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Department of Neurophysiology (K.F., H.A., T.N., M.O., T.S., N.A., M.Se., T.A.), First Department of Surgery (K.F., H.A., M.U., M.O., T.O., K.S., M.Su., S.M.), Department of Pediatrics (T.N., Y.K., K.I.), Second Department of Internal Medicine (T.S., S.I.), Third Department of Internal Medicine (N.A., T.S.), Department of Molecular Pharmacology (K.N.), Tohoku University School of Medicine, Sendai 980-8575, Japan; Analytical and Metabolic Research Laboratories, Sankyo Co., Ltd. (T.T.), Tokyo 140-8710, Japan; and Division of Nephrology, Faculty of Medicine, University of Tokyo (M.T.), Tokyo 113-8655, Japan
Address all correspondence and requests for reprints to: Dr. Takaaki Abe, Division of Nephrology, Endocrinology and Vascular Medicine, Department of Medicine, Tohoku University Graduate School of Medicine, 1-1 Seiryo-cho, Aoba-ku, Sendai 980-8574, Japan. E-mail: takaabe{at}mail.cc.tohoku.ac.jp
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In humans, two organic anion transporters have been reported: liver-specific organic anion transporter (LST-1) (12) and human OATP (13). Compared with rat oatps, the expression of these isolated organic anion transporters is much organ specific. LST-1 is exclusively expressed in the liver, and OATP in the brain (12). In addition, although LST-1 transports thyroid hormones (12), no molecules transporting thyroid hormone have been identified in other tissues.
Here, we report the isolation and pharmacological characterization of human and rat novel organic anion transporters, OATP-E/oatp-E, which transport thyroid hormone in various peripheral tissues. Combined with our data demonstrating that human OATP transports thyroid hormone in the brain, these results suggest that thyroid hormone transport is mediated by different molecules with distinct expression patterns in humans.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and
p-aminohippuric acid (PAH) were purchased from
Sigma (St. Louis, MO).
[125I]T3,
[125I]T4, and
[125I]rT3 were purchased
from NEN Life Science Products (Boston, MA), and
[3H]taurocholate from Amersham Pharmacia Biotech (Arlington Heights, IL).
Isolation of human and rat complementary DNA (cDNA)
The GenBank database dbEST was searched for all known
mammalian oatp family and PG transporters. As a result, a clone that
had weak to moderate similarity to both the oatp family and the PG
transporters was identified (GenBank accession no. H85940). A human
hippocampus cDNA library was constructed in a 
ZAPII vector
(14). Independent clones (5 x
105) were screened with the EST clone under high
stringency (12). In a series of screenings, two clones
were isolated, and the clone that had the largest insert (pH8) was
chosen for further analysis. The sequences were determined using
ABI PRISM 377 DNA sequencer (Perkin-Elmer Corp.,
Foster City, CA). A rat retina cDNA library (5 x
105 independent clones) was also screened with
the same EST fragment.
Homology analysis
The hydropathy profile analysis was performed according to the
method reported by Kyte and Doolittle (15). Multiple
sequence alignments of amino acid sequences were carried out using
CLUSTAL W (16). The phylogenetic tree was described by
TreeView (17).
Northern blot analysis
Human multiple tissue Northern blots containing 2 µg
polyadenylated RNA were purchased (CLONTECH Laboratories, Inc., Palo Alto, CA). The coding region of pH8
(EcoRI-EcoRI, 2125 bp) was used as a probe.
Filters were hybridized with the 32P-labeled
fragment in a buffer containing 50% formamide, 5 x SSC (standard
saline citrate), 5 x Denhardt’s solution, and 1% SDS at 42 C
overnight; washed in 0.1 x SSC and 0.1% SDS at 65 C for 1
h; and exposed to film at -80 C overnight (11, 12, 14).
Human ß-actin was also used to qualify the quality of the mRNA.
RT-PCR
The tissue distribution of human OATP-E was further
characterized by RT-PCR. The primers used are located in the exon 1
(forward) and exon 2 of human OATP-E genome (GenBank accession no.
AL357033; forward, 5'-CGGCCGGGCCCTCGAGAC-3'; reverse, 5'-
GCAGGGCACGTCCTGACA-3') to avoid nonspecific amplification from the
genomic DNA. A human multiple tissue cDNA panel (CLONTECH Laboratories, Inc.) was used as a template. As a control,
glyceraldehyde-3-phosphate dehydrogenase primers were used (forward,
5'-TCCACCACCCTGTTGCTGTAG-3'; reverse, 5'-GACCACAGTCCATGCCATCACT-3').
