Molecular Cloning and Characterization of a New Multispecific
Organic Anion Transporter from Rat Brain*
Hiroyuki
Kusuhara
§,
Takashi
Sekine§,
Naoko
Utsunomiya-Tate§,
Minoru
Tsuda§,
Ryoji
Kojima¶,
Seok Ho
Cha§,
Yuichi
Sugiyama
,
Yoshikatsu
Kanai§, and
Hitoshi
Endou§
From the § Department of Pharmacology and Toxicology,
Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo
181-8611, the
Department of Biopharmaceutics, Graduate
School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo,
Bunkyo-ku, Tokyo 113-0033, and the ¶ Department of Pharmacology,
Faculty of Pharmacy, Meijo University, 150, Yagotoyama, Tenpaku-ku,
Nagoya-shi, Tokyo 468-8503, Japan
 |
ABSTRACT |
A cDNA encoding the new member of the
multispecific organic anion transporter family, OAT3, was isolated by
the reverse transcription-polymerase chain reaction cloning method.
Degenerate primers were designed based on the sequences conserved among
OAT1, OAT2, and organic cation transporter 1 (OCT1), and reverse
transcription-polymerase chain reaction was performed using rat brain
poly(A)+ RNA. The 536-amino acid protein sequence
encoded by OAT3 showed 49, 39, and 36% identity to those of OAT1,
OAT2, and OCT1, respectively. Northern blot analysis revealed that rat
OAT3 mRNA is expressed in the liver, brain, kidney, and eye. When
expressed in Xenopus laevis oocytes, OAT3 mediated the
uptake of organic anions, such as p-aminohippurate
(Km = 65 µM), ochratoxin A
(Km = 0.74 µM), and estrone sulfate
(Km = 2.3 µM) and a cationic compound, cimetidine. OAT3-mediated uptake of [3H]estrone
sulfate was sodium-independent. para-Aminohippuric acid, estrone sulfate or ochratoxin A did not show any
trans-stimulatory effect on either influx or efflux of
[3H]estrone sulfate via OAT3. Organic anions such as
sulfobromophthalein, probenecid, indocyanine green, bumetanide,
piroxicam, furosemide, azidodeoxythymidine,
4,4'-diisothiocyanostilbene-3,3'-disulfonic acid, and
benzylpenicillin inhibited OAT3-mediated estrone sulfate uptake, while
ouabain and digoxin did not. Organic cations such as
tetraethylammonium, guanidine, verapamil, and quinidine did not
interact with OAT3. Acidic metabolites of neurotransmitters derived
from dopamine, epinephrine, norepinephrine, and serotonin inhibited the
uptake of estrone sulfate via OAT3. These results suggest an important
role of OAT3 in the excretion/detoxification of endogenous and
exogenous organic anions, especially from the brain.
 |
INTRODUCTION |
The liver and kidney are organs central to the elimination of
endogenous and exogenous organic anions (1-5). Previous physiological and pharmacological experiments have revealed that the uptake of
organic anions in the liver and kidney across the basolateral membrane
occur via transporters possessing a wide substrate selectivity (1-5).
In the liver, the organic anion transporting polypeptide 1 (oatp1)1 and oatp2 are
multispecific organic anion transporters that transport structurally
unrelated organic anions in a sodium-independent manner (1, 2,
6, 7).
In regard to the kidney, organic anion transporter 1 (OAT1/ROAT1) has
recently been isolated from the rat kidney (8, 9). OAT1 is a
multispecific organic anion transporter that interacts with a variety
of organic anions such as p-aminohippurate (PAH), dicarboxylates, cyclic nucleotides, prostaglandin E2,
urate,
-lactam antibiotics, nonsteroidal anti-inflammatory drugs,
and diuretics (8). The transport characteristics of OAT1 are identical
to those of the classical organic anion transporter on the basolateral membrane of the proximal tubule (3-5, 8, 9). A search of a DNA data
base revealed that the amino acid sequence of novel liver-specific
transport protein (NLT) (10) shows 42% identity to that of OAT1 (11).
We demonstrated that NLT also mediated the transport of organic anions
such as PAH, salicylate and acetylsalicylate, prostaglandin
E2, and dicarboxylates and concluded that NLT is a
liver-specific organic anion transporter (OAT2) (11).
