (27Krenning E.P. Docter R. Bernard B. Visser T. Hennemann G. FEBS Lett. 1982; 140: 229-233Crossref PubMed Scopus (79) Google Scholar). In our study, the oatp2-mediated uptake of taurocholate was inhibited by some of these compounds, but no oatp3-mediated uptake was found for tryptophan, phenylalanine, tyrosine, and indomethacin. In the oatp1-expressing mammalian cells, T4 did not exhibit acis-inhibitory effect for BSP uptake (28Kanai N. Lu R. Bao Y. Wolkoff A.W. Schuster V.L. Am. J. Physiol. 1996; 270: F319-F325PubMed Google Scholar). Thus, further investigations will be required to understand the role and functions of the other members of the organic anion transporter family. The oatp2 mRNA is distributed exclusively in the brain, retina, and liver. In the central nervous system, most of the nuclear T3 is derived from local T4 deiodination (29Crantz F.R. Silvia J.E. Larsen P.R. Endocrinology. 1982; 110: 367-375Crossref PubMed Scopus (292) Google Scholar). However, most of the intracellular T3 in the liver and muscle is derived from plasma (30van Doorn J. van der Heide D. Roelfsema F. J. Clin. Invest. 1983; 72: 1778-1792Crossref PubMed Scopus (102) Google Scholar). Accordingly, the abundant expression of the oatp2 mRNA in the brain and liver implies that this molecule should regulate the availability of thyroid hormone in these tissues. Thyroid hormones also play an essential role in neural function of the mammalian central nervous system, particularly during a critical period of its development (31Vincent J. Legrand C. Rabié A. Legrand J. J. Physiol. ( Paris ). 1982–1983; 78: 729-738PubMed Google Scholar, 32Dussault J.H. Ruel J. Annu. Rev. Physiol. 1987; 49: 321-334Crossref PubMed Google Scholar). The absence of thyroid hormone causes serious damages to structural development and organization of the brain (especially the hippocampus and cerebellum), including biochemical maturation, and leads to irreversible mental retardation (33Porterfield S.P. Hendrich C.E. Endocr. Rev. 1993; 14: 94-106PubMed Google Scholar). In the hippocampus, the oatp2 mRNA is predominantly expressed in the pyramidal cells of CA1–4 and in the granule cells of the dentate gyrus. In the cerebellum, the oatp2 mRNA is highly expressed in Purkinje cells. The prominent expression of the oatp2 mRNA in these thyroid hormone-sensitive neurons further suggests that oatp2 may play a critical role in neuronal development and maintaining cell function. oatp2 mRNA was also moderately distributed in the choroid plexus. This is consistent with the notion that thyroid hormones are transported into the brain via the blood-brain barrier (9Pardridge W.M. Endocrinology. 1979; 105: 605-612Crossref PubMed Scopus (103) Google Scholar) or via the choroid plexus (10Southwell B.R. Duan W. Alcorn D. Brack C. Richardson S.J. Köhrle J. Schreiber G. Endocrinology. 1993; 133: 2116-2126Crossref PubMed Google Scholar). In situ hybridization of rat oatp1 revealed that rat oatp1 mRNA is also expressed in the choroid plexus (34Angeletti R.H. Novikoff P.M. Juvvadi S.R. Fritschy J.-M. Meier P.J. Wolkoff A.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 283-286Crossref PubMed Scopus (128) Google Scholar). Although the probe for rat oatp1 (positions 2031–2090) is 80∼95% identical among organic anion transporter family members, the probe only detected signals in the choroid plexus. In our experiments, to avoid the nonspecific signals, we used the 900-bp 3′-noncoding region that has <48% identity to any organic anion transporter family. Thus, it seems likely that at least two organic anion transporter family subtypes are coexpressed in the choroid plexus: oatp1, which mediates BSP uptake that is not inhibited by T4 (28Kanai N. Lu R. Bao Y. Wolkoff A.W. Schuster V.L. Am. J. Physiol. 1996; 270: F319-F325PubMed Google Scholar); and oatp2, which transports thyroid hormones. Noé et al. (8Noé B. Hagenbuch B. Stieger B. Meier P.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10346-10350Crossref PubMed Scopus (389) Google Scholar) also showed that the oatp2 mRNA is highly expressed in the brain, liver, and kidney. However, in our experiment, no oatp2 expression was detected in the kidney. This discrepancy in the Northern blot analysis may be also attributed to the different nucleotide regions of the probe. We used the 3′-noncoding region of oatp2 that has <48% identity to oatp1, OAT-K1, and oatp3. On the other hand, Noé et al. (8Noé B. Hagenbuch B. Stieger B. Meier P.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10346-10350Crossref PubMed Scopus (389) Google Scholar) used nucleotides 1–360, which has >80% identity to oatp1 (82%) and OAT-K1 (87%). In their report, the size of the major band in the kidney was smaller than that in the brain. It is possible that their probe cross-hybridized to the other member of the organic anion transporter family,e.g. oatp3 or OAT-K1. In addition, since the probes for oatp3 clearly hybridized to the kidney mRNA, the mRNAs used were not degenerate in our study. The same result was obtained using the rat multiple-tissue Northern filter. In both cases, mRNA qualities were confirmed by β-actin (data not shown). Based on our results, we suggest that the oatp2 mRNA is not expressed in the kidney as much as shown for oatp2 (8Noé B. Hagenbuch B. Stieger B. Meier P.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10346-10350Crossref PubMed Scopus (389) Google Scholar). The oatp3 mRNA is exclusively distributed in the kidney. Most of the T4 secreted from the thyroid is deiodinated in peripheral tissues. The liver and kidney are the major peripheral organs producing T3 from T4 (35Berry M.J. Banu L. Larsen P.R. Nature. 1991; 349: 438-440Crossref PubMed Scopus (750) Google Scholar). Thus, the specific expression of oatp3 mRNA in the kidney suggests an essential role for oatp3 in transporting thyroid hormones from the circulation to the deiodination sites in the kidney. Individuals with Refetoff's syndrome, characterized by resistance to thyroid hormone, exhibit reduced clinical and biochemical activities of thyroid hormone action relative to the circulating hormone level (36Refetoff S. Weiss R.E. Usala S.J. Endocr. Rev. 1993; 14: 348-399Crossref PubMed Scopus (610) Google Scholar). Several pathophysiological mechanisms have been suggested to account for the thyroid hormone resistance seen in Refetoff's syndrome patients. One possible mechanism is reduced hormone availability to tissues due to impaired thyroid hormone entry into cells (36Refetoff S. Weiss R.E. Usala S.J. Endocr. Rev. 1993; 14: 348-399Crossref PubMed Scopus (610) Google Scholar). Accordingly, functional and genetic investigations of oatp2 and oatp3 may also help us to understand the etiology of such disorders and to aid in clinical diagnosis. We thank Drs. Kazuo Nunoki and Akira Kobayashi for critical reading of the manuscript and Satoshi Sai for photographic assistance." @default.
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W2125402020 title "Molecular Characterization and Tissue Distribution of a New Organic Anion Transporter Subtype (oatp3) That Transports Thyroid Hormones and Taurocholate and Comparison with oatp2" @default.
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