PCR amplification was performed using a hot start amplification
protocol with LA Taq (Takara) combined with the
Taq Start Antibody (CLONTECH Laboratories, Inc.) according to the following schedule: 94 C for 1 min for
the first cycle, then 94 C for 0.5 min and 67 C for 4 min for 30
cycles. PCR products were transferred to a nylon membrane and
hybridized with the radiolabeled OATP-E full cDNA fragment.
Expression in Xenopus oocytes
The capped RNA of pH8 was transcribed in vitro.
Xenopus laevis oocytes were prepared as described previously
(11, 12, 14). Defolliculated oocytes were microinjected
with 10 ng transcribed RNA and cultured for 72 h in modified
Barth’s medium at 18 C. The uptake of radiolabeled chemicals was
measured at room temperature in a medium containing 100
mM NaCl, 2 mM KCl, 1
mM CaCl2, 1
mM MgCl2, and 10
mM HEPES, pH 7.5. After washing with the same
buffer, each oocyte was dissolved in 500 µl 10% SDS, and the
radioactivity was counted with 4 ml scintillation fluid in a
liquid scintillation counter (Packard, Downers Grove, IL).
Water-injected oocytes were used as controls. The uptake rate of
[3H]taurocholate (5 µM)
by OATP-E-expressing oocytes was determined in the absence or presence
of 0.5, 5, and 50 µM inhibitors. The uptake of
[125I]T3 (1
µM) was further measured in the presence of 100
µM cold thyroid hormone analogs. The
statistical significance was tested by unpaired t test
(Table 1
and Figs. 3, 4A, and 7). In Fig. 4B, statistical analysis of
the data was performed by ANOVA, and significance was determined by
Bonferroni’s test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). In human OATP-E, there are two
putative N-glycosylation sites in the predicted
extracellular loops, and one potential phosphorylation site for
cAMP-dependent protein kinase and seven potential phosphorylation sites
for protein kinase C in the cytosolic portions (18, 19)
(Fig. 1A). The sequence homology analysis revealed a moderate sequence
similarity to the oatp family, human LST-1/rat rlst-1, and the PG
transporter. The overall amino acid sequence identities of OATP-E were
31.2% to human PG transporter (20), 31.0% to rat PG
transporter (21), 30.6% to oatp1 (22),
28.6% to oatp2 (11, 23), 32.2% to oatp3
(11), 29.5% to OAT-K1 (24), 32.4% to OAT-K2
(25), 29.8% to human OATP (13), 30.8% to
human LST-1 (12), 23.2% to rat rlst-1 (26),
28.4% to rat oatp4, which is thought to be a complete form of rlst-1
(27), and 29.5% to rat moat1 (28). We also
isolated the rat counterpart oatp-E from rat retina. The overall
homology between rat oatp-E and human OATP-E was 72.6% at the amino
acid level, and the transmembrane domains and their surrounding area
were highly conserved (Fig. 1A). Phylogenetic tree analysis showed that
human OATP-E and rat oatp-E can be localized separately from
LST-1/rlst-1, the PG transporter, and the oatp family (Fig. 1B). These
data suggest that OATP-E and oatp-E are supposed to be new members of
the organic anion transporter family.
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), both of which are preferable
substrates for the oatp family and LST-1/rlst-1.
The OATP-E-cRNA injected oocytes increased uptake of
[125I]T33-fold above that of the
water-injected oocytes (0.87 ± 0.1 vs. 0.3 ±
0.02 pmol/oocyte/20 min at 1 µM
[125I]T3; P <
0.01; Table 1). These OATP-E-mediated uptakes were saturable with
increasing substrate concentrations. The apparent
Km values for T3 and
taurocholate determined by three independent experiments were
0.9 ± 0.4 and 14.9 ± 3.3 µM,
respectively (Fig. 2). This
OATP-E-mediated uptake was not inhibited by replacing the extracellular
Na+ with choline (data not shown). OATP-E also
transported T4 and rT3
weakly, but significantly (Table 1 and Fig. 7). In addition, we
examined the transport activity of rat oatp-E. The oocytes injected
with transcribed rat oatp-E RNA also transported
T3> and taurocholate (Fig. 3). On the other hand, although
PGE2 was slightly transported, other eicosanoids
(PGD2, PGE1, and
PGF2) and PAH were not transported (Table 1).
These data indicate that OATP-E/oatp-E encode a subfamily of the
organic anion transporter that transports thyroid hormone.