In contrast to the liver and kidney, little is known concerning the
organic anion transport in the brain. The brain possesses two
physiological barriers, namely, blood-brain barrier (BBB) and
blood-cerebrospinal fluid barrier (BCSFB), which prevent the entry of
xenobiotics into the brain (12-14). The tight junction, an anatomical
feature of BBB and BCSFB, connects brain capillary endothelial cells or
choroid epithelial cells to each other and minimizes the influx of
xenobiotics via paracellular routes (12-14). In addition, in
vivo and in vitro kinetic studies have suggested the
presence of efflux transport pathways for organic anions in the BBB and
BCSFB. PAH and benzylpenicillin, typical substrates of the renal
organic anion transporter, have been demonstrated to be effluxed from
the brain (15-17). When injected into the cerebral cortex, the rapid
and saturable elimination of PAH from the brain was observed (15). The
efflux of benzylpenicillin and cimetidine via BCSFB was also reported
to be saturable process (16-18). Based on the results of these
studies, the presence of a kidney-like organic anion efflux
transporter(s) has been predicted in the brain. OAT1 and OAT2 are
expressed specifically in the kidney and liver, respectively (8, 9,
11). Although OAT1 was detected in the brain, its expression level was
very small (8). From these observations, we hypothesized the existence
of a new member of the OAT family in the brain. In the present study,
we report the isolation of the new member of OAT family, OAT3, which is
much more strongly expressed in the brain than OAT1.
 |
EXPERIMENTAL PROCEDURES |
Materials--
[3H]PAH (2.45 Ci/mmol) and
[3H]estrone sulfate (53 Ci/mmol) were purchased from NEN
Life Science Products. [3H]Ochratoxin A (14.8 Ci/mmol)
was purchased from Moravek (Brea, CA). [32P]dCTP and
[3H]cimetidine (18.2 Ci/mmol) were obtained from Amersham
Pharmacia Biotech (Uppsala, Sweden). All other chemicals and reagents
were of analytical grade.
Reverse Transcription-PCR--
Degenerate PCR primers were
designed based on the amino acid sequences that are conserved among
OAT1, OAT2 (NLT) and OCT1 (19): forward primer,
5'-TYMYWGARTCHSCMCGSTGG-3' (corresponding to the nucleotide 803-822 of
OAT1) and reverse primer, 5'-AKSAMWGTRGGGTACARCTC-3' (corresponding to
the nucleotide 1339-1358 of OAT1). One microgram of
poly(A)+ RNA prepared from rat brain was
reverse-transcribed and used as a template for subsequent PCR with the
set of degenerate primers. PCR was performed according to the following
protocol: 94 °C for 10 s, 49 °C for 30 s, and 72 °C
for 30 s; 40 cycles. PCR products were isolated using TA cloning
kit (Invitrogen) and sequenced. A PCR product, whose partial amino acid
sequence showed 55% identity to that of OAT1, was labeled with
[32P]dCTP by random priming method (T7 Quick Prime Kit,
Amersham Pharmacia Biotech) and used for the screening of OAT3 as a
probe. Since the preliminary Northern blot analysis revealed that the mRNA detected by this probe was abundantly expressed in the kidney, we used the rat kidney cDNA library for the screening.
Construction of cDNA Library and Isolation of OAT3--
A
nondirectional cDNA library was prepared from rat kidney
poly(A)+ RNA using the Superscript Choice system (Life
Techonologies, Inc.), and the cDNAs were ligated into
ZipLox
EcoRI arms (Life Techonologies, Inc.). Replicated filters of
a phage library were hybridized overnight at 37 °C in a
hybridization solution (50% formamide, 5 × standard saline
citrate (SSC), 3 × Denhardt's solution, 0.2% SDS, 10% dextran
sulfate, 0.2 mg/ml denatured salmon sperm DNA, 2.5 mM
sodium pyrophosphate, 25 mM MES, and 0.01% Antifoam B, pH
6.5), and washed at 37 °C in 0.1 × SSC and 0.1% SDS. The cDNA inserts in positive
ZipLox phages were recovered in plasmid pZL1 by in vivo excision and further subcloned into
pBluscript II SK
(Stratagene) for sequencing and in vitro transcription.