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, unlabeled taurocholate, BSP, and T3 showed
definite and dose-dependent inhibitory effects on OATP-E-mediated
taurocholate uptake. On the other hand, PGD2,
PGE1, PGE2,
PGF2, and PAH, which were not transported by
OATP-E, did not show significant inhibitory effects at any
concentration tested. We also examined the inhibition of
OATP-E-mediated
[125I]T3 uptake (1
µM) by adding 100 µM unlabeled
T3, T4, and taurocholate.
All of the compounds tested inhibited
[125I]T3 uptake (Fig. 4B). T3 completely inhibited OATP-E-mediated
[125I]T3 uptake.
T4 also significantly inhibited
[125I]T3 uptake. However,
the inhibitory effect of taurocholate was much less than that of the
thyroid hormones tested.
Northern blot analysis and RT-PCR
Northern blot analysis of human OATP-E showed two bands (one major
band at 3.0 kilonucleotides and another minor band at 5.0
kilonucleotides) in heart, placenta, lung, liver, skeletal muscle,
kidney, and pancreas (Fig. 5A). The two
different sized bands of the OATP-E mRNA are probably derived from the
same gene, because both bands were observed under high stringency
filter-washing conditions, although we cannot exclude the possibility
of alternative splicing. To further examine the tissue distribution of
human OATP-E, RT-PCR was performed (Fig. 5B).
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). On the
basis of the organization, we designed a PCR primer set flanking
between exon 1 and exon 2 (Fig. 6). As a result, RT-PCR generated bands
in brain, spleen, thymus, prostate, testis, and small intestine in
addition to the organ detected by Northern blot analysis (Fig. 5B, upper panel). The bands were further identified by
hybridization with human OATP-E radiolabeled probe (Fig. 5B,
middle panel).
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). Human OATP-E also
transports rT3 significantly (0.65 vs.
0.19 pmol/oocyte/20 min; P < 0.01; Fig. 7). This
OATP-mediated uptake was linear for at least 60 min and was independent
of extracellular Na+ (data not shown). This
OATP-mediated transport of thyroid hormone was saturable with
increasing substrate concentrations. The apparent
Km values for T3 and
T4 uptake determined by three independent experiments
were 6.5 ± 2.5 and 8.0 ± 1.7 µM, respectively
(Fig. 8).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In human liver, carrier-mediated transport of thyroid hormone have been
predicted. Our previous data revealed that LST-1, which is exclusively
expressed in the human liver, transported T4 and
T3 in a Na+-independent
manner (12). The pharmacological characterization of
LST-1-mediated thyroid hormone uptake is consistent with the previous
data reported in human liver (29, 30). This is the first
report identifying a human molecule that transports thyroid hormone.
However, thyroid hormone transport systems have been distributed in
various organs [reviewed by Partridge (1)]. What kinds
of molecules are involved in thyroid hormone transport in other
tissues? To answer this, we performed further molecular screening and
pharmacological characterization. As a result, we isolated a novel
organic anion transporter subfamily, OATP-E/oatp-E. The present results
demonstrate that human and rat OATP-E/oatp-E both transport thyroid
hormone in a Na+-independent manner. The apparent
Km of human OATP-E for
T3 was 0.9 ± 0.4 µM. The
pharmacological characterization revealed that nonlabeled
T3, T4, and taurocholate
showed a definite inhibitory effect on OATP-E-mediated
[125I]T3 uptake.
OATP-E-mediated [3H]taurocholate uptake was
also inhibited by taurocholate, BSP, and T3. In
contrast, PGD2, PGE1,
PGE2, PGF2, and PAH
showed no or little inhibitory effect. According to these substrate
specificities, we designated the isolated clone as a subfamily of OATP
according to its substrate specificity rather than its amino acid
sequence homology. These data suggest that OATP-E is the candidate
molecule for thyroid hormone transport in humans.
In this study we also revealed that human OATP, which is exclusively expressed in the brain (12), transports thyroid hormones. To date, there are a few reports about thyroid hormone uptake in the central nervous system (31, 32, 33, 34, 35). The reported Km for thyroid hormone uptake varies from the nanomolar level in mouse neuroblastoma cells (31) to the micromolar level in rat glia cells (32). The Km for OATP obtained in this study is in the same range of values for cultured cerebrocortical neurons and rat glial cells (32). We conclude from these data that in humans different transporting molecules are involved in transporting thyroid hormone in the brain (OATP), liver (LST-1), and peripheral tissues (OATP-E).