Sequence Determination--
Deleted clones obtained by
KiloSequence deletion kit (Takara, Japan), and specially synthesized
oligonucleotide primers were used for sequencing of OAT3 cDNA.
cRNA Synthesis and Uptake Experiments Using X. laevis
Oocytes--
cRNA synthesis and uptake measurements were performed as
described previously (8). The capped cRNAs were synthesized in vitro using T7 RNA polymerase from the plasmid DNAs linearized with XbaI. Defolliculated oocytes were injected with 10 ng
of the capped cRNA and incubated in Barth's solution (88 mM NaCl, 1 mM KCl, 0.33 mM
Ca(NO3)2, 0.4 mM CaCl2,
0.8 mM MgSO4, 2.4 mM NaHCO3, and 10 mM HEPES) containing 50 µg/ml
gentamicin and 2.5 mM pyruvate, pH 7.4, at 18 °C. After
incubation for 2-3 days, uptake experiments were performed at room
temperature in ND96 solution (96 mM NaCl, 2 mM
KCl, 1.8 mM CaCl2, 1 mM
MgCl2, and 5 mM HEPES, pH 7.4). The uptake
experiment was initiated by replacing ND96 solution with that
containing a radiolabeled ligand and terminated by the addition of
ice-cold ND96 buffer after 1 h of incubation. Oocytes were washed
five times with ice-cold ND96 and solubilized with 10% SDS, and the
accumulated radioactivity was determined.
The kinetic parameters for the uptake of PAH, estrone sulfate, and
ochratoxin A via OAT3 were estimated from the following equation:
v = Vmax × S/(Km + S), where v is the uptake rate of
the substrate (picomoles/hour/oocyte), S is the substrate concentration
in the medium (micromolar), Km is the
Michaelis-Menten constant (micromolar), and Vmax
is the maximum uptake rate (picomoles/hour/oocyte). To obtain the
kinetic parameters, the equation was fitted to the OAT3-specific
transport velocity, which was obtained by subtracting the transport
velocity in noninjected oocytes from that in OAT3-expressing oocytes,
by an iterative nonlinear least squares method using a MULTI program.
The input data were weighted as the reciprocal of the observed values,
and the Damping Gauss Newton Method algorithm was used for fitting. The
fitted line was converted to the v/S versus
v form (Eadie-Hofstee plot).
Examination of trans-Stimulatory Effect on the Transport via
OAT3--
In order to examine the trans-stimulatory effet
on the efflux of radiolabeled substrate, oocytes expressing OAT3 were
preloaded with [3H]estrone sulfate (80 nM in
the medium) for 90 min and then transferred into the medium with or
without unlabeled substrates (PAH, ochratoxin A, and estrone sulfate).
Both the radioactivity in the medium and that remaining in oocytes were
determined after 90-min incubation. In the experiment examining
trans-stimulatory effect on the influx of radiolabeled
substrate, oocytes expressing OAT3 were preloaded with unlabeled
substrates for 5 h and then transferred into the medium containing
[3H]estrone sulfate (40 nM). The uptake rate
of [3H]estrone sulfate for 1 h was determined.
Northern Blot Analysis--
Three µg of poly(A)+
RNA prepared from various rat tissues were electrophoresed on a 1%
agarose/formaldehyde gel and transferred onto a nitrocellulose filter.
The filter was hybridized in a hybridization solution overnight at
42 °C with a full-length cDNA of OAT3, which was randomly
labeled with [32P]dCTP. The filter was washed finally in
0.1 × SSC and 0.1% SDS at 60 °C.
 |
RESULTS |
A PCR product was isolated from rat brain mRNA using reverse
transcription-PCR method. And its partial amino acid sequence exhibited
some identity to OAT1. Using this as a probe, we isolated a novel
cDNA (rkl411) from the rat kidney cDNA library. When expressed in X. laevis oocytes, rkl411 mediated the uptake of several
organic anions; we therefore designated rkl411 as OAT3 (organic anion transporter 3). OAT3 cDNA consists of 2191 base pairs encoding a
membrane protein of 536 amino acids. Fig.
1 shows the deduced amino acid sequence
of rat OAT3 and the alignment with those of rat OAT1, OAT2, OCT1, and
OCT2 (20). The amino acid sequence of OAT3 shows 49, 39, 36, and 35%
identity to those of rat OAT1, OAT2, OCT1,and OCT2, respectively (Table
I). Recently, a third member of organic
cation transporter, rat OCT3, was isolated (21). Its amino acid
sequence showed 35% identity to that of OAT3 (Table I). Four putative
N-glycosylation sites and eight protein kinase C-dependent phosphorylation sites are predicted; two
N-glycosylation sites in the first loop between
transmembrane domains 1 and 2 (54 and 102 residues), and two protein
kinase C-dependent phosphorylation sites between
transmembrane domains 6 and 7 (259 and 266 residues) are identical with
those of OAT1 (Fig. 1). Kyte-Doolittle (31) hydropathy analysis
suggests that OAT3 possess 12 putative membrane-spanning domains.

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Fig. 1.
The amino acid sequence of OAT3 aligned with
those of homologous transporters from rat. Boxed
residues denote residues conserved in at least three transporters.
Putative N-linked glycosylation sites of OAT3 are indicated
by stars; putative protein kinase C phosphorylation sites
( ) are also indicated.
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The tissue distribution of OAT3 was investigated by Northern blot
analysis using poly(A)+ RNA from various rat tissues (Fig.
2). A strong 2.7-kilobase mRNA band
was detected in the brain, liver, kidney, and weakly in the eye. No
hybridization signals were detected with mRNA isolated from other
tissues. A slightly short transcript (2.2 kilobases) was detected only
in the liver (Fig. 2).

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Fig. 2.
Localization of OAT3 mRNA in rat tissues
by Northern blot analysis. Three micrograms of
poly(A)+ RNA from various rat tissues (except eye, 1.5 µg) was probed with 32P-labeled cDNA of OAT3.
kb, kilobases.
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Using Xenopus oocytes injected with OAT3 cRNA, we
investigated the transport characteristics via OAT3. OAT3 mediated the
transport of [3H]PAH, [3H]estrone sulfate,
[3H]ochratoxin A, and [3H]cimetidine (Fig.
3), but not salicylate, taurocholate,
digoxin, tetraethylammonium (TEA), guanidine, and daunomycin (data not shown). The uptake of estrone sulfate via OAT3 was not affected by the
replacement of extracellular sodium with lithium, choline, or
N-methyl-D-glucamine (Fig.
4). We examined whether
trans-stimulatory effect was observed in the transport via
OAT3. Estrone sulfate, ochratoxin A, and PAH, when added to the medium,
did not stimulate efflux of the preloaded estrone sulfate via OAT3
(Fig. 5A). Vice versa,
intracellulary accumulated unlabeled PAH, ochratoxin A, or estrone
sulfate did not stimulate the uptake of [3H]estrone
sulfate (Fig. 5B). Concentration dependence of the uptake of
[3H]PAH, [3H]estrone sulfate, and
[3H]ochratoxin A via OAT3 was examined (Fig.
6). OAT3-mediated uptake of these
compounds showed saturable kinetics and followed the Michaelis-Menten
equation. Nonlinear regression analyses yielded Km
values of 64.7 ± 10.0 µM, 2.34 ± 0.20 µM, and 0.739 ± 0.178 µM and
Vmax values of 23.3 ± 2.8 pmol/h/oocyte,
7.60 ± 0.44 pmol/h/oocyte, and 3.08 ± 0.33 pmol/h/oocyte
for PAH, estrone sulfate, and ochratoxin A, respectively.

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Fig. 3.
OAT3-mediated uptake of radiolabeled
compounds. The uptake rates of radiolabeled compounds
([3H]PAH, 3 µM; [3H]estrone
sulfate, 40 nM; [3H]ochratoxin A, 150 nM; and [3H]cimetidine, 150 nM)
by control (open columns) or OAT3-expressing (closed
columns) oocytes were measured after incubation for 1 h
(mean ± S.E.; n = 10).
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Fig. 4.
Effect of extracellular cation on
[3H]estrone sulfate uptake in X. laevis
oocytes expressing OAT3. The uptake rate of
[3H]estrone sulfate (40 nM) by noninjected
oocytes (open columns) or OAT3-expressing oocytes
(closed columns) for 1 h was measured (mean ± S.E.; n = 10) in the presence or absence of
extracellular Na+. Extracellular Na+ was
replaced with equimolar lithium, choline, or
N-methyl-D-glucamine.
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Fig. 5.
Effect of unlabeled substrates on the influx
or efflux of [3H]estrone sulfate via OAT3.