In rats we previously reported that rat oatp2 and oatp3 transport thyroid hormones (11). Subsequently, Friesema et al. reported that rat oatp1 also transports thyroid hormone (36). In contrast to that in humans, the tissue distribution of rat molecules responsible for thyroid hormone transport is completely different. These transporters are distributed in several organs compared with human LST-1 and OATP. Rat oatp1 is expressed in brain, liver, and kidney; oatp2 in brain, retina, and liver; and oatp3 in retina, liver, and kidney. These data further indicate the species difference of the molecules responsible for thyroid hormone transport between humans and rats.
The discovery of OATP-E/oatp-E will also explain the existence of thyroid hormone transport systems in other tissues at the molecular level. OATP-E is slightly expressed in the small intestine. Bile acids secreted by the liver enter the intestine, which are absorbed in large part by the ileum, and return to the liver by way of the portal vein (37, 38). In addition, the existence of an enterohepatic circulation of thyroid hormone in the rat has been well established (39, 40, 41, 42, 43). Because OATP-E and LST-1 transport both thyroid hormone and taurocholate, it is suggested that OATP-E and LST-1 may be involved in the transport of bile acids and thyroid hormone in this circulation. OATP-E mRNA is also expressed in the kidney. Most T4 secreted from the thyroid gland is deiodinated in peripheral tissues. The liver and kidney are the major peripheral organs producing T3 from T4 (44). Thus, the expression of OATP-E mRNA in the kidney suggests the essential role in transporting thyroid hormone from the circulation to the deiodination sites in the kidney, like oatp3 in rats (11). Furthermore, OATP-E mRNA is expressed in skeletal muscle. It is well known that T3 enters skeletal muscle cells by an active transport system (8, 45, 46). Thus, again, OATP-E may transport thyroid hormone by a Na+-independent process in the peripheral tissues.
Individuals with Refetoff’s syndrome, which is characterized by resistance to thyroid hormone, exhibit reduced clinical and biochemical activities of thyroid hormone relative to the circulating hormone level (47). It has been well known that the molecular basis of the syndrome is the abnormality of the nuclear thyroid hormone receptor TR-ß. However, among these patients, other functional defects have been postulated. One possible mechanism is reduced hormone availability to tissues due to impaired thyroid hormone entry into neural cells (47). Thus, our finding might provide a clue for identifying one of the genetic pathogeneses of the disease.
Studies using isolated rat hepatocytes suggested multiple transporters for uptake of T4, T3, and rT3. A role of extracellular Na+ in thyroid hormone uptake has been also suggested. Treatment of hepatocytes with Na+/K+-adenosine triphosphatase inhibitor or replacement of extracellular Na+ results in a marked decrease in thyroid hormone uptake, suggesting that the uptake is Na+ dependent (48). Recently, rat Na+/taurocholate cotransporting polypeptide has been reported to transport thyroid hormone (37), although its mRNA is only expressed in the liver. Further molecular characterization and identification are necessary to clarify this Na+-dependent thyroid hormone uptake fraction in vivo.
In conclusion, candidate molecules responsible for thyroid hormone transport have been identified and have been found to be different in rats and humans. Our findings should aid in understanding the delivery of thyroid hormone to tissue in humans.
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Acknowledgments |
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Footnotes |
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2 The sequences of human OATP-E and rat oatp-E have been deposited
under the GenBank Accession No. AF187817 (human) and AF239262 (rat),
respectively. Human OATP-E has recently been reported by Tamai
(49 ) during the preparation of our manuscript.
3 Present address: Mitsubishi-Tokyo Pharmaceuticals, Inc., Yokohama
227-0033, Japan.
Received July 25, 2000.
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, Uptake in the absence of the compound. Compounds (
, 0.5
µM; 
, 5 µM; 
, 50 µM) of
were added to inhibit 5 µM [3H]taurocholate
uptake. Statistical significance was determined by unpaired
t test (*, P < 0.05; **,
P < 0.01). B, Effects of thyroid hormones and
taurocholate on OATP-E-mediated T3 transport. Thyroid
hormones (100 µM) were added to inhibit 1
µM [125I]T3 uptake. Data
represent the mean ± SEM of 8–15 oocytes.
Statistical significance was determined by Bonferroni’s test (*,
P < 0.05; **, P < 0.01). TCA,
Taurocholate.
) and possible
phosphorylation sites (*) are indicated. B, The Phylogenetic
relationship between OATP-E/oatp-E, LST-1/rlst-1, the oatp family,
moat1, and the PG transporter. Branch lengths are drawn to scale.