A, the lack of trans-stimulated efflux of
[3H]estrone sulfate via OAT3. Oocytes expressing OAT3
were preloaded with [3H]estrone sulfate (80 nM) for 90 min and transferred to the medium with or
without unlabeled substrate as indicated. The effluxed amount of
estrone sulfate during 90 min was shown as percentage of the preloaded
amount. (mean ± S.E.; n = 10) B, the
lack of trans-stimulated influx of [3H]estrone
sulfate via OAT3. Oocytes expressing OAT3 was preloaded unlabeled
substrates for 5 h at the indicated concentration before starting
the uptake experiment. The uptake rate of [3H]estrone
sulfate (40 nM) for 1 h by OAT3-expressing oocytes was
measured (mean ± S.E.; n = 10).
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Fig. 6.
Concentration dependence of OAT3-mediated
uptake of [3H]p-aminohippurate
(A), [3H]estrone sulfate
(B), and [3H]ochratoxin A
(C). The uptake of radiolabeled compounds
([3H]p-aminohippurate,
[3H]estrone sulfate, and [3H]ochratoxin A)
by control or OAT3-expressing oocytes were measured at the
concentration indicated after incubation for 1 h (mean ± S.E.; n = 10). OAT3-mediated transport was obtained by
subtracting the transport velocity in noninjected oocytes from that in
OAT3-expressing oocytes.
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To investigate the substrate selectivity, the inhibitory effect of
various compounds on OAT3-mediated [3H]estrone sulfate
uptake was determined (Fig. 7).
cis-Inhibitory effects were observed for structurally
unrelated organic anions; sulfobromophthalein, probenecid, indocyanine
green, bumetanide, piroxicam, furosemide, azidodeoxythymidine,
benzylpenicillin, and 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic
acid potently inhibited OAT3-mediated [3H]estrone sulfate
uptake (Fig. 7). Inhibition by taurocholate, cholate, cefoperazone, and
methotrexate was moderate, and that by indomethacin, ouabain, digoxin,
and cationic compounds, such as TEA, guanidine, quinidine, and
verapamil was minimal or not observed at all (Fig. 7). Acidic
metabolites of neurotransmitters derived from dopamine, epinephrine,
and norepinephrine, such as 4-hydroxy-3-methoxyphenylacetic acid,
3,4-dihydroxyphenylacetic acid, 4-hydroxy-3-methoxymandelic acid, and
3,4-dihydroxymandelic acid, and those from serotonin, such as
5-methoxyindol-3-acetic acid, 5-hydroxyindol-3-acetic acid and
5-methoxytryptophol potently inhibited the uptake of
[3H]estrone sulfate by OAT3-expressing oocytes, while
3,4-dihydroxymandelic acid did not (Fig.
8).

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Fig. 7.
Inhibition of OAT3-mediated
[3H]estrone sulfate uptake by various compounds. The
concentration used of [3H]estrone sulfate was 40 nM and those of the inhibitors were 1 mM
(except for sulfobromophthalein and digoxin, 250 µM). The
values were expressed as a percentage of OAT3-mediated
[3H]estrone sulfate uptake in the absence of the
inhibitor (mean ± S.E.; n = 10).
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Fig. 8.
Inhibition of OAT3-mediated
[3H]estrone sulfate uptake by acidic metabolites of
neurotransmitters. The concentration used of
[3H]estrone sulfate was 40 nM and those of
the inhibitors were 1 mM. The values were expressed as a
percentage of OAT3-mediated [3H]estrone sulfate uptake in
the absence of the inhibitor (mean ± S.E.; n = 10).
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 |
DISCUSSION |
In the present study, we report the isolation of multispecific
organic anion transporter 3 (OAT3). OAT3 encodes a 536-amino acid
protein, which shows 49, 39, 36, and 35% identity to rat OAT1, OAT2,
OCT1, and OCT2, respectively (Table I). A search of a DNA data base
during the revision of this manuscript revealed that OAT3 shows 92%
identity to a newly published sequence of Roct (reduced in
osteosclerosis transporter) (22) in amino acid level. Although its
function has not been elucidated, the reduced expression level of Roct
was observed in the oc mouse (a model animal for osteopetrosis) (22).
Accordingly, we speculate that Roct is a murine homologue of OAT3.
When expressed in X. laevis oocytes, OAT3 mediated the
uptake of PAH, estrone sulfate, ochratoxin A, and cimetidine in a
sodium-independent manner. In contrast to the fact that OAT1 is
apparently an exchanger, we did not observe any
trans-stimulatory effect in OAT3-mediated transport of
estrone sulfate. trans-Stimulatory effect was not also
observed in the experiments using OAT2 (11); intracellulary accumulated
unlabeled dicarboxylate, a substrate of OAT2, did not stimulate the
uptake of salicylate via OAT2 (11). These results suggest that the
transport characteristic(s) of OAT2 and OAT3 may be different from
those of OAT1. There seems to be the other possibility that
unidentified endogenous substrate(s), which is abundantly existing in
the X. laevis oocytes, may mask the trans-stimulatory effect. Whether OAT2 and OAT3 surely
catalyze one way influx remains to be clarified in further studies.
Since the uptake of estrone sulfate via OAT3 was not
cis-inhibited by organic cations (TEA, guanidine, verapamil,
and quinidine), OAT3 is considered to specifically recognize organic
compounds with anionic moieties. Nonetheless, OAT3 transports
cimetidine, which is a weak base (Fig. 3). Cimetidine is known to be a
bisubstrate, because the uptake of cimetidine into isolated proximal
tubular cells of the rat was inhibited by both probenecid and TEA (23). Ullrich et al. (24) investigated the recognition of
bisubstrates by the renal organic anion and cation transporters using
the stop-flow peritubular capillary microperfusion method. They
demonstrated that hydrophobic imidazole analogues with electronegative
side group, such as cimetidine, can interact with both transporters, although imidazole by itself did not interact with the renal organic anion transporter. From these results, they emphasized on the importance of hydrophobicity and partial charge, but not net charges of
the substrates. Interaction of bisubstrates with both organic anion and
cation transporters may be explained by the structural similarity of
the OAT family and OCT family (Table I). Based on these hypothesis, a
family including both the OAT and OCT family was proposed (8, 25). Now
the molecular structures of three organic anion transporters and three
organic cation transporters have been revealed. The analyses of the
sequence of these transporters may provide clues as to the substrate
binding sites of the transporters, especially the sites for charge(s) recognition.
There exist both similarities and differences in substrate selectivity
and transport properties among members of the OAT family. OAT1, OAT2,
and OAT3 are all multispecific transporters, and PAH is a common
substrate; however, their affinity for, and rates of transport of PAH,
are different among the OAT family. Kinetic analysis revealed that the
Km value of OAT3 for PAH was 65 µM,
which is 4-fold larger than the previously reported value determined
using OAT1-expressing oocytes (8). Although the expression level of
OAT1 and OAT3 in oocytes is not normalized, Vmax
values for PAH were a 10-fold difference between OAT1 and OAT3 (~240
pmol/h/oocyte for OAT1 and 24 pmol/h/oocyte for OAT3). Thus, the
clearance for uptake at tracer concentrations (CLuptake) defined as the Vmax/Km of
PAH, is 40-fold different between OAT1 and OAT3. Another member of the
OAT family, OAT2, also showed relatively small transport activity for
PAH compared with that of OAT1 (11). This difference in the transport
activity for PAH among the members of the OAT family may be related to
the fact that PAH is excreted mainly in the urine. Other than PAH, there are distinct differences in substrate selectivity between OAT1,
OAT2, and OAT3. Indomethacin is a potent inhibitor of renal organic
anion transporter (OAT1); however, their inhibitory activity on the
transport by OAT3 is weak. On the contrary, benzylpenicillin strongly
inhibited OAT3-mediated PAH uptake, but only slightly inhibited
OAT1-mediated transport, suggesting that the affinity of
benzylpenicillin for OAT1 is weaker than that for OAT3. Actually, benzylpenicillin is a good substrate for OAT3 rather than for OAT1 on
the basis of Km value and transport activity of
benzylpenicillin (data not shown). OAT2 transports salicylate, but not
estrone sulfate, and vice versa, OAT3 transports estrone sulfate but
not salicylate. Thus, OAT1, OAT2, and OAT3 have distinct transport
properties, despite the fact that they belong to the same transporter
family. The relatively low structural similarities among OAT1, OAT2,
and OAT3 (less than 50% identity each other) may be relevant to
these functional differences.
A Northern blot analysis revealed the expression of OAT3 in the brain.
OAT1 and OAT2 are expressed predominantly in the kidney and liver,
respectively. The strong expression of OAT3 in the brain suggests its
physiological role in the brain such as in the efflux pathway via the
BBB and/or the BCSFB. It has been demonstrated that PAH, when
microinjected into the cerebral cortex, is eliminated from the brain in
a concentration-dependent manner via the BBB (15).
Carrier-mediated efflux transport of benzylpenicillin and cimetidine
from the cerebrospinal fluid have been also demonstrated (16, 18).
Suzuki et al. (16, 18) reported that the transport of
benzylpenicillin was sodium-independent and inhibited by PAH and that
the elimination of cimetidine from the cerebrospinal fluid was
inhibited by benzylpenicillin and PAH, but not by
N1-methylnicotinamide, a typical substrate for
organic cation transporters. These results suggest that the efflux
transport properties of organic anions via the BBB and/or BCSFB are
consistent with transport by the OAT family, especially OAT3. Since PAH
and cimetidine are transported by OAT3, and benzylpenicillin inhibits
the uptake of estrone sulfate by OAT3, OAT3 may be responsible for the
efflux transport of these organic anions via the BBB and/or BCSFB. In fact, a preliminary immunohistochemical analysis revealed the positive
staining of OAT3 in the choroid plexus (data not shown). In addition to
these exogenous compounds, metabolites of neurotransmitters are also
candidate substrates for OAT3. After being released from presynaptic neurons, certain amounts of neurotransmitters are metabolized by monoamine oxidase and
catechol-O-methyltransferase. Finally, anionic compounds
such as 3,4-dihydroxyphenylacetic acid, 4-hydroxy-3-methoxyphenylacetic
acid, 3,4-dihydroxymandelic acid, 4-hydroxy-3-methoxymandelic acid,
5-methoxyindol-3-acetic acid, 5-methoxytryptophol, and
5-hydroxyindol-3-acetic acid, are produced. Since all the anionic
metabolites, except 3,4-dihydroxymandelic acid, inhibited organic anion
transport via OAT3, they may be endogenous substrates of OAT3. Future
immunohistochemical analysis will reveal the distribution/localization
of OAT3 in the brain and provide clues to understanding the more
precisely physiological role of OAT3.
Oatp1 and oatp2 are also multispecific organic anion transporters
expressed in the brain. Their substrate specificities are quite similar
except digoxin; oatp2 transports digoxin, but oatp1 does not (7). Oatp1
has been demonstrated to be localized specifically on the brush border
membrane of choroid epithelial cells (26), while the localization of
oatp2 in the brain has not been determined. There are common substrates
for OAT3 and the members of oatp family (e.g. estrone
sulfate and ochratoxin A); however, the substrate selectivity of these
two families overlaps little. The transporter(s) responsible for the
efflux transport, via BBB and/or BCSFB, of naphthol glucuronide,
dideoxyinosine, azidodeoxythymidine, and valproic acid has not been
identified (27-30). The functional analysis and localization of oatp1,
oatp2, and OAT3 will clarify the molecular mechanisms of the organic
anion transport in the brain.
In conclusion, the third member of the multispecific organic anion
transporter family was isolated from the rat brain. Among the members
of the OAT family, OAT3 is the most abundantly expressed in the brain.
The substrate selectivity of OAT3 is different from the substrate
selectivities of OAT1 and OAT2, and the characteristics of transport
via this transporter are similar to those of the previously reported
efflux transport system in the brain. Localization and further
functional analysis of OAT3 will provide important information on the
distribution and transport of organic anions, especially in the brain.
 |
FOOTNOTES |
*
This work was supported in part by grants from the Japanese
Ministry of Education Science, Sports and Culture, the Science Research
Promotion Fund of the Japan Private School Promotion Foundation, the
Uehara Memorial Foundation, and CREST (Core Research for Evolutional
Science and Technology) of Japan Science and Technology Corporation (to
J. S. T.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB017446.
To whom correspondence shoud be addressed: Dept. of
Pharmacology and Toxicology, Kyorin University School of Medicine,
6-20-2 Shinkawa, Mitaka, Tokyo 181-8611, Japan. Tel.: 81-422-47-5511 (ext. 3451); Fax: 81-422-79-1321; E-mail: endouh{at}kyorin-u.ac.jp.
 |
ABBREVIATIONS |
The abbreviations used are:
oatp, organic anion
transporting polypeptide;
PAH, para-aminohippuric acid;
TEA, tetraethylammonium;
OAT, organic anion transporter;
MES, 4-morpholineethanesulfonic acid;
PCR, polymerase chain reaction;
OCT, organic cation transporter;
NLT, novel liver-specific transport;
BBB, blood-brain barrier;
BCSFB, blood-cerebrospinal fluid barrier.
 |
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